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.

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.

Estimating the risks of exposure to harmful algal toxins among Scottish harbour seals.

Harmful algal bloom (HAB) toxins consumed by marine predators through fish prey can be lethal but studies on the resulting population consequences are lacking. Over the past approximately 20 years there have been large regional declines in some harbour seal populations around Scotland. Analyses of excreta (faeces and urine from live and dead seals and faecal samples from seal haulout sites) suggest widespread exposure to toxins through the ingestion of contaminated prey. A risk assessment model, incorporating concentrations of the two major HAB toxins found in seal prey around Scotland (domoic acid (DA), and saxitoxins (STX)), the seasonal persistence of the toxins in the fish and the foraging patterns of harbour seals were used to estimate the proportion of adults and juveniles likely to have ingested doses above various estimated toxicity thresholds. The results were highly dependent on toxin type, persistence, and foraging regime as well as age class, all of which affected the proportion of exposed animals exceeding toxicity thresholds. In this preliminary model STX exposure was unlikely to result in mortalities. Modelled DA exposure resulted in doses above an estimated lethal threshold of 1900 µg/kg body mass affecting up to 3.8 % of exposed juveniles and 5.3 % of exposed adults. Given the uncertainty in the model parameters and the limitations of the data these conclusions should be treated with caution, but they indicate that DA remains a potential factor involved in the regional declines of harbour seals. Similar risks may be experienced by other top predators, including small cetaceans and seabirds that feed on similar prey in Scottish waters.

Cyanotoxin Occurrence and Diversity in 98 Cyanobacterial Blooms from Swedish Lakes and the Baltic Sea.

The Drinking Water Directive (EU) 2020/2184 includes the parameter microcystin LR, a cyanotoxin, which drinking water producers need to analyze if the water source has potential for cyanobacterial blooms. In light of the increasing occurrences of cyanobacterial blooms worldwide and given that more than 50 percent of the drinking water in Sweden is produced from surface water, both fresh and brackish, the need for improved knowledge about cyanotoxin occurrence and cyanobacterial diversity has increased. In this study, a total of 98 cyanobacterial blooms were sampled in 2016-2017 and identified based on their toxin production and taxonomical compositions. The surface water samples from freshwater lakes throughout Sweden including brackish water from eight east coast locations along the Baltic Sea were analyzed for their toxin content with LC-MS/MS and taxonomic composition with 16S rRNA amplicon sequencing. Both the extracellular and the total toxin content were analyzed. Microcystin's prevalence was highest with presence in 82% of blooms, of which as a free toxin in 39% of blooms. Saxitoxins were found in 36% of blooms in which the congener decarbamoylsaxitoxin (dcSTX) was detected for the first time in Swedish surface waters at four sampling sites. Anatoxins were most rarely detected, followed by cylindrospermopsin, which were found in 6% and 10% of samples, respectively. As expected, nodularin was detected in samples collected from the Baltic Sea only. The cyanobacterial operational taxonomic units (OTUs) with the highest abundance and prevalence could be annotated to Aphanizomenon NIES-81 and the second most profuse cyanobacterial taxon to Microcystis PCC 7914. In addition, two correlations were found, one between Aphanizomenon NIES-81 and saxitoxins and another between Microcystis PCC 7914 and microcystins. This study is of value to drinking water management and scientists involved in recognizing and controlling toxic cyanobacteria blooms.

Paralytic Shellfish Toxins in Mollusks from Galicia Analyzed by a Fast Refined AOAC 2005.06 Method: Toxicity, Toxin Profile, and Inter-Specific, Spatial, and Seasonal Variations.

