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Chemosphere 120 (2015) 273283

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Chemosphere
journal homepage: www.elsevier.com/locate/chemosphere

Assessment of endosulfan induced genotoxicity and mutagenicity


manifested by oxidative stress pathways in freshwater cyprinid sh
crucian carp (Carassius carassius L.)
Sabzar Ahmad Dar a,, Abdul Rehman Yousuf a, Masood-ul-Hassan Balkhi b, Farooq Ahmad Ganai a,
Farooz Ahmad Bhat b
a
b

Limnology and Fisheries Laboratory, Centre of Research for Development (CORD), University of Kashmir, Srinagar, J & K, India
Division of Fisheries, Sher-e-Kashmir University of Agricultural Sciences and Technology of Kashmir (SKUAST-K), J & K, India

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

 Endosulfan induces mutagenesis;

water contamination can be detected


by DLLME, followed by GCMS.
 Micronucleus and chromosomal
assays are sensitive biological
biomarkers of aquatic pollutants.
 Scanning electron microscopy (SEM),
an efcient tool for authenticating the
genetic damage.
 Mechanism of genotoxicity potential
of endosulfan appears to be
manifested by oxidative stress
pathways.
 Integrated studies in sh model may
be useful for regulatory agencies
entrusted with rational use of
pesticides.

a r t i c l e

i n f o

Article history:
Received 13 February 2014
Received in revised form 9 July 2014
Accepted 14 July 2014

Handling Editor: A. Gies


Keywords:
Endosulfan
Fish
Oxidative stress
Micronucleus
Genetic damage

a b s t r a c t
Over the past few decades, endosulfan, one of the polychlorinated pesticides still in use, has received considerable attention of a number of international regulations and restriction action plans worldwide. This
study aimed to evaluate the cytogenetic effects of endosulfan using robust genotoxicity assays, along
with the oxidative stress pathways in order to understand biochemical mechanism, in Carassius carassius
L. The LC5096 h (95% condence limits) value of endosulfan was 0.070 (0.0460.093) ppm; and on its basis
three test concentrations (sub-lethal I: 0.052, II: 0.035 and III: 0.017 ppm) were selected for 35 d in vivo
exposure. The mean concentration of endosulfan in aquaria was always constant, when analyzed by dispersive liquidliquid micro extraction (DLLME) followed by GCMS. Autopsy was done on days 1, 2, 3, 4,
7, 14, 21, 28 and 35 of endosulfan exposure; the micronucleus formation (MN), authenticated by scanning
electron microscopy, and chromosomal aberrations (CA), were induced signicantly (p < 0.05) in all the
treated groups, including positive control cyclophosphamide (4 ppm), when compared to negative control. Similarly lipid peroxidation (LPO) was induced signicantly with the maximal at higher concentration (SL-I) on 4th day (722.45%; p < 0.01). Antioxidant biomarkers like glutathione reduced, superoxide

Corresponding author. Address: Limnology and Fisheries Laboratory, Centre of


Research for Development (CORD), University of Kashmir, Srinagar, J & K 190006,
India. Cell: +91 09797970976.
E-mail address: sabzar.cord@gmail.com (S.A. Dar).
http://dx.doi.org/10.1016/j.chemosphere.2014.07.031
0045-6535/ 2014 Elsevier Ltd. All rights reserved.

274

S.A. Dar et al. / Chemosphere 120 (2015) 273283

dismutase and catalase also uctuated signicantly (p < 0.01) in all treatment groups. Collective ndings
demonstrated that genotoxic effects were invariably accompanied and correlated with increased
oxidative stress and disturbance of antioxidant enzymes; and the MN and CA assays are useful tools in
determining potential genotoxicity of aquatic xenobiotics and might be appropriate as a part of monitoring program.
2014 Elsevier Ltd. All rights reserved.

1. Introduction
Pesticide residues reach the aquatic environment, where it
poses signicant toxicological risks to a myriad of non target
organisms (Ondarza et al., 2014), and nally nding their way to
the food chain threatening the ecological balance and the biodiversity of the nature (Konstantinou et al., 2006). These contaminators
of surface waters have been well documented worldwide and constitute a major issue that give rise to concerns at local, regional,
national and global scales (Lazartigues et al., 2013). The mutagenic
and carcinogenic action of herbicides, insecticides and fungicides
on experimental animals is well known and several studies have
shown that chronic exposure to low levels of pesticides can cause
mutations and carcinogenicity (Bull et al., 2006). The worldwide
pesticide consumption in the year 2000 was estimated 5.35 billion pounds (USEPA, 2001). In India, the pesticide consumption
was increased by more than fourfold during the post-Green Revolution era (19661999). Although the consumption of pesticides in
India is estimated to be the lowest at 0.5 kg ha1 as against 17 in
Taiwan, 12 in Japan, 6.6 in Korea, 7 in the United States and
2.5 kg ha1 in Europe, the food and agricultural products contain
substantial quantities of pesticide residues (Assocham, 2007). The
major reasons are indiscriminate and nonjudicious use of chemical
pesticides as well as nonobservance of prescribed waiting periods.
Endosulfan is a persistent organochlorine pesticide (OCP), which
along with aldrin, dieldrin, chlordane and heptachlor belongs to the
group of chlorinated cyclodienes. As a commercial pesticide it comprises of two stereoisomers (a- and b-endosulfan) at a ratio of
70:30, and is now being detected as an important cause of pesticide
poisoning in many countries (Pathak et al., 2010; Sabzar et al.,
2014). Endosulfan and its metabolites have regularly been detected
in groundwater, surface water, sediment and rain and snow in
many regions of the world (Tuduri et al., 2006; Kumari et al.,
2007). Aquatic animals periodically suffer from the lethal concentrations of endosulfan ranging from <0.01 to 0.54 mg L1 in surface
waters (Harris et al., 2000). According to the USA environmental
protection agency (EPA), endosulfan has deleterious effect on the
health of aquatic organisms at concentrations above 0.22 lg L1
(acute) and 0.056 lg L1 (chronic) (Mersie et al., 2003). All of the
gures reported below exceed this level. The reports on concentrations of endosulfan in run-off include 66.5 lg L1 in India, 45 lg L1
in Australia and 100 lg L1 in southern USA (Menone et al., 2008).
Furthermore, in India, values from some important places of endosulfan application range from 2.63 to 3.72 lg L1 in surface water of
northern Indo-Gangetic alluvial region (Singh et al., 2007), 2.16
567.49 ng L1 in Gomti River (Malik et al., 2009) and in the Ganga
River it can reach up to 66.5 lg L1 (Selvakumar et al., 2005).
Despite the discontinuation of the use of endosulfan in many countries such as Germany, UK, Sweden, Netherlands, Colombia and Singapore, it is still widely used in most of the developing countries
because of its effectiveness and low application cost (Ondarza
et al., 2014). Therefore, it still remains a serious environmental concern due to its persistence and slow degradation processes in the
environment. In India, endosulfan is classied as an extremely
hazardous pesticide; affecting the central nervous system, reproductive system and immune system (Ganeshwade et al., 2012).

