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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. [ABSTRACT FROM AUTHOR]

The EU Rapid Alert System for Food and Feed (RASFF) is a tool for the rapid exchange of information on food and feed safety issues between EU member states and the European Commission, destined to notify food safety authorities about products posing a risk to human health. Emerging marine and freshwater toxins and their impact on human health and aquatic ecosystems have become a growing concern in the recent years. This is also reflected in the RASFF notifications shared by European countries during the last decade, with the occasional appearance of relevant records. In this work, RASFF notifications related to emerging marine and freshwater toxins from 2012 to date were retrospectively analyzed to discover the patterns of their appearance, as well as to explore their relationship with concurrent occurrence data and/or risk assessment advancements in the field. A total of fifteen notifications involving emerging marine and freshwater toxins were found, which included: ten on ciguatoxins in fish, three on tetrodotoxins in bivalve molluscs, one on microcystins in algae powder, and one on pinnatoxins in bivalve molluscs. This study contributes to a better understanding of the reasons behind the RASFF's provision of notifications on emerging toxins in EU countries. [ABSTRACT FROM AUTHOR]

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.

Pinnatoxins (PnTXs) A-H constitute an emerging family belonging to the cyclic imine group of phycotoxins. Interest has been focused on these fast-acting and highly-potent toxins because they are widely found in contaminated shellfish. Despite their highly complex molecular structure, PnTXs have been chemically synthetized and demonstrated to act on various nicotinic acetylcholine receptor (nAChR) subtypes. In the present work, PnTX-A, PnTX-G and analogue, obtained by chemical synthesis with a high degree of purity (>98%), have been studied in vivo and in vitro on adult mouse and isolated nerve-muscle preparations expressing the mature muscle-type (α1)2β1δε nAChR. The results show that PnTX-A and G acted on the neuromuscular system of anesthetized mice and blocked the compound muscle action potential (CMAP) in a dose- and time-dependent manner, using a minimally invasive electrophysiological method. The CMAP block produced by both toxins in vivo was reversible within 6-8 h. PnTX-A and G, applied to isolated extensor digitorum longus nerve-muscle preparations, blocked reversibly isometric twitches evoked by nerve stimulation. The action of PnTX-A was reversed by 3,4-diaminopyridine. Both toxins exerted no direct action on muscle fibers, as revealed by direct muscle stimulation. PnTX-A and G blocked synaptic transmission at mouse neuromuscular junctions and PnTX-A amino ketone analogue (containing an open form of the imine ring) had no effect on neuromuscular transmission. These results indicate the importance of the cyclic imine for interacting with the adult mammalian muscle-type nAChR. Modeling and docking studies revealed molecular determinants responsible for the interaction of PnTXs with the muscle-type nAChR.

Tetrodotoxins (TTXs), the pufferfish venom traditionally associated with Indo-Pacific area, has been reported during last decades in ever wider range of marine organisms and ever more geographical areas, including shellfish in Europe. Wild mussels (Mytilus galloprovincialis) grown in the Marche Region (N Adriatic Sea, Italy) were shown to be prone to TTX contamination during the warm season, with a suspected role of Vibrio alginolyticus characterized by non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS)-encoding genes. This work aimed to deepen the knowledge about the toxin's origin and the way through which it accumulates in mussels. A two-year study (spring–summer 2020–2021) confirmed the recurrent presence of TTX (11–68 µg kg−1) in the official monitored natural mussel beds of the Conero Riviera. During 2021, a supplementary nonroutine monitoring of a natural mussel bed in the same area was carried out weekly from June until August for TTXs and/or the presence of V. alginolyticus. Biotic (mussels, mesozooplankton, worms and phytoplankton); abiotic (water and sediment) matrices and phytoplankton assemblage characterizations were studied. Mussels showed relevant TTX contamination levels (9–296 µg kg−1) with extremely rapid TTX accumulation/depletion rates. The toxin presence in phytoplankton and its distribution in the different mussel tissues supports its possible exogenous origin. The V. alginolyticus count trend overlaps that of TTX contamination in mussels, and similar trends were reported also for some phytoplankton species. The role of V. alginolyticus carrying NRPS or PKS genes as a possible TTX source and of phytoplankton as a "potential vector" should therefore be further investigated. [ABSTRACT FROM AUTHOR]

