Sea temperature influences accumulation of tetrodotoxin in British bivalve shellfish.

Tetrodotoxin (TTX), a potent neurotoxin mostly associated with pufferfish poisoning, is also found in bivalve shellfish. Recent studies into this emerging food safety threat reported TTX in a few, mainly estuarine, shellfish production areas in some European countries, including the United Kingdom. A pattern in occurrences has started to emerge, however the role of temperature on TTX has not been investigated in detail. Therefore, we conducted a large systematic TTX screening study, encompassing over 3500 bivalve samples collected throughout 2016 from 155 shellfish monitoring sites along the coast of Great Britain. Overall, we found that only 1.1 % of tested samples contained TTX above the reporting limit of 2 µg/kg whole shellfish flesh and these samples all originated from ten shellfish production sites in southern England. Subsequent continuous monitoring of selected areas over a five-year period showed a potential seasonal TTX accumulation in bivalves, starting in June when water temperatures reached around 15 °C. For the first time, satellite-derived data were also applied to investigate temperature differences between sites with and without confirmed presence of TTX in 2016. Although average annual temperatures were similar in both groups, daily mean values were higher in summer and lower in winter at sites where TTX was found. Here, temperature also increased significantly faster during late spring and early summer, the critical period for TTX. Our study supports the hypothesis that temperature is one of the key triggers of events leading to TTX accumulation in European bivalves. However, other factors are also likely to play an important role, including the presence or absence of a de novo biological source, which remains elusive.

Interlaboratory Evaluation of Multiple LC-MS/MS Methods and a Commercial ELISA Method for Determination of Tetrodotoxin in Oysters and Mussels.

BACKGROUND: Given the recent detection of tetrodotoxin (TTX) in bivalve molluscs but the absence of a full collaborative validation study for TTX determination in a large number of shellfish samples, interlaboratory assessment of method performance was required to better understand current capabilities for accurate and reproducible TTX quantitation using chemical and immunoassay methods. OBJECTIVE: The aim was to conduct an interlaboratory study with multiple laboratories, using results to assess method performance and acceptability of different TTX testing methods. METHODS: Homogenous and stable mussel and oyster materials were assessed by participants using a range of published and in-house detection methods to determine mean TTX concentrations. Data were used to calculate recoveries, repeatability, and reproducibility, together with participant acceptability z-scores. RESULTS: Method performance characteristics were good, showing excellent sensitivity, recovery, and repeatability. Acceptable reproducibility was evidenced by HorRat values for all LC-MS/MS and ELISA methods being less than the 2.0 limit of acceptability. Method differences between the LC-MS/MS participants did not result in statistically different results. Method performance characteristics compared well with previously published single-laboratory validated methods and no statistical difference was found in results returned by ELISA in comparison with LC-MS/MS. CONCLUSION: The results from this study demonstrate that current LC-MS/MS methods and ELISA are on the whole capable of sensitive, accurate, and reproducible TTX quantitation in shellfish. Further work is recommended to expand the number of laboratories testing ELISA and to standardize an LC-MS/MS protocol to further improve interlaboratory precision. HIGHLIGHTS: Multiple mass spectrometric methods and a commercial ELISA have been successfully assessed through an interlaboratory study, demonstrating excellent performance.

Diversity and regional distribution of harmful algal events along the Atlantic margin of Europe.

The IOC-ICES-PICES Harmful Algal Event Database (HAEDAT) was used to describe the diversity and spatiotemporal distribution of harmful algal events along the Atlantic margin of Europe from 1987 - 2018. The majority of events recorded are caused by Diarrhetic Shellfish Toxins (DSTs). These events are recorded annually over a wide geographic area from southern Spain to northern Scotland and Iceland, and are responsible for annual closures of many shellfish harvesting areas. The dominant causative dinoflagellates, members of the morphospecies 'Dinophysis acuminata complex' and D. acuta, are common in the waters of the majority of countries affected. There are regional differences in the causative species associated with PST events; the coasts of Spain and Portugal with the dinoflagellates Alexandrium minutum and Gymnodinium catenatum, north west France/south west England/south Ireland with A. minutum, and Scotland/Faroe Islands/Iceland with A. catenella. This can influence the duration and spatial scale of PST events as well as the toxicity of shellfish. The diatom Pseudo-nitzschia australis is the most widespread Domoic Acid (DA) producer, with records coming from Spain, Portugal, France, Ireland and the UK. Amnesic Shellfish Toxins (ASTs) have caused prolonged closures for the scallop fishing industry due to the slow depuration rate of DA. Amendments to EU shellfish hygiene regulations introduced between 2002 and 2005 facilitated end-product testing and sale of adductor muscle. This reduced the impact of ASTs on the scallop fishing industry and thus the number of recorded HAEDAT events. Azaspiracids (AZAs) are the most recent toxin group responsible for events to be characterised in the ICES area. Events associated with AZAs have a discrete distribution with the majority recorded along the west coast of Ireland. Ciguatera Poisoning (CP) has been an emerging issue in the Canary Islands and Madeira since 2004. The majority of aquaculture and wild fish mortality events are associated with blooms of the dinoflagellate Karenia mikimotoi and raphidophyte Heterosigma akashiwo. Such fish killing events occur infrequently yet can cause significant mortalities. Interannual variability was observed in the annual number of HAEDAT areas with events associated with individual shellfish toxin groups. HABs represent a continued risk for the aquaculture industry along the Atlantic margin of Europe and should be accounted for when considering expansion of the industry or operational shifts to offshore areas.

