Microcystin LA Research Articles

Multi-class secondary metabolites in cyanobacterial blooms from a tropical water body: Distribution patterns and real-time prediction

Cyanobacterial blooms that produce toxins occur in freshwaters worldwide and yet, the occurrence and distribution patterns of many cyanobacterial secondary metabolites particularly in tropical regions are still not fully understood. Moreover, predictive models for these metabolites by using easily accessible water quality indicators are rarely discussed. In this study, we investigated the co-occurrence and spatiotemporal trends of 18 well-known and less-studied cyanobacterial metabolites (including [D-Asp3] microcystin-LR (DM-LR), [D-Asp3] microcystin-RR (DM-RR), microcystin-HilR (MC-HilR), microcystin-HtyR (MC-HtyR), microcystin-LA (MC-LA), microcystin-LF (MC-LF), microcystin-LR (MC-LR), microcystin-LW (MC-LW), microcystin-LY (MC-LY), microcystin-RR (MC-RR) and microcystin-WR (MC-WR), Anatoxin-a (ATX-a), homoanatoxin-a (HATX-a), cylindrospermospin (CYN), nodularin (NOD), anabaenopeptin A (AptA) and anabaenopeptin B (AptB)) in a tropical freshwater lake often plagued with blooms. Random forest (RF) models were developed to predict MCs and CYN and assess the relative importance of 22 potential predictors that determined their concentrations. The results showed that 11 MCs, CYN, ATX-a, HATX-a, AptA and AptB were found at least once in the studied water body, with MC-RR and CYN being the most frequently occurring, intracellularly and extracellularly. AptA and AptB were detected for the first time in tropical freshwaters at low concentrations. The metabolite profiles were highly variable at both temporal and spatial scales, in line with spatially different phytoplankton assemblages. Notably, MCs decreased with the increase of CYN, possibly revealing interspecific competition of cyanobacteria. The rapid RF prediction models for MCs and CYN were successfully developed using 4 identified drivers (i.e., chlorophyll-a, total carbon, rainfall and ammonium for MCs prediction; and chloride, total carbon, rainfall and nitrate for CYN prediction). The established models can help to better understand the potential relationships between cyanotoxins and environmental variables as well as provide useful information for making policy decisions.

The effects of water sample treatment, preparation, and storage prior to cyanotoxin analysis for cylindrospermopsin, microcystin and saxitoxin

Cyanobacterial harmful algal blooms occur in freshwater lakes, ponds, rivers, and reservoirs, and in brackish waters throughout the world. The wide variety of cyanotoxins and their congeners can lead to frequent exposure of humans through consumption of meat, fish, seafood, blue-green algal products and water, accidental ingestion of contaminated water and cyanobacterial scum during recreational activities, and inhalation of cyanobacterial aerosols. Cyanotoxins can also occur in the drinking water supply. In order to monitor human exposure, sensitive analytical methods such as enzyme linked immunosorbent assay and liquid chromatography-mass spectrometry are often used. Regardless of the analytical method of choice, some problems regularly occur during sample collection, treatment, storage, and preparation which cause toxin loss and therefore underestimation of the true concentration. To evaluate the potential influence of sample treatment, storage and preparation materials on surface and drinking water samples, the effects of different types of materials on toxin recovery were compared. Collection and storage materials included glass and various types of plastics. It was found that microcystin congeners LA and LF adsorbed to polystyrene, polypropylene, high density polyethylene and polycarbonate storage containers, leading to low recoveries (<70%), cylindrospermopsin and saxitoxin did not adsorb to the containers tested. Therefore, this study shows that glass or polyethylene terephthalate glycol containers are the materials of choice for collection and storage of samples containing the cyanotoxins cylindrospermopsin, microcystins, and saxitoxin. This study also demonstrated that after 15 min chlorine decreased the concentration of microcystin LR to <40%, microcystin LA and saxitoxin to <15%, therefore quenching of drinking water samples immediately upon sample collection is critical for accurate analysis. In addition, the effect of various drinking water treatment chemicals on toxin recovery and the behavior of those chemicals in the enzyme linked immunosorbent assays were also studied and are summarized.