The EU Rapid Alert System for Food and Feed (RASFF) is a tool for the rapid exchange of information on food and feed safety issues between EU member states and the European Commission, destined to notify food safety authorities about products posing a risk to human health. Emerging marine and freshwater toxins and their impact on human health and aquatic ecosystems have become a growing concern in the recent years. This is also reflected in the RASFF notifications shared by European countries during the last decade, with the occasional appearance of relevant records. In this work, RASFF notifications related to emerging marine and freshwater toxins from 2012 to date were retrospectively analyzed to discover the patterns of their appearance, as well as to explore their relationship with concurrent occurrence data and/or risk assessment advancements in the field. A total of fifteen notifications involving emerging marine and freshwater toxins were found, which included: ten on ciguatoxins in fish, three on tetrodotoxins in bivalve molluscs, one on microcystins in algae powder, and one on pinnatoxins in bivalve molluscs. This study contributes to a better understanding of the reasons behind the RASFF’s provision of notifications on emerging toxins in EU countries.

Intense blooms of the toxic dinoflagellate Ostreopsis producing palytoxin (PlTX) analogs, mainly ovatoxins (OVTXs), have been a recurrent phenomenon along several Mediterranean coasts. Although there is evidence of seafood contamination by these toxins, the dynamics of their bioaccumulation during Ostreopsis blooms is not yet clear. Toxin concentrations in wild mussels at two sites in the Conero Riviera, along the northern Adriatic Sea (Portonovo and Passetto), were analyzed from August to October 2021, throughout an Ostreopsis cf. ovata bloom, to investigate their relationships with the bloom pattern and abundances. Contaminated mussels showed the typical toxin profile dominated by OVTX-a and -b, with levels lower than the threshold considered unsafe for human consumption (30 µg PlTX-equivalent kg−1 soft tissue). The toxin content reached 36.4 µg PlTX kg−1 soft tissue only in a single sampling date. A clear correlation between the bioaccumulation of OVTXs in mussels and the abundance of Ostreopsis was observed. Our results highlighted, however, that bioaccumulation in the mussels is not affected merely by the abundance of toxic microalgae, since higher toxin levels were recorded at Portonovo, where the cell abundances were lower than at Passetto. The results from this study indicate that the Italian guidelines for the management of Ostreopsis blooms in bathing waters are also useful in managing the risks of human intoxication through ingestion, as mussel contamination was detected only during the alert phase (10,000–30,000 cells L−1). [ABSTRACT FROM AUTHOR]

Tetrodotoxin (TTX) is a potent neurotoxin naturally occurring in terrestrial and marine organisms such as pufferfish. Due to the risk of TTX poisoning, fish of Tetraodontidae family and other puffer-related species must not be placed in the EU markets. This restriction applies to fish of the family Molidae even though no data on toxins' occurrence is available. In this study, the presence of TTX and its analogues was investigated in the main edible tissue (the white muscle) and the main xenobiotics storage organ (the liver) of ocean sunfish Mola spp. (n = 13) from the South Portuguese coast. HILIC-MS/MS analyses did not reveal TTX in the analyzed samples, suggesting an inexistent or very limited risk of TTX poisoning. [ABSTRACT FROM AUTHOR]

Tetrodotoxins (TTXs), the pufferfish venom traditionally associated with Indo-Pacific area, has been reported during last decades in ever wider range of marine organisms and ever more geographical areas, including shellfish in Europe. Wild mussels (Mytilus galloprovincialis) grown in the Marche Region (N Adriatic Sea, Italy) were shown to be prone to TTX contamination during the warm season, with a suspected role of Vibrio alginolyticus characterized by non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS)-encoding genes. This work aimed to deepen the knowledge about the toxin’s origin and the way through which it accumulates in mussels. A two-year study (spring–summer 2020–2021) confirmed the recurrent presence of TTX (11–68 µg kg−1) in the official monitored natural mussel beds of the Conero Riviera. During 2021, a supplementary nonroutine monitoring of a natural mussel bed in the same area was carried out weekly from June until August for TTXs and/or the presence of V. alginolyticus. Biotic (mussels, mesozooplankton, worms and phytoplankton); abiotic (water and sediment) matrices and phytoplankton assemblage characterizations were studied. Mussels showed relevant TTX contamination levels (9–296 µg kg−1) with extremely rapid TTX accumulation/depletion rates. The toxin presence in phytoplankton and its distribution in the different mussel tissues supports its possible exogenous origin. The V. alginolyticus count trend overlaps that of TTX contamination in mussels, and similar trends were reported also for some phytoplankton species. The role of V. alginolyticus carrying NRPS or PKS genes as a possible TTX source and of phytoplankton as a “potential vector” should therefore be further investigated.

