Dietary beauvericin and enniatin B exposure cause different adverse health effects in farmed Atlantic salmon.

The extensive use of plant ingredients in novel aquafeeds have introduced mycotoxins to the farming of seafood. The emerging enniatin B (ENNB) and beauvericin (BEA) mycotoxins have been found in the novel aquafeeds and farmed fish. Little is known about the potential toxicity of ENNs and BEA in farmed fish and their feed-to-organ transfer. Atlantic salmon (Salmo salar) pre-smolt (75.3 ± 8.10 g) were fed four graded levels of spiked chemical pure ENNB or BEA feeds for three months, in triplicate tanks. Organismal adverse health end-point assessment included intestinal function (protein digestibility), disturbed hematology (red blood cell formation), bone formation (spinal deformity), overall energy use (feed utilization), and lipid oxidative status (vitamin E). Both dietary BEA and ENNB had a low (<∼0.01%) transfer to organs (kidney > liver > brain > muscle), with a higher transfer for ENNB compared to BEA. BEA caused a growth reduction combined with a decreased protein digestion and feed conversion rate- ENNB caused a stunted growth, unrelated to feed utilization capacity. In addition, ENNB caused anemia while BEA gave an oxidative stress response. Lower bench-mark dose regression assessment showed that high background levels of ENNB in commercial salmon feed could pose a risk for animal health, but not in the case of BEA.

Multi-tissue proteogenomic analysis for mechanistic toxicology studies in non-model species.

New approach methodologies (NAM), including omics and in vitro approaches, are contributing to the implementation of 3R (reduction, refinement and replacement) strategies in regulatory science and risk assessment. In this study, we present an integrative transcriptomics and proteomics analysis workflow for the validation and revision of complex fish genomes and demonstrate how proteogenomics expression matrices can be used to support multi-level omics data integration in non-model species in vivo and in vitro. Using Atlantic salmon as an example, we constructed proteogenomic databases from publicly available transcriptomic data and in-house generated RNA-Seq and LC-MS/MS data. Our analysis identified ∼80,000 peptides, providing direct evidence of translation for over 40,000 RefSeq structures. The data also highlighted 183 co-located peptide groups that supported a single transcript each, and in each case, either corrected a previous annotation, supported Ensembl annotations not present in RefSeq, or identified novel previously unannotated genes. Proteogenomics data-derived expression matrices revealed distinct profiles for the different tissue types analyzed. Focusing on proteins involved in defense against xenobiotics, we detected distinct expression patterns across different salmon tissues and observed homology in the expression of chemical defense proteins between in vivo and in vitro liver systems. Our study demonstrates the potential of proteogenomic analyses in extending our understanding of complex fish genomes and provides an advanced bioinformatic toolkit to support the further development of NAMs and their application in regulatory science and (eco)toxicological studies of non-model species.

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

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

Transcriptional effects of dietary chlorpyrifos­methyl exposure in Atlantic salmon (Salmo salar) brain and liver.

Elevated levels of chlorpyrifos­methyl have been detected in plant-based Atlantic salmon feeds. To evaluate the potential negative effects of long-term and continuous dietary exposure to chlorpyrifos­methyl in fish, we fed juvenile Atlantic salmon three concentrations (0.1, 1.0 and 8.0 mg/kg) of the pesticide for about two months. Brain and liver tissues were collected after 30 and 67 days of exposure. Homogenized brain tissue was examined for effects on acetylcholinesterase, and brain and liver tissue from fish exposed to 8.0 mg/kg were used for transcriptional analysis (RNA-seq). The results showed a transient accumulation of chlorpyrifos­methyl in the brain with lower levels after 67 days of exposure compared to after 30 days of exposure. In contrast, the liver showed a time-dependent accumulation pattern. No effect on acetylcholinesterase activity, the primary target of chlorpyrifos­methyl, was seen in the brain. However, after 30 days of exposure, 98 significantly differentially expressed genes (DEGs) were found in brain tissue and 239 DEGs in liver tissue. After 67 days of exposure, two and 258 DEGs were found in brain and liver tissue, respectively. Continuous dietary exposure of chlorpyrifos­methyl most profoundly affected mechanisms associated with protein degradation and lipid metabolism in both brain and liver. Specific for the brain, many of the significant DEGs encode proteins involved in neuron function. In conclusion, this study shows that chlorpyrifos­methyl affects the transcription of genes involved in neurological function in Atlantic salmon brain, even at exposure concentrations below the threshold for systemic toxicity as seen from brain acetylcholinesterase inhibition.

