Plastic Contamination in Seabass and Seabream from Off-Shore Aquaculture Facilities from the Mediterranean Sea.

We characterized the presence of plastics in different organs of the gilthead seabream (Sparus aurata) and European seabass (Dicentrarchus labrax) from some off-shore aquaculture facilities of the Mediterranean Sea. Plastics were detected in 38% of analyzed fish. Higher contamination was observed in fish from Turkey and Greece with respect to Italy, without significant differences between the geographical areas. Plastics accumulated mostly in the gastrointestinal tract and, to a lower extent, in the muscle, which represents the edible part of fish. Based on the particle detected, a maximum amount of 0.01 plastic/g wet weight (w.w.) can occur in muscles, suggesting a low input for humans through consumption. A large portion of the particles identified was represented by man-made cellulose-based fibers. The characterization of the polymeric composition suggests that plastics taken up by fish can have land-based and pelagic origins, but plastics can be introduced also from different aquaculture practices.

First Report of Proteocephalus longicollis (Zeder, 1800) in Alosa fallax lacustris (Fatio, 1890) from Lake Garda (Italy): Morphological and Molecular Study.

Twaite shad (Alosa fallax) includes two subspecies, the anadromous Alosa fallax nilotica, and the landlocked species Alosa fallax lacustris, representing the only Clupeidae inhabiting Lake Garda. Study of the parasitic fauna of this species in this area is still limited. For this reason, the aim of the present study was to evaluate the presence of tapeworms from A. fallax lacustris sampled in Lake Garda. Sixty-six A. fallax lacustris specimens were collected at the Milan fish market (Lombardy, Italy); specifically, an evaluation of the gastrointestinal contents was carried out for the presence of helminths. All parasites found were fixed in 70% ethanol and transferred to the laboratories of the University of Messina for morphological and molecular analysis. Parts of the parasites were stained with Semichon's carmine red technique. Molecular analysis was carried out using LSU rRNA and ITS2 region genes. Eight specimens out of sixty-six (12.1%) were positive for adult cestodes within the pyloric caeca. Morphological and molecular analysis could to identify the parasites found to be Proteocephalus longicollis. This parasite species is not considered a zoonotic agent, representing a low risk of parasitic fish-borne zoonosis for consumers of this appreciated fish from Lake Garda.

Unusual Localization of Hysterothylacium incurvum in Xiphias gladius (Linnaeus 1758) Caught in the Atlantic Ocean.

This study represents the first report of Hysterothylacium incurvum within swordfish (Xiphias gladius) heart chambers. Swordfish is a large pelagic teleost, considered one of the most appreciated fish worldwide. Among swordfish parasites, Anisakis sp. and Hysterothylacium sp. have been used to evaluate biological and ecological aspects of this teleost. Between 2021 and 2022, 364 X. gladius hearts, caught from the Atlantic Ocean (FAO 27.IXa and FAO 34 areas), were collected at the Milan fish market (Lombardy, Italy). Three specimens from FAO 27.IXa was positive for seven adult nematodes (p = 1.55%) within the heart chambers. Of these, three specimens were found within the bulbus arteriosus and 4 in the ventricle. All parasites were stored in 70% ethanol and processed for parasitological and molecular analysis using Cox2, ITS regions/ITS-I-5.8S-ITS-II, and rrnS genes. The analysis allowed us to identify the retrieved parasite as H. incurvum. According to our evaluation, the final localization is due to the movement of L3 larvae from the coelomic cavity to the bloodstream, with consequent development to the adult stage within the heart. Finally, the parasite localization, considered non-marketable fish parts, does not pose a significant risk to consumers, also considering the low zoonotic potential of H. incurvum.

Discrimination between Fresh and Frozen-Thawed Fish Involved in Food Safety and Fraud Protection.

