Nanozyme-assisted molecularly imprinted polymer-based indirect competitive ELISA for the detection of marine biotoxin.

Saxitoxin (STX), which is produced by certain dinoflagellate species, is a type of paralytic shellfish poisoning toxin that poses a serious threat to human health and the environment. Therefore, developing a technology for the convenient and cost-effective detection of STX is imperative. In this study, we developed an affinity peptide-imprinted polymer-based indirect competitive ELISA (ic-ELISA) without using enzyme-toxin conjugates. AuNP/Co3O4@Mg/Al cLDH was synthesized by calcining AuNP/ZIF-67@Mg/Al LDH, which was obtained by combining AuNPs, ZIF-67, and flower-like Mg/Al LDH. This synthesized nanozyme exhibited high catalytic activity (Km = 0.24 mM for TMB and 132.5 mM for H2O2). The affinity peptide-imprinted polymer (MIP) was imprinted with an STX-specific template peptide (STX MIP) on a multi-well microplate and then reacted with an STX-specific signal peptide (STX SP). The interaction between the STX SP and MIP was detected using a streptavidin-coated nanozyme (SA-AuNP/Co3O4@Mg/Al cLDH). The developed MIP-based ic-ELISA exhibited excellent selectivity and sensitivity, with a limit of detection of 3.17 ng/mL (equivalent: 0.317 µg/g). Furthermore, the system was validated using a commercial ELISA kit and mussel tissue samples, and it demonstrated a high STX recovery with a low coefficient of variation. These results imply that the developed ic-ELISA can be used to detect STX in real samples.

What's in a name? Political and economic concepts differ in social media references to harmful algae blooms.

Policies and management decisions in the marine environment are driven in part by public sentiment which can grow more intense during hazard events like Harmful Algae Blooms (HABs). The public conversations on social media sites like Twitter (before X) reveal the polarized nature of HABs through nuanced language and sentiment. This article uses mixed methods of machine learned topic modeling and inductive qualitative coding to describe the ways the long-term 2017-2019 Karenia brevis "red tide" bloom were politicized across Florida's South West coast. It finds that there are topical differences in keywords related to place (e.g. beach, Florida, coast), agent (individual or organization), and epistemic values (reliance on scientific and/or media reports). These topical differences demonstrate different levels of politicization and partisanship in qualitative analysis. Conceptually, this research demonstrates the ways different dimensions of a long-duration marine hazard can be polarized. Regarding management, this research provides insights to political and organizational stakeholders and the gaps in the discourse shaping marine hazards which can be used to strategically guide future social media engagement to manage politicization. What if all the careful work that resource and environmental managers do can be undone by simple, seemingly uncontroversial words? In an era of increased environmental and marine distress-coupled with short format communication-the ways environmental managers choose their words is crucial, even between ostensibly inconsequential nouns like "red tide" or "algae bloom." Policies and management decisions in the marine environment are driven in part by public sentiment which can grow more intense during hazard events like Harmful Algae Blooms (HABs). The public conversations on social media sites like Twitter (before X) reveal the polarized nature of HABs through nuanced language and sentiment. This article relies on mining social media posts, and uses mixed methods of machine-learned topic modeling and human-driven inductive qualitative coding to describe the ways the long-term 2017-2019 Karenia brevis "red tide" blooms were politicized across Florida's South West coast. It finds that there are topical differences in keywords related to place (e.g. beach, Florida, coast), agent (individual or organization), and epistemic values (reliance on scientific and/or media reports). These topical differences demonstrate different levels of politicization and partisanship in qualitative analysis. Conceptually, this research demonstrates the ways different dimensions of a long-duration marine hazard can be polarized. Regarding management, this research provides insights to political and organizational stakeholders and the gaps in the discourse shaping marine hazards which can be used to strategically guide future social media engagement to manage politicization.

Sources and profiles of toxins in shellfish from the south-central coast of Chile (36°â€’ 43° S).

