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Oxidative stress in the freshwater

cyprinid crucian carp (Carassius
carassius L.) upon chronic exposure to
endosulfan
a

Sabzar Ahmad Dar , Abdul Rehman Yousuf , Masood-ul-Hassan

b

Balkhi & Md Niamat Ali

a

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Limnology and Fisheries Laboratory, Centre of Research for

Development (CORD), University of Kashmir, Srinagar 190006,
India
b

Division of Fisheries, Sher-e-Kashmir University of Agricultural

Sciences and Technology of Kashmir (SKUAST-K), Srinagar 190006,
India
c

Molecular and Cytogenetics Laboratory, Centre of Research for

Development (CORD), University of Kashmir, Srinagar, 190006
India
Accepted author version posted online: 30 Oct 2014.Published
online: 24 Nov 2014.

To cite this article: Sabzar Ahmad Dar, Abdul Rehman Yousuf, Masood-ul-Hassan Balkhi & Md Niamat
Ali (2014) Oxidative stress in the freshwater cyprinid crucian carp (Carassius carassius L.) upon
chronic exposure to endosulfan, Toxicological & Environmental Chemistry, 96:6, 906-916, DOI:
10.1080/02772248.2014.982122
To link to this article: http://dx.doi.org/10.1080/02772248.2014.982122

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Toxicological & Environmental Chemistry, 2014

Vol. 96, No. 6, 906
916, http://dx.doi.org/10.1080/02772248.2014.982122

Oxidative stress in the freshwater cyprinid crucian carp (Carassius

carassius L.) upon chronic exposure to endosulfan

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Sabzar Ahmad Dara*, Abdul Rehman Yousufa, Masood-ul-Hassan Balkhib and

Md Niamat Alic
a
Limnology and Fisheries Laboratory, Centre of Research for Development (CORD), University of
Kashmir, Srinagar 190006, India; bDivision of Fisheries, Sher-e-Kashmir University of
Agricultural Sciences and Technology of Kashmir (SKUAST-K), Srinagar 190006, India;
c
Molecular and Cytogenetics Laboratory, Centre of Research for Development (CORD), University
of Kashmir, Srinagar 190006, India

(Received 22 October 2014; accepted 25 October 2014)

Oxidative stress and antioxidant responses of crucian carp, upon chronic exposure to
endosulfan, were evaluated in vivo. The lethal concentration (LC50
96 h) was 70 mg L1;
on its basis, the fish were exposed to endosulfan at 20, 35, and 50 mg L1 and autopsy
was done on days 1, 2, 3, 4, 7, 14, 21, 28, and 35. Lipid peroxidation was induced in a
concentration-dependent manner, being highest at 50 mg L1 (3/4 LC50
96 h, sub-lethal
concentration-I, SL-I) on day 4 (720% versus control), followed in its extent (490%) at
30 mg L1 (1/2 LC50
96 h, sub-lethal concentration-II, SL-II) on day 7 and lowest
(260%) at 10 mg L1 (1/4 LC50
96 h, sub-lethal concentration-III, SL-III) on day 14.
Glutathione showed a concentration- and time-dependent elevation in the initial phase,
with highest level on day 4 (180%) at SL-I, but showed significant reduction in all test
concentrations from day 21 of post-exposure. Superoxide dismutase was decreased
significantly throughout the study, with highest reduction (63%) on day 4 at SL-I;
catalase increased in all test concentrations up to day 14 but showed a significant
decrease from the day 28 of post-exposure. The potential role of these parameters as
indicators of pesticide pollution in aquatic systems is discussed.
Keywords: endosulfan; agricultural runoff; fish; oxidative stress; environmental
monitoring

1. Introduction
Endosulfan, a cyclodiene insecticide, has been classified as highly toxic by the majority of
environmental protection agencies. Endosulfan has been registered and released for use in
the cultivation of soya, cotton, coffee, tobacco, and tea in several countries. In the United
States, an estimated 337,000 kg of endosulfan was applied between 2002 and 2006
(USEPA 2007). In India, endosulfan is classified as an extremely hazardous pesticide
(Ganeshwade et al. 2012). Although its use is restricted to certain crops, residues of this
compound have been detected in the aquatic environment, where it consequently may
affect fish. Therefore, studies that can correlate the effects on fish to the concentration of
this pesticide in water are gaining credence.
Many xenobiotics can induce the formation of reactive oxygen species (ROS) via several mechanisms, such as interference of reactive intermediates with electron transport,
inactivation of antioxidative enzymes, and depletion of antioxidants (Maran et al. 2009).
*Corresponding author. Email: sabzar.cord@gmail.com
2014 Taylor & Francis

