• Amnesic shellfish poisoning in the king scallop, Pecten maximus, from the west coast of Scotland

      Campbell, D.A.; Kelly, M.S.; Busman, M.; Bolch, C.J.S.; Wiggins, E.; Moeller, P.D.R.; Morton, S.L.; Hess, P.; Shumway, S.E. (National Shellfisheries Association, 2001)
      The king scallop, Pecten maximus, is a valuable economic resource in the UK. The industry relies on supplying premium "roe-on" processed scallops to the continental market. In July 1999, king scallops harboring the amnesic shellfish poisoning (ASP) toxin, domnic acid (DA), in gonadal tissue at levels above the regulatory limit (20 μg DA g-1) were detected across a wide area of northern and western Scotland. In response, a survey of the southern extent of the closed harvest areas was initiated to describe variability of ASP toxin levels over varying spatial scales (<5 m to >5 km); determine the anatomical distribution of the toxin, and identify, isolate, and culture causative Pseudo-nitzschia species. Toxin analysis was conducted using a liquid chromatography-tandem mass spectroscopy (LC-MS/MS) procedure. The DA content of tissues followed the predictable rank order: all other tissue -1 gonad -1 adductor. The toxin levels within all other tissue (95% Cl = 580-760 μg DA g-1, n = 170) consistently accounted for 99% of the total individual toxin burden. DA levels in the gonad (95% CI = 8.2-11.0 μg DA g- 1, n = 170) were an order of magnitude below levels in all other tissue and contributed to less than 0.5% of the total individual toxin burden, although levels above the regulatory limit were detected in individual gonad samples. Adductor muscle tissue contained the lowest concentration of DA (95% Cl = 0.38-0.82 μg DA g- 1, n = 170), and was typically within two to three orders of magnitude below levels in all other tissue. None of the scallops examined had DA toxicities in adductor muscle tissue exceeding the regulatory limit. Toxin variability among individuals and sites was high (range of coefficients of variation (CV) in all other tissue = 29&-l20% and gonadal = 45%-85%). The results do give an indication of the scale on which microhabitat differences may influence ASP toxicity in P. maximus populations, because significant differences were found in all other and gonadal tissue toxin levels between groups of individuals only 25-m apart. In total, seven species of Pseudo-nitzschia were identified from west coast waters. A suspected causative species, P. australis, was found to produce high levels of DA, in culture. The high individual variation in toxicities and the occurrence of DA in the gonad at levels above the regula1ory limit clearly demonstrate the complexity of managing the king scallop fishery during ASP events.
    • AZA – the producing organisms – biology and trophic transfer

      Tillmann, U.; Salas, R.; Jauffrais, T.; Hess, P.; Silke, J. (CRC Press, 2014)
      Compared to the knowledge on toxin structure, detection methods, and toxicology, convincing clarification of the aetiology of AZP was seriously lacking behind for quite a long time. Based upon the seasonal and episodic accumulation of AZA toxins in suspension-feeding bivalve molluscs – a situation similar to several other marine biotoxins - a planktonic source has been suspected from the outset. Furthermore, due to their polyether structural features, AZA has been suspected to be a dinoflagellate metabolite. Thus, it was no surprise that is was a dinoflagellate species which was first claimed to be the source of AZA. The link between AZA and P. crassipes, however, remained controversial because production of AZA by P. crassipes could not be verified in spite of numerous attempts based upon field surveys and laboratory investigations of cultured and isolated cells. Moreover, in contrast to other proven producers of phycotoxins, which are all primarily phototrophic, P. crassipes is a heterotrophic dinoflagellate, known to prey upon other dinoflagellates as food. The likelihood, therefore, that another dinoflagellate may produce AZA, which then accumulates in P. crassipes through normal feeding processes, could not be neglected.
    • Irish Shellfish Biotoxin Monitoring Programme

      Silke, J.; McMahon, T.; Hess, P. (Marine Institute, 2006)
      Since its initial development in the early 1970s the Irish aquaculture industry has grown to be an important contributor to the national economy. There has been a steady increase, in both output and value, as well as in job creation. The total production of farmed shellfish has increased from approximately 5,000 tonnes in 1980 to 44,678 tonnes in 2003 (Figure 1), with a first sale value of €41.8m and directly employing some 1100 people (Parsons et al, 2004). Mussels (Mytilus edulis), native oysters (Ostrea edulis), Pacific oysters (Crassostrea gigas), Clams (Tapes semidecussata) and scallops (Pecten maximus) are the main species produced. With a growing recognition and awareness internationally of the potential human health effects of the consumption of shellfish containing algal toxins, a monitoring programme was established in Ireland in the early 1980s and has continued since then. In this paper the evolution and development of the programme is described and discussed.
    • LC-UV and LC-MS methods for the determination of domoic acid

