Metabolic effects of diet containing blue mussel (Mytilus edulis) and blue mussel-fed salmon in a mouse model of obesity.

Alternative feed ingredients for farmed salmon are warranted due to increasing pressure on wild fish stocks. As locally farmed blue mussels may represent an environmentally sustainable substitute with a lower carbon footprint, we aimed to test the potential and safety of substituting fish meal with blue mussel meal in feed for Atlantic salmon. Salmon were fed diets in which fish meal was partially replaced with blue mussel meal in increments, accounting for up to 13.1 % of the ingredients. Fillets from the salmon were subsequently used to prepare obesity-promoting western diets for a 13-weeks mouse feeding trial. In a second mouse trial, we tested the effects of inclusion of up to 8% blue mussel meal directly in a meat-based western diet. Partial replacement of fish meal with blue mussel meal in fish feed preserved the n-3 polyunsaturated fatty acid (PUFA) content in salmon fillets. The observed blue mussel-induced changes in the fatty acid profiles in salmon fillets did not translate into similar changes in the livers of mice that consumed the salmon, and no clear dose-dependent responses were found. The relative levels of the marine n-3 fatty acids, EPA, and DHA were not reduced, and the n-3/n-6 PUFA ratios in livers from all salmon-fed mice were unchanged. The inclusion of blue mussel meal in a meat-based western diet led to a small, but dose-dependent increase in the n-3/n-6 PUFA ratios in mice livers. Diet-induced obesity, glucose intolerance, and hepatic steatosis were unaffected in both mice trials and no blue mussel-induced adverse effects were observed. In conclusion, our results suggest that replacing fish meal with blue mussel meal in salmon feed will not cause adverse effects in those who consume the salmon fillets.

Feed-to-Fillet Transfer of Selenite and Selenomethionine Additives to Plant-Based Feeds to Farmed Atlantic Salmon Fillet.

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.

Erucic Acid (22:1n-9) in Fish Feed, Farmed, and Wild Fish and Seafood Products.

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-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.

Safety of natural mixture of dolomite plus magnesite and magnesium-phyllosilicates (Fluidol) for all animal species.

The additive under assessment consists of a natural mixture, mainly composed of dolomite (~ 30%), magnesite (~ 20%) and magnesium-phyllosilicates (talc (~ 35%) and chlorite (~ 15%)). In 2016, the Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) delivered an opinion on the safety and efficacy of natural mixture of dolomite plus magnesite and magnesium-phyllosilicates. In this opinion, the Panel concluded that 20,000 mg additive/kg complete feed is considered safe for dairy cows and for piglets (weaned). This conclusion was extended to pigs for fattening while no conclusion could be drawn on the safety for poultry or any other species/categories. Following this opinion, the European Commission gave the possibility to the applicant to submit complementary information in order to complete the assessment on the safety for all animal species. The new tolerance studies submitted with cattle for fattening and chickens for fattening showed tolerance of these animal categories to the additive up to approximately fivefold the highest recommended use level. The FEEDAP Panel therefore concluded that 20,000 mg additive/kg complete feed is safe for cattle for fattening and chickens for fattening. The additive at the same dietary concentration was already considered safe in an earlier opinion for piglets and dairy cows. Since the additive at 20,000 mg/kg complete feed is considered safe for four animal categories (three major animal species) with a comparable margin of safety, the conclusion on safety is extrapolated to all animal species. At the safe dietary concentration the additive did not affect the digestibility of the feed (including feed additives).

Safety of natural mixture of dolomite plus magnesite and magnesium-phyllosilicates (Fluidol) for all animal species.

The additive under assessment consists of a natural mixture, mainly composed of dolomite (~ 30%), magnesite (~ 20%) and magnesium-phyllosilicates (talc (~ 35%) and chlorite (~ 15%)). In 2016, the Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) delivered an opinion on the safety and efficacy of natural mixture of dolomite plus magnesite and magnesium-phyllosilicates. The FEEDAP Panel concluded that the additive is safe in complete feed for dairy cows, piglets and pigs for fattening at a maximum concentration of 20,000 mg/kg. However, no conclusions could be drawn for all other animal species/categories. Following this opinion, the European Commission gave the possibility to the applicant to submit complementary information in order to complete the assessment on the safety for all animal species. The applicant answered with a new submission, an analysis of the previous EFSA opinion, but without new data. The FEEDAP Panel considered the arguments made by the applicant, in relation to the tolerance studies with dairy cows and chickens for fattening. No reason was identified to modify the conclusions reached in the previous opinion.

Safety of lactic acid and calcium lactate when used as technological additives for all animal species.

The additives under assessment are lactic acid and calcium lactate. In 2015, the Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) delivered an Opinion on the safety and efficacy of lactic acid and calcium lactate. The FEEDAP Panel could not conclude on the safety of lactic acid in pre-ruminants and poultry. Following this opinion, the European Commission gave the possibility to the applicant to submit complementary information in order to complete the assessment on the safety for all animal species. Based on the studies submitted in chickens for fattening and laying hens, no safe concentration of lactic acid and calcium lactate in complete feed for these species could be identified. Owing to the absence of data on tolerated dietary levels of d-lactic acid, no conclusion on the safety of lactic acid in milk replacer for pre-ruminants is possible. Since a safe concentration of lactic acid (and calcium lactate) was established only for pigs and cattle, and not for a third major animal species, no extrapolation to any other species is possible.

