Domoic Acid In Shellfish Research Articles

Annual variation in domoic acid in phytoplankton and shellfish samples from Daya Bay of the South China Sea

Domoic acid (DA) is a well-known phycotoxin that causes amnesic shellfish poisoning (ASP) and is mainly produced by diatom species belonging to the genus Pseudo-nitzschia. An annual survey was conducted monthly over the period of September 2020 to August 2021 in Daya Bay of the South China Sea to investigate the dynamics of particulate and shellfish DA and their relationships with the abundance of Pseudo-nitzschia spp. and environmental parameters. Pseudo-nitzschia spp. was one of the most dominant phytoplankton taxa, and a Pseudo-nitzschia bloom occurred during the survey with the highest abundance of 1.91 × 106 cells L−1. DA was detected in almost all plankton samples with the highest value of 120.7 ng L−1, and high DA concentrations coincided with the abundant presence of Pseudo-nitzschia. DA is prevalent in Daya Bay throughout the year, with detection rates of 98.3%, 82.6%, and 82.6% in plankton samples, in-situ and purchased shellfish, respectively. Higher DA concentrations were detected in the scallop (Chamys nobilis), with the highest concentration of 5.34 µg g−1. High water temperature and low DSi:DIN ratio promoted the growth of Pseudo-nitzschia and DA production. The results suggest that the increasing nitrogen loading and silicate limitation during Pseudo-nitzschia blooms together with the increase in water temperature may increase the risk of DA contamination in Daya Bay.

Residue Analysis and Assessment of the Risk of Dietary Exposure to Domoic Acid in Shellfish from the Coastal Areas of China.

Harmful algal blooms in Chinese waters have caused serious domoic acid (DA) contamination in shellfish. Although shellfish are at particular risk of dietary exposure to DA, there have been no systematic DA risk assessments in Chinese coastal waters. A total of 451 shellfish samples were collected from March to November 2020. The presence of DA and four of its isomers were detected using liquid chromatography-tandem mass spectrometry. The spatial-temporal distribution of DA occurrence and its potential health risks were examined. DA was detected in 198 shellfish samples (43.90%), with a maximum level of 942.86 μg/kg. DA was recorded in all 14 shellfish species tested and Pacific oysters (Crassostrea gigas) showed the highest average DA concentration (82.36 μg/kg). The DA concentrations in shellfish showed distinct spatial-temporal variations, with significantly higher levels of occurrence in autumn than in summer and spring (p < 0.01), and particularly high occurrence in Guangdong and Fujian Provinces. The detection rates and maximum concentrations of the four DA isomers were low. While C. gigas from Guangdong Province in September showed the highest levels of DA contamination, the risk to human consumers was low. This study improves our understanding of the potential risk of shellfish exposure to DA-residues.

Development of fluorescence sensor and test paper based on molecularly imprinted carbon quantum dots for spiked detection of domoic acid in shellfish and lake water

Excessive levels of domoic acid (DA) in edible shellfish and water can seriously threaten human health and even cause death. Herein, we have developed a fluorescence sensor and test paper based on molecularly imprinted carbon quantum dots (B-CDs@MIPs) for DA analysis. In this research, the prepared carbon quantum dots (B-CDs) with good optical properties were used as the sensitive fluorescent signal probes, and the molecularly imprinted polymers (MIPs) instead of biological antibodies were employed as the specific recognition components because of their high stability and low cost. Due to the shielding effect of imprinted silicon layer and the influence of modified groups introduced in the MIPs preparation process, the fluorescence signal of B-CDs in MIPs is inhibited. Interestingly, the DA can cause the partial recovery of the fluorescence intensity of B-CDs@MIPs by passivating the modified groups on the surface of B-CDs in MIPs. According to this principle, the designed fluorescence sensor reveals outstanding stability, super anti-interference ability, and excellent sensitivity, with the detection limit down to 10 nM. The sensor has been successfully applied to the rapid and accurate spiked analysis of DA in edible shellfish and lake water, providing excellent recoveries. More importantly, the designed inexpensive and easy-to-operate fluorescent test paper can realize preliminary qualitative analysis of DA on site by naked eye, indicating its promising application potential in food and environmental analysis.

