Detection and Characterization of Nodularin by Using Label-Free Surface-Enhanced Spectroscopic Techniques.

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.

Cytotoxic and immunological responses of fish leukocytes to nodularin exposure in vitro.

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-1ß and TGF-ß. 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-1ß of the CLC cells. This study provides evidence that NOD may exert cytotoxicity and immune-toxicity effects depending on cell type and toxin concentration.

Nodularin induced oxidative stress contributes to developmental toxicity in zebrafish embryos.

Nodularin (NOD) is a kind of cyanobacterial toxins. It is of concern due to elicit severe genotoxicity in humans and animals. The comprehensive evaluation of NOD-induced adverse effects in living organisms is urgently needed. This study is aimed to report the developmental toxicity and molecular mechanism using zebrafish embryos exposed to NOD. The embryonic toxicity induced by NOD is demonstrated by inhibition of embryo hatching, increase in mortality rate, abnormal heart rate, embryonic malformation as well as defects in angiogenesis and common cardinal vein remodeling. NOD triggered a decreased rate of angiogenesis through inhibiting endothelial cells migration. NOD induced embryonic cell apoptosis and DNA damage, which can be alleviated by antioxidant N-acetyl-L-cysteine. NOD significantly caused oxidative damage as indicated by changes in reactive oxygen species, superoxide dismutase, catalase, glutathione and malondialdehyde. NOD also altered the expression of vascular development-genes (DLL4, CDH5, VEGFA, VEGFC) and apoptosis-related genes (BAX, BCL-2, P53, CASPASE 3). Taken together, NOD induced adverse effect on zebrafish embryos development, which may be associated with oxidative stress and apoptosis through the activation of P53-BAX/BCL-2-CASPASE 3-mediated pathway.

Specific Chemical and Genetic Markers Revealed a Thousands-Year Presence of Toxic Nodularia spumigena in the Baltic Sea.

In the Baltic Sea, diazotrophic cyanobacteria have been present for thousands of years, over the whole brackish water phase of the ecosystem. However, our knowledge about the species composition of the cyanobacterial community is limited to the last several decades. In the current study, the presence of species-specific chemical and genetic markers in deep sediments were analyzed to increase the existing knowledge on the history of toxic Nodularia spumigena blooms in the Baltic Sea. As chemical markers, three cyclic nonribosomal peptides were applied: the hepatotoxic nodularin, which in the sea was detected solely in N. spumigena, and two anabaenopeptins (AP827 and AP883a) characteristic of two different chemotypes of this species. From the same sediment samples, DNA was isolated and the gene involved in biosynthesis of nodularin, as well as the phycocyanin intergenic spacer region (PC-IGS), were amplified. The results of chemical and genetic analyses proved for the first time the thousands-year presence of toxic N. spumigena in the Baltic Sea. They also indicated that through all this time, the same two sub-populations of the species co-existed.

Toxicity at the Edge of Life: A Review on Cyanobacterial Toxins from Extreme Environments.

Cyanotoxins are secondary metabolites produced by cyanobacteria, of varied chemical nature and toxic effects. Although cyanobacteria thrive in all kinds of ecosystems on Earth even under very harsh conditions, current knowledge on cyanotoxin distribution is almost restricted to freshwaters from temperate latitudes. In this review, we bring to the forefront the presence of cyanotoxins in extreme environments. Cyanotoxins have been reported especially in polar deserts (both from the Arctic and Antarctica) and alkaline lakes, but also in hot deserts, hypersaline environments, and hot springs. Cyanotoxins detected in these ecosystems include neurotoxins-anatoxin-a, anatoxin-a (S), paralytic shellfish toxins, ß-methylaminopropionic acid, N-(2-aminoethyl) glycine and 2,4-diaminobutyric acid- and hepatotoxins -cylindrospermopsins, microcystins and nodularins-with microcystins being the most frequently reported. Toxin production there has been linked to at least eleven cyanobacterial genera yet only three of these (Arthrospira, Synechococcus and Oscillatoria) have been confirmed as producers in culture. Beyond a comprehensive analysis of cyanotoxin presence in each of the extreme environments, this review also identifies the main knowledge gaps to overcome (e.g., scarcity of isolates and -omics data, among others) toward an initial assessment of ecological and human health risks in these amazing ecosystems developing at the very edge of life.

Nodularin induces tumor necrosis factor-alpha and mitogen-activated protein kinases (MAPK) and leads to induction of endoplasmic reticulum stress.

