Survival , growth and metabolic parameters of silver catfish , Rhamdia quelen , juveniles exposed to different waterborne nitrite levels

concentrations (0.06, 0.46, 1.19, and 1.52 mg.L) on growth, and hepatic and muscular lactate, glucose, glycogen and protein. Nitrite LC 50-96h was 20.46 (confidence interval: 16.10-23.68) mg.L. In the growth experiment, exposure to NO 2 did not affect weight, length or specific growth rate, but due to mortality (66.7% and 100% after 20 and 40 days, respectively), biomass of juveniles exposed to 1.52 mg.L NO 2 was significantly lower than the biomass of juveniles exposed to other treatments. Therefore, the safe level of nitrite for growth of silver catfish juveniles is below 1.19 mg.L (2% of LC 50-96h ). Exposure of silver catfish to NO 2 for 40 days reduced lactate levels in muscle, but lactate levels increased in liver tissue of fish maintained at 1.19 mg.L NO 2 . In addition, glucose levels in muscle and liver tissues were significantly lower in silver catfish exposed to the highest NO 2 level. These results indicate that chronic NO 2 exposure causes anaerobic substrate oxidation to meet energy demand.

-was significantly lower than the biomass of juveniles exposed to other treatments.Therefore, the safe level of nitrite for growth of silver catfish juveniles is below 1.19 mg.L -1 (2% of LC 50-96h ).Exposure of silver catfish to NO 2 -for 40 days reduced lactate levels in muscle, but lactate levels increased in liver tissue of fish maintained at 1.19 mg.L -1 NO 2 -.In addition, glucose levels in muscle and liver tissues were significantly lower in silver catfish exposed to the highest NO 2 -level.These results indicate that chronic NO 2 -exposure causes anaerobic substrate oxidation to meet energy demand.

-
) is found in ecosystems as a natural component of the nitrogen cycle (Jensen, 2003).Ammonia is the main nitrogenous waste material produced from amino acid catabolism; in water, ammonia is reduced to NO 2 -by nitrifying bacteria before its conversion into nitrate (Costa et al., 2004).The concentration of NO 2 -is usually low (0.03 -0.1 mg.L -1 ) in tropical fish culture systems with low stocking density (Sipaúba-Tavares et al., 1999;Andrade et al., 2007), but an imbalance in either ammonia production or NO 2 -conversion to nitrate can cause NO 2 -buildup (Jensen, 2003).High NO 2 -levels may develop in aquaculture systems due to high fish density (Hargreaves, 1998) or due to fertilizers added to the water (Lewis & Morris, 1986).In these systems, NO 2 -levels can reach 45 mg.L -1 or more upon establishment of nitrification in biological 1 Departamento de Aquicultura, Universidade Federal de Santa Catarina, Florianópolis, SC, Brazil. 2 Laboratório de Ecofisiologia e Evolução Molecular, Instituto Nacional de Pesquisas da Amazônia, Manaus, AM, Brazil. 3Departamento de Zootecnia, Centro de Educação Superior Norte do Rio Grande do Sul, Palmeira das Missões, RS, Brazil. 4Departamento de Zootecnia, Universidade Federal de Santa Maria, Santa Maria, RS, Brazil. 5Departamento de Química, Universidade Federal de Santa Maria, Santa Maria, RS, Brazil. 6Departamento de Fisiologia e Farmacologia, Universidade Federal de Santa Maria, Santa Maria, RS, Brazil.bernardo@smail.ufsm.brfilters or during imbalance in the process (Collins et al., 1975;Avnimelech et al., 1986;Kamstra et al., 1996).
An increase of waterborne NO 2 -levels induces NO 2 accumulation in fish blood and tissues from 10 to 100 times the environmental concentration (Holt & Arnold, 1996;Costa et al., 2004) , which produces toxic derivatives with deleterious effects on physiological processes (Jensen et al., 1987).Despite the importance of such effects, the lethal concentration at 96 h (LC 50-96h ) for NO 2 -was determined only in a few neotropical freshwater fish species (Avilez et al., 2004;Costa et al., 2004), and the effect of a single level of this nitrogenous compound for 8 h on biochemical parameters was analyzed in traira, Hoplias malabaricus (Moraes et al., 1998), pacu, Piaractus mesopotamicus and the hybrid tambacu (Piaractus mesopotamicus x Colossoma macropomum) (Moraes et al., 2006).
Nitrite reduces growth in silver perch, Bidyanus bidyanus (Frances et al., 1998) and rainbow trout, Onchorhynchus mykiss (Kroupova et al., 2008).Exposure to NO 2 -increases methemoglobin, a form of hemoglobin incapable of binding oxygen, in channel catfish (Urrutia & Tomasso, 1987), but not in rainbow trout (Kroupova et al., 2008).Long term exposure to NO 2 -provokes lamellar hyperplasia and increases plasma glucose levels in rainbow trout (Kroupova et al., 2008), but does not induce any histopathological changes in the gills of silver perch (Frances et al., 1998).Because of those variable effects on different fish species, the objective of this study was to verify the LC 50-96h for NO 2 -in silver catfish, Rhamdia quelen.As knowledge of the effect of NO 2 -on growth is scarce, and its chronic effect on metabolic parameters (glucose, glycogen, lactate) is unknown, in the present study it was also tested the effect of NO 2 -on these parameters in silver catfish.This species was used because it is one of the main native species raised in southern Brazil (Baldisserotto, 2009).As exposure to high NO 2 -levels compromises oxygen transport (Spotte, 1979;Aggergaard & Jensen, 2001), the study of the selected biochemical parameters in liver and muscle is important to analyze the metabolic behavior of the fish against internal hypoxia and its consequences to growth.

