Microcystins YR Research Articles

Cyanotoxins in Epipelic and Epiphytic Cyanobacteria from a Hypersaline Coastal Lagoon, an Environmental Hazard in Climate Warming Times and a Potential Source of New Compounds.

Cyanobacterial biodiversity and potential toxicity in coastal lagoons have barely been studied despite these transitional water systems being very important in conservation and for the preservation of economic resources. Most of these transitional systems have been affected by eutrophication, and climate change will severely affect them by promoting cyanobacteria growth, especially in Mediterranean areas. This study aims to characterize the diversity of epipelic and epiphytic cyanobacteria species in a Mediterranean coastal lagoon and their potential for toxins production (microcystins and saxitoxins). Strains were isolated and genetically identified. Toxins were extracted and quantified by LC/MS-MS. All the taxa belong to the former Oscillatoriales. The presence of Nodosilinea and Toxifilum is reported for the first time for Spanish waters, but Pseudanabaena, Phormidium, Geitlerinema and Synechococcus also formed part of benthic mats. All the strains contained Microcystin-YR (MC-YR), but saxitoxin (STX) was present only in the extracts of Nodosilinea and Pseudanabena. MC-LY, MC-LW and [D-Asp3] MC-LR were detected in the extracts of Synechococcus and MC-LF in Toxifilum, but at concentrations that did not permit quantification. Toxins production by epipelic and epiphytic strains in coastal lagoons may represent a hazard, but also an opportunity to obtain potentially interesting compounds that should be further studied.

Self-assembled flower-like carbon nanosheets for magnetic solid-phase extraction of microcystins from aquatic organism

Adsorbents with good dispersibility and high efficiency are crucial for magnetic solid-phase extraction (MSPE). In this study, flower-like magnetic nanomaterials (F-Ni@NiO@ZnO2-C) were successfully prepared by calcination of metal-organic framework (MOF) precursors that was stacked by two-dimensional (2D) nanosheet. The synthesized F-Ni@NiO@ZnO2-C has a flower-like layered structure with a large amount of pore space, promoting the rapid diffusion of targets. In addition, Zn2+ doped in MOF precursors was still retained that further produced strong metal chelation with targets. The unique structure of F-Ni@NiO@ZnO2-C was used as MSPE adsorbent, and combined with high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) for extraction of three microcystins (MCs) detection, including microcystin-LR (MC-LR), microcystin-RR (MC-RR), microcystin-YR (MC-YR). The resulting method has a detection limit of 0.2–1.0 pg mL−1, a linear dynamic range of 0.6–500.0 pg mL−1 and has good linearity (R ≥ 0.9996). Finally, the established method was applied to the highly selective enrichment of MCs in biological samples, successfully detecting trace amounts of MCs (8.4–15.0 pg mL−1) with satisfactory recovery rates (83.7–103.1 %). The results indicated that flower-like magnetic F-Ni@NiO@ZnO2-C was a promising adsorbent, providing great potential for the determination of trace amounts of MCs in biological samples.

Effects of the catastrophic 2020 Yangtze River seasonal floods on microcystins and environmental conditions in Three Gorges Reservoir Area, China.

During July and August 2020, Three Gorges Reservoir Area (TGRA) suffered from catastrophic seasonal floods. Floods changed environmental conditions and caused increase in concentration of microcystins (MCs) which is a common and potent cyanotoxin. However, the effects and seasonal variations of MCs, cyanobacteria, and environmental conditions in TGRA after the 2020 Yangtze River extreme seasonal floods remain largely unclear, and relevant studies are lacking in the literature. A total of 12 representative sampling sites were selected to perform concentration measurement of relevant water quality objectives and MCs in the representative area of the TGRA. The sampling period was from July 2020 to October 2021, which included the flood period. Organic membrane filters were used to perform the DNA extraction and analyses of the 16S rRNA microbiome sequencing data. Results showed the seasonal floods result in significant increases in the mean values of microcystin-RR (MCRR), microcystin-YR (MCYR), and microcystin-LR (MCLR) concentration and some water quality objectives (i.e., turbidity) in the hinterland of TGRA compared with that in non-flood periods (p < 0.05). The mean values of some water quality objectives (i.e., total nitrogen (TN), total phosphorus (TP), total dissolved phosphorus (TDP), and turbidity), MC concentration (i.e., MCRR, MCYR, and MCLR), and cyanobacteria abundance (i.e., Cyanobium_PCC-6307 and Planktothrix_NIVA-CYA_15) displayed clear tendency of increasing in summer and autumn and decreasing in winter and spring in the hinterland of TGRA. The results suggest that seasonal floods lead to changes in MC concentration and environmental conditions in the hinterland of TGRA. Moreover, the increase in temperature leads to changes in water quality objectives, which may cause water eutrophication. In turn, water eutrophication results in the increase in cyanobacteria abundance and MC concentration. In particular, the increased MC concentration may further contribute to adverse effects on human health.

