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.

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.

Chemical characterization of 21 species of marine macroalgae common in Norwegian waters: benefits of and limitations to their potential use in food and feed.

BACKGROUND: In the past few years, much effort has been invested into developing a new blue economy based on harvesting, cultivating and processing marine macroalgae in Norway. Macroalgae have high potential for a wide range of applications, e.g. as source of pharmaceuticals, production of biofuels or as food and feed. However, data on the chemical composition of macroalgae from Norwegian waters are scant. This study was designed to characterize the chemical composition of 21 algal species. Both macro- and micronutrients were analysed. Concentrations of heavy metals and the metalloid arsenic in the algae were also quantified. RESULTS: The results confirm that marine macroalgae contain nutrients which are relevant for both human and animal nutrition, the concentrations whereof are highly dependent on species. Although heavy metals and arsenic were detected in the algae studied, concentrations were mostly below maximum allowed levels set by food and feed legislation in the EU. CONCLUSION: This study provides chemical data on a wide range of algal species covering the three taxonomic groups (brown, red and green algae) and discusses both benefits of and potential limitations to their use for food and feed purposes. © 2017 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Uptake of heavy metals and arsenic in black soldier fly (Hermetia illucens) larvae grown on seaweed-enriched media.

BACKGROUND: The black soldier fly (Hermetia illucens) is one of the most promising insect species for use in animal feed. However, studies investigating feed and food safety aspects of using black soldier fly as feed are scarce. In this study, we fed black soldier fly larvae feeding media enriched with seaweed, which contains naturally high concentrations of heavy metals and arsenic. The aim of this study was to investigate the potential transfer of such undesirable substances from the feeding media to the larvae. RESULTS: The larvae accumulated cadmium, lead, mercury and arsenic. Concentrations of these elements in the larvae increased when more seaweed was added to the feeding media. The highest retention was seen for cadmium (up to 93%) and the lowest for total arsenic (up to 22%). When seaweed inclusion exceeded 20% in the media, this resulted in larval concentrations of cadmium and total arsenic above the current European Union maximum levels for these elements in complete feed. CONCLUSION: Our results confirm that insect larvae can accumulate heavy metals and arsenic when present in the feeding media. A broader understanding of the occurrence of these undesirable substances in processed larvae products is needed to assess feed and food safety. © 2017 Society of Chemical Industry.

Methylmercury Induced Neurotoxicity and the Influence of Selenium in the Brains of Adult Zebrafish (Danio rerio).

The neurotoxicity of methylmercury (MeHg) is well characterised, and the ameliorating effects of selenium have been described. However, little is known about the molecular mechanisms behind this contaminant-nutrient interaction. We investigated the influence of selenium (as selenomethionine, SeMet) and MeHg on mercury accumulation and protein expression in the brain of adult zebrafish (Danio rerio). Fish were fed diets containing elevated levels of MeHg and/or SeMet in a 2 × 2 full factorial design for eight weeks. Mercury concentrations were highest in the brain tissue of MeHg-exposed fish compared to the controls, whereas lower levels of mercury were found in the brain of zebrafish fed both MeHg and SeMet compared with the fish fed MeHg alone. The expression levels of proteins associated with gap junction signalling, oxidative phosphorylation, and mitochondrial dysfunction were significantly (p < 0.05) altered in the brain of zebrafish after exposure to MeHg and SeMet alone or in combination. Analysis of upstream regulators indicated that these changes were linked to the mammalian target of rapamycin (mTOR) pathways, which were activated by MeHg and inhibited by SeMet, possibly through a reactive oxygen species mediated differential activation of RICTOR, the rapamycin-insensitive binding partner of mTOR.

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.

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.

Risk assessment of rare earth elements, antimony, barium, boron, lithium, tellurium, thallium and vanadium in teas.

In recent years, a great intensification in the use of various elements especially in modern technology can be observed. However, the anthropogenic activities, including industrialisation, urbanisation or intensive agriculture, have led to the release of many of the elements into the environment. The consequence of the accumulation of the elements both in soil and water systems is their presence in the food chain. Inhalation and consumption of the contaminated food and beverages have been indicated as the main pathways of the exposure to many elements. Due to the fact, that tea is considered the second most popular beverage worldwide and its consumption is constantly increasing, it is crucial to evaluate the safety of the product, especially for toxic elements contamination. Thus, the aim of the project was to evaluate the contamination levels of rare earth elements (REEs) including lanthanides, scandium (Sc) and yttrium (Y) and also antimony (Sb), barium (Ba), boron (B), lithium (Li), tellurium (Te), thallium (Tl) and vanadium (V) in teas. Subsequently, the risk assessment was carried out. Additionally, the Fellowship provided hands-on training on the evaluation of applications of new biocides and participation in the science-based advises given to the Danish Food and Veterinary Administration, Danish Environment Protection Agency and Danish Medical Agency.

