Rapid and sensitive detection of domoic acid in shellfish using a magnetic bead-based competitive ELISA with a high-affinity peptide as a molecular binder.

Addressing the critical health concerns posed by domoic acid (DA), a neurotoxic compound produced by toxic marine algae and bioaccumulated in shellfish, necessitates the development of a rapid, precise, and robust detection system. Traditional DA detection methods have stability and sensitivity issues, which hinder effective toxin detection. To overcome these limitations, we developed a novel direct competitive enzyme-linked immunosorbent assay (dc-ELISA) platform that utilizes peptide-immobilized magnetic beads (MGBs/peptide). The affinity peptides identified through phage display and chemically synthesized with biotin labels present an innovative alternative to conventional antibodies for ELISA applications. Streptavidin-modified MGBs were used as the bioreceptor carriers to facilitate magnetic separation and simplify sample preparation, making the MGB/peptide-based dc-ELISA platform an ideal tool for comprehensive monitoring efforts. The developed platform exhibits a detection range of 0.5-10 ng mL-1 and a low limit of detection of 0.29 ng mL-1, offering enhanced sensitivity and cost-effectiveness. Moreover, our developed dc-ELISA demonstrated a high recovery rate when validated with DA-spiked CRM-mussel samples. This method overcomes the limitations of traditional detection techniques and offers a scalable and efficient approach to marine toxin surveillance with improved marine environmental monitoring and public health management.

The queen scallop Aequipecten opercularis: A slow domoic acid depurator?

Domoic acid (DA) is a dangerous phycotoxin produced by several strains of diatoms of the genus Pseudo-nitzschia, and responsible for Amnesic Shellfish Poisoning (ASP) in humans. The increasingly intense ASP-outbreaks along the English Channel over the last three decades have forced persistent harvest closures of economically important and highly contaminated bivalve stocks exhibiting slow DA-depuration rates, like the king scallop Pecten maximus. Under this scenario, other pectinid species, such as the queen scallop Aequipecten opercularis have been empirically proposed as alternative resources to redress the high economic losses due to the banning of the exploitation of P. maximus. Nevertheless, the kinetics of DA depuration in A. opercularis have not been assessed so far, and its direct extraction after ASP-episodes could represent a serious threat to public health. Hence, the main objective of this work was to estimate the DA-depuration rate in the digestive gland (DG) of naturally contaminated scallops A. opercularis after a toxic Pseudo-nitzschia australis bloom subjected to experimental depuration in the laboratory for 30 days. This study also intended to go further in the knowledge about the anatomical distribution of DA in scallop tissues, and corroborate the implications of autophagy in DA-sequestration in the DG of this species as recently hypothesized. In the DG, the DA-depuration rate (0.018 day-1) suggested that even with toxin burdens as low as 40 mg⋅kg-1 in the DG, queen scallops may remain contaminated for about 70 days, thus longer under intensely contamination scenarios. The subcellular analyses corroborated DA-sequestration mainly through late-autophagy within residual bodies in the DG, without differences in the frequencies of anti-DA labeled residual bodies across the entire depuration process. These results revealed that A. opercularis cannot be considered a fast DA-depurator, and represent a baseline knowledge for decision-making about harvesting natural beds of queen scallops after toxic Pseudo-nitzschia blooms. The findings of this work also represent a cornerstone for further research to accelerate DA-depuration in this species.

A biosensor monitoring approach for toxic algae: Construction of calibration curves to infer cell numbers in field material.

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.

Molecular monitoring of Dinophysis species assemblage in mussel farms in the Northwestern Adriatic Sea.

