Diarrhetic Shellfish Poisoning Research Articles

Stability of okadaic acid and 13-desmethyl spirolide C in seawater and sediment

Persistence of lipophilic toxins in different environmental compartments determines the way in which their concentrations can be used to evaluate temporal trends and associated risks. Short term stability of two lipophilic toxins, representing two chemically different groups of toxins — okadaic acid (OA) (Diarrhetic Shellfish Poisoning toxin) and 13-desmethyl spirolide C (13desmSPXC) (cyclic imine) — in seawater, interstitial water and sediment were analyzed. OA was found to be very stable in seawater, interstitial water and sediment, without significant changes over a 23-day period. Contrarily, 13desmSPXC was readily degraded, but faster in interstitial water than in seawater. In sediments, its degradation was also fast and dependent on the origin of the samples. 13desmSPXC was found in natural sediments, and this toxin was mostly associated to large particles (>10 μm) in the sediment, that are or include resting cysts of the producer species. Therefore, it appears that high concentrations of 13desmSPXC in seawater would indicate recent blooms of the producer species, while those of OA could be due to recent blooms or to others that took place long before detection. The absence of OA and 13desmSPXC indicates a low impact of those toxins in the study area.

Reliable quantification of trace diarrhetic shellfish poisoning toxins in high-lipid bivalves by UHPLC-ESI-MS/MS in time segment polarity switching mode: Comparison of three extraction methods

Analysis of marine biotoxins in high-lipid bivalves has several limitations due to the coexistence of lipid matrices, low biotoxin concentrations, and the different physiochemical properties of the various biotoxins. In this study, a sensitive and reliable method to simultaneously determine six diarrhetic shellfish poisoning (DSP) toxins (five acidic and one neutral biotoxins) in oysters and mussels was developed based on UHPLC-MS/MS-multiple ion reaction monitoring (MRM) using time segment polarity switching. For effective sample pretreatment, three protocols including syringe filtration, Strata™-X solid phase extraction (SPE), and freezing lipid filtration combined with SPE (FLF + SPE) were evaluated, and FLF + SPE method was shown to be the most effective with high lipid removal efficiency. Aging effects of the UHPLC mobile phase on MS detection sensitivity and retention time shift of DSP toxins were carefully examined. The developed method provided good recoveries ranging from 81.0 to 119.6%, showing no significant matrix effect and assay was linear with coefficients of determination above R2 > 0.99 for all analytes. Validation tests using a certified reference material (CRM) and spiking DSP toxins (OA and DTX-1 at 1.07 μg/g, and DTX-2 at 0.86 μg/g) showed this method to be acceptable, with a relative standard deviation of <0.17% and recovery ranging from 87.29 to 109.97%. Finally, the established method was successfully applied to quantify DSP toxins in 40 oyster and mussel samples collected from Korean fishery markets, ensuring sea food safety.

A taxonomical study of benthic Prorocentrum species (Prorocentrales, Dinophyceae) from Anse Dufour (Martinique Island, eastern Caribbean Sea)

About 30 benthic Prorocentrum species have been described, some of which producing okadaic acid and derivatives involved in diarrhetic shellfish poisoning. The western Caribbean has been extensively studied for benthic dinoflagellates associated with ciguatera, and fifteen Prorocentrum species were described from mangroves and coral reefs of Belize. In contrast, no study reported the diversity of this genus in the Eastern Caribbean, especially in the Lesser Antilles. This study adds to the biodiversity knowledge in Martinique Island by investigating one site of the Caribbean coast from 2010 to 2017. Sediment samples were collected each year in March and studied taxonomically. Identification was realized morphologically by scanning electron microscopy, while the partial large subunit (LSU) ribosomal DNA was sequenced for 42 isolated specimens (single-cells) and one strain in culture. A molecular phylogenetic analysis revealed 11 OTUs from Martinique, identified morphologically as P. concavum, P. cf. foraminosum, P. cf. tropicale, P. lima, P. hoffmannianum, P. cf. norrisianum, P. glenanicum, P. panamense, P. cf. sculptile, P. cf. fukuyoi, and P. rhathymum. Two morphospecies were also identified (P. cf. maculosum and P. cf. ruetzlerianum) but with no sequence obtained. Some species like P. cf. tropicale and P. cf. norrisinamum are sequenced for the first time. Our analysis reveals probable former misidentifications of P. cf. foraminosum and P. cf. sculptile since the sequences from Martinique form new clades and their geographical origin are closer from the type locality than any other previous studies. Further studies and sequences from the type localities are yet required to assess identifications.