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907

Endogenous antioxidative enzymes and antioxidants are essential for the conversion of
ROS to harmless metabolites, as well as for protection and restoration of normal cellular
metabolism and functions. Oxidative stress occurs when ROS overwhelm cellular
defenses and damage proteins, membranes, and nucleic acids (Velisek et al. 2012). Estimation of lipid peroxidation (LPO) has been found to have predictive value for oxidative
stress. Measures of oxidative stress have been used as early warning indicators of adverse
effects on contaminated aquatic habitats (Toni et al. 2010).
Fish are frequently used as bioindicators since they are sensitive to changes in their
environment and play significant roles in assessing potential risks associated with contamination. Some characteristics make Carassius carassius L. an excellent model in ecotoxicology, such as its ready availability, cost effectiveness, and easiness of maintenance
(Dar et al. 2015). Since there is growing concern over the presence of pesticides in the
aquatic environment, it is important to develop or standardize existing methods for
assessing the deleterious effects of xenobiotics in aquatic organisms.
Negative effects of endosulfan on fish have been documented including histological,
physiological, hematological, neurological, behavioral, immunological, and mutagenic
(Ganeshwade et al. 2012; Velisek et al. 2012; Dar et al. 2014), but there is a dearth of
data on the chronic toxicity of endosulfan manifested by oxidative stress to crucian carp
at environmentally realistic concentrations. This is the aim of this study.

2. Materials and methods

2.1. Experimental fish and chemicals
Healthy fish specimen of C. carassius L. (Family: Cyprinidae and Order: Cypriniformes)
of a length of 12.5 1.6 cm and weight of 33 5 g were procured with the help of a local
fisherman from the Dal Lake (34 070 N 74 520 E) in the vicinity of the University of Kashmir, Srinagar, India, transported to the laboratory, and subjected to a prophylactic treatment by bathing in a 0.05% aqueous solution of potassium permanganate for 2 min to
avoid dermal infection. The fish stock was then acclimatized for at least three weeks to a
1:1 diurnal photoperiod in well-aerated 60 L glass aquaria at 19.7 2.6  C with 24 h
aged dechlorinated tap water (pH 7.6
8.4) and fed ad libitum daily with commercially
available fish food (Feed Royal , Maa Agro Foods, Visakhapatnam, Andhra Pradesh,
India). Waste products were siphoned off daily to prevent increase of ammonia in the
water. Every effort as suggested by Bennett and Dooley (1982) was taken to maintain
optimal conditions during acclimatization: no fish died during this period. The acclimatized fish were used for the experiments, conducted in accordance with the principles of
the Institutional Ethical Committee (IEC) for the protection of research animals at the
University of Kashmir. Endosulfan and cyclophosphamide were purchased from the
Sigma Aldrich, Bengaluru, India.

2.2. Determination of acute toxicity

Determination of the LC50
96 h of endosulfan to C. carassius was conducted in a semistatic system with 60 L glass aquaria, changing the endosulfan solution (99.5% pure)
every alternate day to maintain its similar concentration. Briefly, triplicate sets of 10 fish
each were exposed to endosulfan at concentrations of 10, 30, 50, 70, 90, 110, 500, and
1000 mg L1 derived from a range finding test. Fish were not fed throughout the experiment and lethality was the toxicity end-point. Fish were visually examined daily and

908

S.A. Dar et al.

considered dead when no respiratory movements or no sudden swimming in response to

gentle touching were observed. The LC50
96 h of endosulfan was determined by probit
analysis (Finney and Stevens 1948). The study was conducted following the United States
Environmental Protection Agency Report No. EPA 821-R-02-012 (EPA 2002).

