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Pesticide Mixture Induced DNA Damage in Peripheral Blood Erythrocytes of Freshwater Fish, Oreochromis niloticus




Pesticide Mixture Induced DNA Damage in Peripheral Blood Erythrocytes of Freshwater Fish, Oreochromis niloticus

Faiza Ambreen*1 and Muhammad Javed2

1Department of Zoology, GC Women University Faisalabad, Pakistan

2Department of Zoology, Wildlife and Fisheries, University of Agriculture, Faisalabad-38040, Pakistan


The present study was undertaken to examine the DNA damage induced by pesticide mixture (endosulfan+chlorpyrifos) in peripheral blood erythrocytes of freshwater fish, Oreochromis niloticus by using Comet assay. The 96-h LC50 value of pesticide mixture was estimated for 180-old fingerlings of O. niloticusin a static system and then four sub-lethal concentrations viz. 1/3rd of LC50, 1/4th of LC50, 1/5th of LC50 and 1/6th of LC50 were calculated and fish were exposed to these concentrations, separately in glass aquaria for 70 days at constant laboratory conditions. Peripheral blood erythrocytes were taken on 14, 28, 42, 56 and 70 day of exposure for the DNA damage assessment in-terms of percentage of damaged cells. In general, a dose dependent response was observed in fish erythrocytes with induction of maximum DNA damage at highest concentration (1/3rd of LC50) of pesticide mixture. Statistically significant effects for both concentrations and time of exposure in terms of DNA damage were observed in treated fish as compared to control group. The results supported the use of Comet assay for evaluating the toxicity of pollutants which may be used as part of environmental monitoring programs.

Article Information

Received 12 August 2015

Revised 03 July 2017

Accepted 23 September 2017

Available online 16 January 2018

Authors’ Contribution

AF designed the experiments, collected and analyzed the data and wrote the manuscript. JM reviewed the final version of manuscript.

Key words

Comet assay, Insecticides, Genotoxicity, Sub-lethal effects.


* Corresponding author:

0030-9923/2018/0001-0339 $ 9.00/0

Copyright 2018 Zoological Society of Pakistan



Different classes of pesticides are now frequently used against a number of pests, to increase the production of crops even though these are highly toxic to other non-target species in the environment (Pandey et al., 2008; Naqvi et al., 2016). An increase in global food demand has resulted in a significant increase in the use of pesticides in agriculture. Residues of pesticides reach the aquatic environment, representing a risk for the non-target organisms threatening the ecological balance and biodiversity (Asita and Makhalemele, 2008). As a result non-target organisms are exposed to a mixture of pesticides which may induce genetic damage (Konen and Cavas, 2008), representing major problem at local, regional, national and global scale (Cerejeira et al., 2003). Organochlorine and organophosphate are commonly detected pesticides classes in freshwater. Endosulfan belongs to organochlorine class of pesticides which are hazardous for the survival of life. This may modulate antioxidant defense system and cause oxidative damage in aquatic organisms due to production of reactive oxygen species (Liu et al., 2006). Among all forms of organophosphate pesticides, chlorpyrifos is one of the most widely used pesticides in the world. It is neurotoxic and irreversible inhibitor of the cholinesterase. Chlorpyrifos is highly toxic to aquatic organisms, and is among the most detected pesticide in rivers, streams, ponds and reservoirs (Ensminger et al., 2011). Genotoxicity of pesticides for non-target organisms and their influence on ecosystems are of worldwide concern. Fishes are being used as useful genetic model for the evaluation of pollution in aquatic environment (Nagrani et al., 2009). The DNA integrity has been proposed as a sensitive biomarker for monitoring environmental mutagens (Lourenço et al., 2013). Xenobiotics inducing strand breaks over extended time periods represent a constant threat to genomic integrity. Therefore, there exists need for rapid and sensitive assays that can be able to detect the foremost DNA lesions (Mohanty et al., 2013). Among different techniques used for the determination of genotoxic damage, the Comet assay (single cell gel electrophoresis) can detect DNA damage in single cell (Singh et al., 1988). This is very rapid and sensitive assay that can be successfully applied to the nucleated red blood cells of many fish species, exposed to different genotoxicants and environmental stressors (Mustafa et al., 2011). A number of studies have shown that Comet assay is the most effective assay for the detection of genotoxic effect of xenobiotics under the field (Polard et al., 2011) and laboratory conditions (Ali et al., 2008). Comet assay in alkaline conditions is a highly sensitive biomarker that can quantify and detect DNA damage such as single strand breaks, double strand breaks and alkali labile sites. These types of damages can be induced directly by the contaminants or indirectly through inactivation of repairing process (Tice et al., 2000). Therefore, the present study endeavor to investigate the DNA damage in terms of the percentage of damaged DNA in the freshwater fish, Oreochromis niloticus after in vivo chronic exposure to a binary pesticide mixture.


