Paralytic Shellfish Toxins Research Articles

Validation of LC-MS/MS Method for Analysis of Paralytic Shellfish Toxins in Shellfish and Tunicates

Validation of LC-MS/MS Method for Analysis of Paralytic Shellfish Toxins in Shellfish and Tunicates

Predicting the Effects of Climate Change on the Occurrence of the Toxic Dinoflagellate Alexandrium catenella Along Canada’s East Coast

Alexandrium catenella produces paralytic shellfish toxins that affect marine fisheries and aquaculture as well as ecosystem and human health worldwide. This harmful algal species is extremely sensitive to environmental conditions and potentially to future climate change. Using a generalized additive mixed model (GAMM) we studied the potential effects of changing salinity and temperatures on A. catenella bloom (≥1000 cells L–1) occurrence along Canada’s East Coast throughout the 21st century. Our GAMM was applied to two high greenhouse gas emissions scenarios (RCP 8.5) and one mitigation scenario (RCP 4.5). Under present-day conditions, our model successfully predicted A. catenella’s spatio-temporal distribution in Eastern Canada. Under future conditions, all scenarios predict increases in bloom frequency and spatial extent as well as changes in bloom seasonality. Under one RCP 8.5 scenario, A. catenella bloom occurrences increased at up to 3.5 days per decade throughout the 21st century, with amplified year-to-year variability. Blooms expanded into the Gulf of St. Lawrence and onto the Scotian Shelf. These conditions could trigger unprecedented bloom events in the future throughout our study region. In all climate scenarios, the bloom season intensified earlier (May–June) and ended later (October). In some areas of the Gulf of St. Lawrence, the thermal habitat of A. catenella was exceeded, thereby locally reducing bloom risk during the summer months. We conclude that an increase in A. catenella’s environmental bloom window could further threaten marine fauna including endangered species as well as fisheries and aquaculture industries on Canada’s East Coast. Similar impacts could be felt in other coastal regions of the globe where warming and freshening of waters are intensifying.

The Latin America and Caribbean HAB status report based on OBIS and HAEDAT maps and databases

Harmful Algae Blooms (HAB) have been documented for at least fifty years in Latin America and the Caribbean (LAC), however, their impacts at social, ecological and economic levels are still little known. To contribute to the impact assessment of HABs in LAC region, the available information in HAEDAT, OBIS, CAREC, and CARPHA databases, and scientific literature was analyzed. This historical analysis allows identification of the main syndromes and causal organisms. Considering the existence of two regional working groups of the Intergovernmental Oceanographic Commission (IOC): Algas Nocivas del Caribe (ANCA) and Floraciones Algales Nocivas en Sudamérica (FANSA), representing Central American/Caribbean and South American countries, respectively, the analysis is presented both globally and subregional. For the FANSA region, the HAEDAT data base listed 249 records from 1970 to 2019, with a total of 1432 human intoxications, including 37 fatalities. The majority of these events comprised Paralytic Shellfish Toxins (49%), Diarrhetic Shellfish Toxins (34%), Cyanotoxins (12%) and 6 % other toxins. The total number of harmful taxa in the OBIS database includes 79 species distributed over 25 genera. The most commonly reported species are Alexandrium catenella/tamarense, Gymnodinium catenatum and the Dinophysis acuminata complex. Two new species Prorocentrum caipirignum Fraga, Menezes and Nascimento and Alexandrium fragae Branco and Menezes were newly described from Brazilian waters. In the ANCA region, HAEDAT listed 131 records from 1956 to 2018. The main problems are PSP and Ciguatera and common HAB taxa are Gambierdiscus, Gymnodinium, Pyrodinium, Alexandrium and Dinophysis. The most reported HAB forming species are Gymnodinium catenatum, Pyrodinium bahamense and Gambierdiscus spp. In recent years Margalefidinium polykrikoides blooms have become frequent, causing fish and invertebrates massive mortalities and impacts on touristic activities. In the LAC region, the greatest economic losses were produced by ichthyotoxic massive events causing salmon deaths associated to Pseudochattonella verruculosa and Alexandrium catenella in Chile and tuna deaths related to Tripos furca and Chattonella spp. in the Mexican Pacific. In the last decade, several studies in LAC have linked HAB events with local mesoscale oceanographic and atmospheric phenomena. Trends analyzed up to 2019 are related to the increasing awareness about presence of toxic species, the geographical expansion of already known species, the detection of new toxins for the region, and HAB events duration and/or impacts.