Several Dinophysis species can produce potent lipophilic toxins that pose a risk to human health when contaminated seafood is consumed, especially filter-feeding bivalve mussels. In the mussel farms of the Northwestern Adriatic Sea, seawater and seafood are regularly monitored for the presence of Dinophysis species and their associated toxins, but the current methodological approaches, such as light microscopy determinations, require a long time to make results available to local authorities. A molecular qPCR-based assay can be used to quantify various toxic Dinophysis species in a shorter timeframe. However, this approach is not currently employed in official testing activities. In this study, field samples were collected monthly or bi-weekly over one year from various mussel farms along the Northwestern Adriatic coast. The abundance of Dinophysis species in the seawater was determined using both traditional microscopy and qPCR assays. In addition, the concentration of lipophilic toxins for DSP in mussel flesh was quantified using LC-MS/MS focusing on the okadaic acid group. Dinophysis spp. site-specific single cells were isolated and analysed by qPCR yielding a mean rDNA copy number per cell of 1.21 × 104 ± 1.81 × 103. The qPCR assay gave an efficiency of 98 % and detected up to 10 copies of the rDNA target gene. The qPCR and light microscopy determinations in environmental samples showed a significant positive correlation (Spearman rs = 0.57, p-value < 0.001) with a ratio of 2.24 between the two quantification methods, indicating that light microscopy estimates were generally 44.6 % lower than those obtained by the qPCR assay. The qPCR approach showed several advantages such as rapidity, sensitivity and efficiency over conventional microscopy analysis, showing its potential future role in phytoplankton monitoring under the Official Controls Regulations for shellfish.

First record of paralytic shellfish toxins in marine pufferfish from the Spanish Mediterranean coast using cell-based assay, automated patch clamp and HPLC-FLD.

Pufferfish is one of the most poisonous marine organisms, responsible for numerous poisoning incidents and some human fatalities due to its capability to accumulate potent neurotoxins such as tetrodotoxins (TTXs) and paralytic shellfish toxins (PSTs). In this study, tissue extracts (muscle, skin, liver, intestinal tract and gonads) obtained from sixteen pufferfish specimens of the Lagocephalus lagocephalus and Sphoeroides pachygaster species, collected along the Spanish Mediterranean coast, were analysed for the presence of voltage-gated sodium channel (also known as Nav channel) blockers using cell-based assay (CBA) and automated patch clamp (APC). No toxicity was observed in any of the S. pachygaster specimens, but toxicity was detected in the liver of most L. lagocephalus specimens. Instrumental analysis of these specimens, as well as in one Lagocephalus sceleratus specimen, by high-performance liquid chromatography coupled to fluorescence detection (HPLC-FLD) was performed, which confirmed the presence of PSTs only in L. lagocephalus specimens. This analysis reported the presence of saxitoxin (STX) and decarbamoylsaxitoxin (dcSTX) in all positive samples, being dcSTX the major analogue. These results demonstrate the ability of this species to accumulate PSTs, being the first report of the presence of PSTs in Mediterranean L.lagocephalus specimens. Furthermore, the presence of high PSTs contents in all five tested tissues of one L. lagocephalus specimen pointed the risk that the presence of this toxic fish in the Mediterranean Sea may represent for seafood safety and human health in case of accidental consumption.

Nationwide seasonal monitoring of lipophilic marine algal toxins in shellfish and causative microalgae along the coasts of South Korea.

In this study, lipophilic marine algal toxins (LMATs)-producing microalgae were identified at 23 sites along the coasts of Korea, and distribution characteristics of LMATs in phytoplankton and mussels were investigated. The causative microalgae, including Gonyaulux spinifera, Dinophysis acuminata, D. caudata, and D. fortii, were observed in the study area, with notably higher densities during the summer. Significant correlations were found between the densities of these microalgae and the water temperature. Seasonal distribution patterns of LMATs in phytoplankton closely matched those observed in mussels. Notably, LMAT concentrations in mussels from the Yellow Sea were relatively high. PTX2 was detected predominantly in phytoplankton, and homo-yessotoxin was found mainly in mussels. Overall, LMAT concentrations were elevated in the summer, raising concerns about biotoxin contamination in shellfish. These results provide important insights into the dynamics of unmanaged marine biotoxins in Korea and offer baseline data for future safety management policies and inflow surveillance.

Dye-sensitized NiO photocathode sensor based on signal-sensitive change strategy for MC-LR detection.

