Azaspiracid poisoning (AZP) toxins in shellfish: Toxicological and health considerations

It has been almost a decade since a previously unknown human toxic syndrome, azaspiracid poisoning (AZP), emerged as the cause of severe gastrointestinal illness in humans after the consumption of mussels (Mytilus edulis). Structural studies indicated that these toxins, azaspiracids, were of a new unprecedented class containing novel structural features. It is now known that the prevalent azaspiracids in mussels are AZA1, AZA2 and AZA3, which differ from each other in their degree of methylation. Several hydroxylated and carboxylated analogues of the main azaspiracids have also been identified, presumed to be metabolites of the main toxins. Since its first discovery in Irish mussels, the development of facile sensitive and selective LC-MS/MS methods has resulted in the discovery of AZA in other countries and in other species. Mice studies indicate that this toxin class can cause serious tissue injury, especially to the small intestine, and chronic exposure may increase the likelihood of the development of lung tumours. Studies also show that tissue recovery is very slow following exposure. These observations suggest that AZA is more dangerous than the other known classes of shellfish toxins. Consequently, in order to protect human consumers, proper risk assessment and regulatory control of shellfish and other affected species is of the utmost importance.     

The first identification of azaspiracids in shellfish from France and Spain.

Incidents of human intoxications throughout Europe, following the consumption of mussels have been attributed to Azaspiracid Poisoning (AZP). Although first discovered in Ireland, the search for the causative toxins, named azaspiracids, in other European countries has now led to the first discovery of these toxins in shellfish from France and Spain. Separation of the toxins, azaspiracid (AZA1) and analogues, AZA2 and AZA3, was achieved using isocratic reversed-phase liquid chromatography coupled, via an electrospray ionisation source, to an ion-trap mass spectrometer. Azaspiracids were identified in mussels (Mytilus galloprovincialis), 0.24 microg/g, from Galicia, Spain, and scallops (Pecten maximus), 0.32 microg/g, from Brittany, France. Toxin profiles were similar to those found in the equivalent shellfish in Ireland in which AZA1 was the predominant toxin.     

First evidence of an extensive northern European distribution of azaspiracid poisoning (AZP) toxins in shellfish.

Azaspiracids have recently been identified as the toxins responsible for a series of human intoxications in Europe since 1995, following the consumption of cultured mussels (Mytilus edulis) from the west coast of Ireland. Liquid chromatography-mass spectrometric (LC-MS) methods have been applied in the study reported here to investigate the new human toxic syndrome, azaspiracid poisoning. Separation of azaspiracid (AZA1) and its analogues, 8-methylazaspiracid (AZA2) and 22-demethylazaspiracid (AZA3), was achieved using reversed-phase LC and coupled, via an electrospray ionisation source, to an ion-trap mass spectrometer. These azaspiracids have now been identified in mussels from Craster (north-east England) and Sognefjord (south-west Norway) using source collision induced dissociation-MS and multiple tandem MS detection. AZA1 was the predominant toxin and toxin profiles were similar to those found in contaminated Irish shellfish. This is the first report of the occurrence of these azaspiracids outside Ireland with the significant implications that these toxins may occur in shellfish throughout northern Europe.     

Detection of five new hydroxyl analogues of azaspiracids in shellfish using multiple tandem mass spectrometry.

The polyether dinoflagellate toxins, azaspiracids, are responsible for azaspiracid poisoning (AZP), a new human toxic syndrome arising from the consumption of shellfish. To date, five azaspiracids have been isolated and fully structurally elucidated, including, AZA1, its 8-methyl and 22-demethyl analogues, AZA2 and AZA3, respectively, and two hydroxyl derivatives of AZA3, named AZA4 and AZA5. Using a recently developed method involving liquid chromatography with multiple tandem mass spectrometry (LC-MS(n)), five new azaspiracids, AZA7-AZA11, have been found in mussels (Mytilus edulis). AZA6 is a positional isomer of AZA1 and four of the new compounds are isomers with a mass of 857.5 amu. AZA7 and AZA8 are hydroxyl analogues of AZA1 while AZA9 and AZA10 are hydroxyl analogues of AZA6. AZA11 is a hydroxyl analogue of AZA2. The separation of all 11 azaspiracids was achieved using isocratic reversed phase liquid chromatography using a combination of eluent additives, trifluoroacetic acid and ammonium acetate. The ion-trap MS experiments, with electrospray ionisation, involved the fragmentation of the protonated molecule [M+H](+), trapping and fragmenting the product ions due to the loss of a water molecule [M+H-H(2)O](+), together with mass spectral data analysis that included the characteristic A-ring fragmentation for each compound.     

