Contamination Status and Risk Assessment of Paralytic Shellfish Toxins in Shellfish along the Coastal Areas of China.

Paralytic shellfish toxins (PSTs) are widely distributed in shellfish along the coast of China, causing a serious threat to consumer health; however, there is still a lack of large-scale systematic investigations and risk assessments. Herein, 641 shellfish samples were collected from March to November 2020, and the PSTs' toxicity was detected via liquid chromatography-tandem mass spectrometry. Furthermore, the contamination status and potential dietary risks of PSTs were discussed. PSTs were detected in 241 shellfish samples with a detection rate of 37.60%. The average PST toxicities in mussels and ark shells were considerably higher than those in other shellfish. The PSTs mainly included N-sulfonylcarbamoyl toxins (class C) and carbamoyl toxins (class GTX), and the highest PST toxicity was 546.09 µg STX eq. kg-1. The PST toxicity in spring was significantly higher than those in summer and autumn (p < 0.05). Hebei Province had the highest average PST toxicity in spring. An acute exposure assessment showed that consumers in Hebei Province had a higher dietary risk, with mussels posing a significantly higher dietary risk to consumers. This research provides reference for the green and sustainable development of the shellfish industry and the establishment of a shellfish toxin prevention and control system.

Developing a detection strategy for ten paralytic shellfish poisonings in urine, combining high-throughput DESI-MS screening and accurate UPLC-QqQ/MS quantification.

Paralytic shellfish poisoning (PSP) is the most widespread and harmful form of shellfish poisoning with high mortality rate. In this study, a combined desorption electrospray ionization mass spectrometry (DESI-MS) and ultra-performance liquid chromatography triple quadrupole mass spectrometry (UPLC-QqQ/MS) method was established for the detection of PSPs in urine. The method was optimized using a spray solution of methanol and water (1:1, v/v) containing 0.1 % FA, at a flow rate of 2.5 µL·min-1 and an applied voltage of 3 kV. The limit of detection (LOD) for PSPs detection by DESI-MS was in the range of 87-265 µg·L-1, which basically meets the requirements for the rapid screening of PSPs. The LOD for UPLC-QqQ/MS was in the range of 2.2-14.9 µg·L-1, with a limit of quantification (LOQ) of 7.3-49.7 µg·L-1, thus fulfilling the quantitative demand for PSPs in urine. Finally, after spiking the urine samples of six volunteers with PSPs to a concentration of 100 µg·L-1, DESI-MS successfully and efficiently detected the positive samples. Subsequently, UPLC-QqQ/MS was employed for precise quantification, yielding results in the range of 84.6-95.1 µg·L-1. The experimental findings demonstrated that the combination of DESI-MS and UPLC-QqQ/MS enables high-throughput, rapid screening of samples and accurate quantification of positive samples, providing assurance for food safety and human health.

Clam calamity: five concurrent cases of neurotoxic shellfish poisoning with varying presentations following ingestion of clams from Gulf of Mexico water contaminated with Karenia brevis confirmed by serum brevetoxin assays.

INTRODUCTION: Karinia brevis, a marine dinoflagellate, is the causative organism for "red-tide" on the east coast of Florida.This microbe produces brevetoxins, which bioaccumulate in filter feeding bivalve shellfish. In humans, inhalational exposure is common, while ingestion of contaminated shellfish is more rare. Ingested brevetoxin causes gastrointestinal and neurological symptoms collectively known as neurotoxic shellfish poisoning. CASE CLUSTER: A group of tourists collected clams from a beach during a red tide event. The clams were soaked in brine, microwaved, and consumed for lunch. The index patient experienced seizure-like activity postprandially prompting the cohort to present for medical attention. Five people presented to the emergency department with neurotoxic shellfish poisoning-related symptoms. All patients received supportive care only. Symptoms resolved within 24 hours. Serum brevetoxin concentrations were reported for four patients. DISCUSSION: Ingestion of brevetoxin is rare but may become more common as the frequency and severity of "red-tide" events increase. In our cluster, each person consumed a different number of clams and presented with classic and some "non-classic" symptoms. A trend toward more severe symptoms with a larger number of clams ingested was observed. CONCLUSIONS: This case cluster describes the clinical course of individuals after consumption of brevetoxin contaminated shellfish.

