Monica Cangini

The main objective of the present study was to assess the contamination level and nature of microplastics and their role as vectors of trace elements in Mediterranean mussels (Mytilus galloprovincialis). A total of 39 samples were collected from four farming areas (1–4) located in Sardinia (Italy) and selected according to the principles of the risk assessment. The concentrations of 22 trace elements (Be, Tl, Sb, Ag, Hg, Th, U, Sn, Co, Cd, Li, Mo, Cr, Ba, V, Ni, Pb, Se, Mn, Cu, As, Zn) in mussels and plastic particles extracted from mussels was determined by inductively coupled plasma mass spectrometry (ICP-MS/MS). The isolated plastic particles were characterized by Fourier Transform Infrared Spectroscopy (µ-FTIR). All samples were compliant with the European Union official limits set for Hg, Cd and Pb (Reg. UE 915/2023). Plastic particles were identified in all the samples with a total of 1007 isolated microfibers. Qualitative characterization of microplastics showed that natural polymers predominated, accounting for over 85% of the isolated microfibers. Cellulose was the most abundant polymer (55.29%), followed by cotton (30.59%). Synthetic polymers were present only as a minor fraction (~15%). These results support the use of Mytilus galloprovincialis as a reliable bioindicator of microplastics pollution. Heavy metal concentrations associated with microplastics ingested by Mytilus galloprovincialis were markedly lower than those measured directly in the edible tissues of the mussels, highlighting that microplastics likely contribute minimally as vectors of metals to human consumers. The results of the present study allowed for a comprehensive evaluation of both microplastics contamination and the distribution of heavy metals in mussels reared in Sardinia, providing a robust framework for subsequent risk assessment studies.

Tetrodotoxin (TTX) is a potent marine neurotoxin responsible for severe seafood poisoning in humans, characterized by neurological symptoms that may be fatal. Originally identified as a natural contaminant of pufferfish (Tetraodontidae family), over the last few years TTX and its analogs have also been detected in other edible marine organisms, including mollusks, gastropods and crustaceans. Consequently, there is a need for rapid, sensitive, and reliable methods for TTX detection in seafood. In this study, a functional assay based on the use of mouse neuroblastoma Neuro-2a cells has been optimized and characterized for TTX detection in mussels. The assay is based on the toxin’s ability to block voltage-gated sodium channels, thereby counteracting the sodium-dependent cytotoxicity induced by veratridine and ouabain. The linear range of the TTX standard curve fell between 0.44 and 33 ng/mL, with limits of TTX detection (LOD) and quantitation (LOQ) of 0.132 ng/mL and 0.439 ng/mL, respectively, and good intra- and inter-day repeatability (RSDr= 15 and 11%, respectively). The assay also detected saxitoxin, which shares the same mechanism of action as TTX, but was less sensitive towards 4,9-anhydro-TTX. The minimum mussel extract dilution of 1:100 did not result in matrix-related interference, allowing accurate TTX quantitation, with a LOQ of 0.54 µg TTX equivalents/kg mussel meat. Given its sensitivity, the optimized Neuro-2a assay represents a promising tool for toxicity-based TTX quantitation in mussels before their consumption.

The detection of tetrodotoxins (TTXs) in European shellfish led the European Union to request a risk assessment from the European Food Safety Authority (EFSA). EFSA set a reference limit of 44 µg TTX equivalents kg⁻¹ and called for more data on TTX occurrence, especially in gastropods, which can accumulate in TTXs but remain poorly studied. Recently, preliminary monitoring has revealed the recurrent presence of TTXs in mussels in three areas along the North–Central Adriatic coast of Italy, while research on non-bivalve organisms has not yet been carried out. This study presents a preliminary survey, conducted from January 2023 to March 2025, on the presence of TTXs in gastropods, echinoderms, and arthropods collected from this area. A method in Hydrophilic Interaction Liquid Chromatography coupled with tandem Mass Spectrometry (HILIC-MS/MS) for detecting TTXs in bivalve mollusks was first tested through an international proficiency test, then optimized for the other invertebrates, the object of this study. TTX levels in all gastropods and arthropod samples were undetectable, while traces (~5 µg kg⁻¹) were found in one echinoderm sample (Paracentrotus lividus), marking the first reported occurrence of TTX in this species. Sea urchins are widely consumed in Italy; therefore, this finding is of particular importance from a public health perspective and deserves further investigation. Some gastropod genera or species sampled (e.g., Nassarius, Rapana venosa) have been identified as TTX carriers in other regions; therefore, the negative results obtained in this study may be related to seasonal or geographic variability. These results provide valuable data to EFSA’s call for monitoring emerging risks, particularly as climate change may increase TTX prevalence in European waters as well as worldwide.

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.

Background: Saxitoxin (SXT) can cause the paralytic shellfish poisoning (PSP) with severe impacts on both human health and marine ecosystems. Dinoflagellates are the major marine producers of STX and other variety of secondary toxic metabolites that can gather in molluscs becoming a risk for consumers’ poisoning. The aim of this work was to investigate the presence of the main algal species producing SXT in an area intended for molluscs breeding. Methods: During 2020-2023, 40 seawater samples were collected from two macro-areas in the Adriatic Sea (North-Western) at different distances from the coast. After water filtration, DNA was extracted (DNeasy PLANT Mini Kit) and analysed with Real Time PCR for Alexandrium minutum and Gymnodinium catenatum detection. The possible ability to produce SXT was confirmed by sxt4A gene detection. Molecular results were compared with the microscopy gold-standard method. Results: A. minutum was detected in 62.5% (CI 95% 0.47-0.75) of the samples, with 84% (CI 95% 0.65-0.94) of them possibly producing SXT. G. catenatum was found in 95% (CI 95% 0.83-0.99) of samples, with 73.7% (CI 95% 0.58-0.85) of them presenting STX gene. By microscopy, only Alexandrium spp. were identified without successful species identification. No other saxitoxin-producing genera or species were observed. Conclusions: Our results underlined that A. minutum and G. catenatum can play an important role in the production of SXT, confirming their harmful presence in the areas intended for molluscs breeding. Microscopic identification can lead to an underestimation of the presence of potentially toxic algal species, stressing the relevance of improving molecular tools also in this field. These findings highlighted the importance of reporting and monitoring toxic algae presence, given the saxitoxin impact for food safety, and its significance for public health. Key messages: • Saxitoxin-producing algae detection through molecular methods in molluscs breeding areas may represent an early warning useful to improve food safety and human health. • Harmful algal monitoring can improve water management in a One Health perspective.