Acute toxicity of ammonia on juvenile cobia (Rachycentron canadum, Linnaeus, 1766) according to the salinity Edison Barbieri &

Sonia Assami Doi

Aquaculture International Journal of the European Aquaculture Society ISSN 0967-6120 Volume 20 Number 2 Aquacult Int (2012) 20:373-382 DOI 10.1007/s10499-011-9467-3

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Aquacult Int (2012) 20:373382 DOI 10.1007/s10499-011-9467-3

Acute toxicity of ammonia on juvenile cobia (Rachycentron canadum, Linnaeus, 1766) according to the salinity
Edison Barbieri Sonia Assami Doi

Received: 4 February 2011 / Accepted: 23 July 2011 / Published online: 25 August 2011 Springer Science+Business Media B.V. 2011

Abstract Juvenile cobia (Rachycentron canadum) (total length 15.16 0.92 cm and weight 19.26 4.5 g) were exposed to different concentrations of ammoniaN (unionized plus ionized ammonia as nitrogen), using the static renewal method at different salinity levels of 5, 20, and 35% at pH 8.1 and 25C. The 24, 48, 72, 96 h LC50 values of ammoniaN for R. canadum juveniles were 60.28, 48.57, 37.42, 22.73 mg l-1 at 35%; 51.25, 43.63, 28.17, 19.05 mg l-1 at 20%; and 39.48, 25.31, 19.50, 8.13 mg l-1 at 5%, respectively. The 24, 48, 72, 96 h LC50 values of NH3N (unionized ammonia as nitrogen) were 1.81, 1.46, 1.12, and 0.68 mg l-1 at 35%; 1.75, 1.49, 0.96, and 0.65 mg l-1 at 20%; and 1.52, 0.97, 0.71, and 0.31 mg l-1 at 5%, respectively. As the salinity decreased from 35 to 5%, susceptibility of ammoniaN increased by 34.5, 47.88, 50.56, and 64.23% after 24, 48, 72, and 96 h exposure, respectively. Furthermore, we found that exposure of sh to ammoniaN caused an increase in oxygen consumption of 129.1, 157.5, and 192% and a decrease in the ammonia excretion level of 53.4, 38.2, and 23.3% with respect to the control. Keywords Cobia Ammonia Rachycentron canadum Salinity Toxicity

Introduction The cobia (Rachycentron canadum) population is distributed throughout the tropical and subtropical waters of Atlantic, Pacic, and Indian Oceans (Shaffer and Nakamura 1989). They are found in areas of the Atlantic and Pacic Oceans where water temperatures average 24C or greater (Smith 1995). In the Atlantic, cobia is an important commercially
E. Barbieri (&) Instituto de Pesca, APTA, SAA/SP, Caixa Postal 61. Av. Prof. Besnard s/n, ia, SP 11990-000, Brazil Canane e-mail: edisonbarbieri@yahoo.com.br S. A. Doi s-graduac o do Instituto de Pesca, APTA, SAA/SP, Av. Francisco Matarazo, Programa de Po a o Paulo CEP: 05001-900, Brazil 455, Sa

