Research Journal of Animal, Veterinary and Fishery Sciences ___________________________ ISSN 2320 – 6535 Vol. 2(8), 11-16, August (2014) Res. J. Animal, Veterinary and Fishery Sci. International Science Congress Association 11 Acute toxicity of Calcium chloride on different stages (Egg, Spawn, Fry and Fingerling) of rohu (Labeo rohita Hamilton)Mallick A.1,2*, Mohapatra B.C. and Sarangi N.2 Department of Environmental Science, University of Kalyani, Kalyani, West Bengal -741235, INDIA Central Institute of Freshwater Aquaculture, (Indian Council of Agricultural Research), Bhubaneswar- 751 002, Odisha, INDIA Available online at: www.isca.in, www.isca.me Received 21st July 2014, revised 6th August 2014, accepted 21st August 2014 AbstractThe acute toxicity of calcium chloride (CaCl) was studied on the early stages of Indian major carp rohu, Labeo rohita (egg, spawn, fry and fingerling) through bioassay tests. Percentage of dead or damaged egg, spawn, fry and fingerling at 6, 12, 18, 24, 36, 48, 60, 72 and 96 hours were recorded for the calculation of LC50. The increase in CaCl concentration in water increased the toxicity and reduced the duration to damage 50% of the eggs, spawn, fry and fingerling. Low hatchability, delayed hatching, poor survival observed in the test solutions �1000ppm concentration. The deformed and dead larvae were observed through microscopic examinations. The behavioural studies such as swimming, surfacing, activeness and survival were also observed in spawn, fry and fingerling. The LC50 values of CaCl for egg, spawn, fry and fingerling were 3743.79-28.93ppm, 7249.78-1260.73ppm, 9872.44-6384.12ppm and 12276.52-9987.67ppm in 6 - 96 hours respectively. Keywords: Acute toxicity, calcium chloride, Rohu, Bioassay, LC50IntroductionAquatic organisms differ from terrestrial organisms in their metabolic requirements for calcium that typically can be met by absorbing this mineral from the water in which they live. Both shrimp and fish can absorb some minerals from the water via drinking (primarily marine organisms), and by direct absorption via gills, fins and skin. The gills are the most important sites of calcium regulation in freshwater and marine fish. Freshwater of moderate hardness (50 mg/l as CaCO) as well as brackish water and sea water, contain much higher levels of dissolved calcium; therefore, dietary supplementation of calcium for various fish and crustacean species is generally not necessary. The calcium helps to protect the freshwater fish against environmental toxicants, osmotic and ionic losses. The excessive levels of dietary calcium should be avoided because it can negatively affect the utilization of other minerals. The uptake of ions from the water may be driven by the perivitelline potential,ion exchange, transporting enzyme or by any combination of these1-3Calcium has a marked effect on sodium fluxes in both marine and freshwater organisms4-6. In few fish species, which have so far been examined, the effects of external calcium appear to be primarily on sodium efflux, which is reduced in the presence of calcium4,6. Fish embryos maintain the constancy in the ion concentrations and osmolality through their body fluids and the skin chloride is responsible for the active transport of ions7-9. The physicochemical parameters of the water body affects the fish production and diversity10,11. The acute toxicity of mercury to Clarias gariepinus and tannery chromium to Labeo rohita were studied by Guedenon et al and Praveena et al respectively12,13. Eknath14 studied the toxicity of detergents on Mystus montanusfish and the behavioural changes. In the present investigation studied the toxicity of calcium chloride concentrations on different stages of rohu and the LC50 dose over a period of 6-96 hours. Material and Methods Test organism: The rohu (Labeo rohita) was selected as test organism. The egg, spawn, fry and fingerling of appropriate quality of rohu were selected for the experiment. Spawn, fry and fingerlings were collected from the hatchery of Central Institute of Freshwater Aquaculture (CIFA), Kausalyaganga, Bhubaneswar. The organisms were brought to the laboratory for in vitro study. The spawn were acclimatized in the glass aquaria for one day, but in case of fry and fingerlings they were acclimatized in plastic pool for a period of two days before bioassay. Test container: Glass jar tanks of 20 liters capacity were used as test containers. Before using these were cleaned with laboratory detergents then with 100% acetone and tap water. After each test, the containers were washed appropriately with acid to remove metals, bases and organic compounds. Each of the test containers were provided with facilities of continuous aeration and covered with velon screen netting to prevent the test organisms from jumping out. Test concentration: Selections of test concentrations were made following the APHA method15. At least five different concentrations were taken in each experiment. While designing, minimum of five exposure (treatment) concentrations of a test substance and one control was taken for bioassay test. In the present study the test organisms were exposed to a wide range of Research Journal of Animal, Veterinary and Fishery Sciences ________________________________________ ISSN 2320 – 6535 Vol. 2(8), 11-16, August (2014) Res. J. Animal, Veterinary and Fishery Sci. International Science Congress Association 12 calcium concentrations in different stages of rohu. The concentrations were as far as possible selected in logarithmic or geometric scale. For egg, spawn, fry and fingerling 0.5- 10000, 20-20000, 0.75-11250, and 200-11250ppm of CaCl were selected respectively. Bioassay: The bioassay was conducted for different stages of rohu (egg, spawn, fry and fingerling). Four experiments were conducted for bioassay test. These experiments were EGG bioassay (experiment-1), spawn bioassay (experiment-2), Fry bioassay (experiment-3) and Fingerling bioassay (Experiment-4). Different concentrations of CaCl were used as the test solutions. Data analysis: The data obtained from the experiments were processed by Probit analysis for determination of LC50 values using SPSS statistical software16-18. The lethal concentrations were plotted against time in hours to get "Toxicity curve"19. Results and Discussion The embryonic development and LC50 values were determined by applying CaCl as a toxic agent to the developmental stages of rohu (figure 1-4). The toxic effect of CaCl was observed in different concentrations and exposure time. The percentages of damaged or dead eggs were varied in different concentrations. In higher concentration (�1000 ppm) the percentage of damaged eggs were increased, eggs became smaller, whitish just before death and coelomic content turned opaque or white. In the hatchling stage the tail bended towards the back (figure 5a-d). Early life stages of the teleosts are known to be very sensitive stages. The sensitivity depends on the developmental stages and the time of exposure, physico-chemical characteristic of water, toxicant concentration and fish species20-22. In higher salt concentrations, high percentage of embryos exhibited deformation in the spinal cord and larval tail bended towards backside. The present findings corroborated with the observation of Hodson et al23, in rainbow trout exposed to lead (Pb) at different pH. The exchange of cations like Ca2+ and Mg2+ is crucial for normal embryonic development24. A decrease in heart rate and pigmentation has also been reported for early life stages of freshwater rainbow trout exposed to a low pH or to a low ambient calcium concentration25Figure-1 Toxicity curve of rohu eggs exposed to different lethal concentration of CaCl solution Figure-2 Toxicity curve of rohu spawn exposed to different lethal concentrations of CaCl solution 0.51.52.500.511.522.533.54Log time (hrs)Log CaCl2 Conc. (ppm)Egg 0.51.52.5012345Log time (hrs)Log CaCl2 Conc. (ppm)Spawn Research Journal of Animal, Veterinary and Fishery Sciences ________________________________________ ISSN 2320 – 6535 Vol. 2(8), 11-16, August (2014) Res. J. Animal, Veterinary and Fishery Sci. International