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 50 ng·g-1 spiking levels. LOQs were 2 ng·g-1 for most compounds, and LODs ranged from 0.5 to 2 ng·g-1. Analysis of real-world samples revealed the presence of naphthalene, fluorene, phenanthrene, fluoranthene and pyrene at concentration levels ranging from 4.8 to 187 ng·g-1. No PCBs, DDTs and pesticides were found in fillets from salmon and sea bream

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

Risk assessment of the use of alternative animal and plant raw material resources in aquaculture feeds

A wide range of raw materials are now used routinely in aquaculture feeds throughout the world, primarily to supply protein and energy in the form of lipid from edible oils. Protein meals and oils used can generally be divided into those of plant or animal origin and many have considerable potential to supply the required dietary nutrients required by aquaculture species. However, the use of any raw material introduces a suite of risks that need to be considered to enable the production of safe, sustainable and functional feeds to underpin this sector. A lack of understanding of some of those risks can result in failure of dietary specifications being met and/or negative nutritional elements being introduced (e.g. antinutritional factors). Importantly, it is this feed that when fed to food‐producing animals is such an important element of food safety, and as such any undesirable aspects relating to feed production can also have a negative impact on the rest of the food chain. However, there is some disparity internationally among raw materials that are used and the perceptions surrounding the risk of their use. It is the scientific assessment of these risks that is the basis of this review

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. © 2014 American Chemical Society.This work has been (partly) funded under the EU Seventh Framework Programme by ARRAINA Project 288925: Advanced Research Initiatives for Nutrition and Aquaculture. The views expressed in this work are the sole responsibility of the authors and do not necessarily reflect the views of the European Commission. This work has been developed within the framework of the Research Unit of Marine Ecotoxicology (IATS (CSIC)-IUPA (UJI)). We also acknowledge the financial support of Generalitat Valenciana (research group of excellence PROMETEO/2009/054; ISIC 2012/016).Peer Reviewe

Occurrence and potential transfer of mycotoxins in gilthead sea bream and Atlantic salmon by use of novel alternative feed ingredients

Plant ingredients and processed animal proteins (PAP) are suitable alternative feedstuffs for fish feeds in aquaculture practice, although their use can introduce contaminants that are not previously associated with marine salmon and gilthead sea bream farming. Mycotoxins are well known natural contaminants in plant feed material, although they also could be present on PAPs after fungi growth during storage. The present study surveyed commercially available plant ingredients (19) and PAP (19) for a wide range of mycotoxins (18) according to the EU regulations. PAP showed only minor levels of ochratoxin A and fumonisin B1 and the mycotoxin carry-over from feeds to fillets of farmed Atlantic salmon and gilthead sea bream (two main species of European aquaculture) was performed with plant ingredient based diets. Deoxynivalenol was the most prevalent mycotoxin in wheat, wheat gluten and corn gluten cereals with levels ranging from 17 to 814 and μgkg-1, followed by fumonisins in corn products (range 11.1-4901μgkg-1 for fumonisin B1+B2+B3). Overall mycotoxin levels in fish feeds reflected the feed ingredient composition and the level of contaminant in each feed ingredient. In all cases the studied ingredients and feeds showed levels of mycotoxins below maximum residue limits established by the Commission Recommendation 2006/576/EC. Following these guidelines no mycotoxin carry-over was found from feeds to edible fillets of salmonids and a typically marine fish, such as gilthead sea bream. As far we know, this is the first report of mycotoxin surveillance in farmed fish species. © 2015 Elsevier Ltd.This work has been (partly) funded under the EU Seventh Framework Programme by ARRAINA Project 288925: Advanced Research Initiatives for Nutrition and Aquaculture. The views expressed in this work are the sole responsibility of the authors and do not necessarily reflect the views of the European Commission. The authors also acknowledge the financial support (partly) of the project SAFE-PAP, Research Council Research and Development Project (227387) National Institute of Nutrition and Seafood Research (NIFES, Norway). The authors acknowledge the financial support of Generalitat Valenciana, as research group of excellence (PROMETEO II/2014/023, PROMETEO II/2014/085, and Collaborative Research on Environment and Food-Safety, ISIC/2012/016).Peer Reviewe

Dietary pesticide chlorpyrifos-methyl affects arachidonic acid metabolism including phospholipid remodeling in Atlantic salmon (Salmo salar L.)

The pesticide chlorpyrifos-methyl (CLP-m) has been identified in plant ingredients intended for aquaculture feed production. To investigate systemic effects of CLP-m with emphasis on lipid metabolism, post-smolt Atlantic salmon were fed in duplicate (n = 2) either diets with no CLP-m (Control) or CLP-m at different concentrations (0.1, 1.0 or 8.0 mg kg(-1)) for a total of 67 days (Low, Medium, High). Fish in all groups almost doubled their weight during the feeding trial from 262 +/- 26 g (mean +/- SD) to 465 +/- 64 g (overall mean), with no significant effects on any growth parameters. There was a significant dose-dependent inhibition of plasma cholinesterase activity (BuChE) after 67 days. The CLP-m biotransformation metabolite, TCP was detected in liver and bile, with low levels of the parent compound in the organs. Spleen somatic index decreased significantly with increasing dietary CLP-m intake. Hematocrit (%) decreased linearly with increasing dietary exposure to CLP-m after 30 days of exposure, but this decrease was less at 67 days of exposure. A significantly reduced content of arachidonic acid (ARA 20:4n - 6), accompanied by a significantly increased content of the saturated fatty acid, palmitic acid (PA 16:0), was observed in liver phospholipids (PLs) with increasing dietary content of CLP-m. Major effects were seen on the PL classes in liver which showed a significantly decreased absolute content, possibly indicating inhibition of PL remodeling pathways or other membrane perturbation effects from CLP-m exposure. In conclusion, this study shows that the pesticide CLP-m is a relatively potent toxicant in Atlantic salmon, especially affecting liver PLs and ARA metabolism

Tolerance and dose-response assessment of subchronic dietary ethoxyquin exposure in Atlantic salmon (Salmo salar L.)

