Tolerance of Nile tilapia juveniles to highly acidic rearing water

The present work aimed at determining the tolerance of Nile tilapia juveniles to highly acidic rearing waters and the effects of water acidity on the quality of tank effluents. The experimental design consisted of four treatments with different water pH values (4.12 ± 0.84; 5.13 ± 0.74; 6.14 ± 0.64 and 8.06 ± 0.48), with five replicates each. No exchange of water was performed throughout the study, only water replenishment to maintain the initial level. Variables of water quality, soil, growth performance, metabolism and effluents were monitored for eight weeks. Despite the lower total ammonia nitrogen (TAN) concentration in the pH 8 tanks, their levels of non-ionized ammonia (NH3) were the highest ones. At the end, the lowest body weight of fish was observed in the pH 8 tanks. There was a significant improvement in feed conversion ratio (FCR) and protein efficiency ratio (PER) due to to water acidification. There were reduced concentrations of NH3 in the acidified tanks’ effluents. It was concluded that the gradual water acidification up to pH 4 can improve the Nile tilapia juveniles’ growth performance.


Introduction
The animal physiology works within certain species-specific environmental conditions.The water pH variations that deviate from the ideal range for the species may affect fish survival and performance (White et al., 2014).Fish try to adapt its behavior and physiology when subjected to stressful pH conditions.Some groups of fish exhibit greater body growth in waters with pH values away from neutrality.These species of fish adapted to live in acidic (Duarte et al., 2013)et al., andalkaline (Saha et al., 2002) waters are called acidophilic and basophilic, respectively.
The pirapitinga, Piaractus brachypomus, for instance, has greater resistance to stressful pH values when the dietary level of crude protein is high (Garcia et al., 2014).The walking catfish, Clarias batrachus, can survive in very alkaline waters, with pH up to 10.In that species, the stimulation of ureogenesis is a major physiological strategy for survival in alkaline waters (Saha et al., 2002).
The Mozambique tilapia, Oreochromis mossambicus, has some mechanisms for adaptation to acidic waters.Under acidity, that fish species increases the release of α-melanophore stimulating hormone that darkens the skin of the animal, changing its pattern of ionic regulation.This adjustment is usually successful if the pH reduction is slow (Van der Salm et al., 2005).The tambaqui, Colossoma macropomum, has increased growth in acidic waters, and is negatively affected by waters with pH above 8.Hematological changes, ionic regulations and mucus production are some of the adaptations of tambaqui to life in an acidic medium (Aride et al., 2007).
Although freshwater fish can adapt to stressful water pH, the farming of those animals should be conducted in their optimal environmental conditions to prevent metabolic stress.According to El-Sherif and El-Feky (2009), the optimal range of water pH for rearing Nile tilapia, Oreochromis niloticus, is between 7 and 8.However, recent data obtained in our laboratory by Nobre et al. (2014) suggest that the optimal range of water pH for farming Nile tilapia juveniles in green waters is wider than that reported by El-Sherif & El-Feky (2009), ranging from 5 to 8. The Nile tilapia, therefore, adapts and grows well in moderately acidic waters (Nobre et al., 2014).In that work, however, the growth performance of tilapia was not evaluated under stronger water acidity (pH< 5).
This study aimed to determine the tolerance of Nile tilapia juveniles to high acidity waters in an eutrophic culture tanks, based on variables of water and soil quality; and growth and metabolic performance.In addition, we determined the effects of water acidification on the quality of tank effluents.

