Investigation of the genotoxicity of

endosulfan to freshwater Cyprinid fish

Crucian carp (Carassius carassius L.)
using the micronucleus and chromosomal
aberration as biomarkers
Sabzar Ahmad Dar, Abdul Rehman
Yousuf, Masood-ul-Hassan Balkhi,
Farooq Ahmad Ganai & Farooz Ahmad
Bhat
The Nucleus
An International Journal of Cytology
and Allied Topics
ISSN 0029-568X
Volume 57
Number 2
Nucleus (2014) 57:87-98
DOI 10.1007/s13237-014-0110-3

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Author's personal copy

Nucleus (August 2014) 57(2):8798
DOI 10.1007/s13237-014-0110-3

ORIGINAL ARTICLE

Investigation of the genotoxicity of endosulfan to freshwater

Cyprinid fish Crucian carp (Carassius carassius L.) using
the micronucleus and chromosomal aberration as biomarkers
Sabzar Ahmad Dar & Abdul Rehman Yousuf &
Masood-ul-Hassan Balkhi & Farooq Ahmad Ganai &
Farooz Ahmad Bhat

Received: 11 February 2014 / Published online: 14 May 2014

# Archana Sharma Foundation of Calcutta 2014

Abstract This study was aimed to evaluate the in vivo

genotoxic effects of endosulfan in freshwater fish crucian
carp, Carassius carassius L. The LC5096h value of endosulfan (with 95 % confidence limits), determined by
probit analysis, was 0.070 (0.0460.093) ppm L1 and
the estimated safe levels of endosulfan varied from 00.70
102 to 00.70 106 ppm L1. Based on the LC5096h
value, three test concentrations (viz. sub-lethal I, II, and
III) were determined to be 0.052, 0.035 and
0.017 ppm L1, respectively, and the fish specimens were
exposed to these concentrations for 35 days. The mean
concentration of endosulfan in water samples (aquaria)
was always within 5 % of the intended concentration
when analyzed by dispersive liquid-liquid micro extraction (DLLME) followed by gas chromatographymass
spectrometry (GC-MS). Autopsy was done on days 1,
2, 3, 4, 7, 14, 21, 28 and 35 of endosulfan exposure for
assessment of micronucleus (MN) formation and chromosomal aberrations (CA). The MN formation in the
peripheral erythrocytes, authenticated by scanning electron microscopy (SEM), was found to be significantly
higher (p < 0.05) in all the treated groups, including
positive control cyclophosphamide (4 ppm L1) compared to negative control. Similar significant effects (p
< 0.05) were also observed on CA in the head kidney
cells. In general, both MN and CA exhibited a
S. A. Dar (*) : A. R. Yousuf : F. A. Ganai
Limnology and Fisheries Laboratory, Centre of Research for
Development (CORD), University of Kashmir, Srinagar 190006,
J & K, India
e-mail: sabzar.cord@gmail.com
M.<u.<H. Balkhi : F. A. Bhat
Division of Fisheries, Sher-e-Kashmir University of Agricultural
Sciences and Technology of Kashmir (SKUAST-K), Srinagar, J & K,
India

concentration and time-dependent response. The current

study revealed the potential genotoxicity of endosulfan in
fish; and that the micronucleus and chromosomal assays
are useful tools in determining genotoxicity of water
pollutants and might be appropriate as a part of environmental monitoring program.
Keywords Pesticide residues . Fish . Genotoxicity . SEM .
GC-MS