Distribution of Tetrodotoxin in Pacific Oysters (Crassostrea gigas).

A potent and heat-stable tetrodotoxin (TTX) has been found to accumulate in various marine bivalve species, including Pacific oysters (Crassostrea gigas), raising a food safety concern. While several studies on geographical occurrence of TTX have been conducted, there is a lack of knowledge about the distribution of the toxin within and between bivalves. We, therefore, measured TTX in the whole flesh, mantle, gills, labial palps, digestive gland, adductor muscle and intravalvular fluid of C. gigas using liquid chromatography-tandem mass spectrometry. Weekly monitoring during summer months revealed the highest TTX concentrations in the digestive gland (up to 242 µg/kg), significantly higher than in other oyster tissues. Intra-population variability of TTX, measured in the whole flesh of each of twenty animals, reached 46% and 32% in the two separate batches, respectively. In addition, an inter-population study was conducted to compare TTX levels at four locations within the oyster production area. TTX concentrations in the whole flesh varied significantly between some of these locations, which was unexplained by the differences in weight of flesh. This is the first study examining TTX distribution in C. gigas and the first confirmation of the preferential accumulation of TTX in oyster digestive gland.

Isolation and characterization of potentially pathogenic Vibrio species in a temperate, higher latitude hotspot.

The recent emergence of Vibrio infections at high latitudes represents a clear human health risk attributable to climate change. Here, we investigate the population dynamics of three Vibrio species: Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio cholerae within a British coastal estuarine site, with contrasting salinity and temperature regimes during an intense heatwave event. Water samples were collected weekly through the summer of 2018 and 2019 and filtered using membrane filtration and subsequently grown on selective media. Suspected vibrios were confirmed using a conventional species-specific PCR assay and further analysed for potential pathogenic markers. Results showed that Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio cholerae were present at high concentrations throughout both years, with their populations at substantially greater abundances corresponding to conditions of higher water temperatures during the heatwave of 2018 and at lower salinity sites, which is comparable to the results of previous studies. A subset of strains isolated during the extreme heatwave event in 2018 (46 Vibrio parahaemolyticus, 11 Vibrio cholerae and 4 Vibrio vulnificus) were genomically sequenced. Analysis of these 63 sequenced strains revealed a broad phenotypic and genomic diversity of strains circulating in the environment. An analysis of pathogenicity attributes identified a broad array of virulence genes across all three species, including a variety of genes associated with human disease. This study highlights the importance of the need for an increased Vibrio spp. surveillance system in temperate regions and the potential impact warming events such as heatwaves may have on the abundance of potentially pathogenic bacteria in the environment.

Ultrahigh-Performance Hydrophilic Interaction Liquid Chromatography with Tandem Mass Spectrometry Method for the Determination of Paralytic Shellfish Toxins and Tetrodotoxin in Mussels, Oysters, Clams, Cockles, and Scallops: Collaborative Study.

