Global food security is an increasing challenge due to population growth and the limited availability of natural resources, driving the search for sustainable protein sources. In this context, edible insects such as yellow mealworms (Tenebrio molitor) have emerged as a promising alternative, while probiotics have been widely applied in animal production to enhance growth performance and nutritional quality. This study aimed to evaluate the effects of probiotic supplementation on the growth performance, biomass yield, and nutritional composition of yellow mealworm larvae at laboratory and pilot scales. Three probiotic strains-Bacillus velezensis, Bacillus coagulans, and Pediococcus pentosaceus-were tested at four different dosage levels, using wheat bran and brewer's spent grain as feed substrates. Larval growth was monitored weekly, and total harvested biomass, proximate composition (dry matter, protein, fat, and ash), amino acid profile, and mineral composition were determined using standardized analytical methods. At the laboratory scale, probiotic supplementation did not result in significant differences in mean larval weight or total biomass (p > 0.05). In contrast, at the pilot scale, significant improvements in larval growth and biomass were observed for specific probiotic treatments, with mean larval weights reaching approximately 140-150 mg and total harvest biomass increases of up to similar to 15% compared to the control (p < 0.05). Growth curves at both scales followed a sigmoidal pattern with a high correlation between laboratory and pilot experiments (R-2 = 0.98). Probiotic supplementation did not significantly affect crude protein content, but alterations in fat content, specific amino acid concentrations, and mineral composition were observed at the pilot scale, depending on strain and dosage. Overall, the results demonstrate that probiotic supplementation can enhance yellow mealworm production under pilot-scale conditions, while laboratory-scale trials may not fully capture these effects. These findings highlight the importance of scale when evaluating probiotic strategies and support the potential application of Bacilli-based probiotics to improve the efficiency and nutritional quality of industrial insect production systems.

As diets shift toward sustainable options, plant-based products are gaining popularity, though dairy remains a key source of essential micronutrients. Nutritional value depends not only on content but also on bioaccessibility. This study evaluated the bioaccessibility of riboflavin, vitamin K, calcium, phosphorus, zinc, and iodine in dairy products, their plant-based alternatives, and selected plant foods. Riboflavin bioaccessibility was high in liquid dairy and similar in a fortified plant-based beverage, but lower in more complex plant-based foods. Vitamin K showed moderate bioaccessibility in dairy and comparable or higher values in some plant-based products. Calcium bioaccessibility was moderate in dairy but often higher in plant-based alternatives, while phosphorus was highly bioaccessible in dairy and lower in plant-based matrices. Zinc and iodine were largely undetectable in plant-based foods. Overall, bioaccessibility was strongly nutrient- and matrix-dependent, highlighting the importance of food structure and the need for confirmation in vivo.

Nitrite, nitrate, and N-nitrosamines (NAs) have been extensively studied for their potential health risks; however, significant gaps remain in dietary exposure assessments, particularly due to the lack of occurrence data for many food categories including vegetables. Most studies have focused on processed meats, creating a need to explore other food sources to better estimate total dietary exposure. Leafy green vegetables, particularly when processed and stored, may accumulate significant nitrite and NA concentrations due to their naturally high nitrate content, contributing to dietary exposure underestimated in current risk assessments. This study examined the occurrence of nitrite, nitrate, and NAs in nine leafy green vegetables that were purchased from Danish supermarkets, pureed, and stored under varying conditions including ambient temperature, 5 °C, -20 °C and - 80 °C. Nitrite concentrations increased in most vegetables during ambient storage, with the highest concentration found in arugula after 48 h (2448 ± 213 mg·kg-1). Simultaneously, nitrate levels decreased, indicating conversion to nitrite. The NAs N-nitrosodimethylamine (NDMA), N-nitrosopiperidine (NPIP), N-nitrosopyrrolidine (NPYR), N-nitrosomethylethylamine (NMEA), and N-nitrosomorpholine (NMOR) were detected in arugula, spinach, kale, cabbage, and lettuce under various conditions following pureeing. Among them, NPYR was found at the highest level in arugula after 72 h at ambient temperature, reaching 21.7 μg·kg-1. These results emphasize the need to consider vegetables in NA exposure assessments, particularly those processed and stored under varied conditions. Future studies should include microbial, amine precursor or nitrate reductase expression data to provide further mechanistic insights into nitrite and NAs formation in leafy green vegetables.

