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.

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

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 Consequently, concern related to traditional persistent contaminants typically associated with fish-based feed has been replaced by other potential contaminants not previously associated with salmon farming.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

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

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

In vitro toxicity of pirimiphos-methyl in Atlantic salmon hepatocytes.

Recent screening of Atlantic salmon feeds has uncovered residues of several pesticides, including pirimiphos-methyl. Pirimiphos-methyl is an organophosphate (OP) insecticide, causing inhibition of acetylcholinesterase in target organisms. The aim of this study was to examine pirimiphos-methyl non-targeted mode of action toxicity in Atlantic salmon using in vitro exposure. Hepatocyte cells were exposed to pirimiphos-methyl (control-0.1-1.0-10-100-1000µM) for 48h. Transcriptomics (RNA-seq) and non-targeted metabolomics were used to screen for effects of the pesticide. The results showed that the compound acts cytotoxic and impacts accumulation of lipids (steatosis) at 1000µM. Metabolomics screening revealed effects on lipid metabolism (diHOME fatty acids, cholesterol and lysophospholipids), glutathione (depletion), glycolysis and tryptophan metabolism, as well as on several vitamins. At 1000µM, vitamin E levels increased, while folate and thiamine derivate levels decreased. Surprisingly few transcripts were affected by the treatment, with only 64 differentially expressed genes (DEGs) showing a clear dose-dependent response. Several DEGs encoding proteins in cholesterol biosynthesis showed negative correlations with pirimiphos-methyl exposure. Other affected DEGs indicate an estrogenic effect, and points to mitochondrial dysfunction at the highest dose. The finding suggests that glutathione and glycine conjugation reactions are involved in the detoxification process. In conclusion, this study shows that pirimiphos-methyl is a relatively potent toxicant in Atlantic salmon hepatocytes affecting lipid and vitamin metabolism as well as glutathione turn-over.

Ion-Mobility-Derived Collision Cross Section as an Additional Identification Point for Multiresidue Screening of Pesticides in Fish Feed.

Ion mobility spectrometry allows for the measurement of the collision cross section (CCS), which provides information about the shape of an ionic molecule in the gas phase. Although the hyphenation of traveling-wave ion mobility spectrometry (TWIMS) with high-resolution quadrupole time-of-flight mass spectrometry (QTOFMS) has been mainly used for structural elucidation purposes, its potential for fast screening of small molecules in complex samples has not yet been thoroughly evaluated. The current work explores the capabilities of ultrahigh-performance liquid chromatography (UHPLC) coupled to a new design TWIMS-QTOFMS for the screening and identification of a large set of pesticides in complex salmon feed matrices. A database containing TWIMS-derived CCS values for more than 200 pesticides is hereby presented. CCS measurements showed high intra- and interday repeatability (RSD < 1%), and they were not affected by the complexity of the investigated matrices (ΔCCS ≤ 1.8%). The use of TWIMS in combination with QTOFMS was demonstrated to provide an extra-dimension, which resulted in increased peak capacity and selectivity in real samples. Thus, many false-positive detections could be straightforwardly discarded just by applying a maximum ΔCCS tolerance of ±2%. CCS was proposed as a valuable additional identification point in the pesticides screening workflow. Several commercial fish feed samples were finally analyzed to demonstrate the applicability of the proposed approach. Ethoxyquin and pirimiphos-methyl were identified in most of the analyzed samples, whereas tebuconazole and piperonil butoxide were identified for the first time in fish feed samples.

Screening of pesticides and polycyclic aromatic hydrocarbons in feeds and fish tissues by gas chromatography coupled to high-resolution mass spectrometry using atmospheric pressure chemical ionization.

This paper reports a wide-scope screening for detection and identification of pesticides and polycyclic aromatic hydrocarbons (PAHs) in feeds and fish tissues. QuEChERS sample treatment was applied, using freezing as an additional cleanup. Analysis was carried out by gas chromatography coupled to hybrid quadrupole time-of-flight mass spectrometry with atmospheric pressure chemical ionization (GC-(APCI) QTOF MS). The qualitative validation was performed for over 133 representative pesticides and 24 PAHs at 0.01 and 0.05 mg/kg. Subsequent application of the screening method to aquaculture samples made it possible to detect several compounds from the target list, such as chlorpyrifos-methyl, pirimiphos-methyl, and ethoxyquin, among others. Light PAHs (≤4 rings) were found in both animal and vegetable samples. The reliable identification of the compounds was supported by accurate mass measurements and the presence of at least two representative m/z ions in the spectrum together with the retention time of the peak, in agreement with the reference standard. Additionally, the search was widened to include other pesticides for which standards were not available, thanks to the expected presence of the protonated molecule and/or molecular ion in the APCI spectra. This could allow the detection and tentative identification of other pesticides different from those included in the validated target list.

