impact of latex and plant extract of and the recovery of latex toxicity ...

IMPACT OF LATEX AND PLANT EXTRACT OF 

Calotropis gigantea(L.)R.Br. ON Anabas testudineus (Bloch) 

AND THE RECOVERY OF LATEX TOXICITY WITH 

ADDITIVE NUTRIENTS 

KANCHI MAMMUNIVAR CENTRE FOR P.G 

STUDIES, LAWSPET, PUDUCHERRY-605008 

INDIA 

March - 2007

se8icate8 

%o dY helove8 Zhinanna 

A.Narayan Menon

Dr. KADEM RAMUDU 

~ecturer in Zoology, 

TAGORE ARTS COLLEGE, 

Lawspet, 

PUDUCHERRY- 605 008 

CERTIFICATE 

This is to certify that Mrs. K. Sree Latha carried out the work ot her thesis 

entitled "The Impact of latex and plant extract of Calotropis gigantea(L.)R.Br, on 

Anabas testudineus (Bloch) and recovery of latex toxicity with additive 

nutrients" for the degree of Doctor of Philosophy in Zoology of the Pondicherry 

University for the requisite period under regulation in force and that the thesis is a 

bonafide record of the work done by her under my supetvision and guidance. The 

work is original and has not fomed the basis of the award to the candidate of any 

degree, diploma, associateship, fellowship, or other similar title. 

I further state that the entire thesis represents the independent work of Mrs. 

K. Sree Latha and all the experimental techniques employed in this work were 

actually undertaken by the candidate herself under my guidance. 

(Dr. K. RAMUDU) 

Research Su~ervisor 

Place PUDUCHERRY 

~ p ~ a a m - x J 

___* 

DIRBCIO& 

SMCH~MAMUNW~' .. 

FOI mar-m~nurn 8ruom 

C9YIKYC8~Y,

Mrs. K. Sree Latha 

Research Scholar 

Department of Zwlogy 

Kanchl Marnun~var Centre For Post Graduate Stud~es 

Lawspet. Puducherry-605008 

DECLARATION 

I hereby declare that the thesis entitled "The Impact of latex and plant 

extract of Calotropis gigantea(L.)R.Br. on Anabas testudineus(Bloch) and 

recovery of latex toxicity with additive nutrients" is the result of a study 

originally carried out by me under the guidance and supervision of 

Dr. K. Ramudu, Lecturer in Zoology, Tagore Arts College, Lawspet, 

Puducherry -605008. 

This work has not been submitted earlier in full or in part for any diploma 

or degree in this or any other university. I also declare that no part of the thesis is 

a reproduction from any other source, published or unpublished without 

acknowledgement, 

Station: Puducherry, 

Date.15.3.07 

k-. Lg+Ab= 

(K. Sree ~a

ACKNOWLEDGEMENTS 

1 eprers y dup sense offlatid to my K~qeardi Superviror Dr. Tadem Gmudu. 

M.~c.,PhD.,SeniorLecturer, Department of Zoo&, 

7agorc Arts Coilege, Lawspet, 

r~uducherry, for h vaLabkguidance, incpiritg advice andconstant enrouryement tfzrougibut 

I &ti 

to thn(Dr.T.Uma ,I&-Directod,jor pennittiy me to registerfor my ~ h.9.~ 

my sincere thudis to Dr.Ba[(I fali&,(~Directofl andDr. V. BaMramanian, 

pprent 

Director, XaCnnd;' Mamunivar Centerfor Post Graduate Studies, fuwspet, Fududieny ,for 

thir emuragement andsupport. 

1 owe my ptitude and simre that& to Dr.yV.Lu, $ientist-T and Dr.5.xam 

xnihna, Scientist-E., Dept. of NaturalPoducts and Phharmacoiogy, Mr.~acnb Vemn, IICT- 

Hyderabad and Dr.C.Sures/, Scientkt, Dept. oj Bwdamisty, N1g-Hyderabadfor thir 

mynanimous support andvafuabh suagulions tfiroylhou t y course. 

1 am pent4 idbted to firof.Nataraja,(Xetd , %of .A%hi.Zlagovan(xetd ,Prof Mrs. 

.Tnpura Sundan(Ketri) ,Pro$'Drd.Passopathy, Head; Department of Zuohyy, 

Dr./dna&n, 

Vrd.Guidigh, Dr.5.Xumaran, Dr.Ka(pvi Chndrane, Dr.G.Xric/rna 

Moorthy, Dr.S.~+La(shmi mCF(iS, Prof. $ Xarunanih, A0.V Zooh~y, Mrs. $.Lafitha 

Kumari Amm, Mr. M. Firummhne, and Mrs.Ma&rvizhi, Dept of Zaob~y, 73.C and 

DrA.Tragll~am, Vr.V.Kamasamy., Vept. of 2btan$,m@GS 

H0.D Botay, Mr. D. Rajarajan, Dr. I: Gaman, Dr. 

and Trrof. 2! .layacJinndran, 

Xajendiran, Dr. 2! 3&mnresan 

Depamnmt of Botay, I3.C for Air constant keie[p, prodig a[l dePurtmentaf ditk, 

uafira61 guid~nce andfnritfuf dicclrrrion ttirougibut y research work uhkh ha* 

peat &a[ inaviy shpe to 

t/wis wort. 

hbcd a 

1 &ti to than( Prof.Vr.$aui Shanker Pista ,!Vcad ,Department of Fisheries., 

Rof.Dr.5Vp~a $uju, 

Deportment of Pysiology, Osmania Uniwrsiy, Hyderabad and llie 

Director, Centre for Advanced Studies, Annamahi University, l'araypettai., for their he4 

andmoperalion duriy y eperimcntaf work I am akogratefulto Dr.Venu Gopal Dinctor, 

and the %dinua[pf+-rs , WE, 

Xabnada for &ir va~uableguidanu andpennitlitg me for 

literature milction

I simnly &a& My IPrindpd, Dr. S.$K 

Guut Arts. Co[Lge, yanam and +of. 

x,QGopa(a xu, !W@D, !Department ofhbJy, Dr. Dhnwwrr Pasad; X0.D. Carputer 

science, and all my collengues, !Dr.S,$$r Govt. Arts Colkg, yonam., for their patient 

suaqestioru, constant support and encouragement. I evrers my deep appreciation and regark 

to Mrs..$Knjfina 'Vetti, Sr.Lecturer, 'Deparhnent of Mathematics, Dr. S.$P.r Gow, arts 

cob, ~anam,jorguiding me in doing statictkaimrk 

I wish to thn& the Lab %&ant 

the k[p redredduring my qerimentalmrk 

amfJittender, Depamnt ofZooiogy, @LTPGSjor 

I qrus y special regark to my Mother and aU my fami4 meders for pviy me 

freedom and neressay support, andfnendr Dr.S.xajeswari, Mi.Josphene %u[ina, M~.Z'rc~tth 

Xumari, !M'~.s.lPS/ierin, Mr.S.qa$ar andMr.Bhrathi for their help: cnrouraflemnt andgood 

wirhes. 

I wish to &a& tfie Librariaru of ~C!PGS,CIF'E-Ka@mda, 'VCRC ~nnamahi 

University, %ndLheny University Bw-Infonnatirs Centre, Osmania Uniztrsity L$ sciences 

Department, 1ICI;,C~M%%~Ikra6ad for their hip and Co-operation in prorn'diy the 

informationfor literature survey. 

I sincere4 tha& Mr.K'Tuh Ram, my elder bro&-for 

support, withgood wick. 

My &ads are due to my brother Mr. 

hk time4 hek, care and' 

Suya Narayana, Suya CCEandra Dgtalc, d o 

he[pcdme inphtcyraplry andcomputer operatiom andMr.xambabu she helpedme in typing 

m'th pmrnptms. 

It is with gnat joy that I uhh to a&o~&dgL y sincrn than& to my behd 

huband Mr.%Uma yl&er and my 6ebved Son Muter $Vishifor the encouragement, 

palience,filco-operation andalcofor being a piahr of strength andsupport duriy period 

of stress end strain and for hviy taken vey specid efforts in helping me to compkte the 

tl;ecis. 

research mrk 

a6m all I &a& the ahghty for hdgrau uhkh h[ped me at every stage of y 

Kanuru Sreelatha

CONTENTS 

PAGE NO 

1. INTRODUCTION 

1-1 0 

2. REVIEW OF LITERATURE 11-29 

3. MATERIALS AND METHODS 30-58 

4. RESULTS 59-74 

5. DISCUSSION 75-98 

6. SUMMARY AND CONCLUSION 99-1 03 

7. REFERENCES i-xxv

LIST OF TABLES 

Table.1 Plant resources of Biopesticides. 

Table.2 Characteristics of Water used in Bioassays 

Table.3 Percentage Mortality of Anabas testudineus exposed to different 

concentrations of Latex of Calotropis gigantea 

Table.4 Percentage Mortality of Anabas testudineus exposed to different 

concentrations of Plant Extract of Calotropis gigantea. 

Table.5 Safe Concentration of Latex and Plant Extract of Calotropis 

igantea in Anabas testudineus based on 96 hr (LC5o) and Application 

Factor. 

Table.6 Optomotor Behavioural changes of Anabas testudineus exposed 

to sublethal concentrations of Latex,Latex+Supplements and Plant extract 

of Calotropis gigantea for 96 hrs. 

Table.7 Behavioural changes of Anabas testudineus exposed to sublethal 

concentrations of Latex,Latex+Supplements and Plant extract of Calotropis 

gigantea for 96 hrs. 

Table.8 Protein levels in Anabas testudineus exposed to sublethal 



Table.9 Glycogen levels in Anabas testudineus exposed to sublethal 

concentrations of Latex, Latex+Supplements and Plant extract of 

Calotropis gigantea for 96 hrs.

Table.10 Acid Phosphatase Activity in Anabas testudineus exposed to 

sublethal concentrations of Latex, Latex+Supplements and Plant extract of 

Calotropis gigantea for 96 hrs. 

Table.11 Alkaline Phosphatase Activity in Anabas testudineus exposed to 

sublethal concentrations of Latex,Latex+Supplements and Plant extract of 


Table.12 Acetyl Cholinesterase Activity in Anabas testudineus exposed to 

sublethal concentrations of Latex,Latex+Supplements and Plant extract of 


Table.13 Adenosine Triphophatase Activity in Anabas testudineus exposed 

to sublethal concentrations of Latex,Latex+Supp\ements and Plant extract 

of Calotropis gigantea for 96 hrs. 

Table.14 Effect of Latex on Haematolog~cal Parameters in fish Anabas 

testudineus exposed for 96 hrs. 

Table.15 Effect of Latex+Supplements on Haematological Parameters in 

fish Anabas testudineus exposed for 96 hrs. 

Table.16 Effect of Plant Extract Haematological Parameters in fish 

Anabas testudineus exposed for 96 hrs.

LIST OF FIGURES 

Figure.1: Percentage Mortality of Anabas testudineus exposed to different 

concentrations of Latex of Calotropis gigantea. 

Figure.2: Percentage Mortality of Anabas testudineus exposed to different 

concentrations of Plant Extract of Calotropis gigantea. 

Figure.3: Optornotor Behavioural changes of Anabas testudineus 

exposed to sublethal concentrations of Latex, Latex+Supplements and 

Plant extract of Calotropis gigantea for 96 hrs. 

Figure.4: Behavioural changes of Anabas testudineus exposed to sublethal 



Figure.5: Protein levels in Anabas testudineus exposed to sublethal 



Figure.6: Glycogen levels in Anabas testudineus exposed to sublethal 



Figure.7: Acid Phosphatase Activity in Anabas testudineus exposed to 

sublethal concentrations of Latex, Latex+ Supplements and Plant extract of 


Figure.8: Alkaline Phosphatase Activity in Anabas testudineus exposed to 


Calotropis gigantea for 96 hrs.

Figure.9: Acetyl Cholinesterase Activity in Anabas testudineus exposed to 



Figure.10: Adenosine Triphophatase Activity in Anabas testudineus 

exposed to sublethal concentrations of Latex, of Calotropis gigantea for 96 

hrs. 


exposed to sublethal concentrations of Latex+ Supplements of Calotropis 



exposed to sublethal concentrations of Plant extract of Calotropis gigantea 

for 96 hrs. 

Figure.13: Effect of Latex of Calotropis gigantea on Haematological 

Parameters in fish Anabas testudineus exposed for 96 hrs. 

Figure.14: Effect of Latex+Supplements on Haematological Parameters in 

fish Anabas testudineus exposed for 96 hrs. 

Figure.15: Effect of Calotropis gigantea Plant Extract Haematological 

Parameters in fish Anabas testudineus exoosed for 96 hrs.

LIST OF PLATES 

Plate. A Calotropis gigantea: a-plant, b- T.S of Stem, c- Stem Laticiferous 

tissue, d- leaf Laticiferous tissue. 

Plate.1 Effect of Latex on Brain of Anabas testudineus. 

Plate.2 Effect of Plant Extract on Brain of Anabas testudineus 

Plate.3 Effect of Latex on Gill of Anabas testudineus 

Plate.4 Effect of Plant Extract on Gill of Anabas testudineus 

Plate.5 Effect of Latex on Liver of Anabas testudineus. 

Plate.6 Effect of Plant Extract on Liver of Anabas testudineus. 

Plate.7 Effect of Latex on Red Blood Corpuscles of Anabas testudineus 

Plate.8 Effect of Plant Extract on Red Blood Corpuscles of Anabas 

testudineus. 

Plate.9 Effect of Latex + Supplements on Red Blood Corpuscles of 

Anabas testudineus.

INTRODUCTION 

The word 'aquaculture' IS used widely for eyer a decade to denote all fonns of 

culture of aquatic animals and plant: in fresh. brackish and marine environments 

Man's pcrslstent struggle over hunger resulted In search of food 111 the resources both 

terrestrial as well as aquatlc, and depended on hunting and gathering for subsistence 

until the Neolithic perlod. Fish developed as part of this basic subsistence activity. 

but has witnessed considerable technolo_elcal advances in modem times In methods 

of capture and utilization of aquatic products. Fisheries have been recogn~~ed as a 

powerful income and employment generator as it stimulates growth of a number of 

subsidiary ~ndustries and is a source of cheap animal protein. 

It is instrument of 

I~velihood for a large section of economically backward population of the country 

and source as 

means to ensurlng national food security (Pillay ,1977). Aquatic 

Pdmilng existed in inland areas from ancient times, most likely from the tlme of 

evolution to pastoralism and land cultivation. The classic of fish culture believed to 

have been written around 500 BC by Fan Lei, a Chinese politician turned fish 

culturist, is considered proof that commercial fish culture time, as he cited his fish 

ponds as the source of wealth. Fish existed in Roman times and later in monastic 

houses in the Midd!e Ages (2500BC) in Egypt ,Tilapia were believed to be raised in 

a pond, the earliest form of fish culture appears to be of the common carp (Cyprinus 

carpio), a native of china. Different species of carp caught from rivers gave rise to 

the celebrated system of polyculture. A number of other fish has been added to the 

species comb~nations, w~th the expectation of increas~ng productivity in polyculture 

ponds after 6th century AD. Indigenous systems of Indian carp culture have existed 

In eastern parts of Indian subcontinent in I llh Century AD. After World War I1 that is 

in the year 1948 onwards World estimates of fish production and trade started by 

1950, the trends showed increased average rate about 3% from 1950 to 1956 and the 

steady increased in total world catch averaging 6.6%. or more than double the ra:e of 

Increase in terrestrial food products. About 86% of the world fishery production

comes from the sea. Much of the recent interest in aquaculture has arisen from the 

large possible increases in yield of animals per unit of area, the potential ,the animal 

yield from natural waters ranges from about 10 kg per ha for the most barren to a 

maximum of about 200 -5OOkgiha for the most fertile. Private aquaculture supplies 

food, bait, recreational angling, and ornamental fish at profit to the producers 

(William Royce, 1972).Wastelands unsuitable for agriculture are utilized for landbased 

aquaculture, and foreshore or protected coastal areas for other types of open 

water farming, and the increase in production, of the more valuable products. Fish 

have relatively low energy requirements, except for metabolism and maintenance of 

body functions, use little energy for maintenance of body temperature and normal 

locomotion, that result in higher growth rates and greater production per unit area, 

taking full benefit of the three dimensional nature of water bodies. The protein 

efficiency ratio (weight gain per unit of protein intake) is either equal to or higher 

than that for poultry, swine, sheep and steers. Many tpes of proteins that are not 

consumed by man can be upgraded through aquaculture to produce highly acceptable 

and well-relished products. Culture techniques have to be used to prevent the 

extinction of species that are ecologically or economically important to the 

environment. 

Global aquaculture production increased from 0.64 million metric tons (mmt) in 1950 

to 51.39 mmt in 2002. Asia accounted for the bulk of this production (about 90%) in 

this present centaury. China accounted for 70% of global farmed fish production, 

while India's share was only about 5%). India is blessed with abundant fishery 

resources both marine and inland. Ponds are generally considered to be extensive 

culture systems. According to latest FA0 Report, nearly half the fish consumed as 

food world wide, are raised on fish farms, in 2004 global production of farmed fish 

was about 60million tones,59% was produced in China and 22% from rest of 

Asia(Down to Earth,2006-Report).Many species that could not be spawned or 

reared a few decades ago are now being produced in large quantities around the

world. This indeed is an enviable position. The country has a coastline of 8085 km 

and an exclusive economic zone of over Zmillion sq.km. It is studded with perennial 

rivers having a total length exceeding 64,12Ikm, 1097 million ha of reservoirs, 1.3 

million hectares of beel's, 1.4 million ha of brackish waters and innumerable tanks 

and ponds with an extent of over 2.12 million ha of water area. The nation's potential 

for fishing production is exceptionally vast and is now on the threshold of a major 

breakthrough in fisheries development (Din'tulu and Paparao ,1994 

1.1 PESTICIDE USE-AN OVER VIEW 

The aquaculture industry has been experiencing growing pains as a result of water 

quality deterioration, and poor water quality has in turn contributed to disease 

outbreaks. Man's ever increasing use of water and his contamination of natural 

waters pose serious problems for the fisheries. There is growing concern over the 

safety of ingesting fish and other aquatic life taken from polluted waters. Over onethird 

of all commercial and recreational fish species have dramatically declined in 

population and have completely disappeared in the past 15 years. Toxic pollutants 

rendered almost lifeless by a lethal combination of agri:ultural fertilizers, pesticides, 

industrial pollutants, sewage runoff, and diminished oxygen. The process kills the 

food chain from the bottom up, rendering the area virtually lifeless. Even though the 

contaminant level may be very low in the water source itself, have a tendency to bioaccumulate 

when a predator eats prey, the contaminants are deposited in the tissue of 

the predator and over time, it can build up to unsafe levels. In the past 50 Years, 

many kinds of chemical pesticides have been released into the environment and are 

considered pollutants. Pesticides do pose a possible pollutant source, most likely 

during the rainy season when ~ noffrom farming may occur. Aquatic life is exposed 

to these compounds because the rivers, estuaries, bays, and oceans often behave as a 

sink or receptacle for these compounds. The excessive use of fertilizers in intensive 

agriculture and phosphate rich sewage emuents increasingly, causing problems on 

worldwide basis affecting marine as well as freshwater ecosystems. The interaction

among various chemical pollutants in the aquatic system may be synergistic, 

antagonistic or additive, and may cause acute toxic effect on different species 

(Stickney, 1994). 

TWO 

general categories of pesticides have dominated the market for the past 

fewdecades, chlorinated hydrocarbons and organophosphates. Chlorinated 

hydrocarbons are persistent in nature. They or their breakdown products can remain 

active and lethal for many years. Many other chlorinated hydrocarbons aher 1970s, 

banned for public use. Biopesticides of any type have been approved for use by 

producers in the developing world where chemical use is largely or totally 

uncontrolled They can be insecticidal or antimicrobial, some are herbicidal. Naturally 

occumng insecticide chemicals such as Pyrethrum, Nicotine, Rotenone, Hellebore, 

Ryania, Sabadilla could also be potential pollutants. Rotenone is a crystalline ketone 

extracted from species of bean family used in ponds to kill fish, is restricted use 

pesticide in U.S. The bio active chemical substances are found in plants species that 

show deleterious effects on fishes are Saponins, which lower the surface tension 

preventing uptake of oxygen leading to death of fi~h. Glycosides are colorless, 

crystalline compounds, soluble in water, bitter in taste, optically active, considered to 

be as waste products dunng plant metabolism, use to remove harmful substances 

such as phenols by detoxification mechanism. Like alkaloids, they possess 

physiological activity (Cardiac glycosides). They are poisonous compounds 

(Aganval,l992;Gurdeep Chahual ,I98l)Alkaloids generally b~tter in taste and has 

pronounced physiological activity, although, many possess curative properties , they 

are powerful poisons, protective substances against animal or insect anack. 

(Aganval, 1992) Calotropis alkaloids are namely uscharin and voruscharin (Roben 

RaffauLl970) Terpenoids are hydrocarbons as wells as oxygenated derivatives 

readily volatile in steam. They are optically active, various terpenoids are 

biologically active namely, insecticidal, antihelmintic or antiseptic in action, and are 

also useful in pharmacy these compounds induce histomorphological changes in

gills, liver, RBC's, stomach, kidneys and brain (Agarwal ,1992;Patole and 

Mahajan,2006). 

Some naturally occumng insecticide chemicals mentioned in Table-l as a potential 

pollutants, if not used properly. The large scale discharge of pollutants into aquatic 

system may alter the physical, chemical and biological nature of water. Balancing the 

goal of increasing sustainability with the need to generate a reasonable profit, and 

maintenance of environmental quality will contlnue to challenge aquaculturist (Tht 

Wealth of India, 1992; Savita and Anandhi ,2006).

TABLE.l 

PLANTS - POTENTIAL BIOPESTICIDES 

Common 

name 

Botanic~l Name Family Effective range Plant part 

used 

Insedic~dal , larv~cidal 

Akanda Colorropisggon,eo Asclcped~ccae antffcedant. ~nfantlcidc , 'Iem 

and 

homtctdal polson and plqcidal 

leaf' 

Annona 

Mamcey 

Nccm 

Perstan L~lac 

Pyrethrum 

Quassta 

Copstcum onnusm 

Deris elliprrpaco 

Annonaceae 

Solanaceas 

Lcgumlnosease 

Composltae 

Simambaccac 

Contact and stomach pa~son. 

~nsect~c~dal, larvicidal. 

rcpcllcnt, antifeedant 

Contact and stomach polson, 

~nscctlc~dal and repellent 

Insccuc~dal, repellent, 

anttfccdanc, bacter~c~dal, 

qung,adal, nornat~rldal and 

eficct~ve agalnst ocks 

Contact and stomach poison, 

inscct~cidal, rcpcllcnt, 

nemattc~dal and effentve 

agamst tlcks 

Inscctic;dai, rcpellcnt, 

antlfeedant, ncmattctdal 

Contact and stomach polson. 

~nscctictdal, rrpcllcnr, 

antifcnlant, growih tnhibit~ng 

cffcnlvc againn t~cks 

Pure contact poision 

Insecticidal, rcpcllcnt, 

antlfcedant 

Contacl and stomach poision 

Insecticidal, ianicidal, 

ncmatlc~dal, Quassia also acts. 

Secds 

Stomach poison, ~nsecticldal 

rcpcllcnt, ant~fcedanl, fumigant, F~lt 

vlroid. 

Rwts 

Sceds 

All plant pans 

Drlcd leaves 

Flowers 

Ryanta Ryonlaspedoso Flacourtiac~sc Contact and stomach poison ~ ~ ~ s r ~ ~ a l k q 

Sabadllla 

Schoennocaulon 

oflcinole 

Contact and stomach polson. 

Lilaccasc Insdicidal, repellent, Secds 

rodennc~dal 

Sacet flag Acorns colomus Aracease lnSccticidal rrpcilent' 

ant~fedant , antlfertlie 

Turmenc Curcuma domertrco Zlngibcraccae lnscct~cldal and repellent 

Root 

Anllfiudant Ncmatfc~dal 

lupin Lupinus murabills 

Ocimum bar,lcum Lam~acac fungictdal Sccds 

rh'zOmcs

16 Basd ocimum bar,llc,i,,r Lam,acae Insccc"idal, repellent gmwth "" 

~nh~bttingagainst ticks 


esscni~al oil 

I7 Tomato Lkcoperricon Soianaceasc Rcpcllcnt, prevents egg laytng Lcavcs 

I8 Malabar nut Adhorodo varlco Acanthaceae Insecticidal, fung~cidal Lcavcs 

I9 Oleandcr h'erfanr oleonde Apocyanaceae Rat polson, grain protectant Lcavcs 

20 Mugwort Arlemi~ia vulyorls Composite BacC'e"c'dal, fung'c'da' and 

tnsectictdal propcnlcs 

Leavcs 

21 swect 

plgweed Chenopod~um Chenopod~acae Gram protectant 011 

22 Melon tree Coricopopaaa Fungictdal Lervcs 

23 Physic nut Jorropha curcas Euphorbiacae Mo"usclclde, rodcn"clde and leave and 

as an inscct repcllcnt 

sccds 

24 Madre da 

cacao 

Glrnnla septum Legumlnosac Rat polson Snds or 

powdcrs bark 

25 Karanla Pongomtoptntloro Legumlnosae 'nsectlc'dal Sccds 011 

26 Bystropogon Lab~atae Protectant for stored products Leave5 

27 Lavcnder Lab~atac Inscct repellen1 Stcrn, leaves 

anglrsr~lol! 

and imi 

28 Lavender Lab~atac lnscct repcllcni 011 

29 W~id sage Laniotia cvniura Verbcnacac inSect'cldal propcmes' 

against storage pests 

30 Nirgundl Viler negundo Vcrbcnacac lrsCniC'da' propm'cs' 

againa aoragc pesls 

Leaves 

Lcavcs 

1.2 CHOICE OF TEST ORGANISM: 

Rationale Behind Selecting Anabas testudineus(Bloch), as test organism: 

Anabus testdineus (Blochj, commonly known as climbing perch is popular table fish 

in 1ndia.So also indeed in the third world. Anabas particularly the airbreathing 

species, are attracting attention of the piscisulturists owing to their high production 

potential from paddy fields and stagnant shallow ponds (Dehadrai and 

Mukhapadhaya, 1979). The fish is easy to rear and are hardy, these attributes add to 

~ts demand. Hence it is necessary to study the immediate and chronic effects of 

insecticides on the fish, which form a part of human diet.

Anabus testudineus was chosen for the present study due to the following reasons: 

It is an edible fish. 

It is easily available everywhere. 

I: is a hardy fish, resistance to diseases, hence suitable of chronic study 

It can be easily transported without large scale mortality. It is a freshwater as well as 

brackish water species and tolerates adverse condition largely compared to several 

other species of fish. 

Taxonomic Position: 

The taxonomic Position of the fish is as follows 

Order : Terciformes 

Suborder : Percoidei 

Family : Anabantidae 

Genus : Anabas 

Species : testudineus 

Food and Feeding: 

Anabus is reported to be a carnivore Pandey Ashu,(1987) but mentions are available 

in the literature of the omnivorous feeding habit of the fish. In the laboratory, the 

fishes are generally maintained well on goat liver and trash fish. 

1.3 CHOICE OF THE TEST ORGANS: 

Toxic effects are greatly variable in nature, target organs and mechanism of action. 

All toxic effects results from biochemical interaction between the toxicants (and 1 or 

their metabolites) and certain structure of the organisms . The nature of effects may 

vary from organ to organ(Frank, 1985u).Gills, liver and brain are the most 

vulnerable organs of a fish exposed to the medium containing any type of toxicants 

(Jana and Bondyopadhyaya, 1987).

GUls: 

In take of the pesticides variously affect the fish physiology and metabolism. Gills 

are one of the vital organs, which come in direct contact with pollutant. Entry of the 

pesticides into a fish is largely through the gills (Holden,l972).Many water and fat 

soluble substances including organophosphorus pesticides freely pass across the gills, 

causing irreversible damage (Johanson, 1968). Herbert (1962) has pointed out that 

chemical variables can influence toxicity by affecting respiratory rate and thereby the 

amount that accumulates and be absorbed at the gills. Gills play an important role in 

osmoregulation. Change in structure and biochemistry may in turn affect the normal 

physiology of the organism and its survival. Hence, they often serve as the most 

sensitive index of environmental perturbations caused by pesticides (Bimber et 

aL,1976). Hence, gill is chosen as one of the focal organ for the present study. 

Liver: 

Liver is one of the most multifaceted and active organs in higher animals. It is the 

site for numeroas and varied metabolic activities, including synthesis of bile which 

contains bile salts, bile pigments, cholesterol and lecithin. Liver also has other 

important functions like metabolism of carbohydrates, fats and proteins. Liver is also 

involved in detoxification. Other functions of liver are excretion of certain substances 

discharged along with bile, formation of red blood corpuscles in embryos, and 

destruction of old erythrocytes in adults. It also helps in the synthesis of fibrinogen, 

which is essential for clotting of blood. Livei is the principle site of detoxification in 

verterbates(Bhatacharaya and Mukherjee,1976).Taking into consideration the 

myriad function performed by the liver, especially its pivotal role in detoxification, 

liver is chosen as one of the focal organ for our study. Furthermore, as all toxins pass 

through liver at some point or the other, the liver may manifest the highest toxin 

concentration so also the most clearly discemable struchlral and functional impacts.

Brain: 

Brain is the highest coordinating and integrating centre of central nervous system. It 

is a specialized organ of the body with complex structure and function. Brain is 

unique in regulating the functions of all the organs. The alteration in the normal 

structure and biochemistry of brain disturb the homeostatic regulations of the body. 

Since brain plays an important role in fish physiology and it is a rather interesting 

tissue in fish toxicology (Fmnk,l985).The study of brain was camed out to elucidate 

the biochemical and structural changes in this tissue during latex and plant extract of 

C, glgantea exposure.

REVIEW OF LITERATURE 

Aquatlc an~mals have become lmponant as surrogate specles for tox~colog~cal testing 

matenal that may have adverse b~ologlcal effect In man The fish mortal~ty IS largely 

due to the penetration of pollutants Into the fi sh body affecting vanous organ systems 

acutely or chronically causlng permanent damage and resulting In degraded growth 

and lor populat~on deplet~on Pestlcldes vary In thelr chemlcal formulat~ons as well as 

toxlc~ty, env~ronmental persistence and pathways of actlon 

The presence of 

pestlcldes In the aquatlc system can obv~ously lead to mult~fold lnteractlon w~th other 

forms of pollut~on In Ind~a, scores of stud~es have been undertaken to estlmate the 

acute toxlc~ty level of vanous pestlcldes on aquatlc fauna (Arora el aL,1971; Basak 

and Konar 1977, Shorrna el aL,1979) Vanous b~opestlclde compounds affect almost 

every physlolog~cal system They may act on the skln, In the d~gest~ve system, on the 

blood, on the var~ous parts of the nervous system, on the metabolism, In the 

nutntional value of food, and the hormonal and reproductive system of an~mals 

Presence of these toxlc chemicals (pestlc~des, fung~cldes and fert~l~zers) In fresh 

water med~a, may cause death or sublethal effects on the non-target oryanlsms 11ke 

fish, rats etc (Bhanacharp,i985 ; Desai and Joshi,1985; Sashy and MaIik,1979; 

Bergeri el aL,1984; Patole and Mahajan ,2006). In vlew of th~s there 1s a great 

mntenslty for testlng the toxic~ty effects of commonly used pest~c~des on the 

commerc~ally Important fishes to comprehens~vely w~thstand the effect of pestlclde 

on fishes Hence ~t IS proposed to study the toxlc effects of Calotropis gigantea 

(L )R Br latex , plant extract and ~ ts recovery w~th add~tlve nutnents 

2.1 CaloIropis giganlea : 

The specles of Calotroprs such as Calofroprs gigantea(L)R Br and Calotropts 

procera belong to family Asclep~adaceae A genus of glabrous or hoary, latlc~ferous

shrubs or small trees, commonly known as the swallow wort or milkweed.The plant 

is poisonous.(Azariah el a1,.1988 ;The Wealth of India,1992) . 

