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Effects of elevated nitrate on endocrine function in Atlantic salmon, Salmo salar
Aquaculture 436 (2015) 8–12
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Effects of elevated nitrate on endocrine function in Atlantic salmon, Salmo salar Alyssa R. Freitag a, LeeAnne R. Thayer a, Christopher Leonetti b, Heather M. Stapleton b, Heather J. Hamlin a,⁎ a b
Aquaculture Research Institute, The University of Maine, School of Marine Sciences, Orono, ME 04469, USA Duke University, Nicholas School of the Environment, Durham, NC 27708, USA
a r t i c l e
i n f o
Article history: Received 26 August 2014 Received in revised form 16 October 2014 Accepted 27 October 2014 Available online 31 October 2014 Keywords: Atlantic salmon Endocrine disruption Nitrate Non-monotonic Steroids Thyroid
a b s t r a c t Recirculating aquaculture systems (RAS) have recently emerged as a sustainable alternative to traditional net pen or flow-through aquaculture. These systems reduce the environmental impact of fish production by treating and recycling culture water, and could be used in the large-scale commercial production of a variety of fish species including Atlantic salmon (Salmo salar). One major concern surrounding RAS is the natural accumulation of nitrate in the culture water. An experiment was conducted to investigate the sublethal effects of elevated nitrate on juvenile Atlantic salmon, with emphasis on thyroidal and steroidogenic endpoints. Animals were exposed to 5.2, 10.3 or 101.8 mg/L nitrate-N for 27 days. Upon completion of the trial, the animals were euthanized and bled by puncture of the caudal vein. Mean plasma nitrate/nitrite concentrations increased significantly with increasing ambient nitrate-N concentration. Plasma testosterone concentrations displayed a highly significant non-monotonic dose response to increasing nitrate-N concentration, and were elevated at 10.3 mg/L nitrate-N. Plasma 11-ketotestosterone, total thyroxine and total triiodothyronine concentrations did not differ significantly between treatments. These results suggest that elevated nitrate can interfere with the synthesis or metabolism of sex steroids, but that Atlantic salmon may be relatively insensitive, in terms of growth and most endocrine endpoints examined, to nitrate-N concentrations up to 101.8 mg/L, and are a promising candidate for production in RAS. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Recirculating aquaculture is a relatively new approach to large-scale commercial fish farming. It reduces many of the concerns surrounding traditional aquaculture, including environmental degradation and the threat of fish escapes, by raising animals through one or all life stages in a contained, land-based system (reviewed by Martins et al., 2010). Recirculating aquaculture systems (RAS) are designed to conserve water and produce little or no waste by continuously treating and reusing the culture water. While these systems are appealing to farmers and could be used to produce a variety of species, very few commercial scale RAS exist due to high start-up costs and initial time requirements. In addition, culture parameters for farming in RAS have not been optimized for many commercial fish species. One important issue surrounding RAS is the natural accumulation of substances such as minerals, orthophosphate, and nitrogenous wastes (Martins et al., 2009). Several studies have reported adverse responses by fishes grown in RAS, including decreased body size, increased egg and larval mortality, abnormal swimming behaviors and skeletal deformities (Davidson et al., 2011, 2014; Deviller et al., 2005; Martins et al., ⁎ Corresponding author. Tel.: +1 207 581 2563. E-mail address: [email protected] (H.J. Hamlin).
http://dx.doi.org/10.1016/j.aquaculture.2014.10.041 0044-8486/© 2014 Elsevier B.V. All rights reserved.
2009; van Bussel et al., 2012). It is difficult to determine whether one or several substances are responsible for these issues, but one naturally accumulating substance that has received recent attention is nitrate, NO− 3 . Within RAS, a biological filter uses oxidizing bacteria to convert ammonia (NH3) produced by the animals to nitrite (NO− 2 ), and subsequently to NO− 3 (Timmons and Ebeling, 2007). The most common method of NO− 3 removal is via water exchange, however, restrictions on water use encourage the accumulation of NO− 3 to potentially hazardous concentrations. Denitrification technology is available but is still being developed for large-scale commercial aquaculture operations (van Rijn et al., 2006). As a result, NO− 3 concentrations in RAS can reach 500 mg/L NO3-N or more (van Rijn et al., 2006). Water quality recommendations for fish culture are highly variable with regard to NO− 3 , and the toxicity of this ion has largely been ignored due to 96hour LC50 of greater than 1000 mg/L NO− 3 reported for some species (Colt, 2006; Hamlin, 2006; Westin, 1974). Recent studies, however, have shown significant sublethal effects of elevated NO− 3 in nature and intensive aquaculture. Nitrate is a known cause of methemoglobinemia, which affects both human and non-human vertebrates, and reduces the capacity of hemoglobin to bind oxygen and carry it through the body (reviewed by Guillette and Edwards, 2005). It has received recent
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attention as an endocrine disrupting chemical (EDC) due to its effect on reproductive and thyroid hormone systems (Edwards and Guillette, 2007; Edwards et al., 2006; Guillette and Edwards, 2005; Hamlin et al., 2008). Nitrate is a goitrogen and interferes with the production of thyroid hormones (THs) by competitively binding to the sodium/iodide symporter (NIS) on thyroid follicles, decreasing the availability of iodide (I −) for use in TH synthesis (De Groef et al., 2006; Lahti et al., 1985; Tonacchera et al., 2004). Female Wistar rats exposed to NO − 3 in their drinking water for 30 weeks experienced significant reductions in total triiodothyronine (TT3) at doses of 50, 250 and 500 mg/L NO− 3 (Eskiocak et al., 2005). In fishes, expo125 I) sure to elevated NO− 3 resulted in significantly lower iodine-125 ( uptake by a number of tissues including thyroid (Lahti et al., 1985). Whitespotted bamboo sharks (Chiloscyllium plagiosum) raised in a 70 mg/L NO3-N environment for 29 days did not experience a significant reduction in plasma free thyroxine (FT4), but did show signs of mild to moderate hyperplasia and hypertrophy of the thyroid gland (Morris et al., 2011). Several studies have also reported significant changes in sex steroid concentrations and other reproductive endpoints after exposure to elevated NO− 3 . Male rats exposed to 50 mg/L NaNO3 in their drinking water for four weeks experienced a highly significant reduction in circulating testosterone (T) concentration (Panesar and Chan, 2000). Conversely, female Siberian sturgeon (Acipenser baerii) grown in water with 57 mg/L NO3-N experienced a significant increase in T compared to females grown at 11.5 mg/L NO3-N. 11-Ketotestosterone (11-KT) concentrations were also significantly elevated in animals exposed to high environmental NO− 3 (Hamlin et al., 2008). Elevated NO3-N was also associated with reduced sperm count and reduced likelihood of pregnancy in male and female mosquitofish (Gambusia holbrooki), respectively (Edwards and Guillette, 2007; Edwards et al., 2006). Although the complete mechanism is still unclear, NO− 3 is thought to interfere with key steroidogenic enzymes by its conversion to nitric oxide (NO) within the body (Panesar and Chan, 2000). The present study investigates hormonal responses to elevated NO− 3 by juvenile Atlantic salmon (Salmo salar), with emphasis on androgens (11-KT and T) and THs (thyroxine (T4) and triiodothyronine (T3)). This species was selected as a model because it is a highly valued commercial fish species and a candidate for use in RAS (Martins et al., 2010). The goal of this study is to understand both the responses and the sensitivity of Atlantic salmon to elevated NO− 3 during the juvenile life stage, and to provide new information to commercial salmon farmers as they optimize RAS for this species. In addition, the findings of this project may be applied to natural systems, as NO− 3 from agricultural sources is a growing concern in coastal waterways. 2. Materials and methods 2.1. Experimental animals Juvenile, pre-smolt Atlantic salmon were obtained from Cooke Aquaculture, Inc. in Oquossoc, ME, USA and transported to the Aquaculture Research Center at the University of Maine, Orono, ME, USA. 2.2. Experimental design Three independent systems were used and each system was assigned a nominal treatment of 0 (control), 10 (moderate) or 100 (elevated) mg/L NO3-N. Each system consisted of four rearing tanks (70 L each), one header tank (150 L) and one sump tank (130 L). Within each sump tank was one cartridge filter to remove suspended solids and a mesh bag filled with polyethylene biofilter media. Animals were randomly assigned to a treatment and tank so that each tank held 17 or 18 individuals. Average initial weight ± S.E. was 102.3 ± 1.1 g. Animals were fed 0.75% initial body weight once a day,
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six times per week with a commercial pellet feed (Skretting, Tooele, Utah, USA), and uneaten food was removed daily. An artificial photoperiod of 14 h light:10 h dark was provided. The NO 3-N concentration in each system was measured daily with an ion-specific probe (Hanna Instruments, Woonsocket, RI, USA), and was maintained through the addition of sodium nitrate (NaNO 3 ) (Rocky Mountain Reagents, Golden, CO, USA) and well water. The control treatment was operated as a flow-through system, but because the incoming well water contained NO− 3 , the mean ± S.E. concentration of the control treatment was 5.2 ± 0.15 mg/L NO3-N. Concentrations in the 10 and 100 mg/L treatments averaged 10.3 ± 0.14 and 101.8 ± 1.6 mg/L NO3-N, respectively, and at least 50% of the water in these systems was replaced daily. Water temperature and dissolved oxygen content (DO) were measured twice daily with a Traceable™ portable dissolved oxygen meter (Fisher Scientific, Pittsburgh, PA, USA) and maintained at approximately 10.3 °C and 9.3 mg/L, respectively. Additional water chemistry parameters (NH3-N, NO2-N and hardness) were measured with a SMART3 colorimeter (LaMotte Company, Chestertown, Maryland, USA), and pH was measured with a pHTestr® 20 (Oakton Instruments, Vernon Hills, IL, USA). After 27 days, all animals were euthanized with MS-222 (Western Chemical, Inc., Ferndale, WA, USA) and weighed. Blood was collected by puncture of the caudal vein with a 3-mL syringe and a 21-gauge needle, and immediately transferred to lithium heparin Vacutainer™ tubes (BD, Franklin Lakes, NJ, USA). Blood was then centrifuged at 2800 ×g for 10 min. Plasma was stored at −80 °C until assayed.