Biomarkers in natural fish populations indicate adverse biological effects of offshore oil production.

BACKGROUND: Despite the growing awareness of the necessity of a sustainable development, the global economy continues to depend largely on the consumption of non-renewable energy resources. One such energy resource is fossil oil extracted from the seabed at offshore oil platforms. This type of oil production causes continuous environmental pollution from drilling waste, discharge of large amounts of produced water, and accidental spills. METHODS AND PRINCIPAL FINDINGS: Samples from natural populations of haddock (Melanogrammus aeglefinus) and Atlantic cod (Gadus morhua) in two North Sea areas with extensive oil production were investigated. Exposure to and uptake of polycyclic aromatic hydrocarbons (PAHs) were demonstrated, and biomarker analyses revealed adverse biological effects, including induction of biotransformation enzymes, oxidative stress, altered fatty acid composition, and genotoxicity. Genotoxicity was reflected by a hepatic DNA adduct pattern typical for exposure to a mixture of PAHs. Control material was collected from a North Sea area without oil production and from remote Icelandic waters. The difference between the two control areas indicates significant background pollution in the North Sea. CONCLUSION: It is most remarkable to obtain biomarker responses in natural fish populations in the open sea that are similar to the biomarker responses in fish from highly polluted areas close to a point source. Risk assessment of various threats to the marine fish populations in the North Sea, such as overfishing, global warming, and eutrophication, should also take into account the ecologically relevant impact of offshore oil production.

Carry-over of dietary organochlorine pesticides, PCDD/Fs, PCBs, and brominated flame retardants to Atlantic salmon (Salmo salar L.) fillets.

Information on carry-over of contaminants from feed to animal food products is essential for appropriate human risk assessment of feed contaminants. The carry-over of potentially hazardous persistent organic pollutants (POPs) from feed to fillet was assessed in consumption sized Atlantic salmon (Salmo salar). Relative carry-over (defined as the fraction of a certain dietary POP retained in the fillet) was assessed in a controlled feeding trial, which provided fillet retention of dietary organochlorine pesticides (OCPs), dioxins (PCDD/Fs), polychlorinated biphenyls (PCBs), and brominated flame retardants (BFRs). Highest retention was found for OCPs, BFRs and PCBs (31-58%), and the lowest retentions were observed for PCDD/Fs congeners (10-34%). National monitoring data on commercial fish feed and farmed Atlantic salmon on the Norwegian market were used to provide commercially relevant feed-to-fillet transfer factors (calculated as fillet POP level divided by feed POP level), which ranged from 0.4 to 0.5, which is a factor 5-10 times higher than reported for terrestrial meat products. For the OCP with one of the highest relative carry-over, toxaphene, uptake and elimination kinetics were established. Model simulations that are based on the uptake and elimination kinetics gave predicted levels that were in agreement with the measured values. Application of the model to the current EU upper limit for toxaphene in feed (50 µg kg(-1)) gave maximum fillet levels of 22 µg kg(-1), which exceeds the estimated permissible level (21 µg kg(-1)) for toxaphene in fish food samples in Norway.

Effects of refining and removal of persistent organic pollutants by short-path distillation on nutritional quality and oxidative stability of fish oil.

