Natural Organic Matter and Humic Substances Interactions

ADVANCES IN NATURAL ORGANIC MATTER 

AND HUMIC SUBSTANCES RESEARCH 

2008-2010 

XV Meeting of the 

International Humic Substances Society 

Puerto de la Cruz, Tenerife, Canary Islands, 27 June - 2 July 2010 

Proceedings 

Vol 3 

J.A. González-Pérez, F.J. González-Vila, G. Almendros Eds

15th IHSS Meeting- Vol. 3 

ADVANCES IN 

NATURAL ORGANIC MATTER 

AND 

HUMIC SUBSTANCES RESEARCH 

2008-2010 

Vol. 3 

POSTER PRESENTATIONS 

Proceedings Book of the Communications presented to the 

15 th Meeting of the International Humic Substances Society 

Tenerife - Canary Islands. June 27- July 2, 2010 

Editors: 

J.A. González-Pérez, F.J. González-Vila & G. Almendros 

Vol. 3 Page - 1 -


Maquetación: Carlos Marfil Daza 

Published on-line in: Digital.CSIC (http://digital.csic.es/), the Institutional 

Repository of “Consejo Superior de Investigaciones Científicas” (CSIC). 

© 15 th Meeting of the IHSS, Tenerife, Canary Islands. 2010 

URI: 

Vol. 3 Page - 2 -


CONTENT Vol. 3 

POSTER PRESENTATIONS SESSION B 

Natural Organic Matter and Humic Substances in 

Aquatic Systems and Sediments 

WAT1 (1) Characterization of humic acid from the river bottom sediments of 

Burigonga: complexation studies of metals with humic acid. M.A. Rahman, A. Hasan, A. 

Rahim, A.M. Shafiqul Alam 

WAT2 (5) Adsorption of fulvic acids by activated carbon. O. Samsoni-Todorova, L. 

Savchyna, N. Klymenko 

WAT3 (19) Examining the effect of humic acid on gag pipe corrosion in sea water. 

A.R.Sardashti, R.Kafian 

WAT4 (33) Color removal by coagulation from water containing aquatic humic 

substances with different apparent molecular size. E. Sloboda, C. Tolledo Santos, A. Di 

Bernardo Dantas, L. Di Bernardo, E.M. Vieira 

WAT5 (57) pH effect in aquatic fulvic acid from Brazilian river. S. da Costa Saab, E.R. 

Carvalho, R.B. Filho, M. R. de Moura Aouada, L. Martin-Neto, L.H.C. Mattoso 

WAT6 (75) The role of organic matter in the transport of suspended minerals in the 

estuarine zone. E.V. Lasareva, A.M. Parfenova, E.A. Romankevich 

WAT7 (86) Organic material of uneven-age anthropogenic origin lakes. S. Zalmanova 

WAT8 (99) Comparative measurement of hydrophobic organic matter dissolved in 

water by the XAD resin method and the polarity rapid assessment method (PRAM). M. 

Philibert, A. Revchuk, D. Quiros, A. Roh, M. Suffet 

WAT9 (122) Microbial changes in the spectroscopic characteristics and molecular 

weight of dissolved organic matters extracted from diverse source materials. J. Hur, B.- 

M. Lee, T.-H. Lee, K.-Y. Jung 

WAT10 (165) Dynamics of humic matters in fen bog water in conditions of climate 

change. E.S. Ivanova, E.S. Voistinova, J.A. Kharanzhevskaya 

WAT11 (168) Structural characteristics of deep groundwater humic substances in 

Horonobe area, Hokkaido, Japan. M. Terashima, S. Nagao, T. Iwatsuki, Y. Sasaki, Y. 

Seida, H. Yoshikawa 

WAT12 (197) Browning of stream water during hydrological events. D.O. Andersen 

WAT13 (199) Fluxes of natural and combustion-derived organic matter into the coastal 

ocean off Southern Brazil. D.C. Podgorski, J.Y. Paeng, T. Dittmar, M.S.M.B. Salomao, C.E. 

Rezende, M.C. Bernardes, B. Cooper 

WAT14 (203) The changes of water organic contamination under the influence of 

ultrasounds. L. Stepniak, E. Stanczyk-Mazanek, U. Kepa 

WAT15 (238) Unexpected uniformity of humic substances in thermal waters. K. Kovács, 

C. Sajgó, A. Brukner-Wein, Z. Kárpáti, A. Gáspár, E. Tombácz, P. Schmitt-Kopplin 

Vol. 3 Page - 3 -


WAT16 (240) Basic by-products formation during chlorination of water containing 

humic substances. E.V. Trukhanova, M.Y. Vozhdaeva, L.I. Kantor, E.A. Kantor 

WAT17 (261) Research of the physics and chemical properties on sediments of the 

Lobelia lakes in West Pomeranian region of Poland. L. Mielnik, J. Czekała 

WAT18 (282) Ratio of color to chemical oxygen demand as an indicator of quality of 

dissolved organic matter in surface waters. A.I. Konstantinov, N.S. Latyshev, P.A. Ivkin, 

I.V. Perminova 

WAT19 (313) UV-Vis spectrometry and size-exclusion chromatography study of 

seasonal dynamics of quality of dissolved organic matter. A.I. Konstantinov, E.V. 

Trukhanova, M.Y. Vozhdaeva, L.I. Kantor, I.V. Perminova 

WAT20 (324) Humin contribution to sedimentary organic matter the Adriatic Sea. F. 

Rampazzo, D. Berto, M. Giani, L. Langone 

WAT21 (342) Influence of pre-ozonation of solutions of fulvic acid on equilibrium 

adsorption on activated carbon. I. Kozyatnyk, L. Savchyna, N. Klymenko 

WAT22 (358) Study of estuarine sediments in Galway Bay. R. Mylotte, M.H.B. Hayes, C. 

Dalton 

WAT23 (364) Effect of river floods on marine organic matter fluorescence. E. Parlanti, S. 

Relexans, F. Ibalot, S. Huclier-Markai, R. Nicolau, S. Mounier, Y. Lucas 

WAT24 (365) Study of colloidal organic matter transformation processes at superficial 

sediment interfaces. E. Parlanti, S. Relexans, D. Amouroux, R. Bridou, S. Bouchet, G. Abril, 

H. Etcheber 

WAT25 (369) Resolving ahthropogenic and natural organic matter using hypy. X. 

Zhang, C.E. Snape, W. Meredith, Y. Sun 

WAT26 (370) Organomineral association patterns of humic substances in different 

Venezuelan estuarine mangroves. A. Méndez, Z. Hernández, G. Almendros, X.L. Otero, F. 

Macías, W. Meléndez 

Natural Organic Matter and Humic Substances Interactions 

INT1 (7) Relationship between organic carbon forms and selected trace elements in 

grassland soils. L. Pospíšilová, P. Škarpa, V. Petrášová, M. Konečná" 

INT2 (9) Investigation of humic substances by particle size distribution of soils and by 

determination of zeta potential. S. Joó, J. Tóth, G. Samu, R. Földényi 

INT8 (41) Interactions of organic compound with NOM need water: strong waterinduced 

enhancement of carbamazepine sorption on peat. M. Borisover, M. Sela, B. 

Chefetz 

INT9 (44) Adsorption of metal ions on humic acid derived from Turkish lignite. B.Z. 

Uysal, D. Öztan, U.G. Zafer, Ö.M. Doğan, S. Anaç, M. Özdingiş, Z. Olgun 

INT10 (48) Characteristics of humic acids isolated from heavy metals contaminated soils 

at the copper-smelter “Legnica” (S-W Poland). A. Maciejewska, J. Kwiatkowska-Malina 

Vol. 3 Page - 4 -


INT11 (50) The interaction of Cu 2 + with humic acids of different soils. G.V. Motuzova, 

H. Dergam, A.A. Stepanov 

INT12 (53) Influences of humic acids on the pattern of oxidation products of 

tetrabromobisphenol A derived from a catalytic system using Iron(III)-tetrakis(psulfophenyl)porphyrin 

and KHSO5. M. Fukushima, Y. Ishida, S. Shigematsu 

INT13 (54) Adsorption of trihalomethanes by humin: batch and fixed bed column 

studies. G. da Costa Cunha, L.P. Cruz Romão, M. Cardoso Santos, B.R. Araújo, S. 

Navickiene, V.L. de Pádua 

INT14 (56) Combined effects of humic matter and surfactants on PAH solubility: Is 

there a mixed micellization? H. Lippold 

INT16 (76) Complexation of Copper(II) ions with humic acids and EDTA studied by 

high resolution ultrasonic spectrometry. M. Klucakova, M. Pekar 

INT17 (80) CE-ICP-MS as speciation technique to analyze the complexation behavior of 

Europium, Gadolinium and Terbium with humic acid. C. Möser, R. Kautenburger, H.P. 

Beck 

INT19 (92) Determinations of ability of extracted HSs coordinated with metal ions. K.-F. 

Ding, Q.-H. Fan, Y.-Y. Zhang, W.-S. Wu 

INT22 (102) Characterization of zinc binding ability of dissolved hydrophilic organic 

matter from the Seine River and major wastewater effluents. Y. Louis, B. Pernet- 

Coudrier, G. Varrault 

INT23 (108) Molecular size distribution of metal complexes with pore water dissolved 

organic matter determined by HPSEC and ICP-MS. N. Makarõtševa, V. Lepane, T. 

Alliksaar 

INT24 (160) Influence of Pb(II) ions on semiquinone radicals of humic acids and modle 

compounds. J.M. Jerzykiewicz, M. Witwicki 

INT25 (164) The effect of natural organic matter on the formation and solubility of 

M(OH)4 solid phases (Th(OH)4, Zr(OH)4 Ce(OH)4). S. Antoniou, I. Pahalidis 

INT27 (172) Adsorption of polycyclic aromatic hydrocarbons (PAHs) onto engineered 

and natural nanoparticles. L. Marino, D. Mondelli, N. Senesi 

INT28 (174) The challenge of building a humic-metal binding constants database. M. 

Filella, W. Hummel, P.M. May, J. Puy, F. Quentel 

INT29 (175) Heavy metal compounds with organic substance and methods of their 

definition. T.M. Minkina, G.V. Motuzova, O.G. Nazarenko, S.S. Mandzhieva 

INT30 (183) Concentrations of iron in the interactions of some acid ones organic with 

minerals. C.F.D. Bassan; A.A. Paccola; P. de Magalhães Padilha 

INT31 (215) Fluorescence study of adsorption mechanisms of flubendiamide onto humic 

acids. I. Cavoski, V. D’Orazio, T. Miano 

INT33 (243) Size exclusion characterization of dissolved organo-mineral complexes in 

soils of the Southern Far East. T.N. Lutsenko, A.S. Volk 

INT35 (251) Sorption of pharmaceuticals to humic substances. H. Mori, T. Ohtani, I. 

Fukuda, H. Ashida, N. Fujitake 

Vol. 3 Page - 5 -


INT36 (258) Influence of aromaticity degree on the aggregation of Humic Substances. 

M. Drastík, J. Kučerík, O. Zmeškal, A. Čtvrtníčková, F. Novák 

INT37 (283) Aggregation of humic acids in solution. Vapor pressure osmometry, 

conductivity and mass spectrometric study. E.M. Peña-Méndez, D. Fetsch, J. Havel 

INT38 (290) Sorption of silanol-modified humic acids onto different solid supports 

including silica gel, clay and sand. I.V. Dubinenkov, A.B. Volikov, V.A. Kholodov, E.M. 

Garanin, I.V. Perminova 

INT39 (294) Imprinted humics-based sorbents as selective trap for metal Ions. E. 

Kasymova, R.P. Koroleva, E.M. Khudaibergenova, N. Hertkorn, S.J. Jorobekova, A.D. 

Pomogailo, K. Kydralieva 

INT41 (307) Flow injection analysis (FIA) for fast monitoring of gold nanoparticles 

formation from various precursors and theirs separation by using humic acids. E.M. 

Peña-Méndez, A.I. Jiménez Abizanda, J.J. Arias León, J. Havel 

INT42 (312) Spectrofluorimetric study of the interaction of Gold (III) and humic acids 

under the formation of gold nano-particles. E.M. Peña-Méndez, F. Jiménez Moreno, J.E. 

Conde González, J. Havel 

INT43 (329) Metal binding by humic acids extracted from recent sediments from the 

SW Iberian coastal area. J.M. de la Rosa, M. Santos, F.J. González Vila, H. Knicker, J.A. 

González Pérez, M.F. Araújo 

INT44 (363) Polycyclic aromatic hydrocarbons (PAHs) - dissolved organic matter 

(DOM) interactions studied by solid phase microextraction (SPME). C. De Perre, K. Le 

Menach, A.-M Dorthe, C. Béchemin, H. Budzinski, E. Parlanti 

Environmental Applications of Natural Organic Matter 

and Humic Substances 

ENV1 (32) Use of humin for removal of phosphorus from sewage treatment station 

effluents: influences of time and pH. L. Camargo de Oliveira, W.G. Botero, A.G. Ribeiro 

Mendonça, J.C. Rocha, A. dos Santos, A.H. Rosa 

ENV2 (37) Phytoremediation of a soil polluted with multiple heavy metals using MSW 

compost as organic carbon source. K. Farrag, G. Brunetti, P. Soler, F. Nigro 

ENV3 (55) Hydrogels filled with humic-rich lignite for various environmental 

applications. M. Pekař 

ENV4 (125) Mitigation of GHGs emission from soils by a catalyzed in situ photooxidative 

polymerization of soil humic molecules. A. Piccolo, R. Spaccini 

ENV6 (155) Sorption of endocrine disruptors by humic substances from sediment 

samples collected on Guarapiranga reservoir, São Paulo state-Brazil. B. Barboza Cunha, 

W.G. Botero, L. Camargo de Oliveira, G. Carvalho Leite, D. Goveia, V. Moschini Carlos, 

M.L. Martins Pompêo, L. Cardoso de Morais, L. Fernandes Fraceto, A. Henrique Rosa 

ENV7 (180) Dual effect of humic acid on the degradation of pentachlorophenol by 

Iron(II) and H2O2. K.C. Christoforidis, M. Louloudi, Y. Deligiannakis 

Vol. 3 Page - 6 -


ENV8 (191) Radiotracer method in the investigation of humic substances sorption on 

carbon-based nanomaterials. M.G. Chernysheva, G.A. Badun 

ENV9 (195) Study of flow-through sample preparation methods for group of pesticides 

determination in soil by reversed-phase high-performance liquid chromatography. M. 

Chalányová, M. Hutta, I. Procházková 

ENV11 (227) Humus substance role in technogenic soil formation at Priokhotie mining 

industry developments. A.F. Makhinova, A.N. Makhinov 

Industrial Production and Commercial Applications 

IND2 (49) Study of copper extraction efficiency by humic acid/polypyrrole on paraffinimpregnated 

graphite electrode. M. Antilén, M. Araus, M. Pérez, F. Armijo, R. del Río, 

M.A. del Valle 

IND3 (71) Sorption of silanized humic derivatives onto montmorillonite clay. V.A. 

Kholodov, V.M. Zelikman, K. Hatfield, I.V. Perminova 

IND4 (87) Peat humic acids as the redox mediators for textile technologies. I.Y. 

Vashurina, Y.A. Kalinnikov, I.V. Perminova 

IND5 (124) Peat humic acids as surfactants. O. Purmalis, M. Klavins 

IND6 (128) Efficiency and application prospect of humatized mineral fertilizers. 

O.Gladkov, R. Poloskin. G.O. Andreevich, P.R. Borisovich 

IND7 (139) Physical-chemical properties and application potential of humates prepared 

from regenerated lignites. J. David, J. Kučerík 

IND9 (178) Removal of tributyltin biocide by using black carbon. L. Fang, O.K. 

Borggaard, H. Marcussen, P.E. Holm, H.C.B. Hansen 

IND10 (221) Evaluation of the efficiency of fulvic and humic acids (Agrolmin Bravo and 

Cerrado) in soybean production in the Brazilian savanna. L.T. Dias Cappelini, D. 

Cordeiro, L.A. Artimonte Vaz, L.F. Artimonte Vaz, E.B. Azevedo, E.M. Vieira 

IND11 (274) Pyrolisis parameters evaluation in the biochar preparation process. E.I.P. 

de Rezende, A.P. Mangoni, I. Messerschmidt, A.S. Mangrich, E.H. Novotny, M.H. R. 

Velloso 

IND12 (277) Extraction of high-value lipids from Irish peats. R. McInerney, D.J. Hayes, 

J.J. Leahy, M.H.B. Hayes 

IND13 (278) Fluorescence of aqueous solutions of commercially produced humic 

substances. O. Yakimenko, A. Izosimov, D. Shubina, V. Yuzhakov, S. Patsaeva 

IND17 (326) Assessment of the oil shale byproducts use as soil conditioner: study of 

sorption and biodegradation of phenol models with soil. R. Garrett Dolatto, G. Abate, I. 

Messerschmidt, B. Fraga Pereira, A.S. Mangrich, C. Posser Silveira, C.N. Pillon 

IND18 (330) Humic acids from fines of residual coal type material: preparation and 

characterization. G.M. Maurício, A.C.S. Wimmer, E.A. Brocchi, A.C. Vidal, R.A. Nunes 

Vol. 3 Page - 7 -


IND19 (350) Assessing the effect of a bio-accelerated composting process using 

analytical pyrolysis (Py-GC/MS). F. Pérez-Barrera, K. Akdi, F.J. González-Vila, J.A. 

González-Pérez, T. Verdejo 

Natural Organic Matter and Humic Substances Biological 

and Physiological Effects 

PHY1 (10) A turning point of wheat breeding and humic substances. R. Shahryari 

PHY2 (17) Response of maize genotypes to changes in chlorophyll content at presence of 

two types humic substances. R. Shahryari, B.S. Moghanlou, A.M. Pour Khaneghah 

PHY4 (31) Bioactivity of chemically transformed humic matter on plant root growth. 

L.P. Canellas, L.B. Dobbss, F.L. Olivares, N.O. Aguiar, L.E.P. Peres, R. Spaccini, A. Piccolo, 

A.R. Façanha 

PHY5 (35) Effect of two humic substances as bifertilizers on germination and seedling 

growth of maize genotypes. R. Shahryari, N. Bahari, M. Khayatnejad 

PHY6 (107) Comparative evaluation of the inhibitory action of compost humic fractions 

on two soil-borne phytopathogenic fungi. A. Traversa, E. Loffredo, N. Senesi 

PHY9 (147) Effect of liquid humic compounds extracted from plant based-compost to 

soil microorganisms. F. Suárez-Estrella, M.C. Vargas-García, G. Guisado, M.J. López, J. 

Moreno 

PHY11 (235) Analysis of the sorption properties soils after the application of sewage 

sludges and conventional organic fertilizers. E. Stańczyk-Mazanek, L. Stępniak, U. Kępa 

PHY13 (273) Interactions between plant-root exudates and soils in extracting humic-like 

substances. D. Pizzeghello, A. Muscolo, A. Ertani, S. Nardi 

PHY14 (276) Bioactivity of humic acids from vermicompost at increasing maturity 

stages. N.O. Aguiar, L.P. Canellas, F.L. Olivares, J.G. Busato, LG.JR.S. Silva; E.H. Novotny, 

A.R. Façanha 

PHY15 (281) Root Growth promotion by humic acids from urban organic residues. K. 

Jindo, C. García-Izquierdo, L.P. Canellas 

PHY16 (284) Direct and indirect effects of humic substances of different origin on the 

green algae Monoraphidium braunii. C.E. Gattullo, H. Bährs, J. Qianru, C.E.W. Steinberg, 

E. Loffredo 

PHY17 (318) Effects of compost water-extracts on the germination and growth of 

slickspot peppergrass (Lepidium papilliferum). E. Loffredo, A.J. Palazzo, A. Traversa, T.L. 

Bashore, N. Senesi 

PHY18 (328) The action of humic acids promoting plant shoot development are 

associated with nitrate-related changes on the plant hormonal balance. V. Mora, E. 

Bacaicoa, E. Aguirre, R. Baigorri, M. Garnica, M. Fuentes, A.M. Zamarreño, J.C. Yvin, J.M. 

García-Mina 

Vol. 3 Page - 8 -


Health and Medical Applications of Natural Organic Matter 


MED1 (2) Inclusion complexes of aspirin in fulvic acid offer enhanced dissolution, 

permeability, stability and better pharmacodynamics. K. Anwer, A. Mirza, S.P. Agarwal, 

A. Ali, Y. Sultana 

MED3 (84) The effect of fulvic and humic acid supplementation on the intensity of the 

immune response in rats. A.V. Vucskits, I. Hullár, E. Andrásofszky, N. Hetényi, J. Csicsor, 

A. Móré, J. Szabó 

MED4 (85) Evaluating potential nephrotoxicity of compost derived humic acid to 

African mud catfish (Clarias gariepinus) grown in static water culture. I.M. Adekunle, 

O.R. Ajuwon 

MED5 (145) Treatment of pilonidal sinus by humic acid salts. M. Dizman, A. Tutar 

MED6 (194) Stabilization of iron oxide magnetic nanoparticles with different 

morphology in aqueous suspensions using humic substances. A.Y. Polyakov, A.E. Goldt, 

T.A. Sorkina, E.A. Goodilin, I.V. Perminova 

MED7 (241) Neutralisation of the anticoagulant effect of naturally occurring humic 

acids and synthetic humic acid-like polymers by protamine sulphate. H.P. Klöcking, N. 

Mahr, S. Kunze, R. Klöcking 

MED8 (289) Protolytic properties of alkoxysilylated versus natural humic materials 

aimed at use as stabilizers for magnetic fluids. T. Sorkina, A. Goldt, A. Polyakov, A. 

Dubov, I. Toth, A. Hajdu, E. Goodilin, E. Tombacz, I. Perminova 

MED9 (321) Halogen-free preparation and preliminary characterization of humic 

substances from different substrates. C. Kleiner, C. Barthel, R. Junek, R. Schubert, J.I. 

Schoenherr, R. Klöcking 

Young Researchers in Humic Substances and 

Natural Organic Matter IHSS Travel Award) 

TA1 (4) Influence of surface chemistry and structure of activated carbon on adsorption 

of fulvic acids from water solution. T.V. Poliakova, L.A. Savchynaand, N.A. Klymenko 

TA2 (16) Studies by chimiometric methods of the interaction between Pb(II) and humic 

acids. S. Orsetti, E. Andrade; F. Molina 

TA3 (193) Seasonal dynamics of biomass and copper concentrations in ectohumus of 

forest soils impacted by copper industry in South-West Poland. A. Medyńska, C. Kabala 

TA4 (207) Limitation for study of humic substances or NOM using high resolution and 

accuracy mass-spectrometry. G. Vladimirov, E. Nikolaev 

TA5 (220) A study of interaction between pharmaceuticals and humic substances. L. 

Ansone, M. Klavins 

Vol. 3 Page - 9 -


TA6 (222) Characterization of soil organic matter of treated sewage effluent irrigated 

areas. B.H. Martins, L. Macedo dos Santos, D.M.B.P. Milori, L. Martin-Neto, C.R. Montes 

TA7 (234) Modelling differential absorbance spectra of SRFA during complexation with 

copper and lead. D.J. Dryer, G.V. Korshin, M.F. Benedetti 

TA8 (255) Behavior of soil carbon in amended areas with sewage sludge. B.H. Martins, 

T.L. de Almeida, S. Gaiad, D.M.B.P. Milori, L. Martin-Neto 

TA9 (257) Gold(III) and nanogold interaction with humic acids: spectrophotometry, 

capillary electrophoresis and mass spectrometric study. N.R. Panyala, E.M. Peña-Méndez, 

J. Havel 

TA10 (259) Abiotic treatment of rice bran using an accelerator including organo-iron 

compound. H. Kanno, N. Tachibana, M. Fukushima 

TA11 (266) Effect of humic substances on uranium removing by bacterium Bacillus 

polymyxa IMV 8910 from aqueous solution. I. Shevchuk, N. Klymenko 

TA12 (360) Humic acids inspired hybrid materials as heavy-metal adsorbents. P. Stathi, 

Y. Deligiannakis 

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Vol. 3 Page - 11 -


Natural Organic Matter and Humic Substances in Aquatic Systems 

and Sediments 

Vol. 3 Page - 12 -

Characterization of Humic Acid From the River Bottom Sediments of 

Burigonga: Complexation Studies of Metals with Humic Acid 

Mohammad Arifur Rahman * , Abu Hasan, Abdur Rahim and A. M. Shafiqul Alam 

1. Introduction 


Department of Chemistry, University of Dhaka, Dhaka-1000, Bangladesh 

E-mail: marahman76@yahoo.com 

Humic substances are ubiquitous in waters, being found wherever organic matter is decomposed or 

has been transported. Their importance in agriculture and soil sciences has been acknowledged for 

over 150 years. Aquatic scientists have been slower in appreciating their importance, but now realize 

that they may constitute as much as 95% of the total dissolved organic matter in aquatic systems and 

often are equal to or greater than the concentrations of inorganic ions present. Moreover, the 

structures, molar masses and functional groups of humic acid vary depending on origin and age [1, 2]. 

The river Buriganga, which runs by the side of the Dhaka city, is at present one of the most polluted 

rivers in Bangladesh. The amount of untreated wastes, both domestic and industrial, being released in 

the Buriganga is tremendous and increasing day by day. So, the structure of humic acid in the river 

Buriganga would be different. Therefore, it is required to determine the structure of humic acid of the 

Buriganga River in Bangladesh. 

Iron is a transition metal. High concentration of iron in the river water made scaling in the boiler of 

water purification system. Cadmium is not only heavy metal but also toxic in nature. It is one of the 

most harmful elemental pollutants and is of particular concern because of its toxicities to humans. 

Pollutant cadmium in water may arise from industrial discharges. 

Humic acid represent the dominant part of dissolved organic matter in freshwater supplies [3, 4]. The 

elimination of metals and humic acid upon drinking water treatment is mainly performed coagulation 

with hydrolyzed metal species. Considerable attention has then been focused on this removal step as 

uncoagulated humic materials lead to severe in the following treatment stages. Indeed. Membrane 

fouling, trihalomethanes formation during chlorine disinfection, or biological regrowth in the 

distribution network, have all been linked to the presence of residual humic substance in the clarified 

water [5]. Three main mechanisms are generally invoked to explain the removal of humic acid and 

metals by coagulation: charge neutralization/complexation preferentially applies at acidic pH and finds 

experimental support from stoichiometric relationships between coagulant demand and dissolved 

organic matter concentration, and from suspension restablization upon overdosing [6]. On the other 

hand, under conditions favouring metal hydroxide precipitation, physical enmeshment and/ or 

adsorption onto the freshly formed precipitate are assumed to play a major role in humic substance 

elimination [7]. So, complexation of iron and cadmium with humic acid would be helpful to reduce 

iron and cadmium concentration from the surface water of the river. 

Vol. 3 Page - 13 -

There are many investigations of complexation of metals with humic acid were carried out [1, 8, 9]. 

Moreover, many characterization studies also carried out in different countries. However, molar 

masses and functional groups of humic acid varied with origin and its surrounding environment. Since 

the Buriganga is very much polluted river in Bangladesh its humic acid structure might be different. 

So, the structure of humic acid and its interaction with metals would be different. No information is 

available regarding the structure of humic acid and complexation study of humic acid with metals of 

the Buriganga River. Therefore, it is urgently required to determine the structure of humic acid and 

study of the metals and humic acid complexation of the Buriganga River. This work involves with the 

characterization of humic acid of the Buriganga River and its complexation study with iron (III) and 

cadmium (II). 

2. Materials and Methods 


Sampling: The river bottom sediments in the Burigonga River were collected in 0.15m depth from the 

surface of river bottom sludge at 24 th July (rainy season) in 2008 at 30 o C atmospheric temperature 

from five sampling stations. The sampling areas(23 o 42’N 90 o 24’E to 23 o 92’N 90 o 53’E) are as follows: 

a. Bibi Shaheb’s Ghat, b. Forashgonj Ghat, c. Shahid Shaheb’s Ghat, d. Shagorer Dock and e. 

Talukder Ghat. The sampling sites are located near about to Dhaka (Capital city of Bangladesh). 

Methods: In order to characterize and study of the complexation with metal ions, sediment samples 

were collected from five different places in Burigonga River. The Humic Acids were extracted with 

the following standard procedure [10]. The extracted Humic Acids were characterized with the help of 

IR, FTIR, SEM, EDX and CHNS analyzer. A complexation study of the Humic Acid with iron and 

cadmium was carried out by using SEM, EDX, UV-Visible spectrophotometer and AAS. 

Characterization of Humic Acid and Study of the Metal Complexes: Fourier Transform Infrared 

(FTIR) Spectroscopy (SHIMADZU KN S72-120, Japan), UV-Visible Spectroscopy ((UV-160A, 

SHIMADZU, Japan), Scanning Electron Microscopy (SEM, HITACHI S-3400N) combined with 

Energy Dispersive X-ray (EDX, Princeton Gamma Tech Imix-PC with ultra-thin window detector) 

and CHNS Analyzer (‘Elementar’ Germany) were used to evaluate the structure of humic acid and its 

complexation with metals. Moreover, pH meter (Hanna 210), Conductometer (EYLA) and Atomic 

Absorption Spectrophotometer (AA Analyst 800, Perkin Elmer, USA) were also used to measure pH, 

conductance and the metal concentration in the river Buriganga. 

Complexation Study of Humic Acid and Metals: In order to evaluate the complexation efficiency of 

humic acid with metals accurately 25mg standard humic acid (BDH, England) was taken in 1L 

deionized water for preparing 25 mg/L humic acid. Then 0.5 M NaHCO3 (E Merck, Germany) was 

also added into the suspension to provide a carbonate alkalinity similar to that of natural waters [1]. 

Before the coagulant injection, the pH of synthetic waters was adjusted to pH 6 by drop wise addition 

of 0.1 M HCl [1]. The coagulant, commercial Ferric Chloride (FeCl3) (BDH, England) and Cadmium 

Vol. 3 Page - 14 -

Sulfate (3CdSO4. 7H2O) (BDH (England) of various concentrations 3x10 -4 M, 4x10 -4 M, 5x10 -4 M, 

6x10 -4 M, 7x10 -4 M were injected into 20.0 mL humic acid solution. A rapid mix period for 5 minutes 

at 200 rpm followed by slow stirring 50 rpm for 40 min was done for the mixture. At the end of the 

mixing, the coagulated suspension was allowed to settle in graduated conic plastic holder for 24 hours. 

About 10 mL of supernatant was withdrawn with graduated pipette from upper of solution retained 

after complex formation. pH, Conductivity, Concentration and absorbance were monitored of the 

supernatant with the pH meter, Conductometer, AAS and UV- visible spectrometer with a resonance 

of 254.0 and 436.0 nm respectively [12]. For the comparison study, pH and molar conductance of 

3×10 -4 M, 4×10 -4 M, 5×10 -4 M, 6×10 -4 M, 7×10 -4 M of ferric chloride and cadmium sulfate solution 

were monitored before and after complexation. Finally, the complex of humic acid and metals were 

dried with oven at 50–60 o C. Then the complexes were characterized with SEM and EDX analyzer. 

3. Results and Discussion 


From the characterization study of extracted humic acid with FTIR, it was revealed that humic acid of 

the Buriganga River contain aromatic hydrocarbons, enolic aldehyde/ ketone, carboxylate anions, 

carboxylic acids, intermolecular hydrozen bond (polymeric form) and aryl carboxylic acids etc. Humic 

acid analysis by CHNS showed that the higher C/N ration. These higher C/N values in sediment 

containing higher organic carbon may be produced due to the degradation of organic matter (waste 

materials) present in the Buriganga River. A complexation study of the humic acid with iron and 

cadmium was carried out. The Fe(III):humic acid and Cd(II):humic acid complex were analyzed and 

characterized with AAS, UV-Visible spectrophotometer, SEM, EDX respectively. The complexation 

reaction is mainly responsible for charge neutralization of humic acid entities thus reducing HA 

colloid stability [13]. From this study, it was found that iron and cadmium could make a complex at 

pH 6.0 which was confirmed by EDX spectrum. Since iron and cadmium can make a complex with 

humic acid of the Buriganga River, this complexation or coagulation process can be used to remove 

the high concentration of iron and cadmium from the surface water of this river. We simply assume 

that humic acid can be existed in a low-density network of hydrophobic and hydrophilic moieties. 

Such nanocolloids change conformation according to their ionization state, can be built either from 

amphiphilic polymers or assemblies of small molecules, and accordance with previous models of the 

secondary structure of humic substances [14, 15, 16]. The formation of humic acid aggregates is then 

controlled by the number of coagulant species and two dynamic aspects: (i) the reconformation of 

humic network, and (ii) the collision rate of destabilized particles. An overall shrinkage of anionic 

humic network is indeed expected upon binding cationic coagulant species, which promotes the 

formation of intra- and inter-particle hydrophobic moieties particle hydrophobic domains according to 

the extent of neutralization. This suggests that, in addition to coagulant species, hydrophobic moieties 

participate in the floc build up. The classical aggregation mechanisms proposed in the literature to 

explain the coagulation of humic acid include charge neutralization/precipitation at acidic pH, and 

Vol. 3 Page - 15 -

adsorption and /or sweep-flocculation in a hydroxide precipitate at alkaline pH. From our work, it can 

assumed that a similar charge neutralization/complexation with hydrolyzed iron and cadmium species 

occurs at the acidic pH, thus increasing the number of carboxylic groups available for complexation. 

It should be noted that hydrolyzed iron and cadmium species are known to strongly interact with NOM 

carboxylic groups, and that the humic network remain unaffected even in the overdosages range at 

acid pH with highly charged. As most of features of Buriganga River humic acid are similar to 

freshwater humic acid. The complexation/aggreagation model described above should be general. 

Acknowledge 


The authors acknowledge the International Foundation for Science (IFS) for their financial support to 

carry out this research work. 

References 

1. J.M. Siéliéchi, B.S. Lartiges, G.J.Kyem, S. Hupont, C. Frochot, J.Theime, J.Ghanbaja, J.B. d’Espinose de la 

Caillerie, O.Barres, R.Kamga, P.Levitz,L.J.Michot ;Changes in humic acid conformation during coagulation 

with ferric chloride: Implication for drinking water treatment; Water Res. 42,2111–2123, 2008. 

2. G.Davies and E.A Ghabbor, Humic Substances: Structures, Properties and uses, Royal Society of 

Chemistry, Cambridge, 1998. 

3. E. Tripping, Cation Binding by humic substances: Cabridge University Press: Cambridge, UK, 2002. 

4. J. Zumstein, J. Buffle, Circulation of pedogenic and aquagenic organic matter in a eutrophic lake. Water 

Res. 23 (2), 229–239. 

5. N. Narkis, M. Rebhun, Stoichioche,istry relationship between humic and fulvic acids and flocculants. J. 

AWWA, 325–328, 1977. 

6. C.F. Lin, T.Y., O.J. Hao, Effects of humic substances characterization on UF performance. Water Res. 34, 

1097–1106, 2000. 

7. P. Bose, D. A. Reckhow, Adsorption of natural organic matter on preformed aluminum hydroxide flocs, J. 

Envirn. Eng. 124, 803–811, 1998. 

8. J. Dries, L. Bastiaens, D. Springael, S. Kuypers, S. N. Agathos, L. Diels, Effect of humic acids on heavy 

metal removal by zero-valent iron in batch and continuous flow column systems, Water Res.39, 3531–3540, 

2005. 

9. S. Suteerapataranon, M. Bouby, H. Geckeis, Interaction of trace elements in acid mine drainage solution 

with humic acid, Water Res. 40, 2044–2054, 2006. 

10. R.S Swift, Organic matter characterization. In: D. L. (1996); D.L. Sparks et al. (Ed.), Methods of Soil 

Analysis, Part 3: Chemical methods. SSSA Book, Series No. 5. SSSA and ASA, Madison. 

11. Regina M.B.O. Duarte, Eduarda B.H. Santos, Armando C.Duarte, Spectroscopic characteristics of ultra 

filtration fractions of fulvic and humic acids isolated from an eucalyptus bleached Kraft pulp mill effluent, 

Water Res.37, 4073–4080, 2003. 

12. M. A. Rahman., S. Kaneco, T. Suzuki, H. Katsumata, K. Ohta, A. M. Shafiqul Alam, Development of 

sintering materials by sea sediments and TiO2 for the cleaning technology, Pak. J. Anal. Environ. Chem. 8 

(1 & 2), 2007. 

13. V. Jung, V. Chanudet, J. Ghanbajo, B. S. Lartiges, J. –L. bersillon, Coagulation of humic substances and 

dissolved organic matter with ferric salt: an electron energy loss spectroscopy investigation, Water Res. 39, 

3849–3862, 2005. 

14. K. Ghosh, M. Schnitzer, Macromolecular structures of of humic substances, Soil Sci.129, 266–276, 1980. 

15. Piccolo, The supramolecular structure of humic substances, Soil Sci. 166, 810–832, 2001 

16. J. F. L. Duval, K. J. Wilkinson, H. P. Van Leeuwen, J. Buffle, Humic substances are soft and permeable: 

evidence from their electrophoretic mobilities, Eniviron. Sci. Technol. 39, 2005. 

Vol. 3 Page - 16 -

Adsorption of Fulvic Acids by Activated Carbon 

Olena Samsoni-Todorova a* , Liudmyla Savchyna b , Natalia Klymenko b 

a* National Technical University of Ukraine “ Kyiv Polytechnic Institute”, 37 Peremogy Pr., 

Kyiv 03056, Ukraine; b Institute of Colloid Chemistry and Chemistry of Water, Ukrainian 

National Academy of Sciences, 42 Vernadsky Avenue, Kyiv 03680, Ukraine 

E-mail: samsoni@online.ua 


It is known that the presence of humic substances in nature water complicates of its treatment 

for drinking purposes and it is reason for generation a number of toxic, mutagenic, or 

carcinogenic substances [1, 2]. Unfortunately a little information is available today on the 

molecular structure of fulvic acids, their associativity in the presence of salt ions and their 

molecular weight than on humic acids. This substantially hampers generalization of the 

various experimental results and theoretical substantiation of the most effective operating 

procedure for adsorption units, whether in facilities used for the production of drinking water or 

in technological systems designed to prepare deionized water. 

Our purpose was to obtain the model conceptions of fulvic acids molecules and degree of their 

ionization on the basis of adsorption measurements. The final object is to improve the 

activated carbon use in the water treatment plant. 


We used activated carbon of Akant-meso trade mark that had made by high-temperature water 

steam activation of anthracite modification of the Donetsk Basin deposit. The surface and the 

porous structure of Akant-meso were determined by nitrogen adsorption at 77 K with 

Quantachrom device. 

A fulvic acid preparation was obtained from peat by the Forsyth method [3]. Solutions of 

sodium fulvates at pH 7 and 12 were obtained by adding corresponding quantities of sodium 

hydroxide. Concentrations of fulvic acid solutions were established by the organic carbon 

content which was determined by Shіmadzu TOC-V CSN device. 



It is known that the model conceptions of the chemical structure of fulvic acid molecules can 

be obtained by adsorption measurements. This is achieved by comparing of the limiting 

equilibrium adsorption of fulvic acid (а∞) on activated carbon with a known surface and pore 

Vol. 3 Page - 17 -

size (rp) with the value а∞ calculated by model. It is obvious, that the pore size of activated 

carbon must be equal to the minimum or greater than effective size of researched fulvic acid 

molecules (rmin). 

Sizes of fulvic acid molecules depend on their aggregation and wettability. In fresh water (pH 

5–10, salt content 0.8 nm, meaning that all this surface must be 

Vol. 3 Page - 18 -

accessible to such fulvic acid molecules. The limiting specific adsorption of fulvic acids at pH 

2, which we found from the adsorption isotherm for Akant-meso, was 207.7 mgC/g. The 

quantity of adsorbed fulvic acids corresponding to this value depends on the carbon content in 

the molecule (Table 2). 

Table 2: Relationship between limiting value of fulvic acid adsorption on Akant-meso at pH 2 and 

carbon concentration in molecule and molecular weight 

Carbon concentration 

in molecule, % 

а∞, 

а∞ at (Мw)n, mmol/g 

mg FA/g 800 900 950 980 1000 1100 

48.0 432.7 0.54 0.48 0.45 0.44 0.43 0.39 

49.0 423.8 0.53 0.47 0.45 0.43 0.42 0.38 

50.0 414.5 0.52 0.46 0.44 0.42 0.41 0.38 

Based on the data in Table 2, we calculated, for an Akant-meso surface area of 950m 2 /g, areas 

screened by a fulvic acid molecule ωFA ("landing surface" of a molecule): 

The results obtained are listed in Table 3. 

ωFA = SBEТ(r ≥ 0,8 nm) ⋅ 10 18 / а∞ ⋅ NА. 

Table 3: Areas of "landing surfaces" of fulvic acid molecules in compact adsorption layer 

Carbon concentration in 

ωFA at (Мw)n, nm 2 

fulvic acid molecule, % 800 900 950 980 1000 1100 

48.0 2.92 3.28 3.46 3.57 3.65 4.00 

49.0 2.98 3.35 3.54 3.65 3.72 4.09 

50.0 3.04 3.42 3.62 3.73 3.81 4.19 

As can be seen from the data in Table 3, the area of the "landing surface" varies from 2.92 to 

4.19 nm 2 corresponding to a Van der Waals volume of the molecule of 1.16 to 1.68 nm 3 . These 

values well conform with those reported in the literature. 

At pH 7 and 12 respectively, the carboxyl and the phenol groups of the fulvic acid molecules 

were ionized resulting in an electrostatic repulsion of the anions and an increase in their 

hydration, which reduced the anion packing density and, consequently, the а∞ value. The а∞ 

values for fulvic acids adsorbed on the activated carbon at pH 2, 7, and 12 and their 

proportions are shown in Table 4. 


Vol. 3 Page - 19 -

Table 4: Values of а∞ of fulvic acids adsorbed on activated carbons 

Adsorbent 

а∞, mg C/g at pH 

2 7 12 

Akant-meso 207.7 136.6 91.1 

The largest value of fulvic acid adsorption was registered at pH 2 (see Table 4). Such 

conditions are unacceptable for the production of drinking water but can be realized at thermal 

power plants if the adsorptive water purification of natural organic compounds is carried out 

after the H-cationization stage. As can be seen from Table 4, this would increase the 

efficiency of Akant-meso by 50%. 

4. Conclusions 


It was shown that the adsorption measurements of values of the limiting equilibrium specific 

adsorption of fulvic acids on activated carbon can get conceptions of the fulvic acids 

molecules size in the aqueous solution. It was found that the most efficient activated carbons 

for a thorough removal of fulvic acids from natural waters will be those similar to Akant-meso 

in porous structure, i.e., having an average effective mesopore radius >0.8 nm. 

References 

1. V. V. Goncharuk, N. A. Klimenko, L. A. Savchina, T. L. Vrubel and I. P. Kozyatnik, Journal of 

Water Chemistry and Technology, 28 (2006) 2–49. 

2. G. Garnier, S. Mouner and J. Y. Benaim, Water Res., 38 (2004) 3685–3692. 

3. L. N. Aleksandrova, Organic Soil Matter and Processes of its Transformation [in Russian], 

Nauka, Leningrad, 1980, p. 288. 

4. D. Buffle, Complexation Reactions in Aquatic Systems: An Analytical Approach, Ellis Horwood 

Limited, New York, 1988, p. 692. 

5. A. M. Koganovskii, T. M. Levchenko and V. F. Kirichenko, Adsorption of Dissolved Substances 

[in Russian], Nauk. Dumka, Kiev, 1977, p. 223. 

Vol. 3 Page - 20 -

Examining the Effect of Humic Acid on Gag Pipe Corrosion in Sea Water 

A.R.Sardashti * , R.Kafiana 

Department of Chemistry,Faculty of Science, University of Sistan and Baluchestan, P.O.Box 

98135-674, zahedan, IRAN 

E-mail: sardasht@hamoon.usb.ac.ir 


Corrosion, is an electrochemical process in which a potential –difference happens either 

between two metals or two different parts of a same metal. the created potential-difference 

can be measured in relation to standard electrod.electrical potential of this metal may be more 

or less than standard limit [1].organic acids dissolved in water, such as Humic acid, exist 

mostly with high concentration in water of marsh as well as, in waters which were not taken 

from such places. The lands, covered with plants mainly, and surface-fleeing waters, partly, 

possess such material [2]. One of new projects of gas-pipe lines passage, cross countries 

because of security reasons, may be performed through the bottom(depth)ce, of seas and 

ociens.Since the Humic substances of certain water of humic acid have the properties of 

Rodox and ionic exchange ,corrosion ukelihood by humic substances is very great [3]. 

Because, pH of sea water, in most areas varies, ranging from 7.2 to 7.6, therefor, in usual 

temperature degrees and small changes pH has no effect of usual rate of steel corroding. This 

can be an economic loss. So, in this project along with examining the rate of corrosion, for 

preventing purposes tests of electrochemical polarization and SEM on water of Oman Sea as 

well as a few preventers, were conducted 



To conduct the examination of electrochemical polarization and cycle voltamer, providing a 

surface-having electrode which plays the role of work-procedure electrode, is necessary. At 

first, we prepare an electrode sample which is made of the materials involved in gas pipe, 

with surface of 1 cm 2 and set it with (twins) glue, then smooth it sand paper, wash it with 

distilled water and dry it, finally. Then we sink the electrode-sample in sea-water or other 

mixture with stable pH, or having inhibtator, so that the corrosion takes place.Now, we put 

the electrode sample along with reference electrode (Ag/Agcl) and opposite electrode in 50 ml 

water of sea or other mixtures that form our electrochemical cell. At this time, we provide the 

cell with gas for 15 minutes. Now draw the polarization curve and record the voltamgram CV 

of corroded piece. in experiments of weight-reduction, we do as the same and then, get out the 

Vol. 3 Page - 21 -

sample from solution and after drying, we will weigh it depositions have brown colour).in 

element decomposition of electrode sample, by solving 0.02g of it, in concentrated 

clorohydric acid inside a joujet balloon and in water of two-times distillation, we reach it to 

desired volume. Content elements of prepared solution are defined by technique of Flame 

atomic absorption 


The results of elemental Analysis of steel sample (forming substances of gas pipe) are showed 

in Table 1. In measuring the weight reduction in sea –water, it is 10 mg. In mixture of seawater 

and 5mg extracted humic acid, the reduction reaches to 20mg. Average potential of 

corrosion, resulted from some electrochemical polarization curves, in sea-water is E= 0.98 

volts and I=51 µA (time of half-hour) (Fig 1) .By adding Benzoic acid(02.g) as a inhibitor 

potential of corrosion in a time of half-hour, is E=- 0.92volts(Fig2) and I=32µA and in 

period of one-hour, becomes E=-0.84 volts I=1.9µA. Brown-colour deposits would 

disappear(Fig3).By adding 10 mg humic acid to 50 ml sea water, corrosion potential becomes 

E=-1.04volts I=90 µA (Fig 4).in other word, by increasing humic acid, corrosion increases. 

This result can be approved by experiments of weight-reduction. average potential of 

corrosion, resulted from few curves of polarization of electrode sample, in Buffer pH=7, will 

be 0.817 volts .By putting 5 mg humic acid in Buffered solution ,corrosion potential becomes 

0.805 volts.pH shows the effect of humic acid on corrosion of electrode sample[4, 5]. 

Element 

%(w/w) 

Al 

19.8 

Cr 

19 

Table1: Elemental Analysis of steel sample 

Fe Ni Cu Zn 

63.92 


0.005 

Vol. 3 Page - 22 - 

0.76 

0.52 

Cd 

0 

Pb 

0


Figure1: Electrochemical polarization of electrode sample in sea water (in period of half hour) 

Figure2: Electrochemical polarization of electrode sample in sea water content 0.20 g of Benzoic acid 

(in period of half-hour) 

Figure 3: Electrochemical polarization of electrode sample in sea water content 0.20 g of Benzoic acid 

(in period of one-hour) 

Vol. 3 Page - 23 -

Figure4: Electrochemical polarization of electrode sample in sea water content 10mg of humic acid 

(in period of half-hour) 

Acknowledgments 


The author’s is grateful to Dr. Hussein from university of Kerman (Faculty of Science) for 

corporation in this work 

References 

1. L.FP.dick and L.M.Rodrigues, corrosion science journal, 62, 1 (2006) 35 

2. A.Mshams El Din, Desalination, 238(2009)166-173 

3. S.M.A.Hossenini, A.Azimi, corrosion science, 51(2009)728-732 

4. G.A.Zhang, Y.F.cheng, corrosion science (2009) 

5. Z.Y.Lit, X.G.Li, C.W.Du, G.L.Zhaj, Y.F.cherg, corrosion science 50(2008)2251-2257 

Vol. 3 Page - 24 -

Color Removal by Coagulation from Water Containing Aquatic Humic 

Substances with Different Apparent Molecular Size 

Eliane Sloboda a* , Camila Tolledo Santos b , Angela Di Bernardo Dantas b , Luiz Di Bernardo b , 

Eny Maria Vieira a 

a Instituto de Química de São Carlos, Universidade de São Paulo, São Carlos-SP, Brazil; 

b Escola de Engenharia de São Carlos, Universidade de São Paulo, São Carlos-SP, Brazil 

E-mail: elisloboda@hotmail.com 


Humic substances originate from soil humic material and aquatic plants. In surface waters 

humic substances generally account for 50 to 70% of the dissolved organic matter (DOM) [1]. 

When aquatic humic substances (AHS) are not efficiently removed during the water treatment 

steps, they can cause several problems such as serving as substrate for the growth of 

microorganisms, complexing with metals such as Fe, Mn, Pb and others and making their 

removal difficult, causing corrosion in piping, and producing substances with unpleasant 

tastes and odors, some of which are toxic and potentially carcinogenic when preoxidation is 

performed using free chlorine [2]. 

The coagulation of humic acid seems to be brought about two major mechanisms, depending 

on the pH conditions: adsorption of the humic acid on the precipitate of Al(OH)3(s) at pH 

above 6; and precipitation of the humic acid by the neutralization of charge through a soluble 

or incipient solid-phase aluminum hydrolysis species in the range of pH 4.0 to 5.5 [3, 4]. This 

study aimed to verify the influence of the apparent molecular size of AHS on the effectiveness 

of coagulation with aluminum sulfate and characterize the DOM in different fractions de 

AHS. 



The water samples were collected from the Itapanhaú river (true color intensity in the order of 

300 Hazen units), Bertioga, São Paulo State, Brazil. Extraction of humic substances was made 

according to the procedure adopted by the International Society of Humic Substances (IHSS) 

[5]. The extracted humic substances were filtered through a membrane with 0.45 μm pores 

(Millipore) and this fraction was denoted as F1. Fraction F1 was then separated by 

ultrafiltration using polyethersulfone membranes (Vivalflow50) into 3 apparent molecular 

size fractions: F2: from 100 kDa to 0.45 μm, F3: from 30 to 100 kDa and F4: < 30 kDa. 

Water from an artesian well was used to prepare 4 experimental samples: water sample I (F1), 

water sample II (F2): water sample III: (F3) and water sample IV (F4). Water sample color 

Vol. 3 Page - 25 -

intensity was 100 ± 5 Hazen units, turbidity 5.0 ± 0.5 NTU with added kaolinite, temperature 

20 ± 1°C. Solutions of NaOH and HCl were used to adjust the coagulation pH. 

Jar test equipment (ETICA) and direct filtration, was used to carry out the tests. The following 

parameters were adopted to perform the tests: rapid mixing time = 30 s; velocity gradient = 

1000 s -1 ; filtration time = 20 min; filtration rate = 60 m/d; each bench-scale sand filters 

consisted of a 15 cm layer of sand with grain sizes of 0.30 to 0.59mm. The coagulant 

employed was a commercial liquid aluminum sulfate with 7.28% (w/w) of Al2O3. Color 

measurements were based on the procedure recommended by the Standard Methods for the 

Examination of Water and Wastewater (1998) [6]. 

The characterization of DOM in Itapanhaú river was made as outlined in Figure 1 and 

described in the sequence. 

Elution with 

NaOH 0.1 M 

HPOA 

Elution with 

NaOH 0.1 M 


TPHA 

2 L of river water 

filtered through 

0.45μm at pH 2 

XAD-8 

XAD-4 

HPI 

HPON 

Figure 1: Fractionation of DOM 

Extraction with 

acetonitrile/water 

for 48 h, 50ºC in 

Soxhlet extraction 

TPHN 

Extraction with 

acetonitrile/water 

for 48 h, 50ºC in 

Soxhlet extraction 

The hydrophobic fraction (HPOA) and transphilic (TPHA), respectively adsorbed on XAD-8 

and XAD-4 resins were eluted according to the methodology proposed by Malcolm and 

MacCarthy (1992) [7]. After the water passed through the two resins, samples were collected 

and were identified as hydrophilic fraction (HPI). The acetonitrile was removed from extracts 

in rotaevaporador at 90 °C. The extracts were identified as hydrophobic neutral fractions 

(HPON) and neutral transphilic (TPHN). Measurements of total organic carbon (TOC) in the 

fractions were made by the spectrophotometric method (TOC Analyzer 5000ª). 

Vol. 3 Page - 26 -



Considering only the relationship of the mass of each of the fractions isolated in the MOD 

XAD-8 and XAD-4, it was found that the largest proportion of the DOM Itapanhaú River 

water is represented by the fraction HPOA (46%), and smaller quantities HPI fractions (18%), 

TPHA (15%), TPHN (11%) and HPON (10%). It justifies the use of the methodology of 

Thurman and Malcolm (1981) [8] to obtain the SHA River water Itapanhaú. 

The results of the coagulation filtration tests on water samples I to IV using aluminum sulfate 

as a coagulant are shown in Figures 2a to 2d. 

a) Results of Water Sample I b) Results of Water Sample II 

c) Results of Water Sample III d) Results of Water Sample IV 

Figure 2: Diagram of aluminium coagulation and color removal domains for water samples I to IV 

In water sample I (Figure 2a), Zone 1 for ≥ 95% removal is defined by the pH ranging from 

4.7 to 5.3 and aluminum dosages between 1.9 and 2.7mg L -1 , Zone 1.1 for ≥ 95% removal is 

defined by the pH ranging from 5.5 to 6.0 with aluminum dosages of > 3.5mg L -1 Al. Zone 2 

Vol. 3 Page - 27 -

(color removal ≥ 90%) is defined by the pH ranging from 4.4 to 6.2 and aluminum dosages of 

> 1.9mg L -1 Al. 

Figures 2b and 2c show similar results, but for water sample IV (Figure 2d), indicating only 

one zone for ≥ 95% removal efficiency, which was defined by the pH ranging from 4.9 to 5.5 

and aluminum dosages of > 4.2mg L -1 Al; Zone 2 (≥ 90% removal efficiency) was defined by 

the pH ranging from 4.5 to 5.6 and aluminum dosages of > 2.7mg L -1 Al. 



To achieve the same degree of color removal, the water samples with lower apparent 

molecular sizes required higher doses of aluminum sulfate. The coagulation probably 

occurred as a result of charge neutralization through a positively charged hydrolyzed 

aluminum species at lower pH values, while at higher pH values it probably occurred due to 

the adsorption of humic and fulvic acids on the Al(OH)3(s) precipitate. 

References 

1. G.R. Aiken, in G.R. Aiken et al. (Eds.), John Wiley and Sons, New York, 1985, p. 363–385. 

2. L.D. Bernardo and A.D.B. Dantas (2005). Methods and Techniques for Water Treatment, RiMa, 

São Carlos, 2005. 

3. P.N. Johnson and A. Amirtharajah, Res. and Techn. J. AWWA., 75 (1983), 239. 

4. G. A. Edwards and A. Amirtharajah, J. AWWA., 77 (1985), 57. 

5. http://www.ihss.gatech.edu, acessed in jun 2008. 

6. Standards Methods for the Examination of Water and Wastewater 1998 20th Edition, APHA, 

AWWA, AWPCF, Washington, DC, USA. 

7. R.L. Malcolm and P. MacCarthy, Environ. Intern., 18(1992), 607. 

8. E.M. Thurman and R.L. Malcolm, Environ. Sci. Technol., 15 (1981), 466. 

Vol. 3 Page - 28 -

pH Effect in Aquatic Fulvic Acid From Brazilian River 

Sérgio da Costa Saab a* , Eduarda Regina Carvalho b , Rubens Bernardes Filho b , Márcia Regina 

de Moura Aouada b , Ladislau Martin-Neto b , Luiz Henrique C. Mattoso b 

a Departamento de Física, UEPG, Av. Carlos Cavalcanti 4748, CEP 84030-999, Ponta Grossa 

PR Brazil; b Embrapa Instrumentação Agropecuária Rua XV de novembro 1452, CEP 13560- 

970, São Carlos,SP, Brazil 

E-mail: scsaab@uepg.br 


Presence of humic substances (HS) in a water supply is undesirable for several reasons, for 

instance: it produces esthetical problems as color in the water; stabilizes dispersed and 

colloidal particles during coagulation processes; leads to formation of biodegradable organic 

compounds during ozonation and thereby enhances regrowth of microorganisms within the 

water-distribution systems [1]. 

Atomic Force Microscopy (AFM) technique can image surfaces with atomic resolution by 

scanning a sharp tip across the surface at forces smaller than the forces between atoms [2]. 

AFM is a technique which has been employed to study the morphologies of humic and fulvic 

acid [3–5]. It is a powerful tool to characterize small colloids, as well as colloid 

agglomeration, adsorption onto surfaces, or modification in morphologies affected by changes 

in the physical-chemical properties. 

The objective of this work was to get AFM images of aquatic acid fulvic (AFA) Brazilian 

river, and zeta potential with pH change to verify the structural and morphologic change of 

the AFA. 



The aquatic HS were isolated from a sample collected from a tributary stream of River 

Itapanhaú within of the State Park called "Serra do Mar". This is an environmental protection 

area located in the seaboard, 7 th UGRHI of 11 th group of UGRHI from São Paulo State, 

Brazil. 

The extraction of fulvic acids from the river samples was made followng the methodology 

suggested by International Humic Substances Society (IHSS) [6]. The imaging of AFM of 

AFA samples was carried out at two pH values 3.0 and 9.0. These were used to identify 

structural changes of FA when the pH varies. Images were obtained using the AFM 

microscope Didimension V, Veeco. Tapping mode was used and Silicon SPM. 

Vol. 3 Page - 29 -

For the study of zeta potential in function of the pH, AFA samples in a suspension of 100mg 

sample in 1L distilled water milliQ were used. The suspension was sonificated for 30 min in a 

60W bath ultrasound in 20 mL parts. pH was adjusted with the addition of 0.1M HCl or 

NaOH at 20 o C and after 24 h the pH was readjusted. The equipment used was Malvern 

Instruments, Zeta sizer nano ZS model Zen 3600. 


Figure 1a (up) shows de AFA AFM images at pH 3.0 and in figure 1a (down) the height and 

diameter of AFA particles on the mica sheet measured from the two straight lines indicated in 

figure 1a (up). Agglomerates in the shape of pyramids can with diameter around 150–300 nm 

and 10–55 nm high are observed. 

height / nm 

60 

50 

40 

30 

20 

10 

0 

2 

1 

0.1 0.2 0.3 0.4 0.5 0.6 

X / μm 

0.7 0.8 0.9 1.0 1.1 1.2 1.3 

1 

2 


a) pH 3.0 b) pH 9.0 

height / nm 

4 

3 

2 

1 

0 

0.3 

X / μm 

Figure 1: AFA AFM image at pH a) 3.0 and b) 9.0Figure 1b (up) shows the AFA AFM images at pH 

9.0. Figure 1b image indicates a more open distribution of AFA on the mica sheet when compared 

with figure 1a image at pH 3.0. Dimensions can be observed in figure 1b (below) where the AFA 

height on the mica at pH 9.0 was between 2.5–4.0 nm and diameter between 100–300 nm. With pH 

increase, AFA particles expand and repel one another electrostratically. Strengths become weaker, due 

to H bonding, van der Waals interactions and interactions of π electrons from adjacent molecules, with 

dissociation of carboxylic and phenolic groups, generating negative charges [7] as shown in figure 2 

(zeta potential) 

Figure 2 shows the zeta potential variation with the pH of the river AFA sample. Zeta 

potential becomes more negative with the increase in pH. There is a sharp increase in the 

negative charge from pH 7.0, this fact coincides with the beginning of phenolic acid groups 

ionization, with a gradual increase of these groups from pH 7.0. Thus, the contribution of 

0.1 

Vol. 3 Page - 30 - 

0.2 

0.4 

0.5 

1 

2 

3 

0.6

phenolic acids for the formation of AFA negative charges in solution is more important than 

the carboxylic groups. 


ZP / mV 

0 

-30 

-60 


-90 

2 4 6 8 10 12 

Mean 

Figure 2: Zeta potential variation with the AFA sample pH 

This work showed that through atomic force microscopy techniques, structural change in 

fulvic acid of a Brazilian river was identified when the solution pH varied. Results suggest 

that in acid pH weak electrostatic interactions and hydrogen bonding are responsible for 

aggregates formation while in alkaline pH electrostatic interactions are strong due to increase 

in the phenolic groups ionization and low hydrogen interaction forming more open structures. 

References 

1. E. R. Carvalho; L. Martin-Neto; D.M.B.P. Milori; J. C. Rocha; A. H. Rosa, J. Braz. Chem. Soc., 

15 (2004) 421. 

2. F. L. Leite; P. S. P. Herrmann; J. Adhesion, Sci. Technol., 19 (2005) 365. 

3. K. Namjesnik-Dejanovic and P. A. Maurice, Colloids and Surf. A: Physicochem. Eng. Aspects, 

120 (1997) 77. 

4. M. Plaschke; J. Rothe; T. Schäfer; M. A. Denecke; K. S. Dardenne; K. Pompe; H. Heise, Colloids 

Surf. A: Physicochem. Eng. Aspects, 197 (2002) 245. 

5. J. M. Gorham; J. D. Wnuk; M. Shin; H. Fairbrother, Environ. Sci. Technol., 41 (2007) 1238. 

6. R. S. Swift; in Sparks (Ed), Methods of Soil Analysis Part 5, Soil Sci.Soc.Am.:, Madison, 1996, 

p.1018. 

7. R. A Alvarez-Puebla. and J .J. Garrido, Chemosphere, 59 (2005) 659. 

Vol. 3 Page - 31 -

The Role of Organic Matter in the Transport of Suspended Minerals in the 

Estuarine Zone 

Lasareva E.V. a , Parfenova A.M. a , Romankevich E.A. b 

a Chemistry Department Moscow State University, 1 Leninskie gory, 119991 Moscow, Russia; 

b Shirshov Institute of Oceanology, 36 Nachimovski prospect, 117997 Moscow, Russia 

E-mail: elasareva@ya.ru 


The idea of V.I. Vernadsky, concerning the important role of boundary zones in the ocean, as 

zones of considerable biogeochemical activity, especially takes place in the marginal filter — 

zone of river and seawater mixing in river mouth. Large-scale processes of flocculation and 

coagulation of dissolved (colloidal) and suspended matter, sorption on newly formed surfaces 

take place in this zone. All these processes result in that 93–95% of suspended matter and 20– 

40% of dissolved matter of river discharge (pollution included) is deposited in the zone [1]. 

Our observations of the seasonal distribution of particulate and dissolved organic matter (OM) 

concentrations in the Severnaya Dvina river and the Amazon river estuaries showed a 

tendency to inverse patterns of distribution of particulate (POC) and dissolved (DOC) organic 

carbon. It was shown that the decrease in DOC concentrations is accompanied by the growth 

of POC concentrations. This pattern is observed at salinity 1–8 ‰ and may be considered as a 

result of coagulation and flocculation processes [2]. 

2. Material and methods 


To confirm the nonconservative behavior of DOM and to understand the role of OM in the 

coagulation and flocculation processes in estuarine zone we conducted experiment, using 

model systems – suspensions of different clay and carbonate minerals under increasing 

salinity from 1 up to 35‰. Montmorillonite and kaolinite (1 g/L) were studied as clay 

minerals, carbonate minerals —calcite and aragonite were prepared in laboratory [3]. The 

addition of model organic substances —chitosan (as flocculent and fulvic type OM) and 

humic acids (HA) under varying salinity values allows us to study the role of OM (humic and 

fulvic types) on the processes of coagulation and flocculation. In thre study 1mL of 0.05 % 

chitosan solution (Mw=300000) and 0.1 mL of 1 g/L HA (Humintech Ltd., Germany) 

solution were added to the mineral suspensions. The optical density of suspensions after 

mixing of minerals with salt (NaCl) and OM was used as an indicator of stability of the model 

systems. 

Vol. 3 Page - 32 -

3. Results 

The laboratory experiments showed that kaolinite suspensions are more stable under different 

salinity conditions than montmorillonite ones. The addition of chitosan solution to clay 

suspensions leads to flocculation of montmorillonite suspensions just at small salinity (1 ‰) 

and does not effect kaolinite flocculation under studied conditions. The addition of HA 

solution to clay and carbonate suspensions does not change greatly the behaviors of 

suspensions at different salinity. It is interesting to mention that the joint action of HA and 

chitosan solutions leads to the flocculation of kaolinite at 7‰ (Fig. 1). 

D 

1,2 

1,0 

0,8 

0,6 

0,4 

0,2 

0 2 4 6 8 10 12 14 16 18 20 22 

Figure 1: Dependence between the optical density of the kaolinite suspension and the salinity (1), the 

same 3 hours after of НА addition (2), the same 20 hours after of НА addition, (3), the same with 

addition of НА and chitosan (4) 


The laboratory data allow to explain in-situ investigations concerning the difference in clay 

and carbonate behavior in estuarine systems and show that the joint action of OM of humic 

and fulvic type may lead to removal of clay mineral in estuarine zone, while the presence only 

humic or fulvic type OM may stabilize suspensions and allow them migrate to long distance. 

The mechanisms of flocculation and coagulation of clay and carbonate minerals under 

increasing salinity in estuarine zone are considered. The further investigation may clarify a 

number of related problems, as the transport of mineral, nutrients and pollutants from river to 

ocean, the explanation of sedimentation rates, and the role of colloidal fraction in the 

mechanisms of dissolved and suspended matter removal. 

References 


1. A.P. Lisitsyn. The marginal filter of the ocean. Oceanology. 1995, V. 34, № 5. P.671. 

2. A. Vetrov, E. Romankevich. Carbon Cycle in the Russian Arctic Seas. 2004, Springer, 331p. 

3. J.L. Wray, F. Daniels. Precipitation of calcite and aragonite. J. Am. Chem. Soc. 1957, V. 79, № 9. 

P.2031. 

S, ‰ 

Vol. 3 Page - 33 - 

1 

2 

3 

4

Organic Material of Uneven-Age Anthropogenic Origin Lakes 

Sofia Zalmanova 

St. Petersburg State Agrarian University, Faculty of Soil Science and Agroecology, Soil 

Science and Soil Ecology Department name of Pr. L. N. Aleksandrova, Petersburg’s road, 2, 

Pushkin, St. Petersburg, Russian Federation 196601 

E-mail: lisofang@yandex.ru 


Global stability of biosphere depends on preservation and maintenance of water ecosystem 

functioning. In this case artificial lakes should possess ability to self-restoration and dynamic 

adaptation to external influences, and also not to cause basic changes in developed 

ecosystems, surrounding lakes. In this connection the questions linked to studying water 

ecosystems are actual. The accent of the research work has been made on a problem of 

organic material (basically submitted by HS) and fresh lake bottom sediments, as to the most 

important characteristic of an ecological condition lake ecosystem. 

For the description of a lake ecosystem ecological condition the following researches have 

been carried out: the chemical estimation of water in investigated reservoirs (definition of 

oxidation-reduction potential, pH, quantities of humic organic substances, the contents of 

heavy metals); the chemical estimation of lake sediments (definition of oxidation-reduction 

potential, pH, losses of ignition, hygroscopic humidity, chemodestruction fractionating of 

organic material); the estimation of an ecological condition of reservoirs on biological 

parameters (the maximum(supreme) water vegetation and macrozoobenthos). 



In this research work the problem of artificial lakes is considered by the example of the park 

Suoranda (Leningrad region). Investigated lakes have appeared on a place of extraction of 

sand. It is the most widespread reason of occurrence of lakes of an anthropogenic origin in 

our region. As a result of biological succession the career landscape to the external attributes 

becomes similar to typical for Northwest of Russia moraine landscapes with a characteristic 

combination of lakes and woody hills. The given territory functions as technogenic 

disturbance of natural environment landscape and represents interest for research as allows 

expanding representations about restoration of natural ecosystem after anthropogenic 

intervention [2]. 

Vol. 3 Page - 34 -


Humic substances (HS) are extracted from lake water by the sorption method. For this aim the 

lake water with HS was acidified by a sulfuric acid up to рН = 1-2, then it was filtrated 

through special gel sorbent, on which humic substances were sorbed. Desorption of HS from 

a sorbent have been made by using an alkaline solution (0.1 M NaOH). 

For an estimation of qualitative composition of lake sediment organic matter the method of 

chemodestruction fractionating (CDF) was used. Various components of bottom sediments 

organic matter (BSOM) or various parts of organic macromolecules (including HS) have a 

different resistance to biota enzymes. Relatively difficult (stable) and easy degraded (labile) 

organic compounds (or fragments of macromolecules) have different role in BSOM system. A 

ratio of stable and labile BSOM parts is an important characteristic of ecosystem [1]. 

The method of CDF concerns the physic-chemical analysis of soils. It is based on different 

susceptibility of SOM components to an oxidizing agent. That the higher a rate of organic 

matter decomposition in soil, the higher an ability of its destruction by mineral oxidizers. The 

stronger the oxidizing solutions, the more the oxidation of BSOM components. On this basis, 

we can divide the SOM into labile and stable parts. The resistance of BSOM components to 

oxidation was connected with both the chemical composition and the spatial three-dimension 

structure of macromolecules, especially so far as concerns the native humic substances. The 

easily oxidized organic compounds and/or fragments of macromolecules actively take part in 

trophical cycle of the biota of lake sediment and higher plants, as main source of substances 

and energy. This fraction stipulates the biochemical properties. In turn, the difficult oxidized 

organic material influence on physical and physic-chemical properties. The biota of lake 

sediments can decays the difficult degraded material to the easily degraded organic 

compounds [1]. 

For an establishment of red-ox potential of lakes the contents of iron in sediments and water 

has been determined: the ratio of ferrous and ferric iron presented aerobic and anaerobic 

conditions and character of oxidation-reduction processes. Iron was determined by 

colorimetric method as the painted complex with С7Н6О6·2Н2О. 

Measurements of concentration of metals in samples of water have been made by a method 

atomic absorption spectrometry [3]. 

The ecological status of artificial lakes of various age was estimated by O.N. Mandryka and 

L.V. Kulangievoj in indissoluble connection with character of a landscape and inherent in its 

plant associations various type [2]. 

Vol. 3 Page - 35 -



In the table 1 the integrated ecological estimation of the investigated reservoirs based on the 

characteristic of properties lake sediments and water, and also on biological parameters of 

reservoirs is resulted are presented. 

Table 1: The comparative characteristic of Marsh lake and Career - 1 

The characteristic of research objects Lake Marsh Career-1. 

Age of a reservoir About 50 years About 6-7 years 

рН of waters 5,95 (poorly sour) 6,09 (poorly sour) 

рН of lake bottom sediments 6,85 (neutral) 6,67 (neutral) 

The contents humic substances in water of 

reservoirs, mg TOС/l 

~ 1 No 

Distribution of organic substance in lake Non-uniform between Uniform, than 0,25 mm 

bottom sediments 

fractions, are present by the 

semidecomposed plant 

remains 

are dated for fraction less 

The contents of organic substance in lake 

sediments, % 

8,7 8,4 

Geochemical conditions Regenerative, there are Regenerative, but with 

attributes of bogging the big enrichment by 

oxygen 

Prevailing processes of transformation of 

organic substance in lake sediments 

destructive Synthetic 

Type of a reservoir Dystrophic Oligotrophic 

Saprobity [2] 1.504 (between oligo-and β- 1.805 (between 

mesosaprobic) 

oligosaprobic and βmesosaprobic) 

In organic matter of Career - 1 sediments synthetic processes dominate above destructive, and 

over lake Marsh on the contrary — prevalence of destructive processes. 

On the data obtained from CDF of organic substance of lake adjournment is received, that as a 

result of the processes of transformation of organic substance the unstable and unbalanced 

system of components of organic substance lake sediments of both lakes is formed kinetically. 

In sediments of lake Marsh the quantity ferrous iron was more, than in lake sediments of 

Career - 1, in this lake is more expressed development marsh process. In lake sediments of 

Career - 1 prevails quantity ferric iron that apparently, specifies enrichment of oxygen in this 

reservoir. 

Concentration of copper, lead, cadmium, iron, zinc in the samples of water of both lakes 

corresponded to the established ecological norms. In the samples of water of both reservoirs 

Vol. 3 Page - 36 -

concentration of manganese and iron exceeds maximum concentration limit, therefore under 

the contents of manganese and iron in waters of the investigated objects the given lakes are 

suitable only for the recreational purposes. 

Less than 0.25 mm Career - 1 contained sediments of fraction of more fine-material an 

organic material, than lake Marsh, except for that in sediments of lake Marsh there was a 

significant amount of large fossils that corresponds to the type of these lakes: oligotrophic – 

Career -1 and dystrophic - Marsh lake. 



On the basis of the carried out researches, it is possible to tell, that organic matter of water 

and bottom sediments of lakes is in interaction with other characteristics of lakes and together 

they form representation about ecological conditions of lakes. The lake Marsh in which 

contents of organic matter prevails can be attributed to lakes of the dystrophic type, Career-1 

— oligotrophic type. The ecological condition Career-1 and lakes Marsh is characterized as 

poorly polluted. 

References 

1. A. I. Popov, V. P. Tsiplenkov, M. A. Nadporozhskaya., G. T. Frumin An estimate of qualitative 

humus composition by the chemodestruction fractioning// Humic Substances in the Global 

Environment and Implications in Human Health / Abstracts, 6th Int. Meeting IHSS. Italy. 

Monopoli (Bari), 1992, p. 240. 

2. O.N. Mandryka., L.V. Kulangieva, The Concept of transformation technogenic reservoirs in 

ecologically steady complexes for recreation the population near of megapolis // The Theory and 

practice of restoration of internal reservoirs. Works of the international scientific practical 

conference. (St.-Petersburg, October, 15-18, 2007). — SPb., 2007, p. 248-253. 

3. I. Havesov, D. Tsalev The Method of atomic absorption spectrometry/ Translation. from 

Bulgarian. — Leningrad, 1983. 

4. I. V. Baranov The Organic materials in lake bottom sediments and water basins and its 

bioproductive value, Theses of reports of IV congress All-Union Geobiotic Association. In 3 

parts, Ch. 1.-Kiev, 1981, p. 98-99. 

Vol. 3 Page - 37 -

Comparative Measurement of Hydrophobic Organic Matter Dissolved in 

Water by the XAD Resin Method and the Polarity Rapid Assessment 

Method (PRAM) 

Marc Philibert, Alex Revchuk, David Quiros, Arthur Roh, Mel Suffet * 

Environmental Science and Engineering Program - UCLA, School of Public Health, Room 

46-081-CHS, Charles E. Young Drive South Los Angeles, CA 90095-1772, USA 

E-mail: msuffet@ucla.edu 


Dissolved organic matter (DOM) is a mixture of thousands of molecules prevalent in water 

bodies. Dissolved organic matter is responsible for multiple water treatment issues including 

trihalomethanes formation and membrane fouling. A better understanding of the physical and 

chemical characteristics of DOM (e.g. polarity and size fractions) is necessary to find ways of 

controlling these water treatment problems. Multiple measurements over time are needed at 

ambient pH to define how these problems occur. Therefore, a quick probing polarity method 

is needed for the evaluation. The polarity rapid assessment method (PRAM) 1.2 was chosen to 

study these problems at ambient pH and ionic strength with the PRAMs solid phase extraction 

cartridges of different polarity in parallel. The classical methods of evaluating the polarity of 

DOM is the XAD resin series column adsorption method 3 . The resin method has been used 

for over two decades. The objective of this study was a comparison of Resin 3 and PRAM 1,2 

methods to try to relate data in the literature using the XAD resin method and the PRAM 

method. During the evaluation, the PRAM method was run in series as the XAD resin method 

is completed to develop a more consistent evaluation of comparative polarity. Also, the pH 

was run at ambient pH and adjusted to


Figure 1: represents the comparison between C18 and the XAD-8 retention for DOC at the Fort 

Collins Water Utility at pH

Figure 3 shows the fluorescence results of UF fractions and of their respective C18 cartridge 

effluents. Once again we see that the C18 cartridge increases the importance of the region I. 

This means that that region is the least hydrophobic (most hydrophilic) as it completely goes 

through the cartridge. The other regions all decrease in importance, with regions III and V 

decreasing the most. This highlights that these two regions are the most hydrophobic as they 

are retained by the C18. The effect of the UF is different. The peaks retain their importance 

through the different membranes though they don’t react in the same way. The main peak of 

region III increases through the three membranes. Region III representing molecules of fulvic 

acid-like functionality. This increase of region III is counterbalanced by decreases in region I 

and IV. This shows that regionsI and IV are responsible for the production of larger 

molecules. Molecules in these regions are representative of microbial by-products and 

aromatic proteins that are indeed likely to be larger. 


% intensity 

FCWTF influent 

Raw 

80 

1k 1k C18 5k 5k C18 10k 10k C18 

60 

40 

20 

0 


I II III IV V 

region 

Figure 3. Fluorescence characteristics of UF fractions and their C18 fraction 

This study shows that the two methods used here: the XAD resin adsorption and PRAM 

though used for similar purposes are not directly comparable to define hydrophobicity. The 

comparison of the two methods focused mainly on the C18 cartridge and the XAD-8 and 

XAD-8/XAD-4 resins. All three of these are used in the literature to evaluate the hydrophobic 

character of organic matter. We show here that these two methods are not equivalent and 

retain different fractions of the organic matter. This emphasizes the difficulty of defining the 

hydrophobic fraction of a sample. Both methods assume that the material they are retaining is 

hydrophobic as this fraction is retained by a hydrophobic adsorbing material though there is 

no correlation between the retention of these materials. The difference between the retention 

of these two methods is seen more clearly in the fluorescence intensity measurements. We 

notice that the C18 cartridge’s effluent prominently lets region I (aromatic protein) material 

through whereas the XAD method removes that region. This fluorescent region is defined by 

Vol. 3 Page - 40 -

aromatic protein and is consistent for interpretation by both methods. Also, XAD resins are 

more partial to the region type IV molecules. This could be due to the interpretation of 

fluorescence data from a point of view of XAD isolation for region IV. Can the differences 

and potential complementary use of the two methods be utilized for evaluation of polarity is 

an open question? 

Finally this study shows that the use of the PRAM method coupled with fluorescence and/or 

ultrafiltration can be used to gather more insight into the hydrophobic fraction of organic 

matter. The ultrafiltration method described here allows one to interpret the size distribution 

of the samples. Once the UF method is run, the collected effluent is run by PRAM and finally 

the PRAM effluent material is run on fluorescence. This allows a comparison of the 

hydrophobic fractions of each size fraction as well as their fluorescence signature. This 

method can be used to evaluate correlations between these fractions and different parameters 

of concern such as THM formation or membrane fouling. 

Acknowledgements 

The authors would like to thank Dr. Judy Billica of the City of Fort Collins Utilities for her 

help in obtaining all the samples. We thank the partial support of research funding from the 

City of Fort Collins, City of Greeley, Tri-Districts, Northern Colorado Water Conservancy 

District and the Metropolitan Water District of Southern California. Many thanks also to Dr. 

Eric M. V. Hoek of the Civil and Env. Eng. Dept. at UCLA for use of his XAD Resin set-up. 

References 


1. Rosario-Ortiz, F. L., Snyder, S. and Suffet, I. H. (2007a). Characterization of dissolved organic 

matter in drinking water sources impacted by multiple tributaries. Water Res. 41: 4115–4128. 

2. Rosario-Ortiz, F. L., Snyder, S. and Suffet, I. H. (2007b). Characterization of the polarity of 

natural organic matter under ambient conditions by the polarity rapid assessment method (PRAM). 

Environ. Sci. Technol. 41: 4895–4900. 

3. Singer, P.C, Schneider, M., Brandt, J.E. and Budd, G.C. (2007). "MIEX for removal of DBP 

precursors: pilot-plant findings". J. Amer. Water Works Assoc. 99: 128–139. 

4. Revchuk, A. D. and I. H. Suffet, 2009. Quality Assurance of Ultrafiltration Separation for Humic 

Substances by Chemical Probes. Water Res., In press. 

5. Chen, W., Westerhoff, P., Leenheer, J.A., Booksh, K. Fluorescence Excitation-Emission Matrix 

Regional Integration to Quantify Spectra for Dissolved Organic Matter. Environ. Sci, Technol. 

2003, 37 (24). 

Vol. 3 Page - 41 -

Microbial Changes in the Spectroscopic Characteristics and Molecular 

Weight of Dissolved Organic Matters Extracted from 

Diverse Source Materials 

Jin Hur * , Bo-Mi Lee, Tae-Hwan Lee, Ka-Young Jung 

Dept. of Earth and Environmental Sciences, Sejong University, Seoul, 143- 747, Korea 

E-mail: jinhur@sejong.ac.kr 


Dissolved organic matter (DOM) plays important roles in aquatic ecosystems by supplying 

nutrients and energy to heterotrophic organisms, by functioning as a carbon carrier in a carbon 

cycle, and by altering light environments. Many environmentally related DOM reactivities are 

known to be correlated with the physico-chemical properties and the composition of DOM, 

which highly depend on the precursor materials (i.e., sources). The temporal and the spatial 

variations in the apparent characteristics of DOM in rivers and lakes are likely to be attributed 

to the respective mixing of different DOM sources and the combination of several natural 

fractionation and/or transformation processes. In general, the degree of the variations 

observed in watersheds tends to be much smaller than those among the DOM freshly 

produced from diverse source materials [1]. Microbial transformation has been suggested as a 

crucial factor to explain the relatively smaller variations. Despite a number of the studies that 

investigated microbial transformation on DOM characteristics, little effort has gone to explore 

potential microbial changes in correlations among DOM characteristics. The objectives of this 

study were as follows: (1) investigate microbial changes in the selected characteristics of the 

DOM prepared from diverse source materials and (2) compare the correlations among some 

selected DOM characteristics before versus after microbial incubation. 



The DOM sources for this study included diverse organic materials that may serve as the 

precursor materials for organic constituents in watersheds (i.e., treated sewage, suspended 

algae, attached algae, paddy field soil, field soil, sediment, reed, leaf, and weed). A constant 

volume (250 mL) of DOM samples for microbial incubation was prepared in sterile 300-mL 

Erlenmeyer flasks by diluting the DOM extract solutions to 30 mg C/L. Aliquots (4 mL) of 

the prepared inoculum solution were added to each incubation flask. The flasks were sealed, 

incubated in the dark at 20 °C for 28 days, and gently shaken every day. Concentrations of 

dissolved organic carbon (DOC) were determined using a Shimadzu V-CPH analyzer. 

Absorption spectra were measured at 1-nm increments over the wavelength range 200–600 

Vol. 3 Page - 42 -

nm with a spectrophotometer (Evolution 60, Thermo Scientific). Synchronous fluorescence 

spectra of the samples were recorded with a luminescence spectrometer (Perkin-Elmer LS- 

50B). Excitation and emission slits were adjusted to 10 nm and 10 nm, respectively. The 

excitation wavelengths ranging from 250 to 600 nm were used with constant offsets (Δλ = 30 

nm). Size exclusion chromatography (SEC) was used to determine apparent weight- and 

number-average molecular weight (MWw and MWn) values of DOM samples following the 

methodology reported by Hur and Schlautman [2]. 


Microbial changes in synchronous fluorescence spectra of DOM: Synchronous fluorescence 

spectra of the DOM after incubation are presented in Fig. 1. 

DOC-normalized QSE 

(QSE-L mg C -1 ) 







6.0 

5.0 

4.0 

3.0 

2.0 

1.0 

0.0 

(a) Effluent 

(before incubation) 

250 300 350 400 450 500 550 600 

6.0 

5.0 

4.0 

3.0 

2.0 

1.0 

Wavelength (nm) 

Lake algae 

Attached algae 

(c) Algal-derived 


0.0 

250 300 350 400 450 500 550 600 

6.0 

5.0 

4.0 

3.0 

2.0 

1.0 


0.0 

250 300 350 400 450 500 550 600 

6.0 

5.0 

4.0 

3.0 

2.0 

1.0 

Leaf litter 

(e) Terrestrial 


Field soil 


Weed Reed 

Lake sediment 

Paddy soil 

0.0 

250 300 350 400 450 500 550 600 



(g) Plant-derived 










6.0 

5.0 

4.0 

3.0 

2.0 

1.0 

0.0 

250 300 350 400 450 500 550 600 

6.0 

5.0 

4.0 

3.0 

2.0 

1.0 

0.0 

250 300 350 400 450 500 550 600 

6.0 

5.0 

4.0 

3.0 

2.0 

1.0 


(d) Algal-derived 

(after incubation) 

Lake algae 

Attached algae 

0.0 

250 300 350 400 450 500 550 600 

6.0 

5.0 

4.0 

3.0 

2.0 

1.0 



Reed 

Weed 

(f) Terrestrial 


Field soil 

Lake sediment 

Paddy soil 

0.0 

250 300 350 400 450 500 550 600 


(b) Effluent 


(h) Plant-derived 


Leaf litter 

Figure 1: Comparison of DOC-normalized synchronous fluorescence spectra for dissolved organic 

matters (DOM) from diverse sources prior to versus after incubation 

Vol. 3 Page - 43 -

Substantial changes were observed for the algal- and the plant-derived DOM groups. For both 

DOM groups, the major changes were the decrease of protein-like fluorescence (PLF) region 

and the enhancement of other HS-associated fluorescence regions. In contrast, relatively small 

changes were observed for the spectra of the other DOM samples. Irrespective of the DOM 

investigated, the fluorescence shifted to longer wavelengths after incubation. The trend of the 

fluorescence shift may be associated with enrichment of more condensed aromatic structures 

with electron-withdrawing substituents and/or more conjugation of aliphatic chains [3]. The 

simultaneous occurrence of the PLF disappearance and the enhancement of HS-like 

fluorescence characteristics imply the possibility of the mutual relationship between the two 

fluorescence regions. 

Changes in the correlations among DOM characteristics by microbial incubation: For this 

study, no correlation was found between SUVA and fluorescence index (FI) values before 

incubation (Fig. 2a). The result was not consistent with the previous report of a negative 

correlation between aromatic carbon content and FI values for aquatic fulvic acids. After 

incubation, however, the correlation became slightly negative although it was not statistically 

significant (P = 0.117) (Fig. 2b). This comparison suggests that the typical trend of decreasing 

FI with higher SUVA values may not be applied to such freshly produced DOM samples and 

also that the typical trend may be limited to the DOM collected in natural waters, in which 

various natural transformation processes are occurring. 

Fluorescence Index (FI) 

MWn (g/mol as PSS) 

2.5 

2.0 

1.5 

Treated sewage 

1.0 

Before incubation 

Algal-derived 

Terrestrial 

0.5 

Plant-derived 

0.00 0.50 1.00 1.50 2.00 2.50 

1600 

1200 

800 

400 

SUVA 280 (L mg C -1 m -1 ) 

r = 0.848, p = 0.004 

(c) 

0 

0.00 0.01 0.02 0.03 0.04 0.05 0.06 

Spectral slope (S 275-295) 



Algal-derived 

Terrestrial 

Plant-derived 

(a) 

Before incubation 

Fluorescence Index (FI) 

MWn (g/mol as PSS) 

2.50 

2.00 

1.50 

1.00 

0.50 

1600 

1200 

800 

400 


Algal-derived 

Terrestrial 

Plant-derived 

r = 0.483, p = 0.188 

r = 0.561, p = 0.117 

After incubation 

0 1 2 3 4 5 

SUVA 280 (L mg C -1 m -1 ) 

(d) 

0 

0.00 0.01 0.02 0.03 0.04 0.05 0.06 

Spectral slope (S 275-290) 


Algal-derived 

Terrestrial 

Plant-derived 

(b) 

After incubation 

Figure 2: Correlations between SUVA280 values and fluorescence index for diverse sources of DOM 

prior to incubation (a) and after incubation (b). Correlations between spectral slope (275–290 nm) and 

MWn values for diverse sources of DOM prior to incubation (c) and after incubation (d). 

Vol. 3 Page - 44 -

Several previous studies have shown that spectral slope is negatively correlated with MW of 

DOM [4]. For this study, however, the opposed trends were observed between spectral slope 

and MW differed for the DOM samples before and after incubation (i.e., freshly produced 

versus biodegraded DOM). Before incubation, a significant negative correlation was observed 

between the two DOM descriptors whereas the correlation became weakly positive after 

incubation (Figs. 2c and 2d). The results suggest that although the typical trend between 

spectral slope and DOM MW may be applied to diverse sources of freshly produced DOM, it 

is impossible to generalize the trend for aquatic environments with microbial degradation 

occurring. 


In general, the extent of microbial changes in the selected DOM characteristics including 

SUVA, fluorescence, and MW values was more pronounced for the DOM types containing 

higher biodegradable carbon content (i.e., algal-derived and plant-derived DOM). Irrespective 

of the DOM sources, microbial changes resulted in the enhancement of HS-like structures and 

the associated characteristics. Microbial utilization of biodegradable organic substances 

appears to be prerequisite for the enhancement. This study provided significant evidences that 

microbial transformation affected the correlation among several DOM descriptors. The results 

imply that the correlations established based on freshly produced DOM may not be applied to 

aquatic environments where microbial degradation is occurring. 



This work was supported by National Research Foundation of Korea Grant funded by the 

Korean Government (No. 2009-0058569). 

References 

1. K.P. Wickland, J.C. Neff, G.R. Aiken, Ecosystems 10 (2007) 1323–1340. 

2. J. Hur, J. and M.A. Schlautman, Environ. Sci. Technol. 37 (2003) 880–887. 

3. J. Hur, J., M.-H. Park, M.A. Schlautman, Environ. Sci. Tech. 43 (2009) 2315–2321. 

4. J.R. Helms, A. Stubbins, J.D. Ritchie, E.D. Minor, D.J. Kieber, K. Mopper, Limnol. Oceanogr. 53 

(2008) 955–969. 

Vol. 3 Page - 45 -

Dynamics of Humic Matters in Fen Bog Water in Conditions of Climate 

Change 

Ivanova E.S. * , Voistinova E.S., Kharanzhevskaya J.A. 

Siberian Research Institute of Agriculture and Peat, Gagarina st. 3, Tomsk, 634050, Russia 

E-mail: Ivanova_e_s@bk.ru 


Processes of organic substance transformation are one of the main phases of the biological 

cycle. They ensure general stability of the biosphere. Main processes of organic substance 

transformation are decomposition with liberation of СО2 and СН4 and humification, process 

of formation of a special class of compounds —humic acids. Despite the fact that there is a 

wide range of materials dedicated to this subject, there are still some not yet fully investigated 

issues. This work presents the study of correlation dependencies of humic substance content 

in swamp waters on main hydrometeorological parameters: levels of swamp waters, 

temperature of peat beds, oxidation-reduction potential and humidity of peat beds. Analysis 

was carried out using the data for vegetative periods of seven years. 



A native fen situated on a low river terrace of Bakchar river in Bakhchar district of Tomsk 

region, Russia, has been chosen as a model for study of humic substances content in bog 

waters. Average thickness of peat deposit in the central open part of the fen is 3–4 m, 

maximal depth is marked in near-slope part of the terrace basin – 5,5 m. The peat deposit of 

the central part of the fen mass to the depth of 2–2,5 meters in the top is composed of swamp 

hypnum and sedge-hypnum types of peat. Fraction of sedge peat in composition of the peat 

deposit of fen increases from the flood-plain part to near-terrace slope as well as diversity of 

peat types it’s composed of. 

In terms of their composition waters of the studied fen pertain to geochemical type of 

hydrocarbonated calcium-magnesium waters. Values of bog water mineralization range from 

32.67 to 110.81 mg/L. Average content of macro-components in bog waters: HCO3 (87.82 

mg/L). Ca (20.98 mg/L). Mg (8.88 mg/L). Fe (5.17mg/L). Medium reaction is weakly acidic. 

close to neutral (рН 5.28–7.07). 

Research methods included the study of organic substance content depending on main factors 

of natural processes that occur in swamps: peat humidity, peat bed temperature, level of 

swamp waters and oxidation-reduction potential. Analysis of humic substances was carried 

Vol. 3 Page - 46 -

out according to [1]. Samples for chemical analysis, determination of humic substances, 

measurement of bog water levels were taken from stilling wells according to [2]. 

Observations of oxidation-reduction potential (ORP) and temperature regimes were conducted 

per layers of 10 centimeters down to mineral ground be means of fixed measuring elements 

[3, 4]. Peat samples for humidity measurement were taken from all depth levels according to 

[5]. Observations of ORP processes, peat deposit temperature, its humidity, and taking of 

samples for chemical analysis and determination of humic substances were conducted once a 

month from May to September 2002 to 2008. 



Bog waters are characterized by high concentrations of fulvic acids (from 33.8–137.9 mg/L) 

and low concentration of humic acids (from 1.2 to 19.81 mg/L). Monthly average 

concentration of humic acids in bog water for a year over a period of study didn’t change and 

amounted to 7.5±1.3mg/L in contrast to fulvic acids whose content was more variable – 

70.3±20 mg/L. Changes of average concentration of humic acids over the period of 

observation were cyclic. Maximum values of humic acids were registered in 2004, in the 

conditions of the highest bog water levels (up to +12 in August), the highest amount of 

precipitation (190 mm in July) over the period of study and high air temperatures. In 2006 the 

content of humic acids in the bog waters was the lowest, in the conditions of the lowest bog 

water levels over the period of seven years (average level for vegetative period -28 cm). 

Concentration of fulvic acids from 2004 to 2007 was gradually decreasing. In 2004 content of 

fulvic acids was at its maximum. Decrease of concentration over the following years of 

observations was registered at the time when the bog water level decreased. Fulvic acids are 

very soluble in comparison to humic acids and easily migrate both in forms of free acids and 

as parts of compounds. 

The conducted analysis indicated the necessity of investigation of correlation and regression 

dependencies of humic substance content in bog waters on main hydrometeorological 

parameters. Within limits of one parameter, coefficients of correlation not only differ in 

magnitude but also are of different signs. Differences in dependencies of humic substances on 

peat deposit temperature and ORP are, first of all, connected with non-uniformity of water 

regime, consequently, with differences in limiting factors (water level, air temperature, etc.). 

It’s hard to distinguish parameters that are crucial in determining the concentration of humic 

substances of bog water. High correlation of fulvic acids with levels of bog waters is observed 

during high-water years, whereas during other years either the coefficient of correlation 

Vol. 3 Page - 47 -

changes its sign or this relationship becomes weaker. No stable positive relationship was 

revealed for any of the introduced parameters. Dependencies between the amount of summer 

precipitation and humic acids are weaker and not so unambiguous: some of correlations are 

positive while some are negative. At the same time a close interrelation between precipitation 

and fulvic acids was registered over a period of several years. 



A peat deposit is a multi-component system where reaction of oxidation and reaction of 

different nature and of different speed proceed simultaneously. We have registered close 

dependency between the amount of humic acids and ORP of peat deposit: quantitative 

characteristic of humic acids increases (R 2 =0,85) as conditions change from oxidizing to 

reducing ones. Close dependency was also recorded with hydrothermal regime and humidity 

of peat deposit. Consequently, indirect interrelation was observed between humic acids and 

the above mentioned parameters. Besides, we registered close dependency between 

concentration of fulvic acids and levels of bog waters. 

References 

1. Y.Y. Lurje, Unified Methods of Water Analysis, 1973, p.376. 

2. А.А. Reznikov, Е.P. Mulikovskaya, Y.I. Sokolov, Methods of Natural Water Analysis, Nedra, 

Moscow, 1970, p. 488. 

3. L.I. Inisheva, V.I. Yukhlin, F.F. Zelinger, Determination of ORP by means of ESK-1 device, 

Tomsk CTIC, Тоmsk, 1975, № 35-75, p. 2. 

4. L.I. Inisheva, N.G. Inishev, F.F. Zelinger, V.I. Yukhlin, Determination of soil and peat 

temperature by means of ММТ-4, Tomsk CTIC, Тоmsk, 1975, № 36-75, p. 4. 

5. GOST 11305-83. Peat. Moisture Determination Methods, Standards Publishing House, Moscow, 

1983, p. 7. 

Vol. 3 Page - 48 -

Structural Characteristics of Deep Groundwater Humic Substances 

in Horonobe Area, Hokkaido, Japan 

Motoki Terashima a* , Seiya Nagao b , Teruki Iwatsuki c , Yoshito Sasaki a , Yoshimi Seida a , 

Hideki Yoshikawa a 

a Geological Isolation Research and Development Directorate, Japan Atomic Energy Agency (JAEA), 

4-33 Muramatsu, Tokai-mura, Naka-gun, Ibaraki 319-1194, Japan; b Institute of Natural and 

Environmental Technology, Kanazawa University, Wake, Nomi, Ishikawa 923-1224, Japan; 

c Horonobe Underground Research Center, Japan Atomic Energy Agency (JAEA), 432-2 Hokushin, 

Horonobe-cho, Teshio-gun, Hokkaido 098-3224, Japan 

E-mail: terashima.motoki@jaea.go.jp 


In geological disposal system of high level radioactive waste, carrier effects of humic substances 

(HSs) on migration of radionuclides are one of concerns, because HSs have a binding ability to metal 

ions and a possibility to be mobile in geological medium. In general, the metal-ion binding ability and 

mobility of HSs can strongly depend on their structural characteristics. For example, the metal-ion 

binding ability of HSs is related to the amount and types of acid functional groups of HSs. In addition 

the mobility (i.e., sorption and diffusion) of HSs in geological mediums can be dominated by their 

size. On the other hand, the characteristics of HSs depend on their origin. In Japan, the geological 

disposal system has been planned to be constructed at deep underground below 300 m depth. Thus, 

information of the structural characteristics of HSs in deep groundwaters is required for a better 

understanding of the effects of HSs on migration of radionuclide in geological disposal system. 

However, information on the characteristics of deep groundwater HSs in Japan is lacking. Nagao et al. 

showed the characteristics of HSs from the saline groundwater in argillaceous rock layer (790 – 1200 

m depth) in the Mobara area [1,2], and the groundwaters in sedimentary rock layer (ca. 160 m depth) 

and granitic rock layer (c.a. 180 m depth) in the Tono area [3]. Ueda and Sakamoto characterized the 

HSs isolated from the shallow groundwater in sandy soil layer (ca. 50 m depth) [4]. Among these, only 

characteristics of Mobara groundwater HSs are provided as the information on the deep groundwater 

HSs below 300 m depth. 

In this study, dissolved HSs, i.e., fulvic (FA) and humic acids (HA), were isolated from deep 

groundwater at a depth of ca. 500 m in the Horonobe area, Hokkaido, Japan. The isolated groundwater 

HSs were characterized by elemental analyses, spectroscopic analyses (UV-Vis, Fluorescence, FT-IR, 

NMR), and size fractionation analysis. The structural characteristics evaluated were discussed on the 

basis of the comparisons with HSs from surface waters and other groundwaters. 



The groundwater in sedimentary rock (495 – 550 m depth) was collected from the borehole (HDB-10) 

near the Horonobe Underground Research Center by using the packer type groundwater sampling 

Vol. 3 Page - 49 -

system. The chemical components of the Horonobe groundwater are shown in Table 1. To extract 

dissolved HSs, the groundwater was pumped up and passed through DAX-8 column after filtration 

and acidification. After the extraction, the HSs were purified in our laboratory, according to the IHSS 

method. Their weights that were finally obtained as a powder were as follows: 300 mg FA and 100 

mg HA for total treated water of 2929 L in October 2007, 615 mg FA and 12 mg HA for total treated 

water of 1743 L in October 2008. 

Elemental compositions of C, H and N were determined with an elemental analyzer (Yanagimoto, 

MT-6), and S was analyzed by ion chromatography after transformation to SO4 2- . Ash contents were 

also determined by means of combustion at 550 ºC. 1 H and 13 C NMR spectra were recorded by a 

Bruker AVANCE K500 spectrometer. A pre-saturation method and an inverse gated decoupling 

method were applied for the 1 H and 13 C NMR measurements, respectively. UV-Vis spectra were 

measured by Hitachi U-3300 spectrophotometer using 1-cm quartz cell. Three-dimensional excitation 

emission matrix (3-D EEM) spectra were measured by a Hitachi F-4500 fluorescence 

spectrophotometer. Relative fluorescence intensity is expressed in terms of standard quinine unit 

(QSU). A QSU corresponds to fluorescence intensity of standard quinine sulfate (10 μg L -1 in 0.05 M 

H2SO4) at an excitation / emission wavelength of 345 / 450 nm. Molecular size distribution was 

determined by using ultrafiltration method. The ultrafiltration was sequentially conducted using the 

series of ultrafilters with molecular weight cut-off of 100k, 30k, 10k, and 5k Daltons. 

Table 1: Chemical components of Horonobe groundwater, Mobara and Tono 

K + Na + Cl - SO4 2- HCO3 - Aquifers Sampling date Depth / m pH 

mg L 

TOC 

-1 

Horonobe Oct, 2007 495.89~550.00 6.9 100 5300 7100 < 1.0 2230 47.3 

Oct, 2008 495.89~550.00 6.9 129 4930 7300 < 2.0 2290 22.0 

Mobara 1 792~1202 7.9 3020 10700 18800 22 903 55.7 

Tono 2 160 9.6 --- --- --- --- --- 0.11 



The elemental compositions of the Horonobe HSs are given in Table 3. The CHN compositions are 

almost same compared with those of Mobara and Tono HSs. However, remarkable differences were 

observed for O. In the Horonobe HSs, the values of O are evaluated to be 25 – 32%, while the values 

of 38 – 46% are observed for the Mobara and Tono HSs. This relatively lower percentage of O implies 

that the contents of acid functional groups (e.g., carboxyl and phenolic hydroxyl) in the Horonobe 

groundwater HSs are lower than the others. 

1 13 

H and C NMR spectra of the Horonobe FA are shown in Figure 1. Both spectra of Horonobe FA 

exhibited large broad peaks assigned to aliphatic proton and carbon species. In addition, 13 C NMR 

spectrum showed large sharp signal originating from carboxyl carbon at 165 – 190 ppm and 

considerably small broad signal assigned to phenol carbon at 145 – 165 ppm. These results indicate 

that the Horonobe FA is mainly composed of aliphatic carbon and most of the acid functional groups 

Vol. 3 Page - 50 -


are carboxyl groups. These results were also supported by the UV-Vis and FT-IR measurements. The 

composition of carbon species estimated from 13 C NMR spectrum in Horonobe FA is summarized in 

Table 4. When the composition was compared with that of the Mobara FA originated from deep 

groundwater, it was found that the Horonobe FA has relatively low carboxyl content and aromaticity. 

Based on the results of elemental analyses and 13 C NMR, the carboxyl contents were calculated. In the 

Horonobe FA, the carboxyl content was evaluated to be 7.08 meq g -1 . This value was comparable to 

that of Suwannee River fulvic acid (6.47 meq g -1 ), but was slightly lower than that of Mobara FA (8.15 

meq g -1 ). These results suggest that the Horonobe FA has a capability of binding to radionuclide. 

Table 3: Elemental composition (ash-free basis, %) in Horonobe groundwater HSs and those in other 

groundwater HSs from literatures 

Aquifers Depth / m HSs C% H% N% O% S% Ash% 

Horonobe 07 495.89~550.00 FA 59.54 6.85 1.77 29.68 2.16

phenomenon in RFI trend of HSs. Thus this indicates that the Horonobe HSs may have a unique 

structural characteristic. 

The size distributions of Horonobe HSs and those of 

Mobara and Tono HSs are shown in Figure 2. 

Regardless of their origin, more than 80% of FA was 

distributed into size fraction below 10 kDa. In 

addition, 50 – 60% of FAs was found in size fraction 

below 5 kDa. In contrast, the distributions of HAs 

depended on the origin. The 80% fraction in the 

Horonobe HA was distributed below 10 kDa, while 

about 35% fractions in the Mobara and Tono HAs 

were found in same size range, indicating that the 

size of Horonobe HA is smaller than those of 

Mobara and Tono HAs. In previous study, it is 

Figure 2: Size distribution of Horonobe 

proposed that Mobara FA was distributed into 

groundwater HSs. Data for the Mobara and Tono 

smaller size fraction, compared to Nordic Lake FA HSs are referred from Nagao et al., 2009 

[2]. Thus, this indicates that, in same manner as Mobara and Tono groundwater FAs, the deep 

groundwater FA in Horonobe area is relatively smaller than surface water FA. 


The dissolved HSs in deep groundwater at depth of 495 – 550 m were extracted, and their structural 

characteristics were investigated with regard to acid functional groups and molecular size. Based on 

the series of analysis, following findings were obtained, (i) FA is dominant fraction of HSs in 

Horonobe deep groundwater, (ii) the deep groundwater FA has carboxyl groups of which content is 

the comparable amount as that of surface water FA, and (iii) more than 50% of the deep groundwater 

FA is found in the size fraction below 5kDa. These findings can be useful for understanding the metalion 

binding ability and mobility of deep groundwater HSs in geological disposal systems. 



We thank Prof. N. Fujitake at Kobe University for the measurement of NMR spectra. This study was 

partly funded by the Ministry of Economy, Trade and Industry of Japan. 

References 

1. S. Nagao, Y. Nakaguchi, N. Fujitake and H. Ogawa, Entering the Third Millennium with a Common 

Approach to Humic Substances and Organic Matter in Waters, Soil and Sediments. IHSS, Toulouse, 2000, 

p.1143. 

2. S. Nagao, Y. Sakamoto, R.R. Rao and N. Fujitake, Humic Substances Research, 5/6 (2009) 9. 

3. S. Nagao, T. Iwatsuki and K. Hama, Journal of Nuclear Fuel Cycle and Environment, 15 (2009) 77. 

4. M. Ueda and Y. Sakamoto, Journal of Nuclear Fuel Cycle and Environment, 12 (2006) 31. 

Vol. 3 Page - 52 -

Browning of Stream Water During Hydrological Events 

Dag Olav Andersen 

University of Agder, Department of Natural Sciences, Service box 422, 4604 Kristiansand, 

Norway 

E-mail: dag.o.andersen@uia.no 


Long-term data series show increased water colour and dissolved organic carbon (DOC) 

concentrations in Norwegian surface waters [1, 2] and more generally in northern Europe and 

North America [3]. Several mechanisms have been proposed to explain these observations but 

isolating single factors is notoriously difficult [4]. In this context seasonal variations in water 

colour, DOC and molecular size were studied in the two inlet streams of Lake Terjevann, 

southernmost Norway. 



Lake Terjevann is about 2.5 km from the coastline, about 8 km west of Kristiansand in the 

southernmost part of Norway. The catchment consists of four sub-catchments A, B and C+D 

of 0.50, 0.24 and 0.35 km 2 , respectively. The altitude ranges from 22 to 128 m above sea 

level. The soil cover is thin (0 – 70 cm) with abundant outcrops of bedrock, mainly felsic 

augen-gneiss. The vegetation is dominated by conifers, mainly Scots pine, with some oak and 

birch. About 50 % of sub-catchment A was forested with Norway spruce about 50 years ago. 

The climate is maritime with monthly mean temperatures commonly just below 0 o C in 

December, January and February and just above 14 o C in June, July and August. The annual 

mean deposition is about 1300 mm with maxima during late autumn and winter. Sea-salts as 

well as long-range transported acidic sulphur and nitrogen compounds are common. 

Samples of drainage water from sub-catchments A and B were collected almost weekly in 

clean glass bottles and transported to the laboratory where conductivity, pH, absorbance (200- 

700 nm) and HPSEC fractions were measured a few hours after sampling. Conductivity and 

pH were measured with standard Radiometer equipment calibrated against KCl and 

Radiometer buffers of 4.01 and 7.00 respectively. The UV-VIS spectra were obtained on 

filtered (0.45 µm) samples by a Shimadzu Multispec photodiode array spectrophotometer and 

a 10 mm quartz cuvette. The same instrument with a 10 mm quartz flow-cell was used as a 

detector (254 nm) in the HPSEC system in combination with a Perkin Elmer Series 200 pump 

and a Tosoh TSK-G3000SW column (7.5 x 600 mm with a 7.5 x 75 mm guard). A sodium 

Vol. 3 Page - 53 -

acetate buffer with pH 7.0 was used as mobile phase. DOC was measured on filtered (0.45 

µm) and acidified (pH 2) samples by a Shimadzu TOC-V total organic carbon analyzer with 

an ASI-V sampler unit. 

3. Results 

During the autumn and winter of 2004 the weekly depths of rainfall varied between 0 and 

about 45 mm (Fig. 1a) while the deposited amounts of sea-salts were relatively low. During 

this period, the stream water quality fluctuated slightly in conductivity and pH (Fig. 1b) while 

water colour and DOC (Fig. 2a) co-varied with rainfall and stream flow. During higher flow, 

higher molecular weight (HMW) materials dominated the DOC (Fig. 2b). 

Rainfall (mm) 

Conductivity; pH 

50 

45 

40 

35 

30 

25 

20 

15 

10 

5 

0 

13,0 

12,0 

11,0 

10,0 

9,0 

8,0 

7,0 

6,0 

5,0 

4,0 

a) 

01.09.2004 29.09.2004 27.10.2004 24.11.2004 22.12.2004 19.01.2005 16.02.2005 16.03.2005 13.04.2005 11.05.2005 08.06.2005 06.07.2005 

b) 

01.09. 

04 

29.09. 

04 

27.10. 

04 

24.11. 

04 


22.12. 

04 

19.01. 

05 

16.02. 

05 

16.03. 

05 

13.04. 

05 

Cond. (mS/m) 

pH 

m3/s 

Figure 1: a) Rainfall (mm) measured at Kjevik airport (bar chart) and discharge (m 3 /s) and b) 

conductivity (mS/m), pH and discharge in stream water from sub-catchment B. 

