Distribution of toxigenic cyanobacteria in Alpine lakes and rivers as revealed by molecular screening.

The increasing frequency of cyanobacteria blooms in waterbodies caused by ecosystem eutrophication could endanger human health. This risk can be mitigated by effective monitoring incorporating molecular methods. To date, most molecular studies on toxigenic cyanobacteria have been limited to microcystins (MCs), disregarding other cyanotoxins, to freshwater planktic habitats while ignoring benthic habitats, and to limited geographic areas (usually one or a few specific waterbodies). In this study, we used PCR-based methods including PCR product sequencing and chemical-analytical methods (LC-MS/MS) to screen many plankton (n = 123) and biofilm samples (n = 113) originating from 29 Alpine lakes and 18 rivers for their cyanotoxin production potential. Both mcyE (indicating MC synthesis) and anaC (indicating anatoxin (ATX) synthesis) gene fragments were able to qualitatively predict MC or ATX occurrence. The abundance of mcyE gene fragments was significantly related to MC concentrations in plankton samples (R2 = 0.61). mcyE gene fragments indicative of MC synthesis were most abundant in planktic samples (65 %) and were assigned to the genera Planktothrix and Microcystis. However, mcyE rarely occurred in biofilms of lakes and rivers, i.e., 4 % and 5 %, respectively, and were assigned to Microcystis, Planktothrix, and Nostoc. In contrast, anaC gene fragments occurred frequently in planktic samples (14 % assigned to Tychonema, Phormidium (Microcoleus), and Oscillatoria), but also in biofilms of lakes (49 %) and rivers (18 %) and were assigned to the genera Phormidium, Oscillatoria, and Nostocales. The cyrJ gene fragment indicating cylindrospermopsin synthesis occurred only once in plankton (assigned to Dolichospermum), while saxitoxin synthesis potential was not detected. For plankton samples, monomictic and less eutrophic conditions were positively related to mcyE/MC occurrence frequency, while oligomictic conditions were related to anaC/ATX frequency. The anaC/ATX frequency in biofilm was related to the lake habitats generally showing higher biodiversity as revealed from metabarcoding in a parallel study.

Differences in Phytobenthic Diatom Community between Natural and Channelized River Sections.

The structure of phytobenthic diatom communities was studied to reveal differences between natural and channelized river sections in Slovenia. As part of the national monitoring of surface waters, samples of phytobenthos were collected at 85 sites throughout the country according to standard protocols. At the same time, basic environmental parameters were also assessed. Trophic (TI) and saprobic (SI) indices were calculated based on diatoms and other algae, while diversity indices and gradient analyses were performed only for the diatom community. The results showed that channelized rivers harbor significantly more diverse benthic diatom communities than natural sections, mainly due to the significantly higher number of motile diatom taxa that are able to take advantage of more nutrient-rich and less-shaded river sections because of their high adaptability. Selected environmental parameters explained 34% of the variability in diatom community structure when taxa were classified into ecological types. The removal of Achnanthidium minutissimum yielded clearer results (24.1%) than the total species matrix (22.6%). Therefore, we suggest excluding this taxon from calculations of TI, SI, or other indices when it is determined as A. minutissimum complex, because A. minutissimum complex was most abundant in both types of reaches in our study and has a wide ecological amplitude, which reduces the indicative power of the diatom community in the evaluation of environmental conditions and ecological status.

Genotoxic activity of endocrine disrupting compounds commonly present in paper mill effluents.

In the present study we evaluated cytotoxic and genotoxic activities of endocrine disrupting compounds (EDCs), including dimethyl phthalate (DMP), diethyl phthalate (DEP), dibutyl phthalate (DBP), benzyl butyl phthalate (BBP), di(2-ethylhexyl) phthalate (DEHP), bisphenol A (BPA), and nonylphenol (NP), which have been previously identified in effluents from two paper mills with different paper production technologies (virgin or recycled fibres). Moreover, we evaluated genotoxic activity of the effluents from these two paper mills and compared it to the activity of artificial complex mixtures consisting of the seven EDCs at concentrations detected in corresponding paper mill effluents. None of the EDCs was genotoxic in Salmonella typhimurium (SOS/umuC assay), while all induced DNA damage in human hepatocellular carcinoma (HepG2) cells (comet assay). After 4 h of exposure genotoxic effects were determined at concentrations ≥ 1 µg/L for BBP and DEHP, ≥10 µg/L for DMP, DEP, DBP, and BPA, and ≥100 µg/L for NP, while after 24 h of exposure DNA damage occurred at ≥10 µg/L for DBP, BPA and NP, and ≥100 µg/L for DMP, DEP, BBP and DEHP. The effluents and corresponding artificial mixtures of EDCs from paper mill that uses virgin fibres did not induce DNA damage in HepG2 cells, while the effluents and corresponding artificial mixtures for the paper mill that uses recycled fibres were genotoxic. Genotoxic activity of effluents was significantly higher compared to corresponding artificial mixtures suggesting the presence of further unknown compounds contributing to the effect. Wastewater monitoring based on chemical analysis is limited to determination of targeted compounds and does not take into account possible interactions between chemicals in mixtures. Therefore, it alone cannot provide an adequate information on potential toxic effects required for the assessment of genotoxic activity of real environmental samples and their potential threats to the environment and human health.