Nodularin is produced by the cyanobacterium Nodularia spumigena. It is of concern due to hepatotoxicity in humans and animals. Here we investigated unexplored molecular mechanisms by transcription analysis in human liver cells, focusing on induction of pro-inflammatory cytokines, the tumor necrosis factor α (TNF-α), endoplasmic reticulum (ER) stress and components of the activator protein-1 complex in human hepatoma cells (Huh7) exposed to non-cytotoxic (0.1 and 1µM) and toxic concentrations (5µM) for 24, 48, and 72h. Transcripts of TNF-α and ER stress marker genes were strongly induced at 1 and 5µM at all time-points. TNF-α led to induction of mitogen-activated protein kinases (MAPK), as demonstrated by induction of CJUN and CFOS, which form the AP-1 complex. Human primary liver cells reacted more sensitive than Huh7 cells. They showed higher cytotoxicity and induction of TNF-α and ER stress at 2.5nM, while HepG2 cells were insensitive up to 10µM due to low expression of organic anion transporting polypeptides. Furthermore, nodularin led to induction of TNF-α protein, and CCAAT/enhancer-binding protein-homologous (CHOP) protein. Our data indicate that nodularin induces inflammation and ER stress and leads to activation of MAPK in liver cells. All of these activated pathways, which were analysed here for the first time in detail, may contribute to the hepatotoxic, and tumorigenic action of nodularin.

Production of monoclonal antibodies with broad specificity and development of an immunoassay for microcystins and nodularin in water.

Microcystins (MCs) and nodularin (NOD) are cyanobacterial hepatotoxins that can greatly harm human health. Multi-analyte immunoassays provide efficient and cheap methods of screening these toxins. To develop a multi-analyte immunoassay, an antibody with both broad specificity and high affinity for structurally similar algal toxins is urgently needed. In this study, microcystin-leucine-arginine (MC-LR) and NOD were conjugated to carrier proteins using a one-step active ester (AE) method and multistep thiol-ene click chemistry and glutaraldehyde method, respectively. The immunogens obtained from these two conjugation methods were evaluated for their effectiveness in producing antibodies. The results demonstrated that the antisera derived from AE immunogens showed better performance in terms of affinity and titer. Using this simple AE method, we prepared a new immunogen for NOD and successfully produced a monoclonal antibody (mAb), 2G5, which could recognize not only NOD but also all eight of the tested MCs (MC-LR, MC-RR, MC-YR, MC-WR, MC-LA, MC-LF, MC-LY, and MC-LW) with high sensitivity and improved uniform affinities (0.23 ≤ IC50 ≤ 0.68 ng mL(-1)) compared with previously described mAbs. Under optimal conditions, one indirect competitive enzyme-linked immunosorbent assay was developed based on mAb2G5 for the detection of MC-LR and NOD, with limits of detection of 0.16 and 0.10 µg L(-1), respectively, and a recovery of 62-86 % with a coefficient of variation below 12.6 % in water samples.

Cell Death Inducing Microbial Protein Phosphatase Inhibitors--Mechanisms of Action.

Okadaic acid (OA) and microcystin (MC) as well as several other microbial toxins like nodularin and calyculinA are known as tumor promoters as well as inducers of apoptotic cell death. Their intracellular targets are the major serine/threonine protein phosphatases. This review summarizes mechanisms believed to be responsible for the death induction and tumor promotion with focus on the interdependent production of reactive oxygen species (ROS) and activation of Ca(2+)/calmodulin kinase II (CaM-KII). New data are presented using inhibitors of specific ROS producing enzymes to curb nodularin/MC-induced liver cell (hepatocyte) death. They indicate that enzymes of the arachidonic acid pathway, notably phospholipase A2, 5-lipoxygenase, and cyclooxygenases, may be required for nodularin/MC-induced (and presumably OA-induced) cell death, suggesting new ways to overcome at least some aspects of OA and MC toxicity.

Cyanobacterial blooms in surface waters - Nature-based solutions, cyanotoxins and their biotransformation products.

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.

Cyanotoxin exposure and hepatocellular carcinoma.

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.

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.

Environmental Exposure to Cyanobacteria Hepatotoxins in a Pacific Island Community: A Cross-Sectional Assessment.

(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.

Preparation of Specific Nanobodies and Their Application in the Rapid Detection of Nodularin-R in Water Samples.

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.

Oral exposure to environmental cyanobacteria toxins: Implications for cancer risk.

BACKGROUND: 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. OBJECTIVES: 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. METHODS: 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. RESULTS: 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. DISCUSSION: 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.

Cyanobacterial Akinete Distribution, Viability, and Cyanotoxin Records in Sediment Archives From the Northern Baltic Sea.

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.

How Copepods Can Eat Toxins Without Getting Sick: Gut Bacteria Help Zooplankton to Feed in Cyanobacteria Blooms.

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.

A sensitive and accurate method for simultaneous analysis of algal toxins in freshwater using UPLC-MS/MS and 15N-microcystins as isotopically labelled internal standards.