Aluminium (Al) is the third most common element in the Earth’s crust and occurs naturally in drinking water and agricultural products, and humans are consequently exposed to the element from dietary sources. A tolerable weekly intake of 1 mg/kg has been established by the European Food Safety Authority (EFSA); however, no maximum levels for aluminium in foodstuffs have so far been established in the European Union (EU) legislation. Official food control requires validated methods for the determination of aluminium. Acid digestion assisted by microwaves is the main sample preparation technique used for the determination of aluminium, usually in combination with atomic spectrometry for quantification. In the present study, different parameters in the digestion step were investigated including test portion, digestion temperature, the reagent used and duration of the digestion to assess the aluminium extraction. The presented work is following up on an observation from a proficiency test (PT) on trace elements (including aluminium) in cocoa powder organised in 2020 by the European Union Reference Laboratory for metals and nitrogenous compounds in feed and food (EURL-MN), where the participant results for aluminium showed an unexpectedly large variation. In addition to the PT material, different certified reference materials were included in the present study, and the results highlighted that the temperature and reagent used are the most critical parameters to obtain a satisfactory sample digestion prior to aluminium determination. Based on the obtained results, it is recommended to digest food samples with a mix of ultrapure water and nitric acid for 25 min at a temperature of at least 240 °C with a mix of HNO3 and H2O to achieve satisfactory microwave-assisted digestion.

