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Seafood, such as mussels is rich of essential nutrients, but it could be also a source of contaminants that may cause adverse health effects. This article aimed to identify safety status of mussels harvested from Bulgarian coast. Wild and cultivated mussels (N = 17) were collected from their breeding sites in the period throughout 2021, along the Bulgarian coast of the Black Sea. Among all six regulated marine biotoxins (domoic acid, dinophysistoxin-1 (DTX1), dinophysistoxin-2 (DTX2), azaspiracid-1 (AZA1), pectenotoxin-2 (PTX2) and yessotoxin (YTX)) were determined via high performance liquid chromatography-tandem mass spectrometry. Safety of investigated samples was assessed by calculating the human exposure to detected toxins and comparing it with the legislated acute reference doses. For calculations a body weight (66 kg) and mean portion size of 400 g, recommended by EFSA were used. The presence of only PTX2, yessotoxin and some of their analogues (PTX2-seco acid (PTX2sa) and hydroxy-YTX) was confirmed in all samples. Highest calculated exposure was to YTX – 0,00013 microgram/kg bw, which is much lower than the accepted acute reference dose of 25 microgram/kg bw. Calculated exposures to pectenotoxins were more than thousand times lower than the acute reference dose of 0,8 microgram/kg bw. Estimated results indicate that no human health risk could be expected based on the analyzed mussels. Although, no human health risk was identified, a running monitoring of marine biotoxins is important as mass proliferations of harmful microalgal are hard to predict. Thus, high levels of yessotoxins and pectenotoxins might be a certain danger to consumers. The insidiousness lies in the mechanism of action of these toxins, as it is still unknown and does not allow us to have reliable information on the symptoms and problems that it develops in humans. This work presents that in mussel samples from Bulgarian coast from 2021, low levels of marine biotoxins were detected. No human health risk might be expected if mussels were consumed. [ABSTRACT FROM AUTHOR]

EFSA was asked by the European Commission to provide information on levels of lipophilic shellfish toxins in whole scallops that would ensure levels in edible parts below the regulatory limits after shucking, i.e. removal of non-edible parts. This should include the okadaic acid (OA), the azaspiracid (AZA) and the yessotoxin (YTX) groups, and five species of scallops. In addition, EFSA was asked to recommend the number of scallops in an analytical sample. To address these questions, EFSA received suitable data on the three toxin groups in two scallop species, Aequipecten opercularis and Pecten maximus, i.e. data on individual and pooled samples of edible and non-edible parts from contamination incidents. The majority of the concentration levels were below limit of quantification (LOQ)/limit of detection (LOD), especially in adductor muscle but also in gonads. Shucking in most cases resulted in a strong decrease in the toxin levels. For Pecten maximus, statistical analysis showed that levels in whole scallops should not exceed 256 lg OA eq/kg or 217 lg AZA1 eq/kg to ensure that levels in gonads are below the regulatory limits of 160 lg OA or AZA1 eq/kg with 99% certainty. Such an analysis was not possible for yessotoxins or any toxin in Aequipecten opercularis and an assessment could only be based on upper bound levels. To ensure a 95% correct prediction on whether the level in scallops in an area or lot is correctly predicted to be compliant/non-compliant, it was shown that 10 scallops per sample would be sufficient to predict with 95% certainty if levels of OA-group toxins in the area/lot were 25% below or above the regulatory limit. However, to predict with a 95% certainty for levels between 140 and 180 lg OA eq/kg, a pooled sample of more than 30 scallops would have to be tested. [ABSTRACT FROM AUTHOR]

Harmful Algal Blooms (HABs) are massive proliferations of toxic algal species that occur under appropriate climatic and environmental conditions. HABs can exert their adverse impacts both on the environment and on particular living organisms through the production of marine biotoxins. Consumption of contaminated seafood or direct exposure to marine biotoxins (swimming, aerosols etc.) can result in human illness or even death. Thousands of cases of marine-toxin-related human poisoning are reported every year on a global basis. In Italy, research on marine biotoxins has shown a continuously changing toxin profile, with most of the toxin classes known so far being found at some point in seafood and seawater. Starting in the late 1980s, with the first finding of a marine biotoxin, okadaic acid, along the Italian coastline, a number of other classes of marine toxins - namely, yessotoxins, pectenotoxins, saxitoxins, domoic acid, spirolides, palytoxins and azaspiracids - have been found. Here we provide an overview, in chronological order, of the many classes of marine biotoxins that have infested Italian seawater over the past 25 years. [ABSTRACT FROM AUTHOR]

