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The floating freshwater fern Azolla is the only plant that retains an endocyanobiont, Nostoc azollae (aka Anabaena azollae), during its sexual and asexual reproduction. The increased interest in Azolla as a potential source of food and its unique evolutionary history have raised questions about its cyanotoxin content and genome. Cyanotoxins are potent toxins synthesized by cyanobacteria which have an anti-herbivore effect but have also been linked to neurodegenerative disorders including Alzheimer's and Parkinson's diseases, liver and kidney failure, muscle paralysis, and other severe health issues. In this study, we investigated 48 accessions of Azolla–Nostoc symbiosis for the presence of genes coding microcystin, nodularin, cylindrospermopsin and saxitoxin, and BLAST analysis for anatoxin-a. We also investigated the presence of the neurotoxin β-N-methylamino-L-alanine (BMAA) in Azolla and N. azollae through LC-MS/MS. The PCR amplification of saxitoxin, cylindrospermospin, microcystin, and nodularin genes showed that Azolla and its cyanobiont N. azollae do not have the genes to synthesize these cyanotoxins. Additionally, the matching of the anatoxin-a gene to the sequenced N. azollae genome does not indicate the presence of the anatoxin-a gene. The LC-MS/MS analysis showed that BMAA and its isomers AEG and DAB are absent from Azolla and Nostoc azollae. Azolla therefore has the potential to safely feed millions of people due to its rapid growth while free-floating on shallow fresh water without the need for nitrogen fertilizers. [ABSTRACT FROM AUTHOR]

Establishing reliable and cost-effective tools to identify the occurrence of toxin-producing cyanobacteria could lead to an early warning system to prevent water consumption and recreational activities, especially for low-income agricultural countries. The current study utilised a two-method approach, an enzyme-linked immunosorbent assay and polymerase chain reaction, to enumerate commonly occurring cyanotoxins: microcystin (MC), cylindrospermopsin (CYN), nodularin (NOD), anatoxin-a (ANA-a), and saxitoxin (SAX), and to evaluate the presence of the genes responsible for producing these toxins. Water samples from 43 drinking, recreational and irrigational water bodies in Sri Lanka were collected between November 2019 and July 2020. Results indicated that 24 of the sampled water bodies contained at least one of the toxin-producing genes screened. Notably, the overall MC concentration detected for Beira Lake in Colombo, a recreational waterbody in the country's largest and most populous city, was 3.31 μg/L. Among the different cyanotoxins analysed, MC was found to be the most dominant (37%) in the collected water samples compared to CYN (10%), SAX (10%), NOD (10%), and ANA-a (10%). This is the first such study where molecular approaches were combined with immunological assays to provide an extensive survey highlighting an alarming level of contamination of cyanotoxins in intensively used water bodies. [ABSTRACT FROM AUTHOR]

Over the last two decades, proliferations of benthic cyanobacteria producing derivatives of anatoxin-a have been reported in rivers worldwide. Here, we follow up on such a toxigenic event happening in the Areuse river in Switzerland and investigate the diversity and genomics of major bloom-forming riverine benthic cyanobacteria. We show, using 16S rRNA-based community profiling, that benthic communities are dominated by Oscillatoriales. We correlate the detection of one Microcoleus sequence variant matching the Microcoleus anatoxicus species with the presence of anatoxin-a derivatives and use long-read metagenomics to assemble complete circular genomes of the strain. The main dihydro-anatoxin-a-producing strain in the Areuse is distinct from strains isolated in New Zealand, the USA, and Canada, but forms a monophyletic strain cluster with them with average nucleotide identity values close to the species threshold. Compared to the rest of the Microcoleus genus, the toxin-producing strains encode a 15 % smaller genome, lacking genes for the synthesis of some essential vitamins. Toxigenic mats harbor a distinct microbiome dominated by proteobacteria and bacteroidetes, which may support cyanobacterial growth by providing them with essential nutrients. We recommend that strains closely related to M. anatoxicus be monitored internationally in order to help predict and mitigate similar cyanotoxic events.

Anatoxin-a (ATX-a) is a potent neurotoxin produced by several species of cyanobacteria whose exposure can have direct consequences, including neurological disorders and death. The increasing prevalence of harmful cyanobacterial blooms makes the detection and reliable assessment of ATX-a levels essential to prevent the risk associated with public health. Therefore, the aim of this review is to compile the analytical methods developed to date for the detection and quantification of ATX-a levels alone and in mixtures with other cyanotoxins and their suitability. A classification of the analytical methods available is fundamental to make an appropriate choice according to the type of sample, the equipment available, and the required sensitivity and specificity for each specific purpose. The most widely used detection technique for the quantification of this toxin is liquid chromatography–tandem mass spectrometry (LC-MS/MS). The analytical methods reviewed herein focus mainly on water and cyanobacterial samples, so the need for validated analytical methods in more complex matrices (vegetables and fish) for the determination of ATX-a to assess dietary exposure to this toxin is evidenced. There is currently a trend towards the validation of multitoxin methods as opposed to single-ATX-a determination methods, which corresponds to the real situation of cyanotoxins' confluence in nature. [ABSTRACT FROM AUTHOR]

Cyanotoxins, produced from cyanobacteria-dominated harmful algal blooms (cHABs), pose a threat to ecosystems, as well as drinking water systems. Disinfection via chlorine is the last step in a conventional drinking water treatment plant (DWTP) to remove any extracellular toxins present in the water. This research investigated the impact of pH on the oxidation of different saxitoxin (STX) and anatoxin-a (ANTX-a) concentrations in the post-filtration water collected from two DWTPs. STX degradation was the most efficient at higher pH levels. For instance, pH 6 resulted in 13–43% degradation and pH 9 resulted in 56–74% degradation of STX. The presence of microcystin-LR (MC-LR) did not significantly inhibit the removal of STX, with STX removal (at pH 6–8) higher than when MC-LR was not present. A dose of 1.8 mg/L chlorine and 235-min contact achieved 38% removal of ANTX-a while the dose of 3.2 mg/L chlorine and the contact time of 118 min removed 75% of ANTX-a at pH 6. [ABSTRACT FROM AUTHOR]

Sustainable agriculture, which requires clean water, healthy soil, and adequate nutrients, is crucial to meet the growing demand for food and fiber. Over application of nutrients to meet demand has degraded surface water quality, leading to accelerated eutrophication. Cultural eutrophication is a process by which aquatic ecosystems such as ponds, lakes, and estuaries become so enriched with nutrients--primarily nitrogen and phosphorus--as to become unusable for safe consumption and ecological purposes. Eutrophication has intensified due to climate change. Increased temperatures, intense storms, and drought can drive the formation of eutrophic and hypereutrophic conditions. Hypereutrophication results in the rapid proliferation of algae and phytoplankton, resulting in algal blooms. Harmful algal blooms (HABs), including the proliferation of cyanobacteria, which can produce cyanotoxins such as microcystins and cylindrospermopsin, can also grow in hypereutrophic conditions. These toxins can detrimentally impact humans, wildlife, and agricultural systems (e.g., fish, livestock, and crops). Fish that ingest algal toxins may suffer from liver damage and oxidative stress, with varying effects depending on the type of fish, the amount of exposure, and the duration of exposure to toxins. Additionally, a few studies report livestock death after drinking water contaminated with byproducts of HABs such as microcystin, nodularin, cylindrospermopsin, anatoxin-a, guanitoxin, and saxitoxin. The available research on the effects of microcystin on crops, vegetables, and fruits consistently demonstrates their negative impact. This short communication article summarizes the literature published between 2012 and 2022 documenting the impact of microcystins on agricultural commodities, particularly livestock and various crops. Although awareness has increased, few publications have discussed microcystin-related livestock deaths in recent years. However, numerous global studies have highlighted the harmful effects of microcystins on crops, fruits, and vegetables. The researchers suggest that in cases where the levels of microcystin exceed the standards established by the World Health Organization, careful monitoring is needed. Human exposure to microcystin may occur through the consumption of livestock, crops, and vegetables contaminated by microcystins through drinking or irrigation. More research is needed to understand the fate and transport mechanisms of microcystins in various agricultural settings, including controlled, simulated, and field experiments. [ABSTRACT FROM AUTHOR]