Metabolomic Profile of Arthrospira platensis from a Bulgarian Bioreactor-A Potential Opportunity for Inclusion in Dietary Supplements.

The present study aims to elucidate the metabolomic profile of Arthrospira platensis grown in a bioreactor in Bulgaria. The results show that Arthrospira platensis has a high content of mannose, 137.02 mg g-1, and vitamin A (retinol)-10.3 μg/100 g. High concentrations of calcium, sulfur, and zinc distinguish its elemental composition. The freeze-dried powder contained 15.81 ± 0.45% dietary fiber, 50.16 ± 0.25% total protein content, and 1.22 ± 0.11% total fat content. Among the unsaturated fatty acids with the highest content is α-linolenic acid (25.28%), while among the saturated fatty acids, palmitic acid prevails (22.55%). Of the sterols in the sample, β-sitosterol predominated. There is no presence of microcystins LR, RR, YR, and nodularin. Therefore, Arthrospira platensis grown in a Bulgarian bioreactor is safe for use in the pharmaceutical and food industries. Many of the organic compounds found have applications in medicine and pharmacology and play an important role in biochemical processes in the body. Therefore, Arthrospira platensis grown in Bulgaria has a high potential for use as an independent food supplement or in combination with other natural products.

Management of the Risk of Blue-Green Algae Blooms in the Iskar Dam for Drinking Water Quality of Sofia City

The introduction of systems for ensuring quality and safe drinking water and risk management is the World Health Organization (WHO) good practice introduced as a legislative requirement for all water supply organizations (Directive EU 2020/2184) on the quality of water intended for human consumption. A specific object of this research is the implemented risk assessment and management system, part of the drinking water safety plan for Sofia city. The water supply system from the water source to the end user, operated by Sofiyska voda JSC and part of Veolia, was analyzed. The hazards and risk events were reviewed for each component of the system—the catchment, water source, supply water pipes, drinking water treatment plants (DWTPs) and distribution water network. The results of the risk evaluation show that the risk of an algal bloom falls from medium risk, with a value of 36 in 2018. This risk was new for the Iskar Dam—the main water source for Sofia city. The purpose of the current study is to analyze the risk of blue-green algae blooming and the potential release of toxins in the Iskar Dam. A data analysis of phytoplankton counts, temperature of the water in the dam, precipitation, algal diversity and quantity of microcystins for a period of three years shows that there is a low probability of algae blooming. The concentration of microcystin LR was below 0.6 µg/L and of microcystin YR was below 0.1 µg/L. The highest phytoplankton count was 70,000 cells/mL in 2018, and it decreased to 30,000 cells/mL in 2020. The impact of the risk of “blue-green algae blooms in the Iskar Dam” is assessed as moderate (3) and that has not changed between 2018 and 2022. The likelihood of occurrence of this risk decreased from possible (3) to unlikely (2) after risk reassessment, and the current control effectiveness changed from weakly controlled (4) in 2018 to well controlled (2) in 2022, because more risk management actions were included in Sofiyska voda JSC. The results of the risk evaluation show that the risk of an algal bloom falls into the low-risk category with a value of 12, after measures were considered and a reassessment. As a preventive measure for better management and risk minimization, it is necessary to continue monitoring the phytoplankton count, species composition, concentration of algae toxins (microcystin LR) and nutrients in raw water from the Iskar Dam. This research has added value to the global database for surface water sources intended for human consumption and is focuses on a potential problem, valid for agricultural as well as for urban territories rich in diffused sources of pollution. The specific risk evaluation and management steps could be used and adapted by other water supply companies.

