Rapid in situ assessment of high-resolution spatial and temporal distribution of cyanobacterial blooms using fishery echosounder.

Cyanobacterial blooms are increasing in frequency, magnitude, and duration globally because of enhanced eutrophication and climate change. Thus, comprehensive investigation and systematic monitoring of the spatial and temporal distribution of cyanobacteria in aquatic environments are urgently needed to better understand bloom development and complex interactions within a dynamic environment. Various methods have been used to investigate the distribution of cyanobacteria, however, none of them can provide high-resolution data for the three-dimensional spatial structure of the bloom and its dynamics in real time. In the present study, we investigated the applicability of a high-frequency (200 kHz) fishery echosounder, a type widely used in fisheries acoustics, to detect and estimate the cyanobacterial genus Microcystis bloom distribution and biomass in a shallow lake (Sulejów Reservoir, Poland). Verification of the usefulness of in situ acoustic quantification of bloom-forming cyanobacteria was based on a comparison of acoustic estimates of cyanobacterial biomass with the ground truth-that is, fluorometric measurements and chlorophyll a concentrations. We compared the acoustic estimates with other methods for continuous measurements along 10 predetermined parallel transects and point samples at 14 stations situated on the transects. In vertical hydroacoustic measurements at night, we observed that cyanobacterial biomass was highest in the uppermost layer and diminished continuously with depth. For both horizontal and vertical continuous measurements, we found significant positive correlations between acoustic and fluorometric estimates of cyanobacterial biomass. The traditional point samples measurements, however, did not agree equally well with the acoustic estimates, especially for vertical beam. We argue that the point measurements have more stochastic character and less adequately describe dynamic changes in the cyanobacteria distribution than continuous acoustic estimates. More studies are required to explore the cyanobacteria distribution patterns under different biological, physical, and meteorological conditions.

Temporal and functional interrelationships between bacterioplankton communities and the development of a toxigenic Microcystis bloom in a lowland European reservoir.

The cyanobacteria-associated microbiome is constantly reshaped by bloom development. However, the synergistic-antagonistic nature of the relationships between Microcystis and its microbiome still remains unclear. Therefore, temporal changes of bacterioplankton communities and their functional potential through different developing stages of a Microcystis toxigenic bloom were investigated, considering bacterioplankton assemblages as particle-attached (PAB) and free-living (FLB) bacteria. 16S rRNA sequencing revealed that PAB were represented by Proteobacteria and Cyanobacteria, while FLB by Proteobacteria and Actinobacteria. Network and ordination analyses indicated that PAB inter-relationships were more complex-numerous connections between taxa with stronger correlations, than FLB-rather influenced by physico-chemical parameters. PAB in pre-summer was diverse with Proteobacteria containing potential taxa involved in nitrogen-transforming processes. In mid-summer, PAB presented a mix-bloom dominated by Snowella, Aphanizomenon, and Microcystis, which were succeeded by toxigenic Microcystis in post-summer. Both periods were associated to potential taxa with parasitic/predatory lifestyles against cyanobacteria. In post-summer, Sutterellaceae were recognized as poor water quality indicators, and their strong association with Microcystis could have represented an increased threat for that period. Microcystis was a major factor significantly reducing PAB diversity and evenness, suggesting that it negatively influenced bacterioplankton assemblages, probably also altering the overall community functional potential.

Cyanophage Distribution Across European Lakes of the Temperate-Humid Continental Climate Zone Assessed Using PCR-Based Genetic Markers.