Classical and Common names: 

Sanskrit - Arka; Alarka; Mandara; Surya pattra; Eng - Gigantic; Swallowwort; 

Mudar; H~nd.- Madar; Ak. Ben.BrBom. - Akanda. Pers. - Khok; Kark. Guj.-Akado. 

Mab - Ruvi; Akda; Akra. Te1.-Mandaramu; Ekke; Jilledu; Arkamu. Tam - Badabada; 

Erukku; Yercum. Mal-Erikka. Can. Ekkemale. Sind. - Byclospa. Fr -Arbre-a - Soie. 

Geographical distribution 

Calotropis is distributed in tropical and sub-tropical Asia and Africa ,such as Sri 

Lanka, India, tropical Himalaya east to west and central China, Malaysia, Nepal. In 

dry, sandy parts of Africa extending into Mediterranean belt, Jordan, Arabia, 

Palestine, Abu Dhabi, the west Indies and tropical South and Central Arnenca. 

Found in all plains in waste places and on roadsides, often on black cotton so~ls. 

Description 

There are two common species of Calofropis snch as Cgiganfea(L.)R.Br. and 

C,procera. Both the species easily grow from seeds; even root and shoot-cutting is 

recommended. They do not required specific cultivation practices or imgation they 

are good soil-binders, and are recommended for desserts. They have a life span of 12 

years. The plants flower during December-July and fruit during February-June; in 

some regions such as in the cotton belt of Vidarbha and in many parts of South India, 

they flower and fruit throughout the year. Both species are used as substitutes for one 

another and are said to have similar effects. Although in South India Calotroprs 

giganlea(L.)R.Br. is most common and both the species are known by the same 

vernacular name. 

Calotropis gigan~ea(L.)R.Br.(Plate:A) A tall shrub reaching 2.4-3 m. high; bark 

yellowish white, furrowed; branches stout; more or less covered (especially the

younger ones) with fine appressed cottony pubescence. Leaves 10 -20 by 3.8-10 

cm., sessile or nearly so, elliptic-oblong or obovate-oblong, acute, thick, glaucousgreen, 

clothed beneath and more or less above with fine cottony tomentum; base 

narrow, cordate, sometimes amplexicaul. Flowers inodourous. purplish or white, 3.8- 

5 cm. diam., in umbellate lateral cymes; peduncles from between the petioles, 5-9 

cm. Long, dilated at the base; pedicels much longer that the flowers, covered with 

cottony wool; buds ovoid. Calyx divided to the base: sepals 6 by 4mm., ovate, acute, 

cottony. Corolla 2 cm. Long or more; lobes 1.3.1.6 by 4 mm ., long, deltoid -ovate. 

sub acute, revolute and twisted in age; lobes of the corona 1.3cm. Long by 5mm. 

Broad in the middle, shorter than the column, the back much curved towards the 

column above the obtuse spur, pubescent on the slightly thickened margin, the apex 

rounded 9not bifid) with 2 obtuse auricles just below it. Follicles 9-10 cm., long, 

broad, thick, fleshy, ventricose, and green. Seeds numerous, 6 by 5 mrn., broadly 

ovate, flattened, narrowly margined, minutely tomentose, brown; coma 2.5-3.2 cm. 

long (Nadkarni,l991 ;The Wealth ofIndia,l992). 

Medicinal Importance: 

Calotropis plants are used as medicinal source since vedic times. All parts of the 

plant dried and taken with milk act as a good tonic, expzctorant, and 

anthelmintic.The levels are applied to paralysed parts, painful joints, swellings, 

healing wounds.The milk is caustic, acrid; expectorant, depilatory; useful ill leprosy, 

scabies, ringworm , piles, eruptions on the body, asthma, enlargement of spleen and 

liver, dropsy.The tribal and rural people of Gujarat used to treat directly as and when 

required for ailments such as to painful joints, swellings ,boils , on wounds, cuts, 

eczema, ring worm, corn and orally for mouth ulcers, sore throat, tooth ache, 

pyorrhea, tonsilitis. The flowers are good for the liver (Yunnani).Oil, in which the 

leaves have been boiled, is applied to paralysed parts, a powder of the dried leaves is 

dusted upon wounds to destroy excessive granulation and promote healthy action. 

The root bark and juice of this plant are used in mediclne for their emetic, diaphoretic

and purgative properties. In the treatment of dysentery, the dried bark of the root is 

stated to be an excellent substitute for Ipecacusnha. The bark, root, and dried milky 

sap may be used in small doses in certain cutaneous infections, such as leprosy and 

secondary syphilis; the root-bark, in large doses, is an emetic. It is administered to 

promote secretions, and is stated to be useful in enlargements of the abdominal 

viscera, intestinal worms, cough, ascities, anasarca, etc. The flowers are considered 

digestive, useful in asthma, catarrh, and loss of appetite. The powder of the root in 3 

to 5 grains promotes gastric secretion and acts as a mild stimulant and may be given 

with carminatives in dyspepsia. It is also given as a febrifuge. The tincture from the 

leaves was tried in cases of intermittent fever. The powdered root bark in doses of 

five grains was given to several cases of dysentery and was generally found to give 

relief. The plant is a popular remedy for snake bite and scorpion sting (The Wealth of 

lndia,1992). 

The milk is bitter, heating, oleagenous, violent purgative and abortifacient, cures 

leucoderama, tumours, ascities, diseases of the abdomen, posses antiseptic 

properties-antibacterial, anti fungal, antlprotozoal and astringent activity(Jagid and 

Bhan,2005). In La1 Eela, the warmed leaves are used as a poultice. In Cianibla, the 

plant is said to be a good cure for sprains, headaches, and other pains; the leaves are 

applied warm to the affected part. The Hausas and Northern Territories people use 

this plant greatly in medicine. The leaves are used to cure headache, eye troubles. 

The leaves and fruits are boiled together and are used in the extraction of guinea 

won infection. It is used as for enema. In the Gold Coast, the leaves cure swollen 

legs and also wounds caused by rusty nails. The leaves are said to cure catarrh, being 

warmed first of all and then the juice is dropped into the nose (Nadkarni,l991).The 

flower extract exhibited anti bacterial activity namely Bacillus 

subtilis,Staphylococcus aureus,Pseudornonas aeruginosa and Escherichia coli 

(Akhtar et a1,1992;lorhsini et ~1,2002) antiulcer activity. The plant extract shows

cytotoxic activity (Smith et aL.1995) anti plasmodial and larvicidal (Sharma and 

Sharma,2001;Moursy.199 7) 

Prolonged high doses cause headache, burning in micturition and leucorrhoea, wide 

spread testicular necrosis and damage to liver when admin~stered orally to desert 

gerbil. The drug is highly toxic. Higher doses cause vomiting diarrhea, bradycardia 

and convulsions These medicinal plants produce toxic effect son the animal system, 

if they are not used carefully or in regulated amount (Khare,2004). 

Other Uses 

Cgigantea (L.)R.Br.and Cprocera is similarly used. The leaves are used as green 

manure for betelnut, paddy and wheat; they are reported to correct alkalinity in soil. 

Compost can also be made out of it. The Chinese prepare a sweetmeat from the 

flowers by boiling them in sugar solution after removing the latex. Akund floss (35- 

40mm long) is mostly used as substitute for or as an adulterant of the Indian kapok 

since it has visual resemblances to it, although it is much inferior. The stem bark 

yields resins and wax (The Wealth of India, 1992). 

2.2 Latex: 

The term latex refers to the fluid that can be extracted from laticifers(P1ate A) which 

varies in appearance and in composition. It is milky (clear or colorless or Brown 1 

Orange).Components found commonly are terpenoids, polyterpenes which occur as 

particulars in cytoplasm are in vesicles. Others are alkaloids, sugar, waxes, proteins , 

enzymes crystals, tannins , starch. The cells appeat like enlarged parenchyma cells 

and or known to occur in vascular and ground tissues of stem and leaf. Latex is a 

fluid of complex composition .All parts of the plants of both the species of 

Calotropts yields latex. It is also a source of hydrocarbons and can be used as a 

renewable source of energy. Both the species can be mixed with other organic refuse 

for producing biogas.

2.3 ACTIVE PRINCIPLES 

Both [he Calolropis species contains the physiologically active components 

namely cardiac glycosides (Cardenolides) - calotropin, uscharin, voruscharin 

(dihydrousacharin), calotoxin, calactin, uscharidin, gigantin, triterpenes, pentacyclic 

triterpenoids and flavonoid triglycosides (VonBrusehweiler et aL,1969;Martin et 

aL,1979) These compounds are highly toxic (Kiuchi ef al,l998;Havagiray et 

~1,2004). They have direct effect on heart and central nervous system. 

Calotropagenin is the common aglycone of all the glycosides calotropin, gigantin and 

uscharin show digitalis-like action on the heart. Many members of Asclepiadacea 

family are toxic and cardiac glycosides are often the culprit, cause gastro-intestinal 

toxicosis, inhibit cellular membrane ATPase enzyme system activity .During early 

course of poisoning, animals exhibit rapid breathing ,convulsions, irregular hean 

activity. Cattle and Horses consuming cardiac glycoside-containing plants are often 

found dead, postmortem findings include hemorrhages, congestion, edema and cell 

degeneration of the organs including multifocal myocardial degeneration and 

necrosls (Knight and Walter, 2OO2).A bacterioytic principle, capable of lysing 

Micrococus lysodeikticus was also found in the latex, anti inflammatory (Singh ef 

a1,2000)analgesic effect (Dewan et 01,2000) and Cardiotoxic (Ahmed et 01,2001). A 

non-toxic proteolytic enzyme, calotropain isolated from the latex, it is more 

proteolytic than papain, ficin and bromelain, coagulates milk and digests meat, 

gelatin and casein. (Kartikrr and Basu, 1984; The Wealth of India, 1992).The fresh 

milk is employed in the Punjab for the purposes of infanticide. In a drachm dose the 

fresh juice will kill a large dog in 15 minutes; its action, though slower, resembles 

that of hydrocyanic acid, but commences with foaming at the mouth. The latex is 

used as a poison by the Danoa or Haddad in the South-Eastern portion of the Kanem 

(Nadkarni, 1991). 

Latex of C.gigantea(L.)R.Br. and the extracts of aerial parts of the plant are used as 

arrow poison, infanticide,slows anti implantation activity.homicida1 poison. Toxic to

abbit, dog,donkey which showed increased hean beat and respiration leading to 

distress and death. And also effective fish poison, ~nsecticidal, good ovicidal and 

larvictdal in naturefThe Wealth of India,1992 ;Khare,ZOOQ). 

2.4 Acute Toxicity Tests: 

Acute toxicity tests are useful in screening large number of chemicals and in 

evaluating the relative sensitivity of different organisms to the same chemical. Thus 

they have been highly related in their present utility use in assessing the hazard to 

aquattc env~ronments (Macek et aL, 1976). The lrterature coocemtng toxtcrty test 

with pesticides was reviewed by Holden (1973). It was found that the 96hr.LCro to 

monocrotophos for Nile tilapia fish was 4.9 mg! I (Thanginipon et a1 1995) and 6.5 

ppm for edible mudskipper (Patilet aL, 1990). Pickering et aL, (1962) conducted a 

variety of acute tox~ctty test with 13 organophosphate pesticides. Dutta et 

(19921) reported the 24hr. LCso value of Malathion of Anabas lestudineus was 

28mgi I . LCro value to parathion for fish Oreochromis hornomm.was 0.147 mg i I 

(hura Martinez-Tabche el a:, 1992) LCso value of the pesttcides malathion and 

sevlll for Cyprinus carpio- was 0.0020 mg/ land 3.7 mgil (Dhanapakiam and Ju!ief 

Premalatha, 1994). 966hr LCso Value for fish Herreropneustes foss111Ls exposed to 

malathion was 12mg I htre (Durn et al., 1992a)The variat~on in toxicity depends 

upon number of factors such as size, age, sex, physiological state, ecological 

peculiarities (temperature, pH, C02, hardness of water) and pesticide specialities 

(rate of absorption, rate of degeneration, techntcal grade or commercial 

grade).Tolerance to pesticide increase with size. Various repons are available on the 

combined toxic effects of chemicals. It was 

al., 

found out that out of twelve 

combinations of chlordane, malathion and furdane were synergistic, two were 

antagonistic and one was additive in nature.96hr LCso Value for fish Herteropneustes 

fossil~s exposed to malathion was 12mg ! I (Dutta et al., 199211.) 

in most acute 

toxicity studies it is found that most compounds are increasingly toxic at high 

temperature. Endrin was several hundred times as toxic to Cyprinus carpio at 27@ C 

'age. 17. 96 hours LC 50: It defines the Lethal coneentr~tion at which 50 % of 

'ORaliQ for 96 hours duntion. 

fish

than at 7 ' ~ (Iyatomiet aL, 1958~) expression of dosage is less if the body weight or 

surface area is high 

reported that dosage profoundly influences the pattern of 

distribution of some chemical elements. According to Roy and Munshi (1987), 

technical grade malathion is less toxic than commercial grade. Young fish as 

compared to older ones have larger respiratory area per unit body we~ght and a higher 

metabolic rate. Therefore pollutants are more toxic to younger fish than to older ones 

(Duffa et al., 1992a) Fate head minnows fry 2-30days old were 10 times more 

sensitive to dioxthion than adults (Piekering et aL, 1962). 

2.5 Bebavioural Toxicity: 

Aquatic organisms exhibit a broad range of responses to insectic~des depending on 

the compound, exposure time, water conditions and specles; coppage and Mathews, 

1974). Behavioural modification is one of the most sensitive indicators of 

environmental stress (Olla et al., 1980) and may affect survival rate. 

Many 

pesticides affect both instincitive and learned behaviour in aquatic organisms .The 

concern of neurotoxicity in human population has motivated a large number of 

psychophysiological studies on the effect of toxic substance on animals (Duna el aL, 

1934).It is believed that behavioural changes are the most sensitive measures of 

neurotoxicity A single behavioural parameter IS more comprehensive than a 

physiological or biochemical parameter (Warner er al., 1966). by their experiments 

on the effects of sublethal doses of Hydrazine on the behaviour of blue gills 

supported the concept that toxicant caused stress upon organisms can be quantified 

by methods other than mortality.Changes in behaviour have been suggested for use 

as a sensitive indicator of chronic sublethal toxicant exposure. Some fish behaviours 

(locomotor activity and avoidance)are extremely sensitive to pollutant chemicals, 

whereas others (aggression) seem to be rather refractory(Richmondo and Duffa 

1992; Duffa et al, 1992b).Conditioned learning is a sensitive identification of 

chemically induced stress in aquatic organisms.Warner et al., (1966) trained gold 

fish in a conditioned avoidance response apparatus and showed 96hr.exposure of 1-

Bmgllitre of toxaphene produced changes in its conditioned behaviour.The locomotor 

behavior of fish has been altered by sublethal concentration of pesticides. 

Heath(l987)showed that some fish behav~ours as locomotion actlv~ty and avoidance 

response are extremely sensitive to pollutants. Swimming perfonnance(or endurance) 

was also altered in Juvenile Coho Salmon exposed to sumithlon (Bull and MC 

Inerney, 1974).Rand (19770, b) found out a decline in the activity in blue gills and 

large-mouth bass exposed to sublethal concentration of parathion. The optomotor 

response is considered to be essential for maintenance of position wlthin the habit 

and for schooling in fish (Duna et a/., 19926). This response is defined as a 

movement of the animal in the direction moving reference polnts In the field of 

vision (Scherer and Harrision, 1979). Macmillan (1987) recorded the changes in the 

optomotor responses of fat head minnows exposed to herbicide alachlor and atrazine. 

Change in the optomotor behaviour was observed Indian carp, hbeo rohita exposed 

to malathion (Dutta et al., 1992b).Pesticides have an alteration effect on the 

temperature selection of fishes. Fish living in the suitable temperature after treated 

with a specific concentration changes its temperature range. Atlantic salmon when 

treated with DDT and methoxychlor changed its temperature rang' A fish treated 

with pesticide may fail to feed.Fishes have different mechanism or countering their 

predators. Exposure for 24 hr.to l.0ppm of sumithion, Atlantic Salmon purr were 

more vulnerable to predation by large brook trout than unexposed fish (Hatfeld land 

Anderson,l972).Numerous study was reported on avoidance response and 

behavioural changes in fishes exposed to paper mill, kmft mill and sulphite mill 

efluent. It was observed the avoidance by both susceptible and resistant populations 

of mosquito fish of sublethal level of endrin, toxaphene and parathion. 

2.6 Haematology: 

When an animal system is toxicated, its blood oAen manifests pathological changes 

before external signs of poisoning are noticed.Haematological observations are also 

perhaps the simplest and quickest of diagnostic tool in physiological studies (Dewraj

et aL, 1990). Previous research on the blood of fish has shown that haematological 

parameters are influenced by toxic substances (Bakthavathasalam 1991; Dufta et al., 

1992c; Gill er al., 1991a, b;Singh and Srivastava,l993).Numerous Laboratory 

studies have reported changes in red blood cells (RBC), haemoglobin (Hbj and PCV 

of fish exposed to pesticides Some reporting sign~ficant increase Rao and Murthy 

1983) and some reporting significant decrease (Reddy et al., 1992; Ahamad Figar et 

al., 1995; Narh Rabindra and Banerjee 1996; Ibrahim el al., 1995). Haemolysis of 

red blood cells provides a simple and rapid way of studying the effects of pollutants 

on b~ological membranes (Harington et a1.,1971). Numerous investigations have 

considered membrane models for a measure of a pollutant have considered 

membrane model for a measure of pollutants's cytotox~city (Allision et al., 1966). 

The haemolysis of red blood cell membrane has proved to be a simple and rapid way 

of attempting to find the possible correlation between cytotoxicity and haemolytic 

activity (Macnab and Hnrringion, 1967). Abbasi and Sujata Krishnan (1993) 

reported haemolysis of RBC in fish Tilopra mossarnbim~exposed to pesticide cartap. 

The WBCs help in protecting the body against microbes. The WBC counts may rise 

abnormally in acute infections and in some other cases Heterouneusres Jossllir 

exposed to malathion similar increase in WBCs was noted when fish Tilapia exposed 

to cartap (Abbasi and Sujatha Krishnan,1993). Mukhopadhyay and Dehadrai 

(1979) reported hematological abnormalities in catfish Clarias bcrrachus exposed to 

malathion.Gil1 et al., (1991a) observed change's in leukocyte subpopulation and 

erythrocytes in fish (Puntius containr, Hamilton) exposed to aldicarb. Pesticide 

exposures were known to cause lymphopenia in Cyprinur carpio), Clarias barrachus 

(Dalela et a[., 1980, Srivastava and Mishra, 1985) reported thrombocytopenia in 

different species exposed to organophosphate pesticides. 

2.7 Biochemical Studies 

Pesticides modify the general metabol~c stage of the organisms by influencing 

different metabolic segments (Swamy et al., 1992b).lt is known that tissue protein, 

O. Me&lic 

me Mummobules Pmtein, Culah~bte. ~ i ~ i d Nucleic aids 

KNA ) Metabolism are the metabolic segments.

carbohydrate and lipids play a major role as energy precursors for fishes exposed to 

stress conditions (Mortata et al., 1982; Puviani et al., 1990) .Proteins constitute a 

large part of tbe structure of cells and are present in all tissues. Many proteins have 

also special physiological functions such as structural components of cell 

membranes, enzymes, hormone, bloodproteins and nucleoproteins. 

Organophosphates cause changes in protein metabolism (Sandhu and Malik 1988~; 

Sandhu el al., 1991). Very meager information is available about changes in protein 

patterns as an indicator of contamination or the nature of pollutants. Monocrotophos 

has significant effect on the brain protein and phosphatases of fish (Joshi and Desai, 

1983). Sandhu and Malik (19886) reported change in protein level was also reported 

by Patil et a!., (1990)in Boloeopthalmus dussurneri exposed to monocrotophos. 

Heterpneustes fossilis when subjected to BHC contamination showed a remarkable 

variat~on in serum protein fraction (Reeta Pandey et al., 1991)) found a similar 

variation in the blood protein fractions in Channapunctuatus treated with Malathion. 

It was reported reduction in protein fractions in Channa punctatus treated exposed to 

5 ppm, of BHC. In Anabar testudineus(B1och) protein level in muscle and liver was 

reduced on exposure to disystron (Bhaktavathasalam 1987). In rainbow trout 

exposed to sublethal concentration of dietary endrin of 165 days, significant changes 

occurred in total protein Changes in protein level and glycogen were also reported in 

various tissues of fish exposed to pesticides. Ihrahim et al, (1995);: Ahamad Figar 

el aL, 11995); Ikhatair- Ud- Din et al., (1996); Tazeem Arasta et al., (1996) 

Anusha el al., (1996).Studies on the effect of pesticides on phosphatases activity are 

very much limited in fishes.Alkaline and acid phosphatases are known as "inducible 

enzymes" whose activity in animal tissues goes up where there is a toxic impact and 

the enzymes begin to counter act.Subsequently the enzyme activity may begin to 

drop either as a result of having partly or fully countered the toxin or as a result of 

cell damage (Abassi and Sujata Krishnan,l993).Changes in the acid and alkaline 

phosphatase activity were also reported in fishes exposed to pesticides Ahamadfigar 

et al., (1995); Abassi and Sujota Krishnan (1993); Satheesh Kumar Reddy,

(1994).The brain Acetyl cholinesterase (AchE) activity is inversely proportional to 

ACh content in toxicant treated fishes. The measurement of AchE activity is taken as 

a good indicator of the extent of aquatic pollution by toxicants Accumulation of Ach 

and inhibition of AchE activity in brain and other tissues have been reported in fishes 

.The parameter appear to be reliable ~ndicator of induced toxicity to Ash and decrease 

in activity is found to be dose dependent.ATPase activity in general transport speaks 

about transport of sodium and potassium ions and as well synthesis of ATP.The 

active transport involves magnesium ion dependent ,sodium and potassium ion 

activated ATPase which provides the largest contribution to the maintenance of ionic 

trans membrane gradients .Inhibition of enzyme activity was reported by many, for 

the exposure to pesticides and their by interfering with membrane ionic conductance. 

2.8 Histopathology: 

Histopathology studies along with physiological and biochemical data provide to 

unravel the mode of action of the toxicants(Satheesh Kumar Reddy, 1994).1n aquatic 

environment th:: pesticides are diluted to sublethal levels producing chronic 

histopathological effects on organs.Kumar and Pant (1994)have stated that 

histopathological studics are useful to evaluate the pollution potential of pesticides 

since trace levels of pesticides, which do not cause animals mortelity over a given 

period, are capable of producing considerable organ damage. lnsecticides have been 

shown to induce many pathological changes in the organs of variotls species of fishes 

(El-Elaimy et al., 1990; Desai er aL, 1984). 

Gills: 

Gills become more vulnerable because of their location, and constant intimate contact 

with the water.Gills are liable to damage by any irritant material, whether dissolved 

or suspended in water(Lemke and Mount,l963).Anabas testudineuc (Bloch) exposed 

to sublethal dose of Saponin 50ppm showed swelling of gill filaments and 

]amellae,increased mucous production, inflammatory alterations in epithelium, loss

of microridges,shrinkage of blood capillaries and reduction of interlamellar 

spaces(Roy el a1.,1986).Epithelial desquamation, epithelial separation from basement 

membrane, necrosis, telangiectasia, epithelial hypertrophy and epithelial hyperplasia 

with lamellar fusion have occurred following exposure to toxicants-Malathion 

(Areechon and Plumbe, 1990); endosulfan (Sateesh Kumar Reddy, 1994) ; lindane 

(Zayapragassarazan, 1993);Chloradane (Jyofsna Shrivatava and Srinivastava, 

1984);Sewage (Arun shanker Narain,l990)monocrotophos (Vijayalakshmi and 

Tilak 1996) ;heptachlor (Moses Cirija and Jayantha Rao, 1995) and carbofuran 

(Karpagaganapafhy and Sukumar, 1988). 

Liver: 

Hepatotoxic les~ons of fatty ~nfiltration, nuclear or general hypertrophy of 

hepatocytes, other degenerative changes in parenchyma (cytoplasmic vacuolation, 

cellular pleomorphism, deposition of bile or ceroid pigments, hydropic degeneration), 

loss of hepatic glycogen,coagulative hepatocyte necrosis, sinusoidal and vascular 

congestion, degeneration or necrosis of biliary epithelium have been reported 

following exposure to several pesticide-malathion(Areechcn and Plumb, 

199O);carbofuran (Karapagaganapafhy and Sukumar, 1988; endosulfan (Sateesh 

Kumar Reddy, 1994); lindane (Zayapragassarazan, 1993); aldrin (Mathur er al., 

1981). Kulshresrha and Lakhmi Jauhar (1984) found that thiodon exposure 

produced rapid degeneration, hypertrophy, necros~s of hepatocytes in Channa 

slriatus. 

Brain: 

Reports of hyperanemia, hemorrhage, vascular congestion and dilation, fraction, 

cerebral oedema, nuclear pyknosis, rupture and haemorrhage of meninu primitive and 

swelling of myelin sheaths around nerve fibers have occurred with the exposure of 

pesticides like malathion(Wa1sh and Ribelin, 1975); 2,4-D(Cope el al., 1970); 

methoxychlor (Kennedy et a[., 1970), endosulfan (Sajitha Bhaskar, 1994).

2.9 Scope of the present work 

Residual pesticides in soil or on crops are transported from fields to surface and 

ground waters. The leaves and stems of Calotropis giganiea (L.)R.Br. are used In 

agriculture for soil fertil~ty. If they are released in environment In adequate before 

degradation ,they kill non-target and other aquatic species ,some toxic substances b ~o 

accumulate in tissues of fish and other species thus poslng health risks to human 

beings and others, who consume the fish and contaminated water. The nutntive 

value of fishes gets reduced; fish population gradually diminishes because of the 

toxicants. It is been known in medicinal importance in Shrimps (SEMBV viral 

infection) and prevents viral white spot disease (Raman, 1997).So far, no body has 

made an attempt to study the latex and plant extract of Calotropis gigantea(L.)R.Br. 

on fish as it is known for medicinal importance, However, the supplements such as 

Glucose,Fnrctose,Vitamin C, Vitamin E including egg albumin and glycine are 

considered as nutrients, which enhances the metabolic activity and provide resistance 

also, protection against the fish mortality. Addition of Glucose and Fructose will 

enhance energy source and the metabolic activity thereby protected from stress 

condition. The addition of Albumin, Glycine, Vitamin C and Vltamin E appears to be 

the first of the defecce agalnst peroxidation of cellular and sub cellular membrane 

phospholipids., prevention of peroxidative damage to cellular and sub-cellular 

elements and provides tensile strength thereby preserve the organs or organelles 

potential savlor against degenerative diseases, necessary to cope with the disease, 

physical and chemical environmental insults and other stress, The findings may be 

helpful to the farmers in their day-today profession with special reference to 

aquaculture industry and to counsel the farmers and thereby, the bio-toxicity will be 

aware of the mortally which shrunk the industry as a whole.

CHARACTERISTICS OF TOXIC BIOACTIVE COMPOUNDS OF 

GIGANTICINE 

C.gigantea(L.)R.Br. 

CHAPMAN&HALL NO: DYCI 5-1 

CAS NO: 199665-81-1 

COMPOUND CODE: VVOIOOVV9999 

MOLECULAR FORMULA: CllHlaN:O~ 

MOLECULAR WEIGHT: 280 

USE: INSECT ANTIFEEDENT 

PHYSICAL DESCRIPTION: CRYSTALLINE (MeOH) 

MELTlNG POIKT: 159-162

GOMPHOSIDE 

CHAPMAN&HALL NO: HGL38-G 

CAS NO: 36597-51-0 

COMPOUND CODE: VT0750XA2'60 

MOLECULAR FORMULA: CoKI:08 

MOLECULAR WEIGIIT: 518.287 

BIOLOGICAL SOURCE: CARDIAC GLYCOSlDE 

PHYSICAL DESCRIPTION: CRYSTALLWE(Mc0H) 

MELTING POINT: 268-271 

GAMPHOSIDE DERIVATII'ES 

CALACTIN 

CHAPMAN&HALL NO: BZK24 

CAS NO: 2030447-6 

COMPOUND CODE: AJ0750\T0750XA2760 

MOLECULAR FORMULA: C3&@ 

MOLECULAR WEIGHT: 532.63 



GOMPHOTOXIN (or) CALOTROPIN 

CHAPMAN&BALL NO: HDG2-E 

CAS NO: 1986-70-5 

COMPOUND CODE: AJ0750VT0750AJ5000 

MOLECULAR FORMULA: Cx&Oq


USE: AFRICAN ARROW POISON 

PHYSICAL DESCRIPTION: CRYSTALLME 

TOXICITY(CAT): O.lm&Kg 

CALOTOXIN 

CHAPMAN&HALL SO: HDD88-M 

CAS NO: 2030449-8 

COhZPOUHD CODE: AJ0750VT0750 

MOLECULAR FORMULA: C.bOlo 


PHYSICAL DESCRIPTION: CRYSTALLINE (McOH) 


USCHARIDIN 

CHAPMAN&HALL NO: HDR40-K 

CAS NO: 2030447-7 

COMPOUND CODE: VT0750AJ0750 

MOLECULAR FORMULA: CtpHlaOp 



MELTING POINT: 290 

ASCLEPZN 

CHAPMAN&HALL NO: .WR-38-P 

CAS NO: 36573-634 

MOLECULAR FORMULA: C,,&zO,, 


PHYSICAL DESCRIPTION: CRYSTALLINE (McOH) 


(Abel, 2000;Abdcl azim ,1998).

C ALOTROPAGENIN 

HO.. 

HO 

do 

CHAPMAN&HALL NO: HGP48-D 

CASNO: 24211-64-1 

COMPOUND CODE: AJ0750UT0750 

MOLECULAR FORMULA: C23H320a 

hlOLECULAR WEIGHT: 404.502 

BlOLOGlCAL SOURCE: AGLYCONE 

PHYSICAL DESCRIPTION: CRYSTALLINE 


(Sing., 1972;Lardon ,I 969;Lardon,1970). 

USCHARIN 

CHAPMAN&HALL NO: HGK16-T 

CASNO: 24211-81-2 

COMPOUND CODE: AF77M)AJ0750VX6790W7700VT0750 

MOLECULAR FORMULA: C,,It,NOaS

MOLECULAR WEIGHT: 587 733 

BlOLOGlCAL SOURCE: LATEX-ALKAIOID 


TOXICITY: CARDIAC POISON 

VORUSCHARIN (dihydrouscharin) 

(DERIVATIVE OF USHARIN) 

CHAPMAN&HALL KO: BSK97-K 

CAS NO: 27892-03-1 

COMPOUND CODE: VX679OAJO750W0750 

MOLECULAR FORMULA: C,,%INO~S 

MOLECULAR WEIGHT: 589 749 

BIOLOGICAL SOURCE: LATEX-ALKALOLD 

PHYSICAL DESCRIPTION: CRYSTALLINE 

MELTING POINT: 165.166 

(Abe,ZOOO;Abdel- Azim. ,1998: Ware Sinha ,1994;Komissarenko ,1997).

