Microcystin Congeners Research Articles

Simultaneous electrochemical removal of three microcystin congeners and sulfamethoxazole in natural water

Microcystins (MCs), frequently detected in freshwater ecosystems, have raised significant human health and ecological concerns. New approaches are being developed to control and remove MCs. In this study, we examined factors influencing the efficacy of electrochemical oxidation as a means of control. Anode material (Pt/Ti, Ta2O5-IrO2/Ti, SnO2-SbO2/Ti, boron-doped diamond (BDD)/Si), anode surface area ratios and solution volumes, initial pollutant concentrations, and the co-existing antibiotic sulfamethoxazole (SMX) were investigated. MCs and SMX were dissolved in filtered Taihu Lake water to simulate the natural aquatic environment. The results showed that non-active anodes, lower initial concentration of MC, larger surface area ratio of anode to cathode, and smaller ratio of reaction solution volume to anode surface area could promote the degradation target pollutants. Under optimal conditions in this study, the degradation rates of MC-LR, MC-YR, MC-RR, and SMX each reached more than 90% within 6 hours, and the removal efficiency of MC-YR was the highest among three congeners. The effect of SMX on the degradation of MC congeners depended mainly on their concentration differences, such that when the initial concentration of SMX was one to two orders of magnitude lower than microcystin, the presence of SMX would promote the degradation of MCs. In contrast, when the initial concentration of SMX was higher than that of microcystin by approximately an order of magnitude, sulfamethoxazole would inhibit the degradation of MCs by between 4.6% and 24.5%. Ultra-high-performance liquid chromatography tandem mass spectrometry analysis revealed that the three MC congeners were electrochemically degraded through aromatic ring oxidation, alkene oxidation, and bond cleavage on the ADDA (3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid) side chain. Notably, the removal of MCs was accompanied by a decline in the hardness of the reaction water. This study provided insights into electrochemical degradation of microcystins and antibiotics in natural water, offering suggestions for its practical application.

Effects of Ciprofloxacin on the Production and Composition of Cellular Microcystins in Microcystis aeruginosa.

Antibiotics can affect the photosynthetic system of Microcystis, potentially altering the balance of carbon and nitrogen, which may influence the synthesis of different microcystin (MC) congeners. However, the regulatory mechanisms by which antibiotics affect the synthesis of various MC congeners in Microcystis remain unknown. In this study, the effects of ciprofloxacin (CIP) on the growth, carbon and nitrogen balance, amino acid composition, mcyB gene expression, and production of different MC congeners were investigated in two toxin-producing strains of Microcystis aeruginosa. The results show that CIP exposure significantly inhibited the growth of both strains, achieving an inhibition rate of 71.75% in FACHB-315 and 41.13% in FACHB-915 at 8 μg/L CIP by the end of the cultivation. The intracellular C:N ratio in FACHB-315 increased by 51.47%, while no significant change was observed in FACHB-915. The levels of leucine, tyrosine, and arginine, as identified and quantified by UPLC-MS/MS, were significantly altered at higher CIP concentrations, leading to a reduction in leucine percentage and a notable increase in tyrosine in both strains, which contributed to a reduction in MC-LR proportion and an increase in MC-RR and MC-YR proportion. Additionally, the expression of the mcyB gene was upregulated by as much as 5.57 times, indicating that antibiotic stress could enhance MC synthesis at the genetic level, contributing to the increased toxicity of cyanobacteria. These findings emphasize the significant role of CIP in the biochemical processes of M. aeruginosa, particularly in MC synthesis and composition, providing valuable insights into the ecological risks posed by antibiotics and harmful cyanobacteria.