Anatoxin-a and its analogues are potent neurotoxins produced by several genera of cyanobacteria. Due in part to its high toxicity and potential presence in drinking water, these toxins pose threats to public health, companion animals and the environment. It primarily exerts toxicity as a cholinergic agonist, with high affinity at neuromuscular junctions, but molecular mechanisms by which it elicits toxicological responses are not fully understood. To advance understanding of this cyanobacteria, proteomic characterization (DIA shotgun proteomics) of two common fish models (zebrafish and fathead minnow) was performed following (±) anatoxin-a exposure. Specifically, proteome changes were identified and quantified in larval fish exposed for 96 h (0.01–3 mg/L (±) anatoxin-a and caffeine (a methodological positive control) with environmentally relevant treatment levels examined based on environmental exposure distributions of surface water data. Proteomic concentration - response relationships revealed 48 and 29 proteins with concentration - response relationships curves for zebrafish and fathead minnow, respectively. In contrast, the highest number of differentially expressed proteins (DEPs) varied between zebrafish (n = 145) and fathead minnow (n = 300), with only fatheads displaying DEPs at all treatment levels. For both species, genes associated with reproduction were significantly downregulated, with pathways analysis that broadly clustered genes into groups associated with DNA repair mechanisms. Importantly, significant differences in proteome response between the species was also observed, consistent with prior observations of differences in response using both behavioral assays and gene expression, adding further support to model specific differences in organismal sensitivity and/or response. When DEPs were read across from humans to zebrafish, disease ontology enrichment identified diseases associated with cognition and muscle weakness consistent with the prior literature. Our observations highlight limited knowledge of how (±) anatoxin-a, a commonly used synthetic racemate surrogate, elicits responses at a molecular level and advances its toxicological understanding.

Anatoxins (ATXs) are a potent class of cyanobacterial neurotoxins for which only a handful of structural analogues have been well characterized. Here, we report the development of an LC–HRMS/MS method for the comprehensive detection of ATXs. Application of this method to samples of benthic cyanobacterial mats and laboratory cultures showed detection of several new ATXs. Many of these result from nucleophilic addition to the olefinic bond of the α,β-unsaturated ketone functional group of anatoxin-a (ATX) and homoanatoxin-a (hATX), analogous to the conjugation chemistry of microcystins, which contain similar α,β-unsaturated amide functionality. Conjugates with glutathione, γ-glutamylcysteine, methanethiol, ammonia, methanol and water were detected, as well as putative C-10 alcohol derivatives. Structural confirmation was obtained by simple and selective analytical-scale semisynthetic reactions starting from available ATX standards. Methanol, water and ammonia conjugates were found to result primarily from sample preparation. Reduction products were found to result from enzymatic reactions occurring primarily after cell lysis in laboratory cultures of Kamptonema formosum and Cuspidothrix issatschenkoi. The relative contributions of the identified analogues to the anatoxin profiles in a set of 22 benthic-cyanobacterial-mat field samples were estimated, showing conjugates to account for up to 15% of total ATX peak area and 10-hydroxyanatoxins up to 38%. The developed methodology, new analogues and insight into the chemical and enzymatic reactivity of ATXs will enable a more comprehensive study of the class than possible previously. [ABSTRACT FROM AUTHOR]

Anatoxin-a (ATX-a) is a potent neurotoxin produced by several species of cyanobacteria whose exposure can have direct consequences, including neurological disorders and death. The increasing prevalence of harmful cyanobacterial blooms makes the detection and reliable assessment of ATX-a levels essential to prevent the risk associated with public health. Therefore, the aim of this review is to compile the analytical methods developed to date for the detection and quantification of ATX-a levels alone and in mixtures with other cyanotoxins and their suitability. A classification of the analytical methods available is fundamental to make an appropriate choice according to the type of sample, the equipment available, and the required sensitivity and specificity for each specific purpose. The most widely used detection technique for the quantification of this toxin is liquid chromatography–tandem mass spectrometry (LC-MS/MS). The analytical methods reviewed herein focus mainly on water and cyanobacterial samples, so the need for validated analytical methods in more complex matrices (vegetables and fish) for the determination of ATX-a to assess dietary exposure to this toxin is evidenced. There is currently a trend towards the validation of multitoxin methods as opposed to single-ATX-a determination methods, which corresponds to the real situation of cyanotoxins’ confluence in nature.

The frequency of dogs becoming ill or dying from accidental exposure to cyanotoxins, produced by cyanobacteria, is increasing throughout the United States. In January and February of 2021, two dogs died and five dogs became ill after swimming in Lake Travis, central Texas, USA; one deceased dog (C1) was subjected to pathological testing. Algal materials, sediment samples, zebra mussel viscera, periphyton from shells, as well as fluids and tissues from the digestive tract of C1 were investigated for the following cyanotoxins: anatoxin-a, homoanatoxin-a, dihydroanatoxin-a (dhATX), cylindrospermopsin, saxitoxin, and microcystins. Necropsy results of C1 indicated neurotoxicosis with significant levels of dhATX in the duodenum tissues (10.51 ng/g dry weight (DW)), jejunum tissue (6.076 ng/g DW), and stomach contents (974.88 ng/g DW). Algae collected near the site of C1's death contained levels of dhATX, ranging from 13 to 33 µg/g. By comparison, dhATX was detected at much lower concentrations in sediment samples (310.23 ng/g DW) and the periphyton on zebra mussel shells (38.45 ng/g DW). While dhATX was suspected in the deaths of canines from an event in Texas in 2019, this is the first report linking dhATX neurotoxicosis through pathological findings in Texas and potentially in the United States. [ABSTRACT FROM AUTHOR]

Anatoxin-a (ATX-a) is a cyanobacterial toxin whose occurrence has been reported worldwide and has attracted increasing scientific interest due to its toxicity. Moreover, in nature, ATX-a usually appears together with other cyanotoxins, such as cylindrospermopsin (CYN), so possible interaction phenomena could happen and should be considered for risk assessment purposes. For this reason, the aim of this work was to explore the potential mutagenicity and genotoxicity of pure ATX-a and an ATX-a/CYN mixture using a battery of in vitro assays, including the bacterial reverse-mutation assay in Salmonella typhimurium (OECD 471) and the micronucleus test (MN) (OECD 487) on L5178Y Tk+/− cells. The results showed that ATX-a was not mutagenic either alone or in combination with CYN under the conditions tested. Nevertheless, genotoxic effects were observed for both ATX-a and its mixture with CYN following the in vitro MN assay. The genotoxicity exhibited by ATX-a was only observed in the absence of S9 mix, whereas in the cyanotoxin mixture the concentration-dependent genotoxicity of ATX-a/CYN in vitro was observed only in the presence of S9. Thus, the toxicity induced by cyanotoxin mixtures may vary from that produced by toxins alone, and consequently more studies are necessary in order to perform more realistic risk assessments. [ABSTRACT FROM AUTHOR]

