Int. J. Aquat. Biol. (2014) 2(3): 155-163 E-ISSN: 2322-5270; P-ISSN: 2383-0956 Journal homepage: www.ij-aquaticbiology.com © 2014 Iranian Society of Ichthyology Original Article Acute toxicity of alkali and alkaline earth metals on Rohu, Labeo rohita (Hamilton) egg and hatchlings Anusaya Mallick*1, 2, Bikash Chandra Mohapatra2, Niranjan Sarangi2 1Department of Environmental Science, University of Kalyani, Kalyani, West Bengal -741235, India. 2Central Institute of Freshwater Aquaculture, (Indian Council of Agricultural Research), Bhubaneswar- 751 002, Orissa, India. Article history: Received 14 November 2013 Accepted 18 April 2014 Available online 2 5 June 2014 Keywords: Alkali and alkaline earth metals Bioassay LC50 Rohu Abstract: The acute toxicity of salts of alkali and alkaline earth metals, such as sodium (Na), potassium (K), calcium (Ca) and magnesium (Mg) were studied on the egg and larval stages of Indian major carp Labeo rohita (Hamilton). The acute toxicity experiments were conducted followed by the range finding bioassay tests. The experiments were conducted in triplicates. The cumulative percentage of dead or damaged eggs at the end of 6, 12, 18, 24, 36, 48, 60, 72 and 96 hours was recorded for the calculation of LC50. The increase in salt concentrations in water increased their toxicity and reduced the duration to damage 50% of the eggs. The eggs became smaller than their normal size and whitish before being damaged in the test solutions. Most of the exposed eggs and hatchlings tended to lay on the floor of the tank. The toxicity of the metals was in the order of K>Na>Mg>Ca. The 96 hours LC50 values were 3.25, 2.73, 28.9 and 20.52 ppm for sodium, potassium, calcium and magnesium, respectively. Introduction Indian major carps (IMC) are the most important groups of fishes cultured in Indian subcontinent. Rohu (Labeo rohita) belongs to the family cyprinidae and is found commonly in rivers and freshwater lakes, mainly in South-East Asia (Gupta et al., 1997). In order to bridge the gap between the ever- increasing demand and supply of the seed of the species, induced breeding plays a major role. Alkali and alkaline earth metals affect fish breeding, water hardening of eggs, growth and survival of hatchlings (Mallick et al., 2010). The embryological stages of an organism have important considerations, when examining the effect of heavy metals and concentration of these in the body tissues. It can vary with the age or size of the organism (Bennett and Dooley, 1982; Newman and Mitz, 1988). The health of fish may be affected, either directly through uptake from the water or indirectly through their diet of vegetation, * Corresponding author: Anusaya Mallick E-mail address: anusaya.cifa@gmail.com invertebrates or smaller fish (Kime et al., 1996). Metals released into aquatic ecosystems are responsible for several fish physiological irregularities (Sehgal and Saxena, 1986). These can also disturb the ion regulatory mechanism in aquatic organisms (Hansen et al., 1996). In an aquatic environment, metal toxicity can be influenced by various abiotic factors such as oxygen, calcium/water hardness (Skidmore, 1964; Cairns and Mount, 1990; Ghillebaert et al., 1995), pH and temperature (Cairns and Mount, 1990; Kotze et al., 1999). According to Rose et al. (1993) abiotic factors are defined as variables that exert a direct effect on individuals in the population. The sodium level in water bodies is quite variable. Wide ranges of seasonal fluctuations of sodium in freshwater bodies have been reported by Khan and Siddqui (1974), Goel et al. (1986) and Khatavkar et al. (1990). High concentrations of sodium chloride have strong local effects in fish (Metelev et al., 156 Mallick et al./ acute toxicity of alkali and alkaline earth metals on rohu International Journal of Aquatic Biology (2014) 2(3): 138-146 1983). Potassium is a naturally occurring element, which remains in lesser concentration than calcium, magnesium and sodium in freshwater. The potassium level in aquatic habitats is quite variable from 0.2-35.0 ppm. Mohan and Zafar (1986) demonstrated that the potassium is fast accumulated with the passage of time and doubling up in every fifteen years in the aquatic habitat. According to Metelev et al. (1983) and Mohapatra (1999) the symptoms of poisoning due to potassium compounds are analogous to those of poisoning with sodium compounds. Calcium is a very important cation for the animal body because not only takes part in the structural formation, but also in the various metabolic and physiological activities in the body. The increase in environmental calcium rises the tolerance of aquatic animals to ammonia toxicity (Tomasso et al., 1980). Calcium is a primary structural component of hard tissues such as bones, exoskeleton, scales and teeth of aquatic organisms. Freshwater fish obtain magnesium ions through active uptake from the environment or from dietary sources. Fish eggs contain a significant amount of magnesium associating with the yolk (Hayes et al., 1946). Therefore, the yolk may serve as a magnesium source for the developing embryo during the early developmental stage. Magnesium is essential for cellular respiration and neuromuscular transmission (Lall, 1989). Egg hatchability and hatching time are more sensitive indicators of toxicity than “standard” end point, like mortality and growth (Pyle et al., 2002). Except one report of Mohapatra (1999) on the toxicity studies on fingerlings of Catla catla, no work has been done on the determination of tolerance limits of alkali and alkaline earth metals on egg and larvae of Indian major carps viz., Catla catla, Labeo rohita and Cirrihinus mrigala. Therefore, the present study was planned to investigate the comparative sensitivity of rohu egg and hatchlings to alkali and alkaline earth metals through toxicity tests. Materials and methods Test containers: The glass jar tanks with 20 L capacity were used as test containers after 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 was provided with facilities of continuous aeration. Test solutions: The test solutions were prepared by dissolving the calculated amount of salts, such as sodium chloride, potassium chloride, magnesium chloride and calcium chloride as per the experiments in the dilution water of the jars. The solutions were prepared immediately prior to the initiation of the experiments. De-chlorinated tap water was used as dilution water for control tests and for making concentrations of the test substances. The dilution water was clean, uncontaminated and of constant quality. Test concentrations: Selection of test concentrations was made as per the protocol of APHA (1998). The concentrations of the metals ware expressed as parts per million (ppm). The test organisms were exposed to a range of salt concentrations in logarithmic scale, such as 0.0001, 0.001, 0.01, 0.1, 1.0, 10, 100, 1000 and 10,000 ppm. Based on the results of the range finding bioassay, the concentrations 0.5-10,000; 0.05-1000; 1-10,000 and 1-5000 ppm for the salts of sodium, potassium, magnesium and calcium, respectively were selected as