This study aims to discriminate fresh fish from frozen/thawed by identification of the key metabolites that are altered during the freezing/thawing processing. Atlantic salmon (Salmo salar) and bullet tuna (Auxis rochei) were selected as they are representative of broad consumption, and susceptible to pathogen contamination. Atlantic salmon samples were subjected to the following regimes: -20 °C (24h) and -35 °C (15 h) freezing, then thawed respectively in the blast chiller and in the cold room and analyzed immediately or after 10 days; (2) bullet tuna samples were frozen at -18 °C and thawed after 15, 30 and 90 days. High resolution mass spectrometry based on untargeted metabolomic analyses and statistical data treatment confirmed significant variations in the quantity of certain metabolites: the amount of l-phenylalanine in salmon increased immediately after thawing while that of anserine decreased. The concentration of l-arginine and its metabolites was altered at the 10th day after thawing rendering them promising markers of salmon freezing/thawing. As regards bullet tuna, compounds resulting from lipid degradation (l-α-Glyceryl-phosphoryl-choline and N-methyl-ethanolamine phosphate) increased notably during the storage period. This approach could be used to reveal common fraudulent incidents such as deliberate replacement of fresh fish with frozen/thawed, with food safety risks as the primary implication.

Molecular Characterization of Clistobothrium sp. Viable Plerocercoids in Fresh Longfin Inshore Squid (Doryteuthis pealeii) and Implications for Cephalopod Inspection.

Cephalopods, an appreciated seafood product, are common hosts of marine cestodes. The aim of this work is to report visible alive plerocercoids in longfin inshore squid (Doryteuthis pealeii), a cephalopod species commercialized as fresh and whole in Italy. Seventy D. pealeii from the Northwest Atlantic (FAO area 21) were collected and visually inspected. In total, 18 plerocercoid larvae were found in the viscera of 10 host specimens (P: 14.3% 95% CI 7.1-24.7; MI: 1.8, MA: 0.26; range 1-4) and molecularly analyzed targeting the variable D2 region of the large subunit (LSU) rRNA gene and the cytochrome c oxidase subunit I (COI) gene. The molecular characterization allowed to identify all the plerocercoids as Clistobothrium sp., a cestode of the Phyllobothriidae family with Lamnidae sharks as definitive hosts, and cephalopods as second intermediate hosts. These findings represent the first molecular record of Clistobothrium sp. in D. pealeii, thus contributing to elucidate its poorly known life cycle. Even if not affecting consumer's health, these visible parasites may represent a reason for disgust for consumers. Therefore, the results suggest that Food Business Operators should also check for the presence of these visible parasites during inspection and underline the importance of a correct consumers' education.

Exposure to metals and arsenic from yellow and red tuna consumption.

Tuna is one of the most frequently consumed fish and, as a predator, can biomagnify pollutants. Metal and other elements pollution is an important worldwide concern. Based on these considerations, the aim of this work was to investigate the occurrence of As, Cd, Cr, Ni, Hg and Pb in tuna coming from different FAO areas to evaluate human exposure. The analysis was performed on muscle tissues through a quadrupole inductively coupled mass spectrometry. One hundred thirty-one samples were analysed. One red tuna from the Adriatic Sea and 11 yellow tunas exceeded Pb maximum levels (MLs) with a concentration ranging 0.31-0.86 mg kg-1; three red tunas from different Mediterranean sub-areas exceeded Hg MLs, with a concentration range 1.19 to 1.80 mg kg-1. All the Hazard Indexes (HIs) were lower than one, indicating that only a negligible health hazard could derive from the ingestion of tuna, for both average and high consumers. The risk of carcinogenicity from Cr is still under debate at the concentrations detectable in food.

Persistent organic pollutants in fish: biomonitoring and cocktail effect with implications for food safety.

The impact of anthropogenic wastes of persistent organic pollutants (POPs) on the marine environment has increased in the last decades. POPs include polychlorinated biphenyls (PCBs) and organochlorine pesticides (OCs). To assess the levels of these POPs in the wild fish population, pelagic and benthopelagic predator fish species were selected as biomonitors. For detection and quantification of POPs in muscular tissues, a simple extraction through Accelerated-Solvent-Extraction (ASE) with an 'in-line' clean up purification approach was applied, followed by a GC-MS/MS analysis. Concentrations of sum DDT, sum HCH and endrin correlated with all PCB concentrations. Significant differences among fish species were found for all OCs and all PCBs except PCB 31 and 101. Blackspot seabream had the highest PCB concentrations; OCs were highest in tuna. Due to major concerns regarding fish population losses and the possible human chronic exposure to contaminated fish, studies addressing combined effects of multiple POPs ('cocktail effect') should be implemented. Our data motivate further experimental and observational studies in fish to define adequate baseline levels for cumulative human exposure and potential role of these contaminants for food safety.