Is there a risk of human poisoning by azaspiracids from shellfish harvested at the Portuguese coast?

Azaspiracid poisoning (AZP), the most recently discovered human gastrointestinal illness resulting from consumption of contaminated shellfish, so far has been found in coastal areas of northern Europe. This is the first report of a survey carried out for contamination of shellfish harvested in costal areas of Portugal for the presence of azaspiracids. The study design covered the commercial species usually more contaminated by toxins from dinoflagellates (blue mussel, common cockle, donax clam) in coastal areas representative of the NW, SW and south coasts. A method based on liquid chromatography–mass spectrometry was setup for the first time for this purpose. No azaspiracids were found on 300 samples tested between 2002 and 2003. On at least three samples a peak with a retention time matching that of AZA2 was found, never surpassing one tenth of the current EU limit. Unambiguous identification of any known AZA did not occur yet. The risk for human outbreaks of AZP seems to be very low, comparatively with amnesic shellfish poisoning (ASP), where levels close to the allowance level are found sparsely, or to diarrhetic shellfish poisoning (DSP) and paralytic shellfish poisoning (PSP), where high levels and registered human outbreaks have been found in recent years.     

First evidence of azaspiracids (AZAs): A family of lipophilic polyether marine toxins in scallops (Argopecten purpuratus) and mussels (Mytilus chilensis) collected in two regions of Chile

Azaspiracids are a family of lipophilic polyether marine biotoxins that have caused a number of human intoxication incidents in Europe since 1995 following the consumption by consumers of intoxicated shellfish (Mytilus edulis). These azaspiracids have now been identified in mussels (Mytilus chilensis) and scallops (Argopecten purpuratus) from two Chilean locations. This is the first report of the occurrence of azaspiracid toxins in these species (Mytilus chilensis and Argopecten purpuratus) from Chile. The areas studied were Bahía Inglesa (III Region, 27° SL) and Chiloé Archipelago, both important scallop and mussels farming areas. Separation of azaspiracid (AZA1), azaspiracid isomer (AZA6) and its analogues, 8-methylazaspiracid (AZA2) and 22-demethylazaspiracid (AZA3), was achieved using reversed-phase LC and toxins were identified using a turbo electrospray ionisation (ESI) source, to a triple quadrupole mass spectrometer.In mussels, AZA1 was the predominant toxin in mussel hepatopancreas with AZA2, AZA3 and AZA6 present in approximate equivalent amounts in the remaining tissues, 20-30% of the AZA1 level. AZA2 predominated in the scallop samples with the toxin almost entirely present in the hepatopancreas (digestive gland). AZA1 was only observed in some of the scallop samples and was present at 12-15% of the AZA2 levels.Whilst the levels of AZAs in Chilean samples are below the EU regulatory limit of 160 μg/kg, it is significant that this toxin is present in Pacific Ocean species. Consequently measures should be taken by regulatory authorities to implement regular seafood monitoring to ensure safety of harvested product.     

Evidence of Methylobacterium spp. and Hyphomicrobium sp. in azaspiracid toxin contaminated mussel tissues and assessment of the effect of azaspiracid on their growth

A flagellar protein belonging to the genus Methylobacterium or Agrobacterium was previously observed by proteomics in azaspiracids (AZA) toxic mussels. Here, we report the isolation of two different Methylobacterium spp. (NTx1 and Tx1) from non-toxic and AZA toxic mussels, respectively, which when co-cultured with AZA exhibited significantly different growth responses - isolate Tx1 growth rate was enhanced, whereas growth of isolate NTx1 was adversely affected, compared to non-AZA supplemented control cultures. A Hyphomicrobium sp. (Tx2) also isolated from the toxic mussels achieved greater cell density in AZAs supplemented cultures.     

Structural confirmation and occurrence of azaspiracids in Scandinavian brown crabs (Cancer pagurus).

