Shellfish Toxins Research Articles

Frequent accumulation of diarrheic shellfish toxins by different bivalve species in a shallow subtropical estuary

Harmful blooms of Dinophysis spp. have been frequently reported along the southern Brazilian coast. This study intended to determine the accumulation of diarrheic shellfish toxins (DST) in co-occurring wild Anomalocardia brasiliana clams and farmed Perna perna mussels, and relate it to the mesoscale distribution of Dinophysis spp., Mesodinium rubrum and toxins over 13 months in a shallow and vertically homogeneous estuary. Bivalves and depth-integrated water samples were collected from two regions (shallower and deeper areas) representative of the outer section of Babitonga Bay estuary. Cells belonging to the Dinophysis acuminata complex (99% of the total Dinophysis abundance) and those of M. rubrum occurred frequently over the studied period. The DST okadaic acid (OA) was detected both in the particulate fraction of the seawater and in the soft tissues of suspension-feeding bivalves, even when Dinophysis cell density was extremely low (e.g. 30 cell.L−1). There was a marked seasonal variation in both the abundance of D. acuminata complex cells in suspension and the DST levels in bivalve tissues, with higher values recorded from late autumn to spring. Farmed mussels accumulated 3- to 13-fold greater levels of DST than co-occurring wild clams, even though no significant variations in Dinophysis abundance or particulate toxin concentration were observed between the areas where each bivalve species was sampled. Non-esterified OA levels in hanged mussels were not linked to Dinophysis cell density, suggesting that part of the toxin recently incorporated by these bivalves could be adsorbed onto organic and/or inorganic particles. Considering the potential chronic effects of OA, the frequent accumulation of moderate toxin levels (up to 100 μg.Kg−1 in mussels) may thus represent a health risk for frequent consumers of bivalves in this area. Additionally, the risks for episodes of acute human intoxication in this area should be monitored at shorter sampling intervals over multiple years.

Evaluation of different strategies to minimize the matrix effects on LC-MS/MS analysis of multiple lipophilic shellfish toxins in both acidic and alkaline chromatographic conditions

Lipophilic shellfish toxins (LSTs) accumulated by shellfish pose a potential threat to consumer health. A mandatory routine monitoring of LSTs has been adopted for seafood products by liquid chromatography–mass spectrometry (LC-MS) in many countries. In this study, two methods developed on liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) under acidic and alkaline chromatographic conditions were assessed for the determination of multiple LSTs. Different strategies including matrix solid-phase dispersion (MSPD), solid phase extraction (SPE) and sample dilution were applied and evaluated the matrix effects of mussel, scallop, clam, and oyster samples on the signal response of mass spectrometry. Results showed that the alkaline method achieved a lower limit of detection (LOD) and more robust compared to the acidic method. The obvious signal suppression of OA and DTX1 (55%–76%) and signal enhancement of PTX2 (27%–34%) occurred in the crude extracts of shellfish under acidic chromatography. In the alkaline method, no remarkable matrix effects of crude extracts were found except for the scallop matrix on the signal intensity of DTX1, AZA3 and GYM-A (121%–130%). Clean-up methods MSPD, SPE and sample dilution obviously reduced the inhibition of shellfish matrices on the signal response of OA and DTX1, however, which were still subject to signal inhibition under acidic condition. Sample dilution was more effective than SPE and MSPD in minimizing the matrix interference in both acidic and alkaline methods. Furthermore, sample dilution in combination with the alkaline chromatography was the most effective method. Bivalve mollusks harvested from Beibu Bay, South China Sea, were generally contaminated by GYM-A and SPX1 at low concentrations.

Toxicity and histopathological effects of toxic dinoflagellate, Alexandrium catenella exudates on larvae of blue mussel, Mytilus galloprovincialis, and Pacific oyster, Crassostrea gigas

HighlightToxicity and pathological effects of A. catenella were investigated on shellfish larvaeUnfiltered exudates of A. catenella caused significant mortality of blue mussel larvaeApplication of 0.22 mm filtration on A. fundyense exudates potentially decrease the toxicity effectsPathological effects of A. catenella occurred as early as 3 h after exposureThe prevalence and intensity of necrosis increased with exposure duration to A. catenella exudatesAbstractBlooms of the toxic dinoflagellate Alexandrium catenella have affected shellfish industries globally due to their capacity to produce paralytic shellfish toxins(PST). This study aimed to investigate the toxicity effect of exudate A. catenella on larvae of blue mussel Mytilus galloprovincialis and Pacific oyster Crassostrea gigas and filtration methods to reduce the toxic effect. Blue mussel and Pacific oyster larvae were assessed their survival and histopathological changes after exposure to extracellular exudates of A. catenella ranging from 100 to 1,000 cells ml-1 . The results showed that exposure to exudate A. catenella caused significantly higher larval mortality (39 to 52%) than exposure to an equivalent biovolume of the nontoxic species, Tisochrysis lutea (33%) or unfed controls (17%). Filter-sterilization (0.22 µm) of exudates and activated carbon filtration decreased the mortality of Pacific oyster larvae to a level similar to controls (unfed), with the exception of the highest concentrations (600 and 1,000 cells ml-1 ) and mortality of bluemussel larvae mortality by 32% respectively. Blue mussel larvae exposed to exudate A. catenella showed pathological changes mainly in the stomach (digestive gland and style sac) as early as three hours after onset of exposure. The findings of this study suggest that early detection of blooms in the vicinity of mussel and Pacific oyster hatcheries and taking steps to mitigate their effects, is important to reduce the effects of A. catenella blooms on shellfish larval rearing.