Abstract: The basal L1 medium was found to be unsatisfactory for culturing the red tide dinoflagellate Protoceratium reticulatum at a high growth rate and biomass yield. The L1 medium enhanced with phosphate to a total concentration of 217μM supported the highest attainable growth rate and biomass yield. Once the phosphate concentration exceeded 6× L1, phosphate inhibited the dinoflagellate growth and negatively affected cell viability. At the optimal phosphate concentration of 217μM, an increase in nitrate concentration over the range of 882–8824μM, did not affect cell growth and yield. Nitrate did not inhibit growth at any of the concentrations used. Clearly, the basal nitrate level in L1 is sufficient for effectively culturing P. reticulatum. At the ranges of phosphate and nitrate concentrations tested, cell volume was not sensitive to the concentration of nutrients but the concentration of phosphate affected both the specific cell number and cell volume growth rates. Elevated levels of nutrients supported their intracellular accumulation. Cell-specific production of yessotoxin was not influenced by concentration of phosphate in the culture medium, but elevated (>1764μM) nitrate concentration did enhance the yessotoxin level. Phosphate concentration that maximized biomass yield also maximized volumetric production of yessotoxin in the culture broth. [Copyright &y& Elsevier]

Abstract: Yessotoxin (YTX) and its analogues are a newly recognized group of toxins with increased presence in shellfish in recent years. They can be quantified by various functional assays due to their interaction with phosphodiesterases (PDEs). One of these assays detects the binding between the YTX and the fluorescently labeled PDE I using fluorescence polarization, a spectroscopic technique based on exciting a fluorescent molecule with plane-polarized light and measuring the polarization degree of the emitted light. The aim of this study was to develop a YTX extraction procedure from mussels that does not interfere with this detection method. YTX concentrations were measured in spiked mussel extracts obtained through use of different extraction methods and cleaning procedures. The percentages of toxin recovery in various steps of the processes were calculated using these concentrations. Six extraction methods and two cleaning steps were used and no matrix effects and high toxin recoveries were obtained in two cases. One case used acetone as extraction solvent followed by three dichloromethane partitions and the other case used methanol. The cleaning procedure includes a silica cartridge and a 10,000 NMWL filter. Finally these two extraction–cleaning–detection methods were applied to a naturally contaminated mussel sample and results showed that not only YTX but also homoYTX and hydroxyYTX can be quantified with a 85–90% recovery. [Copyright &y& Elsevier]

A simple and sensitive in situ method for monitoring the occurrence of toxic algal blooms and shellfish contamination events has been developed. The technique involves the passive adsorption of biotoxins onto porous synthetic resin filled sachets (SPATT bags) and their subsequent extraction and analysis. The success of the method is founded on the observation that during algal blooms significant amounts of toxin, including the low polarity lipophilic compounds such as the pectenotoxins and the okadaic acid complex toxins, are dissolved in the seawater. The results of field trials during Dinophysis acuminata and Protoceratium reticulatum blooms are presented. These data prove the concept and demonstrate that the technique provides a means of forecasting shellfish contamination events and predicting the net accumulation of polyether toxins by mussels. As an early warning method it has many advantages over current monitoring techniques such as shellfish-flesh testing and phytoplankton monitoring. In contrast to the circumstantial evidence provided by genetic probe technologies and conventional phytoplankton monitoring methods, it directly targets the toxic compounds of interest. The extracts that are obtained for analysis lack many of the extraneous lipophilic materials in crude shellfish extracts so that many of the matrix problems associated with chemical and biological analysis of these extracts are eliminated. Analyses can confidently target parent compounds only, because analytical and toxicological uncertainties associated with the multiplicity of toxin analogues produced by in vivo biotransformation in shellfish tissues are reduced. Time integrated sampling provides a good simulation of biotoxin accumulation in filter feeders and the high sensitivity provides lengthy early warning and conservative estimates of contamination potential. The technique may reduce monitoring costs and provide improved spatial and temporal sampling opportunities. When coupled with appropriate analytical techniques (e.g. LC–MS/MS multi-toxin screens, ELISA assays, receptor binding assays), the technique has the potential to offer a universal early warning method for marine and freshwater micro-algae toxins. [Copyright &y& Elsevier]

