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Anthropogenic releases of plastics, persistent organic pollutants (POPs), and heavy metals can impact the environment, including aquatic ecosystems. Nanoplastics (NPs) have recently emerged as pervasive environmental pollutants that have the ability to adsorb POPs and can cause stress in organisms. Among POPs, DDT and its metabolites are ubiquitous environmental pollutants due to their long persistence. Despite the discontinued use of DDT in Europe, DDT and its metabolites (primarily p,p′-DDE) are still found at detectable levels in fish feed used in salmon aquaculture. Our study aimed to look at the individual and combined toxicity of NPs (50 mg/L polystyrene) and DDE (100 μg/L) using zebrafish larvae as a model. We found no significant morphological, cardiac, respiratory, or behavioural changes in zebrafish larvae exposed to NPs alone. Conversely, morphological, cardiac and respiratory alterations were observed in zebrafish larvae exposed to DDE and NPs + DDE. Interestingly, behavioural changes were only observed in zebrafish larvae exposed to NPs + DDE. These findings were supported by RNA-seq results, which showed that some cardiac, vascular, and immunogenic pathways were downregulated only in zebrafish larvae exposed to NPs + DDE. In summary, we found an enhanced toxicological impact of DDE when combined with NPs. publishedVersion

Assessing the availability of dietary micro-minerals is a major challenge in mineral nutrition of fish species. The present article aims to describe a systematic approach combining different methodologies to assess the availability of zinc (Zn) in Atlantic salmon (Salmo salar). Considering that several Zn chemical species can be present in an Atlantic salmon feed, it was hypothesised that Zn availability is influenced by the Zn chemical species present in the feed. Thus, in this study, the first protocol is about how to extract the different Zn chemical species from the feed and to analyze them by a size exclusion chromatography-inductively coupled plasma mass spectroscopy (SEC-ICP-MS) method. Subsequently, an in vitro method was developed to evaluate the solubility of dietary Zn in Atlantic salmon feeds. The third protocol describes the method to study the impact of changing Zn chemical species composition on the uptake of Zn in a fish intestinal epithelial model using a rainbow trout gut cell line (RTgutGC). Together, the findings from the in vitro methods were compared with an in vivo study examining the apparent availability of inorganic and organic sources of Zn supplemented to Atlantic salmon feeds. The results showed that several Zn chemical species can be found in feeds and the efficiency of an organic Zn source depends very much on the amino acid ligand used to chelate Zn. The findings of the in vitro methods had less correlation with that outcome of the in vivo study. Nevertheless, in vitro protocols described in this article provided crucial information regarding Zn availability and its assessment in fish feeds. publishedVersion

Background: Antimicrobials revolutionized human as well as animal medicine in the 20th century by providing effective treatment of diseases caused by pathogenic microorganisms. However, microorganisms have the ability to develop antimicrobial resistant strains. This occurs when microorganisms mutate or when resistance genes are exchanged between them. The use of antimicrobial drugs accelerates the emergence of drug-resistant strains. A priority is to safeguard the efficacy of antimicrobial drugs we depend on for treatment of infectious diseases in humans. Use of antimicrobials in food animals can create a source of antimicrobial resistant bacteria that can spread to humans both by direct contact and through the food supply. Coccidiosis is an intestinal disease in animals caused by unicellular parasites called coccidia. As most of the damage of this infection is done by the time signs of the disease are widespread, preventive measures are preferred. Coccidiostats are animal feed additives used to prevent coccidiosis by inhibiting or killing coccidia. There are two major groups of coccidiostats; ionophores and non-ionophores, the latter also referred to as “non-ionophore coccidiostats” (but also called chemicals). One main difference between these groups is that ionophores also inhibit or kill some bacterial species, whereas non-ionophore coccidiostats do not. Consequently, some bacterial infections may also be controlled by ionophore coccidiostats, e.g. the poultry disease necrotic enteritis caused by the bacterium Clostridium perfringens (C. perfringens). Eleven different coccidiostats have been authorised for use in the EU, both ionophores and non-ionophore coccidiostats. Norway has been exempted from the EEA Agreement in this field and has approved only five; all ionophores. The two ionophore coccidiostats currently used in Norway are narasin for broilers and monensin for turkeys. Resistance to coccidiostats in coccidia and bacteria: Development of resistance in coccidia to all eleven coccidiostats has been described in the scientific literature, but the prevalence of resistance is unknown. Cross-resistance between various ionophore coccidiostats has also been shown, i.e. development of resistance to one ionophore may also render the coccidia resistant to another ionophore. Various rotation and shuttle programmes with exchange between ionophores and non-ionophore coccidiostats are believed to prevent or delay development of resistance in coccidia. In Norway, such programmes will have little effect as long as only ionophores and not non-ionophore coccidiostats are approved for use. Development of resistance against ionophores has also been observed in bacteria. In the Norwegian surveillance programme NORM-VET during the years 2002 - 2013, between 50 - 80% of the tested flocks had narasin resistant faecal enterococci, which are bacteria that are part of the normal intestinal microbiota. However, the pathogenic bacterium C. perfringens has not been shown to be resistant against any ionophore. Cross-resistance in bacteria to more than one ionophore has been observed. In addition, a limited amount of data may indicate an association between narasin and resistance to the antibacterials bacitracin and vancomycin. As these are antibacterials used for treatment in humans, more research should be performed to validate these results. Non-ionophore coccidiostats, which do not have antibacterial effect, are not approved in Norway. If such coccidiostats were approved in Norway, coccidiostats with negligible probability of inducing resistance in bacteria would be available. Human exposure to resistant bacteria and coccidiostats: Humans may theoretically be exposed to coccidiostat resistant bacteria from poultry in a number of ways, e.g. by handling live animals and their manure, through slaughtering and processing, and by preparation and consumption of poultry meat. Furthermore, bacteria of the human normal microbiota, which cover all skin and mucosal surfaces, might develop resistance if they are exposed to coccidiostats. In this assessment, the probabilities of exposure are classified as: Negligible (extremely low), Low (possible, but not likely), Medium (likely), High (almost certain) and Not assessable. The Panel has estimated the following probabilities of human exposure: Handling manure from coccidiostat fed poultry without sufficient risk-reducing measures entails a high probability of exposure to both resistant bacteria and coccidiostats. Without proper protection, the probability of exposure to coccidiostats is also high when handling coccidiostat premixes and feeds containing coccidiostats without proper protection measures. Various treatments, e.g. composting, of the manure may reduce the probability. The probability of exposure to resistant bacteria is medium for workers handling carcasses and raw meat on a daily basis if risk-reducing measures are not applied, whereas the probability of exposure to coccidiostats is negligible. For consumers, the probability of exposure to coccidiostats is negligible. The probability for exposure to resistant bacteria is also negligible in heat treated food since heat treatment kills the bacteria. The probability of exposure to coccidiostat resistant bacteria is low to medium if handling raw meat without proper hygienic procedures, because raw meat may harbour resistant bacteria. Risk-reducing measures will lower the probabilities. However, little is known concerning the consequences of human exposure to coccidiostat resistant bacteria or to to coccidiostats. There is little information in scientific literature indicating whether such bacteria in fact will colonize the human body, either transitionally or permanently. Furthermore, there is no information on the probability of exchange of resistance genes from transferred bacteria to bacteria of the human natural microbiota or to pathogens. Likewise, the Panel has no information on the level of exposure, e.g. the amount of coccidiostats and their metabolites, or the time period, necessary for the various bacteria to give rise to resistant variants. As coccidiostats are not used to treat infectious diseases in humans, concern of resistance is related to possible cross- or co-resistance with antibacterials considered important in human medicine. Such resistance has so far not been confirmed. Use of therapeutic antibacterials for poultry: If the ionophore coccidiostats used in Norway are replaced by one or more non-ionophore coccidiostat with no antibacterial effect and no other changes are done, the coccidiostats used will no longer inhibit the bacterium Clostridium perfringens, which is the cause of necrotic enteritis. Over time this will likely to lead to a need for intermittent or continuous use of higher levels of therapeutic antibacterials due to increased incidence of this desease in poultry production. The magnitude of the increase is difficult to predict. Alternatives to in-feed antimicrobials: Eradication from the birds’ environment of coccidia causing coccidiosis is difficult to achieve because the coccidia form oocysts that survive outside the host and resist commonly used disinfectants. Vaccination with non-pathogenic vaccines is now used increasingly in commercial Norwegian broiler farms, instead of in-feed coccidiostats. So far coccidiosis has not been reported as a problem in this transition process to broiler rearing without in-feed coccidiostats in Norway. Non-antimicrobial feed additives with purported health-promoting benefits, i.e. acid-based products, probiotics, prebiotics, synbiotics, yeast-based products, plant-derived products, combinations of these, and other products have been developed and used in feed. These products have been tested for efficacy against coccidia with conflicting, non-consistent or non-convincing results. The majority of these products appear to target the bacterial microbiota rather than coccidia. The Panel has not assessed possible effects of other types of management changes.

