Aquaculture sludge as feed for black soldier fly: Transfer of chemical and biological contaminants and nutrients.

Aquaculture sludge (uneaten feed and faeces) is nutrient rich and has potential as feed for insects. The aim of this study was to investigate the transfer of chemical and biological contaminants, as well as nutrients, from aquaculture sludge to black soldier fly larvae. The larvae were reared on a sludge mixture made of different sludges collected from Norwegian freshwater salmonid facilities. The sludge was spiked with four common salmon pathogens: Infectious Pancreatic Necrosis Virus, Infectious Salmon Anemia virus, Yersinia ruckeri or Mycobacterium salmoniphilum. During the 15 days of growth on sludge, the black soldier fly larvae accumulated valuable nutrients including protein, fat, eicosapentaenoic acid, iron, manganese, zinc and selenium. The larvae also accumulated undesirable substances including cadmium, mercury, dioxins and polychlorinated biphenyls. The concentrations of dioxins exceeded the EU maximum level set for animal feed. None of the salmon pathogens that were spiked to the sludge were detected in the black soldier fly larvae. This study reports low risk of transfer of salmon pathogens from sludge to insect larvae, and showed that the transfer of heavy metals, minerals and metalloids are in accordance with earlier studies. The large variations in levels of heavy metals between batches of sludge can cause levels in BSF exceeding the EU maximum levels, and thus indicate a need for monitoring of the proposed value chain. The transfer of dioxins from sludge to insects, reported for the first time in this paper, would be of special interest for future research, with special focus on risk mitigation.

Characterization of nutrients and contaminants in fish sludge from Atlantic salmon (Salmo salar L.) production sites - A future resource.

A total of 47 fish sludge samples from commercial land-based Atlantic salmon (Salmo salar) farms in Norway were assessed for their nutrient composition, presence of various legacy contaminants and a wide spectrum of contaminants of emerging concern, veterinary medicines as well as selected salmonid pathogenic bacteria and virus. The aim was to document the levels of desirable and undesirable components in fish sludge in relation to a potential future use of sludge as invertebrate feed. The samples had variable, but relatively high protein and fat contents, indicating a high load of undigested feed in some of the sludge samples. Fatty acid analysis showed the presence of essential omega-3 fatty acids. In terms of undesirable substances, 43% and 84% of the sludge samples contained levels of arsenic and cadmium, respectively, which exceeded the EU Maximum Levels established for complete animal feed. The concentrations of copper, zinc, iron and aluminum were highly variable in the sludge samples. The concentrations of dioxins, sum PCB6, and chlorinated pesticides were all below the Maximum Levels for animal feed. Of the 18 per- and polyfluoroalkyl substances (PFAS) only one compound (L-PFOS) was present at measurable levels. None of the samples had detectable levels of veterinary medicines, salmonid virus or bacteria. Performing a suspect and non-target screening of the sludge samples identified 18 compounds, including four pharmaceuticals, plastic-related products and the UV filter benzophenone, warranting further investigations. Overall, the results from this study show that fish sludge is a nutrient-rich resource; however, undesirable substances, originating from the feed or from treatment of sludge may be present.

Organ-specific mercury stable isotopes, speciation and particle measurements reveal methylmercury detoxification processes in Atlantic Bluefin Tuna.

Identifying metabolism and detoxification mechanisms of Hg in biota has important implications for biomonitoring, ecotoxicology, and food safety. Compared to marine mammals and waterbirds, detoxification of MeHg in fish is understudied. Here, we investigated Hg detoxification in Atlantic bluefin tuna Thunnus thynnus using organ-specific Hg and Se speciation data, stable Hg isotope signatures, and Hg and Se particle measurements in multiple tissues. Our results provide evidence for in vivo demethylation and biomineralization of HgSe particles, particularly in spleen and kidney. We observed a maximum range of 1.83‰ for δ202Hg between spleen and lean muscle, whereas Δ199Hg values were similar across all tissues. Mean percent methylmercury ranged from 8% in spleen to 90% in lean muscle. The particulate masses of Hg and Se were higher in spleen and kidney (Hg: 61% and 59%, Se: 12% and 6%, respectively) compared to muscle (Hg: 2%, Se: 0.05%). Our data supports the hypothesis of an organ-specific, two-step detoxification of methylmercury in wild marine fish, consisting of demethylation and biomineralization, like reported for waterbirds. While mass dependent fractionation signatures were highly organ specific, stable mass independent fractionation signatures across all tissues make them potential candidates for source apportionment studies of Hg using ABFT.

