A Summer of Cyanobacterial Blooms in Belgian Waterbodies: Microcystin Quantification and Molecular Characterizations

In the context of increasing occurrences of toxic cyanobacterial blooms worldwide, their monitoring in Belgium is currently performed by regional environmental agencies (in two of three regions) using different protocols and is restricted to some selected recreational ponds and lakes. Therefore, a global assessment based on the comparison of existing datasets is not possible. For this study, 79 water samples from a monitoring of five lakes in Wallonia and occasional blooms in Flanders and Brussels, including a canal, were analyzed. A Liquid Chromatography with tandem mass spectrometry (LC-MS/MS) method allowed to detect and quantify eight microcystin congeners. The mcyE gene was detected using PCR, while dominant cyanobacterial species were identified using 16S RNA amplification and direct sequencing. The cyanobacterial diversity for two water samples was characterized with amplicon sequencing. Microcystins were detected above limit of quantification (LOQ) in 68 water samples, and the World Health Organization (WHO) recommended guideline value for microcystins in recreational water (24 µg L⁻¹) was surpassed in 18 samples. The microcystin concentrations ranged from 0.11 µg L⁻¹ to 2798.81 µg L⁻¹ total microcystin. For 45 samples, the dominance of the genera Microcystis sp., Dolichospermum sp., Aphanizomenon sp., Cyanobium/Synechococcus sp., Planktothrix sp., Romeria sp., Cyanodictyon sp., and Phormidium sp. was shown. Moreover, the mcyE gene was detected in 75.71% of all the water samples.     

The effects of Microcystis aeruginosa cells lysate containing microcystins on physiological and molecular responses in the nematode Caenorhabditis elegans

Microcystins (MCs) are potent toxins produced by environmental cyanobacterial blooms. The present study evaluated the effects of a Microcystis aeruginosa cyanobacterial lysate containing 0.1, 1, and 10 μg L^?1 MC‐LR equivalent in the C. elegans Bristol N2 wild‐type and the effects caused by equivalent concentrations of a MC‐LR standard. The lysate was prepared from a culture of toxic strain (RST9501) originated from the Patos Lagoon Estuary (RS, Brazil). The minimal concentration necessary to cause significant effects in C. elegans under exposure to M. aeruginosa lysate or to MC‐LR standard were, respectively, 10 and 0.1 μg L^?1 MC‐LR equivalent for growth and 10 and 1 μg L^?1 MC‐LR equivalent for fertility. Reproduction (ie, brood size) was only affected by the exposure to 10 μg L^?1 MC‐LR standard and was not affected by the lysate. The nematodes that were exposed to lysate containing 1 μg L^?1 MC‐LR equivalent or MC‐LR were also analyzed for pharyngeal pumping and gene expression using RT‐qPCR. The worms’ rhythmic contractions of the pharynx were similarly affected by the lysate containing 1 μg L^?1 of MC‐LR equivalent and the MC‐LR standard. The MC‐LR standard caused down‐regulation of genes related to growth (daf‐16), fertility (spe‐10), and biotransformation (gst‐2). This is the first study to evaluate the effects of a toxic cyanobacterial lysate using the C. elegans model. This study suggests the organism as a potential biotest to evaluate toxicity of natural waters containing M. aeruginosa cells and to environmental risk assessment associated to cyanobacterial bloom events.     

Analysis of factors affecting the ratio of microcystin to chlorophyll‐a in cyanobacterial blooms using real‐time polymerase chain reaction

Chlorophyll‐a concentration has been used as an indicator to estimate microcystin levels in water bodies. This study examined the microcystin to chlorophyll‐a ratio in a fishpond in Japan. The ratio varied spatially and temporally during the six‐month field survey, which is consistent with reports by other researchers. We investigated the causes of the variability of the ratio by quantifying microcystin synthetase (mcy) A gene with real‐time PCR, so as to observe the growth of microcystin‐producing cyanobacteria and Microcystis strains in natural cyanobacterial blooms. The application of real‐time PCR enabled corroboration of the relationship between the toxigenicity and the toxicity of the blooms. The microcystin to chlorophyll‐a ratio was influenced by the combined effects of the durability of the toxic bloom, and the quantity of microcystin‐producing cyanobacteria carrying the mcy A gene, especially toxic Microcystis strains. Cyanobacterial blooms produced more microcystin when high concentrations of microcystin‐producing Microcystis aggregated in a stationary state with low growth rates. The variable toxicity of blooms needs to be reflected in accurate and efficient alert systems for toxic cyanobacteria and cyanotoxins. © 2009 Wiley Periodicals, Inc. Environ Toxicol 26: 21–28, 2011.     