Modelling of the feed-to-fillet transfer of ethoxyquin and one of its main metabolites, ethoxyquin dimer, to the fillet of farmed Atlantic salmon (Salmon salar L.).

Ethoxyquin (EQ) is an antioxidant supplemented to feed ingredients, mainly fish meal, which is currently under re-evaluation for use in the food production chain. EQ is partly metabolized into several metabolites of which the ethoxyquin dimer (EQDM) accumulates most in the farmed fish fillet. In this study, the feed-to-fillet transfer of dietary EQ and EQDM in Atlantic salmon fillet was investigated, and a physiologically based pharmacokinetic (PBPK-) two-compartmental model was developed, based on experimental determined EQ and EQDM uptake, metabolism, and elimination kinetics. The model was verified with an external data-set and used to simulate the long term (>1.5 years) EQ and EQDM feed-to fillet transfer in Atlantic salmon under realistic farming conditions such as the seasonal fluctuations in feed intake, growth, and fillet fat deposition. The model predictions showed that initial EQDM levels in juvenile fish are the driving factor in final levels found in food-producing animals, while for EQ the levels in feed, and seasonal variations were the driving factor for food EQ levels.

The partial pressure of oxygen affects biomarkers of oxidative stress in cultured rainbow trout (Oncorhynchus mykiss) hepatocytes.

Oxidative stress, the imbalance between production of reactive oxygen species and the cellular detoxification of these reactive compounds, is believed to be involved in the pathology of various diseases. Several biomarkers for oxidative stress have been proposed to serve as tools in toxicological and ecotoxicological research. Not only may exposure to various pro-oxidants create conditions of cellular oxidative stress, but hyperoxic conditions may also increase the production of reactive oxygen species. The objective of the current study was to determine the extent to which differences in oxygen partial pressure would affect biomarkers of oxidative stress in a primary culture of hepatocytes from rainbow trout (Oncorhynchus mykiss). Membrane integrity, metabolic activity, levels of total and oxidized glutathione (tGSH/GSSG) was determined, as well as mRNA expression levels of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione reductase (GSSG-R), gamma-glutamyl-cystein synthetase (GCS) and thioredoxin (TRX). The results show that different biomarkers of oxidative stress are affected when the cell culture is exposed to atmospheric oxygen, and that changes such as increased GSSG content and induction of GSSG-R and GSH-Px can be reduced by culturing the cells under lower oxygen tension. Oxygen tension may thus influence results of in vitro based cell research and is particularly important when assessing parameters in the antioxidant defence system. Further research is needed to establish the magnitude of this effect in different cellular systems.

Multi-class determination of undesirables in aquaculture samples by gas chromatography/tandem mass spectrometry with atmospheric pressure chemical ionization: A novel approach for polycyclic aromatic hydrocarbons.

In this work, a method for the analysis of 24 PAHs in 19 different matrices, including fish tissues, feeds and feed ingredients, has been developed using gas chromatography coupled to triple quadrupole tandem mass spectrometry with atmospheric pressure chemical ionization source (GC-APCI-MS/MS). The method is based on a modification of the unbuffered QuEChERS method, using freezing as an additional clean-up step and applying a 20-fold dilution factor to the final extract. The procedure was also tested for 15 pesticides and 7 polychlorinated biphenyl (PCB) congeners in order to widen the scope of the method. The excellent sensitivity and selectivity provided by GC-APCI-MS/MS allowed the dilution of the sample extracts and quantification using calibration with standards in solvent for all the 19 matrices tested. The developed method was evaluated at 2, 5 and 50ngg-1 spiking levels. LOQs were 2ngg-1 for most compounds, and LODs ranged from 0.5 to 2ngg-1. Analysis of real-world samples revealed the presence of naphthalene, fluorene, phenanthrene, fluoranthene and pyrene at concentration levels ranging from 4.8 to 187ngg-1. No PCBs, DDTs and pesticides were found in fillets from salmon and sea bream.

Sensitivity and toxic mode of action of dietary organic and inorganic selenium in Atlantic salmon (Salmo salar).

