Natural Cyanobacterial Bloom Research Articles

Succession of particle-attached and free-living bacterial communities in response to microalgal dynamics induced by the biological cyanocide paucibactin A

Microalgae, including cyanobacteria and eukaryotic algae, are hotspots of primary production and play a critical role in global carbon cycling. However, these species often form blooms that poses a threat to aquatic ecosystems. Although the use of bacteria-derived cyanocides is regarded as an environmentally friendly method for controlling cyanobacterial blooms, only a few studies have examined their potential impact on ecosystems. This study is the first to explore the response of particle-attached (PA) and free-living (FL) bacteria to the dynamics of microalgal communities induced by the biological cyanocide paucibactin A. The microalgal community dynamics were divided into two distinct phases [phase I (days 0–2) and phase II (days 3–7)]. In phase I, paucibactin A caused a sudden decrease in the cyanobacterial concentration. Phase II was characterized by increased growth of eukaryotic microalgae (Scenedesmus, Pediastrum, Selenastrum, and Coelastrum). The stability of the bacterial community and the contribution of stochastic processes to community assembly were more pronounced in phase II than in phase I. The microalgal dynamics triggered by paucibactin A coincided with the succession of the PA and FL bacterial communities. The lysis of cyanobacteria in phase I favored the growth of microbial organic matter degraders in both the PA (e.g., Aeromonas and Rheinheimera) and FL (e.g., Vogesella) bacterial communities. In phase II, Lacibacter, Phycisphaeraceae, and Hydrogenophaga in the PA bacterial community and Lacibacter, Peredibacter, and Prosthecobacter in the FL bacterial community showed increased relative abundances. Overall, the FL bacterial community exhibited greater sensitivity to the two sequential processes compared with the PA bacterial community. These results highlight the need for studies evaluating the impact of biological cyanocides on aquatic ecosystems when used to control natural cyanobacterial blooms.

Eco-friendly management of harmful cyanobacterial blooms in eutrophic lakes through vertical flow multi-soil-layering technology

Eutrophication has led to the widespread occurrence of cyanobacterial blooms. Toxic cyanobacterial blooms with high concentrations of microcystins (MCs) have been identified in the Lalla Takerkoust reservoir in Morocco. The objective of this study was to evaluate the efficiency of the Multi-Soil-Layering (MSL) ecotechnology in removing natural cyanobacterial blooms from the lake. Two MSL pilots were used in rectangular glass tanks (60 × 10 × 70 cm). They consisted of permeable layers (PLs) made of pozzolan and a soil mixture layer (SML) containing local soil, ferrous metal, charcoal and sawdust. The main difference between the two systems was the type of local soil used: sandy soil for MSL1 and clayey soil for MSL2. Both MSL pilots effectively reduced cyanobacterial cell concentrations in the treated water to very low levels (0.09 and 0.001 cells/mL). MSL1 showed a gradual improvement in MC removal from 52 % to 99 %, while MSL2 started higher at 90 % but dropped to 54% before reaching 86%. Both MSL systems significantly reduced organic matter levels (97.2 % for MSL1 and 95.8 % for MSL2). Both MSLs were shown to be effective in removing cyanobacteria, MCs, and organic matter with comparable performance.

Use of decomposition products of Pontederia crassipes Mart., Solms (water yacinth) for inhibition of cyanobacteria growth: A full-scale study