2.3. Plasma nitrate measurements Plasma samples were thawed on ice and filtered using 10 kDa centrifugal filter columns (Merck KGaA, Darmstadt, Germany). Plasma − NO− 3 + NO2 was quantified according to the instructions provided with a commercial colorimetric assay kit (Cayman Chemical, Ann Arbor, MI, USA). Samples were run in duplicate, and to remain within detection limits of the assay, 5 μl of plasma was assayed from animals from the elevated NO− 3 treatment, and 40 μl of plasma was used from animals from the remaining treatments.
2.4. Plasma hormone measurements To quantify steroid hormone concentrations, plasma was extracted twice with diethyl ether, dried under air and reconstituted in phosphate buffered saline (PBS), pH 7.0 (T), or buffer provided with the commercial enzyme immunoassay kit (11-KT). For T, samples were concentrated by reconstituting in 77% of the initial plasma volume and for 11-KT, samples were diluted 1:8. All samples and standards were run in duplicate. Plasma T was quantified by solid-phase radioimmunoassay (previously validated for Atlantic salmon in our lab) using 96-well Protein A coated FlashPlate® PLUS microplates (PerkinElmer, Waltham, MA, USA). Antibody (Fitzgerald Industries, Acton, MA, USA, Cat #20-TR05T), samples, standards and radiolabel were prepared and added to the plate as previously described (Hamlin et al., 2011). Standards ranged from 0 to 800 pg mL−1 and 3H-Testosterone (PerkinElmer) was added to the plate at approximately 12,500 cpm per 100 μl. Plates were read using a MicroBeta2 Plate Counter (PerkinElmer). Average interassay variation was 34.5%. Plasma 11-KT concentrations were measured according to the instructions included with a commercial enzyme immunoassay (EIA) kit (Cayman Chemical). Average interassay variation was 17.4%. Total T4 (TT4) and total T3 (TT3) were extracted and measured in plasma by liquid chromatography tandem mass spectrometry (LC/MS/ MS) according to previously published methods (Noyes et al., 2014).
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2.5. Statistics
3.4. Plasma hormones
Water quality parameters and average weight gain per system were − compared with a one-way ANOVA. Plasma NO− 3 + NO2 and plasma hormone concentrations were compared using a Restricted Maximum Likelihood (REML) model with tank as a random effect. Significant effects (p b 0.05) were further analyzed using Tukey's HSD. In all analyses besides water quality, tanks were used as replicates, and raw data were log10 transformed when necessary to meet assumptions of normality and homogeneity of variance. All statistical analyses were conducted using JMP 10.0.0. (SAS Institute, Inc., Cary, NC, USA).
There was no significant difference in mean plasma T concentration between males and females (p = 0.1854). Animals exposed to 10.3 mg/L NO3-N had significantly greater plasma T concentrations than animals exposed to 5.2 and 101.8 mg/L NO3-N (p = 0.0073) (Fig. 2). Mean plasma T concentrations were not significantly different between animals exposed to 5.2 and 101.8 mg/L NO3-N (Fig. 2). The variation between tanks explained only 20% of the total variation. Mean ± S.E. plasma 11-KT concentrations differed significantly for males (318.5 ± 20.6 pg/mL) and females (26.6 ± 1.5 pg/mL) (p b 0.0001) and were analyzed separately. There were no significant differences in plasma 11-KT concentrations between NO 3-N treatments for males (p = 0.2768) or females (p = 0.3476). Variation between tanks explained 0% of the total variation for both sexes. There was no significant difference in plasma TT4 concentration between males and females (p = 0.4144), nor was there an interaction between sex and treatment (p = 0.6799). There was no significant difference in TT4 concentration between treatments (p = 0.3053) (Fig. 3). Similarly, there was no significant difference in plasma TT3 between males and females (p = 0.2745), and no interaction between sex and treatment (p = 0.9739). There was no statistically significant difference in plasma TT3 between NO3-N treatments (p = 0.3431) (Fig. 3). For TT4 and TT3, random variation between tanks explained 0 and 14.5% of the total variation, respectively.
3. Results 3.1. Water quality Maximum concentrations of ammonia-N (NH3-N unionized) and nitrite-N (NO2-N) were maintained within acceptable limits for Atlantic salmon (Timmons and Ebeling, 2007; Wolters et al., 2009). Maximum ± S.E. nitrite-N (NO2-N) concentrations for the control, moderate and elevated nitrate treatments were 0.02 ± 0.003 mg/L, 0.03 ± 0.003 mg/L and 0.14 ± 0.008 mg/L, respectively. It should be noted that the maximum concentration of NO2-N in the elevated NO3-N treatment (101.8 mg/L) was significantly greater (p b 0.05) than that in the control and moderate NO3-N treatments. Mean ± S.E. pH and total hardness for all systems were 7.98 ± 0.05 and 276 ± 1.8 mg/L CaCO3, respectively. 3.2. Average weight gain After 27 days, there were no significant differences in average weight gain (n = 4, p = 0.1895) between systems. Mean ± S.E. weight gain per individual for all treatments was 43.3 ± 0.8 g. − 3.3. Plasma NO− 3 + NO2
Increasing NO3-N concentration induced a highly significant, dose− dependent increase in plasma NO− 3 + NO2 (p b 0.0001) (Fig. 1). Mean − − plasma NO3 + NO2 in animals exposed to 101.8 mg/L NO3-N was more than 23 times that of animals exposed to the control treatment of 5.2 mg/L NO3-N, and more than 14 times that of animals exposed to 10.3 mg/L NO3-N. According to REML variance component estimates, tank variation explained only 7% of the total variation, and it was concluded that there were no significant tank effects on the response. 350
c
4. Discussion Plasma T responded to increasing NO 3-N in a significant, nonmonotonic manner. Plasma T concentrations were significantly elevated in animals from the 10.3 mg/L NO3-N treatment compared to animals from the control (5.2 mg/L) and elevated (101.8 mg/L) NO3-N treatments. Nitrate is thought to interfere with steroidogenesis by its reduction to nitric oxide (NO), particularly within the gut (reviewed by Jensen, 2003). While most studies report an inhibition of steroidogenesis by NO− 3 and NO (Del Punta et al., 1996; nee Pathak and Lal, 2008; Panesar and Chan, 2000; Pomerantz and Pitelka, 1998), recent work on female Siberian sturgeon showed a significant elevation in plasma T after exposure to 57 mg/L NO3-N for 30 days (Hamlin et al., 2008). Additionally, a biphasic response to administration of NO-donors was demonstrated in rat Leydig cells. At low concentrations, the NO-donors caused a significant increase in T secretion, and at higher doses, an inhibitory effect of NO was apparent (Valenti et al., 1999). There are a number of potential explanations for the non-monotonic dose response presented in the current study. First, elevated plasma T
250 200 150 100 50 0
b
60
Plasma testosterone (pg/mL)
PlasmaNO3-+ NO2-(µ µM)
300
a
b
5.2
10.3
101.8
NO3-N concentration (mg/L) Fig. 1. Mean ± 1 S.E. plasma nitrate + nitrite concentrations of juvenile Atlantic salmon exposed to 5.2, 10.3 or 101.8 mg/L NO3-N for 27 days (n = 4). Different letters indicate statistically significant differences between means (p b 0.0001).
