The Prevalence of Marine Lipophilic Phycotoxins Causes Potential Risks in a Tropical Small Island Developing State.

Tropical small island developing states (SIDS), with their geographical isolation and limited resources, heavily rely on the fisheries industry for food and revenue. The presence of marine lipophilic phycotoxins (MLPs) poses risks to their economy and human health. To understand the contamination status and potential risks, the Republic of Kiribati was selected as the representative tropical SIDS and 55 species of 256 coral reef fish encompassing multiple trophic levels and feeding strategies were collected to analyze 17 typical MLPs. Our results showed that the potential risks of ciguatoxins were the highest and approximately 62% of fish species may pose risks for consumers. Biomagnification of ciguatoxins was observed in the food web with a trophic magnification factor of 2.90. Brevetoxin-3, okadaic acid, and dinophysistoxin-1 and -2 were first reported, but the risks posed by okadaic acid and dinophysistoxins were found to be negligible. The correlation analysis revealed that fish body size and trophic position are unreliable metrics to indicate the associated risks and prevent the consumption of contaminated fish. The potential risks of MLPs in Kiribati are of concern, and our findings can serve as valuable inputs for developing food safety policies and fisheries management strategies specific to tropical SIDS contexts.

Spatiotemporal variations of domoic acid: New findings in the sedimentary environment of a typical nearshore mariculture bay, China.

Domoic acid (DA) is a neurotoxin produced by marine microalgae. It tends to accumulate in marine shellfish and fish, posing a threat to aquaculture and seafood consumers' health. In this study, DA in the surface and bottom seawater, sediment, and porewater of the Jiaozhou Bay, a typical mariculture bay in China, was systematically investigated for the first time over different seasons. Surprisingly, a high concentration of DA was discovered in the marine sediment porewater (maximum detected concentration: 289.49 ng/L) for the first time. DA was found to be extensively distributed in the water body and sedimentary environment of the Jiaozhou Bay. DA in the surface and bottom seawater of Jiaozhou Bay in spring was uniformly distributed, whereas DA showed obvious spatial variations in summer and winter. The high concentration areas of DA are located in the north of Jiaozhou Bay and decreased to the south areas. DA was also distributed in the sediment (spring mean: 316.57 ng/kg; summer mean: 10.22 ng/kg; winter mean: 237.08 ng/kg) and porewater (spring mean: 129.70 ng/L; summer mean: 53.54 ng/L; winter mean: 19.90 ng/L) of Jiaozhou Bay. The DA concentrations in the surface sediment and porewater were higher in the spring than in the winter and summer, contrary to the seasonal variation pattern observed in the surface and bottom seawater. The DA concentration in porewater was significantly higher than in the surface and bottom seawater, indicating that the risk of pollution contamination from DA to benthic fishery organisms may be underestimated. Overall, DA is widely distributed in the seawater and also in the benthic environment of Jiaozhou Bay and exhibited potential harm to fishery organisms varied greatly with seasons. It is an important discovery for marine algae toxins and has important guiding significance and important indicative role for the routine monitoring and management of DA pollution in water and benthic environment.

Cytotoxicity and intestinal permeability of phycotoxins assessed by the human Caco-2 cell model.

Phycotoxins are a class of multiple natural metabolites produced by microalgae in marine and freshwater ecosystems that bioaccumulate in food webs, particularly in shellfish, having a great impact on human health. Phycotoxins are mainly leached and absorbed in the small intestine when human consumers accidentally ingest toxic aquatic products contaminated by them. To assess the intestinal uptake and damage of phycotoxins, a typical in vitro model was developed and widely applied using the human colorectal adenocarcinoma Caco-2 cell line. In this review, the application cases were summarized for multiple phycotoxins, including microcystins (MCs), cylindrospermopsins (CYNs), domoic acids (DAs), saxitoxins (STXs), palytoxins (PLTXs), okadaic acids (OAs), pectenotoxins (PTXs) and azaspiracids (AZAs). The results of the previous studies showed that each group of phycotoxins presented different cytotoxicity and mechanisms to Caco-2 cells, and significant discrepancies in the transport of phycotoxin across the Caco-2 cell monolayers. Therefore, this review describes the evaluation assays of the Caco-2 cell monolayer model, illustrates the principles of several primary cytotoxicity evaluation assays, and summarizes the cytotoxicity of each group of phycotoxins to Caco-2 cells line and their cellular transport, and finally proposes the development of multicellular intestinal models for future comprehensive studies on the toxicity and absorption of phycotoxins in the intestine. It will improve the understanding of Caco-2 cell monolayer models in the toxicology studies on phycotoxins and the potentially detrimental effects of microalgal toxins on the human intestine.