Studies of the diversity and distribution of freshwater cyanophages are generally limited to the small geographical areas, in many cases including only one or few lakes. Data from dozens of various lakes distributed at a larger distance are necessary to understand their spatial distribution and sensitivity to biotic and abiotic factors. Thus, the objective of this study was to analyze the diversity and distribution of cyanophages within the infected cells using marker genes (psbA, nblA, and g91) in 21 Polish and Lithuanian lakes. Physicochemical factors that might be related to them were also analyzed. The results demonstrated that genetic markers representing cyanophages were observed in most lakes studied. The frequently detected gene was psbA with 88% of cyanophage-positive samples, while nblA and g91 were found in approximately 50% of lakes. The DNA sequence analyses for each gene demonstrated low variability between them, although the psbA sequences branched within the larger cluster of marine Synechoccocuss counterparts. The principal component analysis allowed to identify significant variation between the lakes that presented high and low cyanobacterial biomass. The lakes with high cyanobacterial biomass were further separated by country and the different diversity of cyanobacteria species, particularly Planktothrix agardhii, was dominant in the Polish lakes and Planktolyngbya limnetica in the Lithuanian lakes. The total phosphorous and the presence of cyanophage genes psbA and nblA were the most important factors that allowed differentiation for the Polish lakes, while the pH and the genes g91 and nblA for the Lithuanian lakes.

Cyanobacterial cell-wall components as emerging environmental toxicants - detection and holistic monitoring by cellular signaling biosensors.

Cyanobacterial blooms constitute a recognized danger to aquatic environment and public health not only due to presence of main group of cyanotoxins, such as microcystins, cylindrospermopsin or anatoxin-a, but also other emerging bioactivities. An innovative approach identifying such bioactivities is the application of cellular biosensors based on reporter genes which detect the impact of cyanobacterial cells and components on actual human cells in a physiological-like setting. In the present study biosensor cell lines detecting four different types of bioactivities (ARE - oxidative stress, NFKBRE - immunomodulatory pathogen-associated molecular patterns, AHRE - persistent organic pollutants, GRE - endocrine disruptors) were exposed to concentrated cyanobacterial cells from 21 environmental bloom samples and from eight cultures (Microcystis aeruginosa, Aphanizomenon flos-aquae, Planktothrix agardhii and Raphidiopsis raciborskii). The AHRE and GRE biosensors did not detect any relevant bioactivity. In turn, ARE biosensors were significantly activated by bloom samples from Jeziorsko (180-250%) and Sulejów (250-400%) reservoirs with the highest cyanobacterial biomass, while activation by cultures was weak/undetectable. The same biosensors were stimulated by microcystin-LR (250%) and anatoxin-a (150%). The NFKBRE biosensors were activated to varying extent (140-650%) by most bloom and culture samples, pointing to potential immunomodulatory toxic effects on humans. Lipopolysaccharide and lipoproteins were identified as responsible for NFKBRE activation (probably via pattern recognition receptors), while peptidoglycan had no bioactivity in this assay. Thus, the holistic approach to sample analysis with the application of cellular biosensors geared towards 4 separate pathways/bioactivities was validated for identification of novel bioactivities in organisms with recognized public health significance (e.g. this study is the first to describe cyanobacterial lipoproteins as potential environmental immunomodulators). Moreover, the ability of cellular biosensors to be activated by intact cyanobacterial cells from blooms provides proof of concept of their direct application for environmental monitoring, especially comparison of potential threats without need for chemical analysis and identification of toxicants.

Culturable nitrogen-transforming bacteria from sequential sedimentation biofiltration systems and their potential for nutrient removal in urban polluted rivers.

Novel heterotrophic bacterial strains-Bzr02 and Str21, effective in nitrogen transformation, were isolated from sequential sedimentation-biofiltration systems (SSBSs). Bzr02, identified as Citrobacter freundii, removed up to 99.0% of N-NH4 and 70.2% of N-NO3, while Str21, identified as Pseudomonas mandelii, removed up to 98.9% of N-NH4 and 87.7% of N-NO3. The key functional genes napA/narG and hao were detected for Bzr02, confirming its ability to reduce nitrate to nitrite and remove hydroxylamine. Str21 was detected with the genes narG, nirS, norB and nosZ, confirming its potential for complete denitrification process. Nitrogen total balance experiments determined that Bzr02 and Str21 incorporated nitrogen into cell biomass (up to 94.7% and 74.7%, respectively), suggesting that nitrogen assimilation was also an important process occurring simultaneously with denitrification. Based on these results, both strains are suitable candidates for improving nutrient removal efficiencies in nature-based solutions such as SSBSs.

