Hiding in plain sight: Shellfish-killing phytoplankton in Washington State.

Summer bivalve shellfish mortalities have been observed in Puget Sound for nearly a century and attempts to understand and mitigate these losses have been only partially successful. Likewise, the understanding of the environmental conditions triggering shellfish mortalities and successful strategies for their mitigation are incomplete. In the literature, phytoplankton have played only a cursory role in summer shellfish mortalities in Washington State because spawning stress and bacteria were thought to be the primary causes. In recent years, the occurrence of Protoceratium reticulatum (Claparede & Lachmann) Buetschli and Akashiwo sanguinea (Hirasaka) Hansen & Moestrup, have been documented by the SoundToxins research and monitoring partnership in increasing numbers and duration and have been associated with declining shellfish health or mortality at various sites in Puget Sound. Blooms of these species occur primarily in summer months and have been shown to cause mass mortalities of shellfish in the U.S. and other parts of the world. In 2016-2017, yessotoxins (YTX) were measured in several species of Puget Sound bivalve shellfish, with a maximum concentration of 2.20 mg/kg in blue mussels, a value below the regulatory limit of 3.75 mg/kg established by the European Union for human health protection but documented to cause shellfish mortalities in other locations around the world. In July 2019, a bloom of P. reticulatum coincided with a summer shellfish mortality event, involving a dramatic surfacing of stressed, gaping Manila clams, suggesting that YTX could be the cause. YTX concentrations in their tissues were measured at a maximum of 0.28 mg/kg and histology of these clams demonstrated damage to digestive glands. A culture of P. reticulatum, isolated from North Bay during this massive bloom and shellfish mortality event, showed YTX reaching 26.6 pg/cell, the highest recorded toxin quota measured in the U.S. to date. Concentrations of YTX in phytoplankton samples reached a maximum of 920 ng/L during a P. reticulatum bloom in Mystery Bay on 13 August 2019 when cell abundance reached 1.82 million cells/L. The highest cellular YTX quota during that bloom that lasted into September was 10.8 pg/cell on 3 Sept 2019. Shellfish producers in Washington State have also noted shellfish larvae mortalities due to A. sanguinea passing through filtration intake systems into hatchery facilities. Early warning of shellfish-killing harmful algal bloom (HAB) presence in Puget Sound, through partnerships such as SoundToxins, provides options for shellfish growers to mitigate their effects through early harvest, movement of shellstock to upland facilities, or enhanced filtration at aquaculture facilities.

Determination of lipophilic marine biotoxins by liquid chromatography-tandem mass spectrometry in five shellfish species from Washington State, USA.

Low extraction efficiency (60-81%) of okadaic acid (OA) and dinophysistoxin 1 (DTX1) was obtained for 4 out of 5 shellfish species from Washington State (WA), USA, during application of a standard extraction method for determination of lipophilic marine biotoxins by LC-MS/MS as recommended by the European Union Reference Laboratory for Marine Biotoxins (EURLMB). OA and total OA including esters, DTX1, DTX2, and total DTX including esters, azaspiracid 1, 2, and 3 (AZA1, AZA2, and AZA3), pectenotoxin 2 (PTX2), and yessotoxin (YTX) were the toxins examined. Matrix-matched standards prepared from the same control samples used for spike-and-recovery tests were employed to evaluate toxin extraction efficiency and sample clean-up procedures. We adjusted the EURLMB extraction method by either using an acidified methanol extraction or pre-cooking shellfish homogenates at 70 °C for 20 min before EURLMB extraction. Extraction efficiency was improved markedly for OA and DTX1 with both modified methods and for YTX with the pre-cooking step included. However, recoveries were lower for YTX using the acidified methanol extraction and for PTX2 in non-mussel samples with the pre-cooking step. A hexane wash was applied to clean water-diluted non-hydrolyzed samples and a hexane wash was combined with solid-phase extraction for cleaning hydrolyzed samples. Improved sample clean-up, combined with LC-MS/MS adjustments, enabled quantification of U.S. Food and Drug Administration-regulated toxins in five shellfish species from WA with acceptable accuracy using non-matrix matched calibration standards.

Diversity and toxicity of Pseudo-nitzschia species in Monterey Bay: Perspectives from targeted and adaptive sampling.