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exploited species (Arnold et al. 2002). In recent years, cobia has been considered an important sh for aquaculture because of its rapid growth and effective feed utilization (Chou et al. 2001; Chen et al. 2009). This species inhabits a wide range of salinity: from brackish water of 515% up to salt concentrations of 37% (Denson et al. 2006) and is found inhabiting localities up to 47 m deep (Smith 1995). The cobia juveniles survive in temperatures between 20 and 30C and salinities greater than 20% (Chen et al. 2009). The optimal temperature for growth is 2328C (Shaffer and Nakamura 1989). Chen et al. (2009) demonstrated that salinity can have an immediate and signicant effect on the growth of juvenile cobia, and a salinity of about 30% is recommended for the culture of juvenile cobia at this development stage. In an intensive culture system, ammonia is among the most common toxicant resulting from the excretion of cultured animals and mineralization of organic detritus such as unconsumed food and feces (Lin and Chen 2003). Accumulation of ammonia in pond water may deteriorate the quality of the water, reduce growth, decrease ammoniaN excretion, increase oxygen consumption, alter concentrations of free amino acid levels, and even cause high mortality (Arana 2000). The lethal effects of ammonia in sh juveniles have been studied in Bidyanus bidyanus (Frances et al. 2000), Arapaiama gigas (Cavero et al. 2004), Morone saxatilis (Hargreaves et al. 2004), Opsanus tau (Wang and and Kucuk 2001), Dicentrarchus labrax (Lamarie Walsh 2000), Mugil platanus (Sampaio et al. 2002), Oncorhynchus kisutch (Wicks and Randall 2002) and Odontesthes bonariensis (Piedras et al. 2006). However, little information is available on the lethal effect of ammonia at different salinity levels in Rachycentron canadum (Rodrigues et al. 2007). Exposure to ammonia in an aquatic environment produces many physiological changes in sh, including alterations to their metabolism (Barbieri 2010). The metabolic rate of an organism is a useful and sensitive indication of its daily consumption of energy. Therefore, in aerobic organisms, the quantication of the rate of oxygen consumption can be directly associated with the amount of energy released from the oxidation of food substratum. Based on the amount of oxygen consumed by an animal for a certain period of time, it is possible to calculate the energy spent during the same period to maintain its vital processes (Barbieri 2009). Evaluation of oxygen consumption and ammonium excretion was used, for example, to study toxic effects caused by aromatic compounds (Lemaire et al. 1996), heavy metals (Barbieri and Paes 2011), detergents (Barbieri et al. 2002; Christiansen et al. 1998), and a variety of toxicants (Boudou and Ribeyre 1989; Barbieri and Ferreira 2011). The purpose of the present study is to estimate ammonia toxicity, oxygen consumption, and ammonia excretion at salinity levels of 5, 20, and 35% at temperature 25C, in Rachycentron canadum juveniles.

Materials and methods Toxicity experiment (LC50) Cobia juvenile, cultivated in the laboratory of the Instituto de Pesca, were exposed to different concentrations of ammoniaN and NH3N for periods up to 96 h and then the acute toxicity was measured. A total of 360 juvenile of cultivated sh, with an average wet weight of 19.26 4.5 g and total length of 15.16 0.92 cm, were used. The sh were divided randomly into three groups with the same salinity (35%), and then the

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Table 1 Percent mortality (%) of cobia exposed to various ammoniaN and NH3N concentrations for 96 h and its medium lethal concentration (LC50 with 95% condence limits) calculated by Spearman Karber estimates, for 35% salinity Exposure time (h) Values of ammoniaN (unionized plus ionized ammonia as nitrogen) and NH3N (unionized ammonia as nitrogen) (mg/l) 0.0 24 48 72 96 0.0 0.0 0.0 0.0 1 0.0 0.0 0.0 0.0 5 0.0 0.0 0.0 6.66 10 0.0 0.0 6.66 13.33 20 0.0 6.66 66.6 20 50 20 40 60 100 100 100 100 100 100 120 100 100 100 100 60.28 (50.9871.08) 48.57 (38.6561.04) 37.42 (28.7648.70) 22.73 (17.6429.30) 1.81 (1.532.13) 1.46 (1.161.83) 1.12 (0.861.46) 0.68 (0.500.88) LC50 mg NH4N/L LC50 mg NH3N/L