Paralytic shellfish poisoning is an important concern for mollusk fisheries, aquaculture, and public health. In Galicia, NW Iberian Peninsula, such toxicity has been monitored for a long time using mouse bioassay. Therefore, little information exists about the precise toxin analogues and their possible transformations in diverse mollusk species and environments. After the change in the European PSP reference method, a refinement of the Lawrence method was developed, achieving a 75% reduction in chromatogram run time. Since the beginning of 2021, when this refinement Lawrence method was accredited under the norm UNE-EN ISO/IEC 17025, it has been used in the area to determine the toxin profiles and to estimate PSP toxicity in more than 4500 samples. In this study, we have summarized three years of monitoring results, including interspecific, seasonal, and geographical variability of PSP toxicity and toxin profile. PSP was detected in more than half of the samples analyzed (55%), but only 4.4% of the determinations were above the EU regulatory limit. GTX1,4 was the pair of STX analogs that produced the highest toxicities, but GTX2,3 was found in most samples, mainly due to the reduction of GTX1,4 but also by the higher sensitivity of the method for this pair of analogs. STX seems to be mainly a product of biotransformation from GTX2,3. The studied species (twelve bivalves and one gastropod) accumulated and transformed PSP toxins to a different extent, with most of them showing similar profiles except for Spisula solida and Haliotis tuberculata. Two seasonal peaks of toxicity were found: one in spring-early summer and another in autumn, with slightly different toxin profiles during outbreaks in relation to the toxicity during valleys. In general, both the total toxicity and toxin profiles of the southernmost locations were different from those in the northern part of the Atlantic coast and the Cantabrian Sea, but this general pattern is modified by the PSP history of some specific locations.

Temporal variation in the concentrations and profiles of paralytic shellfish toxins and tetrodotoxin in scallop (Mizuhopecten yessoensis) and bloody clam (Anadara broughtonii) collected from the coast of Miyagi Prefecture, Japan.

For food safety, the concentrations and profiles of paralytic shellfish toxins (PSTs) and tetrodotoxin were examined in economically important scallops and bloody clams collected from the coast of the Miyagi Prefecture, Japan. PSTs were the major toxins in both species. The tetrodotoxin concentration in scallops increased in summer, although the highest value (18.7 µg/kg) was lower than the European Food Safety Authority guideline threshold (44 µg/kg). This confirmed the safety for tetrodotoxin in this area.

Spatial and Temporal Variability of Saxitoxin-Producing Cyanobacteria in U.S. Urban Lakes.

Harmful cyanobacterial blooms (HCBs) are of growing global concern due to their production of toxic compounds, which threaten ecosystems and human health. Saxitoxins (STXs), commonly known as paralytic shellfish poison, are a neurotoxic alkaloid produced by some cyanobacteria. Although many field studies indicate a widespread distribution of STX, it is understudied relative to other cyanotoxins such as microcystins (MCs). In this study, we assessed eleven U.S. urban lakes using qPCR, sxtA gene-targeting sequencing, and 16S rRNA gene sequencing to understand the spatio-temporal variations in cyanobacteria and their potential role in STX production. During the blooms, qPCR analysis confirmed the presence of the STX-encoding gene sxtA at all lakes. In particular, the abundance of the sxtA gene had a strong positive correlation with STX concentrations in Big 11 Lake in Kansas City, which was also the site with the highest quantified STX concentration. Sequencing analysis revealed that potential STX producers, such as Aphanizomenon, Dolichospermum, and Raphidiopsis, were present. Further analysis targeting amplicons of the sxtA gene identified that Aphanizomenon and/or Dolichospermum are the primary STX producer, showing a significant correlation with sxtA gene abundances and STX concentrations. In addition, Aphanizomenon was associated with environmental factors, such as conductivity, sulfate, and orthophosphate, whereas Dolichospermum was correlated with temperature and pH. Overall, the results herein enhance our understanding of the STX-producing cyanobacteria and aid in developing strategies to control HCBs.

An Occurrence and Exposure Assessment of Paralytic Shellfish Toxins from Shellfish in Zhejiang Province, China.

