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.

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.

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.