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ISSN: 0148-0545 (print), 1525-6014 (electronic)
RESEARCH ARTICLE
Abstract Keywords
Chlorpyrifos is a widely used insecticide of organophosphate group, which causes severe Chlorpyrifos, comet assay, Labeo rohita,
toxicological effects in non target aquatic organisms especially in fish. In the present study the DNA damage, behavior
genotoxic effects of sublethal concentrations of chlorpyrifos were observed in the erythrocytes
and gill cells of Labeo rohita (commonly known as rohu) using the Alkaline Single-Cell Gel History
Electrophoresis (Comet) assay. Effects of chlorpyrifos on the behavior of the fish were also
investigated. The 96 h LC50 value of chlorpyrifos, estimated by Trimmed Spearman-Karber (TSK) Received 21 March 2013
in static bioassay, was found to be 442.8 mg/L. On the basis of LC50 value, the fish were exposed Revised 24 July 2013
to three sublethal concentrations of chlorpyrifos (SL-I 221.4 mg/L, SL- II 110.7 mg/L and SL-III Accepted 21 January 2014
73.8 mg/L) for 96 h. Blood and gill samples were collected at every 24 h and were subjected Published online 12 February 2014
to the Comet assay. The observed DNA damage was concentration dependent and time
For personal use only.
dependent and those levels of DNA damage in between the tested concentrations and times
were significantly different (p50.01). It was also found that the gill cells are more sensitive to
chlorpyrifos, though; it revealed more DNA damage as compared to the erythrocytes of fish.
Fish exposed to different concentrations of chlorpyrifos showed different neurotoxic behavioral
responses. It was concluded that chlorpyrifos is a genotoxic and neurotoxic insecticide causing
DNA damage and neurotoxic effects in Labeo rohita.
organisms (Ateeq et al., 2005; Jha, 2004, 2008; Kassie et al., Experimental fish specimen
2000; Pandey et al., 2006; Ventura Campos de et al., 2008;
Freshwater fish Rohu (Labeo rohita, Family: Cyprinidae,
Yin et al., 2008). The Comet assay has a variety of advantages
Order: Cypriniformes) was obtained from Faisalabad fish
over other cytogenetic methods like micronucleus test, sister
hatchery and shifted to the glass aquaria after treating
chromatid exchanges and chromosome aberrations test to
with potassium permanganate (KMnO4) solution (0.05 %) for
detect DNA damage. The cells used in this assay do not need
2 min to remove any dermal contaminant. The fish specimens
to be mitotically active (Ali et al., 2011; Collins, 2004; Tice
had an average body weight and body length 15.5 ± 0.9 g and
et al., 2000). As the red blood cells of the fish are nucleated
12.5 ± 1.2 cm. These specimens were acclimatized for 15 days
with DNA, the Comet assay has been effectively used in the
prior to the pesticide exposure with a 16:8 h (light:dark)
erythrocytes of the fish (Cavas & Konen, 2007; Ventura
photoperiod. The specimens were fed with pelleted feed
Campos de et al., 2008).
(containing fish meal, Charoen Pokphand Foods Public
Chlorpyrifos was not found to be genotoxic in any of these
Company Limited, Thailand) at the rate of 3% of their body
assays (Ames test, rat lymphocyte chromosomal aberration
weight once in a day and feeding was stopped 24 h prior to the
test, CHO/HGPRT assay, mouse bone marrow Micronucleus
pesticide exposure till the end of the experiment.
