The diatom genus Pseudo-nitzschia (Peragallo) associated with the production of domoic acid (DA), the toxin reposnsible for amnesic shellfish poisoning, is abundant in Scottish waters. A two year study examined the relationship between Pseudo-nitzschia cells in the water column and DA concentration in blue mussels (Mytilus edulis) at two sites, and king scallops (Pecten maximus) at one site. The rate of DA uptake and depuration differed greatly between the two species with M. edulis whole tissue accumulating and depurating 7 mu g g(-1) (now expressed as mg kg(-1)) per week. In contrast, it took 12 weeks for DA to depurate from P. maximus gonad tissue from a concentration of 68 mu g g(-1) (now mg kg(-1)) to <20 mu g g(-1) (now mg kg(-1)). The DA depuration rate from P. maximus whole tissue was <5% per week during both years of the study. Correlations between the Pseudo-nitzschia cell densities and toxin concentrations were weak to moderate for M. edulis and weak for P. maximus. Seasonal diversity on a species level was observed within the Pseudo-nitzschia genus at both sites with more DA toxicity associated with summer/autumn Pseudo-nitzschia blooms when P. australis was observed in phytoplankton samples. This study reveals the marked difference in DA uptake and depuration in two shellfish species of commercial importance in Scotland. The use of these shellfish species to act as a proxy for DA in the environment still requires investigation. Crown Copyright (C) 2017 Published by Elsevier B.V. All rights reserved.

Proliferations of toxic Alexandrium spp. have adversely affected the shellfish aquaculture industry worldwide. A. ostenfeldii can produce several biotoxins, including the recently characterised fast-acting toxins spirolides (SPX). A dual labelling fluorescent in situ hybridisation (FISH) assay was developed for discriminating simultaneously between the closely related taxa A. ostenfeldii and A. peruvianum. Surveys were undertaken throughout the summers of 2006, 2007 and 2008 in Cork Harbour, Ireland, where a mixed community of Alexandrium spp. develops annually. A. peruvianum was not detected but the presence of A. ostenfeldii was confirmed by FISH and morphological analysis. The species never reached high concentrations (max. similar to 200 cells l(-1)) and contributed on average to only 0.4% of the Alexandrium community, usually dominated by A. minutum and A. tamarense (Group III). Although cell concentrations were several orders of magnitude lower, the dynamics of A. ostenfeldii were similar to those of other Alexandrium spp. during the 3 consecutive summers, suggesting a common response to environmental forcing. Analytical chemistry performed on extracts from passive solid-phase adsorption samplers identified lipophilic toxins dominated by okadaic acid, but also 13-desmethyl SPX C and 20-methyl SPX G, with dynamics generally congruent with those of A. ostenfeldii. The passive samplers enabled the quantification of background toxin levels at very low A. ostenfeldii concentrations, showing potential for forecasting of toxic events. The ability to quantify toxic A. ostenfeldii cells within high density microalgal populations of morphologically similar species makes the dual FISH assay valuable for phytoplankton monitoring programs and future biogeographical and population dynamics studies.

A computationally designed polymer (CDP), based on the functional monomer ethylene glycol methacrylate phosphate (EGMP), with a reported high specific affinity for the neurotoxic paralytic shellfish poisoning (PSP) toxins saxitoxin (STX) and neosaxitoxin (neoSTX) was evaluated with a view to it being used in a solid phase adsorption toxin tracking (SPATT) system for deployment in the marine environment. In addition, a synthetic resin adsorbent (SEPABEADS (R) SP700) which had previously shown to adsorb lipophilic shellfish toxins (LSTs) from seawater was also assessed. Adsorption and desorption of the PSP toxin analogues on and from the CDP polymer and the SP700 resin were investigated. Both adsorbents were contained within dialysis membrane bags and deployed in PSP toxins-spiked seawater and in cultures of Alexandrium tamarense known to be producing PSP toxins. Additionally, some bags holding SP700 resin were also deployed in a mixture of dinoflagellate cultures composed of A. tamarense and Prorocentrum lima, the latter being a diarrheic shellfish toxins (DSP) producer.The polymer and the resin were extracted and analysed for PSP toxins using high performance liquid chromatography (HPLC). In addition, the SP700 resin was also analysed for DSP toxins using liquid chromatography-mass spectrometry (LC-MS).The results indicated that both CDP and SP700 are suitable for the adsorption of a wide range of PSP toxins for a limited period of time (3-7 days). The CDP appears to be more appropriate because it adsorbs higher quantities of PSP toxins, however SP700 adsorbing PSP and DSP toxins over the same range seems to be more useful. PSP toxins can be easily removed from both materials with rinse water or in a toxin-free medium. In summary, either CDP or SP700 could be used as an early warming system for the monitoring of harmful algal blooms (HAB) responsible for PSP poisoning found in European coastal waters. (C) 2011 Elsevier B.V. All rights reserved.