Monterey Bay, California experiences near-annual blooms of Pseudo-nitzschia that can affect marine animal health and the economy, including impacts to tourism and commercial/recreational fisheries. One species in particular, P. australis, has been implicated in the most toxic of events, however other species within the genus can contribute to widespread variability in community structure and associated toxicity across years. Current monitoring methods are limited in their spatial coverage as well as their ability to capture the full suite of species present, thereby hindering understanding of HAB events and limiting predictive accuracy. An integrated deployment of multiple in situ platforms, some with autonomous adaptive sampling capabilities, occurred during two divergent bloom years in the bay, and uncovered detailed aspects of population and toxicity dynamics. A bloom in 2013 was characterized by spatial differences in Pseudo-nitzschia populations, with the low-toxin producer P. fraudulenta dominating the inshore community and toxic P. australis dominating the offshore community. An exceptionally toxic bloom in 2015 developed as a diverse Pseudo-nitzschia community abruptly transitioned into a bloom of highly toxic P. australis within the time frame of a week. Increases in cell density and proliferation coincided with strong upwelling of nutrients. High toxicity was driven by silicate limitation of the dense bloom. This temporal shift in species composition mirrored the shift observed further north in the California Current System off Oregon and Washington. The broad scope of sampling and unique platform capabilities employed during these studies revealed important patterns in bloom formation and persistence for Pseudo-nitzschia. Results underscore the benefit of expanded biological observing capabilities and targeted sampling methods to capture more comprehensive spatial and temporal scales for studying and predicting future events.

Morphology and toxicity of Pseudo-nitzschia species in the northern Benguela Upwelling System.

The Benguela upwelling system, considered the world's most productive marine ecosystem, has a long record of potentially toxic diatoms belonging to the genus Pseudo-nitzschia. Species of Pseudo-nitzschia were reported as early as 1936 from the northern Benguela upwelling system (nBUS). For the current study, long-term phytoplankton monitoring data (2004-2011) for the Namibian coast were analysed to examine inshore and offshore temporal distribution of Pseudo-nitzschia species, their diversity and ultrastructure. The potentially toxigenic P. pungens and P. australis were the dominant inshore species, whereas offshore Pseudo-nitzschia showed a higher diversity that also included potentially toxic species. During a warming event, a community shift from P. pungens and P. australis dominance to P. fraudulenta and P. multiseries was documented in the central nBUS. A case study of a toxic event (August 2004) revealed that P. australis and P. pungens were present at multiple inshore and offshore stations, coincident with fish (pilchard) and bird mortalities reported from the central part of Namibia. Toxin analyses (LC-MS/MS) of samples collected from June to August 2004 revealed the presence of particulate domoic acid (DA) in seawater at multiple stations (maximum ∼180 ng DA/L) in the >0.45 µm size-fraction, as well as detectable DA (0.12 µg DA/g) in the gut of one of two pilchard samples tested. These findings indicate that DA may have been associated with the fish and bird mortalities reported from this event in the nBUS. However, the co-occurrence of very high biomass phytoplankton blooms suggests that other explanations may be possible.

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.

Use of biosensors for the detection of marine toxins.

Increasing occurrences of harmful algal blooms (HABs) in the ocean are a major concern for countries around the globe, and with strong links between HABs and climate change and eutrophication, the occurrences are only set to increase. Of particular concern with regard to HABs is the presence of toxin-producing algae. Six major marine biotoxin groups are associated with HABs. Ingestion of such toxins via contaminated shellfish, fish, or other potential vectors, can lead to intoxication syndromes with moderate to severe symptoms, including death in extreme cases. There are also major economic implications associated with the diverse effects of marine biotoxins and HABs. Thus, effective monitoring programmes are required to manage and mitigate their detrimental global effect. However, currently legislated detection methods are labour-intensive, expensive and relatively slow. The growing field of biosensor diagnostic devices is an exciting area that has the potential to produce robust, easy-to-use, cost-effective, rapid and accurate detection methods for marine biotoxins and HABs. This review discusses recently developed biosensor assays that target marine biotoxins and their microbial producers, both in harvested fish/shellfish samples and in the open ocean. The effective deployment of such biosensor platforms could address the pressing need for improved monitoring of HABs and marine biotoxins, and could help to reduce their global economic impact.

Recovery and identification of Pseudo-nitzschia (Bacillariophyceae) frustules from natural samples acquired using the environmental sample processor.

