Systemic and strict regulation of the glutathione redox state in mitochondria and cytosol is needed for zebrafish ontogeny.

BACKGROUND: Redox control seems to be indispensable for proper embryonic development. The ratio between glutathione (GSH) and its oxidized disulfide (GSSG) is the most abundant cellular redox circuit. METHODS: We used zebrafish harboring the glutaredoxin 1-redox sensitive green fluorescent protein (Grx1-roGFP) probe either in mitochondria or cytosol to test the hypothesis that the GSH:GSSG ratio is strictly regulated through zebrafish embryogenesis to sustain the different developmental processes of the embryo. RESULTS: Following the GSSG:GSH ratio as a proxy for the GSH-dependent reduction potential (EhGSH) revealed increasing mitochondrial and cytosolic EhGSH during cleavage and gastrulation. During organogenesis, cytosolic EhGSH decreased, while that of mitochondria remained high. The similarity between EhGSH in brain and muscle suggests a central regulation. Modulation of GSH metabolism had only modest effects on the GSSG:GSH ratios of newly hatched larvae. However, inhibition of GSH reductase directly after fertilization led to dead embryos already 10 h later. Exposure to the emerging environmental pollutant Perfluorooctane Sulfonate (PFOS) disturbed the apparent regulated EhGSH as well. CONCLUSIONS: Mitochondrial and cytosolic GSSG:GSH ratios are almost identical in different organs during zebrafish development indicating that the EhGSH might follow H2O2 levels and rather indirectly affect specific enzymatic activities needed for proper embryogenesis. GENERAL SIGNIFICANCE: Our data confirm that vertebrate embryogenesis depends on strictly regulated redox homeostasis. Disturbance of the GSSG:GSH circuit, e.g. induced by environmental pollution, leads to malformation and death.

Arsenic speciation and arsenic feed-to-fish transfer in Atlantic salmon fed marine low trophic feeds based blue mussel and kelp.

BACKGROUND: Aquaculture aims to reduce the environmental and climate footprints of feed production. Consequently, low trophic marine (LTM) resources such as blue mussels and kelp are potential candidates to be used as ingredients in salmon feed. It is relevant to study potential undesirables associated with their use, as well as assessing food safety by investigating their transfer from feed-to-fish. The marine biota is well known to contain relatively high levels of arsenic (As), which may be present in different organic forms depending on marine biota type and trophic position. Thus, it is important to not only obtain data on the concentrations of As, but also on the As species present in the raw materials, feed and farmed salmon when being fed novel LTM feed resources. METHODS: Atlantic salmon were fed experimental diets for 70 days. A total of nine diets were prepared: four diets containing up to 4 % fermented kelp, three diets containing up to 11 % blue mussel silage, and one diet containing 12 % blue mussel meal, in addition to a standard reference diet containing 25 % fish meal. Concentrations of As and As species in feeds, faeces, liver and fillet of Atlantic salmon were determined by inductively coupled plasma mass spectrometry (ICP-MS) and high-performance liquid chromatography coupled to ICP-MS (HPLC-ICP-MS), respectively. RESULTS: The use of kelp or blue mussel-based feed ingredients increased the concentration of total As, but maximum level as defined in Directive 2002/32 EC and amendments was not exceeded. The concentrations found in the experimental feeds ranged from 3.4 mg kg-1 to 4.6 mg kg-1 ww. Arsenic speciation in the feed varied based on the ingredient, with arsenobetaine dominating in all feed samples (36-60 % of the total As), while arsenosugars (5.2-8.9 % of the total As) were abundant in kelp-included feed. The intestinal uptake of total As ranged from 67 % to 83 %, but retention in fillet only ranged from 2 % to 22 % and in liver from 0.3 % to 0.6 %, depending on the marine source used. Fish fed feeds containing blue mussel showed higher intestinal uptake of total As when compared with fish fed feeds containing fermented kelp. Fish fed fermented kelp-based feeds had higher retained concentrations of total As when comparing with fish fed feeds containing blue mussel. Despite relatively high intestinal uptake of total As, inorganic and organic As, the retained concentrations of As did not reflect the same trend. CONCLUSION: Although the use of LTM feed ingredients increased the level of total As in this feeds, salmon reared on these diets did not show increased total As levels. The well-known toxic inorganic As forms were not detected in salmon muscle reared on LTM diets, and the non-toxic organic AsB was the dominant As species that was retained in salmon muscle, while the organic AsSug forms were not. This study shows that speciation analysis of the LTM resources provides valuable information of the feed-to-fish transfer of As, needed to assess the food safety of farmed Atlantic salmon reared on novel low trophic feeds.