Development and validation of a LC-MS method for the quantification of microcystins in fish for their monitoring in environmental and food context

Cyanobacterial blooms in aquatic ecosystems can result in the accumulation of cyanotoxins in other organisms. Fish can potentially accumulate large amounts of cyanotoxins through their direct interaction with contaminated water and the ingestion of cyanobacterial cells. As a human food source, this can result in a potential health risk. Investigating and monitoring this risk requires analytical methods sufficiently robust to be suitable for different fish species. This study, therefore, aimed to optimize and validate an UHPLC-MS/MS method for the quantification of eight microcystin congeners and nodularin in five different fish species. The limits of detection and quantification were 1 and 3 μg kg−1, respectively. The linearity of the method was very good, with calculated R2 values above 0.99. The mean recovery of the method was within the acceptable range of 70.0%–120.0 %, the repeatability was not higher than 12.6 %, and the highest intra-laboratory reproducibility was 18.7 %. The estimated range for the expanded measurement uncertainty was between 29 % and 58 %. Moreover, multiple fish from Flemish waters were analyzed to show the method's applicability. Multiple microcystin congeners were detected (up to 88.3 μg kg−1 sum of analyzed microcystins) in the liver and viscera of perch and sander. Small amounts of MC-LR and MC-YR (up to 6.1 μg kg−1) were also detected in perch muscle tissue.

Evaluation of Carbonized Corncobs for Removal of Microcystins and Nodularin-R from Water

Microcystins (MCs) and nodularins (NODs) are cyanotoxins that can be found in water bodies during cyanobacterial harmful algal blooms (cyanoHABs). Consumption of water contaminated with cyanotoxins leads to health risks for humans and animals. Herein, corncob-based biochar and activated carbon (AC) were initially investigated for the sorption of six common MC congeners (MC-RR, MC-YR, MC-LR, MC-LA, MC-LW, and MC-LF) and nodularin-R (NOD-R) from spiked water. Biochar was prepared by refluxing commercial corncob with HCl and heating it to 250, 300, or 350 °C. AC was prepared by chemical activation of corncob with H3PO4 at 500 °C under a nitrogen atmosphere. Low-temperature nitrogen adsorption measurements confirmed that H3PO4-AC has a higher specific surface area (≈1100 m2/g) and total pore volume (≈0.75 cm3/g) than biochar and commercial AC. H3PO4-AC showed the maximum efficacy, among all corncob-based sorbents, to remove MCs and NOD-R from water as confirmed by experiments that involved sample analyses by ultrahigh-pressure liquid chromatography-mass spectrometry (UHPLC-MS). The effect of natural organic matter (NOM) on the adsorption of MCs was checked by incubating sorbents with Lake Erie water collected during cyanoHABs from 2020 to 2022. The total concentration (extracellular and intracellular) of studied MC congeners ranged from 1.37 µg/L to 438.51 µg/L and 50 mg of H3PO4-AC completely removed them from 3 mL of lake water. The effect of water pH on cyanotoxin adsorption was studied at pH values of 5.5, 7.0, and 8.5 at both a lower (10 μg/L each) and a higher (50 μg/L each) toxin concentration. Removal was influenced by solution pH at both concentrations when using biochar, while only at higher toxin concentration when using H3PO4-AC. At higher MC and NOD-R concentrations, competitive adsorption was prominent, and overall, the adsorption increased at acidic pH (5.5). The study results suggest that processed corncobs can remove a significant amount of MCs and NOD-R from water, and the measured sorption capacity of H3PO4-AC was ~20 mg of MC-LR and NOD-R per g of this sorbent.

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.

Efficient removal of microcystin-LR from contaminated water using water-stable MIL-100(Fe) synthesized under HF-free conditions.

Cyanobacterial algal hepatotoxins, called microcystins (MCs), are a global health concern, necessitating research on effective removal methods from contaminated water bodies. In this study, we synthesized non-fluorine MIL-100(Fe) using an environmentally friendly room-temperature method and utilized it as an adsorbent to effectively remove microcystin-LR (MC-LR), which is the most toxic MC congener. MIL-100(Fe) was thoroughly characterized, and its adsorption process was investigated under various conditions. Results revealed rapid MC-LR adsorption, achieving 93% removal in just 5min, with the pseudo-second-order kinetic model indicating chemisorption as the primary mechanism. The Langmuir isotherm model demonstrated a monolayer sorption capacity of 232.6µgg-1 at room temperature, showing favorable adsorption. Furthermore, the adsorption capacity increased from 183µgg-1 at 20°C to 311µgg-1 at 40°C, indicating an endothermic process. Thermodynamic parameters supported MC-LR adsorption's spontaneous and feasible nature onto MIL-100(Fe). This study highlights MIL-100(Fe) as a promising method for effectively removing harmful biological pollutants, such as MC-LR, from contaminated water bodies in an environmentally friendly manner.