Identification of Perna viridis contaminated with diarrhetic shellfish poisoning toxins in vitro using NIRS and a discriminative non-negative representation-based classifier.

Diarrhetic shellfish poisoning (DSP) toxins are one of the most widespread marine biotoxins that affect aquaculture and human health, and their detection has become crucial. In this study, near-infrared reflectance spectroscopy (NIRS) with non-destructive characteristics was used to identify DSP toxins in Perna viridis. The spectral data of the DSP toxin-contaminated and non-contaminated Perna viridis samples were acquired in the 950-1700 nm range. To solve the discrimination of spectra with crossover and overlapping, a discriminative non-negative representation-based classifier (DNRC) has been proposed. Compared with collaborative and non-negative representation-based classifiers, the DNRC model exhibited better performance in detecting DSP toxins, with a classification accuracy of 99.44 %. For a relatively small-scale sample dataset in practical applications, the performance of the DNRC model was compared with those of classical models. The DNRC model achieved the best results for both identification accuracy and F-measure, and its detection performance did not significantly decrease with decreasing sample size. The experimental results validated that a combination of NIRS and the DNRC model can facilitate rapid, convenient, and non-destructive detection of DSP toxins in Perna viridis.

Human poisonings by neurotoxic phycotoxins related to the consumption of shellfish: study of cases registered by the French Poison Control Centres from 2012 to 2019.

CONTEXT: In June 2019, a paralytic shellfish poisoning (PSP) case related to the consumption of mussels contaminated by saxitoxins at a concentration below the regulatory threshold came to the attention of the French Agency for Food, Environmental and Occupational Health and Safety (ANSES). This pointed to probable undetected human cases of poisoning by neurotoxic phycotoxins. METHODS: We conducted a retrospective study of poisoning cases by bivalve shellfish (oysters, mussels and scallops) recorded by the French Poison Control Centres (PCC) from 2012 to 2019. All medical records were reviewed by a toxicologist.Cases that could be related to neurotoxic phycotoxins were selected and described. Diagnosis was based on symptoms compatible with ingestion of contaminated shellfish and on contamination data for the shellfish production area (analysed by the French Research Institute for Exploitation of the Sea, Ifremer), or notifications to the European Rapid Alert System for Food and Feed when the origin of the shellfish was known. RESULTS: Among the 619 shellfish poisoning cases recorded by the PCCs from 2012 to 2019, 22% (n = 134) had reported at least one neurological symptom (headache, dizziness or paraesthesia). Review of medical records for the 134 patients led to suspicion of 14 cases of PSP and one case of amnesic shellfish poisoning. Five patients experienced persistent neurological symptoms. Marine toxins were not tested for in the blood or urine of these patients. CONCLUSION: This retrospective identification of cases strongly suspected of being related to neurotoxic phycotoxins led ANSES, PCCs and Ifremer to develop a specific questionnaire and to recommend actions to take when neurological symptoms related to shellfish consumption are reported to a PCC. Daily monitoring of shellfish poisoning cases registered in the national PCCs database was also implemented in order to rapidly detect any suspicious cases, alert the competent authorities, and warn the general population.

Drastic hypothermia after intraperitoneal injection of okadaic acid, a diarrhetic shellfish poisoning toxin, in mice.

The mouse bioassay for diarrhetic shellfish poisoning (DSP) toxins had been used as the official method in Japan and also used in the world. In this study, hypothermia, one of the symptoms observed in mice after inoculation with DSP toxins, were characterized. Lethal and sublethal doses of okadaic acid (OA), a representative component of DSP toxins, were inoculated intraperitoneally into mice. Body-temperature changes over time were measured by an electronic thermometer or monitored by an infrared camera. Drastic hypothermia (<30°C in some mice) was observed in a few hours after administration of a lethal dose of OA. Dose-dependency was clearly seen between doses of OA inoculated and body-temperature decrease. Drastic hypothermia was also detected by using an infrared camera. These results suggest that hypothermia could be used as an index for the humane endpoint in experimental animal toxicological studies.