Risk assessment indexes for shellfish poisoning outbreak caused by red tide

To establish the indexes and weights of risk assessment of shellfish poisoning outbreak caused by red tide. The risk assessment indexes were developed with the methods of literature review, brainstorm and expert consultation, and the weights of indexes were calculated by the method of analytic hierarchy process. The established indexes contained the risk possibility, impacts of public health, population vulnerability and resilience. The relative risk indexes(integrated risk indexes) of different shellfish poisoning were computed by combining hierarchy process and TOPSIS methods. Moreover, the weights of indexes were further used to generate absolute risk values by multiplying indexes. Four primary indexes and 17 secondary indexes were identified for risk assessment of shellfish poisoning outbreak. Of 17 secondary indexes, the knowing rate of shellfish poisoning, medical accessibility, the number of people being affected, laboratory testing capacity and the habits of eating seafood of local residents had relatively large weights (0.0876, 0.0840, 0.0716, 0.0703 and 0.0644, respectively), which accounted for nearly 38% of the total weight. All consistency ratio (CR) were less than 0.1. The index system was applied in Cangnan county of Zhejiang province. The results showed the relative risk indexes of paralytic shellfish poisoning (PSP), diarrhetic shellfish poisoning (DSP), neurotoxic shellfish poisoning (NSP) and amnesic shellfish poisoning (ASP) were 0.4526, 0.7116, 0.1657 and 0.2884, and the absolute risk values were 0.2542, 0.2668, 0.1907 and 0.2184, respectively. The risk orders of the 4 kinds of shellfish poisoning sorted by relative risk indexes and absolute risk values were consistent. The indexes and weights of risk assessment of shellfish poisoning outbreak caused by red tide are established, which can provide scientific advice for prevention and control of shellfish poisoning outbreak.

A Dual Functional Cardiomyocyte-based Hybrid-biosensor for the Detection of Diarrhetic Shellfish Poisoning and Paralytic Shellfish Poisoning Toxins.

Okadaic acid (OA) and saxitoxin (STX) are typical toxins of diarrhetic shellfish poisoning (DSP) and paralytic shellfish poisoning (PSP), respectively, which are highly toxic marine toxins threatening human health and environmental safety. OA is a potent inhibitor of serine/threonine protein phosphatases that can cause cellular death, while STX is an inhibitor of sodium channel that can lead to neurological damage. In this work, a dual functional cardiomyocyte-based biosensor was proposed to detect DSP and PSP toxins by monitoring the viability and electrophysiology of cardiomyocytes. The results showed that the viability of cardiomyocytes was sensitive to the OA and STX, resulting in significant changes of the electrophysiological properties, including amplitude, firing rate and duration of the extracellular field potential (EFP). The detection limits of the hybrid-biosensor are as low as 7.16 ng/mL for OA and 5.19 ng/mL for STX. In summary, all of the results indicate that the dual functional cardiomyocyte-based hybrid-biosensor will be a promising and utility tool for shellfish toxin detection.

Evaluation of Rapid, Early Warning Approaches to Track Shellfish Toxins Associated with Dinophysis and Alexandrium Blooms.

Marine biotoxin-contaminated seafood has caused thousands of poisonings worldwide this century. Given these threats, there is an increasing need for improved technologies that can be easily integrated into coastal monitoring programs. This study evaluates approaches for monitoring toxins associated with recurrent toxin-producing Alexandrium and Dinophysis blooms on Long Island, NY, USA, which cause paralytic and diarrhetic shellfish poisoning (PSP and DSP), respectively. Within contrasting locations, the dynamics of pelagic Alexandrium and Dinophysis cell densities, toxins in plankton, and toxins in deployed blue mussels (Mytilus edulis) were compared with passive solid-phase adsorption toxin tracking (SPATT) samplers filled with two types of resin, HP20 and XAD-2. Multiple species of wild shellfish were also collected during Dinophysis blooms and used to compare toxin content using two different extraction techniques (single dispersive and double exhaustive) and two different toxin analysis assays (liquid chromatography/mass spectrometry and the protein phosphatase inhibition assay (PP2A)) for the measurement of DSP toxins. DSP toxins measured in the HP20 resin were significantly correlated (R2 = 0.7–0.9, p < 0.001) with total DSP toxins in shellfish, but were detected more than three weeks prior to detection in deployed mussels. Both resins adsorbed measurable levels of PSP toxins, but neither quantitatively tracked Alexandrium cell densities, toxicity in plankton or toxins in shellfish. DSP extraction and toxin analysis methods did not differ significantly (p > 0.05), were highly correlated (R2 = 0.98–0.99; p < 0.001) and provided complete recovery of DSP toxins from standard reference materials. Blue mussels (Mytilus edulis) and ribbed mussels (Geukensia demissa) were found to accumulate DSP toxins above federal and international standards (160 ng g−1) during Dinophysis blooms while Eastern oysters (Crassostrea virginica) and soft shell clams (Mya arenaria) did not. This study demonstrated that SPATT samplers using HP20 resin coupled with PP2A technology could be used to provide early warning of DSP, but not PSP, events for shellfish management.

