Dinophysistoxin-1 Research Articles

Lipophilic marine toxins discovered in the Bohai Sea using high performance liquid chromatography coupled with tandem mass spectrometry

Some dinoflagellates can produce lipophilic marine toxins, which pose potent threats to seafood consumers. In the Bohai Sea, an important semi-closed inland sea with intensive mariculture industry in China, there is little knowledge concerning lipophilic marine toxins and their potential threats. In this study, net-concentrated phytoplankton samples were periodically collected from 5 typical mariculture zones around the Bohai Sea, including Laishan (LS), Laizhou (LZ), Hangu (HG), Qinhuangdao (QHD) and Huludao (HLD) in 2013 and 2014, and a method using high performance liquid chromatography (HPLC) coupled with a Q-Trap mass spectrometer was applied to analyze seven representative lipophilic marine toxins, including okadaic acid (OA), dinophysistoxin-1 (DTX1), pectenotoxin-2 (PTX2), yessotoxin (YTX), azaspiracid-1 (AZA1), gymnodimine (GYM), and 13-desmethyl spirolide C (desMeC). The method had high sensitivity and repeatability, and exhibited satisfactory recoveries for most of the lipophilic marine toxins (92.1–108%) except for AZA1 (65.8–68.9%). Nearly all the lipophilic marine toxins could be detected in phytoplankton samples from the Bohai Sea. OA, DTX1 and PTX2 were predominant components and present in most of the phytoplankton samples. The maximum content of lipophilic marine toxin in phytoplankton samples concentrated from seawater (OA 464 pg L−1; DTX1 783 pg L−1; YTX 86.6 pg L−1; desMeC 15.6 pg L−1; PTX2 1.11 × 103 pg L−1) appeared in June 2014. Based on toxins present in phytoplankton samples, it is implied that seafood in the Bohai Sea is more likely to be contaminated by OA group and PTX group toxins, and spring is the high-risk season for toxin contamination.

Occurrence of marine algal toxins in oyster and phytoplankton samples in Daya Bay, South China Sea

The occurrence and seasonal variations of marine algal toxins in phytoplankton and oyster samples in Daya Bay (DYB), South China Sea were investigated. Two Dinophysis species, namely, D. caudata and D. acuminata complex, were identified as Okadaic acid (OA)/pectenotoxin (PTX) related species. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis demonstrated that 2.04–14.47 pg PTX2 per cell was the predominant toxin in single-cell isolates of D. caudata. D. acuminata was not subjected to toxin analysis. The occurrence of OAs in phytoplankton concentrates of net-haul sample coincided with the presence of D. accuminata complex, suggesting that this species is most likely an OA producer in this sea area. OA, dinophysistoxins-1 (DTX1), PTX2, PTX2sa, gymnodimine (GYM), homoyessotoxin (homoYTX), and domoic acid (DA) demonstrated positive results in net haul samples. To our best knowledge, this paper is the first to report the detection of GYM, DA, and homoYTX in phytoplankton samples in Chinese coastal waters. Among the algal toxins, GYM demonstrated the highest frequency of positive detections in phytoplankton concentrates (13/17). Five compounds of algal toxins, including OA, DTX1, PTX2, PTX2sa, and GYM, were detected in oyster samples. DA and homoYTX were not detected in oysters despite of positive detections for both in the phytoplankton concentrates. However, neither the presence nor absence of DA in oysters can be determined because extraction conditions with 100% methanol used to isolate toxins from oysters (recommended by the EU-Harmonised Standard Operating Procedure, 2015) would likely be unsuitable for this water-soluble toxin. In addition, transformation of DA during the digestion process of oysters may also be involved in the negative detections of this toxin. GYM exhibited the highest frequency of positive results in oysters (14/17). OAs were only detected in the hydrolyzed oyster samples. The detection rates of PTX and PTX2sa in oysters were lower than those in the net haul samples.

