• An investigation into the detection and identification of OsHV-1 µvar virus and associated risk factors causing mortalities in Crassostrea gigas in Ireland.

      Morrissey, Teresa (Galway-Mayo Institute of Technology (GMIT), 2014)
      The Oyster Herpes Virus micro variant, OsHV-1μvar, is an emerging disease affecting Crassostrea gigas oysters which has caused significant mortalities in France, Ireland, and the UK (Jersey and Kent, England) between the summer of 2008 and 2012.The present MSc. study was carried out in 2 parts. The first part of the study aimed to characterize and further elucidate the strains of OsHV-1 present in C. gigas oysters sampled in Ireland by further examining sequence data obtained from animals collected from 30 bays during mortality outbreaks between 2003-2012. Part two of this study investigated the detection of OsHV-1 µvar and associated risk factors during mortality outbreaks. The current study provides some insights into mortality events affecting farmed Pacific oyster production in Ireland. The epidemiological study conducted found that batch mortality was lower in 2011, compared with earlier years, in association with lower OsHV-1 µvar viral loads.
    • Optimisation of isolation methods for the azaspiracid group of marine biotoxins and the development of accurate and precise methods of analysis

      Kilcoyle, J. (Dublin Institute of Technology, 2015)
      The two main groups of biotoxins which affect the Irish shellfish industry are azaspiracids (AZAs) and the okadaic acid (OA) group (OA, DTX2, DTX1 and their esters) toxins. Since AZAs were first identified in 1998, well over 30 analogues have been reported. Structural and toxicological data have been described for AZA1–5 (isolated from shellfish). LC-MS/MS is the EU reference method for detection of the AZAs (AZA1, -2 and -3) and the OA group toxins in raw shellfish with the regulatory limit set at 160 μg/kg for each toxin group. Limited supplies of purified toxins for certified reference materials (CRMs) were available for AZA1−3. Little knowledge was also available on the relevance of the additional AZA analogues that had been reported, in terms of human health protection. The analysis of marine biotoxins by LC-MS/MS can be severely affected by matrix interferences. Here, a study was performed on two instruments; a quadrapole time of flight (QToF) and a triple stage quadrupole (TSQ) to assess matrix interferences for AZA1 and OA using a number of tissue types. Enhancement was observed for OA on the QToF while matrix suppression was observed for AZA1 on TSQ. The enhancement on the QToF was overcome by use of an on-line SPE method and matrix matched calibrants, while the suppression on the TSQ was found to be due to late eluting compounds from previous injections and was overcome by employing either a column flush method or an alkaline mobile phase.The isolation of 11 AZA analogues (AZA1−10 and 37-epi-AZA1) from shellfish using an improved procedure (7 steps) is described. Recoveries increased ~2-fold (~ 52%) from previously described isolation procedures. The preparative isolation procedure developed for shellfish was optimised for Azadinium spinosum bulk culture extracts such that only four steps were necessary to obtain purified AZA1 and -2. A purification efficiency of ~70% was achieved, and isolation from 1,200 L of culture yielded 9.3 mg of AZA1 and 2.2 mg of AZA2 (purities >95%). This work demonstrated the feasibility of sustainably producing AZA1 and -2 from A. spinosum cultures. In addition to AZA1 and -2, the novel analogues AZA33, -34 were isolated (also from A. spinosum).Sufficient quantities were purified to enable full structural elucidation, the preparation of reference standards and CRMs, and toxicity studies. Nine of these analogues were fully characterised for the first time (Table 7.1). Structural determination was achieved by NMR and chemical analysis, while toxicity was assessed using the Jurkat T lymphocyte cell assay, mouse intraperitoneal (AZA1−3 and -6) and mouse oral (AZA1−3) administration.The preparation of reference standards for the analogues AZA4−10, 37-epi-AZA1, AZA33 and -34 enabled their relevance in terms of human health protection to be determined. The in vitro and in vivo toxicity studies performed confirmed AZA toxicity.The results from the oral and intraperitoneal mice studies correlated very well, contradicting previous reports and showing that AZA1 is more toxic than AZA2 and -3 and that AZA6 is slightly less toxic than AZA1. Analysis of shellfish (Mytilus edulis) submitted to the Irish biotoxin monitoring programme using the reference standards confirmed previous reports showing that levels of AZA3, -4, -6 and -9 increase following cooking due to heat induced decarboxylation of AZA17, -21, -19 and -23. Very high levels of AZA3 (up to 3-fold that of AZA1) and -6 (up to 3- fold that of AZA2) were detected in some samples (with levels varying most likely due to different rates of metabolism and time of harvesting). As the concentrations of AZA3 and -6 are negligible in raw mussels, yet can increase significantly during the cooking of mussels, the overall concentrations are underestimated by methods used according to current legislation. In cooked shellfish the AZA analogues -4, -5, -7–10, as well as AZA33 and -34, comprise on average ~5% of the total AZA content, however in some samples levels of AZA4 were higher than AZA6. Levels of the 37-epimers in the cooked shellfish extracts were ~ 15% that of the parent analogues.
    • Passive Sampling for Quality Monitoring of Irish Marine Waters

