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There is a high regard for peracetic acid (PAA)-based disinfectants in recirculating aquaculture systems (RAS) because of the low risk of bioaccumulation, fast degradation with neutral residuals and minimal impact on biofilter performance. However, the no-observed-effect concentration in Atlantic salmon parr is unknown. The present study evaluated the effect of an acute PAA exposure on Atlantic salmon parr health and welfare by evaluating survival, swimming behaviour, appetite and histopathological alterations in the gills and skin. Nine experimental RAS units were employed, where each unit was dedicated for one PAA concentration (0.0, 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2 and 6.4 mg/L). Fish were exposed to the target PAA concentration in a static system for 1 h and the exposure protocol was repeated after a 52 h recovery. The fish survival was 100%, 80% and 0%, respectively ≤1.6, 3.2 and 6.4 mg/L. Fish swimming behaviour was normal in PAA ≤ 1.6 mg/L, whereas it become erratic with air gasping for PAA ≥ 3.2 mg/L. The fish appetite did not change among the different PAA treatment groups. Skin and gill histopathological alterations were pronounced in PAA ≥ 3.2 mg/L, characterized by a poorer skin condition and necrotic gill lamella. The skin acidic mucous cells density was 55% lower in the 6.4 mg/L group than the 0 mg/L group. The sub-lethal water pH values observed in the 6.4 mg/L group after PAA administration may have played a confounding and compounding factor to the PAA toxicity response in this group. In conclusion, the current study identified the no-observed-effect concentration for PAA to be below 1.6 mg/L for Atlantic salmon parr and provided insights into its use as a water prophylactic strategy in RAS. Toxicity of PAA-based disinfectants is influenced by its acidified nature, which can interfere with the water pH of low alkalinity aquaculture systems. Further studies should evaluate the health and welfare consequences of a long-term PAA exposure in Atlantic salmon parr.

Simple Summary Atlantic salmon (Salmo salar) has become a commodity worldwide. The culture of Atlantic salmon is by far the most well-developed branch of marine finfish aquaculture, with this species ranking among the top ten most highly produced in global aquaculture. While Atlantic salmon has been commonly farmed in sea cages located in colder waters (e.g., in Norway, Chile and Tasmania), these regions can experience the negative impacts of heat waves that push seawater temperature above values tolerated by this species. These climate-change-driven shifts in water temperature can be associated with mass mortality events and urgent actions are needed to cope with a changing ocean. This paper reviews the thermal limits of adult Atlantic salmon and lists the negative effects driven by heat stress. We highlight how biotechnology and the genetic diversity of wild populations may help producers to tackle this challenge. Selective breeding programs and other more advanced biotechnological solutions (e.g., gene editing) may play a key role in this quest to produce new strains of Atlantic salmon that more readily tolerate higher water temperatures, without compromising productivity and profitability. Abstract Atlantic salmon (Salmo salar) cage farming has traditionally been located at higher latitudes where cold seawater temperatures favor this practice. However, these regions can be impacted by ocean warming and heat waves that push seawater temperature beyond the thermo-tolerance limits of this species. As more mass mortality events are reported every year due to abnormal sea temperatures, the Atlantic salmon cage aquaculture industry acknowledges the need to adapt to a changing ocean. This paper reviews adult Atlantic salmon thermal tolerance limits, as well as the deleterious eco-physiological consequences of heat stress, with emphasis on how it negatively affects sea cage aquaculture production cycles. Biotechnological solutions targeting the phenotypic plasticity of Atlantic salmon and its genetic diversity, particularly that of its southernmost populations at the limit of its natural zoogeographic distribution, are discussed. Some of these solutions include selective breeding programs, which may play a key role in this quest for a more thermo-tolerant strain of Atlantic salmon that may help the cage aquaculture industry to adapt to climate uncertainties more rapidly, without compromising profitability. Omics technologies and precision breeding, along with cryopreservation breakthroughs, are also part of the available toolbox that includes other solutions that can allow cage farmers to continue to produce Atlantic salmon in the warmer waters of the oceans of tomorrow.