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.

Thyroid function and urinary concentrations of iodine, selenium, and arsenic in vegans, lacto-ovo vegetarians and pescatarians.

PURPOSE: Populations following a plant-based diet may be at particular risk of thyroid dysfunction due to low iodine and selenium intakes. The main purpose was to assess thyroid function and urinary concentration of iodine, selenium, and arsenic, in subjects following a vegan, lacto-ovo vegetarian, or pescatarian diet. METHODS: In Norway, a country without mandatory dietary iodine fortification, 205 adults, following vegan (n = 115), lacto-ovo vegetarian (n = 55) and pescatarian diet (n = 35) were included. Thyroglobulin (Tg), thyroid-stimulating hormone (TSH), free triiodothyronine (fT3), free thyroxine (fT4), and serum anti-TPO (S-anti-TPO) were measured in a venous blood sample and concentrations of iodine (UIC), creatinine (UCC), selenium, and arsenic were measured from single spot urine samples. RESULTS: Subclinical hypothyroidism (TSH > 4.0 mU/L) was observed in 3% of subjects. The overall median (p25, p75) Tg was 17 (9, 30) µg/L and vegans had higher Tg compared to pescatarians. Vegans not consuming iodine-containing supplements (n = 43) had higher Tg, than supplement users (n = 72), 27 (11, 44) vs. 16 (8, 25) µg/L and higher fT4, 16 (15, 17) vs. 15 (14, 17) pmol/L, respectively. The overall median UIC was 57 (28, 130) µg/L, all dietary groups had median UIC below WHO thresholds. Median urinary selenium and arsenic concentration was 13 (6, 22) and 3 (2, 8) µg/L, respectively. CONCLUSION: The prevalence of subclinical hypothyroidism was low and fT4 and fT3 were within the normal range for all dietary groups. Vegans had significantly increased Tg compared to pescatarians.

Arsenic speciation in low-trophic marine food chain - An arsenic exposure study on microalgae (Diacronema lutheri) and blue mussels (Mytilus edulis L.).

Microalgae and blue mussels are known to accumulate undesirable substances from the environment, including arsenic (As). Microalgae can biotransform inorganic As (iAs) to organoarsenic species, which can be transferred to blue mussels. Knowledge on As uptake, biotransformation, and trophic transfer is important with regards to feed and food safety since As species have varying toxicities. In the current work, experiments were conducted in two parts: (1) exposure of the microalgae Diacronema lutheri to 5 and 10 µg/L As(V) in seawater for 4 days, and (2) dietary As exposure where blue mussels (Mytilus edulis L.) were fed with D. lutheri exposed to 5 and 10 µg/L As(V), or by aquatic exposure to 5 µg/L As(V) in seawater, for a total of 25 days. The results showed that D. lutheri can take up As from seawater and transform it to methylated As species and arsenosugars (AsSug). However, exposure to 10 µg/L As(V) resulted in accumulation of iAs in D. lutheri and lower production of methylated As species, which may suggest that detoxification mechanisms were overwhelmed. Blue mussels exposed to As via the diet and seawater showed no accumulation of As. Use of linear mixed models revealed that the blue mussels were gradually losing As instead, which may be due to As concentration differences in the mussels' natural environment and the experimental setup. Both D. lutheri and blue mussels contained notable proportions of simple methylated As species and AsSug. Arsenobetaine (AB) was not detected in D. lutheri but present in minor fraction in mussels. The findings suggest that low-trophic marine organisms mainly contain methylated As species and AsSug. The use of low-trophic marine organisms as feed ingredients requires further studies since AsSug are regarded as potentially toxic, which may introduce new risks to feed and food safety.

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.

Seasonal variations in mercury, cadmium, lead and arsenic species in Norwegian blue mussels (Mytilus edulis L.) - Assessing the influence of biological and environmental factors.