Sustainable ingredients for animal feed are becoming scarcer. Insects have emerged as a promising protein and lipid ingredient for fish feed, and black soldier fly (BSF; Hermetia illucens) larvae in particular have great potential to efficiently convert organic matter into high value protein and fat. Seaweeds are a sustainable source of organic matter and complex carbohydrates, but can also provide marine long chain polyunsaturated fatty acids for fly larvae, and therefore could offer a commercially attractive alternative to traditional aquafeeds. However, pathogenic bacteria and faecal indicator organisms (FIOs) readily attach to seaweeds, therefore before this novel BSF larvae feed ingredient is advocated, microbiological risk assessments are warranted to ensure animal and public health protection from farm-to-fork. In this study, screening of raw materials and finished products during formulation of experimental insect meal fish feed was undertaken to evaluate the potential for the introduction of selected bacterial pathogens and FIOs via seaweed substrate to BSF larvae, and subsequent survival during multiple manufacturing processing stages. Processed seaweed powder was found to be a microbiologically safe feed substrate for BSF larvae. Low levels of FIOs were associated with larvae at the point of harvest, although larvae meal and extracted lipids were free of FIOs immediately after processing. During handling, distribution and storage the larvae meal and other externally sourced raw feed ingredients for larvae rearing and feed pellet formation became contaminated with FIOs and Listeria spp. FIOs were also present, albeit at very low levels, in the finished feed pellets. Processing treatments provided effective decontamination, and FIO and pathogen concentrations in finished products never exceeded microbiological quality standards for insect processed animal proteins. Microbiological contamination of raw materials and finished products during packaging and distribution, or originating from production environments, were identified as critical control points, requiring assessment to ensure good hygiene practices.

Seafood can be a source of contaminants, which may raise health concerns. The aim of this study was to analyse the levels of inorganic contaminants in commercially available seafood products and assess consumer exposure. Commercially available samples were collected from 2015-2018 and analysed as composite samples for mercury, lead, arsenic, and cadmium, using accredited methods. Levels of cadmium, lead, and arsenic were low and human exposure of these metals would be minimal from consumption of the analysed seafood products. Mercury levels were well below the EU maximum limit for mercury in fish. However, children, who are high consumers, might be at risk of exceeding the tolerable weekly intake for methyl mercury, when eating products with the highest mercury levels. The collected data can be used for future risk-benefit assessments as intake of processed seafood products represent a large proportion of the populations' seafood intake in Europe.

Chemical contaminants are present in all foods. Data on the occurrence of contaminants in foods that are often consumed or contain high contaminant concentrations are critical for the estimation of exposure and evaluation of potential negative health effects. Due to limited resources for the monitoring of contaminants and other chemical substances in foods, methods for prioritisation are needed. We have developed a straightforward semi-quantitative method to rank chemical substances in foods for monitoring as part of a risk-based food control. The method is based on considerations of toxicity, level of exposure including both occurrence in food and dietary intake, vulnerability of one or more population groups due to high exposure because of special food habits or resulting from specific genetic variants, diseases, drug use or age/life stages, and the adequacy of both toxicity and exposure data. The chemical substances ranked for monitoring were contaminants occurring naturally, unintentionally or incidentally in foods or formed during food processing, and the inclusion criteria were high toxicity, high exposure and/or lack of toxicity or exposure data. In principle, this method can be used for all classes of chemical substances that occur in foods, both unintended contaminants and deliberately added chemical substances. Foods considered relevant for monitoring of the different chemical substances were also identified. The outcomes of ranking exercises using the new method including considerations of vulnerable groups and adequacy of data and a shortened version based on risk considerations only were compared. The results showed that the resolution between the contaminants was notably increased with the extended method, which we considered as advantageous for the ranking of chemical substances for monitoring in foods.

Farmed insects can offer an environmentally sustainable aquafeed or livestock feed ingredient. The value of black soldier fly (Hermetia illucens) (BSF) larvae could be improved by enrichment in omega-3 through the dietary inclusion of seaweed. However, the industry practice of drying seaweed at low temperatures to retain nutritional properties may benefit the survival of human pathogenic bacteria, particularly if the seaweed has been harvested from contaminated water. Here we have demonstrated that E. coli and E. coli O157:H7 died-off in seaweed dried at 50 °C, although both were detected in the dried powder following 72 h storage. V. parahaemolyticus fell below the level of detection in stored seaweed after drying at ≥ 50 °C, but L. monocytogenes remained detectable, and continued to grow in seaweed dried at ≤60 °C. Therefore, drying seaweed at low temperatures risks pathogen carry-over into insects destined for animal feed. BSF larvae reared on an artificially contaminated seaweed-supplemented diet also became contaminated by all four bacteria present in the supplement. Water quality at seaweed harvesting sites, seaweed desiccation, and insect rearing practices, represent critical points where development of regulatory standards could achieve targeted control of pathogenic hazards.

Background The determination of dietary mineral solubility is one of the main steps in the evaluation of their availability for a given species. Methods This study proposed an in vitro digestion method (acidic and alkaline hydrolysis). The method was applied to evaluate the solubility of inorganic and organic forms of zinc (Zn), selenium (Se) and manganese (Mn) in salmonid diets. An inorganic mineral (IM) diet was supplemented with zinc sulphate, sodium selenite and manganous sulphate and an organic mineral (OM) diet was supplemented with zinc chelate of glycine, l -selenomethionine and manganese chelate of glycine. Results The solubility of Zn was similar in both diets tested. The amount of soluble Zn was low in the acidic hydrolysis (3–8%) and lower in the alkaline hydrolysis (0.4–2%). The solubility of Se was higher in the OM diet (7–34%) compared with the IM diet (3–12%). Regarding Mn, after the acidic hydrolysis the solubility was higher in the IM diet (6–25%) than the OM diet (4–17%). The in vitro solubility were compared with in vivo availability of Zn, Se and Mn. Data obtained for solubility (%) of Zn, Se and Mn was lower when compared with apparent availability (%) of Zn, Se and Mn. Conclusion Data obtained demonstrated that solubility of Zn, Se and Mn was influenced by the mineral chemical form supplemented to the diet and by the gastrointestinal environment. The solubility of Zn, Se and Mn was not comparable with the apparent availability of Zn, Se and Mn. Nevertheless, the effect of the chemical form of the minerals was similar for the solubility of Zn, Se and Mn and the apparent availability of Zn, Se and Mn. Considering the overall results of this study, the in vitro method could replace some of the in vivo studies for a qualitative evaluation but not for a quantitative evaluation.

