Detection of SARS-CoV-2 RNA in the Danube river in Serbia associated with the discharge of untreated wastewaters

In Serbia less than 13 % of collected municipal wastewaters is being treated before their release in the environment. This includes all municipal wastewater discharges from Belgrade (capital city of Serbia; population 1,700,000). Previous research has identified the impacts of raw wastewater discharges from Belgrade on the Danube River, and this study investigated if such discharges also provided a pathway for SARS-CoV-2 RNA material. Samples were collected during the most critical circumstances that occurred so far within the COVID-19 pandemics in Serbia. Grab and composite samples were collected in December 2020, during the peak of the third wave (in terms of reported cases) at the site which receives the wastewater loads in Belgrade. Grab samples collected upstream and downstream of Belgrade were also analyzed. RNA was quantified using RT-qPCR with primer sets targeting nucleocapsid (N1 and N2) and envelope (E) protein genes. SARS-CoV-2 RNA (5.97×103 to 1.32×104 copies/L) was detected only in samples collected at the site strongly impacted by the wastewaters where all three applied primer sets gave positive signals. Determined concentrations correspond to those reported in wastewater influents sampled at treatment plants in other countries indicating an epidemiological indicator function of used approach for rivers with high pollution loads in countries with poor wastewater treatment

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

Seasonal variations of microbiological parameters of water quality of the Velika Morava river Serbia

In this study, we investigated the level of sanitary pollution and organic contamination of the Velika Morava River, the largest river in central Serbia. Samples of water for microbiological analysis were collected at 5 sites, monthly, from April 2010 to February 2011. Sanitary analysis, i.e. enumeration of total and fecal coliforms and intestinal enterococci, indicated moderate to critical fecal contamination, while organic load assessment (oligotroph to heterotroph ratio, index of phosphatase activity) revealed the category of moderately polluted water. We also investigated seasonal variations of these groups of bacteria and the factors that could contribute to these differences. Our results showed that the micro- biological quality of the water in the Velika Morava River during different seasons is affected by numerous factors such as unequal loading of wastewaters, solar irradiation, and relations of flow/dilution and rainfall/runoff. [Acknowledgments. This study was financially supported by the Ministry of Education and Science of the Republic of Serbia: projects No 173025, No 177045 and No 043002.

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

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

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

Evaluating the transmission risk of SARS-CoV-2 from sewage pollution

The presence of SARS-CoV-2 in untreated sewage has been confirmed in many countries but its incidence and infection risk in contaminated waters is poorly understood. The River Thames in the UK receives untreated sewage from 57 Combined Sewer Overflows (CSOs), with many discharging dozens of times per year. This study investigated if such discharges provide a pathway for environmental transmission of SARS-CoV-2. Samples of wastewater, surface water, and sediment collected close to six CSOs on the River Thames were assayed over eight months for SARS-CoV-2 RNA and infectious virus. Bivalves were also sampled as an indicator species of viral bioaccumulation. Sediment and water samples from the Danube and Sava rivers in Serbia, where raw sewage is also discharged in high volumes, were assayed as a positive control. No evidence of SARS-CoV-2 RNA or infectious virus was found in UK samples, in contrast to RNA positive samples from Serbia. Furthermore, this study shows that infectious SARS-CoV-2 inoculum is stable in Thames water and sediment for <3 days, while SARS-CoV-2 RNA is detectable for at least seven days. This indicates that dilution of wastewater likely limits environmental transmission, and that detection of viral RNA alone is not an indication of pathogen spillover

Evaluating the transmission risk of SARS-CoV-2 from sewage pollution

The presence of SARS-CoV-2 in untreated sewage has been confirmed in many countries but its incidence and infection risk in contaminated waters is poorly understood. The River Thames in the UK receives untreated sewage from 57 Combined Sewer Overflows (CSOs), with many discharging dozens of times per year. This study investigated if such discharges provide a pathway for environmental transmission of SARS-CoV-2. Samples of wastewater, surface water, and sediment collected close to six CSOs on the River Thames were assayed over eight months for SARS-CoV-2 RNA and infectious virus. Bivalves were also sampled as an indicator species of viral bioaccumulation. Sediment and water samples from the Danube and Sava rivers in Serbia, where raw sewage is also discharged in high volumes, were assayed as a positive control. No evidence of SARS-CoV-2 RNA or infectious virus was found in UK samples, in contrast to RNA positive samples from Serbia. Furthermore, this study shows that infectious SARS-CoV-2 inoculum is stable in Thames water and sediment for <3 days, while SARS-CoV-2 RNA is detectable for at least seven days. This indicates that dilution of wastewater likely limits environmental transmission, and that detection of viral RNA alone is not an indication of pathogen spillover