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2.3. Experimental design for chronic exposure

The experiments consisted of five treatments each with five replicates, in total 25 aquaria,
containing 60 L dechlorinated and well-aerated tap water with 10 fish in each aquarium.
Fish maintained in dechlorinated tap water served as negative (treatment 1) and those

exposed to cyclophosphamide at a concentration of 4 mg L1 (Ozkan

et al. 2011) as positive control (treatment 2). In treatments 3
5, the fish were kept in water containing endosulfan at concentrations of 20, 35, and 50 mg L1. The mean concentration of endosulfan
in the water samples was always within 5% of the intended concentration when determined by dispersive liquid
liquid micro extraction followed by gas chromatographymass spectrometry (Dar et al. 2014). The experiment was a semi-static assay conducted
over a period of 35 days. From each group, five fish were sacrificed on days 1, 2, 3, 4, 7,
14, 21, 28, and 35 of endosulfan exposure for determination of biochemical parameters.
To carry out the whole experiment with ease and authenticity and keeping into consideration the guidelines of the IEC that generally not more than hundred fish were allowed at
once, the experiment was carried out in five parts, i.e., only one treatment group was carried out once at a time, and the whole experiment was completed in 175 days. Fish behavior of C. carassius exposed to endosulfan resulted in the exhibition of aggressive
behavior, rapid gulping of water with circular swimming, and signs of respiratory distress
such as rapid ventilation, increased rate of gill opercular movements, or fish floating at
the water surface. However, the mortality did not exceed 10% during the trial period,
indicating that the treatments were sufficient to cause the changes described hereafter.
Water temperature was 19.7 2.6  C, pH 7.5
8.4, dissolved oxygen 7.9
8.4 mg L1,
total alkalinity 69
73 mg L1, ammonical nitrogen 25
29 mg L1, and conductivity
was 0.9
1.2 mScm1 (APHA, AWWA, and WPCF 2005).
2.4. LPO
LPO in fish blood, obtained by caudal puncture, was determined as described (Uchiyama
and Mihara 1978) by the thiobarbituric acid (TBA) method, with some modifications. A
500 mL aliquot of blood was mixed with 1 mL of 30% (w/v) trichloroacetic acid (Sigma,
Bengaluru, India) and centrifuged for 10 min at 5000 rpm. An aliquot of 25 mL of supernatant was mixed with 25 mL of an aqueous solution 10 mmol L1 of butylated hydroxytoluene (Himedia, Mumbai, India), 3 mL of an aqueous 1% solution of o-phosphoric acid
(Merck, Mumbai, India), and 1 mL of an aqueous 0.67% solution of 2-TBA (Merck). The
mixture was incubated for 45 min at 90  C. The concentration of TBA reactive substances
(TBARS) was calculated from the absorption at 535 nm and a molar extinction coefficient
of 1.56 105 L mol1cm1.
2.5. Antioxidative enzyme assays
2.5.1. Glutathione (GSH)
The assay mixture for the estimation of reduced GSH (Beutler, Duron, and Kelly 1963)
consisted of 100 mL blood, 900 mL double distilled water, and 1.5 mL precipitating

Toxicological & Environmental Chemistry

909

reagent (m-phosphoric acid, ethylene diamine tetra acetic acid, and sodium chloride). The
mixture was incubated at room temperature for 5 min and then centrifuged at 4000 rpm
for 15 min at 4  C. An aliquot of 1 mL of the supernatant was taken and mixed with 4 mL
of 0.3 mol L1 disodium hydrogen phosphate solution (Hi-Media) and 0.5 mL of
dithio-bis-2-nitrobenzoic acid (Sigma) prepared in 1% sodium citrate. The color intensity
of reaction product was measured at 412 nm using a UV
Vis spectrophotometer
(Shimadzu, Kyoto, Japan). The GSH content was expressed as nmol GSH/mg protein
(Lowry et al. 1951).