MATERIALS AND METHODS 

Experimental Animals: 

Air-breathing fish, Anabas testudineus(B1och) of uniform size (11i 2.2 grams 

weight, 8cm length) was collected from Velarampet fish farm, two kilometers south 

of Puducherry. All the fishes were collected from the same stocking pond. Test 

organisms are transported in circular plastic containers to prevent the animals from 

crowding or damaging themselves by striking the walls as suggested by Cox (1974). 

During transport to the laboratory the test organisms were kept free from 

overcrowding, bruising, stress, thereby decreasing their susceptibility to disease. 

They were disinfected with 0.1% solution of potassium permanganate for 5 minutes 

to avoid dermal infection. Further the active and healthy ones were selected for 

acclimation during which they were kept in quarantine for 16 days The species were 

fed with fresh goat liver during acclimation. The best among the acclimated were 

chosen and subjected to f~rther acclimation in the fiber -glass aquarium for 48 hours 

before the toxicity test. Test animals were not fed 24 hr before commencement of the 

experiment. 

Fish Tanks: 

Plastic tanks of 20 liters capacity were chosen for maintaining the fish. Each tank 

was filled with 15 liters of water to avoid the fish jumping out of the tank. 

Water Supply: 

Thorough scrubbing and cleaning of each tank were carried out and tap water stored 

for 48 hr. was used for complete replenishment in the morning, and is used for 

bioassay experiment The quality of water was determined periodically according to 

Standard methods (APHA, 1998).0nly slight variation in water quality was observed 

during the experiment (Table :2).

Disinfection: 

All the tanks were disinfected once a week with 5% Povidone iodine (Alphadine) 

diluted to Iml I liter of water. 

Tenure: 

The experiment with latex of C. gigantea (L.)R.Br. latex with feed and the plant 

extract was run for 96hrs continuously, with all parameters being maintained the 

same through out the period. 

Feeding: 

Both the control and experimental fish were fed with fresh goat liver only once a day 

in the morning at 10.00hr. 

Chemicals and Glassware: 

Thz chemicals, reagents, subslrates and standards used m the present investigation 

were of analytical grade purchased from British Drug House, England; Fluke A.G., 

Switzerland; Loba Chemie, Austria and Sigma Chemical Company, USA .Distilled 

and deionised water was used for all analytical work and for preparing stock 

solutions. Acid and alkali resistant glassware manufactured by MIS Borosil, India 

was employed. 

Latex and Plant extract coUeetion: 

Latex of C. gigantea (L.)RBr is used for the present study. A cut was made at the tip 

of the stem of C. giguntea (L.)RBr. and the oozed latex was collected in glass 

container and used for the analytical work (Rooj et ~1,1984). And for plant extract 

preparation, C. gigantea (L.)RBr. plant material were collected from nearby places 

of Puducheny and authenticated in our Botany department. The stem and leaves was 

taka and dried at room temperature and then pulverized using a grinder. Usual 

solvent water was used to prepare the aqueous extract. This extract was prepared by 

heating the material(5 grams powder) with IOOrnl of water at 50-60 C on water

ath over a period of 6-8hrs.lt was then filtered through whatman filter paper and 

concentrated on water bath by slow evaporation(Mcrhajan and PatoIe,2003;Tiwari 

and Singh,2003). The requisite quantity was exposed to animal by preparing dose in 

water, while control animals exposed with only same quantity of water. 

Latex with feed supplements: 

Feed alone with water as control and Supplements (VitaminC,Vitamin E,Glucose, 

Fructose and Egg albumin plus glycine) added was 100mgi51itre volume15 number 

of fishes along with feed, in combination with three sublethal concentrations of latex 

of C. gigantea(L.)R.Br. was done separately. 

Methods: 

Toxicity test both acute (Short-term) and chronic (long -term) are necessary in water 

pollution evaluations because chemical and physical tests alone are not sufficient to 

assess potential effects on aquatic life.Since no much information and data on 

toxicity of C. gigantea (L.)R.Br. on fish were reported in the literature, WHO (1977) 

has recommended the use of short -term toxicity studies of pesticides in the living 

organisms. 

Acute Toxicity Tests 

Acute Toxicity tests were done by time dependent (96 hours) static renewal 

technique in conformity with guidelines suggested by APh2 (1998). 

Sub lethal Studies and exposure periods: 

In natural or experimental conditions a sub lethal concentration of a pesticide is most 

likely to produce sub lethal effects to alter the morphological, physiological, 

histological and ethological conditions of the fish though it may not cause immediate 

death of the individual. Hence, the study of sub lethal dose effect of a pesticide is 

comparatively more rational than lethal or fatal dose, as in most of the polluted 

natural environment, sub lethal concentration is met which may cause the alteration

in the normal survival of organisms over a prolonged period of time. Accordingly, 

the present study was taken up with sub lethal dose response in the experimental fish. 

The 96 hours LCso was considered as One Toxic Unit (ITU) (Sprague and Ramsay, 

1965) and based on this value, three different sub lethal concentrations viz., 

1/~'~,1/10" and 1120'~ of LC50 values (TUl(0.005 mU5litre); TU2(0.01 mlI5lihe) and 

TU3 (0.02 ml/Slihe) of 

latex, for plant extract TUl(0.5 mUSlitre);TU2(1 

ml/Slitre);TU3(2 mU5litre) and three different sub lethal concentrations viz., 

115'~,1/10" and 1120~ of LC50 values (TUl(O.(X)S mV5lihe); TU2(0.01 mU5litre) and 

TU3 (0.02 mV5lihe) of latex plus supplements was taken for biochemical, 

behavioural, haematological, histopathological studies. Behavioural study, 

biochemical, enzyme assay and haematological studies were performed for 96hrs. 

Histopathological studies were also carried out for 96hrs of exposure at three 

sublethal concentrations as mentioned above. A detailed description of the 

methodology for acute toxicity test and the sublethal tests is described in the 

respective chapters. 

Statistical Analysis: 

The data are presented as the Mean * SEM (Standard Error of Mean).Variations 

among the sample means is due to sampling errors. Standard Error is an important 

instrument which measures sampling variability due to chance or random forces, with 

this we determine limits with in which the parameter values are expected to lie. Here 

significant treatment effect was found, the students "t" test was used to compare the 

experimental groups with their respective controls, for each exposure period (Ode 

,1966).From the degree of freedom, values of probability were obtained from the 

standard table given by Fisher and Yates (1948). If the calculated value is more 

than the table value, it is significant at the probability level. The following levels of 

significance were used p

3.1 TOXlCITY TESTS with Anubas lesfudineus 

The "work horse" in monitoring the effects of water pollution is the assessment of 

toxicity of pollutant(s) for the specimen concerned (Buikemu er aL, 1982). Toxicity 

threshold is commonly assessed by bioassay test Acute bioassay test has been used 

to determine the actual impact of various pesticides on aquatic life. Static acute 

toxicity test provides rapid and reproducible concentration response curves for 

estimating toxic effect of chemicals on aquatic organisms. These tests provide a 

database for determining relative toxicity to a variety of species (Ferrundo and 

Adrew-Moliner, 1991).LCso or the median tolerance limit (TLM) is the concentration 

in which 50 percent of the population tested for a specific period of exposure died 

(Duffa d aL, 1992~).LCro data are useful for calculation of application factor (AP). 

Maximum Allowable toxicant concentration (MATC) and for producing a toxicity 

curve (APHA, 1989). The purpose of the present study is to estimate the 96hr LCJo 

value of latex and the plant extract of C. giganrean (L.)RBr. to an air-breathing 

teleost fish Anabas testudineur(Bloch). 

Method: 

Static renewal test as suggested by APHA (1989) has been undertaken for finding the 

acute toxicity of latex of C. gigantea. &.)RBr. The test material was added to the 

dilution water to produce the desired test concentration.A range finding bioassay test 

was done fmt which indicated that the "no lethal level" and 'Total lethal level" of 

latex and plant extract .These trial runs were made to delineate the pmbable 

concentration range. Accordingly for the definitive test latex of C.gigantea(L.)R.Br 

concentrations of O.OIrnl, 05ml 071111, IOml, 151x11, 20ml, 25ml, 5hl, 75 rnV5litre 

were employed. Ten fishes, randomly picked, were introduced in each of the nine test 

chambers containing these concentrations, a tenth test chamber also containing ten 

fishes but no latex, sewed as control. The above procedure is also employed for plant 

extract of C.giganrea, (L.)RBr. taking concentrations of Iml, 5ml, 7ml, IOml, 15m1, 

20ml, 25ml. SOml, 7SmV5litre. Each test chamber was observed continuously for the 

tests.: page. 34 The coaceatrations of latex corrected a9 tableJ. 

Concentratioas of latex cormt doga: 0.01,0.0~,0.07,0.1,0.1~,0.20~25,0.5~0.75 are the doses#

I" day (when mortality in higher concentration was high) and subsequently four 

times a day. This test was conducted in static water but the latex, plant extract in 

water and control waters were renewed periodically (usually at 24 hr intervals) by 

removing and replacing the materials in the original containers. This avoids the 

accumulation of excretory wastes and the death of test animals due to oxygen 

depletion. Fishes were not fed through out the toxicity test (Sprague, 1973). 

Observations of mortality were made at 12, 24,48,72,96 and 120 hr. The data were 

analysed using the probit analysis (Finney, 1981). The probit mortality were plotted 

on graph against log concentrations. The concentrations producing a 50% mortality 

and the slope of the probit lines were drawn for 96 hr exposure of latex and plant 

extract of Calotropis gigantea(L.)R.Br. in Anabas testudineus {Bloch) (Fig: 1 and 2). 

3.2 BEKAVIOURAL TOXICITY: 

Behavior is the recordable and observable actlvity of the living organisms. The Fish 

depends on an intact nervous system for mediating relevant behavior. The nervous 

system is most vulnerable and icjuries to its elements through pesticides or toxicants 

may drastically change the behavior and conseque~tly the survival of the fish. A 

behavioral changes may, in fact be the first response of an organism to environmental 

perturbation (Slobodkin, 1968). Aquatic organisms exhibit a broad range of 

responses to insecticides depending on the compound, exposure time, water condition 

and species (Coppage and Manhews, 1974) . It has been shown that some fish 

behaviors (e.g., locomotor activity and avoidance) are extremely sensitive to 

pollutants (Heath, 1987). The changes in the locomotor activity of bluegills exposed 

to sublethal doses of DDT, Cadmium and Zinc were observed by Effgaard et al., 

(1977,1978). Macmillan (1987) recorded the changes in the optomotor responses of 

fat head minnows exposed to the herbicides Arachlor and Atrazine. The optomotor 

responses are wide spread throughout the animal kingdom. It is considered to be 

essential for maintenance of position within the habitat and for schooling in fish. 

This response is defined as a movement of the animal in the direction of moving

eference points in the field of vision (Scherer and Harrison, 1979). Scherer and 

Harrison (1979) described a technique to elicit the optomotor response in fish and 

invertebrates, by rotating circular screens marked with black and white vertical 

stripes around a cylindrical test chamber. This response is considered to be an 

essential one for maintaining position within the habitat, and for schooling in fish. 

This optomotor response test can detect the lmpaiment of visual orientational 

function. Since the optomotor response can be quantified, it was selected for this 

study. Changes in the respiratory metabolism are one of the early symptoms of acute 

pesticide poisoning (Holden, 1972, 1973). Many pesticides increase respiratory 

metabolism (Bhaktavathslam and Srinivasa Reddy, 1983). Pesticide contamination 

of water may decrease the oxygen intake of fishes (Vasanthy and Ramswamy, 1987). 

Besides gills and skin, in Anabac tesiudineus,(Bloch) The branchial chambers are 

present that acts as the accessory respiratory organ that aids the fish to consume 

atmospheric air at times of oxygen depletion due to pesticide contamination. Huges 

and Singh (1970) reported that Anabas consumes about 54% of air by surfacing than 

from water (About 46%) of its total Volume of oxygen by bimodal respiration. The 

ventilator movement of gill depends on the oxygen availability and its consumption. 

An over all decline in the activity of fish was also reported (Rand, 1977a, b). The 

present work was undertaken to point out the inter- relationship of various behavioral 

patterns and its modification in the behavior due to introduction of latex and plant 

extract of C. gigantea(L.)RBr. in Anabas restdineus (Bloch). 

Materials and Methods: 

The procedure employed in collecting, maintaining and acclimatizing and the latex, 

latex with supplements and plant extract of C. gigantea (L.)RBr.used in the present 

work is described in detail in the earlier chapters. Anabas teshrdineus(B1och) 

weighing I l* 2.2 grams, 8 cm body length was kept in a square test container that 

effectively minimizes the horizontal swimming of the fish. The depth of the water 

was kept constant at 25 cm in all aquariums and therefore the fish traveled 0.5 m

when surfaced to breathe. The fish was subjected to fasting which involved 24 hr 

periods of food deprivation followed by repeated feeding with goat liver once in 

every day for 2 hr. All the fish accepted the feed, ate readily and showed no fright 

response. Each experimental aquarium conta~ned only one fish and 12 such aquarium 

is used, 3 for control, 3 for TU1. 3 for TU2, and 3 for TU3 and were maintained for 

96hrs. The Experiment was conducted In the laboratory when there was no 

disturbance except for feeding and routine observation. The laboratory was under 

natural illumination for 12 hrl day, the temperature fluctuated 25' C to 27' C and 

averaged 26'c.~he number of times each test individuals surfaced was observed for a 

known period (5-l0min.) at 9.30 A.M., 11.30 A.M. and 3.30 P.M. The distance 

traveled by the individuals per day was estimated by multiplying the mean number of 

visits. Observations were made on three individuals in each group belonging to 4 

units. One control and three latex of C. gigantea(L.)R.Br. treated and also with the 

plant extract separately. 

Enumeration of Optomotor Behanour: 

The optomo!or response or the control and experimental fish were measured using 

the optomotor response test apparatus (Macrnil/an, 1987; Duffa el al., 199211; 

Richmonds and Duna, 1992).The apparatus consisted of a plastic bucket, a turntable 

and 2.2 L Meson Jar. The inside of the bucket wall contained 1.2cm wide stripes of 

black electrical tape placed 7cm apart at an angle of 55 (D). The jar was suspended 

inside a plastic bucket. The glass jar was filled with water containing the same 

concentration of latex, plant extract to which the fish were exposed. The bucket was 

made to rotate at a speed of 20 rpm. With the direction of the movement being in the 

downward slope of the stripes. The bucket was made to rotate and the fish was 

allowed to settle down with the bucket rotating for 6 minutes. Rotation of the bucket 

was stopped for 3 minutes, during "OW period. This was followed by an observation 

and recording period of 3 minutes with the bucket rotating. Every 90' turn or 

movement of the fish, referred as "Quarter turn" was recorded as one movement.

Quarter turns in the direction of the drum movements were recorded as "followings" 

and quarter turns in the opposite direction of the rotation were recorded as 

'*reversals". The mean scores of 6 readings of 3 fishes for each concentration, with 

two trials for control were calculated and the SEM of the means was also derived. 

Considering the mean scores, graphs were drawn for the "following" and "reversals" 

movements of the fish (Control and at different concentrations of latex of C gigantea 

(L.)R.Br.were studied on the 96 hrs of exposure and also with plant extract of C 

gigantea (L.)R.Br.). 

Enumeration of Surfacing, Distance Moved and Opercular Movement 

AAer acclimation, the frequency of surfacing per minute was studied and enumerated 

in control and all the three test compounds of C. gigantea (L.)R.Br exposed fishes for 

different concentrations and periods of exposure. The number of opercular 

movenients was observed for 1 minute using a stop watch. The observation was done 

again for 1 minute and the mean values were taken to get the behavioral action of the 

particular fish. 

3.3 TOXICITY IMPACT ON HEMATOLOGICAL PARAMETERS 

Numerous biochemical indices of stress have been proposed to assess the health of 

non-target organisms exposed to toxic chemicals in aquatic ecosystems (Niimi, 

1990).The physical properties of fish blood are very sensitive to environmental 

changes (Hugcs and Nemcsok, 1988) and are commonly used (Wedemeyer and 

Yasfuke, 1977). The use of hematological methods as indicators of sub lethal stress 

can supply valuable information concerning the physiological reaction of fish in a 

changing environment. The reason for this is the close association between the 

circulatory system of the fish and the external environment (Cassilas and smith, 

1977). The present study was undertaken to trace the effects of sub-lethal 

Concentrations of latex, latex with supplements and plant extract of Calotropis 

giganrea(L.)R.Br. on blood of Anabas testudineus.(Bloch). The parameters studied

includes hemoglobin content (Hb), oxygen combining capacity, total red blood 

corpuscles count (RBC), Total white blood cell count, Haematocrait (Ht). Mean 

corpuscular hemoglobin (MCH),Mean corpuscular volume (MCV), Mean 

corpuscular hemoglobin concentration (MCHC), blood smear and to explain how 

latex and the plant extract produces changes in erythropoietic activity. These 

parameters were studied in fish exposed to TU1 (0.005rn1151itre); TU2 (.Olml15litre) 

and TU3 (.02mlI5litre) of 96hr LCso concentration of latex, latex with supplements 

and for plant extract TU1 (0.5rnlISlitre); TU2 (ImlISlitre); TU3 (2mllSlitre) was 

taken. 


The collection, maintenance and acclimation of the fish Anaba resrudineus(B1och) in 

the present study are according to the details given in the earlier chapters. 

Collecting Blood from Fish: 

Blood samples were obtained directly from ductus cuvies, situated beneath the 

operculum, which was seen clearly as a bulging ventral to the last branchla] arch. 

The blood was collected using 2 m! sterile disposable syringe with No: 21 needle. 

The use of glass syringes was avoided because it may quicken coagulation (Smith et 

aL, I95Z).The syringe was rinsed with anticoagulant (potassium salt of ethy1er.e 

diamine tetra acetic acid, EDTA). 

The needle was inserted directly into the ductus cuvies and was very slightly 

aspirated during penetration. Blood was taken under gentle aspiration and then the 

needle was withdrawn. AAer detaching the needle from the syringe, the blood was 

mixed well in a vial containing anticoagulant (EDTA) at the concentration of 5 mg 

EDTA per ml of blood (Blahall and Daisley, 1973).For preparing blood smear 

blood was taken without the anticoagulant.

Blood Smear: 

Two clean slides were taken. A drop of blood was placed at the edge of one end of 

the slide. With another slide, held at 45 degrees the drop was spread evenly. The 

blood film was allowed to dry. Then the blood film was covered with 15 drops of 

Giemsa stain for one minute. Then 30 drops of distilled water were added mixed well 

and allowed to stand for five minutes. Then the slide was washed with distilled 

water. The stained film was dried in air at room temperature. 

Determination of Heamoglobin by Sahli's Method: 

Haemoglobin content of blood was determined by Sahli's method. The hemometer 

tube was filled with 0.1 N Hydrochloric acid upto the level of lowest graduation 

mark.The blood was drawn into the Sahli's pipette up to 20 Mark. Then the pipette 

was dipped into the Sahli's tube with the tip below the surface of the hydrochloric 

acid. The blood was gently blown into the acid. The acidified blood was sucked 

back again into the pipette and let into the tube. This was repeated 2-3 times when 

the blood colour changed, the tube was inserted into the Haemoglsbino meter. The 

solution was diluted drop by drop with distilled water and mixed thoroughly until the 

colour matches with the colour of the standard. After 3 minutes, the Haemoglobin 

value in grm % was read on the precalibrated scale of the tube at the level of the fluid 

meniscus. 

Estimation of oxygen combining capacity of blood: 

Oxygen combining capacity of blood was calculated by multiplying the haemoglohin 

content with the oxygen combining power of 1.25 ml of oxygen per gram of 

Haemoglobin @ecie and Lewis, 1963). 

Total Erythrocyte Count: 

Blood was drawn into the blood diluting pipette of Haemocytometer upto the 0.5 

mark and the Hayenm's fluid was drawn up to the 101 mark. Blood mixing the 

pipette was carried out gently by swinging the hand for 2-3minutes during which the

pipette was kept in a horizontal position. AAer thorough mixing, blood was released 

from the pipette to fill the counting chamber. Under a microscope, the erythrocytes 

were counted in 80 small squares of the Neubauer chamber. The corpuscles touching 

the boundary lines at the upper and right sides were counted. The total number of 

corpuscles counted was multiplied by 10,000(Volume factor X dilution factor 200) to 

get the total number of red cells in 1 mm3 for whole blood. 

Total Leukocyte Count: 

The blood was drawn up to the 0.5 mark of the diluting pipette of haemocytometer 

and the modified Dacies fluid was drawn upto the 11 mark. The filled pipette was 

gently revolved of 2-3 minutes and the Neubauer counting chamber was filled. Cells 

were allowed to settle for 3 minutes. The cells were counted in the comer blocks, 

with a total area of 4 mm2.rhe leukocytes on the upper and right sides were also 

counted. The total number of leukocytes counted was multiplied by 50 (Volume 

factor 2.5X dilution factor 20) to obtain total number of leukocytes in 1 mm3 of 

blood. 

Determination of Haematocrit: 

The blood was drawn into a haematocrit tube and one end of the tube was sealed. 

The tube was then centrifuged for 15 minutes at 3500 rpm. The reading was made 

from the graduation of the tube and expressed as the volume of the erythrocytes per 

100cm' (Snieszko, 1960). 

Mean Corpuscular Volume (MCV) (Dacie and Lewis, 1963) 

The average volume of a single red cell in cubic micrometers was determined 

MCV = 

Packed cell volume per litre of blood 

Erythrocytes in millions per mm'

Mean corpuscular Haemoglobin (MCH) (Decie and Lewis, 1963) 

Mean heamoglobin content of single red cell in micro grams was determined 

MCH = 

Heamoglobin m grams per litre of blood 

Erythrocytes in millions per mm' 

Mean corpuscular Heamoglobin Concentration (MCHC) (Deeie and Lewis, 

1963) 

The haemoglobin content of 100 ml of packed cells as a percentage as opposed to the 

percentage of haemoglobin of whole blood was determined. 

Haemoglobin in g per 100 ml 

MCHC = X 100 

Packed cell Volume in I00 ml 

3.4 BIOCHEMICAL PARAMETERS 

Toxicity Impact on Protein Metabolism 

Proteins are "building blocks of life" found every where in an organism (William er 

aL, 1984). Proteins constitute a large part of the structure of cells and are present in 

all tissues. Many proteins have also special physiological functions such as structural 

components of cell membranes enzymes, hormones, blood proteins (Plasma proteins 

and hemoglobin),antibodies and nucleoproteins.(Sandhu er aL,1991). Besides, the 

energy needed to mitigate and physiological stress is first obtained from 

carbohydrates followed by fat and lastly by proteins.Indeed on of the most active 

fields of research in biochemistry today is the study of protein structure and 

metabolism, important for toxicological studies.The study of protein fractions by 

Polyacrylamide gel electrophoresis is another well-established research field that is 

going to be the long tun. Even then there is scarce literature available about the

changes in protein as a function of the extent of contamination on the nature of the 

pesticide. However some show alterations is the mobility of protein patterns in 

response to pesticides (Reeta Pundey et uL, 1991).The impact of the latex, latex with 

supplements and plant extract of C.g~gantea(L.)R.Br. was thus taken for present 

to find if any alteration in protein content had taken place, because depletion of 

the protein content could be due to its util~zation as an energy source, thus ind~cating 

that other energy source had already been used up to a formidable extent and the 

organisms is on the brink of death; cell damage, thus influencing protein synthesis 

and causing disruption of many physiological and metabolic processes. 

hlaterials and Methods: 

The details of collection, maintenance and acclimation of the fish Anabas 

resludineus, the experimental procedures followed In the present study are detailed in 

the earlier chapters. The fishes were exposed to TUI (0.005m1/51itre); 

TU2(.0lml/5litre) and TU3 (.02mll5litre) of 96hr LC50 concentration of latex, latex 

w~th supplements and for plant extract TU1(0.5ml/5litre); TU2(lml/5litre); 

TU3(2mlISlitre) of 96 hr LC50 was taken. The Proteln concentrations in gill, liver 

and brain are determined by analyzing the tissues of 96hrs exposure. 

Preparation of Tissue Homogenate: 

The fishes after subject to sacrifice, the organs were dissected. The tissue was 

weighed for 100 mg, minced and cleaned in saline and homogenized in 2 ml of Tris- 

HCL buffer @H 7.5, 0.1 M) using a homogenizer at 4'~.~he homogenate was 

centrifuged at 12,000 rpm, for 30 minutes. The supernatant was carefully removed 

Into another tube and used as tissue extract. 

Estimation of protein: 

The estimation of protein concentration was done by the method of Lowry et uL, 

(1951) using crystalline bovine serum albumin (BSA) as the standard.

Principle: 

Protein forms a complex with copper ions in alkaline solution and this copper protein 

complex reacts with Folin's Ciocalteau reagent to give a blue colour that is due to the 

reduction of phosphomolybdate by colour developed was read at a wave length of 

720 nm. 

Reagents: 

1. 0.1N Sodium Hydroxide: 

0.4g of sodlum Hydroxide in 100 ml of distilled water. 

2. Reagent A: 

2 % Sodium carbonate in 0.1 N sodium hydroxide (2g of sodium carbonate In 100 

ml of 0.1 N Sodium hydroxide). 

3. Reagent B: 

0.5 % Copper sulphate in 1.35% Sodium potassium tartrate. 

4. Reagent C: 

Alkaline copper reagent: This was prepared jvst beforz use by mixing 50 ml of 

reagent A with 1 ml of reagent B. 

5. Folin Ciocalteau Phenol Reagent (lN): 

Commercially available 2 N Folin's reagent was diluted to 1 N by adding equal 

volume of distilled water 

6. Protein Standard : 

The standard was prepared with the help of Bovine serum albumin (BSA). The 

Stock solution containing 250 pg 1 ml of BSA was prepared in 0.1 N Sodium 

Hydroxide. From this stock solution 0.1 ml, 0.21111, 0.3mI and 0.4ml were pipetted 

out into series of test tubes and final volume was made up to lml with distilled 

water.These series of standards were subjected to further analysis as described 

under procedure and the standard graph was plotted with the concentration 

against absorbance.

Procedure: 

From the tissue extract 0.01ml was taken by micropipette into clear glass tubes and 

was made up to I ml with distilled water.5 ml of alkaline copper reagent was added, 

mixed and allowed to stand at room temperature for 10 minutes. Then 0.5 ml of folin 

ciocalteau reagent was added to this and shaken well. The blue colour that developed 

was read against reagent blank at 720 nm after 20 minutes using in a 

spectrophototneter. The amount of protein present in the aliquot of the sample was 

calculated by refemng to the standard curve obtained. The protein concentration was 

expressed in mgi 100 mg of tissue. 

Toxicity Impact on Glycogen 

Carbohydrates are one of the important building blocks of the biosphere, they 

provide energy on oxidation to body tissues which utilize for metabolic activ~t~es of a 

living being.Carbohydrates in excess are stored in the form of glycogen in liver and 

muscle. Glycogen, the polymer compound is broken down to glucose by the process 

of glycolysis, which is influenced both by intrinsic and extrinsic factors which in turn 

govern the physiology of the organism. Glycogen also maintains blood glucose level 

(Martin el aL,1981) and helps in muscle contraction for a finite time. Certain 

chemical pesticides are seem to be affecting several physiological Functions in 

fsh(Johnson,1968;Kah er al,l969;Henderson et al,1959).Various pesticides cause 

stress in fish 

when they are exposed(Pickering,I981).Stress response primary 

response(hormona1 changes)and secondary responses(changes in enzymes, substrates 

and ions),including 

blood glucose and lactate placing fatty acids. Electrolytes 

balznce, liver glycogen, immunosupression (Mazeaud et aL, 1977). Intermediary 

metabolism of carbohydrates shown to be impaired by the pesticide chemicals, and 

reported to alter carbohydrate metabolism. Under toxic tress conditions, the glycogen 

is broken down to glucose, primary fuel utilized for energy production. (Nakano and 

Tomlinson, 1967; Larson, 1973; Mukhopadyay and Dehadrai,l980; Srivrrrtava, 

1981; Vinod 1989).

Estimation of glycogen 

Fishes were exposed to three sublethal concentrations of latex, latex with 

supplements and plant extract. The tissues of gill, liver and brain excised and are 

used for the following experiment. 

Principle 

The dissected tissue were digested with 30% KOH and glycogen was precipitated 

with ethanol. The precipitate was further treated with anthrone reagent and the 

glucose content was determined calorimetrically (Klicpera et ~1,1975). 

Reagents 

1. 30.% (wiv) KOH. 

30g of Potassium hydroxide was dissolved in 100 ml of distilled water. 

2. 95% ethanol (viv) 

95 ml of 100% alcohol was made 100 ml with distilled water. 

3. 95% sulphuric acid (viv) 

95 ml of concentrated sulfuric acid was made 100 ml with distilled water. 

4. 0.2% anthrone reagent. 

This reageni was prepared by dissolving 0.2 of anthrone in 100 ml of 95% 

sulfuric acid and just befor: use and stored in the refrigerator. 

5. A standard glucose solution containing 100 mg ilitre of glucose per ml. 

Procedure: 

The dissected out tissues were weighed in an electronic weighing balance and 

then it is taken in a centrifuge tube for centrifugation 1 ml of KOH was used for 

dissolving the tissue. Digestion of the tissues was done by keeping the centrifuge 

tube in a water batch for 20 minutes. It was cooled and 1.25 ml of 95% ethanol was 

added. After a through mixing the contents were boiled in water bath. It was cooled 

and then subjected to centrifugation for 15 minutes of 3000 g. The precipitate was 

carefully taken by decanting the supernatant and allowing the tube to drain on a filter

paper. To the precipitate 1 ml of distilled water was added and re-dissolved in 1.25 

ml of 95% ethanol. The Supernatant was decanted off and the tube was drained on 

filter paper as before. The precipitate was then dissolved in 5 ml of distilled water 

and placed in ice bath to this 10 ml of anthrone reagent was added and boiled in a hot 

water bath. Glass marbles were kept on the tubes while boiling to avoid the overflow 

of reagents. A green colour was developed which was then red in a 

spectropbotometer at 620nm after cooling the tube. Similar procedure was employed 

for preparing "blank" and standard solutions.5 ml of water and 5 ml of standard 

solution containing 100 pg 1 I of glucose treated with 10 ml of anthrone reagent 

sewed as blank and standard respectively. 

The glycogen content in aliquot was calculated by using the foilowing 

equation: 

Glycogen in aliquot (mg ) I00 X U 

1.11 X5 

Where, U - Optical density of unknown solution 

S - Optical density of 10 pg 11 glucose standard 

1.11 - the factor determined by Moms, 1948. 

For the convesion of glucose to glycogen with this equation. The glycogen content 

of the tissue was expressed as mg / gram tissue. 