Fate of Planktothrix-derived toxins in aquatic food webs: A case study in Lake Mindelsee (Germany)

Blooms of the red, filamentous cyanobacterium Planktothrix rubescens occur frequently in pre-alpine lakes in Europe, often with concomitant toxic microcystin (MC) production. Trophic transfer of MCs has been observed in bivalves, fish, and zooplankton species, while uptake of MCs into Diptera species could facilitate distribution of MCs into terrestrial food webs and habitats. In this study, we characterized a Planktothrix bloom in summer 2019 in Lake Mindelsee and tracked possible trophic transfer and/or bioaccumulation of MCs via analysis of phytoplankton, zooplankton (Daphnia) and emergent aquatic insects (Chaoborus, Chironomidae and Trichoptera). Using 16 S rRNA gene amplicon sequencing, we found that five sequence variants of Planktothrix spp. were responsible for bloom formation in September and October of 2019, and these MC-producing variants, provisionally identified as P. isothrix and/or P. serta, occurred exclusively in Lake Mindelsee (Germany), while other variants were also detected in nearby Lake Constance. The remaining cyanobacterial community was dominated by Cyanobiaceae species with high species overlap with Lake Constance, suggesting a well-established exchange of cyanobacteria species between the adjacent lakes. With targeted LC–HRMS/MS we identified two MC-congeners, MC-LR and [Asp3]MC-RR with maximum concentrations of 45 ng [Asp3]MC-RR/L in lake water in September. Both MC congeners displayed different predominance patterns, suggesting that two different MC-producing species occurred in a time-dependent manner, whereby [Asp3]MC-RR was clearly associated with the Planktothrix spp. bloom. We demonstrate an exclusive transfer of MC-LR, but not [Asp3]MC-RR, from phytoplankton into zooplankton reaching a 10-fold bioconcentration, yet complete absence of these MC congeners or their conjugates in aquatic insects. The latter demonstrated a limited trophic transfer of MCs from zooplankton to zooplanktivorous insect larvae (e.g., Chaoborus), or direct transfer into other aquatic insects (e.g. Chironomidae and Trichoptera), whether due to avoidance or limited uptake and/or rapid excretion of MCs by higher trophic emergent aquatic insects.

Identification of Serine-Containing Microcystins by UHPLC-MS/MS Using Thiol and Sulfoxide Derivatizations and Detection of Novel Neutral Losses.

Microcystins (MCs) are hepatotoxic cyclic heptapeptides produced by cyanobacteria, and their structural diversity has led to the discovery of more than 300 congeners to date. However, with known amino acid combinations, many more MC congeners are theoretically possible, suggesting many remain unidentified. Herein, two novel serine (Ser)-containing MCs were putatively identified in a Lake Erie cyanobacterial harmful algal bloom (cyanoHAB), using high-resolution UHPLC-MS as well as thiol and sulfoxide derivatization procedures. These MCs contain an α,β-unsaturated carbonyl on methyl dehydroalanine (Mdha) residue that undergoes Michael addition to produce a thiol-derivatized MC. Derivatization reactions using various thiolation reagents were followed by MS/MS, and two Python codes were used for data analysis and structural elucidation of MCs. Two novel MCs containing Ser at position 1 (i.e., next to Mdha) were putatively identified as [Ser1]MC-RR and [Ser1]MC-YR. Using thiol- and sulfoxide-modified [Ser1]MCs, identifications were confirmed by the observation of specific neutral losses of the oxidized thiols or sulfoxides in CID-MS/MS spectra in both positive and negative electrospray ionization (ESI) modes. These novel neutral losses are unique for MCs with Mdha and an adjacent Ser residue. Data suggest that a gas-phase reaction occurs between oxygen from adjacent Ser residue and sulfur of the Mdha-bonded thiol or sulfoxide, which leads to the formation and detection of stable cyclic MC ions in MS/MS spectra at m/z values corresponding to the loss of oxidized thiols or oxidized sulfoxides from Ser1-containing MCs.

Aerosolized Cyanobacterial Harmful Algal Bloom Toxins: Microcystin Congeners Quantified in the Atmosphere.

