Cyanobacterial blooms are expected to become more frequent and severe in surface water reservoirs due to climate change and ecosystem degradation. It is an emerging challenge that especially countries relying on surface water supplies will face. Nature-based solutions (NBS) like constructed wetlands and biofilters can be used for cyanotoxin remediation. Both technologies are reviewed and critically assessed for different types of water resources. The available information on cyanotoxins (bio)transformation products (TPs) is reviewed to point out the potential research gaps and to disclose the most reliable enzymatic degradation pathways. Knowledge gaps were found, such as information on the performance of the revised NBS in pilot and full scales, the removal processes covering different cyanotoxins (besides the most widely studied microcystin-LR), and the difficulties for real-world implementation of technologies proposed in the literature. Also, most studies focus on bacterial degradation processes while fungi have been completely overlooked. This review also presents an up-to-date overview of the transformation of cyanotoxins, where degradation product data was compiled in a unified library of 22 metabolites for microcystins (MCs), 7 for cylindrospermopsin (CYN) and 10 for nodularin (NOD), most of them reported only in a single study. Major gaps are the lack of environmentally relevant studies with TPs in pilot and full- scale treatment systems, information on TP\'s toxicity, as well as limited knowledge of environmentally relevant degradation pathways. NBS have the potential to mitigate cyanotoxins in recreational and irrigation waters, enabling the water-energy-food nexus and avoiding the degradability of the ecosystems.

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.

Cyanobacteria are ubiquitous in aquatic and terrestrial environments worldwide and include a number of species producing tumor-promoting hepatotoxins. Human exposure to cyanobacteria and cyanotoxins primarily occurs though ingestion of contaminated drinking water and food sources. In a Northeast U.S. population, we recently reported an independent association of oral cyanobacteria with risk of hepatocellular carcinoma (HCC). In a cross-sectional study of 55 HCC patients in Hawaii, U.S.A., serum microcystin/nodularin (MC/NOD), cylindrospermopsin (CYN), and anabaenopeptin (AB) were measured by ELISA. In a subset of 16 patients, cyanotoxin levels were compared by tumor expression of over 700 genes analyzed via the Nanostring nCounter Fibrosis panel. MC/NOD, CYN, and AB were detected in all HCC patients. MC/NOD and CYN levels significantly varied by etiology with the highest levels in cases attributed to metabolic risk factors, specifically, hyperlipidemia, type 2 diabetes, and non-alcoholic fatty liver disease/non-alcoholic steatohepatitis. Cyanotoxin levels were significantly positively correlated with tumor expression of genes functioning in PPAR signaling and lipid metabolism. Our study provides novel albeit limited evidence that cyanotoxins may a role in the pathogenesis of HCC through the dysregulation of lipid metabolism and progression of hepatic steatosis.

Nodularin (NOD) is a potent toxin produced by Nodularia spumigena cyanobacteria. Usually, NOD co-exists with other microcystins in environmental waters, a class of cyanotoxins secreted by certain cyanobacteria species, which makes identification difficult in the case of mixed toxins. Herein we report a complete theoretical DFT-vibrational Raman characterization of NOD along with the experimental drop-coating deposition Raman (DCDR) technique. In addition, we used the vibrational characterization to probe SERS analysis of NOD using colloidal silver nanoparticles (AgNPs), commercial nanopatterned substrates with periodic inverted pyramids (KlariteTM substrate), hydrophobic Tienta® SpecTrimTM slides, and in-house fabricated periodic nanotrenches by nanoimprint lithography (NIL). The 532 nm excitation source provided more well-defined bands even at LOD levels, as well as the best performance in terms of SERS intensity. This was reflected by the results obtained with the KlariteTM substrate and the silver-based colloidal system, which were the most promising detection approaches, providing the lowest limits of detection. A detection limit of 8.4 × 10-8 M was achieved for NOD in solution by using AgNPs. Theoretical computation of the complex vibrational modes of NOD was used for the first time to unambiguously assign all the specific vibrational Raman bands.