In 2005 and 2006, azaspiracids were for the first time detected in brown crabs (Cancer pagurus) from the west coast of Sweden and the north and north-west coast of Norway. Azaspiracids are marine toxins that have been detected in blue mussels in Europe in recent years. On some occasions, they have been responsible for human intoxications with symptoms similar to those occurring by consumption of shellfish contaminated with okadaic acid group toxins. While the latter toxin group has been reported to accumulate in green crabs and brown crabs, azaspiracids have previously only been reported to occur in bivalve molluscs. LC-MS analysis of the hepatopancreas (HP) and roe of brown crabs revealed the presence of azaspiracid-1, -2 and -3, but only very low levels were detected in the white meat from the claws or the main shell. Mass spectral data were recorded using two different mass spectrometers, one with a triple-quadrupole mass analyzer and one with a linear ion-trap mass analyzer. The identities of the toxins were confirmed by comparing retention times and mass spectra of azaspiracid standards and the detected toxins. Levels detected ranged from 1.4 microg/kg tissue up to as much as 733 microg/kg tissue, although the majority of samples analyzed were below the suggested regulatory limit of 170 microg/kg HP. Higher levels were detected in HP compared with roe. Very little azaspiracids were detected in mussels from the same locations at the same time, and no proposed microalgal source of azaspiracids was reported in the water previous to or at the time of collection of the toxic crabs.     

Confirmation by LC–MS/MS of azaspiracids in shellfish from the Portuguese north-western coast

The search for azaspiracids (AZAs) in shellfish on the Portuguese coast started in 2002, but the presence of these toxins could not be demonstrated until the summer of 2006. Analysis by liquid-chromatography-tandem mass spectrometry (LC-MS/MS) allowed the confirmation of AZA2 as a dominant compound, followed by AZA1, in blue mussel (Mytilus galloprovincialis), common cockle (Cerastoderma edule), clams (Venerupis senegalensis, Ruditapes decussatus), razor clam (Solen marginatus) and oyster (Crassostrea spp). Traces of AZA3 were found only in blue mussel. Total levels of AZA1-3 determined in the whole flesh by LC-MS/MS ranged from 1.6 to 6.1 microg/kg. The finding of low levels of AZAs since 2002 suggests a low risk level when compared with the highest risks posed by diarrhetic shellfish poisoning (DSP) and paralytic shellfish poisoning (PSP) toxins. However, the limited number of years studied might generate a misleading conclusion. The contamination with PSP is an example, as no contamination occurred for an extended period of time between 1996 and 2004, despite high levels having occurred outside this period. Thus, there appears overall a moderate likelihood of occurrence of AZAs in the range that may be relevant to consumers.     

Azaspiracid accumulation, detoxification and biotransformation in blue mussels (Mytilus edulis) experimentally fed Azadinium spinosum

Azadinium spinosum (Elbrächter and Tillmann), a small marine dinoflagellate, has been recently described as a de novo producer of azaspiracid-1 and -2 (AZA1 and -2) diarrhoeic toxins. A culture of A. spinosum was established in our laboratory and optimised for pilot-scale production of this organism, to evaluate and understand AZA1 and -2 accumulation and biotransformation in blue mussels (Mytilus edulis) fed with A. spinosum.Adult mussels were continuously exposed to A. spinosum over 1 week in 160 L cylindrical conical tanks. Three different diets were tested for contamination: 5000, 10 000 cells mL⁻¹ of A. spinosum and a mixture of 5000 cells mL⁻¹ of A. spinosum with 5000 cells mL⁻¹ of Isochrysis aff. galbana (T-Iso, CCAP 927/14). During the subsequent period of detoxification (2 weeks), contaminated mussels were continuously fed with 5000 cells mL⁻¹ of T-Iso. Kinetics of accumulation, detoxification and biotransformation were evaluated, as well as the toxin distribution and the effect of A. spinosum on mussel digestive gland tubules.M. edulis fed on A. spinosum in the three tested conditions; this finding confirmed our recent experiments feeding A. spinosum to mussels. The original algal toxins AZA1 and -2, as well as mussel metabolites AZA3 to 12, -17, -19, -21 and -23 were found during these trials. After as little as 6 h, azaspiracid contents in mussels reached the EU regulatory limit, and metabolites were observed in all conditions at approximately 25% of the total AZA content. This fraction exceeded 50% after 24 h, and continued to increase until the end of the study. AZA17 and -19 were found to be the main metabolites, with AZA17 concentrations estimated in the same order of magnitude as that of the main algal toxin, AZA1.     