Safety and efficacy of natural mixture of illite, montmorillonite and kaolinite for all animal species.

The additive under assessment is a natural mixture mainly composed of illite (~ 53%), montmorillonite (~ 16%) and kaolinite (~ 17%). In 2016, the Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) delivered an Opinion on the safety and efficacy of a natural mixture of illite, montmorillonite and kaolinite (MIMK). The FEEDAP Panel concluded that the additive is safe for piglets and pigs for fattening at a maximum concentration of 20,000 mg/kg, and for cattle for fattening at a maximum concentration of 50,000 mg/kg. However, no conclusion could be drawn for all other species/categories. The Panel also concluded that the additive is efficacious at a minimum concentration of 50,000 mg/kg. Following this opinion, the European Commission gave the possibility to the applicant to submit complementary information in order to complete the assessment on the safety for all animal species and on its efficacy at a minimum concentration of 20,000 mg/kg. The applicant submitted a new tolerance study in piglets and an analysis of the previous EFSA opinion regarding poultry and milk-producing animals. The FEEDAP Panel concluded that the additive is safe for piglets and pigs for fattening at 50,000 mg/kg. The Panel concluded that the safe level (50,000 mg/kg) found for cattle for fattening could be extrapolated to minor growing ruminants. The conclusion could be extrapolated to dairy cows and minor ruminant species for milk production. The FEEDAP Panel confirmed that no safe concentration of MIMK in feed for chickens for fattening could be identified. No conclusions could be drawn for all the other animal species/categories. The additive is effective as a pellet binder and an anticaking agent at the lowest level tested of 5,000 mg/kg feed.

Marine fatty acids aggravate hepatotoxicity of α-HBCD in juvenile female BALB/c mice.

Oily fish, a source of long-chain omega-3 polyunsaturated fatty acids (LC n-3 PUFAs), may contain persistent organic pollutants (POPs), including α-hexabromocyclododecane (α-HBCD). In experimental studies, marine LC n-3 PUFAs ameliorate fatty liver development while HBCD exposure was found to cause liver fatty acid (FA) changes. The present study investigated interactions of FAs and α-HBCD in juvenile female BALB/c mice using a factorial design. Mice (n = 48) were exposed for 28 days to a low (100 µg*kg body weight (BW)-1*day-1) or high dose (100 mg*kg BW-1*day-1) of α-HBCD in diets with or without LC n-3 PUFAs. High dose α-HBCD affected whole body lipid metabolism leading to changes in body weight and composition, and pathological changes in hepatic histology, which surprisingly were aggravated by dietary LC n-3 PUFAs. Hepatic FA profiling and gene expression analysis indicated that the dietary modulation of the hepatotoxic response to the high dose of α-HBCD was associated with differential effects on FA ß-oxidation. Our results suggest that in a juvenile mouse model, marine FAs accentuate hepatotoxic effects of high dose α-HBCD. This highlights that the background diet is a critical variable in the risk assessment of POPs and warrants further investigation of dietary mediated toxicity of food contaminants.

Dietary selenomethionine influences the accumulation and depuration of dietary methylmercury in zebrafish (Danio rerio).

Methylmercury (MeHg) is a toxicant of concern for aquatic food chains. In the present study, the assimilation and depuration of dietary MeHg and the influence of dietary selenium on MeHg toxicokinetics was characterised in zebrafish (Danio rerio). In a triplicate tank experimental design (n=3 tanks per treatment group), adult zebrafish were exposed to dietary MeHg (as methylmercury-cysteine) at 5 and 10 µg/g and with or without selenium (as selenomethionine) supplemented to the diets at a concentration of 5 µg/g for 8 weeks followed by a 4-week depuration period. Methylmercury accumulated in muscle, liver and brain of zebrafish; with higher mercury concentrations in liver and brain than in muscle following 8 weeks of exposure. In muscle, the mercury concentrations were 3.4±0.2 and 6.4±0.1 µg/g ww (n=3) in zebrafish fed the 5 and 10 µg Hg/g diets, respectively. During the depuration period, mercury concentrations were significantly reduced in muscle in both the 5 and 10 µg Hg/g diet groups with a greater reduction in the high dose group. After depuration, the mercury concentrations were 2.4±0.1 and 4.0±0.3 µg/g ww (n=3) for zebrafish fed the 5 and 10 µg Hg/g diets, respectively. Data also indicated that supplemented dietary selenium reduced accumulation of MeHg and enhanced the elimination of MeHg. Lower levels of mercury were found in muscle of zebrafish fed MeHg and SeMet compared with fish fed only MeHg after 8 weeks exposure; the mercury concentrations in muscle were 5.8±0.2 and 6.4±0.1 µg/g ww (n=3) for zebrafish fed the 10 µg Hg/g+5 µg Se/g diet and the 10 µg Hg/g diet, respectively. Furthermore, the elimination of MeHg from muscle during the 4-week depuration period was significantly greater in the fish fed the diet containing SeMet compared to a control diet; the mercury concentrations were 3.3±0.1 and 4.0±0.3 µg/g ww (n=3) for zebrafish fed the 5 µg Se/g and the control diets, respectively. In summary, dietary SeMet reduces the accumulation and enhances the elimination of dietary MeHg in muscle of zebrafish.