assay, cytogenetic abnormalities) (Gollapudi et al., 1995). But
later on, it was reported that chlorpyrifos induce in vivo
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genotoxicity in leucocytes of Swiss albino mice using Comet Determination of acute toxic and sublethal
assay (Rahman et al., 2002). Genotoxicity of chlorpyrifos concentrations
has been reported in the lymphocytes and gill cells of Stock solution of the insecticide was prepared by dissolving
freshwater fish (Channa punctatus) (Ali et al., 2008a, 2009). chlorpyrifos in acetone. Five working chlorpyrifos concen-
Chlorpyrifos has also been found genotoxic in root meri- trations (420, 430, 440, 450 and 460 mg/L) were prepared
stematic cells of Smooth Hawksbeard (Crepis capillaris), in acetone (solvent) from the stock solution. To determine
in erythrocytes and liver cells of Chinese toad (Bufo bufo the acute LC50 value of chlorpyrifos, static bioassay was
gargarizans) (Dimitrov & Gadeva, 1997; Yin et al., 2009). employed. Six groups of fish, each containing 10 individuals,
Although acute toxicity and genotoxicity of chlorpyrifos were selected at random and placed in the aquaria. Glass
have been evaluated earlier in freshwater fish Channa aquaria (with dimensions of 30 cm depth, 30 cm width and
punctatus using semi-static system, there is a need for 45 cm length), with the capacity of about 40 L was filled with
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studying the acute toxicity and genotoxicity of chlorpyrifos 30 L of water. The above mentioned concentrations were
in freshwater fish Rohu (Labeo rohita), which is a most added to different aquaria containing specimens, keeping one
preferred fish species in Pakistan, using static system. as solvent control receiving no pesticide but maximum
Therefore, the present study was conducted for the assessment acetone that any dosing solution contain. Acute toxicity
of the acute toxicity of chlorpyrifos in a static system and the assay was performed following the guidelines of Organization
genotoxic effects of sublethal concentrations of chlorpyrifos for Economic Co-operation and Development (OECD) for
in Labeo rohita. This study may also lead to reinforce the fact testing of chemicals (OECD, 1992).
of the efficacy of the Comet assay as a sensitive biomarker Acute LC50 (96 h) value of chlorpyrifos for L. rohita
of DNA damage in the erythrocytes and gill cells of Labeo was calculated as 442.8 mg/L (95% Confident limits: 437.77–
rohita. 447.93 mg/L) using a computer program, TSK (Trimmed
Spearman–Karber, 1991, Version 1.5). Using the LC50 value,
Materials and methods three nominal sub-lethal concentrations viz., sub-lethal I
Chemicals (SL-I, 1/2nd of LC50 ¼ 221.4 mg/L), sub-lethal II (SL- II,
1/4th of LC50 ¼ 110.7 mg/L) and sub-lethal III (SL-III, 1/6th
Methyl methanesulfonate (MMS) and Ethidium bromide of LC50 ¼ 73.8 mg/L) were prepared.
were purchased from Sigma-Aldrich, (St. Louis, MO).
Normal melting point (NMP) agarose was obtained from
In vivo sub lethal exposure experiment
Invitrogen Life Technologies Ltd. (Paisley, UK). Low melting
point (LMP) agarose was supplied by Promega Corporation The fish specimens were exposed to three above mentioned
(Madison, WI). Ethylenediamine tetraacetic acid (EDTA) sublethal concentrations of chlorpyrifos in a static bioassay
disodium salt was purchased from GIBCO Life technologies system for 96 h with a 16:8 h (light:dark) photoperiod.
Inc. (Grand Island, NY). Phosphate buffered saline (PBS) was Tissue samples were procured at the intervals of 24, 48, 72
supplied by Invitrogen (Carlsbad, CA). Dimethyl sulphoxide and 96 h from the two fish per concentration per interval.
(DMSO) was obtained from Labscan Asia Co Ltd. (Bangkok, Dechlorinated tap water and methyl methanesulfonate
Thailand). Triton X-100 was supplied by Applichem GmbH (MMS) (5 mg/L) were used as negative and positive controls,
(Darmstodt, Germany). Trizma base, NaOH, HCl, NaCl, respectively.
MgSO47H2O, K2HPO4, and all other chemicals used were Physico-chemical properties of water (Temperature, pH,
of analytical grade. dissolved oxygen, electrical conductivity and total hardness)
Commercial formulation of chlorpyrifos product (40% were analyzed before and after the pesticide exposure.