Distribution of invasive Cylindrospermopsis raciborskii in the East-Central Europe is driven by climatic and local environmental variables.

Mechanisms behind expansion of an invasive cyanobacterium Cylindrospermopsis raciborskii have not been fully resolved, and different hypotheses, such as global warming, are suggested. In the East-Central Europe, it is widely occurring in western part of Poland but only in single locations in the East due to some limiting factors. Therefore, broad-scale phytoplankton survey including 117 randomly selected lakes in Poland and Lithuania was conducted. The results showed that C. raciborskii occurred widely in western part of Poland but was absent from other regions and Lithuania except one lake. The regions in which C. raciborskii was present had higher annual mean air temperature, higher maximum air temperature of the warmest month and higher minimum temperature of the coldest month, demonstrating that average air temperature, and indirectly, the duration of growing season might be more important factor driving C. raciborskii distribution than measured in situ water temperature. In turn, the presence of C. raciborskii in single localities may be more related to physiological adaptations of separated ecotype. Collectively, these results provide novel evidence on the influence of temperature on C. raciborskii distribution in East-European regions but also indicate high ecological plasticity of this species.

Potential for Phytoremediation of PCDD/PCDF-Contaminated Sludge and Sediments Using Cucurbitaceae Plants: A Pilot Study.

The current study evaluates the impact of sewage sludge and urban reservoir sediment on changes in total and Toxic Equivalency (TEQ) PCDD/PCDF concentration in soil and phytotoxicity measured using three test species: Lepidium sativum, Sinapis alba, and Sorghum saccharatum, during 5 weeks of Cucurbita pepo L. cv 'Atena Polka' (zucchini) cultivation. 'Atena Polka' was found to reduce total PCDD/PCDF concentration by 37 % in soil amended with sludge and 32 % in soil treated with sediment from an urban reservoir. The TEQ reduction was almost twofold greater: 68 % in soil amended with sludge and 52 % with urban sediment. Addition of sludge increased root growth inhibition of L. sativum, S. alba and S. saccharatum, from 44 % to 90 %. Observed inhibitions were, however, reduced by 'Atena Polka' cultivation, and as high as 32 % promotion in root length was noted. Amendment with urban sediment, in turn, resulted in an initial 1 %-36 % promotion of root growth, while 'Atena Polka' cultivation reduced this positive effect by inhibition as high as 26 %. Results demonstrated positive influence of 'Atena Polka' on the phytotoxicity alleviation and mitigation of total and TEQ PCDD/PCDF concentrations in soil treated with bio-solids from sewage sludge and an urban reservoir.

Effects of Stormwater and Snowmelt Runoff on ELISA-EQ Concentrations of PCDD/PCDF and Triclosan in an Urban River.

The aim of the study was to determine the effects of stormwater and snowmelt runoff on the ELISA EQ PCDD/PCDF and triclosan concentrations in the small urban Sokolówka River (Central Poland). The obtained results demonstrate the decisive influence of hydrological conditions occurring in the river itself and its catchment on the quoted PCDD/PCDF ELISA EQ concentrations. The lowest PCDD/PCDF values of 87, 60 and 67 ng EQ L-1 in stormwater, the river and its reservoirs, respectively, were associated with the highest river flow of 0.02 m3 s-1 and high precipitation (11.2 mm) occurred five days before sampling. In turn, the highest values of 353, 567 and 343 ng EQ L-1 in stormwater, the river and its reservoirs, respectively, were observed during periods of intensive snow melting (stormwater samples) and spring rainfall preceded by a rainless phase (river and reservoir samples) followed by low and moderate river flows of 0.01 and 0.005 m3 s-1. An analogous situation was observed for triclosan, with higher ELISA EQ concentrations (444 to 499 ng EQ L-1) noted during moderate river flow and precipitation, and the lowest (232 to 288 ng EQ L-1) observed during high river flow and high precipitation preceded by violent storms. Stormwater was also found to influence PCDD/PCDF EQ concentrations of the river and reservoirs, however only during high and moderate flow, and no such effect was observed for triclosan. The study clearly demonstrates that to mitigate the high peaks of the studied pollutants associated with river hydrology, the increased in-site stormwater infiltration and purification, the development of buffering zones along river course and the systematic maintenance of reservoirs to avoid the accumulation of the studied micropollutants and their subsequent release after heavy rainfall are required.

