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A variety of shellfish toxin-producing Harmful Algal Blooms (HABs) occur every year in coastal temperate waters worldwide. These toxic HABs may cause lengthy (months) harvesting bans of mussels and other suspension feeding bivalves exposed to their blooms. To safeguard public health and the shellfish industry, European Union regulations request periodic monitoring of potentially toxic microalgae in seawater and phycotoxins in live bivalve molluscs from shellfish production areas. Monitoring of other toxic microalgae, e.g., fish killers, is based solely on cell counts. Morphological identification and quantification of microalgal cells with light microscopy is time-consuming, requires a good expertise, and accurate identification to species level (e.g., Pseudo-nitzschia species) may require electron microscopy. Toxicity varies among morphologically similar species; there are toxic and non-toxic strains of the same species. Molecular techniques using ribosomal DNA sequences offer a possibility to identify and detect precisely the potentially toxic genus/species. In an earlier project (MIDTAL), specific probes against rRNA sequences of all HAB taxa, known at the time of the project, affecting shellfish areas worldwide were designed, and those affecting Europe were tested and calibrated against rRNA extracts of clonal cultures and field samples. Microarray technology was adopted to relate to cell numbers the fluorescence signal from the reaction of all target species probes spotted in the microarray slides with those present in a single sample extract. The EMERTOX project aimed to develop a more automatic "Lab on a chip" (LOC) technology, including a non- (cell) disruptive water concentration system and biosensors for HAB cells detection. Here, calibration curves are presented against toxic microalgae (cultures and field samples) causing endemic and emerging toxicity events in Galicia (NW Spain) and Portugal. Results here relating cell numbers to electrochemical signals will be used in an early warning biosensor for toxic algae., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Monitoring drinking water quality is an important public health issue and pathogenic organisms present a particularly serious health hazard in freshwater bodies. However, many pathogenic bacteria, including cyanobacteria, and pathogenic protozoa can be swept into coastal lagoons and into near-shore marine environments where they continue to grow and pose a health threat to marine mammals and invertebrates. In this study, we tested the suitability of a phylochip (microarray for species detection) developed for freshwater pathogenic organisms to be applied to samples taken across a marine/freshwater interface at monthly intervals for two years. Toxic cyanobacteria and pathogenic protozoa were more numerous in a coastal lagoon than at the freshwater or marine site, indicating that this microarray can be used to detect the presence of these pathogens across a marine/freshwater interface and thus the potential for toxicity to occur within the entire watershed., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019. Published by Elsevier B.V.)

Blooms of ichthyotoxic microalgae pose a great challenge to the aquaculture industry world-wide, and there is a need for fast and specific methods for their detection and quantification in monitoring programs. In this study, quantitative real-time PCR (qPCR) assays for the detection and enumeration of three ichthyotoxic flagellates: the dinoflagellate Karenia mikimotoi (Miyake & Kominami ex Oda) Hansen & Moestrup and the two raphidophytes Heterosigma akashiwo (Hada) Hada ex Hara & Chihara and Fibrocapsa japonica Toriumi & Takano were developed. Further, a previously published qPCR assay for the dinoflagellate Karlodinium veneficum (Ballantine) Larsen was used. Monthly samples collected for three years (Aug 2009-Jun 2012) in outer Oslofjorden, Norway were analysed, and the results compared with light microscopy cell counts. The results indicate a higher sensitivity and a lower detection limit (down to 1 cell L -1 ) for both qPCR assays. Qualitative and semi-quantitative results were further compared with those obtained by environmental 454 high throughput sequencing (HTS, metabarcoding) and scanning electron microscopy (SEM) examination from the same samplings. All four species were detected by qPCR and HTS and/or SEM in outer Oslofjorden (Aug 2009-Jun 2012); Karlodinium veneficum was present year-round, whereas Karenia mikimotoi, Heterosigma akashiwo and Fibrocapsa japonica appeared mainly during the autumn in all three years. This is the first observation of Fibrocapsa japonica in Norwegian coastal waters. This species has previously been recorded off the Swedish west coast and German Bight, which may suggest a northward dispersal., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Harmful cyanobacterial blooms are a major threat to water quality and human health. Adequate risk assessment is thus required, which relies strongly on comprehensive monitoring. Here, we tested novel multi-probe RNA chips developed in the European project, μAqua, to determine the abundance of harmful cyanobacterial species and expression of selected toxin genes in six Dutch lakes. All of the targeted cyanobacterial genera, except for Planktothrix, were detected using the microarray, with predominance of Dolichospermum and Microcystis signals, of which the former was found across all sites and detected by the probes for Anabaena where it was formerly placed. These were confirmed by microscopic cell counts at three sites, whereas at the other sites, microscopic cell counts were lower. Probe signals of Microcystis showed larger variation across sites but also matched microscopic counts for three sites. At the other sites, microscopic counts were distinctly higher. We detected anatoxin-a in the water at all sites, but unfortunately no genes for this toxin were on this generation of the toxin array. For microcystins, we found none or low