Materials and Methods Collection and maintenance of test fish Channa punctata were collected from culture pond near Serispore T. E., Hailakandi, Assam, India and brought to the laboratory in aerated containers. In the laboratory, the fishes were maintained in an aquarium with constant aeration. Fishes were fed with commercially available fish meal twice daily and were acclimatized in the aquarium for a week before the experiment. Experimental design of fish bio-assay For test setup, Channa punctata were kept in a tank (Group-I) with heavy metal (CdCl 2) for seven days. A control with no treatment was also run simultaneously in another tank. After seventh day, the protein content of liver was examined. Experimental Design The fishes were randomly divided into three groups viz., control, low dose, sub-lethal- 1 (SL-I, 5 ppm) and high dose sub-lethal 2 (SL-II, 10 ppm). They were kept in three different buckets containing 5 litres of water obtained from a pond and were labelled as control, SL- 1 (5 ppm) and SL- 2 (10 ppm). The experiment was run for seven days. Fishes were taken out from all the tanks after seven days and were anesthetized and the total length and body weight were recorded. Test fishes were sacrificed and tissues like gills and liver were dissected and stored at refrigerator until further analysis. Procedure for estimation of total protein content Estimation of protein content Total protein content was estimated by the protocol developed by Lowry et al. (1951). Reagents used A. Alkaline copper Reagent. Reagent A: 2 % Na 2 CO 3 in 0.1 N NaOH. Reagent B: 0.5 % CuSO 4. 5 H 2 O in sodium potassium tartarate The alkaline copper reagent was made by mixing 50 ml of reagent A and 1 ml of reagent B. B. Folin Ciocalteu phenol reagent (It is commercially available which is diluted with distilled water in the ratio of 1: 2 C. 0.1 N NaOH D. 70 % alcohol E. Protein (stock) Standard solution: 100 mg % Bovine Serum Albumin in 0.1 N NaOH. Working standard 10 ml of the stock was diluted to 100 ml with distilled water. The protocol 1 ml of 70 % alcohol was added to the test tube after 0.2 ml of tissue homogenate was pipetted out. For ten minutes, the tubes were centrifuged at 5,000 rpm. After discarding the supernatant, 1 ml of 0.1 N NaOH was used to dissolve the precipitate. 5 ml of Alkaline Copper Reagent was added, and it was left at room temperature for ten minutes. The Folin-Ciocalteu phenol reagent was added after ten minutes, and it was left in the dark for thirty minutes. The UV-Vis Spectrophotometer was used to measure the absorbance at 620 nm against a blank for the reagent. Simultaneously, a series of graded protein standard volumes were run. The data are given as milligrams of protein per gram of tissue weight that is moist.