Simultaneous detection of three biotoxins causing diarrhetic shellfish poisoning (okadaic acid, dinophysistoxin-1, dinophysistoxin-2) in oyster by LC-MS/MS

Simultaneous detection of three biotoxins causing diarrhetic shellfish poisoning (okadaic acid, dinophysistoxin-1, dinophysistoxin-2) in oyster by LC-MS/MS

Distribution of Diarrhetic Shellfish Toxins in Mussels, Scallops, and Ascidian.

Diarrhetic shellfish toxins (DST) are a group of phycotoxins that include Okadaic acid (OA) and structurally related toxins. In Japan, the regulatory limit of DST in shellfish for human consumption is a total OA equivalent of 0.16 mg per kg of edible tissue. Distribution and individual differences of DST in scallops collected in Aomori Prefecture were investigated. Fourteen to 20 individual scallops were divided into hepatopancreas, gonads, mantles, gills, adductor muscles, and the concentrations of diarrhetic shellfish poisoning (DSP) in each tissue were quantified by LC/MS/MS after hydrolysis. The dominant toxin in the scallops was Dinophysis toxin 1 (DTX1). More than 97% of the observed DTX1 in the scallop tissue was detected in the hepatopancreas and the average level of DTX1 was higher in mussels than the scallops. The number of individual scallops or mussels required to correctly reflect the DTX1 content of a sample group was estimated by resampling. In scallops, using 10 individuals fell within ± 20% of 30 individual's average with a probability of 99.8%. On the other hand, in the blue mussel, an average of 19 individuals fell within ± 20% of 30 individual's average with 98% probability. In addition, the analysis of the DST in ascidians collected from Miyagi Prefecture was carried out. The muscles, gills, hepatopancreas and intestines were analyzed. High concentration of both DTX1 and OA were detected in the hepatopancreas after hydrolysis. Low levels of DST were detected from other tissues, indicating that DST are primarily accumulated in the hepatopancreas in the ascidians.

A method combining a kit with the Bionic e-Eye for rapid on site detection of diarrhetic shellfish poisoning

A method combining a kit with the Bionic e-Eye for rapid and portable on site detection of diarrhetic shellfish poisoning.

Feeding of scallop mantle epithelial cell layer causes subacute toxicity against rodents

Japanese scallops Patinopecten yessoensis are one of the main marine products of Hokkaido, Japan. In addition to adductor muscle, scallop mantle tissue is also eaten in Japan. We fed Wistar rats diets containing the scallop mantle epithelial cell layer for 3–8 weeks and compared the outcomes with a control group. No differences in general appearance or behavior were observed between the groups for the first 2 weeks. Thereafter, a significant decrease in food consumption was observed, and the treated rats died between 6 and 8 weeks. Serum biochemistry and histological studies revealed nephrotoxicity and hepatotoxicity in rats fed scallop mantle epithelial cells. Glucose tolerance tests showed that feeding scallop mantle epithelial cells to rats causes a glucose metabolism disorder. A diet containing a water extract from scallop mantle epithelial cells caused toxicity in mice, but a diet containing a methanol extract, which dissolves diarrhetic shellfish poisoning (DSP) toxin, or an extract in heated 0.1 M HCl, which dissolves paralytic shellfish poisoning (PSP) toxin, did not cause toxicity. Mice fed a diet containing pronase-treated water extract neither showed a decrease in their food intake nor death. These results suggest that a water-soluble component other than DSP or PSP toxin causes toxicity.