Toxicity Impact on Acid and Alkaline Phosphatase Activity 

Acid Phosphatase (E.C 3.1.3.2) and Alkaline Phosphatase (E.C. 3.1.3.1) are group of 

widely distributed enzymes of very broad specificity. They act on a wide range of 

nonoesten of orthophosphoric acid, both aliphatic and aromatic (Fernley and 

Walker, 1971). They are known as "inducible" enzymes whose activity in animal 

tissue goes up when there is a toxic impact and the enzyme begins to counter act. 

Subsequently the enzyme's activity may begin to drop either as result of having

partly or fully countered the toxin or as a result of cell damage. Further lysosomes 

are sub-cellular membrane-bound organelles active in the catabolism of cellular and 

extracellular material. 

Exposure to toxicants causes changes in size, quality, 

membrane, liability and lysosomal stability (Love,1980; Leland, 1983). The 

membrane prevents indiscriminate cell autophagy depending on the property of 

lysosomal enzyme .Acid Phosphatases act on a wide variety of synthetic substrates 

such as a and P-glycerophophate, a-napthylphosphate and b-napthylphosphate . It 

has been shown that purified alkallne phosphatase from number of source can also 

hydrolyze creatinephosphate, inorganic pyrophosphate and a number of 

Polyphosphates including ATP and metaphosphate of average chain length (Moss er 

of., 1967). Lysosomal Enzyme Release Assay (LERA) is a sens~tive indicator of 

environmental stress on invertebrates and fish (Widdows et al., 1982; Moore and 

Clarke, 1982). The nature of the present work was aimed to study the level of the 

acid and alkaline phosphatase in various tissues of fish Anabas testudinew(B1och) 

exposed to sublethal concentration of latex and plant extract. 


Materials, method of collection, acclimation and experimental condition were the 

same as those described in the earlier chapters. Fishes were exposed to three 

sublethal concentrations of latex, latex with supplements and plant extract. The 

tissues of gill, liver and brain were excised and washed in saline, blotted on a filter 

paper,lO% tissue homogenate was prepared in 0.5 M Tria HCI buffer @H 7.5) using 

a motor driven glass homogenizer. Then they were centrifuged at 10000 g for 10 min. 

The supernatant was used for enzyme assay. 

Estimation of Acid Phospbatase ( E C 3.1.3.2) 

Acid Phosphatase was estimated following the method of Andersh and 

Szaypinski (1974) as modified by Tennis Wood et al, (1976).

Principle: 

Para nitrophenyl Phosphate is a colourless solution but upon hydrolysis the 

phosphatase liberate paranitrophenol that is highly coloured in an alkaline solution. 

The rate of hydrolysis of Para nitrophenyl Phosphate is proportional to the enzyme 

present in the tissues. 

p-nitrophenyl phosphate t H20 - 

(Colourless in acid and alkal~) 

Reagents: 

I: Citrate buffer, 0.1M @H 4.85) 

Phospharuse 

Paranitrophenol + H,PO, 

(Yellow colour) 

21.014 g of citric acid was dissolved in 600 ml of distilled water. To this 180 

ml of I N Sodium Hydroxide 100 ml and 0.1 N Hydrochloric acid was added and 

made up to 1000 ml with distilled water. 

2. Para nitrophenylphospha~e (Substrate) 

This was prepared freshly by dissolving 40 mg of para - n~trophenyl phosphate in 10 

ml of distilled water. 

3. 0.1 N Sodium Hydroxide 

4 g of Sodium Hydroxide was dissolved in 1000 ml of distilled water 

Procedure: 

To the clean test tuhes labeled 'test' and 'blank' 0.5 ml of substrate was pipetted. To 

this 0.5 ml of citrate buffer was added. The tuhes were placed in water bath at 37' C 

for 5 minutes. The reaction was initiated by the addition of 0.1 ml of the sample to 

the test and 0.1 ml of distilled water to the blank exactly after 30 minutes of 

incubation at 37' C. The reaction was arrested by addition of 3.8 ml of 0.1 N NaOH. 

The reaction product, para-nitrophenol was measured at 415 nm against the blank in 

the spectrophotometer suitable standards were run along with the assay. The enzyme

activity is calculated by referring the calibration curve obtained using free paranitrophenol. 

Acid Phosphatase activity is expressed in p moles of para nitrophenol 

formed per hour per mg protein. 

Estimation of Alkaline Phosphatase (E C 3.1.3.1) 

Alkaline phosphatase was assayed following the method of Bessey et a/., (1946). 

Principle: 

Para nitrophenylphosphate is a colourless solution but upon hydrolysis. The 

phosphate group liberated paranitrophenol .that IS highly coloured in an alkaline 

solution.The rate of hydrolysis of p-nltrophenyl phosphate is proportional to the 

enzyme present. 

- 

p-nltrophenyl phosphate t H20 

(Colourless in acid and alkali) 

Phosphatase 

Paranitrophenol + H,PO, 

(Yellow colour) 

Reagents: 

1.GIycine buffer 0.1 M @H 10.5) 

7.8 g of glycine and 0.095 g of magnesium chloride was dissolved in 750 ml of 

distilled water. To this 85 ml of 1 N sodium hydroxide (4 g of sodium hydroxide 

dissolved in 100 rnl of distilled water) was added and made up to 1000ml. 

2 para-nitrophenylphosphate: 

40 g of p-nitrophenyl phosphate was dissolved in 10 ml of distilled water just before 

use. 

3 0.02 NNaOH: 

0.8 g of NaOH was dissolved in 100 ml of distilled water.

Procedure: 

To the tubes labeled 'test' and 'blank' 0.5 ml of para-nitrophenyl phosphate and 0.5 

ml of glycine buffer was added. The tubes were placed in a water bath at 37'~ 

for 5 

minutes. The reaction was initiated by the addition of 0.1 ml of sample to the test 

and 0. I ml of distilled water to the blank. Exactly after 30 minutes of incubation at 

37'~.~he reaction was arrested by addition of 10 ml of NaOH. The tubes were mixed 

well and the colour developed was read at 410 nm, in a spectrophotometer, against 

the blank. 0.1 ml of concentrated Hydrochloric acid was added, mixed and the optical 

density was read at 410 nm against the blank. The difference in the two absorbance 

was taken as the measure of enzyme activity. Suitable standards were mn along with 

the assay. The alkaline Phosphatase activity was calculated from the calibration 

curve obtained using para-nitrophenol standard. The enzyme activity is expressed as 

p moles of para nitrophenol formed per hour per mg protein. 

Toxicity Impact on Acetylcholinesterase 

The important toxic property of pesticides is inhibiting their target enzyme acetyl 

cholinesterase (AChE), and as well studied (O'Brein, lP67;Corbelt,1974),most of 

the chemical pesticides are similar with the ester part of the acetylcholine and they 

react with esterase part of AChE after entering into the exposed fish The conversion 

of acetic acid and choline catalysed by AChE is considered to be the reaction in 

synaptic transmission(Bachelard,l976).Decrease in AChE activity and increased 

accumulation of AChE was observed in fresh water mussel treated with 

demacron(Vijendrababu and Vasudev,l984),with Fenotrothion in Senex senex 

(Bagyalarmi and Ramamur~,l980) and many organophosphates in fishes were 

reported(March et a1 .,1956;Murphy el aL,1968).A drop in AChE in vertebrates 

caused various neurobiological changes that reduce the animal survival ability. 

Fishes were exposed to three sublethal concentrations of latex, latex with 

supplements and plant extract. The tissues of gill, liver and brain excised and are 

used for the following experiment.

Estimation of Acetylcholine esterase (EC 3.1.1.7) 

Acetylcholine esterase (AChE) is a specific cholinesterase that catalyses the 

hydrolysis of a neurotransmitter acetylcholine. The acetylcholine esterase 

concentrations were assayed following the method adopted by Ellman et aL, (1961). 

Principle 

Th~s analogue of acetylcholine 1s used as a substrate and the SH groups 

released are titrated with 5, 51 dithiohis (2-nitrobenzoic acid) (DTNB).The change in 

optical density can be followed spectrophotometrically at 412 nm . 

Reagents 

1. 1MNaC1 : 5.845 g 1 100 ml distilled water 

2. lM MgC12 : 20.33 g 1 I00 ml distilled water 

3. 0.5 M Tris - HCl (pH. 7.5) 

4. 0.2 M Ethylene d~amine tetra acetic acid (EDTA): Mix 7.44 g of EDTA in 50 

ml of water and add about 16 ml of IN NaOH to bring to neutral pH Make 

upto 100 ml with water. Dilute lin 100 to get 2 mM solution. 

Preparation of cocktail Mix the following 

1. 1.OMNacl. 13. ml 

2. 1.0 M Mgclz 2.0 ml 

3. 0.5 M Tris Hcl I0 ml 

4. 0.2 M EDTA 10 ml 

0.1 Acetyl choline chloride: 181.66 mg / 10 ml of distilled water. To be preserved 

Frozen in amber coloured bottle,l mM DTNB: Dissolve 0.396 mg 1 ml in distilled 

water 

Preparation of Reaction Mixture: 

Mix the following: 

Cocktail .I03 ml,l mM DTNB - 3.0 ml (to be added freshly), Water -6.5 ml

Procedure 

The assay mixture in a final volume of 3 ml contains the following. 

Reaction mixture: 2 ml 

Substrate (0.1 M Acetylchohne chloride): 0.03 ml distilled water. 0.92 MI 

Sample: 0.05ml 

The reaction is initiated by the addition of sample and change in optical density was 

noted for every 30 seconds for 5-10 minutes.A substrate blank without enzyme is 

necessary since acetylcholine always has 2-5 % of free SH groups and also it is 

unstable. 

Toxicity Impact on Adenosine Triphosphatases 

Adenosine triphosphatases are membrane bound enzymes which funct~on as a major 

caniers of chemical energy in all physiological activities of the cell.They have 

profound effect on hio-membrane, modulates the activity of integral membrane 

protein in a variety of organisms.The terminal phosphate group of ATP is transferred 

to variety of acceptor aolecules which are there by activated for further chemical 

transfer i.e., ADP recycled to ATP.ATPases has role in inter cellular functions (Skou, 

1957). They are the sensitive indicators of toxicity. These membrane bound enzymes 

are worthy,catalyses movement of ions and coinpounds across cellular 

membrane.Na' K' ATPases is a transmembrane protein responsible for maintaining 

~ a and ' K' gradient essential for uptake by cells of metabolites like glucose, amino 

acids and regeneration of transmembrane potential. ~a', M~* ions is used for 

phosphorylation, where as dephosphorylation requires K' ions, when ionic movement 

take place in and out of cellular membrane (Yutes,1980; Towle,1981). caw, ~ g * 

ions act as factor for several enzymatic reactions.caU ions are utilized mainly for 

signal transduction in second messenger system. ~ g * ions have vital role in 

activation of enzymes thus activating the membrane related ATPases. Xenobiotic

substances may induce disturbance(inhibiti0n) in intracellular ion homeostasis 

(Desaiah,1982) and also blocks ion channels resulting in deleterious effect on vital 

cellular functions and also greater reduction of solute transport (Bignani and 

Palladini,1966 ;Gonzal et aL,1983;DnIeIa et nL,l978).Significant inhibition of M ~" 

, Ca" and Na' K' ATPase was observed in brain gill and liver tissues of blue gill 

treated with organochlorins (Cutkomp et aL,1971), Mugil cephalus wlth IindaneAn 

attempt is made to evaluate the sub lethal effect s of latex and plant extract on gill, 

liver and brain ATPases in Anabas 

restudineus. Fishes were exposed to three 

sublethal concentrations of latex, latex with supplements and plant extract. The 

tissues of gill, liver and brain exc~sed and are used for the following experiment. 

Estimation of Adenosine Triphosphatases (EC 3.6.1.3) 

The calcium, magnesium, sodium and potassium dependent adenosine 

triphosphatases were estimated according to the method of Takeo and Snkanashi, 

(1985). 

Principle: 

Adenosine Triphosphatase catalyses the conversion of adenosine 

triphosphate(ATP) into adenosine disphosphate (ADP). During the conversion one 

- 

molecule of phosphorous is liberated. 

ATPase 

ATP 

ADPt Pi 

The inorganic phosphorous is estimated 

The proteins are precipitated with trichloro acetic acid ,the free filtrate reacts with an 

acid molybdate solution to phosphomolybdic acid which is reduced by the addition 

of 1,2,3 aminaphtho sulphonic acid reagent, to produce a blue colours the intensity of 

colour is proportinonal to the amount of phosphate present. 

Reagents 

Histidine 50 mM . pH. 7.4 (wlv)

775-800 mg of histidine was dissolved in 10 ml of distilled water and the pH was 

adjusted to7.4, Ethylene diamine tera acetic acid (EDTA) ,0.25 mM wlv: 

9.306 mg of EDTA disodium salt was dissolved in 100 ml of distilled water 

Magnesium chloride .4Mm(w/v) 

81.24 mg of magnesium chloride was dissolved in 100 ml of d~stilled water. 

Calcium chloride, 4 mm (wlv): 44.400 mg of calcium chloride was dissolved in 

100 ml distilled water. 

Sodium chloride, 20 mm (wlv): 168 mg of Sodium chloride was dissolved in 100 

ml of distilled water. 

Potassium chloride,lO mm (wlv) :7.4 ,560 mg of potassium chloride was dissolved 

in 100 ml of distilled water. 

Trichloroacetic acid (TCA), 12%(wlv) :12, g of trichloacetic acid was dissolved in 

100 ml of dist~lled water. 

Ammonium molybdate, 0.1 M (wlv). 

25 g of ammonium molybdate was dissolved in 200 ml of distilled water. To this 

solution 300 ml of 10 N sulphuric acid was added and made upto one litre with 

distiiled water. 

Sulphuric acid, 10 N (wlv) 

83.1 ml of concentrated sulphuric acid was made up to 300 ml with distilled water. 

Sodium bisulphate, 15 % (wlv): 15 g of sodium bisulphate was dissolved in 100 ml 

of distilled water. It the solution was turbid, it was allowed to stand for several hours 

and filtered. 

Sodium sulphlte, 20% (w/v):20 g of anhydrous sodium sulphite was dissolved in 

100 ml of distilled water.

~minonsphtho sulphonic acid (ANSA) reagent (wiv): 

195 ml of I5 % sodium bisulphate solution and 0.5 g of ANSA were mixed and 5 ml 

of 20% sodium sulphite solution was also mixed in it. This solution was transferred 

to a brown bottle and stored at 4' C this solution was prepared freshly. 

Sodium azide, 5 mM (wlv) 

32.5 mg of sodium azide was dissolved in I00 ml of distilled water. 

Procedure 

To the tubes labeled as "test" and "nonspecific " 0.5 ml of histidine buffer was added 

To the 'test' tubes 0.1 ml of 4 mM magnesium chloride or 0.1 of 4 mm calcium 

chloride or 0.1 ml of 20 mm sodium chloride plus 10 ml mm potassium chloride was 

added, according to the specific assays.To the 'non specific tubes' 0.1 ml of distilled 

water was added .To this mixture a known a liquot of extract was added 0.1 ml of 

EDTA and 0.1 ml of sodium azide were also added to all the tubes. Afier 3 minutes 

of pre incubation at room temperature, the reaction was initiate by 0.1 ml of ATP the 

reaction mixture was incubated at 373 C for 15 minutes. The reaction was termmated 

by adding 1 ml of 12% TCA; the proteins were precipitated with TCA. After 5 

minutes the resulting mixture was centrifuged at IOOOrpm for 5 minutes and the 

supernatant was analyzed for phosphorous content This method is highly sensitive 

for calorimetric estimation of phosphorous. 

TO the supernatant 1 ml of ammonium molybdate (0.1M) and 0.4 ml of ANSA 

reagent were added. After 10 minutes the intensity of the blue colour was read at 680 

nlm against reagent blank.Appropriate standard (Sodium dihydrogen phosphate) was 

also run in each batch of assay.The non specific activity was subtracted from each 

activity detected .The enzyme activity was calculated from the standard graph. The 

enzme activity is expressed as units I mg protein.

3.5 HISTOPATHOLOGY 

Living organisms have tremendous capacity to concentrate pesticides, thereby 

altering histological changes in fish. Fish exposed to pollutants will gradually die due 

to malfunctioning of cells (Mount and Putnicki, 1966).Hence, the study of such 

pathological lesions has been considered as an important dev~ce in predicting the 

mode of action of a particular pesticide at different tissue levels (Sprague, 1971 

Though several studies have reported the effects of pesticides on aquatic organisms 

but they are mostly restricted to mortality studies. The htstopathological information 

along with biochemical data may provide complete and accurate description of the 

activity of pesticides. Meagre information on histopathological effects of pesticides 

on tissues is available. The tissue damages were mostly caused by an array of 

chemical compoundsThe susceptibilities of d~fferent species of fish to various 

toxicants differ considerably (Eisler et aL, 1972). The extent of damage or nature of 

lesion may be high in one species than other.Even the slightest damage to a tissue 

structure results in considerable stress on the behavi0r.A slight destruction of the gills 

may lead to considerable decrease in oxygen consumption.The nervous system is one 

of the most vulnerable parts of the animal body, and injuries to its elements influznce 

the behavior and survival of the organisms (Smith, 1984). Pesticides absorbed across 

the skin or the gills enter the blood stream there by reaching internal sense organs 

and other nervous tissues, including the brain (Baatrup, 1990).The brain is more 

susceptible to toxicants that may alter :he complete behavior and reduce the fitness of 

the fish in its environment. In the present study gill, liver and brain were chosen to 

study histopathological transformations in Anobas testudinem exposed to sublethal 

concentrations of latex Fishes were exposed to three sublethal concentrations of 

latex, latex with supplements and plant extract for 96 hrs exposures. 

Materials and Methods 

The materials, collection, maintenance and acclimation were the same according to 

detailed in the earlier chapter. A.testudineus(B1och) was exposed to sublethal

concentrations. The fishes atter exposure to sub lethal concentrations of latex and 

plant extract were killed and tissues like gill, liver and brain were dissected, washed 

In physiological saline and fixed in Bouin's fluid. The gills were decalcified by 

treating with 5% concentrated nitric acid in 70% alcohol for an hcur. The tissues 

were embedded in paraffin wax (56'~ to 58'~) and sectioned at 6-7p.The sectians 

for gill were taken in such a way that the primary lamellae of gill were cut 

longitudinally and across the secondap lamellae, whereas for other tissues cross 

sections were taken. The sect~ons were stained with hematoxylin and eosin 

(Humason, 1972).

RESULTS 

TOXICITY TESTS with Anabas testudineus 

The major concern in performing the acute toxlclty test with pestlc~de on different 

tishes 

IS to analyze the safe concentration. The maintenance of water qual~ty and 

acceptance of fishery regulation rules are possible only w~th adequate knowledge 

about the "safe concentration" of an insecticide and its mode of action. The test 

organisms may appear tolerant to the pest~clde, but a particular stage In 11s life-cycle 

may be highly sensitive and bnng the crucial state for the population The larval 

stages are more sensitwe to the pesticide than adults. Change in water quality 

parameters deteriorates the quality of environment leading to suffocat~on and shows 

adverse impact on fish health (Table-2). Chronic bioassays were conducted 

throughout the Life-cycle of the fish includ~ng the reproduction period and subsequent 

development of the eggs and juven~les of the test organisms (Mount and Stephen, 

1967). Chronic bioassays have primary importance Ln deteminmg the level of a 

pesticide. Many suggestions regarding the LCIo value and prediction of safe 

concentration are given by d~fferent authors. The Aquatic Life Advisory Committee 

(ALAC) of the Ohio River (Anon, 1955) suggested the use of 0.1 factor times the 2 

day LCso as the mean for calculating the safe concentration. The safe concentrations 

found out by the Committee of Water Quallty Critena (CWQC, 1972) and Indian 

Standard Institution (Rao , 1974) was 0.01 of the 96 hr LCIO. In the present study a 

.alue of 0.01 was analyzed to calculate the safe concentration. The results are 

summarized in Table: 3 for latex and Table: 4 for plant extract of Calolrop~s 

giganlea. It was observed that in all case's percentage mortality increased with 

Increasing concentrations of the pesticide and duratlon of exposure. The mortality of 

fish occurred steadily with the passage of time in each of the concentrations. The 

toxicity to the fish Increases with increased concentration of pesticides. Singh and 

Sahai (1984) repofled sim~lar findings. The median lethal concentration (LCIo) 

value calculated by probit analysis for latex was O.lml dlit. for 96 hr of exposure 

'Fig:l) and for plant extract of Calotropis gigonlea was lOml 151it. for 96 hr 

IPter. 4. Results. Sentence 5 is corrected against the table 2 

Concentrations of later corrected on page 34 as shown in table.. The correct doses 

are 0.01,0.05,0.07,0.1,0.15,0.20.25,0.5,0.75. 

The sample sizes were only 5 it is corrected in table 3 and 4.

of exposure (Fig:Z).The safe concentration was found to be 0.001ml Nit for latex 

and O.lmll5lit for plant extract (Table : 5) It has been proved that the sensitivity of 

the individuals of a particular species to a pesticide may be influenced by internal 

factors like sex, age and size (Williams et aL, 1984). The present study was taken up 

with sub lethal dose response in the experimental fish. The 96 hours LCro was 

considered as One Toxic Unit (ITU) (Sprague and Ramsay, 1965) and based on this 

value, three different sub lethal concentrations viz., l/20'~, 11l0'~ and 115'~~ of LCIo 

values -TU1(0.005m1151it); TU2(O.OlrnlISlit) and TU3(0.02m1151it) of 

latex, for 

plant extract TUl(O.Srnli5lit); TU2(lrnllSlit); TU3(2m1/51it) and three different sub 

lethal concentrations viz., 1/20'. 1110'~ andl15'~ of LCso values- TU1(0.005mlISI); 

TU2(0.01rn1151) and TU3 (0.02m1151) of latex plus supplements was taken for the 

filllowing experiments. 

BEHAVIOURAL TOXICITY 

Optornotor Behavioural Changes 

Table-6 shows the mean scores of the optomotor response at different exposure 

concentration of latex , latex plus supplements and plant extract. The mean score for 

'.following" during "on" position bucket rotating was significantly (P

Impact on Surfacing behaviour, Distance traveled and Opercular movement 

The data showing the influence of the latex , latex plus supplements and plant 

extract on surfacing behaviour of Anabm testudineus are given in the Table:7 and 

Fig:4. The number of surfacing was more in the treated fish compared to control. 

The increase was sig'nificant (P

Impact on Haemoglobin content 

The haemoglobin content was lower in the pesticide exposed fish with respect to 

controls was observed but the decrease was significant in TUI (P

fish;their was an increase in value from TUI to TU3 in latex+supplements treated 

fish was observed(Tab1e 14.1 5,16, fig.5,6,7). 

Impact on Ht, MCV, MCH and MCHC 

Gradual decrease in haematocrait (Ht) value was observed in sublethal concentrat~on 

of latex compared with that of control ,the decrease In haematocra~t value was 

s~gnificant (P

Impact on Total White Blood Cell (WBC) count 

Gradual increase in total WBC level was observed in fishes exposed to the three 

sublethal concentrations ,when compared to the control but the increase was 

significant (P

Brain: 

Significant changes in protein concentration were observed in the brain of fish. The 

decrease was significant in TU1,TUZ of latex(P

glycogen concentration varies from - 1.82 to -43.8 for latex and -14.43 to -15.95 for 

plant extract treated fish (Table 9, fig.9). 

Brain: 

Significant changes in glycogen concentration were observed in the hra~n of fish. The 

decrease was significant (P

Brain : 

The Acid Phosphatase activity in the brain of the fish exposed to different sublethal 

concentrations along with statistical data and the percentage of changes is ylven in 

Table:lO, fig:l0. The brain Ac~d Phosphatase activity showed an increase at 

different exposure levels with respect to controls except with latex+supplements. 

which showed a decreasing trend. In the fish exposed to TUI ,TU2,TU3 of latex,TUl 

of latextsupplements and TUI,TU2,TU3 of plant extract treated fish showed 

significant changes (P

Increase in Alkaline Phosphatase activity varies from 47.1 I to 50.84 for latex and 

I 1.5 to 24.35 for plant extract treated fish. 

Brain: 

There was an increase in the alkaline phosphatase activity in all the treatments on 

0bservation.Significant increase (P

fish were significant at P

significant(P

Liver : 

Trend observed in liver was similar to the one observed In gills. In TU2 ,TU3 of latex 

and in TUI,TU3 of plant extract treated fish ,changes observed was 

sgnificant(P

the primary gill lamellae. The primary gill lamellae consist of centrally placed rod 

like supporting axis with blood vessels on either side.The secondary lamellae also 

termed as respiratory lamellae are highly vascularised and covered with a thin layer 

of epithelial cells. The epithelial layers of two sides are separated by pillar cells or 

pilaster cells. Blood vessels are extended into each of the secondary gill filaments. 

The blood cells of the secondary gill lamellae have a single nucleus, which are 

flattened in appearance. The region between the two adjacent secondary gill lamellae 

is known as inter lamellar region. (Plate 3 and 4). 

Alterations in gill on exposure to sublethal concentrations 

Latex has induced marked pathological changes in fish gills. The changes include 

the bulging of tips of primary gill filaments. The secondary gill filaments lost their 

original shape and curling of secondary gill filaments was observed. The pillar cells 

nucleus showed necrosis and development of vacuoles in the secondary gills 

epithelium. There is tendency of fusion of disorganized secondary lamellae were 

very much affected wirh loss of the respiratory epithe!ium in TU2 as well as TU3. 

The arrangement of the pillar cells was greatly disturbed. Necrosis were seen in TUI 

,TU2 and severe in TU3 in the lamellar region. At some places a fusion of adjacent 

gill lamellae was seen.A number of secondary gill lamellae were damaged in fish 

exposed to TU3. The pillar cell system also appeared to collapse. The pilaster 

columns were seen to be curled up and their blood spaces were engorged with 

stagoant masses of blood in TU1 and TU3. Pools of congested blood were also 

visible within the sub-epithelial spaces.The respiratory epithelia were slightly 

swollen in the TU2 whereas it was either swollen or lost in the case of TUI, and TU3 

exposed fish.Respiratory epithelium was ruptured at different points ,so that the 

capillaries were exposed to water and hemorrhage exudates could be seen at many 

places over the lamellar surface and in the branchial cavity in TU3.At the filament tip 

Iwge masses of glycoprotein clogs were noticed at several sites in the blood vessels 

of gills filaments and filament axis was also thrombosed.The spatial anangement had

ecome irregular while the epithelial cells were distended. Swollen nuclei were also 

observed. Inflammatory alternations were seen in the epithelium.Shrinkage of blood 

capillaries and loss of micro ridges were seen in TU1 (Plate3 and 4). 

Liver : 

Normal structure 

In Anabus tesrudineus there are 3 slender and leaf like liver lobes which are reddish 

brown in colour. The outer protective membranous covering is seen intact. A typical 

tubulosinusoid pattern of arrangement of parenchymal cells is seen, the liver cords 

are characteristically two cells thick and are alternated with sinusoids. They are 

polygonal, vacuolated and uniform in size.Vacuoles are varying in size and indistinct 

as cytoplasm in less granular. The nuclei are spherical and uniform in size. The blood 

vessels with red blood cells are found in good condition (Plate 5 and 6). 

Changes due :o exposure to sublethal concentrations 

Diffuse necrosis in parenchymal cells with cytoplasmic vacuolation was found, 

Increased cytoplasmic granularity was seen in TU2 and TU3 exposed fish.Blood 

cdngestion in the central vein was easily noticed.Significant loss of radial orientation 

and parenchymal shrinkage were readily observed in TU2 and TU3 exposed fish 

than in TU1 exposed fishes.The liver of treated fish appeared necrotic with vanous 

levels of accumulation of remnants of dead cells giving the liver a foamy appearance. 

Pycnotic nuclei were seen scattered throughout the liver. The fibrosis was also 

observed in TU3 exposed fishes. The tubulosinusoid arrangement was partly or 

completely lost in TU3 exposed fishes when compared toTUl and TU2 exposed 

fishes the loss of tubulosinusoid arrangement was scarcely seen. The liver of the 

fishes exposed to higher sublethal concentrations ( TU2 and TU3) was conspicuously 

damage. The degree of damage could be illustrated as TUI < TU2< TU3 (Plate5 

and 6).

Brain : 

Normal Structure 

The study of brain sections of the control fish showed the mode of arrangement of 

different layers in the cerebral cortex. It consists of an outer region, the molecular 

iayer and an inner region the granular layer. At the junction between the two are the 

large purkinji cells that are characteristic of the cerebellum. Optic tectum lies within 

the molecular layer. The cells of the molecular layer are widely spaced, in contrast, 

the cells of granular layer are closely packed. The purkinjl cells are large and 

extends to the molecular layer. The granular layer consists of many small cells called 

granule cells. They appear as dark bodies. The granular layer shows clear areas 

callcd glomureli.Nucleus genicultaum is present within this. To the centre of the 

cortex lies the nucleus rotundus. Besides the purkinji, within the granular layer, is 

the small opening called torus semicircularis efferent. (platel and 2). 

Changes due to exposure to sublethal concentrations 

The outer layer of the cortex was ruptured at many polnts .Remarkable difference 

was noticed in TU3 treated fish and the atrophy of the malecular layer was deserved 

leavmg intracellular spaces.Nucleus rotundus of the TU3 treated brain was 

degenerated.Necrosis of the neurofibrillar region had also been observed. The 

granular layer also became atrophied in TU2.A complete change in the cellular make 

up due to vascular dilation in the granular layer had been observed. Cells lying in the 

molecular layer degenerated especially in the brain treated with TU2. The Purkinji 

cells differentiating the molecular layer get closely packed in TU land TU2, the 

granular layer becomes reduced in size and cells seemed to be more adjacently 

packed. The torus semicircularis efferent when compared with control was found to 

be widened inTUl treated fish brain section and also in TU2 and TU3 treated one. In 

contrast to the control section, the various layers of the brain cortex were marked in

TABLE-2 : CHARACTERISTIC OF WATER USED IN BIOASSAYS 

I S.NO 

1 I 

I 

1 2 

13 

Parameters 

Water Temperature 

Oc 

Dissolved oxygen 

pH Range 

Control Latex 

27OC 27' C + 3 

I 

I 

7 ppm 7-4.2 ppm 

6.5 6.5 - 8 

Control 

25.C 

7.4 ppm 

7 

4 

Plant Extract 

25.5' C + 3,7 

7.4 - 4.6 ppm 

7- 8 

4 

i. 

5 

Free carbondioxide 

Hardness(CaCo,) 

18 ppm 

132ppm 

18 - 24.4 ppm 

132-180 ppm 

16 ppm 

140 ppm 

16 - 22.4 ppm 

140 - 170 ppm 

6 

Alkalinity 

106ppm 

106-180 ppm 

I02 ppm 

102 - 180 ppm

TABLE-3 : MortaliQ of Anabas testudineus at Different Concentrations of Latex 

of Calotropis 

- 

gigantea after 96 hrs exposure, Mortality expressed both 

in Percent and Probit kill. 

j Number of ' 

No. ( (ml15 liters ) I ' Concentration 

1 Fishy KiN 1 

~i.-l 

-- 

-1 (I 

51. Concentration 1 Number of Percent Probit , 

TABLE-I : Mortality of Anabas testudineus at Different Concentration~ of Plant 

Extract of Calotropis gigantea after 96 hrs exposure, Rltrrtalit> 

expressed both in Percent and Probit kill. 

