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The presence of yessotoxins (YTXs) was analyzed in 10,757 samples of Galician bivalves from 2014 to 2022. Only YTX and 45-OH YTX were found. YTX was detected in 31% of the samples, while 45-OH YTX was found in 11.6% of them. Among the samples containing YTX, 45-OH YTX was detected in 37.3% of cases. The maximum recorded levels were 1.4 and 0.16 mg of YTX-equivalentsg−1, for YTX and 45-OH YTX, respectively, which are well below the regulatory limit of the European Union. The YTX and 45-OH YTX toxicities in the raw extracts and extracts subjected to alkaline hydrolysis were strongly and linearly related. Due to the lack of homo-YTX in Galician samples, the effect of alkaline hydrolysis on homo-YTX and 45OH-Homo-YTX was only checked in 23 additional samples, observing no negative effect but a high correlation between raw and hydrolyzed extracts. Hydrolyzed samples can be used instead of raw ones to carry out YTXs determinations in monitoring systems, which may increase the efficiency of those systems where okadaic acid episodes are very frequent and therefore a higher number of hydrolyzed samples are routinely analyzed. The presence of YTX in the studied bivalves varied with the species, with mussels and cockles having the highest percentages of YTX-detected samples. The presence of 45-OH YTX was clearly related to YTX and was detected only in mussels and cockles. Wild populations of mussels contained proportionally more 45-OH YTX than those that were raft-cultured. Spatially, toxin toxicities varied across the sampling area, with higher levels in raft-cultured mussels except those of Ría de Arousa. Ría de Ares (ARE) was the most affected geographical area, although in other northern locations, lower toxin levels were detected. Seasonally, YTX and 45-OH YTX toxicities showed similar patterns, with higher levels in late summer and autumn but lower toxicities of the 45-OH toxin in August. The relationship between the two toxins also varied seasonally, in general with a minimum proportion of 45-OH YTX in July–August but with different maximum levels for raft-cultured and wild mussel populations. Interannually, the average toxicities of YTX decreased from 2014 to 2017 and newly increased from 2018 to 2021, but decreased slightly in 2022. The relationship between 45-OH YTX and YTX also varied over the years, but neither a clear trend nor a similar trend for wild and raft mussels was observed. [ABSTRACT FROM AUTHOR]

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]

In late summer and early autumn 2022, an intense bloom of Protoceratium reticulatum —the main yessotoxin (YTX) producer along Chilean coasts and a major threat to artisanal fisheries, the aquaculture industry, and environmental health—was recorded in the Patagonian fjord system. The high YTX levels (>3.75 mg kg−1) resulted in the first ban of shellfish collection in Chile. At Puyuhuapi Fjord, a global "hotspot" of harmful algal bloom events, the cell density of P. reticulatum determined in integrated tube samples (0–10 m) at the end of April 2022 reached 407,000 cells L−1. At the same time, YTX levels well exceeded the regulatory limit by roughly 2.5-fold, with concentrations as high as 9.42 mg kg−1 measured in native populations of the blue mussel Mytilus chilensis. Five different YTX analogues, 45-OH-YTX, COOH-45-keto-YTX, COOH-45-OH-YTX, COOH-YTX, and 45,55-diOH-YTX, were also detected in relevant amounts. While the ban lasted close to 3 months, accumulation and detoxification processes were monitored over a 1-year period. This study assessed the implications of high levels of YTXs and their analogues on the local economy and ecosystem health, given the increase in P. reticulatum blooms predicted for NW Patagonia in the context of a changing climate. [Display omitted] • Protoceratium reticulatum reached a maximum cell density of 407,000 cells L−1 in integrated tube samples (0–10 m). • First report of YTX levels in mussels exceeding the regulatory limit in Chile. • A maximum yessotoxin of 9.42 mg YTX kg−1 was detected in native populations of Mytilus chilensis. • Five different YTX analogues were identified. • Investigate accumulation and detoxification processes were followed over a 1-year period. [ABSTRACT FROM AUTHOR]

The presence of yessotoxins (YTXs) was analyzed in 10,757 samples of Galician bivalves from 2014 to 2022. Only YTX and 45-OH YTX were found. YTX was detected in 31% of the samples, while 45-OH YTX was found in 11.6% of them. Among the samples containing YTX, 45-OH YTX was detected in 37.3% of cases. The maximum recorded levels were 1.4 and 0.16 mg of YTX-equivalentsg−1, for YTX and 45-OH YTX, respectively, which are well below the regulatory limit of the European Union. The YTX and 45-OH YTX toxicities in the raw extracts and extracts subjected to alkaline hydrolysis were strongly and linearly related. Due to the lack of homo-YTX in Galician samples, the effect of alkaline hydrolysis on homo-YTX and 45OH-Homo-YTX was only checked in 23 additional samples, observing no negative effect but a high correlation between raw and hydrolyzed extracts. Hydrolyzed samples can be used instead of raw ones to carry out YTXs determinations in monitoring systems, which may increase the efficiency of those systems where okadaic acid episodes are very frequent and therefore a higher number of hydrolyzed samples are routinely analyzed. The presence of YTX in the studied bivalves varied with the species, with mussels and cockles having the highest percentages of YTX-detected samples. The presence of 45-OH YTX was clearly related to YTX and was detected only in mussels and cockles. Wild populations of mussels contained proportionally more 45-OH YTX than those that were raft-cultured. Spatially, toxin toxicities varied across the sampling area, with higher levels in raft-cultured mussels except those of Ría de Arousa. Ría de Ares (ARE) was the most affected geographical area, although in other northern locations, lower toxin levels were detected. Seasonally, YTX and 45-OH YTX toxicities showed similar patterns, with higher levels in late summer and autumn but lower toxicities of the 45-OH toxin in August. The relationship between the two toxins also varied seasonally, in general with a minimum proportion of 45-OH YTX in July–August but with different maximum levels for raft-cultured and wild mussel populations. Interannually, the average toxicities of YTX decreased from 2014 to 2017 and newly increased from 2018 to 2021, but decreased slightly in 2022. The relationship between 45-OH YTX and YTX also varied over the years, but neither a clear trend nor a similar trend for wild and raft mussels was observed.

