Background: An ultrahigh-performance LC (UHPLC)–tandem MS (MS/MS) method for determination of paralytic shellfish poisoning toxins and tetrodotoxin (TTX) in bivalve molluscs was developed. To be used for regulatory testing, it needed to be validated through collaborative study. Objective: The aim was to conduct a collaborative study with 21 laboratories, using results to assess method performance. Methods: Study materials incorporated shellfish species mussels, oysters, cockles, scallops, and clams and were assessed to demonstrate stability and homogeneity. Mean concentrations determined by participants for blind duplicate samples were used to assess reproducibility, repeatability, and trueness. Results: Method performance characteristics were excellent following statistical assessment of participant data, with method trueness showing excellent method accuracy against expected values. No significant difference was found in the trueness results determined by different chromatographic column types. Acceptability of the between-laboratory reproducibility for individual analytes was evidenced by >99% of valid Horwitz ratio values being less than the 2.0 limit of acceptability. With excellent linearity and sensitivity fit-for-purpose over a range of mass spectrometer instruments, the UHPLC-MS/MS method compared well against other detection methods. It includes additional paralytic shellfish toxin (PST) analogues as well as TTX, which, to date, have not been incorporated into any other hydrophilic marine toxin official method of analysis. Conclusions: The results from this study demonstrate that the method is suitable for the analysis of PST analogues and TTX in shellfish tissues and is recommended as an official alternative method of analysis for regulatory control. Highlights: A new mass spectrometric method for PST and TTX has been validated successfully through collaborative study.

In 2015, tetrodotoxins (TTXs) were considered a potential threat in Europe since several studies had shown the presence of these toxins in European bivalve molluscs. In this study, we investigated the occurrence of TTXs in 127 bivalve samples (mussels and oysters) and in 66 gastropod samples (whelks) collected all along the French mainland coasts in 2017 and 2018. Analyses were carried out after optimization and in-house validation of a performing hydrophilic interaction liquid chromatography associated with tandem mass spectrometry (HILIC-MS/MS) method. The concentration set by European Food Safety Authority (EFSA) not expected to result in adverse effects (44 µg TTX equivalent/kg) was never exceeded, but TTX was detected in three mussel samples and one whelk sample (1.7–11.2 µg/kg). The tissue distribution of TTX in this whelk sample showed higher concentrations in the digestive gland, stomach and gonads (7.4 µg TTX/kg) than in the rest of the whelk tissues (below the limit of detection of 1.7 µg TTX/kg). This is the first study to report the detection of TTX in French molluscs.

Pinnatoxins (PnTXs) are a group of emerging marine biotoxins produced by the benthic dinoflagellate Vulcanodinium rugosum, currently not regulated in Europe or in any other country in the world. In France, PnTXs were detected for the first time in 2011, in mussels from the Ingril lagoon (South of France, Mediterranean coast). Since then, analyses carried out in mussels from this lagoon have shown high concentrations of PnTXs for several months each year. PnTXs have also been detected, to a lesser extent, in mussels from other Mediterranean lagoons and on the Atlantic and Corsican coasts. In the French data, the main analog is PnTX G (low levels of PnTX A are also present in some samples). No cases of PnTXs poisoning in humans have been reported so far in France or anywhere else in the world. In mice, PnTXs induce acute neurotoxic effects, within a few minutes after oral administration. Clinical signs of toxicity include decreased mobility, paralysis of the hind legs, tremors, jumps and breathing difficulties leading to death by respiratory arrest at high doses. The French agency for food safety (ANSES) recently conducted a review of the state of knowledge related to PnTXs and V. rugosum. Based on (i) the clinical signs of toxicity in mice, (ii) the mode of action of PnTXs as nicotinic acetylcholine receptor competitive antagonists and (iii) knowledge on drugs and natural toxins with PnTX-related pharmacology, potential human symptoms have been extrapolated and proposed. In this work, a provisional acute benchmark value for PnTX G of 0.13 μg/kg bw per day has been derived from an oral Please note that this is an author-produced PDF of an article accepted for publication following peer review. The definitive publisher-authenticated version is available on the publisher Web site.acute toxicity study in mice. Based on this value and a large shellfish meat portion size of 400g, a concentration lower than 23 μg PnTX G/kg shellfish meat is not expected to result in adverse effects in humans. ANSES recommends taking into account PnTXs in the French official monitoring program for shellfish production and identified data gaps to refine health risk assessment. Highlights► A state of knowledge of PnTXs and Vulcanodinium rugosum is proposed. ► A provisional acute healthbased guidance value for PnTX G of 0.13 μg/kg bw per day has been derived. ► PnTXs in shellfish should not exceed 23 μg PnTX G/kg. ► PnTXs should be included in national official monitoring programs for shellfish production. ► This work is a major step forward in taking into account this emerging hazard.

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When considering the geographical expansion of marine toxins, the emergence of new toxins and the associated risk for human health, there is urgent need for versatile and efficient analytical methods that are able to detect a range, as wide as possible, of known or emerging toxins. Current detection methods for marine toxins rely on a priori defined target lists of toxins and are generally inappropriate for the detection and identification of emerging compounds. The authors describe the implementation of a recent approach for the non-targeted analysis of marine toxins in shellfish with a focus on a comprehensive workflow for the acquisition and treatment of the data generated after liquid chromatography coupled with high resolution mass spectrometry (LC-HRMS) analysis. First, the study was carried out in targeted mode to assess the performance of the method for known toxins with an extended range of polarities, including lipophilic toxins (okadaic acid, dinophysistoxins, azaspiracids, pectenotoxins, yessotoxins, cyclic imines, brevetoxins) and domoic acid. The targeted method, assessed for 14 toxins, shows good performance both in mussel and oyster extracts. The non-target potential of the method was then challenged via suspects and without a priori screening by blind analyzing mussel and oyster samples spiked with marine toxins. The data processing was optimized and successfully identified the toxins that were spiked in the blind samples.