Potentially Toxic Planktic and Benthic Cyanobacteria in Slovenian Freshwater Bodies: Detection by Quantitative PCR.

Due to increased frequency of cyanobacterial blooms and emerging evidence of cyanotoxicity in biofilm, reliable methods for early cyanotoxin threat detection are of major importance for protection of human, animal and environmental health. To complement the current methods of risk assessment, this study aimed to evaluate selected qPCR assays for detection of potentially toxic cyanobacteria in environmental samples. In the course of one year, 25 plankton and 23 biofilm samples were collected from 15 water bodies in Slovenia. Three different analyses were performed and compared to each other; qPCR targeting mcyE, cyrJ and sxtA genes involved in cyanotoxin production, LC-MS/MS quantifying microcystin, cylindrospermopsin and saxitoxin concentration, and microscopic analyses identifying potentially toxic cyanobacterial taxa. qPCR analyses detected potentially toxic Microcystis in 10 lake plankton samples, and potentially toxic Planktothrix cells in 12 lake plankton and one lake biofilm sample. A positive correlation was observed between numbers of mcyE gene copies and microcystin concentrations. Potential cylindrospermopsin- and saxitoxin-producers were detected in three and seven lake biofilm samples, respectively. The study demonstrated a potential for cyanotoxin production that was left undetected by traditional methods in both plankton and biofilm samples. Thus, the qPCR method could be useful in regular monitoring of water bodies to improve risk assessment and enable timely measures.

Raw and biologically treated paper mill wastewater effluents and the recipient surface waters: Cytotoxic and genotoxic activity and the presence of endocrine disrupting compounds.

Paper mill effluents are complex mixtures containing different toxic compounds including endocrine-disrupting (EDCs) and genotoxic compounds. In the present study non-concentrated raw and biologically treated wastewaters from two paper mill plants with different paper production technologies i) Paper mill A uses virgin fibres, and ii) Paper mill B uses recycled fibres for paper production and the corresponding receiving surface waters, were assessed for their cytotoxic/genotoxic activity with SOS/umuC, Ames MPF 98/100 Aqua, and comet assay with human hepatoma HepG2 cells. In addition the levels of seven selected EDCs were quantified in wastewater samples and receiving surface waters. All investigated EDCs were confirmed in raw and biologically treated effluents from both paper mills with concentrations being markedly higher in Paper mill B effluents. In the receiving surface waters three of the studied EDCs were determined downstream of both paper mills effluent discharge. The wastewater samples and the recipient surface water samples from Paper mill A were not mutagenic for bacteria and did not induce DNA damage in HepG2 cells. On the contrary, half of the raw wastewater samples from Paper mill B were mutagenic whereas biologically treated wastewater and the recipient surface water samples were negative. In HepG2 cells most of the raw and biologically treated wastewater samples from Paper mill B as well as surface water samples collected downstream of Paper mill B effluent discharge induced DNA damage. The results confirmed that genotoxic contaminants were present only in wastewaters from Paper mill B that uses recycled fibres for paper production, and that the combined aerobic and anaerobic wastewater treatment procedure efficiently reduced contaminants that are bacterial mutagens, but not those that induce DNA damage in HepG2 cells. This study highlights that in addition to chemical analyses bioassays are needed for a comprehensive toxicological evaluation of complex wastewater samples.

Use of hydrodynamic cavitation in (waste)water treatment.

The use of acoustic cavitation for water and wastewater treatment (cleaning) is a well known procedure. Yet, the use of hydrodynamic cavitation as a sole technique or in combination with other techniques such as ultrasound has only recently been suggested and employed. In the first part of this paper a general overview of techniques that employ hydrodynamic cavitation for cleaning of water and wastewater is presented. In the second part of the paper the focus is on our own most recent work using hydrodynamic cavitation for removal of pharmaceuticals (clofibric acid, ibuprofen, ketoprofen, naproxen, diclofenac, carbamazepine), toxic cyanobacteria (Microcystis aeruginosa), green microalgae (Chlorella vulgaris), bacteria (Legionella pneumophila) and viruses (Rotavirus) from water and wastewater. As will be shown, hydrodynamic cavitation, like acoustic, can manifest itself in many different forms each having its own distinctive properties and mechanisms. This was until now neglected, which eventually led to poor performance of the technique. We will show that a different type of hydrodynamic cavitation (different removal mechanism) is required for successful removal of different pollutants. The path to use hydrodynamic cavitation as a routine water cleaning method is still long, but recent results have already shown great potential for optimisation, which could lead to a low energy tool for water and wastewater cleaning.