Persistence of cyclic peptide toxins in dried Microcystis aeruginosa crusts from lake Mokoan,Australia

Dried Microcystis aeruginosa Kuetzing emend. Elenkin crusts estimated to be 5–6 months old from the shore of Lake Mokoan were toxic by mouse bioassay (LD₁₀₀ 100–140 mg dry wt/kg mouse). Fresh bloom material from the lake was also highly toxic (LD₁₀₀ 25–35 mg dry wt/kg mouse). Microcystin high performance liquid chromatography (HPLC) profiles of the crust and fresh material were very similar, with 24 compounds having UV spectra consistent with microcystin LR. Five of the major microcystins were purified and analysed by electrospray/mass spectrometry. The molecular weights of these microcystins [910, 924, 982, 982 (two compounds), and 986] do not correspond with known microcystins. All five compounds were hepatotoxic to mice with LD₁₀₀ values ranging from 85 to 140 μg microcystin/kg mouse). Total microcystin contents (expressed as microcystin LR aquivalents) determined by HPLC correlated with the mouse bioassay analyses (crust 2.1 μg microcystin/mg dry wt; fresh 4.1 μg microcystin/mg dry wt). These results suggest that microcystin is protected from degradation while encapsulated within the dried Microcystis crusts. Leaching experiments demonstrated that re-wetting of the crust material leads to rapid release of microcystins into the surrounding water. These observations have important management implications for lakes and reservoirs where crusts of cyanobacterial material form on the shoreline. © by John Wiley & Sons, Inc.     

Effect of different cyanobacterial biomasses and their fractions with variable microcystin content on embryonal development of carp (Cyprinus carpio L.)

While numerous studies focused on the effects of microcystins, the role of other components of complex cyanobacterial water blooms in toxicity is poorly understood. In this study we have evaluated effects of various fractions of cyanobacterial biomass with different composition and microcystin content on embryolarval development of carp (Cyprinus carpio). The following samples (fractions) of four natural water blooms were prepared and tested: complex cyanobacterial biomass, crude aqueous extract of biomass, cellular pellet remaining from aqueous extract, permeate (i.e. microcystin-free fraction prepared during C-18 solid-phase extraction; SPE), and eluate (i.e. fraction prepared by SPE containing mostly microcystins). Complex biomass and the crude aqueous extract (regardless of microcystin content and/or microcystin variants present) in the sample were the most toxic. On the other hand, eluate fractions of all samples containing microcystins in concentrations 8-255 microgL(-1) induced no or only weak toxic effects. Exposures of fish to permeate fractions (with removed microcystins) of two samples dominated by Aphanizomenon sp. and Planktothrix sp. resulted in significant mortality, while other two samples dominated by Microcystis spp. induced minor effects. We have also observed significant inhibition of glutathione S-transferases (GST) at most fractions of the Aphanizomenon sp. and Planktothrix sp. dominated samples. Our data indicate that cyanobacterial water blooms as well complex biomass extracts induce significant embryolarval toxicity in common carp. However, these effects were independent of microcystin content, and the most pronounced effects were observed with the non-Microcystis dominated samples. Therefore, a critical examination of microcystin role in overall ecotoxicology of complex cyanobacterial blooms is needed.     

Comparative assessment of the specificity of the brine shrimp and microtox assays to hepatotoxic (microcystin-LR-containing) cyanobacteria

Specific, straightforward, and rapid procedures are required for the detection, identification, and quantification of the potent low molecular weight toxins that are produced by blooms and scums of cyanobacteria (blue-green algae) in waterbodies. Use of the Microtox bioluminescence assay and the brine shrimp (Artemia salina) has been advocated for the initial screening of cyanobacterial blooms for microcystin hepatotoxins. Inhibition of bacterial luminescence in the Microtox assay and brine shrimp mortality were determined with microcystin-containing and nonmicrocystin-containing cyanobacteria. Extraction and fractionation of test samples was undertaken to select and isolate microcystincontaining fractions and reduce interference from other fractions. Maximal inhibition of bacterial luminescence in the Microtox assay occurred with fractions from Microcystis strains and an Anabaena bloom that did not contain microcystins. By contrast, the bioassay of fractions using brine shrimps correlated with the distribution of microcystin-LR in the fractionated Microcystis extracts. © 1994 by John Wiley & Sons, Inc..     