Food Risk Assess EuropeVolume 2, Issue 2 0024E Technical reportOpen Access Risk assessment of grilled and barbecued food Espen Mariussen, Espen MariussenSearch for more papers by this authorJan Alexander, Jan AlexanderSearch for more papers by this authorBarbara A. Bukhvalova, Barbara A. BukhvalovaSearch for more papers by this authorLisbeth Dahl, Lisbeth DahlSearch for more papers by this authorAnn-Karin Hardie Olsen, Ann-Karin Hardie OlsenSearch for more papers by this authorHelen Engelstad Kvalem, Helen Engelstad KvalemSearch for more papers by this authorMartin Schlabach, Martin SchlabachSearch for more papers by this authorHeidi Amlund, Heidi AmlundSearch for more papers by this authorRita Hannisdal, Rita HannisdalSearch for more papers by this authorAnders Ruus, Anders RuusSearch for more papers by this authorIngunn Anita Samdal, Ingunn Anita SamdalSearch for more papers by this authorHelle K Knutsen, Helle K KnutsenSearch for more papers by this author Espen Mariussen, Espen MariussenSearch for more papers by this authorJan Alexander, Jan AlexanderSearch for more papers by this authorBarbara A. Bukhvalova, Barbara A. BukhvalovaSearch for more papers by this authorLisbeth Dahl, Lisbeth DahlSearch for more papers by this authorAnn-Karin Hardie Olsen, Ann-Karin Hardie OlsenSearch for more papers by this authorHelen Engelstad Kvalem, Helen Engelstad KvalemSearch for more papers by this authorMartin Schlabach, Martin SchlabachSearch for more papers by this authorHeidi Amlund, Heidi AmlundSearch for more papers by this authorRita Hannisdal, Rita HannisdalSearch for more papers by this authorAnders Ruus, Anders RuusSearch for more papers by this authorIngunn Anita Samdal, Ingunn Anita SamdalSearch for more papers by this authorHelle K Knutsen, Helle K KnutsenSearch for more papers by this author First published: 17 April 2024 https://doi.org/10.2903/fr.efsa.2024.FR-0024 Scientific Opinion of the Panel on Contaminants of the Norwegian Scientific Committee for Food and Environment VKM has assessed the health risk from grilled food consumption and summarized the knowledge on formation of several carcinogenic process contaminants in grilled food. Based on exposure scenarios for polycyclic aromatic hydrocarbons (PAH) in grilled food VKM concludes that the risk is low for most consumers. VKM noted a public health concern for those who often consume fat rich meat that is grilled in a way leading to high PAH formation, i.e., when fat burns on the heat source, food is grilled very well-done on charcoal or particularly campfire. Such conditions may also increase formation of other heat-induced contaminants. VKM Report 2024:2 Risk assessment of grilled and barbecued food Scientific Opinion of the Panel on Contaminants of the Norwegian Scientific Committee for Food and Environment 21.03.2024 Phone: +47 21 62 28 00 Email: [email protected] vkm.no Cover photo: Helen Engelstad Kvalem Suggested citation: Espen Mariussen, Jan Alexander, Barbara Alexandra Bukhvalova, Lisbeth Dahl, Ann-Karin Hardie Olsen, Helen Engelstad Kvalem, Martin Schlabach, Heidi Amlund, Rita Hannisdal, Anders Ruus, Ingunn Anita Samdal, Helle K Knutsen. Risk assessment of grilled and barbequed food. Scientific Opinion of the Panel on Contaminants of the Norwegian Scientific Committee for Food and Environment. VKM Report 2024:2, ISBN: 978-82-8259-438-7, ISSN: 2535-4019. Norwegian Scientific Committee for Food and Environment (VKM), Oslo, Norway. Preparation of the opinion: The Norwegian Scientific Committee for Food and Environment (Vitenskapskomiteen for mat og miljø, VKM) appointed a project group to draft the opinion. The project group consisted of five VKM members, 1 VKM staff. Three referees commented on and reviewed the draft opinion. The Committee, by the Panel on Contaminants assessed, and approved the final opinion. Authors of the opinion: The authors have contributed to the opinion in a way that fulfils the authorship principles of VKM (VKM, 2019). The principles reflect the collaborative nature of the work, and the authors have contributed as members of the project group and/or the VKM Panel on Contaminants. Members of the project group: (in alphabetical order after chair of the project group): Espen Mariussen – Chair of the project group. Member of the Panel on Contaminants. Affiliation: 1) VKM; 2) Norwegian Institute of Public Health Jan Alexander - Member of the Panel on Contaminants. Affiliation: 1) VKM; 2) Retired, former Norwegian Institute of Public Health Barbara Alexandra Bukhvalova, VKM staff. Affiliation: VKM Lisbeth Dahl - Member of the Panel on Contaminants. Affiliation: 1) VKM; 2) Institute of Marine Research Helen Engelstad Kvalem – Project leader, VKM staff. Affiliation: VKM Ann-Karin Hardie Olsen - Member of the Panel on Contaminants. Affiliation: 1) VKM; 2) Norwegian Institute of Public Health Martin Schlabach - Member of the Panel on Contaminants. Affiliation: 1) VKM; 2) Retired, former NILU Members of the Panel on Contaminants: (in alphabetical order before chair of the Panel/Scientific Steering Committee): Heidi Amlund – Member of the Panel on Contaminants. Affiliation: 1) VKM; 2) National Food Institute, Technical University of Denmark Rita Hannisdal - Member of the Panel on Contaminants. Affiliation: 1) VKM; 2) Institute of Marine Research Anders Ruus - Member of the Panel on Contaminants. Affiliation: 1) VKM; 2) Norwegian Institute for Water Research Ingunn Anita Samdal - Member of the Panel on Contaminants. Affiliation: 1) VKM; 2) Norwegian Veterinary Institute Espen Mariussen – Chair of the project group. Member of the Panel on Contaminants. Affiliation: 1) VKM; 2) Norwegian Institute of Public Health Jan Alexander - Member of the Panel on Contaminants. Affiliation: 1) VKM; 2) Retired, former Norwegian Institute of Public Health Lisbeth Dahl - Member of the Panel on Contaminants. Affiliation: 1) VKM; 2) Institute of Marine Research Helen Engelstad Kvalem – Project leader, VKM staff. Affiliation: VKM Ann-Karin Hardie Olsen - Member of the Panel on Contaminants. Affiliation: 1) VKM; 2) Norwegian Institute of Public Health Martin Schlabach - Member of the Panel on Contaminants. Affiliation: 1) VKM; 2) Retired, former NILU Helle Knutsen – Chair of the Panel on Contaminants. Affiliation: 1) VKM; 2) Norwegian Institute of Public Health Acknowledgement: VKM would like to thank the referees Wim Mennes (retired, RIVM, Netherlands), David Gott (Food Standards Agency, UK) and Peter Fürst (retired, Chemical and Veterinary Analytical Institute, Germany) for their valuable comments through critical review of the draft opinion. VKM emphasises that the referees are not responsible for the content of the final opinion. In accordance with VKM's routines for approval of a risk assessment (VKM, 2018), VKM received their comments before evaluation and approval by VKM Panel on Contaminants and before the opinion was finalised for publication. Trine Husøy is acknowledged for advice on probabilistic exposure assessment. Sagnik Sengupta (VKM secretariat) is acknowledged for his contributions with compiling the occurrence data on PAH and the data analysis in R. Gro Haarklou Mathisen (VKM secretariat) is acknowledged for her contribution with initiating work on the protocol and kind advice along the