The intake of paralytic shellfish toxins (PSTs) may adversely affect human health. Therefore, this study aimed to show the prevalence of PSTs from commercially available shellfish in Zhejiang Province, China, during the period of frequent red tides, investigate the factors affecting the distribution of PSTs, and assess the risk of PST intake following the consumption of bivalve shellfish among the Zhejiang population. A total of 546 shellfish samples were collected, 7.0% of which had detectable PSTs at concentrations below the regulatory limit. Temporal, spatial, and interspecific variations in the occurrence of PSTs were observed in some cases. The dietary exposure to PSTs among the general population of consumers only was low. However, young children in the extreme scenario (the 95th percentile of daily shellfish consumption combined with the maximum PST concentration), defined as 89-194% of the recommended acute reference doses, were possibly at risk of exposure. Notably, Arcidae and mussels were the major sources of exposure to toxins. From the public health perspective, PSTs from commercially available shellfish do not pose a serious health risk; however, more attention should be paid to acute health risks, especially for young children, during periods of frequent red tides.

An innovative passive sampling approach for the detection of cyanobacterial gene targets in freshwater sources.

Cyanotoxins pose significant human health risks, but traditional monitoring approaches can be expensive, time consuming, and require analytical equipment or expertise that may not be readily available. Quantitative polymerase chain reaction (qPCR) is becoming an increasingly common monitoring strategy as detection of the genes responsible for cyanotoxin synthesis can be used as an early warning signal. Here we tested passive sampling of cyanobacterial DNA as an alternative to grab sampling in a freshwater drinking supply lake with a known history of microcystin-LR. DNA extracted from grab and passive samples was analyzed via a multiplex qPCR assay that included gene targets for four common cyanotoxins. Passive samples captured similar trends in total cyanobacteria and the mcyE/ndaF gene responsible for microcystin production when compared to traditional grab samples. Passive samples also detected genes associated with the production of cylindrospermopsin and saxitoxin that were not detected in grab samples. This sampling approach proved a viable alternative to grab sampling when used as an early warning monitoring tool. In addition to the logistical benefits of passive sampling, the detection of gene targets not detected by grab samples indicates that passive sampling may allow for a more complete profile of potential cyanotoxin risk.

Metabolic inhibitor induces dynamic changes in saxitoxin biosynthesis and metabolism in the dinoflagellate Alexandrium pacificum (Group IV) under in vivo labeling condition.

In paralytic shellfish toxin-producing dinoflagellates, intracellular levels of saxitoxin and its analogues (STXs) are controlled by a balance between degradation and biosynthesis in response to marine environmental fluctuations and stresses. The purpose of this study was to demonstrate the utility of statistical analysis of in vivo labeling data for the dynamic analysis of variations in toxin production under stress. A toxic strain of the dinoflagellate Alexandrium pacificum (Group IV) was cultured in colchicine-containing 15N-labeled sodium nitrate-medium and metabolite levels were analyzed over time by liquid chromatography-mass spectrometry. Quantitative values of all isotopomers of precursor amino acids, biosynthetic intermediates, and major STXs were subjected to statistical analysis. The decrease of the nitrogen incorporation rates for all compounds suggested that colchicine decreased nitrate assimilation upstream of glutamate biosynthesis. In colchicine-treated cultures, the per-cell content of total STX analogues did not change significantly over time; however, the production rate of each pathway varied greatly. De novo STX biosynthesis was decreased by colchicine until Day 3, while the salvage pathway was not. Subsequently, biosynthesis by both pathways was enhanced. This analysis of dynamic metabolism provides new insights into the complex mechanisms regulating STX metabolism in dinoflagellates.

Biotransformation and detoxification of saxitoxin by Bacillus flexus in batch experiments.

