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Aquaculture Research, 2012, 116

doi:10.1111/j.1365-2109.2012.03170.x

Inuence of long term ammonia exposure on Atlantic salmon (Salmo salar L.) parr growth and welfare
Jelena Kolarevic1, Roger Selset1, Olga Felip3, Christopher Good4, Kevin Snekvik5, Harald Takle2, Elisabeth Ytteborg2, Grete Bverfjord1, Torbjrn Asgard1 & Bendik Fyhn Terjesen1
1 2 3 4 5

Noma AS, Sunndalsra, Norway Noma AS, As, Norway University of Barcelona, Department of Animal Physiology, Barcelona, Spain The Conservation Funds Freshwater Institute, Shepherdstown, WV, USA Washington Animal Disease Diagnostic Laboratory, Department of Veterinary, Microbiology and Pathology, College of

Veterinary Medicine, Washington State University, Pullman, WV, USA


Correspondence: J Kolarevic, Noma AS, NO-6600 Sunndalsra, Norway. E-mail: jelena.kolarevic@noma.no

Abstract
The objective of this study was to determine the long-term effects of ambient unionized ammonia nitrogen (NH3-N) combined with different feeding regimes on Atlantic salmon Salmo salar L parr growth, welfare and smoltication. Previous studies on the parr stage of Atlantic salmon have mostly focused on acute exposure, or at low temperatures. Atlantic salmon parr were exposed for 105 days (at 12C, pH 6.8) to four sublethal ammonia concentrations ranging from 0.1 to 35 lg L1 NH3-N (0.125 mg L1 TAN) at two feeding levels: full feed strength (+20% overfeeding) and 1/3 of full feed strength. After 21 days, it was observed that 32 lg L1 NH3-N reduced growth rate of parr fed full ration, but this effect was not evident at the end of the exposure. Feed utilization was not affected by ammonia exposure at any sampling point. Increasing ammonia levels were associated with a higher prevalence and severity of gill damage at 22 days but not at the end of the exposure. The examination of welfare indicators revealed only a few pathologies, not related to ammonia exposure. In addition, higher ammonia concentrations did not appear to inuence the development of hypoosmoregulatory ability during parr-smolt transformation.

Introduction Ammonia is the main nitrogenous waste product in most teleosts and it is mainly excreted through gills to the surrounding water (Randall & Wright 1987; Fivelstad, Kellevik, Iversen, Mretr, Vage & Binde 1993; Randall & Tsui 2002; Wilkie 2002; Eddy 2005). Ammonia is a toxicant with negative effects on teleosts dependent on e.g. the species, developmental stage, feeding rate, swimming activity and other factors (Wright & Fyhn 2001; Finn 2007; Terjesen 2008). In intensive high-density ow-through systems ammonia build-up can cause reduced growth and mortality (Person-Le Ruyet, Galland, Le Roux & Chartois 1997). In water, total ammonia exists as the sum of the un-ionized (NH3) and ionized (NH4+) forms whose equilibrium varies with pH, temperature and salinity. Toxicity, of total ammonia nitrogen (TAN) in the environment, increases with the concentration of the more toxic NH3-N (Randall & Tsui 2002; Eddy 2005). Recommended safe levels of TAN and NH3-N are given for a number of sh species based on acute toxicity studies in controlled environmental conditions (USEPA 1998; Norwegian Food Safety Authority 2004). Knoph (1995) conducted a series of experiments to determine effects of acute ammonia toxicity on Atlantic salmon parr and post-smolts. However, the effects of chronic exposure to sublethal ammonia levels have not been well documented, especially for Atlantic salmon (Salmo salar) in freshwater at relevant production temperatures (Terjesen, Ulgenes, Fjra, Summerfelt,

Keywords: ammonia exposure, Atlantic salmon, growth, welfare, gill histology, smoltication

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Brunsvik, Bverfjord, Nerland, Takle, Norvik & Kittelsen 2008). Recently it was shown in two separate experiments that long-term exposure to low levels of exogenous ammonia can promote growth in rainbow trout (Oncorhynchus mykiss) without a change in feed intake (Wood 2004). This nding challenges the traditional view that ammonia is always damaging for sh. Several authors have emphasized the effect of feeding on acute ammonia toxicity (Wicks & Randall 2002a; Zimmer, Nawata & Wood 2010). Wicks and Randall (2002b) showed that feeding protects juvenile rainbow trout from ammonia toxicity during the rst 24 h of exposure to high environmental ammonia and that fasting exacerbates ammonia toxicity. Restricted feeding, unlike feeding to satiation, did not induce the growth promoting effect of exogenous ammonia in Woods (2004) long-term ammonia exposure study. Inuences of different feeding regimes on ammonia tolerance of Atlantic salmon are largely unknown. The question of sh welfare in aquaculture and scientic research is becoming increasingly important (EFSA 2008). The welfare concept is complex, with several denitions that have focus on the animals condition, on its subjective experience of that condition and/or on whether it can live a natural life (Turnbull, Bell, Adams, Bron & Huntingford 2005; Huntingford, Adams, Brauthwaite, Kudri, Pottinger, Sande & Turnbull 2006). A number of recommendations have been made regarding the welfare of sh under production conditions, and on methods to assess welfare indicators (The Farm Animal Welfare Council 1996; Johansen, Needham, Colquhoun, Poppe & Smith 2006). However, there is still a need to validate the most commonly used welfare indicators in relation to gender, life stage, time of the year and the environment to which sh are exposed to (Huntingford et al. 2006; Johansen et al. 2006; The Research Council of Norway 2009). The objective of this study was to examine the effects of long-term ammonia exposure, feeding and combination of these two factors on growth, welfare and smoltication of farmed Atlantic salmon in fresh water. Materials and methods Experimental animals and rearing conditions Atlantic salmon used in this experiment originated from the Salmobreed strain 3.V-06 MB (L) located

