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1 Available online at International Journal of Research in Environmental Science and Technology Universal Research Publications. All rights reserved ISSN Original Article Impact of Dichlorvos on tissue glycogen and protein content in freshwater fingerlings, Oreochromis mossambicus (Peters) Lakshmanan, S 1*, A. Rajendran 2, C. Sivasubramaniyan 2 1 Department of Zoology, Poompuhar College, Melaiyur, Poompuhar, Nagapattinam District, Tamil Nadu. India. 2 Department of Botany, Bharathiar University, Coimbatore , Tamilnadu, India. 3 Department of Environmental Science, Tamil University, Thanjavur , Tamilnadu, India. * professorlakshmanan@gmail.com Received 20 January 2013; accepted 02 February 2013 Abstract Dichlorvos is an organophosphate (OP) insecticide widely used in developing countries. Because of its high acute toxicity and the consequent dangers to workers, there are concerns whether safe use is possible under such conditions. Hence, the present investigation was designed to assess the impact of Dichlorvos on tissue glycogen, total protein and albumen content in the selected tissues. Perusal of the data pertaining to the present experiments clearly indicates that there is a great changes in the selected parameters of the experimental fishes than control fishes. In the present study, when O. mossambicus is treated with sub lethal doses of Dichlorvos for all the exposure periods, it shows a significant decrease in the liver, kidney and muscle protein content and it is suggested that depletion of tissue total proteins after 7 days exposure period may be due to increased proteolysis thereby contributing to the availability of free amino acids that may be fed to the tricarboxylic acid (TCA) cycle and further possible utilization of its products for metabolic process. Several workers have observed the decrease in protein content in liver when organisms were subjected to pesticide treatments. But the present investigation provides the first report on the effect of Dichlorvos in fish fingerlings of O. mossambicus 2013 Universal Research Publications. All rights reserved Key words: Glycogen, Albumen, total protein, Oreochromis INTRODUCTION The Tamilnadu is a transitional environment on the Indian coast influenced by such human activities as agriculture, industry and tourism. The sludge of Tamilnadu and the rivers from the hinterland pour into the lagoon, where sediments trap pollution. Moreover, Trichy, Karur and Selam are still the site of one of the most important chemical industries in Tamilnadu and its industrial activities affect the surrounding environment, by contamination of air, soil groundwater and inner tidal canals. Bio-monitoring in the lagoon environment plays an important role in strategies and actions to identify, control and reduce the environmental threats. Bio-monitoring programs usually involve the use of biomarkers, which represent biochemical, physiological or behavioural variations measured in tissues, biological fluids or whole organisms [1,2]. Furthermore, Dichlorvos is an organophosphate (OP) insecticide widely used in developing countries. Because of its high acute toxicity and the consequent dangers to workers, there are concerns whether safe use is possible under such conditions. Dichlorvos is an insecticide of the organophosphate (OP) group. It has been in use since about 1955 and is used in the UK both professionally and in homes and gardens in a number of areas: in agriculture and horticulture it is used: in mushroom houses against mushroom flies; against various insects and beetles in poultry houses; and on protected ornamentals, protected vegetables and herbs and brassica seedlings; as a veterinary medicine, in protecting farmed salmon against salmon lice; and as an aerosol against cat and dog fleas; in public hygiene as an aerosol insecticide and space spray [3,4 & 5]. All these toxicological studies indicate that if these conditions of aquatic media prolong further, the survival of fishes will become a matter of question mark and indeed it is now very essential to understand the toxicity nature of various pollutants for proper management of fish culture. However, it is noticed that the toxic effect of Dichlorvos 20% EC on Oreochromis mossambicus has not yet been elucidated. Therefore now, an attempt has been made to study the possible impact and a complete systematic evaluation of Dichlorvos on the experimental freshwater fish Oreochromis mossambicus because of its wide 19