50 40 30
a
a
20 10 0
5.2
10.3
101.8
NO3-N concentration (mg/L) Fig. 2. Mean ± 1 S.E. plasma testosterone (T) concentrations in juvenile Atlantic salmon exposed to 5.2, 10.3 or 101.8 mg/L NO3-N for 27 days (n = 4). Means with the same letter are not significantly different (p N 0.05).
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4.5
Plasma TT4and TT3(ng/mL)
4.0
TT4
TT3
3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0
5.2
10.3
101.8
NO3-N concentration (mg/L) Fig. 3. Mean ± 1 S.E. plasma total thyroxine (TT4) and total triiodothyronine (TT3) concentrations in juvenile Atlantic salmon exposed to 5.2, 10.3 or 101.8 mg/L NO3-N for 27 days (n = 3 or 4).
concentrations elicited at 10.3 mg/L NO3-N may represent a physiological response by Atlantic salmon, whereas the reduction in plasma T at 101.8 mg/L NO3-N represents a toxicological response. Moderate concentrations of NO− 3 could induce an up-regulation of steroidogenic activity while elevated concentrations of NO− 3 may inhibit steroid hormone synthesis. In the latter scenario, elevated NO-3 may result in increased NO within the gonadal tissue. Nitric oxide binds to the heme group on several key steroidogenic enzymes, including the cholesterol side-chain cleavage enzyme (P450scc) and P450 17α-hydroxylase, inhibiting their activity and subsequent steroid production (Del Punta et al., 1996; Drewett et al., 2002; Pomerantz and Pitelka, 1998). Conversely, NO could inhibit aromatase, another P450 enzyme, at moderate NO− 3 concentrations, thus reducing the amount of T that is converted to estradiol (E2). Secondly, NO could interfere with liver clearance of T at 10.3 mg/L NO3-N, as similar cytochrome P450 enzymes play a role in hepatic function (reviewed by Morgan et al., 2001). It is also possible that the plasma T concentration in animals exposed to 101.8 mg/L NO3-N initially increased, but by the end of the experiment, plasma T had returned to concentrations more similar to control animals. It would be interesting to measure plasma T at different time points during the exposure to NO− 3 to determine if there is an interaction between time and dose. Plasma 11-KT concentrations differed significantly between males and females in the present study. Similar results were found in a number of salmonids, including rainbow trout (Oncorhynchus mykiss) and coho salmon (Oncorhynchus kisutch) (reviewed by Borg, 1994). 11Ketotestosterone is considered to be the major androgen in teleost fishes and is involved in the development of secondary sex characteristics in males (reviewed by Borg, 1994). Circulating concentrations of 11-KT did not differ significantly between NO3-N treatments for males or females, suggesting that the interaction between 11-KT and elevated NO− 3 differs from the interaction between T and NO− 3 . In duplicated experiments on female Siberian sturgeon, plasma 11-KT concentrations were significantly elevated after exposure to 57 mg/L NO3-N in the first study, but were not in the second. Thus, the effect of NO− 3 on 11-KT synthesis may be controlled by a number of factors including age, time of year or reproductive state (Hamlin et al., 2008). Plasma TH concentrations were not significantly affected by elevated NO− 3 . Similar results were reported in whitespotted bamboo sharks exposed to 70 mg/L NO3-N for 29 days. Despite changes in thyroid morphology that were suggestive of impaired thyroid function, plasma T4 concentrations did not differ significantly between the beginning and end of the exposure (Morris et al., 2011). In zebrafish (Danio rerio), exposure to perchlorate, another NIS inhibitor, caused clear changes in thyroid histology, but no significant reduction in T4 concentration
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(Mukhi et al., 2005). In contrast, female Wistar rats exposed to various concentrations of NO− 3 in their drinking water for 30 weeks showed depressed TH concentrations at exposures as low as 50 mg/L NO− 3 (Eskiocak et al., 2005). It is possible that a 27-day exposure to elevated NO3-N was not sufficient to elicit a response in TT4 or TT3 in the current study. Because T4 can be stored within the colloid of thyroid follicles, a reduction in plasma TH may not be manifested until those stores are depleted (Mukhi et al., 2005). More sensitive measures of thyroid function, including histological analyses of follicle structure and immunostaining for T4 within the colloid, have been suggested (Mukhi et al., 2005). While histological endpoints were not included in the present study, recent work on nitrate exposure during embryonic development suggests that early thyroid tissue development in Atlantic salmon is not impaired at concentrations up to 93 mg/L NO3-N (unpublished data). Water chemistry parameters were maintained within acceptable limits for Atlantic salmon culture (Timmons and Ebeling, 2007; Wolters et al., 2009). While the maximum concentration of NO2-N was significantly greater in the 101.8 mg/L NO3-N treatment than in the control and moderate NO3-N treatments, it was comparable to NO2-N concentrations observed in RAS (Timmons and Ebeling, 2007; van Bussel et al., 2012; Wolters et al., 2009). A study on elevated NO− 3 in RAS found significantly elevated concentrations of NO2-N in the highest NO3-N treatment, and explained that NO− 3 was likely converted back to NO− 2 by passive denitrification (Davidson et al., 2014). There was no significant effect of elevated NO3-N on average weight gain after 27 days. A similar result was reported in juvenile rainbow trout grown in RAS with 30 and 91 mg/L NO3-N for three months (Davidson et al., 2014). In contrast, the average final weights of juvenile turbot (Scophthalmus maximus) exposed to 125, 250 and 500 mg/L NO3-N in RAS for six weeks were significantly reduced compared to control animals. In the same study, elevated NO− 3 caused a significant increase in food conversion ratio (FCR) and reduction in specific growth rate (SGR) (van Bussel et al., 2012). A significant reduction in weight or body size in response to a stressor may indicate that energy is being used in detoxification rather than growth. Because elevated NO− 3 did not affect average weight gain in the present study, it may be speculated that animals grown in even the highest NO3-N treatment were not using excess energy to compensate for adverse physiological effects such as methemoglobinemia (van Bussel et al., 2012). − Plasma NO− 3 + NO2 increased significantly with increasing NO3-N exposure. Assuming that plasma NO− 2 concentrations were negligible due to low ambient concentrations, the average plasma NO− 3 in the highest NO3-N treatment was approximately 297 μM or 18.4 mg/L NO− 3 (equivalent to approximately 