Co-localisation of Azaspiracid Analogs with the Dinoflagellate Species Azadinium spinosum and Amphidoma languida in the Southwest of Ireland.

Phytoplankton and biotoxin monitoring programmes have been implemented in many countries to protect human health and to mitigate the impacts of harmful algal blooms (HABs) on the aquaculture industry. Several amphidomatacean species have been confirmed in Irish coastal waters, including the azaspiracid-producing species Azadinium spinosum and Amphidoma languida. Biogeographic distribution studies have been hampered by the fact that these small, armoured dinoflagellates share remarkably similar morphologies when observed by light microscopy. The recent releases of species-specific molecular detection assays have, in this context, been welcome developments. A survey of the south west and west coasts of Ireland was carried out in August 2017 to investigate the late summer distribution of toxic amphidomataceans and azaspiracid toxins. Azadinium spinosum and Am. languida were detected in 83% of samples in the southwest along the Crease Line and Bantry Bay transects between 20 and 70 m depth, with maximal cell concentrations of 7000 and 470,000 cells/L, respectively. Azaspiracid concentrations were well aligned with the distributions of Az. spinosum and Am. languida, up to 1.1 ng/L and 4.9 ng/L for combined AZA-1, -2, -33, and combined AZA-38, -39, respectively. Although a snapshot in time, this survey provides new insights in the late summer prominence of AZAs and AZA-producing species in the southwest of Ireland, where major shellfish aquaculture operations are located. Results showed a substantial overlap in the distribution of amphidomatacean species in the area and provide valuable baseline information in the context of ongoing monitoring efforts of toxigenic amphidomataceans in the region.

Future HAB science: Directions and challenges in a changing climate.

There is increasing concern that accelerating environmental change attributed to human-induced warming of the planet may substantially alter the patterns, distribution and intensity of Harmful Algal Blooms (HABs). Changes in temperature, ocean acidification, precipitation, nutrient stress or availability, and the physical structure of the water column all influence the productivity, composition, and global range of phytoplankton assemblages, but large uncertainty remains about how integration of these climate drivers might shape future HABs. Presented here are the collective deliberations from a symposium on HABs and climate change where the research challenges to understanding potential linkages between HABs and climate were considered, along with new research directions to better define these linkages. In addition to the likely effects of physical (temperature, salinity, stratification, light, changing storm intensity), chemical (nutrients, ocean acidification), and biological (grazer) drivers on microalgae (senso lato), symposium participants explored more broadly the subjects of cyanobacterial HABs, benthic HABs, HAB effects on fisheries, HAB modelling challenges, and the contributions that molecular approaches can bring to HAB studies. There was consensus that alongside traditional research, HAB scientists must set new courses of research and practices to deliver the conceptual and quantitative advances required to forecast future HAB trends. These different practices encompass laboratory and field studies, long-term observational programs, retrospectives, as well as the study of socioeconomic drivers and linkages with aquaculture and fisheries. In anticipation of growing HAB problems, research on potential mitigation strategies should be a priority. It is recommended that a substantial portion of HAB research among laboratories be directed collectively at a small sub-set of HAB species and questions in order to fast-track advances in our understanding. Climate-driven changes in coastal oceanographic and ecological systems are becoming substantial, in some cases exacerbated by localized human activities. That, combined with the slow pace of decreasing global carbon emissions, signals the urgency for HAB scientists to accelerate efforts across disciplines to provide society with the necessary insights regarding future HAB trends.

Biosensors for the monitoring of harmful algal blooms.

Harmful algal blooms (HABs) are a major global concern due to their propensity to cause environmental damage, healthcare issues and economic losses. In particular, the presence of toxic phytoplankton is a cause for concern. Current HAB monitoring programs often involve laborious laboratory-based analysis at a high cost and with long turnaround times. The latter also hampers the potential to develop accurate and reliable models that can predict HAB occurrence. However, a promising solution for this issue may be in the form of remotely deployed biosensors, which can rapidly and continuously measure algal and toxin levels at the point-of-need (PON), at a low cost. This review summarises the issues HABs present, how they are difficult to monitor and recently developed biosensors that may improve HAB-monitoring challenges.

Forecasting the risk of harmful algal blooms.