Vol. 3 Page - 54 - 

11.05. 

05 

08.06. 

05 

06.07. 

05 

0,40 

0,35 

0,30 

0,25 

0,20 

0,15 

0,10 

0,05 

0,00 

0,40 

0,35 

0,30 

0,25 

0,20 

0,15 

0,10 

0,05 

0,00 

-0,05 

Discharge (m 3 /s) 

Discharge

DOC; Colour 

HMW-fractions (%) 

Absorbance / DOC 

80 

70 

60 

50 

40 

30 

20 

10 

20,0 

16,0 

12,0 

8,0 

4,0 

0,0 

a) 

DOC (mg/L) 

mg Pt/L 

01.09.04 29.09.04 27.10.04 24.11.04 22.12.04 19.01.05 16.02.05 16.03.05 13.04.05 11.05.05 08.06.05 06.07.05 

b) 

01.09.04 29.09.04 27.10.04 24.11.04 22.12.04 19.01.05 16.02.05 16.03.05 13.04.05 11.05.05 08.06.05 06.07.05 

0,050 

0,045 

0,040 

0,035 

0,030 

0,025 

0,020 

0,015 

0,010 

c) 

01.09. 

04 

29.09. 

04 

27.10. 

04 

24.11. 

04 


22.12. 

04 

19.01. 

05 

16.02. 

05 

16.03. 

05 

13.04. 

05 

11.05. 

05 

240-280 

280-320 

320-400 

400-700 

Figure 2: a) DOC (mgC/L), water colour (mgPt/L), b) part HMW fractions (%) and c) normalized 

specific absorbance values for UV and VIS light in stream water from sub-catchment B. 

Vol. 3 Page - 55 - 

08.06. 

05 

06.07. 

05

In early January 2005 strong south-westerly winds entrained increased sea-salts into the 

atmosphere. Combined with relatively large amounts of rainfall, the catchments were heavily 

loaded. As a result the conductivity of the stream water increased from about 7 to 11 mS/m 

without a significant decrease in pH and the high conductivity persisted for several months 

(Fig. 1b). A concomitant decrease in water colour and DOC that also lasted for several 

months occurred in spite of the heavy rain and increased flow, e.g. January 11 (Fig. 1b). 

During this period, lower molecular weight materials dominated the DOC in the stream water 

(Fig. 2b). 

Rain low in sea-salts in May and June diluted the salt concentration in the soil indicated by 

the decrease in stream water conductivity that returned to about pre-event levels (Fig. 1b) as 

well as increased water colour and DOC (Fig. 2a), with higher proportions of HMW materials 

(Fig. 2b). 

4. Discussion 

The results indicate an increase in coloured HMW stream water DOC absorbing at longer 

wavelengths during hydrological events. Shifts in peak maxima to longer wavelengths are 

generally ascribed to conjugation and increasing numbers of chromophores [5], i.e. 

unsaturated HMW materials. Plant and soil organic matter are the two major sources for DOC 

in streams and rainwater percolating plant organic matter has been shown to obtain higher 

concentrations of coloured organic acids than from soil organic matter [6]. Flushing of the 

upper soil horizons seems to increase the export of coloured DOC from the catchment. 



Rainfall amounts define the hydrological flow paths in soils. Increased DOC and more 

coloured HMW DOC characterise the drainage from the upper soil horizons. Generally, 

browning of downstream lake waters may be explained, at least partly, by this mechanism. 

References 

1. H. Liltved, R. Wright and E. Gjessing, Vann, 1 (2001) 70. 

2. D. Hongve, G. Riise and J.F. Kristiansen, Aquat. Sci. 66 (2004) 231. 

3. B.L. Skjelkvåle et al., Envir. Pollut., 137 (2005) 165. 

4. N. Roulet and T.R. Moore, Nature, 444 (2006) 283. 

5. D.A. Skoog, D.M. West, F.J. Holler and S.R. Crouch. Fundamentals of analytical chemistry, 

Thomson Brooks/Cole, USA, 2004, Chapter 26, p. 784. 

6. E.M. Thurman, Organic geochemistry of natural waters, Martinus Nijhoff/Dr W. Junk Publishers, 

Dordrecht, 1985, Chapter 2, p. 67. 

Vol. 3 Page - 56 -

Fluxes of natural and combustion-derived organic matter into the coastal 

ocean off Southern Brazil 

David C. Podgorski a , JiYoung Paeng b , Thorsten Dittmar c , Marcos S.M.B. Salomao d , Carlos E. 

Rezende d , Marcelo C. Bernardes e , Bill Cooper a* 

a Florida State University, Department of Chemistry and Biochemistry, Tallahassee, Florida 

USA; b Florida State University, Department of Oceanography, Tallahassee, Florida, USA; 

c Max Planck Research Group for Marine Geochemistry, University of Oldenburg (ICBM), 

Oldenburg, Germany; d Universidade Estadual do Norte Fluminense, Laboratório de Ciências 

Ambientais, Campos dos Goytacazes, Brazil; e Universidade Federal Fluminense, 

Departamento de Geoquímica, Niterói, Brazil 

E-mail: cooper.chem.fsu.edu 


Combustion-derived black carbon is a potentially significant source of terrestrial organic 

matter in the ocean, especially in South Brazilian catchments where the burning of sugar cane 

is common practice. In this presentation we describe our efforts to identify and quantitate 

black carbon in riverine DOM fluxes and the fate of these fluxes in the estuaries and coastal 

zone of the North Fluminense region of Brazil. We utilized a variety of analytical tools to 

identify the major source terms of dissolved organic matter (DOM), including ultrahigh 

resolution mass spectrometry. 


Riverine and estuarine water samples were obtained during a series of cruises in May, 2008 

along the North coast of Rio de Janeiro State. The DOM was extracted using solid-phaseextraction 

(SPE) methods described previously [1]. The salt free extracts were oxidized with 

nitric acid in a microwave digestion system and the benzenepolycarboxylic acids (BPCAs) 

formed from condensed aromatic structures were separated and quantified with highperformance 

liquid chromatography and diode-array detection [2]. 

Ultrahigh resolution mass spectra were acquired on the home-built 9.4 T FT-ICR mass 

spectrometer located at the National High Magnetic Field Laboratory, Tallahassee, Florida 

[3]. Negative-Ions were produced by an external electrospray ionization source. 



The first analysis of ultrafiltered water samples from Rio de Janeiro state showed that the 

residues of pasture and mainly sugar cane burning are transported in the river system to a 

large degree as low-molecular weight solutes (

zone. BCPA analyses confirmed the presence of condensed aromatic structures (i.e. 

combustion-derived organic matter) in waters all along the coast of Rio de Janeiro. 

Bulk compositional information on this same DOM was obtained by FT-ICR mass 

spectrometry. We identify combustion-derived organic matter by assigning each molecular 

formula obtained from the mass spectrum a number to be used in a modified aromaticity 

index (A.I.mod) proposed by Koch and Dittmar [4]. The formulas, and those in the same 

homologous series, are considered to be aromatic if AImod > 0.5 and condensed aromatic if 

A.I.mod ≥ 0.67 [4]. The data is also projected on traditional van Krevelen Diagrams for further 

visual interpretation. 


Analyses of benzenepolycarboxylic acids (BPCAs) formed from the oxidation of condensed 

aromatic structures identified significant amounts of black carbon being transported down 

rivers and into the coastal zones of Southeastern Brazil. Molecular compositional information 

obtained by ultrahigh resolution mass spectrometry confirmed the presence of both aromatic 

and condensed aromatic compounds in these same samples. 



Financial support for sampling and analyses was provided by the National Science 

Foundation (NSF-OISE-0710744). Mass spectra were obtained at the National High Field FT- 

ICR Facility (NSF-DMR-06-54118). The Brazilian researchers are fellows of the National 

Institute for Science and Technology (TMCOcean, CNPq 573.601/2008-9) and Bilateral 

Cooperation supported by CNPq (490658/2006-7) and NSF. 

References 

1. T. Dittmar, B. Koch, N. Hertkorn, G. Kattner, Limnol. Oceanogr. Meth. 6, 230 (Jun, 2008). 

2. M. P. W. Schneider, R. H. Smittenberg, T. Dittmar, M. W. I. Schmidt, Geochim. Cosmochim. Acta 

73, A1181 (Jun, 2009). 

3. M.W. Senko, C. L. Hendrickson, L. PassaTolic, J. A. Marto, F. M White, S. H. Guan, A. G. 

Marshall, Rap. Comm. Mass Specrom. 10, 1824-1828 (1996) 

4. B. P. Koch, T. Dittmar, Rap. Comm. Mass Spectrom. 20, 926 (2006). 

Vol. 3 Page - 58 -

The Changes of Water Organic Contamination under the 

Influence of Ultrasounds 

L. Stepniak * , E. Stanczyk-Mazanek, U. Kepa 

Częstochowa University of Technology, Institute of Environmental Engineering 

Brzeźnicka 60a, 42-200 Czestochowa, Poland 

E-mail: stepniak@is.pcz.czest.pl 


Humic substances (HS) constitute of about 60–80% of the total content of the organic matter 

which is present in natural water. The indirect share of HS within the accumulation of heavy 

metals, the creation of stable suspensions and toxic substances (DBPs) determine the 

necessity of their removal. The type and the sequence of the processes in the contemporary 

water treatment systems are often selected in order to optimize the removal of natural organic 

matter. In this context, the unconventional ultrasonic method is considered as the removal 

mode of HS from water [1]. The literature-based research indicates that this method was 

described on the basis of the effects obtained with the use of prepared water, most frequently 

with the commercial humic acids (HA) preparation. The effectiveness of the ultrasonic impact 

on HA model solutions is evaluated on the basis of the TOC index decrease. The results 

showed that the effectiveness of reducing the TOC increased together with the intensity of the 

ultrasound and exposure time (35% for example at the intensity of 42 W/cm 2 and the time 

of 20 min.) [2]. The best effect was observed at pH 3 in comparison with the effect obtained 

at pH 5 and pH 11 [3]. It is believed that the sonochemical effects are associated with radical 

oxidation reactions (chemical degradation of HA) as well as with the impact forces generated 

during the annihilation of cavitation bubbles (mechanical fragmentation of HA) [2]. Due to 

the composition of HS in surface water (where fulvic acid are predominant), the verification 

of these effects in natural water environment is justified. 



The substrate for the research was surface and prepared water. The analysis of the natural 

surface water showed that the increased organic contamination indices: colour, oxygen 

consumption, TOC (DOC), UV254. High intensity of water colour occured mainly because 

of the presence of soluble HSs which predominated in the content of water samples.As the 

prepared water we used a solution of commercial HA (Fluka) in deionized water. These 

samples had the HA concentration of 30 mg/l, the content of which, determined by the TOC 

index, was similar to one given for the natural water (Tab.1). 

Vol. 3 Page - 59 -

Table .1: The physico-chemical analysis of the investigated water samples 

Index Unit Prepared Natural 

pH 

colour 

turbidity 

oxygen consumption 

total iron 

absorbance UV254 

TOC 

DOC 

– 

mg/l(Pt) 

NTU 

mgO2/l 

mg/l 

1/cm 

mgC/l 

mgC/l 

5.12 

80 

10.3 

12.14 

– 

0.68 

12.2 

9.66 

6.55–7.36 

40–45 

6.02–14.09 

7.07–10.04 

0.75–1.05 

0.17–0.43 

10.69–14.20 

7.91–11.97 

The samples of both waters underwent sonification at the natural pH solution, as well as at pH 

3 and pH 9. The water samples (500 ml volume) were sonificated with the use of the high- 

power ultrasonic generator of Sonics&Materials VC-750 (20 kHz) equipped with a sonotrode 

of 1.9 cm diameter (2.83 cm 2 surface). As a variable process parameter the vibration 

amplitude (12–60 μm) was taken, influencing the intensity of ultrasonic field. The maximum 

intensity was high–37.8 W/cm 2 (the density of power–0.2 W/ml). Changes in water organic 

impurity were controlled mainly by the TOC index analyses. The TOC were determined with 

the use of Analyzer Multi N/C 2100S (according to PN-EN 1484:1999). The selected results 

of the research presenting the effects of the investigated process throughout the changes of the 

TOC index depending on the amplitude, pH and the type of water were evaluated. 



Studying the prepared water pH influence on the HA removal with the ultrasound method, the 

highest effect was obtained at pH 3 (Fig. 1a). The decrease in the process effect at higher pH 

level (natural pH 5.12) is related to the increase of the HA dissociation level. In strongly 

alkaline medium, HA make proper solutions. In the case of natural water the effect of the 

process at the acidic and non-corrected reading was similar. At the alkaline habitat the 

decrease of the TOC has not been observed almost either. The effect of ultrasonic parameters 

visible in the examination results indicates that the vibration amplitude has a major 

importance for the process effects. Increasing the vibration amplitude enables an increase in 

ultrasonic field intensity to be obtained, which for A=60 μm amounted to approx. 40 W/cm 2 . 

For the greatest amplitude value and a sonification time of 10 minutes, the most favourable 

reduction of the TOC index (by 4 mgC/dm 3 ) was noted. The effect of removing water organic 

contaminants in the sonochemical oxidation processes was also indicated by a reduction in the 

oxygen consumption index. 

Vol. 3 Page - 60 -

TOC, mgC/dm 3 

15 

14 

13 

12 

11 

10 

9 

8 

7 

6 

5 

4 

3 

2 

1 

0 

a) prepared water 

0 10 20 30 40 50 60 70 

Amplitude A, μm 

acid 

alkaline 

natural 

TOC, mgC/dm 3 

15 

14 

13 

12 

11 

10 

9 

8 

7 

6 

5 

4 

3 

2 

1 

0 

b) natural water 

0 10 20 30 40 50 60 70 

Amplitude A, μm 

acid 

alkaline 

natural 

Figure 1: The influence of the vibration amplitude (t=10 min.) and pH on the TOC index changes in 

the water 

In order to verify his dependence the further tests were made for the higher mount of natural 

water samples. The samples were taken from the same source and were slightly varied 

in terms of the initial TOC value. The most efficient maximum amplitude of vibrations 60 

μm, was assumed. The average effectiveness of the process obtained in these tests was 

compared with the effectiveness for the prepared water from the previous series (Fig.1b), 

which is presented in Fig.2. 

Effectiveness, % 

35 

30 

25 

20 

15 

10 

5 

0 


A=60 μm, t=10 min. 

acid 

natural 

alkaline 

prepared w ater natural w ater 

Figure 2: The effectiveness of the TOC reduction for the prepared water and the mean effectiveness 

for the natural water (SD= 2.1–3.1) in relation to the pH 

Vol. 3 Page - 61 -

The obtained results reveal that the effectiveness of the process for the researched natural 

water was the highest at the natural pH (6.55–7.36) and amounted to over 25%. These 

incompatibilities need to be explained in the research conducted on fulvic acids (FA) 

solutions, dominating in natural surface types of water. As it was stated in other research, that 

as smaller-sized and more dissociated molecules, are removed in ultrasound field mainly with 

the involvement of radical mechanism. Simultaneously, the results of this research indicate 

the lower ultrasonic effect for FA in comparison with HA [2, 3]. Regardless of the kind 

of water, the disadvantageous influence of the alkalinity, which inhibits the radical processes 

of oxidation, was proved. 


The effect of ultrasonic field intensity, as defined by the vibration amplitude, on process 

effectiveness was confirmed for water investigated. The maximum effectiveness of the 

ultrasonic method, i.e. 32% (60 μm, 10 min), was achieved for prepared water at the acid 

reaction. The effect of pH on the process effectiveness for natural water may be connected 

with the share of KH and KF in the composition of HSs. The sort of the fraction of the acids 

in water depending on the pH influences the intensity of the effectiveness 

of ultrasounds. The intensity depends on the share of mechanical and chemical degradation 

of the researched humic compounds. What influences the ultimate effect is the content 

of different organic compounds in natural water, revealed by the amount of organic carbon. 

As a result of this, the effectiveness of the sonochemical influence, at the non-corrected 

natural water reaction was higher than for the prepared water. 



This investigation was supported by the statutory research fund No. BW/401/203/07 of the 

Częstochowa University of Technology. 

References 

1. T. J. Mason, E. Joyce, S.S. Phull, J.P. Lorimer, Ultrasonics Sonochemistry, 10 (2003) 319–323. 

2. V. Naddeo, V. Belgiorno, R. Napoli, Desalination, 210 (2007) 175–182. 

3. F. Chemat, P.G. Teunissen, S. Chemat, P.V. Bartels, Ultrasonics Sonochemistry, 8 (2001) 247– 

250. 

Vol. 3 Page - 62 -

Unexpected Uniformity of Humic Substances in Thermal Waters 

Krisztina Kovács a* , Csanád Sajgó b , Alice Brukner-Wein b , Zoltán Kárpáti d , András Gáspár c , 

Etelka Tombácz a , Philippe Schmitt-Kopplin c 

a University of Szeged, Department of Physical Chemistry and Material Science, 6720 Szeged, 

Aradi Vt. 1., Hungary; b Institute for Geochemical Research, Hungarian Academy of Sciences, 

1112 Budapest, Budaörsi St. 45., Hungary; c Helmholtz Zentrum München, German Research 

Center for Environmental Health, Institute of Ecological Chemistry, Department of 

BioGeoChemistry and Analytics, 85764 Neuherberg, Ingolstädter Landstr. 1., Germany; 

d Budapest Sewage Works Ltd., North-Pest Wastewater Treatment Plant; 1044 Budapest, 

Timár St. 1, Hungary 

E-mail: kkriszta@chem.u-szeged.hu 


Thermal water is warm, hot groundwater tapped from deeper aquifers. According to the local 

geothermal gradient value (50 ºC km -1 in Pannonian Basin) the water temperature increases in 

a function of aquifer depth. Traditionally groundwater from natural springs and wells has 

supplied drinking water and fed artificial spas for a long time. Humic substances are proven to 

have biologic effects (e.g. anti-inflammatory and anti-viral activity), so the balneological use 

of thermal waters is of great importance. In addition, heat of thermal waters provides local, 

import independent, renewable energy source, which is free from pollution emission and 

unaffected by weather conditions. Low temperature (

wells in February 2008. However, the acidification was performed immediately after the 

sampling in the field. The acidified samples were stored below 5 ºC until processing. 

Isolation of humic substances was according to the procedure of International Humic 

Substance Society “Method for Preparation of IHSS Aquatic Humic and Fulvic Acids” [1]. 

The isolated humic and fulvic acids were characterized by potentiometric acid-base titration 

(equilibrium titration, CO2-free condition, 0.01M NaCl as background electrolyte), FTIR 

spectroscopy (KBr technique, Perkin-Elmer 1600 Series) and ESI-FT-ICR mass spectrometry 

(Bruker APEX Qe Fourier transform ion cyclotron resonance mass spectrometer (FTICR/MS) 

equipped with a 12 Tesla superconducting magnet and an APOLLO II ESI source, negative 

ionization mode). The samples were dissolved in mixture of methanol and water (in the 

volume ratios 99:1 and 99.5:0.5 MeOH:H2O, total volume 2 mL) directly before the analysis 

and analyzed at 10 mg L -1 concentration. Humic acid solutions were prepared using trace 

amount of NH4OH for complete dissolution (40 μL 28w% NH4OH). Additional details on 

calibration, molecular formulae determination can be found in a recent paper [2]. 


Isolation method permits the gravimetric determination of humic and fulvic acid 

concentration in water on the basis of the amounts of isolated material in the reference to the 

volume of the water sample. The results of the same sample in each year show only a small 

difference in the concentration of humic fractions (Table 1). 

Table 1: Humic and fulvic acid concentration in thermal waters determined by gravimetry. 

Depth (m) 


Sampling year Humic acid 

(mg L -1 ) 

Fulvic acid 

(mg L -1 ) 

993 2006 1.9 1.2 

2007 1.9 0.9 

2103 2006 6.7 2.7 

2008 7.0 2.6 

The acid-base properties of the isolated organic acids were investigated by potentiometric 

acid-base titration. Some pH-dependent ionization curves are illustrated in Fig. 1. The amount 

of charged groups is equivalent to the dissociated acidic groups of the acids. The amount of 

charged groups across the pH range and the total acidity value at pH 10 are very similar for 

both humic and fulvic acids from different samplings. Before the titration NaOH was not 

added to the solutions of humic acids, so the complete dissolution of the humic acids took 

Vol. 3 Page - 64 -


place in the course of titration. Due to the hindered dissolution in acidic region, the measured 

points of humic acids did not run smoothly in succession until pH 7–8 as those of fulvic acids. 

Figure 1: The pH-dependent dissociation of acidic groups on humic and fulvic acids isolated from 

different samplings of thermal waters tapped from 2103 and 993 m 

The infrared (IR) absorbance values belonging to different wave numbers, i.e., 2925, 1710 

and 1620 cm -1 can be used for representing the aliphatic, carbonyl and aromatic content of the 

humic substances (Fig. 2). Ratios of these absorbance values are suited to illustrate the 

differences and changes in aliphatic, carbonyl and aromatic content [3]. The differences 

between the ratios of humic and fulvic acids from different samplings are negligible. 

Figure 2: Ratios of IR absorbance values for humic and fulvic acids from different samplings (e.g. Aal 

/ Acar expresses the ratio of absorbance at 2925 cm -1 to tt at 1710 cm -1 ) 

The ultrahigh-resolution FT-ICR mass spectrometric data was converted to molecular 

formulae. The elemental compositions for these peaks can be calculated from mass spectra, 

allowing their H/C and O/C atomic ratios to be calculated and plotted on the van Krevelen 

diagram [4]. The positions of the patterns occupied by points differ slightly in case of humic 

acids obtained from 993 m depth thermal water. Additional detailed investigation on 

molecular composition is needed. 

Vol. 3 Page - 65 -

Figure 3: van Krevelen diagrams of humic acids from different samplings of thermal waters tapped 

from 993 and 2103 m 


The characteristics of humic and fulvic acids isolated from thermal waters of various depths 

are different. However, these properties do not show considerable changes depending on the 

sampling method, i.e. the date of acidification of the water sample. It can be supposed that the 

changes in conditions such as temperature, pressure, presence of oxygen do not affect the 

properties of humic and fulvic acids after outcropping groundwater. This observation may 

confirm the recalcitrant feature of humic substances in groundwater, too and facilitate the 

design and operation of geothermal works from these materials of point of view. 



The research was supported by the Hungarian National Science Foundation (OTKA) through 

grant T-48829 and IHSS Training Award 2007. 

References 

1. E.M. Thurman and R.L. Malcolm, Environ. Sci. Technol., 15 (1981) 463. 

2. A. Gaspar, E.V. Kunenkov, R. Lock, M. Desor, I. Perminova, Ph. Schmitt-Kopplin, Rapid 

Commun. Mass Sp., 23 (2009) 683. 

3. G.P. Lis, M. Mastalerz, A. Schimmelmann, M.D. Lewan, B.A. Stankiewicz, Org. Geochem., 36 

(2005) 1533. 

4. S. Kim, R.W. Kramer, P.G. Hatcher, Anal. Chem., 75 (2003) 5336. 

Vol. 3 Page - 66 -

Basic By-Products Formation During Chlorination of Water Containing 

Humic Substances 

Ekaterina V. Trukhanova a* , Margarita Yu. Vozhdaeva a , Lev I. Kantor a , Evgeniy A. Kantor b 

a Municipal enterprise “Ufavodokanal”, Rossiyskaya str., 157/2, 450098 Bashkortostan, 

Russia; b Ufa State Oil Technical University, Komarova str., 1, 450089, Bashkortostan, 

Russia 

E-mail: e.truhanova@mail.ru 



Organic composition of natural water is formed by soil and peat humus, plankton, higher 

aquatic vegetation, animal organisms, as well as by organic compounds introduced into water 

reservoirs by urban settlements being developed, by industrial and agricultural facilities [1]. 

Organic materials forming water composition may be both of natural and anthropogenic 

origin. Humic compounds are natural materials that have the most significant effect on water 

quality (by amount and by composition) being precursors of formation of numerous water 

treatment by-products, particularly after chlorine disinfection of water. 

Chlorination of natural water containing humic materials is followed by formation of 

chlorine-bearing toxic, mutagenic and cancerogenic substances [2]. Trihalomethanes (THMs) 

and haloacetic acids (HAAs) [3] are main by-products of chlorine disinfection of water. 

Therefore, problem of HAAs and THMs formation during water treatment (Fig. 1) and 

problem of control of their content are topical, especially for regions with a high humic 

content in water. 

Figure 1: Scheme of HAAs and THMs formation during natural waters chlorination [4] 

No data is currently available in Russia on pattern of HAAs distribution in regional drinking 

water, as well as no approved control procedures. In this connection, this research is focused 

on studying dynamics of THMs and HAAs formation in drinking water of the city of Ufa. 

Vol. 3 Page - 67 -


Determination of HAAs in water: A procedure of HAAs determination in water has been 

developed by us on the basis of EPA 552.1 and 552.2 methods. This procedure is developed 

in order to determinate monochloroacetic acid (MCAA) (within the range of 0.005–0.04 

mg/dm 3 ), dichloroacetic acid (DCAA), thichloroacetic acid (TCAA), monobromoacetic acid 

(MBAA), chlorobromoacetic acid (CBAA) (0.001–0.04 mg/dm 3 ), dibromochloroacetic acid 

(DBCAA), dichlorobromoacetic acid (DCBAA) (0.001–0.02 mg/dm 3 ). This procedure is 

based on a twofold liquid-liquid extraction of HAAs with methyl-tert-butyl ether (MTBE), 

followed by conversion of HAAs into methyl ethers with acid methanol and gas 

chromatography with electron-capture detector (GC-ECD). 

Determination of THMs in water: Determination of THMs in water was carried out with a 

static mode sample preparation, followed by a Headspace Analysis. This procedure allows to 

identify: chloroform (within the range of 0.0006–0.007 mg/dm 3 ), bromodichloromethane and 

dibromochloromethane (0.0003–0.03 mg/dm 3 ), bromoform (0.001–0.1 mg/dm 3 ). 



Water samples of two water supply systems – river water intake system (RWIS) and 

infiltration water intake system (IWIS) – were used to study HAAs and THMs content in 

drinking water monthly from October 2006 through October 2009. 

Diverse composition of HAAs forming in drinking water of RWIS and IWIS depends mainly 

on source water quality, as well as on composition of organic and inorganic impurities in 

water. 

As Fig. 2 shows, THMs and HAAs content in RWIS water is approximately equal, while in 

IWIS water the amount of HAAs formation predominates over the amount of THMs 

formation. Total THMs content in RWIS drinking water within the entire period of the study 

exceeds approximately 4 times total THMs content in IWIS water – 0.022 and 0.0056 mg/dm 3 

respectively. In the case of HAAs this difference amounts to 2.5 times – 0.024 and 0.0095 

mg/dm 3 . 

Average seasonal THMs concentrations indicated that during autumn drinking water sampling 

THMs content is twice higher in water systems under study than during winter-spring period. 

This can be explained by diverse humic composition in source water during these periods. 

Vol. 3 Page - 68 -

С, mg/dm 3 

0,06 

0,05 

0,04 

0,03 

0,02 

0,01 

0 

October 

November 

December 

January 

February 

March 

April 

May 

Total THMs 

a) b) 

Total HAAs 

С, mg/dm 3 

0,06 

0,05 

0,04 

0,03 

0,02 

0,01 

0 

October 

November 

December 

January 

February 

Figure 2: Total THMs and HAAs from October 2008 through May 2009: a) in RWIS drinking water, b) 

in IWIS drinking water 



Dynamics of formation of water chlorination by-products (haloacetic acids and 

trihalomethanes) which occur during chlorination of water containing humus, is different, 

depending on season and on water treatment procedure. Total content of by-products under 

study occurring in RWIS water after a complex treatment exceeds several times similar 

content in water from an infiltration water intake. This fact is explained by a high content of 

humic substances, which are precursors of THMs and HAAs formation, in river water, 

comparing to water from under-river wells that has passed filtering gravel beds. It is possible 

to note a clearly remarkable seasonal prevalence in forming of chlorination by-products. 

Therefore, study of pattern of THMs and HAAs distribution and their content in drinking 

water is important while organizing drinking water quality analytical control. 

References 

1. A.V. Slipchenko, L.A. Kulskiy, E.S. Matskevich. Modern condition of water impurities oxidation 

methods and chlorination prospects. Water Chemistry and Technique, 1990, vol. 12, №4, p. 334– 

336. 

2. L. Ts. Bonter., L. P. Alekseyeva, Ya. L. Khromchenko. Effect of organic impurities in natural 

water on formation of toxic volatile halogen-alkanes during chlorination. Water Chemistry and 

Technique, 1986, vol. 8, №1, p.37–41. 

3. V.V. Goncharuk, N.A. Klimenko, L.A. Savchina, et al. Modern problems of drinking water 

treatment technique. Water Chemistry and Technique, 2006, №1, vol. 28. 

4. Rook J.J., Formation of galoforms during chlorination of natural waters. J. AWWA. 1976, V.68, 

№3, p. 168 

Vol. 3 Page - 69 - 

March 

April 

May

Research of the Physics and Chemical Properties on Sediments of the 

Lobelia Lakes in West Pomeranian Region of Poland 

Lilla Mielnik a* , Jacek Czekała b 

a West Pomeranian University of Technology in Szczecin, Department of Physics and 

Agrophysics, ul. Papieża Pawła VI 3, 71-459 Szczecin, Poland; b Department of Soil Science 

and Land Protection, University of Life Sciences in Poznań, ul. Szydłowska 50, 60-656 

Poznań, Poland 

E-mail: lilla.mielnik@zut.edu.pl 


Bottom sediments are the important element of all lakes. These sediments are considered to be 

a valuable and unique source of processes occurring in the aquatic environment. The quality 

of organic matter is an important feature with respect to the physicochemical conditions on 

the lake bottom. The approximate value of the amount and quality of the organic matter, as 

well as its decomposition, can be determined by the organic carbon to total nitrogen ratio 

Corg/Ntot [Kamaleldin et al., 1997, Meyers, 2003, Twichell et al., 2002, Mielnik, 2005, 

Mielnik et all 2009]. The Corg/Ntot ratio in lake sediments is often used as an indicator of 

time changes in the organic matter cycles in aquatic ecosystems [Kamaleldin et al. 1997]. 

The work presents and discusses the results of research on the physics-chemical composition 

of bottom sediments of Lobelia lakes located in West Pomeranian Region in Poland. 


The research material was the bottom sediments of the Lobelia lakes. To do the investigations 

one chose reservoirs, which differ in morfometric parameters and drainage basin development 

The lake sediments were sampled from the superficial layer (at the depth of up to 25 cm) in 

duration of the summer stagnation. The samples were collected in two fields: (i) in the costal 

area in the littoral zone (samples designated L), (ii) at the maximum depth of the basin in the 

profundal zone (samples designated P). In the air dried sediments there was determined the 

content of organic carbon Corg by Orlov and Grindel method and the content of total nitrogen 

Ntot by Kiejdahl method. The Corg/Ntot values were determined based on the results obtained. 



Values of analysed parameters Corg and Ntot were various and changed in terms of lake 

character, as well as in terms of the field where the sediments were collected in the lake basin 

(Table 1). Due to the distinct distribution of depth, sedimentation takes place in a different 

Vol. 3 Page - 70 -

way in each lake in the shallow zone as well as in the deeper one. In the bottom sediments of 

the investigated lakes the Corg contents ranged from 0.4 to 29.5 %. This indicates there is a 

different contribution of organic and mineral substances to the examined sediments. 

This showing at different participation of organic and mineral substances in examined 

sediments. 

Examining the total nitrogen Ntot content in sediments provides important information about 

the quality of sedimentary organic matter. The concentration of Ntot in the investigated 

sediments is significantly high from 0.05 to 3.13 %. Bottom sediments from littoral zone in 

two lakes: Morskie Oko i Ciemino were exception (the concentration of Ntot in this sediments 

0.03 and 0.05 %). High nitrogen content in lake sediments results from high protein content in 

the organic matter. This is a product of the life activity of aquatic organisms, as well as the 

decomposition of plant and animal residues. 

Table 1. Elemental composition of the sediments of the studied lakes 

Lake Zone Maximum depth[m] pH Corg 

[%] 

Jelonek 

Kociołek 

Wielkie 

Oczko 

Morskie Oko 

Ciemino 

Ntot 

[%] 

Ptot 

[g/kg] 

Corg/ Ntot 

L 3.5 5.9 21.0 1.76 1.82 11.9 

P 

20.1 1.78 1.80 11.3 

L 16.3 5.7 2.4 0.10 0.12 21.1 

P 

22.0 1.61 1.94 13.7 

L 10.0 7.6 20.8 1.50 0.55 13.8 

P 

20.3 1.60 2.18 12.7 

L 19.2 7.0 0.4 0.03 0.05 13.4 

P 

29.5 3.13 2.55 9.4 

L 12,6 8.1 0.7 0.05 0.18 13.3 

P 


13.0 1.59 1.52 8.2 

L – litoral zone, P – profundal zone. 

Calculated values of the Corg/Ntot ratio are be reflected of differentation Corg and Ntot contents 

in the examined sediments too. The Corg/Ntot ratio for examined sediments was practically the 

same for all the lakes at the range of 8.2 to 13.8. This indicates the predominant contribution 

of plankton to organic matter production, and the rather low contribution of higher plants 

containing lignin and cellulose. The sediments in littoral zone of Kociołek lake are the 

exception. In this sediments the Corg/Ntot ratio were 21.1 and suggests the big participation of 

the macrophytes and higher plants rich in lignin and cellulose and poor in protein [Meyers and 

Ishiwatari 1993, Meyers 1997, Mielnik et all 2009]. 

When analyzing the Corg/Ntot ratio, it should be taken into consideration that the selective 

decomposition of the organic matter by microorganisms during its slow diagenesis may be 

Vol. 3 Page - 71 -

modified by its elemental composition as well as by the Corg/Ntot ratio of the organic matter in 

sediments [Kamaleldin et al. 1997 Routh et al. 1999]. 

The biological mineralization of the phosphorus depends on biochemical transformations of 

carbon and nitrogen. The tendency of changes in Ptot content in the examined sediments is 

similar to that observed for Corg and Ntot content. 


The differences in sediments in respect of their chemical properties result from the individual 

features of each lake, including the morphometric differences, and the character of the 

drainage basin for which the lake is a sedimentation tank. The flora growing over the water 

body is a significant factor differentiating the properties of the sediments. 



This work was supported by the Polish Ministerial Research Project: No.0547/P01/2008/34. 

References 

1. M. H. Kamaleldin, J.B. Swinehart, R.F. Spalding, Journal of Paleolimnology, 18, (1997), 121– 

130. 

2. P.A. Meyers, R. Ishiwatari, Organic Geochemistry, 20 (1993,) 867-90 

3. P.A. Meyers. Organic Geochemistry 34, (2003) 261–289. 

4. L. Mielnik, (in polish), Inżynieria Rolnicza 4(64), (2005) 31-36. 

5. L. Mielnik, R. Piotrowicz, P. Klimaszyk, Oceanological and Hydrobiological Studies, XXXVIII, 

3, (2009), 69-76. 

6. J. Routh, T.J. McDonald., E.L. Grossman, Organic Geochemistry, 30, (1999), 1437-53. 

7. S.C. Twichell, P.A. Meyers, Organic Geochemistry 33, (2002) 715–722. 

Vol. 3 Page - 72 -

Ratio of Color to Chemical Oxygen Demand as an Indicator of Quality of 

Dissolved Organic Matter in Surface Waters 

Andrey I. Konstantinov a* , Nikolay S. Latyshev b , Petr A. Ivkin b , Irina V. Perminova a 

a Department of Chemistry, Lomonosov Moscow State University, Leninskie Gory, 1-3, 

119991 Moscow, Russia; b OJSC «NII VODGEO», Komsomolskii prospect, 42-2, 119048 

Moscow, Russia 

E-mail: konstant@org.chem.msu.ru 


The majority of surface waters contain significant amounts of dissolved organic matter 

(DOM). To assess the content of DOM, different parameters are used, such as color, chemical 

oxygen demand and total organic carbon (TOC). Those parameters give direct or indirect of 

the content of DOC, but they do not characterize its quality. The important parameter of DOM 

which defines its hydrophobic-hydrophilic balance is the content of aromatic carbon. It has 

been numerously shown [1] that the absorbance value at 254 nm normalized to mass 

concentration of DOM expressed on organic carbon (OC) basis (known as SUVA parameter) 

gives a good indirect estimate of aromaticity of DOM. In this study, we have hypothesized 

that a ratio of two bulk parameters traditionally used to characterize water quality such as 

color and chemical oxygen demand (COD) may serve as an analogue of SUVA in 

characterizing quality of DOM, nominally, of its aromaticity as well as of the contribution of 

humic matter into the total pool of DOM To prove this hypothesis, the existence of the 

relationship was tested between direct aromaticity estimates provided by 13 C NMR and 

values of color to COD parameters for a set of well characterized humic materials. The 

obtained relationship was further extended to aromaticity assessment of DOM present in the 

samples of medium and high colored surface waters. 



Two samples of medium and highly colored surface water (the Volkhov River (Russia) and 

Orsha River (Belarus) were used in this study sampled according to standard procedure [2]. 

Seven samples of humic substances (HS) isolated from different sources according to IHSS 

technique [3] were studied: fulvic acids (FA) and humic acids (HA) of sod-podzolic soil 

(Moscow region, Russia) – SFA and SHA; HA and non-fractionated humic substances (HS) 

of high-land sphagnum peat (Tver region, Russia) – PHA and PHF; coal HA of leonardite 

(Humintech Ltd, Germany) – CHA; HS of the Istra River (Moscow region, Russia) and the 

Suwannee River (IHSS standard, USA) – AHF and SRDOM, respectively. 

Vol. 3 Page - 73 -

Color of water was determined using chromate-cobalt units according to standard procedure 

[4]. Chemical oxygen demand (COD) was determined by the Kubel method [5]. UV-Vis 

analysis was conducted using spectrophotometer Cary-50 (“Varian”, USA) using 1 cm quartz 

cuvettes. Organic carbon content in the model humic materials was determined using Carlo 

Erba Strumentazione elemental analyzer (Italy). Quantitative 13 C solution state NMR spectra 

were acquired using Avance 400 spectrometer (Bruker, Germany) operating at 100 MHz 

carbon-13 frequency. The spectra were recorded on the samples dissolved in 0,3 M 

NaOD/D2O at concentration of 80 mg/mL. Carbon-13 NMR spectra were acquired with a 5 

mm broadband probe, using CPMG pulse program with 7,8 s relaxation delay and acquisition 

time about 0,2 s and INVGATE procedure. Aromatic carbon content (CAR) was determined as 

integral intensity in the spectral region from 110 to 165 ppm according to [6]. 


To characterize SUVA values of model humic materials used, UV absorbance was registered 

at 254 nm and normalized to the concentration of solution expressed on organic carbon basis. 

To explore if the color to COD ratio can be used as a parameter of aromaticity of DOM, the 

model set of humic materials was analyzed for color and COD values as well. The obtained 

results are given in Table 1. The corresponding correlation plots are shown in Figure 1. 

Samples DOC 

(mg/L) 

Table 1: Obtained physicochemical characteristics of the model HS samples 

Abs254 


SUVA254 

(L/[mgС*cm]) 

CAR 

(%) 

Color 

(Cr-Co 

units) 

COD 

(mgO2/L) 

Color/COD 

(Cr-Co 

units*L/mgO2) 

SFA 7,00 0,476 0,043 31 55,2 7,6 7,3 

SHA 8,02 0,406 0,042 34 128,0 10,2 12,6 

PHF 8,97 0,493 0,044 34 135,4 12,2 11,1 

PHA 6,69 0,742 0,085 38 202,9 18,0 11,2 

CHA 5,07 0,682 0,100 56 185,1 9,8 18,8 

IRDOM 11,65 0,472 0,032 26 39,9 9,0 4,4 

SRDOM 10,48 0,493 0,038 30 99,8 13,1 7,6 

As follows from the obtained results, there are statistically significant relationships observed 

between the values of color to COD ratio and Car as well as between color to COD ratio and 

SUVA254. 

Vol. 3 Page - 74 -


Figure 1: Correlation plots of CAR vs Color/COD (left) and SUVA254 vs Color/COD (right) 

At the next stage of this study Color/COD values were used to estimate quality of DOM after 

water treatment. For this purpose, the medium colored water samples from the Volkhov River 

and highly colored samples from the Orsha River were treated with the coagulants and the 

floculant. Water treatment efficiency coefficients (η) were calculated according to the 

equation (1). 

η (%) = 100% . (Xorigin – X)/Xorigin, (1) 

where: X – Color, COD and Color/COD values; index “original” means non-treated water sample 

Characteristics of water quality measured and η values calculated are shown in Table 2. As it 

can be seen, water treatment efficiency is slightly less for medium colored water samples as 

compared to the highly colored ones. This corroborates well the lower value of Color/COD 

parameter indicating less aromaticity of the medium colored water sample and its lesser 

susceptibility to the action of coagulants and flocculants. 

Hence, the parameter of Color/COD can be used for prediction of efficiency of water 

treatment. The higher values of this parameter may be indicative of better suitability of 

traditional coagulants-flocculants technologies to treatment of the corresponding water 

samples. This can be the case because the higher Color/COD values can be provided by the 

considerable content of high-molecular weight HS that have a high negative charge and are 

able to form stable water insoluble compounds with coagulant molecules. Low values of 

Color/COD can indicate high concentration of low-molecular fractions of DOM. In this case, 

water treatment with coagulants is less efficient. 

Vol. 3 Page - 75 -

Sample 

№ 

1.0 

(original) 

1.1 

1.2 

1.3 

2.0 

(original) 

2.1 

2.2 

2.3 

Table 2: The quality assessment of the original and treated samples of the river waters 


Source 

The 

Volkhov 

River 

The 

Orsha 

River 


Coagulant 

amounts, mg/l 

Color COD Color/COD 

AOC PS Cr-Co η, % mgO2/L η, % Cr-Co η, 

units 

units % 

L/mgО2 

- - 93.4 - 19,9 - 4,7 - 

8 - 16,6 82 9,7 51,4 1,7 

- 8 14,0 85 11,3 43,3 1,2 

4 4 27,2 71 15,3 23,1 1,8 

- - 288,7 - 38,5 - 7,5 - 

30 - 19,5 93 10,3 73,2 1,9 

- 18 27,6 90 13,2 65,5 2,1 

5 15 34,8 88 16,2 57,9 2,2 

1. Color/COD parameter is developed to characterize the content of aromatic carbon in DOM. 

Its applicability for this purpose was proven using a set of well characterized HS. 

2. The introduced parameter was used to asses efficiency of water treatment technology. High 

values of the parameter indicate a high efficiency of water treatment based on coagulantflocculant 

approach, low values indicate that alternative approach (for example, oxidation) 

should be used. 

References 

1. S.J. Traina, J. Novak, N.E. Smeck, J. Environ. Qual., 41 (1990) 151–153. 

2. GOST R 51592-2000 Water. The general sampling requirements (in Russian). 

3. R.S. Swift, Organic Matter Characterization, in D.L. Sparks (Ed.), Methods of Soil Analysis, Part 

3. Chemical Methods, Soil Sci. Soc. of America, Madison, WI, 1996, p. 1018–1020. 

4. GOST 3351-74 Drinking water. Techniques to determine taste, odor, chromaticity and turbidity 

(in Russian). 

5. Yu.V. Novikov, K.O. Lastochkina, Z.N. Boldina, Techniques to research water quality of water 

reservoirs, Meditsina, Moscow, 1990 (in Russian). 

6. D.V. Kovalevskii, A.B. Permin, I.V. Perminova, V.S. Petrosyan, Moscow State University 

Bulletin, Series 2 (Chemistry), 41 (2000) 39–42 (in Russian). 

Vol. 3 Page - 76 - 

63, 

6 

73, 

6 

62, 

1 

74, 

8 

72, 

3 

71, 

3

UV-Vis Spectrometry and Size-Exclusion Chromatography Study of 

Seasonal Dynamics of Quality of Dissolved Organic Matter 

Andrey I. Konstantinov a* , Ekaterina V. Trukhanova b , Margarita Yu. Vozhdaeva b , 

Lev I. Kantor b , Irina V. Perminova a 

a Department of Chemistry, Lomonosov Moscow State University, Leninskie Gory 1-3, 

119991 Moscow, Russia; b Municipal enterprise “Ufavodokanal”, Rossiyskaya str. 157/2, 

450098 Ufa, Bashkortostan Republic, Russia 

E-mail: konstant@org.chem.msu.ru 


The necessity of upgrading natural water treatment techniques which would improve 

performance of conventional techniques is urged by new achievements in understanding 

nature of dissolved organic matter (DOM) as well as by development of new analytical 

instrumentation [1, 2]. The important issue for research is elucidating the feature of DOM 

which impacts the most efficiency of water treatment technology used for drinking water 

production. This feature might be connected to the source of natural water, as well as to 

seasonal conditions. The objective of this research was to study seasonal dynamics of the 

quality of DOM in surface and infiltration waters using UV-Vis spectrometry and sizeexclusion 

chromatography (SEC). 



Nineteen raw and treated water samples were studied. The samples were taken from the 

surface water intake system (SWIS): four samples of surface water (SW) were taken from the 

River Ufa in July and November 2008, and in February and April 2009, four samples of 

filtered water (FW) were taken after the surface water passed several treatment steps (primary 

chlorination with low doses of chlorine, chemical treatment and high-rate filters with burnt 

rock filtering medium), and four samples of drinking water (FWCl) (filtered water undergone 

secondary chlorination). The water samples from infiltration water supply system (IW) were 

also included into the experimental data set represented by four samples of non-chlorinated 

water samples taken in July and November 2008. and in February and April 2009, and three 

samples of drinking water taken from the infiltration water supply system that passed 

chlorination in November 2008, and February and April 2009. 

UV-Vis spectrometry analysis was conducted using spectrometer Cary-50 (Varian, USA) 

equipped with quartz cuvettes with optical path length of 1 cm. 

Vol. 3 Page - 77 -

SEC system consisted of a solvent pump, a packed column and a UV-detector with variable 

wavelength as described elsewhere [3]. The UV-absorbance was measured at 254 nm. The 

SEC column was 15x250 mm packed with Toyopearl HW-55S (“Toso-Haas”, Japan). 0,03M 

phosphate buffer with pH 6,8 was used as a mobile phase at a flow rate of 1 ml/min. The 

column was calibrated using sodium polystyrenesulfonates (PSS) (Da): 4480, 14000, 20700, 

45100, and 80840 (Polymer Standard Service, Mainz, Germany). Blue dextran (2000 kDa) 

served as a void volume probe, acetone – as a permeation volume probe. 

Dissolved organic carbon (DOC) content was measured photometrically with Skalar SAN plus 

Segmented Flow Analyzer («Skalar», Netherlands). The method is essentially based on 

decomposition of dissolved organic matter using ultraviolet irradiation and potassium 

persulphate oxidation to carbon dioxide. Carbon dioxide diffuses through special gaspermeable 

silicone membrane and is absorbed by phenolphthalein solution. Optical density of 

the phenolphthalein solution is measured at a wavelength of 550 nm. 


UV-Vis absorbance spectra of the samples studied are given in Figure 1. They are typical for 

natural DOM. To characterize UV absorptivity of the water samples, specific UV absorbance 

(SUVA) was calculated at 254 and 280 nm which correspond to wavelengths characteristic of 

absorbance of aromatic groups. SUVA is defined as the UV absorbance of a water sample at a 

given wavelength normalized to DOC concentration. The results obtained are given in Table 

1. 

0,25 

0,2 

0,15 

0,1 

0,05 


0 

200 300 

wavelength, nm 

400 500 

IW-nov 

IWCl-nov 

SW-nov 

FW-nov 

FWCl-nov 

Figure 1: Typical UV-Vis absorbance spectra of the samples studied 

Vol. 3 Page - 78 -


As it can be seen from Table 1, the filtered river water sample (FW) has the highest SUVA 

value at both wavelengths, though, at the same time, its DOC concentration decreased 1,7 

times compared to water from surface water supply (the River Ufa) (SW). 

Table 1: UV absorbance (А) and SUVA of the autumn samples studied (at 254 and 280 nm) 

Sample Dissolved organic carbon A254 SUVA254, A280 SUVA280, 

(DOC), mg/L 

L/(mgС*cm) 

L/(mgС*cm) 

IW-nov 1,7 0,0293 0,0173 0,0215 0,0126 

IWCl-nov 1,5 0,0232 0,0154 0,0165 0,0110 

SW-nov 4,2 0,0622 0,0148 0,045 0,0107 

FW-nov 2,5 0,0511 0,0204 0,038 0,0153 

FWCl-nov 2,7 0,0429 0,0159 0,030 0,0111 

The results obtained indicate that water treatment steps (filtration of water samples) mostly 

cause a decrease in aliphatic matter and increase in aromatic one. Secondary chlorination 

causes partial decomposition of aromatics-rich organics, which is reflected as a decrease in 

SUVA values for chlorinated water samples (FWCl and IWCl) as compared to less 

chlorinated (FW) and non-chlorinated (IW) samples. The similarity of DOC values for the 

SW samples might indicate an increase in low-molecular chloroorganic compounds – 

chlorination by-products. 

Typical SEC chromatograms in Kd scale are given in Figure 2. Molecular weight 

characteristics calculated from the chromatograms obtained are given in Table 2. 

Figure 2: Typical chromatograms of the samples studied: surface (left) and infiltrated water 

IW-samples taken in autumn are characterized with the lowest Mw and Mp values, summer 

samples have the highest ones, and winter and spring values – medium values. Maximum 

molecular weight (MW) of spring samples is caused by the high rate of humus formation in 

the spring. Increased microbiologic activity in summer is accompanied by degradation of 

organic matter and a decrease in average MW of DOM of IW. In autumn and winter, when 

the rate of humus formation is the lowest. Mw and Mp are the lowest. 

Spring samples have maximum Mw and Mp for SW which is caused by floods. Provided that 

DOM being highly concentrated in river waters is more susceptible to decomposition during 

water treatment, it is reflected in maximum decrease in Mw and Mp of spring samples after 

Vol. 3 Page - 79 -


filtration and chlorination. Decreasing Mw and Mn after chlorination in autumn, winter and 

spring water samples might indicate their enrichment with low-molecular weight chlorination 

byproducts. This is in sync with the results of UV-Vis study of autumn water samples. For a 

summer sample (FW-jul) after chlorination (FWCl-jul) an increase in Mw and Mn was 

observed that might be indicative of more complete decomposition of low-molecular weight 

components of DOM in summer samples as compared to those of other seasons. 

Table 2: Molecular-weight characteristics of the samples studied 

Sample 

Molecular weights, Da 

Polydispersity 

Weight-average (Mw) Number-average (Mn) Peak (Mp) (Mw/Mn) 

IW-jul 6050 4050 6700 1,5 

IW-nov 5070 4130 5820 1,2 

IW-feb 5750 4290 6260 1,3 

IW-apr 5860 4460 6230 1,3 

IWCl-nov 4740 3500 5260 1,4 

IWCl-feb 5110 3430 5950 1,5 

IWCl-apr 4990 2590 6110 1,9 

SW-jul 5780 3590 6440 1,6 

SW-nov 5830 4340 6560 1,3 

SW-feb 5960 3510 6870 1,7 

SW-apr 6730 4350 7310 1,5 

FW-jul 6070 4150 6770 1,5 

FW-nov 5480 3970 6280 1,4 

FW-feb 5620 3690 6420 1,5 

FW-apr 5270 3140 6190 1,7 

FWCl-jul 6510 5150 6890 1,3 

FWCl-nov 5160 3560 5820 1,5 

FWCl-feb 4160 2120 5210 2,0 

FWCl-apr 4010 2410 4730 1,7 


The seasonal dynamics of structural and molecular weight characteristic of dissolved organic 

matter is determined by seasonal changes in microbiological activity and in the rate of humus 

formation including seasonal floods for surface water. In accordance with the results of UV- 

Vis and SEC studies, efficiency of surface water treatment during spring season drops 

substantially. 

References 

1. E.M. Perdue, in G.E. Likens (Ed.), Encyclopedia of Inland Waters, Academic Press, NY, 2009, p. 

806-819. 

2. J. Peuravuori and K. Pihlaja, in L.M.L. Nollet (Ed.), Handbook of Water Analysis, CRC Press, 

NY, 2nd edn., 2007, p. 435-447. 

3. I.V. Perminova, F.H. Frimmel, A.V. Kudryavtsev, N.A. Kulikova, G. Abbt-Braun, S. Hesse, V.S. 

Petrosyan, Environ. Sci. Technol., 37 (2003) 2477-2485. 

Vol. 3 Page - 80 -

Humin Contribution to Sedimentary Organic Matter of The Adriatic Sea 

F. Rampazzo a* , D. Berto a , M. Giani b , C. Baldin a , L. Langone c 

a Istituto Superiore per la Protezione e la Ricerca Ambientale, Brondolo Chioggia (Ve), Italy; 

b Istituto Nazionale di Oceanografia e di Geofisica Sperimentale, Trieste, Italy; c Istituto di 

Scienze Marine, ISMAR-CNR, Bologna 

E-mail: f.rampazzo@icram.org 


A substantial proportion of the so-called refractory organic matter (OM) in waters and 

sediments is constituted by humic substances (HS) that are structurally complex 

polyelectrolytic, dark coloured organic acids which are formed from the decomposition of 

plant, animal and microbial tissues and tend to be more recalcitrant than their precursors [1]. 

They are defined according to fractionation schemes, based on solubility, in humic acids 

which are soluble in dilute alkaline solution and precipitate under acid condition, fulvic acids 

which are soluble in both base and acids and humin (HM) insoluble at all pH conditions. 

Due to its insolubility in alkali, HM is difficult to study and few investigations were 

performed in marine environments. 

The aim of this study is to quantify the contribution of HM to sedimentary organic matter and 

to investigate its origin (autochthonous or allochthonous sources) and biogeochemical 

features in order to investigate the formation pathway of HS in the Adriatic sediments. 



Sediments cores were sampled in three sites located respectively in a coastal area of central 

Adriatic sea (0-25 cm), in the middle Adriatic pit (0-25 cm) and in the southern Adriatic pit 

(0-35 cm) during the SIT-1 cruise carried out in the framework SESAME project during 

February 2008. The extraction of HS from sediments was performed following the method of 

the International Humic Substance Society modified following Moreda-Pineiro [2]. 

Organic carbon (Corg) and total nitrogen were determined in sediments and residual HM by a 

CHN Elemental Analyzer. Corg was determined after removal of carbonates with HCl. Weight 

percentages of nitrogen were determined following the same procedure. The content of HM in 

the sediments was expressed as mg C/g sediment (dry weight) and as % of HM-carbon on the 

total Corg in sediment. Stable isotopic ratio of organic carbon ( 13 C/ 12 C) was determined by a 

CHNS-O analyzer coupled with an Isotope Ratio Mass Spectrometer. The results were 

expressed in parts per mil from the international standard VPDB (Vienna Pee Dee Belemnite). 

Vol. 3 Page - 81 -


Corg content in sediments was lower in the southern Adriatic pit (0.35 ± 0.20 %), while higher 

values were observed in the coastal central Adriatic (0.65 ± 0.04%) and in the meso Adriatic 

pit (0.61 ± 0.08 %). 

HM contribution to the sedimentary organic matter was relevant (Table 1) in all stations, 

increasing with the depth along the core, in particular in the southern Adriatic pit. 

HS can constitute up to 40-68% of the organic carbon in other coastal and oceanic sediments 

[3] or up to 64-100% of organic matter in some estuaries [4] where the humic acids are 4-28% 

of the total HS. 


Table 1: Corg/N ratios and humin concentration in the Adriatic sediments. Coastal Central Adriatic Sea 

(CCA), Middle Adriatic Pit (MAP) and Southern Adriatic Pit (SAP) sediments 

Coastal central 

Adriatic CCA 

Middle Adriatic Pit 

MAP 

Southern Adriatic 

Pit SAP 

Parameters N Mean Dev.Std. N Mean Dev.Std. N Mean Dev.Std. 

Corg/N (mol/mol) 4 8.9 0.4 4 7.4 0.6 6 6.7 0.9 

Chumin in sediment (mg/g) 4 4.7 0.7 4 4.2 0.3 6 2.8 1.4 

Chumin/Corg % 4 72.6 13.6 4 69.2 9.0 6 86.1 11.1 

Corg/N ratios in the sediments of the southern Adriatic pit were in the range typical of 

phytoplankton. These values are comparable with respect to those of the residual HM (Fig.1) 

suggesting a common origin of the sedimentary organic matter. This hypothesis is also 

confirmed by the isotopic ratio (δ 13 C) of sediments. 

In the station located in the coastal zone of the middle Adriatic, more subjected to continental 

organic matter input, the highest Corg/N ratios of HM fractions were observed (Fig.1). 

The increase of Corg/N ratio in the HM of the coastal station with respect to the values 

reported for the bulk sedimentary organic matter could indicate possible higher degradation 

processes of the HS during sedimentation in the water column and burial in the sediments. It 

cannot be excluded that a part of this HM could derive from allocthonous material refractory 

to degradation like vascular plants compounds rich in lignin that may be preserved or only 

partially altered by oxidation. 

Vol. 3 Page - 82 -

C org/N molar ratio 

10.0 

9.0 

8.0 

7.0 

6.0 

5.0 

4.0 

SAP MAP CCA 

Stations 

C org/N sediments (mol/mol) 

C org/N humin (mol/mol) 

Figure 1: Corg/N molar ratio in sediments and sedimentary humin of the. Coastal Central Adriatic Sea 

(CCA), Middle Adriatic Pit (MAP) and Southern Adriatic Pit (SAP). Mean values of all the core 

standard deviation and standard error are represented 


A large amount of sedimentary organic matter (up to 80%) in Adriatic Sea is due to the HM 

fraction pointing out the possible relevance both of the different origin of the organic matter 

depending on the zone and of the humification processes. 



We thank the captain and the crew of the R/V Urania of CNR for the assistance during the 

VECSES1 cruise. The research activity has been possible thanks to the financial support of 

the VECTOR Italian national project and the SESAME EU-FP6 project (contract n. 36949). 

References 

1. J.D. Coates, K.D.Cole, R., Chakraborty, S.M.O’Connor and L.A. Achenbach, Appl. Environ. 

Microbiol. 68 (2002) 2445. 

2. A. Moreda-Piñeiro, A.B. Barrera and P.B, Barrera, Anal. Chim. Acta 524 (2004) 97. 

3. A. Nissenbaum and I.R.Kaplan, Limnol. Oceanogr. 17 (1972) 570. 

4. L. Tremblay and J.P. Gagné, Org. Geochem. 38 (2007) 682. 

Vol. 3 Page - 83 -

Influence of Pre-Ozonation of Solutions of Fulvic Acid on Equilibrium 

Adsorption on Activated Carbon 

I. Kozyatnyk * , L. Savchyna, N. Klymenko 

a Institute of Colloid Chemistry and Chemistry of Water, National Academy of Sciences of 

Ukraine, 42 Vernadsky Avenue, Kyiv 03680 

E-mail: koziatnik@ukr.net 



Adsorption on activated carbon (AC) is one of the best methods for removing of natural organic 

matters (NOM) in the technology of high quality drinking water preparation. Activated carbon is used 

in the technological scheme for the final removal of the NOM before chlorination to prevent the 

formation of chlorinated disinfection by products in most water supply [1]. 

In [2] it was proposed to use the constants of adsorption equilibrium applying to the concentration of 

the mixture of organic substances “conditional component” for quantify the absorbability of a 

multicomponent mixture of organic substances from aqueous solutions. Using “conditional 

component” approach [2] is most successfully accomplished through the application of ideal 

adsorption solution theory [3–5] for estimation of equilibrium adsorption parameter of 

multicomponent NOM. 

Different ability to the adsorption for factions of FA on the AC is stipulated primarily by the value of 

their hydrophilic-lipophilic balance and the availability of the porous space of the AC for the 

penetration of molecules of FA with different molecular weight. It is shown the higher the degree of 

hydrophilicity of the adsorbate, the lower the change of its Gibbs free energy of adsorption [2]. 

The effectiveness of adsorption of NOM on the biologically active carbon is determined by the value 

of biodegradable organic carbon (BDOC) in the content of total organic carbon in solution. It is known 

that the biologically degradable organic matters more hydrophilic compared with bioresistant. So 

perhaps there should be a correlation between the BDOC content in the solution of FA and the 

magnitude of the free energy of adsorption of FA and products of their destruction on the BAC. 

In [6] it was proposed an approach for assessing the rational degree of organic matter oxidation in 

water before to subsequent stages of treatment. It is known that the effectiveness of biofiltration of 

organic matter on the AC depends on the change of Gibbs free energy of adsorption (-ΔG 0 а): the 

higher the rate, the lower the contribution of the biodegradable component in the overall efficiency of 

the process of biofiltration and vice versa [7]. Ozonation converts dissolved organic carbon from 

hydrophobic to hydrophilic organic carbon without significant removal of dissolved organic carbon. 

The positive role of this transformation is particularly effective when filtering water through BAC [8]. 

Thus, the purpose of this study was to evaluate the parameters of equilibrium adsorption of NOM (for 

example, FA) from aqueous solutions and their changes after ozonation solutions before adsorption. 

Vol. 3 Page - 84 -


As the object of study were used fulvic acids (FA), obtained by the Forsyth method [9] from highmoor 

peat. The object of investigation was KAU carbon, which is obtained by treatment of crushed 

fruit stones with concentrated alkali and hot hydrochloric acid (after washing with water), washing, 

carbonization and activation with steam. The major adsorptive characteristics of that carbon were as 

follows: the total specific surface of 1036.4 m 2 /g, the limiting adsorptive pore volume of 0.51 cm 3 /g, 

the microporous space volume of 0.16 cm 3 /g. The investigations were conducted using the AC fraction 

of 0.5–1.0 mm. 

The ozonation of the FA solutions has been performed as follows: 5 L of the FA solution have been 

treated with the ozone/airy mixture. The ozone concentration in that mixture was 5 mg/L. The delivery 

rate of that mixture was 2.5 L/min. The solutions were ozonized for 2, 4 and 6 min, and the 

corresponding ozone dosages were 6, 12 and 18 mg/L. 

The BDOC part in the TOC has been determined using technique represented in [10–12]. It is pointed 

out in these papers that, at the BDOC determining, it is the best thing to fix the microorganisms at the 

inorganic carriers (quartz sand) and to use the microorganisms from the potable water treatment 

stations and the distributing systems. 

We investigated the adsorption equilibrium in systems with non-ozonized and ozonized solutions of 

FA at pH 6. For ozonation were used solutions of FA with different initial concentration of dissolved 

substances: a)1 series of experiments - the content of TOC 12.7–14.7 mg C/L; b) 2 series of 

experiments - the content of TOC 31.5–33.7 mg C/L. Assessment of adsorption characteristics of AC 

samples at adsorption from aqueous solutions of FA was carried out by the Freundlich model, using 

the approaches given in [13,14], and the method of “conditional component” [2]. 



At the first it is necessary to evaluate the equilibrium adsorption capacity of the AC for qualitative and 

quantitative assessment of the effectiveness of activated carbon for water purification from NOM. 

Removing of the NOM leads to a decrease in colour and content of BDOC, and removal of precursor 

of chlorine disinfection by-products formation. Adsorption isotherms can be used to determine the 

minimum dose of adsorbent and the ratio of the liquid-sorbent in the dynamic experiments. 

At the different content of TOC in the initial solution, the change of the adsorption is different in 

ozonated solutions at the same equilibrium concentration. The dependence of the equilibrium 

adsorption on the concentration of the initial solution is a feature of adsorption from multi-component 

NOM solutions, as noted in several studies [15, 16]. The change in ozone dose has different effects on 

reducing the adsorption capacity of the AC. The smallest decrease in the magnitude of adsorption is 

observed at a dose of 12 mg O3/L, the largest – with 6 mg O3/L. This shows the different chemical 

nature and molecular size of the products of the NOM oxidation. 

Vol. 3 Page - 85 -


Freundlich equation constants was defined in order to 

evaluate the influence of ozonation on the parameters 

of equilibrium adsorption. Furthermore, it was 

estimated the change in free energy of adsorption of 

FA ozonation products using the method of conditional 

component. Figure shows the adsorption isotherms of 

FA from aqueous solutions in the coordinates of the 

modified Freundlich equation [2–4] when the initial 

concentration of TOC in solution was 31.5–33.7 mg/L. 

Isotherms at initial concentration of TOC 12.7–14.7 

mg/L have the same shape. 

а, mg С/g 

1000 

100 

10 

1 

0,1 

○ – 1 

■ – 2 

▲ – 3 

♦ – 4 

1 10 100 

С , mg С/l 

eq 

Isotherms of FA adsorption from ozonated and nonozonated 

solutions: 

1 – non-ozonated; 2 – 6 mg O3/l; 

3 – 12 mg O3/l; 4 – 18 mg O3/l 

As it is seen from Fig. the adsorption isotherm of non-ozonized FA solution at initial TOC 

concentrations 31.5–33.7 mg C/L in logarithmic coordinates of the Freundlich equation has two 

distinct areas that are characterized the adsorption capacity of different FA factions in solution. As a 

result of ozonation two distinct areas disappear on the FA adsorption isotherms in the Freundlich 

equation coordinates, and adsorption isotherms are described by a straight line. That is, the 

composition of the FA becomes more uniform with respect to the adsorption capacity. 

Table 1: Changing in the Freundlich equation constants, BDOC and (-ΔGa 0 ) values at the ozonation of 

FA solutions 

TOC, mg 

C/L 

Ozone dose, 

mg C/L 

BDOC (part of the TOC),% 

Freundlich equation constants а 

КF 

1/n 

(-∆Gа 0 ), 

kJ / mol 

14.7 0 48.2 0.33 1.73 19.08 

12.7 6 56.6 0.69 0.94 17.37 

13.7 12 51.0 0.64 1.27 18.08 

13.6 18 49.8 1.52 0.77 19.05 

31.6 0 52.6 0.54 1.52 18.85 

31.5 6 47.9 0.91 1.28 19.05 

32.1 12 45.8 1.56 1.02 19.76 

33.7 18 47.2 0.84 1.40 19.76 

As seen from Table 1, ozonation leads to an increase in the BDOC content with lower initial 

concentrations of TOC in solution. Change of free energy of adsorption (-ΔGа 0 ) also decrease that 

confirms our assumption about the correlation of these values. Change the dose of ozone has 

practically no effect on the change in the percentage BDOC in ozonated solutions when we ozonated 

of solutions with higher initial content of the BDOC. Biodegradable organic carbon value decreases 

from 52.6% in non-ozonated solutions to 47.2–47.9% in ozonated solutions. According to these the 

value of (-ΔGа 0 ) changes from 18.85 kJ/mol up to 19.05 – 19.76 kJ/mol. 

Increasing of the ozone dose leads to an increase in the value of the coefficient KF and reduce 1/n, due 

to a change in the chemical properties of ozonation products compared to initial multi-component 

solution of FA. The constants KF and 1/n for non-ozonized solution were calculated for the first plot, 

which characterizes the adsorption of low-adsorbed faction of FA. Averaging properties of FA 

ozonation products in relation to the adsorption capacity generally leads to an increase in the constant 

Vol. 3 Page - 86 -

KF and decrease the constant 1/n. Ozonation of FA solutions by dose 12 mg/L probably leads to the 

formation of by-products with properties different from those in other ozonated solutions. We can 

conclude that it is difficult to judge unambiguously about the influence of ozonation on the adsorption 

of FA from aqueous solutions on the AC on the basis of analysis of the Freundlich equation constants 

changes. Therefore it is more appropriate to use a another approach, associated with the definition of 

the correlation between the percentage of BDOC in solutions of FA and the change in Gibbs free 

energy of adsorption of products of FA ozonation. 

It is evident from present data that ozonation of the FA solutions with high relative BDOC content 

leads to a reduction of its value in total organic carbon. This fact can have positive effects in the 

subsequent process of coagulation because of hydrophobization of FA ozonation products. However, it 

is expedient to increase the value BDOC in the content of TOC at the sequential combination of 

ozonation and biofiltration through activated carbon to improve the process. 



Thus, it can be concluded that ozonation of FA solutions with high initial BDOC content by ozone 

doses that are economically and technologically acceptable, leads to a decrease in the BDOC value 

compared with non-ozonized solution. Ozonation of FA solutions leads to equalization of the 

adsorption ability of FA factions compared with non-ozonized solution. Ozonation of FA solutions 

increases the adsorption energy of FA in the most of the investigated systems with a high initial 

BDOC content. This may worsen the conditions for biofiltration through biologically active carbon, 

but also improve the conditions of coagulation treatment of water. Prediction of the effectiveness of 

ozonation NOM solutions before filtration through BAC is more appropriate by determination the 

value of the free energy of adsorption. 

References 

1. S.D Faust and Aly O.M. Chemistry of Water Treatment, Lewis Publishers, Boca Raton, 1999, p.581. 

2. A.M. Koganovskij, N.A. Klymenko, T.M. Lievchenko and I.G. Roda, Adsorption of Organic Matters from 

Water, Khimia, Leningrad, 1990, p. 256. In Russian. 

3. G.W. Harrington and F.A. DiGiano, J. Amer. Water Works Assoc., 81 (1989) 93. 

4. E.H. Smith and W.J.Jr. Weber, Water Air Soil Pollut., 53 (1990) 279. 

5. H. Sontheimer, J.C. Crittenden and R.S. Summers, Activated Carbon for Water Treatment, DVGW– 

Forschungstelle, Karlsruhe, 1988, p. 690. 

6. N.A. Klymenko, L.V. Nevynna, Yu.V. Sydorenko, O.G. Shvidenko, and Yu. O. Shvadshina, J. Water 

Chem. Technol., 29, 1 (2007), 15. 

7. N.A. Klimenko, M. Winther-Nielsen, S. Smolin, L. Nevynna and Y. Sydorenko Water Res., 36 (2002) 5132 

8. W. Nishijima, W.H. Kim, E. Shoto and M. Okada, Water Sci. Technol., 38, 6 (1998) 163. 

9. L.N. Alexandrova, Soil Organic Matter and Processes of its Transformation, Nauka, Leningrad, 1980. In 

Russian. 

10. J. Joret, Y. Levi, T. Dupin and M. Gilbert, Proc. AWWA Annual Conference, Orlando, FL., 1998, 1715. 

11. S. Trulleyova and M. Rulik, Science of the Total Environment, 332 (2004) 253. 

12. U. Raczyk-Stanislawiak, J. Swietlik, A. Dabrowska and J. Nawrocki, Water Res., 38 (2004) 1044. 

13. E.H. Smith, Water Res, 28 (1994) 1693. 

14. E.H. Smith and W.I. Weber, Water Air Soil Pollut., 53 (1990), 279. 

15. S. Qi and L. Schideman, Water Res., 42 (2008), 3353. 

16. F.S.Li, A. Yuasa, H. Chiharada and Y. Matsui, J. Colloid Interface Sci., 265, 2 (2003), 265. 

Vol. 3 Page - 87 -

Study of Estuarine Sediments in Galway Bay 

R. Mylotte a* , M. H. B. Hayes a , C. Dalton b 

a Chemical and Environmental Science Dept., University of Limerick, Ireland; b Dept. of 

Geography, Mary Immaculate College, Limerick, Ireland 

E-mail: rosaleen.mylotte@ul.ie 



Oceans are the largest global carbon pool and are estimated to hold approximately 38,000 

PgC (petagrams of carbon) (Mahli, 2002). The oceanic sediments contain 150 Pg of organic 

matter (OM) (Ridgewell and Edwards, 2007). The oceans potentially could absorb and store 

vast quantities of anthropogenic carbon dioxide (CO2), a potent greenhouse gas. Cold 

turbulent waters dissolve CO2 while warm waters dissolve less CO2 and can even release 

CO2, switching the oceans to a source of CO2. Global warming has focused attention on 

oceans in an effort to reduce atmospheric CO2 and to help prevent acidification of the ocean 

surface. 

Core samples are being studied from the transitional waters in Galway Bay. A main focus of 

the study is the effect that the estuary is having on the bay especially, with regards to the 

organic matter (OM) present. OM is washed into the Bay from the River Corrib and its 

tributary streams. OM is a reservoir of carbon (in sediments) and an important sink. Studying 

the organic and inorganic colloidal components contained within the estuarine sediments can 

give indications of changes that have occurred over time to the composition of the matter 

transported to the estuary and will provide an insight into the composition of carbon 

sequestered in the sediments. 

The humic substances will be extracted from each of the four cores at different depths. Humic 

substances (HS) are the refractory end products from the degradation of plant and microbial 

organic material (Lepane, 1999). HS is composed of humic acid (HA), fulvic acid (FA) and 

humin. HS were traditionally thought to be high molecular weight molecules (Laird, 2008). 

Work by Piccolo (2001) as elaborated Sutton and Sposito (2005) has suggested that HS are a 

supramolecular association of many relatively small and chemically diverse organic 

molecules that form clusters linked together by hydrogen bonds and by hydrophobic 

interactions. The project is studying in detail the compositions of the HS at different depths 

and their associations with the sediments. 

Vol. 3 Page - 88 -

2. Location 

Galway Bay is located in the west of Ireland between Co. Galway and Co. Clare. The River 

Corrib flows from Lough Corrib into Galway Bay. Core samples were taken at increasing 

distances from the River Corrib estuary (see Fig. 1). Four cores were taken using a vibrocorer 

(Geo-corer 6000) supplied by the Geological Survey of Ireland aboard the Marine Institute’s 

ship the Celtic Explorer (Cruise No. CE09-04). Core samples are up to 6m in depth. Surface 

grab samples were also taken using a Day grab. 



Figure 1: Map of Coring Positions in Galway Bay 

The sediment samples are pretreated with HCl to remove any CaCO3 present (H + exchanged). 

This involves adding 1M HCl to the sediment and leaving it to stand until the reaction is 

complete. The OM is extracted and analysed using methods developed by the Carbolea 

Research Group. The methods employed involved exhaustive extraction using 0.1M NaOH 

(at pH 7, 10.5 and 12.6), 0.1M NaOH + 6M Urea, and DMSO + 6% H2SO4. All extractions are 

carried out under nitrogen. The humic substances (HS) are isolated using the XAD-8 and 

XAD-4 resin in tandem procedure (Hayes et al. 2008). Solid and liquid state NMR will be 

employed to investigate the compositions of the organic fractions (Song et al., 2008). The 

sediment (core 1 0-25cm) has being fractionated into sand, silt and clay. The isolated clay 

will be analyzed using X-ray diffraction to determine the minerals present. The Walkley 

Black procedure (Allison, 1965) will be used to determine the total organic matter in the 

sediments at different depths and different fraction. Elemental analysis and XRF core 

scanning will determine what elements are present in the sediment. The Itrax XRF can also 

take high resolution images allowing visualization of the layers present in the sediment. Dr. 

Catherine Dalton will investigate the palaeontology of the sediments at different depths. 

Vol. 3 Page - 89 -

Changes in chemical composition can be related to palaeontology differences observed. A 

master core will be dated radiometrically. Microfossils will be dated using 14 C Accelerator 

Mass Spectrometry. Diatoms and foraminifera will be examined to investigate biological 

diversity. The quality of the intact core, sedimentation rates, permeability and chemistry will 

be analysed using the Geotek multi sensor core logger. Sediment core lithology will be 

examined to determine the rock present and the associated minerals. 

4. Results/Discussion/Future Work 

From the extractions (Grab samples A1001, D1004 and core 1 0-25 cm), pH 7 and pH 10.5 

(adjusted using 0.1M NaOH) yielded no OM in the supernatent. Extractions at pH 12.6 and 

6M urea + 0.1M NaOH resulted in breaking of the hydrogen bonds and OM was released. 

Therefore the sediment has been exhaustively extracted at this pH. The particulate matter was 

removed by centrifugation and filteration. Further extractings with DMSO + 6% H2SO4 are 

ongoing.The humic fractions will be isolated using resins. Core’s 1 to 4 have been scanned, 

the results are being analysed. 

0 

0.5 

1 

1.5 

2 

2.5 

3 

3.5 

4 

4.5 

5 

5.5 

Core Thickness (cm) 

P Wave Amp 


P Wave Vel (m/s) 

gamma density (gm/cc) 

10.0 10.5 11.0 11.5 12.00.0 

0.2 0.4 0.6 0.8 1.0 0 400 800 1200160020000.0 

1.0 2.0 3.00. 

0 2.0 4.0 6.0 

Figure 1: GeoTek MSCL Plot for Core 1 

Magnetic 

Susceptibility (S 

The preliminary results from core 1 (Fig. 1) have been compiled. Further analysis of the 

results is necessary. Pictures were taken with a digital camera of the cores (see Fig. 2) as they 

were split using the GeoTek core splitter. 

Top Bottom 

Figure 2: Picture of a split core 1 (2-3 metres) showing visable layers 

Vol. 3 Page - 90 -

Cores 1 to 4 have been scanned with the Itrax XRF scanning instrument. This generates a vast 

amount of information which needs to be analysed. The clay has been found to be aggregrated 

in the sediment. Currently there are experiments underway to disaggregrate the clay. The 

efficacies of sonication and the addition of a deflocculant (sodium hexametaphosphate), or a 

combination of both, are being determined. Clay will be analysed by XRD to identify the 

minerals present that are associated with carbon sequestration. Soil minerology can help to 

protect HS from degradation. This highlightes the importance of investigating the clay 

minerology. This work will examine the composition of the carbon sequestered in oceanic 

sediments. 



This work is funded by the HEA through the PRTLI IV scheme. Thanks to my supervisors, 

Prof. Michael Hayes and Dr. Catherine Dalton. Thanks to the Marine Institute, Dr. D. Toal, 

Dr. C. Dalton and F. Melligan, all of whom were involved in collecting the core samples. I 

also thank Dr. S. McCarron and Dr. J. Turner who are technicians in NUI Maynooth and 

UCD. 

References 

1. Allison, L.E., (1965) ‘Organic carbon’ In: Method of Soil Analysis, Part 2, Chemical and 

Microbiological Properties (eds. Black, C.A., et al.), pp. 1367-1378. American Society of 

Agronomy, Inc., Madison, WI. 

2. Hayes, T. M., Hayes, M. H. B., Skjemstad, J. O., Swift. R.S., (2008), 'Studies of compositional 

relationships between organic matter in a grassland soil and its drainage waters', European 

Journal of Soil Science. 59: pp. 603-616. 

3. Laird, D. A., Chappell, M. A., Martens, D. A., Wershaw, R. L. and Thompson, M, (2008), 

'Distinguishing black carbon from biogenic humic substances in soil clay fractions', Geoderma, 

143 : page 115-122 

4. Lepane, V., (1999), 'Comparison of XAD resins for the isolation of humic substances from 

seawater', Journal of Chromatography A, 845: pp. 329-335 

5. Mahli, Y., (2002), ‘Carbon in the atmosphere and terrestrial biosphere in the 21st century’, 

Philosophical Transactions of The Royal Society, 360: pp. 2925-2945 

6. Piccolo, A., (2001), 'The Supramolecular Structure of Humic', Soil Science, 166: pp.810-832 

7. Ridgewell, A. and Edwards, U., (2007), 'Geological Carbon Sinks', p.76, in Raey, D., Hewitt, C. 

N., Smith, K and Grace, J., (eds.), Greenhouse Gas Sinks, UK, CABI Publishing 

8. Song, G., Novotny, E. H., Simpson, A. J., Clapp, C. E., Hayes, M. H. B., (2008), ‘Sequential 

exhaustive extractions, and characterisations using solid and solution state NMR, of the humic, 

including humin, components in a Mollisol soil’, European Journal of Soil Science, 59: pp. 505- 

516. 

9. Sutton, R. and Sposito, G, (2005), 'Molecular Structure in Soil Humic Substances: The New 

View', Environmental Science and Technology, 39 : pp. 9009-9015 

Vol. 3 Page - 91 -

Effect of River Floods on Marine Organic Matter Fluorescence 

Edith Parlanti a,b* , Stéphane Relexans a,b , Fabienne Ibalot a,b , Sandrine Huclier-Markai c,d , Rudy 

Nicolau c , Stéphane Mounier c , Yves Lucas c , 

a Université de Bordeaux, UMR 5255, ISM, Groupe LPTC, 351 Cours de la Libération 

Talence, F-33405 France; b CNRS, UMR 5255, ISM, Groupe LPTC Talence, F-33405 

France; c Université du Sud Toulon-Var ; Laboratoire PROTEE-CAPTE, B.P. 132, La Garde 

Cedex, F-83597 France; d Université de Nantes, Laboratoire Subatech, Ecole des Mines de 

Nantes, CNRS/IN2P3, 4 Rue A. Kastler, BP 20722, 44307 Nantes Cedex 3, France. 

E-mail: e.parlanti@ism.u-bordeaux1.fr 


Dissolved organic matter (DOM) consists of a mixture of macromolecular compounds with 

wide ranging chemical properties and diverse origins. Fluorescence spectroscopy has been 

applied for characterizing fluorescence properties of coloured dissolved organic matter 

(CDOM) for several decades. This technique yields important information on the dynamics 

and chemical nature of bulk CDOM as a function of its fluorescence intensity and fluorescent 

functional groups. The monitoring of the fluorescent DOM has often been used to distinguish 

between water masses from various sources [1, 2], to follow the distribution of water masses 

[3] or, equally to study the mixing processes in coastal and estuarine waters [4, 5]. In the 

1990s, three-dimensional excitation emission matrix (EEM) spectroscopy came into more 

common use for characterizing fluorescence properties of CDOM. EEM spectroscopy 

provides highly detailed information and the data can be analysed as excitation spectra, 

emission spectra or synchronous scan spectra. This technique reveals the complete 

photophysical system of the complex multi-chromophore macromolecular CDOM and is now 

largely used for the characterization of fluorescent organic material in aquatic environments. 



The fluorescence spectra were recorded with a Fluorolog SPEX FL3-22 Jobin Yvon 

Fluorometer. The fluorescence EEM spectroscopy involved scanning and recording of 17 

individual emission spectra (260-700 nm) at sequential 10 nm increments of excitation 

wavelength between 250 and 410 nm. 

In order to discuss the results of the fluorescence analysis of the different samples, we 

considered on the one hand the ratios of the intensities of the main fluorescence bands. On the 

other hand, we applied to aquatic environments the humification index (HIX) [6] in order to 

estimate the maturation of DOM in soils. Referring to this humification index HIX, we also 

Vol. 3 Page - 92 -

uilt another parameter BIX (biological index) to characterize the autochthonous inputs 

(biological origin) to DOM. 

The goal of this study was to characterise the organic matter inputs from a small river 

(Eygoutier River) into the Mediterranean Sea (Toulon Bay). We studied the organic matter 

inputs during the lowest water levels of the river and the flood events for two years (2004 and 

2005). Raw water samples were filtered through a 0.7 µm GF/F glass fibre filter. Samples 

were treated against bacterial growth by adding 100 µL of 98 % sodium azide. 


The increase of terrestrial DOM inputs in seawater was very well correlated with the increase 

of the river flow. Samples were collected every hour from the very beginning of the first rain 

for each flood. The influence of floods on Mediterranean coastal water CDOM was mainly 

detected on surface water samples. Major modifications of the quality and quantity of DOM 

were observed. DOM was then in particular characterized by higher HIX values. This index 

appeared to be a good indicator of the impact of the floods in seawater. Even if the effects 

were less significant for the deep water samples collected, we could observe some 

modifications of DOM due to the flood inputs. The terrestrial inputs did not reach however 

the most remote sites studied. 


This work showed that the fluorescence intensity ratios as well as the HIX and BIX indexes 

were particularly well adapted to the characterization and classification of CDOM in marine 

and coastal environments. The HIX index appeared to be a good indicator of the impact of the 

floods in seawater. Even though some CDOM modifications due to the flood events were 

observed for the deep water samples, they were really less impacted than surface waters. 



This work was supported by the French national program ECODYN. 

References 

1. B.J.H. Matthews, A.C. Jones, N.K. Theodorou, A.W. Tudhope, Mar. Chem., 55 (1996) 312- 317. 

2. P.G. Coble, Mar. Chem., 51 (1996) 325-346. 

3. R.F.Chen, J.L.Bada, Mar. Chem., 37 (1992) 191-221. 

4. M.M. De Souza-Sierra, O.F.X. Donard, M. Lamotte, Mar. Chem., 58 (1997) 51-58. 

5. C.E. Del Castillo, P.G. Coble, J.M. Morell, J.M. Lopez, J.E. Corredor, Mar. Chem., 66 (1999) 35– 

51. 

6. A. Zsolnay, E. Baigar, M. Jimenez, B; Steinweg, F. Saccomandi, Chemosphere, 38-1 (1999) 45- 

50. 

Vol. 3 Page - 93 -

Study of Colloidal Organic Matter Transformation Processes at Superficial 

Sediment Interfaces 

E. Parlanti a,b* , S Relexans a,b , D Amouroux c , R Bridou c , S Bouchet c , G Abril d , H Etcheber d 

a Université de Bordeaux, UMR 5255, ISM, Groupe LPTC, 351 Cours de la Libération 


France; c Université de Pau et des Pays de l’Adour, CNRS, UMR 5034, LCABIE, av P. 

Angot, Pau, F-64053 France; d Université de Bordeaux, CNRS, UMR 5805, EPOC, Avenue 

des Facultés, Talence, F-33405 France 

E-mail: e.parlanti@ism.u-bordeaux1.fr 


The maximum turbidity zone (MTZ) of an estuary is especially characterized by intense 

cycles of settling and resuspension of anoxic mud fluid. Moreover dissolved organic matter 

(DOM) accumulates in the MTZ where it has a longer residence time and is then submitted to 

flocculation and sedimentation processes that modify the size distribution of the 

macromolecules during the transit of organic material to the marine medium [1]. Due to 

intense diurnal, tidal and seasonal cycles as well as to high organic matter amounts, 

superficial sediments are submitted to a lot of redox oscillations and show a great reactivity. 

The role played by the colloidal fractions is of great interest to understand the variability of 

sediment reactivity during these oxic/anoxic cycles. The aim of this study was to simulate in 

vitro series of oxic/anoxic cycles in coastal and estuarine superficial sediments. A detailed 

study of DOM fluorescence behaviour during oscillating oxic/anoxic conditions is reported. 



The incubator used for this work (Fig.1) was developed by Commarieux and Abril [2]. 

Figure 1: Description of the incubator used to simulate oxic/anoxic oscillations in suspended sediment 

samples 

The forcing parameters studied in the experiments were mainly aerobic and anaerobic 

conditions. Gas allowed moving suspended sediment from oxic to anoxic conditions, and vice 

Vol. 3 Page - 94 -

versa. Continuous measures were possible in both gaseous and dissolved phases. Fine 

superficial sediments from the Arcachon Bay and from the Adour Estuary (South western 

France) were collected as marine and estuarine samples respectively. As DOM is a mixture of 

organic macromolecules with a broad range of molecular size and weight it was fractionated 

according to molecular size by using tangential-flow ultrafiltration using a molecular size cutoff 

membrane of 500 Da. Each isolated fraction (filtrate and retentate) was then characterized 

using EEM spectroscopy. 


This study showed significant modifications of DOM during oxic/anoxic oscillations. During 

anoxic/oxic transitions we observed a decrease of DOM fluorescence intensity and a relative 

increase of the proportion of components of molecular size >500Da. A global decrease in 

fluorescence intensity of filtrates was observed all along the experiments indicating a decrease 

of the relative proportion of small molecules (< 500 Da). A light increase of this fraction 

proportion was however observed at the anoxic/oxic transition. Weak variations of the 

fluorescence intensity ratios were observed: increase of Iγ/Iα and Iα'/Iα during the 

anoxic/oxic transition and in oxic phase. Similar trends were observed for the two sediments 

with an increase of DOM fluorescence intensity in oxic phases. The fluorescence indices HIX 

and BIX variations highlighted rapid modifications of fluorescent DOM in response to the 

redox oscillations. 


A great reactivity of sediment organic matter was observed during redox oscillations. The 

results show global changes of the intensity of fluorescent dissolved organic matter during the 

transition phases, as well as rapid modifications of its quality. Tangential ultrafiltration allows 

to observe differences in the size of molecules between the redox phases. The proportion of 

molecules greater than 500 Da notably increases during the transition to oxic phases. 



This work was supported by the French national program ECODYN 

References 

1. G. Abril, H. Etcheber, P. Le Hir, P. Bassoullet, B. Boutier, M. Frankignoulle, Limnology and 

Oceanography, 44 (1999) 1304-1315. 

2. M.V. Commarieux, G. Abril European Geoscience Union 1st meeting. Nice, April 25-30 Abstract 

book (2004) 

Vol. 3 Page - 95 -

Resolving Ahthropogenic and Natural Organic Matter Using “hypy” 

Xiaoyu Zhang a* , Colin. E. Snape b , Will Meredith b , Yongge Sun a 

a Earth Science Department, Zhejiang University, 38, Zheda Road, Hangzhou, China, 310027; 

b Department of Chemical and Environmental Engineering, University of Nottingham, 

University Park, Nottingham NG7 2RD, UK 

E-mail: xiaoyu.zhang@nottingham.ac.uk 


Completed in the Sui dynasty (581-618BC), the 1800 kilometre-long Beijing–Hangzhou 

Great Canal is under consideration for designation as world cultural heritage site. Due to the 

industrial output from the 18 major cities along the canal, representing one fifth of China's 

industrial output and heavy use for transportation, parts of canal have been subject to severe 

industrial pollution. The severe ecological deterioration endangers its potential for world 

cultural heritage. Systematic study of roles of highly heterogeneous of natural organic matter 

(NOM) in sediments play on the mixed combustion residues (black carbon) derived both 

naturally from biomass and anthropogenic sources, together with coal and oil-derived 

contaminants is key to better understand not only the biogeochemistry behavior of man-made 

pollution in natural environment, but also the effects on the pollutant transportation and 

transformation. 

To help resolve and apportion the various possible inputs of macromolecular material and 

black carbon (BC) in sediments collected from Great Canal, extraction and liquid 

chromatography are being used. Temperature staged hydropyrolysis (hypy), which has great 

power to convert biomass and labile OM completely into volatile products while providing 

extremely good preservation of hydrocarbon moieties in kerogens is also utilized. The residue 

of hypy comprising black carbon, together with extremely thermally mature kerogen are 

important fraction of the structure of sediment and play significant role to the absorption and 

transfer of man-made pollutants. GC-MS was used to help get information of products from 

sequential steps. 

2. Methods 


The total organic carbon (TOC) of the samples in sequential stages were measured with 

Elemental Analyser to get the organic carbon loss during sequential processing. The samples 

were extracted using Soxhlet apparatus for 72 hours with a mixture of dichloromethane and 

methanol (70:30). The resulting extracts were fractionated using open column liquid 

Vol. 3 Page - 96 -

chromatography (silica: alumina) into aliphatic, aromatic and polar fractions by sequential 

elution with 15 ml hexane, 15 ml hexane:DCM (60:40), 15 ml DCM: methanol (50:50). 

The saturated and aromatic hydrocarbon fractions were then analyzed by gas 

chromatography-mass spectrometry (GC-MS), with components were identified on the basis 

of their mass spectra, GC retention times and comparison with literature mass spectra. 

The extraction residue is then subjected to hypy to 550 o C in order to release the hydrocarbons 

bound within solvent insoluble organic matter which can then be characterized by GC-MS to 

resolve the NOM and anthropogenic inputs. The carbon residue remaining after hypy is 

defined as the BC content of the sediment. 

3. Data and discussion 


The Canal Sediment sample contains total carbon (TC) of 3.35%, total inorganic carbon (TIC) 

of 0.28%. The relatively high proportion of BC in the sediment may due to the extensive 

distribution of coal combustion residue. 

Table 1: Carbon content in sequential stages 

Samples TOC (%) Proportion of original TOC (%) 

Starting carbon 3.07 - 

EOM carbon 0.70 22.8 

Hypy released carbon 1.16 37.8 

Residual carbon (BC) 1.21 39.4 

The extractable organic matter (EOM) was then fractionated by liquid column chromatograph 

and analyzed with GC-MS. The Carbon Preference Index (CPI) value of aliphatic was 

calculated as the following formula (Zheng et al., 2007): 

( ∑ i i+ 2 L i+ 8) ( ∑ i− 1 i+ 1 L i+ 7) ( ∑ i i+ 2 L i+ 8) ( ∑ i+ 1 i+ 3 L i+ 

9) 

CPI = 1/2 ⎡ 

⎣ 

In which, i=25 

C + C + C / C + C + C + C + C + C / C + C + C ⎤ 

⎦ 

Usually n-alkanes derived from the cuticular waxes of higher plants have strong odd/even 

predominance and give CPI values >5. In contrast, n-alkanes from bacteria and algae give low 

CPI values, around 1 (Cranwell et al., 1987). The CPI for the Canal Sediment is 1.08, 

suggesting that the pollutants in the Canal Sediment are largely derived from crude oil. The 

distribution of n-alkanes in the the aliphatic hydrocarbon fraction extracted from the sediment 

is shown in Figure 1 and suggests that the oil input has undergone significant biodegradation, 

as evidenced by high Unresolved Complete Mixture (UCM) below the chromatogram 

Vol. 3 Page - 97 -


baseline and Pristane/C17 ratio of 2.74. The slight predominance of odd n-alkanes over even 

combined with the apparent elevated abundance of C25, C27 and C29 which usually derived 

from cuticular waxes are illustrated with terrestrial plant sources. (Powell, 1988; Murray and 

Boreham, 1992; Sarmiento and Rangel, 2004; Basant et al., 2005). 

Figure 1: Total ion chromatogram (TIC) of the aliphatic hydrocarbon fraction extracted from sediment 

from the Great Canal, China 

Figure 2: Total ion chromatogram (TIC) of the aromatic hydrocarbon fraction extracted from sediment 

from the Great Canal, China 

The polycyclic aromatic hydrocarbon (PAH) distribution shown in figure 2 display a 

dominance of “parent” PAHs such as phenanthrene and pyrene, with relatively low abundance 

of alkyl substituted PAH as methyl phenanthrene and methyl pyrene. Such a distribution is 

typical of biodegradation oil (Volkman, 1984) or high temperature coal tar (Sun et al., 2003). 

Vol. 3 Page - 98 -

Further work on the characterizing the hypy generated fraction, together with soils and 

sediments samples collected from eastern China as references will be carried out to get more 

information on the structure of NOM to the fate of man-made pollutants. 

4. Summary 


The Beijing-Hangzhou Great Canal sediment is found to be highly heterogeneous comprising 

various complex macromolecules. Organic matter in the Canal sediment were extracted 

sequentially according to their distinctive properties. The measurement for the organic matters 

suggested that the Canal sediment is characterized by the highly man-made pollution. The 

high BC content in the sample suggests combustion residues derived mainly from 

anthropogenic combustion residues. Significant biodegradation of the crude oil derived input 

is evidenced by the UCM distribution and hydrocarbon ratios. CPI and elevated C25, C27 and 

C29 n-alkanes suggest the input of plant derived organic matter. Such systematic study of the 

structural composition of the above organic matter continuum in the Canal sediments is 

significant to better understand not only the biogeochemistry behavior in natural environment 

but also the effects on the pollutant transportation and transformation and could give 

instruction to the treatment. 

References 

1. Basant et al., 2005 G.G. Basant, S.B. Rajendra, K.B. Ashok, K. Dinesh, L.P. Kusum, K.M. 

Adarsh, P.G. Jagdish, C.D. Gaur and J.T. Nizhat, Geochemical characterization and source 

investigation of oils discovered in Khoraghat–Nambar structures of the Assam–Arakan Basin, 

India, Organic Geochemistry 36 (2005), pp. 161–181. 

2. Murray and Boreham, 1992 A.P. Murray and C.J. Boreham, Organic Geochemistry in Petroleum 

Exploration, Australian Geological Survey Organization, Canberra (1992) 230 p. 

3. Powell, 1988 T.G. Powell, Pristane/phytane ratio as environmental indicator, Nature 333 (1988), 

p. 604. 

4. Sarmiento and Rangel, 2004 L.F. Sarmiento and A. Rangel, Petroleum systems of the Upper 

Magdalena Valley, Colombia, Marine and Petroleum Geology 21 (2004), pp. 373–391. 

5. Volkman, J.K., Alexander, R., Kagi, R.I., Rowland, J. & Sheppard, P.N., 1984. Biodegradation of 

aromatic hydrocarbons in crude oil from the Barrow Sub-basin of Western Australia. Organic 

Geochemistry, 6, 619-632. 

6. Zheng, Y., Zhou, W., Meyers, P.A., Xie, S., 2007. Lipid biomarkers in the Zoige-Hongyuan peat 

deposit: indicators of Holocene climate changes in West China. Organic Geochemistry 38, 1927- 

1940. 

Vol. 3 Page - 99 -

Organomineral Association Patterns of Humic Substances in Different 

Venezuelan Estuarine Mangroves 

Adriana Méndez a* , Zulimar Hernández b , Gonzalo Almendros b , 

Xosé Luis Otero a , Felipe Macías a , Williams Meléndez c 

a Dpto. Edafología y Química Agrícola, Universidad de Santiago de Compostela (USC) 15782, 

Santiago de Compostela (Spain); b Centro de Ciencias Medioambientales (CSIC), Serrano 115B, 

28006-Madrid (Spain); c Instituto de Ciencias de la Tierra (ICT), Universidad Central de Venezuela 

(UCV), Caracas 1010 A (Venezuela) 

E-mail: adriana.mendez.moreno@rai.usc.es 


Mangroves have been the subject of interest by several researchers concerned with humic 

substances. The physicochemical conditions of these hydromorphic soils (low redox potential, 

anaerobic conditions…) together with a large variety of high intensity seasonal environmental 

factors may have a large impact on the geochemical processes determining its dynamics. In 

general, these ecosystems store a high amount of organic matter (OM) as a consequence of 

inputs from fast-growing tropical vegetation and the constant contribution by riverine 

sediments. This represents a suitable scenario to study not only factors related to the 

performance of soil C sequestration, but also the nature, origin, dynamics, evolution and 

organomineral interactions of its OM. Assuming the criterion that the extent to which plant 

and microbial biomass are transformed into humic substances ought to be considered as 

objective measurement of the performance of the mangrove as an active C sink, this study 

focuses on assessing the quality of the soil OM in three estuarine mangroves in Venezuela, 

aiming to establish its potential contribution to terrestrial C balance. 



A total of eight mangrove soil samples were collected at different depths within the 

Venezuelan insular territory (Margarita Island, Nueva Esparta State) and coastal region 

(Falcón State) [1]. The sampling was carried out in three estuarine systems: i) plant 

communities of Rhizophora (soil profile labelled as P09) in the Laguna de La Restinga, ii) 

plant communities of Thalassia (P05) in the Laguna de Las Marites and iii) plant 

communities of Avicennia and Rhizophora (P10 and P11) in the Golfete de Cuare. 

Standard procedures were employed for the sequential isolation of the different humus 

fractions [2]. A previous physical separation of the partially decomposed organic particles 

was carried out by flotation in CHBr3-ethanol mixture (free organic matter, FOM (ρ< 1.8 

Vol. 3 Page - 100 -

g·mL -3 ). The isolation of the extractable humic fractions was carried out by treating the soil 

with 0.1M Na4P2O7 and 0.1M NaOH, five times each. The total humic extract was then 

precipitated by the addition of 6M HCl to separate the humic acids (HA) from the fulvic acids 

(FA). The alkali-insoluble soil residue contains the humin. The content of total organic carbon 

(TOC), HA and FA were determined by wet partial oxidation with K2Cr2O7. The HA was 

purified for its further chemical characterization. 

The optical densities of the HAs were measured at 465 (E4) and 665 (E6) nm. The visible 

spectra were acquired in a Hewlett-Packard model 8452A VIS-UV spectrophotometer. The E4 

is often taken as an index for progressive humification [3] and the E4/E6 ratio was used as 

index mainly of HA molecular size [4]. Infrared spectra were determined by a Shimadzu 

FTIR-8400 PC and KBr pellets with 2 mg sample. 

3. Results and discussion 


The content of TOC varies over a wide range, between 4 and 280 g · kg -1 , depending on soil 

profile depth and the type of vegetation. Upper horizons, under vegetation of Rhizophora 

(samples P11, P09 and to a lesser extent, P05) showed high TOC values, whereas deep 

horizons, under Avicennia vegetation (sample P10), showed comparatively lower TOC values. 

The mangroves with comparatively high OM concentration (soils which a priori could be 

considered as behaving as potential C sinks) were also associated to high amounts of 

particulate organic fractions (Fig. 1), i.e., the FOM, which is the case of mangrove plant 

communities of Rhizophora (P11 and P09), whereas soils under plant communities of 

Avicennia or Thalassia displayed a low content of FOM. The distribution of total C along to 

soil depth evidenced an abrupt decrease of the particulate organic fraction and a significant 

increase in the concentration of humin (strongly linked to mineral fraction) in all soil profiles. 

The HA/FA ratio also changed with depth, depending on the area and type of plant 

community. In mangroves developed from Avicennia (P10) the increase the concentration of 

HA-type substances suggested active soil OM accumulation processes attributed to anaerobic 

conditions in this zone, whereas in mangroves of Rhizophora (P11 and P09) the content of 

HAs decreased in comparison with the amount of FA-type substances. In the latter case 

approximately 20% of TOC consisted of colloidal fractions (HA+FA), with a low molecular 

weight and low HA/FA ratio (~ 0.4). 

Vol. 3 Page - 101 -


Figure 1: Visible and infrared spectra of the HAs, and distribution of the TOC in the different organic 

matter fractions in soil from Venezuelan mangroves. Bars height (and the numbers above) corresponds 

to total soil C in samples from different soil depths (in cm). P11 and P09: Rhizophora, P05: Thalassia, 

P10: Avicennia. HA: humic acid, FA: fulvic acid, FOM: free organic matter, Humin 

In general, the values of E4 in HAs from mangrove soil (Fig. 2) were relatively low (< 1 AU), 

which could indicate weakly condensed humic substances, derived mainly from aquatic 

biomass. The lack of a gradient of progressive aromatization with depth could be related with 

the continuous contribution of sediments and plant debris of different origin. In this sense, the 

E4 could be used as an indicator of OM quality, with independence of its value as source 

indicator (allochthonous or autochthonous biomass) and evolution into the mangrove soil. 

Some differences in the condensation processes of the HA are suggested by the E4/E6 ratio. 

The high values of E4/E6 are observed in Rhizophora profile (P09), indicate small molecular 

weight of HAs as regards the other samples, coinciding with the concentration of HA 

observed in the Fig 1. 

Vol. 3 Page - 102 -

Infrared spectra (not shown) suggested significant differences between HAs mainly as regards 

the intensity of the peak centred at 1720 cm -1 , indicating oxidative processes and/or terrestrial 

origin. To lesser extent, the spectra differ in intensity of alkyl structures (2920 and 1460 cm -1 ) 

inversely paralleling the E4 values and indicating less degree of humification in HAs with 

more marked aquatic signature also reflected in the intensity of the amide (1660, 1540 cm -1 

bands) revealed in the resolution-enhanced spectra. 


In Venezuelan mangroves, the OM show high amount of particulate organic fractions in the 

uppermost horizon, whereas the amount of humin (or C strongly linked to minerals) increase 

with the depth of the soil profile. Nevertheless, the soil OM quality (which could be 

considered as a surrogate indicator of the time of residence) is not correlated with depth or 

vegetal community. In fact, the E4 and the carboxyl content of the HAs seem to be suitable 

indicators to evaluate the local soil C quality. Mangroves with noticeable amount of soil C (in 

depth) and high OM quality could be considered as the most active C sinks, as may be the 

case of Golfete de Cuare. 



We acknowledge with thanks the technical staff of the laboratories of Soil Science of the USC 

and the UCV their help in the analysis and preparation of the samples. 

References 

1. M.B. Barreto, Acta Biológica Venezuelica, 4, (2004), 24. 

2. P. Duchaufour and Jacquin, Bulletin de l'Association Française pour l' Étude du Sol, 1, (1975) 29– 

36. 

3. S.J. Traina, J. Novak and N.E. Smeck, Journal of Environmental Quality, 19 (1990) 151–153. 

4. J.S. Chen and C.Y. Chiu, Geoderma, 117 (2003) 129–141. 

Vol. 3 Page - 103 -


Vol. 3 Page - 104 -


Natural Organic Matter and Humic Substances Interactions 

Vol. 3 Page - 105 -

Relationship Between Organic Carbon Forms and Selected Trace Elements 

in Grassland Soils 

Pospíšilová Lubica * , Škarpa Petr, Petrášová Veronika, Konečná Marie 

Mendel University of Agriculture and Forestry in Brno, Department of Agrochemistry, Soil 

Science, Microbiology and Plant Nutrition, Zemědelská 1, 613 00 Brno, Czech Republic 

E-mail: lposp@mendelu.cz 


Type of management is main factor influencing organic carbon content and its sequestration in soils. 

Every type of farming should fulfil some basic conditions: it should be optimal with respect to yields, 

considerate with respect to natural environment and should unified new and traditional forms of 

farming with respect to regions [1]. Labile carbon content is supposed to be one of important factor for 

anthropogenic effect evaluation. In our contribution content of stabile carbon forms (total organic 

carbon, humic substances carbon, humic acids and fulvic acids carbon) and labile carbon form (hot 

water extractable carbon) in different grassland soils will be discussed. Further we would like to 

follow correlation between different carbon forms and selected trace elements. 


Objects of our study were following grassland soils: Haplic Cambisol (locality Tři Kameny), Haplic 

Cambisol (locality Rapotin), Haplic Stagnosol (locality Sluneční)), and Haplic Stagnosol (locality 

Bílčice). Total organic carbon content was determined by oxidimetric titration method according to 

[2]. Humic substances carbon, humic acids carbon and fulvic acids carbon were detected by short 

fractionation method [3]. Labile organic carbon was determined by hot water extraction method [4]. 

Total and labile trace elements content was determined by flame or electrothermal atomic absorption 

spectrometry after extraction of the soil samples in the aqua regia (total content) and in the solution of 

0.01M CaCl2 (labile form). Description of the methods applied, and of the materials studied including 

references [5]. 



Average values of different carbon forms determined in spring 2008 and spring 2009 are showed in 

Table 1. Results showed that the highest total organic carbon was in Haplic Cambisol (Tři Kameny). 

The highest amount of HA was determined in Haplic Stagnosol (Bílčice). FA amount was higher to 

compare with HA content. Quality of humic substances was low. Ratio HA/FA was less than 1. Labile 

carbon content was the highest in Haplic Stagnosol (Sluneční)) and decreased in order: Haplic 

Stagnosol (Sluneční) > Haplic Cambisol (Tři Kameny) > Haplic Stagnosol (Bílčice) > Haplic 

Cambisol (Rapotín). Determined trace element content is given in Table 2. Correlations between labile 

carbon form and labile Zn was found. Labile Zn and Cd well correlated with humic substances carbon 

(stabile form). 

Vol. 3 Page - 106 -

Soil types Locality 

Table 1: Stabile and labile carbon forms in studied soils 

TOC C labile HS HA FA HA/FA 

% mg/kg mg/kg mg/kg mg/kg 

Spring 2008 

H. Stagnosol Sluneční 2.2 2, 200 7.0 2.5 6.5 0.4 

H. Cambisol Tři Kameny 2.9 1,720 9.0 3.1 5.9 0.5 

H. Cambisol Rapotín 1.34 240 6.0 2.4 3.6 0.6 

H. Stagnosol Bílčice 1.96 690 10.75 4.1 6.6 0.6 

Spring 2009 

H. Stagnosol Sluneční 1.8 2,300 9.0 3.0 6.0 0.5 

H. Cambisol Tři Kameny 2.2 1,750 13.0 4.0 9.0 0.45 

H. Cambisol Rapotín 1.30 270 5.5 2.1 3.4 0.6 

H. Stagnosol Bílčice 2 750 9.0 4.0 5.0 0.8 

Soil types Locality 

Table 2: Trace elements content in studied soils (spring 2008) 

Total 

content 

mg/kg 

Labile 

forms 

Zn Co Cu Pb Cd Mo Zn Co Cu Cd 

H. Stagnosol Sluneční 72.33 5.99 8.28 29.78 0.153 0.230 0.039 0.012 0.034 0.028 

H. Cambisol Přemyslov 81.18 5.61 7.92 22.04 0.133 0.186 0.535 0.051 0.028 0.049 

H. Cambisol Rapotín 59.69 13.87 20.26 7.55 0.212 0.366 0.127 0.081 0.040 0.024 

H. Stagnosol Bílčice 94.93 13.97 20.75 27.22 0.205 0.252 0.519 0.098 0.060 0.055 


We can conclude that grassland soils contained low amount of humic acids. Sorption and mobility of 

trace elements was mainly influenced by fulvic acids. Correlation between labile Zn and Cd and 

carbon content was detected. 



This work was supported by the project MŠMT No. 2B08039. 

References 

1. J. Bouma, Implementing soil quality knowledge in land-use planning. In: P. Schjonning, S. 

Elmholt, B.T. Christensen (eds.), CABI Publ., 2004, 283–295. 

2. D. W. Nelson and L. E. Sommers, Total carbon, organic carbon, and organic matter. Page A. L., 

Miller R. H., Keeny D. R. (Eds.). SSSA Publ., Wisconsin, 1982, 539–579. 

3. D. S. Orlov, Soil Chemistry, MGU, Moscow, 1985, p. 376. 

4. M. Körschens, A. Wiegel, E. Schulz, J. Plant Nutr. Soil Sci. 4/98 (1998), 409–424. 

5. F. J. Stevenson, Humus Chemistry – Genesis, Composition, Reactions. New York: Wiley & Sons, 

1982, p. 443. 

Vol. 3 Page - 107 -

Investigation of Humic Substances by Particle Size Distribution of Soils and 

by Determination of Zeta Potential 

Szilvia Joó a , Judit Tóth b , Gyöngyi Samu a , Rita Földényi a * 

a University of Pannonia, Department of Earth and Environmental Sciences, 8200 Veszprém, 

Egyetem u. 10., Hungary; b Institute of Materials and Environmental Chemistry, Chemical 

Research Center, Hungarian Academy of Sciences, Hungary 

E-mail: foldenyi@almos.uni-pannon.hu 



Soils are characterized by their particle size distribution basically. Particle size distribution 

(PSD) influences the specific surface of materials hereby the adsorption capacity of the 

adsorbent [1]. Adsorption processes have special role in the fate of pollutants in the 

environment. The composition of the aqueous media can influence the particle size 

distribution of soil and the transport processes of chemicals [2–3]. 

