Analyzing the neurotoxic effects of anatoxin-a and saxitoxin in zebrafish larvae.

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.

A cohesive Microcoleus strain cluster causes benthic cyanotoxic blooms in rivers worldwide.

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.

Risk quick sketch: Soil captured most anatoxin-a and microcystin-RR rather than cylindrospermopsin and microcystin-LA/-LY.

Cyanobacteria proliferate in warm, nutrient-rich environments and release toxic secondary metabolites into natural waters. Using cyanotoxin-contaminated water to irrigate crops could expose humans and biota, but the risk may be low if agricultural soils can sorb and retain cyanotoxins. In this report, we compared the sorption and desorption capacities of multi-class cyanotoxins/anabaenopeptins in soils of variable properties with a batch sorption procedure. The target compounds were anabaenopeptin-A, anabaenopeptin-B, anatoxin-a, cylindrospermopsin, and microcystins -LR, -RR, -LA, -LY, -LW, and -LF. Based on solid-liquid distribution coefficients (Kd), we classified cylindrospermopsin and microcystin-LA/-LY as "very low sorptivity", anabaenopeptin-A, -B and microcystin-LR, -LF, and -LW as "low sorptivity", and anatoxin-a and microcystin-RR as "medium sorptivity". We remain concerned about irrigating agricultural land with water contaminated with high levels of CYN and MC-LA/-LY because of their relatively low affinity and high desorption proportion in soils. The results also suggest that soil sorption can be an effective immobilization pathway for anatoxin-a and microcystin-RR. The generated data will be useful for prioritizing research on the most bioavailable cyanotoxins/anabaenopeptins that are likely to be released by the soil matrix, for environmental risk assessment.

Inhibitory effects of toxic Dolichospermum flos-aquae and anatoxin-a on inducible defenses of Daphnia magna.

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.

Analytical Methods for Anatoxin-a Determination: A Review.

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.

Proteome changes in larval zebrafish (Danio rerio) and fathead minnow (Pimephales promelas) exposed to (±) anatoxin-a.

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.

A systematic review on guanitoxin: General characteristics and ecological risks.

Guanitoxin (GNT) is a potent cyanotoxin, with a relatively low number of publications (n = 51) compared to other cyanotoxins. Among the published studies, 35 % were on the effect of the toxin in animals, mainly in rodents and in vitro testing, followed by studies that identified species of cyanobacteria that produce GNT in aquatic systems and consequently accidental poisoning in wild and domestic animals (27 %). Studies that developed or tested methods for identifying the molecule, based on colorimetric and analytical techniques, represented 14 %, while 8 % were on GNT biosynthesis. Review articles and chemical isolation (6 %) and on the stability of the molecule (4 %) were the topics with the lowest number of publications. The results show the occurrence of GNT was identified mainly in eutrophic environments with a higher incidence in the American continent. Chemical characteristics of the molecule, such as short half-life in the environment, instability in solutions with alkaline pH values, temperature >23 °C, added to the lack of an analytical standard, are factors that make it difficult to identify and quantify it. However, GNT monitoring can be performed using LC-MS-MRM methods or genes specific to the newly discovered molecule.

An exploration of the binding prediction of anatoxin-a and atropine to acetylcholinesterase enzyme using multi-level computer simulations.

Acetylcholinesterase (AChE) is crucial for the breakdown of acetylcholine to acetate and choline, while the inhibition of AChE by anatoxin-a (ATX-a) results in severe health complications. This study explores the structural characteristics of ATX-a and its interactions with AChE, comparing to the reference molecule atropine for binding mechanisms. Molecular docking simulations reveal strong binding affinity of both ATX-a and atropine to AChE, interacting effectively with specific amino acids in the binding site as potential inhibitors. Quantitative assessment using the MM-PBSA method demonstrates a significantly negative binding free energy of -81.659 kJ mol-1for ATX-a, indicating robust binding, while atropine exhibits a stronger binding affinity with a free energy of -127.565 kJ mol-1. Umbrella sampling calculates the ΔGbindvalues to evaluate binding free energies, showing a favorable ΔGbindof -36.432 kJ mol-1for ATX-a and a slightly lower value of -30.12 kJ mol-1for atropine. This study reveals the dual functionality of ATX-a, acting as both a nicotinic acetylcholine receptor agonist and an AChE inhibitor. Remarkably, stable complexes form between ATX-a and atropine with AChE at its active site, exhibiting remarkable binding free energies. These findings provide valuable insights into the potential use of ATX-a and atropine as promising candidates for modulating AChE activity.

