• Acidification and its effect on the ecosystems of the ICES Area

      Fernand, L.; LeQuesne, W.; Silke, J.; Li, B.; Kroeger, S.; Pinnegar, J.; Fossä, J.H.; Morán, X.A.G. (ICES, 2011)
      This focuses on the impacts of ocean acidification (OA) on ecosystems and higher trophic levels in the ICES Area. One of ICES distinguishing features is its access to scientists across the entire marine field. This report is based on the Report of the Workshop on the Significance of Changes in Surface CO2 and Ocean pH in ICES Shelf Sea Ecosystems (WKCpH; ICES, 2007c), updated to include recent research, using inputs from the chairs of ICES working groups. Oceanic uptake of atmospheric CO2 has led to a perturbation of the chemical environment, primarily in ocean surface waters, which is associated with an increase in dissolved inorganic carbon (DIC). The increase in atmospheric CO2 from ca. 280 ppmv (parts per million by volume) 200 years ago to 390 ppmv today (2011) has most probably been caused by an average reduction across the surface of the oceans of ca. 0.08 pH units (Caldeira and Wickett, 2003) and a decrease in the carbonate ion (CO32−) of ca. 20 μmol kg −1 (Keshgi, 1995; Figure 5.1). It has been estimated that the level could drop by a further 0.3 – 0.4 pH units by the year 2100 if CO2 emissions are not regulated (Caldeira and Wickett, 2003; Raven et al., 2005). A study of potential changes in most of the North Sea (Blackford and Gilbert, 2007) suggests that pH change this century may exceed its natural annual variability. Impacts of acidity induced change are likely, but their exact nature remains largely unknown, and they may occur across the whole range of ecosystem processes. Most work has concentrated on open‐ocean systems, and little research has been applied to the complex systems found in shelf‐sea environments.
    • 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.
    • Bioactive agents from marine mussels and their effects on human health

      Grienke, U.; Silke, J.; Tasdemir, D. (Elsevier, 2014)
      The consumption of marine mussels as popular seafood has increased steadily over the past decades. Awareness of mussel derived molecules, that promote health, has contributed to extensive research efforts in that field. This review highlights the bioactive potential of mussel components from species of the genus Mytilus (e.g. M. edulis) and Perna (e.g. P. canaliculus). In particular, the bioactivity related to three major chemical classes of mussel primary metabolites, i.e. proteins, lipids, and carbohydrates, is evaluated. Within the group of proteins the focus is mainly on mussel peptides e.g. those obtained by bio-transformation processes, such as fermentation. In addition, mussel lipids, comprising polyunsaturated fatty acids (PUFAs), are discussed as compounds that are well known for prevention and treatment of rheumatoid arthritis (RA). Within the third group of carbohydrates, mussel polysaccharides are investigated. Furthermore, the importance of monitoring the mussel as food material in respect to contaminations with natural toxins produced by microalgae is discussed
    • Creating a weekly Harmful Algal Bloom bulletin

      Leadbetter, A.; Silke, J.; Cusack, C. (Marine Institute, 2018)
      This document describes the procedural steps in creating an information product focused on toxic and harmful phytoplankton. The product is an online Harmful Algal Bloom (HAB) bulletin for aquaculturists, who can face serious operational challenges in the days after a HAB event. Data from satellite, numerical hydrodynamic models and In-situ ocean observations are organised and presented into visual information products. These products are enhanced through local expert evaluation and their interpretation is summarised in the bulletin. This document aims to provide both process overviews (the “what” of the Best Practice in producing the bulletins) and detail procedures (the “how” of the Best Practice”) so that the bulletins may be replicated in other geographic regions.
    • Development & implementation of the Phytotest project

      Kavanagh, S.; Brennan, C.; Lyons, C.; Chamberlain, T.; Salas, R.; Moran, S.; Silke, J.; Maher, M. (Marine Institute, 2008)
      Phytotest is a 3-year research and development project funded through the Marine Institute Strategic Research Programme in Advanced Technologies as part of the National Development plan 2000-2006. The project is a collaboration between the National Diagnostics Centre at NUI Galway and the MI and involves the development of real-time PCR assays for Dinophysis and Pseudo-nitzschia species that are important in Irish waters. In the current final phase of the project, the real-time PCR assays are being transferred to the MI to support the phytoplankton monitoring service.
    • Dinoflagellate cysts in Irish coastal sediments - a preliminary report