Application of cellular biosensors for detection of atypical toxic bioactivity in microcystin-containing cyanobacterial extracts.

Despite the focus of most ecotoxicological studies on cyanobacteria on a select group of cyanotoxins, especially microcystins, a growing body of evidence points to the involvement of other cyanobacterial metabolites in deleterious health effects. In the present study, original, self-developed reporter gene-based cellular biosensors, detecting activation of the main human xenobiotic stress response pathways, PXR and NFkappaB, were applied to detect novel potentially toxic bioactivities in extracts from freshwater microcystin-producing cyanobacterial blooms. Crude and purified extracts from cyanobacteria containing varying levels of microcystins, and standard microcystin-LR were tested. Two cellular biosensor types applied in this study, called NHRTOX (detecting PXR activation) and OXIBIOS (detecting NFkappaB activation), successfully detected potentially toxic or immunomodulating bioactivities in cyanobacterial extracts. The level of biosensor activation was comparable to control cognate environmental toxins. Despite the fact that extracts were derived from microcystin-producing cyanobacterial blooms and contained active microcystins, biosensor-detected bioactivities were shown to be unrelated to microcystin levels. Experimental results suggest the involvement of environmental toxins (causing a response in NHRTOX) and lipopolysaccharides (LPS) or other cell wall components (causing a response in OXIBIOS) in the potentially harmful bioactivity of investigated extracts. These results demonstrate the need for further identification of cyanobacterial metabolites other than commonly studied cyanotoxins as sources of health risk, show the usefulness of cellular biosensors for this purpose and suggest a novel, more holistic approach to environmental monitoring.

Role of environmental factors and toxic genotypes in the regulation of microcystins-producing cyanobacterial blooms.

The aim of this study was to understand: (1) how environmental conditions can contribute to formation of Microcystis-dominated blooms in lowland, dam reservoirs in temperate climate-with the use of quantitative molecular monitoring, and (2) what is the role of toxic Microcystis genotypes in the bloom functioning. Monitoring of the Sulejow Reservoir in 2009 and 2010 in two sites Tresta (TR) and Bronislawow BR), which have different morphometry, showed that physicochemical conditions were always favorable for cyanobacterial bloom formation. In 2009, the average biomass of cyanobacteria reached 13 mg L(-1) (TR) and 8 mg L(-1) (BR), and in the second year, it decreased to approximately 1 mg L(-1) (TR and BR). In turns, the mean number of toxic Microcystis genotypes in the total Microcystis reached 1% in 2009, both in TR and BR, and in 2010, the number increased to 70% in TR and 14 % in BR. Despite significant differences in the biomass of cyanobacteria in 2009 and 2010, the mean microcystins (MCs) concentration and toxicity stayed at a similar level of approximately 1 µg L(-1). Statistical analysis indicated that water retention time was a factor that provided a significant difference between the two monitoring seasons and was considered a driver of the changes occurring in the Sulejow Reservoir. Hydrologic differences, which occurred between two studied years due to heavy flooding in Poland in 2010, influenced the decrease in number of Microcystis biomass by causing water disturbances and by lowering water temperature. Statistical analysis showed that Microcystis aeruginosa biomass and 16S rRNA gene copy number representing Microcystis genotypes in both years of monitoring could be predicted on the basis of total and dissolved phosphorus concentrations and water temperature. In present study, the number of mcyA gene copies representing toxic Microcystis genotypes could be predicted based on the biomass of M. aeruginosa. Moreover, MCs toxicity and concentration could be predicted on the basic of mcyA gene copy number and M. aeruginosa (biomass, 16S rRNA), respectively. Present findings may indicate that Microcystis can regulate the number of toxic genotypes, and in this way adjust the whole bloom to be able to produce MCs at the level which is necessary for its maintenance in the Sulejow Reservoir under stressful hydrological conditions.