Ethoxyquin (EQ; 6-Ethoxy-2,2,4-trimethyl-1,2-dihydroquinoline) has been used as an antioxidant in feed components for pets, livestock and aquaculture. However, possible risks of EQ used in aquafeed for fish health have not yet been characterized. The present study investigated the toxicity and dose-response of subchronic dietary EQ exposure at doses ranging from 41 to 9666 mg EQ/kg feed in Atlantic salmon (Salmo salar L.). Feed at concentrations higher than 1173 mg EQ/kg were rejected by the fish, resulting in reduced feed intake and growth performance. No mortality was observed in fish exposed to any of the doses. A multi-omic screening of metabolome and proteome in salmon liver indicated an effect of dietary EQ on bioenergetics pathways and hepatic redox homeostasis in fish fed concentrations above 119 mg EQ/kg feed. Increased energy expenditure associated with an upregulation of hepatic fatty acid â-oxidation and induction and carbohydrate catabolic pathways resulted in a dose-dependent depletion of intracytoplasmic lipid vacuoles in liver histological sections, decreasing whole body lipid levels and altered purine/pyrimidine metabolism. Increased GSH and TBARS in the liver indicated a state of oxidative stress, which was associated with activation of the NRF2-mediated oxidative stress response and glutathione-mediated detoxification processes. However, no oxidative DNA damage was observed. As manifestation of altered energy metabolism, the depletion of liver intracytoplasmic lipid vacuoles was considered the critical endpoint for benchmark dose assessment, and a BMDL10 of 243 mg EQ/kg feed was derived as a safe upper limit of EQ exposure in Atlantic salmon

Occurrence and potential transfer of mycotoxins in gilthead sea bream and Atlantic salmon by use of novel alternative feed ingredients

Plant ingredients and processed animal proteins (PAP) are suitable alternative feedstuffs for fish feeds in aquaculture practice, although their use can introduce contaminants that are not previously associated with marine salmon and gilthead sea bream farming. Mycotoxins are well known natural contaminants in plant feed material, although they also could be present on PAPs after fungi growth during storage. The present study surveyed commercially available plant ingredients (19) and PAP (19) for a wide range of mycotoxins (18) according to the EU regulations. PAP showed only minor levels of ochratoxin A and fumonisin B1 and the mycotoxin carry-over from feeds to fillets of farmed Atlantic salmon and gilthead sea bream (two main species of European aquaculture) was performed with plant ingredient based diets. Deoxynivalenol was the most prevalent mycotoxin in wheat, wheat gluten and corn gluten cereals with levels ranging from 17 to 814 and μg kg−1, followed by fumonisins in corn products (range 11.1–4901 μg kg−1 for fumonisin B1 + B2 + B3). Overall mycotoxin levels in fish feeds reflected the feed ingredient composition and the level of contaminant in each feed ingredient. In all cases the studied ingredients and feeds showed levels of mycotoxins below maximum residue limits established by the Commission Recommendation 2006/576/EC. Following these guidelines no mycotoxin carry-over was found from feeds to edible fillets of salmonids and a typically marine fish, such as gilthead sea bream. As far we know, this is the first report of mycotoxin surveillance in farmed fish species.EU Seventh Framework Programme by ARRAINA Project, 288925. project SAFE-PAP, Research Council Research and Development Project National Institute of Nutrition and Seafood Research (NIFES, Norway), 227387. Generalitat Valenciana, PROMETEO II/2014/023 PROMETEO II/2014/085 ISIC/2012/01

Safe limits of selenomethionine and selenite supplementation to plant-based Atlantic salmon feeds

The use of plant-based feeds warrants the supplementation with selenium (Se) to cover the requirement for Atlantic salmon. Depending on its chemical form, Se is a trace element with a narrow range between requirement and toxicity for most vertebrates. Information on safe upper limit for Atlantic salmon feed supplementation is lacking. Atlantic salmon (147 g) were fed a low natural background organic Se diet (0.45 mg Se kg−1, wet weight (ww)) fortified with 5 graded levels of inorganic sodium selenite (0.45, 5.4, 11.0, 29.4, or 60.0 mg kg−1 ww) or organic selenomethionine (SeMet) (0.45, 6.2, 16.2, 21, or 39 mg kg−1 ww), in triplicate for 3 months. Excess Se supplementation was assessed by targeted biomarkers of Se toxicity pathways (e.g. markers of oxidative stress and lipid metabolism), as well as general adverse effect parameters (plasma biochemistry, hematology, liver histopathology, and growth). Safe limits were set by model-fitting the effect data in a dose-response (lower bound) bench mark dose (BMDL) evaluation. Fish fed the two highest selenite levels showed mortality while fish fed SeMet had no mortality. Fish fed 5.4–11 mg selenite kg−1 feed showed significantly (ANOVA, Tukey's t-test,

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

This is a pre-print of an article published in Analytical and Bioanalytical Chemistry. The final authenticated version is available online at:https://doi.org/10.1007/s00216-019-02104-6The 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