Self-assembled monolayer-based immunoassays for okadaic acid detection in seawater as monitoring tools

Rapid and cost-effective methods to monitor the presence of diarrhetic shellfish poisoning (DSP) toxins in seawater samples in an easy and reliable manner are required to protect human health and avoid economic losses to shellfish industry. Immunoassays for the detection of okadaic acid (OA) and dinophysistoxin-1 and dinophysistoxin-2 are developed by immobilising OA on self-assembled monothiols or dithiols in an ordered and oriented way, providing an effective limit of detection of ∼1 ng OA equiv./mL seawater. The immunoassays are applied to the analysis of the particulate fraction of seawater samples from two Catalan harbours (NW Mediterranean) and samples collected periodically from the Galician Rias (E Atlantic), as well as a reference mussel sample. Results are in agreement with LC-MS/MS and the certified values. OA concentration in seawater correlates with Dinophysis cell abundance, with a 1–2 weeks lag. The immunoassays provide powerful high-throughput analytical methods potentially applicable as alternative monitoring tools.

Pulsed light reduces the toxicity of the algal toxin okadaic acid to freshwater crustacean Daphnia pulex.

This constitutes the first study to report on the reduction in toxicity of the dinoflagellate algal toxin okadaic acid after novel pulsed light (PL) treatments where ecotoxicological assessment was performed using a miniaturised format of the conventional in vivo freshwater crustacean Daphnia sp. acute toxicity test. Bivalves accumulate this toxin, which can then enter the human food chain causing deleterious health effects such as diarrhetic shellfish poisoning. This miniaturised toxicological bioassay used substantially less sample volume and chemical reagents. Findings revealed a 24-h EC50 of 25.87μg/L for PL-treated okadaic acid at a UV dose of 12.98μJ/cm2 compared to a 24-h EC50 of 1.68μg/L for the untreated okadaic acid control, suggesting a 15-fold reduction in toxicity to Daphnia pulex. The bioassay was validated in this study and correlated well with the "classic" ISO format (r=0.98) using the traditional reference chemical potassium dichromate (K2Cr2O7). Reduction by up to 65% in PL-treated okadaic acid concentration was confirmed by LC-MS/MS analysis. Findings from this study have positive ecological, societal and enterprise implications, such as the development of PL technology for the prevention or reduce algal contamination of fisheries and aquaculture industries.

Adsorption of marine phycotoxin okadaic acid on a covalent organic framework

Phycotoxins, compounds produced by some marine microalgal species, can reach high concentrations in the sea when a massive proliferation occurs, the so-called harmful algal bloom. These compounds are especially dangerous to human health when concentrated in the digestive glands of seafood. In order to generate an early warning system to alert for approaching toxic outbreaks, it is very important to improve monitoring methods of phycotoxins in aquatic ecosystems. Solid-phase adsorption toxin tracking devices reported thus far based on polymeric resins have not been able to provide an efficient harmful algal bloom prediction system due to their low adsorption capabilities.In this work, a water-stable covalent organic framework (COF) was evaluated as adsorbent for the hydrophobic toxin okadaic acid, one of the most relevant marine toxins and the parental compound of the most common group of toxins responsible for the diarrhetic shellfish poisoning. Adsorption kinetics of okadaic acid onto the COF in seawater showed that equilibrium concentration was reached in only 60min, with a maximum experimental adsorption of 61mgg−1. Desorption of okadaic acid from the COF was successful with both 70% ethanol and acetonitrile as solvent, and the COF material could be reused with minor losses in adsorption capacity for three cycles. The results demonstrate that COF materials are promising candidates for solid-phase adsorption in water monitoring devices.