Intense blooms of the toxic dinoflagellate Ostreopsis producing palytoxin (PlTX) analogs, mainly ovatoxins (OVTXs), have been a recurrent phenomenon along several Mediterranean coasts. Although there is evidence of seafood contamination by these toxins, the dynamics of their bioaccumulation during Ostreopsis blooms is not yet clear. Toxin concentrations in wild mussels at two sites in the Conero Riviera, along the northern Adriatic Sea (Portonovo and Passetto), were analyzed from August to October 2021, throughout an Ostreopsis cf. ovata bloom, to investigate their relationships with the bloom pattern and abundances. Contaminated mussels showed the typical toxin profile dominated by OVTX-a and -b, with levels lower than the threshold considered unsafe for human consumption (30 µg PlTX-equivalent kg−1 soft tissue). The toxin content reached 36.4 µg PlTX kg−1 soft tissue only in a single sampling date. A clear correlation between the bioaccumulation of OVTXs in mussels and the abundance of Ostreopsis was observed. Our results highlighted, however, that bioaccumulation in the mussels is not affected merely by the abundance of toxic microalgae, since higher toxin levels were recorded at Portonovo, where the cell abundances were lower than at Passetto. The results from this study indicate that the Italian guidelines for the management of Ostreopsis blooms in bathing waters are also useful in managing the risks of human intoxication through ingestion, as mussel contamination was detected only during the alert phase (10,000–30,000 cells L−1).

Tetrodotoxin (TTX) is a potent neurotoxin naturally occurring in terrestrial and marine organisms such as pufferfish. Due to the risk of TTX poisoning, fish of Tetraodontidae family and other puffer-related species must not be placed in the EU markets. This restriction applies to fish of the family Molidae even though no data on toxins’ occurrence is available. In this study, the presence of TTX and its analogues was investigated in the main edible tissue (the white muscle) and the main xenobiotics storage organ (the liver) of ocean sunfish Mola spp. (n = 13) from the South Portuguese coast. HILIC-MS/MS analyses did not reveal TTX in the analyzed samples, suggesting an inexistent or very limited risk of TTX poisoning.

The recent emergence of ciguatera in the eastern Atlantic, particularly in the Canary Islands (Spain) and Madeira (Portugal) prompted the development and implementation of liquid chromatography tandem–mass spectrometry (LC/MS-MS) methods for the detection of ciguatoxins in fish. The complexity of fish tissue matrices, low concentrations of ciguatoxins in hazardous fish, and the scarcity of ciguatoxin standards present challenging issues for successful implementation of routine ciguatoxin analysis. A laboratory reference material of Caribbean Ciguatoxin-1 (C-CTX1), which was previously confirmed in fish responsible for ciguatera outbreaks in the Canary Islands, was used to assess the toxin's stability under strongly acidic conditions and solvent systems commonly used in LC-MS/MS. It was observed that strongly acidic conditions caused the transformation of C-CTX1 to a C56 methoxy congener, C-CTX1-Me. C-CTX1 was structurally characterised by LC-MS/MS and fragmentation pathways are presented showing the same fragmentation pattern as C-CTX1-Me. These results suggest that the use of strongly acidic conditions during sample pretreatment for C-CTX analysis, might produce significant artefacts, and risks failing to detect the presence of C-CTX1. [ABSTRACT FROM AUTHOR]