4.2 mg/L NO3-N). This is well below the ambient NO3-N concentration of 101.8 mg/L. Numerous studies have reported that branchial permeability to NO− 3 is relatively low compared to that of NO− 2 (Jensen, 1996; Stormer et al., 1996; reviewed by Camargo et al., 2005). In general, circulating concentrations − of NO− 3 remains below ambient concentrations whereas NO2 is concentrated within the blood (Jensen, 1996; Stormer et al., 1996). Nitrite appears to compete with chloride (Cl−) at the Cl−/HCO− 3 exchanger that facilitates ion uptake at the gills, while NO− 3 uptake is likely passive (Jensen, 1996; Stormer et al., 1996; reviewed by Jensen, 2003). This is a reasonable explanation as to why NO− 2 is toxic to aquatic animals at low concentrations, and why many species can tolerate very high concentrations of NO− 3 in their environment. In conclusion, juvenile Atlantic salmon exposed to increasing concentrations of NO3-N for 27 days did not demonstrate significant reductions in average weight gain, or significant changes in plasma 11-KT, TT4 or TT3 concentrations. There was a significant non-monotonic dose response to increasing NO3-N by T. Interestingly, T was significantly elevated at 10 mg/L NO3-N, which is the current maximum contaminant level set for drinking water by the U.S. Environmental Protection Agency (EPA, 2012). It would be worthwhile to repeat this study with a longer exposure time, and to look at a variety of additional endpoints including mRNA expression of steroidogenic enzymes and colloidal T4 stores. The
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preliminary results of this study suggest that Atlantic salmon is a promising candidate for production in RAS because it is relatively unaffected by elevated nitrate. However, the long-term effects of elevated NO− 3 on this species remain unclear, and a similar experiment should be conducted to understand the effects of NO− 3 in a commercial scale RAS. In addition, the sensitivity of Atlantic salmon to elevated NO− 3 should be evaluated at other life stages to determine how chronic exposure might impact growth, reproductive success and harvest quality. Acknowledgments We extend gratitude to Cooke Aquaculture, Inc. for supplying the experimental animals. We thank Scarlett Tudor, Sarah Barker, Deborah Bouchard, Kevin Neves, Rachelle Mason and Ashley Donor for their assistance in system maintenance and sample collection, and William Halteman for his assistance in statistical analysis. Maine Agricultural and Forest Experiment Station publication #3391. References Borg, B., 1994. Androgens in teleost fishes. Comp. Biochem. Physiol. C Pharmacol. Toxicol. Endocrinol. 109, 219–245. Camargo, J.A., Alonso, A., Salamanca, A., 2005. Nitrate toxicity to aquatic animals: a review with new data for freshwater invertebrates. Chemosphere 58, 1255–1267. Colt, J., 2006. Water quality requirements for reuse systems. Aquac. Eng. 34, 143–156. Davidson, J., Good, C., Welsh, C., Summerfelt, S.T., 2011. Abnormal swimming behavior and increased deformities in rainbow trout Oncorhynchus mykiss cultured in low exchange water recirculating aquaculture systems. Aquac. Eng. 45, 109–117. Davidson, J., Good, C., Welsh, C., Summerfelt, S.T., 2014. Comparing the effects of high vs. low nitrate on the health, performance, and welfare of juvenile rainbow trout Oncorhynchus mykiss within water recirculating aquaculture systems. Aquac. Eng. 59, 30–40. De Groef, B., Decallonne, B.R., Van der Geyten, S., Darras, V.M., Bouillon, R., 2006. Perchlorate versus other environmental sodium/iodide symporter inhibitors: potential thyroid-related health effects. Eur. J. Endocrinol. 155, 17–25. Del Punta, K., Charreau, E.H., Pignataro, O.P., 1996. Nitric oxide inhibits Leydig cell steroidogenesis. Endocrinology 137, 5337–5343. Deviller, G., Palluel, O., Aliaume, C., Asanthi, H., Sanchez, W., Franco Nava, M.A., Blancheton, J.P., Casellas, C., 2005. Impact assessment of various rearing systems on fish health using multibiomarker response and metal accumulation. Ecotoxicol. Environ. Saf. 61, 89–97. Drewett, J.G., Adams-Hays, R.L., Ho, B.Y., Hegge, D.J., 2002. Nitric oxide potently inhibits the rate-limiting enzymatic step in steroidogenesis. Mol. Cell. Endocrinol. 194, 39–50. Edwards, T.M., Guillette, L.J., 2007. Reproductive characteristics of male mosquitofish (Gambusia holbrooki) from nitrate-contaminated springs in Florida. Aquat. Toxicol. 85, 40–47. Edwards, T.M., Miller, H.D., Guillette, L.J., 2006. Water quality influences reproduction in female mosquitofish (Gambusia holbrooki) from eight Florida springs. Environ. Health Perspect. 114, 69–75. Environmental Protection Agency (US EPA), 2012. 2012 Edition of the Drinking Water Standards and Health Advisories, (Washington, DC). Eskiocak, S., Dundar, C., Basoglu, T., Altaner, S., 2005. The effects of taking chronic nitrate by drinking water on thyroid functions and morphology. Clin. Exp. Med. 5, 66–71. Guillette, L.J., Edwards, T.M., 2005. Is nitrate an ecologically relevant endocrine disruptor in vertebrates? Integr. Comp. Biol. 45, 19–27. Hamlin, H.J., 2006. Nitrate toxicity in Siberian sturgeon (Acipenser baerii). Aquaculture 253, 688–693.
Hamlin, H.J., Moore, B.C., Edwards, T.M., Larkin, I.L.V., Boggs, A., High, W.J., Main, K.L., Guillette, L.J., 2008. Nitrate-induced elevations in circulating sex steroid concentrations in female Siberian sturgeon (Acipenser baerii) in commercial aquaculture. Aquaculture 281, 118–125. Hamlin, H.J., Milnes, M.R., Beaulaton, C.M., Albergotti, L.C., Guillette, L.J., 2011. Gonadal stage and sex steroid correlations in Siberian sturgeon, Acipenser baerii, habituated to a semitropical environment. J. World Aquacult. Soc. 42, 313–320. Jensen, F.B., 1996. Uptake, elimination and effects of nitrite and nitrate in freshwater crayfish (Astacus astacus). Aquat. Toxicol. 34, 95–104. Jensen, F.B., 2003. Nitrite disrupts multiple physiological functions in aquatic animals. Comp. Biochem. Physiol. A Mol. Integr. Physiol. 135, 9–24. Lahti, E., Harri, M., Lindqvist, O.V., 1985. Uptake and distribution of radioiodine, and the effect of ambient nitrate, in some fish species. Comp. Biochem. Physiol. A Physiol. 80, 337–342. Martins, C.I.M., Pistrin, M.G., Ende, S.S.W., Eding, E.H., Verreth, J.A.J., 2009. The accumulation of substances in Recirculating Aquaculture Systems (RAS) affects embryonic and larval development in common carp Cyprinus carpio. Aquaculture 291, 65–73. Martins, C.I.M., Eding, E.H., Verdegem, M.C.J., Heinsbroek, L.T.N., Schneider, O., Blancheton, J.P., d'Orbcastel, E.R., Verreth, J.A.J., 2010. New developments in recirculating aquaculture systems in Europe: a perspective on environmental sustainability. Aquac. Eng. 43, 83–93. Morgan, E.T., Ullrich, V., Daiber, A., Schmidt, P., Takaya, N., Shoun, H., McGiff, J.C., Oyekan, A., Hanke, C.J., Campbell, W.B., Park, C.S., Kang, J.S., Yi, H.G., Cha, Y.N., Mansuy, D., Boucher, J.L., 2001. Cytochromes P450 and flavin monooxygenases — targets and sources of nitric oxide. Drug Metab. Dispos. 