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2.5.2. Superoxide dismutase (SOD)

SOD activity was determined by modifying the method of Marklund and Marklund
(1974), which depends on the auto-oxidation of pyrogallol. A sample of 200 mL blood
was centrifuged at 3000 rpm for 15 min at 4  C and the supernatant aspirated. The erythrocyte rich precipitate was washed thrice using physiological saline (3:1 v/v); lysed by
distilled water and lysate was used for measuring the enzyme activity. The assay mixture
consisted of 8.7 mL of 50 mmol L1 phosphate buffer (pH 8.24), 100 mL lysate, and
300 mL of pyrogallol dissolved in 10 mmol L1 HCl. The rate of pyrogallol autooxidation was read at 420 nm. The SOD activity was expressed as units/mg protein. One
unit of SOD activity is defined as the amount of the enzyme that inhibit 50% of the
oxidation rate of 0.1 mmol L1 pyrogallol in 1 mL of solution at 25  C.
2.5.3. Catalase (CAT)
The method described by Johansson and Borg (1988), with few modifications, was used
for the estimation of CAT activity. The erythrocyte rich precipitate obtained by centrifuging 500 mL blood at 3000 rpm for 15 min at 4  C was washed thrice using physiological
saline (3:1 v/v) and lysed by distilled water. Briefly, three replicates of 20 mL lysate were
mixed with 100 mmol L1 potassium phosphate (pH 7.0) and 30 mL methanol. The reaction was initiated by adding 20 mL of 35 mmol L1 hydrogen peroxide (H2O2) and the
mixture was incubated for 20 min on shaker at room temperature. The reaction was terminated by adding 30 mL of 10 mol L1 potassium hydroxide. The triplicates of each sample were added with 30 mL of 4-amino-3-hydrazino-5-mercapto-1, 2, 4-triazole, a
chromogen (Sigma), and incubated for 10 min at room temperature on a shaker. After
incubation, 10 mL of potassium periodate (Sigma) was added to the mixture and again
incubated for 5 min. Finally, the absorbance of assay mixture was read at 540 nm using a
UV
Vis spectrophotometer. Enzyme activity was expressed as nmol L1 H2O2 decomposed/min/mg protein.
2.6. Statistical analysis
Probit analysis was performed with the SPSS (version 16.0) computer program (SPSS
Inc. Chicago, IL, USA), with the consultation of Finneys table (Finney and Stevens
1948). Data were compared for statistically significant difference between control and
treatment groups using one-way analysis of variance (ANOVA). Significant difference in
ANOVA were further analyzed by post-hoc Bonferronis, Newman
Keuls and Dunnetts
multiple comparison test using Graph Pad Prism 5 software (Graph Pad Software, Inc.
San Diego, CA). The p-values less than 0.05 were considered statistically significant. Furthermore, the increase or reduction percentage in case of biochemical biomarkers was

910

S.A. Dar et al.

calculated according to the following formula (Dar et al. 2013):

Increase=reduction% Control Test=Control100:

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3. Results and discussion

3.1. LC50 and application factor
In acute toxicity bioassay, the LC50 values (with 95% confidence limits) of endosulfan to
C. carassius were found to be 215 (158
272), 150 (112
191), 95 (75
114), and 70
(46
93) mg L1 for 24, 48, 72, and 96 h, respectively (Figure 1). A dose-dependent
increase and time-dependent decrease were observed in mortality rate such that as the
exposure time increases from 24 to 96 h, the median concentration required to kill the
fish was reduced. The estimated safe levels of endosulfan, as calculated by multiplying
the LC50
96 h with different application factors (AF), are given in Table 1. The LC50
96 h
value of the endosulfan in this study was 70 mg L1, which indicated that it is very toxic
to fish. Our estimate is higher than the LC50
96 h value of 35 mg L1 for Channa striatus
(Ganeshwade et al. 2012). The variation may be due to the difference and hardiness of
the test species and water quality parameters. The estimated safe levels of endosulfan in
C. carassius varied from 00.70 102 to 00.70 106 mg L1. However, the large variation in safe levels determined by different methods has resulted in controversy over its
acceptability (Pandey et al. 2005).

Figure 1. Probit line graph (95% confidence intervals) of acute toxicity of endosulfan to
C. carassius.

Toxicological & Environmental Chemistry

911

Table 1. Estimate of safe levels of endosulfan at 96 h exposure time.

