Document Type

Theses, Ph.D

Rights

This item is available under a Creative Commons License for non-commercial use only

Publication Details

Thesis submitted in fulfilment of requirement leading to the award of PhD to the School of Chemical and Pharmaceutical Sciences, College of Sciences and Health, Dublin Institute of Technology, 2015.

Abstract

Food poisoning incidences relating to marine biotoxins are a global phenomenon and have the potential to severely impact the aquaculture industry. As a result, and as a legislative requirement in the European Union (EU), many countries have implemented monitoring programmes for these compounds but their success relies on the availability of certain quality assurance tools, two of which are reference materials (RMs) and proficiency testing. The limited amounts of RMs, in particular matrix certified reference materials (CRMs) for paralytic shellfish poisoning (PSP) toxins has been a limiting factor in the implementation of alternatives to the mouse bioassay for routine monitoring programmes. Various stabilisation procedures were investigated to ascertain the applicability of each for preparing RMs for various uses including internal QA/QC, proficiency testing and as candidates for certification. The beginning of these studies coincided with a large PSP toxic event in Icelandic waters. During that period mussels from two production sites on the north and north-west coasts of Iceland accumulated PSP toxins to levels many times over the EU regulatory limit. Mussels sampled during this period were characterised and presented along with phytoplankton data from the same period and presented as a first report of PSP toxins from Icelandic waters. Large quantities of naturally contaminated mussel tissues were harvested during this period for use in these studies. Various stabilisation procedures were investigated, such as thermal treatment, the use of preserving additives, high pressure processing (HPP) and freeze drying, for their applicability in preparing RMs for PSP toxins. Extensive characterisation of the materials was performed through homogeneity and short and long-term stability studies using two LC-FLD methods to evaluate each technique in reducing levels of degradation, biotransformations or epimerization. Freeze drying proved the most effective technique evaluated and this procedure was used to prepare RMs in two shellfish species, Pacific oyster (Crassostrea gigas) and blue mussels (Mytilus edulis). The technique improved the stability of all toxins assessed compared to untreated controls. A successful feasibility study was carried out in a C. gigas matrix which resulted in the production of a medium scale RM as a candidate CRM. Certification of this material was not carried out during the course of these studies however. Although freeze drying proved the most effective in stabilising both the tissue matrix and the PSP toxins themselves, the labour intensive nature of the procedure as well as the significant per unit production costs led to alternative RM techniques being investigated. HPP of a C. gigas tissue matrix was performed and it provided an extremely effective technique for stabilising the toxins through a reduction of microbial activity. Stability studies showed the technique reduces or eliminates toxin degradation and epimerisation compared to untreated control materials with the technique potentially having applications in CRM development, although a full feasibility study was not conducted. The use of heat treatment and preserving additives provided the simplest and most cost effective stabilisation procedure investigated with both techniques, particularly when combined, improving toxin and matrix stability compared to untreated controls. Each technique was evaluated separately and combined in one tissue matrix, M. edulis and applications for materials prepared by this procedure were examined. Combining the use of preserving additives with a thermal pre-treatment step provided sufficiently stable and homogenous RMs which were used as an internal QA/QC tool in the Irish National Monitoring Programme (NMP) and in proficiency testing (PT) schemes operated by QUASIMEME and VEREFIN. Data from a RM prepared by the combined techniques above and used in the Irish NMP over a two year period provided evidence for the long-term stability of the material using a classical stability model and was used to calculate an expanded uncertainty of measurement for the method used for official control purposes at the Marine Institute. Materials prepared by these combined techniques were also used to develop the first commercially available PT scheme for PSP toxins. Data is presented from the first six years of this development exercise from participants using a wide array of methodologies. Data showed the improvement of participants over this period but found statistical differences in the datasets of both LC-FLD methods employed by some subscribers in determining dcSTX and GTX2,3. A material was also prepared for use in a separate PT scheme operated by VEREFIN which highlights the wide applicability of these RMs. The techniques investigated during these studies have multiple applications in method development, as QA/QC tools, in CRM preparation and in proficiency testing schemes.

DOI

10.21427/D7MK5J

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