No transfer of the non-regulated mycotoxins, beauvericin and enniatins, from feeds to farmed fish reared on plant-based diets.

Concern about the risk of exposure to emerging plant-derived mycotoxins such as beauvericin and enniatins has been addressed by the European Commission who requested the European Food Safety Authority for a scientific opinion on their risk to human and animal health. The studied mycotoxins were found in feeds with enniatin B and beauvericin at average concentrations of 19.9 µg/kg and 30 µg/kg, respectively. In all cases, concentrations of all the mycotoxins analyzed were below quantification limits (<0.1 µg/kg) in fish samples (n = 82). The present work provides comprehensive and traceable data of emerging mycotoxins in plant-based aquafeeds and fish reared on the feeds, responding to increasing concerns about safety of farmed fish fed on sustainable feeds. On the basis of data reported, there was no transfer of the emerging mycotoxins, beauvericin and enniatins, from feeds to fish and so, no risk for human consumption.

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.

Untargeted metabolomics approach for unraveling robust biomarkers of nutritional status in fasted gilthead sea bream (Sparus aurata).

A metabolomic study has been performed to identify sensitive and robust biomarkers of malnutrition in farmed fish, using gilthead sea bream (Sparus aurata) as a model. The metabolomic fingerprinting of serum from fasted fish was assessed by means of ultra-high performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry. More than 15,000 different m/z ions were detected and Partial Least Squares-Discriminant analysis allowed a clear differentiation between the two experimental groups (fed and 10-day fasted fish) with more than 90% of total variance explained by the two first components. The most significant metabolites (up to 45) were elucidated on the basis of their tandem mass spectra with a broad representation of amino acids, oligopeptides, urea cycle metabolites, L-carnitine-related metabolites, glutathione-related metabolites, fatty acids, lysophosphatidic acids, phosphatidylcholines as well as biotin- and noradrenaline-related metabolites. This untargeted approach highlighted important adaptive responses in energy and oxidative metabolism, contributing to identify robust and nutritionally-regulated biomarkers of health and metabolic condition that will serve to assess the welfare status of farmed fish.

Investigation of pharmaceuticals in processed animal by-products by liquid chromatography coupled to high-resolution mass spectrometry.

There is an on-going trend for developing more sustainable salmon feed in which traditionally applied marine feed ingredients are replaced with alternatives. Processed animal products (PAPs) have been re-authorized as novel high quality protein ingredients in 2013. These PAPs may harbor undesirable substances such as pharmaceuticals and metabolites which are not previously associated with salmon farming, but might cause a potential risk for feed and food safety. To control these contaminants, an analytical strategy based on a generic extraction followed by ultra-high performance liquid chromatography coupled to high resolution mass spectrometry (UHPLC-HRMS) using quadrupole time-of-flight mass analyzer (QTOF MS) was applied for wide scope screening. Quality control samples, consisting of PAP commodities spiked at 0.02, 0.1 and 0.2 mg/kg with 150 analytes, were injected in every sample batch to verify the overall method performance. The methodology was applied to 19 commercially available PAP samples from six different types of matrices from the EU animal rendering industry. This strategy allows assessing possible emergent risk exposition of the salmon farming industry to 1005 undesirables, including pharmaceuticals, several dyes and relevant metabolites.

Atmospheric-pressure chemical ionization tandem mass spectrometry (APGC/MS/MS) an alternative to high-resolution mass spectrometry (HRGC/HRMS) for the determination of dioxins.