The use of plant-based feeds warrants the supplementation with selenium (Se) to cover the requirement for Atlantic salmon. Depending on its chemical form, Se is a trace element with a narrow range between requirement and toxicity for most vertebrates. Information on safe upper limit for Atlantic salmon feed supplementation is lacking. Atlantic salmon (147 g) were fed a low natural background organic Se diet (0.45 mg Se kg−1, wet weight (ww)) fortified with 5 graded levels of inorganic sodium selenite (0.45, 5.4, 11.0, 29.4, or 60.0 mg kg−1 ww) or organic selenomethionine (SeMet) (0.45, 6.2, 16.2, 21, or 39 mg kg−1 ww), in triplicate for 3 months. Excess Se supplementation was assessed by targeted biomarkers of Se toxicity pathways (e.g. markers of oxidative stress and lipid metabolism), as well as general adverse effect parameters (plasma biochemistry, hematology, liver histopathology, and growth). Safe limits were set by model-fitting the effect data in a dose-response (lower bound) bench mark dose (BMDL) evaluation. Fish fed the two highest selenite levels showed mortality while fish fed SeMet had no mortality. Fish fed 5.4–11 mg selenite kg−1 feed showed significantly (ANOVA, Tukey's t-test, p

The use of the synthetic antioxidant ethoxyquin (6-ethoxy-2,2,4-trimethyl-1,2-dihydroquinoline; EQ) in animal feed results in the presence of EQ residues and metabolites, including the EQ dimer (1,8′-bi(6-ethoxy-2,2,4-trimethyl-1,2-dihydroquinoline); EQDM) in animal food products. To investigate the toxicity and dose-response of dietary exposure to EQDM, male BALB/c mice were exposed to one of six dietary doses of EQDM, ranging from 0.015 to 518 mg/kg body weight/day for 90 days. Doses above 10 mg/kg body weight/day affected whole body lipid metabolism resulting in increased liver weights and decreased adipose tissue mass. Metabolomic screening of livers revealed alterations indicating incomplete fatty acid β-oxidation and hepatic oxidative stress. Histopathological evaluation and biochemical analyses of the liver confirmed the development of microvesicular steatosis and activation of the glutathione system. Hepatic protein profiling and pathway analyses suggested that EQDM-induced responses are mediated through activation of CAR/PXR nuclear receptors and induction of a NRF2-mediated oxidative stress response. Based on the development of microvesicular steatosis as the critical endpoint, a Reference Point for dietary EQDM exposure was established at 1.1 mg/kg body weight/day (BMDL10) from benchmark dose modelling. Applying an uncertainty factor of 200, an Acceptable Daily Intake of 0.006 mg EQDM/kg body weight was proposed.

Selenium (Se) is an essential element for animals, including fish. Due to changes in feed composition for Atlantic salmon (Salmo salar), it may be necessary to supplement feeds with Se. In the present work, the transfer of Se and Se species from feed to muscle of Atlantic salmon fed Se supplemented diets was studied. Salmon were fed basal fish feed (0.35 mg Se/kg and 0.89 mg Se/kg feed), or feed supplemented either with selenised yeast or sodium selenite, at low (1–2 mg Se/kg feed) and high (15 mg Se/kg feed) levels, for 12 weeks. For the extraction of Se species from fish muscle, enzymatic cleavage with protease type XIV was applied. The extraction methods for Se species from fish feed were optimised, and two separate extraction procedures were applied, 1) enzymatic cleavage for organic Se supplemented feeds and 2) weak alkaline solvent for inorganic Se supplemented feeds, respectively. For selenium speciation analysis in feed and muscle tissue anion-exchange HPLC-ICP-MS for analysis of inorganic Se species and cation-exchange HPLC-ICP-MS for analysis of organic Se species, were applied. In addition, reversed phase HPLC-ICP-MS was applied for analysis of selenocysteine (SeCys) in selected muscle samples. The results demonstrated that supplemented Se (organic and inorganic) accumulated in muscle of Atlantic salmon, and a higher retention of Se was seen in the muscle of salmon fed organic Se diets. Selenomethionine (SeMet) was the major Se species in salmon fed basal diets and diets supplemented with organic Se, accounting for 91–118% of the total Se. In contrast, for muscle of salmon fed high inorganic Se diet, SeMet accounted for 30% of the total Se peaks detected. Several unidentified Se peaks were detected, in the fish fed high inorganic diet, and analysis showed indicated SeCys is a minor Se species present in this fish muscle tissue.

Contaminants and fatty acid levels in farmed- versus wild Atlantic salmon have been a hot topic of debate in terms of food safety. The present study determined dioxins (polychlorinated dibenzo-p-dioxin and dibenzofuran), polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs), organochlorine pesticides (OCPs), metals and fatty acids in wild and farmed Atlantic salmon. Contaminant levels of dioxins, PCBs, OCPs (DDT, dieldrin, lindane, chlordane, Mirex, and toxaphene), and mercury were higher in wild salmon than in farmed salmon, as were the concentrations of the essential elements selenium, copper, zinc and iron, and the marine omega-3 fatty acid docosahexaenoic acid (DHA). PBDE, endosulfan, pentachlorobenzene, hexachlorobenzene, cadmium and lead levels were low and comparable in both wild and farmed fish, and there was no significant difference in the marine omega-3 fatty acid eicosapentaenoic acid (EPA) concentration. The total fat content was significantly higher in farmed than wild salmon due to a higher content of both saturated and monounsaturated fatty acids, as well as a higher content of omega-6 fatty acids. The omega-3 to omega-6 fatty acid ratio was considerably lower in farmed than wild salmon due to the high level of omega-6 fatty acids. Contaminant concentrations in Atlantic salmon were well below maximum levels applicable in the European Union. Atlantic salmon, both farmed and wild, is a good source of EPA and DHA with a 200g portion per week contributing 3.2g or 2.8g respectively, being almost twice the intake considered adequate for adults by the European Food Safety Authority (i.e. 250mg/day or 1.75g/week).

Insects are promising sources of protein and lipid in feeds for farmed animals. In the European Union, the use of insect meal (IM) and insect oil is permitted in fish feed. However, the European Food Safety Authority has highlighted the lack of data regarding the chemical safety of insects and products thereof. In this study, Atlantic salmon (

Ethoxyquin (EQ) is an antioxidant supplemented to feed ingredients, mainly fish meal, which is currently under re-evaluation for use in the food production chain. EQ is partly metabolized into several metabolites of which the ethoxyquin dimer (EQDM) accumulates most in the farmed fish fillet. In this study, the feed-to-fillet transfer of dietary EQ and EQDM in Atlantic salmon fillet was investigated, and a physiologically based pharmacokinetic (PBPK-) two-compartmental model was developed, based on experimental determined EQ and EQDM uptake, metabolism, and elimination kinetics. The model was verified with an external data-set and used to simulate the long term (>1.5 years) EQ and EQDM feed-to fillet transfer in Atlantic salmon under realistic farming conditions such as the seasonal fluctuations in feed intake, growth, and fillet fat deposition. The model predictions showed that initial EQDM levels in juvenile fish are the driving factor in final levels found in food-producing animals, while for EQ the levels in feed, and seasonal variations were the driving factor for food EQ levels.