Blooms of marine planktonic dinoflagellates are usual along the Pacific coast of Mexico, sometimes producing toxic red tides. The thecate dinoflagellate Gonyaulax areolata was found to produce a red tide event in the tropical Mexican Pacific. The species was originally described in 1911 by Kofoid and Michener without an illustration. The morphology of G. areolata was studied here by LM and SEM. Photosynthetic, solitary cells were fairly small, polyhedral, with a short apical protrusion and 'shoulders' on the epitheca, and two short, asymmetrical antapical spines. The cingulum was approximately median, cavozone, with the ends fairly displaced but with no, or only slight, overhang. The thecae were strongly ornamented with striae, areolae and pores. The plate formula was Po, 4ʹ, Q, 6ʹʹ, 6c, 4s, 6ʹʹʹ, 1p, 1ʹʹʹʹ, as recently attributed to some species of Gonyaulax. Details of important plates are given. Gonyaulax areolata seems to be morphologically closely related to other small species with a slight cingulum displacement and overhang, such as G. minima and G. striata, and more distantly related to G. spinifera. The maximum concentration of G. areolata was 1.74 × 106 cells l–1, in association with lower numbers of other dinoflagellates and diatoms. Yessotoxins were not detected. Oceanographic conditions during the bloom indicated moderate to weak upwelling, confirmed by both relatively low surface temperature and high Chl a concentration (14 µg l–1). [ABSTRACT FROM AUTHOR]

• An intense P. reticulatum summer bloom occurred in Puyuhuapi Fjord, Southern Chile. • Surface aggregation of the P. reticulatum cells was modulated by the light cycle, suggesting daily vertical migration (DVM). • P. reticulatum cell numbers and yessotoxins followed a synchronic distribution pattern. • The yessotoxin content per P. reticulatum cell ranged from 9.4 to 52.2 pg. Protoceratium reticulatum is the main yessotoxin-producer along the Chilean coast. Thus far, the yessotoxin levels recorded in this region have not posed a serious threat to human health. However, a bloom of P. reticulatum during the austral summer of 2022 caused the first ban of shellfish collection, due to the high toxin levels. A bloom of P. reticulatum during the austral summer of 2020 allowed an evaluation of the fine-scale distribution of the dinoflagellate during a tidal cycle. High-resolution measurements of biophysical properties were carried out in mid-summer (February 18–19) at a fixed sampling station in Puyuhuapi Fjord, Chilean Patagonia, as part of an intensive 24-h biophysical experiment to monitor the circadian distributions of P. reticulatum vegetative cells and yessotoxins. High P. reticulatum cell densities (>20 × 103 cells L–1) were found in association with a warmer (14.5–15 °C) and estuarine (23.5–24.5 g kg–1) sub-surface water layer (6–8 m). P. reticulatum cell numbers and yessotoxins followed a synchronic distribution pattern consistent with the excursions of the pycnocline. Nevertheless, the surface aggregation of the cells was modulated by the light cycle, suggesting daily vertical migration. The yessotoxin content per P. reticulatum cell ranged from 9.4 to 52.2 pg. This study demonstrates both the value of fine-scale resolution measurements of biophysical properties in a highly stratified system and the potential ecosystem impact of P. reticulatum strains producing high levels of yessotoxins. [ABSTRACT FROM AUTHOR]

Abstract 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 μg OA eq/kg or 217 μg AZA1 eq/kg to ensure that levels in gonads are below the regulatory limits of 160 μg 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 μg OA eq/kg, a pooled sample of more than 30 scallops would have to be tested.

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]

Yessotoxins (YTXs) are polycyclic toxic ether compounds produced by phytoplanktonic dinoflagellates which accumulate in filter-feeding organisms. We know that the water temperature in our areas Northwestern Adriatic Sea is optimal for the growth of potentially toxic algae (around 20 °C). In recent years, these temperatures have remained at these levels for longer and longer periods, probably due to global warming, which has led to an excessive increase in toxin levels. The interruption of mussel harvesting caused by algae negatively affects farmers’ revenues and the availability of local fish, causing a major economic loss in Italy’s main shellfish sector. Methods: In the nine years considered, 3359 samples were examined: 1715 marine waters, 73 common clams; 732 mussels; 66 oysters; and 773 veracious clams. Bivalve molluscs were examined for the presence of marine biotoxins, including YTXs, while potentially toxic algae, including those producing YTXs, were searched for and counted in marine waters. The method adopted for the quantification of lipophilic toxins involves the use of an LC-MS/MS system. The enumeration of phytoplankton cells was performed according to the Utermhöl method. Results: Between 2012 and 2020, 706 molluscs were tested for YTXs. In total, 246 samples tested positive, i.e., 34.84%. Of the positive samples, 30 exceeded the legal limit. Conclusion: In this regard, it is essential to develop and activate, as soon as possible, an “early warning” system that allows a better control of the production areas of live bivalve molluscs, thus allowing an optimal management of the plants in these critical situations.