Introduction
The use of pesticides has greatly improved agricultural yield
through eradication of pests in the fields and during storage
[48]. However, the agricultural runoff introduces pesticide
residues into the aquatic environment, where it poses a high
risk for aquatic organisms and consequently the entire food
chain including human beings [10, 34]. Although the consumption of pesticides in India is estimated to be the lowest at
0.5 kg/ha as against 17 in Taiwan, 12 in Japan, 6.6 in Korea, 7
in the United States and 2.5 kg/ha in Europe, the food and
agricultural products contain substantial quantities of pesticide
residues [7]. The major reasons are indiscriminate and
nonjudicious use of chemical pesticides as well as nonobservance of prescribed waiting periods. 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 [4, 9].
Introduced in the 1950s, endosulfan (6,7,8,9,10,10hexachloro-1,5,5a,6,9,9a-hexahydro-6,9-methano-2,4,3benzodioxa-thiepin-3 oxide) emerged as a leading chemical used against a broad spectrum of insects and mites
in agriculture and allied sectors. But now it is being

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88

detected as an important cause of pesticide poisoning in

many countries [39]. Endosulfan is a persistent organochlorine pesticide (OCP), commercially comprising of
two isomers (- and -endosulfan) at a ratio of 70:30,
belong to the group of chlorinated cyclodienes [11]. Due
to its toxic effect, the World Health Organisation (WHO)
and the United States Environmental Protection Agency
(USEPA) have classified endosulfan as Class IB and
Class II pesticides, respectively [17, 25]. Although endosulfan was banned in countries such as Germany, UK,
Sweden, Netherlands, Colombia and Singapore, but it is
still widely used in most of the developing countries
because of its effectiveness and low application cost
[34]. In India, endosulfan is classified as an extremely
hazardous pesticide; affecting the central nervous system, reproductive system and immune system [21].
The significance of assessment of pesticide genotoxicity in
fish lies in the fact that fish is an important source of diet all
across the world. Hence, the genotoxic agents are easily
exposed to higher vertebrates and ultimately, the humans,
when millions of tons of fish are caught and consumed per
year [22]. The pesticide, owing to its stability, contaminates
the aquatic environment even at sub-lethal concentrations and
tends to accumulate in tissues of aquatic organisms [28].
Ecotoxicological characteristics of freshwater fish, 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.
The genotoxic effects of environmental pollutants can be
monitored using a broad range of both the in vitro and in vivo
biomarker assays but several studies have shown that micronucleus and chromosomal aberration tests are efficient, cost
effective and popular techniques for showing clastogenic and
aneugenic effects [1, 5, 45]. The study of DNA damage at the
chromosomal level is an essential part of genotoxicology
because chromosomal aberration is an important event in the
development of mutations. The use of negative and positive
control groups is recommended in all mutagenicity tests and
cyclophosphamide, a clastogenic agent for various animal
species, is recommended as positive control in chromosome
aberration tests, sister chromatid exchanges and micronucleus
(MN) formation in vitro and in vivo [14].
Endosulfan, one of the chlorinated cyclodienes, has high
toxicity to a broad spectrum of aquatic organisms and thus, it
is imperative to understand its eco-genotoxicological implication and susceptibility to aquatic biota. Most of the studies on
the endosulfan effect are confined to the doseresponse and
physiological changes of the fish. There is relatively less
attention being given to the genotoxic modulation induced
by the endosulfan. Thus, the objective of the present study was
to study the genotoxic effect of endosulfan in freshwater fish
crucian carp, C. carassius.

Nucleus (August 2014) 57(2):8798

Materials and methods

Experimental fish specimens and chemicals
Carassius carassius L. (Family: Cyprinidae and Order:
Cypriniformes), having chromosome number (2n) 100
[27], was selected as the experimental model. Specimens
were procured with the help of a local fisherman, from
the pollution free area of the Dal Lake (3407N 7452
E), in the vicinity of University of Kashmir, Srinagar,
India. Juvenile healthy fish specimens were transported
to the laboratory and were subjected to a prophylactic
treatment by bathing in 0.05 % potassium permanganate
(KMnO4) for 2 min to avoid any dermal infection. The
fish stock in good number was then acclimatized for at
least 3 weeks to 1:1 diurnal photoperiod in artificially
aerated 60 L glass aquaria at 19.66 2.58 C, with aged
dechlorinated tap water (pH 7.68.4), and fed ad libitum
daily with commercially available fish food (Feed Royal, Maa Agro Foods, Andhra Pradesh, India). The metabolic waste products were siphoned off daily to reduce
ammonia content in water and no fish mortality occurred
during this period. The acclimatized fish were then used
for the experiments in accordance with the principles of
the Institutional Ethical Committee (IEC) for the Protection of research animals in the University of Kashmir.
For the present study, endosulfan (CAS No. 45852) and
cyclophosphamide (CAS No. C3250000-1EA) were purchased from the Sigma Aldrich, India. The other
chemicals used in the study were of high purity.

