Dihydrodinophysistoxin-1 Produced by Dinophysis norvegica in the Gulf of Maine, USA and Its Accumulation in Shellfish.

Dihydrodinophysistoxin-1 (dihydro-DTX1, (M-H)-m/z 819.5), described previously from a marine sponge but never identified as to its biological source or described in shellfish, was detected in multiple species of commercial shellfish collected from the central coast of the Gulf of Maine, USA in 2016 and in 2018 during blooms of the dinoflagellate Dinophysis norvegica. Toxin screening by protein phosphatase inhibition (PPIA) first detected the presence of diarrhetic shellfish poisoning-like bioactivity; however, confirmatory analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS) failed to detect okadaic acid (OA, (M-H)-m/z 803.5), dinophysistoxin-1 (DTX1, (M-H)-m/z 817.5), or dinophysistoxin-2 (DTX2, (M-H)-m/z 803.5) in samples collected during the bloom. Bioactivity-guided fractionation followed by liquid chromatography-high resolution mass spectrometry (LC-HRMS) tentatively identified dihydro-DTX1 in the PPIA active fraction. LC-MS/MS measurements showed an absence of OA, DTX1, and DTX2, but confirmed the presence of dihydro-DTX1 in shellfish during blooms of D. norvegica in both years, with results correlating well with PPIA testing. Two laboratory cultures of D. norvegica isolated from the 2018 bloom were found to produce dihydro-DTX1 as the sole DSP toxin, confirming the source of this compound in shellfish. Estimated concentrations of dihydro-DTX1 were >0.16 ppm in multiple shellfish species (max. 1.1 ppm) during the blooms in 2016 and 2018. Assuming an equivalent potency and molar response to DTX1, the authority initiated precautionary shellfish harvesting closures in both years. To date, no illnesses have been associated with the presence of dihydro-DTX1 in shellfish in the Gulf of Maine region and studies are underway to determine the potency of this new toxin relative to the currently regulated DSP toxins in order to develop appropriate management guidance.

Acute and chronic dietary exposure to domoic acid in recreational harvesters: A survey of shellfish consumption behavior.

Domoic acid (DA) is a neurotoxin that is naturally produced by phytoplankton and accumulates in seafood during harmful algal blooms. As the prevalence of DA increases in the marine environment, there is a critical need to identify seafood consumers at risk of DA poisoning. DA exposure was estimated in recreational razor clam (Siliqua patula) harvesters to determine if exposures above current regulatory guidelines occur and/or if harvesters are chronically exposed to low levels of DA. Human consumption rates of razor clams were determined by distributing 1523 surveys to recreational razor clam harvesters in spring 2015 and winter 2016, in Washington, USA. These consumption rate data were combined with DA measurements in razor clams, collected by a state monitoring program, to estimate human DA exposure. Approximately 7% of total acute exposures calculated (including the same individuals at different times) exceeded the current regulatory reference dose (0.075mgDA·kgbodyweight-1·d-1) due to higher than previously reported consumption rates, lower bodyweights, and/or by consumption of clams at the upper range of legal DA levels (maximum 20mg·kg-1 wet weight for whole tissue). Three percent of survey respondents were potentially at risk of chronic DA exposure by consuming a minimum of 15 clams per month for at 12 consecutive months. These insights into DA consumption will provide an additional tool for razor clam fishery management.

Screening tests for the rapid detection of diarrhetic shellfish toxins in Washington State.

The illness of three people due to diarrhetic shellfish poisoning (DSP) following their ingestion of recreationally harvested mussels from Sequim Bay State Park in the summer of 2011, resulted in intensified monitoring for diarrhetic shellfish toxins (DSTs) in Washington State. Rapid testing at remote sites was proposed as a means to provide early warning of DST events in order to protect human health and allow growers to test "pre-harvest" shellfish samples, thereby preventing harvest of toxic product that would later be destroyed or recalled. Tissue homogenates from several shellfish species collected from two sites in Sequim Bay, WA in the summer 2012, as well as other sites throughout Puget Sound, were analyzed using three rapid screening methods: a lateral flow antibody-based test strip (Jellett Rapid Test), an enzyme-linked immunosorbent assay (ELISA) and a protein phosphatase 2A inhibition assay (PP2A). The results were compared to the standard regulatory method of liquid chromatography coupled with tandem mass spectroscopy (LC-MS/MS). The Jellett Rapid Test for DSP gave an unacceptable number of false negatives due to incomplete extraction of DSTs using the manufacturer's recommended method while the ELISA antibody had low cross-reactivity with dinophysistoxin-1, the major toxin isomer in shellfish from the region. The PP2A test showed the greatest promise as a screening tool for Washington State shellfish harvesters.