Cyanobacterial harmful algal blooms (cHABs) have the potential to adversely affect public health through the production of toxins such as microcystins, which consist of numerous molecularly distinct congeners. Microcystins have been observed in the atmosphere after emission from freshwater lakes, but little is known about the health effects of inhaling microcystins and the factors contributing to microcystin aerosolization. This study quantified total microcystin concentrations in water and aerosol samples collected around Grand Lake St. Marys (GLSM), Ohio. Microcystin concentrations in water samples collected on the same day ranged from 13 to 23 μg/L, dominated by the d-Asp3-MC-RR congener. In particulate matter <2.5 μm (PM2.5), microcystin concentrations up to 156 pg/m3 were detected; the microcystins were composed primarily of d-Asp3-MC-RR, with additional congeners (d-Asp3-MC-HtyR and d-Asp3-MC-LR) observed in a sample collected prior to a storm event. The PM size fraction containing the highest aerosolized MC concentration ranged from 0.44 to 2.5 μm. Analysis of total bacteria by qPCR targeting 16S rDNA revealed concentrations up to 9.4 × 104 gc/m3 in aerosol samples (≤3 μm), while a marker specific to cyanobacteria was not detected in any aerosol samples. Concentrations of aerosolized microcystins varied even when concentrations in water were relatively constant, demonstrating the importance of meteorological conditions (wind speed and direction) and aerosol generation mechanism(s) (wave breaking, spillway, and aeration systems) when evaluating inhalation exposure to microcystins and subsequent impacts on human health.

Simultaneous Detection and Quantification of Aflatoxin M1, Eight Microcystin Congeners and Nodularin in Dairy Milk by LC-MS/MS

Dairy milk holds a prominent position as a widely consumed food, particularly among infants and children. However, it is crucial to address the presence of multiple natural toxic compounds that may co-occur in dairy milk to ensure its safety prior to consumption. Aflatoxin M1 (AFM1), an emerging mycotoxin of interest, is a potential contaminant in the milk of animals who ingest aflatoxin B1 (AFB1). The toxin is regulated in the European Union under Commission Regulation No 1881/2006. Unfortunately, there is a notable lack of data concerning the transfer of various emerging microbial contaminants into dairy milk and, therefore, their natural occurrences. In this study, a simple and sensitive LC-MS/MS method was developed and validated for the quantification of multiple cyanotoxins (microcystin congeners and nodularin) and AFM1 by the main analytical guidelines. Toxins are extracted with methanol 80%, followed by an SPE clean-up step before LC-MS/MS analysis. The LOQ was fixed at 1 µg/L for the nine cyanotoxins and 0.05 µg/L for AFM1. Recoveries were measured between 82.67% and 102%. To the best of our knowledge, there are no other LC-MS/MS methods available for the simultaneous quantification of cyanotoxins and mycotoxins in milk.

Simultaneous Analysis of Cyanotoxins β-N-methylamino-L-alanine (BMAA) and Microcystins-RR, -LR, and -YR Using Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS).

β-N-methylamino-L-alanine (BMAA) and its isomers, 2,4-diaminobutyric acid (2,4-DAB) and N-(2-aminoethyl)-glycine (AEG), along with microcystins (MCs)-RR, -LR, and -YR (the major MC congeners), are cyanotoxins that can cause detrimental health and environmental impacts during toxic blooms. Currently, there are no reverse-phase (RP) LC-MS/MS methods for the simultaneous detection and quantification of BMAA, its isomers, and the major MCs in a single analysis; therefore, multiple analyses are required to assess the toxic load of a sample. Here, we present a newly developed and validated method for the detection and quantification of BMAA, 2,4-DAB, AEG, MC-LR, MC-RR, and MC-YR using RP LC-MS/MS. Method validation was performed, assessing linearity (r2 > 0.996), accuracy (>90% recovery for spiked samples), precision (7% relative standard deviation), and limits of detection (LODs) and quantification (LOQs) (ranging from 0.13 to 1.38 ng mL-1). The application of this combined cyanotoxin analysis on a culture of Microcystis aeruginosa resulted in the simultaneous detection of 2,4-DAB (0.249 ng mg-1 dry weight (DW)) and MC-YR (4828 ng mg-1 DW). This study provides a unified method for the quantitative analysis of BMAA, its isomers, and three MC congeners in natural environmental samples.