The composition of salmonid diets has changed from the use of mainly marine ingredients to increased use of plant ingredients, and this has an impact on the mineral content and availability. Minerals, like zinc (Zn), selenium (Se) and manganese (Mn), are supplemented to diets as inorganic or organic forms to cover the nutritional requirements of fish. This study compared the apparent availability (AA) of Zn, Se and Mn from inorganic metal salts and their organic forms in Atlantic salmon. Sixteen diets were prepared based on a two-level factorial design (24). The tested factors were Zn additive source (A), Se additive source (B), Mn additive source (C) and phytic acid level (D). The diets were fed to Atlantic salmon for 11 days, faeces were collected by stripping, and the total content of mineral and yttrium in diets and faeces were determined by inductively coupled plasma mass spectrometry. Data obtained were used to estimate the AA for the minerals. Zinc additive source had no significant effect on the AA of Zn. However, the Se and Mn additive source had significant effects on the AA of Se and Mn, respectively. Higher AA of Se was achieved with selenomethionine than with selenite, and Mn sulphate was more available than Mn chelate of glycine. The phytic acid level did not significantly affect the AA of Zn, Se or Mn. However, several interactions between mineral additive sources and the phytic acid level significantly affected the AA of Zn, Se and Mn. publishedVersion

Ethoxyquin (EQ; 6-Ethoxy-2,2,4-trimethyl-1,2-dihydroquinoline) has been used as an antioxidant in feed components for pets, livestock and aquaculture. However, possible risks of EQ used in aquafeed for fish health have not yet been characterized. The present study investigated the toxicity and dose-response of subchronic dietary EQ exposure at doses ranging from 41 to 9666 mg EQ/kg feed in Atlantic salmon (Salmo salar L.). Feed at concentrations higher than 1173 mg EQ/kg were rejected by the fish, resulting in reduced feed intake and growth performance. No mortality was observed in fish exposed to any of the doses. A multi-omic screening of metabolome and proteome in salmon liver indicated an effect of dietary EQ on bioenergetics pathways and hepatic redox homeostasis in fish fed concentrations above 119 mg EQ/kg feed. Increased energy expenditure associated with an upregulation of hepatic fatty acid β-oxidation and induction and carbohydrate catabolic pathways resulted in a dose-dependent depletion of intracytoplasmic lipid vacuoles in liver histological sections, decreasing whole body lipid levels and altered purine/pyrimidine metabolism. Increased GSH and TBARS in the liver indicated a state of oxidative stress, which was associated with activation of the NRF2-mediated oxidative stress response and glutathione-mediated detoxification processes. However, no oxidative DNA damage was observed. As manifestation of altered energy metabolism, the depletion of liver intracytoplasmic lipid vacuoles was considered the critical endpoint for benchmark dose assessment, and a BMDL10 of 243 mg EQ/kg feed was derived as a safe upper limit of EQ exposure in Atlantic salmon.

This study investigated the transfer kinetics of dietary selenite and selenomethionine (SeMet) to the fillet of farmed Atlantic salmon (Salmo salar). The uptake and elimination rate constants of the two selenium (Se) forms were determined in Atlantic salmon fed either selenite- or SeMet-supplemented diets followed by a depuration period. The fillet half-life of selenite and SeMet was 779 ± 188 and 339 ± 103 days, respectively. The elimination and uptake rates were used in a simple one-compartmental kinetic model to predict levels in fillet based on long-term (whole production cycle) feeding with given dietary Se levels. Model predictions for Atlantic salmon fed plant-based feeds low in natural Se and supplemented with either 0.2 mg of selenite or SeMet kg-1 gave a predicted fillet level of 0.042 and 0.058 mg Se kg-1 wet weight, respectively. Based on these predictions and the European Food Safety Authority risk assessment of Se feed supplementation for food-producing terrestrial farm animals, the supplementation with 0.2 mg of selenite kg-1 would likely be safe for the most sensitive group of consumers (toddlers). However, supplementing feed to farm animals, including salmon, with 0.2 mg of SeMet kg-1 would give a higher (114%) Se intake than the safe upper intake limit for toddlers.

The impact of dietary manganese (Mn) levels and sources on the growth and mineral composition of post-smolt Atlantic salmon (Salmo salar) fed practical diets was studied. Seven experimental diets were prepared with graded supplementation level of Mn; basal diet had a Mn concentration of 15 mg kg−1, four diets with 5, 15, 35 and 65 mg kg−1 supplementation of Mn as MnSO4 and two diets with at 5 and 15 mg kg−1 supplementation of Mn as Mn-glycine (Mn-gly). Atlantic salmon (initial weight, 307 ± 25 g) were distributed to 21 tanks (35 fish/tank). These fish were randomly fed with one of the experimental diets, in triplicate groups to apparent satiation for 8 weeks. At week 4 and 8, samples of whole body, plasma, bile, liver and vertebrae were collected, and their mineral concentration determined. At week 8, feaces were collected by stripping for measuring apparent availability coefficient (AAC). Neither the Mn inclusion level, nor the source had a significant impact on growth and other performance indicators. Plasma and bile Mn concentrations were significantly affected by dietary treatments at both sampling points, at week 4 and 8; whereas, whole body, liver and vertebrae responded significantly only at the end of 8 weeks. Dietary Mn level needed to meet the requirement of post-smolt Atlantic salmon was estimated by non-linear regression models using saturation of whole body or tissue Mn status as response criteria. The estimates based on Mn concentration in whole body, vertebrae, plasma and apparent availability were 29.5 ± 5.3, 26.2 ± 2.7, 26.3 ± 4.9 and 34 ± 3.4 mg Mn kg−1 diet, respectively. Analysis of relative bioavailability index showed that low inclusion of Mn-gly (5 mg kg−1 supplementation) was 2.6 to 4.5-fold more efficient than MnSO4 to attain whole body or tissue Mn saturation levels. On the contrary, high inclusion of Mn-gly (15 mg kg−1 supplementation) reduced the AAC of Mn, zinc (Zn) and copper (Cu), along with lower Mn and Zn status in tissues. The mean estimate of dietary Mn required to maintain tissue Mn saturation ranged between 26 and 34 mg Mn kg−1 diet; the corresponding Mn supplementation (as MnSO4) ranged from 14 to 22 mg Mn kg−1 diet. The supplementation level can be reduced to 4.9 to 5.7 mg Mn kg−1 diet by using Mn-gly as Mn source without compromising growth or Mn status of Atlantic salmon. High inclusion levels of Mn-gly (15 mg kg−1 diet) was found to be not beneficial.

Insects are promising sources of protein and lipid in feeds for farmed animals. In the European Union, the use of insect meal (IM) and insect oil is permitted in fish feed. However, the European Food Safety Authority has highlighted the lack of data regarding the chemical safety of insects and products thereof. In this study, Atlantic salmon (Salmo salar) were fed diets in which fish meal (FM) was partially or fully substituted with IM, resulting in four diets with an FM replacement of 0%, 33%, 66% and 100% by IM. The IM was produced from Black soldier fly (Hermetia illucens) larvae fed media containing 60% seaweed (Ascophyllum nodosum). After 16 weeks of feeding, fish fillet samples were collected. The concentrations of undesirable substances, e.g., heavy metals, arsenic, dioxins, mycotoxins, pesticides, in the IM, the diets and fillets were determined. The concentrations of the analysed compounds in the IM were all below EU maximum levels for feed ingredients, except for arsenic. However, for complete feeds the concentrations of these compounds in the feeds, including arsenic, were all below EU MLs. Arsenic was transferred from seaweed to IM, resulting in arsenic levels in IM similar to what has been documented for FM. Transfer of arsenic from feed to fillet was observed; however, total arsenic concentrations in the fillet significantly decreased when fish were fed diets with more IM and less FM. Arsenic speciation analysis of the diets showed that although total arsenic levels were similar, the arsenic species were different. Arsenobetaine was the major organoarsenic species in the diets containing FM, while in diets containing IM several unidentified arsenic species were detected. The results suggest that the lower feed-to-fillet transfer of arsenic when FM is replaced by IM may be due to the presence of arsenic species with low bioavailability in the IM. publishedVersion

Ethoxyquin (EQ) is an additive present in fish feed and its fate in fish should be carefully characterized due to food safety concerns regarding this compound. Therefore, the objective of this work was to identify the transformation products (TPs) of EQ in Atlantic salmon. Salmon in independent tanks were given feed containing ethoxyquin concentrations of 0.5 mg/kg, 119 mg/kg or 1173 mg/kg for 90 days. After the feeding trial, salmon fillets were extracted in acetonitrile and analyzed by liquid chromatography with traveling-wave ion mobility spectrometry coupled to high resolution mass spectrometry (UHPLC-TWIMS-QTOFMS). EQ was transferred from the feed to salmon fillets and 23 TPs were characterized, resulting from dimerization, oxygenation, cleavage, cleavage combined with oxygenation, cleavage combined with conjugation, and other uncategorized alterations. Moreover, EQ and some TPs were also detected in commercial salmon randomly sampled from different Norwegian fish farms. This study confirmed that the dimer 1,8′-EQDM was the main TP of EQ and, together with previous research, brought the overall number of characterized TPs to a total of 47.