Application of Six Detection Methods for Analysis of Paralytic Shellfish Toxins in Shellfish from Four Regions within Latin America.

With the move away from use of mouse bioassay (MBA) to test bivalve mollusc shellfish for paralytic shellfish poisoning (PSP) toxins, countries around the world are having to adopt non-animal-based alternatives that fulfil ethical and legal requirements. Various assays have been developed which have been subjected to single-laboratory and multi-laboratory validation studies, gaining acceptance as official methods of analysis and approval for use in some countries as official control testing methods. The majority of validation studies conducted to date do not, however, incorporate shellfish species sourced from Latin America. Consequently, this study sought to investigate the performance of five alternative PSP testing methods together with the MBA, comparing the PSP toxin data generated both qualitatively and quantitatively. The methods included a receptor binding assay (RBA), two liquid chromatography with fluorescence detection (LC-FLD) methods including both pre-column and post-column oxidation, liquid chromatography with tandem mass spectrometry (LC-MS/MS) and a commercial lateral flow assay (LFA) from Scotia. A total of three hundred and forty-nine shellfish samples from Argentina, Mexico, Chile and Uruguay were assessed. For the majority of samples, qualitative results compared well between methods. Good statistical correlations were demonstrated between the majority of quantitative results, with a notably excellent correlation between the current EU reference method using pre-column oxidation LC-FLD and LC-MS/MS. The LFA showed great potential for qualitative determination of PSP toxins, although the findings of high numbers of false-positive results and two false negatives highlighted that some caution is still needed when interpreting results. This study demonstrated that effective replacement methods are available for countries that no longer wish to use the MBA, but highlighted the importance of comparing toxin data from the replacement method using local shellfish species of concern before implementing new methods in official control testing programs.

Dihydrodinophysistoxin-1 Produced by Dinophysis norvegica in the Gulf of Maine, USA and Its Accumulation in Shellfish.

Dihydrodinophysistoxin-1 (dihydro-DTX1, (M-H)-m/z 819.5), described previously from a marine sponge but never identified as to its biological source or described in shellfish, was detected in multiple species of commercial shellfish collected from the central coast of the Gulf of Maine, USA in 2016 and in 2018 during blooms of the dinoflagellate Dinophysis norvegica. Toxin screening by protein phosphatase inhibition (PPIA) first detected the presence of diarrhetic shellfish poisoning-like bioactivity; however, confirmatory analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS) failed to detect okadaic acid (OA, (M-H)-m/z 803.5), dinophysistoxin-1 (DTX1, (M-H)-m/z 817.5), or dinophysistoxin-2 (DTX2, (M-H)-m/z 803.5) in samples collected during the bloom. Bioactivity-guided fractionation followed by liquid chromatography-high resolution mass spectrometry (LC-HRMS) tentatively identified dihydro-DTX1 in the PPIA active fraction. LC-MS/MS measurements showed an absence of OA, DTX1, and DTX2, but confirmed the presence of dihydro-DTX1 in shellfish during blooms of D. norvegica in both years, with results correlating well with PPIA testing. Two laboratory cultures of D. norvegica isolated from the 2018 bloom were found to produce dihydro-DTX1 as the sole DSP toxin, confirming the source of this compound in shellfish. Estimated concentrations of dihydro-DTX1 were >0.16 ppm in multiple shellfish species (max. 1.1 ppm) during the blooms in 2016 and 2018. Assuming an equivalent potency and molar response to DTX1, the authority initiated precautionary shellfish harvesting closures in both years. To date, no illnesses have been associated with the presence of dihydro-DTX1 in shellfish in the Gulf of Maine region and studies are underway to determine the potency of this new toxin relative to the currently regulated DSP toxins in order to develop appropriate management guidance.

Dose-response assessment for impaired memory from chronic exposure to domoic acid among native American consumers of razor clams.