concentration was decreased by 5% a day to obtain the two different salinity levels of 20 and 5%. The seawater was reconstituted using sea-salt from Cabo Frio, Rio de Janeiro, Brazil. Three replicates of groups of 15 individuals were exposed to each one of the following concentrations of ammoniaN and NH3N: 0.00, 1.0, 5.0, 10, 20, 50, 100, and 120 mg/l, in semi-static text. Dead sh were removed from the tanks (300 l) and counted after 24, 48, 72, and 96 h of exposure. During the experiment, sh were fed a diet of 40% protein twice a day (at 09:00 and 16:00) at 15% of body weight per day. Water temperature was maintained at 25C (0,6), dissolved oxygen at 7.14 0.15 mg l-1, pH ranged from 7.00 to 8.23 with an average of 7.76 (Table 1). The lethal concentration (LC50 with 95% condence limits) of ammoniaN and NH3N was calculated by SpearmanKarber estimates (Hamilton et al. 1977). Death was presumed when sh were immobile and showed no response to touch with a glass rod. Dead sh were removed daily. Seawater concentration was adjusted to 35, 20, and 5%, with distilled water, and aerated for 3 days before use. Stock solution of ammoniaN at 10,000 mg l-1 was prepared with ammonia chloride and then diluted to the desired concentrations of ammoniaN with 35, 20, and 5% seawater. The nominal concentration of ammoniaN for test solution ranged from 5 to 80 mg l-1. The actual concentrations of ammoniaN in the test solutions were measured using the method described by Solorzano (1969). The concentration of NH3N (unionized ammonia as nitrogen) was calculated according to the equations of Khoo et al. (1977) based on pH 8.00, temperature 25C, and salinity levels of 5, 20, and 35%. The concentration of NH3N was 3.0, 3.4, and 3.8% of ammoniaN concentration at 5, 20, and 35%, respectively (Khoo et al. 1977). Oxygen consumption and ammonia excretion experiment One hundred and fty cobia, weighing an average of 1.57 (0.5) g and measuring an average of 1.62 (0.36) cm in length, were used for the routine metabolism measurements using sealed respirometers at temperature 25C (0.6) according to the three salinity (5, 20 and 35%). The respirometer was made in our laboratory, with a tube of acrylic and PVC covers. Ten sh were subjected individually to oxygen consumption measurements in one of the concentrations of ammonium chloride (0.0, 5, 10, 20, and 40 mg/l). The pH and oxygen concentration of the test solution at the different concentrations of ammonium chloride were measured: the average of pH values were 8.19 (0.02), 7.99 (0.06), and 7.09 (0.064) at salinity 35, 20, and 5, respectively. The range of oxygen values was 6.957.25 ml oxygen/l.

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Before beginning the experiments, the animals were maintained in the respirometer with continuous water circulation for at least 90 min to attenuate the handling stress. Then, the water supply was suspended and the respirometer closed, so that the sh could consume the oxygen in the known water volume for a period of 1 h. The respirometers were protected by a barrier to isolate the animals from movement in the laboratory. The difference between the oxygen and ammonia concentration determined at the beginning and end of the connement was used to calculate the consumption and excretion during the period. To minimize the effect of low oxygen concentration and metabolite accumulation on the metabolism, the length of the experiment was regulated, so that the oxygen concentration at the end of experiments was 70% of its initial concentration. The dissolved oxygen was determined through the Winkler Method (Winkler 1888). To obtain the desired concentration of ammonia, the necessary volume of the master substance (stock solution of ammoniaN at 10,000 mg l-1 was prepared with ammonium chloride) was calculated for each volume of respirometer and added with a micropipet at the end of the acclimation. As soon as the substance was added, the entry orice was immediately sealed. Additionally, the seawater in the bottle was sampled at the beginning and at the end of the oxygen consumption analysis. Determination of ammonium-nitrogen in the seawater was based on the phenolhypochlorite method (Solorzano 1969). Statistics analyzes The average of the cobia oxygen-specic consumption and ammonia excretion was tested, using analysis of variance (ANOVA). Before statistical tests, all data tested using Tukey test (P \ 0.05).

Results Mortality Actual concentrations of ammonia in the test solution were within 5% of nominal concentrations. No sh died in the control solutions at any salinity levels. No sh died in 0.1mg l-1 ammoniaN solution at 35% nor at 20% when exposed for 72 h or in 0.1-mg l-1 ammoniaN solution at 5% when exposed for 24 h. Survival of the R. canadum juveniles for the three interactions, concentration of ammoniaN/salinity, concentration of ammonia/ exposure time, and salinity/exposure time, was signicantly different (13). Acute toxicity The LC50 values of ammoniaN and NH3N at different exposure times for R. canadum juveniles are shown in Tables 1, 2, 3. At 24, 48, 72, and 96 h, LC50 values of ammoniaN were 60.28, 48.57, 37.42, and 22.73 mg l-1 at 35%; 51.25, 43.63, 28.17, and 19.05 mg l-1 at 20%; and 39.48, 25.31, 18.50, and 8.13 mg l-1 at 5%, respectively. Those of NH3N were 1.81, 1.46, 1.12, and 0.68 mg l-1 at 35%; 1.75, 1.49, 0.96, and 0.65 mg l-1 at 20%; and 1.52, 0.97, 0.71, and 0.31 mg l-1 at 5%. The LC50 values of ammoniaN and NH3N declined sharply during the rst 96 h. As the salinity decreased from 35 to 5%, susceptibility of ammoniaN increased by 34.5, 47.88, 50.56, and 64.63% after 24, 48, 72, and 96 h of exposure, respectively (Tables 1, 2, 3).