Impact of temperature on the temporal dynamics of microcystin in Microcystis aeruginosa PCC7806.

Cyanobacterial blooms pose a serious threat to water quality and human health due to the production of the potent hepatotoxin microcystin. In microcystin-producing strains of the widespread genus Microcystis, the toxin is largely constitutively produced, but there are fluctuations between the cellular and extracellular pool and between free microcystin and protein-bound microcystin. Here we addressed the question of how different temperatures affect the growth and temporal dynamics of secondary metabolite production in the strain Microcystis aeruginosa PCC7806 and its microcystin-deficient ΔmcyB mutant. While the wild-type strain showed pronounced growth advantages at 20°C, 30°C, and 35°C, respectively, the ΔmcyB mutant was superior at 25°C. We further show that short-term incubations at 25°C-35°C result in lower amounts of freely soluble microcystin than incubations at 20°C and that microcystin congener ratios differ at the different temperatures. Subsequent assessment of the protein-bound microcystin pool by dot blot analysis and subcellular localization of microcystin using immunofluorescence microscopy showed re-localization of microcystin into the protein-bound pool combined with an enhanced condensation at the cytoplasmic membrane at temperatures above 25°C. This temperature threshold also applies to the condensate formation of the carbon-fixing enzyme RubisCO thereby likely contributing to reciprocal growth advantages of wild type and ΔmcyB mutant at 20°C and 25°C. We discuss these findings in the context of the environmental success of Microcystis at higher temperatures.

Microcystin congeners in Lake Erie follow the seasonal pattern of nitrogen availability

Cyanobacteria harmful algal blooms produce many toxic secondary metabolites called cyanotoxins. The most studied group of cyanotoxins are microcystins (MC), with over 300 congeners reported. MC-LR is the most studied congener because of its abundance and toxicity. Recent toxicology studies suggest that more hydrophobic MC congeners such as MC-LA, MC-LF, and MC-LW may be less abundant but up to seven times more toxic than MC-LR, whereas, MC-RR's toxicity is only one-fifth that of MC-LR. Hence, understanding the environmental stressors that change the MC congener profile is critical to assessing the negative impact on environmental and human health. A two-year field and experimental study investigated seasonal and spatial changes of MC congener profiles in the western basin of Lake Erie. Both studies showed that nitrogen enrichment favored the production of nitrogen-rich MC-RR (C49H75N13O12). The field study showed that nitrogen depletion favored the low-nitrogen MC-LA (C46H67N7O12). MC-LR (a medium N level, C49H75N10O12) accounted for ∼30% to 50% of the total MC concentration and was stable across nitrogen concentrations. Using the relative toxicity and concentrations of each MC congener, both LC-MS/MS and ELISA overestimated the toxicity early bloom (July) and underestimated it late bloom (September). On 24 July 2019, highly toxic MC-LW and MC-LF were detected at nearshore stations with relative toxicity exceeding drinking water standards. This study demonstrated that the less toxic, high nitrogen MC-RR dominated under nitrogen-replete conditions in the early season, whereas the more toxic, less nitrogen MC-LA dominated under nitrogen-limited conditions later in the season.

Microcystin concentrations and congener composition in relation to environmental variables across 440 north-temperate and boreal lakes

Understanding the environmental conditions and taxa that promote the occurrence of cyanobacterial toxins is imperative for effective management of lake ecosystems. Herein, we modeled total microcystin presence and concentrations with a broad suite of environmental predictors and cyanobacteria community data collected across 440 Canadian lakes using standardized methods. We also conducted a focused analysis targeting 14 microcystin congeners across 190 lakes, to examine how abiotic and biotic factors influence their relative proportions. Microcystins were detected in 30 % of lakes, with the highest total concentrations occurring in the most eutrophic lakes located in ecozones of central Canada. The two most commonly detected congeners were MC-LR (61 % of lakes) and MC-LA (37 % of lakes), while 11 others were detected more sporadically across waterbodies. Congener diversity peaked in central Canada where cyanobacteria biomass was highest. Using a zero-altered hurdle model, the probability of detecting microcystin was best explained by increasing Microcystis biomass, Daphnia and cyclopoid biomass, soluble reactive phosphorus, pH and wind. Microcystin concentrations increased with the biomass of Microcystis and other less dominant cyanobacteria taxa, as well as total phosphorus, cyclopoid copepod biomass, dissolved inorganic carbon and water temperature. Collectively, these models accounted for 34 % and 70 % of the variability, respectively. Based on a multiple factor analysis of microcystin congeners, cyanobacteria community data, environmental and zooplankton data, we found that the relative abundance of most congeners varied according to trophic state and were related to a combination of cyanobacteria genera biomasses and environmental variables.

