Exploration of Vulcanodinium rugosum Toxins and their Metabolism Products in Mussels from the Ingril Lagoon Hotspot in France.

Over the year 2018, we assessed toxin contamination of shellfish collected on a monthly basis in Ingril Lagoon, France, a site known as a hotspot for Vulcanodinium rugosum growth. This short time-series study gave an overview of the presence and seasonal variability of pinnatoxins, pteriatoxins, portimines and kabirimine, all associated with V. rugosum, in shellfish. Suspect screening and targeted analysis approaches were implemented by means of liquid chromatography coupled to both low- and high-resolution mass spectrometry. We detected pinnatoxin-A and pinnatoxin-G throughout the year, with maximum levels for each one observed in June (6.7 µg/kg for pinnatoxin-A; 467.5 µg/kg for pinnatoxin-G), whereas portimine-A was detected between May and September (maximum level = 75.6 µg/kg). One of the main findings was the identification of a series of fatty acid esters of pinnatoxin-G (n = 13) although the levels detected were low. The profile was dominated by the palmitic acid conjugation of pinnatoxin-G. The other 12 fatty acid esters had not been reported in European shellfish to date. In addition, after thorough investigations, two compounds were detected, with one being probably identified as portimine-B, and the other one putatively attributed to pteriatoxins. If available, reference materials would have ensured full identification. Monitoring of these V. rugosum emerging toxins and their biotransformation products will contribute towards filling the data gaps pointed out in risk assessments and in particular the need for more contamination data for shellfish.

Five Years Monitoring the Emergence of Unregulated Toxins in Shellfish in France (EMERGTOX 2018-2022).

Shellfish accumulate microalgal toxins, which can make them unsafe for human consumption. In France, in accordance with EU regulations, three groups of marine toxins are currently under official monitoring: lipophilic toxins, saxitoxins, and domoic acid. Other unregulated toxin groups are also present in European shellfish, including emerging lipophilic and hydrophilic marine toxins (e.g., pinnatoxins, brevetoxins) and the neurotoxin ß-N-methylamino-L-alanine (BMAA). To acquire data on emerging toxins in France, the monitoring program EMERGTOX was set up along the French coasts in 2018. Three new broad-spectrum LC-MS/MS methods were developed to quantify regulated and unregulated lipophilic and hydrophilic toxins and the BMAA group in shellfish (bivalve mollusks and gastropods). A single-laboratory validation of each of these methods was performed. Additionally, these specific, reliable, and sensitive operating procedures allowed the detection of groups of EU unregulated toxins in shellfish samples from French coasts: spirolides (SPX-13-DesMeC, SPX-DesMeD), pinnatoxins (PnTX-G, PnTX-A), gymnodimines (GYM-A), brevetoxins (BTX-2, BTX-3), microcystins (dmMC-RR, MC-RR), anatoxin, cylindrospermopsin and BMAA/DAB. Here, we present essentially the results of the unregulated toxins obtained from the French EMERGTOX monitoring plan during the past five years (2018-2022). Based on our findings, we outline future needs for monitoring to protect consumers from emerging unregulated toxins.

First Detection of Tetrodotoxin in Bivalves and Gastropods from the French Mainland Coasts.

In 2015, tetrodotoxins (TTXs) were considered a potential threat in Europe since several studies had shown the presence of these toxins in European bivalve molluscs. In this study, we investigated the occurrence of TTXs in 127 bivalve samples (mussels and oysters) and in 66 gastropod samples (whelks) collected all along the French mainland coasts in 2017 and 2018. Analyses were carried out after optimization and in-house validation of a performing hydrophilic interaction liquid chromatography associated with tandem mass spectrometry (HILIC-MS/MS) method. The concentration set by European Food Safety Authority (EFSA) not expected to result in adverse effects (44 µg TTX equivalent/kg) was never exceeded, but TTX was detected in three mussel samples and one whelk sample (1.7-11.2 µg/kg). The tissue distribution of TTX in this whelk sample showed higher concentrations in the digestive gland, stomach and gonads (7.4 µg TTX/kg) than in the rest of the whelk tissues (below the limit of detection of 1.7 µg TTX/kg). This is the first study to report the detection of TTX in French molluscs.

First Evidence of the Presence of Anatoxin-A in Sea Figs Associated with Human Food Poisonings in France.