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Table 2 Percent mortality (%) of cobia exposed to various ammoniaN and NH3N concentrations for 96 h and its medium lethal concentration (LC50 with 95% condence limits) calculated by Spearman Karber estimates, for 20% salinity Exposure time (h) Values of ammoniaN (unionized plus ionized ammonia as nitrogen) and NH3N (unionized ammonia as nitrogen) (mg/l) 0.0 24 48 72 96 0.0 0.0 0.0 0.0 1 0.0 0.0 0.0 0.0 5 0.0 0.0 6.66 13.3 10 0.0 0.0 13.3 20 20 6.66 6.66 13.3 26.6 50 33.3 53.3 80 100 100 100 100 100 100 120 100 100 100 100 51.25 (41.0663.97) 43.63 (34.6055.01) 28.17 (21.0737.67) 19.05 (14.0025.93) 1.75 (1.402.18) 1.49 (1.181.87) 0.96 (0.711.28) 0.65 (0.470.88) LC50 mg NH4N/L LC50 mg NH3N/L

Table 3 Percent mortality (%) of cobia exposed to various ammoniaN and NH3N concentrations for 96 h and its medium lethal concentration (LC50 with 95% condence limits) calculated by Spearman Karber estimates, for 5% salinity Exposure time (h) Values of ammoniaN (unionized plus ionized ammonia as nitrogen) and NH3N (unionized ammonia as nitrogen) (mg/L) 0.0 24 48 72 96 0.0 0.0 0.0 0.0 1 0.0 0.0 0.0 0.0 5 0.0 6.66 6.66 20 10 6.66 13.3 20 46.6 20 20 26.6 33.3 100 50 46.6 80 100 100 100 100 100 100 100 120 100 100 100 100 39.48 (29.8352.26) 25.31 (18.5034.62) 18.50 (14.6725.91) 8.13 (6.0410.94) 1.52 (1.152.02) 0.97 (0.711.33) 0.71 (0.561.00) 0.31 (0.230.42) LC50 mg NH4N/L LC50 mg NH3N/L

Oxygen consumption For the sh acclimated at 25C, the specic oxygen consumption increased with respect to the ammoniaN concentration for the three salinities. The specic oxygen consumption in any ammoniaN concentration increased with a decrease in salinity. The specic oxygen consumption of sh from the acclimated control group was measured at 25C (Fig. 1), for 35, 20, and 5% salinity. The values were 2.4, 2.33, and 2.6 mlO2/g/min, respectively. For the sh exposed to the concentration of 40 mg/l of ammoniaN, the consumption was 5.5, 6.0, and 7.6 ml O2/kg/min for the tested salinities. These values represent an increase in the metabolic level of 129.1, 157.5, and 192% with respect to the control. Using the Tukey (P \ 0.05) statistical test, it was veried that the averages of the oxygen-specic consumption for ammoniaN at 20 and 40 mg l-1 concentration in salinity 35 and 20 are different with respect to the control (F = 1.684, P = 0.229, 1.134, 0.394, respectively). We were veried that the averages of the oxygen-specic consumption for the ammoniaN at 10, 20, and 40 mg l-1 concentrations in salinities 5 were different in relation to the control (F = 0.734; P = 0.589). The same test showed that there is a difference between the specic consumption averages for the salinities 5 and 35% and for the ammoniaN concentrations of 10, 20, and 40 mg l-1 (F = 1.84998; P = 0.19603).

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Specific oxygen consumption (mgO2/Kg/min)

Salinity 35 Salinity 20 Salinity 5

0 0 5 10 20 40

Ammonia-N concentration (mg/L)

Fig. 1 Variation in specic oxygen consumption of cobia at different AmmoniaN concentrations. The bars are the respective standard deviations (n = 5)

0,55

Ammonia-N excretion (g/g/min)

0,50 0,45 0,40 0,35 0,30 0,25 0,20 0,15 0,10 0,05 0,00 0 5 10 20

Salinity 35 Salinity 20 Salinity 5

40

Ammonia-N concentration (mg/L)

Fig. 2 Variation in ammonia excretion of cobia at different ammoniaN concentrations. The bars are the respective standard deviations (n = 5)

Ammonia excretion The ammonia excretion decreased as the ammoniaN concentration increased. Cobia acclimated at 25C (Fig. 2) and exposed to 5, 20, and 35% salinities, excreted, on average, 0.43, 0.34, and 0.30 lg/g/min of ammonia. Comparing these results with the averages of ammonia excretion at the highest ammoniaN (40 mg l-1) concentration employed in the test, we found that the ammonia excretion average decreased to 0.20, 0.21, and 0.23 lg/g/ min, for the salinities 5, 20, and 35%, respectively. These values represent a decrease in the metabolic level of 53.4, 38.2, and 23.3% with respect to the control.

