Assessment of estrogenic potential from exudates of microcystin-producing and non-microcystin-producing Microcystis by metabolomics, machine learning and E-screen assay.

Cyanobacterial blooms, often dominated by Microcystis aeruginosa, are capable of producing estrogenic effects. It is important to identify specific estrogenic compounds produced by cyanobacteria, though this can prove challenging owing to the complexity of exudate mixtures. In this study, we used untargeted metabolomics to compare components of exudates from microcystin-producing and non-microcystin-producing M. aeruginosa strains that differed with respect to their ability to produce microcystins, and across two growth phases. We identified 416 chemicals and found that the two strains produced similar components, mainly organoheterocyclic compounds (20.2%), organic acids and derivatives (17.3%), phenylpropanoids and polyketides (12.7%), benzenoids (12.0%), lipids and lipid-like molecules (11.5%), and organic oxygen compounds (10.1%). We then predicted estrogenic compounds from this group using random forest machine learning. Six compounds (daidzin, biochanin A, phenylethylamine, rhein, o-Cresol, and arbutin) belonging to phenylpropanoids and polyketides (3), benzenoids (2), and organic oxygen compound (1) were tested and exhibited estrogenic potency based upon the E-screen assay. This study confirmed that both Microcystis strains produce exudates that contain compounds with estrogenic properties, a growing concern in cyanobacteria management.

An assessment of a biosensor system for the quantification of microcystins in freshwater cyanobacterial blooms.

Microcystin-producing cyanobacterial blooms are a global issue threatening drinking water supplies and recreation on lakes and beaches. Direct measurement of microcystins is the only way to ensure waters have concentrations below guideline concentrations; however, analyzing water for microcystins takes several hours to days to obtain data. We tested LightDeck Diagnostics' bead beater cell lysis and two versions of the quantification system designed to give microcystin concentrations within 20 min and compared it to the standard freeze-thaw cycle lysis method and ELISA quantification. The bead beater lyser was only 30 % effective at extracting microcystins compared to freeze-thaw. When considering freeze-thaw samples analyzed in 2021, there was good agreement between ELISA and LightDeck version 2 (n = 152; R2 = 0.868), but the LightDeck slightly underestimated microcystins (slope of 0.862). However, we found poor relationships between LightDeck version 2 and ELISA in 2022 (n = 49, slopes 0.60 to 1.6; R2 < 0.6) and LightDeck version 1 (slope = 1.77 but also a high number of less than quantifiable concentrations). After the quantification issues are resolved, combining the LightDeck system with an already-proven rapid lysis method (such as microwaving) will allow beach managers and water treatment operators to make quicker, well-informed decisions.

Investigation of dissolved organic matter's influence on the toxicity of cadmium to the cyanobacterium Microcystis aeruginosa by biochemical and molecular assays.

Rapid economic development has increased the accumulation of dissolved organic matter (DOM) and heavy metals in aquatic environments. In addition, Microcystis aeruginosa can cause the outbreak of cyanobacteria bloom and can produce microcystin, which poses a threat to human water safety. Therefore, this study analyzed the biochemical and molecular assays of DOM (0, 1, 3, 5, 8, 10 mg C L-1) extracted from four different sources on the toxicity of cadmium (Cd) to M. aeruginosa. The results showed that the addition of different concentrations of DOM from sediment, biochar, and humic acid alleviated the toxicity of Cd to M. aeruginosa. But the addition of rice hulls DOM at high concentrations (8 and 10 mg L-1) significantly reduced the normal growth and metabolic activities of M. aeruginosa. DOM from four different sources promoted the expression level of microcystin-related gene mcyA and the production of microcystin-leucine-arginine (MC-LR), and mcyA was positively correlated with MC-LR. DOM from biochar, sediment, and humic acid were able to bind Cd through complexation. The results will help to understand the toxic effects of heavy metals on toxic-producing cyanobacteria in the presence of DOM, and provide certain reference for the evaluation of water environmental health.

State-of-the-art review on the ecotoxicology, health hazards, and economic loss of the impact of microcystins and their ultrastructural cellular changes.