Material and Methods
Experimental animals and management conditions.Silver catfish juveniles were obtained from fish farmers near Santa Maria, southern Brazil, and transported to the Fish Physiology Laboratory at the Universidade Federal de Santa Maria (voucher specimen catalogued as UFRGS 14114 in the fish collection at Departamento de Zoologia, Universidade Federal do Rio Grande do Sul, Brazil).Fish were maintained in continuously aerated 250-L tanks for a period of 3 weeks prior to each experiment for acclimation.The experimental diet was composed mainly of yeast and soybean meal and 320 g.kg -1 crude protein, 0.17 g NaCl.kg -1 food and was prepared according to Garcia et al. (2007).Fish were fed once per day (5% of body mass).Uneaten food, as well as other residue and feces, were siphoned 30 min after feeding.The measured Na + , K + , and Cl -concentrations of the food were (mean ± SEM): 3 ± 0.1, 56 ± 4.4, and 10.8 ± 3.5 mmol.kg - , respectively.Waterborne Na + , K + , and Cl -were 21.85, 1.95 and 11.3 mg.L -1 , respectively.

-lethal concentration (LC 50-96h
).After the acclimation period silver catfish (7.16 ± 0.27 g) were transferred to 2 L aquaria (10 fish each, 3 replicates) and were exposed for 96 h to the following waterborne NO 2 -levels (in mg.L -1 ): 0.08 ± 0.07, 5.33 ± 0.07, 10.66 ± 0.05, 21.33 ± 0.09, and 42.66 ± 0.04.These levels were maintained through the addition of sodium nitrite (NaNO 2 , Merk, 99.5% purity) and were chosen based on previous LC 50-96h experiments with neotropical species (Bianchini et al., 1996;Avilez et al., 2004;Costa et al., 2004).All feces and residues were removed daily by suction, and consequently approximately 40% of the water in the boxes was replaced by water with previously adjusted nitrite levels.Fish were observed every 12 h and removed when immobile and respiratory movements ceased.Mortality was determined after 96 h exposure.LC 50-96h was calculated by the probits method (Finney, 1971).As some fish change swimming behavior in response to hypoxia (Braun et al., 2006), swimming behavior of the juveniles was qualitatively observed through the experimental period.
Growth of silver catfish exposed to NO 2 -.After acclimation, juveniles (3.01± 0.19 g and 58.9 ± 1.3 mm) were placed in continuously aerated 40-L polypropylene tanks and kept for 40 days (10 juveniles in each tank).The fish were exposed to 4 different levels of waterborne NO 2 -(in mg.L -1 ): 0.06 (control), 0.46, 1.19, and 1.52 (0, 2.2, 5.8 and 7.4% of the LC 50-96h , respectively -values can be seen in the results) in triplicate.These values were chosen because minimum values of nitrogenous compounds that affect fish growth are in the 3-12% of LC 50-96h range (Tomasso, 1994).Fish were fed with the same diet of the acclimation, and approximately 20% of the water in the tanks was replaced by water with previously adjusted nitrite levels.Swimming (position in the water column) and alimentary (fed or not) behaviors of the juveniles were observed throughout the experimental period.Ten fish were collected at 0, 20 and 40 days for biometry.Specific growth rate (SGR) was calculated by the equation: SGR = (ln initial medium weight -ln final medium weight) x 100/ time in days.Biomass = medium weight x number of final survivors in each analyzed period.