      O'Mahony, J.H.; Silke, J. (1993)
      Since the mid 1970's the production of bivalve shellfish in Ireland has increased annually to a present level of some 17,000 tonnes. Several problems limit the continued expansion of the industry, most notably the problem of natural biotoxins. These toxins are accumulated in the product by the ingestion of toxic phytoplankton. This causes no obvious ill effects to the shellfish themselves but upon consumption may be transferred to human or other vertebrate consumers causing illness and sometimes death. In Ireland the most common of the toxins are those associated with Diarrhetic Shellfish Poisoning (DSP) which causes diarrhoea. Other more serious toxins which to date have not been confirmed in Ireland are those associated with Paralytic Shellfish Poisoning (PSP) which causes paralysis or even death and Amnesic Shellfish Poisoning (ASP) which causes short term memory loss. Of the phytoplankton species which can result in toxicity, under both bloom and non bloom conditions, the dinoflagellates play an important role. Many of these dinoflagellates have been shown to include a dormant benthic cyst stage in their life cycle. Therefore a better understanding of the dynamics of toxic events may be obtained by studying the distribution and abundance of benthic cysts. There is growing international concern about the transport of harmful aquatic organisms, including cysts, into new areas via the discharge of ships ballast water. Also, as a result of EC directive 91/67/EEC permitting the free movement of shellfish between EU member states there is now increasing concern in Ireland that harmful cysts may be introduced with shipments of imported shellfish. Little research has been carried out on the distribution of dinoflagellate cysts in Irish marine sediments. In this paper preliminary results of a study designed to map the distribution and undertake taxonomic studies on dinoflagellate and other cysts in Ireland are presented and discussed. Also presented are the results of the examination of cysts associated with imported shellfish.
    • Dinophysis species in Irish waters 1990 - 1993

      Jackson, D.; Silke, J. (ICES, 1993)
      The distribution and abundance of Dinophysis species as recorded in the national phytoplankton monitoring programme are described. An apparent spread in the occurrence of Dinophysis to the west coast of Ireland is reported. The lack of correlation between the concentrations of Dinophysis in the water and DSP toxicity in shellfish is reported on and discussed.
    • Establishing boundary classes for the classification of UK marine waters using phytoplankton communities

      Devlin, M.; Best, M.; Coates, D.; Bresnan, E.; O'Boyle, S.; Parke, R.; Silke, J.; Cusack, C.; Skeats, J. (Elsevier, 2007)
      This paper presents a description of three of the proposed phytoplankton indices under investigation as part of a classification framework for UK and ROI marine waters. The three indices proposed for the classification process are (i) phytoplankton biomass measured as chlorophyll, (ii) the frequency of elevated phytoplankton counts measuring individual species and total cell counts and (iii) seasonal progression of phytoplankton functional groups through the year. Phytoplankton biomass is calculated by a 90th percentile measurement of chlorophyll over the growing season (April to September) compared to a predetermined reference value. Calculation of functional groups and cell counts are taken as proportional counts derived from the presence of the indicator species or group as compared to the total phytoplankton count. Initial boundary conditions for the assessment of high/good status were tested for each index. Chlorophyll reference conditions were taken from thresholds developed for previous EU directives with the setting of offshore concentrations as a reference condition. Thresholds for elevated counts of phytoplankton taxa were taken from previous EU assessments describing counts that could be impact negatively on the environment. Reference seasonal growth curves are established using phytoplankton counts from ‘‘high status’’ waterbodies. To test the preliminary boundaries for each index, a risk assessment integrating nutrient enrichment and susceptibility for coastal and transitional waters was carried out to identify WFD waterbodies in England and Wales at different levels of risk. Waterbodies assessed as having low or medium risk from nutrient enrichment were identified as type 1 and type 2 waterbodies, and waterbodies assessed as high risk were identified as type 3 waterbodies. Phytoplankton data was extracted from the risk assigned waterbodies and applied to each phytoplankton index to test the robustness of the preliminary classification ranges for each phytoplankton index.
    • Harmful and nuisance algal blooms in Irish coastal waters 1990 - 1993

      Silke, J.; Jackson, D. (ICES, 1993)
      Algal blooms occur naturally around our coast. These high concentrations of planktonic algae are associated with favourable conditions of light and nutrients, and often occur at stratification/ mixing fronts. Many blooms are completely harmless, and form the diet of shellfish and zooplankton. Some colour the water red or brown. A few species are toxic and can cause fish kills or make shellfish unsafe to eat. The Fisheries Research Centre monitors phytoplankton in order to detect any toxic or potentially harmful blooms. The harmful and nuisance algal events from 1990 to 1993 are described.
    • Impacts of climate change on harmful algal blooms