The study of marine toxins in shellfish is of the utmost importance to ensure people's food safety. Marine toxins in shellfish and microalgae in the water column off the south-central coast of Chile (36°â€’43° S) were studied in a network of 64 stations over a 14-month period. The relative abundance of harmful species Alexandrium catenella, Alexandrium ostenfeldii, Protoceratium reticulatum, Dinophysis acuminata, Dinophysis acuta, Pseudo-nitzschia seriata group and P. delicatissima group was analyzed. The detection and quantification of lipophilic toxins and domoic acid (DA) in shellfish was determined by UHPLC-MS/MS, and for Paralytic Shellfish Toxins (PSTs) by HPLC-FD with post-column oxidation, while for a culture of A. ostenfeldii a Hylic-UHPLC-MS/MS was used. Results showed that DA, gonyautoxin (GTX)-2, GTX-3 and pectenotoxin (PTX)-2 were detected below the permitted limits, while Gymnodimine (GYM)-A and 13-desmethylespirolide C (SPX-1) were below the limit of quantitation. According to the distribution and abundance record of microalgae, DA would be associated to P. seriata and P. delicatissima-groups, PTX-2 to D. acuminata, and GTX-2, GTX-3, GYM-A, and SPX-1 to A. ostenfeldii. However, the toxin analysis of an A. ostenfeldii culture from the Biobío region only showed the presence of the paralytic toxins C2, GTX-2, GTX-3, GTX-5 and saxitoxin, therefore, the source of production of GYM and SPX is still undetermined.

New azaspiracid analogues detected as bi-charged ions in Azadinium poporum (Amphidomataceae, Dinophyceae) isolated from Japanese coastal waters.

Lipophilic marine biotoxin azaspiracids (AZAs) are produced by dinoflagellates Azadinium and Amphidoma. Recently, several strains of Azadinium poporum were isolated from Japanese coastal waters, and detailed toxin profiles of two strains (mdd421 and HM536) among them were clarified by several detection techniques on liquid chromatography-tandem mass spectrometry (LC-MS/MS) and liquid chromatography-quadrupole time of flight mass spectrometry (LC-QTOFMS). In our present study, AZA analogues in seven strains of A. poporum from Japanese coastal waters (including two previously reported strains) were determined by these detection techniques. The dominant AZA in the seven strains was AZA2 accompanied by small amounts of several known AZAs and twelve new AZA analogues. Eight of the twelve new AZA analogues discovered in our present study were detected as bi-charged ions on the positive mode LC/MS/MS. This is the first report describing AZA analogues detected as bi-charged ions with hexose and sulfate groups in their structures.

Determination of lipophilic marine biotoxins (azaspiracids, brevetoxins, and okadaic acid group) and domoic acid in mussels by solid-phase extraction and reversed-phase liquid chromatography with tandem mass spectrometry.

An accurate and efficient method was developed for the determination of azaspiracid shellfish toxins (azaspiracids-1, -2, and -3), neurotoxic shellfish toxins (brevetoxins-2 and -3), diarrhetic shellfish toxins (okadaic acid and dinophysistoxins-1 and -2), and the amnesic shellfish toxin (domoic acid) in mussels (Mytilus galloprovincialis). Lipophilic marine biotoxins (azaspiracids, brevetoxins, and okadaic acid group) were extracted with 0.5 % acetic acid in methanol under heating at 60°C to improve the extraction efficiency of okadaic acid group toxins and then cleaned up with a C18 solid-phase extraction cartridge. Domoic acid was extracted with 50 % aqueous methanol and then cleaned up with a graphitized carbon solid-phase extraction cartridge. Lipophilic marine biotoxins and domoic acid were quantified by reversed-phase liquid chromatography coupled to electrospray ionization tandem mass spectrometry. The developed method had insignificant matrix effects for the nine analytes and good recoveries in the range of 79.0 % to 97.6 % at three spiking levels for all analytes except brevetoxin-2 (43.8-49.8 %). The developed method was further validated by analyzing mussel tissue certified reference materials, and good agreement was observed between certified and determined values.

Using solid phase adsorption toxin tracking and extended local similarity analysis to monitor lipophilic shellfish toxins in a mussel culture ranch in the Yangtze River Estuary.