Characterization of the dinophysistoxin-2 acute oral toxicity in mice to define the Toxicity Equivalency Factor

Ingestion of shellfish with dinophysistoxin-2 (DTX2) can lead to diarrheic shellfish poisoning (DSP). The official control method of DSP toxins in seafood is the liquid chromatography-mass spectrometry analysis (LC-MS). However in order to calculate the total toxicity of shellfish, the concentration of each compound must be multiplied by individual Toxicity Equivalency Factor (TEF). Considering that TEFs caused some controversy and the scarce information about DTX2 toxicity, the aim of this study was to characterize the oral toxicity of DTX2 in mice.A 4-Level Up and Down Procedure allowed the characterization of DTX2 effects and the estimation of DTX2 oral TEF based on determination of the lethal dose 50 (LD50). DTX2 passed the gastrointestinal barrier and was detected in urine and feces. Acute toxicity symptoms include diarrhea and motionless, however anatomopathology study and ultrastructural images restricted the toxin effects to the gastrointestinal tract. Nevertheless enterocytes microvilli and tight junctions were not altered, disconnecting DTX2 diarrheic effects from paracellular epithelial permeability. This is the first report of DTX2 oral LD50 (2262 μg/kg BW) indicating that its TEF is about 0.4. This result suggests reevaluation of the present TEFs for the DSP toxins to better determine the actual risk to seafood consumers.

Lipophilic Toxin Profile in Mytilus galloprovincialis from the North Atlantic Coast of Morocco: LC-MS/MS and Mouse Bioassay Analyses

Forthe Moroccan Phycotoxins Monitoring that is part of theSafety of theCoastal Monitoring Network(RSSL),shellfish sampleswereharvestedfromdifferentlocations at NorthAtlantic of Morocco whereharmful algaeblooms were known to have occurred. Forallshellfishsamples found positive by themouse bioassay fordiarrheicshellfishpoisoning(DSP)toxins,liquid chromatography coupled to tandem massspectrometry(LC-MS-MS) in order to search thefollowinglipophilictoxins: okadaic acid (OA), dinophysistoxins(DTXs), pectenotoxins(PTXs), azaspiracids(AZAs), yessotoxins(YTXs), spirolides(SPXs) and gymnodimines (GYMs).Theresultsrevealeddifferentlipophilictoxinprofiles as a function of theshellfish sampling location. It has beennoticed that allthesamples contained OA and its derivativesnamed dinophysistoxins(DTXs). In addition, other lipophilictoxinswerefound in shellfishsamples:(YTXs), and pectenotoxins(PTX-2, PTX-2-seco-acid and 7- epi-PTX-2-seco-acid) on theNorthAtlantic coast .This paper reports on thefirstdetection of YTXs,PTXsGYMs and their derivatives in Moroccan shellfish.

Sublethal doses of dinophysistoxin-1 and okadaic acid stimulate secretion of inflammatory factors on innate immune cells: Negative health consequences

One of the proposed mechanisms to explain why Diarrhetic Shellfish Poison (DSP) toxins are tumor promoters is founded on the capacity of these toxins to increase TNF-α secretion. Although macrophages are the principal cells in the activation of the inflammatory response, the immune profile that Okadaic acid (OA) and Dinophysistoxin-1 (DTX-1) trigger in these cells has not been fully explored. We have therefore investigated the effect of various concentrations of both toxins on the activity of several inflammatory factors. Our results demonstrate that OA and DTX-1, at sublethal doses, stimulate secretion of inflammatory factors. Nevertheless DTX-1 was more potent than OA in increasing TNF-α and IL-6 as well as their dependent chemokines KC, MCP-1, LIX, MIP-1 α, MIP-1 β and MIP-2. On the other hand, secretion of IFN-γ and the anti-inflammatory cytokines, IL-4 and IL-10, was unaffected. In addition, DTX-1 also raises matrix metalloproteinase-9 (MMP-9) activity. In this study, for the first time the effect of OA and DTX-1 over the secretion of pro-inflammatory and carcinogenic signals in macrophages are compared, showing that DTX-1 is ten times more potent that OA. The inflammatory profile produced by DTX-1 is shown for the first time. The safe limit regulation should be changed to DSP toxins zero tolerance in the shellfish to be consumed by humans.

