Comparative toxicology of algal cell extracts and pure cyanotoxins: insights into toxic effects and mechanisms of harmful cyanobacteria Raphidiopsis raciborskii.

Ongoing research on cyanotoxins, driven by the socioeconomic impact of harmful algal blooms, emphasizes the critical necessity of elucidating the toxicological profiles of algal cell extracts and pure toxins. This study comprehensively compares Raphidiopsis raciborskii dissolved extract (RDE) and cylindrospermopsin (CYN) based on Daphnia magna assays. Both RDE and CYN target vital organs and disrupt reproduction, development, and digestion, thereby causing acute and chronic toxicity. Disturbances in locomotion, reduced behavioral activity, and weakened swimming capability in D. magna have also been reported for both RDE and CYN, indicating the insufficiency of conventional toxicity evaluation parameters for distinguishing between the toxic effects of algal extracts and pure cyanotoxins. Additionally, chemical profiling revealed the presence of highly active tryptophan-, humic acid-, and fulvic acid-like fluorescence compounds in the RDE, along with the active constituents of CYN, within a 15-day period, demonstrating the chemical complexity and dynamics of the RDE. Transcriptomics was used to further elucidate the distinct molecular mechanisms of RDE and CYN. They act diversely in terms of cytotoxicity, involving oxidative stress and response, protein content, and energy metabolism, and demonstrate distinct modes of action in neurofunctions. In essence, this study underscores the distinct toxicity mechanisms of RDE and CYN and emphasizes the necessity for context- and objective-specific toxicity assessments, advocating nuanced approaches to evaluate the ecological and health implications of cyanotoxins, thereby contributing to the precision of environmental risk assessments.

Burden of recreational water illness due to exposure to cyanobacteria and their toxins in freshwater beaches in Canada: protocol of a prospective cohort study.

INTRODUCTION: Cyanobacterial blooms are increasingly common in freshwater sources used for swimming and other recreational water contact activities in Canada. Many species of cyanobacteria can produce toxins that affect human and animal health, but there are limited data on the risk of illness associated with water contact at impacted beaches. METHODS AND ANALYSIS: This study will investigate the incidence of recreational water illness due to exposure to cyanobacterial blooms and their toxins in four targeted and popular freshwater beaches in Ontario, Manitoba and Nova Scotia, Canada. A prospective cohort design and One Health approach will be used. On-site recruitment of recreational water users will be conducted at two beaches per year during the summers of 2024 and 2025. The population of interest includes recreational water users of any age and their pet dogs. After enrolment, an in-person survey will determine beach exposures and confounding factors, and a 3-day follow-up survey will ascertain any acute illness outcomes experienced by participants or their dogs. The target sample size is 2500 recreational water users. Water samples will be taken each recruitment day and analysed for cyanobacterial indicators (pigments), cell counts and toxin levels. Bayesian regression analysis will be conducted to estimate the association with water contact, cyanobacterial levels and risks of different acute illness outcomes. ETHICS AND DISSEMINATION: This study has been approved by the Toronto Metropolitan University Research Ethics Board (REB 2023-461). Study results will be published in a peer-reviewed journal and as infographics on a project website.

Bacterial community and cyanotoxin gene distribution of the Winam Gulf, Lake Victoria, Kenya.

The Winam Gulf (Kenya) is frequently impaired by cyanobacterial harmful algal blooms (cHABs) due to inadequate wastewater treatment and excess agricultural nutrient input. While phytoplankton in Lake Victoria have been characterized using morphological criteria, our aim is to identify potential toxin-producing cyanobacteria using molecular approaches. The Gulf was sampled over two successive summer seasons, and 16S and 18S ribosomal RNA gene sequencing was performed. Additionally, key genes involved in production of cyanotoxins were examined by quantitative PCR. Bacterial communities were spatially variable, forming distinct clusters in line with regions of the Gulf. Taxa associated with diazotrophy were dominant near Homa Bay. On the eastern side, samples exhibited elevated cyrA abundances, indicating genetic capability of cylindrospermopsin synthesis. Indeed, near the Nyando River mouth in 2022, cyrA exceeded 10 million copies L-1 where there were more than 6000 Cylindrospermopsis spp. cells mL-1. In contrast, the southwestern region had elevated mcyE gene (microcystin synthesis) detections near Homa Bay where Microcystis and Dolichospermum spp. were observed. These findings show that within a relatively small embayment, composition and toxin synthesis potential of cHABs can vary dramatically. This underscores the need for multifaceted management approaches and frequent cyanotoxin monitoring to reduce human health impacts.