• Microcystin-LR improves removal of saxitoxin and anatoxin-a. • Anatoxin-a adsorption on PAC was higher in source water than distilled water. • Initial saxitoxin concentration heavily impacts removal rates. • Cyanobacteria cells in solution lower removal efficiencies of toxins. • Higher PAC contact time and dosage removed more saxitoxin. Cyanobacteria can produce cyanotoxins such as microcystin-LR (MC), saxitoxin (STX), and anatoxin-a (ANTX-a) which are harmful to humans and other animals. Individual removal efficiencies of STX and ANTX-a by powdered activated carbon (PAC) was investigated, as well as when MC-LR and cyanobacteria were present. Experiments were conducted with distilled water and then source water, using the PAC dosages, rapid mix/flocculation mixing intensities and contact times of two drinking water treatment plants in northeast Ohio. At pH 8 and 9, STX removal was 47%-81% in distilled water and 46%-79% in source water, whereas it was 0-28% for pH 6 in distilled water and 31%-52% in source water. When 1.6 µg/L or 20 µg/L MC-LR was present with STX, STX removal was increased with PAC simultaneously removing 45%-65% of the 1.6 µg/L MC-LR and 25%-95% of the 20 µg/L MC-LR depending on the pH. ANTX-a removal at pH 6 was 29%-37% for distilled water and 80% for source water, whereas it was 10%-26% for pH 8 in distilled water and 28% for pH 9 in source water. The presence of cyanobacteria cells decreased ANTX-a removal by at least 18%. When 20 µg/L MC-LR was present with ANTX-a in source water, 59%-73% ANTX-a and 48%-77% of MC-LR was removed at pH 9 depending on the PAC dose. In general, a higher PAC dose led to higher cyanotoxin removals. This study also documented that multiple cyanotoxins can be effectively removed by PAC for water at pH's between 6 and 9. [ABSTRACT FROM AUTHOR]

Pesticides are widely used in a variety of applications, resulting in significant discharge into the terrestrial environment. Hence, the present study was performed to evaluate the effect of two pesticides (Glufosinate-ammonium and Anatoxin- a) on biochemical parameters, oxidative stress, nuclear factor kappa B (NF-KB) activity, and histopathology of the digestive gland of the land snail Monacha cartusiana, which is often employed as a bioindicator of terrestrial pollution. There are no further studies on the effect of Anabaena flos-aquae extract on land snails. The results showed that LCR 50R of Glufosinate-ammonium and Anatoxin-a after exposure to land snail Monacha cartusiana for 96 hrs were 66.6 and 5.3 mg LP −1P, respectively. LC 50 Rvalues reveal the potential activity of Anatoxin-a than Glufosinate-ammonium against the tested snails. Within four days of exposure to glufosinate-ammonium, there were no deaths among treated snails. After the second day of exposure to Anatoxin-a, mortality percentages appeared and gradually rose with increasing concentrations and exposure time. After 28 days of exposure to LCR 25R of tested pesticides, AST, ALT, ALP, MDA, CAT, GPX, and GST levels increased while TAC levels were decreased. NF-KB immunopositivity was much more severe in the cells of tested snails treated with Anatoxin-a than Glufosinate-ammonium. Histopathological examination of the digestive gland showed that sublethal concentration of Glufosinate-ammonium produced excessive secretion and vacuolation. Anatoxin-a induced severe cellular damage in digestive tubules. In conclusion, two pesticides caused alterations in oxidative stress, NF-KB activity, and histological nature of M. Cartusiana that confirmed the toxicity of both pesticides for the living species in the terrestrial environment. [ABSTRACT FROM AUTHOR]

Cyanobacteria, ancient prokaryotes, interfere with ecosystem water quality through the production of cyanotoxins and bloom formation. Therefore, for water safety and public health reasons, the application of faster, sensitive, and specific tools on its risk assessment is demanded. Polymerase chain reaction (PCR) coupled with DNA sequencing can be a helpful tool for the presence and potential to cyanotoxicity. To achieve these, seven waterbodies located on the North and Center regions of Portugal were sampled for two monitoring periods (2017 and 2018). Thus, given the five risk levels proposed (none up to four cyanotoxins — mcyA, cyrC, anaC, sxtI — being detected per risk level), results showed that the great majority of the ecosystems analyzed on the presence of blooms and under climate change phenomenon (heat waves) had an elevated risk (up to four cyanotoxins being detected) corresponding to a situation of high potential of cyanotoxicity. In the opposite conditions (i.e., absence of blooms and heat waves), the risk was lowered to none or only one cyanotoxin being detected. Two ecosystems escaped this trend and demonstrated little to no alterations among risk levels from 1 year to another corresponding to a high potential of cyanotoxicity and cyanotoxins persistence in comparison to other studied ecosystems. Overall, the risk assessment undertaken suggests that other ecosystems ecological variables (physical, hydrological, or chemical) are interfering on the occurrence and persistence of cyanotoxins biosynthesis genes. Given the observed conditions (eutrophic status, bloom occurrence, and heat waves) of the analyzed ecosystems, cyanobacterial potential for toxicity seems to have increased, suggesting a need of the incorporation of other cyanotoxins apart of the regulated microcystins-LR on cyanotoxins surveillance programs of Portugal. [ABSTRACT FROM AUTHOR]

Toxic cyanobacterial blooms are a well-known problem in eutrophic water bodies, but cyanobacterial toxicity in ambient springs is unexplored. Therefore, we studied the toxin content and effects of two extracts obtained from epilithic and benthic microbial mats containing different algae and filamentous cyanobacteria, Phormidium breve and Oscillatoria limosa, respectively, on fresh biomass, number of roots, and pigment content in duckweed Spirodela polyrhiza and on survivorship of Daphnia magna (Cladocera). Mat samples, used to prepare extracts for bioassays, were collected in the anthropogenically transformed limnocrenic Górecko spring, located (50°31′08″ N and 22°57′10″ E) in the Roztocze region (Eastern Poland). It drains an abundant aquifer built with Cretaceous sediments. Neither anatoxin-a (ANTX) nor microcystins (MCs) were detected in the extracts using HPLC techniques; however, negative effects of the extracts on tested organisms were observed. The Phormidium extract contained concentrations of cyanobacterial metabolites a few times higher than the Oscillatoria extract. In general, both extracts affected plants in a dose-dependent manner; however, the Oscillatoria extract influenced pigment production without a clear trend. The highest concentrations of Phormidium extract (p < 0.05) significantly decreased the number of roots and the content of chlorophylls and carotenoids in S. polyrhiza. The Oscillatoria extract caused a statistically significant (p < 0.05) decrease in biomass and number of roots; however, it generally positively influenced the production of pigments. The extract containing O. limosa metabolites was more toxic to D. magna than the extract containing higher amounts of metabolites of P. breve. Cyanobacteria inhabiting temperate springs may negatively affect hydrobionts by producing secondary metabolites other than ANTX and MCs; however, the contribution of algae cannot be excluded. Extensive research on cyanobacteria in springs, their metabolites, and their effects on living organisms should be conducted. [ABSTRACT FROM AUTHOR]

The presence of cyanotoxins and its bioaccumulation in the food chain is an increasingly common problem worldwide. Despite the toxic effects produced by Anatoxin-a (ATX-a), this neurotoxin has been less studied compared to microcystins (MCs) and cylindrospermopsin (CYN). Studies conducted under laboratory conditions are of particular interest because these provide information which are directly related to the effects produced by the toxin. Currently, the World Health Organization (WHO) considers the ATX-a toxicological database inadequate to support the publication of a formal guideline reference value. Therefore, the aim of the present work is to compile all of the in vitro and in vivo toxicological studies performed so far and to identify potential data gaps. Results show that the number of reports is increasing in recent years. However, more in vitro studies are needed, mainly in standardized neuronal cell lines. Regarding in vivo studies, very few of them reflect conditions occurring in nature and further studies with longer periods of oral exposure would be of interest. Moreover, additional toxicological aspects of great interest such as mutagenicity, genotoxicity, immunotoxicity and alteration of hormonal balance need to be studied in depth. [ABSTRACT FROM AUTHOR]