Molecular diversity patterns among various phytoplankton size-fractions in West Greenland in late summer

Arctic regions have experienced pronounced biological and biophysical transformations as a result of global change processes over the last several decades. Current hypotheses propose an elevated impact of those environmental changes on the biodiversity, community composition and metabolic processes of species. The effects on ecosystem function and services, particularly when invasive or toxigenic harmful species become dominant, can be expressed over a wide range of temporal and spatial scales in plankton communities. Our study focused on the comparison of molecular biodiversity of three size-fractions (micro-, nano-, picoplankton) in the coastal pelagic zone of West Greenland and their association with environmental parameters. Molecular diversity was assessed via parallel amplicon sequencing the 28S rRNA hypervariable D1/D2 region. We showed that biodiversity distribution within the area of Uummannaq Fjord, Vaigat Strait and Disko Bay differed markedly within and among size-fractions. In general, we observed a higher diversity within the picoplankton size fraction compared to the nano- and microplankton. In multidimensional scaling analysis, community composition of all three size fractions correlated with cell size, silicate and phosphate, chlorophyll a (chl a) and dinophysistoxin (DTX). Individually, each size fraction community composition also correlated with other different environmental parameters, i.e. temperature and nitrate. We observed a more homogeneous community of the picoplankton across all stations compared to the larger size classes, despite different prevailing environmental conditions of the sampling areas. This suggests that habitat niche occupation for larger-celled species may lead to higher functional trait plasticity expressed as an enhanced range of phenotypes, whereas smaller organisms may compensate for lower potential plasticity with higher diversity. The presence of recently identified toxigenic harmful algal bloom (HAB) species (such as Alexandrium fundyense and A. ostenfeldii) in the area points out the potential risk for this vulnerable ecosystem in a changing world.

Structure-Activity Relationship Studies Using Natural and Synthetic Okadaic Acid/Dinophysistoxin Toxins.

Okadaic acid (OA) and the closely related dinophysistoxins (DTXs) are algal toxins that accumulate in shellfish and are known serine/threonine protein phosphatase (ser/thr PP) inhibitors. Phosphatases are important modulators of enzyme activity and cell signaling pathways. However, the interactions between the OA/DTX toxins and phosphatases are not fully understood. This study sought to identify phosphatase targets and characterize their structure–activity relationships (SAR) with these algal toxins using a combination of phosphatase activity and cytotoxicity assays. Preliminary screening of 21 human and yeast phosphatases indicated that only three ser/thr PPs (PP2a, PP1, PP5) were inhibited by physiologically saturating concentrations of DTX2 (200 nM). SAR studies employed naturally-isolated OA, DTX1, and DTX2, which vary in degree and/or position of methylation, in addition to synthetic 2-epi-DTX2. OA/DTX analogs induced cytotoxicity and inhibited PP activity with a relatively conserved order of potency: OA = DTX1 ≥ DTX2 >> 2-epi-DTX. The PPs were also differentially inhibited with sensitivities of PP2a > PP5 > PP1. These findings demonstrate that small variations in OA/DTX toxin structures, particularly at the head region (i.e., C1/C2), result in significant changes in toxicological potency, whereas changes in methylation at C31 and C35 (tail region) only mildly affect potency. In addition to this being the first study to extensively test OA/DTX analogs’ activities towards PP5, these data will be helpful for accurately determining toxic equivalence factors (TEFs), facilitating molecular modeling efforts, and developing highly selective phosphatase inhibitors.