BACKGROUND: An ultrahigh-performance LC (UHPLC)-tandem MS (MS/MS) method for determination of paralytic shellfish poisoning toxins and tetrodotoxin (TTX) in bivalve molluscs was developed. To be used for regulatory testing, it needed to be validated through collaborative study. OBJECTIVE: The aim was to conduct a collaborative study with 21 laboratories, using results to assess method performance. METHODS: Study materials incorporated shellfish species mussels, oysters, cockles, scallops, and clams and were assessed to demonstrate stability and homogeneity. Mean concentrations determined by participants for blind duplicate samples were used to assess reproducibility, repeatability, and trueness. RESULTS: Method performance characteristics were excellent following statistical assessment of participant data, with method trueness showing excellent method accuracy against expected values. No significant difference was found in the trueness results determined by different chromatographic column types. Acceptability of the between-laboratory reproducibility for individual analytes was evidenced by >99% of valid Horwitz ratio values being less than the 2.0 limit of acceptability. With excellent linearity and sensitivity fit-for-purpose over a range of mass spectrometer instruments, the UHPLC-MS/MS method compared well against other detection methods. It includes additional paralytic shellfish toxin (PST) analogues as well as TTX, which, to date, have not been incorporated into any other hydrophilic marine toxin official method of analysis. CONCLUSIONS: The results from this study demonstrate that the method is suitable for the analysis of PST analogues and TTX in shellfish tissues and is recommended as an official alternative method of analysis for regulatory control. HIGHLIGHTS: A new mass spectrometric method for PST and TTX has been validated successfully through collaborative study.

Variability and profiles of lipophilic toxins in bivalves from Great Britain during five and a half years of monitoring: azaspiracids and yessotoxins.

Cefas has been responsible for the delivery of official control biotoxin testing of bivalve molluscs from Great Britain for just over a decade. Liquid chromatography tandem mass spectrometric (LC-MS/MS) methodology has been used for the quantitation of lipophilic toxins (LTs) since 2011. The temporal and spatial distribution of okadaic acid group toxins and profiles in bivalves between 2011 and 2016 have been recently reported. Here we present data on the two other groups of regulated lipophilic toxins, azaspiracids (AZAs) and yessotoxins (YTXs), over the same period. The latter group has also been investigated for a potential link with Protoceratium reticulatum and Lingulodinium polyedra, both previously recognised as YTXs producing phytoplankton. On average, AZAs were quantified in 3.2% of all tested samples but notable inter-annual variation in abundance was observed. The majority of all AZA contaminated samples were found between July 2011 and August 2013 in Scotland, while only two, three-month long, AZA events were observed in 2015 and 2016 in the south-west of England. Maximum concentrations were generally reached in late summer or early autumn. Reasons for AZAs persistence during the 2011/2012 and 2012/2013 winters are discussed. Only one toxin profile was identified, represented by both AZA1 and AZA2 toxins at an approximate ratio of 2 : 1, suggesting a single microalgal species was the source of AZAs in British bivalves. Although AZA1 was always the most dominant toxin, its proportion varied between mussels, Pacific oysters and surf clams. The YTXs were the least represented group among regulated LTs. YTXs were found almost exclusively on the south-west coast of Scotland, with the exception of 2013, when the majority of contaminated samples originated from the Shetland Islands. The highest levels were recorded in the summer months and followed a spike in Protoceratium reticulatum cell densities. YTX was the most dominant toxin in shellfish, further strengthening the link to P. reticulatum as the YTX source. Neither homo-YTX, nor 45-OH homo-YTX were detected throughout the monitored period. 45-OH YTX, thought to be a shellfish metabolite associated with YTX elimination, contributed on average 26% in mussels. Although the correlation between 45-OH YTX abundance and the speed of YTX depuration could not be confirmed, we noted the half-life of YTX was more than two-times longer in queen scallops, which contained 100% YTX, than in mussels. No other bivalve species were affected by YTXs. This is the first detailed evaluation of AZAs and YTXs occurrences and their profiles in shellfish from Great Britain over a period of multiple years.

New Invasive Nemertean Species (Cephalothrix Simula) in England with High Levels of Tetrodotoxin and a Microbiome Linked to Toxin Metabolism.

The marine nemertean Cephalothrix simula originates from the Pacific Ocean but in recent years has been discovered in northern Europe. The species has been associated with high levels of the marine neurotoxin Tetrodotoxin, traditionally associated with Pufferfish Poisoning. This study reports the first discovery of two organisms of C. simula in the UK, showing the geographical extent of this species is wider than originally described. Species identification was initially conducted morphologically, with confirmation by Cox 1 DNA sequencing. 16S gene sequencing enabled the taxonomic assignment of the microbiome, showing the prevalence of a large number of bacterial genera previously associated with TTX production including Alteromonas, Vibrio and Pseudomonas. LC-MS/MS analysis of the nemertean tissue revealed the presence of multiple analogues of TTX, dominated by the parent TTX, with a total toxin concentration quantified at 54 µg TTX per g of tissue. Pseudomonas luteola isolated from C. simula, together with Vibrio alginolyticus from the native nemertean Tubulanus annulatus, were cultured at low temperature and both found to contain TTX. Overall, this paper confirms the high toxicity of a newly discovered invasive nemertean species with links to toxin-producing marine bacteria and the potential risk to human safety. Further work is required to assess the geographical extent and toxicity range of C. simula along the UK coast in order to properly gauge the potential impacts on the environment and human safety.