Cyanotoxin cylindrospermopsin disrupts lipid homeostasis and metabolism in a 3D in vitro model of the human liver.

Cylindrospermopsin, a potent hepatotoxin produced by harmful cyanobacterial blooms, poses environmental and human health concerns. We used a 3D human liver in vitro model based on spheroids of HepG2 cells, in combination with molecular and biochemical assays, automated imaging, targeted LC-MS-based proteomics, and lipidomics, to explore cylindrospermopsin effects on lipid metabolism and the processes implicated in hepatic steatosis. Cylindrospermopsin (1 µM, 48 h) did not significantly affect cell viability but partially reduced albumin secretion. However, it increased neutral lipid accumulation in HepG2 spheroids while decreasing phospholipid levels. Simultaneously, cylindrospermopsin upregulated genes for lipogenesis regulation (SREBF1) and triacylglycerol synthesis (DGAT1/2) and downregulated genes for fatty acid synthesis (ACLY, ACCA, FASN, SCD1). Fatty acid uptake, oxidation, and lipid efflux genes were not significantly affected. Targeted proteomics revealed increased levels of perilipin 2 (adipophilin), a major hepatocyte lipid droplet-associated protein. Lipid profiling quantified 246 lipid species in the spheroids, with 28 significantly enriched and 15 downregulated by cylindrospermopsin. Upregulated species included neutral lipids, sphingolipids (e.g., ceramides and dihexosylceramides), and some glycerophospholipids (phosphatidylethanolamines, phosphatidylserines), while phosphatidylcholines and phosphatidylinositols were mostly reduced. It suggests that cylindrospermopsin exposures might contribute to developing and progressing towards hepatic steatosis or metabolic dysfunction-associated steatotic liver disease (MASLD).

Temperature rise and its influence on the toxic effects caused by cyanotoxins in a neotropical catfish.

Potentially toxic cyanobacterial blooms (cyanoHABs) have become a problem in public water supply reservoirs. Temperature rise caused by climate change can increase the frequency and intensity of blooms, which may influence the cyanotoxins concentration in the environment. This study aimed to evaluate the effect of the temperature on the responses of a Neotropical catfish exposed to a neurotoxin-rich cyanobacterial crude extract (Raphidiopsis raciborskii T3). Juveniles of Rhamdia quelen were exposed to four treatments, based on study data: control at 25 °C (C25), control at 30 °C (C30), crude extract equivalent to 105 cells.mL-l of R. raciborskii at 25 °C (CE25) and 30 °C (CE30). After 96 h of exposure, the fish were anesthetized and blood was taken. After euthanasia, the gill, posterior kidney, brain, muscle, liver and gonad were sampled for hematological, biochemical, genotoxic and histopathological biomarker analysis. Liver was sampled for proteomic analysis for identification of proteins related to energy production. Water samples were collected at the beginning and the end of the experiment for neurotoxins quantification. Different parameters in both males and females were altered at CE25, evidencing the effects of neurotoxins in freshwater fish. At CE30, a water warming scenario, more effects were observed in females than at 25 °C, such as activation of saxitoxin metabolism pathway and genotoxicity. More damage to macromolecules was observed in females at the higher temperature, demonstrating that the increase in temperature can aggravate the toxicity of neurotoxins produced by R. raciborskii T3.

Cyanotoxin Occurrence and Diversity in 98 Cyanobacterial Blooms from Swedish Lakes and the Baltic Sea.