Depending on its chemical form, selenium (Se) is a trace element with a narrow range between requirement and toxicity for most vertebrates. Traditional endpoints of Se toxicity include reduced growth, feed intake, and oxidative stress, while more recent finding describe disturbance in fatty acid synthesis as underlying toxic mechanism. To investigate overall metabolic mode of toxic action, with emphasis on lipid metabolism, a wide scope metabolomics pathway profiling was performed on Atlantic salmon (Salmo salar) (572±7g) that were fed organic and inorganic Se fortified diets. Atlantic salmon were fed a low natural background organic Se diet (0.35mg Se kg-1, wet weight (WW)) fortified with inorganic sodium selenite or organic selenomethionine-yeast (SeMet-yeast) at two levels (∼1-2 or 15mgkg-1, WW), in triplicate for 3 months. Apparent adverse effects were assessed by growth, feed intake, oxidative stress as production of thiobarbituric acid-reactive substances (TBARS) and levels of tocopherols, as well as an overall metabolomic pathway assessment. Fish fed 15mgkg-1 selenite, but not 15mgkg-1 SeMet-yeast, showed reduced feed intake, reduced growth, increased liver TBARS and reduced liver tocopherol. Main metabolic pathways significantly affected by 15mgkg-1 selenite, and to a lesser extent 15mgkg-1 SeMet-yeast, were lipid catabolism, endocannabinoids synthesis, and oxidant/glutathione metabolism. Disturbance in lipid metabolism was reflected by depressed levels of free fatty acids, monoacylglycerols and diacylglycerols as well as endocannabinoids. Specific for selenite was the significant reduction of metabolites in the S-Adenosylmethionine (SAM) pathway, indicating a use of methyl donors that could be allied with excess Se excretion. Dietary Se levels to respectively 1.1 and 2.1mgkg-1 selenite and SeMet-yeast did not affect any of the above mentioned parameters. Apparent toxic mechanisms at higher Se levels (15mgkg-1) included oxidative stress and altered lipid metabolism for both inorganic and organic Se, with higher toxicity for inorganic Se.

Comprehensive strategy for pesticide residue analysis through the production cycle of gilthead sea bream and Atlantic salmon.

Plant ingredients and processed animal proteins are alternative feedstuffs for fish feeds in aquaculture. However, their use can introduce contaminants like pesticides that are not previously associated with marine Atlantic salmon and gilthead sea bream farming. This study covers the screening of around 800 pesticides by gas chromatography (GC) and liquid chromatography (LC) coupled to high resolution time-of-flight mass spectrometry in matrices throughout the entire marine food production chain. Prior to analysis of real-world samples, the screening methodology was validated for 252 pesticides to establish the screening detection limit. This was 0.01 mg kg-1 for 113 pesticides (45%), 0.05 mg kg-1 for 73 pesticides (29%) and >0.05 mg kg-1 for 66 pesticides (26%). After that, a quantitative methodology based on GC coupled to tandem mass spectrometry with atmospheric pressure chemical ionization source (GC-APCI-MS/MS) was optimized for the pesticides found in the screening. Although several polar pesticides, of which pirimiphos methyl and chlorpyriphos-methyl were most dominant, were found in plant material and feeds based on these ingredients, none of them were observed in fillets of Atlantic salmon and gilthead sea bream fed on these feeds.

Species and tissues specific differentiation of processed animal proteins in aquafeeds using proteomics tools.

UNLABELLED: The rapidly growing aquaculture industry drives the search for sustainable protein sources in fish feed. In the European Union (EU) since 2013 non-ruminant processed animal proteins (PAP) are again permitted to be used in aquafeeds. To ensure that commercial fish feeds do not contain PAP from prohibited species, EU reference methods were established. However, due to the heterogeneous and complex nature of PAP complementary methods are required to guarantee the safe use of this fish feed ingredient. In addition, there is a need for tissue specific PAP detection to identify the sources (i.e. bovine carcass, blood, or meat) of illegal PAP use. In the present study, we investigated and compared different protein extraction, solubilisation and digestion protocols on different proteomics platforms for the detection and differentiation of prohibited PAP. In addition, we assessed if tissue specific PAP detection was feasible using proteomics tools. All work was performed independently in two different laboratories. We found that irrespective of sample preparation gel-based proteomics tools were inappropriate when working with PAP. Gel-free shotgun proteomics approaches in combination with direct spectral comparison were able to provide quality species and tissue specific data to complement and refine current methods of PAP detection and identification. SIGNIFICANCE: To guarantee the safe use of processed animal protein (PAP) in aquafeeds efficient PAP detection and monitoring tools are required. The present study investigated and compared various proteomics workflows and shows that the application of shotgun proteomics in combination with direct comparison of spectral libraries provides for the desired species and tissue specific classification of this heat sterilized and pressure treated (≥133°C, at 3bar for 20min) protein feed ingredient.

