Evaluation of the maximum limits for selenium in Atlantic salmon feeds

publishedVersio

Quality of fish sludge as fertiliser to spring cereals: Nitrogen effects and environmental pollutants

The aim of this study was to contribute to development of organic fertiliser products based on fish sludge (i.e. feed residues and faeces) from farmed smolt. Four dried fish sludge products, one liquid digestate after anaerobic digestion and one dried digestate were collected at Norwegian smolt hatcheries in 2019 and 2020. Their quality as fertilisers was studied by chemical analyses, two 2-year field experiments with spring cereals and soil incubation combined with a first-order kinetics N release model. Cadmium (Cd) and zinc (Zn) concentrations were below European Union maximum limits for organic fertilisers in all products except one (liquid digestate). Relevant organic pollutants (PCB7, PBDE7, PCDD/F + DL-PCB) were analysed for the first time and detected in all fish sludge products. Nutrient composition was unbalanced, with low nitrogen/phosphorus (N/P) ratio and low potassium (K) content relative to crop requirements. Nitrogen concentration in the dried fish sludge products varied (27–70 g N kg-1 dry matter), even when treated by the same technology but sampled at different locations and/or times. In the dried fish sludge products, N was mainly present as recalcitrant organic N, resulting in lower grain yield than with mineral N fertiliser. Digestate showed equally good N fertilisation effect as mineral N fertiliser, but drying reduced N quality. Soil incubation in combination with modelling is a relatively cheap tool that can give a good indication of N quality in fish sludge products with unknown fertilisation effects. Carbon/N ratio in dried fish sludge can also be used as an indicator of N quality.publishedVersio

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.publishedVersio

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.publishedVersio

Use of (Q)SAR genotoxicity predictions and fuzzy multicriteria decision-making for priority ranking of ethoxyquin transformation products

Ethoxyquin (EQ; 6-ethoxy-2,2,4-trimethyl-1,2-dihydroquinoline) has been used as an antioxidant in feed for pets and food-producing animals, including farmed fish such as Atlantic salmon. In Europe, the authorization for use of EQ as a feed additive was suspended, due to knowledge gaps concerning the presence and toxicity of EQ transformation products (TPs). Recent analytical studies focusing on the detection of EQ TPs in farmed Atlantic salmon feed and fillets reported the detection of a total of 27 EQ TPs, comprising both known and previously not described EQ TPs. We devised and applied an in silico workflow to rank these EQ TPs according to their genotoxic potential and their occurrence data in Atlantic salmon feed and fillet. Ames genotoxicity predictions were obtained applying a suite of five (quantitative) structure–activity relationship ((Q)SAR) tools, namely VEGA, TEST, LAZAR, Derek Nexus and Sarah Nexus. (Q)SAR Ames genotoxicity predictions were aggregated using fuzzy analytic hierarchy process (fAHP) multicriteria decision-making (MCDM). A priority ranking of EQ TPs was performed based on combining both fAHP ranked (Q)SAR predictions and analytical occurrence data. The applied workflow prioritized four newly identified EQ TPs for further investigation of genotoxicity. The fAHP-based prioritization strategy described here, can easily be applied to other toxicity endpoints and groups of chemicals for priority ranking of compounds of most concern for subsequent experimental and mechanistic toxicology analyses.publishedVersio

A ranking method of chemical substances in foods for prioritisation of monitoring, based on health risk and knowledge gaps

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.publishedVersio

Zinc uptake in fish intestinal epithelial model RTgutGC: Impact of media ion composition and methionine chelation

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 < 0.01), but not at 12 h or 24 h. Half effective concentration of Zn exposure over 12 h (EC50, in μM) was not differentially affected by media composition (FW, 59.7 ± 12.1 or SW, 83.2 ± 7.2; mean ± SE, P = 0.43). Zinc (65Zn) influx in RTgutGC was not different between FW or SW treated cells, but increased significantly in the presence of methionine (2 mM, L-Met or DL-Met). An interaction effect was observed between Zn concentration and media ionic composition on the impact of Met on apical Zn uptake (L-met, P < 0.001; DL-met, P = 0.02). In the presence of Met, apical Zn uptake in SW medium was significantly lower compared to FW, but only at higher Zn concentrations (12 and 25 μM, P < 0.01). Further, Met facilitated Zn uptake was reduced in cells treated with an amino acid transport system blocker with the effect being more significant and stereospecific in SW ionic conditions. The findings of this study showed that (i) Zn speciation in the presence of Met improved apical Zn uptake in RTgutGC cells and Zn-Met species were possibly taken up through Met uptake system. (ii) The effect was differentially affected by the ionic composition of the medium. Implications and limitations of the observations towards practical Zn nutrition of salmonids are discussed.publishedVersio

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 â-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