The "Applied Simulations and Integrated Modelling for the Understanding of Harmful Algal Blooms" (Asimuth) project sought to develop a harmful algal bloom (HAB) alert system for Atlantic Europe. This was approached by combining, at a national or regional level, regulatory monitoring phytoplankton and biotoxin data with satellite remote sensing and other information on current marine conditions, coupled with regional scale models that included a representation of HAB transport. Synthesis of these products was achieved by expert interpretation within HAB risk alert bulletins that were prepared on a regular basis (typically weekly) for use by the aquaculture industry. In this preface to the Asimuth Special Issue we outline the main HAB species of concern in the region and the strengths and limitations of different methodologies to provide early warning of their blooms.

Applied simulations and integrated modelling for the understanding of toxic and harmful algal blooms (ASIMUTH): Integrated HAB forecast systems for Europe's Atlantic Arc.

Reasons for the emergent interest in HABs are abundant, including concerns associated with human health, adverse effects on biological resources, economic losses attributed to recreation, tourism and seafood related industries, and the cost of maintaining public advisory services and monitoring programs for shellfish toxins and water quality. The impact of HABs can potentially be mitigated by early warning of their development. In this regard the project ASIMUTH (Applied Simulations and Integrated Modelling for the Understanding of Toxic and Harmful algal blooms) was borne in order to develop short term HAB alert systems for Atlantic Europe. This was achieved using information on the most current marine conditions (weather, water characteristics, toxicity, harmful algal presence etc.) combined with high resolution local numerical predictions. This integrated, multidisciplinary, trans-boundary approach to the study of HABs developed during ASIMUTH led to a better understanding of the physical, chemical and ecological factors controlling these blooms, as well as their impact on human activities. The outcome was an appropriate alert system for an effective management of areas that are usually associated with HAB events and where these episodes may have a more significant negative impact on human activities. Specifically for the aquaculture industry, the information provided enabled farmers to adapt their working practices in time to prevent mortalities in finfish farms and/or manage their shellfish harvest more effectively. This paper summarises the modelling and alert developments generated by the ASIMUTH project.

Harmful algal bloom forecast system for SW Ireland. Part I: Description and validation of an operational forecasting model.

A 3D primitive equation coastal ocean model for southwest Ireland, called the Bantry Bay model, was developed and implemented operationally. Validated model outputs have multiple uses. One of the incentives to develop the model was to explore the possible transport pathways that carry harmful algae blooms (HAB) into Bantry Bay. The model is nested offline in a regional North East Atlantic operational model. Surface forcing is taken from the half-degree Global Forecasting System, available at three-hourly intervals. Heat fluxes are calculated from the bulk formulae. Surface freshwater fluxes are obtained from the prescribed rainfall rates and the evaporation rates calculated by the model. Freshwater discharges from five rivers are included in the model. Model validation and the model skill in representing the water level, currents, temperature and salinity in the bay are reported. A scoring system based on the average adjusted relative mean absolute error for the predicted currents was used. An upgrade to a higher score was achieved through the incorporation of local winds into the surface forcing and by varying the bottom roughness coefficient. The model, designed to work in forecast mode, can replicate the main oceanographic features in the region. The model forecast is used in a decision support system for HAB alerts. An operational HAB alert system did not exist in Ireland prior to the use of this model.

Potential impact of an exceptional bloom of Karenia mikimotoi on dissolved oxygen levels in waters off western Ireland.

In the summer of 2005 an exceptional bloom of the dinoflagellate Karenia mikimotoi occurred along Ireland's Atlantic seaboard and was associated with the mass mortality of both benthic and pelagic marine life. Oxygen depletion, cellular toxicity and physical smothering, are considered to be the main factors involved in mortality. In this paper we use a theoretical approach based on stoichiometry (the Anderson ratio) and an average K. mikimotoi cellular carbon content of 329pgCcell-1 (n=20) to calculate the carbonaceous and nitrogenous oxygen demand following bloom collapse. The method was validated against measurements of biochemical oxygen demand and K. mikimotoi cell concentration. The estimated potential oxygen utilisation (POU) was in good agreement with field observations across a range of cell concentrations. The magnitude of POU following bloom collapse, with the exception of three coastal areas, was considered insufficient to cause harm to most marine organisms. This indicates that the widespread occurrence of mortality was primarily due to other factors such as cellular toxicity and/or mucilage production, and not oxygen depletion or related phenomena. In Donegal Bay, Kilkieran Bay and inner Dingle Bay, where cell densities were in the order of 106cellsL-1, estimated POU was sufficient to cause hypoxia. Of the three areas, Donegal Bay is considered to be the most vulnerable due to its hydrographic characteristics (seasonally stratified, weak residual flow) and hypoxic conditions (2.2mgL-1 O2) were directly observed in the Bay post bloom collapse. Here, depending on the time of bloom collapse, depressed DO levels could persist for weeks and continue to have a potentially chronic impact on the Bay.

