International Research Journal of Environment Sciences________________________________ ISSN 2319–1414Vol. 2(6), 88-90, June (2013) Int. Res. J. Environment Sci. International Science Congress Association 88 Short Communication Histopathological changes in the Liver of Indian Flying Barb (Esomus danricus) exposed to Organochlorine Pesticide, Endosulfan (EC 35) Das Suchismita and Gupta AbhikDepartment of Life science and Bioinformatics, Assam University, Silchar-788011, INDIA Department of Ecology and Environmental Science, Assam University, Silchar-788011, INDIA Available online at: www.isca.in Received 9th May 2013, revised 21st May 2013, accepted 20th June 2013 AbstractIndian flying barb (Esomus danricus) was exposed to sublethal concentrations of endosulfan (EC 35) for 28 days and liver histopathology was observed by light microscopy after staining with Haematoxylin-Eosine. The hepatocytes showed pathological changes ranging from mild inflammatory infiltration, swelling and vacuolation to nuclear picnosis and necrosis. Higher dose had more severe effect on liver. Keywords: Hepatocyte, chronic, necrosis, vacuolation, picnosis. Introduction Endosulfan, an organochlorine pesticide, is used to control insects and mites infesting crops including vegetables, fruits, tea, coffee, cotton, rice and grains. It is a neurotoxic pesticide that attacks the gamma aminobutyric acid receptor complex in the central nervous system. Endosulfan is still used in many areas of the developing countries irrespective of ban or restricted use in many countries. This fuels concern about potential pesticide run off into water bodies which harbours, amongst other, fishes. Endosulfan is partially degraded into endosulfan sulfate, which often enters the water bodies via surface runoff of contaminated soil. Endosulfan sulfate is highly toxic. The Indian flying barb, Esomus danricus (Hamilton-Buchanan) inhabits shallow water bodies of Northern India and is a cyprinid that is economically important both as an ornamental and food fish. This fish runs the risk of ending up as the non target victims of such pesticides that are being sprayed in agricultural crops. Although liver plays crucial role in metabolism5, yet exposure of excess xenobiotics often cause structural changes of hepatocytes. Endosulfan is reported to be hepatotoxic and therefore, liver can serve as a potent biomarker for assessing endosulfan toxicity in fish. The present study was thus, aimed to determine the histopathological effects of sublethal concentrations of endosulfan to Indian flying barb liver after 28 days of exposure. Material and MethodsFishes of similar length (46.77 ± 4.30 mm) and weight (0.86 ± 0.16 g) were collected from unpolluted, freshwater ponds near Assam University campus, Barak valley, South Assam, India. They were acclimatized under laboratory conditions seven days prior to experimentation. Temperature, pH, hardness and dissolved oxygen under laboratory condition were 29C, 6.8, 30 mg l-1 and 5.5 mg l-1 respectively. A stock solution of commercial grade endosulfan (Thiodan- endosulfan 35% EC, Bayer Crop Science, India) was prepared using double distilled water. Serial dilutions of stock solutions were prepared using tap water as per dilution techniques10. Static-with-renewal acute toxicity tests were conducted with ten fish in each graded concentration and 96 hours LC50 value was found to be 4.9 gl-1 in a prior study. Three sub-lethal test concentrations viz., 0.49, 0.049 and 0.0049 gl-1 were selected for inducing histological changes in fish liver. Ten fish for each concentration of test chemical were kept separately in three litres of toxicant treated media for 28 days. Food was given during the study period. Test water was renewed every 24 hrs. After 28 days of exposure, fish were sacrificed and liver were removed immediately and kept in 10% Formalin, as fixative, for 24 h, dehydrated, embedded in paraffin and sections cut at 5 µm thickness and stained with Harris Haematoxylin and Eosin. Changes induced by treatment in the liver tissues were photographed and analyzed by light microscope at 10X eye piece magnification and 40X objective magnification {Olympus (model U-CMAD3) with Camera attachment of Samsung (model SDC-313B)}. Results and DiscussionOn analysis of control liver (figure 1) of Esomus at 400X magnification, a normal morphology with no pathological abnormalities was observed. The hepatocytes present a homogenous cytoplasm and a large central or subcentral spherical nucleus. The qualitative liver histology in fish exposed to 0.49µgl-1 endosulfan for 28 