A novel photoelectrochemical (PEC) sensor for the detection of microcystic toxins (MC-LR) was developed on the basis of signal-sensitive change strategy. NiO nanoarray as a basic photoactive material was grown directly on the ITO glass electrode via calcination after hydrothermal reaction, while dye N719 was used to sensitize the electrode for enhancing visible light absorption, and the first signal-on stage was obtained. In the meantime, p-type Cu2O was applied as the signal probe attached to probe DNA (DNA2) to improve the sensitivity, and the second "signal-on" stage appeared because of its synergistic effect with NiO nanoarrays. The PEC signal decreases after the target analyte MC-LR is modified on the electrode due to the stronger affinity between MC-LR and its complementary aptamer DNA; part of the Cu2O-DNA2 will dissociate from the electrode. This sensitive signal change strategy allows the detection limit of the MC-LR sensor to be as low as 1.7 pM, which offers an optional method for the sensitive and selective detection of other target molecules, with potential applications in environmental monitoring and toxin determination.

Variability in Paralytic Shellfish Toxin Profiles and Dinoflagellate Diversity in Mussels and Seawater Collected during Spring in Korean Coastal Seawater.

Paralytic shellfish toxins (PSTs) are potent neurotoxins produced by certain microalgae, particularly dinoflagellates, and they can accumulate in shellfish in coastal seawater and thus pose significant health risks to humans. To explore the relationship between toxicity and PST profiles in seawater and mussels, the spatiotemporal variations in PST concentrations and profiles were investigated along the southern coast of Korea under peak PST levels during spring. Seawater and mussel samples were collected biweekly from multiple stations, and the toxin concentrations in the samples were measured. Moreover, the dinoflagellate community composition was analyzed using next-generation sequencing to identify potential PST-producing species. The PST concentrations and toxin profiles showed substantial spatiotemporal variability, with GTX1 and GTX4 representing the dominant toxins in both samples, and C1/2 tending to be higher in seawater. Alexandrium species were identified as the primary sources of PSTs. Environmental factors such as water temperature and salinity influenced PST production. This study demonstrates that variability in the amount and composition of PSTs is due to intricate ecological interactions. To mitigate shellfish poisoning, continuous monitoring must be conducted to gain a deeper understanding of these interactions.

Biogeochemical conditions controlling the intensity of paralytic shellfish poisoning (PSP) outbreak caused by Alexandrium blooms: Results from 6-year field observations in Jinhae-Masan Bay, Korea.

Previous field observations from 2018 to 2019 revealed that paralytic shellfish poisoning (PSP) caused by the blooms of toxic dinoflagellate Alexandrium species occurred under low concentrations of dissolved inorganic nitrogen (DIN) and high concentrations of dissolved organic nitrogen (DON) and humic-like fluorescent dissolved organic matter (FDOMH) in Jinhae-Masan Bay, Korea. In this study, we obtained more data for DIN, DON, FDOMH, and Alexandrium cell density from 2020 to 2023 to further validate environmental conditions for the PSP outbreak. We also measured total hydrolyzed amino acids (THAA) to determine the bioavailability of DON fueling the PSP outbreak. Over the 6-year observations, there was a consistent pattern of low DIN concentrations and high DON and FDOMH concentrations during the PSP outbreak periods. The Alexandrium cell densities, together with the PSP toxin concentrations, increased rapidly under this environmental condition. The PSP outbreak occurs when a large amount of DIN originating from the stream waters near the upstream sites is transformed into DON by biological production before entering the PSP outbreak area. The produced DON is characterized by high bioavailability based on the various AA-derived indices (enantiomeric ratio, degradation index, non-protein AA mole%, and nitrogen-normalized AA yield). In addition, the intensities of PSP outbreaks are mainly dependent on the conversion stage of DIN to DON and enhanced FDOMH. We found that the strong PSP outbreak occurred consistently under a low level of DIN (<1.0 µM) and high levels of DON (>9.0 µM) and FDOMH (>1.5 R.U.). Thus, our results suggest that the monitoring data of environmental conditions can be used to predict the PSP outbreak in the coastal oceans.

Cyanotoxins in Epipelic and Epiphytic Cyanobacteria from a Hypersaline Coastal Lagoon, an Environmental Hazard in Climate Warming Times and a Potential Source of New Compounds.