Enniatin B and beauvericin affect intestinal cell function and hematological processes in Atlantic salmon (Salmo salar) after acute exposure.

Unintentional use of mold-infested plant-based feed ingredients are sources of mycotoxins in fish feeds. The presence of the emerging mycotoxins ENNB and BEA in Norwegian commercial fish feeds and plant-based feed ingredients has raised concerns regarding the health effects on farmed Atlantic salmon (Salmon salar). Atlantic salmon pre-smolts were exposed to non-lethal doses of BEA and ENNB (ctrl, 50 and 500 µg/kg feed for 12 h), after which total RNA sequencing of the intestine and liver was carried out to evaluate gut health and identify possible hepatological changes after acute dietary exposure. ENNB and BEA did not trigger acute toxicity, however ENNB caused the onset of pathways linked to acute intestinal inflammation and BEA exposures caused the onset of hepatic hematological disruption. The prevalence and concentration of ENNB found in today's commercial feed could affect the fish health if consumed over a longer time-period.

Arsenic species in mesopelagic organisms and their fate during aquafeed processing.

A responsible harvest of mesopelagic species as aquafeed ingredients has the potential to address the United Nations Sustainable Development Goal 14, which calls for sustainable use of marine resources. Prior to utilization, the levels of undesirable substances need to be examined, and earlier studies on mesopelagic species have reported on total arsenic (As) content. However, the total As content does not give a complete basis for risk assessment since As can occur in different chemical species with varying toxicity. In this work, As speciation was conducted in single-species samples of the five most abundant mesopelagic organisms in Norwegian fjords. In addition, As species were studied in mesopelagic mixed biomass and in the resulting oil and meal feed ingredients after lab-scale feed processing. Water-soluble As species were determined based on ion-exchange high-performance liquid chromatography coupled to inductively coupled plasma mass spectrometry (HPLC-ICP-MS). This was supplemented by extracting arsenolipids (AsLipids) and determining total As in this fraction. The non-toxic arsenobetaine (AB) was the dominant form in mesopelagic crustaceans and fish species, accounting for approximately 70% and 50% of total As, respectively. Other water-soluble species were present in minor fractions, including carcinogenic inorganic As, which, in most samples, was below limit of quantification. The fish species had a higher proportion of AsLipids, approximately 35% of total As, compared to crustaceans which contained 20% on average. The feed processing simulation revealed generally low levels of water-soluble As species besides AB, but considerable fractions of potentially toxic AsLipids were found in the biomass, and transferred to the mesopelagic meal and oil. This study is the first to report occurrence data of at least 12 As species in mesopelagic organisms, thereby providing valuable information for future risk assessments on the feasibility of harnessing mesopelagic biomass as feed ingredients.

Processing Mixed Mesopelagic Biomass from the North-East Atlantic into Aquafeed Resources; Implication for Food Safety.