Science Congress Association 13 Figure-3 Toxicity curve of rohu fry exposed to different lethal concentrations of CaCl solution Figure-4 Toxicity curve of rohu fingerlings exposed to different lethal concentrations of CaCl solution The results of toxicity studies of CaCl2 expressed in terms of LC50 values obtained from probit analysis shown in figure 1-4. The lethal concentration (LC50) of CaCl decreased gradually with the increase in exposure time from 6 to 96 hours (table 1). The LC50 values of CaCl in case of egg were 3743.79, 2326.36, 1075.07, 255.95, 64.47, 58.89, 45.34, 42.72 and 28.93ppm after 6, 12, 18, 24, 36, 48, 60, 72 and 96hrs respectively. In case of spawn the LC50 values of CaCl were 7249.78, 5507.77, 3738.12, 3192.77, 2343.94 and 1260.73ppm after 6, 12, 24, 48, 72 and 96 hrs respectively. The LC50 values of CaCl in case of fry were 9872.44, 9770.46, 8858.51, 8605.69 and 6384.12ppm after 12, 24, 48, 72 and 96hrs respectively and in case of fingerling were 12276.52, 11407.75, 10481.15, 10229.91, 10110.34 and 9987.67ppm after 6, 12, 24, 48, 72 and 96hrs respectively. Metelev et al25 observed that10000ppm CaCl is toxic for white salmon, carp and perch after 16 to 29 hrs. Symptoms of poisoning in carp begin to appear at a concentration of 7000ppm CaCl. 13900ppm CaCl2 is toxic after 10 days for juvenile brown trout. At 15000ppm CaCl2 proved toxic for all fish in period ranging from one hour to several days26. According to Mohapatra27 CaCl2 is toxic to Catla catla at 7500, 6300, 4950, 4100 and 3950ppm after 12, 24, 48, 72 and 96hrs respectively and 24400, 23000, 21800, 21700 and 21400ppm after 12, 24, 48, 72 and 96hrs respectively in case of S. mossambicus. Eel can tolerate 27000ppm CaCl 2H25. There are close inverse relationships between calcium concentration of water and chloride cell density in teleosts28. 0.51.52.53.753.83.853.93.9544.05Log time (ppm)Log CaCl2 Conc. (ppm)Fry 0.51.52.53.9844.024.044.064.084.1Log time (hrs)Log CaCl2 Conc.(ppm)Fingerling Research Journal of Animal, Veterinary and Fishery Sciences ________________________________________ ISSN 2320 – 6535 Vol. 2(8), 11-16, August (2014) Res. J. Animal, Veterinary and Fishery Sci. International Science Congress Association 14 Figure-5 (a) Normal egg; (b) Deformed egg; (c) Normal spawn with fully developed yolk sac and other internal organs; (d) Deformed spawn with irregular yolk sac and bended tailTable-1 LC50 values of calcium chloride (CaCl) for rohu egg, spawn, fry and fingerling Exposure periods (hr) LC 50 values of CaCl 2 (ppm) Egg Spawn Fry Fingerling 6 3743.79 7249.78 - 12276.52 12 2326.36 5507.77 9872.44 11407.75 18 1075.07 - - - 24 255.95 3738.12 9770.46 10481.15 36 64.47 - - - 48 58.89 3192.77 8858.51 10229.91 60 45.34 - - - 72 42.72 2343.94 8605.69 10110.34 96 28.93 1260.73 6384.12 9987.67 ConclusionEarly larval stages are the most crucial and vulnerable one in the life cycle of fish. Hence, the rate of survival in these stages depends on the maintenance of water quality parameters such as alkalinity, pH and hardness in the hatchery system. Hardness (calcium and magnesium) is very important in hatchery system. The optimum water quality should be maintained by the farmer before going to operate a hatchery system. It is essential to know the calcium content in pond water as it is one of the important water quality parameter for fish culture. From the findings of this study it was concluded that � 1000 ppm of calcium in the aquatic environment can affect the larval stage (hatchling stage), but the LC50 values are varied in different stages. Acknowledgement The authors acknowledge the DBT (GoI) for fund supporting and Research Journal of Animal, Veterinary and Fishery Sciences ________________________________________ ISSN 2320 – 6535 Vol. 2(8), 11-16, August (2014) Res. J. Animal, Veterinary and Fishery Sci. International Science Congress Association 15 Central Institute of Freshwater Aquaculture, Bhubaneswar, Odisha, India for providing laboratory and infrastructural facilities. 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