Laboratory studies of the algaecide GreenTD: stability, algaecidal activity and reduction of microcystin production

GreenTD (Dichlorobenzyl derivative, MW 362.17) is a new dichlorobenzyl derivative algaecide. It is effective and selective against harmful algal blooms (HABs). HABs cause serious problems for public health and fishery industries. Algae that cause HABs include Microcystis spp., Anabaena spp., and Aphanizomenon spp. Blooms of toxin-producing Microcystis aeruginosa occur regularly in fresh water where is rich in nitrogen and phosphorus nutrients. Environmental fate studies are needed to investigate the degradation of GreenTD. In the present study, we studied the persistence of GreenTD (90% aqueous solution) in water and toxic effects GreenTD on M. aeruginosa and reduction of microcystin production in the culture media. GreenTD was added in the water pots and microcystis media tubes at levels of 25 g/0.1 ha (0.5 mg/kg) and 50 g/0.1 ha (1.0 mg/kg). Samples were collected after 1, 3, 5, 7 and 14 days. The residues of GreenTD and microcystins in water and the media were determined using high performance liquid chromatography-diode array detection and ultra-performance liquid chromatography-tandem mass spectrometry, respectively. The half-life of GreenTD at concentrations of 0.5 and 1.0 mg/kg was 4.5 and 3.6 days, respectively. This result presents a safety level suitable for the acceptable guideline of water residue. The average recoveries of microcystins RR, YR, LR, and LA were 106–115%, 103–110%, 96–105% and 89–113% in the microcystis media, respectively. The limit of detection of (LOD) the microcystins was 0.1 µg/kg. No microcystins in the media were detected at the LOD (0.1 µg/kg). GreenTD at concentrations of 0.2 and 0.5 µg/kg had a 100% of control efficacy of M. aeruginosa. No growth of the blue-green algae was observed after 14 days of GreenTD application.

A new method for the simultaneous determination of cyanotoxins (Microcystins and Cylindrospermopsin) in mussels using SPE-UPLC-MS/MS

The aim of this study was to optimize the extraction conditions of Microcystin-LR (MC-LR), Microcystin-RR (MC-RR), Microcystin-YR (MC-YR) and Cylindrospermopsin (CYN) simultaneously from mussels by using response surface methodology (RSM) and to validate the method by a dual solid phase extraction (SPE) system combined with ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The optimal parameters were: 90% MeOH (% v/v) for the extraction, a solvent/sample ratio of 75 and 15% MeOH in the extract before loading onto SPE. Mussels were spiked at 10; 37.5 and 75 ng g−1 fresh weight (f.w) of the 4 toxins, showing linear ranges of 0.5–75 ng g−1 f.w; low values for the limits of detection (0.01–0.39 ng g−1 f.w.) and quantification (0.23–0.40 ng g−1 f.w.); acceptable recoveries (70.37–114.03%) and relative standard deviation (%RSDIP) values (2.61–13.73%). The method was successfully applied to edible mussels exposed to cyanobacterial extracts under laboratory conditions, and it could allow the monitoring of these cyanotoxins in environmental mussel samples.

