Investigating the influence of nitrate nitrogen on post-smolt Atlantic salmon Salmo salar reproductive physiology in freshwater recirculation aquaculture systems

Accepted Manuscript Title: Investigating the influence of nitrate nitrogen on post-smolt Atlantic salmon Salmo salar reproductive physiology in freshwater recirculation aquaculture systems Author: Christopher Good John Davidson Luke Iwanowicz Michael Meyer Julie Dietze Dana W. Kolpin David Marancik Jill Birkett Christina Williams Steven Summerfelt PII: DOI: Reference:

S0144-8609(16)30143-1 http://dx.doi.org/doi:10.1016/j.aquaeng.2016.09.003 AQUE 1864

To appear in:

Aquacultural Engineering

Received date: Accepted date:

2-9-2016 25-9-2016

Please cite this article as: Good, Christopher, Davidson, John, Iwanowicz, Luke, Meyer, Michael, Dietze, Julie, Kolpin, Dana W., Marancik, David, Birkett, Jill, Williams, Christina, Summerfelt, Steven, Investigating the influence of nitrate nitrogen on post-smolt Atlantic salmon Salmo salar reproductive physiology in freshwater recirculation aquaculture systems.Aquacultural Engineering http://dx.doi.org/10.1016/j.aquaeng.2016.09.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Investigating the influence of nitrate nitrogen on post-smolt Atlantic salmon Salmo salar reproductive physiology in freshwater recirculation aquaculture systems Christopher Good1*, John Davidson1, Luke Iwanowicz2, Michael Meyer3, Julie Dietze3, Dana W. Kolpin4, David Marancik5, Jill Birkett6, Christina Williams1, and Steven Summerfelt1 1

The Conservation Fund’s Freshwater Institute, Shepherdstown, West Virginia 25443, USA

2

United States Geological Survey, National Fish Health Laboratory, Leetown, West Virginia, USA

3

United States Geological Survey, Kansas Water Science Center, Lawrence, Kansas, USA

4

United States Geological Survey, Iowa Water Science Center, Iowa City, Iowa, USA

5

Fish Vet Group, Portland, Maine, USA

United States Department of Agriculture – Agriculture Research Service, National Center for Cool and Cold Water Aquaculture, Leetown, West Virginia, USA 6

*

Corresponding author’s contact information: [email protected] Phone: +1 304 876-2815 ext.279; Fax: +1 304 870-2208

Abstract A major issue affecting land-based, closed containment Atlantic salmon Salmo salar growout production in water recirculation aquaculture systems (RAS) is precocious male maturation, which can negatively impact factors such as feed conversion, fillet yield, and product quality. Along with other water quality parameters, elevated nitrate nitrogen (NO3-N) has been shown to influence the reproductive development and endogenous sex steroid production in a number of aquatic animal species, including Atlantic salmon. We sought to determine whether elevated NO3-N in RAS can influence early maturation in post-smolt Atlantic salmon in an 8-month trial in replicated freshwater RAS. Post-smolt Atlantic salmon (102 ± 1 g) were stocked into six RAS, with three RAS randomly selected for dosing with high NO3-N (99 ± 1 mg/L) and three RAS set for low NO3-N (10 ± 0 mg/L). At 2-, 4-, 6-, and 8-months post-stocking, 5 fish were randomly sampled from each RAS, gonadosomatic index (GSI) data were collected, and plasma was sampled for 11-ketotestosterone (11-KT) quantification. At 4- and 8-months post-stocking, samples of culture tank and spring water (used as “makeup” or replacement water) were collected and tested for a suite of 42 hormonally active compounds using liquid chromatography / mass spectrometry, as well as for estrogenicity using the bioluminescent yeast estrogen screen (BLYES) reporter system. Finally, at 8-months post-stocking 8-9 salmon were sampled from each RAS for blood gas and chemistry analyses, and multiple organ tissues were sampled for histopathology evaluation. Overall, sexually mature males were highly prevalent in both NO3-N treatment groups by study’s end, and there did not appear to be an effect of NO3-N on male maturation prevalence based on grilse identification, GSI, and 11-KT results, indicating that other culture parameters likely instigated early maturation. No important differences were noted