2 availability and suitability as a model for toxicity testing. MATERIALS AND METHODS Live specimen of Oreochromis mossambicus fingerlings procured from fish farm at Poompuhar Village Nagapattinam Districts were collected and brought to the laboratory without any mechanical injury. The collected fishes were given a dip treatment in 1% KMnO 4 solution for a few seconds to avoid any dermal microbial infection and then were rinsed with pure water. The fishes were screened for pathological signs, if any and kept in a big sterilized plastic trough containing well aerated and dechlorinated tap water for a period of two weeks at 27 ± 2 C room temperature under the laboratory conditions [6]. A commercial grade of Dichlorvos 20% EC manufactured by ACCO Industries, Haveli, has been used in the present investigation. (Dichlorvos technical based on 94% w/w ai, 215 w/w, emulsifier anionic/non ionic (Alkyl aryl sulphonate and polyoxyethylene ether), 6% w/w Aromex 72.5% w/w, Total 100% w/w]. The stock solution was prepared by adding 1.0 ml of Dichlorvos 20% EC to 19.0 ml of distilled water, which denoted 10,000ppm of pesticide dissolved. From this solution, 1.0 ml was pipetted out and added to 9.0 ml of distilled water, which denoted 1,000 ppm of pesticide dissolved. From this solution, 1.0 ml was taken out and added to 9.0 ml of water, which showed 100ppm of pesticide dissolved. Required amount of test solution as described in Standard methods.only healthy and active fish fingerlings of particular size of 6-7 cm in length and 9-10 g in weight were collected from the same fish farm and put in use for all the tests to find out the potency of pesticide pollutant Dichlorvos and to get information for the design and selection of dose levels. Static but renewal type of acute toxicity study was conducted after feeding the fish, to prevent any fungal infection and also to maintain the optimum dissolved oxygen level. Analysis of experimental work was mainly based on the techniques as recommended by [7] and APHA, [6]. Colorimetric micro method by [8] was adopted for the quantitative estimation of tissue glycogen. The liver, kidney and muscle tissues were isolated, weighed and homogenized in 5.0 ml of 80 percent methanol and centrifuged at 3000 rpm for 15 minutes. The residue was set apart for the quantitative estimation of glycogen. For the estimation of glycogen, the residue left after methanol extraction was homogenized in 5.0 ml of deprotenizing silver sulphate solution (5.0 gm TCA and 10 mg Ag 2 S0 4 in 100 ml distilled water) and heated at l00 C in water bath for 15 minutes. The mixture was then cooled and made up to 5.0 ml with deproteinizing silver sulphate solution once again and centrifuged at 2000 rpm for 10 minutes. The clear supernatant was collected for the estimation of glycogen. 1.0 m1 of respective tissue sample was taken in a separate test tube and 3.0 ml of concentrated sulphuric acid was added to it. The mixture was heated in a boiling water bath for 8 minutes and subsequently cooled under running tap water. The intensity of colour developed was measured in a spectrophotometer against the regent blank (3.0 ml con. sulphuric acid) at 520 nm. The quantitative amount of glycogen present in the respective tissues samples was read from the standard graph drawn previously from known quantitative amount of glycogen. The glycogen values are expressed as mg/g wet weight of tissue/ h. Total protein content in the tissues was estimated by the method of [9]. The liver, kidney and muscles tissues were isolated and homogenised in 10 percent TCA. The homogenate was centrifuged at 3000 rpm for 5 minutes. The supernatant was decanted and the residue was suspended in 1.0 ml of 0.1 N Sodium hydroxide solutions. 0.5 ml of this solution was transferred into a clean test tube and 4 ml of copper carbonate reagent solution was added and allowed to react for 5 min. The contents were mixed by lateral shaking and 0.4 ml of Folin Phenol (1: 1 dilution) reagent was added. The thoroughly mixed contents were kept at room temperature for 30 min. The colour developed was read at 600 nm against the reagent blank (0.5 ml Sodium hydroxide (1 N), 4.0 ml copper carbonate reagent and 0.4ml Folin reagent) in Spectrophotometer. The total protein contents in the tissues were expressed in mg / g wet wt. of tissue / h. For the estimation of tissue albumin Biuret method by [10] was adopted. The liver, kidney and muscles tissue were isolated and homogenised in 10 per cent TCA. The homogenate was centrifuged at 3000 rpm for 5 minutes. 