In this study, an attempt was made to determine the acute toxicity and medial lethal concentrations of cypermethrin (25%EC) for Sharp tooth African catfish Clarias Gariepinus by Finney’s static bioassays probit analysis method at 24, 48, 72 and 96 hours and LC50 values were found to be 0.367 ± 0.85 µl Lit-1, 0.333 ± 0.93µl Lit-1, 0.317 ± 0.99 µl Lit-1 and 0.273 ± 0.35 µl Lit-1 respectively. Average length and weight of fishes was 19 ± 1.5 cm and 170 ± 18.5 gms. respectively. Different concentrations of Cypermethrin to which the fishes were exposed were 0.2 µl Lit-1, 0.225 µl Lit-1, 0.25 µl Lit-1, 0.3 µl Lit-1, 0.325 µl Lit-1, 0.35 µl Lit-1 and 0.4 µl Lit-1. Increase in time of exposure of fish to cypermethrin showed the decrease in LC50 value. Change in the behaviour of the fish was noted after exposure to cypermethrin. Fishes become restless, showed more opercular movements, loss of equilibrium, gulping the air by mouth, discolouration of skin. Thick mucous covering was noted all over the skin and gills. Fishes become hyperactive and showed the respiratory distress. Kewords: acute toxicity, Cypermethrin, LC50, Clarias gariepinus,
Cypermethrin is widely used synthetic pyrethroid against wide range of agricultural insect pests like cotton, wheat, paddy, cabbage, brinjal, sugarcane, sunflower and some ectoparasites like fish lice, Argulus. It is also used to control the many horticultural, animal husbandry insect pests and domestic pests (Jahanbakhshi et al., 2012 ; Rose et al., 2015). It is a 4th generation halogenated type II pyrethroid (Kaviraj and Gupta, 2014). Evaluation of acute toxicity is an important tool to observe the effect of pesticide on the fishes and other aquatic organisms. Inappropriate use of pesticides on agricultural crops has led to the serious problem. When these pesticides are washed out by rainwater, through surface water runoff it contaminates the water bodies. These uncontrolled and unregulated chemical discharges cause a serious threat to aquatic organisms and ecosystem (Kamble and Muley, 2000). Increased use of cypermethrin resulted in the increased concentration of cypermethrin discharge into suface run off water which causes deleterious effect on non target organism due to indiscriminate killing (Santhakumar and Balaji, 2000; David et al., 2004; Mishra et al., 2010; Singh et al., 2010; Neelima et al., 2016). Toxicity of the pesticide differs with respect to concentration, duration of exposure, species and size of fish (Dutta et al., 1995; Khare, 2015; Mishra and Verma, 2016). Cypermethrin is lipophilic in nature and thus it is highly toxic to fishes. Cypermethrin from the water media get absorbed by gills and then enters within the body of the fish by blood vascular system (Polat. et al., 2002). The Clarias gariepinus, is omnivorous and bottom feeder fish which is commonly reared for food purpose therefore it a economically important fish. Thus in the present study an attempt has been made to assess acute toxicity of cypermethrin on Clarias gariepinus.