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Using the Tukey (P \ 0.05) statistical test, it was veried that the averages of the ammonia excretion for the ammoniaN 40 mg l-1 concentrations in all salinities are different with respect to the control. For the ammoniaN concentration at 10 mg l-1, there was a signicant difference with respect to the control for 5% salinity (F = 0.248; P = 0.904). The same test showed that there is difference between the ammonia excretion averages for the salinities 5 and 35%, at 5 mg/l ammoniaN concentration.

Discussion The toxicity of ammoniaN for sh juveniles has been studied by several authors (Frances et al. 2000; Hargreaves and Kucuk 2001; Wang and Walsh 2000; Wicks and Randall 2002; et al. 2004; Piedras et al. 2006). The Sampaio et al. 2002; Cavero et al. 2004; Lamarie results of this study conrm that the ammoniaN and NH3 are toxic to cobia, an ecologically and economically important sh in coastal waters. Toxic effects of ammonia on various sh species have been reported: freshwater teleosts (Alabaster and Lloyd 1982), Ictalurus punctatus (Colt and Tchobanoglous 1976), Sciaenops ocellatus (Holt and Arnold 1983), Odontesthes argentinensis (Ostrensky and Brugger 1992), Salmo salar (Fivelstad et al. 1993), Perca uviatilis (Ball 1967), Dicen et al. 2004). It was reported that the 96 h LC50 of NH3 trarchus labrax juveniles (Lamarie N ranged from 0.40 mg l-1 for Salmo mykiss (Ball 1967) to 0.80 mg l-1 for Odontesthes argentinensis (Ostrensky and Brugger 1992). At 35% salinity, cobia juveniles are considered to be more tolerant to ammonia as compared to 5% salinity. Ammonia is acutely toxic for marine sh in the range of 0.54 ppm NH3N for Centropristis striata (Weirich and Riche 2006) to 1.77 ppm NH3N for Menidia beryllina (Miller et al. 1990) and 1.13 ppm (LC50 96 h) NH3N for cobia (Rodrigues et al. 2007). In this work, the toxicity (LC50 96 h) of NH3N to cobia was 1.25 mg l-1 at 35%; 1.04 mg l-1 at 20%; and 0.44 mg l-1 at 5%, respectively. The toxicity of ammoniaN to cobia juveniles increased with exposure time and decreased with salinity. The tolerance of cobia juveniles to ammoniaN decreased sharply by 19.4, 37.9, and 62.2% after 48, 72, and 96 h as compared to 24 h LC50 at 35% salinity. The tolerance of cobia juveniles to ammoniaN decreased sharply by 35.89, 50.6, and 79.4% after 48 and 96 h as compared to 24 h LC50 at 5% salinity. Similar results were obtained previously with other sh, for example, Boardman et al. (2004) observed that the tolerance of Cyprinodon variegatus juveniles decreased by 23% after 96 h as compared to 48 h LC50 at 25% salinity. Boardman et al. (2004) reported that the tolerance of Paralichthys dentatus juveniles to ammoniaN decreased by 40.16% after 96 h as compared to 48 h LC50 at 25% salinity. The increase in ammoniaN toxicity over time was abrupt in cobia as compared to that in Cyprinodon variegatus or in Paralichthys dentatus. Costa et al. (2008) record that toxicity of NH3N for juvenile pompano (Trachinotus marginatus) was inuenced by salinity. They noted that juvenile pompano were signicantly less sensitive to NH3N when maintained at an isosmotic environment, compared to lower or higher salinities. However, Weirich and Riche (2006) record that, with the exception of LC50 values determined at 48 h, median lethal concentrations of NH3N to black sea bass (Centropristis striata) were not inuenced by environmental salinity; 24, 48, and 96 h LC50 values ranged from 0.81 to 0.85, 0.650.77, and 0.460.54 mg-NH3N/L, respectively. The effects of salinity on the pattern of acute toxicity of ammonia to juvenile mullet (Mugil platanus) were different when considering total ammonia or NH3 (Sampaio et al. 2002). Mullet was more sensitive to total ammonia when acclimated to salinity 15 or 30.