Carbon-based TiO2 materials for the degradation of Microcystin-LA

The photocatalytic degradation of a cyanobacterial toxin, microcystin-LA (MC-LA), was studied in aqueous solutions under both simulated solar light and visible light irradiation. Neat TiO2 and carbon-based TiO2 materials, prepared with carbon nanotubes (CNT) or graphene oxide (GO), were compared. The highest photocatalytic activity was obtained with a GO-TiO2 composite comprising 4wt.% of carbon content (GO-TiO2-4). Complete conversion of MC-LA was achieved under solar light irradiation in 5min. GO-TiO2-4 was also active under visible light illumination, with 88% of MC-LA removal in 2h. The high photocatalytic activity of GO-TiO2-4 was attributed to the optimal assembly and interfacial coupling between the TiO2 nanoparticles and the GO sheets that can effectively inhibit electron/hole recombination. Reaction intermediates of MC-LA photocatalytic degradation were also identified by LC/Q-TOF and LC/MS/MS, most of them resulting from the attack of hydroxyl radicals to the MC-LA molecule under solar light irradiation.

Fate and Persistence of Particulate and Dissolved Microcystin-LA from Microcystis Blooms

ABSTRACTFew studies have estimated fate and persistence of the hepatotoxic microcystins (MCs) in situ, making ecological and human health risk assessments challenging. We determined fate and persistence of MC congeners during 2 years of Microcystis blooms in a small, shallow, closed-basin lake in Ontario, Canada. In situ half-lives were compared to estimates obtained in vitro under controlled temperature and light. The blooms produced elevated microcystin-LA (MC-LA) (maximum ∼4.2 mg L−1) with minor concentrations of MC-LR, -RR, and -YR. Dissolved MC-LA declined more slowly and persisted longer than particulate MC-LA with in situ half-lives (total 1.5–8.5 days) shorter than in vitro (total 6.8–60.0 days). Half-lives in 2010 were two to eight times shorter compared to 2009, likely due to differences in bloom phenology and species/strain composition. In vitro, higher temperature (4°C → 25°C in dark), and irradiance (dark → 45 → 260 μE m−2s−1 at 25°C) accelerated particulate and dissolved MC-LA decline, respectively. MC-RR accumulated in surface sediments while MC-LA was near detection despite elevated surface water concentrations. MC-LA appears to persist longer in surface waters than the equally toxic MC-LR, requiring almost the entire recreational season (9.5 weeks) to reach guideline concentrations (20 μg L−1).

Simultaneous quantification of microcystins and nodularin in aerosol samples using high‐performance liquid chromatography/negative electrospray ionization tandem mass spectrometry

Cyanobacteria are a small group of photosynthetic planktonic bacteria, producing a large group of strong hepatotoxins called microcystins (MCs). Many studies have been conducted to evaluate the presence of MCs and nodularin (NOD) in water or in marine organisms, but little research has been done on the atmospheric environment. Waterborne toxins can be found in the aerosol phase due to bubble-bursting processes. The aim of this study was to obtain a sensitive method for the simultaneous determination of trace concentrations of individual cyanotoxins in aerosol samples, using liquid chromatography coupled with a triple quadrupole (HPLC/MS/MS). During method development improved electrospray ionization was found in negative ion mode. In contrast with other authors, we have developed a chromatographic separation using alkaline conditions, thus achieving good resolution, improved electrospray ionization and therefore better sensitivity. A sensitive analytical method was set up to simultaneously measure trace concentrations of cyanotoxins in aerosol samples in a single chromatographic analysis using the internal standard method. The limit of detection for all the toxins was determined to be between 1 fg/μL (MC LA and LF) and 9 fg/μL (NOD). The method was applied to ten aerosol samples from the Venice Lagoon. In these samples, trace concentrations of MC-LA ranging between 90 fg m(-3) and 706 fg m(-3), MC-LF between n.d. and 369 fg m(-3) and MC-LW between n.d. and 262 fg m(-3). This is the first study to quantify the cyanotoxins in Venetian aerosol samples using the HPLC/(-)ESI-MS/MS.