Determination of domoic acid in shellfish by liquid chromatography- tandem mass spectrometry coupled with dispersive solid phase extraction

A method utilizing liquid chromatography-tandem mass spectrometry(LC-MS/MS) coupled with dispersive solid phase extraction for quantitative analysis of domoic acid in four kinds of shellfish was established. The sample of 0. 1 g was extracted with 25% methanol aqueous solution, the extract was purified by dispersive solid phase extraction with 50 mg HLB and 5 mg GCB, and then filtered through a PTFE membrane. The analytes were separated on a C_(18) column(100 mm×2. 1 mm, 1. 9 μm), and detected in selected reaction monitoring(SRM) mode via positive electrospray ionization. The matrix matching and external standard method was used for quantitation. Domoic acid showed good linearity in the concentration range between 1. 0 ng/mL and 50. 0 ng/mL with correlation coefficients higher than 0. 9994. The detection limits of domoic acid in shellfish was 5 μg/kg. The inter-and intra-day recoveries were 91. 6%-109. 2% and 90. 9%-109. 3%, respectively. The inter-and intra-day ralitive standard deviations(RSDs) were lower than 8. 2% at spiked concentrations of 20, 50 and 100 μg/kg. The method is accurate, fast, easy to operate, which can satisfy the requirements of public health emergency testing or routine testing.

Toxic marine microalgae and noxious blooms in the Mediterranean Sea: A contribution to the Global HAB Status Report

We review the spatial distribution of toxic marine microalgal species and the impacts of all types of harmful algal events (Harmful Algal Blooms, HABs) in the Mediterranean Sea (MS), including the Black Sea, the Sea of Marmara, coastal lagoons and transitional waters, based on two databases compiled in the Ocean Biogeographic Information System (OBIS). Eighty-four potentially toxic species have been detected in the MS (2,350 records), of which 16 described from these waters between 1860 and 2014 and a few suspected to have been introduced. More than half of these species (46) produce toxins that may affect human health, the remainders ichthyotoxic substances (29) or other types of toxins (9). Nevertheless, toxicity-related events are not frequent in the MS (308 records in 31 years), and mainly consist of impacts on aquaculture, caused by the dinoflagellates Dinophysis and Alexandrium, along with a few actual shellfish poisoning cases. Pseudo-nitzschia blooms are widespread, but domoic acid in shellfish rarely exceeds regulatory levels. Fish kills are probably less sporadic than reported, representing a problem at a few places along the southern MS coasts and in the Ebro River Delta. Since the last decade of the 20th century, blooms of the benthic dinoflagellates Ostreopsis cf. ovata have regularly occurred all along rocky shores of the MS, at times with human health problems caused by toxic aerosol. New records of Gambierdiscus and Fukuyoa, until now reported for the westernmost and easternmost MS coasts, raise concerns about the risk of ciguatera, a syndrome so far known only for subtropical and tropical areas. Recent discoveries are the dinoflagellates Vulcanodinium rugosum, responsible for the presence of pinnatoxins in French lagoons’ shellfish, and the azaspiracid-producers Azadinium spp. Mucilages and discolorations have a major impact on tourism in summer. Reports of toxic species and HABs have apparently increased in the MS over the last half century, which is likely related to the increased awareness and monitoring operations rather than to an actual increase of these phenomena. Indeed, while the case of Ostreopsis appears as a sudden upsurge rather than a trend, no actual increase of toxic or noxious events has so far emerged in intensively studied areas, such as the French and Spanish coasts or the Adriatic Sea. Moreover, some cases of decrease are reported, e.g., for Alexandrium minutum blooms disappearing from the Harbour of Alexandria. Overall, main HAB risks derive from cases of massive development of microalgal biomass and consequent impacts of reduced coastal water quality on tourism, which represents the largest part of the marine economy along the MS coasts.