Eutrophication exacerbates the growth of primary producers that may reduce water quality, such as macrophytes and cyanobacteria. The floating macrophyte Pontederia crassipes and the cyanobacterium Raphidiopsis raciborskii occur worldwide in eutrophic environments, but data on the inhibition of R. raciborskii by floating macrophytes are scarce. We aimed to test the inhibitory activity of P. crassipes on cyanobacterial growth, especially R. raciborskii. First, we tested the effect of live P. crassipes exudates and decomposing macrophyte extracts on a R. raciborskii strain under culture conditions. Cyanobacterial growth was not affected by the exudate from the live macrophyte. In contrast, exposure to decaying macrophyte extracts containing 4.0 and 6.0 mg L−1 total phenols completely inhibited cyanobacterial growth after five days and decreased photosynthetic activity. Mass spectrometry FT-IRC analysis led to the identification of some molecules in P. crassipes extracts, but no association with the inhibitory effect of the was identified. We then tested the inhibitory effect of P. crassipes in mesocosms containing a natural cyanobacterial bloom. The slow release of chemicals from the dry biomass over 12 days decreased chlorophyll concentrations. Finally, in a field experiment, we applied tons of P. crassipes dry biomass to large areas (900 m3) of a reservoir to test the release of chemicals into the water during one month. At the final time, the phenol concentration in the backwater region was close to the inhibitory value found for cyanobacterial growth and an inverse correlation was observed between chlorophyll and phenol concentrations. Thus, under natural conditions, the release of phenolic compounds and other allelochemicals from the decomposing material of macrophyte mats inhibits cyanobacterial growth. Macrophyte biomass, which is usually considered waste, might be used to mitigate cyanobacterial blooms and may be a promising new and sustainable biotechnological tool to improve water quality.

Investigation on cyanobacterial production of the proposed neurotoxin β-N-methylamino-L-alanine (BMAA)

β-N-methylamino-L-alanine (BMAA) is an environmental neurotoxin thought to be produced by cyanobacteria. However, the cyanobacterial origin of BMAA remains controversial. The detection method and culture conditions of cyanobacteria are often cited as factors behind the discrepancy of published results. We showed previously that BMAA was highly toxic to the cyanobacterium Nostoc PCC 7120, and it is taken up via an amino acid transport system. Using a mutant ΔnatAΔbgtA deficient in amino acid transport as a genetic control, we show here that BMAA taken up from the medium can be detected quantitatively at a threshold similar to, or below those reported, but was undetectable in the mutant. The BMAA isomer, 2,4-diaminobutanoic acids (DAB), but not BMAA, could be detected in cell free extracts of Nostoc PCC 7120. Long-term (20 ​days) diazotrophic growth or non-limiting supply of phosphate, conditions reported to enhance BMAA synthesis, did not lead to the detection of BMAA. An UPLC-MS/MS signal with a similar retention time to BMAA was found after prolonged diazotrophic incubation, but did not have fragment ions of BMAA after further analysis. When extended to 29 different cyanobacterial strains and 6 natural cyanobacterial bloom samples, none of them was found to produce BMAA. The cytotoxicity of BMAA to cyanobacteria, and the lack of a cellular protective mechanism against such toxicity, contradict the presence of a BMAA synthesis pathway in these organisms. More specific methods for BMAA detection in vivo need to be developed to clarify the cyanobacterial origin of BMAA.

The characteristics and algicidal mechanisms of cyanobactericidal bacteria, a review.

Cyanobacterial blooms are a worldwide problem, especially in freshwaters. As one of the most abundant co-existing organisms of algae, bacteria play critical roles in cyanobacteria growth, particularly the cyanobactericidal bacteria which can efficiently kill cyanobacteria. Recent years, cyanobactericidal bacteria are highly recognized as a method that could potentially block cyanobacterial blooms. Many studies have been conducted to assess their effects on the termination of cyanobacteria blooms and explore their cyanobactericidal mechanisms, e.g., attacking by cell to cell or releasing specific compounds, the physiological, metabolic, and transcriptional disturbance on cyanobacteria. In this review, the present state of research on cyanobactericidal bacteria for the bloom-causing cyanobacteria species is summarized. The challenges in applying cyanobactericidal bacteria in the control of natural cyanobacterial blooms are discussed.