Materials and methods

Experimental fish and chemicals

The fingerlings of freshwater fish O. niloticus were purchased from local suppliers and transported to the Fisheries Research Farm, University of Agriculture, Faisalabad, Pakistan. Fish fingerlings were acclimatized under laboratory conditions in cemented tanks for two weeks and fed with pellet diet. The fecal matter and other waste materials were siphoned off daily to reduce ammonia contents in water. Endosulfan and chlorpyrifos of equal parts were dissolved, separately, in 95% analytical grade methanol (J.T Baker) as a carrier solvent to prepare the stock-I solutions (1g/100 ml) while binary mixture of pesticides were prepared by its further dilutions in deionized water (stock-II).

Determination of sub-lethal concentrations

The 96-h LC50 value of the pesticide mixture (endosulfan+chlorpyrifos) i.e., 5.64 µgL-1 for the present study on Oreochromis niloticus was determined. Based on this value, four sub-lethal concentrations viz. 1/3rd of LC50 (1.88 µgL-1), 1/4th of LC50 (1.41 µgL-1), 1/5th of LC50 (1.13 µgL-1) and 1/6th of LC50 (0.94 µgL-1) were calculated and used for in vivo genotoxicity experiments.

Comet assay

Twelve fingerlings (180-day old with an average weight of 16.26±0.79 g) of O. niloticus were exposed to each of the four aforementioned test concentrations, separately, in glass aquaria having 70 L water capacity. Simultaneously, one group of fish was maintained in tap water also, which was considered as “control” (unstressed group). During 70 days of exposure period, the fish were fed daily small quantity of food. Water temperature (30 ºC), pH (7.75) and hardness (225 mgL-1) were kept constant throughout the experiment. The exposure was continued for 70 days by using static water system and peripheral blood slides were prepared on day 14, 28, 42, 56 and 70 of exposure period and subjected to Comet assay. Aquaria water was renewed after every one week of interval. Experiment was conducted with three replications for each sub-lethal concentration. Blood samples were collected from caudal vein of fish, immediately transferred to eppendorf and treated with anticoagulants (heparin salt). Comet assay was performed as three steps procedure, followed by lysis, electrophoresis and staining (Singh et al., 1988). Two slides were prepared and one hundred and fifty cells per slide were randomly scored and analyzed by using an image analysis system attached to Epi-Fluorescence microscope (N-400M, American Scope; USA) at 400 X magnification and low lux (MD-800, American Scope; USA) camera. The DNA damage was quantified by visual classification of cells into the five categories “comets” corresponding to the tail length (measured through TriTekCometScore™) as undamaged (Type 0); low level damage (Type I); medium level damage (Type II); high level damage: (Type III) and complete damage (Type IV). The extent of DNA damage was examined as mean percentage of cells with medium, high and complete damaged DNA, which was calculated as the sum of cells with Types II+ III + IV. Statistical analyses were performed by using MSTATC computer software. Results were expressed as Means±SD. Means of data were compared for the statistical differences by using Duncan Range Multiple tests (Steel et al., 1996). A p-value less than 0.05 were considered statistically significant.