Harmful effects of Dinophysis to the ciliate Mesodinium rubrum: Implications for prey capture

Toxigenic Dinophysis spp. are obligate mixotrophic dinoflagellates that require a constant supply of prey—Mesodinium rubrum—to achieve long-term growth by means of kleptoplasty. Mesodinium rubrum is, however, a fast moving, jumping ciliate exhibiting an effective escape response from suspensivorous predators. In the present study, a series of laboratory experiments evaluating the motility and survival of M. rubrum in the presence of Dinophysis cells and/or substances contained in their culture medium was designed, in order to assess the mechanisms involved in prey capture by Dinophysis spp. Cell abundance of M. rubrum decreased in the presence of Dinophysis cf. ovum cells producing okadaic acid (OA; up to 7.94±2.67pgcell−1) and smaller amounts of dinophysistoxin-1 (DTX-1) and pectenotoxin-2 (PTX-2). Prey capture was often observed after the ciliate had been attached to adhesive “mucus traps”, which only appeared in the presence of Dinophysis cells. Before being attached to the mucus traps, M. rubrum cells reduced significantly their swimming frequency (from ∼41 to 19±3 jumps min−1) after only 4h of initial contact with D. cf. ovum cells. M. rubrum survival was not affected in contact with purified OA, DTX-1 and PTX-2 solutions, but decreased significantly when the ciliate was exposed to cell-free or filtered culture medium from both D. cf. ovum and D. caudata, the latter containing moderate concentrations of free eicosapentaenoic acid and docosahexaenoic acid. The results thus indicate that Dinophysis combines the release of toxic compounds other than shellfish toxins, possibly free PUFAs, and a “mucus trap” to enhance its prey capture success by immobilizing and subsequently arresting M. rubrum cells.

The Mechanism of Diarrhetic Shellfish Poisoning Toxin Production in Prorocentrum spp.: Physiological and Molecular Perspectives.

Diarrhetic shellfish poisoning (DSP) is a gastrointestinal disorder caused by the consumption of seafood contaminated with okadaic acid (OA) and dinophysistoxins (DTXs). OA and DTXs are potent inhibitors of protein phosphatases 2A, 1B, and 2B, which may promote cancer in the human digestive system. Their expression in dinoflagellates is strongly affected by nutritional and environmental factors. Studies have indicated that the level of these biotoxins is inversely associated with the growth of dinoflagellates at low concentrations of nitrogen or phosphorus, or at extreme temperature. However, the presence of leucine or glycerophosphate enhances both growth and cellular toxin level. Moreover, the presence of ammonia and incubation in continuous darkness do not favor the toxin production. Currently, studies on the mechanism of this biotoxin production are scant. Full genome sequencing of dinoflagellates is challenging because of the massive genomic size; however, current advanced molecular and omics technologies may provide valuable insight into the biotoxin production mechanism and novel research perspectives on microalgae. This review presents a comprehensive analysis on the effects of various nutritional and physical factors on the OA and DTX production in the DSP toxin-producing Prorocentrum spp. Moreover, the applications of the current molecular technologies in the study on the mechanism of DSP toxin production are discussed.

Development of Certified Reference Materials for Diarrhetic Shellfish Poisoning Toxins, Part 1: Calibration Solutions.

Okadaic acid (OA) and its analogs dinophysistoxins-1 (DTX1) and -2 (DTX2) are lipophilic polyethers produced by marine dinoflagellates. These toxins accumulate in shellfish and cause diarrhetic shellfish poisoning (DSP) in humans. Regulatory testing of shellfish is essential to safeguard public health and for international trade. Certified reference materials (CRMs) play a key role in analytical monitoring programs. This paper presents an overview of the interdisciplinary work that went into the planning, production, and certification of calibration-solution CRMs for OA, DTX1, and DTX2. OA and DTX1 were isolated from large-scale algal cultures and DTX2 from naturally contaminated mussels. Toxins were isolated by a combination of extraction and chromatographic steps with processes adapted to suit the source and concentration of each toxin. New 19-epi-DSP toxin analogs were identified as minor impurities. Once OA, DTX1, and DTX2 were established to be of suitable purity, solutions were prepared and dispensed into flame-sealed glass ampoules. Certification measurements were carried out using quantitative NMR spectroscopy and LC-tandem MS. Traceability of measurements was established through certified external standards of established purity. Uncertainties were assigned following standards and guidelines from the International Organization for Standardization, with components from the measurement, stability, and homogeneity studies being propagated into final combined uncertainties.

