First Report of Two Gymnodimines and Two Tetrodotoxin Analogues in Invertebrates from the North Atlantic Coast of Spain.

Gymnodimine D (GYM D), 16-desmethyl gymnodimine D (16-desmethyl GYM D), and two tetrodotoxin analogues have been found in invertebrates obtained from the north Atlantic coast of Spain from May 2021 to October 2022. It is the first report of GYMD and 16-desmethyl GYM D in invertebrates worldwide and of the tetrodotoxin analogues, 5,6,11 trideoxy tetrodotoxin (5,6,11 trideoxy TTX) and its isomer (referred to as 5,6,11 trideoxy-epi-TTX), in the north Atlantic Coast of Spain. In this study, we also report for the first time the detection of tetrodotoxin (TTX) in three species (the cnidaria Calliactis parasitica, an unidentified species, and the bivalve Tellina donacina). The prevalence was medium for GYM D and 16-desmethyl GYM D and low for TTXs overall. The concentrations recorded were variable, with maximum values of GYM D in the bivalve Cerastoderma edule (8.8 µg GYM A equivalents kg-1), of 16-desmethyl GYM D in the bivalve Magellana gigas (10 µg GYM A equivalents kg-1) and of TTX and 5,6,11 trideoxy TTX in the cnidaria C. parasitica (49.7 and 233 µg TTX equivalents kg-1, respectively). There is very scarce information about these compounds. Therefore, the reporting of these new detections will increase the knowledge on the current incidence of marine toxins in Europe that the European Food Safety Authority (EFSA), in particular, and the scientific community, in general, have. This study also highlights the importance of analyzing toxin analogues and metabolites for effective monitoring programs and adequate health protection.

Tetrodotoxins (TTXs) and Vibrio alginolyticus in Mussels from Central Adriatic Sea (Italy): Are They Closely Related?

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.

Detection of Tetrodotoxin Shellfish Poisoning (TSP) Toxins and Causative Factors in Bivalve Molluscs from the UK.

Tetrodotoxins (TTXs) are traditionally associated with the occurrence of tropical Pufferfish Poisoning. In recent years, however, TTXs have been identified in European bivalve mollusc shellfish, resulting in the need to assess prevalence and risk to shellfish consumers. Following the previous identification of TTXs in shellfish from southern England, this study was designed to assess the wider prevalence of TTXs in shellfish from around the coast of the UK. Samples were collected between 2014 and 2016 and subjected to analysis using HILIC-MS/MS. Results showed the continued presence of toxins in shellfish harvested along the coast of southern England, with the maximum concentration of total TTXs reaching 253 µg/kg. TTX accumulation was detected in Pacific oysters (Crassostreagigas), native oysters (Ostreaedulis) common mussels (Mytilusedulis) and hard clams (Mercenariamercenaria), but not found in cockles (Cerastodermaedule), razors (Ensis species) or scallops (Pectenmaximus). Whilst the highest concentrations were quantified in samples harvested during the warmer summer months, TTXs were still evident during the winter. An assessment of the potential causative factors did not reveal any links with the phytoplankton species Prorocentrumcordatum, instead highlighting a greater level of risk in areas of shallow, estuarine waters with temperatures above 15 °C.

Potential Threats Posed by Tetrodotoxins in UK Waters: Examination of Detection Methodology Used in Their Control.

Tetrodotoxin is a neurotoxin responsible for many human fatalities, most commonly following the consumption of pufferfish. Whilst the source of the toxin has not been conclusively proven, it is thought to be associated with various species of marine bacteria. Whilst the toxins are well studied in fish and gastropods, in recent years, there have been a number of reports of tetrodotoxin occurring in bivalve shellfish, including those harvested from the UK and other parts of Europe. This paper reviews evidence concerning the prevalence of tetrodotoxins in the UK together with methodologies currently available for testing. Biological, biomolecular and chemical methods are reviewed, including recommendations for further work. With the recent development of quantitative chromatographic methods for these and other hydrophilic toxins, as well as the commercial availability of rapid testing kits, there are a number of options available to ensure consumers are protected against this threat.

Tetrodotoxin and analogues in two local pufferfish species from Inhaca Island - South of Mozambique: First report in the Mozambican coast.

Tetrodotoxins (TTXs) were investigated in two local pufferfish species, Diodon hystrix and Arothron hispidus, from Mozambican coast. TTX and analogues 4-epiTTX, 4,9-anhydroTTX, 11-deoxyTTX, and 11-norTTX-6-(R/S)-ol were found in both species and high level of TTX was found in A. hispidus (9522.0 µg TTX kg-1) than in D. hystrix (350.9 µg TTX kg-1). The distribution of TTX and their analogues in A. hispidus was intestine > liver > skin â‰« muscle > gonads. This is the first report of TTXs in Mozambican coast.