Aphanizomenon gracile (Nostocales), a cylindrospermopsin-producing cyanobacterium in Polish lakes.

The cyanobacterial cytotoxin cylindrospermopsin (CYN) has become increasingly common in fresh waters worldwide. It was originally isolated from Cylindrospermopsis raciborskii in Australia; however, in European waters, its occurrence is associated with other cyanobacterial species belonging to the genera Aphanizomenon and Anabaena. Moreover, cylindrospermopsin-producing strains of widely distributed C. raciborskii have not yet been observed in European waters. The aims of this work were to assess the occurrence of CYN in lakes of western Poland and to identify the CYN producers. The ELISA tests, high-performance liquid chromatography (HPLC)-DAD, and HPLC-mass spectrometry (MS)/MS were conducted to assess the occurrence of CYN in 36 lakes. The cyrJ, cyrA, and pks genes were amplified to identify toxigenic genotypes of cyanobacteria that are capable of producing CYN. The toxicity and toxigenicity of the C. raciborskii and Aphanizomenon gracile strains isolated from the studied lakes were examined. Overall, CYN was detected in 13 lakes using HPLC-MS/MS, and its concentrations varied from trace levels to 3.0 µg L(-1). CYN was widely observed in lakes of western Poland during the whole summer under different environmental conditions. Mineral forms of nutrients and temperature were related to CYN production. The molecular studies confirmed the presence of toxigenic cyanobacterial populations in all of the samples where CYN was detected. The toxicity and toxigenicity analyses of isolated cyanobacteria strains revealed that A. gracile was the major producer of CYN.

Preliminary molecular identification of cylindrospermopsin-producing Cyanobacteria in two Polish lakes (Central Europe).

The presence of toxigenic cyanobacteria capable of biosynthesis of cylindrospermopsin (CYN) was measured in 24 water samples collected from the lakes Bytynskie (BY) and Bninskie (BN) in the Western Poland. The study also covered analysis of toxigenicity and production of CYN by the culture of Cylindrospermopsis raciborskii isolated from BY. The cyrJ gene associated with CYN production was identified in 22 water samples collected in the summer seasons of 2006 and 2007. The presence of CYN was confirmed in 16 samples. The homology searches revealed that amplified sequences of four water samples, which were selected from among all the samples, displayed a strong 99% homology to cyrJ gene of Aphanizomenon sp. 10E6. The culture of C. raciborskii did not contain the cyrJ gene nor the CYN. The specificity of C. raciborskii was confirmed by application of a fragment of the rpoC1. These first genetic analyses have shown that Aphanizomenon seems to be the main cyanobacterial genus responsible for the production of CYN in the Polish lakes. The lack of toxigenicity of the isolated C. raciborskii suggests that it is possible that this invasive species does not demonstrate toxigenic activity in Polish water bodies.

Perennial toxigenic Planktothrix agardhii bloom in selected lakes of Western Poland.

The presence of toxigenic blooms dominated by filamentous cyanobacterium Planktothrix agardhii with estimation of microcystins (MCs) concentration and toxicity was measured in two lakes: Bytynskie and Lubosinskie situated in Western Poland. Investigations were carried out in summer, autumn, and winter of 2007/2008 and early spring of 2008. In both lakes, a domination of P. agardhii in relation to the total cyanobacterial biomass oscillated, throughout the year, almost on the same level between 75 and 99%. The PCR analysis of mcyE gene indicated a presence of toxigenic strains in all collected samples. In addition, the result of semiquantification of mcyE gene band showed that both lakes seem to have variable, throughout the seasons, toxigenic potential with the highest density of mcyE gene in spring. Two separate methods were used: protein phosphatase inhibition assay for estimation of MCs toxicity (biological activity) and high-performance liquid chromatography for determination of MCs concentration (quantity). The highest seasonal MCs toxicity (15.8 µg/L Bytynskie and 21.9 µg/L Lubosinskie) and concentration (34.6 µg/L Bytynskie and 52.2 µg/L Lubosinskie) were determined in autumn and indicated on a Second Alert Level, according to WHO guidelines for bathing water. The results showed the ability of toxigenic strains of cyanobacteria dominated by P. agardhii to remain and produce MCs during the whole year. This was confirmed by significant correlations between P. agardhii biomass and MCs concentrations in both lakes (r = 0.84, Bninskie and r = 0.79, Lubosinskie; P < 0.05).