Tetrodotoxins (TTXs) are potentially lethal paralytic toxins that have been identified in European shellfish over recent years. Risk assessment has suggested comparatively low levels (44 µg TTX-equivalent/kg) but stresses the lack of data on occurrence. Both bacteria and dinoflagellates were suggested as possible biogenic sources, either from an endogenous or exogenous origin. We thus investigated TTXs in (i) 98 shellfish samples and (ii) 122 bacterial strains, isolated from French environments. We optimized a method based on mass spectrometry, using a single extraction step followed by ultrafiltration without Solid Phase Extraction and matrix-matched calibration for both shellfish and bacterial matrix. Limits of detection and quantification were 6.3 and 12.5 µg/kg for shellfish and 5.0 and 10 µg/kg for bacterial matrix, respectively. Even though bacterial matrix resulted in signal enhancement, no TTX analog was detected in any strain. Bivalves (either Crassostrea gigas or Ruditapes philippinarum) were surveyed in six French production areas over 2.5–3 month periods (2018–2019). Concentrations of TTX ranged from ‘not detected’ to a maximum of 32 µg/kg (Bay of Brest, 17 June 2019), with events lasting 2 weeks at maximum. While these results are in line with previous studies, they provide new data of TTX occurrence and confirm that the link between bacteria, bivalves and TTX is complex.

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.

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.

Highlights • LC-MS/MS allowed to confirm the presence of Ciguatoxins in fish from Atlantic coasts. • The evaluation of the efficiency of sample pretreatment is critical. • Caribbean Ciguatoxin-1 has been the main responsible for the contamination. • This is the first identification of a hydroxy derivative of C-CTX1. Abstract The increased emergence of Ciguatera Fish Poisoning in the Canary Islands and Madeira demanded the development of confirmatory methods by liquid chromatography coupled to tandem mass spectrometry to characterize the risk. Efforts were particularly focused on the optimization of sample pretreatment, especially in the sample cleanup step, to efficiently remove matrix interferences as a critical factor to consider in mass spectrometry detection. Two different LC-MS/MS approaches have been used for confirmation purposes, the first one using the sodium adduct as precursor and product ion to allow an increased sensitivity in the detection, whereas additional fragments were also monitored for further confirmation. The optimized conditions above mentioned allowed the confirmation of Caribbean Ciguatoxin-1 as the main responsible for the samples analyzed from these geographical areas, while the presence of a new hydroxyl metabolite of C-CTX1 was also confirmed in one sample analyzed in this study. [ABSTRACT FROM AUTHOR]

Microalgal species growing in marine and aquaculture environments can be responsible for harmful events because of their ability to produce potent natural toxins that can accumulate in edible mollusc species. Their consumption can cause severe illness and even be lethal. The European Union provides comprehensive regulations covering various general food safety aspects to manage the risk of contamination in shellfish farms. Many analytical methods have been proposed to evaluate algal toxins presence in the environment and in food products, for conducting surveillance studies of the main molluscs production sites and, where necessary, immediate monitoring of possible contamination of shellfish. In this work, a one-year analytical surveillance study was carried out to verify the possible presence of algal biotoxins in molluscs from a Mediterranean breeding area. Water and molluscs were sampled from a district of the North-East coast of Sicily, consisting of a unique brackish ecosystem of two lakes connected to each other and to the sea by narrow canals. Water samples were collected to investigate phytoplankton i by microscope analysis to assess the presence of potentially toxin-producing species, such as Pseudo-nitzschia spp, Alexandrium spp and Gonyaulax spinifera, although the presence of toxic phytoplankton has never reached alert levels. Mussels and clams samples were submitted to analysis of paralytic shellfish poisoning toxins, amnesic shellfish poisoning toxins and lipophilic toxins by liquid chromatography-based methods Only a few yessotoxins were detected, having concentrations always below the regulation limits. An existing liquid chromatography-tandem mass spectrometry-based multiresidue method for lipophilic biotoxins was adopted and extended to cover emerging biotoxins such as cyclic imines. The performance of the analytical method for Gymnodimine A and Spirolide 13-desMeC was assessed, obtaining respective quantitation limits of 20 and 10 µg kg−1, a precision always lower than 13% and trueness in the 81–120% range. Method applicability was confirmed using certified materials and a naturally contaminated sample. [ABSTRACT FROM AUTHOR]

