The aim of this study was to capture and explain changes in antibiotic resistance gene (ARG) presence and concentration internationally across the Rhine river. Intl1 concentrations and national antibiotic usage were investigated as proxies to predict anthropogenic ARG pollution. Newly-developed multiplex qPCR assays were employed to investigate ARG profiles across 8 locations (L1-L8) in three countries (Switzerland, Germany, the Netherlands) and to detect potential regional causes for variation. Two of these locations were further monitored, over the duration of one month. A total of 13 ARGs, Intl1 and 16S rRNA were quantified. ARG presence and concentrations initially increased from L1(Diepoldsau) to L3(Darmstadt). A continuous increase could not be observed at subsequent locations, with the large river volume likely being a major contributing factor for stability. ARG presence and concentrations fluctuated widely across different locations. L2(Basel) and L3 were the two most polluted locations, coinciding with these locations being well-developed pharmaceutical production locations. We draw attention to the characteristic, clearly distinct ARG profiles, with gene presence being consistent and gene concentrations varying significantly less over time than across different locations. Five genes were Rhine-typical (ermB, ermF, Intl1, sul1 and tetM). Intl1 and sul1 were the genes with highest and second-highest concentration, respectively. Aph(III)a and bla_OXA were permanently introduced downstream of L1, indicating no source of these genes prior to L1. We highlight that correlations between Intl1 and ARG concentrations (R² = 0.72) were driven by correlations to sul1 and disappeared when excluding sul1 from the analysis (R² = 0.05). Intl1 therefore seems to be a good proxy for sul1 concentrations but not necessarily for overall (anthropogenic) ARG pollution. Aminoglycoside usage per country correlated with concentrations of aph(III)a and several unrelated antibiotic resistance genes (bla_OXA ermB, ermF and tetM). This correlation can be explained by co-resistance caused by mobile genetic elements (MGEs), such as Tn1545.

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High pressure membranes are increasingly used for the treatment of contaminated water for various purposes including irrigation and drinking water. The lack of a fast and easy to implement membrane integrity test method with a log removal value (LRV) >3 hampers the implementation of these membranes. Current on-line methods include conductivity, TOC (total organic carbon) and turbidity measurements and can monitor a maximum LRV of 3. Furthermore, challenge tests using chemical or bacteriological virus surrogates such as bacteriophage MS2 show that RO and NF systems can reach LRVs of 6-7, but dosing of these surrogates is not feasible and desirable in full scale drinking water plants. This study describes the identification and use of indigenous viruses, naturally present in surface waters to monitor the integrity of RO membranes in a pilot installation. Natural viruses were identified from fresh source water using metagenomics and qPCR primers developed for a selected set of viruses that were present in high numbers in surface water. The qPCR assays were used to determine the number of gene copies of these viruses in the feed and permeate of the pilot RO installation, and the LRV of these natural viruses was compared with the LRV of spiked MS2 and with on-line conductivity. The concentration of the selected natural viruses in the source water was sufficient to demonstrate a LRV of >7 and was comparable to the results of the spiked MS2 bacteriophage. Furthermore, after inflicting damage to the membrane element by drilling small holes of 1 and 4 mm, both MS2 and the natural viruses detected the damage to the membrane with a nearly identical decrease of LRV, while conductivity lacked sensitivity to monitor any integrity loss. This novel method enables monitoring of the RO membrane integrity at a high sensitivity (LRV > 7), without the addition of chemical or biological virus surrogates. Furthermore, the high concentration of viruses in source water simplifies detection without laborious sample concentration procedures. The implementation of this method facilitates monitoring of the integrity of RO membranes in full scale operation with a much higher sensitivity than current methods.

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This study quantified antibiotic and antibiotic resistance gene (ARG) concentrations in hospital and communal wastewaters as well as the influents and effluents of the receiving urban wastewater treatment plants (UWWTP) in two Dutch cities. In only one city, hospital wastewater was treated on-site using advanced technologies, including membrane bioreactor treatment (MBR), ozonation, granulated activated carbon (GAC) and UV-treatment. On-site hospital wastewater (HWW) treatment reduced gene presence of hospital-related antibiotic resistance genes and antibiotic concentrations in the receiving urban wastewater treatment plant. These findings support the need for on-site treatment of high-risk point sources of antibiotic resistance genes. 13 antibiotic resistance genes, Integrase Class 1 and 16S rRNA concentrations were quantified using multiplex quantitative real-time PCR (qPCR) assays and the presence and/or concentration of 711 antibiotics were analyzed. Hospital wastewater contained approximately 25% more antibiotics and gene concentrations between 0.4 log to 1.8-fold higher than communal wastewater (CWW). blaKPC and vanA could be identified as hospital-related genes and were reduced to under the limit of detection (LOD) during on-site treatment. Advanced on-site treatment removed between 0.5 and 3.6-fold more genes than conventional biological urban wastewater treatment (activated sludge). Advanced on-site treatment was able to eliminate 12 out of 19 detected antibiotics, while urban waste water treatment eliminated up to 1 (out of 21 detected). Different advanced treatment technologies were able to target different pollutants to varying extents, making sequential alignment more effective. MBR treatment was most efficient in antibiotic resistance gene reduction and ozonation in antibiotic reduction. blaKPC could only be detected in the influent of the urban wastewater treatment plant receiving untreated hospital wastewater. Similarly, vanA was only consistently detected in this treatment plant. These results indicate a positive effect of on-site treatment of hospital wastewater on the communal sewage system.@en