Alzheimer's Disease and Toxins Produced by Marine Dinoflagellates: An Issue to Explore.

This paper examined the toxins naturally produced by marine dinoflagellates and their effects on increases in ß-amyloid plaques along with tau protein hyperphosphorylation, both major drivers of Alzheimer's disease (AD). This approach is in line with the demand for certain natural compounds, namely those produced by marine invertebrates that have the potential to be used in the treatment of AD. Current advances in AD treatment are discussed as well as the main factors that potentially affect the puzzling global AD pattern. This study focused on yessotoxins (YTXs), gymnodimine (GYM), spirolides (SPXs), and gambierol, all toxins that have been shown to reduce ß-amyloid plaques and tau hyperphosphorylation, thus preventing the neuronal or synaptic dysfunction that ultimately causes the cell death associated with AD (or other neurodegenerative diseases). Another group of toxins described, okadaic acid (OA) and its derivatives, inhibit protein phosphatase activity, which facilitates the presence of phosphorylated tau proteins. A few studies have used OA to trigger AD in zebrafish, providing an opportunity to test in vivo the effectiveness of new drugs in treating or attenuating AD. Constraints on the production of marine toxins for use in these tests have been considered. Different lines of research are anticipated regarding the action of the two groups of toxins.

Marine invertebrate interactions with Harmful Algal Blooms - Implications for One Health.

Harmful Algal Blooms (HAB) are natural atypical proliferations of micro or macro algae in either marine or freshwater environments which have significant impacts on human, animal and ecosystem health. The causative HAB organisms are primarily dinoflagellates and diatoms in marine and cyanobacteria within freshwater ecosystems. Several hundred species of HABs, most commonly marine dinoflagellates affect animal and ecosystem health either directly through physical, chemical or biological impacts on surrounding organisms or indirectly through production of algal toxins which transfer through lower-level trophic organisms to higher level predators. Traditionally, a major focus of HABs has concerned their natural production of toxins which bioaccumulate in filter-feeding invertebrates, which with subsequent trophic transfer and biomagnification cause issues throughout the food web, including the human health of seafood consumers. Whilst in many regions of the world, regulations, monitoring and risk management strategies help mitigate against the impacts from HAB/invertebrate toxins upon human health, there is ever-expanding evidence describing enormous impacts upon invertebrate health, as well as the health of higher trophic level organisms and marine ecosystems. This paper provides an overview of HABs and their relationships with aquatic invertebrates, together with a review of their combined impacts on animal, human and ecosystem health. With HAB/invertebrate outbreaks expected in some regions at higher frequency and intensity in the coming decades, we discuss the needs for new science, multi-disciplinary assessment and communication which will be essential for ensuring a continued increasing supply of aquaculture foodstuffs for further generations.

Aerosolizable Marine Phycotoxins and Human Health Effects: In Vitro Support for the Biogenics Hypothesis.

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.

Monitoring and warning of lipophilic marine algal toxins in mariculture zone based on toxin profiles of phytoplankton.

Some toxigenic dinoflagellates can produce lipophilic marine algal toxins (LMATs), which are potent threats to marine breeding industries. In this study, a new method based on the profiling analysis of six LMAT classes in phytoplankton was developed for the monitoring and warning of LMATs in mariculture zones. This method was applied to monitor and evaluate LMATs in the Jiaozhou Bay and the Changjiang estuary in China. Results demonstrated that the occurrence and spatiotemporal variations of LMATs in mariculture zones can be revealed by the toxin profiles of phytoplankton, indicating the method's effectiveness for the comprehensive monitoring of the composition and levels of various LMATs in coastal aquaculture zones. The method was further used as an alarm for potential pollution risk from LMATs in mariculture zones at an early stage. The "alert" thresholds of LMAT pollution in the mariculture zones were preliminarily proposed based on the statistical data analysis of LMATs in phytoplankton in three typical mariculture areas in China. This study is the first to conduct simultaneous monitoring and warning of multi-class LMATs based on toxin profiles of phytoplankton, thereby providing new insight into the monitoring and early warning of natural poisonous pollutants in coastal aquaculture zones around the world.