Highlights • Death of several million farmed abalone linked to YTX-producing dinoflagellates. • YTX analogues homo-YTX and 45-hydroxy-YTX were dominant in plankton and abalone. • Gonyaulax spinifera implicated as the likely source of YTX. • Abalone gills were characterized by degeneration and necrosis of epithelial cells. Abstract A large dinoflagellate bloom in Walker Bay (South Africa) in January 2017 impacted 3 land-based abalone farms resulting in the death of several million animals. Satellite-derived images of Chl- a from the Ocean and Land Colour Imager (OLCI) on board the European Space Agency Sentinel-3 A showed bloom initiation in late December 2016 and dispersal in mid-February 2017. The bloom was dominated by two dinoflagellate species identified by light microscopy as Gonyaulax spinifera (Claparède & Lachmann) Diesing, 1866 and Lingulodinium polyedrum (Stein) Dodge, 1989. These morphologically based identifications were confirmed by phylogenetic analysis using partial sequences of the large subunit rDNA of both dinoflagellates. The appearance of yessotoxins (YTX) in abalone clearly coincided with increases in dinoflagellate concentrations. Yessotoxins in both the plankton and abalone were dominated by the two analogues homo-YTX and 45-hydroxy-YTX. The absence of toxins in a clonal culture of L. polyedrum implicated G. spinifera as the likely source of YTX. Toxin concentrations were found to be highest in the gills which showed the most significant pathology, including severe, generalized disruption of the gill epithelium characterized by degeneration and necrosis of epithelial cells accompanied by a modest inflammatory response. Some farms undertook pre-emptive or emergency harvesting to reduce financial losses. [ABSTRACT FROM AUTHOR]

Respiratory exposure to marine phycotoxins is of increasing concern. Inhalation of sea spray aerosols (SSAs), during harmful Karenia brevis and Ostreopsis ovata blooms induces respiratory distress among others. The biogenics hypothesis, however, suggests that regular airborne exposure to natural products is health promoting via a downregulation of the mechanistic target of rapamycin (mTOR) pathway. Until now, little scientific evidence supported this hypothesis. The current explorative in vitro study investigated both health-affecting and potential health-promoting mechanisms of airborne phycotoxin exposure, by analyzing cell viability effects via cytotoxicity assays and effects on the mTOR pathway via western blotting. To that end, A549 and BEAS-2B lung cells were exposed to increasing concentrations (ng·L−1−mg·L−1) of (1) pure phycotoxins and (2) an extract of experimental aerosolized homoyessotoxin (hYTX). The lowest cell viability effect concentrations were found for the examined yessotoxins (YTXs). Contradictory to the other phycotoxins, these YTXs only induced a partial cell viability decrease at the highest test concentrations. Growth inhibition and apoptosis, both linked to mTOR pathway activity, may explain these effects, as both YTXs were shown to downregulate this pathway. This proof-of-principle study supports the biogenics hypothesis, as specific aerosolizable marine products (e.g., YTXs) can downregulate the mTOR pathway.

• An unprecedented bloom of Lingulodinium polyedra was observed on the French Atlantic coast during summer 2021. • Unusually high precipitation during mid-July led to increased river discharges, which likely triggered the bloom. • The bloom deposited a significant seed bank. • The bloom caused hypoxia events, high biomass and increased concentrations of yessotoxins. At the end of July 2021, a bloom of Lingulodinium polyedra developed along the French Atlantic coast and lasted six weeks. The REPHY monitoring network and the citizen participation project PHENOMER contributed to its observation. A maximum concentration of 3,600,000 cells/L was reached on the 6th of September, a level never recorded on French coastlines. Satellite observation confirmed that the bloom reached its highest abundance and spatial extension early September, covering about 3200 km2 on the 4th of September. Cultures were established, and morphology and ITS-LSU sequencing identified the species as L. polyedra. The thecae displayed the characteristic tabulation and sometimes a ventral pore. The pigment composition of the bloom was similar to that of cultured L. polyedra , confirming that phytoplankton biomass was dominated by this species. The bloom was preceded by Leptocylindrus sp., developed over Lepidodinium chlorophorum , and was succeeded by elevated Noctiluca scintillans concentrations. Afterwards, relatively high abundance of Alexandrium tamarense were observed in the embayment where the bloom started. Unusually high precipitation during mid-July increased river discharges from the Loire and Vilaine rivers, which likely fueled phytoplankton growth by providing nutrients. Water masses with high numbers of dinoflagellates were characterized by high sea surface temperature and thermohaline stratification. The wind was low during the bloom development, before drifting it offshore. Cysts were observed in the plankton towards the end of the bloom, with concentrations up to 30,000 cysts/L and relative abundances up to 99%. The bloom deposited a seed bank, with cyst concentrations up to 100,000 cysts/g dried sediment, particularly in fine-grained sediments. The bloom caused hypoxia events, and concentrations of yessotoxins up to 747 μg/kg were recorded in mussels, below the safety threshold of 3,750 μg/kg. Oysters, clams and cockles also were contaminated with yessotoxins, but at lower concentrations. The established cultures did not produce yessotoxins at detectable levels, although yessotoxins were detected in the sediment. The unusual environmental summertime conditions that triggered the bloom, as well as the establishment of considerable seed banks, provide important findings to understand future harmful algal blooms along the French coastline. [ABSTRACT FROM AUTHOR]