Harmful algal bloom forecast system for SW Ireland. Part II: Are operational oceanographic models useful in a HAB warning system.

This study investigated the application of a three-dimensional physical hydrodynamic model in a harmful algal bloom forecast system for Bantry Bay, southwest Ireland. Modelled oceanographic conditions were studied and used to help understand observed changes in the chemical and biological patterns from the national biotoxins and phytoplankton monitoring program. The study focused on two toxic events in 2013. An upwelling event was predicted by the model prior to the appearance and population increase of potentially toxic diatoms, Pseudo-nitzschia, and associated domoic acid in shellfish. A downwelling episode was provided as a forecast in the model prior to the arrival of a Dinophysis bloom and detection of its associated biotoxins in Bay shellfish. The modelled forecast products developed included expected surface, mid-depth and bottom current pathways at the mouth of the Bay and on the adjacent shelf. The rate and direction of water volume flow at the mouth and mid-bay sections were produced by the model to examine predicted upwelling and downwelling pulses. The model also calculated the evolution of water properties (temperature, salinity and density) with depth along the Bay axis and on the adjacent continental shelf. Direct measurements of water properties at a fixed point, mid-bay, were comparable to model calculations. The operational model for southwest Ireland produces a reliable 3-day physical hydrodynamic forecast of the dominant regional physical processes that result in water exchange events between Bantry Bay and its adjacent shelf. While simulated physical hydrodynamics were provided as a 3-day forecast, the upwelling and downwelling signals from the model, closely linked to toxic HAB episodes, were evident up to 10 days prior to the contamination of shellfish in the Bay.

Bioactive compounds from marine mussels and their effects on human health.

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.

Real-time PCR detection of Dinophysis species in Irish coastal waters.

Diarrhetic shellfish toxin-producing Dinophysis species occur in Irish coastal waters throughout the year. Dinophysis acuta and Dinophysis acuminata are the most commonly occurring species and are responsible for the majority of closures of Irish mussel farms. This study describes the development of a qualitative real-time polymerase chain reaction (PCR) assay for identification of D. acuta and D. acuminata in Irish coastal waters. DNA sequence information for the D1-D2 region of the large ribosomal sub-unit (LSU) was obtained, following single-cell PCR of D. acuta and D. acuminata cells isolated from Irish coastal locations. PCR primers and hybridization probes, specific for the detection of D. acuta, were designed for real-time PCR on the LightCycler™. The LightCycler™ software melt curve analysis programme determined that D. acuta was identified by a melt-peak at 61°C, while D. acuminata cells produced a melt peak at 48°C. The limit of detection of the real-time PCR assay was determined to be one to ten plasmid copies of the LSU D1-D2 target region for both species and one to five D. acuminata cells. Lugol's preserved water samples were also tested with the assay. The real-time PCR assay identified Dinophysis species in 100% of samples found to contain Dinophysis species by light microscopy and had a greater than 90% correlation with light microscopy for identification of D. acuta and D. acuminata in the samples. The assay can identify and discriminate D. acuta and D. acuminata at low numbers in Irish waters and has the potential to add value to the Irish phytoplankton monitoring programme.

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.

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. 2.24mg/kg in hepatopancreas, which corresponds to a maximum whole flesh AZA1-equivalent of ca. 0.34mg/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 LC(50) of 0.70mg 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 0.16mg/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 0.16mg/kg for Azaspiracids in shellfish.

Establishing boundary classes for the classification of UK marine waters using phytoplankton communities.

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.