Comparison of comet assay parameters for estimation of genotoxicity by sum of ranking differences

Genotoxic potential of waters in six rivers and reservoirs from Serbia was monitored in different tissues of chub (Squalius cephalus L. 1758) with the alkaline comet assay. The comet assay, or a single cell gel electrophoresis, has a wide application as a simple and sensitive method for evaluating DNA damage in fish exposed to various xenobiotics in the aquatic environment. Three types of cells, erythrocytes, gills and liver cells were used for assessing of DNA damage. Images of randomly selected cells were analyzed with a fluorescence microscope Leica and image analysis by software (Comet Assay IV Image analysis system, PI, UK). Three parameters (tail length - l, tail intensity - i and Olive tail moment – m) were analyzed on 1750 nuclei per cell type. The procedure for sum of ranking differences (SRD) was implemented to compare different types of cells and different parameters for estimation of DNA damage. Regarding our nine different estimations of genotoxicity: tail length, intensity and moment in erythrocytes (rel, rei, rem), liver cells (rll, rli, rlm) and gill cells (rgl, rgi, rgm) SRD procedure has shown that the Olive tail moment and tail intensity are (almost) equally good parameters; the SRD value was lower for the tail moment and tail intensity than for tail length in case of all types of cells. The least reliable parameter was rel; close to the borderline case were rei, rll, and rgl (~5% probability of random ranking)

Determination of the level and source of microbial pollution in the Sava River Basin

Introduction. The contamination of water bodies by sewage or manure is generally determined by fecal indicator bacteria. Specific differentiation between sources of contamination is of particular importance, since the health risk to humans is usually considered higher from human, than from animal fecal contamination. Quantitative PCR (qPCR)-based assays for analysis of human- or animal-associated genetic Bacteroidetes fecal markers have gained increased popularity in the field of microbial source tracking (MST). Aim. The aim of the present study was to assess the level and source of microbial pollution in the Sava River Basin (SRB). Materials and methods. The sampling at the SRB was performed in September 2015, at 15 locations. Additional samples were collected from 4 wastewater outlets. Quantification of total coliforms, E. coli and enterococci was performed with Colilert Quanti-Tray 2000 and MPN approach. The human-associated BacHum and HF183II, the ruminant-associated BacR and the pig-associated Pig2Bac fecal markers were enumerated by quantitative PCR (qPCR). Results. According to the concentrations of fecal indicator bacteria, the river samples were scattered within the classes I and II (slight to moderate pollution). According to all monitored indicators, samples collected at 3 wastewater outlets were excessively contaminated. The results of MST revealed the presence of human-associated fecal markers BacHum and HF183II at 13 locations. The ruminant-associated BacR and the pig-associated Pig-2-Bac markers were not detected. High correlation was observed between the standard fecal indicators and human associated fecal markers. Conclusions. The results reveal human origin of fecal pollution in the SRB and indicates the urgent need for effective wastewater treatment plants in water management

Application of sos/umuc assay in eco/genotoxicology

Introduction. The SOS/umuC assay is used for assessment of water genotoxicity. Salmonella typhimurium TA1535/pSK1002 is used in this test, but the results can be extrapolated on higher eukaryotic organisms with the introduction of enzymatic S9 fraction in experimental procedure. The test is standardised for the determination of the genotoxic potential of water and wastewater (ISO/DIS 13829, 2000). Aim. Sensitivity of the assay was challenged by using parallel in situ and in vitro approach in evaluation of the genotoxic potential in the basins of significant tributaries of the Danube River: the Sava River and the Velika Morava River. Materials and methods. Within in vitro testing, native water samples were analysed by SOS/umuC test. DNA damage in situ was assessed in bleak (Alburnus alburnus) erythrocytes by the comet and micronucleus assays. The concentration of heavy metals in fish tissue and the data of the physico-chemical parameters measured in water were used as a measure of the pollution pressure at the sites. Results. Results showed that applied in vitro tests with native water samples are less sensitive in comparison with in situ tests. None of 20 investigated samples showed genotoxic potential in SOS/umuC assay while in situ analyses indicated variation of genotoxic potential among the investigated sites. Conclusions. The results of our study point towards low sensitivity of the SOS/umuC test when processing un-concentrated (native) water samples; the results of in vitro tests should be taken with precaution when making predictions on the status of the ecosystem

Determination of the level and source of microbial pollution in the Sava River Basin