Many species within the diatom genus Pseudo-nitzschia are difficult to distinguish without applying molecular analytical or microscopy-based methods. DNA, antibody and lectin probes have previously been used to provide rapid and specific detection of species and strains in complex field assemblages. Recently, however, well-documented cryptic genetic diversity within the group has confounded results of DNA probe tests in particular. Moreover, the number of species descriptions within the genus continues to increase, as do insights into toxin production by both new and previously described species. Therefore, a combination of classical morphological techniques and modern molecular methodologies is needed to resolve ecophysiological traits of Pseudo-nitzschia species. Here, we present an approach to recover and identify frustules from sample collection filters used for toxin analysis onboard the Environmental Sample Processor (ESP), an in situ sample collection and analytical platform. This approach provides a new and powerful tool for correlating species presence with toxin detected remotely and in situ by the ESP, and has the potential to be applied broadly to other sampling configurations. This new technique will contribute to a better understanding of naturally occurring Pseudo-nitzschia community structure with respect to observed domoic acid outbreaks.

Global solutions to regional problems: Collecting global expertise to address the problem of harmful cyanobacterial blooms. A Lake Erie case study.

In early August 2014, the municipality of Toledo, OH (USA) issued a 'do not drink' advisory on their water supply directly affecting over 400,000 residential customers and hundreds of businesses (Wilson, 2014). This order was attributable to levels of microcystin, a potent liver toxin, which rose to 2.5µgL-1 in finished drinking water. The Toledo crisis afforded an opportunity to bring together scientists from around the world to share ideas regarding factors that contribute to bloom formation and toxigenicity, bloom and toxin detection as well as prevention and remediation of bloom events. These discussions took place at an NSF- and NOAA-sponsored workshop at Bowling Green State University on April 13 and 14, 2015. In all, more than 100 attendees from six countries and 15 US states gathered together to share their perspectives. The purpose of this review is to present the consensus summary of these issues that emerged from discussions at the Workshop. As additional reports in this special issue provide detailed reviews on many major CHAB species, this paper focuses on the general themes common to all blooms, such as bloom detection, modeling, nutrient loading, and strategies to reduce nutrients.

The Vibrio harveyi bioassay used routinely to detect AI-2 quorum sensing inhibition is confounded by inconsistent normalization across marine matrices.

The Vibrio harveyi autoinducer-2 (AI-2) bioassay is used routinely to screen for inhibition of the AI-2 quorum sensing system. The present study utilizes three well-described bacterial strains to demonstrate that inconsistent normalization across matrices undermines the assay's use in screening marine samples for AI-2 inhibition.

Marine algal toxin azaspiracid is an open-state blocker of hERG potassium channels.

Azaspiracids (AZA) are polyether marine dinoflagellate toxins that accumulate in shellfish and represent an emerging human health risk. Although human exposure is primarily manifested by severe and protracted diarrhea, this toxin class has been shown to be highly cytotoxic, a teratogen to developing fish, and a possible carcinogen in mice. Until now, AZA's molecular target has not yet been determined. Using three independent methods (voltage clamp, channel binding assay, and thallium flux assay), we have for the first time demonstrated that AZA1, AZA2, and AZA3 each bind to and block the hERG (human ether-à-go-go related gene) potassium channel heterologously expressed in HEK-293 mammalian cells. Inhibition of K(+) current for each AZA analogue was concentration-dependent (IC(50) value range: 0.64-0.84 µM). The mechanism of hERG channel inhibition by AZA1 was investigated further in Xenopus oocytes where it was shown to be an open-state-dependent blocker and, using mutant channels, to interact with F656 but not with Y652 within the S6 transmembrane domain that forms the channel's central pore. AZA1, AZA2, and AZA3 were each shown to inhibit [(3)H]dofetilide binding to the hERG channel and thallium ion flux through the channel (IC(50) value range: 2.1-6.6 µM). AZA1 did not block the K(+) current of the closely related EAG1 channel. Collectively, these data suggest that the AZAs physically block the K(+) conductance pathway of hERG1 channels by occluding the cytoplasmic mouth of the open pore. Although the concentrations necessary to block hERG channels are relatively high, AZA-induced blockage may prove to contribute to the toxicological properties of the AZAs.

Determination of paralytic shellfish toxins in shellfish by receptor binding assay: collaborative study.