Development of a silicone-membrane passive sampler for monitoring cylindrospermopsin and microcystin LR-YR-RR in natural waters

Silicone membrane tubes were functionalised by filling them with synthesised γ-Fe2O3 nanoparticles and used as a passive sampling device for monitoring microcystins and cylindrospermopsin in aquatic environments. This novel device was calibrated for the measurement of microcystin and cylindrospermopsin concentrations in water. The effect of temperature and hydrodynamics on the sampler performance was studied in a flow-through system under controlled conditions. The chemical uptake of microcystins (MCs) and cylindrospermopsin (CYN) into the passive sampler remained linear and integrative throughout the exposure period. The rate of accumulation of most of the MC compounds tested was dependent on temperature and flow velocity. The use of 13C labelled polychlorinated biphenyls as performance reference compounds (PRCs) in silicone membrane/γ-Fe2O3 nanoparticle passive sampler, Chemcatcher and polar organic chemical integrative sampler (POCIS) was evaluated. The majority of PRCs improved the semi quantitative nature of water concentration estimated by the three samplers. The corrected sampling rate values of model biotoxin compounds were used to estimate the time-weighted average concentrations in natural cyanobacterial water blooms of the Hartbeespoort dam. The corrected sampling rates RScorr values varied from 0.1140 to 0.5628 Ld−1 between samplers with silicone membrane having the least RScorr values compared to the Chemcatcher and POCIS. The three passive sampling devises provided a more relevant picture of the biotoxin concentration in the Hartbeespoort dam. The results suggested that the three sampling devices are suitable for use in monitoring microcystins and cylindrospermopsin concentrations in aquatic environments.

Inhibition equivalency factors for microcystin variants in recombinant and wild-type protein phosphatase 1 and 2A assays.

In this work, protein phosphatase inhibition assays (PPIAs) have been used to evaluate the performance of recombinant PP1 and recombinant and wild-type PP2As. The enzymes have been compared using microcystins-LR (MC-LR) as a model cyanotoxin. Whereas PP2ARec provides a limit of detection (LOD) of 3.1 μg/L, PP1Rec and PP2AWild provide LODs of 0.6 and 0.5 μg/L, respectively, lower than the guideline value proposed by the World Health Organization (1 μg/L). The inhibitory potencies of seven MC variants (-LR, -RR, -dmLR, -YR, -LY, -LW and -LF) have been evaluated, resulting on 50 % inhibition coefficient (IC50) values ranging from 1.4 to 359.3 μg/L depending on the MC variant and the PP. The PPIAs have been applied to the determination of MC equivalent contents in a natural cyanobacterial bloom and an artificially contaminated sample, with multi-MC profiles. The inhibition equivalency factors (IEFs) have been applied to the individual MC quantifications determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, and the estimated MC-LR equivalent content has been compared to PPIA results. PPIAs have demonstrated to be applicable as MC screening tools for environmental applications and to protect human and animal health.

Behaviour of cyanobacterial bloom material following coagulation and/or sedimentation

The global increase in detection of cyanobacteria and their metabolites has prompted greater emphasis in optimizing water treatment options for their effective removal. In particular, conventional coagulation and clarification processes have been shown to be effective in removing whole cyanobacterial cells, and consequently a majority of the metabolites. However, the resultant cyanobacterial-laden sludge is often not closely monitored in treatment plants. This study has shown that the sludge supernatant derived from the coagulation/sedimentation of a range of natural cyanobacterial blooms contained extremely high concentrations of metabolites (up to ~8,000 ng L-1 of geosmin and ~90 μg L-1 of microcystins), which was attributed to cell lysis. Furthermore, the fate of the metabolites in the sludge supernatants differed with geosmin shown to be biodegradable, while the microcystins were released at different stages with some variants shown to be more persistent. The release and biodegradation of the metabolites followed pseudofirst-order kinetics with rate constants comparable to experiments using laboratory-cultured cyanobacteria. A key finding from this study was that the identification of cyanobacterial cell type is critical in making informed operational decisions, as not only did the cells behave differently within the sludge, but also the released metabolites behaved quite differently within the time frames studied. © IWA Publishing 2013.