Recent trends in development of biosensors for detection of microcystin

Increased cyanobacterial blooms, a source of cyanotoxins are linked with climate change and eutrophication in aquatic bodies, a major concern worldwide. Microcystins are potently hepatotoxic, nephrotoxic as well as carcinogenic. Thus microcystins are threat to tourism, agriculture and animal's health. However, there is a still lacuna in the knowledge of regulation of microcystins production. Presence of toxic and non-toxic cyanobacterial strains together and occurrence of various microcystin variants in aquatic bodies compounded the problem. Although several analytical techniques for microcystin detection such as bioassay, ELISA, HPLC and LC-MS etc. have been already prevalent, the development of biosensors offered rapid and accurate detection, high reproducibility and portability. Sequencing of Microcystis spp., opened the new vistas towards the development of biosensor at molecular and genetic level. This review incorporates the current trends in the development of biosensors for microcystin detection in the light of state-of-the-art techniques.     

Changes in concentrations of microcystins in rainbow trout, freshwater mussels, and cyanobacteria in Lakes Rotoiti and Rotoehu

Microcystin concentrations in cyanobacteria and their accumulation in rainbow trout (Oncorhynchus mykiss) and freshwater mussels (Hyridella menziesi) in Lakes Rotoiti and Rotoehu (New Zealand) were investigated. Hatchery rainbow trout were added to an enclosure in Lake Rotoiti where concentrations of microcystins in the phytoplankton and cyanobacterial cell concentrations could be closely monitored. Rainbow trout that were free to roam in the entire area of each lake were also included in the study. Freshwater mussels were suspended subsurface in cages in the enclosure. Phytoplankton samples, rainbow trout liver and muscle tissue, and the tissues of mussels were analyzed for microcystins using the ADDA-ELISA method, and selected samples were analyzed using LC-MS. A maximum concentration of microcystins in the phytoplankton samples of 760 microg L(-1) was recorded in Te Weta Bay, Lake Rotoiti, in March 2004. ELISA results confirmed microcystin immunoreactivity in rainbow trout liver and muscle tissues and in freshwater mussels. The microcystin congeners LR, YR, RR, AR, FR, LA, and WR were detected by LC-MS in caged freshwater mussels in Lake Rotoiti but were not detected in either muscle or liver tissue of rainbow trout. The daily tolerable intake limit of microcystins for human consumption recommended by the World Health Organisation is 0.04 microg kg(-1) day(-1). Modeling was carried out for the human intake of microcystin compounds from rainbow trout muscle tissue, and the potential health risks were estimated, assuming the ADDA-ELISA was determining compounds of toxicity equivalent to microcystin-LR.     

Effects of lead on Na+, K+‐ATPase and hemolymph ion concentrations in the freshwater mussel Elliptio complanata

Freshwater mussels are an imperiled fauna exposed to a variety of environmental toxicants such as lead (Pb) and studies are urgently needed to assess their health and condition to guide conservation efforts. A 28‐day laboratory toxicity test with Pb and adult Eastern elliptio mussels (Elliptio complanata) was conducted to determine uptake kinetics and to assess the toxicological effects of Pb exposure. Test mussels were collected from a relatively uncontaminated reference site and exposed to a water‐only control and five concentrations of Pb (as lead nitrate) ranging from 1 to 245 μg/L in a static renewal test with a water hardness of 42 mg/L. Endpoints included tissue Pb concentrations, hemolymph Pb and ion (Na⁺, K⁺, Cl^?, Ca²⁺) concentrations, and Na⁺, K⁺‐ATPase enzyme activity in gill tissue. Mussels accumulated Pb rapidly, with tissue concentrations increasing at an exposure‐dependent rate for the first 2 weeks, but with no significant increase from 2 to 4 weeks. Mussel tissue Pb concentrations ranged from 0.34 to 898 μg/g dry weight, were strongly related to Pb in test water at every time interval (7, 14, 21, and 28 days), and did not significantly increase after day 14. Hemolymph Pb concentration was variable, dependent on exposure concentration, and showed no appreciable change with time beyond day 7, except for mussels in the greatest exposure concentration (245 μg/L), which showed a significant reduction in Pb by 28 days, suggesting a threshold for Pb binding or elimination in hemolymph at concentrations near 1000 μg/g. The Na⁺, K⁺‐ATPase activity in the gill tissue of mussels was significantly reduced by Pb on day 28 and was highly correlated with tissue Pb concentration (R² = 0.92; P = 0.013). The Na⁺, K⁺‐ATPase activity was correlated with reduced hemolymph Na⁺ concentration at the greatest Pb exposure when enzyme activity was at 30% of controls. Hemolymph Ca²⁺ concentration increased significantly in mussels from the greatest Pb exposure and may be due to remobilization from the shell in an attempt to buffer the hemolymph against Pb uptake and toxicity. We conclude that Na⁺, K⁺‐ATPase activity in mussels was adversely affected by Pb exposure, however, because the effects on activity were variable at the lower test concentrations, additional research is warranted over this range of exposures. © 2010 Wiley Periodicals, Inc. Environ Toxicol, 2012.     