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.

Water Quality, Toxicity and Diversity of Planktonic and Benthic Cyanobacteria in Pristine Ancient Lake Khubsugul (Hövsgöl), Mongolia.

For the first time, microcystin-producing cyanobacteria have been detected in Khubsugul, which is ancient, pristine and one of the world's largest lakes. The microcystin synthetase genes belonged to the genera Nostoc, Microcystis and possibly Snowella spp. No microcystins were found in the water of the lake. Using the HPLC-HRMS/TOF, five microcystin congeners were identified in biofilms from stony substrates sampled in the coastal zone. The concentration of microcystins in biofilms was low: 41.95 µg g-1 d. wt. by ELISA and 55.8 µg g-1 d. wt. using HPLC. The taxonomic composition of planktonic and benthic cyanobacterial communities was determined by means of microscopy and high-throughput sequencing of 16S rDNA amplicons. Nostocales cyanobacteria dominated benthos of Lake Khubsugul and Synechococcales-plankton. The abundance of cyanobacteria was low both in plankton and benthos; there was no mass development of cyanobacteria. Hydrochemical and microbiological analyses showed that the water in the lake was clean; the number of faecal microorganisms was significantly below the acceptable guideline values. Hydrochemical and hydrophysical parameters, and the concentration of chlorophyll a, were low and within the range of values recorded in the 1970s to 1990s, and corresponded to the oligotrophic state of the lake. There were no signs of anthropogenic eutrophication of the lake and no conditions for the cyanobacterial blooms.

Determination of Microcystins in Fish Tissue by ELISA and MALDI-TOF MS Using a Highly Specific Single Domain Antibody.

The development of simple, reliable, and cost-effective methods is critically important to study the spatial and temporal variation of microcystins (MCs) in the food chain. Nanobodies (Nbs), antigen binding fragments from camelid antibodies, present valuable features for analytical applications. Their small antigen binding site offers a focused recognition of small analytes, reducing spurious cross-reactivity and matrix effects. A high affinity and broad cross-reactivity anti-MCs-Nb, from a llama antibody library, was validated in enzyme linked immunosorbent assay (ELISA), and bound to magnetic particles with an internal standard for pre-concentration in quantitative-matrix-assisted laser desorption ionization-time of flight mass spectrometry (Nb-QMALDI MS). Both methods are easy and fast; ELISA provides a global result, while Nb-QMALDI MS allows for the quantification of individual congeners and showed excellent performance in the fish muscle extracts. The ELISA assay range was 1.8-29 ng/g and for Nb-QMALDI, it was 0.29-29 ng/g fish ww. Fifty-five fish from a MC-containing dam were analyzed by both methods. The correlation ELISA/sum of the MC congeners by Nb-QMALDI-MS was very high (r Spearman = 0.9645, p < 0.0001). Using ROC curves, ELISA cut-off limits were defined to accurately predict the sum of MCs by Nb-QMALDI-MS (100% sensitivity; ≥89% specificity). Both methods were shown to be simple and efficient for screening MCs in fish muscle to prioritize samples for confirmatory methods.

Genetic Diversity of Microcystin Producers (Cyanobacteria) and Microcystin Congeners in Aquatic Resources across Africa: A Review Paper.