Variability and profiles of lipophilic toxins in bivalves from Great Britain during five and a half years of monitoring: Okadaic acid, dinophysis toxins and pectenotoxins.

Official control biotoxin testing of bivalve molluscs from Great Britain has been conducted by Cefas for over a decade. Reflecting the changes in legislation, bioassays were gradually replaced by analytical methods, firstly for analysis of Paralytic shellfish toxins, followed by introduction of liquid chromatography tandem mass spectrometric (LCMS/MS) method for lipophilic toxins (LTs) in 2011. Twelve compounds, representing three main groups of regulated lipophilic toxins, as well as two non-regulated cyclic imines were examined in over 20,500 samples collected between July 2011 and December 2016. The toxins belonging to Okadaic acid (OA) group toxins were the most prevalent and were quantified in 23% of samples, predominantly from Scotland. The temporal pattern of OA group occurrences remained similar each year, peaking in summer months and tailing off during autumn and winter, however their abundance and magnitude varied between years significantly, with concentrations reaching up to 4993 µg OA eq./kg. Three toxin profiles were identified, reflecting the relative contribution of the two main toxins, OA and dinophysis toxin-2 (DTX2). Dinophysis toxin-1 (DTX1) was less common and was never detected in samples with high proportions of DTX2. Inter-annual changes in profiles were observed within certain regions, with the most notable being an increase of DTX2 occurrences in north-west Scotland and England in the last three years of monitoring. In addition, seasonal changes of profiles were identified when OA, the dominant toxin in early summer, was replaced by higher proportions of DTX2 in late summer and autumn. The profile distribution possibly reflected the availability of individual Dinophysis species as a food source for shellfish, however persistence of DTX2 during autumn and winter in mussels might have also been attributed to their physiology. Mussels were the only species with higher average proportions of non-esterified toxins, while Pacific oysters, cockles, surf clams, razors and queen scallops contained almost exclusively ester forms. In addition, a temporal change in proportion of OA and DTX2 free form was observed in mussels. Pectenotoxin-2 (PTX2) was quantified only on rare occasions.

Fatal Canine Intoxications Linked to the Presence of Saxitoxins in Stranded Marine Organisms Following Winter Storm Activity.

At the start of 2018, multiple incidents of dog illnesses were reported following consumption of marine species washed up onto the beaches of eastern England after winter storms. Over a two-week period, nine confirmed illnesses including two canine deaths were recorded. Symptoms in the affected dogs included sickness, loss of motor control, and muscle paralysis. Samples of flatfish, starfish, and crab from the beaches in the affected areas were analysed for a suite of naturally occurring marine neurotoxins of dinoflagellate origin. Toxins causing paralytic shellfish poisoning (PSP) were detected and quantified using two independent chemical testing methods in samples of all three marine types, with concentrations over 14,000 µg saxitoxin (STX) eq/kg found in one starfish sample. Further evidence for PSP intoxication of the dogs was obtained with the positive identification of PSP toxins in a vomited crab sample from one deceased dog and in gastrointestinal samples collected post mortem from a second affected dog. Together, this is the first report providing evidence of starfish being implicated in a PSP intoxication case and the first report of PSP in canines.

Analysis of Microcystins in Cyanobacterial Blooms from Freshwater Bodies in England.

Cyanobacterial blooms in freshwater bodies in England are currently monitored reactively, with samples containing more than 20,000 cells/mL of potentially toxin-producing species by light microscopy resulting in action by the water body owner. Whilst significantly reducing the risk of microcystin exposure, there is little data describing the levels of these toxins present in cyanobacterial blooms. This study focused on the quantitative LC-MS/MS analysis of microcystins in freshwater samples, collected across England during 2016 and found to contain potentially toxin-producing cyanobacteria. More than 50% of samples contained quantifiable concentrations of microcystins, with approximately 13% exceeding the WHO medium health threshold of 20 µg/L. Toxic samples were confirmed over a nine-month period, with a clear increase in toxins during late summer, but with no apparent geographical patterns. No statistical relationships were found between total toxin concentrations and environmental parameters. Complex toxin profiles were determined and profile clusters were unrelated to cyanobacterial species, although a dominance of MC-RR was determined in water samples from sites associated with lower rainfall. 100% of samples with toxins above the 20 µg/L limit contained cell densities above 20,000 cells/mL or cyanobacterial scum, showing the current regime is suitable for public health. Conversely, with only 18% of cell density threshold samples having total microcystins above 20 µg/L, there is the potential for reactive water closures to unnecessarily impact upon the socio-economics of the local population. In the future, routine analysis of bloom samples by LC-MS/MS would provide a beneficial confirmatory approach to the current microscopic assessment, aiding both public health and the needs of water users and industry.