Aquaculture produces most of the world's seafood and is a valuable food source for an increasing global population. Low trophic mesopelagic biomasses have the potential to sustainably supplement aquafeed demands for increased seafood production. The present study is a theoretical whole-chain feed and food safety assessment on ingredients from mesopelagic biomass and the resulting farmed fish fed these ingredients, based on analysis of processed mesopelagic biomass. Earlier theoretical estimations have indicated that several undesirable compounds (e.g., dioxins and metals and fluoride) would exceed the legal maximum levels for feed and food safety. Our measurements on processed mesopelagic biomasses show that only fluoride exceeds legal feed safety limits. Due to high levels of fluoride in crustaceans, their catch proportion will dictate the fluoride level in the whole biomass and can be highly variable. Processing factors are established that can be used to estimate the levels of undesirables in mesopelagic aquafeed ingredients from highly variable species biomass catches. Levels of most the studied undesirables (dioxins, PCBs, organochlorine pesticides, brominated flame retardant, metals, metalloids) were generally low compared to aquafeed ingredients based on pelagic fish. Using a feed-to-fillet aquaculture transfer model, the use of mesopelagic processed aquafeed ingredients was estimated to reduce the level of dioxins and PCBs by ~30% in farmed seafood such as Atlantic salmon.

Do background levels of the pesticide pirimiphosmethyl in plant-based aquafeeds affect food safety of farmed Atlantic salmon?

The substitution of fish oil and fishmeal with plant-based ingredients in commercial aquafeeds for Atlantic salmon, may introduce novel contaminants that have not previously been associated with farmed fish. The organophosphate pesticide pirimiphos-methyl (PM) is one of the novel contaminants that is most prevalent in commercial salmon feed. In this study, the feed-to-fillet transfer of dietary PM and its main metabolites was investigated in Atlantic salmon fillet. Based on the experimental determined PM and metabolite uptake, metabolisation, and elimination kinetics, a physiologically based toxicokinetic (PBTK) compartmental model was developed. Fish fed PM had a relatively low (~4%) PM retention and two main metabolites (2-DAMP and Desethyl-PM) were identified in liver, muscle, kidney and bile. The absence of more metabolised forms of 2-DAMP and Desethyl-PM in Atlantic salmon indicates different metabolism in cold-water fish compared to previous studies on ruminants. The model was used to simulate the long term (>1.5 years) feed-to-fillet transfer of PM + metabolite in Atlantic salmon under realistic farming conditions including seasonal fluctuations in feed intake, growth, and fat deposition in muscle tissue. The model predictions show that with the constant presence of the highest observed PM concentration in commercial salmon feed, fillet PM+ metabolite levels were approximately 5 nmol kg-1, with highest levels for the metabolite 2-DAMP. No EU maximum residue levels (MRL) for PM and its main metabolites exist in seafood to date, but the predicted levels were lower than the MRL for PM in swine of 32.7 nmol kg-1.

Undesirables in Mesopelagic Species and Implications for Food and Feed Safety-Insights from Norwegian Fjords.

The increase in the global population demands more biomass from the ocean as future food and feed, and the mesopelagic species might contribute significantly. In the present study, we evaluated the food and feed safety of six of the most abundant mesopelagic species in Norwegian fjords. Trace elements (i.e., arsenic, cadmium, mercury, and lead), organic pollutants (i.e., dioxins, furans, dioxin-like polychlorinated biphenyls, and polybrominated flame-retardants), and potentially problematic lipid compounds (i.e., wax esters and erucic acid) were analyzed and compared to existing food and feed maximum levels and intake recommendations. Furthermore, contaminant loads in processed mesopelagic biomass (protein, oil, and fish meal) was estimated using worst-case scenarios to identify possible food and feed safety issues. While most undesirables were low considering European food legislation, we identified a few potential food safety issues regarding high levels of fluoride in Northern krill, wax esters in glacier lanternfish, and long-chain monounsaturated fatty acids in silvery lightfish. Our estimates in processed biomass indicated high levels of undesirable trace elements in the protein fraction, frequently exceeding the maximum levels for feed ingredients. However, in fish meal, almost no exceedances were seen. In the oil fraction, dioxins and furans were above the maximum levels, given for food and feed ingredients. The present study is crucial to enable an evaluation of the value of these species; however, more data is needed before proceeding with large-scale harvesting of mesopelagic biomass.

No transfer of the non-regulated mycotoxins, beauvericin and enniatins, from feeds to farmed fish reared on plant-based diets.