Microcystins are produced by multifaceted organisms called cyanobacteria, which are integral to Africa's freshwater environments. The excessive proliferation of cyanobacteria caused by rising temperature and eutrophication leads to the production and release of copious amounts of microcystins, requiring critical management and control approaches to prevent the adverse environmental and public health problems associated with these bioactive metabolites. Despite hypotheses reported to explain the phylogeography and mechanisms responsible for cyanobacterial blooms in aquatic water bodies, many aspects are scarcely understood in Africa due to the paucity of investigations and lack of uniformity of experimental methods. Due to a lack of information and large-scale studies, cyanobacteria occurrence and genetic diversity are seldom reported in African aquatic ecosystems. This review covers the diversity and geographical distribution of potential microcystin-producing and non-microcystin-producing cyanobacterial taxa in Africa. Molecular analyses using housekeeping genes (e.g., 16S rRNA, ITS, rpoC1, etc.) revealed significant sequence divergence across several cyanobacterial strains from East, North, West, and South Africa, but the lack of uniformity in molecular markers employed made continent-wise phylogenetic comparisons impossible. Planktothrix agardhii, Microcystis aeruginosa, and Cylindrospermopsis raciborskii (presently known as Raphidiopsis raciborskii) were the most commonly reported genera. Potential microcystin (MCs)-producing cyanobacteria were detected using mcy genes, and several microcystin congeners were recorded. Studying cyanobacteria species from the African continent is urgent to effectively safeguard public and environmental health because more than 80% of the continent has no data on these important microorganisms and their bioactive secondary metabolites.

Different environmental factors drive the concentrations of microcystin in particulates, dissolved water, and sediments peaked at different times in a large shallow lake

Global distribution and health threats of microcystins (MCs) have received much more attention, but there are still significant knowledge gaps in the peak periods and driving factors of MC in different phases of freshwater ecosystems. Thus, we systematically analyzed the annual variation of different MC congeners (-LR, -RR, and -YR, where L, R, and Y respectively represent leucine, arginine, and tyrosine) in particulates, dissolved water, and sediments in three eutrophic bays of Lake Taihu, China. The results indicated that particulate MCs concentration was the highest, followed by dissolved and sediment MC, with the mean concentration of 7.58 μg/L, 1.48 μg/L, and 0.15 μg/g (DW), respectively. Except for particulate MC, the concentrations of the other two types of MC showed significant differences among the three bays. The dominant congeners of the three types of MCs were different, with the highest proportion of MC-LR being observed in sediment MCs and the lowest in particulate MCs. The peak period of the three types of MC was also different, with particulate MCs reaching their peak in July and October, dissolved MCs in May to July and October, and sediment MCs reaching their peak in September. Consistent with our hypothesis, the dynamics of different types of MCs were driven by different environmental factors. Particulate MCs were primarily related to biological parameters, followed by TP and dissolved carbon. By contrast, dissolved MCs strongly correlated with water temperature and dissolved oxygen. While sediment MCs were primarily driven by properties of sediments, followed by different forms of nitrogen in the water column. Our results suggested that particulate and dissolved MCs in northern Lake Taihu pose high health threats, especially in the peak period. Moreover, a more detailed and targeted risk management strategy should be designed to prevent the possible hazards posed by different types of MC.

Measurement of Microcystin Activity in Human Plasma Using Immunocapture and Protein Phosphatase Inhibition Assay.

Microcystins are toxic chemicals generated by certain freshwater cyanobacteria. These chemicals can accumulate to dangerous levels during harmful algal blooms. When exposed to microcystins, humans are at risk of hepatic injury, including liver failure. Here, we describe a method to detect microcystins in human plasma by using immunocapture followed by a protein phosphatase inhibition assay. At least 279 microcystins have been identified, and most of these compounds share a common amino acid, the Adda side chain. We targeted this Adda side chain using a commercial antibody and extracted microcystins from human samples for screening and analysis. To quantitate the extracted microcystins, we fortified plasma with microcystin-LR, one of the most well-studied, commonly detected, and toxic microcystin congeners. The quantitation range for the detection of microcystin in human plasma using this method is 0.030-0.50 ng/mL microcystin-LR equivalents. This method detects unconjugated and conjugated forms (cysteine and glutathione) of microcystins. Quality control sample accuracies varied between 98.9% and 114%, with a precision of 7.18-15.8%. Finally, we evaluated plasma samples from a community health surveillance project of Florida residents living or working near harmful algae blooms.