Presence of Cyanotoxins in a Mexican Subtropical Monomictic Crater Lake

Microcystins (MCs) produced by cyanobacteria are a ubiquitous worldwide problem because some MCs can cause tumor formation and are hepatotoxic. In the Santa María del Oro crater lake, Mexico, plankton scums are recurrent during most of the year and are associated with cyanobacteria of the genera Microcystis spp. and Lyngbya spp. As some of these species are associated with the production of MCs and paralytic shellfish toxins (PSTs), samples from these scums and particulate matter were collected and analyzed for the main bloom species and toxins by a ultrahigh performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) and high performance liquid chromatography with fluorescence detection (HPLC-FLD). Results showed that the main bloom-forming species were Limnoraphis robusta and Microcystis aeruginosa, the presence of at least seven MC congeners and the absence of PSTs in the algae scums. The MCs identified were MC-WR, MC-LR, MC-LA, MC-HilR, MC-LF, MC-YR, and MC-LY. On a dry mass weight basis, MC concentrations were low and ranged between 0.15 and 6.84 μg/kg. Toxin profiles were dominated by MC-WR, MC-LR, and MC-LA, representing 94.5% of the total sample, with each analog contributing 39.8%, 38.1% and 16.5% by relative concentration, respectively. Two of the more hazardous congeners, MC-LR and MC-LA, represented 54.6% of the total MC concentration. MCs in particulate matter along the depth profile were not detected. The MC profile is linked to M. aeruginosa, and it represents the first quantitative MC congener description for this species from a Mexican water ecosystem. Since these mats are recurrent yearly, their effects on humans and wild fauna, and the possible role of anthropogenic activities that favor their presence and proliferation, need to be evaluated.

DNA damage and oxidative stress responses of mussels Mytilus galloprovincialis to paralytic shellfish toxins under warming and acidification conditions – Elucidation on the organ-specificity

Commonly affected by changes in climate and environmental conditions, coastal areas are very dynamic environments where shellfish play an important ecological role. In this study, the oxidative stress and genotoxic responses of mussels (Mytilus galloprovincialis) exposed to paralytic shellfish toxin (PST) - producing dinoflagellates Gymnodinium catenatum were evaluated under i) current conditions (CC: 19 °C; pH 8.0), ii) warming (W: 24 °C; pH 8.0), iii) acidification (A:19 °C; pH 7.6) and iv) combined effect of warming and acidification (WA: 24 °C; pH 7.6). Mussels were fed with G. catenatum for 5 days, and to a non-toxic diet during the following 10 days. A battery of oxidative stress biomarkers and comet assay was performed at the peak of toxin accumulation and at the end of the post-exposure phase. Under CC, gills and hepatopancreas displayed different responses/vulnerabilities and mechanisms to cope with PST. While gills presented a tendency for lipid peroxidation (LPO) and genetic damage (expressed by the Genetic Damage Indicator - GDI), hepatopancreas seems to better cope with the toxins, as no LPO was observed. However, the mechanisms involved in hepatopancreas protection were not enough to maintain DNA integrity. The absence of LPO, and the antioxidant system low responsiveness, suggests DNA damage was not oxidative. When exposed to toxic algae under W, toxin-modulated antioxidant responses were observed in both gills and hepatopancreas. Simultaneous exposure to the stressors highlighted gills susceptibility with a synergistic interaction increasing DNA damage. Exposure to toxic algae under A led to genotoxicity potentiation in both organs. The combined effect of WA did not cause relevant interactions in gills antioxidant responses, but stressors interactions impacted LPO and GDI. Antioxidant responses and LPO pointed out to be modulated by the environmental conditions in hepatopancreas, while GDI results support the dominance of toxin-triggered process. Overall, these results reveal that simultaneous exposure to warming, acidification and PSTs impairs mussel DNA integrity, compromising the genetic information due to the synergetic effects. Finally, this study highlights the increasing ecological risk of harmful algal blooms to Mytilus galloprovinciallis populations.