Material and methods
The experiment was conducted in a green water rearing system in tanks without mechanical aeration at the Laboratório de Ciência e Tecnologia Aquícola -LCTA (Departamento de Engenharia de Pesca, Centro de Ciências Agrárias, Universidade Federal do Ceará), located at Fortaleza, Ceará State, Brazil (03°44'39,9"S; 38°34'55,9"W).Masculinized juvenile Nile tilapia, weighing between 1-2 g, were purchased from a fish farmer of the region and transported to the laboratory.At the LCTA, fish initially remained for four days in a 1,000-L circular tank for acclimation to the experimental conditions of 1 fish L -1 .During this period, a balanced commercial diet for omnivorous tropical fish was given at 8, 11, 14 and 17h, containing 39.4% crude protein, at 10% of the stocked biomass.
At the onset, eight tilapia juveniles (body weight = 1.61 ± 0.06 g) were stocked in 250-L circular polyethylene tanks, where they remained for eight weeks.Fish feed was delivered daily at 10, 13, 15 and 17h.The feeding rates started with 7.9% body weight with a crumbled diet, and finished with 3.7% with a pelleted diet (0.8 to 1.2 mm).There were four treatments with five replicates each, totaling twenty experimental tanks.In the control group, no management of water quality was carried out, allowing the water pH vary freely over the experiment.In each of the other tanks, a daily water acidification with a 3.6 N HCl solution was performed, varying the volume of acidic solution according to the treatment.The application of the acidic solutions was discontinued when the desired values of water pH were reached.The tanks of the first treatment received applications of the HCl solution to achieve a water pH between 5.5 and 6.5; the tanks of the second treatment received applications of the HCl solution to achieve a water pH between 4.5 and 5.5; finally, the tanks of the third treatment received the acidic solution to achieve a water pH between 3.5 and 4.5.The pH readings (mPA210 -Tecnopon MS pH meter) were performed daily in the morning (7h30min) and afternoon (15h).The daily water pH was calculated averaging those two records.The daily total HCl dose was divided into three equal sub doses starting at 8h with a 30 minutes break between them.Over the entire experiment, no water exchange was performed in the tanks, only replenishment to maintain the initial volume.The bottom of the tanks was covered with a 5-cm layer of gross sand.
Variables of water quality, soil quality, growth performance and metabolic performance were analyzed.The pH of water was measured daily, while the temperature and the electrical conductivity of water (CD-850 conductivimeter) were determined twice a week, in the morning and in the afternoon, at 8h and 15h.Weekly, the concentrations of nitrite (sulfanilamide method) and total ammonia nitrogen (TAN, indophenol method) were determined.The NH 3 concentrations were calculated by the use of the NAT, pH and water temperature results, obtained at 15h, in the Emerson's Formula (El-Shafai et al., 2004).
Fortnightly, it was determined the concentrations of dissolved oxygen (DO 2 ; YSI-55 oximeter), free CO 2 (Na 2 CO 3 standard solution titration), total alkalinity (H 2 SO 4 standard solution titration), total hardness (EDTA standard solution titration), dissolved iron (Herapath method), reactive phosphorus (molybdenum blue method), nitrate (reducing cadmium column), organic matter (oxygen consumed), total dissolved sulfides (TDS; Na 2 S 2 O 3 standard solution titration) and hydrogen sulfide.The H 2 S concentration was obtained by applying the TDS, pH and water temperature data obtained at 7h30min to the formula presented by Boyd (2000).Variables of water quality were determined according to APHA (1999) soil pH and the tank soil organic carbon (K 2 Cr 2 O 7 digestion and FeSO 4 standard solution titration) were determined at the beginning, middle and end of the experiment (Boyd, 2000).The variables of growth performance analyzed were the following: final body weight, weekly weight gain, specific growth rate (SGR = [Ln (final weight) -Ln (initial weight)] /days of rearing) x 100, fish yield (g m -3 day -1 ), feed conversion ratio (FCR = feed allowed/body weight gain) and protein efficiency ratio (PER = fish weight gain/dietary protein allowed).The crude protein content of the diets was determined in laboratory (Laboratório de Nutrição Animal, Departamento de Zootecnia, UFC), by the micro Kjeldahl method.
The metabolic performance of fish was observed by using four 2.5-L respirometers, sealed with lids and composed of PET containers, as proposed by Barbieri et al. (2005).The consumption of dissolved oxygen after 4h by one fish placed inside the respirometer was measured weekly, beginning at the third experimental week (one fish per respirometer).Initially, the water used in each respirometer was taken from the rearing tanks, and filtered through filter paper.Fish that would be used were maintained in a separate tank with non-acidified water.Previously to fish allocation, mechanical aeration was provided to the respirometers for one hour.Afterwards, the initial concentrations of DO 2 were measured and the juveniles were placed inside their respective chambers.After a 4-h period, the final concentrations of DO 2 were determined in each respirometer by the Winkler's method with azide modification.The consumption of DO 2 inside the respirometer (μg L -1 DO 2 g -1 fish h -1 ) was estimated by the following equation: [(DO 2i -DO 2f )/fish body weight (g)]/ test duration (h), where DO 2i = initial concentration of DO 2 in the respirometer, and DO 2f = final concentration of DO 2 in the respirometer.
Statistical analyses of water quality, soil quality, growth and metabolic performances were made through the one-way ANOVA, using a significance level of 5%.In the case of significant differences between treatments, the means were compared by the Tukey's test.Prior to analysis, the results were tested for normal distribution (Shapiro-Wilk's test) and for homogeneity of variance (Levene's test).The statistical analyses were run with the softwares BioStat 5.0 and Excel 2010.