Purification and identification of microcystin-RR

An effective method based on the combination of solid phase extraction and gel chromatography to extract and purify microcystin-RR (MC-RR) from natural cyanobacteria bloom was developed. Thirty five gram natural cyanobacterial bloom scum was extracted with 70% aqueous methanol. After the methanol in the crude extract was removed by a rotary evaporator, the solid phase extraction was used for preliminary purification and concentration. And then 7.5 mL eluent was concentrated to 2 mL and loaded onto a Sephadex LH-20 gel chromatographic column for further purification. The eluate was collected by tubes and detected at 238 nm. The elution curve was plotted by tube numbers as X-axis and the absorbance at 238 nm as Y-axis. The purity and the spectral characteristics of the final extract were identified with high performance liquid chromatograph (HPLC) and spectrophotometer, respectively. A total of 3.65 mg of MC-RR with a final yield of 74. 1% was obtained, and the purity was more than 90% with characteristic UV absorption spectra at 238 nm.

Accumulation of Microcystins in Nile Tilapia, Oreochromis niloticus L., and Effects of a Complex Cyanobacterial Bloom on the Dietetic Quality of Muscles

The aim of this study was to investigate the kinetics of accumulation and elimination of microcystins in the tissues of Nile tilapia (Oreochromis niloticus) and to evaluate the effect of cyanobacterial exposure on fish muscle quality (levels of total fat and ash, protein, dry matter and the composition of saturated, monounsaturated and polyunsaturated fatty acids). Fish were exposed for 28 days to a natural cyanobacterial bloom with total microcystin concentration around 1,200 μg g⁻¹ biomass dry weight. The hepatopancreas accumulated microcystins up to 350 ng g⁻¹ fresh weight, but concentrations in muscle were generally below the detection limit (2 ng g⁻¹ fresh weight). Following the exposure, fish were moved to the clean water, but only minor microcystin removal from the hepatopancreas was observed during a 4 week depuration period. Exposures of tilapia to the complex cyanobacterial bloom had only minor and temporary impacts on the investigated parameters of dietetic quality.

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.

Effects of microcystins-containing cyanobacteria from a temperate ecosystem on human lymphocytes culture and their potential for adverse human health effects

The present study confirmed the domination of two cyanobacterial genera Microcystis aeruginosa and Aphanizomenon sp. in the summer season in Jeziorsko Reservoir (Central Poland). On the basis of mcyE gene analysis, toxigenic cyanobacterial strains were detected throughout the monitoring period. The highest concentration and toxicity of microcystins (above 2μgL−1), with domination of the hydrophobic variant microcystin-YR, appeared in August and reached the cell concentrations for The First Alert Level, according to WHO guidelines for recreational water. For the estimation of the potential threat to human health from regular exposure to microcystins-containing cyanobacteria, in the second phase of study, cultured human lymphocytes were assessed by: XTT reduction test, comet assay and micronucleus test. The effects of three different variants of cyanobacterial extracts including: (i) crude microcystins-containing extracts and (ii) purified microcystins-containing and (iii) nonmicrocystins-containing extracts were investigated. The present study indicated the highest cytotoxicity and genotoxicity of the purified extract containing microcystins (MC-LR, MC-RR and MC-YR). Additionally, no significant effect or clear relation between microcystins concentration in crude extracts and human lymphocytes mortality and DNA damage was observed. The data indicated the influence of other compounds, apart from microcystins, present in the extracts. This research supports the necessity of application of additional tests for determination of total threat from natural cyanobacterial blooms, apart from determination of concentration and toxicity of the main group of cyanotoxins.

Gene expression and activity of digestive proteases in Daphnia: effects of cyanobacterial protease inhibitors.