BACKGROUND: Blue mussels (Mytilus edulis L.) can accumulate undesirable substances, including the potentially toxic elements (PTEs) cadmium (Cd), mercury, (Hg), lead (Pb), arsenic (As) and As species. In this study, the levels of PTEs and As species were determined in samples of blue mussels to assess the influence of environmental and biological factors, and evaluate the potential risk associated with blue mussels in terms of food and feed safety. METHODOLOGY: Blue mussels were collected monthly from one location in Western Norway from February 2018 to December 2018, and from April 2019 to April 2020. Samples were analyzed for PTEs using inductively coupled plasma mass spectrometry (ICP-MS), and high-performance liquid chromatography (HPLC) coupled to ICP-MS. Temperature, salinity and fluorescence (chlorophyll a) were monitored in the seawater column by STD/CTD, to assess the potential influence of these environmental factors on the PTE levels in the mussels. RESULTS: The results showed seasonal variations in the PTEs, with somewhat higher concentrations in spring and winter months. Unusually high levels of total As (101.2 mg kg-1 dw) and inorganic As (53.6 mg kg-1 dw) were observed for some of the time points. The organic As species arsenobetaine was generally the major As species (17-82% of total As) in the mussels, but also simple methylated As species and arsenosugars were detected. Principal components analysis (PCA) did not show a consistent relationship between the environmental factors and the PTE concentrations, showing contrary results for some elements for the periods studied. The condition index (CI) could explain variations in element concentration with significant correlations for Cd (r = -0.67, p = 0.009) and Pb (r = -0.62, p = 0.02 in 2019/20 and r = -0.52, p = 0.02 in 2018), whereas the correlation between As and CI was not significant (r = 0.12 in 2018, and r = -0.06 in 2019/20). Higher concentrations of iAs and arsenosugars coincided with increased signals of chlorophyll a, suggesting that phytoplankton blooms could be a source of As in the blue mussels. CONCLUSION: To our knowledge, this is the first study of As species in blue mussels collected over a time period of two years, providing an insight into the natural variations of these chemical forms in mussels. In terms of mussel as food and future feed material, concentrations of Cd, Hg and Pb were below the maximum levels (MLs) established in the EU food and feed legislation. However, levels of As and iAs in mussels at some time points exceeded the MLs for As in the feed legislation, and the margin of exposure (MOE) was low if these mussels were for human consumption, highlighting the importance of determining the chemical forms of As in feed and food.

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.

Speciation analysis of organoarsenic species in marine samples: method optimization using fractional factorial design and method validation.

Organoarsenic species in marine matrices have been studied for many years but knowledge gaps still exist. Most literature focuses on monitoring of arsenic (As) species using previously published methods based on anion- and cation-exchange high-performance liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICP-MS). These studies are often limited to few As species and/or only specific method performance characteristics are described. Most marine certified reference materials (CRMs) are only certified for arsenobetaine (AB) and dimethylarsinate (DMA), making it difficult to evaluate the accuracy of analytical methods for other organoarsenic species. To address these gaps, the main objective of this work was to develop and validate a method for speciation analysis of a broad range of organoarsenic species in marine matrices. Optimum extraction conditions were identified through a 27-3 fractional factorial design using blue mussel as test sample. The effects of sample weight, type and volume of extraction solution, addition of H2O2 to the extraction solution, extraction time and temperature, and use of ultrasonication were investigated. The highest As recoveries were obtained by using 0.2 g as sample weight, 5 mL of aqueous methanol (MeOH:H2O, 50% v/v) as extractant, extraction carried out at 90 °C for 30 min, and without ultrasonication. Anion- and cation-exchange HPLC-ICP-MS settings were subsequently optimized. The method detected a total of 33 known and unknown As species within a run time of 23 and 20 min for cation-exchange and anion-exchange, respectively. A single-laboratory validation was conducted using several marine CRMs: BCR 627 (tuna fish tissue), ERM-CE278k (mussel tissue), DORM-4 (fish protein), DOLT-5 (dogfish liver), SQID-1 (cuttlefish), TORT-3 (lobster hepatopancreas), and CRM 7405-b (hijiki seaweed). Method performance characteristics were evaluated based on selectivity, limits of detection and quantification, linearity, trueness, precision, and measurement uncertainty. This work proposes an extraction procedure which allowed satisfactory quantification of As species with low solvent and energy consumption, supporting "Green Chemistry" principles. The study also presents a new set of As speciation data, including methylated arsenic species and arsenosugars, in recently issued marine CRMs, which will be valuable for future speciation studies on As. This work is the first to report a total of 33 different As species in marine CRMs.