process with this report. The Library at the Norwegian Institute of Public Health is acknowledged for the valuable help an insight in the systematic literature search. Inger Therese Lillegaard (VKM secretariat) is acknowledged for her reflections and advice on food consumption and scenarios. We also express gratitude to all of our European colleagues that responded to our call for data through the EFSA focal point network on occurrence data of process contaminants; Finland, Italia, Poland, Slovenia, Spain, Sweden, Belgium and Hungary. Competence of VKM experts: Persons working for VKM, either as appointed members of the Committee or as external experts, do this by virtue of their scientific expertise, not as representatives for their employers or third-party interests. The Civil Services Act instructions on legal competence apply for all work prepared by VKM. AboutPDF ToolsExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat Summary Background It is well-known that heat treatment of food, such as grilling and frying may give rise to unwanted, harmful substances in food, so-called process contaminants. VKM summarized this in the previous risk assessment of grilled food from 2007. In 2022, the Norwegian Food Safety Authority asked VKM to update and summarize the knowledge on formation of processed contaminants in various food by different grilling methods, and to assess the risk this may pose. More specifically the tasks were: Identify process contaminants which are formed to a greater extent by grilling than by frying and create an overview of reported amounts of these process contaminants in various types of grilled food. Elucidate factors (for example grill type, grill method and food) that are important for the formation of the identified process contaminants in grilled food. If possible, based on available information, assess the health risks associated with the consumption of grilled food compared to fried food. Methods VKM prepared a protocol, published in February 2023, describing the scope and methods for this risk assessment. New knowledge since 2006 about the relationship between the consumption of grilled food and health outcomes in humans was collected through an umbrella review of systematic summaries on this topic. Information on which contaminants are formed in grilled food, the amount formed, and factors important for their formation were collected using non-systematic literature searches. Characterization of health hazards linked to the contaminants was obtained from assessments carried out by international risk assessment bodies. VKM considered that the identified data were considered sufficient for a quantitative risk assessment of exposure to polycyclic aromatic hydrocarbons (PAH), but not for other contaminants. The PAH exposure was estimated by a probabilistic approach, with two scenarios for the consumption of grilled food. This involved modelling e the content of PAHs in different grilled food items and exposure to PAHs. The total risk from dietary PAH exposure from was described by the margin of exposure (MOE) approach. Results Identification of process contaminants VKM identified several process contaminants formed during grilling. These were PAHs, PAH analogues such as chlorinated-PAHs and nitrated PAHs, polyphenols, heterocyclic aromatic amines (HAAs), 3- monochloropropane-1,2-diol (3-MCPD) and glycidyl esters, acrylamide, nitrosamines, nitrite, harmful Maillard reaction products, biogenic amines, 7-ketocholesterol, acridine derivatives, anthraquinone (ATQ), pyrazines, advanced glycation end products (AGE). For two groups of compounds, PAHs and HAAs, there was substantial or plausible evidence for higher concentration in grilled meat and fish than in fried food. There was few data available on process contaminants in other grilled food than meat and fish, such as e.g. vegetables and bread. Concentration data for PAHs in grilled food (mostly meat and meat products) was collected from a total of 81 studies. The highest concentrations of PAHs (BaP and PAH4) were found in campfire grilled sausages. Concentration data for BaP, regardless of grilling method, indicated variability ranging from levels below quantification limits to more than 100 ng/g in fatty pork meat and sausages. Most of the grilled food items had median BaP concentrations below approximately 1 ng/g but varied from 0.1 to 4.1 ng/g across all food items. The occurrence of the different HAAs in grilled food varied greatly, from below detection limits to about 50 ng/g. The concentrations could, however, be up to 240 ng/g for PhIP in very well-done grilled chicken, and with higher levels in meat than in fish. Factors (for example grill type, grill method and food) that are important for the formation of the identified process contaminants in grilled food. Main types of heat sources for grilling are electricity, gas, charcoal (lump charcoal, briquettes) and firewood. The type of heat source may influence the content of PAHs and HAAs differently. Other factors that may influence the formation of PAHs and HAAs include type of fuel, direct or indirect grilling, type of meat, distance from the heat source, grilling temperature, and grilling time. Contamination of food with PAHs may primarily occur through: (1) deposition on the food of fuel-related PAHs in smoke. Electric heating and gas emit no or little PAHs, while charcoal and in particular firewood may contribute significantly to PAH contamination. When using charcoal, the PAH emission is higher in the initial period after lighting the grill. (2) deposition of smoke from incomplete combustion (pyrolysis) of dripping fat onto the heating source or hot surfaces. Dripping fat on the heat source is a significant source of PAHs in grilled food and is related to the fat content of the food. Avoiding fat dripping directly on the heat source and diverting the smoke from the food may reduce PAH deposition on the food. Studies on the impact of marination are conflicting, as marination may both reduce and increase the content of PAHs. (3) over-heating the food resulting in burned surface during cooking. Temperature, proximity to the heat source, frequent flipping of the food, and cooking time are important for avoiding burning the food during grilling. HAAs are mainly formed in the crust or gravy by heating. Variables that affect the formation are (1) temperature and cooking time. Concentrations of HAAs increase with rising temperature, while regular turning of the meat during cooking reduce the surface temperature and mitigate HAA formation. (2) presence of precursors, such as creatine, reducing sugars and free amino acids in the food. More HAA is formed in lean meat than in fatty meat, fish or mixed products. (3) method of cooking, such as marination, may affect the formation of HAAs. Microwaving beef and chicken prior to charcoal grilling can reduce the content of HAAs. Health risk associated with consumption of grilled food The umbrella review of systematic reviews of epidemiological studies published since 2006 did not reveal additional knowledge on the association between consumption of grilled food and health outcomes in humans. Hence, the conclusion drawn in the previous VKM 2007 assessment remains pertinent, suggesting a possible