Effect of steel plant effluent on acid and alkaline - Integrated ...

INTERNATIONAL JOURNAL OF ENVIRONMENTAL SCIENCES Volume 1, No 6, 2011
© Copyright 2010 All rights reserved Integrated Publishing Association
Research article ISSN 0976 – 4402
<strong>Effect</strong> <strong>of</strong> <strong>steel</strong> <strong>plant</strong> <strong>effluent</strong> on acid and alkaline phosphatases <strong>of</strong> gills, liver
and gonads <strong>of</strong> Cyprinus carpio Linn. (1758)
Chetna Bakde, Aditi Niyogi Poddar
School <strong>of</strong> Studies in Life sciences, Pt.Ravishankar Shukla University, Raipur (CG)
adinpod@yahoo.co.in
ABSTRACT
Fishes are aquatic and poikilothermic animals. Hence, their existence and performance is
dominated by the quality <strong>of</strong> their environment. Pollution <strong>of</strong> water bodies forces them to
acclimatize to various factors thus imposing a considerable amount <strong>of</strong> stress on their lives.
This ability to detect sudden changes in environment and monitoring short or long term
changes in water quality makes the fish efficient biomarkers. The Bhilai Steel Plant is
situated 30 kilometers (west) <strong>of</strong> Raipur, the capital <strong>of</strong> Chhattisgarh. Besides good quality
<strong>steel</strong>, it also produces important by products, such as, Coal tar, Naphthalene and Benzol.
Effluents generated are dumped into the two local rivers, the Kharoon and the Sheonath
through various channels. Analysis <strong>of</strong> the physico­chemical constituents <strong>of</strong> the different
<strong>effluent</strong>s reveals the coke oven <strong>effluent</strong> to contain a high amount <strong>of</strong> phenol, besides the
presence <strong>of</strong> other toxic substances and hence is not expected to be congenial for the existence
<strong>of</strong> fish. Common Carp (Cyprinus carpio Linn, 1758) were exposed to different concentrations
(10,20,30%)<strong>of</strong> this <strong>effluent</strong> for a short term duration and changes in acid and alkaline
phosphatases recorded in the three organs,viz.,gills,liver and gonads at different time points
(48,96 and 160 hours).The various concentrations <strong>of</strong> the <strong>effluent</strong> were found to alter the
acidic and alkaline phosphatase activity in the three organs. Hence, the results from present
investigations may be useful in the assessment <strong>of</strong> environmental stress in the aquatic
ecosystem.
Key words: Steel <strong>plant</strong> <strong>effluent</strong>, Alkaline phosphatases, Acid phosphatases, Cyprinus carpio
Linn.
1. Introduction
Water, a universal solvent, is an essence for life on earth. Hence, discharge <strong>of</strong> various toxic
chemicals and substances into water makes life difficult. Fishes are aquatic and
poikilothermic animals. Hence, their existence and performance is dominated by the quality
<strong>of</strong> their environment. Conditions in large bodies <strong>of</strong> water although are relatively stable, a
greater magnitude <strong>of</strong> environmental change exists in smaller bodies <strong>of</strong> water, particularly in
the presence <strong>of</strong> man ­made stressors. Thus, in the aquatic environment, life goes on under
dynamic and unstable circumstances, forcing fishes to acclimatize to various factors, such as,
changes in population density, pressure, temperature, dissolved gases, light, pH, etc, which
impose a considerable amount <strong>of</strong> stress on their lives and predispose them to diseases.
The term ‘Stress’ means the sum <strong>of</strong> all the physiological responses by which an animal tries
to maintain or reestablish a normal metabolism in the face <strong>of</strong> physical and chemical forces
(Selye, 1950). In other words, stress is a state produced by an environmental or other factor,
which extends the adaptive response <strong>of</strong> an animal beyond the normal range or which disturbs
the normal functioning to such an extent that in either case the chances <strong>of</strong> survival are
Received on January, 2011 Published on April 2011 1305

<strong>Effect</strong> <strong>of</strong> <strong>steel</strong> <strong>plant</strong> <strong>effluent</strong> on acid and alkaline phosphatases <strong>of</strong> gills, liver and gonads <strong>of</strong> Cyprinus carpio
Linn. (1758)
significantly reduced (Brett, 1958). A series <strong>of</strong> morphological, biochemical and
physiological changes occur as a result <strong>of</strong> stress and constitute the General Adaptation
Syndrome (Wedemeyer et al,1999). Hence, studying the changes in biochemical constituents
and enzyme activities <strong>of</strong> tissues are important to determine the nature and extent <strong>of</strong> toxicants
effects on organisms (Murthy, 1985; Heath, 1987) and can provide an early warning that
pollution is reaching harmful levels. With their ability to detect sudden changes in
environment and monitor short or long term changes in water quality, fishes thus make
efficient biomarkers.