Averaged 7-day composite effluent wastewater samples from twelve wastewater treatment plants (WWTPs) in nine countries (Romania, Serbia, Hungary, Slovenia, Croatia, Slovakia, Czechia, Austria, Germany) in the Danube River Basin were collected. WWTPs' selection was based on countries' dominant technology and a number of served population with the aim to get a representative holistic view of the pollution status. Samples were analyzed for 2248 chemicals of emerging concern (CECs) by wide-scope target screening employing LC-ESI-QTOF-MS. 280 compounds were detected at least in one sample and quantified. Spatial differences in the concentrations and distribution of the compounds classes were discussed. Additionally, samples were analyzed for the possible agonistic/antagonistic potencies using a panel of in vitro transactivation reporter gene CALUX® bioassays including ERα (estrogenics), anti-AR (anti-androgens), GR (glucocorticoids), anti-PR (anti-progestins), PPARα and PPARγ (peroxisome proliferators) and PAH assays. The potency of the wastewater samples to cause oxidative stress and induce xenobioticmetabolism was determined using the Nrf2 and PXR CALUX® bioassays, respectively. The signals from each of the bioassays were compared with the recently developed effect-based trigger values (EBTs) and thus allowed for allocating the wastewater effluents into four categories based on their measured toxicity, proposing a putative action plan for wastewater operators. Moreover, samples were analyzed for antibiotics and 13 antibiotic-resistant genes (ARGs) and one mobile genetic element (intl1) with the aim to assess the potential for antibiotic resistance. All data collected from these various types of analysis were stored in an on-line database and can be viewed via interactive map at https://norman-data.eu/EWW_DANUBE.@en

To ensure delivery of microbiologically safe drinking water, the physical integrity of the distribution system is an important control measure. During repair works or an incident the drinking water pipe is open and microbiologically contaminated water or soil may enter. Before taking the pipe back into service it must be cleaned. The efficacy of flushing and shock chlorination was tested using a model pipe-loop system with a natural or cultured biofilm to which a microbial contamination (Escherichia coli, Clostridium perfringens spores and phiX174) was added. On average, flushing removed 1.5-2.7 log microorganisms from the water, but not the biofilm. In addition, sand added to the system was not completely removed. Flushing velocity (0.3 or 1.5 m/s) did not affect the efficacy. Shock chlorination (10 mg/L, 1-24 h) was very effective against E. coli and phiX174, but C. perfringens spores were partly resistant. Chlorination was slightly more effective in pipes with a natural compared to a cultured biofilm. Flushing alone is thus not sufficient after high risk repair works or incidents, and shock chlorination should be considered to remove microorganisms to ensure microbiologically safe drinking water. Prevention via hygienic working procedures, localizing and isolating the contamination source and issuing boil water advisories remain important, especially during confirmed contamination events.@en

Soil passage of (pretreated) surface water to remove pathogenic microorganisms is a highly efficient process under oxic conditions, reducing microorganism concentrations about 8 log¹⁰ within tens of meters. However, under anoxic conditions, it has been shown that removal of microorganisms can be limited very much. Setback distances for adequate protection of natural groundwater may, therefore, be too short if anoxic conditions apply. Because removal of microorganisms under suboxic conditions is unknown, this research investigated removal of bacteriophage MS2 and PRD1 by soil passage under suboxic conditions at field scale. At the field location (dune area), one injection well and six monitoring wells were installed at different depths along three suboxic flow lines, where oxygen concentrations ranged from 0.4 to 1.7 mg/l and nitrate concentrations ranged from 13 to 16 mg/L. PRD1 and MS2 were injected directly at the corresponding depths and their removal in each flow line was determined. The highest bacteriophage removal was observed in the top layer, with about 9 log removal of MS2, and 7 log removal of PRD1 after 16 meters of aquifer transport. Less removal was observed at 12 m below surface, probably due to a higher groundwater velocity in this coarser grained layer. MS2 was removed more effectively than PRD1 under all conditions. Due to short travel times, inactivation of the phages was limited and the reported log removal was mainly associated with attachment of phages to the aquifer matrix. This study shows that attachment of MS2 and PRD1 is similar for oxic and suboxic sandy aquifers, and, therefore, setback distances used for sandy aquifers under oxic and suboxic conditions provide a similar level of safety. Sticking efficiency and the attachment rate coefficient, as measures for virus attachment, were evaluated as a function of the physico-chemical conditions.

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