First Finding of Ostreopsis cf. ovata Toxins in Marine Aerosols

Since the late 1990s, a respiratory syndrome has been repetitively observed in humans concomitant with Ostreopsis spp. blooms (mainly O. cf. ovata) in the Mediterranean area. Previous studies have demonstrated that O. cf. ovata produces analogues of palytoxin (ovatoxins and a putative palytoxin), one of the most potent marine toxins. On the basis of the observed association between O. cf. ovata blooms, respiratory illness in people, and detection of palytoxin complex in algal samples, toxic aerosols, containing Ostreopsis cells and/or the toxins they produce, were postulated to be the cause of human illness. A small scale monitoring study of marine aerosol carried out along the Tuscan coasts (Italy) in 2009 and 2010 is reported. Aerosols were collected concomitantly with O. cf. ovata blooms, and they were analyzed by both PCR assays and LC-HRMS. The results, besides confirming the presence of O. cf. ovata cells, demonstrated for the first time the occurrence of ovatoxins in the aerosol at levels of 2.4 pg of ovatoxins per liter of air. Given the lack of toxicological data on palytoxins by inhalation exposure, our results are only a first step toward a more comprehensiveunderstanding of the Ostreopsis-related respiratory syndrome

In-house validation of a liquid chromatography tandem mass spectrometry method for the analysis of lipophilic marine toxins in shellfish using matrix-matched calibration

A liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the quantitative analysis of lipophilic marine toxins in shellfish extracts (mussel, oyster, cockle and clam) was validated in-house using European Union (EU) Commission Decision 2002/657/EC as a guideline. The validation included the toxins okadaic acid (OA), yessotoxin (YTX), azaspiracid-1 (AZA1), pectenotoxin-2 (PTX2) and 13-desmethyl spirolide-C (SPX1). Validation was performed at 0.5, 1 and 1.5 times the current EU permitted levels, which are 160 µg kg-1 for OA, AZA1 and PTX2 and 1,000 µg kg-1 for YTX. For SPX1, 400 µg kg-1 was chosen as the target level as no legislation has been established yet for this compound. The method was validated for determination in crude methanolic shellfish extracts and for extracts purified by solid-phase extraction (SPE). Extracts were also subjected to hydrolysis conditions to determine the performance of the method for OA and dinophysistoxin esters. The toxins were quantified against a set of matrix-matched standards instead of standard solutions in methanol. To save valuable standard, methanolic extract instead of the homogenate was spiked with the toxin standard. This was justified by the fact that the extraction efficiency is high for all relevant toxins (above 90%). The method performed very well with respect to accuracy, intraday precision (repeatability), interday precision (within-laboratory reproducibility), linearity, decision limit, specificity and ruggedness. At the permitted level the accuracy ranged from 102 to 111%, the repeatability from 2.6 to 6.7% and the reproducibility from 4.7 to 14.2% in crude methanolic extracts. The crude extracts performed less satisfactorily with respect to the linearity (less than 0.990) and the change in LC-MS/MS sensitivity during the series (more than 25%). SPE purification resulted in greatly improved linearity and signal stability during the series. Recently the European Food Safety Authority (EFSA) has suggested that to not exceed the acute reference dose the levels should be below 45 µg kg-1 OA equivalents and 30 µg kg-1 AZA1 equivalents. A single-day validation was successfully conducted at these levels. If the regulatory levels are lowered towards the EFSA suggested values, the official methods prescribed in legislation (mouse and rat bioassay) will no longer be sensitive enough. The validated LC-MS/MS method presented has the potential to replace these animal tests

New Approach Using the Real-Time PCR Method for Estimation of the Toxic Marine Dinoflagellate Ostreopsis cf. ovata in Marine Environment

Background: We describe the development and validation of a new quantitative real time PCR (qrt-PCR) method for the enumeration of the toxic benthic dinoflagellate Ostreopsis cf. ovata in marine environment. The benthic Ostreopsis sp. has a world-wide distribution and is associated during high biomass proliferation with the production of potent palytoxin-like compounds affecting human health and environment. Species-specific identification, which is relevant for the complex of different toxins production, by traditional methods of microscopy is difficult due to the high morphological variability, and thus different morphotypes can be easily misinterpreted. Methodology/Findings: The method is based on the SYBR I Green real-time PCR technology and combines the use of a plasmid standard curve with a ‘‘gold standard’’ created with pooled crude extracts from environmental samples collected during a bloom event of Ostreopsis cf. ovata in the Mediterranean Sea. Based on their similar PCR efficiencies (95% and 98%, respectively), the exact rDNA copy number per cell was obtained in cultured and environmental samples. Cell lysates were used as the templates to obtain total recovery of DNA. The analytical sensitivity of the PCR was set at two rDNA copy number and 8.061024 cell per reaction for plasmid and gold standards, respectively; the sensitivity of the assay was of cells g21 fw or 121 in macrophyte and seawater samples, respectively. The reproducibility was determined on the total linear quantification range of both curves confirming the accuracy of the technical set-up in the complete ranges of quantification over time. Conclusions/Significance: We developed a qrt-PCR assay specific, robust and high sample throughput for the absolute quantification of the toxic dinoflagellate Ostreopsis cf. ovata in the environmental samples. This molecular approach may be considered alternative to traditional microscopy and applied for the monitoring of benthic toxic microalgal species in the marine ecosystems