Assessing the presence of marine toxins in bivalve molluscs from southwest India.

The south west coast of India has been showing a steady increase in shellfish cultivation both for local consumption and fishery export, over recent years. Perna viridis and Crassostrea madrasensis are two species of bivalve molluscs which grow in some selected regions of southern Karnataka, close to the city of Mangalore. In the early 1980s, shellfish consumers in the region were affected by intoxication from Paralytic Shellfish Poison present in local bivalves (clams and oysters) resulting in hospitalisation of many, including one fatality. Since then, there have been no further reports of serious shellfish intoxication and there is little awareness of the risks from natural toxins and no routine monitoring programme in place to protect shellfish consumers. This study presents the findings from the first ever systematic assessment of the presence of marine toxins in mussels and oysters grown in four different shellfish harvesting areas in the region. Shellfish were collected and subjected to analysis for ASP, PSP and lipophilic toxins, as well as a suite of non-EU regulated toxins such as tetrodotoxin and selected cyclic imines. Results revealed the presence of low levels of PSP toxins in oysters throughout the study period. Overall, total toxicities reached a maximum of 10% of the EU regulatory limit of 800 µg STX eq/kg. Toxin profiles were similar to those reported from the 1980 outbreak. No evidence was found for significant levels of ASP and lipophilic toxins, although some cyclic imines were detected, including gymnodimine. The results indicated that the risk to shellfish consumers during this specific study period would have been low. However, with historical evidence for extremely high levels of PSP toxins in molluscs, there is a strong need for routine surveillance of shellfish production areas for marine toxins, in order to mitigate against human health impacts resulting from unexpected harmful algal blooms, with potentially devastating socio-economic consequences.

Detection of Tetrodotoxin Shellfish Poisoning (TSP) Toxins and Causative Factors in Bivalve Molluscs from the UK.

Tetrodotoxins (TTXs) are traditionally associated with the occurrence of tropical Pufferfish Poisoning. In recent years, however, TTXs have been identified in European bivalve mollusc shellfish, resulting in the need to assess prevalence and risk to shellfish consumers. Following the previous identification of TTXs in shellfish from southern England, this study was designed to assess the wider prevalence of TTXs in shellfish from around the coast of the UK. Samples were collected between 2014 and 2016 and subjected to analysis using HILIC-MS/MS. Results showed the continued presence of toxins in shellfish harvested along the coast of southern England, with the maximum concentration of total TTXs reaching 253 µg/kg. TTX accumulation was detected in Pacific oysters (Crassostreagigas), native oysters (Ostreaedulis) common mussels (Mytilusedulis) and hard clams (Mercenariamercenaria), but not found in cockles (Cerastodermaedule), razors (Ensis species) or scallops (Pectenmaximus). Whilst the highest concentrations were quantified in samples harvested during the warmer summer months, TTXs were still evident during the winter. An assessment of the potential causative factors did not reveal any links with the phytoplankton species Prorocentrumcordatum, instead highlighting a greater level of risk in areas of shallow, estuarine waters with temperatures above 15 °C.

Assessment of a semiquantitative liquid chromatography-fluorescence detection method for the determination of paralytic shellfish poisoning toxin levels in bivalve molluscs from Great Britain.