Alexandrium is detected throughout Scottish coastal waters on a near annual basis, and corresponding paralytic shellfish poisoning (PSP) toxins are found in Scottish shellfish. Previous studies at selected Scottish sites have shown diversity within the genus Alexandrium. In order to examine the distribution, diversity and toxicity of Alexandrium populations around the Scottish coast, historic cyst and cell data were compiled and cultures established from sediment and water samples. Historic data showed high cell densities of Alexandrium in Shetland, Orkney, the Western Isles and mainland east coast. Low abundances of Alexandrium cysts were observed along the west coast. Four species of Alexandrium (A. tamarense, A. ostenfeldii, A. minutum and A. tamutum) were established in laboratory culture and identified using morphological criteria. Sequencing of LSU rDNA from isolates of A. ostenfeldii, A. minutum and A. tamutum confirmed their identification and showed them to be similar to other European strains. Alexandrium tamarense, identified by morphological criteria, was observed to have a widespread distribution around the coast. Both toxin- and non toxin-producing strains of this species were isolated, suggesting the presence of A. tamarense Groups I and III. Alexandrium ostenfeldii was isolated from the east coast and Shetland Isles and was observed to produce both spirolide and PSP toxins. Alexandrium tamutum was identified from cultures isolated from Shetland and Orkney, the most northerly observation of this species to date. PSP toxins were not detected in isolates of A. minutum from the east coast and Orkney or of A. tamutum under the culture conditions used. This study has highlighted the diversity of Alexandrium in Scottish waters and reveals the requirement for temporal and regional studies on a species level in order to understand the variation in cell densities and PSP toxicity that is observed on an annual and decadal scale.

Contamination of shellfish with paralytic shellfish poisoning (PSP) toxins produced by Alexandrium species poses a potential threat to the sustainability of the Scottish aquaculture industry. Routine LM analysis of water samples from around the Scottish coast has previously identified Alexandrium (Dinophyceae) as a regular part of the spring and summer phytoplankton communities in Scottish coastal waters. In this study, Alexandrium tamarense (M. Lebour) Balech isolated from sediment and water samples was established in laboratory culture. Species identification of these isolates was confirmed using thecal plate dissections and by molecular characterization based on their LSU and, in some cases, ITS rDNA sequence. Molecular characterization and phylogenetic analysis showed the presence of two ribotypes of A. tamarense: Group I (North American ribotype) and Group III (Western European ribotype). Assessment of PSP toxin production using hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC–MS/MS) showed that A. tamarense Group I produced a complex array of toxins (∼2,000 fg STX equivalents · cell−1) with the major toxins being C2, neosaxitoxin (NEO), saxitoxin (STX), gonyautoxin‐4 (GTX‐4), and GTX‐3, while A. tamarense Group III did not produce toxins. Historically, it was considered that all Alexandrium species occurring in Scottish waters produce potent PSP toxins. This study has highlighted the presence of both PSP toxin‐producing and benign species of A. tamarense and questions the ecological significance of this finding.

The combination of hydrophilic interaction liquid chromatography (HILIC) and liquid chromatography/mass spectrometry (LC/MS) for the determination of paralytic shellfish poisoning (PSP) toxins has been proposed for use in routine monitoring of shellfish. In this study, methods for the detection of multiple PSP toxins [saxitoxin (STX), neosaxitoxin (NEO), decarbamoyl saxitoxin (dcSTX), decarbamoyl neosaxitoxin (dcNEO), gonyautoxins 15 (GTX1, GTX2, GTX3, GTX4, GTX5), decarbamoyl gonyautoxins (dcGTX2 and dcGTX3), and the N-sulfocarbamoyl C toxins (C1 and C2)] were optimized using single (MS) and triple quadrupole (MS/MS) instruments. Chromatographic separation of the toxins was achieved by using a TSK-gel Amide-80 analytical column, although superior chromatography was observed through application of a ZIC-HILIC column. Preparative procedures used to clean up shellfish extracts and concentrate PSP toxins prior to analysis were investigated. The capacity of computationally designed polymeric (CDP) materials and HILIC solid-phase extraction (SPE) cartridges to retain highly polar PSP toxins was explored. Three CDP materials and 2 HILIC cartridges were assessed for the extraction of PSP toxins from aqueous solution. Screening of the CDPs showed that all tested polymers adsorbed PSP toxins. A variety of elution procedures were examined, with dilute 0.01 acetic acid providing optimum recovery from a CDP based on 2-(trifluoromethyl)acrylic acid as the monomer. ZIC-HILIC SPE cartridges were superior to the PolyLC equivalent, with recoveries ranging from 70 to 112 (ZIC-HILIC) and 0 to 90 (PolyLC) depending on the PSP toxin. It is proposed that optimized SPE and HILIC-MS methods can be applied for the quantitative determination of PSP toxins in shellfish.