Draft genome sequence of the anatoxin-a producing cyanobacterium Tychonema bourrellyi B0820 isolated from the epilimnion of the deep Alpine Lake Garda.

We report the draft genome sequence of strain B0820 of the cyanobacterium Tychonema bourrellyi isolated from the epilimnion of Lake Garda and assembled from a metagenome of a non-axenic culture. The strain analyzed was shown to produce anatoxin-a, a potent neurotoxin that can cause fatal intoxication in exposed organisms.

First Confirmed Case of Canine Mortality Due to Dihydroanatoxin-a in Central Texas, USA.

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.

Liquid chromatography-high-resolution tandem mass spectrometry of anatoxins, including new conjugates and reduction products.

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,ß-unsaturated ketone functional group of anatoxin-a (ATX) and homoanatoxin-a (hATX), analogous to the conjugation chemistry of microcystins, which contain similar α,ß-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.

In Vitro Mutagenic and Genotoxic Assessment of Anatoxin-a Alone and in Combination with Cylindrospermopsin.

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.

Present and future potential role of toxin-producing Synechococcus in the tropical region.

As anthropogenic induced temperature rises and nutrient loadings increase in fresh and brackish environments, the ecological function of the phytoplankton community is expected to favour the picocyanobacteria, of the genus Synechococcus. Synechococcus is already a ubiquitous cyanobacterium found in both freshwater and marine environments, notwithstanding that the toxigenic species still remains unexplored in many freshwaters. Their fast growth rate and their ability to produce toxins make Synechococcus a potential dominant player in harmful algal blooms under climate change scenarios. This study examines the responses of a novel toxin-producing Synechococcus (i.e., one belonging to a freshwater clade; the other belonging to a brackish clade) to environmental changes that reflect climate change effects. We conducted a series of controlled experiments under present and predicted future temperatures, as well as under various N and P nutrients loadings. Our findings highlight how Synechococcus can be altered by the differing reactions to increasing temperature and nutrients, which resulted in considerable variations in cell abundance, growth rate, death rate, cellular stoichiometry and toxin production. Synechococcus had the highest growth observed at 28 °C, and further increases in temperature resulted in a decline for both fresh and brackish waters. Cellular stoichiometry was also altered, where more nitrogen (N) per cell was required, and the plasticity of N:P was more severe for the brackish clade. However, Synechococcus become more toxic under future scenario. Anatoxin-a (ATX) saw the greatest spike when temperature was at 34 °C especially under P-enrichment conditions. In contrast, Cylindrospermopsin (CYN) was promoted at the lowest tested temperature (25 °C) and under N-limitation. Overall, both temperature and external nutrients are the dominant control over Synechococcus toxins production. A model was also created to assess Synechococcus toxicity to zooplankton grazing. Zooplankton grazing was reduced by two folds under nutrient limitation, but temperature accounted for very insignificant change.

Cyanotoxins dissipation in soil: Evidence from microcosm assays.

Cyanobacteria proliferate in warm, nutrient-rich environments, and release cyanotoxins into natural waters. If cyanotoxin-contaminated water is used to irrigate agricultural crops, this could expose humans and other biota to cyanotoxins. However, cyanotoxins may be degraded by the diverse microbial consortia, be adsorbed or otherwise dissipate in agricultural soil. This study investigates the disappearance and transformation of 9 cyanotoxins in controlled soil microcosms after 28 d. Six soil types were exposed to factorial combinations of light, redox conditions and microbial activity that influenced the recovery of anabaenopeptin-A (AP-A), anabaenopeptin-B (AP-B), anatoxin-a (ATX-a), cylindrospermopsin (CYN), and the microcystin (MC) congeners -LR, -LA, -LY, -LW, and -LF. Cyanotoxins estimated half-lives were from hours to several months, depending on the compound and soil conditions. Cyanotoxins were eliminated via biological reactions in aerobic and anaerobic soils, although anaerobic conditions accelerated the biological dissipation of ATX-a, CYN and APs. ATX-a was sensitive to photolytic degradation, but CYN, and MCs were not reduced through photochemical transformation. MC-LR and -LA were recovered after exposure to light, redox conditions and low microbial activity, suggesting that they persisted in extractable forms, compared to other cyanotoxins in soil. Cyanotoxin degradation products were identified using high-resolution mass spectrometry, revealing their potential degradation pathways in soil.