Concern about the risk of exposure to emerging plant-derived mycotoxins such as beauvericin and enniatins has been addressed by the European Commission who requested the European Food Safety Authority for a scientific opinion on their risk to human and animal health. The studied mycotoxins were found in feeds with enniatin B and beauvericin at average concentrations of 19.9 µg/kg and 30 µg/kg, respectively. In all cases, concentrations of all the mycotoxins analyzed were below quantification limits (<0.1 µg/kg) in fish samples (n = 82). The present work provides comprehensive and traceable data of emerging mycotoxins in plant-based aquafeeds and fish reared on the feeds, responding to increasing concerns about safety of farmed fish fed on sustainable feeds. On the basis of data reported, there was no transfer of the emerging mycotoxins, beauvericin and enniatins, from feeds to fish and so, no risk for human consumption.

LC-MS/MS method for the determination of organophosphorus pesticides and their metabolites in salmon and zebrafish fed with plant-based feed ingredients.

The composition of Atlantic salmon feed has changed considerably over the last two decades from being marine-based (fishmeal and fish oil) to mainly containing plant ingredients. Consequently, concern related to traditional persistent contaminants typically associated with fish-based feed has been replaced by other potential contaminants not previously associated with salmon farming. This is the case for many pesticides, which are used worldwide to increase food production, and may be present in plant ingredients. Earlier studies have identified two organophosphorus pesticides, chlorpyrifos-methyl and pirimiphos-methyl, in plant ingredients used for aquafeed production. In the present study, we developed a reliable and sensitive analytical method, based on liquid chromatography coupled to tandem mass spectrometry, for the determination of these pesticides and their main metabolites in warm water (zebrafish) and cold water (Atlantic salmon) species, where possible differences in metabolites could be expected. The method was tested in whole zebrafish and in different salmon tissues, such as muscle, bile, kidney, fat, and liver. The final objective of this work was to assess kinetics of chlorpyrifos-methyl and pirimiphos-methyl and their main metabolites in fish tissue, in order to fill the knowledge gaps on these metabolites in fish tissues when fed over prolonged time.

Effects of Agricultural Pesticides in Aquafeeds on Wild Fish Feeding on Leftover Pellets Near Fish Farms.

Screening has revealed that modern-day feeds used in Atlantic salmon aquaculture might contain trace amounts of agricultural pesticides. To reach slaughter size, salmon are produced in open net pens in the sea. Uneaten feed pellets and undigested feces deposited beneath the net pens represent a source of contamination for marine organisms. To examine the impacts of long-term and continuous dietary exposure to an organophosphorus pesticide found in Atlantic salmon feed, we fed juvenile Atlantic cod (Gadus morhua), an abundant species around North Atlantic fish farms, three concentrations (0.5, 4.2, and 23.2 mg/kg) of chlorpyrifos-methyl (CPM) for 30 days. Endpoints included liver and bile bioaccumulation, liver transcriptomics and metabolomics, as well as plasma cholinesterase activity, cortisol, liver 7-ethoxyresor-ufin-O-deethylase activity, and hypoxia tolerance. The results show that Atlantic cod can accumulate relatively high levels of CPM in liver after continuous exposure, which is then metabolized and excreted via the bile. All three exposure concentrations lead to significant inhibition of plasma cholinesterase activity, the primary target of CPM. Transcriptomics profiling pointed to effects on cholesterol and steroid biosynthesis. Metabolite profiling revealed that CPM induced responses reflecting detoxification by glutathione-S-transferase, inhibition of monoacylglycerol lipase, potential inhibition of carboxylesterase, and increased demand for ATP, followed by secondary inflammatory responses. A gradual hypoxia challenge test showed that all groups of exposed fish were less tolerant to low oxygen saturation than the controls. In conclusion, this study suggests that wild fish continuously feeding on leftover pellets near fish farms over time may be vulnerable to organophosphorus pesticides.

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.

Species-Specific Discrimination of Insect Meals for Aquafeeds by Direct Comparison of Tandem Mass Spectra.