between treatment groups for whole blood gas and chemistry parameters, and no significant tissue changes were noted on histopathology. No hormones, hormone conjugates, or mycotoxins were detected in any water samples; phytoestrogens were generally detected at low levels but were unrelated to NO3-N treatment. Finally, low-level estrogenicity was detected in RAS water, but a NO3-N treatment effect could not be determined. The major findings of this study are i) the NO3-N treatments did not appear to be related to the observed male maturation, and ii) the majority of hormonally active compounds were not detectable in RAS water.

Keywords: Recirculation aquaculture; Atlantic salmon; nitrate nitrogen; sexual maturation; waterborne hormones

1. Introduction

The plasticity of the Atlantic salmon Salmo salar life cycle is well known (Saunders and Schom, 1985). The timing of sexual maturation in this species is highly variable and is considered to be an evolutionary adaptation to maximize overall reproductive success (Taranger et al., 2010). This variability in maturation onset can pose a significant problem to aquaculturists, given that early maturing salmon (i.e. “grilse”) exhibit decreased growth and feed conversion efficiency (McClure et al., 2007), reduced product quality (Aksnes et al., 1986), and increased susceptibility to opportunistic infections (St-Hilaire et al., 1998). The traditional salmon farming industry has adopted various strategies to reduce grilsing, including photoperiod manipulation (Bromage et al., 2001), selective breeding, (Gjedrem, 2000), and triploidy induction (Benfey, 1999). These efforts have largely been successful; however, grilsing remains a significant issue for Atlantic salmon grown to market size in land-based, closed containment operations utilizing water recirculation aquaculture system (RAS) technologies, for reasons that are presently unclear. Atlantic salmon growout trials in freshwater have demonstrated early maturation in males as high as 80% by harvest (22-26 months post-hatch, 4-5 kg average weight) (Davidson et al., 2016). Given the considerable capital investment required to design, construct and commission closed containment growout facilities, it is imperative to investigate risk factors and/or husbandry practices that are involved in instigating early maturation. By remediating grilsing, the economic viability of the growing land-based, closed containment aquaculture sector should improve significantly.

There are numerous documented and putative environmental variables that are associated with Atlantic salmon maturation (Taranger et al., 2010). Recently, the possibility of waterborne steroid hormones accumulating in RAS, and influencing the reproductive physiology of production fish, has been investigated (Good et al., 2014; Mota et al., 2014). All fish release steroid hormones into their surrounding environment, either through urine and feces in conjugated forms (Vermeirssen and Scott, 1996) or through the gills in unconjugated “free” forms (Sorensen et al., 2000; Ellis et al., 2005). Steroid hormones such as testosterone (T), 11ketotestosterone (11-KT), and estradiol (E2) have been shown to accumulate in RAS (Good et al., 2014; Mota et al., 2014), although much more research, utilizing more refined methodologies, is needed to confirm these findings and to determine the specific effects that these and other hormones have on fish in RAS. Hormonally active compounds (HACs) have also been speculated to accumulate in RAS and influence fish physiology; however, as with steroid hormones, research in the area of HACs in RAS aquaculture is still in its infancy. Nitratenitrogen (NO3-N) has been identified as an HAC of aquatic species (Hamlin et al., 2008). This could be of importance in RAS because, in the absence of denitrification unit processes, NO3-N can accumulate as the end-product of biofiltration as water reuse rates increase. Freitag et al. (2015) tested the effects of NO3-N on the endocrine function of pre-smolt Atlantic salmon, and determined that T was significantly elevated in salmon exposed to 10.3 mg/L NO3-N, compared to fish in 5.2 and 101.8 mg/L NO3-N treatment groups; however, this study was relatively short in duration (27-days) and carried out on juvenile fish. The long-term exposure to elevated NO3-N as this species grows toward market size remains an area that requires further research.