the test concentrations for definite bioassay experiment. To avoid contamination, the controls were maintained little away from bioassay tanks. Bioassay of egg: The rohu eggs of appropriate quality were selected for the experiment. Eggs were received from CIFA carp hatchery at Bhubaneswar and were transferred to the experimental tanks with test media on arrival at laboratory, and the containers were calibrated to determine the approximate number of eggs for experimentation. The eggs were measured volumetrically before release into the test containers including control tanks. The percentage of dead/ damaged eggs at the end of every 6, 12, 18, 24, 48, 72 and 96 hours were recorded and tabulated. To avoid contamination, the dead eggs were removed 157 Int. J. Aquat. Biol. (2014) 2(3): 155-163 from the experimental tanks immediately. Data analysis: The data obtained from the experiments were processed by probit analysis (Finney, 1971; Reish and Oshida, 1987; Mohapatra and Rengarajan, 1995) for determination of LC50 values in computer using SPSS software. The lethal concentrations were plotted against time in hours to get "Toxicity curve"(Seegertet al., 1979). Determination of toxicity of elementary form of metals: Grams of compound containing 1.0 g element= Molecular weight of compound/Molecular weight of element i.e., 1.0 g of NaCl, KCl, CaCl2 2H2O and MgCl2 6H2O contain 0.39, 0.524, 0.273 and 0.12 g of Na+, K+, Ca++ and Mg++, respectively. Results Range finding bioassay: In range finding bioassay a wide range of concentrations i.e. 0.0001, 0.001, 0.01, 0.1, 1.0, 10, 100, 1000 and 10,000 ppm were selected for each salt solution. No mortality was observed in Table 1. Percentage death of eggs or hatchlings of rohu exposed to different concentrations of sodium chloride (NaCl) salt solution Conc. (ppm) % death of eggs and hatchlings of rohu in different exposure time (hours) 6 12 18 24 36 48 60 72 96 0.5 30 35 40 40 40 40 40 40 40 1 45 50 50 50 50 50 50 50 50 10 50 50 50 50 55 55 55 55 55 100 50 50 50 50 55 55 60 70 70 1000 60 75 80 90 100 100 100 100 100 10000 65 85 100 100 100 100 100 100 100 Control 10 15 15 20 20 25 30 35 40 Table 2. Percentage death of eggs or hatchlings of rohu exposed to different concentrations of potassium chloride (KCl) salt solution Conc. (ppm) % death of eggs and hatchlings of rohu in different exposure time (hours) 6 12 18 24 36 48 60 72 96 0.05 20 25 30 30 45 45 45 45 50 0.1 20 30 35 45 45 45 45 45 45 1 30 30 40 50 50 50 50 50 50 10 35 45 50 50 60 60 65 65 75 100 40 55 70 90 100 100 100 100 100 1000 45 60 80 100 100 100 100 100 100 Control 10 20 20 30 30 30 30 30 35 Table 3. Percentage death of eggs or hatchlings of rohu exposed to different concentrations of magnesium chloride (MgCl2) salt solution Conc. (ppm) % death of eggs and hatchlings of rohu in different exposure time (hours) 6 12 18 24 36 48 60 72 96 1 30 35 40 40 45 45 45 45 45 10 40 40 45 50 50 50 50 50 50 100 45 45 50 50 50 60 60 60 65 1000 50 50 55 70 90 100 100 100 100 10000 50 55 65 100 100 100 100 100 100 Control 20 20 20 30 30 30 40 40 40 Table 4. Percentage death of eggs or hatchlings of rohu exposed to different concentrations of calcium chloride (CaCl2) salt solution Conc. (ppm) % death of eggs and hatchlings of rohu in different exposure time (hours) 6 12 18 24 36 48 60 72 96 1 30 35 45 40 40 40 40 40 40 10 40 45 50 50 50 50 50 50 50 100 40 45 50 50 60 60 65 65 70 1000 50 60 80 90 100 100 100 100 100 5000 55 70 80 100 100 100 100 100 100 Control 20 20 20 20 20 30 40 40 40 158 Mallick et al./ acute toxicity of alkali