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.

Iodine determination in animal feed by inductively coupled plasma mass spectrometry - results of a collaborative study.

A collaborative study was conducted in order to fully validate the performance characteristics and to evaluate the suitability of a method for determination of iodine in animal feed. The method consists of an alkaline extraction in tetramethylammonium hydroxide (TMAH) solution followed by the determination of iodine by inductively coupled plasma-mass spectrometry (ICP-MS). The method was validated for different types of feed and feed materials with a broad concentration range of 0.65-622 mg I/kg. Good agreement was found between the overall mean mass fraction values from the collaborative trial (13.8 ± 1.3 mg I/kg and 0.657 ± 0.228 mg I/kg) and the values previously determined in proficiency tests for two of the test materials (12.65 ± 2.47 mg I/kg and 0.72 ± 0.22 mg I/kg) indicating satisfactory accuracy of the method. Reproducibility standard deviations were between 7.85% and 34.65% and the HorRat values were under the acceptable limit of 2 so the between-laboratory precision was considered acceptable. Based on the statistical evaluation of the results it was concluded that the method is suitable for its intended purpose; it has been accepted as European Standard EN17050:2017 by the European Committee for Standardisation (CEN).

Assessing Mineral Availability in Fish Feeds using Complementary Methods Demonstrated with the Example of Zinc in Atlantic Salmon.

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

Bioavailability of a natural lead-contaminated invertebrate diet to zebrafish.

Dietary metals are increasingly recognized as key determinants of total metal burdens in fish, yet their ecotoxicological significance remains unclear. In this study, a pairwise experimental design was used to assess reproductive performance of zebrafish (Danio rerio) fed diets supplemented with a natural Pb-enriched polychaete, Nereis diversicolor. Zebrafish were fed 1% flake food (dry wt diet/wet wt fish/d), 1% brine shrimp, and 1% N. diversicolor collected from either Gannel estuary, Cornwall, United Kingdom (UK), an estuary with legacy Pb contamination, or Blackwater estuary, Essex, UK, a reference site with low background metal concentrations, for 63 d. Mean daily dietary doses of Pb were 0.417 and 0.1 mg/kg/d (dry wt feed:wet wt fish) for fish fed N. diversicolor from Gannel and Blackwater estuaries, respectively. With the exception of Ag, which was higher for fish fed N. diversicolor from Gannel estuary, there were no differences in daily dietary exposures to other metals (As, Cd, Cu, Fe, and Zn) between treatment groups. Fish fed Pb-enriched Gannel N. diversicolor exhibited no significant impairment to incidence of spawning, numbers of eggs per breeding pair or hatch rate of embryos compared with pre-exposure levels, when N. diversicolor was omitted from the dietary regimen. Nevertheless, metal analysis revealed significant increases in whole-body Pb burdens of male fish fed polychaetes from Gannel estuary, Ag in female fish fed Gannel worms, and Ag and Cd in male fish fed the Blackwater worms. These data demonstrate that Pb naturally incorporated in N. diversicolor is bioavailable to fish, and fish exhibit sex-dependent dietary metal accumulation patterns, but after 63 d of the experimental feeding regimen, reproductive performance was unaffected.

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.

Growth performance, bioavailability of toxic and essential elements and nutrients, and biofortification of iodine of rainbow trout (Onchorynchus mykiss) fed blends with sugar kelp (Saccharina latissima).

Aquaculture production is demanding novel feed ingredients that reflect natural marine nutrient levels, that are also essential to humans. In this regard, biofortification through addition of iodine-rich sugar kelp in feed formulations was assessed in a 12 week rainbow trout trial. Yttrium inclusion in feed allowed determinations of apparent absorption coefficients of essential and potentially toxic elements and apparent digestibility coefficient of nutrients. E.g. apparent absorption coefficients in trouts fortified feed with 1-4% dw kelp were 67-61% As, 32-40% Cd, <5% Fe; 80-83% I; 66-58% Se. Iodine concentrations in feed up to 239 mg/kg (~4% kelp) was proportional to iodine accumulation in trout fillets (R2 = 1.00) with 0.5% transfer ratio. Feed iodine concentrations up to 117 mg/kg (~2% kelp) did not affect growth performance negatively, but increased significantly protein efficiency ratio after eight weeks feeding. However, 4% kelp meal inclusion affected final growth and hepato somatic index, and caused histomorphological changes in the intestine. All fillets had low toxic element concentrations (As, Cd, Hg, Pb). The potential applicability of Saccharina latissima as feed ingredient to tailor iodine concentration in farmed fish is evident. Consuming of a 160 g fillet (2% kelp) contributes ~60% of recommended daily iodine intake for adults.

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.

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.