In vitro digestion method to evaluate solubility of dietary zinc, selenium and manganese in salmonid diets.

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.

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.

Replacing fish meal with insect meal in the diet of Atlantic salmon (Salmo salar) does not impact the amount of contaminants in the feed and it lowers accumulation of arsenic in the fillet.

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.

A Validated Routine Method for Butyltin Determination in Seafood by Gas Chromatography Inductively Coupled Plasma Isotope Dilution Mass Spectrometry.

Organotin compounds are anthropogenic metal species with multiple uses as pesticides, preservatives, antifouling agents, biocides, and catalysts. Butyltins are the main organotin compounds found in biota, and the highest levels are found in marine foodstuffs. In this paper, we present the figures of merit for an in-house validated method for routine analysis of butyltins in seafood using GC inductively coupled plasma isotope dilution MS. The working range of the method spanned several orders of magnitude from 3.3-1013, 2.4-785, and 0.3-900 ng Sn/g dry weight for monobutyltin (MBT), dibutyltin (DBT), and tributyltin (TBT), respectively. The trueness of the method was evaluated by analyzing Certified Reference Materials (CRMs) ERM CRM 477 (Mussel Tissue) and NIES CRM 15 (Scallop). Recoveries, with RSD % in parentheses, were 78 (±14), 80 (±6), and 88% (±8%) for MBT, DBT, and TBT in ERM CRM 477 and 96% (±5%) for TBT in NIES CRM 15. Good agreements were found between experimental uncertainties and uncertainties predicted for single-laboratory validated methods calculated from the maximum standard measurement uncertainty function. The method has proven to be robust, and the wide range of seafood validated ensures that the method is applicable for measuring butyltins in marine tissue.

A Validated Routine Method for Butyltin Determination in Seafood by Gas Chromatography Inductively Coupled Plasma Isotope Dilution Mass Spectrometry.

Organotin compounds are anthropogenic metal species with multiple uses as pesticides, preservatives, antifouling agents, biocides, and catalysts. Butyltins are the main organotin compounds found in biota, and the highest levels are found in marine foodstuffs. In this paper, we present the figures of merit for an in-house validated method for routine analysis of butyltins in seafood using GC inductively coupled plasma isotope dilution MS. The working range of the method spanned several orders of magnitude from 3.3-1013, 2.4-785, and 0.3-900 ng Sn/g dry weight for monobutyltin (MBT), dibutyltin (DBT), and tributyltin (TBT), respectively. The trueness of the method was evaluated by analyzing Certified Reference Materials (CRMs) ERM CRM 477 (Mussel Tissue) and NIES CRM 15 (Scallop). Recoveries, with RSD % in parentheses, were 78 (±14), 80 (±6), and 88% (±8%) for MBT, DBT, and TBT in ERM CRM 477 and 96% (±5%) for TBT in NIES CRM 15. Good agreements were found between experimental uncertainties and uncertainties predicted for single-laboratory validated methods calculated from the maximum standard measurement uncertainty function. The method has proven to be robust, and the wide range of seafood validated ensures that the method is applicable for measuring butyltins in marine tissue.

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.

A study of lipid- and water-soluble arsenic species in liver of Northeast Arctic cod (Gadus morhua) containing high levels of total arsenic.

In the present study liver samples (n=26) of Northeast Arctic cod (Gadus morhua), ranging in total arsenic concentrations from 2.1 to 240mg/kg liver wet weight (ww), were analysed for their content of total arsenic and arsenic species in the lipid-soluble and water-soluble fractions. The arsenic concentrations in the lipid fractions ranged from 1.8 to 16.4mg As/kg oil of liver, and a linear correlation (r(2)=0.80, p<0.001) was observed between the total arsenic concentrations in liver and the total arsenic concentrations in the respective lipid fractions of the same livers. The relative proportion of arsenolipids was considerably lower in liver samples with high total arsenic levels (33-240mg/kg ww), which contained from 3 to 7% of the total arsenic in the lipid-soluble fraction. In contrast liver samples with low arsenic concentrations (2.1-33mg/kg ww) contained up to 50% of the total arsenic as lipid-soluble species. Arsenic speciation analysis of the lipid-soluble fractions of the livers, using reversed-phase high performance liquid chromatography coupled to inductively coupled plasma mass spectrometry (HPLC-ICP-MS), revealed the presence of several arsenolipids. Three major arsenic-containing hydrocarbons (C17H39AsO, C19H41AsO and C23H37AsO) and five arsenic-containing fatty acids (C17H35AsO3, C19H39AO3, C19H37AsO3, C23H37AsO3 and C24H37AsO3) were identified using HPLC coupled to quadrupole time-of-flight mass spectrometry (qTOF-MS). Arsenobetaine was the major arsenic species in the water-soluble fraction of the livers, while dimethylarsinate, arsenocholine and inorganic arsenic were minor constituents. Inorganic arsenic accounted for less than 0.1% of the total arsenic in the liver samples.