Water Blooms and Toxins Produced by Cyanobacteria in the Lower Suzdalskoeskoe Lake (Saint-Petersburg, Russia)

The analysis of the freeze-dried phytoplankton biomass extracts collected during the water bloom in the Lower Suzdalskoe Lake (St. Petersburg) was performed using a high efficiency liquid chromatography method (HPLC). The analysis revealed the presence of hepatotoxic microcystin LA and LF in the biomass, as well as other biologically active compounds - protease inhibitors microcin SF608 and anabenopeptids A and F. Dangerous phytotoxins were not found in the biomass extract of the plankton dominant Planktothrix agardhii (Gom.) Anagn. et Kom. CALU1306. The presence of microcystins in the biomass is apparently due to the populations of other cyanobacteria (Microcystis (Kütz.) Elenk, Anabaena Bory, etc.). It has been concluded that the water bloom conditions require the effective control of environmental factors that cause eutrophication using chromatographic techniques, immunosorbent assays, and protein phosphatase tests.

Analysis of dissolved microcystins in surface water samples from Kovada Lake, Turkey

Dissolved (extracellular) microcystin (MC) concentrations were determined at 3 sampling stations on Lake Kovada, Turkey. The dominant species of cyanobacteria found in August and September of 2006 were Microcystis aeruginosa, Synechococcus sp., Phormidium limosum, Phormidium formosa and Planktothrix limnetica. MC concentrations in water were measured by ELISA and MC variants were examined by HPLC-PDA. Quantitative analysis by HPLC indicated that five MC variants (MC–LR, –RR, –LA, –LW, –LF) were identified in water samples from Kovada Lake. The maximum concentration of dissolved MC–LW was 98.9 µg l − 1 in October. MC–LR was only detected in May at a concentration of 0.5 µg l − 1 . The cross reactivity of the antibody (MC10E7) to variants such as MC–LA MC–LW & MC–LF was low. Hence the results determined by ELISA were lower than those determined by HPLC in September and October samples due to differences in the specificity of the antibody to MC variants. Total extraceullar MCs was quantified by ELISA and ranged from 0.73 to 48.5 µg MC–LR equivalents l − 1 , which in some cases exceeded the WHO provisional Guideline Value for MC–LR in drinking water. This study confirms that the lakes of Turkey should be monitored for toxic cyanobacteria and for MCs to avoid or reduce the potential exposure of people to these health hazards.

Decrease in toxicity of microcystins LA and LR in drinking water by ozonation

Unchlorinated treated waters from two Australian reservoirs were spiked with microcystin-LA and -LR extracted from a toxic scum of Microcystis aeruginosa. The two waters had considerably different water quality and therefore ozone demands. The spiked sample waters were ozonated using the batch method of ozonation at a range of doses and the samples were analysed for toxins using high-performance liquid chromatography (HPLC). The toxin content of the samples was also determined using a protein phosphatase type 2A inhibition assay (PP2A) and toxicity via the standard mouse bioassay. The HPLC results correlated well with the PP2A results and toxicity tests for both waters. A loss of both toxins and toxicity was observed with increasing ozone dose, resulting in a complete loss of toxicity for both waters once an ozone residual had been achieved. At this ozone residual no toxin was detected using HPLC. The results indicate that microcystins are not transformed into toxic by-products.

Water treatment options for dissolved cyanotoxins

When treating water subject to a cyanobacterial bloom the first priority should be removal of intact cells using separation techniques such as coagulation or membrane filtration. Chlorination and ozonation are effective for the destruction of residual dissolved microcystins and cylindrospermopsin. Anatoxin-a can be effectively removed using ozone, although chlorine is relatively ineffective. Oxidation techniques do not appear to be the best method for the treatment of saxitoxins under normal treatment plant operating conditions. Powdered activated carbon can be effective for the removal of all toxins, except, perhaps, microcystin LA, provided the appropriate carbon and the correct dose is applied. Granular activated carbon filters show a limited lifetime for the adsorption of most microcontaminants, including cyanotoxins. The biodegradation of cyanotoxins across GAC filters shows great potential as a treatment process.