Screening-level evaluation of marine benthic dinoflagellates toxicity using mammalian cell lines.

Complementary studies at different levels of the biological organization are fundamental to fully link environmental exposure to marine benthic dinoflagellate toxins and their effects. In order to contribute to this transdisciplinary evaluation, and for the first time, the present study aims to study the effects of Gambierdiscus excentricus, Ostreopsis cf. ovata, Prorocentrum hoffmannianum and Prorocentrum lima extracts on seven functionally different mammalian cell lines: HEK 293, HepG2, HNDF, H9c2(2-1), MC3T3-E1, Raw 264.7 and SH-SY5Y. All the cell lines presented cell mass decrease in a concentration-dependence of dinoflagellate extracts, exhibiting marked differences in cell toxicity. Gambierdiscus excentricus presented the highest effect, at very low concentrations with EC50,24h (i.e., the concentration that gives half-maximal response after a 24-h exposure) between 1.3 and 13 cells mL-1, followed by O. cf. ovata (EC50,24h between 3.3 and 40 cells mL-1), and Prorocentrum species (P. lima: EC50,24h between 191 and 1027 cells mL-1 and P. hoffmannianum: EC50,24h between 152 and 783 cells mL-1). Cellular specificities were also detected and rat cardiomyoblast H9c2(2-1) cells were in general the most sensitive to dinoflagellate toxic compounds, suggesting that this cell line is an animal-free potential model for dinoflagellate toxin testing. Finally, the sensitivity of cells expressing distinct phenotypes to each dinoflagellate extract exhibited low relation to human poisoning symptoms.

Novel Insights on the Toxicity of Phycotoxins on the Gut through the Targeting of Enteric Glial Cells.

In vitro and in vivo studies have shown that phycotoxins can impact intestinal epithelial cells and can cross the intestinal barrier to some extent. Therefore, phycotoxins can reach cells underlying the epithelium, such as enteric glial cells (EGCs), which are involved in gut homeostasis, motility, and barrier integrity. This study compared the toxicological effects of pectenotoxin-2 (PTX2), yessotoxin (YTX), okadaic acid (OA), azaspiracid-1 (AZA1), 13-desmethyl-spirolide C (SPX), and palytoxin (PlTX) on the rat EGC cell line CRL2690. Cell viability, morphology, oxidative stress, inflammation, cell cycle, and specific glial markers were evaluated using RT-qPCR and high content analysis (HCA) approaches. PTX2, YTX, OA, AZA1, and PlTX induced neurite alterations, oxidative stress, cell cycle disturbance, and increase of specific EGC markers. An inflammatory response for YTX, OA, and AZA1 was suggested by the nuclear translocation of NF-κB. Caspase-3-dependent apoptosis and induction of DNA double strand breaks (γH2AX) were also observed with PTX2, YTX, OA, and AZA1. These findings suggest that PTX2, YTX, OA, AZA1, and PlTX may affect intestinal barrier integrity through alterations of the human enteric glial system. Our results provide novel insight into the toxicological effects of phycotoxins on the gut.

Sediment as a Potential Pool for Lipophilic Marine Phycotoxins with the Case Study of Daya Bay of China.

Marine sediments can reserve many environmental pollutants. Lipophilic marine phycotoxins (LMPs) are natural toxic substances widespread in the marine environment; however, evidence of their existence in sediment is scarce. In the present study, in order to explore the occurrence and distribution characteristics of LMPs in sediment, surface sediment samples collected from a tropical area of Daya Bay (DYB) at different seasons, were analyzed using liquid chromatography with tandem mass spectrometry (LC-MS/MS). According to the results, up to six toxin compounds were detected in sediment samples from DYB, OA and DTX1 had the highest levels, followed by PTX2, homo-YTX, AZA2, and GYM. Although AZA2 and GYM were found in most of the sediment, OA, DTX1, homo-YTX, and PTX2 were the predominant toxin compounds, and PTX2 was the most ubiquitous toxin in sediment. The spatial distribution of LMP components in the sediment fluctuated with sampling times, partially according to the physical-chemical parameters of the sediment. There are likely several sources for LMPs existing in surface sediments, but it is difficult to determine contributions of a specific toxin-source in the sediment. Therefore, marine sediments may be a toxin reservoir for LMPs accumulation in benthic organisms via food chains.

Synthetic Pinnatoxins A and G Reversibly Block Mouse Skeletal Neuromuscular Transmission In Vivo and In Vitro.