Detected cyanotoxins by UHPLC MS/MS technique in tropical reservoirs of northeastern Colombia

This study focused on the detection and quantification of eight cyanotoxins in water samples in three reservoirs located in the eastern department of Antioquia, Colombia. The reservoirs are a source of water supply and hydroelectricity, and also generate economic activities in fishing and recreation. Between May 2015 and October 2016, 8 samplings were carried out at times of high temperatures, which ranged from 20 to 29 °C. This period was selected because of a significant or strong El Niño phenomenon, according to the World Meteorological Organization. For the study, 270 integrated samples were taken from the photic zone (PZ) and the surface of the reservoirs, at each of the three sampling points. The samples were analyzed by the analytical technique of ultra-high-performance liquid chromatography coupled to triple quadrupole mass spectrometry (UHPLC MS/MS). The quantification performed for six microcystins (MCs), a nodularin (NOD) and a cylindrospermopsin (CYN), showed positive results well above 1 μg L−1. In the water of the Abreo Malpaso and Peñol reservoirs, microcystin-LR (MC-LR), microcystin-YR (MC-YR) and [D-Asp3,(E)-Dhb7]- microcystin-RR toxins were detected at levels of considerable concentration, especially between May and September 2015, when there was no rainfall in this region. In the Playas reservoir, positive results for [D-Asp3,(E)-Dhb7]-MC-RR were detected from May to November 2015, with the highest concentrations being reached in dry season. The temperatures reached and the changes in climatic conditions witnessed during the monitoring period of this study were important factors in the production of cyanotoxins. This was evidenced in this work by the high concentrations of detected cyanotoxins and their absence in periods of rain, as happened from the second quarter of 2016 until the end of the study in October. This is the first study of the detection and quantification of cyanotoxins in tropical reservoirs of northeastern Colombia using the UHPLC MS/MS analytical technique, which allowed the toxins to be unequivocally detected and confirmed. A method was developed and validated, proving to be sensitive, reproducible and accurate. For each of the toxins (microcystin-LR (MC-LR), microcystin-RR (MC-RR), microcystin-YR (MC-YR), [D-Asp3,(E)-Dhb7]- microcystin-RR, microcystin-LW (MC-LW), microcystin-LF (MC-LF), nodularin (NOD) and cylindrospermopsina (CYN)) the correlation coefficients (R2) were in a range between 0.9907 and 0.9999. Verification of the accuracy of the method was performed through a calibration curve in solvent. The recovery percentages of the accuracy and precision tests of the method for low level, medium level and high level were in a range between 64% and 115% for all the cyanotoxins. The validation of the cyanotoxin method shows that it is possible to detect them individually in natural water with a quantification limit (LOQ) of approximately 0.05 μg L−1.

Determination of cylindrospermopsin, nodularin and microcystins in freshwater fish by dispersive solid phase extraction-liquid chromatography-tandem mass spectrometry

A method was developed for the determination of cylindrospermopsin (CYN), nodularin (NOD), microcystin-RR (MC-RR), microcystin-YR (MC-YR) and microcystin-LR (MC-LR) in freshwater fish by dispersive solid phase extraction-liquid chromatography-tandem mass spectrometry (DSPE-LC-MS/MS). The analytes were extracted from fish tissues with acetonitrile-water-formic acid (89:10:1, v/v/v), and purified by DSPE using C18 as the adsorbent. The separation of analytes was performed on an Agilent ZORBAX Eclipse XDB C18 column with the gradient elution of acetonitrile and water as mobile phases. Qualitative analysis was performed using the multiple reaction monitoring (MRM) mode. The analytes were quantified by matrix-matched external standard curves. The chromatographic and MS parameters were optimized. Major factors affecting the extraction and cleanup efficiencies including the type of extraction solvent and cleanup sorbent were investigated. The linear correlation coefficients (R2) of the five target compounds were no less than 0.9954. The limits of detection (LODs, S/N=3) and limits of quantification (LOQs, S/N=10) of the five target compounds were 5-10 μg/kg and 15-40 μg/kg, respectively. The spiked recoveries of the five target compounds ranged from 62.3% to 101.2%. The developed method is simple, rapid, accurate, sensitive, and is suitable for the determination of cylindrospermopsin, nodularin and microcystins in freshwater fish.

New Method for Simultaneous Determination of Microcystins and Cylindrospermopsin in Vegetable Matrices by SPE-UPLC-MS/MS.

