ACUTE TOXICITY OF CHLORPYRIFOS TO ZEBRAFISH, DANIO RERIO (CYPRINIDAE)

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1 Research Article NBU J. Anim. Sc. 11: (2017) ISSN ACUTE TOXICITY OF CHLORPYRIFOS TO ZEBRAFISH, DANIO RERIO (CYPRINIDAE) Swati Singh 1, Min Bahadur 1 *, Soumen Bhattacharjee 2 and Joydeb Pal 3 1 Genetics and Molecular Biology Lab, Department of Zoology, University of North Bengal, 2 Cell and Molecular Biology Lab, Department of Zoology, University of North Bengal, 3 Ecology and Toxicology Lab, Department of Zoology, University of North Bengal, Received: September 4, 2017 Revised & Accepted: November 17, 2017 ABSTRACT Chlorpyrifos is a widely used organophosphate insecticide which causes toxic effects to non-target aquatic organisms, especially fishes. In this study experiments were made in the laboratory to determine median lethal concentration ( ) of chlorpyrifos to Zebrafish, Danio rerio (Cyprinidae) and behavioural effects of the insecticide on the fish. The 96 hour value of chlorpyrifos, determined from the mortality data using Probit analysis, was found as 289 µg/l for the Zebrafish, D. rerio. The fish exhibited respiratory distress such as gasping in air, loss of balance and erratic swimming behaviour prior to death. The results indicate that chlorpyrifos is extremely toxic to fish and should be used with extreme caution. Keywords: Zebrafish, toxicity, chlorpyrifos, Probit, behaviour, INTRODUCTION Pesticides are used extensively in agriculture to control pests. After application, less than 0.1% of the pesticides reach the specific target organisms whilst the rest 99.9% enters the environment (Pimentel, 1995) causing environmental contamination and toxicity to non-target organisms, both terrestrial and aquatic. Pesticides cause chronic and acute fish poisoning and damage the vital organs (Joshi and Sahu, 2007), reduce reproductive ability (Sharma and Ansari, 2010), skeletal deformities (Kumar and Ansari, 1984) and induce various biochemical changes in fish. Chlorpyrifos (CPF), an organophosphate insecticide, is used worldwide for the control of agricultural and non-agricultural insect pests (Lemus and Abdelghani, 2000). It is an acetylcholine esterase (AChE) inhibitor, and affects the nervous system (Sun and Chen, 2008). This insecticide is useful for the control of a wide range of important insects and other arthropod pests. Chlorpyrifos, being a nonsystemic insecticide, is effective by ingestion, inhalation and direct contact (Tomlin, 2006). Chlorpyrifos is activated to its oxon metabolite by cytochrome P450 to become toxic and also inhibits the AChE activity (Barata et al., 2004). Half life of chlorpyrifos is 25.6 days (water ph 7.0) (Shi et al., 2000), 30 days (soil), 60 days (less alkaline soils) and weeks or months (indoors) (Arcury et al., 2007). Chlorpyrifos passes via air drift of surface run-off into the surrounding waters and gets accumulated in different aquatic organisms, particularly fishes, adversely affecting them. The occurrence of chlorpyrifos in freshwater bodies is harmful to the fish and other non-target aquatic *Author for correspondence: Dr. Min Bahadur: bahadurmin@rediffmail.com / min.b@rediffmail.com 45