Response of fatty acids and lipid metabolism enzymes during accumulation, depuration and esterification of diarrhetic shellfish toxins in mussels (Mytilus galloprovincialis)

Bivalve mollusks accumulate diarrhetic shellfish toxins (DSTs) from toxigenic microalgae, thus posing a threat to human health by acting as a vector of toxins to consumers. In bivalves, free forms of DSTs can be esterified with fatty acids at the C-7 site to form acyl esters (DTX3), presumably a detoxification mechanism for bivalves. However, the effects of esterification of DSTs on fatty acid metabolism in mollusks remain poorly understood. In this study, mussels (Mytilus galloprovincialis) were fed the DST-producing dinoflagellate Prorocentrum lima for 10 days followed by an additional 10-days depuration in filtered seawater to track the variation in quantity and composition of DST acyl esters and fatty acids. A variety of esters of okadaic acid (OA) and dinophysistoxin-1 (DTX1) were mainly formed in the digestive gland (DG), although trace amounts of esters also appeared in muscle tissue. A large relative amount of OA (60%–84%) and DTX1 (80%–92%) was esterified to DTX3 in the visceral mass (referred to as digestive gland, DG), and the major ester acyl chains were C16:0, C16:1, C18:0, C18:1, C20:1 and C20:2. The DG and muscle tissues showed pronounced differences in fatty acid content and composition during both feeding and depuration periods. In the DG, fatty acid content gradually decreased in parallel with increasing accumulation and esterification of DSTs. The decline in fatty acids was accelerated during depuration without food. This reduction in the content of important polyunsaturated fatty acids, especially docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), would lead to a reduction in the nutritional value of mussels. Enzymes involved in lipid metabolism, including acetyl-coenzyme A carboxylase (ACC), fatty acid synthase (FAS), lipoprotein lipase (LPL) and hepatic lipase (HL), were actively involved in the metabolism of fatty acids in the DG, whereas their activities were weak in muscle tissue during the feeding period. This study helps to improve the understanding of interactions between the esterification of DSTs and fatty acid dynamics in bivalve mollusks.

A Mediterranean Alexandrium taylorii (Dinophyceae) Strain Produces Goniodomin A and Lytic Compounds but Not Paralytic Shellfish Toxins.

Species of the dinophyte genus Alexandrium are widely distributed and are notorious bloom formers and producers of various potent phycotoxins. The species Alexandrium taylorii is known to form recurrent and dense blooms in the Mediterranean, but its toxin production potential is poorly studied. Here we investigated toxin production potential of a Mediterranean A. taylorii clonal strain by combining state-of-the-art screening for various toxins known to be produced within Alexandrium with a sound morphological and molecular designation of the studied strain. As shown by a detailed thecal plate analysis, morphology of the A. taylorii strain AY7T from the Adriatic Sea conformed with the original species description. Moreover, newly obtained Large Subunit (LSU) and Internal Transcribed Spacers (ITS) rDNA sequences perfectly matched with the majority of other Mediterranean A. taylorii strains from the databases. Based on both ion pair chromatography coupled to post-column derivatization and fluorescence detection (LC-FLD) and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis it is shown that A. taylorii AY7T does not produce paralytic shellfish toxins (PST) above a detection limit of ca. 1 fg cell−1, and also lacks any traces of spirolides and gymnodimines. The strain caused cell lysis of protistan species due to poorly characterized lytic compounds, with a density of 185 cells mL−1 causing 50% cell lysis of cryptophyte bioassay target cells (EC50). As shown here for the first time A. taylorii AY7T produced goniodomin A (GDA) at a cellular level of 11.7 pg cell−1. This first report of goniodomin (GD) production of A. taylorii supports the close evolutionary relationship of A. taylorii to other identified GD-producing Alexandrium species. As GD have been causatively linked to fish kills, future studies of Mediterranean A. taylorii blooms should include analysis of GD and should draw attention to potential links to fish kills or other environmental damage.

Biokinetics and biotransformation of paralytic shellfish toxins in different tissues of Yesso scallops, Patinopecten yessoensis