association between intake of well-done fried or grilled meat and cancer in the colon, rectum, prostate, breast, and pancreas. It was beyond the scope of the present assessment to review original studies on the health effects of consumption of grilled food compared to other cooking methods. VKM therefore assessed the risk from exposure to process contaminants formed by grilling. VKM estimated PAH exposure in grilled food quantitatively based on scenarios of consumption of grilled food also including a background exposure of PAH from the rest of the diet. Due to lack of data, the risk from HAA exposure and other heat-induced contaminants could not be characterised. The PAH concentration distributions in various grilled food items based on the extracted information from the 81 identified publications were generated from all types of grilling methods, of which more than 60% of the data were from charcoal grilled food. Due to limitations in the database the data did not allow for differentiation among grilling methods. The cumulative concentration distribution in most of the foods showed a sharp increase in the PAH concentrations above the 75 percentiles. The simulated concentration at the 90- and 95-percentile may represent grilling methods known to increase formation of PAHs in the food. The highest concentrations of BaP were found in sausages grilled on campfire, and in hamburgers and fatty pork. The two grilled food consumption scenarios include only meat and salmon because of lack of occurrence data in other grilled food items, such as vegetables, bread and meat imitate. The scenarios illustrate possible consequences of different food preferences on the intake of PAHs from grilled foods. The scenario plate of 200 g grilled fat-rich meat contained fat rich pork meat, hamburger, sausages, and chicken with skin. The other plate with 200 g lean meat and fish contained beef, lean pork meat, chicken without skin and salmon. Based on concentration distribution of BaP and PAH4 in each food item on the plate the distribution of the contents of BaP and PAH4 on each plate was simulated. The exposure scenarios based on the two plates included background PAH exposure from the rest of the diet and 1 to 100 servings of each plate per year. Average daily intakes of BaP or PAH4 for one year were calculated. Risk was characterized by the MOE approach using BMDL10s for BaP and PAH4 from the EFSA risk assessment of PAHs in food from 2008 as reference points. VKM considers that exposure to PAH resulting in MOE below 10,000 is of public health concern. For the plate with lean meat and salmon the use of mean, median and 75 percentile exposure to BaP and PAH4 up to 100 servings per year, resulted in MOEs above 10,000. At the 90 percentile and 100 servings the MOEs were approximately 10,000 for BaP and slightly below for PAH4. For the plate with fat rich meat the MOEs for the scenario related exposures remained above 10,000 when consuming grilled fat rich meat with a mean BaP and PAH4 content up to 100 times a year. At concentrations equivalent to the 95 percentile of BaP and PAH4 the MOEs were below 10,000 when consuming grilled fat rich meat approximately 15 and 25 times a year, respectively. Using PAH4 as an indicator of total PAH exposure, instead of BaP, gave approximately the same result as that of BaP, with slightly higher MOEs for the fat rich meat plate and slightly lower for the lean meat and salmon plate. The impact of campfire grilling on exposure estimates and the associated MOEs was examined in a sensitivity analysis. Data on campfire grilled food was only available for food items on the fat rich meat plate. Excluding campfire grilled food from the fat rich plate substantially reduced impact on the mean, median and higher percentiles values for the contents of both PAH4 and BaP on the plate. The resulting MOEs were above 10,000 when consuming more than 100 servings per year of plates with a mean, median and 75 percentile content of BaP and PAH4. At a 95-percentile content of BaP and PAH4 the MOEs were above 10,000 for up to 30 servings per year. Due to limitations the database did not allow creation of exposure scenarios reflecting various grilling methods. The MOEs calculated from the mean, median and 75 percentile exposure to PAHs likely represent the use of varied grill methods and food not causing substantial PAH formation. This will probably apply to most grilling situations. The MOEs calculated for the 90- and 95-percentile exposures to PAH may represent frequent use of grilling methods known to increase formation of PAHs in the food, such as high temperature grilling with charcoal or even campfire for a longer grill time leading to well done food. Due to lack of data, the risk from HAA exposure and other heat-induced contaminants could not be characterised. The concentrations of HAAs seem to be higher in grilled meat than in fried meat, particularly when well done. Grilling of food is associated with higher and less controllable surface temperature than frying. As most of the HAAs are genotoxic and carcinogenic in rodents and some have been classified as possible human carcinogens, their formation during grilling may be of concern. Although not precisely known, it is plausible that several other heat-induced contaminants, such as of PAH-analogues, can occur in higher concentrations in grilled food than in fried food, as the mechanism of formation is presumed to be similar to that of the PAHs or due to a presumably higher and less controllable temperature in grilling. Uncertainty The uncertainties in this assessment are large. Exposure to PAH was estimated from simulated occurrence data and consumption scenarios. The health risk from exposure to PAHs in consumed grilled food characterised by the MOE approach may both be over- and underestimated. Due to lack of data, the health risk associated with exposure to heat-induced contaminants in grilled food other than PAH could not be assessed. Due to high and less controllable temperature during grilling, it is likely that HAAs and some other heat-induced contaminants may be present in higher concentrations in grilled food than in fried food. Therefore, the total risk associated with the presence of process contaminants in grilled food is likely to be higher than for PAH alone. Data gaps The main data gaps related to the health risk assessment of grilled food identified by VKM are the following: 1) systematic reviews on health outcomes related to consumption of grilled food in comparison with other cooking methods are lacking, 2) data on consumption of grilled food, food items, frequency of grilling and grilling method applied are missing, 3) data on occurrence of PAH and other heat-induced contaminants in food prepared by different grilling methods and by other comparable cooking methods are missing, 4) toxicological data on individual HAAs for better hazard characterization considering toxicokinetic differences between rodents and humans are missing, and 5) occurrence data on HAAs in grilled food analysed with controlled and validated analytical methods are missing. References Abramsson-Zetterberg, L., Darnerud, P. 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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.