Live transport of red king crab from the fishing grounds to the market is an activity that is increasing and the future sustainability of this practice depends on maintaining optimal animal welfare during transport. The current study evaluated the physiological response of red king crab (Paralithodes camtschaticus) to live air transport, mimicking current commercial practices. Specifically, this study assessed stress and osmoregulatory haemolymph metabolite variation, together with free amino acids (FAAs) pool variation in the haemolymph of red king crab. Forty male crabs (2136 ± 800 g) were caught in the North Cape area in the Barents Sea during April 2019. They were transported by the fishing boat directly to a land-based live holding facility in Honningsvarg, Norway where they were held for

Ozone is a strong oxidant, and its use in aquaculture has been shown to improve water quality and fish health. At present, it is predominantly used in freshwater systems due to the high risk of toxic residual oxidant exposure in brackish water and seawater. Here, we report the effects of ozone on Atlantic salmon (Salmo salar) post-smolts (~100 g), in a brackish water (12 ppt) flow-through system. Salmon were exposed to oxidation reduction potential concentrations of 250 mV (control), 280 mV (low), 350 mV (medium), 425 mV (high) and 500 mV (very high). The physiological impacts of ozone were characterized by blood biochemical profiling, histopathologic examination and gene expression analysis in skin and gills. Fish exposed to 425 mV and higher showed &ge, 33% cumulative mortality in less than 10 days. No significant mortalities were recorded in the remaining groups. The skin surface quality and the thickness of the dermal and epidermal layers were not significantly affected by the treatments. On the other hand, gill histopathology showed the adverse effects of increasing ozone doses and the changes were more pronounced in the group exposed to 350 mV and higher. Cases of gill damages such as necrosis, lamellar fusion and hypertrophy were prevalent in the high and very high groups. Expression profiling of key biomarkers for mucosal health supported the histology results, showing that gills were significantly more affected by higher ozone doses compared to the skin. Increasing ozone doses triggered anti-oxidative stress and inflammatory responses in the gills, where transcript levels of glutathione reductase, copper/zinc superoxide dismutase, interleukin 1&beta, and interleukin were significantly elevated. Heat shock protein 70 was significantly upregulated in the skin of fish exposed to 350 mV and higher. Bcl-2 associated x protein was the only gene marker that was significantly upregulated by increasing ozone doses in both mucosal tissues. In conclusion, the study revealed that short-term exposure to ozone at concentrations higher than 350 mV in salmon in brackish water resulted in significant health and welfare consequences, including mortality and gill damages. The results of the study will be valuable in developing water treatment protocols for salmon farming.

Optimal water quality is vital for the growth of Atlantic salmon aquaculture production. Recent data showed that Atlantic salmon feed intake and growth reduce linearly with increasing water carbon dioxide (CO2) concentrations, suggesting that even relatively low concentrations may impact fish performance. This study evaluated the molecular and physiological responses of Atlantic salmon (Salmo salar) to long-term CO2 exposure. For this purpose, Atlantic salmon post-smolts (N = 900; 67 ± 8 g) were exposed to six CO2 treatments (5, 12, 19, 26, 33 and 40 mg/L) for 12-weeks (RAS phase) followed by non-CO2 exposure for a (

Steroid hormones accumulate in recirculation aquaculture systems (RAS) and may influence the reproductive physiology of farmed fish. Ozone reduces hormone concentrations in freshwater RAS used to rear Atlantic salmon, but its effect on reproductive development is unknown. Accordingly, an 8-month trial was carried out to evaluate the growth, health, and maturation of post-smolt Atlantic salmon (296 ± 4 g initial weight) reared in six replicated freshwater RAS (9.5 m3 total volume) operated with or without ozone (N = 3/treatment). Residual ozone was controlled with an oxidation reduction potential (ORP) of 300–320 mV, and mean water temperature was maintained at 14.7 °C. Atlantic salmon growth was generally faster in ozonated RAS. Salmon from RAS with and without ozone weighed 2156 ± 101 and 1810 ± 15 g, respectively, by the end of the study. Caudal, anal, and pelvic fin damage was greater (P < 0.05) for salmon in ozonated RAS early in the trial but improved thereafter. No statistical differences in gill, skin, and skeletal muscle histopathology were observed between treatments at the end of the study. Waterborne estradiol, testosterone, and 11-ketotestosterone levels were periodically lower (P < 0.05) in ozonated RAS, but maturing salmon were more prevalent in these systems. At the end of the trial, percent maturation of salmon populations reared in RAS with and without ozone was 63 ± 7 and 48 ± 1%, respectively; however, maturity appeared to be related to fish size. Improved water quality was observed in ozonated RAS including reduced dissolved copper, iron, and zinc levels, total heterotrophic bacteria counts, and true color, and increased ultraviolet transmittance, which may have supported improved Atlantic salmon growth. Overall, ozone did not inhibit the onset or prevalence of Atlantic salmon maturation, but significant improvements in water quality and salmon growth performance resulted from its use.