BackgroundThe frequency of cyanobacterial blooms has increased worldwide, and these blooms have been claimed to be a major factor leading to the decline of the most important freshwater herbivores, i.e. representatives of the genus Daphnia. This suppression of Daphnia is partly attributed to the presence of biologically active secondary metabolites in cyanobacteria. Among these metabolites, protease inhibitors are found in almost every natural cyanobacterial bloom and have been shown to specifically inhibit Daphnia's digestive proteases in vitro, but to date no physiological responses of these serine proteases to cyanobacterial protease inhibitors in Daphnia have been reported in situ at the protein and genetic levels.ResultsNine digestive proteases were detected in D. magna using activity-stained SDS-PAGE. Subsequent analyses by LC-MS/MS and database search led to the identification of respective protease genes. D. magna responded to dietary protease inhibitors by up-regulation of the expression of these respective proteases at the RNA-level and by the induction of new and less sensitive protease isoforms at the protein level. The up-regulation in response to dietary trypsin- and chymotrypsin-inhibitors ranged from 1.4-fold to 25.6-fold. These physiological responses of Daphnia, i.e. up-regulation of protease expression and the induction of isoforms, took place even after feeding on 20% cyanobacterial food for only 24 h. These physiological responses proved to be independent from microcystin effects.ConclusionHere for the first time it was shown in situ that a D. magna clone responds physiologically to dietary cyanobacterial protease inhibitors by phenotypic plasticity of the targets of these specific inhibitors, i.e. Daphnia gut proteases. These regulatory responses are adaptive for D. magna, as they increase the capacity for protein digestion in the presence of dietary protease inhibitors. The type and extent of these responses in protease expression might determine the degree of growth reduction in D. magna in the presence of cyanobacterial protease inhibitors. The rapid response of Daphnia to cyanobacterial protease inhibitors supports the assumption that dietary cyanobacterial protease inhibitors exert a strong selection pressure on Daphnia proteases themselves.

Selected Haematological and Biochemical Indices of Nile Tilapia (Oreochromis niloticus) Reared in the Environment with Cyanobacterial Water Bloom

The aim of this study was to evaluate the influence of toxic cyanobacterial water blooms on blood indices in the Nile tilapia (Oreochromis niloticus). Experimental fish were exposed to natural cyanobacterial water blooms (consisting mainly of Microcystis aeruginosa and M. ichthyoblabe) which contained microcystins (total concentration 1187 - 1211 μg g-1 of dry weight and 17.4 - 25.4 μg l-1 of water) for 28 days without additional feeding. Control groups of fish were kept in another pond without apparent cyanobacterial bloom formation. Experimental and control rearing ponds had the same water source. After exposure, fish were placed in dechlorinated potable water for the same period. Statistical evaluation of the influence of cyanobacterial water bloom on biochemical indices of experimental fish showed a distinct increase of alkaline phosphatase (p ⪬ 0.05), total bilirubin (p ⪬ 0.001), creatinine (p ⪬ 0.01), lactate (p ⪬ 0.01) and urea (p ⪬ 0.01) when compared to controls. After transfer to the dechlorinated potable water the experimental group showed significantly lower values of phosphorus (p ⪬ 0.001), urea (p ⪬ 0.01) and cholinesterase (p ⪬ 0.05) and higher values of lactate (p ⪬ 0.05) and iron (p ⪬ 0.05) compared to controls. It may be concluded that the exposure of the Nile tilapia to the environment containing cyanobacterial water bloom influenced only some biochemical indices. However, this modulation is to a much lower degree compared to the common carp and silver carp.

Biochemical parameters of blood plasma and content of microcystins in tissues of common carp (Cyprinus carpioL.) from a hypertrophic pond with cyanobacterial water bloom