and alkaline earth metals on rohu International Journal of Aquatic Biology (2014) 2(3): 138-146 0.0001-0.01 ppm for all the tested salts i.e. sodium chloride, potassium chloride, calcium chloride and magnesium chloride, but 75% mortality occurred at 10,000 ppm in case of salts of sodium and magnesium, 1000 ppm for potassium and 5000 ppm for calcium in 96 hour of exposure. Hence, the concentrations of 0.5, 1, 10, 100, 1000 and 10,000; 0.05, 0.1, 1, 10, 100 and 1000; 1, 10, 100, 1000 and 10,000; and 1, 10, 100, 1000 and 5000 ppm were selected for the salts of sodium, potassium, magnesium and calcium, respectively for conducting definitive bioassay. Bioassay results: The percentage of damaged or dead eggs was not similar in all experimental concentrations. In higher concentrations, the percentage of damaged eggs was high. Egg became smaller, whitish just before death and coelomic content turned opaque or white. Most of the exposed eggs and hatchlings after death were settled on the aquarium floor. The cumulative percentage of dead or damaged eggs; or hatchlings after hatching out from eggs at 6, 12, 18, 24, 36, 48, 60, 72 and 96 hours of exposure was recorded for the calculation of LC50 (Tables 1-4). The results of toxicity studies expressed in terms of LC50 values obtained from probit analysis for different salts are given in Table 5. The median lethal concentration (LC50) decreased gradually with the increase in exposure time from 6 to 96 hours. The rank order of toxicity of metal salts for rohu egg and hatchlings was found to be potassium chloride > sodium chloride > magnesium chloride > calcium chloride. The toxicity of inorganic solids such as sodium, potassium, calcium and magnesium were calculated from the results of bioassay studies with their salts and presented in table 6. Based on 96 h LC50 values, potassium was found to be more toxic to rohu larval stages and the least was the calcium. The toxicity order was K>Na>Mg>Ca (Table 6). Toxicity curve: The toxicity curves were obtained for different salts by plotting the log time against the log LC50 values (Fig. 1A-D). The toxicity curve may not pass through all the LC50 points on the graph at all times. It gives the overall picture of the progress Table 5. LC50 values of salts of alkali and alkaline earth metals on eggs and hatchlings of rohu, Labeo rohita Exposure Period (hours) LC50 (ppm)a NaCl KCl MgCl2 CaCl2 6 5561 1170 9122.5 3743.8 12 2471.1 560.6 6890.1 2326.4 18 345.7 188.8 4413.8 1075.1 24 353.1 17 387.8 256.1 36 93.2 7.2 193.4 64.5 48 21.6 6 58.8 59.1 60 13.9 6 38.8 45.4 72 4.1 4.6 36.2 42.7 96 3.3 2.7 20.5 28.9 a After 18 hours of exposure the eggs hatched to hatchlings Table 6. LC50 values of elementary forms of alkali and alkaline earth metals on eggs and hatchlings of rohu, Labeo rohita Exposure Period (hours) LC50 (ppm) Na+ K+ Mg++ Ca++ 6 2168.8 613.1 1094.7 1022.1 12 963.7 293.7 826.8 635.1 18 134.8 98.9 529.6 293.5 24 137.7 8.9 46.5 69.9 36 36.3 3.8 23.2 17.6 48 8.4 3.2 7.1 16.13 60 5.4 3.2 4.6 12.4 72 1.6 2.4 4.3 11.6 96 1.3 1.4 2.5 7.9 159 Int. J. Aquat. Biol. (2014) 2(3): 155-163 Figure 1. Toxicity curve for rohu eggs hatchlings exposed to different lethal concentrations of NaCl, KCl, MgCl2, and CaCl2 up to 96 hours. Table 7. LC50 values of elementary forms of alkali and alkaline earth metals on eggs and hatchlings of rohu, Labeo rohita Salts Species Value (ppm) Toxicity Source NaCl Roach and Tench 10,000-11,000 Not toxic within 24 hrs Metelev et al.,1983 13,000 Mortality after 1 day Carp (100-150g) 15,000 Toxic Trout (fry and