II. Materials and Methods
Collection of fishes For experiment, Clarias gariepinus (average length 19 ± 1.5 cm, average weight 170 ± 18.5 g) were procured from the local fisherman of Sakkardara Market, Nagpur. Fishes were brought to the laboratory in well oxygenated polythene bags without injury. Maintenance of fishes Fishes were acclimatized to the laboratory conditions in aquarium for about 8 days. No mortality was observed during the acclimatization. In each aquarium 10 fishes were kept in 30 litres of unchlorinated well water. During acclimatization fishes were fed with freeze-dried prawns in the morning and at evening, twice a day. Feeding was stopped 24 hrs. prior to the commencement of the experimental setup. Water was exchanged within every 24 hours. Experimental setup and Determination of lethal concentration (LC50) For determining the acute toxicity of cypermethrin to Clarias gariepinus Finney’s probit bioassay test was performed (Finney,1971). Stock solution of Cypermethrin was prepared by dissolving 10 µl of Cypermethrin (25% EC) in one litre of water. From the stock solution different concentrations of Cypermethrin, 0.2 μl Lit-, 0.225 μl Lit-, 0.25 μl Lit-, 0.3 μl Lit-, 0.325 μl Lit-, 0.35 μl Lit- and 0.4 µl Lit-1 were prepared. For the acute toxicity test, ten fishes were kept in a each 45 litre glass aquaria filled with 30 litres of unchlorinated well water with different concentrations of Cypermethrin (25% EC). Fishes were randomly selected and kept in the aquaria having different concentrations of Cypermethrin (25% EC).Three replicas were performed for each concentration. Along with experimental fishes, the control fishes were also kept in the aquarium for 96 hours. The observations were made continuously up to 96 hours. Dead fishes were promptly removed from the aquaria to avoid the deterioration of the water. Mortality of fishes in each concentration was noted down for 24, 48, 72 and 96 hours. Statistical Analysis: Data was analysed to calculate the percent mortality, probit mortality, log concentration, LC50, slope, regression equation, 95% conﬁdence limits, goodness of fit by using the Statistical Package for Social Sciences (SPSS 10.0).
Behavioral Changes in Clarias gariepinus after exposure to Cypermethrin (25% EC): Several changes were observed in the behaviour of Clarias gariepinus when exposed to the sublethal doses of the Cypermethrin (25%EC) for 96 hours. When the fishes were exposed to the sublethal dose of the Cypermethrin, these become hyperactive and restless. Loss of buoyancy and equilibrium was noted. Fishes showed the more opercular movements and tried to gulp the air by mouth, by facing the mouth in upward direction in vertical position. Discolouration of skin was observed. Thick mucous covering was noted all over the skin and gills. Fishes become hyperactive and showed the respiratory distress. Sr. No. Conc. of Cypermetrin (25% EC)
Median lethal concentration (LC50): Finney’s probit bioassay median lethal concentration (LC50) test was performed to determine the LC50 values to Clarias gariepinus at different exposure periods to synthetic pyrethoid Cypermethrin (25%EC). The mortality, percent mortality and probit mortality of fishes for 24, 48,72 and 96 hours of Cypermethrin (25%EC) exposure at 0.2 μl Lit-, 0.225 μl Lit-, 0.25 μl Lit-, 0.3 μl Lit-, 0.325 μl Lit-, 0.35 μl Lit- and 0.4 µl Lit-1 concentrations is presented in the table 1. Table 1: Mortality, Percent mortality and probit mortality of Clarias gariepinus at different concentrations of Cypermethrin (25% EC) upto 96 hours of exposure period. (Note- M-Mortality, %M- Percent mortality, PM- Probit mortality). The LC50 value ± SE of Estimate, slope, y-intercept, regression equation, R2, 95% confidence limits, i.e. upper confidence limit (UCL) and lower confidence limit (LCL) for LC50 value, ratio of confidence limits (R = UCL/LCL) was calculated for 24, 48.72 and 96 hours of exposure is shown in table 2. The regression equations for 24, 48.72 and 96 hours of exposure were found to be Y= 21.59X +14.35, Y = 21.97X + 15.49, Y = 19.10X +14.49 and Y = 11.37 X + 11.40 respectively. Medial lethal concentrations ± standard error calculated by probit analysis for 24, 48.72 and 96 hours were found to be 0.367 ± 0.85 µl lit -1, 0.333 ± 0.93 µl lit -1, 0.317 ± 0.99 µl lit -1 and 0.273 ± 0.35 µl lit -1 respectively. Present investigation revealed that LC50 value get decreased as the exposure time increased. The ratio of lower and upper confidence limits for 24, 48.72 and 96 hours were found to be 1.44, 1.52, 1.87 and 1.26 respectively, which are significant and represents the good performance of the acute toxicity test. Histograph showing percent mortality at 24, 48.72 and 96 hours against different concentrations of cypermethrin (25% EC) is presented in figure 1. The Linear regression equations showing probit mortality of Clarias gariepinus against log concentration for 24, 48.72 and 96 hours are presented in figures 2, 3, 4 and 5 respectively. Chi Square Probit Pearson goodness of fit test was performed for 96 hours of exposure to observe the significance and is shown in table 3. Observed Chi-square for the bioassay test is 0.29 which is highly significant (0.999).\n\n \nTable 2: Showing the LC50 value ± SE of Estimate, slope, y-intercept, regression equation, R2, 95% confidence limits, i.e. upper confidence limit (UCL) and lower confidence limit (LCL) for LC50 value upto 96 hours of exposure period.