The aim of this study was to test the efficacy of a new marine P ingredient from the bone fraction of herring by-products by evaluating the dietary impacts on growth, mineralization and skeletal development in Atlantic salmon fry. Five experimental diets were produced from a fish meal based diet, only differing in the dietary P source and level; low P control (5.5 g/kg soluble P), intermediate P (6.5 g/kg soluble P) and high P (8.0 g/kg soluble P) from either fish bone hydrolysate (FBH1, FBH2) or from NaH 2 PO 4 (NaP1, NaP2). The diets were given for 168 days from start of feeding (0.17 g fish) until 24 weeks of feeding (33 g). Weight and specific growth rate did not reveal diet dependent differences except in the initial 10 week feeding period where fish fed FBH1 showed lower growth as compared to fish fed the Na–P diets. Mineralization of fish evaluated by whole body and bone ash and mineral contents showed diet dependent differences that were related to dietary P level, but not to P source. Fish fed the low P diet showed clear P deficiency signs with significantly reduced tissue ash and mineral content, reduced whole body Ca:P ratio and morphological deviation from the normal. Histological evaluation of the vertebrae after 19 weeks of feeding (15 g) revealed stagnation in cartilage development, with accumulation of mature chondrocytes in fish fed low P control and FBH1 diet. FTIR showed that fish fed FBH1 had lower mineralization and increased cross binding in the vertebral end plates. Fish fed FBH2 resembled the positive NaP1 control. Real time qPCR analyses confirmed the histological results, by showing up-regulating of col10a1 (a marker for mature cartilage) in fish fed low P diet and FBH1. Osteocalcin (a marker for mineralization) was also activated in fish fed the low P diet and FBH1, possibly indicating a compensatory regulation in response to inadequate P. The dietary impacts on histology, FTIR and qPCR analyses in the FBH1 fed fish disappeared at 24 weeks of feeding (33 g). In conclusion, the results showed that P solubilized from bone fraction of herring can be efficiently utilized for growth, mineralization and bone development in Atlantic salmon fry. However, in the initial weeks of feeding, the FBH was a less efficient P source compared to NaP. Overall, the data suggest that P from fish bone hydrolysate is more suited for fish of minimum 15 g size. Statement of relevance The phosphate (P) rock reserve is a limited resource worldwide. Increased efficiency in the utilization of P and improved recycling of P from waste and manures are examples that could reduce the industry's vulnerability to the limited P supply while also reducing the negative environmental impacts. Recycling of P from fishery offal and development of new available P ingredients to farmed fish will significantly improve the sustainability of the aquaculture industry and reduce the environmental loss. In this paper we show that P hydrolysed from herring bone by-product is as efficient and good for salmon fry as commercially available NaP-salts.

The European Food Safety Authority (EFSA) published a risk assessment of erucic acid (22:1n-9) in 2016, establishing a Tolerable Daily Intake (TDI) for humans of 7 mg kg&minus, 1 body weight per day. This report largely excluded the contribution of erucic acid from fish and seafood, due to this fatty acid often not being reported separately in seafood. The Institute of Marine Research (IMR) in Norway analyzes erucic acid and has accumulated extensive data from analyses of fish feeds, farmed and wild fish, and seafood products. Our data show that rapeseed oil (low erucic acid varieties) and fish oil are the main sources of erucic acid in feed for farmed fish. Erucic acid content increases with total fat content, both in farmed and wild fish, and it is particularly high in fish liver, fish oil, and oily fish, such as mackerel. We show that the current TDI could be exceeded with a 200 g meal of mackerel, as at the maximum concentration analyzed, such a meal would contribute 143% to the TDI of a 60 kg person. These data cover a current knowledge gap in the scientific literature regarding the content of erucic acid in fish and seafood.

Apical uptake of zinc as ionic Zn(II) or as Zn-methionine (Zn-Met) was studied in RTgutGC cell line in vitro under media compositions mirroring the gut luminal ionic concentration of freshwater (FW) and seawater (SW) acclimated salmonids. Viability of the RTgutGC cells exposed to experimental media preparations showed a time-dependent decrease in SW treated cells, with the effect being significant at 48 h (P

Access to marine feed ingredients is considered a limiting factor for the rapidly growing aquaculture industry. With increasing inclusion of vegetable feed ingredients in aquafeeds, new challenges have arisen. Substituting marine oils and meals with vegetable ingredients has decreased the level of persistent organic pollutants (POPs) in marine farmed fish such as Atlantic salmon. Consequently, this shift in feed ingredients has led to increased levels of polyaromatic hydrocarbons (PAHs) in aquafeeds. Several of the PAHs listed are on EFSAs and EPAs list of priority substances. Not only are the carcinogenic and genotoxic properties of PAHs of concern, but also their ability to interact with and disrupt metabolic pathways such as vitamin metabolism and signalling. The interactions, and the potential depletion of vitamin stores that follows, are of particular interest as marine ingredients are naturally rich in micronutrients such as vitamins A and D. This study has summarized the current knowledge on the effect of PAHs commonly found in vegetable feed ingredients on vitamin metabolism/signalling and their possible implications in fish.

Triplicate groups of triploid and diploid Atlantic salmon were fed diets with a low (LP, total P: 7.1 g kg−1), medium (MP, total P: 9.4 g kg−1) or high (HP, total P: 16.3 g kg−1) phosphorous (P) level from first feeding (0.18 g) to transfer to sea water (~50 g, duration: 203 days) and subsequently fed a commercial diet in sea water for 426 days (~3 kg). This study examined the short- and long-term effects of dietary P on freshwater performance (mortality, growth), vertebral deformities (radiology), bone cell activity (ALP and TRACP enzyme activity in vertebrae and scales, and fgf23, bgp and igf-I relative gene expression in vertebrae), bone mineralization (ash content) and some parameters related to fish condition (heart and liver size). Irrespective of ploidy, at seawater transfer, fish fed the MP diet had significantly highest length and weight and those fed the LP diet significantly lowest length and weight, while those fed the HP diet had intermediate lengths and weights. Increased dietary phosphorus reduced deformities in both ploidies at seawater transfer; however, triploids fed the LP and MP diets had more deformities than diploids fed the respective diets, while there was no ploidy effect observed for fish fed the HP diet. The vertebral bone ash content at seawater transfer was significantly higher in diploids than in triploids when fed the MP diet only. Alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRACP) enzyme activities and relative gene expression of bone hormones involved in metabolism of plasma phosphate (fgf23) and bone growth (bgp) were not affected by ploidy at seawater transfer, but by dietary P level; LP increased ALP activity and reduced TRACP activity and fgf23 and bgp expression levels in vertebral bone. In scales, LP increased both ALP and TRACP activity. At the termination of the seawater period, the group-wise pattern in occurrence of vertebral deformities was the same as at seawater transfer. The present results on mortality, growth, bone mineralization and development of skeletal deformities all demonstrate that triploids have a higher P requirement than diploids in fresh water. This study shows that an optimalization of P nutrition for triploid Atlantic salmon can improve health and welfare and reduce down-grading of triploid salmon.