Cyanobacterial biodiversity and potential toxicity in coastal lagoons have barely been studied despite these transitional water systems being very important in conservation and for the preservation of economic resources. Most of these transitional systems have been affected by eutrophication, and climate change will severely affect them by promoting cyanobacteria growth, especially in Mediterranean areas. This study aims to characterize the diversity of epipelic and epiphytic cyanobacteria species in a Mediterranean coastal lagoon and their potential for toxins production (microcystins and saxitoxins). Strains were isolated and genetically identified. Toxins were extracted and quantified by LC/MS-MS. All the taxa belong to the former Oscillatoriales. The presence of Nodosilinea and Toxifilum is reported for the first time for Spanish waters, but Pseudanabaena, Phormidium, Geitlerinema and Synechococcus also formed part of benthic mats. All the strains contained Microcystin-YR (MC-YR), but saxitoxin (STX) was present only in the extracts of Nodosilinea and Pseudanabena. MC-LY, MC-LW and [D-Asp3] MC-LR were detected in the extracts of Synechococcus and MC-LF in Toxifilum, but at concentrations that did not permit quantification. Toxins production by epipelic and epiphytic strains in coastal lagoons may represent a hazard, but also an opportunity to obtain potentially interesting compounds that should be further studied.

Risk quick sketch: Soil captured most anatoxin-a and microcystin-RR rather than cylindrospermopsin and microcystin-LA/-LY.

Cyanobacteria proliferate in warm, nutrient-rich environments and release toxic secondary metabolites into natural waters. Using cyanotoxin-contaminated water to irrigate crops could expose humans and biota, but the risk may be low if agricultural soils can sorb and retain cyanotoxins. In this report, we compared the sorption and desorption capacities of multi-class cyanotoxins/anabaenopeptins in soils of variable properties with a batch sorption procedure. The target compounds were anabaenopeptin-A, anabaenopeptin-B, anatoxin-a, cylindrospermopsin, and microcystins -LR, -RR, -LA, -LY, -LW, and -LF. Based on solid-liquid distribution coefficients (Kd), we classified cylindrospermopsin and microcystin-LA/-LY as "very low sorptivity", anabaenopeptin-A, -B and microcystin-LR, -LF, and -LW as "low sorptivity", and anatoxin-a and microcystin-RR as "medium sorptivity". We remain concerned about irrigating agricultural land with water contaminated with high levels of CYN and MC-LA/-LY because of their relatively low affinity and high desorption proportion in soils. The results also suggest that soil sorption can be an effective immobilization pathway for anatoxin-a and microcystin-RR. The generated data will be useful for prioritizing research on the most bioavailable cyanotoxins/anabaenopeptins that are likely to be released by the soil matrix, for environmental risk assessment.

Spatiotemporal distribution of lipophilic shellfish toxins in plankton and shellfish in the offshore regions of Shandong province, China.

Lipophilic shellfish toxins (LSTs) threaten the ecosystem health and seafood safety. To comprehensively investigate the spatiotemporal distribution of common LSTs in phytoplankton, zooplankton and economic shellfish, three cruises were conducted in five typical offshore aquaculture regions of Shandong province, China, including Haizhou Bay, Jiaozhou Bay, Sanggou Bay, Sishili Bay and Laizhou Bay, in spring (March-April), summer (July-August) and autumn (November-December). This study revealed significant variability in the composition and content of LSTs in phytoplankton samples collected from different regions. Pectenotoxin-2 (PTX2), dinophysistoxin-1 (DTX1) and okadaic acid (OA) were mainly detected in the ranges of not detected (nd)-5045 pmol g-1 dry weight (dw), nd-159 pmol g-1 dw, and nd-154 pmol g-1 dw, respectively. In zooplankton, DTX1 and OA were the predominant components of LSTs, with the highest levels of ∑LSTs in spring ranging from nd to 406 pmol g-1 dw. Spearman's correlation analysis between LSTs and environmental factors indicated significant correlations for the contents of homo-yessotoxin (hYTX), gymnodimine-A (GYM-A), and spirolide-1 (SPX1) with these factors. Totally relatively low levels of LSTs with dominative DTX1 were detected in economic shellfish, which showed a low risk to seafood safety for human health.

Modulable 3D-printed plantibody-laden platform enabling microscale affinity extraction and ratiometric front-face fluorescence detection of microcystin-LR in marine waters.

