Occurrence and Exposure Assessment of Lipophilic Shellfish Toxins in the Zhejiang Province, China.

Although lipophilic shellfish toxins (LSTs) pose a significant threat to the health of seafood consumers, their systematic investigation and risk assessment remain scarce. The goals of this study were as follows: (1) analyze LST levels in commercially available shellfish in Zhejiang province, China, and determine factors influencing LST distribution; (2) assess the acute dietary risk of exposure to LSTs for local consumers during the red tide period; (3) explore potential health risks of LSTs in humans; and (4) study the acute risks of simultaneous dietary exposure to LSTs and paralytic shellfish toxins (PSTs). A total of 546 shellfish samples were collected. LSTs were detected in 89 samples (16.3%) at concentrations below the regulatory limits. Mussels were the main shellfish species contaminated with LSTs. Spatial variations were observed in the yessotoxin group. Acute exposure to LSTs based on multiple scenarios was low. The minimum tolerable exposure durations for LSTs calculated using the mean and the 95th percentile of consumption data were 19.7 and 4.9 years, respectively. Our findings showed that Zhejiang province residents are at a low risk of combined exposure to LSTs and PSTs; however, the risk may be higher for children under 6 years of age in the extreme scenario.

Contamination Status and Risk Assessment of Paralytic Shellfish Toxins in Shellfish along the Coastal Areas of China.

Paralytic shellfish toxins (PSTs) are widely distributed in shellfish along the coast of China, causing a serious threat to consumer health; however, there is still a lack of large-scale systematic investigations and risk assessments. Herein, 641 shellfish samples were collected from March to November 2020, and the PSTs' toxicity was detected via liquid chromatography-tandem mass spectrometry. Furthermore, the contamination status and potential dietary risks of PSTs were discussed. PSTs were detected in 241 shellfish samples with a detection rate of 37.60%. The average PST toxicities in mussels and ark shells were considerably higher than those in other shellfish. The PSTs mainly included N-sulfonylcarbamoyl toxins (class C) and carbamoyl toxins (class GTX), and the highest PST toxicity was 546.09 µg STX eq. kg-1. The PST toxicity in spring was significantly higher than those in summer and autumn (p < 0.05). Hebei Province had the highest average PST toxicity in spring. An acute exposure assessment showed that consumers in Hebei Province had a higher dietary risk, with mussels posing a significantly higher dietary risk to consumers. This research provides reference for the green and sustainable development of the shellfish industry and the establishment of a shellfish toxin prevention and control system.

Lipophilic Toxins in Chile: History, Producers and Impacts.

A variety of microalgal species produce lipophilic toxins (LT) that are accumulated by filter-feeding bivalves. Their negative impacts on human health and shellfish exploitation are determined by toxic potential of the local strains and toxin biotransformations by exploited bivalve species. Chile has become, in a decade, the world's major exporter of mussels (Mytilus chilensis) and scallops (Argopecten purpuratus) and has implemented toxin testing according to importing countries' demands. Species of the Dinophysis acuminata complex and Protoceratium reticulatum are the most widespread and abundant LT producers in Chile. Dominant D. acuminata strains, notwithstanding, unlike most strains in Europe rich in okadaic acid (OA), produce only pectenotoxins, with no impact on human health. Dinophysis acuta, suspected to be the main cause of diarrhetic shellfish poisoning outbreaks, is found in the two southernmost regions of Chile, and has apparently shifted poleward. Mouse bioassay (MBA) is the official method to control shellfish safety for the national market. Positive results from mouse tests to mixtures of toxins and other compounds only toxic by intraperitoneal injection, including already deregulated toxins (PTXs), force unnecessary harvesting bans, and hinder progress in the identification of emerging toxins. Here, 50 years of LST events in Chile, and current knowledge of their sources, accumulation and effects, are reviewed. Improvements of monitoring practices are suggested, and strategies to face new challenges and answer the main questions are proposed.

A High Throughput Screening HPLC-FLD Method for Paralytic Shellfish Toxins (PSTs) Enabling Effective Official Control.

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.

Development of a Fast Liquid Chromatography Coupled to Mass Spectrometry Method (LC-MS/MS) to Determine Fourteen Lipophilic Shellfish Toxins Based on Fused-Core Technology: In-House Validation.