Results and discussion
Water and soil quality The pH of the rearing waters differed significantly between the treatments (Figure 1).Tanks with no HCl application showed the highest pH value (pH = 8.06 ± 0.48).Tanks that received the HCl solution at different doses exhibited the following pH values: 4.12 ± 0.84 (30 mL 3.6 N HCl solution/tank); 5.13 ± 0.74 (21 mL HCl solution 3.6 N/tank) and 6.14 ± 0.64 (15 mL HCLl solution 3.6 N/tank).Hereafter, those values of water pH will be represented as pH 8, pH 4, pH 5 and pH 6, respectively.As expected, water acidification reduced significantly the total alkalinity of water (Table 1).The total hardness of pH-8 tanks was lower than that found in the pH-4 tanks (p < 0.05; Table 1).The higher concentrations of free CO 2 in the pH-4 tanks probably favored the dissolution of calcium carbonate present in the tank soil that raised the hardness of water.The pH-8 tanks had the lowest concentrations of free CO 2 .The electrical conductivity (EC) of the water was not significantly different between the tanks (Table 1).Likewise, the concentrations of DO 2 were not significantly different between the experimental tanks.This indicates that the artificial acidification of water did not impair the photosynthesis in rearing tanks.A CO 2 increase in the water can promote phytoplankton growth due to increased availability of nutrients and thus change the composition of phytoplankton communities.In that context, there can be an increased frequency of green algae (Low-Décarie et al., 2014).
That assumption is supported by the results of organic matter (Table 1), which were not different between the experimental treatments (p > 0.05).
At the end of the experimental period, the concentrations of total ammonia nitrogen (TAN) in the pH-4, pH-5 and pH-6 tanks were significantly higher than those observed in pH-8 tanks (Figure 2).The TAN concentrations in pH-8 tanks remained low throughout the experiment, except for a peak of 0.97 mg L -1 in the 3 rd week.The main source of TAN in fish tanks is the ammonification of organic matter (animal feces, unconsumed feed and dead plankton).As the amount of feed supplied to the tanks varied with the fish biomass, and because of the lower fish biomass in the pH-8 tanks, the input of artificial diet to those tanks was lower when compared to the others, thus explaining the lower concentrations of TAN.Garcia et al. (2013) reported that both ammonia and nitrite tend to accumulate in Nile tilapia cages with higher stocking densities.On the other hand, NH 3 concentrations were higher in pH-8 tanks compared to the others (p<0.05).The highest NH 3 concentration (0.12 ± 0.04 mg L -1 ) was found at the 3 rd experimental week in the pH-8 tanks.The TAN is composed by the ammonium ion (NH 4 + ) and non-ionized ammonia (NH 3 ).A shift between NH 4 + and NH 3 that favors more NH 3 occurs at high pH values.The NH 3 is the toxic form of TAN to aquatic animals (Pereira & Mercante, 2005).Lower values of TAN and pH (pH<8) are the ideal conditions for efficient excretion of NH 3 by fish.Under those conditions, there is a fast diffusion of NH 3 from fish blood to the water (Silva et al., 2013).
The concentrations of nitrite and nitrate in water were low and not significantly different between the experimental tanks (Table 2).Low values of nitrite were probably related to the high nitrification rate happening in the rearing tanks.The nitrification process comprises the oxidation of ammonia to nitrite (NO 2 -) and that to nitrate (NO 3 -).Different groups of microorganisms catalyze each of those steps (Füssel et al., 2012).Low nitrate in water is probably related to the high algal productivity observed in the tanks.Phytoplankton absorb nitrate from water for their growth (Masclaux et al., 2015).The highest concentrations of reactive phosphorus were observed in the pH-4 tanks (Table 2; p < 0.05).The concentrations of reactive phosphorus in the other tanks (pH 5, pH 6 and pH 8) were not significantly different to each other.Similarly, the highest concentrations of soluble iron were observed in the pH-4 and pH-5 tanks (p <0.05).Under acidic conditions, it may occur internal fertilization of water, in which phosphates are released from sediments to the water column (Falagán et al., 2014).There was a pronounced H2S increase in the water as the pH decreased.With the pH reduction, the formation of H 2 S was favored through the following reaction: H 2 S ↔ H + + HS - .Another possibility for the presence of H 2 S in the water is the occurrence of sulfate reduction (Koschorreck et al., 2003).As there was no loss in performance of tilapia stocked in the pH-4 tanks, it is inferred that O. niloticus is quite tolerant to high concentrations of H 2 S in water as the Poecilia mexicana, a freshwater fish species (Tobler et al., 2014).
The pH of soil in the pH-8 tanks was higher than in the pH-4 tanks (Table 2; p < 0.05).The differences in concentrations of organic carbon of tank soils were not significantly different between the treatments.Over time, the water column and the sediments tend to reach a chemical-physical balance (Queiroz et al., 2004).