EC), named as ‘‘Chlorpyrifos’’ (manufactured by M/s. K & N Whole blood and the gills were taken at each sampling
Efthymiadis, Greece), was purchased from the local market. event and were immediately processed for the Comet assay,
It was observed that the chlorpyrifos of this grade is mostly subsequently. Blood sampling was carried out from the
employed in the fields. cardiac puncture using heparinized syringe.
468 M. Ismail et al. Drug Chem Toxicol, 2014; 37(4): 466–471
Alkaline single-cell gel electrophoresis (SCGE) and were not considered for the analysis. The length of the
migrated DNA in the Comet tail was measured using an
The alkaline single-cell gel electrophoresis (SCGE)/
ocular micrometer (Grover et al., 2003). DNA damage
Comet assay was performed using three-layer procedure
was measured at individual cell level with the help of the
(Singh et al., 1988) with slight modifications (Klaude et al.,
following formula:
1996). The gill tissue (about 50 mg) was cut into small
pieces using scissors and homogenized in ice-cold homogen- Comet tail length ðmmÞ ¼ total comet length
ization buffer (1X Hanks’ balanced salt solution, 20 mM head diameter
EDTA, 10% dimethyl sulphoxide (DMSO), pH 7.0–7.5).
The cell suspension was centrifuged at 3000 rpm at 4 C
for 5 min and the cell pellet was finally suspended in Behavioral study
chilled phosphate buffered saline solution for the Comet Behavioral responses of the fish were observed right after
assay. the application of the pesticide till the end of experiment.
The blood samples (500 ml) were collected from each The behavioral changes were also monitored in the control
fish by cardiac puncture using a heparinized syringe and group.
diluted with 1 ml phosphate buffered saline solution (pH 7.0–
7.5). Viability of both the erythrocytes and gill cells was Statistical analysis
Drug and Chemical Toxicology Downloaded from informahealthcare.com by Gunda Reddy on 09/08/14
lysing) and refrigerated at 4 C for 2 h. The slides were then ments recorded as temperature: 26 ± 1 C; pH: 8.2 ± 0.2;
immersed in fresh cold alkaline electrophoresis buffer dissolved oxygen: 8.7 ± 0.2 mg/L; electrical conductivity:
(300 mM NaOH, 1 mM Na2 EDTA, pH413) in a horizontal 2.62–2.75 mS/m; hardness: 85–96 as mg CaCO3/L were
gel electrophoresis unit and left in the solution at 4 C for according to USEPA specifications (USEPA, 2002). Using the
20 min for salt equilibrium and DNA unwinding. Comet assay significantly higher levels of DNA damage
Electrophoresis was performed in the same solution at 25 V (p50.01) were detected in fish tissues exposed to different
(0.73 V/cm), 300mA for 25 min at 4 C. The slides were sublethal concentrations of chlorpyrifos than in control
neutralized three times with 0.4 M Tris buffer (pH 7.5) for samples (Table 1). The observed DNA damage was concen-
5 min each to remove excess alkali and finally were stained tration dependent and those levels of DNA damage in
with 20 ml of ethidium bromide (20 mg/ml). Slides were between the tested concentrations were significantly different
observed under an epifluorescent microscope (Labomed (p50.01). The highest DNA damage was observed at the
Lx400, Labo America, Inc, Fremont, CA) equipped with an highest sublethal chlorpyrifos concentration III (i.e. 221.4 mg/
excitation filter 515–560 nm and emission filter 590 nm. Total L) followed by sublethal concentration II (i.e. 110.7 mg/L) and
100 individual cells were observed per fish per concentration sublethal concentration I (i.e. 73.8 mg/L).
(50 cells per slide). Cells with no DNA damage have The observed DNA damage was time dependent and those
nucleoids, whereas the cells with DNA damage have levels of DNA damage in between the times were signifi-
Comet-like appearance. The cells having DNA damage with cantly different (p50.01) (Table 1). The lowest DNA damage
no head or a dispersed head were regarded as apoptotic cells was observed at 24 h of exposure, whereas the highest DNA
Table 1. Mean comet tail length (mm), ±SD, of erythrocytes and gill cells of Labeo rohita exposed to different concentrations of chlorpyrifos
at different time intervals.