Through an 8-month study conducted in replicated RAS, we sought to investigate the influence of elevated NO3-N on Atlantic salmon health and sexual development, through qualitative morphological assessments (e.g. grilse identification), quantitative physical measurements (e.g. gonadosomatic indices), physiological parameters (e.g. plasma 11-KT), and RAS water characterization of steroid hormones, hormonally active compounds, and estrogenicity.

2. Materials and Methods

2.1. Atlantic salmon Eyed Atlantic salmon eggs (mixed sex, diploid) were purchased from a commercial producer (SalmoBreed, Bergen, Norway) and hatched in an incubation system maintained at 7.6 oC mean water temperature. Hatched fry were acclimated to 13.0 oC and transferred to a freshwater flowthrough system where fish were reared under constant (i.e., LD24:0) photoperiod until approximately 40 g in size, at which point they were exposed to a 6-week LD12:12 “winter” photoperiod prior to resumption of constant lighting. When the salmon reached approximately 80 g mean weight, they were transferred to the experimental freshwater RAS (see below). A total of 336 salmon was added to each RAS culture tank. The salmon were acclimated in the experimental RAS for a period of one month prior to study commencement, at which point they were 102 ±1 g in mean weight and were at a stocking density of 6-7 kg/m3. By study’s end, densities in the high and low NO3-N treatment groups were 60.4 and 60.5 kg/m3, respectively.

2.2. Recirculation aquaculture systems

The replicated experimental RAS utilized in this study have been previously described in detail (Davidson et al., 2009). Briefly, six identical RAS consisting of 5.3 m3 circular “Cornell-type” dual-drain tanks, fluidized sand biofilters, packed column degassers, low head oxygenators (LHOs), and drum filters (with 60 µm sieves), were used in this study. Total system water volume was 9.5 m3; recirculation flow was set at 380 L/min, with new (i.e., “makeup”) water from a spring source being added at 3.7 L/min. With this level of water reuse, the total system volume was exchanged approximately once every 1.7 days. Throughout the study, salmon were fed a standard commercial diet via automated feeders, with one feeding per hour. Feeding rates were based on industry-standard charts and regularly fine-tuned based on observations of feeding activity and wasted feed. Study fish were kept under a constant LD24:0 photoperiod, as is standard protocol at our research facility in order to maintain relatively constant water quality conditions for optimal biofiltration. Over the 8 month study period, alkalinity, carbon dioxide, dissolved oxygen, salinity, hardness, pH, and temperature averaged approximately 240 mg/L, 4 mg/L, 9.5 mg/L, <1.0 mg/L, 297 mg/L, 8.1, and 14.3 oC, respectively.

2.3. Nitrate-nitrogen treatments Three RAS were randomly selected to receive additional NO3-N, which was continuously dosed via a peristaltic pump with a stock solution of sodium nitrate (NaNO3; Tilley Chemical Company, Inc., Baltimore, Maryland, USA) to target a mean concentration of 100 mg/L NO3-N. The high NO3-N RAS were slowly brought up to target NO3-N concentrations during the first month of the study, and were then maintained at target concentrations for the remaining seven study months. The actual mean concentration in the high NO3-N RAS during these last seven months was 99 ± 1 mg/L. The remaining RAS did not receive NaNO3 and, as such, were

operated at a mean concentration of 10 ± 0 mg/L NO3-N which resulted from biofiltration. Sodium sulfate (Na2SO4) was continuously dosed to the low NO3-N systems in order to achieve comparable conductivity with the high NO3-N RAS. The replicated RAS were operated under these conditions for a period of 8 months.

