Harbor Porpoise Deaths Associated with Erysipelothrix rhusiopathiae, the Netherlands, 2021.

In August 2021, a large-scale mortality event affected harbor porpoises (Phocoena phocoena) in the Netherlands. Pathology and ancillary testing of 22 animals indicated that the most likely cause of death was Erysipelothrix rhusiopathiae infection. This zoonotic agent poses a health hazard for cetaceans and possibly for persons handling cetacean carcasses.

Interlaboratory Evaluation of Multiple LC-MS/MS Methods and a Commercial ELISA Method for Determination of Tetrodotoxin in Oysters and Mussels.

BACKGROUND: Given the recent detection of tetrodotoxin (TTX) in bivalve molluscs but the absence of a full collaborative validation study for TTX determination in a large number of shellfish samples, interlaboratory assessment of method performance was required to better understand current capabilities for accurate and reproducible TTX quantitation using chemical and immunoassay methods. OBJECTIVE: The aim was to conduct an interlaboratory study with multiple laboratories, using results to assess method performance and acceptability of different TTX testing methods. METHODS: Homogenous and stable mussel and oyster materials were assessed by participants using a range of published and in-house detection methods to determine mean TTX concentrations. Data were used to calculate recoveries, repeatability, and reproducibility, together with participant acceptability z-scores. RESULTS: Method performance characteristics were good, showing excellent sensitivity, recovery, and repeatability. Acceptable reproducibility was evidenced by HorRat values for all LC-MS/MS and ELISA methods being less than the 2.0 limit of acceptability. Method differences between the LC-MS/MS participants did not result in statistically different results. Method performance characteristics compared well with previously published single-laboratory validated methods and no statistical difference was found in results returned by ELISA in comparison with LC-MS/MS. CONCLUSION: The results from this study demonstrate that current LC-MS/MS methods and ELISA are on the whole capable of sensitive, accurate, and reproducible TTX quantitation in shellfish. Further work is recommended to expand the number of laboratories testing ELISA and to standardize an LC-MS/MS protocol to further improve interlaboratory precision. HIGHLIGHTS: Multiple mass spectrometric methods and a commercial ELISA have been successfully assessed through an interlaboratory study, demonstrating excellent performance.

A Generic LC-HRMS Screening Method for Marine and Freshwater Phycotoxins in Fish, Shellfish, Water, and Supplements.

Phycotoxins occur in various marine and freshwater environments, and can accumulate in edible species such as fish, crabs, and shellfish. Human exposure to these toxins can take place, for instance, through consumption of contaminated species or supplements and through the ingestion of contaminated water. Symptoms of phycotoxin intoxication include paralysis, diarrhea, and amnesia. When the cause of an intoxication cannot directly be found, a screening method is required to identify the causative toxin. In this work, such a screening method was developed and validated for marine and freshwater phycotoxins in different matrices: fish, shellfish, water, and food supplements. Two LC methods were developed: one for hydrophilic and one for lipophilic phycotoxins. Sample extracts were measured in full scan mode with an Orbitrap high resolution mass spectrometer. Additionally, a database was created to process the data. The method was successfully validated for most matrices, and in addition, regulated lipophilic phycotoxins, domoic acid, and some paralytic shellfish poisoning toxins could be quantified in shellfish. The method showed limitations for hydrophilic phycotoxins in sea water and for lipophilic phycotoxins in food supplements. The developed method is a screening method; in order to confirm suspected compounds, comparison with a standard or an additional analysis such as NMR is required.

Transfer of pyrrolizidine alkaloids from ragwort, common groundsel and viper's bugloss to milk from dairy cows.