The Drinking Water Directive (EU) 2020/2184 includes the parameter microcystin LR, a cyanotoxin, which drinking water producers need to analyze if the water source has potential for cyanobacterial blooms. In light of the increasing occurrences of cyanobacterial blooms worldwide and given that more than 50 percent of the drinking water in Sweden is produced from surface water, both fresh and brackish, the need for improved knowledge about cyanotoxin occurrence and cyanobacterial diversity has increased. In this study, a total of 98 cyanobacterial blooms were sampled in 2016-2017 and identified based on their toxin production and taxonomical compositions. The surface water samples from freshwater lakes throughout Sweden including brackish water from eight east coast locations along the Baltic Sea were analyzed for their toxin content with LC-MS/MS and taxonomic composition with 16S rRNA amplicon sequencing. Both the extracellular and the total toxin content were analyzed. Microcystin's prevalence was highest with presence in 82% of blooms, of which as a free toxin in 39% of blooms. Saxitoxins were found in 36% of blooms in which the congener decarbamoylsaxitoxin (dcSTX) was detected for the first time in Swedish surface waters at four sampling sites. Anatoxins were most rarely detected, followed by cylindrospermopsin, which were found in 6% and 10% of samples, respectively. As expected, nodularin was detected in samples collected from the Baltic Sea only. The cyanobacterial operational taxonomic units (OTUs) with the highest abundance and prevalence could be annotated to Aphanizomenon NIES-81 and the second most profuse cyanobacterial taxon to Microcystis PCC 7914. In addition, two correlations were found, one between Aphanizomenon NIES-81 and saxitoxins and another between Microcystis PCC 7914 and microcystins. This study is of value to drinking water management and scientists involved in recognizing and controlling toxic cyanobacteria blooms.

Comprehensive analysis of cyanobacterial secondary metabolites distribution and toxicity in urban water bodies.

This study addresses the increasing concern regarding cyanotoxin contamination of water bodies, highlighting the diversity of these toxins and their potential health implications. Cyanobacteria, which are prevalent in aquatic environments, produce toxic metabolites, raising concerns regarding human exposure and associated health risks, including a potential increase in cancer risk. Although existing research has primarily focused on well-known cyanotoxins, recent technological advancements have revealed numerous unknown cyanotoxins, necessitating a comprehensive assessment of multiple toxin categories. To enhance the cyanotoxin databases, we optimized the CyanoMetDB cyanobacterial secondary metabolites database by incorporating secondary fragmentation patterns using the Mass Frontier fragmentation data prediction software. Water samples from diverse locations in Shanghai were analyzed using high-resolution mass spectrometry. Subsequently, the toxicity of cyanobacterial metabolites in the water samples was examined through acute toxicity assays using the crustacean Thamnocephalus platyurus. After 24 h of exposure, the semi-lethal concentrations (LC50) of the water samples ranged from 0.31 mg L-1 to 1.78 mg L-1 (MC-LR equivalent concentration). Our findings revealed a critical correlation between the overall concentration of cyanobacterial metabolites and toxicity. The robust framework and insights of this study underscore the need for an inclusive approach to water quality management, emphasizing continuous efforts to refine detection methods and comprehend the broader ecological impact of cyanobacterial blooms on aquatic ecosystems.

Cylindrospermopsin enhances the conjugative transfer of plasmid-mediated multi-antibiotic resistance genes through glutathione biosynthesis inhibition.

Cylindrospermopsin (CYN), a cyanobacterial toxin, has been detected in the global water environment. However, information concerning the potential environmental risk of CYN is limited, since the majority of previous studies have mainly focused on the adverse health effects of CYN through contaminated drinking water. The present study reported that CYN at environmentally relevant levels (0.1-100 µg/L) can significantly enhance the conjugative transfer of RP4 plasmid in Escherichia coli genera, wherein application of 10 µg/L of CYN led to maximum fold change of ∼6.5- fold at 16 h of exposure. Meanwhile, evaluation of underlying mechanisms revealed that environmental concentration of CYN exposure could increase oxidative stress in the bacterial cells, resulting in ROS overproduction. In turn, this led to an upregulation of antioxidant enzyme-related genes to avoid ROS attack. Further, inhibition of the synthesis of glutathione (GSH) was also detected, which led to the rapid depletion of GSH in cells and thus triggered the SOS response and promoted the conjugative transfer process. Increase in cell membrane permeability, upregulation of expression of genes related to pilus generation, ATP synthesis, and RP4 gene expression were also observed. These results highlight the potential impact on the spread of antimicrobial resistance in water environments.

Neuronal control of microglia through the mitochondria.