Monitoring toxigenic Microcystis strains in the Missisquoi bay,Quebec, by PCR targeting multiple toxic gene loci

The increasing incidence of mixed assemblages of toxic and nontoxic cyanobacterial blooms in Quebec's freshwater bodies over the last decade, coupled with inherent inadequacies of current monitoring approaches, warrants development of sensitive and reliable tools for assessing the toxigenic potential of these water blooms. In this study, we applied three independent polymerase chain reaction (PCR) assays that simultaneously target the microcystin synthetase (mcy) genes A, E, and G to rapidly and reliably detect and quantify potentially toxic Microcystis genotypes in the Missisquoi bay, Quebec, Canada. Linear regressions of quantitative PCR threshold cycles (C_t) against the logarithm of their respective Microcystis cell number equivalents resulted in highly significant linear curves with coefficients of determination (R²) greater than 0.99 (p < 0.0001, n = 6) and reaction efficiencies of 91.0, 95.8, and 92.7%, respectively, for the mcyA, mcyE, and mcyG‐based quantitative real‐time PCR (qPCR) assays. The three assays successfully estimated potential microcystin‐producing Microcystis genotypes from all field samples. The proportions of MicrocystismcyA, mcyE, and mcyG genotypes to total Microcystis cell counts showed substantial spatial variability ranging between 1.7–21.6%, 1.9–11.2%, and 2.2–22.6%, respectively. Correlation of microscopically determined total Microcystis counts to qPCR‐based MicrocystismcyA, mcyE, or mcyG cell number equivalents resulted in highly significant associations with R² > 0.90. Thus, PCR‐based assays targeting the mcyA, mcyG, and/or mcyE genes can serve as powerful screening tools for rapid and sensitive estimation of microcystin‐producing Microcystis genotypes in freshwater water bodies. © 2012 Wiley Periodicals, Inc. Environ Toxicol 29: 440–451, 2014.     

Recreational exposure to microcystins during algal blooms in two California lakes

We conducted a study of recreational exposure to microcystins among 81 children and adults planning recreational activities on either of three California reservoirs, two with significant, ongoing blooms of toxin-producing cyanobacteria, including Microcystis aeruginosa (Bloom Lakes), and one without a toxin-producing algal bloom (Control Lake). We analyzed water samples for algal taxonomy, microcystin concentrations, and potential respiratory viruses (adenoviruses and enteroviruses). We measured microcystins in personal air samples, nasal swabs, and blood samples. We interviewed study participants for demographic and health symptoms information. We found highly variable microcystin concentrations in Bloom Lakes (<10 μg/L to >500 μg/L); microcystin was not detected in the Control Lake. We did not detect adenoviruses or enteroviruses in any of the lakes. Low microcystin concentrations were found in personal air samples (<0.1 ng/m³ [limit of detection]-2.89 ng/m³) and nasal swabs (<0.1 ng [limit of detection]-5 ng). Microcystin concentrations in the water-soluble fraction of all plasma samples were below the limit of detection (1.0 μg/L). Our findings indicate that recreational activities in water bodies that experience toxin-producing cyanobacterial blooms can generate aerosolized cyanotoxins, making inhalation a potential route of exposure. Future studies should include collecting nasal swabs to assess upper respiratory tract deposition of toxin-containing aerosols droplets.     