A 3D-printed stereolithographic platform for selective biorecognition is designed to enable convective microscale affinity extraction of microcystin-LR (MC-LR) followed by direct solid-phase optosensing exploiting ratiometric front-face fluorescence spectroscopy. For this purpose, a recombinant monoclonal plantibody (recAb) is covalently attached to a 3D-printed structure for sorptive immunoextraction, whereupon the free and unbound primary amino moieties of the recAb are derivatized with a fluorescent probe. The fluorophore-recAb-MC-LR laden device is then accommodated in the cuvette holder of a conventional fluorometer without any instrumental modification for the recording of the solid-phase fluorescence emission. Using Rodbard's four-parameter sigmoidal function, the 3D-printed bioselective platform features a limit of detection (LOD) of 28 ng L-1 using a sample volume of 500 mL, device-to-device reproducibility down to 12%, and relative recoveries ranging from 91 to 100% in marine waters. Printed prototypes are affordable, just 0.4 € per print and ≤ 10 € per device containing recAb. One of the main assets of the miniaturized immunoextraction device is that it performs comparably well in terms of analytical figures of merit with costly mass spectrometric-based analytical methodologies, such as HPLC-MS/MS. The device is readily applicable to high-matrix samples, such as seawater, as opposed to previous biosensing platforms, just applied to freshwater systems.

Spatial distribution of lipophilic shellfish toxins in seawater and sediment in the Bohai Sea and the Yellow Sea, China.

Lipophilic shellfish toxins (LSTs) are widely distributed in marine environments worldwide, potentially threatening marine ecosystem health and aquaculture safety. In this study, two large-scale cruises were conducted in the Bohai Sea and the Yellow Sea, China, in spring and summer 2023 to clarify the composition, concentration, and spatial distribution of LSTs in the water columns and sediments. Results showed that okadaic acid (OA), dinophysistoxin-1 (DTX1) and/or pectenotoxin-2 (PTX2) were detected in 249 seawater samples collected in spring and summer. The concentrations of ∑LSTs in seawater were ranging of ND (not detected) -13.86, 1.60-17.03, 2.73-17.39, and 1.26-30.21 pmol L-1 in the spring surface, intermediate, bottom water columns and summer surface water layers, respectively. The detection rates of LSTs in spring and summer seawater samples were 97% and 100%, respectively. The high concentrations of ∑LSTs were mainly distributed in the north Yellow Sea and the northeast Bohai Sea in spring, and in the northeast Yellow Sea, the waters around Laizhou Bay and Rongcheng Bay in summer. Similarly, only OA, DTX1 and PTX2 were detected in the surface sediments. Overall, the concentration of ∑LSTs in the surface sediments of the northern Yellow Sea was higher than that in other regions. In sediment cores, PTX2 was mainly detected in the upper sediment samples, whereas OA and DTX1 were detected in deeper sediments, and LSTs can persist in the sediments for a long time. Overall, OA, DTX1 and PTX2 were widely distributed in the water column and surface sediments in the Bohai Sea and the Yellow Sea, China. The results of this study contribute to the understanding of spatial distribution of LSTs in seawater and sediment environmental media and provide basic information for health risk assessment of phycotoxins.

Toward Sensitive and Reliable Immunoassays of Marine Biotoxins: From Rational Design to Food Analysis.

Marine biotoxins are metabolites produced by algae that can accumulate in shellfish or fish and enter organisms through the food chain, posing a serious threat to biological health. Therefore, accurate and rapid detection is an urgent requirement for food safety. Although various detection methods, including the mouse bioassay, liquid chromatography-mass spectrometry, and cell detection methods, and protein phosphatase inhibition assays have been developed in the past decades, the current detection methods cannot fully meet these demands. Among these methods, the outstanding immunoassay virtues of high sensitivity, reliability, and low cost are highly advantageous for marine biotoxin detection in complex samples. In this work, we review the recent 5-year progress in marine biotoxin immunodetection technologies such as optical immunoassays, electrochemical immunoassays, and piezoelectric immunoassays. With the assistance of immunoassays, the detection of food-related marine biotoxins can be implemented for ensuring public health and preventing food poisoning. In addition, the immunodetection technique platforms including lateral flow chips and microfluidic chips are also discussed. We carefully investigate the advantages and disadvantages for each immunoassay, which are compared to demonstrate the guidance for selecting appropriate immunoassays and platforms for the detection of marine biotoxins. It is expected that this review will provide insights for the further development of immunoassays and promote the rapid progress and successful translation of advanced immunoassays with food safety detection.