For evaluation of Acute toxicity bioassay test upto 96 hours is commonly performed to evaluate the effect of pesticide on the organisms. Acute toxicity test is performed to observe the medial lethal concentration (LC50) and deleterious effects of toxicant on the behaviour and histology of the vital organs of the animal (Muniyan and Veeraragahavan, 1999). Pyrethroids act on GABA receptors in the synapse and cause the neurotoxicity in organisms (ICPS, 1990). According to Reddy and Yellamm (1991) and Veeraiah and Prasad (1998) pyrethroids causes toxicity to the many economically significant and beneficial organisms like fishes, crustaceans and honey bees. Cypermethrin does not cause the problem of biomagnification in ecosystem due to its unstable nature. In aquatic habitat, cypermethrin have higher absorption rate through respiratory organ, that is gills due to its lipophilic nature. In the present investigation 24, 48, 72 and 96 hours LC50 value for Clarias gariepinus were found to be 0.367 ± 0.85 µl lit -1, 0.333 ± 0.93 µl lit -1, 0.317 ± 0.99 µl lit -1 and 0.273 ± 0.35 µl lit -1 respectively. Saha and Kaviraj (2003) has calculated the LC50 values of organophosphate pesticide Cypermethrin (Ustad) 0.96, 0.79, 0.67,0.67 µg/L for 24, 48, 72 and 96 hours respectively to the freshwater cat fish Heterpneustes fossilis. Anita Susan et al. (2010) had studied the acute toxicity (LC50 ) of fenvalerate to the Indian major carps L. rohita, C. catla and C. mrigala and reported the concentrations as 0.014, 0.016 and 0.006 mg/L for 24, 48 and 96 hours respectively. Kumar and Dhakad (2017) has studied the LC50 value of cypermethrin on catfish Clarias batrachus 1µg/lit for 24 hours. Padmanabha et al. (2015) had determined the acute toxicity of chlorpyrifos to the freshwater fish, Oreochromis mossambicus and noted the 96 hour LC50 value as 0.022 ppm. Adewumi et al. (2018) has studied the toxicity of Chlorpyrifos and DDforce on C. gariepinus and noted the 96h LC50 value as 0.30mg/l for Chlorpyrifos and 0.18mg/l for DDforce. Suja and Williams (2018) had noted the 4.521 ppm, 96 hours LC50 of organophosphate chlorpyrifos for C. striatus. According to Soderlund et al. (2002) pyrethroids mainly act on nervous system, but in mammals, inhalation of Cypermethrin above or near lethal dose affects the respiratory tract leading to massive haemorrhages and oedema of the lungs.
When the fishes were exposed to sublethal dose of Cypermethrin, change in the behavior of the fish was noted. Fish breaths rapidly and try to gulp the air by mouth (Anita Susan et al., 2010). According to Schmidt et al. (2005) gulping the air may be for avoiding the contact with the toxic water media. Fish becomes hyperactive due to physiological stress. Loss of balance and hanging vertically might be due to destruction of neurotransmitters, opening of sodium ion channel for longer duration and inhibition of acetylcholine secretion (Narahashi, 1996; Tilak et al., 2001; Anita Susan et al., 2010; Yaji et al., 2011). Increased opercular movement may occur for taking the atmospheric oxygen with the help of accessory respiratory organs (Santhakumar et al., 2000). To avoid and minimize the contact with the toxicant, there is increased amount of mucous all over the body and the gills (Tilak et al., 2001; Rao et al., 2003). Colour of the body of fish fade away on exposure to the cypermethrin (Wasu et al., 2009: Dube and Hosetti, 2010).