29, 1366–1376. Morris, A.L., Hamlin, H.J., Francis-Floyd, R., Sheppard, B.J., Guillette, L.J., 2011. Nitrateinduced goiter in captive whitespotted bamboo sharks Chiloscyllium plagiosum. J. Aquat. Anim. Health 23, 92–99. Mukhi, S., Carr, J.A., Anderson, T.A., Patino, R., 2005. Novel biomarkers of perchlorate exposure in zebrafish. Environ. Toxicol. Chem. 24, 1107–1115. nee Pathak, N.D., Lal, B., 2008. Nitric oxide: an autocrine regulator of Leydig cell steroidogenesis in the Asian catfish, Clarias batrachus. Gen. Comp. Endocrinol. 158, 161–167. Noyes, P.D., Lema, S.C., Roberts, S.C., Cooper, E.M., Stapleton, H.M., 2014. Rapid method for the measurement of circulating thyroid hormones in low volumes of teleost fish plasma by LC-ESI/MS/MS. Anal. Bioanal. Chem. 406, 715–726. Panesar, N.S., Chan, K.W., 2000. Decreased steroid hormone synthesis from inorganic nitrite and nitrate: studies in vitro and in vivo. Toxicol. Appl. Pharmacol. 169, 222–230. Pomerantz, D.K., Pitelka, V., 1998. Nitric oxide is a mediator of the inhibitory effect of activated macrophages on production of androgen by the Leydig cell of the mouse. Endocrinology 139, 922–931. Stormer, J., Jensen, F.B., Rankin, J.C., 1996. Uptake of nitrite, nitrate, and bromide in rainbow trout, Oncorhynchus mykiss: effects on ionic balance. Can. J. Fish. Aquat. Sci. 53, 1943–1950. Timmons, M.B., Ebeling, J.M., 2007. Recirculating Aquaculture, 2nd ed. Cayuga Aqua Ventures, LLC, Ithaca. Tonacchera, M., Pinchera, A., Dimida, A., Ferrarini, E., Agretti, P., Vitti, P., Santini, F., Crump, K., Gibbs, J., 2004. Relative potencies and additivity of perchlorate, thiocyanate, nitrate, and iodide on the inhibition of radioactive iodide uptake by the human sodium iodide symporter. Thyroid 14, 1012–1019. Valenti, S., Cuttica, C.M., Fazzuoli, L., Giordano, G., Giusti, M., 1999. Biphasic effect of nitric oxide on testosterone and cyclic GMP production by purified rat Leydig cells cultured in vitro. Int. J. Androl. 22, 336–341. van Bussel, C.G.J., Schroeder, J.P., Wuertz, S., Schulz, C., 2012. The chronic effect of nitrate on production performance and health status of juvenile turbot (Psetta maxima). Aquaculture 326, 163–167. van Rijn, J., Tal, Y., Schreier, H.J., 2006. Denitrification in recirculating systems: theory and applications. Aquac. Eng. 34, 364–376. Westin, D.T., 1974. Nitrate and nitrite toxicity to salmonid fishes. Prog. Fish Cult. 36, 86–89. Wolters, W., Masters, A., Vinci, B., Summerfelt, S., 2009. Design, loading, and water quality in recirculating systems for Atlantic Salmon (Salmo salar) at the USDA ARS National Cold Water Marine Aquaculture Center (Franklin, Maine). Aquac. Eng. 41, 60–70.
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Effects of elevated nitrate on endocrine function in Atlantic salmon, Salmo salar Alyssa R. Freitag a, LeeAnne R. Thayer a, Christopher Leonetti b, Heather M. Stapleton b, Heather J. Hamlin a,⁎ a b
Aquaculture Research Institute, The University of Maine, School of Marine Sciences, Orono, ME 04469, USA Duke University, Nicholas School of the Environment, Durham, NC 27708, USA
a r t i c l e
i n f o
Article history: Received 26 August 2014 Received in revised form 16 October 2014 Accepted 27 October 2014 Available online 31 October 2014 Keywords: Atlantic salmon Endocrine disruption Nitrate Non-monotonic Steroids Thyroid
a b s t r a c t Recirculating aquaculture systems (RAS) have recently emerged as a sustainable alternative to traditional net pen or flow-through aquaculture. These systems reduce the environmental impact of fish production by treating and recycling culture water, and could be used in the large-scale commercial production of a variety of fish species including Atlantic salmon (Salmo salar). One major concern surrounding RAS is the natural accumulation of nitrate in the culture water. An experiment was conducted to investigate the sublethal effects of elevated nitrate on juvenile Atlantic salmon, with emphasis on thyroidal and steroidogenic endpoints. Animals were exposed to 5.2, 10.3 or 101.8 mg/L nitrate-N for 27 days. Upon completion of the trial, the animals were euthanized and bled by puncture of the caudal vein. Mean plasma nitrate/nitrite concentrations increased significantly with increasing ambient nitrate-N concentration. Plasma testosterone concentrations displayed a highly significant non-monotonic dose response to increasing nitrate-N concentration, and were elevated at 10.3 mg/L nitrate-N. Plasma 11-ketotestosterone, total thyroxine and total triiodothyronine concentrations did not differ significantly between treatments. These results suggest that elevated nitrate can interfere with the synthesis or metabolism of sex steroids, but that Atlantic salmon may be relatively insensitive, in terms of growth and most endocrine endpoints examined, to nitrate-N concentrations up to 101.8 mg/L, and are a promising candidate for production in RAS. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Recirculating aquaculture is a relatively new approach to large-scale commercial fish farming. It reduces many of the concerns surrounding traditional aquaculture, including environmental degradation and the threat of fish escapes, by raising animals through one or all life stages in a contained, land-based system (reviewed by Martins et al., 2010). Recirculating aquaculture systems (RAS) are designed to conserve water and produce little or no waste by continuously treating and reusing the culture water. While these systems are appealing to farmers and could be used to produce a variety of species, very few commercial scale RAS exist due to high start-up costs and initial time requirements. In addition, culture parameters for farming in RAS have not been optimized for many commercial fish species. One important issue surrounding RAS is the natural accumulation of substances such as minerals, orthophosphate, and nitrogenous wastes (Martins et al., 2009). Several studies have reported adverse responses by fishes grown in RAS, including decreased body size, increased egg and larval mortality, abnormal swimming behaviors and skeletal deformities (Davidson et al., 2011, 2014; Deviller et al., 2005; Martins et al., ⁎ Corresponding author. Tel.: +1 207 581 2563. E-mail address: [email protected] (H.J. Hamlin).
http://dx.doi.org/10.1016/j.aquaculture.2014.10.041 0044-8486/© 2014 Elsevier B.V. All rights reserved.
