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.

A green-footprint approach for parallel multiclass analysis of contaminants in roasted coffee via LC-HRMS.

The development and validation of a simple, comprehensive, and environment-friendly procedure to determine pesticide residues, naturally occurring and processing contaminants in roasted coffee is presented. A solid-liquid extraction of pesticides and mycotoxins with ethyl acetate and the concurrent partition of acrylamide to an aqueous phase follows a parallel analytical strategy that requires a single analytical portion to determine contaminants that are typically analyzed by dedicated single residue methods. The partition rules the lipids out of the aqueous extract before an "in-tube" dispersive solid phase microextraction (dSPME) for acrylamide retention. This is followed by the elution with buffer prior to injection. This extract is independently introduced into the system front end followed by the injection of the compounds from the organic phase, yet all spotted in the same run. A novel liquid chromatography high-resolution mass spectrometry (LC-HRMS) method setup enables the quantification of 186 compounds at 10 µg/kg, 226 at 5 µg/kg, and the acrylamide at 200 µg/kg for a total of 414 molecules, with acceptable recoveries (70-120%) and precision (RSD < 20%) making this strategy significantly faster and cost-effective than the dedicated single residue methods. Even though the presence of chlorpyrifos, acrylamide, and ochratoxin A was confirmed on samples of different origins, the findings were below the limit of quantification. During the storage of raw coffee, no proof of masking of OTA was found; however, condensation with glucose was evidenced during thermal processing experiments with sucrose by using stable isotope labeling (SIL). No detected conjugates were found in roasted nor in commercial sugar-added torrefacto samples, an industrial processing usually carried out above the decomposition temperature of the disaccharide.

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).

New efficient methodology for screening of selected organic micropollutants in raw- and drinking water from 90 Swedish water treatment plants.

A new, efficient method for analysis of organic micropollutants (OMPs) in water samples was developed, validated and applied in a nationwide survey. The goal with the survey was to identify common compounds with relatively high concentrations to be used as markers e.g. for routine monitoring of removal efficiencies. The method comprises sample concentration by evaporation, and large volume injection on a standard UHPLC-MS/MS system. The OMPs selected for this approach were mainly semi-polar and non-volatile, with molecular weights below 300 Da. Except one outlier, the limit of detection (LoD) ranged from 0.01 to 1 ng/L which is sufficient for most surface waters, and also useful for many ground waters. The method requires low manual labor and comparably small sample volumes, enabling a cost-efficient nationwide screening. Raw- and drinking water from 90 Swedish water treatment plants (WTPs) were investigated for occurrence of the selected OMPs. Carbamazepine and tramadol were the most widespread compounds, detected in around 50% of the surface waters. Ground water from rock aquifers were least contaminated, while soil aquifers were more similar to surface waters. Due to the frequent use of ground water in Sweden, many samples did not contain any of the investigated compounds (i.e. below LoD). In the positive samples, the median estimated concentrations of individual OMPs were generally <1 ng/L in raw water and <0.5 ng/L in drinking water. Swedish waters were in general less contaminated than those investigated in similar Brazilian, Chinese, pan-European and US studies. Altogether, the presented methodology gave a cost-efficient overview on the occurrence and estimated concentrations of OMPs in raw- and drinking water on a national level in Sweden. From the data, WTPs can find suitable OMPs to monitor at their site, for example for measuring removal efficiencies on a routine basis.

Microcystins activate nuclear factor erythroid 2-related factor 2 (Nrf2) in human liver cells in vitro - Implications for an oxidative stress induction by microcystins.

Microcystins, a potential threat to drinking water quality, are hepatotoxic but it has remained unclear if microcystins induce oxidative stress. We investigated if four microcystins could activate the Nrf2 pathway, a regulator of oxidative stress response. Nrf2 activity was significantly increased by microcystin-LR and -RR at 10 µM, by microcystin-LY at 3 µM, by [D-Asp3]-LR and by microcystin-LR at 1 µM. Our results lend support to the suggestion that microcystins may induce oxidative stress response.

Fast, rugged and sensitive ultra high pressure liquid chromatography tandem mass spectrometry method for analysis of cyanotoxins in raw water and drinking water--First findings of anatoxins, cylindrospermopsins and microcystin variants in Swedish source waters and infiltration ponds.

Freshwater blooms of cyanobacteria (blue-green algae) in source waters are generally composed of several different strains with the capability to produce a variety of toxins. The major exposure routes for humans are direct contact with recreational waters and ingestion of drinking water not efficiently treated. The ultra high pressure liquid chromatography tandem mass spectrometry based analytical method presented here allows simultaneous analysis of 22 cyanotoxins from different toxin groups, including anatoxins, cylindrospermopsins, nodularin and microcystins in raw water and drinking water. The use of reference standards enables correct identification of toxins as well as precision of the quantification and due to matrix effects, recovery correction is required. The multi-toxin group method presented here, does not compromise sensitivity, despite the large number of analytes. The limit of quantification was set to 0.1 µg/L for 75% of the cyanotoxins in drinking water and 0.5 µg/L for all cyanotoxins in raw water, which is compliant with the WHO guidance value for microcystin-LR. The matrix effects experienced during analysis were reasonable for most analytes, considering the large volume injected into the mass spectrometer. The time of analysis, including lysing of cell bound toxins, is less than three hours. Furthermore, the method was tested in Swedish source waters and infiltration ponds resulting in evidence of presence of anatoxin, homo-anatoxin, cylindrospermopsin and several variants of microcystins for the first time in Sweden, proving its usefulness.