Supernatants from Macrophages Stimulated with Microcystin‐LR Induce Electrogenic Intestinal Response in Rabbit Ileum

Microcystin‐LR is a cyclic heptapeptide hepatotoxin produced by the cyanobacterium Microcystis aeruginosa. This microorganism often forms toxic blooms in freshwater lakes and reservoirs for drinking water supply, producing serious disorders in humans and animals. Some have suggested that certain biological activities of microcystin may depend upon the stimulation of immune cells. Therefore, the aims of this research were to examine electrogenic intestinal secretion, in vitro, caused by the supernatants from macrophages stimulated with microcystin‐LR, as well as to investigate the presence of interleukin‐1β and tumour necrosis factor‐α in these supernatants. We found that the supernatants of macrophages stimulated with microcystin‐LR (0.1, 0.3 and 1.0 μg/ml) caused electrogenic intestinal effects (change in short‐circuit currents (Δ SCC)=57.6, 50.8 and 73.3, respectively, versus control=19.6 μA.cm^?2) in a time‐dependent way (microcystin‐LR (1.0 μg/ml)=63.2, 108.8, 120.4 and 132.3 μA.cm^?2 at time 0, 40, 50 and 60 min., respectively). In addition, the intestinal secretory activity present in these supernatants was blocked (57%) by the prior treatment of macrophages with dexamethasone. We also demonstrated that microcystin‐LR (0.1, 0.3 and 1.0 μg/ml) is capable of stimulating the synthesis of tumour necrosis factor‐α (375.4, 369.0 and 610.8 pg/ml, respectively, versus control=165.0 pg/ml) and interleukin‐1β (198.9, 189.3 and 522.1 pg/ml, respectively, versus control=39.7 pg/ml). These findings demonstrate that microcystin‐LR induces the release of interleukin‐1β and tumour necrosis factor‐α by peritoneal macrophages in vitro, and that the supernatants from these macrophages induce electrogenic secretion in rabbit ileal mucosa.     

Temporal variabilities of the concentrations of intra- and extracellular microcystin and toxic Microcystis species in a hypertrophic lake,Lake Suwa,Japan (1991–1994)

Temporal variability in the concentration of toxic heptapeptide microcystin was studied during the warm season of four years (1991–1994) in a hypertrophic lake (Lake Suwa) in central Honshu, Japan. Lake water samples (ca. 5 L) were filtered to separate intracellular microcystin (cell fraction) from extracellular microcystin (filtered lake water fraction). These fractions were analyzed to measure the total quantity of microcystin in lake water. Total amounts of extra- and intracellular microcystin were measured with high performance liquid chromatography. Concentrations of intracellular microcystin usually exceeded concentrations of extracellular microcystin (24 out of 26 times). High concentrations of intracellular microcystin were found during the exponential growth phase of the blooms, whereas concentrations of extracellular microcystin were highest at the end of the blooms. However, concentrations of extracellular microcystin remained very small (<4μg/L) compared to the levels of intracellular microcystin. The relatively higher percentages of microcystin in filtered lake water ( > 20%) at the end of blooms suggests that release of microcystin from cells occurs during senescence and the decomposition period of Microcystis cells. © 1998 John Wiley & Sons, Inc. Environ Toxicol Water Qual 13 : 61–72, 1998     

Effects of low‐dose exposure to pesticide mixture on physiological responses of the pacific oyster,Crassostrea gigas

This study investigated the effects on the physiology of Pacific oyster, Crassostrea gigas, of a mixture of pesticides containing 0.8 μg L^?1 alachlor, 0.6 μg L^?1 metolachlor, 0.7 μg L^?1 atrazine, 0.6 μg L^?1 terbuthylazine, 0.5 μg L^?1 diuron, 0.6 μg L^?1 fosetyl aluminum, 0.05 μg L^?1 carbaryl, and 0.7 μg L^?1 glyphosate for a total concentration of 4.55 μg L^?1. The total nominal concentration of pesticides mixture corresponds to the pesticide concentrations in the shellfish culture area of the Marennes‐Oleron basin. Two varieties of C. gigas were selected on the foreshore, based on their characteristics in terms of resistance to summer mortality, to assess the effects of the pesticide mixture after 7 days of exposure under controlled conditions. The early effects of the mixture were assessed using enzyme biomarkers of nitrogen metabolism (GS, glutamine synthetase), detoxification metabolism (GST, glutathione S‐transferase), and oxidative stress (CAT, catalase). Sublethal effects on hemocyte parameters (phagocytosis and esterase activity) and DNA damages (DNA adducts) were also measured. Changes in metabolic activities were characterized by increases in GS, GST, and CAT levels on the first day of exposure for the “resistant” oysters and after 3–7 days of exposure for the “susceptible” oysters. The formation of DNA adducts was detected after 7 days of exposure. The percentage of hemocyte esterase‐positive cells was reduced in the resistant oysters, as was the hemocyte phagocytic capacity in both oyster varieties after 7 days of exposure to the pesticide mixture. This study highlights the need to consider the low doses and the mixture of pesticides to evaluate the effects of these molecules on organisms. © 2011 Wiley Periodicals, Inc. Environ Toxicol 28: 689–699, 2013.     