Removal of saxitoxin and anatoxin-a by PAC in the presence and absence of microcystin-LR and/or cyanobacterial cells.

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.

Risk assessment of cyanobacteria toxic metabolites on freshwater ecosystems applying molecular methods.

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.

Elevated CO2 significantly increases N2 fixation, growth rates, and alters microcystin, anatoxin, and saxitoxin cell quotas in strains of the bloom-forming cyanobacteria, Dolichospermum.

The effect of rising CO2 levels on cyanobacterial harmful algal blooms (CHABs) is an emerging concern, particularly within eutrophic ecosystems. While elevated pCO2 has been associated with enhanced growth rates of some cyanobacteria, few studies have explored the effect of CO2 and nitrogen availability on diazotrophic (N2-fixing) cyanobacteria that produce cyanotoxins. Here, the effects of elevated CO2 and fixed nitrogen (NO3-) availability on the growth rates, toxin production, and N2 fixation of microcystin, saxitoxin, and anatoxin-a - producing strains of the genus Dolichospermum were quantified. Growth rates of all Dolichospermum spp. were significantly increased by CO2 or both CO2 and NO3- with rates being highest in treatments with the highest levels of CO2 and NO3-for all strains. While NO3- suppressed N2 fixation, diazotrophy significantly increased when NO3--enriched Dolichospermum spp. were supplied with higher CO2 compared to cultures grown under lower CO2 levels. This suggests that diazotrophy will play an increasingly important role in N cycling in CO2-enriched, eutrophic lentic systems. NO3- significantly increased quotas of the N-rich cyanotoxins, microcystin and saxitoxin, at ambient and enriched CO2 levels, respectively. In contrast, elevated CO2 significantly decreased cell quotas of microcystin and saxitoxin, but significantly increased cell quotas of the N-poor cyanotoxin, anatoxin. N2 fixation was significantly negatively and positively correlated with quotas of N-rich and N-poor cyanotoxins, respectively. Findings suggest cellular quotas of N-rich toxins (microcystin and saxitoxin) may be significantly reduced, or cellular quotas of N-poor toxins (anatoxin) may be significantly enhanced, under elevated CO2 conditions during diazotrophic cyanobacterial blooms. Finally, in the future, ecosystems that experience combinations of excessive N loading and CO2 enrichment may become more prone to toxic blooms of Dolichospermum.

Toxic Effects Produced by Anatoxin-a under Laboratory Conditions: A Review.

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.

Confirmation Using Triple Quadrupole and High-Resolution Mass Spectrometry of a Fatal Canine Neurotoxicosis following Exposure to Anatoxins at an Inland Reservoir.

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.

Cyanobacteria and Their Metabolites in Mono- and Polidominant Shallow Eutrophic Temperate Lakes.

Monodominant (one species dominates) or polidominant (multiple species dominate) cyanobacterial blooms are pronounced in productive freshwater ecosystems and pose a potential threat to the biota due to the synthesis of toxins. Seasonal changes in cyanobacteria species and cyanometabolites composition were studied in two shallow temperate eutrophic lakes. Data on cyanobacteria biomass and diversity of dominant species in the lakes were combined with chemical and molecular analyses of fifteen potentially toxin-producing cyanobacteria species (248 isolates from the lakes). Anatoxin-a, saxitoxin, microcystins and other non-ribosomal peptides formed the diverse profiles in monodominant (Planktothrix agardhii) and polidominant (Aphanizomenon gracile, Limnothrix spp. and Planktolyngbya limnetica) lakes. However, the harmfulness of the blooms depended on the ability of the dominant species to synthesize cyanometabolites. It was confirmed that P. agardhii produced a greater amount and diverse range of MCs and other NRPs. In the polidominant lake, isolates of the co-dominant A. gracile, L. planctonica and P. limnetica synthesized no or only small amounts of cyanometabolites. In general, the profile of cyanometabolites was greater in cyanobacteria isolates than in environmental samples, indicating a high potential for toxic cyanobacteria bloom.