The use of sh as a bio-indicator of pollutant effects is being


more and more used since sh are very sensitive to changes in
their environment and play signicant roles in assessing potential
risk associated with contamination of new chemicals in aquatic
environment (Lakra and Nagpure, 2009). Ecotoxicological characteristics of freshwater sh, Carassius carassius L., such as its wide
distribution and availability throughout the year, easy maintenance in the aquaria and commercial importance make this species
an excellent model for toxicity studies. Since there is growing concern over the presence of genotoxins in the aquatic environment, it
is important to develop or standardize the existing methods for
detection of genotoxic chemicals in aquatic organisms (Pandey
et al., 2006).
Genetic damage at the chromosomal level entails alterations in
either chromosome structure or chromosomal number and such
alterations can be measured as chromosomal aberrations or micronucleus frequency. Several studies have shown that the micronucleus and chromosomal aberration test are the two sensitive,
rapid and extensively used methods in the detection of mutagenicity and genotoxicity of xenobiotics under eld and laboratory conditions (Ansari et al., 2011).
Chlorinated cyclodienes, including endosulfan have high toxicity to a broad spectrum of aquatic organisms and thus, it is imperative to understand the eco-genotoxicological implication of
endosulfan in water bodies and its susceptibility to aquatic biota.
Many classes of pesticides or their residues may exert toxicity
related to oxidative stress and can cause oxidative damage in sh
(Velisek et al., 2012). Biochemical constituents, like lipid peroxidation and antioxidant enzymes, are the potential biomarkers of oxidative stress in different organisms including sh (Bechard et al.,
2008). They have the advantage of being more sensitive, less variable, highly conserved between species and often easier to measure as stress indices (Agrahari et al., 2007). Most of the studies
on the endosulfan effect are conned to the doseresponse and
physiological changes of the sh. There is relatively less attention
being given to the genotoxic modulation induced by the endosulfan at environmentally realistic concentrations. The objective of
the present study was to study the chronic genotoxic and mutagenic effects of endosulfan, along with its mechanism of action,
in freshwater sh C. carassius. These considerations have prompted
interest in the development of such techniques and its use as bioindicators for monitoring the genomic damage from environmentally hazardous contaminants in the aquatic environment.

2. Materials and methods


2.1. Experimental sh specimen and chemicals
C. carassius L. (Family: Cyprinidae and Order: Cypriniformes),
having chromosome number (2n) 100 with four types of chromosomes: submetacentric, metacentric, subtelocentric and acrocentric (Knytl et al., 2013), was selected as the test organism. Live
juvenile specimen, procured with the help of hand nets from the
pollution free area of the Dal Lake (34070 N 74520 E), were transported to the laboratory and subjected to a prophylactic treatment

S.A. Dar et al. / Chemosphere 120 (2015) 273283

by bathing in 0.05% potassium permanganate (KMnO4) for 2 min to


avoid any dermal infection. Their average length and wet weight
(SD) were recorded as 12.5 1.64 cm and 33 4.94 g, respectively. The sh stock in good number was then acclimatized for
at least 3 weeks to 1:1 diurnal photoperiod in articially aerated
60 L glass aquaria at 19.66 2.58 C, with aged dechlorinated tap
water (pH 7.68.4), and fed ad libitum once in 24 h (10% bw) with
commercially available sh food (Feed Royal, Maa Agro Foods,
Andhra Pradesh, India). Every effort, as suggested by Bennett and
Dooley (1982), was made to maintain optimal conditions during
acclimatization. For the present study, endosulfan with a and b isomers in the ratio of 7:3 (CAS No. 45852), cyclophosphamide (CAS
No. C3250000-1EA), 4-amino-3-hydrazino-5-mercapto-1,2,4-triazole (chromogen) (CAS No. 162892), potassium periodate (CAS
No. 210056), trichloroacetic acid (CAS No. 76039) and dithiobis-2-nitrobenzoic acid (CAS No. D218200) were purchased from
the Sigma Aldrich, India. o-phosphoric acid (CAS No. 100563),
pyrogallol (CAS No. 1006120050) and thiobarbituric acid (TBA,
CAS No. 108180) were purchased from, Merck, Germany. 2-butylated hydroxytoluene (BHT, CAS No. 128370) was purchased from
Hi-Media Labs, Mumbai, India. All other chemicals, used in the
study were also of analytical grade.
2.2. Acute toxicity and determination of sublethal concentrations
The acute toxicity bioassay of endosulfan in C. carassius was
conducted in a semi-static system, to determine its LC5096 h value.
Since endosulfan was emulsiable concentrate, it was directly
added to the system. The LC5096 h value of endosulfan was determined following the probit analysis method as described by
Finney (1971). Based on the LC5096 h value, three test concentrations of test chemical, namely sublethal concentration I (SL-I; 1/
4th of LC5096 h), II (SL-II; 1/2nd of LC5096 h) III (SL-III; 3/4th of
LC5096 h), were determined for the in vivo experiment. The estimated safe levels of endosulfan were calculated by multiplying
the LC5096 h with different application factors (AF) (Sprague,
1971; CWQC, 1972; NAS/NAE, 1972). This and other animal experiments were in accordance with the principles of the Institutional
Ethical Committee (IEC) for the Protection of research animals in
the University of Kashmir, and were conducted under the United
States Environmental Protection Agency Report No. EPA 821-R02-012 (EPA, 2002).
2.3. In vivo exposure experiment
The semi-static assay consisted of ve treatments each with 9
replicates (total 45 aquaria), containing 60 L dechlorinated and
well-aerated tap water with 10 sh specimen in each aquaria
(n = 450). The specimen maintained in dechlorinated tap water
and those exposed to cyclophosphamide (4 ppm, concentration
selection based on previous investigation; zkan et al., 2011) were
considered as the negative and positive controls, respectively. In
treatments 35, the sh specimen were kept in water containing
the three aforementioned test concentrations of the endosulfan
and the samplings were done on days 1, 2, 3, 4, 7, 14, 21, 28 and
35. On each sampling interval, ten sh specimen were sacriced;
ve sh were processed for the chromosomal aberration test
(0.05% colchicine treatment was given prior to 3 h of autopsy)
and the micronucleus and biochemical assay were carried out from
the blood of the rest ve sh as per standard protocols. Some
important physico-chemical properties of test water like temperature 18.223.3 C, pH 7.58.4, dissolved oxygen 7.98.4 mg L1,
total alkalinity 6973 mg L1, ammonical nitrogen 2529 lg L1
and conductivity 211239 lM cm1 were analyzed throughout
the study by standard methods (APHA, AWWA and WPCF, 2005).
The sh behavior was observed according to the standard method