in Mjaneset (Norway). The sh were hatched in December 2008 at the Noma research station in Sunndalsra (Norway). Alevins were rst fed with a formulated commercial diet (Ewos, Bergen, Norway) in February 2009 under conditions of continuous light (LD 24:0) and at a water temperature ranging from 13.0 to 13.5C. One month prior to the experiment, Atlantic salmon parr (6.20 0.03 g) were transferred to the experimental facilities at Noma, Sunndalsra and randomly distributed into 24 similar experimental tanks (55 sh per tank). The sh were reared in 150 L tanks with fresh water from ground wells (ion concentrations in mg L1: Mg2+ 3.9, Ca2+ 4.4, Na+ 48.5, K+ 2.2, SO42 21.5) at 12.11C 0.02 (SEM), pH of 6.84 0.02 and 24 h continuous light. These conditions were maintained during the whole experiment. Feeding was done by automatic band feeders for 45 min every 8 h using a commercial pelleted feed that varied in size during the trial according to the sh size (Table 1). Water ow was set to 3 L min1 for each of the experimental units and ow was monitored on a monthly basis until the end of the experiment. The use of experimental animals for this trial was approved by the Norwegian Animal Research Authority. The 3Rs principle (Russell & Burch 1959) was applied and humane end points were established prior to the start of the experiment. Experimental design and sampling Prior to the start of the experiment, a system for ammonia dosage was set up and tested. A peristaltic pump (520DuN, Watson Marlow Bredel,Wilmington, NC, USA) was used to deliver a stock solution of (NH4)2SO4 from the mixing tank (Iwaki Unit; 21 g (NH4)2SO4 L1 fresh water) into three header tanks at a constant speed of 150 rpm. Different water ammonia concentrations were achieved by using silicone and marprene tubes with appropriate diameter sizes delivering on average 26.2, 12.0 and 7.0 mL min1 of (NH4)2SO4 to header tanks for high, medium and low ammonia concentration treatments respectively. Water from each header tank was supplied to six experimental tanks. Delivered doses of the stock solution were measured ve times a week and the tubes were replaced if the observed oscillations exceeded 10% of the desired ow. At the start of exposure, ten sh from each tank were euthanized using 100 mg L1 of tricaine
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Effects of ammonia exposure on Atlantic salmon J Kolarevic et al.

Table 1 Pellet sizes and analysed chemical composition of the experimental diets (in g kg1)
Diet Pellet size 1 Pellet size 2 Pellet size 3
2.8 926.4 459.9 240.4 84.4 77.3 26.7 11.5 20.0 32.9 33.3 12.1 20.3 17.7 4.4 22.1 24.5 40.1 4.3 25.5 68.2 21.3 17.8 13.7

Pellet size (mm) 1.7 2.2 Dry matter (DM) 947.4 926.5 Crude protein 505.2 478.5 Crude lipid 207.1 212.1 Starch 80.4 79.9 Ash 90.1 82.9 Hydrolized indispensable amino acids Arg 29.7 28.4 His 12.8 11.8 Ile 22.3 21.6 Leu 36.9 34.5 Lys 37.2 34.7 Met 13.8 12.5 Phe 22.4 21.5 Thr 19.5 18.3 Trp 5.6 4.9 Val 24.7 23.4 Hydrolized dispensable amino acids Ala 27.5 25.1 45.0 43.6 Asx* Cys 5.1 4.6 Gly 27.9 25.3 Glx 75.1 70.3 Pro 22.9 19.9 Ser 20.2 18.5 Tyr 15.6 14.3

daily and feed intake was determined according to Helland, Grisdale-Helland and Nerland (1996). During the experiment, the sh were counted and weighed in bulk each third week. Five sh from each tank were individually measured for length and weight after 21, 42 and 63 days, while ten sh were removed after weighing at 84 and 105 days, respectively, to reduce the biomass and prevent the oxygen saturation from falling below 85%. Prior to weighing, the sh were not fasted (Wood 2001) and calculated biomass was therefore corrected for removed gut content. Ten sh from the initial sampling and ten sh from each tank after 105 days were sampled for body composition analysis and stored at 20C. Gill samples were also collected at day 22 and day 105 from three sh from each tank and subsequently xed in phosphate-buffered formalin (4%, pH 7.2). Ten sh from each experimental tank were used for external assessment of sh welfare at the end of the exposure period. Immediately after euthanasia with 100 mg L1 of MS 222, the sh were inspected for n condition, cataract, skin lesions, gill condition and externally visible/palpable deformities. Smoltication and seawater challenge test To examine if the long-term ammonia exposure inuenced development of hypo-osmoregulatory ability during parr-smolt transformation, the remaining sh were subjected to a photoperiod manipulation treatment following the exposure experiment. After 105 days of exposure, ten sh from each tank were individually PIT-tagged and placed in a circular tank (2 m2) with running freshwater and subjected to a photoperiod treatment to induce smoltication. During 7 weeks, sh were exposed to a photoperiod with 12 h of light and 12 h of darkness (LD 12:12), followed by 6 weeks of continuous light regime (LD 24:0). At the start of the continuous light period, the sh were distributed in three circular tanks, containing three or four sh from each of the previous ammonia treatments. After 4 weeks of continuous light, sh from the rst tank were exposed to a 72-h seawater challenge test (SWT1). The same procedure was repeated after week ve (SWT2) and week six (SWT3), with sh from the remaining two tanks. The static sea water used in this test was made by adding articial sea-salt to reach 34.535& salinity.