The microbial toxin ß-N-methylamino-L-alanine (BMAA), which is derived from cyanobacteria, targets neuronal mitochondria, leading to the activation of neuronal innate immunity and, consequently, neurodegeneration. Although known to modulate brain inflammation, the precise role of aberrant microglial function in the neurodegenerative process remains elusive. To determine if neurons signal microglial cells, we treated primary cortical neurons with BMAA and then co-cultured them with the N9 microglial cell line. Our observations indicate that microglial cell activation requires initial neuronal priming. Contrary to what was observed in cortical neurons, BMAA was not able to activate inflammatory pathways in N9 cells. We observed that microglial activation is dependent on mitochondrial dysfunction signaled by BMAA-treated neurons. In this scenario, the NLRP3 pro-inflammatory pathway is activated due to mitochondrial impairment in N9 cells. These results demonstrate that microglia activation in the presence of BMAA is dependent on neuronal signaling. This study provides evidence that neurons may trigger microglia activation and subsequent neuroinflammation. In addition, we demonstrate that microglial activation may have a protective role in ameliorating neuronal innate immune activation, at least in the initial phase. This work challenges the current understanding of neuroinflammation by assigning the primary role to neurons.

Microbial Diversity Impacts Non-Protein Amino Acid Production in Cyanobacterial Bloom Cultures Collected from Lake Winnipeg.

Lake Winnipeg in Manitoba, Canada is heavily impacted by harmful algal blooms that contain non-protein amino acids (NPAAs) produced by cyanobacteria: N-(2-aminoethyl)glycine (AEG), ß-aminomethyl-L-alanine (BAMA), ß-N-methylamino-L-alanine (BMAA), and 2,4-diaminobutyric acid (DAB). Our objective was to investigate the impact of microbial diversity on NPAA production by cyanobacteria using semi-purified crude cyanobacterial cultures established from field samples collected by the Lake Winnipeg Research Consortium between 2016 and 2021. NPAAs were detected and quantified by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) using validated analytical methods, while Shannon and Simpson alpha diversity scores were determined from 16S rRNA metagenomic sequences. Alpha diversity in isolate cultures was significantly decreased compared to crude cyanobacterial cultures (p < 0.001), indicating successful semi-purification. BMAA and AEG concentrations were higher in crude compared to isolate cultures (p < 0.0001), and AEG concentrations were correlated to the alpha diversity in cultures (r = 0.554; p < 0.0001). BAMA concentrations were increased in isolate cultures (p < 0.05), while DAB concentrations were similar in crude and isolate cultures. These results demonstrate that microbial community complexity impacts NPAA production by cyanobacteria and related organisms.

Cyanobacterial Harmful Algal Mats (CyanoHAMs) in tropical rivers of central Mexico and their potential risks through toxin production.

Cyanobacteria inhabiting lotic environments have been poorly studied and characterized in Mexico, despite their potential risks from cyanotoxin production. This article aims to fill this knowledge gap by assessing the importance of benthic cyanobacteria as potential cyanotoxin producers in central Mexican rivers through: (i) the taxonomic identification of cyanobacteria found in these rivers, (ii) the environmental characterization of their habitats, and (iii) testing for the presence of toxin producing genes in the encountered taxa. Additionally, we introduce and discuss the use of the term "CyanoHAMs" for lotic water environments. Populations of cyanobacteria were collected from ten mountain rivers and identified using molecular techniques. Subsequently, these taxa were evaluated for genes producing anatoxins and microcystins via PCR. Through RDA analyses, the collected cyanobacteria were grouped into one of three categories based on their environmental preferences for the following: (1) waters with high ionic concentrations, (2) cold-temperate waters, or (3) waters with high nutrient enrichment. Populations from six locations were identified to genus level: Ancylothrix sp., Cyanoplacoma sp., and Oxynema sp. The latter was found to contain the gene that produces anatoxins and microcystins in siliceous rivers, while Oxynema tested positive for the gene that produces microcystins in calcareous rivers. Our results suggest that eutrophic environments are not necessarily required for toxin-producing cyanobacteria. Our records of Compactonostoc, Oxynema, and Ancylothrix represent the first for Mexico. Four taxa were identified to species level: Wilmottia aff. murrayi, Nostoc tlalocii, Nostoc montejanii, and Dichothrix aff. willei, with only the first testing positive using PCR for anatoxin and microcystin-producing genes in siliceous rivers. Due to the differences between benthic growths with respect to planktonic ones, we propose the adoption of the term Cyanobacterial Harmful Algal Mats (CyanoHAMs) as a more precise descriptor for future studies.