Progress on the investigation and monitoring of marine phycotoxins in China.

Marine phycotoxins associated with paralytic shellfish poisoning (PSP), diarrhetic shellfish poisoning (DSP), amnesic shellfish poisoning (ASP), neurotoxic shellfish poisoning (NSP), ciguatera fish poisoning (CFP), tetrodotoxin (TTX), palytoxin (PLTX) and neurotoxin ß-N-methylamino-L-alanine (BMAA) have been investigated and routinely monitored along the coast of China. The mouse bioassay for monitoring of marine toxins has been progressively replaced by the enzyme-linked immunosorbent assay (ELISA) and liquid chromatography tandem mass spectrometry (LC-MS/MS), which led to the discovery of many new hydrophilic and lipophilic marine toxins. PSP toxins have been detected in the whole of coastal waters of China, where they are the most serious marine toxins. PSP events in the Northern Yellow Sea, the Bohai Sea and the East China Sea are a cause of severe public health concern. Okadaic acid (OA) and dinophysistoxin-1 (DTX1), which are major toxin components associated with DSP, were mainly found in coastal waters of Zhejiang and Fujian provinces, and other lipophilic toxins, such as pectenotoxins, yessotoxins, azaspiracids, cyclic imines, and dinophysistoxin-2(DTX2) were detected in bivalves, seawater, sediment, as well as phytoplankton. CFP events mainly occurred in the South China Sea, while TTX events mainly occurred in Jiangsu, Zhejiang and Fujian provinces. Microalgae that produce PLTX and BMAA were found in the phytoplankton community along the coastal waters of China.

First Detection of Tetrodotoxin in Bivalves and Gastropods from the French Mainland Coasts.

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.

Bioaccessibility of lipophilic and hydrophilic marine biotoxins in seafood: An in vitro digestion approach.

This study aimed to assess the bioaccessibility of different marine biotoxins in naturally contaminated shellfish and fish gonads using an in vitro digestion methodology. In general, hydrophilic toxins (domoic acid, paralytic shellfish poisoning toxins and tetrodotoxins) showed higher bioaccessibility than lipophilic ones (okadaic acid and azaspiracids). The bioaccessibility of toxins from the okadaic acid group ranged from 69% (raw European razor clams) to 74% (raw donax clams). Regarding azaspiracids, 47% of the initial content was bioaccessible in steamed blue mussel. As for hydrophilic toxins, 100% of the initial content was bioaccessible after digestion in raw shellfish and puffer fish gonads. The total tetrodotoxin bioaccessibility in puffer fish gonads decreased significantly after steaming. The profile of tetrodotoxins changed during the digestion process: TTX and 11-norTTX-6S-ol analogues decreased significantly after digestion, but the 5,6,11-trideoxy TTX analogue increased in both raw and steamed puffer fish gonads. These preliminary findings confirm the need to consider bioaccessibility data in future seafood risk assessment, as such information enables a more accurate and realistic estimation of potential seafood hazards, particularly in what concerns lipophilic toxins, therefore, constituting a crucial tool in the refinement of regulatory limits for the presence of biotoxins in seafood.

First Report on the Occurrence of Tetrodotoxins in Bivalve Mollusks in The Netherlands.

Tetrodotoxin (TTX) is traditionally associated with seafood from tropical regions, but recently TTX was detected in bivalve mollusks in more temperate European waters. In The Netherlands it was therefore decided to monitor TTX in shellfish harvested from Dutch production areas. All shellfish production areas were monitored in 2015, 2016 and 2017. Samples were analyzed using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). In total 1063 samples were investigated, and the highest concentrations were observed in 2016, i.e., 253 µg TTX/kg in oysters and 101 µg TTX/kg in mussels. No TTX analogues, with the exception of 4-epi-TTX in one single sample, were found and contaminated samples also showed positive results in the neuro-2a bioassay. The occurrence of TTX seems to be consistent over the last three years with the highest concentrations observed annually in late June. The causative organism and the reasons why specific Dutch production areas are affected while others are not, are still unclear. Initially in The Netherlands an action limit of 20 µg TTX/kg was used to ensure the safety of consumers (2016), but recently The European Food Safety Authority (EFSA) established an acute reference dose, and based on a high portion size of consuming 400 g mussels, this dose was translated into a safe concentration of 44 µg TTX per kg for shellfish. This concentration is now used as an action limit and TTX is formally included in the Dutch shellfish monitoring program.