High carbon dioxide (CO2) concentrations negatively impact fish, which makes data on its tolerance especially relevant for production systems that can accumulate CO2 such as recirculating aquaculture system (RAS). The current study evaluates the effect of CO2 on the growth performance, welfare, and health of Atlantic salmon post-smolts in RAS. This study consisted of two phases. The first was a CO2 exposure phase, where eighteen tanks were used with six treatments in triplicate: 5, 12, 19, 26, 33 and 40 mg/L of CO2 during 12 weeks in a 12 ppt salinity RAS (hereafter RAS phase). In the second phase, PIT-tagged fish were transferred to a 34 ppt salinity single flow-through tank at CO2 < 5 mg/L (hereafter seawater phase) for an additional 6-week experimental period mimicking a seawater phase. Overall, mortality of fish exposed to CO2 was low and not related to treatments. The mean final body weight was significantly higher in the 5 mg/L treatment compared to CO2 treatments ≥12 mg/L at the end of RAS phase and to CO2 treatments ≥33 mg/L at the end of seawater phase. Moreover, regressions showed that growth significantly decreased linearly with increasing CO2 in the water. Eye cataracts and visible external damage on skin, operculum, and fins were inexistent and similar among CO2 treatments. Kidneys showed no signs of mineral deposits in any of the structures of the tissue. However, skin analysis showed that fish exposed to high CO2 concentrations had a significantly thinner dermis layer (both at the end of RAS and seawater phase) and a significantly thinner epidermis layer and lower mucus cells count (at the end of seawater phase). In conclusion, Atlantic salmon post-smolts cultured in brackish water RAS showed a maximum growth performance at CO2 concentrations below 12 mg/L. Except skin, no major effects of health and welfare were observed, including cataracts and nephrocalcinosis. Further studies should evaluate the molecular and physiological responses to both short-term and long-term carbon dioxide exposure.

We evaluated the effect of low pH and low and high total ammonia nitrogen (TAN) concentrations on the physiology, stress status and the growth performance of turbot in RAS. Two experiments were conducted. In Experiment 1, turbot (466 g) were grown at control (pH 7.5; TAN ~0.5 mg/L) or low pH and high TAN (pH 5.7; TAN ~50 mg/L) for 55 days. In Experiment 2, turbot (376 g) were grown at control (pH 7.5; TAN ~0.5 mg/L), low pH and low TAN (pH 5.7; TAN ~5 mg/L) or low pH and high TAN (pH 5.7; TAN ~50 mg/L) for 59 days. In Experiment 1, final body weight, feed intake and growth were significantly lower and FCR significantly higher in turbot exposed to low pH and high TAN. In Experiment 2, only growth was significantly lower in turbot exposed to treatment low pH and high TAN as compared to fish in the control treatment and low pH and low TAN. Osmoregulation and stress indicators measured were within normal levels. In conclusion, turbot grew equally well in a water pH of 7.5 or 5.7 provided a low TAN. In contrast, low pH combined with a high TAN impaired turbot performance. The effect of low pH on physiology, stress status and growth performance of turbot (Psetta maxima L.) cultured in recirculating aquaculture systems

The accumulation of steroids released by fish in recirculating aquaculture systems (RAS) may potentially influence their physiology and behavior. The present study examined the release rate of cortisol and testosterone by Nile tilapia, Oreochromis niloticus, and their accumulation in six identical lab scale RAS operated at different water exchange rates (150 L/kg feed/day, (LowRAS) and 1500 L/kg feed/day, (HighRAS)) and how steroid accumulation is affected by grading and weighing induced stress. Water cortisol and testosterone concentrations during the experimental period ranged between 1.0 and 5.1 ng/L and between 1.4 and 9.4 ng/L, respectively. Water cortisol concentration was 34% and 43% significantly higher in LowRAS when compared with HighRAS at week 3 and week 4, whereas for water testosterone concentration the two treatments did not differ. Overall steroid release rates were 0.02 +/- 0.02 and 0.04 +/- 0.02 ng/g body weight/h for cortisol and testosterone, respectively. Two hours after grading and weighing water cortisol concentration increased 30% in the LowRAS whereas water testosterone concentration remained unchanged. Calculated cortisol and testosterone discharge from the system were, respectively, 87% and 89% lower in LowRAS than in HighRAS. These findings show that reduced water usage and acute stressors can induce significant accumulation of cortisol in the rearing water at levels close to olfactory detection. Accumulation of metabolites, in particular steroids, should be taken in consideration when designing and managing RAS to prevent exceeding allowable concentrations. Statement of relevance: RAS containing Nile tilapia, cortisol and testosterone are released to the culture water. Water cortisol concentration depends on the water exchange rate that is used and higher concentrations are expected in lower water exchange rates. Fish grading and weighing has a short-term effect on the water cortisol concentrations, after which concentrations return to the basal levels. These findings show that reduction in water usage and fish handling may lead to a significant increase of hormones in the rearing water, which emphasizes the importance of accounting for steroids in the design and management of RAS. (C) 2016 Elsevier B.V. All rights reserved. Portuguese institution FCT - Fundacao para a Ciencia e Tecnologia (QREN-POPH - Advanced Training - European Social Fund) [SFRH/BD/65673/2009] Portuguese institution FCT - Fundacao para a Ciencia e Tecnologia (QREN-POPH - Advanced Training - national funds MEC) [SFRH/BD/65673/2009]