Cyanobacteria are ubiquitously globally present in both freshwater and marine environments. Ample reports have been documented by researchers worldwide for pros and cons of cyanobacterial toxins. The implications of cyanobacterial toxin on health have received much attention in recent decades. Microcystins (MCs) represent the unique class of toxic metabolites produced by cyanobacteria. Although the beneficial aspects of cyanobacterial are numerous, the deleterious effect of MCs overlooked. Several studies on MCs evidently reported that MCs exhibit a plethora of harmful effect on animals, plants, and cell lines. Accordingly, numerous histopathological studies have also found that MCs cause detrimental effects to cells by damaging cellular organelles, including nuclear envelope, Golgi apparatus, endoplasmic reticulum, mitochondria, plastids, flagellum, pilus membrane structures and integrity, vesicle structures, and autolysosomes and autophagosomes. Such ultrastructural cellular damages holistically influence the morphological, biochemical, physiological, and genetic status of the host. Indeed, MCs have also been found to cause the deleterious effect to different animals and plants. Such deleterious effects of MCs have greater impact on agriculture, public health which in turn influences ecotoxicology and economic consequences. The impairments correspond to oxidative stress, organ failure, carcinogenesis, aquaculture loss, with an emphasis for blooms and respective bioaccumulation prospects. The preservation of mortality among life forms is addressed in a critical cellular perspective for multitude benefits. The comprehensive cellular assessment could provide opportunity to develop strategy for therapeutic implications.

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

Cyanobacteria, ancient prokaryotes, interfere with ecosystem water quality through the production of cyanotoxins and bloom formation. Therefore, for water safety and public health reasons, the application of faster, sensitive, and specific tools on its risk assessment is demanded. Polymerase chain reaction (PCR) coupled with DNA sequencing can be a helpful tool for the presence and potential to cyanotoxicity. To achieve these, seven waterbodies located on the North and Center regions of Portugal were sampled for two monitoring periods (2017 and 2018). Thus, given the five risk levels proposed (none up to four cyanotoxins - mcyA, cyrC, anaC, sxtI - being detected per risk level), results showed that the great majority of the ecosystems analyzed on the presence of blooms and under climate change phenomenon (heat waves) had an elevated risk (up to four cyanotoxins being detected) corresponding to a situation of high potential of cyanotoxicity. In the opposite conditions (i.e., absence of blooms and heat waves), the risk was lowered to none or only one cyanotoxin being detected. Two ecosystems escaped this trend and demonstrated little to no alterations among risk levels from 1 year to another corresponding to a high potential of cyanotoxicity and cyanotoxins persistence in comparison to other studied ecosystems. Overall, the risk assessment undertaken suggests that other ecosystems ecological variables (physical, hydrological, or chemical) are interfering on the occurrence and persistence of cyanotoxins biosynthesis genes. Given the observed conditions (eutrophic status, bloom occurrence, and heat waves) of the analyzed ecosystems, cyanobacterial potential for toxicity seems to have increased, suggesting a need of the incorporation of other cyanotoxins apart of the regulated microcystins-LR on cyanotoxins surveillance programs of Portugal.

Protective Role of Native Rhizospheric Soil Microbiota Against the Exposure to Microcystins Introduced into Soil-Plant System via Contaminated Irrigation Water and Health Risk Assessment.

Microcystins (MCs) produced in eutrophic waters may decrease crop yield, enter food chains and threaten human and animal health. The main objective of this research was to highlight the role of rhizospheric soil microbiota to protect faba bean plants from MCs toxicity after chronic exposure. Faba bean seedlings were grown in pots containing agricultural soil, during 1 month under natural environmental conditions of Marrakech city in Morocco (March-April 2018) and exposed to cyanobacterial extracts containing up to 2.5 mg·L-1 of total MCs. Three independent exposure experiments were performed (a) agricultural soil was maintained intact "exposure experiment 1"; (b) agricultural soil was sterilized "exposure experiment 2"; (c) agricultural soil was sterilized and inoculated with the rhizobia strain Rhizobium leguminosarum RhOF34 "exposure experiment 3". Overall, data showed evidence of an increased sensitivity of faba bean plants, grown in sterilized soil, to MCs in comparison to those grown in intact and inoculated soils. The study revealed the growth inhibition of plant shoots in both exposure experiments 2 and 3 when treated with 2.5 mg·L-1 of MCs. The results also showed that the estimated daily intake (EDI) of MCs, in sterilized soil, exceeded 2.18 and 1.16 times the reference concentrations (0.04 and 0.45 µg of microcysin-leucine arginine (MC-LR). Kg-1 DW) established for humans and cattle respectively, which raises concerns about human food chain contamination.

