Date of Award


Document Type

Doctoral Thesis

Degree Name

Doctor of Philosophy



First Advisor

Prof. Kevin J. James


Azaspiracids (AZAs) are a family of lipophilic polyether marine biotoxins, that have caused a number of human intoxication incidents in Europe since 1995, which was the first incident reported following the consumption by consumers of intoxicated shellfish {Mytilus edulis). This class of phycotoxins has been responsible for extended closures of shellfisheries in various locations around Europe, where levels of AZAl-3 are regulated in shellfish. Since their discovery in 1995, AZAs have become the focus of extensive research, resulting in the discovery of numerous analogues. Only AZAl and AZA2 have been found in phytoplankton and all the other analogues are believed to be products of biotransformation in shellfish, mussels (M edulis). During feeding studies that were conducted on AZA free mussels (M edulis), the suspected biotransformation of AZAl and AZAl were confirmed and mechanism proposed. Mussels were assimilated and depurated by keeping in 100 litre tanks assisted with an algae suspension and supplemented with fresh sea water for up to 12 months. When mussels were free of toxin, they were than exposed to two feeding experiments, fhese involved administering isolated AZAl and AZA2 at a fixed concentration over a set number of days. At the end of the feeding experiment, mussels were removed, rinsed and dissected immediately into HP, gills and rest of tissue in order to avoid leaching of toxin into different tissue compartments which can happen as a result of cold storage. Liquid Chromatography-tandem-Mass Spectrometry (LC-MS/MS) was used for AZA analysis. It was observed that AZAl undergoes a rare C-demethylation via the formation of a carboxylate intermediate AZA 17 to form AZA3. Since AZAl 7 is 22- carboxy-AZA3, produced by oxidative metabolism of the 22-methyl of AZAl in the mussel, this also predicted that the levels of AZA6 would also increase through an equivalent series of transformations via AZA 19 and was the basis of isolating AZA2 and administering to live shellfish. From the AZA2 feed experiment the most likely explanation for the observed conversion to AZA6 upon administration of AZA2 was the presence of a thermally labile AZA6 precursor in the shellfish and their extracts, and was observed that AZA2 undergoes C-demethylation in position C22 producing AZA 19.

Distribution of AZAs was investigated in three of the dissected compartments of M. edulis, (hepatopancreas (HP), gills and rest of tissue) and demonstrates that AZAs are not solely confined to the HP as is widely accepted. In order to confirm the distribution observed, 20 mussels, naturally contaminated with AZA, were dissected into their individual compartments and analysed. The study showed significant levels of AZAs in all three compartments when the mass of the tissue was taken into account. This demonstrated the importance of extracting toxin from whole tissue, not just from the HP, for a truer appraisal of the levels of AZA within the mussel. This finding is of great significance in terms of procedures used in the processing of shellfish for regulatory analysis, and it exemplifies the role of taking whole tissue and not just HP.

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