Saxitoxins (STXs) are carbamate alkaloid neurotoxins produced by some species of cyanobacteria. They are water soluble and relatively stable in the natural environment, and thereby represent a risk to animal and human health through a long-time exposure. STXs cannot be sufficiently removed by conventional water treatment methods. Therefore, this study investigates the potential STX biodegradation and detoxification by bacteria as a promising method for toxin removal. STX biodegradation experiments were conducted using Bacillus flexus SSZ01 strain in batch cultures. The results revealed that SSZ01 strain grew well and rapidly detoxified STX, with no lag phase observed. STX detoxification by SSZ01 strain was initial-toxin-concentration-dependent. The highest biotransformation rate (10 µg STX L-1 day-1) the pseudo-first-order kinetic constant (0.58 d-1) were obtained at the highest initial toxin concentration (50 µg L-1) and the lowest ones (0.06 µg STX L-1 day-1 and 0.14 d-1, respectively) were recorded at the lowest initial concentration (0.5 µg L-1). STX biotransformation rate increased with temperature, with highest occurred at 30 ºC. This rate was also influenced by pH, with highest obtained at pH8 and lowest at higher and lower pH values. HPLC chromatograms showed that STX biotransformation peak is corresponding to the least toxic STX analog (disulfated sulfocarbamoyl-C1 variant). The Artemia-based toxicity assay revealed that this biotransformation byproduct was nontoxic. This suggests the potential application of this bacterial strain in slow sand filters for cyanotoxin removal in water treatment plants. Being nontoxic, this byproduct needs to be assayed for its therapeutic effects toward neurodegenerative diseases.

Paralytic Shellfish Toxins in the Gastropod Concholepas concholepas: Variability, Toxin Profiles and Mechanisms for Toxicity Reduction.

Harmful algal blooms of toxin-producing microalgae are recurrent in southern Chile. Paralytic shellfish poisoning (PSP) outbreaks pose the main threat to public health and the fishing industry in the Patagonian fjords. This study aims to increase understanding of the individual and spatial variability of PSP toxicity in the foot of Concholepas concholepas, Chile's most valuable commercial benthic invertebrate species, extracted from the Guaitecas Archipelago in Chilean Patagonia. The objective is to determine the effect of pigment removal and freezing during the detoxification process. A total of 150 specimens (≥90 mm length) were collected from this area. The live specimens were transferred to a processing plant, where they were measured and gutted, the foot was divided into two equal parts, and pigment was manually removed from one of these parts. The PSP toxicity of each foot (edible tissue) was determined by mouse bioassay (MBA) and high-performance liquid chromatography with fluorescence detection and postcolumn oxidation (HPLC-FLD PCOX). The individual toxicity per loco, as the species is known locally, varied from <30 to 146 µg STX diHCL eq 100 g−1 (CV = 43.83%) and from 5.96 to 216.3 µg STX diHCL eq 100 g−1 (CV = 34.63%), using MBA and HPLC, respectively. A generalized linear model showed a negative relation between individual weight and toxicity. The toxicological profile showed a dominance of STX (>95%), neoSTX and GTX2. The removal of pigment produced a reduction in PSP toxicity of up to 90% and could represent a good detoxification tool moving forward. The freezing process in the muscle with pigment did not produce a clear pattern. There is a significant reduction (p < 0.05) of PSP toxicity via PCOX but not MBA. Furthermore, the study discusses possible management and commercialization implications of the findings regarding small-scale fisheries.

Contamination status of paralytic shellfish toxins in shellfish from Southeastern China in 2017-2021.