Paralytic shellfish toxins (PSTs) are a group of natural toxic substances often found in marine bivalves. Accumulation, anatomical distribution, biotransformation and depuration of PSTs in different tissues of bivalves, however, are still not very well understood. In this study, we investigated biokinetics and biotransformation of PSTs in six different tissues, namely gill, mantle, gonad, adductor muscle, kidney, and digestive gland, in Yesso scallops Patinopecten yessoensis exposed to a toxic strain of dinoflagellate Alexandrium pacificum. High daily accumulation rate (DAR) was recorded at the beginning stage of the experiment. Most of the PSTs in toxic algae ingested by scallops were retained and the toxicity level of PSTs in scallops exceeded the regulatory limit within 5 days. At the late stage of the experiment, however, DAR decreased obviously due to the removal of PSTs. Fitting results of the biokinetics model indicated that the amount of PSTs transferred from digestive gland to mantle, adductor muscle, gonad, kidney, and gill in a decreasing order, and adductor muscle, kidney, and gonad had higher removal rate than gill and mantle. Toxin profile in digestive gland was dominated by N-sulfocarbamoyl toxins 1/2 (C1/2), closely resembled that of the toxic algae. In contrast, toxin components in kidney were dominated by high-potency neosaxitoxin (NEO) and saxitoxin (STX), suggesting that the kidney be a major organ for transformation of PSTs.

Temporal and spatial distribution of Azadinium species in the inland and coastal waters of the Pacific northwest in 2014–2018

Azaspiracids, produced by some species of the dinoflagellate genera Azadinium and Amphidoma, can cause a syndrome in humans called azaspiracid shellfish poisoning (AZP). In 1995, mussels from the Irish west coast contaminated with azaspiracids were, for the first time, linked to this human illness that has symptoms of nausea, vomiting, severe diarrhea, and stomach cramps. The only confirmed cases of AZP to date in the United States occurred in Washington State in 2008 from mussels imported from Ireland. Shortly after this case, several others involving similar gastrointestinal symptoms were reported by shellfish consumers from Washington State. However, no detectable diarrhetic shellfish toxins or Vibrio contamination were found. Cursory analysis of Solid Phase Adsorption Toxin Tracking (SPATT) samplers suggested the presence of azaspiracids in Washington State waters and motivated a study to evaluate the presence and distribution of Azadinium species in the region. During the spring and summer months of 2014–2015, quantitative polymerase chain reaction (qPCR) analyses detected the presence of the toxigenic species Azadinium poporum and A. spinosum on the outer coast and throughout the inland waters of Washington State. In 2016–2018, standard curves developed using A. poporum isolated from Puget Sound and A. spinosum isolated from the North Sea were used to quantify abundances of up to 10,525 cells L−1 of A. poporum and 156 cells L−1 of A. spinosum at shore-based sites. Abundances up to 1,206 cells L−1 of A. poporum and 30 cells L−1 of A. spinosum were measured in the coastal waters of the Pacific Northwest in 2017. Other harmful genera, including Alexandrium, Dinophysis, and Pseudo-nitzschia, were observed using light microscopy at coastal sites where A. poporum was also observed. In some samples where both A. poporum and A. spinosum were absent, an Amphidomataceae-specific qPCR assay indicated that other species of Azadinium or Amphidoma were present. The identification of Azadinium species in the PNW demonstrates the need to assess their toxicity and to incorporate their routine detection in monitoring programs to aid resource managers in mitigating risks to azaspiracid shellfish poisoning in this region.

Effects of paralytic shellfish toxins on the middle intestine of Oncorhynchus mykiss: Glutathione metabolism, oxidative status, lysosomal function and ATP-binding cassette class C (ABCC) proteins activity

We studied the absorption, cytotoxicity and oxidative stress markers of Paralytic Shellfish Toxins (PST) from three extracts from Alexandrium catenella and A. ostenfeldii, in middle Oncorhynchus mykiss intestine in vitro and ex vivo preparations. We measured glutathione (GSH) content, glutathione-S transferase (GST), glutathione reductase (GR) and catalase (CAT) enzymatic activity, and lipid peroxidation in isolated epithelium exposed to 0.13 and 1.3 μM PST. ROS production and lysosomal membrane stability (as neutral red retention time 50%, NRRT50) were analyzed in isolated enterocytes exposed to PST alone or plus 3 μM of the ABCC transport inhibitor MK571. In addition, the concentration-dependent effects of PST on NRRT50 were assayed in a concentration range from 0 to 1.3 μM PST. We studied the effects of three different PST extracts on the transport rate of the ABCC substrate DNP-SG by isolated epithelium. The extract with highest inhibition capacity was selected for studying polarized DNP-SG transport in everted and non-everted intestinal segments. We registered lower GSH content and GST activity, and higher GR activity, with no significant changes in CAT activity, lipid peroxidation or ROS level. PST exposure decreased NRRT50 in a concentration-depend manner (IC50 = 0.0045 μM), but PST effects were not augmented by addition of MK571. All the three PST extracts inhibited ABCC transport activity, but this inhibition was effective only when the toxins were applied to the apical side of the intestine and DNP-SG transport was measured at the basolateral side. Our results indicate that PST are absorbed by the enterocytes from the intestine lumen. Inside the enterocytes, these toxins decrease GSH content and inhibit the basolateral ABCC transporters affecting the normal functions of the cell. Furthermore, PST produce a strong cytotoxic effect to the enterocytes by damaging the lysosomal membrane, even at low, non-neurotoxic concentrations.