Acute toxicity test and sub-lethal concentrations

The 96-h LC50 value of endosulfan+chlorpyrifos mixture was calculated from the data obtained in acute toxicity bioassay by using Probit analyses method. The 96-h LC50 value for O. niloticus was 5.64 µgL-1. Genotoxicity evaluation needs the live samples therefore; the sub-lethal concentrations were selected for Comet assay. Accordingly, four sub-lethal test concentrations were calculated as 1/3rd of LC50 (1.88 µgL-1), 1/4th of LC50 (1.41 µgL-1), 1/5th of LC50 (1.13 µgL-1) and 1/6th of LC50 (0.94 µgL-1) at which fish were exposed separately, for the detection of DNA damage.

DNA damage assessment

Proportions of undamaged nuclei (Type 0), damaged nuclei (Type I to IV) and percentage of damaged cells (Type II+III+IV) induced in the peripheral erythrocytes of O. niloticus under 70 day exposure of endosulfan+chlorpyrifos mixture are presented in Table I and Figure 1 (A-F).

Results showed that fish exposed to different concentrations of pesticide mixture exhibited significantly (p<0.05) higher DNA damage in their erythrocytes than the control specimens. Among all test concentrations, proportions of Type 0 cells were observed higher in control group on all sampling days. However, percentage of Type I damaged cells were maximum on day 14 under 1/4th of LC50 while the maximum percentage of Type I damaged cells was observed on day 28, 42, 56 and 70 in fish exposed to 1/6th of LC50 as evident from their mean values of 30.00±2.00, 32.00±4.00, 34.00±2.00, 32.67±4.16 and 37.33±5.03%, respectively. However, Type I damaged nuclei exhibited non-significant differences between 1/5th and 1/6th of LC50 on 56th day of exposure. Percentage of Type II damaged cells ranged from 26.67 – 48.00% and exhibited concomitant increase in damage with exposure time from day 14 to 70 due to 1/3rd of LC50 exposure as compared to control group. Among different test concentrations, Type III damaged nuclei in peripheral blood erythrocytes of O. niloticus followed the sequence: 1/4thof LC50 ≥ 1/3rdof LC50> 1/5th of LC50> 1/6th of LC50> control on day 14; 1/3rd of LC50> 1/4th of LC50> 1/5th of LC50 ≥ 1/6th of LC50> control on day 28; 1/3rd of LC50 ≥ 1/4th of LC50> 1/6th of LC50> 1/5th of LC50> control on day 42; 1/3rd of LC50> 1/4th of LC50> 1/6th of LC50> 1/5th of LC50> control on day 56 and 1/3rd of LC50> 1/4th of LC50> 1/5th of LC50> 1/6th of LC50> control on day 70, respectively. However, on day 14 and 42, the proportion of Type III damaged cells showed non-significant differences between 1/3rd and 1/4th of LC50 exposures.



Table I.- DNA damage in peripheral erythrocytes of Oreochromis niloticus exposed to endosulfan + chlorpyrifos mixture.

Exposure duration


Test concent rations

Undam aged nuclei (%)

Comet class damaged nuclei (%)

Damaged cells (%) (II+ III+ IV)