Pinnatoxins (PnTXs) A-H constitute an emerging family belonging to the cyclic imine group of phycotoxins. Interest has been focused on these fast-acting and highly-potent toxins because they are widely found in contaminated shellfish. Despite their highly complex molecular structure, PnTXs have been chemically synthetized and demonstrated to act on various nicotinic acetylcholine receptor (nAChR) subtypes. In the present work, PnTX-A, PnTX-G and analogue, obtained by chemical synthesis with a high degree of purity (>98%), have been studied in vivo and in vitro on adult mouse and isolated nerve-muscle preparations expressing the mature muscle-type (α1)2ß1δε nAChR. The results show that PnTX-A and G acted on the neuromuscular system of anesthetized mice and blocked the compound muscle action potential (CMAP) in a dose- and time-dependent manner, using a minimally invasive electrophysiological method. The CMAP block produced by both toxins in vivo was reversible within 6-8 h. PnTX-A and G, applied to isolated extensor digitorum longus nerve-muscle preparations, blocked reversibly isometric twitches evoked by nerve stimulation. The action of PnTX-A was reversed by 3,4-diaminopyridine. Both toxins exerted no direct action on muscle fibers, as revealed by direct muscle stimulation. PnTX-A and G blocked synaptic transmission at mouse neuromuscular junctions and PnTX-A amino ketone analogue (containing an open form of the imine ring) had no effect on neuromuscular transmission. These results indicate the importance of the cyclic imine for interacting with the adult mammalian muscle-type nAChR. Modeling and docking studies revealed molecular determinants responsible for the interaction of PnTXs with the muscle-type nAChR.

Mixtures of Lipophilic Phycotoxins: Exposure Data and Toxicological Assessment.

Lipophilic phycotoxins are secondary metabolites produced by phytoplanktonic species. They accumulate in filter-feeding shellfish and can cause human intoxication. Regulatory limits have been set for individual toxins, and the toxicological features are well characterized for some of them. However, phycotoxin contamination is often a co-exposure phenomenon, and toxicological data regarding mixtures effects are very scarce. Moreover, the type and occurrence of phycotoxins can greatly vary from one region to another. This review aims at summarizing the knowledge on (i) multi-toxin occurrence by a comprehensive literature review and (ii) the toxicological assessment of mixture effects. A total of 79 publications was selected for co-exposure evaluation, and 44 of them were suitable for toxin ratio calculations. The main toxin mixtures featured okadaic acid in combination with pectenotoxin-2 or yessotoxin. Only a few toxicity studies dealing with co-exposure were published. In vivo studies did not report particular mixture effects, whereas in vitro studies showed synergistic or antagonistic effects. Based on the combinations that are the most reported, further investigations on mixture effects must be carried out.

Cyclic imine toxins from dinoflagellates: a growing family of potent antagonists of the nicotinic acetylcholine receptors.

We present an overview of the toxicological profile of the fast-acting, lipophilic macrocyclic imine toxins, an emerging family of organic compounds associated with algal blooms, shellfish contamination and neurotoxicity. Worldwide, shellfish contamination incidents are expanding; therefore, the significance of these toxins for the shellfish food industry deserves further study. Emphasis is directed to the dinoflagellate species involved in their production, their chemical structures, and their specific mode of interaction with their principal natural molecular targets, the nicotinic acetylcholine receptors, or with the soluble acetylcholine-binding protein, used as a surrogate receptor model. The dinoflagellates Karenia selliformis and Alexandrium ostenfeldii / A. peruvianum have been implicated in the biosynthesis of gymnodimines and spirolides, while Vulcanodinium rugosum is the producer of pinnatoxins and portimine. The cyclic imine toxins are characterized by a macrocyclic skeleton comprising 14-27 carbon atoms, flanked by two conserved moieties, the cyclic imine and the spiroketal ring system. These phycotoxins generally display high affinity and broad specificity for the muscle type and neuronal nicotinic acetylcholine receptors, a feature consistent with their binding site at the receptor subunit interfaces, composed of residues highly conserved among all nAChRs, and explaining the diverse toxicity among animal species. This is an article for the special issue XVth International Symposium on Cholinergic Mechanisms.

Growth, Toxin Production and Allelopathic Effects of Pseudo-nitzschia multiseries under Iron-Enriched Conditions.