Development of Certified Reference Materials for Diarrhetic Shellfish Poisoning Toxins, Part 2: Shellfish Matrix Materials.

Okadaic acid (OA) and its analogs, dinophysistoxins-1 (DTX1) and -2 (DTX2) are lipophilic biotoxins produced by marine algae that can accumulate in shellfish and cause the human illness known as diarrhetic shellfish poisoning (DSP). Regulatory testing of shellfish is required to protect consumers and the seafood industry. Certified reference materials (CRMs) are essential for the development, validation, and quality control of analytical methods, and thus play an important role in toxin monitoring. This paper summarizes work on research and development of shellfish tissue reference materials for OA and DTXs. Preliminary work established the appropriate conditions for production of shellfish tissue CRMs for OA and DTXs. Source materials, including naturally incurred shellfish tissue and cultured algae, were screened for their DSP toxins. This preliminary work informed planning and production of a wet mussel (Mytilus edulis) tissue homogenate matrix CRM. The homogeneity and stability of the CRM were evaluated and found to be fit-for-purpose. Extraction and LC-tandem MS methods were developed to accurately certify the concentrations of OA, DTX1, and DTX2 using a combination of standard addition and matrix-matched calibration to compensate for matrix effects in electrospray ionization. The concentration of domoic acid was also certified. Uncertainties were assigned following standards and guidelines from the International Organization for Standardization. The presence of other toxins in the CRM was also assessed and information values are reported for OA and DTX acyl esters.

Prorocentrum lima from the South Atlantic: Morphological, molecular and toxicological characterization

Morphological descriptions using light and scanning electron microscopy and molecular characterization of two Prorocentrum lima strains (UNR-01 and UNR-09) isolated from Armação dos Búzios, Rio de Janeiro, Brazil are provided. Okadaic acid (OA), dinophysistoxin-1 (DTX1) and DTX2 production by strain UNR-01 was investigated using liquid chromatography with mass spectrometry. Toxins were extracted from heat-treated (boiled) and non-boiled cell pellets to obtain respective quantities of free and total OA and DTX1. Growth parameters (growth rate and mean generation time) were determined for strain UNR-01. Prorocentrum lima cells were oblong-to-ovate in shape, broad in the middle region, and narrow at the anterior end. The periflagellar area was triangular, set into a V-shaped depression and was composed of eight periflagellar platelets of different sizes. The morphology fits well the characterization of the species isolated from elsewhere. Phylogenetic analysis based on internal transcribed spacer – ITS – and D1–D3 large subunit – LSU – of ribosomal RNA gene sequences revealed that both strains were identical and closely related to P. lima isolates from the Caribbean Sea and USA. The growth rate of strain UNR-01 was 0.24divday−1. OA concentrations were on average 15.2 and 38.5pg[OA]cell−1 for heat-treated and non-treated cells respectively, while DTX1 mean concentration was 0.5pg[DTX1]cell−1 for both heat-treated and non-treated cells. DTX2 was not detected. To date, these are the first strains of P. lima from the south Atlantic that have been characterized.

Early Genotoxic and Cytotoxic Effects of the Toxic Dinoflagellate Prorocentrum lima in the Mussel Mytilus galloprovincialis.