10 ml centrifuge tube was taken and 5.5ml of Sodium sulphite (28% solution) was added. Then 0.5 ml of tissue extract was added and mixed thoroughly by inversion. Then 1.0 ml of ether was added and closed with a stopper and inverted gently for 20 times. Then the stopper was removed and centrifuged for 10 minutes. Then to the 3.0 ml of supernatant layer, 5.0 ml of Biuret reagent was added, mixed thoroughly and kept in water bath at 37 C for 10 minutes. 3.0mI of Standard solution was prepared by using 2.0 ml of Bovine Albumin Protein Standard Solution in 25 ml of distilled water. 3.0 ml distilled water was taken as Blank solution. After cooling, the colour developed was compared in a colorimeter using a Green filter. The values were expressed in mg / g wet weight of tissue I h. RESULTS Figure 1 gives the data on changes in liver glycogen level during sub-lethal Dichlorvos treatment and statistical analysis has revealed that these decreased changes are significant. At the end of 7 th day, an decrease in liver glycogen has been observed in all the three sub-lethal treatment. The analysis for liver glycogen level has shown a significant variation in the treated groups whereas the mean of control groups such as , and for 7, 14 and 21 days respectively are on par with each other. The analysis for kidney glycogen level has shown a significant variation in the treated groups whereas the mean of control groups such as 0.322, and for 7, 14 and 21 days respectively are on par with each other (Fig.2). Figure 3 shows the changes in muscle glycogen level during sub-lethal Dichlorvos treatment and statistical analysis has revealed that these decreased changes are found in treated groups whereas the mean of control groups such as 3.223, and for 7, 14 and 21 days respectively are on par with each other. The data in total protein content of liver in treated fish at sub-lethal concentrations of ppm, 20

3 Figure 1. Effect of Dichlorvos on the glycogen level (mg /g wet wt of tissue) in liver of Oreochromis mossambicus Figure 6. Effect of Dichlorvos on the total protein level (mg /g wet wt of tissue) in muscle of Oreochromis Figure 2. Effect of Dichlorvos on the glycogen level (mg /g wet wt of tissue) in kidney of Oreochromis Figure 7. Effect of Dichlorvos on the Albumin content (mg /g wet wt of tissue) in liver of Oreochromis Figure 3. Effect of Dichlorvos on the glycogen level (mg /g wet wt of tissue) in muscle of Oreochromis Figure 8. Effect of Dichlorvos on the Albumin content (mg /g wet wt of tissue) in kidney of Oreochromis Figure 4. Effect of Dichlorvos on the total protein level (mg /g wet wt of tissue) in liver of Oreochromis Figure 5. Effect of Dichlorvos on the total protein level (mg /g wet wt of tissue) in kidney of Oreochromis Figure 9. Effect of Dichlorvos on the Albumin content (mg /g wet wt of tissue) in muscle of Oreochromis ppm and 0.015ppm of Dichlorvos after 7 th, 14 th and 21 st day's exposure period are shown in figure 4. In the control fish, the mean value for protein content observed for 7, 14 and 21 treated periods are , and mg/g wet wt of tissue respectively. But under long term exposure to Dichlorvos, a decreasing trend in the liver protein content is recorded. The fish when exposed to 21

4 ppm of Dichlorvos, a gradual reduction in the protein content over to control has been in liver for 7, 14 and 21 days. Figure 5 gives the data in total protein content of kidney in treated fish at sub-lethal concentrations of ppm, ppm and 0.015ppm of Dichlorvos after 7 th, 14 th and 21 st day's exposure period. Similarly, the fishes when exposed to ppm of Dichlorvos, a maximum reduction in percentage on protein content over to control is in kidney for 7, 14 and 21 days. Figure 6 shows the data in total protein content of muscle in treated fish at sub-lethal concentrations of ppm, ppm and 0.015ppm of Dichlorvos after 7 th, 14 th and 21 st day's exposure period. The data in albumin content of liver in treated fish at sublethal concentrations of ppm, ppm and 0.015ppm of Dichlorvos after 7 th, 14 th and 21 st day's exposure period. In the control fish, the mean value for albumin content observed for 7, 14 and 21 treated periods are 0.346, and mg/g wet wt of tissue respectively (Figure 7). But under long term exposure to Dichlorvos, a decreasing trend in the liver albumin content is recorded. The fish when exposed to ppm of Dichlorvos, a gradual reduction in the albumin content over the control has been observed. The data depicted in Figure 