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.

Multiparametric monitoring of microbial faecal pollution reveals the dominance of human contamination along the whole Danube River.

The microbial faecal pollution of rivers has wide-ranging impacts on a variety of human activities that rely on appropriate river water quality. Thus, detailed knowledge of the extent and origin of microbial faecal pollution is crucial for watershed management activities to maintain safe water use. In this study, the microbial faecal pollution levels were monitored by standard faecal indicator bacteria (SFIB) along a 2580 km stretch of the Danube, the world's most international river, as well as the Danube's most important tributaries. To track the origin of faecal pollution, host-associated Bacteroidetes genetic faecal marker qPCR assays for different host groups were applied in concert with SFIB. The spatial resolution analysis was followed by a time resolution analysis of faecal pollution patterns over 1 year at three selected sites. In this way, a comprehensive faecal pollution map of the total length of the Danube was created, combining substantiated information on both the extent and origin of microbial faecal pollution. Within the environmental data matrix for the river, microbial faecal pollution constituted an independent component and did not cluster with any other measured environmental parameters. Generally, midstream samples representatively depicted the microbial pollution levels at the respective river sites. However, at a few, somewhat unexpected sites, high pollution levels occurred in the lateral zones of the river while the midstream zone had good water quality. Human faecal pollution was demonstrated as the primary pollution source along the whole river, while animal faecal pollution was of minor importance. This study demonstrates that the application of host-associated genetic microbial source tracking markers in concert with the traditional concept of microbial faecal pollution monitoring based on SFIB significantly enhances the knowledge of the extent and origin of microbial faecal pollution patterns in large rivers. It constitutes a powerful tool to guide target-oriented water quality management in large river basins.

Variability in DNA damage of chub (Squalius cephalus L.) blood, gill and liver cells during the annual cycle.

In this work the genotoxic potential of water in three localities in Serbia, which differ by the nature and degree of pollution, was determined in tissues of European chub (Squalius cephalus L.) on monthly basis over the 2011/2012 year season using the alkaline comet assay. Specimen samples of chub were taken from Special Nature Reserve "Uvac", as control site, and Pestan and Beljanica Rivers, as polluted sites at Kolubara basin, surrounded with coal mines. Three tissues, blood, gills and liver were used for assessing the level of DNA damage. Analysis was done by software (Comet Assay IV). The control site at Reserve "Uvac" showed the lowest DNA damage values for all three tissues compared to Pestan and Beljanica. Blood has the lowest level of DNA damage in comparison with liver and gills. Decreased damage for all three tissues was observed at summer, while during the spring and autumn damage increased.

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

The 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 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, gill cells, and liver cells, were used for assessing DNA damage. Images of randomly selected cells were analyzed with a Leica fluorescence microscope 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 1,700 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), the 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 the 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).