Insect protein has the potential to become a sustainable feed ingredient for the rapidly growing aquaculture industry. In the European Union, insect derived protein is placed under the same legislation as processed animal proteins (PAP). It is therefore of interest to develop methods for regulatory use, which unambiguously identify the species origin of insect-based ingredients. We performed (i) total protein quantification of insect samples using the traditional nitrogen-to-protein conversion factor of 6.25 and the sum of anhydrous amino acids, (ii) quantitative amino acid profiling and (iii) high-throughput tandem mass spectrometry to describe and differentiate 18 different commercial-grade insect meal samples derived from Hermetia illucens (8), Tenebrio molitor (5), Alphitobius diaperinus (3) and Acheta domesticus (2). In addition, we investigated and compared different protein extraction and digestion protocols for proteomic analysis. We found that irrespective of sample preparation, shotgun proteomics in combination with direct spectral comparison were able to differentiate insect meal according to their taxonomic classification. The insect specific spectral libraries created in the present work can in future be used to develop more sensitive targeted methods of insect PAP identification and quantification in commercial feed mixtures.

A Refined Nontarget Workflow for the Investigation of Metabolites through the Prioritization by in Silico Prediction Tools.

The application of nontargeted strategies based on high-resolution mass spectrometry (HRMS) directed toward the discovery of metabolites of known contaminants in fish is an interesting alternative to true nontarget screening. To reduce prolonged and costly laboratory experiments, recent advances in computing power have permitted the development of comprehensive knowledge-based software to predict the metabolic fate of chemicals. In addition, machine-based learning tools allow the prediction of chromatographic retention times (RT) or collision cross section (CCS) values when using ion mobility spectrometry (IMS). These tools can ease data evaluation and strengthen the confidence in the identification of compounds. The current work explores the capabilities of in silico prediction tools, refined by the use of RT and CCS prediction, to prioritize and facilitate nontarget liquid chromatography (LC)-IMS-HRMS data processing. The fate of the insecticide pirimiphos-methyl (PM) in farmed Atlantic salmon exposed to contaminated feed was used as a case study. The theoretical prediction of 60 potentially relevant biological PM metabolites permitted the prioritization of screening in different salmon tissues (liver, kidney, bile, muscle, and fat) of known and unknown PM metabolites. An average of 43 potential positives was found in the sample matrixes based on the accurate mass of protonated molecules (mass error ≤5 ppm). The application of different tolerance filters for RT (Δ ≤ 2 min) and CCS (Δ ≤ 6%) based on predicted values permitted us to reduce this number up to 66% of the features. Finally, five PM metabolites could be identified; two known metabolites (2-DAMP and N-desethyl PM) were confirmed with a standard, whereas three previously unknown metabolites (2-DAMP glucuronide, didesethyl PM, and hydroxy-2-DAMP glucuronide) were tentatively identified in different matrixes, allowing the first proposition of a metabolic pathway in fish.

Identification of ethoxyquin and its transformation products in salmon after controlled dietary exposure via fish feed.

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.

Tolerance and dose-response assessment of subchronic dietary ethoxyquin exposure in Atlantic salmon (Salmo salar L.).

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 ß-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.

Effects of dietary deoxynivalenol or ochratoxin A on performance and selected health indices in Atlantic salmon (Salmo salar).

Post-smolt Atlantic salmon (Salmo salar) were fed with standard feed added one of five concentrations of either pure deoxynivalenol (DON; 0.5-6 mg/kg) or pure ochratoxin A (OTA; 0.2-2.4 mg/kg), or no added toxins for up to 8 weeks. Performance effects (feed intake, feed efficiency, gain, length and condition factor), various clinical biochemical parameters, packed cell volume and vaccination response against Aeromonas salmonicidae were all inversely correlated with DON dose, whereas relative liver weight increased with DON dose. In fish fed OTA, however, the effects at the doses tested were rather small. We observed no effects of OTA exposure on performance parameters, but some clinical biochemical parameters tended to increase with OTA dose primarily at 3 weeks, and compared with controls OTA exposure caused increased mRNA expression of two immune markers in the spleen. No liver histopathological effects were found from DON or OTA exposure. For DON, we derived a BMDL20 of 0.3 mg/kg feed for reduced total protein in plasma, a BMDL5 of 0.5 mg/kg feed for reduced condition factor, and a NOAEL of 1 mg/kg feed for DON. For OTA, a BMDL or NOAEL could not be derived (>2.4 mg/kg).