Insights into the nature of ichthyotoxins from the Chrysochromulina leadbeateri blooms in Northern Norwegian fjords.

In May-June 2019, the microalga Chrysochromulina leadbeateri caused a massive fish-killing event in several fjords in Northern Norway, resulting in the largest direct impact ever on aquaculture in northern Europe due to toxic algae. Motivated by the fact that no algal toxins have previously been described from C. leadbeateri, we set out to investigate the chemical nature and toxicity of secondary metabolites in extracts of two strains (UIO 393, UIO 394) isolated from the 2019 bloom, as well as one older strain (UIO 035) isolated during a bloom in Northern Norway in 1991. Initial LC-DAD-MS/MS-based molecular networking analysis of the crude MeOH extracts of the cultivated strains showed that their profiles of small organic molecules, including a large number of known lipids, were very similar, suggesting that the same class of toxin(s) were likely the causative agents of the two harmful algal bloom (HAB) events. Next, bioassay-guided fractionation using the RTgill-W1 cell line and metabolomics analysis pointed to a major compound affording [M + H]+ ions at m/z 1399.8333 as a possible toxin, corresponding to a compound with the formula C67H127ClO27. Moreover, our study unveiled a series of minor analogues exhibiting distinct patterns of chlorination and sulfation, together defining a new family of compounds, which we propose to name leadbeaterins. Remarkably, these suspected toxins were detected in situ in samples collected during the 2019 bloom close to Tromsø, thereby consistent with a role in fish kills. The elemental compositions of the putative C. leadbeateri ichthyotoxins strongly indicate them to be long linear polyhydroxylated polyketides, structurally similar to sterolysins reported from a number of dinoflagellates.

Burden of recreational water illness due to exposure to cyanobacteria and their toxins in freshwater beaches in Canada: protocol of a prospective cohort study.

INTRODUCTION: Cyanobacterial blooms are increasingly common in freshwater sources used for swimming and other recreational water contact activities in Canada. Many species of cyanobacteria can produce toxins that affect human and animal health, but there are limited data on the risk of illness associated with water contact at impacted beaches. METHODS AND ANALYSIS: This study will investigate the incidence of recreational water illness due to exposure to cyanobacterial blooms and their toxins in four targeted and popular freshwater beaches in Ontario, Manitoba and Nova Scotia, Canada. A prospective cohort design and One Health approach will be used. On-site recruitment of recreational water users will be conducted at two beaches per year during the summers of 2024 and 2025. The population of interest includes recreational water users of any age and their pet dogs. After enrolment, an in-person survey will determine beach exposures and confounding factors, and a 3-day follow-up survey will ascertain any acute illness outcomes experienced by participants or their dogs. The target sample size is 2500 recreational water users. Water samples will be taken each recruitment day and analysed for cyanobacterial indicators (pigments), cell counts and toxin levels. Bayesian regression analysis will be conducted to estimate the association with water contact, cyanobacterial levels and risks of different acute illness outcomes. ETHICS AND DISSEMINATION: This study has been approved by the Toronto Metropolitan University Research Ethics Board (REB 2023-461). Study results will be published in a peer-reviewed journal and as infographics on a project website.

Development of a Multiplexed Electrochemical Aptasensor for the Detection of Cyanotoxins.