Acute toxicity and determination of sublethal concentrations

The acute toxicity bioassay of endosulfan in C. carassius
was conducted in a semi-static system, with the change of
test solution after every 24 h to maintain the similar concentration of the chemical, to determine its LC5096h value.
Since endosulfan was emulsifiable concentrate, it was directly added to the system. Briefly, a set of 10 specimens
were randomly exposed to each of the eight endosulfan
concentrations (0.01, 0.03, 0.05, 0.07, 0.09, 0.11, 0.5 and
1 ppmL1), obtained after the range finding test, and the
experiment was repeated in triplicate to obtain the LC5096h
value of the test chemical for the target species. Fish were
not fed throughout the experiment and lethality was the
toxicity end-point. The LC5096h value of endosulfan was
determined following the probit analysis method as described by Finney [20]. Based on the LC5096h value, three
test concentrations of test chemical, namely sublethal concentration I (SL-I; 1/4th of LC5096h), II (SL-II; 1/2nd of
LC5096h) III (SL-III; 3/4th of LC5096h), were determined
for the in vivo experiment.

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Nucleus (August 2014) 57(2):8798

In vivo exposure experiment

The experiment consisted of five groups each with four replicates (total 20 aquaria), containing 60 L dechlorinated and
well-aerated tap water with 11 1 fish specimens in each
aquaria. The specimens maintained in dechlorinated tap water
and those exposed to cyclophosphamide (ppmL1, concentration selection based on previous investigation [36]) were
considered as the negative and positive controls, respectively.
In groups 35, the fish specimens were kept in water containing the three aforementioned test concentrations of the endosulfan. From each group, the samplings were done on days 1,
2, 3, 4, 7, 14, 21, 28 and 35 after endosulfan exposure for
performing the cytogenetic markers. The fish behavior and
physico-chemical properties of test water, namely temperature, pH, dissolved oxygen, total alkalinity and ammonical
nitrogen were analysed throughout the study by standard
methods [6]. To ensure an agreement between nominal and
actual test chemical concentrations in the aquaria, water samples were analyzed during the experimental period by a simple, fast and economical method, dispersive liquid-liquid micro extraction (DLLME) followed by gas chromatography
mass spectrometry (GCMS-QP2010 Plus, Shimadzu, Japan)
[46].

89

oil immersion at 1000 magnification 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. Only the cells clearly isolated from the
surrounding cells were scored. The criteria for the identification of micronuclei were as follows: diameter less than 1/5-1/
20 of the main nucleus; no connection or link with the main
nucleus; must be on the same plane of focus and staining
intensity similar to that of the main nucleus; non refractability;
round or oval with a nuclear membrane; no overlap with the
principal nucleus and is intracytoplasmic [19].
Scanning electron microscopy (SEM)
The SEM was carried out by standard procedure [41, 53].
Briefly, the aforementioned micronucleated slides were
reshaped, sputter-coated with a gold and platinum to a layer
of 35 nm and were 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).
Chromosomal aberration test