The presence of YTX was confirmed in Protoceratium reticulatum cultures and detected for the first time in Lingulodinium polyedrum cultures, mainly in the cells but also, to a lesser extent, dissolved in the culture medium. The production of yessotoxins (YTXs) by cultures of different strains of P. reticulatum and L. polyedrum was studied with liquid chromatography coupled to fluorometric detection using the dienophile reagent DMEQ-TAD and liquid chromatography–mass spectrometry. When comparing toxin production at different stages of culture growth, larger amounts of toxins were observed in the cellular fraction and in the culture medium at the last stage of the culture (day 21) in both species. Although YTX was detected in culture medium, with this study it was not possible to explain which is the release mechanism of the toxin in the medium. [Copyright &y& Elsevier]

This study reports, for the first time, immunocytochemical evidence of the distribution of algal yessotoxins (YTXs) in the mussel Mytilus galloprovincialis. Immunopositivity to YTXs was found in immunocytes and in the digestive gland. With regards the gland, the positivity was mainly present in the lumen of both tubules and ducts. No YTXs were detected in the gonads, while the presence of toxins in the gills cannot be excluded. The data are supported by both HPLC analysis and functional assays. [Copyright &y& Elsevier]

Yessotoxins are a group of large polyether toxins, produced by marine dinoflagellates, which cause widespread contamination of filter-feeding shellfish. A new, sensitive liquid chromatography–mass spectrometry (LC–MS) method has been developed for the determination of yessotoxin (YTX) and 45-hydroxy-yessotoxin (45-OHYTX), a major metabolite in shellfish. The LC system was coupled, via an electrospray ionisation (ESI) source, to an ion-trap MS in negative mode. The molecular related ion species at m/z 1141 [M-2Na+H]− was used as the parent ion for multiple MS experiments. MS–MS and MS3 gave major fragment ions at m/z 1061 [1141-SO3H]− and m/z 945 [1061-C9H12O]−. Predominant ions, that are due to the fragmentation of the backbone structure of YTXs, were observed at the MS4 stage. Reversed-phase LC using a C16 amide column was preferable to C18 phases for the separation of YTX and 45-OHYTX. Optimum calibration and reproducibility data were obtained for YTX using LC–MS–MS; r2=0.9960, RSD≤6.3% at 0.25 μg YTX/g (n=5). The detection limit (S/N=3) was 30 pg YTX on-column which corresponded to 3 ng/g shellfish tissue. [Copyright &y& Elsevier]

Toxin profiles were determined in phytoplankton cell concentrates and Greenshell mussels (Perna canaliculus) exposed to a dinoflagellate bloom dominated by Dinophysis acuta and Protoceratium reticulatum. This was achieved by using a method for the simultaneous identification and quantification of a variety of micro-algal toxins by liquid chromatography–tandem mass spectrometry (LC–MS/MS) with electrospray ionisation (+/−) and monitoring of daughter ions in multiple reaction modes. Plankton concentrates and shellfish contained high levels of yessotoxins (YTXs) and pectenotoxins (PTXs) and low levels of okadaic acid (OA). A high proportion (>87%) of the OA in both plankton and shellfish was released by alkaline hydrolysis. An isomer of pectenotoxin 1 (PTX1i) was nearly as abundant as pectenotoxin 2 (PTX2) in the plankton and shellfish, and the latter contained high levels of their respective seco acids. DTX1, DTX2, and PTX6 were not detected. MS–MS experiments revealed that the shellfish contained several other oxygenated metabolites of YTX in addition to 45-hydroxy yessotoxin (45OH-YTX). Gymnodimine (GYM) was present in the shellfish but not plankton and it was probably the residue from a previous GYM contamination event. Unlike the other toxins, GYM was concentrated in tissues outside the digestive gland and levels did not decrease over 5 months. The depuration rates of YTX and PTXs from mussels were modelled. [Copyright &y& Elsevier]