-- T ------- -- - 

, , 

Sl Concentrution Log I of 1 umber of i Percent i Probit 

ho. (mli5 liters) Concentration 

Fish Dead i K 7 i 

-. 1 Exposed ! 

I 

i 

- --- I 0 

,' 5 0.698 

1- 

> 7 1 

10 ;, .-.- 0 : -- 

lo ! 2 20 I 4.1 6 

--7- 

1 

' 0.845 1 10 , 4 40 1.75 I

TABLE5 : SAFE CONCENTRATION OF LATEX AND PLANT EXTRACT OF 

C. gignntea IN Anabas testudineus BASED ON 96 Hr (LCSO) AND 

APPLICATION FACTOR 

Samples 

Application 

96 hr LC50 

( m ~ , ~ ~ f a c t o I 

* 

Safe concentration 

(m1151) 

Plant extract 

I0 

0.01 0. I 

* CWQC (1972)

Table-14 : Effect Of Latex On Haematological Parameters In Fish Anabas 

tesludineus Exposed For 96 Hn 

1 Hb P5C OCC H MCV MCH MCHC WBC 

Treatment 

1 

Groups ' Hb'lOO 

\~~awmmc~ 

8 ml 

- 

j Ozhy :i ' yml pg i Ui 

I 

mm- 

1448 4.04 I I8 16 

1 

36 18 8925 36.69 38.12 46 

Control + i * I f + = 

0.18 0.12 0.12 0.58 4.6 2.76 1.12 015 

I 

I 

13.16** 3.63 ' 1 16.46'" 34.98' 98.06' 38.65"' 35.66. 498'. 

; * I * i i i * * 

TL I 

0:8 0.16 1 0.26 1.05 5.04 166 1.72 0.06 

( 9 1 (-10 14) 1 (-9.36) (-3.31) (9.87) (5 34) (-6.45) (8.26) 

+ - 

12.5*** 32I*** I5.18*** 34 i* 108.48** 40.50' 

36:3 

';j5 

i i2 0!2 Oy2 3;4 l:6 * * 

1.0 0.14 

I (-13.67) (-20.5) (-13.10) (-4.47) ! (21.5) (10.3) (-4.171 951 

-- 

-- 

--- 

12 20"' 2.94;" 1 15.2'** 33 68.' 115.4 ** ! 40.88' 36.1' ',10 

i * i- * 

Of6 0 l I 024 0.76 5.84 18 126 0,i84 

(-15.74) (-27.22) (-16.29) (-6 9) (29.2) (1 1.42) (-5.3) 113,04) 

Each value is mean + SEM (n = 5) 

Slgnlficant values : *(P

able-15 : Effect Of Latex+ Supplements on Haematological Parameters in Fish 

Anabas Testudineus exposed Tor 96 Hrs 

I I I I I I I 

I 

Hb RBC OCC Ht MCV MCH MCHC WBC 

I 

I 

I 

Control * i I 

Each value is mean + SEM (n = 5) 

Significant values :*(P

Table-16 : Effect of Plant Extract on Haematological Parameters in Fish Anabas 

Testudineus Exposed For 96 Hrs 

Hb 

RBC 

OCC 

Ht 

MCV 

MCH 

MCHC 

WBC 

Hb/ 100 

rnl 

OCC/W I 

M , ~ ~ , ~ % ~ ~ ~ ~ 

ofhb 

vm1 PB % 

104! 

nim' 

14.42 

4 08 

i 

0.18 

18.00 

i 

0.26 

I 

36 1 89 82 

0.96 5.85 

* / * 

36.66 

i 

2.01 

39.8 

i 

2 25 

4 8 

0 12 

Each value is mean * SEM (n = 5) . 

Significant values : *(P

Figure-] : Prohit Mortality Plotted Against Latex Concentration For 96 Hrs. 

Figure-2 : Probit Mortality Plotted Against Plant Extract Concentration for 96 hrs. 

100 

90 - 

80 - 

70 - 

= 60 - 

Y 

6 soe 

a 40- 

30 - 

20 - 

10 - 

0, 

1.11 1.38 1.69 1.85 194 2 

Log Concentratton (mli5l)

5 r Ern 

1 m m 

< 8

LIVER BRAIN GILL LIVER BRAIN GILL LIVER 8 ~ ~ 1 MTU ~ I 

Latex Latex + Supplements .TU-2 

Plant extract .TU 3

ll.l'w I" frrrg 1 a#-. I.JII.\ + 5t.pplrmrnl\

ABBREVIATIONS USED IN PLATES 

ASL 

BC 

BCL 

BCL 

BH 

EM 

BV 

CHI 

CL 

cs 

CSGL 

DGL 

DH 

DL 

DPL 

DSL 

EIL 

F 

FSG 

GL 

HC 

HE 

L 

LS 

MD 

MH 

MI. 

NC 

NR 

OT 

PL 

ROT 

SL 

SN 

ST 

VC 

VH 

WTSE 

atropy of secondary lamellae 

blood capillary 

blood clots 

broken gill lamellae 

bile ductule hyperplasia 

basement membrane 

blood vessels 

cloudy swelling of hepatocytes 

clumping 

cell shrinkage 

curling of secondary lamellae 

damaged gill lamellae 

degenerated hepatocyte 

damaged lamellae 

damaged primary lamellae 

damaged secondary lamellae 

eroded interlamellar epithelium 

hepatocyte fibrosis 

fusion of secondary gill lamellae 

granular layer 

hepatocyte 

hernarrhagic exudates 

Laticiferous tissue 

lysis of cell membrane 

membrane damage 

megalocytosis of hepatocytes 

molecular layer 

necrosis 

nucleus rotundus 

optic tectum 

purkinji layer 

ruptured optic tectum 

secondary gill lamellae 

swollen nucleus 

splitting of tissue 

vacuolation 

vacuolated hepatocyte 

widened torus semicircularis

PLATE-A 

Calotropis gigantea 

a-Plant 

Photomicrographs 

b-T S of Stem (X 100) 

c-Stem Lat~ciferous tissue (X 200) 

d-Leaf iat~ciferous tissue (X 100)

PLATE .A

Photomicrographs of sections through Brain. 

(Haematoxylin and Eosin Stain) 

CONTROL -section through Brain of control fish(>: 200) 

TUI -sections through Brain of test unit-1 latex treated fish (X 100) 

TU2 -sections through Brain of test unit-2 latex treated fish (X 200) 

TU3 -sections through Brain of test unit-3 latex treated fish (X 100)

PLATE ,I 

CONTROL TU 1

Photomicrographs of sections through Brain 


CONTROL -section through Brain of control fish(X 40) 

TUI -sections through Brain of test unit-1 plant extract treated fish (X 100) 

TU2 -sections through Brain of test unit-2 plant extract treated fish (X 100) 

TU3 -sections through Brain of test unit-3 plant extract treated fish(X 100)

CONTROL 

PLATE. 2

Photomicrographs of sections through Gills 


CONTROL -section through Gill of control fish(X 100) 

TUI -sections through Gill of test un~t-I latex treated fish (X 100) 

TU2- sections through Gill of test unit-2 latex treated fish (X 200) 

TU3 -sections through Gill of test unit-3 latex treated fish (X 100)

PLATE .3 

CONTROL TU 1

Photomicrographs of sections through Gills 


CONTROL -section through Gill of control fish(X 200) 

TUI -sections through Gill of test unit-I Plant extract treated fish (X 400) 

TU2 -sections through Gill of test unit-2 Plant extract treated fish (X 100) 

TU3- sections through Gill of test unit-3 Plant extract treated fish (X 100)

PLATE .4 

CONTROL TU 1

Photomicrographs of sections through Liver 


CONTROL -sect~on through Llver of control fish(X 40) 

TUI -sections through Liver of test unit-I latex treated fish (X 100) 

TU2-sections through Liver of test unit-2 latex treated fish (X 200) 

TU3-sections through Liver of test unit-3 latex treated fish (X 100)

PLATE .5 

CONTROL TU 1

Photomicrographs of sections through Liver 

(Haernatoxylin and Eosin Stain) 

CONTROL -section through Liver of control fish(X 40) 

TUI -sections through Liver of test unit-I Plant extract treated fish (X 40) 

TU2-sections through Liver of test unit-2 Plant extract treated fish (X 100) 

TU3-sections through Liver of test unit-3 Plant extract treated fish (X 100)

PLATE .6 

CONTROL TU 1

Photomicrographs of Blood smear showing Red Blood Cells 

(Haernatoxylin and Eosin Stain) 

CONTROL - Red Blood Cells of control fish(X 40) 

TUI - Red Blood Cells of test unit-1 latex treated fish (X 100) 

TU2- Red Blood Cells of test unit-2 latex treated fish (X 100) 

TU3- Red Blood Cells of test unit-3 latex treated fish (X 100)

PLATE .7 

CONTROL 

TII r




TUI - Red Blood Cells of test unit-I Plant extract treated fish (X 100) 

TU2- Red Blwd Cells of test unit-2 Plant extract treated fish (X 100) 

TU3- Red Blood Cells of test unit-3 Plant extract treated fish (X 100)

PLATE .8


(Haematoxyl~n and Eosin Stain) 


TUI - Red Blood Cells of test unit-1 latex +supplements treated 

fish (X 100) 

TU2- Red Blood Cells of test unit-2 latex +supplements treated 

fish (X 100) 

TU3- Red Blood Cells of test unit-3 latex +supplements treated 

fish (X 100)

PLATE .9 

CONTROL TU 1

DISCUSSION 

Anabas restudineus became excited after exposure to different concentrations of latex 

and the~r surfac~ng frequencies increased for a few minutes. The colour of thz body 

became pale after about 6hrs.The observed behavioral changes in LCSu and 

subsequent higher doses, such as erratic jumping movements, changes In opercular 

movements rate, irregular swimming activity of the body, hyper and hypo activity, 

increase in surfacing activity, loss in equilibrium, spiraling, jerky movements, 

vertical movements spreading of excess mucus all over the surface of the body lead 

ultimately death occurred as the result of exposure to pesticide. These findings are in 

conformity with the earlier findings of Sabita borah and Yadav (1996);Pravakar et 

aL, (1993); Sadhu (1993); Santha Kumar(l998);Prasanth et aL,(2005);Mahajan 

and Patole(2003);Patole and Mahajan,(2006).Duration of latent period depend on 

the nature and concentrations of toxicant used and fish species (with other conditions 

remaining uniform).But in general,organic pesticides have a short latent period, thus 

the symptoms of poisoning start when the fishes are exposed .Changes in ventilation 

rate and surfacing frequencies are the general symptoms noticed in the fish after 

exposure to the pesticide and these activities help the fish to avoid the contact with 

poison and fight against the stress (Chowdhary et aL, 1981; Roy and Munshi, 1987) 

Fishes that have water-breathing and air-breathing organs are bimodal regarding 

breathing.Bimoda1 species are noted for their resistance to environmental stress and 

aquatic hypoxia (Dehadrai and Tripati, 1976). The LCs0 Value differs from species 

to species for the same pesticide and different pesticides due to the mode of their 

action on fish (King, 1962).The purified principle of Calotropis gigantea,0.02-0.04 G 

when injected subcutaneously kills rabbit in 30 minutes, guinea pig in 15 minutes, in 

pigeon there results vomiting, in frogs causes systolic arrest with in 6 

minutes(Nadkarni ,1991) .96 hr LCso value for bio pesticide of Calotropis gigantea 

was found to be IOmlIS liters (Bharathi,2005), and latex was 0.lmliSlitres 

(JosphenePaulina, 2003; Preethakumari,ZOO4) in the Anabm testudinew. Air

eathing fish have a higher LCSO value compared to water-breathing fish (Duna et 

aL, 1992 a). In the presence of toxin, these fishes reduce the opercular movement rate 

but increase the surfacing frequency leading to more air breathing .The observed 

mucus accumulation on the gills and skin of the fishes exposed to pesticides were 

probably due to toxic effects of pesticides, because respiratory epithelium might be 

the main target site of toxicity during the period of experiment. The mucus may be an 

adaptive response perhaps providing additional protection against corrosive nature of 

pesticides This agrees with the earlier findings of Sadhu (1993), Pravakar et aL, 

(1993) and pesticides of plant origin (Patole and Mahajan, 2004). The increased 

secretion of mucous by the skin made by body slippety of quick movement in the test 

solution and avoids the absorption of the toxicant by the general body surface (Sabita 

Borah and Yadav, 1995). lcthyo toxins like saponins, cardiac glycosides, alkaloids , 

isoflavonoids, tannins, cynogenic compounds proved highly icthyo toxic and these 

chemicals lower surface tension of water interfering respiration and central nervous 

system(acetylcho1ine inhibitors). Death of the fish may be due to the acetylcholine 

inhibition and the respiratory failure. It was noted that 90% of inhibiton of Acetyl 

cholinesterase resulted in death of the test fish (Sinho andDafra Munshi,1996;Singh 

and Singh,2005). All the organophosphate insecticides also act as a nerve poisons 

by blocking synaptic transmission and the cholinergic parts of a nerve (O'Brien et 

aL, 1974).This results in the accumulation of Acetylcholine as synapses that produce 

severe physiological disturbances leading to titanic paralysis, respiraiory failure vla 

respiratory centre and death. 

BEHAVIOURAL TOXICITY 

Optornotor behavioural Changes 

The optomotor response is essential for behaviour such as searching for food, 

orientation towards food,odor, location of a mate, escaping from a predator and 

avoidance of an adverse situation. Two major behavioral changes observed were 

"hypoactive" and "lethargy" exposed to different sublethal concentrations of latex,

latex plus supplements and plant extract. In Anabas teshrdineus hypo activity and 

lethargy was seen at the higher concentrations TU2(0.01ml/Sliters) and 

TU3(0.02m1151iters) of latex, TU2(lml/Sliters); TU3(2mliSliters) of plant extract 

treated fish. The lethargic condition that results due to pesticide exposure would 

affect fish in several ways.The fish that became lethargic would fall easy prey to 

predators.Feeding and food capture will be hampered by lethargy and loss of 

orientation caused by the action of the pollutants. Fish living in streams may not be 

able to maintain their position and may be swept downstream. Sharma et aL, (1983) 

reported erratic swimming movements followed by lethargy in Clarias botrachus 

exposed to 0.25 to 2 mg /I Malathion. Sim~lar hypoactive and lethargic conditions 

were observed in fish Labeo rohila and Anabas restltdineus exposed to malathion, 

(Dufta et aL,19926,1994), Anabas testudineus exposed to monocrotophos(Santha 

kumar,1998), Anabas testudineus exposed to latex of Calotropis gigantea 

(Josephine pauIina,2003;Preetha kumari,2004). Anabas testudineus exposed to 

biopesticide prepared from Calotropis gigantea (Bharathi, 2005), Cirrihnus mrigala 

exposed to cqpermethrin 

(Prasanrh et aL, 2005). From the present studies it 

appeared that the deviation from normal activity is pronounced, however, certain 

amount of recovery could be seen in latex plus supplements treated fish, enabling to 

withstand stress in toxic cnvironment. 

Impact on Surfacing behaviour, Distance traveled and Opercular movement 

In the present study, the fish Anabas testudineus showed a continuous decline in the 

opercular movements and increase in surfacing behaviour when exposed to sublethal 

exposure of latex and plant extract. This coincides with the work done by Anbu and 

Ramaswamy (1991) in Channa srraiatus exposed to sevin, Zayapragassarazan 

(1993) in Anabas testudineus exposed to lindane and Sajitha Bhaskar (1934) in 

Anabas tesrudineus exposed to endosulfan, Anabas restudineus exposed to 

monocrotophos (Sanlha Kumar,1998), Anabas testudinelcr exposed to latex of 

Calotropis gigantea (Josephine Paulina,2003) Anabas leshrdineus exposed to latex

of Calotropis gigantean (Preetha Kumari,2004) Anabas testudineus exposed to 

biopesticide prepared from Calotropir gigantea (Bharathi,200S).Changes in 

ventilation rate and surfacing frequencies are the several symptoms noticed in the 

fish aAer exposure to the pesticide and these activities help the fish to avsid the 

contact with poison and fight against the stress (Chowdhary ef aL, 1981; Roy and 

Munshi, 1987) .Attabas testudineus have water breathing and air breathing 

organs(bimodai regarding breathing). Bimodal species are noted for their resistance 

to environmental stress and aquatic hypoxia (Dehadrai and Tripathi, 1976). These 

are because, air breathing supplements the oxygen requirements in these fishes. 

Further, they have reduced aquatic respiratory surfaces with a thicker barrier 

providing a lesser toxicant. It is possible that air breathing renders fish more 

resistance to toxicants by permitting the reduction of gill ventilation, thereby 

reducing contact with toxicant at a major site of uptake (Anbu and Ramaswamy, 

1991). In these fishes opercular movement rate decreased, but surfacing frequency 

increased leading to more air breathing. Bimodal fishes respond to toxicants by 

reducing the proportion of oxygen uptake via gills (Kulakkatolickal and Karmer, 

1988). The capacity to reduce the uptake of noxious substances should be added to 

the list of other possible advantages of bimodal respiration in fishes. Bull and hfc 

Inerney (1974) reported that many juveniles of Coho Solmon were unable to 

maintain position and where swept downstream after being exposed to sumithion, an 

organophosphorus insecticide, in a stream aquarium. The results of the present study 

show that exposure of fish to latex, plant extract in the aquatic environment may not 

cause immediate death, hut it certainly can cause some undesirable behavioural 

changes. These undesirable behavioral changes may lead to a reduction in the 

number of fish population that will result in a disturbance in the aquatic ecosystem . 

However, certain amount of recovery could be seen in latex plus supplements treated 

fish, enabling to withstand stress in toxic environment and the nutrients, that provide 

strength and support to fish.

TOXICITY IMPACT ON HEMATOLOGICAL PARAMETERS 

Haemolysis of red blood cells provides simple and rapid way of studying the effect of 

pollutants on biological membranes (Harington et aL, 1971) numerous investigations 

have considered membrane model a measure of pollutant's cyotoxicity (Alliso~i el 

aL, 1966). The red blood cell membrane haemolysis has proved to be a simple and 

rapid way of attempting to find the possible correlation between toxicity and 

haemolytic activity (Mncnab and Harington, 1967).1n the present study a clear trend 

was observed linking latex and plant extract concentration with membrane damage. 

The impact was the most severe on fishes exposed to the highest of the three sub- 

lethal concentrations of latex, plant extract of Calotropis gigantea 

.In this 

concentration severe lysis was also observed. The next lower concentration of latex 

and in the plant extract exposed fishes caused lysis of a less severe magnitude. The 

present experiments revealed that Haemoglobin content, RBC,Ht and MCHC were 

significantly lesser in the latex, plant extract exposed fishes. The most pronounced 

depression in RBC count, Ht, MCHC and Hb content were in the fishes exposed to 

the highest of three sublethal concentrations and decrease was progressively lesser in 

the lesser conce~trations. Oxygen combining capacity of blood is direct function of 

its Hb content. The trends observed with this parameter were exactly parallel to the 

trends observed for Hb content. The similar decrease in Hb, RBC count, Ht and 

MCHC was observed in rats exposed to monocrotophos and its analogues (Siddiqui 

et aL, 1991) and 

in Anabas testudineus exposed to monocrotophos (Santha 

Kumar,1998), Anabar testudinew exposed to biopesticide prepared from Calofropis 

giganfea (Bharathi,IOOS) . Decrease in Kb content, RBC count, Ht and MCHC was 

also observed in fishes exposed to other pesticides: fenproparthrin (Ahamad Figar et 

aL, 1995) ; paraquant (Ibrahim et aL, 1995); malathion (Ikhair -ud- Din et aL, 

1996) praquant (Ibrahim et aL, 1995) ; Methyl parathion (Nath Rabindra and 

Banerjee, 1996). Haematological studies disclose possible reaction of blood and 

blood forming organs to the pesticide treatment (Siddiqui el aL, 1987; Srinivasan 

and Radha Krishnamunhy, 1983). 

Janardhan and Sisodia (1990) reported

decrease in the Hb concentration, erythrocyte counts in rats exposed to 

monocrotophos. In the present study total RBC, oxygen combining capacity of blood 

and Hb content showed a decreasing trend with increasing exposure time. The 

reduction in RBC number may be due to microcytic or nolmocytic anaemia 

(Tuschiya, 1973). Janardhan and Sisodia (1990) observed decrease in haemoglohin 

concentration with concomitant increase in bilirubin concentration indicates 

haemolytic condition that could be the result of d~rectoxic effect of monocrotophos 

on erythrocytes.ln most vertebrates, including, fish erythropoietic act~vity is 

regulated by erythropoietin produced in the kidney (Gordon et aL, 1967). 

Erythropoietin, besides promoting erythropoiesis by indicating; hemopoietic stem 

cells to differentiate into erythroblasts, which forms RBC, also makes pyidoxal 

phosphate active in developing RBC'S inducing Hb synthesis. Hypoxia constitutes 

the fundamental stimulus for erythropoiesis with the kidney as the probable sensing 

organ for low blood oxygen tensions (Jacobsen and Krantz, 1968). Latex, plant 

extract. of Calorropis gigantea like several other organophosphates, impairs 

neuromuscular transmission through acetylchol~neesterase inhibition (Preetha 

Kumari,ZOO4) resulting in a reduction or cessatioji of respiratory movements as 

observed in behaviorual studies and a decrease in oxygen uptake. In the present 

studies, Anabus testudineus showed a decrease in RBC counts suggesting a decrease 

in erythropoietic activity. A structurally intact and normally functioning kidney is 

essential for erythropoietin revealed progressive dystrophic changes in the kidney 

tubules of Anabus testudineus exposed to the three sublethal concentrations of 

monocrotophos(Santha Kumar,1998). Kidney damage usually causes decrease in 

erythropoietin levels which in turn decrease RBC production and Hb synthesis even 

under pesticide induced stress condition. The significant decrease in Ht value in fish 

is correlated with a decrease in RBC, which might he due to its effect on blood 

forming organs (Srinivasan and Radhakrshnamurthy, 1983). The slight increase 

and decrease observed in MCHC and MCH values cannot be attributed to cells 

shrinking or swelling but rather to a proportional decrease in Red blood cells and

haemoglobin concentration. The significant increase in MCV result of swelling may 

be due to beta adrenergic stimulation caused by 

exposed stress condition 

experienced by the test organisms to latex and plant extract of Calotropis gigantea 

(Butler el 01, 1978 and Santha Kumar,1998 ).An increase in MCV value may be 

considered as 

RBC destruction leading to anaemia (Johansson- Sjobeck and 

Larson, 1978). The decreased MCHC also points to the fact that cells swelling 

occurred. These findings in Anabas restudineus exposed to latex and plant extract of 

Calotropis gigantea are in partial agreement with the results of other researches 

(Dalela el aL, 1981 ; Mishra and Srivaslava, 1983; El-Doimary 1987; Gill et aL, 

1991a, b; Duna et aL, 1992c, Santha kumar,l998;Josphene pauIina,2003). The 

leukocytes or white blood corpuscles are colorless due to the absence of 

haemoglobin. They play a crucial role in defending the animal against the invading 

toxins. They contribute to the immune response by destroying harmful bacteria and 

inactivating harmful foreign materials in the tissues and blood. The results of our 

experiment reveals that exposure to latex and plant extract of Calotropis gigantea 

caused increase in WBC count .In all the cases the increase in the WBC count was 

thc highest in the higher of the sub-lethal concentration of latex and was 

progressively lower in the lower concentrations. The increase in WBC count may be 

attributed to the response of the fish to latex and plant extract of Calotropis gigonlea 

, where latex and plant extract may act as an antigen. The significant increase in the 

total leukocyte count corroborates the earlier work on fishes (Ahamad Figar et al, 

1995; Ibrahim et aL, 1995; lkhair-Ud-Din el al, 1996; Nath Rabindra and 

Banerjee, 1996). WBC is inextricably involved in the regulation of immunoiogical 

function and prolonged exposure of Anabas testudineus to latex and plant extract 

may inflict immunological deficiency. The WBC count decreased slightly in the 

fishes exposed to three sublethal concentrations of latex plus supplements treated fish 

however, certain amount of recovery could be seen protecting from anaemic 

condition, enhancing immune power enabling to withstand 

stress in toxic 

environment. The present findings are in agreement with Santha

Kumar,1998;JosphenePau1ina,2003;PreethaKumri,2004;Bharathi,2005in Anabas 

testudineus. 

TOXICITY IMPACT ON BIOCHEMICAL PARAMETERS 

Protein Levels 

Proteins play a vital role in the biological functions and are, hence aptly called the 

building blocks for cellular components. In fish, proteins are the primary energy 

source and are involved in regulating physiological and metabolic processes in the 

body through hormones, enzymes etc., They play a vital role as energy precursors in 

fishes exposed to stress conditions (Jones Nelson and Sunil Kumar, 1996; Anitha 

Kumari and Sree Ram Kumar, 1996; Ramalingam, 1980). The Present experiment 

indicates that sub-lethal concentrations of latex and plant extract of Calotropis 

gigantea have a cognizable impact on the brain, liver, gill protein levels of Anabas 

tesfudineus. Protein levels were found to decrease and the significant decrease was 

observed in almost all organs. The decrease in protein levels was in the order of liver, 

gill and brain. Latex treated fishes were more affected than plant extract treated 

fishes. The absence or slight changes in alterations in the early periods of exposure 

supports the concept of Fry (1971) that fishes tend to resist stress or a specific period 

but may eventually succumb as result of their inability, to endure further. Umminger 

(1970) attributes this to the fact that proteins are reserves of energy, which are used 

up gradually during the periods of stress. Rapid loss of brain proteins during pesticide 

toxicity was reported (Richardosn, 1981). The decrease in total tissue proteins 

indicates their metabolic utilization (Swamy et aL, 19926). The sub-lethal 

concentrations of latex and plant extract of Calotropis gigantea reduced the brain 

protein content (Josephinepaulina,2003;Prectha kumari,2004) and Bharathi(2005) 

in Anabm resfudineus with biopesticide of Calotropis gigantea Santha 

kumar(1998) with monocrotophos in Anabas testudineus gill,liver and brain .Swamy 

el aL, (19926) reported decrease in protein content in rat brain, Tilapia mossambica 

(Joshi and Desai , 1983) exposed to monocrotophos. Patil el aL, (1990) reported in

estuarine edible Mudskipper Boleophthalmus dussumieri that the muscle protein and 

liver protein were reduced on exposure to sublethal concentrations of monocrotophos 

as observed in the present study. This decline in protein synthesizing capacity of the 

liver receives support for significant decline in the protein content of gills,liver and 

brain. The loss of protein under latex and plant extract stress not~ced in the present 

study may be attributed to utilization of amino acids in the various catabolic 

reactions. The amount of protein becomes reduced when the concentration and the 

period of exposure increases. Lesion may result in the loss of protein and lead to the 

death of fish. Swamy et aL, (19926) reported that increase in the acid, neutral and 

alkaline proteases in rat brain and the increase in the activity of proteases correlated 

with the decrease of total protein. Zayaprgassarazan and Anandan (1996) reported 

significant decline in protein level and decrease in protein patterns in gills, liver, and 

brain of Anabas testudineus exposed to Iindane. Gray and Meckenzie (1970) have 

stated that protein levels in fishes are not influenced by Inherent factors like sex, 

stage of maturation and geograph~cal location. Therefore, the alterations in the 

protein levels, which are noticed in the present study, may be due to the influence of 

exogenous factor like toxic environment as has been suggested by CasteN et aL, 

(1970). Nammalwar (1984), Anita Kumari and Sree Ram Kumar (1996 )reported 

changes in protein fraction in the gill, liver, and brain in mullets exposed to DDT and 

BHC and in pollution stressed fish as observed in brain, liver and gills in the present 

study. The altered mobility and low content of proteins in muscles reflect a change in 

the rate of synthesis and degradation of protein, lowered working capacity under the 

impact of stress. Fractions from the liver of the pesticide treated fish also displayed 

significant changes. These changes may be due to the accumulation of pollutants in 

the liver leading to an alteration in its function indicating the vulnerability of the 

organ. In the tissues, Lysosomal (acidic) proteases found to have role in protein 

degradation (Marzella et aL, 1981). It was reported that impaired energy supply 

leads to the break down of tissue proteins then susceptible to the action of tissue 

Proteolytic enzymes and leading to their consequent degradation by proteases

(Berger et aL, 1983). Depletion of tissue proteins has been reported by Several 

investigators in fishes exposed pesticides: Quinalphos (Anusha Amali et aL,1996): 

ekalux (Jones Nelson and Sunil Kumar, 1996); aldrin (Singh and Srivastava, 

1995); nuvan (Tazeem Arasta, 1996;Anuradha,1993)); aldrin and propoxur (Singh 

et a/., 1996, ) reported significant decrease in proteln levels in Tilapto mossambica 

exposed to monocrotophos. Significant decline in the nucleic acid content has been 

reported earlier in the fish, Tilapia mossambica subjected to monocrotophos (Joshi 

and Desai, 1988). The lower proteln synthesis indicating lower metabolism in these 

fishes.In the present study decrease in protein could be attributed to the enhanced 

activities of proteases, lower protein synthesis at the transcriptional level of impaired 

incorporation of amino acids into polypeptide chain. In latex plus supplements 

treated fi shes, the difference in values were less significant. However, certain amount 

of recovery could be seen. It was interesting to note that, there was a tendency of 

these values to come nearer to control values of selected tissues, This may also be 

due to the capacity of fish to restore protein and because of additive supplements 

which might have given strength, support and protection against toxic stress to fish in 

environment. 

Glycogen Levels 

Carbohydrates are the first substrates to be utilized in metabolism more so under 

toxic stress conditions. Glucose as the primary fuel is utilized during biological 

oxidations for energy production. Glycogen and other polysaccharides are broken 

down by glycolysis into glucose or any other intermediates. The present study 

provides the evidence that like other organochlorine , carbamate, organophosphorus 

and synthetic pyrethroids, biopest~cides also effect carbohydrate metabolism in 

different tissues by altering the levels of metabolites and their associated enzyme 

levels in Anabus testudineus. The glycogen content decreased with the increase in 

sub-lethal concentrations of latex and plant extract of Calotropis gigantea in gills, 

liver and brain, this decrease suggested greater mobilization of glycogen from tissues

as to meet the toxic stress. The decrease in glycogen levels was in the order of liver, 

gill and brain. Latex treated fishes were rather more affected than plant extract 

treated fishes. Fish being subjected lo a situation of stress, both catecholamines 

(Nakano and Tomlison, 1967) and adrenocorticostero~ds (Fagerland, i967; 

Wedemeyer, 1969) were secreted in increased amounts causing marked changes in 

carbohydrate reserves in fish (Stimpson, 1965; Nakano and Tomlison, 1967; 

Swallow and Flemming, 1970; larson, 1973). These catacholamines increase the 

process of glycogenolysis leading to decrease in glycogen content which helps to 

meet the energy demand caused by the toxic environment (Wasserman et, aL, 1970). 

