Diatoms of the genus Pseudo-nitzschia H.Peragallo are known to produce domoic acid (DA), a toxin involved in amnesic shellfish poisoning (ASP). Strains of the same species are often classified as both toxic and nontoxic, and it is largely unknown whether this difference is also genetic. In the Northern Adriatic Sea, there are virtually no cases of ASP, but DA occasionally occurs in shellfish samples. So far, three species—P. delicatissima (Cleve) Heiden, P. multistriata (H. Takano) H. Takano, and P. calliantha Lundholm, Moestrup, & Hasle—have been identified as producers of DA in the Adriatic Sea. By means of enzme-linked immunosorbent assay (ELISA), high-performance liquid chromatography with UV and visible spectrum detection (HPLC-UV/VIS), and liquid chromatography with tandem mass spectrometry (LC-MS/MS), we reconfirmed the presence of DA in P. multistriata and P. delicatissima and detect for the first time in the Adriatic Sea DA in P. galaxiae Lundholm, & Moestrup. Furthermore, we attempted to answer the question of the distribution of DA production among Pseudo-nitzschia species and strains by sequencing the internal transcribed spacer (ITS) phylogenetic marker and the dabA DA biosynthesis gene and coupling this with toxicity data. Results show that all subclades of the Pseudo-nitzschia genus contain toxic species and that toxicity appears to be strain dependent, often with geographic partitioning. Amplification of dabA was successful only in toxic strains of P. multistriata and the presence of the genetic architecture for DA production in non-toxic strains was thus not confirmed.

Yessotoxins (YTXs) are polycyclic toxic ether compounds produced by phytoplanktonic dinoflagellates which accumulate in filter-feeding organisms. We know that the water temperature in our areas Northwestern Adriatic Sea is optimal for the growth of potentially toxic algae (around 20 °C). In recent years, these temperatures have remained at these levels for longer and longer periods, probably due to global warming, which has led to an excessive increase in toxin levels. The interruption of mussel harvesting caused by algae negatively affects farmers’ revenues and the availability of local fish, causing a major economic loss in Italy’s main shellfish sector. Methods: In the nine years considered, 3359 samples were examined: 1715 marine waters, 73 common clams; 732 mussels; 66 oysters; and 773 veracious clams. Bivalve molluscs were examined for the presence of marine biotoxins, including YTXs, while potentially toxic algae, including those producing YTXs, were searched for and counted in marine waters. The method adopted for the quantification of lipophilic toxins involves the use of an LC-MS/MS system. The enumeration of phytoplankton cells was performed according to the Utermhöl method. Results: Between 2012 and 2020, 706 molluscs were tested for YTXs. In total, 246 samples tested positive, i.e., 34.84%. Of the positive samples, 30 exceeded the legal limit. Conclusion: In this regard, it is essential to develop and activate, as soon as possible, an “early warning” system that allows a better control of the production areas of live bivalve molluscs, thus allowing an optimal management of the plants in these critical situations.

Background: Given the recent detection of tetrodotoxin (TTX) in bivalve molluscs but the absence of a full collaborative validation study for TTX determination in a large number of shellfish samples, interlaboratory assessment of method performance was required to better understand current capabilities for accurate and reproducible TTX quantitation using chemical and immunoassay methods.Objective: The aim was to conduct an interlaboratory study with multiple laboratories, using results to assess method performance and acceptability of different TTX testing methods.Methods: Homogenous and stable mussel and oyster materials were assessed by participants using a range of published and in-house detection methods to determine mean TTX concentrations. Data were used to calculate recoveries, repeatability, and reproducibility, together with participant acceptability z-scores.Results: Method performance characteristics were good, showing excellent sensitivity, recovery, and repeatability. Acceptable reproducibility was evidenced by HorRat values for all LC–MS/MS and ELISA methods being less than the 2.0 limit of acceptability. Method differences between the LC–MS/MS participants did not result in statistically different results. Method performance characteristics compared well with previously published single-laboratory validated methods and no statistical difference was found in results returned by ELISA in comparison with LC–MS/MS.Conclusion: The results from this study demonstrate that current LC–MS/MS methods and ELISA are on the whole capable of sensitive, accurate, and reproducible TTX quantitation in shellfish. Further work is recommended to expand the number of laboratories testing ELISA and to standardize an LC–MS/MS protocol to further improve interlaboratory precision.Highlights: Multiple mass spectrometric methods and a commercial ELISA have been successfully assessed through an interlaboratory study, demonstrating excellent performance.

Pufferfish (Tetraodontidae) inhabiting the Mediterranean Sea may represent an emerging public health risk due to the possible accumulation of marine neurotoxins such as tetrodotoxin (TTXs) and saxitoxin (STXs) in their tissues. In this study, the presence of pufferfish species in the Strait of Sicily (Lampedusa Island, Italy) was investigated using a citizen science (CS) approach, involving local fishermen. Samples (liver, intestine, gonads, muscle, skin) from 20 specimens were sent to the National Reference Laboratory on Marine Biotoxins for TTXs detection using a validated HILIC-MS/MS method on fish tissue. The presence of STXs was also screened in part of the specimens. Overall, 56 specimens identified as Sphoeroides pachygaster (Müller &Troschel, 1848) were collected. Data on their total length, body weight, fishing method and catch area (with relative depth temperature and salinity) were analyzed and compared with the S. pachygaster records reported in literature which were updated to 2022. All the analysed tissues were found to be negative for both TTXs and STXs. CS played an essential role in monitoring potentially toxic marine species in this investigation. Outcomes from this study, which is the first investigating S. pachygaster toxicity in Italian waters, may provide useful data for the proper assessment of this emerging risk.

This study reports the first detection of the marine neurotoxin pinnatoxin-G (PnTX-G) in clams collected in the northwestern Adriatic Sea (Italy). It also represents the first report of the potential toxin-producing dinoflagellate, Vulcanodinium rugosum, in Italian seas. This result, from the coasts of the Emilia-Romagna Region, indicates a successful colonization process, reflecting conditions in France where V. rugosum was initially documented. In this case, the concentration of PnTXs was very low, making further sampling necessary to fully understand the extent of the phenomenon. Discussions on the need to obtain more data to support a proper risk assessment and the need to implement a monitoring program that includes emerging marine biotoxins are also included.

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.