Comparative effects of the marine algal toxins azaspiracid-1, -2, and -3 on Jurkat T lymphocyte cells

Azaspiracids (AZA) are polyether marine toxins of dinoflagellate origin that accumulate in shellfish and represent an emerging human health risk. Although monitored and regulated in many European and Asian countries, there are no monitoring programs or regulatory requirements in the United States for this toxin group. This did not prove to be a problem until June 2009 when AZAs were identified in US seafood for the first time resulting in human intoxications and further expanding their global distribution. Efforts are now underway in several laboratories to better define the effects and mechanism(s) of action for the AZAs. Our investigations have employed Jurkat T lymphocyte cells as an in vitro model to characterize the toxicological effects of AZA1, AZA2, and AZA3. Cytotoxicity experiments employing a metabolically based dye (i.e., MTS) indicated that AZA1, AZA2, and AZA3 each elicited a lethal response that was both concentration- and time-dependent, with EC(50) values in the sub- to low nanomolar range. On the basis of EC(50) comparisons, the order of potency was as follows: AZA2 > AZA3 > AZA1, with toxic equivalence factors (TEFs) relative to AZA1 of 8.3-fold and 4.5-fold greater for AZA2 and AZA3, respectively. Image analysis of exposed cells using Nomarski differential interference contrast (DIC) imaging and fluorescent imaging of cellular actin indicated that the morphological effects of AZA1 on this cell type are unique relative to the effects of AZA2 and AZA3. Collectively, our data support the growing body of evidence suggesting that natural analogues of AZA are highly potent and that they may have multiple molecular targets.     

Toxic dinoflagellates (Dinophyceae) from Rarotonga, Cook Islands

Dinoflagellate species isolated from the green calcareous seaweed, Halimeda sp. J.V. Lamouroux, growing in Rarotongan lagoons, included Gambierdiscus australes Faust & Chinain, Coolia monotis Meunier, Amphidinium carterae Hulburth, Prorocentrum lima (Ehrenberg) Dodge, P. cf. maculosum Faust and species in the genus Ostreopsis Schmidt. Isolates were identified to species level by scanning electron microscopy and/or DNA sequence analysis. Culture extracts of G. australes isolate CAWD149 gave a response of 0.04 pg P-CTX-1 equiv. per cell by an N2A cytotoxicity assay (equivalent to ca 0.4 pg CTX-3C cell⁻¹). However, ciguatoxins were not detected by LC-MS/MS. Partitioned fractions of the cell extracts potentially containing maitotoxin were found to be very toxic to mice after intraperitoneal (i.p.) injection. A. carterae was also of interest as extracts of mass cultures caused respiratory paralysis in mice at high doses, both by i.p. injection and by oral administration. The Rarotongan isolate fell into a different clade to New Zealand A. carterae isolates, based on DNA sequence analysis, and also had a different toxin profile. As A. carterae co-occurred with G. australes, it may contribute to human poisonings attributed to CTX and warrants further investigation. A crude extract of C. monotis was of low toxicity to mice by i.p. injection, and an extract of Ostreopsis sp. was negative in the palytoxin haemolysis neutralisation assay.     

Pectenotoxin-2 in single-cell isolates of Dinophysis caudata and Dinophysis acuta from the Galician Rías (NW Spain).

Dinophysis acuta and Dinophysis caudata are seasonal components of the dinoflagellate community in the Galician Rías Bajas (NW Spain). These species can be the main contributors to the occurrence of Lipophilic Shellfish Toxins (LST) in September-October, leading to prohibition of harvesting in an area of intensive mariculture (250 x 10(3)t of cultured mussels, 60 x 10(3)t of other shellfish in natural banks, per year). Previous analyses of okadaic acid (OA) and related toxins in these two species by HPLC revealed significant amounts of OA and DTX2 in D. acuta, but only trace amounts of OA in D. caudata cells, and led to the erroneous conclusion that the contribution of the latter species to autumn LST events was negligible. Recent analyses by LC-MS/MS of individually picked cells of D. acuta and D. caudata have shown that both species may have high levels of PTX2 (up to 30 pg and 130 pg cell(-1), respectively) and that this toxin can be the dominant toxin during toxic outbreaks associated with Dinophysis spp. Although the quick conversion of PTX2 to PTX-2SA in mussels may reduce the risks for human health, these results have important implications for monitoring programmes. The complex toxin profile of shellfish exposed to Dinophysis spp. populations should be taken into account when making decisions on the toxin control methods to be used as the basis of the programme. Conclusive toxicological studies are required to elucidate the public health relevance of the different PTXs derivatives and to provide the scientific basis for regulations.     