275

(Rao et al., 2005). Briey, before recording, sh from control and


exposed groups were acclimatized individually for ten minutes in
a recording glass aquarium. The internal three sides of the aquarium and bottom were made opaque to avoid the mirror image of
the sh and visual disturbances. The sh behavior was recorded
for ve minutes (n = 10) with the help of a high resolution HD
camera (SONY, DCR-DVD650E, Japan), mounted 20 cm away from
the left over plain side of the recording aquarium. To ensure an
agreement between nominal and actual test chemical concentrations in the aquaria, water samples were analyzed during the
experimental period by dispersive liquidliquid micro extraction
(DLLME) followed by gas chromatography-mass spectrometry
(GCMS) (Shegefti et al., 2010). Because of innitely large surface
area between extraction solvent and aqueous sample, the equilibrium state is achieved quickly and extraction is independent of
time. So, under optimum conditions, 2 mL of each sample was
placed in a 10 mL screw cap glass tube with conical bottom, and
500 lL of methanol (as disperser solvent) containing 40 lL chloroform (as extraction solvent) was injected rapidly into each sample
solution using a 1 mL syringe. The mixture was centrifuged for
5 min at 5000 rpm using the centrifuge. The dispersed ne particles of extraction solvent separated and settled at the bottom of
conical tube. 1 lL of the separated phase was removed using a
micro syringe and injected into the GCMS (GCMS-QP2010 Plus,
Shimadzu, Japan).
2.4. Genotoxicity biomarkers
2.4.1. Micronucleus test
Prior to blood collection, sh were anaesthetized with
0.12 g L1 benzocaine (Marques de Miranda Cabral Gontijo et al.,
2003). Blood samples were withdrawn by cardiac puncture and
peripheral blood smears were immediately made by applying
two micro drops of blood on precleaned, grease free slides, using
the standard sh micronuleated erythrocytes method of Al-Sabti
and Metcalfe (1995). For every sampling time, replicate slides per
specimen were prepared and a minimum of 10000 erythrocytes
scored in each treatment group, were examined for the presence
of MN. The frequency of MN/sh was calculated per 1000 cells
(Raisuddin and Jha, 2004), and was evaluated by scoring the slides
under oil immersion at 1000 magnication using Olympus BX 50
microscope (Tokyo, Japan). Coded and randomized slides were
scored using blind review by a single observer to avoid any technical variation. The criteria for the identication of micronuclei were
according to standard procedures (Fenech et al., 2003).
2.4.2. Scanning electron microscopy (SEM)
The SEM was carried out by standard procedure (Reimer, 1985).
Briey, the aforementioned micronucleated slides were reshaped,
sputter-coated with platinum to a layer of 35 nm, and exclusively
examined in the secondary electron mode, at an accelerating voltage of 10 kV, with a scanning electron microscope (JSM6510LV,
JEOL, Japan). The images were recorded simultaneously with
Digiscan hardware and processed with Digital Micrograph 3.4.4
software (Gatan, Inc., Pleasantdon, CA, USA).
2.4.3. Chromosomal aberration test
Chromosome preparations were made from the highly haemapoietic and mitotically active head kidney cells, following the techniques of Cucchi and Baruffaldi (1990), and observed with light
microscope (100) for chromosomal aberrations. Replicate slides
were selected per sh and a minimum of 25 metaphases were
scored from each slide in each group including control (since
n = 5 per group/exposure time, minimum 250 metaphases).
Notwithstanding the conventional method of scoring, the CA was
recorded under two broad categories i.e. classical aberration and

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S.A. Dar et al. / Chemosphere 120 (2015) 273283

non classical aberration. In the classical aberrations, both chromosome and chromatid type breaks, including acentric fragments,
sister chromatid union and multiple aberrations (polyploidy,
aneuploidy, rings etc.) were counted and non-classical aberration
comprised of stickiness, pulverization and c-metaphases. Other
aberrations have not been counted for the statistical analysis of
the data due to controversial genetic signicance and also because
some deletions and rearrangements (translocation, inversion) may
not be lethal (DHSS, 1982).
2.4.4. Oxidative stress biomarkers
The thiobarbituric acid (TBARS) method described by Uchiyama
and Mihara (1978) was used to evaluate lipid peroxidation (LPO) in
the sh blood, with little modication. The assay consisted of, 25 lL
of supernatant mixed with 25 lL of 10 mM butylated hydroxytoluene (BHT), o-phosphoric acid (3 mL of 1% solution) and 2-thiobarbituric acid (TBA, 1 mL of 0.67% solution). The incubation time for the
mixture was 45 min at 90 C. The concentration of TBARS was
calculated from the absorption at 535 nm (Shimadzu, Kyoto, Japan)
and a molar extinction coefcient of 1.56  105 M1 cm1.

2.4.6. Protein estimation


Protein content was determined using colorimetric method and
bovine serum albumin (BSA) as standard (Lowry et al., 1951).
2.5. 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 was compared for statistically signicant difference between control and
treatment groups using one-way analysis of variance (ANOVA).
Signicant differences in ANOVA were further analyzed by post
hoc Bonferronis, Newman-Keuls and Dunnetts multiple comparison tests. Correlation analysis was performed to analyze relationship between data from MN test (a cytogenetic genotoxic test)
and LPO (a robust oxidative stress biomarker) by using SPSS (version 16.0). Furthermore, the increase or reduction percentage in
case of biochemical biomarkers was calculated according to the
following formula (Sabzar et al., 2013):

Increase=reduction% Control  Test=Control  100:


2.4.5. Antioxidant enzyme assays
The method of Beutler et al. (1963) was used for the estimation
of glutathione reduced (GSH) in sh erythrocytes. The assay mixture consisted of 100 lL of blood, 900 lL of double distilled water
and 1.5 mL of precipitating reagent (m-phosphoric acid, EDTA and
NaCl, Merck). The mixture was incubated at room temperature for
5 min, and then centrifuged at 4000 rpm for 15 min at 4 C. From
this mixture, 1 mL of supernatant was taken out to mix with
4 mL of phosphate solution (0.3 M disodium hydrogen phosphate)
and 0.5 mL of dithio-bis-2-nitrobenzoic acid (prepared in 1%
sodium citrate; Sigma). The color intensity of reaction product
was measured at 412 nm using UVvis spectrophotometer. The
GSH content was expressed as nmol GSH/mg protein using standard calibration.
SOD activity was determined by the method of Marklund and
Marklund (1974), which depends on the auto-oxidation of pyrogallol. 200 lL of blood sample was centrifuged at 3000 rpm at 4 C for
15 min and the supernatant aspirated. The erythrocyte rich precipitate was washed thrice using physiological saline (3:1 v/v) and
lysed by distilled water. The lysate was used for enzyme activity
measurement. The assay mixture consisted of 8.7 mL of 50 mM
phosphate buffer (pH 8.24), 100 lL lysate and 300 lL of pyrogallol
(dissolved in 10 mM HCl, Merck, Germany). The rate of pyrogallol
auto-oxidation was read at 420 nm. The SOD activity was
expressed as units/mg protein.
The CAT activity was determined by centrifuging 500 lL of
blood at 10 000 rpm for 30 min at 4 C; the supernatant from each
sample was used as the enzyme source and analyzed according to
the method described by Johansson and Borg (1988) with few
modications. The principle of the assay is based on the reaction
of the CAT with methanol in the presence of an optimal concentration of hydrogen peroxide (H2O2), and the measurement of formaldehyde produced. Briey, three replicates of 20 lL supernatant
were mixed with 100 mM potassium phosphate (pH 7.0) and
30 lL methanol. The reaction was initiated with 20 lL of 35 mM
H2O2 and the mixture was incubated for 20 min on shaker at room
temperature. The reaction was terminated by adding 30 lL of 10 M
potassium hydroxide. 30 lL of 4-amino-3-hydrazino-5-mercapto1,2,4-triazole (chromogen) was added to the three replicates of
each sample, and incubated for 10 min at room temperature on a
shaker. After incubation, 10 lL of potassium periodate was added
to the mixture and again incubated for 5 min at room temperature.
Finally, the absorbance of assay mixture was read at 540 nm using
UVvis spectrophotometer. Enzyme activity was expressed as nM
H2O2 decomposed/min/mg protein.

3. Results and discussion


3.1. Acute toxicity
In acute toxicity bioassay, the LC5096 h value (with 95% condence limits) of endosulfan in C. carassius was found to be 0.070
(0.0460.093) ppm (Table 1). Our estimate is higher than the
LC5096 h value of 0.0035 ppm 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. Based on the LC50
96 h value, the SL-I, II and III were determined as 0.052, 0.035 and
0.017 ppm respectively, which were further used for in vivo exposure. The estimated safe levels of endosulfan in the sh species under
study varied from 00.70  102 to 00.70  106 ppm. However, the
large variation in safe levels determined by different methods has
resulted in controversy over its acceptability (Pandey et al., 2005).
The exposure of C. carassius 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 sh oating at
the water surface. These characteristics have also been reported
in Oreochromis niloticus, Chrysichthyes auratus and C. auratus
(Hussein et al., 1996; Saglio and Trijasse, 1998; Oropesa et al.,
2009) following acute poisoning with various xenobioitics. The
symptoms exhibited correspond to the physical deformity syndrome (PDS), an indicator of neurotoxicity described by
Drummond and Russom (1990) in fathead minnows (Pimephales
promelas) exposed to several toxic agents. The exact pathway that
leads endosulfan to exert its neurotoxic effect on sh is still
unknown. Endosulfan exposure was found to up regulate histamine
receptors sub types 3 and 1 in localized areas of the brain (pre-optic
and hypothalamic areas) involved predominantly on endocrine
dependent activities in the ornate wrasse (Thalassoma pavo) leading
to a higher hyperventilation activity and decreased feeding behavior (Giusi et al., 2005). However, in the present study the exposed
sh were not facing such conditions like the deciency of food.
Therefore, the PDS of sh may be due to the toxicant induced stress,
which is an indication of neurotoxicity. One way toxicants can
induce a PDS is through the inhibition of acetylcholinesterase
(AChE) (Drummond and Russom, 1990). This enzyme is involved
in several key optomotor behaviors of sh, including locomotion,
orientation, feeding, and predator evasion (Scott and Sloman,
2004). Reduction of brain AChE enzyme activity leads to an

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S.A. Dar et al. / Chemosphere 120 (2015) 273283


Table 1
Lethal concentration (LC) of endosulfan (ppm) (95% condence intervals) depending on exposure time for Carassius carassius.
LC

Exposure Time (h)


24

LC5
LC10
LC30
LC50
LC70
LC90
LC95

0.080
0.135
0.176
0.215
0.240
0.303
0.361

48
(0.0260.133)
(0.0750.194)
(0.1290.223)
(0.1580.272)1
(0.1560.323)2
(0.2510.355)3
(0.2640.458)3

0.070
0.085
0.120
0.151
0.210
0.273
0.310

72
(0.0030.136)
(0.0450.124)A
(0.0570.182)
(0.1120.191)A
(0.1350.284)2
(0.1800.366)2
(0.2380.381)2

96

0.040
0.060
0.080
0.095
0.160
0.251
0.293

(0.0030.076)
(0.0170.102)A
(0.0270.132)A
(0.0750.114)C
(0.0940.225)A2
(0.2340.268)2
(0.2220.363)2

0.030
0.040
0.050
0.070
0.130
0.233
0.270

(0.0110.048)
(0.0170.062)B
(0.0220.077)C
(0.0460.093)C
(0.0740.185)B2
(0.1780.287)A2
(0.2020.337)A2

Values with different alphabet superscript differ signicantly (Ap < 0.05: signicant. Bp < 0.01: highly signicant. Cp < 0.001: extremely signicant.) between exposure time
within lethal concentrations, whereas values with different numeric superscripts differ signicantly (1p < 0.05: signicant. 2p < 0.01: highly signicant. 3p < 0.001:extremely
signicant) between concentrations within duration (Dunnetts multiple comparison test).