The humic substances (HS) which are very important components of the soils can adsorb 

pollutants. Their solubility depends on the pH of the soil solution while the type of the 

dissolved HS is determined by the type of the soil [4–5]. 

Surfactants are used everywhere and form one group of the main water and soil pollutants. 

Since their role is to dissolve the non- or hardly water soluble materials as well as to form 

stable emulsion, suspension etc. these compounds seem to be very harmful to the 

environment. 

The electrocinetic (zeta) potential let us conclude the stability of solution or suspension [6] 

which affects the fate of the suspension in natural waters. Stability of solution affects 

sedimentation, adsorption, aggregation, transportation of contaminants in soils. Better 

stability means further transport. 

The aim of the present work is to compare the PSD of the soils (determined in suspensions), 

the composition as well as the stability of the soil solution, and to find relation among them. 

Our investigations were focused on the role of anionic and cationic surfactants in the soil 

solution. The model compounds chosen for investigations are used most frequently as 

detergent in cosmetics (sodium dodecylsulphate: SDS), as forming agents in pesticide 

formulations (Supragil WP) or in industry (alkyltrimethylammonium bromide: Cetrimide). 

SDS and Supragil WP represent the anionic, while Cetrimide the cationic type of surfactants. 

Vol. 3 Page - 108 -


The particle size distribution of different soils being typical in Carpathian Basin was 

investigated by laser diffraction meaning a quite new method in PSD measurements [7]. In 

this case suspensions made from the appropriate soil (hernozem, brown forest and sandy soil) 

and alginite were analyzed by Master Sizer 2000 equipment. Every sample was studied in 

distilled water. Furthermore sandy soil suspensions were made not only in pure water but 

even in different aqueous solutions as follows: 

HA. 0.1 mol/dm 3 NaCl; HB. 0.1 mol/dm 3 phosphate buffer (pH=6.6); HC. 0.01 mol/dm 3 

phosphate buffer (pH=6.84); HD. 0.1 mol/dm 3 NaCl and 0.01 mol/dm 3 phosphate buffer 

(pH=6.6); HE. 0.1 g/dm 3 Supragil WP; HF. 0.1 g/dm 3 Supragil WP and 0.1 g/dm 3 NaCl; HG. 

0.1 g/dm 3 Cetrimide; HH. 0.1 g/dm 3 Cetrimide in 0.1 mol/dm 3 NaCl solution; HI. 0.01 

mol/dm 3 CaCl2; HJ. 0.1 g/dm 3 SDS; HK. 0.1 g/dm 3 SDS in 0.01 mol/dm 3 CaCl2 solution; HL. 

0.1 g/dm 3 SDS in 0.1 mol/dm 3 NaCl solution; HV. distilled water. 

The sandy soil suspensions were measured in different media after removing organic matter 

by H2O2, too. 

Critical micelle concentration (CMC) of surfactants was determined by measuring surface 

tension by Traube stalagmometer in 0.01 mol/dm 3 phosphate buffer (pH=7). 

Adsorption of surfactants was investigated on sandy soil by static equilibrium experiments. 

The analysis of equilibrated solutions was carried out by the so-called two-phased titration 

[8]. Solutions of fulvic acid (FA, Organit Ltd.), sodium humate (HANa, Roth+Co, Karlsruhe) 

with 35 mg C/dm 3 concentration and suspensions of sandy soil were examined at different pH 

values (5; 6; 7) by Zeta Sizer equipment (Malvern) using dynamic light scattering. Solution is 

instable if zeta potential (ζ) 30 mV. 



Different soils in pure water have different particle size distribution (Fig. 1). Surfactants 

strongly influenced the PSD (Fig. 2.), while the salt solutions have less effect. 

In Supragil WP (HE) and Cetrimide (HG) containing suspensions the size of soil particles 

decreased slightly while SDS (HJ) had similar but more significant effect. In the presence of 

surfactants the smaller, colloidal particles - like the humic substances - got into solution by 

means of dispergation named as solubilization effect [5,9] which can enhance the transport of 

contaminants. In salty media (HK, HL) SDS promoted formation of aggregates, which cannot 

Vol. 3 Page - 109 -


be observed at the other investigated tensides. The proportion of bigger particles in the 

suspension increased with higher SDS concentration. It can be explained by formation of 

aggregates which were arisen either from the HS molecules or from HS and SDS molecules. 

CaCl2 also assisted aggregate formation. 

Figure 1: Particle size distribution of different soil suspensions obtained in pure water 

Figure 2: Particle size distribution of sandy soil in surfactant containing suspensions 

After removing organic matter (mostly FA, [5]) of the sandy soil by H2O2, surfactants in 

themselves had no effect on smaller inorganic particles, however, in the presence of salts 

aggregates could be observed. 

According to the results of static equilibrium experiments surfactants adsorbed on sandy soil 

in more layers caused by hydrophobic interaction between the solute and the mostly 

hydrophobic surface (organic matter, quartz) of the adsorbent. 

The CMC of surfactants as well as the pH influenced the zeta potential and the stability of the 

suspension. If the tenside concentration was higher than the CMC the suspension proved to be 

stable in the case of anionic surfactants (Fig. 3.a), if their concentration was lower, the 

suspension was instable. 

Cetrimide as a cationic surfactant could result in positive zeta potential of the system (Fig. 

3.b). ζ=0 was observed at different tenside concentrations depending significantly on the type 

of the dissolved HS. 

Vol. 3 Page - 110 -

Zeta potential (mV) 

0 

-10 

-20 

-30 

-40 

-50 

-60 

-70 

CMC (Supragil WP at pH=7): 646 mg/l 

-80 

10 100 1000 10000 


Supragil WP concentration (mg/l) 

Fig 3.a 

sandy soil, pH=7 

FA, 35 mg C/l, pH=7 

HANa, 35 mg C/l, pH=7 

boundary of stability 


CMC (Cetrimide at pH=7): 1820 mg/l 

Zeta potential (mV) 

40 

30 

20 

10 

0 

-10 0 500 1000 1500 2000 2500 3000 

-20 

-30 

-40 

-50 

Cetrimide concentration (mg/l) 

Fig 3.b 

sandy soil, pH=7 

FA, 35 mg C/l, pH=7 

HANa, 35 mg C/l, pH=7 

boundary of stability 

Figure 3: Investigation of zeta potencial in different surfactant containing solutions 

The investigated surfactants influenced the particle size distribution. Especially significant 

was the solubilization effect of SDS used widely as a detergent. The removal of organic 

matter content of the sandy soil did not resulted in similar process proving the special 

interaction between the HS and surfactant molecules. 

Adsorption of surfactants on sandy soil was mostly governed by hydrophobic interaction. 

If the tenside concentration was higher than its CMC, the suspension became stable with 

Supragil WP and SDS. The cationic surfactant Cetrimide behaved as a counter ion of HS 

polyanions and was able to form even stable colloid system with positive zeta potential while 

the stability caused by anionic tensides (SDS and Supragil WP) could be observed due to the 

repulsive power of the negative charges. 

According to these results the surfactants can influence material transport in the soil, 

including hardly dissolving contaminants that may be adsorbed on soil particles, too. 

References 

1. D. Baenninger, P. Lehmann, H. Flühler, Eur. J. Soil. Sci. 57 (2006) 906. 

2. R. Haque, V. H. Freed (Eds.), Environmental Dynamics of Pesticides. Plenum Press, New York 

and London, (1975) 115. 

3. M. S. Wilson (Ed.), Advances in Soil Organic Matter Research: The Impact on Agriculture and 

the Environment. Redwood Press Ltd., Wiltshire (1991) 121. 

4. E. Illés, E. Tombácz, Colloids Surf., A: Physicochem. Eng. Asp., 230 (2004) 99. 

5. T. Ertli, A. Marton, R. Földényi, Chemosphere, 57 (2004) 771. 

6. Malvern Instruments: Zetasizer Nano Series User Manual, MANO 317, 3.1, 2007. 

7. G. Eshel, G. J. Levy, U. Mingelgrin, M. J. Singer, Soil Sci. Soc. Am. J. 68 (2004) 736. 

8. Á. Patzkó, Laboratory Practices in Colloid Chemistry (in Hungarian), JATEPress, Szeged (1996) 

54. 

9. J .P.Gao, J. Maguhn, P. Spitzauer, A. Kettrup, Water Res. 32 (1998) 2089. 

Vol. 3 Page - 111 -

Interactions of Organic Compound with NOM Need Water: Strong Water- 

Induced Enhancement of Carbamazepine Sorption on Peat 

Mikhail Borisover a* , Maggie Sela a,b , Benny Chefetz a 

a Institute of Soil, Water and Environmental Sciences, Agricultural Research Organization, 

The Volcani Center, Bet Dagan, Israel; b The Department of Soil and Water Sciences, The 

Hebrew University of Jerusalem, Rehovot, Israel. 

E-mail: vwmichel@volcani.agri.gov.il 


Natural organic matter (NOM) controls distributions of multiple organic compounds between 

different environmental compartments. The role of NOM-associated water in sorbate-NOM 

interactions is not well understood, in contrast to solute-bulk water interactions. Recently, a 

concept was suggested to explain the role of NOM-bound water in sorption of organic 

compounds which included consideration of cooperative water-induced disruption of intra- 

NOM interactions upon a penetration of organic sorbate [1, 2]. Yet, there is no clarity how a 

type a NOM hydration effect (i.e. enhancement or suppression of sorbate interactions) and its 

magnitude are related to the structure of organic sorbates. In this research, we examined the 

effect of a hydration of a model NOM sorbent, Pahokee peat, on interactions of a probe 

organic compound, carbamazepine (CBZ; 5H-dibenzo[b,f]azepine-5-carboxamide). The CBZ 

characterized by relatively large molar volume (186.5 cm 3 /mol) was compared, in terms of 

interactions with NOM, with phenanthrene (PHEN) having similar molar volume (178.2 

cm 3 /mol), and, in terms of NOM hydration effect, with smaller specifically interacting 

sorbate, phenol (87.8 cm 3 /mol). This comparison provided an insight in a relation between an 

involvement of water in sorbate-NOM interactions and a size of organic sorbate. 


Pahokee peat (supplied by IHSS) was freeze-dried and used as a model (dehydrated) NOM 

sorbent. Sorption of CBZ by peat was examined in batch experiments from water and, at 

different extents of peat hydration, from inert solvent, n-hexadecane (HD), according to the 

protocols similar to that described in Refs. [2, 3]. 



Sorption of CBZ from water on fully hydrated NOM is stronger than its sorption on 

dehydrated NOM from HD (Fig. 1A). To eliminate the differences in CBZ-solvent 

interactions, solution concentrations were normalized by the CBZ solubility in a specific 

Vol. 3 Page - 112 -

solvent. Thus, interactions of CBZ with NOM are enhanced at the presence of NOM-bound 

water by factor of 20-40 (Fig. 1B). This profound effect of NOM hydration on CBZ-NOM 

interactions is associated with specific CBZ-NOM interactions (elucidated by comparing the 

distribution coefficients of CBZ and PHEN between fully hydrated NOM sorbent and the 

inert reference state, [3]). Water-induced CBZ sorption enhancement appears also in HD 

environment when the partial NOM hydration is increased. The CBZ sorption enhancement 

starts at significantly higher water activities as compared with earlier observations of NOM 

hydration effect on phenol sorption [2]. This enhancement was modeled, using Link Solvation 

Model [1, 2], to provide an estimate for a number of water molecules involved in a 

penetration of a large-size organic sorbate into the NOM interior. 

Sorbed concentration, mg/kg 


1000 

100 

A. 

0.1 1 10 

Solution concentration, mg/L 

1000 

100 

1E-3 0.01 0.1 1 

Sorption from water Sorption from n-C 16 H 34 

B. 

X 20-40 times 

Solution concentration/solubility (~activity) 

Figure 1: Sorption isotherms of carbamazepine on differently solvated model NOM 

A strong water-induced enhancement of sorbate-NOM interactions is associated with greater 

hydration of NOM moieties upon penetration of CBZ molecules. 



This research was supported by Research Grant No. IS-3322-06 from BARD, The United 

States-Israeli Binational Agricultural Research and Development Fund. Help from Nadezhda 

Bukhanovsky (The Volcani Center, Israel) is greatly appreciated. 

References 

1. M. Borisover and E.R. Graber, Langmuir, 18 (2002) 4775. 

2. E.R. Graber, L. Tsechansky and M. Borisover, Environ. Sci. Technol. 41 (2007) 547. 

3. M. Borisover and E.R. Graber, Environ. Sci. Technol., 37 (2003) 5657. 

Vol. 3 Page - 113 -

Adsorption of Metal Ions on Humic Acid Derived from Turkish Lignite 

Bekir Zühtü Uysal a* , Duygu Öztan a , Ufuk Gündüz Zafer a , 

Özkan Murat Doğan a , Selahaddin Anaç b , Mustafa Özdingiş b , Zeki Olgun b 

a Chemical Engineering Department, Faculty of Engineering and Clean Energy Research and 

Application Center (CERAC) , Gazi University, Maltepe, 06570 Ankara, Turkey; b Turkish 

Coal Enterprises, Hipodrom Cad., No:12, 06330 Ankara, Turkey 

E-mail: bzuysal@gazi.edu.tr 

Abstract 

Adsorption of some metal ions (Pb +2 , Cu +2 , Zn +2 , Cr +6 , Ni +2 ) on humic acid produced in a 

pilot plant from Turkish Ilgın lignite was investigated in this work. The effects of adsorption 

time, solution pH, metal concentration and temperature on humic acid adsorption capacity 

were investigated. The humic acid adsorption capacity was found to follow an order as Pb +2 > 

Cu +2 > Zn +2 > Cr +6 > Ni +2 . 


Environmental contamination with heavy metals represents a potential threat to humans, 

animals and plants. Many of them are soluble in water, therefore become more available for 

living systems and accumulate in the environment. Removal of heavy metals from waste 

streams employs various technologies, which are often expensive. Use of inexpensive natural 

sorbents such as zeolites, fly ash, coal has been considered as a promising alternative for this 

purpose [1]. A large number of organic humic substances are also increasingly being applied 

worldwide especially in agricultural applications. Turkish Coal Enterprises (TKI) of Turkey 

has been producing humic acid and other humic containing substances in a pilot plant since 

2008. The potential of using the humic acid produced by TKI for removal of heavy metal ions 

from liquid waste streams was aimed to be assessed in this research. 



Humic acid, produced from Ilgın lignites, was provided from the pilot plant of Turkish Coal 

Enterprises (TKİ). The kits used for the analyses of Pb +2 , Cu +2 , Zn +2 , Cr +6 , Ni +2 were 

purchased from Hach-Lange. A Hach DR/4000 spectrophotometer and quartz cuvettes were 

used for all absorbance measurements. Calibration curves for all the metal ions were first 

developed employing Lambert-Beer Law and used in the analyses later. 

Vol. 3 Page - 114 -


Optimum temperature and pH intervals, adsorption time and maximum adsorption capacities 

(g metal / kg Humic Acid) were determined for each metal ion as shown in Table 1 and Fig 1. 

Table 1: Optimum operating conditions and humic acid adsorption capacity 

Metal Ion Temperaure 

( o pH Adsorption time to Adsorption Capacity 

C) 

reach equilibrium, min (g metal / kg Humic Acid) 

Nickel 20 6 15 12.64 

Chrome 22 2.8 240 37.93 

Zinc 20 4.5 120 97.50 

Copper 15 3, 4, 6 15 154.33 

Lead 15 3 5 196.90 

Adsorption capacity 

(g metal/kg HA) 

200 

150 

100 

50 

0 

Amount of HA (kg) 

Figure 1: Humic acid adsorption capacities for different heavy metals 

Nickel 

Chrome 

Zinc 

Copper 

It can be seen in Figure 1, the maximum adsorption capacity of humic acid was observed for 

lead. For the others an order in the sequence of Pb +2 > Cu +2 > Zn +2 > Cr +6 > Ni +2 was obtained. 

A similar order (Pb > Fe > Cu > Zn > Ni ) and similar magnitudes of adsorption capacities 

were reported by Arctech Inc for their Humasorb-cs product [2]. 



In this work, the adsorption of heavy metal cations, Pb +2 , Cu +2 , Zn +2 , Cr +6 and Ni +2 from 

aqueous solutions on humic acid derived from Turkish Ilgın lignite was studied. The results 

show that humic acid adsorption capacity followed the order of Pb +2 > Cu +2 > Zn +2 >, Cr +6 > 

Ni +2 . Optimum pH values were determined to be different for each metal as reported earlier 

[3] and found to be between approximately 3 and 6. 

References 

1. M. Havelcova, J. Mizera, Sorption of metal ions on lignite and the derived humic substances, 

Journal of Hazardous Materials, 161 (2009) 559. 

2. www.arctech.com visited in December 2009. 

3. P. A. Brown, S. A. Gill, Metal Removal from wastewater using peat,Wat. Res., 34 

a. (2000) 3907. 

Vol. 3 Page - 115 - 

Lead

Characteristics of Humic Acids Isolated from Heavy Metals Contaminated 

Soils at the Copper-Smelter “Legnica” (S-W Poland) 

Alina Maciejewska, Jolanta Kwiatkowska-Malina * 

Department of Spatial Planning and Environmental Sciences, Warsaw University of 

Technology, 1 Politechniki Sq., 00-661Warsaw, Poland 

E-mail: J.Kwiatkowska@gik.pw.edu.pl 


The presence of organic matter in soil is important not only with respect to plant nutrition but 

to environmental pollution, as well. Heavy metals, such as: Cu, Pb, Zn and Cd can accumulate 

in top soils. Humic substances (HS) and humic acids (HAs) of soil organic matter are 

important in trapping and subsequent transport of heavy metals in the environment, due to the 

presence of functional groups in their structures. The S-W region of Poland, particularly the 

Legnicko-Głogowski district, is heavily contaminated with heavy metals [1], with the 

maximum contents of Cu and Pb measured in the vicinity of the Copper-Smelter “Legnica”, 

that amount to several grams per kilogram of soil. Despite considerable improvements of the 

air quality in the last 2 decades, soil contamination with heavy metals in this region have not 

considerably decreased , and it is likely to pose a long-term hazard to the food chain, ground 

and surface waters and soil microorganisms [2]. Phytoextraction is commonly used to remove 

heavy metals from soil by concentrating them in the harvestable parts of plants [4]. However, 

in some cases it may be more efficient to limit the biological activity of heavy metals in soils 

by transferring heavy metals into forms not available for plants. Organic matter present in soil 

can form organometallic compounds, the so-called chelates, with heavy metals lowering their 

accessibility for plants and soil micro-organisms, as well as their potential leaching to the 

adjacent ground and surface waters. 

The aim of this study was to compare the influence of soil contamination level on the quantity 

and selected qualitative parameters of HAs extracted from soils from the area of the Copper– 

Smelter “Legnica” heavily contaminated with heavy metals (mostly Cu and Pb). 

2. Material and Methods 


The research was made on the soil developed from silt loam on clay (WRB – Deluvial 

Brown) from zone I and II (0.1 km and 1.0 km from the main emitter, respectively) 

contaminated with heavy metals: zone I - 4985 (Cu), 1236 (Pb), 294.6 (Zn), 2.82 (Cd) [mg 

Vol. 3 Page - 116 -

kg −1 ] and zone II - 1008 (Cu), 413.0 (Pb), 194.5 (Zn), 1.51 (Cd) [mg kg −1 ]. The soil from zone 

I showed neutral, while from zone II - alkaline reaction (Table 1). 

Soil samples were taken from the 0-30 cm horizon, air-dried, mixed and sieved (φ = 1-mm) 

prior to analyses. The total organic carbon (TOC) was determined by the TOC analyzer 

(Shimadzu 5000), the total nitrogen (Nt) - using the standard Kjeldahl method, pH - 

potentiometrically in H2O and 1 M KCl, and the hydrolytic acidity (Hh) - using the Kappen's 

method. 

Humic acids were extracted from soil samples according to the IHSS standard method [6]. 

The values of absorbance at wavelength: 280 (A280), 400 (A400), 465 (A465), 665 nm (A665) 

were determined for separated HAs. VIS spectra were performed for 0.02% HAs solutions in 

0.1 M NaOH, and UV-spectra were determined after fivefold dilution by the Lambda 20 

Perkin-Elmer Analyzer. Based on the determined absorbance values at wavelengths of: 280 

(A280), 465 (A465), 600 (A600), and 665 nm (A665), the following absorbance ratios were 

calculated: A2/4 - at wavelengths of 280 and 465 nm, A2/6 - at wavelengths of 280 and 665 nm, 

A4/6 - at wavelengths of 465 and 665 nm. Simultaneously, the values of the coefficient Δlog K 

= log A400 - log A600 were calculated for HAs. The FT-IR spectra were obtained in the 4000 to 

400 cm -1 wavelength range by the Nicolet 5PC FTIR spectrophotometer on KBr pellets 

obtained by pressing, under reduced pressure, uniformly prepared mixtures of 1 mg sample 

and 400 mg KBr, spectrometry grade, with precaution taken to avoid moisture uptake. The 

elemental composition of HAs was determined using the CHN 2400 Perkin-Elmer Analyser. 

The oxygen content was calculated from the difference [100% - (%C+%H+%N)], in relation 

to the ashless sample weight. Based on the HAs elemental composition, the values of atomic 

ratios (H:C, O:C, N:C, O:H) were calculated, and the internal oxidation degree was 

determined referring to the formula [3]: ω = (2O+3N-H):C (where: C, H, O, N - represent 

contents of carbon, hydrogen, oxygen and nitrogen in atomic percentage, respectively). 

3. Results and Discussions 


The properties of examined soils (Table 1), particularly: reaction, hydrolytic acidity and the 

organic carbon content, may cause a decrease of solubility (mobility) of heavy metals in top 

soil, and consequently of their phytoavailability. The contamination levels of soil had no 

influence on contents of organic carbon and nitrogen. The ratios of organic carbon to nitrogen 

for both zones were similar and characteristic for polish arable soils. 

Vol. 3 Page - 117 -


Table 1: Physicochemical and chemical properties of soils at the Copper-Smelter “Legnica” 

pH Hh TOC Nt TOC:Nt 

Sample 

H2O 1 M KCl cmol(+) kg −1 of soil g . kg -1 of soil 

zone I 7.30 7.05 0.44 0.94 0.091 10.33 

zone II 7.55 7.30 0.39 0.88 0.091 9.67 

The absorbance values at wavelengths of 280, 465 and 665 nm of the HAs extracts are 

presented in Table 2. Extracts from the soil samples (zone I) had higher absorbance values as 

compared to zone II indicating higher carbon contents. However, it is not clearly reflected in 

the elemental composition of HAs (Table 3). Although the TOC contents in soil samples from 

zone I were higher compared to zone II, however, the absorbance values do not reflect it. The 

values of A2/4 in both cases were similar, but the A4/6 and A2/6 absorbance ratios of HAs 

extracts from zone II were lower than for zone I. HAs extracted from soil from zone II 

compared with these from zone I had higher oxygen content, which indicates evolution 

towards oxidation, was supported by an increase in O:C ratio. Humic acids extracted from 

soils from both zones had similar carbon contents, as well as H:C and N:C ratios. HAs from 

zone II were richer in nitrogen and oxygen, and were characterised by lower value of the ω 

parameter, as compared to these from zone I. The data show that HAs in both zones have the 

elemental composition similar to these from arable soils [5]. 

Table 2: Spectral properties of soil alkali extracts - the Copper-Smelter “Legnica” 

Sample A280 A465 A600 A665 A2/4 A2/6 A4/6 ΔlogK 

zone I 5.3 0.94 0.304 0.161 5.62 32.9 5.86 0.763 

zone II 4.3 0.76 0.261 0.142 5.66 30.0 5.34 0.728 

Table 3: The eelemental composition of humic acids (in atomic percentage) extracted from soils at the 

Copper Smelter “Legnica” 

Sample C H N O H:C O:C Ο:Η N:C ω 

zone I 34.54 43.52 2.81 18.14 1.26 0.525 0.417 0.081 0.032 

zone II 34.55 43.98 2.84 18.24 1.27 0.528 0.415 0.082 0.029 

The FT-IR spectra of HAs from soils from both zones feature the following common bands 

(Fig. 1): 3380 cm -1 (O–H stretching of various functional groups); 2925 and 2850 cm -1 

(aliphatic C–H group stretching); 1720 cm -1 (C=O stretching of carboxyl groups); 1650 cm -1 

(aromatic C=C stretching and COO - symmetric stretching); 1508 cm -1 (amide II band); 1424 

Vol. 3 Page - 118 -

cm -1 (amide III band); 1380 cm -1 (O–H deformation and C-O stretching of phenolic OH 

and/or antisymetric stretching of COO - groups); a broad band at 1225 cm -1 (C-O stretching 

and O-H deformation of carboxyl and C-O stretching of aryl ethers and phenols); 1040 cm -1 

(C-O stretching of polysaccharides). The major differences are in the relative intensity of 

peaks assigned to carboxyl groups, which are higher in the case of HAs from zone II than 

from I. 

%T 

4000 

3500 

3000 

2500 

2000 

Wavenumbers 

1500 

1000 

I zone 

II zone 

Figure 1: FT-IR-spectra of HAs extracted from soils at the Copper–Smelter “Legnica” 



The lower absorbance ratios of HAs from the soil samples from zone II as compared with 

zone I indicate higher carbon contents. However, it is not clearly reflected in the elemental 

composition of HAs. Higher oxygen content in HAs from zone II indicates evolution towards 

oxidation. Examination of FT-IR spectra showed that metal ions react mainly with carboxylic 

groups of the HAs. The contamination level of heavy metals (mostly Cu and Pb) has not the 

influence on the quantity and selected qualitative parameters of HAs extracted from soils. 

References. 

1. A. Karczewska, Zesz. Naukowe AR Wrocław, 432 (2002). 

2. B.J. Alloway, Heavy Metals in Soils (2nd ed.), Blackie Academic and Professional, 1995. 

3. J.A. Zdanov, Biochimija, 30, 6, (1965) 1257. 

4. M.W.H. Evangelou, M. Ebel and A. Schaeffer, Chemosphere, 63 (2006), 996 

5. N. Szombathova, B. Debska, M. Lacko-Bartosova, A. Zaujec, S.S. Gonet, Acta Sci.Pol., 

Agricultura 3(2), 37 (2004). 

6. S.S. Gonet, B. Debska, Environ. Int., 24 (1998) 603. 

Vol. 3 Page - 119 - 

500 

cm -1

The Interaction of Cu 2+ with Humic Acids of Different Soils 

Motuzova G.V., Dergam H., Stepanov A.A. 

Moscow State University, Faculty of Soil Science, 119991, Moscow, Leninskie Gory, 

Moscow University, Faculty of Soil Science 

E-mail: motuzova@mail.ru 


The growth of productive human activity leads to the increase of the environmental pollution 

by heavy metals. Heavy metals are accumulated in the soils, interacting with soil components. 

The most important are the interactions of heavy metals with humic substances. Recent 

studies demonstrated the ability of humic substances to bind the ions of heavy metals strongly 

and loosely, namely in the exchangeable state, in the form of outer-and inner-sphere 

complexes. But insufficient attention is paid to the changes that occur with humic acids (HA) 

themselves under the influence of heavy metals. These processes deserve the special attention, 

because of their danger for ecosystem. 

The objects of the investigation were the samples of upper humus horizons of soils, that have 

been developed in different conditions: 1) soddy-podzolic (S-P) heavy loamy soil, developed 

on the moraine deposits (fallow, Leningradskij region); 2) chernozem (CH) heavy loamy on 

the loess deposits (Voronezh region), 3) meadow serozem (MS) light loamy on the loess 

deposits (Syria,Mesopotamia). 


The HA samples were received throw extractions by 0, 1 N. NaOH solution. There were 

determined the elemental composition of HA, their molecular mass distribution (by gel 

filtration); IR spectra, hydrophilic-hydrophobic properties, 1 H NMR spectra, acid-base 

properties (by the reverse titration). There were determined some parameters of complexes 

formation of the HA with Cu 2+ ions: a) the content of Cu in fractions 10 kDa; 

b) the distribution of Cu between fractions HA + Cu with different properties (molecular 

masses, amphiphilic properties; c) the properties of the HA in the fractions- products of their 

interaction with Cu 2+ (C content in the fractions, molecular masses distribution, amphiphilic 

properties, 1 H NMR spectra). 


Vol. 3 Page - 120 -



Humic acids of the investigated soils are characterized as by the general, so by some specific 

properties. They are united by the elemental composition, dominance of aliphatic structures, 

consisting of components, that exhibit the hydrophobic properties, by the predominance of 

molecules with average masses (18000-25000 a.m.u.), the uniform set of the structures of 

aliphatic and aromatic parts of the HA. The greatest differences are noticed between the HA 

of chernozem and HA of serozem. Humic acids of chernozem are richer with carbon, they 

content less nitrogen, molecular-mass distribution is more homogeneous, their aliphatic part is 

less developed (the number of methyl groups is less, the number of carboxyl groups is more 

than in HA of serozem). Humic acids of soddy-podzolic soils occupy an intermediate position 

between the HA of chernozem and serozem. 

At the next stage the complexes of HA-Cu were obtained by the interaction of HA with the 

solution of Cu (NO3) 2 at pH 7,0. The properties of HA-Cu complexes of three soils have also 

both the general and specific features. There were revealed two types of ligands in the 

structure of HA, that are able for complexation with Cu 2+ . The complexation capacity of the 

centers of the first type of HA ligands of three soils is low and close in magnitude (24 -26 

mmol/100g HA), but the stability of complexes, formed by them is relatively high (log K1 

4,8-5,9) and changes in order CH> S-P> MS. The complexation capacity of the second type 

of HA ligands of three soils is 2,3-3 times higher (75-59 mmol/100g HA ) and changes in 

order: CH> S-P> MS. But the sustainability of these complexes is not large (lg K2 3,2-2.8). 

The difference of complexation ability of three soils is in accordance with the content of the 

functional groups in HA and their degree of dissociation (HA of CH> HA of S-P> HA of 

SER).By the adding of copper salts to the solution of HA the most part of Cu2+ in the soddypodzolic 

soil (79%) and chernozem (59%) is bound in complexes with HA. In the serozem the 

free forms of Cu 2+ , their complexes with inorganic ligands and with organic ligands with low 

molecular masses are dominated (63%). The highest specific activity in the complexes 

formation with Cu 2+ show the HA species with low molecular masses (especially in HA of 

serozem), the least active show the species of HA with high molecular masses. But due to the 

domination of the species with average molecular masses in the composition of HA of all 

three soils just namely these species are predominantly involved in the complexation with 

Cu 2+ (for soddy-podzolic soil and chernozem 81-88%, for serozem, -65% from HA total). 

Vol. 3 Page - 121 -

The highest specific activity in the complexes with Cu 2+ show the species of HA from three 

soils that exhibit the hydrophylic properties, especially in HA of chernozem. The share of 

these complexes constitutes 65 % and 55% in chernozem and soddy-podzolic soils. HA of 

serozem occupy the intermediate positions; the portion of the complexes consists of 72%. 

As a result some properties of HA have changed after their interaction with Cu 2+ . It is 

attested by the changes of 1 HNR spectra, molecular mass distribution of HA, their 

amphiphilicity, the change of metal concentration in the dialysate. It is assumed that the 

restructuring of the initial metal-humus complexes under the influence of the ions Cu 2+ takes 

place, accompanied by the separation of the long aliphatic chains from the aromatic rings of 

the HA, the appearance in their composition of the unsubstituted structures, the changing in 

the molecular-mass distribution of HA and increasing of their hydrophobicity. 

4. Conclusion 


Interaction of humic acids with Cu 2+ leads to changes of the state as the metal, so the 

properties of humic acids. The last can be classified as the sign of humus degradation. 

Vol. 3 Page - 122 -

Influences of Humic Acids on the Pattern of Oxidation Products of 

Tetrabromobisphenol a Derived from a Catalytic System using Iron(III)tetrakis(p-sulfophenyl)porphyrin 

and KHSO5 

Masami Fukushima * , Yosuke Ishida, Satoko Shigematsu 

Laboratory of Chemical Resources, Division of Sustainable Resources Engineering, Graduate 

School of Engineering, Hokkaido University, Sapporo 060-8628, Japan 

E-mail: m-fukush@eng.hokudai.ac.jp 


Tetrabromobisphenol A (TBBPA) is the most widely used brominated flame retardant. The 

leaching of the TBBPA from landfills is facilitated in the presence of humic acids (HAs), 

which can enhance the water solubility of hydrophobic organic pollutants [1]. TBBPA is 

known to be able to act as an endocrine disruptor [2]. Thus, studies on the transformation of 

TBBPA are important to elucidate its fate in environments. While oxidation may be major 

degradation processes in environments, there have been a few reports on the oxidation of 

TBBPA [3, 4]. In addition, influences of HAs on the oxidation of TBBPA have not been 

investigated. On the other hand, it had been reported that an iron(III)-tetrakis(psulfonatophenyl)porphyrin 

(FeTPPS) can oxidize chlorophenols [5]. This suggests a 

possibility for applying the FeTPPS/KHSO5 catalytic system to the oxidation of TBBPA. In 

the present study, we studied the influences of HAs on the pattern of byproduct of TBBPA, 

derived from the catalytic oxidation in the FeTPPS/KHSO5 system. 

2. Materials and Methods. 


Humic acids. An HA was extracted from a Shinshinotsu peat soil (Hokkaido, Japan) and 

purified according to a method, recommended by the International Humic Substances Society 

[6]. To alter the amounts of phenolic moieties, the HA was treated with hydroquinone (HQ- 

HA) according to the method, as reported by Perminova and coworkers [7]. The results of 

elemental and acidic functional group analyses for the HAs are summarized in Table 1. 

Table 1: The results of elemental and acidic functional group analyses for HAs 

Elemental Composition (%) 

Functional Groups 

(meq g -1 Samples 

C) 

%C %H %N %O %S %ash COOH Phenol-OH 

HA 54.5 5.35 2.17 35.1 0.66 2.22 3.2 ± 0.1 7.3 ± 0.3 

HQ-HA 59.2 5.01 2.24 30.7 0.87 2.01 4.1 ± 0.4 11 ± 0.1 

Vol. 3 Page - 123 -

Oxidation tests for TBBPA. A 25 mL aliquot of 0.02 M NaH2PO4/Na2HPO4/citrate buffer at 

pH 4 – 8, which contained 0 or 50 mg L -1 of HAs, was placed in a 100-mL Erlenmeyer flask. 

Stock solutions of TBBPA (0.01 M in acetonitrile) and FeTPPS (0.2 mM) were added to set 

the final concentrations at 50 μM and 5 μM, respectively. After mixing vigorously, aqueous 

0.01 M KHSO5 was added to set the final concentration at 125 μM, and the flask was then 

shaken in a thermostatic shaking water bath at 25±0.1. After a 1, 5, 15 or 30 min reaction 

period, 1 mL of 1 M ascorbic acid aqueous was added, and pH of the solution was adjusted to 

11 – 11.5 by the addition of aqueous K2CO3. Subsequently, 5 mL of acetic anhydride was 

added dropwise to the solution, and 0.5 mL of a 1 mM anthracene hexane solution was added 

as an internal standard for the GC/MS analyses. This mixture was doubly extracted with 15 

mL of n-hexane, and the extract dehydrated with Na2SO4 anhydride. After filtration, the 

extract was evaporated under a stream of dry N2, and the residue was then dissolved in 0.25 

mL of n-hexane. A 1 μL aliquot of the extract was introduced into a GC-17A/QP5050 GC/MS 

system (Shimadzu). A Quadrex methyl silicon capillary column (0.25 mm i.d. × 25 m) was 

employed in the separation. The temperature ramp was as follows: 65 for 1.5 min, 65 – 120 at 

35 min -1 , 120 – 300 at 4 min -1 and a 300 hold for 10 min. Concentrations of TBBPA before 

and after the oxidation were analyzed by an HPLC, as described in a previous report [5]. 



Influence of solution pH of TBBPA degradation. Figure 1 shows the influence of solution pH 

on percentages of the degradation of TBBPA. The percent of TBBPA degradation increased 

with an increase in solution pH. In particular, more than 90% of TBBPA was degraded at pH 

8 in the absence and presence of HAs. Because the pKa value of TBBPA is reported to be 7.4 

[4], one of reasons for this trend can be attributed to the fact that water solubility of TBBPA is 

enhanced in weak-alkaline condition. The range of pH for leachates from landfills is reported 

to be 7 – 12 [1]. Thus, oxidation products from TBBPA were investigated at pH 8. Although 

Br - and BrO3 - were analyzed by ion chromatography, these were not detected in all reaction 

mixtures. Thus, debromination is not considered in the catalytic oxidation of TBBPA via the 

FeTPPS/KHSO5 system. This suggests that further polymerized brominated compounds are 

produced as a result of catalytic oxidation. 

Vol. 3 Page - 124 -

TBBPA degradation (%) 

100 

80 

60 

40 

20 

0 

FeTPPS + KHSO 5 

FeTPPS + HA + KHSO 5 

FeTPPS + HQ-HA +KHSO 5 

4 5 6 7 8 

Figure 1: Influence of solution pH on percentages of the TBBPA degradation 

Influences of HAs on the oxidation products of TBBPA. To identify the oxidation products, n- 

hexane extracts from the reaction mixtures were analyzed by means of GC/MS. Figure 2 

shows the chromatograms in the absence and presence of HA. In the absence of HA 

(FeTPPS+KHSO5), no clear peaks for the oxidation products were observed. However, in 

FeTPPS + HA + KHSO5 (and FeTPPS + HQ-HA+ KHSO5, data not shown), clear peaks for 

4-(2-hydroxyisopropyl)-2,6-dibromophenol acetate (2HIP-2,6DBP, m/z 352) and trimer (m/z 

836) of 2,6DBP appeared. Because debromination cannot occur in the catalytic oxidation of 

TBBPA, more polymerized byproducts of brominated phenol may be formed in FeTPPS + 

KHSO5. However, in FeTPPS + HA+ KHSO5, the [2HIP-2,6DBP] were much smaller than 

those of degraded TBBPA. 

Br 


O 

C CH 3 

O 

Br 

H H3C 3C C CH 3 

OH 

m/z 352 

H H3C 3C 

pH 

C O 

TBBPA 

m/z 836 

15 20 25 30 35 40 45 50 55 

O 

Retention time (min) 

Br Br 

Br 

CH 3 

Br 

Br 

O C 

CH 3 

Br 

FeTPPS+HA+KHSO 5 

FeTPPS + KHSO 5 

Figure 2: GC/MS chromatograms of n-hexane extract from the reaction mixtures (pH 8) 

Vol. 3 Page - 125 - 

OH

To estimate the mass balance of bromine in the FeTPPS/HAs/KHSO5 catalytic system, the 

HA fractions in the reaction mixtures were separated, as described in a previous report [5]. 

The contents of bromine in the separated HA and HQ-HA were 7.6% and 9.6%, respectively. 

Considering the amounts of remained TBBPA in the reaction mixture, it was estimated that 

64% and 91% of the oxidized TBBPA were incorporated into HA and HQ-HA, respectively. 

From the pyrolysis-GC/MS analysis of HA fractions in reaction mixtures, the bound 

brominated species in HAs were identified as bromophenols that can be derived from the 

oxidation intermediates of TBBPA. 


Oxidation of TBBPA via the FeTPPS/KHSO5 catalytic system was not significantly enhanced 

or inhibited in the presence of HAs. However, more polymerized byproducts may be formed 

in the absence of HA. In the presence of HA, low-molecular-weight byproduct, such as 2HIP- 

2,6DBP, was produced and approximately 64 – 91% of oxidized TBBPA were bound to 

polymeric structures of HAs. Although more polymerized compounds from TBBPA only are 

difficult to soluble in water, low-molecular-weigh byproducts and HA-bound byproducts are 

relatively soluble in water. Thus, transportability of brominated byproducts from TBBPA is 

decelerated in the absence of HA. However, the transportability of brominated byproducts 

may be enhanced in the presence of HAs. These results suggest that the HAs can serve as 

carrier of brominated byproducts, derived from catalytic oxidation of TBBPA, to aquatic 

environments. 



This work was supported by Grants-in-Aid for Scientific Research from the JSPS (21310048). 

1. References. 

2. M. Osako, Y.-J. Kim and S. Sakai, Chemosphere, 57 (2004) 1571. 

3. S. Kitamura, T. Suzuki, S. Sanoh, R. Kohta, N. Jinno, K. Sugihara, S. Yoshihara, N. Fujimoto, H. 

Watanabe and S. Ohta, Toxicol. Sci., 84 (2005) 249. 

4. K. Lin, W. Liu and J. Gay, Environ. Sci. Technol., 43 (2009) 4480. 

5. S.-K. Han, P. Bilski, B. Karriker, R.H. Sik and C.F. Chignell, Environ. Sci. Technol., 42 (2008) 

166. 

6. M. Fukushima, H. Ichikawa, M. Kawasaki, A. Sawada, K. Morimoto, K. Tatsumi, Environ. Sci. 

Technol., 37 (2003) 386. 

7. R.S. Swift, In, Methods of Soil Analysis Part 3, Soil Science Society of America, Madison, 1996, 

p. 1018. 

8. I.V. Perminova, A.N. Kovalenko, P. Schmitt-Kopplin, K. Hatfield, N. Hertkorn, E.Y. Belyaeva, 

V.S. Petrosyan, Environ. Sci. Technol., 39 (2005) 8518. 

Vol. 3 Page - 126 -

Adsorption of Trihalomethanes by Humin: Batch and Fixed Bed Column 

Studies 

Graziele da Costa Cunha a , Luciane Pimenta Cruz Romão a *, Mônica Cardoso Santos a , 

Bruno Rafael Araújo a , Sandro Navickiene a and Válter Lucio de Pádua b 

a Department of Chemistry, Federal University of Sergipe, 49100-000 Aracaju, SE, Brazil; 

b Department of Sanitary and Environmental Engineering, Federal University of Minas Gerais, 

30110-001 Belo Horizonte, MG, Brazil 

E-mail: luciane@ufs.br 



During the end of the 19th century and beginning of the 20th century, diseases caused by pathogenic 

microorganisms, such as typhoid fever, dysentery and cholera, increasingly became a focus of 

attention, especially in relation to potable water quality. To address this problem, in 1908 chlorine was 

first used as a disinfectant in New Jersey, USA, and has continued to be widely used in water 

treatment stations (WTSs) worldwide. Its various compounds destroy and/or inactivate the organisms 

responsible for many illnesses hence greatly reducing human mortality caused by diseases 

disseminated via hydric systems [1]. Despite the benefits provided by disinfection, use of chlorine and 

other compounds has attracted the attention of the scientific community, due to reactions with natural 

organic matter (NOM) that can generate subproducts that may be undesirable from the human health 

perspective. The trihalomethanes are frequently found in water treatment systems, with chloroform 

(CHCl3), dichlorobromomethane (CHBrCl2), dibromochloromethane (CHBr2Cl) and bromoform 

(CHBr3) being the most common. Various studies have reported on the high carcinogenic and 

mutagenic potentials of these compounds. Commercial activated carbon is the material most widely 

used for adsorption of THMs in water treatment stations (WTSs) worldwide, due to its high removal 

capacity. Nevertheless, its efficiency is dependent on the need for large dosages in short time periods, 

and its use is limited by the associated cost. To reduce costs of treatment, alternative adsorbents are 

sought that are more economically viable, easily disposed of, and above all may be readily regenerated 

without losing their properties. Bioadsorbents have merited special attention, due to their availability 

and abundance, and significantly lower cost compared to synthetic adsorbents. Amongst these 

adsorbents is peat, an organic soil formed continuously by a complex process of decomposition and 

humification of plant residues by microbiological oxidation in flooded environments. Humin is the soil 

organic matter that remains after removal of humic and fulvic acids, defined as the fraction that is 

insoluble in aqueous solution at any pH, and possesses higher molecular weight and carbon content 

compared with other peat humic fractions. High porosity and surface area are indicative of humin’s 

potential as an adsorbent [2]. The objective of the present work was therefore to assess the 

performance, in batch and fixed bed column systems, of humin used either in natura or immobilized 

on sodium silicate, respectively, for adsorption of the main trihalomethanes found in water supply 

systems. 

Vol. 3 Page - 127 -


The peat sample was collected from a peat bog in the vicinity of Santo Amaro das Brotas, Sergipe 

State, Brazil. The sample was air-dried, ground using a pestle and mortar, and sieved first through a 9mesh 

grid to remove roots and twigs, and then through a 48-mesh grid to obtain a uniform particle 

size. The in natura peat was not used as an adsorbent, because of the dissolution of humic and fulvic 

acids at the pH range of 6.5-9.0 typically found for water from treatment stations. The procedure for 

immobilization of the humin biomass on sodium silicate was similar to that described by [3]. Batch 

tests to investigate the influence of initial concentration, the kinetics and the adsorption isotherm were 

conducted individually for each of the THMs studied, with agitation (150 rpm, 25 ± 0.2 ºC) in amber 

glass flasks. All tests were performed using 10 mL of THM solution and 0.1 g of humin. A Millex-HV 

0.45 μm syringe-driven filter unit was used for removal of the supernatant for subsequent analysis. 

After filtration, the THM concentrations were quantified using a gas chromatograph (Varian, USA) 

fitted with an electron capture detector (GC-ECD), coupled to a purge and trap (PT) system. Internal 

standard was added at the time of injection, in order to avoid any losses. A blank solution was 

prepared without the humic material, and all experiments were performed in triplicate. Humin 

immobilized on sodium silicate was packed into a glass column (20 cm × 2.0 cm i.d.), through which 

the THM solution was percolated, in descending flow, using a peristaltic pump. The influences of 

height and flow rate on THM adsorption were monitored. Breakthrough curves (C/C0 vs time, where C 

is the concentration exiting the column, and C0 the initial concentration) were obtained by collection 

of aliquots in amber flasks after different time intervals (up to 360 min). The concentrations of THMs 

remaining after exiting the column, and in the solution at the end of the experiment (to identify 

possible losses by volatilization) were determined by GC/ECD-PT. 



The effect of contact time on the adsorption of the THMs was studied using the concentration (250 μg 

L -1 ) that showed highest percentage removal. There was rapid removal in the first few minutes, with 

equilibrium achieved at around 240 min, indicative of fairly fast kinetics. The percentage removals of 

bromoform, dibromochloromethane, dichlorobromomethane and chloroform were 83.2 ± 0.1%, 78.0 ± 

0.1%, 77.0 ± 0.1% and 74.6 ± 0.1%, respectively. The selectivity of adsorption followed the order 

CHBr3 > CHBr2Cl > CHBrCl2 > CHCl3, however without any significant differences (P > 0.05), 

suggesting that the active sites on the humin did not exhibit any preference in adsorption of the THMs 

studied. In addition, significant correlation (P < 0.05) was obtained between the adsorption 

percentages of the THMs by humin, as a function of time (bromoform, r = 0.98; 

dibromochloromethane, r = 0.96; chloroform, r = 0.99; dichlorobromomethane, r = 1.0). The validity 

of the pseudo first and second order kinetics, and intra-particle diffusion models was used to analyze 

the linear equations (t/qt) vs. t, log (qe – qt) vs. t, and qt vs. t 0.5 , respectively. The experimental data 

obeyed pseudo second order kinetics, as can be seen in Table 1. The values of k2 2 were greater than 

Vol. 3 Page - 128 -

those of k1 2 and ki 2 , as well as those of R2 2 . Intra-particle diffusion was also a contributing factor. 

However, the lines do not pass through the origin, indicating that this mechanism did not determine 

the rate of the overall process, and the existence of a complex mechanism consisting of adsorption and 

intra-particle transport. The values of qe (experimental) and qt (theoretical, obtained from the angular 

coefficient of the straight line) show good agreement. 

Table 1: Pseudo first order, pseudo second order and intra-particle diffusion kinetic parameters for 

adsorption of THMs on humin 

qt qe 

2 -5 

k1 · 10 

2 

R1 2 -4 

k2 ·10 

2 

R2 2 -4 

ki · 10 

2 

Ri min -1 

(µg·g -1· min) µg·g -1· min 0.5 

THMs µg·mg 

Bromoform 19.60 19.51 6.67 0.477 3.38 0.999 2.60 0.984 

Chlorodibromomethane 18.81 17.73 1.53 0.389 7.34 0.999 3.90 0.989 

Bromodichloromethane 18.05 17.89 8.90 0.804 1.67 0.999 1.57 0.817 

Chloroform 21.01 20.45 3.18 0.508 1.39 0.997 1.00 0.963 

-1 

The experimental data fitted the Freundlich model, since in addition to giving the highest correlation 

coefficient (R 2 ) value (Table 2), the separation factor (RL), an essential characteristic of the Langmuir 

isotherm, was not favorable (RL > 1). The constants Kf and n are related to the maximum adsorption 

capacity and the adsorption intensity, respectively. The Kf value showed the following order of 

selectivity of humin for the THMs: CHBr3 > CHBr2Cl > CHBrCl2 > CHCl3. This behavior was also 

confirmed by the experimental values, with the constant, n, showing favorable adsorption of the 

THMs. Humin was efficient for THM removal, with concentrations reduced by at least 83.0% 

following treatment. 

The influence of flow rate on adsorption of THMs by humin immobilized on sodium silicate was 

investigated using two different flows (2 and 3 mL min -1 ). From the results presented in Figures 1a 

and 1b, increased flow caused a significant (P < 0.05) reduction in the adsorption capacity of the 

humin for the THMs. This was because increased flow reduced the residence time of the solute in the 

adsorbent bed, which then reduced solute diffusion into the pores of the adsorbent. Figures 1c and 1d 

show the breakthrough curves obtained for different bed heights (2 and 4 cm), at a constant flow rate 

of 2 mL min -1 . 



The humin has been shown to be effective for adsorption of the main THMs found in water supply 

systems. Adsorption was relatively fast, with a maximum adsorption of 83.2% in batch experiments. 

Using a fixed bed column, adsorption results demonstrated the efficacy of humin as an adsorbent, 

extracting 99.7% under optimized conditions of TTHMs in the systems studied. The adsorption 

capacity of a fixed bed employing humin immobilized on sodium silicate showed the same selectivity 

as batch adsorption. 

Vol. 3 Page - 129 -

Figure 1: Breakthrough curves for adsorption of THMs by humin immobilized on sodium silicate in 

column experiments. Conditions: Bed height 2 cm, flow rate a) 3 and b) 2 ml min -1 ; flow rate 2 mL 

min -1 , bed height c) 2 and d) 4 cm; e) adsorption of TTHMs; T = 25 ± 0.1°C 



The authors thank MCT/CNPq (proc. n° 550062/2007-2) for financial support of this study. 

References 

1. A.D. Nikolaou, S.K. Golfinopoulos, G.B. Arhonditsis, V. Kolovoyiannis, T.D. Lekkas, 

Chemosphere. 55 (2004) 409. 

2. G. De La Rosa, J.R. Peralta-Videa, J.L. Gardea-Torresdey, J. Hazard. Mater. 97 (2003) 207. 

3. A.P.S. Batista, L.P.C. Romão, M.L.P.M. Arguelho, C.A.B. Garcia, J.P.H. Alves, E.A. Passos, 

A.H. Rosa, J. Hazard. Mater. 163 (2009) 517. 

4. O. Ozdemira, M. Turana, A.Z. Turanb, A. Fakia, A.B. Enginc, J. Hazard. Mater. 166 (2009) 647. 

Vol. 3 Page - 130 -

Combined Effects of Humic Matter and Surfactants on PAH Solubility: 

Is There a Mixed Micellization? 


Holger Lippold a* 

a Forschungszentrum Dresden-Rossendorf, Institut für Radiochemie 

E-mail: h.lippold@fzd.de 

It has been recognized that solid-liquid distribution and transport of hydrophobic 

contaminants such as PAH (polycyclic aromatic hydrocarbons) are governed by their 

interaction with humic matter, which is present on sediment surfaces as well as in solution, 

acting as a sink or a mobilizing agent, respectively [1, 2]. As surface-active compounds, 

humic substances are often compared to surfactants. Emerging environmental technologies 

involve a deliberate application of surfactants to enhance the sorption capacity of soils and 

aquifer materials [3, 4], or to increase the efficiency of soil washing procedures and pumpand-treat 

operations for groundwater decontamination [5]. Whereas contaminant binding to 

humics as well as to surfactants has been extensively studied, there is a notable lack of 

literature on their combined action in mixed systems. This topic is, however, important 

because environmental influences of surfactants are inevitably associated with the effects of 

the ubiquitous natural organics. Since both are amphiphilic, it seems conceivable that mixed 

micelles can be formed, involving synergistic or antagonistic effects in the solubilization of 

organic compounds. 

In this study, we have examined the joint influence of humic acid (HA) and surfactants 

(cationic, anionic) on the water solubility of pyrene as a representative of PAH, at surfactant 

concentrations below and above the critical micelle concentration (CMC). In order to detect 

and characterize interaction processes, we have investigated the octanol-water partitioning of 

HA in the presence of various surfactants, using radiolabelled humic material. In particular, 

the hypothesis of a micellar nature of dissolved humic substances has been addressed. 



All chemicals and HA were purchased from Sigma-Aldrich (Germany). The HA was purified 

by acid-base treatment, followed by dialysis. For experiments on octanol-water partitioning, it 

was radiolabelled with 131 I (Amersham, Germany), adopting the Iodogen method [6]. 

Surfactants and 14 C-labelled pyrene were used as received. 

Vol. 3 Page - 131 -

Pyrene solubilities were determined by liquid scintillation counting (Beckman LS 6000, GMI, 

USA) after shaking an excess amount with surfactant / HA solutions in glass vials for 7 days 

and passing the supernatants through membrane syringe filters. The results were corrected for 

adsorption losses and colour quenching. 

For determining octanol-water partition ratios, solutions of 131 I-labelled HA and surfactants 

(pre-equilibrated for 15 h) were overlaid with octanol (2 mL / 2 mL in PP test tubes) and then 

gently shaken for 24 h. Samples of both phases were analyzed by gamma counting, using a 

1480 Wallac Wizard 3” (Perkin Elmer, USA). 

Size exclusion chromatography was performed by means of an HPLC equipment HP 1100 

(Hewlett-Packard, USA) with a TSKgel G3000 PWXL column (Phenomenex, USA), run with 

phosphate buffer as eluent. 



The water solubility of pyrene is increased in the presence of HA, which acts as a carrier due 

to hydrophobic interaction of both components. When adding the cationic surfactant 

dodecyltrimethylammonium bromide (DTAB), this solubility enhancement was found to be 

cancelled; the humic colloids were precipitated as a consequence of charge compensation by 

the organo-cations. 

Interestingly, an antagonistic effect was also observed on addition of an anionic surfactant, 

sodium dodecylsulfate (SDS). While no precipitation was induced in this case, the solubility 

of pyrene was reduced by half and remained constant on further addition. Only at surfactant 

concentrations above the CMC, the solubility increased sharply owing to micellar 

incorporation. The presence of HA did not cause any change in the CMC of SDS, as is 

normally observed on addition of a second amphiphilic compound. Furthermore, the effects of 

HA and micellar SDS on pyrene solubility turned out to be strictly additive. Consequently, 

they are based on distinct processes, occurring independently of each other, i.e., there is no 

mixed micellization with humic molecules acting as a co-surfactant. 

The octanol-water partition ratios of HA changed significantly in the presence surfactants. 

The partitioning equilibrium was shifted towards the organic phase on addition of cationic 

surfactants, and towards the aqueous phase on addition of anionic surfactants. Based on these 

findings, different modes of interaction could be identified, as shown in Fig. 1. 

Vol. 3 Page - 132 -

(a) 

(b) 

Figure 1: Schematic representation of association modes between humic colloids and cationic (a) and 

anionic surfactants (b) as derived from octanol-water partitioning experiments 

The binding mechanism shown in Fig. 1(b) provides an explanation for the decline in pyrene 

solubilization in systems of HA and SDS. Obviously, a competitive situation arises in the 

hydrophobic binding of the PAH and the surfactant tail groups. The fact that the pyrene 

molecules cannot be displaced completely supports the proposition that different binding sites 

exist in humic colloids: weak near-surface sites and strong inner sites [7]. 

The size distribution of the colloids was found to be unaffected by the association with 

anionic as well as with cationic surfactants. A general micellar character (as originally 

suggested by Wershaw [8]) is thus unlikely since a co-aggregation should then entail 

substantial disruptions and rearrangement processes. 



A humic-bound mobilization of hydrophobic pollutants is not facilitated by surfactants that 

may be present at contaminated sites, and surfactant-aided flushing procedures are neither 

impaired nor enhanced by dissolved organic matter. Both cationic and anionic surfactants 

associate with humic colloids, which could be demonstrated in octanol-water partitioning 

experiments with radiolabelled HA, but our studies did not provide any indication of a 

formation of mixed micelles. It is thus questionable whether dissolved humic substances are 

organized in micelle-like aggregates. 

Vol. 3 Page - 133 -

Acknowledgement 


This research has been supported by funding from the German Federal Ministry of Education 

and Research (BMBF), contract number 0330537. 

References 

1. S.W. Karickhoff, Chemosphere, 10 (1981) 833. 

2. C.T. Chiou, P.E. Porter, D.W. Schmedding, Environ. Sci. Technol., 17 (1983) 227. 

3. S.A. Boyd, J.F. Lee, M.M. Mortland, Nature, 333 (1988) 345. 

4. J. Wagner, H. Chen, B.J. Brownawell, J.C. Westall, Environ. Sci. Technol., 28 (1994) 231. 

5. C.C. West and J.H. Harwell, Environ. Sci. Technol., 26 (1992) 2324. 

6. P.J. Fraker and J.C. Speck, Biochem. Biophys. Res. Commun., 80 (1978) 849. 

7. J.J. Pignatello and B. Xing, Environ. Sci. Technol., 30 (1996) 1. 

8. R.L. Wershaw, J. Contam. Hydrol., 1 (1986) 29. 

Vol. 3 Page - 134 -

Complexation of Copper(II) Ions With Humic Acids and EDTA Studied by 

High Resolution Ultrasonic Spectrometry 

Martina Klucakova * , Miloslav Pekar 

Brno University of Technology, Faculty of Chemistry, Purkynova 118, 612 00 Brno, Czech 

Republic 

E-mail: klucakova@fch.vutbr.cz 


Ultrasound spectroscopy is based on observing interactions of ultrasound wave with studied 

system. Two principal characteristics are measured – ultrasound velocity and attenuation 

(decrease in amplitude). Velocity reflects local elasticity and density of material, which are 

determined by molecular arrangements, conformation and solvation shell. Attenuation reflects 

in homogeneous samples fast relaxation (chemical) processes and in heterogeneous samples 

scattering of ultrasound wave, which can be used e.g. for particle sizing. Considering the 

measured parameters are strongly sensitive to molecular conformation and inter- and intramolecular 

interactions ultrasound spectroscopy is used as method for investigation of 

chemical reactions [1–3]. 

Metal binding of humic acids (HA) is the subject of many studies. In our previous works [4– 

7] complexation of humic acids and by metal ions has been studied by conductometry, 

potentiometry, UV/VIS and FT-IR spectrometry. This study utilizes High Resolution 

Ultrasound Spectrometry (HRUS) for analysis of interactions between copper(II) ions and HA 

and EDTA as a humic-like model. 


HA were isolated from South Moravia lignite by standard alkaline extraction as described 

elsewhere [4, 8]. More details on the chemical structure of the initial lignite matrix, as well as 

that of the isolated HA, can be found in previous papers [8-10]. The basic characteristics of 

used humic sample are listed in Tab. 1. 

Table 1: Characterization of used humic sample (normalized on dry ash-free HA) 

C 

(at. %) 

H 

(at. %) 


N 

(at. %) 

S 

(at. %) 

O 

(at. %) 

total acidity 

(mmol/g) 

43.9 40.2 0.7 0.2 15.0 4.94 

Vol. 3 Page - 135 -

Humic sol was prepared by dissolution in concentrated NaOH and mixing with 15% (wt,) 

solution of HEPES (Aldrich) up to pH = 7, The content of HA in final solution was 14.23 g/L 

EDTA was dissolved in the solution of HEPES, Final concentration of EDTA in HEPES 

buffer was 5.72 mmol/L (pH = 7). 

Ultrasonic spectrometer with high resolution HR-US 102 (Ultrasonic Scientific, Ireland), was 

utilized for measurement of basic ultrasonic parameters, The device consists of two 

independent cells tempered at 25 °C, Reference cell was filled by HEPES buffer (EDTA 

measurement) or HEPES with NaOH (HA measurement), Measuring cell was filled by EDTA 

or HA solution prepared by ways described above and titrated by CuCl2 up to saturation of 

their acidic groups, Velocity (U) and attenuation (N) in both cells was measured, the resulting 

differences ΔU and ΔN between reference and sample cell we can see at graphs, Blank 

experiments with NaOH in HEPES, EDTA and HA versus HEPES were carried out to 

correction of data obtained from titration experiments, All HRUS measurements were done at 

four different frequencies. 



The dependencies of measured ultrasonic velocity and attenuation on degree of saturation of 

acidic groups in HA and EDTA by CuCl2 are shown in Figs, 1 and 2, According the results 

both compounds are able to bind the bivalent Cu 2+ ions by two functional groups, In case of 

EDTA all binding sites can be occupied and saturation is represented by ratio Cu 2+ /H + = 0.5. 

Complexation of HA is more complicated, HA contains many various binding sites with 

different strength which can be also gradually occupied, On the other hand, each Cu 2+ ion 

may not be able to find two suitable acidic groups for its binding for structure and 

conformation of HA particles, The steric effects are the reason, why some acidic groups can 

remain vacant, A part of Cu 2+ ions can be bonded only by one acidic group but our quantumchemical 

computing showed that such arrangement is hardly likely [11], The maximum of 

measured ultrasonic parameters then quadrates with ratio Cu 2+ /H + = 0.4. 

The differences between complexation behaviour of HA and EDTA are clearly evident also 

from slopes of measured dependencies, The slope of decrease of ΔU with degree of saturation 

of binding sites is -1.31 ± 0,04 m/s for HA and -0.58 ± 0,01 m/s for EDTA, It shows on more 

rigid structure of Cu-HA complexes, which can be caused by formation of inter- and intramolecular 

bridges. 

Vol. 3 Page - 136 -

ΔU (m/s) 

0 

-0,1 

-0,2 

-0,3 

-0,4 

-0,5 

-0,6 

Cu 2+ /H + (mol/mol) 

0 0,2 0,4 0,6 0,8 1 1,2 

Figure 2: The dependence of ΔU on saturation of acidic groups in HA (circles) and EDTA (triangles) 

in their titration by CuCl2 

ΔN (1/m) 

10 

8 

6 

4 

2 

0 

-2 


Cu 2+ /H + (mol/mol) 

0 0,2 0,4 0,6 0,8 1 1,2 

Figure 3: The dependence of ΔN on saturation of acidic groups in HA (circles) and EDTA (triangles) 

in their titration by CuCl2 

The stronger influence of complexation on HA is probably caused by its aggregation and 

formation of much bigger particles in comparison with EDTA, which corresponds with data 

obtained for titration of the saturated organic compounds, because Cu-HA aggregates can 

sediment in contrast to Cu complexes with EDTA, The marked HA aggregation was also 

confirmed by strong increase of ultrasonic attenuation for Cu 2+ /H + > 0,4. 

Vol. 3 Page - 137 -


Measurement of ultrasonic velocity and attenuation in titration of HA and EDTA as “humiclike” 

model by CuCl2 showed that HRUS is very sensitive method for detection and analysis 

of complexation of organic compounds, It was confirmed that saturation of acidic groups in 

HA is not 100 % probably due to their structure and steric effects (in contrary to EDTA), The 

formation of relatively big Cu-HA particles (able to sediment) was observed, On the other 

hand, aggregation of EDTA caused only indistinctive increase of attenuation. 



This work was supported by government funding – Czech Science Foundation, project, Nr, 

104/08/0990. 

References 

1. B,I, Kankia, T, Funck, H, Uedaira and V,A, Buckin, J, Solution Chem,, 26 (1997) 877, 

2. E,D, Kudryashov, C, Smyth, B, O’Driscoll and V,A, Buckin, Spectroscopy, 18 (2003) 26, 

3. W,-B, Ko and V,A, Buckin, Elastomer, 41 (2006) 57, 

4. M, Klucakova, M, Kalab, M, Pekar and L, Lapcik, J, Polym, Mater,, 19 (2002) 287, 

5. M, Klucakova and M, Pekar, J, Polym, Mater,, 20 (2003) 145, 

6. M, Klucakova and M, Pekar, in E,A, Ghabbour and G, Davies (Eds,), Humic Substances: 

Molecular Details and Applications in Land and Water Conservation, Taylor & Francis, New 

York 2005, Chapter 11, p,167, 

7. M, Klucakova and M, Pekar, Colloid, Surface, A, 286 (2006) 126, 

8. M, Klucakova and M, Pekar, Colloid, Surface, A, 252 (2005) 157, 

9. J, Kucerik, M, Klucakova and M, Pekar, Petrol, Coal, 45 (2003) 58, 

10. P, Peuravuori, P, Zbankova and K, Pihlaja, Fuel Proc, Technol,, 87 (2006) 829, 

11. M, Klucakova, P, Pelikan, L, Lapcik, B, Lapcikova, J, Kucerik and M, Kalab, J, Polym, Mater,, 17 

(2000) 337. 

Vol. 3 Page - 138 -

CE-ICP-MS as Speciation Technique to Analyze the Complexation 

Behavior of Europium, Gadolinium and Terbium with Humic Acid 

Christina Möser * , Ralf Kautenburger, Horst Philipp Beck 

Institute of Inorganic and Analytical Chemistry and Radiochemistry, Saarland University, 

Campus C4.1, 66123 Saarbrücken, Germany 

E-mail: c.moeser@mx.uni-saarland.de 



For the long-term disposal of radioactive waste, detailed information about geochemical 

behavior of radioactive and toxic metal ions under environmental conditions is necessary. The 

work with the radioactive actinides needs specialized high security standards. In order to 

avoid these standards the lanthanides europium, gadolinium and terbium were used as 

homologues of the actinides americium, curium and berkelium. Their similar chemical and 

physical properties permit a conferment to the behavior of the actinides. 

In the spotlight of the investigations stand the sorption and desorption behavior of the 

lanthanides onto Opalinus clay. Natural organic matter can play an important role in the 

immobilization or mobilization of metal ions due to complexation and colloid formation. This 

complexation behavior could interfere the sorption of metal ions onto Opalinus clay. In 

addition to humic acid (HA), in Opalinus clay natural appear organics like lactate, formiate or 

propionate were used. As a selected technique, capillary electrophoresis (CE) was hyphenated 

with inductively coupled plasma mass spectrometry (ICP-MS). With this method, both the 

uncomplexed metal ions and metal organic complexes can be simultaneous detected in one 

analysis step. As medium for these experiments additionally to 10mM sodium perchlorate 

solution synthetic prepared pore water was used. The influence of the high concentration of 

cations like Magnesium and Calcium in the pore water is very important and needs specific 

notice. 


For preparation of all used solutions MilliQ deionised water (18,2 MΩ) was used. The humic 

acid is commercially available from Aldrich (St. Louis, USA; HA sodium salt) and was 

purified through some precipitation and dissolution steps with 0.1 M HCl and 0.1 M NaOH / 

0.01 M NaF. For detection in the ICP-MS the humic acid became iodinated with iodine, iodic 

acid and sodium iodide (ultrapure, Merck) [1]. The single element standards of Eu, Gd, and 

Tb were of ultrapure quality (nitrates) and receivable from Merck. As electropheric buffer a 

mixture of 100 mM acetic acid / 10 mM Na-acetate was used. Before starting experiments the 

Vol. 3 Page - 139 -

uffer was filtered and degassed in an ultrasonic bath. The complexation experiments were 

carried out with a lanthanide concentration of 500 ppb, 25 mg/L HA and 10mM NaClO4 / 

synthetic pore water. The used salts for pore water preparation were of p. a. quality or better. 

The NaClO4 was p. a. quality. The samples were equilibrated in a horizontal shaker for 72 h at 

25 °C. As speciation technique a CE was hyphenated with ICP-MS. Beside excellent 

separation performance we obtained a high sensitivity for the determination of rare earth 

metals like europium (representative for lanthanides) and its organic complexes. The 

connecting piece between CE and ICP-MS was a homemade interface. The fused-silica 

capillary of the CE was flexibly fitted into a MicroMist 50 µl nebulizer with a Cinnabar 

cyclonic spray chamber. The chamber can be chilled to a temperature of 4 °C for best 

sensitivity. 200 ppb of cesium (ultrapure, Merck) was added to the CE separation buffer to 

observe the capillary flow. A make-up fluid including 4 ppb Ho (ultrapure, Merck) as an 

internal standard was combined with the flow from the capillary within the interface to obtain 

a fluid throughput high enough to maintain a continuous nebulization [2]. The conditions for a 

optimal separation were 1.5 psi for sample injection and 3 psi with 30 kV for the separation 

process. A description of the analytic conditions and operating parameter can be found in [3]. 



In these report we show the influence of the high cation concentration in the synthetic pore 

water on the complexation behavior of humic acid. Fig. 1 shows a typical electropherogram 

with three Eu species (red line). The green line pictured the run of the Cs curve. The first 

decrease of the curve corresponds to the start of the sample migration. The second minimum 

of the curve shows the point of neutrality (EOF). With these minimum neutral species 

migrate; behind this point migrate the negative species. The first Eu signal (peak1) represents 

the uncomplexed metal before CE separation. The aquatic Eu 3+ ion was complexed with the 

acetate in the electrolyte buffer and migrates during the separation as EuAc 2+ towards the CE 

cathode. Peak 2 and peak 3 pictured the HA complexed metal ions. Peak 3 shows the strong 

bound Eu, peak 2 the weak bound Eu. During the separation the weak bound Eu 3+ dissociates 

due to the attached voltage out of the HA complex and migrates as peak 2 towards the 

cathode. The strong bound Eu is stabile during the separation and migrates as negative 

charged complex behind the other peaks. 

Vol. 3 Page - 140 -

[counts] 

153 Eu 

9000 

8000 

7000 

6000 

5000 

4000 

3000 

2000 

1000 

Peak 2 Peak 3 

Peak 1 

133 Cs 

0 

0 

0 50 100 150 200 

Migration time [sec] 

60000 

50000 

40000 

30000 

20000 

10000 

Figure 1: Typical electropherogram of uncomplexed and HA-compexed Eu species and Cs as CE flow 

marker in 10 mM NaClO4 solution 

The electropherogram with synthetic pore water (Fig. 2) shows a completely different run of 

the Eu and Cs curve. In this case peak 2 represents the free Eu 3+ ions which are complexed 

with the acetate in the electrolyte buffer and migrate during the separation as EuAc 2+ towards 

the CE cathode. Peak 1 and 3 pictured the HA complexed Eu-ions. The blue curve represents 

the migration of the iodated HA. This curve shows the correlation between the Eu- and the 

HA-migration. 

[counts] 

12000 

10000 

8000 

6000 

4000 

2000 

153 Eu 

127 I 


Peak 1 

Peak 3 

Peak 2 

0 

0 

0 50 100 150 

Migraton time [sec] 

133 

Cs 

140000 

120000 

100000 

80000 

60000 

40000 

20000 

Figure 2: Typical electropherogram of uncomplexed and HA-compexed Eu species, Cs as CE flow 

marker and iodide as HA migration marker in synthetic pore water 

Vol. 3 Page - 141 -

The first Eu-humate complex migrates with the sample front (Cs signal, green line) and 

presents the most positive charged complex. The Eu-humate complex in peak 3 migrates with 

the EOF. Due to the high amount of competing cations in the pore water the Eu-humate 

complex is uncharged. 

4. Conclusion 

In summary, the results show that the competing cations in the synthetic pore water have a 

great influence of the complexation behavior of the HA. From this it follows that 

investigations under natural conditions are very important for the comprehension of the 

complex system. 



The author thanks the BMWi for financial support (grant no. 02 E 9683 and 02 E 10196). 

References. 

1. D. Braun, H. O. Wirth, Patentschrift 1 088 227, 1961, Deutsches Patentamt. 

2. R. Kautenburger, H.P. Beck, J. Chromatogr. A, 1159, 2007, 75. 

3. R. Kautenburger, H. Anal. At. Spectrom., 24, 2009, 934. 

Vol. 3 Page - 142 -

Determinations of Ability of Extracted HSs Coordinated with Metal Ions 

Ke-fei Ding, Qiao-hui Fan, Yu-ying Zhang, Wang-suo Wu * 

Radiochemistry Laboratory, School of Nuclear Science and Technology, Lanzhou University, 

Lanzhou, Gansu P.R. China 

E-mail: wuws@lzu.edu.cn 

Humic substances (HSs) are the major fraction of dissolved organic compounds present in 

water and soils, which could be separated into humic acids (HAs), fulvic acids (FAs) and 

humin according to their solubility in acid and alkaline solution [1]. HSs are considered 

natural polyelectrolytic organic compound of complex structure involving a proportion of 

aromatic rings with a larger number of attached –OH and –COOH groups, which result in the 

interaction with metal ions to form complexes easily [2]. Since the strong complex ability, 

HAs and FAs play important roles as ligands in the mobilization of metal ions in soils and 

aqueous of environment, and they also affect the bioavailability and toxicity of these metal 

ions. In most cases, the free metal ion, rather than all species of metal, is correlated to 

toxicity, so complexes with humic substances as ligands generally reduces metal toxicity [3]. 

The objectives of this research are to model and predict the complexation behavior of HSs 

with metal ions and radionuclides. Ion exchange method was used to determine the 

conditional stability constants of complexes (i.e., β) between HA or FA with selective cations, 

i.e., Ni 2+ , Cu 2+ , UO2 2+ and Th 4+ [4]. 

HA and FA were extracted from the soil of Huajia county (Gansu province, China) according 

to a references standard of the International Humic Substances Society (IHSS), which were 

used in following all the experimental studies. Cross-polarization magic angle spinning 

(CPMAS) 13 C NMR spectra of humic substance was divided into six chemical shift regions, 

0-60, 61-80, 81-110, 111-160, 161-180 and 181-210 ppm (Figure 1). These regions were 

referred to as aliphatic, heteroaliphatic, acetal, aromatic, carboxyl, and carbonyl regions. In 

addition, predictive weight-average molecular (Mw) to the method of Moriguchi et al. [5], in 

which absorbance coefficients (ε) of humic substance at 280 nm on UV-vis spectroscopy are 

applied to the empirical equation: 

M w = 3.99 ε +490 

(1) 


The result shows that the molecular weights of HA and FA were ~3132.18 and ~1691.15, 

respectively. 

Vol. 3 Page - 143 -

Figure 1: Cross-polarization magic angle spinning (CPMAS) 13 C NMR spectra of HA and FA 

In this study, the Schubert method was used to determine the metal complexes of HS. The 

fundamental assumption was made, namely, the metal ion was the central group, thus the 

complexes ML1, ML2, ML3….were formed in the aqueous solution and the conditional 

stability constants are defined by: 

[ ML ] 

[ M ][ L] 

i β i = i=1, 2, 3 …… (2) 

i 

Where M is the metal ion, L is the free ligand and [ ] stands for the concentration. The 

distribution coefficient (λ0) of metal between resin and aqueous solution in the absence of 

ligand could be defined by: 

[ M ] ads λ 0 = 

(3) 

[ M ] 

In the presence of hydrolyzed species (M(OH)i), buffer-complexed species (MBj), bicarbonate 

and carbonate species M(HCO3)m and M(CO3)n in the aqueous phase, Eq(3) convert to Eq.(4): 

+ 

[ M ] ads 

λ 1 = 

(4) 

[ M ] 

[ M ( HCO ) ] 

∑ ∑ ∑ ∑ + 

+ 

+ 

i[ 

M ( OH ) i ] b[ 

MBb] 

m[ 

M ( CO3 

) n ] 

Eq(4) was also further modified to include the effect of complexation by HSs to form ML1, 

ML2, ML3,. . . 


Vol. 3 Page - 144 - 

n 

3 

n

[ M] 

+ 

ads 

λ 2 = 

(5) 

[ M] 

k[ 

MLk 

] 

∑ ∑ ∑ ∑ ∑ + 

+ 

+ 

+ 

i[ 

M( 

OH) 

i ] b[ 

MBb] 

m[ 

M( 

CO3 

) n] 

n[ 

M( 

HCO3 

) n] 

The concentration of HSs complexes can be substituted by conditional stability constants �1; 

� 2; �3. At a constant pH, a constant buffer concentration, and a constant bicarbonate or 

carbonate concentration, combination of Eqs. (3)–(5) leads to the following equation: 

The plots of 

1 

λ 

2 

1 

λ 

1 

1 1 1 

2 

− = ( β1[ 

L] + β 2[ 

L] 

+ β3[ 

L] 

λ λ λ 

2 

1 

0 


3 

+ ⋅⋅⋅ 

⋅⋅⋅ 

− vs. [L] were achieved and the conditional stability constants would be 

obtained by using quadratic polynomial equation fitting method. The results have been listed 

in Table 1. It is clear that the complexes of metal ions with HSs mainly formed 1:1 and 1:2, 

and the mono-molecular formation is predominant. The value of � of HA-M was larger than 

that of FA-M, since the larger macular weight of HA results in the larger volume and more 

coordinate groups than FA. In addition, the complex ability of HSs with metal ions was 

obviously influenced by the kind of metal ions and reaction temperature. 

Vol. 3 Page - 145 - 

(6)

Ni 2+ 

Cu 2+ 

UO2 2+ 

Th 4+ 

Table1: Conditional stability constants of metal ions-HA and FA complexes 

Metal Ligand pH 


Log�1 

Log�2 

HA 4.70 5.932 5.451 0.99356 

FA 4.80 3.537 - 0.99373 

HA 4.80 5.555 4.819 0.99870 

FA 4.77 4.084 1.315 0.99932 

HA 4.60 5.223 3.711 0.99874 

FA 4.60 4.540 3.464 0.97537 

HA 2.69 6.290 5.631 0.98384 

FA 2.64 4.218 3.733 0.99216 

References 

1. C.L. Chen, X.K. Wang, H. Jiang, W.P. Hu, Colloid. Surf. A. 302 (2007) 121. 

2. F.J. Stevenson, Humus Chemistry: Genesis, Composition, Reactions, 2nd ed. Wiley, New York 

1994. 

3. D. Gondar, R. López, S. Fiol, J.M. Antelo, F. Arce, Geoderma 135(2006) 196. 

4. W.M. Dong, H.X. Zhang, M.D. Huang, Z.Y. Tao, Appl. Radiat. Isot. 56(2002)959. 

5. T. Moriguchi, M. Tahara, K. Yaguchi, J. Colloid Interf. Sci. 297(2006) 678. 

Vol. 3 Page - 146 - 

r

Characterization of Zinc Binding Ability of Dissolved Hydrophilic Organic 

Matter from The Seine River and Major Wastewater Effluents 

Louis Yoann a , Pernet-Coudrier Benoît a,b , Varrault Gilles a* 

a Laboratoire Eau Environnement et Systèmes Urbain (LEESU), Université Paris-Est, 61 

Avenue du General de Gaulle, F 94010 Créteil Cedex, France ; b Eawag, department water 

resources and drinking water, Überlandstrasse 133, 8600 Dübendorf, Switzerland 

E-mail: varrault@univ-paris12.fr; yoann.louis@leesu.enpc.fr 



Complexation of trace metals in aquatic ecosystems by natural organic matter (NOM) and 

inorganic ligands, i.e. their chemical speciation, determines their mobility, bioavailability and 

toxicity [1, 2]. Natural organic matter, which is ubiquitous in the environment, is known to 

play important roles in the fate of many contaminants due to its complexing properties. A 

better determination of NOM structural and functional properties can greatly improve our 

understanding of the underlying mechanisms responsible for heavy metals complexation. 

Over the past few decades, many studies have been published regarding the capacity of 

dissolved organic matters (DOM) to complex metals and especially copper. It is interesting to 

note however that the published data pertain mainly to the so called “humic substances” (HS) 

and demonstrate the ability of these substances to complex metals. HS are derived from 

oxidative and hydrolytic biodegradation of plants and animals [3] and they make up 40–60% 

of DOC in natural surface water [4]. 

In urbanized aquatic system, the hydrophobic characteristic of DOM is weaker as a result of 

various urban DOM discharges and the strong primary productivity induced by these 

discharges. Previous study has shown that non-hydrophobic organic matter represents more 

than 50% of DOC in urbanized aquatic system [5]. However, because of the difficulty in 

isolating the hydrophilic fraction of DOM, no information is available regarding hydrophilic 

DOM in urbanized aquatic system and its influence on Zn complexation. 

As a matter of fact, this work focus on zinc interactions with 3 isolated DOM fractions 

fractionated according to polarity criteria, i.e. hydrophilic (HPI), hydrophobic (HPO) and 

transphilic (TPH) fractions from four sampling sites located on the urbanized basin of the 

Seine River (France) upstream and downstream Paris city. Suwannee River Fulvic Acid 

(SRFA) obtained from the International Humic Substances Society (IHSS) has also been 

characterised in order to compare properties of DOM from urban aquatic system with natural 

humic substances 

Vol. 3 Page - 147 -



Among the four sites studied on the Seine River basin, the first one located upstream of Paris 

city (called Méry sur Marne) will be considered as our natural reference because of little 

anthropic impact and those downstream of Paris (called Andrésy and Méricourt) as the most 

impacted by urbanization as well as the treated effluent of the biggest wastewater treatment 

plant (WWTP) of Parisian conurbation (Seine-Aval WWTP). For each sample, from 250 L to 

1000 L were collected during a dry weather period (Fig. 1). 

Figure 1: Sampling site on the Seine River basin (France) with on site filtration, softening and 

concentration by reverse osmosis 

Samples were filtered onsite through subsequent 10 µm and 0.45 µm polypropylene cartridge. 

The samples were then softened on sodium cation-exchange and concentrated by reverse 

osmosis (RO) in order to reduce the volume. Sample filtration, softening and concentration 

were carried out in line and onsite so as to limit process duration and potential DOM 

biodegradation (Fig. 1). The RO concentrate was then acidified and filtered back at the 

laboratory on non-ionic mac roporous Amberlite® DAX-8 resins (acrylate ester) and 

Supelite® 

XAD-4 (divynil benzene) combined with one another. This allows us to fractionate 

DOM into three fractions according to polarity criteria: hydrophobic (HPO), transphilic (TPI) 

and hydrophilic (HPI) fraction [5]. 

Among the techniques allowing trace metals speciation in natural waters, voltammetry, and in 

the case of this study differential pulse anodic stripping voltammetry (DPASV), is really 

appropriate because it offers high sensitivity and enough selectivity for zinc measurement. 

Obtained voltammetric data are related to the physico-chemical characteristics of the 

electroactive metal species, and information on speciation parameters of the investigated 

metal could be obtained [6]. 

Vol. 3 Page - 148 -

The pseudopolarography [7] technique, based on successive DPASV measurements at 

different deposition potentials, was also employed in this study. This technique allows 

discrimination/separation of different fractions of metals, from the electrochemically labile to 

the inert ones, regarding the analytical window of the method. It is successfully applied for 

the speciation of metals in model solutions and in natural waters, both for the labile and for 

the inert metal complexes. According to the theory and to experimental evidences [7 and ref 

therein], one or more reduction 

waves could be obtained (similarly like in classical 

polarography) which correspond to one labile and/or to one or more inert metal complexes. 

In order to work on a wider analytical window, zinc additions were performed in a 

logarithmic mode [8] (from nano-molar to about 14 micro-molar) which also allows a better 

accuracy of the complexing parameters determination. 


Depending on the studied DOM fraction, pseudopolarography experiments point out different 

behaviour with one (Figure 2 on the right) or two wave (on the left) showing the specificity of 

each extracted fraction. 


Figure 2: Pseudopolarography experiments representative of a HPO (on the left) and a HPI (on the 

right) DOM fraction. x axis: different deposition potentials with step of 0.05V from -0.8 (labelled 1) 

to -1.6V (labelled 17), y axis: scanning potential for stripping, z axis: Intensity for [ZnT]=13.8µM 

Vol. 3 Page - 149 -

However for all the studied fractions, a deposition potential of -1.05 V give a good sensitivity 

to analyse the labile zinc. Experiments carried out with logarithmic zinc additions give new 

data (stability constant and ligands concentration) concerning the zinc-NOM interactions in an 

urbanized area especially with HPI fraction. From the analysis, it is emerging a tendency 

showing that the HPI fraction has a higher affinity for zinc than respectively the TPH and the 

HPO fractions. This tendency was also observed on the same samples for copper and mercury 

on previous study carried out in our lab. 


Thanks to this study and the news determined complexing parameters usable in 

m odelization/prediction study, the behavior of zinc (which is a metal always present in 

urbanized 

aquatic system mainly due to its use in roof and gutter) will be better understood in 

such complex 

ecosystem than the urbanized rivers with especially the hydrophilic DOM 

fraction. 



We would like to thanks the French Ministry of agriculture for the post doctoral grant of Y. 

Louis, and B. Pernet-Coudrier for the DOM fractionation made during his PhD with the 

financial support of the French Ministry of research and higher education. 

References 

1. J. Buffle, Complexation Reactions in Aquatic Systems: An Analytical Approach, M. Masson and J. 

F. Tyson, Ellis Horwood, New York, 1988, p.692. 

2. A. Tessier and D. R. Turner, Metal Speciation and Bioavailability in Aquatic Systems, J. Buffle 

and H. P. Van Leeuwen, John Wiley & sons, Chichester, 1995, p. 696. 

3. F.J. Stevenson, Humus Chemistry – Genesis, Composition, Reactions, John Wiley & Sons, New 

York, 1994. 

4. B. Martin-Mousset, J.P. Croué, E. Lefebvre, B. Legube, Distribution et caractérisation de la 

matière organique dissoute d’eaux naturelles de surface, Water Res., 31 (1997), 541. 

5. B. Pernet-Coudrier et al., Chemosphere, 73 (2008) 593. 

6. Y. Louis et al., Mar. Env. Res., 67 (2009) 100. 

7. R. Nicolau et al., Anal. Chim. Acta., 618 (2008) 35. 

8. C. Garnier et al., Anal. Chim. Acta., 505 (2004) 263. 

Vol. 3 Page - 150 -

Molecular Size Distribution of Metal Complexes with Pore Water Dissolved 

Organic Matter Determined by HPSEC and ICP-MS 

Natalja Makarõtševa a* , Viia Lepane a , Tiiu Alliksaar b 

a Institute of Chemistry, Tallinn University of Technology, Akadeemia tee 15, Tallinn, 

Estonia; b Institute of Geology, Tallinn University of Technology, Ehitajate tee 5, Tallinn, 

Estonia 

E-mail: natalja.makarotseva@ttu.ee 


It is widely known that natural organic matter is influencing the fate and behavior of 

contaminants in aquatic environment; however, the interaction mechanisms are relatively 

poorly understood. The goal of the present study was to investigate which organic fractions 

bind to specific metals. 


In the present 

study pore water extracted from the sediments from Lake Peipsi, Estonia, was 

investigated using high-performance 

size exclusion liquid chromatography (HPSEC) with 

fluorescence detection and inductively-coupled plasma mass-spectrometry (ICP-MS). 

Sediment core was collected on ice using Russian type peat corer from the central part of 

Lake Peipsi (58°47.213´; 27°19.299´) in March, 2007. The core was sliced 

into 1 cm thick 

sub-samples. 

Before the analysis, pore water was extracted from the solid phase by 

centrifugation 

and filtration. 

Pore water dissolved organic matter (DOM) from different depths was separated and 

fractionated using HPSEC. 100 mM phosphate buffer was used as a mobile phase at the flow 

rate 0.5 mL/min. Injected volume was 20 µL. Fluorescence signal was detected at 340/420 nm 

(humic-like fluorescence). 

HPSEC column was calibrated using polystyrene sulphonate 

standards (PSS) with molecular weights of 17, 13, 6.8, and 4.3 kDa dissolved in the mobile 

phase. Collected HPSEC fractions were analyzed with ICP-MS for selected metals, such as 

Al, Ca, Mn, Ni, Cu, Zn, Ag, Cd, and Pb, in order to reveal which molecular weight fractions 

complexed 

them. Mentioned metals were also measured directly in not fractionated pore 

water samples by ICP-MS. 



Common chromatogram of pore water DOM with marked collected fractions is shown on Fig. 

1. Phosphate buffer is widely used for DOM separations but its usage in case of ICP-MS 

Vol. 3 Page - 151 -

analysis could be problematic, because phosphates itself may contain metal impurities. In the 

present study the attempt to minimize buffer contamination influence was done by injecting 

the buffer as a blank sample to the HPSEC column and fractionating it at the same retention 

times 

as the collected pore water samples. 

Results showed 

that Ca was mostly complexed with DOM eluted at 19.5-23.0 min (3rd 

fraction). The molecular wei ghts of that fraction corresponded to number average molecular 

weight Mn of 600 Da, and weight average molecular weight Mw of 700 Da. The presence of 

other investigated metals was detected in all collected fractions and their contents were not 

differing much. Thus, under the used conditions it was not possible to distinguish which 

fraction contains more metals. The possible reason for it was that the content of metals was 

very low and it was close 

to ICP-MS LOD. 

Detector 

signal, mV 

1 

0,8 

0,6 

0,4 

(a) 

0,2 

1 2 3 

0 

4 

0 5 10 15 20 25 30 

-0,2 

Retention time, min 

800 

600 

400 

Ca, ug/L 1000 

200 

0 

0 1 2 3 4 

Fraction 

Figure 1: (a) HPSEC example chromatogram of pore water DOM, with marked fractions; (b) Ca 

content in the separated fractions by ICP-MS 

In this study Ca was determined in fractionated pore water samples from different depths. 

Other metals distributions in depth profile were also constructed. 


The obtained information is valuable for understanding the binding and transport of metals in 

aquatic water bodies. Also, this knowledge enables to draw conclusions about the 

bioavailability of metals towards the organisms living in lake ecosystems. 

Acknowledgements. 


The authors thank the Department 

of Analytical Chemistry of Masaryk University, Brno, 

Czech Republic, for kind possibility to use ICP-MS. 

Vol. 3 Page - 152 - 

(b)

Influence of Pb(II) Ions on Semiquinone Radicals of Humic Acids and 

Model Compounds 

Maria Jerzykiewicz * , Maciej Witwicki 

Faculty of chemistry, Wroclaw University, F. Joliot-Curie 14 St., Wroclaw, Poland 

E-mail: mariaj@wchuwr.pl 


The complexation of Pb 2+ with humic acids macromolecules leads to the formation of a new 

kind of radical species characterized by unusually low giso values (~ 2.001) [1,2]. Noteworthy, 

these new radicals appears to be very stable. 

In this paper we decided to investigate the interaction of Pb(II) ions with humic acids and 

their model compounds in details. We hoped to obtain an insight into the components of the 

g-tensor and how they change upon the interactions of the parent radicals 

with the Pb(II) ions. 

On the other hand, the systematic theoretical DFT study of the model complexes between 

Pb(II) ions and semiquinone radicals was preformed, examining different possible 

deprotonation forms of the ligands and different possible binding schemes with the metal 

cation. We expected that the correlation between theoretically predicted and experimentally 

obtained g-tensors would reveal the structural types responsible for the low giso values 

observed by the EPR spectroscopy, allowing for char acterization of the best model mimicking 

the Pb(II) coordination to HA which is an important problem for environmental chemistry [3]. 


3,4-dihydroxybenzoic acid (34dhb) and gallic acid (3,4,5-trihydroxybenzoic acid, 345thb) 

were purchased from Aldrich. Humic acids were extracted from peat (HAP) of Odra river 

lowland near Wroclaw 

(Lower Silesia, Poland) and from compost (HAC) derived from the 

Municipal Composting Plant in Zabrze (Upper Silesia, Poland). The isolation was carried out 

using standard IHSS procedure [4]. To obtain the complexes of Pb(II) ions with HAC, HAP, 

34dhb, and 345thb in powder form, a constant amount of a solid ligand L (1 mmol of 34dhb 

and 345thb or 50 mg of HAC and HAP) was treated with a fixed volume of lead acetate 

solution (20 mL) of concentration appropriate to obtain various initial Pb(II):L molar ratios 

from 1:1 to 5:1. The solid product was filtered and dried at room temperature, and the EPR 

spectra of powder samples were recorded. 


Vol. 3 Page - 153 -

X-band EPR (9.6 GHz) spectra were recorded at the room temperature on a Bruker ESP 300E 

spectrometer equipped with a Bruker NMR gauss meter ER 

035 M and a Hewlett-Packard 

microwave frequency counter HP 5350B. As investigated systems 

contain lead, all single- 

point calculations of the g-tensor were preformed within the two-component approach using 

spin-orbit ZORA relativistic form an Lenthe in alism, as implemented by v Amsterdam Density 

unctional (ADF) version 2007.01 . F 

Three different functionals were used: (a) local density 

approximation of Vosko, Wilk and Nusair (VWN) with the exchange gradient correction 

proposed by Becke (B) and the correlation term developed by Perdew (P86); (b) VWN local 

density approximation with the exchange gradient correction and the correlation term by 

Perdew and Wang ient correction and the correlation term by Lee, 

(PW91); (c) B exchange grad 

Yang and Parr (LYP). More about computational approach in [5]. 



Formation of low-g radicals upon the Pb(II) interaction. Treatment of solid HAC and HAP 

with lead acetate solution results in a characteristic modification of the EPR signal. Total 

signal intensity increases as compared to that recorded for the pure HAs. The native radical 

concentration in the HAC was found to be about 0.3 × 10 18 spins per gram. After formation of 

Pb(II)-HAC complex the concentration increases even to 1.7 × 10 18 spins per gram. 

Subtraction of the HAs signal from the signal of Pb-treated HAs reveals a new radical line, as 

reported previously [2]. The g values for new radicals in both Pb-HAs systems are 

significantly smaller than those for native radicals that occur in HAs: geff = 2.0012 for Pb- 

HAC compared to geff = 2.0033 

for HAC and 2.0013 for Pb-HAP compared to 2.0036 for 

HAP. Nevertheless, no significant dependence of EPR parameters on concentration of Pb(II) 

was observed. It needs to be noticed that the presence of indigenous signal due to HAs in the 

spectra of Pb(II)-treated HAs proves that the native radicals, which are probably located in the 

humic acid macromolecular matrix, are not affected by the Pb(II) ions. Pb(II) seems to be 

bonded mainly to the outer macromolecular fragments. Furthermore, if the Pb(II) ions were 

involved in the interaction with the native radical sites in HAs giving the g ~ 2.001 signals, 

the complexes could not be generated from model dihydroxybenzoic acids (since 

dihydroxybenzoic acids do not contain the native radicals). Therefore, it can be assumed that 

the radical complexes formation is preceded by the Pb(II) ions coordination to the 

diamagnetic ligand. In contrast to HAs, the initial concentration of Pb(II) ions appeared to be 

a major factor determining the EPR parameters observed for radical complexes with 34dhb 

and 345thb ligands. Two types of spectra were observed depending on Pb(II) concentration 

Vol. 3 Page - 154 -


(see Figure 1). For the system prepared using molar ratio Pb(II):L = 3:1, the powder EPR 

spectra indicating formation of complex 1 are similar to those reported previously (geff ~ 

2.001) [1]. However, the spectra of the Pb(II):L= 2:1 system differ distinctly from those 

observed when molar ratio Pb(II):L=3:1 was used. They are stronger and slightly anisotropic, 

with larger geff, but still below the giso values for the radical anions derived from the parent 

hydroxybenzoic acids. In addition, the powder EPR spectra for the 2:1 system show satellite 

splitting due to the anisotropic hyperfine interaction of an unpaired electron with the 207 Pb 

nucleus (I= 1/2, 22.1% abundance)- figure 1. Therefore, the second complex (complex 2) has 

been identified. The parameters of hyperfine couplings with the 207 Pb nucleus strongly 

suggest a covalent interaction between the Pb(II) ion and a radical ligand in complex 2. 

Figure 1: EPR spectra of radicals in Pb(II)-34dhb powders with different metal ion to ligand ratio 

DFT Computations for Model Complexes of Pb(II) with Semiquinone Radical Species. Three 

different forms of ligand were considered: L2-•, HL-•, and H2L•. The radical derived from 

34dhb is an ambidentate ligand, and many possible isomers exist for every form. Two binding 

sites (carboxylic or phenolic oxygens) were taken into account for the ligand acting as either 

mono- or bidentate. First, the geometry of the Pb(II):L 1:1 model complexes and their g 

parameters were calculated. Next, analogous calculations were performed for model 1:2 

complexes that were derived from those 1:1 complexes in which a reasonable agreement 

between the theoretical and experimental g-tensors was achieved. 

Systematic theoretical studies for the Pb(II)-34dhb system has shown that the structures with 

a significant accumulation of the spin population on the Pb atom cannot explain the shifts of 

experimentally observed g-tensor components. DFT investigations show, that decrease of the 

Vol. 3 Page - 155 -

spin population on all oxygen atoms in favour of the carbon atoms of a benzoic ring, observed 

for the complexation via carboxyl oxygen atoms while both hydroxyl oxygen atoms are 

protonated, can reproduces the experimental results and therefore can be responsible for the 

observed shift of the g-tensor. The binding mode and the form of ligands present in Pbsemiquinone 

complexes have been determined and the reaction mechanism, which may lead 

to the formation of such complexes, has been proposed. 



X-band and high field EPR spectroscopy has been applied to investigate the complexes of 

Pb(II) ions with the semiquinone radicals of HA and their model compounds (34dhb and 

345thb). They are characterized by unusually low g-parameters in comparison to the parent 

dicals. For the model compounds, a formation of two complexes (complex 1 and complex 

2) has been revealed by two different EPR spectra. For complex 2, a splitting of the spectrum 

due to the anisotropic hyperfine interaction with the 207 Pb nucleus (I= 1 ra 

/2, 22.1%) has been 

observed. 

References 

1. E.Giannakopoulos, K. C. Christoforidis, A Tsipis, M. Jerzykiewicz,Y. J. Deligiannakis, Phys. 

Chem. A 2005, 109, 2223 

2. M. Jerzykiewicz, Geoderma 2004, 122, 305 

3. E. Tipping, E. Cation Binding by Humic Substances, 1st ed.; Cambridge University Press: 

Cambridge, U.K., 2002 

4. R.S. Swift. Organic matter characterization, In Methods of Soil Analysis. Part 3 Chemical 

Methods - SSSA Book Series no. 5, Madison, WI, 1996, pp.1011–1069, 

5. 

M. Witwicki, M. Jerzykiewicz, A.R. Jaszewski, J. Jezierska, A. Ozarowski, J. Phys. Chem. A 

2009, 113, 14115–14122. 

Vol. 3 Page - 156 -

The Effect of Natural Organic Matter on the Formation and Solubility of 

M(OH)4 Solid Phases (Th(OH)4, Zr(OH)4 Ce(OH)4) 

Stella Antoniou, Ioannis Pashalidis * 

Chemistry Department, University of Cyprus, P.O. Box 20537, 1678 Nicosia, Cyprus 

E-mail: pspasch@ucy.ac.cy 


The chemical behavior and mobility of metal ions in aquatic environments depends strongly 

on the solubility of the solid phases/minerals present. (Oxo)hydroxide solid phases are usually 

the predominant secondary phases of tetravalent metal ions in natural aquatic systems, and are 

also important sinks for (radio)toxic metals (e.g. Th(IV), Pu(IV), Zr(IV)) in the near field of 

nuclear waste repositories and heavily contaminated water systems. Therefore, in order to 

understand the chemistry and mobility of (radio)toxic metals, knowledge of the solubility and 

stability of relevant solid phases/minerals 

is of fundamental importance. In natural water 

systems where humic acid is present, the complexation with humic acid plays a significant 

role 

in the geochemical behavior and migration of thorium and other actinide ions in the 

geosphere [1 - 4]. Hence, the impact of humic acid on the stability of M(IV) solid phases and 

species predominantly formed is of particular interest. 

To ascertain the effect of humic acid complexation on the solid phase formation 

and 

subsequently the chemical behavior of M(IV) (e.g. Th(IV) and Zr(IV)) in aqueous solutions, 

the stability of M(OH) 4 has been studied as a function of the humic acid 

concentration in 0.1 

M NaClO4, in the pH range from 3 to 5. The solid phase formation of redox sensitive metal 

ions has been investigated for Ce(IV) hydroxides formed by alkaline precipitation from 

aqueous solution containing various amounts of humic acid. 



M(IV) and humic acid stock solutions were prepared by dissolution of Th(NO3)4 5H2O 

(Merck Co), ZrO(NO3)2 xH2O (Aldrich) and Ce(NO3)4 (Kristallhandel Kelpin) and humic acid 

sodium salt (Aldrich) in de-ionized water, respectively. M(OH)4 solid phases were prepared 

by alkaline precipitation of M(IV) from pure aqueous solutions or solutions containing 

different amounts of NOM (0, 0.1, 0.3 and 0.5 g l -1 humic acid), under normal atmospheric 

conditions at 25 o C. For solubility studies Th(OH)4 and Zr(OH)4 solids were conducted with 

40 ml of pure 0.1 M NaClO4 solutions or 0.1 M NaClO4 solutions containing 0.1 g l -1 humic 

acid under normal atmospheric conditions at 25 o C. pH was adjusted by 0.1 M NaOH or 0.1 

M HClO4 and was measured using a glass electrode (Hanna Instruments pH 211). The 

Vol. 3 Page - 157 -

a nalytical M(IV) concentration in solution was determined by spectrophotometry (UV 2401 

PC Shimadzu) using arsenazo III according to a previously described method [5] . 

For physicochemical characterization, the precipitates were separated 

from the solutions by 

centrifugation, washed (2 times) with de-ionized water, air-dried and characterized by 

Thermogravimetry (TGA-50, Shimadzu) and x-ray diffraction (XRD 6000 Shimadzu). 

FTIR- 

ATR spectroscopy (IR Prestige-21 Shimadzu) 

and XPS measurements were performed on 

solid samples after separation and thermal treatment of the samples at 100 o C under vacuum 

conditions. The XPS measurements were carried out at the ICE/HT-FORTH Labs in Patras, 

Greece. 



Investigation of pure Th(OH)4 and Zr(OH)4 solid phases and the same solids in equilibrium 

with aquatic solutions of 0.1 M NaClO4 containing 0.1 g/L of humic acid by ATR-FTIR 

spectroscopy shows similar features independent of the presence of humic acid. In the 

spectrum predominate two intensive absorption bands at 3610 cm -1 and 1629 cm -1 in the FTIR 

spectrum, which correspond to O-H stretching and to H-O-H bending, respectively. The 

absorption band at 1081 cm d to the Th-OH and Zr-OH stretching, 

-1 and 1074 cm -1 correspon 

respectively. Thermogravimetric analysis of the Th(OH) 4 and Zr(OH) 4 solids prior contacting 

with humic acid solutions, show mass losses (about 32%) with temperature, which according 

to stoichiometric calculation data correspond to M(OH)4 solid phases. After equilibration with 

humic acid solutions, the thermogravimetric data indicate only on a higher mass loss (about 

5%), which is attributed to excess physisorbed water on the humic acid treated solids [4]. Xray 

diffraction analysis of pure Th(OH)4 and Zr(OH)4 solid phases and the same solids in 

equilibrium with aquatic solutions of 0.1 M NaClO4 containing 0.1– 0.5 g/l of humic acid 

indicates also only the presence 

of M(OH)4 solid phases. Based on the Scherrer equation [6] 

the particle/crystallite sizes have been evaluated and are summarized in Table 1. 

Table 1: Particle size of Zr(OH)4 and Th(OH)4 solid phases prior and after contact with aqueous 

solutions of 0.1M NaClO4 containing 0.1-0.5 g/l of humic acid 

[HA], mg/l Particle size, nm 

Th(OH)4 Zr(OH)4 

0 8 9.4 

0.1 7.5 9.0 

0.3 7.6 9.3 

0.5 7.7 9.2 

According to data summarized in Table 1, the presence of humic acid in solution does not 

affect the particle size of the Th(OH)4 solid phase. The absence of any effect on the crystallite 

Vol. 3 Page - 158 -

size of Th(OH)4, which in contrast to the effect of humic acid on the crystallite size of U(VI) 

solid phases (e.g. UO2(OH)2 [3] and UO2CO3 [4]), can be attributed to the higher stability of 

the M(IV) solid phases compared to the U(VI) solid phases. 

Solubility measurements were performed with Th(OH)4 under normal atmospheric conditions 

in aquatic solutions of 0.1 M NaClO4 containing 0 and 0.1 g/L of humic acid. According to 

solubility measurements corresponding to the systems with or without humic acid, the 

Τh(OH)4 solid phase determines solubility. Fluctuations which are observed in the thorium 

concentration were attributed to the formation of Th(IV) colloids in the pH range 3-5 [2]. In 

this study the formation of Th(IV) colloids has been confirmed by ultrafiltration experiments 

using membrane filters of different 

pore size (0.45μm, 0.22μm, 30kD, 100kD). According to 

the ultrafiltration experiments, decreasing pore size of the ultrafiltration mebranes results in a 

significant decrease of the total Th(IV) concentration in solution. Furthermore, lower Th(IV) 

concentration in humic acid - containing solution after filtration indicates the formation of 

larger particles generated upon humic acid - Th(IV) colloid interaction. 

To investigate the effect of NOM to the solid formation of redox-sensitive (oxidizing) 

tetravalent ions, Ce(IV) ions have been precipitated upon addition of alkaline aqueous 

solutions containing different amounts (0 - 0.5 g/l) of humic acid. The resulting solid phases 

have been separated, vacuum dried and characterized by means of XPS analysis regarding the 

relative amount of Ce(IV) in the solid phase as a function of the humic acid (NOM) 

concentration in solution. The evaluation of the XPS spectra (Figure 1) resulted in a linear 

correlation between the humic acid concentration in the test solution and the reduced amount 

of Ce(IV) in the precipitated solid phase, indicating that in the presence of NOM oxidizing 

ions may be reduced affecting solid phase composition. The latter is important for the 

chemical behavior and migration of actinide (e.g. Pu(IV) ions) in the geosphere. 


The results obtained from this study lead to the conclusions that (a) M(OH)4 is stable and 

remains the solubility limiting solid phase even in the presence of 

increased 

NOM (humic 

acid) concentrations in solution, (b) the presence of NOM (humic ac id) results in increased 

hydrophylicity of the solids but doesn’t affect the crystallite size and the solubility product of 

M(OH)4, (c) M(OH)4 solubility is basically pH depended and governed by the presence of 

colloidal species and (d) NOM (humic acid) may reduce redox-sensitive (oxidizing) metal 

ions and result in increased amount of the reduced species in the solid phase affecting its 

solubility behavior. 


Vol. 3 Page - 159 -

Figure 1: Relative amount of Ce(IV) in the solid phase as a function of the humic acid (NOM) 

concentration in the test solution 


The research leading to these results has received funding from the Cyprus Research 

Promotion Foundation (Grant agreement No. ΠΕΝΕΚ/ΕΝΙΣΧ/0308/05). The authors thank 

Dr Elina Siokou for the XPS measurements. 


References 

1. J.I. Kim, Mat. Res. Soc. Symp. Proc., 294 (1993) 3. 

2. V. Neck and J.I. Kim, Radiochim. Acta, 89 (2001) 1. 

3. C. Kolokassidou and I. Pashalidis, J. Radioanal. Nuclear Chem., 279 (2009) 523. 

4. S. Antoniou, C. Kolokassidou, K. Polychronopoulou and I. Pashalidis, I., J. Radioanal. 

Nuclear Chem., 279 (2009) 863. 

5. S.B. Savvin, Talanta, 8 (1961) 673. 

6. Jenkins, R., Snyder, R.L., Introduction to x-Ray Powder Diffractometry, J. Wiley & Sons, 

N. York, 1996. 

Vol. 3 Page - 160 -

Adsorption of Polycyclic Aromatic Hydrocarbons (PAHs) onto Engineered 

and Natural Nanoparticles 

Lina Marino, Donato Mondelli, Nicola Senesi 

University of Bari, Dip. Biologia e Chimica Agrof. Amb., Via G. Amendola 165/A, 70126, 

Bari, Italy 

E-mail: senesi@agr.uniba.it 


Nanoparticles have been demonstrated to have a very high sorption capacity for a variety of 

organic contaminants from water and soil (soil remediation) [1]. However, there are 

unan swered questions about the impact of engineered nanomaterials and nanoproducts on 

human 

health and the environment [2]. Among nanoparticles, fullerene C60 has received 

considerable 

attention due to its unique characteristics and numerous potential applications 

[3]. Fullerene C60 is virtually 

insoluble in water [4], where it forms clusters that present closed 

interstitial spaces within the aggregates into which organic compounds can diffuse and remain 

trapped [5]. Adsorption of organic compounds by fullerene have been shown to depend to a 

great extent on its dispersion state in water [ 6]. Fullerenes are reported as weak sorbents for 

organic compounds including PAHs, but are very efficient for the removal of organometallic 

compounds 

[7]. 

The specific objective of this study is to evaluate the use 

of fullerene C60 as an adsorbant of 

organic pollutants, such as polycyclic aromatic hydrocarbons (PAHs), from aqueous 

solutions, compared with the adsorbent effect of different types of soil with various organic 

carbon (OC) and clay contents, texture, pH and cation exchange capacity (CEC), as such and 

with the addition of suitable amounts of fullerene (F), compost (C) and humic acid from 

compost (HAC). 


Soils. ES (Elliott Soil), a clay soil of the IHSS (International Humic Substances Society) 

collection with 2.9% OC; S2, a silt loam soil with 1.1% OC; S3, a loamy sand soil with 0.1% 

OC (Table 1). 


The soil samples were homogenized in a ball mill (Retsch MM 200) and used both as such 

and added with 5% F or 1% C or 1% HAC. 