8 represent that the albumin content of kidney in treated fish and figure 9 gives the data in albumin content of muscle in treated fish at sub lethal concentrations of ppm, ppm and 0.015ppm of Dichlorvos toxicity stress after 7 th, 14 th and 21 st day's exposure period. DISCUSSION Glucose is an important metabolite transported by blood to different parts of the body. Further, blood is the first tissue being affected by a foreign compound and hence offers a sensitive and reliable indicator, which could be effectively used to assess the magnitude of biochemical stress. Hyperglycemia is a typical response caused by the exposure of fish to several pesticides [11&12]. The increase in serum glucose in Clarias batrachus exposed to carbaryl and phorate observed by [13], has established that the carbohydrate metabolism is adversely affected in tissues by the treated pesticides. The blood glucose level of the fish O. mossambicus subjected to different sub lethal concentrations of Dichlorvos for 7, 14 and 21days, has increased significantly at all periods. An increase in the rate of glycogenolysis has caused an increase in the blood sugar level and the results observed are in agreement with the results obtained in Barbus conchonius exposed to endosulfan for 4 weeks [14] in Clarias batrachus, Saccobranchus fossilis and Mystus vittatus exposed to sub lethal concentrations of thiotox and dichlorvos for 30 days [15], in Clarius batrachus exposed to malathion [16]. [17] have confirmed the observations that the higher blood glucose level in the treated fish might be due to impairment in the carbohydrate metabolism and due to the increase in the rate of glycogenolysis. [18] and [19] reported that hyperglycemic condition in the pesticide treated fish, blood plasma might be due to non- specific response to pesticide induced stress. The tissue-specific depletion of glucose is observed in the present study and it may be due to its rapid utilization to meet the energy demands for more muscular activities under toxic manifestation. A decrease in the liver glucose content has been reported for Heteropneustes fossilis exposed to formothion by [20] who suggested that the decrease in glucose content in the liver might be due to its transport to the muscle via blood to meet the energy requisites for muscular activity. In the present study also, the reduction in carbohydrate content in all the tissue may be due to the rapid depletion of stored glycogen to provide energy for O. mossambicus under stress. The induction of hypoglycemia in the tissues and hyperglycemia in blood in the present study may be due to the hormonal imbalance as a result of toxicity effect, which probably blocks the absorption of glucose into the tissue with consequential rise in the blood glucose levels. The glycogen content in liver, kidney and muscle is considered as one of the important sensitive biochemical indicators that reflects the various functional changes in the systems of an organism [21]. In the present study, the glycogen content of liver, kidney and muscle tissue of treated fish, Cyprinus carpio var. communis fingerling shows a decrease at all sub-lethal doses of Dichlorvos for 7, 14 and 21 days exposure period. A stressful situation in an animal elicits neuroendocrine responses, which in turn induces disturbances in carbohydrate metabolism [22]. [23] have observed liver glycogen depletion in C. carpio var. communis and indicated that liver, a primary vital organ for carbohydrate metabolism was badly affected by the effect of sub lethal concentration of Cypermethrin in the affected fish might be the cause for the depletion of glycogen reserve in the liver. The results of the present study clearly reveal a dose dependent decrease in the glycogen content of the liver, kidney and muscle in all the treated concentrations for all the treated periods. [24] has reported that the decreased liver glycogen content in Cirrhinus mrigala exposed to lead acetate was solely due to the increased cellular activities to meet the excess energy demand under toxic stress through increased glycogenolysis and inhibited glycolytic pathway. This finding has also been supported by [25]. In the present investigation, the higher protein content in liver of control fish may be due to the greater concentration of enzymes in the liver, since liver is the site for metabolic activities. Liver is also the important detoxifying organ, capable of doing biotransformation of foreign chemicals into nontoxic materials and also acts as a