AOAC Official Method 2005.06 precolumn oxidation LC-fluorescence detection method has been used for many years for the detection and quantitation of paralytic shellfish poisoning (PSP) toxins in bivalve molluscs. After extensive single- and multiple-laboratory validation, the method has been slowly gaining acceptance worldwide as a useful and practical tool for official control testing. In Great Britain, the method has become routine since 2008, with no requirement since then for reverting back to the bioassay reference method. Although the method has been refined to be semiautomated, faster, and more reproducible, the quantitation step can be complex and time-consuming. An alternative approach was developed to utilize the qualitative screening results for generating a semiquantitative results assessment. Data obtained over 5 years enabled the comparison of semiquantitative and fully quantitative PSP results in over 15 000 shellfish samples comprising eight different species showed that the semiquantitative approach resulted in over-estimated paralytic shellfish toxin levels by an average factor close to two in comparison with the fully quantified levels. No temporal trends were observed in the data or relating to species type, with the exception of surf clams. The comparison suggested a semiquantitative threshold of 800 microg saxitoxin (STX) eq/kg should provide a safe limit for the determination of samples to be forwarded to full quantitation. However, the decision was taken to halve this limit to include an additional safety factor of 2, resulting in the use of a semiquantitative threshold of 400 microg STX eq/kg. Implementation of the semiquantitative method into routine testing would result in a significant reduction in the numbers of samples requiring quantitation and have a positive impact on the overall turnaround of reported PSP results. The refined method would be appropriate for any monitoring laboratory faced with high throughput requirements.

Variability of paralytic shellfish toxin occurrence and profiles in bivalve molluscs from Great Britain from official control monitoring as determined by pre-column oxidation liquid chromatography and implications for applying immunochemical tests.

As the official control monitoring laboratory in Great Britain for the analysis of marine biotoxins in shellfish, Cefas have for the past five years conducted routine monitoring for paralytic shellfish poisoning toxins (PST) using a non-animal alternative method to the mouse bioassay reference method; a refined version of the AOAC 2005.06 pre-column oxidation liquid chromatography method. Application of this instrumental methodology has enabled the generation of data not only on the occurrence and magnitude of PST events, but also the quantitation and assessment of different PST profiles. Since implementation of the method in 2008, results have shown huge variabilities in the occurrence of PSTs, with large spatial and temporal variabilities around the coastline. Mean PST profiles were not found to correlate either with total PST content of the shellfish, the year of sampling or with a few notable exceptions, the shellfish species. Toxin profiles were found to fall into one of four distinct profile types, with one relating solely to the exclusive presence of decarbamoyl toxins in surf clams. The other profile types contained variable proportions of gonyautoxins, N-sulfocarbamoyl toxins, neosaxitoxin and saxitoxin. While some indications of geographical repeatability were noted, this was not observed for all profile types. Consequently, the application of rapid immunochemical testing methods to end product testing would need to be considered carefully given the large differences in PST congener cross-reactivities.

Investigations into matrix components affecting the performance of the official bioassay reference method for quantitation of paralytic shellfish poisoning toxins in oysters.

Significant differences previously observed in the determination of paralytic shellfish poisoning toxins (PSTs) in oysters using official method AOAC 2005.06 and 959.08 were investigated in detail with regard to possible matrix effects. Method AOAC 2005.06 gave results 2-3 times higher than the mouse bioassay method, 959.08, differences thought to be due to underestimation of PSTs by the mouse bioassay. In order to prove the cause of these large differences, work was conducted here to examine the presence and effects of matrix components on the performance of each of the two assays. A range of oyster, cockle and mussel samples were extracted using the AOAC 959.08 hydrochloric acid (HCl) extraction method and analysed for PSP by both MBA and LC-FLD. In addition, extracts were analysed by Inductively Coupled Plasma Mass Spectrometry (ICP-MS) for metals as well as being subjected to a range of nutritional testing methods. Whilst there was no evidence for effect of nutritional components on either assay, ICP-MS analysis revealed a relationship between samples exhibiting the largest differences in relative method performance, specifically those with the largest LC-FLD/MBA toxicity ratio, and samples containing the highest concentrations of zinc and manganese. In order to prove the potential effect of the metals on either the LC-FLD and/or MBA assays, HCl extracts of a range of shellfish were subjected to a number of matrix modifications. Firstly, a number of PSP-positive oyster samples were processed to reduce the concentrations of metals within the extracts, without significantly reducing the concentrations of PSTs. Secondly, a range of mussel and cockle extracts, plus a standard solution of saxitoxin di-hydrochloride were spiked at variable concentrations of zinc. All treated and non-treated extracts, plus a number of controls were subjected to ICP-MS, LC-FLD and MBA testing. Results proved the absence of any effect of metals on the performance of the LC-FLD, whilst showing a large suppressive effect of the metals on the MBA. As such, the results show the performance of the official MBA is potentially unsafe for application to the routine monitoring of PSP toxicity in oysters or in any other shellfish found to contain high concentrations of metal ions.