Micronucleus test
Slides were prepared using the standard fish micronuleated
erythrocytes method of Al-Sabti and Metcalfe [3], with few
modifications. Prior to blood collection, fish were anaesthetized with 0.12 g L1 benzocaine [30]. Blood samples were
withdrawn by caudal puncture with heparinized syringes and
peripheral blood smears, two for each fish specimen, were
immediately made by applying two drops of blood on
precleaned, grease free slides. The smeared slides were left
to air dry at room temperature for overnight in a dust and
moisture free environment. The next day slides were fixed by
dipping in cold absolute methanol (4 C) for 15 min and again
left to air dry at room temperature for 1 h. Finally, the slides
were stained in May-Grunwald stain (Sigma) for 510 min
followed with 6 % Giemsa (Sigma) in phosphate buffer for
30 min. The slides were then washed thoroughly in double
distilled water, dried and made permanent with DPXmounting.
Scoring criteria for micronuclei (MN)
For every sampling time (five fish specimens were used),
replicate slides per specimen were prepared and 20002400
cells from each specimen (minimum of 10,000 erythrocytes
were scored in each treatment group) were examined for the
presence of MN. The frequency of MN/fish was calculated per
1000 cells [40], and was evaluated by scoring the slides under

Chromosome preparations were made from the highly

haemapoietic and mitotically active head kidney cells, following the standard techniques [2, 16]. The fish of all the groups
(five fish/group/esposure) were injected with 0.05 % colchicine intramuscularly at 1 ml/100 g body weight 3 h prior to
dissection, to arrest the metaphase stage. The head kidney was
dissected out, macerated and homogenized in 2 ml of 0.56 %
KCl, in glass tissue homogenizer, to prepare cell suspension.
The cell suspension was poured into eppendorf tubes and
incubated for 2030 min at room temperature for hypotonic
treatment. The cell suspension was fixed in chilled Cornoys
fixative (methanol:glacial acetic acid, 3:1 v/v), mixed gently
with Pasteur pipette, centrifuged at 1,500 rpm for 10 min and
supernatant was discarded. The pellet was resuspended in
chilled Cornoys fixative and the above process was repeated
34 times until the whitish pellet was obtained. Chromosome
slides were prepared by dropping one or two drops of cell
suspension onto pre-cold slides in 70 % alcohol. The slides
were then air dried and stained with 5 % Giemsa prepared in
Sorensens buffer (pH- 6.8) for 20 min. Finally, the slides were
cleared in xylene and permanently mounted in DPX.
Scoring criteria for chromosomal aberrations (CA)
The slides having brightly stained well spread metaphase
chromosomes were independently coded and observed at
magnification of 100 under oil immersion with light

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Nucleus (August 2014) 57(2):8798

Fig. 1 Comparison of GC-MS chromatograms of aquaria water samples at 1 h (black line) and 24 h (pink line) after the renewal of endosulfan
concentration. Peaks 1 and 2 are - and -endosulfan, respectively

microscope for chromosomal aberrations. Replicate slides

were selected per fish 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 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.
Statistical analysis
Probit Analysis was performed with the SPSS (version
16.0) computer program (SPSS Inc. Chicago, IL, USA).
Data was compared for statistically significant difference
between control and treatment groups using one-way
analysis of variance (ANOVA). Significant difference

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

Exposure Time (h)

24

48

72

96

LC5
LC10
LC30
LC50
LC70
LC90

0.080 (0.0260.133)
0.135 (0.0750.194)
0.176 (0.1290.223)
0.215 (0.1580.272)1
0.240 (0.1560.323)2
0.303 (0.2510.355)3

0.070 (0.0030.136)
0.085 (0.0450.124)A
0.120 (0.0570.182)
0.151 (0.1120.191)A
0.210 (0.1350.284)2
0.273 (0.1800.366)2

0.040 (0.0030.076)
0.060 (0.0170.102)A
0.080 (0.0270.132)A
0.095 (0.0750.114)C
0.160 (0.0940.225)A2
0.251 (0.2340.268)2

0.030 (0.0110.048)
0.040 (0.0170.062)B
0.050 (0.0220.077)C
0.070 (0.0460.093)C
0.130 (0.0740.185)B2
0.233 (0.1780.287)A2

LC95

0.361 (0.2640.458)3

0.310 (0.2380.381)2

0.293 (0.2220.363)2

0.270 (0.2020.337)A2

Values with different alphabet superscript differ significantly (A p<0.05: significant. B P<0.01: highly significant. C p<0.001: extremely significant.)
between exposure time within lethal concentrations, whereas values with different numeric superscripts differ significantly (1 p<0.05: significant.
2
P<0.01: highly significant. 3 p<0.001:extremely significant) between concentrations within duration (Dunnetts multiple comparison test)

