Supplementary data for article : Kolarević, S.; Milovanović, D.; Kračun-Kolarević, M.; Kostić, J.; Sunjog, K.; Martinović, R.; Đorđević, J.; Novaković, I.; Sladić, D.; Vuković-Gačić, B. Evaluation of Genotoxic Potential of Avarol, Avarone, and Its Methoxy and Methylamino Derivatives in Prokaryotic and Eukaryotic Test Models. Drug and Chemical Toxicology 2019, 42 (2), 130–139. https://doi.org/10.1080/01480545.2017.1413108

Supplementary material for: [https://doi.org/10.1080/01480545.2017.1413108]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/322

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

Supplementary data for article : Kolarević, S.; Milovanović, D.; Kračun-Kolarević, M.; Kostić, J.; Sunjog, K.; Martinović, R.; Đorđević, J.; Novaković, I.; Sladić, D.; Vuković-Gačić, B. Evaluation of Genotoxic Potential of Avarol, Avarone, and Its Methoxy and Methylamino Derivatives in Prokaryotic and Eukaryotic Test Models. Drug and Chemical Toxicology 2019, 42 (2), 130–139. https://doi.org/10.1080/01480545.2017.1413108

Supplementary material for: [https://doi.org/10.1080/01480545.2017.1413108]Related to published version: [http://cherry.chem.bg.ac.rs/handle/123456789/322

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

Impact of pollution on rivers in Montenegro: Ecotoxicological perspective

Montenegrin surface water and groundwater are important for the Balkan Peninsula since they are connected by the transboundary Dinaric Karst Aquifer System with the waters of additional five countries. The pollution from the surface water can rapidly infiltrate in aquifer and endanger this sensible ecosystem and the health of humans through drinking water supply. This chapter gives insights in the pressures of pollution on Montenegrin waters and in a limited literature data regarding freshwater ecotoxicological studies in Montenegro. Also, this chapter provides new ecotoxicological data obtained during survey in 2019, with a focus on the sites which are identified as hotspots of fecal pollution. The highest responses of biomarkers which indicate embryotoxic, genotoxic, and phytotoxic effects in zebrafish embryo test and in roots of Allium cepa were obtained at Ćehotina – downstream of Pljevlja. Similar results were detected at the site downstream Mojkovac at Tara, yet this site is affected by different type of pollution. Genotoxic endpoints in zebrafish stressed out sites on Morača and Lim rivers which are under pressures of fecal pollution. The data in this chapter provides an insight into current status obtained by the ex situ bioassays and indicates need for more comprehensive in situ assessment

Evaluation of genotoxic potential of avarol, avarone, and its methoxy and methylamino derivatives in prokaryotic and eukaryotic test models

In this study, mutagenic and genotoxic potential of anti-tumor compounds avarol, avarone, and its derivatives 3′-methoxyavarone, 4′-(methylamino)avarone and 3′-(methylamino)avarone was evaluated and compared to cytostatics commonly used in chemotherapy (5-fluorouracil, etoposid, and cisplatin). Mutagenic potential of selected hydroquinone and quinones was assessed in prokaryotic model by the SOS/umuC assay in Salmonella typhimurium TA1535/pSK1002. Genotoxic potential was also assessed in eukaryotic models using comet assay in human fetal lung cell line (MRC-5), human adenocarcinoma epithelial cell line (A549), and in human peripheral blood cells (HPBC). The results indicated that avarol and avarone do not exert mutagenic/genotoxic potential. Among the studied avarone derivatives, mutagenic potential was detected by SOS/umuC test for 3′-(methylamino)avarone, but only after metabolic activation. The results of comet assay indicated that 3′-methoxyavarone and 3′-(methylamino)avarone have a significant impact on the level of DNA damage in the MRC-5 cell line. Genotoxic potential was not observed in A549 cells or HPBC probably due to a different uptake rate for the compounds and lower in metabolism rate within these cells.Supplementary material: [http://cherry.chem.bg.ac.rs/handle/123456789/3033