Cyanotoxins are a large group of noxious metabolites with different chemical structure and mechanisms of action, with a worldwide distribution, producing effects in animals, humans, and crop plants. When cyanotoxin-contaminated waters are used for the irrigation of edible vegetables, humans can be in contact with these toxins through the food chain. In this work, a method for the simultaneous detection of Microcystin-LR (MC-LR), Microcystin-RR (MC-RR), Microcystin-YR (MC-YR), and Cylindrospermopsin (CYN) in lettuce has been optimized and validated, using a dual solid phase extraction (SPE) system for toxin extraction and ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) for analysis. Results showed linear ranges (5–50 ng g−1 f.w.), low values for limit of detection (LOD) (0.06–0.42 ng g−1 f.w.), and limit of quantification (LOQ) (0.16–0.91 ng g−1 f.w.), acceptable recoveries (41–93%), and %RSDIP values for the four toxins. The method proved to be robust for the three variables tested. Finally, it was successfully applied to detect these cyanotoxins in edible vegetables exposed to cyanobacterial extracts under laboratory conditions, and it could be useful for monitoring these toxins in edible vegetables for better exposure estimation in terms of risk assessment.

Effects of lanthanum on Microcystis aeruginosa: Attention to the changes in composition and content of cellular microcystins

Algal blooms threaten human health and aquatic ecosystem through the production of microcystins (MCs) by toxic strains. The accumulation of rare earth elements (REEs) in water affects the growth and physiological activities of algae. However, whether or how REEs affect cellular microcystins (MCs) is largely unknown. In this study, the effects of lanthanum ion [La(III)], a type of REE, on the MCs in Microcystis aeruginosa were investigated, and the mechanism of the effect was analyzed using ecological stoichiometry. The different concentrations of La(III) were selected to correlate environmental pollution status. Low-dose La(III) (0.2, 2.0, and 4.0 μM) exposure increased the total content of MCs and the percentage contents of microcystin-YR (MC-YR) and microcystin-LW (MC-LW) and decreased the percentage content of microcystin-LR (MC-LR). High-dose La(III) (8.0, 20, 40, and 60 μM) exposure decreased the total content of the MCs, increased the percentage content of MC-LR, and decreased the percentage contents of MC-YR and MC-LW. The changes in the total MCs content were positively associated with the ratios of C:P and N:P in algal cells. The composition of MCs was dependent on the ratio of C:N in algal cells; for example, the percentage content of MC-LR decreased and the percentage content of MC-YR and MC-LW increased as the ratio of C:N in algal cells increased. In conclusion, La(III) could affect the content and composition of MCs via changes in the growth and chlorophyll-a content of Microcystis aeruginosa, and these effects depended on the ratios of C:P, N:P, and C:N in Microcystis aeruginosa. Such changes may influence the toxicity of Microcystis blooms. The results provides a new insight into the mechanism of REEs effects on algal toxins and provide references for evaluating environmental risks of REEs pollution in aquatic ecosystems.

Hepatotoxicity and metabolic effects of cellular extract of cyanobacterium Radiocystis fernandoi containing microcystins RR and YR on neotropical fish (Hoplias malabaricus)

The toxicological effect of cellular extract of cyanobacterium Radiocystis fernandoi strain R28 containing RR and YR microcystins was analyzed in the fish Hoplias malabaricus with emphasis on the liver structure and energetic metabolism, after short-term exposure. Fish were intraperitoneally (i.p.) injected with 100 μg of equivalent MC-LR kg−1 body mass containing in the cellular extract of R. fernandoi strain R28. Twelve and 96 h post-injection, the plasma, liver and white muscle were sampled for biochemical analyses and liver was also sampled for morphological analyses. After i.p. injection, the activity of acid phosphatase (ACP), alanine aminotransferase (ALT) and direct bilirubin increased in the plasma, while ALT and aspartate aminotransferase (AST) decreased in the liver. Glucose, lactate and pyruvate increased while protein decreased in the plasma; glycogen, pyruvate and lactate decreased in the liver; and glycogen and glucose increased in the muscle. Ammonia increased in the plasma, liver and muscle. The hepatocyte cell shape changed from polyhedral to round after cellular extract injection; there was loss of biliary canaliculus organization, but the biliary duct morphology was conserved in the liver parenchyma. In conclusion, microcystins present in the cellular extract of R. fernandoi strain R28 affect the liver structure of H. malabaricus, but the liver was able to continuously produce energy by adjusting its intermediate metabolism; glycogenolysis and gluconeogenesis maintained glucose homeostasis and energy supply.