2.4. Fish sampling and data collection Salmon study populations were sampled at 2-, 4-, 6-, and 8-months into the study period (corresponding to 10-, 12-, 14-, and 16-months post-hatch in fish age), with sample sizes for each RAS determined by the following formula (based on determining mean fish weight in grams): n = (Z * (stdev. g /accepted error g)) 2, where Z = 1.65 (relative to a 90% confidence interval), and with an accepted error of 5 grams (Kitchens, 1998). All sampled fish were examined qualitatively by designated personnel for signs of early male maturation, which included color changes (bronzing) and kype (hooked jaws) development that are typical phenotypic signs of sexual development in male salmon. Tallies were kept of fish identified as mature males in each RAS, and percentage male maturation was calculated for each treatment group. Additionally, during each sampling event five random fish per RAS were euthanized with an overdose (200 mg/L) of tricaine methanesulfonate (MS-222; Western Chemical, Ferndale, Washington, USA), and weight and gonad weight data were collected from each fish. The sex of sampled fish was determined based on gonad morphology, and gonadosomatic indices (GSI = gonad weight/whole fish weight*100) were calculated from fish and gonad weight data; males were identified as sexually maturing if GSI >0.1 (cut-off based on previous on-site experience assessing male salmon maturation in RAS). Whole blood was collected via caudal venipuncture using 1.5-inch 21.5-gauge needles on 3mL syringes; blood was immediately transferred to

heparinized 1.5mL centrifuge tubes, and all tubes were kept chilled prior to centrifugation for 10 minutes at 10,000 rpm. Plasma was carefully pipetted from centrifuge tubes and transferred to sterile 2mL cryovials for longer term storage at -80oC. At a later date, these frozen plasma samples were thawed and 11-KT concentrations were quantified at the USDA-ARS National Center for Cool and Cold Water Aquaculture (Leetown, WV, USA) using enzyme-immunoassay (EIA) kits (Cayman Chemicals Inc., Ann Arbor, Michigan, USA) following EIA kit instructions. Plasma samples were extracted three times with ethyl ether (Fisher Scientific, Fair Lawn, New Jersey, USA) for 11-KT measurement (Schultz et al., 2005); the intra-assay variability for 11-KT was CV 13.9%, and the inter-assay variability was CV 12.7%. For histopathology evaluation, representative samples of gill, heart, spleen, liver, kidney, skeletal muscle and skin were carefully collected from each fish and fixed in 10% neutral buffered formalin. Tissues were processed routinely, embedded in paraffin, sectioned at 5 µm, and stained with hematoxylin and eosin (H&E). Gill and skin were examined and semi-quantitatively scored based on signs of irritation; heart, spleen, liver, kidney and skeletal muscle were examined for overt signs of cellular degeneration or inflammation (Table 1). All histopathology scoring was carried out by a single veterinary pathologist who was blinded to the treatment groups origins of all tissues examined. Finally, during the 8-month sampling event whole blood samples were also analyzed using an i-Stat 1 portable analyzer (Abbott Laboratories, Abbott Park, Illinois, USA) with CG4+ and CHEM8+ cartridges. The CG4+ blood gas cartridges assessed a suite of blood parameters including pH, pCO2, pO2, HCO3, total CO2, O2 saturation, and lactate, while CHEM8+ blood chemistry cartridges provided data for whole blood sodium, potassium, chloride, calcium, glucose, hematocrit, and hemoglobin levels.

2.5 Water sampling and analyses At the 4- and 8-month study points, water from each RAS tank and from two randomly selected makeup water influent pipes was sampled for hormones, hormonally active compounds, and estrogenicity. Water samples were collected in replicate 125mL and 1L amber, baked glass bottles and care was taken to avoid environmental contamination. Field blanks and field replicates were also taken in order for quality assurance / quality control to be assessed. The water samples were shipped overnight on ice to the USGS Organic Geochemistry Research Laboratory (Lawrence, Kansas, USA) for processing prior to analysis via liquid chromatography / mass spectrometry (LC/MS) assessing a broad range of hormonally active compounds, including hormones, hormone conjugates, phytoestrogens, and mycotoxins (Table 2). All compounds had detection limits of 0.5 ng/L, with the exception of 17-α Estradiol (1.0 ng/L). Processed samples originating from the 1L bottles were sent to the USGS Leetown Science Center (Leetown, West Virginia, USA) and screened for estrogenicity using the bioluminescent yeast estrogen screen (BLYES) (Sanseverino et al., 2005) or chemiluminescent yeast nuclear receptor assays for androgens or glucocorticoids.