In order to assess the effects of Fe-enrichment on the growth and domoic acid (DA) production of the toxigenic diatom Pseudo-nitzschia multiseries, static cultures that received the addition of different iron (Fe) concentrations were maintained for 30 days. Intra- and extracellular DA concentrations were evaluated over time, and growth and chain-formation were compared to those of non-toxic diatoms, Bacillaria sp. Growth rates of P. multiseries (µ = 0.45-0.73 d-1) were similar among cultures containing different Fe concentrations. Likewise, the similar incidence and length of P. multiseries stepped cell chains (usually 2-4; up to 8-cell long) among the treatments reinforces that the cultures were not growth-inhibited under any condition tested, suggesting an efficient Fe acquisition mechanism. Moreover, DA concentrations were significantly higher under the highest Fe concentration, indicating that Fe is required for toxin synthesis. Bacillaria sp. reached comparable growth rates under the same Fe concentrations, except when the dissolved cell contents from a P. multiseries culture was added. The 50-70% reduction in cell density and 70-90% decrease in total chlorophyll-a content of Bacillaria sp. at early stationary growth phase indicates, for the first time, an allelopathic effect of undetermined compounds released by Pseudo-nitzschia to another diatom species.

Effects of okadaic acid, azaspiracid-1, yessotoxin and their binary mixtures on human intestinal Caco-2 cells.

Phycotoxins are responsible for foodborne intoxications. Symptoms depend on the ingested toxins but mostly imply gastro-intestinal and neurological disorders. Importantly, humans are exposed to combinations of several phycotoxins, resulting in possible mixture effects. Most previous studies, however, have been focused on single toxin effects. Thus, the aim of this study was to examine the effects of binary mixtures of three main phycotoxins, okadaic acid (OA), azaspiracid-1 (AZA1) and yessotoxin (YTX), on human intestinal Caco-2 cells. The focus was placed on cell viability studies and inflammation responses using a multi-parametric approach to assess cell population (nuclei staining), cell metabolism/viability (reductase activity and lysosomal integrity), and release of inflammation markers (e.g., interleukins). Mixture effects were evaluated using the concentration addition (CA) and independent action (IA) models. Our assays show that none of the toxins had an impact on the cell population in the tested concentration range. Only OA modulated reductase activity, while all three toxins had strong effects on lysosomal integrity. Furthermore, all toxins triggered the release of interleukin 8 (IL-8), with OA being most potent. Mixture effect analysis showed additivity in most cases. However, supra-additivity was observed in regards to IL-6 and IL-8 release for combinations implying high concentrations of OA. This study extends the knowledge on mixture effects of phycotoxins in human cells.

Marine toxins in seafood: Recent updates on sample pretreatment and determination techniques.

Marine toxins can lead to varying degrees of human poisoning, often resulting in fatal symptoms and causing significant economic losses in seafood-producing regions. To gain a deeper comprehension of the role of marine toxins in seafood and their impact on the environment, it is imperative to develop rapid, cost-effective, environmentally friendly, and efficient methods for sample pretreatment and determination to mitigate adverse impacts of marine toxins. This review presents a comprehensive overview of advancements made in sample pretreatment and determination techniques for marine toxins since 2017. The advantages and disadvantages of various technologies were critically examined. Additionally, the current challenges and future development strategies for the analysis of marine toxins are provided.

Occurrence and distribution of phycotoxins in the Antarctic Ocean.

Lipophilic phycotoxins (LPTs) and domoic acid (DA) in Antarctic seawater, as well as parts of the South Pacific and the Southern Indian Oceans were systematically investigated. DA and six LPTs, namely pectenotoxin-2 (PTX2), okadaic acid (OA), yessotoxin (YTX), homo-yessotoxin (h-YTX), 13-desmethyl spirolide C (SPX1), and gymnodimine (GYM), were detected. PTX2, as the dominant LPTs, was widely distributed in seawater surrounding Antarctica, whereas OA, YTX, and h-YTX were irregularly distributed across the region. The total concentration of LPTs in surface seawater ranged from 0.10 to 13.57 ng/L (mean = 2.20 ng/L). ∑LPT levels were relatively higher in the eastern sea areas of Antarctica than in the western sea areas. PTX2 was the main LPT in the vertical profiles, and the PTX2 concentration was significantly higher in the epipelagic zone than water depths below 200 m. The predominant sources of PTX2 and OA in Antarctic sea areas are likely to be Dinophysis.

Simultaneous determination of trace marine lipophilic and hydrophilic phycotoxins in various environmental and biota matrices.