      White, Philip (Dublin Institute of Technology, 2014)
      This study details the steps involved in fabrication, deployment and retrieval of mainly polydimethyl siloxane (PDMS) passive sampling devices deployed in a number of locations in and around Ireland in an attempt to derive dissolved water concentrations of contaminants in-situ. PDMS samplers were initially deployed in the Burrishoole catchment, Co. Mayo in conjunction with the collection of biological tissues and sediment to investigate the source of elevated dioxins in the catchment. Passive samplers were used to generate dissolved water concentrations of persistent organic pollutants (POPs) and also to successfully screen for the presence of dioxins in the water column. The dioxin profile present was also found in sediment and biological tissue and through statistical profiling potential sources were identified as being possibly related to the use of technical pentachlorophenol in the catchment though no direct evidence was found. Passive samplers (PDMS and SPMD) were then deployed at various depths on the M6 weather buoy, 400 miles off the West Coast of Ireland, in conjunction with temperature and salinity monitors to test how the technology would fare over a long period deployment (585 days) in a harsh, dynamic environment. The PDMS samplers were almost completly lost where the SPMDs last better (80 % recovered). Dissolved water concentrations estimated using both sampler types were found to be very low (<ppb) with polyaromatic hydrocarbons found in higher levels than polychlorinated biphenlys, and organochlorine compounds. The use of statistical analysis suggests that passive samplers can also be used to differentiate different water masses by investigating contaminant loadings at each depth sampled. Finally passive samplers were deployed in various inshore and inland waters across Ireland with the results indicating that the remote West of Ireland had the lowest levels of dissolved water concentrations estimated. Many estuaries and inland water bodies had levels of contaminants higher than the west of Ireland with the heavily industrialised Cork and Dublin sites having the highest levels estimated. The separation of sites based on concentrations found indicated that assessment criteria could be generated in an Irish context (IRef) which could be used to classify a site in relation to ‘background’ levels found in the West of Ireland and at M6. The results generated during this study were then assessed based on various legislative requirements and assessment criteria such as the Water Framework Directive (WFD) and the Oslo Paris convention (OSPAR). Results from the WFD assessment indicate that concentrations found at all sites were below the EQS values set down. However this EQS value is based on total water concentration hence the EQS was modified to a dissolved water concentration basis. This reduced the total water EQS values by up to 80 % for some analytes however in most locations the dissolved water concentrations found were at or below this dissolved water EQS value indicating that the levels of contaminants from across Ireland are below the EQS values generated as part of the WFD. Assessments were also made on the concentrations found across Ireland using background assessment criteria (BAC) suggested by OSPAR. The results indicate that the levels across Ireland are above the BAC for most compounds with the M6 weather buoy faring better. Concentrations from “pristine” Irish sites were then chosen to generate reference criteria on an Irish basis (IRef) which were found to be below the concentration levels suggested as part of the BAC assessment criteria in the majority of locations.
    • Phytoplankton precision trials in the enumeration and identification of marine microalgae through the scheme "Biological Effects Quality Assurance in Monitoring Programmes (BEQUALM)"