In this study, we report a multiplexed platform for the simultaneous determination of five marine toxins. The proposed biosensor is based on a disposable electrical printed (DEP) microarray composed of eight individually addressable carbon electrodes. The electrodeposition of gold nanoparticles on the carbon surface offers high conductivity and enlarges the electroactive area. The immobilization of thiolated aptamers on the AuNP-decorated carbon electrodes provides a stable, well-orientated and organized binary self-assembled monolayer for sensitive and accurate detection. A simple electrochemical multiplexed aptasensor based on AuNPs was designed to synchronously detect multiple cyanotoxins, namely, microcystin-LR (MC-LR), Cylindrospermopsin (CYL), anatoxin-α, saxitoxin and okadaic acid (OA). The choice of the five toxins was based on their widespread presence and toxicity to aquatic ecosystems and humans. Taking advantage of the conformational change of the aptamers upon target binding, cyanotoxin detection was achieved by monitoring the resulting electron transfer increase by square-wave voltammetry. Under the optimal conditions, the linear range of the proposed aptasensor was estimated to be from 0.018 nM to 200 nM for all the toxins, except for MC-LR where detection was possible within the range of 0.073 to 150 nM. Excellent sensitivity was achieved with the limits of detection of 0.0033, 0.0045, 0.0034, 0.0053 and 0.0048 nM for MC-LR, CYL, anatoxin-α, saxitoxin and OA, respectively. Selectivity studies were performed to show the absence of cross-reactivity between the five analytes. Finally, the application of the multiplexed aptasensor to tap water samples revealed very good agreement with the calibration curves obtained in buffer. This simple and accurate multiplexed platform could open the window for the simultaneous detection of multiple pollutants in different matrices.

Occurrence and Exposure Assessment of Lipophilic Shellfish Toxins in the Zhejiang Province, China.

Although lipophilic shellfish toxins (LSTs) pose a significant threat to the health of seafood consumers, their systematic investigation and risk assessment remain scarce. The goals of this study were as follows: (1) analyze LST levels in commercially available shellfish in Zhejiang province, China, and determine factors influencing LST distribution; (2) assess the acute dietary risk of exposure to LSTs for local consumers during the red tide period; (3) explore potential health risks of LSTs in humans; and (4) study the acute risks of simultaneous dietary exposure to LSTs and paralytic shellfish toxins (PSTs). A total of 546 shellfish samples were collected. LSTs were detected in 89 samples (16.3%) at concentrations below the regulatory limits. Mussels were the main shellfish species contaminated with LSTs. Spatial variations were observed in the yessotoxin group. Acute exposure to LSTs based on multiple scenarios was low. The minimum tolerable exposure durations for LSTs calculated using the mean and the 95th percentile of consumption data were 19.7 and 4.9 years, respectively. Our findings showed that Zhejiang province residents are at a low risk of combined exposure to LSTs and PSTs; however, the risk may be higher for children under 6 years of age in the extreme scenario.

Effect of temperature, salinity and nutrients on the growth and toxin content of the dinoflagellate Gymnodinium catenatum from the southwestern Mediterranean.

The dinoflagellate Gymnodinium catenatum is considered the primary cause of recurrent paralytic shellfish toxins (PSTs) in shellfish on the Moroccan Mediterranean coasts. The impacts of key environmental factors on the growth, cell yield, cell size and PST content of G. catenatum were determined. Results indicated that increasing salinity from 32 to 39 and nitrate concentrations from 441 µM to 1764 µM did not significantly (ANOVA, P-value >0.63) modify the growth rate of the studied species. Gymnodinium catenatum exhibited the highest growth rate at 24 °C. Cells arrested their division at 15 °C and at ammonium concentration above 441 µM, suggesting that this nitrogen form is toxic for G. catenatum. Furthermore, G. catenatum was unable to assimilate urea as a nitrogen source. In G. catenatum cells, eight analogues of saxitoxin were detected, belonging to the N-sulfocarbamoyl (C1-4, B1 and B2) and decarbamoyl (dc-GTX2/3) toxins. C-toxins contributed 92 % to 98 % of the molar composition of the PSTs. During the exponential growth, C2 tended to dominate, while C3 prevailed during the stationary phase. Toxin content per cell (ranging from 5.5 pg STXeq.cell-1 to 22.4 pg STXeq.cell-1) increased during the stationary growth phase. Cell toxin content increased with the concentrations of nitrate, ranging from 12.1 pg STXeq.cell-1 at 441 µM to 22.4 pg STXeq.cell-1 at 1764 µM during the stationary growth phase. The toxin content of G. catenatum showed the highest values measured at the highest tested temperatures, especially during the stationary phase, where toxicity reached 17.8 pg STXeq.cell-1 and 16.4 pg STXeq.cell-1 at 24 °C and 29 °C, respectively. The results can help understand the fluctuations in the growth and PST content of G. catenatum in its habitat in response to changing environmental variables in the Mediterranean Sea when exposed to increases in warming pressure and eutrophication.