Variability of Amnesic Shellfish Toxin and Pseudo-nitzschia occurrence in bivalve molluscs and water samples–Analysis of ten years of the official control monitoring programme

As the official control laboratory for marine biotoxins within Great Britain, the Centre for Environment, Fisheries and Aquaculture Science, in conjunction with the Scottish Association for Marine Science, has amassed a decade’s worth of data regarding the prevalence of the toxins associated with Amnesic Shellfish Poisoning within British waters. This monitoring involves quantitative HPLC-UV analysis of shellfish domoic acid concentration, the causative toxin for Amnesic Shellfish Poisoning, and water monitoring for Pseudo-nitzschia spp., the phytoplankton genus that produces domoic acid. The data obtained since 2008 indicate that whilst the occurrence of domoic acid in shellfish was generally below the maximum permitted limit of 20 mg/kg, there were a number of toxic episodes that breached this limit. The data showed an increase in the frequency of both domoic acid occurrence and toxic events, although there was considerable annual variability in intensity and geographical location of toxic episodes. A particularly notable increase in domoic acid occurrence in England was observed during 2014. Comparison of Scottish toxin data and Pseudo-nitzschia cell densities during this ten-year period revealed a complex relationship between the two measurements. Whilst the majority of events were associated with blooms, absolute cell densities of Pseudo-nitzschia did not correlate with domoic acid concentrations in shellfish tissue. This is believed to be partly due to the presence of a number of different Pseudo-nitzschia species in the water that can exhibit variable toxin production. These data highlight the requirement for tissue monitoring as part of an effective monitoring programme to protect the consumer, as well as the benefit of more detailed taxonomic discrimination of the Pseudo-nitzschia genus to allow greater accuracy in the prediction of shellfish toxicity.

Development and Application of Immunoaffinity Column Purification and Ultrahigh Performance Liquid Chromatography-Tandem Mass Spectrometry for Determination of Domoic Acid in Shellfish.

Domoic acid (DA) is a neurotoxin associated with amnesic shellfish poisoning (ASP). Though LC coupled to tandem mass spectrometry (LC-MS/MS) has become the preferred method for DA determination, traditional sample pretreatment is still labor-intensive. In this study, a simple, efficient and selective method for LC-MS/MS analysis of DA in shellfish was established by optimizing clean-up procedures on a self-assembly immunoaffinity column (IAC). Shellfish was extracted with 75% methanol twice and diluted with phosphate buffered saline (PBS, 1:2). The mixture was purified on IAC as follows: preconditioned with PBS, loaded with sample, washed by 50% MeOH, and eluted with MeOH containing 2% ammonium hydroxide. Concentrated analyte was monitored by multiple reaction monitoring (MRM) using electrospray (ESI) positive ion mode throughout the LC gradient elution. Based on the post-extraction addition method, matrix effects for various shellfish matrices were found to be less than 8%. The developed method was fully validated by choosing mussel as the representative matrix. The method had a limit of detection (LOD) of 0.02 µg·g−1, showed excellent linear correlation in the range of 0.05–40 µg·g−1, and obtained ideal recoveries (91–94%), intra-day RSDs (6–8%) and inter-day RSDs (3–6%). The method was successfully applied to DA determination in 59 shellfish samples, with a detection rate of 10% and contaminated content of 0.1–14.9 µg·g−1.

Taxonomical diversity of Pseudo‐nitzschia from the Central Adriatic Sea

SUMMARYThe aim of our research was to study the composition of Pseudo‐nitzschia species during a period when neurotoxin domoic acid (DA) was present in shellfish. Sampling was conducted in Kaštela Bay (Central Adriatic Sea), between November 2015 and January 2016. Concentrations of DA analyzed in various shellfish species were low, below the regulatory limit, while the highest abundance of Pseudo‐nitzschia spp. reached 1.85 × 105 cells L−1 in the surface layer, at the beginning of November. Within the temperature and salinity range recorded during the investigated period, a positive correlation of Pseudo‐nitzschia spp. abundance was recorded with temperature. Morphological analyses by scanning electron microscopy revealed the presence of five Pseudo‐nitzschia species that had already been reported in the Adriatic Sea – P. calliantha, P. delicatissima, P. fraudulenta, P. pseudodelicatissima /P. cuspidata and P. subfraudulenta – as well as an unknown Pseudo‐nitzschia sp. The composition of the Pseudo‐nitzschia assemblage changed over the investigated period. The species P. pseudodelicatissima/P.cuspidata was found throughout the entire period and the highest diversity was noticed in January, when all six observed species were recorded. These results represent the first taxonomical investigation of the genus Pseudo‐nitzschia in Kaštela Bay, as well as the first report of DA in shellfish from this area.