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91

Fig. 2 Probit line graph (95 %

confidence intervals) of acute
toxicity of endosulfan to crucian
carp (Carassius Carassius)

in ANOVA were further analyzed by post-hoc Bonferronis,

Newman-Keuls and Dunnetts multiple comparison test and
the p-values less than 0.01 were considered statistically
significant.

Results
Physicochemical properties of the test water
The physicochemical characteristics of the test water measured during experimentation were temperature 18.2
23.3 C, pH 7.58.4, dissolved oxygen 7.98.4 mg L1, total
alkalinity 6973 mg L 1 and ammonical nitrogen 25
29 g L1. The conductivity of the water ranged from 211 to
239 Mcm1. 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 (Fig. 1), when
analyzed by DLLME followed by GC-MS.

Table 2 Estimate of safe levels

of endosulfan at 96 h exposure
time

Fish behavior
During the experiment, both control and exposed carp showed
normal feeding behavior. 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 fish floating at the water surface.
Fish was progressively stressed, became lethargic and exhibited
transient hyperactivity before collapsing. However, the mortality did not exceed 10 % during the 35 day trial period in both
control and the treatment groups, indicating that the treatments
were sufficient to cause the changes described hereafter.

LC50 and application factor

In acute toxicity bioassay, the LC50 values (with 95 % confidence
limits) of different concentration of endosulfan in C. carassius
(Table 1, Fig. 2) were found to be 0.215 (0.1580.272), 0.15
(0.1120.191), 0.095 (0.0750.114) and 0.070 (0.0460.093)

Chemical

LC5096 h (ppm L1)

Method

AF

Safe level (ppm L1)

Endosulfan

0.070

Sprague [47]
CWQC [15]
NAS/NAE [32]
IJC [24]

0.1
0.01
0.10.00001
5 % LC5096 h

00.70 102
00.70 103
00.70 102 to 00.70 106
00.35 102

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Values with different letter superscripts differ significantly from the negative control (Newman-Keuls and Dunnetts multiple comparison test), whereas values with different numeric superscripts differ
significantly between exposure times within concentration (Bonferronis test)

NC Negative control (tap water), PC Positive control (cyclophosphamide: 4 ppm L1 ), SL I Sub lethal I (1/25 of LC50: 0.052 ppm L1 ), SL II Sub lethal II (1/50 of LC50: 0.035 ppm L1 ), SL III Sub lethal
(1/75 of LC50: 0.017 ppm L1 )

3.733 (0.041)C2
2.021 (0.065)C1
1.113 (0.075)A2
4.096 (0.066)C3
2.756 (0.070)1
1.693 (0.064)A1
6.071 (0.058)C3
2.550 (0.033)C2
1.406 (0.025)A3
Endosulfan
SL I
0.052
SL II
0.035
SL III
0.017

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

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

0.225 (0.025)1
4.840 (0.084)C3
0.240 (0.016)
5.198 (0.035)C3
0.203 (0.012)1
5.738 (0.110)C3
0.288 (0.018)2
6.901 (0.055)C3
0.300 (0.015)3
7.526 (0.113)C3
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

0.250 (0.012)
6.325 (0.056)C3

28
7
4
1

NC
PC

Concentration (ppm)

Exposure time (days)

14

21

35

Nucleus (August 2014) 57(2):8798

Chemical

Table 3 Mean (S.E.) percentage micronucleus frequency in blood erythrocytes of Carassius carassius exposed to three concentrations of endosulfan (n=~10,000 cells/concentration/exposure time)