fingerlings) 10,000 Non-toxic for several hrs Catla catla 15,600 6 hr LC50 Mohapatra, 1999 13,700 12 hr LC50 10,900 24 hr LC50 10,800 48 hr LC50 10,100 72 hr LC50 9,000 96 hr LC50 Sarotherodon mossambicus 31,300 24 hr LC50 30,300 48 hr LC50 29,700 72 hr LC50 27,600 96 hr LC50 Labeo rohita (Egg) 5561 6 hr LC50 Present study 2472.1 12 hr LC50 Labeo rohita (Hatchling) 345.7 18 hr LC50 353.1 24 hr LC50 93.2 36 hr LC50 21.6 48 hr LC50 13.9 60 hr LC50 4.1 72 hr LC50 3.3 96 hr LC50 KCl Perch and white salmon 10,000 Toxic after 18 hrs Metelev et al.,1983 Fish 1000 Toxic 500 Non toxic Emerald shiner, Feathead minnow, Golden shiner, White bass, Carp 10,000 No mortality up to 24 hrs Snyder et al., 1991 Catla catla 3350 12 hr LC50 Mohapatra, 1999 1830 24 hr LC50 1270 48 hr LC50 800 72 hr LC50 3400 96 hr LC50 160 Mallick et al./ acute toxicity of alkali and alkaline earth metals on rohu International Journal of Aquatic Biology (2014) 2(3): 138-146 Table 7. Continued. Sarotherodon mossambicus 3,400 12 hr LC50 1,750 24 hr LC50 920 48 hr LC50 900 72 hr LC50 810 96 hr LC50 Labeo rohita (Egg) 1170 6 hr LC50 Present study 560.6 12 hr LC50 Labeo rohita (Hatchling) 188.8 18 hr LC50 17 24 hr LC50 7.2 36 hr LC50 6 48 hr LC50 6 60 hr LC50 4.6 72 hr LC50 2.7 96 hr LC50 MgCl2 6H2O Three-spined stickle back 2,300 Can withstand up to 6 months without damage Metelev et al.,1983 Perch and White salmon 10,000 Can tolerate up to 24 hours Carp (100g) 6000 Can tolerate for 3-4 days without damage Brown trout fry 4900 Can live up to 8 days Barbel 10,000 Can withstand for 4 weeks 15,000 A percentage die after 5 days 20,000 Die after 1hrs Carp 1000-15,000 Can tolerate for 4 weeks 20,000 A percentage die after 5 days 30,000 Die after 4 hours Eel 10,000 Live for 4 weeks 15-20,000 Die after 14 days Catla catla 18,500 12 hr LC50 Mohapatra, 1999 18,300 24 hr LC50 18,200 48 hr LC50 18,000 72 hr LC50 17,900 96 hr LC50 Sarotherodon mossambicus 38,000 12 hr LC50 36,500 24 hr LC50 36,100 48 hr LC50 35,600 72 hr LC50 35,100 96 hr LC50 Labeo rohita (Egg) 9122.5 6 hr LC50 Present study 6890.1 12 hr LC50 Labeo rohita (Hatchling) 4413.8 18 hr LC50 387.8 24 hr LC50 193.4 36 hr LC50 58.8 48 hr LC50 38.8 60 hr LC50 36.2 72 hr LC50 20.5 96 hr LC50 CaCl2 6H2O White Salmon, carp and perch 10,000 Toxic after 16-29 hours Metelev et al.,1983 Juvenile brown trout 13,900 Toxic after 10 days Carp, rainbow trout and barbel 5,000 Not toxic in 4 weeks Fishes 15,000 Toxic in 1 hr to several days Eel 27,000 Can tolerate Catla catla 7,500 12 hr LC50 Mohapatra, 1999 6,350 24 hr LC50 4,950 48 hr LC50 4,100 72 hr LC50 3,950 96 hr LC50 161 Int. J. Aquat. Biol. (2014) 2(3): 155-163 of the test and also indicates when acute lethality has stopped. With longer exposure times, the curve tends to be parallel to the time axis. Discussion Acute toxicity test is necessary in water pollution control to determine whether a potential toxicant is dangerous to aquatic life and if so, to find the relationship between the toxicant concentration and its effect on aquatic animals. Bioassay is necessary to determine the concentration of a toxicant, which may be allowed in receiving waters without adverse effects on the living resources (Standing Committee of Analysts, 1981; Ward and Parrish, 1982; Reish and Oshida, 1987). Bioassay technique has been the cornerstone of programmes on environmental health and chemical safety (Ward and Parrish, 1982; Mohapatra and Saha, 2000). The median lethal concentration (LC50) of the metals sodium, potassium, calcium and magnesium on rohu eggs and hatchlings decreased