Harmful algal blooms is a widespread problem in aquatic ecosystems, in particular dinoflagellates that produce PSTs which are harmful to animal and human health. To explore the contamination status of PSTs in shellfish in the Southeastern China, a total of 2355 shellfish samples were analyzed by ultra high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) to study the toxin profiles of the 10 PSTs collected from the southeast coast of China from 2017 to 2021. From 2355 shellfish samples, 257 were detected (10.91%), with the highest value in samples of Perna viridis. Among the six source areas in China, the samples from Fujian recorded the highest detected rate (15.28%). PSTs were found in Fuzhou, Ningde, Quanzhou, Putian, Zhangzhou, and Xiamen, with Quanzhou and Fuzhou having the highest and lowest detection rates of 15.28% and 4.23%, respectively. Saxitoxin (STX), neosaxitoxin (neoSTX), gonyautoxin (GTX1, GTX2, GTX3, GTX4), N-sulfocarbamoyl toxin (GTX5), and decarbamoyl toxin (dcSTX, dcGTX2, dcGTX3) were detected, and GTX5 and dcGTX2 were dominant. In addition, the samples containing PSTs were mostly concentrated in May to August. The study confirms the risks of PSTs to shellfish consumers in the region. It will offer a great foundation for future monitoring of marine toxins and protecting the health of seafood consumers in China. This is the first detailed evaluation of PSTs occurrences and their profiles in shellfish from the Southeastern China over a period of multiple years. HIGHLIGHTS: 2355 mussels from China were analyzed by UPLC-MS/MS for PSTs in 2017-2021. The predominant PSTs were GTX5, neoSTX and dcGTX2. Arca granosa and Crassostyea gigas exhibited higher levels than other shellfish. Shellfish containing PSTs were mostly concentrated in May to August. Maximum detected level in shellfish was 2137.10 ug STXeq/kg.

Marine Biotoxins in Whole and Processed Scallops from the Argentine Sea.

Harmful algal blooms are an increasing worldwide threat to the seafood industry and human health as a consequence of the natural production of biotoxins that can accumulate in shellfish. In the Argentine Sea, this has been identified as an issue for the offshore fisheries of Patagonian scallops (Zygochlamys patagonica), leading to potentially harmful effects on consumers. Here we assess spatial and temporal patterns in marine biotoxin concentrations in Patagonian scallops harvested in Argentinian waters between 2012-2017, based on analyses for paralytic shellfish toxins, lipophilic toxins, and amnesic shellfish toxins. There was no evidence for concentrations of lipophilic or amnesic toxins above regulatory acceptance thresholds, with trace concentrations of pectenotoxin 2, azaspiracid 2 and okadaic acid group toxins confirmed. Conversely, paralytic shellfish toxins were quantified in some scallops. Gonyautoxins 1 and 2 dominated the unusual toxin profiles (91%) in terms of saxitoxin equivalents with maximum concentrations reaching 3985 µg STX eq/kg and with changes in profiles linked in part to seasonal changes. Total toxin concentrations were compared between samples of the adductor muscle and whole tissue, with results showing the absence of toxins in the adductor muscle confirming toxin accumulation in the digestive tracts of the scallops and the absence of a human health threat following the processing of scallop adductor meat. These findings highlight that paralytic shellfish toxins with an unusual toxin profile can occur in relatively high concentrations in whole Patagonian scallops in specific regions and during particular time periods, also showing that the processing of scallops on board factory ships to obtain frozen adductor muscle is an effective management process that minimizes the risk of poisonings from final products destined for human consumption.

Stability of Saxitoxin in 50% Methanol Fecal Extracts and Raw Feces from Bowhead Whales (Balaena mysticetus).