Liver is the largest vital organ in the body which mainly stores glucose in the form of 

glycogen and has function of detoxifying xenobiotic substances. The decrease in 

glycogen content may be due to increased glycogenolysis and also due to decreased 

glycogen synthesis. There are reports to show that organochlorine insecticides 

depressed the activity of glycogen and the activity of glycogen synthetase 

(Hickenbottom and Yau 1974). Increase in glycogen phosphorylase activity in liver 

and muscle was reported by Koundinya and Ramamurthi (1979) and depletion of 

hepatic glycogen content in Tilapia mossainbica exposed to sumithion was also 

observed.The decrease in glycogen content of all the tissues was observed due to 

Malathion and Nuvan exposure in Labeo rohita (AnuradhaJ993). In muscle the 

depletion is next to brain, this may be due to increased muscular activity as fish tries 

to move fast after exposing it to pesticides. Depletion in glycogen gill tissue might 

be due to increased respiratory rate which requires more energy supply to the tissues. 

Shafj7 (1979) reported damage to the surface cells and blood capillaries of gill 

filaments extensively when fresh water fishes were exposed to heptachlor. A similar 

fall in glycogen content was observed in the tissues of T mossarnbica exposed to 

Methyl parathion, Malathion and Lindane (Sivaprasada Rao and Ramana Rao, 

1979; Kabir Ahmed et aL, 1983; Vasanfhi, 1983) Depletion of glycogen was also 

reported in C. Punctaius when exposed to Malathion, Similar reports were available 

to show the depletion of glycogen in fish, snail and fresh water muscle due to

insecticide toxic stress (Koundinya and Ramomurthy 1979; Srinivasa Murrhy el aL, 

1983). Reduction in the glycogen was also observed in tissues of S. mossambicus 

when exposed to DDT, Malathion and mercury (Ramalingam, 1988). The decrease 

in glycogen might be due to hypoxic condition which Increases carbohydrate 

consumption. In latex plus supplenlents treated fishes, the difference in values were 

less significant. However. certain amount of recovery could be seen. It was 

interesting to note that, there was a tendency of these values to come nearer to control 

values, this may also is due to the capaclty of fish to restore glycogen and because of 

additive supplements which might have given strength, support and protection 

against toxic stress to fish in environment. 

Acid and Alkaline Phosphatase Activity 

Acid and Alkaline phosphates also serve as diagnost~c tool to assess the toxicity 

stress of chemicals in the living organisms (Harper, 1991). Acid phosphatases and 

Alkaline phosphatases are hydrolytic lysosomal enzymes and are released by the 

lysosomes for the hydrolysis of foreign material. Hence,they play a role in certair. 

de:oxification functions. It is known as ~nducibl enzymes, whose activity in animal 

tissue goes up when there is a toxic impact and the enzyme begins to counter act. 

Subsequently the enzyme activity may begin to drop either as a result of having 

partly or fully encountered the toxin or as a result of cell damage. In the present study 

on acid and alkaline phosphatase activity, were observed in gills, liver and brain. 

Similar findings have been reported with other pesticide. Keshavan and Kamble 

(1984). Lysosomal hydrolases are thought to contribute to the degradation of 

damaged cells and hence to facilitate their replacement by normal tissue (DeDuve, 

1963). Acid phosphatase is an enzyme of lysosomal origin, which hydrolysis the 

phosphorous esters in acidic medium moreover help in autolysis of the cell after its 

death. The impact was the highest on the fishes exposed to the highest of the three 

sublethal concentrations of latex and plant extract of Calotropis gigantea. The 

increase in acid and alkaline phosphatase activity was in the order of liver, gill and

ain. Latex treated fishes were rather more affected than plant extract treated fishes. 

The fluctuation and increase of acid phosphatase activity encountered in the three 

sublethal concentrations of latex and plant extract of Calorropts giga~i~ea corresponds 

to the observation made by Singh and Singh(2OOS);Usha Kaisabhatjao and Vibhuti 

Rai (1993). Sabita Borah and Yadav (1996) reported increased in acid and alkaline 

phosphatase activity in gills exposed to pest~cide rogor In Heteropneustes fossrlis. 

Similar observations were reported by Josephine Paulina(2003) In the sub-lethal 

concentrations of latex of Calotropis grgantea and in Anabas testudineus exposed to 

monocrotophos(Santha Kumar,1998) .The enhanced acid phosphatase might be due 

to the increase in active uptake of ions through gills.Mullainathan (1982) reported 

that gill form a major site of accumulation of foreign substances. The hlgher activity 

of the acid phosphatase might have ~nfluenced the changes in the energy supply of 

metabolites and it was associated w~th carbohydrate metabolisrnSwamy et aL, 

(1992a) reported increased acid and alkaline phosphatase activity in rat brains, 

exposed to monocrotophos.Alkaline phosphatase is brush border enzyme that splits 

various phospho esters at alkaline pH and mediates membrane transport (Goldfisher 

et aL, 1964) and involved in transphvsphorylation reactions (Srinivasulu Reddy et 

aL, 199l).Alkaline phosphatase has also been shown to be involved in active 

transport (Danielli, 1972), glycogen metabolism (Reddy and Rao, 1988), protein 

synthesis (Pilo et aL, 1972), secretory activity (Ibrahim et aL, 1974) and in synthesis 

of certain enzymes (Summer, 1965). Any change in the alkalina phosphatase activity 

will affect the physiological and biochemical pathways of animals .Increased alkaline 

phosphatase activity may be indicative of an adaptive rise in enzyme activity to the 

persistent stress (Murphy and Porter,1966) as observed in giils, liver and brain .Acid 

and alkaline phosphatase present in nucleolus are reported to be involved in the 

synthesis of nucleic acid (Con and Griffin, 1965) and thus any change in the 

activities of these enzymes also disturbs the protein synthesis (Srinivasulu Reddy et 

al, 1991). The behavior of phosphatase activity observed in gills,liver and brain in 

the present study may be due to the toxic effect of 

latex and plant extract of

Calolropis gigantea, by which the cellular membranes and lysosomal membrane 

might have been ruptured (Singh and Singh,tOOS; Dalela et aL, 1978) or due to 

tissue inflammatory reaction of toxin , increased transphophorylation activity of the 

tissue (Sastry and Sharma, 1978) may also be a factor, elevation in both 

phosphatase enzyme activity was observed in gills, liver and brain. Alkaline 

phosphatase contains a serine residue at its active site (Mahendra and Aganval, 

1983) and organo phosphorous insecticides are reported to the potent inhibitors of 

serine containing enzymes.Bel1 er aL,(l970) and Rama Rao et al(1996) reported 

increase in activity of alkaline phosphatase in the liver in fish, Sartherodon 

mossambicus exposed to sublethal concentration of carbon tetrachloride .The 

phosphatases can be consider as metabolic scavengers which break down the nonfunctional 

biomolecules to their respective monomeric units to be utilised for other 

physiological and metabolic functions(Rama Rao et aL, 1996). The increased 

activity of alkaline phosphatase indicated increased cleavage of high energy bonds. 

The phosphate system comes into operation when the tissue is facing energy crisis 

(Rama Rao er aL, 1996). Gill er aL, 0990).1t was reported increased in acid and 

alkaline phosphatase zctivity in fish, Ciarim batrachus exposed to acephate. 

Ahamad Figar et al, (1995) reported increased hepatic acid phosphate increase after 

two weeks and decreased at the end of four weeks in fish, Chinese grass carp exposed 

to Danitol. The most probable reason for the decrease in the activity of acid 

phosphatase observed in liuer,in present study could be uncoupling of oxidative 

phosphorylation (Dalela et aL, 1980: Vermo et aL, 1980). Simon (1953) stated that 

concentrations higher than those needed to prevent oxidative phosphorylation could 

injure mitochondrial system so as to block the action of enzymes concerned with 

oxidative metabolism.Action of uncoupling of oxidative phosphorylation has been 

pointed out on the basis of chemical (Pressman, 1963) and chemiosmotic (Mitchel, 

1961) interactions. According to Pressman (1963) uncoupling promote the 

conductivity of protons within mitochondrial membranes and subsequently prevent a 

gradient across the membrane. Weinbach and Garbus (1969) suggested that

uncoupling traverse through lipoprotein layer of mitochondrial membrane and 

interact with protein groups that then undergo structural changes. 

Further, the 

uncouplers bind tightly with mitochondrial proteins that are lnvolved in aminoacid 

metabolism. It may be suggested that alteration In membrane permeability, disruption 

of normal functioning of cell organelles like lysosome and mitochondria, and 

different repressor mechanisms associated with pesticide tox~city together resulted in 

significant changes in the level of the enzyme acid phosphatase in the tissues 

examined. In latex plus supplements treated fishes, the difference in values were less 

significant. However, certain amount of recovery could be seen. It was interesting to 

note that, there was a tendency of these values to come nearer to control values, this 

may also be due to the capacity of fish to restore phosphatase activity and because of 

additwe supplements which might have given strength, support and prevention 

against toxic stress to fish in environment. The changes observed in the present study 

with respect to acid and alkaline phosphatase activities in different organs, are due 

to the toxic effect of latex and plant extract of Calotropis gigantea. 

Acetyl cholinesterase Activity 

The most important toxic property of pesticide compounds is inhibiting their target 

enzyne Acetyl cholinesterase activity (0' Brein, 1967; Corbeit, 1974). Most of the 

organophosphate compounds are similar with the ester part of acetylocholine and 

they react with esterase part of AchE after entering into the exposed animal .The 

conversion of acetylcholine into acetic acid and choline catalyzed by AchE is 

considered to be the key reaction in synaptic transmission (Bachelard 1976). 

A significant decrease in acetyl cholinesterase activity was observed in the present 

study during short term exposure. It was recorded in the selected tissues. 

decrease in AchE activity was in the order of brain, gill and liver. Latex treated 

fishes were rather more effected than plant extract treated fishes.The serine hydroxyl 

group is blocked by op insecticides leading to the inhibition of AchE which may 

The

esult in the excessive accumulation of Ach at the synapse, disrupting the transfer of 

nerve impulse. Similar decrease in AchE activity and increased accumulation of Ach 

was observed when treated with Roger and Di~necron in fresh water muscle, 

L Marginalis (Vijayaendrababu and Vasudev, 1984). Similar observations were 

made in fresh water crab, O.Senex senex by Fenitrothion (Bhagyalakshmi and 

Ramamurthi, 1980) and in snails exposed to different organophosphorus compounds 

(Ramana Rao and Ramurthi, 1979; Anuradha,1993; Singh and Singh,2005;Singh 

and Aganual, 1982). The accumulated Ach may induce the synthesis of increased 

amounts of AchE leading to the revival of affected fishes (Kabir Ahmed e&aL, 1980). 

AchE was inhibited in both short term and long term. Exposure to Nuvan has 

resulted in significant changes only during the short term and insignificant during the 

long term.It was well documented that highly purified phosphorothionates (P= S 

form ) are not direct 'inhibitors of cholinesterase' but when they are metabolized to 

their corresponding oxygen analogues (P=o form) become highly potent 

inhibitors(March e& aL, 1956 and Murphy e&aL, 1968), the susceptibility of animals 

to poisoining by organophosphorus insecticides will be dependent, at least in one art 

upon the rate at which the analogues are made available to inhibit cholinesterase at 

critical site in nerve tissue or organs innervated by cholinergic nerves. Pesticides 

which inhibit normal function of AchE are known as "Anticholinesterases" agents. 

With regard to nature of inhibition some of the pesticides are characterized by the 

nature of inhibition for eg. Carbamate compounds are "Reversible inhibitors" of 

AchE, as they are less potent and shorter acting anti cholinesterase agents, in contrast 

OP compounds are known to be "irreversible inhibitors" because of long acting 

cholinesterase agents (Koelle, 1975). Based on the inhibitory activity of AchE , the 

results indicated that latex was relatively more effective than plant extract. And in 

latex plus supplements treated fishes, the difference in values were less significant. 

However, certain amount of recovery could be seen. It was interesting to note that, 

there was a tendency of these values to come nearer to control values, this may also 

is due to the capacity of fish to restore AchE activity and because of additive

supplements which might have given strength, support and protection against toxic 

stress to fish in environment 

Adenosine Triphosphatase Activity: 

M~~ and N~'K' ATPase Activity 

ATPase activity in general speaks about the transport of Na' and K' ions and as well 

synthesis of ATP. Nat,K' ATPase is responsible for active cation transport (Skou, 

1957). Generally most of the animals maintain high intracellular K' concentration 

and a low Na' concentration and transport of these ions across the cell membrane 

against an electro chemical gradient takes place by an active transport process. The 

active transport involves Mg" dependent, Nai and K' 

activated ATPase which 

provides the largest contribution to the maintenance of these ionic Transmembrane 

gradients (Trachtenberg et aL. 1981).Ki ATPase is an intrinsic membrane bound 

protein which hydrolyses ATP to ADP releasing an inorganic phosphate.In this 

process energy requirement is more (Ya!es, 1980; Schurmans Stekhovan and 

Bonting, 1981; Towle, 1981). An important ene:.gy produc~ng enzyme in 

mitochondria is the Mg* ATPase.Which is shown to be irvolved in the coupling of 

ADP + Pi in the biosynthesis of ATP (Boyer et aL, 1977). In the present study ~ a ,* 

K' ATPase and MgH ATPase were estimated. All the three above stated enzyme 

activities were seen to be reduced during 96 hr exposure. The impact was the hlghest 

on the fishes exposed to the highest of the three sublethal concentrations of latex and 

plant extract of Calotropis gigantea 

The decrease in ~ a', K' ATPase and 

ATPase activity was in the order of liver, gill and brain.Latex treated fishes were 

rather more effected than plant extract treated fishes.Among the percentage obtained 

liver has the maximum amount of decrease followed by gill and brain.lhe values 

obtained(Table:13) were statistically significant.These results clearly showed the 

disturbances in the enzyme activity due to the exposure of the fish to latex and plant 

extract. In latex plus supplements treated fishes, the difference in values were less

significant. However, certain amount of recovery could be seen .It was interesting to 

note that, there was a tendency of these values to come nearer to control values, This 

may also be due to the capacity of fish to restore Na', K' ATPase and Mg" ATPase 

activity and because of additive supplements which might have given strench and 

support against toxic stress to fish in environment.Relatively high concentration of 

~ a and ' K' ATPase were recorded in Brain of latex and plant extract treated fish. 

The inhibition of this enzynle in brain indicates its neurotoxic action and their 

interference with membrane ionic sonductance.There are certain reports from 

literature that these symptoms can be caused by the inhibition of Na', K' ATPase 

similar symptoms were observed when rats were injected intracranialy with aubain 

(Bignani and Palladini, 1966) in fish (Anuradha,l993)the well known inh~bitor of 

active Na' , K' transport. In liver due to the impainent of ATPase enzyme activity, 

there was greater reduction in intracellular metabolism, resulting from the greater 

reduction of solute transport (Gonzalezcalivin el aL, 1983).Considerable inh~bition of 

ATPase in gill, suggested the reduction in osmoregulatory mechanism. 

epithelium and tight junctions which are supposed to function as channels for salt 

excretion and osmoregulation might be affected by the toxicants.For uptake of 

pollutants, gills play primary role and it is the first organ to exhibit symptoms of sub- 

lethal toxicity. Towle (1981) concluded that Nau, K' ATPase plays central role in 

whole body ionic regulation. Thus any toxicant which interferes with 

G~ll 

ion~c 

homeostasis will also alter ~ a*, K' ATPase and was reported by many authors, after 

exposure of a fresh water teleost, Channa gachua to sub lethal level of endosulfan; 

gill Nai , Kt ATPase activity was reduced by 20-50% @alela et aL,1978) .~g" 

ATPase is associated with oxidative phosphorylation and synthesis of ATP (Desaiah 

et aL,1979).Considerable decrease of Mg* ATPase activity was observed after 96 

hrs exposure to latex and plant extract . This might be due to the disturbance in the 

transport of ions through mitochondrial membrane, resulting in a marked effect on 

the coupling of oxidation to phosphorylation Wlrich, 1963) the decrease in ~ g " 

ATPase also indicated decreased ATP synthesis.lt was generalized that both in vivo

and in vitro studies showed that the pesticides whether organophosphates, 

organochlorides or biopesticides altered the activity of ATPases. In Vitro inhibition 

of mitochondria1 Mg*' ATPase and Na' , K- ATPase in the brain tissue of blue gill 

fish under the influence of organochlorines was reported by Cutkomp er aL, (1971) 

Significant inhibition of ~ g and " Na' , K' ATPase was observed in the brain and 

gill tissues of Juvenile Mugil cephalus under the influence of three sublethal 

concentrations of Lindane (Anuradha,l993) Although the exact mechanism of action 

is not clearly understood, the inhihit~on of ATPase suggests that membrane bound 

enzymes and receptor activities may be partly involved in latex and plant extract 

exposed fishes. Membrane bound ATPase system is reported to be the prime target 

for the action of various xenobiotics (Desaiah, 1981; Cutkomp et. aL, 1982; 

Desaiah, 1982; Bansal and Desaiah, 1982;Anuradha,1993). Decrease in Mg* 

ATPase in 96hr exposure clearly indicated that the synthesis of ATP was affected in 

a pronounced way. From these studies it appeared that the decrease in ATPase 

activity is pronounced, however, certain amount of recovery could be seen in latex 

plus supplements treated fish. 

HISTOPATHOLOGY 

The pesticide present in water reaches the fish body through ,water taken in with 

food, mucosa of the mouth or gills, they may reach liver, brain, kidney through blood 

circulation. The percentage of pesticide poisoning was high in the brain. This alters 

the physiological and behavioural functions of the fish. Various regions in fish brain 

are concerned with many functions. The impairment of tissues of a region may lead 

into the curtailment of the particular function. This has become proved from the 

classical work of Ariens Kappers et aL, (1963) relating the functions of brain in 

various fishes. It is described that, the gross morphology of the brain of some Indian 

fishes and correlated its structure with the feeding habit and habitat of fishes. Ito 

(1970) discussed the functional significance of the carp optic tectum. On the basis of 

the various experimental evidences, it has been established that brain is the

controlling centre for all functions and movements in the body serving as a relay 

station. Signs of brain toxicity due to exposure to Fenvalarate in fish include tremors 

which progress to convulsion(Bradbury et aL,1987).The effect of latex and plant 

extract on brain showed vascular dilation that is corresponding to eadier observation 

by Cope er aL, (1970) and Sajitha Bhaskar (1994) .The rupture of the wall of brain 

found in the present works coincides with the work done by Kennedy er aL, (1970) in 

methoxychlor and Di Michele and Taylor (1978) on naphthalene eoki(1978) on 

heavy metals, Cardner and La Roche (1973) on Copper chloride, Reichenback 

Klinke(l975) on chemotherapeuticagents,Josephinepaulina,2003;Preetha Kumari, 

2004; Bharathi(2005) in Anabas testudineus with latex and biopesticide of 

Caiotropis gigantea .Santha Kumar(1998) with monocrotophos in 

testudineus were consistent with the present observation(p1ate I 

Anabas 

and 2).The 

pathological examinations, of intracellular space and irregularly shaped nuclear 

arrangement coincides with the work done on industrial eMuent by Joshi and Dubey 

(1984). However, the fish exposed to latex plus supplements showed less damage to 

the tissue due to the presence of additive nutrients, which might act as 

antioxidants(savior against free radical induced oxidative damage) enhancing proper 

coordination and thus, protects from fish mortality. 

Histological observation on gills showed that latex and plant extract induced fish 

showed remarkable pathological changes after 96hr exposure. In fish, the gill is the 

most important organ for respiration and osmoregulation and it is the first organ to 

which the pollutant comes into contact. Hence, it is more vulnerable to damage than 

any other tissue. The pathological conditions include separation of epithelial layer 

over secondary gill filaments, necrosis in inter lamellar spaces and fusion of 

secondary gill lamellae (Plate: 3 and 4 ) , swelling of secondary gill filaments and 

bulging of primary gill lamellae.Necrosis is in the central axis region and atrophy or 

secondary gill filaments. Some of these changes were noticed in fishes under 

exposure to different pesticides by Vijayalakshmi and Tilak (1996); Moses Cirija

and Jayantha Rao (1995); Chakrabarthy and Konar (1974).1n the fish, malathion 

has been shown to induce histopathological changes in gills (Singh and Sahai, 

1984). Josephine paulina,2003;Preetha kumari,2004 with latex and 

Bharathi(2005) in Anabas testudineus with biopestlcide of Calotropis gigantea 

.Santha kumar(1998) with monocrotophos in Anabas testtld~neus were consistent 

with the present observation.Hyperplasia of primary filaments and secondary 

lamellae were reported in the gills Puntius exposed to Khan River water with 

industrial sewage (Chouhan and Pandey, 1987).Hqperplasia of brachlal epithelium 

was found to be the most pronounced damage caused to Anabas testudineus because 

of toxicants. Osburn (1910) has concluded that proliferation of gill epithelium 

protects the gill filaments from constant irritation in silver salmon fingerlings that 

lack adequate gill covers.But. since proliferative thickening of gill epithelium 

produced by most kind's environmental toxicity (Skidmore and Toveii, 

1972; Roberts, 1978). This response considered as general safety measure against 

irrigation by environmental toxicants. Hqperplasia of branchial epithelium has been 

observed commonly in fish facing low tension of oxygen and high concentrations of 

wastes in the environment (Roberts, 1978). Lifting oi lamellar epithelium usually 

occurs when the lymphoid space between the epithehm and its supporting elements 

gets enlarged by accumulation of fluid on account of factors like increased capillary 

permeability (Roberts, 1978) or lowered efficiency of the epithelial cells in 

maintaining normal water balance (Skidmore and Tovell, 1972). Inflammatory 

alterations of lamellar epithelium and hyperplasia were reported in the gills of fresh 

water major carp Cirrhinus mrigala ('Hamilton) during 48 hr exposure to sublethal 

dose of Malathion (Roy and Dana Munshi, 1991). Oedema with liRing lamellar 

epithelium and hyperplasia of epithelium was observed in the gills of all cat fish 

containing residues of Endosulfan (Nowk and Barbara, 1992).The changes that took 

place in the gill of latex and plant extract exposed fish result from the direct contact 

with the organ. In latex and plant extract of Calotropis gigantea exposed fish bulging 

of tip of primary gill filaments, curling of secondary gill filaments, necrosis in the

cells and development vacuoles are observed. Similar observations were found in the 

gills of Puntius aurilus (Bengeri Patti, 1987) and in Tilapia mossambica exposed to 

malathion an organophosphrous insecticide (Rao er aL, 1983). In fish, the respiratory 

epithelium is the banier between the blood and the surrounding water through which 

respiratory gases and other materials needed for sustenance are exchanged. Any 

damage to this epithelium impairs not only the ventilator process but also other vital 

processes, like ion-exchange, during the secretory and excretory functions of the 

gills. Fusion of gill lamellae and lifting of gill epithelium from the supporting 

elements are such effect which reduce the surface area available for gaseous and 

other exchange and lengthen the distance over which the exchange diffusion occurs 

(Jargoe and Haines, 1983); ultra structural features like loss of surface macroridges 

of secondary lamellar epithelial cells also produce similar reduction in the exchange 

surface(Jagoe md Haines, 1983). When this hampers the exchange of respiratory 

gases across the gill epithelium then respiratory insufficiency becomes a natural 

consequence. Respiratory distress also results when stress-induced damage in the gill 

epithelium lead to events like increased influx of hydrogen ions that reduce the pH of 

the blood and thus decrease the oxygen carrying capacity of haemoglobin (Haines 

and Schofield, 1980). Study on hematological parameters has shown the oxygen 

combining capacity of the blood was reduced in fishes exposed to latex and plant 

extract. On the other hand, the ion regulatory and excretory functions of the gill are 

hampered when epithelial damage disturbs the exchange of ammonium and 

bicarbonate ions of the blood with sodium and chloride ions of the medium that 

normally occurs across the gill epithelium of fish (Love, 1980). Mucus cells in the 

secondary lamellae of the experimental fish are more than in control fish, indicating 

the responses of the fish to the toxicant. Effects like collapse of pillar cell system and 

rupture of gill epithelium tend to stagnate or even stop the lamellar blood flow 

(Skimore and Tovell, 1972). This consequence is also likely to limit the respiratory 

capacity of the gills. Degeneration of epithelial cells indicates the damage in the gill 

lamellae that reduces the activity. Undoubtedly, therefore, as stipulated by ENer

(1975), gill alterations, such as those observed presently, would represent basic 

physiological problems that the fish under stress may not ultimately be able to cope 

with. The fish exposed to latex plus supplements showed less damage to the tissue 

and the presence of additive nutnents might have protected from damage of gill 

epithelium increasing respiratory capacity of gills of fish preventing distress and 

mortality. 

The liver showed marked cytoplasmic granularity, periportal atropy and radial 

disorientation. These cond~tions are similar to the cirrhosis (Boyd, 1949) and earlier 

observations of Eller (1971) and Verma el aL, (197S).The loss in the tubulosinusoid 

arrangement in the present study is in the liver of L. Macrochirus treated with 

heptachlor also corresponds to the find~ngs of Eller (1971) in S. Clarkr treated with 

10 ppb endrin for 5 months. Hyperophy of the hepatic cells as reported by Mathur 

(1965) Sastry el aL, (1977) is noted in the present study. There was increased 

cytoplasmic granularity and might be due to the loss of vacuolation and shrinkage of 

hepatic cells as reported by Kennedy et aL, (1970) in I. Macrochinus exposed to 

methoxychlor. Pyconsis of nuclei was previously described by Cope et al., (1969) in 

dichloben exposed in blue gills and Bhanacharya et aL, (1975) in endrin treated 

Channa punctatus while in DDT exposed trout (King, 1962), endosulfan treated 

Anabar testudineus (Satheesh Kumar Reddy, 1994); lindane treated Anabas 

testudineus (Zayapragasaarazan, 1993); tri-aromatic hydrocarbon treated cat fish, 

Heteropneusres fossilis (Dwivedi and Rajakamal Sarin, 1996) and latex treated 

Tilapia mossambica (Desai et aL, 1984). And in the histological and cytochemical 

indices that lysosomal perturbations in dab Limanda limanda indicated early tissue 

les~ons, Impairment of the lysosomal membrane stability, pathological lipid 

accumulation and Liver histopathology was also reported by them along the North 

sea. The early fibrosis as reported by Cope et aL, (1970) in blue gills also noticed in 

the present study. The deformations of hepatocytes as observed in endrin treated cut 

throat trout (Eller, 1971), Clarios batrachus treated with alloxan (Goel and Agrawal,

1977) and Myslur tengara exposed to carbary and endrin (Toor et al., 1977) is one of 

the observation of this study. The development of intercellular spaces and large 

gaps(Plate:S and 6) in the parenchymal mass might be the factors of disorientation of 

liver cords. Congestion of blood might be the factors for disorientation of liver cords. 

Congestion of blood in the central vein and partial loss of radial orientation, 

parenchymal shrinkage and increased cytoplasmic granularity are consistent with 

hepatitis described by Boyd (1949). The results in the present study coincide with 

observations made by Santha kumar(1998) with monocrotophos ,Josephine 

Paulina, 2003; PreethaKkumari, 2004; Bharathi, 2005 in Anabas tesrudineus with 

latex and biopesticide of Calotropisgigantea in Anabas testudineus (Plate: 5 and 6). 

Walsh and RibeIin.(1975) in fishes on exposure to endosulfan and other pesticide 

reports. King (1962) reported necrosis of the tubular epithelium that coincides in the 

present observations. The interstitial cells were found as foamy masses with deeply 

stained nuclei. Similar observation was also reported by Areechon and Plumb (i990) 

in Channel catfish. Ictalurus puncratus exposed to malathion. Results in this work 

showed severe necrosis, pqknosis and disintegration of hepatocytes, were very much 

evident in this test level. These responses can cause severe physico-metabol~c 

dysfunction leading to death. Fish mortalities observed in such situation may then be 

related to complete hepatocyie degeneration, Hemorrhage of blood vessels followed 

by clumping and scattering of erythrocytes were also seen. However, in the present 

studies, the fish exposed to latex plus supplements showed less damage to the tissue. 

This may be due to the presence of immuno-modulators and additive nutrients, which 

might have prevented from hepatic abnormalities and disturbance in liver functions, 

promoting normalacy, protecting fish from mortality.

exposed to the sublethal concentrations of latex, plant extract and the recovery of 

latex toxicity with the additive nutrients were studied. 

1. The LC~~value of latex was O.lml15litres for 96 hrs of exposure and for plant 

extract of Caiotropis gigantea(L.)R.Br. was lOml ISlitres for 96 hr of 

exposure. The safe concentration was found to be 0.001ml ISlitres for latex 

and 0. lmllSlitres for plant extract for Anabas testudrneus (Bloch)(weighing I I 

* 2.2 grams , average length 8 cm).The mortality of fish occurred steadily 

with the passage of time in each of the latex and piant extract concentrations, 

including that the pesticide had not either substantially degraded with time or 

the degradation products themselves were toxic. Further the concentrations 

considered safe in the present study may help In determining the water quality 

criteria and standards for aquatic environment protection works and 

management. The latex in combination with the additive nutrients with feed 

brings resistance and protection to fish against toxic stress. 

2. The behavicural response of the fish varied in accordance with the tcst 

concentrations of the toxicant above LCSO value, to which the fishes were 

exposed during the range finding test, the fishes showed erratic jumping 

movements, changes in opercular movements rate, irregular swimming 

activity of the body, hyper and hypo active, increase in surfacing act~vity, loss 

in equilibrium, spiraling jerky movements, vertical movements spreading of 

excess mucus secretion all over the surface of the body. Some fishes 

frequently dashed against walls of the test chamber suggesting impairment of 

the motor ability and sense of balance. Typically before dying the fishes 

would suddenly show extreme agitations, twisting and diving in spiral-like 

movements before they become stationary at the bottom of the aquarium. 

3. The behavioral study in iishes exposed to sub lethal concentrations showed a 

significant increase in surfacing, distances traveled and decrease in opercular

movements in the treated than in the control. The significant change in 

optomotor response was observed in the treated fishes. 

4. Sublethal Concentrations of latex and plant extract have a cognizable impact 

on the protein suggests the domination of proteolysis during pesticidal 

toxicosis and,also glycogen levels in liver, brain and gills. And showed a 

general decrease.The impact of glycogen was high in liver. 

5. Exposure of latex and plant extract caused a significant elevation in the 

activity of acid phosphatase and alkaline phosphatase activity in gills, liver 

and brain. The impact was highest on the fishes exposed to the highest of the 

three sublethal concentrations. 

6. A significant decrease in acetyl cholinesterase activity was observed in the 

present study during short term exposure in the selected tissues leading to the 

inhibition of AchE which may result in the excessive accumulation of 

Acetylcholine at the synapse, disrupting the transfer of nerve impulse. Latex 

treated fishes were rather more effected than plant extract treated fishes. 

7. In the present study prime target enzymes Na', KA ATPase and ~ g ATPase * 

were estimated. All the three above stated enzyme activities were seen to be 

reduced during 96 hr exposure. The impact was the highest on the fishes 

exposed to the highest of the three sublethal concentrations of latex and plant 

extract of Calotropis gigantea(L.)R.Br. Latex treated fishes were rather more 

affected than plant extract treated fishes. Among the percentage obtained liver 

has the maximum amount of decrease followed by gill and brain.The values 

obtained were statistically significant and suggest that fish resorted 

predominantly to anaerobic metabolism so as to overcome toxic stress and the 

physiological process of tissues including neuronal conduction are disturbed 

by xenobiotics. 

8. Anabas was exposed to sublethal concentrations of latex and plants extract to 

asses the impact on hematological parameters. The blood smear showed a 

number of morphological changes in nucleus and cell shape. Nuclear

morphological changes and membrane damage was directly related to the 

concenhation. The fishes exposed to the highest of the three sublethal 

concentrations showed the most severe Impact. 