Phytotoxicity and bioconcentration of microcystins in agricultural plants: Meta-analysis and risk assessment.

Microcystins are cyanotoxins produced by many species of cyanobacteria. They are specific inhibitors of serine/threonine protein phosphatases and are phytotoxic to agricultural plants. This study used a formal meta-analysis to estimate the phytotoxicity and bioconcentration rates of agricultural plants exposed to microcystins, and the human health risk from consuming microcystin-contaminated plants. Among the 35 agricultural plants investigated, microcystins were most phytotoxic to durum wheat, corn, white mustard and garden cress. Leafy vegetables such as dill, parsley and cabbage could bioconcentrate ∼3 times more microcystins in their edible parts than other agricultural plants. Although the human health risk from ingesting microcystins could be greater for leafy vegetables than other agricultural plants, further work is needed to confirm bioconcentration of microcystins in realistic water-soil-plant environments. Still, we should avoid growing leafy vegetables, durum wheat and corn on agricultural land that is irrigated with microcystins-contaminated water and be attentive to the risk of microcystins contamination in the agricultural food supply.

Biomarkers in bivalve mollusks and amphipods for assessment of effects linked to cyanobacteria and elodea: Mesocosm study.

The effects of cyanobacteria (Aphanizomenon flos-aquae (90%), Microcystis aeruginosa) and dense Elodea canadensis beds on the health endpoints of the amphipod Gmelinoides fasciatus and bivalve mollusc Unio pictorum were examined in mesocosms with simulated summer conditions (July-August 2018) in the environment of the Rybinsk Reservoir (Volga River Basin, Russia). Four treatments were conducted, including one control and three treatments with influencing factors, cyanobacteria and dense elodea beds (separately and combined). After 20 days of exposure, we evaluated the frequency of malformed and dead embryos in amphipods, heart rate (HR) and its recovery (HRR) after stress tests in molluscs as well as heat tolerance (critical thermal maximum or CTMax) in both amphipods and molluscs. The significant effect, such as elevated number of malformed embryos, was recorded after exposure with cyanobacteria (separately and combined with elodea) and presence of microcystins (MC) in water (0.17 µg/l, 40% of the most toxic MC-LR contribution). This study provided evidence that an elevated number (>5% of the total number per female) of malformed embryos in amphipods showed noticeable toxicity effects in the presence of cyanobacteria. The decreased oxygen under the influence of dense elodea beds led to a decrease in HR (and an increase in HRR) in molluscs. The notable effects on all studied biomarkers, embryo malformation frequency and heat tolerance in the amphipod G. fasciatus, as well as the heat tolerance and heart rate in the mollusc U. pictorum, were found when both factors (elodea and cyanobacteria) were combined. The applied endpoints could be further developed for environmental monitoring, but the obtained results support the importance of the combined use of several biomarkers and species, especially in the case of multi-factor environmental stress.

Estimation of production and sedimentation of cyanobacterial toxins (microcystin) based on nutrient budgets in the reservoir of Isahaya Bay, Japan.

The increasing eutrophication of freshwater and brackish habitats globally has led to a corresponding increase in the occurrence of harmful cyanobacterial blooms. Cyanobacteria can produce highly toxic substances such as microcystins (MCs) that affect the health of livestock, wildlife, and humans. The present study broaden the understanding of cyanobacteria ecology and MC dynamics in the field, focusing on the estimation of the production and sedimentation rates of MCs in a natural habitat. The nutrient concentrations of the reservoir water and sediment pore water were monitored at 3-h intervals for 24 h during the summer cyanobacterial bloom. The DIN uptake rate of Microcystis in the Isahaya reservoir was estimated and the large-scale blooms in the reservoir were largely controlled by the interactions between rainfall and nutrient levels in the warm season. By using calculations based on the nitrogen budgets and tracking changes of the MC concentrations in the water column, the total MC production and sedimentation rates were estimated to be 52.2 kg MCs d-1 and 21.5 kg MCs d-1, respectively. Although MCs could be degraded in the environment, the MC sedimentation still comprised 41% of the in-water production.

Simple and practical on-site treatment of high microcystin levels in water using polypropylene plastic.