Cyanobacterial blooms, resulting from serious eutrophication, can produce various cyanotoxins and severely disrupt aquatic ecosystems. Inducible defenses are adaptive traits developed by prey in response to predation risks. However, the effects of the increasing proportion of cyanobacteria and cyanotoxins produced during cyanobacterial blooms on the inducible defenses of cladocerans, particularly in terms of behavioral defenses, remain unclear. In this study, we selected Daphnia magna and investigated the defensive traits against predation risks by the predator Rhodeus ocellatus under different ratios of cyanobacteria (Dolichospermum flos-aquae) and green algae (Scenedesmus obliquus), as well as varying concentrations of anatoxin-a (ATX), a cyanotoxin. We recorded the inducible defensive traits involving to morphology, behavior, and offspring production of D. magna. Results showed that the body length of D. magna at sexual maturity and the number of offspring in the first brood were significantly reduced by the presence of D. flos-aquae. Moreover, when the proportion of D. flos-aquae reached 75% and 100%, D. magna did not develop to sexual maturity. Furthermore, D. flos-aquae inhibited the formation of inducible behavioral defense of D. magna , with a stronger inhibitory effect as the proportion of D. flos-aquae increased. In this experiment, the effects of ATX on the morphological traits at sexual maturity and offspring production of D. magna were minor, but ATX still had the potential to inhibit the formation of inducible behavioral defense. We confirmed that changes in the proportion of cyanobacteria and green algae as well as the production of ATX by cyanobacteria during cyanobacterial blooms can affect the growth, development, and inducible defensive traits of cladocerans, potentially altering their population dynamics during such events. [Display omitted] • Body length and reproductive capacity of Daphnia magna were reduced by D. flos-aquae. • D. flos-aquae inhibited the formation of inducible behavioral defense of D. magna. • The inhibitory effect became stronger as the proportion of D. flos-aquae increased. • Anatoxin-a had no effect on the morphological defense and reproduction of D. magna. • Anatoxin-a inhibited the formation of inducible behavioral defense of D. magna. [ABSTRACT FROM AUTHOR]

Cyanotoxins produced by blue-green algae in lakes are among the most serious threats to water quality worldwide. As global warming rapidly extends the locations and timing of blue-green algae blooms, a simple and accessible method for the detection and quantification of cyanotoxins in fresh water is increasingly necessary. Here, a quick, simple and accessible simultaneous analytical method for five cyanotoxins (cylindrospermopsin, anatoxin-a, microcystin-RR, YR and LR) is reported. This method has three advantages. First, it does not require complicated operations, such as a concentration operation. Second, it employs an HPLC column without high pressure. Third, the use of stable isotope-labeled surrogates enables correct identification and precise quantification of cyanotoxins. The method was applied to the lakes of Fukuoka Prefecture in Japan, and four of the five above-named cyanotoxins (i.e., all but cylindrospermopsin) were detected. The limits of quantification were 20–43 ng/L, which were considerably lower than the WHO guideline values. The recovery levels were 97–104%. Microbial flora analysis revealed that the sources of anatoxin-a were Pseudanabaena limnetica and Cuspidothrix issatschenkoi, and the source of microcystins was the group A1 of Microcystis aeruginosa. This study provides a quick, easy and accessible method for the worldwide monitoring of cyanotoxin levels.

Increasing prevalence of cyanotoxins in surface water bodies worldwide threatens groundwater quality when contaminated water recharges an aquifer through natural or artificial means. The subsurface fate of anatoxin-a (ATX) is not well studied. Laboratory batch experiments were performed to expand the current knowledge of ATX sorption affinities to geologic media, with a focus on natural soil (Vertisol, Ultisol, Alfisol, and Inceptisol) and physical, chemical, and mineralogical characteristics. For a range of aqueous ATX concentrations (0.3–14 μg/L), linear, Freundlich, and Langmuir isotherms fit observed data well (r2 = 0.92–1.00, RMSE = 0.4–6.3 μg/kg). Distribution coefficient (Kd) and retardation factor (Rf) values were computed for the linear isotherm, giving Kd of 22.3–77.1 L/kg and Rf of 62–256. Average percent removals were 85.0–92.2%. The strongest predictors of Kd were kaolinite and smectite group mineral abundances and for Rf were smectite group and silt and clay abundances. Results indicate that loamy, silty, or clayey soils—particularly Vertisols—tend to substantially slow migration of ATX through natural soil systems. Where implemented as a functionalized amendment in an engineered pollution control media, such soils may enhance natural ATX attenuation processes, thereby supporting the protection of in situ and extracted groundwater during irrigation, natural and managed aquifer recharge, or riverbank filtration. [ABSTRACT FROM AUTHOR]

While freshwater cyanobacteria are traditionally thought to be limited by the availability of phosphorus (P), fixed nitrogen (N) supply can promote the growth and/or toxin production of some genera. This study characterizes how growth on N2 (control), nitrate (NO3-), ammonium (NH4+), and urea as well as P limitation altered the growth, toxin production, N2 fixation, and gene expression of an anatoxin-a (ATX-A) -- producing strain of Dolichospermum sp. 54. The transcriptomes of fixed N and P-limited cultures differed significantly from those of fixed N-deplete, P-replete (control) cultures, while the transcriptomes of P-replete cultures amended with either NH4+ or NO3- were not significantly different relative to those of the control. Growth rates of Dolichospermum (sp. 54) were significantly higher when grown on fixed N relative to without fixed N; growth on NH4C was also significantly greater than growth on NO3-. NH4+ and urea significantly lowered N2 fixation and nifD gene transcript abundance relative to the control while cultures amended with NO3- exhibited N2 fixation and nifD gene transcript abundance that was not different from the control. Cultures grown on NH4+ exhibited the lowest ATX-A content per cell and lower transcript abundance of genes associated ATX-A synthesis (ana), while the abundance of transcripts of several ana genes were highest under fixed N and P - limited conditions. The significant negative correlation between growth rate and cellular anatoxin quota as well as the significantly higher number of transcripts of ana genes in cultures deprived of fixed N and P relative to P-replete cultures amended with NH4+ suggests ATXA was being actively synthesized under P limitation. Collectively, these findings indicate that management strategies that do not regulate fixed N loading will leave eutrophic water bodies vulnerable to more intense and toxic (due to increased biomass) blooms of Dolichospermum. [ABSTRACT FROM AUTHOR]

Some of the most commonly identified freshwater toxins are anatoxin-a (ATX-a), cylindrospermopsin (CYN), and microcystin-LR (MC-LR). The aim of this paper was to compare different methods of extracting and concentrating these cyanotoxins and check the impact of selected physical factors on the accumulation of biomass of Dolichospermum flos-aquae, Microcystis aeruginosa, and Raphidiopsis raciborskii. The effect of different cyanobacteria cultivation conditions on the amount of cyanotoxins synthesized showed no significant changes over time in the average concentration of all tested toxins in the medium compared to the control. Mixing cultures increases the intracellular content of ATX-a. Aerating also positively affects the concentration of MC-LR intracellularly. In order to optimize the solid phase extraction (SPE) process of toxins, the C18 phase or activated carbon was used. In general, higher toxin recoveries were achieved when using the C18 phase. The best result was achieved for ATX-a, 94% recovery with elution using methanol with 0.1% trifluoroacetic acid (TFA). For MC-LR, the best recovery was 59%, and for CYN 22%. The study evaluated the various methods to release cyanotoxins from cyanobacteria showed that: the highest ATX-a concentration (0.60 μg/mg d.w) was obtained using MilliQ water and microwave treatment for 10 to 15 seconds. For MC-LR, the highest extracted amount (6.73 μg/mg d.w) resulted from methanol treatment and boiling at 100°C for 15 minutes. CYN extraction was the most effective by using MilliQ water and alternative freezing/thawing (1.54 μg/mg d.w). In conclusion, changing the optimal parameters of cyanobacterial cultivation, only slightly affects the increase in biomass accumulation and synthesis of cyanobacterial toxins. In the case of ATX, the key is the use of the TFA additive in the SPE process. No single method has been identified as the ideal approach for isolating various intracellular cyanotoxins., Competing Interests: Declaration of Competing Interest ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:, (Copyright © 2024. Published by Elsevier Ltd.)