A new Pentaplacodinium species with six precingular plates is described from Bahía Concepción and Bahía de la Paz, Gulf of California. The non-fossil motile stage is described as Pentaplacodinium lapazense , whilst the fossil stage is described as Operculodinium lapazense. The cyst morphology is compared to topotype material of Operculodinium israelianum , which is larger, has longer processes and has a different wall structure. The motile cells display a plate formula of Po, Pt, X, 2′+*2′, 6′′, 6c, 7s, 5′′′, 1p, 1′′′′. A typical gonyaulacalean fission line and plate overlap are observed. SSU-ITS-LSU ribosomal DNA sequences demonstrate that Pentaplacodinium saltonense is its closest relative. The species is homothallic. This species occurs in relatively shallow and restricted coastal areas, and has a preference for higher sea-surface temperatures and salinities. Micro-FTIR spectra of the cysts are compared to spectra of cysts of other gonyaulacaleans and suggest very similar compositions. No yessotoxins were detected in any of the analyzed strains, hence, this species is unlikely to be responsible for the elevated yessotoxin concentration observed in shellfish on the southern and central coastal region of the Gulf of California. • A new species Pentaplacodinium lapazense is described from the southwestern coastal region of the Gulf of California, Mexico. • The plate formula is: Po, Pt, X, 2′+*2′, 6′′, 6c, 7s, 5′′′, 1p, 1′′′′. • The corresponding cyst stage is similar to Operculodinium israelianum and is named Operculodinium lapazense. • No yessotoxins were detected in the analyzed ten strains. [ABSTRACT FROM AUTHOR]

Harmful algal blooms (HABs) are recurrent in the NW Patagonia fjords system and their frequency has increased over the last few decades. Outbreaks of HAB species such as Alexandrium catenella , a causal agent of paralytic shellfish poisoning, and Protoceratium reticulatum , a yessotoxins producer, have raised considerable concern due to their adverse socioeconomic consequences. Monitoring programs have mainly focused on their planktonic stages, but since these species produce benthic resting cysts, the factors influencing cyst distributions are increasingly gaining recognition as potentially important to HAB recurrence in some regions. Still, a holistic understanding of the physico-chemical conditions influencing cyst distribution in this region is lacking, especially as it relates to seasonal changes in drivers of cyst distributions, as the characteristics that favor cyst preservation in the sediment may change through the seasons. In this study, we analyzed the physico–chemical properties of the sediment (temperature, pH, redox potential) and measured the bottom dissolved oxygen levels in a "hotspot" area of southern Chile, sampling during the spring and summer as well as the fall and winter, to determine the role these factors may play as modulators of dinoflagellate cyst distribution, and specifically for the cysts of A. catenella and P. reticulatum. A permutational analysis of variance (PERMANOVA) showed the significant effect of sediment redox conditions in explaining the differences in the cyst assemblages between spring-summer and fall-winter periods (seasonality). In a generalized linear model (GLM), sediment redox potential and pH were associated with the highest abundances of A. catenella resting cysts in the spring-summer, however it was sediment temperature that most explained the distribution of A. catenella in the fall-winter. For P. reticulatum , only spring-summer sediment redox potential and temperature explained the variation in cyst abundances. The implications of environmental (physico-chemical) seasonality for the resting cysts dynamics of both species are discussed. [Display omitted] • Physico-chemical drivers explain differences in dinoflagellate resting cyst assemblages modulated by seasonality. • Anoxic sediments are associated with higher abundances of A. catenella and P. reticulatum cysts. • There is no evidence of a large seedbed of A. catenella cysts after the 2016 bloom event. • Sediment grain size composition is not a good predictor of A. catenella cysts abundance. • The abundance and richness of cysts was higher for heterotrophic dinoflagellate species than for autotrophic species. [ABSTRACT FROM AUTHOR]

Since the late 1980s, the North-central Adriatic Sea has frequently experienced blooms of harmful algal species, producing marine lipophilic toxins (MLTs) which accumulate in mussels and pose a serious threat to consumer health. Here, we present a 2-year LC-MS/MS study (2012–2014) of the MLT profile in mussels from the North-central Adriatic Sea in the context of the presence of toxic phytoplankton concentrations in seawater. Okadaic acid increased in mussels from all areas during the summer and autumn-winter periods with a rising trend between 2012 and 2014. In the same periods, Dinophysis sp. increased in abundance in seawater, but the highest densities of algae did not always coincide with the highest levels of toxins in mussels. Yessotoxins (YTXs) content in mussel increased sharply in the autumn-winter periods even exceeding the legal limit; although this accumulation did not always correlated with the YTX-producers in water (such as Lingulodinium polyedrum and Protoceratium reticulatum ) a massive bloom of Gonyaulax spinifera was reported in November 2013, suggesting the role of this species in YTXs shellfish contamination. Traces of Azaspiracid 2 (AZA-2) were observed often in mussels during the study period, confirming for the first time the presence of this biotoxin in Mediterranean seafood. [ABSTRACT FROM AUTHOR]