Quantitative determination by screening ELISA and HPLC-MS/MS of microcystins LR, LY, LA, YR, RR, LF, LW, and nodularin in the water of Occhito lake and crops.

The occurrence of harmful cyanobacterial blooms in surface waters is often accompanied by the production of a variety of cyanotoxins, and these toxins are designed to target in humans specific organs on which they act. When introduced into the soil ecosystem by spray irrigation of crops, they may affect the same molecular pathways in plants having identical or similar target organs, tissues, cells, or biomolecules. There are also several indications that terrestrial plants, including crops, can bioaccumulate cyanotoxins and present, therefore, potential health hazards for humans. During this project, for monitoring purposes, water samples were collected from lake Occhito, in which there was an algal bloom (Planktothrix rubescens) in 2009, and from three tanks which acted as hydraulic junctions. In addition, crop samples irrigated with water from the three tanks mentioned above were also picked. Finally, the characterization of principal cyanobacteria was performed, to determine the presence of cyanotoxins such as microcystins and validate a method of screening ELISA for the determination of microcystins in vegetable samples and a confirmatory method by HPLC-ESI-MS/MS. Graphical abstract Occhito lake (left), microcystin LR (center), Tomato field in Foggia (right); figures below: ELISA (left), HPLC-MS/MS (right).

High diversity of microcystins in a Microcystis bloom from an Algerian lake

Microcystins (MCs) are cyanobacterial heptapeptides, produced by several genera and species of cyanobacteria, which have been involved in poisoning of animals throughout the world and have also been implicated in human health problems. They are regarded as the most frequently occurring and widespread of the cyanotoxins, with more than 100 MC variants reported to date including the present study. The lake des Oiseaux is a shallow permanent freshwater lake located in north-eastern Algeria. It is an important natural reserve playing a major role for the migratory birds after the crossing of the Mediterranean Sea and from the Sahara desert. In recent years, possibly related to increased eutrophication of the lake, massive blooms of cyanobacteria identified as Microcystis spp. have been observed. A bloom sample collected in September 2013 was analyzed by the serine/threonine phosphatase PP2A inhibition assay and liquid chromatography-mass spectrometry to determine respectively, the total concentration of MCs and the different variants of these toxins present. The results revealed that the Microcystis spp. bloom sample contained microcystins of which 21 putatively congeners were detected. Among these, 12 known microcystins (MC-RR, MC-LR, MC-FR, MC-WR, MC-YR, MC-LA, MC-(H4)YR, MC-HilR, [Asp3]MC-RAba, and [Glu(OCH3)6]MC-LR) and two new congeners ([Asp3]MC-HarAba and [Glu(OCH3)6]MC-FR) were characterized, considering their molecular mass and the fragment ions produced by collision-induced dissociation of the [M+H]+ ions. MC-RR was the major (43.4%) in the bloom sample.

Calibration of a passive, in situ, integrative sampler for monitoring of microbial biotoxins in aquatic environments

In this work, an integrative passive sampler based on a silicone membrane filled with a suspension of γ-Fe2O3 at pH 3.5 was developed. The novel device was calibrated for the measurement of microcystin concentrations in water. Laboratory calibration studies of the passive sampling devises under controlled conditions of temperature, water turbulence, and analyte concentration were conducted in order to establish how variable environmental conditions affect the novel sampler's performance. The chemical uptake of microcystin (MC)-RR, -LR, and -YR into the passive sampler remained linear and integrative throughout the 28-day exposure. The relative standard deviations of mean concentrations obtained using silicone-based sampler ranged from 1.42 to 3.74% for microcystin-LR, -RR, and -YR. The values for reproducibility from triplicate samplers ranged from 3.5 to 7.1% for microcystin-LR, -RR, and -YR. The detection limits on high performance liquid chromatography (HPLC) with PDA detection for microcystins LR, RR, and YR were 24.7, 17.2, and 23.8 μg L−1 respectively, calculated as three times the signal to noise ratio. The rate of accumulation of most of the MC compounds tested was dependent on temperature and flow velocity. Furthermore, the sample matrix, e.g. humic substances, had no significant effect on the concentration of compounds trapped in the acceptor solution and once these MC compounds were trapped in the acceptor phase they did not diffuse back during the deployment period.