Depending on its chemical form, selenium (Se) is a trace element with a narrow range between requirement and toxicity for most vertebrates. Traditional endpoints of Se toxicity include reduced growth, feed intake, and oxidative stress, while more recent finding describe disturbance in fatty acid synthesis as underlying toxic mechanism. To investigate overall metabolic mode of toxic action, with emphasis on lipid metabolism, a wide scope metabolomics pathway profiling was performed on Atlantic salmon (Salmo salar) (572±7g) that were fed organic and inorganic Se fortified diets. Atlantic salmon were fed a low natural background organic Se diet (0.35mg Se kg-1, wet weight (WW)) fortified with inorganic sodium selenite or organic selenomethionine-yeast (SeMet-yeast) at two levels (∼1-2 or 15mgkg-1, WW), in triplicate for 3 months. Apparent adverse effects were assessed by growth, feed intake, oxidative stress as production of thiobarbituric acid-reactive substances (TBARS) and levels of tocopherols, as well as an overall metabolomic pathway assessment. Fish fed 15mgkg-1 selenite, but not 15mgkg-1 SeMet-yeast, showed reduced feed intake, reduced growth, increased liver TBARS and reduced liver tocopherol. Main metabolic pathways significantly affected by 15mgkg-1 selenite, and to a lesser extent 15mgkg-1 SeMet-yeast, were lipid catabolism, endocannabinoids synthesis, and oxidant/glutathione metabolism. Disturbance in lipid metabolism was reflected by depressed levels of free fatty acids, monoacylglycerols and diacylglycerols as well as endocannabinoids. Specific for selenite was the significant reduction of metabolites in the S-Adenosylmethionine (SAM) pathway, indicating a use of methyl donors that could be allied with excess Se excretion. Dietary Se levels to respectively 1.1 and 2.1mgkg-1 selenite and SeMet-yeast did not affect any of the above mentioned parameters. Apparent toxic mechanisms at higher Se levels (15mgkg-1) included oxidative stress and altered lipid metabolism for both inorganic and organic Se, with higher toxicity for inorganic Se.

In the present study, zebrafish (Danio rerio) were fed casein/gelatin-based diets containing either 19 % Bt (Bacillus thuringiensis)-maize or its parental non-Bt (nBt)-maize control for two generations (F0: sixty fish; F1: forty-two to seventy fish per treatment). The study focused on growth and reproductive performance, liver CuZn superoxide dismutase (SOD) enzyme activity, gene transcript levels targeting important cellular pathways in the liver and mid-intestine, histomorphological evaluation of the intestine, differential leucocyte counts, offspring larva swimming activity and global DNA methylation in offspring embryos. No significant effects were observed in the parental generation. The offspring were either fed the same diets as those fed to their parents (Bt–Bt or nBt–nBt) or switched from the Bt diet to the nBt diet (Bt–nBt). The Bt–Bt offspring exhibited a significantly higher body mass increase, specific growth rate and feed utilisation than fish fed the nBt–nBt diet and/or fish fed the Bt–nBt diet. Liver and mid-intestinal gene transcript levels of CuZn SOD were significantly higher in fish fed the nBt–nBt diet than in those fed the Bt–Bt diet. Liver gene transcript levels of caspase 6 were significantly lower for the nBt–nBt group than for the Bt–Bt group. Overall, enhanced growth performance was observed in fish fed the Bt diet for two generations than in those fed the nBt diet for one and two generations. Effects observed on gene biomarkers for oxidative stress and the cell cycle (apoptosis) may be related to the contamination of nBt-maize with fumonisin B1 and aflatoxin B1. In conclusion, it is suggested that Bt-maize is as safe and nutritious as its nBt control when fed to zebrafish for two generations.

The vitamin A (VA) concentration in salmon aquaculture feeds is varying and may lead to sublethal adverse effects. In this study, 135 g Atlantic salmon postsmolts were given eight diets in duplicates with 6, 12, 26, 55, 82, 112, 360 and 749 mg retinol (ROL) kg−1 for 116 days. Subsequently, fish given 6, 82 and 749 mg ROL kg−1 were transferred to a common net pen and given a standard commercial diet for further 28 weeks. Feed conversion rate, liver functionality and markers of VA homoeostasis were not negatively affected by dietary VA level, but chronic high VA intakes led to adverse effects on growth and bone health. In plasma, there was an antagonistic effect of dietary ROL on circulating 1,25 (OH)2 vitamin D3 (calcitriol). Moreover, a dose–response of VA on craniofacial deformities, condition factor and vertebral morphometry and mechanical strength was observed. Vertebral deformities were observed after 28 weeks on a standard diet and not immediately after the 116 days on the experimental diet. Elevated VA is a risk factor for bone deformities, and the dietary intake of VA should not exceed 37 mg ROL kg−1 body weight day−1 in Atlantic salmon postsmolts.

Feed additives are typically used in intensive farming production over long periods, and hence, they can accumulate in farmed animal tissues. Concerns regarding the use of ethoxyquin as an antioxidant feed additive, have recently arisen due to its potential conversion into a series of transformation products (TPs). The aim of this work was to characterize the TPs of ethoxyquin in fish feed by a novel approach based on the use of traveling-wave ion mobility spectrometry (TWIMS) coupled to high-resolution quadrupole time-of-flight mass spectrometry (QTOFMS). First, ethoxyquin was oxidized under controlled conditions and the generated TPs were added to a comprehensive database. Atlantic salmon feeds were then screened for ethoxyquin TPs using both targeted and untargeted approaches. Twenty-seven TPs were tentatively identified during the oxidation experiments, fifteen of them also being present in the feed samples. In addition, ten other potential TPs were detected in fish feed following the untargeted approach. Thirty-one of these TPs have been reported for the first time in this work through the oxidation experiments and the feed samples. Therefore, this study provides valuable information on the oxidative fate of ethoxyquin in feed, which can be used for future evaluations of potential risk related to this additive.

Several studies have reported on the interaction between vitamin A (VA) and aryl hydrocarbon receptor (AhR)-binding toxicants, including poly-aromatic hydrocarbons (PAHs). In aquaculture, the use of plant oils in novel aquafeeds can increase PAH levels while simultaneously lowering natural VA background levels, causing the need to supplement plant oil-based feeds with synthetic VA. To study dietary VA-PAH interactions, Atlantic salmon (initial weight 195±0.15g) were fed four identical plant-based diets that were supplemented with PAHs (100 and 10mgkg(-1) benzo[a]pyrene (BaP) and phenanthrene (Phe), respectively) or VA (retinyl acetate 8721IUkg(-1)) separately or combined for 2.5 months in a 2×2 factorial design, with triplicate net-pens per diet. Dietary PAH significantly reduced hepatic VA storage, and VA-enriched diets restored hepatic VA. There was a significant PAH-VA interaction effect on hepatic BaP, but not Phe, accumulation, with reduced hepatic BaP concentrations in fish fed VA+PAH compared to fish fed PAH alone. Concurrently, PAH and VA significantly interacted in their effects on CYP1A phase I biotransformation as observed from increased ethoxyresorufin-O-deethylase (EROD) activity, increased CYP1A protein concentration, and elevated transcription (cyp1a1 gene expression) in fish fed PAH+VA compared to PAH alone. Dietary VA supplementation alone had no significant effect on CYP1A phase I biotransformation. Metabolomic assessment showed that dietary VA caused a restoration of metabolic intermediates involved in energy metabolism that were affected by dietary PAH. Moreover, a PAH-induced growth inhibition was partially ameliorated by dietary VA supplementation. In conclusion, dietary VA interacted with PAH toxicity on the level of CYP1A-mediated detoxification, hepatic PAH accumulation, energy allocation, and growth.