9. Haemoglobin content, oxygen combining capacity, total erythrocyte count, 

Ht, MCHC all lower than in control fishes and the decrease was statistically 

significant while there was a consistent vis-a-vis increase in the value of 

MCV, MCH and total leukocfle count in the exposed fishes compared to 

control. 

10.The damage caused by latex and plant extract exposure by histological 

procedures was highest of the three sublethal concenh-ations of the exposed 

fishes than in observed in lower concentration. Organs such as gill, brain and 

liver exhibited severe damage at the highest concentrations. Many changes 

observed in the present study appear to indicate that latex and plant extract of 

Calotropis gigantea (L.)R.Br. reduces the survival of an organism. 

11,The present studies clearly revealed that the latex and plant extract has 

discernible adverse effects on non-target organisnis due to deterioration of 

water quality. Anabas teshrdineus (Bloch) is a very hardy fish and yet latex 

and plant extract even at a low concentration had cognizable impact on the 

fish with short-term exposure causes severe damage at biochemical, 

physio!ogical, haematological and histological levels in the fish Anabus 

teshtdinacs within this period. 

12.It was interesting to note that, recovery could be seen in latex plus 

supplements treated fish, there was a tendency of the values(comparatively 

was insignificant) to come nearer to control values of selected tissues, 

enabling to withstand stress in toxic environment and the nutrients ,that 

provide strength and support to fish, protecting from anaemic condition, 

enhancing immune power, increased amounts of AchE leading to the 

revival of affected fishes, ATPases for maintenance of the ionic 

Transmembrane gradients, restoring biochemical, haematological, behavioural

and histopathological parameters in the air breathing fish, Anabas resrudineus 

(Bloch). This response is considered as general safety measure against latex 

toxicity. 

13. However, the supplements such as Glucose. Fructose, Vitamin C, Vitamin E 

including egg albumin and glycine which are considered as nutrients, will 

enhance the metabolic activity and provlde resistance and protect against the 

fish mortality. Addition of Glucose and Fructose will enhance energy source 

and thereby protected from stress condition. The additlon of Albumin, 

Glycine, Vitamin C and Vitamin E makes the tissue resistant and also disease 

resistant including anti-oxidant property preventing tissue damage and 

providing tensile strength. Hence, a timely study was made on the toxic 

impact of latex and plant extract, also recovery with additive nutrients in 

Anabar resrudineus (Bloch) under sub lethal exposure. 

14.The data achieved of this work may go into the addition of information to the 

toxicology field and to draw strategic plans to protect aquaculture industry and 

biodiversity, more so of fish which influence the economy of man, from 

po!lutants. From this study a suggestion could be extended to farmers tu make 

use of sublethal dose as measurement before the pesticide and fertilizer 

application on crops and to control harmful diseases in fish.

& efe reen ces

REFERENCES 

Abassi and Sujata Krishnan (1993)The new Japanese pesticide cartap -1s environmental 

friendly as claimed.Global Enviromental Perceptions .Val-2 Asish publ. 

House,lndia,l-145. 

Abdel - AzIm,N.S.(1998)Phyto Chemistry, 48,273-275 

Abe,F. (2000)Chem Pharm Bu11,48,991-993 

Aganval , 0. P ,(199% Chemrsfty of Organic Natural Prodt~cts, Gael Publishing House, 

Meemt, 1ndia.Vol. 1 : 74. 

Ahamad Figar ,Ali Syed .S and ShakooriAbdul(l995) Sub lethal effects of Danitol a 

synthetic pyrethrinoid on Chinese grass carp,Folia.Eiol.43(3-4): 151-159. 

Ahmed H.H(2001) Phytochemical investigation of Calotropis procera and the study of 

influence of cardiac glycoside (uscharin)on adult male rats.JPhar.Sc1ences,27:297- 

333. 

Akhtarpsrgis,MalilqAbdu1(1992) Proceragenin,an antibacterial cardenolide from 

C.procera.Phyrochemistty 31(8):2821-2824. 

Allision .A.C,Harigtan.J.S,and Birbeck(1966) Jolrrnal of rxp.Mep.,24:1141-153. 

Anbu,RB. and Hamaswamy,M. (1991) Adaptive changes in respiratory 

movements of an air breathing fish Channa straiatus exposed to carbamate 

pesticide sevin J.Ecobiol.,3(J):I 1-16, 

Andersh and Szaypinski (1974) Use of P-nitrophenyl phosphate as the substrate in 

determination of serum acid phosphotaseArn.J.CIin Path01 17.571-574. 

Andrew Cheng, Franeis.C.K.Chiu, Thomas.r(l983) Cardenolide Glycosides of 

AsclepiadaceaeJChem.Soc.Perkin Trans.l:2827-2835. 

Anita Kumari ,S and Sree Ram Kumar,N. (1996)Effect of water pollution on the 

biochemical parameters of the selected tissues in Channa striatus 

J.Ecobio1.,8(3):221-227. 

Anon (1955) Aquatic life water quality criteria- First progress report. Aquatic life 

Advisory committee of the Ohio River valley water sanitation commission. 

Sewage lnd. Wastes,27:321-331.

Anuradha,G.(1993) Impact of organophosphate peticides on Labeo rohita, Ph.D 

Thesis ,Osmania University, Hyderabad, India. 

Anusha Amali.A,Cyril Arun Kumar.L ,Elizabeth Jayanthi (1996) Quinolphos induced 

biochemical anomalies in Cirrrhnus mrigala JEnviron.Bzol.l7(2): 121-124. 

Aoki ,K.(1978) Effects of cadmium on embryos and fry of the medaka, Oryziar 

latipes.Zoo1 Mag.,87:YI-97 

APHA(1989) Standard methods for exammation of water and waste water(revised 

edition) publ,67,American Water works Association Water Pollution Control 

Federation ,Washington DC,USA,1268. 

APHA(1998) Standard methods for examination of water and waste water.20~ edition. 

American Public Health Association, New York. 

Areechon ,N. and PIumb,J.A. (1990) Sublethal effects of malathion on Icfalurus 

punctattrs. Bull. Environ. Conlam. Toxicol., 44:435-442. 

Areecbon and Plumb J.A (1990)Sublethal effects of Ictal~rru~ lnmclatus. Bull Environ. 

Conlam. Toxicol.44:435-442. 

Arora (1971) Bioassay studies of some commercial organic pesticides with an Exotic carp. 

Indian J.Envir Hlfh.13.226-233. 

Arun shanker Narain ,Anil kumar Srivastava and Braj Bushan Singh(1990) Gill lesions 

in Perch, Anabas lestudineus subjected to sewage toxicity.Bul1. 

Envir.Confam. Toxrcol.45:235-242. 

Azariah J,Azariah.H,Mnllikesan.Sumathi,M.V,Sunderraj.C(1988 ) Toxicology of plant 

Calorroprs giganfean. Toxicon.26(1): 15. 

Baatrup E.(1990) Structural and functional effects of heavy metals on nervous 

system including sense organs of fish.Comp.Biochem.Physiol.,IOOC(12):237- 

257. 

Bachelard, H.S(l976) 1n:Brain ~iochemistry,3~ Edition-Cambridge University 

Press. 

Bagyalaxmi A. and Ramamurty R(1980)Recovery of AchE activity from 

fenitrothion induced inhibition in fresh water field crab. Bull. Env. Contam. 

Toxicol., 24:866-869. 

Bakthavathasalam R(1991) Haematology of Anabas testud~neus exposed to lindane and 

carbofuran.Env.Ecol.,9(1)124-127.

Bansal S.K.and Desaiah,D.( 1982) Chlorodocone toxic~ty:Effect of withdrawl on 

ATPase inhibition.Fed.Proc.41: 1582. 

Basak ,P.K and S.K.Konar (1977) A new method for determrnation of safe concentration 

of insecticides to protect fishcs Indian J.Envir Hlrh 19.283-293 

Bell ,G.H., Davidson,J.N. and Smith,D.E.(1970) Text book of physiology and 

biochemistry ,8Ih ed., Pub:Churchill,London. 

Bengeri ,K.V and Patil,H.S.( 1987) Lead induced histopathalogical changes in the 

liver of Puntius aurilius .J.Animal.Morpho Phjarol..33(1/2):147-150. 

Bengeri .K.V ,Shivraj.K.M and Patil.H.S(l984) Toxicity of drmethyl parathion to Labeo 

rohita and oxygen uptake rate. Envi.Eco1.2O:l-4. 

Berger ,D.,Vischer,T.L and Micheli,A( 1983) Inrroduction of proteolytic activity in 

serum by treatment with anion in detergents and organic solvents 

Experent~a, 30: 1109-1110. 

Bessey,O.A.,Lowry,O.H and Brock,M.S(1946) A method for rapid determination 

of alkaline Phosphatase with five cubic m~llimeters of serum. J Biol. Chem., 

164:321-330. 

Bhaktavathasalam (1987) Protein metabol~sm durir~g disystron 

lestudineus .Pollution Research.6:l-4. 

exposure to Anabas 

Bhaktavathsalam and Srinivasa Reddy,M.R(1983) Changes in bimodal oxygen 

Uptake in Anabas testudineus exposed to lindane. Water Research.,/7:1221- 

1226. 

Bharathi,M.(ZOOS) The impact of biopesticide prepared from Calotropis gigantea on 

fish Anabac testudineus(Bloch), M.Phil Thesis-Pondicheny Univeaity, 

Puducheny, India. 

Bhataeharaya ,S. and Mukherjee,K. (1976)Activity of hepatopancreatic protease and 

esterase in Fish exposed to industrial pollutants,Comp.Physio.E~01.,1,45-56. 

Bhattachary(l985) Toxicity of Carbobran and Phehthoate on Channa punctatus and 

Anabas testudineus.J.Env Biol, 6:129-13 7. 

Bhattacharya S.,Mukerjee,S, and Bhattacharya S (1975) Toxic effect of endrin 

on hepatopancreas of Clarias batracus ,Linn.lndian J. Expertl.Bio1.. l3:185- 

186.

Bignani A. and Palladini,G(1966) Experimentally produced cerebral status 

spongiosus and continuous pseudorhythmic electroencephalographic 

discharges with a membrane-ATPase inhibitor In rat. A'ature 209:413. 

Bimher,O.L.,Boeing,R.W and Sharma,M.L, (1976) Resp~ratory stress in yellow perch 

induced by diquat .OhioJ,Sci.76(2).87-90. 

Blaxhall P.C and Daisley(1973) Routine heamatological methods for use with fish 

blood.J.Fish Bml.,R, 771-781 

Boyd,W.(1949) A text book of pathology .Pub. Lea and Fabier,Philadelphia.1464. 

Boyer,D.D., Chanda,B., Ernester,L. Mitchel,P.,Racker,E. and Slater,E.C (1977). 

Oxidative phosphorylation and photophosphorylation. Ann. Rev. Biochem 

46.955-1026. 

Bradbury,S.P,Mc Kuin,J.M. and Coats,J.R(1987) Physiological response of 

raibow trout to active fenvalerate in toxicat~on .Pesticide 

Biochem.Physiol.,2 7.2 75-288. 

Buikema,A.L, Niederlehnar, B.R and CairnsJ(1982) Biological Monitoring:part 

iv.Toxicity testing. WaterRes., 16:239-262. 

Bul1.C.J and Me lnerney .J.E( 1974) Behavior of juvenile Coho salmon exposed to 

sur~11thionJ.Res.Bd.Can.31:1867-1872. 

Butler ,P.J, Taylor,E.W.,Capra,M.F,and Dandson.( 1978) The effect of hpoxia 

on the level of circulating catacholamines in Scylivrhinus canrcula. J 

Comp.Physiol., 127:325-330. 

Cassilas E. and Smith,L.S(1977) Effect of stress on blood coagulation and 

hematology in rain bow troutJFishBiol.,10:481-491. 

Castell C.H.,Smith,B. and Neal. (1970) Effects of transition metal ions on the 

extractable proteins of fish muscle J.Fish.Res.Bd.Can..27:701-714. 

Chakrabarthy G. and Konar,S.K (1974) Chronic effects of pesticides on fish . 

Proc. Natl. Acad,Sci..446:241-246.

Chowdhary B.P.,Pandy,P.K and Dubey, N.K(l981) Effect of malathion on the 

behavior and body composition of Heleroneusles fossilis. J. Envtron. Btol., 

2(1).21-36. 

Cope .O.B, McCraren d.P and Eller.L(l969) Effects of dichloben on two fish pond s 

Weedscience, 17/2).158-165. 

Cope .O.D, Waod.W.M, Wallen(1970) Some chronic effects of 2,4-D on the blue 

gills Trans Amer.Fish.Soc,99:1-12. 

Coppage D.L and Matthews .E(1974) Short term effects of organophosphates 

pesticides on cholinesterase of estuarine fishes and pink 

shrimp.Bull.env.Confam Toxicol.,l1:483-488. 

Corbeit,J.R(1974) Insecticides inhibiting acetyl cholinesterase.ln:Biochemical mode of 

action.Academic press,New York.107-164. 

Cox (1974) Marine bioassays work shops proceedings 1974,American petroleum institute, 

EPA,Manne Tech. Society, Washington DC. 

Cox ,R.P and Griffin,M.J( 1965) Ppophosphate activity of mammalian alkaline 

phosphatase .Lancef,2:10IR. 

Cutkomp .L.K,Yap,H.H,Ves.E.V and Koch( 1971) Inhibition of Oligomycin 

sensitive (Mitochondria)Mg++ ATPase by DDT and selected analogs in fish 

and insect tissues Life Sci. 10.1201-1209. 

CWQC(1972)A report of the committee of Water Quality Criteria. Ecological 

Research Series .EPA-R3-73-003. 

Dalela, RC.,Bhatnagnr,M.C,Tyagi and Verma(1978) ATPase activ~ty in few 

tissues of Channa gachua following in viva exposure to endosulfan 

.Toxicology 11:361-368. 

Dalela RC,Ravi.S, and Verma(1980) Physiological stress induced by sub lethal conc. of 

Phenol and Pentachlorophenol in Noioptems.Env.Pollu.2l:3-8. 

Dalela.RC, Bhatnagar,M.C and Verma,S.R(1981) Effects of in viva 

haernatological alteration in Mysrw vitfarus following subacute to pesticide 

and their combinations,Envir.PoNu.21:3-8. 

Danielli, J.F(l972)Structural factors in cell permeability and secretion. 

Symp.Soc.Exp.Biol.,6.1-15.

De Duve, C.(1963) Intracellulw distributive pattern of enzymes.In "Lysosomes"De 

Reuck A.V.S.,Gamenran.M.P(Eds),Churchill Ltd.,London. 

Decie J.V and Lewis,S.M(1963) Practical Heamatology: J&A Ltd., Churchill ., 

London. 

Dehadrai. Vand Mukhapadhaya,P.K (1979) Fish culture in paddy 

tields.lndFarm Dig.. 12(6)21-24 

Dehadrai P.V and Tripathi S.D( 1976) Environment and ecology of fresh water air 

breathing teleosts .In Respiration of amphibious Vertebrates, G.H .Huges 

(ed).,Acadernic press,London.39-72. 

Desai ,A.K and Joshi U.M(l985) Histoiog~cal observation of Tilapia mossambica after 

exposure to monocrotophos.Toxicol.Le11er,21:325-331. 

Desai,A.K.,Joshi,U.M and Ambedkar,P.M(1984) Histological observation if liver 

of Tilapia mossambica exposure to monocrotophos.ToxicoI Letrer.,21.325- 

331. 

Desaiah ,D., H0,l.K and Mahendale,H.M( 1979) Possible molecular mechanisms 

of mirex and kepone induced hepatobiliary disfunctions. 1n:Environmental 

hiology (Eds),The Academy of Environmental biology, Muzaffamagar, 

India.113.131. 

Desaiah D.(1982)Biochemical mechanism of chioro decone neurotoxiclty: A review. 

Neuroioxicology 3:103-110. 

Desaiah,D.(1981) Interaction of chlordecone with biological membranes. 

J. ToxicolEnvir.HeaNh. 8: 719- 730. 

Desaiah,D.(1982) Biochemical parameters of chlorodocone on 

neurotoxicity:Areview,Neurotoxicologv. 3: 103-110. 

DevarajKV, Krlsnemurthy.RV, Srikanth(l990) Progress in pollution research,Seminar 

on environmental pollution ,Agricultural sciences,Bangalore. 

Dewan .S, Songrau1a.H and Kumar(2000) Preliminary studies on the Analgesic activity of 

Calolropisprocera .J.Ethano Pharm. 73(1-2):307-311. 

Dhanapakiam and Juliet Premalatha( 1994) Histopathological changes in kidney of 

qprinu carpio exposed to malathion and sevin. JEnv.Bio1.. l5(4)283-287.

Di Michele .L and Taylor,M.H (1978) Histopathological and physiological 

responses of Fundulur heleroclitus on naphthalene exposure. J.Fish Res. Ed, 

Can.. 35:1060-1066. 

Dixitulu J.V.H.and Paparao (1994), Hand book of fishenes ,ICAR.,lndia 

Down to Earth(oct-ZOO6)Fact file,Monthly Sc~ence Magazine(lnd1a) pg-56. 

Dutta H.M, Datta munsbl.J.S,Roy.P.K,Singh(1992a) Variation in toxic~ty of malathion to 

air and water breathing teleosts. Bull. Envir.Contum.Toxicol.4Y.27Y-R4 

Dutta H.M, Doger.J.V.V, Singh.N.K, Roy(1992c) Malathion induced changes in serum 

protein and haematological parameters of ffeferoneustes fossilis Bull. 

Envir. Conram. Toxicol,49:91-97. 

Dutta H.M, Naser.S.S.T, Munshi.J.S.D and Richmonds.C ( 1994) Behavioural changes 

in Anabos lesrudineus exposed to malathion. Bull. Envir Contom. Tox1col.52:RO-86. 

Dutta M.M,Naser,S.S.T ,Munshi J.S.D ,Richmonds(l992b) Malathion induced changes 

in optomotor behavior in lndian carp,Labeo rohita Bull. 

Envir Contam Toxico1,49:552-568. 

Dwivedi H.R and Rajakamal Sarin(1996) Histological changes in the liver of 

Heferopneustes fossilis induced by Triaromatic hydrocarbons. Eco~oxicol. 

Environ. Monit, 6(4):2 73-278 

Eisler,R.,Gardner,G.R, Hennekey,La Roche,Walsh(l972) Acute toxicity of NTC 

and NTC detergents to marine organisms. Wafer.Res. 6:1009-1027. 

El-Doimaty,N.A (1987) Stress response of juvenile Clarias larva elicited by 

copper.Comp.Biochem.Physiol.,88C:259-262. 

El-Elaimy I.A,ELSadamy,M.M Cabr S.A and Sahr( 1990) Ultra structural alterations of 

intestine of Tilapia nilotica exposed to diazinon and neopybuthrin, 

Eller ,L.C(1971) Histopathological lesions in Salmo clarki exposed chronically to 

endrin.4rn.J Patho.,64:321. 

Eller, L.L(l975) Gill lesions in fresh water teleosts. In Ribelin E and M Igaki 

G.(Eds). The Pathology of fishes .Univ.of Winconsin Press,Madison.305-330. 

Ellgaard.E.G,Tusa.J.E,Maliuia(l978) Locomotor activity in the blue gill,sun fish 

by sub lethal conc.of cadmium,chromium and zinc LFish.Bio1.. 1:lY-23.

Ellman G.L. Courtney,K.D, Andres V, and Feather stone (1961) A new and rapid 

Calorimetric determination of Acetyl Cholinesterase activity. Biochemical 

Pharmacolog)~, 7:88-95. 

Eagerland, U.H.M.(1967) Plasma cortisol concentratton in relation to stress in adult 

sockeye salmon .Gen.Comp.Endocrin 8,197-207. 

Fernley H.N and Walker,P.G(1971) Acid and Alkaline Phosphatases as inducible 

enzymes.Biochem.J, 104: 1071. 

Ferrando, M.D and Adrew Moliner, E(1991) Acute lethal toxicity of some 

pesticides to Branchionus calyciflonts and Branchion~~splicafilis. Bull. Env. 

Contam. Toxico1.47:479-484. 

Finney D.J(l981)Probit Analysis.Cambridge University Press,Cambridge.333 

Fisher R.A and Yates,T(1948) 1n:Staistical tables for biological, agriculrural and medical 

research, Oliver and Boyd. London. 

Frank, C.Lu. (1985) Toxic effects, Basic Tox~cology ,Fundamentals, Target organs and 

Risk Assesment.40-53. 

Frank,C.Lu.(l985)Toxicology of Brain.Baslc Toxicology,Fundarnentals,Target organs and 

Rrsk Assesment.185-195. 

Fry F.E.J(l971) 1n:Fish Physiology.W.S.Hoar and D.J.Randal(eds),Academic press 

Vol:6,P.l.New York. 

Gardner, G.R and La Roche,G(1973)Copper induced lesions 

teleostsJ.Fish.Res.Bd.Can., 30.363-368. 

In estuarine 

Gill T.S,Pande.J, and Tewari (1991,b) Haemo pathological changes associated with 

aldicarb poisoning in fish. . Bull. Envir.Conrarn.Toxico1,47(4)628-633. 

GiU ,S.Tejendra,S.,Pande,J. and Tewari,H (1990) Enzyme modulation by 

Sublethal concentrations of aldicrab, phosphomidon and endosulfan in fish 

tissues.Pestec.Biochem. Physiol..38(3):231-224. 

Gill T.S, Pande.J, and Tewari(l99la) Effects of endosulfan and phosphomidon poisoning 

on the peripheral blood of Barbus conchoniiis.J.Env.Sci.Heal!h.26(2):249-255. 

Goe1,K.A and Agrawal,P(1977) Histopathological effects of alloxan treatment on 

liver and kidney in Clarias batracus. All India seminar in Icthyology,Meerut 

University ,Meerut.pp:3

Goldiisher,S. Osser,E. and Novikoff,A.B.(1964)1n :Use of histological and 

histochemical assessment in prognosisof the effects of aquatic pollutants . 

Kendal1,M.W and Silver,B.B(Eds)dmer.Soc.Test.Mat.,Philadelphia,pp 194- 

208. 

Gonzalezcalivin .J.L.,Saunders,J.B and Williams,R.C( 1983) Effects of ethanol 

and acetaldehyde on hepatic plasma membrane ATPase. 

Biochemical.Pharmacol.32(11/: 1723-1 728. 

Gordon,A.S.,Cooper,G.W.and Zanjani,E.D(1967) The kidney and 

Erythropoiesis.Sem.Haema~ .4:337-343. 

Gray,RW and Meckenzie,J.A (1970) Muscle protein electrophoresis in Salmon of 

East CanadaJ.Fish.Res.Bd. Can..27:2109-2112. 

Gurdeep Chahval (l981), Organic Chernisr~ oJNatural Prodrrcts , Himalaya Publishing 

House.Mumbai,india,Vol. I: I I I. 

Harington, J.S, Miller,K. and Mac Nab,G.( 1971) Environ Res.,4:95-117. 

Harington J.S,Miller.K,Macnab( 1971) Environmen~al research.,4:95-117. 

Harper, H.A.(1991) Harper's Review of Biochemistry. Murrzy R.K. Mayes 

P.A.Granner , D.K and Rodeel V.W(eds)Lange Medical Publ,London,pp:400- 

458. 

Hattield land C.T and Andenon(l972) Effects of two insecticides solmon par to learn 

and retain a simple conditioned reflexJ.Fivh.Res.BdCan..29:315-321. 

Heath A.G(l987) Behavior and nervous system function in water pollution and fish 

physiology.RC press.lnc Boca Raton,Florida,l81-196. 

Henderson ,C.Pickering,Q.H and Tarnve(1959) Relative toxicity of ten 

chlorinated hydrocarbon insecticides to four species of fish .Trans 

Am.Fish.Soc.,88:22-23 

Herbert ,D.W.M (1962) A preliminary shldy of the effect of physiological activity on 

resistance of rainbow trout to two poisonsAnn.Appl.Biol.52,321-326. 

Hickenbottom,J.P and Yau,E.T.( 1974) Effects of DDT on liver glycogen 

metabolism in rats.Pharmacologist. 16:35.

Holden ,A.V(1972) The Effects of pesticides on life in fresh waters.Proc.LR 

Soc.Lond.B,180,383-394. 

Holden A.V(l973) Effects of pesticides on fish.ln: Env pollut~on by pesticides. C.A 

Edwards(ed),Plenum Press,London.Z 13-249. 

Huges G.Mand Nemcsok J.(1988)Effect of low pH alone and combined copper 

sulphate on blood parameters of rain bowtrout.Env.Pollu..55 89-95. 

Huges and Singh (1970) Respirat~on in Anabas restudineus,oxygen uptake and 

carbondioxide release into air and water.J.Exp.Biol.53:265-2R0. 

Hughes ,G.M and Morgan .M (1973) The structure of fish gills in relation to their 

respiratory functions.BioI.Rev.,48:41Y-475. 

Humason,G.L,( 

Francisco. 

1972) Animal tissue Techniques.W H Fremand and Co San 

Ibrahim ,A.M Gigazi,M.G and Deimaian,E.S(1974) Histochemlcal localization of 

alkaline phosphatase activity in alimentary tract of Mariso carnuanctus(Lj 

Zool.Soc.Egyp1.Bull.,26:94-105. 

Ibrahim I.G,Mohsmad.A.M,Zaki.ZT (1995) Changes induced by paraquat herbicide in 

muscle protein and blood of two fishes from EgyptAl-AzarBul1.S c1..6(1)637-647. 

Ikhstair-Ud-Din K,Hafeez and Mohmad Abdul (1996) Eifect of malathion on blood of 

ryprinion.Pak.JZool.,28(1):45-49. 

Ito,H.(197O)Fine structure of carp tectum opticumJ.Fur.Himforschung.12(4-5):325- 

354. 

lyatomi .K,Tsnura.T,ltazawa( 1958) Toxicity of endnn to fish.Proc.Fish.Cul.20:155-162. 

Jacobsen,L.D. and KrantzS.B(l968) Summary on 

erythropoietin,Am.Ny.Ac.Sci., 149;578-583. 

Jagid and Bhstt(2OOS) Antiseptic value of some ethno botanical plants of 

Gujaratddv.Biol.Sci. Vo1.4:91-93. 

Jana ,S.nnd Bandyopadhyaya, S. .(1987) Effect of metals on some biochemical parameters 

in Channopunc~atus,Env .Eco1.,5,488-493. 

Janardhan,A. and Sisodia,P (1990) Monocrotophos :Short term toxicity in 

rats.Bull.Environ.Contaminar. Toxicol..44:230-239.

Johanson,D.W.( 1968) Pesticides and Fishes-A Review Traans.Arn.Fish.Soc..97(4),398-427. 

Johansson-Sjobeck and Larson(1978) Effect of cadmium on haematology 

haemopoeitic tissue of Pleuronecfesf[uses.L.Environ.Res. 17:191-204. 


Jones Nelson and Sunil Kumar(1996) Effects of ekalux on biochemical parameters 

in Etroplus maculates.J.Ecotoxicol.Environ.Monit.,6(1):6.(-67. 

Joshi ,P.K and Dubey,G.K (1984) Histopathological lesions in brain(optic 

tectum)of Oxygasler bacaila exposed to industrial effluent. Proc. Sem. Ejf 

Pest.Aq. Fau., 45-49. 

Joshi ,lJ.M. and Desai A.K,(1988) Biochemical changes in the liver of T~lapia 

mossarnbica during continuous exposure to monocrotophos. Ecotoxicol. Env. 

Saj, 15:272-276. 

Joshi ,U.Z. and Desai,A.K.(1983) Effects of monocrotophos on some phosphatases 

and ATPases in the brain of Tilapia mossambica ' Anim. Morpho 

Physiol..30(1-2):201-207. 

Joshi U.M and Desai A.K(l983) Biochemical changes in her of Tilapia mossambica 

dunng exposure to monochrotophos.Ecofoxicol.En~,. 15:272-276. 

Josphene Paulina,C.(2003) The toxic effect of latex cf Calotropis gigantea and its 

recovery with additive nutrients on Anabar testudineus (Bloch). M.Phil.thesis, 

Pondicheny University ,Puducheny,lndia. 

Jyotsna Shrivastava and Srivastava (1984) Histopathology of gills in Channa gachua 

exposed to sub lethal conc. of malathion and chlordane.Proc.Sem Ef Pest. Aq. 

Fau.,37-44. 

Kabir Ahmed ,I.,Sailatha,D. and Ramana Rao,K.V(1980) Impact of malathion on 

Acetyl cholinesterase in Tilapia rnossambica.J,Biosci.2:37-41. 

Kabir Ahmed ,I.,Sambasiva Ra0,K.R.S and Ramana Rao K.V(l983) 

Dehydrogenase system of Tilapia mossarnbica under Sublethal malathion 

stress J.Anim. Morphol. Physiol.30:lOl-106. 

Karapagaganapathy .P.R and Sukumar.A(l988) Effects of carbofuran on the histology of 

gill and liver of Colis lalia. J.Annamalai Univ,Parf-B.34:51-58 . 

Kartikar and Basu(1984) hdian medicinal plants ,Val-3,Lalit Mohan Basu 

publ,India..1606-1611.

Katz .M.,Pederson, G.L,Yoshinaka,M and Sjolseth,D(1969) Effects of pollution 

on fish 1ife.J. Water Pollu. Contro.Feder 4 1:994. 

Kennedy H.D ,Eller L.L, and Walsh(1970) Chronic effects of methoxychlor on blue g~lls 

and Aquatic inveriebrates.US.Bur.Spori.F~sh. Wild Tech.p.53. 

Keshavan,R and Kamble,S.M(1984) Effect of Hildane on ac~d and alkaline 

phosphastase activity in hepatopancreas and muscle of fresh water crab 

Barytelphusa guirina.J.Curr.Bloscl, 1.145-148. 

Khare,C.P(ZOO4) Encyclopedia of Ind~an Medicinal Plants, Springer 

Publications,lndia.,120-121. 

King, S.F,(1962)Some effects on DDT on guppy and brown trout.US 

Fish. Wild.Sew.,Serv.Spec.Sci Rep.,399-1-20 

Kiuchl .F, Fukao.Y, Maruyama, T(1998) Cytotoxic principles of a Bangladeshi crude 

drug, akondmul (Caluiropis giganiea).Chem. Pliarm.Bu11,46.528-530 

Klicpera .M, Droahota .Z and ZakR(197S)Notes on the determination of muscle 

glycogen.Physiol.Bohemos1iov.. 6.569. 

Knight .A.P and Walter .K.G(ZOOt) Flants affecting Cardio-vascular system. A guide to 

plant poisoning in Animals in N. America ,International Info. Sew. New York. 

Koelle,G.B.(1975)Anticholinesterase agents.ln:The Phanna cological basis of 

Therapeutics(eds.L.S.Good man and A.Gilman).Macmilllan pub.Inc.,New 

York. 

Komissarenko ,A.N, (1997)., Kasfit .Resur, 33.29-41 

Koundinya P.R and Ramamurthi,R (1979) Effect of sumithion on some aspects of 

carbohydrate metabolism in Sarotherodon mossambica.Experimentia.3:1632- 

1633. 