      Bresnan, E.; Davidson, K.; Edwards, M.; Fernand, L.; Gowen, R.; Hall, A.; Kennington, K.; McKinney, A.; Milligan, S.; Raine, R.; et al. (Marine Climate Change Impacts Partnership, 2013)
      High biomass Harmful Algal Blooms (HABs) such as Karenia mikimotoi and shellfish toxin producing HAB species continue to be observed in UK and Republic of Ireland waters. Regional differences continue to be seen in the distribution of HABs in UK and RoI waters with impacts mainly observed in the south and west coast of Ireland and regions in the UK with a strong Atlantic influence, e.g. Regions 1, 3, 4, 6 and 7. There is little monitoring aside from the continuous plankton recorder (CPR) in Region 8. The impacts from HABs in Wales, Northern Ireland and the Isle of Man are generally low. Since the last MCCIP report card was issued, blooms of Karenia mikimotoi have caused problems in Ayrshire, Scotland, and also in the north-west coast of Ireland where concerns about the quantity of dead wild fish washing on shore during an event in Ireland in 2012 resulted in two beaches being closed to the public. No clear trend that can be attributed to climate change can be observed in the incidence of shellfish toxin producing HABs since the last report card was issued. During the last two years the incidence of some shellfish toxins has continued to decrease (e.g. paralytic shellfish poisoning toxins in Scotland). High concentrations of yessotoxins (YTX) and azaspiracids (AZAs) have been recorded for the first time in Scotland. Northern Ireland enforced its first shellfish harvesting closure for high concentrations of domoic acid (the toxin responsible for amnesic shellfish poisoning, ASP) in 2012. A recent survey in Scottish waters (Regions 1, 6 and 7) has revealed the presence of domoic acid in the urine and faeces of harbour seals (Phoca vitulina). The impacts of these toxins on the health of marine mammals are unknown and a more detailed study is currently being undertaken. Many of the future impacts of climate change are unknown. Increasing sea surface temperatures as a result of climate change may increase the potential for blooms of species that are not currently found in UK and RoI waters through range expansion or human mediated introduction. There is evidence that no new HAB species have become established during the last two years. An increase in the duration of stratification of the water column may influence the abundance of HABs in UK and RoI waters. This is particularly relevant in shelf areas and Region 8, an area where offshore high biomass K. mikimotoi blooms have been hypothesized to initiate and impact coastal areas along the west of Ireland and Regions 6, 7 and 1. Conversely, an increase in wind speed and duration may reduce the duration of stratification in the water column. This may result in a decrease of some HAB dinoflagellate species and an increase in HAB diatom species. Little is known about the impacts of ocean acidification or changes in offshore circulation on the incidence of HABs. The role of offshore blooms in seeding coastal blooms (e.g. of K. mikimotoi) remains unknown and the lack of monitoring in Region 8 and on the shelf edge compounds this knowledge gap.
    • The implications of Alexandrium tamarense resting cysts in an area of shellfish aquaculture in Ireland

      Silke, J.; McMahon, T. (1998)
      The Irish Marine Institute's Fisheries Research Centre carry out a monitoring programme for the detection of algal toxins in shellfish. This programme is carried out under EU Directive 91/492. During the course of this programme the North Channel area of Cork Harbour has been the only location in Ireland where toxins causing Paralytic Shellfish Poisoning (PSP) have been detected in shellfish above the regulatory limit. For short periods during each of the summers of 1996,1997 and 1998, PSP toxins were found in mussels{Mytilus edulis) from this area above the regulatory limit period necessitating a ban on harvesting. Oysters {Crassostrea gigas) from the same area remained below the regulatory threshold. The dinoflagellate Alexandrium tamarense, a known vector of PSP toxins, was observed in the area during each of the toxic events. The exact origin of the populations of A. tamarense was unknown. A. tamarense is known to produce a cyst stage as part of its life cycle. These cysts can remain viable in the sediments for several years. A survey of the distribution of cysts of A. tamarense in the surface sediments in Cork Harbour was carried out in order to determine if they were potentially seeding the area. They were detected in 6 sites, and successfully germinated to yield vegetative cells. The results of the survey are presented and discussed.
    • Irish National Phytoplankton Monitoring (Sites 41–45)

