Exploring Perfluoroalkyl Substances (PFASs) in Aquatic Fauna of Lake Trasimeno (Italy): Insights from a Low-Anthropized Area.

This study investigated the concentrations and profiles of 19 perfluoroalkyl substances (PFASs) in the muscle and liver of four freshwater species from Lake Trasimeno (Italy): Anguilla anguilla (European eel), Carassius auratus (goldfish), Perca fluviatilis (European perch), and Procambarus clarkii (red swamp crayfish). In livers, the amount of PFASs ranged from 3.1 to 10 µg kg-1, significantly higher than that in muscle (0.032-1.7 µg kg-1). The predominant PFASs were perfluorooctane sulfonic acid (PFOS) and long-chain carboxylic acids (C8-C14). Short-chain compounds (C4-C5), as well as the long-chain sulfonic acids (C9-C12), were not quantified. The contamination patterns were similar among species with few differences, suggesting the influence of species-specific accumulation. The PFAS concentrations in livers were comparable among species, while in muscle, the higher values were measured in European eel, followed by goldfish, European perch, and red swamp crayfish. The levels were generally lower than those reported for fish from Northern Italian lakes and rivers. The concentrations of regulated PFASs were lower than the maximum limits set by Regulation EU 2023/915 and did not exceed the Environmental Quality Standards (PFOS in biota). This study provides the first valuable insights on PFASs in freshwater species from Lake Trasimeno.

Brominated flame retardants (PBDEs and HBCDs) and perfluoroalkyl substances (PFASs) in wild boars (Sus scrofa) from Central Italy.

Twenty-six samples of wild boar liver and muscle from the Central Apennine Mountain (Italy) were analysed for 19 perfluoro-alkyl substances (PFASs), 10 polybrominated diphenylethers (PBDEs) and 3 hexabromocyclododecanes (HBCDs). All samples were analysed by gas chromatography-tandem mass spectrometry for PBDEs and liquid chromatography-tandem mass spectrometry for PFASs and HBCDs, using an in-house developed analytical procedure. The brominated flame retardants (BFR) levels in livers were negligible: Σ10PBDEs reached a maximum value of 0.079 µg/kg, whereas HBCDs were not quantified in almost all of the samples analysed. BFR concentrations in muscles were higher, but not significantly therefore, for Σ10PBDEs lower bound, a mean value of 0.045 µg/kg (0.005-0.155 µg/kg range) was measured, while α-HBCD was quantified with a maximum of 0.084 µg/kg in 9 of the samples. Only two muscles contained all 3 HBCD isomers at concentrations of approximately 0.200 µg/kg. Σ19PFAS in the 26 wild boar livers was in the range 31.9-228 µg/kg, with a mean value of 87.7 µg/kg, reaching levels significantly higher than in muscles, which exhibited a mean concentration of 3.08 µg/kg (0.59-9.12 µg/kg range). Perfluorooctanesulfonic acid (PFOS) was the most prevalent compound in all liver samples, accounting for more than half of the total PFASs contamination, confirming that the liver is the primary target organ for PFOS exposure Perfluorotridecanoic acid (PFTrDA), which accounts for 25-30-% of the total contamination, was the most abundant compound in the muscle, followed by PFOS. The estimated daily intake (EDIs) of BFRs remained below the estimated chronic human daily dietary intake (Dr,h) defined from European Food Safety Authority (EFSA). Furthermore, the exposure to PFASs in muscle was 7.7 times lower than the EFSA's tolerable daily intake (TDI). In contrast, exposure due to liver consumption was significant: the EDI exceeded the EFSA's 2020 TDI by approximately 7 times.

A High Throughput Screening HPLC-FLD Method for Paralytic Shellfish Toxins (PSTs) Enabling Effective Official Control.