Treatment options for microcystin toxins: Similarities and differences between variants

Over sixty variants of the blue‐green algal toxin microcystin have been identified. The two microcystin variants LR and LA vary in only one amino group ie. arginine for microcystin LR and alanine for microcystin LA. In the literature to date, the general consensus has been that m‐LR and m‐LA should respond similarly to a range of water treatment processes. This is the case for ozonation and biodegradation by organisms colonising granular activated carbon filters; there is negligible difference in the response to these processes between the two variants. However, the adsorption of m‐LR onto activated carbon is significantly higher than that of m‐LA. This result is surprising as m‐LA has a lower molecular weight, and is more hydrophobic, factors that would be expected to favour the adsorption of this compound over m‐LR. This trend is also seen for the variants RR and YR. The effect is seen on both negatively and positively charged carbons, indicating that the difference between the variants is not caused by electrostatic interactions with the carbon surface. Electrostatic shielding experiments suggest that electrostatic repulsion between the adsorbed m‐LA molecules, with a net charge of ‐2, may be responsible for the low adsorption. The other variants tested have a lower net charge and therefore experience lower intermolecular repulsion in the adsorbed state.

Comparison of Liquid Chromatography/Mass Spectrometry, ELISA, and Phosphatase Assay for the Determination of Microcystins in Blue-Green Algae Products

More than 100 samples of blue-green algae products (consisting of Aphanizomenon, Spirulina, and unidentified blue-green algae) in the form of pills, capsules, and powders were collected from retail outlets from across Canada. The samples were extracted with 75% methanol in water and centrifuged to remove solids. Aliquots of the extracts along with spiked blank sample extracts were sent to each participating laboratory and independently analyzed for microcystins by enzyme-linked immunosorbent assay (ELISA), protein phosphatase inhibition assay, and by liquid chromatography-tandem mass spectrometry (LC-MS/MS) after sample cleanup using C18 solid-phase extraction. The results obtained by ELISA and LC-MS/MS agreed very well over a concentration range of about 0.5-35 microg/g. The colorimetric phosphatase results generally agreed with the other 2 methods. While the 2 biochemical assays measured total microcystin content compared with a standard of microcystin LR, the LC-MS/MS method measured specific microcystins (LA, LR, RR, YR) using external standards of these for identification and quantitation. Microcystin LR was found in all positive samples by LC-MS/MS. Microcystin LA was the only other microcystin found in the samples analyzed. These 2 microcystins represent essentially all the microcystins that were present in the extracts. Otherwise, the LC-MS/MS results would have been significantly lower than the results of the biochemical assays had other unknown microcystins been present.

Isolation and preparative purification of microcystin variants

Preparative reversed-phase liquid chromatography was successfully used to purify two microcystins (microcystin LR and microcystin LA) from a cyanobacterial process waste. The separation protocol involved extraction of lyophilized cells by methanol, isolation and concentration by solid-phase extraction, and purification by reversed-phase HPLC. Milligram-level loading of microcystins was obtained on a solid-phase extraction cartridge packed with 0.5 g of C 18 stationary phase. The separations were first carried out on an analytical column and then scaled-up to a preparative column. The microcystins were quantified by HPLC and enzyme-linked immunosorbent assay. A method to remove microcystins rapidly and economically from the cyanobacterial process waste is also described.

The design, synthesis, and biological evaluation of analogues of the serine-threonine protein phosphatase 1 and 2A selective inhibitor microcystin LA: rational modifications imparting PP1 selectivity

Based on the results from previously reported molecular modeling analyses of the interactions between the inhibitor microcystin and the serine-threonine protein phosphatases 1 and 2A, we have designed analogues of microcystin LA with structural modifications intended to impart PP1 selectivity. The synthesis of several first generation analogues followed by inhibition assays revealed that all three are PP1-selective, as predicted. Although the observed selectivities are modest, one of the designed analogues is more selective for PP1 than any known small molecule inhibitor.

Microcystin composition of an axenic clonal strain of Microcystis viridis and Microcystis viridis-containing waterblooms in Japanese freshwaters

Toxic cyclis heptapeptides (microcystin) in cells of an axenic clonal strain of Microcystis viridis were analyzed quantitatively. Cells from the logarithmic, stationary and death phases of batch culture contained 670, 618 and 372 μg toxic cyclic heptapeptides per g dry cells, respectively. The toxic peptides of the cells from the stationary phase consisted of microcystin RR (65%), microcystin LR (22%), microcystin YR (10%) and microcystin LA (3%). The composition of the toxic peptides changed only slightly through the phases of batch culture.