Diarrhetic shellfish poisoning, Washington, USA, 2011.

Diarrhetic shellfish poisoning is a gastrointestinal illness caused by consumption of bivalves contaminated with dinophysistoxins. We report an illness cluster in the United States in which toxins were confirmed in shellfish from a commercial harvest area, leading to product recall. Ongoing surveillance is needed to prevent similar illness outbreaks.

Diarrhetic shellfish toxins and other lipophilic toxins of human health concern in Washington State.

The illness of three people in 2011 after their ingestion of mussels collected from Sequim Bay State Park, Washington State, USA, demonstrated the need to monitor diarrhetic shellfish toxins (DSTs) in Washington State for the protection of human health. Following these cases of diarrhetic shellfish poisoning, monitoring for DSTs in Washington State became formalized in 2012, guided by routine monitoring of Dinophysis species by the SoundToxins program in Puget Sound and the Olympic Region Harmful Algal Bloom (ORHAB) partnership on the outer Washington State coast. Here we show that the DSTs at concentrations above the guidance level of 16 µg okadaic acid (OA) + dinophysistoxins (DTXs)/100 g shellfish tissue were widespread in sentinel mussels throughout Puget Sound in summer 2012 and included harvest closures of California mussel, varnish clam, manila clam and Pacific oyster. Concentrations of toxins in Pacific oyster and manila clam were often at least half those measured in blue mussels at the same site. The primary toxin isomer in shellfish and plankton samples was dinophysistoxin-1 (DTX-1) with D. acuminata as the primary Dinophysis species. Other lipophilic toxins in shellfish were pectenotoxin-2 (PTX-2) and yessotoxin (YTX) with azaspiracid-2 (AZA-2) also measured in phytoplankton samples. Okadaic acid, azaspiracid-1 (AZA-1) and azaspiracid-3 (AZA-3) were all below the levels of detection by liquid chromatography tandem mass spectrometry (LC-MS/MS). A shellfish closure at Ruby Beach, Washington, was the first ever noted on the Washington State Pacific coast due to DSTs. The greater than average Fraser River flow during the summers of 2011 and 2012 may have provided an environment conducive to dinoflagellates and played a role in the prevalence of toxigenic Dinophysis in Puget Sound.

Parallel analyses of Alexandrium catenella cell concentrations and shellfish toxicity in the Puget Sound.

Alexandrium catenella is widespread in western North America and produces a suite of potent neurotoxins that cause paralytic shellfish poisoning (PSP) in humans and have deleterious impacts on public health and economic resources. There are seasonal PSP-related closures of recreational and commercial shellfisheries in the Puget Sound, but the factors that influence cell distribution, abundance, and relationship to paralytic shellfish toxins (PSTs) in this system are poorly described. Here, a quantitative PCR assay was used to detect A. catenella cells in parallel with state shellfish toxicity testing during the 2006 bloom season at 41 sites from April through October. Over 500,000 A. catenella cells liter(-1) were detected at several stations, with two main pulses of cells driving cell distribution, one in June and the other in August. PSTs over the closure limit of 80 mug of PST 100 per g of shellfish tissue were detected at 26 of the 41 sites. Comparison of cell numbers and PST data shows that shellfish toxicity is preceded by an increase in A. catenella cells in 71% of cases. However, cells were also observed in the absence of PSTs in shellfish, highlighting the complex relationship between A. catenella and the resulting shellfish toxicity. These data provide important information on the dynamics of A. catenella cells in the Puget Sound and are a first step toward assessing the utility of plankton monitoring to augment shellfish toxicity testing in this system.