92

ppm L1 for 24, 48, 72 and 96 h, respectively. 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 96 h LC5096 h with different application factors (AF), are
given in Table 2. Based on the LC5096h value, the SL-I, II and III
were determined as 0.052, 0.035 and 0.017 ppm L1, which were
further used for in vivo exposure.
Micronuclei induction
The results of MN induction in peripheral blood erythrocytes of
C. carassius after exposure to different concentrations of endosulfan are presented in Table 3. It caused one, two and three
micronucleated cells but single MN was predominant in the
erythrocytes analyzed. The induction was significantly (P
0.05) higher in all the treatment groups compared to the control
at all the exposure durations except SL-II on 28th day. The
maximum MN frequency was observed on day 4 (6.071 %; p <
0.001) at the highest concentration (SL-I), whereas the MN
formation for SL-II and SL-III concentrations, was highest on
day 7 (4.440 %; p < 0.001) and day 14 (2.978 %; p < 0.05),
respectively. Treatment with genotoxic agent (cyclophosphamide; positive control) also resulted in an extremely significant
increase (p < 0.001) in the MN frequencies at all the sampling
intervals. A concentration-dependent response in MN induction
was observed. The results of MN were more authenticated by
SEM analyses as shown in figure Fig. 3.
Analysis of chromosomal aberrations
The typical diploid metaphase complements of fish,
C. carassius, were found to consist of 100 chromosomes of
four types such as submetacentric, metacentric, subtelocentric
and acrocentric. Various forms of chromosome damage recorded were chromosome and chromatid breaks, fragments,
sister chromatid union, dicentric, multiple aberrations, stickiness, pulverization and c-metaphases; whereas gaps were
excluded. The frequency of CA observed in C. carassius after
exposure to different concentrations of test chemical and
standard genotoxic agent, cyclophosphamide, were significantly (P 0.05) higher when compared to the control 1
(Table 4, Fig. 4), at all the exposure durations, and the chromatid and chromosome breaks were more frequent than the
other types of aberrations. 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. In general, a concentration-dependent response was
also observed in case of CA. At the termination of the

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Nucleus (August 2014) 57(2):8798

93

Fig. 3 Scanning electron

microscopy image used to show
micronucleus formation in
erythrocytes of Carassius
carassius. a Image of normal a.1
and micronucleated a.2
erythrocyte (5000x, scale bar =
5 m). b Image from negative
control group. c Image from the
positive control group
(cyclophosphamide 4 ppm L1).
d, e and f images from fish
exposed to SL I: 0.052, SL II:
0.035 and SL III: 0.017 ppm L1
concentrations of endosulfan,
respectively for 96 h (1000x,
scale bar = 10 m)

experiment, the frequency of CA was observed to be more or

less similar for all test concentrations.

Discussion
At present, more than 1000 chemicals have been classified as
pesticides and studies using different models have indicated
that some of them have genotoxic properties [52]. Fish are
often used as sentinel organism for ecotoxicological studies
because they play a number of roles in the trophic web,
accumulate toxic substances and respond to low concentration
of mutagens in a similar way to higher vertebrates [35].

In the present study, pre- treatment of KMnO4 solution (0.05 %) was given to the specimens for 2 min to
avoid any dermal infections, and after that, the specimens were acclimatized for at least 3 weeks under laboratory condition, prior to start of the post treatment, to
remove the residual effects of other chemicals. Several
investigators [38, 45, 1, 33], have used 0.05 % KMnO4
solution for prophylactic treatment before starting their
experiments, and they did not report any adverse effect
in test organisms due to prophylactic treatment.
The LC5096h value of the endosulfan in the present study
was 0.070 ppm L1which indicated that it is very toxic to fish.
Our estimate is higher than the LC 5096h value of

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Nucleus (August 2014) 57(2):8798

Table 4 Chromosomal aberration frequencies induced by endosulfan in Carassius carassius kidney cells
Exp. (days)