Kulakkatolickal 4.T. and Karmer,D.L(1988) The role of air breathing in the 

resistance on bimodally respiring fish to waterborne toxins 

J.Fish.Bio1.,32:119-127. 

Kulshrestha S.K and Lakbmi Jauhar (1984) Effects of sublethal dose of thiodan and 

sevin on liver of Channa stroiatus.Proc.Sem.EffPest.Aq.Fau.. 71-78. 

Kumar .S and Pant .S.C (1984) Organa1 damage caused by aldicarbto Borbus 

chonchonirus. Bull. Envir.Conlam. Toxico1..33:50-55.

Larhsini .M,Markoak.M, Jaouhari.J.T (2OOZ)The anti pyretic activity of some Moroccan 

medicinal plants,Ph.ylotherapy Research. 16(1):397-398. 

Larson,A.(1973) Metabolic effects of epinephrine and nor epinephrine in 

eel.Gen. Comp.Endocrin.20:155-167. 

Laura Martinez-Tabche,BeatrizEstrada.M and Igncio(1992) Parathion and salinity 

effect on gills on Oreochromis hornorun Bt(lLEnv.Conf. Toxico1..49:929-934. 

Lemke.E and Mount .J( 1963) Some effects of alkyl bezenesulfonate on blue 

gill.Trans.Amer.Fish.Soc.,92:372-378. 

Love,R.M(1980).The chemical biology of fishes. Acad.Press.Inc(London)Ltd 

Lowry,O.H.,Rosenbough,N.J,Farr~.L and Randall RI(I951)Protein 

measurement with Folin phenol reagent JBiol.Chem. 193:265-275. 

Macek K.J, Lindberg.M .A, Santers bucton( 1976) Toxicity to fcur water fleas and fat 

head minnows to heptachlor, acrolein, endosulfan and bifluralin.EPA 600.73-76-009 

US EPA. 

Macmillan S.J(l987)Effects of sublethal conc. of alachlor and atrazine on the 

optomotor responses in fat head minnows.Masier thesis,Kent state university, 

Kent, Ohio. 

Macnab and Harrington(l967) Nanire (London),214:522-523. 

Mahajan,RT and Patole S.S(Z003) Effect of plant extracts on oxygen consumption rate 

in fish: Nemacheilus evezardi.J.Fresh water Biol.l5(1-4):109-113. 

Mahendra,V.K, Agarwal,RA(1983) Phorate and mexacarbate induced changes in 

enzymes of snailJymanaea acurninafe.Arch.Env.Contam.Toxicol.,l2: 77-82. 

March,RB, Fukut0,T.h Metcalf, R.L and Maron(1956) Fate of P-32 labeled 

malathion in laying hen, white mouse and American cockroach. J. Econ. 

Enromo1.,49:185-195. 

Martin,D.W ,Mayes,P.A and Rodwell,V.W(1981) Review of Biochemistry 18'~ 

ed.lange Medical Publ.California. 

Martin.A.J.P,Istvan balsz,Heinz(1979) Flip-Flop Chromatography-Recovery of heart 

glycoside (uscharin)of Calorropis gigantea.JChromarography, 186: 15-24.

Marzella,L.,Allbug,J. and Glanmaun,H(198l) Autophagy ,hepatophagy and 

rnicrophagy as the means for intracellular degradation. Virchows. Arch. B. 

CellPathol.. 36:219-234. 

Mathur,D.S(1965) Histopathological changes induced by DDT in liver ,kidney and 

intestine of certain fishes .Expermentin, 18:506-509. 

Mazeaud,M.M, Mazeaud ,F and Donaldson .E( 1977) Primary and secondary 

effects of stress in fish: Some new data with a general 

review.Trans.Amer.Fish.Soc. 106:201-212. 

Mishra ,J and Srivastava,A.K(1983) Malathion induced haematological changes in 

Clariar batracus exposed to malathion .Env.Pollu.Ser.Ecol.Biol.,22:I49-158. 

Mitchel,P(1961) Coupling of phosphorylation by a chemiosmotic type of 

mechanism. Noture(Londonj, I91 :l44-148. 

Moore M.N and Clarke, K.R (1982) Use of macrosterology and qualitative 

cytochemistry to determine the effects of crude oil derived aromatic 

hydrocarbons on lysosomal shucture and functions in Mytilus 

edilis.Hislochem.J.14: 713-718. 

Morta P,Vargese.A.M and Sanchaz(1982) Evolution of gluconeogenlc enzyme activities 

during starvation in liver and kidney of rainbow trout.Comp.Biochern.Phvsio(71:65- 

70. 

Muses Girija and Jayantha Rao( 1995) Histopathological changes induced by hepatochlor 

in gills of fresh water fish Tilapia mossambica.Env.Ecol,3(~):74-76. 

Moss P.W., Eaton,R.M, Smith .J.M and Whitney( 1967)Sources of Alkaline 

Phosphatase.Biochem.J. 102:53. 

Mount ,D.I and Putnicki,G.J(1966) Summary report of 1963-Mississipi river fish 

kill.Trans.31 st NAmer. Wild.LJConJ. 177:31. 

Mount 9.1. and Stephen,C.E.(1967)A method for establishing acceptable toxicant 

toxicant limit for fish malathion and hutoxy ethanol ester of 2,4- 

D.Trans.Amer.Fish.S0~.,96:185-193. 

M0ursy.L.E (1997) Insecticidal activity of Calotropis procera extracts of flash fly. 

/.Egyptian Society ofParasitologv27(2)505-514.

Mukbopadbyay and Dehadrai (1979) Fish culture in paddy fields.lnd.Farm.Dig.,12(6):21- 

24. 

Mukhopadyay ,P.K and Dehadrai P.V,(1980)BiochemicaI changes in C.barracus 

exposed to malathion Envir. Po/lu.(Sereis A)22:149-158, 

Mullainathan,P(1982) Studies on the clearance of foreign substances from the 

haemolymph of Scylla serrafa ,Ph.D.Thesis,Univ of Madras,ppl71. 

Murphy and Porter .S(1966) Effects of some toxic chemicals on some adaptive 

liver enzymes,glycogen and blood glucose in fasted rats 

.Biochem.Pharm, 15:1665-1676. 

Murphy S.D,Laywerys,R.R and Cheever,K.L(1968) Comparative anti choline 

esterase of organophosphorus insecticides in vertebrates. Toxicol. Appl. 

Pharmacol. 12:22-35. 

Nadkaroi K.M(l991) Indian materia medica,Popular Book Depot,Bombay,India 

Nakano T.and Tomlinson,N(1967) Catacholamines and carbohydrate conc. in 

rainbow trout in relation to physical disturbance. J.Fish Res.Bd.Can.24:1701- 

1715. 

Nammalwar,P.(1984) Biochemical changes in hioassays of pesticides DDT and 

BHC to Liza rnacrolepis.Proc.Sem. EffPesl.Aq.Fau.. 139-145. 

Natb Rabindra and Banerjee (1996) Effect of methyl parathion and cypermethrin on 

Heteropneurtes fossilis .Env.Ecol. 14(1):163-165. 

Niimi AJ(1990) Review of biological methods and other indicators to assess fish 

health in aquatic ecosystenis containing toxic chemicals. J.Great Lakes 

Res.,16:529-541. 

Nowk and Barbara (1992)Histological changes in gills of fishes induced by 

residues of endosulfan Aqua.Toxi..23(1):65-83. 

O'Brein, R.D.(1967)ln:lnsectiside 

York. 

action and metabolism.,Academic Press, New 

O'Brien,R.D.,Hetnarski,B,Tripathi,R.K and Hart,A.J.(1974) Recent studies on 

acetylcholinesterase inhibition.1n:Mechanisms of pesticide 

action.Kohn,A.K(Ed),Amer.Chem.Soc.,Washinon DC,PPI -13.

Olla B.I,Pearson W.H. and Standholme(l980) Applicability of behavioral measures in 

environmental stress assessment.Rupp.Pv.Revn. Cons.lnt.ExporMer. 179:162- 173. 

Ostle,B(1966) In: Statistics and Research, Ostle.B(ed),Oxford and IBH Publicatio~ New 

Delhi. 

Pandey Asha, (1987) Some aspects of ecophysiology of Anabus restudineus. Ph.D.thesis 

Bhagalpur University, India. 

Patil V.T, Shinde S.V,and Kulkarni(l990) Biochemical changes induced by 

monocrotophos on mudskipper.Env.Ecol..8(4):1168-I173 

Patole ,S.S and Mahajan,R.T.(ZOOb), Comparative lcthyotox~c activ~ty of some Indigenous 

plants in fish: Lepidocephalichthys, J.Agua.Bio1.. Vo/.21(1),2006:168-172. 

Patole,S.S and Mahajan,R.T (2004) Effect of the aqueous extract of the leaves of 

Cestium noc!urnum(Linn) on Histomorphological changes in of gill of 

fish:Nemacheilus evezardi. J.Ayua.B1ol.,Vol19(2j.2004:135-140. 

Pickering A.S(l981)Stress and Fish. Academic Press,NewYork, 367 

Pickering O.H,Henderson.C, Lemke(1962) Toxicity of organophosphate pesticides to 

different sps. of warm water fishes. nuns Amer.Fish.Sci.,PI:l75-184. 

Pillay T.V.R,(1977),Planning of aquaculture in fishery development and management. 

J.Fi.sh.Res.Board Can.,30(12)2202- 17. 

Pilo ,B., Asnani,M,V and Shah,RV(1972) Studies on wound healing and repair in 

pigeon liver II1,Histochemical studies on acid and alkaline phosphatase 

activities during the processJ.Anim.MorphoI.Physiol. 19:205-212. 

Prasanth,M.S.,David,M and Mathed ,S.G(2005) Behavioral changes in Cirrhinus 

mrigala exposed to cqpermethrin J.Environ.Biol..26(1)141-144. 

Pravakar J.D .,Martin,RE.Jaypat,N.R and Kshemikalyani,S.B (1993) Effect of 

endosulfan and Hexachlorocyclohexane(HCH) on Labeo rohita. J.Ecobiol., 

5(2):149-150. 

Preethakumari,V.M(2004) Role of additive supplements on latex of Calotropis 

gigantea induced toxicity in fish Anabas teshrdinew(Bloch).M.Phil- 

Thesis,Pondicherry University,Puducheny,India.

Pressman,B.C(1963) 1n:Energy linked functions of mitochondria.Chance 

B(ed),Academic press,New York.pp: 188- 191. 

Puviani A.C,Ottolengi .C,Gavioli( 1990) Action of glucogons on glycogen metabolism of 

trout.CompBioChem Physiol.,Y6B:387-391. 

Rama Rao ,S.V.,Padmavathi,B.,SatyaVelu Reddy K and Indira,K.( 1996) Carbon 

tetrachloride induced hepatotoxicity in Sartherodon mossambicus 

Poll.Res.,15(4):385-387. 

Ramalingam, K(1980) Studies on toxic effects of few toxicants on 

biochemistry,physiology and histology of Ttlapiamossambica. Ph.D.,Thesls, 

University of Madras, Madras. 

Ramalingam,K.(1988)Toxic effects of DDT,Malathion and Mercury on the tissue of 

Sarorherodo mosssambinrs.Proc.Indtan.Acad.Sci. 97:443-445. 

Raman(1997) Management of Brackish Water Aquaculture Production Technology, 

Course Manual, ARTC-Central Institute of Fisheries 

Education,Kakinada,India. 

Ramana Rao ,K.V and Ramurthi,R.(1979)Studies on metabolism of Apple snail In 

relation to pesticide impact.Indian .J.Heredity.lI:llO 

Rand G.M (1977 b) Effect of sub acute parathion on the locomotor behavior of blue plll 

sunfish and large mouth bass. In :Aquatic toxicology and hazard 

ev3luation.F.L.Mayer,J.L.Hanclink (eds).ASM STP 634,Philadelphia astm.253-268. 

Rand G.M (1977a) Effect of parathion on general locomotor behavior of the gold fish. 

Bull. Envir.Conlam.Toxicol.,l8:259-274. 

Rao K.J and Murthy V.S.R( 1983) Hypoxia induced hsemopoeitic tissue in Tilapia 

mossabica exposed to phosphomidon.Geo Bios.,10:204-207. 

Rao(1974)The comparative toxicities of organophosphorus and carbarnate pesticides 

in Mahasagar.BulI.Natl.lns.Oceonogr., 7: 79-81. 

Reddy D.C ,Vijayakumari ,Kalarani and Davis( 1992) Changes in elythropoeitic activity 

of S.mossambicus exposed to sublethal conc. of diuron. Bull. 

Envir. Contam. Toxicol., 33:50-55. 

Reddy, M.S and Raa,K.V.R( 1988) Modulation of carbohydrate metabolism in 

Peneus indicus under phosphomidon induced stress. Ecotoxicol Env.Safre@.. 

I5:212-240.

Reeta Pandey .,Saraf,D.K and Bhergarai.S(l991) Variations in serum proteins 

under the stress of BHC in Heteroneusfes,fossilis.Him.J.Env Zool.,S: 75-78 

Reeta Pandey, Sharaf.D.K, Bargarai(l991) Variations in serum proteins to BHC in 

Heteropneustes fossilis.H~m.J,Env.Zooi .5.7.(-78. 

Reichenback Klinke,H.H (1975) Lesions due to drugs .In :Pathology of 

fishes,W.ERibeiin,G.Migake(eds).,Madson,W~sUniv of Wisconsin 

Press,pp:647-656. 

RichardsonRJ(1981) Toxicology of nervous systen1,In: Toxicology,Pncipies ,and 

Practice,A.L Reeves(ed),Wiley Inter science -Pub.,Toranto. 

Richmonds .C and Dutta.H(l992) Effect of malathion on the optomotor behavior 

of blue gill sunfish.Comp.Biochem.Physiol., 102C:523-526. 

Richmonds and Duma (1992) Effect of malathion on optomotor behav~or in blue gill 

Comp.Biochem. Physio. 102:523-526. 

Robert F.Raffauf , (1970 )A hand book of Alkaloids and alkaloid containing plants, Wiley 

-inter science. PlewYork. 

Roberts, RJ(1978) The pathophysiology and systemic pathology of teleosts.ln:Fish 

pathology, Baillera tindal1,London ,pp:55-91. 

Rooj M2,Ojha.J. and Datta Munshi(l984) Effect of certain biocidal plant sap on gills of 

certain Hillstream fishes of Ranchi (Lndia).Biol.ofIndia,6:320-322. 

Roy P.K and Munshi J.S.D(l987) Toxicity of malathion on Cirrhinus mrigala. Bid. Bull. 

India.. 9(1):50-56. 

Rey P.K, Datta Munshi and Jayashree(l986) Scanning electron microscopic evaluation 

of effect of saponin on gills of Anabas testudineus. 1nd.J. of Expti Biol.Voi.24:511- 

516. 

Roy,P.K and Datta MunshiJ.S(l991) Malathion induced structural and 

morphometric changes of gills of Cirrhinus mrigaia.J.Environ.Biol.,I2(1):79- 

87. 

Sabita Borah and Yadav,RN.S (1996) Effect of rogor on LDH,acid and alkaline 

phosphatase in muscle of Heteropneusles fossilis.J.Environ.Biol.,17(4)~279- 

283.

Sadhu,D.N(1993)Toxicity of monocil to Channa punctarus. J Ecotox~col. 

EnvironMonit.. 3(2):133-135. 

Sajitha Bhaskar(l994) Toxicological evaluation of endosulfan in Anabas resrttdtnerts 

.M.phil thesis Univ of Pondicheny 

Sandhu H.S and Malik J.K(l988b) Acute toxic~ty of monocrotophos and its ~nfluence on 

blood enzymes of buffalo calves. Ind.JAnim.Sci.61(5):530-531 

Sandhu H.S and Malik J.K( 1988a) Sub acute toxlcity of monocrotophos and its ~nfluence 

on circulating enzymes of Bubalu.Pharmacol and Ti~xico1.,62:290-292. 

Sandhu H.S, Brar.RS, and Gupta.R( 1991) Acute toxicity and anticholinesterase effecr of 

monocrotophos in domestic fowls .Ind J.Anim.Sci.,61(5):530-531. 

Santha kumar ,M(1998) Studies on toxicological effects of monocrotophos in an air 

breathing fish Anabas tesrudineus(B1och) under Sublethal exposure.M.Phi1- 

Thesis,Pondcherry University,Puducheny,India. 

Sastry K.V and Melik P.V(l979) Studies on effect of demacron on digestive system of 

channapunc!arus.Env. Conlam. Toxtco1,8:397-398. 

Sastry K.V.,Agarwal,V.P.,and Sharma,S.K (1977) Histopathological effects of 

methyl parathion on liver and kidney of Hereropneurtes fossilis.All India 

seminar on lcthyology Meemt University,Meemt.pp:S. 

Sastry,K.V. and Sharma ,S.K.( 1978)In vivo effect of endrin phosphatases in 

kidney and liver of Ophiocephalrrs punctottts. Bull. Env Conram. Tox~col. 

21:185. 

Satheesh Kumar Reddy,C (1994) Toxicity an physiological impact on endosulfan on 

Anabm resrudineus. Mphil thesis Univ. of Pondicheny. 

Savita and Anandhi. P, (2006), Agrobios, Vol. 5 issue. 06 :19. 

Scherer .E and Harrision S.E( 1979) The optomotor response: Toxicity tests for fresh 

water organisms.Canadian special publ. of fisheries sciences. 

Schurmans.,Stekhovan F.M.A.H and Bonting, ,S.L(1981)Sodium Potassium 

activated Triphosphatase.1n:Membrane transport,Eds.,ElsevierMorth Holland 

Biomedical Press,Amsterdam,ppl59-182. 

Sen .T, Basu. and Chaudari(l998)S~dies on the possible mechanism of gastric mucosal 

protection by Calotropis procera. Fundamental and clinical Pharmacolo~i20):82- 

87.

Sharma P and Shnrma .J.D(2001) In vitro haemolysts of human erythrocytes by plant 

extracts of anti plasmodia1 activity J EthonoPharm .74(3):239-243. 

Sharma RK.,Shandilya,S. and Sharma,S. (1983)Observations of malathion on 

Clarias balracus. Comp. Physiol.Eco1.. 9/21: 155-156. 

Sharma S.K, Chaturvedi, K.V.Sastry(l979) Acute Endrin toxicity on Ophiocephalus 

punctancs.Bull.Env. Conlam. Toxicol, 23:153-15 7. 

Sherekar P.V and Kulkharni,K.M (1987) Studies on acid and alkaline phosphatase 

activity of methylparathion exposed fish,Channa orientalis.JZool., 7:154-159 

Siddiqui M.K.J.,Rahaman,M.F., Mustafa,M and Balerao,U.T.(1991) 

Comparative study of blood changes and brain acetylcholinesterase inhibition 

by monocrotophos in rats.Ecotoxicol and Env.Safetj 21,283-289 

Siddiqui M.K.J.,Rahaman,M.F.,hlahabole,hl.,Anjum F and Quadri,S.S.H( 

1987) Effect of oral administration of endosulfan on some haematological 

parameters and serum enzymes in rats..Pesticides,21:25-27. 

Simon E.W (1953) Mechanism of dinitrophenol toxicity .Biol. Rev. Cambridge 

Phil.Soc.,28:453-470. 

Singh N.N and Snvastava A.K( 1993)Biochemical changes in Hereroneusres fossilis due 

to sublethal exposure to aldrin.Proc.Nat.Acad.SciiIndia,65A:33-38. 

Singh,D. and Singh.A.(2005)The toxicity of four Indian native p1ants:effect on 

AchE and Acidialkaline phosphastase activity in Channa marulius. 

Chemoshhere, 60(1):135-140. 

Singh.D.K and Agarwal, RA(1982)In vitro inhibition of acetyl cholinesterase by 

carbarnate and organophosphorus pesticide in Lymnea acuminara. Comp. 

Physiol.Ec01, 7: 177-178. 

Singh.H, Kumar.S, Dewan.S and Kumar(2000) Inflammation induced by latex of 

Calotropisprocera .J.Pharm and Toxicol Melhods.43(3):219-224. 

Singh N.N and Srivastava,A.K( 1995)Sublethal physiological effects of on 

Heteropneusres fossilis due to exposure of aldrin and subsequent 

recovery.Proc. Naf.Acad.SciIndio. 65@):33-38.

Singh N.N,Das.V.K and Srivsstsva A.K (1996) Effects of aldnn on carbohydrate .protein 

and ionic metabolism of Heteroneustes/ossilis. Biomedical and Env.Sc~. 10.408-414. 

Singh S. and Sahai S.(1984) Effects of malathion on gills ,liver and kidneyof 

Rarbor dnniconius .Proc.7Is' Ind.Scr.Congress.pp:Y2. 

Sinha and Datta Munshi,J.(1996)Toxicity of alkaloid extract of Agremone 

maicana on Indian air-breathing fishes.Ad.Bios ,15(11)6Y-78 

Sivaprasada Rao,K and Ramana Rao,K.V.( 1979 )Effect of methylparathion in 

selected oxidative enzymes and organic constituents in Tilapia 

mossambica.Curr.Sci 48(12):526-528. 

Skidmore,J.F and Tovell,P.W.A( 1972)Toxic effects of zinc sulphate on the gills of 

rain bow trout. Water Research.6:217-230. 

Skou,J.C(1957).Influence of some catikons on an ATPase from peripheral 

Slobodkin ,L.B(1968) Towards a predictive theory of evolution, population biology 

and evolution,edi!ed by R.C Lewanotir.Syracuse Univ Press.187-203. 

Smit.H.F,Weordenbag.H.J,Singh.R.H(1995) Ayun8ed1c herbal drugs w~th possible 

cytostatic act1vityJEthnnoPhnrrn.47(2J75-84. 

Smith,G.C,Lewis,W.M and Kaplan(1952)A comparative morphologic and 

physiologic study of fish blood.Progre.Fish. Cui. 14:169-172. 

SmithC.B,(1984)Aging and changes in cerebral energy metabolism. 

Trends.Neuro.Sci, 7:203-208. 

Snieszko S.F(1960)Microhaematocnt as a tool in fishery research and management. 

Spec.Scient.Rep.U.S.Fish.Wild If.Sew.N0:341. 

Sprague J.B. and Ramsay,B.A.( 1965) Lethal levels of mixed copper-zinc solution 

for juveniles of salmon. J.Fish Res.Bd.Can.,22:425-432. 

Sprague J.B and Ramsay, B.A(l965) Lethal levels of mixed copper-zinc solution for 

juveniles of Salmon.Fish Res.Bd.Can.,22:425-432. 

Sprague J.B( 1971) Measurement of pollutanr toxicity to fish. Sublethal effects and safe 

conc. Wal,Res..5:245-246.

Sprague J.B(l973) The ABC's of pollutants bioassay using fish biological methods 

for the assessment of watn quality.AmerSoc /or Testing materials, ASTM 

STP 528:6-30. 

Srinivasa Murthy,K.,Dhananjaya Naidu,M.,Chetty,C.S and Swami,K.S.(1983) 

Changes in carbohydrate metabolism of L.marginalis exposed to 

phosphomidon.Bull.Environ .Contam.Toxicol,30:219-222. 

Srinivasan ,K. and Radha Krishnamurthy,R.(1983)Effect of HCH isomers on 

some haematological parameters J. Food Sci.Techl20:322 

Srinivasulu Reddy,M. ,Jayaprada.P,and Ramana Rao,K.V( 1991) Toxicity 

impact of aldrin ion acid and alkaline phosphatase activity of Metapeneus 

monoceros:in vitro study.Bull.of Ennviron. Toxrco/46:479-484. 

Srivastava ,O.P and Srivastava,G.J(1981) Urea induced histopathology in kidney 

of the fresh water teleost Channa punctatus .Bull. Soc. Sci. Vet. Med. 

Compare.,83:213-217. 

Srivastava A.K and Mishra J,(1985) Lindane induced hematologocal changes in 

Hereroneurtes fossilis.Nat.AcadSci.LettR.373-375. 

Stickn ey R.k(l994) Principles of Aquaculture, Jhon W~ley & Sons,New York,h45-657 

Stimpson,J.H.( 1965)Comparative aspects of the control of glycogen utilization in 

vertebrate liver.Comp.Biochem.P~ysioi.15: 187-197. 

Summer,A+T(1965) The cytology and histochemistly of digestive gland cells of 

Helix.Quart.J.Microsc,Sc., 106:173-192. 

Swallow R.L. and Flemming,W.R( 1970)The effect of oxaloacetate on liver 

glycogen of Tilapia mossambica.CompBiochem.Physio1.36:93-98. 

Swamy KV, Ravikumar.R and Murali(l992) Changes in cholinesterase in different brain 

areas to monochrotophos toxicity in male albino rats. Biochem Int1.,27(4):661-669. 

Swamy K.V, Ravikumar.R, and Murali Mohan .P( 19926) Effects of daily dosing of 

monocrotophos on some aspects of protein metabolism in rat 

brain.Bul1. Environ.Cont Toxicol.,49: 723- 729.

Take0.S and Sakanashi .M(1985) Characterization of membrane bound Adenosine 

Triphosphatase activity of enriched fraction from vascular smooth 

muscle.Enzyme.34: 152-165. 

Tazeem Arasta , Bais.V.S, Preeti Thakur (1996) Effects of nuvan on some biochemical 

parameters of Mys/us.J EnviBiol.. 167.169. 

Tennis Wood ,M., Biri,C.E and Clark,A.F (1976)Acid phosphatases:Androgen 

dependent markers of rat prostrate.Can j.Biochem.,54.350. 

Thanginipon.W, Luangpaihoon.P, Thannipan(l995)Effects of monacroiophos on 

acetylcholinesterase activity on in N~le Tilapia brain. Neuro chernicol 

Research20(5):515-519. 

The Wealth of India(1992) Raw materials Publications and Information Directorate,CSIR, 

lndia.78-84. 

Tiwari S, and Singh. A(2003) Control of common predatory fish, Channa punctatus, 

through Nerium indicum leaf extract~.Chemosphere,53(8):865-75 

Toor,H.S.,SatinderpaI,K.and Kamalfleep,K.(1977) Histopathological changes in 

digestive tract and liver of Mystus tengara exposed to carbaryl and endrin.Al1 

India seminar on Ichthyology,Meerut Univ.,Meemt,pp:21-23. 

Towle,D.W(1981) Role of ~ a K' ' ATPase in ionic regulation by marine and 

estuarine animals.MarBiol.Lett.2:107. 

Trachtenberg ,M.C.,Packey,D.J. and Sweeney,T(1981) In vivo fi~nctioning of 

Sodtudotassium ATPase activity.ln:Current topics in cellular 

regulation,Vol: l9.Academic press ,New York,pp: 159-217. 

Tuschiya, K(1973) Lead.ln:Friberg L,NordbergG A ,Vonk VB(Eds),Hand book on 

toxicology of metals.Elsvierh'orth Holland Biomedical Press ,Amsterdam. 

Ulrich, F.(1963)The inhibition of mitochondria1 ATPase-effects of alkali metal 

ions.Biochem. J, 88: 193-206, 

Umminger B.L (1970) Physiological studies on super cooled hill fish Pundulus 

hetroclitus I11 carbohydrate metabolism and survival at sub zero tempemture. 

J. Exp. Zoo1,173:159-174.

Usha Kaisabhatia and Vibhuti Rai (1993) Changes in plasma level of phosphatases 

and transaminases of rabbits following vanadium exposure. Poll. Res., 

12(1).19-27. 

Vasanthi,R.( 1983)Effect of lindane on glycogen content of Tilapia mossambica 

.M.Sc .,dissertation .University of Madras,Madras. 

Vasanthy M. and Ramswamy.M,(1987)A shiR in metabolic pathway of 

Sarotherodon mossambicus exposed to thiodon. Proc. Indian. Acad. Sci., 

96(1):55-61. 

Verma ,S,R..,Gupta,S.P and Tyagi,M.P(1975)Studies on histopathological changes 

in Colisa fasciatus.Morph.Jahrb.Liepzig, 121:38-54. 

Verma ,S.R,Rani,S. and Dalela.R.C( 1980 )Effect of phenol and dinitrophenol on 

phophatases in Noroprem notoprerus.Arch.Envir.Contam. Toxicol.9:451-4.59. 

Vijayaendrababu .K.V.K.and Vasudev,T.( 1984)Effect of dimecron ,rogor and 

cumin-L on AchE and phosphatases in L.marginalis,Curr.Sci.,53(17):935- 

936. 

Vijayalakshmi .S and Tilak K.S (1996) Effect of pesticides on gill morphology of Labro 

rohita.JEcoroxicol.A4onit.,6(1):59-64. 

Vijendrababu K.V.K and Vasudev, T(1984) Effect of demecron ,rogor and cumin- 

Lon AchE and phosphatases in L,marginalts,Curr.Sci.53@~7/935-936. 

Vinod V.G(l989). In vivo effects of BHC on some aspects of physiology and 

histopathology of fish ,Channa punctahrs .PhD thesis, Osmania University, 

India. 

Walsh A.H and Ribelin W.E( 1975) The pathology of pesticides poisoning:Pathology of 

fishes, Wis:Univ.of Wisconsin Press.,5 15-557. 

Warn Sinba,T.(1994) Phyro Chemistry. .37.217-226 

Warner A.H, Paterson K. K and Borgman (1966) Behavioral pathology in fish: A 

qualitative study of sub lethal pesticide toxicationJ.Appl.Ecol.3:223-247. 

Wasserman,M., Wasserman, D., and AronovaskiJ(1970) Effect of 

organochlorine pesticides on serum cholesterol levels in people 

ccupationally exposed, Bull. Environ. Contam. Toxicol.5:368.

Wedemeyer, G.(1969) Stress induced ascorhate depletion and cortisol production in 

two salmonid fishes,Comp.Biochem.Physiol.29: 1247. 

Wedemeyer,G.A and Yastuke,W.T(1977)Clinical methods for assessment of the 

effects of environmental stress in fish health.U.S.Fish and Wild life 

service, Wnshingron DC.89. 

Weinbach ,E.C .and Garbus,J. (1969) Mechanism of action of reagents 

uncouple oxidative phosphorylation.Natrrre(Londonj.221:1016-1018. 

that 

WHO (1977) Pestic~des resldues in food. Repon of the 1976.Joint FAOlWHO meeting, 

Funher work or information required desirable No 612:29. 

Widdows , J.J,Bakke, B.L.,Bayne P,Donkin(l982) Responses in Mytilus edulis in 

exposure to water accumulated fraction of NorthSea.Mar.Biol67:15-31. 

William Royce .F (1972) Introduction to Fishery Sciences.,Academic Press, New York 

Williams,K. Green,D. and Pascode,D.(1984)Toxicity testing with fresh water 

macroinvertebrates: methods and application in environmental management. 

In: fresh water biological monitoring. Pascode D and Edwards, R.W. (ed)., 

Pergamas,Oxford.pp:81-91. 

Yates, F.E.(1980) Biology of chlorides cell: lean Maetz memorial 

symposium.Am J.Phisiol. 7:R13Y-R276. 

Za japragassarazan,Z.(1993) Some observations on toxicological of lindane in Anabas 

resfudinetc~.Envir.Ecol..I4(I):35-5Y. 

Zayaprgassarazan ,Z .and Anandan,V. (1996) Effect of g-BHC on protein profiles 

of selected tissues of Anabas testudineus(Bloch).MPhil-Thesis ,Pondicherry 

University,Puducherry.

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