Toxicokinetic model assessment on the dechlorination of dietary toxaphene CHB-62 into CHB-44 in Atlantic salmon (Salmo salar L.).

European Union legislation on the upper limits of toxaphene in feed and food include the congeners CHB-26, CHB-62 and CHB-50 and is set at 50 µg kg⁻¹ feed for the sum of these three congeners. However, due to their elevated presence in fish, the congeners CHB-40 and CHB-41, CHB-44, and CHB-42 should also be included according to the European Food Safety Authority (EFSA) in 2005. Earlier trials with model zebra fish have shown in vivo dechlorination of dietary CHB-62 to CHB-44 and, to a lesser degree, of CHB-50 to CHB-40. Biomagnification patterns of Atlantic salmon, fed with technical toxaphene-enriched feeds, indicated that Atlantic salmon have a similar dechlorination. In the present study, a serial one-compartment physiological kinetic model, which includes differentiated growth of body components, is used to quantify the contribution of dechlorination to the congener-specific fillet accumulation of a mixture of dietary toxaphene congeners in Atlantic salmon. The model is assessed from experimental uptake and elimination kinetics of Atlantic salmon smolt fed with technical toxaphene for 122 days followed by a depuration period of 75 days in which the fish were fed toxaphene-free control feed. The serial one-compartment model shows that about 31% of CHB-44 that accumulated in the fillet originated from dietary CHB-62. In contrast, dechlorination of CHB-50 into CHB-40 is not significant. The results show that previously demonstrated in vivo dechlorination of CHB-62 into CHB-44 in zebra fish also occurs in the farmed fish species Atlantic salmon. This dechlorination can at least partly explain the relatively elevated CHB-44 observed in toxaphene fish surveys.

Dechlorination of the dietary nona-chlorinated toxaphene congeners 62 and 50 into the octa-chlorinated toxaphene congeners 44 and 40 in zebrafish (Danio rerio) and Atlantic salmon (Salmo salar).

The relative feed-to-fish accumulation and possible biotransformation of the nona-chlorinated toxaphene congeners currently included in EU-legislation (CHB-50 and -62) and the octa-chlorinated congeners recommended by the European Food Safety Authority to be included in future surveillance of fish samples (CHB-40, 41, and 44) were investigated in the present study. Model fish Danio rerio were fed either (a) diets spiked with a combination as well as the pure individual toxaphene congeners CHB-50 or 62 or (b) diets spiked with the combination of CHB ∑50+62 and/or CHB ∑40+41+44. In addition, seawater adapted Atlantic salmon smolts were fed technical toxaphene enriched feeds for 62 days. Zebrafish fed a diet containing CHB-50 and CHB-62 accumulated newly formed CHB-40&41 and CHB-44, respectively. The biomagnifications factors (BMF) of the toxaphene congeners in Atlantic salmon muscle from the feeds spiked with technical toxaphene were significantly correlated with their relative lipophilicity (expressed as logK(ow)). An exception was CHB-44 which had a higher BMF than could be expected from its specific logK(ow), reflecting that CHB-44 is a metabolite formed under dietary exposure to CHB-62. This paper reports the in vivo dechlorination of nona-chlorinated toxaphene congeners into octa-chlorinated congeners in feeding trials with a model fish (zebrafish) and an oily food fish (Atlantic salmon).

Toxicokinetics and carry-over model of α-hexabromocyclododecane (HBCD) from feed to consumption-sized Atlantic salmon (Salmo salar).

A two-compartmental model for the kinetics of carry-over of the brominated flame retardant α-hexabromocyclododecane (HBCD) from feed to the fillet of farmed harvest-sized Atlantic salmon (Salmo salar L.) was developed. The model is based on a fat compartment for storage of the lipophilic α-HBCD and a central compartment comprising all other tissues. Specific for this model is that the salmon has a continuous growth and that fillet contaminant levels are explained by both the fat and the central compartments. The uptake and elimination kinetics are obtained from experimental data where consumer sized (start weight approximately 1 kg) Atlantic salmon was fed α-HBCD spiked feed (280 ± 11 µg kg(-1)) for 2 months followed by a depuration period of 3 months. The model was used to simulate the HBCD feed-to-fillet transfer in Atlantic salmon under realistic farming conditions such as the seasonal fluctuations in feed intake, growth and fillet fat deposition. The model predictions gave fillet concentrations of 0.2-1.8 µg kg(-1) depending on the level of fish oil inclusion in the salmon diets when using fish oil with high POP background levels. Model simulations show that currently farmed Atlantic salmon can contribute to a maximum of 6% of the estimated provisional food reference dose for HBCD.