Evaluation of cyanotoxin L-BMAA effect on α-synuclein and TDP43 proteinopathy.

The complex interplay between genetic and environmental factors is considered the cause of neurodegenerative diseases including Parkinson's disease (PD) and Amyotrophic Lateral Sclerosis (ALS). Among the environmental factors, toxins produced by cyanobacteria have received much attention due to the significant increase in cyanobacteria growth worldwide. In particular, L-BMAA toxin, produced by diverse taxa of cyanobacteria, dinoflagellates and diatoms, has been extensively correlated to neurodegeneration. The molecular mechanism of L-BMAA neurotoxicity is still cryptic and far from being understood. In this research article, we have investigated the molecular pathways altered by L-BMAA exposure in cell systems, highlighting a significant increase in specific stress pathways and an impairment in autophagic processes. Interestingly, these changes lead to the accumulation of both α-synuclein and TDP43, which are correlated with PD and ALS proteinopathy, respectively. Finally, we were able to demonstrate specific alterations of TDP43 WT or pathological mutants with respect to protein accumulation, aggregation and cytoplasmic translocation, some of the typical features of both sporadic and familial ALS.

Cyanotoxins in food: Exposure assessment and health impact.

The intricate nature of cyanotoxin exposure through food reveals a complex web of risks and uncertainties in our dietary choices. With the aim of starting to unravel this intricate nexus, a comprehensive review of 111 papers from the past two decades investigating cyanotoxin contamination in food was undertaken. It revealed a widespread occurrence of cyanotoxins in diverse food sources across 31 countries. Notably, 68% of the studies reported microcystin concentrations exceeding established Tolerable Daily Intake levels. Cyanotoxins were detected in muscles of many fish species, and while herbivorous fish exhibited the highest recorded concentration, omnivorous species displayed a higher propensity for cyanotoxin accumulation, exemplified by Oreochromis niloticus. Beyond fish, crustaceans and bivalves emerged as potent cyanotoxin accumulators. Gaps persist regarding contamination of terrestrial and exotic animals and their products, necessitating further exploration. Plant contamination under natural conditions remains underreported, yet evidence underscores irrigation-driven cyanotoxin accumulation, particularly affecting leafy vegetables. Finally, cyanobacterial-based food supplements often harbored cyanotoxins (57 % of samples were positive) warranting heightened scrutiny, especially for Aphanizomenon flos-aquae-based products. Uncertainties surround precise concentrations due to methodological variations (chemical and biochemical) and extraction limitations, along with the enigmatic fate of toxins during storage, processing, and digestion. Nonetheless, potential health consequences of cyanotoxin exposure via contaminated food include gastrointestinal and neurological disorders, organ damage (e.g. liver, kidneys, muscles), and even elevated cancer risks. While microcystins received significant attention, knowledge gaps persist regarding other cyanotoxins' accumulation, exposure, and effects, as well as combined exposure via multiple pathways. Intriguing and complex, cyanotoxin exposure through food beckons further research for our safer and healthier diets.

Presence of the neurotoxin ß-N-methylamino-L-alanine in irrigation water and accumulation in cereal grains with human exposure risk.