      Hess, P.; Morris, S.; Stobo, L.A.; Brown, N.A.; McEvoy, J.D.G.; Kennedy, G.; Young, P.B.; Slattery, D.; McGovern, E.; McMahon, T.; et al. (Elsevier, 2005)
      Under European legislation, domoic acid (DA), the main constituent of amnesic shellfish poisoning, is monitored to protect the shellfish consumer. To ensure comparability amongst analytical data, it was deemed necessary to undertake performance assessments of the methods conducted by monitoring laboratories of the United Kingdom and Ireland. In phase I of a two-phase inter-comparison, three laboratories used high-performance liquid chromatography and ultraviolet detection (HPLC-UV). Concentration data for a DA standard solution, a crude extract of whole scallops and a scallop-homogenate fell within internationally accepted limits, demonstrating good agreement for these matrices. Between-laboratory analyses of a scallop gonad showed a higher variation (>16%). In phase II, a second gonad homogenate containing DA one order of magnitude higher in concentration gave results acceptable to internationally set criteria. The efficiency of the strong anion-exchange cartridges used in sample-extract clean-up should be monitored as part of a laboratory quality control system. From a recovery study, it is suggested that recovery correction should also be applied. There was no difference in the quantitation of DA in standard solutions or shellfish using either LC-UV or LC with mass spectrometric (MS) detection, and between-laboratory MS data for a gonad homogenate were also equivalent. Variations of the published method practised by the monitoring laboratories were found not to compromise results, thus demonstrating an acceptable degree of ruggedness, as well as comparability between the participants.
    • Performance of the EU Harmonised Mouse Bioassay for Lipophilic Toxins for the Detection of Azaspiracids in Naturally Contaminated Mussel (Mytilus edulis) Hepatopancreas Tissue Homogenates Characterised by Liquid Chromatography coupled to Tandem Mass Spectrometry

      Hess, P.; Butter, T.; Petersen, A.; Silke, J.; McMahon, T. (Elsevier, 2009)
      Azaspiracids (AZAs) are a group of lipophilic polyether toxins that were discovered in shellfish from Ireland in 1995, following a food poisoning incident. Both the limited availability of pure AZAs and the co-occurrence in shellfish of other toxins in combination with AZAs have so far prevented an in-depth evaluation of the performance of the EU reference test, the mouse bioassay (MBA), for this toxin group at the regulatory limit. The present study evaluated the performance of the mouse bioassay at the example of a mussel tissue homogenate, naturally contaminated with AZAs, diluted with uncontaminated tissues to appropriate concentration levels. Concentrations were determined using liquid chromatography coupled to tandem mass spectrometry (LC-MS-MS) (7 levels ranging from levels less than the limit of quantification to a maximum of ca. 2.24 mg/kg in hepatopancreas, which corresponds to a maximum whole flesh AZA1-equivalent of ca. 0.34 mg/kg). Replicate homogenates of each concentration level were analysed by MBA on 7 independent occasions over 6 weeks. Inhomogeneity between replicate aliquot portions was evaluated using LC-MS-MS and ranged from 1.8 to 6.6% RSD for the six levels contaminated above quantification limits. This variation was similar to the variability of the LC-MS-MS method within a batch, and the difference between replicate aliquots could thus be considered negligible. Other uncertainties considered in the study included the short- and long-term variability of the LC-MS-MS method, toxic equivalence factors, relative response factors in mass spectrometric detection, additional analogues and matrix effects. A concentration-response curve was modelled as a 4-parametric logistic fit to a sigmoidal function, with an LC50 of 0.70 mg AZA1-equivalent/kg hepatopancreas tissue. Furthermore, the mathematical model of the lethality data from this study suggests that occasional negative mouse assays at high concentrations, previously observed in the Irish statutory monitoring, are at least partly due to the biological variation of mice and can be understood on a statistical basis. The mathematical model of the concentration-response curve also describes the probability of a positive mouse bioassay at the current regulatory limit of 0.16 mg/kg to be ca. 95%. Therefore, it appears that the mouse bioassay performs very well in the implementation of this limit. Hence, the present study very strongly suggests that the MBA and LC-MS-MS techniques can be considered equivalent in the implementation of the current regulatory limit of 0.16 mg/kg for Azaspiracids in shellfish.
    • Use of LC-MS testing to identify lipophilic toxins, to establish local trends and interspecies differences and to test the comparability of LC-MS testing with the mouse bioassay: an example from the Irish biotoxin monitoring programme 2001

      Hess, P.; McMahon, T.; Slattery, D.; Swords, D.; Dowling, G.; McCarron, M.; Clarke, D.; Gobbons, W.; Silke, J.; O'Cinneide, M. (Conselleria de Pesca e Asuntos Maritimos da Xunta de Galicia and Intergovernmental Oceanographic Commission of UNESCO, 2003)
      During 2001, the Marine Institute has extended its range of chemical tests to include the analysis of DSP toxins by Liquid Chromatography coupled to Mass Spectrometry (LC-MS). Thus the range of compounds determined extends from domoic acid over DSP compounds (okadaic acid and DTXs) to azaspiracids (AZAs). These tests complement the mouse bioassay, which is the current reference method for lipophilic toxins within the European Community. The development and performance characteristics of the LC-MS method are discussed. Isomer patterns and interspecies differences are discussed as well as local trends in time and variability at one production site at a given time. Comparison of the LC-MS results with the results from the mouse bioassay showed good agreement (93%), and a small but significant number of discrepancies (7%). Overall, the chemical testing has proven to be an invaluable tool in the assessment of shellfish toxicity in Ireland. Lacks of standards and reference materials are discussed as well as the need for further research into the equivalence of methods.