Phenol and phenolic compounds are stressful environmental factors which because <strong>of</strong> their
lipophilic properties present a threat against natural environment and also to human health
(Hori et al., 2006).They have been found to induce genotoxic (Jagetia and Aruna, 1997),
carcinogenic (Tsutsui et al., 1997) and immunotoxic effects (Taysse et al., 1995)on fish
health, besides causing reduction in fish weight and fertility (Saha et al., 1999). Some
phenolic compounds, such as, Polycyclic Aromatic Hydrocarbons and Polychlorinated
biphenyls are Endocrine disrupters (Kashiwada et al., 2002; Pait and Nelson, 2003; Tollefsen,
2006; Barse et al., 2006; Martin­Skilton et al., 2006) and adversely affect fish metabolism
(Gupta et al., 1983; Abdel­Hameid, 1994). Most importantly, phenol has been listed in the
NRWQC as a priority pollutant with an organoleptic effect criterion <strong>of</strong> 300µg/l
(USEPA,2009).Hence, it is very toxic to fish and has a unique quality <strong>of</strong> tainting the taste <strong>of</strong>
fish if present in marine environments at 0.1­1.0 ppm (Kirk and Othmer,1982;
Neff,2002).Thus, the importance <strong>of</strong> taking into consideration phenol intoxication in natural
aquatic habitats becomes evident.
Alkaline phosphatase (ALP) (EC 3.1.3.1) is a hydrolase enzyme responsible for removing
phosphate groups from many types <strong>of</strong> molecules, such as, nucleotides, proteins and alkaloids.
It is a P­stress marker enzyme most effective in an alkaline environment, that catalyzes the
hydrolysis <strong>of</strong> phosphorous compounds and the transfer <strong>of</strong> phosphoryl groups to an acceptor
molecule. The rate <strong>of</strong> catalytic activity <strong>of</strong> the enzyme is inversely proportional to the
concentration <strong>of</strong> inorganic phosphate in the ambient environment (Dyhrman & Palanik 1999).
This enzyme could serve as a good indicator <strong>of</strong> intoxication because <strong>of</strong> its sensitivity to
metallic salts (Boge et al, 1992).Acid phosphatase,on the other hand is a phosphatase which
frees attached phosphate groups from other molecules during digestion. It is a
lysosomal ,hydrolytic enzyme with an acid pH optimum .It takes part in the dissolution <strong>of</strong>
dead cells and as such serves as a good indicator <strong>of</strong> stress condition in the biological system
(Gupta et al, 1983; Verma et al., 1984).
The gills are the most delicate structures <strong>of</strong> the teleost body and their vulnerability has thus
considerable importance because <strong>of</strong> their external location and necessarily intimate contact
with water, which means that they are liable to damage by any irritant material in the water
whether dissolved or suspended (Roberts ,1978). Therefore, they are potentially useful to
monitor the health <strong>of</strong> fish (Pawert et al, 1998; Mallatt ,1985 and Mallatt et al,1995).