Prevalence and incidence of the marine toxins (paralytic, amnesic, and lipophilic toxins) including the so-called emerging toxins (these are, gymnodimines, pinnatoxins, or spirolides among others) have increased in recent years all over the world. Climate change, which is affecting the distribution of their producing phytoplankton species, is probably one of the main causes. Early detection of the toxins present in a particular area, and linking the toxins to their causative phytoplankton species are key tools to minimize the risk they pose for human consumers. The development of both types of studies requires fast and highly sensitive analytical methods. In the present work, we have developed a highly sensitive liquid chromatography-mass spectrometry methodology (LC-MS/MS), using a column with fused-core particle technology, for the determination of fourteen lipophilic toxins in a single run of 3.6 min. The performance of the method was evaluated for specificity, linearity, precision (repeatability and reproducibility) and accuracy by analysing spiked and naturally contaminated samples. The in-house validation was successful, and the limit of detection (LOD) and quantification (LOQ) for all the toxins were far below their regulatory action limits. The method is suitable to be considered in monitoring systems of bivalves for food control.

Guidance Level for Brevetoxins in French Shellfish.

Brevetoxins (BTXs) are marine biotoxins responsible for neurotoxic shellfish poisoning (NSP) after ingestion of contaminated shellfish. NSP is characterized by neurological, gastrointestinal and/or cardiovascular symptoms. The main known producer of BTXs is the dinoflagellate Karenia brevis, but other microalgae are also suspected to synthesize BTX-like compounds. BTXs are currently not regulated in France and in Europe. In November 2018, they have been detected for the first time in France in mussels from a lagoon in the Corsica Island (Mediterranean Sea), as part of the network for monitoring the emergence of marine biotoxins in shellfish. To prevent health risks associated with the consumption of shellfish contaminated with BTXs in France, a working group was set up by the French Agency for Food, Environmental and Occupational Health & Safety (Anses). One of the aims of this working group was to propose a guidance level for the presence of BTXs in shellfish. Toxicological data were too limited to derive an acute oral reference dose (ARfD). Based on human case reports, we identified two lowest-observed-adverse-effect levels (LOAELs). A guidance level of 180 µg BTX-3 eq./kg shellfish meat is proposed, considering a protective default portion size of 400 g shellfish meat.

Field Validation of the Southern Rock Lobster Paralytic Shellfish Toxin Monitoring Program in Tasmania, Australia.

Paralytic shellfish toxins (PST) are found in the hepatopancreas of Southern Rock Lobster Jasus edwardsii from the east coast of Tasmania in association with blooms of the toxic dinoflagellate Alexandrium catenella. Tasmania's rock lobster fishery is one of the state's most important wild capture fisheries, supporting a significant commercial industry (AUD 97M) and recreational fishing sector. A comprehensive 8 years of field data collected across multiple sites has allowed continued improvements to the risk management program protecting public health and market access for the Tasmanian lobster fishery. High variability was seen in toxin levels between individuals, sites, months, and years. The highest risk sites were those on the central east coast, with July to January identified as the most at-risk months. Relatively high uptake rates were observed (exponential rate of 2% per day), similar to filter-feeding mussels, and meant that lobster accumulated toxins quickly. Similarly, lobsters were relatively fast detoxifiers, losing up to 3% PST per day, following bloom demise. Mussel sentinel lines were effective in indicating a risk of elevated PST in lobster hepatopancreas, with annual baseline monitoring costing approximately 0.06% of the industry value. In addition, it was determined that if the mean hepatopancreas PST levels in five individual lobsters from a site were <0.22 mg STX equiv. kg-1, there is a 97.5% probability that any lobster from that site would be below the bivalve maximum level of 0.8 mg STX equiv. kg-1. The combination of using a sentinel species to identify risk areas and sampling five individual lobsters at a particular site, provides a cost-effective strategy for managing PST risk in the Tasmanian commercial lobster fishery.

Temporal Variation of the Profile and Concentrations of Paralytic Shellfish Toxins and Tetrodotoxin in the Scallop, Patinopecten yessoensis, Cultured in a Bay of East Japan.