*Asx represents Asp and Asn. Glx represents Glu and Gln.

methane sulphonate, MS 222 (Argent Chemical Laboratories, Redmond, WA, USA) and individually measured for weight (0.1 g accuracy) and length (0.1 cm accuracy). In addition, bulk weight for each tank was noted before, and after, sh sampling. After the initial sampling, Atlantic salmon parr (45 sh per tank) with an average individual weight (SEM) of 17.4 (0.1) g, were exposed to four different ammonia concentrations ranging from 0.1 to 35 lg L1 NH3-N (0.125 mg L1 TAN) during 105 days. Each ammonia treatment (Table 2) was represented with six tanks; three tanks in which the sh received full feed strength (+20% overfeeding) at expected growth capacity according to ClubN 2002 (Skretting 2007) and three tanks in which sh received only 1/3 of the above mentioned feed strength. Full randomization of sh tanks was prevented by system infrastructure limitations; however, each of the four blocks of tanks contained two of eight randomly selected treatments. Waste feed was collected and weighed
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Table 2 Feeding regimes, measured total ammonia (TAN) and calculated average un-ionized ammonia (NH3-N) and approximate partial pressure of NH3 (PNH3) for all treatments
Feeding level
Reduced feeding (1/3 of full feed strength)

TAN (mg L1)*


0.1 6.5 11.2 23.9 0.1 6.7 11.3 25.1 0.1 0.1 0.1 0.6 0.0 0.1 0.1 0.6

NH3-N (g L1)*
0.1 9.3 16.6 34.7 0.1 8.1 14.4 32.2 0.0 0.2 0.4 1.2 0.0 0.2 0.4 1.1

TAN (mol L1)*


4 465 800 1704 4 478 809 1794 2 4 8 44 1 37 8 46

PNH3 (torr)
0 13 22 46 0 13 22 48

Full feed strength (+20% overfeeding)

*TAN and NH3-N values are given as means SEM for all treatments.

The oxygen levels were kept above 85% with continuous aeration. After 72 h, blood samples were collected from the caudal vessel using heparinised vacutainers (Terumo Europe, Leuven, Belgium). Samples were centrifuged for 10 min at 3000 rpm (Allegra 6R centrifuge, Beckman) and plasma stored at 20C for subsequent ion analysis. Measurements and analytical procedures Water quality was monitored throughout the experimental period. Conductivity was measured ten times throughout the experiment in all sh tanks using a portable HQ40D system (Hach Lange, Dusseldorf, Germany) with HACH Intelli CAL CDC401 Standard Conductivity probe. Daily conductivity values were calculated as the average of two consecutive conductivity measurements taken before and after the day in question. Water samples were collected and analysed daily for total ammonia nitrogen (TAN mg L1) with an automated chemistry analyser (Flow Solution IV, OI Analytical, College Station, TX, USA). Values for NH3-N were calculated using the temperature, conductivity, pH and TAN values for the day in question, using formulas at the American Fisheries Society website (Colt 2011), based on Emerson, Russo, Lund and Thurson (1975). The effect of the added (NH4)2SO4 between control and the highest exposure concentration (25.1 mg L1 TAN) on ionic strength and ammonia pKa represented only up to 2%, in terms of lg L1 of NH3-N and was therefore disregarded. Daily measurements of pH were done in the water outlet of sh tanks using a portable HQ40D system (Hach Lange, Germany) with HACH PHC101 IntelliCAL pH electrode. Periodic measurements of samples were also done in the laboratory (720A; Thermo Orion, Beverly, MA,

USA) for cross reference. Due to discrepancies between values obtained using the two different pH metres, especially for the control group (ground well water with low buffering capacity), a PHC28103 Intellical pH ultra electrode (Hach Lange, Germany) designed for fast response in lowionic strength samples was used for verication. The pH values were adjusted for the last one and a half months of the experiment, according to measurements using the Intellical pH ultra electrode. Oxygen was measured weekly, and on a daily basis during the last month of the exposure period, in the efuent water of all tanks with HQ40D system and Intellical LDO outdoor sensor (LDO101-5). The oxygen saturation was kept above 85% at all times. At the end of the ammonia exposure, ten to 11 sh from each treatment group were euthanized using 100 mg L1 of tricaine methane sulphonate, MS 222 (Argent Chemical Laboratories, Redmond, WA, USA). Blood samples were collected from caudal vessels using 1 mL heparinized syringes (~37 USP units of Li Heparin, SigmaAldrich, St. Louis, MO, USA). An i-STAT Portable Clinical Analyser with EC8 + disposable cartridges (Abbott Laboratories, Abbott Park, IL, USA) was used to measure plasma sodium, potassium and chloride levels, glucose (Glc), haematocrit (Htc) and haemoglobin (Hb). All diets and whole-body samples were analysed for dry matter (105C until constant weight), nitrogen (Kjeltec Auto System, Tecator, Hoganas, Sweden), ash (550C until constant weight), energy (Parr 6300 Bomb calorimeter; Parr, Moline, IL, USA) and crude fat (Soxtec 2055; Tecator, Hoganas, Sweden). In addition, dry matter was also analysed in the waste feed (105C until constant weight). After the seawater challenge test, plasma Cl concentration was measured with an electrolyte
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analyser (Biolyte 2000, BioCare Corporation, Taoyuan County, Taiwan). Several welfare indicators, such as n condition, cataract, skin lesions and skin coloration, gill condition and externally visible/palpable deformities, were examined at the end of the exposure period by a veterinarian not otherwise involved in this study. Each of the observed welfare indicators were given a welfare index between 0 and 2, with 0 indicating good and 2 indicating bad condition. Based on the scores for all examined welfare indicators, each sh was then given an overall welfare index between 0 and 2 with a low index value indicating good welfare. Each tank received a total score (between 0 and 20) that was the sum of the individual scores for all ten examined sh. Gill tissues collected from 72 sh after 22 and 105 days of exposure were embedded in parafn, cut into 5 lm sections after surface decalcication (decalcifying solution light, SigmaAldrich) and stained according to standard haematoxylin-eosin (H&E) histological protocol. H&E stained slides of gill tissues were evaluated for evidence of damage or tissue change and the severity of lesions observed was scored according to criteria stated in Table 3. Total gill RNA was isolated from 15 individuals per treatment using TRIzolTM and Micro to Midi Kit (Invitrogen, MD, USA) and DNAse1 treated (Invitrogen). Purity and quantity of isolated RNA were measured by spectrophotometry (Nanodrop ND-1000 Spectrophotometer; NanoDrop Technologies, Wilmington DE, NC, USA). For all samples, 0.5 mg total RNA was reverse transcribed to