Nanoparticles. Fullerene C60 (F) with purity>99,5% was obtained from Sigma-Aldrich and 

used as received. 

Vol. 3 Page - 161 -

Compost 

(C) from vegetable wastes with 38.5% OC. 

Humic acid from compost (HAC) 

with about 50% OC. 

Chemicals. Pyrene (Sw: 135 µg/L at 25 °C, purity>99,0%) was purchased from Fluka 

A nalytical and used as received. Its stock solutions were prepared in methanol (HPLC grade). 

Adsorption Experiments. The adsorption kinetics and adsorption 

isotherms of pyrene onto the 

various soil substrates 

were determined using a Batch Equilibrium Method (OECD, 2000) and 

the high-performance liquid chromatography (HPLC) technique with a fluorescence-detector 

operating at 337 nm in excitation and 377 nm in emission. 

Soil 

Table 1. Some physical and chemical properties of soils examined 

Textural 

Class 

(USDA) 


pH 

(1:2.5 

H2O) 

Organic 

carbon 

(%) 

Total 

nitrogen 

(%) 

Total 

carbonate 

(%) 

ES Clay 6.8 2.9 0.25 < 0.1 

S2 Silt Loam 8.1 1.1 0.12 5.9 

S3 Loamy sand 8.5 0.1 0.01 8.9 

Adsorption kinetics. 50 mg of soil were suspended in 40 mL of 0.01 M CaCl2 aqueous 

solutions containing pyrene at a concentration of 100 µg/L in glass flasks. A blank sample 

(without soil) at the same concentration of pyrene was prepared. The mixtures were then 

mechanically shaken for five different time periods: 12, 24, 48, 72 e 96 h. At the end of each 

time period, the suspensions were centrifuged at 12000 rpm for 20 min, and the supernatants 

were analyzed by HPLC to determine the residual concentration of pyrene in solution. All 

experiments were conducted in triplicate at a temperature of 25 °C. 

Adsorption isotherms. Aliquots of 50 mg of the various soil substrates were added in glass 

flasks with 40 mL of 0.01 M CaCl2 aqueous solutions containing pyrene at concentrations of 

40, 60, 80, 100, and 120 µg/L. Equilibration was achieved by mechanical shaking of mixtures 

for 72 h at 25 °C in the dark. All experiments were conducted in five replicates. Suspensions 

were then centrifuged at 12000 rpm for 20 min, and the supernatants were analyzed for the 

equilibrium concentrations, Ce, of pyrene by HPLC. Three standard samples (without soil) at 

concentrations of 30, 60 and 120 µg/L were also analyzed in each set of sorption isotherm 

experiments. The amount of pyrene adsorbed onto substrate (x/m, in µg g 

ion and the equilibrium concentration (Ce, in µg 

-1 

. 

ngmuir equations. 

-1 ) was calculated as 

the difference between the initial concentrat 

mL ) of pyrene in solution Experimental data were fitted to both a linear and non linear 

Freundlich and La 

Vol. 3 Page - 162 -


Adsorption kinetics. Adsorption of pyrene onto the examined soils occurs in two phases, a 

rapid one in less than 48 hours of contact amounting to more than 90% of total adsorption, 

and a slow one that may need several hours until attainment of equilibrium. The rapid 

adsorption phase would occur on the most reactive and/or accessible sites of substrate, 

whereas sites less reactive and/or less sterically accessible would be involved subsequently in 

the slow phase. 


Adsorption isotherms. Generally, experimental adsorption 

data for pyrene onto the various 

substrates fit better in Langmuir isotherms. The adsorption isotherms indicate that pyrene has 

a moderately high affinity for the substrate in the initial stages of adsorption, 

whereas it has 

increasing difficult y in finding vacant sites, un til reaching a maximum of adsorption. 

The KD values (Table 2) indicate that addition of compost and HAC to soils is more 

efficient 

than fulleren e for pyrene adsorption. 

Further, the KOC values (Table 2) differ for the samples examined, thus indicating that the 

structural and chemical properties of the organic matter in the samples, and not only its 

amount, affect markelly the extent of adsorption. 

Table 2. Distribution coefficients (Kd) and organic carbon partition 

coefficient (Koc), for Langmuir adsorption isotherms of pyrene onto 

substrates studied 

Substrate KD Koc (x10 3 ) 

ES 1324 46 

ES+5% F 1459 53 

ES+1% C 2226 69 

ES+1% HAC 2175 66 

S2 275 25 

S2+5% F 304 29 

S2+1% C 473 43 

S2+1% HAC 411 27 

S3 0.2 0.2 

S3+5% F 6.2 6.5 

S3+1% C 141 29 

S3+1% HAC 112 19 

C 9721 25 

HAC 3995 8 

Vol. 3 Page - 163 -


References 

1. L. Dai (Ed.), Carbon Nanotechnology: Recent Developments in Chemistry, Physics, Materials 

Science and Applications, Elsevier, Boston, 2006. 

2. V. L. Colvin, The Potential Environmental Impact of Engineered Nanomaterials, Nat. Biotechnol. 

21, pp. 1166-1170, 2003. 

3. H. W. Kroto, J. R. Heath, S. C. Obrien, R. F. Curl, R. E. Smalley, C60-Buckminsterfullerene, 

Nature 318, pp. 162–163, 1985. 

4. D. Heymann, Fullerene, Sci. Technol., 4, p. 509, 1996. 

5. X. Cheng, A. T. Kan, M. B. Tomson, J. Nanopart. Res., 7, p. 555, 2005. 

6. X. Cheng, A. T. Kan, M. B. Tomson, 

J. Chem. Eng. Data, 49, p. 675, 2004. 

7. E. Ballesteros, M. Gallego, M. Valcarcel, J. Chromatogr. A 2000, p. 869, 101. 

Vol. 3 Page - 164 -


The Challenge of Building a Humic-Metal Binding Constants Database 

Montserrat Filella a* , Wolfgang Hummel b , Peter M. May c , Jaume Puy d , François Quentel e 

a 

Institute F.-A. Forel, University of Geneva, 10 route de Suisse, CH-1290 Versoix, 

Switzerland; b Paul Scherrer Institut, Laboratory for Waste Management, CH-5232 Villigen, 

Switzerland; 

nt de Química, Universitat de Lleida, Rovira Roure 191, Ele, 

CNRS UMR 6521, 6 avenue V. Le 

c School of Mathematical and Physical Sciences, Murdoch University, Murdoch, 

WA 6150 Australia; d Departame 

25198 Lleida, Spain; e Université de Bretagne Occidenta 

Gorgeu, F-29238 Brest Cedex, France 

E-mail: montserrat.filella@unige.ch 

The fraction of natural organic matter (NOM) more refractory to degradation, often known as 

fulvic and humic compounds, plays a decisive role in trace element chemistry in 

environmental 

and engineering systems. Accordingly, a significant amount of research has 

been devoted to its characterization as well as to the determination of binding constants to 

quantify its interaction with trace elements. However, in spite of the effort deployed, the 

difficulties encountered when trying to compare complexation constants reported in the 

literature or to find constant values for less studied elements remain well-known problems. 

The reason is that our ability to measure and interpret the complexation equilibria of humic 

substances is severely constrained by their ill-defined nature that, together with certain of 

their characteristics, hinder the application of the experimental and interpretation methods 

usually applied in the field of stability constant determination. This has led to the 

development of a wide range of interpretation models for the representation and quantification 

of the binding properties of humics, which adds a further difficulty for the practical 

application of existing data. 

Since no systematic compilation of published data exists, we have undertaken the gathering of 

all data published on the complexation of trace elements with humic substances over the past 

50 years. Our ultimate goal is the critical analysis and the interpretation of all existing data 

with the objective of providing a robust framework for further research as well as a useful tool 

for practical applications. 

The first step in this endeavour is the object of an IUPAC-sponsored project: the development 

of a comprehensive database of published values of humic-metal binding constants. The 

building of such database represents a considerable challenge. On the one hand, it has to face 

problems common with ‘classical’ thermodynamic equilibrium constant databases such as the 

need of gathering information published in journals and reports not always readily available 

or the adoption of criteria that minimize the inherent degree of subjectivity associated with 

Vol. 3 Page - 165 -

any evaluation of data quality. On the other hand, new aspects need to be carefully considered 

before deciding which type of information needs to be extracted and included in the database. 

These further requirements are a direct consequence of the above-mentioned ill-defined nature 

o f the humic substances that has led to the use of a wide variety of experimental methods and 

interpretation approaches. 

The IUPAC project has started in 2009 with the collection of publications 

in electronic form 

for subsequent information extraction by the members of this project. 

Currently this collection 

comprises some 600 files and is expected to increase further. The rate of ’hard-to-find‘ journal 

articles and reports until now stayed below 3% but may also increase when we will screen our 

collected papers for further references with the goal of an exhaustive compilation. 

Interesting and immediate spin-offs of the project are: (i) the exhaustive compilation of all the 

studies ever published, irrespective of the quality or applicability of the data contained; (ii) the 

identification of the elements for which little or no information exists; (iii) the elaboration of 

recommendations for authors and editors concerning the information that needs to be included 

in any publication for the data to be meaningful. 



The work described is sponsored by IUPAC (project 2008-025-1-500). 

Vol. 3 Page - 166 -

Heavy Metal Compounds with Organic Substance and Methods for their 

Definition 

Tatiana M. Minkina a , Galina V. Motuzova b , Olga G. Nazarenko c , Saglara S. Mandzhieva a* 

a 

Department of Biology and Soil Science, Southern federal University, ul. Bolshaya Sadovaya 

105, Rostov-on-Don, 344006 Russia; b 

Faculty of Soil Science, Moscow State University, 

Vorob'evy gory, Moscow, 

119992 Russia; c Agroecological Faculty, Don State Agrarian 

University, Persianovskii, Rostov-on-Don oblast, 346493 Russia 

E-mail: msaglara@mail.ru 


Heavy metals (HMs) are related to the chemical properties of soils in various ways. The 

uptake of heavy metals by soils was found to be dependent on the soil composition and 

properties [1]. Special attention is paid to the participation of organic matter in these 

processes. Heavy metals interacted with the soil organic matter forming various compounds. 

The aim of this work was to study the interaction of heavy metals with the soil organic matter 



A pot experiment was conducted with the upper (0- to 20-cm) layer of an arable calcareous 

clay loamy ordinary chernozem with the following properties: pH water, 7.2; particles


pr esence of 2 M HNO3 and heating to 85 

extracted with an ammonium acetate buf h pH 4.8 [3] and a mixture of AAB and 

1% 

de 

º C). From separate samples, HM compounds were 

fer (AAB) wit 

EDTA with pH 4.8 [4]. In both cases, the extraction was performed at a soil to solution 

ratio of 1 : 5; the time of the extraction was 18 h. The content of the metals in all extracts was 

termined by atomic absorption spectrophotometry. 

We suppose th 4 h 

30% H O in the presence of 2 M HNO3 and heating to 85 o at the content of metal in the 3.2 M CH3COONH 

extract after oxidation wit 

2 2 

C characterizes the metal 

compounds strongly bound to organic components 

of the soil. Hydrogen peroxide in an acid 

environment is an active oxidant of organic substances in the soil. According to different 

authors, this treatment oxidizes 80–95% of the organic substances in the soil [5, 6]. Isolation 

of these forms was preceded by the displacement of the equilibrium-exchangeable metal ions 

bound to carbonates and nonsilicate compounds of iron, aluminum, and manganese [2]. 

The AAB with pH 4.8 presumably solubilizes all the potentially exchangeable ions, among 

which are those retained by organic substances as the main carriers of exchangeable positions 

in the soil exchange complex [7]. Metal compounds extractable by an AAB + EDTA solution 

are classified among the potentially mobile forms. Along with the exchangeable ions, these 

compounds include the organomineral complexes of metals. Solov'ev [4] and McLaren and 

Crawford [8] also believe that the treatment of soils with this mixed reagent predominantly 

solubilizes the metals loosely bound to organic substances. 

We calculated the contents of the following groups of compounds [9]: 

(1) metal compounds strongly bound to organic and mineral soil components (from the 

difference between the total metals 

in the soil and their potentially mobile compounds), and 

(2) metal compounds bound in complexes with organic matter (from the difference 

between the contents of metals in the AAB + EDTA and AAB extracts). 

The relationships between the analyzed metal compounds are shown in Fig. 1. 

Vol. 3 Page - 168 -

Figure 1: The scheme of metal compounds distribution bound to organic matter 


Lead, copper, and zinc interacted with the soil organic matter when applied to an ordinary 

chernozem in a pot experiment. Two years after the treatment, an appreciable part of the 

metals applied was found in the organic substances, predominantly in a loosely bound state. 

These organic substances were supposed to be organomineral complexes, the formation of 

which resulted in the partial destruction of humic acid molecules. In the contaminated soils, 

the organomineral compounds made up about half the total content of metals in the organic 

matter; in the uncontaminated soils, their share was no more than 10% (Table 2). 

Table 2. Contents of Pb, Cu, and Zn in the organic substances and 

the potentially mobile compounds 

(mg/kg, above the line; % of the total content in the 

soil, under the line) 

Experimental 

Pb Cu 

Zn 

treatment 1 2 1 2 1 2 

Control 

100 mg/kg 

6.8/29 

57.4/49 


0.8/4 

13.0/10 

4.4/10 

55.7/40 

0.5/1 

13.9/10 

1.3/2 

6.4/4 

0.7/1 

8.0/5 

300 mg/kg 

156.0/49 89.0/18 131.0/38 76/17 21.6/6 156/12 

Note: (1) content of metal strongly bound to organic matter; (2) potentially mobile metal compounds 

The series of metals arranged in accordance with their content in the potentially 

mobile 

organomineral compounds is similar to that found for the metals strongly bound to organic 

matter: Pb > Cu > Zn. humus substances. This series also corresponds to that of the metal ion 

radii values (Pb > Cu > Zn), which suggests that the ion size of the metals affects their 

retention by the organic substances. 

It is notable that, in the contaminated soils, the content of zinc in the potentially mobile form 

was even higher than that in the compounds strongly bound to organic matter. This could be 

an artifact, in part, caused by the transition of metal ions retained by the amorphous Al and 

Vol. 3 Page - 169 -

Mn compounds (if they were not completely removed at the pretreatment stage) into the AAB 

+ EDTA extract, along with the organomineral zinc compounds. In fact, zinc predominantly 

accumulated in the sesquioxide-bound fractions of the contaminated soils. 

In the control treatment, the content of metals in the AAB + EDTA extract varied from 1% of 

the total content for Cu and Zn to 4% for Pb. The increase in the content of potentially mobile 

compounds in the contaminated soils depended not only on the nature of the metal and the 

rate applied, but also on its application mode. At the separate application, the share of 

potentially mobile Pb and Cu, which are more active complexing agents, increased from 3– 

6% of the total content at a rate of 25 mg/kg 

to 17–18% at 300 mg/kg; the share of Zn 

increased from 3–12%. 



This work was supported in part by the Ministry of education and science of Russian 

Federation (project nos. № 2.1.1/3819). 

References 

1. T.M. Minkina, G.V. Motuzova and O.G. Nazarenko, Interaction of heavy metals with the organic 

matter of an ordinary chernozem, Eurasian Soil Sci., 2006, № 7, P. 702–710. [in Russian]. 

2. A. Tessier, P. G. C. Campbell and M. Bisson, Sequential Extraction Procedure for the Speciation 

of Particulate Trace Metals, Anal. Chem., 1979, 51, 844–850. 

3. N.K. Krupskii and A.M. Aleksandrova, Determination of Mobile Forms of Trace Elements, 

in 

Trace Elements in the Life of Plants, Animals, and Humans, Kiev, 1964, pp. 34-36 [in Russian]. 

4. Laboratory Manual for Agricultural Chemistry, Ed. by V.G. Mineev, Mosk. Gos. Univ., 

Moscow, 

1989, [in Russian]. 

5. N.G. Zyrin, 

G.V. Motuzova, V.D. Simonov and A.I. Obukhov, Trace Elements in Soils of 

Western Georgia, in Forms of Trace Elements in Soils, Moscow, 1979, pp. 3–160 [in Russia n]. 

6. J.A. Omuite, Sodium Hypochlorite Treat ment for Organic Matter Destruction in Tropical Soils of 

Nigeria, Soil. Soc. Am. J., 1983, 44, 878–888. 

7. G.V. Motuzova and N.Yu. Barsova, Reserve of Mobile Metal Compounds 

in Carbonate-Free Soils 

and Its Determination, 

in 

Proceedings of the II International 

Conference " Heav y Metals, 

Radionuclides, 

and Biophilic 

Elements in the Environment", 

Semipalatinsk, 

Kazakhs 

tan, 2002, 

Vol. 

1, pp. 173–178. 

8. R.G. McLaren and D.W. Crawford, Studies on Soil Copper: 1. The fractionation of copper in 

soils, J. Soil Sci., 1973, 4, p. 172. 

9. D.L. Ladonin and S.E. Margolina, Interaction between Humic Acids and Heavy Metals, 

Pochvovedenie, 1997, No. 7, 806–811 [Eur. Soil Sci. 30 (7), 710–715 (1997)]. 

Vol. 3 Page - 170 -

Concentrations of Iron in the Interactions of Some Acid Ones 

Organic with Minerals 

Cassia Fernanda Domingues Bassan a , Ademércio Antonio Paccola b , Pedro de Magalhães 

b 

Padilha 

a University of Marília, Department of Agrarian Sciences; b Department of Natural resources 

UNESP-Botucatu; c Department of Chemistry UNESP-Botucatu. Brazil 

E-mail: cfbassan@unimar.br 



The external and superficial portion of the terrestrial crust is formed by several types of rocky 

bodies, subjects to conditions that alter its physical form and chemical composition, that is to 

say, the disintegration 

and decomposition. In certain cases, organic acids formed by the 

decomposition of the organic matter, aid in the chemical processes of meteriorization, 

together with other chemical factors and 

with the alteration of original minerals in other 

secondary 

ones due to the temperature variation, oxide-reduction reactions and chemical 

substances secreted 

by micro-organism and unicellular alga. 

The iron meets in the soil in appreciable amounts in the primary minerals, as in the 

ferromagnesians and in the biotite; in the accessories as ilmenite (FeTiO 3), magnetite (Fe3O 4) 

e pyrite (FeS2); in the secondary minerals, as in the hydrated oxides (limonite – Fe2O3.nH2O; 

turgite – 2Fe2O3.H2O e goethite – Fe2O3.H2O). It also happens in having composed organic 

(humate of Fe), in salts, as phosphate and in small amounts, as exchangeable ion and in the 

solution. The Fe it is one of the most abundant elements of the terrestrial crust (about 5,1% ), 

being just overcome by the oxygen, for the siliceous and for the aluminum. In the soils, 

however, it can suffer extreme variations. The iron is absorbed in the ferrous or ferric forms. 

Deficiencies of iron have been associated the soils of high pH, being common in calcareous or 

alkaline soils. "Results of the conversion of the Fe 

and it reaches a 

2+ to Fe 3+ and subsequent precipitation as 

Fe(OH) 3 ". The solubility of the iron 

in the soil is, largely, controlled by the solubility of the 

hydrated oxides of Fe 3+ . The Fe 3+ inorganic in solution it varies with the pH 

minimum between 6,5 and 8,0. The species hydrolytic constitutes most of the ions Fe 3+ in 

solution. Above pH 8,0 Fe(OH) 4- it is the predominant ion. The activity of the ions of Fe 3+ in 

the solution of the soil is difficult of being calculated by virtue of the great amount of 

colloidal iron. It is increased that the presence of the natural quelates in the soils. The activity 

of the Fe 3+ in solution decreases 1000 times for each increase of an unit of pH. Where the 

drainage and the aeration are good the ferric compositions they prevail and where is 

Vol. 3 Page - 171 -

inadequate they are formed more soluble ferrous compositions. In addition to the effects 

of 

the oxygen on the state of oxidation of the Iron, it should be increased 

that certain bacterium 

are capable to accomplish the transformation of the Fe y 

microorganisms and the CO2 liberated by of the plants, they can contribute to the 

so 

2+ in Fe 3+ . Organic acids produced b 

the roosts 

lution of the precipitate iron. 

The present work studies the behavior of minerals of iron that 

composes part of the soils of 

the 

humid tropical areas and its interaction with some organic acids. 



The used minerals were: hematite (Fe2O3), goethite (HFeO2) e magnetite (Fe3O4) that 

commonly they happen in areas humid intertropical, being coming of the following Brazilian 

areas: Itabira-MG, Botucatu-SP and Registration-SP, respectively. 

Prepare of the samples: The minerals were triturated and sifted separately in mesh of 

0,05mm. It leaves of the mineral samples they were used in I prepare it of sheets for 

mineralogical determinations for diffratometryc of ray-X and other part, 1 g for sample, was 

conditioned to react with organic acids, separately. For a collection of glass flasks with cover, 

properly sterilized, 1g of mineral were transferred, 0,5g of inoculate * and 250 mL of solution 

of organic acid 0,02 mol.L -1 . 

The experiment was driven under weekly soft agitations for 53 days. 

Experimental determinations: It leaves of the samples of minerals they were examined with 

relationship its composition for Diffractometryc of Ray-X (XRD), seeking the confirmation of 

the studied mineral. It leaves of the solutions they suffered digestion with acid nitric and 

peroxide of hydrogen after 53 days, and they were analyzed in ICP – Inductively Coupled 

Plasma. 


The largest extracted concentration of iron was of the oxide of iron goethite for the oxalic 

acid, followed by the mineral magnetite for the citric acid and the hematite for the oxalic acid. 

*inoculate: Solution microbiological inoculate with extract of soil of earth purple (TE) under forest vegetation, according to 

NISHIGUCHI (1999), presenting a relationship respectively C:N:P:K 250:1:0,2:0,2, for the microorganisms development. 

Vol. 3 Page - 172 -

Table 1: Concentrations (mg.Kg -1 ) of iron (Fe) presents in the extracts of organic acids and water, 

determined by ICP 

ÁCIDS e WATER 

MINERALS Acetic Butiric Citric Fenic Latic Malic Oxalic Propionic Tanic Water 

Hematite 2,15 1,48 80,53 5,18 56,00 65,73 2335,36 1,73 116,02 23,18 

Goethite 17,90 57,85 659,93 451,18 177,88 171,65 12076,88 29,73 1487,52 101,78 

Magnetite 31,60 28,20 4613,00 61,70 1017,20 1229,20 1798,28 116,33 2955,00 11,10 

-, below or very close of the limit of detection of the technique (0,004 mg.Kg -1 ) 

Figure 1 – Concentrations of Fe (mg.Kg -1 ) extracted or solubilized for the organic acids and 

water, determined by ICP 



The results suggest that, in most of the systems, it happened the quelatization of the metallic 

ions for the organic molecules, proposing the formation of a salt of the respective 

minerals; 

the particularity of acid rights in the solubilization of the iron, if showing more efficient those 

with more than a carboxyl a and that they present hydroxyls, that is to say, with larger amount 

of hydrogen you ionized and larger carbonic chain. 

References 

1. CHAPMAN, 

P. M. et al. Evoluation of bioaccumulation factors in regulating metals. 

Environmental Science & Technology News, 

v. 30, p. 448-452, 1996. 

2. DANA, J. D. Manual de mineralogia. 

Rio de Janeiro: Ao Livro Técnico, 1969, p. 271. 

3. EIRA, P. A.; CARVALHO, P. C. T. A decomposição da matéria orgânica pelos microrganismos 

do solo e sua influência nas variações de pH. Revista de Agricultura, v. 45, p. 15-21, 1970. 

4. 

HART, B. Trace metal complexing capacity of natural Waters: a review. Environmental 

Technology Letters, v. 2, p. 95-110, 1981. 

5. 

MALCOM, R. The uniqueness of humic substances in each of loll, stream and marine 

environments. Analytical Chemical Acta, v. 232, p. 19-30, 1990. 

6. 

NISHIGUCHI, I; PACCOLA, A. A. Estudo da degradação de resíduos de endosulfan em palha 

de café (Coffea arábica L.. 1999. 39f. Dissertação (Mestrado em Agronomia) – Faculdade de 

Ciências Agronômicas da Universidade Estadual Paulista de Botucatu, Botucatu, 1999. 

7. ROCHA, J. C. et al. Relative liability of trace metals complexed in aquatic humic substances 

using ion-exchanger cellulose-htphan. J. Brazilian Chem. Soc., v. 8, p. 239-243, 1997. 

Vol. 3 Page - 173 -

A Fluorescence Study of Adsorption Mechanisms of Flubendiamide onto 

Humic Acids Ivana Cavoski 

a 

, Valeria D’Orazio eo ian 

b* , T doro M o b 

a 

CIHEAM-IAM B, Ist. Agron. Mediterr. di Bari,Via Ceglie 9, 70010, Valenzano, Bari, Italy; 

b 

Univ. of Bari, Dip. Biol. Chim. Agrof. Amb ., Via G. Amendola 165/A, 70126, Bari, Italy 

E-mail: dorazio@agr.uniba.it 


Flubendiamide, N0-[1,1-dimethyl-2-(methylsulfonyl)ethyl]-3-iodo-N-{4-[2,2,2 tetrafluoro-1- 

(trifluoromethyl)ethyl]-0-tolyl} phthalimide is a powerful insecticide belonging to a new 

chemical class (the phthalic acid diamides), widely used against lepidopteran pests on a large 

variety of annual and perennial crops. Its residues and metabolite, the desiodo flubendiamide, 

were determined in a number of crops. Flubendiamide 

is almost insoluble in water, and since 

soils exhibit a marked affinity for hydrophobic organic compounds, they exert an essential 

role in controlling the environmental fate 

of these chemicals. In addition, soil sorption of most 

hydrophobic 

organic compounds is directly related to soil organic matter (SOM) content and 

especially to its humic fractions (HA). 

In facts, they show a large reactivity 

towards these 

compounds, mainly as a function of their functional 

groups as well as their molecular and 

structural arrangements [1, 2]. In this study, fluorescence spectroscopy was utilized to 

investigate on the physico-chemical mechanisms involved in flubendiamide adsorption onto 

HAs of different origin. 



Flubendiamide (FLU) (CAS N°: 272451-65-7), purity 

98%, was purchased from Sigma 

Aldrich (Steinheim, 

Germany) and used to prepare the initial working solution (1 mg/mL in 

ACN) was prepared. The two humic acids used in this study, were isolated from a) a graybrown 

podzolic soil (Normandy, France) according 

to 

the International Humic Substances Society (IHSS) 

procedures (HA S); and purchased from b) the IHSS, 

(lot 1S101H), Suwannee River (HA SR). A stock 

solution of 5 mg HA in 50 mL 0.05M NaOH at pH 8 

was prepared for both HAs; 4 mL of each HA solution 

Figure 1: Flubendiamide structure were added with 0.05, 0.1 and 0.2 mL of FLU solution, 

respectively FLU1, FLU2 and FLU3, were mechanically shaken for 24 hours at room 

T. A 

blank solution for each HA was prepared as control at the same conditions. Both controls and 

Vol. 3 Page - 174 -

interaction solutions were directly analyzed by fluorescence spectroscopy, using a Perkin- 

Elmer (Norwalk, CT) LS 55 luminescence spectrophotometer 

equipped with the WinLab 

4.00.02 software for data processing. Total luminescence spectra, in the form of excitatione 

mission matrices (EEMs, contour maps), were recorded over the emission wavelength range 

fro m 300 to 600 nm, increasing sequentially by 5 mm steps the excitation wavelength from 

2 50 to 500 nm. The EEM plots were generated as contour maps 

from spectral data by using 

Surfer 8.0 software 

(Golden Software, Inc., 2002, Golden, CO). 



Data reported on Table 1 indicate significant differences between the two HA. On the whole, 

the higher O content measured for the HA SR is mainly due to the occurrence of acidic 

functional groups, whereas in the HA S the 

Table 1: Elemental composition (g kg 

oxygen-containing groups seem to be related to 

non acidic groups, such as hydroxyl, methoxyl, 

ethers, ketones, and quinones. Further, HA S 

sample appears to be characterized by a greater 

aromaticity degree and lower polarity with 

respect to the first one, with higher H/O and 

lower (O+N) /C ratio. In general, chemical data 

suggest a more aromatic character for the HA S 

−1 ), 

atomic ratios, acidic functional group 

content (cmol kg −1 ) and E4/E6 ratios of the 

examined HAs 

and a mainly aliphatic and acidic character for the 

HA SR, characteristics confirmed by E4/E6 

values. Such properties are expected to result in a 

well different chemical reactivity and residual 

adsorbing capacity of these HAs toward the nonpolar 

flubendiamide molecule. Figures 2 and 3 

show the contour maps (EEMs) of HA S and HA 

SR, respectively, and their interaction products with flubendiamide at different 

concentrations. The EEMs spectra of both HAs show two common peaks, 1 and 2, identified 

by the excitation/emission wavelenght pairs (EEWPs) 

430-440ex/510-522em and 390- 

400ex/476-500em, respectively, and characterized by greater 

relative fluorescence intensity 

( RFI) values in the HA S sample with respect to HA SR one. 

Vol. 3 Page - 175 -


The excitation wavelengths of the 

fluorophore 1 are in the range of those 

generally ascribed to extensively 

conjugated quinones and phenols with an 

elevated polycondensation degree, 

Figure 2: EEMs of HA S and its interaction products 

whereas those of the peak 2 can be 

correlated to components like esculetin 

and/or scopoletin structures [3]. The 

slightly higher values of the HA S EEWP 

can be likely related to a greater 

condensation degree of these aromatic 

groups. Finally, a third additional peak (3) 

appears in the HA SR sample, centered in 

the shorter wavelength region (EEWP 

345-355ex/450-464em) and generally 

ascribed to flavones and isoflavones like 

structures [3]. 

The changes occurring in the EEMs 

spectra of the HA-FLU products suggest, 

for both HAs, the involvement of all 

fluorophores in the interaction mechanism 

at various extent, as a function of FLU 

Figure 3: EEMs of HA SR and its interaction products concentration and HA sample. In 

particular, the peak 1 seems not involved 

in the interaction with the lowest FLU 

concentration, especially in the HA S sample, 

whereas the RFI value decrease and the EEWPs 

shift indicate its involvement with FLU both 

at intermediate and at highest concentration. On 

the contrary, the peak 2 appears involved in the interaction regardless of FLU concentration in 

the HA S, and only marginally in the HA SR sample. Finally, the peak 3 (HA SR) strongly 

appears to interact with FLU for each concentration tested, as remarked by the RFI and 

EEWPs values changes. In order to better evaluate the changes occurring in the EEMs spectra 

of interaction products, the EEMs spectra of FLU molecule dissolved in three solvents with 

different polarity indexes were recorded, and the RFI values changes were evaluated as a 

function of the medium (Fig. 4). As shown in Fig. 4, the RFI value of both FLU peaks 

Vol. 3 Page - 176 -

increases markedly with decreasing medium polarity, 

and this effect is due to the presence of 

CF3 substituent, that lead to an efficient quenching 

Figure 4: EEMs of FLU in acetonitrile (ACN), methanol (MeOH) and ethyl acetate (Ethyl Acet) 

because of the turning of the aromatic amide moiety of FLU (Fig. 1); this 

effect results more 

evident in the HA SR sample because of the polar properties of this latter. Additionally, 

the 

greatest RFI value measured in the sample HA SR FLU 3 can be likely ascribed to decreased 

rotation of the macromolecular aggregate occurring at the highest 

FLU concentration. In the 

case of HA S FLU no significant changes were observed in the RFI values. The FLU 

substituents were not effective, FLU molecule may result entrapped 

inside the HA. 


On the whole, fluorescence data seem to suggest that flubendiamide adsorption can occur 

onto HA as a function both of the aromatic degree by means of hydrophobic bonds, as it 

happened for HA S, and of acidic functional groups availability by means of hydrogen bonds, 

as it happened for HA SR. Further studies may help in revealing further insights on the 

suggested mechanisms. 

References 


1. R.P. Schwarzenbach, P.M. Gschwend, D.M. Imboden, Environmental Organic Chemistry, Wiley, 

New York, 1993. 

2. R.R. Engebretson, T. Amos and R.V. Wandruszka, Environ. Sci. Technol., 30(1996) 990. 

3. O.S. Wolfbeis, in S.G. Schulman SG (Ed.), Molecular Luminescence Spectroscopy, Part I, Wiley, 

New York, 1985, p 167. 

Vol. 3 Page - 177 -

Size Exclusion Characterization of Dissolved Organo-mineral Complexes in 

Soils of the Southern Far East 

Tatiana N. Lutsenko a* , Alexandra S. Volk b 

a Pacific Institute of Geography, Far Eastern Branch of the Russian Academy of Sciences, 

Radio St. 7, 690041 Vladivostok, Russia; b Institute of Chemistry, Far Eastern Branch of the 

Russian Academy of Sciences, 100-letiya Vladivostoka Av. 159, 690022 Vladivostok, Russia 

E-mail: luts@tig.dvo.ru 


The illuviation processes of the dissolved organo-mineral species are quite expressed in soils 

of the Southern Far East. In our research we aim to study DOC relationship with Fe and Al in 

mountain-taiga brown soils of the Sikhote-Aline applying a method of size exclusion 

chromatography (SEC). On the basis of these efforts we expected to determine differences in 

binding properties of DOC exposed to different ecological conditions of synthesis. 



The study area is one of the west slopes of the Sikhote-Aline mountain ridge (South of the 

Russian Far East). Lysimetric waters were collected in zero-tension lysimeters (for the period 

from July to September) below organic and mineral horizons of the main types of mountaintaiga 

brown soils: humic Cambisol (750 and 950 m a.s.l.); and dystric Cambisol (1250 and 

1400 m a.s.l.). The lysimetric waters were filtered through 0,23 μm filters. 

Table 1: Characterization of the lyzimetric waters of mountain-taiga brown soils [1] 

Soil, 

elevation Horizon рН DOC, mM Fe, mM Al, mM 

Humic Сambisol, 750 m А1 5.63 3.45 0.0091 0.0126 

B2hf 5.25 1.47 0.0034 0.0119 

Humic Сambisol, 950 m А1 4.71 4.10 0.0127 0.0167 

В1hf 4.70 2.65 0.0118 0.0210 

Dystric Сambisol, 1250 m А2 4.19 4.86 0.0127 0.0141 

В4hf 5.16 1.28 0.0118 0.0211 

Dystric Сambisol, 1450 m А1А2 4.50 1.92 0.0095 0.0200 

BhfC 5.17 1.06 0.0039 0.0222 

The filtrates were concentrated by freezing and rotary evaporation by a factor of 5-10. 

Fractionation of DOС and study of its binding with Fe and Al were performed on Sephadex 

G-25. A set of standard organic substances was taken for column calibration. Absorbance of 

Vol. 3 Page - 178 -

Conductivity 

DOC, mM 

Fe, mM 

Al, mM 

DOС fractions was measured at 665 and 465 nm to determine E4/E6 ratios. DOC 

concentrations were determined 

by the dichromate technique followed 

by colorimetry. The 

content of Fe was analyzed 

by AAS-method. Aluminum was 

determined by a 

spectrophotometry with the organic reagent anthrazochrom after decomposition of organic 

matter. Potentiometric titrations of DOC fractions were performed in a PC-controlled system 

under constant argon flow. DOC fractions, accumulated in 7 runs of SEC, after rotary 

evaporation 

were dissolved in 10 ml of 0.1M KCl adjusted to pH=10 by addition of 0,1M 

KOH for complete 

solubilization. Prior to titration 

the solution was readjusted to pH=2,5 with 

0,1M HCl to attain complete protonation of functional groups. Results of potentiometric 

titration were processed using the density function method which yields pK-spectra of DOC. 


During fractionation on the Sephadex G-25, the DOC of soil waters is separated into three 

major molecular weight fractions. The first fraction has a high molecular weight (HMW) 


is characterized by a limit of MM > 5000 Da. MW 

for the middle molecular weight (MMW) 

fraction spans 650-1500 Dа; the low-molecular weight (LMW) fraction spans 200-400 Dа. 

200 

100 

0 

100 

10 

150 200 250 

8 

6 

4 

2 

0 

DOC 

100 150 200 250 

Fe 

0,08 

0,04 

0 

100 150 200 

250 

0,2 

0,1 

0 

C, μS/cm 

Al 

100 150 200 250 

Elution volume, ml 

Figure1: SEC of the lyzimetric waters 


Vol. 3 Page - 179 - 

Ratio D4/D6 testifies that the HMW fraction 

contains more aromatic molecules (D4/D6 

5,2-6,2), but MMW fraction (D4/D6 6,3- 

10,5) and LMW fraction (D4/D6 10,3-15,0) 

are more aliphatic. Investigation of the 

degree of dissociation and the 

concentrations of functional groups has 

also 

shown that the fr actions of DOC differ in 

the chemical nature. Potentiometric titration 

data indicated that the content of the 

most 

acidic groups ( pK 2-4) in the HMM 

fr action is al most three times 

higher than in 

the MMW fraction. For the LMM fraction, 

the concentr ation of acidic groups is 

lower 

than the detection limit of the method 

because of insufficient amount of the 

fraction organic matter.

SEC of the natural water samples is complicated by the fact that the peaks of MMW and 

LMW fractions overlap the elution peaks of dissolved salts, which leads to some uncertainty 

in interpretation. However, we can estimate the complexation ability of the HMW fraction of 

DOC since its elution peak does not coincide with the elution of dissolved salts. 

A portion of DOC, Fe and Al is sorbed by Sephadex. We take this fourth fraction into account 

as labile compounds possessing affinity for gel. A share of fraction 4 (Figure 2) is calculated 

by difference between introduced and eluted amounts of DOC, Fe and Al. 

There is a tendency of decreasing of the amount of HMM fraction in the distribution of DOC 

organic horizons with the increase of elevation. This tendency can be also tracked in the 

distribution of dissolved Fe and Al in the organo-mineral forms of its migration. In the 

warmest conditions (elevation 

750 m), DOC consisting of more than 65 % of HMW 

substances migrates. More than 98 % of dissolved Fe and 68 % of Al are associated with this 

fraction. In the colder conditions (elevation 950 m), the amount of HMW fraction goes down 

to 60 %, the quantity of Fe and Al presented by this form simultaneously reaches to 71,3 % 

and 72 %, respectively. The smallest amount of the HMW fraction (53,8 %) is characteristic 

for the most acidic solutions (pH 4,19) of the soil 

at elevation of 1250 m. The solution pH 

also limits the binding of the metals: about 60 % of Fe and 40 % of Al are associated in HMW 

complexes. In connection with the pH of waters 

and, correspondingly, reduction in the 

stability of DOC and Fe complexes, a share of labile 

fraction (4) of the latter exceeds 25 %. 

DOC, Fe, Al, % 

100 

80 

60 

40 

20 

0 

80 

60 

40 

20 

0 

Humic cambisol (750 m) 

A1 

1 2 3 4 

B2hf 

1 2 3 4 


Humic cambisol (950 m) 

A1 

1 2 3 4 

B1hf 

1 2 3 4 

Dystric cambisol (1250 m) 

1 2 3 4 

Figure 2: Distribution of DOC, Fe, Al (% of total) in SEC fractions of soil waters: 1–HMW fraction, 

2–MMW fraction, 3–LMW, 4-fraction of DOC and metals 

absorbed by Sephadex 

Vol. 3 Page - 180 - 

A1 

B4hf 

1 2 3 4 

Dystric cambisol (1400 m) 

A1A2 

1 2 3 4 

Bhfc 

1 2 3 4 

Fractions 

DOC 

Fe 

Al

From studying elevation dynamics of Al speciation, it is possible to see that it, to a lesser 

degree, reflects dynamics of the speciation of DOC than does dissolved Fe, although, on the 

whole, the tendency is similar. Aluminium forms weaker complexes with DOC than Fe, thus 

the dependence of Al behaviour on changes in DOС speciation is not so obvious. 

A stoichiometric ratio Мe:С for the HMW complexes is 3-4 atoms of Fe and 6-10 atoms of Al 

per a chain of thousand carbon atoms. In the course of migration in the soil, the fractions of 

DOC are saturated by Fe and Al and coagulate as well as are sorbed at the surface 

of minerals. 

In the organo-mineral complexes of the lower horizons, absolute shares of metals increase up 

to 4-9 atoms of Fe and 13-45 atoms of Al per a chain of thousand carbon atoms, respectively. 

In the mineral horizons the multiformity of DOC, Fe and Al speciation is more expressed. 

The capacity of DOC to bind Fe and Al becomes less pronounced, their migration is 

supported by fractions of DOС with МW 200-400 Dа as well as fraction of labile, maybe, 

hydrolytic forms. 



From the results of lysimetric waters SEC in the present study, DOC was separated into three 

major fractions. Differences between the DOC production and transformation conditions are 

expressed in different ratios of its fractions and binding Fe and Al. As shown by us earlier, a 

share of humic acids reduces and of fulvic acids grows in composition of DOC from the lower 

to upper parts of the slope. In the elevation series of soils, an acidity of produced acids 

increases which results in increasing the acidity of solutions. As a result of action of these 

factors, basic identified elevation tendency is a reduction in formation of the high-molecularweight 

fractions and intensity of precipitation processes of organo-mineral complexes in the 

soil profile and intensification of processes of their illuviation as the low-molecular weight 

and labile forms. 


The author wish to thank Dr. V.S. Arzhanova, senior researcher of Pacific Institute of 

Geography of FEB RAS for the samples of lysimetric waters and discussion of the data 

obtained. The author thanks Dr. S. Yu. Bratskaya, senior researcher of Institute of Chemistry 

of FEB RAS for the potentiometric titrations of the concentrated SEC fractions of DOC. 

References 

1. V.S. Arzhanova and P.V. Yelpatievsky, Mountain geosystems: geochemistry, functioning and 

dynamics (Sikhote-Aline Mountains, Southern Russian Far East), Dalnauka, Vladivostok, 2005, 

p.68-69 (in Russian). 

Vol. 3 Page - 181 -

Sorption of Pharmaceuticals to Humic Substances 

Hisayo Mori, Tomoya Ohtani, Itsuko Fukuda, Hitoshi Ashida, Nobuhide Fujitake 

Graduate School of Agricultural Science, Kobe Univ., Rokkodai 1, Kobe 657-8501, Japan 

E-mail: fujitake@kobe-u.ac.jp 


Pharmaceuticals and personal care products (PPCPs) in the environment have received much 

attention during the last decade. Approximately 100 compounds have been detected in 

wastewaters, streams, ground-waters, and drinking waters in several countries (e.g.[1]). These 

substances enter the aquatic environment via the effluent of sewage-treatment plants as a 

result of their incomplete removal. Not only in aquatic environment, but some medicines from 

the application of sludge or manure were persistent in agricultural soils [2] and they were 

absorbed by vegetables [3]. Therefore, these compounds may act as allergens for human 

beings, if not actually below the acute toxicity level. In addition, it is concerned 

that PPCPs, 

especially antibiotics, 

could induce drug-resistant bacteria in aquatic or soil ecosystems. 

In this study, we focus the interactions between pharmaceuticals and humic substances. 

Humic substances are ubiquitous in virtually all terrestrial, and coexist with pharmaceuticals 

in environments. Little is known, however, of the behavior of these compounds in the 

presence of humic substances. There is a possibility that the bioactivity of pharmaceuticals is 

changed if they interact with humic substances. So we examined the sorption experiments 

with 4 pharmaceuticals and 10 humic substances isolated from different environmental 

conditions. 



atenolol ibuprofen 

ciprofloxacin fluoxetine 

Figure 1: Structural formulae of 4 pharmaceuticals 

Vol. 3 Page - 182 -

The pharmaceuticals, such as an antihypertensive (atenolol), a nonsteroid anti-inflammatory 

drug (ibuprofen), an antidepressant (fluoxetine 

hydrochloride), and an antibiotic 

(c iprofloxacin) were selected. Humic materials were purchased or isolated from freshwater, 

sediment, peat, and soils using the quasi-IHSS methods, and 1 aquatic fulvic acid, 7 soil 

humic 

acids, and 2 soil fulvic acids were obtained. All humic samples were characterized by 

liquid-state MR spectroscopy. 

13 C N 

The organic carbon normalized sorption coefficient (Koc) was determined according to the 

fluorescence quenching method [4]. The experimental aqueous phase was prepared with fixed 

pharmaceutical concentrations and varied concentrations of humic substances (1–5 mg C L -1 ) 

in phosphate buffer, pH 7.2, and 0.01 ionic strength. The final concentrations of 

pharmaceuticals used were as follows: atenolol, 500 μg L -1 ; ibuprofen, 700 μg L -1 ; fluoxetine, 

1500 μg L -1 ; ciprofloxacin, 150 μg L -1 . The solutions were shaken for 24 hours at 25 ºC in the 

dark and the fluorescence intensity was measured by Jasco FP-6200 spectrofluorometer. 

The excitation / emission wavelengths used were 230/302 nm for atenolol, 230/293 nm for 

ibuprofen, 230/294 nm for fluoxetine, and 270/410 nm for ciprofloxacin with slit widths of 5 

nm. The correction factor for inner filter effects and the concentration quenching of humic 

materials were calculated with absorbance values of excitation and emission wavelengths for 

each solutions [4]. The corrected fluorescence intensity of pharmaceuticals in the presence of 

humic substances (F) and absence (F0) was used in the Stern-Volmer equation, which 

described the decrease in fluorescence in the presence of quencher (humic substances). Values 

of F0/F formed linear plots against concentration of humic substances, with the Koc values 

calculated from 

the slopes. 

Fo/F 

Stern-Volmer equation: F0/F = 1 + K [Humic substance] 


The Stern-Volmer plots for fluorescence 

quench- 


ing of atenolol with 10 humic substances are 

given in Fig. 2. It shows linear relationships for 

Concentration of humic substances (mgC L -1 ) 

any combinations, and similar results were Figure 2: Stern-Volmr plot for 

quenching atenolol with 10 

obtained 

on all the other target pharmaceuticals 


(not 

all shown in Figure). The differences of slopes between humic materials indicate that 

Vol. 3 Page - 183 -

they have different binding affinities to pharmaceuticals respectively. 

LogKoc 


Table 1: Log Koc between 10 humic substances and 4 pharmaceuticals 

Soil humic acids 

Soil fulvic acids 

Aquatic fulvic acid 

Suwannee NOM a 

a Reference [5] 

atenolol ibuprofen fluoxetine ciprofloxacin 

Figure 3: Log Koc between 10 humic substances and 4 pharmaceuticals 

The LogKoc values between humic materials and pharmaceuticals shows in Table 1. They 

ranged widely from 4.42 to 6.82 for ciprofloxacin, while ranging from 4.8 to 5.86 for the 

other three drugs. These results suggest the different ranges of values should depend on the 

chemical properties of pharmaceuticals used. Then, the most of values were plotted in the 

range from 5 to 6 (Fig. 3) and comparable binding affinities to PAHs (LogKoc=3.7- 5.4) ( e.g. 

Vol. 3 Page - 184 -

[6]), whereas some exceeded these values. In addition, our data were higher than the values of 

Suwannee 

river NOM with atenolol, ibuprofen, and fluoxetine (shown in Fig. 3 as the 

asterisk) 

[5]. 

ll humic samples were characterized by liquid-state 13 A 

C NMR spectroscopy (Fig. 4), and the 

correlation 

coefficients between LogKoc and the distribution of carbon species in humic 

substances 

were calculated. The result indicated the LogKoc values correlated with the Aryl, 

O-Aryl, and alkyl carbons (e.g. Aryl carbon: r 2 = 0.86, O-Aryl carbon: r 2 =0.78, and alkyl 

r 2 carbon: = -0.81, for ciprofloxacine). 


Aquatic fulvic acid 

Soil fulvic acids 

Soil humic acids 


Figure 4: The distribution percentage of carbon species by py 

13 C NMR spectrosco 

Ten humic 

substances were interacted with 4 pharmaceuticals such as atenolol, 

ibuprofen, 

fluoxetine, and ciprofloxacin. The LogKoc 

values between humic materials and 

pharmaceuticals 

were a comparable level of interactions with PAHs, whereas some 

combinations 

led to exceed these values. The result indicated the Koc values 

correlated with 

th e Aryl, O-Aryl, and alkyl carbon moieties of humic substances. 

References 

1. 

B. Halling-Sorensen, S.N. Nielsen, P.F. Lanzky, F. Ingerslev, H.C. Holten Lutzhoft, and S.E. 

Jorgensen, Chemosphere., 36(1998) 357. 

2. M. Rabolle and N.H. Spliid, Chemosphere, 40 (2000) 715. 

3. K. Kumar, S.C. Gupta, S.K. Baidoo, Y. Chamder, and C.J. Rosen, J. Environ. Qual., 34 (2005) 

2082. 

4. T.D. Gauthier, E.C. Shane, W.F. Guerin, W.R. Seitz, and C.L. Grant, Environ. Sci. Technol., 20 

(1986) 1162. 

5. H. Yamamoto, A. Hayashi, Y. Nakamura, and J. Sekizawa, Environ. Sci., 12 (2005) 347. 

6. I.V. Perminova, N.Y. Grechishcheva, and V.S. Petrosyan, Environ. Sci. Technol., 33 (1999) 3781. 

Vol. 3 Page - 185 - 

(%)

Influence of Aromaticity Degree on the Aggregation of Humic Substances 

Martin Drastík a , Jiří Kučerík a , Oldřich Zmeškal a , Anna Čtvrtníčková a , František Novák b 

a Faculty of Chemistry, Brno University of Technology, Purkyňova 118, 612 00, Brno, Czech 

Republic; b Biology Centre AS CR, v. v. i., Institute of Soil Biology, Na Sádkách 7, 370 05 

České Budějovice, Czech Republic 

E-mail: xcdrastik@fch.vutbr.cz; kucerik@fch.vutbr.cz 


Humic substances are for a long period classified according to the isolation procedure (which 

reflects also the solubility in water according to pH) into three groups – humic acids (HA), 

fulvic aci ds (FA) and humin. 

Distinguishing between HA and FA is basically just formal 

because 

as shown several times, there is no strict line which separates these two groups. As 

published by Kučerík 

et al. [1] even the aggregation patterns are very similar and can be 

described 

by the same mathematical apparatus. 

Aggregation of HAs in solutions was studied intensively 

by various techniques such as small- 

angle neutron scattering, small-angle 

X-ray scattering, turbidimetry or scanning electron 

microscopy 

and consequently utilizing fractal analysis [2, 3]. However, those techniques have 

undisputable 

limitations which are associated mainly with the limited concentration range and 

inconvenient possibility of changing the concentration or composition of measured solution 

during 

running experiment. Recently, it has been demonstrated that this problem can be 

overcame by the coupling of ultrasonic spectrosc opy and consequent fractal analysis [1]. The 

purpose of this work is to exten d the recent results which suggested the correlation between 

aggregation behavior represented by fractal dimension 

of sodium humates and fulvates and 

their chemical composition. 


Humic and fulvic acids were isolated from individual soil horizons of long-term research stands 

by standard procedure and recommendations published in [4] were taken into account. Freeze- 

+ 

dried samples 

were converted to Na salts by titration to pH 7.2 using 0.1 M NaOH. 

To monitor ultrasonic velocity High Resolution Ultrasonic Spectroscopy HRUS 102 device 

(Ultrasonic-Scientific, Dublin, Ireland) was employed. All measurements were carried out the 

same way as published in [1, 5 ,6]. The ultrasonic velocity (U) was measured in the 

concentration range from 0.001 to 3.5 g/L. For easier observation of potential interactions, the 

concentration increment of ultrasonic velocity (I) was determined using the relation published 

in [5], i.e. I=(U–U )/(U mρ ). 

0 0 0 


Vol. 3 Page - 186 -

3. 

Results and Discussion 

Measured data were processed as publishe d elsewhere [1] utilizing non-linear fitting. 

Obtained 

parameters will be correlated with primary characteristics (elemental composition, 

ar omaticity, carboxyl carbon) and treated by fractal dimension analysis developed by Drastík 

et al. [6]. Figure1 represents the dependence of increment of ultrasonic velocity I (non-linear 

fitting included) and fractal dimension D on concentration. It was already demonstrated that 

fractal dimension D can reveal the differences in the way of aggregates formation for various 

samples. The validity of observations done in references [1] and [6], where samples of IHSS 

were analyzed, will be examined. 

Figure 1: Dependence of fractal dimension D and increment of ultrasonic velocity I on concentration 


The financial support of Ministry of Education of the Czech Republic, project MSM 

0021630501. 


References 

1. J. Kučerík, M. Drastík, O. Zmeškal, A. Čtvrtníčková, WSEAS Transactions on Environment and 

Development, 5 (2009) 705. 

2. J. Rice, J.S. Lin, Environ. Sci. Technol. 27 (1993) 413. 

3. N. Senesi, F.R. Rizzi, P. Dellino, P. Acquafredda, Colloid Surface A, 127 (1997) 57. 

4. R.L. Malcolm in B. Allard, H. Borén and A. Grimvall (Ed.), Humic substances in the aquatic and 

terrestrial environment, Springer Berlin/Heidelberg, 1991, Chapter 16. 

5. J. Kučerík , D. Šmejkalová, H. Čechovská, M. Pekař, Org. Geochem., 38 (2007) 2098. 

6. M. Drastík, A. Čtvrtníčková, O. Zmeškal, J. Kučerík, Energy and Environmental Engineering 

Series, WSEAS Press, 2009, 163–168. 

Vol. 3 Page - 187 -

Aggregation of Humic Acids in Solution. Vapor Pressure Osmometry, 

Conductivity and Mass Spectrometric Study 

E. M. Peña-Méndez a* , D. Fetsch b b, c 

, J. Havel 

a 

Department of Analytical Chemistry, Nutrition and Food Chemistry, Faculty of Chemistry, 

University of La Laguna, Campus de Anchieta, 38071 – La Laguna, Tenerife, Spain; 

b 

Department of Chemistry, Faculty of Science, Masaryk University, Kotlarska 2, 61137 

Brno, Czech Republic; c Department of Physical Electronics, Faculty of Science, Masaryk 

University, Kotlarska 2, 61137 Brno, Czech Republic 

E-mail: empena@ull.es 


During last two decades there was a considerable change in view of the structure and 

molecular weight of humic acids (HA). HA were found to be supramolecular 

associations/aggregates of relatively low molecular weight components [1,2]. The knowledge 

of aggregation is quite fundamental because its important role in the processes taking place in 

the environment. 

The aim of this work was to evaluate the possibilities to apply non invasive methods such as 

vapour pressure osmometry (VPO), conductivity and matrix assisted laser 

desorption/ionization time of flight mass spectrometry (MALDI) for studying the aggregation 

of HA in solution and to elucidate the process. 


The Vapor Pressure Osmometer from Knauer (Berlin, Germany) was used to measure vapor 

pressure of solutions. Conductometry 

experiments were performed on the conductometer OK- 

104 of Radelkis (Budapest, 

Hungary). Mass spectra were measured via Laser Desorption 

Ionization (LDI), i.e. without the use of any matrix. Time of Flight (TOF) MS measurements 

were carried out 

on the AXIMA CFR from Kratos Analytical (Manchester, UK) mass 

spectrometer equipped with the nitrogen laser (wavelength 337 nm) and spectra were 

measured either in the linear or positive ion modes. 



Vapour pressure osmometry measurements of HA aqueous solution prove aggregation of HA 

when increasing their concentration (Fig. 1); after the diluting of the solutions the aggregation 

was shown as reversible. It is also evident from 

Fig. 1 that several aggregates are formed. 

Vol. 3 Page - 188 -

Aggregation, due to hydrophobic interactions of alkyl groups, π–π interactions, hydrogen 

bonds, and supramolecular interactions, etc. explains for decades suggested 

high molecular 

weights of HA. 

VPO Res 

ist ance Difference 

Fig. 1 Vapour pressure osmometry 

measurements of HA aqueous solution 

as a function of HA concentration. 

Humic acid aggregation was also 

studied by MALDI TOF MS. The 

aggregates, due to weak interactions 

through the different functional 

groups present in their structure (hydrophobic and/or π–π, etc.), are broken down by the 

action of the laser. The mass spectra are complex, showing many components with low m/z 

values in the range 

~200–1000 Da while mostly no peaks are observed for m/z values greater 

than 1000 Da. 


The results obtained by VPO, conductivity and confirmed by MALDI TOF MS show that 

studied HA consist of a mixture of numerous 

number of compounds with much lower 

molecular weight than still often suggested in the literature. Also the direct soil analysis by 

MALDI yield almost the same mass spectra like those for isolated HAs. 


This work was supported by the Grant Agency of the Czech Republic (project KAN 

101630651 and the Ministry of Education, Youth and Sports of the Czech Republic (projects 

MSM0021622411 and LC06035). E.M. Peña Méndez thanks for the partial support from the 

University of La Laguna. 

References 

HA (mgl-1 HA (mgl ) -1 ) 


1. J. Havel, D. Fetsch, E.M. 

Peña-Méndez, P. Lubal, P. and J. Havliš. In Understanding and 

Managing Organic Matter in Soils, Sediments and Waters (Swift R.S. and Spark K.M., Eds.). 

IHSS, Australia. 1991. 

2. E.B. Kujawinski, M.A. Freitas, X. Zang, P.G. Hatcher, K.B. Green-Church, and R.B. Jones. Org. 

Chem. 33 (2002) 171. 

Vol. 3 Page - 189 -

Sorption of Silanol-Modified Humic Acids onto Different Solid Supports 

Including Silica Gel, Clay and Sand 

Ivan V. Dubinenkov a , Alexander B. Volikov a , Vladimir A. Kholodov b , Evgeny M. Garanin a , 

Sergey A. Ponomarenko c a* 

, Irina V. Perminova 

a 

Department of Chemistry, Lomonosov Moscow State University, 

119991, Moscow, Russia; 

b 

Dokuchaev Soil Institute, Pyzhevskiy per. 1, 119017 

Moscow, Russia 

c 

Institute of Synthetic Polymeric Materials of the Russian Academy of Sciences, 117393 

Profsoyuznaya street 70, Moscow, Russia 

E-mail: iperm@org.chem.msu.ru 


Restoration of the polluted and degraded soil belongs to crucial environmental problems of 

the 21 st century. It is well-known that humic substances (HS) play a key role in soil fertility 

by forming stable microaggregates which, retain water and structurize soil [1]. In addition, HS 

regulate geochemical fluxes of metals due to high chelating ability and play protective role in 

the polluted environments 

by binding heavy metals and organic pollutants into non- 

bioavaileble complexes. 

In our previous research, we have demonstrated that the directed modification of HS is a 

powerful tool for manufacturing humic materials with controlled properties [2]. In this study 

we demonstrate that incorporation of silanol groups into the structure of HS allows for 

production of the derivatives that show considerable promise as soil conditioners. 

This is due 

to intense formation of stable soil aggregates stimulated by an increased content of soil 

organic matter. The soil conditioners of the art will bring about formation of the stable 

organic coating on the soil mineral surfaces. 

The goal of this work was to estimate sorption of silanol-modified HS derivatives on the 

different inorganic supports including silica gel and soil minerals – quartz sand and bentonite 

and kaolinite clay. 



Leonardite humic acids (CHP) isolated from the commercial potassium humate (Powhumus, 

Humintech Ltd., Germany) were used for all modifications. 3-amino-propyltriethoxy-silane 

(APTES) was used for treatment 

of CHP. The choice of APTES was provided by the presence 

of reactive amino groups in its structure which can yield amide bonds upon reaction with 

carboxyl and carbonyl groups [3, 4]. In addition, APTES is commercially available 

organosilane suitable for preparative production of the corresponding derivatives. 

Vol. 3 Page - 190 -


Silanol derivatives of CHP were obtained by its condensation with APTES. The reaction was 

run at three different APTES-to-humics ratios, nominally: 0.2, 0.5, and 

1.0 g of APTES per g 

o f CHP. These ratios corresponded to different extents of modification associated with 

carboxyl groups available within the humic backbone. The corresponding 

amounts of APTES 

were 

calculated in accordance with 1:1 stoichiometry of modification reaction on the basis of 

the amount of COOH groups present in the CHP sample (3.8 mmol per g). Depending on 

modification degree, the corresponding samples were designated CHP-APTES-20, CHP- 

APTES-50, and CHP-APTES-100. One sample was synthesized with tetraethoxysilane (TES) 

substituting 10% of APTES out of the amount 

needed for 100% modification. TES was added 

for increasing the number of silanol groups in the sample. The corresponding sample was 

designated CHP-APTES-TES. The reaction was carried out at 130-140 °C for 5 hours. 

Sorption of the modified HA was carried out on silica gel, quartz sand, bentonite and kaolin 

clays. Quartz sand and silica gel were used as obtained from the manufacturer. Clays were 

thoroughly soaked in 0.001 M CaCl2 prior to use in the experiments for saturation with Ca 

50 mg, 100 mg and 500 mg for silica, clays and sand, 

respectively. The total volume of experimental solution was 10 mL, concentration of humic 

2+ . 

A weight of sorbent accounted 

samples was set in the range from 0.1 to 4 g/L. All sorption experiments were conducted in 

phosphate buffer (0,03 M, pH 6.0). Equilibrium time was 24 hours. 


To achieve maximum sorption affinity of the silanol modified humic derivatives for the 

different Si-containing supports carrying hydroxyl groups, the sorption experiments were run 

at pH 6. This pH was shown to provide for the maximum 

binding of silanol-HS to silica gel. 

This finding corroborates well the reported data on the optimum conditions for formation of 

siloxane bonds between silanol groups [5]. 

Figure 1 shows sorption isotherms for the silanol derivatives samples with different 

modification degree on silica gel. As it was expected, the maximum sorption was observed for 

the sample with maximum modification degree (CHP-APTS-100), which displayed the 

maximum affinity for silica gel surface. 

Vol. 3 Page - 191 -

q, mg/g 

350 

300 

250 

200 

150 

100 

50 

0 


0 0,5 1 1,5 2 2,5 3 3,5 

Ceq, g/l 

. 

Figure 1: Sorption isotherms of the silanol derivatives of leonardite HA onto silica gel. 

▲ - CHP-APTES-100; ● - CHP-APTES-TES; ■ - CHP-APTES-50; ♦ - CHP-APTES-20 

The CHP-APTES-100 isotherm fits to Lengmuir isotherm model. The sample showed high 

affinity and sorption activity within a wide range of concentrations. At the same time, the 

other samples tested: CHP-APTES-50, CHP-APTES-20 and CHP-APTES-TES displayed Sshaped 

isotherms. This might indicate differences in filling in the silica surface with the 

humic derivatives. It should be noted, however, that 100% modified sample showed a rather 

low water solubility. The optimal 

ratio between sorption affinity and solubility was observed 

for 50%-modified sample – CHP-APTES-50. 

Given these properties of CHP-APTES-50, it was selected for conducting comparative 

sorption studies on different solid matrices. Figure 2 shows sorption isotherms for CHP- 

APTES-50 onto different sorbents. It can be seen that it displays the maximum sorption 

affinity for silica gel and activated bentonite clay. The sorption isotherms on clays were of 

Langmuir shape, while the sorption isotherms on silica gel were of S-shape. It might be 

indicative of different sorption mechanisms. The sorption values at maximum concentration 

of the silanol-derivative tested – 4 g/L - reached about 100 mg/g for silica gel and activated 

bentonite. For non-activated bentonite, it was much less and accounted for 50 mg/g. For 

kaolinite it did not exceed a value of 30 mg/g. There was no considerable sorption of the 

tested derivative observed on quartz sand. This was true for all other samples. 

Vol. 3 Page - 192 -

q, mg/g 

160 

140 

120 

100 

80 

60 

40 

20 

0 

0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 

Figure 2. Sorption isotherms of CHP-APTES-50 derivative onto different solid supports. 

▲ - activated bentonite; + - silica gel;* - kaolinite; ● - quartz sand; ♦ - non activated bentonite 


The sorption of silanol-modified HS on different solid supports including silica gel, quartz 

sand and clays was studied. It was shown that the silanolized humic derivatives had high 

affinity for sorption on silica gel and clays which are characterized with highly developed 

surface area. At the same time, very limited sorption was observed on quartz sand with low 

surface area. Maximum sorption achieved was 300 mg per gram of silica gel for CHP- 

Ceq, g/l 

APTES-100 derivative at 4 g/L concentration and 

a weight of silica gel of 50 mg. 

The conclusion could be made that the silanolized HS can be used as soil conditioners for 

increasing the pool of stable soil organic carbon due to sorption on clay particles. 



This research was supported by the grant of RFBR 10-03-00803 and of the NATO CLG 

983197. 

References 

1. Martin J.P. and Waksman S.A., Soil Sci., 52 (1941) p. 381. 

2. Perminova I.V., Ponomarenko S.A., Karpiouk L.A., and Hatfield K. PCT application № 

/RU2006/000102. 

3. Prado, A. G. S., Sales, J. A. A., Airoldi, C. J., Therm. Anal. Calorim. 70 (2002) p.191. 

4. Koopal, L. K., Yang, Y., Minnaard., A. J. Coll. Surf. 141 (1998) p.385 

5. Shabanova N.A., Sarkisov P.D, Fundamentals of sol-gel technologies, 2004 

Vol. 3 Page - 193 -

Imprinted Humics-Based Sorbents as Selective Trap for Metal Ions 

Elvira Kasymova a , Rozalina P.Koroleva a , Elnura M.Khudaibergenova a , Norbert Hertkorn c , 

Sharipa J. Jorobekova a , Anatolii D.Pomogailo b , Kamila Kydralieva a 

a Institute of Chemistry and Chemical Technology, National Academy of Sciences, Chui ave. 

267, Bishkek 720071, Kyrgyz Republic; b Institute of Problems of Chemical Physics, RAS, 

Ac. Semenov ave, 1, Chernogolovka, Moscow region, 142432; c Helmholtz Zentrum 

München, German Research Center for Environmental Health, Institute of Ecological 

Chemistry, Ingolstädter Landstr. 1, 85764 Neuherberg, Germany 

E-mail: k_kamila@mail.ru 


Selective binding of the traced metals with humic acids (HA) has very important value for the 

removal of heavy metals and radio-nuclides from migration cycles. Additional 

functionalization of HA causes the increase of their sorption capacity and the selectivity of 

binding 

of metal atoms. 


To increase the efficacy of the proposed sorbents we have been used the technique of 

“adjustment” of polymeric complexes to the template on the stage of their synthesis or 

formation of three-dimensional structure. The essence of this technique is the use of 

"imprinted polymers," molecular chains imprinted with empty binding sites that match the 

size and shape of specific kinds of metal ions. These polymers can be used to selectively trap 

and contain a desired species of ion for removal from solution. Previously such approach was 

used for the selective binding of strontium ions in the regions contaminated as a consequence 

of Chernobyl disaster, adjusted polymeric sorbents on the basis of co-polymers of 

diacrylate 

strontium with sterol, methylmetacrylate, acrylic acid and cross-linking agent – dimethacryl 

ether ethyleneglycol, etc. [1]. 

3. Results 

and Discussion 


To create imprinted 

sorbents, a target metal ion such as zinc, cobalt, nickel or copper was 

sandwiched between a pair of ligands as humic acids and m-aminophenol (shortly – HAA). 

After these sandwich complexes are cross-linked into a polymer, the metal ions are 

washed 

away with HCl, leaving empty sites of the right size to fit similar ions. 

The hollow spaces are 

able to encase and rebind the target molecule if encountered with for example 

a water sample 

containing the molecule. The rebinding has a specificity connected to both the geometrical 

structure of the hollow site and the chemically functional groups within the site. 

Vol. 3 Page - 194 -

A set of imprinted sorbents were synthesized and characterized in terms of elemental and 

functional groups composition and molecular weight distribution, FTIR and NMR 

sp ectroscopy. Batch equilibrium studies using imprinted and non-imprinted polymer solutions 

were conducted 

to determine metal-binding capacities. 

"Template" polymer 

Figure 1: Scheme for template synthesis (“template” arrangement of polymer sorbent) 

Imprinted sorbents possess 

significant speed of sorption (the balance is set up in several 

minutes), sorption capacity 

“adjusted” sorbents. 

(0.5–3.0 mg-eq M/g) and selectivity of sorption onto the 

In a solution with equal concentrations of two different ion species including both copper and 

zinc, the polymer captured 23 copper ions for every ion of zinc. The HAA imprinted with zinc 

much prefers the zinc ions over the other metal ions, grabbing zinc over nickel, the nearest 

competitor, in a ratio of five to one. 


This research has demonstrated that molecular imprinting of metals using humics-based 

sandwiches can be used to selectively induce binding of target metals based on shape 

differences of their topologies. 


M 1 M1 M 1 


Cross-linking 

M 1 + M 2 + M 3 ... 

This work supported by grant of the International Science and Technology Centre (#ISTC 

KR-1316). 

References 

1. A.D. Pomogailo, et al. Doklady Akademii nauk, 1994, 335, 749–752. 

M 1 

Vol. 3 Page - 195 - 

M 1 

M 1 

M 1 

Removing of M 1 

M 1 

M 1

Flow Injection Analysis (FIA) for Fast Monitoring of Gold Nanoparticles 

Formation from Various Precursors and Theirs Separation by Using Humic 

Acids 

Eladia María Peña-Méndez a , Ana I. Jiménez Abizanda a* , Juan José Arias León a and Josef 

b,c 

Havel 

a Department of Analytical Chemistry, Nutrition and Food Science, Faculty of Chemistry, 

University of La Laguna, 38071 La Laguna, Tenerife, Spain; b Department of Chemistry, 

Faculty of Science, Masaryk University, Kotlarska 2, 61137 Brno, Czech Republic; 

c Department of Physical Electronics, Faculty of Science, Masaryk University, Kotlarska 2, 

61137 Brno, Czech Republic 

E-mail: aijimene@ull.es 


Unique properties of nanomaterials accelerate the development in biotechnology, industry, 

medicine, etc. The delocalized electrons in the metal clusters can undergo a collective 

excitation called surface plasmon resonance. Gold nanoparticles (GNP) represent one of the 

most widely studied nanoparticles systems [1, 2]. Many different chemical and physical 

methods to produce nanogold particles with different size and shape are described. Thus, 

GNP can be obtained reducing gold (I, III) salts with reducing agents like AlBH4, citric acid 

or sodium citrate, hydroxylamine, tin (II) chloride and organic compounds such as amines, 

hydroquinone and/or natural organic compounds. Unfortunately, nanoparticles tend to form 

aggregates in solution as a consequence of their small size. One of the most effective 

strategies to avoid this fact is to protect 

the colloids obtained with protecting agents. In 

addition, high molecular 

weight polymers improve function of the particle surface for their 

application in bioanalytical methods. 

In this work auric acid (HAuCl4) and reducing agents like gallic or H2O2, etc. were used to 

produce GNP in aqueous solution. 

The HA can also react with Au(III) in solution, generating 

different size GNP. The aim was to study possibility of FIA for the separation of GNP and 

the influence of HA in the formation 

and separation of nanoparticles with different size. 



Hewlett–Packard 

HP 8453A diode array spectrophotometer equipped with quartz cuvettes of 

1 cm light path and 4 ml inner volume. Gilson Minipuls-2 peristaltic 

pump fitted with PVC 

tubes. All other tubing and connectors were made of Teflon. Crison digital pH-meter 

furnished with a glass–saturated calomel double electrode. 

Vol. 3 Page - 196 -

T he Suwannee river standard HA (1S101H). The 200 mg·l 

from Sigma-Aldrich (Steinheim, Germany hydroxide was from Merck (Darmstadt, 

G o 

prepare all solutions was double distilled fr l ore system. 

-1 stock solution was prepared by 

dissolving the corresponding weight in 36 mM NaOH. Auric acid, HAuCl4 trihydrate was 

). Sodium 

ermany). All other reagents were of analytical grade purity. De-ionized water used t 

om Mi liP 

For 

FIA studies, 10–50 μL of solutions was injected in the FIA system. 

3. 

Results and Discussion 


Classical FIA was used e.g. for rapid, highly sensitive and routine automatic determination 

of ionic forms of gold in different samples [4] and to study the catalysis of GNP in some 

reactions [5]. In this work, FIA with Diode Array Detector (DAD) was used to follow the 

nanoparticles formation and separation of various size GNP. 

Instrumental set up as the length of the capillary, temperature, flow rate, additives, etc. were 

optimized. Even if only partial separation of the GNP was obtained, it enables fast 

characterization of the particles produced. Specifically, separation enables to get UV/Vis 

spectra of individual GNP plasmons and monitoring the kinetics of GNP formation. 

A B 

0 

0 200 600 1000 1400 

Figure 1: Flow Injection Analysis set up (A) and an example of GNP separation and effect of HA (B) 

Suwannee river standard (SW), +HA, +2HA, +4 HA: different additions of SW to GNP 

As can be seen from Fig. 1, the addition of HA to GNP improves the separation of different 

size nanoparticles. An increase 

in the HA concentration produces a displacement in the FIA 

peak to early times. Thus, HA not only act as a reducing agent but also contribute to modify 

the rate of displacement of GNP formed. 

Vol. 3 Page - 197 - 

Absorbance 

0.16 

0.12 

0.08 

0.04 

GNP 

HA 

GNP+HA 

2GNP+2HA 

GNP+4HA 

time (s)


It was found that FIA with long path capillary can be used to separate GNP. The separation 

mechanisms is suggested to be similar to turbulent chromatography. Additions of HA to 

GNP improve the separation. Humic acids are probably adsorbed on the GNP surface 

stabilizing them and/or GNP are simultaneously encapsulated with HA forming 

supramolecular complexes. 

Flow Injection Analysis technique developed enable fast 

monitoring of GNP formation. 



Grant Agency of the Czech Republic, projects no. 525/06/0663 and 202/07/1669, Academy of 

Sciences of the Czech Republic (project KAN 101630651) and the Ministry of Education, 

Youth and Sports of the Czech Republic (projects MSM0021622411 and LC 06035) are 

acknowledged. Canary Autonomic Government by research project PI 2007/011 is 

acknowledged. E.M.P., A.I.J. and J.J.A. thank the partial support of the University of La 

Laguna (Spain). 

References 

1. 

Y. Sun, Y. Xia, Science 298, (2002), 2176. 

2. 

B.K. Jena, C.R. Raj, Biosens. Bioelectron. 23, (2008),1285. 

3. 

E.M. Peña-Méndez, J.R. Hernández-Fernaud, R. Nagender, J. Houška and J. Havel, Chem. Listy 

102,(2008), 1394. 

4. 

D.G. Themelis, A.V. Trellopoulos, P.D.Tzanavaras, M. Sofoniou, Talanta 72, (2007), 277. 

5. L. Wang, P. Yang, Y. Li, H. Chen, L. Maoguo, L. Fabao, Talanta 72 , (2007), 1066. 

Vol. 3 Page - 198 -

Spectrofluorimetric Study of the Interaction of Gold (III) and Humic Acids 

under the Formation of Gold Nano-Particles 

a a 

Eladia María Peña-Méndez , Francisco Jiménez Moreno , Jose Elías Conde González, 

Josef Havel b,c 

a Department of Analytical Chemistry, Nutrition and Food Science, Faculty of Chemistry, 

University of La Laguna, 38071-La Laguna, Tenerife, Spain; b Department of Chemistry, 

Faculty of Science, Masaryk University, Kotlarska 2, 61137 Brno, Czech Republic; 

c Department of Physical Electronics, Faculty of Science, Masaryk University, Kotlarska 2, 

61137 Brno, Czech Republic 

E-mail: fjimenez@ull.es 


Humic acids are a complex mixture of partially "decomposed" and otherwise transformed 

organic materials from different sources. The chemistry of their formation is quite complex 

and still it is not completely understood. There are several subclasses of humic acids (tannins, 

lignins, fulvic acids, etc.). A substantial fraction of the mass of the humic acids is containing 

carboxylic acid functional groups which endow these molecules with the ability to chelate 

positively charged multivalent ions (Mg 

The fluorescent structures are minor components in humic substances. 

luorescence spectroscopy is fast, relatively easy and powerful method to follow such 

structures but also method for providing knowledge about the chemistry and 

eduction of Au(III) may be due to 

ts for 

2+ , Ca 2+ , Fe 2+ , Fe 3+ , Al 3+ and most other "trace 

elements") [1]. 

F 

fluorescence 

nature of the interactions between gold (III) and HA. The r 

functional groups such as e.g. amino, hydroquinones and phenolics groups normally present 

on humic acids; functional groups which are recognized to be efficient reducing agen 

gold cations [2]. 

The 

aim of the work is to investigate the interaction taking place between gold (III) and soil 

humic acids (HA). 


Perkin-Elmer (Beaconsfield, Buckinghamshire, UK) spectrofluorimeter equipped with a 

xenon 

lamp and quartz cuvettes of 1 cm path length and 4 mL inner volume. Crison 

(Barcelona, 

Spain) digital pH-meter furnished with a combined glass–saturated calomel 

double electrode. Lauda (Königshofen, Germany) MS6 thermostat. Ultrasonic cleaner 

(Selecta, Seville, Spain) was also used. 


The HA soil IHSS standard stock solution (200 mg·L -1 ) was prepared by dissolving the 

corresponding weight in 36 mM NaOH. Auric acid, HAuCl4·3H2O, was purchased from 

Vol. 3 Page - 199 -

Sigma-Aldrich 

(Steinheim, Germany). Sodium hydroxide was from Merck (Darmstadt, 

Germany). All reagents were of analytical grade purity. All aqueous solutions 

were made 

using 

ultrahigh purity water purified using a Mill-Q Plus system (Millipore Co). 


The current study employed fluorescence spectroscopy to investigate the interactions of 

h umic acid (HA) with gold (III). The results of kinetics show that gold (III) is in the first stage 

bound to HA, most probably complexed to- 

160 

SH and/or amino groups present in the humic 

structures and later on the reducing groups 

140 

pH = 3.02, HA + Au(III) 

pH = 8.58, HA + Au(III) 

pH = 5.05, HA + Au(III) 

from the HA are reducing Au(III) to Au (0) 

120 

pH = 3.02, HA 

pH = 3.02, HA + AuNP 

generating gold nano-particles of various size 

100 

pH = 3.02, AuNp 

pH = 3.35, Au(III) 

of. The redox reaction is pH dependent (cf. 

80 

Fig. 1). 

60 

The nano-particles were also characterized by 

scanning electron microscopy (SEM). Because 

fluorescence of GNP is also influenced by HA, 

the interaction GNP-HA can also be suggested. 


0 

250 350 450 


550 650 

Humic acids are interacting with Au(III) under some kind of complexation. Finally, humic 

acids are reducing Au (III) to gold nano-particles in a reaction which consists of several steps. 

Also, the interaction between humic acid and gold nano-particles was proved. Highly 

homogeneous in size gold nano-particles were prepared but at different conditions GNP of 

varied size are formed. 



Grant Agency of the Czech Republic, projects no. 525/06/0663 and 202/07/1669, Academy of 

Sciences of the Czech Republic (project KAN 101630651) and the Ministry of Education, Youth and 

Sports of the Czech Republic (projects MSM0021622411 and LC 06035) are acknowledged. Canary 

Autonomic Government by research project PI 2007/011 is acknowledged. E.M.P-M., J.E.C. and F.J. 

thank the partial support of the University of La Laguna (Spain). 

References 

1. V.L. Pallem, H.A. Stretz and M.J.M. Wells, Environ. Sci. Technol., 43 (2009) 7531. 

2. R.A. Alvarez-Puebla, D.S. dos Santos, and R.F. Aroca, Analyst, 132 (2007) 1210. 

Fluorescence 

40 

20 

Vol. 3 Page - 200 -

Metal Binding by Humic Acids Extracted from Recent Sediments from the 

SW Iberian Coastal Area 

De la Rosa, J.M. a* , Santos, M. a , González Vila, F.J. b , Knicker, H. b , González Pérez, J. A. b , 

Araújo, M.F. a 

a 

Instituto Tecnológico e Nuclear, Estrada Nacional 10, 2686-953, Sacavém, Portugal; 

b 

IRNAS-CSIC, Av. Reina Mercedes, 10, 41012-Sevilla, Spain 

E-mail: jmrosa@itn.pt 


It is well known that humic substances (HS) play 

a key role in a range of environmental 

issues, such as soil and water acidification, nutrient control, 

weathering, mobility and 

distribution of heavy metals, ecosystem buffering, etc [1]. In almost 

all of those issues, cation 

binding is recognized to be an important factor, which 

has been studied in many papers [1–4]. 

These macromolecules 

are also interesting because of their structural features, which includes 

binding sites with different complexing strength, able to form inert and labile complexes with 

inorganic cations (metals) [5] and organic compounds. 

The information on the compositions 

and functional groups of HAs is critical for understanding 

their reactivity with organic and 

inorganic contaminants. However, due to their complexity 

and heterogeneity, it is difficult to 

determine the HAs structures. Bearing in mind the fact that cation-humic interactions depend 

on the presence of reactive acidic functional groups, such as carboxylic and phenolic groups 

[6], their characterization and quantification may enlighten the metal-humic interactions. The 

major aim of this study is to report information about the amounts of metals bound in “not 

labile” forms to sedimentary humic acids from estuarine and coastal sediments taken in the 

Guadiana estuary, Tinto and Odiel River mouth areas, and in the adjacent continental shelf, 

within the northern Gulf 

of Cadiz (Spain). The analytical strategies followed included the 

total concentration of organic carbon (TOC), inorganic carbon (TIC), nitrogen, sulphur and a 

series of fundamental heavy metals 

(Fe, As, Cr, Cu, Hg, Ni, Pb and Zn) in both, bulk 

sediments and humic acids (HA). In order to check the relationship between the presence of 

carboxylic and phenolic groups and the amount of metals “captured” by HA, they were 

characterized by using analytical Pyrolysis-GC/MS and solid state 13 C-NMR spectroscopy. 



Area of study 

and sediment sampling. Surface sediment samples (0 to 20 cm depth) were 

collected on northern of the Gulf of Cadiz under the auspices of the Spanish BACH-Project 

[7]. It is a site of geological and environmental interest, highly influenced by water run-off 

Vol. 3 Page - 201 -



sediment load derived from the Guadiana River, which is a major river of the Iberian 

Peninsula (742 km of length). The flow of the Guadiana River has 

been extensively modified 

du ring last century due to a range of anthropogenic activities, including damming, mining, 

urbanisation, deforestation and dredging. A set of representative 

samples was selected for this 

study, they consist of three samples from the Guadiana estuary (GE209; GE220; GE226), one 

sample from the Tinto estuary (TE25), 

one sample from the 

Odiel estuary (OE39), and two 

samples 

from the adjacent continental shelf (S131; S155). All samples were stored frozen in 

glass containers to avoid microbial growth. Before analysis, samples were freeze-dried, 

thoroughly ground in a mortar mill and homogenized. 

Elemental Analysis. Carbon, nitrogen and sulphur contents were determined in triplicates by 

using an elemental analyzer. TOC was measured on decarbonised samples (HCl) and TIC was 

calculated from the difference between TC and TOC [8]. 

Humic acids extraction. HAs were extracted with a mixture 0.1 M Na4P2O7 and 0.1 M NaOH 

and the dark brown supernatant (total humic extract) was precipitated by adjusting the pH to 

2, de-ashed, redissolved in 0.1 M NaOH and centrifuged. The residue was discarded, and the 

brown of sodium humate supernatant was reprecipitated with HCl and dialysed in to remove 

the salts introduced during the extraction procedure. The HA was then freeze dried and kept 

for further chemical characterization. 

13 13 

C NMR spectroscopy. Solid-State C-NMR spectroscopy analyses of HAs were performed 

on a Bruker DSX 200 spectrometer, operating at a 13 C resonance frequency of 50.3 MHz. A 

commercial Bruker double bearing probe and phase-stabilized zirconium dioxide rotor was 

used. The cross-polarisation (CP) technique was applied during magic angle spinning (MAS) 

of the rotor. For quantification, the spectra were divided into different chemical shift regions 

according to [9]. 

Pyrolysis-gas chromatography-mass spectrometry (Py-GC/MS). Pyrolysis of HAs was 

performed in a double shot Frontier Laboratories pyrolyzer (model 2020) directly connected 

to a GC-MS system Agilent 6890 equipped with a fused silica capillary column HP 5MS (30 

m × 250 μm × 0.25 μm inner diameter). The detector consisted of an Agilent 5973 mass 

selective detector (EI at 70 eV). Standard chromatographic conditions and identification of 

individual compounds procedure were carried out according to [10]. 

Metal analysis. The analytical determination of major and minor metals (Fe, As, Cr, Cu, Hg, 

Ni, Pb and Zn) in both bulk sediments and HA samples were performed by digesting 10 mg of 

Vol. 3 Page - 202 -

samples in a microwave oven (5 min 300W) with a mixture of H2O2 and HNO3. All the 

samples were diluted to 50 mL with ultra pure water (18MΏ) and keep refrigerated before 

analysis. The resulting solutions were analyzed by ICP-MS (Perkin Elmer ELAN DRCe) and 

the metals quantified by external calibration. For correcting any instrument drifts Rh 10 ppb 

was added as an internal standard to all the samples and standard reference materials. 

3. Results and Discussion. 

The TC values ranged from 12.8 to 31.1 g kg -1 , which are typical values for coastal and 

marine sediments [8]. Greater TOC values were found in riverine 

ppm 

3750 

3500 

3250 

3000 

1750 

1500 

1250 

1000 

750 

500 

250 

0 

and in the Guadiana 

estuarine samples. The TN values were found to be very similar and ranged between 0.9 and 

2.0 g kg -1 . The C/N ratios in the sediments studied ranged between 7 and 12. Larger C/N 

ratios (≥ 12) were observed for the estuarine samples, indicative of a large contribution of 

s ranged from 1.8 to 13.5 g kg -1 terrestrial detritus. Sulphur (TS) contents in the sample 

. 

Larger values were observed in the Odiel estuary sample (OE39), and the smallest value was 

in the marine sample S131. 


Total contents of Fe, Cu, Zn, Pb and Cr metals in bulk sediments were significantly increased 

in samples located in the Tinto and Odiel river 

Figure 1: Contents of selected metals in Humic acids 

mouths (TE25; OE39), which is explained by 

the fact that those rivers drain a catchment 

area 

that traverses the pyritic belt in the Southwest 

of the Iberian Peninsula. Guadiana estuarine 

sediments presented lower metal contents 

compared to the samples located in the 

continental shelf, pointing to a transport of 

sedimentary material 

due to the influence of 

strong river discharges. 

Figure 1 shows the contents of several metals 

TE 25 OE 39 S 131 S 155 GE209 GE220 GE226 

in HAs. Retention capacity of particular metals on HAs may be ordered in the following 

sequence Fe>>>Cu>>Zn>Cr. This preferential binding order of metal-HA doesn’t correlate 

with the metal content order in bulk sediments. 

The CP-MAS major peaks at 30 ppm 

13 C NMR spectrum of the seven HAs all exhibited 

(alkyl C), 55 ppm and 71 ppm (O-alkyl carbons), 130 ppm and 152 ppm (aromatic carbons), 

173 ppm (carboxylic carbon), and 196 ppm (carbonyl carbon). Aromaticity was significantly 

Vol. 3 Page - 203 - 

Cu 

Zn 

Cr 

Ni 

Pb

higher in the Tinto estuarine sample (TE25; 40.9 %) and Guadiana estuary (GE209; 34.4 %) 

HAs, contrasting with the aliphaticity of the marine HAs (S131 and S155; 70.2 and 74.3 % 

respectively), in which aquatic plants are the dominant contributors to the HS. The carboxylic 

and phenolic C parameter accounts the % of reactive acidic functional groups in the HAs [6], 

an increase was shown for HAs isolated from estuarine samples, so they might have further 

binding sites able to form complexes 

with metals. 

Pyrolysis-GC/MS experiments revealed similar compositions of pyrolysates generated from 

all samples. These were characterized by the presence of a complex mixture of phenols, 

benzenes, naphthalenes, indanes, pyrroles, alkylated homologues with a low degree of 

alkylation and a scarce contribution of fatty acids and low relative abundances of n-alkanes 

and n-alkenes. A slight increase in the aromatic and carboxylic compounds released from 

riverine HAs was observed confirming results obtained by 13 C NMR spectroscopy. 



Overall, the results of this study demonstrate that the combination 

of techniques used yield 

valuable information concerning the nature of HAs in recent sediments. There was a positive 

correlation between metal content and functionality 

(acidic carboxylic and phenolic groups). 

Nevertheless this study in still in progress, so hopeful 

results are expected with the aim of 

increase the knowledge in the metal-humic interactions. 

Acknowledgements. We thank Ms. Trinidad Verdejo 

(IRNAS-CSIC) for assisting us in the 

Py-GC/MS spectrometry. 

References 

1. E. Tipping, Cation Binding by Humic Substances, 

Cambridge University Press, Cambridge, 2002. 

2. J.A. Marinsky and J. Ephraim, Environ. Sci. Technol., 

20 (1986) 349. 

3. F.J. Stevenson, Humus Chemistry: Genesis, Composition, 

Reactions, John Willey & Sons, New 

York, 1994. 

4. A.E. Martell and R.D. Hancock, Metal Complexes in Aqueous Solutions, Kluwer, New York, 

1996. 

5. R. Yamamoto and S. Ishiwatary, Sci. Total Environ., 

279 (1992) 117. 

6. J.C. Masini, G. Abate, E.C. Lima, L.C., Hahn, M.S. Nakurama, J. Lichtig, and H.R. Nagatomy, 

Anal. Chim. Acta, 364 (1998) 223. 

7. BACH project, REN 2002-04602-C02-01. Environmental Geochemistry of Sediments from the 

Huelva Coast. 

8. J. Nieuwenhuize, Y.E. Maas, J. Middelburgh, Mar. 

Chem., 45 (1994) 217. 

9. H. Knicker, H.-D. Lüdemann, Org. Geochem., 23 (1995) 329. 

10. J.A.González Pérez, C.D. Arbelo, F.J. González Vila, A. Rodríguez, G. Almendros, C.M. Armas, 

O. Polvillo. J. Anal. Appl. Pyrolysis, 80 (2007) 369. 

Vol. 3 Page - 204 -

Polycyclic Aromatic Hydrocarbons (PAHs) - Dissolved Organic Matter 

(DOM) Interactions Studied by Solid Phase Microextraction (SPME) 

Chloé De Perre a,b , Karyn Le Menach a,b , Anne-Marie Dorthe c,d , Christian Béchemin e , 

Hélène Budzinski a,b* , Edith Parlanti a,b* 

a 

Université de Bordeaux, UMR 5255, ISM, Groupe LPTC, 351 Cours de la Libération 


France; c Université de Bordeaux, UMR 5255, ISM, Groupe NSysA, ENSCPB, 16 Avenue 

d 

Pey Berland Pessac, F-33607 France; CNRS, UMR 5255, ISM, Groupe NSysA Pessac, F- 

33607 France; e IFREMER, LER/PC, BP 7, L’Houmeau, F-17137 France 

E-mail: e.parlanti@ism.u-bordeaux1.fr; h.budzinski@ism.u-bordeaux1.fr 


Polycyclic Aromatic Hydrocarbons (PAHs) are highly toxic pollutants, with carcinogenic 

properties for some of them (US-EPA priority substances). Dissolved organic matter (DOM) 

in aquatic environments 

is well known to play an important role in the fate of organic 

pollutants. Indeed, DOM 

should bind these compounds modifying their distribution, 

bioavailability, biodegradation and subsequently their toxicity towards aquatic organisms. In 

order to calculate the partitioning coefficient (KDOC) of each pollutant to DOM, it is necessary 

to measure concentrations of free and DOM bound pollutant fraction. Few analytical 

techniques allow the measurement of only free compound concentration but most of them 

may modify the interactions during the analysis, making the study of these interactions a real 

challenge. The goal of this study was therefore to develop a reliable technique that permits to 

quantify rapidly total and free organic pollutant concentrations: solid-phase microextraction 

coupled 

to gas chromatography-mass spectrometry (SPME-GC-MS). We aimed also at 

highlighting environmental parameters which influence PAH/DOM interactions. 



SPME-GC-MS was chosen for the analysis of PAHs, especially since it allows the 

quantification of both freely dissolved and DOM-bound PAH concentrations simultaneously, 

with a negligible disturbance of the equilibrium DOM-PAH [1, 2]. SPME technology is 

constituted of a polymeric fiber that 

is introduced into the water samples in order to 

adsorb/absorb, according 

to their affinity with the fiber coating, the only free PAHs that are 

then desorbed by heating in the GC injector. KDOC values were calculated 

for individual 

compounds and for a mixture of 4 and 16 PAHs, with variations of DOC and PAH 

concentrations. DOM samples were characterized by their optical properties by means 

of 

excitation-emission matrix (EEM) spectroscopy and UV-visible absorption. Moreover, full 

Vol. 3 Page - 205 -

and fractional factorial designs were performed to investigate the effect of environmental 

parameters 

like salinity, pH, DOC and PAH concentrations on the interactions. 

3. Results 

and Discussion 

A series of tests was performed to optimize SPME parameters for the analysis of PAHs. 

Results showed that fiber coating in polydimethylsiloxane (PDMS) seems to provide the best 

e fficiency with a time of analysis of only one hour, low limits of detection and quite good 

reproducibilities. It was shown that the strength of interactions highly depends on 

DOM 

o rigin and structure and on type of PAHs. Humification and aromaticity of DOM were 

not the 

driving factors of 

interactions and fresh material of aquatic DOM could have stronger 

interactions with PAHs than humified aquatic fulvic acids. Salinity was not an important 

factor since it modified significantly neither KDOC values nor optical properties of DOM. On 

the contrary, pH and DOC concentrations could strongly affect interactions and had appeared 

to be interrelated for some samples. PAH concentration was shown to affect interactions: 

KDOC values tended to decrease at high concentrations of PAHs. 


SPME is an appropriate tool to study interactions of many PAHs simultaneously at low 

concentrations with DOM. This study showed that interactions of PAHs with DOM are very 

complex and depend on many factors. Some of them have no effect on interactions, such as 

salinity, while some others highly affect KDOC values such as pH and DOC concentrations. 

Nevertheless, interactions and environmental factor effects have been shown to depend on 

PAHs and DOM origin and structure and appear to be difficult to apprehend. Indeed, in this 

study three different DOM were studied and three different complex behaviors were 

pointed 

out, so the phenomena that occur 

in natural waters should be a challenge to model. 



"Region Aquitaine", FEDER, ORQUE program (CPER) and INSU (EC2CO program- 

IMOTOX project) are acknowledged for financial support and the French Ministry of 

Research for the PhD grant of C. de Perre. We also gratefully acknowledge C. Vérité 

(IFREMER LER/PC) for the dissolved organic carbon analyses, R. Brizard and J.C. Billy 

(IFREMER LGP, La Tremblade) for the algae cultures. 

References 

1. J. Poerschmann, Z. Zhang, F.D. Kopinke, J. Pawliszyn, Analytical Chemistry, 69 (1997) 597-600. 

2. M.B. Heringa, J.L.M. Hermens, TrAC Trends in Analytical Chemistry, 22 (2003) 575-587. 

Vol. 3 Page - 206 -


Vol. 3 Page - 207 -


Environmental Applications of Natural Organic Matter and Humic 

Substances 

Vol. 3 Page - 208 -

Use of Humin for Removal of Phosphorus from Sewage Treatment Station 

Effluents: Influences of Time and pH 

Luciana Camargo de Oliveira *a , Wander Gustavo Botero a,b , André Gustavo Ribeiro 

Mendonça a , Julio Cesar Rocha a , Ademir dos Santos a , André Henrique Rosa c 

a Institute of Chemistry of Araraquara – UNESP, Araraquara, SP, Brazil; b Federal University 

of Alagoas, Arapiraca, AL, Brazil; c Department of Environmental Engineering – UNESP, 

Sorocaba, SP, Brazil 

E-mail: lcamargo@iq.unesp.br 



Phosphorus-containing substances have widespread applications in industry, agriculture and 

in the home. These chemicals are ultimately transported to waterways where, despite 

treatment of effluents, they can cause serious pollution problems. Although phosphate is not 

considered to be toxic, it is a nutrient that in excess can cause algal overgrowth and 

eutrophication. Under conditions of low oxygen content it is anaerobic bacteria that are 

mainly responsible for decomposition of organic matter, so that instead of oxidation, 

reduction takes place, producing noxious compounds such as hydrogen sulphide, 

methanethiol and ammonia. In addition, phosphates can produce dense layers of foam, 

reducing the surface tension of water, to the detriment of aquatic fauna. Removal of 

phosphorus from freshwaters is therefore of environmental interest [1, 2]. 

For many years, control of phosphorus in surface waters has focused on point sources, such as 

detergents in domestic effluents, however more recently attention has shifted to diffuse 

sources. In Europe, it is estimated that 50 % of the phosphorus present in surface waters 

derives from diffuse sources, due to its use in agriculture. Large annual inputs of phosphate 

fertilizer, some of which leaches to waterways, are responsible for substantial environmental 

impacts. Hence, there is a need for substances that could assist in retention of phosphate, and 

that could ideally be subsequently used as natural fertilizers [1]. 

Humic substances (HS) are promising agents that might be able to perform this dual function. 

Enriched with nutrients, they have already been marketed as inorganic fertilizers. Humin, the 

fraction of HS that is insoluble throughout a wide range of pH, has received less scientific 

attention than other HS fractions [3]. Nonetheless, there have been reports in the literature 

concerning its interaction with organic compounds (herbicides, insecticides, fungicides, PAHs 

and PCBs) [3, 4], as well as a small number of studies involving inorganic substances [5]. 

Vol. 3 Page - 209 -

The present work examines humin-phosphorus interactions from the perspective of the possible 

use of humin to remove phosphorus from sewage treatment station effluents, for later application 

in agriculture as a natural fertilizer. 

2. Materials and methods 

Extraction of humin. Humin was isolated from peat samples collected in the vicinity of the Mogi 

River, in the municipality of Rincão, São Paulo State, Brazil, using alkaline extraction procedure 

recommended by the International Humic Substances Society (IHSS) [4]. 

Preparation of standard solutions.A 1000 mg L -1 phosphorus (as phosphate-P) stock standard 

solution was prepared, and dilutions made as required immediately prior to the experiments 

concerning pH and temperature, as well as for construction of calibration curves. 

Influence of pH on humin-phosphorus interactions. One hundred mg quantities of humin were 

weighed out into polypropylene flasks, and 50 mL of 10 mg L -1 phosphorus standard solution 

added, at pHs varying from 2 to 8. After pH adjustment, the solutions were left under agitation for 

24 hours, and then filtered and the phosphorus contents quantified by inductively coupled plasma 

atomic emission spectrometry (ICP-OES). 

Influence of time on humin-phosphorus interactions. Mixtures of humin and phosphorus were 

prepared as above, and the pH adjusted to the value at which greatest adsorption of phosphorus 

occurred. After pH adjustment, the solutions were agitated for periods of 5, 10, 30, 180, 1440 and 

4320 minutes, then filtered and the phosphorus contents measured. 

Use of humin with sewage treatment station effluents. Adsorption of phosphorus was investigated 

by adding 10 mg of humin to 50 mL portions of effluent, either as received or after adjustment of 

pH to 3.0. The labile phosphorus contents of the samples were measured before and after humin 

addition. The mixtures were filtered after the maximum adsorption time, and the phosphorus 

contents quantified. 



The maximum adsorption of phosphorus by humin occurred at pH 3.0 (Fig. 1). At pH 8.0, which 

is typical of sewage treatment station effluents, adsorption was 24.2 %. Experiments were then 

performed at these two pHs (3.0 and 8.0), to determine the effect of time on the adsorption 

process (Fig. 2a). At pH 3.0, the humin adsorbed ~84 % of the added phosphorus after 72 hours, 

after which no further changes in adsorption were observed. At pH 8.0, the humin adsorbed ~31 

% of the phosphorus after 72 hours. The adsorption process was more efficient at pH 3.0. 

Vol. 3 Page - 210 -

Phosphorus adsorption (%) 

90 

80 

70 

60 

50 

40 

30 

20 

10 

0 

Adsorbed phosphorus (mg L -1 ) 

6 

5 

4 

3 

2 

1 

0 

Figure 1: Influence of pH on phosphorus adsorption by humin. Initial phosphorus concentration: 

3 4 5 6 7 8 

pH 

10 mg L -1 

Concentration ratio (C -C)/C l l 

0,9 

0,6 

0,3 

0,0 

b) 

pH 3.0 

pH 8.0 

0 2000 4000 

Time (min) 

Figure 2: a) Influence of time on phosphorus adsorption by humin; b) Kinetic study of the interaction 

between humin and phosphorus 

The data obtained were analysed from the perspective of chemical kinetics [6, 7]. Results are 

illustrated in Figure 2b, where Cl is the limit concentration of phosphorus, and C the 

concentration after different time intervals. From this, it was possible to estimate the times 

required to reach equilibrium between the phosphorus ions and humin. At pH 3.0, the time 

required for equilibrium to be achieved was 180 minutes, while at pH 8.0 it was 160 minutes, 

indicating that the kinetic behaviour of phosphorus-humin interactions is similar at different 

pHs. 

a) 

) 

pH 3.0 

pH 8.0 

0 1000 2000 3000 4000 5000 

Time (min) 


When humin was added to samples of sewage treatment station effluent, adsorptions varied 

from 22 to 32 % at pH 8.0, and from 89 to 92 % at pH 3.0 (Table 1), in agreement with the 

values previously obtained using phosphorus standard solutions (Fig. 2a). 

Vol. 3 Page - 211 -

Table 1: Adsorption by humin of phosphorus in sewage treatment station effluent samples 

Effluent 

sample 

pH 

Initial phosphorus content, 

prior to humin addition 

(mg L –1 ) 

Final phosphorus 

content, after humin 

addition (mg L –1 ) 

Removal (%) 

1 3.0 5.54 ± 0.07 0.51 ± 0.09 89.3–92.3 

2 8.0 3.16 ± 0.08 2.29 ± 0.10 22.4–32.4 



Preliminary studies of phosphorus-humin interactions indicate that humin is able to adsorb 

phosphorus across a range of pHs. Humin is therefore a promising material for removal of 

phosphorus from effluents, with an additional potential benefit being the ability to use the 

products as sources of nutrients in agriculture. This work is in development and as soon as 

possible the authors will present major data as full paper. 

References 

1. J.C. Rocha, A.H. Rosa, A.A. Cardoso, Introdução à Química Ambiental, Bookman, Porto Alegre, 

2009. 

2. I.R.S. Chao, T.H. Ferraz, H2O Água, (2007) 40. 

3. J. Rice, Humin. Soil Science, 166 (2001) 848. 

4. J. Zhang, M. He, Y. Shi, J. Hazard. Mat., 166 (2009) 802. 

5. G. De La Rosa, J.L. Gardea-Torresdey, J.R. Peralta-Videa, I. Herrera, C. Contreras, Bioresour. 

Technol., 90 (2003) 11. 

6. J.C. Rocha, É. Sargentini Júnior, L.F. Zara, A. H. Rosa, A. Santos, P. Burba, Talanta, 53 (2001) 

551. 

7. P. Burba, Van Den Bergh, Anal. Bioanal. Chem., 378 (2004) 1637. 

Vol. 3 Page - 212 -

Phytoremediation of a Soil Polluted with Multiple Heavy Metals Using 

MSW Compost as Organic Carbon Source 

Karam Farrag a , Gennaro Brunetti b *, Pedro Soler-Rovira c , Franco Nigro d 

a Central Lab for Environmental Quality Monitoring (CLEQM), National Water Research 

Center, Ministry of Water Resources and Irrigation, Egypt; b Dip. Biologia e Chimica Agro- 

Forestale ed Ambientale, University of Bari, Via Amendola 165/A, Bari, Italy; c Centro de 

Ciencias Medioambientales (C.S.I.C.), Serrano 115 bis, Madrid, Spain; d Dip. Protezione delle 

Piante e Microbiologia Applicata, University of Bari, Italy 

E-mail: brunetti@agr.uniba.it 


Phytoremediation can be defined as the combined use of plants, soil amendments and 

agronomic practices to remove pollutants from the environment or to decrease their toxicity 

(1). Finding the optimum plant species for remediation of a specific soil is a key point 

affecting the achievement of the objective, as well as the selection of appropriate soil 

amendments which would improve soil conditions, allowing plant survival and growth (2). 



Analytical methods: pH and EC were measured in 1:2.5 and 1:2 sample:water ratio, 

respectively. Organic carbon (OC) was determined by dichromate oxidation and titration with 

ferrous ammonium sulphate; total nitrogen (N) was assessed by Kjeldahl method. Soil 

available phosphorus (P) was quantified by Olsen method. Heavy metals contents were 

determined by ICP in extracts from samples digested in HNO3: H2O2 (7 mL+1 mL). 

Chromium oxidation test (Bartlett test) was performed by addition of CrCl3 and UV-VIS 

spectrophotometer measure (540 nm) of coloured complex between Cr (VI) and 

diphenylcarbazide (3). 

Soil samples were collected from a polluted area located at Altamura area (Apulia, Southern 

Italy), where industrial wastes increased soil heavy metals content up to following levels (mg 

kg -1 ): Cd 2, Cr 1277, Cu 89, Ni 132, Pb 166, and Zn 497. Main characteristics of soil were: 

silty loamy texture; pH (H2O) = 8.5; EC = 0.32 dS m -1 ; OC = 66.1 g kg -1 ; N (total) = 7.2 g kg - 

1 -1 

; C/N ratio = 9.1; P = 83.5 g kg . 

A greenhouse experiment was performed using Brassica napus as accumulator plant and 

testing the addition to the soil of organic amendment (MSW compost) and a bacterial strain 

(Bacillus licheniformis BLMB1). Compost analysis showed the following main values: pH 

Vol. 3 Page - 213 -

(H2O) = 8.7; EC = 1.25 dS m -1 ; OC = 232 g kg -1 ; N = 14.8 g kg -1 . Treatments were as follows: 

T1 (soil), T2 (soil + compost 10%), T3 (soil + B. licheniformis BLMB1 10%), T4 (soil + 

Compost 10% + B. licheniformis BLMB1 10%). Bacterial strain was applied to the soil as 

aqueous suspension (10%, w/v) containing 10 8 cells ml -1 . Data statistical analysis, regression 

models and correlations matrixes were performed using Statgraphics Plus 5.1 software. 


The values of Cr 3+ oxidation test resulted lower in the treatments with highest amounts of OC, 

and a significant (P

correlation between soil OC content and plant height (0.904***), foliar area (0.902***), and 

dry weight (0.955***). Therefore, treatments with OC inputs (T1 and T3) improved plant 

biomass (Fig 2) due to the direct and indirect benefits of organic matter fractions on soil and 

plants. 

g OC kg -1 soil 

80 

60 

40 

20 

0 

T1 T2 T3 T4 

TOC Plant DM 

Figure 2: Results for soil OC content and Brassica napus biomass production 

Our data also indicate that supplying soil with organic C and microorganisms Bacillus 

licheniformis BLMB1 enhance the extraction of some heavy metals and their accumulation in 

B. napus. This effect was more evident for copper and lead (Fig. 3). 

mg kg -1 

150 

120 

90 

60 

30 


0 

3,3 

3,2 

3,1 

3 

2,9 

2,8 

T1 T2 T3 T4 

Pb Cu 

g (dry weight) 

Figure 3: Copper and lead contents in shoots of Brassica napus 

Vol. 3 Page - 215 -

Significantly high correlations were found between soil OC and metal content in shoots 

(0.660**, and 0.658 * , for Cu and Pb, respectively), as compared to the untreated control. 

Similar results were reported by other (2) for Cu, in a site polluted by several heavy metals. In 

the case of copper, compost organic fractions, such as humic acids, would allow the formation 

of surface complexes (7) thus facilitating the release to soil solution and maintaining Cu 

available for plant extraction. These aspects have particular relevance in the phytoremediation 

practices, considering that not only metals extraction by plant but also vigorous growth and 

higher biomass production need to be pursued in order to obtain the highest metal 

phytoextraction. 


The preliminary results from our experiment confirm the advantages of using appropriate 

amendment in combination with an optimum plant species for phytoremediation purposes. 

Addition of exogenous organic carbon and B. licheniformis BLMB1 reduced the tendency of 

Cr 3+ to be oxidized, preventing the accumulation and the environmental damage of the Cr 6+ 

form. Compost organic matter increased plant biomass and enhanced the phytoextraction of 

some heavy metals. The combination of both effects (soil immobilization and plant 

extraction) will be the target for our next research activities on phytoremediation, in order to 

obtain deepest knowledge about these processes 



This work was founded by Regione Puglia (Italy) through the research project POR Puglia 

2000-2006, Misura 1.8 – Azione 4: “Monitoraggio siti inquinati”. Supporto scientifico alle 

attività di recupero funzionale ed il ripristino ambientale del sito inquinato dell’Alta Murgia. 

References 

1. D.E. Salt, R.D. Smith, L. Raskin, Phytoremediation Annual Review of Plant Physiology and Plant 

Molecular Biology 49, (1998) 643-668. 

2. R. Clemente, D.J. Walker, M.P. Bernal, Environmental Pollution 138, (2005) 46-58. 

3. Metodi ufficiali di analisi chimica del suolo. Decreto Ministeriale del 13 Settembre 1999. 

4. H. Kerndorff and M Schnitzer, Geochim. Cosmochim. Acta, 44 (1980) 1701-1708. 

5. R.J. Bartlett, Environmental Health Perspectives 92, (1991) 17-24. 

6. N.S. Bolan, D.C. Adriano, R. Natesan, B.J. Koo, J. Environ. Qual. 32, (2003) 120-128. 

7. N. Senesi, G. Sposito, J.P. Martin, Sci. Tot. Environ. 55, (1986) 351-362. 

Vol. 3 Page - 216 -

Hydrogels Filled with Humic-Rich Lignite for Various Environmental 

Applications 

Miloslav Pekař 

Institute of Physical and Applied Chemistry, Faculty of Chemistry, Brno University of 

Technology, Purkyňova 118, 612 00 Brno, Czech Republic 

E-mail: pekar@fch.vutbr.cz 


Lignite is a young coal with a high content of organic matter, especially humic acids. It could 

not be therefore viewed as a fuel but rather as a valuable natural product and chemical raw 

material [1–3]. Lignite can be used also in its natural state which represents the most costeffective 

way, e.g. as a sorbent, in-situ remediation agent or soil conditioner. Direct 

application of lignite in its natural state is not “user-friendly” and, further, very small lignite 

particles (dust) can suffer from stability problems, e.g. in soil. This work aimed at 

incorporating lignite into poly(vinyl alcohol) (PVA) hydrogels to obtain easy usable material 

capable of uptake of sufficient amount of water or aqueous solutions. 