Domoic acid (DA) is a marine neurotoxin that accumulates in filtering shellfish during harmful algal blooms. A health protection limit of 20 ppm DA in razor clams (RC) has been set based principally upon an episode of acute DA toxicity in humans that included Amnesic Shellfish Poisoning among survivors. The objective of this study was to determine the dose-response relationship between estimated DA exposure through RC consumption and memory loss in Washington state Native Americans from 2005 to 2015. Results from total learning recall (TLR) memory scores were compared before and after the highest DA exposures. A decrease in TLR was related to DA dose (p < 0.01) regardless whether the effect was assumed to be transient or lasting, and whether the dose was expressed as an average daily dose or an average dose per meal. Benchmark dose modeling identified BMDL10 values of 167 ng/kg-day and 2740 ng/kg-meal assuming a transient effect, and 196 ng/kg-day and 2980 ng/kg-meal assuming no recovery of function occurs. These DA dose thresholds for a measurable memory function reduction observed in this study of clam consumers are well below the safe acute dose underpinning the current regulatory DA limit of 20 ppm (ca. 60 µg/kg).

Paralytic shellfish poisoning and palytoxin poisoning in dogs.

BACKGROUND: Fatal cases of exposure to paralytic shellfish toxins and palytoxins have occurred in companion animals but are poorly described. METHODS: We describe one case of paralytic shellfish poisoning (PSP) and three cases of palytoxin poisoning in dogs. RESULTS: Mild PSP occurred following ingestion of crab while walking on a beach. Analysis confirmed the presence of paralytic shellfish toxins, particularly decarbamoyl saxitoxin, in clinical samples and marine organisms. This case occurred shortly after an outbreak of PSP in dogs on the eastern coast of England. Palytoxin poisoning occurred in a dog after it chewed coral removed from an aquarium. Signs included collapse, hypothermia, bloody diarrhoea and respiratory distress. The dog was euthanised due to rapid deterioration and poor prognosis. Palytoxin was not detected in a premortem blood sample. Two other dogs in a separate incident developed only mild signs (fever and respiratory distress) after suspected exposure to aerosolised palytoxin and recovered within a few hours. CONCLUSION: Cases of PSP are episodic and not common in dogs. Cases of palytoxin exposure are reportedly increasing in humans, and there is presumably also an increased risk to pets. There is no specific treatment for PSP or palytoxin poisoning.

First detection of tetrodotoxin and high levels of paralytic shellfish poisoning toxins in shellfish from Sicily (Italy) by three different analytical methods.

Paralytic shellfish toxins (PST) and tetrodotoxin (TTX) are naturally-occurring toxins that may contaminate the food chain, inducing similar neurological symptoms in humans. They are co-extracted under the same conditions and thus their combined detection is desirable. Whilst PST are regulated and officially monitored in Europe, more data on TTX occurrence in bivalves and gastropods are needed before meaningful regulations can be established. In this study, we used three separate analytical methods - pre-column oxidation with liquid chromatography and fluorescence detection, ultrahigh performance hydrophilic interaction liquid chromatography (HILIC) tandem mass spectrometry (MS/MS) and HILIC high resolution (HR) MS/MS - to investigate the presence of PST and TTX in seawater and shellfish (mussels, clams) collected in spring summer 2015 to 2017 in the Mediterranean Sea. Samples were collected at 10 sites in the Syracuse Bay (Sicily, Italy) in concomitance with a mixed bloom of Alexandrium minutum and A. pacificum. A very high PST contamination in mussels emerged, unprecedentedly found in Italy, with maximum total concentration of 10851 µg saxitoxin equivalents per kg of shellfish tissue measured in 2016. In addition, for the first time TTX was detected in Italy in most of the analysed samples in the range 0.8-6.4 µg TTX eq/kg. The recurring blooms of PST-producing species over the 3-year period, the high PST levels and the first finding of TTX in mussels from the Syracuse bay, suggest that monitoring programmes of PST and TTX in seafood should be activated in this geographical area.

Outbreak of Haff disease caused by consumption of crayfish (Procambarus clarkii) in nanjing, China.

