Joanna Mankiewicz-Boczek

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

ABSTRACT 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.

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.

The ecology of the invasive cyanobacterium Cylindrospermopsis raciborskii (Nostocales, Cyanophyta) in two hypereutrophic lakes dominated by Planktothrix agardhii (Oscillatoriales, Cyanophyta)

Biological invasions have attracted particular attention since they often result in serious consequences for natural ecosystems. One planktonic invasive species is Cylindrospermopsis raciborskii, a cyanobacterium originally reported to occur exclusively in the tropics. Over the last few decades its range has extended to temperate regions and it occupies shallow highly eutrophic lakes previously dominated by other cyanobacteria. The purpose of this study was to examine the ecology of C. raciborskii during Planktothrix agardhii blooms in two shallow lakes in western Poland and to determine whether these species have different environmental preferences. Multiple linear regression showed that the biomass of P. agardhii was significantly negatively related to Secchi depth in Lake Bnińskie. In Lake Bytyńskie, P. agardhii was significantly positively related to concentrations of , chlorophyll a and total phosphorus and negatively related to Secchi depth, , and total nitrogen. Cylindrospermopsis raciborskii was significantly positively related only to concentrations of . There was a negative correlation between the biomass of P. agardhii and C. raciborskii perhaps showing different responses to environmental variables. Moreover, the biomass of P. agardhii was negatively correlated with Shannon–Wiener diversity of the phytoplankton assemblages. Our results support the concept that these cyanobacterial species have different environmental preferences and their niches differ from each other. These results suggest that light is an important driver of phytoplankton community structure resulting in shifts from a community dominated by P. agardhii in very turbid waters to more diverse communities perhaps including the invasive C. raciborskii in clearer waters.

Ecohydrological system solutions to enhance ecosystem services: the Pilica River Demonstration Project

Abstract The application of ecohydrology principles as part of Integrated Water Resources Management (IWRM) has the potential to enhance the resilience of a catchment to anthropogenic impacts. Linking this approach with an understanding of water users and social and economic conditions in a given region, provides a foundation for the development of system solutions. Improving the quality of the environment, and the ecosystem services provided, can be a driver of new employment opportunities that contribute to both the overall economy of a region and sustainability. With these goals in mind, the paper presents a four-step approach for implementation of ecohydrology principles in IWRM, including a) monitoring of threats, b) analysis of the cause-effect relationships, c) development of methods, and d) system solutions. This approach was formulated and tested within a UNESCO-IHP and UNEP-IETC Demonstration Project on the Pilica River in Poland. This project aims to support fulfilment of Polands obligations resulting from the EU Water Framework Directive and other European directives, and constitutional obligations for sustainable development. Attempts to transfer lessons learned to other catchments and socio-ecological systems (such as urban catchments) are highlighted.

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).

Bacteria homologus to Aeromonas capable of microcystin degradation

Abstract Water blooms dominated by cyanobacteria are capable of producing hepatotoxins known as microcystins. These toxins are dangerous to people and to the environment. Therefore, for a better understanding of the biological termination of this increasingly common phenomenon, bacteria with the potential to degrade cyanobacteria-derived hepatotoxins and the degradative activity of culturable bacteria were studied. Based on the presence of the mlrA gene, bacteria with a homology to the Sphingopyxis and Stenotrophomonas genera were identified as those presenting potential for microcystins degradation directly in the water samples from the Sulejów Reservoir (SU, Central Poland). However, this biodegrading potential has not been confirmed in in vitro experiments. The degrading activity of the culturable isolates from the water studied was determined in more than 30 bacterial mixes. An analysis of the biodegradation of the microcystin-LR (MC-LR) together with an analysis of the phylogenetic affiliation of bacteria demonstrated for the first time that bacteria homologous to the Aeromonas genus were able to degrade the mentioned hepatotoxin, although the mlrA gene was not amplified. The maximal removal efficiency of MC-LR was 48%. This study demonstrates a new aspect of interactions between the microcystin-containing cyanobacteria and bacteria from the Aeromonas genus.

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.

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.

The characterization of a novel bacterial strain capable of microcystin degradation from the Jeziorsko reservoir, Poland: a preliminary study

Abstract The paper describes the isolation and characterization of a novel pelagic bacterial strain capable of hepatotoxic microcystin (MC) degradation. From a total of 24 bacterial strains isolated from water samples collected during the cyanobacteria blooming period in Jeziorsko reservoir, Poland, only the strain JEZ-8L was found to completely degrade microcystin-LR (MC-LR). The strain JEZ-8L was found to include the full mlrABCD gene cluster associated with the biological degradation of MC. To our knowledge, the strain JEZ-8L may represent the first report of an isolated bacterium to contain the full mlrABCD gene cluster in an aquatic ecosystem from central Europe. The nucleotide sequence analysis of each mlr gene and their translated amino acid sequences revealed a close similarity to other MC-degrading bacteria belonging to the Sphingomonadaceae family. Phylogenetic analysis using the bacterial gene marker 16S rRNA revealed 99% sequence similarity with nine strains from the Sphingosinicella genus. The nucleotide sequence similarity between the strain JEZ-8L and Sphingosinicella microcystinivorans Y2 was probably the most representative, since the strain Y2 is known to be a MC-degrading bacterium with confirmed detection from the full mlrABCD gene cluster. It is important to mention that our findings represent a preliminary study on the microbiota involved in MC degradation in water bodies from central Poland. The MC-degrading activity of the strain JEZ-8L could be promising for further research towards the elaboration of biotechnological solutions to reduce the harmful effects of MCs in water.

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 Sokołówka 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.