A liquid chromatography mass spectrometry (LC–MS) method is proposed for the sensitive, specific and direct detection of yessotoxin and its analogues, marine biotoxins which are associated with diarrhetic shellfish poisoning (DSP) and which have been found in the North Adriatic sea since 1995. The LC–MS method provided a detection limit of 70 pg for yessotoxin in full scan mode and was applied to determine the toxic profiles of a number of extracts or partially purified fractions of toxic mussels collected along the Emilia Romagna coasts (Italy) in the period 1995–1999. Detection of a desulfo-yessotoxin derivative from Mytilus galloprovincialis collected in 1998 is also reported. [ABSTRACT FROM AUTHOR]

In the last decades, due to monitoring programs and strict legislation poisoning incidents occurrence provoked by ingestion of naturally contaminated marine organisms has decreased. However, climate change and anthropogenic interference contributed to the expansion and establishment of toxic alien species to more temperate ecosystems. In this work, the coasts of Madeira, São Miguel islands and the northwestern Moroccan coast were surveyed for four groups of lipophilic toxins (yessotoxins, azaspiracids, pectenotoxins, and spirolides), searching for new vectors and geographical tendencies. Twenty-four species benthic organisms were screened using UHPLC-MS/MS technique. We report 19 new vectors for these toxins, six of them with commercial interest (P. aspera , P. ordinaria , C. lampas , P. pollicipes , H. tuberculata and P. lividus). Regarding toxin uptake a south-north gradient was detected. This study contributes to the update of monitoring routines and legislation policies, comprising a wider range of vectors, to better serve consumers and ecosystems preservation. Unlabelled Image • 19 new vectors were detected for the screened toxins in edible and commercial interest species. • Monitoring practices regarding lipophilic toxins should comprise a wider range of vectors. • Geographical tendencies regarding lipophilic toxins distribution were unravelled. [ABSTRACT FROM AUTHOR]

• AZA and YTX group toxin results from over 5 years of monitoring in bivalves. • Large interannual variation in AZA occurrences. • Winter occurrences of AZAs noted in some years. • AZA1 the most dominant in the AZA group. • YTXs present mainly during summer months in focused areas. • Protoceratium reticulatum as the likely source of YTXs in British bivalves. Cefas has been responsible for the delivery of official control biotoxin testing of bivalve molluscs from Great Britain for just over a decade. Liquid chromatography tandem mass spectrometric (LC–MS/MS) methodology has been used for the quantitation of lipophilic toxins (LTs) since 2011. The temporal and spatial distribution of okadaic acid group toxins and profiles in bivalves between 2011 and 2016 have been recently reported. Here we present data on the two other groups of regulated lipophilic toxins, azaspiracids (AZAs) and yessotoxins (YTXs), over the same period. The latter group has also been investigated for a potential link with Protoceratium reticulatum and Lingulodinium polyedra , both previously recognised as YTXs producing phytoplankton. On average, AZAs were quantified in 3.2% of all tested samples but notable inter-annual variation in abundance was observed. The majority of all AZA contaminated samples were found between July 2011 and August 2013 in Scotland, while only two, three-month long, AZA events were observed in 2015 and 2016 in the south-west of England. Maximum concentrations were generally reached in late summer or early autumn. Reasons for AZAs persistence during the 2011/2012 and 2012/2013 winters are discussed. Only one toxin profile was identified, represented by both AZA1 and AZA2 toxins at an approximate ratio of 2 : 1, suggesting a single microalgal species was the source of AZAs in British bivalves. Although AZA1 was always the most dominant toxin, its proportion varied between mussels, Pacific oysters and surf clams. The YTXs were the least represented group among regulated LTs. YTXs were found almost exclusively on the south-west coast of Scotland, with the exception of 2013, when the majority of contaminated samples originated from the Shetland Islands. The highest levels were recorded in the summer months and followed a spike in Protoceratium reticulatum cell densities. YTX was the most dominant toxin in shellfish, further strengthening the link to P. reticulatum as the YTX source. Neither homo-YTX, nor 45−OH homo-YTX were detected throughout the monitored period. 45−OH YTX, thought to be a shellfish metabolite associated with YTX elimination, contributed on average 26% in mussels. Although the correlation between 45−OH YTX abundance and the speed of YTX depuration could not be confirmed, we noted the half-life of YTX was more than two-times longer in queen scallops, which contained 100% YTX, than in mussels. No other bivalve species were affected by YTXs. This is the first detailed evaluation of AZAs and YTXs occurrences and their profiles in shellfish from Great Britain over a period of multiple years. [ABSTRACT FROM AUTHOR]