Paralytic shellfish toxins (PSTs) are the major neurotoxic contaminants of edible bivalves in Japan. Tetrodotoxin (TTX) was recently detected in bivalve shellfish around the world, drawing widespread attention. In Japan, high levels of TTX were reported in the digestive gland of the scallop, Patinopecten yessoensis, in 1993; however, no new data have emerged since then. In this study, we simultaneously analyzed PSTs and TTX in scallops cultured in a bay of east Japan using hydrophilic interaction chromatography (HILIC)-MS/MS. These scallops were temporally collected from April to December 2017. The highest concentration of PSTs (182 µmol/kg, total congeners) in the hepatopancreas was detected in samples collected on May 23, lined to the cell density of the dinoflagellate, Alexandrium tamarense, in seawater around the scallops, whereas the highest concentration of TTX (421 nmol/kg) was detected in samples collected on August 22. Contrary to the previous report, temporal variation of the PSTs and TTX concentrations did not coincide. The highest concentration of TTX in the entire edible tissues was 7.3 µg/kg (23 nmol/kg) in samples obtained on August 22, which was lower than the European Food Safety Authority (EFSA)-proposed threshold, 44 µg TTX equivalents/kg shellfish meat. In addition, 12ß-deoxygonyautoxin 3 was firstly identified in scallops.

Outbreak of diarrhetic shellfish poisoning associated with consumption of mussels, United Kingdom, May to June 2019.

We report on six cases of diarrhetic shellfish poisoning following consumption of mussels harvested in the United Kingdom. Dinophysis spp. in the water column was found to have increased rapidly at the production site resulting in high levels of okadaic acid-group lipophilic toxins in the flesh of consumed mussels. Clinicians and public health professionals should remain aware of algal-derived toxins being a potential cause of illness following seafood consumption.

Preliminary Results on the Evaluation of the Occurrence of Tetrodotoxin Associated to Marine Vibrio spp. in Bivalves from the Galician Rias (Northwest of Spain).

Tetrodotoxins (TTX) are a potent group of natural neurotoxins putatively produced by symbiotic microorganisms and affecting the aquatic environment. These neurotoxins have been recently found in some species of bivalves and gastropods along the European Coasts (Greece, UK, and The Netherlands) linked to the presence of high concentrations of Vibrio, in particular Vibrio parahaemolyticus. This study is focused on the evaluation of the presence of Vibrio species and TTX in bivalves (mussels, oysters, cockles, clams, scallops, and razor clams) from Galician Rias (northwest of Spain). The detection and isolation of the major Vibrio spp. and other enterobacterial populations have been carried out with the aim of screening for the presence of the pathways genes, poliketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) possibly involved in the biosynthesis of these toxins. Samples containing Vibrio spp. were analyzed by biochemical (API20E-galery) and genetic tests (PCR-RT). These samples were then screened for TTX toxicity by a neuroblastoma cell-based assay (N2a) and the presence of TTX was further confirmed by LC-MS/MS. TTX was detected in two infaunal samples. This is the first confirmation of the presence of TTX in bivalve molluscs from the Galician Rias.

Phycotoxins in Marine Shellfish: Origin, Occurrence and Effects on Humans.

Massive phytoplankton proliferation, and the consequent release of toxic metabolites, can be responsible for seafood poisoning outbreaks: filter-feeding mollusks, such as shellfish, mussels, oysters or clams, can accumulate these toxins throughout the food chain and present a threat for consumers' health. Particular environmental and climatic conditions favor this natural phenomenon, called harmful algal blooms (HABs); the phytoplankton species mostly involved in these toxic events are dinoflagellates or diatoms belonging to the genera Alexandrium, Gymnodinium, Dinophysis, and Pseudo-nitzschia. Substantial economic losses ensue after HABs occurrence: the sectors mainly affected include commercial fisheries, tourism, recreational activities, and public health monitoring and management. A wide range of symptoms, from digestive to nervous, are associated to human intoxication by biotoxins, characterizing different and specific syndromes, called paralytic shellfish poisoning, amnesic shellfish poisoning, diarrhetic shellfish poisoning, and neurotoxic shellfish poisoning. This review provides a complete and updated survey of phycotoxins usually found in marine invertebrate organisms and their relevant properties, gathering information about the origin, the species where they were found, as well as their mechanism of action and main effects on humans.

A Strategy to Replace the Mouse Bioassay for Detecting and Identifying Lipophilic Marine Biotoxins by Combining the Neuro-2a Bioassay and LC-MS/MS Analysis.