2 Swati Singh, Min Bahadur, Soumen Bhattacharjee and Joydeb Pal organisms (Starner et al., 2005). Chlorpyrifos has been reported to be used in the tea plantations of the Terai and the Dooars region of the Darjeeling foothills to curb the insect pest menace (Bishnu et al., 2009, Singh et al., 2015). Water bodies have been reported to be contaminated with this insecticide with a residue level of ± ppm (Singh et al., 2015). Fish muscles of Puntius sp. showed a concentration of chlorpyrifos as high as ± ppm in river Deomoni of Terai region of West Bengal (Singh et al., 2015). Zebrafish is used as model organism in various researches including toxicological research (Spitsbergen and Kent, 2003). The International Organization for Standardization and the Organization for Economic Co-operation and Development (OECD) recommends the fish for toxicological researches (OECD, 1992) because large clutch sizes, small body sizes reduce the experimental costs. Objectives of the present study were to determine median lethal concentration ( ) of chlorpyrifos to Zebrafish D. rerio and changes in the behaviour of the fish exposed to the insecticide. MATERIALS AND METHODS Insecticide: The chlorpyrifos 20% EC was purchased from the local agrochemicals supplier (Dursban, Trade Mark of Dow AgroSciences LLC, USA). Test animal: Danio rerio having average body weight of 289 mg and total body length 2.55 cm was used in the present study. The fish specimens were collected from local hatchery, stocked at appropriate density in 20 litre glass aquaria containing de-chlorinated tap water and were acclimatized in this condition for at least 10 days before they were subjected to investigations. The fish were fed with commercial artificial feed equal to 1/10 th of their body weight. Renewal of water, clearing of unconsumed feed and fecal wastes were done daily by siphoning out 80% of water and refilling with fresh water and the aquaria screens were also cleaned daily (Sreedevi et.al, 2014). The test fishes were starved for 24 hrs before the start of the experiment (OECD, 1992). Experimental procedure: Static bioassays were conducted in the laboratory in a series of glass aquaria of 20 litres capacity. Aquaria were filled with water. A stock solution of the insecticide was prepared by dissolving Dursban (Chlorpyrifos 20% EC) in acetone. For the toxicity test, four concentrations of Chlorpyrifos (100, 200, 300, 400 µg/l) previously diluted in acetone were used. Three replicates of 8 fishes for each concentration of the pesticide were used. Eight (8) acclimatized fishes of nearly uniform size were stocked in each aquarium as soon as the test material (chlorpyrifos) was mixed. Each experiment was accompanied with a control having the same volume of acetone but without the pesticide. The acute toxicity assay was performed following the guidelines of the Organization for Economic Co-operation and Development (Test no. 203, OECD, 1992) for testing of chemicals. A fish was considered dead when its gill movements ceased and it did not respond to gentle prodding. Dead fish was removed from the aquarium to avoid deterioration. The behaviour of each test fish was observed and the mortality of the Zebrafish was recorded for different exposure periods, 24, 48, 72 and 96 hr at different concentrations. The dead fishes were removed as soon as they were seen. Probit analysi: Data obtained in each experiment were evaluated by the use of probit analysis statistical method (Finney, 1964) in Microsoft Excel RESULTS AND DISCUSSION Behavioural study: Fishes showed abnormal behaviour when exposed to the various concentrations of chlorpyrifos. No abnormal response was observed in the control group. Some 46

3 Acute toxicity of chlorpyrifos to zebrafish of the pesticide exposed fishes aggregated at the corner of the aquarium, took rest at the bottom of the aquarium laterally, frequently came to the surface to gulp air, showed stronger opercular movements, and occasionally turned upside down. They also showed erratic and darting swimming behaviour and lost their equilibrium compared to the controls. Their body colour darkened, pectoral and pelvic fins got expanded with frequent movement of fins and the fish rolled vertically with erratic swimming prior to death. Erratic/darting swimming behaviour and loss of equilibrium after the pesticide exposure was likely due to inhibition of Acetylcholine Esterase (AChE) activity. Similar results have also been reported by Kristen et al. (2009) in hybrid striped bass. Inhibition of AChE activity is a typical characteristic of organophosphate pesticides (Timchalk et al. 2002). The observed behavioural responses in the present study are in accordance with the previous reports of Ismail et al. (2014). and probit analysis: The 96 hr of Chlorpyrifos for the fish, Danio rerio was found to be 289 µg/l. Fig. 1 shows the regression line between the Probit kill of Danio rerio and log concentration of Chlorpyrifos. Tiwari and Ansari (2014) have reported the 24, 48, 72 and 96 hour values for Chlorpyrifos in Zebrafish as 0.40, 0.29, 0.21 and 0.16 µg/l, respectively, which is very less as compared to the present study. Reports by Rao et al. (2005) to chlorpyrifos toxicity in Oreochromis mossambicus and Gambusia affinis by semi-static method was found to be mg/ L and mg/l, respectively, which is comparable to results of the present study. Jeon et al., 2016 reported value of µg/L for chlorpyrifos in adult Zebrafish after 96 hr exposure which is much higher as compared to results of the present study (289 µg/l). The values of chlorpyrifos vary with species of fish. Johnson and Finley (1980) reported 96 hour value of chlorpyrifos as mg/l and 2.4 µg/l for channel catfish, Ictalurus punctatus Fig.1. Regression line between the Probit kill of Danio rerio and log concentration of Chlorpyriphos and Lepomis michrochirus, respectively. In another study conducted by Ali et al. (2008) the 96 hr values of chlorpyrifos for the freshwater fish, Channa punctatus was found as µg/l. Similarly, Werner et al. (2008) have shown that the 96 hr value of chlorpyrifos for fathead minnow (Pimephales promelas) was 311 µg/l. The value of chlorpyrifos to Cyprinus carpio was reported as 0.16 mg/l and 0.58 mg/l by Ramesh and Munniswamy (2009) and Xing et al. (2011) respectively. The 96 hr values of chlorpyrifos in juvenile and adult of Oreochromis niloticus were determined as µg/l and µg/l, respectively, which reveal that chlorpyrifos can be rated as highly toxic to fish (Oruc, 2010). The 96 hr value of chlorpyrifos to Poecila reticulata was found as ppm/l (Sharbidre et al., 2011). In a study conducted by Ismail et al. (2014) the acute (96hr) value of Chlorpyrifos for Rohu (Labeo rohita) was found as µg/l. Such variations in the values among different fish species may be attributed to the different behaviour of the species due to differences in their genetic makeup that may have a role in influencing the ability to detoxify certain chemicals. This may also be due to the resistance of different fish species to the pesticide and water quality parameters. It can be concluded that chlorpyrifos and other organophosphate pesticides are highly toxic to fish species including Zebrafish. CONCLUSION The behavioural changes observed in the Zebrafish due to exposure to the chlorpyrifos indicate 47