Steroids accumulate in recirculating aquaculture system (RAS), although explanatory factors for such accumulation are still poorly explored. This study investigated the effect of water exchange rate and pH in six replicated RAS on the concentration of the stress hormone cortisol in rainbow trout blood plasma and in the holding water and of the sex steroids testosterone, 11-ketotestosterone (11-KT) and 17,20-dihydroxypregn-4-en-3-one (17,20-P) over a 70-day experimental period. Three combinations of water exchange rate and pH were used each treatment, with two replications: (i) high water exchange rate (+/- 1700Lkg(-1) feed) and neutral pH (+/- 7.3), (ii) low water exchange rate (+/- 500Lkg(-1) feed) and neutral pH (+/- 7.3) and (iii) low water exchange rate (+/- 500Lkg(-1) feed) and low pH (+/- 5.8). Plasma cortisol concentrations at day 70 were higher (24.4 +/- 9.5ngmL(-1)) for fish kept at low pH when compared to fish kept at neutral pH (12.0 +/- 0.1 and 8.7 +/- 0.2ngmL(-1)). Water cortisol and testosterone concentrations at day 35 were higher at low pH than at neutral pH, whereas water 11-KT and 17,20-P did not differ among treatments. At day 70, there were no significant differences between low and high pH. These results demonstrate that low pH contributes to increased plasma cortisol concentrations and to its accumulation in water, possibly indicating a stress response to low pH. The higher concentration of testosterone but not of the other sex hormones point to unspecified reproductive effects that need further investigation. PhD scholarship - FCT - the National Foundation for Science and Technology of Portugal through QREN-POPH Advanced Training European Social Fund and National funds (MEC) [SFRH/BD/65673/2009] info:eu-repo/semantics/publishedVersion

One of the challenges that Recirculating Aquaculture Systems (RAS) are still facing is the risk that in RAS fish grow less than in flow-through systems due to the accumulation of substances originating from feed, fish or bacteria associated with the water re-use. The present study investigated whether RAS with high and low accumulation levels of these substances affect feed intake and growth of Nile tilapia Oreochromis niloticus, African catfish Clarias gariepinus, and European eel Anguilla Anguilla. One-hundred and twenty individuals of each species were used (start body weights: Nile tilapia 264.8 ± 8.3 g; African catfish 253.2 ± 2.1 g and European eel 66.6 ± 1.3 g). For a period of 39 days, growth and feed intake were compared between high and low accumulation RAS. HIGH accumulation RAS was designed for maximal accumulation of substances in the water by operating the system at nearly-closed conditions (30 L/kg feed/d), using mature biofilters and high feed loads; and (2) LOW accumulation RAS was designed to be a proxy for flow-through systems by operating at high water exchange rates (1500 L/kg feed/d), new biofilters and low feed load. HIGH accumulation RAS induced a reduction in feed intake (42%) and growth (83%) of Nile tilapia, as compared to systems that are a proxy for flow-through conditions. This effect was not observed in European eel and African catfish. The cause of this reduced feed intake and growth rate of Nile tilapia is still unclear and should be addressed in further studies.