To investigate the transfer of pyrrolizidine alkaloids (PAs) from feed to milk, rumen-cannulated dairy cows were intra-ruminally fed with 200 g/day of dried plant material of either ragwort (mixture of Jacobaea vulgaris and Senecio inaequidens), common groundsel (Senecio vulgaris) or viper's bugloss (Echium vulgare) for a period of 4 days. PA levels in the plant materials were 3767, 2792 and 1674 µg g-1 respectively. Feed intake, milk yield and several blood parameters indicative for liver function were not influenced by the treatment. When fed ragwort, increased levels of PAs were detected in the milk, in particular jacoline and an unidentified cyclic diester, possibly a hydroxylated metabolite from retrorsine. The latter was the most important PA in milk from cows fed common groundsel. For viper's bugloss, echimidine was the most abundant identified PA but in addition several hydroxylated PA metabolites were detected. For ragwort, the overall PA transfer was estimated at 0.05% and 1.4% for jacoline (N-oxide). Transfer rates were similar for viper's bugloss (0.05%) but lower for common groundsel (0.01%). Only a small portion of the administered PAs was quantified in milk, urine and faeces, with an overall balance of 4.5%, 2.9% and 5.8%, for ragwort, common groundsel and viper's bugloss, respectively. Samples taken from the rumen indicated that the N-oxides were converted into the free bases, which was confirmed by in vitro studies with the same plant species incubated with ruminal fluid. These results confirm that the transfer of PAs to milk is relatively low but may be of concern for human health regarding the genotoxic and carcinogenic properties of these compounds. The transfer rate depends on the type of PAs present in the weeds. The incomplete balance of input vs output stresses the need to further investigate the metabolism and the potential transfer of metabolites into edible products.

BPA, BADGE and analogues: A new multi-analyte LC-ESI-MS/MS method for their determination and their in vitro (anti)estrogenic and (anti)androgenic properties.

Information on the occurrence and endocrine potencies of analogues of bisphenol A (BPA) and diglycidyl ester derivatives (BDGEs) of BPA and BPF is limited. Such information is, however, important as the current debate on BPA and the lowered BPA migration limit in Europe may provide an incentive for application of structural analogues. A new sensitive multi-analyte LC-ESI-MS/MS method was developed to measure 17 bisphenols (BPs) and 6 BDGEs in food, beverages and drinkware. Yeast based bioassays were used to determine the in vitro (anti)estrogenic and (anti)androgenic properties of these and 7 additional BPs and BDGEs. Drinkware of polycarbonate and other materials were analysed for BPs and BDGEs. Only BPA and BPS and both at trace levels were found in a few containers. A limited number of (canned) foods and beverages were also analysed. BPA was the most frequently detected BP (ranged from 0.03 ng mL-1 in a beverage sample to 68 ng g-1 in food). Other BPs detected were BPS, 2,2-BPF and 4,4-BPF. In addition BADGE, BADGE.HCl, BADGE.H2O and BADGE.2H2O were detected from 0.08 ng mL-1 in a beverage sample to 3.3 ng g-1 in food. In vitro testing showed that most BPs exhibited an equal or higher estrogenic potency than BPA and most of them also showed a higher anti-androgenic potency, i.e. BPB, BPCl, BPC, BPE, 4,4-BPF, BPP, BPAF, and BPTMC. Some BPs and BDGEs were not estrogenic, but showed an anti-estrogenic effect and were anti-androgenic too. BPS was only weakly estrogenic and BADGE.2H2O and BFDGE.2H2O showed no in vitro activity. The present data show that in addition to BPA, other BPs and BDGEs can be present in food and drinks, some displaying in vitro endocrine activities.

A Sensitive LC-MS/MS Method for Palytoxin Using Lithium Cationization.

Palytoxin (PlTX) and analogues are produced by certain dinoflagellates, sea anemones, corals and cyanobacteria. PlTX can accumulate in the food chain and when consumed it may cause intoxication with symptoms like myalgia, weakness, fever, nausea, and vomiting. The analysis of PlTXs is challenging, and because of the large molecular structure, it is difficult to develop a sensitive and selective liquid chromatography-mass spectrometry (LC-MS/MS) method. In this work, an LC-MS/MS method was developed to analyse PlTXs with use of lithium iodine and formic acid as additives in the mobile phase. For method development, initially, LC-hrMS was used to accurately determine the elemental composition of the precursor and product ions. The main adduct formed was [M + H + 2Li]3+. Fragments were identified with LC-hrMS and these were incorporated in the LC-MS/MS method. A method of 10 min was developed and a solid phase extraction clean-up procedure was optimised for shellfish matrix. The determined limits of detection were respectively 8 and 22 µg PlTX kg-1 for mussel and oyster matrix. Oysters gave a low recovery of approximately 50% for PlTX during extraction. The method was successfully in-house validated, repeatability had a relative standard deviation less than 20% (n = 5) at 30 µg PlTX kg-1 in mussel, cockle, and ensis, and at 60 µg PlTX kg-1 in oyster.