Marine harmful algal blooms, human health and wellbeing : challenges and opportunities in the 21st century

Author Posting. © Marine Biological Association of the United Kingdom, 2015. This is the author's version of the work. It is posted here by permission of Marine Biological Association of the United Kingdom for personal use, not for redistribution. The definitive version was published in Journal of the Marine Biological Association of the United Kingdom 96 (2016): 61-91, doi:10.1017/S0025315415001733.Microalgal blooms are a natural part of the seasonal cycle of photosynthetic organisms in marine ecosystems. They are key components of the structure and dynamics of the oceans and thus sustain the benefits that humans obtain from these aquatic environments. However, some microalgal blooms can cause harm to humans and other organisms. These harmful algal blooms (HABs) have direct impacts on human health and negative influences on human wellbeing, mainly through their consequences to coastal ecosystem services (valued fisheries, tourism and recreation) and other marine organisms and environments. HABs are natural phenomena, but these events can be favoured by anthropogenic pressures in coastal areas. Global warming and associated changes in the oceans could affect HAB occurrences and toxicity as well, although forecasting the possible trends is still speculative and requires intensive multidisciplinary research. At the beginning of the 21st century, with expanding human populations, particularly in coastal and developing countries, there is an urgent need to prevent and mitigate HABs’ impacts on human health and wellbeing. The available tools to address this global challenge include maintaining intensive, multidisciplinary and collaborative scientific research, and strengthening the coordination with stakeholders, policymakers and the general public. Here we provide an overview of different aspects to understand the relevance of the HABs phenomena, an important element of the intrinsic links between oceans and human health and wellbeing.The research was funded in part by the UK Medical Research Council (MRC) and UK Natural Environment Research Council (NERC) for the MEDMI Project; the National Institute for Health Research Health Protection Research Unit (NIHR HPRU) in Environmental Change and Health at the London School of Hygiene and Tropical Medicine in partnership with Public Health England (PHE), and in collaboration with the University of Exeter, University College London and the Met Office; and the European Regional Development Fund Programme and European Social Fund Convergence Programme for Cornwall and the Isles of Scilly (University of Exeter Medical School). EB was supported by the CTM2014-53818-R project, from the Spanish Government (MINECO). KDA was in receipt of funding from the BBSRC-NERC research programme for multidisciplinary studies in sustainable aquaculture: health, disease and the environment. P. Hess was supported by Ifremer (RISALTOX) and the Regional Council of the Pays de la Loire (COSELMAR). Porter Hoagland was supported by the US National Science Foundation under NSF/CNH grant no. 1009106.2016-05-2

Marine Toxins: Chemistry, Toxicity, Occurrence and Detection, with Special Reference to the Dutch Situation

Various species of algae can produce marine toxins under certain circumstances. These toxins can then accumulate in shellfish such as mussels, oysters and scallops. When these contaminated shellfish species are consumed severe intoxication can occur. The different types of syndromes that can occur after consumption of contaminated shellfish, the corresponding toxins and relevant legislation are discussed in this review. Amnesic Shellfish Poisoning (ASP), Paralytic Shellfish Poisoning (PSP), Diarrheic Shellfish Poisoning (DSP) and Azaspiracid Shellfish Poisoning (AZP) occur worldwide, Neurologic Shellfish Poisoning (NSP) is mainly limited to the USA and New Zealand while the toxins causing DSP and AZP occur most frequently in Europe. The latter two toxin groups are fat-soluble and can therefore also be classified as lipophilic marine toxins. A detailed overview of the official analytical methods used in the EU (mouse or rat bioassay) and the recently developed alternative methods for the lipophilic marine toxins is given. These alternative methods are based on functional assays, biochemical assays and chemical methods. From the literature it is clear that chemical methods offer the best potential to replace the animal tests that are still legislated worldwide. Finally, an overview is given of the situation of marine toxins in The Netherlands. The rat bioassay has been used for monitoring DSP and AZP toxins in The Netherlands since the 1970s. Nowadays, a combination of a chemical method and the rat bioassay is often used. In The Netherlands toxic events are mainly caused by DSP toxins, which have been found in Dutch shellfish for the first time in 1961, and have reoccurred at irregular intervals and in varying concentrations. From this review it is clear that considerable effort is being undertaken by various research groups to phase out the animal tests that are still used for the official routine monitoring programs

Marine mollusks from Australia and New Zealand: Chemical and Ecological Studies

Marine mollusks contain structurally diverse terpenes, polyketides, polypropionates and nitrogenous metabolites that may confer an ecological advantage on the host organism. From a chemical perspective, the most studied Australian taxa include representatives of the nudibranchs and sea hares, which are characterised by terpenes acquired from their specialised diets of sponges and algae, respectively. In contrast, siphonariid limpets that are prevalent on temperate seashores carry out de novo biosynthesis of polypropionate metabolites. Nitrogenous compounds isolated from Australian marine mollusks include precursors to the first commercially significant marine bioproduct, Tyrian Purple, and metabolites that are characteristic of ingested cyanobacteria