days showed inflammatory cell infiltration, picnotic nucleus, swelled hepatocytes and necrotic areas where hepatocytes have lost the membrane with dissolved nucleus (figure 2). In fish exposed to exposed to 0.049µgl-1endosulfan for 28 days, the hepatocytes show inflammatory cell infiltration, picnotic nucleus, swelled hepatocytes and necrotic International Research Journal of Environment Sciences______________________________________________ ISSN 2319–1414 Vol. 2(6), 88-90, June (2013) Int. Res. J. Environment Sci. International Science Congress Association 89 areas (figure 3). In fish exposed to exposed to 0.0049µgl-1endosulfan for similar duration, the hepatocytes show mild inflammatory infiltration, swelling with peripheral nucleus and necrotic areas (figure 4). In the liver, histological changes observed were more pronounced in fish exposed to higher endosulfan concentrations. Figure-1 T.S. of control liver of Esomus danricus Figure-2 T.S. of liver of Esomus danricus exposed to 0.49 µgl-1 endosulfan (400X). The hepatocytes show inflammatory cell infiltration (a), picnotic nucleus (b), swelled hepatocytes (c) and necrotic areas (d) Fish liver has the ability to detoxify pesticides but high concentrations of these compounds can alter hepatic enzyme activities that can result in damage of hepatocytes11,12. Several studies demonstrated that alterations in number, size and shape of the hepatocyte nucleus can be due to contaminants. In the present study, the flying barb hepatocytes show swelling and vacuolation, the nucleus is pushed towards periphery; some necrotic areas were also observed where hepatocytes have lost the membrane with dissolved nucleus. The swelling of hepatocytes can be indicative of increased activity of xenobiotic biotransformation enzymes as observed in perch, Perca fluviatilis13 and sculpin, Myoxocephalus scorpius14. Alterations in size and shape of nucleus have often been regarded as signs of increased metabolic activity as well as pathological origin15. In the present study, higher doses of endosulfan caused picnosis of hepatocyte nucleus. Extensive degeneration of cytoplasm with piknosis of nuclei and loss of glycogen in liver tissue of Heteropneustes fossilis due to acute endosulfan (Thiodan) toxicity was also observed in another study16. In Brachydanio rerio, alterations in the size of nucleus have been reported to be influenced by contaminant12 The present study also showed that all the exposure doses caused inflammatory infiltration in the portal areas. Sublethal concentrations of endosulfan can cause behavioural disturbances and hepatic injury17-18. The liver histology in flying barb showed that endosulfan caused some alterations of the hepatocytes like vacuolization and necrosis. These alterations are often associated with a degenerative-necrotic condition19. In Channa punctata, endosulfan caused lipid peroxidation and disrupted antioxidant enzymes in liver20which in turn might lead to hepatic dysfunction. Besides, it appears that the degree of structural heterogeneity is enhanced with increasing concentration of the toxicant21. As seen in the present study, the liver histological changes observed were more evident in fish exposed to higher dose of endosulfan. Similar hepatic histopathological effects were observed in fish due toother chlorinated pesticides such ashexachlorocyclohexane 22and lindane23. Figure-3 T.S. of liver of Esomus danricus exposed to 0.049 µgl-1 endosulfan (400X). The hepatocytes show inflammatory cell infiltration (a), picnotic nucleus (b), swelled hepatocytes (c) and necrotic areas (d) Figure-4 T.S. of liver of Esomus danricus exposed to 0.0049 µgl-1 endosulfan (400X). The hepatocytes show mild inflammatory infiltration (a), swelling with peripheral nucleus (b) and necrotic areas (c) International Research Journal of Environment Sciences______________________________________________ ISSN 2319–1414 Vol. 2(6), 88-90, June (2013) Int. Res. J. Environment Sci. International Science Congress Association 90 ConclusionThus, it is evident from this study that endosulfan, at very low doses, was able to induce pathological lesions in the liver of Indian flying barb. References 1.Thangavel P., Sumathiral K., Maheswari S., Rita S. and Ramaswamy M., Hormone profile of an edible, freshwater teleost, Sarotherodon mossambicus (Peters) under endosulfan toxicity, Pestic. Biochem. Physiol, 97(3), 229– 234 (2010)2.Hassall K.A., The biochemistry and uses of pesticides structure, metabolism, mode of action and uses in crop protection, 2nd ed. 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