Harmful algal blooms (HABs) and their associated phycotoxins are increasing globally, posing great threats to local coastal ecosystems and human health. Nutrients have been carried by the freshwater Yangtze River and have entered the estuary, which was reported to be a biodiversity-rich but HAB-frequent region. Here, in situ solid phase adsorption toxin tracking (SPATT) was used to monitor lipophilic shellfish toxins (LSTs) in seawaters, and extended local similarity analysis (eLSA) was conducted to trace the temporal and special regions of those LSTs in a one-year trail in a mussel culture ranch in the Yangtze River Estuary. Nine analogs of LSTs, including okadaic acid (OA), dinophysistoxin-1 (DTX1), yessotoxin (YTX), homoyessotoxin (homoYTX), 45-OH-homoYTX, pectenotoxin-2 (PTX2), 7-epi-PTX2 seco acid (7-epi-PTX2sa), gymnodimine (GYM) and azaspiracids-3 (AZA3), were detected in seawater (SPATT) or rope farmed mussels. The concentrations of OA + DTX1 and homoYTX in mussels were positively correlated with those in SPATT samplers (Pearson test, p < 0.05), indicating that SPATT (with resin HP20) would be a good monitoring tool and potential indicator for OA + DTX1 and homoYTX in mussel Mytilus coruscus. The eLSA results indicated that late summer and early autumn were the most phycotoxin-contaminated seasons in the Yangtze River Estuary. OA + DTX1, homoYTX, PTX2 and GYM were most likely driven by the local growing HAB species in spring and summer, while Yangtze River diluted water may impact the accumulation of HAB species, causing potential phycotoxin contamination in the Yangtze River Estuary in autumn and winter. Together, the results showed that the mussel harvesting season, late summer and early autumn, would be the season with the greatest phycotoxin risk and would be the most contaminated by local growing toxic algae. Routine monitoring sites should be set up close to the local seawaters.

Marine toxins in seafood: Recent updates on sample pretreatment and determination techniques.

Marine toxins can lead to varying degrees of human poisoning, often resulting in fatal symptoms and causing significant economic losses in seafood-producing regions. To gain a deeper comprehension of the role of marine toxins in seafood and their impact on the environment, it is imperative to develop rapid, cost-effective, environmentally friendly, and efficient methods for sample pretreatment and determination to mitigate adverse impacts of marine toxins. This review presents a comprehensive overview of advancements made in sample pretreatment and determination techniques for marine toxins since 2017. The advantages and disadvantages of various technologies were critically examined. Additionally, the current challenges and future development strategies for the analysis of marine toxins are provided.

Effect of temperature on growth and yessotoxin production of Protoceratium reticulatum and Lingulodinium polyedra (Dinophyceae) isolates from the Portuguese coast (NE Atlantic).

The dinoflagellates Protoceratium reticulatum and Lingulodinium polyedra are potential yessotoxin (YTX) producers, which have been associated with blooms responsible for economic, social, and ecological impacts around the world. They occur in Iberian waters, but in this region, little is known of their ecophysiology and toxin profiles. This study investigated the growth and toxin production of two strains of each species, from the Portuguese coast, at 15 °C, 19 °C, and 23 °C. Growth curves showed higher growth rates at 19 °C, for both species. YTX and three analogs (homo YTX; 45-OH YTX; 45-OH homo YTX) were investigated by Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS), and the presence of other analogs was investigated by Liquid Chromatography-High-Resolution Mass Spectrometry (LC-HRMS). No evidence of toxin production was found in L. polyedra. By contrast, YTX and 45,55-diOH-YTX were detected in both strains of P. reticulatum. These results confirm P. reticulatum as a source of yessotoxins along the Portuguese coast and add to the observed high intraspecific variability on YTX production of both species, at a global scale.

Seasonal occurrence of toxic phytoplankton and phycotoxins at a mussel aquaculture site in Nova Scotia, Canada.

A three-year field study at a mussel (Mytilus edulis) aquaculture site in Ship Harbour, Nova Scotia, Canada was carried out between 2004 and 2006 to detect toxic phytoplankton species and dissolved lipophilic phycotoxins and domoic acid. A combination of plankton monitoring and solid phase adsorption toxin tracking (SPATT) techniques were used. Net tow and pipe phytoplankton samples were taken weekly to determine the abundance of potentially toxic species and SPATT samplers were deployed weekly for phycotoxin analysis. Mussels were also collected for toxin analysis in 2005. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to analyse the samples for spirolides (SPXs), pectenotoxins (PTXs), okadaic acid group toxins (OA, DTXs) and domoic acid (DA). Phycotoxins were detected with SPATT samplers beginning from the time of deployment until after the producing organisms were no longer observed in pipe samples. Seasonal changes in toxin composition occurred over the sampling period and were related to changes in cell concentrations of Alexandrium Halim, Dinophysis Ehrenberg and Pseudo-nitzschia (Hasle) Hasle. Spirolides peaked in late spring and early summer, followed by DA in mid-July. Okadaic acid, DTX1 and PTXs occurred throughout the field season but peaked in late summer. Concentrations of some phycotoxins detected in SPATT samplers deployed within the area where mussels were suspended on lines were lower than in those deployed outside the mussel farm. The SPATT samplers provided a useful tool to detect the presence of phycotoxins and to establish trends in their appearance in the Ship Harbour estuary.