Lignite mined in the South Moravia (Czech Republic; for details see [1,2,4]) was used in its 

natural state after the removal of water (105 °C) to the equilibrium value in ambient 

laboratory atmosphere, i.e. to about 7%. 

Poly(vinyl alcohol hydrogels were prepared by the freeze-thaw procedure [5]. PVA (Mowiol 

28–99, Fluka) was prepared as 5% aqueous solution. Part of the solution was used directly to 

prepare control (lignite-free) samples. Part of the solution was thoroughly mixed with defined 

amount of milled lignite (particle size below 0.2 mm). Solution and suspensions were poured 

into microtitration plates and placed to freezer (about –18 °C) for 16 h. They were then left to 

thaw at laboratory temperature for 8 h. The freeze-thaw cycle was repeated four times. 

Resulting hydrogels were left to dry in the air at laboratory temperature. In this way, pellets of 

diameter of about 4 mm were prepared containing up to 90% (by weight) of lignite in the dry 

state (xerogel). 

Xerogels swelling was tested using deionized water until the constant mass was attained. 

Swelling degree was calculated as the weight difference between the swelled gel and initial 

xerogel relatively to the weight of the xerogel. 

Vol. 3 Page - 217 -

Sorption capabilities were tested by two simple batch tests. First one used methylene blue in 

the concentration of 100 mg/L, second one applied cupric ions in the concentration of 0.025 

mol/L. Ratio hydrogel:solution was 0.1 g per 10 mL of the solution in the case of methylene 

blue and 0.5 g per 10 mL in the case of copper. Concentrations of the dye and copper ions 

were determined spectrophotometrically. 


Filling with lignite lowered the swelling degree as can be seen in Fig. 1 but even the gel 

containing about 90% of lignite was able to swell about a half of its weight. Lignite in the 

freshly mined state contains about 50% of water, consequently, hydrogel swelling can be 

attributed both to PVA network and lignite. 

Sorption tests were made on hydrogel containing 33, 83, and 89% of lignite (in xerogel). 

Sorption on the natural lignite gave final concentration of methylene blue of 6 mg/L and 

cupric ions of 0.0055 mol/L in these tests. Control hydrogel and hydrogel with the lowest 

amount of lignite showed no measurable sorption affinity for methylene blue. Hydrogels 

containing 83 and 89% of lignite decreased the methylene blue concentration to 7 and 2 mg/L, 

respectively. This indicates that at sufficiently high lignite filling in hydrogels the lignite 

active sites for methylene blue are well accessible to the methylene blue solution penetrating 

the hydrogel structure and hydrogel sorption capability is close to that of natural lignite. 

Example of results obtained for copper sorption test is given in Fig. 2. Control hydrogel 

showed only weak sorption capacity for copper which then increased with increasing filling 

by lignite but still was appreciable lower than for the natural lignite. Active sites for copper 

sorption are probably partially blocked by interactions of lignite functional groups with 

hydroxyl groups of PVA, i.e. participate in forming the hydrogel network. 



In summary, physical PVA hydrogels can be successfully filled by natural lignite particles 

while still possessing acceptable swelling properties and sorption capabilities, especially for 

organic sorptives. The optimum lignite filling is about 80% (by weight in the dry gel form). 

Lignite can be thus immobilized and prepared in the more user-friendly form that combines 

water retention and sorption capacities as well as contents of humic substances for various 

sorption, remediation or agricultural applications. 

Vol. 3 Page - 218 -

swelling degree (%) 

Cu 2+ concentration (mol/l) 

200 

150 

100 

50 

0.025 

0.02 

0.015 


0 33 37 43 83 89 

lignite content (% in xerogel) 

Fig..1. Influence of lignite on hydrogel swelling 

0 33 83 89 

lignite contents (% in xerogel) 

Fig. 2. Influence of lignite on copper concentration in equilibrium after the sorption from 

the 0.025 mol/l copper solution 

Vol. 3 Page - 219 -



Assistance of M.Dvořáková in experimental work is gratefully acknowledged. This work was 

supported by government funding – Czech Science Foundation, project. Nr. 105/05/0404. 

References 

1. M. Klučáková, L. Doskočil, P. Bušinová and M. Pekař, in International Conference on Coal 

Science & Technology (ICCS & T), Conference Proceedings CD. 2009, P12, p. 1–13. 

2. M. Pekař, I. Sýkorová and I. Koutník, in Twenty-Fourth Annual International Pittsburgh Coal 

Conference, CD-ROM Proceedings. PCC: Pittsburgh, 2007, P3–4, 13 pp. 

3. M. Pekař, M. Klučáková, L. Omelka and P. Zedníčková, in Humic Substances – Linking Structure 

to Functions, Proceedings of the 13th Meeting of the International Humic Substances Society. 

F.H. Frimmel, G. Abbt-Braun, Eds., Schriftenreihe Bereich Wasserchemie Engler-Bunte-Institut 

der Universität Karlsruhe, 2006, vol. 45-II, p. 1029–1032. 

4. I. Sýkorová and O. Michna, Zesz. Nauk. Polit. Slas., Gornictwo, 249 (2001) 177. 

5. I. Galeska, T.K. Kim, S.D. Patil, U. Bharwaj, D. Chattopadhyay, F. Papadimitrakopoulos and D.J. 

Burgess, AAPS Journal, 7 (2005) E231. 

Vol. 3 Page - 220 -

Mitigation of GHGs Emission from Soils by a Catalyzed in situ Photooxidative 

Polymerization of Soil Humic Molecules 

Alessandro Piccolo * , Riccardo Spaccini 

Dipartimento di Scienze del Suolo, della Pianta, dell’Ambiente e delle Produzioni Animali, 

Università di Napoli Federico II, Via Università 100, 80055 Portici, Italy 

E-mail: alessandro.piccolo@unina.it 


In 2005, agriculture accounted for an estimated emission of 5.1 to 6.1 GtCO2-eq/yr (10– 12% 

of total global anthropogenic emissions of greenhouse gases (GHGs)). However, measures to 

mitigate GHGs emission from agricultural soils are limited to improved cropland practices 

such as crop rotation, nutrient management, tillage/residue management, agroforestry, and 

return to natural vegetation. These practices are not only far from substantially reducing 

GHGs emissions from soils or permanently stabilizing soil organic matter, but are also 

predicted to hardly match more than a maximum of 25% of the GHGs reductions required by 

the Kyoto Protocol within 2050. Despite the knowledge that GHGs release from soil largely 

derives from biochemical transformations of plant litter and soil humus (SH), no new and 

much wished biotechnological measures are adopted so far to augment mitigation. Here we 

propose an innovative approach to mitigate GHGs emissions from soils based on the in situ 

photo-polymerization of SH under biomimetic catalysis. Three Mediterranean soils of 

different physical and chemical properties were added with a synthetic watersoluble ironporphyrin, 

irradiated by solar light, and subjected to 15, and 30 wetting and drying cycles. 



Soil Samples and Characterization. Soil samples were collected from the surface layers (0-30 cm) 

of three agricultural plots from south-central Italy: 1. Porrara (Avellino), 2, Colombaia (Caserta), 

3. Itri (Latina). Samples were air dried, sieved through a 4.75 mm sieve, and used for 

characterization and incubation experiments. 

Photo-polymerization experiments. For each replicate (n = 3), 60 g of air dried soil sample was 

placed on a Petri dish (12 cm diameter) and soil moisture was kept at 40% of water holding 

capacity (WHC) by adding X mL of water (X = 20, 17, 10 mL, for Porrara, Colombaia and Itri 

soil, respectively) in order to obtain a control series. The polymerized series were similarly 

prepared (n = 3) and added with 0.24 μmol of synthetic water-soluble iron-porphyrin (meso- 

tetra(2,6-dichloro-3-sulfonatophenyl)porphyrinate of iron(III) chloride, Fe-(TDCPPS)Cl) 

dissolved in the X mL of water pertaining to WHC of each soil. After preparation, both control 

and polymerized series were left under natural solar radiation throughout the following 

Vol. 3 Page - 221 -

treatments: (i) covered with a Petri dish and incubated for 5 d; (ii) submitted to 15 wetting/drying 

cycles; (iii) submitted to 30 wetting/drying cycles. During wetting/drying cycles, samples were 

uncovered after 5 d incubation and distilled water was added, whenever samples became dry 

(approximately once a week), to reestablish WHC. 

Aggregate stability. An air–dried sub–sample (30 g) was placed on the top sieve of a set of three 

nested sieves (1.0, 0.50, and 0.25 mm) and submerged into 2 cm of distilled water for 30 min. 

After this time, the sieves were manually oscillated (up and down 4 cm) for 30 times during 1 

min. Recovered aggregate fractions were oven–dried at 60°C, weighed, and stored at room 

conditions. The mean weight diameter index in water (MWDw) used for the determination of 

aggregate stability was calculated according to the equation: 

MWDw = 

n 

∑ 

i= 

1 

X iWi 

where Xi is the mean diameter of each aggregate fraction and Wi is the proportion of the total 

sample weight occurring in the i–th fraction. The amount of OC (%) in each aggregate fraction 

was normalized to the weight of each fraction: OC content in fraction (g kg –1 ) × mass of 

recovered fraction (g kg–1) / total OC recovered (g kg –1 ). 

Soil respiration. Soil respiration was evaluated by a dynamic absorption method. Briefly, 9 g of 

air dried and rewetted soil sample (< 2 mm) were placed on a air-tight soil respiration flask in 

which a CO2-free air was continuously fluxed by a peristaltic pump. The CO2 emitted from soil 

was then captured in a trap containing a 0.01 M NaOH solution. The amount of CO2 absorbed in 

this solution was determined after 27 days by back titration with 0.01 M HCl after addition of 7 

mL of 0.5 M BaCl2. The ambient CO2 concentration was determined by inserting blank samples 

(i.e.: no soil) into the respiration system. 

Statistical analysis. A Student’s t-test was used to compare values obtained for control and 

treatments, and difference was considered to be significant at the level of P ≤ 0.05. 



The humified organic matter in soil (70–80% of SOM) represents the principal potential C 

sink in the biosphere, whose advanced comprehension may help to mitigate CO2 emissions 

from soil. Soil humus is composed by the hydrophobic and heterogeneous aliphatic and 

aromatic molecules progressively surviving the microbial transformation of dead biological 

tissues [1]. Recent scientific evidence shed new light on the chemical nature of humus by 

describing humic molecules as heterogeneous but relatively small in mass (≤ 1000 Da) [2], 

rather than the previously assumed macropolymers [3]. Humic molecules were shown to be 

Vol. 3 Page - 222 -


tightly associated in supramolecular structures, which are prevalently stabilized by 

noncovalent hydrophobic bonds [1]. Based on this, it is reasonable that small aromatic humic 

molecules could be covalently linked to each other by oxidative coupling reactions under 

appropriate catalysis, thereby enhancing their molecular size and complexity. It is already 

shown that larger and more chemically-stable humic molecules were obtained by treating 

humic solutions in oxidative (H2O2) conditions, with a phenoloxidase enzyme, such as 

peroxidase [1], or a biomimetic catalyst, such as an iron-porphyrin [4]. Both the enzymatic 

and biomimetic catalysts accelerate the oxidative coupling of phenols via a free-radical 

mechanism. Moreover, the biomimetic catalysis increased the molecular dimension of humic 

matter in solution simply by photo-oxidation under solar radiation, without the need of an 

additional oxidant [5]. By applying photo-oxidative catalysed conditions on humic phenolic 

monomers, a rapid formation of new intermolecular C-C and C-O-C bonds led to several 

identified (up to tetramers) and unidentified oligomers [6–8]. 

The photo-polimerization technology can be applied in situ on soils. The catalyzed photo- 

oxidative formation of covalent bonds among soil phenolic molecules would chemically 

stabilize SH by increasing the content of chemical energy in humic structures and 

consequently reducing the extent of SH biomineralization. Moreover, increasing the mass of 

humus molecules would result in linking together soil particles to larger soil aggregates and 

thus improving soil physical quality. 

Figure 1: Mean Weight Diameter in water (MWDw) for the three soils, Porrara, Colombaia, Itri, 

before and after photo-polymerization treatment for 5 d incubation, and 15 and 30 wetting and drying 

cycles. Error bars indicate standard error (n = 3). The asterisks denote a significant difference between 

control and treatment at the level of P ≤ 0.05 

The occurrence of SOM photo-polymerization was suggested by the mean-weight diameter of soil 

aggregates in water (MWDw), an index of aggregate stability, that significantly increased over control 

for all the three soils after the 5 d incubation (Fig. 1), though to a different extent depending on the 

intrinsic composition of each soil. This indicates that the photo-polymerization treatment, by 

Vol. 3 Page - 223 -


increasing the molecular mass and cross-linking of humic molecules, promoted a tighter association 

among soil particles and formation of larger water-stable aggregates. An enhanced soil aggregate 

stability in the photo-polymerized samples was still kept after 15 w/d cycles, but it was lost for all 

soils after 30 w/d cycles. 

Figure 2: Soil respiration (mg CO2.g -1 of soil) from soils, Porrara, Colombaia, Itri, before and after 

photo-polymerization treatment for 5 d incubation, and 15 and 30 wetting and drying cycles. Error 

bars indicate standard error (n = 3). The asterisks denote a significant difference between control and 

treatment at the level of P ≤ 0.05 

A stronger chemical and physical stabilization of OC after the catalytic photo-polymerization of soils 

can be inferred by the amount of respired CO2 (Fig. 2). Microbial mineralization of SH was 

significantly inhibited in the photo-polymerized samples, as compared to control, for all soils after 5 d 

incubation and even after 15 w/d cycles. While the reduction of CO2 emission was significant for 

Porrara and Itri soils also after 30 w/d cycles, this was no longer true for the Colombaia soil. The CO2 

respiration behaviour of Porrara and Itri soils confirmed that the catalytic photo-polymerization 

strongly stabilized their SH. The different result for the Colombaia soil after 30 w/d cycles may be 

attributed to its larger amount of OC, thus limiting the treatment effect. The OC stabilization obtained 

in Porrara, Colombaia, and Itri soils mitigated the CO2 emission by, respectively, 12.8, 7.4, and 29.4 % 

after 5 d incubation, 8.3, 5.6, 18.7 % after 15 w/d cycles, and 7.0, -2.0, 15.9 % after 30 w/d cycles. 

Such mitigation corresponded to 0.34, 0.16 and 0.20 Mg of CO2.ha -1 for Porrara, Colombaia, and Itri 

soils, respectively, after 5 d incubation, and still to 0.18 and 0.20 Mg of CO2.ha -1 for Porrara and Itri 

soils, respectively, even after severe disaggregation of 30 w/d cycles. The results confirm the 

effectiveness of the proposed technology. 

References 

1. A. Piccolo, Adv. Agron. 75 (2002) 57. 

2. A. Piccolo and M. Spiteller, Anal. Bioanal. Chem. 377 (2003) 1047. 

3. A. Piccolo, P. Conte and A. Cozzolino. Soil Sci. 166 (2001) 174. 

4. A. Piccolo, P. Conte and P. Tagliatesta, Biomacromolecules 6 (2005) 351. 

5. D. Smejkalova and A. Piccolo, Biomacromolecules 6 (2005) 2120. 

6. D. Smejkalova and A. Piccolo, Environ. Sci. Technol., 40 (2006) 1644. 

7. D. Smejkalova, A. Piccolo and M. Spiteller, Environ. Sci. Technol., 40 (2006) 6955. 

8. D.Smejkalova, P. Conte and A. Piccolo, Biomacromolecules, 8 (2007) 737. 

Vol. 3 Page - 224 -

Sorption of Endocrine Disruptors by Humic Substances from Sediment 

Samples Collected on Guarapiranga Reservoir, São Paulo State-Brazil 

Bruno Barboza Cunha a,b , Wander Gustavo Botero a , Luciana Camargo de Oliveira a , 

Guilherme Carvalho Leite b , Danielle Goveia a,b , Viviane Moschini Carlos b , Marcelo Luiz 

Martins Pompêo c , Leandro Cardoso de Morais b , Leonardo Fernandes Fraceto b , André 

Henrique Rosa *b 

a Institute of Chemistry – UNESP, Araraquara-SP, Brazil; b Department of Environmental 

Engineering – UNESP, Sorocaba-SP, Brazil; Institute of Biosciences – USP, São Paulo, 

Brazil 


E-mail: ahrosa@sorocaba.unesp.br 

Endocrine disrupting chemicals (EDCs) are defined as exogenous substances that alter the 

functions of the endocrine system and consequently cause adverse health effects in an intact 

organism, or its progeny. Researches about EDCs have been increasing due to their 

widespread occurrence, persistence, bioaccumulation and potential adverse effects on 

ecosystem functioning and human health [1–3]. 

The EDCs are considering an emergent and dangerous class of contaminants in aquatic 

systems because already are present in high concentrations natural waters and the actual 

wastewater treatment plants can’t remove them. In aquatic systems there are many factors that 

can control the transport, availability and reactivity of EDCs like: pH, redox potential and 

specially content/characteristics of humic substances present in the water and sediment. 

However, there are only a few papers in the literature associated with the interactions between 

Humic Substances (HS) from sediments and EDCs [1, 3, 5]. Then, the primary objective of 

this study was to characterize the interactions beetwen Bisphenol A (BA), Estrone (E), 17β– 

Estradiol (E2) and 17α–Ethinylestradiol (EE) in two different sediments collected on 

Guarapiranga Reservoir, São Paulo City-Brazil. The adsorption process was evaluated and 

Langmuir/Freudlich parameters were obtained from sorption experiments. 



Sorption experiments. The sorption experiments were done with two different sediments 

collected at Guarapiranga Reservoir, São Paulo State, Brazil. All equilibrium batch 

experiments were conducted in triplicates in 100-mL glass vials. Between 0.1 and 1 g of 

sorbent were weighed into the vials, which were filled with 50 mL of milli-Q water with 2 mg 

L -1 of each endocrine disruptor (BA, E, E2 and EE). Vials were shaken on a horizontal plate 

Vol. 3 Page - 225 -

shaker at 180 rpm for 1 day at a constant temperature of 25 o C. Blanks were set up using the 

same solid-to-water ratios as the samples but without adding BA, E, E2 and EE. Similar 

experiments were done to check the equilibrium sorption with the same conditions, except 

that 1 g of sorbent were weighed into the vials and were taken off aliquots of 1 mL at 5, 10, 

15, 30, 60, 120, 240,360, 480 and 1440 min. 

Chromatography analysis. Optimization of SPE extraction of analytes using Nexus cartridges 

(Varian) was performed according to the Nexus applications recommendations for endocrine 

disruptors. The derivatization of standard solutions and samples was performed in a test tube, 

according to the procedure proposed by Jeannot [4] and the Varian GC-MS system was 

employed for analysis. 


Sorption experiments. Adsorption kinetics curves measured for BA, E, E2 and EE in two 

sediments with different contents or organic matter are shown in Figure 1. In all cases, 

adsorption of EDC onto sediments examined appears to occur in two steps, a rapid one 

occurring in the first few hours of contact (in this case less than 2 h), which generally 

corresponds to more than 80% of total adsorption, and a slow one that may need several hours 

until the attainment of the equilibrium. 

q (μg g -1 ) 

100 

90 

80 

70 

60 

50 

40 

30 

20 

10 

BA 

E 

E2 

EE 

0 

0 

0 

a) 

200 400 600 800 1000 

Time (min) 

1200 1400 1600 

0 200 

b) 

400 600 800 1000 

Time (min) 

1200 1400 1600 

Figure 1: Adsorption kinetics curves of EDCs in a) sediment with less organic matter content and b) 

sediment with more organic matter content 

EE appears to be the most adsorbed EDC onto all substrates. The rapid adsorption phase 

would occur on the most reactive and/or accessible sites of the sediment, whereas the slower 

adsorption may reflect the involvement of less reactive and/or more sterically hindered sites. 

In all cases, an equilibration time of 24 h has been considered adequate and has been used 

for the measurements of adsorption isotherms. 


q (μg g -1 ) 

100 

90 

80 

70 

60 

50 

40 

30 

20 

10 

Vol. 3 Page - 226 - 

BA 

E 

E2 

EE

Sorption isotherms. In this work were tested Langmuir and Freudlich sorption models. The 

Langmuir parameter were calculated by regression analysis using the equation 

q 

e 

o 

bQ Ce 

= , where qe is the concentration of a chemical adsorbed in the solid phase (µg 

( 1+ 

bCe 

) 

g 1 ); b is affinity; Q o is the Langmuir coefficient; Ce is the equilibrium solution concentration 

(µg L -1 ) Freundlich parameters were calculated by regression analyses using the equation 

q = k + C 

e 

e 

( 1/ 

n) 

e , where qe and Ce has the same meaning of Langmuir equation; kF is the 

Freundlich coefficient((µg g -1 )/(µg L -1 ) 1/n ) and 1/n the Freundlich exponent, and are 

summarized in Table 1. 


Table 1: Langmuir and Freudlich parameters obtained from sorption experiments 

Isotherm Langmuir parameters Freudlich parameters 

Sediment Compound Q o 

b r kF n r 

P1 BA 0.125 -0.344 0.7926 4.29x10 11 -0.033 0.7416 

P1 E 0.200 -1.174 0.5528 9.17 -0.482 0.2935 

P1 E2 0.399 -0.418 0.7561 57.00 -0.239 0.5537 

P1 EE 0.510 -0.429 0.9936 7.42 -0.593 0.9631 

P2 BA 0.193 -0.370 0.9432 5.63x10 -7 0.123 0.9700 

P2 E 0.243 -0.371 0.9601 2.98x10 -6 0.140 0.9826 

P2 E2 0.255 -0.388 0.9824 8.09x10 -6 0.148 0.9920 

P2 EE 0.668 -0.466 0.9966 5.58x10 -4 0.225 0.9999 

Table 2 – Pseudo second order kinetics parameters obtained from sorption experiments 

Kinetic Parameters 

Sediment Compound h 

qe K r 

P1 BA 0.525 30.93 5.49×10 -4 0.9953 

P1 E 3.866 24.21 6.59×10 -3 0.9992 

P1 E2 0.952 26.30 1.38×10 -3 0.9968 

P1 EE 4.359 73.86 7.99×10 -4 0.9978 

P2 BA 17.844 27.89 2.30×10 -2 0.9986 

P2 E 2.748 36.44 2.07×10 -3 0.9993 

P2 E2 4.697 59.21 1.34×10 -3 0.9998 

P2 EE 23.952 86.80 3.18×10 -3 0.9999 

Data presented in Table 1 shows that the sorption performed with the sample containing higher humic 

substances content is more suited to the Freudlich model. All kinetics experiments have a good fit of 

pseudo second order kinetics model, with correlation coefficients grater than 0.9953. It is possible to 

see in Table 2 that there is a higher rate of adsorption for the samples with higher content of HS, 

except for estrone that can interact preferentially with organominerals present in the sediment samples. 

Vol. 3 Page - 227 -


These results demonstrate that the availability of endocrine disruptors could be directly 

related to the presence of humic substances in aquatic systems. Consequently, studies of the 

interactions between AHS and endocrine disruptors are vital for a better understanding of the 

transport and/or reactivity of this type of contaminant in the environment. 



The authors gratefully acknowledge the funding of their work by FAPESP and CNPq 

(Brazilians Agencies). 

References 

1. R. Liu, J.L. Zhou, A. Wilding, Journal of Chromatography A, 1038 (2004) 19. 

2. G.R. Aiken, R.L. Wershaw, P. MaCcarthy, Humic Substances in Soil, Sediment and Water— 

Geochemistry, Isolation and Characterization, Wiley, New York, 1985. 

3. J. Lintelmann, A. Katayama, N. kurihara, L. Shore, A. Wenzel, Pure Appl. Chem., 75, 5, (2003) 

631. 

4. R. Jeannot, H. Sabik, E. Sauvard, T. Dagnac, K. Dohrendorf, J. Chromatogr., 974 (2002) 143. 

5. E. Loffredo, N. Senesi, Developments in Soil Science, 28A (2002), 143. 

6. A. Navarro, S. Endo, T. Gocht, J. A. C. Barth, S. Lacorte, D. Barceló, P. Grathwohl, 

Environmental Pollution, 157 (2009) 698. 

7. L.P.C. Romão, G.R. Castro, A.H. Rosa, J.C. Rocha, P.M. Padilha, H.C. Silva, Anal. Bioanal. 

Chem., 375 (2003) 1097. 

8. D.J. Hawke, K.J. Powell, J.E. Gregor, Marine Fresh. Res.,47 (1996) 11. 

9. M.L. Pacheco, E.M. Pena-Méndes, J. Havel, Chemosphere, 51 (2003) 95. 

10. S. Chen, W.P. Inskeep, S.A. Williams, P.R. Callis, Soil Sci. Soc. Am. J., 56 (1992) 67. 

11. R.S. Swift, in: D.L. Sparks, (Ed), Methods of Soil Analysis: Chemical Methods, SSSA, Maddison, 

1996, p.1011. 

Vol. 3 Page - 228 -

Dual Effect of Humic Acid on the Degradation of Pentachlorophenol by 

Iron(II) and H2O2 

Konstantinos C. Christoforidis, Maria Louloudi, Yiannis Deligiannakis 

Laboratory of Physical Chemistry, Department of Environmental and Natural Resources 

Management, University of Ioannina, Seferi 2, 30100 Agrinio, Greece 

E-mail: kchristofo@gmail.com; mlouloud@cc.uoi.gr; ideligia@cc.uoi.gr 


Pentachlorophenol (PCP) is a well known organochlorine compound used extensively as 

wood preservative. Chlorophenols are highly toxic and are poorly biodegradable [1] therefore 

efficient abiotic methods are needed for their removal from the environment [2]. 

Fenton reaction [3] has been extensively used for the degradation of a broad range of organic 

pollutants. The HO· radicals generated from the Fenton reaction are highly reactive radicals 

[4]. Since HO· radicals are nonselective, they can react with non-pollutant substances such as 

natural organic mater. This postulates a pervasive effect of humic acid (HA) on a Fenton 

system which can be crucial for the detoxification of natural waters. However the effect of 

HA on the catalytic efficiency of the Fenton reaction still remains controversial, since the 

degradation of organic pollutants has been reported to be either inhibited [5,6] or enhanced 

[6,7] in the presence of humic materials. For example, recently has been reported that the 

initial catalytic conditions may either increase or decrease HO· production [8]. 

In the present study, the efficiency of the Fenton reaction on the decomposition of PCP in the 

presence of HA was studied by catalytic and EPR methods. The data reveal that ratio [HA/Fe] 

is the determining factor which can result in either enhancement or inhibition of PCP 

decomposition. An EPR-based method is proposed for the estimate of the optimal [HA/Fe] 

ratio in order to enhance the catalytic performance. 



HA used was a lignite sample extracted from a mining site of Greece according to the 

protocols of International Humic Substances Society (IHSS) [9]. PCP was purchased from 

Aldrich and H2O2 was obtained as a 30% solution from Fluka. FeSO4·7H2O (Merck) was used 

in the Fenton reactions. For the EPR study of Fe-HA complexation Fe2(SO4)3·xΗ2Ο (Riedelde 

Haën) was used. 2-isopropanol was obtained from Merck. 

Vol. 3 Page - 229 -

All aquatic stock solutions were prepared in ultra-pure Milli-Q water. An 8g/L HA stock 

solution was incubated for 24 h at pH 12 adjusted with NaOH. Afterwards, the solution was 

brought to pH 3 by adding H2SO4 and stored at 4 o C. Aquatic stock solutions of FeSO4·7H2O 

(100 mg/L, at pH 1 adjusted with H2SO4) and of H2O2 (3330 mg/L) were prepared. A stock 

solution of 2000 mg/L PCP was prepared in acetonitrile. Catalytic mixtures were prepared at 

pH 3.5, in the absence and presence of 20 mg/L HA, containing 13 mg/L PCP and varied 

concentration of H2O2 (4.35–54 mg/L) and FeSO4·7H2O (0.87–32.5 mg/L). All reactions 

were kept in dark during the reaction time. Quantification of PCP was performed with HPLC. 

For the EPR experiments 0.4mM of Fe2(SO4)3·xΗ2Ο were incubated with 230, 1710 and 5110 

mg/L HA for 2 h at pH 3.5. 


PCP decomposition. Control experiments have shown that [H2O2] does not affect 

significantly the degradation of PCP in either the classic or the HA-modified Fenon reaction. 

On the other hand, [FeSO4·7H2O] appeared to have a decisive effect on PCP degradation. 

Specifically, depending on the concentration of Fe II , 60–100% and 35–100% of PCP was 

removed by the classic and HA-modified Fenton reaction. Control experiments in the 

presence of 2ml isopropanol in all HA-modified Fenton reactions resulted to no conversion of 

PCP within 9 days (data not shown). This suggests that the degradation of PCP in the 

presence of HA is due to the formation of OH· like in the classic Fenton reaction. However, 

for [FeSO4·7H2O] below 19.5 mg/L in the presence of HA the kinetics is severely lower 

compared to the unmodified Fenton reaction. 

% [PCP] Remained 

100 

80 

60 

40 

20 

0 

A 

Decrease Increase 

0 5 10 15 20 25 30 


100 

80 

Decrease Increase 

60 

40 

20 

[FeSO 4 �7H 2 O] (ppm) 

0 

B 

0 5 10 15 20 25 30 

Figure 1: Effect of [FeSO4·7H2O] on PCP removal in the absence (�) and in the presence of 20 mg/L 

HA (�). Reaction time (A) 3 days and (B) 9 days. Catalytic conditions: 13 mg/L PCP, 52 mg/L H2O2 

Vol. 3 Page - 230 -


Reaction Yield. The effect of varying [FeSO4·7H2O] on the decomposition of PCP at 3 or 9 

days in the presence and absence of HA is compared in Fig. 1. The data show that there is a 

crossing in the reaction yield vs. [FeSO4·7H2O] which occurs at [FeSO4·7H2O] = 6.5 mg/L. 

This crossing implies the existence two –or more- interfering mechanisma. In particular, in 

the presence of 0.87 and 2.6 mg/L FeSO4·7H2O the presence of 20 mg/L HA inhibited PCP 

removal -at any reaction time- under the conditions of our experiment (Fig. 1). On the other 

hand, at higher FeSO4·7H2O concentration (i.e. >6.5 mg/L), the decomposition of PCP 

increased dramatically compared to the reaction without HA. At 6.5 mg/L of FeSO4·7H2O a 

characteristic point appeared where the performance of the catalytic reaction was the same in 

the presence or absence of HA. These results show that HA has a rather complex effect on the 

removal of PCP, i.e. it can act either as an enhancer or as an inhibitor of the Fenton reaction 

depending on the FeSO4·7H2O concentration. 

Overall, under the conditions of our experiments, HA appears to play a significant dual role 

on the decomposition of PCP by Fenton reaction. The data revealed that HA can act either as 

an inhibitor or as an enhancer. A key observation is that the function of HA is correlated with 

the iron concentration. The beneficial role of HA in total PCP conversion is observed only 

when [FeSO4·7H2O]>6.5 mg/L. Based on the results presented in Figure 1, the total 

conversion of PCP is determined by the ratio HA/Fe. In particular when [HA 

(mg)]/[Fe(μmol)]2.7 Fe III forms hydroxylated species Fek(OH)m which are EPR silent. 

However, at low pH values (pH=1) a signal with g=4.3 and E/D=0.33 -characteristic of 

mononuclear high spin Fe III - was observed. On the other hand, in the presence of HA at pH 

3.5 a high spin Fe III with g=4.3 was detected. This shows that HA mentains mononuclear Fe III 

species, acting as chelator of Fe. 

Vol. 3 Page - 231 -

dX''/dH (au) 

A 

(e) 

(d) 

(c) 

(b) 

(a) 

Signal Intensity 

400 800 1200 1600 2000 2400 

Magnetic Field (G) 

% [Fe III Adsorbed + (EPR silent species)] 

40 

30 

20 

No HA 

10 

0 

B 

Increase 

of PCP 

oxidation 

Decrease 

of PCP 

oxidation 

[FeSO 4 •7H 2 O]=0.87 ppm 

[FeSO 4 •7H 2 O]=2.6 ppm 

[FeSO 4 •7H 2 O]=19.5 ppm 

0 1500 3000 4500 

[HA] (ppm) 

Figure 2: A) EPR spectra of 0.4mM Fe2(SO4)3·xH2O incubated in the absence of HA, spectrum (a) 

and (b) (spectrum (b) has 30 % v/v glycerol) and in the presence of (c) 230, (d) 1710 and (e) 5110 

mg/L HA for 2 h at pH 3.5. B) [Fe III -HA+ Fe-SL] % vs. [HA] used for each sample. Each case is 

labelled with the [FeSO4·7H2O] of the corresponding Fenton reaction 

Figure 2B shows the percent of the sum of Fe III that has been adsorbed by HA (Fe-HA) along 

with the EPR silent Fe species (Fe-SL), i.e. Fe III reduced to Fe II by HA [10] or dimers of iron. 

It is observed that increase of the HA concentration resulted to an increase of [Fe-HA + Fe- 

SL]. Based on the data in Figure 2B, the % of [Fe-HA + Fe-SL] where the oxidation of PCP 

is increased in the presence of HA is estimated to be less than 7.5%, otherwise it is decreased 

(areas indicated by the arrows). 

Comparing the catalytic with the spectroscopic EPR data we observe that (a) in cases where 

the presence of HA inhibits the decomposition of PCP, the [Fe-HA + Fe-SL] ratio is 40% and 

25% ([FeSO4·7H2O]=0.87 and 2.6 mg/L) respectively, while it is only 1% for the case where 

HA improves the modified Fenton reaction ([FeSO4·7H2O]=19.5 mg/L). Furthermore, HA 

could act as a sink of HO·. In this way HA inhibits the oxidation of PCP by the Fenton 

reaction, competing in this way with PCP and resulting to a significant degrease of the 

reaction’s efficiency. Reaction of HA with OH· has been reported in the literature in a photo- 

Fenton system [11]. 

Overall, our results indicate that Fe III EPR intensity, along with high [Fe-HA + Fe-SL] ratio 

determines the catalytic efficiency depending on the HA/Fe ratio. 



Based on the catalytic results, the only way that HA may affect catalytic removal of PCP is: 

(I) to chelate strongly Fe without allowing it to react with H2O2 and 

(II) to promote the Fenton redox cycle of Fe [12]. 

Specifically, case (I) would hold when [HA(mg)]/[Fe(μmol)]>0.85 while case (II) when 

Vol. 3 Page - 232 -


[HA(mg)]/[Fe(μmol)]

Radiotracer Method in the Investigation of Humic Substances Sorption on 

Carbon-Based Nanomaterials 

Maria G. Chernysheva, Gennadii A. Badun 

Lomonosov Moscow State University, Leninskie Gory, 119992, Moscow, Russia 

E-mail: masha.chernysheva@gmail.com 


Nanomaterials of carbon are widely use in different fields. The physical, chemical, and 

electronic properties of carbonaceous nanomaterials are strongly coupled to carbon’s 

structural conformation and, thus, its hybridization state. Mutable hybridization states of 

carbon account for the diversity of organic compounds as well as the considerable differences 

among carbon’s bulk configurations, which changes from sp 3 for nanodiamond to sp 2 +π for 

graphene. Studying the properties of carbonaceous nanomaterials is the important field of 

modern science of material. Now days carbon nanomaterials particularly carbon nanotubes 

(CNT) are tested as a unique substrate for sorption of biomolecules including peptides [1] and 

proteins [2, 3]. In review [4] single-walls carbon nanotubes were also tested for removal of 

contaminants in drinking water. It is appear to be perspective to study the binding of humic 

substances with different types of nanocarbon. The goal of this research was to compare the 

sorption ability of nanodiamonds and graphene unto humic materials by means of 

radiochemical approach [5] using tritium labeled humic materials and liquid scintillation 

spectrometry. 



Brown coal humic acids (CHA) (commercially available preparation Powhumus (Humintech, 

Germany)) was used. Tritium label was introduced in CHA by means of thermal activation 

method. The labeling technique and purification procedures were previously described in Ref. 

[6]. 

Sorption experiments with carbon nanomaterials were conducted at room temperature in 

0.028 M phosphate buffer (pH 6.8) for the initial concentrations from 1 to 360 mg/L. Sample 

of nanomaterial was placed in the test-tube followed by the addition of [ 3 H]-CHA solution in 

phosphate buffer. Mixture was sonicated during 15 min. Then a bit of suspension was picked 

out, centrifuged and its counting rate was measured by liquid scintillation counter RackBeta 

1215 (Finland). Equilibrium concentration of CHA solution was calculated from the specific 

Vol. 3 Page - 234 -

adioactivity of CHA and radioactivity of the solution. Adsorption amount of CHA was 

calculated as a difference between initial and equilibrium amount of CHA in the solution. 


Equilibrium CHA concentrations were from 0.2 to 150 mg/L. Comparative analysis of 

adsorption isotherms obtained shows that on both nanodiamonds and graphene CHA 

adsorption increase with concentration growth until 100±10 for nanodiamonds and 30±5 

mg/L for graphene. Then it archive plateau and does not change for nanodiamonds, but start 

increase again in case of graphene at 120±10 mg/L. The value of adsorption per gram of 

nanocarbon in plateau region was 20±5 mg/g for both materials. It has to be noted that 

desorption under the same conditions was ca. 30 % for HS on nanodiamonds and less then 

10 % in case of graphene. It was also found that graphene with adsorbed CHA forms stable 

suspension in aqueous solution while graphene itself is to hydrophobic to be placed in water. 


Radiochemical assay for the first time was applied for studying sorption of HS on carbonbased 

nanomaterials. This approach allow conduction of sorption experiment with HS in wide 

range of concentrations from ultra low (less then 1 mg/L) to hundreds mg/L. The difference in 

the adsorption isotherms results in the influence of carbon structure. 



This work was supported by Russian Federal Agency of Education (project # 2351P). 

References. 

1. G.R. Dieckmann et al., J. Am. Chem. Soc. 125 (2003) 1770. 

2. L. Song et al., Colloids and Surfaces B. 49 (2006) 66. 

3. L.E. Valenti et al., J. Colloid Interface Sci. 307 (2007) 349. 

4. V.K.K. Upadhyayula et al., Sci. Total Environment. 408 (2009) 1. 

5. A.V. Severin, G.A. Badun, Z.A. Tyasto. Radiochemistry, 51 (2009) 55. 

6. G.A. Badun et al, Radiochimica Acta, (2010) (in press). 

Vol. 3 Page - 235 -

Study of Flow-Through Sample Preparation Methods for Group of 

Pesticides Determination in Soil by Reversed-Phase High-Performance 

Liquid Chromatography 


Mária Chalányová * , Milan Hutta, Ivana Procházková 

Department of Analytical Chemistry, Faculty of Natural Sciences, 

Comenius University, Mlynská dolina CH-2, 842 15 Bratislava, Slovakia 

E-mail: chalanyova@fns.uniba.sk 

Soil is a heterogeneous system containing organic and inorganic matter. It contains solid 

particles, soil colloids and gases among which equilibrium is established [1]. Soil from this 

point-of-view is a complex matrix and the isolation of analytes from soil is a complex 

analytical problem [2]. From literature survey clearly follows that the soil sample pretreatment 

is usually a critical and time consuming step also due to presence of humic 

substances. The basis of successful analytical method for determination of pollutant residues 

in complex matrices at trace concentration levels is the use of selective isolation techniques 

[3, 4] enabling isolation of analytes with high recovery and with minimum quantity of matrix 

co-extracts. Flow-through methods, namely matrix solid phase dispersion (MSPD) and offline 

flow-through solid-liquid extraction (FSSLE) for isolation eight common pesticides 

(atrazine, propazine, simazine, terbutrine, metoxuron, cloquintocet-mexyl, cypermethrin 

and permethrin) from soils before their RP-HPLC analysis were studied. 



Procedure of MSPD pre-treatment of soil sample:The bottom of plastic column was filled by 

0.05 g silica or florisil and mixture of 0.50g of contaminated and uncontaminated soil sample 

homogenized with around 0.50 g of sorbent. The mixture of 8 pesticides was eluted with 2.00; 

2.50 and 3.00 ml of 100% of methanol, resp. Collected extract volume was reduced to 0.20 or 

0.50 ml and reconstituted in 0.50 or 1.00 mL volume in the mobile phase methanol:water 1:1 

(v/v). 

Procedure of off-line flow-through solid-liquid extraction (FTSLE) pretreatment of soil 

sample: bottom part of CGC column (150x3 mm) was filled with mass of Silica L 40/100 

ranging from 0.05 g to 0.20 g. This layer was topped by fill and tap method by 1.00 g of soil 

sample (alternatively, original soil or soil fortified by pesticides at various concentration 

levels). The rest of the column volume was filled up by glass beads (0.5 mm diameter). 

Studied analytes were extracted by various volumes of methanol by flow rates alternatively 

Vol. 3 Page - 236 -

switched between 0.5 and 0.3 mL/min. Further processing of the extract was the same as is 

described above. 


Influence of mobile phase pH to the pesticides retention was thoroughly studied for definition 

of optimal separation conditions. Methanolic mobile phase pH was adjusted by aqueous 

phosphate buffer in the pH range 2.5-5.5. Dependence of retention times of individual 

investigated pesticides on pH is shown in Fig. 1. 

35 

30 

25 

20 

15 

10 

5 

0 


2 2,5 3 3,5 4 4,5 5 5,5 

pH 

metoxuron 

simazine 

atrazine 

propazine 

terbutrine 

cloq 

cypermethrin 

permethrin 

Figure1: Dependence of retention time of selected pesticides on methanolic mobile phase pH 

The optimal chromatographic conditions chosen for the separation of 8 pesticides were: 

Purospher Star RP 18e (50x4mm) analytical colum, separation of analytes was achieved by 

linear gradient elution from methanol:aqueous pH buffer 50:50 (v/v) to 100% methanol in 35 

minutes. The injection volume was 0.02 mL. UV spectrophotometric detection was done at 

235 nm wavelength. The mobile phase flow rate was 0.5 ml/min. 

The work is presenting results of the study of several parameters affecting the extraction 

efficiency of 8 pesticides at concentration levels around 1-2.5 mg / kg dry soil. 

MSPD: For MSPD pretreatment of soil sample optimal conditions for given pesticides were 

achieved by use of silica L 40/100. Experimental conditions for isolation of pesticides are as 

follows: desorption by 3.00 mL of 100% methanol, reduction of the extract volume to 0.50 

mL by evaporation under dry air stream, make-up the volume to 1.00 ml. Extraction recovery 

for the mixture of 8 studied pesticides achieved 2.5 mg/g dry soil, within the range 56-94%. 

Vol. 3 Page - 237 -


Table 1: Extraction recovery of selected pesticides from soil contaminated by pesticides in 

comparison to their standard addition into the extract of the same soil MP/20 at 2 

concentration levels 

Recovery (%) 

1.0 µg/g 2.5 µg/g 

Analytes 

metoxuron 

Extract Std. 

dd 

96,1 ± 3,9 

Contam. Soil 

62,6 ± 1,8 

Extract Std. 

dd 

107,2 ± 2,9 

Contam. Soil 

67,5 ± 0,3 

simazine 108,4 ± 3,3 78,3 ± 1,2 109,5 ± 2,6 94,3 ± 4,2 

atrazine 102,1 ± 6,4 71,3 ± 0,7 107,3 ± 8,3 77,8 ± 1,1 

propazine 82,0 ± 2,9 64,5 ± 0,7 102,7 ± 2,5 71,3 ± 4,4 

terbutrine 79,6 ± 0,1 65,6 ± 0,1 90,1 ± 2,9 69,1 ± 1,4 

cloq 67,7 ± 0,5 48,3 ± 1,4 89,5 ± 0,6 56,4 ± 1,2 

cypermethrin 68,5 ± 5,7 57,7 ± 1,1 89,4 ± 2,1 69,2 ± 1,6 

permethrin 69,8 ± 8,3 63,5 ± 1,2 81,7 ± 0,5 60,9 ± 3,2 

FTSLE: For FTSLE soil pretreatment the optimal mass of silica bottomed in the column is 

0.50 g. Experimental conditions for the isolation of pesticides are as follows: desorption by 

3.00 mL of 100% methanol, flow rate 0.30 ml/min, reduction of the extract volume to 0.50 

mL by evaporation under dry air stream, make-up the volume to 1.00 mL. Extraction recovery 

for the mixture of studied pesticides achieved 2.5 mg/g dry soil was within the range 61-88%. 

Table 2: Recovery of pesticides using FTSLE at 0.3 mL/min from soil contaminated by 

pesticides in comparison to their standard addition into the extract of the same soil MP/20 at 2 

concentration levels 

Recovery (%) 

2.5 µg/g 1.0 µg/g 

analytes 

metoxuron 

Extract Std. 

105,1 

dd 

± 1,8 

Contam. Soil 

73,3 ± 2,4 

Extract Std. 

106,4 

dd 

± 5,7 

Contam. Soil 

67,3 ± 1,0 

simazine 105,5 ± 4,2 81,6 ± 2,4 109,3 ± 3,0 78,2 ± 2,5 

atrazine 102,4 ± 3,9 81,1 ± 0,7 104,5 ± 4,8 72,8 ± 6,2 

propazine 103,9 ± 2,5 81,8 ± 1,4 106,1 ± 4,2 73,3 ± 4,0 

terbutrine 98,6 ± 0,9 83,5 ± 0,2 101,1 ± 3,2 75,4 ± 6,1 

cloq 91,6 ± 0,2 88,3 ± 0,5 84,8 ± 2,2 68,4 ± 5,0 

cypermethrin 62,6 ± 2,4 61,1 ± 3,0 65,0 ± 2,8 56,5 ± 1,7 

permethrin 62,8 ± 3,3 63,2 ± 2,1 62,1 ± 0,4 54,6 ± 5,6 

Volumes: Vfraction=3.0 mL; Vafter evaporation=0.5 mL; Vfinal= 1.0 mL. 

Vol. 3 Page - 238 -


Under the defined conditions both studied sample pre-treatment methods gave comparable 

recoveries of pesticides investigated by RP-HPLC. 



The work was supported by grants VEGA 1/0870/09, APVV-0595-07 and VVCE-0070-07. 

References 

1. C.Sánchez-Brunette, B. Albero, J. L. Tadeo, Determination of Pesticides in Soil, in J. L. Tadeo, 

(Ed.,) Analysis of Pesticides in Food and Environmental Samples, Chapter 8., CRC Press, Boca 

Raton, 2008, pp. 207-230. 

2. T. A. Howell, in: D. Hillel, (Ed.), Encyclopaedia of Soils in the Environment, Oxford, UK, 

Elsevier Press, 2004, pp. 379–386. 

3. M. Chalányová, M. Paulechová, M. Hutta, J. Sep. Sci., 29 (2006) 2149. 

4. I. Rybár, R. Góra, M. Hutta, J. Sep. Sci., 30 (2007) 3164. 

Vol. 3 Page - 239 -

Humus Substance Role in Technogenic Soil Formation at Priokhotie Mining 

Industry Developments 

Makhinova А.F., Makhinov А.N. 

Institute of Water and Ecology Problems FEB RAS, 65, Kim Yu Chen st., Khabarovsk, 

Russia 

E-mail: mahinova@ivep.as.khb.ru 


Open mining and intensification of mineral extraction in Priokhotie cause destruction of 

enormous forest massifs. Forest soil resources being a deficit, the problem of soil reclamation 

and cultural technogenic landscapes formation becomes most urgent. Technologies of mine 

reclamation and technogenic soil formation first of all involve intensive reclamation of a 

humus-organogenic horizon and in particular covering the planned surface of the dump with 

humus-accumulative horizons of mountain-taiga soils removed at the initial stage of field 

development [1]. Reclamation of technogenic landscapes still remains a single-action activity. 

At present actually no ecological monitoring is implemented [5]. However, a technogenic soil 

profile shows significant changes in physical and chemical characteristics and soil-formation 

processes, which cause soil degradation and which mechanisms are weakly-studied. 

The work was focused on the studies of physical and chemical specifics of recultivated soils 

and the role of humus substances in soil profile degradation. 



The objects of study were technogenic soils formed at reclaimed dumps in the Taryng-Lata 

River basin. Observation sites were chosen on the 12-8 о gradient mountain slope of NE 

position in Ayano-Maisky District of Priokhotie. Dump reclamation with an organogenic 20 

cm-layer was performed immediately after the stripping operations were completed. Two 

observation sites were made on the reclaimed land down the slope 50 meters apart. The 

distance between the site profiles was 6 meters. An alluvium-diluvium layer was 48 cm-think 

at the upper site and 56 cm-thick at the lower site. To prevent slipping down of filled-up and 

organogenic layers the section wall, perpendicular the slope lengths, was enforced with metal 

mesh pieces 3 meters long and up to 45 cm deep. The thickness of upper layers was 20 cm. 

When a stable grass cover appeared three years after reclamation, two types of technogenic 

soils at different slope gradients were studied: 1) with a filled-up mineral-organogenic layer 

on a metal mesh with D-3 cm cells (Profiles 1 and 2); 2) with a filled-up organogenic weakly- 

Vol. 3 Page - 240 -

decomposed layer (Profiles 3 and 4). Standard methods were used to estimate technogenic 

soil density, chemical and physical characteristics, granulometric composition and carbon 

concentrations [3]. 

Observation site 1 (P-1 and P-3). Analytical studies showed no differentiation of organic 

carbon content in the technogenic soil profile. In Profile 1, characterized with a rich mineralorganogenic 

layer, inner-ped pores were colored with a dark-grey humus cutan. In P-3 (20-30 

cm layer) brown films were found on the sides of ped structures and water filtration rate was 

increased. 

Some experimental studies were undertaken to describe pore space and prevailing mode of 

soil water migration. 100 cm-long glass pieces were fixed of the metal mesh on the profile 

walls along the slope. Water was poured on the profile surface till a 2 cm-water column was 

formed on the frozen rock layer. It was found out that compared to technogenic soil with a 

filled up mineral-organogenic layer (P-1), in technogenic soil with a filled up organogenic 

layer (P-3) the descending water flow rate was 1.21 times higher and the inner-lateral 

discharge of water solution was 1.35 times more rapid (Table 1). All the measurements were 

repeated three times. 

Table 1: ome Physical and Chemical Characteristics of Technogenic Soils 

№ 

РР 

Р1 

Р3 

Р2 

Р4 


Layer 

thickness, 

cm 

РН 

water salt 

Unit 

weight 

g/сm 

Sum of 

fractions , % 

3 

Density 

g/сm 

3 

%Po 

-res 

in 

volume 

(V) 

filtr. 

cm/ 

sec 

>0,01

cm. Field studies also revealed humus-ferrous cutans in the mineral layer (20-30 cm) of P1 

under the organo-mineral layer; brown films on the sides of ped structures were rarely 

observed. 



Morphological studies at the observation sites revealed that a) thickness of upper filled up 

mineral organogenic and organogenic layers decreased; b) all mineral layers of technogenic 

soils became more dense; c) mechanical washing out of filled up layers changed their color 

from brown into dark-brown; structural fragments became more solid and clayey fractions in 

them increased. Land reclamation with definite boundaries between the soil layers caused the 

formation of technogenic soils different from original forest soils. Soil layer transformation 

degree in the whole profile primarily depends on the character of humus-organogenic layers, 

used for soil recultivation, as well as on the degree and duration of their watering and the 

slope gradient. Warm-time rains accelerate washing out of filled up organogenic-mineral soil 

into the supporting layers [2]. High concentrations of washed out humus increase the 

formation of peds, different in size and surface specifics, and cause their “glueing” and layer 

packing. 

Morphological studies of ped surfaces show that in P-3 a brown film on the aggregate surface 

is ferriferous, that indicates the presence of more aggressive fractions of fulvic acids, which 

form an organogenic layer, while being decomposed. Ferric hydroxide prevalence in the 

upper 20-30 cm layer restrains their mobility and excludes alluvial genesis of cutans 

(penetrated from the upper layer). The main formation mechanism of differences in 

technogenic soils may be of a microbiological nature on the background of heterogeneity of 

organic matter in the filled up layers. Increased concentrations of Fe ions that form ion 

bounds of a crystallization type result in the formation of solid aggregates, and thus in a wellmarked 

inter-aggregate porosity in the technogenic soil layers. That is why, the rate of 

filtration through inter-ped cracks increases (Table 1). Similar changes of soil filtration 

characteristics were also described by several other authors [4]. 

There are no big differences between profiles 2 and 4 (site 2). Still, the following differences 

from the first (upper) site should be noted: a) increase of technogenic profile thickness and 

density of a 20-40 cm layer; b) water capillary disperse indicates weaker permeability of the 

layers; c) permafrost melting goes much slower in the lower layers due to weak inner-lateral 

water discharge; lower layers are temporary over wet; d) sod pedogenesis prevails in P-2; 

e)mycelium appears at the bottom of the upper matted and organogenic layers. 

Vol. 3 Page - 242 -



Technogenic landscape reclamation in Priokhotie causes irreversible physical and chemical 

changes and technogenic soil formation. Dump reclamation with organogenic (organo- 

mineral and other) soil layers accelerates sod pedogenesis and technogenic soil formation, in 

most cases characterized with formation processes, different from those of disturbed soils. 

Due to the absence of sod pedogenesis on the steep slopes (>10 о ) at the initial stage of 

technogenic soils formation, the filled up layer thinners to 7 cm: a) in P-3 an organogenic 

layer is rapidly decomposed; b) in P-1 an organo-mineral layer is rapidly washed out through 

a large pore space into the supporting layers. Direction of soil forming processes depends on 

the specifics of a layers used for recultivation. The use of coarse organogenic layers 

intensifies the formation of aggressive humus fulvic acids, processes of fersiallitization and 

the formation of ferriferous films on the sides on peds. Organo-mineral complexes in humous 

soil layers used for land reclamation foster the processes of alluvial and humus discharge and 

formation of drip humus-ferriferous inter-aggregate films. Thickness of diluvia deposits plays 

a significant role in technogenic soil formation on the gentle slopes and impacts the length of 

permafrost processed and the formation of a zone of capillary-suspended moisture. Appearing 

anaerobic processes most often cause gleization inside the peds and misbalanced Fe secretion 

on the ped sides. 

References 

1. Е.V. Abakumov, E.I. Gagarina. Land reclamation in post-technogenic landscapes and physical 

characteristics of dump ground // Proc. All-Russia Conf. “Fundamental Physical Studies in Soil 

Science and Melioration” М., 2003. P. 262-264. 

2. J.L. Anderson, J. Bouma. Relation between hydraulic conductivity and morphometric data of an 

argillic horizon //Soil Sci. Soc, Am. Proc. 1973. V.37. P. 408-413. 

3. А.F. Vadunina, Z.А. Korchagina. Methods for studying physical characteristics of soil. М.: 

Agropomizdat, 1986. 241p. 

4. J. Bouma. Hydropedology as a powerful tool for environmental policy research // Geoderma. V. 

131. #3-4. P. 275-286. 

5. A.F. Makhinova, A.N. Makhinov. Risk assessment of soil degradation and possible soil 

recultivation in mining in Priokhotie region //V.1. From Molecular Understanding to Innovative 

Applications of Humic Substances. Proceeding of the 14 th Meeting of Internati-onal Humic 

Substance Society. September 14-19, 2008, Moscow-Saint Petersburg, Russia, Editors: I.V. 

Perminova, N. A. Kulikova, Vol.P, Sapiens, Moscow, 2008, P.273-278. 

Vol. 3 Page - 243 -


Vol. 3 Page - 244 -


Industrial Production and Commercial Applications 

Vol. 3 Page - 245 -

Study of Copper Extraction Efficiency by Humic Acid/Polypyrrole on 

Paraffin-Impregnated Graphite Electrode 

Mónica Antilén * , Miguel Araus, Mauricio Pérez-Ponce, Francisco Armijo, Rodrigo del Río, 

M.A. del Valle 

Pontificia Universidad Católica de Chile, Vicuna Mackenna 4860, 7820436, Chile 

E-mail: mantilen@puc.cl 


Due to its polyfunctionality, humic acid, HA, is one of the most powerful chelating agents 

among the existing natural organic substances: they are able to complex heavy metals [1], 

inorganic anions and halogens [2], organic acids [3], aromatic compounds [4], pesticides and 

herbicides [5]. This allows establishing that HA may alter the availability, transport, fixation, 

and toxicity of environmental contaminants. On the other hand, it is well known that the 

electrochemical activity of conducting polymers, such as polypirrole (Ppy) is accompanied by 

the insertion and ejection of anions from the electrolytic solution according to the reaction: 

Ppy + xA − 


oxidation 

⎯⎯⎯→ reduction 

←⎯⎯⎯ 

[(Ppy) x+ (A − )x] + xē 

where A is a dopant anion to compensate the positive charges generated during the oxidation 

process and x is the (level) dopant [6]. Because in this case polypyrrole is the type of polymer 

that can be p doped/undoped, it has been previously utilized to remove contaminants such as 

arsenic species [7], and results showed that the matrix has a preference for AsO4 3− species. 

Moreover, the natural properties of PPy as anion exchanger explain why it has been widely 

employed for film preparation in solid phase micro-extraction coupled to chromatographic 

techniques [8], an advanced technique to obtain analytical samples of volatile and 

semivolatile organic compounds. The formation of composite by the insertion of polyethylene 

glycol (PEG) has been previously reported to improve Ppy properties. The results 

demonstrated that at low PEG concentration an open structure of the film is obtained and 

consequently the insertion and ejection of ions to the electrolyte is accelerated [6]. From the 

abovementioned background about the different approaches by which an electrode can be 

modified to incorporate HA in a reproducible way, obtaining devices with improved 

sensitivity and within the spectrum allowed by this kind of matrix, a novel alternative for the 

preparation of electrodes in a simple, reproducible and using chelating properties of humic 

substances, along with the of p-doping of conducting polymers properties, predicting a 

synergistic effect, is proposes in this article. 

Vol. 3 Page - 246 -



Apparatus: All electrochemical experiments were performed on a VoltaLab PGZ100 

potentiostat system using a three-compartment/three-electrode glass cell under an argon 

atmosphere. Paraffin-impregnated graphite (0.28cm 2 ) was used as working electrode (PIGE). 

A Pt wire (20 cm 2 ) was the counter electrode. All potentials quoted in this article were 

measured versus a Ag|AgCl reference electrode. PIGE was prepared by impregnating 

spectroscopic graphite rods with melted paraffin wax under vacuum [9]. 

Chemicals and solutions: HA (Sigma–Aldrich) was purified by precipitation as previously 

described [9]. Stock solutions containing Cu(II) in 20 mM HCl + 100 mM KCl (solution B) 

were prepared by dissolution of the appropriate amount of CuCl2 in twice-distilled water 

according to standard procedures. Pyrrole (Sigma–Aldrich) was purified by distillation, stored 

at 4ºC, and protected from light. All other reagents used were analytical grade (Merck). All 

solutions were deaerated with pure Ar for at least 20 min 

Preparation of modified PIGE: As for PIGE, four electrodic surfaces were characterized by 

cyclic voltammetry: (1) PIGE, (2) PIGE electrochemically modified by pyrrole (PIGE/PPy), 

(3) PIGE modified with HA transferred and immobilized onto the electrode surface using the 

abrasive transfer technique [10] and then electropolymerized with pyrrole (PIGE/HA/PPy), 

and (4) PIGE modified with HA and then electropolymerized with pyrrole-polyethylene 

glycol (PIGE/HA/PPy-PEG). Electrochemical growth of PPy or Ppy-PEG films was carried 

out on PIGE or PIGE/HA from a 1.4 mM pyrrole + solution A (50 mM Na2SO4 + 30 mM 

H2SO4) by potentiodynamic methods with the application of five successive cycles between − 

0.2 and 1.0 V [9], while PPy-PEG films were prepared with 1.0 g L −1 PEG (M.W. 1000). 

Extractions: Three electrode surfaces, PIGE/PPy, PIGE/HA/PPy, and PIGE/HA/PPy-PEG, 

were studied in solutions containing 22.32 mM CuCl2 + 20 mM HCl + 100 mM KCl (solution 

C , cell A) and 20 mM HCl + 100 mM KCl (solution B, cell B) to select the doping–undoping 

potentials to perform the extractions. The total volume of electrolyte in each cell was always 

10 mL. Each extraction was carried out as follows: the modified PIGE was immersed in 

solution C and potentiostatically perturbed at 0.4 V for 5 min, after which it was dipped into 

solution B, wherein it was potentiostated at -0.6 V for 5 min. Initially and after several 

extractions, the copper concentration in solutions A and B was determined by inductively 

coupled plasma/optical emission spectrometry (ICP–OES) on a Varian Liberty series II 

instrument. 

Vol. 3 Page - 247 -


Figure 1 depicts the stable voltammetric profile of PIGE without modification, PIGE 

modified with Ppy, PIGE modified with HA-Ppy, and the same PIGE modified with HA- 

PEG-Ppy. The comparative response of each electrode clearly shows that all modifications 

were successfully accomplished, causing an increase in the capacitive response. However, the 

incorporation of HA led to a current drop (not showed) indicating clearly that it is a non- 

conducting solid, where no redox processes occur, demonstrating also that adsorption of HA 

on the PIGE surface takes place [9]. Consequently, PIGE-HA-PPy and PIGE-HA-Ppy-PEG 

modifications produce the highest current values. This has been ascribed to the type of 

porosity and morphology of the obtained polymer as a result of HA and PEG incorporation [6, 

9]. 


Figure 1: Potentiodynamic response of 

PIGE; PIGE/Ppy; PIGE/HA/Ppy and 

PIGE/HA/Ppy-PEG in 20 mM HCl and 

100 mM KCl between -0.6 V and 0.4 V 

at 0.1 V s −1 (I is current intensity and E is 

the voltage) 

Table 1 summarize the results obtained with the modified electrodes tested for copper 

extraction, where the electrode possessing HA and in addition a composite of PPY-PEG is the 

most efficient to extract Cu(II) species. This high efficiency could be explained due to a HA− 

Cu(II) species interaction as 1:1 complexes, which would be boosted with the help of the 

doping-undoping process of the conductive composite Ppy-PEG. The chemical speciation 

obtained by Geochem [11], indicated that about 95% of Cu(II) is as CuCl4 2- which is essential 

in order that Ppy-PEG exchanges these anions from its film. Because both species (HA and 

CuCl4 2- ) bear negative charge, weak interactions are expected. However, the results indicate 

that this is not the case, because it was electrochemically demonstrated that a drop of the 

Cu(II) signal (data not shown) occurs in the presence of HA. Since copper chloride ion has a 

formal charge of 2+, a phenolate addition to the electrophilic center followed by protonation 

and water release might take place in a similar manner to other anions [9]. Therefore, 

adsorption mechanisms other than electrostatic ones (charge) may occur, since the possibility 

of forming cross-bridges between acid molecules has been suggested [12]. 

Vol. 3 Page - 248 -

Table 1: Copper (II) extracted as a function of modified PIGE electrode 

Modified 

electrode 

Extraction 

number 

Initial 

Cu(II) 

(g L -1 ) 

Extracted 

Cu(II) 

(g L -1 ) 

PIGE/PPy 10 1.267 ± 0.013 0.00160 ± 0.00002 

0.00177 ± 0.00004 

PIGE/HA/PPy 

10 

1.398 ± 0.014 0.00281 ± 0.00003 

1.262 ± 0.013 0.00290 ± 0.00003 

PIGE/HA/PPy-PEG 10 1.530 ± 0.015 0.00810 ± 0.00003 

1.532 ± 0.015 0.00830 ± 0.00003 

On the other hand, average charge results for all modified electrodes indicated the existence 

of a reversible doping/undoping process in acid medium, which means that the stability of the 

electrode would remain unchanged even after 10 extractions. 


Finally, the use of a composite (PPy-PEG) has enabled improving the porosity of the polymer 

which in turn, in the presence of HA, has proved to be the most efficient Cu(II) extractor. 



The authors thank to DIPOG, PUC-Chile and Financiamiento Basal para Centros Científicos 

y Tecnológicos de Excelencia, under grant FB-0807 for funding this research project. 

References 

1. P.Lubal, D. Široky, D. Fetsch, J. Havel, Talanta, 47 (1998) 401. 

2. S.C. Myneni, Science ,295 (2002) 1039. 

3. A. Cozzolino, P. Conte, A. Piccolo, Soil Biol.& Biochem., 33 (2001) 563. 

4. K. Nam and J.Y. Kim, Environ. Pollution, 118 (2002) 427. 

5. U. Klaus, T. Pfeifer, M. Spiteller , Environ. Sci. Technol., 34 (2000) 3514. 

6. R. Schrebler, P. Cury, H. Gómez, R. Córdova, L.M. Gassa, Bol. Soc. Chil. Quím., 47 (2002) 537. 

7. M.A. del Valle, G.M. Soto, L. Guerra, J. Vélez, F.R. Díaz, Polymer Bull., 51 (2004) 1436. 

8. J.Wu and J. Pawliszyn, J. Chromatogr A, 909 (2001) 37. 

9. M. Antilén and F. Armijo, J. Appl. Polym. Sci., 113 (2009) 3619. 

10. F. Scholz and B. Meyer, Electroanalytical Chemistry, A series of Advances, Marcel Dekker: New 

York, 1998, p 8. 

11. D. R., Parker, W.A. Norvell, and R. L. Chaney. Chemical equilibrium and reaction models, SSSA 

Spec. Publ. 42, ASA and SSSA, Madison,1995, p 253. 

12. K.M. Sparks, J.D. Wells, B.B. Johnson, Aust. J.Soil Res., 35 (1997) 89. 

Vol. 3 Page - 249 -

Sorption of Silanized Humic Derivatives onto Montmorillonite Clay 

Vladimir A. Kholodov a* , Vladimir M. Zelikman b , Kirk Hatfield c , Irina V. Perminova b 

a Dokuchaev Soil Science Institute, Pyzhevskiy per. 7, 119017 Moscow, Russia; b Department 

of Chemistry, Lomonosov Moscow State University, Leninskie Gory 1-3, 119991, Moscow, 

Russia, c University of Florida, Gainesville, Florida, USA 

E-mail: vkholod@mail.ru 


Composite materials on the basis of humic substances (HS) and alumosilicate might be used 

in the field of environmentally sound technologies. However, relative low sorption capability 

of HS toward alumosilicate confines application of these materials. Affinity of HS to 

alumosilicate could be increased by treatment of HS with organosilanes [1]. The goal of this 

work was to compare sorption capability toward montmorillonite clay of silanized and native 

HS. 


The montmorillonite clay from the deposit “10 khutor”, Khakassia, Russia was used. Clay 

was dispersed by ultrasonic treatment several times with separation of coarse fraction by 

decantation. After that, montmorillonite was Ca 2+ saturated by fivefold treatment with 0.001 

M CaCl2. 

The silanized humic materials were derived by treatment of HS from leonardite (the 

preparation “Powhumus”, Humintech Ltd., Germany) by aminopropyltroethoxysilane as 

described in [2]. 

Sorption capability of HS was estimated in batch adsorption experiments. Stock solution of 

HS (silanized or native) containing 0.001 Ca 2+ at pH 5.5 was added to 50 mg of 

montmorillonite to the final concentrations of 25-4000 ppm. Solutions were shaken during 

48 h. Equilibrium concentrations of humic substances ([HS], ppm) were determined by 

optical density at 465 nm. HS amount adsorbed onto montmorillonite (Cads, mg×g -1 ) was 

calculated as a difference between initial and equilibrium HS concentrations. 



General adsorption isotherms curves are shown on Fig. 1. 

Initial parts of both isotherms had L-form. Sorption of native HS on montmorillonite reached 

plateau near 20 mg×g -1 . At the same time, sorption of silanized HS linearly increased up to 80 

Vol. 3 Page - 250 -

mg×g -1 . After equilibrium concentration of 1500 ppm, the inflection point was observed on 

the sorption isotherm of the silanized HS. It might be explained by the beginning of 

multilayer sorption of HS. 

180 

160 

140 

120 

100 

80 

60 

40 

20 

0 

C ads , mg×g -1 

Alkoxysilylated HS 

Native HS 

[HS], ppm 

0 500 1000 1500 2000 2500 

Figure 1: Adsorption isotherm silanized and native HS onto montmorillonite. 

Thus sorption capacity of silanized HS was much higher as compared to the native HS. The 

obtained data suggest considerable increase in mineral affinity of HS as a result of 

introduction of silanolic groups. 


The higher sorption capacity toward montmorillonite clay of alkoxysilylated HS as compared 

to the native HS was demonstrated. Synthesis of silanized humic materials is a powerful tool 

for manufacturing new hybrid materials. 



The research was supported by NATO CLG #983197. 

References 

1. Karpiouk L.A., Perminova I.V., Ponomarenko S.A., Muzafarov A.M., Hatfield K. In: From 

Molecular Understanding to Innovative Applications of Humic Substances; Proceedings of the 

14th International Meeting of the International Humic Substances Society, I.V. Perminova, N.A. 

Kulikova, (Ed.), Vol. II, Humus Sapiens, Moscow, 2008. p. 521-524. 

2. Perminova I.V., Ponomarenko S.A., Karpiouk L.A., Hatfield K. Humic derivatives, methods of 

preparation and use. PCT application. Pub. No.: #WO/2007/102750. 

Vol. 3 Page - 251 -


Peat Humic Acids as the Redox Mediators for Textile Technologies 

Irina Yu. Vashurina a* , Yuri A. Kalinnikov a , Irina V. Perminova b 

a Ivanovo State University of Chemistry and Technology, Engels Prosp., 7, 153000 Ivanovo, 

Russia; b Department of Chemistry, Lomonosov Moscow State University, Leninskie Gory, 1- 

3, 119991, Moscow, Russia 

E-mail: irina-new@bk.ru, 

I. Introduction 

One of the essential qualities of humic acids (HA) is their high catalytic activity in diverse 

redox transformations that occur in soils, peats, coals etc. It is attributable to the quinonoid 

redox-active units of HA as well as to transition metals that form stable complexes with 

functional groups of HA and participate in electron transfer reactions. Scientific interest to 

humic substances as redox mediators is steadily growing due to the progress in the 

understanding of their role in abiotic and biotic redox transformations of ecotoxicants in 

contaminated environments. At the same time, there is almost no information available on the 

application of humic substances in industrial technologies that rely on redox reactions. 

In the present study, the possibility is assessed to use peat HA as redox mediators for textile 

technologies, namely, vat printing of cellulose fabrics and preparation of native starch gels for 

cellulose yarn sizing. 


HA were extracted from lowland peat mined in the centre of European Russia (Kostroma 

region) as described elsewhere [1]. UV-VIS spectroscopy was used to study the reduction 

kinetics of HA and dye in aqueous solutions. Viscometry and rheology was used to 

characterize starch gels. Potentiometric titration, other analytical and specific textile protocols 

were also used in our studies. 


The kinetic and potentiometric data obtained have revealed that the reduction of HA by 

sodium hydroxymethanesulfinate and their follow up oxidation by benzene chlorosulfamide 

(the reagents used in the tested textile technology) in aqueous alkaline media is reversible 

from 88 to 92%. The activation energy of redox transformations in HA is about tenfold less as 

compared to that of vat dyes reduction and native starch oxidation. 

Using 1-4-diaminoderivatives of anthraquinone (the dyes Acid Green 27 and Acid Blue 80) as 

the water soluble models of vat dyes, catalytic activity of peat HA in vat dye reduction by 

sodium hydroxymethanesulfinate and sodium dithionite was shown. The effect is comparable 

to that of conventional redox catalysts (such as β-anthraquinone sulfonic or 1,2-dihydroxy 

Vol. 3 Page - 252 -

anthraquinone-3-sulfonic acids), but achieved using concentrations an order of magnitude 

lower. The addition of 0.05-0.10 gOC·kg -1 of HA causes 2-20-fold increase in the dye 

reduction rate constant and an increase in reaction output up to 97-98%. 

It was revealed that at high pH values (10-13) peat HA effectively dismutated supeoxide ionradical 

and peroxide ions formed in vat dyeing and printing compositions because of 

interactions of reducing agents with oxygen, thus precluding the accumulation of superoxide 

and peroxide and favoring dye reaction with the reducing agent. This allowed for a twofold 

reduction in the amount of sodium hydroxymethanesulfinate in printing pastes. 

HA were recognized as chemical and structural modifiers of starch hydrogels used for cotton 

yarn sizing. Chemical modification consists in deeper starch oxidation (the content of 

oxidized groups in corn starch increased by 50%), full consumption of oxidizing agents and 

less than half the gelation period. Structural modification reveals in essential decrease in 

starch gel viscosity and a transformation to a Newtonian flow regime. 

Due to their enhanced fluidity and adhesion to cellulose together with better elasticity of 

formed films, HA modified starch gels provide complex improvement of the properties of 

sized yarn and reduce yarn break while spinning. 


This investigation produced new HA containing compositions for textile materials vat dyeing 

and printing and for cotton yarn sizing that markedly differ from the existing ones by less 

consumption of aggressive chemicals (reducing and oxidizing substances, alkali), full 

utilization of vat dyes, starch, reducing and oxidizing agents, replacement of some toxic 

textile auxiliaries by biocompatible HA, shortening the duration of thermal treatments. This 

makes the HA-based technologies more efficient and it introduces elements of green 

chemistry into textile industry. 


This research was supported by the Russian Foundation for Basic Research (grants 05-04- 

96405 and 06-04-08048) and by the Foundation for the Assistance to Scientific Innovative 

Enterprises (contract No 4302 р/6530). 

References 


1. Lowe L.E. Studies on the nature of sulphur in peat humic acids from the Fraser River Delta, 

British Columbia // Sci. Total Environ., 1992, 113 (1-2), P. 133-145. 

Vol. 3 Page - 253 -


Peat Humic Acids as Surfactants 

Oskars Purmalis, Maris Klavins 

University of Latvia, Latvia 

E-mail: oskars.purmalis@lu.lv; maris.klavins@lu.lv 

Humic substances are a general category of naturally occurring, biogenic, heterogeneous 

organic substances that can be characterised as being yellow to black in colour, of high 

molecular weight and refractory. They form most of the organic component of soil, peat and 

natural waters, they influence the process of formation of fossil fuels, and they play a major 

role in the global carbon geochemical cycle. Structure of humic substances is characterised by 

presence of numerous aromatic, carboxylic and phenolic functionalities, linked together with 

alkylmoieties, imparting a measure of flexibility to the polymer chains [1]. Surface tension 

measurement defined humic substances as surface active substances [2, 3]. Unless micellar 

structural model of humic substances has been suggested, in the same time there are only a 

few studies about the factors that affect the surface tension of humic solutions. 

The ability to influence surface tension of solutions of humic substances depends on 

decomposition degree and botanical composition of the peat. The surface tension-pH curves 

of humic substances featured a minimum for all solutions, declining steeply from higher and 

lower pH values. Surface tension of solutions of humic substances decreased with increasing 

concentration. The objective of this study was to study changes in surface activity variation in 

peat profile of humic substances. 



The peat samples were collected in the Kronu Dzelves Bog (0 – 350 cm) and Ploce bog (0 - 

130 cm) (Latvia), both bogs botanical origin – fuscum peat. Humic acids were extracted and 

purified using procedures recommended by the International Humic Substances Society 

(IHSS) [4]. 

Surface tension measurements were taken with tensiometer Krüss K6 (Krüss GmbH), fitted 

with a 19 mm diameter platinum ring. Samples were prepared for measurement diluting till 

the appropriate concentration standard solution at humic substances (1000 mg/l) and 

equilibrating for 24 hours. Solutions were placed in a shallow glass dish of 50 mm diameter 

and the platinum ring was inserted in the middle of the container to avoid edge effects and 

equilibrated for 90 min. The ring was raised through manual operation of the torsion 

Vol. 3 Page - 254 -

mechanism and the tension readings at the instant of surface detachment were noted. All 

measurements were taken in triplicate at a temperature of 22 0 C and mean results are used in 

the figures with standard deviation not more than ±2 mN/m. 

Hydrophobicity of humic substances has been characterized by their distribution between 

water and polyethylene (PEG) phases (PEG 20000, Fluka) [5] as distribution coefficient 

KPEGW (analogous to octanol water distribution coefficient – Kow). 

Elemental analysis (C, H, N, S, and O) was carried out using an Elemental Analyzer Model 

EA-1108 (Carlo Erba Instruments). 


To study the changes of ability of humic substances to influence surface tension, we have 

used well characterised humic substances (Table 1), isolated from bogs in Latvia. 

Studied humic substances demonstrate ability to influence surface tension of their solutions 

(Figure 1, 2). In concentration intervals from 50 mg/l to 1000 mg/l γ dropped from 57,6 – 62,9 

mN/m to 53 – 55,2 mN/m. The surface tension of solution of humic substances at fixed 

concentration so can be used to describe their surfactant properties and among studied humic 

substances comparatively high variability could be observed. 

Surface tension, mN/m . 

64 

62 

60 

58 

56 

54 

52 


0 200 400 600 800 1000 

HA concentration, mg/l 

Figure 1: Variation of surface tension of humic acid (isolated from Ploce bog) solutions 

depending on the location depth (cm): 

0-30 cm (▲); 31-60 cm (●); 61-85 cm (○); 86-110 cm (■); 111-130 cm (♦) 

Vol. 3 Page - 255 -

There exist significant differences in the ability of humic acids to influence surface tension of 

their solutions. Peat humic substances isolated from Ploce bog (Latvia) from consecutively 

increasing depths do have major differences in their ability to modify tension of aquatic 

solutions (Figure 1). The surface tension of solutions of humic substances is decreasing with 

increasing depth of their location in peat column (age of the peat and their humification 

degree). 

Surface tension, mN/m 

63 

62 

61 

60 

59 

58 

57 

56 

55 

54 

Ploce 0.0 - 0.30 

Ploce 0.30 - 0.60 

Ploce 0.60 - 0.85 

Ploce 0.85 - 1.10 

Ploce 1.10 - 1.30 

Dzelve HA 0.0 - 0.10 

Dzelve HA 0.10 - 0.20 


Dzelve HA 0.20 - 0.30 

Dzelve HA 0.30 - 0.40 

Dzelve HA 0.40 - 0.50 

Dzelve HA 0.50 - 0.60 

Dzelve HA 0.60 - 0.70 

Dzelve HA 0.70 - 0.80 

Dzelve HA 0.80 - 0.90 

Dzelve HA 0.90 - 1.00 

Figure 2: Variation of surface tension of humic acid (isolated from Ploce and Kronu Dzelves 

bog) solutions: concentration – 100 mg/l 

Table 1: Elemental and functional composition of humic substances used in the study 

Humic Acid C, % H, % N, % mEq COOH/g KPEG/W 

Ploce 0.0 – 0.30 51.51 4.80 2.12 5.66 6.35 

Ploce 0.30 – 0.60 51.13 4.81 2.06 5.80 6.23 

Ploce 0.60 – 0.85 51.12 4.87 2.05 6.01 5.77 

Ploce 0.85 – 1.10 52.11 4.61 1.60 5.91 10.75 

Ploce 1.10 – 1.30 57.50 4.94 1.92 5.10 8.98 

Dzelve 0.0 - 0.10 42.36 4.19 2.30 3.90 4.98 

Dzelve 0.10 - 0.20 50.62 4.56 2.61 5.00 7.37 

Dzelve 0.20 - 0.30 51.62 4.85 2.77 4.70 9.30 

Dzelve 0.30 - 0.40 51.92 4,48 2.57 4.60 8.63 

Dzelve 0.40 - 0.50 53.16 4.84 2.24 4.30 7.15 

Dzelve 0.50 - 0.60 50.88 4.56 2.25 4.50 6.65 

Dzelve 0.60 - 0.70 48.05 4.05 2.04 4.70 7.04 

Dzelve 0.70 - 0.80 53.95 4.94 2.32 4.50 7.19 

Dzelve 0.80 - 0.90 52.55 4.82 2.17 4.30 2.78 

Dzelve 0.90 - 1.00 53.34 4.87 2.39 4.30 2.71 

Vol. 3 Page - 256 -

Humic acids isolated from Kronu Dzelve bog, demonstrates higher variability, particulary in 

first 50 cm of peat column. It depends on hydrological regime fluctuations and peat 

humification effect irregularity on the top layer of peat bog. In the deeper layers Kronu 

Dzelve bog peat humic acids demonstrates the same results as Ploce bog peat humic acids. 

The effect of humic substances on surface tension depends on their amphiphilic character and 

tendency to accumulate at the water-air interface. It is known that the behaviour of humic 

substances in aquatic solutions depends on their concentration, pH, metal ion concentrations 

[6, 7] and the same factors determines the influence of humic substances on the surface 

tension and the formation of pseudomicelles, since both are manifestations of the same 

solution properties. 



The surface tension-pH curves of humic substances featured a minimum for all solutions, 

declining steeply from higher and lower pH values. The decrease in surface tension with 

decreasing pH reflects the gradual neutralization of acidic sites, which created amphiphilic 

species that migrated to the surface. Surface tension of solutions of humic substances 

decreased with increasing concentration. Thus there exist direct links between peat 

decomposition degree, botanical composition, structure of humic substances and their ability 

to influence surface tension of aquatic solutions. 

References 

1. Engebretson, R.R., von Wandruszka, R. (1994) Microorganization in dissolved humic acids. 

Environ. Sci. Technol., 28, 1934 – 1941 

2. Engebretson, R.R., von Wandruszka, R. (1996) Quantitative approach to humic acid associations. 

Environ. Sci. Technol., 30, 990 – 997 

3. Gašparovic, B., Cosovic, B. (2003) Surface-active properties of organic matter in the North 

Adriatic Sea. Estuarine, Estuar. Coast. Shelf. S., 58, 555 – 566 

4. Guetzloff, T.F., Rice, J.A. (1994) Does humic acid form a micelle? Sci. Total Environ., 152, 31 – 

35 

5. Klavins, M. (1998) Aquatic humic substances. University of Latvia, Riga, 234 pp. 

6. Tan, K. H. (2005) Soil sampling, preparation, and analysis - second edition. N.Y.: Taylor & 

Francis group, 623 pp. 

7. Zavarzina, A.G., Demin, V.V., Nifanteva, T.I., Shkinev, V.M., Danilova, T.V., Spivakov, B.Ya. 

(2002) Extraction of humic acids and theit fractions in poly(ethylene glycol)-based aqueous 

biphasic systems. Anal.Chim.Acta, 452, 95-103. 

Vol. 3 Page - 257 -

Efficiency and Application Prospects of Humatized Mineral Fertilizers 

Oleg Gladkov * , Rodion Poloskin 

Open Company "SPA" “AET” Ltd (RET Ltd Company), Saint-Petersburg, Russia, 195112 

E-mail: gladkov@humate.spb.ru 


Development of a new market niche of the fertilizers combining for end users the tempting 

properties of organic and mineral fertilizers draws attention of scientists and consumers for a 

long time. Using of such fertilizers allows to raise plant uptake of nutrients on 20-50 % and, 

accordingly, to decrease the application rates, or it is essential to raise productivity at the same 

application rates. Thus the general expenses for application of fertilizers essentially decrease, 

and the yield of non-polluted agricultural production and the maintenance of soil fertility at 

the minimum impact on the environment are provided. 


The Company "SPA" "AET" Ltd (RET Ltd Company) is engaged in the development of 

"know-how" and researches of the efficiency of application of industrial humates and 

humatized organic-mineral fertilizers (HOMF), including the phosphoric and complex 

fertilizers, more than 15 years. In 1999 after the long-term researches the company let out on 

the market the concentrated humic product under the trade mark "Lignohumate R ". The 

product is made on the unique patented technology of accelerated humification of commodity 

ligno-sulfonates. The product is a 20% water solution or completely soluble powder. The 

comparison of "Lignohumate" with the products of other leading world manufacturers has 

shown that their physical and chemical characteristics do not concede to the analogues. 

At the same time Lignohumates have a number of positive features such as increased content 

of fulvic and other low-molecular acids, and also of macro- and microelements. 



The basic marks of Lignohumate have been examined and registered in different countries, 

they are widely used in the agriculture of Russia, the CIS countries, EU, the North America, 

China. The constructed typical industrial module allows to let out more than 1500 tons/year of 

humic product in recalculation on a solid. It has already made the company one of the largest 

manufacturers of humates in Russia. 

In the present report we give the information only about one of the perspective directions of 

Vol. 3 Page - 258 -


application of Lignohumate - in a composition with the mineral fertilizers. For the realization 

of this target our company cooperates with the variety of the Russian scientists and experts of 

the industrial companies, manufacturers of mineral fertilizers, together with the foreign 

companies among which are CSC "ARVI" (the Lithuanian Republic), "Amagro" (Czechia), 

«RЕTRIVALL» Inc. (Canada), "Radostim" (Germany), etc. At the factories were fulfilled the 

technologies of humatizing fertilizers with the humic additive component directly “in melt” 

as well as covering of ready granules with humic additive “on the surface” 

The tests of physical and chemical properties of such fertilizers have shown that the addition 

of Lignohumate to mineral fertilizers considerably improves the basic functional properties of 

fertilizers (dusting and blocking) in comparison with the standard mineral analogues. The 

entering of humic organic component gives new properties to the mineral fertilizers. First of 

all, their structure changes that causes the changes of physical and chemical properties, such 

as dusting, durability of granules, blocking, for example, drawing on ammophos of 0,5-1 % of 

Lignohumate in the form of a cover reduces the dusting on 37 %. The drawing of 

Lignohumate on a carbamide granule reduces by 30 % its blocking and by 30-35 % increases 

the durability of granules (1,3 kg/g, at the durability of standard fertilizer of 0,95 kg/g). Some 

information on the most important and indicative researches of agricultural efficiency of 

application of such fertilizers is resulted 

The Efficiency of Application of Humatized Carbamide:For carrying out of tests two parties of 

humatized carbamide with the various maintenance of Lignohumate have been made. The 

first trial party was with drawing of humic additive on the surface and the industrial party 

with placing of Lignohumate “in melt” before granulation was made on the Open Society 

"Akron". Both parties have been directed to the different organizations for the tests on grain 

crops. The results received by the experts are well correlated that guarantees their objectivity. 

Some results of these tests are shown here. 

Vol. 3 Page - 259 -

The conducted tests have allowed to conclude the following: 

- The use of both parties of humatized carbamide leads to the increase of productivity, quality 

of production and nitrogen operating ratio; 

- The optimum concentration of Lignohumate as a part of fertilizer for the efficiency and 

profitability is within 0,36-0,5 %, 

- The bigger efficiency is received using the fertilizer with the put humic cover; 

- The technological tests of industrial party of humatized carbamide have shown that the use 

of Lignohumate “in melt” improves the durability and water resistance of granules, reduces 

the washing out of easily soluble nutrients of fertilizers. 

Other model experiments of application of humatized fertilizers have been spent on the basis 

of three types of complex NPK fertilizers of the Companies CSC "ARVI" (Lithuania). The 

manufacture of the trial parties was carried out in a real production cycle with the minimum 

technological completions. Some results of agricultural tests of three types of humatized NPK 

fertilizers at the cultivation of rape are resulted on the figures. 

Fig.3, Fig.4, Fig.5 



The carried out researches have allowed to draw the following conclusions: 

- At the manufacture of fertilizers the improvement of durability of granules and the quality of 

the technological processes of granulation is noted; 

- On all tested cultures the trial fertilizers provide the reduction of the vegetative period, the 

Vol. 3 Page - 260 -

increase in productivity and quality of production in relation to the standard analogue; 

- The application of humatized NPK fertilizers allows to recommend the decrease of norms of 

their entering in relation to the standard NPK not less than for 25-30 % without productivity 

decrease; 


- The major result of researches is the acknowledgement of the possibility of reception of high 

effect at rather small concentration of Lignohumate in the fertilizer within 0,1-0,3 %. 

The last circumstance does not so considerably change the fertilizer cost price in comparison 

with the base analogue (for 15-20 EURO/TON) that is considerably below the economic 

efficiency of application of such fertilizers. The received results have allowed the company 

CSC "ARVI" & CO to register the new kind of NPK fertilizers with the additive of 

Lignohumate under the mark “ARVI Extra plus” on EU market. The first industrial party of 

these fertilizers was put to Germany in the end of 2008. Now long-term contracts are being 

coordinated. 

The carried out researches and the reached technological experience allow to develop the 

new, much more capacious market of application of humic products in different countries. At 

the same time the necessity of development of joint scientific researches with the 

manufacturers of mineral fertilizers for the expansion of this direction of application of 

humates amplifies. 

References 

1. Oleg Gladkov, Iren Sokolova “Lignohumate - Newly Developed Humic Preparation for 

Recultivation and Restoration of Humic soil”. NATO Advanced Research Workshop “Use of 

Humates to Remediate Polluted Environments: from Theory to Practice” Zvenigorod, Russia, 

September 23-29 2002. Soil Science Department, Moscow State University, 119992, Moscow, 

Russia 

2. Olga Yakimenko, Oleg Gladkov, Rodion Poloskin, Vera Кing « LIGNOHUMATE – A NEW 

MEMBER OF HUMATES FAMILY». Humic Science & Technology Conference IX. March 22 to 

24, 2006, Northeastern University, Boston, MA, USA 

3. Oleg Gladkov, Rodion Poloskin, Vera King, Olga Yakimenko “Commercial Humates from 

Lignosulfonate: Production, Properties and Use”. Humic Science & Technology Conference IX. 

March 22 to 24, 2006, Northeastern University, Boston, MA, USA 

4. Wolfgang Novik, Uwe Bёm, Oleg Gladkov, Virginius Streimikis, “The Pilot Project on 

Manufacture of Humatized Fertilizer “ARVI Extra” and “ARVI Extra plus” and the First Results 

of its Application in Germany”. Fifth International Conference “Humic Preparations and Phytohormones 

in Agriculture”, February, 16-18th, 2010, Dnepropetrovsk, Ukraine. 

Vol. 3 Page - 261 -

Physical-Chemical Properties and Application Potential of Humates 

Prepared from Regenerated Lignites 

Jan David, Jiří Kučerík 

Faculty of Chemistry, Brno University of Technology, Purkyňova 118, 612 00, Brno, Czech 

Republic 

E-mail: kucerik@fch.vutbr.cz; xcdavid@fch.vutbr.cz 



At the beginning of the 21 st century, our world is facing a set of challenges which are overall 

related to the state of soils and to the old environmental loads. The search of new resources 

for the chemical industry and agriculture is also of a great interest. Humic substances, 

primarily humic acids (HAs) and their salts (i.e. humates) are thought to be helpful in 

designing of the particular solutions of those problems. 

HAs are heterogeneous mixture of organic compounds, the dark and refractory alkali-soluble 

fraction of soil organic matter [1]. Recently, the scientific discussion was brought up on the 

structure of HAs, resulting in the recognition, that HAs represent the collections of diverse 

low-molecular-mass molecules held together by hydrophobic bonds and hydrogen-bonding 

interactions [2, 3]. Using various techniques, it was also proved, that from very low 

concentration HAs in aqueous solutions form aggregates of various dimensions, which may 

be crucial for the future HAs’ industrial and agricultural applications [4, 5]. 

The aim of this research is focused on the production and characterization of HAs originating 

from the pre-treated South-Moravian lignite (Czech Republic). Lignite is the youngest type of 

coal, usually called as brown coal. It is the first product of coalification and the intermediate 

between peat and subbituminous coal [6]. The most frequent lignite pretreatment described in 

literature is wet regeneration/oxidation with solutions of strong oxidizers; it has been used 

both for coal [7] and lignite [8]. This regeneration may increase the yield of extractable HAs 

and modify their structure (in particular by increasing the ratio of aromatic and semiquinone 

structures and content of polar elements such as O and N). That modification is indisputably a 

way how to extend the application potential of the HAs. Following research is the 

continuation of our previous pilot study [9] with the aim to characterize regenerated HAs via 

various methods as thoroughly as possible and to design the particular applications of 

regenerated HAs (especially in agriculture, environmental protection and applied chemistry). 

Even though recently a progress has been done in the research of the physical chemistry of 

HAs in diluted solutions by means of Dynamic Light Scattering (DLS) and High Resolution 

Vol. 3 Page - 262 -

Ultrasonic Spectrometry [10, 11], the knowledge about behavior of diluted humates is still 

limited. The combination of results obtained from those techniques with other methods and 

approaches (see Discussion) would provide knowledge on the relations between physical 

properties such as hydration and conformation, chemical structure (elemental analysis) and 

their biological activity and sorption capacity. Such approach should bring the deeper insight 

into the chemistry of lignite humic substance as well as to promote their technological and 

agricultural applications. 


Regeneration of lignite and extraction of HAs. South-Moravian lignite (Mine “Mír”, 

Mikulčice, Lignit Hodonín, Czech Republic) was regenerated in the similar way as in 

previously published research [7, 8] (50 g of lignite, 500 mL of agent, 40 °C, stirring for 20 

minutes) by (10; 20; 30; 40; 50; 65 wt%) nitric acid and by (5; 10; 20; 30 wt%) hydrogen 

peroxide agents. The HAs were extracted using the modified IHSS alkali extraction (0.5 

mol·L -1 sodium hydroxide and 0.1 mol·L -1 Na4P2O7) method. The HAs were purified by 5 

wt% hydrofluoric acid (and then dialyzed against deionized water until no Cl – anions were 

present. Finally, the samples were freeze-dried using Labconco Freezone 4.5 system, with the 

Vacuubrand RZ6 pump. When needed from the point of water solubility, obtained HAs were 

transferred to the water soluble potassium salt via titration with 0.1 mol·L -1 potassium 

hydroxide on the Schott TitroLine Alpha plus automatic titrator to static pH 7.2 and 

ammonium salt (titrated with an excess of diluted ammonium hydroxide to pH 7–9). 

Samples were characterized by techniques described in the Discussion. 



As the first step, results obtained by elemental analysis (Perkin Elmer 2400 elemental 

analyzer) showed that regeneration of lignite has significant influence on the elemental 

composition of the resulting HA. Regeneration with nitric acid increased the H/C, N/C and 

O/C ratio, while regeneration with hydrogen peroxide increased the H/C ratio. 

The research continues in combining the data from techniques such as High Resolution 

Ultrasonic Spectrometry and densitometry (Ultrasonic Scientific HR-US 102 and Anton Paar 

DMA 4500 Density meter) determining the number of water molecules hydrating a humic 

molecule in the humate solution at specific concentration. The results were correlated with 

aggregate dimensions obtained from DLS (Coulter N4 Plus Particle sizer). Considerations are 

supported by Diode Array Detector (DAD) detected High Performance Size Exclusion 

Vol. 3 Page - 263 -

Chromatography (HPSEC) (Gilson UltiMate 3000 Chromatography station). Final 

conclusions will be done with respect to the chemical characteristics gained from not only 

from elemental analysis, but also from solid state 13 C Nuclear Magnetic Resonance and 

Excitation-Emission Fluorescence Spectrometry, i.e. the efficiency of the pre-treatment of 

parental lignite with respect to the concentration and character of oxidant and physicochemical 

properties of obtained HAs. 

The extraction and regeneration of supra mentioned HAs was done with the purpose of further 

practical application. From this point of view, two main purposes for HAs are strongly 

emerging. The first one lies in agriculture exploiting the humics “hormone-like” biological 

activity or just simply the stimulating effects on the plant growth [12–14], the second one is in 

the possibility to adsorb or bind metals and adsorb or solubilize organic pollutants [15, 16]. 

The environmental applicability (interactions with selected contaminants) of south-Moravian 

lignite and HAs derived from it are being tested according to Conte et al. [17] and von 

Wandruszka and Newell [16]. 

Finally, the root growth enhancement possibilities are being tested on the germination and 

root growth of maize (Zea mays) in the defined light and temperature conditions [9]. 

For all these applications, the surface activity of HAs seems to be one of important 

parameters, therefore before the application tests, surface tension of samples of various 

concentrations were measured via KSV Sigma 700 Surface tension meter with du Noüy ring. 

Selected results will be presented and discussed on the conference. 



The funding of this research by the Ministry of Education, Youth and Physical Training of the 

Czech Republic in terms of the MSM0021630501 research project is acknowledged. 

Presenting author thanks for all the support for his travelling costs. 

References 

1. F.J. Stevenson, Humus Chemistry, John Wiley & Sons, New York, 1994. p. 33. 

2. R.L. Wershaw, Environ. Sci. Technol., 27 (1993), 814–816. 

3. A. Piccolo, Adv. Agron., 75 (2002) 57–134. 

4. R.R. Engebretson, R. von Wandruszka, Org. Geochem., 26 (1997), 759–767. 

5. R. von Wandruszka, Geochem. Trans., 2 (2000), DOI: 10.1039/b001869o. 

6. B. Mikulášková, et al., Chem. Listy, 91 (1997), 160–168. 

7. R. Rausa et al., in N. Senesi, T. M. Miano (Ed.), Humic Substances in the Global Environment 

and Implication on Human Health, Amsterdam: Elsevier, 1994, 1225–1244. 

8. Kučerík et al., Petroleum and Coal, 45 (2003), 58–62. 

Vol. 3 Page - 264 -


9. Z. Vlčková et al., Soil Biol. Biochem., 41 (2009), 1894–1901. 

10. N.E. Palmer, R. von Wandruszka, Fresenius J. Anal. Chem. 371 (2001), 951-954. 

11. J. Kučerík et al., Org. Geochem., 38 (2007), 2098–2110. 

12. A. Muscolo et al., Soil Sci. Soc. Am. J., 71 (2006), 75–85. 

13. L.P. Canellas et al., Ann. Appl. Biol., 153 (2008), 157–166. 

14. B. Antošová et al., in M. I. Barroso (Ed.), Reactive and Functional Polymers Research Advances, 

NovaScience Publishers, 2007, 191–203. 

15. M. Havelcová, et al., J. Haz. Mat., 161 (2009), 559–564. 

16. R. von Wandruszka, J.D. Newell, Environ. Prog., 21 (2002), 209–214. 

17. P. Conte et al., Environ. Pollut., 135 (2005), 515–522. 

Vol. 3 Page - 265 -

Removal of Tributyltin Biocide by Using Black Carbon 

Liping Fang * , Ole K. Borggaard, Helle Marcussen, Peter E. Holm and Hans Chr. B. Hansen 

Department of Basic Sciences and Environment, Faculty of Life Sciences, University of 

Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C., Denmark 

E-mail: fang@life.ku.dk 


Black carbon (BC) is the product of incomplete combustion of fossil fuel and biomass, which 

is ubiquitously in environment [1]. Charcoal and soot have been distinguished as two main 

forms of BC [2]. BC exhibits strong sorption with hydrophobic organic compounds (HOCs), 

highly contributing to total sorption of HOCs to soils and sediments [3]. Therefore, the 

application of BC as sorbent becomes a promising and low-cost option for purifying industrial 

wastewater and groundwater. 

In this study, we i) characterized two BCs, e.g., soot and charcoal; ii) surveyed the sorption 

capacity of tributyltin biocide by BCs (soot and charcoal) at different pH values. 



Tributyltin chloride ((C4H9)3SnCl) (96%) was purchased from Sigma Aldrich (Denmark). All 

other reagents used were pro analysis. A 1000 mg L -1 TBT stock solution was prepared by 

dissolving the corresponding amounts of TBT in methanol. 

Wheat charcoal was produced as follows: finely ground wheat straw was combusted by in the 

muffle furnace at 300 °C for approximate 24 h. Soot as carbon black was donated by 

TIMCAL (Switzerland). Wheat charcoal was ground with ball mill to fine powder. The 

resulting powder was washed with acids in order to remove silica and salts. 

The element composition of the three BCs was determined on a CHN elemental analyzer 

(Flash EA 1112, Thermo Scientific). The contents of carboxylic acid groups and phenolic 

groups on the BCs were determined by titration of BC suspensions. 

Sorption experiments were determined by adding appropriate amounts of tributyltin (TBT) 

stock solution into suspensions of the charcoal (50-250 mg/L) and soot ( 60- 400 mg/L) in pH 

4, 6 and 8 buffers to obtained initial TBT concentrations in the range 0.42 to 8.42 µmol L -1 . 

The suspensions were shaken for 24 h in the dark at room temperature (22±1 ºC). Finally, 

each suspension was filtrated into10 mL volumetric flask with a mixed cellulose ester syringe 

filter, and added 30 µL 69-70% HNO3. The loss of TBT during the whole sorption and 

pretreatment processes was estimated by performing both types of experiments without 

Vol. 3 Page - 266 -

adding BC in triplicate. Less than 5 % and 10 % were lost during sorption and filtration, 

respectively. The acidified filtrates were analyzed by GFAAS (Perkin Elmer, Denmark). 



The selected characteristics of two BCs are listed in Table 1, it shows wheat charcoal contains 

relatively high oxygen comparing with that of soot (Table 1), which also could be reflected 

from their substantial contents of oxygen - contained functional groups (-COOH, Ar-OH). In 

addition, the ash is blow 6 (Figure 1). Soot possesses no charged functional 

groups (Table 1), and in aqueous solution TBT exists as TBT + ((C4H9)3Sn + ) and TBTOH 

((C4H9)3Sn OH) depending on pH (Eq. 1, [4]). Therefore, hydrophobic sorption of uncharged 

TBT (TBTOH) is considered the only sorption mechanism. 

pKa =6.3 (1) 

The sorption edge for wheat charcoal also increase with increasing pH from limited sorption 

at pH < 3 to maximum sorption around pH 7 but the raising curves are less steep than the soot 

sorption edge and the sorption maxima are followed by slight declines (Fig. 1). The sorption 

edge resembles those reported for TBT sorption by minerals and humic substances [5, 6], but 

higher relative sorption (Cs, pH/Cs, pH 3) at high pH. Since the charcoal possess charged surface 

sites, both TBT + and TBTOH may be sorbed by charcoal. The shape of the sorption edges 

may therefore reflect a combination of two kinds of sorption including hydrophobic sorption 

of TBTOH on uncharged sites and electrostatic sorption of TBT + on negative surface sites. 

Vol. 3 Page - 267 - 

Figure: 1. Sorption edge of wheat charcoal, and 

soot versus pH, respectively (long dash, solid 

line); TBTOH proportion versus pH (dotted line) 

simulated by Visual Minteq (50 μg L -1 TBTCl, 

I= 0.02 M NaNO3).


The empirical nonlinear Langmuir model was applied to fit the observed data from the 

sorption experiments (Fig. 2). In agreement with sorption edge results (Fig. 1), the Langmuir 

sorption maximum (Cs,max) increases from about 5×10 3 µmol kg -1 at pH 4 to approx. 1×10 5 

µmol kg -1 at pH 8 with soot. 

The Langmuir binding constant, KL may indicate trends in the affinity between sorbents and 

sorbate. It can be seen that KL of the charcoal is considerably higher than that of soot, 

especially at pH 4. At this pH, sorption is almost restricted to reaction with TBT + , as this form 

constitutes >99% of the TBT at pH 4 (Figure 1). The larger KL for wheat charcoal than for 

soot may therefore indicates stronger bonding of the electrostatic than hydrophobic sorption, 

since soot is limited to hydrophobic sorption, which is very little at pH 4. With increasing the 

pH value, the KL of wheat charcoal decreases showing the decline of bonding between 

charcoal and TBT. Nevertheless, the Cs, max of charcoal increases owing to the increase of 

charged sites on surface. In contrast, soot shows higher KL at pH 8 than that at pH 4 and 6, 

which elucidates that stronger bonding between soot and TBT at pH 8. It could be ascribed to 

the hydrophobic form TBTOH dominates at high pH values (>99% at pH 8). Consequently, it 

could strongly bind with the hydrophobic surface of soot, resulting in higher sorption 

capacity. 

Figure 2: Langmuir sorption isotherms of soot and wheat charcoal at three constant pH values (filled 

circles: pH 4, empty circles: pH 6, filled triangles: pH 8). All the curves were fitted with all raw data 

obtained in triplicates. Vertical bar: standard error 

4. Conclusion 

This work has been to survey the sorption of TBT to BCs at different pHs, showing the uptake 

ability of TBT by BCs much relays on pH factor. At pH 4, both of hydrophobic and 

electrostatic sorption are relatively weak, whereas, the sorption reaches maxima at pH 6 for 

both soot and charcoal. Moreover, due to the decreasing of TBT + , the TBT sorption to 

Vol. 3 Page - 268 -

charcoal appears to be slight attenuated. As an important component in environment, BCs 

therefore could influence the bioavailability, transformation, etc of TBT. However, by taking 

the advantage of those properties, the application of BCs on purification of wastewater shows 

to be an alternative choice. 


References 

1. M.W.I. Schmidt and A.G. Noack, Global Biogeochemical Cycles, 14 (2000) 777. 

2. M. Elmquist., G. Cornelissen, Z. Kukulska, and O. Gustafsson, Global Biogeochemical Cycles 20 

(2006) GB2009. 

3. T.D. Bucheli and O. Gustafsson, Chemosphere 53 (2003) 515. 

4. C.G. Arnold, A. Weidenhaupt, M.M. David, S.R. Muller, S. B. Haderlein and R. P. 

Schwarzenbach, Environmental Science & Technology 31 (1997) 2596. 

5. M. Hoch, J. Onso-Azcarate and M. Lischick, Environmental Toxicology and Chemistry 21(2002) 

1390A. 

6. Weidenhaupt, C. G. Arnold, S.R. Muller, S.R. Haderlein and R.P. Schwarzenbach, Environmental 

Science & Technology 31(1997) 2603. 

Vol. 3 Page - 269 -

Evaluation of the Efficiency of Fulvic and Humic Acids (Agrolmin Bravo 

and Cerrado) in Soybean Production in the Brazilian Savanna 

L.T. Dias Cappelini a , D. Cordeiro a , L.A. Artimonte Vaz b , L.F. Artimonte Vaz b , E. Bessa 

Azevedo b , E.M. Vieira b* 

a Instituto de Química de São Carlos, Universidade de São Paulo, São Carlos-SP, Brazil; 

b Agrolatino Ltda, Rua João Pessoa, 996 – Matão – São Paulo 

E-mail: eny@iqsc.usp.br 

Introduction 

The average yield for the soya culture in Brazil is 2,629 kg/hectare or 43.80 bag/hectare [1], a 

very low value when compared to the national agricultural productivity potential, in real 

conditions of cultivation, approx. 4,000 kg of grain per hectare or 66.70 bag/hectare [2]. 

Therefore, new alternatives and technologies that promote agricultural productivity have been 

developed respecting the concepts of environmental sustainability and enabling high annual 

production without promoting deforestation. There is a growing interest in the technology of 

applying humic substances by using products made from humic and/or fulvic acids in 

agriculture. 

This study has the scope of stimulating research in this sector and evaluating the existent new 

resources, focusing on products made from humic and fulvic acids, with the aim of increasing 

productivity of the soya culture in tropical climate, complying with the new environmental 

requirements. 


The present experiment was conducted in Oxisol III on October 21 st , 2008, and the harvest 

occurred on February 23 rd , 2009. The treatments were: Agrolmin with Bravo Cerrado and 

conventional fertilization application to the soil during the planting operation and Bravo 

Cerrado with conventional fertilization application, applied to the leaves, which were 

compared with standard treatments adopted in the property. 

A randomized block design was used with 3 treatments and 11 repetitions. In the experiment, 

each parcel consisted of six lines, spaced by 0.45 m, with a length of 2 m, and a total area of 

5.4 m 2 for each parcel. Since the total number of parcels equals to 33, the area occupied by the 

soya experiment was equal to 178.2 m 2 . 

The description of treatments, products, doses and time of application on the soya crop are 

listed in Table 1. 


Vol. 3 Page - 270 -

Table 1: Description of treatments, products, doses and timing of application for soya 

Treatments 

(T1) (02-20-20) + 0,2% 

B + Agrolmin 

+150 kg KCl/hectare 

(T2) (02-20-20) + 0,2% 

B 

+ 150 kg KCl/hectare 

(T3) (02-20-20) + 0,2% 

B 

+ 150 kg KCl/hectare 

Dose 

(kg/hectare) 

350 

350 

350 

Foliar Dose 

Bravo 

Cerrado 

Bravo 

Cerrado 

Zn 

Mn 

Cu 

B 

S 

N 

3 L/hectare 

3 L/hectare 

90 g/hectare 

150 g/hectare 

1 g/hectare 

1 g/hectare 

135 g/hectare 

150 g/hectare 

Applicatio 

Period 

Soil sampling was performed in the whole area, collecting the samples in the depth of 0 to 20 

cm. Leaf samples were collected at the R1 stage – beginning of flowering – to assess the 

nutritional status of the culture. 

The application of Agrolmin with Bravo Cerrado and conventional fertilization application 

was performed together with the operation of planting in spray jet directed at seed pre- 

coverage. However, the application of Bravo Cerrado with conventional fertilization was 

made on the leaves. 


The results for roots dry mass (RDM) and shoot dry mass (SDM) production of the plants and 

the yield of soybeans are shown in Figures 1 to 3. Comparing the treatments, it can be 

observed that the conventional fertilization with 300 kg/hectare of the 02-20-20 formulation 

plus 150 kg/hectare of KCl (T1) presented the lowest RDM. The application of conventional 

fertilizer plus 3 L/hectare of Bravo Cerrado (T2) increased the RDM in 7.4%. However, this 

result is not statistically different from the conventional fertilization. On the other hand, the 

combination of the conventional fertilization plus Bravo Cerrado and 20 L/hectare of 

Agrolmin increased significantly (P ≤ 0.05) the production of RDM. This increase was 0.5 

g/plant, corresponding to 16.6%. Considering that Agrolmin is a product made from humic 

substances containing significant amounts of humic and fulvic acids that play an important 

role in the induction of root growth [3,4], this result was expected. It was also observed a 

significant increase in the amount, size, and persistence of nodules, indicating higher rates of 

biological nitrogen fixation biological [5]. 


Vol. 3 Page - 271 - 

V4, V8 e R4

The analysis of the leaves indicated that increment, showing nitrogen average levels of 57.1, 

48.8, and 45.3 g/kg for Agrolmin with Bravo Cerrado and conventional fertilization (T1), 

Bravo Cerrado with conventional fertilization (T2), and conventional (T3) treatments, 

respectively. 