Table 2
Mean (S.E.) percentage micronucleus frequency in blood erythrocytes of Carassius carassius exposed to three concentrations of endosulfan (n = 10000 cells/concentration/
exposure time).
Chemical

Concentration
(ppm)

Exposure time (days)


1

14

21

28

35

NC

0.181
(0.007)
3.483
(0.126)C

0.230
(0.023)
4.100
(0.060)C3

0.191
(0.016)
4.386
(0.045)C3

0.300
(0.015)3
7.526
(0.113)C3

0.288
(0.018)2
6.901
(0.055)C3

0.250
(0.012)
6.325
(0.056)C3

0.203
(0.012)1
5.738
(0.110)C3

0.240
(0.016)
5.198
(0.035)C3

0.225
(0.025)1
4.840
(0.084)C3

2.590
(0.080)C
1.270
(0.021)C
0.671
(0.017)A

3.171
(0.065)C3
1.801
(0.022)C3
0.911
(0.016)A3

3.575
(0.028)C2
2.168
(0.046)C2
1.010
(0.028)A2

6.071
(0.058)C3
2.550
(0.033)C2
1.406
(0.025)A3

5.611
(0.052)C3
4.440
(0.054)C3
1.866
(0.036)A3

5.270
(0.050)C3
3.975
(0.089)C3
2.978
(0.059)A3

4.800
(0.052)C3
3.306
(0.082)C3
2.410
(0.087)A3

4.096
(0.066)C3
2.756
(0.070)1
1.693
(0.064)A1

3.733
(0.041)C2
2.021
(0.065)C1
1.113
(0.075)A2

PC
Endosulfan
SL I
0.052
SL II

0.035

SL III

0.017

NC: negative control (tap water). PC: positive control (cyclophosphamide: 4 ppm). SL I: sub lethal I (1/25 of LC50: 0.052 ppm). SL II: sub lethal II (1/50 of LC50: 0.035 ppm). SL
III: sub lethal (1/75 of LC50: 0.017 ppm). Values with different letter superscripts differ signicantly from the negative control (NewmanKeuls and Dunnetts multiple
comparison test), whereas values with different numeric superscripts differ signicantly between exposure times within concentration (Bonferronis test).

accumulation of acetylcholine in the brain tissue, interfering with


energy metabolism in the nervous system and preventing transmission of nerve impulses, thereby causing behavioral alterations
(Dutta et al., 1992; Rao et al., 2005). Furthermore, the primary site
of action of some cyclodienes insecticides, including endosulfan, is
the interference with GABA receptors in the brain of target insects
(Bloomquist, 2003). An inhibition of the GABA-induced chlorine
ux was also observed in cultured cerebellar granule cells of mice
treated with a-endosulfan, probably due to binding to picrotoxinin
sites (Vale et al., 2003), so it is possible that the neurotoxic effect of
endosulfan in sh may also follows this pathway.
Water samples were collected from the aquaria 1 h and 24 h
after renewing the test solutions. The mean concentration of endosulfan in the water samples was always within 5% of the intended
concentration (sub-lethal I: 0.052, II: 0.035 and III: 0.017 ppm),
when analyzed by DLLME followed by GCMS.
3.2. Genotoxicity biomarkers
The results of MN induction in peripheral blood erythrocytes of
C. carassius after exposure to different concentrations of endosulfan
are presented in Table 2. It caused one, two and three micronucleated cells but single MN was predominant, when analyzed by SEM
analyses (Fig. 1), which provides efcient results as compared to
simple microscopy. The maximum induction of MN frequency
was observed on day 4 (261.3%; p < 0.001) at the highest concentration (SL-I), whereas the MN formation for SL-II and SL-III concentrations, were highest on day 7 (170%; p < 0.001) and day 14
(158.8%; p < 0.05), respectively. The time required for induction
of MN increased with the decrease in test concentration of the
endosulfan, in general. These results are more environmentally

relevant than previous studies, which have typically used injection


as the route of exposure, because waterborne exposure is more
realistic of what occurs in nature. Fish absorb and accumulate
xenobiotics from water and food particularly those with water
solubility and high lipophilicity. The uptake from water occurs
because of the very intimate contact with the medium that carries
the chemicals in solution or suspension and also because sh have
to exchange oxygen from the medium by passing enormous volumes of water over the gills (Murty, 1986). Presumably, endosulfan
has affected the genetic material by absorption through the gill
epithelium. Earlier, it has been emphasized that exposure of sh
to genotoxic chemicals, for various interval of time, by the respiratory route following the absorption of chemicals through gill epithelium could be occurred (Rishi and Grewal, 1995; Farah et al.,
2006). Our results of MN are in agreement with some earlier studies (Bahari et al., 1994; Ali et al., 2008).
The frequency of CA observed in C. carassius after exposure to
different concentrations of test chemical and standard genotoxic
agent, cyclophosphamide, were signicantly (p < 0.05) higher
when compared to the negative control (Table 3), at all the exposure durations. The maximum CA, like MN frequency, was
observed on day 4 (12.14%; p < 0.01) at the highest concentration
(SL-I), whereas the CA for SL-II and SL-III concentrations, reached
the maximum value on day 7 (10.18%; p < 0.01) and day 14
(10.25%; p < 0.01), respectively. The CA were more at higher as
compared to lower test concentrations (Fig. 2), throughout the post
exposure, except at the termination of the experiment where the
CA showed the constancy effect at all the tested concentrations,
as reported by Rishi and Grewal in case of dichlorvos impacts on
Channa punctatus (Rishi and Grewal, 1995). The chromatid and
chromosome breaks were more frequent than the other types of

278

S.A. Dar et al. / Chemosphere 120 (2015) 273283

Fig. 1. Scanning electron microscopy image used to show micronucleus formation in erythrocytes of Carassius carassius. (A) Image of normal erythrocytes. (B) Image from the
positive control group (cyclophosphamide 4 ppm). (C, D and E) images from sh exposed to SL I-0.052, SL II-0.035 and SL III-0.017 ppm concentrations of endosulfan,
respectively for 96 h (1000, scale bar = 10 lm). (F) shows the normal (F1) and micronucleated erythrocyte (F2) (5000, scale bar = 5 lm).