Oxidative stress responses in rainbow trout (Oncorhynchus mykiss) hepatocytes exposed to pro-oxidants and a complex environmental sample.

A wide range of pollutants in the aquatic environment have the capacity to induce toxic effects expressed as cellular oxidative stress. In the current study, the potential of an in vitro toxicity testing system was therefore investigated using rainbow trout (Oncorhynchus mykiss) hepatocytes to assess different endpoints of oxidative stress. The pro-oxidants CuSO(4) and paraquat were used as models for comparison to a complex environmental sample. Results following 6, 24, 48 and 96h exposure to different concentrations of these substances show cellular effects on intracellular ROS formation, glutathione levels and redox status, expression of the antioxidant enzymes superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, gamma-glutamyl-cysteine synthetase (GCS) and thioredoxin, as well as cytotoxicity parameters. The most consistent effects (maximum values within brackets), observed in dose and time parameters for both model compounds and environmental sample, were the depletion of total glutathione (9.4% of control), induced levels of oxidized glutathione (695% of control), and gene expression regulation depicted relative to the control gene beta-actin of GCS mRNA (239% of control) and catalase (29% of control). In conclusion, the responses on several antioxidant defence system parameters demonstrated the validity of the in vitro toxicity testing system. Not only could multiple effects be detected at sub-lethal exposure concentrations, but these effects also gave valuable insight to the toxic mechanisms at the molecular level.

Reducing persistent organic pollutants while maintaining long chain omega-3 fatty acid in farmed Atlantic salmon using decontaminated fish oils for an entire production cycle.

Oily fish are an important source of health promoting nutrients such as the very long chain marine omega-3 (VLC-n3) fatty acids and simultaneously a source of potentially hazardous contaminants. Fish oils that are used in fish feed are the main source for both contaminants and VLC-n3. Decontamination techniques have recently been developed to effectively remove persistent organic contaminants from fish oils. The aim of the present study was to assess the level of potentially hazardous contaminants and the health beneficial fatty acids in Atlantic salmon reared on novel decontaminated feeds. Atlantic salmon were fed for 18 months (an entire seawater production cycle) on diets based on decontaminated or non-treated (control) fish oils until market size (approximately 5 kg). The level of known notorious persistent organic pollutants (POPs, i.e. dioxins, dioxin-like polychlorinated biphenyls (DL-PCBs), non dioxin-like PCBs, poly brominated diphenyl ethers (PBDE), and organochlorine pesticides), as well as fatty acid composition were analysed in fish oils, the two diets, and Atlantic salmon fillet. The oil decontamination process was a two-step procedure using active carbon and short path distillation. The fillet levels of POPs in market size fish were reduced by 68-85% while the concentration of very long chain omega-3 fatty acids was reduced by 4-7%. No differences in biomarkers of dioxin-like component exposures, such as hepatic gene expression of CYP1A or AhR2B, CYP1A protein expression and 7-ethoxyresorufin O-deethylase (EROD) activity, were observed between salmon raised on normal or decontaminated feeds, thus indicating that the difference in POPs levels were of no biological significance to the fish. Atlantic salmon reared on decontaminated feeds had sum polychlorinated dibenzodioxins/furans (PCDD/Fs) and DL-PCB concentrations that were comparable with terrestrial food products such as beef, while the level of marine omega-3 fatty acids remained as high as for commercially farmed Atlantic salmon.

An examination of the intestinal tract of Atlantic salmon, Salmo salar L., parr fed different varieties of soy and maize.