Type 0

Type I

Type II

Type III

Type IV

14-day Control

97.33± 1.15a

2.67± 1.15 e

0.00±0.00 e

0.00±0.00 e

0.00± 0.00 d

0.00± 0.00 e


1/3rd of LC50

22.67± 2.31d

24.00± 2.00 d

26.67±3.06 a

15.33±1.15 b

11.33± 1.15 ab

53.33± 1.15 a


1/4th of LC50

22.67± 2.31d

30.00± 2.00 abc

21.33±1.15 b

16.67±2.31 ab

9.33± 1.15 b

47.33± 1.15 b


1/5th of LC50

35.33± 1.15c

26.00± 2.01 cd

18.00±2.00 c

11.33±1.15 c

9.33± 1.15 b

38.67± 1.15 c


1/6th of LC50

50.67± 1.15b

28.00± 1.11 bcd

7.33±1.15 d

7.33±1.15 d

6.67± 1.15 c

21.33± 1.15 d

28-day Control

96.00± 3.46 a

2.67± 1.15 e

0.67±1.15 e

0.67±1.15 d

0.00± 0.00 e

1.33± 2.31 e


1/3rd of LC50

17.33± 2.31 e

19.33± 1.15 d

30.67±1.15 a

19.33±1.15 a

13.33± 1.15 b

63.33± 1.15 a


1/4th of LC50

24.67± 1.15 d

20.00± 2.00 cd

22.00±2.00 bc

15.33±1.15 b

18.00± 4.00 a

55.33± 3.06 b


1/5th of LC50

32.00± 2.00 c

27.33± 3.06 b

19.33±1.15 c

12.00±2.00 c

9.33± 1.15 c

40.67± 2.31 c


1/6th of LC50

40.67± 3.06 b

32.00± 4.00 a

12.00±2.00 d

10.00±2.00 c

5.33± 1.15 d

27.33± 3.06 d

42-day Control

97.33± 1.15 a

2.00± 0.00 e

0.67±1.15 e

0.00±0.00 d

0.00± 0.00 c

0.67± 1.15 e


1/3rd of LC50

14.67± 3.06 d

16.67± 3.06 d

38.00±2.00 a

20.00±2.00 a

10.67± 1.15 a

68.67± 1.15 a


1/4th of LC50

21.33± 2.31 c

22.00± 2.00 c

28.00±2.04 c

19.33±2.31 a

9.33± 2.31 a

56.67± 1.15 b


1/5th of LC50

22.00± 4.00 c

30.00± 2.00 b

30.00±3.46 bc

8.67±1.15 c

9.33± 1.15 a

48.00± 5.29 c


1/6th of LC50

29.33± 2.31 b

34.00± 2.00 ab

20.00±2.00 d

11.33±1.15 b

5.33± 1.15 b

36.67± 3.06 d

56-day Control

96.00± 2.00 a

3.33± 1.15 d

0.67±1.15 e

0.00±0.00 e

0.00± 0.00 e

0.67± 1.15 e


1/3rd of LC50

8.67± 1.15 e

20.00± 2.00 c

40.67±3.06 a

22.67±1.15 a

8.00± 2.00 bc

71.33± 3.06 a


1/4th of LC50

14.67± 1.15 d

23.33± 3.06 bc

32.67±3.06 c

20.00±2.00 b

9.33± 1.15 ab

62.00± 2.00 b


1/5th of LC50

18.67± 3.06 cd

30.00± 2.00 a

35.33±1.15 bc

10.00±2.00 d

6.00± 2.00 c

51.33± 3.06 c


1/6th of LC50

25.33± 4.16 b

32.67± 4.16 a

22.67±1.15 d

16.00±2.00 c

3.33± 1.15 d

42.00± 2.00 d

70-day Control

94.67± 1.15 a

4.67± 1.15 e

0.67±1.15 e

0.00±0.00 e

0.00± 0.00 e

0.67± 1.15 e


1/3rd of LC50

15.33± 4.16 cd

16.00± 4.00 d

48.00±2.00 a

18.00±2.00 a

2.67± 1.15 cd

68.67± 1.15 a


1/4th of LC50

10.67± 1.15 e

25.33± 1.15 c

39.33±1.15 b

15.33±1.15 b

9.33± 1.15 ab

64.00± 0.00 b


1/5th of LC50

12.00± 2.00 de

32.67± 3.06 b

34.67±1.15 c

12.00±2.00 c

8.67± 1.15 b

55.33± 1.15 c


1/6th of LC50

35.33± 4.16 b

37.33± 5.03 a

19.33±1.15 d

7.33±1.15 d

0.67± 1.15 de

27.33± 1.15 d

Means with similar letters in a single column for each variable are statistically non-significant at p<0.05. n, 100 cells per treatment; Type I-IV, extent of DNA damage.


