Acute toxicity of nitrate in Litopenaeus vannamei juveniles at low salinity levels

Different technologies have been developed to improve the performance of Litopenaeus vannamei in low salinity, mainly in super-intensive systems like recirculation and BFT (Biofloc Technology System) systems. However, there is an accumulation of toxic nitrogenous compounds to the shrimps such as nitrate, that at high concentrations and depending on the salinity of the culture water can be lethal. Acute toxicity tests allow to analyze the relationship between the compound and other abiotic or biotic variables. The aim of this research was to determine the acute toxicity and safety level of nitrate (N-NO3 -) for juveniles of L. vannamei at salinities of 5 and 10g.L-1. For salinity of 5g.L-1, a control and 5 treatments were tested, with nitrate concentrations of 100, 500, 1500, 2500 and 3500mg.L-1.For salinity of 10mg.L-1, a 4500mg.L-1nitrate concentration was added. Juveniles were exposed to concentrations during 24, 48, 72, 96 hours in static system. The Mean Lethal Concentration (LCC50) was calculated and the recommended safety level for L. vannamei cultivation is 60.05 and 127.61mg.L 1 of nitrate for salinities 5 and 10g.L-1, respectively.


INTRODUCTION
The Pacific white shrimp Litopenaeus vannamei is an euryhaline species that tolerates a wide range of salinity (0.5 to 40g.L -1 ), which allows it to be reared in low salinity and is already established in several countries, including the USA, Thailand, Ecuador and Israel (BOYD 2001, SAMOCHA et al., 2001).For this reason, studies on the minimum ionic concentrations required for its good zootechnical performance have been developed (CHONG-ROBLES et al., 2014;ROY et al., 2010) and together with super-intensive cultivation systems such as recirculation and Biofloc Technology System (BFT) provide ability to expand the activity.However, superintensive systems of aquaculture production require high feed input and stocking densities of animals, increasing the deposition of deleterious compounds of water quality and toxic to the shrimp (MUHLERT et al., 2013).Among these compounds, generation and accumulation of nitrogenous products, which are inserted into the system through the degradation of the organic matter, unconsumed food and from the excretion resulting from the metabolism of the animals, are highlighted (BIANCHINI et al., 1996;EBELING et al., 2006).
The main product generated by the protein catabolism of most aquatic organisms and the Ciência Rural, v.49, n.1, 2019.Alves Neto et al. decomposition of organic matter from over-feeding is ammonia (MAILLARD et al., 2005;REGNAULT, 1987).In water it is found in the ionized (NH 4 + ) and non-ionized (NH 3 ) forms which together form the total ammoniacal nitrogen.In systems that reuse water, this compound goes through a nitrification process, resulting in a final form of nitrate-N through nitrite oxidation (SCHULER et al., 2010).
Nitrate is considered the less toxic form among nitrogen compounds, but at high concentrations and depending on the salinity of the water, it can be lethal.The conversion of pigments responsible for the transport of oxygen, such as hemocyanin, in forms incapable of carrying out transportation (metahemocyanin) is the main toxic action of nitrate in aquatic animals, reducing the availability of oxygen for metabolism and may induce hypoxia and mortality (CHENG & CHEN, 2002;JENSEN, 1996;SCOTT &CRUNKILTON, 2000;TAHON et al., 1988).
Acute toxicity tests represent a standard method for quantifying and comparing the relative toxicities of pollutants and are defined by the concentration that kills 50% of the test population, known as the LC 50 value (APHA, 1998).The LC 50 values can be presented from 12 to 24 hours exposure to pollutant concentrations at intervals of up to 96 hours.In addition to providing comparable data for other species or pollutants, these values allow us to analyze the relationships between the compound and other abiotic or biotic variables.
Several studies have been conducted to determine the acute toxicity of nitrogenous compounds in shrimp (CAMPOS et al., 2012;GROSS et al., 2004;SCHULER et al., 2010;TSAI & CHEN, 2002;WICKINS, 1976).However, until the present study, data for low salinities is still scarce.The aim of this research was to determine the acute toxicity and safety level for nitrate (N-NO 3 -) in juveniles of Litopenaeus vannamei at salinities 5 and 10g.L -1 .