OMICs Approaches in Diarrhetic Shellfish Toxins Research.

Diarrhetic shellfish toxins (DSTs) are among the most prevalent marine toxins in Europe’s and in other temperate coastal regions. These toxins are produced by several dinoflagellate species; however, the contamination of the marine trophic chain is often attributed to species of the genus Dinophysis. This group of toxins, constituted by okadaic acid (OA) and analogous molecules (dinophysistoxins, DTXs), are highly harmful to humans, causing severe poisoning symptoms caused by the ingestion of contaminated seafood. Knowledge on the mode of action and toxicology of OA and the chemical characterization and accumulation of DSTs in seafood species (bivalves, gastropods and crustaceans) has significantly contributed to understand the impacts of these toxins in humans. Considerable information is however missing, particularly at the molecular and metabolic levels involving toxin uptake, distribution, compartmentalization and biotransformation and the interaction of DSTs with aquatic organisms. Recent contributions to the knowledge of DSTs arise from transcriptomics and proteomics research. Indeed, OMICs constitute a research field dedicated to the systematic analysis on the organisms’ metabolisms. The methodologies used in OMICs are also highly effective to identify critical metabolic pathways affecting the physiology of the organisms. In this review, we analyze the main contributions provided so far by OMICs to DSTs research and discuss the prospects of OMICs with regard to the DSTs toxicology and the significance of these toxins to public health, food safety and aquaculture.

The occurrence of lipophilic toxins in shellfish from the Middle Adriatic Sea

The first survey of the phycotoxin profile in mussels (Mytilus galloprovincialis) from the coastal waters of Bosnia and Herzegovina (The Bay of Neum, Middle Adriatic Sea) in correlation to the Makarska City Bay (Croatia, Middle Adriatic Sea) was conducted in 2017. Throughout the monitoring period, occasions of gymnodimine (GYM) and azaspiracid (AZA2) shellfish toxicity were recorded in concentrations that do not endanger human health. The occurrence of yessotoxins (YTXs), the most common toxins found in the Adriatic Sea, was correlated to the presence of the Gonyaulax species, a potential source of YTX. The DSP group of toxins is represented by the ester-OA. Phytoplankton analysis confirmed the presence of dinoflagellates from the Prorocentrum genus, a species associated with DSP toxicity. Occurrence frequency and variability of toxin composition were investigated in conjunction to physico-chemical parameters in the surrounding sea water. In the central Adriatic Sea, the infestation period ranges in general from June to August. However, the depuration phase extended beyond September in the Bay of Neum, increasing the length of the decontamination period.

Multiple New Paralytic Shellfish Toxin Vectors in Offshore North Sea Benthos, a Deep Secret Exposed.

In early 2018, a large easterly storm hit the East Anglian coast of the UK, colloquially known as the ‘Beast from the East’, which also resulted in mass strandings of benthic organisms. There were subsequent instances of dogs consuming such organisms, leading to illness and, in some cases, fatalities. Epidemiological investigations identified paralytic shellfish toxins (PSTs) as the cause, with toxins present in a range of species and concentrations exceeding 14,000 µg STX eq./kg in the sunstar Crossaster papposus. This study sought to better elucidate the geographic spread of any toxicity and identify any key organisms of concern. During the summers of 2018 and 2019, various species of benthic invertebrates were collected from demersal trawl surveys conducted across a variety of locations in the North Sea. An analysis of the benthic epifauna using two independent PST testing methods identified a ‘hot spot’ of toxic organisms in the Southern Bight, with a mean toxicity of 449 µg STX eq./kg. PSTs were quantified in sea chervil (Alcyonidium diaphanum), the first known detection in the phylum bryozoan, as well as eleven other new vectors (>50 µg STX eq./kg), namely the opisthobranch Scaphander lignarius, the starfish Anseropoda placenta, Asterias rubens, Luidia ciliaris, Astropecten irregularis and Stichastrella rosea, the brittlestar Ophiura ophiura, the crustaceans Atelecyclus rotundatus and Munida rugosa, the sea mouse Aphrodita aculeata, and the sea urchin Psammechinus miliaris. The two species that showed consistently high PST concentrations were C. papposus and A. diaphanum. Two toxic profiles were identified, with one dominated by dcSTX (decarbamoylsaxitoxin) associated with the majority of samples across the whole sampling region. The second profile occurred only in North-Eastern England and consisted of mostly STX (Saxitoxin) and GTX2 (gonyautoxin 2). Consequently, this study highlights widespread and variable levels of PSTs in the marine benthos, together with the first evidence for toxicity in a large number of new species. These findings highlight impacts to ‘One Health’, with the unexpected sources of toxins potentially creating risks to animal, human and environmental health, with further work required to assess the severity and geographical/temporal extent of these impacts.