Factors influencing risk assessments of brominated flame-retardants; evidence based on seafood from the North East Atlantic Ocean.

Brominated flame-retardants (BFRs) such as polybrominated diphenyl ethers (PBDE) and hexabromocyclododecane (HBCD) are considered hazardous to human health. Due to their persistence, they are still present in the environment and in biota and seafood is major contributor of BFRs to human exposure. Here, we used data from >9700 samples of wild and farmed fish, fish feed and fish feed ingredients collected from the North Atlantic between 2006 and 2016 aiming to investigate factors influencing the risk assessments of BFRs. Due to most representative number of analyses, PBDEs were the main focus of investigation. Mean ∑PBDE in fillet samples ranged from below quantification in Atlantic cod fillet to 2.0 µg kg-1 in Atlantic halibut. The main congener contributing to the ∑PBDE in all species was BDE 47. Factors affecting the level of BFR in seafood were multifaceted, and the levels were within species mainly determined by fish age, geographical origin and time of sampling. BDE 47, 99, 153 and HBCD were selected for margin of exposure (MOE) evaluation. When other sources of BFR than seafood were excluded, our risk assessment showed low risk at the current dietary intake of seafood. However, the dietary intake of BDE 99 may be of concern for toddlers when all sources are considered. The choice of fish species, dietary studies, choice of statistics, as well as exposure from other sources than seafood, were all factors that influenced the final MOE of BFRs. We propose the use of regression on order statistics as a tool for risk assessment, to illustrate means and spreads in large surveillance datasets to avoid the issue of measurements below the limit of quantification. A harmonized, updated evaluation of the risk associated with exposure to BFRs from diet, air and dust is warranted, where the fish species most commonly consumed also is taken into consideration.

Modelling the long-term feed-to-fillet transfer of leuco crystal violet and leuco malachite green in Atlantic salmon (Salmo salar).

Leuco crystal violet (LCV) and leuco malachite green (LMG) are the main metabolites of two dyes that are forbidden for use in food production, but can be present at low background concentration in novel Atlantic salmon feed ingredients such as processed animal proteins (animal by-product [ABP]). In this study, the potential transfer of dietary LCV or LMG to the fillet of farmed Atlantic salmon was investigated. The uptake and elimination rate kinetics were determined in seawater-adapted Atlantic salmon (initial weight 587 ± 148 g) fed two levels of either LCV- or LMG-enriched diets (~500 and 4000 µg kg-1, respectively) for 40 days, followed by a 90-day depuration period with feeding on control diets (<0.15 µg kg-1 LCV and LMG). A three-compartmental model was developed, based on a fillet fat, fillet muscle and a central body compartment comprising all other tissues. Model calibrations showed a good fit with measured values during overall uptake and elimination period; however, the model poorly predicted the short-term (days) peak measured values at the end of the exposure period. The model was used to simulate the long-term (>16 months) LCV and LMG 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 gave highest expected LCV and LMG fillet concentrations of approximately 0.12 and 0.45 µg kg-1, depending on the dietary levels of ABP and background level of LCV and LMG contamination. These levels are under the reference point for action of 2 µg kg-1 for the sum of MG and LMG that EFSA assessed as adequate to protect public health. However, for LCV, the predicted highest levels exceed the analytical decision limit (CCα) of 0.15 µg kg-1 for the method used in this paper.

Subchronic dietary exposure to ethoxyquin dimer induces microvesicular steatosis in male BALB/c mice.

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 ß-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 and selenium species in feeds and muscle tissue of Atlantic salmon.

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.