Okadaic acid (OA) and dinophysistoxins (DTXs) are the main toxins responsible for diarrhetic shellfish poisoning (DSP) intoxications during harmful algal blooms (HABs). Although the genotoxic and cytotoxic responses to OA have been evaluated in vitro, the in vivo effects of these toxins have not yet been fully explored. The present work fills this gap by evaluating the in vivo effects of the exposure to the DSP-toxin-producing dinoflagellate Prorocentrum lima during the simulation of an early HAB episode in the mussel Mytilus galloprovincialis. The obtained results revealed that in vivo exposure to this toxic microalgae induced early genotoxicity in hemocytes, as a consequence of oxidative DNA damage. In addition, the DNA damage observed in gill cells seems to be mainly influenced by exposure time and P. lima concentration, similarly to the case of the oxidative damage found in hemocytes exposed in vitro to OA. In both cell types, the absence of DNA damage at low toxin concentrations is consistent with the notion suggesting that this level of toxicity does not disturb the antioxidant balance. Lastly, in vivo exposure to growing P. lima cell densities increased apoptosis but not necrosis, probably due to the presence of a high number of protein apoptosis inhibitors in molluscs. Overall, this work sheds light into the in vivo genotoxic and cytotoxic effects of P. lima. In doing so, it also demonstrates for the first time the potential of the modified (OGG1) comet assay for assessing oxidative DNA damage caused by marine toxins in marine invertebrates.

Effect of carbon chain length in acyl coenzyme A on the efficiency of enzymatic transformation of okadaic acid to 7-O-acyl okadaic acid

Okadaic acid (OA), a product of dinoflagellate Prorocentrum spp., is transformed into 7-O-acyl OA in various bivalve species. The structural transformation proceeds enzymatically in vitro in the presence of the microsomal fraction from the digestive gland of bivalves. We have been using LC–MS/MS to identify OA-transforming enzymes by detecting 7-O-acyl OA, also known as dinophysistoxin 3 (DTX3). However, an alternative assay for DTX3 is required because the OA-transforming enzyme is a membrane protein, and surfactants for solubilizing membrane proteins decrease the sensitivity of LC–MS/MS. The present study examined saturated fatty acyl CoAs with a carbon chain length of 10 (decanoyl), 12 (dodecanoyl), 14 (tetradecanoyl), 16 (hexadecanoyl) and 18 (octadecanoyl) as the substrate for the in vitro acylation reaction. Saturated fatty acyl CoAs with a carbon chain length of 14, 16 and 18 exhibited higher yields than those with a carbon chain length of 10 or 12. Acyl CoAs with carbon chain lengths from 14 to 18 and containing either a diene unit, an alkyne unit, or an azide unit in the carbon chain were synthesized and shown to provide the corresponding DTX3 with a yield comparable to that of hexadecanoyl CoA. The three functional units can be conjugated with fluorescent reagents and are applicable to the development of a novel assay for DTX3.

Accumulation, transformation and breakdown of DSP toxins from the toxic dinoflagellate Dinophysis acuta in blue mussels, Mytilus edulis

Okadaic acid (OA), dinophysistoxins (DTX) and pectenotoxins (PTX) produced by the dinoflagellates Dinophysis spp. can accumulate in shellfish and cause diarrhetic shellfish poisoning upon human consumption. Shellfish toxicity is a result of algal abundance and toxicity as well as accumulation and depuration kinetics in mussels. We mass-cultured Dinophysis acuta containing OA, DTX-1b and PTX-2 and fed it to the blue mussel, Mytilus edulis under controlled laboratory conditions for a week to study toxin accumulation and transformation. Contents of OA and DTX-1b in mussels increased linearly with incubation time, and the net toxin accumulation was 66% and 71% for OA and DTX-1b, respectively. Large proportions (≈50%) of both these toxins were transformed to fatty acid esters. Most PTX-2 was transformed to PTX-2 seco-acid and net accumulation was initially high, but decreased progressively throughout the experiment, likely due to esterification and loss of detectability. We also quantified depuration during the subsequent four days and found half-life times of 5–6 days for OA and DTX-1b. Measurements of dissolved toxins revealed that depuration was achieved through excreting rather than metabolizing toxins. This is the first study to construct a full mass balance of DSP toxins during both accumulation and depuration, and we demonstrate rapid toxin accumulation in mussels at realistic in situ levels of Dinophysis. Applying the observed accumulation and depuration kinetics, we model mussel toxicity, and demonstrate that a concentration of only 75 Dinophysis cells l−1 is enough to make 60 mm long mussels exceed the regulatory threshold for OA equivalents.