The present study demonstrates the presence of the neurotoxin ß-N-methylamino-L-alanine and its cyanobacterial producers in irrigation water and grains of some cereal plants from farmlands irrigated with Nile River water in Egypt. BMAA detected by LC-MS/MS in phytoplankton samples was found at higher concentrations of free form (0.84-11.4 µg L-1) than of protein-bound form (0.16-1.6 µg L-1), in association with the dominance of cyanobacteria in irrigation water canals. Dominant cyanobacterial species isolated from these irrigation waters including Aphanocapsa planctonica, Chroococcus minutus, Dolichospermum lemmermanni, Nostoc commune, and Oscillatoria tenuis were found to produce different concentrations of free (4.8-71.1 µg g-1 dry weight) and protein-bound (0.1-11.4 µg g-1 dry weight) BMAA. In the meantime, BMAA was also detected in a protein-bound form only in grains of corn (3.87-4.51 µg g-1 fresh weight) and sorghum (5.1-7.1 µg g-1 fresh weight) plants, but not in wheat grains. The amounts of BMAA accumulated in these grains correlated with BMAA concentrations detected in relevant irrigation water canals. The presence of BMAA in cereal grains would constitute a risk to human and animal health upon consumption of contaminated grains. The study, therefore, suggests continuous monitoring of BMAA and other cyanotoxins in irrigation waters and edible plants to protect the public against exposure to such potent toxins.

Impact of the neurotoxin ß-N-methylamino-L-alanine on the diatom Thalassiosira pseudonana using metabolomics.

The neurotoxin ß-N-methylamino-L-alanine (BMAA) has emerged as an environmental factor related to neurodegenerative diseases. BMAA is produced by various microorganisms including cyanobacteria and diatoms, in diverse ecosystems. In the diatom Phaeodactylum tricornutum, BMAA is known to inhibit growth. The present study investigated the impact of BMAA on the diatom Thalassiosira pseudonana by exposing it to different concentrations of exogenous BMAA. Metabolomics was predominantly employed to investigate the effect of BMAA on T. pseudonana, and MetaboAnalyst (https://www.metabo-analyst.ca/) was used to identify BMAA-associated metabolisms/pathways in T. pseudonana. Furthermore, to explore the unique response, specific metabolites were compared between treatments. When the growth was obstructed by BMAA, 17 metabolisms/pathways including nitrogen and glutathione (i.e. oxidative stress) metabolisms, were influenced in T. pseudonana. This study has further determined that 11 out of 17 metabolisms/pathways could be essentially affected by BMAA, leading to the inhibition of diatom growth.

Fungal removal of cyanotoxins in constructed wetlands: The forgotten degraders.

Harmful cyanobacterial blooms have increased globally, releasing hazardous cyanotoxins that threaten the safety of water resources. Constructed wetlands (CWs) are a nature-based and low-cost solution to purify and remove cyanotoxins from water. However, bio-mechanistic understanding of the biotransformation processes expected to drive cyanotoxin removal in such systems is poor, and primarily focused on bacteria. Thus, the present study aimed at exploring the fungal contribution to microcystin-LR and cylindrospermopsin biodegradation in CWs. Based on CW mesocosms, two experimental approaches were taken: a) amplicon sequencing studies were conducted to investigate the involvement of the fungal community; and b) CW fungal isolates were tested for their microcystin-LR and cylindrospermopsin degradation capabilities. The data uncovered effects of seasonality (spring or summer), cyanotoxin exposure, vegetation (unplanted, Juncus effusus or Phragmites australis) and substratum (sand or gravel) on the fungal community structure. Additionally, the arbuscular mycorrhizal fungus Rhizophagus and the endophyte Myrmecridium showed positive correlations with cyanotoxin removal. Fungal isolates revealed microcystin-LR-removal potentials of approximately 25 % in in vitro biodegradation experiments, while the extracellular chemical fingerprint of the cultures suggested a potential intracellular metabolization. The results from this study may help us understand the fungal contribution to cyanotoxin removal, as well as their ecology in CWs.

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.

Detection of Anatoxins in Human Urine by Liquid Chromatography Triple Quadrupole Mass Spectrometry and ELISA.

Harmful cyanobacterial blooms are becoming more common and persistent around the world. When in bloom, various cyanobacterial strains can produce anatoxins in high concentrations, which, unlike other cyanobacterial toxins, may be present in clear water. Potential human and animal exposures to anatoxins occur mainly through unintentional ingestion of contaminated algal mats and water. To address this public health threat, we developed and validated an LC-MS/MS method to detect anatoxins in human urine to confirm exposures. Pooled urine was fortified with anatoxin-a and dihydroanatoxin at concentrations from 10.0 to 500 ng/mL to create calibrators and quality control samples. Samples were diluted with isotopically labeled anatoxin and solvent prior to LC-MS/MS analysis. This method can accurately quantitate anatoxin-a with inter- and intraday accuracies ranging from 98.5 to 103% and relative standard deviations < 15%, which is within analytical guidelines for mass spectrometry methods. Additionally, this method qualitatively detects a common degradation product of anatoxin, dihydroanatoxin, above 10 ng/mL. We also evaluated a commercial anatoxin-a ELISA kit for potential diagnostic use; however, numerous false positives were detected from unexposed individual human urine samples. In conclusion, we have developed a method to detect anatoxins precisely and accurately in urine samples, addressing a public health area of concern, which can be applied to future exposure events.