Three decades of Canadian marine harmful algal events: Phytoplankton and phycotoxins of concern to human and ecosystem health

Spatial and temporal trends of marine harmful algal events in Canada over the last three decades were examined using data from the Harmful Algal Event Database (HAEDAT). This database contains the most complete record of algal blooms, phycotoxins and shellfish harvesting area closures in Canada since 1987. This 30-year review of 593 Canadian HAEDAT records from 1988 to 2017, together with other Canadian data and publications, shows that recurring harmful algal events have been widespread throughout both the Atlantic and Pacific coastal regions. The 367 paralytic shellfish toxin (PST) reports revealed annual and frequent recurrence throughout both the Atlantic and Pacific regions, including multi-year PST events in the Bay of Fundy, the Estuary and Gulf of St. Lawrence and the Strait of Georgia. The 70 amnesic shellfish toxin (AST) records revealed no recognizable trend, as these events were usually area specific and did not recur annually. The increasing frequency of diarrhetic shellfish toxin (DST) events over the period of this review, in total 59 records, can be at least partially explained by increased sampling effort. Marine species mortalities caused by harmful algae (including diatoms, dictyochophytes, dinoflagellates, and raphidophytes), were a common occurrence in the Pacific region (87 reports), but have been reported much less frequently in the Atlantic region (10 reports). Notable Canadian records contained in HAEDAT include the first detection worldwide of amnesic shellfish poisoning (ASP), attributed to the production of domoic acid (an AST) by a diatom (Pseudo-nitzschia multiseries) in Prince Edward Island in 1987. The first proven case of diarrhetic shellfish poisoning (DSP) in Canada and North America was recorded in 1990, and the first closures of shellfish harvesting due to DST (associated with the presence of Dinophysis norvegica) occurred in Nova Scotia in 1992, followed by closures in Newfoundland and Labrador in 1993. In 2008, mass mortalities of fishes, birds and mammals in the St. Lawrence Estuary were caused by Alexandrium catenella and high levels of PST. During 2015, the Pacific coast experienced a large algal bloom that extended from California to Alaska. It resulted in the closure of several shellfish harvesting areas in British Columbia due to AST, produced by Pseudo-nitzschia australis. Data from the Canadian Arctic coast is not included in HAEDAT. However, because of the emerging importance of climate change and increased vessel traffic in the Arctic, information on the occurrence of harmful algal species (pelagic and sympagic = sea ice-associated) in that region was compiled from relevant literature and data. The results suggest that these taxa may be more widespread than previously thought in the Canadian Arctic. Information in HAEDAT was not always robust or complete enough to provide conclusions about temporal trends. Compilation of spatial and temporal information from HAEDAT and other records is nevertheless important for evaluating the potential role of harmful algae as a stressor on Canadian marine ecosystems, and will support the next step: developing a knowledge gap analysis that will establish research priorities for determining their consequences on human and ecosystem health.

Multi-Toxin Quantitative Analysis of Paralytic Shellfish Toxins and Tetrodotoxins in Bivalve Mollusks with Ultra-Performance Hydrophilic Interaction LC-MS/MS-An In-House Validation Study.

Ultra-performance hydrophilic interaction liquid chromatography tandem mass spectrometry system (UP-HILIC–MS/MS) was used in multi-toxin analysis of paralytic shellfish toxins (PSTs) and tetrodotoxins (TTXs) in sample matrices from bivalve molluscan species commercially produced for human consumption in Sweden. The method validation includes 17 toxins of which GTX6 and two TTX analogues, TTX and 4,9-anhydroTTX, were previously not analyzed together with hydrophilic PSTs. 11-deoxyTTX was monitored qualitatively with a non-certified reference standard. The performance of the method was evaluated for selectivity, repeatability, and linearity by analyzing spiked samples which generated linear calibration curves across the concentration ranges used (R2 > 0.99). The in-house reproducibility (RSD) was satisfactory including the LOD and LOQ for both PST and TTX toxins being far below their regulatory action limits. The major advantage of the method is that it allows direct confirmation of the toxin identity and specific toxin quantification using a derivatization-free approach. Unlike the PST-chemical methods used in routine regulatory monitoring until now for food control, the UP-HILIC-MS/MS approach enables the calibration set-up for each of the toxin analogs separately, thereby providing the essential flexibility and specificity in analysis of this challenging group of toxins. The method is suitable to implement in food monitoring for PSTs and TTXs in bivalves, and can serve as a fast and cost-efficient screening method. However, positive samples would, for regulatory reasons still need to be confirmed using the AOAC official method (2005.06).