cDNA using a 50:50 mix of random hexamer and oligo(dT) primers and Taqman Gold RT-PCR kit (Applied Biosystems, CA, USA). All reactions were performed in accordance with the manufacturers protocol. Primers for expression analysis were based on known Atlantic salmon sequences. The Hsp70 (Genbank no. AJ632154) primers (forward: TGA CGTGTCCATCCTGACCAT and reverse: CTGAAGA GGTCGGAACACATCTC) were designed using the Vector NTI Advance 10 (Life technologies, MD, USA), and NetPrimer (PREMIER Biosoft, CA, USA) software, while primers against the internal standard gene 18S rRNA (forward: TGTGCCGCTAGA GGTGAAATT and reverse: GCAAATGCTTTCGC TTTCG) were found in Jorgensen, Hetland, Press, Grimholt and Gjen (2007). The PCR products from both primers were cloned using pGEM T-easy (Promega, WI, USA) and sequenced with Big Dye Terminator chemistry and the ABI 3730 automated sequencer, both delivered by Applied Biosystems. Triplicate real-time qPCR reactions were performed using the Light cycler 480 and SYBR Green chemistry (Roche, Basel, Switzerland) at the following thermal cycling conditions: 95C for 10 min, followed by 45 cycles at 95C for 15 s, 60C for 15 s and 72C for 15 s. Specicity was assessed by the melting curves and on EDTA stained agarose gel. Relative Hsp70 mRNA was normalized to relative 18S rRNA mRNA levels. The transcription ratios of Hsp70 were tested by using the Relative Expression Software Tool, REST, including exact PCR efciency of each amplicon according to Pfaf, Horgan and Dempe (2002).

Table 3 Scoring criteria for determination of lesion severity used in the histopathological evaluation of Atlantic salmon gills
Lesion severity
Absent Very minimal Minimal Moderate Marked Severe

Score
0 1 2 3 4 5

Description
Normal tissue <5% of examined tissue or microanatomical regions affected/corresponding very minimal numbers of inammatory inltrates 25% of examined tissue or microanatomical regions affected/corresponding minimal numbers of inammatory inltrates 50% of examined tissue or microanatomical regions affected/corresponding moderate numbers of inammatory inltrates 75% of examined tissue or microanatomical regions affected/corresponding very marked numbers of inammatory inltrates Essentially 100% of examined tissue affected/obliteration of normal architecture/numerous inammatory inltrates

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Calculations Cumulative feed intake was calculated by dividing the total amount of feed eaten per tank by the total number of sh present at that moment (g feed individual1). Fish individual weight was calculated by dividing bulk weight (corrected for the estimated gut content weight) with the number of sh in the tanks (g). Specic growth rates (SGR, % day1) between two sampling points (t, days) were calculated using measured body weight at the start (BW1) and at the end (BW2): SGR lnBW2 lnBW1 100=t Body weight measurements, BW1 and BW2 were also used to calculate feed conversion ratios (FCR) between two consecutive sampling points: FCR TFI=BW2 BW1 where TFI represents the total dry feed intake over the experimental period in question. Condition factors (CF) at the end of the ammonia exposure period and after the three saltwater tests were calculated from the sh body weight (W, g) and corresponding length (L, cm) (Handeland, Wilkinson, Sveinsb, McCormick & Stefansoon 2004): CF W L3 100

To examine statistically whether ammonia concentration and feeding regime were associated with overall gill damage, an ordinal logistic regression was carried out in SAS using the GENMOD procedure, with an overall gill pathology score (i.e., the sum of scored pathologies observed per tissue specimen) serving as the dependent variable. Ammonia exposure and feed regime (plus an interaction term for these variables) were included as categorical dependent variables. This modelling was repeated using data from each sampling event. Differences between the transcription ratios of Hsp70 mRNA were tested for signicance by the Pair Wise Fixed Reallocation Randomization Test (Pfaf et al. 2002) and data are presented as means SEM. Results Growth and body composition There were no treatment-related mortalities during the trial. Mean individual weight increased ve and threefold in groups with full strength feeding and restricted feeding, respectively, at the end of the long-term exposure to sublethal ammonia concentrations (Fig. 1 c,d). Apart from signicant difference in body mass (P < 0.0001), groups with restricted feeding also had a signicantly lower cumulative feed intake (Fig. 1 a,b) (P < 0.0001), due to the difference in feeding regime. Among the groups with full feed strength, the highest environmental ammonia concentration induced a negative effect on growth rate after 22 days of exposure. A signicantly lower SGR (P < 0.05) (Fig. 2) and average individual weight (P = 0.02) was noted for the group exposed to 32 lg L1 NH3-N compared with the control group. At the end of exposure, however, individual weight, SGR and FCR (Fig. 2) did not differ among the four groups. The groups with restricted feeding showed reduced growth without any additional effects of differential ammonia exposure (Fig. 1). Among the groups that received full feed strength, the whole-body composition analysis revealed signicantly higher energy and lipid levels in the group exposed to 14 lg L1 NH3-N compared with the corresponding control group (0.1 lg L1 NH3-N) (Table 4). Among treatments with restricted feeding levels, there was no effect of

