Evaluation of the Energy Budget of the Fish Cyprinus Carpio in Acid Waters

The present study was focused on the inuence of acid waters on the energy budget of the sh Cyprinus carpio. The experimental sh were tested in the experimental media (pH 5.0, 5.8, 6.6, and control 7.2) for 21 days for the bioenergetics evaluation. The shes were fed by the fresh beef liver. The pH was upheld vigilantly throughout the investigation, after the experimental period, the results revealed the declined rates of food consumption (69.75 J/g/day), absorption (96.28 (J/g/day), conversion ( -30.76 Rate of energy conversion (J/g/day), and the hyper-metabolic rate (5.09 Energy metabolized KJ/animal) was observed in the shes exposed to low pH 5.0, 5.8, and 6.6 respectively.


Introduction
The acidic environs drastically altered the amount of food consumption, absorption, and conversion, of the Oreochromis mossambicus (Ibrahim 2003). The hyper acidic stress diminishes the energetic matters of the carbohydrate, lipid, and proteins of the tissues of shes ( Ibrahim 2003). The deleterious feat of acidity adversely upsets the various tissues of the sh C.Carpio (Ibrahim 2020) The effect of acid waters affects the nutritional physiology of the shes ( Ibrahim 2003, Mota et al 2018). The acidic pH waters disturb the standard and routine metabolisms of the sh O.mossambicus ( Ibrahim 2003) O.niloticus (Mota et al., 2014), Environmental stress in the form of high acidic levels has resulted in decreased growth rates (Ibrahim, 2003, Beamish et al., 1975, Ryan and Harvey, 1977, Abbink et al., 2012, Kennedy & Picard, 2012. Growth inhibition appears to be a common response in some sh species to acid stress and such inhibition is usually reported as an actual decrease in body weight (Ibrahim 2003, Beamish et al., 1974. Though many works are available on the effect of low pH on the growth of brook trout (Fromm, 1980, Wood andMc Donald, 1982) (Ryan, 1980) yellow perch, (Frost, 1940) Norwegian salmonids (Jenson Snekvik, 1972) brook charr (Scho eld, 1976) and in rock bass (Ryan and Harvey, 1977), McKim and Benoit (1971) reported that the food consumption was found to decrease in rainbow trout Salmo gairdneri when exposed to pH 6.0.  exhibited the reduced food consumption of Atlantic salmon when exposed to environmental low pH. Lemly and Smith (1985) disclosed the declined food consumption of Fathead minnows at pH 5.5. Cleveland et al. (1989) showed the decreased food consumption in brook trout. Tam et al. (1988) reported the reduced food consumption on brook trout when exposed to pH 4.54 and Denny Buckler (1995) also reported the reduction in food consumption in Atlantic salmon when exposed to pH 4.5 and growth reduction of 40% for seabass cultured at a pH of 5.5 (Lemarié et al. 2000) also reported. Rosseland (1980) reported accumulation of uneaten food when Atlantic salmon was exposed to pH 5.2.

Materials And Methods
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 sacri ce method of Maynard and Loosli (1962) was followed in the present investigation to estimate sh growth. After the complete evacuation of their alimentary canal by starving them for at least 24 hrs (Mohanty, 1990) healthy shes were selected and the wet weight of the experimental shes were determined at the beginning of the experiment by electronic analytical balance (@0.1mg accuracy). The feces released by the shes were ltered 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 sh were starved for a period of 24 hrs. The nal weights of the individual sh of each experimental series were taken and the shes were oven-dried. The dried shes 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 uctuations. The pH was monitored vigilantly. Already several experiments were conducted on the effect of acidity and acidic trauma on the various physiological modi cations in laboratory and eld animals. In the laboratory observations, researchers have used acids, such as sulphuric acid (H 2 So 4 ), hydrochloric acid (HCl), and nitric acid (HNo 3 ) 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(Ibrahim , 2020 Harvey, 1972, Scho eld, 1976). To reduce the water pH sulphuric acid was used by Fromm (1980), Ultsch (1981), Louisemilligan and , 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 nd the effect of acidic (low pH) stress on the selected experimental shes 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 shes 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 sh could tolerate above pH 4.9. The results revealed that pH 4.9 to 4.7 was acutely lethal to the test shes. Based on this different pH media were selected (pH 5.0, 5.8, and 6.6) for studying the in uence 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 sh tissue samples were done by plain jacket oxygen bomb calorimeter (Toshniwal, India) and feces energy was estimated by wet combustion method, Dried sh samples were blended into a homogeneous mixture for energy estimation, The necessary

Energy budget
The energy budget followed here is the slightly modi ed 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 sh at the commencement of the experiment from the energy of sh 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 sh (g) and the duration of the experiment (21 days).
Consumption rate (KJ / g / day) = Energy consumed (KJ) / Initial wet wt. of sh (g) × days Absorption rate (KJ / g / day) = Energy absorbed (KJ) / Initial wet wt. of sh (g) × days Metabolic rate (KJ / g / day) = Energy metabolised / Initial wet wt. of sh (g) × days . 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 signi cant differences were indicated at the 5% level.