2.6 Statistical analyses A p-value of 0.05 was used to identify significant statistical associations. Gonadosomatic indices and 11-KT data were analyzed via ANCOVA for each sampling event, with NO3-N treatment group as the independent variable and RAS as a covariate. For i-Stat whole blood data, MANCOVA was performed for all dependent variables (i.e. blood parameters), with treatment (NO3-N) as the primary independent variable and RAS included as a covariate (to control for tank effect, capture effect, and time-of-day effect in the analysis). Using Wilk’s λ, the overall

MANCOVA model was highly significant (p=0.0071), with both treatment and tank demonstrating significant association with the panel of outcome variables (p=0.0089 and p=0.0143, respectively); therefore, a subsequent multivariate multiple regression model was used to determine treatment effects on individual parameters. All statistical procedures were performed using STATA 9 software (StataCorp, College Station, TX, USA). Group mean comparison tests were run in STATA to compare phytoestrogen levels between treatment groups, and for each water sampling event, when sufficient detectable concentrations were available to perform these tests.

3. Results

Prior to study commencement, no maturation was observed in the salmon population, based on qualitative assessment of sampled fish and quantified GSI values; histopathological lesions were also not noted in sampled fish at this time. By study’s end, grilsing (based on visual assessment) was highly prevalent in both NO3-N treatment groups, with 40% and 38% of sampled fish observed to be mature males in the low and high NO3-N groups, respectively (Figure 1). Grilse identified through GSI assessment (i.e., GSI >0.1) were likewise high in both treatments by study’s end, with 100% and 78% of sampled males categorized as maturing/mature in the low and high NO3-N groups, respectively (Table 3). Mean male gonadosomatic indices gradually increased over time in both treatment groups, with the largest increase in mean GSI occurring between the 2- and 4- month sampling events (Figure 2). Female GSI was relatively low throughout the study, although an upward trend was observed at the 8-month sampling event (Figure 2); no sexually mature females were observed among sampled fish. Similar to GSI,

plasma 11-KT in males also increased during the study period (Figure 3), although no statistical differences were determined at any sampling point between low and high NO3-N treatment groups.

Histopathologic findings were similar in all fish. Semi-quantitative scoring of gill and skin showed no grades beyond a 1 in any category for any fish. There were no appreciable changes in the heart, spleen, liver, kidney and skeletal muscle tissues examined that could be attributed to nitrate toxicity, or believed to compromise fish health.

Whole blood gas and chemistry values (Table 4) were consistent with those obtained from healthy Atlantic salmon on-site using the i-Stat 1 analyzer, and were generally within published normal ranges for freshwater salmonids (Stoskopf, 1993; Wedemeyer, 1996). Both chloride and bicarbonate were determined to be significantly higher in sampled fish from the high NO3-N treatment group, although the absolute differences between values of these parameters were small and were likely of little biological significance.

Aside from phytoestrogens, all hormonally active compounds listed in Table 2 were not detectable in RAS water from either treatment group, nor from the spring makeup water samples, during either the 4- or 8-month sampling events. Specific phytoestrogens (Table 5) were detectable in RAS water (and in the case of formonetin, in the spring makeup water during the 4month sampling event); however, no statistical associations between phytoestrogen concentrations and NO3-N treatments were detected at either sampling event.

Water estrogenicity, in estrogen equivalence (EEQ, ng/L), was only detectable in one RAS in the high NO3-N treatment group (0.44 ng/L), and detectable in two RAS in the low NO3-N group (0.24 ng/L and 0.35 ng/L), using the BLYES reporter system. No androgenicity was detectable in any processed water samples collected during the study.