Snapshot of cyanobacterial toxins in Pakistani freshwater bodies.

Cyanobacteria are known to produce diverse secondary metabolites that are toxic to aquatic ecosystems and human health. However, data about the cyanotoxins occurrence and cyanobacterial diversity in Pakistan's drinking water reservoirs is scarce. In this study, we first investigated the presence of microcystin, saxitoxin, and anatoxin in 12 water bodies using an enzyme-linked immunosorbent assay (ELISA). The observed cyanotoxin values for the risk quotient (RQ) determined by ELISA indicated a potential risk for aquatic life and human health. Based on this result, we made a more in-depth investigation with a subset of water bodies (served as major public water sources) to analyze the cyanotoxins dynamics and identify potential producers. We therefore quantified the distribution of 17 cyanotoxins, including 12 microcystin congeners using a high-performance liquid chromatography-high-resolution tandem mass spectrometry/mass spectrometry (HPLC-HRMS/MS). Our results revealed for the first time the co-occurrence of multiple cyanotoxins and the presence of cylindrospermopsin in an artificial reservoir (Rawal Lake) and a semi-saline lake (Kallar Kahar). We also quantified several microcystin congeners in a river (Panjnad) with MC-LR and MC-RR being the most prevalent and abundant. To identify potential cyanotoxin producers, the composition of the cyanobacterial community was characterized by shotgun metagenomics sequencing. Despite the noticeable presence of cyanotoxins, Cyanobacteria were not abundant. Synechococcus was the most abundant cyanobacterial genus found followed by a small amount of Anabaena, Cyanobium, Microcystis, and Dolichospermum. Moreover, when we looked at the cyanotoxins genes coverage, we never found a complete microcystin mcy operon. To our knowledge, this is the first snapshot sampling of water bodies in Pakistan. Our results would not only help to understand the geographical spread of cyanotoxin in Pakistan but would also help to improve cyanotoxin risk assessment strategies by screening a variety of cyanobacterial toxins and confirming that cyanotoxin quantification is not necessarily related to producer abundance.

Experimental accumulation and depuration kinetics and natural occurrence of microcystin-LR in basil (Ocimum basilicum L.).

Microcystin-LR (MC-LR) is a hepatotoxic metabolite that naturally occurs during some cyanobacterial blooms in eutrophic waterbodies, and irrigation of edible plants with MC-LR-contaminated water causes bioaccumulation of the toxin. However, sufficient information about accumulation and depuration mechanics in hydroculture-grown herb plants is still lacking. This work aimed at 1) investigating bioaccumulation and depuration of MC-LR in basil, 2) verifying the possible MC-LR detoxification mechanisms in the plant, and 3) detecting the natural occurrence of MC-LR in basil (n = 50) collected from the Belgian market. Basil plants grown in a hydroculture were exposed to MC-LR (5, 20, and 50 µg L-1) spiked in a Hoagland solution for seven days. MC-LR depuration was also studied by transferring the plants to a non-contaminated Hoagland solution after exposure to MC-LR for another seven days. MC-LR concentrations in Hoagland solution, basil leaves, and roots were quantified using a validated UHPLC-MS/MS method. In addition, ELISA and LC-HRMS (only basil leaves) were used for confirmation. The results showed an increase in the accumulated levels of MC-LR at higher exposure doses, with higher MC-LR levels in roots than in leaves for all the treatment conditions. For MC-LR depuration, significant reductions were observed in all the treatment conditions for roots only. No MC-LR conjugates, potentially related to metabolism, were detected by LC-HRMS. Finally, MC-LR was detected in one store-bought basil sample, representing the first occurrence of cyanotoxins in an edible crop from Belgium.