The Bhilai Steel Plant is an integrated <strong>steel</strong> <strong>plant</strong> situated 30 kilometers (west) <strong>of</strong> Raipur, the
capital <strong>of</strong> Chhattisgarh. Besides the major marketable product that is good quality <strong>steel</strong>, it
also produces important by products, such as, Coal tar, Naphthalene and Benzol, which is
further rectified to Benzene, Toluene and Xylene. Effluents generated in the three main units
are further dumped into the two local rivers, the Kharoon and the Sheonath through various
channels. The coke oven <strong>effluent</strong> has strong phenolic odour and contains a high amount <strong>of</strong>
phenol, besides the presence <strong>of</strong> other toxic substances (Sinha, 1999, PhD Thesis). It is
Chetna Bakde, Aditi Niyogi Poddar
International Journal <strong>of</strong> Environmental Sciences Volume 1 No.6, 2011
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<strong>Effect</strong> <strong>of</strong> <strong>steel</strong> <strong>plant</strong> <strong>effluent</strong> on acid and alkaline phosphatases <strong>of</strong> gills, liver and gonads <strong>of</strong> Cyprinus carpio
Linn. (1758)
primarily used for irrigation purposes by the local villagers, since it causes skin rashes in
humans and cattle. The soil texture <strong>of</strong> the embankment appears to be oily black. All these
factors raise many questions about the extent <strong>of</strong> toxicity <strong>of</strong> the <strong>effluent</strong> on food fishes. Hence,
this work was undertaken in an attempt to study the stress response <strong>of</strong> fishes to the presence
<strong>of</strong> this <strong>effluent</strong>. Cyprinus carpio Linn.1758 (Common carp):Family Cyprinidae, a widespread
freshwater fish <strong>of</strong> Europe and Asia was taken as the model fish.
2. Materials and Methods
Healthy and disease free Carps (Cyprinus carpio Linn., 1758) within the range <strong>of</strong> 80­100 gm
weight and 18­22 cms. length were acclimatized for a period <strong>of</strong> 7 days at 28±1 o C in an
aquarium tank <strong>of</strong> size (4x3 ft.) and fed with fish food during the period. Effluent generated
by the coke oven batteries <strong>of</strong> the Bhilai Steel <strong>plant</strong> was collected from the discharge point at
Purena nala, near Bhilai­ 3 <strong>of</strong> district Durg (Chhattisgarh) and its various physico­chemical
characteristics viz., pH, Conductivity, Turbidity, Total solids, Total Dissolved
Solids(TDS),Total Suspended Solids(TSS),Oil­Grease, Total Alkalinity, Chlorides, Sulphates,
Total Hardness, Ca, Mg, Na, K, Fe, Phenol, Nitrates measured by the expertise available at
the Pollution laboratory, School <strong>of</strong> Studies in Chemistry,Pt.Ravishankar Shukla
University,Raipur.
Short term exposure <strong>of</strong> the fishes to three different concentrations (10, 20 and 30 %) <strong>of</strong> the
whole <strong>effluent</strong>, taken separately in glass aquaria <strong>of</strong> size (3x2 ft) along with normal tap water
for control, was done for a period <strong>of</strong> 7days. Collection <strong>of</strong> fresh samples <strong>of</strong> liver, gills and
gonads was done after sacrificing the fishes at 3 time points, ie.,48,96 and 160 hours.
Aeration and feeding at fixed time was done throughout the experiment. Homogenate <strong>of</strong> the
fresh tissue was prepared in 0.9% ice cold saline to approximately 10% w/v and supernatant
used for enzyme assay after centrifugation at 3000 rpm at 4 o C for 15 minutes. Estimation <strong>of</strong>
Alkaline and acid phosphatases was done by the method <strong>of</strong> Fiske & Subba Rao
(1925).Protein was determined by the method <strong>of</strong> Lowry et al,1951. The results obtained were
expressed as µg Pi/mg protein/hour and statistically analyzed by ANOVA using COSTAT.