4. Discussion

The first major finding of this study is that NO3-N, at the tested concentrations and exposure duration, does not appear to be a primary factor driving early male Atlantic salmon maturation in freshwater RAS, as grilsing levels were quite high in both low and high NO3-N treatment groups (100% and 78%, respectively, based on GSI data). Clearly, there were other instigators of maturation under our study conditions that impacted these populations; however, these factors remain unidentified. An assessment of NO3-N’s effects on salmon maturation in the absence of other inciting factors will be required to complete our understanding of the influence of NO3-N as salmon are grown to market size in RAS. Previous studies have demonstrated that NO3-N can disrupt endocrine function in aquatic species, although published results have been variable and more research is required on this subject. Female Siberian sturgeon Acipenser baerii exposed to 57 mg/L NO3-N demonstrated elevated plasma T, 11-KT, and E2 compared to females grown at 11.5 mg/L NO3-N (Hamlin et al., 2008). As mentioned previously, Freitag et al. (2015) exposed pre-smolt Atlantic salmon (~100g) to 5.3, 10.3, and 101.8 mg/L NO3-N for 27 days; plasma T concentrations were significantly elevated in those exposed to 10.3 mg/L NO3-N, but not in the other two exposure groups (other measured steroid hormones, including 11-KT, did not vary between treatments). Nitrate nitrogen has also been shown to inhibit reproductive function in

some fish, such as the common carp Cyprinus carpio (Folmar et al., 1996) and mosquitofish Gambusia holbrooki (Edwards et al., 2006). With the recent trend towards culturing fish in RAS (Bergheim et al., 2009; Summerfelt and Christianson, 2014), where NO3-N can accumulate as an end-product of nitrification (Camargo, 2005; Davidson et al., 2009; 2011), more research is needed to understand the effects of moderately elevated NO3-N, as is often observed in RAS culture, on salmon reproductive performance.

The second major finding of this study is that high rates of early maturation took place without appreciable build-up of hormonally active compounds, including sex steroids. Many of the compounds assessed have been previously reported in RAS (Good et al., 2014; Mota et al., 2014); however, the detection limits were much lower in those studies, such that the reported levels were below the detection limits of the assays in our present study and, in the case of 11KT, were considerably lower than the levels detected in immature male plasma (i.e., >200ng/L; Figure 3). Taken together, the previous observations of hormone levels in the water and male salmon maturation rates made by Good et al. (2014) are likely a result, as opposed to a cause, of early maturation. Nevertheless, evidence that waterborne hormones can affect sexual maturation of aquatic species has been previously reported. For example, it has been demonstrated that maturing male European eels Anguilla anguilla are able to influence maturation in cohabitating immature males through what appeared to be waterborne chemical communication (Huertas et al., 2006; Huertas et al., 2007). In theory, uptake of steroid hormones or hormonally active compounds through the gills, gastrointestinal tract, or other routes should influence maturation if sufficient quantities and duration of exposure are provided. In male teleosts, 11-KT is the major androgen produced by the testes (Taranger et al., 2010) and instigates maturation in numerous

fish species (e.g. Cavaco et al., 2001; Schulz and Miura, 2002; Campbell et al., 2003; Rodriguez et al., 2005). In female fish, rising E2 levels are associated with the onset of secondary oocyte growth (Chadwick et al., 1987; King and Pankhurst, 2003). When assessing the effects of waterborne hormones, however, the issue is complicated by the fact that male and female hormones can exert influence on either sex. For example, T has been shown to stimulate early oogenesis and female maturation in coho salmon (Forsgren and Young, 2012) and milkfish Chanos chanos (Marte et al., 1988). As such, given that estrogenicity was detectable in certain RAS water samples (albeit at low concentrations), it is conceivable that long-term exposure to even low levels of EEQ or androgenic sex steroids could influence the development of male maturation. More research is needed to test this hypothesis before such a relationship can be established.