Risk characterisation from the presence of environmental contaminants and antibiotic residues in wild and farmed salmon from different FAO zones.

Salmon consumption is increasing year by year. Salmon aquaculture is the fastest growing food production system in the world, and often uses feed mixed with antibiotics or other drugs. Feed can be also contaminated by environmental contaminants like persistent organic pollutants and organophosphorus pesticides that usually accumulate in fatty tissue, or emerging contaminants such as perfluoroalkyl substances (PFASs), that instead bioaccumulate in protein tissues. Therefore, there is the need to investigate the presence of antibiotics and environmental contaminants, with multi-class and multi-residue liquid chromatography-high resolution mass spectrometry and gas chromatography tandem mass spectrometry methods to monitor a broad spectrum of residues comparing between wild and farmed salmons. The presence of residues was encountered at a concentration range of 0.35-51.52 ng g-1 for antibiotics only in farmed salmon, 0.19-34.51 ng g-1 for PFASs and 0.26-9.01 ng g-1 for (polybrominated diphenyl ethers) PBDEs, and 0.19-5.91 ng g-1 for organochlorine pesticides with higher frequencies and concentrations in farmed fish. Finally, the risk deriving from salmon intake is low, being of minor concern only for PBDE 99 and perfluorooctanoic acid.

Validated multiclass targeted determination of antibiotics in fish with high performance liquid chromatography-benchtop quadrupole orbitrap hybrid mass spectrometry.

High performance liquid chromatography, coupled with a benchtop Q-Exactive Orbitrap high-resolution mass spectrometer, was successfully applied for the determination of 24 target antibiotics (selected beta-lactams, tetracyclines, fluoroquinolones, sulfonamids, phenicols, macrolides, cephalosporins, lincosamides, diaminopyrimidine) in fish matrices. The Q-Exactive parameters were carefully studied to accomplish the best compromise between a suitable scan speed and selectivity, considering the restrictions associated with generic sample preparation methodology. Retention time, an exact mass with tolerance of 2 ppm and data-dependent MS2 spectra were the main identifiers. The method was validated through specificity, linearity, recovery, intra- and inter-day repeatability, decision limit (CCα) and detection capability (CCß), according to 2002/657/EC. The values of CCα and CCß ranged from 29.2 to 36.8 and 32.5 to 48.9, respectively, while overall recovery ranged from 91.1 to 105.6%. Fifty fish samples were analysed, showing the sporadic incidence of enrofloxacin, chlortetracycline, oxytetracycline, amoxicillin and trimethoprim, albeit below the maximum residual levels.

Food safety traits of mussels and clams: distribution of PCBs, PBDEs, OCPs, PAHs and PFASs in sample from different areas using HRMS-Orbitrap® and modified QuEChERS extraction followed by GC-MS/MS.

Reviewing the presence of contaminant residues is important both for food safety and for monitoring of environmental pollution. Here, the occurrence of 6 polychlorinated biphenyls (PCBs), 15 organochlorine pesticides (OCPs), 7 polybrominated diphenyl ethers (PBDEs), 4 polycyclic aromatic hydrocarbons (PAHs) and 17 perfluoroalkyl substances (PFASs) was evaluated in mussels and clams. A liquid chromatography-high resolution mass spectrometry (HPLC-HRMS) and an innovative QuEChERS extraction followed by gas chromatography-tandem mass spectrometry (GC-MS/MS) methods were developed, validated and applied. We demonstrate good linearity, repeatability and accuracy of these methods, confirming that they are suitable for the analyses of mollusc samples. The prevalence of PCBs, OCPs and PAHs was higher in mussels than in clams. For PFASs, contamination was higher in clams than in mussels. The samples were all compliant with the regulations, and, for the compounds without legislative limits, a risk assessment confirmed that the values were lower than the tolerable intakes.

Occurrence of antibiotics in mussels and clams from various FAO areas.

Filter feeders, like mussels and clams, are suitable bioindicators of environmental pollution. These shellfish, when destined for human consumption, undergo a depuration step that aims to nullify their pathogenic microorganism load and decrease chemical contamination. Nevertheless, the lack of contamination by drugs may not be guaranteed. Antimicrobials are a class of drugs of particular concern due to the increasing phenomenon of antibiotic resistance. Their use in breeding and aquaculture is a major cause of this. We developed a multiclass method for the HPLC-MS/MS analysis of 29 antimicrobials, validated according to the Commission Decision 2002/657/UE guidelines, and applied it to 50 mussel and 50 clam samples derived from various Food and Agricultural Organisation marine zones. The results obtained, indicate a negligible presence of antibiotics. Just one clam sample showed the presence of oxytetracycline at a concentration slightly higher than the European Union Maximum residue limit set for fish.