Tissue-Biased and Species-Specific Regulation of Glutathione Peroxidase (GPx) Genes in Scallops Exposed to Toxic Dinoflagellates.

Marine bivalves could accumulate paralytic shellfish toxins (PSTs) produced by toxic microalgae, which might induce oxidative stress. Glutathione peroxidases (GPxs) are key enzymes functioning in the antioxidant defense, whereas our understanding of their roles in PST challenge in bivalves is limited. Herein, through genome-wide screening, we identified nine (CfGPx) and eight (PyGPx) GPx genes in Zhikong scallop (Chlamys farreri) and Yesso scallop (Patinopecten yessoensis), respectively, and revealed the expansion of GPx3 sub-family in both species. RNA-Seq analysis revealed high expression of scallop GPx3s after D stage larva during early development, and in adult hepatopancreas. However, in scallops exposed to PST-producing dinoflagellates, no GPx was significantly induced in the hepatopancreas. In scallop kidneys where PSTs were transformed to higher toxic analogs, most CfGPxs were up-regulated, with CfGPx3s being acutely and chronically induced by Alexandrium minutum and A. catenella exposure, respectively, but only one PyGPx from GPx3 subfamily was up-regulated by A. catenella exposure. Our results suggest the function of scallop GPxs in protecting kidneys against the oxidative stresses by PST accumulation or transformation. The tissue-, species-, and toxin-dependent expression pattern of scallop GPxs also implied their functional diversity in response to toxin exposure.

Características ambientales y algas nocivas en un área de producción de moluscos bivalvos del Canal Beagle, Argentina

En diciembre de 2013 se realizó un estudio para conocer las especies de microalgas tóxicas presentes en el área, las distintas toxinas que producen y los factores ambientales que condicionan la distribución de las comunidades de fitoplancton y su estructura. Se observó una alteración en la estructura de la comunidad fitoplantónica respecto al mismo período de años anteriores que indicó un retraso en las primeras etapas de la sucesión clásica. Dicho retraso influyó en el patrón anual de formación de floraciones de especies tóxicas en el canal y, en consecuencia, en los niveles de toxicidad de los bivalvos que normalmente se registran en esa época. El análisis de HPLC mostró escaso número de toxinas paralizantes de moluscos que coincidió con una muy baja concentración de células tóxicas. En las muestras analizadas se detectaron principalmente gonyautoxinas, sin presencia de toxina amnésica de moluscos. Los resultados aportan un conocimiento más detallado del perfil tóxico de los bivalvos de interés comercial en el área, que complementa el programa de monitoreo en curso. El análisis de los parámetros ambientales mostró que el sector interior de Bahía A. Brown es un área con circulación restringida que puede funcionar como reservorio potencial de los estadios bentónicos de resistencia de algunos dinoflagelados, proveyendo un inóculo inicial para la formación de floraciones o de eventos de toxicidad locales. La ausencia de floraciones de especies tóxicas y el bajo nivel de toxicidad de los moluscos del área en ese período se asociaron con las inusuales condiciones climáticas que se registraron en la región durante los meses previos, lo cual advierte sobre la necesidad de contar con un registro continuo de las variables meteorológicas para tratar de predecir la ocurrencia de floraciones de algas nocivas en el canal.