Microcystin (MC) is a hepatotoxin produced by various cyanobacteria during harmful algal blooms (HAB's) in freshwater environments. Advanced treatment methods can remove MC from drinking water, but are costly and do not address recreational water exposure and ecosystem health concerns. Here we investigate the feasibility of utilizing plastics as a MC-adsorbing material, for use in water resources used for recreation, agriculture, aquaculture and drinking water. Water containing 20 µg/L MC-LR was exposed to polypropylene (PP) plastic for a six-day period at varying temperatures (22, 37, 65°C). Water samples were then collected at 0, 1, 2, and 6 hour-intervals to examine short term treatment feasibility. Samples were also taken at 24 hours, 3 days, and 6 days to determine long-term treatment effectiveness. MC concentrations were analyzed using ELISA. Results showed a maximal reduction of nearly 70% of MC-LR after a 6-day treatment with PP at 65°C. Temperature enhanced MC-LR reduction over a 6-day period: 70% reduction at 65°C; 50% at 37°C; 38% at 22°C. We propose an inexpensive intervention strategy which can be deployed rapidly on-site in various source waters, including in resource-limited settings. During the high peak of HAB season, the strategy can be applied in source waters, alleviating water treatment burden for treatment plants, lowering treatment costs and reducing chemical usage.

First report on free and covalently bound microcystins in fish and bivalves from Vietnam: Assessment of risks to humans.

The free and covalently bound microcystins (MCs) in 3 fish and 2 bivalves from the Dau Tieng Reservoir in Vietnam were investigated for the first time in the present study. The results showed that all species were contaminated with MCs. Our findings indicate that eating the muscle of fish from the Dau Tieng Reservoir is safe but that eating the bivalves is not safe during toxic cyanobacterial bloom episodes. Environ Toxicol Chem 2017;36:2953-2957. © 2017 SETAC.

Assessment of uptake and phytotoxicity of cyanobacterial extracts containing microcystins or cylindrospermopsin on parsley (Petroselinum crispum L.) and coriander (Coriandrum sativum L).

Blooms of harmful cyanobacteria that synthesize cyanotoxins are increasing worldwide. Agronomic plants can uptake these cyanotoxins and given that plants are ultimately ingested by humans, this represents a public health problem. In this research, parsley and coriander grown in soil and watered through 7 days with crude extracts containing microcystins (MCs) or cylindrospermopsin (CYN) in 0.1-1 µg mL-1 concentration range were evaluated concerning their biomass, biochemical parameters and uptake of cyanotoxins. Although biomass, chlorophylls (a and b), carotenoids and glutathione-S-transferase of parsley and coriander exposed to the crude extracts containing MC or CYN had shown variations, these values were not statistically significantly different. Protein synthesis is not inhibited in coriander exposed to MC or CYN and in parsley exposed to MC. Also, glutathione reductase (GR) and glutathione peroxidase (GPx) in parsley and coriander was not affected by exposure to MC, and in coriander, the CYN did not induce statistically significant differences in these two antioxidative enzymes. Only parsley showed statistically significant increase in protein content exposed to 0.5 µg CYN mL-1 (3.981 ± 0.099 mg g-1 FW) compared to control (2.484 ± 0.145 mg g-1 FW), statistically significant decrease in GR exposed to 0.1 µg CYN mL-1 (0.684 ± 0.117 nmol min-1 mg-1 protein) compared to control (1.30 ± 0.06 nmol min-1 mg-1 protein) and statistically significant increase in GPx exposed to 1 µg CYN mL-1 (0.054 ± 0.026 nmol min-1 mg-1 protein) compared to 0.5 µg CYN mL-1 (0.003 ± 0.001 nmol min-1 mg-1 protein). These changes may be due to the induction of defensive mechanisms by plants by the presence of toxic compounds in the soil or probably to a low generation of reactive oxygen species. Furthermore, the parsley and coriander leaves and stems after 10 days of exposure did not accumulate microcystins or cylindrospermopsin.

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, [Asp(3)]MC-RAba, and [Glu(OCH3)(6)]MC-LR) and two new congeners ([Asp(3)]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.

Microcystin-LR bioaccumulation and depuration kinetics in lettuce and arugula: Human health risk assessment.