During the last decade, a significant increase in the occurrence of harmful algal blooms (HABs), linked to repetitive cases of shellfish contamination has become a public health concern and therefore, accurate methods to detect marine toxins in different matrices are required. In this study, we developed a method for profiling lipophilic marine microalgal toxins based on ultra-high-performance liquid chromatography coupled to high-resolution Orbitrap mass spectrometry (UHPLC-HR-Orbitrap MS). Extraction of selected toxins (okadaic acid (OA), dinophysistoxin-1 (DTX-1), pectenotoxin-2 (PTX-2), azaspiracid-1 (AZA-1), yessotoxin (YTX) and 13-desmethyl spirolide C (SPX-1)) was optimized using a Plackett-Burman design. Three key algal species, i.e., Prorocentrum lima, Protoceratium reticulatum and Alexandrium ostenfeldii were used to test the extraction efficiency of OA, YTXs and SPXs, respectively. Prorocentrum micans, fortified with certified reference solutions, was used for recovery studies. The quantitative and confirmatory performance of the method was evaluated according to CD 2002/657/EC. Limits of detection and quantification ranged between 0.006 and 0.050 ng mL and 0.018 to 0.227 ng mL, respectively. The intra-laboratory reproducibility ranged from 6.8 to 11.7 %, repeatability from 6.41 to 11.5 % and mean corrected recoveries from 81.9 to 119.6 %. In addition, algae cultures were retrospectively screened for analogues and metabolites through a homemade database. Using the ToxID software programme, 18 toxin derivates were detected in the extract of three toxin producing microalgae species. In conclusion, the generic extraction and full-scan HRMS approach offers an excellent quantitative performance and simultaneously allows to profile analogues and metabolites of marine toxins in microalgae. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

Protoceratium reticulatum is a dinoflagellate with a life cycle that includes a motile planktonic stage and a resting cyst stage in benthic habitat, both with a wide geographical distribution, including southern South America. P. reticulatum produces yessotoxins (YTX) – these can be accumulated in shellfish and show potent cytotoxicity, posing a risk to human health if contaminated shellfish is consumed. YTX have been reported from coastal shellfish of many localities, but until now it was unknown if they were present in the austral Southwestern Atlantic and also if local populations of P. reticulatum have the ability to produce these toxins. In this study we report the presence of YTX in plankton samples and its production in culture by two P. reticulatum strains isolated from the San Jorge Gulf (SJG). In addition, we describe the geographical distribution and seasonal abundance of this species based on data collected over the past two decades. The YTX cell quotas calculated from net hauls (∼10 pg cell −1 ) are in the same range as the toxin cell quotas observed in these two isolates. The phylogenetic analysis of sequences of the hypervariable region of the large subunit (LSU) 28S rDNA showed that the two clonal strains from the SJG were part of a monophyletic clade that subdivides P. reticulatum into two well-supported, divergent sub-clades. The sequences of the two strains of P. reticulatum from the SJG fell in the same clade as the majority of sequences of P. reticulatum , which belong to a geographically widely distributed evolutionary clade. P. reticulatum was occasionally observed from about 35° S in Uruguayan shelf waters up to 53° S on the Patagonian shelf and north of Tierra del Fuego, and it was present from coastal areas up to the shelf break zone. We recorded P. reticulatum in plankton samples during spring, summer and autumn but invariably in low abundance (maximum: 560 cells L −1 ). Viable cysts of the species in surface sediments also showed a wide geographical distribution. Together, the high total abundances and high relative numerical contribution to planktonic dinoflagellate assemblages near frontal areas, emphasize the necessity to pay attention to the dynamics of this species in areas of potential risk of harmful algal bloom development. [ABSTRACT FROM AUTHOR]

Lipophilic marine toxins are produced by harmful microalgae and can accumulate in edible filter feeders such as shellfish, leading to an introduction of toxins into the human food chain, causing different poisoning effects. During the last years, analytical methods, based on liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), have been consolidated by interlaboratory validations. However, the main drawback of LC-MS/MS methods remains the limited number of compounds that can be analyzed in a single run. Due to the targeted nature of these methods, only known toxins, previously considered during method optimization, will be detected. Therefore in this study, a method based on ultra-high-performance liquid chromatography coupled to high-resolution Orbitrap mass spectrometry (UHPLC-HR-Orbitrap MS) was developed. Its quantitative performance was evaluated for confirmatory analysis of regulated lipophilic marine toxins in shellfish flesh according to Commission Decision 2002/657/EC. Okadaic acid (OA), dinophysistoxin-1 (DTX-1), pectenotoxin-2 (PTX-2), azaspiracid-1 (AZA-1), yessotoxin (YTX), and 13-desmethyl spirolide C (SPX-1) were quantified using matrix-matched calibration curves (MMS). For all compounds, the reproducibility ranged from 2.9 to 4.9 %, repeatability from 2.9 to 4.9 %, and recoveries from 82.9 to 113 % at the three different spiked levels. In addition, confirmatory identification of the compounds was effectively performed by the presence of a second diagnostic ion (C). In conclusion, UHPLC-HR-Orbitrap MS permitted more accurate and faster detection of the target toxins than previously described LC-MS/MS methods. Furthermore, HRMS allows to retrospectively screen for many analogues and metabolites using its full-scan capabilities but also untargeted screening through the use of metabolomics software. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