A solid phase extraction column based on SiO2@ZIF-8 for efficient analysis of domoic acid toxins in the seawater environment: experiments and DFT calculations on adsorption behaviour.

Algal toxins are important metabolites of toxic harmful algal blooms (HABs), and their qualitative and qualitative detection can serve as early warning indicators for toxic HABs, complementing traditional HAB monitoring and improving the accuracy of early warning. Therefore, this work took the detection of domoic acid (DA) as an example and prepared zeolitic imidazolate framework-8 (ZIF-8) with high enrichment performance and high water stability and its core-shell composite material SiO2@ZIF-8 as an adsorbent filler. Density functional theory (DFT) calculations and interference experiments verified that Zn2+ on SiO2@ZIF-8 played a crucial role in enriching DA on SiO2@ZIF-8. By using it as a solid-phase extraction (SPE) filler, it showed excellent performance compared with other SPE columns (C18/HLB/SAX/ZIF-8). Therefore, the SiO2@ZIF-8 column was coupled to high-performance liquid chromatography-mass spectrometry (SPE-HPLC-MS/MS) to establish a highly sensitive detection method for algal toxins in seawater, which had a wide linear range (12.0-5000.0 ng L-1), good reproducibility (RSD) and low limit of detection (4.0 ng L-1), and realized the monitoring of trace DA in the Pingtan sea area of Fujian Province from 2021 to 2022. By comparing other HAB early warning indicators such as salinity and pH and combining them with the information released by the Fujian Provincial Ocean and Fisheries Bureau, the content of DA in seawater measured by the established SPE-HPLC-MS/MS method can provide reference information for HAB monitoring and early warning.

Lipophilic Shellfish Poisoning Toxins in Marine Invertebrates from the Galician Coast.

For the purpose of assessing human health exposure, it is necessary to characterize the toxins present in a given area and their potential impact on commercial species. The goal of this research study was: (1) to screen the prevalence and concentrations of lipophilic toxins in nine groups of marine invertebrates in the northwest Iberian Peninsula; (2) to evaluate the validity of wild mussels (Mytilus galloprovincialis) as sentinel organisms for the toxicity in non-bivalve invertebrates from the same area. The screening of multiple lipophilic toxins in 1150 samples has allowed reporting for the first time the presence of 13-desmethyl spirolide C, pinnatoxin G, okadaic acid, and dinophysistoxins 2 in a variety of non-traditional vectors. In general, these two emerging toxins showed the highest prevalence (12.5-75%) in most of the groups studied. Maximum levels for 13-desmethyl spirolide C and pinnatoxin G were found in the bivalves Magallana gigas (21 µg kg-1) and Tellina donacina (63 µg kg-1), respectively. However, mean concentrations for the bivalve group were shallow (2-6 µg kg-1). Okadaic acid and dinophysistoxin 2 with lower prevalence (1.6-44.4%) showed, on the contrary, very high concentration values in specific species of crustaceans and polychaetes (334 and 235 µg kg--1, respectively), to which special attention should be paid. Statistical data analyses showed that mussels could be considered good biological indicators for the toxicities of certain groups in a particular area, with correlations between 0.710 (for echinoderms) and 0.838 (for crustaceans). Polychaetes could be an exception, but further extensive surveys would be needed to draw definitive conclusions.

Yessotoxins in Mollusks of the Galician Coast from 2014 to 2022: Variability, Biotransformation, and Resistance to Alkaline Hydrolysis.