The aim of this study was to evaluate the dynamics of the blood plasma parameters and the content of microcystins in the tissues of common carp (Cyprinus carpio L.) in relation to the toxic cyanobacterial water bloom. Fish (average body mass 2176±697 g) in the hypertrophic pond were exposed to natural water bloom (dominated by Planktothrix agardhii, Pseudanabaena limnetica and Limnothrix redekei), which contained microcystins (concentration in biomass 20–181 μg g−1 dry wt, concentration in water 0.3–9.5 μg L−1). Biochemical parameters in fish blood plasma were analysed in 89 fish at 14-day intervals during the whole season (nine sampling periods). Our results demonstrated high variability and fluctuations in the investigated parameters. The content of microcystins and density of cyanobacterial cells correlated with some haematological indices as lipase, alanine–aminotransferase, albumin, magnesium and chlorides. The concentrations of microcystins in the muscle and liver of the fish (determined by high-performance liquid chromatography with mass spectrometer) were below the limit of detection during the monitored period [0.31 ng g−1 fresh weight (f.w.) for the liver and 0.13 ng g−1 f.w. for muscle]. Our study demonstrates that although known cyanobacterial toxin microcystins were not detected in the fish tissues, several biochemical parameters important for the fish physiology were modulated by natural cyanobacterial bloom.

Oral administration of cyanobacterial bloom extract induced the altered expression of the PP2A, Bax, and Bcl‐2 in mice

The frequent occurrences of the toxic cyanobacterial (specifically Microcystis aeruginosa) bloom are becoming a global environmental issue. Lots of researches have been focused on the pure cyanobacterial toxins, but little on the natural cyanobacterial bloom. This study was undertaken to investigate the effect of the natural cyanobacterial bloom extract on the expression of proteins, which have been shown to be affected by pure microcystins. In current study, the cyanobacterial bloom extract has been administered orally to ICR mice for 7 days with different dosages. The expression level of PP2A, Bcl-2, and Bax was measured via western blotting. The results showed that after 7 days of exposure to cyanobacteria extract, in mice liver tissue, the expression level of PP2A and Bax was increased significantly between the control and treatment groups, but there is no significant change on the Bcl-2 expression. This is the first report to describe the altered expression of PP2A in vivo when mice exposure to natural water blooms extract that means many cellular pathways would be interfered via the change of PP2A activity.

Field and Laboratory Studies on Pathological and Biochemical Characterization of Microcystin-Induced Liver and Kidney Damage in the Phytoplanktivorous Bighead Carp

Field and experimental studies were conducted to investigate pathological characterizations and biochemical responses in the liver and kidney of the phytoplanktivorous bighead carp after intraperitoneal (i.p.) administration of microcystins (MCs) and exposure to natural cyanobacterial blooms in Meiliang Bay, Lake Taihu. Bighead carp in field and laboratory studies showed a progressive recovery of structure and function in terms of histological, cellular, and biochemical features. In laboratory study, when fish were i.p. injected with extracted MCs at the doses of 200 and 500 μg MC-LReq/kg body weight, respectively, liver pathology in bighead carp was observed in a time dose-dependent manner within 24 h postinjection and characterized by disruption of liver structure, condensed cytoplasm, and the appearance of massive hepatocytes with karyopyknosis, karyorrhexis, and karyolysis. In comparison with previous studies on other fish, bighead carp in field study endured higher MC doses and longer-term exposure, but displayed less damage in the liver and kidney. Ultrastructural examination in the liver revealed the presence of lysosome proliferation, suggesting that bighead carp might eliminate or lessen cell damage caused by MCs through lysosome activation. Biochemically, sensitive responses in the antioxidant enzymes and higher basal glutathione concentrations might be responsible for their powerful resistance to MCs, suggesting that bighead carp can be used as biomanipulation fish to counteract cyanotoxin contamination.