The fine scale vertical distribution of Dinophysis spp. and Protoceratium reticulatum (potential producers of lipophilic shellfish toxins, LSTs) and its relation with LSTs in shellfish was studied in Reloncaví fjord, a strongly stratified system in Southern Chile. Samples were taken over two years from late spring to early autumn (2007–2008 period) and from early spring to late summer (2008–2009 period). Dinophysis spp., in particular Dinophysis acuminata, were always detected, often forming thin layers in the region of the salinity driven pycnocline, with cell maxima for D. acuminata of 28.5×103 cellsL−1 in March 2008 and 17.1×103 cellsL−1 in November 2008. During the 2008–2009 sampling period, blooms of D. acuminata co-occurred with high densities of cryptophyceans and the ciliate Mesodinium spp. The highest levels of pectenotoxin-2 (PTX-2; 2.2ng L−1) were found in the plankton in February 2009, associated with moderate densities of D. acuminata, Dinophysis tripos and Dinophysis subcircularis (0.1–0.6×103 cellsL−1). However, only trace levels of PTX-2 were observed in bivalves at that time. Dinophysistoxin (DTX-1 and DTX-3) levels in bivalves and densities of Dinophysis spp. were not well correlated. Low DTX levels in bivalves observed during a major bloom of D. acuminata in March 2008 suggested that there is a large seasonal intraspecific variability in toxin content of Dinophysis spp. driven by changes in population structure associated with distinct LST toxin profiles in Reloncaví fjord during the study period. A heterogeneous vertical distribution was also observed for P. reticulatum, whose presence was restricted to summer months. A bloom of this species of 2.2×103 cellsL−1 at 14m depth in February 2009 was positively correlated with high concentrations of yessotoxins in bivalves (51–496ngg−1) and plankton samples (3.2ngL−1). Our results suggest that a review of monitoring strategies for Dinophysis spp. in strongly stratified fjord systems should be carried out. They also indicate that early warning of LST events based on Dinophysis cell numbers are not reliable for seafood control. [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]

In this paper, we extend an existing approach to biotechnologically assess grazer-prey interactions between the crustacean Artemia salina (grazer) and the toxic dinoflagellate Protoceratium reticulatum (prey). The applied strategy is presented as a bioprocessing tool for enhancing the production of toxins and bioactive compounds in dinoflagellate cultures. Interactions were based on direct and indirect contact between the grazer and the prey, as well as on the use of different extracts from A. salina cysts and supernatants from cultures in which A. salina had been grown. Several treatments were found to stimulate the growth and yessotoxin production of P. reticulatum mainly due to the action of dissolved excreted substances and/or metabolites released and/or extracted from A. salina. One of the best results was obtained with a culture medium formulation containing 10 % ( v/ v) supernatant from a culture of A. salina nauplii. This treatment was scaled up to a 15-L photobioreactor. The average maximum specific growth rate ( μ) of P. reticulatum in this photobioreactor, operated in batch mode, increased by 27 %, whereas the maximum cell concentration ( C) decreased by 20 % relative to the corresponding control culture. An average increase in yessotoxin production of 50 % with respect to the control culture was observed. [ABSTRACT FROM AUTHOR]

Abstract: Yessotoxins (YTXs) production along the culture growth of three strains of the dinoflagellate Protoceratium reticulatum isolated from seawater of Galician Rias Baixas, Spain was investigated. Quantification and toxin profile determination in both cells and culture medium along the growth curve were performed by liquid chromatography–mass spectrometry (LC–MS3) analysis. The YTX profile was very similar among strains, the three algal strains produce mainly YTX and also some YTX analogs. Among the strains the maximum toxin production ranged between 416 and 576ngmL−1. This is the first report about YTX production by P. reticulatum isolated in Galician coast, NW Spain. [Copyright &y& Elsevier]

Abstract: A genetic algorithm stochastic search strategy was used to optimize the culture medium for producing the toxic marine dinoflagellate microalga Protoceratium reticulatum. The optimized medium contained 26 different components (macronutrients, trace elements, vitamins). The use of this medium allowed a 60% enhancement in the final cell concentration relative to the control culture based on L1 medium. The final titer of yessotoxins was improved by 40% relative to the control medium. [Copyright &y& Elsevier]

Abstract: Large scale culture of marine dinoflagellate microalgae has proved difficult owing to their extreme sensitivity to hydrodynamic forces in photobioreactors. This work discusses the scale up of Protoceratium reticulatum dinoflagellate culture from a successful 2L stirred-tank photobioreactor operation to a 15L stirred photobioreactor. Both bioreactors were equipped with internal spinfilters for cell retention. A semicontinuous perfusion culture in the 15L photobioreactor proved to be more productive than fed-batch perfusion culture. Under the best operational conditions, the average cell productivity in the 15L photobioreactor was 5228cellmL−1 day−1, or nearly 3.7-fold greater than the best attainable value in static flask cultures, but similar to the results obtained in the 2L stirred tank. At 9.16μgL−1 day−1 the average volumetric productivity of yessotoxin in the scaled up operation in the semicontinuous perfusion mode was comparable to the results obtained in the small bioreactor. P. reticulatum has a cell damage threshold shear rate of as low as 0.1s−1. Notwithstanding this extreme shear sensitivity, the results suggest a good potential for mass scale culture of this dinoflagellate in suitably designed photobioreactors. [Copyright &y& Elsevier]