Cerebral gene expression in response to single or combined gestational exposure to methylmercury and selenium through the maternal diet.

Controversy remains regarding the safety of consuming certain types of seafood, particularly during pregnancy. While seafood is rich in vital nutrients, it may also be an important source of environmental contaminants such as methylmercury (MeHg). Selenium (Se) is one essential element present in seafood, hypothesised to ameliorate MeHg toxicity. The aim of the present study was to ascertain the impact of Se on MeHg-induced cerebral gene expression in a mammalian model. Microarray analysis was performed on brain tissue from 15-day-old mice that had been exposed to MeHg throughout development via the maternal diet. The results from the microarray analysis were validated using qPCR. The exposure groups included: MeHg alone (2.6 mg kg(-1)), Se alone (1.3 mg kg(-1)), and MeHg + Se. MeHg was presented in a cysteinate form, and Se as Se-methionine, one of the elemental species occurring naturally in seafood. Eight genes responded to Se exposure alone, five were specific to MeHg, and 63 were regulated under the concurrent exposure of MeHg and Se. Significantly enriched functional classes relating to the immune system and cell adhesion were identified, highlighting potential ameliorating mechanisms of Se on MeHg toxicity. Key developmental genes, such as Wnt3 and Sparcl1, were also identified as putative ameliorative targets. This study, utilising environmentally realistic forms of toxicants, delivered through the natural route of exposure, in association with the power of transcriptomics, highlights significant novel information regarding putative pathways of selenium and MeHg interaction in the mammalian brain.

Chemical contaminants in aquafeeds and Atlantic salmon (Salmo salar) following the use of traditional- versus alternative feed ingredients.

Marine feed ingredients, traditionally used in commercial fish feeds, are the source of these pollutants in farmed fish. The aim of the study was to assess the chemical contaminant load in Atlantic salmon (Salmo salar L.) raised on novel sustainable feeds based on a combination of alternative ingredients. Atlantic salmon were reared on feeds based on either traditional or alternative feed ingredients for an entire seawater production cycle up to approximately 4kg, which is a typical market-size for this species. The levels of several notorious contaminants were analysed in feed ingredients, feed, and skin-off fillets. These included persistent organic pollutants (POPs) such as polychlorinated biphenyls (PCBs), dioxins and furans (PCDD/Fs), organochlorine pesticides (OCPs), and polybrominated diphenyl ethers (PBDEs), as well as elements such as arsenic, mercury, cadmium, lead, copper, zinc and fluorine. In addition, poly-aromatic hydrocarbons (PAHs) were analysed. The use of alternative feed ingredients reduced the fillet load of POPs by 51-82% and the level of arsenic and mercury by 80-96%. In contrast, the PAH levels in fish reared on the novel feeds were significantly (p<0.05) higher than the PAH concentrations in traditionally-raised fish. The present study shows that developments in feed formulation will reduce the load of most persistent organic pollutants in farmed salmon, but may increase concentrations of other contaminants such as PAHs, which are normally not associated with Atlantic salmon. The paper also compares the levels of contaminants in farmed Atlantic salmon expected in the future with those levels reported in the literature and currently on the market.

Higher faecal excretion and lower tissue accumulation of mercury in Wistar rats from contaminated fish than from methylmercury chloride added to fish.

A short-term low level exposure experiment was conducted on rats in order to determine urinary and faecal excretion, accumulation, and biological responses to methylmercury from fish products. Male Wistar rats were fed fish-meal diets containing methylmercury contaminated fish (1.45 or 2.61 mgHg/kg as methylmercury), uncontaminated fish supplemented with methylmercury chloride (CH3HgCl) at similar levels (1.24 and 2.49 mgHg/kg, respectively) or uncontaminated fish as a control (0.052 mgHg/kg) for 4 weeks (n=6 rats per treatment). After 2 and 4 weeks of exposure, rats were placed in metabolic chambers for 48 h to assess overall faecal and urinary excretion of mercury. The overall faecal excretion in rats fed fish supplemented with CH3HgCl (12%) was significantly lower (P <0.05) than rats fed methylmercury in fish muscle (19%) or rats fed control diet (76%). Urinary excretion did not differ among the experimental groups. Rats fed the highest level of CH3HgCl had a significantly higher (P <0.05) blood, liver, kidney and brain mercury contamination compared to rats fed methylmercury contaminated fish or rats fed control diet. Metallothionein levels in kidney were significantly higher in CH3HgCl-fed rats compared to rats fed contaminated fish. The results indicate a higher faecal excretion and lower tissue accumulation, and metallothionein induction in rats following exposure to methylmercury naturally incorporated in fish compared to methylmercury chloride added to the same matrix.