HPLC determination of domoic acid in shellfish based on magnetic molecularly imprinting polymers

ABSTRACTA magnetic molecularly imprinting polymer for domoic acid was fabricated. Synthesis conditions were optimized. The polymer particles have high magnetization for rapid magnetic separation. The apparent maximum absorption amount and dissociation constant of the polymer were 1,600 µg g−1 and 20.6 µg mL−1, respectively. The polymer retained 90% of adsorption amount after 5 times of repeated use. It was used as an adsorbent for purification and HPLC detection of domoic acid in shellfish with a detection limit of 0.050 µg g−1. Thus, the polymer could be applied to the sample pretreatment of aquatic products.

Climatic regulation of the neurotoxin domoic acid

Domoic acid is a potent neurotoxin produced by certain marine microalgae that can accumulate in the foodweb, posing a health threat to human seafood consumers and wildlife in coastal regions worldwide. Evidence of climatic regulation of domoic acid in shellfish over the past 20 y in the Northern California Current regime is shown. The timing of elevated domoic acid is strongly related to warm phases of the Pacific Decadal Oscillation and the Oceanic Niño Index, an indicator of El Niño events. Ocean conditions in the northeast Pacific that are associated with warm phases of these indices, including changes in prevailing currents and advection of anomalously warm water masses onto the continental shelf, are hypothesized to contribute to increases in this toxin. We present an applied domoic acid risk assessment model for the US West Coast based on combined climatic and local variables. Evidence of regional- to basin-scale controls on domoic acid has not previously been presented. Our findings have implications in coastal zones worldwide that are affected by this toxin and are particularly relevant given the increased frequency of anomalously warm ocean conditions.

Laser ablation electrospray ionization high-resolution mass spectrometry for regulatory screening of domoic acid in shellfish.

RationaleDomoic acid (DA) is a potent neurotoxin that accumulates in shellfish. Routine testing involves homogenization, extraction and chromatographic analysis, with a run time of up to 30 min. Improving throughput using ambient ionization for direct analysis of DA in tissue would result in significant time savings for regulatory testing labs.MethodsWe assess the suitability of laser ablation electrospray ionization high‐resolution mass spectrometry (LAESI‐HRMS) for high‐throughput screening or quantitation of DA in a variety of shellfish matrices. The method was first optimized for use with HRMS detection. Challenges such as tissue sub‐sampling, isobaric interferences and method calibration were considered and practical solutions developed. Samples included 189 real shellfish samples previously analyzed by regulatory labs as well as mussel matrix certified reference materials.ResultsDomoic acid was selectively analyzed directly from shellfish tissue homogenates with a run time of 12 s. The limits of detection were between 0.24 and 1.6 mg DA kg−1 tissue, similar to those of LC/UV methods. The precision was between 27 and 44% relative standard deviation (RSD), making the technique more suited to screening than direct quantitation. LAESI‐MS showed good agreement with LC/UV and LC/MS and was capable of identifying samples above and below 5 mg DA kg−1 wet shellfish tissue, one quarter of the regulatory limit.ConclusionsThese findings demonstrate the suitability of LAESI‐MS for routine, high‐throughput screening of DA. This approach could result in significant time savings for regulatory labs carrying out shellfish safety testing on thousands of samples annually. © 2016 The Authors. Rapid Communications in Mass Spectrometry Published by John Wiley & Sons Ltd.

Harmful algal bloom forecast system for SW Ireland. Part II: Are operational oceanographic models useful in a HAB warning system

This study investigated the application of a three-dimensional physical hydrodynamic model in a harmful algal bloom forecast system for Bantry Bay, southwest Ireland. Modelled oceanographic conditions were studied and used to help understand observed changes in the chemical and biological patterns from the national biotoxins and phytoplankton monitoring program. The study focused on two toxic events in 2013. An upwelling event was predicted by the model prior to the appearance and population increase of potentially toxic diatoms, Pseudo-nitzschia, and associated domoic acid in shellfish. A downwelling episode was provided as a forecast in the model prior to the arrival of a Dinophysis bloom and detection of its associated biotoxins in Bay shellfish. The modelled forecast products developed included expected surface, mid-depth and bottom current pathways at the mouth of the Bay and on the adjacent shelf. The rate and direction of water volume flow at the mouth and mid-bay sections were produced by the model to examine predicted upwelling and downwelling pulses. The model also calculated the evolution of water properties (temperature, salinity and density) with depth along the Bay axis and on the adjacent continental shelf. Direct measurements of water properties at a fixed point, mid-bay, were comparable to model calculations. The operational model for southwest Ireland produces a reliable 3-day physical hydrodynamic forecast of the dominant regional physical processes that result in water exchange events between Bantry Bay and its adjacent shelf. While simulated physical hydrodynamics were provided as a 3-day forecast, the upwelling and downwelling signals from the model, closely linked to toxic HAB episodes, were evident up to 10 days prior to the contamination of shellfish in the Bay.