Statistical analyses Data were analysed using JMP 7.0 (SAS 2007; SAS Institute Inc, Cary, NC, USA) where tank was used as the statistical unit. Square root and angular transformations were performed for all percentage and scoring data to normalize variances prior to analysis. Normality of distributions was assessed by the ShapiroWilk test and homogeneity of variances was tested with Levenes F-test. A multivariate analysis of variance (MANOVA) was used to examine the inuence of ammonia treatment and feed regime and their interaction on measured parameters. If signicant, a one-way analysis of variance (ANOVA) was performed to determine differences between treatments, and Students t-test to establish difference between control and each other experimental group. All data are expressed as means standard error of the mean (SEM), apart from molecular data, and difference between treatments was judged signicant if P 0.05.

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(a)

(b)

(c)

(d)

Figure 1 Cumulative feed intake per sh in (a) full fed groups exposed to 0.1, 8, 14 and 32 lg Ll NH3-N and in (b) groups with restricted feeding exposed to 0.1, 9, 17 and 35 lg Ll NH3-N. Individual body weight development over period of 105 days for fully fed and groups with restricted feeding are presented in graphs (c) and (d). All values are given as tank means + SEM (n = 3 for each treatment).

ammonia exposure on any examined body composition parameter. Measured blood parameters At the end of the ammonia exposure Htc and Hb values were higher in the full feed groups compared with sh fed restrictedly (Table 5). In addition, full fed sh exposed to 8 lg L1 NH3-N had signicantly higher Htc and Hb compared with the control group (0.1 lg L1 NH3-N). Among treatments with restricted feeding levels, sh exposed to 9 lg L1 NH3-N and 35 lg L1 NH3-N had lower Htc and Hb values than control group sh (0.1 lg L1 NH3-N). Blood glucose (Glu) was inuenced by an interaction between ammonia treatment and feeding regime (MANOVA, P < 0.05). Among groups with full feed strength regime,
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exposure to 8 lg L1 NH3-N and 32 lg L1 NH3N signicantly increased Glu values in comparison with the control group (0.1 lg L1 NH3-N). Fish fed restrictedly, and exposed to 9 lg L1 NH3-N also showed signicantly higher Glu values compared with the corresponding control group (0.1 lg L1 NH3-N). The highest levels of blood Na+ (142 1 mM) were found for the 14 lg L1 NH3-N treatment, among the groups with full feed strength. This value was signicantly higher than blood Na+ of Atlantic salmon parr exposed to 0.1 lg L1 NH3N and 8 lg L1 NH3-N. Welfare indicators The average overall welfare index for the groups with restricted feeding levels were signicantly

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Figure 2 Feed conversion ratio (FCR) and specic growth rate (SGR) in full fed groups exposed to 0.1, 8, 14 and 32 lg Ll NH3-N at day 22 and at day 105 (tank mean + SEM; n = 3). Different letters denote signicant difference between treatments on day 22 (P 0.05).

different compared with the full feed strength treatments (Fig. 3). The highest overall score of six (lowest welfare) was given to sh of the control group with restricted feeding, followed by a score of ve in the group with restricted feeding exposed to 17 lg L1 NH3-N and 35 lg L1 NH3-N and a score of four for the sh exposed to 9 lg L1 NH3N. The best sh condition was found in the treatment groups with full feed strength. Among these the best scores were given to groups with exposure to 0.1 lg L1 NH3-N, 14 lg L1 NH3-N and 32 lg L1 NH3-N (score = one), while the group exposed to 8 lg L1 NH3-N received an overall score of two. The most commonly observed abnormalities were short or crippled ns and slightly shortened opercula noted mostly for the restricted feed ration treatments. Histopathological evaluations of gill tissue identied four distinct lesion types: epithelial hypertrophy, epithelial hyperplasia, secondary lamellae fusion and lymphohistiocytic inammation (Fig. 4). After 22 days of exposure, increasing ammonia levels were signicantly (P < 0.05) associated with increased prevalence and severity of gill damage. The prevalence of each lesion type

was low among all gills evaluated (Table 6), and the severity of observed lesions was never more than minimal, i.e. lesion score = 2. Feeding regime was not associated statistically with gill damage. At the end of the ammonia exposure period, no statistical associations between gill damage and ammonia exposure were identied. The transcriptional levels of the stress marker Hsp70 in the gills were not signicantly up-regulated after 105 days of ammonia exposure (Fig. 5). However, down-regulation of the mRNA expression was evident for groups exposed to 17 lg L1 NH3-N (P = 0.06) and 14 lg L1 NH3-N (P = 0.09). The transcription of Hsp70 in sh of the 35 lg L1 NH3-N group with restricted feeding, was signicantly higher compared with the 32 lg L1 NH3-N with full feeding. In contrast, feeding did not signicantly affect the ratio of Hsp70 transcription at the lower ammonia exposure concentrations. Smoltication indicators After 13 weeks of light manipulation, hypo-osmoregulatory ability of the remaining experimental
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3.4 3.0 0.5 0.1

Values are given as mean SEM for all treatments at the end of ammonia exposure period. Signicantly different values from comparable control group values are marked with an asterisk (P < 0.05). Signicant differences between rationed and full fed treatments exposed to the same ammonia levels are presented with black bullet sign (P < 0.05).