4 Swati Singh, Min Bahadur, Soumen Bhattacharjee and Joydeb Pal that Zebrafish is highly sensitive to chlorpyrifos. The 96hr value of chlorpyrifos to zebrafish also indicates that chlorpyrifos is highly toxic to fish and its application in the agricultural fields should be made judiciously so that non-target organisms are protected from the hazards of the insecticide. ACKNOWLEDGEMENTS Authors are thankful to the Head, Department of Zoology for providing the Departmental Central Instrument facility which is supported by the Fund for Improvement of Science and Technology Infrastructure programme (FIST), Department of Science and Technology, New Delhi, India and the Special Assistance Programme (SAP) of the University Grants Commission, New Delhi. Department of Biotechnology, Government of West Bengal, EN 24, Sector V, Salt Lake, Kolkata is sincerely acknowledged for funding the Project (Sanction no. 241/Sanc)-BT (Estt.) RD-19/13 dated ). DISCLOSURE STATEMENT The authors report no declaration of interest. FUNDING This work is supported by the funding agency Department of Biotechnology, Government of West Bengal (Sanction no. 241/Sanc)-BT (Estt.) RD-19/13 dated ) Ali, D., Nagpure, N., Kumar, S., Kumar, R. & Kushwaha, B Genotoxicity assessment of acute exposure of chlorpyrifos to freshwater fish Channa punctatus (Bloch) using micronucleus assay and alkaline singlecell gel electrophoresis. Chemosphere 71: Arcury, T. A. Grzywacz, J. G. Barr, D. B. Tapia, J. Chen, H. & Quandt, S. A Pesticide urinary metabolite levels of children in eastern North Carolina farm worker households. Environ. Health Perspect. 115: Barata, C., Solayan, A., & Porte, C Role of b- esterases in assessing toxicity of OP (Chlorpyriphos, Malathion) and carbamate (Carbofuran) pesticides to Daphnia magna. Aquat. Toxicol. 66: Bishnu, A., Chakrabarti, K., Chakraborty, A. & Saha, T Pesticide residue level in tea ecosystems of Hill and Dooars regions of West Bengal, India. Environ. Monit. Assess.149: Finney, D. J Probit Analysis: A statistical treatment of sigmoid response curve. Cambridge University Press, U.K Ismail, M., Khan, Q. M., Ali, R., Ali, T. & Mobeen, A Genotoxicity of chlorpyrifos in freshwater fish Labe o rohi ta using Alkali ne Si ngle- Cell Gel Electrophoresis (Comet) assay. Drug Chem. Toxico, Early Online: 1-6. Jeon, H. J., Lee, Y. H., Kim, M. J., Choi, S. D., Park, B. J. & Lee, S. E Integrated biomarkers induced by REFERENCES chlorpyrifos in two different life stages of (Danio rerio) for environmental risk assessment. Environ. Toxicol. Pharmacol. 43: Johnson, W. W. & Finley, M. T Handbook of Acute toxicity of Chemicals to fish and aquatic invertebrates. U.S. Fish and Wildlife Service Resource Publications. 71: Joshi, N. & Dharml ata Sahu, A. P Histopath ological cha nges in the liver of Heteropneustes fossilis exposed to Cypermethrin. J. Environ. Biol. 28: Kristen, M. G., Roberts, A. P., Ellis, N., Swers, A. D. & Klain, S. J Biochemical and behavioral effects of Diazinon exposure in hybrid striped bass. Environ. Toxicol. Chem. 28: Kumar, K. & Ansari, B. A Malathion Toxicity: Skeletal deformities in Zebrafish, (Brachydanio rerio). Pestic. Sc. 15: Lemus, R. & Abdelghani, A Chlorpyrifos: an unwelcome pesticide in our homes. Rev. Environ. Health 15: Organisati on of Economi c Coope ration and Development (OECD) Guidelines for testing of chemicals, Guideline 210 fish, Early- life stage Toxicity Test. Oruç, E. O Oxidative stress, steroid hormone 48

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