Similarly, percentage of Type IV damaged nuclei were observed maximum and minimum on 14th day exposure to 1/3rd of LC50 and control treatment, respectively. However, on day 28, 42, 56 and 70 the same was maximum due to 1/4th of LC50 exposure with significant differences among them (p<0.05). Among four test concentrations, 1/3rd, 1/4th and 1/5th of LC50 showed non-significant difference for the induction of Type IV damaged cells in peripheral erythrocytes of O. niloticus on all sampling days, except on 28th day. The extent of DNA damage was examined as the mean percentage of cells with medium, high and complete damaged DNA, calculated as sum of Type II, III and IV. Statistically significant (p<0.05) DNA damage was observed during whole exposure period due to different test concentrations. Regarding different treatments (control, 1/3rd of LC50, 1/4th of LC50, 1/5th of LC50 and 1/6th of LC50) the extent of DNA damage was observed significantly higher (p<0.05) due to 1/3rd of LC50 exposure followed by that of 1/4th of LC50, 1/5th of LC50 and 1/6th of LC50 as compared to control group on all sampling days indicating dose dependent DNA damage. Statistically significant time based DNA damage was observed in treated fish as compared to control group under four different sub-lethal concentrations. Comparing the DNA damage during all sampling days, the peripheral blood erythrocytes of O.niloticus exhibited concomitant increase in damage from 14th to 56th day, followed by decline on day 70 due to 1/3rd of LC50 exposure (Fig. 2A). However, at 1/4th and 1/5th of test concentrations, damage was significantly increased with the passage of time from day 14th to 70 (Fig. 2B, C). Similarly, 1/6th of LC50 exposure exhibited gradual increase in DNA damage from day 14th to 56th, followed by sharp decline on day 70 (Fig. 2D).




The present study elucidates the genotoxicity of pesticides mixture (endosulfan+chlorpyrifos) in freshwater fish, O. niloticus by using Comet assay. During the present experiments water temperature (ranged from 30.01 to 30.04ºC), pH (ranged from 7.75 to 7.77) and total hardness (ranged from 224 to 226 mgL-1) were kept constant because toxicity of pesticides to the aquatic organisms has been reported to be affected by water temperature, pH, hardness, size, species and age of fish (Young, 2000). Effects of pesticides on organism were often evaluated by using single toxicant, however, under present environmental conditions pesticides are present in multiple combinations in aquatic ecosystems. The contamination of aquatic ecosystems by pesticides has gained increasing attention in recent decades (Byer et al., 2011). Biological monitoring using a target species could allow a sensitive approach to predict the potential risk of persistent pollutants in aquatic environment. Tested sub-lethal concentrations in the present study could be environmentally relevant concentrations, although repeated applications of the pesticides in most developing countries may be higher, suggesting the relevance of test concentrations. Pesticides in sub-lethal concentration present in water are too low to cause rapid death directly but may affect the functioning of organisms, disrupt normal behavior and reduce the fitness of natural population (Susan et al., 2010).