2009; van Bussel et al., 2012). It is difficult to determine whether one or several substances are responsible for these issues, but one naturally accumulating substance that has received recent attention is nitrate, NO− 3 . Within RAS, a biological filter uses oxidizing bacteria to convert ammonia (NH3) produced by the animals to nitrite (NO− 2 ), and subsequently to NO− 3 (Timmons and Ebeling, 2007). The most common method of NO− 3 removal is via water exchange, however, restrictions on water use encourage the accumulation of NO− 3 to potentially hazardous concentrations. Denitrification technology is available but is still being developed for large-scale commercial aquaculture operations (van Rijn et al., 2006). As a result, NO− 3 concentrations in RAS can reach 500 mg/L NO3-N or more (van Rijn et al., 2006). Water quality recommendations for fish culture are highly variable with regard to NO− 3 , and the toxicity of this ion has largely been ignored due to 96hour LC50 of greater than 1000 mg/L NO− 3 reported for some species (Colt, 2006; Hamlin, 2006; Westin, 1974). Recent studies, however, have shown significant sublethal effects of elevated NO− 3 in nature and intensive aquaculture. Nitrate is a known cause of methemoglobinemia, which affects both human and non-human vertebrates, and reduces the capacity of hemoglobin to bind oxygen and carry it through the body (reviewed by Guillette and Edwards, 2005). It has received recent
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attention as an endocrine disrupting chemical (EDC) due to its effect on reproductive and thyroid hormone systems (Edwards and Guillette, 2007; Edwards et al., 2006; Guillette and Edwards, 2005; Hamlin et al., 2008). Nitrate is a goitrogen and interferes with the production of thyroid hormones (THs) by competitively binding to the sodium/iodide symporter (NIS) on thyroid follicles, decreasing the availability of iodide (I −) for use in TH synthesis (De Groef et al., 2006; Lahti et al., 1985; Tonacchera et al., 2004). Female Wistar rats exposed to NO − 3 in their drinking water for 30 weeks experienced significant reductions in total triiodothyronine (TT3) at doses of 50, 250 and 500 mg/L NO− 3 (Eskiocak et al., 2005). In fishes, expo125 I) sure to elevated NO− 3 resulted in significantly lower iodine-125 ( uptake by a number of tissues including thyroid (Lahti et al., 1985). Whitespotted bamboo sharks (Chiloscyllium plagiosum) raised in a 70 mg/L NO3-N environment for 29 days did not experience a significant reduction in plasma free thyroxine (FT4), but did show signs of mild to moderate hyperplasia and hypertrophy of the thyroid gland (Morris et al., 2011). Several studies have also reported significant changes in sex steroid concentrations and other reproductive endpoints after exposure to elevated NO− 3 . Male rats exposed to 50 mg/L NaNO3 in their drinking water for four weeks experienced a highly significant reduction in circulating testosterone (T) concentration (Panesar and Chan, 2000). Conversely, female Siberian sturgeon (Acipenser baerii) grown in water with 57 mg/L NO3-N experienced a significant increase in T compared to females grown at 11.5 mg/L NO3-N. 11-Ketotestosterone (11-KT) concentrations were also significantly elevated in animals exposed to high environmental NO− 3 (Hamlin et al., 2008). Elevated NO3-N was also associated with reduced sperm count and reduced likelihood of pregnancy in male and female mosquitofish (Gambusia holbrooki), respectively (Edwards and Guillette, 2007; Edwards et al., 2006). Although the complete mechanism is still unclear, NO− 3 is thought to interfere with key steroidogenic enzymes by its conversion to nitric oxide (NO) within the body (Panesar and Chan, 2000). The present study investigates hormonal responses to elevated NO− 3 by juvenile Atlantic salmon (Salmo salar), with emphasis on androgens (11-KT and T) and THs (thyroxine (T4) and triiodothyronine (T3)). This species was selected as a model because it is a highly valued commercial fish species and a candidate for use in RAS (Martins et al., 2010). The goal of this study is to understand both the responses and the sensitivity of Atlantic salmon to elevated NO− 3 during the juvenile life stage, and to provide new information to commercial salmon farmers as they optimize RAS for this species. In addition, the findings of this project may be applied to natural systems, as NO− 3 from agricultural sources is a growing concern in coastal waterways. 2. Materials and methods 2.1. Experimental animals Juvenile, pre-smolt Atlantic salmon were obtained from Cooke Aquaculture, Inc. in Oquossoc, ME, USA and transported to the Aquaculture Research Center at the University of Maine, Orono, ME, USA. 2.2. Experimental design Three independent systems were used and each system was assigned a nominal treatment of 0 (control), 10 (moderate) or 100 (elevated) mg/L NO3-N. Each system consisted of four rearing tanks (70 L each), one header tank (150 L) and one sump tank (130 L). Within each sump tank was one cartridge filter to remove suspended solids and a mesh bag filled with polyethylene biofilter media. Animals were randomly assigned to a treatment and tank so that each tank held 17 or 18 individuals. Average initial weight ± S.E. was 102.3 ± 1.1 g. Animals were fed 0.75% initial body weight once a day,
9
six times per week with a commercial pellet feed (Skretting, Tooele, Utah, USA), and uneaten food was removed daily. An artificial photoperiod of 14 h light:10 h dark was provided. The NO 3-N concentration in each system was measured daily with an ion-specific probe (Hanna Instruments, Woonsocket, RI, USA), and was maintained through the addition of sodium nitrate (NaNO 3 ) (Rocky Mountain Reagents, Golden, CO, USA) and well water. The control treatment was operated as a flow-through system, but because the incoming well water contained NO− 3 , the mean ± S.E. concentration of the control treatment was 5.2 ± 0.15 mg/L NO3-N. Concentrations in the 10 and 100 mg/L treatments averaged 10.3 ± 0.14 and 101.8 ± 1.6 mg/L NO3-N, respectively, and at least 50% of the water in these systems was replaced daily. Water temperature and dissolved oxygen content (DO) were measured twice daily with a Traceable™ portable dissolved oxygen meter (Fisher Scientific, Pittsburgh, PA, USA) and maintained at approximately 10.3 °C and 9.3 mg/L, respectively. Additional water chemistry parameters (NH3-N, NO2-N and hardness) were measured with a SMART3 colorimeter (LaMotte Company, Chestertown, Maryland, USA), and pH was measured with a pHTestr® 20 (Oakton Instruments, Vernon Hills, IL, USA). After 27 days, all animals were euthanized with MS-222 (Western Chemical, Inc., Ferndale, WA, USA) and weighed. Blood was collected by puncture of the caudal vein with a 3-mL syringe and a 21-gauge needle, and immediately transferred to lithium heparin Vacutainer™ tubes (BD, Franklin Lakes, NJ, USA). Blood was then centrifuged at 2800 ×g for 10 min. Plasma was stored at −80 °C until assayed.
2.3. Plasma nitrate measurements Plasma samples were thawed on ice and filtered using 10 kDa centrifugal filter columns (Merck KGaA, Darmstadt, Germany). Plasma − NO− 3 + NO2 was quantified according to the instructions provided with a commercial colorimetric assay kit (Cayman Chemical, Ann Arbor, MI, USA). Samples were run in duplicate, and to remain within detection limits of the assay, 5 μl of plasma was assayed from animals from the elevated NO− 3 treatment, and 40 μl of plasma was used from animals from the remaining treatments.
2.4. Plasma hormone measurements To quantify steroid hormone concentrations, plasma was extracted twice with diethyl ether, dried under air and reconstituted in phosphate buffered saline (PBS), pH 7.0 (T), or buffer provided with the commercial enzyme immunoassay kit (11-KT). For T, samples were concentrated by reconstituting in 77% of the initial plasma volume and for 11-KT, samples were diluted 1:8. All samples and standards were run in duplicate. Plasma T was quantified by solid-phase radioimmunoassay (previously validated for Atlantic salmon in our lab) using 96-well Protein A coated FlashPlate® PLUS microplates (PerkinElmer, Waltham, MA, USA). Antibody (Fitzgerald Industries, Acton, MA, USA, Cat #20-TR05T), samples, standards and radiolabel were prepared and added to the plate as previously described (Hamlin et al., 2011). Standards ranged from 0 to 800 pg mL−1 and 3H-Testosterone (PerkinElmer) was added to the plate at approximately 12,500 cpm per 100 μl. Plates were read using a MicroBeta2 Plate Counter (PerkinElmer). Average interassay variation was 34.5%. Plasma 11-KT concentrations were measured according to the instructions included with a commercial enzyme immunoassay (EIA) kit (Cayman Chemical). Average interassay variation was 17.4%. Total T4 (TT4) and total T3 (TT3) were extracted and measured in plasma by liquid chromatography tandem mass spectrometry (LC/MS/ MS) according to previously published methods (Noyes et al., 2014).
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2.5. Statistics
3.4. Plasma hormones
Water quality parameters and average weight gain per system were − compared with a one-way ANOVA. Plasma NO− 3 + NO2 and plasma hormone concentrations were compared using a Restricted Maximum Likelihood (REML) model with tank as a random effect. Significant effects (p b 0.05) were further analyzed using Tukey's HSD. In all analyses besides water quality, tanks were used as replicates, and raw data were log10 transformed when necessary to meet assumptions of normality and homogeneity of variance. All statistical analyses were conducted using JMP 10.0.0. (SAS Institute, Inc., Cary, NC, USA).