Summary The present review sums up and discusses the current literature on occurrence, causation and pathology of vertebral deformi- ties in farmed Atlantic salmon, and also gives a brief introduction into the normal ontogeny and anatomy of the vertebral column of Atlantic salmon. Skeletal development and growth are sensitive processes that can be affected by many factors. Many of these factors can be manipulated under farming conditions, and are thus regarded as risk factors. Several risk factors that relate to environmental conditions and to feed composition have been identified. Elevated tempera- tures and photoperiod manipulation to speed up growth are likely the most important environmental factors that cause skeletal deformities. Among the nutritional factors, optimal phosphorus nutrition during specific periods, for example after transfer to sea water, appears to be critical for development of deformity at later stages. More research is needed to under- stand the interdependency of genetics, development, aging, phosphorus nutrition, temperature and photoperiod, in order to establish the best practice procedures for salmon farming that improve fish welfare.

This study investigated the combined effects of photoperiod and dietary P level on bone osteoclast and osteoblast activity, and morphology, as well as plasma vitamin D status in Atlantic salmon post-smolts. The fish were reared under continuous light (LL) or 12 h light/dark (LD) per day, and fed diets with insufficient phosphorous (P) (4 g kg−1 available dietary P, LP) or sufficient P (8 g kg−1 available dietary P, HP) for 79 days in seawater. LL significantly increased plasma 25(OH)D3 level and bone tartrate resistant acid phosphatase (TRACP) activity, and decreased bone collagen XI α1 chain (col11a1) mRNA transcription. The LP diet significantly increased plasma 1,25(OH)2D3 level and bone alkaline phosphatase activity, but decreased bone TRACP activity and matrix metalloproteinase 13 (mmp13) mRNA transcription, and vertebral mineral content, stiffness and length/dorso-ventral diameter (l/d) ratio. A significant interaction between light and P was only observed on the l/d ratio, and the LP-LL group was the only group that developed vertebrae with a compressed morphology (significantly lowest l/d ratio). In practical terms, these results show that Atlantic salmon post-smolts under continuous light may need P supplemented diets to support normal bone development.

Vitamin K belongs to the lipid soluble vitamins, and occurs naturally as phylloquinone (vitamin K1) and menaquinone (vitamin K2). In addition, there is a synthetic provitamin, menadione (vitamin K3), primarily used as a vitamin K source in animal feed. Menadione is unstable during feed processing and storage and the dietary content may reach critically low levels. Recent publications also question the availability of menadione in feed for salmonids. Vitamin K plays vital roles in blood coagulation and bone mineralization in fish, but the suggested minimum requirement varies considerably depending on the vitamin K source used. Vitamin K deficiency is characterized by mortality, anaemia, increased blood clotting time and histopathological changes in liver and gills. However, one should assess both inherent and supplemented forms of vitamin K in feeds for exact determinations, as relevant novel feed ingredients of plant origin may be sufficient to meet the requirement for vitamin K. The current review gives an overview of the biochemical role of vitamin K, and discusses vitamin K requirement in fish in light of updated literature, with special emphasis on salmonids.

Inflammation is a non-specific protective mechanism towards injury known to affect bone remodelling. This study aimed to investigate the effect of Freunds complete adjuvant (FCA) induced-inflammation on the prevalence of spinal deformities of Atlantic salmon postsmolts fed with two different dietary P levels. Sextuple groups of salmon postsmolts were fed with either a low-phosphorous (6 g kg-1 available P, LP) or a high-phosphorous (9 g kg-1 available P) diet for a period of 101 days. On Day 102, individually tagged fish were subjected to (i) single injection with FCA (0.125 mg kg-1 BW) dissolved in phosphate-buffered saline (PBS) (ii) placebo injection with PBS or (iii) sham injection (insertion of needle only) or (iv) remained untreated. On Day 103, fish were given a common diet for 174 days in seawater. No significant differences in body weight were observed. Injected fish, particularly the FCA group, had more compressions in the injection site than untreated fish. No effect of diet and no interaction between treatment and diet were observed. Severe scoliosis was observed in similar to 7% of FCA-injected individuals, corresponding to a mixture of bone malformations in the tail region. In conclusion, experimentally induced inflammation may be an independent risk factor for bone deformities in Atlantic salmon.

Vegetable oils (Vo) are an alternative to fish oil (Fo) in aquaculture feeds. This study aimed to evaluate the effect of dietary soybean oil (Vo diet), rich in linoleic acid, and of dietary fish oil (Fo diet) on the development of spinal deformities under bacterial lipopolysaccharide (LPS)-induced chronic inflammation conditions in Atlantic salmon, Salmo salar L. Fish [25 g body weight (BW)] were fed the experimental diets for 99 days. On day 47 of feeding (40 g BW), fish were subjected to four experimental regimes: (i) intramuscular injections with LPS, (ii) sham-injected phosphate-buffered saline (PBS), (iii) intraperitoneally injected commercial oil adjuvant vaccine, or (iv) no treatment. The fish continued under a common feeding regime in sea water for 165 more days. Body weight was temporarily higher in the Vo group than in the Fo group prior to immunization and was also affected by the type of immunization. At the end of the trial, no differences were seen between the dietary groups. The overall prevalence of spinal deformities was approximately 14% at the end of the experiment. The Vo diet affected vertebral shape but did not induce spinal deformities. In groups injected with LPS and PBS, spinal deformities ranged between 21% and 38%, diet independent. Deformed vertebrae were located at or in proximity to the injection point. Assessment of inflammatory markers revealed high levels of plasma prostaglandin E₂ (PGE₂) in the Vo-fed and LPS-injected groups, suggesting an inflammatory response to LPS. Cyclooxigenase 2 (COX-2) mRNA expression in bone was higher in fish fed Fo compared to Vo-fed fish. Gene expression of immunoglobulin M (IgM) was up-regulated in bone of all LPS-injected groups irrespective of dietary oil. In conclusion, the study suggests that Vo is not a risk factor for the development of inflammation-related spinal deformities. At the same time, we found evidence that localized injection-related processes could trigger the development of vertebral body malformations.

The aim of the present study was to elucidate if increasing levels of added vitamin K in the feed influenced fish growth, health or the incidences of bone deformities in Atlantic salmon (Salmo salar L.) from onset of start feeding to ∼100 g in freshwater. Duplicate groups of Atlantic salmon fry (∼0.20 g) were fed eight levels (0, 2.5, 5, 7.5, 10, 15, 20 and 50 mg menadione kg−1) of the vitamin K derivate menadione nicotinamide bisulphite (MNB) in a regression design for 28 weeks. All fish maintained high growth rates throughout the experiment, and showed no significant differences in specific growth rate, condition factor, whole body proximate analysis, blood coagulation time, vertebra morphology or mechanical properties of vertebrae. We found a dose-response between whole body vitamin K concentration and the dietary MNB supplementation level. Analysis of liver γ-glutamylcarboxylase activity revealed significant dose-dependent differences between groups given the 0, 10 and 50 mg MNB kg−1 diets. In conclusion, Atlantic salmon seems to require low levels of dietary vitamin K, and the amount of vitamin K found naturally in the presently used feed ingredients may be enough to maintain optimal growth, health and bone strength in Atlantic salmon fry from start feeding.