From January 2011 to March 2018, 26 patients aged from 20 to 80 years old reported being sick in France after eating sea figs of the genus Microcosmus. The patients had symptoms evoking a cerebellar syndrome: blurred or double vision, ataxia and dizziness, asthenia, headache, muscle cramps, paresthesia and digestive disorders (nausea, vomiting and diarrhea). Three of the 18 food poisoning events recorded by the Poison Control Center in Marseille and involving four patients were further investigated as the meal leftovers were collected and analyzed. A previous study ruled out the presence of the regulated lipophilic marine toxins after high-resolution mass spectrometry, but further analyses were required to look for hydrophilic cyanotoxins. The sea fig leftovers from food poisoning case Numbers 1 (January 2011), 6 (December 2012) and 17 (March 2018) of this published case series were analyzed by hydrophilic interaction liquid chromatography coupled to low- and high-resolution mass spectrometry to investigate the presence of hydrophilic cyanotoxins. The sea fig samples showed anatoxin-a (ATX-a) concentrations ranging from 193.7 to 1240.2 µg/kg. The sea fig control sample analyzed was also contaminated with ATX-a but in a much smaller concentration (22.5 µg/kg). To the best of our knowledge, this is the first report of human food poisoning involving ATX-a as the possible causative toxin where the cyanotoxin could be unequivocally identified.

Extended Targeted and Non-Targeted Strategies for the Analysis of Marine Toxins in Mussels and Oysters by (LC-HRMS).

When considering the geographical expansion of marine toxins, the emergence of new toxins and the associated risk for human health, there is urgent need for versatile and efficient analytical methods that are able to detect a range, as wide as possible, of known or emerging toxins. Current detection methods for marine toxins rely on a priori defined target lists of toxins and are generally inappropriate for the detection and identification of emerging compounds. The authors describe the implementation of a recent approach for the non-targeted analysis of marine toxins in shellfish with a focus on a comprehensive workflow for the acquisition and treatment of the data generated after liquid chromatography coupled with high resolution mass spectrometry (LC-HRMS) analysis. First, the study was carried out in targeted mode to assess the performance of the method for known toxins with an extended range of polarities, including lipophilic toxins (okadaic acid, dinophysistoxins, azaspiracids, pectenotoxins, yessotoxins, cyclic imines, brevetoxins) and domoic acid. The targeted method, assessed for 14 toxins, shows good performance both in mussel and oyster extracts. The non-target potential of the method was then challenged via suspects and without a priori screening by blind analyzing mussel and oyster samples spiked with marine toxins. The data processing was optimized and successfully identified the toxins that were spiked in the blind samples.

Ochratoxin A determination in swine muscle and liver from French conventional or organic farming production systems.

Consumers generally considered organic products to be healthier and safer but data regarding the contamination of organic products are scarce. This study evaluated the impact of the farming system on the levels of ochratoxin A (OTA) in the tissues of French pigs (muscle and liver) reared following three different types of production (organic, Label Rouge and conventional). Because OTA is present at trace levels in animal products, a sensitive ultra-high performance liquid chromatography-tandem mass spectrometry method using stable isotope dilution assay was developed and validated. OTA was detected or quantified (LOQ of 0.10 µg kg-1) in 67% (n = 47) of the 70 pig liver samples analysed, with concentrations ranging from <0.10 to 3.65 µg kg-1. The maximum concentration was found in a sample from organic production but there were no significant differences in the content of OTA between farming systems. OTA was above the LOQ in four out of 25 samples of the pork muscles. A good agreement was found between OTA levels in muscle and liver (liver concentration = 2.9 × OTA muscle concentration, r = 0.981).

Micropollutants and chemical residues in organic and conventional meat.

The chemical contamination levels of both conventional and organic meats were assessed. The objective was to provide occurrence data in a context of chronic exposure. Environmental contaminants (17 polychlorinated dibenzodioxins/dibenzofurans, 18 polychlorinated biphenyls (PCBs), 3 hexabromocyclododecane (HBCD) isomers, 6 mycotoxins, 6 inorganic compounds) together with chemical residues arising from production inputs (75 antimicrobials, 10 coccidiostats and 121 pesticides) have been selected as relevant compounds. A dedicated sampling strategy, representative of the French production allowed quantification of a large sample set (n=266) including both conventional (n=139) and organic (n=127) raw meat from three animal species (bovine, porcine, poultry). While contamination levels below regulatory limits were measured in all the samples, significant differences were observed between both species and types of farming. Several environmental contaminants (Dioxins, PCBs, HBCD, Zn, Cu, Cd, Pb, As) were measured at significantly higher levels in organic samples.