aberrations at all the exposure durations in our study. Similar


results have also been reported by Rita and Milton (2008) in C.
punctatus and Yadav and Trivedi (2009) in Orechromis mosambicus
on exposure to various xenobiotics. The current study, thus,
emphasized that the CA and MN assays are sensitive biological
makers for evaluating the mutagenic and genotoxic effects of various clastogenic agents, especially in the aquatic environment.
3.2.1. Oxidative stress biomarkers
Oxidative stress, is an adverse reaction resulting from the exposure of molecules, cells or tissues to excess levels of free radical
oxidants, especially reactive oxygen species (ROS) (Li et al., 2009;
Velisek et al., 2012). Under normal conditions, antioxidant systems
within the cell minimize the perturbations caused by ROS (Kavitha
and Rao, 2009). To neutralize ROS, animals including aquatic

organisms have evolved antioxidant defense pathway comprising


of enzymes such as SOD, CAT and as well as non-enzymatic antioxidants such as GSH, which prevent oxidative damage (Modesto and
Martinez, 2010).
A general pathway of toxicity for many environmental pollutants is mediated by the enhancement of intracellular ROS, which
modulate the occurrence of cell damage via initiation and propagation of LPO (Oruc and Usta, 2007). LPO is a complex process in
which polyunsaturated fatty acids in the biological membrane system undergo changes by chain reactions and form lipid hydroperoxides, which decompose double bonds of unsaturated fatty acids
and disrupt membrane lipid (Radwan et al., 2010). The oxidative
stress was reected by signicant increase (p < 0.05) in LPO in all
the three test concentrations of endosulfan when compared to
the negative control (Table 4). The peroxidative damage was found

279

S.A. Dar et al. / Chemosphere 120 (2015) 273283


Table 3
Chromosomal aberration frequencies induced by endosulfan in Carassius carassius head kidney cells.
Exp. (days)

Treatment

TMS

Csb

Ctb

Frg

Scu

Dic

Mla

Stp

Cmt

NC
PC
Endosulfan
SL I
SL II
SL III

103
105

1
3

1
2

1.94 0.132
8.57 0.41B

113
101
107

2
2
1

1
1
2

1
1

1
1

6.19 0.30B2
4.95 0.25B2
3.73 0.23A2

NC
PC
Endosulfan
SL I
SL II
SL III

105
109

2
3

1
2

2.85 0.212
9.17 0.46B

117
108
113

2
1
1

2
2
2

2
1

2
1
1

1
1

7.69 0.37B1
5.55 0.26B2
4.42 0.20A2

NC
PC
Endosulfan
SL I
SL II
SL III

104
115

1
3

1
2

1
2

110
106
109

2
3
2

2
1
2

1
1
1

1
1

2
1

NC
PC
Endosulfan
SL I
SL II
SL III

119
116

2
3

1
3

1
2

3.36 0.222
15.51 0.58B

107
102
112

3
2
2

2
1
1

1
1
1

2
1
1

2
1

1
1
1

1
1

12.14 0.47B2
7.84 0.30B2
6.25 0.27B2

NC
PC
Endosulfan
SL I
SL II
SL III

106
105

2
2

2
3

3.77 0.272
13.33 0.50B

114
108
104

3
3
2

3
2
1

1
1
2

1
1
1

1
1
1

1
2

1
1
1

9.64 0.42B2
10.18 0.43B2
7.69 0.33B2

NC
PC
Endosulfan
SL I
SL II
SL III

101
106

2
3

1
3

1
1

3.96 0.242
12.26 0.51B

112
119
117

2
2
2

1
2
1

2
1
2

2
1
1

1
2
2

1
1
2

1
1
1

9.82 0.37B2
8.40 0.35B2
10.25 0.40B2

NC
PC
Endosulfan
SL I
SL II
SL III

119
113

1
4

2
2

1
2

1
1

4.20 0.232
12.38 0.51B

115
109
107

2
2
2

1
2
1

2
1
2

2
1
1

1
2
1

2
1
1

1
1
1

10.43 0.40B2
9.17 0.37B2
8.41 0.33B2

NC
PC
Endosulfan
SL I
SL II
SL III

104
108

2
3

2
3

1
1

4.80 0.292
13.88 0.53B

113
115
111

2
3
2

2
2
3

1
1
2

1
2
1

2
2
1

2
1

2
1
1

11.50 0.43B2
10.43 0.44B2
9 0.41B2

NC
PC
Endosulfan
SL I
SL II
SL III

117
106

1
2

2
3

1
1

1
2

4.27 0.282
14.15 0.52B

103
114
118

2
2
2

4
3
2

1
1
1

1
2
2

1
1
2

1
2
1

1
1
1

11.65 0.44B2
10.52 0.42B2
9.32 0.39B2

14

21

28

35

Classical aberrations

Non-classical aberrations

Total aberr. mean (%) S.D.

2.88 0.162
10.43 0.44B
9.09 0.37B
6.60 0.34B2
5.50 0.29B2

Exp: exposure time in days. TMS: total metaphasic plates studied. NC: negative control (tap water). PC: positive control (cyclophosphamide: 4 ppm). SL I: sub lethal I (1/25 of
LC50: 0.052 ppm). SL II: sub lethal II (1/50 of LC50: 0.035 ppm). SL III: sub lethal (1/75 of LC50: 0.017 ppm). Csb: chromosome break. Ctb: chromatid break. Frg: fragment. Scu:
sister chromatid union. Dic: dicentric. Mla: multiple aberrations. Stp: stickiness and pulverization. Cmt: c-metaphase. Values with different letter superscripts differ
signicantly from the negative control (NewmanKeuls and Dunnetts multiple comparison tests), whereas values with different numeric superscripts differ signicantly
from the positive control (Dunnetts multiple comparison test).

to be time and concentration dependent with the highest at SL-I


concentration on 4th day (722.45%; p < 0.01). Previous investigations have also reported the induction of LPO by pesticides such
as chlorpyrifos (Bebe and Panemangalore, 2003), methyl parathion
(Celik and Suzek, 2008) and tebuconazol (Toni et al., 2010). This
increase in TBARS levels can be a result of the impairment in
antioxidant enzymes due to ROS formation that attacks the cell
membrane, with direct consequences on cell integrity and cell

function. However, the levels of LPO may differ among sh species.