Marine biotoxins in fish and shellfish can cause several symptoms in consumers, such as diarrhea, amnesia, or even death by paralysis. Monitoring programs are in place for testing shellfish on a regular basis. In some countries testing is performed using the so-called mouse bioassay, an assay that faces ethical concerns not only because of animal distress, but also because it lacks specificity and results in high amounts of false positives. In Europe, for lipophilic marine biotoxins (LMBs), a chemical analytical method using LC-MS/MS was developed as an alternative and is now the reference method. However, safety is often questioned when relying solely on such a method, and as a result, the mouse bioassay might still be used. In this study the use of a cell-based assay for screening, i.e., the neuro-2a assay, in combination with the official LC-MS/MS method was investigated as a new alternative strategy for the detection and quantification of LMBs. To this end, samples that had been tested previously with the mouse bioassay were analyzed in the neuro-2a bioassay and the LC-MS/MS method. The neuro-2a bioassay was able to detect all LMBs at the regulatory levels and all samples that tested positive in the mouse bioassay were also suspect in the neuro-2a bioassay. In most cases, these samples contained toxin levels (yessotoxins) that explain the outcome of the bioassay but did not exceed the established maximum permitted levels.

Heliotropiumides A and B, new phenolamides with N-carbamoyl putrescine moiety from Heliotropium foertherianum collected in Hawaii and their biological activities.

Two new compounds heliotropiumides A (1) and B (2), phenolamides each with an uncommon carbamoyl putrescine moiety, were isolated from the seeds of a naturalized Hawaiian higher plant, Heliotropium foertherianum Diane & Hilger in the borage family, which is widely used for the treatment of ciguatera fish poisoning. The structures of compounds 1 and 2 were characterized based on MS spectroscopic and NMR analysis, and DP4+ calculations. The absolute configuration (AC) of compound 1 was determined by comparison of its optical rotation with those reported in literature. Compound 2 showed inhibition against NF-κB with an IC50 value of 36µM.

[Poisoning caused by marine biotoxins]. / Intoxikation durch marine Biotoxine.

This paper presents a short summary of the knowledge on marine biotoxins. As toxins are known for their acute effects, they have been classified here according to the effects they cause in acute human poisoning incidents. Toxins may thus be distinguished into those that affect the nervous system (paralytic and other neurotoxins), memory (amnesic poisons), and the digestive system (diarrhetic toxins). Furthermore, newly emerging toxins, such as ciguatoxins or shark toxins, and factors that lead to the introduction of toxins in new areas, are presented. Relevant suggestions for further reading are given.

Potential Threats Posed by New or Emerging Marine Biotoxins in UK Waters and Examination of Detection Methodologies Used for Their Control: Cyclic Imines.

Cyclic imines (CIs) are a group of phytoplankton produced toxins related to shellfish food products, some of which are already present in UK and European waters. Their risk to shellfish consumers is poorly understood, as while no human intoxication has been definitively related to this group, their fast acting toxicity following intraperitoneal injection in mice has led to concern over their human health implications. A request was therefore made by UK food safety authorities to examine these toxins more closely to aid possible management strategies. Of the CI producers only the spirolide producer Alexandrium ostenfeldii is known to exist in UK waters at present but trends in climate change may lead to increased risk from other organisms/CI toxins currently present elsewhere in Europe and in similar environments worldwide. This paper reviews evidence concerning the prevalence of CIs and CI-producing phytoplankton, together with testing methodologies. Chemical, biological and biomolecular methods are reviewed, including recommendations for further work to enable effective testing. Although the focus here is on the UK, from a strategic standpoint many of the topics discussed will also be of interest in other parts of the world since new and emerging marine biotoxins are of global concern.

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.

Potential threats posed by new or emerging marine biotoxins in UK waters and examination of detection methodology used in their control: brevetoxins.

Regular occurrence of brevetoxin-producing toxic phytoplankton in commercial shellfishery areas poses a significant risk to shellfish consumer health. Brevetoxins and their causative toxic phytoplankton are more limited in their global distribution than most marine toxins impacting commercial shellfisheries. On the other hand, trends in climate change could conceivably lead to increased risk posed by these toxins in UK waters. A request was made by UK food safety authorities to examine these toxins more closely to aid possible management strategies, should they pose a threat in the future. At the time of writing, brevetoxins have been detected in the Gulf of Mexico, the Southeast US coast and in New Zealand waters, where regulatory levels for brevetoxins in shellfish have existed for some time. This paper reviews evidence concerning the prevalence of brevetoxins and brevetoxin-producing phytoplankton in the UK, together with testing methodologies. Chemical, biological and biomolecular methods are reviewed, including recommendations for further work to enable effective testing. Although the focus here is on the UK, from a strategic standpoint many of the topics discussed will also be of interest in other parts of the world since new and emerging marine biotoxins are of global concern.