CONTEXT: This paper reports a detailed epidemiological investigation of a large Haff disease outbreak in Nanjing, China, including laboratory analysis of water, river sediment, and fish. METHODS: Structured questionnaires were administered to all case-patients. An unmatched case-control study was conducted to identify the factors affecting the incidence of Haff disease. Thirteen samples of crayfish, together with four samples of water and four samples of sediment were collected and sent for laboratory analysis. RESULTS: The disease onset of 494 patients occurred between 2 am on 30 June 2016 and 3 am on 29 August 2016. The median incubation time for onset of symptoms after crayfish ingestion was 7.1 hours (range: 1-20 hours). All patients presented with or developed local or diffuse myalgia. However, no kidney injury was observed. Serum creatinine kinase was elevated in all patients (mean: 4614 U/L, median: 2000 U/L, range: 81-55200 U/L). The average number of crayfish consumed by patients on the day of illness was 20 (2-80). The case-control study revealed that eating a large quantity of crayfish, drinking alcohol, and eating the crayfish head and/or intestines were associated with an increased risk of disease. Chemical analysis of crayfish, river water and sediment did not identify known or novel toxins including anticoccidiosis drugs, niclosamide, organophosphorus pesticides, and microcystins. CONCLUSION: An outbreak of Haff Disease was associated with consumption of crayfish from the Yangtze River and its surrounding water system. Eating a large quantity of crayfish, eating the head and/or intestines, and consuming alcohol with the crayfish were associated with the development of Haff Disease.

A Dual Functional Cardiomyocyte-based Hybrid-biosensor for the Detection of Diarrhetic Shellfish Poisoning and Paralytic Shellfish Poisoning Toxins.

Okadaic acid (OA) and saxitoxin (STX) are typical toxins of diarrhetic shellfish poisoning (DSP) and paralytic shellfish poisoning (PSP), respectively, which are highly toxic marine toxins threatening human health and environmental safety. OA is a potent inhibitor of serine/threonine protein phosphatases that can cause cellular death, while STX is an inhibitor of sodium channel that can lead to neurological damage. In this work, a dual functional cardiomyocyte-based biosensor was proposed to detect DSP and PSP toxins by monitoring the viability and electrophysiology of cardiomyocytes. The results showed that the viability of cardiomyocytes was sensitive to the OA and STX, resulting in significant changes of the electrophysiological properties, including amplitude, firing rate and duration of the extracellular field potential (EFP). The detection limits of the hybrid-biosensor are as low as 7.16 ng/mL for OA and 5.19 ng/mL for STX. In summary, all of the results indicate that the dual functional cardiomyocyte-based hybrid-biosensor will be a promising and utility tool for shellfish toxin detection.

Exposure assessment to paralytic shellfish toxins through the shellfish consumption in Korea.

Paralytic shellfish poisoning is caused by saxitoxin and its analogues. The paralytic shellfish toxins (PSTs) are produced by marine dinoflagellates and can be accumulated in filter feeding shellfish, such as mussel, clam, oyster and ark shell. The worldwide regulatory limits for PSTs in shellfish are set at 80 µg STX eq./100 g meat and this is widely accepted as providing adequate public health protection. In this study, we have determined five individual PSTs (STX, GTX1, GTX2, GTX3 and GTX4) in shellfish using LC-MS/MS and assessed the human acute and chronic exposures to PSTs through shellfish consumption. Food consumption data was obtained from the Korea National Health and Nutrition Examination Survey (KNHANES 2010-2015). The acute exposure using a large portion size of 88 g/day (95th percentile for consumers only) with maximum toxin level of 198.7 µg/kg was 0.30 µg/kg bw. Even though we estimated the acute exposure with a conservative manner, it was below the ARfDs (0.5 or 0.7 µg STX eq./kg bw) proposed by the international organizations, representing 43-60% of the ARfDs. The chronic exposures using mean consumption data for whole population with mean concentration of PSTs were ranged from 0.002 to 0.026 µg STX eq./kg bw/day. For consumers only, the chronic exposures were in the range of 0.012-0.128 µg STX eq./kg bw/day.

Saxitoxin Exposure Confirmed by Human Urine and Food Analysis.