Table 4 Whole-body composition in Atlantic salmon at starting point and at the end of a long-term ammonia exposure (g or MJ kg1 of wet weight)

35 g L1 NH3-N

101.3 178.2 20.8 8.0

sh was tested. None of the examined treatments resulted in statistically different plasma chloride levels compared with the control group with full feed strength regime. Plasma chloride levels increased signicantly after SWT1 (sea water test 1, Fig. 6a) in all treatments. In subsequent tests (SWT2 and SWT3), however, plasma chloride levels were lower (<150 mM). At SWT3 the highest chloride level (146 mM) was observed for sh originating from the group with restricted feeding and exposure to 9 lg L1 NH3-N, while sh that were previously exposed to 14 lg L1 NH3-N with full feed strength had signicantly lower chloride levels (135 mM, P < 0.05). A signicant decrease in CF (Fig. 6b) was evident in sh from all original ammonia treatments after 4, 5 and 6 weeks of exposure to continuous daylight (LD 24:0). There was no signicant difference in CF between groups fed full feed strength and groups with restricted feeding levels, or due to ammonia exposure, at the end of the photo manipulation period. Discussion The results of this study suggest that growth, welfare and smoltication of the Atlantic salmon parr used in this study were not affected by long-term exposure to sublethal ammonia levels up to 35 lg L1 NH3-N (25 mg L1 TAN). Furthermore, no visible physical damages or mortalities were attributed to increased environmental ammonia levels at the end of the experiment. The growth promoting effect of ammonia seen in the rainbow trout (Wood 2004), however, was not evident for the Atlantic salmon strain used in this study. Negative effects of ammonia on growth of juvenile Atlantic cod, Gadus morhua (Foss, Siikavupio, Sther & Evensen 2004) and juvenile turbot, Scophthalmus maximus (Foss, Imsland, Roth, Schram & Stefansson 2009) were documented after 96 and 64 days of exposure to 60 lg L1 and 130 lg L1 NH3-N respectively. One month long sublethal exposure of Atlantic salmon to ammonia in freshwater and seawater at very low temperature (4C; Fivelstad et al. 1993; Fivelstad, Schwarz & Strmsnes 1995) did not have any effect on growth parameters. Positive effects of moderately elevated water ammonia on growth and protein production, however, were documented for rainbow trout fed to satiation (Linton, Reid & Wood 1997, 1999; Wood

32 g L1 NH3-N 14 g L1 NH3-N 8 g L1 NH3-N 0.1 g L1 NH3-N 17 g L1 NH3-N 9 g L1 NH3-N 0.1 g L1 NH3-N Starting point Experimental treatments (feeding regimes/environmental ammonia concentration) Full feed strength (+20% overfeeding) Reduced feeding (1/3 of full feed strength)

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Crude lipid Crude protein Ash Gross energy

90.3 164.1 20.3 7.4

102.0 176.7 21.3 8.1

3.5 2.6 0.3 0.1

107.5 170.6 20.3 8.4

7.6 2.2 0.6 0.4

108.7 170.7 21.2 8.6

2.3 3.2 0.6 0.4

2.8 2.0 0.7 0.1

106.1 180.2 20.8 8.4

1.5 1.1 0.7 0.1

115.3 175.9 20.5 8.7

8.6 0.6 0.4 0.3

123.4 178.7 21.6 9.1

5.3* 0.9 0.4 0.1*

111.5 179.9 21.6 8.6

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Table 5 Measured blood parameters at the end of the 105-day long-term ammonia exposure in Atlantic salmon parr
NH3-N (lg L1)
0.1 9 17 35 0.1 8 14 32

Feeding level
Reduced feeding (1/3 of full feed strength) Full feed strength (20% overfeeding)

Na+ (mmol L1)


141.64 140.22 141.53 140.89 139.39 138.58 142.22 141.86 0.70 0.83 1.02 0.59 0.63bc 0.46c 0.76a 0.70ab

K+ (mmol L1)
3.38 3.44 3.59 3.11 3.34 3.34 3.18 2.96 0.19 0.46 0.11 0.25 0.04 0.36 0.11 0.17

Cl (mmol L1)
130.67 131.89 130.83 131.92 132.61 132.22 130.69 131.47 0.88 0.95 0.73 0.46 0.63 0.40 0.86 0.60

Glu (mg dL1)


4.95 4.28 4.53 4.73 4.56 4.84 4.47 4.96 0.31* 0.31 0.02* 0.14* 0.10b 0.03a 0.05b 0.16a

Htc (%PCV)
37.22 34.28 35.33 33.08 37.36 40.81 36.92 38.03 0.40* 0.60 1.21* 0.51t 0.65b 1.18a 1.52b 1.67ab

Hb (g L1)
126.47 116.61 120.03 112.47 127.22 138.83 125.47 129.39 1.30* 2.12 4.12* 1.72t 2.22b 4.11a 5.12b 5.55ab

Values are given as tank means SEM (n = 3 for each treatment). Different letters denote signicant differences (Student t-test) between full feed treatments and and * denote signicant differences (Student t-test) between reduced feeding regimes.

Figure 3 Welfare index of full feed treatments exposed to 0.1, 8, 14 and 32 lg Ll NH3-N and treatments with restricted feeding exposed to 0.1, 9, 17 and 35 lg Ll NH3-N at day 105 of exposure (tank mean + SEM; n = 3); Different letters denote signicant difference (Student t-test, P 0.05) between treatments.

(a)

(b)

Figure 4 Gill tissue of Atlantic salmon with rationed feeding (a) exposed to 35 lg L1 NH3-N and (b) exposed to 32 lg L1 NH3-N. Small letters denote identied gill lesions: (a) epithelial hyperplasia, (b) secondary lamellae fusion and (c) general inamation (H&E stain, 2009 magnications).