(1) Background: Cyanobacteria produce a wide range of secondary metabolites, including tumor-promoting hepatotoxins. We recently reported evidence of an independent association between oral cyanobacteria and hepatocellular carcinoma in a U.S. population. We sought to characterize the nature, sources, and health correlates of cyanotoxin exposure in the U.S. Pacific Island territory of Guam, which has a high incidence of liver cancer. (2) Methods: Seventy-four adult males and females were enrolled in a cross-sectional study to quantify cyanotoxins in saliva, urine, and blood and their correlation with health behaviors, medical history, and environmental exposures. Plant samples were collected from locations throughout the island. Microcystin/nodularin (MC/NOD), cylindrospermopsin (CYN), and anabaenopeptin (AB) were measured in biospecimens and in plant extracts by ELISA. (3) Results: Overall, among study participants MC/NOD were detected in 53.9% of saliva, 7.5% of urine, and 100% of serum.; CYN in 40.0% of saliva, 100.0% of urine, and 70.4% of serum; AB in 30.8% of saliva, 85% of urine, and 92.6% of serum. Salivary MC/NOD levels were significantly higher in individuals using municipal tap water as their primary source of drinking water; both salivary and urinary MC/NOD levels were higher in those not using store-bought/commercial water. Urine MC/NOD levels were highest among individuals consuming fruits and vegetables exclusively from local sources. Urine MC/NOD levels were elevated in individuals with hypertension and hyperlipidemia and salivary MC/NOD in those with recent alcohol consumption. Cyanotoxins were prevalent in plant samples including MC/NOD (46.6%), CYN (35.1%), and AB (51.7%). (4) Conclusions: Our study provides evidence that exposure to cyanobacterial hepatotoxins, including tumor promoters, may be prevalent in Guam and may originate from environmental sources. Population-based epidemiologic studies are needed to investigate the role of cyanotoxins in liver cancer development.

Nanobodies have several advantages, including great stability, sensibility, and ease of production; therefore, they have become important tools in immunoassays for chemical contaminants. In this manuscript, nanobodies for the detection of the toxin Nodularin-r (NOD-R), a secondary metabolite of cyanobacteria that could cause a safety risk for drinks and food for its strong hepatotoxicity, were for the first time selected from an immunized Bactrian camel VHH phage display library. Then, a sensitive indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) for NOD-R, based on the nanobody N56 with great thermostability and organic solvent tolerance, was established under optimized conditions. The results showed that the limit of detection for NOD-R was 0.67 µg/L, and the average spike recovery rate was between 84.0 and 118.3%. Moreover, the ic-ELISA method was validated with spiked water sample and confirmed by UPLC-MS/MS, which indicated that the ic-ELISA established in this work is a reproducible detection assay for nodularin residues in water samples.

Cyanobacteria of the order Nostocales, including Baltic Sea bloom-forming taxa Nodularia spumigena, Aphanizomenon flosaquae, and Dolichospermum spp., produce resting stages, known as akinetes, under unfavorable conditions. These akinetes can persist in the sediment and germinate if favorable conditions return, simultaneously representing past blooms and possibly contributing to future bloom formation. The present study characterized cyanobacterial akinete survival, germination, and potential cyanotoxin production in brackish water sediment archives from coastal and open Gulf of Finland in order to understand recent bloom expansion, akinete persistence, and cyanobacteria life cycles in the northern Baltic Sea. Results showed that cyanobacterial akinetes can persist in and germinate from Northern Baltic Sea sediment up to >40 and >400 years old, at coastal and open-sea locations, respectively. Akinete abundance and viability decreased with age and depth of vertical sediment layers. The detection of potential microcystin and nodularin production from akinetes was minimal and restricted to the surface sediment layers. Phylogenetic analysis of culturable cyanobacteria from the coastal sediment core indicated that most strains likely belonged to the benthic genus Anabaena. Potentially planktonic species of Dolichospermum could only be revived from the near-surface layers of the sediment, corresponding to an estimated age of 1-3 years. Results of germination experiments supported the notion that akinetes do not play an equally significant role in the life cycles of all bloom-forming cyanobacteria in the Baltic Sea. Overall, there was minimal congruence between akinete abundance, cyanotoxin concentration, and the presence of cyanotoxin biosynthetic genes in either sediment core. Further research is recommended to accurately detect and quantify akinetes and cyanotoxin genes from brackish water sediment samples in order to further describe species-specific benthic archives of cyanobacteria.