Identification and quantification of tetrodotoxin in the marine gastropod Nassarius by LC-MS.

Tetrodotoxin (TTX) has been usually analyzed in marine and other sources by HPLC separation, followed by alkaline treatment and fluorescence assay. However, the applications of such methods were limited because of their tedious extraction and derivation procedures. A simpler, rapid LC-MS method, using selected ion-monitoring, has now been developed, and used to identify and quantitate TTX in a marine gastropod of the genus Nassarius, collected from Fijian province of China. The toxin was confirmed comparable to authentic TTX by MS and MSn. In addition, a moderate amount of TTX was detected in the Nassarius by this novel approach, suggesting that Nassarius would be a potential food poison source.     

Occurrence of paralytic shellfish poison (PSP) in the starfish Asterina pectinifera collected from the Kure Bay, Hiroshima Prefecture, Japan.

Assays were made for paralytic toxicity of marine invertebrates inhabiting at the coasts of Hiroshima Bay, where the infestation of bivalves such as cultured oysters with paralytic shellfish poison (PSP) has been occurred. The starfish Asterina pectinifera collected at the estuary of Nikoh River, Hiroshima Bay, was found to contain moderate levels of paralytic toxicity. Its highest toxicities as PSP found on July 30, 1999 were 12.5 MU/g for whole body, 11.0 MU/g for integument tissues and 3.9 MU/g for viscera, respectively. The toxicity of integument was changed from 3.6 to 11.0 MU/g in 1 year. Its paralytic toxin principles were identified as PSP toxins, composing mainly from saxitoxin (STX) group toxins such as carbamoyl-N-hydroxy neosaxitoxin (hyneoSTX), and STX, by HPLC and LC-MS, accounting for over 90 mol%. The PSP toxins contained in the starfish A. pectinifera considered to be transferred from bivalves or detritus living in the same area, which were contaminated with PSP. However, the involved pathway may be different from that of Asterias amurensis which was infested directly through food chain from its food bivalves, for its toxin pattern.     

Occurrence of the cyanobacterial toxin cylindrospermopsin in northeast Germany

The frequent occurrence of the cyanobacterial toxin cylindrospermopsin (CYN) in the (sub)tropics has been largely associated with cyanobacteria of the order Nostocales of tropical origin, in particular Cylindrospermopsis raciborskii. C. raciborskii is currently observed to spread northwards into temperate climatic zones. In addition, further cyanobacteria of the order Nostocales typically inhabiting water bodies in temperate regions are being identified as CYN-producers. Therefore, data on the distribution of CYN in temperate regions are necessary for a first assessment of potential risks due to CYN in water used for drinking and recreation. A total of 127 lakes situated in the north-eastern part of Germany were investigated in 2004 for the presence of the toxin CYN and the phytoplankton composition. The toxin could be detected in half of the lakes (n = 63) and in half of 165 samples (n = 88). Concentrations reached up to 73.2 microg CYN/g DW. CYN thus proved more widely distributed than previously demonstrated. The analyses of phytoplankton data suggest Aphanizomenon sp. and Anabaena sp. as important CYN producers in Germany, and confirm recent findings of Aphanizomenon flos-aquae as CYN-producing species frequently inhabiting water bodies in temperate climatic regions. The data shown here suggest that CYN may be an important cyanobacterial toxin in German water bodies and that further data are needed to assess this.     