Animals and location
The study was conducted at the Shrimp Production Laboratory of Marine Aquaculture Station (Estação Marinha de Aquacultura -EMA) belonging to the Oceanography Institute of the Federal University of Rio Grande -FURG, located in the city of Rio Grande, Cassino -RS, Brazil (32º 19 'S, 52º 15 'W).Prior to the experiments, the animals were acclimated to the salinities of 5.0 and 10.0g.L -1 .L. vannamei juveniles (1.8±0.5g) were stocked in two 700L tanks in a stocking density of 700 shrimps/m³.The initial salinity of 30g.L -1 was gradually reduced, five parts per day until reaching the desired final values.Water quality parameters were maintained at suitable values for species, with the temperature controlled by thermostatic water heaters (Aquarium Heater H-606) and dissolved oxygen kept near saturation point.Water renewals were performed (50%) whenever total ammonia nitrogen reached concentrations close to 0.8mg.L - 1 .During acclimation, animals were fed 2x per day considering a feed rate of 10% of the biomass.
In the same period, toxicity pre-tests were carried out, exposing shrimp at the concentrations of 500, 1000, 2000 and 3000mg.L -1 of N-NO 3 -, in order to determine the minimum and maximum concentrations of nitrate and its effects on survival.From these data it was possible to delineate the experimental treatments.

Experimental design
Experimental units with 20 liters of useful volume were used with aeration supplied by radial blower and distributed by two air stones diffusers inside each experimental unit.Sea water was diluted with chlorinated and dechlorinated fresh water for each desired salinity.For salinity 5g.L -1 , a control (without addition of sodium nitrate) and 5 treatments were tested, with concentrations of N-NO 3 -: 100, 500, 1500, 2500 and 3500mg.L -1 .For salinity of 10g.L -1 , a control and 6 treatments in differentconcentrations of N-NO 3 -were determined: 100, 500, 1500, 2500, 3500 and 4500mg.L -1 .For both salinities, nitrate concentration was obtained through the dilution of 8. 5, 42.52, 127.57, 212.62, 297.66 and 382.70g (for 4500mg.L -1 treatment in salinity 10) of Sodium nitrate (NaNO 3 --P.A. Synth) in the water, respectively.To maintain the Na: K ratio (28: 1),0.156,0.78, 2.34, 3.9 and 5.46g of Potassium Chloride (KCl -), respectively, was added in both experiments and 7.02g in the treatment with the highest concentration of nitrate in the salinity 10g.L -1 .Treatments were completely randomized distributed.
Forty-five juveniles were stocked per treatment and monitored daily to verify mortalitydeath criteria adopted was the absence of any type of movement or reaction to mechanical stimuli when touched by a glass rod.Dead animals were immediately removed from experimental units.The exposure times at concentrations were 24, 48, 72, 96 hours in static system.During exposure times, shrimps were not fed.

Water quality
The parameters of water quality were monitored daily.Temperature and dissolved oxygen were measured with the aid of multiparameter water quality meter (YSI -ProODO).The salinity was measured using digital refractometer (Atago, PAL-UREA).A table top pHmeter (Mettler Toledo) was used to measure pH and alkalinity was measured according to the methodology proposed by APHA (1998).Concentrations of ammonia (NAT) were monitored daily and nitrate (N-NO 3 -) was measured to verify the concentrations of the compound after addition of NaNO 3 , according to methodologies proposed by UNESCO (1983) and AMINOT & CHAUSSEPIED (1983), respectively.

Statistical analysis
Water quality data were submitted to Levene's homoscedasticity and Shapiro-Wilk normality tests and after confirmation of these premises a One-Way Analysis of Variance (ANOVA) was performed to verify the significant differences in water quality parameters.When differences were reported (p<0.05),Tukey's test for comparison was applied (ZAR, 1996).
Median Lethal Concentration (LC 50 ) and confidence interval (95% CI) were calculated using the Trimmed Spearman-Karber Method software (HAMILTON et al., 1977).To calculate the safety levels (concentration of the compound that does not have an adverse effect on the organisms) for each salinity, the value of LC 50 96h was multiplied by an application factor proposed by SPRAGUE (1971) (LC 50 96h * 0.1).

RESULTS
Water quality parameters remained within the ranges considered appropriate for the species studied (LIN AND CHEN, 2003, 2001;WYK et al., 1999) and presented differences only in dissolved oxygen levels.The data are presented in tables 1 and 2.
Actual concentrations of nitrate measured were similar to the nominal concentrations, with small variations between nominal concentrations and replicates.The mean values of NO 3 -ranged from -0.39% to 2.38% of the nominal concentrations (99.61±0.83,503.15±2.77,1507.92±7.95,2504.57±26.41,3547.13±55.43 and 4606.94±104.75mg.L -1 of nitrate).To obtain compound concentrations in the experimental units, high purity reagents were used which was representative by comparing actual and nominal concentrations.The differences reported in this study for the variations between nominal and real concentrations were smaller than those described in other studies such as TSAI & CHEN (2002) and SOUCEK & DICKINSON (2012).
Figures 1 and 2 illustrate that there was mortality in the control group (without addition of nitrate) in both experiments starting from 60h.In 24 hours, the treatment with 3500mg.L -1 of nitrate reached 100% of mortality when in salinity of 5g.L -1 .For the same period and at the highest concentration, 4,500mg.L -1 , 93.33% mortality was observed in salinity 10.
Figures 3 and 4 a linear negative relationship between the LC 50 lethal concentration and exposure time was shown in two salinities, 5 and 10g.L -1 .Results of Mean Lethal Concentration (LC 50 ) as well as the confidence intervals for each salinity (95% CI) are presented in table 3. The acute toxicity of nitrate was tested for several species of crustaceans, especially for penaeids, as shown in table 4.