5. Conclusions

Overall, NO3-N concentration did not appear to be related to male Atlantic salmon maturation under the conditions of our study, and the majority of hormonally active compounds examined were not detectable in RAS water or makeup water. Long-term exposure to elevated NO3-N (100 mg/L) did not appear to negatively influence the health of Atlantic salmon. More research is required to identify the factor(s) responsible for early male maturation in our experimental and semi-commercial scale RAS, as well as in commercial land-based, closed-containment salmon growout facilities.

6. Acknowledgements

The authors wish to thank Dr. Gregory Weber (USDA-ARS) for advice during study planning and data analyses, as well as for critical review of the manuscript. Our gratitude is also extended to the following individuals for assistance with this study: Karen Schroyer, Susan Glenn, Brianna Taylor, Natalie Monacelli, Michael “Wells” Larson, Ryan Snader, and Jeremy Ruffner. This research was supported by the USDA Agricultural Research Service under Agreement No. 598082-5-001. The experimental protocols and methods described are in compliance with the Animal Welfare Act (9CFR) requirements and were approved by The Conservation Fund Freshwater Institute’s Institutional Animal Care and Use Committees. Use of trade names is for identification purposes only and does not imply endorsement by the U.S. Government.

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Table 1. Scoring system for lesions observed during histopathology evaluation of tissues collected at study’s end (8 months post-stocking).

Parameter

(1)

Score (2)

(3)

Epithelial hyperplasia No or scattered pockets of thickened epithelial cells piling up to two cell layers deep

Coalescing areas of thickened epithelial cells piling up to two cell layers deep

Diffuse hyperplasia throughout gill or skin with piling greater than two cell layers

Lamellar adhesion/fusion (gill only)

No, or scattered, lamellae are joined forming intralamellar pockets

Coalescing areas where lamellae are joined forming intralamellar pockets

Widespread joining of lamellae throughout the gill

Inflammation

Influx of less than 10 mononuclear or granular cells within a single filament or section of skin

Influx of 10-50 mononuclear and/or granular cells within a single filament or section of skin

Greater than 50 mononuclear and/or granular cells within a single filament or section of skin

Edema

No, or rare, epithelial lifting or ballooning degeneration and presence of proteinaceous fluid

Epithelial lifting and proteinaceous fluid is present in scattered lamellae

Epithelial lifting and proteinaceous fluid is widespread and diffuse throughout the gill

Goblet cell hyperplasia

Rare or scattered goblet cells within the apical portion of the epithelial layer

Increased proximity of goblet cells within the epithelium

Goblet cells are prominent and often clustered together

Microorganisms present (bacteria, protozoa, fungi)

No or rare organisms are seen

Multiple pockets of organisms are present

Organisms are widespread and can be seen invading into the tissue

Table 2. The suite of hormones, hormone conjugates, mycotoxins, and phytoestrogens assessed using the liquid chromatography / mass spectrometry panel. All compounds had detection limits of 0.5 ng/L, with the exception of 17-α Estradiol (1.0 ng/L). Class

Compound

Hormones

11-Ketotestosterone, 17-α Estradiol, 17-α Ethynylestradiol, 17-β Estradiol, 19Norethindrone, 4-Androstene-3,17-dione, Trans-diethylstilbestrol, Equilin, Equilenin, Estriol, Estrone, Norgestimate, Progesterone, 6-α-methyl-17-αhydroxyprogesterone, Testosterone, Epitestosterone, Trenbolone, Trenbolone acetate

Hormone conjugates

17-β-estradiol-3-sulfate, 17-β-estradiol-17-sulfate, Androsterone sulfate, Equilenin sulfate, Equilin sulfate, Estriol-3-sulfate, Estriol-17-sulfate, Estrone3-sulfate, Ethinylestradiol-3-sulfate, Testosterone sulfate, 17-β-estradiol-17glucuronide, Androsterone glucuronide, Diethylstilbesterol glucuronide, Estriol-3-glucuronide, Estrone glucuronide, Ethenylestradiol-3-glucuronide, Testosterone glucuronide