Abstract: Most liquid chromatography (LC) mass spectrometric (MS) methods used for routine monitoring of lipophilic marine toxins focus on the analysis of the 13 toxins that are stated in European Union legislation. However, to date over 200 lipophilic marine toxins have been described in the literature. To fill this gap, a screening method using LC coupled to high resolution (HR) orbitrap MS (resolution 100000) for marine lipophilic toxins has been developed. The method can detect a wide variety of okadaic acid (OA), yessotoxin (YTX), azaspiracid (AZA) and pectenotoxin (PTX) group toxins. To build a library of toxins, shellfish and algae samples with various toxin profiles were obtained from Norway, Ireland, United Kingdom, Portugal and Italy. Each sample extract was analyzed with and without collision induced dissociation fragmentation. Based on their mass and specific fragmentation pattern, 85 different toxins were identified comprising 33 OA, 26 YTX, 18 AZA and 8 PTX group toxins. A major complication of full scan HRMS is the huge amount of data generated (file size), which restricts the possibility of a fast search. A software program called metAlign was used to reduce the orbitrap MS data files. The 200-fold reduced data files were screened using an additional software tool for metAlign: ‘Search_LCMS’. A search library was constructed for the 85 identified toxins. The library contains information about compound name, accurate mass, mass deviation (<5ppm), retention time (min) and retention time deviation (<0.2min). An important feature is that the library can easily be exchanged with other instruments as the generated metAlign files are not brand-specific. The developed screening procedure was tested by analyzing a set of known positive and blank samples, processing them with metAlign and searching with Search_LCMS. A toxin profile was determined for each of the contaminated samples. No toxins were found in the blank sample, which is in line with the results obtained for this sample in the routine monitoring program (rat bioassay and tandem LC–MS). [Copyright &y& Elsevier]

Abstract: This paper reports on the determination of toxin profile of mussels (Mytilus galloprovincialis) collected in November–December 2003 along the Emilia Romagna coasts (Italy) when a high concentration of Alexandrium ostenfeldii cells was detected in seawater. Detailed liquid chromatography–mass spectrometry (LC–MS) analyses were performed on the crude extracts in both selected ion monitoring (SIM) and multiple reaction monitoring (MRM) modes. They revealed that M. galloprovincialis had accumulated the three major spirolides produced by the alga, namely 13-desMethyl spirolide C, 13,19-didesMethyl spirolide C and 27-hydroxy-13,19-didesMethyl spirolide C, which fully accounted for toxicity of lipophilic extracts shown in mouse bioassay. Interestingly, yessotoxin (YTX) and its analogues were still present in mussel polar extracts but YTX itself was not the major toxin contained in mussels. The presence of pectenotoxin-2 seco acid (PTX-2sa) and its putative epimer was also assessed. The presence of azaspiracids, never reported from the Adriatic sea, as well as of diarrhetic shellfish poisoning toxins (okadaic acid, dinophysistoxins and OA esters) and domoic acid, long known as contaminants of Adriatic mussels, was also investigated. [Copyright &y& Elsevier]

Conditions for the determination of lipophilic marine toxins, such as yessotoxins and pectenotoxins (PTX)-6, were investigated with capillary electrophoresis coupled to mass spectrometry (MS) with an electrospray ionization source. After optimization, a simple and MS compatible alkaline volatile buffer solution of ammonium acetate was selected as background electrolyte, with isopropanol/water (80/20, v/v) sheath liquid modified with ammonium acetate used at the electrospray ionization (ESI) source. Previously to capillary electrophoresis (CE) separations, the application of an on-line sample pre-concentration approach based on field-amplified sample stacking was accomplished to increase sensitivity. As a result, the limits of detection provided by capillary electrophoresis-electrospray ionization-mass spectrometry (CE-ESI-MS) were 0.02 µg ml-1 (0.01 µg g-1), which corresponded to 1.25 pg for yessotoxin and 0.25 µg ml-1 (0.13 µg g-1 and 13.25 pg on capillary) for PTX-6. Accuracy tests showed 97.7% recovery from spiked blank mussel samples that showed no significant matrix influence running under optimal conditions. Intermediate precision was close to 4% relative standard deviation (RSD) for the migration time, and an RSD of 7.5% for peak areas. The method was successfully applied to naturally contaminated seafood samples in which yessotoxins and pectenotoxins-6 were clearly determined. This work demonstrated the potential of CE-ESI-MS to be applied for a sensitive determination of lipophilic toxins from the marine environment as alternative to liquid chromatography-electrospray ionization-single quadrupole mass spectrometry (LC-ESI-MS) for this purpose. [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: Yessotoxins (YTXs) are a group of polyether toxins which have been previously reported as responsible for seafood contamination in several places worldwide. Despite their toxicity, which is not yet fully discussed, YTXs have been reported as an interference in the success of mouse bioassay for the determination of diarrhetic shellfish poisoning (DSP) toxins, and therefore, efficient and reliable analytical methodologies are required to evaluate their presence, avoiding false positives for DSP. High-performance capillary electrophoresis (HPCE) is presented in this work as an alternative to HPLC technique widely used for the analysis of YTXs. Improvements in the applicability of HPCE have been carried out through the development of different CE modes as well as different detection modes. With this aim, micellar electrokinetic chromatography (MEKC) has been considered for an increased selectivity while an increased sensitivity was achieved by using sample stacking. Moreover, the coupling of CE with mass spectrometry allowed the confirmation of YTXs present in the contaminated samples evaluated in this work. The results obtained showed the potential of CE as an alternative to HPLC for the analysis of YTXs present in naturally contaminated samples. [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]