There was no significant difference in mean plasma T concentration between males and females (p = 0.1854). Animals exposed to 10.3 mg/L NO3-N had significantly greater plasma T concentrations than animals exposed to 5.2 and 101.8 mg/L NO3-N (p = 0.0073) (Fig. 2). Mean plasma T concentrations were not significantly different between animals exposed to 5.2 and 101.8 mg/L NO3-N (Fig. 2). The variation between tanks explained only 20% of the total variation. Mean ± S.E. plasma 11-KT concentrations differed significantly for males (318.5 ± 20.6 pg/mL) and females (26.6 ± 1.5 pg/mL) (p b 0.0001) and were analyzed separately. There were no significant differences in plasma 11-KT concentrations between NO 3-N treatments for males (p = 0.2768) or females (p = 0.3476). Variation between tanks explained 0% of the total variation for both sexes. There was no significant difference in plasma TT4 concentration between males and females (p = 0.4144), nor was there an interaction between sex and treatment (p = 0.6799). There was no significant difference in TT4 concentration between treatments (p = 0.3053) (Fig. 3). Similarly, there was no significant difference in plasma TT3 between males and females (p = 0.2745), and no interaction between sex and treatment (p = 0.9739). There was no statistically significant difference in plasma TT3 between NO3-N treatments (p = 0.3431) (Fig. 3). For TT4 and TT3, random variation between tanks explained 0 and 14.5% of the total variation, respectively.
3. Results 3.1. Water quality Maximum concentrations of ammonia-N (NH3-N unionized) and nitrite-N (NO2-N) were maintained within acceptable limits for Atlantic salmon (Timmons and Ebeling, 2007; Wolters et al., 2009). Maximum ± S.E. nitrite-N (NO2-N) concentrations for the control, moderate and elevated nitrate treatments were 0.02 ± 0.003 mg/L, 0.03 ± 0.003 mg/L and 0.14 ± 0.008 mg/L, respectively. It should be noted that the maximum concentration of NO2-N in the elevated NO3-N treatment (101.8 mg/L) was significantly greater (p b 0.05) than that in the control and moderate NO3-N treatments. Mean ± S.E. pH and total hardness for all systems were 7.98 ± 0.05 and 276 ± 1.8 mg/L CaCO3, respectively. 3.2. Average weight gain After 27 days, there were no significant differences in average weight gain (n = 4, p = 0.1895) between systems. Mean ± S.E. weight gain per individual for all treatments was 43.3 ± 0.8 g. − 3.3. Plasma NO− 3 + NO2
Increasing NO3-N concentration induced a highly significant, dose− dependent increase in plasma NO− 3 + NO2 (p b 0.0001) (Fig. 1). Mean − − plasma NO3 + NO2 in animals exposed to 101.8 mg/L NO3-N was more than 23 times that of animals exposed to the control treatment of 5.2 mg/L NO3-N, and more than 14 times that of animals exposed to 10.3 mg/L NO3-N. According to REML variance component estimates, tank variation explained only 7% of the total variation, and it was concluded that there were no significant tank effects on the response. 350
c
4. Discussion Plasma T responded to increasing NO 3-N in a significant, nonmonotonic manner. Plasma T concentrations were significantly elevated in animals from the 10.3 mg/L NO3-N treatment compared to animals from the control (5.2 mg/L) and elevated (101.8 mg/L) NO3-N treatments. Nitrate is thought to interfere with steroidogenesis by its reduction to nitric oxide (NO), particularly within the gut (reviewed by Jensen, 2003). While most studies report an inhibition of steroidogenesis by NO− 3 and NO (Del Punta et al., 1996; nee Pathak and Lal, 2008; Panesar and Chan, 2000; Pomerantz and Pitelka, 1998), recent work on female Siberian sturgeon showed a significant elevation in plasma T after exposure to 57 mg/L NO3-N for 30 days (Hamlin et al., 2008). Additionally, a biphasic response to administration of NO-donors was demonstrated in rat Leydig cells. At low concentrations, the NO-donors caused a significant increase in T secretion, and at higher doses, an inhibitory effect of NO was apparent (Valenti et al., 1999). There are a number of potential explanations for the non-monotonic dose response presented in the current study. First, elevated plasma T
250 200 150 100 50 0
b
60
Plasma testosterone (pg/mL)
PlasmaNO3-+ NO2-(µ µM)
300
a
b
5.2
10.3
101.8
NO3-N concentration (mg/L) Fig. 1. Mean ± 1 S.E. plasma nitrate + nitrite concentrations of juvenile Atlantic salmon exposed to 5.2, 10.3 or 101.8 mg/L NO3-N for 27 days (n = 4). Different letters indicate statistically significant differences between means (p b 0.0001).
50 40 30
a
a
20 10 0
5.2
10.3
101.8
NO3-N concentration (mg/L) Fig. 2. Mean ± 1 S.E. plasma testosterone (T) concentrations in juvenile Atlantic salmon exposed to 5.2, 10.3 or 101.8 mg/L NO3-N for 27 days (n = 4). Means with the same letter are not significantly different (p N 0.05).
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4.5
Plasma TT4and TT3(ng/mL)
4.0
TT4
TT3
3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0
5.2
10.3
101.8
NO3-N concentration (mg/L) Fig. 3. Mean ± 1 S.E. plasma total thyroxine (TT4) and total triiodothyronine (TT3) concentrations in juvenile Atlantic salmon exposed to 5.2, 10.3 or 101.8 mg/L NO3-N for 27 days (n = 3 or 4).
concentrations elicited at 10.3 mg/L NO3-N may represent a physiological response by Atlantic salmon, whereas the reduction in plasma T at 101.8 mg/L NO3-N represents a toxicological response. Moderate concentrations of NO− 3 could induce an up-regulation of steroidogenic activity while elevated concentrations of NO− 3 may inhibit steroid hormone synthesis. In the latter scenario, elevated NO-3 may result in increased NO within the gonadal tissue. Nitric oxide binds to the heme group on several key steroidogenic enzymes, including the cholesterol side-chain cleavage enzyme (P450scc) and P450 17α-hydroxylase, inhibiting their activity and subsequent steroid production (Del Punta et al., 1996; Drewett et al., 2002; Pomerantz and Pitelka, 1998). Conversely, NO could inhibit aromatase, another P450 enzyme, at moderate NO− 3 concentrations, thus reducing the amount of T that is converted to estradiol (E2). Secondly, NO could interfere with liver clearance of T at 10.3 mg/L NO3-N, as similar cytochrome P450 enzymes play a role in hepatic function (reviewed by Morgan et al., 2001). It is also possible that the plasma T concentration in animals exposed to 101.8 mg/L NO3-N initially increased, but by the end of the experiment, plasma T had returned to concentrations more similar to control animals. It would be interesting to measure plasma T at different time points during the exposure to NO− 3 to determine if there is an interaction between time and dose. Plasma 11-KT concentrations differed significantly between males and females in the present study. Similar results were found in a number of salmonids, including rainbow trout (Oncorhynchus mykiss) and coho salmon (Oncorhynchus kisutch) (reviewed by Borg, 1994). 11Ketotestosterone is considered to be the major androgen in teleost fishes and is involved in the development of secondary sex characteristics in males (reviewed by Borg, 1994). Circulating concentrations of 11-KT did not differ significantly between NO3-N treatments for males or females, suggesting that the interaction between 11-KT and elevated NO− 3 differs from the interaction between T and NO− 3 . In duplicated experiments on female Siberian sturgeon, plasma 11-KT concentrations were significantly elevated after exposure to 57 mg/L NO3-N in the first study, but were not in the second. Thus, the effect of NO− 3 on 11-KT synthesis may be controlled by a number of factors including age, time of year or reproductive state (Hamlin et al., 2008). Plasma TH concentrations were not significantly affected by elevated NO− 3 . Similar results were reported in whitespotted bamboo sharks exposed to 70 mg/L NO3-N for 29 days. Despite changes in thyroid morphology that were suggestive of impaired thyroid function, plasma T4 concentrations did not differ significantly between the beginning and end of the exposure (Morris et al., 2011). In zebrafish (Danio rerio), exposure to perchlorate, another NIS inhibitor, caused clear changes in thyroid histology, but no significant reduction in T4 concentration