Differences observed in TBARS levels could reect variation in the
antioxidant mechanisms of sh species, duration of exposure and
the pesticide tested (Marigoudar et al., 2009).
3.2.2. Antioxidant response
Endosulfan exposure caused a signicant increase in GSH levels
during the rst 14 d of post exposure Table 4. Signicant elevations

280

S.A. Dar et al. / Chemosphere 120 (2015) 273283

Fig. 2. Metaphase plates prepared from kidney cell of Carassius carassius showing (A) normal chromosomes (2n = 100), (B, C and D) chromosomal aberrations from endosulfan
exposed sh (SL I-0.052, SL II-0.035, SL III-0.017 ppm), respectively for 96 h.

in the GSH content, compared to the control group, were found in


SL-I up to 7th day of the experiment, with the highest level on 4th
day (181.5%; p < 0.01). SL-II and SL-III also showed increasing trend
up to 14st day with the maximum elevation on 7th (183.8%;
p < 0.01) and 14th day (159%; p < 0.01) respectively. Increase in
GSH levels is one of the protective mechanisms that sh adopt in
the initial phases of exposure to aquatic pollutants and are in
agreement with many previous studies (Hasspielar et al., 1994;
Stephensen et al., 2002). However, there was a signicant decrease
in the GSH content in all the test concentrations of endosulfan from
the 21st day of the post exposure. A considerable decline in GSH
content in the present experimental model may be due to its utilization to challenge the prevailing oxidative stress under the inuence of ROS generated from endosulfan exposure. Reduced GSH
and its metabolizing enzymes provide the foremost defense
against ROS-induced cellular damage (Celik and Suzek, 2008).
The SOD activity in erythrocytes of C. carassius, exposed to various concentrations of endosulfan showed a signicant decrease
throughout the period of study (Table 4). Signicantly highest
reduction (62.75%; p < 0.01) in SOD activity with respect to control
was observed in SL-I on 4th day of post-exposure. SL-II and SL-III
also showed a signicant reduction, compared to control, with
the maximal on 7th (57.57%; p < 0.01) and 14th day (44.56%;
p < 0.01) respectively. The SODs are a group of metallo-enzymes
that play a crucial antioxidant role and constitute a rst line
defense system against the natural or chemically induced production of ROS (Deviller et al., 2005). The concentration and time

dependent reduction in the SOD activity, as observed in the present


study, could be an indicator of compensatory cell response to
dismutase the superoxide radical to H2O2, which itself is an important ROS as well (Li et al., 2009). Similar results were veried in O.
niloticus (Oru and ner, 2000) and in Cyprinus carpio (Oruc and
Usta, 2007).
Table 4 summarizes the CAT activity in the blood of test organism exposed to various concentrations of endosulfan. SL-I showed
a signicant increase in the rst half of post exposure (up to 14th
day), but showed a signicant reduction (p < 0.05) at the end of
second half of the post exposure. SL-II and SL-III also showed a
signicant elevation up to 14th and 21st day, with the highest increase on 7th (77.45%; p < 0.01) and 14th day (64.45%;
p < 0.01), respectively. The increase in CAT activity, as depicted
by our study, may be in response to H2O2 produced by SOD activity, since CAT is responsible for the conversion of H2O2 to water. A
signicant increase in CAT activity has been observed in some
studies after sh exposure to pesticides (Glusczak et al., 2011;
Stara et al., 2012). The reduction of catalase activity by higher concentration at the termination of the experiment could be justied
by the ux of superoxide radicals, which have been reported to
inhibit CAT activity (Ballesteros et al., 2009). Taken together, CAT
and SOD results indicate a disruption of normal oxidative process,
indicating an impairment of antioxidant defense system.
The mechanism of genotoxicity of endosulfan largely remains
less understood. When sh were exposed to endosulfan, an
increased oxidative stress was reected by increase in LPO in sh

281

S.A. Dar et al. / Chemosphere 120 (2015) 273283

Table 4
Levels of thiobarbituric acid reactive substances (TBARS, nm/h/mg protein) and activity of reduced glutathione (nm GSH/mg protein), superoxide dismutase (SOD, nm NBT/min/
mg protein) and catalase (CAT, nm H2O2/min/mg protein) in Carassius carassius (mean S.D.) after chronic exposure to endosulfan.
Marker

Exp. (days)

Test groups
NC

PC

SL I

SL II

SL III

296.8 5.41B
304.8 4.83B1
313.2 3.86B3
343.5 5.32B3
339.2 4.83B3
335.2 5.84B3
326.2 5.03B3
319.8 7.41B3
318.3 8.66B3

206.3 4.88B
224.2 5.07B3
232.5 4.68B3
249.3 6.94B3
247.5 6.89B3
238.2 5.67B3
234.2 4.40B3
226.7 5.46B3
231.2 4.4 B3

157.8 6.27B
162.3 4.45B
169.3 5.24B2
177.7 5.46B3
190.8 5.11B3
186 6.72B3
180.8 7.38B3
184.2 4.57B3
174.3 5.46B3

83.33 6.12B
87.16 7.30B
98.16 6.24B2
105.2 5.87B3
112.5 8.19B3
121.3 6.68B3
117 6.35B3
123.5 7.14B3
114.8 6.30B3

TBARS

1
2
3
4
7
14
21
28
35

29 4.19
31 3.89
32 3.16
33 3.16
36.1 3.711
40 4.053
39 2.283
41 3.683
43 4.053

GSH

1
2
3
4
7
14
21
28
35

1.9 0.37
1.95 0.28
2.3 0.26
2 0.38
1.8 0.54
1.93 0.6
2 0.52
2.1 0.54
2.18 0.48

3.03 0.74A
4.3 0.71B1
5.2 0.91B3
6.08 0.93B3
5.9 0.70B3
4.25 1.00B2
2.9 0.70
1.3 0.31A3
1.6 0.632

2.91 0.95
3.3 0.96A
4.15 1.39A
5.63 1.35B2
5.1 1.96B1
3.3 1.38
1.1 0.48 A
1 0.36B1
0.9 0.40 B3

SOD

1
2
3
4
7
14
21
28
35

16.2 0.50
16.26 0.74
16.22 0.48
16.38 0.58
16.31 0.80
16.29 0.59
16.11 0.60
16 0.59
16.15 0.70