In recent decades, harmful algal blooms (HABs) producing paralytic shellfish toxins (including saxitoxin, STX) have become increasingly frequent in the marine waters of Alaska, USA, subjecting Pacific Arctic and subarctic communities and wildlife to increased toxin exposure risks. Research on the risks of HAB toxin exposures to marine mammal health commonly relies on the sampling of marine mammal gastrointestinal (GI) contents to quantify HAB toxins, yet no studies have been published testing the stability of STX in marine mammal GI matrices. An understanding of STX stability in test matrices under storage and handling conditions is imperative to the integrity of toxin quantifications and conclusions drawn thereby. Here, STX stability is characterized in field-collected bowhead whale feces (stored raw in several treatments) and in fecal extracts (50% methanol, MeOH) over multiple time points. Toxin stability, as the percent of initial concentration (T0), was reported for each storage treatment and time point. STX was stable (mean 99% T0) in 50% MeOH extracts over the 8-week study period, and there was no significant difference in STX concentrations quantified in split fecal samples extracted in 80% ethanol (EtOH) and 50% MeOH. STX was also relatively stable in raw fecal material stored in the freezer (mean 94% T0) and the refrigerator (mean 93% T0) up to 8 weeks. STX degraded over time in the room-temperature dark, room-temperature light, and warm treatments to means of 48 ± 1.9, 38 ± 2.8, and 20 ± 0.7% T0, respectively, after 8 weeks (mean ± standard error; SE). Additional opportunistically analyzed samples frozen for ≤4.5 years also showed STX to be relatively stable (mean 97% T0). Mean percent of T0 was measured slightly above 100% in some extracts following some treatments, and (most notably) at some long-term frozen time points, likely due to evaporation from samples causing STX to concentrate, or variability between ELISA plates. Overall, these results suggest that long-term frozen storage of raw fecal samples and the analysis of extracts within 8 weeks of extraction in 50% MeOH is sufficient for obtaining accurate STX quantifications in marine mammal fecal material without concerns about significant degradation.

Nontoxic Enantiomeric Reference Materials for Saxitoxins.

Saxitoxin (STX) is a potent neurotoxin that is biosynthesized by toxic dinoflagellates and accumulated in shellfish via the food chain. STX and its various analogues are now monitored in shellfish by the hygiene authorities in many countries with instrumental analytical methods, which require calibration with standards. Unfortunately, STX is registered as a chemical warfare agent in Schedule 1 of the Chemical Weapons Convention, and this has made it difficult to import calibration standards into some countries. We aimed to avoid violation of the Chemical Weapons Convention and facilitate analyses by preparing calibration standards based on unnatural nontoxic antipodal STXs (ent-STXs) with the same physicochemical properties as natural STXs. Our findings demonstrate that the nontoxic ent-STXs can be safely utilized as alternative reference materials of STXs in the routine monitoring program by the local authorities and consequently can lead to reduced usage of STX.

Effect of Different N:P Ratios on the Growth, Toxicity, and Toxin Profile of Gymnodinium catenatum (Dinophyceae) Strains from the Gulf of California.

The harmful microalgae Gymnodinium catenatum is a unique naked dinoflagellate that produces paralytic shellfish poisoning toxins (PSTs). This species is common along the coasts of the Mexican Pacific and is responsible for paralytic shellfish poisoning, which has resulted in notable financial losses in both fisheries and aquaculture. In the Gulf of California, G. catenatum has been related to mass mortality events in fish, shrimp, seabirds, and marine mammals. In this study, the growth, toxin profiles, and toxin content of four G. catenatum strains isolated from Bahía de La Paz (BAPAZ) and Bahía de Mazatlán (BAMAZ) were evaluated with different N:P ratios, keeping the phosphorus concentration constant. All strains were cultivated in semi-continuous cultures (200 mL, 21.0 °C, 120 µmol photon m-2s-1, and a 12:12 h light-dark cycle) with f/2 + Se medium using N:P ratios of: 4:1, 8:1, 16:1, 32:1, and 64:1. Paralytic toxins were analyzed by HPLC with fluorescence detection. Maximum cellular abundance and growth were obtained at an N:P ratio of 64:1 (3188 cells mL-1 and 0.34 div day-1) with the BAMAZ and BAPAZ strains. A total of ten saxitoxin analogs dominated by N-sulfocarbamoyl (60-90 mol%), decarbamoyl (10-20 mol%), and carbamoyl (5-10 mol%) toxins were detected. The different N:P ratios did not cause significant changes in the PST content or toxin profiles of the strains from both bays, although they did affect cell abundance.

Revisiting the HPLC-FLD Method to Quantify Paralytic Shellfish Toxins: C3,4 Quantification and the First Steps towards Validation.

