Cyanotoxin Occurrence and Diversity in 98 Cyanobacterial Blooms from Swedish Lakes and the Baltic Sea.

The Drinking Water Directive (EU) 2020/2184 includes the parameter microcystin LR, a cyanotoxin, which drinking water producers need to analyze if the water source has potential for cyanobacterial blooms. In light of the increasing occurrences of cyanobacterial blooms worldwide and given that more than 50 percent of the drinking water in Sweden is produced from surface water, both fresh and brackish, the need for improved knowledge about cyanotoxin occurrence and cyanobacterial diversity has increased. In this study, a total of 98 cyanobacterial blooms were sampled in 2016-2017 and identified based on their toxin production and taxonomical compositions. The surface water samples from freshwater lakes throughout Sweden including brackish water from eight east coast locations along the Baltic Sea were analyzed for their toxin content with LC-MS/MS and taxonomic composition with 16S rRNA amplicon sequencing. Both the extracellular and the total toxin content were analyzed. Microcystin's prevalence was highest with presence in 82% of blooms, of which as a free toxin in 39% of blooms. Saxitoxins were found in 36% of blooms in which the congener decarbamoylsaxitoxin (dcSTX) was detected for the first time in Swedish surface waters at four sampling sites. Anatoxins were most rarely detected, followed by cylindrospermopsin, which were found in 6% and 10% of samples, respectively. As expected, nodularin was detected in samples collected from the Baltic Sea only. The cyanobacterial operational taxonomic units (OTUs) with the highest abundance and prevalence could be annotated to Aphanizomenon NIES-81 and the second most profuse cyanobacterial taxon to Microcystis PCC 7914. In addition, two correlations were found, one between Aphanizomenon NIES-81 and saxitoxins and another between Microcystis PCC 7914 and microcystins. This study is of value to drinking water management and scientists involved in recognizing and controlling toxic cyanobacteria blooms.

LC-MS/MS Analysis of Cyanotoxins in Bivalve Mollusks-Method Development, Validation and First Evidence of Occurrence of Nodularin in Mussels (Mytilus edulis) and Oysters (Magallana gigas) from the West Coast of Sweden.

In this paper, an LC-MS/MS method for the simultaneous identification and quantification of cyanotoxins with hydrophilic and lipophilic properties in edible bivalves is presented. The method includes 17 cyanotoxins comprising 13 microcystins (MCs), nodularin (NOD), anatoxin-a (ATX-a), homoanatoxin (h-ATX) and cylindrospermopsin (CYN). A benefit to the presented method is the possibility for the MS detection of MC-LR-[Dha7] and MC-LR-[Asp3] as separately identified and MS-resolved MRM signals, two congeners which were earlier detected together. The performance of the method was evaluated by in-house validation using spiked mussel samples in the quantification range of 3.12-200 µg/kg. The method was found to be linear over the full calibration range for all included cyanotoxins except CYN for which a quadratic regression was used. The method showed limitations for MC-LF (R2 = 0.94), MC-LA (R2 ≤ 0.98) and MC-LW (R2 ≤ 0.98). The recoveries for ATX-a, h-ATX, CYN, NOD, MC-LF and MC-LW were lower than desired (<70%), but stable. Despite the given limitations, the validation results showed that the method was specific and robust for the investigated parameters. The results demonstrate the suitability of the method to be applied as a reliable monitoring tool for the presented group of cyanotoxins, as well as highlight the compromises that need to be included if multi-toxin methods are to be used for the analysis of cyanotoxins with a broader range of chemical properties. Furthermore, the method was used to analyze 13 samples of mussels (Mytilus edulis) and oysters (Magallana gigas) collected in the 2020-2022 summers along the coast of Bohuslän (Sweden). A complementary qualitative analysis for the presence of cyanotoxins in phytoplankton samples collected from marine waters around southern Sweden was performed with the method. Nodularin was identified in all samples and quantified in bivalve samples in the range of 7-397 µg/kg. Toxins produced by cyanobacteria are not included in the European Union regulatory monitoring of bivalves; thus, the results presented in this study can be useful in providing the basis for future work including cyanotoxins within the frame of regulatory monitoring to increase seafood safety.

Multi-Toxin Quantitative Analysis of Paralytic Shellfish Toxins and Tetrodotoxins in Bivalve Mollusks with Ultra-Performance Hydrophilic Interaction LC-MS/MS-An In-House Validation Study.

Ultra-performance hydrophilic interaction liquid chromatography tandem mass spectrometry system (UP-HILIC-MS/MS) was used in multi-toxin analysis of paralytic shellfish toxins (PSTs) and tetrodotoxins (TTXs) in sample matrices from bivalve molluscan species commercially produced for human consumption in Sweden. The method validation includes 17 toxins of which GTX6 and two TTX analogues, TTX and 4,9-anhydroTTX, were previously not analyzed together with hydrophilic PSTs. 11-deoxyTTX was monitored qualitatively with a non-certified reference standard. The performance of the method was evaluated for selectivity, repeatability, and linearity by analyzing spiked samples which generated linear calibration curves across the concentration ranges used (R2 > 0.99). The in-house reproducibility (RSD) was satisfactory including the LOD and LOQ for both PSP and TTX toxins being far below their regulatory action limits. The major advantage of the method is that it allows direct confirmation of the toxin identity and specific toxin quantification using a derivatization-free approach. Unlike the PST-chemical methods used in routine regulatory monitoring until now for food control, the UP-HILIC-MS/MS approach enables the calibration set-up for each of the toxin analogs separately, thereby providing the essential flexibility and specificity in analysis of this challenging group of toxins. The method is suitable to implement in food monitoring for PSTs and TTXs in bivalves, and can serve as a fast and cost-efficient screening method. However, positive samples would, for regulatory reasons still need to be confirmed using the AOAC official method (2005.06).

Quantitative analysis of cereulide toxin from Bacillus cereus in rice and pasta using synthetic cereulide standard and 13C6-cereulide standard - a short validation study.

A single laboratory validation study of a rapid and sensitive quantitative method for the analysis of cereulide toxin produced by Bacillus cereus using ultra high performance liquid chromatography-electrospray-tandem mass spectrometry is presented. The analysis of this cyclic peptide toxin was validated for pasta and rice samples using a newly presented synthetic cereulide peptide standard, together with 13C6-cereulide that previously have not been commercially available. The use of cereulide standard was also compared to the most frequently used surrogate standard, the antibiotic valinomycin. The performance of the method was evaluated by analyzing spiked sample pools from different types of rice and pasta, as well as 21 individual rice and pasta samples from differently prepared meals. Inoculation of samples with three cereulide toxin-producing strains of Bacillus cereus was finally used to mimic naturally contaminated foods. The quantification range of the method was 1-500 ng/g (R2 = 0.999) and the limits of detection and quantification were 0.1 and 1 ng/g, respectively. The precision varied from 3% to 7% relative standard deviation and the trueness from -2% to +6% relative bias at different concentration levels in cooked rice and pasta.