A Strategy to Replace the Mouse Bioassay for Detecting and Identifying Lipophilic Marine Biotoxins by Combining the Neuro-2a Bioassay and LC-MS/MS Analysis.

Marine biotoxins in fish and shellfish can cause several symptoms in consumers, such as diarrhea, amnesia, or even death by paralysis. Monitoring programs are in place for testing shellfish on a regular basis. In some countries testing is performed using the so-called mouse bioassay, an assay that faces ethical concerns not only because of animal distress, but also because it lacks specificity and results in high amounts of false positives. In Europe, for lipophilic marine biotoxins (LMBs), a chemical analytical method using LC-MS/MS was developed as an alternative and is now the reference method. However, safety is often questioned when relying solely on such a method, and as a result, the mouse bioassay might still be used. In this study the use of a cell-based assay for screening, i.e., the neuro-2a assay, in combination with the official LC-MS/MS method was investigated as a new alternative strategy for the detection and quantification of LMBs. To this end, samples that had been tested previously with the mouse bioassay were analyzed in the neuro-2a bioassay and the LC-MS/MS method. The neuro-2a bioassay was able to detect all LMBs at the regulatory levels and all samples that tested positive in the mouse bioassay were also suspect in the neuro-2a bioassay. In most cases, these samples contained toxin levels (yessotoxins) that explain the outcome of the bioassay but did not exceed the established maximum permitted levels.

First Report on the Occurrence of Tetrodotoxins in Bivalve Mollusks in The Netherlands.

Tetrodotoxin (TTX) is traditionally associated with seafood from tropical regions, but recently TTX was detected in bivalve mollusks in more temperate European waters. In The Netherlands it was therefore decided to monitor TTX in shellfish harvested from Dutch production areas. All shellfish production areas were monitored in 2015, 2016 and 2017. Samples were analyzed using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). In total 1063 samples were investigated, and the highest concentrations were observed in 2016, i.e., 253 µg TTX/kg in oysters and 101 µg TTX/kg in mussels. No TTX analogues, with the exception of 4-epi-TTX in one single sample, were found and contaminated samples also showed positive results in the neuro-2a bioassay. The occurrence of TTX seems to be consistent over the last three years with the highest concentrations observed annually in late June. The causative organism and the reasons why specific Dutch production areas are affected while others are not, are still unclear. Initially in The Netherlands an action limit of 20 µg TTX/kg was used to ensure the safety of consumers (2016), but recently The European Food Safety Authority (EFSA) established an acute reference dose, and based on a high portion size of consuming 400 g mussels, this dose was translated into a safe concentration of 44 µg TTX per kg for shellfish. This concentration is now used as an action limit and TTX is formally included in the Dutch shellfish monitoring program.

Development and validation of a maleimide-based enzyme-linked immunosorbent assay for the detection of tetrodotoxin in oysters and mussels.