A collaborative study was conducted on a microplate format receptor binding assay (RBA) for paralytic e shellfish toxins (PST). The assay quantifies the composite PST toxicity in shellfish samples based on the ability of sample extracts to compete with (3)H saxitoxin (STX) diHCl for binding to voltage-gated sodium channels in a rat brain membrane preparation. Quantification of binding can be carried out using either a microplate or traditional scintillation counter; both end points were included in this study. Nine laboratories from six countries completed the study. One laboratory analyzed the samples using the precolumn oxidation HPLC method (AOAC Method 2005.06) to determine the STX congener composition. Three laboratories performed the mouse bioassay (AOAC Method 959.08). The study focused on the ability of the assay to measure the PST toxicity of samples below, near, or slightly above the regulatory limit of 800 (microg STX diHCl equiv./kg). A total of 21 shellfish homogenates were extracted in 0.1 M HCl, and the extracts were analyzed by RBA in three assays on separate days. Samples included naturally contaminated shellfish samples of different species collected from several geographic regions, which contained varying STX congener profiles due to their exposure to different PST-producing dinoflagellate species or differences in toxin metabolism: blue mussel (Mytilus edulis) from the U.S. east and west coasts, California mussel (Mytilus californianus) from the U.S. west coast, chorito mussel (Mytilus chiliensis) from Chile, green mussel (Perna canaliculus) from New Zealand, Atlantic surf clam (Spisula solidissima) from the U.S. east coast, butter clam (Saxidomus gigantea) from the west coast of the United States, almeja clam (Venus antiqua) from Chile, and Atlantic sea scallop (Plactopecten magellanicus) from the U.S. east coast. All samples were provided as whole animal homogenates, except Atlantic sea scallop and green mussel, from which only the hepatopancreas was homogenized. Among the naturally contaminated samples, five were blind duplicates used for calculation of RSDr. The interlaboratory RSDR of the assay for 21 samples tested in nine laboratories was 33.1%, yielding a HorRat value of 2.0. Removal of results for one laboratory that reported systematically low values resulted in an average RSDR of 28.7% and average HorRat value of 1.8. Intralaboratory RSDr based on five blind duplicate samples tested in separate assays, was 25.1%. RSDr obtained by individual laboratories ranged from 11.8 to 34.9%. Laboratories that are routine users of the assay performed better than nonroutine users, with an average RSDr of 17.1%. Recovery of STX from spiked shellfish homogenates was 88.1-93.3%. Correlation with the mouse bioassay yielded a slope of 1.64 and correlation coefficient (r(2)) of 0.84, while correlation with the precolumn oxidation HPLC method yielded a slope of 1.20 and an r(2) of 0.92. When samples were sorted according to increasing toxin concentration (microg STX diHCl equiv./kg) as assessed by the mouse bioassay, the RBA returned no false negatives relative to the 800 microg STX diHCl equiv./kg regulatory limit for shellfish. Currently, no validated methods other than the mouse bioassay directly measure a composite toxic potency for PST in shellfish. The results of this interlaboratory study demonstrate that the RBA is suitable for the routine determination of PST in shellfish in appropriately equipped laboratories.

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.

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.

Optimization of solid-phase extraction and liquid chromatography-tandem mass spectrometry for the determination of domoic acid in seawater, phytoplankton, and mammalian fluids and tissues.

We previously reported a solid-phase extraction (SPE) method for determination of the neurotoxin domoic acid (DA) in both seawater and phytoplankton by liquid chromatography-tandem mass spectrometry (LC-MS/MS) with the purpose of sample desalting without DA pre-concentration. In the present study, we optimized the SPE procedure with seawater and phytoplankton samples directly acidified with aqueous formic acid without addition of organic solvents, which allowed sample desalting and also 20-fold pre-concentration of DA in seawater and phytoplankton samples. In order to reduce MS contamination, a diverter valve was installed between LC and MS to send the LC eluant to waste, except for the 6-min elution window bracketing the DA retention time, which was sent to the MS. Reduction of the MS turbo gas temperature also helped to maintain the long-term stability of MS signal. Recoveries exceeded 90% for the DA-negative seawater and the DA-positive cultured phytoplankton samples spiked with DA. The SPE method for DA extraction and sample clean-up in seawater was extended to mammalian fluids and tissues with modification in order to accommodate the fluid samples with limited available volumes and the tissue extracts in aqueous methanol. Recoveries of DA from DA-exposed laboratory mammalian samples (amniotic fluid, cerebrospinal fluid, plasma, placenta, and brain) were above 85%. Recoveries of DA from samples (urine, feces, intestinal contents, and gastric contents) collected from field stranded marine mammals showed large variations and were affected by the sample status. The optimized SPE-LC-MS method allows determination of DA at trace levels (low pg mL(-1)) in seawater with/without the presence of phytoplankton. The application of SPE clean-up to mammalian fluids and tissue extracts greatly reduced the LC column degradation and MS contamination, which allowed routine screening of marine mammalian samples for confirmation of DA exposure and determination of fluid and tissue DA concentrations in experimental laboratory animals.