      Silke, J.; Cusack, C. (ICES, 2012)
      The Marine Institute in Ireland carries out a national phytoplankton monitoring programme which extends back to the late 1980s. This includes a harmful algal blooms (HABs) monitoring service that warns producers and consumers of concentrations of toxic plankton in Irish coastal waters that could contaminate shellfish or cause fish deaths. This programme is primarily located along the Atlantic seaboard and Celtic Sea. Scientists working on this monitoring programme have developed an understanding of phytoplankton populations and dynamics around the Irish coastline, especially in relation to those that cause shellfish toxicity. Particular emphasis is put on the detection and enumeration of harmful species. The importance of phytoplankton as an indicator of water quality is also studied and is a key component of the European Water Framework.
    • 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.
    • Karenia mikimotoi: An Exceptional Dinoflagellate Bloom in Western Irish Waters, Summer 2005

      Silke, J.; O'Beirn, F.X.; Cronin, M. (Marine Institute, 2005)
      A protracted bloom of Karenia mikimotoi was present in summer 2005 along the northern half of the western Irish coastline. The onset of this bloom was identified in late May / early June. This event subsequently dissipated over the month of July and was succeeded by a bloom of the same species in the southwest in late July. The bloom was very intense and resulted in discolouration of seawater and foaming in coastal embayments. Major mortalities of benthic and pelagic marine organisms were observed and a complete decimation of marine faunal communities was reported and observed in several locations. Deaths of echinoderms, polychaetes and bivalve molluscs were observed in County Donegal and Mayo, while farmed shellfish and hatchery raised juvenile bivalve spat suffered significant mortalities along the Galway and Mayo coasts. Reports of dead fish and crustacea were received from Donegal, Galway, West Cork and Kerry. Karenia mikimotoi is one of the most common red tide causative dinoflagellates known in the Northeast Atlantic region, and is also common in the waters around Japan. Blooms of this species often reach concentrations of over several million cells per litre and these densities are often associated with marine fauna mortalities. Although cytotoxic polyethers have been extracted from cultures of the species, the exact mechanism of the toxic effect and resultant devastating damages yet remains unclear. It is known in the literature under several different names as the taxonomy and genetics have been studied. It is now known that previously reported names including Gyrodinium aureolum, G. cf. aureolum, G. nagasakiense and G. mikimotoi are synonymous with the current name given to the organism. The visible effects following the mortalities included noticeable quantities of dead heart urchins (Echinocardium cordata L.) and lugworms (Arenicola marina L.) deposited on beaches. Several species of wild fish were also found dead. The bloom coincided with a period of fine weather and tourists visiting the seaside were concerned about the safety of swimming in waters that were obviously harmful to marine organisms on this scale. A public awareness programme was mounted by the Marine Institute with several radio broadcasts, press releases and a website provided to give up to date pronouncements on the event. While there have been several instances of Karenia mikimotoi blooms reported in Ireland over the past 30 years, this scale of mortalities associated with the 2005 bloom were not previously observed. Recording the scale of this event was facilitated by satellite imagery while direct counts of the cells in seawater by the Marine Institute monitoring programme gave very useful information regarding the size and intensity of this event. The mortalities of marine organisms were documented from reports made by various observers and by Marine Institute field surveys.
    • Monitoring of Shellfish Growing Areas - 1993

      Nixon, E.; Rowe, A.; Smith, M.; McLoughlin, D.; Silke, J. (Department of the Marine, 1994-08)
      During 1993, water and shellfish from 19 major growing areas were monitored for chemical parameters in accordance with the 1979 Council Directive 79/923/EC. At each site temperature, salinity, pH, dissolved oxygen and suspended solids measurements were taken and shellfish samples were returned to the laboratory for metal, chlorinated hydrocarbon and algal biotoxin determinations. Generally, water quality in all areas was good and conformed to the guidelines of the Directive. The highest levels of metals recorded were: cadmium in Tralee Bay (0.4 to 0.7µg/g) and Carlingford Lough (0.3 to 0.7µg/g) and lead in Wexford Harbour (0.5µg/g). Mercury in all cases was low with the exception of Cromane during November when levels of 0.3µg/g were detected. Chlorinated hydrocarbons levels were extremely low and indicate the clean nature of Irish shellfish, unpolluted by these synthetic organic compounds. Algal biotoxins were not detected in any samples.
    • New insights into the causes of human illness due to consumption of azaspiracid contaminated shellfish