As occurred with the RDM production of soya plants, the addition of 3 L/hectare of Bravo 

Cerrado with conventional fertilization (T2) and 20 L/hectare of Agrolmin with Bravo 

Cerrado and conventional fertilization (T1) increased the SDM production. This increase was 

statistically significant and corresponds to 7.7% and 21.0% for Bravo Cerrado with 

conventional fertilization and Agrolmin with Bravo Cerrado and conventional fertilization, 

respectively, when compared to conventional fertilizer (T3). Given that there was an increase 

in root growth, the volume of soil explored by the roots was greater, increasing the absorption 

of water and nutrients, fact that is reflected on SDA production of soya plants. Furthermore, 

the combination of mineral sources of nutrients with Bravo Cerrado and Agrolmin increases 

the usage efficiency of those nutrients, which can be seen by gains in the SDM production. 

The soybeans production significantly increased [6] (P ≤ 0.05) with the application of 20 

L/hectare of Agrolmin with Bravo Cerrado and conventional fertilization (T1), when 

compared to the conventional treatment (T3). This increase was of 1,133 kg/hectare from 

soybeans, accounting for 24.5%. The leaf analysis indicated that 5.5 g/kg of phosphorus in the 

treatment that received the application Agrolmin with Bravo Cerrado and conventional 

fertilization (T1) against 2.7 g/kg for treatment with Bravo Cerrado with conventional 

fertilization (T2) and 2.87 g/kg for conventional treatment (T3). These effects indicate higher 

rates of recovery of the applied mineral fertilizers, resulting in greater agricultural 

productivity. 


The application of Bravo Cerrado with conventional fertilization promoted a better foliar 

nutrition compared to the conventional treatment. This fact can be explained by the chelating 

effect promoted by humic and fulvic acids added to the nutrient sources of Bravo Cerrado. A 

better nutritional balance was also reflected in the grains productivity of parcels treated with 

Bravo Cerrado with conventional fertilization (T2), which produced 8.7% more (335 kg/ha) 

than the control area (T3). Although this production increase was not significant, it clearly 

shows the effect of a better nutrition on production. 

Vol. 3 Page - 272 -


The application of Bravo Cerrado with conventional fertilization promoted an increase of 

7.4% in the production of RDM, 7.7% in the production of SDM, and 8.7% in soybean 

production. On the other hand, the application of the commercial product Agrolmin with 

Bravo Cerrado increased the roots dry mass production of soya plants by 16.6%, the shoots 

dry mass by 6.7%, and consequently the grain yield by 24.5%. 

The productivity increase with the application of Bravo Cerrado and Agrolmin with the same 

doses of mineral fertilizers is a viable alternative to reducing production costs, as well as 

reducing environmental impacts due to maintaining the extent of the cultivated area. 



The authors thank CAPES, FAPESP, CNPq, for financial support and Agrolatino Ltda. 

References 

1. Ministério da Agricultura, Pecuária e Abastecimento. Companhia Nacional de Abastecimento 

Superintendência. Regional do Paraná. Gerência de Desenvolvimento e Suporte Estratégico / Setor 

de Apoio à Logística e Gestão da Oferta. Soja, (2009). 

http://www.conab.gov.br/conabweb/download/sureg/PR/Soja%20Junho%202009.pdf (12/01/10). 

2. III Simpósio de Plantas Oleaginosas. Realidades e Potencialidades Brasileiras, (2009). 

ESALQ/USP - Piracicaba/SP. http://www.pecege.esalq.usp.br/soja/ (12/01/10). 

3. CHEN, Y; AVIAD, T. (1990), Effects of humic substances on plant growth. In: McCARTHY P.; 

CLAPP, C.E.; MALCOLM, R.L. & BLOOM, P.R., (Eds.) Humic substances in soil and crop 

sciences: selected readings. Madison: SSSA. 161-186. 

4. VAUGHAN, D.; ORD, B.G. (1976), Uptake and incorporation of C14-labelled soil organic matter 

by roots of Pisum sativum L. Journal of Experimental Botany, Oxford, 32, 679-687. 

5. LEE, Y. S.; BARTLETT, R. J. (1976), Stimulation of plant growth by humic substances. Soil 

Science Society of America Journal, Madison, 40, 876-879. 

6. BENITES, V. de M.; POLIDORO, J.C.; MENEZES, C.C.; BETTA, M. (2006). Aplicação foliar 

de fertilizante organo-mineral e soluções de ácido húmico em soja sob plantio direto. Embrapa, 

Rio de Janeiro. 6p. (Circular Técnica, 35). 

Vol. 3 Page - 273 -

Pyrolisis Parameters Evaluation in the Biochar Preparation Process 

E. Inayve P. de Rezende a , A.P. Mangoni a , I. Messerschmidt a , A.S. Mangrich a* , 

E.H. Novotny b , M.H.R. Velloso b 

a Universidade Federal do Paraná, Centro Politécnico, Jardim das Américas, C.P. 19081, 

81531-990, Curitiba, PR, Brazil; b EMBRAPA Solos, R. Jardim Botânico, 1024, 22460-000, 

Rio de Janeiro, RJ, Brazil 

E-mail: mangrich@quimica.ufpr.br 


Considerable efforts are being proposed to mitigate the environmental problems caused by the 

significant increase of CO2 concentration in the atmosphere. The methodology of using 

biochar, obtained by pyrolysis of biomass, as a organic soil conditioner to mitigate this 

problem has gained support of a considerable number of components of the scientific 

community. The use of biochar in soil to produce positive effects on soil fertility was applied 

in the Amazon region by pre-Columbian indigenous community 1 . David R. Montgomery in 

its 2020 visions about care with world soil´s sad: “Over the next few decades, approaches 

such as low-till and organic methods could restore native soil fertility and store enough soil 

organic matter to offset global fossil-fuel emissions by 5–15%. Offsets, and soil fertility, 

could be further increased through adding biochar — charcoal made by heating organic 

wastes” 2 . Biochar presents as structural features characteristic condensed aromatic 

compounds, hydrogen-deficient, highly resistant to oxidation, and therefore the action of soil 

microorganisms, thereby contributing to carbon sequestration. Yet it may be partially oxidized 

in their peripherals aromatic groups, producing carboxylic and phenolic groups that contribute 

to soil CEC, buffering the acidity, complexing ions and inorganic structures, retaining water 

via hydrogen bonds and, consequently, increasing security (stabilization) and fertility of soil 

3,4 

. In the pyrolysis of biomass study in our laboratory, methods have been developed not only 

to produce biochar, but also aim to produce bio-fuels. Towards the development of scientific 

knowledge, technology and innovation in the use of organic by-products, especially derived 

from the biofuel industries, this work has been carried out to prepare "biochar" from the 

castor oil cake, through the pyrolysis at low temperatures ( 300-350 ° C) and deficiency of air. 



The castor oil cake, gridding in ball mills to a particle size of 80 meshes was placed in 

porcelain boats in the inner glass tube furnace EDG FT-40 microprocessor-controlled. The 

factors assessed by a 2 3 factorial design were: heating rate (V), final temperature (T) and 

warm-up period (P), at the levels of 5 and 10 ° C min -1 , 300 and 350 ° C and 30 and 60 min, 

Vol. 3 Page - 274 -

espectively. The solid material obtained was characterized by EPR, FTIR and NMR 

spectroscopy. 

EPR spectroscopy. The EPR spectra were obtained at room temperature (~ 300 K) on a 

Bruker EMX spectrometer operating at X-band (~ 9.5 GHz) using 100 kHz modulation 

frequency and 0.05 mT amplitude modulation. The parameters values were obtained by the 

treatment of experimental spectra with the aid of WinEPR software and weak pitch standard 

from Bruker Company. 

FTIR spectroscopy. The FTIR analyses were performed on a spectrophotometer model FTIR 

Biorad Excalibur Series (FTS-3500 GX) with the spectra resolution of 4 cm -1 in the region 

from 4000 to 400 cm -1 . The sample pellets for analyses were made using approximately 1 mg 

of the biochar sample and 99 mg of KBr spectroscopic grade and submitting the homogenized 

mixture to pressure. For each spectrum 32 scans were summed. 

NMR spectra. Solid-state 13 C NMR experiments were carried out using a Varian VNMRS 500 

MHz spectrometer at 13 C and 1 H frequencies of 125.7 and 500.0 MHz, respectively. The 

technique used was variable amplitude cross-polarization (VACP). 



EPR spectra. By EPR spectroscopy the g-factor, spin density and power saturation of the 

signal values were obtained. The values of the g-factor of EPR found are around 2.003, 

indicating the presence of free radicals in organic structures (OFR). The parameter values of 

spin density and power saturation of the signal can be seen in Figure 1. For the spin density 

parameter, the highest values are of those samples with longer periods of heating (P1, P2, P5, 

P6). For the power saturation of the EPR signal, the sample P2 (V = 30 0 C min -1 , T = 350 0 C, 

P = 60 min and D = 11.81 x 1018 spins g-1) sustained the greatest power of EPR while the 

sample P4 (V = 30 0 C min -1 , T = 350 0 C, P = 30 min and D = 4.60 x 10 18 spins g -1 ) showed a 

lower saturation power. Thus, the sample P2, by EPR spectroscopy, is presented with more 

integration spin arrangement of aromatic structures (better dissipates the energy resulting 

from the relaxation of spins), supporting more power, suggesting that the heating time was the 

most important factor for the formation of stable internal structure of the sample. 

FITR spectra. The FTIR spectra of the samples were very similar, all featuring mainly a broad 

band at 3680-3300 cm -1 , associated with the O-H stretching from alcohol carboxylic acids and 

water. Energy absorption in 1625 cm -1 attributed to the structural vibrations of C = C 

Vol. 3 Page - 275 -

aromatics, asymmetric stretching of C = OO- and bending of O-H groups. Shoulder at 1706 

cm -1 is assigned to C = O stretching of ketone, esther and carboxylic acid. 

Eixo z 

300 

350 

P2 

11,81 

5,66 

P4 

4,60 

1,74 

P (min) 

Eixo y 

P6 

12,16 60 

2,04 

P1 

9,31 

4,11 

P8 

8,72 

1,93 

30 

P3 

7,70 

4,48 

P5 

P7 

13,88 

2,68 

4,03 

1,76 

5 10 Eixo x 

v (\ C min ‐1 ) 

Figure 1: Diagram for the effects in planning 2 3 interpretation (Bold values inside the circles 

correspond to spin density (x10 16 spins g -1 ) and those below without bold are power (x10 -4 W)) 

NMR spectra. To aid in the analyses of the results, the Multivariate Curve Resolution (MCR) 

procedure was carried out using the software ‘The Unscrambler® v9.7’ (CAMO Software 

AS). The basic goals of MCR are: the determination of the number of components co-existing 

in the chemical system; the extraction of the pure spectra of the components (qualitative 

analysis); and extracting the concentration profiles of the components (quantitative analysis). 

The results of this analysis indicate that the set of analysed samples can be modelled by 

means of a two component mixture – binary, one of a partially carbonised material 

(Component 1, Fig. 2a), with aromatic groups presenting poor ring condensation (129 ppm) 

and alkyl groups. The other estimated compound is still less carbonised, presenting features of 

the precursors, like O-alkyl (72 ppm) and di-O-alkyl (shoulder at ~110 ppm) from cellulose; 

O-aryl (143 ppm) from lignin and aliphatic carboxyl (173 ppm). 

240 210 180 150 120 90 60 30 0 -30 

13 C Chemical Shift (ppm) 


Compound 1 

Compound 2 

Estimated Concentration (%) 

100 

80 

60 

40 

20 

0 

Compound 1 

Compound 2 

P1 P2 P3 P4 P5 P6 P7 P8 

Samples 

Figura 2 – Results of multivariate curve resolution (MCR) analysis. (a) Estimated spectra; (b) 

Estimated concentrations 

Vol. 3 Page - 276 -


The samples treated at lower temperature (300 °C - P3, P4, P7 and P8) were those that 

showed signals from the precursor (castor oil cake). In other words, the cited oxygen 

substituted groups present in the compound 2 are preserved from the castor oil cake due to the 

low carbonisation temperature employed, since this temperature avoided that these 

thermolabile compounds were decomposed or altered, unlike the samples treated at 350 °C. 


CNPq, DQ/UFPR, Ao Professor Dr. Ronny R. Ribeiro. 

References 

1. S. Bruun and J. Luxhoi, Environ. Sci. & Technology, March 1 (2008); 

2. D. R. Montgomery, Nature, 463, p. 26-32 (2010); 

3. E. H. Novotny at al., J. Braz. Chem. Soc., 20 (6), p. 1003-1010 (2009); 

4. T. Whitman and J. Lehmann, Environ. Sci. & Policy, p. 1024-1027 (2009). 

Vol. 3 Page - 277 -


Extraction of high-value lipids from Irish Peats 

Raymond McInerney a , Daniel John Hayes a , J.J. Leahy a , Michael HB. Hayes a 

a University of Limerick, Limerick, Ireland 

E-mail: Raymond.McInerney@ul.ie 

Lipids, which range from simple n-fatty acids or n-alcohols to more complex cyclic 

terpenoids and steroids, are insoluble in water but extractable with non-polar solvents e.g. 

hexane, chloroform, benzene or ether [1]. Lipids are generally not components of humic 

substances but they occur in associations with humic molecules. They can, however, be 

incorporated in humic structures through esterification with the carboxyl and hydroxyl 

components in humic acids, especially [2]. 

For the characterization of lipids in complex mixtures it is important to isolate the 

components freed from contamination, as evidenced by well resolved peaks in 

chromatographic procedures [3]. 

Organic solvent extraction of peat can give wax yields ranging from 5 to 15%. The amounts 

depend on the source of the peat, its pretreatment, and the solvent used [4]. 

The components of the waxes have relatively wide molecular weight profiles, and 

functionalities that range from n-alkanes to a variety of structures with reactive functionalities. 



Low density (LD), medium density (MD), and high density (HD) peat samples from raised 

bogs were provided by Bord na Mona (the Irish Peat Board). Samples were air dried and 

sieved. 

Solvent Extraction of Peat Samples. Each of the peat samples was extracted under reflux for 

4.5 hours in a 1:1 toluene/ethanol (1:1 v/v) mixture, then filtered under reduced pressure. The 

filtrate was rotary evaporated to remove the bulk of the solvent, and the remaining solvent 

was evaporated in a fume cupboard. The residual wax was then dried in a vacuum oven at 40 

o 

C for at least 3 hours. 

Derivatization of Sample for Gas Chromatography-Mass Spectrometry Analyses. The 

procedure described by Jansen et al. [5] was used for the clean-up and derivatization of the 

wax samples. The temperature programme for the GC-MS analysis was: 50 °C, 2 min; heating 

at 40 °C/min to 80 °C; holding at 80 °C for 2 min; heating at 20 °C/min to 130 °C; 

Vol. 3 Page - 278 -

immediately followed by heating at 4 °C/min to 300°C; and finally holding at 300 °C for 10 

min. Elemental analysis of the peat waxes used an Elemental Analyzer. 


The elemental analyses of the peat waxes are given in Table 1. Hydrogen to carbon atomic 

ratio of 1.7 and higher indicates that these components are long chain aliphatic compounds; 

i.e. mainly fatty acids, alcohols and sterols. 


Figure 1 shows the chromatogram of the wax from the HD peat sample. The compositions of 

the different peaks were identified using the GC/MS library and their relevant yields were 

quantified by the introduction to the filtered extracts of internal standards of the deuterated 

lipids, n-eicosane-d42 and eicosanoic-d39 acid. 

Table 1: Elemental composition of the peat wax samples 

C H O N S H/C a O/C a 

HD Wax 75.02 11.56 11.0 0.45 1.98 1.85 0.11 

MD Wax 73.91 10.90 12.86 0.38 1.94 1.77 0.13 

LD Wax 72.76 10.33 14.16 0.51 2.24 1.70 0.15 

a: Atomic ratio 

Figure 1: GC Analysis of wax extracted from the high density peat using ethanol/toluene 

A list of the possible components that are to be found in the wax extracted from the HD peat 

samples are given in Table 2. The waxes extracted from the MD and LD peat samples 

contained a similar list of components (not shown here). The major products identified, and 

their potential uses were: 

Docosanoic acid, also known as behenic acid, is an n-carboxylic acid, a fatty acid, formula 

C21H43COOH. In appearance. It is a white to cream coloured crystalline or powder material, 

Vol. 3 Page - 279 -

MP 74–78 °C, BP 306°C. Commercially, it is often used to give smoothing properties to hair 

conditioners and moisturizers. 

Table 2: Identifications of the different peaks in the wax of the high density peat 

Retention 

Time (min) 

Name of Compound % mass (dry mass) wax 

11.00 n-Butanoic acid 0.02 

23.56 Hexadecanoic acid 0.26 

27.69 Decanoic acid 0.14 

33.69 1-Docosanol 0.12 

35.23 Docosanoic acid 0.20 

37.18 Tetracosan-1-ol 0.12 

38.72 Tetracosanoic acid 1.08 

42.13 Hexacosanoic acid 1.59 

45.46 7,8, D-hydro-1-biopterin 0.13 

1-Docosanol is a long chain 22-carbon primary alcohol. It is also known as behenyl alcohol. 

It is used as an emollient in skin care. It has a major use as an antiviral agent, specifically for 

treatment of "cold sores" caused by the herpes simplex virus. It acts by inhibiting fusion 

between the human plasma cell membrane and the viral envelope. 

β-sitosterol is a common phytosterol, MP 139 o C. It is either used alone or in combination 

with similar phytosterols in reducing blood levels of cholesterol, and it is sometimes used in 

treating hypercholesterolemia. It has a positive effect on androgenetic alopecia (causing hair 

loss in males), and is also used in the treatment of prostatic carcinoma and breast cancer. 

Decanoic acid or capric acid, a saturated fatty acid, CH3(CH2)8COOH. It is used in organic 

synthesis and industrially in the manufacture of perfumes, lubricants, greases, rubber, dyes, 

plastics, food additives and pharmaceuticals. 

Tetracosanoic acid (or lignoceric acid), is the saturated fatty acid, C23H47COOH. It is a 

byproduct of lignin production. It is the most abundant fatty acid in skin ceramides [6]. 

Tetracosanol is a constituent of polycosanol, a mixture of alcohols. Tetracosanol and other 

long chain fatty alcohols, and their esters are known to improve the physical performance of 

athletes. Components comprising such alcohols and esters are contained in vegetable oil. 

4. Conclusion 

Long-chain aliphatic alcohols (polycosanols) and fatty acids (with more than 20 carbon 

atoms) are of considerable interest as healthcare and personal care products. Distillation and 

fractionated by short-path distillation would provide fractions that are rich in docosanol (C22) 

and tetracosanol (C24) and other long chain fatty alcohols that may be found in different peats. 



We acknowledge the financial and sample contributions of Bord na Mona. 

Vol. 3 Page - 280 -


References 

1. Dinel, H., M. Schnitzer and G.R. Mehuys, 1990. Soil lipids: origin, nature, content, decomposition 

and effect on soil physical properties. In: Bolag, J.M., Stotzky, G. (Eds.), Soil Biochemistry, Vol. 

6. Marcel Dekker, New York, pp. 397–429. 

2. Mathur, S.P. and R.S. Farnham, 1985. Geochemistry of humic substances in natural and cultivated 

peatlands. In: Aiken, G.R., McKnight, D.M., Wershaw, R.L. and MacCarthy, P., Editors, 1985. 

Humic Substances in Soil, Sediment and Water, Wiley, New York, pp. 53–85. 

3. Wiesenberg, G.L.B., L. Schwark and M.W.I. Schmidt, 2004. Improved automated extraction and 

separation procedure for soil lipid analyses. Eur. J. Soil Sci. 55, pp. 349–356. 

4. Howard A.J., and D. Hame, 1962. The extraction and constitution of peat wax. Chromatographic 

fractionation of wax, Journal of the American Oil Chemists' Society, 39, 5 

5. Jansen, B., K.G.J. Nierop, M.C. Kotte, P. de Voogt and J.M. Verstraten, 2006. The applicability of 

accelerated solvent extraction (ASE) to extract lipid biomarkers from soils. Appl. Geochem., 21: 

1006–1015. 

6. Vávrová, K., J. Zbytovská, K. Palát, T. Holas, J. Klimentová, A. Hrabálek and P. Dole al, 2004. 

Ceramide analogue 14S24 ((S)-2-tetracosanoylamino-3-hydroxypropionic acid tetradecyl ester) is 

effective in skin barrier repair in vitro, Eur. J. Pharm. Sciences 21(5), 581–587 

Vol. 3 Page - 281 -

Fluorescence of Aqueous Solutions of Commercially Produced Humic 


Olga Yakimenko a* , Aleksei Izosimov a , Daria Shubina b , Viktor Yuzhakov b , Svetlana Patsaeva b 

a Soil Science Department, Moscow State University, Moscow 119991, Russia; 

b Department of Physics, Moscow State University, Moscow 119991, Russia 

E-mail: ola-yak@mail.ru 


Humic substances (HS) and especially their water-soluble fraction play a very important role 

in environmental biogeochemistry [1-2]. Usage of commercially produced HSs in agriculture 

and soil remediation is very perspective nowadays. However diversity of commercial HS 

sources and technological know-how of their production cause the problem of their 

characterization and classification. 

Fluorescence spectroscopy is a powerful tool for rapid characterization of organic substances, 

in particular dissolved organic matter (DOM) in natural waters of different origin [3-4]. 

Fluorescence emission spectra of humic substances naturally occurring in water are generally 

characterized by a unique broad band, so called “humic-type fluorescence”, showing a 

maximum wavelength (λem) around 420-460 nm depending on both the sample origin and 

excitation wavelength (λex) [5-7]. Typically fluorescence of humic acids isolated from various 

soils and soil-related materials is shifted to longer wavelength region (500-520 nm) [8-9] 

comparably to humic substances of natural waters. Fluorescence spectra of commercial HS 

differ depending on genesis of source material: coal humates show the emission maximum at 

470 nm whether humates originated from peat, lake sediments and lignin derivatives 

demonstrate maximum emission shifted towards shorter wavelengths at excitation 

wavelengths 355 nm [10]. 

This objective of this work was to study the fluorescence properties of commercially available 

HSs produced from various organic resources, and to find spectral parameters useful for their 

rapid characterization and discrimination. 



Fluorescence spectra were examined for commercially available sodium and potassium 

humates manufactured in Russian Federation, USA, Canada and China from a number of 

source materials differ in their origin and humification conditions: coalified materials (brown 

coal, lignite, leonardite and humalite), peat, lake bottom sediment (sapropel) and organic 

waste material (lignosulphonate). 

Vol. 3 Page - 282 -

Fluorescence emission spectra were measured by luminescence spectrometer Solar CM 2203 

under excitation at 270, 310 and 355 nm for the samples diluted in 10 times. The choice of 

excitation wavelengths was based on our previous reports on DOM fluorescence [5-7]. Both 

absorption and fluorescence measurements were made under room temperature for aqueous 

solutions of humates at concentration 0.02 g l -1 and pH 6.0, placed in quartz cuvettes with 1 

cm optical path length and 5 ml volume. Fluorescence quantum yield was estimated using 

quinine sulphate solution as a reference. 

3. Results 

Fluorescence emission excited at different wavelength. Fig.1 illustrates that fluorescence 

emission maximum position differs for commercial HS of different origin. Most of HS from 

coals show the maximum near 500 nm, whether humates from young caustobioliths peat and 

sapropel demonstrate emission maximum at shorter wavelength area (430-480 nm). Humic 

product from lignosulphonate subjected to “artificial humification” has emission maximum in 

UV-area (360 nm). 

Intensity, rel. un. 

2 

1 


0 

300 400 500 

Wavelength, nm 

600 700 

Sa-Plod from sapropel 

BC-Hum from brown coal 

Pe-IXP from peat 

Pe-Eda from peat 

Hu-Usa from humalite 

Hu-Bsol from humalite 

Le-Sah from leonardite 

Le-Sp100 from leonardite 

Li-Ion from lignite 

Li-Sol80 from lignite 

OW-LhNa from lignosulphonate 

Figure 1: Fluorescence spectra excited at 310 nm for aqueous solutions of commercial HSs 

In contrast to studied earlier samples of DOM in natural waters or soil extracts [5-7], 

commercially available HSs did not exhibit noticeable shift of fluorescence maximum 

position along with increasing excitation wavelength: emission wavelength keeps practically 

constant for all commercial HS, except for HS produced from peat. 

Vol. 3 Page - 283 -

Fluorescence quantum yield (QY). Fluorescence QY for commercial HSs (see Fig. 2) is 

typically smaller than that of DOM in water (0.02-0.04), and is comparable for that of soil 

water-extractable DOM (0.001-0.003). Fluorescence QY depends essentially on excitation 

wavelength for natural HS, its value is increasing along with λex rising. In contrast, for 

commercial HSs the QY value is either decreasing with rising of excitation wavelength, or 

keeps constant. The former phenomenon is especially noticeable for coal humates, produced 

from mature caustobioliths, whether the latter one was observed for HS from peat and 

sapropel. 

We explain this observation considering that natural HSs differed from commercial HSs in 

bigger heterogeneity of fluorophores composition as evidenced by the behavior of their 

emission maximum and QY changes along with increasing of excitation wavelength. 

0,04 

0,03 

0,02 

0,01 

0,00 

Sa-Plod 

Fluorescence quantum yeild ex 270 

ex 310 

ex 355 

Pe-IXP 

Pe-Eda 

BC-Hum 

Li-Ion 

Li-BGHa 

Li-Sol80 

Le-Sah 

Le-HPA 

Le-Sp100 

Hu-Usa 

Hu-Dsol 

Hu-Bsol 

OW-LhNa 

Figure 2: Fluorescence QY measured at excitation wavelengths 270, 310 and 355 nm for different 

commercial HS samples (see Fig.1 for details of HSs) 



UV-excited fluorescence spectra for aqueous solutions of commercially produced humic 

substances were compared with that for earlier studied natural HSs of riverine and marine 

origin. Two parameters such as fluorescence emission maximum (λem) and fluorescence 

quantum yield were found very useful for rapid characterization of commercially available 

HSs. Both parameters measured along with excitation wavelength rising from 270 to 355 nm 

for solutions of commercially available HSs differs much from that for substances of natural 

origin. In contrast to natural HS, commercially available humates do not exhibit noticeable 

shift of fluorescence maximum position along with increasing excitation wavelength 270 to 

Vol. 3 Page - 284 -

355 nm. For commercial HSs the fluorescence quantum yield value is either decreasing with 

rising of λex or does not depend on it, demonstrating at opposite pattern comparably to natural 

HS, for which QY is rising with increasing excitation wavelength. Distinct fluorescence 

properties of humic substances observed along with variation of λex provide useful diagnostic 

criteria for distinguishing between commercial and natural humic substances. 


Financial supports of Russian Foundation of Basic Research (project 07-04-01510) and 

Presidium of Russian Academy of Science (grant of Biodiversity Program) are deeply 

appreciated. 

References 


1. D.S. Orlov, Humic Substances of Soils and General Theory of Humification. Balkema, Brookfield, 

1995, p.266 

2. I.V. Perminova, et al. in I. Twardowska, H.E Allen, M.H. Haggblom, S. Stefaniak, (Eds.), Viable 

Methods of Soil and Water Pollution Monitoring, Protection and Remediation. Series IV: Earth 

and Environmental Sciences, Springer, Netherlands, 69 (2005) 249-274. 

3. M.М.D. Sierra , et al., Marine Chemistry, 47 (1994) 127-144. 

4. P.G. Coble, et al., Marine chemistry, 51(1996) 325-346. 

5. S.V. Patsaeva, EARSeL Advances in Remote Sensing, 3 (1995) 66-70. 

6. A.S. Milyukov, et al., Moscow University Physics Bulletin, 6 (2007) 368-372. 

7. O.M. Gorshkova, A.S. Milyukov, S.V. Patsaeva, V.I. Yuzhakov. Proc. SPIE 6263 (2006) 248- 

255. 

8. N. Senesi, et al., Soil Sci. 152 (1991) 259-271. 

9. A. Zsolnay, et al., Chemosphere, 38 (1999) 45-50. 

10. P. Volkov, O.Yakimenko. in Humic Substances – Linking Structure to Functions. Proc. of 13th 

Meeting of the International Humic Substances Society (2006) 45-I: 261-265. 

Vol. 3 Page - 285 -

Assessment of the Oil Shale Byproducts Use as Soil Conditioner: Study of 

Sorption and Biodegradation of Phenol Models with Soil 

Rafael Garrett Dolatto a , Gilberto Abate b , Iara Messerschmidt b , Betânia Fraga Pereira a* , 

Antonio Salvio Mangrich b , Carlos Posser Silveira c , Clenio Nailto Pillon c 

a FAPEG/Embrapa Clima Temperado, BR 392, km 78, CP 403, 96001-970, Pelotas-RS, 

Brazil; b Univ. Federal do Paraná, Depto. de Química, CP 19081, 81531-990, Curitiba-PR, 

Brazil; c Embrapa Clima Temperado, BR 392, km 78, CP 403, 96001-970, Pelotas-RS, Brazil 

E-mail: betaniapereira@yahoo.com.br 


A reasonable oil shale water volume is generated in the oil shale industrialization, arising 

from the shale pyrolysis process. This oil shale water byproduct can be used as a soil 

conditioner, due to be rich in important micronutrients to plants and crops. Nevertheless, a 

high content of other not desirable compounds, such phenol and the derivatives o-cresol and 

p-cresol, can be present in this oil shale water. Although the water would be diluted 

previously to the application, it is very important to be aware of the phenolic compounds’ 

behavior in the presence of the soil sample in order to prevent and avoid the leaching of it to 

the groundwater . In this way, in the first moment, the aim of this work was to study, the 

interaction of phenol, o-cresol, and p-cresol models with a soil sample rich in clay fraction 

that was collected in an experimental area near an oil shale industry. A comparison of 

mercuric chloride and sodium azide methods was performed to investigate the existence of 

phenolic compounds models biodegradation. The following studies will considerer the 

interaction of the real oil shale water with soil samples. 



Tests in Batch: One gram (± 0.1 mg) of soil was transfer to glass flask of with 30.00 mL of 

CaCl2 0.01 mol L -1 [1]. The isotherms were obtained from 10 points in increasing 

concentrations of each phenolic compound model in the range of 0.00 to 500.0 mg L -1 . For 

excitement, it was used an orbital bench, operating at 170 oscillations min -1 for 48 hours. 

After this period the samples were centrifuged at 3000 rpm and concentrations remaining of 

the phenolic compound models in the supernatants phases were determined by the method of 

4-AAP [2]. 

Contact time: The time of apparent equilibrium between the phenol model and the soil sample 

was studied using a mass of five grams (± 0.1 mg) of soil volume in 150.0 mL of phenol at a 

concentration of 50.00 mg L -1 in an ionic medium of CaCl2 0.01 mol L -1 . Similarly, blank 

Vol. 3 Page - 286 -

evidence was prepared in the absence of phenol. 

Microbial Inhibition: For this test similar conditions of the experiments performed at the 

contact time were used. Some glass bottles were prepared only with soil samples (5.0000 g) in 

150.0 mL of phenol at a concentration of 50.0 mg L -1 and in another group of bottles beyond 

the soil it was also added HgCl2 100 mg L -1 acting as a microbial inhibitor [3]. The vials were 

kept under agitation of 170 oscillations min -1 for 10 days. During this time aliquots of 5.00 

mL of the suspensions were withdrawn, centrifuged at 3000 rpm for 5 minutes and the 

concentration of phenol was determined by absorption spectrophotometry in the UV region - 

λ fixed at 270 nm. Experiments were performed similarly to o-cresol and p-cresol. When the 

phenol was not more detected in the supernatants, the samples were fortified with a new 

injection of phenol (fortifications) from the stock solution, providing a concentration close to 

50.0 mg L -1 . All the inhibition experiments were performed in triplicate. 

Also another study was conducted using a second type of microbial inhibitor. It was used the 

solution of NaN3 drawing upon the same conditions of those studied with HgCl2, however, it 

was used NaN3 solution with a concentration of 1% (w / v) in the middle of CaCl2 0.01 mol L - 

1 as an inhibitor of microbial activity. 



The soil sample (62.3% clay, 31.1% silt and 2.6% sand) was collected in the surface layer 

between 0–18 cm, which is classified as clayey. The results of the sorption capacity of 

phenolic compounds from the soil are shown in Fig. 1. 

Figure 1 show that the isotherms for phenol and p-cresol are very similar, tending to linearity. 

For phenol and p-cresol, the curves showed a very similar behavior and are almost linear, and 

a removal between 10 and 4% was verified for phenol and one of between 25 and 4% for p- 

cresol, for the first and the last point of the sorption isotherms, respectively. For o-cresol the 

sorption was not significant, being near 3% for the first point of the curve and 0.7% for the 

last point. The decrease of sorption for the three compounds suggests saturation of sorption 

sites of soil, probably due to high concentration of phenols. 

However, other processes can proceed in parallel to the sorption, such as, biological 

degradation of phenol throughout the experiment. 

Vol. 3 Page - 287 -

Amount sorbed (μg g -1 ) 

600 

500 

400 

300 

200 

100 

0 

0 100 200 300 400 500 

Equilibrium concentration (mg L -1 ) 

fenol 

o-cresol 

p-cresol 

Figure 1: Sorption isotherms of phenolic compounds after 24 h of contact time with 1.0000 g of soil in 

30.0 ml of CaCl2 0.01 mol L -1 . Initial concentration of phenolic species between 5.00 and 500.0 mg L - 

1 . The points shown the average result of three experiments 

In this way, it was investigated the possibility of biodegradation of compounds in the soil 

sample, solutions of HgCl2 100 mg L -1 and sodium azide NaN3 1% (m/v) were used as the 

inhibitors microbial, and the results are shown in Fig. 2. 

phenol (mg L -1 ) 

60 

55 

50 

45 

40 

35 

30 

25 

20 

15 

10 


spike 1 

spike 2 

spike 3 

5 

0 

L.O.Q. 

0 1 2 3 4 5 6 7 8 9 10 

contact time (days) 

Figure 2: Monitoring of phenol concentration versus time in the absence of inhibitor (A), in the 

presence of 100 mg L -1 HgCl2 solution (B), and in the presence of 10 g L -1 azide solution (C). Initial 

phenol concentration and spike values, 50.0 mg L -1 ; soil mass, 5.0000 g; initial volume, 150.0 mL; 

ionic medium, 0.01 mol L -1 CaCl2 solution. All points represent the medium result of three 

experiments 

Initially the suspension containing HgCl2 over 10 days of monitoring showed no reduction in 

the concentration of phenol presentation that the effects of sorption and volatilization are not 

very pronounced in these samples. Concomitantly, the suspensions containing phenol in NaN3 

showed a similar behavior to the suspensions in HgCl2, but it was observed a decrease in the 

concentration of phenol in relation to the concentration initially added. It suggests the 

possibility of phenol adsorption by the soil around 20% when the sodium azide was used as 

Vol. 3 Page - 288 - 

(a) 

(b) 

(c)

an inhibitor. And it could be some influence of sodium in order to facilitate the process of 

phenol adsorption in the soil. Also, the effect of sorption is not significant in the presence of 

HgCl2, which could somehow block the adsorption sites of the soil, preventing the interaction 

of phenol, through electrostatic interactions between phenol and soil. Moreover, in the 

suspension with no addition of an inhibitor, the phenol is no longer detected in solution after 

48 hours of contact in the experimental conditions studied. Although it was tested the ability 

of soil in to degrade a new quantity of phenol in the solution (fortification 1), again, the 

compound was not detected in the suspension after 72 hours. A second phenol fortification 

was made in the same bottle (fortification 2) and it was again observed degradation. A new 

fortification (fortification 3) was performed and after 48 hours, 50 % of the added phenol was 

detected, indicating that possibly half of it was being degraded. The degradation of phenol 

after new injections was considered a strong indication of the degradation of the compound 

instead of the sorption process. Similar experiments were conducted using o-cresol and p- 

cresol and similar results were observed in the microbial inhibition tests for the compound 

phenol. 


The study conducted with microbial inhibitors HgCl2 and NaN3 indicate the biodegradation of 

phenol and o-cresol in the studied soil sample. In the following researches it will be studied 

the sorption or biodegradation of phenolic compounds in Brazilian soil samples with different 

characteristics (texture, organic matter, clay content, pH, CEC) where it was added solid and 

liquid (oil shale retort water) by-products from the industrialization of oil shale in order to 

verify the environmental safety these. 



FAPEG/Embrapa Clima Temperado/PETROBRAS, DQ/UFPR, CNPq, Brazil. 

References 

1. OECD-Organization for Economic Co-Operation And Development. Guideline for the testing of 

Chemicals. Adsorption-Desorption Using a Batch Equilibrium Method; Adopted: 21st January 

2000, OECD/OCDE 106. 

2. APHA – American Public Health Association. Standard Methods for the Examination of Water 

and Wastewater. 1995. 19th Edition. Washington, DC, USA. 

3. Viotti, P.; Papini, M., P.; Stracqualursi, N.; Gambá, C. Ecological Modelling, 182 (2005) 131-148. 

Vol. 3 Page - 289 -

Humic Acids from Fines of Residual Coal Type Material: Preparation and 

Characterization 


G.M.Maurício a , A.C.S. Wimmer a , E.A.Brocchi a* , A.C.Vidal a , R.A.Nunes a 

a Catholic University of Rio de Janeiro, PUC-Rio, Brazil 

E-mail: ebrocchi@puc-rio.br 

The United States have the biggest reserves of xystus in the world, followed by Brazil. The 

major part of the Brazilian xystus is distributed along Irati, an area that contains one of the 

largest amounts of xystus in the world. Irati comprises several Brazil states such as São Paulo, 

Paraná, Santa Catarina, Rio Grande do Sul and Goiás. 

In Goiás it has been developed by Petrobás a continuous xystus oil removing process which is 

well recognized by both a low water consumption and a fine solid waste generation. 

These fines are residual coal type and, as such, can be considered as a source for humic 

substances (HS) production. In that context, several laboratorial scale methods were tested in 

order to obtain a kind of HSs. The obtained material was characterized and has been tested in 

environmental applications such as degraded soil recovery, production water treatment and 

fertilization. 

This work presents an initial evaluation of a selected extraction method applied on the 

residual fines based on a preliminary infrared analysis of the obtained humic substance and its 

comparison with the starting material original spectrum. 



The material employed for this study was a fine coal type obtained by a xystus processing 

industry. The residue pretreatment consisted of a brief granulometric adjustment, where the 

material was triturated in a mill and screened to particles finer than 150 mesh (0.105 mm). 

Then it was characterized and analyzed in order to determine C, H, O, N and S concentrations 

as well as humidity and ashes content. The Humic Acid (HA) preparation was carried out 

through different chemical treatment such as those using H2O2, formic acid, formic acid + 

H2O2, KOH, NaOH and HNO3. Among the studied treatments, the oxidation method with 

HNO3 was the one which showed the best result. 

It has been carried out by placing 5.0 grams in a 1000 mL flask with 100 mL HNO3 25%. The 

procedure was based on the method described by Trompowsy (2006) in which the whole 

Vol. 3 Page - 290 -

sample is heated up to boiling but kept under reflux for 4 hours. After this the cold material 

was placed in a number of a centrifuge tubes and left to rest for 12 hours. Then a 5000 rpm 

centrifuge has been used for 20 minutes to produce both a solution and a precipitated. The 

latter one has been put to react with 100 mL KOH 1M solution under mechanical agitation for 

12 hours in a N2 atmosphere. The former (solution) has been again put to rest for 12 hours in a 

set of centrifuge tubes before being centrifuged in the same previously conditions. The 

generated solution was taken to pH about 1.0 by adding 6M HCl and then placed in 

centrifuge tubes. After 12 hours it has been centrifuged in the same condition (5000 rpm, 20 

minutes) in order to obtain a precipitated which was to be the humic acid (HA). This material 

has been left to dialysis before being freeze-dried. 

The characterization of the obtained material was performed through conventional methods, 

typically employed for the humic substances studies, such as visible UV, Infrared and ICP. 

C, H, N e S analysis was implemented as described in ASTM-5291 method and the humidity 

concentration was determined through drying procedure up to a constant weight in a stove at 

105 ºC. The ashes were obtained in an oven at 550 ºC in order to evaluate its concentration. 

The UV-Vis equipment has been employed to determine the E4/E6 ratio (Abs 

465nm/665nm). The HA was dissolved at 20 mg/L in a NaHCO3 0.05 mol/L solution while 

the pH solution was kept at 8.5. A spectrophotometer was used to perform the sample 

scanning in the 190-800 nm range. An infrared spectrometer with Fourier transformed was 

also employed (2500 to 25000 nm). 



The starting material prior to the chemical treatments presented 2.61% and 86.20% humidity 

and ashes level, respectively. The oxidation method with HNO3 has produced a HA weight 

recovery, as compared with the initial sample mass, of about 6.5%. The sample 

characterization results of the obtained material are presented below. 

Table 1: Elementary analysis of the starting material (-150 mesh) and resulting HA 

Species 

% (m/m) 

Fines HA 

C 6.4 18.2 

H 0.9 2.3 

N 0.3 1.2 

S 3.2 < 0.3 

Vol. 3 Page - 291 -


The humification number, given by the E4/E6 relation, was 0.3371. Figs. 1 and 2 show the 

infrared spectra originated from the residual fine samples and from the obtained HA. 

Figure 1 – Xystus fines infrared spectrum. 

Figure 1. Infrared spectrum of the residual fine 

Figure 2: Resulting infrared spectrum of HA obtained from the residual fines 

Comparing the two infrared spectra one can notice that in the HA spectrum new bands 

appeared in the 1400-3400 cm -1 range. These bands are related to the carboxyls and hidroxyls 

bonds. According to literature data, the peaks indicated below have the following 

relationships. 

Vol. 3 Page - 292 -

Conclusions 


• 3354.20 cm -1 → O-H and N-H 

• 2287.35 cm -1 – 1838.11 cm -1 → C-H 

• 1626.53 cm -1 – 1680.64 cm -1 → C=O 

• 1510.80 cm -1 , 1534.80 cm -1 and 1575.95 cm -1 → COO - 

• 1285.29 cm -1 – 1424.55 cm -1 → deformation of O-H carboxyls 

An alternative starting material has been used in order to extract humic acid from it. The 

material presents some coal characteristics as it is consisted of fine particles generated as 

residue in a xystus oil removing process. 

Among several studied reactants and process it was clear that the HNO3 oxidation followed 

by a number of separation steps was the one which gave best results. The obtained material, 

having some HA features, was related to a weight recovery of about 6.5 % in respect to the 

initial material mass. 

In the infrared spectrum of the obtained material it was detected new band appearances in the 

range between 1400 and 3400 cm -1 which can be related to typical HS existing bonds (O–H 

and N–H). 

These preliminary results allow us to conclude that the product obtained through chemical 

oxidation with HNO3 has shown some compatible characteristics with those presented by a 

typical HA. This fact is a great incentive to carry on the investigation on applying this residue 

as an alternative starting material for extracting humic substances such as the humic acid. The 

obtained HS also opens new possibilities of environmental interest since it has been tested in 

the recovery of spoiled soils and in the production water treatment. Also, it has been planned 

to apply some HS samples as agriculture fertilizer. 

The whole idea is now being considered to be applied on materials related to the coal industry 

as a mean of giving some use for the concentration process tailings. 

References 

1. GIEGUZYNSKA, E. et al. Compositional differences between soil humic acids extracted by 

various methods as evidenced by photosensitizing and electrophoretic properties. Chemosphere 1 

(2009) . Consulting: 12 /March/ 2009. 

2. HAUMAIER, L.and ZECH, W. Black carbon – possible source of highly aromatic components of 

soil humic acids. Org. Geochem. 23 (1995) 191–196. 

3. RAJ, S. The nature and composition of coal humic acids. Ebasco services incorporated. Available 

at: :http://www.anl.gov/PCS/acsfuel/preprint%20archive/Files/25_4_SAN%20FRANCISCO_08- 

80_0058.pdf. Consulting: 23/Jan/2009. 

4. TROMPOWSKY, P.M. Síntese e caracterização de substâncias húmicas semelhantes aos ácidos 

húmicos de carvão de eucalipto, e sua interação com diclorofenol, cálcio, manganês e alumínio. 

MSc Thesis.Viçosa University. MG. 2006. 107 p. 

Vol. 3 Page - 293 -

Assessing the Effect of a Bio-accelerated Composting Process Using 

Analytical Pyrolysis (Py-GC/MS) 

F. Pérez-Barrera a *, K. Akdi a , F.J. González-Vila b , J.A. González-Pérez b , T. Verdejo b 

a A.M.C. Chemical/Trichodex S.A., P.I. La Isla, Avda. Rio Viejo, 44-45, 41700 Dos 

Hermanas, Sevilla, Spain, b IRNAS, CSIC, P.O. Box 1052, 41080 Sevilla, Spain 

E-mail: info@amcchemical.com 


The use of recycled materials in agriculture as fertiliser and as an organic amendment in 

intensively cropped and organic matter-depleted soils is an important environmental strategy 

[1]. There is a large variety of composted materials for which quality must be guaranteed to 

ensure a safe agricultural use. Appropriate management of the composting process of urban 

wastes is needed to avoid harmful effects caused by non-matured compost application [2]. At 

the same time, excessive composting could lead to i) loss of N and polysaccharides with a role 

in soil aggregation, and ii) immobilization of nutrients (mainly N and P), hence it has been 

reported that such a “postmature” compost may be less favourable to plant nutrients uptake 

than are less-matured composts [3]. 

Pyrolysis coupled to gas chromatography and mass spectrometry (Py-GC/MS) is a powerful 

tool widely applicable in the characterization of complex organic mixtures with diverse origin 

and is mainly used for the direct study of materials which, owing to their complexity, are 

difficult to analyse by conventional methods like composted organic matter (OM) [4]. Py- 

GC/MS. Pyrolysis of composted OM generates a wide range of products with diverse 

chemical properties that can be related to their biochemical origin (aliphatic compounds and 

methoxyphenols derived from lignin, cyclic ketones and furans from polysaccharides, Ncontaining 

molecules from proteins, organic acids...). 

In this work Py-GC/MS has been applied to the study of the composting evolution of organic 

a organic material composed of urban pruning residues and sewage sludge with and without 

the addition of a microbial bio-accelerator (CBB). 



The bio-accelerator CBB is a natural product developed by A.M.C. Chemical/Trichodex Co. 

(www.amcchemical.com, Seville, Spain) and based in a mixture of organisms that, when 

applied to fresh organic materials, is able to accelerate the composting process. Among 

known catalytic activity of the selected microbial mix favoring the degradation of 

lignocellulosic materials include xylanase and glucanase activities. 

Vol. 3 Page - 294 -

The organic material used was composed of urban pruning residues and sewage sludge at a 

ratio 30:70; 2) and was composted in the presence of CBB at the recommended dose and 

without CBB using a “slow process” composting i.e, without using enforced aeration of the 

windrow during the degradation phase. During the first 4 weeks of composting, the windrow 

temperature was monitored periodically every 2–3 days. 

Compost piles were sampled in different seasons and at regular time intervals during 

composting. After sampling, fractions were sieved at a particle size < 5 mm, air-dried and 

grinded before analysis. 

Analytical pyrolysis (Py-GC/MS) was performed with a double-shot pyrolyzer (model 2020, 

Frontier Laboratories) directly connected to a GC/MS system Agilent 6890 equipped with a 

fused silica capillary column (J&W Scientific 5MS 30 m × 250 µm × 0.25 µm inner 

diameter). The detector consisted of an Agilent 5973 mass selective detector (EI at 70 eV). 

The analysis was performed at pyrolysis temperature 500 ºC with final temperature achieved 

at a rate of 20 ºC min -1 and the end temperature was maintained for 1 min. The GC–MS 

conditions were as follows: oven temperature was held at 50 ºC for 1 min and then increased 

up to 100 ºC at 30 ºC min -1 , from 100 to 300 ºC at 10 ºC min -1 and isothermal at 300 ºC for 10 

min. The carrier gas used was He with a controlled flow of 1 ml min -1 . Pyrolysis products 

were identified using the Wiley and NIST computer libraries and attending to the relative 

retention times and spectra reported in the literature. The chromatograms were then integrated 

and the relative contents of the different products calculated on the basis of peak areas. 

3. Results 


The evolution of the temperature in compost piles amended with CBB (recommended BIO 1 

and double recommended dose BIO 2) and 

unamended (0) is shown in Figure 1. It is 

apparent that untreated piles are unable to 

sustain an adequate thermophilic stage to 

appropriately catalyze the degradation 

Figure 1: Evolution of the temperature in piles of 

compost SS amended and not with CBB with an 

indication of the different composting stages 

processes. In the pile amended with CBB, 

even at lower dose (BIO 1), a shoulder in 

the curve is evident indicating the growth 

of fungi and lignin degradation. 

Vol. 3 Page - 295 -


For practical purposes the chromatograms of 

the products released by pyrolysis can be 

analyzed at first sight when divided in three 

main domains: Domain I (elution time c. 2–9 

min): low molecular weight (MW) products 

dominated by polysaccharides (Ps) + proteins 

(Pr) derived products; Domain II (elution time 

c. 9–19 min): where most lignin (Lg) derived 

products (methoxyphenols) are included; 

Domain III (elution time >c. 19 min): high 

MW compound where sterols and compounds 

derived from lipids (Lip) elute [4]. 

When studying the pyrograms produced by the 

un-composted wastes (starting material) and 

composted material with and without addition 

of CBB at thermophilic stage (Fig.2), the 

effect of the bio-accelerator is apparent. The 

non-treated piles show a very similar 

pyrogram to that of the starting material 

indicating a low degree of transformation. This 

is dominated by alkyl structures presumably 

Figure 3: Evolution of the relative abundance of the 

different families of compounds (% of total 

chromatographic areas) as inferred by Py-GC/MS during 

the composting process 

Vol. 3 Page - 296 - 

Figure 2: Pyrograms from un-composted wastes 

(starting material) and composted material with 

and without CBB at thermophylic stage 

present as esters, or physically 

entrapped in a matrix consisting of 

carbohydrate and protein (Domain 

I) which, after pyrolysis, yields only 

small amount of furan derivatives, 

cyclohexene, cyclopentadiene, Nbearing 

fragments and some 

aromatic products (mainly 

alkylbenzenes). The CBB treated 

compost showed conspicuous 

changes with respect to the starting 

and un-treated compost, in

particular a decrease in fragments arising from polysaccharides and an increase in the relative 

yields of aromatic products and methoxyphenols derived from lignin (lignin residues) is 

observed. Finally, the yields of heterocyclic compounds (indole derivatives) were higher in 

the starting materials decreasing progressively with composting time. The CBB bacterial 

product effectively seems to favour composting also shortening composting times. 

Analytical pyrolysis can be used as a semi-quantitative technique when working with relative 

abundances of the different families of compounds as percentage of total chromatographic 

areas. In Fig.3 the evolution of the main components of organic matter is depicted. At the 

early stage of composting (after 3 weeks), in untreated piles only a decrease in the relative 

abundance of polysaccharide constituents is observed whereas the effect of the bio-accelerator 

is again apparent in the treated piles where a sharp decrease of polysaccharide, polypeptides 

and lignin derived compounds is evident. This is in agreement with the degradation process of 

complex lignocellulosic materials where hemicellulose and lignin are degraded and partly 

transformed during thermophilic stage after easily degradable carbon sources have been 

consumed [5 and references therein]. 



The pyrolysis technique used (double shot Py-GC/MS) is a valid tool to assess compost 

evolution and was also informative in assessing to which extent compost transformation 

reached an acceptable stabilization when final compost is sufficiently mature. In this respect, 

Py-GC/MS was the efficacy of CBB compound in accelerating the composting process. The 

technique also provides information about the main biogenic structures affected during 

composting (polysaccharides, polypeptides, lipids and lignins). Furthermore, by calculating 

percentual values this technique could also be developed into a semi-quantitative tool to study 

composting. 

References 

1. C.García, T.Hernández, F.Costa, Waste Manage. Res. 10 (1992) 445. 

2. I.Déportes, J.L.Benoit-Guyod, D.Zmirou, Sci. Total Environ. 172 (1995) 197. 

3. M.J.Blanco, G.Almendros, Plant Soil 196 (1997) 15. 

4. F.J.González-Vila, J.A.González-Pérez, K.Akdi, M.D.Gómis, F.Pérez-Barrera, T.Verdejo, Biores. 

Technol. 100 (2009) 1304. 

5. M.Tuomela, M.Vikman, A.Hatakka, M.Itävaara, Biores. Technol. 72 (2000) 169. 

Vol. 3 Page - 297 -


Vol. 3 Page - 298 -


Natural Organic Matter and Humic Substances Biological 

and Physiological Effects 

Vol. 3 Page - 299 -



A Turning Point of Wheat Breeding and Humic Substance 

Reza Shahryari * 

Islamic Azad University, Ardabil branch, IRAN 

E-mail: shahryari@iauardabil.ac.ir 

Organic matter in the soil exists in three different forms: 1) Living plant and animal matter, 2) Dead 

plant and animal matter and 3) decomposed plant and animal matter (humic substances)(4). 

Differences in SOM and its turnover are related to changes in climate, parent rock, and vegetation and 

to numerous complexes biological, chemical and physical soil processes (7). Humus is defined by 

Stevenson (1994) as the total organic fraction in soils exclusive of non-decomposed plant and animal 

material, their partial decomposition products, and the soil biomass. Thus humic substances (e.g. 

humic acids and fulvic acids) make up the bulk of humus (7). The concentration of HS varies from 

place to place; the values in seawater being normally from two to three mg/l (8). HS in soils and 

sediments can be divided into three main fractions: humic acids (HA or HAs), fulvic acids (FA or 

FAs) and humin (3). Humic acids are the fraction of humic substances that is not soluble in water 

under acidic conditions (pH < 2) but is soluble at higher pH values. They can be extracted from soil by 

various reagents and which is insoluble in dilute acid. Humic acids are the major extractable 

component of soil humic substances. They are dark brown to black in colure. Fulvic acids are the 

fraction of humic substances that is soluble in water under all pH conditions. They remain in solution 

after removal of humic acid by acidification. Fulvic acids are light yellow to yellow-brown in color. 

Humin is the fraction of humic substances that is not soluble in water at any pH value and in alkali. 

Humins are black in color. Many investigators now believe that all dark colored humic substances are 

part of a system of closely related, but not completely identical, high - molecular - weight polymers. 

According to this concept, diferences between humic acids and fulvic acids can be explained by 

variations in molecular weight, numbers of functional groups (carboxyl, phenolic OH) and extent of 

polymerization (12). 

In regard to the potential of the HA, continuous development has led to availability of various 

commercial humic acid based products and they are widely marketed. The HA products are usually 

available in the form of inexpensive soluble salts, referred to as potassium humate (2). Humates of 

brown coal, peat and soils are mostly studied while sapropel humates (bottom organic sediments) are 

much less known (6). 

The following principal ways of HS action could be proposed: organism development, hormone-like 

activity, nutrient carriers, catalysts of biochemical reactions and antioxidant activity (5). The 

biological activity of HS encompasses all the activities of HS in regulating plant biochemical and 

physiological processes, irrespective of their stimulatory or inhibitory roles. Mitigating activity of HS 

Vol. 3 Page - 300 -

is observed under various stress conditions including both biotic and abiotic ones (7). 

Nowadays, humic preparations are increasingly applied as stimulators in plant breeding (6 and 10). 

Shahryari et al (11) believed that major aim of each wheat breeding program is to increase yield and 

improve quality. Potassium Humate causes increase in crop quality and quantity (10). 

Application of HA in agriculture as soil fertilizer and soil conditioner has been extensively discussed 

in the literatures. To date, numerous researches have demonstrated conclusively that HS have 

significant impacts on the soil structure and plant growth (2). Farmers use humates to accelerate seed 

germination and improve rhizome growth. These materials are able to stimulate oxygen transport, 

accelerate respiration and promote efficient utilization of nutrient by plants. These observations 

prompted scientists to study the specific properties of humates and their possible benefits in improving 

health and well being of humans and animals (4). Nevertheless, HA in proper concentrations can 

enhance plant and root growth (1). 

Seyedbagheri (9) evaluated commercial humic acid products derived from lignite and leonardite in 

different cropping systems from 1990 to 2008. The results of those evaluations differed as a result of 

the source, concentration, processing, quality, types of soils and cropping systems. Under their 

research, crop yield increased from a minimum 9.4 percent to a maximum 35.8 percent. Also, 

Shahryari et al (10) observed potassium humate increased average grain yield of bread wheat from 

2.49 to 3.61 ton/ha in a well watered condition. This was 45% increase in yield. 

In present study, we try answer to the question how humic substance increase wheat grains yield. 


Six bread wheat genotypes (Gascogen, Sabalan, 4057, Ruzi-84, Qobustan and Saratovskaya-29) 

planted in Agricultural Research Station of Islamic Azad University, Ardabil branch, Iran. 

Experimental design was split plot on the basis of completely randomized block design with three 

replications. Factor A was application of potassium humate or not; and factor B was genotypes. 

Treatments by a sapropel derived potassium humate (1ml/l) were done at four stages: pre-planting on 

seeds, tillering stage, stem elongation and after anthesis. Yield and some of yield related characters 

measured. Those were grain yield, biomass per plant, spike number per square meter, seeds number 

per spike, 1000 seed weight, Spike length, peduncle length and plant height. 

Cause and effect of characters studied by use of the path coefficient analyses where grain yield was 

kept as resultant variable and other contributing characters as causal variables. The most suit model 

formed for grain yield, separately, in the both conditions of with potassium humate or without it. 

Characters with non significant correlations removed and Path analysis were done for remained 

characters. 



Vol. 3 Page - 301 -


Increasing of grain yield by 45% under effect of potassium humate was unbelievable (10). Kulikova et 

al. (5) expressed that in spite of numerous studies on the biological effects of HS, the mechanism of 

their action remains unclear. But there must be a logical reason. A biometric procedure Such as Path 

analysis leads us to understanding of the genetic association of traits and their contribution to yield. 

Comparison of path coefficients in two different conditions of this study revealed there were more 

complex relations between characters at presence of potassium humate (Figure 1). Cumulative effects 

(significantly direct and indirect effects) of traits caused increase in yield. 

In the first step of path analysis; peduncle length, plant height, 1000 seed weight and spike number per 

square meter removed from model. Therefore these four traits had not effect on increasing of yield at 

presence of potassium humate. In the other steps, traits with non significant regression removed from 

regression model and continued path analysis. Resulted model revealed that seed weight per spike had 

the most effect on yield increase with direct effect (r = 0.576) at presence of potassium humate. After 

that spike length (r = 0.337), biomass (r = 0.254) and seed number per spike (r = 0.175) had total 

correlation effects on increasing of grain yield. 

Researchers must be tried to improve quality, while maintaining the yield level of standard cultivars in 

breeding programs. Plant breeders focused on increasing of yield, pay attention to genetic of alleles 

such as dominance or recessive; co dominance, incomplete dominance, polygenic, pleiotropic and 

epistasis effects. On the other hand may be a linkage between a desirable and a non-desirable gene. 

Such relations as these induce difficulties in breeding program. There are negative correlations 

between crop yield and some of traits. For example in a breeding program, we want to increase protein 

content of wheat flour but yield may decrease. And for successfully breeding for increasing of these 

two characters together, we make an effort for a long time. But we can succeed to this important 

subject by application of humic substance as a miracle natural biological material. 

Figure 1: Diagrammatic representation of direct and indirect effects of variables on grain yield with 

potassium humate treatment 


Vol. 3 Page - 302 -

Potassium humate increased grain yield by the way of seed weight per spike. After that spike length, 

biomass and seed number per spike were notable. These traits should be attending breeding programs 

for extra increasing of yield at presence of potassium humate. This could be a turning point between 

wheat breeding and humic substances application. 



I would like appreciate from my Professors A. Gadimov (Institute of Botany at the Azerbaijan 

National Academy of Sciences), E. Gurbanov (Faculty of Biology at the Baku State University, 

Azerbaijan) and M. Valizadeh (Agriculture Faculty at the Tabriz University in IRAN) are greatly 

acknowledged. The author also thanks R. Talai (Ardabil Agricultural and Natural Resources Research 

Centre, IRAN) for assistance in statistical analysis. 

References 

1. M. Bacilio, P. Vazquez and Y. Bashan. Alleviation of noxious effects of cattle ranch composts on 

wheat seed germination by inoculation with Azospirillum spp. Biol Fertil Soils. 2003. 38: 261– 

266. 

2. S.S. Fong, L. Seng and H. B. Mat. Reuse of Nitric Acid in the Oxidative Pretreatment Step for 

Preparation of Humic Acids from Low Rank Coal of Mukah, Sarawak. J. Braz. Chem. Soc. 2007. 

Vol: 18. No: 1. 41-46. 

3. International Humic Substances Society. What are humic substances? Focus on form: Retrieved 

May 27, 2009, from http://ihss.gatech.edu/ihss2/whatarehs.html 

4. K.M.S. Islam, A. Schuhmacher and J.M. Gropp. Humic Acid Substances in Animal Agriculture. 

Pakistan Journal of Nutrition. 2005. 4 (3): 126-134. 

5. Kulikova N. A, E. V. Stepanova and O.V. Koroleva. Mitigating Activity of humic substances: 

direct influence on biota. In: I.V. Perminova, et al. (ed.). Use of humic substances to remediate 

polluted environments: from theory to practice. Springer Netherlands. 2005. Vol: 52. 285–309. 

6. A.A. Perk and Popov A.A. Growth and development of plants under the action of different 

fractions of sapropel humates. 2nd International symposium plant growth substances: intercellular 

hormonal signaling and applying agriculture Abstracts. Kyiv, Ukraine. 2007. P: 149. 

7. D. Pizzeghello, G. Nicolini and S. Nardi. Hormone-like activity of humic substances in Fagus 

sylvaticae forests. New Phytologist. 2001. 151: 647–657. 

8. A. Al-Rasheed Radwan. Water treatment by heterogeneous photocatalysis an overview. 4 th SWCC 

Acquired Experience Symposium held in Jeddah. 2005. 

9. Mir-M. A. Seyedbagheri. Perspective on Over a Decade of On-Farm Research on the Influence of 

Humates Products on Crop Production. Proceedings of the 14th meeting of International Humic 

Substances Society. From molecular understanding to innovative applications of humic 

substances. I.V. Perminova and N. A. Kulikova. (eds). 2008. 603- 604. 

10. R. Shahryari, A. Gadimov, E. Gurbanov and M. Valizadeh. 2009. Application of potassium 

humate in wheat for organic agriculture in Iran. Abstracts Book of Go Organic International 

Symposium. The Approach of Organic Agriculture: New Market, Food Security and a Clean 

Environment. Bangkok, Thailand. P: 59. 

11. R. Shahryari, E. Gurbanov, A. Gadimov, M. Valizadeh, H. A. Hosseinpour, J. Bargiyan Khiyabani 

and B. Teymuri. Wheat genotypes quality affected by potassium humate under terminal drought. 

(In Persian). Proceedings of the 11 th National Iranian Soil Sciences Congress. 2009. Gorgan, Iran. 

12. Weber, J. 2009. Definition of soil organic matter. Focus on form: Retrieved Oct 10, 2009, from: 

http://www.humintech.com/001/articles/article_definition_of_soil_organic_matter.html. 

Vol. 3 Page - 303 -

Response of Maize Genotypes to Changes in Chlorophyll Content at 

Presence of Two Types Humic Substances 

Reza Shahryari a* , Babak Shahmorad Moghanlou b , Ali Mohammad Pour Khaneghah b 

a Islamic Azad University, Ardabil Branch, Iran; b Young Researchers Club, Islamic Azad 

University, Ardabil Branch, Iran 

E-mail: rz_shahriari@yahoo.com 



Corn (Zea mays L.) grows in more countries than any other cultivated crops. It is a major 

source of food for both Human and animals through the world [8]. Also, it is one of the major 

crops in Iran. 

According to the classical definition, Humic substances (HS) are "a general category of 

naturally occurring heterogeneous organic substances that can generally be characterized as 

being yellow to black color, of high molecular weight and refractory" [4]. The concentration 

of HS varies from place to place. Their size, molecular weight, elemental composition, 

structure, and the number and position of functional groups vary, depending on the origin and 

age of the material [7]. HS are major components of the natural organic matter (NOM) in soil 

and water as well as in geological organic deposits such as lake sediments, peats, brown coals 

and shales. They make up much of the characteristic brown color of decaying plant debris and 

contribute to the brown or black color in surface soils. HS in soils and sediments can be 

divided into three main fractions: humic acids (HA or HAs), fulvic acids (FA or FAs) and 

humin [3]. 

Nowadays, humic preparations are increasingly applied as stimulators in plant breeding [6 and 

9]. Shahryari et al [10] believed that major aim of each wheat breeding program is to increase 

yield and improve quality. Potassium humate causes increase in crop quality and quantity [9]. 

Research has confirmed that humic substances can indirectly and directly affect the 

physiological processes of plant growth [11]. The following principal ways of HS action 

could be proposed: organism development, hormone-like activity, nutrient carriers, catalysts 

of biochemical reactions and antioxidant activity. In spite of numerous studies on the 

biological effects of HS, the mechanism of their action remains unclear [4]. Yang et al [12] 

expressed it is unknown how humic substances decrease chlorophyll accumulation. They may 

inhibit the biosynthetic pathway of chlorophyll; stimulate the degradative pathway of 

chlorophyll, or both [12]. This reduces chlorophyll accumulation and photosynthesis, which, 

in turn, diminishes total plant growth [11]. 

Vol. 3 Page - 304 -

Liu et al. [5] evaluated the effect of a commercial preparation of humic acid on the 

chlorophyll concentration of creeping bentgrass. Badsar et al [1] conducted an experiment for 

determination effect of a sapropel derived HS on wheat genotypes under drought stress. 

The aim of this investigation was to find-out the effects of two type humic substances derived 

from different origins on chlorophyll content of maize genotypes. 


An investigation was conducted for determination effect of two HS on chlorophyll content of 

different maize genotypes. Twenty six grains of every seven maize genotypes cultured three 

replicated in Petri dishes. Solution of 0.067 % w/v prepared from the both of liquid HS (Table 

1). The volume of preparations was 1.5 L. Then, 15 ml solution was put in each Petri dish. 

Petri dishes had put in the dark place within the laboratory temperature. Solution consumed 

by seed, was to provide equal value during the germination. 

Table 1: Compounds of liquid humic fertilizers based on peat and leonardite 

Humic substances Humic acids Fulvic acids Total humic extracts 

(% w/v) 

(% w/v) 

(% w/v) 

Peat based 3.3 0.9 4.2 

Leonardite based 13.2 3.3 16.5 

After completion of germination period (15 days later), three healthy germinated seeds from 

every Petri dish transferred to small plastic pots into the greenhouse soil. 

Factorial experiment used on the basis of completely randomized design in the three 

replications. Factor A was solutions (peat and leonardite HS; and water as control). Factor B 

was maize genotypes. 

Chlorophyll content index (CCI) measured by a CCM-200 (made by Opti-Science Company) 

for Fifteen-day seedlings. Measurements were done in the beginning, middle and bottom of 

leaves. Mean of collected data for three seedlings per replication used for analysis of variance 

by MSTATC software. Comparison of means was made by Duncan's Multiple Range Test. 



ANOVA (table 2) revealed high significant differences between solutions, maize genotypes 

and their interactions. Leonardite, water and peat produced 7.06, 6.31 and 5.45 CCI, 

respectively. Comparison of means for genotypes was known that genotypes grouped to two 

classes at probability level of 5 %. Genotypes with high CCI were ZP 434, OS 499 and 505. 

Genotypes with low CCI were Golden West, Single Cross 704, ZP 677 and 500. 

Vol. 3 Page - 305 -

S.O.V Df MS 

13.611 ** 

Solutions 2 

8.743 ** 

Genotypes 6 

2.644 ** 

Table 2: ANOVA for maize genotypes 

CCIunder treatment of two types 


S × G 12 

Error 

CV (%) 

content. 

42 

- 

0.794 

14.20 

**: significant at probability level of 1% 

Comparison of CCI means for G ×S presented at 

Table 3 and Figure 1. Genotypes Single Cross 704 

and 505 had the highest CCI in leonardite HS 

treatment. After those genotypes OS 499 in water 

and leonardite, ZP 434 in water and leonardite, 505 

in water were placed. Peat HS caused to decrease 

CCI in 500, OS 499 and 505. This was similar to 

result of Ferreti et al [2]. They reported that humic 

substances apparently decreased the chlorophyll 

Leonardite HS increased CCI in Golden West and Single Cross 704 but Peat HS had not 

effect on these two maize genotypes. Leonardite HS had not effect on 500, OS 499 and 505. 

Genotypes ZP 677 and ZP 434 had not responses to application of both two types of HS. This 

result was accordant with Liu et al [5] and Badsar et al [1]. They reported that chlorophyll 

content was unaffected by HS. It is unknown how HS affect on chlorophyll content of leaves. 

Table 3: Comparison of CCI means for G ×S 

(W: water, P: Peat & L: Leonardite derived HS) 

Genotype W 

P 

L 

ZP 677 5.67 CD 5.14 CD 5.51 CD 

Golden West 

500 

OS 499 

ZP 434 

505 

Single Cross 704 

Figure 1: Chlorophyll content index of maize 

genotypes in humic substances derived from 

peat (p) and leonardite (l); and water (w) 


5.49 CD 

5.56 CD 

8.01 A 

7.71 A 

7.51 A 

4.21 D 

Vol. 3 Page - 306 - 

5.85 BCD 

4.29 D 

5.78 BCD 

7.35 AB 

5.43 CD 

4.32 D 

6.61 ABC 

5.38 CD 

7.96 A 

7.54 A 

8.20 A 

8.22 A



Effects of HS on chlorophyll content of leaves are related to origin of HS and genetic 

response of genotypes. Also, concentration of humic acids and fulvic acids in HS preparations 

could be effective on chlorophyll contents. This should be studied. 

References 

1. M. Badsar, R. Shahryari and V. MollaSadegi. Effect of potassium humate on chlorophyll content 

of wheat leaf under terminal drought condition. (In Persian). Proceedings of the 11th National 

Iranian Soil Sciences Congress. 2009. 

2. M. Ferretti, R. Ghisi, S. Nardi and C. Passera. Effect of humic substances on photosynthetic 

sulphate assimilation in maize seedlings. Can. J. Soil Sci. 1991. 71:239–242. 

3. International Humic Substances Society. What are humic substances? Focus on form: Retrieved 

May 27, 2009, from http://ihss.gatech.edu/ihss2/whatarehs.html 

4. N. A. Kulikova, E. V. Stepanova and O.V. Koroleva. Mitigating Activity of humic substances: 


polluted environments: from theory to practice. Springer Netherlands. 2005. Vol: 52. 285–309. 

5. C. Liu, R. J. Cooper and D. C. Bowman. Humic acid application affects photosynthesis, root 

development, and nutrient content of creeping bentgrass. Hort. Sci. 33: 1998. 1023–1025. 

6. A. A. Perk and A. A. Popov Growth and development of plants under the action of different 

fractions of sapropel humates. 2nd International symposium plant growth substances: intercellular 

hormonal signaling and applying agriculture Abstracts. Kyiv, Ukraine. 2007. P: 149. 

7. A. Al-Rasheed. Radwan. Water treatment by heterogeneous photocatalysis an overview. 4th 

SWCC Acquired Experience Symposium held in Jeddah. 2005. 

8. M. El-Khallal Samia, Tahani A. Hathout, Abd El Raheim A. Ashour and Abd-Almalik A. Kerrit. 

Brassinolide and Salicylic Acid Induced Growth, Biochemical Activities and Productivity of 

Maize Plants Grown under Salt Stress. Research Journal of Agriculture and Biological Sciences. 

2009. 5(4): 380-390. 

9. R. Shahryari, A. Gadimov, E. Gurbanov and M. Valizade. Application of potassium humate in 

wheat for organic agriculture in Iran. Abstracts Book of Go Organic International Symposium. 

The Approach of Organic Agriculture: New Market, Food Security and a Clean Environment. 

Bangkok, Thailand. 2009. P: 59. 

10. R. Shahryari, E. Gurbanov, A. Gadimov, M. Valizade, H. A. Hosseinpour, J. Bargiyan Khiyabani, 

B. Teymuri. 2009. Wheat genotypes quality affected by potassium humate under terminal drought. 

(In Persian). Proceedings of the 11th National Iranian Soil Sciences Congress. Gorgan, Iran. 

11. C. M. Yang, M. H. Wang, Y. F. Lu, I. F. Chang and C. H. Chou. Humic substances affect the 

activity of chlorophyllase. J. Chem. Ecol. 30(5): 2004. 1057-1065. 

12. C. M. Yang, C. N. Lee and C. H. Chou. Effects of three allelopathic phenolics on the chlorophyll 

accumulation of rice (Oryza sativa) seedling: II. Stimulation of consumption-orientation. Bot. 

Bull. Acad. Sin. 45: 2002. 119-125. 

Vol. 3 Page - 307 -

Bioactivity of Chemically Transformed Humic Matter 

on Plant Root Growth 

Luciano P. Canellas a* , Leonardo B. Dobbss a , Fábio L. Olivares a , Natália O. Aguiar a , Lázaro 

E. P. Peres b , Riccardo Spaccini c , Alessandro Piccolo c , Arnoldo R. Façanha a 

a UENF-NUDIBA Campos dos Goytacazes 28602-013, Rio de Janeiro, Brazil; b USP-ESALQ, 

Universidade de São Paulo (USP), Piracicaba, Brazil; c Dipartimento di Scienze del Suolo, 

della Pianta, dell’Ambiente e delle Produzioni Animali (R.S., A.P.) Università di Napoli 

Federico II, Portici, Italy 

E-mail:canellas@uenf.br 


The application of products derived from humic substances (HS) at low concentration on crop 

plants and their potential to act as plant growth promoters have been creating increased 

interest among farmers. However, there is little information about the mechanisms by which 

HS influence biological activities in plants. Evidence of the physiological mechanism through 

which HS exert their effects may depend on hormones and, in particular, on the presence of 

auxin or auxin-like components in their structure. Chemical modification of HSs has been 

widely used as a tool to understand their chemical structure [1]. Studies of chemical 

transformation of HS followed by biomonitoring can provide new insights on humus 

bioactivity. The aim of this studied was to evaluate the influence of chemical modifications of 

humic structure on root stimulation. The HS were evaluated by elemental composition, 

HPSEC, CP-MAS 13 C NMR and DOSY H NMR spectroscopies, and their bioactivities were 

monitored by following the morphological and biochemical traits of arabidopsis (Arabidopsis 

thaliana eco col. 4), tomato (Licopersicum esculentum), and maize (Zea mays). 



Humic substances were isolated from vermicompost produced with cattle manure and E. 

foetida using 0.1M NaOH. The humic derivates were produced by acidic oxidation with 

KMnO4 (D1); basic oxidation with KMnO4 (D2); Reduction with sodium borohydride (D3); 

alkaline methanolic hydrolysis (D4); acid hydrolysis with H2SO4 (D5); acid hydrolysis by 

dioxane in 2M HCl (D6); extraction of free lipids (D7); methylation (D8). The complete 

description of all chemical reaction used can be found in the different chapters of reference 

book edited by Hayes et al. [1]. Humic derivatives were characterized by elemental 

composition, CP-MAS 13 C NMR, DOSY-H NMR, HPSEC and their effects on lateral root 

emergence were evaluated using Arabidopsis, tomato and maize [2–3]. The activity of plasma 

membrane H+-ATPase isolated from maize root seedlings was used as biochemical marker of 

Vol. 3 Page - 308 -

humic bioactivity [2]. Four-day-old DR5::GUS transgenic Micro Tom tomato plants were 

treated with HS and their chemical derivatives for easier detection of auxin-like activity. 


The main results of HS characterization are showed in Table 1 and Fig. 2. All humic 

derivatives promotes induction of lateral root emergence on Arabidopis, tomato and maize 

(data not showed). The effect of HS and their derivatives on plasma membrane (PM) H+- 

ATPase activity isolated from maize roots vesicles can be observed in Fig. 1B. The evidence 

of auxin-like presence on HS structure are showed in Fig 2 as revealed by DR5::GUS gene 

reporter. Table 2 shown the significance of hydrophobicity of HS on H+-ATPase induction. 

These findings seem to indicate that, although the relationship between bioactivity and 

molecular size varies considerably, other chemical features such hydrophobicity appears to 

play a combined role with size in providing a bioactivity to the modified humic matter from 

vermicompost. The root acidification mediated by plasma membrane H + -ATPase is important 

for the regulation of cytoplasmic pH and the activation of cell wall-loosing enzymes and 

proteins through acidification of apoplast [4]. This effect is closely related to the auxininduced 

cell growth as proposed by the acid-growth theory. It has been earlier postulated that 

some HS may include compounds similar to indolacetic acid in their structure, and the 

capacity of HS to promote root growth was attributed to these compounds [5–6]. In fact, 

humic complex structures can be disrupted by simple organic acids exuded by plant roots and 

microbes and small auxin-like molecules may then be released and act on the cell receptors in 

plasma membrane [7]. Induction of the auxin responsive synthetic reporter DR5::GUS by 

Micro Tom-type plants by all humic derivatives is a clear evidence that physiological 

response of humic matter is due hormonal action (Fig. 2). 

Table 1 – Elemental composition and area integration from CP-MAS 13 C-NMR spectra 

Sample 


Elemental composition (%) Chemical shift (CP-MAS 13 C NMR) % 

C H N H/C C/N 0–40 40–110 110–160 160–200 HB/HI Aromaticity c 

Bulk HS 25.24 2.38 2.74 1.30 12.37 23.20 42.90 23.90 10.00 0.89 4.18 

D1 28.14 2.80 4.32 1.84 11.71 25.40 38.70 26.20 9.80 1.06 3.82 

D2 31.25 3.02 4.07 1.56 12.09 24.70 39.90 25.10 10.20 0.99 3.98 

D3 25.00 2.24 3.69 1.77 13.01 22.00 43.90 24.20 9.90 0.86 4.13 

D4 21.64 1.99 2.04 1.13 12.69 21.20 42.40 25.80 10.60 0.89 3.87 

D5 31.60 2.94 3.37 1.28 12.54 25.70 35.10 30.70 8.50 1.29 3.25 

D6 47.84 3.08 2.71 0.68 18.12 20.80 42.70 27.30 9.30 0.93 3.66 

D7 27.60 2.38 2.69 1.17 13.53 20.30 43.30 25.60 10.80 0.85 3.90 

D8 33.23 2.98 4.40 1.59 13.02 25.00 41.20 23.90 9.90 0.96 4.18 

a) C-alkyl + C-aromatic /C-polysaccharides + COOH 

Vol. 3 Page - 309 -

Fig. 1A: Mw dimension calculated by DOSY H NMR data; B: Effect on maize root PM H+-Activity 

Figure.2A: MicroTom DR5::GUS gene reporter seedlings treated with HS and their chemical derivatives 

(D); Control plants=C 

4. Conclusion 


Here, we found that chemical modifications of a humic structure affect its root growth in 

three plant species. The chemical modifications varied the composition, hydrophobicity, and 

components molecular sizes in humic superstructures, and this was reflected in their capacity 

to interact with plant cells. While a certain relation was shown between molecular size and 

humic bioactivity, we found that this may not be the only criterion to evaluate humic effects 

on root growth, and should be combined with the material hydrophobic character. It thus 

appears that HS bioactivity on plants depend on sufficient hydrophobicity to allow 

interactions with plant root cells, but the hydrophobic domains should concomitantly possess 

a conformation sufficiently labile to release, possibly by the action of acidic root exudates, 

Vol. 3 Page - 310 -


auxin-like molecules which exert a biological stimulation. 

References 

1. Hayes, M.H. et al. Humic Substances II: In Search of Structure. Chichester, Wiley, 1989. 

2. Canellas, L. P.; Façanha, A. O.; Olivares, F. L.; Façanha, A. R.. Plant Physiol. 2002, 130, 1951– 

1957 

3. Dobbss, L., Medici, L.O., Peres, L.E.P., Pino-Nunes, L.E., Rumjanek, V.M., Façanha, 

4. A.R., Canellas, L.P., Ann. Appl. Biol. 2008, 153, 157–166. 

5. Sze, H.; Li, X.; Palmgren, M. G. Plant Cell. 1999, 11, 677–689. 

6. Muscolo, A.; Cultrupi, S.; Nardi, S. Soil Biol. Biochem. 1998, 30, 1199–1201 

7. Nardi, S., Pizzeghello, D., Muscolo, A., Vianello, A., Soil Biol. Biochem. 2002, 34, 1527–1536. 

8. Canellas, L. P.; Teixeira Junior, L. R. L.; Dobbss, L. B.; Silva, C. A.; Medici, L. O.; Ann. Appl. 

Biol. 2008, 153, 157–166. 

Vol. 3 Page - 311 -

Effect of Two Humic Substances as Bifertilizers on Germination and 

Seedling Growth of Maize Genotypes 

Reza Shahryari a* , Niknam Bahari a , Majid Khayatnejad b , 

a Islamic Azad University, Ardabil branch; Iran; b Young Researchers Club, Islamic Azad 

University, Ardabil Branch, Iran 

E-mail: rz_shahriari@yahoo.com 



Humic substances (HS) are the result of organic decomposition and the natural organic 

compounds comprising 50 to 90 % of the organic matter of peat, lignites, sapropels, as well as 

of the no- living organic matter of soil and water ecosystems. According to the classical 

definition, HS are "a general category of naturally occurring heterogeneous organic 

substances that can generally be characterized as being yellow to black color, of high 

molecular weight and refractory [1]. The biological activity of HS encompasses all the 

activities of HS in regulating plant biochemical and physiological processes, irrespective of 

their stimulatory or inhibitory roles. Humic matter from forest soils has a very complex 

biological activity and depending on its origin, molecular size, and concentration exhibits 

high or low stimulations of plant metabolic parameters. In forest soils, litter composition does 

not influence the chemical characteristics of humic fractions greatly, as revealed by nuclear 

magnetic resonance spectra, but it does influence biological activity and as a result forest 

species are affected in different ways by their HS. HS are known to possess bioactivating 

properties in relation to plants [2]. In spite of numerous studies on the biological effects of 

HS, the mechanism of their action remains unclear [1]. However, farmers use humates to 

accelerate seed germination and improve rhizome growth. These materials are able to 

stimulate oxygen transport, accelerate respiration and promote efficient utilization of nutrient 

by plants [3]. Nevertheless, humic acid in proper concentrations can enhance plant and root 

growth [4]. Presence of HS is important during all stages of plants’ development but 

particularly vital in the early stages. That is why the pre-planting treatment of seeds is very 

important. Even before germination begins, vital forces are awakened, and the immune 

system is stimulated [5]. To date, numerous researches have demonstrated conclusively that 

HS have significant impacts on the soil structure and plant growth [6]. Gadimove et al [9] 

concluded that humates are miraculous natural substances for increasing quantity and quality 

of crop yields. They expressed that a practical- scientific perspective and programming need 

to application of this technology in the world; especially in the developing countries. Also, 

they expressed action rate of these materials are related to origin and quality of HS. Quality of 

Vol. 3 Page - 312 -

commercial humates is related to procedure of extraction and percent of humic acids and 

fulvic acids. We must note to these subjects for select and application of those for agricultural 

applications. 

In this investigation, effect of two types HS as biofertilizers were studied on germination and 

seedling growth of different maize genotypes under cultivation in Moghan plain, Ardabil 

province, Iran. 



A laboratory study was conducted to evaluate the influence of HS on germination and early 

growth of seven maize genotypes which were include of ZP 677, Golden West, 500, OS 499, 

ZP 434, 505 and Single Cross 704. Solution of 0.067 % w/v prepared from the two type liquid 

HS (Table 1). 

Table 1. Compounds of liquid humic based on peat and leonardite 

Compounds Humic acids Fulvic acids Total humic extract 

Humics 

(% w/v) (% w/v) (% w/v) 

Peat based 3.3 0.9 4.2 

Leonardite based 

13.2 

3.3 

16.5 

Factorial experiment used on the basis of completely randomized design in the three 

replications. Factor A was solutions (peat and leonardite HS; and water as control). Factor B 

was maize genotypes. Twenty six seeds of maize genotypes were allowed to germinate in 

Petri dishes in the dark room temperature. Solution consumed by seed, was to provide equal 

value during the germination. Final germination percent measured after 15 days. Then three 

germinated seeds of uniform appearance was transferred to separate small plastic pots into the 

greenhouse soil and then grown in the room temperature. Watering only was made by water. 

Fifteen-day seedlings were measured for seminal root lengths and coleoptiles length. Mean of 

collected data for three seedlings per replication used for analysis of variance by MSTATC 

software. Comparison of means was made by Duncan's Multiple Range Test. Linear 

regression between seminal root lengths, coleoptiles length and germination percent 

separately calculated for experimental solutions. Relative seminal root elongation percent and 

relative shoot elongation percent calculated by following formula: 

Relative root elongation % = {mean root length in test solution/ mean root length in control} 

× 100. 

Vol. 3 Page - 313 -

Relative shoot elongation % = {mean shoot length in test solution/ mean shoot length in 

control} × 100. 

3. Results and Discussion. 


ANOVA (Table 2) showed that solutions produced significant differences for seminal root 

lengths (p

Table 3. Mean comparisons of seminal root lengths and germination percent for under study maize 

genotypes 

Character 

Genotypes 

seminal root lengths(cm) 

Relative root elongation % 

Germination(%) 

1.6 

1.4 

1.2 

1 

0.8 

0.6 

0.4 

0.2 

0 

ZP 

677 

3.55 c 

81.22 ab 

Golde 

n West 

3.26 c 

63.68 c 

500 

3.68 bc 

84.20 a 

OS 

499 

4.41 ab 

87.62 a 

ZP 434 

3.43 c 

79.06 abc 

505 

2.62 d 

65.39 bc 

Table 4: Mean comparisons of seminal root lengths for G ×S at probability level of 1% 

(W: water, P: Peat & L: Leonardite HS) 

zp 677 

Golden west 

500 

OS 499 

Genotype 

ZP 677 

Golden West 

500 

OS 499 

ZP 434 

505 

Single Cross 704 

zp434 

505 

single cross 704 

W 

3.42 defgh 

3.77 cdef 

3.03 fgh 

3.06 fgh 

2.95 fgh 

2.57 gh 

4.59 abc 

Potassium humate 

based on peat 


based on leonardite 

Relative shoot elongation % 

1.4 

1.2 

1 

0.8 

0.6 

0.4 

0.2 

0 

P 

4.27 bcde 

3.03 fgh 

4.47 abcd 

4.24 bcde 

4.01 cdef 

2.94 fgh 

3.37 efgh 

zp 677 

Golden west 

500 

OS 499 

L 

2.96 fgh 

3.18 fgh 

3.56 cdefg 

5.15 ab 

3.34 efgh 

2.35 h 

5.36 a 

zp434 

505 

single cross 704 

Single 

Cross 

704 

4.44 a 

73.51 abc 


based on peat 


based on leonardite 

Figure 1: Relative root elongation % Figure 2: Relative shoot elongation % 

Peat HS created significantly positive correlation (r = 0.82*) between seminal root length and 

germination percent at probability level of 5%. There was similar positive correlation (r = 

0.70ns) for leonardite HS. Control (water) treatment was not able to create significant 

correlations between measured characters. 


The results of this study showed that applied humic substances had effect on biological 

characters of maize in early growth stage. There are similar reports about effect of HS on 

Vol. 3 Page - 315 -

crops germination. Some of them expressed by Gadimove et al [7] and Sasaki et al [8]. They 

were noted that treatment of tomato seeds with 0.01 % Potassium humate solution before 

planting for 24 hours related to variety increased production by 20 – 25 %. Also, Pre- planting 

seed treatment of cucumber with 0.01 % potassium humate solution for 24 hours increased 

production by 38 %. And, time and percentage of germination investigated for hazel-nut seeds 

in three concentrations of Potassium Humate (0.01, 0.02 and 0.03 ml/seed) and two times (12 

and 24 hours). Related to variety, germination percentage was increased between 37.08 and 

64.14 % and the highest germination percentage (53%) observed in concentration of 0.02 

relative to control (43%). 



Types of humic substances could play the same or a different role as a biofertilizer on maize. 

In this study, applied Peat and leonardite HS with each other produced more seminal root 

lengths than control. But coleoptiles length of control and leonardite HS treatments was 

higher than treated by peat HS. These HS biofertilizers in small amounts have many effects 

on plant growth. Thus need to continue researches by different amounts of desired HS 

biofertilizres for information on how they impact. 

References 

1. N. A. Kulikova, E. V. Stepanova and O.V. Koroleva, Mitigating Activity of humic substances: 


polluted environments: from theory to practice. Springer Netherlands, 2005, Vol: 52. 285–309. 

2. D. Pizzeghello, G. Nicolini and S. Nardi, Hormone-like activity of humic substances in Fagus 

sylvaticae forests, New Phytologist, 2001, 151: 647–657. 

3. K. M. S Islam, A. Schuhmacher and J.M. Gropp, Humic Acid Substances in Animal Agriculture. 

Pakistan Journal of Nutrition, 2005, 4 (3): 126-134. 

4. M. Bacilio, P. Vazquez and Y. Bashan, Alleviation of noxious effects of cattle ranch composts on 

wheat seed germination by inoculation with Azospirillum spp, Biol Fertil Soils, 2003, 38:261– 

266. 

5. B. Levinsky, All about humates, Focus on form: Retrieved May 27, 2009, from 

http://www.teravita.com/Humates/HumateIntro.htm 

6. S. S. Fong, L. Seng and H. B. Mat, Reuse of Nitric Acid in the Oxidative Pretreatment Step for 

Preparation of Humic Acids from Low Rank Coal of Mukah, Sarawak, J. Braz. Chem. Soc, 2007, 

Vol: 18. No: 1. 41-46. 

7. A. Gadimov, N. Ahmaedova and R. C. Alieva, Symbiosis nodules bacteria Rhizobium 

leguminosarum with Peas (Pisum Sativum) nitrate reductase, salinification and potassium humus, 

2007, Azerbaijan National Academy of Sciences. 

8. O. Sasaki, I. Kanai, Y. Yazawa and T. Yamaguchi, Relationship between the chemical structure of 

humic substances and their hygroscopic properties, Annals of Environmental Science, 2007, Vol: 

1. 17-22. 

9. A. G. Gadimov, R. Shahryari and A. G. Garayeva, A perspective on humic substances as natural 

technological products with miraculous biological effect on crops, Transaction of the Institute of 

Microbiology of Azerbaijan national Academy of Sciences, 2009, V. 7. p. 118-126. 

Vol. 3 Page - 316 -

Comparative Evaluation of the Inhibitory Action of Compost Humic 

Fractions on Two Soil-Borne Phytopathogenic Fungi 

Andreina Traversa, Elisabetta Loffredo * , Nicola Senesi 

Dipartimento di Biologia e Chimica Agro-forestale e Ambientale, University of Bari, 

Via G. Amendola 165/A 70126 Bari, Italy 

E-mail: loffredo@agr.uniba.it 


The biological activity of a compost humic acid (C-HA) depends mainly on the type of 

substrate used for composting and the type and duration of the process. Nowadays, the needs 

of sustainable agriculture and the increasing tendency to adopt soil-less cultivation suggest 

new management practices, such as an increasing use of compost as partial substitute of peat 

in potting media for ornamental plants and in greenhouse nurseries of horticultural plants. The 

recent literature supports the positive effects exerted by compost addition to growing media, 

which especially originates from its humic fraction, that improves plant growth and protects 

plants from different soil-borne phytopathogens such as fungi [1-5]. This latter evidence, 

besides the ascertained organic matter benefits to soil and other growing media, will add value 

to compost application and encourages deeper research in this aspect. The objective of this 

study was to evaluate comparatively the capacity of different C-HAs to inhibit the growth and 

activity of two widespread plant pathogenic fungi, Pythium ultimum and Sclerotinia 

sclerotiorum, and to possibly relate this ability to some C-HA properties. 



The HA samples were isolated from a mixed compost (MC-HA), a green compost (GC-HA) 

and a coffee compost (CC-HA) by using the conventional procedure and characterized by 

means of a series of chemical and spectroscopic analyses. Some properties of the HA samples 

examined are referred in Table 1. Each HA sample suspended in PDA (potato dextrose agar) 

substrate was tested at concentrations of 10, 50 e 200 mg/L on the growth in vitro in Petri 

dishes of P. ultimum and S. sclerotiorum and, in the case of the latter fungus, the time of 

appearance and the number of sclerotia (non active fungal structures) formed. All experiments 

were performed in controlled conditions and replicated eight times. All data were statistically 

analyzed by one-way analysis of variance (ANOVA) and the least significant differences test 

(LSD). Further, in order to evaluate the possible relationships existing between C-HA activity 

on the two fungi and C-HA chemical properties (Table 1), the correlation coefficients were 

calculated between C-HA properties and the average of inhibition degree (percentage of 

Vol. 3 Page - 317 -

decrease of radial mycelial growth with respect to the control) and, in the case of S. 

sclerotiorum, of the number of sclerotia at 168 h. 

HA sample 

Table 1: Some properties of C-HAs examined 

Ash a 

% 

COOH a 

meq/g 

Phenolic OH a 

meq/g 

Total acidity a 

meq/g 

E4/E6 

ratio 

MC-HA 3.2 3.38 2.94 6.32 8.08 

GC-HA 3.4 3.34 1.80 5.14 8.24 

CC-HA 4.9 3.08 2.55 5.63 8.54 

a on moisture free basis 


With respect to the corresponding control (PDA alone), no morphological changes were 

observed for P. ultimum and S. sclerotiorum mycelia as a function of the presence of any C- 

HA examined at any concentration in the growing medium. These results are in agreement 

with previous results observed in vitro tests for two formae speciales of Fusarium oxysporum 

in the presence of soil and compost humic fractions [3]. Differently, since the first hours after 

fungal inoculation and during the entire experimental time, the presence of any C-HAs in the 

PDA medium produced in general an inhibition of the radial mycelial growth of P. ultimum. 

Different growth decreases were measured with respect to the control as a function of the dose 

and the type of C-HA. The maximum inhibition was exerted by MC-HA (Fig. 1). 

Radial mycelial growth (mm) 

45 

40 

35 

30 

25 

20 

15 

10 

5 

0 

** 

* 

*** 

* 

18 24 

Hours 

40 


*** 

*** ** 

* P ≤ 0.05; ** P ≤ 0.01 and *** P ≤ 0.001 according to LSD test 

Figure 1: Effects of MC-HA at 

concentrations of 10 mg/L (light grey 

bars), 50 mg/L (dark grey bars) and 200 

mg/L (black bars) on the radial mycelial 

growth of P. ultimum on PDA, with 

respect to the control (white bars) as a 

function of time 

In the case of S. sclerotiorum, none of the three C-HAs reduced significantly the growth of 

the fungus during the experimental period (88 h). Conversely, sclerotial development 

Vol. 3 Page - 318 -

appeared more sensitive than the radial growth to all C-HAs. Apparently, any C-HA sample at 

any concentration stimulated markedly sclerotial initiation and greatly increased the number 

of sclerotia in the plates (Fig. 2). After 168 h from the fungus inoculation, the number of 

sclerotia resulted generally much higher in C-HA treatments, especially in CC-HA, with 

respect to the control. These results indicated that the presence of C-HA determined the 

occurrence of adverse nutritional conditions that arrest the fungal activity, with evident 

benefit for plant health. 

Number of sclerotia 


35 

30 

25 

20 

15 

10 

5 

0 

35 

30 

25 

20 

15 

10 

5 

0 

MC-HA 

136 144 

Hours 

160 168 

CC-HA 

136 144 160 168 

Hours 

C 10 mg/L 50 mg/L 200 mg/L 



35 

30 

25 

20 

15 

10 

5 

0 

GC-HA 

136 144 160 168 

Hours 

Figure 2: Effects of C-HAs at concentrations of 

10, 50 and 200 mg/L on the sclerotia formation 

of S. sclerotiorum on PDA, with respect to the 

control, as a function of time 

Some significant relationships were found between C-HA properties and fungal inhibition. In 

the case of P. ultimum, a significant positive correlation occurred between phenolic OH 

content and mycelial growth inhibition. The number of sclerotia formed were positively 

correlated with ash and phenolic OH content, total acidity and E4/E6 ratio, and negatively 

correlated with COOH group content. These results, which are in good agreement with 

findings of a recent study performed in vitro on the fungus F. oxysporum [3], apparently 

confirm what already hypothesized in that work, i.e., the inhibitory action of humic acids on 

the growth of the fungi tested might be related more to hydrophobic than hydrophilic 

properties of HA. 

Vol. 3 Page - 319 -



This study confirmed what already demonstrated by the authors and other researchers with 

different fungi and different humic fractions, that is the general depressing activity of humic 

compounds on some soil-borne plant pathogenic fungi. The C-HA samples examined in our 

work exerted a different action on the two phytopathogenic fungi considered. P. ultimum 

appeared to be significantly inhibited by any C-HA at any concentration adopted, especially 

by HA originated from the mixed compost. In the case of S. sclerotiorum, a marked 

enhancement of sclerotial formations was observed in all C-HA treatments, whereas the 

mycelial growth of this fungus was not significantly altered. The efficiency of C-HA studied 

in controlling fungal growth was apparently related to the substrate of origin, i.e. the different 

HA properties, the HA concentration and the type of fungus examined. 

References 

1. A. Bernal-Vicente, M. Ros, F. Tittarelli, F. Intrigliolo and J.A Pascual, Biores. Technol. 99 (2008) 

8722. 

2. A.M..Litterick, L. Harrier, P. Wallace, C.A. Watson and M. Wood, A review. Crit. Rev. Plant Sci. 

23 (2004) 453. 

3. E. Loffredo, M. Berloco, F. Casulli and N. Senesi, Biol. Fert. Soils 43 (2007) 759. 

4. E. Loffredo, M. Berloco and N. Senesi, Ecotoxicol. Environ. Safe. 69 (2008) 350. 

5. E. Loffredo and N. Senesi, Sci. Hortic. 122 (2009) 432. 

Vol. 3 Page - 320 -

Effect of Liquid Humic Compounds Extracted from Plant Based-Compost 

to Soil Microorganisms 

F. Suárez-Estrella * , M.C. Vargas-García, G. Guisado, M.J. López, J. Moreno 

Department of Applied Biology, Microbiology Division, CITE IIB, University of Almería, La 

Cañada de San Urbano s/n, 04120 Almería, Spain 

E-mail: fsuarez@ual 


The effects of humic compounds (HCs), the major component of soil organic matter, on plant 

growth have been examined in recent works [1] (Nardi et al. 2002). Humic colloids also affect 

the growth of soil microbial populations. Many soil microorganisms from different taxonomic 

and functional groups respond favourably to the presence of HCs in in vivo or in vitro 

experiments [2, 3]. 

The present study compares the effects of HCs extracted from compost based on horticultural 

waste and those from a commercial liquid fertilizer on soil microbial populations using a pot 

experiment carried out with tomato plants (Solanum lycopersicum L.). 



Humic-like substances. A concentrated humic extract from leonardite (LHs) provided from an 

agricultural company were used to represent humates of fossil origin. On the other hand, 

liquid HCs from compost based on horticultural waste (WCHs) were used. 

Plants and substrates used in pot trials. Two varieties of tomato were used: certified cv. 

‘Raf’, type Marmande and certified cv. ‘Durinta’. Both sandy soil (SS) and a semi-inert 

substrate (IS) on the basis of mainly “vermiculite” were used separately to compare the effect 

of HCs on microbial growth. 

Plants were supplied weekly with LHs and WCHs in aqueous solutions at 0.7% but control 

plants were not amended with HCs. Plants were located in a randomized block in the 

greenhouse and grown for 60 days at constant temperature of 24 ± 1°C and relative humidity 

of 75%. 

Vol. 3 Page - 321 -

Estimation of microbial growth. After 7, 14, 28, 45 and 60 days, microbial counts were taken 

on 3 separate soil samples from each experimental block. Total number of aerobic bacteria 

(TB), actinomycetes (TA) and fungi (TF), cellulolytic population (CEL), hemicellulolytic 

microorganisms (HEM), ligninolytic microorganisms (LIG) and nitrogen-fixing bacteria (NF) 

were determined by plate counts. Ammonifiers (AM) and nitrifying bacteria (NIT) were 

determined by the most probable number technique. 

Statistical analysis. Data were subjected to one multifactorial analysis of variance (ANOVA) 

in which counts of the different groups were compared for the different levels of humic 

treatment (LHs, WCHs and Control), sampling time and pot substrate type (IS and SS). In 

order to determine which means were significantly different (p < 0.05), multiple comparison 

tests (Fisher’s least significant difference) were used. All experiments were carried out twice. 



Microbial populations were affected when different HCs were added to all experimental 

blocks. The effect observed depended on the origin of the amendment applied (LHs or 

WCHs) as well as the tomato cultivar used (‘Raf’ or ‘Durinta’). 

Table 1 shows the significant influence of the different factors on all microbial groups 

analysed as well as the influence of the interactions between them. In general, several 

differences were observed when humic extracts were added to different cultivars. In this 

sense, the humic treatment significantly influenced on TB, TF, CEL, LIG and NF populations 

when ‘Raf’ was used while this effect was observed on TB, TF, HEM, NF and NIT when 

‘Durinta’ was used. On the other hand, in both cultivars, the interactions between the different 

factors significantly influenced TA, TF, CEL, LIG and NF populations. 

In the ‘Raf’-soil system, when HCs were added to the soil, populations of TA, HEM, AM and 

NIT did not differ from those observed in the control treatment. On the other hand, counts of 

TB, TF, NF and CEL populations in LHs and WCHs treatments were, in general, higher than 

those obtained in control soils (data not shown). The population of nitrogen-fixing bacteria 

(NF) was particularly higher in the WCHs treatment than in the LHs, and values from control 

fell in between them. This effect was more evident from 28 days and when IS was used (data 

not shown). 

In the ‘Durinta’-soil system, microbial counts of TA, CEL, LIG and AM were not affected by 

the addition of HCs to the soil. On the other hand, as observed in the case of ‘Raf’, counts of 

TB, TF and NF populations were higher when LHs or WCHs were added. Contrary to the 

Vol. 3 Page - 322 -

esults obtained from the ‘Raf’-soil system, microbial counts of HEM and NIT were also 

higher in amended substrates. NIT showed higher counts than those obtained in the case of 

substrates added with LHs. These differences were higher during the second month and when 

IS was used rather than SS (data not shown). 

Table 1: Effect of Sampling time, substrate and humic treatment on microbial populations from 

a plant-soil system. Significant differences are observed at 95% confidence level (p < 0.05). 

Factors 

TB 

‘Raf’ 

1 

p < 0.05 

TA 2 

p < 0.05 

TF 3 

p < 0.05 

CEL 4 

p < 0.05 

HEM 5 

p < 0.05 

LIG 6 

p < 0.05 

NF 7 

p < 0.05 

NIT 8 

p < 0.05 

AM 9 

p < 0.05 

Sampling time 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0001 0.0000 

Substrate kind 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0158 0.0010 

Humic treatment 0.0087 ns 10 0.0009 0.0000 ns 0.0000 0.0001 ns ns 

Interactions p < 0.05 p < 0.05 p < 0.05 p < 0.05 p < 0.05 p < 0.05 p < 0.05 p < 0.05 p < 0.05 

Sampling time X 

Substrate kind 

0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0001 0.0000 

Sampling time X Humic ns ns 0.0357 0.0000 0.0005 0.0000 0.0029 ns 0.0000 

treatment 

Humic treatment X 


ns ns ns 0.0000 

‘Durinta’ 

0.0000 0.0000 ns 0.0010 0.0029 

Factors 

TB 1 

p < 0.05 

TA 2 

p < 0.05 

TF 3 

p < 0.05 

CEL 4 

p < 0.05 

HEM 5 

p < 0.05 

LIG 6 

p < 0.05 

NF 7 

p < 0.05 

NIT 8 

p < 0.05 

AM 9 

p < 0.05 

Sampling time 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 

Substrate kind 0.0001 0.0000 0.0000 0.0294 0.0000 0.0000 0.0000 0.0048 0.0001 

Humic treatment 0.0005 ns 0.0060 ns 0.0000 ns 0.0002 0.0000 ns 

Interactions p < 0.05 p < 0.05 p < 0.05 p < 0.05 p < 0.05 p < 0.05 p < 0.05 p < 0.05 p < 0.05 

Sampling time X 


0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 

Sampling time X Humic 

treatment 

0.0393 ns 0.0036 0.0000 0.0197 0.0159 0.0025 0.0000 ns 

Humic treatment X ns ns ns 0.0000 ns 0.0000 ns 0.0005 ns 


1 Total Bacteria 

2 Total Actinomycetes 

3 Total Fungi 

4 Cellulolytic Microorganisms 

5 Hemicellulolytic Microorganisms 

6 Ligninolytic Microorganisms 

7 Nitrogen-Fixing Bacteria 

8 Ammonifiers Microorganisms 

9 Nitrifying Bacteria 

10 non significant 


The results obtained in this work have shown that the application of HCs has a significant 

impact on several soil microbial groups. This response could be attributed to the nutritive 

value of humates from WCHs and LHs. Also, Valdrighi et al. (1996) [4] suggest that 

potassium added to WCHs has no stimulatory effects on microbial populations. Several 

authors have confirmed that molecular characteristics of HCs may result in higher biological 

activity due to enzymatic activation of nutrient uptake or modification of bacterial cell 

permeability to nutrients [2, 5]. Counts of total aerobic bacteria, fungi and nitrogen-fixing 

bacteria were higher in soils treated with HCs than in control soils (data not shown). This 

effect was observed in both plant cultivars tested (‘Raf’ and ‘Durinta’). However, treatment 

with WCHs promoted the highest counts of nitrogen-fixing (NF) bacteria with ‘Raf’, while 

the highest counts of nitrifying bacteria were obtained with ‘Durinta’ (data not shown). It is 

therefore possible that different plant-soil systems react differently to the presence of HCs [6]. 

Vol. 3 Page - 323 -

Soil microorganisms involved in nitrogen cycling have previously been studied as regards 

their response to the application of HCs. Populations of autotrophic ammonia and nitrite 

oxidizers increased in soil or axenic cultures amended with humates from composted 

vegetable waste, especially at high rates [2, 4]. On the other hand, Acea et al. (2003) [7] 

confirmed the importance of nitrogen-fixing bacteria to promote microbial crust formation, 

enhancing C and N cycling microorganisms and increasing organic matter and nutrient 

content in deteriorated soils. 



Therefore, results derived from this preliminary work showed the potential for improving the 

utilization of HCs extracted from compost based on plant waste (WCHs). The extraction of 

these substances (WCHs) produced an extract which behaved as a stimulatory substance on 

some microorganisms related to the plant roots. 

References 

1. S. Nardi, G. Concheri and G. Dell’Agnola G, in: Piccolo A (Ed) Humic Substances in Terrestrial 

Ecosystems, Elsevier, Amsterdam, 1996, pp 361-406 

2. M.M. Valdrighi, A. Pera, S. Scatena, M. Agnolucci and G. Vallini, Compost Science Utilization 3 

(1) (1995) 30-38 

3. G. Vallini, A. Pera, M. Agnolucci and M.M. Valdrighi, Biology and Fertility of Soils 24 (1997), 

243-248 

4. M.M. Valdrighi, A. Pera, M. Agnolucci, S. Frassinetti, D. Lunardi and G. Vallini, Agriculture 

Ecosystem and Environment 58 (1996), 133-144 

5. M. Tejada, C. García, J.L. González, M.T. Hernández, Soil Biology and Biochemistry 38 (2006) 

1413-1421 

6. D. Vaughan and R.E. Malcom, In: Vaughan D, Malcom RE (Eds) Soil Organic Matter and 

Biological Activity, Martinus Nijhoff/Junk W, Dordrecht, 1985, pp 37-76 

7. M.J. Acea, A. Prieto-Fernández and N. Diz-Cid, Soil Biology and Biochemistry 35 (2003) 513-524 

Vol. 3 Page - 324 -

Analysis of the Sorption Properties Soils After the Application of Sewage 

Sludges and Conventional Organic Fertilizers 

Stańczyk-Mazanek Ewa * , Stępniak Longina, Kępa Urszula 

Institute of Environmental Engineering, Czestochowa University of Technology, 

ul. Brzeznicka 60a, 42-200 Częstochowa, Poland 

E-mail: stanczykewa@wp.pl; stanczyk@is.pcz.czest.pl 


The sorption capacity of soil is regarded as one of the most important factors influencing the 

fertility of the soil and the properties of plants grown. An extended sorption complex is an 

element that retains and absorb various soil contaminants. The state of the sorption complex 

is influenced, inter alia, by the following: organic matter content, humus compounds forming 

during organic matter decomposition, silt minerals, pH, and hydrolytic acidity. The pH value 

of soil very strongly determines its physical, chemical and biological properties. The reaction 

of the soil influences the stability of the structure and associated air-water relationships. All 

these factors provide optimal growing and yielding conditions for plants [1, 2]. The authors of 

the present work undertake the analysis of the effect of application of sewage sludges and 

selected organic fertilizers on the changes in the sorption properties of thus treated soils. 



Sewage sludges and, for comparison purposes, also manure were introduced to sandy soils 

(P). Tests were conducted under pot experiment conditions. The following doses of organic 

fertilizers were applied: 0, 10, 50, 100 and 200 tonnes per hectare. Particular sewage sludges 

and manure were mixed, respectively, with both soil types. The amounts of fertilizers were 

calculated for 10 kg of soil (which was held in experimental pots) so that they corresponded 

to the following doses: 10, 50, 100 and 200 tonnes of sewage sludge or manure (PO) per 

hectare. Unfertilized sand constituted the control soil. So prepared soil samples were left for a 

period of about 6 months, while maintaining constant humidity. After this period, test samples 

were taken for analysis. The tests were carried out in 3 replications. The results represent the 

mean of these replications. After 6 months from the fertilization, variations in active and 

hydrolytic acidity in the grounds treated were analyzed. The content of organic matter; the 

sum of bases, S, in the sorption complex (by the Kappen method); sorption capacity, T; and 

the contents of humic acids in the soils treated were also determined. 