Monitoring Oregon Coastal Harmful Algae: Observations and implications of a harmful algal bloom-monitoring project

The accumulation of domoic acid (DA) and saxitoxins (STX), phycotoxins produced by some species of Pseudo-nitzschia and Alexandrium, respectively, in coastal food webs are a focus of research on the West Coast of the United States due to the deleterious effects they have on coastal ecosystems and economies. Results are presented from the 2007–2012 Monitoring Oregon Coastal Harmful Algae (MOCHA) project, the Oregon coast's first HAB monitoring and research program. Both historical toxin databases and more detailed case-study observations of individual HAB events are compiled to provide the first detailed overview of HAB occurrence in this region. These results are also presented in the context of informing future HAB monitoring in this and other upwelling regimes affected by STX and DA. A 2009–2010 warming event was associated with the greatest HAB activity during the MOCHA project, including anomalously high sea surface temperatures and shellfish harvesting closures due to STX and DA in 2009 and 2010, respectively. In regards to HAB monitoring, it is shown that (1) razor clams are a more sensitive indicator of DA than mussels; (2) water column concentrations of particulate domoic acid greater than 103ngL−1 can be used as a threshold for early-warning of shellfish DA toxicity and (3) approximately bi-weekly, or shorter, monitoring of Alexandrium in the surf zone and/or offshore can provide advance notice of STX contamination of shellfish. Both of the latter two metrics gain added value when coupled with local wind stress, a proxy of downwelling/relaxation events that facilitate greater interaction between offshore blooms and shellfish.

Detection of domoic acid in Mytilus galloprovincialis and Ostrea edulis linked to the presence of Nitzschia bizertensis in Bizerte Lagoon (SW Mediterranean)

Seasonal variation of DA in bivalves was monitored from August 2008 to March 2009 at a shellfish station in Bizerte Lagoon, one of the most important shellfish areas in Tunisia. At the same time, dynamics of potentially toxic diatoms was studied at six stations in the lagoon. Several diatom strains were isolated, maintained in culture and their identification as well as the toxicity was examined.During the study period, domoic acid (DA) was detected in Tunisia, in both mussels (Mytilus galloprovincialis: 0.13–0.86 μg DA g−1 tissue) and oysters (Ostrea edulis: 0.42–1.04 μg DA g−1 tissue). The amount of DA in the two species was below the EU guideline limits for shellfish (20 μg DA g−1).Within the phytoplankton community, potentially toxic diatoms were observed in high densities (104–106 cells l−1). Among the 9 established cultured strains, seven were identified as the recently described species Nitzschia bizertensis sp. nov, which is apparently a commonly occurring species in the Bizerte Lagoon. Among tested five strains of N. bizertensis, four were confirmed to be toxin producers, containing 2–7 × 10−3 pg DA cell−1 during stationary growth phase.Our results indicate for the first time a Nitzschia species, here Nitzschia bizertensis, could be in relation to accumulation of DA in shellfish. Monitoring of DA in phytoplankton and shellfish should be considered for Bizerte Lagoon in order to gain knowledge about the ecology and toxin-production of N. bizertensis and to explore the risk of DA accumulation in the local shellfish industry caused by this species.