Profiling of Extracellular Toxins Associated with Diarrhetic Shellfish Poison in Prorocentrum lima Culture Medium by High-Performance Liquid Chromatography Coupled with Mass Spectrometry

Extracellular toxins released by marine toxigenic algae into the marine environment have attracted increasing attention in recent years. In this study, profiling, characterization and quantification of extracellular toxin compounds associated with diarrhetic shellfish poison (DSP) in the culture medium of toxin-producing dinoflagellates were performed using high-performance liquid chromatography–high-resolution mass spectrometry/tandem mass spectrometry for the first time. Results showed that solid-phase extraction can effectively enrich and clean the DSP compounds in the culture medium of Prorocentrum lima (P. lima), and the proposed method achieved satisfactory recoveries (94.80%–100.58%) and repeatability (relative standard deviation ≤9.27%). Commercial software associated with the accurate mass information of known DSP toxins and their derivatives was used to screen and identify DSP compounds. Nine extracellular DSP compounds were identified, of which seven toxins (including OA-D7b, OA-D9b, OA-D10a/b, and so on) were found in the culture medium of P. lima for the first time. The results of quantitative analysis showed that the contents of extracellular DSP compounds in P. lima culture medium were relatively high, and the types and contents of intracellular and extracellular toxins apparently varied in the different growth stages of P. lima. The concentrations of extracellular okadaic acid and dinophysistoxin-1 were within 19.9–34.0 and 15.2–27.9 μg/L, respectively. The total concentration of the DSP compounds was within the range of 57.70–79.63 μg/L. The results showed that the proposed method is an effective tool for profiling the extracellular DSP compounds in the culture medium of marine toxigenic algae.

Detection of okadaic acid (OA) using ELISA and colloidal gold immunoassay based on monoclonal antibody

Okadaic Acid (OA), a small seafood-borne toxin secreted by Dinophysis and Prorocentrum dinoflagellates, is generally distributed in various species of shellfish and has caused diarrhetic shellfish poisoning (DSP). In view of OA toxin threat to humans and animals, it is essential to develop a rapid, accurate and sensitive method for the detection and quantification of OA in real samples. In this study, a monoclonal antibody named 10E8 was screened by cells fusion of Sp2/0 with spleen cells isolated from immunized mouse, and the isotype of McAb 10E8 was belonged to IgG1. The resulted McAb 10E8 displayed higher specificity to OA antigen, with the highest affinity of 2.66×109L/moL until now. Indirect competitive ELISA (ic-ELISA) indicated that the linear range to detect OA was 20–750ng/mL. The limit of detection (LOD) was 12pg/mL, and the recovery average was (84.04±5.08)%. The LOD of colloidal gold immunoassay by naked eye and strip reader was 1ng/mL and 100pg/mL, respectively, with an average recovery of (88.0275±4.4225)%. Therefore, the developed ELISA and colloidal gold immunoassay based on this McAb can be used for OA detection in real samples.

Transcriptional and biochemical analysis of antioxidant enzymes in the mussel Mytilus galloprovincialis during experimental exposures to the toxic dinoflagellate Prorocentrum lima

The genotoxic and cytotoxic effects of Diarrhetic Shellfish Poisoning (DSP) toxins have been widely investigated in bivalve molluscs, representing the main vectors of these compounds in the Atlantic coast of Europe. DSP toxins are produced by Harmful Algal Blooms (HABs) of Dinophysis and Prorocentrum dinoflagellates, being subsequently accumulated by marine organisms and biomagnified throughout trophic webs. Yet, bivalves display increased resistance to the harmful effects of these toxins during HAB episodes. While previous reports have suggested that such resilience might be the result of an increased activity in the bivalve antioxidant system, very little is still known about the specific mechanism underlying the protective effect observed in these organisms. The present work aims to fill this gap by studying transcriptional expression levels and biochemical activities of antioxidant enzymes in different tissues the mussel Mytilus galloprovincialis during experimental exposures to DSP toxins produced by the dinoflagellate Prorocentrum lima. Results are consistent with the presence of a compensatory mechanism involving a down-regulation in the expression of specific genes encoding antioxidant enzymes [i.e., SuperOxide Dismutase (SOD) and CATalase (CAT)] which is counterbalanced by the up-regulation of other antioxidant genes such as Glutathione S-Transferase pi-1 (GST-pi) and Selenium-dependent Glutathione PeroXidase (Se-GPx), respectively. Enzymatic activity analyses mirror gene expression results, revealing high antioxidant activity levels (consistent with a protective role for the antioxidant system) along with reduced lipid peroxidation (increasing the defense against oxidative stress).