Growth performance
There were no significant differences for the survival of fish stocked in the different experimental tanks.Overall, they exceeded 95% (Table 3).This evidences the strength of tilapia, which are able to tolerate water acidity as low as pH = 4. Wangead et al. (1988) observed that juvenile and adult tilapias did not survive when subjected to pH 2 and 3 waters.These same authors, at the same time, reported survival rates of 57.8; 82.2; 84.5% for juvenile tilapia maintained in water pH 4, 5 and 7, respectively.The survival rates for the same pH values were equal to 86.6; 100 and 100%, respectively, for adult tilapias.
Fish final weight in the pH-8 tanks was significantly lower than that observed in the other tanks (pH 4, pH 5 and pH 6; Table 3).That result points out that the water acidification carried out was beneficial to tilapia growth.This may be associated with the lower concentrations of NH 3 in the more acidic tanks (Figure 2).Sakala and Musuka (2014) observed that high concentrations of ammonia affected both the growth and survival of Tilapia rendalli.Similarly, the lowest results for specific growth rate values (SGR) and fish yield were observed in the pH-8 tanks (p < 0.05).Therefore, the results obtained in the present study suggest that Nile tilapia juveniles present similar growth performance when reared in waters with pH 4, 5 or 6.
The water pH reduction significantly improved the feed conversion ratio (FCR; Table 3).Similarly, the protein efficiency ratio (PER) increased with reducing water pH.The best FCR and PER results were obtained in the pH-4 tanks.Those results suggest that Nile tilapia juveniles tolerate waters with strong acidity, having a pH around 4. If future works confirm such suggestion, O. niloticus could be reclassified as an acidophilus species of freshwater fish.Supporting that argument, Wangead et al. (1988) concluded that O. niloticus has the ability to adapt itself well to pH-4 waters.The same authors, however, argued that O. niloticus does not support waters with higher acidity (pH<4).Likewise, the tambaqui, C. macropomum, shows better growth performance when reared in acidic waters (pH 4 -6; Aride et al. (2007)).

Effluent quality
The tank effluents' pH followed the same trend of the rearing waters' pH.Hence, there was a progressive lowering in the effluent pH through the artificial water acidification carried out in the present work (Table 4).Therefore, if water acidification is performed to achieve a better growth performance, it is important to correct the effluents' pH before discharging them into the environment.One known method to lower the pH of the rearing water is to apply ammonium fertilizers to the water (Tsadilas et al., 2005).According to the CONAMA's Resolution n. 430, the effluents from any pollution sources can only be discharged directly into the receiving water bodies if they have a pH between 5 and 9.The concentrations of organic matter in the effluents were not significantly different between the treatments (p>0.05;Table 4).Although high TAN concentrations were observed in the acidic tanks' effluents, their levels of NH 3 were significantly low or even zero.However, there will be an increase in the NH 3 levels when the effluent pH values are corrected upwards.For that, the effluent should be aerated in the treatment basin for ammonia removal by nitrification and volatilization after the pH correction.The nitrite concentrations in the tank effluents were not different between the treatments.On the other hand, higher concentrations of reactive phosphorus were found in the pH-4 tanks.The use of aquatic macrophytes, such as Eichhornia crassipes and Lemnaceae minor, can be used to remove phosphate from the wastewater (Shilton et al., 2012).Therefore, while the water acidification can improve tilapia growth performance, it causes at the same time environmental problems that should be considered by the responsible farmer.

Metabolic performance
The DO 2 consumption rates by fish inside the respirometers were not significantly different between the treatments (p>0.05; Figure 3).In the second week of observation, fish of all treatments showed a rise in the DO 2 consumption inside the respirometers.Afterward, fish of the pH 6 and pH 8 treatments showed a decline in DO 2 consumption.A DO 2 reduction was also registered for all treatments in the fourth monitoring week.
The results of the fourth week (Figure 3), along with the downward trend in the DO 2 consumption observed throughout the study, suggest that a better adaptation to acidic conditions is achieved when fish has a greater body weight.The relationship between the metabolic rate and the body size of fish varies according to intrinsic and extrinsic factors, such as the animal physiology and environmental conditions, respectively (Urbina & Glover, 2013).

Conclusion
Juvenile O. niloticus is a fish species tolerant to high concentrations of H 2 S in water (up to 4.4 mg L -1 ).Besides, a gradual acidification of water to pH 4 is advantageous to its growth performance.However, the management of water acidification deteriorates the quality of the effluents from the rearing tanks.

Figure 2 .
Figure 2. Total ammonia nitrogen (TAN) and non-ionized ammonia (NH 3 ) of water for rearing of juvenile Nile tilapia for eight weeks in 250-L outdoor tanks, subjected to different pH values.Lines with different letters indicate significantly different means in the last observation by Tukey's test (p < 0.05).

Table 3 .
Growth performance of juvenile Nile tilapia (body weight = 1.61 ± 0.06 g) stocked in 250-L outdoor tanks for eight weeks and subjected to different pH values (mean ± SD; n = 5).

Table 4 .
Effluent quality of Nile tilapia's tanks one week after harvest.