Global warming due to climate change, as well as freshwater eutrophication caused by anthropogenic activities are responsible, among other factors, for an increasing occurrence of harmful algal blooms (HABs) in aquatic systems. These can lead to the generation of cyanotoxins, secondary metabolites coming from cyanobacteria, producing adverse effects in living organisms including death. This research aims to study the effects that two neurotoxins, anatoxin-a (ATX-a) and saxitoxin (STX), have on living organisms. Once the stability of both compounds in water was determined for a 24 h period using ultra-high-performance liquid chromatography coupled to a triple quadrupole mass spectrometer (UPLC-MS/MS), zebrafish larvae were exposed to different levels of toxins (1 ng L -1 , 10 ng L -1 , 100 ng L -1 and 1 μg L -1 ) during 24 h. Behavioral studies including vibrational startle response (VSR), habituation to vibrational stimuli, basal locomotor activity (BLM) and visual motor response (VMR) were performed using Danio Vision system, and neurotransmitters (NTs) from 15-head pools of control and exposed zebrafish larvae were extracted and analyzed by UPLC-MS/MS. Both compounds induced hypolocomotion in the individuals, while 10 and 100 ng L -1 of ATX-a significantly increased methionine (120 % and 126 %, respectively) and glutamate levels (118 % and 129 %, respectively). Saxitoxin enhanced 3-metoxytyramine (3-MT) levels at 1 ng L -1 by 185 %. The findings of this study show that both studied cyanotoxins influence the behavior of zebrafish larvae as well as their metabolism., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Cristian Gomez Canela reports financial support was provided by Spain Ministry of Science and Innovation. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Over the last two decades, proliferations of benthic cyanobacteria producing derivatives of anatoxin-a have been reported in rivers worldwide. Here, we follow up on such a toxigenic event happening in the Areuse river in Switzerland and investigate the diversity and genomics of major bloom-forming riverine benthic cyanobacteria. We show, using 16S rRNA-based community profiling, that benthic communities are dominated by Oscillatoriales. We correlate the detection of one Microcoleus sequence variant matching the Microcoleus anatoxicus species with the presence of anatoxin-a derivatives and use long-read metagenomics to assemble complete circular genomes of the strain. The main dihydro-anatoxin-a-producing strain in the Areuse is distinct from strains isolated in New Zealand, the USA, and Canada, but forms a monophyletic strain cluster with them with average nucleotide identity values close to the species threshold. Compared to the rest of the Microcoleus genus, the toxin-producing strains encode a 15 % smaller genome, lacking genes for the synthesis of some essential vitamins. Toxigenic mats harbor a distinct microbiome dominated by proteobacteria and bacteroidetes, which may support cyanobacterial growth by providing them with essential nutrients. We recommend that strains closely related to M. anatoxicus be monitored internationally in order to help predict and mitigate similar cyanotoxic events., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Diego Gonzalez reports financial support was provided by Swiss National Science Foundation. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Author(s).)

Cyanobacterial harmful algal blooms (CHABs) are increasing at an alarming rate in different water bodies worldwide. In India, CHAB events in water bodies such as Dal Lake have been sporadically reported with no study done to characterize the cyanobacterial species and their associated toxins. We hypothesized that this Lake is contaminated with toxic cyanobacterial species with the possibility of the presence of cyanotoxin biosynthetic genes. We, therefore, used some of the molecular tools such as 16S ribosomal DNA, PCR, and phylogenetic analysis to explore cyanobacterial species and their associated toxins. A 3-year (2018–2020) survey was conducted at three different sampling sites of Dal Lake namely, Grand Palace Gath (S1), Nigeen basin (S2), and Gagribal basin (S3). Two strains of Dolichospermum sp. AE01 and AE02 (S3 and S1 site) and one strain of Microcystis sp. AE03 (S2 site) was isolated, cultured, and characterized phylogenetically by 16S ribosomal DNA sequencing. The presence of cyanotoxin genes from the isolates was evaluated by PCR of microcystins (mcyB), anatoxins (anaC), and cylindrospermopsins (pks) biosynthesis genes. Results revealed the presence of both mcyB and pks gene in Microcystis sp. AE03, and only anaC gene in Dolichospermum sp. AE02 strain. However, Dolichospermum sp. AE01 strain was not found to harbor any such genes. Our findings, for the first time, reported the coexistence of pks and mcyB in a Microcystis AE03 strain. This study has opened a new door to further characterize the unexplored cyanobacterial species, their associated cyanotoxin biosynthetic genes, and the intervention of high-end proteomic techniques to characterize the cyanotoxins.

The frequency of dogs becoming ill or dying from accidental exposure to cyanotoxins, produced by cyanobacteria, is increasing throughout the United States. In January and February of 2021, two dogs died and five dogs became ill after swimming in Lake Travis, central Texas, USA; one deceased dog (C1) was subjected to pathological testing. Algal materials, sediment samples, zebra mussel viscera, periphyton from shells, as well as fluids and tissues from the digestive tract of C1 were investigated for the following cyanotoxins: anatoxin-a, homoanatoxin-a, dihydroanatoxin-a (dhATX), cylindrospermopsin, saxitoxin, and microcystins. Necropsy results of C1 indicated neurotoxicosis with significant levels of dhATX in the duodenum tissues (10.51 ng/g dry weight (DW)), jejunum tissue (6.076 ng/g DW), and stomach contents (974.88 ng/g DW). Algae collected near the site of C1’s death contained levels of dhATX, ranging from 13 to 33 µg/g. By comparison, dhATX was detected at much lower concentrations in sediment samples (310.23 ng/g DW) and the periphyton on zebra mussel shells (38.45 ng/g DW). While dhATX was suspected in the deaths of canines from an event in Texas in 2019, this is the first report linking dhATX neurotoxicosis through pathological findings in Texas and potentially in the United States.

Anatoxin-a (ATX-a) is a cyanobacterial toxin whose occurrence has been reported worldwide and has attracted increasing scientific interest due to its toxicity. Moreover, in nature, ATX-a usually appears together with other cyanotoxins, such as cylindrospermopsin (CYN), so possible interaction phenomena could happen and should be considered for risk assessment purposes. For this reason, the aim of this work was to explore the potential mutagenicity and genotoxicity of pure ATX-a and an ATX-a/CYN mixture using a battery of in vitro assays, including the bacterial reverse-mutation assay in Salmonella typhimurium (OECD 471) and the micronucleus test (MN) (OECD 487) on L5178Y Tk+/− cells. The results showed that ATX-a was not mutagenic either alone or in combination with CYN under the conditions tested. Nevertheless, genotoxic effects were observed for both ATX-a and its mixture with CYN following the in vitro MN assay. The genotoxicity exhibited by ATX-a was only observed in the absence of S9 mix, whereas in the cyanotoxin mixture the concentration-dependent genotoxicity of ATX-a/CYN in vitro was observed only in the presence of S9. Thus, the toxicity induced by cyanotoxin mixtures may vary from that produced by toxins alone, and consequently more studies are necessary in order to perform more realistic risk assessments.