Two soil types with the grain-size composition of loose sand were used for the fertilization 

tests. The reaction of the first soil (which was designated as soil P1) was 8.31, while that of 

Vol. 3 Page - 325 -

the other (soil P2) was 4.79. The sewage sludges used for the fertilization of selected soils 

originated from 2 sewage treatment plants. These were designated by P and R, being the first 

letters of the names of locations where the sewage treatment plants were situated. Sewage 

sludge P was oxygen-stabilized. Sewage sludge R came from a biological sewage treatment 

plant. Two sewage sludge types were taken from the sewage treatment plant. One of them was 

dewatered by gravity without the addition of a polyelectrolyte. This was designated as sewage 

sludge R (plot). The other sewage sludge was dewatered on a belt press with the use of a 

polyelectrolyte, and was designated as sewage sludge R (press). 


The results of the testing of the sandy soil (P1) treated with sewage sludges and manure, 

respectively, are shown in Figures 1 and 2. Whereas, the results of the analysis of soil P2 are 

illustrated in Figures 3 and 4. 

pH 

9 

8,5 

8 

7,5 

7 

6,5 

Kontrola 10 50 100 200 

dose [t/ha] 

P1P(prasa) 

P1R(prasa) 

P1R(poletka) 

P1O 

hydrolytic acidity 

2 

1,8 

1,6 

1,4 

1,2 

1 

0,8 

0,6 

0,4 

0,2 

0 

Kontrola 10 50 100 200 

dose [t/ha] 

P1P(prasa) 

P1R(prasa) 

P1R(poletka) 

Fig. 1 Variations in the pH of sandy soil (P1) Fig. 2 Variations in the Hh of sandy soil (P1) 

after treatment after treatment 

pH 

10 

8 

6 

4 

2 

0 

Kontrola 10 50 100 200 

dose [t/ha] 


P2P(prasa) 

P2R(prasa) 

P2R(poletka) 

P2O 

hydrolytic acidity 

2,5 

2 

1,5 

1 

0,5 

0 

Kontro la 1 0 50 100 20 0 

dose [t/ha] 

P1O 

P2P(prasa) 

P2R (pra sa) 

P2R(poletka) 

Fig. 3 Variations in the pH of sandy soil (P2) Fig. 4 Variations in the Hh of sandy soil (P2) 

after treatment after treatment 

Vol. 3 Page - 326 - 

P2O

The properties of the sorption complex of soils treated (soil P2 is shown) are summarized in 

Table 1. 

Soil mix type 

Table 1. Test results for the properties of the sorption complex of P2 soil mixes 

Organic matter 

content [%] 

Sum of bases S 

[meq/100g soil] 

Determination 

Sorption capacity 

T[meq/100g soil ] 

Humic acid 

content [%] 

Control (P2) 0.6 2.55 3.63 0.025 

PP 10 0.6 3.35 4.63 0.050 

PP 50 1 6.86 8.4 0.165 

PP 100 2.2 10.4 12.31 0.285 

PP 200 4.4 20.2 22.53 0.495 

PR 10 press 0.6 2.95 4.19 0.070 

PR 50 press 0.8 11.25 12.6 0.155 

PR 100 press 1.6 12.6 14.6 0.245 

PR 200 press 2.8 25.3 27.63 0.515 

PR 10 plot 0.6 5.5 6.59 0.065 

PR 50 plot 1 8.55 9.83 0.195 

PR 100 plot 1.8 12.5 13.93 0.250 

PR 200 plot 3 27.9 29.81 0.620 

PO 10 0.6 3.1 4.11 0.065 

PO 50 0.8 6.1 6.93 0.135 

PO 100 1.4 9.3 9.94 0.250 

PO 200 3 15.7 16.19 0.435 

4. Conclusion 


In summing up the obtained investigation results it can be concluded that the application of 

the organic fertilizers, both manure and sewage sludge, significantly influenced the sorption 

properties of soil. As a result of organic fertilization, the physicochemical properties of the 

soil are improved, the acidification is reduced, and the sum of exchange bases, humic 

compounds, and sorption complex capacity increase [1,3,4]. Similar results have been 

obtained in the present work. However, only manure used in the tests did always cause a 

reduction in the acidity of soils treated with it. This phenomenon was observed for the 

treatment of both basic and acidic sandy soils, alike. The application of manure reduced both 

active and potential (hydrolytic) acidity. The acidity-reducing tendency of this fertilizer was 

Vol. 3 Page - 327 -


visible for a long time after its application. Different responses of soil treated were observed 

after the application of sewage sludge. A basic soil upon treatment with all sewage sludge 

types underwent acidification, which was the greater, the higher was the sewage sludge dose. 

It was observed that a higher increase in hydrolytic acidity in soils was caused by sewage 

sludge with the addition of polyelectrolytes used for dewatering. Sewage sludges from plots 

exhibited lower acidifying properties. This is probably dependent on the variable properties of 

sewage sludges themselves. Navas et al. [3] observed a decrease in the pH of soils after the 

application of sewage sludge at a rate of 0, 40, 80, 160 and 320 tonnes/hectare, respectively. 

The reported increase in acidity (similarly as in the present work) was the higher, the larger 

was the sewage sludge dose introduced to the soil. Similar results were obtained by Wong et 

al. [5]. A drop in the pH of soil after the application of sewage sludge was also observed by 

Forsberg and Ledin [6]. Other investigation results [7] indicate an increase in the pH of soil 

after the introduction of sewage sludge. The differences are most likely to be due to the 

properties of particular sewage sludges, which indicates very variable and often unpredictable 

chemical and biological properties of sewage sludge. 

References 

1. Gorlach E., Mazur T.: Chemia rolna (Agricultural chemistry), Wydawnictwo Naukowe PWN, 

Warsaw 2001. 

2. Wang A.S., Scott Angle J., Chaney R.L., Delorme T.A., McIntosh M.: Changes in soil biological 

activities under reduced soil pH during Thlaspi caerulescens phytoextraction, Soil Biology and 

Biochemistry 38 (2006), 1451–1461. 

3. Navas A., Bermudez F.,Machin J.: Influence of sewage sludge application on physical and 

chemical properties of Gypsisols, Geoderma 87 (1998) 123–135. 

4. Bieniek B., Różańska E., Bieniek A.: Wpływ ścieków przemysłu rolno-spożywczego na 

właściwości sorpcyjne gleb mineralno-organicznych (The effect of agricultural & food industry 

wastes on the sorption properties of mineral-organic soils), Zeszyty Problemowe Postępów Nauk 

Rolniczych 2000, z. 472: 97–102. 

5. Wong J.W.C., Fang L.M., Ma K.K.: Effects of sewage sludge amendment on soil microbial 

activity and nutrient mineralization, Environment International, 24, No 8, 1998: 935-943. 

6. Forsberg L.S., Ledin S.: Effects of sewage sludge on pH and plant availability of metals in 

oxidising sulphide mine tailings, Science of the Total Environment 358 (2006) 21–35. 

7. Bramryd T.: Effects of liquid and dewatered sewage sludge applied to a Scots pine Stand (Pinus 

sylvestris L.) in Central Sweden, Forest Ecology and Management 147 (2001), 197–216. 

Vol. 3 Page - 328 -

Interactions Between Plant-Root Exudates and Soils in Extracting Humiclike 


Diego Pizzeghello a* , Adele Muscolo b , Andrea Ertani a , Serenella Nardi a 

a Department of Agricultural Biotechnologies, University of Padova, Agripolis, Viale dell’ 

Universita` 16, 35020 Legnaro, Padova, Italy; b Department of Agricultural and Forest 

Systems Management “Agriculture Faculty, Mediterranea” University of Reggio Calabria, 

Feo di Vito 89126 Reggio Calabria, Italy 

E-mail: diego.pizzeghello@unipd.it 


Plants have evolved with roots in close contact with the solid phase of the soil, therefore 

organic acid root exudates, in plant–root–microbial interactions, have attracted much interest 

not so much in terms of carbon source but as ‘signals’ for recognition or as precursors of 

phytohormone production [1]. Recent studies have demonstrated that organic acids (i.e. 

fumaric and succinic acids) occurring in the root exudates can dissociate humic substances 

(HS) in low and high molecular weight structures. This interpretation may support the 

hypothesis that the conformational behavior of dissolved humus in the rhizosphere, and 

therefore also the interaction of humic components with plant-root cells, may be controlled by 

the presence of root-exuded or microbe-released organic acids in the soil solution [2, 3]. 

Therefore, root exudates may be a better medium for extracting low molecular size (LMS) 

organic fractions (humic-like substances) than currently used alkaline solutions (i.e. NaOH, 

KOH). Our objective was to compare the chemical and biological activity of LMS organic 

fractions extracted using maize (Zea mays L.) and two forest species (Picea abies and Pinus 

sylvestris) root exudates to humic substances extracted by KOH [4]. The identification of 

some organic acids species present in the LMS organic fractions have been investigated by 

GC/MS technique and the biological activity of the humic-like substances extracted was 

evaluated by determining their hormone-like activity. 



Humic substances (HS) were extracted with common alkaline (KOH) extraction procedures 

(4) from the A horizons of a Eutric Cambisol (EC) [8] and a Rendzic Leptosol (RL) [8]. The 

EC developed under a field of Bermuda grass (Cynodon dactylon) located near the College of 

the Faculty (Legnaro, Padova, Italy), and the RL was covered by a Scotch pine forest and 

located near Cortina d'Ampezzo (Belluno, Italy).Two forest species (Picea abies Karst. and 

Pinus sylvestris L.) and two commercial maize hybrids (Zea mays L. cultivars Mytos and 

Vol. 3 Page - 329 -

Samantha; Dekalb, Italy) were used for the root exudates production. The root exudates were 

collected from forest plants raised in sterile conditions, as described in a previous work [5], 

and from maize seedlings treated with sterile Hoagland’s solution [6, 7]. The LMS 

humic/organic substances were obtained by treating 2 g of soil (A horizon) with 20 ml of 

water or 20 ml of root exudates and shaken them at room temperature for 16 h under a N2 

atmosphere. The suspensions were centrifuged at 10 °C and 5000 g for 30 min and the 

supernatants (extracts) were analyzed for total carbon. The GC/MS analysis was made on 2 

ml freeze-dried exudate or extract by an HP 5890 gas chromatograph coupled with a 

quadrupole HP 5971 A [9]. The separation and determination of the low molecular weight 

organic and phenolic acids were made by HPLC following the procedures reported in 

previous papers [9, 10]. The auxin-like and gibberellin-like activity of the LMS 

organic/humic fractions was assessed by checking the reduction in the growth of watercress 

(Lepidium sativum L.) roots and the increase in the length of lettuce (Lactuca sativa L.) 

epicotyls [11]. 



Agrarian and forest seedlings released different types of exudates in the rhizosphere, and they 

have higher extracting abilities towards carbon then water when in contact with poorer 

substrates [7, 9]. The concentrations of oxalic and succinic carboxylic acids were always 

higher in the P. abies and P. sylvestris than in the two commercial maize hybrid root 

exudates. The two forest tree exudates differed in that the P. abies had a high content of 

oxalic and L-malic acids, whereas the P. sylvestris contained citric acid. GC/MS spectra 

revealed that the LMS organic/humic-like fractions had a greater variety of fatty acids than 

the HS. Mytos extracted C15H31COOH and C16H23COOH from EC, whereas Sandek extracted 

only C15H31COOH. The P. abies extracted C11H23COOH and C13H27COOH from both soils. 

The fraction extracted from the EC by P. sylvestris revealed C13H27COOH and C17H35COOH. 

All soil extracts by exudates exhibited a hormonal activity that was not present in either water 

extracts or in the original exudates. Moreover, the extracts from the agrarian soil exhibited a 

higher hormone-like activity with respect to the extracts from forest soil. The extracting 

ability of the different species appear to be related to their different environmental conditions 

[12]. Concerning the content in phenolics, benzoic acid and in minor extent phtalic acid, 

resulted to be the more present in the LMS organic/humic-like components extracted by using 

exudates. Analysis of the phenolic pool demonstrated specificity on the extracted molecules 

not directly correlated with the phenolics present in the pure exudates. Studies have shown 

Vol. 3 Page - 330 -

that phenolic compounds have been found to be highly effective in plant defense against 

pathogens, nematodes, phytophagous insects [13]. In particular, benzoic acid is one of the 

most active phenolic compound inhibitors of fungi [14]. 



This paper reconfirms that many different regulatory signals affect rhizosphere interactions. 

Among these, the role of exudates in breaking out from the bulk humus the active humic 

components endowed with hormone-like activity and pest inhibitors is evident. This suggest 

that an appropriate combination of factors may be useful for the best use of humic substances 

on plant's health and plant capacity to adapt to different environmental conditions. 

References 

1. H. Marschner, Mineral Nutrition of Higher Plants, Academic Press, London, 1995, p. 889. 

2. S. Nardi, G. Concheri and G. Dell'Agnola, in A. Piccolo (Ed.), Humic Substances in Terrestrial 

Ecosystems, Elsevier, Amsterdam, p. 361-406. 

3. A. Piccolo, P. Conte, R. Spaccini and M. Chiarella, Biol. Fertil. Soils, 37 (2003) 255–259. 

4. F.J. Stevenson, Humus Chemistry, Genesis, Composition, Reaction, second ed. Wiley, New York, 

1994, p. 496. 

5. S. Nardi, F. Reniero and G. Concheri, Chemosphere, 35 (1997) 2237-2244. 

6. D.R. Hoagland and D.I. Arnon DI, Agricultural Experiment Station, Circular 347 (1950). 

7. S. Nardi, E. Sessi, D. Pizzeghello, A. Sturaro, A. Rella and G. Parvoli, Chemosphere, 46 (2002) 

1075–108. 

8. FAO, Soil Map of the World: revised legend, FAO, Rome, 1990. 

9. S. Nardi, M. Tosoni, D. Pizzeghello, M.R. Provenzano, A. Cilenti, A. Sturaro, R. Rella and A. 

Vianello, Soil Sci. Soc. Am. J., 69 (2005) 2012-2019. 

10. S. Nardi, D. Pizzeghello, L. Bragazza and R. Gerdol, J. Chem Ecol., 29 (2003) 1549-1564. 

11. L.J. Audus, Plant Growth Substances, Leonard Hill, London, 1972. 

12. T. Ingestad, Physiol. Plantarum, 13 (1960) 513–533. 

13. F.D. Dakora and D.A. Phillips, Physiol. Mol. Plant Pathol., 49 (1996) 1-20. 

14. M., Weidenborner, H. Hindorf, H.C. Jha and P. Tsotsonos, Phytochemistry, 29 (1990) 1103-1105. 

Vol. 3 Page - 331 -

Bioactivity of Humic Acids from Vermicompost at Increasing Maturity 

Stages 

Natalia O. Aguiar a* , Luciano P. Canellas a , Fabio L. Olivares a , Jader G. Busato a , Luis Gonzaga 

JR. S. Silva. a , Etelvino H. Novotny b , Arnoldo R. Façanha a 

a Núcleo de Desenvolvimento de Insumos Biológicos para Agricultura - Universidade Estadual 

do Norte Fluminense Darcy Ribeiro. Av. Alberto Lamego, 2000, Campos dos Goytacazes 

28013-602, Rio de Janeiro, Brasil; b Embrapa Centro Nacional de Pesquisa de Solos (CNPS), 

R. Jardim Botânico, 1024, Rio de Janeiro, Brasil 

E-mail: nattyaguiar@gmail.com 


The application of humic substances (HS)-derived products at low concentration and their 

effects as plant growth promoters have been creating increased interest among farmers. 

Despite of the technological potential, little information have been accumulated about the 

mechanism by which HS influences biological activities in plants. It has been widely 

demonstrated that humic acid (HA) can affect plant growth and metabolism, but scientific 

efforts linking HA structure to biological activity have so far produced divergent results. The 

relationship between different levels of bioactivity and variation at chemical structure of HS 

persists as a challenge for scientific and technological purposes into the direction of 

improvement of organic fertilizers based on humic matter. It was previously observed that HA 

induced changes in the developmental program of root growth and in plant development by 

proliferation of lateral emerging sites and induction of a plasma membrane (PM) H + -ATPases 

in different plant species [1]. The aim of this work was to evaluate chemical changes and 

plant growth-promoting effect of HA from different maturing stages isolated from 

vermicompost, relating bioactivity and its chemical characteristics. 

2. Material and methods 


Vermicompost (VC) was prepared using cattle manure putted on concrete cylinder with 150 L 

of capacity and humidity was maintained at 65–70%. After approximately one month, the 

earthworm was introduced (Eisenia foetida) at a ratio of 5 kg worms per m 3 of organic 

residue. Two cylinders of each organic residue were used to sample at different time: 0, 30, 

60, 90 and 120 days. Before sampling, the content of cylinder was vigorously mixed by 

manual spade. The VC was chemically characterized (organic carbon, C-N ratio, CEC and 

HA content). HA were isolated with 0.5M NaOH under N2 and precipitated with 6M HCl. 

The diluted HF:HCl was used for 16 hours to decrease the ash content and after that HA was 

washed with water, dialyzed (membrane cutoff 1000 Da) and freeze dried. The relative Mw 

Vol. 3 Page - 332 -

index was obtained by the ratio of low: high and characterized by size exclusion (Polysep- 

GFC-P 3000 (600 mm per 7.8 mm i.d.) column (Phenomenex) and reverse-phase (Supelco C- 

18 column) high performance chromatography for hydrophobicity. Maize seedlings with 

0.5 cm of root length was treated for 48 h with HA solution (20 mg C L -1 of AH and 2 mM 

CaCl2 at pH 7.0). After this time, the seedlings were transferred to 40 mL of 2 mM CaCl2 at 

pH 7.0. After 48 h the pH was evaluated using Thermo Orion pHmeter. A preliminary assay 

was carry-out to verify a putative relationship between H + extrusion and PM H + -ATPase 

activity [2]. 

3. Results 

Parameters related to organic matter evolution during vermicompost maturation stages. The 

C-N ratio and lignin content, decreased and CEC and HA content increase (Fig. 1). The 

relative Mw had shown no change and the hydrophobic content, determined by NMR, showed 

small changes with the maturation process (Fig. 2). However the hydrophobic content, 

determined by RP-HPLC showed changes with the maturation process (Fig. 3). 

CEC (Cmolc kg -1 ) 

C/N ratio 

80 

70 

60 

50 

40 

30 

A 

20 

0 30 60 90 120 

15.5 

C 

15 

14.5 

14 

13.5 

13 

12.5 

12 

11.5 

11 

0 30 60 90 120 

vermicomposting time (days) 


lignin (%) 

gCHA kg -1 VC 

20 

18 

16 

14 

12 

10 

8 

6 

4 

2 

0 

4 

3.5 

3 

2.5 

2 

B 

D 

VC1 

0 30 60 90 120 

1.5 

0 20 40 60 80 100 120 

vermicomposting time (days) 

Figure 1: Parameters related to organic matter stabilization according vermicompost maturity time 

Vol. 3 Page - 333 -


Figure 2: 13 C-CPMAS-NMR spectra from vermicompost at different maturing stages 

Figure 3: HPSEC chromatograms - Mw distribution (A), RP-HPLC Hydrophobicity (B) index Mw (C) 

and index hydrophobicity (D) of HA from vermicompost at different maturing stages (0, 30, 60, 90 

and 120 days) 

Vol. 3 Page - 334 -


Figure 4: A: Proton extrusion (mM H + g -1 dry root) for 2 mM CaCl2 solution of maize root seedlings 

treated with 48 hours of HA isolated from cattle manure vermicompost at different maturing stages (0, 

30, 60, 90 and 120 days). B: relationship between H + extrusion and PM H + -ATPase activity 

At the end of vermicompost maturation it was observed a selective increase of C-aryl, O-aryl 

and C-carboxyl species as a consequence of carbohydrates decrease (Fig. 2). The relative 

molecular size/weight (Mw) revealed no changed with HA maturation (Fig. 3A and C), but 

the hydrophobicity evaluated by RP-HPLC increase at 60-d and after this time decrease (Fig, 

3 B and D). After 60-d of vermicomposting process the HA displayed bioactive effect 

compared with control manifested by increaser of maize root seedlings H + extrusion (Fig. 

4A), which gave a good correlation with PM H + -ATPase activity (Fig. 4B). 

We concluded that at the initial stages of vermicompost maturing process the HA had 

diminished plant growth promoting effect. Increased bioactivity of HA evaluated by H + 

extrusion at the solution and PM H + -ATPase activity by HA was achieved with enhanced 

vermicompost maturing coupled with increase of hydrophobic domains on its structure, 

mainly until 60 days. However the form of protection of bioactive molecules in the structure 

of AH is not even well understood and needs to be more study. Furthermore the process of 

humification by itself is not a sufficient to explain the promotion of plant growth promoted. 

References. 

1) Canellas, L.P., Façanha, A.O., Olivares, F.L., Façanha, A.R., Humic acids isolated from 

earthworm compost enhance root elongation, lateral root emergence, and plasma 

membrane H+-ATPase activity in maize roots. Plant Physiol. 130, 1951– 658 1957. 2002. 

2) Canellas, L.P., Piccolo, A., Spaccini, R., Dobbss, L., Zandonadi, D., Olivares, F. and 

Facanha, A.. Chemical composition and bioactivity properties of size-fractions separated 

from a vermicompost humic acid. Chemosphere (2009), doi: 

10.1016/j.chemosphere.2009.10.018. 

Vol. 3 Page - 335 -

Root Growth promotion by humic acids from urban organic residues 

Keiji Jindo, Carlos García Izquierdo, Luciano Pasqualoto Canellas 

CEBAS-CSIC. Campus Universitario de Espinardo. Apartado de correos 164. Murcia. E- 

30100, Spain 

E-mail: keijindo@cebas.csic.es 


The effect of HA on plant physiology is generally recognized to result in root growth 

enhancement (Vaughan et al., 1985; Nardi et al., 2002) and enhance of nutrients uptake (Chen 

et al., 2004; Pinton et al., 2007). Humic acids (HA) promotes plasma membrane (PM) H + 

ATPase activity and it synthesis (Pinton et al., 1987; Canellas et al., 2002; Quaggiotti et al., 

2004). The major role of this enzyme on energetic metabolism PM H + -ATPase activity was 

found to be a useful physiological indicator of HA bioactivities (Canellas et al., 2006).The 

aim of this work was to evaluate the root seedling growth and PM H + -ATPase activity on 

maize treated with HA isolated from sewage sludge and municipal solid wastes at initial and 

final stage of composting process. 

2. material and Methods 


Organic materials from urban origin: Four different organic wastes were used in this study: 

Sewage sludge (SS) from a municipal wastewater treatment plant in El Raal—Murcia, 

compost produced from the abovementioned SS (SSC), organic fraction of MSW collected 

from the treatment plant, and the compost produced from this organic material (MSWC). 

HA extraction: The method of extraction of humic acid (HA) was followed by Stevenson. The 

dialyzate was lyophilized and characterized chemically. Later, these 4 samples were 

chemically characterized by solid-state nuclear magnetic resonance (13C CPMAS-NMR), 

Maize: Maize seeds (var UENF 506) provided were surface-sterilized by soaking in 0.5% 

NaCl for 30 min, followed by rinsing and then soaking in water for 6 h. The seeds were then 

sown on wet filter paper and germinated in the dark at 28°C. After regression analysis, a new 

experiment was carried out using 2mM C of each HA. Four-day-old maize seedlings with 

roots approximately 0.5 cm long were transferred into a solution containing 2 mM CaCl2 (to 

avoid any interference with nutrients contsittuents (Pinton et al.1999) and either 0 or 2 mM C 

L-1 of HA . Roots were collected on the seventh day and scanned at 300 dpi to estimate their 

length and area using Delta-T Scan image analysis software (Cambridge, UK) (Bouma et al., 

2000). Additional samples of root seedlings were collected for mitotic sites experiments and 

lateral roots (all graphics of results are demonstrated with comparison with the control). 

Vol. 3 Page - 336 -


1. References 

2. Canellas, L. P., D. B. Zandonadi, F. L. Olivares, and A. R. Façanha. 2006. In Nutrição Mineral de 

Plantas. M. S. Fernandes (ed.). Sociedade Brasileira de Ciência do Solo, Viçosa, Brazil, pp. 175- 

200. 

3. Bouma, T. J., K. L. Nilsen, and B. Koutstaal. 2000. Plant Soil. 218:185-196. 

4. Canellas, L. P., F. L. Olivares, A. L. Okorokova-Façanha, and A. R. Façanha. 2002. Plant Physiol. 

130:1951-1957. 

5. Vaughan, D., R. E. Malcolm, and B. G. Ord. 1985. Soil Organic Matter and Biological Activity. 

Kluwer Academic Publishers, Dordrecht, The Netherlands, pp. 77-108. 

6. Chen Y., Clapp C.E., Magen H. (2004) Soil Science & Plant Nutrition. 50, 1089 

7. Pinton R., Varanini Z., Nannipieri P. (2007) The rhizosphere: biochemistry and organic 

substances at the soil–plant interface, CRC Press, Madison, USA: pp. 447. 

8. Quaggiotti, S., B. Ruperti, D. Pizzeghello, O. Francioso, V. Tugnoli, and S. Nardi. 2004. J. Exper. 

Bot. 55:803-813. 

9. Nardi, S., D. Pizzeghello, A. Muscolo, and A. Vianello. 2002. Soil Biol. Biochem. 34:1527 

Vol. 3 Page - 338 -

Direct and Indirect Effects of Humic Substances of Different Origin on the 

Green Algae Monoraphidium braunii 

C. Eliana Gattullo a* , Hanno Bährs b , Ji Qianru b , Christian E.W. Steinberg b , Elisabetta 

Loffredo a 

a Dipartimento di Biologia e Chimica Agro-forestale ed Ambientale, University of Bari,Via 

Amendola 165/A, 70126 Bari, Italy; b Institute of Biology, Freshwater and Stress Ecology, 

Humboldt University at Berlin, Späthstr. 80/81, 12437 Berlin, Germany 

E-mail: e.gattullo@agr.uniba.it 



Dissolved natural organic matter (NOM) is widespread and abundant in freshwater 

ecosystems, exceeding the total living organic carbon by one order of magnitude [1, 2]. A 

consistent part of NOM (50–80%) is made up by humic substances (HS) [1, 3]. It’s well 

known that HS, because of their physical and chemical properties, modify the underwater 

light climate, alter the bioavailability of inorganic micronutrients and xenobiotics and, after 

their photodegradation, supply carbon and/or energy to heterotrophic organisms. New 

ecophysiological studies, without disregarding the traditional conception that HS affect 

indirectly the aquatic biocenosis, consider HS as natural environmental chemicals able to 

interact also directly with organisms [4]. Due to the low molar mass of their building blocks, 

HS can be taken up by organisms. Once internalized, they act like xenobiotics inducing 

several specific or not specific stress responses. Specific reactions include reduction of 

photosynthetic oxygen production, estrogenicity and chemical attraction. Non specific 

reactions comprise physical and chemical membrane irritation, induction and modulation of 

biotransformation enzymes, induction of stress defense proteins and internal oxidative stress 

[5]. According to recent theories of aging and evolution, a mild stress may be benefit for 

individuals, training their chemical defense system [5, 6]. Therefore, the study of interactions 

between HS and organisms represents the first step to clarify the possible role of HS as mild 

stressors able to modify the biochemical and physiological properties of individuals and, on a 

larger scale, structure the biocenosis. Much information are reported in the literature on the 

interactions between dissolved organic carbon and animals or aquatic plants. With regard to 

phytoplankton, previous works [7, 8] revealed contrasting effects of HS on algal growth rate. 

Moreover, HS lower the oxygen production in various green algae, but the mechanism of 

action remains still obscure [9]. The present work is a part of a larger study on stress 

responses of various green algae to different organic fractions. Here, the authors measured the 

growth and some fluorescence parameters on the green algae Monoraphidium braunii (MB) 

Vol. 3 Page - 339 -

exposed to two HS sources. A parallel experiment was carried out in the same conditions, but 

using a nitrogen-free culture medium, to assess the attitude of HS to supply nitrogen to MB. 



Monoraphidium braunii was obtained from the Algal Culture Collection, Göttingen, and 

cultivated in axenic condition in FW04-medium [10]. The Suwannee river NOM, acquired 

from the International Humic Substances Society, and the HuminFeed ® (HF), a commercial 

HS isolated from leonardite by alkaline extraction, were tested at the concentrations of 2, 5 

and 20 ppm DOC. Algae in the logarithmic growth phase were used to inoculate FW04medium 

alone (control) or added with each concentration of NOM or HF, up to an initial 

concentration of about 10 5 cells/mL. Algae solutions were cultivated in axenic conditions in 

glass flasks, at 20 °C, under a cool white light set to 150 µM photons/m 2 s, using a 

photoperiod of 12 hours. In each flask, a polyethylene bag filled by a solution of KHCO3 and 

K2CO3 ensured the necessary CO2 supply to algae. After 2 and 4 days, algae cell number and 

size were measured by a particle counter. Small aliquots of MB solutions were washed twice, 

through centrifugation at 4000 rpm for 10 minutes and replacement of surnatant by fresh 

culture medium, then adapted to the dark for 30 minutes and analyzed by the Phytoplankton 

Analyzer (Walz Effeltrich, Germany) to determine the chlorophyll a content (chl) and various 

fluorescence parameters like the maximum quantum yield of PSII in the dark adapted state 

(Fv/Fm), the quantum yield of PSII in the light adapted state (ΦPSII), the photochemical (PQ) 

and not-photochemical (NPQ) quenching. After 4 days, when algae reached the maximum 

growth, the experiment was stopped and the antioxidative capacity of the water soluble 

compounds (ACW) was measured by the Photochem (Analytik Jena, Germany), an 

instrument which uses the method of photochemiluminescence. For this analysis, algae 

samples were concentrated, then the pellet was suspended in a 0.1 M NaH2PO4 buffer, mixed 

with glass beads (Ø 0.3 µm) and cleft by a speed mill. The protein content was measured 

according to Bradford [11]. The ACW was not determined for algae treated with NOM. A 

similar experiment was performed growing algae in FW04-medium nitrogen-free, alone 

(control) or added with HF at the concentration of 1,5,10 and 20 ppm DOC. For both 

experiments, treatments were replicated 4 times and data were statistically analyzed by oneway 

analysis of variance (ANOVA) and the SNK test. 

Vol. 3 Page - 340 -


Neither NOM or HF modified algae cell number. HuminFeed determined a decreasing of cell 

size, but only at the higher concentration. In previous works, Steinberg and other Authors 

[7,8] assessed that HS usually modulate algae growth, but they also observed that algae 

response pattern to HS exposure is species-specific. Therefore, it’s not surprising that MB 

reaction to HS differed from what is reported in literature for other algae species. After two 

days of exposure to NOM, fluorescence parameters like chl, Fv/Fm and NPQ increased at each 

NOM concentration, with respect to the control. However, no significative differences were 

revealed between the control and the different NOM treatments on the fourth day. The 

interaction MB-HF did not affect any fluorescence parameters. These indexes are indicative 

of the overall photosynthetic rate [12]. In particular, Fv/Fm refers to the maximum efficiency 

of PSII, i.e. the efficiency if all PSII reaction centers are opened. NPQ express the efficiency 

by which energy is dissipated in heat. Initially, MB reacted positively to each concentration of 

NOM, showing an higher chlorophyll content and, consequently, a combination of both high 

photosynthetic light use and heat dissipation. To the best of our knowledge, the only studies 

in the literature to which compare our results refers to the aquatic plant Ceratophyllum 

demersum [13]. Unlike MB, PSII electron transport chain of C. demersum was inhibited 

significantly by much lower concentration of several NOM. The exposure to HF reduced the 

ACW (%) 

120 

100 

80 

60 

40 

20 

0 

* 

** 


** 

Control 

HF 2 ppm DOC 

HF 5 ppm DOC 

HF 20 ppm DOC 

* P≤ 0.05; ** P ≤ 0.01; according to SNK test 

Figure 1: ACW of algae exposed to different HF concentration 

ACW of MB (Fig.1). In particular, this 

stress response increased with increasing 

HF concentrations. Probably, HF is taken 

up by algae and then metabolized with 

oxidizing oxygen and nitrogen species as 

byproducts, causing an internal oxidative 

stress. 

When a nitrogen-free medium was used for all treatments, included the control, algae 

concentration rose in the presence of HF, especially at 5, 10 and 20 ppm DOC (Fig.2), while 

cell size slowly decreased. This result represents the evidence that HS supply nitrogen and, 

probably, also other nutrients to algae, accelerating their growth rate. With regard to 

chlorophyll content and fluorescence parameters, all treatments were not significative 

different to the control. In general, MB was not deeply stressed neither by NOM nor HF. 

Vol. 3 Page - 341 -

Maybe, because of its origin from a DOC-rich environment (Grosse Fuchskuhle), this algae 

species was well adapted to HS. 

Cell concentration (%) 

300 

250 

200 

150 

100 

50 

0 

** 

*** *** *** 

Control 

HF 1 ppm DOC 

HF 5 ppm DOC 

HF 10 ppm DOC 

HF 20 ppm DOC 

** P≤ 0.01; *** P≤ 0.001; according to SNK test 

Figure 2: Cell concentration after 5 days of exposure to differentconcentrations of HF 



This study confirmed the recent theory that HS, upon their uptake, affect organisms directly. 

HF provoked the internal oxidative stress, but did not interfere within the photosynthetic 

electron chain. The experiment confirmed also that algae benefit of the exposure to HF if they 

are cultivated in a nitrogen-poor medium. Hence, HS play an important role in the structuring 

of water biocenosis, suppressing the most sensitive species and favoring the less sensitive. 

References 

1. E.M. Thurman, Organic Geochemistry of Natural Waters, Dr W. Junk Publishers, Dordrecht, 

1985, p. 497. 

2. R.G. Wetzel, Limnology. Lake and River Ecosystems, Academic Press, San Diego, 3rd edn., 2001, 

p. 1006. 

3. C.E.W. Steinberg and U. Münster, Humic Substances in Soil, Sediment and Water. Geochemistry, 

Isolation and Characterisation, G.R. AikenD.M., McKnight, R.L. Wershaw and P. MacCarthy 

(Eds.), Wiley, New York, 1985, 105–145. 

4. C.E.W. Steinberg, A. Paul, S. Pflugmacher, T. Meinelt, R. Clöcking and C. Wiegand, Fresenius 

Env. Bull., 12 (2003) 391–401. 

5. C.E.W. Steinberg, T. Meinelt, M.A. Timofeyev, M. Bittner and R. Menzel, Env. Sci. Pollut. Res., 

15 (2008) 128–135. 

6. E.J. Calabrese, Env. Pollut., 138 (2005) 379–412. 

7. T.A. Karasyova, E.O. Klose, R. Menzel and C.E.W. Steinberg, Env. Sci. Pollut. Res., 14 (2) 

(2007) 88-93. 

8. V.Y. Prokhotskaya and C.E.W. Steinberg, Env. Sci. Pollut. Res., 14 (2007) 11–18. 

9. C.E.W. Steinberg, S. Kamara, V. Prokhotskaya, et Al., Freshwater Biol., 51 (2006) 1189–1210. 

10. A. Nicklisch, T. Shatwell and J. Köhler, J. Plankt. Res., 30 (2008) 75-91. 

11. M.M. Bradford, Anal. Biochem., 72 (1976) 248-254. 

12. K. Maxwell and G.N. Johnson, J. Experim. Botany, 51 (2000) 659-668. 

13. S. Pflugmacher, C. Pietsch, W. Rieger and C.E.W. Steinberg, Sci. Tot. Env., 357 (2006) 169– 175. 

Vol. 3 Page - 342 -

Effects of Compost Water-Extracts on the Germination and Growth of 

Slickspot Peppergrass (Lepidium papilliferum) 

Elisabetta Loffredo a* , Antonio J. Palazzo b , Andreina Traversa a , Terry L. Bashore c , 

Nicola Senesi a 

a Dipartimento di Biologia e Chimica Agro-forestale e Ambientale, University of Bari, 

Via G. Amendola 165/A 70126 Bari, Italy; b ERDC-CRREL, Hanover, NH 03755-1290, USA; 

c HQ ACC/A3A, Airspace, Ranges, Airfield Operations Division, Langley AFB, VA 23665-2789, 

USA 

E-mail: loffredo@agr.uniba.it 


Compost addition to soil is a common practice, and amendment of soils low in organic 

content can be used to improve soil fertility. Slickspot peppergrass (Lepidium papilliferum) is 

a rare plant species of high conservation concern in southwestern Idaho, and grows in 

depressed soils called slickspots. Slickspot soils are low in organic content and additions of 

composts may be used to help establish new stands of this rare species. Compost waterextracts 

(C-WE) contain the portion of organic material that can pass through a 0.45 μm filter 

membrane and is constituted of an heterogeneous mixture of molecules with different 

molecular size and complexity, ranging from simple sugars and organic acids to relatively 

high molecular weight humic colloids. The biological activity of C-WE depends mainly on 

the type of substrate used for composting and the type and duration of the process. In some 

cases, however, phytotoxic effects may originate from compost application depending on the 

plant species type and age and environmental conditions. The aim of this work, which is part 

of a more extensive research on the restoration of rare plant species, was to evaluate the 

effects of C-WE from different composts on the germination and growth of slickspot 

peppergrass, and possibly relate its biological activity to chemical and physico-chemical 

characteristics of C-WE. 



The three composts used in this work were a green compost (GC), a mixed compost (MC) and 

a coffee compost (CC). The C-WE were obtained from each compost by extracting the matrix 

with distilled water (1/10, w/v), centrifuging at 6000 rpm and filtering sequentially through 

Whatman filters with particle size retention decreasing from 11 to 0.45 μm [1]. The C-WE 

samples were characterized by means of chemical and physico-chemical methods, such as pH, 

electrical conductivity (EC), total organic carbon (TOC) and E4/E6 ratio. The C-WE were 

diluted with Nitch nutrient solution in the ratios of 1:2 (C-WE1:2), 1:5 (C-WE1:5) and 1:10 (C- 

Vol. 3 Page - 343 -

WE1:10) (v/v) and tested on the germination and growth of slickspot peppergrass. In 

germination experiments, the nutrient solution alone (control) and each diluted C-WE sample 

were separately added in agar solution (1.5 % agar) to 60 seeds of slickspot peppergrass in 

Petri dishes. Seed germination was achieved in a Phytotron growth chamber at 18 ± 1 °C 

during the illumination period and at 10 ± 1°C in the dark with 8-h photoperiod. The 

germination percentage was measured after 7 and 15 days, and primary shoot and root lengths 

were measured on germinated seeds collected after 7 days. Subsequently, growth experiments 

were performed on the 7-day germinated seeds, which were transplanted on new plates 

containing the same C-WE samples and let to grow with a 12-h photoperiod and a 

temperature of 23 ± 1 °C. After 15 and 30 days, live seedlings were counted and root and 

shoot lengths were measured. Then, the 30-day grown seedlings were transplanted in plastic 

pots containing peat, and irrigated periodically either with the nutrient solution alone or with 

each diluted C-WE sample. All experiments were replicated three times and all data obtained 

were analyzed statistically by one-way analysis of variance (ANOVA) and the least 

significant difference test (LSD). 



Some properties of the three C-WE samples examined are shown in Table 1. The pH and EC 

values were similar for GC-WE and MC-WE whereas they resulted much higher or lower, 

respectively, for CC-WE. The TOC content, the molar absorptivity at 280 nm (ε280) and the 

E4/E6 ratio increased in the order: GC-WE < MC-WE < CC-WE. The relatively high values 

measured for ε280 and E4/E6 ratio and other spectroscopic evidence would suggest, especially 

for CC-WE, the occurrence of relatively low molecular weight aromatic molecules, such as 

phenolic-like units, aniline-derived compounds, benzoic acid derivatives, polyenes, and polycyclic 

aromatic hydrocarbons, which are generally present in C-WE samples. A more detailed 

presentation and discussion of the properties of the three C-WE can be found in Traversa et al. 

[2]. 

Table 1: Some characteristics of the C-WE samples used 

EC TOC 

Sample pH 

(dS/m) (mg L -1 ε280 

) (L cm -1 mol -1 ) E4/E6 ratio 

GC-WE 7.4 3.14 140 26.8 3.6 

MC-WE 7.0 3.28 186 32.5 4.3 

CC-WE 8.3 1.66 375 38.9 6.9 

Vol. 3 Page - 344 -

Results of germination experiments indicated that after 7 days, GC-WE and MC-WE at the 

higher dilution and CC-WE at any dilution did not alter the germination percentage with 

respect to the control, whereas a reduction of germination was observed in all the other cases. 

After 15 days, a significant reduction of the germination percentage was measured only for 

GC-WE and MC-WE at the lower dilution. Probably, the relatively high EC of the latter two 

samples was responsible for the decrease of the germination percentage when a scarce 

dilution was adopted. The effects of the three C-WE treatments at three dilutions on the shoot 

and root length of seedlings after 7 and 30 days are shown in Figs. 1-2. No significant effect 

was observed on primary shoot length for any C-WE treatment at the three dilutions, whereas 

only in the case of GC-WE and MC-WE at the lower dilution a significant reduction of 

primary root length was measured with respect to the control (Fig. 1). Also in this case, it can 

be hypothesized that the higher EC of the two samples inhibited root elongation. After 15- 

(data not shown) and 30-days growth, no differences of shoot and root lengths were observed 

between the C-WE treatments and the control (Fig. 2). After 7-month growth, the plants 

treated with the different C-WE showed better growth and health conditions with respect to 

the control. At the lower dilution, the maximum benefit for plant growth was caused by GC- 

WE treatment with respect to the other two samples (Fig. 3). The GC-WE produced the 

highest plant stimulation at the lower dilution, whereas MC-WE and CC-WE showed the best 

effects at the highest dilution (Fig. 3). Results obtained suggest that the molecular 

composition of the C-WE plays an important role in their biological activity on plants. 



The addition of C-WE to the germination and growth medium of slickspot peppergrass 

generally did not alter the rate of germination and early growth when a low or moderate dose 

was used. In particular, among the three different composts used, the coffee compost 

presented a water soluble organic fraction completely tolerable by the seedlings also at a very 

high dose. After a prolonged period of growth, favorable effects of any C-WE were observed 

on slickspot peppergrass, and particularly by the WE from the green compost. This 

information will be used, along with other environmental and genetic factors that affect seed 

germination and early root and leaf growth, to better understand how restore this rare plant 

species. 

Vol. 3 Page - 345 -

Length (%) 

Length (%) 

140 

120 

100 

80 

60 

40 

20 

0 

160 

140 

120 

100 

80 

60 

40 

20 

0 

SHOOT ROOT 

C GC-WE MC-WE CC-WE C GC-WE MC-WE CC-WE 

C-WE1:10 

C-WE1:5 

* 

C-WE1:2 

SHOOT ROOT 

C GC-WE MC-WE CC-WE C GC-WE MC-WE CC-WE 


C-WE1:10 

C-WE1:5 


C-WE1:2 

** 

Figure 1: Effects of C-WE at 

different dilutions on primary shoot 

and root lengths of germinated 

seeds after 7 days, expressed as 

percentages of the control (100 %). 

The vertical line on each bar 

indicates the standard error for 3 

replicates 

Figure 2: Effects of C-WE at 

different dilutions on shoot and root 

lengths of seedlings after 30 days, 

expressed as percentages of the 

control (100 %). The vertical line 

on each bar indicates the standard 

error for 3 replicates 

Figure 3: Effects of MC-WE1:2, 

GC-WE1:2 and CC-WE1:2 (from left 

to right) on the growth of slickspot 

peppergrass after about 7 months 

This work was supported by the Research Contract n. W911NF-08-1-0076 of the US Army 

RDECOM ACQ CTR – W911NF, Durham NC, USA. Project title: “Effects of Quality 

Composts and Other Organic Amendments and Their Humic and Fulvic Acid fractions on the 

Germination and Early Growth of Slickspot Peppergrass (Lepidium papilliferum) and 

Switchgrass in Various Experimental Conditions”, funded by the Airspace, Ranges, and 

Airfield Operations Division, HQ Air Combat Command, Langley AFB, VA. 

References 

1. A. Traversa, V. D’Orazio and N. Senesi, Forest Ecol. Manag., 256 (2008) 2018. 

2. A. Traversa, E. Loffredo, C.E. Gattullo and N. Senesi, Geoderma (under revision) (2010). 

Vol. 3 Page - 346 -

The Action of Humic Acids Promoting Plant Shoot Development are 

Associated with Nitrate-Related Changes on the Plant Hormonal Balance 

V. Mora, E. Bacaicoa, E. Aguirre, R. Baigorri, M. Garnica, M. Fuentes, A.M. Zamarreño, J.C. 

Yvin, J.M. Garcia-Mina * 

CIPAV- Roullier Group and Department of Chemistry and Soil Chemistry, Faculty of 

Sciences, University of Navarra 

E-mail: jgmina@timacagro.es 



A number of studies have shown the ability of humic substances (HS) to enhance the growth 

of different plant species cultivated both in soils or inert substrate (1). However, the 

mechanism or mechanisms responsible for these effects of HS are not well understood. Some 

authors propose that this action of HS is principally indirect, by improving soil texture and the 

availability of certain nutrients such as Fe and Zn (1). Other authors suggest that, besides 

these HS effects on soil properties, there exist direct effects of HS on plant metabolism (2). 

The question is whether these metabolic effects of HS are the consequence of previous effects 

on nutrient uptake and translocation, or they are independent of them. 

Several studies have demonstrated that the growth promoting effects of certain nutrients, such 

as nitrate, are linked to effects on the distribution of specific plant hormones within the plant 

(3). On the other hand, several studies describe the ability of HS to increase H+-ATPase 

activity and nitrate root uptake (4). It is, thus, possible that the action of HS on plant growth is 

related to nitrate root uptake and further root to shoot translocation and the effects of this 

nitrate-related action on the hormonal balance. In turn, this HA effect might be related to a 

previous action on root functionality (ATPase activity and root architecture). 

In this presentation we analyze this working hypothesis. To this end we have investigated in 

cucumber plants the time-course effect of the root application of a purified leonardite humic 

acid (HA) on: (i) the growth of the root and the shoot; (ii) root H+-ATPase activity and nitrate 

concentrations in roots and shoot; (ii) the concentrations of ethylene and IAA in the root; (iii) 

the concentrations of polyamines (PAs), cytokinins (CKs) and ABA in root and shoot; (iv) the 

root-shoot distribution of the main mineral nutrients. 

Vol. 3 Page - 347 -


The experimental design and the methodology used for measuring the main plant hormones 

are described in (5). The analytical methods for analysing ethylene and polyamines are 

described in (6). 

The humic acid used in the experiments was extracted from a leonardite obtained from 

Czechia. The extraction and characterization methods are described in (5). The analysis of the 

main plant phytoregulators in HA revealed that their presence was negligible. 



The application of HA caused a significant a significant increase in root growth and root 

growth rates that was associated with significant increases in the root concentration of IAA 

(after 24 and 72 h from the onset of treatments) and the root production of ethylene (after 24 

and 72 h from the onset of treatments). These results were obtained in three independent 

experiments. 

On the other hand, HA root application caused a significant increase in the root activity of the 

H+-ATPase that was also associated with changes in the root-shoot distribution of nitrate 

concentration, which decreased in roots and increased in the shoot. 

These changes in nitrate root-shoot distribution were associated with concomitants changes in 

the root-shoot distribution of the main cytokinins (principally, isopentenyladenine and t- 

Zeatine Riboside) and polyamines (principally, putrescine), which increased in the shoot and 

decrease in the roots. These effects were well correlated to significant increases in shoot 

growth and shoot relative rates. A transient increase in ABA shoot concentration was also 

observed. 

Finally, it was noteworthy that all these effects, principally those concerning CK plant 

distribution were also associated with changes in the root to shoot distribution of the 

concentration of the main mineral nutrients, which experience a descrease in the root and an 

increase in the shoot. 

In consequence all these results, taken together, indicate that in the shoot promoting effect of 

HA are directly involved significant effects on the plant distribution of CKs and PAs, which 

in turn seem to be linked to a previous action on nitrate plant distribution. This effect of HA 

on nitrate plant distribution is associated with significant increases in the root H+-ATPase 

activity. 

Vol. 3 Page - 348 -

Regarding the mechanism by which HA acts on H+-ATPase activity, it could be associated 

with the above-mentioned effects on IAA, NO and / or ethylene root concentrations. 

All these effects are presented in Figure 1. 


Figura 1: HA action mechanism on cucumber development: working model 

References 

1. Y. Chen, M. De Nobili, and T. Aviad. Stimulatory effects of humic substances on plant growth. 

Soil Organic Matter in Sustainable Agriculture. Boca Raton, Florida: CRC Press; (2004). p. 103- 

29. 

2. S. Nardi, D. Pizzeghello, A Muscolo, and A. Vianello. Soil Biol Biochem;34 (2002) 1527. 

3. V. Rubio, R. Bustos, ML. Irigoyen, X. Cardona-Lopez, M. Rojas-Triana, and J. Paz-Ares. Plant 

Mol Biol 69(2009) 361. 

4. R. Pinton, S. Cesco, G. Iacolettig, S. Astolfi, and Z. Varanini. Plant Soil 215 (1999) 155. 

5. E. Aguirre, D. Leménager, E. Bacaicoa, M. Fuentes, R. Baigorri, AM. Zamarreño , and JM 

García-Mina. Plant Physiol Biochem 47 (2009) 215. 

6. M. Garnica, F. Houdusse, JC. Yvin , and JM Garcia-Mina. J Plant Physiol 166 (2009) 363. 

Vol. 3 Page - 349 -


Health and Medical Applications of Natural Organic Matter 


Vol. 3 Page - 350 -

Inclusion Complexes of Aspirin in Fulvic Acid Offer Enhanced Dissolution, 

Permeability, Stability and Better Pharmacodynamics 

Khalid Anwer a, b* , Aamir Mirza a , Suraj P. Agarwal a , Asgar Ali a , Yasmin –Sultana a 

a Dept. of Pharmaceutics, Jamia Hamdard (University), Hamdard Nagar, New Delhi-110062, 

India; b Al-kharj College of Pharmacy, King Saud University, Saudi Arabia 

E-mail: mkanwer2002@yahoo.co.in 


Shilajit, a wonder medicine of ayurveda, neither a plant nor animal origin, it is a mineral pitch 

that comes out from the rocks of the Himalayas, as they become warm during summer months 

[1–2]. Shilajit contains a variety of organic compounds that can be broadly classified into 

humic and non-humic substances [2]. Humic substances are further classified into humic and 

fulvic acid. Humic acid (HA) and fulvic acid (FAs) have relatively open, flexible structure 

punctured by voids (micropores) of different diameters (200-1000Å) as reported in literature 

[3-4]. The interior of these humic and FA hydrophobic and thus are capable of forming 

inclusion complexes with non-polar solutes and unstable drug molecules [4]. 

Aspirin (acetylsalicylic acid) is very old drug but still havimg a very high market value. It 

possesses antipyretic, anti-inflammatory, analgesic and anti-aggregatory activity due to 

decreased production of prostaglandins and thromboxanes. The acetylsalicylic acid molecule 

has a carboxyl group and an ester group. The ester group can be easily hydrolyzed, which 

reduces the medicinal value and causes side effects on humans. A strategy designed how to 

inhibit the hydrolytic decomposition and enhancement of dissolution of aspirin inside the void 

of HA of shilajit. We propose to investigate the effects of FA as carrier on aspirin 

(acetylsalicylic acid) in enhancing the dissolution rate and bioavailability, increasing the 

stability and decreasing the toxicity of aspirin through complexation. 

2. Experimental 


Extraction of humic acid form Shilajit . Humic acid is extracted from shilajit by increasing the 

polarity of solvent as reported in literature (6). The method consisted of successive extraction 

of raw shilajit with hot organic solvents of increasing polarity to remove the bioactive 

components. The residue (marc) was dissolved in 0.1 M NaOH with intermittent shaking in 

the presence of nitrogen. The suspension was filtered and the filtrate was acidified to a pH of 

less than 3 to precipitate the HAs. The resulting HA is dried, pulverized in glass mortar pestle 

and stored in dessicator. 

Vol. 3 Page - 351 -

Preparation of inclusion complexes. Complexes of Aspirin-FA in the molar ratio of 1:0.5, 1:1 

and 1:2 were prepared by using solvent evaporation (rota evaporator), freeze drying 

(lyophilizer) and spray drying (mini spray dryer) methods. 

Characterization of Complexes. The complexes were characterized by using differential 

scanning calorimetry (DSC), X-Ray diffraction (XRD), Fourier transform infra red 

spectroscopy (FT-IR), scanning electron microscopy (SEM) and proton nuclear magnetc 

resonance ( 1 HNMR) methods. 

Release study of aspirin from their complexes. The sample corresponding to 100 mg of aspirin 

were placed in hard gelatin capsules. Dissolution medium was acetate buffer (pH 4.5). The 

stirring speed was 50 rpm and temperature 37 ± 0.5 ºC. 5 mL samples were withdrawn at a 

settled time interval using a syringe and analyzed by HPLC method. 

Stability studies. All the complexes and ASA alone were packaged in well labeled sealed 

polythene lined aluminium pouches and stored in stability chamber at 40 ± °C and 75 ± 5% 

RH for 120 days. Samples were analyzed by HPLC for salicylic acid content at 0, 30, 60, 90 

and 120 days. 

Drug Permeation Study Across Rat Everted Gut Sac. In order to study the effect of 

complexation on the intestinal permeability of aspirin and the permeability of aspirin-FA 

spray dried complex (1:1) was compared with aspirin alone by the rat everted gut sac 

technique. 

Anti-Inflammatory and Gastric Ulceration studies. Anti-inflammatory activity was performed 

using carrageenan induced rat hind paw edema model. However, pyloric ligation ulcer model 

was used for gastric ulceration study. 



Inclusion complex formation resulted in the production of an amorphous powder with 

improved solubility, dissolution, permeability and stability of aspirin. Aspirin-FA system 1:1 

spray dried complex was optimized according to spectral characterization, in vitro release and 

stability studies. It is evident from the results that the complexation showed a significant 

increase in the solubility of the drug, with the maximum increase in solubilization is observed 

in the case of spray dried (1:1) ASA-FA complex (43 times) as compared to aspirin alone in 

0.1 M HCl. The dissolution data indicated only 31.32% release was obtained with aspirin 

alone at 30 minutes and a maximum of 99.7% release was obtained from 1:1 spray dried 

fulvic acid complex in 25 minutes. The overall profile of ASA degradation in the complexes 

Vol. 3 Page - 352 -

of fulvic acid were studied at 40 ± 2 °C and 75 ± 5% RH for 120 days as indicated by the rate 

of appearance of salicylic acid. However, content of salicylic acid 4.31% was determined 

after 120 days. The permeation of aspirin from aspirin -fulvic acid complex (1:1) prepared by 

spray drying was found to be significantly higher (about 8 times) as compared to aspirin 

alone. The anti-inflammatory effect of aspirin caused an inhibition of 22.92 % after four hours 

while their optimized complex of fulvic acid inhibited edema 35.4% after four hours of 

treatment. The spray dried complex prepared with fulvic acid (1:1) gave the lowest score of 

ulcer index; 0.48 ± 0.08 as compared to aspirin alone 1.12 ± 0.08. Tablets prepared with 

aspirin-FA (1:1 spray dried complex) have greater dissolution as compared to marketed 

formulation containing aspirin in an uncomplexed form. The optimized tablets of aspirin-FA 

complex wase subjected to accelerated stability studies to ascertain the chemical and physical 

stability of formulations. The optimized tablets were kept at 40 0 ± 0.5 0 C and 75% ± 5% RH. 

No significant changes in properties like hardness and disintegration time of formulation was 

observed. 



Figure 1: Comparative stomach lesions after treatment 

Desirable enhancement in dissolution and stability of aspirin can be achieved through FA 

complexation. However, such a novel approach appears to be beneficial to overcome the 

problem of poor bioavailability. A highly significant anti-inflammatory and anti-ulcerogenic 

action were evidenced by the treatment of optimized complex. This has potential for industrial 

application in developing and improved dosage form of aspirin 

Vol. 3 Page - 353 -


References 

1. S. Ghosal, Shilalit: its origin and significance. Indian. J. Indg. Med 9 (1992) 1. 

2. S.P. Agarwal, R. Khanna, R. Karmarkar, M.K. Anwer, and R. Khar, Shilajit: A Review. 

Phytother. Res. 21 (2007) 401. 

3. M.K. Anwer, S.P. Agarwal, A. Ali and Y. Sultana, Influence of fulvic acid and hydroxy propyl-βcyclodextrin 

on aspirin degradation”. Drug dev Indus Pharm. 2009, in press. 

4. R. Khanna, M. Witt, M.K. Anwer, S.P. Agarwal, and B.P. Koch, Spectroscopic characterization of 

fulvic acids extracted from the rock exudate Shilajit. Org. Geochem. 39 (2008) 1719. 

Vol. 3 Page - 354 -

The Effect of Fulvic and Humic Acid Supplementation on the Intensity of 

the Immune Response in Rats 

Vucskits A. V. a* , Hullár I. a , Andrásofszky E. a , Hetényi N. a , Csicsor J. b , Móré A. c , Szabó J. a 

a Department of Animal Breeding, Nutrition and Laboratory Animal Science, Faculty of 

Veterinary Science, Szent István University, H-1077 Budapest, István u. 2, Hungary; b Organit 

Ltd., H-8175 Balatonfűzfő, Ipari Park hrsz.: 1498/278; c Alpha-Vet Ltd., H-8000 

Székesfehérvár, Homoksor 7 

E-mail: vucskits.andras@aotk.szie.hu 


The immune system is one of the most important mechanisms for preserving the health of the 

livestock animals. There are a lot of chemical substances that can enhance the immune 

response, such as n-3 and n-6 polyunsaturated fatty acids (PUFA) [7], arginine [2], vitamins 

[9], microelements [1, 4], etc. Humic substances [HSs] are new candidates in the field of 

immune-based nutrition. It has been known that HSs have immune stimulating properties [6]. 

According to the latest findings HSs might help to inhibit the infectivity of certain diseases 

(i.e. HIV) [3]. It is important to state that HSs are not chemically homogenous. Their two 

most important ingredients are humic acid (HA) and fulvic acid (FA). Humic acid is soluble 

in water under alkaline conditions and has a higher molecular weight than FA. It is 

theoretically not absorbed from the intestines. Fulvic acid is soluble in water under all pH 

conditions and it is absorbed well from the intestines [5]. The aim of this experiment was to 

investigate the effect of purified FA and HA - originated from Dudarite - on the humoral 

immune response of rats. 



Thirthy Wistar CRL:[WI] BR, female, SPF rats were used in the experiment. After 4 days of 

adaptation animals were randomly divided into 3 dietary treatment groups (10 animals in each 

group) on the basis of their bodyweight. The diets were composed according to the AIN-93G 

formula of the American Institute of Nutrition [8]. One group received the control diet, 1–1 

treatment groups received FA 0.4% or HA 0.4% supplemented diets respectively. Animals 

were housed in individual cages at 24 °C ambient temperature. Drinking water and diet was 

provided ad libitum during the experiment. On the second day of the experiment animals were 

immunized with ovalbumin incorporated into Freund's incomplete adjuvant (animals were 

injected with 150 µg ovalbumin, 150 µL incomplete freund’s adjuvant 150 µL PBS 

containing suspension sc.) On the 26th day of the experiment animals were euthanized (90 

mg/BW CP Ketamin and 0.5 mg/BW Medetomidin) and insanguinated. Enzyme-linked 

Vol. 3 Page - 355 -

immunosorbent assay (ELISA) was used to analyze the antibody titer of the serum samples. 

Histometrical analysis was carried out on the mucus membrane of the small intestine and the 

lymphoid cell zones if the spleen of 9 animals (3 animals from each group). 

3. Results 

Control FA HA 

Geometrical mean 2. week 709.68 1203.27 1269.92 

Geometrical mean 4. week 544.32 1969.83 1600.00 

Antibody titer against ovalbumin 

Histometrical analysis shows that the size of the lymphoid follicles (average size of the 

centrum germinativum, consisting of lymphoblast cells) in the mucus membrane of the ileum 

was much bigger (800–900 μm) in the FA and HA supplemented groups than in the control 

group (300–400 μm). The average thickness of the marginal (“B”-dependent) cell-zone was 

151–200 μm in the FA and HA supplemented group. This value was 100–150 µm in the 

samples of the control group. 

4. Discussion 


The immunological and histometrical results show that the 0.4% supplementation of either 

fulvic or HA has a strong immune-stimulatory effect. Our results also indicate that this effect 

is mainly focused on the humoral immune response. The antibody titer analysis shows that 

both FA and HA increase the longevity of the immune response, since the antibody titer 

results of the 4th week are higher in the fulvic and HA supplemented groups than at 2 weeks. 

Evaluating the results of the histological and the ELISA examinations together, it can be said 

that both FA and HA are strong humoral immune stimulants and they also increase the 

persistence of antibodies in the system. 

Acknowledgements: The financial support of the Hungarian Scientific Research Fund 

(OTKA, T 049116) is greatfully acknowledged. 

References 

1. A. Favier, Annales Pharmaceutiques Françaises (2006) 64, 390–6. 

2. B. Lewis, B. Langkamp-Henken, J. Nutrition (2000) 130, 1827–1830. 

3. C. E. van Rensburg, T. L. Smith, E. J. van Rensburg, J. Schneider, Chemotherapy (2002) 48, 138– 

143. 

4. K. E. Saker, Vet. Clin. N. A.: Small Animal Practice (2006) 36, 1199–224. 

5. K. M. S. Islam, A. Schumacher, J. M. Gropp, Pak. J. Nutr. (2005) 4, 126–134. 

6. N. Lange, S. Golbs, M. Kühnert, Archiv für experimentelle Veterinärmedizin (1987) 41, 140–146. 

7. P. C. Calder, Lipids (2001) 36, 1007-24 

8. P. G. Reeves, J Nutrit, (1997) 127, 838S-841S. 

9. V. Badmaev, M Majeed, R. A. Passwater, R. A., Alternative Therapies in Health and Medicine 

(1996) 2, 59-62, 65–7 

Vol. 3 Page - 356 -

Evaluating Potential Nephrotoxicity of Compost Derived Humic Acid to 

African Mud Catfish (Clarias Gariepinus) Grown in Static Water Culture 


Iheoma Mary Adekunle* and Olawale Razaq Ajuwon 

Department of Environmental Management and Toxicology, 

University of Agriculture, P.M.B. 2240, Abeokuta, Nigeria 

E-mail: imkunle@yahoo.com 

Composting of organic solid waste materials is seen as an economically feasible waste 

management option in Nigeria and the resulting products (solid and liquid organic fertilizers) 

are utilized in organic farming and horticultural purposes for urban greening in order to boost 

food security, combat global warming and consequent climate change. These organic 

fertilizers inherently contain humic acid (HA), a type of humic substance, which exists in 

decomposed natural organic matter, compost, peat, lignite, soil organic matter, sediment and 

all classes of water (groundwater and surface water). HA consists of closely related complex 

aromatic polymers and oxygenated functional groups but the exact composition varies with 

geographic location amongst other factors. On application of the organic fertilizers to the soil, 

fate processes such as leaching could transfer some humic substance fractions to the 

surrounding water bodies thereby adding to the existing dissolved humic matter in the 

receiving waters and HA is reported to influence a variety of processes in aquatic 

ecosystem[1]. There are no readily available data on the toxicological effect(s) of HA 

isolated from composted organic wastes of Nigeria origin to aquatic organisms. This study, 

therefore, evaluated the potential nephrotoxicity of HA isolated from source segregated 

composted municipal solid wastes generated in Abeokuta, southwest Nigeria, to African mud 

catfish (Clarias gariepinus) grown in static water culture. 



Source separated municipal solid wastes generated from Abeokuta city, Nigeria, consisting 

largely of vegetable matter, crop and food residues were composted for 75 days via in-vessel 

technique after the procedure of [2]. Humic acid was extracted from the cured product using 

alkaline method, purified and characterized using the method of [3] with slight modification. 

Three prognostic markers of kidney disease: plasma creatinine, urea and albumin levels of C. 

garipinus exposed to different concentrations of HA (0, 100, 250, 500 and 1000 mg/L) in 

controlled water culture for 45 days, were determined using standard clinical techniques. 

Vol. 3 Page - 357 -

Creatinine and urea were determined using Cromatest kits while albumin was determined 

using Randox test kit. 

Twenty five juvenile fishes of average weight and length 43.2 ± 0.5g and 18.6 ± 0.8cm 

respectively, fed with 2 mm copen fish feed, were utilized for the experiment at 5 per 

aquarium of 45 L capacity. Temperature, electrical conductivity, nitrate, phosphate, dissolved 

oxygen, total hardness and alkalinity of the process water were also analyzed using standard 

procedures. Data obtained from study were subjected to one-way analysis of variance and 

Pearson correlation using SPSS 15.0. 



Humic acid characteristics: Infrared spectra and results from volumetric analyses showed 

oxygenated functional groups and hydrocarbon skeleton, indicating content of phenolic 

hydroxyl and carboxyl reactive sites, similar to the humic acid properties reported in our 

previous published works [3,4,5]. The process water was suitable for aquaculture (Table 1) 

and conforms to the reports of [6]. 

Serum album: Albumin level in the control group was 0.87 ± 0.19 g/dL and ranged from 0.43 

to 0.87g/dL in the test groups. Correlations gave negative coefficients for albumin versus HA 

concentrations (- 0.114; p > 0.10), indicating albumin depletion (Fig.1) with increasing HA 

concentration in water. Serum albumin is the most abundant plasma protein in humans and 

other mammals, being essential for maintaining the osmotic pressure needed for proper 

distribution of body fluids between intravascular compartments and body tissues. It also acts 

as a plasma carrier by non-specifically binding several hydrophobic steroid hormones and as a 

transport protein for hemin and fatty acids. Albumin (when ionized in water at pH 7.4, as 

found in the body) is negatively charged. The glomerular basement membrane is also 

negatively charged in the body and some studies suggest that this prevents the filtration of 

albumin in the urine. According to this theory, charge plays a major role in the selective 

exclusion of albumin from the glomerular filtrate [7,8]. A defect in this property results in 

nephrotic syndrome leading to albumin loss in the urine. Decreased serum albumin as found 

in this study, may thus be a sign of kidney disease. 

Creatinine: The creatinine level in the control group was 0.36± 0.09 mg/dL and ranged from 

0.20 to 1.53 mg/dL in the test groups. Positive correlation was obtained for creatinine and 

HA concentrations (+ 0.704; p > 0.10), indicating rising blood levels of creatinine (Fig.1) with 

increasing HA concentration in water. Creatinine is produced naturally by the body, being a 

Vol. 3 Page - 358 -

eak-down product of creatine phosphate, which is found in muscle. It is freely filtered by 

the glomerulus. There is little-to-no tubular reabsorption of creatinine. If the filtering of the 

kidney is deficient, blood levels rise [7, 8]. Results from this study are thus predictive of renal 

dysfunction. 

Figure 1: Albumin, creatinine and urea concentrations in C. gariepinus in relation to humic acid 

concentrations in water 

Urea: The urea level in the control group was 5.61 ± 0.07 mg/dL and ranged from 1.95 to 

5.21 mg/dL in the test groups. Correlations gave negative coefficients for urea versus HA 

concentrations (- 0.586; p > 0.10), indicating a decreasing trend (Fig.1) with increasing HA 

concentration in water. Urea is a break down product of amino acid catabolism. Similar to the 

case of creatinine, if kidneys are not able to remove urea from the blood, the level rises, 

predicting nephrotic dysfunction [7, 8] as was found in this study. 

Inter-biomarker ratios: The ratios are presented in Table 1. 

Table 1: Quality of process water used in the aquaria and ratios of the nephrotoxic biomarkers 

Water quality 

Temperature 27 o C 

pH 6.4 

PO4 3- 

NO3 - 

Total 

hardness 

Total 

alkalinity 

Dissolved 

oxygen 


Biomarker ratios 

HA (mg/L) Ur-Cr Cr-Ur Alb-Cr Cr-Alb Alb-Ur Ur-Alb 

20 mg/L 0 15.58 0.064 2.42 0.414 0.155 6.45 

ND 100 26.05 0.038 4.20 0.238 0.161 6.20 

154 g/L 250 7.00 0.143 1.00 1.00 0.143 7.00 

80 mg/L 500 1.27 0.785 0.38 2.040 0.385 2.60 

80 mg/L 1000 2.39 0.419 0.55 1.813 0.231 4.33 

Ur – urea, Cr- creatinine, Alb- albumin, ND – not detected 

Vol. 3 Page - 359 -

A more complete estimation of renal function can be made when interpreting the blood 

(plasma) concentration, using the ratios. This can indicate other problems besides those 

intrinsic to the kidney. As an illustration, a urea level raised out of proportion to that of 

creatinine may indicate a pre-renal problem such as volume depletion [7, 8]. 



The present results show that exposure of C. gariepinus to up to 100 mgHA/L did not evoke 

significant adverse effects on the three biomarker levels in the blood but beyond this 

concentration, from 250 to 1000 mg/L, significant nephrotic dysfunction was predicted from 

albumin and creatinine levels but based on urea levels, nephrotoxicity was not induced. Study 

showed that nephrotic syndrome was HA concentration dependent, indicating safety at levels 

≤ 100 mg/L, which lie in an environmentally realistic range. 

References 

1. F.J. Stevenson, Humus Chemistry, Wiley, New York: 1994. 

2. I.M Adekunle, Temperature effect on water extractability of cadmium, copper, lead and zinc from 

composted organic solid wastes of South-West Nigeria, Int. J. Environ. Res. Public Health 2009, 

6, 2397-2407; doi:10.3390/ijerph6092397 

3. I.M. Adekunle ., T.A. Arowolo., N.P. Ndahi, B. Bello and D.A. Owolabi, Chemical characteristics 

of humic acids in relation to lead, copper and cadmium levels in contaminated soils of southwest 

Nigeria, Annals of Environmental Science, North Eastern University, Boston, Massachusetts USA, 

2007, 1, 23-34. 

4. I.M. Adekunle., S.O. Olagundudu and O.O. Ogunleye, Influence of humic acid on plant metal 

uptake and translocation. Frimmel, F.H., Abbt-Braun, G. (Eds). Proceedings of the 13 th 

International Conference of the International Humic Substance Society (IHSS), held at University 

of Karlsruhe, Germany, July 30 to August 4, 2006, 45 (I), 433-436. 

5. I.M. Adekunle., O.A. Akinola and M.A. Kuyoro, Role of humic acid on metal accumulation in 

plant tissue. Frimmel, F.H., Abbt-Braun, G. (Eds). Proceedings of the 13 th International 

Conference of the International Humic Substance Society (IHSS), held at University of Karlsruhe, 

Germany , July 30 to August 4, 2006, 45 (II), 817- 820. 

6. I.M. Adekunle., T.A. Arowolo., I.T. Omoniyi and Olubambi. O.T, Risk assessment of nile tilapia 

(Oreochromis niloticus) and African mud catfish (Clarias gariepinus) exposed to cassava effluent 

(2007). Chemistry and Ecology 23 (5), 383 -392. 

7. National Kidney Foundation. K/DOQ1 clinical practice guidelines for chronic kidney disease: 

evaluation, classification and stratification. Am J Kidney Dis 2002, 39 (Suppl 1):S1-266. 

8. Spencer K. Analytical reviews in clinical biochemistry: the estimation of creatinine. Ann Clin 

Biochem 1986, 23,1-25. 

Vol. 3 Page - 360 -


Treatment of Pilonidal Sinus by Salts of Humic Acid 

Mümin Dizman 

Oluşum Kimya, Kandıra, 41633, Turkey 

E-mail: mumindizman@hotmail.com 

This study relates to application of curing material containing humic acid and its salts for pilonidal 

sinus disease. In this study humic acid and sodium humate or potassium humate provide to cure 

pilonidal sinus disease by applying topically to skin without any need of surgical application. Humic 

acid containing polyphenol is produced from Afşin-Elbistan lignite gyttja [1]. This treatment method 

guarantees and provides to cure patient with the logic of “healing of intermittent wound” without 

affecting the daily life standard and recurrence probability. There is no study related with pilonidal 

sinus disease which can be rubbed with agent or avoided by a material from the skin. There was no 

other alternative solution suggested except from phenol and silver nitrate (AgNO3) because the only 

medical treatment is known as surgical intervention up until now. 


Humic acids, sodium humate or potassium humate are used as a cosmetic product to solve out 

pilonidal sinus problem. Humic acids and salts that include polyphenolic components are 

prepared from the extraction of Afşin-Elbistan gyttja with sodium hydroxide or potassium 

hydroxide at 70-150 degrees centigrade, pH between 10 to 12 interval and in 12-24 hours with 

continuously stirring. Humic acid originated polyphenols are effective as the products that is 

found as anti-viral, anti-microbial active agent for preventing and reducing microbes and 

viruses inside the human skin [2]. 



Polyphenols that are originated from humic acid treat pilonidal sinus as healing of intermittent 

wound principle by supporting phagocytosis [8] and building up collagen synthesis [3]. If the 

deficient factors in condition deforming wound healing are submitted to wound, healing will 

increase [4]. Because of very high collagen synthesis effect of humic acid polyphenols, 

treatment of damaged skin is quickened [7]. With the impact of the humates containing 

polyphenol hydroxyls, building up three units of α-bond is strengthened before the formation 

of collagen [5]. At this point, three collagen α-bond encounters alteration period. This period 

includes hydroxylization of lysine amino acids by polyphenols and formation of fibrin 

(glicosylation). Polyphenols quickens the linking of α-bonds as knits [6]. This knit is trio 

Vol. 3 Page - 361 -

spiral curve called procollajen monomer. Propeptids of N- and C-terminal are separated by 

the effects of polyphenols to special peptidase enzymes. As a result, processed collagen starts 

to make multiple molecular clusters. These clusters are aligned end to end so as to form 

striped fibers. Cross-links between the special amino acids determines and provides stretching 

resistance of collagen fibers. Skin enriched by the collagen provides to eliminate damaged 

cell by its flexible tough character [7]. 


One hundred-and ninty two patients with chronic and acute pilonidal sinus were treated by a 

new chemical technique [9]. It was found to be simple, time-saving and to minimize the 

postoperative morbidity and without need to stay in hospital. Results of this new technique 

were compared with those of other methods in the literature and were found to be superior to 

them with a minumum recurrence. 



I thank to Prof. Dr. Ahmet Tutar, Department of Chemistry, University of Sakarya, for his 

assistance in the preparation of this article. 

References. 

1. Mümin Dizman, Turkish Patent 2007 00973. 

2. Ricard J. Laub, Laub Biochem. Corp., US Patent 6,534,049. 

3. Gert J. van Klinken and Robert E. M. Hedges, Experiments on Collagen-Humic Interactions: 

Speed of Humic Uptake, and Effects of Diverse Chemical Treatments, Radiocarbon Accelerator 

Unit, University of Oxford, September 19 ,1994, Abstract. 

4. Dr. Sevda CİĞER, Alsancak Devlet Hastanesi Dermatoloji Kliniği Uzmanı, Yara İyileşmesi ve 

Büyüme Faktörleri, www.dermaneturk.com/yara_online/buyume_faktor.doc, p.6. 

5. Scutt, S. Meghji, J. P. Canniff and W. Harvey, Cellular and Molecular Life Sciences, Volume 43, 

Number 4/April, 1987, p.391. 

6. Tamara Stipcevic, Jasenka Piljac and Dirk Vanden Berghe, Plant Foods for Human Nutrition, 

Volume 61, Number 1/March, 2006, p.27. 

7. U. N. Riede, I. Jonas, B. Kirn, U. H. Usener, W. Kreutz and W. Schlickewey, Archives of 

Orthopaedic and Trauma Surgery, Volume 111, Number 5 / September, 1992, p.259. 

8. Inglot AD, Zielińska-Jenczylik J, Sypuła A., Arch Immunol Ther Exp (Warsz). 1993; 41(1):87. 

9. Hasan Mete Aksoy, Berna Aksoy, Didem Egemen, Effectiveness of topical use of natural 

polyphenols for the treatment of sacrococcygeal pilonidal sinus disease: a retrospective study 

including 192 patients, European Journal of Dermatology; Volume 1, Number 1, October 2008. 

Vol. 3 Page - 362 -

Stabilization of Iron Oxide Magnetic Nanoparticles with Different 

Morphology in Aqueous Suspensions Using Humic Substances 

A.Yu. Polyakov a , A.E. Goldt a , T.A. Sorkina b , E.A. Goodilin a, b , I.V. Perminova b* 

a Department of Material Science, Lomonosov Moscow State University, 119991, Moscow, 

Leninskie Gory 1, building 73, Russia; b Department of Chemistry, Lomonosov Moscow State 

University, Leninskie Gory 1-3, 119991, Moscow, Russia 

E-mail: iperminova@gmail.com; iperm@org.chem.msu.ru 


A use of magnetic liquids for therapy of cancer diseases is an important branch of modern 

biomedical technologies. The magnetic nanoparticles suitable for those applications should be 

biocompatible and stable in aqueous suspension. Ferric oxides represent one of the most 

biocompatible magnetic phases which forms a variety of different morphologies. 

Humic acids (HA) are a complex mixture of natural macromolecular compounds with vast 

functional periphery dominated by carboxyl and hydroxyl groups. They possess 

polyelectrolite properties and distinct surface activity. Thereupon, they can bind nanoparticles 

both by electrostatic surface interactions and by complex formation [1]. Humic acids were 

reported to be suitable stabilization agents for magnetite (Fe3O4) suspensions [2]. However 

Fe3O4 could be oxidized to Fe III -compounds in physiological medium and this can lead to 

adverse consequences. At the same time δ-FeOOH and γ-Fe2O3 do not have such 

shortcomings and possess necessary magnetic properties. 

Thereupon, the objective of this work was to synthesize feroxyhyte (δ-FeOOH) and 

maghemite (γ-Fe2O3) nanoparticles and to study the stability of their suspension in the 

presence of HAs. 



Synthesis and characterization of iron oxide nanoparticles. Nanoparticles of δ-FeOOH and 

γ-FeOOH were synthesized by oxidation of “green rust” under different conditions. 

Nanoparticles of γ-Fe2O3 were obtained by annealing of γ-FeOOH at 200-250 o C. Phase 

composition of obtained nanoparticles was proven by X-ray phase analysis (Rigaku 

D/Max-2500 diffractometer). Morphology of the nanoparticles was characterized by using 

Hitachi H-8100 transmission electron microscope (TEM). Magnetic properties of synthesized 

compounds were studied by using Faraday balance magnetometer. 

Vol. 3 Page - 363 -

Preparation of humic materials and suspensions. Humic materials used in this work were 

HAs isolated from leonardite. The HA were dissolved in 1M NaOH under ultrasonic 

treatment. Then the solution was diluted with distilled water and pH was set to 7.00–7.05. 

Magnetic nanoparticles were added directly into HA solutions with subsequent ultrasonic 

treatment. Suspensions were kept at 4–5 o C and their stability was monitored. Suspensions of 

the nanoparticles in distilled water were used as blank experiments. 

Iron (III) concentration in supernatant was monitored as main parameter of suspensions 

stability. Sample preparation was carried out according to conventional thiocyanate technique 

[3]. Optical density were measured by Varian Cary 50 Probe spectrophotometer at λ = 480 

nm. 

Size of colloidal particles and salt tolerance of suspensions were tested by using Zetasizer 

(Malvern, UK) apparatus based on dynamic light scattering (DLS). 



Synthesis of magnetic nanoparticles. Two different morphologies were investigated in this 

work. We have synthesized δ-FeOOH nanospheres (average diameter ~ 30–40 nm, associated 

into 200 nm aggregates) and γ-Fe2O3 nanorods (~ 200–250 nm length, ~ 10–15 nm width) 

(Fig. 1). 

a) b) 

Figure 1: TEM images of δ-FeOOH – a) and γ-Fe2O3 – b) 

Nanoparticles of both iron oxides synthesized in this work were monophase as determined by 

X-ray phase analysis. Monophase δ-FeOOH has lattice parameters a=2.956(2); c=4.519(3) 

and monophase γ-Fe2O3 has lattice parameter a=8.3395(3). 

Magnetic measurements show that both δ-FeOOH and γ-Fe2O3 display ferromagnetic 

properties. Feroxyhyte δ-FeOOH has saturation magnetization (Ms) ~ 18 emu/g and coercive 

force Hc = 110 Oe. Maghemite γ-Fe2O3 has Ms = 45 emu/g and Hc = 182 Oe. At the same 

time magnetite (Fe3O4), which often is synthesized for hyperthermia experiments, has 

Vol. 3 Page - 364 -


saturation magnetization up to 190 emu/g and coercive force from 32 to 65.5 Oe [4]. Thus, the 

synthesized nanoparticles possess magnetic properties comparable to those of magnetite and 

are suitable for biomedical application. 

Study of magnetic suspensions stability in water. During storage magnetic suspensions 

gradually coagulate and large colloid particles precipitate. Thereupon, stability of the prepared 

suspensions was characterized by measurements of iron (III) concentration and colloidal 

particles size in supernatant. 

The stabilisation of the obtained nanoparticles with HAs was studied in the broad range of 

concentrations. It was shown that the concentration of 100 mg/L of HA in solution provides 

the best conditions for stabilization of synthesized magnetic phases. At this concentration, up 

to 14.7 mg/L of iron (III) in the form of feroxyhyte nanoparticles were still stabilized after 4 

days of observation (Fig. 2a). At the same time stabilization of maghemite nanoparticles was 

much worse (Fig. 2b). The difference between two oxides could be explained by the presence 

of large amount of hydroxyl groups on surface of feroxyhyte nanoparticles, whereas hydroxyl 

groups on maghemite surface are lost during annealing. In addition, γ-Fe2O3 nanoparticles 

have rather big size and shape anisotropy. Thus, morphology of magnetic nanoparticles and 

presence of surface hydroxyl groups have a significant impact on stability of suspensions. 

a) b) 

Figure 2: Content of stabilized iron in presence of HA: a) – δ-FeOOH nanospheres, b) – γ-Fe2O3 

nanorods. 

The DLS data show that nanoparticles quickly aggregate in suspensions without HA, whereas 

the presence of HAs sustains initial size of dispersed nanoparticles. Of particular importance 

is that the size of humic colloids without and with iron oxide nanoparticles does not differ 

significantly. This allows for suggestion that magnetic nanoparticles are captured into 

branched structure of HAs which play a role of “nanocontainer” (Fig. 3). 

Vol. 3 Page - 365 -

Figure 3:Supposed mechanism of stabilization of magnetic nanoparticles suspensions by HA 

Magnetic suspensions for biomedical applications must be stable in physiological salt 

solution, whereas ionic strength has a significant impact on stability of suspensions [2]. 

Thereupon, we have characterized salt tolerance of the prepared magnetic suspensions by 

coagulation kinetic measurements in the presence of different concentrations of NaCl. 

Suspensions stabilized by HAs are stable in presence of 150 mmol/L NaCl (concentration of 

physiological salt solution); whereas suspensions of magnetic nanoparticles in distilled water 

possess very low salt tolerance (~ 30 mmol/L NaCl). 



Characterization of magnetic suspensions stability displays that HAs are suitable stabilization 

agent for iron oxide nanoparticles particularly for feroxyhyte (δ-FeOOH) and maghemite 

(γ-Fe2O3). Measurements of iron (III) concentration in supernatant of suspensions show that 

feroxyhyte isotropic (spheroidal) nanoparticles with large amount of hydroxyl groups at their 

surface are stabilized much better than anisotropic annealed maghemite nanorods. According 

to salt tolerance measurements magnetic suspensions stabilized by HAs are stable in 

physiological salt solution. These results show a good promise for development of new 

biologically active magnetic preparations based on ferric-humic interactions. 

References 

1. R.S. Swift, In: Humic Substances II. Hayes M.H.B., MacCarthy P., Swift R.S. (Eds.) John Wiley 

& Sons Ltd., 1989. p. 468-495. 

2. E. Illes, E. Tombacz, The effect of humic acid adsorption on pH-dependent surface charging and 

aggregation of magnetite nanoparticles, Journal of Colloid and Interface Science, 295 (2006), 1, 

115-123. 

3. Pa Ho Hsu, Determination of Iron with Thiocyanate, Soil Sci. Soc. Am. J., 31 (1967), 353–355. 

4. Dong-Lin Zhao, Xian-Wei Zeng, Qi-Sheng Xia, Jin-Tian Tang, Preparation and coercivity and 

saturation magnetization dependence of inductive heating property of Fe3O4 nanoparticles in an 

alternating current magnetic field for localized hyperthermia, Journal of Alloys and Compounds, 

469 (2009), 215–218. 

Vol. 3 Page - 366 -

Neutralisation of the Anticoagulant Effect of Naturally Occurring Humic 

Acids and Synthetic Humic Acid-Like Polymers by Protamine Sulfate 

Hans-Peter Klöcking a *, Nicolle Mahr a , Susanne Kunze a , Renate Klöcking b 

a University of Jena, Institute of Pharmacology and Toxicology/Working Group Erfurt, 

Nordhäuser Str. 78, D-99089 Erfurt, Germany; b Zittau/Görlitz University of Applied 

Sciences, Research Institute for Peat and Natural Products, Theodor-Körner-Allee 16, 

D-02763 Zittau, Germany 

E-mail: hpkloecking@gmx.net 


Previous studies have shown that negatively charged polymers such as naturally occurring 

humic acids (HA) and synthetic HA-like polymers prolong the clotting time of blood in vitro 

and in vivo [1, 2]. This anticoagulant effect is based on their ability to inhibit the coagulation 

factors IIa, VIIa and Xa [3]. Bleeding induced by unfractionated heparin (UFH) can be 

antagonized by protamine sulfate as shown by normalisation of thrombin time and aPTT [4]. 

Protamine sulfate is a positively charged polypeptide widely used to reverse heparin-induced 

anticoagulation by neutralisation negatively charged groups [5]. 

In order to learn about the neutralisation capacity of protamine sulfate against the anticoagulant 

effects of HA and synthetic HA-like polymers, an in vitro study was performed. 



Test substances: Nine negatively charged polymers of different origin were employed: Peat 

humic acid A1 (PAH-A1) and peat humic acid A2 (PAH-A2) isolated according to Kirsch [6] 

from the Altteich Moor in Saxony (Germany); sodium humate (NaHS) isolated according to 

Klöcking et al. [7] from a rainmoor peat of the coastal region of Mecklenburg-Vorpommern 

(Germany); commercially available brown coal humic acid (Aldrich HA; Sigma-Aldrich 

Chemie GmbH, Steinheim, Germany); the synthetic HA-like polymer KOP 409/85 (caffeic 

acid oxidation product) [8]; Melanoidin M1 synthesized from glycine, phenylalanine and 

xylose and Melanoidin M42 synthesized from glutamic acid and xylose, both prepared by 

Pompe et al. [9]; HS 5, an oxidation product of hydroquinone and glycine, and HS 136, an 

oxidation product from hydroquinone and L-lysine, both prepared by Herdering [10]. 

Biochemicals: Thrombin was obtained from Kallies Feinchemie AG Sebnitz, Germany; 

Fibrinogen (Haemokomplettan ® ) from Behringwerke Marburg, Germany, and protamine 

sulfate from Merck, Darmstadt, Germany. 

Vol. 3 Page - 367 -

Test procedure: 50 µl protamine sulfate solution over a concentration range of 0,01 to 100 

µg/ml, 100 ml fibrinogen solution (1%) and 50 µl of a fixed test substance concentration 

which has to exceed the concentration necessary for doubling thrombin time of the control, 

were incubated for 2 minutes at 37°C. After this, 50 µl thrombin solution (3 NIH/ml) were 

added to start the coagulation process. The control value of thrombin time was determined in 

the same way; instead of protamine sulfate solution, 50 µl Tris buffer pH 7.4 were added. The 

coagulometer CL4 (Behnk Elektronik, Norderstedt, Germany) was used for all the 

measurements. 


We started our experiments with investigating the influence of protamine sulfate (0.01-100 

µg/ml) on the thrombin-fibrinogen reaction (Fig. 1). Concentrations below 7 µg/ml protamine 

sulfate did not influence the thrombin-fibrinogen reaction. Higher protamine sulfate 

concentrations caused a thrombin time-shortening activity (procoagulant effect), this mean 

that they could not be used in the neutralisation experiment. 

Thrombin time (s) 

50 

45 

40 

35 

30 

25 

20 

15 

10 

5 

0 

0 0,01 0,1 0,5 1 3 5 7 10 20 40 60 80 100 

Protamine sulfate (µg/ml) 


Figure 1: Influence of protamine sulfate on the thrombin-fibrinogen reaction, n = 4 

In the next experiment, we investigated the effect of protamine sulfate on the thrombin time- 

prolonging activity of HA and HA-like substances. As shown in Table 1, protamine sulfate 

produced varying degrees of neutralisation of the tested HA and HA-like polymers. The 

thrombin time-prolonging activity of NaHS, Aldrich HS, KOP, Melanoidin type M1 and 

M42, HS 5 and HS 136 was completely inhibited by protamine sulfate. The ratios differ from 

1 : 0.2 up to 1 : 1.3 in accordance with the anticoagulant activity of the test substances. HS 5 

Vol. 3 Page - 368 -

Thrombin time (s) 

140 

120 

100 

80 

60 

40 

20 

0 


Blank 0 0,02 0,2 2 

Protamine sulfate (µg/ml) 

Figure 2: Partial neutralisation of the anticoagulant effect of humic acid PHA-A1 (200 µg/ml) by 

protamine sulfate, n = 3 

Table 1: Influence of protamine sulfate on the prolongation of thrombin time by different naturally 

occurring humic acids and synthetic HA-like polymers 

Test substance Substance concen- Test substance Neutralisation ratio 

tration (µg/ml) used for the Test substance : 

necessary for neutralisation protamine sulfate 

doubling clotting time experiment (µg/ml) (w/w) 

PHA-A1 121.9 a) 200 1 : 0.1 

PAH-A2 410.3 a) 600 1 : 0.03 

Na HS 85.7 b) 120 1 : 0.3 

Aldrich HS 28.9 b) 75 1 : 0.4 

KOP 409/85 22.5 b) 30 1 : 0.8 

Melanoidin M1 106.4 b) 130 1 : 0.3 

Melanoidin M42 7.0 b) 25 1 : 0.7 

HS 5 27.2 b) 40 1 : 1.3 

HS 136 135.6 b) 140 1 : 0.2 

a) thrombin-plasma reaction; b) thrombin-fibrinogen reaction 

Vol. 3 Page - 369 -

(1 : 1.3) proved to be the most active substance, in contrast, HS 136 showed only minimal 

activity (1 : 0.2). The thrombin time-prolonging effect of PHA-A1 and PHA-A2 was only 

partially neutralised (70-80 %, see Fig. 2 and Table 1), although the effect of these peat humic 

acids is very weak. 

The ratio between humic acid and protamine sulfate was estimated to be 1 : 0.1 and 1 : 0.03, 

respectively. 



The results suggest that the anticoagulant activities of HA and HA-like polymers can be 

antagonised by protamine sulfate in vitro. These studies warrant further in vivo assessment to 

validate the relative neutralisation profile of HA and HA-like substances. 

References. 

1. H.P. Klöcking, Arch. Toxicol. Suppl. 14 (1991) 166. 

2. H.P. Klöcking, B. Helbig and R. Klöcking, TELMA, 24 (1994) 153. 

3. H.P. Klöcking, N. Mahr, R. Klöcking, K.H. Heise and W. Herdering, in L. Martin-Neto, 

4. D.M.B.P. Milori and W.T.L. da Silva (Eds.), Humic Substances and Soil and Water Environment: 

12 th International Meeting of IHSS, São Pedro, São Paulo, Embrapa Instru-mentação 

Agropecuária, 2004, p. 504. 

5. K. Andrassy, V. Eschenfelder and E. Weber, Thrombosis Research 73 (1994) 85. 

6. A. Racanelli, J. Fareed, J.M. Walenga, E. Coyne, Seminars in Thrombosis and Hemostasis 11 

(1985) 176. 

7. F. Kirsch, Diploma Theses, University of Applied Sciences, Zittau/Görlitz, Germany. 

8. R. Klöcking, B. Helbg, P. Drabke, Pharmazie, 32 (1977) 97. 

9. K.I. Hänninen, R. Klöcking and B. Helbig, Science of the Total Environment, 62 (1987) 201. 

10. S. Pompe, M. Bubner, M.A. Denecke, T. Reich, A. Brachmann, G. Geipel, R. Nicolai, K.H. Heise 

and H. Nitsche, Radiochimica Acta, 74 (1996) 135. 

11. W. Herdering, http://analytik.chemie.uni-hamburg.de/rosig/Modelle.html,1998. Modellsubstanzen 

für ROS. 

Vol. 3 Page - 370 -

Protolytic Properties of Alkoxysilylated versus Natural Humic Materials 

Aimed at Use as Stabilizers for Magnetic Fluids 

Sorkina T. a* , Goldt A. b , Polyakov A. b , Dubov A. b , Toth I. c , Hajdu A. c , Goodilin E. b , 

Tombacz E. c , Perminova I. a 

a Department of Chemistry, Lomonosov MSU, Leninskie Gory 1-3, 119991, Moscow, Russia; 

b Department of Material Science, Lomonosov MSU , Leninskie Gory 1-3, 119991, Moscow, 

Russia; c Department of Colloidal Chemistry, Univ. of Szeged, Aradi Vt. 1, Szeged H-6720 

E-mail: sorkina@org.chem.msu.ru 


Biocompatible magnetic fluids (MF) receive currently a lot of attention due to broad 

applications in biomedical technologies such as hyperthermia, drug delivery, tomography, and 

others. Magnetic fluids of this note are water based colloidal suspensions composed of 

ferromagnetic or superparamagnetic nanoparticles. The main problem to solve is aggregation 

of nanoparticles in aqueous solutions under physiological conditions. The main requirements 

to modifiers are non-toxicity and ability to form stable coating on magnetic nanoparticles. In 

this regard, of particular advantage can be a use of humic substances (HS). Application of HS 

as stabilizing agent for magnetic fluid has been previously reported [1]. However, the humic 

coating obtained was very sensitive to changes in pH and salinity. In this work, the 

proptolytic properties were investigated of native HS against the specifically modified HS 

with incorporated alkoxysilyl-groups providing high affinity of these humic materials for 

mineral surfaces. It was hypothesized that these humic derivatives will form stable coating on 

the surface of iron oxide particles due to formation of Si–O–Fe linkages. The selected 

magnetic nanoparticles were superparamagnetic γ-Fe2O3 and ferromagnetic δ-FeOOH. The 

magnetic nanoparticles possessed different morphology, which will be stable at physiological 

conditions after appropriate coating and possess reliable magnetic properties. 

Thus the goal of the research was to evaluate stabilizing properties of alkoxysilylated humic 

derivative versus natural non-modified HS with respect to magnetic nanoparticles of different 

micromorphology presented by γ-Fe2O3 and δ-FeOOH, and to conclude on their applicability 

for producing biocompatible MF suited for biomedical applications. 



Four samples of HS from different sources were studied as stabilizing agents for MF. Two 

IHSS samples of aquatic SR FA and SR NOM, one sample of natural leonardite humic acids 

(CHA-Pow), extracted from Powhumus (Humintech, Germany) and one sample of modified 

Vol. 3 Page - 371 -

leonardite HA (CHA-APTS-20) were tested as stabilizing agent for biocompatible MF. The 

CHA-APTS-20 was obtained as described in [2] using modification of parent humic materials 

with organosilane 3-aminopropyltriethoxysilane (APTS) in DMF solution. Maghemite 

γ-Fe2O3 nanoparticles were obtained in microspheres using aerosol spray pyrolysis and in 

nanotubes: 200-300 nm long and ~ 10-15 nm thick. Feroxyhyte δ-FeOOH was synthesized in 

the form of spheres with average diameter of ~ 30-40 nm associated into aggregates (~ 200– 

300 nm). 

Humic based MF were obtained by dispersion of iron oxide dry powder in water solution of 

humic samples with pH 7.0–7.15 as described in [3]. The pH-dependent surface charge state 

of HS was determined from acid-base titration under CO2-free condition using background 

electrolytes (NaCl) to maintain the constant ionic strength of 0.01 M as described in [4]. 

The two IHSS samples were dissolved in MQ water; the leonardite samples were converted to 

suspensions using technique described in [5]. Equilibrium titration was performed by means 

of a self-developed titration system (GIMET1) with 665 Dosimat (Metrohm) burets, nitrogen 

bubbling, magnetic stirrer, and high-performance potentiometer. Powder sample was added to 

0.01 M NaCl solution equilibrated with electrolyte to reach a starting pH 3.5. After nitrogen 

purging for 15 min suspensions were titrated by standard NaOH solution up to pH 10.5 and 

then by standard acid solution down to pH 3.5. 

3. Results and discussions 


The set of humic materials used in this study included the samples of aquatic FA and NOM 

with low aromaticity and significant content of aliphatic oxidized structures and the sample of 

leonardite humic acid (CHA-Pow-05) with the highest content of aromatic fragments and 

lowest – of carbohydrate groups and directly modified leonardite humic acid (CHA-APTS-20) 

with 20% modified carboxylic groups. Direct modification of humic backbone using APTS 

increases the affinity of humic materials for mineral surfaces by incorporation of methoxysilyl 

groups into their structure. The latter produce covalent bonds with hydroxyl carrying surfaces 

of silica and metal oxides. Conversion of carboxyl groups into amides can lead to significant 

changes in the acidic properties of HS. 

Furthermore, complexing properties of HS depend on carboxyl groups content. Interaction 

between humic acids and iron oxides occurs via hydroxyl group on the surface of iron oxide 

and acidic groups on HS, so one of the most important parameters is the acidic groups 

content. To characterize acid-base properties of the humic samples used in this study, the 

Vol. 3 Page - 372 -

method of direct potentiometric titration was used. The pH-dependent net proton surface 

excess curves calculated from acid-base titration data are presented in the Fig. 1. 

Net H + excess surface, 

mmol/gC 

2 

0 

-2 

-4 

-6 

-8 

-10 

-12 

-14 

-16 


3 4 5 6 7 8 9 10 11 

pH 

CHA-APTS-20 

CHA-Pow-05 

SR FA 

SR NOM 

Figure 1. pH-dependent net surface H + excess curves for IHSS SR NOM (black rhombuses) and SR 

FA (small white triangles), for the natural leonardite sample CHA-Pow-05 (white rhombuses) and 

modified sample CHA-APTS-20 (grey triangles) 

According to the obtained net proton surface excess vs. pH curves, modified humic material 

has acid-base properties substantially different from those of the parent humic material. The 

negative and positive values of net proton surface excess indicate the presence of negatively 

and positively charged groups such as e.g., deprotoneted –COO - and protonated –NH3 + at the 

given pHs, respectively. In particular, this referred to the appearance of the inflection points 

on the titration curves as well as to the positive values of net proton surface excess. 

Comparing the measured points at pH~8, the given difference might be connected to the 

conversion of carboxyl groups into amide ones, which was caused by the undertaken 

modification. The modified sample had also lower water solubility as compared to the parent 

humic materials that indicates an increase in their hydrophobocity. 

The acidic group content calculated according to Ritchie&Perdue (2003) is given in the Table 

1. The results are presented as quantity of functional group in mmol per mass of carbon in 

samples. 

Table 1: Titration results: humic substances acidic group content 

Sample -COOH, mmol/g C -OH, mmol/gC 

IHSS SR FA 11.57 3.54 

IHSS SR NOM 10.25 4.48 

CHA-Pow-05 6.61 3.52 

CHA-APTS-20 3.32 2.88 

Vol. 3 Page - 373 -

The results of acid-base titration of the parent leonardite humic acids (CHP-Pow-05) and its 

derivative show substantial change in acid-base properties of the modified HS and an increase 

in its hydrophobicity may be caused not only by a significant decrease in –COOH content, but 

also by the presence of residual organic solvent (DMF) in the composition of the obtained 

compound. 

4. Conclusions. 

Natural and modified humic acids are perspective stabilizing agent for water based MF for 

biomedical application. The obtained iron oxides nanoparticles γ-Fe2O3 and δ-FeOOH 

possessed necessary magnetic properties and were suitable for stabilization with HS due to the 

presence of hydroxyl groups on their surface. As APTS modification of parent humic material 

leads to increasing sample’s hydrophobicity and decreasing ability for complexing surface Fe- 

OH sites of iron oxides, efficiency of natural HS as stabilizing agents of water based MF is 

higher in the case of common chemisorption procedure. Directly modified humic samples can 

be applied for preparation of stable humic coating on mineral surfaces in solid organo-mineral 

sorbents. Modification of parent humic material using silicon organic compounds should to be 

provided in other non-toxic and less hydrophobic solvent. At present sample CHA-Pow-05 of 

natural humic acids from leonardite at concentration 100 mg/L have shown to be the most 

effective stabilizing agent against iron oxide nanoparticles in water solutions from tested 

samples. 



Tatiana Sorkina would like to acknowledge the International Humic Substances Society for 

financial support of her stay in the research group of Prof. Etelka Tombacz at the University 

of Szeged (Humgary) within the IHSS Training Award - 2009. 

References 

1. Illés E., Tombácz E. 2006. The effect of humic acid adsorption on pH-dependent surface charging 

and aggregation of magnetite nanoparticles. J Colloid Interface Sci. 295:115–123. 

2. Perminova, I.V., Karpiouk, L.A., Shcherbina, N.S., Ponomarenko, S.A., Kalmykov, St.N. 

Hatfield, K. 2007. Preparation and use of humic coatings covalently bound to silica gel for Np(V) 

and Pu(V) sequestration. J. Alloys Comp., 444–445, 512–517. 

3. Chekanova A.E., Sorkina T.A., Nikiforov V.N., Davidova G.A., Selezneva I.I., Goodilin E.A., 

Dubov A.L., Trusov L.A., Korolev V.V., Aref'ev I.M., Perminova I.V., Tretyakov Y.D. 2009. 

New environmental nontoxic agents for the preparation of core-shell magnetic nanoparticles. 

Mendeleev Commun., 19, 1–4. 

4. Tombacz E., Szekeres M. 2001. Interfacial Acid-Base Reactions of Aluminum Oxide Dispersed in 

Aqueous Electrolyte Solutions. 1. Potentiometric Study on the Effect of Impurity and Dissolution 

of Solid Phase, Langmuir, 17, 1411–1419. 

5. Ritchie J., Perdue M. 2003. Proton-binding study of standard and reference fulvic acids, humic 

acids, and natural organic matter, Geochim.Cosmochim. Acta, Vol. 67, No. 1, pp. 85–96. 

Vol. 3 Page - 374 -

Halogen-free Preparation and Preliminary Characterization of Humic 

Substances from Different Substrates 

Carola Kleiner, Claudia Barthel, Ralf Junek, Roland Schubert, Juergen I. Schoenherr, 

Renate Klöcking* 

Research Institute for Peat and Natural Products, Hochschule Zittau/Görlitz – University of 

Applied Sciences, Theodor-Körner-Allee 16, D-02763 Zittau, Germany 

E-mail: rkloecking@hs-zigr.de 


The potential use of peat-derived humic substances (HS) in medicine, veterinary medicine and 

body care requires adequate peat sources as well as sophisticated methods for isolation of 

active ingredients. Despite various modifications and additional purification steps, F. K. 

Achard’s classical method for isolation of humic acids (HA) from peat, i.e. the alkaline 

extraction of HS followed by the acid precipitation of HA [1], represents the core of most HA 

isolation procedures so far. However, some HS solubilising, precipitating and purifying 

agents currently used are capable to interact with HS and thus may cause problems of 

toxicological concern. For example, different chlorine species including chlorine itself and 

hypochlorite, but also chlorite, chlorate and even chlorides may produce potentially genotoxic 

organic halogen compounds [2, 3]. 

To avoid any contact of halogens with HS during the HA preparation process, the aim of this 

study is twofold: a) to identify halogen-free organic compounds suitable to separate HA and 

fulvic acids (FA) and b) to use these agents for the isolation of HA from different substrates. 

In addition, the isolated HA will be characterized by elemental analysis, AOX determination, 

high performance size exclusion chromatography (HPSEC) and isoelectric focusing (IEF).. 



Humic sources and designation of isolated humic substances (in brackets): Peat from the 

Altteich Peatland situated in the Upper Lusatia, Saxony, Germany (HA-AP), digestate of a 

biogas plant north of Zittau, Saxony, Germany (HA-DBP), autumn foliage from Alnus 

glutinosa (HA-AGL, and commercially obtained roasted coffee (HA-RC) were used. 

IHSS Reference humic substances: Waskish Peat humic acids 1R107H (HA-WP) and Waskish 

Peat fulvic acids 1R107F (FA-WP) were purchased from IHSS. 

Halogen-free isolation procedure for peat humic acids: After homogenization of the peat 

sample, ultrapure water was added at a ratio of 1:10. The pH value of the peat suspension was 

adjusted with one of the usual extracting agents (e.g. NaOH, KOH) to 9 and kept constant for 

Vol. 3 Page - 375 -

2 hours. The extracting temperature was 30° C. After finishing the extraction, the mixture was 

centrifuged at 3500 g for 15 minutes. Peat HA were precipitated in the supernatant by 

addition of the selected halogen-free organic acid (0.5 mol/l) in a ratio of 3:1. After 

precipitation has been completed, HA were separated from FA by centrifugation at 11000 g 

for 10 min. The resultant pellet was washed and centrifuged several times (at least 4 to 5 

times) with water and then freeze-dried. 

Except for the peat HA AP-A which was precipitated with hydrochloric acid according to 

standard procedures (Table 1), the halogen-free isolation method has also been employed for 

HA from the other above-mentioned sources. 

High performance size exclusion chromatography (HPSEC): Freeze-dried HA samples were 

dissolved in a buffered saline solution adjusted with 0.2 mol/l NaOH to pH 10. Water-soluble 

alkali humates were dissolved in ultrapure water. HPSEC analysis was carried out using a 

high-performance liquid chromatograph from Varian (Varian ProStar series) equipped with 

the Diode Array Detector Agilent 1100 (Series G1315B) and fitted with a PSS Hema Bio 

column (Polymer Standard Service Mainz, Germany). Data capture occurred at a wavelength 

of 240 nm. Moreover, UV/vis spectra were recorded from each HA sample and its major SEC 

fractions (Data not shown in the abstract). 

Isoelectric focusing (IEF): Precast SERVALYT ® PRECOTE ® polyacrylamide gels, 

pH 3-10, from SERVA (Heidelberg, Germany) were used. The run was performed in a 

horizontal electrophoresis chamber at 5° C for 3500 Vh and at a final voltage of 1860 V. To 

increase the detection sensitivity for low HA concentrations, HA bands were stained with 

Alcian blue-tetrakis (methylpyridinium) chloride (Sigma-Aldrich Chemie, Steinheim, 

Germany, Cat. No. A4045) dissolved in 10 % acetic acid. 



In search of alternative, halogen-free precipitating agents for HA, we found polyprotic 

organic acids with pKa values between 1.2 and 3.2 the most promising candidates. Two of a 

total of 14 acids were considered appropriate for further investigations. Table 1 contains data 

of the elemental analysis and of the determined adsorbable organic halogen compounds 

(AOX) in peat HA precipitated with 0.5 mol/l hydrochloric acid (A), oxalic acid (B) and citric 

acid, repectively. The results show that the precipitation agents do not considerably influence 

the elemental analysis of the isolated HA. In contrast, insufficient washing of HA after HCl 

precipitation causes a reversible increase of the AOX value. 

Vol. 3 Page - 376 -

Table 1: Elemental analysis and AOX content of three Altteich Peat humic acids (n=3) precipitated 

with different agents (A hydrochloric acid, B oxalic acid, C citric acid); 2 x W, 10 x W = Number 

of washing steps 

Humic acids from Altteich Peat (HA-AP) 

AP-A 

2xW 10xW 


AP-B 

2xW 10xW 

AP-C 

2xW 10xW 

IHSS Reference 

Humic Acids 

from Waskish 

Peat 

C % 58.8 60.7 59.4 57.1 54.9 59.7 54.7 

N % 1.4 1.5 1,3 1.5 1.3 1.3 1.5 

C/N Ratio 41.1 40.1 44.6 38.3 43.2 46,5 36.5 

H % 5.3 5.4 5.2 5.6 5.3 5.1 4.0 

S % 0.3 0.3 0.2 0.4 0.2 0.3 0.2 

AOX (mg/kg) 724 438 424 436 428 434 Not detected 

Figure 1: HPSEC of humic substances isolated from Altteich Peat (HA-AP), from the digestate of a 

biogas plant (HA-DBP) and from autumn foliage of Alnus glutinosa (HA-AGL). Precipitating agent: 

oxalic acid 

Figure 1 exemplifies the molecular size distribution of three HA from different substrates. 

The relation of molecular size classes of HA of the same origin was found to be relatively 

constant and widely independent of the precipitation agents used. 

Vol. 3 Page - 377 -

1 2 3 4 5 6 7 

Figure 2: Isoelectric focusing of HS from different 

substrates. 1, 2 = HA-WP; 3 = HA-DBP; 4, 5 = 

HA-RC; 6, 7 = FA-WP. 


pI 

6.0 

5.3 

5.2 

4.2 

3.5 

Figure 2 provides IEF images of 

HA from the digestate and the 

roasted coffee, both prepared 

according to the halogen-free 

isolation method as described. IEF 

images from IHSS reference 

substances are shown for comparison. 

As a fingerprint technique, 

IEF shows a lot of still unidentified, 

alcianblue-positive bands, the 

position of which allows assessing 

the more or less acid character of 

HS. 

With regard to prospective applications of humic substances in medicine and cosmetics, a 

concept for the halogen-free preparation of HA was developed. Preliminary preparative and 

analytical results make polyprotic organic acids the most promising candidates for replacing 

hydrochloric acid as HA–precipitating agent. 



The authors want to thank Stefanie Mey for their assistance in conducting IEF experiments. 

The financial support of the study by the German Federal Ministry for Education and 

Research (FH³ program, FKZ 1746 X06) is greatly acknowledged. 

References 

1. F.K. Achard, Crells Chem. Annalen 2 (1786) 391-403. 

2. D. Feretti, I. Zerbini, E. Ceretti, M. Villarini, C. Zani, M. Moretti, C. Fatigoni, G. Orizio, F. 

Donato, S. Monarca, Water Res., 42 (2008) 4075-4082. 