Rationale. Iodine is an essential element required for human health and metabolism. Seafood and especially seaweed can accumulate iodine to high amounts. Iodine may exist in different chemical forms (species) in seaweeds. Methodology. The present study describes the development and optimisation of a method for iodine speciation analysis in seaweed based on high performance liquid chromatography-inductively coupled plasma-mass spectrometry (HPLC-ICP-MS). The extraction procedure was conducted in two steps, pancreatic enzymatic extraction followed by alkaline extraction with tetramethylammonium hydroxide for optimum extraction efficiency without compromising species integrity. Results and discussion. Total iodine and iodine species were determined in a range of brown (6 samples), red (6 samples) and green (3 samples) seaweeds. A large variation in the total iodine content of the different seaweeds was observed (33-5611 mu g g(-1) dry weight) with the highest levels encountered in brown seaweed. Iodine speciation analysis revealed differences in the speciation profile of the different types of seaweed. In all seaweeds iodide was the predominant species, and minor contents of MIT (monoiodotyrosine) and DIT (diiodotyrosine) were found in most seaweeds. Furthermore, peaks originating from six unknown iodine-containing species were observed in the chromatograms, especially in red and green seaweeds, while less abundant in brown seaweeds. The speciation method presented here will be valuable in future studies on iodine speciation in seaweed and an important tool for the investigation of iodine speciation and biotransformation in marine algae.

