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.

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.

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.

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.

The Common Sunstar Crossaster papposus-A Neurotoxic Starfish.

Saxitoxins (STXs) are a family of potent neurotoxins produced naturally by certain species of phytoplankton and cyanobacteria which are extremely toxic to mammalian nervous systems. The accumulation of STXs in bivalve molluscs can significantly impact animal and human health. Recent work conducted in the North Sea highlighted the widespread presence of various saxitoxins in a range of benthic organisms, with the common sunstar (Crossaster papposus) demonstrating high concentrations of saxitoxins. In this study, an extensive sampling program was undertaken across multiple seas surrounding the UK, with 146 starfish and 5 brittlestars of multiple species analysed for STXs. All the common sunstars analysed (n > 70) contained quantifiable levels of STXs, with the total concentrations ranging from 99 to 11,245 µg STX eq/kg. The common sunstars were statistically different in terms of toxin loading to all the other starfish species tested. Two distinct toxic profiles were observed in sunstars, a decarbomylsaxitoxin (dcSTX)-dominant profile which encompassed samples from most of the UK coast and an STX and gonyautoxin2 (GTX2) profile from the North Yorkshire coast of England. Compartmentalisation studies demonstrated that the female gonads exhibited the highest toxin concentrations of all the individual organs tested, with concentrations >40,000 µg STX eq/kg in one sample. All the sunstars, male or female, exhibited the presence of STXs in the skin, digestive glands and gonads. This study highlights that the common sunstar ubiquitously contains STXs, independent of the geographical location around the UK and often at concentrations many times higher than the current regulatory limits for STXs in molluscs; therefore, the common sunstar should be considered toxic hereafter.

Acute oral toxicity and tissue residues of saxitoxin in the mallard (Anas platyrhynchos).

Since 2014, widespread, annual mortality events involving multiple species of seabirds have occurred in the Gulf of Alaska, Bering Sea, and Chukchi Sea. Among these die-offs, emaciation was a common finding with starvation often identified as the cause of death. However, saxitoxin (STX) was detected in many carcasses, indicating exposure of these seabirds to STX in the marine environment. Few data are available that describe the effects of STX in birds, thus presenting challenges for determining its contributions to specific mortality events. To address these knowledge gaps, we conducted an acute oral toxicity trial in mallards (Anas platyrhynchos), a common laboratory avian model, using an up-and-down method to estimate the median lethal dose (LD50) for STX. Using an enzyme-linked immunosorbent assay (ELISA), we tested select tissues from all birds and feces from those individuals that survived initial dosing. Samples with an ELISA result that exceeded approximately 10 µg 100 g-1 STX and randomly selected ELISA negative samples were further tested by high-performance liquid chromatography (HPLC). Tissues collected from mallards were also examined grossly at necropsy and then later by microscopy to identify lesions attributable to STX. The estimated LD50 was 167 µg kg-1 (95% CI = 69-275 µg kg-1). Saxitoxin was detected in fecal samples of all mallards tested for up to 48 h after dosing and at the end of the sampling period (7 d) in three birds. In those individuals that died or were euthanized <2 h after dosing, STX was readily detected throughout the gastrointestinal tract but only infrequently in heart, kidney, liver, lung, and breast muscle. No gross or microscopic lesions were observed that could be attributable to STX exposure. Given its acute toxicity, limited detectability, and frequent occurrence in the Alaska marine environment, additional research on STX in seabirds is warranted.

[Rapid Screening System for Paralytic Shellfish Toxins in Bivalves by Oligonucleotide Lateral Flow Immunoassay].

The mouse bioassay (MBA) for paralytic shellfish toxins (PSTs) in bivalves has been used as an official method in Japan. It is necessary to develop an alternative method to animal experiments in PSTs assay because 3Rs (Replacement, Reduction, and Refinement) of animal experiments are required from the animal welfare point of view. Various methods such as HPLC-FL, receptor binding assay, LC-MS/MS and ELISA have been established to detect PSTs without performing animal experiments. The present study was undertaken to develop a screening method using oligonucleotide lateral flow immunoassay (OLFIA) for detecting PSTs in bivalves. The screening level was defined as positive at 2 MU/g of MBA that is the half regulation limit of PSTs monitoring in Japan. All 20 positive (equal to or more than 2 MU/g) samples judged from MBA showed a positive reaction in the OLFIA. No positive samples resulted in a false negative reaction. The OLFIA exhibited high accuracy at 2 MU/g of screening criteria. The authors demonstrated here that the OLFIA can be useful for rapid detection of PSTs in bivalves.

Simultaneous determination of ten paralytic shellfish toxins and tetrodotoxin in scallop and short-necked clam by ion-pair solid-phase extraction and hydrophilic interaction chromatography with tandem mass spectrometry.

