Tetrodotoxins (TTXs), potent neurotoxins, have become an increasing concern in Europe in recent decades, especially because of their presence in mollusks. The European Food Safety Authority published a Scientific Opinion setting a recommended threshold for TTX in mollusks of 44 µg equivalent kg−1 and calling all member states to contribute to an effort to gather data in order to produce a more exhaustive risk assessment. The objective of this work was to assess TTX levels in wild and farmed mussels (Mytilus galloprovincialis) harvested in 2018–2019 along the coastal area of the Marche region in the Central Adriatic Sea (Italy). The presence of Vibrio spp. carrying the non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) genes, which are suspected to be involved in TTX biosynthesis, was also investigated. Out of 158 mussel samples analyzed by hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry (HILIC-MS/MS), 11 (7%) contained the toxins at detectable levels (8–26 µg kg−1) and 3 (2%) contained levels above the EFSA safety threshold (61–76 µg kg−1). Contaminated mussels were all harvested from natural beds in spring or summer. Of the 2019 samples, 70% of them contained V. alginolyticus strains with the NRPS and/or PKS genes. None of the strains containing NRPS and/or PKS genes showed detectable levels of TTXs. TTXs in mussels are not yet a threat in the Marche region nor in Europe, but further investigations are surely needed.

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.

No abstract

Tetrodotoxins (TTXs), the pufferfish venom traditionally associated with Indo-Pacific area, has been reported during last decades in ever wider range of marine organisms and ever more geographical areas, including shellfish in Europe. Wild mussels (Mytilus galloprovincialis) grown in the Marche Region (N Adriatic Sea, Italy) were shown to be prone to TTX contamination during the warm season, with a suspected role of Vibrio alginolyticus characterized by non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS)-encoding genes. This work aimed to deepen the knowledge about the toxin’s origin and the way through which it accumulates in mussels. A two-year study (spring–summer 2020–2021) confirmed the recurrent presence of TTX (11–68 µg kg-1) in the official monitored natural mussel beds of the Conero Riviera. During 2021, a supplementary nonroutine monitoring of a natural mussel bed in the same area was carried out weekly from June until August for TTXs and/or the presence of V. alginolyticus. Biotic (mussels, mesozooplankton, worms and phytoplankton); abiotic (water and sediment) matrices and phytoplankton assemblage characterizations were studied. Mussels showed relevant TTX contamination levels (9–296 µg kg-1) with extremely rapid TTX accumulation/depletion rates. The toxin presence in phytoplankton and its distribution in the different mussel tissues supports its possible exogenous origin. The V. alginolyticus count trend overlaps that of TTX contamination in mussels, and similar trends were reported also for some phytoplankton species. The role of V. alginolyticus carrying NRPS or PKS genes as a possible TTX source and of phytoplankton as a “potential vector” should therefore be further investigated.

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.