3. Results
Analysis <strong>of</strong> the physico­chemical characteristics <strong>of</strong> the whole <strong>effluent</strong> generated by the coke
oven batteries <strong>of</strong> the Bhilai Steel Plant in the month <strong>of</strong> January shows phenol content to be
0.37 mg/l, besides other toxic substances ,such as, Total Dissolved solids ,Total Suspended
Solids ,Oil grease, Total Alkalinity, Chloride, Total Hardness etc.which have exceeded their
Natural recommended water quality criteria values for freshwater fish according to
USEPA,2010 Short term exposure <strong>of</strong> Cyprinus carpio to the different concentrations
(10%,20%,30%) <strong>of</strong> this whole <strong>effluent</strong> and estimation <strong>of</strong> Alkaline and Acid phosphatases in
the Gills, Liver and Gonads <strong>of</strong> exposed fishes vis­avis normal reveal the following features
Table 1: Physico­chemical characteristics <strong>of</strong> coke oven whole <strong>effluent</strong> (Bhilai Steel Plant)
obtained from origin point <strong>of</strong> Purena channel (Jan,2010). (NRWQC for freshwater fish,
USEPA,2010 in parenthesis)
pH 7.189 ± 1.894
Conductivity(milli siemens) 0.529 ± 0.162
Turbidity (NTU) 0.103 ± 0.069
Total solids (mg/l) 0.538 ± 0.255
Total Dissolved solids (mg/l)(15,000) 0.386 ± 0.210
Chetna Bakde, Aditi Niyogi Poddar
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<strong>Effect</strong> <strong>of</strong> <strong>steel</strong> <strong>plant</strong> <strong>effluent</strong> on acid and alkaline phosphatases <strong>of</strong> gills, liver and gonads <strong>of</strong> Cyprinus carpio
Linn. (1758)
Total Suspended Solids (mg/l)(80) 0.158 ± 0.090
Oil grease(mg/l)(0.01) 21.308 ± 6.073
Total Alkalinity(mg/l)(20) 107.137± 43.371
Chloride(mg/l) (86) 107.923 ± 48.911
Sulphates(mg/l) 35.855 ± 24.422
Total Hardness(mg/l)(100) 204.975 ± 52.744
Ca(mg/l) 136.034 ± 37.236
Mg(mg/l) 77.015 ± 30.181
Na(mg/l) 30.011 ± 8.571
K(mg/l) 56.826 ± 37.834
Fe(mg/l) 0.133 ± 0.075
Phenol(mg/l)(0.3) 0.378 ± 0.466
Nitrates(mg/l) 51.249 ± 12.956
Table 2: Alkaline (ALP) and Acid phosphatases (ACP) in organs <strong>of</strong> normal and <strong>effluent</strong>
exposed Cyprinus carpio. Results expressed in µg Pi/mg protein/hour(Mean ± SD).
ORGAN
GILLS
LIVER
GONADS
DURATION
OF
NORMAL
E10
(Effluent 10%)
E20
(Effluent 20%)
E30
(Effluent 30%)
EXPOSURE
( hours) ALP ACP ALP ACP ALP ACP ALP ACP
48 7.121±
0.643
1.187±
0.536
1.229±
0.811
4.752±
5.677
1.399±
0.304
2.207±
2.512
2.476±
0.152
96 1.233± 1.010± 2.781± 0.818± 1.581± 0.527± 0.351±
1.379 0.552 3.779 0.077 0.249 0.083 0.166
160 1.045± 6.042± 0.307± 1.475± 2.032± 0.610± 1.016±
1.267 6.654 0.087 1.043 0.383 0.479 0.862
48 0.461± 0.299± 0.283± 0.660± 0.714± 0.878± 0.195±
0.261 0.033 0.067 0.133 0.078 0.155 0.275
96 0.670± 0.503± 0.385± 0.651± 0.855± 1.179± 0.846±
0.237 0.316 0.042 0.168 0.292 0.333 0.342
160 0.226± 1.391± 1.782± 0.792± 1.570± 0.444± 0.939±
0.046 0.137 0.093 0.187 0.097 0.145 0.000
1.280± 0.753± 1.033± 0.517± 1.509± 2.372± 0.461±
48
1.171 0.426 0.244 0.244 0.305 0.000 0.000
1.390± 1.063± 1.073± 0.626± 2.414± 2.085± 8.612±
96
1.040 0.116 0.506 0.126 0.000 0.155 0.530
3.297± 1.030± 0.678± 1.356± 1.993± 0.664± 0.987±
160
1.166 0.583 0.411 0.274 0.313 0.313 0.465
1.184±
0.457
0.703±
0.331
2.371±
0.479
0.473±
0.354
0.665±
0.085
0.704±
0.221
4.491±
1.140
2.122±
0.530
1.481±
1.164
Chetna Bakde, Aditi Niyogi Poddar
International Journal <strong>of</strong> Environmental Sciences Volume 1 No.6, 2011
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<strong>Effect</strong> <strong>of</strong> <strong>steel</strong> <strong>plant</strong> <strong>effluent</strong> on acid and alkaline phosphatases <strong>of</strong> gills, liver and gonads <strong>of</strong> Cyprinus carpio
Linn. (1758)
Table 3: Percent change vis­avis normal in Alkaline (ALP) and Acid phosphatase (ACP) <strong>of</strong>
organs <strong>of</strong> C.carpio exposed to <strong>steel</strong> <strong>plant</strong> <strong>effluent</strong>.