Endangered North Atlantic right whales (Eubalaena glacialis) experience repeated, concurrent exposure to multiple environmental neurotoxins produced by marine algae.

The western North Atlantic population of right whales (Eubalaena glacialis) is one of the most critically endangered of any whale population in the world. Among the factors considered to have potentially adverse effects on the health and reproduction of E. glacialis are biotoxins produced by certain microalgae responsible for causing harmful algal blooms. The worldwide incidence of these events has continued to increase dramatically over the past several decades and is expected to remain problematic under predicted climate change scenarios. Previous investigations have demonstrated that N. Atlantic right whales are being exposed to at least two classes of algal-produced environmental neurotoxins-paralytic shellfish toxins (PSTs) and domoic acid (DA). Our primary aims during this six-year study (2001-2006) were to assess whether the whales' exposure to these algal biotoxins occurred annually over multiple years, and to what extent individual whales were exposed repeatedly and/or concurrently to one or both toxin classes. Approximately 140 right whale fecal samples obtained across multiple habitats in the western N. Atlantic were analyzed for PSTs and DA. About 40% of these samples were attributed to individual whales in the North Atlantic Right Whale Catalog, permitting analysis of biotoxin exposure according to sex, age class, and reproductive status/history. Our findings demonstrate clearly that right whales are being exposed to both of these algal biotoxins on virtually an annual basis in multiple habitats for periods of up to six months (April through September), with similar exposure rates for females and males (PSTs: ∼70-80%; DA: ∼25-30%). Notably, only one of 14 lactating females sampled did not contain either PSTs or DA, suggesting the potential for maternal toxin transfer and possible effects on neonatal animals. Moreover, 22% of the fecal samples tested for PSTs and DA showed concurrent exposure to both neurotoxins, leading to questions of interactive effects. Targeted studies employing both in vivo and in vitro model systems represent the next logical step in assessing how and to what extent these algal biotoxins might compromise the health and reproduction of this endangered population.

Comparative analysis of bacterioplankton assemblages from Karenia brevis bloom and nonbloom water on the west Florida shelf (Gulf of Mexico, USA) using 16S rRNA gene clone libraries.

The brevetoxin-producing dinoflagellate, Karenia brevis, forms nearly annual blooms off the Florida west coast, severely impacting the region's ecology and economy. Bacteria are often cited as either promoting or interfering with the development of algal blooms, and thus a detailed study of the bacterioplankton assemblages associated with K. brevis was undertaken. We developed sixteen 16S rRNA gene clone libraries from K. brevis bloom and adjacent nonbloom water to determine the bacterial groups present and assess the influence of K. brevis cell number and/or depth on bacterioplankton community composition. Most notably, bacterial groups such as Rhodobacterales (Alphaproteobacteria) and Cytophagales/Sphingobacteriales (Bacteroidetes), reported previously to be associated with other harmful algal species, were often abundant in the presence of K. brevis. Cyanobacteria frequently dominated surface samples containing no detectable K. brevis, consistent with earlier work suggesting that these photosynthetic organisms may be important in promoting the proliferation of these blooms by conditioning the water. Moreover, differences in the abundance/diversity of traditionally more rare and often undocumented phylogenetic groups (e.g. Betaproteobacteria, Deltaproteobacteria, Chloroflexus, Firmicutes) were apparent in bloom vs. nonbloom water. This is the first study to document the association of these phylogenetic groups with natural K. brevis populations and suggests a potential role for these microorganisms in K. brevis bloom dynamics.

Single-laboratory validation of the microplate receptor binding assay for paralytic shellfish toxins in shellfish.