Paralytic Shellfish Toxins (PSTs) are marine biotoxins, primarily produced by dinoflagellates of the genera Gymnodinium spp., Alexandrium spp. They can accumulate in shellfish and, through the food chain, be assimilated by humans, giving rise to Paralytic Shellfish Poisoning. The maximum permitted level for PSTs in bivalves is 800 µg STX·2HCl eqv/kg (Reg. EC N° 853/2004). Until recently, the reference analytical method was the Mouse Bioassay, but Reg. EU N° 1709/2021 entered into force on 13 October 2021 and identified in the Standard EN14526:2017 or in any other internationally recognized validated method not entailing the use of live animals as official methods. Then the official control laboratories had urgently to fulfill the new requests, face out the Mouse Bioassay and implement instrumental analytical methods. The "EURLMB SOP for the analysis of PSTs by pre-column HPLC-FLD according to OMA AOAC 2005.06" also introduced a simplified semiquantitative approach to discriminate samples above and below the regulatory limit. The aim of the present paper is to present a new presence/absence test with a cut-off at 600 µg STX·2HCl eqv/kg enabling the fast discrimination of samples with very low PSTs levels from those to be submitted to the full quantitative confirmatory EN14526:2017 method. The method was implemented, avoiding the use of a large number of certified reference standards and long quantification procedures, resulting in an efficient, economical screening instrument available for official control laboratories. The protocol was fully validated, obtaining good performances in terms of repeatability (<11%) and recovery (53−106%) and accredited according to ISO/IEC 17025. The method was applied to mollusks collected from March 2021 to February 2022 along the Marche region in the frame of marine toxins official control.

Perfluoroalkyl and polyfluoroalkyl substances (PFASs): An optimized LC-MS/MS procedure for feed analysis.

Perfluoroalkyl and polyfluoroalkyl substances (PFASs) are extremely stable highly fluorinated aliphatic compounds, widely used in chemical and industrial applications since 1950s. They are ubiquitously detected in the environment and diet is the main route for human exposure. To ensure human safety, it is necessary to follow the whole food production chain, including animal feed. Still PFASs are not regulated as undesirable substances in feed, although several studies have shown they can be transferred from feed to livestock and thereafter to food. This paper describes the development, optimization and the full validation of a sensitive and reliable analytical protocol enabling the quantification of 19 PFASs in animal feeds by liquid chromatography - mass spectrometry (LC-MS/MS). The method was optimized, assessing the possible matrix interferences, and submitted to comprehensive validation (55 independent spiking experiments). Validation experiments were conducted on blank fish feed samples (natural levels of PFASs < 0.10 ng g-1) spiked at five different concentrations (0.10, 0.50, 1.0, 5.0 and 10 ng g-1). Apparent recoveries (R%) were generally between 88 and 111%; R% < 80% were obtained only at the lower validation levels for those molecules not having the corresponding labelled analogues. Relative standard deviations in repeatability conditions (RSDr) and within-lab reproducibility conditions (RSDwR) were lower or equal to 11% and 22% respectively. Limits of quantification were set for most of the analytes at 0.10 ng g-1 (LOQs) and verified with repeated analysis on fortified samples (0.10 ng g-1). Limits of detection (LODs) were calculated as 1/3 LOQ. Finally the method was applied to 23 feed samples of different type (compound feed, vegetable feed material, complementary feed, pre-mixture and mineral feed) and all of them did not show PFAS above LOQ.

Inorganic Elements in Mytilus galloprovincialis Shells: Geographic Traceability by Multivariate Analysis of ICP-MS Data.

The international seafood trade is based on food safety, quality, sustainability, and traceability. Mussels are bio-accumulative sessile organisms that need regular control to guarantee their safe consumption. However, no well-established and validated methods exist to trace mussel origin, even if several attempts have been made over the years. Recently, an inorganic multi-elemental fingerprint coupled to multivariate statistics has increasingly been applied in food quality control. The mussel shell can be an excellent reservoir of foreign inorganic chemical species, allowing recording long-term environmental changes. The present work investigates the multi-elemental composition of mussel shells, including Al, Cu, Cr, Zn, Mn, Cd, Co, U, Ba, Ni, Pb, Mg, Sr, and Ca, determined by inductively-coupled plasma mass-spectrometry in Mytilus galloprovincialis collected along the Central Adriatic Coast (Marche Region, Italy) at 25 different sampling sites (18 farms and 7 natural banks) located in seven areas. The experimental data, coupled with chemometric approaches (principal components analysis and linear discriminant analysis), were used to create a statistical model able to discriminate samples as a function of their production site. The LDA model is suitable for achieving a correct assignment of >90% of individuals sampled to their respective harvesting locations and for being applied to counteract fraud.