Algal toxin profiles in Nigerian coastal waters (Gulf of Guinea) using passive sampling and liquid chromatography coupled to mass spectrometry

Algal toxins may accumulate in fish and shellfish and thus cause poisoning in consumers of seafood. Such toxins and the algae producing them are regularly surveyed in many countries, including Europe, North America, Japan and others. However, very little is known regards the occurrence of such algae and their toxins in most African countries. This paper reports on a survey of phytoplankton and algal toxins in Nigerian coastal waters.Seawater samples were obtained from four sites for phytoplankton identification, on three occasions between the middle of October 2014 and the end of February 2015 (Bar Beach and Lekki in Lagos State, Port Harcourt in Rivers State and Uyo in Akwa Ibom State). The phytoplankton community was generally dominated by diatoms and cyanobacteria; however several species of dinoflagellates were also identified: Dinophysis caudata, Lingulodinium polyedrum and two benthic species of Prorocentrum.Passive samplers (containing Diaion® HP-20 resin) were deployed for several 1-week periods on the same four sites to obtain profiles of algal toxins present in the seawater. Quantifiable amounts of okadaic acid (OA) and pectenotoxin 2 (PTX2), as well as traces of dinophysistoxin 1 (DTX1) were detected at several sites. Highest concentrations (60 ng OA g−1 HP-20 resin) were found at Lekki and Bar Beach stations, which also had the highest salinities. Non-targeted analysis using full-scan high resolution mass spectrometry showed that algal metabolites differed from site to site and for different sampling occasions. Screening against a marine natural products database indicated the potential presence of cyanobacterial compounds in the water column, which was also consistent with phytoplankton analysis.During this study, the occurrence of the marine dinoflagellate toxins OA and PTX2 has been demonstrated in coastal waters of Nigeria, despite unfavourable environmental conditions, with regards to the low salinities measured. Hence shellfish samples should be monitored in future to assess the risk for public health through accumulation of such toxins in seafood.

In vitro bioaccessibility of the marine biotoxin okadaic acid in shellfish

Okadaic acid (OA) and their derivatives are marine toxins responsible for the human diarrhetic shellfish poisoning (DSP). To date the amount of toxins ingested in food has been considered equal to the amount of toxins available for uptake by the human body. In this study, the OA fraction released from the food matrix into the digestive fluids (bioaccessibility) was assessed using a static in vitro digestion model. Naturally contaminated mussels (Mytilus galloprovincialis) and donax clams (Donax sp.), collected from the Portuguese coast, containing OA and dinophysistoxin-3 (DTX3) were used in this study. Bioaccessibility of OA total content was 88% and 75% in mussels and donax clams, respectively. Conversion of DTX3 into its parent compound was verified during the simulated digestive process and no degradation of these toxins was found during the process. This is the first study assessing the bioaccessibility of OA-group toxins in naturally contaminated seafood. This study provides relevant new data that can improve and lead to more accurate food safety risk assessment studies concerning these toxins.