Tidal and wind-event variability and the distribution of two groups of Pseudo-nitzschia species in an upwelling-influenced Ría

High-resolution physical and biological measurements were carried out in May–June 2007 during the ‘HABIT-Pontevedra 2007’ survey in Ría de Pontevedra (Galician Rías Baixas, NW Spain) to study small-scale physical–biological interactions in the distribution of microphytoplankton, with special emphasis on harmful species. Longitudinal transects from the Ría to the adjacent shelf were sampled to describe the spring–neap tidal and circadian variability. An in situ particle profiler, a moored ADCP, and a towed undulating CTD (Scanfish) were used during the survey, which took place after an upwelling pulse at neap tides during a downwelling–upwelling cycle and coincided with the annual maximum of Pseudo-nitzschia spp. Cell maxima of Pseudo-nitzschia seriata (2×106cellsL−1) and Pseudo-nitzschia delicatissima (6×105cellsL−1) groups were observed during the first half of the cruise during downwelling, and a significant decrease in cell numbers occurred during subsequent relaxation-upwelling conditions. Thin layers were eroded during downwelling and formed again in the subsequent upwelling pulse. Cells of the P. seriata group were always dominant in terms of biomass but the contribution of the P. delicatissima group increased with stratification. Water exchange between the Ría and the adjacent shelf was mainly controlled by the upwelling/downwelling cycle, and tidal (both semidiurnal and spring–neap) variability appeared as a modulation of the response of the Ría circulation to wind variability. The circadian variability was regulated by tidal forcing, and Pseudo-nitzschia spp. maxima were associated with high stratification during low tide. The magnitude of spring–neap tidal and circadian variability has to be considered when designing and implementing water quality and harmful algae monitoring programmes. Blooms of Pseudo-nitzschia ssp. were not associated with the occurrence of domoic acid in shellfish even when Pseudo-nitzschia australis was dominant. These results confirm that just cell densities of the potential toxin producer are not enough for early warning in monitoring of Pseudo-nitzschia events.

Development and validation of the first high performance-lateral flow immunoassay (HP-LFIA) for the rapid screening of domoic acid from shellfish extracts

A lateral flow immunoassay (LFIA) has been developed and fully validated to detect the primary amnesic shellfish poisoning (ASP) toxin, domoic acid (DA). The performance characteristics of two versions of the test were investigated using spiked and naturally contaminated shellfish (mussels, scallops, oysters, clams, and cockles). The tests provide a qualitative result, to indicate the absence or presence of DA in extracts of shellfish tissues, at concentrations that are relevant to regulatory limits. The new rapid assay (LFIA version 2) was designed to overcome the performance limitations identified in the first version of the assay. The improved test uses an electronic reader to remove the subjective nature of the generated results, and the positive cut-off for screening of DA in shellfish was increased from 10ppm (version 1) to 17.5ppm (version 2). A simple extraction and test procedure was employed, which required minimal equipment and materials; results were available 15min after sample preparation. Stability of the aqueous extracts at room temperature (22°C) at four time points (up to 245min after extraction) and across a range of DA concentrations was 100.3±1.3% and 98.8±2.4% for pre- and post-buffered extracts, respectively. The assay can be used both within laboratory settings and in remote locations. The accuracy of the new assay, to indicate negative results at or below 10ppm DA, and positive results at or above 17.5ppm, was 99.5% (n=216 tests). Validation data were obtained from a 2-day, randomised, blind study consisting of multiple LFIA lots (n=3), readers (n=3) and operators (n=3), carrying out multiple extractions of mussel tissue (n=3) at each concentration (0, 10, 17.5, and 20ppm). No matrix effects were observed on the performance of the assay with different species (mussels, scallops, oysters, clams, and cockles). There was no impact on accuracy or interference from other phycotoxins, glutamic acid or glutamine with various strip incubations (8, 10, and 12min). The accuracy of the assay, using naturally contaminated samples to indicate negative results at or below 12.5ppm and positive results at or above 17.5ppm, was 100%. Variability between three LFIA lots across a range of DA concentrations, expressed as coefficient of variation (% CV), was 1.1±0.4% (n=2 days) based on quantitative readings from the electronic reader. During an 8 week stability study, accuracy of the method with test strips stored at various temperatures (6, 22, 37 and 50°C) was 100%. Validation for both versions included comparisons with results obtained using reference LC–UV methods.