Arsenic-containing fatty acids and hydrocarbons in marine oils - determination using reversed-phase HPLC-ICP-MS and HPLC-qTOF-MS.

Arsenolipids are the major arsenic species present in marine oils. Several structures of arsenolipids have been elucidated the last 5 years, demonstrating the chemical complexity of this trace element in the marine environment. Several commercial fish oils and marine oils, ranging in total arsenic concentrations from 1.6 to 12.5 mg kg(-1) oil, were analyzed for arsenolipids using reversed-phase high performance liquid chromatography coupled with inductively coupled plasma mass spectrometry (HPLC-ICP-MS). The arsenolipids were quantified using three different arsenic-containing calibration standards; dimethylarsinate (DMA), triphenylarsinoxide (Ph3AsO) and a synthesized arsenic-containing hydrocarbon (AsHC) (dimethylarsinoyl nonadecane; C21H43AsO). The observed variation in signal intensity for arsenic during the gradient elution profile in reversed-phase HPLC was compensated for by determining the time-resolved response factors for the arsenolipids. Isotopes of germanium ((74)Ge) and indium ((115)In) were suited as internal standards for arsenic, and were used for verification of the arsenic signal response factors during the gradient elution. Dimethylarsinate was the most suitable calibration standard for the quantification of arsenolipids, with recoveries between 91% and 104% compared to total arsenic measurements in the same extracts. A range of marine oils was investigated, including oils of several fish species, cod liver and seal, as well as three commercial fish oils. The AsHCs - C17H38AsO, C19H42AsO and C23H38AsO - were identified as the major arsenolipids in the extracts of all oils by HPLC coupled with quadrupole time-of-flight mass spectrometry (qTOF-MS). Minor amounts of two arsenic-containing fatty acids (AsFAs) (C23H38AsO3 and C24H38AsO3) were also detected in the oils. The sum of the AsHCs and the AsFAs determined in the present study accounted for 17-42% of the total arsenic in the oils.

Detection of arsenic-containing hydrocarbons in a range of commercial fish oils by GC-ICPMS analysis.

The present study describes the use of a simple solid-phase extraction procedure for the extraction of arsenic-containing hydrocarbons from fish oil followed by analysis using gas chromatography (GC) coupled to inductively coupled plasma mass spectrometry (ICPMS). The procedure permitted the analysis of a small sample amount, and the method was applied on a range of different commercial fish oils, including oils of anchovy (Engraulis ringens), Atlantic herring (Clupea harengus), sand eel (Ammodytes marinus), blue whiting (Micromesistius poutassou) and a commercial mixed fish oil (mix of oils of Atlantic herring, Atlantic cod (Gadus morhua) and saithe (Pollachius virens)). Total arsenic concentrations in the fish oils and in the extracts of the fish oils were determined by microwave-assisted acid digestion and ICPMS. The arsenic concentrations in the fish oils ranged from 5.9 to 8.7 mg kg(-1). Three dominant arsenic-containing hydrocarbons in addition to one minor unidentified compound were detected in all the oils using GC-ICPMS. The molecular structures of the arsenic-containing hydrocarbons, dimethylarsinoyl hydrocarbons (C17H38AsO, C19H42AsO, C23H38AsO), were verified using GC coupled to tandem mass spectrometry (MS/MS), and the accurate masses of the compounds were verified using quadrupole time-of-flight mass spectrometry (qTOF-MS). Additionally, total arsenic and the arsenic-containing hydrocarbons were studied in decontaminated and in non-decontaminated fish oils, where a reduced arsenic concentration was seen in the decontaminated fish oils. This provided an insight to how a decontamination procedure originally ascribed for the removal of persistent organic pollutants affects the level of arsenolipids present in fish oils.