ORGANS
GILLS
LIVER
GONADS
DURATION
OF
E10 E20 E30
EXPOSURE
(hours)
ALP ACP ALP ACP ALP ACP
48 ­82.72 +299.16 ­80.34 +85.71 ­65.17 ­0.84
96 +126.02 ­18.81 +28.46 ­47.52 ­71.54 ­30.69
160 ­70.48 ­75.5 +93.33 ­89.9 ­2.86 ­60.76
48 ­39.13 +120 +54.35 +193.33 ­58.7 +56.67
96 ­43.28 +30 +26.87 +136 +26.87 +32
160 +673.91 ­43.17 +582.61 ­68.35 +308.7 ­49.64
48 ­19.53 ­30.67 +17.97 +216 ­64.06 +498.67
96 ­23.02 ­40.57 +73.38 +96.23 +519.42 +100
160 ­79.39 +32.04 ­39.7 ­35.92 ­70 +43.69
Table 4: TWO WAY ANOVA: <strong>Effect</strong> <strong>of</strong> Effluent and Time on Acipase and Alkpase activity
in gills <strong>of</strong> Cyprinus carpio:
Variables Factors F df p
Acipase Activity
Alkpase Activity
Effluent Concentration (EC): 0.497 3,23 0.69 ns
Time: 1.127 2,23 0.36 ns
EC x Time: 0.96 6,23 0.49 ns
Effluent Concentration (EC): 2.61 3,23 0.99 ns
Time: 5.16 2,23 < 0.05
EC x Time: 4.21 6,23 < 0.05
Table 5: TWO WAY ANOVA: <strong>Effect</strong> <strong>of</strong> Effluent and Time on Liver <strong>of</strong> Cyprinus carpio:
Variables Factors F df p
Acipase Activity
Alkpase Activity
Effluent Concentration (EC): 1.102 3,23 0.386 ns
Time: 3.01 2,23 0.087 ns
EC x Time: 6.30 6,23 < 0.01
Effluent Concentration (EC): 10.46 3,23 < 0.01
Time: 28.83 2,23 < 0.001
EC x Time: 11.14 6,23 < 0.001
Chetna Bakde, Aditi Niyogi Poddar
International Journal <strong>of</strong> Environmental Sciences Volume 1 No.6, 2011
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<strong>Effect</strong> <strong>of</strong> <strong>steel</strong> <strong>plant</strong> <strong>effluent</strong> on acid and alkaline phosphatases <strong>of</strong> gills, liver and gonads <strong>of</strong> Cyprinus carpio
Linn. (1758)
Table 6: TWO WAY ANOVA: <strong>Effect</strong> <strong>of</strong> Effluent and Time on Gonads <strong>of</strong> Cyprinus carpio:
Variables Factors F df p
Acipase Activity
Alkpase Activity
Effluent Concentration (EC): 14.22 3,23 < 0.001
Time: 5.27 2,23 < 0.05
EC x Time: 5.89 6,23 < 0.01
Effluent Concentration (EC): 14.23 3,23 < 0.001
Time: 26.91 2,23 < 0.001
EC x Time: 26.77 6,23 < 0.001
3.1 Gills
1. Alkaline phosphatase (Fig.1a &b)
· E10(Effluent 10%)­An initial decline <strong>of</strong> 82.72 % compared from normal is shown in
Alkpase in the first 48 hours, followed by a steep rise <strong>of</strong> 126% in 96 hours and drastically
declining by 70.48% in 160 hours.
· E20(Effluent 20%)­Shows an initial % decline <strong>of</strong> 80.34in 48 hours followed by a % hike
<strong>of</strong> 28.46 in 96 hours and 93.33 in 160 hours.
· E30(Effluent 30%)­ Shows an initial % decline <strong>of</strong> 65.17 in 48 hours followed by a
gradual decline by 71.54% in 96 hours , subsequently rising back to normal(100%) in
160 hours.