Accumulation of paralytic shellfish toxins by Southern Rock lobster Jasus edwardsii causes minimal impact on lobster health

Recurrent dinoflagellate blooms of Alexandrium catenella expose the economically and ecologically important Southern Rock Lobster in Tasmania to paralytic shellfish toxins (PST), and it is unknown if PST accumulation adversely affects lobster performance, health and catchability. In a controlled aquaculture setting, lobsters were fed highly contaminated mussels to accumulate toxin levels in the hepatopancreas (mean of 6.65 mg STX.2HCl equiv. kg−1), comparable to those observed in nature. Physiological impact of PST accumulation was comprehensively assessed by a range of behavioural (vitality score, righting ability and reflex impairment score), health (haemocyte count, bacteriology, gill necrosis and parasite load), nutritional (hepatopancreas index and haemolymph refractive index) and haemolymph biochemical (21 parameters including electrolytes, metabolites, and enzymes) parameters during a 63 day period of uptake and depuration of toxins. Exposure to PST did not result in mortality nor significant changes in the behavioural, health, or nutritional measures suggesting limited gross impact on lobster performance. Furthermore, most haemolymph biochemical parameters measured exhibited no significant difference between control and exposed animals. However, the concentration of potassium in the haemolymph increased with PST, whilst the concentration of lactate and the sodium:potassium ratio decreased with PST. In addition, exposed lobsters showed a hyperglycaemic response to PST exposure, indicative of stress. These findings suggest that PST accumulation results in some measurable indicators of stress for lobsters. However, these changes are likely within the adaptive range for Jasus edwardsii and do not result in a significant impairment of gross performance. Our findings support previous conclusions that crustaceans are relatively tolerant to PST and the implications for the lobster fishery are discussed.

Application of Six Detection Methods for Analysis of Paralytic Shellfish Toxins in Shellfish from Four Regions within Latin America.

With the move away from use of mouse bioassay (MBA) to test bivalve mollusc shellfish for paralytic shellfish poisoning (PSP) toxins, countries around the world are having to adopt non-animal-based alternatives that fulfil ethical and legal requirements. Various assays have been developed which have been subjected to single-laboratory and multi-laboratory validation studies, gaining acceptance as official methods of analysis and approval for use in some countries as official control testing methods. The majority of validation studies conducted to date do not, however, incorporate shellfish species sourced from Latin America. Consequently, this study sought to investigate the performance of five alternative PSP testing methods together with the MBA, comparing the PSP toxin data generated both qualitatively and quantitatively. The methods included a receptor binding assay (RBA), two liquid chromatography with fluorescence detection (LC-FLD) methods including both pre-column and post-column oxidation, liquid chromatography with tandem mass spectrometry (LC-MS/MS) and a commercial lateral flow assay (LFA) from Scotia. A total of three hundred and forty-nine shellfish samples from Argentina, Mexico, Chile and Uruguay were assessed. For the majority of samples, qualitative results compared well between methods. Good statistical correlations were demonstrated between the majority of quantitative results, with a notably excellent correlation between the current EU reference method using pre-column oxidation LC-FLD and LC-MS/MS. The LFA showed great potential for qualitative determination of PSP toxins, although the findings of high numbers of false-positive results and two false negatives highlighted that some caution is still needed when interpreting results. This study demonstrated that effective replacement methods are available for countries that no longer wish to use the MBA, but highlighted the importance of comparing toxin data from the replacement method using local shellfish species of concern before implementing new methods in official control testing programs.

Toxin- and species-dependent regulation of ATP-binding cassette (ABC) transporters in scallops after exposure to paralytic shellfish toxin-producing dinoflagellates

ATP-binding cassette (ABC) transporters are membrane-bound proteins involved in exporting various xenobiotic compounds from living cells. Bivalve mollusks can accumulate large amounts of paralytic shellfish toxins (PSTs) from marine dinoflagellates. For aquatic invertebrates, the importance of ABC proteins in multi-xenobiotic resistance has been demonstrated, however, the systematic identification of ABC transporters is very limited. In this study, 64 and 67 ABC genes containing all eight described subfamilies (A to H) were identified in Yesso scallop (Patinopecten yessoensis) and Zhikong scallop (Chlamys farreri), respectively, with massive gene expansion being observed in the ABCC and ABCG subfamilies. The kidney harbored more specifically expressed ABC genes than other organs/tissues, most of which belonged to ABCB, ABCC, and ABCG subfamilies. After feeding the scallops with PST-producing dinoflagellates, the expression of scallop ABC genes in the kidney was regulated in toxin- and species-dependent manners. In total, 20 and 24 ABC genes in Zhikong scallop (CfABCs) were induced after exposure to Alexandrium minutum and A. catenella, with the up-regulated members from both ABCC and ABCG subfamilies mainly showing acute and chronic induction by A. minutum and A. catenella, respectively, while the up-regulated CfABCBs mainly showing chronic induction by both dinoflagellates. In Yesso scallop, only eight ABC genes (PyABCs) were regulated after A. catenella exposure, and all the five up-regulated PyABCs were acutely induced. Our findings imply the functional diversity of scallop ABC genes in coping with PST accumulation, which may contribute to the lineage-specific adaptation of scallops for dealing with algal toxins challenge.