The presence of emerging contaminants in food and the sources of the contamination are relevant issues in food safety. The impact of climate change on these contaminations is a topic widely debated; however, the consequences of climate change for the food system is not as deeply studied as other human and animal health and welfare issues. Projections of climate change in Europe have been evaluated through the EU Commission, and the impact on the marine environment is considered a priority issue. Marine biotoxins are produced by toxic microalgae and are natural contaminants of the marine environment. They are considered to be an important contaminant that needs to be evaluated. Their source is affected by oceanographic and environmental conditions; water temperature, sunlight, salinity, competing microorganisms, nutrients, and wind and current directions affect the growth and proliferation of microalgae. Although climate change should not be the only reason for this increase and other factors such as eutrophication, tourism, fishery activities, etc. could be considered, the influence of climate change has been observed through increased growth of dinoflagellates in areas where they have not been previously detected. An example of this is the recent emergence of ciguatera fish poisoning toxins, typically found in tropical or subtropical areas from the Pacific and Caribbean and in certain areas of the Atlantic Sea such as the Canary Islands (Spain) and Madeira (Portugal). In addition, the recent findings of the presence of tetrodotoxins, typically found in certain areas of the Pacific, are emerging in the EU and contaminating not only the fish species where these toxins had been found before but also bivalve mollusks. The emergence of these marine biotoxins in the EU is a reason for concern in the EU, and for this reason, the risk evaluation and characterization of these toxins are considered a priority for the European Food Safety Authorities (EFSA), which also emphasize the search for occurrence data using reliable and efficient analytical methods.

Pinnatoxins (PnTXs) A-H constitute an emerging family belonging to the cyclic imine group of phycotoxins. Interest has been focused on these fast-acting and highly-potent toxins because they are widely found in contaminated shellfish. Despite their highly complex molecular structure, PnTXs have been chemically synthetized and demonstrated to act on various nicotinic acetylcholine receptor (nAChR) subtypes. In the present work, PnTX-A, PnTX-G and analogue, obtained by chemical synthesis with a high degree of purity (>98%), have been studied in vivo and in vitro on adult mouse and isolated nerve-muscle preparations expressing the mature muscle-type (α1)2β1δε nAChR. The results show that PnTX-A and G acted on the neuromuscular system of anesthetized mice and blocked the compound muscle action potential (CMAP) in a dose- and time-dependent manner, using a minimally invasive electrophysiological method. The CMAP block produced by both toxins in vivo was reversible within 6−8 h. PnTX-A and G, applied to isolated extensor digitorum longus nerve-muscle preparations, blocked reversibly isometric twitches evoked by nerve stimulation. The action of PnTX-A was reversed by 3,4-diaminopyridine. Both toxins exerted no direct action on muscle fibers, as revealed by direct muscle stimulation. PnTX-A and G blocked synaptic transmission at mouse neuromuscular junctions and PnTX-A amino ketone analogue (containing an open form of the imine ring) had no effect on neuromuscular transmission. These results indicate the importance of the cyclic imine for interacting with the adult mammalian muscle-type nAChR. Modeling and docking studies revealed molecular determinants responsible for the interaction of PnTXs with the muscle-type nAChR.

Aptasensors have a great potential for environmental monitoring, particularly for real-time on-site detection of aquatic toxins produced by marine and freshwater microorganisms (cyanobacteria, dinoflagellates, and diatoms), with several advantages over other biosensors that are worth considering. Freshwater monitoring is of vital importance for public health, in numerous human activities, and animal welfare, since these toxins may cause fatal intoxications. Similarly, in marine waters, very effective monitoring programs have been put in place in many countries to detect when toxins exceed established regulatory levels and accordingly enforce shellfish harvesting closures. Recent advances in the fields of aptamer selection, nanomaterials and communication technologies, offer a vast array of possibilities to develop new imaginative strategies to create improved, ultrasensitive, reliable and real-time devices, featuring unique characteristics to produce and amplify the signal. So far, not many strategies have been used to detect aquatic toxins, mostly limited to the optic and electrochemical sensors, the majority applied to detect microcystin-LR using a target-induced switching mode. The limits of detection of these aptasensors have been decreasing from the nM to the fM order of magnitude in the past 20 years. Aspects related to sensor components, performance, aptamers sequences, matrices analyzed and future perspectives, are considered and discussed.