Endogenous level of acetic acid in yellowfin tuna (Thunnus albacares): a pilot study about a possible controversy on its residue nature.

A method based on headspace solid-phase microextraction (HS-SPME) followed by GC-MS analysis was developed for the determination of underivatised acetic acid in fresh tuna fish muscle. Parameters such as the fibre selected and the extraction time and temperature were optimised and the linearity, detection limits and precision of the whole analytical procedure were assessed. The method was then applied to determine the acetic acid concentration in fresh yellowfin tuna muscles (Thunnus albacares) in order to evaluate the endogenous level and its variations during the shelf life under different storage conditions. A qualitative comparison was also made with variations in histamine levels to evaluate the possibility of the joint monitoring of acetic acid and histamine to identify fish stored in poor conditions. The caudal area always had a lower content of acetic acid than the ventral area, independent of the storage time and temperature. A difference was found between the 6- and 3-day time points and day 0 at a storage temperature of 8°C and between the 6-day time point and day 0 at a storage temperature of 0°C, independent of the anatomical area of the sampled tissue. The evaluation of acetic acid could represent an important approach in the field of food safety to detect the illicit use of acetic acid as an antibacterial preservative treatment or to eliminate the unpleasant smell of trimethylamine.

Evolution of the Anisakis risk management in the European and Italian context.

Due to the social and legislative implications, the presence of Anisakis spp. larvae in fishery products has become a concern for both the consumers and the official Control Authorities. The issuance of a large number of provisions, aimed at better managing fish products intended to be consumed raw or almost raw and the associated risks, resulted in a very complicate legal framework. In this work, we analyzed the evolution of the normative through an overview on the local and international legislations, focusing on issues that are of practical interest for Food Business Operators (FBOs) in the fishery chain. In addition, we performed a survey across the Department of Prevention of the Italian Local Health Authorities (LHA) and the main fish markets in Italy to collect the operating procedures and the monitoring plans. Overall, we found many differences, due to the absence of a national reference standard for the management of the Anisakis risk. From this examination, it turns clear that only a participation of all the involved institutions, a strategy of synergistic interventions, as well as a correct training of FBOs, can result in an effective risk management and a proper risk communication, which should overcome states of confusion and unnecessary negative impacts on the economy.

Authentication of farmed and wild turbot (Psetta maxima) by fatty acid and isotopic analyses combined with chemometrics.

Fatty acid composition and stable isotope ratios of carbon (delta(13)C) and nitrogen (delta(15)N) were determined in muscle tissue of turbot (Psetta maxima). The multivariate analysis of the data was performed to evaluate their utility in discriminating wild and farmed fish. Wild (n=30) and farmed (n=30) turbot of different geographical origins (Denmark, The Netherlands, and Spain) were sampled from March 2006 to February 2007. The application of linear discriminant analysis (LDA) and soft independent modeling of class analogy (SIMCA) to analytical data demonstrated the combination of fatty acids and isotopic measurements to be a promising method to discriminate between wild and farmed fish and between wild fish of different geographical origin. In particular, IRMS (Isotope Ratio Mass Spectrometry) alone did not permit us to separate completely farmed from wild samples, resulting in some overlaps between Danish wild and Spanish farmed turbot. On the other hand, fatty acids alone differentiated between farmed and wild samples by 18:2n-6 but were not able to distinguish between the two groups of wild turbot. When applying LDA isotope ratios, 18:2n-6, 18:3n-3, and 20:4n-6 fatty acids were decisive to distinguish farmed from wild turbot of different geographical origin, while delta(15)N, 18:2n-6, and 20:1n-11 were chosen to classify wild samples from different fishing zones. In both cases, 18:2n-6 and delta(15)N were determinant for classification purposes. We would like to emphasize that IRMS produces rapid results and could be the most promising technique to distinguish wild fish of different origin. Similarly, fatty acid composition could be used to easily distinguish farmed from wild samples.