High densities of cyanobacteria in aquatic ecosystems can cause impacts to ecosystem services because they serve as a poor‐quality food resource, produce toxins and can indirectly cause a variety of other negative impacts to water quality. There are many hypotheses about the potential environmental drivers of variation in cyanobacterial abundance and toxicity, but these hypotheses have rarely been considered in combination and rarely been examined in large river ecosystems. Here, we use monthly data from backwater habitats of the Upper Mississippi River (UMR) to evaluate associations between environmental conditions and cyanobacterial abundance and toxicity (microcystin and anatoxin‐a) that would be expected based on several hypotheses. Backwaters in the Mississippi River vary in flushing rate, temperature, turbidity, nutrient availability, water depth, and vegetative cover. We find support for hypotheses that suggest physical conditions in backwaters (flushing rate, temperature, turbidity, rooted vegetation cover, and water depth) and nutrient availability influence cyanobacterial abundance and toxicity. We then used structural equation modeling to incorporate several hypotheses into a causal modeling framework, which indicated that backwater connectivity (flushing) strongly influences cyanobacterial abundance via the regulation of water temperature, and that nutrient availability strongly influences the presence of microcystin concentrations above our detection limit. Our data suggest that management of backwater connectivity could influence cyanobacterial abundance and toxicity in UMR backwaters. Reconnecting backwaters (via alteration of levees) could serve as a local adaptation to minimize the effects of climate change and excessive nutrient loading. [ABSTRACT FROM AUTHOR]

Cyanobacteria are broadly producing secondary metabolites including cyanotoxins. More than 40 genera of cyanobacteria produce cyanotoxins. The presence of cyanotoxins in water presents a global problem due to environmental deterioration and possible toxic effects to human and animals. Because of anthropogenic activities and climate change, the occurrence of these toxins have been increasing worldwide. The major route of human exposure to cyanotoxins is the oral intake. Increased exposure of cyanotoxins brings some risks related to public health. In this review, possible genotoxic effects of well-known cyanotoxins (Cylindrospermopsin, Microcystin-LR, Nodularin, Anatoxin-a and Homoanatoxin-a) were deeply reviewed. This article aimed to explain genotoxic activity and mechanisms underlying the genotoxic effects of cyanotoxins by reviewing large databases. [ABSTRACT FROM AUTHOR]

Toxin producing cyanobacterial blooms have increased globally in recent decades in both frequency and intensity. Despite the recognition of this growing risk, the extent and magnitude of cyanobacterial blooms and cyanotoxin prevalence is poorly characterized in the heavily populated region of southern California. Recent assessments of lentic waterbodies (depressional wetlands, lakes, reservoirs and coastal lagoons) determined the prevalence of microcystins and, in some cases, additional cyanotoxins. Microcystins were present in all waterbody types surveyed although toxin concentrations were generally low across most habitats, as only a small number of sites exceeded California's recreational health thresholds for acute toxicity. Results from passive samplers (Solid Phase Adsorption Toxin Tracking (SPATT)) indicated microcystins were prevalent throughout lentic waterbodies and that traditional discrete samples underestimated the presence of microcystins. Multiple cyanotoxins were detected simultaneously in some systems, indicating multiple stressors, the risk of which is uncertain since health thresholds are based on exposures to single toxins. Anatoxin-a was detected for the first time from lakes in southern California. The persistence of detectable microcystins across years and seasons indicates a low-level, chronic risk through both direct and indirect exposure. The influence of toxic cyanobacterial blooms is a more complex stressor than presently recognized and should be included in water quality monitoring programs.

Invasive nostocalean cyanobacteria (INC) were first reported in tropical regions and are now globally spreading rapidly due to climate change, appearing in temperate regions. INC require continuous monitoring for water resource management because of their high toxin production potential. However, it is difficult to analyze INC under a microscope because of their morphological similarity to nostocalean cyanobacteria such as the genus Aphanizomenon. This study calculates the gene copy number per cell for each target gene through quantitative gene analysis on the basis of genus-specific primers of genera Cylindrospermopsis, Sphaerospermopsis, and Cuspidothrix, and the toxin primers of anatoxin-a, saxitoxin, and cylindrospermopsin. In addition, quantitative gene analysis was performed at eight sites in the Nakdong River to assess the appearance of INC and their toxin production potential. Genera Cylindrospermopsis and Sphaerospermopsis did not exceed 100 cells mL−1 at the maximum, with a low likelihood of related toxin occurrence. The genus Cuspidothrix showed the highest cell density (1759 cells mL−1) among the INC. Nakdong River has potential for the occurrence of anatoxin-a through biosynthesis by genus Cuspidothrix because the appearance of this genus coincided with that of the anatoxin-a synthesis gene (anaF) and the detection of the toxin by ELISA. [ABSTRACT FROM AUTHOR]

Toxic cyanobacterial blooms are a well-known problem in eutrophic water bodies, but cyanobacterial toxicity in ambient springs is unexplored. Therefore, we studied the toxin content and effects of two extracts obtained from epilithic and benthic microbial mats containing different algae and filamentous cyanobacteria, Phormidium breve and Oscillatoria limosa, respectively, on fresh biomass, number of roots, and pigment content in duckweed Spirodela polyrhiza and on survivorship of Daphnia magna (Cladocera). Mat samples, used to prepare extracts for bioassays, were collected in the anthropogenically transformed limnocrenic Górecko spring, located (50°31′08″ N and 22°57′10″ E) in the Roztocze region (Eastern Poland). It drains an abundant aquifer built with Cretaceous sediments. Neither anatoxin-a (ANTX) nor microcystins (MCs) were detected in the extracts using HPLC techniques; however, negative effects of the extracts on tested organisms were observed. The Phormidium extract contained concentrations of cyanobacterial metabolites a few times higher than the Oscillatoria extract. In general, both extracts affected plants in a dose-dependent manner; however, the Oscillatoria extract influenced pigment production without a clear trend. The highest concentrations of Phormidium extract (p < 0.05) significantly decreased the number of roots and the content of chlorophylls and carotenoids in S. polyrhiza. The Oscillatoria extract caused a statistically significant (p < 0.05) decrease in biomass and number of roots; however, it generally positively influenced the production of pigments. The extract containing O. limosa metabolites was more toxic to D. magna than the extract containing higher amounts of metabolites of P. breve. Cyanobacteria inhabiting temperate springs may negatively affect hydrobionts by producing secondary metabolites other than ANTX and MCs; however, the contribution of algae cannot be excluded. Extensive research on cyanobacteria in springs, their metabolites, and their effects on living organisms should be conducted.

The presence of cyanotoxins and its bioaccumulation in the food chain is an increasingly common problem worldwide. Despite the toxic effects produced by Anatoxin-a (ATX-a), this neurotoxin has been less studied compared to microcystins (MCs) and cylindrospermopsin (CYN). Studies conducted under laboratory conditions are of particular interest because these provide information which are directly related to the effects produced by the toxin. Currently, the World Health Organization (WHO) considers the ATX-a toxicological database inadequate to support the publication of a formal guideline reference value. Therefore, the aim of the present work is to compile all of the in vitro and in vivo toxicological studies performed so far and to identify potential data gaps. Results show that the number of reports is increasing in recent years. However, more in vitro studies are needed, mainly in standardized neuronal cell lines. Regarding in vivo studies, very few of them reflect conditions occurring in nature and further studies with longer periods of oral exposure would be of interest. Moreover, additional toxicological aspects of great interest such as mutagenicity, genotoxicity, immunotoxicity and alteration of hormonal balance need to be studied in depth.

Cyanobacterial blooms are often associated with the presence of harmful natural compounds which can cause adverse health effects in both humans and animals. One family of these compounds, known as anatoxins, have been linked to the rapid deaths of cattle and dogs through neurotoxicological action. Here, we report the findings resulting from the death of a dog at a freshwater reservoir in SW England. Poisoning was rapid following exposure to material at the side of the lake. Clinical signs included neurological distress, diaphragmatic paralysis and asphyxia prior to death after 45 min of exposure. Analysis by HILIC-MS/MS of urine and stomach content samples from the dog revealed the detection of anatoxin-a and dihydroanatoxin-a in both samples with higher concentrations of the latter quantified in both matrices. Detection and quantitative accuracy was further confirmed with use of accurate mass LC-HRMS. Additional anatoxin analogues were also detected by LC-HRMS, including 4-keto anatoxin-a, 4-keto-homo anatoxin-a, expoxy anatoxin-a and epoxy homo anatoxin-a. The conclusion of neurotoxicosis was confirmed with the use of two independent analytical methods showing positive detection and significantly high quantified concentrations of these neurotoxins in clinical samples. Together with the clinical signs observed, we have confirmed that anatoxins were responsible for the rapid death of the dog in this case.