A case of an elderly female with suspected paralytic shellfish poisoning (PSP) is presented. The patient shared a meal of recreationally-harvested shellfish with her family and soon began to experience nausea and weakness. She was taken to the local emergency department and then transported to a larger hospital in Anchorage where she was admitted to the intensive care unit with respiratory depression and shock. Her condition improved, and she was discharged from the hospital 6 days later. No others who shared the meal reported symptoms of PSP. A clam remaining from the meal was collected and analyzed for paralytic shellfish toxins (PST) by the Alaska Department of Environmental Conservation Environmental Health Laboratory; the clam tested positive for saxitoxin (STX; 277 µg/100 g), neosaxitoxin (NEO; 309 µg/100 g), multiple gonyautoxins (GTX; 576-2490 µg/100 g), decarbamoyl congeners (7.52-11.3 µg/100 g) and C-toxins (10.8-221 µg/100 g) using high-pressure liquid chromatography with post-column oxidation (AOAC Method 2011.02). Urine from the patient was submitted to Centers for Disease Control for analysis of selected PSTs and creatinine. STX (64.0 µg/g-creatinine), NEO (60.0 µg/g-creatinine) and GTX1-4 (492-4780 µg/g-creatinine) were identified in the urine using online solid phase extraction with HPLC and tandem mass spectrometry. This was the first time GTX were identified in urine of a PSP case from Alaska, highlighting the need to include all STX congeners in testing to protect the public's health through a better understand of PST toxicity, monitoring and prevention of exposures.

Quantitation of saxitoxin in human urine using immunocapture extraction and LC-MS.

AIM: An immunomagnetic capture protocol for use with LC-MS was developed for the quantitation of saxitoxin (STX) in human urine. MATERIALS & METHODS: This method uses monoclonal antibodies coupled to magnetic beads. STX was certified reference material grade from National Research Council, Canada. Analysis was carried out using LC-MS. RESULTS: With an extraction efficiency of 80%, accuracy and precision of 93.0-100.2% and 5.3-12.6%, respectively, and a dynamic range of 1.00-100 ng/ml, the method is well suited to quantify STX exposures based on previously reported cases. CONCLUSION: Compared with our previously published protocols, this method has improved selectivity, a fivefold increase in sensitivity and uses only a third of the sample volume. This method can diagnose future toxin exposures and may complement the shellfish monitoring programs worldwide.

A cluster of patients with rhabdomyolysis after eating crayfish.

Crayfish or Procambarus clarkii is a freshwater crustacean with worldwide distribution. Tons of crayfish are consumed each year. In this report, four adult patients with rhabdomyolysis after consuming crayfish were described. All of them presented to the emergency department with myalgia. The diagnosis of rhabdomyolysis was supported by an elevated creatine kinase level. All recovered with supportive treatment. The clinical picture of these 4 patients was compatible with Haff disease. Haff disease is a syndrome in which rhabdomyolysis develops subsequent to consumption of certain cooked seafood. Crayfish is a common culprit. Diagnosis depends on obtaining a diet history and creatine kinase level. Most patients recover uneventfully with supportive treatment for rhabdomyolysis.

Case Report: Paralytic Shellfish Poisoning in Sabah, Malaysia.

During the months of January-February and May-June 2013 coinciding with the red tide occurrence in Kota Kinabalu, Sabah, Malaysia, six episodes involving 58 cases of paralytic shellfish poisoning (PSP) or saxitoxin (STX) poisoning and resulting in four deaths were reported. Many of them were intoxicated from consuming shellfish purchased from the markets, whereas others were intoxicated from eating shellfish collected from the beach. Levels of STX in shellfish collected from the affected areas were high (mean 2,920 ± 780 and 360 ± 140 µg STX equivalents/100 g shellfish meat respectively for the two periods). The count of toxic dinoflagellates (Pyrodinium bahamense var compressum) of the sea water sampled around the coast was also high (mean 34,200 ± 10,300 cells/L). Species of shellfish containing high levels of STX were Atrina fragilis, Perna viridis, and Crassostrea belcheri. The age of victims varied from 9 to 67 years. Symptoms presented were typical of PSP, such as dizziness, numbness, vomiting, and difficulty in breathing. Recommended steps to prevent or reduce PSP in future red tide season include better monitoring of red tide occurrence, regular sampling of shellfish for determination of STX level, wider dissemination of information on the danger of eating contaminated shellfish among the communities, fishermen, and fishmongers.