The presence of yessotoxins (YTXs) was analyzed in 10,757 samples of Galician bivalves from 2014 to 2022. Only YTX and 45-OH YTX were found. YTX was detected in 31% of the samples, while 45-OH YTX was found in 11.6% of them. Among the samples containing YTX, 45-OH YTX was detected in 37.3% of cases. The maximum recorded levels were 1.4 and 0.16 mg of YTX-equivalentsg-1, for YTX and 45-OH YTX, respectively, which are well below the regulatory limit of the European Union. The YTX and 45-OH YTX toxicities in the raw extracts and extracts subjected to alkaline hydrolysis were strongly and linearly related. Due to the lack of homo-YTX in Galician samples, the effect of alkaline hydrolysis on homo-YTX and 45OH-Homo-YTX was only checked in 23 additional samples, observing no negative effect but a high correlation between raw and hydrolyzed extracts. Hydrolyzed samples can be used instead of raw ones to carry out YTXs determinations in monitoring systems, which may increase the efficiency of those systems where okadaic acid episodes are very frequent and therefore a higher number of hydrolyzed samples are routinely analyzed. The presence of YTX in the studied bivalves varied with the species, with mussels and cockles having the highest percentages of YTX-detected samples. The presence of 45-OH YTX was clearly related to YTX and was detected only in mussels and cockles. Wild populations of mussels contained proportionally more 45-OH YTX than those that were raft-cultured. Spatially, toxin toxicities varied across the sampling area, with higher levels in raft-cultured mussels except those of Ría de Arousa. Ría de Ares (ARE) was the most affected geographical area, although in other northern locations, lower toxin levels were detected. Seasonally, YTX and 45-OH YTX toxicities showed similar patterns, with higher levels in late summer and autumn but lower toxicities of the 45-OH toxin in August. The relationship between the two toxins also varied seasonally, in general with a minimum proportion of 45-OH YTX in July-August but with different maximum levels for raft-cultured and wild mussel populations. Interannually, the average toxicities of YTX decreased from 2014 to 2017 and newly increased from 2018 to 2021, but decreased slightly in 2022. The relationship between 45-OH YTX and YTX also varied over the years, but neither a clear trend nor a similar trend for wild and raft mussels was observed.

An enhanced LC-MRM-MS platform for sensitive and simultaneous quantification of cyclic imines in shellfish.

Cyclic imines (CIs) produced by microalgae species and accumulating in the food chain of marine organisms are novel biotoxins that do not belong to the classical group of marine biotoxins. In the past, CIs were found only in limited areas, but in recent years, rapid changes in marine ecosystems have led to widespread CIs, increasing exposure to toxic risks. Monitoring of CIs is therefore required, but still analytically challenging due to the presence of high levels of analogues and interference from other lipophilic substances. Herein, we developed the LC/MRM-MS-based quantitative platform that can selectively enrich for marine-derived CIs and monitor seven CIs simultaneously: pinnatoxin (PnTX E, PnTX F, PnTX G), gymnodimine (GYM A), and spirolide (13-desMe SPX C, 13,19-didesMe SPX C, 20-Me SPX G). In particular, the combination of chromatographic separation by the hydrophobic nature of intrinsic residues of CIs with monitoring of CI structure-specific product ions generated by CID-MS/MS significantly improves the selectivity and sensitivity for quantitative analysis. Indeed, three CIs corresponding to PnTX G, GYM A, and 13-desMe SPX C could be successfully determined at the level of part-per-trillion (ppt) in three species of shellfish collected around the Korean Peninsula. Our analysis revealed that the expression of CIs in the Korean Peninsula was more influenced by the season rather than the species. This analytical platform with high sensitivity can be applied not only to marine biology but also to various other fields requiring CI analysis. Key Contribution: A highly sensitive analytical method for the simultaneous quantitation of cyclic imines based on LC/MRM-MS has been developed.

Integrated analysis of marine biotoxins and contaminants of emerging concern in bivalve mollusks from Santa Catarina, Brazil.