Determination of multiple toxins in whelk and clam samples collected from the Chukchi and Bering seas

Buccinidae whelk Neptunea varicifera (Dall), Cardiidae clam Serripes laperousii (Deshayes), and two unknown species of whelk and clam were collected from the Arctic Chukchi Sea and sub-Arctic Bering Sea in July 2014. In this study, the mollusk samples were analyzed by different liquid chromatography-tandem quadrupole mass spectrometry (LC-MS/MS) methods for multiple shellfish toxins, including okadaic acid (OA), pectenotoxin (PTX), yessotoxin (YTX), azaspiracid (AZA), cyclic imines (CI), and saxitoxin (STX) groups. PTX2 (≈2.0 μg kg−1 whole tissues) was detected exclusively in the clam S. laperousii collected from the Chukchi Sea. OA and dinophysistoxin-1 (DTX1) were restricted to mollusk samples collected from the Bering Sea, and OA was the dominant component of the whelk N. varicifera (63 μg kg−1 digestive gland) and an unknown species of whelk (6.8 μg kg−1 digestive gland). Spirolide-1 (SPX1) was confirmed in most samples except for the whelk N. varicifera collected from the Bering Sea. The highest content of SPX1 (≈18.5 μg kg−1 digestive gland) occurred in the whelk N. varicifera collected from the Chukchi Sea, along with the suspected presence of SPX-C, SPX-D and didesMe-SPX-C. YTX, as well as its derivatives 45-OH-YTX and 45,46,47-Trinor-YTX, were found in all samples, with the highest YTX content (66 μg kg−1 digestive gland) present in the whelk N. varicifera collected from the Chukchi Sea. Interestingly, STX and dcSTX were measured only in the whelk N. varicifera and unknown species of clam collected from the Chukchi Sea. No AZA-group toxins, gymnodimine (GYM), or pinnatoxin G were found in any samples analyzed. Results demonstrated that the mollusk samples were contaminated by multiple shellfish toxins in the Chukchi and Bering seas. This study highlights the need to monitor potentially toxic microalgae in the Arctic and sub-Arctic regions, as well as species of mollusk that may be included in future commercial or subsistence harvests.

Assimilation, Accumulation, and Metabolism of Dinophysistoxins (DTXs) and Pectenotoxins (PTXs) in the Several Tissues of Japanese Scallop Patinopecten yessoensis.

Japanese scallops, Patinopecten yessoensis, were fed with the toxic dinoflagellate Dinophysis fortii to elucidate the relative magnitude of assimilation, accumulation, and metabolism of diarrhetic shellfish toxins (DSTs) and pectenotoxins (PTXs). Three individual scallops were separately exposed to cultured D. fortii for four days. The average cell number of D. fortii assimilated by each individual scallop was 7.7 × 105. Dinophysistoxin-1 (DTX1), pectenotoxin-2 (PTX2) and their metabolites were analyzed by liquid chromatography tandem mass spectrometry (LC/MS/MS) and the toxin content in individual tissues (digestive gland, adductor muscle, gill, gonad, mantle, and the others), feces and the seawater medium were quantified. Toxins were almost exclusively accumulated in the digestive gland with only low levels being detected in the gills, mantles, gonads, and adductor muscles. DTX1 and PTX2 were the dominant toxins in the D. fortii cells fed to the scallops, whereas the dominant toxins detected in the digestive gland of scallops were PTX6 and esterified acyl-O-DTX1 (DTX3). In other tissues PTX2 was the dominant toxin observed. The ratio of accumulated to assimilated toxins was 21%–39% and 7%–23% for PTXs and DTXs respectively. Approximately 54%–75% of PTX2 and 52%–70% of DTX1 assimilated by the scallops was directly excreted into the seawater mainly without metabolic transformation.

Detection of Dinophysistoxin-1 in Clonal Culture of Marine Dinoflagellate Prorocentrum foraminosum (Faust M.A., 1993) from the Sea of Japan.

For the first time the presence of dinophysistoxin-1 (DTX-1) in a culture of Prorocentrum foraminosum was revealed in cells and in the culture medium. The clone was isolated from coastal waters of the Sea of Japan and identified by molecular analyses of SSU and D1/D2 regions of LSU rDNA. The concentration of DTX-1 in cells was 8.4 ± 2.5 pg/cell and, in cell-free media, 27.9 ± 14.7 µg/L. The toxin presence was confirmed by HPLC with high-resolution tandem mass-spectrometry.