8.5 0.38B
7.71 0.42B2
6.45 0.51B3
5.21 0.40B3
5.6 0.38B3
5.93 0.51B3
6.2 0.32B3
5.41 0.33B3
6.49 0.36B3

9.16 0.62B
8.75 0.32B
7.91 0.27B3
6.1 0.37B3
6.44 0.32B3
6.88 0.42B3
7.11 0.40B3
6.34 0.23A3
7.21 0.51A3

10.34 0.30B
9.99 0.36B
9.74 0.37B
8.84 0.51B3
6.92 0.31B3
7.34 0.76B3
7.8 0.73B3
7.64 0.45B3
8.3 0.36B3

12.6 0.57B
12.45 0.41B
12.01 0.31B
11.5 0.42B3
11.11 0.38B3
9.03 0.45B3
9.4 0.42B3
10.21 0.36B3
9.51 0.51B3

CAT

1
2
3
4
7
14
21
28
35

4.1 0.28B
4.33 0.34B
4.66 0.64B1
5.87 0.19B3
5.78 0.36B3
5.05 0.31B3
2.8 0.283
1.9 0.31B3
2.15 0.25B3

3.61 0.20B
3.71 0.15B
3.95 0.30B
4.83 0.25B3
4.32 0.24B3
4 0.29B
3.11 0.312
2.29 0.28A3
2.44 0.23A3

3.21 0.23
3.39 0.21A
3.67 0.20A
3.96 0.19B2
4.88 0.26B3
3.79 0.21B1
3.1 0.27
3.35 0.58
3.4 0.28B

3.1 0.27
3.32 0.22
3.49 0.36
3.59 0.36B
3.73 0.23B
4.21 0.34B3
4.03 0.43B2
3.61 0.63
3.33 0.37A

2.94 0.05
3 0.20
3.08 0.05
2.9 0.28
2.75 0.05
2.56 0.063
2.81 0.03
2.98 0.08
2.86 0.09

2.6 1.11
2.8 0.98
3.4 1.17
4.7 1.25B1
5.11 1.26B2
4.6 1.18B1
1.56 1.12
1.4 0.58A
1.11 1.07 A1

2.45 1.07
2.71 1.05
3.1 1.63
3.8 1.02A
4.46 1.09B
5 1.48B1
1.89 1.43
1.72 0.68
1.19 0.89A

NC: negative control (tap water). PC: positive control (cyclophosphamide: 4 ppm). SL I: sub lethal I (1/25 of LC50: 0.052 ppm). SL II: sub lethal II (1/50 of LC50: 0.035 ppm). SL
III: sub lethal III (1/75 of LC50: 0.017 ppm). Values with different letter superscripts (Ap < 0.05: signicant. Bp < 0.01: highly signicant. Cp < 0.001: extremely signicant) differ
signicantly from the negative control, whereas values with different numeric superscripts (1p < 0.05: signicant. 2p < 0.01: highly signicant. 3p < 0.001: extremely signicant) differ signicantly between exposure times within concentration.

Table 5
Correlation analysis between micronucleus frequency (a cytogenetic genotoxic biomarker) and lipid peroxidation (a robust oxidative stress biomarker) in blood erythrocytes of
Carassius carassius exposed to endosulfan (96 h).

LPO NC
MN NC
LPO PC
MN PC
LPO SLI
MN SLII
LPO SLIII

LPO NC

MN NC

LPO PC

MN PC

LPO SL I

MN SL I

LPO SL II

MN SL II

LPO SL III

MN SL III

1
.192
.423
.329
.323
.337
.689*

.192
1
.827**
.866**
.798**
.827**
.636

.423
.827**
1
.988**
.938**
.991**
.851**

.329
.866**
.988**
1
.914**
.996**
.801**

.323
.798**
.938**
.914**
1
.919**
.753*

.337
.827**
.991**
.996**
.919**
1
.817**

.689*
.636
.851**
.801**
.753*
.817**
1

.479
.574
.800**
.759*
.720*
.793*
.921**

.914**
.386
.674*
.594
.549
.612
.893**

.595
.328
.672*
.621
.511
.673*
.794*

LPO: lipid peroxidation. MN: micronucleus frequency. NC: negative control (tap water). PC: positive control (cyclophosphamide: 4 ppm). SL I: sub lethal I. SL II: sub lethal III.
SL III: sub lethal III concentrations of endosulfan respectively.
*
Correlation is signicant at the 0.05 level (2-tailed).
**
Correlation is signicant at the 0.01 level (2-tailed).

erythrocytes in concentration-dependent manner. The increased


LPO was followed by increased incidence of MN and a signicant
positive correlation relation (r2 = 0.9568; p < 0.01) between them
was observed (Table 5). There are number of studies in sh
showing such a relationship (Ahmad et al., 2006; Ansari et al.,
2011). An increase in the frequency of MN in endosulfan-treated
sh is an indication of chromosome damage. Genetic damage at

the chromosome level in either chromosome number or chromosome structure measured as CA or MN assays are useful biomarkers of environmental genotoxicity testing. Previous studies have
also shown that many xenobiotics, such as pesticides are known
to cause oxidative stress through the generation of ROS and can
alter the free oxygen radical scavenging enzyme systems in aquatic
organisms (Figueiredo-Fernandes et al., 2006). ROS can react with

282

S.A. Dar et al. / Chemosphere 120 (2015) 273283

susceptible biological macromolecules and cause LPO, which in


turn leads to DNA damage and protein oxidation (Ansari et al.,
2011). This shows that there is a relationship between genotoxic
and oxidative stress-inducing effects of endosulfan and oxidative
DNA damage may be the most putative mechanism of genotoxicity
of endosulfan in C. carassius. A large-scale use of chlorinated
cyclodienes has been reported in pest control activity in agriculture sector as well as in eradication of insect borne diseases in
several countries including India (Pandey et al., 2006). The ecotoxicological impact of use of endosulfan in eld conditions is not
properly studied. The present study offers some insight into the
eco-genotoxicological consequences of an important member of
chlorinated cyclodienes at consumer level of food chain.
4. Conclusions
Considering the mutagenic and genotoxic effects of endosulfan
on C. carassius obtained in this study by MN and CA assays, there is
serious apprehension about the potential danger of this pesticide
to aquatic organisms, especially to sh, and indirectly to human
beings. Moreover, in the absence of other convenient or practical
methods, the MN and CA will continue to play an important role
in assessing the genotoxicity induced by pesticides. One of the
mechanisms of genotoxic action of endosulfan appears to be its
oxidative stress inducing effect. It may be important to correlate
the assay with other relevant endpoints of ecotoxicity, as a robust
ecotoxicological tool. Information obtained through these integrated studies in sh model may be useful for regulatory agencies
entrusted with rational use of pesticides.
Acknowledgments
This work is part of the Ph.D. thesis of the rst author who
thanks the University Grants Commission (UGC) for his Junior
Research Fellowship (Sr. No. 2061330965; Ref. No: 23/06/2013-iEU-V) and Director of the Centre of Research for Development
(CORD), University of Kashmir, for providing necessary research
facilities. The authors also thank Dr. Ajay kumar, Advanced Instrumentation Research Facility (AIRF), Jawaharlal Nehru University,
New Delhi, for cordially providing the GC-MS facility.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.chemosphere.
2014.07.031.
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