Increasing prevalence of cyanotoxins in surface water bodies worldwide threatens groundwater quality when contaminated water recharges an aquifer through natural or artificial means. The subsurface fate of anatoxin-a (ATX) is not well studied. Laboratory batch experiments were performed to expand the current knowledge of ATX sorption affinities to geologic media, with a focus on natural soil (Vertisol, Ultisol, Alfisol, and Inceptisol) and physical, chemical, and mineralogical characteristics. For a range of aqueous ATX concentrations (0.3–14 μg/L), linear, Freundlich, and Langmuir isotherms fit observed data well (r2 = 0.92–1.00, RMSE = 0.4–6.3 μg/kg). Distribution coefficient (Kd) and retardation factor (Rf) values were computed for the linear isotherm, giving Kd of 22.3–77.1 L/kg and Rf of 62–256. Average percent removals were 85.0–92.2%. The strongest predictors of Kd were kaolinite and smectite group mineral abundances and for Rf were smectite group and silt and clay abundances. Results indicate that loamy, silty, or clayey soils—particularly Vertisols—tend to substantially slow migration of ATX through natural soil systems. Where implemented as a functionalized amendment in an engineered pollution control media, such soils may enhance natural ATX attenuation processes, thereby supporting the protection of in situ and extracted groundwater during irrigation, natural and managed aquifer recharge, or riverbank filtration.

Sutherland JW, Turcotte RJ, Molden E, Moriarty V, Kelly M, Aubel M, Foss A. 2021. The detection of airborne anatoxin-a (ATX) on glass fiber filters during a harmful algal bloom. Lake Reserv Manage. 37:113–119. Anatoxin-a (ATX) is a neurotoxic alkaloid produced by certain cyanobacteria genera that can occur during harmful algal blooms (HABs). This study is the first to report airborne ATX collected outside an aquatic ecosystem. ATX was captured on glass fiber filters using a field-deployed air sampler during an active 2019 HAB at Capaum Pond on Nantucket Island, Massachusetts (USA). These findings indicate that emission of ATX molecules, or cyanobacteria containing ATX, during HABs presents a potential human health exposure not previously examined. [ABSTRACT FROM AUTHOR]

Anatoxin-a and its analogues are potent neurotoxins produced by several genera of cyanobacteria. Due in part to its high toxicity and potential presence in drinking water, these toxins pose threats to public health, companion animals and the environment. It primarily exerts toxicity as a cholinergic agonist, with high affinity at neuromuscular junctions, but molecular mechanisms by which it elicits toxicological responses are not fully understood. To advance understanding of this cyanobacteria, proteomic characterization (DIA shotgun proteomics) of two common fish models (zebrafish and fathead minnow) was performed following (±) anatoxin-a exposure. Specifically, proteome changes were identified and quantified in larval fish exposed for 96 h (0.01–3 mg/L (±) anatoxin-a and caffeine (a methodological positive control) with environmentally relevant treatment levels examined based on environmental exposure distributions of surface water data. Proteomic concentration - response relationships revealed 48 and 29 proteins with concentration - response relationships curves for zebrafish and fathead minnow, respectively. In contrast, the highest number of differentially expressed proteins (DEPs) varied between zebrafish (n = 145) and fathead minnow (n = 300), with only fatheads displaying DEPs at all treatment levels. For both species, genes associated with reproduction were significantly downregulated, with pathways analysis that broadly clustered genes into groups associated with DNA repair mechanisms. Importantly, significant differences in proteome response between the species was also observed, consistent with prior observations of differences in response using both behavioral assays and gene expression, adding further support to model specific differences in organismal sensitivity and/or response. When DEPs were read across from humans to zebrafish, disease ontology enrichment identified diseases associated with cognition and muscle weakness consistent with the prior literature. Our observations highlight limited knowledge of how (±) anatoxin-a, a commonly used synthetic racemate surrogate, elicits responses at a molecular level and advances its toxicological understanding. [ABSTRACT FROM AUTHOR]

Background: Though anatoxin-a (antx-a) is a globally important cyanobacterial neurotoxin in inland waters, information on sublethal toxicological responses of aquatic organisms is limited. We examined influences of (±) antx-a (11–3490 µg/L) on photolocomotor behavioral responses and gene transcription associated with neurotoxicity, oxidative stress and hepatotoxicity, in two of the most common alternative vertebrate and fish models, Danio rerio (zebrafish) and Pimephales promelas (fathead minnow). We selected environmentally relevant treatment levels from probabilistic exposure distributions, employed standardized experimental designs, and analytically verified treatment levels using isotope-dilution liquid chromatography tandem mass spectrometry. Caffeine was examined as a positive control. Results: Caffeine influences on fish behavior responses were similar to previous studies. Following exposure to (±) antx-a, no significant photolocomotor effects were observed during light and dark transitions for either species. Though zebrafish behavioral responses profiles were not significantly affected by (±) antx-a at the environmentally relevant treatment levels examined, fathead minnow stimulatory behavior was significantly reduced in the 145–1960 µg/L treatment levels. In addition, no significant changes in transcription of target genes were observed in zebrafish; however, elavl3 and sod1 were upregulated and gst and cyp3a126 were significantly downregulated in fathead minnows. Conclusion: We observed differential influences of (±) antx-a on swimming behavior and gene transcription in two of the most common larval fish models employed for prospective and retrospective assessment of environmental contaminants and water quality conditions. Sublethal responses of fathead minnows were consistently more sensitive than zebrafish to this neurotoxin at the environmentally relevant concentrations examined. Future studies are needed to understand such interspecies differences, the enantioselective toxicity of this compound, molecular initiation events within adverse outcome pathways, and subsequent individual and population risks for this emerging water quality threat. [ABSTRACT FROM AUTHOR]

Invasive nostocalean cyanobacteria (INC) were first reported in tropical regions and are now globally spreading rapidly due to climate change, appearing in temperate regions. INC require continuous monitoring for water resource management because of their high toxin production potential. However, it is difficult to analyze INC under a microscope because of their morphological similarity to nostocalean cyanobacteria such as the genus Aphanizomenon. This study calculates the gene copy number per cell for each target gene through quantitative gene analysis on the basis of genus-specific primers of genera Cylindrospermopsis, Sphaerospermopsis, and Cuspidothrix, and the toxin primers of anatoxin-a, saxitoxin, and cylindrospermopsin. In addition, quantitative gene analysis was performed at eight sites in the Nakdong River to assess the appearance of INC and their toxin production potential. Genera Cylindrospermopsis and Sphaerospermopsis did not exceed 100 cells mL−1 at the maximum, with a low likelihood of related toxin occurrence. The genus Cuspidothrix showed the highest cell density (1759 cells mL−1) among the INC. Nakdong River has potential for the occurrence of anatoxin-a through biosynthesis by genus Cuspidothrix because the appearance of this genus coincided with that of the anatoxin-a synthesis gene (anaF) and the detection of the toxin by ELISA.