Abstract: We originally developed a functional assay for the detection of yessotoxins (YTX) based on its capacity to induce dose-dependent changes in cellular levels of two marker proteins, consisting of E-cadherin and an E-cadherin fragment (ECRA100) in epithelial cells. The procedure is time-consuming and we have shortened it by a slot blot format, using antibodies recognizing two different epitopes of E-cadherin (HECD-1 and C20820), thereby discriminating those markers. The best performing membrane under our conditions, in terms of binding capacity and even absorption of proteins, was a positively charged nylon membrane. Treatment of the membrane with 0.5μg of Ab/ml was appropriate for maximal detection of antigens by our slot blot procedure with both HECD-1 and C20820 antibodies. The treatment of cells with YTX, resulting in a relative increase in the cellular levels of ECRA100, led to a dose-dependent increase of the signal detected by Ab HECD-1 without a concomitant increase in the signal detected by Ab C20820 in our slot blot format, and the concentrations of YTX were correlated to both the increase of the signal detected through Ab HECD-1 and to the decrease in the ratio of the signals obtained with the two Abs (C20820 over HECD-1). Upon analyses of extracts from cells treated with shellfish samples, we could detect and quantify YTX in naturally contaminated materials. The slot blot format of our functional assay allows a substantial shortening of its analytical step (about seven hr, as compared to the two working days of the original method), providing YTX measurements that are accurate but show large standard deviations. [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]

Yessotoxin (YTX) treatment of MCF-7 cells results in the accumulation of a 100-kDa fragment of E-cadherin (ECRA100) without a parallel loss of the intact protein in cytosoluble extracts. As a consequence, concentration-dependent increases in the total immunoreactivity detectable by anti-E-cadherin antibodies relative to controls (RTI) and in the relative immunoreactivity of ECRA100 (RI) are observed. These responses have been exploited to develop a functional assay to measure YTX in samples from contaminated mussels by a three-step procedure, consisting of (i) treatment of MCF-7 cells with YTX standard in the concentration range 0–1 nM and of unknown samples; (ii) preparation of cellular extracts, fractionation of proteins by polyacrylamide gel electrophoresis under denaturing conditions, and immunoblotting with anti-E-cadherin antibodies, followed by densitometric analyses of autoradiographies and calculation of RI of ECRA100 and of RTI of the samples; and (iii) interpolation of the YTX concentrations in unknown samples on standard curves, by the RI of ECRA100 and the RTI of the samples. The procedure has been used to measure yessotoxins in contaminated mussel samples, and the results obtained show that this functional assay is very sensitive (limit of detection of about 100 ng equivalent YTX/g of digestive gland), and robust, as (i) it is insensitive to matrix effects in the range of toxin concentrations relevant for risk assessment to protect humans from exposure to YTX, (ii) calculations are based on a molecular parameter (the RI of ECRA100) which is not affected by errors in sample preparation, (iii) it can be performed by the use of antibodies commercially available from different companies, and (iv) it does not show an absolute need of calibration by a pure standard within each assay. [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]

Yessotoxins (YTXs) are polycyclic toxic ether compounds produced by phytoplanktonic dinoflagellates which accumulate in filter-feeding organisms. We know that the water temperature in our areas Northwestern Adriatic Sea is optimal for the growth of potentially toxic algae (around 20 °C). In recent years, these temperatures have remained at these levels for longer and longer periods, probably due to global warming, which has led to an excessive increase in toxin levels. The interruption of mussel harvesting caused by algae negatively affects farmers' revenues and the availability of local fish, causing a major economic loss in Italy's main shellfish sector. Methods: In the nine years considered, 3359 samples were examined: 1715 marine waters, 73 common clams; 732 mussels; 66 oysters; and 773 veracious clams. Bivalve molluscs were examined for the presence of marine biotoxins, including YTXs, while potentially toxic algae, including those producing YTXs, were searched for and counted in marine waters. The method adopted for the quantification of lipophilic toxins involves the use of an LC-MS/MS system. The enumeration of phytoplankton cells was performed according to the Utermhöl method. Results: Between 2012 and 2020, 706 molluscs were tested for YTXs. In total, 246 samples tested positive, i.e., 34.84%. Of the positive samples, 30 exceeded the legal limit. Conclusion: In this regard, it is essential to develop and activate, as soon as possible, an "early warning" system that allows a better control of the production areas of live bivalve molluscs, thus allowing an optimal management of the plants in these critical situations. [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]

Respiratory exposure to marine phycotoxins is of increasing concern. Inhalation of sea spray aerosols (SSAs), during harmful Karenia brevis and Ostreopsis ovata blooms induces respiratory distress among others. The biogenics hypothesis, however, suggests that regular airborne exposure to natural products is health promoting via a downregulation of the mechanistic target of rapamycin (mTOR) pathway. Until now, little scientific evidence supported this hypothesis. The current explorative in vitro study investigated both health-affecting and potential health-promoting mechanisms of airborne phycotoxin exposure, by analyzing cell viability effects via cytotoxicity assays and effects on the mTOR pathway via western blotting. To that end, A549 and BEAS-2B lung cells were exposed to increasing concentrations (ng·L−1–mg·L−1) of (1) pure phycotoxins and (2) an extract of experimental aerosolized homoyessotoxin (hYTX). The lowest cell viability effect concentrations were found for the examined yessotoxins (YTXs). Contradictory to the other phycotoxins, these YTXs only induced a partial cell viability decrease at the highest test concentrations. Growth inhibition and apoptosis, both linked to mTOR pathway activity, may explain these effects, as both YTXs were shown to downregulate this pathway. This proof-of-principle study supports the biogenics hypothesis, as specific aerosolizable marine products (e.g., YTXs) can downregulate the mTOR pathway. [ABSTRACT FROM AUTHOR]