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.

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.

The use of plant-based feeds has introduced undesirables, such as pesticides, that have previously not been associated with seawater farming of fish species such as Atlantic salmon. Earlier wide-scope chemical screening showed that the organophosphate (OP) pirimiphos-methyl (PM-m) is one of the most prevalent pesticides present in commercially produced Atlantic salmon feed. Information on the safe upper limit of background levels of PM-m in Atlantic salmon feeds with regards to possible adverse effect on fish health is lacking. Therefore, Atlantic salmon (132 +/- 25 g) were fed graded levels of PM-m spiked feeds (0, 0.35, 1.5, 5, and 22 mg kg(-1)) in triplicate for 3 months. Adverse effects were assessed on OP target toxic exposure such as plasma choline esterase (ChE), and secondary toxic responses such as lipid metabolism and oxidative stress, as well as general adverse effect parameters (plasma biochemistry, haematology, and growth). Safe limits were set by model-fitting the effect data in a dose-response (lower bound) bench mark dose (BMDL) regression analysis. Fish fed 1.5 mg kg(-1) and higher had a significant (p < 0.05) dose-dependent growth reduction, oxidative stress as seen from reduced glutathione ratio, liver damage as seen from plasma alkaline phosphatase, and reduced sum neutral and TAG liver lipids. Inhibition of ChE was observed in fish fed 5 mg kg-1 and above. Disturbance in phospholipid (PL) fatty acid composition and reduced level of liver phosphatidyl choline (PC) occurred at a lower exposure level (0.35 and 1.5 mg kg(-1)), however this was not dose-dependent as at higher exposure levels (5 and 22 mg kg(-1)), no significant differences were observed. A safe feed limit (as BMDL) for dietary PM-m was set at 0.14-0.46 mg kg(-1) ww feed (daily dose 0.49-1.62 ng kg BW-1 day(-1)), based on inhibited growth, ChE, glutathione ratio, and sum neutral and TAG liver lipids. When including an estimated uncertainty factor (UF) of five for inter-species variability and extrapolation to chronic exposure, the safe limits are 0.028-0.092 mg kg(-1), which is lower than the highest PM-m levels observed in commercially produced salmon feeds (0.038 mg kg(-1)). Thus indicating a potential risk for disturbance in lipid metabolism when fish are fed plant-based feeds. For the most sensitive lipid response, disturbance in PL metabolism, no certain BMDL could be assessed due to lack of a clear dose-response relationship.

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.

ABSTRACT 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).

The XyRex® formulations are antimicrobial agents with sodium chlorite as the active substance. The Norwegian Food Safety Authority has received a request to authorise XyRex® formulations for use in refrigerated sea-water (RSW) tanks on board fishing vessels for pelagic fish caught for production of fish meal/fish oil for use in animal feed. The use of XyRex® formulations on fish for human consumption is not approved in Norway. It is a premise that use of food processing aids should not pose a threat to health of humans or animals. The XyRex®-containing RSW will not be completely drained off before the production of fish meal/fish oil. The Norwegian Food Safety Authority has therefore requested The Norwegian Scientific Committee for Food Safety (VKM), Panel on Animal Feed, to assess the safety to humans and animals with regard to the use of XyRex® formulations with 12.5 ppm sodium chlorite, as recommended by the producer, in RSW on board fishing vessels for pelagic fish. The Norwegian Scientific Committee for Food Safety appointed an ad hoc group to answer the request from the Norwegian Food Safety Authority. The report from the ad hoc group has been discussed and approved by VKM’s Scientific Panel on Animal Feed. The use of the XyRex® formulations will mainly result in the release of sodium chlorite and sodium chlorate, which therefore are the components in the XyRex® formulations that may cause concerns for health risks. The amounts of these substances formed and transferred with the fish to the fish meal, fish meal-containing feed, and ultimately to the fish, meats, milk and eggs from animals eating the fish meal, are not known. Therefore, this evaluation is based on a worst-case scenario of transfer of the maximum amounts of these residues from the XyRex®-formulations, and the available information on theoretical transfer in each step from the RSW tanks on board fishing vessels into fish and animal feed and human foods. The estimated exposures of farmed fish and domestic animals to sodium chlorite and sodium chlorate in their fish meal-containing feed are all below the tolerable daily intake (TDI) value of 30 μg/kg body weight (bw)/day, set for both sodium chlorite and sodium chlorate. Therefore, no adverse effects on fish or animal health would be expected by exposure to sodium chlorite and sodium chlorate from the use of XyRex® formulations in RSW on board the fishing vessels catching fish being used for fish meal being included in fish and animal feed. The intakes of sodium chlorite and sodium chlorate for humans from consumption of farmed fish or domestic animals being fed on fish meal from XyRex®-treated fish were estimated assuming the worst-case scenario that all the sodium chlorite and sodium chlorate the fish and animals were exposed to were accumulating in their meats, or transferred to milk and eggs. These estimated average and high exposures to farmed salmon, pork, beef, chicken, turkey, milk and eggs are well below the TDI values for both sodium chlorite and sodium chlorate of 30 μg/kg bw/day. In reality, a significant amount of the sodium chlorite in the XyRex® formulations will be depleted during the disinfection process and not end up in the fish meal and thereafter in the feed, and both sodium chlorite and sodium chlorate will to a large extent be excreted rather than accumulated in the meats of fish and animals, or in milk and eggs. Therefore, the real exposure values are likely to be lower than the values estimated here. The likelihood of by-product formation from the specified use of XyRex® formulations has also been addressed. Because the temperatures used for cooking and drying during fish meal production are well below the temperatures where dioxins and furans are formed, the formation of these substances during production is unlikely. Also, since the amounts of chlorine compounds present with the fish are low, we regard any significant health risks from chlorination by-products to be unlikely. However, further data might be needed to confirm that chlorinated compounds are not generated to a significant extent from the use of XyRex® formulations. Based on the available data on reactions of acidified sodium chlorite (ASC) and chlorine dioxide with proteins and lipids in poultry carcasses or fish, a significant formation of harmful, i.e. mutagenic, by-products from the use of the XyRex® formulations on fish is not expected. A negative effect on the quality of the fish oil, if substantial oxidation is taking place, can not be ruled out. Release of RSW in harbour areas is forbidden by law in Norway. The concentrations of sodium chloride and sodium chlorate in RSW being released from the fishing vessels to open sea during transport will rapidly be diluted around the release point, and are therefore not likely to have any adverse effects. The specified use of XyRex® formulations in RSW tanks on board fishing vessels is therefore not expected to be of significant environmental concern. The Panel on Animal feed has not evaluated the disinfecting activity claimed for the XyRex® products, nor any potential for development of bacterial resistance after use of the XyRex® products, since these questions were not asked by the Norwegian Food Safety Authority.