      Chevallier, O.P.; Graham, S.F.; Alonso, E.; Duffy, C.; Silke, J.; Campbell, K.; Botana, L.M.; Elliott, C.T. (Nature Publishing Group, 2015)
      Azaspiracid (AZA) poisoning was unknown until 1995 when shellfish harvested in Ireland caused illness manifesting by vomiting and diarrhoea. Further in vivo/vitro studies showed neurotoxicity linked with AZA exposure. However, the biological target of the toxin which will help explain such potent neurological activity is still unknown. A region of Irish coastline was selected and shellfish were sampled and tested for AZA using mass spectrometry. An outbreak was identified in 2010 and samples collected before and after the contamination episode were compared for their metabolite profile using high resolution mass spectrometry. Twenty eight ions were identified at higher concentration in the contaminated samples. Stringent bioinformatic analysis revealed putative identifications for seven compounds including, glutarylcarnitine, a glutaric acid metabolite. Glutaric acid, the parent compound linked with human neurological manifestations was subjected to toxicological investigations but was found to have no specific effect on the sodium channel (as was the case with AZA). However in combination, glutaric acid (1mM) and azaspiracid (50nM) inhibited the activity of the sodium channel by over 50%. Glutaric acid was subsequently detected in all shellfish employed in the study. For the first time a viable mechanism for how AZA manifests itself as a toxin is presented.
    • Nucleic acid tests for toxic phytoplankton in Irish waters-phytotest: Marine Strategic RTDI project AT/04/02/02 - research update

      Maher, M.; Kavanagh, S.; Brennan, C.; Moran, M.; Salas, R.; Lyons, J.; Silke, J. (Marine Institute, 2007)
      The Phytotest project is a 3 year collaborative project funded through the Marine Strategic Programme in Advanced Technologies as part of the National Development plan 2000-2006. The project partners include the National Diagnostics Centre at NUI Galway and MI. The overall objective of the project is the development of nucleic acid tests (molecular methods) for the identification of key toxic phytoplankton species in Irish waters. In the final year of the programme the aim is to transfer the molecular methods developed in the project into MI to support their monitoring service. Currently, the monitoring for phytoplankton species in Irish waters is performed by light microscopy which can easily identify some plankton species based on distinctive morphological traits. Other species in particular, Pseudonitzschia spp. and Alexandrium spp. cannot be identified to species level by light microscopy. Identification of these species requires more sophisticated microscopic techniques such as scanning electron microscopy (SEM) and transmission electron microscopy (TEM). These techniques cannot easily be integrated into a routine testing environment. Molecular methods utilise unique information contained within an organism’s genome in order to identify it. This genetic information can be exploited in a range of molecular test platforms enabling microorganisms to be identified to species level. Additionally, there has been a major drive towards the development of highly automated platforms to support molecular tests for high-throughput testing in routine laboratory settings.
    • The occurrence of DSP toxicity in Ireland

      Jackson, D.; Silke, J.; Doyle, J.; Nixon, E.; Taaffe, B. (ICES, 1993)
      The geographical and temporal variations in the occurrence of DSP in Ireland are presented and the implications of the resulting closures on aquaculture operations and fisheries are discussed. Prior to 1992 DSP toxicity had been confined to the southwest and south coasts but in 1992 a protracted occurrence of DSP was recorded in Killary Harbour on the west coast.
    • Overview of trends in plankton communities

      Licandro, P.; Head, E.; Gislason, A.; Benfield, M.C.; Harvey, M.; Margonski, P.; Silke, J. (ICES, 2011)
      Phytoplankton and zooplankton occupy pivotal positions within marine ecosystems. These small organisms fuel and support the foodwebs upon which almost all higher organisms depend. Fisheries and related economic activities are highly dependent on the production, size, and composition of zooplankton which, in turn, rely on primary production by phytoplankton. In addition to their role as prey for herbivorous zooplankton, phytoplankton absorb enormous quantities of dissolved CO2 via photosynthesis. Zooplankton then plays an essential role in the biological pump by consuming phytoplankton and transporting carbon from the upper ocean to the deep ocean, where it is sequestered for hundreds to thousands of years. Given the ecological and economic importance of phyto‐ and zooplankton, it is essential to understand and predict how they are likely to respond to climate change. Climate‐related hydrographic changes may also directly affect the abundance and composition of zooplankton, shifting the distribution of dominant species, changing the structure of the zooplankton community, and altering the timing, duration, and efficiency of zooplankton reproductive cycles. Ocean acidification through increased carbon dioxide dissolution in the upper ocean is lowering the pH in surface waters. A lower pH could impair the physiology and ultimately the abundance of many phytoplankton and zooplankton species. It is important to understand how phytoplankton and zooplankton are likely to respond to climate‐induced changes in the ocean. This section explores what is known about the sensitivity of phytoplankton and zooplankton to climate change and summarizes the trends that are evident in plankton communities within the ICES Area.
    • 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.