Food and feed legislations are implemented to control the level of unwanted persistent organic pollutants (POPs) below health risk concerns. Short-path distillation is established as the most effective industrial process to remove POPs in fish oil. However, the technology involves heating of the oil to high temperature levels (>200 °C) that possibly give unwanted heat-induced side reactions and coevaporation of minor compounds of importance for the nutritional quality of the oil. The effects on retention of vitamins, cholesterol, and unsaponifiable compounds, geometrical isomerization, loss of polyunsaturated fatty acids (PUFA), oxidation level, and oxidative stability have been studied on the basis of experiments designed to optimize and model the effect of process conditions (i.e., evaporator temperature, feed rate, and addition of working fluid) on the reduction of POPs. Loss of volatile nutrients was observed, but the extent will depend on the process conditions needed to obtain target decontamination level, as well as the concentration ratio and difference in vapor pressure between free and esterified forms of the studied compounds. Some reduction in oxidation level was documented with preservation of PUFA level and quality. Oxidative stability was influenced both positively and negatively depending on the applied process conditions. Generally, no adverse negative effects on the nutritional quality of the fish oil could be documented. Optimal process conditions were modeled that ensure removal of POPs to within legislation levels while retaining most of the vitamin levels in fish oil. A 76% reduction of the WHO-PCDD/F-PCB-TEQ level in the used feedstock was needed to be in accordance with the voluntary industrial monograph of GOED. This could be achieved on the basis of operation conditions giving <20% loss of vitamins. A 90% decontamination rate gave vitamin retentions in the 60-90% range.

Modeling of a short-path distillation process to remove persistent organic pollutants in fish oil based on process parameters and quantitative structure properties relationships.

A factorial experimental design based on temperature, feed rate and addition of "working fluid" (WF; fatty acid ethyl esters) was used to model a short-path distillation process applied for removal of persistent organic pollutants (POPs) in fish oil. Decontamination effect was assessed by measuring the level of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs), dioxin-like polychlorinated biphenyls (DL-PCBs) and polybrominated diphenylethers (PBDEs). The average reduction in chemical concentration of the individual PCDD, PCDF, DL-PCB and PBDE congeners was linearly dependent on the number of chlorine or bromine substitutions within each homologue group. DL-PCB and PBDE congeners could also be separated based on ortho-substitution. The quantitative structure properties relationships (QSPR) were combined with process parameters to establish response surface models for each homologue congener group based on partial least squares regression (PLSR). Cross validated predictive ability of the models was in the 4-9% range. Generally high temperature, low feed rate and WF addition improved the decontamination efficiency. The WHO-PCDD/F-PCB-TEQ level could be reduced by up to 98% based on the best experimental settings with residual concentration considerably below present maximum permitted levels in European food and feed legislations.

Effects of decontaminated fish oil or a fish and vegetable oil blend on persistent organic pollutant and fatty acid compositions in diet and flesh of Atlantic salmon ( Salmo salar).

The health benefits of seafood are well documented and based on the unique supply of n-3 highly unsaturated fatty acids (HUFA). Aquaculture now contributes about 50 % of food-grade seafood globally and Atlantic salmon (Salmo salar) is a rich source of n-3 HUFA. However, salmon and other oily fish can accumulate lipophilic persistent organic pollutants (POP), including dioxins (PCDD/F), polychlorinated biphenyls (PCB) and polybrominated diphenyl ethers (PBDE), derived largely from feed. In the present study, triplicate groups of salmon, of initial weight 0.78 kg, were fed one of three experimental diets for 11 weeks. The diets were coated with either a northern fish oil (FO) with a high POP content (cNFO), the same oil that had been decontaminated (deNFO) or a blend of southern fish oil, rapeseed and soyabean oils (SFO/RO/SO). Dietary PCDD/F+dioxin-like PCB (DL-PCB) concentrations were 17.36, 0.45 and 0.53 ng toxic equivalents (TEQ)/kg, respectively. After 11 weeks, the flesh concentrations in fish fed the cNFO, deNFO and SFO/RO/SO diets were 6.42, 0.34 and 0.41 ng TEQ/kg, respectively. There were no differences in flesh EPA and DHA between fish fed the cNFO or deNFO diets although EPA and DHA were reduced by 50 and 30 %, respectively, in fish fed the SFO/RO/SO diet. Thus, decontaminated FO can be used to produce salmon high in n-3 HUFA and low in POP. Salmon produced using deNFO would be of high nutritional value and very low in POP and would utilise valuable fish oils that would otherwise be destroyed due to their high pollutant concentrations.

A study of metal concentrations and metallothionein binding capacity in liver, kidney and brain tissues of three Arctic seal species.

Arctic seals are known to accumulate relatively high concentrations of potential toxic heavy metals in their vital organs, such as livers and kidneys, as well as in their central nervous system. We therefore decided to determine whether mercury, copper, cadmium and zinc levels in liver, kidney and brain tissues of three Arctic seal species were associated with the intracellular metal-binding protein metallothionein (MT) as a sign of toxic exposure. Samples from four ringed (Phoca hispida), five harp (P.groenlandica) and five hooded (Cystophora cristata) seals taken during field trips to Central West Greenland (Godhavn) and the Barents Sea in the spring of 1999 were used for the present study. In all three seal species concentrations of mercury, zinc and copper were highest in the liver, except for cadmium which was highest in the kidneys. Metal concentrations increased significantly in the order: ringed seal

Assessing the sensitivity of Atlantic salmon (Salmo salar) to dietary endosulfan exposure using tissue biochemistry and histology.

