Cyprinus carpio (10.18 ± 0.109 gram) were subjected to four different acidic environments, namely pH 5.0, 5.8, 6.6, and 7.2 (control). The capacity of the experimental trough was 20 L each. All the experiments were carried out in triplicates and conducted at room temperature (29 ± 1°C). The sacrifice method of Maynard and Loosli (1962) was followed in the present investigation to estimate fish growth. After the complete evacuation of their alimentary canal by starving them for at least 24 hrs (Mohanty, 1990) healthy fishes were selected and the wet weight of the experimental fishes were determined at the beginning of the experiment by electronic analytical balance (@0.1mg accuracy). The feces released by the fishes were filtered and oven-dried. The beef liver was kept frozen during the experiment. Every day the frozen beef was taken out, thawed, and the known quantity was weighed and cut into pieces. Fishes were fed with a known quantity of beef. The remnants were collected from the respective experimental troughs the next day before changing the water medium and were oven-dried to calculate the dry weights of the unfed. Thus the dry weight of food consumed can be calculated, which was the difference between the dry weight of food given and that of uneaten food. Feces collected every day (once) were dried powdered and kept in a desiccator for further analysis. All the experiments lasted for three weeks. After the experimental period, the fish were starved for a period of 24 hrs. The final weights of the individual fish of each experimental series were taken and the fishes were oven-dried. The dried fishes were powdered and subjected to estimation of the energy.
Preparation of acid (low pH) media
The pH of the experimental freshwater (control) has gradually reduced to pH 6.6, 5.8, and 5.0 by adding 5% Sulfuric acid (H2So4), The prepared pH experimental media have stirred well by an electric stirrer, and the pH was measured exactly by a high sensitive digital pH meter (Labtronics tabletop pH meter- Model Number: Lt 5001) and the medium was under periodical test with a pen pH meter (Panomex) and ensured the constantly desired pH without the fluctuations. The pH was monitored vigilantly. Already several experiments were conducted on the effect of acidity and acidic trauma on the various physiological modifications in laboratory and field animals. In the laboratory observations, researchers have used acids, such as sulphuric acid (H2So4), hydrochloric acid (HCl), and nitric acid (HNo3) to reduce the pH of the water medium into acid nature. The majority of the researchers used sulphuric acid as it is a mineral acid pollutant in the wild (Ibrahim 2003, 2020) Beamish and Harvey, 1972, Schofield, 1976). To reduce the water pH sulphuric acid was used by Fromm (1980), Ultsch (1981), Louisemilligan and Wood (1982), Hunn et al. (1987), Dheer et al. (1987), Gunn and Noakes (1987), Sadler and Lynam Wood (1987), Tam et al. (1988) and Vanduk et al. (1993). Witters (1986) used nitric acid to reduce the water pH (1986) and hydrochloric acid was used to reduce the water pH by Smith and Haines (1995). According to the researchers' views and their methodology about the preparation of low pH, media were as followed in the present experiments. In this investigation, sulphuric acid was used to prepare various experimental pH media. Based on the earlier reports in the present investigations also, sulphuric acid was used to prepare various experimental pH media (6.6, 5.8, and 5.0).
Acid tolerant bioassay
Preliminary experiments were conducted to find the effect of acidic (low pH) stress on the selected experimental fishes C. carpio, Based on the acute lethality bioassay, it was found that the lethality bioassays were found to be not relevant for the present study. The range of acidity tolerance was very minimum. When the experimental fishes of C. carpio were exposed to below pH 4.9, the mortality begins, at the minimum level, but it gradually increased when the pH was decreased to pH 4.7. For instance, the percentage of mortality for 24 hrs in C. carpio exposed to pH 4.80, 4.85, and 4.90 were 100%, 70%, and 30% respectively (Table 1 and Fig 1). The experiments revealed obviously that the experimental fish could tolerate above pH 4.9. The results revealed that pH 4.9 to 4.7 was acutely lethal to the test fishes. Based on this different pH media were selected (pH 5.0, 5.8, and 6.6) for studying the influence of low pH on the various physiological parameters of experimental animals. Further, it was found that at pH 5.0 and above, there was no mortality for 4 weeks of the experimental period.
Energy estimation
Energy estimations for fish tissue samples were done by plain jacket oxygen bomb calorimeter (Toshniwal, India) and feces energy was estimated by wet combustion method, Dried fish samples were blended into a homogeneous mixture for energy estimation, The necessary corrections were made for the wet combustion method as suggested by Job and Gerald (1969). The energy values are represented here as Joules or Kilo Joules (KJ).
Oxygen Bomb Calorie Meter
The oxygen bomb calorimeter used in the present investigation is a plain jacket calorimeter (Craig et al 1978) and Wet Combustion Method (Karzinkin and Tarkovskaya 1964 and Craig et al 1978).
Energy budget
The energy budget followed here is the slightly modified IBP formula (Petrusewicz and Macfadyen, 1970) represented as C = P + R + F, where C is the energy consumed, P the growth (Conversion), R the energy lost as heat due to metabolism and F the feces.
Energy consumed, was estimated by subtracting the unfed from the energy supplied. Energy absorbed, was calculated by subtracting the feces energy from that of energy consumed. Energy metabolized, Energy metabolized was estimated by subtracting the energy converted from the energy absorbed.
Energy converted, Energy converted was determined by subtracting the energy of fish at the commencement of the experiment from the energy of fish after the termination of the experiment. Rates of energy consumption, absorption, conversion, and metabolism were calculated by dividing the respective quantities of the products of the initial weight of fish (g) and the duration of the experiment (21 days).
- Consumption rate (KJ / g / day) = Energy consumed (KJ) / Initial wet wt. of fish (g) × days
- Absorption rate (KJ / g / day) = Energy absorbed (KJ) / Initial wet wt. of fish (g) × days
- Metabolic rate (KJ / g / day) = Energy metabolised / Initial wet wt. of fish (g) × days
- Conversion rate (KJ / g / day) = Energy converted (KJ) / Initial wet wt. of fish (g) × days
Efficiencies of absorption and conversion
- Absorption efficiency (Ae) (%) = Energy absorbed / Energy consumed × 100
- Gross conversion efficiency (K1) (%) = Energy converted / Energy consumed × 100
- Net conversion efficiency (K2) (%) = Energy converted / Energy absorbed × 100
- Gross conversion efficiency (K1) (%) = Energy converted / Energy consumed × 100
- Net conversion efficiency (K2) (%) = Energy converted / Energy absorbed × 100
Proximate Analysis (Carcass)
Carcass body composition of the experimental fishes were determined as follows: protein by Lowry et al., method (1951), lipid by Bragdon method (1950) and carbohydrate by Anthrone method (Carrol et al., 1956).and Automatic Proximate Analyzer (Model APA2)
Statistical analysis:- the obtained bioenergetics data were statistically analyzed by using Microsoft Excel statistical software and PAST (Paleontological Statistics- Hammer et al 2001). Data are presented as mean ± standard deviation. Statistical analyses were performed by one-way ANOVA which was applied to identify the differences between pH whereas, the significant differences were indicated at the 5% level.