The use of a new atmospheric-pressure chemical ionization source for gas chromatography (APGC) coupled with a tandem quadrupole mass spectrometry (MS/MS) system, as an alternative to high-resolution mass spectrometry (HRMS), for the determination of PCDDs/PCDFs is described. The potential of using atmospheric-pressure chemical ionization (APCI) coupled to a tandem quadrupole analyzer has been validated for the identification and quantification of dioxins and furans in different complex matrices. The main advantage of using the APCI source is the soft ionization at atmospheric pressure, which results in very limited fragmentation. APCI mass spectra are dominated by the molecular ion cluster, in contrast with the high energy ionization process under electron ionization (EI). The use of the molecular ion as the precursor ion in MS/MS enhances selectivity and, consequently, sensitivity by increasing the signal-to-noise ratios (S/N). For standard solutions of 2,3,7,8-TCDD, injections of 10 fg in the splitless mode on 30- or 60-m-length, 0.25 mm inner diameter (id), and 25 µm film thickness low-polarity capillary columns (DB5MS type), signal-to-noise (S/N) ratios of >10:1 were routinely obtained. Linearity was achieved in the region (correlation coefficient of r(2) > 0.998) for calibration curves ranging from 100 fg/µL to 1000 pg/µL. The results from a wide variety of complex samples, including certified and standard reference materials and samples from several QA/QC studies, which were previously analyzed by EI HRGC/HRMS, were compared with the results from the APGC/MS/MS system. Results between instruments showed good agreement both in individual congeners and toxic equivalence factors (TEQs). The data show that the use of APGC in combination with MS/MS for the analysis of dioxins has the same potential, in terms of sensitivity and selectivity, as the traditional HRMS instrumentation used for this analysis. However, the APCI/MS/MS system, as a benchtop system, is much easier to use.

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.

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.

Qualitative screening of undesirable compounds from feeds to fish by liquid chromatography coupled to mass spectrometry.

This paper describes the development, validation, and application of a rapid screening method for the detection and identification of undesirable organic compounds in aquaculture products. A generic sample treatment was applied without any purification or preconcentration step. After extraction of the samples with acetonitrile/water 80:20 (0.1% formic acid), the extracts were centrifuged and directly injected in the LC-HRMS system, consisting of ultra-high performance liquid chromatography coupled to hybrid quadrupole time-of-flight mass spectrometry (UHPLC-QTOF MS). A qualitative validation was carried out for over 70 representative compounds, including antibiotics, pesticides, and mycotoxins, in fish feed and fish fillets spiked at 20 and 100 µg/kg. At the highest level, the great majority of compounds were detected (using the most abundant ion, typically the protonated molecule) and unequivocally identified (on the basis of the presence of two accurate-mass measured ions). At the 20 µg/kg level, many contaminants could already be detected, although identification using two ions was not fully reached for some of them, mainly in fish feed due to the complexity of this matrix. Subsequent application of this screening methodology to aquaculture samples made it possible to find several compounds from the target list, such as the antibiotic ciprofloxacin, the insecticide pirimiphos-methyl, and the mycotoxins fumonisin B2 and zearalenone. A retrospective analysis of accurate-mass full-spectrum acquisition data provided by QTOF MS was also made, without either reprocessing or injecting the samples. This allowed the detection and tentative identification of other organic undesirables different from those included in the validated list.

Non-target screening of organic contaminants in marine salts by gas chromatography coupled to high-resolution time-of-flight mass spectrometry.

Gas chromatography coupled to time-of-flight mass spectrometry (GC-TOF MS) has been applied to characterize the organic pollution pattern of marine salt samples collected in saltworks from the Spanish Mediterranean coast. After dissolving the samples in water, a solid-phase extraction was applied reaching with a 250-preconcentration factor. The screening methodology allowed the detection of sample components without any kind of pre-selection of target pollutants. The identity of components detected was established by accurate mass measurements and comparison of experimental full-acquisition spectra with theoretical MS libraries. Several organic pollutants were identified in the samples, like plasticizers - potentially toxic to humans - and fragrances -included within the group of pharmaceuticals and personal care products-, among others. Our results indicate that these contaminants can be found in the marine salt after the crystallization process. GC-TOF MS is a powerful technique for wide-scope screening of (semi)volatile, low-polar organic contaminants, able to investigate the presence of a large number of compounds. Searching of contaminants is not restricted to a target list of compounds. Therefore, unexpected contaminants can be discovered in an efficient way, with better sensitivity and selectivity than other conventional analytical techniques, and making use of the powerful qualitative information provided by full-spectrum acquisition at accurate mass.

Gas chromatography-mass spectrometric determination of polybrominated diphenyl ethers in complex fatty matrices from aquaculture activities.