DISCUSSION
The material presented in this research adds to the current knowledge more data about the sensitivity of the penaeid shrimp Litopenaeus vannamei to the nitrate.Although, there are studies on acute toxicity to some aquatic organisms (CAMARGO et al., 2005;PANDEY et al., 2011;SOUCEK & DICKINSON, 2012) there is still a demand for more information of the effects on this species, especially at low salinity.In aquaculture it is important to know the limits of tolerance of the species that is aimed to be cultivated since the parameters of water quality are determinant factors for the maintenance of cultivation systems.
CAMPOS et al. ( 2012) tested LC 50 values for juveniles of Farfantepenaeus brasiliensis and their respective confidence intervals at salinity 28g.L -1 and found 912.07mg.L -1 (LC 50 -96h).Based on the application factor proposed by SPRAGUE (1971), the calculated safety level for juveniles of F. brasiliensis is 91.20mg.L -1 .TSAI & CHEN (2002) conducted an acute nitrate toxicity test in Penaeus monodon, using the same empirical application factor presented to estimate safe chronic levels and reported a value of 145mg.L -1 of nitrate in 15g.L -1 of salinity.
In the present study we reported values for salinities 5 and 10g.L -1 of 600.51 and 1276.29mg.L -1 , respectively, with safety levels 60.05 and 127.61, thus showing that L. vannamei is sensitive to nitrate; however, according to the data generated in this The data correspond to mean values of three replicates±standard deviation.Different letters on the same column indicate that means differ significantly (p <0.05).Temperature (°C); DO -Dissolved oxygen (mg.L -1 ); TAN -Total ammonia nitrogen (mg.L -1 ); Alkalinity (mg of CaCO3.L -1 ).study, it is more resistant than Brazil pink shrimp and tiger shrimp.Also, according to LC 50 it was possible to show in this study that salinity is a determining factor for nitrate toxicity, since it is directly proportional to salinity.According to TSAI & CHEN (2002), nitrate toxicity decreases with increasing salinity.KUHN et al. (2010) showed an increase in mortality of L. vannamei when submitted to the reduction of salinity in higher nitrate concentrations.
Studies by FURTADO et al. ( 2014) showed lethal effect on penaeid shrimp exposed  to concentrations above 300mg.L -1 in salinity of 24 and levels below 220mg.L -1 for salinity 11 was reported by KUHN et al. (2010).In addition, the accumulation of this nitrogen compound can cause sublethal effects on organisms, such as affecting antenna length, gill injury, hepatopancreas lesions, and other deleterious effects (FURTADO et al.

2014).
Closed aquaculture systems with little or no water exchange, such as recirculation and bioflocs systems, depend on nitrification for the conversion of ammonia nitrogen to nitrate, which  leads to the accumulation of the last one.Although less toxic, nitrate has lethal effect for organisms when in high concentrations.Some ways have been studied to remove and use this compound, such as the use of biofilters and integrated multitrophic aquaculture systems, which t is the production of food such as fish, shrimp, mollusk, combined with an organism with market value, using plants for nitrate assimilation, reducing and/or recycling this compound (RAKOCY et al., 2006;VAN RIJN, 2013).Another technology under investigation for application in aquaculture is the electrochemical treatment (MOOK et al., 2012), where the nitrate is reduced to the nitrogen gas at the cathode.

CONCLUSION
Based on the values found for LC 50 in 96h, the recommended safety level for L. vannamei cultivation is 60.05 and 127.61mg.L -1 of nitrate for the salinities of 5 and 10g.L -1 , respectively.To reuse water during several cycles of cultivation with low salinity in super-intensive systems, it is recommended the use of mechanisms and management to remove or immobilize this compound so that its toxicity does not compromise the development of the organisms cultivated, avoiding losses in the production.

Figure 3 -
Figure 3 -Linear regression of LC 50 of nitrate-N to juvenile of L. vannamei exposed to different concentrations of nitrate-N at salinity 5g.L -1 .

Table 1 -
Water quality parameters during the LC50 test for salinity 5g.L -1 under different concentrations of nitrate (N-NO3 -).

Table 2 -
Water quality parameters during the LC50 test for salinity 10g.L -1 under different concentrations of nitrate (N-NO3 -).

Table 4 -
LC50 -96h values (mg N-NO3 -.L -1 ) of nitrate for juveniles of different species of crustaceans, including results generated by the present study.