Values with different alphabet superscripts differ significantly (p50.01) between tissues within concentration. Values with different numeric
superscripts differ significantly (p50.01) between concentrations within duration and tissue. Values with different capital alphabet superscripts differ
significantly (p50.01) between durations within concentration and tissue.
DOI: 10.3109/01480545.2014.887093 Genotoxicity of chlorpyrifos 469
Table 2. Behavioral responses of Labeo rohita exposed to different environmental risk assessment of different chemicals
sublethal concentrations of chlorpyrifos for 96 h.
(Bucker et al., 2012). The Comet assay is generally used as
Chlorpyrifos (mg/L)
a biomarker of genotoxicity in animals exposed to environ-
mental pollutants and the increased Comets are associated
Behavioral responses Control 73.8 110.7 221.4 with the serious consequences on the animal health (Fairbairn
Activeness – – + ++ et al., 1995; Pavlica et al., 2001; Steinert, 1999). In past,
Loss of balance – – + +++ a number of studies have explained the genotoxicity
Opercular activity – – + ++
Rate of swimming – – + ++ of chlorpyrifos in animals, for example, chlorpyrifos induced
Lateral side movement – – + ++ the genotoxicity in the lymphocytes and gill cells of
Forward movement with – – + +++ freshwater fish (Channa punctatus) (203.0 mg/L, 406.0 mg/L,
posterior side up 609.0 mg/L and 68.0 mg/L, 102.0 mg/L, 203.0 mg/L) (Ali et al.,
Movement in circular – – + ++
fashion with jerks 2008a, 2009) and in the erythrocytes and liver cells of
Chinese toad (Bufo bufo gargarizans) (80.0 mg/L, 160.0 mg/L,
The increase or decrease in the level of behavioral parameters is shown 320.0 mg/L, 640.0 mg/L) (Yin et al., 2009). Our results
by numbers of (+) sign.
The () sign indicate normal behavioral conditions
showed an increase in DNA damage in erythrocytes and gill
cells of Labeo rohita exposed to sublethal concentrations
Drug and Chemical Toxicology Downloaded from informahealthcare.com by Gunda Reddy on 09/08/14
observed in Rohu (Labeo rohita) exposed to different the variable number of alkali-labile sites in DNA of cells
concentrations of chlorpyrifos throughout the experimental taken from different tissues (Lee & Steinert, 2003). This may
period (Table 2). Fish in the control group showed normal also be attributed to the different physiological nature of the
behavior during the whole exposure time. Exposure of the activities associated with those specific organs, with respect
lowest sublethal concentration of chlorpyrifos (73.8 mg/L) to either detoxification of the toxicants or the repair of
also showed the normal behavior in fish. Fish in the different types of DNA strand breaks (Lee & Steinert, 2003).
sublethal concentration 110.7 mg/L chlorpyrifos showed Furthermore, the significantly higher DNA damage in gill
neurotoxic effects including loss of balance, lateral side cells might be due to the direct and continuous exposure of
movement, movement in circular form with jerks, forward this organ to the genotoxic chemicals dissolved in water
movement with posterior part upward at an angle of around (Dzwonkowska & Hubner, 1986), whereas the erythrocytes
45 and increase in opercular flapping and swimming rate. comes in contact with these DNA damaging chemicals when
At the highest chlorpyrifos concentration (221.4 mg/L), the these genotoxic chemicals enter into the circulatory system.
severity of the previous mentioned effects was increased Our results are in agreement with the previous studies
(Table 2). indicating the higher DNA damage in gill cells as compared
to the blood erythrocytes in different fish species exposed to
different pesticides (Ali et al., 2009; Ateeq et al., 2005;
Discussion
Pandey et al., 2006).