Tetrodotoxins (TTXs), the pufferfish venom traditionally associated with Indo-Pacific area, has been reported during last decades in ever wider range of marine organisms and ever more geographical areas, including shellfish in Europe. Wild mussels ( Mytilus galloprovincialis ) grown in the Marche Region (N Adriatic Sea, Italy) were shown to be prone to TTX contamination during the warm season, with a suspected role of Vibrio alginolyticus characterized by non-ribosomal peptide synthetase (NRPS) and polyketide synthase (PKS)-encoding genes. This work aimed to deepen the knowledge about the toxin's origin and the way through which it accumulates in mussels. A two-year study (spring-summer 2020-2021) confirmed the recurrent presence of TTX (11-68 µg kg -1 ) in the official monitored natural mussel beds of the Conero Riviera. During 2021, a supplementary nonroutine monitoring of a natural mussel bed in the same area was carried out weekly from June until August for TTXs and/or the presence of V. alginolyticus . Biotic (mussels, mesozooplankton, worms and phytoplankton); abiotic (water and sediment) matrices and phytoplankton assemblage characterizations were studied. Mussels showed relevant TTX contamination levels (9-296 µg kg -1 ) with extremely rapid TTX accumulation/depletion rates. The toxin presence in phytoplankton and its distribution in the different mussel tissues supports its possible exogenous origin. The V. alginolyticus count trend overlaps that of TTX contamination in mussels, and similar trends were reported also for some phytoplankton species. The role of V. alginolyticus carrying NRPS or PKS genes as a possible TTX source and of phytoplankton as a "potential vector" should therefore be further investigated., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Abstract The EFSA Panel on Contaminants in the Food Chain (CONTAM Panel) assessed the risks to human health related to the consumption of ciguatoxin (CTX)‐group toxins in fish. CTX‐group toxins occur in fish as a result of biotransformation of precursor gambiertoxins produced by the benthic dinoflagellate Gambierdiscus toxicus. CTX‐group toxins cause ciguatera fish poisoning. They are mainly found in Pacific, Caribbean and Indian Ocean regions and are classified as Pacific (P), Caribbean (C) and Indian Ocean (I) CTX‐group toxins. Recently CTX‐group toxins were identified for the first time in fish in Europe. Currently there are no regulatory limits for CTX‐group toxins in fish in Europe, but the regulation requires that no fish products containing CTX‐group toxins are placed on the market. The toxicological database for CTX‐group toxins is limited, comprising mostly acute toxicity studies. In view of the acute toxicity of CTX‐group toxins the CONTAM Panel considered establishing an acute reference dose (ARfD). However, due to the very limited quantitative data both in experimental animals as well as related to human intoxications, the CONTAM Panel concluded that the establishment of an oral ARfD was not possible. Based on case reports on human intoxications it appears that a concentration of 0.01 μg P CTX‐1 equivalents/kg fish is expected not to exert effects in sensitive individuals when consuming a single fish meal. The mouse bioassay (MBA) has been widely used to detect CTX‐group toxins. However, due to insufficient detection capability and ethical concerns the MBA is not considered an appropriate method. In vitro (cytotoxicity and receptor binding) assays have been developed as alternative, but they need further development. Liquid chromatography‐tandem mass spectrometry methods can be of value for the quantification of CTX‐group toxins, but certified reference standards and reference materials need to be provided to allow method development and (inter‐laboratory) 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. [ABSTRACT FROM AUTHOR]

Tetrodotoxins (TTXs) are potentially lethal paralytic toxins that have been identified in European shellfish over recent years. Risk assessment has suggested comparatively low levels (44 µg TTX-equivalent/kg) but stresses the lack of data on occurrence. Both bacteria and dinoflagellates were suggested as possible biogenic sources, either from an endogenous or exogenous origin. We thus investigated TTXs in (i) 98 shellfish samples and (ii) 122 bacterial strains, isolated from French environments. We optimized a method based on mass spectrometry, using a single extraction step followed by ultrafiltration without Solid Phase Extraction and matrix-matched calibration for both shellfish and bacterial matrix. Limits of detection and quantification were 6.3 and 12.5 µg/kg for shellfish and 5.0 and 10 µg/kg for bacterial matrix, respectively. Even though bacterial matrix resulted in signal enhancement, no TTX analog was detected in any strain. Bivalves (either Crassostrea gigas or Ruditapes philippinarum ) were surveyed in six French production areas over 2.5-3 month periods (2018-2019). Concentrations of TTX ranged from 'not detected' to a maximum of 32 µg/kg (Bay of Brest, 17 June 2019), with events lasting 2 weeks at maximum. While these results are in line with previous studies, they provide new data of TTX occurrence and confirm that the link between bacteria, bivalves and TTX is complex.