First Report of Paralytic Shellfish Toxins in Marine Invertebrates and Fish in Spain.

A paralytic shellfish poisoning (PSP) episode developed in summer 2018 in the Rías Baixas (Galicia, NW Spain). The outbreak was associated with an unprecedentedly intense and long-lasting harmful algal bloom (HAB) (~one month) caused by the dinoflagellate Alexandrium minutum. Paralytic shellfish toxins (PSTs) were analyzed in extracts of 45 A. minutum strains isolated from the bloom by high-performance liquid chromatography with post-column oxidation and fluorescence detection (HPLC-PCOX-FLD). PSTs were also evaluated in tissues from marine fauna (invertebrates and fish) collected during the episode and in dolphin samples. The analysis of 45 A. minutum strains revealed a toxic profile including GTX1, GTX2, GTX3 and GTX4 toxins. With regard to the marine fauna samples, the highest PSTs levels were quantified in bivalve mollusks, but the toxins were also found in mullets, mackerels, starfish, squids and ascidians. This study reveals the potential accumulation of PSTs in marine invertebrates other than shellfish that could act as vectors in the trophic chain or pose a risk for human consumption. To our knowledge, this is the first time that PSTs are reported in ascidians and starfish from Spain. Moreover, it is the first time that evidence of PSTs in squids is described in Europe.

Centrodinium punctatum (Dinophyceae) produces significant levels of saxitoxin and related analogs

Centrodinium punctatum is a fusiform dinoflagellate with a global marine distribution. Due to a close phylogenetic relationship of one C. punctatum strain to Alexandrium species, toxin production of this C. punctatum strain was assessed using liquid chromatography coupled to tandem mass spectrometry. The paralytic shellfish toxin (PST) profile of C. punctatum was dominated by six analogs, i.e. STX (30%), GTX-1 (20%) and neoSTX (24%), followed by GTX-2 (9%), GTX-4 (9%) and GTX-3 (8%); deoxy-STX was also putatively identified while no gymnodimines, spirolides or goniodomins were detected. This is the first record of C. punctatum producing saxitoxins. The estimated cellular toxicity was rather elevated, between 91 and 212 pg cell−1 (or 259 and 601 fmol cell−1). When considering the toxicity equivalent factors, results suggest that this species can produce high cellular toxicity compared to other STX-producing dinoflagellates. Morphological details of the sulcal area and the hypotheca of Centrodinium punctatum were re-examined by scanning electron microscopy (SEM); this revealed that in the sulcal area, the left posterior sulcal plate (Ssp) is larger and longer than the left posterior sulcal plate and extended into the hypotheca. Based on the morphological observation, a revised interpretation of the sulcus and hypotheca is proposed.

Uptake of Paralytic Shellfish Toxins by Blacklip Abalone (Haliotis rubra rubra Leach) from direct exposure to Alexandrium catenella microalgal cells and toxic aquaculture feed