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Studies on the effect of ammoniaN on the respiration of sh demonstrated that the increase in oxygen consumption rates was related to concentration, exposure time, and larval et al. (2004) indicated that ammoniaN as low as 0.370 mg/l stage (Smart 1978). Lamarie would cause an increase in oxygen consumption and ammoniaN excretion of sea bass juveniles (0.079 0.004 g) in 20 h. Arana (2000), in a review of the responses of aquatic organisms in low ambient dissolved oxygen, mentioned that many sh possess an excellent regulatory ability in their oxygen consumption patterns and thus were called oxygen regulators. The present experiments also demonstrated that oxygen consumed by cobia showed no linear relationship to ambient oxygen levels, regardless of whether the sh were exposed to ammoniaN. Despite their regulatory capability, the oxygen consumption rate was indeed increased after cobia was exposed to high concentrations of ammoniaN. Similar results were et al. 2004). also observed in different sh species (Smart 1978; Lamarie Respiratory impairment in sh resulting from exposure to ammoniaN was also studied (Smart 1978; Buckley et al. 1979), and it was concluded that oxygen consumption generally increases when sh are acutely exposed to ammoniaN. Both oxygen uptake and ammonia N excretion of P. juponicus increased with decreasing salinity and increased directly with previous ammoniaN concentration (Chen and Chen 1997). Chen et al. (1991) indicated that ammoniaN as low as 0.678 mg/l would cause increasing oxygen consumption and ammoniaN excretion of P. chinensis juveniles (0.079 0.004 g) in 20 h. The present study indicated that water with 5 mg/l ammoniaN would cause an increase in oxygen consumption of cobia after 1 h of the exposure. Therefore, in an intensive culture system, an increase in ammonia in the water could affect physiological function of cultured sh. Chen and Lei (1990) reported that no mortality occurred when P. chinensis juveniles (0.36 g) were exposed to 10 and 20 mg/l ammoniaN in 33% seawater at pH 7.94 at 26C after 144 h. The present study indicated that ammoniaN as low as 5 mg/l would signicantly increase oxygen consumption and ammoniaN excretion after 1 h. Depending on nutritional conditions, ammonium is one of the nal products following catabolism, principally of amino acids that might have an alimentary or muscular origin (Mayzaud and Conover 1988). Typically, increases in ammonium excretion reect an increase in catabolism of amino acids. However, when exposed to lethal concentrations of ammoniaN, dysfunction of ammonium excretion control produces gill damage. Chen and Nan (1993) registered that ammoniaN excretion increased as P. chinensis juveniles exposed to ambient ammoniaN in the range of 0.0155.167 mg/l in a closed system. However, Arana (2000) registered that ammoniaN excretion decreased as Salmo mykiss and Carassius auratus juveniles were exposed to ambient ammoniaN. The present study indicated also that ammoniaN excretion decreased as cobia juveniles were exposed to ambient ammoniaN in the range of 540 mg/l. Due to the increasing difculty to excrete ammonia, the rst reaction of an aquatic animal may be a reduction or stoppage of the feeding activity to minimize the production of ammonia metabolism. Therefore, one of the most signicant sublethal effects of this compound is that a decrease in excretion may decrease the rate of growth (Arana 2000). Lethal and sublethal concentrations of ammoniaN can cause pathological changes in the kidneys, thyroid tissues, and blood of many sh species (Smith and Piper 1975; Thurston et al. 1984; Fivelstad et al. 1993). In trout, it was found that sublethal concentrations of NH3 (0.04 mg/l) over its life cycle causes pathological damage, such as hypertrophy, necrosis of gill lamellae, and degeneration of renal tubules due to widespread necrosis (Thurston et al. 1984). Diffusion of NH3 from blood to water is the main route for sh and crustaceans to excrete metabolic ammonia, since blood levels are normally much higher than ambient concentration (Kinne 1976; Schmidt-Nielsen 1997).

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Acknowledgments We thank FAPESP (processo 2007/50147-7) and CNPq (processo 308700/2010-4 Bolsa Produtividade) for their support for this work.

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