The recent detection of tetrodotoxins (TTXs) in puffer fish and shellfish in Europe highlights the necessity to monitor the levels of TTXs in seafood by rapid, specific, sensitive and reliable methods in order to protect human consumers. A previous immunoassay for TTX detection in puffer fish, based on the use of self-assembled monolayers (SAMs) for the immobilization of TTX on maleimide plates (mELISA), has been modified and adapted to the analysis of oyster and mussel samples. Changing dithiol for cysteamine-based SAMs enabled reductions in the assay time and cost, while maintaining the sensitivity of the assay. The mELISA showed high selectivity for TTX since the antibody did not cross-react with co-occurring paralytic shellfish poisoning (PSP) toxins and no interferences were observed from arginine (Arg). Moreover, TTX-coated maleimide plates stored for 3 months at -20°C and 4°C were stable, thus when pre-prepared, the time to perform the assay is reduced. When analyzing shellfish samples, matrix effects and toxin recovery values strongly depended on the shellfish type and the sample treatment. Blank oyster extracts could be directly analyzed without solid-phase extraction (SPE) clean-up, whereas blank mussel extracts showed strong matrix effects and SPE and subsequent solvent evaporation were required for removal. However, the SPE clean-up and evaporation resulted in toxin loss. Toxin recovery values were taken as correction factors (CFs) and were applied to the quantification of TTX contents in the analysis of naturally-contaminated shellfish samples by mELISA. The lowest effective limits of detection (eLODs) were about 20 and 50µg/kg for oyster extracts without and with SPE clean-up, respectively, and about 30µg/kg for mussel extracts with both protocols, all of them substantially below the eLOD attained in the previous mELISA for puffer fish (230µg/kg). Analysis of naturally-contaminated samples by mELISA and comparison with LC-MS/MS quantifications demonstrated the viability of the approach. This mELISA is a selective and sensitive tool for the rapid detection of TTX in oyster and mussel samples showing promise to be implemented in routine monitoring programs to protect human health.

Screening for the presence of lipophilic marine biotoxins in shellfish samples using the neuro-2a bioassay.

The neuro-2a bioassay is considered as one of the most promising cell-based in vitro bioassays for the broad screening of seafood products for the presence of marine biotoxins. The neuro-2a assay has been shown to detect a wide array of toxins like paralytic shellfish poisons (PSPs), ciguatoxins, and also lipophilic marine biotoxins (LMBs). However, the neuro-2a assay is rarely used for routine testing of samples due to matrix effects that, for example, lead to false positives when testing for LMBs. As a result there are only limited data on validation and evaluation of its performance on real samples. In the present study, the standard extraction procedure for LMBs was adjusted by introducing an additional clean-up step with n-hexane. Recovery losses due to this extra step were less than 10%. This wash step was a crucial addition in order to eliminate false-positive outcomes due to matrix effects. Next, the applicability of this assay was assessed by testing a broad range of shellfish samples contaminated with various LMBs, including diarrhetic shellfish toxins/poisons (DSPs). For comparison, the samples were also analysed by LC-MS/MS. Standards of all regulated LMBs were tested, including analogues of some of these toxins. The neuro-2a cells showed good sensitivity towards all compounds. Extracts of 87 samples, both blank and contaminated with various toxins, were tested. The neuro-2a outcomes were in line with those of LC-MS/MS analysis and support the applicability of this assay for the screening of samples for LMBs. However, for use in a daily routine setting, the test might be further improved and we discuss several recommended modifications which should be considered before a full validation is carried out.

Fate of pyrrolizidine alkaloids during processing of milk of cows treated with ragwort.

To investigate the fate of pyrrolizidine alkaloids (PAs) during milk processing, milk of cows treated via rumen fistula with a mixture of 84% (w/w) ragwort (Jacobaea vulgaris, syn. Senecio jacobaea) and 16% narrow-leaved ragwort (Senecio inaequidens) was processed using laboratory scale heating systems with industrial settings. Pasteurised and sterilised (UHT) milk were produced, as well as set-type yoghurt and cheese. Samples were analysed for 29 PAs using LC-MS/MS, of which 11 PAs were detected above LOQ in the samples (0.1 µg l-1). Alterations in the PA concentration and composition between the standardised milk and the corresponding end-product(s) were evaluated. The heat treatments applied for pasteurisation and UHT sterilisation to prepare semi-skimmed consumption milk did not affect the PA levels in the end-products. In yoghurt, after fermentation of standardised milk (6 h, pH 4.4), 73% of total PAs were recovered. The PA concentration, specifically dehydrojacoline, was decreased, although not quantifiable, during cheese production. A further decrease of 38% during 6 weeks of ripening was observed. The results show that the PA concentration of natural contaminated cow's milk is not affected by heat treatment applied for pasteurised and sterilised milk, but that microbial fermentation of the milk leads to a lowered PA concentration in yoghurt and cheese. This is probably due to microbiological degradation, since PAs are fairly stable under acidic conditions.