14

21

28

Treatment

NC
PC
Endosulfan
SL I
SL II
SL III
NC
PC
Endosulfan
SL I
SL II
SL III
NC
PC
Endosulfan
SL I
SL II
SL III
NC
PC
Endosulfan
SL I
SL II
SL III
NC
PC
Endosulfan
SL I
SL II
SL III
NC
PC
Endosulfan
SL I
SL II
SL III
NC
PC
Endosulfan
SL I
SL II
SL III
NC
PC
Endosulfan
SL I
SL II

TMS

Classical aberrations

Non-classical aberrations

Total aberr. mean (%) S.D.

Csb

Ctb

Ctb

Ctb

Dic

Mla

Stp

Cmt

103
105

1
3

1
2

1.94 0.132
8.57 0.41B

113
101
107
105
109

2
2
1
2
3

1
1

1
2

1
1

1
1

6.19 0.30B2
4.95 0.25B2
3.73 0.23A2
2.85 0.212
9.17 0.46B

117
108
113
104
115

2
1
1
1
3

2
2
2
1
2

2
1

2
1
1
1
2

7.69 0.37B1
5.55 0.26B2
4.42 0.20A2
2.88 0.162
10.43 0.44B

110
106
109
119
116

2
3
2
2
3

2
1
2
1
3

1
1
1

1
1

2
1

1
2

9.09 0.37B
6.60 0.34B2
5.50 0.29B2
3.36 0.222
15.51 0.58B

107
102
112
106
105

3
2
2
2
2

2
1
1
2
3

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
3.77 0.272
13.33 0.50B

114
108
104
101
106

3
3
2
2
3

3
2
1
1
3

1
1
2

1
1
1

1
1
1

1
2

1
1
1
1
1

9.64 0.42B2
10.18 0.43B2
7.69 0.33B2
3.96 0.242
12.26 0.51B

112
119
117
119
113

2
2
2
1
4

1
2
1
2
2

2
1
2
1
2

2
1
1

1
2
2

1
1
2

1
1
1
1
1

9.82 0.37B2
8.40 0.35B2
10.25 0.40B2
4.20 0.232
12.38 0.51B

115
109
107
104
108

2
2
2
2
3

1
2
1
2
3

2
1
2

2
1
1

1
2
1

2
1
1

1
1
1
1
1

10.43 0.40B2
9.17 0.37B2
8.41 0.33B2
4.80 0.292
13.88 0.53B

113
115

2
3

2
2

1
1

1
2

2
2

2
1

2
1

11.50 0.43B2
10.43 0.44B2

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Nucleus (August 2014) 57(2):8798

95

Table 4 (continued)
Exp. (days)

35

Treatment

SL III
NC
PC
Endosulfan
SL I
SL II
SL III

TMS

Classical aberrations

Non-classical aberrations

Total aberr. mean (%) S.D.

Csb

Ctb

Ctb

Ctb

Dic

Mla

Stp

Cmt

111
117
106

2
1
2

3
2
3

2
1
1

1
1
2

9 0.41B2
4.27 0.282
14.15 0.52B

103
114
118

2
2
2

4
3
2

1
1
1

1
2
2

1
2
1

1
2
1

1
1
1

11.65 0.44B2
10.52 0.42B2
9.32 0.39B2

Exp Exposure time in days, TMS Total metaphasic plates studied, NC Negative control (tap water), PC Positive control (cyclophosphamide: 4 ppm L1 ),
SL I Sub lethal I (1/25 of LC50: 0.052 ppm L1 ), SL II Sub lethal II (1/50 of LC50: 0.035 ppm L1 ), SL III Sublethal (1/75 of LC50: 0.017 ppm L1 ), 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 significantly from the negative control (Newman-Keuls and Dunnetts multiple comparison tests), whereas
values with different numeric superscripts differ significantly from the positive control (Dunnetts multiple comparison test)