Heavy metal spatial and temporal trends (2008-2018) in clams and mussel from Adriatic Sea (Italy): Possible definition of forecasting models.

In 2008-2018, 1458 georeferenced samples of clams and 343 of mussels were harvested in Italy from classified areas along Marche coast and analyzed within food-safety monitoring plans. Pb, Cd, V, Ni, Cr, and As median levels (mg kg-1) were 0.09, 0.08, 0.29, 0.77, 0.35, and 2.35 in clams and 0.16, 0.15, 0.46, 0.48, 0.25, and 3.34 in mussels. The reported levels were comparable with the published ones, and Hg always fell below LOQ (0.025 mg kg-1). Pb and Cd datasets, based on quarterly results, show a decreasing trend in clams and constant baseline value for mussels. Time-trends and forecasting models represent an asset to predict emerging food safety/security risks. The metal levels in mussels and clams from the same area allow environmental safety assessment and thus enable the evaluation of contaminants in both water column and sediment. Monitoring of elements that are not regulated may help in preserving food and environmental safety.

Cyclic Imines (CIs) in Mussels from North-Central Adriatic Sea: First Evidence of Gymnodimine A in Italy.

Cyclic imines (CIs) are emerging marine lipophilic toxins (MLTs) occurring in microalgae and shellfish worldwide. The present research aimed to study CIs in mussels farmed in the Adriatic Sea (Italy) during the period 2014-2015. Twenty-eight different compounds belonging to spirolides (SPXs), gymnodimines (GYMs), pinnatoxins (PnTXs) and pteriatoxins (PtTXs) were analyzed by the official method for MLTs in 139 mussel samples collected along the Marche coast. Compounds including 13-desmethyl spirolide C (13-desMe SPX C) and 13,19-didesmethyl spirolide C (13,19-didesMe SPX C) were detected in 86% of the samples. The highest levels were generally reported in the first half of the year reaching 29.2 µg kg-1 in January/March with a decreasing trend until June. GYM A, for the first time reported in Italian mussels, was found in 84% of the samples, reaching the highest concentration in summer (12.1 µg kg-1). GYM A and SPXs, submitted to tissue distribution studies, showed the tendency to accumulate mostly in mussel digestive glands. Even if SPX levels in mussels were largely below the European Food Safety Authority (EFSA) reference of 400 µg SPXs kg-1, most of the samples contained CIs for the large part of the year. Since chronic toxicity data are still missing, monitoring is surely recommended.

Multimycotoxin Analysis by LC-MS/MS in Cereal Food and Feed: Comparison of Different Approaches for Extraction, Purification, and Calibration.

Twelve different approaches commonly used for the simultaneous LC tandem MS (MS/MS) determination of mycotoxins (deoxynivalenol, aflatoxins, ochratoxin A, T-2 and HT-2 toxins, fumonisins, and zearalenone) were tested in cereals and feed materials. They comprised different extraction solvents, types of cleanup [solid-phase extraction, QuEChERS, and immunoaffinity (IMA)], and calibration approaches (external or matrix-matched). The percentage of mycotoxins with acceptable recovery, according to Regulation (EC) No. 401/2006, ranged from 9 to 100%. The approach giving the highest percentage of acceptable results was selected and further tested for corn, rice, and feed spiked at three different mycotoxin levels (low, medium, and high). The method is based on extraction with MeOH-water (70 + 30, v/v) and cleanup with two multiantibody IMA columns. For corn and rice spiked at low mycotoxin levels, a significant matrix effect was observed and was compensated by using 13C calibration. At higher mycotoxin levels (medium and high), matrix effects were negligible as no significant differences were observed for the majority of recovery results calculated by 13C calibration and external calibration. Although the proposed method still needs improvement in terms of accuracy and, to a lesser extent, precision, it was successfully tested with four proficiency tests in buckwheat, corn, rice, and feed, giving acceptable z-scores for 97% (34 out of 35) of results.