Change in Paralytic Shellfish Toxins in the Mussel Mytilus galloprovincialis Depending on Dynamics of Harmful Alexandrium catenella (Group I) in the Geoje Coast (South Korea) during Bloom Season.

Paralytic shellfish toxins (PSTs) produced by Alexandrium catenella (formerly A. tamarense) in Korean coastal waters caused the deaths of four people (in 1986 and 1996) who consumed contaminated mussels (Mytilus edulis). This led to more detailed consideration of the risks of PST outbreaks and incidents in Korea, including the introduction of shellfish collection bans. In this study, we investigated the relationships between A. catenella population dynamics and PST accumulation in the mussel M. galloprovincialis. Discharges from the Nakdong River affect the environmental conditions along the Geoje coast, resulting in low salinity and high nutrient levels that trigger blooms of A. catenella. At the toxin peak on 24 April 2017, the toxins detected in A. catenella cells were C1, gonyautoxin (GTX)1 and GTX2, whereas the concentrations of PSTs in M. galloprovincialis were high and in the order of GTX4 > GTX1 > GTX3 > saxitoxin (STX) > GTX2 > neoSTX > decarbamoylgonyautoxin (dcGTX)2 > dc GTX3. The PST level in mussels was also high. At 15 °C, the PSTs are constantly found to be higher (10-fold higher in 2017 and 30-fold higher in 2018) than safe levels for human consumption (80 μg STX diHCl equivalents 100 g−1).

Overview of Australian and New Zealand harmful algal species occurrences and their societal impacts in the period 1985 to 2018, including a compilation of historic records

Similarities and differences between Australia and New Zealand in Harmful Algal species occurrences and Harmful Algal Events impacting on human society (HAEDAT) are reported and factors that explain their differences explored. Weekly monitoring of harmful phytoplankton and biotoxins commenced in Australia in 1986 and in New Zealand in 1993. Anecdotal historic HAB records in both countries are also catalogued. In Australia, unprecedented highly toxic Paralytic Shellfish Toxin (PST)-producing blooms of Alexandrium catenella have impacted the seafood industry along the 200 km east coast of Tasmania from 2012 to present. Toxic blooms in 1986-1993 by Gymnodinium catenatum in Tasmania were effectively mitigated by closing the affected area for shellfish farming, while a bloom by this same species in 2000 in New Zealand caused significant economic damage from restrictions on the movement of greenshell mussel spat. The biggest biotoxin event in New Zealand was an unexpected outbreak of Neurotoxic Shellfish Poisoning (NSP) in 1993 in Hauraki Gulf (putatively due to Karenia cf. mikimotoi) with 180 reported cases of human poisonings as well as reports of respiratory irritation north of Auckland. Strikingly, NSP never recurred in New Zealand since and no NSP events have ever been reported in Australia. In New Zealand, Paralytic Shellfish Poisoning (PSP) was the predominant seafood toxin syndrome, while in Australia Ciguatera Fish Poisoning (CFP) was the major reported seafood toxin syndrome, while no CFP has been recorded from consumption of New Zealand fish. In Australia, Diarrhetic Shellfish Poisoning (DSP) illnesses were recorded from two related outbreaks in 1997/98 following consumption of beach harvested clams (pipis) from a previously non-monitored area, whereas in New Zealand limited DSP illnesses are known. No human illnesses from Amnesic Shellfish Poisoning (ASP) have been reported in either Australia or New Zealand. Selected examples of HABs appearing and disappearing (NSP in New Zealand, Alexandrium catenella in Tasmania), species expanding their ranges (Noctiluca, Gambierdiscus), and reputed ballast water introductions (Gymnodinium catenatum) are discussed. Eutrophication has rarely been invoked as a cause except for confined estuaries and fish ponds and estuarine cyanobacterial blooms. No trend in the number of HAEDAT events from 1985 to 2018 was discernible.

Toxic dinoflagellate blooms of Gymnodinium catenatum and their cysts in Taiwan Strait and their relationship to global populations