Microcystins associated with Microcystis dominated blooms in the Southwest wetlands, Western Australia

Potentially toxic cyanobacterial blooms are becoming common in the freshwater wetlands on the Swan Coastal Plain, Western Australia. During summer the dominant bloom-causing species belong to the genera Microcystis and Anabaena and to a lesser extent Aphanizomenon and Nodularia. Although toxic cyanobacteria have been recorded in the Swan-Canning and Peel-Harvey estuaries in Western Australia, very little is known about the blooms in the surrounding freshwater lakes. In this study, a total of 32 natural bloom samples representing 13 lakes were analyzed by HPLC for microcystin (MC)-LR, -RR, and -YR. Twenty-eight samples proved to be toxic. The highest total microcystin concentration ranged from 1645 to 8428.6 microg L(-1), and the lowest concentrations were less than 10 microg L(-1) with some below the detection limit (< 0.05 microg L(-1)). MC-LR (100%) was the predominant microcystin, followed by MC-YR (71.4%) and MC-RR (60.7%). The presence of a Nodularia spumigena bloom in the freshwater Lake Yangebup was associated with the detection of nodularins (1664 microg L(-1)). This is the first study to demonstrate the presence of microcystins and nodularins in urban lakes on the Swan Coastal Plain, Western Australia.     

Effects of 17α‐ethynylestradiol and nonylphenol on liver and gonadal apoptosis and histopathology in Chalcalburnus tarichi

Chalcalburnus tarichi is an endemic cyprinid fish living in the Lake Van basin located in the Eastern Turkey. Fish (3+ ages) were exposed to 17α‐ethynylestradiol (EE₂; 1, 10, 100 ng L^?1; nominal concentrations) and nonylphenol (NP; 10, 60, 200 μg L^?1; nominal concentrations) for 32 days under semistatic daily renewal conditions. The exposure period was followed by an evaluation of liver and gonadal apoptosis and gonad histopathology in males and females. Exposure to the highest concentrations of EE₂ (100 ng L^?1) and NP (200 μg L^?1) caused significant increases in the extent of apoptosis in liver and gonads. Treatment with 100 ng L^?1 of EE₂ and 200 μg L^?1 NP increased the number of TUNEL positive hepatocytes significantly in both sexes compared to controls. The rates of apoptosis in testicular germ cells and ovarian follicular cells were significantly greater at the same concentrations. Exposure to EE₂ (100 ng L^?1) and NP (60 and 200 μg L^?1) caused thickening of interstitial connective tissue (fibrosis) in the seminiferous tubule wall and testis‐ova formation in males. In females treated with 100 ng L^?1 EE₂, increased percentage of atretic ooctytes and fibrotic areas in the ovarian somatic stromal tissue were found in the ovaries. Increase in atresia, without a statistical significance, and fibrotic stromal tissue were also noted in 60 and 200 μg L^?1 NP treatments. Results suggest that EE₂‐ and NP‐dependent hepatotoxicity and gonadotoxicity are causally related to the increase in apoptosis in C. tarichi. © 2010 Wiley Periodicals, Inc. Environ Toxicol, 2011.     

Initial studies on the occurrence of cyanobacteria and microcystins in Irish lakes

We report the results of a synoptic survey at 14 sites across the north of Ireland undertaken to determine the occurrence of cyanobacteria and their constituent microcystin cyanotoxins. Seven microcystin toxins were tested for, and five of which were found, with MC‐LR, MC‐RR, and MC‐YR being the most prevalent. Gomphosphaeria spp and Microcystis aeruginosa were the most dominant cyanobacterial species encountered. Together with Aphanizomenon flos‐aquae, these were the cyanobacteria associated with the highest microcystin concentrations. The occurrence of several microcystin toxins indicates that there may potentially be more than one cyanobacteria species producing microcystins at many sites. Total microcystin concentrations varied over three orders of magnitude dividing the sites into two groups of high (>1000 ngMC/μgChla, six sites) or low toxicity (<200 ngMC/μgChla, eight sites). © 2010 Wiley Periodicals, Inc. Environ Toxicol, 2010.     