gradually with the increase in exposure time from 6 to 96 h. The toxicities of the salts were seen in the order of potassium chloride > sodium chloride > magnesium chloride > calcium chloride. Potassium was found more toxic and calcium was the least. These toxicity values cannot be are compared directly with the available results of other workers, but, a comparative statement is given in Table 7. Because of eggs and larval stages, the toxicity values in the present study for rohu were found less than the values reported by Mohapatra (1999). Metelev et al. (1983) reviewed that symptoms of magnesium poisoning in fish are similar to those with sodium salts. In fish (Crusian carp) the first indication is sluggish eye movement and subsequently they turn on their sides. In the present experiment the damaged eggs and hatchlings before death remained on the floor of the tanks. Acknowledgements We are thankful to the Director, Central Institute of Freshwater Aquaculture, Bhubaneswar, Odisha, India for providing facilities for research work. Reference APHA. (1998). Standard methods for the examination of water and wastewater, 16th ed., American Public Health Association, Water Pollution Central Federation and American Water Works Association, New York, USA. Bennett R.O., Dooley J.K. (1982). Copper uptake by two sympatric species of killifish Fundulus heteroclitus (L.) and L. majalis (Walkbaum). Journal of Fish Biology, 21: 381-398. Cairns J. (Jr)., Mount D.I. (1990). Aquatic toxicology. Environmental Science and Technology, 24 (2): 154- 160. Finney D.J. (1971). Probit Analysis. Univ. Press, Cambridge, 333 p. Ghillebaert F., Chaillou C., Deschamps F., Roubaud P. (1995). Toxic effects at three pH levels of two reference molecules on common carp embryo. Ecotoxicology and Environmental Safety, 32: 19-28. Goel H., Kohli G.S., Lai H. (1986). Serum phosphohexose Sarotherodon mossambicus 24,400 12 hr LC50 23,000 24 hr LC50 21,800 48 hr LC50 21,700 72 hr LC50 21,400 96 hr LC50 Labeo rohita (Egg) 3743.8 6 hr LC50 Present study 2326.4 12 hr LC50 Labeo rohita (Hatchling) 1075.1 18 hr LC50 256.1 24 hr LC50 64.5 36 hr LC50 59.1 48 hr LC50 45.4 60 hr LC50 42.7 72 hr LC50 28.9 96 hr LC50 Table 7. Continued. 162 Mallick et al./ acute toxicity of alkali and alkaline earth metals on rohu International Journal of Aquatic Biology (2014) 2(3): 138-146 isomerase levels in patients with head and neck cancer. Journal of Laryngology and Otology, 100: 581-585. Gupta M.V., Dey M. M., Dunham R., Bimbao G. (1997). Proceedings of the collaborative research and training on genetic improvement of carp species in Asia. Bhubaneswar, India. ICLARM Work, Doc. No. 1. Hayes F.R., Darcy D.A., Sullivan C.M. (1946). Changes in the inorganic constituents of developing salmon eggs. Journal of Biological Chemistry, 163: 621-631. Hansen H.J.M., Olsen A.G., Rosenkilde P. (1996). The effect of Cu on gill and esophagus lipid metabolism in the rainbow trout (Oncorhynchus mykiss). Comparative Biochemistry and Physiology, 113 (C) 1: 23-29. Khan A.A., Siddqui A.Q. (1974). Seasonal change in the limnology of a perennial fish pond at Aligarh. Indian Journal of Fisheries, 21: 463-478 Khatavkar S.D., Kulkarni A.Y., Goel P.K. (1990). Phytoplankton flora of some freshwater bodies in South-western Maharashtra. Environment and Ecology, 8: 267-275. Kime D.E., Ebrahimi M., Nysten K., Roelants I., Rurangwa E., Moore H.D.M., Ollevier F. (1996). Use of computer assisted sperm analysis (CASA) for monitoring the effects of pollution on sperm quality of fish, application to the effects