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Table 6 The prevalence of identied gill lesions at day 22 and 105 of exposure
NH3-N (g L1)
0.1 9 17 35 0.1 8 14 32 0.1 9 17 35 0.1 8 14 32

Day of exposure
Day 22

Feeding level
Reduced feeding (1/3 of full feed strength)

Epithelial hypertrophy
0/9 0/9 0/9 2/9 1/9 1/9 1/9 0/8 1/9 1/3 1/9 1/9 0/9 1/9 1/9 1/9

Epithelial hyperplasia
2/9 4/9 2/3 1/3 2/9 1/3 2/9 1/8 1/9 1/9 2/9 0/9 1/9 2/9 1/9 2/9

Secondary lamellae fusion


1/9 4/9 1/3 2/9 0/9 0/9 0/9 0/8 0/9 1/9 2/9 0/9 1/9 1/9 0/9 1/9

Inammation
0/9 1/3 0/9 5/9 1/9 1/9 1/3 1/8 1/9 0/9 0/9 2/9 1/9 0/9 1/9 2/9

Full feed strength (+20% overfeeding)

Day 105

Reduced feeding (1/3 of full feed strength)

Full feed strength (+20% overfeeding)

n = 9 in all treatments, except for the group exposed to 32 g L1 NH3-N (n = 8).

Figure 5 Relative Hsp70 transcription in Atlantic salmon gills after 105 days of exposure to different levels of ammonia and fed either a restricted or full feed strength diet, and between groups with restricted and full feed strength exposed to the same ammonia level. Data are given as mean values + SEM, n = 15 and signicant differences (P < 0.05 or P < 0.10) are indicated by a and b respectively. Expression ratios are shown in relative mRNA expression along the y-axis, genes along the x-axis.

2004). In two separate experiments at different water temperatures it was concluded that a PNH3 of approximately 23 ltorr signicantly increased body mass after 71 days of exposure (Wood 2004). It was demonstrated that the PNH3 rather than the TAN concentration is critical in initiating the growth response. Although the calculated average PNH3 of 20 ltorr in the present study (~14 lg L1 NH3-N) did not have any signicant effect on growth, it signicantly increased energy content of salmon parr after 105 days of exposure.
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The window of ammonias effectiveness as a growth stimulant may be quite small and it may also depend on the duration of exposure (Wood 2004). The present study suggests that the Atlantic salmon parr used in this study were more tolerant to chronic ammonia exposure than previously assumed for this species. After an initial signicant decrease in specic growth rate at 32 lg L1 NH3-N, no difference was present at the end of the exposure when compared with the control group.

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(a)

(b)

Figure 6 Plasma chloride levels (a) and condition factor (b) at the end of ammonia exposure (EXP) and after three 72 h seawater challenge tests (SW1, SW2 and SW3). Asterisk indicates signicant difference in plasma chloride levels between EXP and SWT1 and signicant difference in condition factor at EXP and at the seawater tests. All values are given as means + SEM. Treatments legend: full feed treatments exposed to 0.1, 8, 14 and 32 lg Ll NH3-N and treatments with restricted feeding exposed to 0.1, 9, 17 and 35 lg Ll NH3-N.

This suggests that a higher level of TAN and NH3N than recommended (Norwegian Food Safety Authority 2004) did not induce any adverse effects on growth of Atlantic salmon. However, follow-up studies on ammonia exposure of other lifestages of Atlantic salmon, and at different physiological and nutritional conditions, should be done. The present results indicate that reared Atlantic salmon are capable of developing an acclimation response to a long-term ambient ammonia exposure. Similar acclimation effects have been observed in juvenile Atlantic cod (Foss et al.