Santa Catarina is the main producer state of oysters and mussels in Brazil, reaching 98 % of national production. To assure the safety of bivalve mollusks production, control programs of marine biotoxins (MBs) have been continuously performed. Herein, the co-occurrence of MBs and contaminants of emerging concern (CECs) in oyster and mussels from the main production sites of Santa Catarina was reported, covering 178 compounds. Samples of wild and non-cultivated oysters and mussels were also assessed. Chemometric tools were used to evaluate and optimize several sample preparation techniques such as solid-liquid, ultrasound assisted, and pressurized liquid extraction. The optimized protocol was based on ultrasound assisted extraction followed by liquid chromatography coupled to tandem mass spectrometry. The results showed the incidence of several CECs and MBs. In the case of MBs, all results were below the regulatory limits for both cultivated and non-cultivated samples. Wild mollusks have shown a higher number of compounds. Regarding CECs, the more frequent compounds were caffeine, diclofenac, meloxicam, and sertraline. Domoic acid and okadaic acid were the main toxins detected. The results highlighted the need of monitoring for MBs and the potential of oyster and mussels as sentinel organisms to risk analysis of CECs in coastal regions. To the best of our knowledge, this is the first method to describe a simultaneous sample preparation and analysis of CECs and MBs in bivalve mollusks, as well as the first report of meloxicam and florfenicol in mussels and oysters.

Rapid determination of domoic acid in seafood by fluorescence polarization immunoassay using a portable analyzer.

Monitoring phycotoxin accumulation in marine products such as edible shellfish is a regulatory requirement in many countries. Therefore, a simple and rapid onsite quantification method is sought. Herein, we present a fluorescence polarization immunoassay (FPIA), a well-known one-step immunoassay, using a portable fluorescence polarization analyzer for domoic acid (DA), widely referred to as the primary toxin of amnesic shellfish poisoning (ASP). To establish FPIA for DA, the matrix effect of methanol, which is widely used to extract DA from shellfish, on FPIA was investigated. To validate this method, we performed a spike recovery test using oysters containing DA at a concentration equivalent to the regulatory limits of North America and the European Union (20 mg/kg). The recovery rate was found to be 79.4-114.7%, which is equivalent to that of the commercially available enzyme-linked immunosorbent assay (ELISA). We expect that this FPIA system will enable the quantitative onsite analysis of DA and significantly contribute to the safety of marine products.

Thecal plate morphology, molecular phylogeny, and toxin analyses reveal two novel species of Alexandrium (Dinophyceae) and their potential for toxin production.

This study describes two novel species of marine dinophytes in the genus Alexandrium. Morphological characteristics and phylogenetic analyses support the placement of the new taxa, herein designated as Alexandrium limii sp. nov. and A. ogatae sp. nov. Alexandrium limii, a species closely related to A. taylorii, is distinguished by having a shorter 2'/4' suture length, narrower plates 1' and 6'', with larger length: width ratios, and by the position of the ventral pore (Vp). Alexandrium ogatae is distinguishable with its metasert plate 1' having almost parallel lateral margins, and by lacking a Vp. Production of paralytic shellfish toxins (PSTs), cycloimines, and goniodomins (GDs) in clonal cultures of A. ogatae, A. limii, and A. taylorii were examined analytically and the results showed that all strains contained GDs, with GDA as major variants (6-14 pg cell-1) for all strains except the Japanese strain of A. limii, which exclusively had a desmethyl variant of GDA (1.4-7.3 pg cell-1). None of the strains contained detectable levels of PSTs and cycloimines.

Variation profile of diarrhetic shellfish toxins and diol esters derivatives of Prorocentrum lima during growth by high-resolution mass spectrometry.

Prorocentrum lima is a widely distributed toxigenic benthic dinoflagellate whose production of diarrhetic shellfish toxins threatens the shellfish industry and seafood safety. Current research primarily assesses the difference between free and post-hydrolysis total toxin methods, ignoring the impact of different detection methods on technical accuracy. After removing matrix interference with SPE extraction, a thorough HRMS strategy was created in this study. Alkaline hydrolysis could release the diol esters and played a crucial role in obtaining an accurate assessment of toxin levels, achieving satisfactory recoveries (74.0-147.0%) and repeatability (relative deviation <12.3%). The HRMS approach evaluated toxin profile variation during the growth of three P. lima strains from China. A total of 24 toxin contents varying in composition, content, and a high proportion were detected. The SHG, HN, and 3XS strains had total toxin contents of 23.3 ± 1.74, 19.8 ± 1.25, and 19.5 ± 1.58 pg cell-1, respectively. The diol esters proportion varied among the strains, with SHG having 58.9-69.9, HN having 75.4-86.5, and 3XS having 91.0-91.7%. The variety of toxins produced by distinct P. lima strains highlighted the significance of this method for appropriately measuring the risks connected with DSTs manufacturing. The proposed approach provides a technical basis for gathering comprehensive and accurate data on the potential risks of P. lima DSTs production, with significant implications for ensuring food safety and preventing harmful toxins from spreading in the marine ecosystem.

