A Comparative Analysis of Methods (LC-MS/MS, LC-MS and Rapid Test Kits) for the Determination of Diarrhetic Shellfish Toxins in Oysters, Mussels and Pipis

Rapid methods for the detection of biotoxins in shellfish can assist the seafood industry and safeguard public health. Diarrhetic Shellfish Toxins (DSTs) are produced by species of the dinoflagellate genus Dinophysis, yet the comparative efficacy of their detection methods has not been systematically determined. Here, we examined DSTs in spiked and naturally contaminated shellfish–Sydney Rock Oysters (Saccostrea glomerata), Pacific Oysters (Magallana gigas/Crassostrea gigas), Blue Mussels (Mytilus galloprovincialis) and Pipis (Plebidonax deltoides/Donax deltoides), using LC-MS/MS and LC-MS in 4 laboratories, and 5 rapid test kits (quantitative Enzyme-Linked Immunosorbent Assay (ELISA) and Protein Phosphatase Inhibition Assay (PP2A), and qualitative Lateral Flow Assay (LFA)). We found all toxins in all species could be recovered by all laboratories using LC-MS/MS (Liquid Chromatography—tandem Mass Spectrometry) and LC-MS (Liquid Chromatography—Mass Spectrometry); however, DST recovery at low and mid-level concentrations (<0.1 mg/kg) was variable (0–150%), while recovery at high-level concentrations (>0.86 mg/kg) was higher (60–262%). While no clear differences were observed between shellfish, all kits delivered an unacceptably high level (25–100%) of falsely compliant results for spiked samples. The LFA and the PP2A kits performed satisfactorily for naturally contaminated pipis (0%, 5% falsely compliant, respectively). There were correlations between spiked DSTs and quantitative methods was highest for LC-MS (r2 = 0.86) and the PP2A kit (r2 = 0.72). Overall, our results do not support the use of any DST rapid test kit as a stand-alone quality assurance measure at this time.

Cinnamaldehyde Could Reduce the Accumulation of Diarrhetic Shellfish Toxins in the Digestive Gland of the Mussel Perna viridis under Laboratory Conditions

Diarrhetic shellfish toxins (DSTs), some of the most important phycotoxins, are distributed almost all over the world, posing a great threat to human health through the food chain. Therefore, it is of great significance to find effective methods to reduce toxin accumulation in shellfish. In this paper, we observed the effects of four phytochemicals including cinnamaldehyde (CA), quercetin, oridonin and allicin on the accumulation of DSTs in the digestive gland of Perna viridis after exposure to the DSTs-producing Prorocentrum lima. We found that, among the four phytochemicals, CA could effectively decrease the accumulation of DSTs (okadaic acid-eq) in the digestive gland of P. viridis. Further evidence demonstrated that CA could reduce the histological alterations of the digestive gland of a mussel caused by DSTs. RT-qPCR showed that CA could suppress the CYP3A4 induction by DSTs, suggesting that the DSTs’ decrease induced by CA might be related to the inhibition of CYP3A4 transcription induction. However, further studies on the underlying mechanism, optimal treatment time, ecological safety and cost should be addressed before cinnamaldehyde is used to decrease the accumulation of DSTs in field.

OMICs Approaches in Diarrhetic Shellfish Toxins Research

Diarrhetic shellfish toxins (DSTs) are among the most prevalent marine toxins in Europe’s and in other temperate coastal regions. These toxins are produced by several dinoflagellate species; however, the contamination of the marine trophic chain is often attributed to species of the genus Dinophysis. This group of toxins, constituted by okadaic acid (OA) and analogous molecules (dinophysistoxins, DTXs), are highly harmful to humans, causing severe poisoning symptoms caused by the ingestion of contaminated seafood. Knowledge on the mode of action and toxicology of OA and the chemical characterization and accumulation of DSTs in seafood species (bivalves, gastropods and crustaceans) has significantly contributed to understand the impacts of these toxins in humans. Considerable information is however missing, particularly at the molecular and metabolic levels involving toxin uptake, distribution, compartmentalization and biotransformation and the interaction of DSTs with aquatic organisms. Recent contributions to the knowledge of DSTs arise from transcriptomics and proteomics research. Indeed, OMICs constitute a research field dedicated to the systematic analysis on the organisms’ metabolisms. The methodologies used in OMICs are also highly effective to identify critical metabolic pathways affecting the physiology of the organisms. In this review, we analyze the main contributions provided so far by OMICs to DSTs research and discuss the prospects of OMICs with regard to the DSTs toxicology and the significance of these toxins to public health, food safety and aquaculture.

A Rapid Method for the Detection of Diarrhetic Shellfish Toxins and Azaspiracid Shellfish Toxins in Washington State Shellfish by Liquid Chromatography Tandem Mass Spectrometry

Background Diarrhetic shellfish toxins (DSTs) in domestic shellfish and azaspiracids (AZAs) in imported products are emerging seafood safety issues in the United States. In addition to causing gastrointestinal illnesses, some of these toxins are also carcinogenic and genotoxic. Efficient analytical strategies are needed for their monitoring in U.S. domestic and imported shellfish. Objective In the US, DSTs and AZAs are the only lipophilic shellfish toxins addressed in regulations. Streamlining of existing methods for several classes of lipophilic toxins, based on liquid chromatography coupled with triple quadrupole mass spectrometry, was pursued. Method The resulting simplified LC-MS/MS method is focused on the separation and detection of just the AZAs and total DSTs using a C18 Hypersil gold column. Filter vials are used to expedite and simplify sample handling. Results The method has a run time of 7.25 min. LOQs for the AZAs and DSTs in shellfish were 0.3–0.4 µg/kg. Recoveries (AZAs and total DSTs) for three spiking levels in three matrixes ranged from 68 to 129%. Trueness was established using certified reference materials. Method equivalence was established using shellfish provided blind by the Washington State Department of Health Public Health Laboratory (WA DOH PHL). Data obtained from these samples agreed well with data from another LC-MS/MS method used in harvest control by WA DOH PHL (R = 0.999; P &lt; 0.0001). Conclusions The LC-MS/MS method described offers more rapid sample handling and has excellent sensitivity, linearity, and repeatability.

Dinophysis Species and Diarrhetic Shellfish Toxins: 20 Years of Monitoring Program in Andalusia, South of Spain

In Andalusia, the official monitoring program for toxic phytoplankton and marine biotoxins was launched in 1994 to comply with European legislation. Since then, there have been numerous episodes of DST (Diarrhetic shellfish toxins) associated with the proliferation of Dinophysis species. This article reviews two decades of time series data and assesses the effectiveness of the program established. The testing of lipophilic toxins and toxic phytoplankton is based on official methods harmonized and accredited since 2007 according to the standard UNE-EN-ISO 17025. The major species of Dinophysis identified were D. acuminata complex, D. caudata, D. acuta and D. fortii, with the main growth season being from early spring until the end of autumn. Both D. acuminata complex and D. acuta have been clearly associated with toxicity in molluscs. Despite the complexity of data obtained through monitoring programs, it is possible to provide early warning of potential health risks for most situations. This is the first report of Dinophysis species and their relation to DST events in a time series from Andalusia.