3. I.J. Fahimi, F. Keppler, H.F. Schöler, Chemosphere. 52 (2003) 513-520. 

Vol. 3 Page - 378 -


Vol. 3 Page - 379 -


Young Researchers in Humic Substances and Natural Organic Matter 

(IHSS Travel Award) 

Vol. 3 Page - 380 -

Influence of Surface Chemistry and Structure of Activated 

Carbon on Adsorption of Fulvic Acids from Water Solution 

T. V. Poliakova, L. A. Savchynaand N. A. Klymenko 

Institute of Colloid Chemistry and Chemistry of Water, Ukrainian National Academy of 

Sciences, 42 Vernadsky Avenue, Kiev 03680, Ukraine 

E-mail: PoliakovaT@ukr.net 

1.Introduction 

The adsorption of fulvic acids (FAs) on activated carbon (AC), which contain fractions of 

different molecular weight with various functional groups, is greatly affected by AC surface 

chemistry. The presence of oxygen-containing groups on the AC surface affects on the 

mechanism of interaction between sorbate and sorbent, changing the value of the free energy 

of adsorption, and creates prerequisites for the manifestation of catalytic properties of AC. 

The adsorption capacity of AC can be changed by modifying its surface by oxygen-containing 

groups. 

Natural organic matters which are containing in surface source of water supply are a mixture 

of compounds with very different adsorption characteristics. 

The characterization of multicomponent adsorption equilibrium for natural organic matters 

using a “conventional component” was successfully effected by applying the ideal adsorption 

solution theory (IAST) [1]. In accordance with the approaches developed in [2], in the case of 

adsorption of natural organic matters from a multicomponent solution, adsorption isotherm in 

the logarithmic coordinates of the Freundlich equation has two or three regions of principle as 

straight lines: (1) nonadsorbable part of NOM, which is expressed by a vertical line even at 

large adsorbent doses; (2) slightly adsorbable part of NOM, where at medium adsorbent doses 

the highly adsorbable component and the proportional part of the fraction of the more slightly 

absorbable part of NOM. Еach region is described by individual values of ni and Kf,i. 

Thus, the aim of this work was to estimate the characteristics of adsorption of FAs from 

aqueous solutions in accordance with the approaches considered and to determine the 

variation of the adsorption characteristics of sorbents as a function of the conditions of 

carrying out the process. 



The FA have been obtained according to the Forsith’s method [3] from a high-moor peat were 

used as a sorbate. 

Vol. 3 Page - 381 -

The object of investigation was KAU carbon, which is obtained by treatment of crushed fruit 

stones with concentrated alkali and hot hydrochloric acid (after washing with water), washing, 

carbonization and activation with steam. 

Carbons were oxidized according to the procedures described in [4]. KAU carbon was 

oxidized with nitric acid during three and nine hours (KAU-N3 and KAU-N9) and hydrogen 

peroxide (KAU-O). 

3.Results and Discussion 

Figure 1 shows adsorption isotherms of FAs on KAU, KAU-O, KAU-N3 and KAU-N9 

active carbons in logarithmic coordinates of the Freundlich equation. 

a (mgC/g) 

100 

10 

1 

0,1 

1 

КАU 

КАU-N9 

КАU-N3 

10 100 

КАU-O 

C eq (mgC/l) 

Figure1: Isotherms of adsorption of fulvic acids on KAU, KAU-O, KAU-N3 and KAU-N9 in the 

coordinates of the Freundlich equation 

In accordance with the proposed approaches [1, 2] we have been divide the adsorption 

isotherm into two regions: (1) for low adsorbable part and (2) for high adsorbable part of FA. 

Table 2 lists values of Freundlich equation constants calculated from a single averaged 

straight line without division into regions (Kav, Hav), and n1 and n2 values corresponding to 

two FA adsorbability regions. 


As it is evident from Table 2, change in surface chemistry, i.e. increase in surface 

heterogeneity, and the appearance of additional lactonic, phenolic groups and basic properties 

on the surface changes greatly the adsorption characteristics of AC concerning adsorption of 

FAs. Although the Freundlich equation is empirical, a physical meaning is attached to its 

constants in some works. KF is regarded as an adsorption capacity factor, and the exponent n 

characterizes the heterogeneity of energy centers on the surface and is related to the driving 

force of adsorption (i.e. adsorption energy). 

Vol. 3 Page - 382 -

Table 2: Change of coordinates of the Freundlich equation in dependence of properties surface of AC 

Constants of the 

Freundlich equation 

Sample КF n n1 n2 

Acidic 

(mg/gAC) 

Basic 

(mg/gAC) 

Carboxy-lic 

(mg/gAC) 

Lacto-nic 

(mg/gAC) 

Phenol-lic 

(mg/gAC) 

KAU 1.41 1.03 0.21 1.13 0.15 0.05 0.46 0.10 0.05 4.8 

KAU-О 0.21 0.74 0.26 1.01 0.85 0.20 0.44 0.55 0.20 2.6 

KAU-N3 0.50 0.81 0.18 1.06 0.85 0.30 0.49 0.50 0.30 3.0 

KAU-N9 0.81 0.75 0.21 0.85 1.0 0.30 0.40 0.70 0.30 3.5 

In accordance with this approach, it can be concluded from Table 2 that the oxidation of AC 

reduces adsorption capacity for FAs as a whole due to an increase of energy surface in- 

homogeneity. In this case, the adsorbability of FA is most likely affected adversely by 

increase in the percentage of lactonic and phenolic surface groups (i.e. negatively charged 

groups). However the adverse effect on the absorbability of FA on KAU-N3 and KAU-N9 is 

weakened as compared with KAU-O. This adverse effect on the adsorbability of FA is 

understandable if the above-mentioned chemical nature of the functional groups of FA is 

taken into consideration. 

The exponent n for the highly adsorbable components (n2) are larger than exponent for the 

low adsorbable components (n1). This points to the fact that increase in energy surface in- 

homogeneity especially of basic properties increases the driving force of adsorption for well 

adsorbable compounds. 

Table 3 lists data concerning the determination of adsorption equilibrium constants Ka and 

changes in the free energy of adsorption -ΔGa 0 for two "conventional component" of FA. 

Table 3: Changes of adsorption equilibrium constants (Ka) and free energy of adsorption (-ΔGa o ) 

for two “conventional component” of FA solutions in dependence of type of AC 

(-ΔGa 0 ), kJ/mol 

Sample 

First “conventional 

component” 

Ka1 

Ka 


Second 

“conventional 

component” 

Ka2 

First “conventional 

component” 

(-ΔGa1 0 ) 

Second 

“conventional 

component” 

(-ΔGa2 0 ) 

KAU 153 4258 11.42 18.97 

KAU-О 35 3881 8.08 18.76 

KAU-N3 150 3767 11.38 18.69 

KAU-N9 251 3761 12.54 18.69 

Vol. 3 Page - 383 - 

pH

As it is evident from Table 3, the lowest adsorption energy of the low adsorbable component 

is characteristic for KAU-O, and for the high adsorbable component this parameter is 

practically invariable for all types of AC. 

Thus, increase in the energy surface in-homogeneity of AC affects mainly on the adsorption 

of the low adsorbable part of FA. This effect may be due to the fact that the adsorption of this 

part of FA is brought about mainly by Van der Waals forces, and that the screening of a part 

of the surface by functional groups reduces the total adsorption energy. The change in surface 

chemistry does not practically affect on the more high adsorbable fraction of FA, indicating 

that there is a specific interaction between the functional groups of FA and the surface. 

Besides, it should be noted that change in the structure and surface chemistry of AC leads to a 

change in concentration limits whereby two FA components. This is evident from the data in 

Table 4. 

Table 4: Change in concentration limits of conditional division FA on slightly adsorbable fraction and 

highly adsorbable fraction 

Samples 

Range of equilibrium concentration on the 

KAU KAU-О KAU-N3 KAU-N9 

isotherm that apropriate slightly adsorbable 

fraction (mg C/l) 

Up to 3.40 Up to 6.25 Up to 4.40 Up to 4.17 

4.Conclusions 


1. The oxidation of AC reduces the adsorption capacity for FAs in whole due to an increase of 

energy surface in-homogeneity. 

2. The data obtained allow the conclusion to be that the change in AC energy surface inhomogeneity 

due to oxidation leads mainly to a decrease in FA adsorption energy and to 

increase of concentration range of the conventional portion of the low adsorbable fraction, 

especially in the case of adsorption on AC oxidized by hydrogen peroxide. 

References 

1. E.H. Smith and W.I. Weber, Water, Air, Soil Pollut., 53 (1990) 279. 

2. E.H. Smith Water Res., 28 (1994) 1693. 

3. L.N. Aleksandrova, Organic Matter of Soil and Processes of its Transformation. Science, 

Leningrad, 1980, p 288. 

4. M.F. Pereira and S.F. Soares, J.J.M. Orfao, J.L. Figueiredo, Carbon. 41 (2003) 811. 

Vol. 3 Page - 384 -

Studies by Chemometric Methods of the Interaction Between 

Pb(II) and Humic Acids 

Silvia Orsetti, Estela Andrade, Fernando Molina * 

INQUIMAE, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires Ciudad 

Universitaria, Pabellón II, Buenos Aires C1428EHA, Argentina 

E-mail: fmolina@qi.fcen.uba.ar 


Humic substances are important components of natural organic matter in groundwaters and 

soils, where they have a fundamental role in the fate of pollutants such as heavy metals. In 

particular, retention and transport of hazardous substances by humic and fulvic acids have 

been frequently studied in the last few years. 

Fluorescence spectroscopy, combined with multi - way data analysis techniques, is a powerful 

tool to obtain information regarding humic substances and their interaction with heavy metals, 

such as Pb(II). In this kind of analysis, excitation emission matrices (EEM) are used: they are 

obtained by combination of emission spectra measured at different excitation wavelengths. In 

general, humic acids (HA) show a broad emission peak between 300 and 700 nm and a broad 

excitation peak between 300 and 450 nm. However, the location and shape of these peaks 

strongly depend of the origin of the humic material. 

Excitation emission matrices provide wide information, and until recently EEMs 

characterization techniques were focused on visual identification and shape of peaks, leading 

to qualitative analysis. Recently, multi - way data analysis techniques have been introduced in 

the study of fluorescence signals of natural organic matter. One of these techniques is parallel 

factor analysis (PARAFAC), which is able to decompose the fluorescence signal into the 

underlying fluorescent individual phenomena [1, 2]. 

PARAFAC models three – way data by using eq. 1, fitting the equation by minimizing the 

sum of squares of the residuals (εijk): 

F 

ijk ijk ∑ if jf kf ijk 

f = 1 

x = a b c + εε 


i = 1,..., I j = 1,..., J k = 1,..., K 

xijk is an element of the three - way array with dimensions I, J and K. In the case of EEMs, xijk 

is the fluorescence intensity of sample i, measured at the emission wavelength j and excitation 

Vol. 3 Page - 385 - 

(1)

wavelength k. The term εijk accounts for the unexplained signal (experimental error). The 

output of the model are the parameters a, b and c. These represent, ideally, the concentration, 

emission spectra and excitation spectra of the underlying fluorophores, respectively, and they 

are usually referred to as scores (a) and loadings (b, c). In this way, important information 

about the number of fluorophores, their fluorescence profiles and their behavior in presence of 

the metal is obtained; the last subject, from the relative concentration of each fluorophore in 

each individual sample, with a characteristic Pb(II) concentration [3]. 

The aim of this work is to study EEMs of HA at several Pb(II) concentrations, analyzing them 

with PARAFAC as a whole to estimate the number of individual components of the sample 

(fluorophores), their emission and excitation fluorescence spectra, as well as the relative 

concentration of those in each sample. 

The effect of pH value and ionic strength is also analyzed. 


The EEMs of two different HA where measured in absence and presence of several 

concentrations of Pb(II). These measurements were conducted for Elliot Soil HA (EHA, 

reference material of the IHSS) and other commercially available from Fluka, previously 

purified (FHA). Experiments were performed at pH 4.0 and 5.5, and at two values of ionic 

strength: NaClO4 0.1 and 0.02 M. The samples were prepared by dissolving the HA in the 

NaClO4 (aprox. concentration 30 mg L -1 ) using a minimum quantity of NaOH, and then the 

pH was adjusted with HClO4. This sample was separated in 25.0 mL aliquots, and different 

volumes of a stock solution of Pb(II) were added to these. The EEMs of those aliquotes were 

measured, as well as the EEM of the HA sample without Pb(II). In all cases, the samples were 

under N2 atmosphere. 

Data processing: after elimination of the scattering zones in the EEMs, PARAFAC was used 

indicating number of components from 2 to 5, adding a non negativity constrain for the b and 

c loadings (which account for emission and excitation spectra respectively). From the values 

of core consistency and SD residuals, the number of components was estimated following Bro 

and Kiers [4], considering that a high core consistency value (over 50%) means that the model 

presents high spectral resolution of components. From there a, b and c were obtained. 



A total of 3 independent components were estimated for both HA. In Fig. 1 the relative 

concentrations of fluorophores (a score) in each set of samples is plotted for EHA. 

Vol. 3 Page - 386 -

From Fig. 1 it can be noticed that all 3 components have a different behavior towards the 

increment of Pb(II) concentration, being the component A the most affected in its 

fluorescence intensity. The difference between components B and C is more marked at higher 

ionic strength (a and b of Fig. 1), component C showing less sensitivity towards Pb(II) at pH 

= 4.0 and I = 0.10 M (a in Fig. 1). The fluorescence decrease does not correlate with the 

fraction of carboxylic and phenolic groups bound to Pb(II) ions as predicted by the NICA – 

Donnan model, thus Pb(II) induced aggregation is proposed as the cause of fluorescence 

quenching. 

Fluorescence Intensity Scores (a. u.) 


2.5x10 6 

2.0x10 6 

1.5x10 6 

1.0x10 6 

5.0x10 5 

8.0x10 6 

6.0x10 6 

4.0x10 6 

2.0x10 6 

a 

1E-5 1E-4 

[Pb 2+ ] added (M) 

c 

1E-5 1E-4 



3.0x10 6 

2.5x10 6 

2.0x10 6 

1.5x10 6 

1.0x10 6 

5.0x10 5 

1E-6 1E-5 

[Pb 2+ ] added (M) 

[Pb 2+ ] added (M) [Pb 2+ ] added (M) 

2x10 6 

1x10 6 

0 

b 

3x10 6 d 

1E-5 1E-4 

Figure 1: a scores (Fluorescence Intensity Scores, in arbitrary units) are shown as a function of Pb(II) 

concentration. squares: component A; circles: component B; triangles: component C 

pH 4.0 and I = 0.10 M (a); pH 5.5 and I = 0.10 M (b); pH 4.0 and I = 0.02 M (c) and pH 5.5 and I = 

0.02 M (d) 

spectral loading 

0.4 

0.3 

0.2 

0.1 


0.0 

200 300 400 500 600 700 

a 

λ (nm) 

λ (nm) 

Figure 2: emission (continuous line) and excitation (dotted line) spectra of components for EHA (a) 

and FHA (b), both at pH = 5.5, I = 0.1 M. Black: component A; red: component B; blue: component C 

spectral loading 

0.3 

0.2 

0.1 

Vol. 3 Page - 387 - 

0.0 

200 300 400 500 600 700 

b

The loadings (b and c of PARAFAC) for EHA at pH = 5.5, I = 0.1 M are shown in Fig. 2. The 

shape and location of these emission and excitation spectra are typical of humic substances 

[5,6]. The spectra are shifted to the red (not shown) as the ionic strength decreases, especially 

at pH = 5.5. This was observed for most components. 



In both EHA and FHA 3 fluorophores can be estimated; one of them is strongly deactivated in 

presence of Pb(II) wile the others show less sensitivity. All present emission and excitation 

spectra typical of humic substances. The fluorophores which are most affected have their 

emission and excitation spectra shifted to the red, thus might be attributed to groups 

containing condensed rings. It is considered that humic molecules would aggregate with the 

metal as a bridge, presenting no–radiative deactivation in the aggregated form and thus 

quenching of their fluorescence intensity. The deactivation depends on the ionic strength and 

pH, especially for the components B and C. 

Regarding the effects of pH and ionic strength values, it was observed that emission and 

excitation spectra shifted towards the red with lower ionic strength, being this shift higher at 

superior pH. 

References 

1. C. M. Andersen and R. Bro, J. Chemometrics, 17, (2003) 200. 

2. C. A.Stedman and R. Bro, Limnol. Oceanogr.: Methods, 6 (2008) 572. 

3. T. Ohno, A. Amirbahman and R. Bro, Environ. Sci. Technol., 42 (2008) 186. 

4. R. Bro and H. A. L. Kiers, Journal of Chemometrics, 17 (2003) 274. 

5. T. Ohno and R. Bro, Soil Sci. Soc. Am. J., 70 (2006) 2028. 

6. M. M. D.Sierra, M. Giovanela, E. Parlanti and E. J. Soriano–Sierra, Chemosphere, 58 (2005) 715. 

Vol. 3 Page - 388 -

Seasonal Dynamics of Biomass and Copper Concentrations in Ectohumus 

of Forest Soils Impacted by Copper Industry in South-West Poland 



Agnieszka Medyńska * , Cezary Kabala 

Institute of Soil Science and Environmental Protection 

University of Environmental and Life Sciences, Wroclaw, Poland 

E-mail: jagamedynska@yahoo.com 

Humus plays an important role in the functioning of forest ecosystems. It acts as a link 

between above- and below-ground ecosystem compartments. The fall and decomposition of 

organic matter return minerals and energy to the soil biota. The forest floor is the most 

dynamic part of soil organic matter (Yanai et al. 2003). Previous studies about nutrient release 

from forest litters, formed under various tree species, show significant differences in 

dynamics of nutrients. The release of nutrient elements during decomposition of forest floor is 

an important internal pathway in nutrient flux of forested ecosystems. Nutrients may be 

released from ectohumus horizons by leaching or mineralization (Regina, 2000). Nutrient 

release from decomposing litter affects the primary productivity of ecosystem, since these 

nutrients become available for plant uptake and are not lost in the ecosystem. The rate at 

which nutrients are released depends on several factors: the chemical composition of the 

forest litter, the structural nature of the nutrient in the litter matrix, the microbial demand for 

the nutrient, and the availability of exogenous substance sources for example from 

anthropogenic activity (Brown et al., 1999). Litter quality affects the rates of mass loss, but 

also the patterns and rates of nutrient immobilization or release. Forest litter highly 

contaminated with heavy metals may effect nutrient cycling throughout the whole ecosystem 

(Tyler, 1974). Many reports have shown that short-term or long-term exposure to toxic metals 

results in the reduction of microbial diversity and activity which is mainly observed in 

inhibiting litter decomposition and increase of undecomposed organic matter in humus layer 

(Berg et al., 1991). From the other hand, forest litter acts like a sink for contaminants, 

regulating the amount of heavy metals leaching to deeper soil layers (Medyńska and Kabała, 

2007 Laskowski et al. 1995). Dynamics of heavy metals in contaminated forest floor is 

exceptionally investigated, and in particular - on recently afforested soils, where the 

ectohumus layer has been already formed. 

The aim of present study was to determine the seasonal dynamics of biomass of forest litters 

in ecosystems impacted by copper industry as an important factor determining actual 

concentration of copper in forest floor. 

Vol. 3 Page - 389 -


Study sites. The investigation was carried on two study areas. The first one was located in a 

surrounding of the large copper smelter near town Legnica, in Lower Silesia region. The 

copper smelter Legnica is a part of the mining and metallurgy complex founded in 1951, 

which currently includes 4 mines, 3 ore enrichment plants and 3 smelters. The complex has 

been producing approximately 500,000 tons of copper annually, one fourth of that is produced 

in the Legnica smelter. Copper smelting was connected in the past with a large emission of 

metal-containing dust, significantly reduced during the 1980s and 1990s (Byrdziak et al. 

2005). Long-term copper smelting in the Legnica area has however resulted in an extensive 

soil contamination with number of trace elements. To exclude crop production in 

neighborhood of the smelter, the so called “protective zone” was delimited in late 80s around 

the smelter. All the area has been successively planted with poplar trees. The other study area 

was located in the surrounding of copper ore tailings impoundment Żelazny Most, located 

near the town Lubin, 30 km north of Legnica smelter. The over-ground impoundment has 

been in operation since 1977. Over 368 mln m 3 of tailings from copper ore flotation are 

assembled on the area of 1,390 ha. Wind expose, dry metal-bearing tailings are blown about 

to surrounding areas leading to soil and plant contamination. 

Methodology. Two study areas were projected as separate experiments. On each study site, 3 

plots were located. The plots at the Cu smelter Legnica were arranged along decreasing 

concentration of Cu in soil, at distances of 0.3, 1.5 and 2.1 km, north of the smelter. All plots 

are on similar soils (Haplic Luvisols) and in the poplar stands of the same age. The plots near 

tailings impoundment are located at the same distance from impoundment, on similar soils 

(Brunic Arenosols), but under pine stands varying in age, from 8 to 50 years old. On each site 

samples of litter were collected in 4 replicates using a stainless-steel cylinder (d=23 cm). 

Samples were collected every month between November 2007 and October 2008. Dry matter 

of litter was determined in all samples, followed by high-pressure sample digestion with aqua 

regia (HCl:HNOs ratio 3:1), and measurements of total Cu concentration by the atomic 

absorption spectroscopy (AAS) technique. 

3. Results 


Forest floor biomass estimation. Biomass of forest floor (as dry matter) and Cu 

concentrations were calculated for standard area of 1m 2 . On both sites biomass of litter varied 

between the plots (Fig. 1 and 2). The highest mass of ectohumus in poplar stands near Cu 

smelter, and the largest thickness (ca 7 cm) was observed on the plot 1 located 0.3 km from 

Vol. 3 Page - 390 -

the smelter. This phenomenon should be considered with the highest concentration of Cu in 

litter – circulating around 11,300 mg/kg (Fig. 3). High concentrations of heavy metals can 

suppress the decomposition rate and lead to accumulation of undecomposed organic matter, 

because of the toxicity effect on soil microorganism (Tyler 1972, Rühling and Tyler 1973; 

Strojan 1978; Berg et al. 1991, Niklińska et al. 1998). The highest mass of ectohumus (litter) 

in forest stands surrounding the tailings impoundment was observed in the oldest (50-years- 

old) oak-pine stand. Changes of a litter mass during studied period of time mostly depended 

on all plots on biomass input pattern. Input of leaf biomass changed seasonally, and was the 

highest in late fall (October-November) and during the drought periods (February, March, 

August, and September), when trees lost leafs partly as a water stress effect. Seasonal changes 

of ectohumus biomass are also considered with the intensity of decomposition process. In 

studied litters, the highest intensity of decomposition process was observed during winter 

time. Unreceptively decomposition played a secondary role in biomass changes, this can be 

considered with an inhibiting effect of heavy metals on decomposers in studied ectohumus 

horizons. 

Fig. 1 Biomass (mg/m 2 of d.m.) of litter layer in poplar 

stands in the surrounding of copper smelter Legnica 

Figure: 3: Copper concentration in litter of forest 

stands in the surrounding of copper smelter Legnica 


Vol. 3 Page - 391 - 

Fig. 2 Biomass (mg/m 2 of d.m.) of litter layer in pine 

stands in the surrounding of tailings impoundment 

Żelazny Most 

Figure 4: Copper concentration in litter of forest 

stands in the surrounding of tailings impoundment 

Żelazny Most


Changes of copper concentration in ectohumus. Significant impacts of copper smelting plant and 

tailings impoundment were observed in studied forest soils and ecosystems. Both emission sources 

cause air pollution, partially filtrated by forest stands planted in surroundings of the industrial objects. 

In studied forest ecosystems through fall of leaf biomass was the main source of copper input into the 

ectohumus horizons. With an increase of through fall, also concentration of studied metal in forest 

litters, increased rapidly. Despite decomposition processes and copper leaching with rain waters 

observed during the year of study, copper concentrations on all studied plots increased successively 

with time (Fig. 3 and 4). This can be concluded as evidence that in young forest ecosystems impacted 

by heavy metal pollution, already developing ectohumus horizon may accumulate heavy metals, 

therefore play a role of a natural sink for anthropogenic contamination. 


1. The dynamics of forest litter biomass and Cu concentrations depend strongly on leaf input during 

seasons. 

2. Decomposition processes play a secondary role in biomass changes and Cu release. Inhibiting 

effect of heavy metals on decomposition rate was observed in poplar stands in the vicinity of 

copper smelter. 

3. In studied young forest ecosystems, developing ectohumus horizons play a role of a natural sink 

for Cu contamination. 

References 

1. Berg B., Ekbohm G., Söderström B., Staff H. 1991: Reduction of decomposition rates of Scots pine needle 

litter due to heavy metal pollution. Water, Air, Soil Pollut. 59: 165–177. 

2. Brown S.L., Schroeder P., Kern J.S. 1999: Spatial distribution of biomass in forests of the eastern USA: 

Forest Ecology and Management 123: 81–90. 

3. Laskowski, R., Niklińska, M., Maryanski, M., 1995. The dynamics of chemical elements in forest litter. 

Ecology 76, 1393–1406. 

4. Byrdziak H., Jędrzejewski J., Kierdel Z., Mizera A. 2005: Environmental Protection – Bulletin 2002–2004. 

KGHM CUPRUM, Lubin, Poland: 1–170. 

5. Medyńska A., Kabała C. 2007: Heavy metal concentration in ectohumus horizons of forest soils under 

impact of copper ore tailings impoundment Żelazny Most: Environmental Protection and natural resources 

31: 137–144. 

6. Niklińska M., Laskowski R., Maryańska M. 1998: Effect of heavy metals and Storage time on two types of 

Forest Litter: Basal respiration rate and exchangeable metals: Ecotoxicol. Environ.Safety 41: 8–18. 

7. Regina I. 2000: Biomass estimation and nutrient pools in four Quercus pyrenaica in Sierra de Gata 

Mountains, Forest Ecology and Management 132: 127–141. 

8. Rühling A, Tyler G. 1973: Heavy metal pollution and decomposition of spruce needle litter. Oikos 24: 402– 

416. 

9. Strojan C.L. 1978: Forest litter decomposition in the vicinity of a zinc smelter. Oecologia 32: 203–212. 

10. Tyler G. 1972: Heavy metal pollution and mineralization of nitrogen in forest soils, Ambio 1(2): 52–57. 

11. Vandecasteele B., De Vos B. , Muys B., Tack F. 2005: Rates of forest floor decomposition and soil forming 

processes as indicators of forest ecosystem functioning on a polluted dredged sediment landfill: Soil Biol. 

Biochem. 37 761–769. 

12. Yanai R.D., Currie W.S., Goodale C.L. 2003: Soil carbon dynamics after forest harvest: an ecosystem 

paradigm reconsidered: Ecosystems 6: 197–212. 

Vol. 3 Page - 392 -

Limitation for Study of Humic Substances or NOM Using High Resolution 

and Accuracy Mass-Spectrometry 

Gleb Vladimirov a,b , Eugene Nikolaev a,b* 

a The Institute for Biochemical Physics RAS, Moscow, Russia; b The Institute for Energy 

Problems of Chemical Physics RAS, Moscow, Russia 

E-mail: ennikolaev@rambler.ru 


Stoichiometric formulas determination using high resolution and accuracy mass-spectrometry 

(ICR-FT mass-spectrometry) for molecules of humic substances (HS) or natural organic mater 

(NOM) is a powerful tool which show full scale of molecular complexity of HS and NOM 

[1,4]. However, using this method you should take into account its limitations: selectivity of 

ionization process and limitations caused by the physics of mass determination processes. 

That leads to correct work of method only within a certain m/z range and for differences in 

components concentrations limited by method dynamic range. 


Suwannee River Reference Fulvic acid (SRFA) spectra (sample id 1S101F) was obtained on 

commercial mass-spectrometer 7 Tesla Finnigan LTQ FT (Thermo Electron, Bremen, 

Germany) equipped with electro spray ion source (Finnigan Ion Max Source) located at the 

facilities of the Emmanuel Biochemphysics Institute of RAS (Russia). The spectra was used 

for determination of ion cloud distribution for estimation of method limitation. The following 

conditions were used for electrospray: flow rate 1 ul/min, negative ion mode, needle voltage 

3.4 kV; tube lens voltage 130 V; heated capillary temperature 250 o C. Filtering linear ion trap 

was set up to collect ions in m/z range from 200 to 2000 Da, the number of accumulated ions 

1·10 6 . The average spectrum was calculated from 100 coadded scans. Time domain was set 

up for resolution R=400000 at m/z 400. LTQ FT calibration mix was used for external mass 

calibration. All acquired spectra were internally recalibrated by solvent impurities to mass 

measurement error

singly charged ions (it is true, because of a little fraction of multiply charged ions) then we 

have: a minimum number of ions observed in ion cloud is 27, the largest number of ions in a 

cloud is 1870 ions. Therefore we observe dynamic range for different SRFA compounds 

equal to 70. (Although dynamic range can be considered as ratio of total number of ions to 

minimum number of ions in observable peak 10 6 /27= 37 000, but that ratio doesn’t reflect 

observable dynamic range of SRFA compounds.) 

Figure 1: Statistics of ion cloud numbers and numbers of elementary charges in ion cloud for spectrum 

of SRFA 

The statistics of cloud charge distribution (see Fig. 1) shows that: in case of SRFA spectrum 

roughly the same number of elementary charge are in ion clouds of different charge value, 

while number of ion clouds with a small number of charges increases greatly during reducing 

of number of charges in ion clouds. High charge ion clouds are not observed - the maximum 

number of elementary charges in a cloud is 1870 charges, and it is ~ 500 times smaller than 

trap capacity. Therefore we can conclude that for spectra like SRFA the main problem is 

detection of clouds with a minimum number of ions (detection limit) and destruction of 

clouds by a huge number of small clouds during detection (dynamic range limit). 

Therefore increasing of dynamic range for work with spectra like SRFA demands decreasing 

of minimum detectable number of ions value: 

There are several different estimations of minimum number of ions [4], which is necessary to 

provide detectable signal. It should be about 50 (Group Dick Smith) or 100 (Marshall) 

elementary charges in a cloud of one m/z to make signal from such m/z detectable for single 

spectrum. 


Minimum number of ions (that is minimum number of elementary charges in the cloud of a 

certain m/z) which is necessary to ensure detectable signal for given m/z can vary for different 

mass-spectrometers, because of influence of amplifier and preamplifier construction, for 

example using of QSUID detector provides an opportunity to receive signal from one single 

charged ion rather than from group of 50 single charges ions for traditional scheme. 

Vol. 3 Page - 394 -


Definition ions minimal number which can provide detectable m/z in spectrum is complicated 

by fact that it depends on level of noise in amplifier system, therefore using sum of several 

different spectra of the same sample obtained at identical conditions allows to reduce noise in 

the system, thus it increases minimum number of ions in which this m/z are detectable. 

Sensitivity of mass-spectrometer depends on the distance from the ion cloud to the walls of 

mass-spectrometer cell, reducing this distance we increase the sensitivity of the mass- 

spectrometer, but the stability of the ion cloud is decreased due to greater anharmonicity of 

trapping potential for higher radius orbit. Anharmonicity Value for trapping potential for cell 

determined by trap design: it could be improved by use of newer harmonized cell design. 

Resolving power requirements of mass-spectrometer for work with complex samples like 

SRFA are determined by necessity to resolve doublet of C13/C12H (dm = 0.00447 Da) for 

work with Cx Hy Oz stoichiometric formulas. Work with Cx Hy Oz Nm Sn stoichiometric 

formulas requires resolving a greater number of doublets for combination of C12=12.00000 

Da, C13=13.00335 Da, H1=1.007825 Da, O16=15.99491, N14=14.00335 Da, S32=31.9720 Da, 

but resolution required for this doublets is comparable with C13/C12H doublet. Resolution of 

mass-spectrometer for work in the same mode decreases with increasing of measured m/z 

therefore for doublet mass difference we have R (C13/C12H) = M (Da) * 224. On other hand 

resolving power on frequency of any m/z is determined by signal duration and m/z frequency. 

Signal duration determined by destruction of ion clouds: due to dependence of cyclotron 

frequency from amplitude for inharmonic trapping potential, collisions of ions with residual 

gas and ion cloud-ion cloud collision. Therefore for B = 7T; t = 1 and 3 s; m/z = 100 Da we 

obtain resolving power about R ~ V [Hz] t [sec], finally we can determine m/z above which 

resolving power will be insufficient for work with complex spectra like SRFA (above this m/z 

impossible to resolve doublets): 

M/z (C13/C12H, B = 7T t = 1 sec) = 750 Da 

M/z (C13/C12H, B = 7T t = 3 sec) = 1250 Da 

Therefore 200–800 Daltons range could be considered suitable for work with such complex 

samples like SRFA on 7 Tesla ICR mass-spectrometers. Bulk part of components distribution 

will fit to this range for majority of complex samples such HS or NOM for such massspectrometers. 

Resolving power is determined by the duration of detection signal, but there is phenomenon 

of coalescence [3] (merging different m/z peaks due to space charge clouds interaction). This 

Vol. 3 Page - 395 -

phenomenon leads to limit of resolution, and it shows itself for ion cloud with large number 

of elementary charges or very close m/z. Coalescence is observed in numerical simulations 

[3], there are several analytical estimations [2] that show the boundary of coalescence 

condition. 

For coalescence limit in case of C13/C12H doublet in 7T, 2 inch sell, 30% of sell cyclotron 

orbit radius according to formula N = 5.6 a 2 R B 2 dm/(m 2 k) [2] we can find m/z above which 

C13/C12H doublet will be unresolved due to coalescence: 

m/z(C13/C12H, cloud size 10000 charges, B=7T ) = 1200 Da 



Therefore in our case resolution limit due to coalescence is less significant compared to 

resolving power limit due to length of detection time. 


Studding of samples like SRFA using ICR-FT mass-spectrometers with 7 Tesla magnetic 

field strength possible in mass range up to 800 Da. Working in higher mass range demands 

using higher magnetic fields. 


Dynamic range for work with HS or NOM is restricted by limiting of minimum number of 

ions needed to make signal detectable. Increasing the minimum number of ions which are 

necessary for detection firstly requires improvement of trap design for harmonization of 

trapping potential. 

References 

1. E.V. Kunenkov, A.S. Kononikhin, I.V. Perminova, N. Hertkorn, A. Gaspar, P. Schmitt-Kopplin, 

I.A. Popov, A.V. Garmash and E.N. Nikolaev, Anal. Chem., (2009) in print. 

2. I.A. Boldin, Nikolaev E.N. Rapid Commun. Mass Spectrom. 2009; 23: 3213–3219. 

3. E.N. Nikolaev, G.N. Vladimirov, I.A. Boldin, R.M. Heeren, C. Hendrickson, G. Blakney, A.G. 

Marshall 57th ASMS Conference on Mass Spectrometry 2009 Instrumentation: FTMS – poster 

278. 

4. A. Kononikhin, G.N. Vladimirov, E.V. Kunenkov, I.A. Popov, I.V. Perminova, A. Garmash, G. 

Karpov, S. Varfolomeev, E.N. Nikolaev 56th ASMS Conference on Mass Spectrometry 

2008 Hydrocarbon and Petrochemical - poster 141. 

Vol. 3 Page - 396 -

A Study of Interaction Between Pharmaceuticals and Humic Substances 

Ansone L b ., Klavins M. a* 

a Faculty of Chemistry, Univ. of Latvia, Kr. Valdemara iela 48, Riga, LV 1013, Latvia; Dept. 

of Environmental Sciences, Univ of Latvia, 19 Raina Blvd., Riga, LV 1586, Latvia 

E-mail: linda_ansone@inbox.lv 


The role of pharmaceuticals in the urban water cycle is of rising concern. The problem is 

increasing with population growth and increased use of pharmaceuticals, with strong impact 

on aquatic ecosystems. It has been found that loading of pharmaceuticals in urban regions can 

be of the same level as loading of pesticides. First degradation stage of excreted 

pharmaceuticals and human metabolism products, takes place in wastewater treatment 

facilities. During treatment of wastewaters as well as in waterways the binding of 

pharmaceuticals and their degradation products to natural organic matter —humic substances 

(HS) plays the major role. 

An important and increasingly used group of pharmaceuticals contains bulky hydrophobic 

structures, however the behavior of these substances in the environment has not been much 

studied. Considering the wide application of some drugs, it may be particularly important to 

study drugs containing tricyclic structures (adamantane derivatives), such as Rimantadine 

(widely used in the prophylaxis and treatment of influenza A virus infections) and many 

others. Substitutions at various locations on the ring will determine the pharmacokinetics and 

biotransformation which could contribute to the overall activity including side effects [1]. 

Adamantane ring-containing substances are extraordinarily stable compounds, thus the study 

of their fate in the environment could be of high importance during intensive use, for example, 

during flu epidemics which may result in high concentrations in the environment [2]. 



The studied pharmaceuticals are summarized in Table. Binding of studied pharmaceuticals to 

humic substances has been studied by fluorescence spectroscopy, FT IR, 1 H NMR. To prove 

the impact of the character of interaction between pharmaceuticals and humic substances also 

biotests has been used. 

To quantitatively characterize the interaction, fluorescence quenching approach has been used [3]. 

Vol. 3 Page - 397 -

Table: The studied pharmaceuticals 

Substance Formula Structure 

Rimantadine hydrochloride 

1-(1-adamantyl)ethylamine 

hydrochloride 

C12H21N · HCl 

2-Aminoadamantane hydrochloride C10H17N · HCl 

Phenibut 

(4-Amino-3-phenyl-butanoic acid 

hydrochloride) 

C10H13NO2 · HCl HCl 

3-Aminoquinuclidine dihydrochloride C7H14N2 · 2HCl 

1-Hydroxyadamantane C10H16O 

The intensity of the fluorescence quenching is supposed to be proportional to the 

concentration of the formed humic-pharmaceutical complex according to the Stern-Volmer 

equation. The fluorescence of the humic acids quenching mechanism by pharmaceuticals is 

considered in the 1:1 interaction model. The binding constants are obtained by steady-state 

fluorescence quenching measurements and are given as a slope in the Stern-Volmer plot and 

can be calculated estimating I0/I - fluorescence intensity ratio of the initial substance and 

fluorescence in the presence of quencher. 



Fluorescence intensity quenching shows significant interaction between pharmaceuticals and 

HS, which increase with HS concentration and solution pH. The character of the relationships 

from modified Stern-Volmer plots (Fig. 1) offer strong support for the 1:1 complex (r 2 > 

0.99). Fluorescence quenching of 4-amino-3-phenyl butanoic acid, 1-hydroxyadamantane and 

2-aminoadamantane by dissolved humic acid was described by nonlinear Stern-Volmer plots. 

Vol. 3 Page - 398 - 

H3C 

H2N 

N 

NH2 

HCl 

NH2 

HCl 

NH2 

2HCl 

OH 

H

I0/I 

45 

40 

35 

30 

25 

20 

15 

10 

5 

0 

R 2 = 0,995 

R 2 = 0,992 

R 2 = 0,990 

0 10 20 30 40 50 60 

CHA, mg/l 

2-aminoadamantane HCl 1-Hydroxyadamantane 

4-amino-3-phenyl-butanoic acid HCl 

Figure 1: Stern-Volmer plots: the ratio of I0/I of Aldrich HA–pharmaceuticals as a function of CHA. 

Cpharmaceuticals = 0.5 mmol/L 

If the linear Stern-Volmer plot is indicative of a single class of fluorophores with equal 

accessibility to the quencher [4], then a combination of dynamic and static quenching 

typically produces a nonlinear Stern-Volmer plots as we found in our case. 

Influence of ionic strength shows salting-out effect. The interaction with pharmaceuticals 

much depends on the origin and structure of humic substances as it has been demonstrated 

comparing humic acids from aquatic sources, soil, peat and reference materials. The 

synchronous-scan fluorescence excitation emission spectra of humic substances are shown in 

Figure 2 and they differ significantly depending on the origin of the humic acids: spectra of 

highly humified HAs (IHSS reference humic acids: Leonardite HA, Pahokee peat HA, as well 

as the industrially produced Aldrich HA) were characterized by two major fluorescence 

peaks. 

Intensity 

160 

140 

120 

100 

80 

60 

40 

20 

0 

Gagu HA 

Daugava HA 


250 300 350 400 450 500 550 600 

Wavelength, nm 

Aldrich HA 

Pahokee HA 

Leonarditte HA 

Figure 2: Synchronous-scan fluorescence spectra of HA used in the study (CHA = 25 mg/L, pH 7) 

Vol. 3 Page - 399 -

Aquatic humic acid isolated from water of the River Daugava was characterized by one peak 

(~ 375 nm), but peat humic acid (isolated from Gagu Sphagnum bog peat) was characterized 

by two peaks (~ 345 and 375 nm). Synchronous scan spectra of humic substances isolated 

from highly humified organic material (leonardite, coal) had an intensive fluorescence peak ~ 

475 nm that was determined by the presence of conjugated unsaturated bond systems bearing 

carbonyl and carboxyl groups (substituting aromatic core structures), but its intensity differed 

with respect to aromaticity of the humic acid selected [5]. 

We have determined the binding constants between HA and selected pharmaceuticals by 

fluorescence quenching technique. It is found that amongst the studied adamantine group 

pharmaceuticals and their precursors the Rimantadine has markedly larger binding constants 

than their structural analogues. This difference is explained by electrostatic interaction 

between HA and studied pharmaceuticals. Our findings suggest that an electrostatic 

interaction plays a dominant role in the complex formation between humic acids and 

pharmaceuticals. The importance of the electrostatic attraction between humic acids and 

pharmaceuticals was also confirmed by a salt effect and pH dependence of the fluorescence 

quenching. The electrostatic interaction between cationic pharmaceuticals (phenibut) and HA 

is weakened at low pH, resulting in decrease in the binding constants. 



It can be supposed the hydrophobic pharmaceuticals can cause aggregation of humic 

molecules, changing their micellar behavior. The obtained results support development of 

understanding of fate of pharmaceuticals in the environment as well as development of 

analytical methods for analysis of pharmaceuticals in waters and medical wastewater 

treatment approaches. 

5. References 

1. Hoffman C.E. Structure, activity and mode of action of amantadine HCl and related compounds. 

Antibiot. Chemother. 27, (1980), 233–250. 

2. Scholtisek C. & Webster R.G. Long-term stability of the anti-influenza A compounds-amantadine 

and rimantadine. Antiviral Res. 38, (3), (1998), 213–215. 

3. Nakashima K., Maki M., Ishikawa T., Yoshikawa T., Gong Y.K. & Miyajima T. Fluorescence 

studies on binding of pyrene and its derivatives to humic acid. Spectrochim. Acta Pt. A. 67, 

(2007), 930–935. 

4. Gadad P., Lei H. & Nanny M.A. Characterization of noncovalent interactions between 6propionyl-dimethylaminonapthalene 

(PRODAN) and dissolved fulvic and humic acids. Water. 

Res. 41, (2007), 4488–4496. 

5. Peuravuori J., Koivikko R. & Pihlaja K. Characterization, differentiation and classification of 

aquatic humic matter separated with different sorbents: synchronous scanning fluorescence 

spectroscopy. Water Res. 36 (18), (2002), 4552–4562. 

Vol. 3 Page - 400 -

Characterization of Soil Organic Matter of Treated Sewage Effluent 

Irrigated Areas 

Bruno Henrique Martins a,b* , Larissa Macedo dos Santos a,c , Débora Marcondes Bastos Pereira 

Milori a , Ladislau Martin-Neto a , Célia Regina Montes d 

a Embrapa Instrumentação Agropecuária, C.P.741, CEP: 13560-970, São Carlos, SP, Brasil; 

b Universidade de São Paulo, Instituto de Química de São Carlos (IQSC/USP), C.P.780, CEP: 

13560-250, São Carlos, SP, Brasil; c Universidade Federal de São Carlos (UFSCar), 

Departamento de Química, CEP: 13565-905, São Carlos, SP, Brasil; d Universidade de São 

Paulo, Centro de Energia Nuclear na Agricultura (CENA/USP), C.P. 96, CEP: 13416-000, 

Piracicaba, SP, Brasil; 

E-mail: brunohm@cnpdia.embrapa.br; larissa@cnpdia.embrapa.br; 

debora@cnpdia.embrapa.br; martin@cnpdia.embrapa.br, crmlauar@usp.br 


The increasing on water sources demand in the cities has done man seek different sources for 

irrigation of crops, since agricultural activity consumes a large amount of this resource. 

According to data from [6], it is estimated that approximately 65% of the water amount 

available in the national territory is targeted to the practice of irrigation of crops, while only 

about 17% is aimed for human consumption. This situation is worrying, once Brazil is a 

country with intense agricultural activity. 

To [4] water represents a development limiting natural resource, both in agricultural and 

industrial activities, and has its quality breakdown by misuse and pollution, largely generated 

by direct discard of raw and treated effluents in water courses. 

According to research accomplished by SABESP (Companhia de Saneamento Básico do 

Estado de São Paulo), only the city of São Paulo, by its STS (sewage treatment station), 

generates nearly 3,000 L·s -1 of TSE (treated sewage effluent). However, in the National Policy 

of Hidric Resources, there is no regulamentation about the use of waste water (such as TSE) 

in any activities. 

In this way, the purpose of the following study is to evaluate the soil organic matter (SOM) of 

irrigated areas, comparing to non-irrigated area, analyzing about the sustainability of the TSE 

use in agricultural soils instead of water, as a contribution in a bigger thematic project. 



This study is part of a multidisciplinary research group of thematic project about use of 

sewage effluent treated by biological process (stabilization pounds, UASB reactor / activated 

sludge) in agricultural soils, sponsored by Fundação de Amparo à Pesquisa do Estado de São 

Vol. 3 Page - 401 -

Paulo – and coordinated by Prof. Dr. Adolpho José Melfi (CENA-ESALQ/USP). 

The project was initiated in January, 2003, and the experimental field was installed near to the 

STS in the city of Lins – SP, operated by SABESP, under cultivation of grazing and 

sugarcane. In this study were analyzed soil samples from the sugarcane area. The city STS is 

Australian kind (primary treatment in anaerobic pounds and secondary treatment in optional 

photosynthetic pounds) with flow rate of 140 L·s -1 and with mostly domestic sewage. The 

experimental arrangement of the sugarcane area was comprised by five treatment blocks with 

four repetitions. The irrigation with TSE was performed according to soil humidity, as it 

follows: SI: soil non-irrigated with TSE; 100: soil irrigated with TSE and soil humidity in the 

same level of field capacity; 125: soil irrigated with TSE and soil humidity 25% above field 

capacity; 150: soil irrigated with TSE and soil humidity 50% above field capacity and 200: 

soil irrigated with TSE and soil humidity 100% above field capacity. In this study were 

analyzed samples of the SI, 100 and 200 conditions. The soil samples were randomly 

collected, in three repetitions by analyzed condition, in May, 2006, in the depth till 100 cm, 

dried at room temperature and subsequently sieved at 0.5 mm mesh. 

The carbon content analyses were carried out by dry combustion [5] in a LECO CN-2000 

instrument, belonging to the Laboratório de Biogeoquímica of CENA/ESALQ. The LIF 

(laser-induced fluorescence) analyses were carried out according [3], in an instrument 

belonging to Embrapa Instrumentação Agropecuária. 



The carbon content analyses for samples for the three different conditions examined showed 

decrease, in all depth consider, being more pronounced in areas subject to TSE irrigation, 

mainly in the 200 condition. The data obtained are illustrated in Table 1. This decrease is 

probably attributed to labile carbon fraction degradation, caused by the increase in microbial 

activity related to the action of TSE in the soil. 

According to [1], the use of TSE as irrigation source may alterate the organic matter 

degradation rate, causing a decrease in the soil carbon content. The authors also remind that it 

may cause an alteration in the soil carbon cycling process. 

Table 1: Carbon content obtained for soil samples subjected to three different types of treatment 

0–10 10–20 20–40 40–60 60–80 80–100 

SI 0.97±0.01 0.96±0.01 0.74±0.01 0.64±0.01 0.54±0.01 0.46±0.01 

100 0.88±0.01 0.86±0.01 0.70±0.02 0.54±0.01 0.53±0.01 0.42±0.03 

200 0.86±0.01 0.82±0.01 0.70±0.01 0.58±0.02 0.52±0.01 0.44±0.01 

Vol. 3 Page - 402 -


This situation is worrying since represents, among other factors, loss of SOM, that may cause 

limitations in soil fertility and structure (taking into consideration its importance to the soil and culture 

and when it comes to a soil with less than 1% carbon content), and possible carbon loss as CO2, 

causing increase in atmospheric greenhouse gases concentration, negatively contributing to the global 

warming scenario. 

The obtained data by LIF spectroscopy, showed by Table 2 and Figure 1, are complementary to the 

carbon content data obtained, achieving excelent data correlation. 

Table 2: Humification degree (HFIL) obtained by Laser-Induced Fluorescence for soil samples 

subjected to three different types of treatment 

0–10 10–20 20–40 40–60 60–80 80–100 

SI 527±5 487±4 718±43 990±7 1254±29 1568±77 

100 548±6 566±9 831±16 1269±6 1289±29 1868±112 

200 574±4 635±5 795±42 1141±15 1328±44 1711±154 

In this way, its suggested that, in this case, the TSE use as irrigation source leads to an alteration in the 

soil organic matter degradation process, probably due to an increase in the soil microbial activity and, 

consequently, more labile carbon fraction degradation (as seen in the carbon content results), 

remaining the most recalcitrant organic matter fraction, harder to degrade. This most recalcitrant 

fraction leads to an increase in the organic matter humification degree, as detected by LIF. 

Figure 1: Humification degree (HFIL) graphics obtained by Laser–Induced Fluorescence for soil 

samples subjected to three different types of treatment 

However, the decrease in the organic matter carbon content was verified even with conventional 

irrigation with water, comparing to irrigation with TSE, in the same experimental field. In this way, it 

may be suggested that the soil itself has this intrinsic characteristic of organic content loss, probably 

due to increase in the soil microbial activity (even when irrigated with water), which is more 

accentuated when irrigated with TSE. 

Otherwise, in his study about stability of organic carbon in deep soil layers controlled by fresh carbon 

Vol. 3 Page - 403 -

supply, [2], observed that changes in agricultural practices, increasing the distribution of fresh carbon 

at depth, could lead to a loss of ancient soil carbon, which affects the present soil carbon content, 

promoting the priming effect in soil. 



Proper weights and discussions about the results for the samples analyzed, it is concluded that, to soil 

conditions analyzed, the employment of TSE replacing the water used in agricultural activities for 

irrigation of crops is worrying and may bring limitations on soil structure and fertility, as evidenced by 

more pronounced decrease on the contents of SOM in samples for the areas under adding TSE, taking 

into account that is a soil with less than 1% carbon content and has negative response even to 

conventional irrigation with water. 

It is suggested that, in this case, irrigation with TSE causes the priming effect in soil, observed by 

more accentuated decrease of carbon content in samples of the irrigated areas, comparing to the nonirrigated 

area, what may become an environmental problem. 

Between the three irrigation conditions analyzed, it is verified a more pronunciated effect in the 

organic matter of samples belonging to the 200 condition (soil irrigated with TSE and soil humidity 

100% above field capacity), as it showed by the lower carbon content and higher humification degree, 

comparing to the SI and 100 conditions. 

However, the experiments must continue to confirm and validate the initials tendencies detected, and 

to search new alternatives for soil and culture tillage to make possible the TSE use and application in 

sustainable conditions. 

References 

1. Falkiner, R.A.; Smith, C.J. 1997. Change in soil chemistry in effluent-irrigated Pinus radiata and 

Eucalyptus grandis. Australian Journal of Soil Research, 35: 131–147. 

2. Fontaine, S.; Barot, S.; Barré, P.; Bdioui, N.; Mary, B.; Rumpel, C. 2007. Stability of organic 

carbon in deep soil layers controlled by fresh carbon supply. Nature, 450: 277–281. 

3. Milori, D.M.B.P.; Galeti, H.V.A.; Martin-Neto, L.; Dieckow, J.; González-Pérez, M.; Bayer, C.; 

Salton, J. 2006. Organic matter study of whole soil samples using laser-induced fluorescence 

spectroscopy. Soil Science Society American Journal, 70: 57–63. 

4. Montes, C. R.; Fonseca, A. F.; Melfi, A. J.; Gloaguen, T.; Mendonça, F. C.; Pivelli, R. P.; Herpin, 

U.; Santos, A. P. R.; Forti, M. C.; Lucas, Y.; Mounier, S.; Carvalho, A.; Almeida, V. V.; 

Cardinalli, C. G.; Steffen, T.; Monteiro R. C. 2004. Agricultural use of stabilization pond effluent: 

a case study in the city of Lins (SP, Brazil). In: Martin-Neto, L.; Milori, D. M. B. P.; Silva, W. T. 

L. (Eds.). Humic substances and soil and water environment, São Carlos: EMBRAPA. p. 732– 

734. 

5. Nelson, D.W.; Sommers, L. E. 1996. Total carbon, organic carbon, and organic matter. In: Sparks, 

D. L. (Ed.). Methods of soil analysis: chemical methods, Madison: Soil Science Society of 

America/American Society of Agronomy. p.961–1010. 

6. Tucci, C. E. M. 2001. Gestão de água no Brasil. Brasília, UNESCO. 156 p. 

Vol. 3 Page - 404 -

Modelling Differential Absorbance Spectra of SRFA During Complexation 

with Copper and Lead 

Deborah J. Dryer a* , Gregory V. Korshin a , Marc F. Benedetti b 

a Department of Civil and Environmental Engineering, University of Washington, Box 

352700, Seattle, Washington, 98195-2700, USA; b Univ. Paris Diderot-Paris 7-IPGP, 

Laboratoire de Géochimie des Eaux, Paris, 75205, France 

E-mail: ddryer@u.washington.edu 



Natural organic matter (NOM) is a component of all natural and engineered environmental 

systems. NOM controls the speciation and distribution of many heavy metals in aquatic 

systems and exerts a strong influence on the release of metal corrosion by-products in 

drinking water systems [1, 2]. In order to understand the interactions of NOM with metals, 

precise measurements of NOM properties like stability constants and binding capacities of 

NOM for protons and metal cations are required. The complex nature of NOM makes this a 

very difficult task. NOM has properties which are strongly dependent on local 

biogeochemical and treatment conditions and contains a diverse array of functional groups 

and molecular sizes [3, 4]. NOM complexation behavior has been studied by a wide variety of 

techniques. However, because these techniques frequently require large amounts of preconcentrated 

NOM samples, a need remains for an efficient means to quantify important 

NOM properties and behaviour in situ. 

Because NOM is the major light-absorbing component of natural waters in the 200-800 nm 

range [5], optical spectroscopy has long been used to assess the NOM content of many water 

sources. However, the wide variety of light-absorbing functional groups (chromophores) in 

NOM results in absorbance spectra which are broad and featureless [6]. Consequently, 

interpretation of these spectra has traditionally been limited to using particular spectral 

parameters (such as absorbance at 254 nm, absorbance ratios, or SUVA254) as surrogates for 

NOM properties like concentration, aromaticity, and molecular size [7-9]. 

Differential absorbance spectroscopy (DAS) is an alternative method for the interpretation of 

absorbance spectral data that focuses on changes in spectra associated with evolving reaction 

conditions which affect NOM chemistry. This technique reveals changes which are not 

apparent, or do not exist, in the raw absorbance spectra. DAS is capable of detecting very 

subtle changes in NOM chemistry and this high sensitivity eliminates the need for sample pretreatment 

and makes it a promising technique for in situ elucidation of NOM behaviour. In 

prior research, this technique has been applied to understanding NOM protonation behaviour 

Vol. 3 Page - 405 -

[10]. In this study, DAS is applied to the exploration of the complexation behaviour of 

standard Suwannee River fulvic acid. 

It is also desirable to have a means to model and predict the metal complexation behaviour of 

fulvic acids. While many models have been developed and utilized, the NICA-Donnan model 

has emerged as a very useful means for fitting and predicting complexation behavior of fulvic 

acids [11, 12]. This model uses a continuous distribution of binding site affinities to model the 

direct complexation of metal cations to NOM binding sites, and models the indirect 

electrostatic interactions between NOM and metal cations through distribution of potential 

throughout a uniform Donnan gel phase. This study uses the NICA-Donnan model for metal 

complexation to fulvic acids to fit the differential absorbance spectra generated during 

complexation of SRFA with metals. 

2. Methods 

A Suwannee River fulvic acid (SRFA) sample was obtained from the International Humic 

Substances Society (Sample # 1S101F). SRFA solutions were prepared at DOC 

concentrations of 5 mg/L, and background ionic strength was established by including 0.01 M 

NaClO4. The pH of NOM solutions during titrations was controlled through addition of 

appropriate amounts of HClO4 or NaOH, and the volume changes associated with these 

additions of acid and base were corrected for in the final data analysis. Metal concentrations 

were adjusted between 0 – 1000 μg/L Cu 2+ or Pb 2+ through addition of small volumes of 

dilute CuSO4 and PbClO4 solutions. Absorbance spectra were recorded in a 5 cm quartz cell 

on a Perkin-Elmer Lambda 18 UV/Vis Spectrophotometer. Differential absorbance data 

fitting to model calculations was done using visual MINTEQA. 



Absorbance spectra for SRFA at varying metal concentrations retain the same broad and 

featureless shape throughout the entire range of the titrations. However, the differential 

spectra for these titrations show the emergence of clear features, as illustrated by Figure 1. 

They indicate that subtle changes of the absorbance of SRFA can be detected even at copper 

concentrations as low as 5 µg/L. At higher CuTOT values, the presence of several bands 

becomes notable, the most prominent of which have maxima at 245 and 390 nm. 

Correlations between the amount of copper bound by SRFA and changes in absorbance were 

calculated using by examining the relative change of the ratio of absorbances at 350 and 390 

nm (denoted as (ΔRA/RA), The selection of the wavelength of 390 nm is due to the presence of 

Vol. 3 Page - 406 -

a distinct Cu-differential spectra feature of SRFA at all pH with maximum close 390 nm. 

Δ A 

390 

⎜ ⎟ 

⎝ A350 

⎠ 0 

The A A parameter is defined as: 

R R / 

A A 

In this expression, terms 350 and 

Δ 

R 

⎛ ⎛ 

⎜ 

A 

⎜ ⎜ 

= 

⎝ 

A 

390 

R A ⎝ 350 ⎠ Cu 

( 390 ) Cu / ( ) 390 / A350 

0 A 

⎞ 

⎟ 

⎛ A 

− 

⎜ 

⎝ A 

⎛ A 

⎜ 

⎞ 

⎟ 

390 

350 

⎞ ⎞ 

⎟ 

⎟ 

⎠ 

⎟ 

0 ⎠ 

correspond to the ratio of absorbances 

at 390 and 350 nm at any selected total copper concentration and its absence, respectively. 

This ratio is introduced in order to more clearly illustrate the emergence of the particular band 

with a maxima near 390 nm which emerges only for Cu concentrations greater than ~40 μg/L. 

differential absorbance (cm -1 mg -1 L) 

0.005 

0.0045 

0.004 

0.0035 

0.003 

0.0025 

0.002 

0.0015 

0.001 

0.0005 

5 μg/L 

200 μg/L 

500 μg/L 

1000 μg/L 

0 

200 250 300 350 400 450 

wavelength (nm) 

Figure 1: Differential absorbance spectra of SRFA with selected copper concentrations at pH 6 

To understand the nature of these phenomena in more detail, the complexation of copper by 

SRFA was modeled using visual MINTEQA program that incorporates generic SRFA 

protonation and complexation constants. The SRFA-bound copper determined in these 

calculations was predominated the copper bound by the operationally defined phenolic and 

carboxylic groups, while the copper bound via Donnan gel interactions was determined to be 

negligible in comparison. The strong correlation between the modeled bound copper 

concentration and the (A390/A350)Cu ratio is illustrated in Figure 2. 

Change of A 390/A 350 ratio 

0.21 

0.18 

0.15 

0.12 

0.09 

0.06 

0.03 

pH 5 

pH 6 

pH 7 

pH 8 


R 2 = 0.97 

0.00 

0.0E+00 2.0E-06 4.0E-06 6.0E-06 8.0E-06 1.0E-05 1.2E-05 

Total NOM-bound copper (M) 

Figure 2: Correlation between total predicted NOM-bound copper and changes in ΔRA/RA index 

Vol. 3 Page - 407 -



This study examined the differential absorbance spectra of metal complexation with 

Suwannee River fulvic acid. While absorbance spectra show a very subtle change in intensity 

with increasing metal concentrations, they retain their characteristic featureless shape 

throughout the titrations. Differential absorbance spectra, on the other hand, show a number 

of features that are not present in the traditionally interpreted absorbance spectra. These 

features can be analyzed to reveal information about the nature of complexation-active 

chromophores in NOM. The differential absorbance spectra of SRFA can be successfully 

correlated to bound metal concentrations calculated based on the NICA-Donnan model 

theory. This study demonstrates that differential absorbance spectroscopy is a useful method 

to examine in situ the complexation behaviour of NOM. DAS can potentially provide detailed 

information about the emergence and contribution of different complexation-active functional 

groups and has the potential to elucidate the presence of functionalities which may not be 

detectable in traditional potentiometric experiments. In principle, this technique could allow 

the obtainment of detailed in situ information about NOM chemistry and reactivity caused by 

a wide variety of environmental and water treatment processes. 

References 

1. Korshin, G. V.et. al. Influence of NOM on copper corrosion,Journal of the American Water 

Works Association 1996, 88, 36–47. 

2. Edwards, M.; Sprague, N. Organic matter and copper corrosion by-product release: A mechanistic 

study,Corrosion Science 2001, 43, 1–18. 

3. Croue, J.-P.et. al. Isolation, fractionation, and characterization of natural organic matter in 

drinking water, AWWA Research Foundation, 2000. 

4. Leenheer, J. A.; Croue, J.-P. Characterizing aquatic dissolved organic matter,Environ. Sci. 

Technol. 2003, 37, 18A–26A. 

5. Stewart, A. J.; Wetzel, R. G. Fluorescence: Absorbance ratios –a molecular-weight tracer of 

dissolved organic matter,Limnol. Oceanogr. 1980, 25, 559–564. 

6. Del Vecchio, R.; Blough, N. V. On the origin of the optical properties of humic 

substances,Environ. Sci. Technol. 2004, 38, 3885–3891. 

7. Chin, Y.-P.et. al. Molecular weight, polydispersity, and spectroscopic properties of aquatic humic 

substances,Environ. Sci. Technol. 1994, 28, 1853–1858. 

8. Novak, J. M.et. al. Estimating the percent aromatic carbon in soil and aquatic humic substances 

using ultraviolet absorbance spectroscopy,J. Environ. Qual. 1992, 21, 144–147. 

9. Peuravouri, J.; Pihlaja, K. Molecular size distribution and spectroscopic properties of aquatic 

humic substances,Analytica Chimica Acta 1997, 337, 133–149. 

10. Dryer, D. J.et. al. In situ examination of the protonation behavior of fulvic acids using differential 

absorbance spectroscopy,Environ. Sci. Technol. 2008, 42, 6644–6649. 

11. Kinniburgh, D. G.et. al. Metal ion binding by humic acid: Application of the NICA-Donnan 

model, Environ. Sci. Technol. 1996, 30, 1687–1698. 

12. Benedetti, M. F.et. al. Metal ion binding by natural organic matter: From the model to the 

field,Geochim. Cosmochim, Acta 1996, 60, 2503–2513. 

Vol. 3 Page - 408 -

Behavior of Soil Carbon in Amended Areas with Sewage Sludge 

Bruno Henrique Martins a,b , Tânia Leme de Almeida a* , Sérgio Gaiad c , Débora Marcondes 

Bastos Pereira Milori a , Ladislau Martin-Neto a 

a Embrapa Instrumentação Agropecuária, C.P.741, CEP: 13560-970, São Carlos, SP, Brasil; 

b Instituto de Química de São Carlos, Univ. de São Paulo (IQSC/USP), C.P. 780, CEP: 13560- 

250, São Carlos, SP, Brasil; c Embrapa Florestas, C.P. 319, CEP: 83411-000, Colombo, PR, 

Brasil 

E-mail: brunohm@cnpdia.embrapa.br; tlalmeida@yahoo.com.br; gaiad@cnpf.embrapa.br; 

debora@cnpdia.embrapa.br; martin@cnpdia.embrapa.br 


The Greenhouse Effect, an Earth’s natural and essential phenomenon, lately has been 

increased by the anthropogenic intervention, by fossil fuel burning, deforestation, and mostly 

wrong agricultural tillage, leading to an increase in the atmospheric gases that causes this 

effect. 

Correct soil tillage and forestry practices are considered important tools to promote decrease 

of emissions of GHG and mitigating of Greenhouse Effect through soil carbon sequestration. 

Soil represents is the third greater pool of carbon in the planet. It is estimated that there is 

approximately 2300 Petagrams of carbon in soils, which represents nearly three times the 

atmospheric carbon concentration. 

Sewage sludge (SS) plays an important role as soil fertility improver because it contains high 

levels of organic matter and nutrients [1]. However, its application requires careful 

monitoring to avoid soil contamination and changes in organic matter that could cause serious 

implications for the crop where it is applied. 

It is related that SS plays an important role as soil fertility improver, hence it is a wastewater 

treatment product and has the potential to enhance soil productivity, as it contains high levels 

of organic matter and nutrients [1]. 

In this way, the purpose of the following study is to evaluate the SOM of SS amended areas, 

comparing to no amended area, analyzing about the sustainability of its use in forestry 

systems as a tool for mitigating GHG emissions and sequestrating atmospheric carbon. 



The experimental field under eucalyptus plantation is installed in two different farms (Entre 

Rios and Areona) in the city of Itatinga, São Paulo state. Each farm was divided in two 

Vol. 3 Page - 409 -

different areas, according to the SS amendment. The soil profile in the Entre Rios farm was 

characterized as an Oxisoil, with a clay content varying between 16 and 20%, while the 

Areona farm soil is characterized as Quartzarenic Neosoil, with a clay content varying 

between 5 and 12%. The sample identification according to the SS amendment was performed 

as it follows: ER 60 (Entre Rios farm and SS amended), ER 228 (Entre Rios farm and no SS 

amended), AN 254 (Areona farm and SS amended) and AN 36 (Areona farm and no SS 

amended). 

In December, 2009, soil samples were collected in three repetitions in the depths 0–10 and 

10–20cm. They were dried at room temperature and subsequently sieved at 0.5 mm mesh. The 

SS amendment occurred ten days before the plantation, at a volume between 1500 and 2000 

kilograms per hectare. 

Carbon content measurements were carried out by an elemental analyzer Carlo Erba EA- 

1110 instrument. The measurements were made in triplicate, taking into account depth and 

condition analyzed in both farms. 



The carbon content results for Entre Rios and Areona farm, as well as some soil physical 

characteristics and eucalyptus plantation beginning, are shown in Table 1. 

In the case of the Entre Rios Farm, probably SS amendment increased the microbial activity 

in soil by high availability of fresh organic matter [4] what, in a second stage, must have 

triggered a soil carbon decrease by degradation of stable fractions [3]. 

According to [3], in their study about stability of organic carbon in soils, increasing of fresh 

organic matter at depth, could lead to a loss of ancient soil carbon, which leads a decreasing 

of total soil carbon content as a function of time, promoting the priming effect in soil. 

This situation in the Entre Rios farm is worrying since represents, among other factors, loss of 

soil organic matter by microbial activity, which may cause limitations in soil fertility and 

structure, and possible carbon loss as CO2, causing increase in atmospheric GHG 

concentration, negatively contributing to the global warming scenario. 

The results obtained for the samples of the Areona farm showed an inverse behavior 

comparing to the samples of the Entre Rios farm. It was observed that in the SS amended area 

the values of carbon content were higher than in the no amended area, in all analyzed depths. 

Vol. 3 Page - 410 -


Table 1: Carbon content results obtained for soil samples from Entre Rios and Areona Farm. ER 228: 

no SS amended, ER 60: SS amended, AN 36: no SS amended and AN 254: SS amended 

SAMPLE C Content Soil Profile Clay Content 

Eucalyptus 

Plantation 

ER 228 0–10 I 1.85±0.02 Oxisoil 16 – 20 % Nov/2004 

ER 228 0–10 II 0.90±0.01 Oxisoil 16 – 20 % Nov/2004 

ER 228 0–10 III 0.98±0.03 Oxisoil 16 – 20 % Nov/2004 

ER 228 10–20 I 1.21±0.01 Oxisoil 16 – 20 % Nov/2004 

ER 228 10–20 II 0.80±0.04 Oxisoil 16 – 20 % Nov/2004 

ER 228 10–20 III 0.67±0.02 Oxisoil 16 – 20 % Nov/2004 

ER 60 0–10 I 0.84±0.03 Oxisoil 16 – 20 % Apr/2004 

ER 60 0–10 II 0.84±0.02 Oxisoil 16 – 20 % Apr/2004 

ER 60 0–10 III 1.18±0.04 Oxisoil 16 – 20 % Apr/2004 

ER 60 10–20 I 0.37±0.02 Oxisoil 16 – 20 % Apr/2004 

ER 60 10–20 II 0.45±0.01 Oxisoil 16 – 20 % Apr/2004 

ER 60 10–20 III 0.60±0.03 Oxisoil 16 – 20 % Apr/2004 

AN 36 0–10 I 0.57±0.03 Quartzarenic Neosoil 5 – 12 % Mar/2008 

AN 36 0–10 II 0.67±0.02 Quartzarenic Neosoil 5 – 12 % Mar/2008 

AN 36 0–10 III 0.70±0.01 Quartzarenic Neosoil 5 – 12 % Mar/2008 

AN 36 10–20 I 0.41±0.05 Quartzarenic Neosoil 5 – 12 % Mar/2008 

AN 36 10–20 II 0.46±0.01 Quartzarenic Neosoil 5 – 12 % Mar/2008 

AN 36 10–20 III 0.45±0.02 Quartzarenic Neosoil 5 – 12 % Mar/2008 

AN 254 0–10 I 0.71±0.01 Quartzarenic Neosoil 5 – 12 % May/2008 

AN 254 0–10 II 0.74±0.03 Quartzarenic Neosoil 5 – 12 % May/2008 

AN 254 0–10 III 0.75±0.02 Quartzarenic Neosoil 5 – 12 % May/2008 

AN 254 10–20 I 0.53±0.03 Quartzarenic Neosoil 5 – 12 % May/2008 

AN 254 10–20 II 0.61±0.04 Quartzarenic Neosoil 5 – 12 % May/2008 

AN 254 10-20 III 0,54±0,01 Quartzarenic Neosoil 5 – 12 % May/2008 

Vol. 3 Page - 411 -

Anyway, these results showed an interesting behavior. The soil in Areona is characterized as 

Quartzarenic Neosoil (a recently generated soil with amounts of quartz in its composition). 

This kind of soil has a major fraction of sand and low carbon concentration in your profile. 

This way, it was not expected strong interaction between SS and the organic matter of this 

soil. However, it was noted that soil tillage with SS amendment in the Areona farm leads to a 

better carbon accumulation when compared with the same tillage in the Entre Rios farm. 

It is important to note that the decrease in the carbon content in the Entre Rios farm is 

potentialized and accentuated by the SS amendment, probably, due to an increase in the 

microbial activity in soil. 

However, it is important to evaluate if this difference was due to different soil properties for 

each farm or if may be attributed to period of the experiment. Eucalyptus plantation began in 

2004 in Entre Rios Farm and in 2008 in the Areona farm. 

4. Conclusion 


Studies of soil amendment using SS are very important to avoid negative environmental 

consequences, as soil contamination or decreasing of carbon content in soil. This kind of 

residues management can be positive depending on the situation, and for this reason each case 

needs to be carefully studied. 

Thus, field experiments must continue in both farms to confirm and validate the initials 

tendencies detected. New alternatives for soil tillage in forestry systems can be suggested to 

make possible SS soil amendment in sustainable conditions with environmental benefits. 

References 

1. Arraigada, C.; Sampedro, I.; Garcia-Romero, I.; Ocampo, J. Sci. Total Environ., 407 (2009). 

2. EMBRAPA, Brazilian System of Soil Classification, National Center of Soil Research, Rio de 

Janeiro, 1999. p. 412. 

3. Fontaine, S.; Barot, s.; Barré, P.; Bdioui, N.; Mary, B.; Rumpel, C., Nature, 450 (2007) 8. 

4. Martins, E. L.; Coringa, J. E. S.; Weber, O. L. S., Acta Amazonica, 39 (2009) 3. 

Vol. 3 Page - 412 -

Gold(III) and Nanogold Interaction with Humic Acids: Spectrophotometry, 

Capillary Electrophoresis and Mass Spectrometric Study 

Nagender Reddy Panyala a , Eladia Mª Peña-Méndez b , Josef Havel a,c * 

a Department of Chemistry, Faculty of Science, Masaryk University, Kotlarska 2, 61137 

Brno, Czech Republic; b Department of Analytical Chemistry, Nutrition and Food Chemistry, 

Faculty of Chemistry, University of La Laguna, Campus de Anchieta, 38071 – La Laguna, 

Tenerife, Spain; c Department of Physical Electronics, Faculty of Science, Masaryk 

University, Kotlarska 2, 61137 Brno, Czech Republic 

E-mail: havel@chemi.muni.cz 


Humic substances (HS) naturally occurring in soils and waters are usually divided into humic 

acids (HA) soluble at high pH and insoluble in acids, fulvic acids (FA) soluble at all pH 

values and insoluble humin. HA are extensively studied [1-3] but their real structure is still 

not completely known. HA easily aggregate, show high complexing ability towards all metal 

ions but also interact with various organics and xenobiotics [3], are also interacting with 

minerals and gold as it is known for a long time [4, 5]. High attention is paid to the role of HA 

in the transport of the elements including platinum group metals and gold in the environment 

[6]. Gold in various forms interacts with HA [7] and the very first remark about this is known 

since 1900 [8]. However, the interaction is by far not sufficiently explained and not 

completely understood. Therefore, the aim of this work is to study and elucidate the 

interaction of HA with Au(III) and metallic gold in the form of gold nano-particles, as this 

knowledge might be quite important to explain gold migration in the environment and for 

gold mining industry. 



The HA used in this work were Soil HA standard (IS102H) and Leonardite HA standard 

(IS101H) from International Humic Substances Society (IHSS) and coal-derived Czech HA 

standard [9]. Gold(III) chloride as HAuCl4·3H2O was from Sigma-Aldrich (Steinheim, 

Germany). Gallic acid was from Lachema (Brno, Czech Republic). Deionized water was 

double-distilled from a quartz apparatus purchased from Heraeus Quartzschmelze (Hanau, 

Germany). All other reagents were of analytical grade purity. Mass spectra were measured 

using MALDI instrumentation of Kratos Shimadzu (Manchester, UK) and/or MALDI-TOF 

Auto-flex mass spectrometer of Bruker Daltonics (Bremen, Germany). CE was carried out on 

the SpectraPhoresis 2000 Thermo Separation Products (Fremont, CA, USA). 

Vol. 3 Page - 413 -


Interaction of Au(III) with HA. HA are reducing Au(III) to metallic gold quite rapidly. An 

example of kinetic spectra concerning reduction process is given in Figure 1 A, B. Figure 1 B 

is showing that in the first stage the reaction is faster and later on slower. Figure 2 illustrates 

details of kinetic spectra (for another HA preparation) [9]. Two isosbestic points and two new 

absorption bands developed (550 and 580 nm) indicate that at least two different reactions 

are going on. 

Absorbance (AU) 

2.5 

2 

1.5 

1 

0.5 

0 

Absorbance 

HA only 

ג ג1 max ג2 max 

1 max ג2 max 

Time 

400 450 500 550 600 650 700 


A 

750 

Absorbance 

2.5 

2 

1.5 

1 

0.5 

0 

0 50 100 150 

time (min) 

200 250 300 

Figure 1: A, B The kinetic spectra concerning of the reduction of HAuCl4 by HA. Conditions: 

pH = 4.6, Total concentrations: HA = 0.12 and Au(III) = 1.6 mM 

1.6 

1.5 

1.4 

1.3 

1.2 

1.1 

1 

1st 1 isosbestic point 

st 1 isosbestic point 

st isosbestic point 

253 258 263 268 



A 

Absorbance 

1.2 

1.1 

1 

0.9 

0.8 

2nd 2 isosbestic point 

nd 2 isosbestic point 

nd isosbestic point 

B 

391nm 

500nm 

568nm 

580nm 

640nm 

B 

265 275 285 295 305 315 325 


Figure 2: A, B. The kinetic spectra concerning the reduction of HAuCl4 by HA (Chemapex standard). 

pH = 2.9; Total concentrations: HA = 0.03 and Au(III) = 0.3 mM 

Electrophoretic study of nanogold interaction with HA. Gold (III) and aqueous nanogold 

solutions were mixed with HA in various ratios. Nanogold was prepared using gallic acid 

(model compound of HA) as a reducing agent. The electrophoretic separation was done in 

borate buffer (pH 9.6). Selected examples of electropherograms are given in Fig. 3. HA 

show mostly just one high “hump” of the HA aggregate, in agreement with the literature 

Vol. 3 Page - 414 -

[9]. Even if HA are negatively charged they migrate towards the cathode due to high 

electroosmotic flow (EOF). Figure 3 B concerning a mixture of nanogold and HA solution 

shows that there are several peaks observed. This is probably due to the interaction of 

different size nanogold with different HA fractions. 

Absorbance (mAU) 

0.017 

0.012 

0.007 

0.002 

EOF 

0.5 1.5 2.5 3.5 4.5 5.5 6.5 7.5 

-0.003 

Migration time (min) 

A 

Absorbance (mAU) 

0.009 

0.007 

0.005 

0.003 

0.001 

- 0.001 0.5 1.5 2.5 3.5 4.5 5.5 6.5 7. 5 

- 0.003 

EOF 

Migration time (min) 

Figure 3: A Capillary electrophoretic separation of HA. B Electropherogram concerning a mixture 

(nanogold+HA). Conditions: 20 mM borate buffer (pH 9.6); Total concentrations: HA = ~ 0.16 and 

Au(III) = ~ 1.6 mM) 

Mass spectrometry. Interaction of gold (III) and nano-gold with HAs was followed by mass 

spectrometry using a commercial MALDI instrumentation where mass spectra were obtained 

in Laser Desorption Ionization (LDI) mode, i.e. using no matrix. However, under these 

conditions in LDI mass spectra only the formation of Aun clusters and adducts of gold with 

low molecular weight HA fragments (215 and 347 Da) were observed. It seems that either the 

stability of {Aun, HA} is low or such adducts are decomposed in the process of ionization. 



It was found by spectrophotometric kinetic study that the reduction of Au(III) with HA is 

relatively fast in wide pH range. The redox reaction proceeds at least in two main steps and 

formed nanogold is stabilized in HA solution similar like with citrate but at elevated gold 

concentration a kind of {Aun, HA} aggregate is anyway precipitated. The interaction of 

nanogold with HA as studied by electrophoresis was found to be relatively weak. But the 

formation of several peaks indicates the complexicity of the reaction mixture. Mass 

spectrometry indicates that nanogold is forming adducts of gold with some low molecular 

weight compounds. Possible explanation is that supramolecular {Aun, HA} aggregates might 

be decomposed in the process of laser desorption ionization. 

Taking into account available literature data and the results achieved we can conclude that the 

both reaction of Au(III) with HA and interaction of nanogold with HA are quite complex. We 

suggest that reduction of Au(III) with HA goes in several steps via Au(III) and Au(II) and 

Vol. 3 Page - 415 - 

B

Au(I) to at least 2 different size of Aun clusters. Complexation reaction with Au(II) and Au(I) 

with HA fractions is not excluded. However, to prove that and to prove also possible 

formation of {Aun, HA}supramolecules needs further intensive research. 



Grant of the Academy of Sciences of the Czech Republic (project KAN 101630651) and of 

the Ministry of Education, Youth and Sports of the Czech Republic (projects 

MSM0021622411 and LC 06035) are acknowledged. 

References 

1. F. J. Stevenson (Ed.), Humus Chemistry, Willey-Interscience, New York, 2nd edn., 1994, Chap. 

12, p. 288. 

2. J. Havel, D. Fetsch, in: Wilson I. (Ed.), Encyclopedia of Separation Science, Academic 

3. Press Ltd., London, UK, 2000, p. 3018. 

4. M. L. Pacheco, E. M. Peña-Méndez, J. Havel, Chemosphere 51 (2003) 95. 

5. F. W. Freise, Econ. Geol., 26 (1931) 421. 

6. W. G. Fetzer, Econ. Geol., 41 (1946) 47. 

7. S. A. Wood, Ore Geol. Rev., 11 (1996) 1. 

8. M. L. Machesky, W. O. Andrade, A. W. Rose, Chem. Geol., 102 (1992) 53. 

9. E. E. Lungwitz, t. prakt. Geol., (1900) 71. 

10. L. Pokorná, D. Gajdošová, S. Mikeska and J. Havel, in: E. A. Ghabbour, G. Davies, (Ed.) 

Versatile Components of Plants, Soils and Water, The Royal Society of Chemistry (RSC), 

Cambridge, 2000, p. 299. 

Vol. 3 Page - 416 -

Abiotic Treatment of Rice Bran Using an Accelerator Including Organo- 

Iron Compound 

Hikari Kanno * , Naoya Tachibana, Masami Fukushima 

Laboratory of Chemical Resources, Division of Sustainable Resources Engineering, Graduate 

School of Engineering, Hokkaido University, Sapporo, Hokkaido 060-8628, Japan 

E-mail: d-kethik@beach.ocn.ne.jp 



Because raw organic waste (ROW) produced during food processing contains organic matter 

and plant nutrients, it represents a potentially useful material for use as a soil amendment. 

Composting typically involves allowing ROW to be converted to compost via the 

fermentation by microbial growth. However, this technique is time consuming and may lead 

to several adverse effects on soils and plants due to the presence of insufficiently digested and 

unstable organic matter in the ROW [1]. These effects include an increase in the rate of 

mineralization of native soil organic carbon through extended microbial oxidation, the 

development of anaerobic conditions as the result of the mineralization of large amounts of 

non-stabilized organic carbon with associated extended oxygen-consumption, and alteration 

in soil pH [2]. Therefore, for optimum use, ROW needs to be converted into chemically stable 

compounds, i.e., humic substances. 

It is generally accepted that humic substances are formed via polycondensation reactions of 

plant, animal and microbial decay products, such as amino acids, phenols and sugars 

(humification processes). Iron in clay minerals can serve as a Lewis acid, which facilitates the 

polycondensation of phenolics and amino acids [3]. In the present study, we describe a system 

that allows the efficient treatment of ROW, in which an organo-iron compound is used to 

accelerate the process and an instrument that reduces the volume of wet ROW by heating. In 

this study, rice bran was used for a model ROW, because of its homogeneous chemical 

composition (e.g., lipids, phenols, proteins and vitamins). The quality of humic acid (HA) in 

the compost-like material (CLM) samples can serve as an indicator of the maturity of a CLM 

[4]. In this study, HAs were extracted from CLM samples and then purified. To optimize the 

conditions for treatment by the proposed technique, the degree of humification for the HAs 

were determined, in terms of elemental composition, acidic functional group content, 

molecular weight, Uv-vis absorption and FTIR spectroscopy. 

Vol. 3 Page - 417 -



The preparation of the CLM sample. The CLM sample was prepared by mixing rice bran, an 

extending agent (sawdust or Akadama soil i.e., tuff loam) and an accelerator (an organ-iron 

compound, Daiso KET Institute Co., Ltd). The mixture was first incubated at 60 ºC for either 

1 day or 5 days. The mixture was subsequently transferred to the instrument for garbage 

processing (MS-N10, Panasonic Co., Ltd.), and the CLM samples were prepared by ustulating 

by incubating for 1, 3 or 15 days. Detailed conditions used for the treatment are summarized 

in Tables 1 and 2. 

Table 1: Ratio (%) of rice bran, extending agents and accelerator 

CLM samples Rice bran Akadama soil Sawdust Accelerator 

A 50 - 50 1 

B 50 50 - 1 

C 100 - - 1 

D 50 - 50 - 

E 50 50 - - 

Table 2:Processing periods (day) for preparing CLM samples 

Condition 

Processing time (day) 

Incubator Garbage processor 

S - 1 

M 1 1 

L 5 3 

LL 5 15 

LL : Half quantity of raw materials were divided to garbage processor 

The refinement and analysis of the HA. The HAs were isolated from the prepared CLMs by 

extraction with aqueous 0.1 M NaOH and purified according to the procedure a recommended 

by the International Humic Substances Society [5]. The HA samples were analyzed by the 

methods described in a previous report [4], for elemental composition (C, H, N, S, and ash), 

acidic functional groups (carboxylic acids and phenolic hydroxyl groups), UV-vis absorption 

spectra, FTIR spectra and molecular weight by HPSEC. 

Vol. 3 Page - 418 -



Yields and elemental compositions of Has. As shown in Fig. 1, the yields of HA that was 

prepared in the presence of Akadama soil as an extending agent (B and E) were significantly 

larger than samples produced in the presence of saw dust (A and D). Thus, the Akadama soil 

is preferable for use as an extending agent. On the other hand, elemental analyses of the HAs 

Figure 1 The yield of HAs in each treatment condition 

(Table 3) showed that the H/C atomic ratio decreased and the O/C and N/C atomic ratios 

increased with increasing incubation time. These results show that a longer incubation results 

in the formation of HAs with a higher degree of humification. 

Table 3:Elemental (C, H, N, O, S and ash) compositions of humic substances 

Sample Condition %C % H % N % O % S % ash 

B S 62.85 9.81 1.15 21.28 0.69 4.22 

M 67.27 10.38 0.95 19.50 0.67 1.24 

L 45.52 6.00 2.27 42.33 0.28 3.60 

LL 39.30 5.31 2.25 49.45 0.80 2.89 

E S 64.04 10.00 1.31 20.97 0.19 3.49 

M 66.94 10.39 1.13 18.75 0.75 2.04 

L 57.28 8.15 1.96 28.85 0.30 3.46 

LL 39.54 5.97 1.18 49.92 0.49 2.90 

Alteration of structural features. Figure 2 shows FTIR spectra of the HAs, isolated from CLM 

samples that contained added Akadama soil (B with the accelerator; E without the 

accelerator). The peaks at 2900–2800 cm -1 , corresponding to alkyl C–H stretching (b in Fig. 

2) decreased with increasing incubation time. In addition, the following peaks also increased 

with incubation time: 3400 cm -1 for phenolic O–H stretching and/or amine N–H stretching (a 

in Fig. 2); 1600 cm -1 for aromatic C=C ring stretching (c in Fig. 2); 1200–1000cm -1 for C–O 

Vol. 3 Page - 419 -

stretching of alcohols and/or ethers (d in Fig. 2). Significant alterations in these peaks were 

observed in the case of the incubation conditions for B-L and E-LL. These results are 

consistent with the elemental analysis data, which suggest that a longer incubation time leads 

to the formation of HAs with a higher degree of humification. 



Figure 2: The FTIR spectra of the HAs from CLM samples 

(i) The extent of humification is dependent on the incubation time used, and adding an 

accelerator is effective in shortening the required incubation time. 

(ii) For incubation pattern B, the intensity of the FT-IR peak for the incubation pattern of L 

was similar to that for LL. Thus, the incubation period for pattern L is sufficient to produce a 

matured CLM. 

(iii) Because of the higher yield of HA, Akadama soil is useful as an extending agent. 

References 

1. F. Sellami, S. Hachicha, M. Chtourou, K. Medhioub and E. Ammar, Bioresour. Technol., 99 

(2008) 6900. 

2. W. Shi, J. M. Norton, B.E. Miller and M.G. Pace, Appl. Soil Ecol., 11 (1999) 17. 

3. A. Miura, R. Okabe, K. Izumo and M. Fukushima, Appl. Clay Sci. 46 (2009) 277. 

4. M. Fukushima, K. Yamamoto, K. Ootusuka, T. Komai, T. Aramaki, S. Ueda, S. Horiya, Biores. 

Technol., 100 (2009) 791. 

5. R.S. Swift, In, Methods of Soil Analysis Part 3, Soil Science Society of America, Madison, 1996, 

p. 1018. 

Vol. 3 Page - 420 -

Effect of Humic Substances on Uranium Removing by Bacterium 

Bacillus polymyxa IMV 8910 from Aqueous Solution 

I. Shevchuk * , N. Klymenko 

A.V. Dumansky Institute of Colloid Chemistry and Chemistry of Water, National Academy 

of Sciences of Ukraine. 42 Vernadsky Avenue, Kyiv 03680, Ukraine 

E-mail: fjord_n@ukr.net 



Uranium is a long-lived radionuclide that is an ecological and human health hazard. The 

mining and processing of uranium for nuclear power plants and nuclear weapons production 

have resulted in the generation of significant amounts of radioactive wastes. The treatment of 

low charge effluents, conciliating economic and technical constraints is impossible with 

traditional physical-chemical processes. It has been suggested that biomass could be used to 

decontaminate these wastes and to concentrate metals. Biological methods are the most 

ecologically appropriate techniques. The major advantages of microbial treatment are selfreproducibility, 

adaptability, recyclisation of bioproducts, specificity, and good cost/benefit 

ratio. 

In most aquatic systems, species of natural organic matter (NOM), such as humic and fulvic 

acids, constitute an important pool of ligands for complexing metals. NOM is a 

polyfunctional, polyelectrolytic, heterogeneous amalgam of organic molecules of varying 

molecular weight and size. Its physical and chemical properties can be a function of the 

nominal molecular weight (e.g., [1]); properties will also vary from one source to the next [2]. 

Although the chemical and physical properties of NOM have been extensively studied and its 

metal binding capability is undisputed [2], there still remain many questions regarding its role 

in metal binding in heterogeneous systems. 

In the process of water purification humic substances (HS) are partially destroyed and can 

form toxic soluble complex compounds with radionuclides, that increase their migration to 

drinking water [3–6]. It is known that, due to the high ability to complexation of actinides, the 

effect of dissolved organic matter on the sorption of these radionuclides is manifested to a 

greater extent than in the case of radionuclides of alkali and alkaline earth elements [5]. 

Existing in the literature are several experimental and modeling studies that have examined 

U(VI) binding by NOM [5–8]. In general, each of these studies [5–8] concluded that NOM 

has a strong affinity for uranium (VI). 

Vol. 3 Page - 421 -

In previous studies [9, 10] was shown that Bacillus polymyxa IMV 8910 cells may be using 

for removing of uranium (VI) from aqueous solutions. In this paper, we present results of 

investigation the influence of humic substances on the efficiency of extraction of uranium 

(VI) from aqueous solutions by biosorbent based on Bacillus polymyxa IMV 8910 cells. 


Bacterium and growth conditions. The strain of Bacillus polymyxa IMV 8910 used in this 

study was obtained from Institute of Microbiology and Virology, National Academy of 

Sciences of Ukraine. The bacteria were subcultured in the laboratory using a meat-peptone 

broth with addition of glucose (30 g/l) at 30°C with agitation. Cultures were harvested at 24 h 

by centrifugation at 10 000×g for 15 min. Cells were triple washed by distilled water (dH2O) 

and resuspended in a minimal volume of dH2O at a concentration of approximately 0.6 g/l. 

Metal uptake experiments. Biosorption experiments were carried out in triplicate series. 

Uranium is provided as uranyl sulfate (UO2SО4·3H2O). The pH was adjusted with 1 M HCl 

and 1 M NaOH before the addition of cells suspension aliquot. The experiments were 

performed at room temperature. As sorption equilibrium was reached, biomass was removed 

by centrifugation at 10 000×g for 15 min. The residual concentration of uranium in solution 

was determined by arsenazo III method. Sorption capacity is calculated by: 

a = (Co-Ce)V/m; 


where a is the sorption capacity (μmol/g of biomass), Co the initial metal concentration, Ce is 

the residual metal concentration in solution (μmol/l), V the volume of solution (l) and m the 

sorbent mass (g). 

Effect of humic substances on sorption of uranium (VI). For investigation of the impact of 

humic substances on sorption of uranium by the microbial sorbent we used humic substances 

isolated from the Dnipro River, which brought in a solution of uranium (CU = 100 μmol/l, I = 

0.01) in concentrations of 10, 25, 50, 100, 200 mg/l; initial pH of humic substances solution 

and pH changes that occurred were recorded. Age of microbial culture was 24 h, duration of 

the experiment – 1 h. 

Vol. 3 Page - 422 -


It is known that complexation of uranium (VI) with HS is almost independent of the type 

(peat, lake, etc.) [11] and ionic strength solution [11, 12], but it is intensified with pH 

increasing [11, 13, 14]. It was shown that within the concentration of humic acids from 10 to 

50 mg/l values of sorption of uranium by B. polymyxa IMV 8910 cells is slightly lower. In 

case of 200-fold increase of their content the intensity of sorption was significantly decreased, 

due to the formation of complex compounds, which are not practically adsorbed by biomass 

(Fig. 1). 

a, μmol/g 

160 

140 

120 

100 

80 

60 

40 

20 

0 

10 25 50 100 200 

C HS, mg/l 

Figure 1: Effect of humic substances on sorption of uranium (VI) by B. polymyxa IMV 8910 cells: 1 - 

curve of U(VI) adsorption; 2 - curve of pH ¡ (CU(VI) = 100 μmol/l, m = 0.03 g, INaCl = 0.01) 

Also, it was observed a slight shift in pH from 6.0 to 7.0 in case of humic substances content 

increasing in the model solution. It also leads to decrease in the values of uranium (VI) 

adsorption by microbial sorbent based on B. polymyxa IMV 8910 cells. 



It is shown that the presence of NOM in natural aqueous systems will significantly influence 

on uranium speciation and must be accounted for in a proper assessment of U(VI) behavior in 

Vol. 3 Page - 423 - 

1 

2 

8 

6 

4 

2 

0 

pH


environmental settings. In the presence of high concentration of humic substances decreasing 

the effectivity of uranium (VI) removal by biosorbent from aqueous solutions. 

References 

1. J. A. Davis and R. Gloor, Environ. Sci. Technol., 15 (1981) 1223. 

2. J. Buffle, Complexation Reactions in Aquatic Systems, Ellis Horwood, Chichester, 1990. 

3. V.Moulin and C. Moulin, Radiochim. Acta, 89 (2001) 773–778. 

4. P. Crancon and J. van der Lee, Radiochim. Acta, 91 (2003) 673–679. 

5. P.K. Appelband, D.C. Baxter and J.O. Thunberg, J. Environ. Monit., 1(1999) 211–217. 

6. W. C. Li, D.M. Victor and C.L. Chakrabarti, Anal. Chem, 52 (1980) 520–523. 

7. V.Moulin, J. Tits and G. Ouzounian, Radiochim. Acta, 58/59 (1992) 179. 

8. J. J. W. Higgo et al., Radiochim. Acta, 61 (1993) 91. 

9. L.M. Spasonova et al., J. Water Chem. Technol., 28 (2006) 61–68. 

10. Shevchuk, N. Klymenko, J. Water Chem. Technol., 31 (2009) 324–328. 

11. M.A. Glaus, M. Hummel and L.R. Van Loon, Appl. Geochem., 15 (2000) 953–973. 

12. G. Montavon et al, Radiochim. Acta., 88 (2000) 17–24. 

13. J.J. Lenhart et al, Radiochim. Acta., 88 (2000) 345–353. 

14. M. Kalin, W.N. Wheeler and G. Meinrath, J. Environ. Radioact., 78 (2005) 151–177. 

Vol. 3 Page - 424 -

Humic Acids Inspired Hybrid Materials as Heavy-Metal Adsorbents 

Panagiota Stathi * , Yiannis Deligiannakis 

Laboratory of Physical Chemistry, Department of Environmental and Natural Resources 

Management, University of Ioannina, Seferi 2, 30100, Agrinio, Greece 

E-mail: me01791@cc.uoi.gr 


Humic substances are naturally occurring biogenic heterogeneous organic materials that 

complex strongly with heavy metals and organic compounds 1,2 . 

In order to study the sorption characteristics, natural humic acid are isolated, purified, and use 

in laboratory studies 1,2 . However, these studies are often thwarted by the fact that it is difficult 

to separate the humic acid from other moieties present in the solution. A way to avoid this 

complication is the immobilization of the humic substances on inorganic or water insoluble 

organic particles that can be separate from their suspension. Covalent grafting of HA on SiO2 

provides a promising technique for the production of a stable zero-leaching hybrid material 3 . 

The aim of the present work was (a) to develop SiO2 materials bearing covalent immobilized 

i.e –COOH and Phenolic group as models for the metal binding groups of humic acids (b) to 

study heavy metal binding by SiO2-COOH, SiO2-phenolic materials (c) to prepare SiO2immobilized 

humic acid SIO2-HA and study the metal ion binding of this material. (d) Based 

o the SiO2–COOH and SiO2-Phenolic materials to parameterize theoretically the role of each 

functional group for metal binding by humic acids. 



Covalent Immobilization of Humic Acid onto SiO2. Humic acid obtained from Aldrich and 

used after purification this humic acid was characterized in detailed previously 4 . Covalent 

immobilization of humic acid on aminopropyl silica has been achieved by the method of 

Koopal et al 3 , by formation of amide bonds between the amino groups of the aminopropyl 

silica and the carboxylic groups of HA activated by EDC. 

Preparation of the SiO2-COOH Material. Activated silica was modified by –(CH2)3CN 

groups following the method of Clark et.al. 5 . Hydrolysis by H2SO4 results in modified silica 

which bears carboxylic acids as functional groups. 

Preparation of SiO2-Gallic Acid. Immobilization of Gallic Acid on silica has been obtained 

by formation of amide bonds between the amino group of the aminopropyl silica and the 

carboxylic group of Gallic Acid activated by EDC 6 . 

Vol. 3 Page - 425 -


Metal uptake. Figures 1-3 present the metal-uptake by the three SiO2-based materials, as a 

function of pH. Control metal-uptake data by unmodified SiO2 are also included for 

comparison. From Figures 1-3 we notice that the metal uptake was significantly increased 

relative to the unmodified silica. Importantly, we observe that the modification of silica 

results in an enhancement of adsorption at all pH values for all metals. Noticeably the 

maximum adsorption by SiO2-HA was 10 times higher than the maximum metal adsorption 

by SiO2-COOH or SiO2-GA. In the following we proceed in detailed analysis of the results 

based theoretical fit of the experimental data. In all Figures the open symbols and lines 

represent the theoretical calculations. Herein the theoretical calculations were performed 

using the program FITEQL 7 . For the fit of the metal adsorption on SiO2-HA we used five 

discrete pK for the protonation reaction of each functional groups i.e. five for carboxyl, five 

for Phenolic. 

% Pb Ads orbed 

100 

90 

80 

70 

60 

50 

40 

30 

20 

10 

Log(Con.)(M) 

(A) 

SiO2-COOH 

4 5 6 7 8 

pH 

SiO2 

-4 

-5 

-6 

SiO -COO-Pb 

2 

-7 

-8 

Free Pb 

-9 

-10 

Pb(OH) 

-11 SiO-Cu 

-12 

-13 

-14 

-15 

-16 

4 5 6 7 8 

pH 


% Cd Adsorbed 

100 

90 

80 

70 

60 

50 

40 

30 

20 

10 

0 

(B) 

SiO2-COOH 

4 5 6 7 8 

pH 

SiO2 

-6 Free Cd 

-7 

SiO -COO-Cd 

2 

-8 

-9 

Cd(OH) 

-10 

SiO-Cd 

4 5 6 7 8 

4 5 6 

Figure 1: Adsorption of Metals on SiO2-COOH . (A) Adsorption edge for Pb(II) onto SiO2-COOH, 

solid squares and on to SiO2 solid circles. (B) Adsorption edge for Cd(II) onto SiO2-COOH, solid 

squares and on to SiO2 solid circles. (C) Adsorption edge for Cu(II) onto SiO2-COOH, solid squares 

and on to SiO2 solid circles. (Initial metal concentration 4.5μM ) 

SiO2-COOH. The Figure 1 (left panel) shows the pH edge adsorption for Pb (Fig 4A), Cd (Fig 

2B) and Cu (Fig 2C) on the SiO2-COOH material. From Figure 2 we notice that the metal 

uptake by SiO2-COOH was significantly increased relative to the unmodified silica i.e. 

compare solid squares with solid circles in Figure 2, left panels. 

log(conc.)(M) 

-5 

-11 

-12 

-13 

The adsorption of metal to silica gel was previously studied for many authors and was 

reported quite low. Our data confirm these observations. 

pH 

Vol. 3 Page - 426 - 

% cu Adsorbed 

100 

90 

80 

70 

60 

50 

40 

30 

20 

10 

0 

log(conc)(M) 

(C) 

SiO2-COOH 

pH 

SiO2 

7 8 

-4 

Free Cu SiO -COO-Cu 

-5 

2 

-6 

-7 

-8 

-9 

-10 

-11 

Cu( OH) 

-12 

-13 

SiO-Cu 

4 5 6 7 8 

pH

SiO2-GA. The SiO2-GA material has been studied for the adsorption Pb 2+ , Cd 2+ , and Cu 2+ . The 

results of adsorption experiments onto to SiO2-GA as a function of pH are presented at Figure 

3. The pH edge on the unmodified silica is included for comparison. From Figure 3 we notice 

that Pb 2+ adsorption was 1.7 times higher than Cd 2+ and Cu 2+ adsorption was 1.46 times 

higher than Cd. For example at pH 8 almost 95% of the 4.5μΜ of Pb 2+ were bound by SiO2- 

GA. 

The experimental data in Figure 2 show that the metal uptake increased at alkaline pH vales, 

for both the SiO2-GA as well as for unmodified SiO2. More particularly, at pH 4 Pb 2+ 

adsorption was 0.52 mmol/Kgr of SiO2-GA and at pH 7 was 2.29 mmol/kgr. For Cd 2+ the 

adsorption at pH 4 was 0.45mmol/Kgr and 1.35 at pH 7, while the adsorption for Cu 2+ was 

1.01mmol/Kgr and 1.98 mmol/Kgr at pH 4 and pH 7 respectively. 

% Pb Adsorbed 

100 

90 

80 

70 

(B) 

60 

50 

40 

30 

SiO2-GA 

20 

10 

SiO2 0 

4 5 6 7 8 

-5 

-6 

pH 

-7 

-8 

Free Pb 

SiO -GA-Pb 2 

-9 

-10 

SiO2-Pb 

-11 

-12 

-13 

-14 

Pb(OH) 

-15 

4 5 6 7 8 

log(Conc.)(M) 

pH 


% Cd Adsorbed 

100 

90 

80 

70 

60 

(B) 

50 

40 

30 

20 

SiO2-GA 

10 

0 

SiO2 

4 

-5 

-6 

5 6 

pH 

7 8 

-7 

Free Cd 

SiO -GA-Cd 

2 -8 

-9 

SiO2-Cd 

-10 

-11 

-12 

-13 

Cd(OH) 

-14 

4 5 6 7 8 

log(Conc.)(M) 

4 5 6 7 

pH 

Figure 2: Adsorption of Metals on SiO2-GA . (A) Adsorption edge for Pb(II) onto SiO2-GA, solid 

squares and on to SiO2 solid circles. (B) Adsorption edge for Cd(II) onto SiO2-GA, solid squares and 

on to SiO2 solid circles. (C) Adsorption edge for Cu(II) onto SiO2-GA, solid squares and on to SiO2 

solid circles. (Initial metal concentration 4.5μM 

SiO2 –HA. Figure 3 shows the adsorption of Pb 2+ , Cd 2+ , Cu 2+ on SiO2-HA as a function pH. 

According to the Figure 4-6 the adsorption of metals to immobilized humic acid was higher 

compared with the adsorption to other materials. For example the Pb 2+ adsorption at pH 7 was 

3.88 mmol/Kgr for the SiO2-COOH material, 2.23mmol/Kgr for SiO2-GA and 34.65 

mmol/Kgr for SiO2-HA material. For Cd 2+ the obtained adsorption on SiO2-COOH was 2.07 

mmol/Kgr at pH 7, 1.35 mmol/Kgr SiO2-GA and 14.22 mmol/Kgr on SiO2-HA. In analogous 

manner the Cu adsorption on three materials was 2.57 mmol/kgr, 1.98 mmol/Kgr, 

35.95mmo/kgr respectively. The experimental data in Figure 3 show that the Pb 2+ by uptake 

SiO2-HA increased at alkaline pH values. 

pH 

Vol. 3 Page - 427 - 

% Ads 

100 

90 

80 

70 

60 

50 

40 

30 

20 

10 

0 

log(conc.)(M) 

(B) 

-5 

-6 

-7 

-8 

-9 

-10 

-11 

-12 

-13 

-14 

-15 

SiO2-GA 

pH 

SiO 2 -GA-Cu 

SiO2 

4 5 6 7 8 

8 

Free Cu 

SiO2-Cu 

Cu(OH)

% Ads 

100 

90 

80 

70 

60 

50 

40 

30 

20 

10 

0 

log(conc.)(M) 

(B) 

SiO2-HA 

4 5 6 

pH 

7 8 

-5.0 

-6.5 

-7.0 

-7.5 

-8.0 

% Cd Adsorbed 

100 

90 

80 

70 

60 

50 

40 

30 

20 

10 

0 

(B) 

SiO2-HA 

4 5 6 

pH 

7 8 

4 5 6 

pH 

7 8 

Figure 3: Adsorption of Metals on SiO2-HA . (A) Adsorption edge for Pb(II) onto SiO2-HA. (B) 

Adsorption edge for Cd(II) onto SiO2-HA, solid squares. (C) Adsorption edge for Cu(II) onto SiO2- 

HA. (D) Adsorption edge for Zn(II) onto SiO2-HA.(E) Adsorption edge for Mg(II) onto SiO2-HA, 

solid squares. (Initial metal concentration 45μM ) 

In analogous manner the adsorption of Cd 2+ at pH 4-6 is due to the deprotonated COO - groups 

while adsorption at pH>6 is due to binding of Cd 2+ to Phenolic groups with pKa 8-10. The 

Cd adsorption is 2.5 times lower than Pb 2+ . For example at pH 7 the adsorption of Cd 2+ was 

14.22mmol/Kgr and the adsorption was 34.64mmol/kgr for Pb 2+ 

Figure 3c shows the adsorption of Cu 2+ adsorption on SiO2-HA the amount of Cu 2+ adsorb 

was 35.95 mmol/Kgr see table 2. The main adsorb species from pH 4-6 are COO-Cu and from 

higher pH the main species is RO - -Cu. 


-4.0 

Free Pb RO 

-4.5 

- -Pb 

-5.5 

COO-Pb 

-6.0 

Pb(OH) 

4 5 6 7 8 

pH 


log(conc.)(M) 

-4 

-5 

-6 

-7 

-8 

-9 

-10 

-11 

Free-Cd 

COO-Cd 

RO - -Cd 

Cd(OH) 

4 5 6 7 8 

pH 

The data presented herein show that all SiO2-based materials show significant improvement 

for metal uptake, compared to unmodified silica. This enhancement was observed at all pH 

values and can be attributed to adsorption of metals to deprotonated form of functional groups 

(COOH, GA, HA). 

Of particular importance is the observation that for SiO2-HA material the adsorption is 10 

times higher compared with other two materials. This is a result of high concentration of 

functional groups per Kgr of humic acid. The maximum amount of metals absorbed per Kgr 

of materials shows in Figure 4 The carboxyl groups appear to be responsible for the 

adsorption at pH 4-7 and phenolic groups for the adsorption at higher pH. In this respect the 

SiO2-HA shows the typical behaviour of HA in solution.In addition, from the adsorption data 

we conclude that SiO2-COOH and SiO2-GA are a good model for the adsorption properties of 

Vol. 3 Page - 428 - 

% cu Adsorbed 

100 

90 

80 

70 

60 

50 

40 

30 

20 

10 

0 

log(conc.)(M) 

(C) 

-4 

-5 

-8 

-10 

-11 

SiO2-HA 

RO 

COO-Cu 

- -Cu 

-6 

Free Cu 

-7 

-9 Cu(OH) 

1 2 3 

pH 

4 5

metals by the –COOH and 

phenolic groups of HA 

respectively. More particularly, we 

observe that metal adsorption on 

the three materials increased by the 

same order [Cu]>[Pb]>[Cd]>[Mg]. 

The present data provide the first 

direct experimental proof that HA 

can be viewed and modeled as a 

combination of -COO and R-OH 

functional groups. 


Ads. Metal (mmol/Kgr) 

35 

30 

25 

20 

15 

10 

5 

0 

SiO 2 

SiO 2 -COOH 

SiO 2 -GA 

SiO 2 -HA 

Pb Cd Cu Zn Mg 

Figure 4: Amounts (mmol/Kgr) a of Heavy Metals 

Adsorbed by Materials at pH=7 

Moreover in natural environment sorption of inorganic and organic compounds to mineral 

bound humic acids is influenced by the chemical properties and the conformation of the 

humic acids. The present work shows that SiO2-COOH, SiO2-GA, and SiO2-HA can be useful 

in physicochemical study of geochemical cycles of metals in natural environment. 

References 

1. Stevenson J. F., Humus chemistry: Genesis, Composition, Reactions, John Willey & Sons Inc: 

New York, 1994. 

2. Tipping E., Cation Binding by Humic Substances, Cambridge University Press, Cambridge, 2002. 

3. Koopal L. K.; Yang Y.; Minnard A .J.; Theunissen P. L. M.; Van Riemsdijk W. H.; Colloids and 

Surfaces A: Physicochem . Eng. Aspects 1998,141, 385. 

4. Drosos M.; Jerlykiewich M.; Deligiannakis Y.; J. Colloid and Interface Sci., 2009, 332, 78. 

5. Butterworth A.J.; Clark J.H.; Walton P. H.; Barlow S.J.; Chem.Comm. 1996, 1859. 

6. Stathi P.; Louloudi M.; Deligiannakis Y.; Chemical Physicis Letters, 2009, 472, 85. 

7. Dzombak D.A.; Morel F.M.M.; Surface Complexation Modeling, Jonh Willey & Son, New York, 

1990. 

Vol. 3 Page - 429 -


Vol. 3 Page - 430 -

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Natural Organic Matter and Humic Substances Interactions

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