Mycotoxins

α-Zearalanol

Phytoestrogens Genestein, Daidzein, Formonectin, Coumesterol, Equol, Biochain A

Table 3. Mature males (% grilse) among 15 random fish, male and female, sampled per treatment group at 2-, 4-, 6-, and 8-month study time points. Grilse were identified as having >0.1 gonadosomatic indices; all sampled fish were determined to be immature at study commencement. Fish Age (months) 10 12 14 16

% Grilse Low NO3-N High NO3-N 0 (0/7) 0 (0/10) 67 (4/6) 70 (7/10) 71 (5/7) 50 (4/8) 100 (6/6) 78 (7/9)

Table 4. Results of whole blood gas and chemistry analyses (mean ± standard error) performed at study’s end (8 months post-stocking). NO3-N Parameter Sodium (mmol/L)

10 mg/L 152

± 1.01

100 mg/L 154

p-value

± 0.62

0.1039

Potassium (mmol/L)

3.16

± 0.11

3.10

± 0.11

0.8573

Calcium (mmol/L)

1.70

± 0.02

1.67

± 0.01

0.6907

± 0.47

0.0006

Chloride (mmol/L)

132

± 0.77

Glucose (mg/dL)

86.6

± 1.59

88.0

± 2.07

0.3542

Hematocrit (%PCV)

41.9

± 1.36

41.0

± 0.84

0.0677

Hemoglobin (g/dL)

14.2

± 0.46

13.9

± 0.29

0.0637

± 0.02

0.2860

pH

6.97

± 0.02

136

7.01

pCO2 (mmHg)

44.0

± 1.20

43.0

± 1.33

0.7087

HCO3 (mmol/L)

10.1

± 0.33

10.9

± 0.31

0.0256

± 0.26

0.0791

± 1.01

0.0967

Total CO2 (mmol/L) pO2 (mmHg)

9.20 10.5

± 0.35 ± 1.01

9.46 12.9

sO2 (%)

5.94

± 0.93

8.47

± 1.10

0.0786

Lactate (mmol/L)

3.41

± 0.24

3.30

± 0.22

0.0897

Table 5. Results of waterborne phytoestrogen testing in low and high NO3-N RAS (and spring makeup water) at 4- and 8-month sampling points. All values (mean ± standard error) are reported in ng/L; limit of detection for individual samples was 0.5 ng/L.

Compound

Low NO3-N

4-Month High NO3-N

Makeup H2O

Low NO3-N

Genestein

419 ±292

140 ±87.2

ND

121 ±39.1

122.3 ±38.4

ND

Daidzein

404 ±239

500 ±404

ND

400 ±226

275 ±111

ND

Formonetin

ND

ND

0.195 ±0.195

Coumesterol

ND

ND

ND

Equol Biochain A

0.600 ±0.600 0.467 ±0.467 ND

ND

ND ND

8-Month High NO3-N

0.293 ±0.293 0.597 ±0.300 ND

ND

0.273 ±0.273 0.530 ±0.272 ND

ND

Makeup H2O

ND ND ND ND

Figure 1. Percentage of sampled fish identified qualitatively as mature males at 2-, 4-, 6-, and 8month study time points. Mean overall fish weight at each sampling point is listed above bars. Error bars represent standard errors.

Figure 2. Gonadosomatic indices for male (top) and female (bottom) Atlantic salmon under high and low NO3-N conditions at 2-, 4-, 6-, and 8-months post stocking, corresponding to 10-, 12-, 14-, and 16-months post-hatch in fish age. Error bars represent standard errors. Number of fish assessed per treatment group at each sampling point was n=15.

Figure 3. Plasma 11-ketotestosterone values for male Atlantic salmon under high and low NO3-N conditions at 2-, 4-, 6-, and 8-months post stocking, corresponding to 10-, 12-, 14-, and 16months post-hatch in fish age. Error bars represent standard errors. Number of males assessed per treatment group at each sampling point ranged from n=15 to n=18.