The Tasmanian abalone fishery represents the largest wild abalone resource in the world, supplying close to 25% of the annual wild-caught global harvest. Prompted by the need to manage Paralytic Shellfish Toxin (PST) contamination of Blacklip Abalone (Haliotis rubra rubra) from east coast Tasmania, the uptake of toxins by this species is investigated in a land-based, controlled aquaculture setting. Abalone were exposed to either live Alexandrium catenella microalgal cultures or PST contaminated feed pellets during a 28 day exposure period and toxins quantified in viscera, foot muscle and epipodium tissues. PST profiles of abalone foot tissues were dominated by saxitoxin and neosaxitoxin, whilst viscera more closely resembled those of the toxin source (A. catenella cells rich in gonyautoxin 1&4 and 2&3 or feed pellets containing A. catenella extracts rich in these analogues). This indicates direct uptake of PST in the viscera via browsing/grazing on the pellet and /or sedimented microalgal cells. After exposure to A. catenella cell culture, PST concentrations in the foot (muscle + epipodium) were on average 8 times higher than in the viscera. Higher toxicity of foot tissue was caused by higher PST content of the epipodium (up to 1,085 µg STX.2HCl equiv. kg−1), which despite its small contribution to total animal weight significantly added to the overall toxin burden. Higher PST levels in the abalone foot suggest that toxin monitoring programmes may not need to routinely analyse both foot and viscera, potentially allowing for a 50% reduction of analytical costs. This option is being further investigated with continuing field studies.

Random forest classification to determine environmental drivers and forecast paralytic shellfish toxins in Southeast Alaska with high temporal resolution

Paralytic shellfish poison toxins (PSTs) produced by the dinoflagellate in the genus Alexandrium are a threat to human health and subsistence lifestyles in Southeast Alaska. It is important to understand the drivers of Alexandrium blooms to inform shellfish management and aquaculture, as well as to predict trends of PST in a changing climate. In this study, we aggregate environmental data sets from multiple agencies and tribal partners to model and predict concentrations of PSTs in Southeast Alaska from 2016 to 2019. We used daily PST concentrations interpolated from regularly sampled blue mussels (Mytilus trossulus) analyzed for total PSTs using a receptor binding assay. We then created random forest models to classify shellfish above and below a threshold of toxicity (80 µg 100 g−1) and used two methods to determine variable importance. We obtained a multivariate model with key variables being sea surface temperature, salinity, freshwater discharge, and air temperature. We then used a similar model trained using lagged environmental variables to hindcast out-of-sample (OOS) shellfish toxicities during April-October in 2017, 2018, and 2019. Hindcast OOS accuracies were low (37–50%); however, we found forecasting using environmental variables may be useful in predicting the timing of early summer blooms. This study reinforces the efficacy of machine learning to determine important drivers of harmful algal blooms, although more complex models incorporating other parameters such as toxicokinetics are likely needed for accurate regional forecasts.

Paralytic shellfish toxins and associated toxin profiles in bivalve mollusc shellfish from Argentina

Paralytic Shellfish Poisoning is a potentially fatal syndrome, resulting from the filter-feeding activities of marine molluscs accumulating harmful neurotoxins naturally occurring in microalgae. Outbreaks are well recognised throughout most regions of the world, but with the highest levels of toxicity to date recorded in mussels from Argentina. Whilst toxicity has been documented for selected outbreaks over the years, testing has been conducted using a mouse bioassay. Consequently there is a need to establish baseline data utilising modern chemical detection methods, which also facilitate the quantification of individual toxin analogues, giving useful data on toxin profiles as well as total sample toxicity. In this study, 151 shellfish samples harvested from the marine waters of Argentina between 1980 and 2012 were subjected to analysis by liquid chromatography with fluorescence detection, since Jan 2019 the European Union reference method for PSP determination. Total PST concentrations were found to vary enormously throughout the coastline of Argentina, with higher levels of toxins found in the central regions of Rio Negro and Chubut. Toxin profiles in terms of molar percentage of total concentrations were dominated by the gonyautoxins GTX1&4 and GTX2&3, followed by C1&2, STX and dcGTX2&3, with minor levels of other analogues previously not reported in the country. Profiles were found to vary significantly, with statistical clusters of profile types associated with a wide range of factors, including species, spatial and temporal differences, as well as likely source microalgae species and potential toxin transformation pathways. Overall application of the chemical detection method has confirmed both the significant risk to shellfish consumers in Argentina with periodic outbreaks of extremely high toxin levels and a large variability in toxin profiles relating in part to previously reported variabilities in microalgal toxin content. The study has demonstrated the potential for the method to systematically study the relationships between toxicity, toxin profile, source phytoplankton and other environmental factors.

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.

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.

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.

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.