We applied the alkaline version of Comet assay to evaluate the DNA strand breaks in the peripheral blood erythrocytes of O. niloticus exposed in vivo to different sub-lethal concentrations of endosulfan+chlorpyrifos mixture for 70 days. Long-term genotoxicity studies can be an important approach for achieving the greater insight into the organism DNA repair ability and other protective mechanisms for excreting the xenobiotics. Present findings showed that exposure of fish to four sub-lethal concentrations of pesticide mixture induced significantly higher DNA damage in blood cells as compared to control group. Mixture of pesticides (endosulfan, chlorpyrifos and thiram) has been reported to cause DNA damage (Tope and Rogers, 2009). Similarly, Polard et al. (2011) observed significant increase in DNA damage under the exposure of five pesticides mixture (metolachlor + isoproturon + chlorotoluron + atrazine + deethylatrazine) as compared to control. Dose and time dependent response of O. niloticus on DNA integrity was also observed by Rani and Kumaraguru (2013) due to exposure of endosulfan. Reactive oxygen species such as hydrogen peroxide, hydroxyl radical and superoxide anion have been shown to produce extensive damage such as DNA strand breaks, enzyme inactivation and apoptosis (Banudevi et al., 2006). It is possible that endosulfan and chlorpyrifos could cause alterations in DNA of O. niloticus resulting in comet induction. Altinoket al. (2012) observed significantly higher DNA damage in terms of tail length, tail intensity, tail moment and tail migration in Oncorhynchus mykiss exposed to various concentrations of carbosulfan for 60 days than that of control group.

Fish specimens exposed to sub-lethal concentrations viz. 1/4th of LC50, 1/2nd of LC50 and 3/4th of LC50 of carbosulfan exhibited significantly higher DNA damage (p<0.01) in erythrocytes and gill cells in terms of percentage of tail DNA than the control group. DNA damage in both tissues was found to be dose and time dependent (Nwani et al., 2010). DNA damage observed in present study might have originated from DNA single strand breaks, double strand breaks, DNA-DNA/DNA-protein cross linking or inhibition of enzymes involved in DNA repair resulting from the interaction of pesticides or their metabolites with DNA (Guilherme et al., 2012). Dose and time related increase in DNA damage in the form of comet induction was observed during present investigation. Similarly, Nwani et al. (2013) observed highest DNA damage on day 14 at 1/10th, 1/8th and 1/5th of LC50 exposure of herbicide, followed by gradual non-linear decline on day 21, 28 and 35. Time dependent decrease in DNA damage may be due to repair of damaged DNA, loss of heavily damaged cells or both (Saleha et al., 2001). Similarly, Banu et al. (2001) observed a time dependent decrease in DNA breaks in erythrocytes of Tilapia mossambica exposed to different concentrations of monocrotophos pesticide by using comet assay. Exposure to different concentrations of chlorpyrifos induced higher DNA damage in fish as compared to controls, indicating genotoxic potential of this pesticide to aquatic organisms (Ali et al., 2008; Yong et al., 2011). Present results are also in accordance with findings of Pandey et al. (2011) who observed concentration dependent DNA damage in Channa punctatus under exposure of organophosphate pesticide. Chronic exposure to mutagenic pollutants, lead to accumulation of DNA strand breaks (Ternjej et al., 2010) because DNA repair capacity of fish cell is low as compared to other species.




Indiscriminate use of pesticides to improve agricultural practices may have impacts on non-target organism’s especially aquatic lives which can ultimately pose a serious threat to the health of human communities. Biological monitoring approach help us to predict the potential risk of these contaminants and to devise the ‘‘safe levels’’ for such chemicals having genotoxic potential. Acute and chronic effects of individual pesticides have been widely studied for different fish species, but the information regarding the genotoxic potential of pesticides in the form of mixture is scant in literature. Our results on O. niloticus elucidate that this pesticide mixture (endosulfan + chlorpyrifos) possesses genotoxic threats. Overall, we focused on organochlorine and organophosphate exposures; however, the capacity of other pesticide classes to induce similar patterns of DNA damage should be investigated. Based on present results, we may suggest careful and sensible use of pesticides to guard against genetic hazards.




The author is grateful to the Higher Education Commission Pakistan for providing funds under the Indigenous Ph. D. Fellowship Program (Pin# 117-4654-BM7-094) to complete this work as a part of Ph. D. research.


Statement of conflict of interest

Authors have declared no conflict of interest.




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Pakistan Journal of Zoology


Vol. 51, Iss. 1, Pages 1-398


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