Microcystin-LR (MC-LR) is one of the most toxic and common microcystins (MCs) variant found in aquatic ecosystems. Little is known about the possibility of recovering microcystins contaminated agricultural crops. The objectives of this study were to determine the bioaccumulation and depuration kinetics of MC-LR in leaf tissues of lettuce and arugula, and estimate the total daily intake (ToDI) of MC-LR via contaminated vegetables by humans. Arugula and lettuce were irrigated with contaminated water having 5 and 10µgL(-1) of MC-LR for 7days (bioaccumulation), and subsequently, with uncontaminated water for 7days (depuration). Quantification of MC-LR was performed by LC-MS/MS. The one-compartment biokinetic model was employed for MC-LR bioaccumulation and depuration data analysis. MC-LR was only accumulated in lettuce. After 7days of irrigation with uncontaminated water, over 25% of accumulated MC-LR was still retained in leaf tissues of plants treated with 10µgL(-1) MC-LR. Total daily toxin intake by adult consumers (60kg-bw) exceeded the 0.04µgMC-LRkg(-1) limit recommended by WHO. Bioaccumulation was found to be linearly proportional to the exposure concentration of the toxin, increasing over time; and estimated to become saturated after 30days of uninterrupted exposure. On the other hand, MC-LR depuration was less efficient at higher exposure concentrations. This is because biokinetic half-life calculations gave 2.9 and 3.7days for 5 and 10µgL(-1) MC-LR treatments, which means 29-37days are required to eliminate the toxin. For the first time, our results demonstrated the possibility of MC-LR decontamination of lettuce plants.

Preliminary Assessment of Cyanobacteria Diversity and Toxic Potential in Ten Freshwater Lakes in Selangor, Malaysia.

Toxic cyanobacteria blooms are increasing in magnitude and frequency worldwide. However, this issue has not been adequately addressed in Malaysia. Therefore, this study aims to better understand eutrophication levels, cyanobacteria diversity, and microcystin concentrations in ten Malaysian freshwater lakes. The results revealed that most lakes were eutrophic, with total phosphorus and total chlorophyll-a concentrations ranging from 15 to 4270 µg L(-1) and 1.1 to 903.1 µg L(-1), respectively. Cyanobacteria were detected in all lakes, and identified as Microcystis spp., Planktothrix spp., Phormidium spp., Oscillatoria spp., and Lyngbya spp. Microcystis spp. was the most commonly observed and most abundant cyanobacteria recorded. Semi-quantitative microcystin analysis indicated the presence of microcystin in all lakes. These findings illustrate the potential health risk of cyanobacteria in Malaysia freshwater lakes, thus magnifying the importance of cyanobacteria monitoring and management in Malaysian waterways.

Assessment of the factors contributing to the variations in microcystins biodegradability of the biofilms on a practical biological treatment facility.

This study revealed the biotic and abiotic parameters driving the variations in microcystins (MCs) biodegradability of a practical biological treatment facility (BTF). Results showed that similar trends of seasonal variation were seen for microcystin-LR (MCLR) biodegradability of biofilms on the BTF and indigenous MCLR-degrader population, where both peaks co-occurred in October, following the peaks of natural MCLR concentration and water temperature observed in August. The lag period might be required for accumulation of MCLR-degraders and MCLR-degrading enzyme activity. The MCLR-degrader population was correlated to temperature, MCLR and chlorophyll-a concentration in water where the biofilms submerged, indicating that these abiotic and biotic parameters exerted direct and/or indirect influences on seasonal variation in MCLR-biodegradability. In comparison, no effect of other co-existing MCs on biodegradation of one MC was observed. However, proliferation of MC-degraders along biodegradation processes positively responded to total amount of MCs, suggesting that multiple MCs contributed additively to MC-degrader proliferation.

Bioaccumulation of microcystins (MCs) in four fish species from Lake Taihu, China: assessment of risks to humans.