The incorporation of plant-based ingredients, and the possible carry-over of pesticides such as endosulfan, in fish feeds may present new toxicological challenges to aquacultural species. Biological responses of Atlantic salmon (Salmo salar) to a 35-day dietary endosulfan exposure at levels ranging from 4 to 710 microgkg(-1) were assessed using tissue histology and biochemistry. Liver 7-ethoxyresorufin-O-deacetylase (EROD) activity was significantly elevated in the highest exposure group (710 microgkg(-1)) by day 35. Other hepatic indicators of stress impacts and responses (glutathione-S-transferase and glutathione peroxidase activities and hepatic alpha-tocopherol content) remained unchanged. Branchial Na(+), K(+)-ATPase activity was significantly reduced at day 14 in the highest exposure group, but returned to control levels by day 35. Conversely, intestinal Na(+), K(+)-ATPase activity was significantly inhibited at day 35, but again only at the highest exposure level. In contrast to the biochemical results, hepatic and intestinal histology revealed effects of exposure even at the lowest dose tested (4 microgkg(-1)). In the posterior intestine, pathology was characterised by vacuolation and fusion of villi, and in the most severe cases, loss of epithelial integrity in villi tips. In the liver the primary effects were glycogen depletion and lipidosis. These changes were typical of a generalised stress response. While histology endpoints may prove to be the most sensitive indicators of dietary endosulfan exposure, the organismal relevance of these structural changes must be considered in the absence of effects in other biomarkers at dietary levels less than 710 microgkg(-1).

Chronic dietary mercury exposure causes oxidative stress, brain lesions, and altered behaviour in Atlantic salmon (Salmo salar) parr.

Atlantic salmon (Salmo salar L.) parr were fed for 4 months on fish meal based diets supplemented with mercuric chloride (0, 10, or 100 mg Hg kg(-1) DW) or methylmercury chloride (0, 5, or 10 mg Hg kg(-1) DW) to assess the effects of inorganic (Hg) and organic dietary mercury on brain lipid peroxidation and neurotoxicity. Lipid peroxidative products, endogenous anti oxidant enzymes, brain histopathology, and overall behaviour were measured. Methylmercury accumulated significantly in the brain of fish fed 5 or 10 mg kg(-1) by the end of the experiment, and inorganic mercury accumulated significantly in the brain only at 100 mg kg(-1) exposure levels. No mortality or growth reduction was observed in any of the exposure groups. Fish fed 5 mg kg(-1) methylmercury had a significant increase (2-fold) in the antioxidant enzyme super oxide dismutase (SOD) in the brain. At dietary levels of 10 mg kg(-1) methylmercury, a significant increase (7-fold) was observed in lipid peroxidative products (thiobarbituric acid reactive substances, TBARS) and a subsequently decrease (1.5-fold) in anti oxidant enzyme activity (SOD and glutathione peroxidase, GSH-Px). Fish fed 10 mg kg(-1) methylmercury also had pathological damage (vacoulation and necrosis), significantly reduced neural enzyme activity (5-fold reduced monoamine oxidase, MAO, activity), and reduced overall post-feeding activity behaviour. Pathological injury started in the brain stem and became more widespread in other areas of the brain at higher exposure levels. Fish fed 100 mg Hg kg(-1) inorganic mercury had significant reduced neural MAO activity and pathological changes (astrocyte proliferation) in the brain, however, neural SOD and GSH-Px enzyme activity, lipid peroxidative products (TBARS), and post feeding behaviour did not differ from controls. Compared with other organs, the brain is particular susceptible for dietary methylmercury induced lipid peroxidative stress at relative low exposure concentrations. Doses of dietary methylmercury in the range of 5 mg kg(-1) induces protective redox defences in the brain as seen from the induction of anti-oxidant enzyme SOD activity. However, above a threshold of 10 mg kg(-1) methylmercury these defences are overcome and lipid peroxidative injury (TBARS) as well as severe pathological damage and adverse behaviour become apparent.