Cyanobacterium producing cylindrospermopsin cause histopathological changes at environmentally relevant concentrations in subchronically exposed tilapia (Oreochromis niloticus)

The acute toxicity of cylindrospermopsin (CYN) has been established in rodents, based on diverse intraperitoneal an oral exposure studies and more recently in fish. But no data have been reported in fish after subchronic exposure to cyanobacterial cells containing this cyanotoxin, so far. In this work, tilapia (Oreochromis niloticus) were exposed by immersion to lyophilized Aphanizomenon ovalisporum cells added to the aquaria using two concentration levels of CYN (10 or 100 μg CYN L^?1) and deoxy‐cylindrospermopsin (deoxy‐CYN) (0.46 or 4.6 μg deoxy‐CYN L^?1), during two different exposure times: 7 or 14 d. This is the first study showing damage in the liver, kidney, hearth, intestines, and gills of tilapia after subchronic exposure to cyanobacterial cells at environmental relevant concentrations. The major histological changes observed were degenerative processes and steatosis in the liver, membranous glomerulopathy in the kidney, myofibrolysis and edema in the heart, necrotic enteritis in the gastrointestinal tract, and hyperemic processes in gill lamellae and microhemorrhages. Moreover, these histopathological findings confirm that the extent of damage is related to the CYN concentration and length of exposure. Results from the morphometric study indicated that the average of nuclear diameter of hepatocytes and cross‐sections of proximal and distal convoluted tubules are useful to evaluate the damage induced by CYN in the main targets of toxicity. © 2013 Wiley Periodicals, Inc. Environ Toxicol 30: 261–277, 2015.     

Microcystis toxigenic strains in urban lakes: a case of study in Mexico City

Microcystis is a bloom-forming, common cyanobacterium in urban lakes of Mexico City. To assess the presence of potentially cyanotoxin-producing Microcystis, molecular techniques were applied and acute toxicity bioassays were performed with Daphnia magna neonates exposed to cyanobacterial crude extracts. Toxigenic potential of isolated strains was inferred by amplifying the mcyA-Cd genes and their identity as Microcystis was confirmed through the 16S rDNA and phycocyanin operon amplification. Microcystins synthesized under culture conditions were quantified through ELISA. The acute toxicity bioassays revealed that mortality was independent from the cyanotoxin concentration in some strains; this suggests the presence of other metabolites (different from microcystins) that also exerted an important biological effect. Isolated strains had the mcyA-Cd gene and most of them produced variable amounts of microcystins in the culture conditions used, confirming their toxigenic potential. Results warn about possible toxic effect risks for aquatic biota, neighboring areas, visitors and users of these sites, due to the constant presence of these blooms in the studied water bodies.     

Inhibition of gap-junctional intercellular communication and activation of mitogen-activated protein kinases by cyanobacterial extracts - Indications of novel tumor-promoting cyanotoxins?

Toxicity and liver tumor promotion of cyanotoxins microcystins have been extensively studied. However, recent studies document that other metabolites present in the complex cyanobacterial water blooms may also have adverse health effects. In this study we used rat liver epithelial stem-like cells (WB-F344) to examine the effects of cyanobacterial extracts on two established markers of tumor promotion, inhibition of gap-junctional intercellular communication (GJIC) and activation of mitogen-activated protein kinases (MAPKs) - ERK1/2. Extracts of cyanobacteria (laboratory cultures of Microcystis aeruginosa and Aphanizomenon flos-aquae and water blooms dominated by these species) inhibited GJIC and activated MAPKs in a dose-dependent manner (effective concentrations ranging 0.5-5 mg d.w./mL). Effects were independent of the microcystin content and the strongest responses were elicited by the extracts of Aphanizomenon sp. Neither pure microcystin-LR nor cylindrospermopsin inhibited GJIC or activated MAPKs. Modulations of GJIC and MAPKs appeared to be specific to cyanobacterial extracts since extracts from green alga Chlamydomonas reinhardtii, heterotrophic bacterium Klebsiella terrigena, and isolated bacterial lipopolysaccharides had no comparable effects. Our study provides the first evidence on the existence of unknown cyanobacterial toxic metabolites that affect in vitro biomarkers of tumor promotion, i.e. inhibition of GJIC and activation of MAPKs.