The presence of emerging contaminants in food and the sources of the contamination are relevant issues in food safety. The impact of climate change on these contaminations is a topic widely debated; however, the consequences of climate change for the food system is not as deeply studied as other human and animal health and welfare issues. Projections of climate change in Europe have been evaluated through the EU Commission, and the impact on the marine environment is considered a priority issue. Marine biotoxins are produced by toxic microalgae and are natural contaminants of the marine environment. They are considered to be an important contaminant that needs to be evaluated. Their source is affected by oceanographic and environmental conditions; water temperature, sunlight, salinity, competing microorganisms, nutrients, and wind and current directions affect the growth and proliferation of microalgae. Although climate change should not be the only reason for this increase and other factors such as eutrophication, tourism, fishery activities, etc. could be considered, the influence of climate change has been observed through increased growth of dinoflagellates in areas where they have not been previously detected. An example of this is the recent emergence of ciguatera fish poisoning toxins, typically found in tropical or subtropical areas from the Pacific and Caribbean and in certain areas of the Atlantic Sea such as the Canary Islands (Spain) and Madeira (Portugal). In addition, the recent findings of the presence of tetrodotoxins, typically found in certain areas of the Pacific, are emerging in the EU and contaminating not only the fish species where these toxins had been found before but also bivalve mollusks. The emergence of these marine biotoxins in the EU is a reason for concern in the EU, and for this reason, the risk evaluation and characterization of these toxins are considered a priority for the European Food Safety Authorities (EFSA), which also emphasize the search for occurrence data using reliable and efficient analytical methods. [ABSTRACT FROM AUTHOR]

Pinnatoxins (PnTXs) A-H constitute an emerging family belonging to the cyclic imine group of phycotoxins. Interest has been focused on these fast-acting and highly-potent toxins because they are widely found in contaminated shellfish. Despite their highly complex molecular structure, PnTXs have been chemically synthetized and demonstrated to act on various nicotinic acetylcholine receptor (nAChR) subtypes. In the present work, PnTX-A, PnTX-G and analogue, obtained by chemical synthesis with a high degree of purity (>98%), have been studied in vivo and in vitro on adult mouse and isolated nerve-muscle preparations expressing the mature muscle-type (α1)2β1δε nAChR. The results show that PnTX-A and G acted on the neuromuscular system of anesthetized mice and blocked the compound muscle action potential (CMAP) in a dose- and time-dependent manner, using a minimally invasive electrophysiological method. The CMAP block produced by both toxins in vivo was reversible within 6–8 h. PnTX-A and G, applied to isolated extensor digitorum longus nerve-muscle preparations, blocked reversibly isometric twitches evoked by nerve stimulation. The action of PnTX-A was reversed by 3,4-diaminopyridine. Both toxins exerted no direct action on muscle fibers, as revealed by direct muscle stimulation. PnTX-A and G blocked synaptic transmission at mouse neuromuscular junctions and PnTX-A amino ketone analogue (containing an open form of the imine ring) had no effect on neuromuscular transmission. These results indicate the importance of the cyclic imine for interacting with the adult mammalian muscle-type nAChR. Modeling and docking studies revealed molecular determinants responsible for the interaction of PnTXs with the muscle-type nAChR. [ABSTRACT FROM AUTHOR]

Aptasensors have a great potential for environmental monitoring, particularly for real-time on-site detection of aquatic toxins produced by marine and freshwater microorganisms (cyanobacteria, dinoflagellates, and diatoms), with several advantages over other biosensors that are worth considering. Freshwater monitoring is of vital importance for public health, in numerous human activities, and animal welfare, since these toxins may cause fatal intoxications. Similarly, in marine waters, very effective monitoring programs have been put in place in many countries to detect when toxins exceed established regulatory levels and accordingly enforce shellfish harvesting closures. Recent advances in the fields of aptamer selection, nanomaterials and communication technologies, offer a vast array of possibilities to develop new imaginative strategies to create improved, ultrasensitive, reliable and real-time devices, featuring unique characteristics to produce and amplify the signal. So far, not many strategies have been used to detect aquatic toxins, mostly limited to the optic and electrochemical sensors, the majority applied to detect microcystin-LR using a target-induced switching mode. The limits of detection of these aptasensors have been decreasing from the nM to the fM order of magnitude in the past 20 years. Aspects related to sensor components, performance, aptamers sequences, matrices analyzed and future perspectives, are considered and discussed. [ABSTRACT FROM AUTHOR]