A single-laboratory validation (SLV) study was conducted for the microplate receptor binding assay (RBA) for paralytic shellfish poisoning (PSP) toxins in shellfish. The basis of the assay is the competition between [3H]saxitoxin (STX) and STX in a standard or sample for binding to the voltage dependent sodium channel. A calibration curve is generated by the addition of 0.01-1000 nM STX, which results in the concentration dependent decrease in [3H]STX-receptor complexes formed and serves to quantify STX in unknown samples. This study established the LOQ, linearity, recovery, accuracy, and precision of the assay for determining PSP toxicity in shellfish extracts, as performed by a single analyst on multiple days. The standard curve obtained on 5 independent days resulted in a half-maximal inhibition (IC50) of 2.3 nM STX +/- 0.3 (RSD = 10.8%) with a slope of 0.96 +/- 0.06 (RSD = 6.3%) and a dynamic range of 1.2-10.0 nM. The LOQ was 5.3 microg STX equivalents/100 g shellfish. Linearity, established by quantification of three levels of purified STX (1.5, 3, and 6 nM), yielded an r2 of 0.97. Recovery from mussels spiked with three levels (40, 80, and 120 microg STX/100 g) averaged 121%. Repeatability (RSD(r)), determined on six naturally contaminated shellfish samples on 5 independent days, was 17.7%. A method comparison with the AOAC mouse bioassay yielded r2 = 0.98 (slope = 1.29) in the SLV study. The effects of the extraction method on RBA-based toxicity values were assessed on shellfish extracted for PSP toxins using the AOAC mouse bioassay method (0.1 M HCI) compared to that for the precolumn oxidation HPLC method (0.1% acetic acid). The two extraction methods showed linear correlation (r2 = 0.99), with the HCl extraction method yielding slightly higher toxicity values (slope = 1.23). A similar relationship was observed between HPLC quantification of the HCI- and acetic acid-extracted samples (r2 = 0.98, slope 1.19). The RBA also had excellent linear correlation with HPLC analyses (r2 = 0.98 for HCl, r2 = 0.99 for acetic acid), but gave somewhat higher values than HPLC using either extraction method (slope = 1.39 for HCl extracts, slope = 1.32 for acetic acid). Overall, the excellent linear correlations with the both mouse bioassay and HPLC method and sufficient interassay repeatability suggest that the RBA can be effective as a high throughput screen for estimating PSP toxicity in shellfish.

Azaspiracid shellfish poisoning: a review on the chemistry, ecology, and toxicology with an emphasis on human health impacts.

Azaspiracids (AZA) are polyether marine toxins that accumulate in various shellfish species and have been associated with severe gastrointestinal human intoxications since 1995. This toxin class has since been reported from several countries, including Morocco and much of western Europe. A regulatory limit of 160 microg AZA/kg whole shellfish flesh was established by the EU in order to protect human health; however, in some cases, AZA concentrations far exceed the action level. Herein we discuss recent advances on the chemistry of various AZA analogs, review the ecology of AZAs, including the putative progenitor algal species, collectively interpret the in vitro and in vivo data on the toxicology of AZAs relating to human health issues, and outline the European legislature associated with AZAs.

Extraction of domoic acid from seawater and urine using a resin based on 2-(trifluoromethyl)acrylic acid.

A new solid-phase extraction (SPE) matrix with high affinity for the neurotoxin domoic acid (DA) was designed and tested. A computational modelling study led to the selection of 2-(trifluoromethyl)acrylic acid (TFMAA) as a functional monomer capable of imparting affinity towards domoic acid. Polymeric adsorbents containing TFMAA were synthesised and tested in high ionic strength solutions such as urine and seawater. The TFMAA-based polymers demonstrated excellent performance in solid-phase extraction of domoic acid, retaining the toxin while salts and other interfering compounds such as aspartic and glutamic acids were removed by washing and selective elution. It was shown that the TFMAA-based polymer provided the level of purification of domoic acid from urine and seawater acceptable for its quantification by high performance liquid chromatography-mass spectrometry (HPLC-MS) and enzyme-linked immunosorbent assay (ELISA) without any additional pre-concentration and purification steps.

Transcriptional profiling and inhibition of cholesterol biosynthesis in human T lymphocyte cells by the marine toxin azaspiracid.

Azaspiracid-1 (AZA-1) is a marine biotoxin reported to accumulate in shellfish from several countries, including eastern Canada, Morocco, and much of western Europe, and is frequently associated with severe gastrointestinal human intoxication. As the mechanism of action of AZA-1 is currently unknown, human DNA microarrays and qPCR were used to profile gene expression patterns in human T lymphocyte cells following AZA-1 exposure. Some of the early (1 h) responding genes consisted of transcription factors, membrane proteins, receptors, and inflammatory genes. Four- and 24-h responding genes were dominated by genes involved in de novo lipid biosynthesis of which 17 of 18 involved in cholesterol biosynthesis were significantly up regulated. The up regulation of synthesis genes was likely in response to the ca. 50% reduction in cellular cholesterol, which correlated with up regulated protein expression levels of the low-density lipoprotein receptor. These data collectively detail the inhibition of de novo cholesterol synthesis, which is the likely cause of cytotoxicity and potentially a target pathway of the toxin.