Little information is available on steroid concentrations in the rearing water of aquaculture systemsand whether they accumulate in recirculating aquaculture systems (RAS). Therefore this study aimedat determining (1) the concentrations and variation of cortisol and sex steroids in RAS, (2) the con-tribution of fish rearing conditions to steroid concentrations in seven commercial RAS. Each RAS wassampled twice at three different points: (1) make-up water; (2) influent and (3) effluent of the rear-ing unit. The results showed significant higher steroid concentrations in the influent and effluent whencompared with the make-up water. On average cortisol concentration was 15.7% higher in the efflu-ent when compared with the influent. Mean steroid concentrations in the rearing unit effluent variedbetween: 3.8–217.0 ng/L for cortisol, 3–12.5 ng/L for testosterone, 0.9–7.1 ng/L for 11-ketoteststerone and1.8–12.8 ng/L for 17,20 -dihydroxypregn-4-en-3-one. Stocking density, Total Ammonia-Nitrogen con-centration and orthophosphate-P concentration (a measure of make-up water usage) showed a positivecorrelation with sex steroids in the water. The steroid concentrations from the present study were ordersof magnitude lower than initial estimations indicating a water treatment efficiency of >99%. The resultssuggest that an intensification of fish production through decrease of make-up water use and increaseof stocking density will lead to a build-up of steroids in the water. Although intensification is critical forthe economical success of RAS, this ultimately could affect fish performance as steroids accumulates inthe water of RAS at levels that can potentially be detected by some fish species. Institution FCT – Fundação para a Ciência e Tecnologia

In allopatric speciation species differentiation generally results from different selective pressures in different environments, and identifying the traits responsible helps to understand the isolation mechanism(s) involved. Male Mozambique tilapia ( Oreochromis mossambicus ) use urine to signal dominance; furthermore, 5β-pregnane-3α,17,20β-triol-3α-glucuronide (and its α-epimer, 5β-pregnane-3α,17,20α-triol-3α-glucuronide), in their urine is a potent pheromone, the concentration of which is correlated with social status. The Nile tilapia ( Oreochromis niloticus ) is a close relative; species divergence probably resulted from geographical separation around 6 million years ago. This raises the question of whether the two species use similar urinary chemical cues during reproduction. The olfactory potency of urine, and crude extracts, from either species was assessed by the electro-olfactogram and the presence of the steroid glucuronides in urine from the Nile tilapia by liquid-chromatography/mass-spectrometry. Both species showed similar olfactory sensitivity to urine and respective extracts from either species, and similar sensitivity to the steroid glucuronides. 5β-Pregnan-3α,17α,20β-triol-3α-glucuronide was present at high concentrations (approaching 0.5 mM) in urine from Nile tilapia, with 5β-pregnan-3α,17α,20α-triol-3α-glucuronide present at lower concentrations, similar to the Mozambique tilapia. Both species also had similar olfactory sensitivity to estradiol-3-glucuronide, a putative urinary cue from females. Together, these results support the idea that reproductive chemical cues have not been subjected to differing selective pressure. Whether these chemical cues have the same physiological and behavioural roles in O. niloticus as O. mossambicus remains to be investigated.

Peracetic acid (PAA), a powerful organic peroxide, is regarded as a rather sustainable disinfectant in aquaculture due to its broad efficacy against various pathogens at low concentrations. PAA-based disinfectants are highly regarded in recirculating aquaculture systems (RAS) due to their low risk of bioaccumulation, rapid degradation with neutral residuals, and minimal impact on biofilter performance. However, the unknown no-observed-effect concentration in Atlantic salmon parr remains a concern. In this study, it is attempted to investigate the decay kinetics of PAA as a disinfecting agent in aquaculture by comparing the consumption of PAA across the analysed samples at two temperatures. The consumption of PAA in two types of sample systems; a. PAA-adapted and PAA-naive RAS water samples, and b. samples with seven levels of feed loads were studied and compared. The effect of PAA treatment on the water quality parameters of TSS, COD and turbidity at two temperatures 12°C and 15°C were quantified and compared as well. Regardless of the treatment temperature, PAA decayed more rapidly in PAA-adapted RAS water samples compared to PAA-naive samples. Higher PAA decay was observed in samples with higher feed loads. Furthermore, PAA treatment resulted in an increase in COD and a significant decrease in TSS and turbidity. The results indicated that the treatment temperature shows a positive temperature-decay correlation for PAA, this was majorly due to the comparatively rapid consumption of PAA immediately after the treatment at 15°C than at 12°C. The effect of temperature on water quality parameters was considerable, with higher temperatures resulting in more significant changes. In summary, the findings of the study can aid in supporting the pulse application of PAA in freshwater RAS because of its implications of an adapted sample system. It also elucidates the contribution of treatment temperature and feed loads in PAA decay.