Gas chromatography coupled to mass spectrometry in negative chemical ionization mode (GC-(NCI)MS) has been applied to the quantification and reliable identification of polybrominated diphenyl ethers (PBDEs) in animal and vegetable samples from aquaculture activities. Matrices analyzed included fish fillet, fish feed, fish oil and linseed oil, their fat content ranged from 5% to 100%. Solid-phase extraction (SPE) (using Florisil and silica cartridges) and normal-phase high performance liquid chromatography were tested for an efficient clean-up in order to obtain sample extracts free of interfering compounds. Combining sulphuric acid digestion and SPE with Florisil led to the highest efficiency in the elimination of interferences from the extracts. The sample procedure developed, together with the application of GC-(NCI)MS for measurement, led to the satisfactory determination of PBDEs at microg kg(-1) levels in complex aquaculture matrices with high lipid content. The use of a short and thin film-thickness fused-silica capillary column allowed to determine the problematic BDE 209 with satisfactory results. Three m/z ions were acquired for each analyte, which ensured a reliable identification of compounds detected in samples.

Bioaccumulation of polycyclic aromatic hydrocarbons in gilthead sea bream (Sparus aurata L.) exposed to long term feeding trials with different experimental diets.

Polycyclic aromatic hydrocarbons (16 EPA list) were determined in oils, fish feed, and fillets from gilthead sea bream fed through a full production cycle (14 months) with feed containing different proportions of fish oil replaced by vegetable oils, followed by a finishing phase with fish oil. At the beginning of the study, fish presented 46.6 microg/kg fresh weight of the sum of PAHs in fillet and a benzo[a]pyrene equivalent value of 9.1 microg/kg fresh weight. These levels decreased after 330 days of rearing to values around 2 microg/kg. Although the concentration increased again during the finishing phase, they remained low. These low concentrations of PAHs could be the result of a dilution process associated with fish growth and with the detoxification pathways, both favored by the low levels of PAHs present in the feeds and the lack of any other potential source of contamination during the whole rearing period.

A reliable analytical approach based on gas chromatography coupled to triple quadrupole and time-of-flight mass analyzers for the determination and confirmation of polycyclic aromatic hydrocarbons in complex matrices from aquaculture activities.

The potential of gas chromatography coupled to tandem mass spectrometry (GC/MS/MS) with a triple quadrupole analyzer (QqQ) has been investigated for the quantification and reliable identification of sixteen polycyclic aromatic hydrocarbons (PAHs) from the EPA priority list in animal and vegetable samples from aquaculture activities, whose fat content ranged from 5 to 100%. Matrices analyzed included fish fillet, fish feed, fish oil and linseed oil. Combining optimized saponification and solid-phase extraction led to high efficiency in the elimination of interfering compounds, mainly fat, from the extracts. The developed procedure minimized the presence of these interfering compounds in the extracts and provided satisfactory recoveries of PAHs. The excellent sensitivity and selectivity of GC/(QqQ)MS/MS in selected reaction monitoring (SRM) allowed to reach limits of detection at pg/g levels. Two SRM transitions were acquired for each analyte to ensure reliable identification of compounds detected in samples. Confirmation of positive findings was performed by GC coupled to high-resolution time-of-flight mass spectrometry (GC/TOFMS). The accurate mass information provided by GC/TOFMS in full acquisition mode together with its high mass resolution makes it a powerful analytical tool for the unequivocal confirmation of PAHs in the matrices tested. The method developed was applied to the analysis of real-world samples of each matrix studied with the result of detecting and confirming the majority of analytes at the microg/kg level by both QqQ and TOF mass spectrometers.

Effects of fish oil replacement and re-feeding on the bioaccumulation of organochlorine compounds in gilthead sea bream (Sparus aurata L.) of market size.

Organochlorine pesticide residues and polychlorinated biphenyls were determined in raw materials, fish feeds and fillets from fish exposed through the productive cycle (14 months) to experimental diets with different percentages of fish oil replacement with vegetable oils. Detectable amounts of organochlorine compounds were found in raw materials derived from fish sources with none being detected in vegetable ingredients. Fish feeds presented trace concentrations of contaminants at the ng/g level, which varied according to the contribution of the different resources used in their manufacture. Contaminants did not accumulate during the first 11 months of exposure, and low concentrations of organochlorine compounds were found both at the start and at the end of this feeding period. Fillets from fish fed the fish oil diet presented the highest concentrations of organochlorine compounds, with these decreasing in proportion to fish oil replacement. Three months of fish oil re-feeding during the finishing phase only produced significant bioaccumulation over the course of the first month. By optimizing fish meal and fish oil replacement with vegetable oils alternative feeds can contribute to significantly reduce the risk of organochlorine uptake by consumers.