Gymnodinium catenatum is able to produce paralytic shellfish toxins (PSTs) and was responsible for a massive bloom in the Taiwan Strait, East China Sea, in June 2017, which resulted in serious human poisoning and economic losses. To understand the origin of the bloom and determine the potential for blooms in subsequent years, water and sediment samples collected in the Taiwan Strait from 2016 to 2019 were analyzed for cells and cysts using light microscopy (LM) and/or quantitative polymerase chain reaction (qPCR). The morphology of both cells and cysts from the field and cultures was examined with LM and scanning electron microscopy (SEM). Large subunit (LSU) and/or internal transcribed spacer (ITS)-5.8S rRNA gene sequences were obtained in 13 isolates from bloom samples and five strains from cysts. In addition, cells of strains TIO523 and GCLY02 (from the Taiwan Strait and Yellow Sea of China, respectively) were subjected to growth experiments, and cysts from the field were used for germination experiments under various temperatures. Our strains shared identical LSU and ITS-5.8S rRNA gene sequences with those from other parts of the world, and therefore belonged to a global population. A low abundance of G. catenatum cells were detected during most of the sampling period, but a small bloom was encountered in Quanzhou on June 8, 2018. Few cysts were observed in 2016 but a marked increase was observed after the bloom in 2017, with a highest density of 689 cysts cm−3. Cysts germinated at temperatures between 14 and 23 °C with a final germination rate over 93%. Strains TIO523 and GCLY02 displayed growth at temperatures between 17 and 26 °C and 14 and 26 °C, respectively, with both strains displaying the highest growth rate of ca. 0.5 divisions d–1 at 23 °C. The PSTs of the three strains and cysts from the sediments were analyzed by liquid chromatography with tandem mass spectrometry (LC-MS/MS). All strains were able to produce PSTs, which were dominated by N-sulfocarbamoyl C toxins (C1/2, 53.0–143.5 pg cell−1) and decarbamoyl gonyautoxins (dcGTX2/3, 26.7–52.1 pg cell−1), although they were not detected in cysts. However, hydroxybenzoyl (GC) toxins were detected in both cells and cysts. Our results suggested that the population in the Taiwan Strait belonged to a warm water ecotype and has a unique toxin profile. Our results also suggested that the persistence of cells in the water column may have initiated the bloom.

Biochemical performance of mussels, cockles and razor shells contaminated by paralytic shellfish toxins

Marine toxins in bivalves pose an important risk to human health, and regulatory authorities throughout the world impose maximum toxicity values. In general, bivalve toxicities due to paralytic shellfish toxins (PSTs) above the regulatory limit occur during short periods, but in some cases, it may be extended from weeks to months. The present study examines whether cockles (Cerastoderme edule), mussels (Mytilus galloprovincialis) and razor shells (Solen marginatus) naturally exposed to a bloom of Gymnodinium catenatum activated or suppressed biochemical responses as result of the presence of PSTs in their soft tissues. Toxins (C1+2, C3+4, GTX5, GTX6, dcSTX, dcGTX2+3 and dcNEO) and a set of biomarkers (ETS, electron transport system activity; GLY, glycogen; PROT, protein; SOD, superoxide dismutase; CAT, catalase; GPx, glutathione peroxidase; GST, glutathione S-transferases; LPO, lipid peroxidation; reduced (GSH) and oxidized (GSSG) glutathione contents and AChE, acetylcholinesterase activity) were determined in the three bivalve species. Specimens were harvested weekly in Aveiro lagoon, Portugal, along thirteen weeks. This period included three weeks in which bivalve toxicity exceeded largely the regulatory limit and the subsequence recovery period of ten weeks. Biochemical performance of the surveyed species clearly indicated that PSTs induce oxidative stress and neurotoxicity, with higher impact on mussels and razor shells than in cockles. The antioxidant enzymes CAT and GPx seemed to be the biomarkers better associated with toxin effects.

Glutathione S-transferase genes in scallops and their diverse expression patterns after exposure to PST-producing dinoflagellates

The glutathione S-transferases (GSTs) are a superfamily of enzymes that function in cellular protection against toxic substances and oxidative stress. Bivalves could accumulate high concentration of paralytic shellfish toxins (PSTs) from harmful algae. To understand the possible involvement of GSTs in protecting bivalves during PST accumulation and metabolism, the GST genes were systemically analyzed in two cultured scallops, Azumapecten farreri and Mizuhopecten yessoensis, which were reported for PST deposition during harmful algae bloom. A total of 35 and 37 GSTs were identified in A. farreri (AfGSTs) and M. yessoensis (MyGSTs) genome, respectively, and the expansion of the sigma class from the cytosolic subfamily was observed. In both scallop species, sigma class GSTs showed higher expression than other members. The high GSTs expression was detected mainly during/after larvae stages and in the two most toxic organs, hepatopancreas and kidney. After ingesting PST-producing dinoflagellates, all the regulated AfGSTs in the hepatopancreas were from the sigma class, but with opposite regulation pattern between Alexandrium catenella and A. minutum exposure. In scallop kidneys, where PSTs transformed into higher toxicity, more AfGSTs were regulated than in the hepatopancreas, and most of them were from the sigma class, with similar regulation pattern between A. catenella and A. minutum exposure. In M. yessoensis exposed to A. catenella, MyGST-σ2 was the only up-regulated MyGST in both hepatopancreas and kidney. Our results suggested the possible diverse function of scallop GSTs and the importance of sigma class in the defense against PSTs, which would contribute to the adaptive evolution of scallops in marine environments.