In this study acute LC50 (96 h) value of chlorpyrifos for Rohu To study of behavioral changes is an important parameter
(Labeo rohita) has been estimated as 442.8 mg/L and is ranked in the evaluation of toxicity of pesticides in fish (Beauvais
as class II (moderately toxic) insecticide (USEPA, 1989). Our et al., 2000; Scholz et al., 2000). In the current study the fish
calculated 96 h LC50 value (442.8 mg/L) of chlorpyrifos in showed concentration and time dependent effects (Table 2).
Labeo rohita is lower than the 96 h LC50 value (811.98 mg/L) The Labeo rohita showed no abnormal response to the control
of chlorpyrifos estimated in freshwater fish (Channa as well as to the least concentration of chlorpyrifos through-
punctatus) (Ali et al., 2008a) and slightly higher than 96 h out the experimental duration (96 h), which shows the severity
LC50 (311 mg/L) in fathead minnow (Pimephales promelas) with the increase of pesticide concentrations and the passage
(Werner et al., 2008). This variation in the LC50 value among of time. The abnormal behavioral responses includes loss of
different fish species may be because of the different behavior balance, lying laterally at bottom, rapid opercular movements
of the species due to difference in their genetic makeup that with opened mouth, swimming forward in circular form
may have role in influencing the ability to detoxify certain with jerks and forward movement with posterior side up.
chemicals. This may also be due to the resistance of different The current behavioral results are in accordance with the
fish species and water quality parameters. previous studies (Fukuto, 1990; Ismail et al., 2009; Sarikaya
Fish can be used as an excellent biological specimen & Yilmaz, 2003; Selvi et al., 2005) of different pesticides
to evaluate the genotoxic potential of toxicants for in other fish species.
470 M. Ismail et al. Drug Chem Toxicol, 2014; 37(4): 466–471
Conclusions to a glyphosate formulation using the micronucleus test and the comet
assay. Mutagenesis 22:263–268.
The current findings reveal that chlorpyrifos seems to be Collins AR. (2004). The comet assay for DNA damage and repair.
moderately toxic to the Labeo rohita in a static bioassay Mol Biotech 26:249–261.
Dimitrov B, Gadeva P. (1997). Genotoxicity studies on the insecticide
system, thus imparting the negative impact on the behavior of dursban in root meristem cells of Crepis capillaris L. Environ Exp Bot
the fish. Chlorpyrifos induces DNA damage, as measured 37:199–209.
with the Comet assay, in erythrocytes and gill cells of Labeo Dzwonkowska A, Hubner H. (1986). Induction of chromosmal aberra-
rohita at sublethal concentrations. Also the increase in tions in the Syrian hamster by insecticides tested in vivo. Arch Toxicol
58:152–156.
DNA damage is concentration and time duration-dependent. Fairbairn DW, Olive PL, O’Neill KL. (1995). The comet assay:
In comparison the gill cells showed more sensibility to a comprehensive review. Mutat Res 339:37–59.
genotoxicity. It can be concluded that the chlorpyrifos is a Fukuto TR. (1990). Mechanism of action of organophosphorus and
genotoxic and neurotoxic insecticide causing DNA damage carbamate insecticides. Environ Health Perspect 87:245–254.
Gilliom R, Barbash J, Crawford C, et al. (2006). The quality of our
and neurotoxic effects in Labeo rohita, and the Comet assay Nation’s waters-Pesticides in the Nation’s streams and ground water,
can be used to evaluate the genotoxic potentials of pollutants 1992–2001: US Geological Survey Circular 1291, 172 p.
in biomonitoring studies using fish (Labeo rohita) as a model Gollapudi BB, Mendrala AL, Ann Linscombe V. (1995). Evaluation of
organism. the genetic toxicity of the organophosphate insecticide chlorpyrifos.
Mutat Res/Gen Toxicol 342:25–36.
Drug and Chemical Toxicology Downloaded from informahealthcare.com by Gunda Reddy on 09/08/14
the acute toxicity of effluents and receiving waters to freshwater and evaluation of chlorpyrifos to amphibian Chinese toad
marine organisms. EPA-821-R-02-012. Office of Water Washington (Amphibian: Anura) by Comet assay and Micronucleus test.
eD. CDC. Mutat Res 680:2–6.
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