Tracing simultaneous cadmium accumulation from different uptake routes in brown crab Cancer pagurus by the use of stable isotopes.

High concentrations of cadmium in brown crab are an issue of food safety, and large variations between different areas have been found. To investigate the relative importance of dietary and aqueous uptake regarding the overall accumulation in brown crab, we used stable isotopes to trace the uptake from both routes simultaneously in the same animals. We demonstrated that the analytical challenges regarding background concentrations of natural isotope distribution and polyatomic interferences in the different matrices can be overcome with an appropriate analytical setup and modern mathematical corrections using a computer software. Cadmium was accumulated via both routes and was found in all measured organs at the end of the exposure phase. The obtained data were used to establish accumulation curves for both uptake routes and estimate accumulation parameters for hepatopancreas, as the most important organ in crab regarding total cadmium body burden. Using the estimated parameters in combination with naturally relevant cadmium concentrations in seawater and diet in a model, allowed us to predict the relative importance of the aqueous and dietary uptake route to the total hepatopancreas burden. According to the prediction, the dietary route is the main route of uptake in brown crab with a minimum of 98% of the accumulated cadmium in hepatopancreas originating from diet. Future studies addressing the source and accumulation of cadmium in crab should therefore focus on the uptake from feed and factors connected to foraging.

Accumulation and elimination of methylmercury in Atlantic cod (Gadus morhua L.) following dietary exposure.

Methylmercury is known to bioaccumulate and biomagnify up the marine food chain. Fish from high levels of the marine food chain may contain relatively high concentrations of mercury, and most (>70%) of the mercury found in muscle is methylmercury. In aquaculture, marine protein (mainly fishmeal) is the dominant source of methylmercury, and this raises some concern with regards to fish welfare and consumer safety. A dietary exposure study, including a depuration period, was carried out in order to study the accumulation and elimination of methylmercury in Atlantic cod (Gadus morhua L.), and to estimate the transfer of methylmercury from feed to fish. Fish were sampled throughout a three month exposure period and a three month depuration period. Muscle samples were fractionated into a protein and a lipid fraction by lipid extraction using methanol and chloroform. Mercury and methylmercury were determined by inductively coupled plasma mass spectrometry (ICPMS) and gas chromatography-inductively coupled plasma mass spectrometry (GC-ICPMS), respectively. A continuous accumulation of methylmercury, after a lag period of 10 days, was observed in muscle tissue during the three months exposure to methylmercury (0.95+/-0.03 microg Hg/g feed, n=6). After three months, the final concentration in muscle was 0.38+/-0.04 microg Hg/g w w (n=6), where methylmercury constituted 90-95% of the mercury present. The elimination of methylmercury from muscle was slow and incomplete (within the three months of depuration) with an estimated elimination half-life (t1/2) of 377 days. The transfer of methylmercury from feed to Atlantic cod, described by the estimated absorption efficiency, was 38%. In muscle more than 99% of the mercury was found in the protein fraction. These results suggest that Atlantic cod readily takes up dietary methylmercury, which is efficiently accumulated into muscle, where it is incorporated into larger peptides or proteins. Comparable results were found for Atlantic salmon (Salmo salar L.). After three months of exposure to dietary methylmercury the final concentration in muscle was 0.33+/-0.01 microg Hg/g w w (n=3), where nearly all (>99%) mercury was found in the protein fraction. This further supports that methylmercury is accumulated in fish muscle, where it is incorporated into larger peptides or proteins.

Accumulation and elimination of dietary arsenobetaine in two species of fish, Atlantic salmon (Salmo salar L.) and Atlantic cod (Gadus morhua L.).

Despite the fact that marine fish contain relatively high concentrations of the naturally occurring arsenic compound arsenobetaine, little is known about the disposition of arsenobetaine in fish. We investigated the accumulation, distribution, and elimination of dietary arsenobetaine in Atlantic salmon (Salmo salar L.) and Atlantic cod (Gadus morhua L.), with the focus on muscle, liver, and kidney tissues. The fish were exposed to dietary arsenobetaine (24.7 +/- 0.6 microg As/g feed) for three months, followed by a three-month depuration period. The two species showed marked differences in the accumulation and elimination of arsenobetaine. Total arsenic concentrations in Atlantic salmon increased significantly in muscle, liver, and kidney, whereas in Atlantic cod, a significant increase in total arsenic concentration was observed only in muscle. Elimination kinetics in muscle were distinct between the two species, with elimination half-lives from muscle tissue estimated at approximately 77 d in Atlantic cod and 37 d in Atlantic salmon, resulting in an absorption efficiency approximately twofold higher in Atlantic cod (15 +/- 1%) compared to that in Atlantic salmon (8 +/- 1%). The differences in arsenobetaine disposition studied in Atlantic salmon and Atlantic cod contribute to explain the differences in arsenic levels observed among marine fish.

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.