Measurement of biochemical parameters.At the end of the exposure period (40 days), all fish were sampled, stunned with a blow to the head and then euthanized by severing the spinal cord.Procedure was run without anesthesia.Liver and muscle tissue were removed, frozen in liquid nitrogen, and then stored at -20 o C. Liver and muscle glycogen were determined according to Bidinoto et al. (1997) and tissue protein levels were determined according to Lowry et al. (1951).Tissue samples were homogenized with 10% trichloroacetic acid using a motordriven Teflon pestle and centrifuged at 1000 xg for 10 min.Deproteinated supernatant was used for the determination of lactate (Harrower & Brown, 1972) and glucose (Park & Johnson, 1949) levels.
Water quality.Water pH was monitored daily with a DMPH-2 pH meter (Digimed, São Paulo, Brazil).Total ammonia levels were verified once a week by nesslerization according to Greenberg et al. (1976) and non-ionized ammonia levels were calculated according to Piper et al. (1982).Dissolved oxygen and temperature were measured daily with a YSI oxygen meter (model Y5512 YSI Inc. Yellow Springs, USA) and laboratory temperature was maintained by an air conditioner.Levels of total alkalinity and nitrite were determined twice per day according to Boyd (1998).Water hardness was determined by the EDTA titrimetric method (Greenberg et al., 1976).Waterborne and dietary Na + and K + concentrations were measured with a Micronal B286 flame photometer (São Paulo, Brazil), and Cl -concentrations were measured according to Zall et al. (1956).
Statistical analysis.Data were reported as mean ± SEM.The relationships between NO 2 -levels with growth parameters (regression-based curve-fitting) were calculated using Sigma Plot 8.0.Homogeneity of variances among groups was tested with the Levene test.Data presented homogeneous variances, and comparisons among different treatments were made by one-way analysis of variance and Tukey test.Analysis was performed using Statistica software (version 5.1) and the minimum significance level was set at p<0.05.
The methodology of this experiment was approved by the Ethical and Animal Welfare Committee of the Universidade Federal de Santa Maria (Proc.24/2007).
Silver catfish maintained at 1.19 mg.L -1 NO 2 -presented significantly higher lactate levels in the liver than control fish and those exposed to 0.46 mg.L -1 NO 2 -.In addition, protein levels in the liver were significantly higher in fish exposed to 1.19 mg.L -1 NO 2 -compared to control fish and those exposed to 0.46 mg.L -1 NO 2 -.Glucose levels in the liver of fish exposed to 1.52 mg.L -1 NO 2 -were significantly lower than in fish exposed to 1.19 mg.L -1 NO 2 -.Glycogen levels in the liver of fish exposed to 1.19 mg.L -1 NO 2 -were significantly lower than in fish exposed to 0.46 mg.L -1 NO 2 -.Lactate levels were significantly lower in the muscle of fish exposed to all NO 2 -levels compared to control fish.Glucose content in the muscle of fish exposed to 1.52 mg.L -1 NO 2 -was significantly lower than in fish exposed to 0.49 and 1.19 mg.L -1 NO 2 -.Glycogen and protein levels in the muscle were not affected by NO 2 -exposure (Table 2).