<strong>Effect</strong> <strong>of</strong> <strong>effluent</strong> concentration on ALP <strong>of</strong> gill was found statistically non significant by
ANOVA. However, duration <strong>of</strong> exposure (F=5.16; P

<strong>Effect</strong> <strong>of</strong> <strong>steel</strong> <strong>plant</strong> <strong>effluent</strong> on acid and alkaline phosphatases <strong>of</strong> gills, liver and gonads <strong>of</strong> Cyprinus carpio
Linn. (1758)
· E10 (Effluent 10%)­Shows a gradual % decline <strong>of</strong> 39.13 to 43.28 followed by a steep rise
by 673.91%.
· E20 (Effluent 20%)­Shows an initial % hike <strong>of</strong> 54.34 followed by 26.86 and a steep rise
by 582.6%.
· E30 (Effluent 30%)­%decline by 58.69 is followed a % hike <strong>of</strong> 26.86 and 308.69%.
<strong>Effect</strong> <strong>of</strong> <strong>effluent</strong> concentration on ALP <strong>of</strong> liver was found statistically significant (F=10.46;
P

<strong>Effect</strong> <strong>of</strong> <strong>steel</strong> <strong>plant</strong> <strong>effluent</strong> on acid and alkaline phosphatases <strong>of</strong> gills, liver and gonads <strong>of</strong> Cyprinus carpio
Linn. (1758)
· E30 (Effluent 30%)­Acpase although shoots by 498.67% ,but declines gradually to
100% and 43.69% above normal.
<strong>Effect</strong> <strong>of</strong> <strong>effluent</strong> concentration on ACP <strong>of</strong> gonad was found to be highly significant
statistically by ANOVA (F=14.22; P

<strong>Effect</strong> <strong>of</strong> <strong>steel</strong> <strong>plant</strong> <strong>effluent</strong> on acid and alkaline phosphatases <strong>of</strong> gills, liver and gonads <strong>of</strong> Cyprinus carpio
Linn. (1758)
Acid phosphatase on the other hand, shows a picture inverse <strong>of</strong> Alkaline phosphatase and
rises (although insignificantly) above normal in the first 48 hours followed by a fall below
normal in 160 hours in gills. Maximum rise above normal followed by resumption <strong>of</strong> normal
values is found to be highest in E10 in gills. Similar picture is shown by liver being
maximum in E20 followed by E10 and E30.In gonads ,maximum rise above normal in first
48 hours, is shown in E30 followed by E20 and E10. This result is in agreement with the
significant increase in ACP in kidney <strong>of</strong> the catfish, Heterpneustes fossilis after intoxication
with cadmium (Sastry and Subhadra, 1985). Similar increase <strong>of</strong> plasma and liver acid
phosphatase has been reported in the teleosts, Brycon cephalus (matrinxã) following exposure
to methyl parathion. The increase was associated either with the decrease in stability <strong>of</strong> liver
lysosome membrane or with liver damage (Moraes et al., 1998). The enzyme ACP is known
to be associated with lyososomal activity. The authors agree with Gill et al., 1992 that the
acid phosphatase elevation reflects proliferation <strong>of</strong> lysosomes in attempt to sequester the
toxic xenobiotic. The present author agrees with Neskovic et al., 1993 and Adham et al.,
1997 that alkaline and acid phosphatases prove to be quite sensitive in assessing sublethal
water intoxicosis in fish . The present data reveal that the different concentrations <strong>of</strong> coke
oven <strong>effluent</strong> i.e. 10%, 20% and 30% alter the acidic and alkaline phosphatase activity <strong>of</strong>
Cyprinus carpio in different organs. Hence, the results from present investigations may be
useful in the assessment <strong>of</strong> environmental stress in the aquatic ecosystem so that attempts
could be made to reduce the chemical compounds and heavy metals in freshwater ecosystem
by pretreatment before discharging in to the aquatic ecosystem.
Acknowledgement
The author expresses her deep sense <strong>of</strong> gratitude to Dr. Shamsh Pervez, Associate Pr<strong>of</strong>essor,
SoS in Chemistry,Pt.Ravishankar Shukla University and his Pollution Lab team for analysis
<strong>of</strong> the physico­ chemical characteristics <strong>of</strong> the <strong>effluent</strong>. Sincere thanks are also due to School
<strong>of</strong> studies in Lifescience for providing laboratory facilities.
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