Retinoid compounds associated with water blooms dominated by Microcystis species

Retinoic acids play a critical role in vital physiological processes and vertebrate development, and their derivatives can be produced by some cyanobacterial species into surface waters. This study presents important environmentally-relevant information on total retinoid-like activity of field cyanobacterial biomasses and their surrounding waters. Intracellular and extracellular levels of total retinoid-like activity and retinoic acids have been investigated at a set of independent sites with the occurrence of water bloom dominated by widespread species Microcystis aeruginosa. Twelve samples of biomass and surrounding water from seven localities affected by blooms were studied in comparison with samples from M. aeruginosa laboratory cultures. The method for biomass extraction was optimized and final extracts and samples of surrounding water concentrated by solid phase extraction were assessed using in vitro reporter gene bioassay and chemical analyses for all-trans-retinoic acid (ATRA), 9-cis retinoic acid (9-cis RA) and microcystins RR, LR and YR. Methanol was the most efficient solvent for the extraction of compounds with retinoid-like activity. An in vitro bioassay with the P19/A15 transgenic cell line revealed retinoid-like activity in all cyanobacterial biomasses in the range of 356–2838ng of retinoid acid equivalents (REQ)/g dry mass (dm), while only three of surrounding water samples exhibited detectable retinoid-like activity, in the range of 12.8–28.7ngREQ/L. Microcystins were detected in all samples, but they elicited no detectable retinoid-like activity up to 10mg/L. Chemical analyses detected concentrations up to 340ng/gdm of all-trans-retinoic acid (ATRA) and 84ng/gdm 9-cis retinoic acid (9-cis RA) in bloom extracts, and up to 19ng/L ATRA and 2.2ng/L 9-cis RA in surrounding water. In most samples, ATRA and 9-cis RA contributed relatively little to the total REQs, which indicates the presence of significant amounts of other compounds with retinoic acid receptor-mediated modes of action. The impact of retinoid-like cyanobacterial metabolites could be of importance namely in smaller water bodies with dense water blooms and low dilution.

Pathway for Biodegrading Microcystin-YR by Sphingopyxis sp. USTB-05.

Harmful cyanobacterial blooms in waters have become a global environmental problem, this mainly due to the production and release of various microalgal toxins, in which microcystins (MCs) are distributed widely. Here, we focused on the study of a typical form of microcystins called microcystin-YR (MC-YR). It was found that initial 14.8 mg/L of MC-YR could be completely eliminated within 10 hr by the crude enzymes (CEs) of Sphingopyxis sp. USTB-05, a promising bacterial strain we isolated and identified in our previous study. During the enzymatic biodegradation of MC-YR with time course, the peaks of two intermediate and two final products were observed on the profiles of HPLC at the wavelengths of 238 nm and 230 nm, respectively. Based on the analysis of m/z ratios of MC-YR and its four products by LC-MS/MS, we suggested that at least four enzymes were involved in the biodegradation of MC-YR by Sphingopyxis sp. USTB-05. The first enzyme microcystinase converted cyclic MC-YR to linear MC-YR as the first product. Then the second enzyme serine protease was found to cleave the target peptide bond between alanine (Ala) and tyrosine (Tyr) of linearized MC-YR, producing a tetrapeptide and a tripeptide as second products, which were Adda-Glu-Mdha-Ala and Tyr-Masp-Arg, respectively. Next, the third enzyme peptidase converted the tetrapeptide of Adda-Glu-Mdha-Ala to Adda. And the fourth enzyme cleaved the tripeptide of Tyr-Masp-Arg to produce Tyr and dipeptide (Masp-Arg), which has never been reported. These findings will help us better understand the biodegradation pathway of MC-YR by Sphingopyxis sp. USTB-05.