Results
Lc 50 value for C. bioassay was found to be not relevant for the present study. The range of acidity tolerance was very minimum. When the experimental shes 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 sh could tolerate above pH 4.9. The results revealed that pH 4.9 to 4.7 was acutely lethal to the test shes. carpio was determined by the lab experiment which shows no mortality above pH 4.95 for 96 hrs ( g. 1) Bray-curtis similarity index illustrates the analogous sway on the existence of the experimental sh.

Consumption rate (Cr)
The rate of energy consumption varied with different degrees of acidic pH. The observed results showed that shes exposed to low pH (pH 5.0, Table 2) exhibited a decrease in consumption rate than those exposed to other pH media (5.8 and 6.6, Table 2). Cr of C. carpio exposed to pH 5.0, 5.8, and 6.6 had lost the energy consumption were 69.75, 63.02 and 11.43 respectively J/g/day ( Table 2). The statistical analysis (ANOVA, Table 6) revealed that the energy consumption rate (j/g/day) of the experimental animals exposed at four different pH are varying signi cantly (F 3, 68 p<0.05, table 6) Absorption rate (Ar) Acidic environments affected the Ar of shes exposed to different acidic pH mediums (5.0, 5.8, and 6.6). The rate of absorption followed the trend of energy consumption. The rate of energy absorption linearly decreased (r=0.98) with increasing acidic pH. The results exhibited the decline of the Ar were 96.28, 73.10, and 13.46 (J/g/day) respectively when the shes were tested with pH 5.0, 5.8, and 6.6 ( Table 2).
The statistical analysis (ANOVA) revealed that the energy absorption rate (j/g/day) of the experimental animals exposed at four different pH are varying signi cantly (F 3, 38, p<0.05, table 6) Absorption e ciency (Ae) The e ciency of absorption was signi cantly in uenced by the acidic media of the experimental sh. The absorption e ciency has ranged from 85.54 ± 0.95 to 94.73 ± 0.41% in the experimental sh (Table 3).

Metabolic rate (Mr)
The maximum metabolic rate was found in the shes tested in pH 5.0, the minimum was found in controls. The maximum metabolic rate of 294.83 ± 3.97 J/g/day was obtained in the shes, which were exposed to pH 5.0. The metabolic rate increased linearly (r=0.94) with increasing acidity. The metabolic rates of the experimental shes exposed to pH 7.2, 6.6, 5.8 and 5.0 were 269.99 ± 4.40, 273.23 ± 7.45, 289.79 ± 1.30, and 294.83 ± 3.97J/g/day respectively (Table 4 and Fig 4). The statistical analysis (ANOVA) revealed that the metabolic rate (j/g/day) of the experimental animals exposed at four different pH are varying signi cantly (F 3, 59 p<0.05, table 6).

Conversion rate (Pr)
Acidic trauma adversely affected the conversion rates in the experimental shes also. Fishes exposed to pH 5.0 and 5.8 invariably showed a decline in conversion rates, For instance, the conversion rate in the experimental shes exposed to pH 7.2, 6.6, 5.8, and 5.0 were 90.41 ± 2.50, 72.94 ± 8.50, -2.54 ± 0.36 and -30.76 ± 2.43 J/g/day respectively (Table 5 and Fig 5). The statistical analysis (ANOVA) revealed that the energy consumption rate (j/g/day) of the experimental animals exposed at four different pH are varying signi cantly (F 3, 54, p<0.05) Gross and net conversion e ciencies (K1 and K2) Acid pH media have affected the gross and net conversion e ciencies of the shes. Fishes exposed to pH 5.0 and 5.8, invariably showed negative conversion e ciencies (K1 and K2), But the shes exposed to pH 6.6 and 7.2 exhibited their conversion e ciencies. The gross conversion e ciencies of the test shes exposed to pH 6.6 and 7.2 were 19.73 ± 2.10 and 23.72 ± 2.18% respectively ( Table 5). The net conversion e ciencies of the shes exposed to pH 6.6 and 7.2 were 20.37 ± 1.95 and 25.08 ± 2.44% respectively (Table 5).