Chemical
Endosulfan

LC50
96 h (mg L1)

Method

AF

Safe level (mg L1)

0.070

Sprague (1971)
CWQC (1972)
NAS/NAE (1973)
IJC (1977)

0.1
0.01
0.1
0.00001
5% LC50
96 h

00.70 102
00.70 103
00.70 102
00.70 106
00.35 102

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3.2. LPO
The LPO in blood of C. carassius increased significantly (p < 0.05) in all the three test
concentrations of endosulfan when compared to the negative control (Figure 2). The peroxidative damage was found to be time and concentration dependent with the highest at
SL-I on day 4 (720%), followed in its extent (490%) at SL-II on day 7 and lowest (260%)
at SL-III on day 14. However, the LPO level of all the test concentrations of endosulfan,
except SL-III, along with the positive control, showed a decreasing trend after day 4 till
termination of the experiment; but they were still significantly (p < 0.01) higher than that
of the negative control. LPO is one of the main manifestations of oxidative damage
induced by various toxic compounds including pesticides; hence, it has been used as an
indicator of pollution. Previous investigations, like our study, have also reported the
induction of LPO by pesticides such as chlorpyrifos, methyl parathion, butachlor, and
tebuconazol (Celik 2008; Toni et al. 2010). However, the level of LPO may differ among
species. Differences observed in TBARS levels could reflect variation in the antioxidant
mechanisms of fish, duration of exposure, and the pesticide tested (Marigoudar, Nazeer,

Figure 2. Effect of endosulfan on LPO in erythrocytes of C. carassius. Fish were exposed to positive control, cyclophosphamide (4 mg L1), endosulfan (SL-I
50, SL-II
35, SL-III
20 mg L1)
for 35 days; fish in tap water served as negative control. Values are expressed as mean SD and
values with different letter superscripts differ significantly (ap < 0.05 D significant; bp < 0.01 D
highly significant; cp < 0.001 D extremely significant) from the negative control (Newman
Keuls
test), whereas values with different numeric superscripts differ significantly (1p < 0.05 D significant; 2p < 0.01 D highly significant; 3p < 0.001 D extremely significant) between exposure times
within concentration (Bonferronis test).

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and David 2009). Many xenobiotics are known to cause oxidative stress through the generation of ROS and can alter the free oxygen radical scavenging enzyme systems in
aquatic organisms. ROS can react with susceptible biological macromolecules and cause
LPO, which in turn leads to nucleic acid damage and protein oxidation (Maran et al.
2009; Velisek et al. 2012). Thus, oxidative nucleic acid damage may be the most putative
mechanism of chronic toxicity of environmental pollutants.

3.3. Antioxidant response

GSH showed significant elevation versus control in SL-I up to day 7, with the highest
level (180%) on day 4. SL-II and SL-III also showed increasing trend till day 14 with the
maximum elevation (180%) on day 7 in case of SL-II followed by SL-III on day 14
(160%). However, there was a significant decrease in all the test concentrations of endosulfan from the day 21 of the post-exposure. SL-I showed the highest reduction (58%)
followed by SL-II (54%) and SL-III (45%) on day 35 (Figure 3). GSH acts as a cellular
reducing reagent and protects against the cellular redox cycling changes induced by
toxic substances. Increase in GSH levels are in agreement with many previous studies
(Hasspielar, Behar, and DiGiulio 1994; Stephensen, Sturve, and Forlin 2002), which
describe it as one of the protective mechanisms that fish adopt in the initial phases of
exposure to aquatic pollutants. GSH and GSH-related processes play a central role in
second line of antioxidant defense by contributing to a number of processes, such as freeradical scavenging, reduction of peroxides, and detoxification of electrophilic compounds. However, under a severe oxidative stress, GSH is consumed to detoxify the
peroxides produced due to increased LPO. A considerable decline in GSH content in the
present fish may be due to its utilization to challenge the prevailing oxidative stress under
the influence of ROS generated from endosulfan exposure (Celik 2008).

Figure 3. Effect of endosulfan on GSH in erythrocytes of C. carassius. Fish were exposed to positive control, cyclophosphamide (4 mg L1) and endosulfan (SL-I
50, SL-II
35, SL-III
20 mg L1)
for 35 days; fish in tap water served as negative control. Values are expressed as mean SD and values with different letter superscripts differ significantly (ap < 0.05 D significant; bp < 0.01 D highly
significant; cp < 0.001 D extremely significant) from the negative control (Newman
Keuls test),
whereas values with different numeric superscripts differ significantly (1p < 0.05 D significant;
2
p < 0.01 D highly significant; 3p < 0.001 D extremely significant) between exposure times within
concentration (Bonferronis test).