This study was conducted to investigate the long-term effects of feeding plant products from both traditional breeding and from biotechnology on intestinal somatic indices, histology and cell proliferation in first-feeding Atlantic salmon, Salmo salar L. (initial weight 0.21 +/- 0.02 g). A standard fishmeal diet (standard fishmeal) was formulated to contain fishmeal as the sole protein source and suprex maize as the main starch source. Six experimental diets were then developed: two in which some of the fishmeal was replaced with commercially available, genetically modified Roundup Ready full-fat soybean meal (GM-soy) or commercially available, non-GM full-fat soybean meal (nGM-soy) at a level of 12.5% of the total diet, and four diets in which the suprex maize was replaced with two lines of GM-maize (Dekalb 1; D1 and Pioneer 1; P1), both products of event MON810, and their half-sibling non-GM counterparts (Dekalb 2; D2 and Pioneer 2; P2), at a level of 12.1% of total diet. Each diet was fed to fish in triplicate tanks and the experiment lasted for 8 months, during which the fish reached a final weight of 101-116 g. There was no significant effect of diet on the intestinal indices, nor were histological changes observed in the pyloric caeca or mid intestine. In the distal intestine, one of nine sampled fish fed nGM-soy showed moderate changes, two of nine sampled fish fed GM-soy showed changes, one with moderate and one with severe changes, and two of nine fish fed nGM-maize D2 had moderate changes. Using a monoclonal antibody against proliferating cell nuclear antigen (PCNA), cell proliferative responses to the experimental diets were assessed. In fish fed both soy diets, a significantly higher (P < 0.05) cell proliferation response was observed in the distal intestine concomitant with an increased localization of PCNA positive cells along the whole distal intestinal folds. The PCNA response among the nGM-soy group was significantly higher compared with all the other diet groups. In contrast, for fish exposed to dietary maize (type D) compared with fish fed the standard fishmeal, the soy-diets (GM-soy and nGM-soy) and maize (type P), a significantly lower (P < 0.05) cell proliferation response was observed in the distal intestine. Results indicated that the GM plant products investigated in this study, at about 12% inclusion level, were as safe as commercially available non-GM products, at least in terms of their effect on indices and histological parameters of the Atlantic salmon intestinal tract.

mRNA expression of antioxidant enzymes (SOD, CAT and GSH-Px) and lipid peroxidative stress in liver of Atlantic salmon (Salmo salar) exposed to hyperoxic water during smoltification.

The mRNA levels of three antioxidant genes, Cu/Zn superoxide dismutase (SOD), catalase (CAT) and phospholipid hydroperoxide glutathione peroxidase (GSH-Px), were quantified with real-time qRT-PCR in liver of Atlantic salmon Salmo salar exposed to 80% (normoxia), 105% and 130% O2 saturation for 54 days. The salmon were then translocated and exposed to 90% and 130% O2 saturation for additional 72 days during smoltification. TBARS and vitamin E levels in liver and the levels of oxidized glutathione (GSSG), total glutathione (GSH) and the resulting oxidative stress index (OSI) in blood were quantified as traditional oxidative stress markers. No significant mean normalized expression (MNE) differences of SOD, CAT or GSH-Px were found in liver after hyperoxia exposure at the two sampling times. Significantly decreased OSI was found in smolt exposed to 130% O2 saturation after 126 days (n = 18, P < 0.0001), indicating hyperoxia-induced oxidative stress. No effects were seen on growth, or on the levels of thiobarbituric reactive substances (TBARS) and vitamin E in liver after the exposure experiment. Overall, the mRNA expression of SOD, CAT and GSH-Px in liver related poorly with the hyperoxic exposure regimes, and more knowledge are needed before the expressed levels of these antioxidant genes can be applied as biomarkers of hyperoxia in Atlantic salmon.

Norwegian monitoring programme on the inorganic and organic contaminants in fish caught in the Barents Sea, Norwegian Sea and North Sea, 1994-2001.

The results from part of a monitoring programme of contaminant levels in fish and other seafood products initiated by the Directorate of Fisheries in Norway in 1994 are presented. Concentrations of 22 elements (four are presented here: As, Cd, Hg and Pb) and HCB, HCH, PCB 28, 52, 101, 105, 118, 138, 153, 156, 180, pp-DDD, p-DDE, p-DDT, sum DDT and (137)Cs were determined in 17 species of fish caught in three sampling locations: the Barents Sea, the Norwegian Sea and the North Sea. The fish species analysed in the survey were limited to species of commercial importance for Norway with catching volumes of at least 10,000 metric tons year(-1). The survey started in 1994 and is expected to continue beyond 2010. The analyses were carried out on 25 individual fish from each species and each sampling location, and the locations were representative of commercial fishing grounds for the species in question. The concentrations of contaminants found were considerably lower than the maximum levels permissible in fish set by CODEX and the European Union for contaminants in seafood products.