Paralytic shellfish toxins (PSTs) are a large group of biotoxins that cause paralytic shellfish poisoning. Their appearance in natural waters and their ingestion by aquatic species have a huge socio-economic impact, whereby their monitoring is of the upmost relevance to minimize the consequences. For earlier detection and faster response/action by stakeholders, validation of adjusted analytical methods, particularly for lower concentration levels, is important. This work proposes a derived High-Performance Liquid Chromatography method, with fluorescence detection (HPLC-FLD). The main differences from the official method are the size of the HPLC column and the gradient elution conditions. It covers the current eleven certified reference materials (CRM) available on the market, including the most recent-C3,4. This first calibration report for C3,4 suggests limits of detection (LOD) and limits of quantification (LOQ) of 6 nM and 19 nM (~5 µg STX.2HCl eqv./kg and 17 µg STX.2HCl eqv./kg), respectively. For the remaining CRM, LODs ranged between 3 and 28 nM (~0.9 and 127 µg STX.2HCl eqv./kg), while LOQs varied between 11 and 94 nM (~3 and 409 µg STX.2HCl eqv./kg, considering toxicity equivalency factors (TEFs) reported by EFSA).

Development of a Method for Detecting Alexandrium pacificum Based on the Quantification of sxtA4 by Chip-Based Digital PCR.

Alexandrium pacificum, which produces the paralytic shellfish toxin (PST) saxitoxin (STX), is one of the causative species of paralytic shellfish poisoning outbreaks in coastal areas of Korea. In this study, we developed a chip-based digital PCR (dPCR) method for A. pacificum detection and tested it for monitoring in Jinhae-Masan Bay. Using the sequence of an A. pacificum strain isolated in 2017, species-specific primers targeting sxtA4 (a STX biosynthesis-related gene) were designed and used in a dPCR, detecting 2.0 ± 0.24 gene copies per cell of A. pacificum. Cell abundance in field samples, estimated by a chip-based dPCR, was compared with the PST content, and measured using a mouse bioassay. A comparison with shellfish PST concentrations indicated that cell concentrations above 500 cells L-1, as measured using the dPCR assay, may cause shellfish PST concentrations to exceed the allowed limits for PSTs. Concordance rates between dPCR and PST results were 62.5% overall in 2018-2021, reaching a maximum of 91.7% in 2018-2019. The sensitivity of the dPCR assay was higher than that of microscopy and sxtA4-based qPCRs. Absolute quantification by chip-based dPCRs targeting sxtA4 in A. pacificum exhibits potential as a complementary approach to mouse bioassay PST monitoring for the prevention of toxic blooms.

Dietary exposure assessment of paralytic shellfish toxins through shellfish consumption in Shenzhen population, China.

Paralytic shellfish toxins (PSTs) produced by certain marine dinoflagellates accumulate in filter-feeding marine bivalves. We used LC-MS/MS to detect and quantify 13 PSTs in 188 shellfish samples of 14 species collected from Shenzhen city's Buji seafood wholesale market from March 2019 to February 2020. Twenty-six of 188 shellfish samples (13.8%) were PSTs detectable. Within 14 species, 10 out of 34 noble clam Chlamys nobilis samples contain detectable PSTs with the highest detection rate 29.4%. Seven out of 17 samples from Nan'ao island contained detectable PSTs with the highest detection rate 41.2% among 11 origins. Samples containing PSTs were concentrated in spring and winter, with the highest levels in March>December>January. Among PSTs detected, C1 was dominant. Acute dietary exposure assessment for Shenzhen residents were based on mean adult body weight, 99th percentile daily shellfish consumption of Shenzhen food consumption survey 2008 and maximum PSTs concentration for each shellfish species. The outcome for Chlamys nobilis was 2.4~3.7-fold higher than recommended ARfDs. Mean PSTs concentration, P99, and mean shellfish consumption were used to assess chronic dietary exposure. The results were lower than recommended ARfDs. In conclusion, residents in Shenzhen are at risk for acute PSTs poisoning, while relatively safe from chronic PSTs exposure.