Microcystins (MCs) are the toxic products of harmful algal blooms and they accumulate in fish. The accumulation of MCs in fish living in different trophic levels from different parts of Lake Taihu was determined. This information was then used to evaluate the risks posed by the MCs in fish to human health. The concentrations of three MCs, MC-LR, MC-YR and MC-RR, were quantified in the following four fish species: silver carp (Hypophthalmichthys molitrix), bighead carp (Aristichthys nobilis), crucian carp (Carassius auratus) and common carp (Cyprinus carpio), using high performance liquid chromatography interfaced with tandem (triple quadrupole) mass spectrometry. The mean concentrations of MCs in the muscle, the kidney, the intestinal wall and the heart were significantly different among the four fishes except in the liver. C. carpio contained the highest mean concentration of MCs in the muscle (31.7 ± 12.1 ng/g, dry mass (dm)), whereas C. auratus had the highest mean concentrations of MCs in the liver (45.4 ± 44.5 ng/g, dm), kidney (114 ± 51.1 ng/g, dm), intestinal wall (2.04 × 10(3)± 4.43 × 10(3)ng/g, dm) and heart (59.5 ± 26.7 ng/g, dm). The mean concentration of MCs in the intestinal walls of the fish species was significantly higher than in other organs (p<0.01). The fish from Meiliang Bay had significantly higher concentrations of MCs than those from the centre, west or south banks of the lake (p<0.01). The body lengths and masses of the fish were negatively correlated with the concentrations of MCs in the kidney (p<0.05) and heart (p<0.01). The average daily intake (ADI) of MCs in the muscle of all fishes exceeded the provisional tolerable daily intake (TDI) set by World Health Organization. The estimated daily intakes of MCs in 55.6% of the muscle samples exceeded the TDI. The MCs in the tissues of the fish from Lake Taihu pose potential risks to the health of humans who consume these four fish species.

Assessment of microcystin concentration in carp and catfish: a case study from Lakshmikund pond, Varanasi, India.

The present study was conducted to analyse microcystin concentrations in Lakshmikund pond, Varanasi, India, as well as in carp and catfish of the pond. The concentrations of microcystin were found well above the WHO guidelines (1 µg/L) both for the dissolved and particulate fractions of bloom samples. The microcystin concentrations in different organs of carp and catfish were in the following sequence; liver > gut > kidney > gall bladder > gills > muscles and gut > liver > kidney > gall bladder > gills > muscles, respectively. The bioaccumulation of microcystin in carp and catfish was negatively correlated with body weight, and showed species specificity. The higher bioaccumulation of microcystin in muscles of catfish (>tenfold) over carp indicates a possible threat to human beings on consumption of catfish. Therefore, to avoid animal and human intoxication, routine analyses of microcystin in pond water as well as fishes are strongly recommended.

Quantitative PCR detection and improved sample preparation of microcystin-producing Anabaena, Microcystis and Planktothrix.

Blooms of toxic cyanobacteria, associated with illness and mortality in humans and animals, are becoming increasingly common worldwide. The safe use of surface waters for drinking water production and recreation necessitates assessment of toxigenic cyanobacteria. We have developed simple and reliable sample preparation and qPCR methods to detect microcystin-producing strains of three major bloom-forming genera, Anabaena, Microcystis and Planktothrix. The mcyB second thiolation motif, previously not recognized as a potential target for qPCR, was used as a basis for primer and genus-specific probe design. Assay specificity and sensitivity was confirmed with cultured cyanobacterial strains and the effect of different sample preparation methods on quantification was investigated. Sample filtration and cell lysis reduced assay time and resulted in more efficient amplification compared to DNA extraction. Positive correlation (p<0.005) between mcyB copy numbers and microcystin concentrations was observed in environmental samples. The results encourage the use of qPCR in water risk management.

Assessment of microcystins in lake water and fish (Mugilidae, Liza sp.) in the largest Spanish coastal lake.

Cyanobacteria dominance and cyanotoxin production can become major threats to humans and aquatic life, especially in warm shallow lakes, which are often dominated by cyanobacteria. This study investigates the occurrence and distribution of microcystins (MCYST) in water, cell-bound and in the tissues of the commercial mugilid Liza sp. in the largest, coastal, Spanish Mediterranean lake (Albufera of Valencia). This is the first report concerning microcystin accumulation in tissues of mugilid fish species. Considerable amounts of microcystins were found in the water and seston, which correlated with development of Microcystis aeruginosa populations in the lake. The MCYST concentrations found in Lake Albufera (mean 1.7 and 17 µg/L and maximum 16 and 120 µg/L in water and seston, respectively) exceeded by one to two orders of magnitude the guideline levels proposed by the World Health Organization and were higher than that reported in other lakes of the Mediterranean zone. The presence of MCYST was found in all the fishes studied and accumulated differently among tissues of the commercial species Liza sp. Toxin accumulation in fish tissues showed that although the target organ for MCYST was the liver, high concentrations of microcystins were also found in other analysed tissues (liver>intestine>gills>muscle). Human tolerable daily intake for microcystins is assessed relative to the WHO guidelines, and potential toxicological risks for humans, wildlife and related ecosystems of the lake are discussed.