Wendy Higman

First evidence of an extensive northern European distribution of azaspiracid poisoning (AZP) toxins in shellfish.

Azaspiracids have recently been identified as the toxins responsible for a series of human intoxications in Europe since 1995, following the consumption of cultured mussels (Mytilus edulis) from the west coast of Ireland. Liquid chromatography-mass spectrometric (LC-MS) methods have been applied in the study reported here to investigate the new human toxic syndrome, azaspiracid poisoning. Separation of azaspiracid (AZA1) and its analogues, 8-methylazaspiracid (AZA2) and 22-demethylazaspiracid (AZA3), was achieved using reversed-phase LC and coupled, via an electrospray ionisation source, to an ion-trap mass spectrometer. These azaspiracids have now been identified in mussels from Craster (north-east England) and Sognefjord (south-west Norway) using source collision induced dissociation-MS and multiple tandem MS detection. AZA1 was the predominant toxin and toxin profiles were similar to those found in contaminated Irish shellfish. This is the first report of the occurrence of these azaspiracids outside Ireland with the significant implications that these toxins may occur in shellfish throughout northern Europe.

Comparison of AOAC 2005.06 LC official method with other methodologies for the quantitation of paralytic shellfish poisoning toxins in UK shellfish species

AbstractA refined version of the pre-column oxidation liquid chromatography with fluorescence detection (ox-LC-FLD) official method AOAC 2005.06 was developed in the UK and validated for the determination of paralytic shellfish poisoning toxins in UK shellfish. Analysis was undertaken here for the comparison of PSP toxicities determined using the LC method for a range of UK bivalve shellfish species against the official European reference method, the PSP mouse bioassay (MBA, AOAC 959.08). Comparative results indicated a good correlation in results for some species (mussels, cockles and clams) but a poor correlation for two species of oysters (Pacific oysters and native oysters), where the LC results in terms of total saxitoxin equivalents were found to be on average more than double the values determined by MBA. With the potential for either LC over-estimation or MBA under-estimation, additional oyster and mussel samples were analysed using MBA and ox-LC-FLD together with further analytical and functional methodologies: a post-column oxidation LC method (LC-ox-FLD), an electrophysiological assay and hydrophilic interaction liquid chromatography with tandem mass spectrometric detection. Results highlighted a good correlation among non-bioassay results, indicating a likely cause of difference was the under-estimation in the MBA, rather than an over-estimation in the LC results. FigureTotal saxitoxin equivalents in oysters (Pacific and native) quantified by ox-LC-FLD, LC-ox-FLD, HPLC-MS/MS and electrophysiological assay as compared with the MBA PSP toxicity reference method

Potential Threats Posed by Tetrodotoxins in UK Waters: Examination of Detection Methodology Used in Their Control

Tetrodotoxin is a neurotoxin responsible for many human fatalities, most commonly following the consumption of pufferfish. Whilst the source of the toxin has not been conclusively proven, it is thought to be associated with various species of marine bacteria. Whilst the toxins are well studied in fish and gastropods, in recent years, there have been a number of reports of tetrodotoxin occurring in bivalve shellfish, including those harvested from the UK and other parts of Europe. This paper reviews evidence concerning the prevalence of tetrodotoxins in the UK together with methodologies currently available for testing. Biological, biomolecular and chemical methods are reviewed, including recommendations for further work. With the recent development of quantitative chromatographic methods for these and other hydrophilic toxins, as well as the commercial availability of rapid testing kits, there are a number of options available to ensure consumers are protected against this threat.

A feasibility study into the provision of Paralytic Shellfish Toxins laboratory reference materials by mass culture of Alexandrium and shellfish feeding experiments