concentrations in the water, despite high population densities of putative microcystin producers (i.e. Microcystis, Dolichospermum). The described method requires further testing with a wider range of cyanobacterial communities and toxin concentrations before implementation into routine cyanobacterial risk assessment. Yet, our results demonstrate a great potential for applying multi-probe RNA chips for species as well as toxins to eutrophic waters with high cyanobacterial densities as a routine monitoring tool and as a predictive tool for toxin potential., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Harmful algal blooms (HABs) are becoming more frequent as climate changes, with tropical species moving northward. Monitoring programs detecting the presence of toxic algae before they bloom are of paramount importance to protect aquatic ecosystems, aquaculture, human health and local economies. Rapid and reliable species identification methods using molecular barcodes coupled to biosensor detection tools have received increasing attention over the past decade as an alternative to the impractical standard microscopic counting-based techniques. This work reports on a PCR amplification-free electrochemical genosensor for the enhanced selective and sensitive detection of RNA from multiple Mediterranean toxic algal species. For a sandwich hybridization (SHA), we designed longer capture and signal probes for more specific target discrimination against a single base-pair mismatch from closely related species and for reproducible signals. We optimized experimental conditions, viz., minimal probe concentration in the SHA on a screen-printed gold electrode and selected the best electrochemical mediator. Probes from 13 Mediterranean dinoflagellate species were tested under optimized conditions and the format further tested for quantification of RNA from environmental samples. We not only enhanced the selectivity and sensitivity of the state-of-the-art toxic algal genosensors but also increased the repertoire of toxic algal biosensors in the Mediterranean, towards an integral and automatic monitoring system., (Copyright © 2016. Published by Elsevier B.V.)

Over the past few decades, there has been an increased frequency and duration of cyanobacterial Harmful Algal Blooms (HABs) in freshwater systems globally. These can produce secondary metabolites called cyanotoxins, many of which are hepatotoxins, raising concerns about repeated exposure through ingestion of contaminated drinking water or food or through recreational activities such as bathing/swimming. An ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) multi-toxin method has been developed and validated for freshwater cyanotoxins; microcystins-LR, -YR, -RR, -LA, -LY and -LF, nodularin, cylindrospermopsin, anatoxin-a and the marine diatom toxin domoic acid. Separation was achieved in around 9min and dual SPE was incorporated providing detection limits of between 0.3 and 5.6ng/L of original sample. Intra- and inter-day precision analysis showed relative standard deviations (RSD) of 1.2-9.6% and 1.3-12.0% respectively. The method was applied to the analysis of aquatic samples (n=206) from six European countries. The main class detected were the hepatotoxins; microcystin-YR (n=22), cylindrospermopsin (n=25), microcystin-RR (n=17), microcystin-LR (n=12), microcystin-LY (n=1), microcystin-LF (n=1) and nodularin (n=5). For microcystins, the levels detected ranged from 0.001 to 1.51μg/L, with two samples showing combined levels above the guideline set by the WHO of 1μg/L for microcystin-LR. Several samples presented with multiple toxins indicating the potential for synergistic effects and possibly enhanced toxicity. This is the first published pan European survey of freshwater bodies for multiple biotoxins, including two identified for the first time; cylindrospermopsin in Ireland and nodularin in Germany, presenting further incentives for improved monitoring and development of strategies to mitigate human exposure., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Harmful algal blooms (HABs) are a natural global phenomena emerging in severity and extent. Incidents have many economic, ecological and human health impacts. Monitoring and providing early warning of toxic HABs are critical for protecting public health. Current monitoring programmes include measuring the number of toxic phytoplankton cells in the water and biotoxin levels in shellfish tissue. As these efforts are demanding and labour intensive, methods which improve the efficiency are essential. This study compares the utilisation of a multitoxin surface plasmon resonance (multitoxin SPR) biosensor with enzyme-linked immunosorbent assay (ELISA) and analytical methods such as high performance liquid chromatography with fluorescence detection (HPLC-FLD) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) for toxic HAB monitoring efforts in Europe. Seawater samples (n=256) from European waters, collected 2009-2011, were analysed for biotoxins: saxitoxin and analogues, okadaic acid and dinophysistoxins 1/2 (DTX1/DTX2) and domoic acid responsible for paralytic shellfish poisoning (PSP), diarrheic shellfish poisoning (DSP) and amnesic shellfish poisoning (ASP), respectively. Biotoxins were detected mainly in samples from Spain and Ireland. France and Norway appeared to have the lowest number of toxic samples. Both the multitoxin SPR biosensor and the RNA microarray were more sensitive at detecting toxic HABs than standard light microscopy phytoplankton monitoring. Correlations between each of the detection methods were performed with the overall agreement, based on statistical 2×2 comparison tables, between each testing platform ranging between 32% and 74% for all three toxin families illustrating that one individual testing method may not be an ideal solution. An efficient early warning monitoring system for the detection of toxic HABs could therefore be achieved by combining both the multitoxin SPR biosensor and RNA microarray., (Copyright © 2016 Elsevier B.V. All rights reserved.)