Fate and effects of CeO2 nanoparticles in aquatic ecotoxicity tests.

Cerium dioxide nanoparticles (CeO2 NPs) are increasingly being used as a catalyst in the automotive industry. Consequently, increasing amounts of CeO2 NPs are expected to enter the environment where their fate in and potential impacts are unknown. In this paper we describe the fate and effects of CeO2 NPs of three different sizes (14, 20, and 29 nm) in aquatic toxicity tests. In each standard test medium (pH 7.4) the CeO2 nanoparticles aggregated (mean aggregate size approximately 400 nm). Four test organisms covering three different trophic levels were investigated, i.e., the unicellular green alga Pseudokirchneriella subcapitata, two crustaceans: Daphnia magna and Thamnocephalus platyurus, and embryos of Danio rerio. No acute toxicity was observed for the two crustaceans and D. rerio embryos, up to test concentrations of 1000, 5000, and 200 mg/L, respectively. In contrast, significant chronic toxicity to P. subcapitata with 10% effect concentrations (EC10s) between 2.6 and 5.4 mg/L was observed. Food shortage resulted in chronic toxicity to D. magna, for wich EC10s of > or = 8.8 and < or = 20.0 mg/L were established. Chronic toxicity was found to increase with decreasing nominal particle diameter and the difference in toxicity could be explained by the difference in surface area. Using the data set, PNEC(aquatic)S > or = 0.052 and < or = 0.108 mg/L were derived. Further experiments were performed to explain the observed toxicity to the most sensitive organism, i.e., P. subcapitata. Toxicity could not be related to a direct effect of dissolved Ce or CeO2 NP uptake or adsorption, nor to an indirect effect of nutrient depletion (by sorption to NPs) or physical light restriction (through shading by the NPs). However, observed clustering of NPs around algal cells may locally cause a direct or indirect effect.

First report of the cyanobacterial toxin cylindrospermopsin in the shallow, eutrophic lakes of western Poland.

Cyanobacterial dominance in eutrophic lakes causes water quality problems due to the production of toxins harmful to humans and animals, as well as a number of odorous compounds. Cylindrospermopsin (CYN) is a potent cytotoxic cyanobacterial metabolite involved in triggering illness in humans. The occurrence of CYN has been mostly associated with tropical and subtropical cyanobacteria. We analyzed CYN concentration and phytoplankton assemblages of three lakes located in western Poland during the summers of 2006 and 2007. CYN was detected in 46% of our samples using the HPLC and LC-MS/MS methods. CYN concentrations were in the range of 0.16-1.8 microg L(-1) and exceeded the drinking water guideline value of 1 microgL(-1) in two samples. This is the first report of CYN occurrence in this part of Europe and provides further evidence that this toxin is common not only in subtropical and tropical regions. The lakes were dominated by Planktothrix agardhii but the occurrence of the CYN investigated here might be associated with the invasive species Cylindrospermopsis raciborskii and/or native Aphanizomenon gracile.

Biomonitoring of cyanobacterial blooms in Polish water reservoir and the cytotoxicity and genotoxicity of selected cyanobacterial extracts.