Species specific LSU rRNA targeted fluorescent oligonucleotide probes, designed by researchers at the Monterey Bay Aquarium Research Institute (USA) for a limited range of Pseudo-nitzschia species, were applied to unialgal cultures and Scottish field samples, to investigate possible applications in Scottish phytoplankton monitoring programmes to detect potential amnesic shellfish poisoning (ASP) toxin producing species. The existing available probe for Pseudo-nitzschia australis gave good results, positively labelling cells from cultures and field samples. However, application of the P. pungens, P. delicatissima and P. fraudulenta probes gave poor results, with little or no fluorescence label observed in field samples, while transmission electron microscopy (TEM) showed these species to be present. Comparison of the same region of the LSU sequence from cultures of P. delicatissima, isolated from Scottish waters, with the probe designed for detection of P. delicatissima isolated from Monterey Bay revealed the presence of a single base difference between the two sequences, which may have prevented the probe from hybridising to Scottish isolates and cells from field samples. In an attempt to assess the potential ASP toxin production by field populations of Pseudo-nitzschia a rapid immunodiagnostic test (the Jellet Rapid Test, JRT) for ASP toxins was examined. Results indicate that additional development of molecular probes for the detection of a range of Pseudo-nitzschia species detected in Scottish coastal waters and the use of JRT for toxin detection could conceivably provide an effective tool for broad-scale mapping of toxin events and management of coastal zone activities.

Some lipophilic shellfish toxins (LSTs) can cause human illness due to eating shellfish that have become naturally contaminated following filter feeding on toxin producing algae. A mouse bioassay (MBA) is widely used to detect LSTs in regulatory monitoring of shellfish. However, the MBA is imprecise giving only a positive or negative result and is prone to interference from other compounds. In this study, liquid chromatography–mass spectrometry (LC–MS) was compared to the MBA, with the aim of substituting the in vivo assay for monitoring shellfish from Scottish waters. Overall, it was not feasible to demonstrate equivalence of LC–MS with the MBA, but due to the detection of a range of LSTs, it is considered that LC–MS methods capable of detecting multiple analogues are accepted by international markets of shellfish to assure consumer protection.

Solid-phase adsorption toxin tracking (SPATT) is founded on the observation that when low levels of toxic dinoflagellates are present in the water column significant amounts of toxins are dissolved in seawater. Previous researchers demonstrated a lag between detection of dissolved toxins adsorbed onto porous synthetic resin, phytoplankton peak cell densities and highest toxin concentrations in shellfish. Here we report on optimised protocols (resin type, type and volume of solvent extraction) for SPATT and subsequent field trials at Loch Ewe (Scotland) which used SPATT in the form of suspended SEPABEADSreg SP700 held within a mesh sachet. Sachets were suspended at 7 m and retrieved weekly with new ones put in and water samples for phytoplankton collected. Phytoplankton cells were identified using conventional light microscopy. The resin from the mesh bags was extracted and analysed for lipophilic toxins using liquid chromatography with mass spectrometry (LC-MS). Results demonstrate the potential of SPATT to provide time integrated sampling to monitor the occurrence of toxic events.

A study was conducted to aid the interpretation of data generated by parallel testing of the qualitative Jellett Rapid Test (JRT) and the mouse bioassay (MBA) for detection of paralytic shellfish poisoning (PSP) toxins within the UK statutory shellfish biotoxin monitoring programme. A selection of stored sample extracts subjected to testing by MBA and/or JRT were further analysed by liquid chromatography with fluorescence detection (LC-FLD) to provide additional information on the concentrations of PSP toxins and toxin profiles.Results, from this study, demonstrate the potential of the JRT to effectively screen out PSP toxin negative shellfish samples and samples containing low concentrations of toxins from UK monitoring programmes. Additionally, data generated using LC-FLD highlights the potential of introducing alternative analytical techniques to completely replace the requirement for the MBA. Crown Copyright (C) 2007 Published by Elsevier B.V. All rights reserved.

Shellfish samples were collected from coastal and offshore aquaculture sites and harvesting areas in Scottish waters between March 2003 and September 2004. Samples were analysed for the presence of algal toxins using traditional mouse bioassays for the detection of paralytic shellfish poisoning (PSP) toxins and diarrhetic shellfish poisoning (DSP) toxins; immuno-lateral flow chromatography for the detection of PSP toxins in the form of the Jellett Rapid Test; high-performance liquid chromatography (HPLC) with UV diode-array for the detection of amnesic shellfish poisoning (ASP) toxins; and liquid chromatography with mass spectrometry (LC–MS) for the detection of multiple lipophilic shellfish toxins (LSTs) including pectenotoxins (PTXs), yessotoxins (YTXs), azaspiracids (AZAs) and toxins from the ‘traditional’ DSP toxin group, okadaic acid (OA) and dinophysistoxins (DTXs). In order to investigate the presence of OA esters, alkaline hydrolysis was performed. All toxin groups were detected with a geographically widespread distribution. ASP toxins were the most prevalent occurring in 69% of samples. Using the PSP mouse bioassay, PSP toxins were detected in 5% of shellfish samples from coastal waters around the islands and the east coast. The Jellett Rapid Test for PSP toxins revealed a wider distribution (24% of samples) including the west coast of Scotland. Toxins from the ‘traditional’ DSP toxin group (OA/DTXs) and/or other LST groups (PTXs, YTXs and AZAs) were detected by LC–MS in 63% of the shellfish analysed. PSP, ASP toxins and LSTs occurred concurrently in a limited sample set, highlighting the importance of using methods capable of detecting multiple algal toxin groups in Scottish shellfish monitoring programmes.