Biochemical (Proximate Analysis)
The acid waters in uence the Protein, Lipid, and carbohydrates quantity of the experimental shes, the obtained results were Hypoproteinemia, Lipolysis, and Hypoglycemia (table 7 and 8) when the experimental shes were experienced with the acid environments (pH 5.0, 5.8, and 6.6) shown the decline in the percentage of the protein was 25.40, 13.40, and 2.00 respectively, similarly the same trends of reduction were found in both lipid and carbohydrate constituents (table, 7 and 8 ), So the experiments ascertained that different concentrations of the acidic pH have suppressed the growth and bioenergetics parameters of the shes living in the acidic environs.

Discussion
Despite ad libitum supply of the food, the acidity of the experimental media affected the Cr, Ar, Pr, and Mr. All the bioenergetics parameters except Mr which increased linearly with increasing acidity. In the present study, C. carpio exposed to different pH media (5.0, 5.8, and 6.6) exhibited a signi cant (P < 0.05) (ANOVA table 6) reduction in Cr. The least Cr was noticed in pH 5.0 followed by 5.8 and 6.6. The observed results clearly showed that low pH considerably upset the Cr in shes. Like the observed results reduction of Cr in shes exposed to low pH is not uncommon in literature (Mota et al 2018). Ibrahim (2003) has registered the results of the decline in the food consumption rate of tilapia, O.mossambicus, when exposed to pH 5.0, 5.8, and 6.6. McKim and Benoit (1971) also reported that food consumption was found to decrease in rainbow trout Salmo gairdneri when exposed to pH 6.0.  exhibited the reduced food consumption of Atlantic salmon when exposed to environmental low pH. Lemly and Smith (1985) disclosed the declined food consumption of fat head minnows at pH 5.5. Cleveland et al. (1989) showed the decreased food consumption in brook trout. Tam et al. (1988) reported the reduced food consumption on brook trout when exposed to pH 4.54 and Denny Buckler (1995) showed the reduced food consumption in Atlantic salmon when exposed to pH 4.5. Rosseland (1980) reported accumulation of uneaten food when Atlantic salmon was exposed to pH 5.2. The observed results of the present study highly support the views of the researchers. Food intake constitutes secondary stress which modulates the direct effect of acid stress (Leivestad and Muniz, 1976). Effect of feeding inhibition especially reduced survival of life stages (Baker et al., 1990). Dennis Lemly (1986) worked on fat head minnows and reported that acidity affected even visual feeding behavior and could be affected by an impairment of chemoreception. This may be the initial point for normal feeding responses because even if the selection of food items is based entirely on visual feeding cues, inadequate gustatory stimulation can lead to the rejection of food. Lemly and Smith (1985) suggested that aquatic acidi cation can affect the chemoreception and modify the normal appropriate behavioral responses of sh to natural stimuli such as food odors. Behavioral studies indicate that the normal attraction of sh to chemical feeding stimuli can be eliminated when pH levels drop by a rather modest pH (from 7.0-8 to 5.5-6.0). Aquatic acidi cation cause disruption of chemically mediated feeding behavior long before other more obvious symptoms of acid stress occur. The observed results of decreased consumption may be due to inhibition in chemoreception and chemical communication which form a very important component of the environmental physiology of sh. The trend obtained for absorption rate (Ar) also paralleled that of energy consumption rate in the tested shes. Ar was highly affected in the experimental shes exposed to pH 5.0, followed by 5.8. the data showed a signi cant (P < 0.05) (ANOVA table 6) reduction from the control when shes were tested in acidic medium pH 5.0. The decline in Ar may be due to physiological stress. The acidic stress the assaults the tissues of the digestive system, especially the intestine showed the abnormalities of chronic in ammation of laminapropria, which led to a massive accumulation of macrophages, necrosis, and atrophy of the intestinal villi when exposed to pH 5.8 experimental media exhibited, the hyper vacuolations of the intestinal mucus membrane, mucosal necrosis of absorptive cells and submucosal edema. and shes were exposed to pH 5.0 test media showed massive sloughing off mucosal epithelium and accumulation of macrophages. This leads to the impairment of the function of the intestinal villi, and the absorptive area of the intestine has failed to absorb the nutrients (Ibrahim 2020), Fishes under acidic stress exhibited hypersensitivity in their physiological process. Jones et al. (1985) strongly reported in Arctic char (Salvelinus alpinus) subjected