Induction of apoptosis pathways in several cell lines following exposure to the marine algal toxin azaspiracid

Azaspiracids (AZAs) are polyether marine dinoflagellate toxins that accumulate in shellfish and represent an emerging human health risk. Although there have been no deaths associated with the AZA toxins, humans exposed to AZAs experience severe gastrointestinal symptoms. This toxin class has been shown to be highly cytotoxic, a teratogen to developing fish, and a possible carcinogen in mice. Just recently, the AZAs have been shown to be potassium channel inhibitors. This report employed multiple human cell lines [Jurkat T lymphocytes, Caco-2 intestinal cells, and BE(2)-M17 neuroblastoma cells] in characterizing cytotoxicity and pathways of apoptosis. Cytotoxicity experiments were consistent with published literature that has shown that AZA1 is cytotoxic in both a concentration- and time-dependent manner to each cell type tested, with mean EC(50) values ranging between 1.1 and 7.4 nM. Despite the absence of morphological indices indicating apoptosis, caspase-3/7 activity was higher in all cell types treated with AZA1. Furthermore, in T lymphocytes, the most sensitive cell type, the activities of initiator caspase-2 and caspase-10 and concentrations of intracellular cytochrome c were elevated. DNA fragmentation was also observed for T lymphocytes exposed to AZA1-AZA3. Collectively, our data confirm that AZA1 was highly cytotoxic to multiple cell types and that cells exposed to AZA1 underwent atypical apoptosis, possibly in conjunction with necrotic cytotoxicity.     

Sub-lethal dosing of azaspiracid-1 in female NMRI mice

The main aim of this study was to examine absorption and pathological effects of a single sub-lethal dose of the marine biotoxin azaspiracid-1 (AZA1) in mice after oral intubation. When the mice received AZA1 at doses of 100, 200 or 300 μg/kg body weight (b.w.), the toxin was absorbed dose-dependently. Highest concentrations after 24 h were detected in kidneys, spleen and lungs, followed by liver and heart. Only trace amounts were seen in the brain. After seven days, the toxin level had dropped significantly in all organs except for the kidneys. The amount of toxin absorbed was highest in the liver, followed by kidneys, lungs, spleen and heart and the total amount of toxin in the internal organs analysed after 24 h was estimated to be only about 2% of the total amount given for all three dose groups. Pathological changes were only detected in the upper part of the small intestine (duodenum), consisting of mild cellular detachment in the tips of the villi, expansion of the crypts and necrotic changes in lamina propria. In a previous study very long persistence of damage to the gastrointestinal tract by repeated exposures to AZA toxins was reported. In our study, full recovery from the pathological changes was observed seven days after a single exposure to AZA1 at the doses applied.     

Studies of polyether toxins in the marine phytoplankton, Dinophysis acuta, in Ireland using multiple tandem mass spectrometry

Diarretic shellfish poisoning (DSP) is a toxic syndrome associated with the consumption of bivalve molluscs. The DSP toxins are polyether compounds, which include okadaic acid (OA), dinophysistoxins (DTXs), pectenotoxins (PTXs) and pectenotoxin seco acids (PTX2SAs). These toxins originate in marine dinoflagellates, including Dinophysis spp. Phytoplankton samples were collected from the southwest coast of Ireland and D. acuta was the predominant species. Monocultures of D. acuta cells were prepared by hand picking from microscope slides in order to confirm their toxin profiles. There was a remarkable consistency in the toxin profiles in all of the phytoplankton samples collected during the summer months, irrespective of location, depth or mesh size. Analysis using liquid chromatography—multiple tandem mass spectrometry (LC-MS/MS) revealed that DTX2 and OA were the predominant toxins at a consistent ratio. The average toxin composition was: DTX2 (53±5%), OA (26.5±2.3%) and total pectenotoxins (20.8±4.7%). Toxin profiles in D. acuta from Europe were distinctly different from those found in New Zealand, where PTX2 was the predominant toxin and DTX2 was absent.     

Toxin composition of a Prorocentrum lima strain isolated from the Portuguese coast

Microalgae of the genus Dinophysis and Prorocentrum are known producers of okadaites, responsible for the human syndrome known as diarrhetic shellfish poisoning (DSP). In temperate regions, only species from the genus Dinophysis are commonly held responsible for shellfish contamination. This is probably related to the different ecological strategies of the two genera, namely the planktonic nature of Dinophysis versus the benthic/epiphytic nature of toxic Prorocentrum species. In recent years, the threat of global warming has drawn attention to the study of benthic toxic microalgae in southern European waters. Here we present results on the toxin production and toxin profile of a Prorocentrum lima strain isolated from the Portuguese coast. This strain, IO66-01, presented a mean growth rate of 0.49 divisions d⁻¹, not common in temperate strains, and only comparable with tropical strains. The parent toxins found were okadaic acid (OA) and dinophysistoxin-1 (DTX1). The major diol esters were D8- and D9- congeners of both OA and DTX1.