Pseudo-nitzschia Peragallo (Bacillariophyceae) diversity and domoic acid accumulation in tuberculate cockles and sweet clams in M’diq Bay, Morocco

Abstract The diversity of Pseudo-nitzschia (Bacillariophyceae) and accumulation of the neurotoxin domoic acid (DA) in two types of shellfish; tuberculate cockles (Acanthocardia tuberculata) and sweet clams (Challista chione) was explored in M’diq Bay,Morocco during 2007. The highest abundances of Pseudo-nitzschia were found during the period from March to October, with peaks occurring in May and September. Toxin analysis showed an accumulation of domoic acid in shellfish sampled during spring and autumn. The maximum toxin concentration was 4.9 mg DAg-1 of the whole tissue recorded in sweet clam during spring. Using transmission electron microscopy, thirteen Pseudo-nitzschia species were identified, eight of which are known as producers of domoic acid: P. multistriata, P. cuspidata, P. galaxiae, P. multiseries, P. pseudodelicatissima, P. pungens var. aveirensis, P. calliantha and P. fraudulenta. The five non- toxic species observed were P. subpacifica, P. arenysensis, P. dolorosa, P. subfraudulenta, and P. cf. caciantha

Sensitive determination of domoic acid in shellfish by on-line coupling of weak anion exchange solid-phase extraction and liquid chromatography–diode array detection–tandem mass spectrometry

An automated method based on the use of on-line solid-phase extraction (SPE) coupled to liquid chromatography with diode array detection and tandem mass spectrometry (LC–DAD–MS/MS) has been developed for the determination of domoic acid in shellfish. The on-line coupling of SPE and liquid chromatography was accomplished by a column switching approach. A weak anion exchange (WAX) sorbent was selected for the SPE procedure, allowing a selective cleanup of shellfish extracts. High sensitivity was achieved by on-line pre-concentration of large volume injections (50–1000μL). A simple protein precipitation cleanup with acetone was used to efficiently remove proteins from shellfish extracts, preventing possible column clogging during chromatographic separation.The proposed method showed good performance in terms of linearity, limits of detection (LODs), precision and accuracy. Quantitative recovery and satisfactory inter-day precision (RSD ⩽7%) was achieved. LODs in the in the sub-nanogram per gram level (0.23–0.32ngg−1) were obtained with MS/MS detection. Matrix effects were assessed by post-extraction addition method and no signal enhancement or suppression was observed during MS detection.This method was successfully applied to the determination of domoic acid in several shellfish samples, including scallops, oysters, mussels, Manila clams, common cockles and razor clams.

Relationships among upwelling, phytoplankton blooms, and phycotoxins in coastal Oregon shellfish

Climatologies derived from satellite data (1998 to 2007) were used to elucidate seasonal and latitudinal patterns in winds, sea surface temperature (SST), and chlorophyll concentrations (chl) over the Oregon shelf. These were further used to reveal oceanographic conditions normally associ- ated with harmful algal blooms (HABs) and toxic shellfish events along the Oregon coast. South of 43° N, around Cape Blanco, summer upwelling started earlier and finished later than north of 43° N. Spring blooms occur when light limitation is relieved, before the initiation of upwelling, and sec- ondary, more intense blooms occur approximately 2 wk after upwelling is established. North of 45° N, SST and chl are heavily influenced by the Columbia River plume, which delays upwelling-driven cooling of the surface coastal ocean in spring, and causes phytoplankton blooms (as indicated by increased chl) earlier than expected. The presence of saxitoxin in coastal shellfish, which causes paralytic shellfish poisoning, was generally associated with late summer upwelling. The presence of domoic acid in shellfish, which leads to amnesic shellfish poisoning, was greatest during the transi- tion between upwelling and downwelling regimes. This work demonstrates that satellite data can indicate physical situations when HABs are more likely to occur, thus providing a management tool useful in predicting or monitoring HABs.

TEM identification of Pseudo-nitzschia species from Lisbon Bay (NE Atlantic)

AbstractSeveral species of the chain-forming marine diatom genus Pseudo-nitzschia produce domoic acid (DA), a neuroexcitant amino acid, responsible for a human amnesic syndrome after consumption of toxified shellfish (ASP). Since 1995, the Portuguese monitoring program has regularly detected the presence of DA in shellfish. So far only P. australis has been implicated, and references therein. Identification of the causative species of Pseudo-nitzchia in natural samples is hampered by difficulties in identification at the light microscope level. Recent studies have shown that ultrastructural details of the wall, observed by transmission electron microscopy, allow the identification of species in good agreement with the phylogenetic and biological species concept.