      Salas, Rafael Gallardo (2011)
      Scientists are coming under increased pressure in recent years to show that results they obtain arising from their scientific work are quality assured and stand up to scrutiny by independent expert auditors. This has meant that the methodologies used by laboratories involved in making these measurements have to be validated and fit for purpose and has led to the adoption of internationally recognised standard protocols. These protocols must be underpinned by robust quality systems and must be accredited to an international standard. In order for laboratories to become accredited in particular methods, they have to fulfil a series of prerequisites but a compulsory one is the participation in a proficiency testing scheme. Proficiency testing schemes are independent assessor organisations which coordinate regular inter-calibration and intercomparative studies between laboratories with a common purpose. What happens, though when proficiency testing schemes do not exist for a particular scientific measurement? This study presents results from two inter-comparison exercises at European level between phytoplankton monitoring laboratories in the enumeration and identification of marine microalgae using the Utermöhl cell counting method. Microalgae are a very important ecological component of the marine ecosystem and have also become important ecological indicators of hydro-climatic change, ocean acidification and eutrophication. Member states of the European Union are obliged to monitor for toxic and harmful algae which can cause problems and devastation in the natural environment, have detrimental effects on human health if contaminated fish and shellfish are eaten, can cause huge economic losses to the aquaculture industry and impact directly in coastal communities. This study shows how an intercomparison of this kind is designed and organised, how samples are set up, materials homogenised and reference values obtained. It demonstrates the importance of using the right technique and best practice, based on experience, to analyse samples and how important it is to design the exercise to be statistically robust, both quantitatively and qualitatively. The taxonomy quiz in 2009 showed that there was no evidence that video clips were better audit trail tools than images or vice versa. The quantitative measure suggested that there was evidence of good agreement between virtually all the analysts and the reference value for all species except one (P.micans). However, there was evidence of lack of reproducibility between and within laboratories. The qualitative measure calculated indicated that analysts are more likely to identify a toxic organism as a non toxic organism than the other way around. The results from the enumeration data in the 2010 exercise showed that there was lack of reproducibility across laboratories using different counting strategies and volume sub-sampled and analysed. These results when compared to a set of hypothesised means used as reference values suggested that cell counts were potentially underestimated by as much as 30% and that this underestimation was most likely due to test method effects.
    • Scale Growth Analysis of Atlantic salmon (Salmo salar Linnaeus) Unlocking Environmental Histories

      Thomas, K. (Galway-Mayo Institute of Technology, 2018)
      Atlantic salmon (Salmo salar L.) populations have declined rapidly in recent years across all geographical ranges with populations becoming extinct within certain areas. Direct observation of the salmon’s life is difficult and costly; therefore, scales remain the most widely used material to indirectly assess and monitor the recent changes in growth. Growth marks (circuli) in scales of Atlantic salmon are used to estimate age and to reconstruct growth histories. This thesis investigated mechanisms of circuli formation and the causes of variation in scale growth measurements. Comparison of scales from multiple body locations (Chapter 2) showed that growth, size and shape measurements varied significantly between body locations. Scale measurements taken from the sampling location recommended by ICES were sufficiently correlated with measurements from two adjacent locations in the posterior body region to facilitate conversion; calibration equations are presented for this purpose. Scale measurements from the anterior body region were highly variable and their use is not recommended. Scale size measurements from the recommended sampling location and from the two adjacent locations in the posterior body region were sufficiently correlated with fish fork length. Differences in scale size could potentially be used to determine the body location from which a scale was most likely sampled if this information has not been recorded (e.g. in archived scale collections); regression equations are presented for this purpose. Analysis of scales from experimentally reared Atlantic salmon post-smolts (Chapters 3 and 4), showed that scale growth and circuli number was proportional to fish growth under a range of different water temperatures and feeding conditions, justifying the use of these measurements as a proxy for growth. The rate of circuli deposition varied between temperature and feeding treatments and circuli number was proportional to cumulative degree day. Narrow inter-circuli spacings were observed during periods of slow growth at low temperatures and during periods of fast growth at high temperatures; therefore, circuli spacing should not be used to infer growth rates. In Chapter 5, scales from Atlantic salmon collected from three Irish rivers (Burrishoole, Moy and the Shannon) between 1954 and 2008 were analysed to determine if marine growth has changed during that period and to establish if trends are consistent across populations. Scale growth measurements and their temporal trends varied between populations. Post-smolt scale growth and circuli number were negatively correlated with SST (Burrishoole and Moy), NAO (Burrishoole) and AMO Burrishoole and Shannon). The results indicate that trends observed in one national index river may not be representative of change across all populations. The new knowledge generated in this thesis supports more accurate interpretation of scale growth measurements, furthers our understanding of this important species and ultimately benefits the future management of this species.