0.0035 ppm L1 for Channa striatus [21]. 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, as calculated by multiplying the LC50
with application factor (AF) as recommended by different
meth ods , va ried fr om 00 .70 10 2 t o 0 0 . 7 0
106 ppm L1. However, the large variation in safe levels
determined by different methods has resulted in controversy
over its acceptability [8, 37].
Fig. 4 Metaphase plates
prepared from kidney cell of
Carassius carassius showing a
normal chromosomes (2n = 100),
b, c and d chromosomal
aberrations from endosulfan
exposed fish (SL I-0.052, SL II0.035, SL III-0.017 ppm L1),
respectively for 96 h

In order to make an accurate assessment of the hazards,

which a contaminant may pose in a natural system, behavioral
indices selected for monitoring must reflect the organisms
behavior in the field [29]. The stressful behavior, as observed
in our study, is in accordance with a number of previous
studies. Velisek et al. [49], reported accelerated respiration
and loss of movement coordination in rainbow trout and carp
following acute poisoning with metribuzin. These characteristics have also been reported in Oreochromis niloticus, and

Author's personal copy

96

Chrysichthyes auratus [23] and in Carassius auratus [44],

following acute poisoning with atrazine.
Micronucleus test, as a genotoxic endpoint, for clastogenic
effects of pollutants has been extensively used in fish such as
prussian carp (Carassius auratus), rainbow trout (Oncorhynchus mykiss), tilapias (Oreochromis mossambicus) and
salmoniform fish (Umbra pygmea) [50]. In the present study,
all concentrations of endosulfan induced significantly (P <
0.01) higher number of MN in erythrocytes and CA in head
kidney cells compared to the control and their frequency
increased in concentrations and time dependent manner. 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. Presumably, endosulfan has affected
the genetic material by absorption through the gill epithelium.
Earlier, it has been emphasized that exposure of fish to
genotoxic chemicals, for various interval of time, by the
respiratory route following the absorption of chemicals
through gill epithelium could be occurred [18, 42]. Our results
are in agreement with some earlier studies [1, 5, 12, 26, 31],
which have reported the induction of MN from exposure to
various xenobiotics present in the aquatic environment.
An advantage of chromosomal studies is that they reveal a
measure of sub-lethal effects of xenobiotics in vivo. The CA
were more at higher as compared to lower concentrations
tested, 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 in case of dichlorvos impacts on Channa punctatus [42]. The chromatid and
chromosome breaks were more frequent than the other types
of aberrations at all the exposure durations in our study. An
increase in chromatid break and chromosomal exchange due
to water pollution has also been reported by Chaurasia et al.
[13]. Similar results have also been reported by Rita and
Milton [43] in Channa punctatus and Yadav and Trivedi
[51] 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
genotoxic effects of various clastogenic xenobiotics, especially in the aquatic environment.

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 fish,
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. Information obtained

Nucleus (August 2014) 57(2):8798

through these integrated studies in fish model may be used

as bioindicators for monitoring the genomic damage from
environmentally hazardous contaminants in the aquatic
environment.
Acknowledgments The authors are grateful to Director of the Centre of
Research for Development (CORD), University of Kashmir, Srinagar,
India for providing laboratory facilities in the course of this research and
University Grants Commission (UGC), New Delhi, India for providing
financial assistance to Mr. Sabzar in the form of Junior Research Fellowship (JRF). The authors also thank the technicians of Advanced Instrumentation Research Facility (AIRF), Jawaharlal Nehru University, New
Delhi and University Sophisticated Instruments Facility (USIF), Aligarh
Muslim University, U.P. for their cordial support in carrying out the GCMS and SEM analysis, respectively.
Conflict of interest The authors report no conflicts of interest.
Author contributions statement First author carried out the experimental work and writing, author second and third designed and calibrated
the experimental setup, fourth author carried out the statistical analysis
and fifth author carried out the writing and critical evaluation, along with
the first author, of the manuscript.

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