Introduction. The contamination of water bodies by sewage or manure is generally determined by fecal indicator bacteria. Specific differentiation between sources of contamination is of particular importance, since the health risk to humans is usually considered higher from human, than from animal fecal contamination. Quantitative PCR (qPCR)-based assays for analysis of human- or animal-associated genetic Bacteroidetes fecal markers have gained increased popularity in the field of microbial source tracking (MST). Aim. The aim of the present study was to assess the level and source of microbial pollution in the Sava River Basin (SRB). Materials and methods. The sampling at the SRB was performed in September 2015, at 15 locations. Additional samples were collected from 4 wastewater outlets. Quantification of total coliforms, E. coli and enterococci was performed with Colilert Quanti-Tray 2000 and MPN approach. The human-associated BacHum and HF183II, the ruminant-associated BacR and the pig-associated Pig2Bac fecal markers were enumerated by quantitative PCR (qPCR). Results. According to the concentrations of fecal indicator bacteria, the river samples were scattered within the classes I and II (slight to moderate pollution). According to all monitored indicators, samples collected at 3 wastewater outlets were excessively contaminated. The results of MST revealed the presence of human-associated fecal markers BacHum and HF183II at 13 locations. The ruminant-associated BacR and the pig-associated Pig-2-Bac markers were not detected. High correlation was observed between the standard fecal indicators and human associated fecal markers. Conclusions. The results reveal human origin of fecal pollution in the SRB and indicates the urgent need for effective wastewater treatment plants in water management

Application of sos/umuc assay in eco/genotoxicology

Introduction. The SOS/umuC assay is used for assessment of water genotoxicity. Salmonella typhimurium TA1535/pSK1002 is used in this test, but the results can be extrapolated on higher eukaryotic organisms with the introduction of enzymatic S9 fraction in experimental procedure. The test is standardised for the determination of the genotoxic potential of water and wastewater (ISO/DIS 13829, 2000). Aim. Sensitivity of the assay was challenged by using parallel in situ and in vitro approach in evaluation of the genotoxic potential in the basins of significant tributaries of the Danube River: the Sava River and the Velika Morava River. Materials and methods. Within in vitro testing, native water samples were analysed by SOS/umuC test. DNA damage in situ was assessed in bleak (Alburnus alburnus) erythrocytes by the comet and micronucleus assays. The concentration of heavy metals in fish tissue and the data of the physico-chemical parameters measured in water were used as a measure of the pollution pressure at the sites. Results. Results showed that applied in vitro tests with native water samples are less sensitive in comparison with in situ tests. None of 20 investigated samples showed genotoxic potential in SOS/umuC assay while in situ analyses indicated variation of genotoxic potential among the investigated sites. Conclusions. The results of our study point towards low sensitivity of the SOS/umuC test when processing un-concentrated (native) water samples; the results of in vitro tests should be taken with precaution when making predictions on the status of the ecosystem

Biology and conservation of freshwater bivalves : past, present and future perspectives

Freshwater bivalves have been highly threatened by human activities, and recently their global decline has been causing conservational and social concern. In this paper, we review the most important research events in freshwater bivalve biology calling attention to the main scientific achievements. A great bias exists in the research effort, with much more information available for bivalve species belonging to the Unionida in comparison to other groups. The same is true for the origin of these studies, since the publishing pattern does not always correspond to the hotspots of biodiversity but is concentrated in the northern hemisphere mainly in North America, Europe and Russia, with regions such as Africa and Southeast Asia being quite understudied. We also summarize information about past, present and future perspectives concerning the most important research topics that include taxonomy, systematics, anatomy, physiology, ecology and conservation of freshwater bivalves. Finally, we introduce the articles published in this Hydrobiologia special issue related with the International Meeting on Biology and Conservation of Freshwater Bivalves held in 2012 in Braganc¸a, Portugal.We would like to express our gratitude to our sponsors and institutions, especially to the Polytechnic Institute of Braganca for all the logistic support. We acknowledge all keynote speakers, authors, session chairpersons and especially to all attendees whose contributions were fundamental for the success of this meeting. We would also like to thank all referees of this special issue and to Koen Martens, Editor-in-Chief of Hydrobiologia, for all the valuable comments and suggestions. The chronogram was built with the help of the expert opinion of fellow colleagues Rafael Araujo, Arthur Bogan, Kevin Cummings, Dan Graf, Wendell Haag, Karl-Otto Nagel and David Strayer to whom we are very grateful. The authors acknowledge the support provided by Portuguese Foundation for Science and Technology (FCT) and COMPETE funds-projects CONBI (Contract: PTDC/AAC-AMB/117688/2010) and ECO-IAS (Contract: PTDC/AAC-AMB/116685/2010), and by the European Regional Development Fund (ERDF) through the COMPETE, under the project "PEst-C/MAR/LA0015/2011"

Antimutagenic effect of essential oil of sage (Salvia officinalis L.) and its fractions against UV-induced mutations in bacterial and yeast cells

The inhibition of spontaneous and UV-induced mutations by essential oil (EO) of sage (Salvia officinalis L.) and its fractions F1-F5 containing different proportions of mono- and sesquiterpenes was studied with the Salmonella/microsome, E. coli K12, and S. cerevisiae D7 reversion assays. The EO, F1, and F2 exhibited antimutagenic potential against UV-induced mutations in all tests. Fractions F3 and F4 produced a toxic, mutagenic, or antimutagenic response, depend­ing on the test organism used. Reduction of spontaneous and UV-induced mutations by F5 was detected only in permeable strains of E. coli. The obtained results demonstrate antimutagenic activity of volatile sage terpenes and recommend them for further antimutagenesis and anticarcinogenesis studies