Persistent Contamination of Octopuses and Mussels with Lipophilic Shellfish Toxins during Spring Dinophysis Blooms in a Subtropical Estuary.

This study investigates the occurrence of diarrhetic shellfish toxins (DSTs) and their producing phytoplankton species in southern Brazil, as well as the potential for toxin accumulation in co-occurring mussels (Perna perna) and octopuses (Octopus vulgaris). During the spring in 2012 and 2013, cells of Dinophysis acuminata complex were always present, sometimes at relatively high abundances (max. 1143 cells L-1), likely the main source of okadaic acid (OA) in the plankton (max. 34 ng L-1). Dinophysis caudata occurred at lower cell densities in 2013 when the lipophilic toxins pectenotoxin-2 (PTX-2) and PTX-2 seco acid were detected in plankton and mussel samples. Here, we report for the first time the accumulation of DSTs in octopuses, probably linked to the consumption of contaminated bivalves. Perna perna mussels were consistently contaminated with different DSTs (max. 42 µg kg-1), and all octopuses analyzed (n = 5) accumulated OA in different organs/tissues: digestive glands (DGs) > arms > gills > kidneys > stomach + intestine. Additionally, similar concentrations of 7-O-palmytoyl OA and 7-O-palmytoly dinophysistoxin-1 (DTX-1) were frequently detected in the hepatopancreas of P. perna and DGs of O. vulgaris. Therefore, octopuses can be considered a potential vector of DSTs to both humans and top predators such as marine mammals.

Toxin profile of Gymnodinium catenatum (Dinophyceae) from the Portuguese coast, as determined by liquid chromatography tandem mass spectrometry.

The marine dinoflagellate Gymnodinium catenatum has been associated with paralytic shellfish poisoning (PSP) outbreaks in Portuguese waters for many years. PSP syndrome is caused by consumption of seafood contaminated with paralytic shellfish toxins (PSTs), a suite of potent neurotoxins. Gymnodinium catenatum was frequently reported along the Portuguese coast throughout the late 1980s and early 1990s, but was absent between 1995 and 2005. Since this time, G. catenatum blooms have been recurrent, causing contamination of fishery resources along the Atlantic coast of Portugal. The aim of this study was to evaluate the toxin profile of G. catenatum isolated from the Portuguese coast before and after the 10-year hiatus to determine changes and potential impacts for the region. Hydrophilic interaction liquid chromatography tandem mass spectrometry (HILIC-MS/MS) was utilized to determine the presence of any known and emerging PSTs in sample extracts. Several PST derivatives were identified, including the N-sulfocarbamoyl analogues (C1-4), gonyautoxin 5 (GTX5), gonyautoxin 6 (GTX6), and decarbamoyl derivatives, decarbamoyl saxitoxin (dcSTX), decarbamoyl neosaxitoxin (dcNeo) and decarbamoyl gonyautoxin 3 (dcGTX3). In addition, three known hydroxy benzoate derivatives, G. catenatum toxin 1 (GC1), GC2 and GC3, were confirmed in cultured and wild strains of G. catenatum. Moreover, two presumed N-hydroxylated analogues of GC2 and GC3, designated GC5 and GC6, are reported. This work contributes to our understanding of the toxigenicity of G. catenatum in the coastal waters of Portugal and provides valuable information on emerging PST classes that may be relevant for routine monitoring programs tasked with the prevention and control of marine toxins in fish and shellfish.

A Natural and Concurrent Interferent in Bolinus brandaris with Consequences on Amnesic Shellfish Poisoning Risk Management.

Regulations aimed to protect public health from amnesic shellfish poisoning (ASP) are focused on the detection and accurate quantification of domoic acid (DA). The reference detection determination used by the different shellfish safety monitoring agencies worldwide is HPLC separation followed by UV detection, in which different chromatographic column lengths or brands are accepted as long as it is C18 column. A laboratory validation of this method showed different performance of two accepted chromatographic columns when analyzing Bolinus brandaris samples. A natural compound, present only in those samples that contained DA, was evidenced by one of the columns. The DA quantification obtained with the column that coelutes both compounds was approximately twice the amount obtained with the column that separates them. This difference has important consequences in the ASP toxins management for this fishery. The identity and toxicity of the compound are still unknown.