Nodularin (NOD) is a cyclic peptide released by bloom-forming toxic cyanobacteria Nodularia spumigena commonly occurring in brackish waters throughout the world. Although its hepatotoxic effects are well known, other negative effects of NOD have not yet been completely elucidated. The present study aims were to evaluate and compare the cytotoxic and immunotoxic effects of the toxin on primary leukocytes (from head kidney [HK]) and stable fish leukocytes (carp leucocyte cell line [CLC] cells). The cells were incubated with the cyanotoxin at concentrations of 0.001, 0.01, 0.05, or 0.1 μg/ml. After 24 h of exposure, the concentrations ≥0.05 μg/ml of toxin resulted in cytotoxicity in the primary cells, while in CLC cells, the toxic effect was obtained only with the highest concentration. Similarly, depending on the concentration, exposure to NOD causes a significant inhibition of chemotaxis of the phagocytic abilities of primary leukocytes and a significant reduction in the proliferation of lymphocytes isolated from the HKs. Moreover, CLC cells and HK leukocytes incubated with this toxin at all the mentioned concentrations showed an increased production of reactive oxygen and nitrogen species. NOD also evidently influenced the expression of genes of cytokine TNF-α and IL-10 and, to a minor extent, IL-1β and TGF-β. Notably, the observed changes in the mRNA levels of cytokines in NOD-exposed cells were evident, but not clearly dose-dependent. Interestingly, NOD did not affect the production and release of IL-1β of the CLC cells. This study provides evidence that NOD may exert cytotoxicity and immune-toxicity effects depending on cell type and toxin concentration.

Toxin-producing cyanobacteria can be harmful to aquatic biota, although some grazers utilize them with often beneficial effects on their growth and reproduction. It is commonly assumed that gut microbiota facilitates host adaptation to the diet; however, the evidence for adaptation mechanisms is scarce. Here, we investigated the abundance of mlrA genes in the gut of the Baltic copepods Acartia bifilosa and Eurytemora affinis during cyanobacteria bloom season (August) and outside it (February). The mlrA genes are unique to microcystin and nodularin degraders, thus indicating the capacity to break down these toxins by the microbiota. The mlrA genes were expressed in the copepod gut year-round, being >10-fold higher in the summer than in the winter populations. Moreover, they were significantly more abundant in Eurytemora than Acartia. To understand the ecological implications of this variability, we conducted feeding experiments using summer- and winter-collected copepods to examine if/how the mlrA abundance in the microbiota affect: (1) uptake of toxic Nodularia spumigena, (2) uptake of a non-toxic algal food offered in mixtures with N. spumigena, and (3) concomitant growth potential in the copepods. The findings provide empirical evidence that the occurrence of mlrA genes in the copepod microbiome facilitates nutrient uptake and growth when feeding on phytoplankton mixtures containing nodularin-producing cyanobacteria; thus, providing an adaptation mechanism to the cyanobacteria blooms.

Areca nut/betel quid (AN/BQ) chewing, a prevalent practice in parts of the Pacific and Asia, is an independent cause of cancers of the oral cavity and esophagus and may be linked to liver cancer. The mechanisms of AN/BQ-associated carcinogenesis are unclear. In a Guam population, we previously demonstrated that AN/BQ chewing alters the oral bacterial microbiome including in chewers with oral premalignant lesions. Enrichment of specific taxa, including Cyanobacteria, was observed.
We undertook an investigation to evaluate Areca catechu and/or Piper betle plants as potential sources of Cyanobacteria and cyanotoxins in AN/BQ chewers in Guam.
We evaluated bacterial 16S rRNA with Illumina MiSeq in 122 oral samples and 30 Areca catechu nut and Piper betle leaf samples. Cyanobacteria sequences were interrogated using the NCBI database to identify candidate species and their reference sequences were evaluated for secondary metabolite toxins using AntiSMASH 5.0. Selected toxins were measured by ELISA in extracts from 30 plant samples and in a subset of 25 saliva samples.
Cyanobacteria was the predominant taxa in Areca catechu and Piper betle plants, comprising 75% of sequences. Cyanobacteria was detected at low levels in oral samples but 90-fold higher in current AN/BQ chewers compared to former/never chewers (p = 0.001). Microcystin/nodularin was detected in saliva (15 of 25 samples) and Piper betle leaves (6 of 10 samples). Cylindrospermopsin was detected in all saliva and leaf samples and 7 of 10 nut/husks. Salivary cylindrospermopsin levels were significantly higher in current chewers of betel quid (i.e., crushed Areca catechu nut wrapped in Piper betle leaf) compared to those chewing Areca nut alone. Anabaenopeptin was detected in saliva (10 of 25 samples), all leaf samples, and 7 of 10 nut/husks. Salivary anabaenopeptin concentration was weakly, albeit significantly, correlated with oral Cyanobacteria relative abundance.
Our study demonstrates that Cyanobacteria can contaminate AN/BQ plants and expose chewers to potent hepatotoxins. With worldwide increases in climate-related overgrowth of Cyanobacteria, our findings have broad implications for cancer risk across populations.