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Treated wastewater irrigation (TWW) releases antibiotics and antibiotic resistance genes (ARGs) into the environment and might thus promote the dissemination of antibiotic resistance in groundwater (GW). We hypothesized that TWW irrigation increases ARG abundance in GW through two potential mechanisms: the contamination of GW with resistant bacteria and the accumulation of antibiotics in GW. To test this, the GW below a real-scale TWW-irrigated field was sampled for six months. Sampling took place before, during and after high-intensity TWW irrigation. Samples were analysed with 16S rRNA amplicon sequencing, qPCR of six ARGs and the class 1 integron-integrase gene intI1, while liquid chromatography tandem mass spectrometry was performed to detect antibiotic and pharmaceutical residues. Absolute abundance of 16S rRNA in GW decreased rather than increased during long-term irrigation. Also, the relative abundance of TWW-related bacteria did not increase in GW during long-term irrigation. In contrast, long-term TWW irrigation increased the relative abundance of sul1 and intI1 in the GW microbiome. Furthermore, GW contained elevated concentrations of sulfonamide antibiotics, especially sulfamethoxazole, to which sul1 confers resistance. Total sulfonamide concentrations in GW correlated with sul1 relative abundance. Consequently, TWW irrigation promoted sul1 and intI1 dissemination in the GW microbiome, most likely due to the accumulation of drug residues.

In the present study, we investigated the impact of treated wastewater (TWW) irrigation on the prevalence of antibiotic resistance genes (ARGs) in subsoil pore-water, a so-far under-appreciated matrix. We hypothesized that TWW irrigation increases ARG prevalence in subsoil pore-water. This hypothesis was tested using a multiphase approach, which consisted of sampling percolated subsoil pore-water from lysimeter-wells of a real-scale TWW-irrigated field, operated for commercial farming practices, and controlled, laboratory microcosms irrigated with freshwater or TWW. We monitored the abundance of six selected ARGs (sul1, blaOXA-58, tetM, qnrS, blaCTX-M-32 and blaTEM), the intI1 gene associated with mobile genetic elements and an indicator for anthropogenic pollution and bacterial abundance (16S rRNA gene) by qPCR. The bacterial load of subsoil pore water was independent of both, irrigation intensity in the field study and irrigation water type in the microcosms. Among the tested genes in the field study, sul1 and intI1 exhibited constantly higher relative abundances. Their abundance was further positively correlated with increasing irrigation intensity. Controlled microcosm experiments verified the observed field study results: the relative abundance of several genes, including sul1 and intI1, increased significantly when irrigating with TWW compared to freshwater irrigation. Overall, TWW irrigation promoted the spread of ARGs and intI1 in the subsoil pore-water, while the bacterial load was maintained. The combined results from the real-scale agricultural field and the controlled lab microcosms indicate that the dissemination of ARGs in various subsurface environments needs to be taken into account during TWW irrigation scenarios.

The occurrence of chemical and biological contaminants of emerging concern (CECs) was investigated in treated wastewater intended for reuse in agriculture. An agarose hydrogel diffusion-based passive sampler was exposed to the outlet of a wastewater treatment plant (WWTP) located in Cyprus, which is equipped with membrane bioreactor (MBR). Passive samplers in triplicate were exposed according to a time-series exposure plan with maximum exposure duration of 28 days. Composite flow-proportional wastewater samples were collected in parallel with the passive sampling exposure plan and were processed by solid phase extraction using HORIZON SPE-DEX 4790 and the same sorbent material (Oasis HLB) as in the passive sampler. The analysis of passive samplers and wastewater samples enabled (i) the field-scale calibration of the passive sampler prototype by the calculation of in situ sampling rates of target substances, and (ii) the investigation of in silico predicted transformation products of the four most ecotoxicologically hazardous antibiotics (azithromycin, clarithromycin, erythromycin, ofloxacin). Additionally, the wastewater samples were subjected to the analysis of seven preselected antibiotic resistant genes (ARGs) and one mobile resistant element (int1). All extracts were analyzed for chemicals in a single batch using a highly sensitive method for pharmaceuticals, antibiotics and illicit drugs by liquid chromatography tandem MS/MS (LC-QQQ) and for various other target compounds (2316 compounds in total) by liquid chromatography high-resolution mass spectrometry (LC-HRMS). 279 CECs and all investigated ARGs (except for blaCTX-M−32) were detected, highlighting potential chemical and biological hazards related to wastewater reuse practices. 16 CECs were prioritized following ecotoxicological risk assessment, whereas sul1 and the mobile resistant element (int1) showed the highest abundance. Comprehensive monitoring efforts using novel sampling methods such as passive sampling, wide-scope target screening and molecular analysis are required to assure safe application of wastewater reuse and avoid spread and crop uptake of potentially hazardous chemicals. Keywords: Hydrogel-based passive sampler, Wastewater reuse, Contaminants of emerging concern, Transformation products, Antibiotics, Antibiotic resistance genes