LeDuc JF, Christensen VG, Maki RP. 2020. Rapid assessment test strips: effectiveness for cyanotoxin monitoring in a northern temperate lake. Lake Reserv Manag, 36:444–453. Precise and rapid methods of determining toxin levels are needed in lakes used for recreation and drinking water to facilitate a quick risk assessment during cyanobacteria blooms. Therefore, we evaluated rapid-assessment test strips, a newer technology for estimating the toxicity of cyanobacterial blooms, in Kabetogama Lake, a popular recreational area of Voyageurs National Park in northern Minnesota (USA). Sixty-seven percent of the test strip results matched results of enzyme-linked immunosorbent assays, with individual toxin results of 75% (anatoxin-a), 80% (cylindrospermopsin), and 64% (microcystin). These results provide some evidence that the test strips may be effective for rapid detection of toxins in northern temperate lakes, although improvements to the test strips may be beneficial. Despite the intensive processing required and uncertainty of some results, the availability of a rapid and inexpensive field method allowed us to sample opportunistically in the fall, when we documented dangerously high toxin concentrations at places where waterfowl-retrieving dogs may be at particular risk of exposure. [ABSTRACT FROM AUTHOR]

Cyanotoxins are a global concern in freshwaters and eutrophication and climate changes can have synergistic effects in exacerbating the problem. Deep Subalpine Lakes (DSL) are a group of lakes of huge economic and naturalistic importance in Italy. Together with eutrophication (occurred during 1960s and 1970s) and re-oligotrophication (from 1990 onward) these lake have been experiencing warming and increase of the water column stability. These changes have influenced the phytoplankton (comprised the cyanobacteria) community of the lakes. Four DSL lakes (Lakes Garda, Iseo, Como and Lugano) have been studied with the aim of comparing their toxic potential. For one of them (Lake Garda) an 8 years survey was conducted, allowing a long term trend analysis. Toxin analysis was conducted on a monthly basis by targeted LC-MS/MS. A screening for anatoxins, cylindrospermopsins, saxitoxins, microcystins (MCs) and nodularins was carried out. Among all the listed toxins, only one anatoxin and five MCs were detected in the lakes. In particular, the alkaloid Anatoxin-a (ATX) was found dominant Lakes Garda, Iseo and Como and absent in lake Lugano; the MC-[D-Asp3]RR was found as the most abundant MC in all four lakes. Four other minoritary MCs were also found. The two major toxins are produced by two different cyanobacteria (Tychonema bourrellyi and Planktothrix rubescens) which have however similar ecological traits. Peaks of these toxins occur in warmer months (typically between May and September) in the thermocline layer (around 20 m, in the considered lakes). In summer 2016, the highest concentrations of ATX and total microcystins (MCs) were registered in Lake Iseo (1100 and 430ngL-1, respectively), while in the other lakes values were approximately twice lower. In the lakes where it is present, ATX peak levels are much higher than MCs, thus highlighting the necessity of considering ATX in the procedures of risk assessment. The importance of ATX is expected to further grow in the future with respect to MCs, as demonstrated by the long term trend analysis carried out in Lake Garda that showed a clear decline for MCs from 2009 till 2016 and a relative constancy for ATX.

Here, we designed a simple, rapid, and ultrasensitive colorimetric aptasensor for detecting anatoxin-a (ATX-a). The sensor employs a DNA aptamer as the sensing element and gold nanoparticles (AuNPs) as probes. Adsorption of the aptamer onto the AuNP surface can protect AuNPs from aggregation in NaCl solution, thus maintaining their dispersion state. In the presence of ATX-a, the specific binding of the aptamer with ATX-a results in a conformational change in the aptamer, which facilitates AuNP aggregation and, consequently, a color change of AuNPs from red to blue in NaCl solution. This color variation is directly associated with ATX-a concentration and can be easily measured using a UV/Vis spectrophotometer. The absorbance variation is linearly proportional to ATX-a concentration across the concentration range of 10 pM to 200 nM, with a detection limit of 4.45 pM and high selectivity against other interferents. This strategy was successfully applied to the detection of ATX-a in lake water samples. Thus, the present aptasensor is a promising alternative method for the rapid detection of ATX-a in the environment.

Blue-green algae produce a wide range of toxic compounds, particularly neurotoxins, which effects have only been shown on the central and peripheral nervous system. The current study was conducted to show the histopathological effects of anatoxin-a purified from local isolates from Shutt Al-Arab river water in Basra governorate / Southern Iraq on some organs including testes, liver and kidneys. Results showed the ability of alga P. limnetica to produce anatoxin-a to reach 35 µg/g D.W. Three groups of laboratory mice (n=8) were injected intraperitonial daily for 15 days under laboratory conditions with (1)-Normal saline (Control group), (2) - low dose group with purified anatoxin-a = 0.5 µg/L and (3) - high dose group =1 µg/L). Purified toxin showed many histopathological effects at low and high doses and the effects increase with increasing dose. The testes showed a decreased number of elongated spermatids in central tubules in low dose group, while in high dose group, good evidence of the gross morphological effect of seminiferous tubules is seen which appear devoid of elongated spermatids in most tubules with predominant rounded spermatids. Liver cells in low dose appear more eosinophilic and without nuclei with fatty degeneration of sinusoids and mild inflammatory infiltrate. The appearance of large vacuoles within hepatocytes, necrotic cells, sinusoids poorly visualized with increased inflammatory cells in liver tissue under high dose. Kidneys at low dose showed increased eosinophilia of renal tubules cells, mild degenerative changes, hemorrhage and decrease in size of the urinary capsule. However, extravasation of red cells, congestion, dilatation of capillaries and chronic inflammatory cells were found under high dose exposure. [ABSTRACT FROM AUTHOR]

The removal of cyanotoxins, microcystin-LR (MLR), anatoxin-a (ANA), and cylindrospermopsin (CYN) in Detroit River water (DRW) was investigated by ozone and advanced oxidation process (AOP). Enhanced removal of all cyanotoxins was observed with increasing applied ozone dose. The reactivity of cyanotoxins with ozone followed the order of MLR>CYN>ANA in DRW at its natural pH of 7.6, as indicated by the calculated pseudo first-order oxidation coefficients (kd) of 17.5, 7.41, and 2.03 L/mg, respectively. The most efficient oxidations of MLR, CYN, and ANA with ozone in DRW were obtained at pH 5, 7.6, and 10, respectively, with kd values of 29.5, 7.41, and 4.65 L/mg. Ozone-based AOP with hydrogen peroxide did not make a significant difference in the removal of cyanotoxins compared to the application of ozone alone. [ABSTRACT FROM AUTHOR]

Cyanobacterial blue–green algal toxins are produced by harmful algal blooms (HABs). Most species of phytoplankton are not harmful, but excessive amounts of certain HAB taxa can cause harm to human and animal health, aquatic ecosystems and local economies. To investigate the prevalence of cylindrospermopsin (CYN) and anatoxin-a (ANA) in Singapore's reservoirs, a hazard analysis was initiated to profile the CYN and ANA levels present. Water samples from 17 reservoirs were monitored monthly over a 12-month period (November 2012–October 2013). Analyses were conducted by liquid chromatography–tandem mass spectrometry (LC-MS/MS) using a triple-stage quadrupole mass spectrometer with a turbo-assisted ion spray source. CYN was more prevalent than ANA. Intracellular CYN concentrations exceeded 0.4 μg L–1 in 6 of 17 man-made reservoirs surveyed, and slightly exceeded the provisional CYN drinking water guidelines of 1 μg L–1 (National Health and Medical Research Council and National Resource Management Ministerial Council 2011) on one occasion (1.1 μg L–1, July 2013) in one reservoir. The dominant cyanobacteria genera during that period were Cylindrospermopsis , Planktolyngbya , Pseudanabaena and Microcystis. For ANA, all 17 reservoirs had concentrations below 0.1 μg L–1. Based on random forest analysis, the most important environmental factors affecting CYN concentrations were total nitrogen (most important), nitrate, total phosphorus and Cylindrospermopsis counts (least important). The findings of this study indicate that reducing total nitrogen concentrations may be useful in minimising CYN concentrations in tropical reservoirs. Cyanobacterial 'blue–green algal' toxins are produced by some harmful algal blooms (HABs). To investigate the prevalence of cylindrospermopsin (CYN) and anatoxin-a (ANA) in Singapore's reservoirs, a hazard analysis was initiated to profile the CYN and ANA levels present. The dominant cyanobacteria genera during that 12-month period (November 2012–2013) were Cylindrospermopsis , Planktolyngbya , Pseudanabaena and Microcystis. [ABSTRACT FROM AUTHOR]