to the exposure of pH 5.5 showed hyperactivity in response to acid exposure. Absorption e ciency has been used by previous workers as energy extraction e ciency or assimilation e ciency (Ibrahim 2003). Pandian and Delvi (1973) conveniently distinguished the energy extraction e ciency from absorption/assimilation e ciency and the discussion of the e ciency of absorption was followed here as it was by Pandian and Delvi (1973). Absorption e ciency varied between 85.54 to 94.73% in individuals of C. carpio. The results of the present investigation were very closer to the reports of Arunachalam (1985) and Sakthivel and Sampath (1989) Ibrahim (2003). However many others have reported that Ae values ranged from 20 -98% for different shes. Absorption e ciency was 20% in Ctenopharyngodon idella (Fischer, 1972) (Manoharam, 1984), 89% in mirror carp Ivlev (1939), 89 to 92.3 in C. straiatus, (Sampath, 1985) 89.76 to 91.65% in H. fossilis, (Arunachalam et al., 1985), 90.33% in S. fontinalis (Job, 1960), 92.77% in Anguila species (Tarr and Hill, 1978), 93.66 to 96.23% in Tilapia mossambica (Narayanan, 1980) and 95.43% in C. carpio (Jeyaseeli, 2000) 65.16 % to 93.88% O.mossambicus (Ibrahim2003), The severe acidic stress effect attributed the reduced growth rates in the shes exposed to the different acidic media of pH 5.0 and 5.8. In addition, the proximate analysis also being evident for the impact of acidity on the growth. The observed results from the experiments revealed the fall in growth. In acidic stress, shes are supplied with an ad libitum diet. But the shes did not consume adequately. So the intake of energy is very less, it was inadequate to maintain their physiological process. In this stressful situation, shes have to expend more of their energy to tackle or overcome the acidic stress. But the consumed energy is very less. To compensate for the depletion of energy from consumption, the shes get their energy from their body reserves and lassitude. Depletion of energy from the body leads to lessened growth. The obtained results of the present study corroborate prior ndings. Ibrahim (2003) registered the declined growth of O. mossambicus tested in pH of 5.0, 5.8 and 6.6. Bucker et al. (1995) reported the signi cant reduction in the growth of Atlantic salmon, exposed to pH 4.5 and 5.0, Baker and Scho eld (1982), Saunders et al. (1983), Lacorix and Townsend (1987) and Perry (1990) reported the reduced growth of Atlantic salmon exposed to pH 4.5, Cleveland et al. (1986Cleveland et al. ( , 1989Cleveland et al. ( , 1991 reported the decreased growth of brook trout when exposed to the acidic environment, Hunn (1987) reported the reduced growth of brook tout exposed to low pH, Geen et al. (1985) reported the decreased growth of Chinook salmon, Saunders et al. (1983) reported the reduction in the growth of Atlantic salmon, Lacorix and Townsend (1985) suggested the decreased growth of Atlantic salmon (Salmo salar), in increased acidity Perry (1990) reported the reduced growth of Atlantic salmon in the experiments of chronic effects of low pH. Beamish (1974 a, b), Ryan and Harvey (1980) reported that the growth of white suckers was reduced in acidi ed lakes. Beamish et al. (1975) reported that growth inhibition appeared to be a common response in some sh species to acidic stress. Such inhibition is usually reported as an actual decrease in body weight. Beamish et al. (1975) reported in white sucker (Catostomus commersoni) was documented that weight loss was associated with linear growth. Inhibition of linear growth suggests a lack of somatotropin and weight loss implies abnormalities in nutrient metabolism. The loss of metabolites from the body leads to the loss of weight. The loss of growth in the present investigation may be due to the loss of metabolites. Again growth may also decrease because of the increased metabolic rate due to the acidic stress.The experimental sh attained the highest rate of metabolism when they were exposed to pH 5.0. The rate of absorption followed the trend of consumption in both species whereas the rate of metabolism increased with increasing acidity. The pH has in uenced the metabolic rate of the animal through consumption. Thus the uctuation in consumption rate is more apparent than those observed in conversion rates.
This again reinforces the results of both species. The in uence of pH is more predominant on the various bio-energetics parameters. Smith et al. (1995) reported that Atlantic salmon showed the increased metabolic rates when exposed to acid environmental pH 5.

Declarations
Funding , The authors did not receive support from any organization for the submitted work.    Figure 1 Bray-Curtis similarity index The consumption and absorption rates of C.Carpio, exposed pH 5.0, 5.8, 6.6, and 7.2