Fish exposed to NO 2 -demonstrate decreased functional hemoglobin blood levels because hemoglobin is converted into methemoglobin (Aggergaard & Jensen, 2001).The increase of methemoglobin levels can be observed by the appearance of asphyxia symptoms (Spotte, 1979).In the present experiment, apparently silver catfish juveniles tried to minimize the effect of asphyxia by swimming near the water's surface.Similar behavior was previously reported by Braun et al. (2006) in silver catfish exposed to hypoxia and in several species exposed to NO 2 -(Parma-de-Croux, 1994; Burleson et al., 2002;Chapman et al., 2002;Delaney & Klesius, 2004).
Lactate is the end product of glycolysis in hypoxic conditions.Exposure of silver catfish to NO 2 -reduce lactate levels in muscle, but lactate levels increase in the liver of fish maintained at 1.19 mg.L -1 NO 2 -.In addition, glucose levels in the muscle and liver is significantly lower in silver catfish exposed to the highest NO 2 -level.Acute exposure (8 h) to 20-30 mg.L -1 NO 2 -also decreased glucose in the liver and white muscle and lactate in the muscle of pacu (Piaractus mesopotamicus) (Moraes et al., 2006), and glucose in the red muscle and heart of traira (Moraes et al., 1998).However, in tambacu the same treatment increased glucose and lactate in the liver and plasma (Moraes et al., 2006), and in traira increased lactate in the red muscle and plasma (Moraes et al., 1998).
The results indicate that chronic NO 2 -exposure in silver catfish causes anaerobic substrate oxidation to meet energy demand.This may be due to the transformation of functional hemoglobin to methemoglobin that may occur in fish exposed to NO 2 -.An increase of lactate content indicates metabolic disorders and may suggest severe respiratory stress (Begum & Vijayaraghavan, 1999).This is the first study to demonstrate metabolic alterations in fish provoked by chronic NO 2 exposure.The need of silver catfish exposed to chronic NO 2 to obtain at least part of its energy through the use of an anaerobic pathway might help explaining a lower growth, but in the present study a relationship with mortality could not be seen because the changes were not proportional to NO 2 levels.
Some metabolic responses are similar to those observed after acute NO 2 -exposure in other neotropical species (Moraes et al., 1998(Moraes et al., , 2006)), but additional studies must be performed to state if the differences found are species-specific or due to length of exposure.In addition, in the present study is showed that silver catfish is comparatively resistant to NO 2 -, since its LC 50-96h is 20.46 mg.L -1 .The safe NO 2 -level for silver catfish juvenile growth is below 1.19 mg.L -1 , i.e., 4.8-5.8%LC 50-96h , and higher NO 2 -levels caused mortality.

Table 1 .
than in the Effect of waterborne nitrite on weight, length, biomass and specific growth rate of silver catfish juveniles.N = 3 tanks.Values are expressed as mean ± SEM.Different letters in the rows indicate significant difference among treatments by one-way ANOVA and Tukey test (p<0.05).*= only one replicate; ** = all fish dead.

Table 2 .
Metabolic parameters of liver and muscle of silver catfish juveniles exposed to different waterborne nitrite levels for 40 days.Lactate, glycogen and glucose in μmol.g -1 .Protein in mg.g -1 .N = 10.Values are expressed as mean ± SEM.Different letters in the rows indicate significant difference among treatments by one-way ANOVA and Tukey test (p<0.05).