An efficient and sensitivity approach, which combines solid-phase extraction or ultrasonic extraction for pretreatment, followed by ultra-performance liquid chromatography-tandem mass spectrometry, has been established to simultaneously determine eight lipophilic phycotoxins and one hydrophilic phycotoxin in seawater, sediment and biota samples. The recoveries and matrix effects of target analytes were in the range of 61.6-117.3 %, 55.7-121.3 %, 57.5-139.9 % and 82.6 %-95.0 %, 85.8-106.8 %, 80.7 %-103.3 % in seawater, sediment, and biota samples, respectively. This established method revealed that seven, six and six phycotoxins were respectively detected in the Beibu Gulf, with concentrations ranging from 0.14 ng/L (okadaic acid, OA) to 26.83 ng/L (domoic acid, DA) in seawater, 0.04 ng/g (gymnodimine-A, GYM-A) to 2.75 ng/g (DA) in sediment and 0.01 ng/g (GYM-A) to 2.64 ng/g (domoic acid) in biota samples. These results suggest that the presented method is applicable for the simultaneous determination of trace marine lipophilic and hydrophilic phycotoxins in real samples.

Did algal toxin and Klebsiella infections cause the unexplained 2007 mass mortality event in Danish and Swedish marine mammals?

An unusual mass mortality event (MME) of harbour seals (Phoca vitulina) and harbour porpoises (Phocoena phocoena) occurred in Denmark and Sweden in June 2007. Prior to this incident, the region had experienced two MMEs in harbour seals caused by Phocine Distemper Virus (PDV) in 1988 and 2002. Although epidemiology and symptoms of the 2007 MME resembled PDV, none of the animals examined for PDV tested positive. Thus, it has been speculated that another - yet unknown - pathogen caused the June 2007 MME. To shed new light on the likely cause of death, we combine previously unpublished veterinary examinations of harbour seals with novel analyses of algal toxins and algal monitoring data. All harbour seals subject to pathological examination showed pneumonia, but were negative for PDV, influenza and coronavirus. Histological analyses revealed septicaemia in multiple animals, and six animals tested positive for Klebsiella pneumonia. Furthermore, we detected the algal Dinophysis toxin DTX-1b (1-115 ng g-1) in five seals subject to toxicology, representing the first time DTX-1b has been detected in marine vertebrates. However, no animals tested positive for both Klebsiella and toxins. Thus, while our relatively small sample size prevent firm conclusions on causative agents, we speculate that the unexplained MME may have been caused by a chance incidence of multiple pathogens acting in parallel in June 2007, including Dinophysis toxin and Klebsiella. Our study illustrates the complexity of wildlife MMEs and highlights the need for thorough sampling during and after MMEs, as well as additional research on and monitoring of DTX-1b and other algal toxins in the region.

Toxic effects of the emerging Alexandrium pseudogonyaulax (Dinophyceae) on multiple trophic levels of the pelagic food web.

The dinoflagellate Alexandrium pseudogonyaulax, a harmful algal bloom species, is currently appearing in increasing frequency and abundance across Northern European waters, displacing other Alexandrium species. This mixotrophic alga produces goniodomins (GDs) and bioactive extracellular substances (BECs) that may pose a threat to coastal ecosystems and other marine resources. This study demonstrated the adverse effects of A. pseudogonyaulax on four marine trophic levels, including microalgae (Rhodomonas salina), microzooplankton (Polykrikos kofoidii) and mesozooplankton (Acartia tonsa), as well as fish gill cells (RTgill-W1, Oncorhynchus mykiss), ultimately leading to enhanced mortality and cell lysis. Furthermore, cell-free supernatants collected from A. pseudogonyaulax cultures caused complete loss of metabolic activity in the RTgill-W1 cell line, indicating ichthyotoxic properties, while all tested GDs were much less toxic. In addition, cell-free supernatants of A. pseudogonyaulax led to cell lysis of R. salina, while all tested GDs were non-lytic. Finally, reduced egg hatching rates of A. tonsa eggs exposed to cell-free supernatants of A. pseudogonyaulax and impaired mobility of P. kofoidii and A. tonsa exposed to A. pseudogonyaulax were also observed. Altogether, bioassay results suggest that the toxicity of A. pseudogonyaulax is mainly driven by BECs and not by GDs, although further research into factors modulating the lytic activity of Alexandrium spp. are needed.