of heavy metals. Aquatic Toxicology, 36: 223-237. Kotze P.J., Dupreez H.H., Van Vuren J.H.J. (1999). Bioaccumulation of copper and zinc in Oreochromis mossambicus and Clarias gariepinus from the Olifants River, Mpumalanga, South Africa. Water SA, 25 (1): 99-110. Lall S.P. (1989). The minerals. In: Fish Nutrition (Ed.) J. H., Halver, Academic Press, London-San Diego- California, pp: 220-252. Mallick A., Mohapatra B.C., Sarangi N. (2010). Effect of alkali and alkaline earth metals on embryonic stages of rohu, Labeo rohita (Ham.) In: A. Sinha, S. Datta & B.K. Mahapatra (Eds.). Diversification of Aquaculture, Narendra Publishers, New Delhi, pp: 229-240. Metelev V.V., Kanaev A.I., Dzasokhova N.G. (1983). Water Toxicology. Amerind Publishing Co. pvt. Ltd., New Delhi, pp: 216. Mohan K.S., Zafar A.R. (1986). Water quality of two drinking water reservoirs of Hyderabad (India). Pollution Research, 5: 111-122. Mohapatra B.C., Rengarajan K. (1995). A manual on bioassays in the laboratory and their techniques. CMFRI, 64:1-75. Mohapatra B.C. (1999). Purna saline tract, Maharastra state: Assessment of ground water quality through fish bioassays. D.Sc. Thesis, Berhampur Univ., Berhampur, Orissa. 101 p. Mohapatra B.C., Saha C. (2000). Aquatic pollution and management. Central Institute of Freshwater Aquaculture, Bhubaneswar, pp: 1-363. Newman M.C., Mitz S.V. (1988). Size dependence of zinc elimination and uptake from water by mosquito fish, Gambusia affinis (Baird and Girard). Aquatic Toxicology, 12: 17-32. Pyle G.B., Wanson S.M., Lehmkuhl D. M. (2002). Toxicity of uranium mine receiving waters to early life stage fathead minnows (Pimaphales promelas) in the laboratory. Environmental Pollution, 16: 243-255. Reish D.L., Oshida P.S. (1987). Manual of methods in aquatic environment research. Part 10. Short-term Static Bioassay. FAO Fish. Technical paper, 247: 1- 62. Rose K.A., Cowan J.H., (Jr)., Houde E.D., Coutant C.C. (1993). Individual based modeling of environmental quality effects on early life stages of fishes: A case study using striped bass. American Fisheries Society Symposium, 14: 125-145. Seegert G.L., Brooks A.S., Castle I.R.V., Gradall K. (1979). The effects of monochloramine on selected riverine fishes. Transactions of the American Fisheries Society, 108: 88-96. Sehgal R., Saxena A.B. (1986). Toxicity of zinc to a viviparous fish Lebistes reticulatus (Peters). Environmental Contamination and Toxicology, 36: 888-894. Snyder F.L., Fisher S.W., Schneider B. (1991). Evaluation of potassium chloride for removal of zebra mussel veligers from commercial fish shipments. Journal of shellfish research, 11 (1): 238-239. Standing Committee of Analysts (1981). Acute toxicity testing with aquatic orgamisms. Methods for the Examination of Waters and Associated Materials. Her majesty’s Stationery Office, London. Skidmore J.F. (1964). Toxicity of zinc compounds to aquatic animals, with special reference to fish. The Quarterly Review of Biology, 39 (3): 227-247. Tomasso J.R., Goudie C.A., Simco B.A., Davis K.B. (1980). Effects of environmental pH and calcium on 163 Int. J. Aquat. Biol. (2014) 2(3): 155-163 ammonia toxicity in channel catfish. Transactions of the American Fisheries Society, 109: 229-234. Vander Merwe M., Van Vuren J.H.J., Du Preez H.H. (1993). Lethal copper concentration levels for Clarias gariepinus (Burchell, 1822) - a preliminary Study. Koedoe, 36 (2): 77-86. Ward G.S., Parrish P.R. (1982). Manual of methods in aquatic environmental research. Part 6. Toxicity Tests. FAO Fisheries Technical Paper, No 185 FIRI/T185.