Paralytic shellfish toxins and tetrodotoxin (puffer-fish toxin), the latter of which was recently found in bivalves from Europe, Japan, and New Zealand, are potent neurotoxins. A simple and effective clean-up procedure was developed for the simultaneous determination of ten paralytic shellfish toxins (gonyautoxins 1-6, decarbamoylgonyautoxins 2 and 3, and N-sulfocarbamoylgonyautoxins 2 and 3) and tetrodotoxin in the scallop, Mizuhopecten (Patinopecten) yessoensis, and the short-necked clam, Ruditapes philippinarum. To reduce matrix effects, 1% aqueous acetic acid extracts of the bivalves were cleaned up by ion-pair solid-phase extraction using a graphite carbon cartridge with tridecafluoroheptanoic acid as the volatile ion-pair reagent, followed by fourfold dilution. The ten paralytic shellfish toxins and tetrodotoxin were then separated on a hydrophilic interaction chromatography column and quantified by tandem mass spectrometry. The limits of detection and the limits of quantification for the ten PSTs ranged from 0.09 to 13.0 µg saxitoxin equivalents/kg and from 0.26 to 39.4 µg saxitoxin equivalents/kg, respectively. The limit of detection and the limit of quantification for tetrodotoxin ranged from 27.4 to 27.9 µg/kg and from 83.1 to 84.4 µg/kg, respectively. The proposed method yielded minimal matrix effects for the 11 analytes, thus allowing their quantification by simple external calibration. The proposed method also gave good mean recoveries of the 11 analytes ranging from 75.7 to 96.2% with relative standard deviations less than 16% at three fortification levels for the ten paralytic shellfish toxins (total concentrations of 277, 554, and 1107 µg saxitoxin equivalents/kg) and tetrodotoxin (100, 200, and 400 µg/kg) in the two bivalve samples. Finally, the proposed method was applied for the determination of the ten paralytic shellfish toxins and tetrodotoxin in scallop and short-necked clam samples.

Marine algal toxins and their vectors in southern California cetaceans.

Published baseline data on biotoxin exposure in cetaceans is sparse but critical for interpreting mortality events as harmful algal blooms increase in frequency and duration. We present the first synthesis of domoic acid (DA), saxitoxin (STX), okadaic acid (OA), and microcystin detections in the feces and urine of stranded and bycaught southern California cetaceans, over an 18 year period (2001-2018), along with corresponding stomach content data. DA was detected in 13 out of 19 cetacean species, most often in harbor porpoise (Phocoena phocoena) (81.8%, n = 22) and long-beaked common dolphins (Delphinus delphis bairdii) (74%, n = 231). Maximum DA concentrations of 324,000 ng/g in feces and 271, 967 ng/ml in urine were observed in D. d. bairdii. DA was detected more frequently and at higher concentrations in male vs. female D. d. bairdii. Higher fecal DA concentrations in D. d. bairdii were associated with a greater proportion of northern anchovy (Engraulis mordax) in the diet, indicating it may be a primary vector of DA. Fecal DA concentrations for D. d. bairdii off Point Conception were greater than those from animals sampled off Los Angeles and San Diego counties, reflecting greater primary productivity and higher Pseudo-nitzschia spp. abundance in that region and a greater abundance of E. mordax in the diet. STX was detected at low levels (fecal max = 7.5 ng/g, urine max = 17 ng/ml) in 3.6% (n = 165) of individuals from 3 out of 11 species. The occurrence of E. mordax in 100% of the 3 examined stomachs suggests this species could be a primary vector of the detected STX. OA was detected in 2.4% of tested individuals (n = 85) at a maximum fecal concentration of 422.8 ng/g. Microcystin was detected in 14.3% (n = 7) of tested individuals with a maximum liver concentration of 96.8 ppb.

Nuclease-assisted target recycling signal amplification strategy for graphene quantum dot-based fluorescent detection of marine biotoxins.

Saxitoxin (STX) is a major marine toxin from shellfish, and it is responsible for paralytic shellfish poisoning (PSP). In this study, a highly sensitive and rapid aptamer assay was developed for STX detection by combining fluorescence resonance energy transfer (FRET) and nuclease-assisted target recycling signal amplification. The aptamer STX-41 conjugated with graphene quantum dots (GQDs) was adsorbed on magnetic reduced graphene oxide (MRGO) to establish a fluorescence quenching system. Then, the binding between STX and aptamer induced the desorption of GQD-aptamer from MRGO and the restoring of fluorescence for the fluorescent determination of STX. The digestion of the target bound aptamer by DNase I could release the target for recycling thus achieving signal amplification. Under the optimized conditions, the aptamer assay showed a wide detection range (0.1-100 ng·mL-1), low detection limit (LOD of 0.035 ng·mL-1), high specificity, good recovery (86.75-94.08% in STX-spiked clam samples) and repeatability (RSD of 4.27-7.34%). Combined with fluorescent detection technology, signal amplification technology, and magnetic separation technology, the proposed method can be used to detect STX in seafood products successfully.