The tissue-specific gene expression of the vitamin K-dependent proteins bone γ-carboxyglutamate-protein (BGP) and matrix γ-carboxyglutamate-protein (MGP) in Atlantic salmon (Salmo salar L.) was investigated. In previous studies, BGP, the most abundant non-collagenous protein of bone, was almost exclusively associated with bone, whereas the non-structural protein MGP has a more widespread tissue distribution. In-situ hybridization of juvenile Atlantic salmon (∼40 g, fresh water) vertebrae demonstrated expression of bgp and mgp mRNA in osteoblasts lining the trabecular bone, whereas no staining was observed in the compact bone. By separating the trabecular and compact bone of both juvenile (∼40 g, fresh water) and adult (∼1000 g, sea water) Atlantic salmon, we observed that the two vertebral bone compartments displayed different levels of bgp, whereas no such differences were seen for mgp. Measurements of the mineral content and Ca/P molar ratio in adult salmon revealed no significant differences between trabecular and compact bone. In conclusion, the osteoblasts covering the salmon vertebrae have unique gene expression patterns and levels of bgp and mgp. Further, the study confirms the presence of mRNA from the vitamin K-dependent proteins BGP and MGP in the vertebrae, fin and gills of Atlantic salmon.

An earlier study demonstrated that under-yearling (0+) Atlantic salmon (Salmo salar L.) smolt had a lower vertebral mineral content and mechanical strength and higher prevalence of vertebral deformities than 1+ smolt during the early seawater (SW) phase. The present study aimed to examine if commercial extruded high-energy diets need to be supplemented additional minerals for proper bone mineralization and prevention of bone deformities in fast growing 0 + smolts. We studied vertebral morphology with radiology, and bone mineral content and mechanical strength in 60 g 0+ smolt fed diets with a normal (NM) or elevated (HM) bone mineral (P and Ca) contents from SW transfer (week 0) until 10 times weight increase at week 17. Thereafter, both groups were fed a commercial diet until a mean slaughter weight of 4100 g after 57 week. There were no differences in body weight and length between the dietary groups during the study, while the condition factor differed significantly at the final sampling (NM 1.40; HM 1.29). The most common bone deformity observed was compressions in the tail region of the vertebral column. Lower incidences of vertebral deformities (percent individuals with one or more deformed vertebrae) was observed in the HM group in week 17 (HM 20%; NM 47%) and week 57 (HM 37%; NM 73%), also reflected by higher vertebral length/dorso-ventral diameter ratio in weeks 17 (HM 0.99; MN 0.92) and 57 (HM 0.97; NM 0.88). The HM group had significantly higher vertebral mineral content (HM 550 g kg−1; NM 480 g kg−1) and mechanical strength (HM 9050 g mm−1; NM 4600 g mm−1) than the NM group after 8 week feeding. Plasma levels of Ca, P and D-vitamin metabolites recorded in week 8 reflected changes in P homeostasis, but could not explain the preventive effect of the HM diet on development of bone deformities. The results suggest that elevated dietary mineral content during the early SW phase may reduce the prevalence of vertebral deformities in fast growing 0 + salmon smolts.

Vitamins A (VA) and D (VD) are metabolised by vertebrates to bioactive retinoic acid (RA) and calcitriol (CTR). RA and CTR involvement in bone metabolism requires fine-tuned regulation of their synthesis and breakdown. In mammals antagonism of VA and VD is observed, but the mechanism of interaction is unknown. We investigated VA–VD interactions in Atlantic salmon (Salmo salar L.) following i.p. injection of RA and/or CTR. VA metabolites, CTR, calcium (Ca), magnesium (Mg) and phosphorus (P) were determined in plasma. Expression of bone matrix Gla protein (mgp), collagen 1 alpha2 chain (col1a2) and alkaline phosphatase (alp) mRNA was quantified to reflect osteogenesis. Branchial epithelial Ca channel (ecac listed as trpv6 in ZFIN Database) mRNA levels and intestinal Ca and P influx were determined to study Ca/P handling targets of RA and CTR. RA-injection (with or without CTR) decreased plasma CTR-levels three- to sixfold. CTR injection did not affect RA metabolites, but lowered CTR in plasma 3 and 5 days after injection. Lowered plasma CTR correlated with decreased mgp and col1a2 expression in all groups and with decreased alp in CTR-injected fish. RA-treated salmon had enhanced alp expression, irrespective of reduced plasma CTR. Expression of ecac and unidirectional intestinal influx of Ca were stimulated following RA–CTR treatment. Plasma Ca, Mg and P were not affected by any treatment. The results suggest cross-talk of RA with the VD endocrine system in Atlantic salmon. Enhanced Ca flux and osteogenesis (alp transcription) in RA-treated fish and inhibition of mgp expression revealed unprecedented disturbance of Ca physiology in hypervitaminosis A.

Seaward migration of Salmo salar is preceded by preparatory physiological adaptations (parr–smolt transformation) to allow for a switch from freshwater (FW) to seawater (SW), which also means a switch in ambient calcium from hypocalcic (2+) to the plasma (~1.25 mM Ca2+) and to strongly hypercalcic (8–12 mM Ca2+). Uptake, storage (skeleton, scales) and excretion of calcium need careful regulation. In fish, the vitamin D endocrine system plays a rather enigmatic role in calcium physiology. Here, we give direct evidence for calcitriol involvement in SW migration. We report the full sequence of the nuclear vitamin D receptor (sVDR0) and two alternatively spliced variants resulting from intron retention (sVDR1 and sVDR2). In FW parr, SW adapting smolts, and in SW adults, plasma concentrations of 25(OH)D3 and 24,25(OH)2D3 did not change significantly. Plasma calcitriol concentrations were lowest in FW parr, doubled during smoltification and remained elevated in SW adults. Increased calcitriol coincided with a twofold decrease in sVDR mRNA levels in gill, intestine, and kidney of FW smolts and SW adults, when compared with parr. Clearly, there was a negative feedback and dynamic response of the vitamin D endocrine system during parr–smolt transformation. The onset of these dynamic changes in FW parr warrants a further search for the endocrines that initiate these changes. We speculate that the vitamin D system plays a crucial role in calcium and phosphorus handling in Atlantic salmon.

A feeding experiment was performed to investigate the possibility of feeding vegetable oils to Atlantic salmon, followed by a wash-out period to maintain salmon fillet as a product highly beneficial for human health, due to the high levels of VLC n −3 PUFA and high n −3/ n −6 ratio. Six groups of Atlantic salmon, initial weight 142 ± 1 g, were fed increasing dietary inclusion of rapeseed oil (RO) in a regression design and one group was fed a 50% olive oil (OO)/50% capelin oil (FO) diet for 42 weeks, followed by 25 weeks, of wash-out, when all groups were fed 100% FO. Muscle gross composition, lipid class and fatty acid composition and astaxanthin were measured at the start and after 22 and 42 weeks of feeding. Fillet fatty acid composition was analysed at the start and throughout the wash-out period of 25 weeks and 1788 day degrees. Growth, total lipid, astaxanthin content, and lipid class composition were not affected by dietary oil source. Muscle and fillet fatty acid composition were highly affected by dietary fatty acid composition. Through the wash-out period, the VLC n −3 PUFAs EPA and DHA was restored already after 1300 day degrees, whereas the wash-out of 18:2 n −6, 18:1 n −9 and 18:3 n −3 was a slower process, requiring a minimum 1788 day degrees for reaching 100% FO levels. For reducing n −6 fatty acids and increasing VLC n −3 PUFA, a period of feeding with 100% FO was sufficient for the groups fed 25% RO, 50% RO and 50% OO prior to wash-out. Considering the recommended n −3/ n −6 ratio and VLC n −3 PUFA intake for human consumption, fillets from fish fed 100% FO, 25% RO, 50% RO and 50% OO prior to wash-out, followed by 1788 day degrees of 100% FO diet, can be considered beneficial for human health promotion.