11
(Mukhi et al., 2005). In contrast, female Wistar rats exposed to various concentrations of NO− 3 in their drinking water for 30 weeks showed depressed TH concentrations at exposures as low as 50 mg/L NO− 3 (Eskiocak et al., 2005). It is possible that a 27-day exposure to elevated NO3-N was not sufficient to elicit a response in TT4 or TT3 in the current study. Because T4 can be stored within the colloid of thyroid follicles, a reduction in plasma TH may not be manifested until those stores are depleted (Mukhi et al., 2005). More sensitive measures of thyroid function, including histological analyses of follicle structure and immunostaining for T4 within the colloid, have been suggested (Mukhi et al., 2005). While histological endpoints were not included in the present study, recent work on nitrate exposure during embryonic development suggests that early thyroid tissue development in Atlantic salmon is not impaired at concentrations up to 93 mg/L NO3-N (unpublished data). Water chemistry parameters were maintained within acceptable limits for Atlantic salmon culture (Timmons and Ebeling, 2007; Wolters et al., 2009). While the maximum concentration of NO2-N was significantly greater in the 101.8 mg/L NO3-N treatment than in the control and moderate NO3-N treatments, it was comparable to NO2-N concentrations observed in RAS (Timmons and Ebeling, 2007; van Bussel et al., 2012; Wolters et al., 2009). A study on elevated NO− 3 in RAS found significantly elevated concentrations of NO2-N in the highest NO3-N treatment, and explained that NO− 3 was likely converted back to NO− 2 by passive denitrification (Davidson et al., 2014). There was no significant effect of elevated NO3-N on average weight gain after 27 days. A similar result was reported in juvenile rainbow trout grown in RAS with 30 and 91 mg/L NO3-N for three months (Davidson et al., 2014). In contrast, the average final weights of juvenile turbot (Scophthalmus maximus) exposed to 125, 250 and 500 mg/L NO3-N in RAS for six weeks were significantly reduced compared to control animals. In the same study, elevated NO− 3 caused a significant increase in food conversion ratio (FCR) and reduction in specific growth rate (SGR) (van Bussel et al., 2012). A significant reduction in weight or body size in response to a stressor may indicate that energy is being used in detoxification rather than growth. Because elevated NO− 3 did not affect average weight gain in the present study, it may be speculated that animals grown in even the highest NO3-N treatment were not using excess energy to compensate for adverse physiological effects such as methemoglobinemia (van Bussel et al., 2012). − Plasma NO− 3 + NO2 increased significantly with increasing NO3-N exposure. Assuming that plasma NO− 2 concentrations were negligible due to low ambient concentrations, the average plasma NO− 3 in the highest NO3-N treatment was approximately 297 μM or 18.4 mg/L NO− 3 (equivalent to approximately 4.2 mg/L NO3-N). This is well below the ambient NO3-N concentration of 101.8 mg/L. Numerous studies have reported that branchial permeability to NO− 3 is relatively low compared to that of NO− 2 (Jensen, 1996; Stormer et al., 1996; reviewed by Camargo et al., 2005). In general, circulating concentrations − of NO− 3 remains below ambient concentrations whereas NO2 is concentrated within the blood (Jensen, 1996; Stormer et al., 1996). Nitrite appears to compete with chloride (Cl−) at the Cl−/HCO− 3 exchanger that facilitates ion uptake at the gills, while NO− 3 uptake is likely passive (Jensen, 1996; Stormer et al., 1996; reviewed by Jensen, 2003). This is a reasonable explanation as to why NO− 2 is toxic to aquatic animals at low concentrations, and why many species can tolerate very high concentrations of NO− 3 in their environment. In conclusion, juvenile Atlantic salmon exposed to increasing concentrations of NO3-N for 27 days did not demonstrate significant reductions in average weight gain, or significant changes in plasma 11-KT, TT4 or TT3 concentrations. There was a significant non-monotonic dose response to increasing NO3-N by T. Interestingly, T was significantly elevated at 10 mg/L NO3-N, which is the current maximum contaminant level set for drinking water by the U.S. Environmental Protection Agency (EPA, 2012). It would be worthwhile to repeat this study with a longer exposure time, and to look at a variety of additional endpoints including mRNA expression of steroidogenic enzymes and colloidal T4 stores. The
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preliminary results of this study suggest that Atlantic salmon is a promising candidate for production in RAS because it is relatively unaffected by elevated nitrate. However, the long-term effects of elevated NO− 3 on this species remain unclear, and a similar experiment should be conducted to understand the effects of NO− 3 in a commercial scale RAS. In addition, the sensitivity of Atlantic salmon to elevated NO− 3 should be evaluated at other life stages to determine how chronic exposure might impact growth, reproductive success and harvest quality. Acknowledgments We extend gratitude to Cooke Aquaculture, Inc. for supplying the experimental animals. We thank Scarlett Tudor, Sarah Barker, Deborah Bouchard, Kevin Neves, Rachelle Mason and Ashley Donor for their assistance in system maintenance and sample collection, and William Halteman for his assistance in statistical analysis. Maine Agricultural and Forest Experiment Station publication #3391. References Borg, B., 1994. Androgens in teleost fishes. Comp. Biochem. Physiol. C Pharmacol. Toxicol. Endocrinol. 109, 219–245. Camargo, J.A., Alonso, A., Salamanca, A., 2005. Nitrate toxicity to aquatic animals: a review with new data for freshwater invertebrates. Chemosphere 58, 1255–1267. Colt, J., 2006. Water quality requirements for reuse systems. Aquac. Eng. 34, 143–156. Davidson, J., Good, C., Welsh, C., Summerfelt, S.T., 2011. Abnormal swimming behavior and increased deformities in rainbow trout Oncorhynchus mykiss cultured in low exchange water recirculating aquaculture systems. Aquac. Eng. 45, 109–117. Davidson, J., Good, C., Welsh, C., Summerfelt, S.T., 2014. Comparing the effects of high vs. low nitrate on the health, performance, and welfare of juvenile rainbow trout Oncorhynchus mykiss within water recirculating aquaculture systems. Aquac. Eng. 59, 30–40. De Groef, B., Decallonne, B.R., Van der Geyten, S., Darras, V.M., Bouillon, R., 2006. Perchlorate versus other environmental sodium/iodide symporter inhibitors: potential thyroid-related health effects. Eur. J. Endocrinol. 155, 17–25. Del Punta, K., Charreau, E.H., Pignataro, O.P., 1996. Nitric oxide inhibits Leydig cell steroidogenesis. Endocrinology 137, 5337–5343. Deviller, G., Palluel, O., Aliaume, C., Asanthi, H., Sanchez, W., Franco Nava, M.A., Blancheton, J.P., Casellas, C., 2005. Impact assessment of various rearing systems on fish health using multibiomarker response and metal accumulation. Ecotoxicol. Environ. Saf. 61, 89–97. Drewett, J.G., Adams-Hays, R.L., Ho, B.Y., Hegge, D.J., 2002. Nitric oxide potently inhibits the rate-limiting enzymatic step in steroidogenesis. Mol. Cell. Endocrinol. 194, 39–50. Edwards, T.M., Guillette, L.J., 2007. Reproductive characteristics of male mosquitofish (Gambusia holbrooki) from nitrate-contaminated springs in Florida. Aquat. Toxicol. 85, 40–47. Edwards, T.M., Miller, H.D., Guillette, L.J., 2006. Water quality influences reproduction in female mosquitofish (Gambusia holbrooki) from eight Florida springs. Environ. Health Perspect. 114, 69–75. Environmental Protection Agency (US EPA), 2012. 2012 Edition of the Drinking Water Standards and Health Advisories, (Washington, DC). Eskiocak, S., Dundar, C., Basoglu, T., Altaner, S., 2005. The effects of taking chronic nitrate by drinking water on thyroid functions and morphology. Clin. Exp. Med. 5, 66–71. Guillette, L.J., Edwards, T.M., 2005. Is nitrate an ecologically relevant endocrine disruptor in vertebrates? Integr. Comp. Biol. 45, 19–27. Hamlin, H.J., 2006. Nitrate toxicity in Siberian sturgeon (Acipenser baerii). Aquaculture 253, 688–693.
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