2004), European seabass, Dicentrarchus labrax (Lamarie, Dosdat, Coves, Dutto, Gasset & PersonLe Ruyet 2004) and spotted wolfsh (Foss, Vollen & iestad 2003). Different physiological and molecular mechanisms are in place to regulate this acclimation response and maintain acceptable tissue ammonia levels in sh (Wright & Wood 2009). When chronically exposed to elevated ammonia, rainbow trout responded with enhanced liver protein turnover through increased ribosomal translational efciency (Reid, Linton, Dockray, McDonald & Wood 1998). Another study demonstrated a stimulative effect of ammonia on net protein synthesis in rainbow trout and an increase in whole-body protein proportion (Wood 2004). Additional investigations of amino acid metabolism will be necessary to elucidate effects of ammonia on this compound class in Atlantic salmon. Feeding conditions and elevated water ammonia levels had a pronounced effect on several blood parameters in the present study. Increased Htc values were observed in sh exposed to 8 lg L1 NH3-N in this study, while Fivelstad et al. 1993 observed a similar effect for Atlantic salmon smolt only at an exposure to 3765 lg L1 NH3-N, but at much lower water temperature (4C). In the present study full feed strength and exposure to 14 lg L1 NH3-N signicantly increased plasma Na+ concentration. A signicant rise in plasma Na+ was also observed in coho salmon, Oncorhynchus kisutch, exposed to 16 lg L1 NH3-N, suggesting some connection between sodium uptake and ammonia excretion (Buckley, Whitmore & Liming 1979). Restricted feeding and increased water ammonia levels can cause stress (Iwama, Afonso, Todgham, Ackerman & Nakano 2004; Peterson & Small 2004), and plasma glucose has been suggested to be a sensitive parameter in detecting a sublethal stress response in Atlantic salmon (Fivelstad et al. 1993, 1995). A signicant effect of the ammonia treatments on plasma glucose levels was found in the present study, but a clear dose-response relationship was not evident. Several studies on marine teleost species have found no effect of long-term sublethal ammonia exposure on plasma glucose levels (Knoph & Thorud 1996; Foss et al. 2004). Many authors have, however, pointed out the danger of using glucose as the only stress indicator, as its levels can be affected by several other factors (Ortuno, Esteban & Meeguer 2002; Iwama et al. 2004; Cheng, Chen, Liou & Chang 2006;
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Martinez-Porchas, Martinez-Cordova & RamosEnriquez 2009). Feed intake is considered to be a reliable criterion to estimate health and welfare in farmed sh (Srum, Toften & Damsgard 2004). In the present study, cumulative feed intake per sh, and FCR, were not affected by ammonia exposure at the end of the trial. Similarly, the appetite of rainbow trout exposed to 23 ltorr pNH3 (Wood 2004) was identical to their control group throughout the 71 days of exposure, although in this case ammonia had a positive effect on gross food conversion efciency. In contrast, increased ammonia reduced total feed intake in juvenile Atlantic cod and turbot and in the latter case it was suggested to be the cause of an observed reduction in growth rate (Person-Le Ruyet et al. 1997; Foss et al. 2004). The present study indicated no direct relationship between long-term ammonia exposure and an increase in the transcription level of Hsp70 in the gills, in sh on full feed strength. This protein plays a critical role in the general stress response in sh (Iwama, Thomas, Forsythe & Vijayan 1998; Basu, Todgham, Ackerman, Bibeau, Nakano, Schulte & Iwama 2002; Takle, Bverfjord, Lunde, Kolstad & Andersen 2005). Its role during stress is related to a cytoprotective function as it can act to prevent and repair protein damage (Ananthan, Goldberg & Voellmy 1986; Geething & Sambrook 1992). The lack of a Hsp70 stress response in the full fed sh suggests that the tested ammonia levels did not reduce the welfare of the sh in this group. In fact, exposure to 14 lg L1 NH3-N (11 mg L1 TAN), regardless of the feeding level, resulted in almost 50% decrease in mRNA expression level of Hsp70, compared to the control group. In contrast, in sh fed restrictedly, we did observe a signicantly higher Hsp70 transcription in gill of the group exposed to 35 lg L1 NH3-N compared with the control group. Feed restriction has previously been shown to induce HSP70 protein expression in sh (Cara, Aluru, Moyano & Vijayan 2005). Hence, the salmon gill hsp70 expression may be upregulated by high ammonia exposure when the sh is kept at a restricted diet. The examination of welfare indicators, such as n condition, cataracts, skin lesions, gill condition and externally visible/palpable deformities, revealed only a few pathologies. Contrary to feeding levels, ammonia exposure did not appear to have signicant negative effects on the welfare parameters in question. The histopathological evaluations of gill
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tissues suggested that higher levels of environmental ammonia, were associated with minor gill damage after 3 weeks of continuous exposure. This relationship, however, was not apparent at 11 weeks after the onset of ammonia exposure. Hyperplasia, hypertrophy and lamellar fusion have been reported to be the most common effects of elevated water ammonia observed in gills of a number of sh species following exposure (Fivelstad et al. 1993; Frances, Nowak & Allan 2000; Lease, Hansen, Bergman & Meyer 2003; Benli, Koksal & Ozkul 2008; Spencer, Pollock & Dube 2008; Schram, Roques, Abbink, Sparings, Vries, Bierman, Vis & Flik 2010). It has been suggested that such structural changes may lead to a reduced gas-exchange capability of the sh and their physiological impairment (Lease et al. 2003). On the other hand, hypertrophy of the epithelium associated with lamellar fusion has been interpreted as an adaptation to reduce the permeability of the gills and therefore ammonia entry (Schram et al. 2010). Although gill damage was noted in all groups after 22 days of exposure, subsequent exposure did not seem to further deteriorate gill status of Atlantic salmon. Instead, the prevalence of almost all noted anomalies was reduced, indicating again an acclimatory response to a long-term presence of environmental ammonia. Poor water quality during early development can have an adverse effect on smoltication and early marine survival (Wedemeyer, Saunders & Clarke 1980). The potential consequences of ammonia exposure on the smoltication capabilities of Atlantic salmon are largely unknown. Together with gill Na+, K+-ATPase activity, condition factor and plasma chloride levels are the most commonly used smoltication indicators (Wedemeyer et al. 1980; Virtanen 1987; Handeland & Stefansson 2001). In the present study, saltwater challenge tests showed that the plasma chloride levels in all groups were below 150 mM and that the condition factors were signicantly reduced. These results suggest that the experimental sh smoltied and that previous exposure of up to 35 lg L1 NH3-N does not seem to have negative effects on parr-smolt transformation in Atlantic salmon. In conclusion, the Atlantic salmon parr used in this study appeared to be more resilient to ammonia than previously thought for this species and were capable of acclimating and developing responses to increased sublethal levels of this compound. Ammonia tolerance of Atlantic salmon is becoming a current issue with increased use of

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recirculating aquaculture systems (RAS) for smolt production, as the ammonia water concentration is one of the critical parameters for design and dimensioning of bioreactors and required water ows in RAS. Further studies on the effects of ammonia on different Atlantic salmon strains, lifestages and at production relevant densities are, however, necessary to establish adequate rearing water quality criteria that will maximize the welfare and performance of farmed Atlantic salmon.

Acknowledgments This research was nanced by the Research Council of Norway as a part of the Strategic Institute Program Fish welfare and performance in recirculating aquaculture systems (project number 186913). Participation of Olga Felip was nanced by a fellowship from the Government of Spain (FPI 2007). The authors would like to thank the tech nical staff at Noma Sunndalsra and As for taking good care of the experimental sh and for help with laboratory analyses.

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