Qualitative analysis of the most toxic and abundant microcystin variants (LR, RR, and YR) by using LCMS–IT–TOF

Contamination of microcystins (a family of heptapeptide hepatotoxins), in eutrophicated water bodies is a worldwide problem. Due to their acute and chronic poisoning of animal and human health even at a low concentration of ppb scale, the accurate analysis is required of all microcystin variants present in a sample. In this study, a sensitive and selective method for the analysis of microcystin-LR (MC-LR), microcystin-RR (MC-RR), and microcystin-YR (MC-YR) using the liquid chromatograph mass spectrometry–ion trap–time of flight (LCMS–IT–TOF) technique has been developed. A new approach to quantify and distinguish microcystin variants by using LCMS–IT–TOF, a novel hybrid mass spectrometer having the multistep tandem mass spectrometry (MSn) ability, excellent resolution, and mass accuracy, could provide highly accurate analysis for evaluation of the toxicity of microcystins in a sample. In addition, chromatographic and spectrometric conditions were optimized for high peak intensity and the fine peak separation of microcystin variants. The results obtained of multistep tandem MS spectra strongly verified the presence of the studied microcystin variants in samples, and offered the lower detection limits of 5pg on a column for MC-RR, and 10pg on a column for MC-LR and MC-YR under the given analytical conditions.

Microcystins-LR, -YR and -RR in Six Bulgarian Water Bodies of Health and Conservational Importance (2012-2014)

This study presented the results of HPLC-DAD microcystin and nodularin analyses from five Bulgarian water bodies (reservoirs Stoudena, Pchelina and Bistritsa, and lakes Dourankoulak and Vaya) carried out in 2012-2014, as a continuation of our work from 2004 to 2005 and in 2011, and first data on microcystins in the lake Momin Brod were detected in the period 2012-2014. The total concentration of microcystins in water samples ranged from 0.1 to 1.8 μg/l and their amount in the concentrated biomasses from net samples ranged from 1.35 to 296 μg/g (d.w.). The presence of the most toxic microcystin-LR was recorded in all studied lakes and reservoirs, where also microcystins RR and YR were detected, but no microcystin-LA and nodularin were found.

Determination of six microcystins and nodularin in surface and drinking waters by on-line solid phase extraction–ultra high pressure liquid chromatography tandem mass spectrometry

Microcystins and nodularin are cyclic peptides hepatotoxins produced by cyanobacterial genera (blue-green algae). Toxic cyanobacterial blooms are a worldwide problem, as reported in several countries, like China, Australia, or the United States. Therefore, it is necessary to develop sensitive and reliable analytical methodology to determine this type of toxins in water at parts per billion levels, or even lower. In this work, the potential of solid-phase extraction coupled on-line to ultra-high-pressure liquid chromatography/electrospray tandem mass spectrometry (SPE–UHPLC–MS/MS) has been investigated for the efficient quantification and confirmation of microcystins LR, RR, YR, LY, LW, LF and nodularin in surface and drinking water samples, at sub-ppb levels. The method developed involves the injection of only 1mL of water sample into the on-line SPE–UHPLC–MS/MS system and allows the rapid determination of the compounds selected (8min of chromatographic run), avoiding laborious sample treatment. The method was validated in surface and drinking water by means of recovery experiments at 0.25 and 1μgL−1. Average recoveries (n=5) ranged from 71 to 116%, with relative standard deviations (RSDs) lower than 15%. For microcystins LR, RR, YR and nodularin, a third level was also assayed (0.1μgL−1) obtaining satisfactory data too. Limits of detection between 0.002 and 0.0405μgL−1 were estimated (0.0005μgL−1 for nodularin). The developed method was applied to the analysis of water samples collected in the province of Castellón (Spain). The acquisition of three MS/MS transitions for each compound allowed the unequivocal confirmation of positive samples, which was supported by the accomplishment of ion intensity ratios and retention time when compared with reference standards.