Short-Term Interactions of Noctiluca scintillans with the Toxic Dinoflagellates Dinophysis acuminata and Alexandrium minutum: Growth, Toxins and Allelopathic Effects.

The Galician Rías (NW Iberian Peninsula) are an important shellfish aquaculture area periodically affected by toxic episodes often caused by dinoflagellates such as Dinophysis acuminata and Alexandrium minutum, among others. In turn, water discolorations are mostly associated with non-toxic organisms such as the heterotrophic dinoflagellate Noctiluca scintillans, a voracious non-selective predator. The objective of this work was to study the biological interactions among these dinoflagellates and their outcome in terms of survival, growth and toxins content. To that aim, short experiments (4 days) were carried out on mixed cultures with N. scintillans (20 cells mL-1) and (i) one strain of D. acuminata (50, 100 and 500 cells mL-1) and (ii) two strains of A. minutum (100, 500 and 1000 cells mL-1). Cultures of N. scintillans with two A. minutum collapsed by the end of the assays. Both D. acuminata and A. minutum exposed to N. scintillans arrested its growth, though feeding vacuoles in the latter rarely contained any prey. Toxin analyses at the end of the experiment showed an increase in intracellular OA levels in D. acuminata and a significant reduction in PSTs in both A. minutum strains. Neither OA nor PSTs were detected in N. scintillans. Overall, the present study indicated that the interactions among them were ruled by negative allelopathic effects.

Sample pooling and incurred samples improve analytical throughput and quality control of lipophilic phycotoxins screening in bivalve mollusks.

Lipophilic marine biotoxins (LMBs) are one of the main risks associated with the consumption of mussels and oysters. Sanitary and analytical control programs are developed to detect the occurrence of these toxins in seafood before they reach toxic levels. To ensure quick results, methods must be easy and fast to perform. In this work, we demonstrated that incurred samples were a viable alternative to validation and internal quality control studies for the analysis of LMBs in bivalve mollusks. These samples were used to optimize, validate, and monitor a simple and fast ultrasound-assisted extraction (UAE) procedure. An internal quality control material containing okadaic acid (227 ± 46 µg kg-1) was produced and characterized. This material had its homogeneity and stability verified and was included as a quality control in all batches of analytical routine. Besides, a sample pooling protocol for extracts analysis was developed, based on tests for COVID-19. Up to 10 samples could be analyzed simultaneously, reducing the instrumental time of analysis by up to 80%. The UAE and sample pooling approaches were then applied to more than 450 samples, of which at least 100 were positive for the okadaic acid group of toxins.

A competitive, bead-based assay combined with microfluidics for multiplexed toxin detection.

The requirement for rapid, in-field detection of cyanotoxins in water resources necessitates the developing of an easy-to-use and miniaturized system for their detection. We present a novel bead-based, competitive fluorescence assay for multiplexed detection of two types of toxins: microcystin-LR (MC-LR) and okadaic acid (OA). To automate the detection process, a reusable microfluidic device, termed toxin-chip, was designed and validated. The toxin-chip consists of a micromixer where the target toxins were efficiently mixed with a reagent solution, and a detection chamber for magnetic retainment of beads for downstream analysis. Quantum dots (QDs) were used as the reporter molecules to enhance the sensitivity of the assay and the emitted fluorescence signal from QDs was reversely proportional to the amount of toxins in the solution. An image analysis program was also developed to further automate the detection and analysis steps. Two toxins were simultaneously analyzed on a single microfluidic chip, and the device exhibited a low detection limit of 10-4 µg ml-1 for MC-LR and 4 × 10-5 µg ml-1 for OA detection. The bead-based, competitive assay also showed remarkable chemical specificity against potential interfering toxins. We also validated the device performance using natural lake water samples from Sunfish Lake of Waterloo. The toxin-chip holds promise as a versatile and simple quantification tool for cyanotoxin detection, with the potential of detecting more toxins.