Tetrodotoxin in live bivalve mollusks from Europe: Is it to be considered an emerging concern for food safety?

Abstract

Tetrodotoxins (TTXs) are a group of potent neurotoxins named after the Tetraodontidae fish family (pufferfish). TTXs have been reported in several animal taxa, both terrestrial and marine. The ingestion of TTX‐contaminated flesh can cause serious neurotoxic symptomatology and can eventually lead to death. Traditionally, TTXs have been associated with Asian countries, in particular with pufferfish consumption. However, they have also been reported in bivalve mollusks farmed in the Pacific area and, recently, in European seas. In Europe, different countries have reported TTXs, especially those bordering the Mediterranean Sea. As a consequence, in 2017 the European Food Safety Authority (EFSA) released an opinion with reference to TTX present in marine gastropods and bivalves, proposing a safety limit of 44 µg/kg TTXs in shellfish meat, below which no adverse effects should be observed in humans. Nevertheless, this limit has been exceeded on many occasions in European shellfish and, while for bivalves there have been no registered human intoxications, that is not the case for marine gastropods. However, TTXs have not yet been included in the list of marine biotoxins officially monitored in live bivalve mollusks within the European Union (EU). Thus, the aims of this manuscript are to discuss the increasing occurrence of TTXs in live bivalve mollusks from European sea waters, to acknowledge the still ongoing knowledge gaps that should be covered and to stimulate constructive debate on the eventuality of adopting a shared regulatory context, at least in the EU, for monitoring and managing this potential threat to food safety.

Interlaboratory Evaluation of Multiple LC–MS/MS Methods and a Commercial ELISA Method for Determination of Tetrodotoxin in Oysters and Mussels

“…Method sensitivities, as quantified by LOD and LOQ, varied enormously, but such data is hard to standardize for MS/MS detection methods, given the notable variation in the algorithms utilized by instrument software for calculating S/N. Notably, LODs quoted by participants in this study were higher than those reported by four previous SLV studies ( 36 , 39 , 40 , 45 ). However, other than the initial validation which was targeting quantitation of TTX at a higher order of magnitude in sea slugs [mg/kg ( 23 )], all methods published and/or utilized in this study are capable of quantifying at concentrations well below the EFSA recommended threshold of 44 µg/kg ( Table 8 ) and consequently are appropriate for regulatory control purposes.…”

“…Whilst numerous reports of TTX in seafood products have been published in recent years using LC–MS/MS, a relatively low number of these methods have been subjected to full validation studies. Table 8 summarizes the main method performance characteristics from this study in comparison with those determined in published LC–MS/MS validation studies to date ( 23 , 34 , 36 , 39–41 , 45 ) together with the performance characteristics for the ELISA method ( 46 ) and a comparative biosensor method ( 52 ). Previous LC–MS/MS single-laboratory validations (SLVs) have demonstrated recoveries which are generally good, ranging from 61 to 121% on average.…”

“…However, TTX was found recently in European seas, in both gastropods ( 26 , 27 ) and bivalves ( 9 , 28–37 ). Consequently, the apparent increase in occurrence of TTX-positive shellfish together with the notable absence of regular monitoring [with the exception of the Netherlands ( 30 )] and TTX regulations for bivalve molluscs is a concern for the safety of seafood consumers globally ( 4 , 36 ).…”

“…Instrumental techniques include LC coupled to fluorescence detection, with detectors subsequently being replaced by single-quadrupole mass spectrometry and more recently by tandem mass spectrometry coupled to ultra high liquid chromatography (UHPLC–MS/MS). The use of UHPLC–MS/MS instruments with hydrophilic interaction liquid chromatography (HILIC) with selected reaction monitoring (SRM) acquisition of characteristic precursor–product ion transitions has ultimately become the most commonly used detection method for TTXs in marine organisms ( 23 , 36 ). Variations of this approach have been subjected to single-laboratory validation studies ( 34 , 38 , 39 ), published as a method standard ( 40 ) and incorporated into a collaborative study for paralytic shellfish toxins (PST) and TTX combined ( 41 ).…”