Microcystin kinetics (bioaccumulation and elimination) and biochemical responses in common carp (Cyprinus carpio) and silver carp (Hypophthalmichthys molitrix) exposed to toxic cyanobacterial blooms

Two species of common edible fish, common carp (Cyprinus carpio) and silver carp (Hypophthalmichthys molitrix), were exposed to a Microcystis spp.-dominated natural cyanobacterial water bloom for two months (concentrations of cyanobacterial toxin microcystin, 182-539 microg/g biomass dry wt). Toxins accumulated up to 1.4 to 29 ng/g fresh weight and 3.3 to 19 ng/g in the muscle of silver carp and common carp, respectively, as determined by enzyme-linked immunosorbent immunoassay. Concentrations an order of magnitude higher were detected in hepatopancreas (up to 226 ng/g in silver carp), with a peak after the initial four weeks. Calculated bioconcentration factors ranged from 0.6 to 1.7 for muscle and from 7.3 to 13.3 for hepatopancreas. Microcystins were completely eliminated within one to two weeks from both muscle and hepatopancreas after the transfer of fish with accumulated toxins to clean water. Mean estimated elimination half-lives ranged from 0.7 d in silver carp muscle to 8.4 d in common carp liver. The present study also showed significant modulations of several biochemical markers in hepatopancreas of fish exposed to cyanobacteria. Levels of glutathione and catalytic activities of glutathione S-transferase and glutathione reductase were induced in both species, indicating oxidative stress and enhanced detoxification processes. Calculation of hazard indexes using conservative U.S. Environmental Protection Agency methodology indicated rather low risks of microcystins accumulated in edible fish, but several uncertainties should be explored.

Dose-dependent effects of extracted microcystins on embryonic development, larval growth and histopathological changes of southern catfish ( Silurus meridionalis)

A laboratory toxic experiment was conducted to examine dose-dependent effects of extracted microcystins (MCs) on embryonic development, larval growth and histopathological changes of southern catfish ( Silurus meridionalis). Fertilized eggs were incubated in solutions with four concentrations of MCs (0, 1, 10, 100 μg MC-LReq l −1). Higher MCs retarded egg development (2–10 h delays) and larval growth, reduced hatching rate (up to 45%), and caused high malformation rate (up to 15%) and hepatocytes damage (characterized by disorganization of cell structure and a loss of adherence between hepatocytes, cellular degeneration with vacuolar hepatocytes and marginal nuclei, even hepatocellular necrosis). A 10 μg MC-LReq l −1 is close to a high concentration in natural cyanobacterial blooms, suggesting a possible existence of such toxic effects in eutrophic waters.

Toxic Effects Produced by Microcystins from a Natural Cyanobacterial Bloom and a Microcystis aeruginosa Isolated Strain on the Fish Cell Lines RTG-2 and PLHC-1

Toxic cyanobacterial blooms are a worldwide problem, causing serious water pollution and public health hazard to humans and livestock. The intact cells as well as the toxins released after cellular lysis can be responsible for toxic effects in both animals and humans and are actually associated with fish kills. Two fish cell lines-PLHC-1 derived from a hepatocellular carcinoma of the topminnow Poeciliopsis lucida and RTG-2 fibroblast-like cells derived from the gonads of rainbow trout Oncorhynchus mykiss were exposed to several concentrations of extracts from a natural cyanobacterial bloom and a Microcystis aeruginosa-isolated strain. After 24 hours, morphologic and biochemical changes (total protein content, lactate dehydrogenase leakage, neutral red uptake, methathiazole tetrazolium salt metabolization, lysosomal function, and succinate dehydrogenase [SDH] activity) were investigated. The most sensitive end point for both cyanobacterial extracts in PLHC-1 cells was SDH activity, with similar EC(50) values (6 microM for the cyanobacterial bloom and 7 microM for the isolated strain). RTG-2 cells were less susceptible according to SDH activity, with their most sensitive end point lysosomal function with an EC(50) of 4 microM for the M. aeruginosa-isolated strain and 72 microM for the cyanobacterial bloom. The lysosomal function was stimulated at low concentrations, although SDH activity increased at high doses, indicating lysosomal and energetic alterations. Increased secretion vesicles, rounding effects, decreased cell numbers and size, hydropic degeneration, esteatosis, and apoptosis were observed in the morphologic study. Similar sensitivity to the M. aeruginosa-isolated strain was observed in both cell lines, whereas the cyanobacterial bloom was more toxic to the PLHC-1 cell line.