Reduction of diarrhetic shellfish poisoning (DSP) toxins accumulation in cultured mussels by means of rope clustering and hydrodynamic barriers

Accumulation of marine toxins in the organisms is one of the most important problems of the bivalve aquaculture. Mitigation methods are difficult to implement especially in species, as the mussel Mytilus galloprovincialis, which have a relatively low commercial value. For this species, one possible way of decreasing the toxin accumulation is to restrict the availability of toxic cells to the mussels by means of the reduction of the water flow around the perimeter of the culture ropes. We have checked the effectivity to decrease the accumulation of DSP toxins (okadaic acid) in mussels of two ways of obtaining that reduction: surrounding the ropes with a fishing net (1cm mesh); and grouping the ropes (groups of four). During a four week period – with relatively high concentrations of particulate matter – those two ways were tested and shown to effectively reduce both, toxin concentration and toxin burden of cultured mussels exposed to toxic populations of Dinophysis acuminata. The observed reductions were important, 41% and 37% for grouping and surrounding with a net, respectively. Both methods can be used for mussel rafts and long lines and it is very likely they could be also effective with other kinds of toxicity and with other environmental conditions. Statement of relevanceIt would allow a substantial reduction of toxin accumulation.

Expanding toxic algal blooms.

Ocean Ecology Ocean temperatures in the North Atlantic and North Pacific oceans have increased in recent decades, particularly in coastal areas. This has been associated with increased algal blooms and, where these blooms include algal species that produce biotoxins, the potential for increases in cases of paralytic and diarrhetic shellfish poisoning. Gobler et al. used high-resolution records of sea surface temperature from 1982 to 2016 and temperature-dependent growth rates of two toxic algal species to create models of harmful algal blooms. These models were validated in areas of the North Atlantic by observations in other studies of increased bloom frequency and range that matched predicted locations. This information could potentially be used to predict the future spread of harmful algal blooms and the consequent impact on human health. Proc. Natl. Acad. Sci. U.S.A. 114 , 4975 (2017).

Effects of algal toxin okadaic acid on the non-specific immune and antioxidant response of bay scallop (Argopecten irradians)

Okadaic acid (OA) is produced by dinoflagellates during harmful algal blooms and is a diarrhetic shellfish-poisoning (DSP) toxin. This toxin is particularly problematic for bivalves that are cultured for human consumption. This study aimed to reveal the effects of exposure to OA on the non-specific immune responses of bay scallop, Argopecten irradians. Various immunological parameters (superoxide dismutase (SOD), acid phosphatase (ACP), alkaline phosphatase (ALP), lysozyme activities, and total protein level) were assessed in the hemolymph of bay scallops at 3, 6, 12, 24, and 48 h post-exposure (hpe) to different concentrations (50, 100, and 500 nM) of OA. Moreover, the expression of immune system-related genes (MnSOD, PrxV, PGRP, and BD) was also measured. Results showed that SOD and ACP activities were decreased between 12 and 48 hpe. The ALP, lysozyme activities, and total protein levels were also modulated after exposure to different concentrations of OA. The expression of immune-system-related genes was also assessed at different time points during the exposure period. Overall, our results suggest that the exposure to OA had negative effects on the antioxidant and non-specific immune responses, and even disrupted the metabolism of bay scallops, making them more vulnerable to environmental stress-inducing agents; they provide a better understanding of the response status of bivalves against DSP toxins.

Sulfated diesters of okadaic acid and DTX-1: Self-protective precursors of diarrhetic shellfish poisoning (DSP) toxins

Many toxic secondary metabolites used for defense are also toxic to the producing organism. One important way to circumvent toxicity is to store the toxin as an inactive precursor. Several sulfated diesters of the diarrhetic shellfish poisoning (DSP) toxin okadaic acid have been reported from cultures of various dinoflagellate species belonging to the genus Prorocentrum. It has been proposed that these sulfated diesters are a means of toxin storage within the dinoflagellate cell, and that a putative enzyme mediated two-step hydrolysis of sulfated diesters such as DTX-4 and DTX-5 initially leads to the formation of diol esters and ultimately to the release of free okadaic acid. However, only one diol ester and no sulfated diesters of DTX-1, a closely related DSP toxin, have been isolated leading some to speculate that this toxin is not stored as a sulfated diester and is processed by some other means. DSP components in organic extracts of two large scale Prorocentrum lima laboratory cultures have been investigated. In addition to the usual suite of okadaic acid esters, as well as the free acids okadaic acid and DTX-1, a group of corresponding diol- and sulfated diesters of both okadaic acid and DTX-1 have now been isolated and structurally characterized, confirming that both okadaic acid and DTX-1 are initially formed in the dinoflagellate cell as the non-toxic sulfated diesters.