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913

Figure 4. Effect of endosulfan on SOD in erythrocytes of C. carassius. Fish were exposed to positive
control, cyclophosphamide (4 mg L1) and endosulfan (SL-I
50, SL-II
35, SL-III
20 mg L1) for
35 days; fish in tap water served as negative control. Values are expressed as mean SD and values
with different letter superscripts differ significantly (ap < 0.05 D significant; bp < 0.01 D highly significant; cp < 0.001 D extremely significant) from the negative control (Newman
Keuls test),
whereas values with different numeric superscripts differ significantly (1p < 0.05 D significant; 2p <
0.01 D highly significant; 3p < 0.001 D extremely significant) between exposure times within concentration (Bonferronis test).

The SOD activity showed a significant decrease throughout the period of study
(Figure 4), with highest reduction (60%) in case of SL-I on day 4 of post-exposure. SL-II
and SL-III also showed a significant reduction compared to control, with maximal (55%)
on day 7 in case of SL-II and lowest (45%) at SL-III on day 14. A concentration and
time-dependent response in the reduction of SOD activity was observed, i.e. higher the
concentration of endosulfan lesser will be the time required for the reduction of SOD
activity and vice-versa. Similar results were verified in Oreochromis niloticus and Cyprinus carpio (Oruc and Usta 2007). This could be an indicator of compensatory cell
response to dismutase the superoxide radical to H2O2, which itself is an important ROS
(Li et al. 2009).
The CAT activity at SL-I showed a significant increase up to day 14 compared to negative control, with the highest induction (65%) on day 4, but showed a significant reduction from day 28 till the termination of the experiment (Figure 5). SL-II showed highest
increase (75%) on day 7 followed by SL-III (65%) on day 14. The values of CAT of both
SL-II and SL-III did not show any significant difference compared to negative control on
day 28, but like SL-I, they were also debased significantly on day 35 of post-exposure.
The increase in CAT activity may be in response to H2O2 produced by SOD activity,
since CAT is responsible for the conversion of H2O2 to water. A significant increase in
CAT activity has been observed in some studies after fish exposure to pesticides (Maran
et al. 2009; Jin et al. 2010). The reduction of CAT activity by higher concentration at the
termination of the experiment could be justified by the flux of superoxide radicals, which
have been reported to inhibit CAT activity. Taken together, CAT and SOD results indicate a disruption of normal oxidative process, indicating an impairment of antioxidant
defense system.

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S.A. Dar et al.

Figure 5. Effect of endosulfan on CAT in erythrocytes of C. carassius. Fish were exposed to positive control, cyclophosphamide (4 mg L1) and endosulfan (SL-I
50, SL-II
35, SL-III
20 mg L1)
for 35 days; fish in tap water served as negative control. Values are expressed as mean SD and values with different letter superscripts differ significantly (ap < 0.05 D significant; bp < 0.01 D highly
significant; cp < 0.001 D extremely significant) from the negative control (Newman
Keuls test),
whereas values with different numeric superscripts differ significantly (1p < 0.05 D significant; 2p <
0.01 D highly significant; 3p < 0.001 D extremely significant) between exposure times within concentration (Bonferronis test).

4. Conclusions
This study revealed that endosulfan is responsible for oxidative stress in the fresh water
fish C. carassius as shown by increase in LPO; in response, the antioxidant defense mechanisms were altered, and oxidative stress appears to be one of the main mechanisms of
toxic action of endosulfan. A large-scale use of chlorinated cyclodienes has been reported
in pest control activity in agriculture sector as well as in eradication of some insect-borne
diseases in several countries including India (Pandey et al. 2005). This study offers some
insight into the eco-genotoxicological consequences of an important member of chlorinated cyclodienes at consumer level of food chain. Information obtained through these
integrated studies in fish model may be useful for regulatory agencies entrusted with
rational use of pesticides.

Acknowledgments
Results showed in this work are part of the PhD thesis of the first author who thanks the University
Grants Commission (UGC) for his Junior Research Fellowship (JRF). The authors also thank Director of the Centre of Research for Development (CORD), University of Kashmir, for providing necessary research facilities.

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