OBJECTIVES: Water pollution with toxic cyanobacterial blooms is a worldwide problem. Cyanobacteria species that mainly produce microcystins predominate in Polish water reservoirs. MATERIALS AND METHODS: In our study, cyanobacterial blooms were monitored during summer of 2004 in the Sulejów reservoir. The concentration of microcystins in water and cyanobacterial cells were determined using liquid chromatography and immunobiotests, while the biological activity of microcystic cyanobacterial extracts was assessed using bacterial tests (SOS Chromotest, UMU test), the comet assay and micronucleus test with human lymphocytes. RESULTS: It was revealed that cyanobacterial bloom was most intensive in mid August and lasted until the end of September. Microcystis aeruginosa and Aphanizomenon flos-aquae dominated in the blooms. The highest concentration of microcystins in cyanobacterial cells was also observed at that time. The concentration of microcystins in water did not exceed 1 microg/l. All cyanobacterial extracts showed weak genotoxicity only for Escherichia coli PQ37. The cyanobacterial extracts prepared at the beginning of September were most toxic to human lymphocytes, the effective microcystin extracts (EC50) concentration was about two or three times lower compared to the other extracts. The level of DNA damage in lymphocytes after short exposure to microcystic extracts (3 and 6 h) was significantly higher than respective levels after longer exposure. The microcystins of cyanobacterial blooms induced a slight increase in micronuclei frequencies in human lymphocytes. CONCLUSION: Phytoplankton biomass and the genotoxicity of massive cyanobacterial blooms should be assessed for eucariotic cells in the Sulejów reservoir to avoid the hazard induced by cyanobacterial blooms.

Detection of microcystin-producing cyanobacteria in Finnish lakes with genus-specific microcystin synthetase gene E (mcyE) PCR and associations with environmental factors.

We studied the frequency and composition of potential microcystin (MC) producers in 70 Finnish lakes with general and genus-specific microcystin synthetase gene E (mcyE) PCR. Potential MC-producing Microcystis, Planktothrixand Anabaena spp. existed in 70%, 63%, and 37% of the lake samples, respectively. Approximately two-thirds of the lake samples contained one or two potential MC producers, while all three genera existed in 24% of the samples. In oligotrophic lakes, the occurrence of only one MC producer was most common. The combination of Microcystis and Planktothrix was slightly more prevalent than others in mesotrophic lakes, and the cooccurrence of all three MC producers was most widespread in both eutrophic and hypertrophic lakes. The proportion of the three-producer lakes increased with the trophic status of the lakes. In correlation analysis, the presence of multiple MC-producing genera was associated with higher cyanobacterial and phytoplankton biomass, pH, chlorophyll a, total nitrogen, and MC concentrations. Total nitrogen, pH, and the surface area of the lake predicted the occurrence probability of mcyE genes, whereas total phosphorus alone accounted for MC concentrations in the samples by logistic and linear regression analyses. In conclusion, the results suggested that eutrophication increased the cooccurrence of potentially MC-producing cyanobacterial genera, raising the risk of toxic-bloom formation.

Detection and monitoring toxigenicity of cyanobacteria by application of molecular methods.

The aim of this study was early genetic identification of microcystin-producing cyanobacteria and monitoring their toxigenicity by determining toxin concentrations in three Polish lakes throughout the summer of 2004. The assessment of cyanobacterial blooms was carried out in shallow, eutrophic water bodies: Lake Jeziorak, Lake Bninskie, and Sulejow Reservoir. Samples for DNA, phycological, and toxin analyses were collected from July till October. Molecular analysis of the 16S rRNA region was used to detect cyanobacteria in water samples. The microscopic analysis was performed to investigate seasonal variation of phytoplankton. Cyanobacteria, with domination by Microcystis, Planktothrix, and Planktolyngbya were detected during the whole monitoring period in Sulejow Reservoir, Lake Bninskie, and Lake Jeziorak, respectively. The presence and identification of toxic strains in water bodies was studied by PCR amplification of mcy genes in the microcystis synthesis pathway. The presence of the mcyA, mcyB, mcyD, and mcyE genes in water samples indicated the genetic potential to produce microcystins. Toxicity of water samples and microcystin concentrations were established by PPIA and HPLC, respectively. The maximum concentration of microcystins was 11.13 microg/L and 4.67 microg/L in samples dominated by P. agardhii and M. aeruginosa, respectively. Molecular analysis showed that toxigenic strains of cyanobacteria occurred in the three lakes throughout the summer season.