sensitive index of toxicants [26]. It possesses all the machinery for detoxification of many toxicants through enzymatic reactions and constantly renews its own intrinsic proteins, which have a very high turnover rate [27]. The liver is also the site for biosynthesis of most of the plasma proteins of blood [28]. Liver, being the first target organ to face any foreign molecule that is carried through hepato-portal circulation is susceptible to more damages due to toxicity of pollutants [29]. In the present study, when O. mossambicus is treated with sub lethal doses of Dichlorvos for all the exposure periods, it shows a significant decrease in the liver, kidney and muscle protein content and it is suggested that depletion of tissue total proteins after 7 days exposure period may be 22

5 due to increased proteolysis thereby contributing to the availability of free amino acids that may be fed to the tricarboxylic acid (TCA) cycle and further possible utilization of its products for metabolic process. Several workers have observed the decrease in protein content in liver when organisms were subjected to pesticide treatments. A progressive decrease in the protein content of the liver was observed in the rainbow trout, Salmo gairdneri exposed to endrin [30] and in Tilapia mossambica exposed to sub lethal dose of fenvalerate for 15 [31&32]. Several investigators have already reported the distinct variations in total protein content of animals affected with different concentrations of toxicants with different exposure periods [18,33&34]. [35] observed that in the liver of fish, Channa orientalis there was a prominent protein decrease exposed to lethal and sub-lethal concentrations of endosulfan and concluded that proteins were utilized in gluconeogenesis for the production of energy to eradicate the pesticide stress. A decrease in total protein content in kidney tissues in Channa punctatus fingerlings due to the impact detergent chemicals [36]. [37] confirmed the above view when Tilapia mossambica was exposed to heptachlor. In the present study, the reduction in muscle protein content in O. mossambicus fingerlings when subjected to the different sub lethal dose of dichlorvos is observed due to the toxic impact of the pesticide. [38] also observed such condition in fish Ctenopharyngodon idellus. [39]reported a perceptible decrease in the total protein of muscle in infected Heteropneustes fossilis resulting in a gross physiological disturbance and decline of growth. The significant decrease in albumin content is observed in all the treated concentrations for all the treatment periods. [40] observed the decrease in albumin content in blood plasma of Channa orientalis when exposed to slaughter house wastes and discussed that this could be definitely related to the pollution stress due to slaughter house wastes, producing intoxication and poisoning in the experimental fish[41, 42, 43, 44 & 45]. These conditions could generally increase the demand for leucocytes at affected sites to initiate mobilization of the cells to cause leucocytosis. Thus the decrease in albumin content particularly in liver may be correlated with the intoxication and poisoning due to dichlorvos pesticide in the experimental fish fingerling, Oreochromis Conclusion: Toxicological and environmental problems resulting from the widespread use of pesticides in agriculture have raised concerns, particularly with respect to the potential toxic effects in humans and animals. The exposure of O. mossambicus to the Dichlorvos was associated with alterations in haematological and biochemical indices as well, resulting in stress to the organism. These organophosphates are therefore classified as belonging to substances strongly toxic for fish. Long-term exposure to Dichlorvos in environmental concentrations can affect the functional aspects of fish. The results observed in the present investigation suggest that the parameters studied in the present study could be used as potential biomarkers for monitoring residual pesticides present in aquatic environments and provide useful parameters for evaluating physiological effects in fish, but the application of these findings will need more detailed laboratory study before they can be established as special biomarkers for monitoring the aquatic environment. Other classical morphologic indices (e.g. condition factor and hepatosomatic index) in fish could provide useful information for evaluating environmental stress. 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