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 2 7–3 fractional factorial design using blue mussel as test sample. The effects of sample weight, type and volume of extraction solution, addition of H 2 O 2 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:H 2 O, 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. Graphical abstract

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.

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 (similar to 4% kelp) was proportional to iodine accumulation in trout fillets (R-2 = 1.00) with 0.5% transfer ratio. Feed iodine concentrations up to 117 mg/kg (similar to 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 similar to 60% of recommended daily iodine intake for adults.

In the last decades, there is an increasing inclusion of various trace metals and metalloids such as thallium, tellurium and rare earth elements (REEs; lanthanides, scandium, and yttrium) in the composition and production of alloys, in agricultural and medicinal applications, as well as in the manufacturing of hi-tech products. All these activities have led to an accumulation of the aforementioned elements both in soil and water bodies and consequently in the food chain, through discharges from mining and mineral processing, liquid industrial waste or disposal of urban and industrial products. It has been demonstrated that chronic exposure to some of these elements, even at low doses, might lead to a wide range of adverse health effects, even from the early stages of life, such as neurotoxicity, neurodevelopmental toxicity and hepatic alterations. Particularly in children, there have been studies suggesting that some of these elements might negatively affect the children's spatial learning and memory ability indirectly. Such effects are triggered by processes like the production of reactive oxygen species (ROS), lipid peroxidation and modulation of antioxidant activities. Nevertheless, the limited data from toxicological studies and their so-far naturally low occurrence levels in the environment acted as a deterrent in measuring their concentrations during routine analyses of metals in foodstuff. Thus, it is important to collect information on their occurrence data both in adults and in children's daily diet. This review sumrises the current knowledge on the concentration of these elements, in plant-based food products to identify whether a potential health risk occurs. As side projects, this Fellowship provided hands-on training on the evaluation of new biocides application and participation in the given advice to the Danish Food and Veterinary Administration, Danish Environmental Protection Agency, the Danish Medical Agency and the European Chemicals Agency.

Seafood and seafood products contain high levels of arsenic, and for most people, seafood is the major source of arsenic exposure. Arsenic has a complex chemistry in the marine environment and more than 100 different arsenic compounds have been identified. The toxicity of the various compounds depends on the chemical form; inorganic arsenic is highly toxic and classified as a class 1 human carcinogen. In seafood, most arsenic is organically bound and arsenic is mainly found as arsenobetaine, which is considered nontoxic. Seafood generally contains low levels of inorganic arsenic, and the consumption of seafood contributes little to the dietary intake of inorganic arsenic. In European adults, the main contributors to the dietary exposure of inorganic arsenic are grain-based processed products, rice, milk and dairy products, and drinking water. The mean dietary exposure to inorganic arsenic in the European population is within the ranges of established BMDLs, and a possible health risk cannot be excluded. Consumption of seafood with elevated levels of inorganic arsenic (e.g., some species of brown algae and bivalves) will increase the exposure to inorganic arsenic and result in an added health risk. The potential risk of elevated levels of inorganic arsenic in some types of seafood must therefore be recognized. Although inorganic arsenic is a human carcinogen, current international regulations on inorganic arsenic in food are scarce.