The development of analytical methods for the detection and accurate quantification of algal toxins is of importance to assess the health risk of exposure to algal toxins in freshwater sources. This study established a sensitive and accurate analytical method for the quantification of 13 algal toxins (microcystins and nodularin) based on solid phase extraction (SPE) coupled with UPLC-MS/MS, in which 15N-microcystins were used as surrogate/internal standards. SPE method was optimized to extract the target algal toxins in freshwater samples. Good SPE efficiencies (84-96%) were achieved for the overwhelming majority of the investigated algal toxins when SPE was performed using HLB (500 mg, 6 mL) under alkaline conditions (pH 11). An accurate quantitative analysis of the algal toxins in real freshwater samples was performed by using 15N-labelled microcystins as isotopically labelled internal standards (ILISs), which compensated for the loss of target toxins during the whole analytical process. In addition, ILISs also helped to correct the effects of environmental matrices and instrument fluctuation in UPLC-MS/MS analysis. The limit of method quantification (MQL) for the algal toxins was <2.0 ng/L that is sensitive enough to quantify extremely low levels of target toxins in freshwater samples.

Effect of the immobilized microcystin-LR-degrading enzyme MlrA on nodularin degradation and its immunotoxicity study.

In freshwater ecosystems with frequent cyanobacterial blooms, the cyanobacteria toxin pollution is becoming increasingly serious. Nodularin (NOD), which has strong biological toxicity, has emerged as a new pollutant and affects the normal growth, development and reproduction of aquatic organisms. However, little information is available regarding this toxin. In this study, a graphene oxide material modified by L-cysteine was synthesized and used to immobilize microcystin-LR (MC-LR)-degrading enzyme (MlrA) to form an immobilized enzyme nanocomposite, CysGO-MlrA. Free-MlrA was used as a control. The efficiency of NOD removal by CysGO-MlrA was investigated. Additionally, the effects of CysGO-MlrA and the NOD degradation product on zebrafish lymphocytes were detected to determine the biological toxicity of these two substances. The results showed the following: (1) There was no significant difference in the degradation efficiency of NOD between CysGO-MlrA and free-MlrA; the degradation rate of both was greater than 80% at 1 h (2) The degradation efficiency of the enzyme could retain greater than 81% of the initial degradation efficiency after the CysGO-MlrA had been reused 7 times. (3) CysGO-MlrA retained greater than 50% of its activity on the 8th day when preserved at 0 °C, while free-MlrA lost 50% of its activity on the 4th day. (4) CysGO-MlrA and the degradation product of NOD showed no obvious cytotoxicity to zebrafish lymphocytes. Therefore, CysGO-MlrA might be used as an efficient and ecologically safe degradation material for NOD.

Blooms of Toxic Cyanobacterium Nodularia spumigena in Norwegian Fjords During Holocene Warm Periods.

In paleoecological studies, molecular markers are being used increasingly often to reconstruct community structures, environmental conditions and ecosystem changes. In this work, nodularin, anabaenopeptins and selected DNA sequences were applied as Nodularia spumigena markers to reconstruct the history of the cyanobacterium in the Norwegian fjords. For the purpose of this study, three sediment cores collected in Oslofjorden, Trondheimsfjorden and Balsfjorden were analyzed. The lack of nodularin in most recent sediments is consistent with the fact that only one report on the sporadic occurrence and low amounts of the cyanobacterium in Norwegian Fjords in 1976 has been published. However, analyses of species-specific chemical markers in deep sediments showed that thousands of years ago, N. spumigena constituted an important component of the phytoplankton community. The content of the markers in the cores indicated that the biomass of the cyanobacterium increased during the warmer Holocene periods. The analyses of genetic markers were less conclusive; they showed the occurrence of microcystin/nodularin producing cyanobacteria of Nostocales order, but they did not allow for the identification of the organisms at a species level.

Occurrence, quantification, and adsorptive removal of nodularin in seawater, wastewater and river water.

The presence of potent hepatotoxic cyanotoxins such as nodularin (NOD) in drinking water, groundwater, surface water, seawater and recreational waters presents a major risks to human and environmental health. Human exposure to cyanotoxins could lead to various health effects such as liver damage, jaundice, neurotoxicity and gastroenteritis. Therefore, it is critical to investigate their occurrence in environmental matrices. This study reports the use of tyre-based activated carbon (WTAC) as an adsorbent for preconcentration and removal of nodularin from environmental matrices prior to high performance liquid chromatographic analysis. The preconcentration and adsorption experiments were carried out in presence of other environmental components to consider the external effect on WTAC adsorption of nodularin. Under optimum conditions, the linear dynamic range was 0.05-70 µg L- 1 with a correlation coefficient of 0.9991. The LOD and LOQ (n = 10) in the absence and in the presence of humic acids were 0.012-0.025 µg L- 1 and 0.040-0.083, n = 10), respectively. The repeatability (n = 10) and reproducibility (n = 5) of the method expressed as relative standard deviation (%RSD) were 3.7 and 5.1%, respectively. The maximum adsorption capacity of WTAC was 345 µg g -1. Furthermore, the results demonstrated that the presence of humic acid has an effect on the nodularin adsorption to WTAC. However, high concentrations other coexisting ions such a Cl-, NO3-, PO43-, HCO3-, SO42- had no significant the effect on the adsorption process. The proposed technique was then used for a preconcentration and elimination of NOD trace levels in different water matrices. The results showed that the WTAC was an effective adsorbent for the preconcentration and removal of NOD from the complex matrices.