As the official control laboratory for biotoxin testing in England, Wales and Scotland, Cefas employs two approaches for the detection of Paralytic Shellfish Poisons (PSP) in bivalve shellfish: a qualitative HPLC method for oysters, whole king scallops and cockles (with PSP bioassay confirmation of positive HPLC samples) with subsequent quantitation of positive samples by mouse bioassay and a quantitative HPLC method for mussels (no PSP bioassay confirmation required). To aid the validation of the quantitative HPLC method for native oysters, Pacific oysters, cockles and king scallops and ultimately remove the need for the PSP bioassay for these species, appropriate contaminated shellfish matrices were required. As it was not possible to obtain naturally contaminated material for these species, shellfish were contaminated in-house through feeding experiments with high concentrations of Alexandrium species. A number of feeding experiments with two Alexandrium strains were performed successfully. The contaminated shellfish materials generated contained a number of different profiles of PSP toxins. This work has demonstrated the feasibility of these methods for the production of laboratory reference materials in a variety of bivalve shellfish species. Based on this study laboratory reference material production via these methods is now undertaken routinely within Cefas. By running two concurrent feeding trials per year for each species, enough laboratory reference material is produced for approximately 1 year of the programme. This removes the necessity for natural contaminated material which is not always available for reference material production. Additionally, such materials enable both the comparative testing of different PSP methodologies and the ongoing generation of long-term precision data for the HPLC method.

Potential Threats Posed by New or Emerging Marine Biotoxins in UK Waters and Examination of Detection Methodology Used in Their Control: Brevetoxins

Regular occurrence of brevetoxin-producing toxic phytoplankton in commercial shellfishery areas poses a significant risk to shellfish consumer health. Brevetoxins and their causative toxic phytoplankton are more limited in their global distribution than most marine toxins impacting commercial shellfisheries. On the other hand, trends in climate change could conceivably lead to increased risk posed by these toxins in UK waters. A request was made by UK food safety authorities to examine these toxins more closely to aid possible management strategies, should they pose a threat in the future. At the time of writing, brevetoxins have been detected in the Gulf of Mexico, the Southeast US coast and in New Zealand waters, where regulatory levels for brevetoxins in shellfish have existed for some time. This paper reviews evidence concerning the prevalence of brevetoxins and brevetoxin-producing phytoplankton in the UK, together with testing methodologies. Chemical, biological and biomolecular methods are reviewed, including recommendations for further work to enable effective testing. Although the focus here is on the UK, from a strategic standpoint many of the topics discussed will also be of interest in other parts of the world since new and emerging marine biotoxins are of global concern.

Testing and application of a refined rapid detection method for paralytic shellfish poisoning toxins in UK shellfish.

The Scotia Rapid Test for PSP is designed for qualitative identification of saxitoxins at levels in shellfish equivalent to the limit of detection of the biological reference method. However, issues with the method have been reported, including the low assay cross reactivity for some toxins, high numbers of false positive results and the subjective test interpretation. This study focussed on approaches to improve each of these issues. A refined test was found to improve GTX1&4 test sensitivity in samples containing high proportions of GTX1&4. The subjectivity of the test was successfully eliminated through use of an automated scanner, which enabled both the reliable identification of test results as well as the provision of a numerical result which could be utilised for more refined results interpretation. Finally the high proportion of false positive results in comparison with the LC-FLD was investigated, with a modified approach incorporating an additional extract dilution applied to a range of shellfish samples with different toxicities. Results showed highly variable limits of detection of the method and no significant reduction in false positive results when applying the additional dilution, which may be of concern to laboratories in receipt of high numbers of samples containing low concentrations of toxins.

Potential Threats Posed by New or Emerging Marine Biotoxins in UK Waters and Examination of Detection Methodologies Used for Their Control: Cyclic Imines

Cyclic imines (CIs) are a group of phytoplankton produced toxins related to shellfish food products, some of which are already present in UK and European waters. Their risk to shellfish consumers is poorly understood, as while no human intoxication has been definitively related to this group, their fast acting toxicity following intraperitoneal injection in mice has led to concern over their human health implications. A request was therefore made by UK food safety authorities to examine these toxins more closely to aid possible management strategies. Of the CI producers only the spirolide producer Alexandrium ostenfeldii is known to exist in UK waters at present but trends in climate change may lead to increased risk from other organisms/CI toxins currently present elsewhere in Europe and in similar environments worldwide. This paper reviews evidence concerning the prevalence of CIs and CI-producing phytoplankton, together with testing methodologies. Chemical, biological and biomolecular methods are reviewed, including recommendations for further work to enable effective testing. Although the focus here is on the UK, from a strategic standpoint many of the topics discussed will also be of interest in other parts of the world since new and emerging marine biotoxins are of global concern.