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ANtibioticS and mobile resistance elements in WastEwater Reuse applications: risks and innovative solutions

Periodic Reporting for period 2 - ANSWER (ANtibioticS and mobile resistance elements in WastEwater Reuse applications: risks and innovative solutions)

Reporting period: 2017-10-01 to 2019-09-30

The major mission of ANSWER was to train 15 ESRs to address the risks associated with chemical/biological contaminants of emerging concern, i.e. antibiotics, antibiotic-resistant bacteria and antibiotic resistance genes (A&ARB&ARGs) and wastewater (WW) reuse. The main scientific outputs of the project were the (i) understanding of the fate of A&ARB&ARGs within urban WW, soil, ground/surface water, and crops (determination of A&ARB&ARGs with highest risk of transfer from WW to soils-crops, assessment of the physicochemical factors favoring the persistence of A&ARB&ARGs in soils-crops, determination of the environmental/public health risk factors related with A&ARB&ARGs in soils-crops), (ii) validation of bioassays for A&ARB&ARGs effects evaluation and hazard identification (development/application of protocols for advanced bioassays for A/TPs, assessment of the effects of A/TPs and relation to AR), (iii) development of a modelling framework capable of predicting the fate and assessing the risks associated with A&ARB&ARGs in the environment (development of models on ARB&ARGs passage in soil/water resources affected by WW reuse schemes, development of models to assess the impact of conventional/advanced technologies on ARB&ARGs), (iv) assessment of the efficiency of innovative technologies to minimize A&ARB&ARGs (evaluation of the efficiency of ozonation to minimize A&ARB&ARGs, development/assessment of the efficacy of new photocatalytic reactors and cost analysis), and (v) development of a database for A, TPs, ARB&ARGs in environmental matrices and development of guidelines for the determination of ELVs.
Along with its scientific goals, ANSWER ensured via a number of dissemination/communication activities that its outcomes and impacts were widely shared with the respective research and innovation community, including industry, stakeholders, scientists, general public, and with EU/national policy makers setting the research agendas of the future. One of the main communication outcomes of ANSWER was the development of the children's book ″The Secret Handbook of the Blue Circle″ (PAPADOPOULOS Publishing (Greek); IWA Publishing (English)).
Determination of A&ARB&ARGs with highest risk of transfer from WW to soils-crops: ♦assessment of the occurrence of A&TPs in WW effluents and of their environmental risk, ♦quantification of ARB&ARGs during subsoil passages from surface water to groundwater and from WW to surface/ground water, ♦assessment of the accumulation of ARB&ARGs in soils/crops, ♦identification of A and of their metabolites in crops, ♦prioritization of A&ARB&ARGs with high probability of being transferred to soil/crops.

Assessment of the physicochemical factors favouring the persistence of A&ARB&ARGs in soils-crops: ♦utilisation of microcosm approaches to evaluate the fate of ARB&ARGs in function of various controlled variables, ♦investigation of the effect of operating parameters on the reduction of A&ARB&ARGs during CAS and MBR, ♦quantification of PI-ARGs in soil/crops at real-scale fields, ♦determination of taxonomic changes of endophytic bacteria, ♦identification of physicochemical properties affecting uptake/translocation of A in field experiments.

Determination of the environmental/public health risk factors related with A&ARB&ARGs in soils-crops: ♦analysis of different crops for the determination of the uptake of A&ARB&ARGs, ♦assessment of the human exposure to A via vegetable consumption, ♦estimation of the inactivation of plant associated-ARB&ARGs under human stomach like conditions.

Assessment of the effects of A and of their TPs and relation to antibiotic resistance: ♦assessment of the biological effects of TPs formed under UV-C/H2O2 oxidation, ♦investigation of biological effects of WW effluents using a battery of bioassays and establishment of effect-based trigger values (EBTs), ♦determination of the MIC of A/TPs.

Models concepts and model development for assessing the passage of ARB&ARGs in soil under WW reuse schemes: ♦identification of parameters affecting the abundance ARB&ARGs, ♦model analysis using relevant data from micro/mesocosm studies on the effect of water quality parameters on the fate of A&ARB&ARGs, ♦collection of data describing the transfer of ARB&ARGs in surface water.

Development of model to assess the impact of conventional/advanced technologies on ARB&ARGs: ♦investigation of AR development in a model CAS system, ♦ determination of parameters promoting the prevalence of ARB&ARGs in UWTPs, ♦assessment of the effect of conditions on the reduction of ARB&ARGs in WW during ozonation (+GAC); ♦assessment of the efficiency of heterogeneous photocatalysis and photocatalytic membrane reactor for the removal of A&ARB&ARGs.

Evaluation of the efficiency of WW technologies to minimize A&ARB&ARGs: ♦optimization of ozonation (batch/continuous) for the removal of A&ARB&ARGs, ♦development/optimization/cost analysis of photocatalytic-based reactors (doped ZnO and PMR) for the minimization of A&ARB&ARGs in WW, ♦evaluation of bacterial regrowth.
Determination of Emission Limit Values (ELVs) of A and their TPs, ARB&ARGs in WW used for irrigation: Guidelines were proposed for the establishment of ELVs of A&ARB&ARGs in WW based on a conceptual approach.

Dissemination/Communication activities: ♦18 journal papers published and 18 under review/preparation; ♦51 oral/poster presentations in 24 conferences; ♦2 awards for the best oral presentations; ♦>70 scientific courses/seminars/workshops attended by ESRs; ♦64 talks by Beneficiaries/partners/ESRs; ♦41 press articles; ♦7 e-newsletters; ♦booklet; ♦poster; ♦4 video clips; ♦3 documentaries; ♦children book.

Outreach activities: ♦5 social media; ♦19 visits to schools [>1500 students]; ♦>100 visits to end-users; ♦3 café scientifique events; ♦2 Open Days [130 students]; ♦XENOWAC II Science Slam (4 ESRs); ♦participation of ESRs in various Science weeks/photo competitions/MSCA campaigns; ♦development of a Greek educational program and a theatrical performance based on the children book.
The consortium was able to contribute towards the understanding of the fate/transmission of A&ARB&ARGs from WW to the environment/humans, leading to advances beyond the current state of the art, including among others: ♦determination of the fate of ARB&ARGs in soil using microcosm assays and evaluate AR phenotypes, genotypes and resistance/mobilization gene expression, in function of various variables; ♦characterization of PI-ARGs in WW and quantification of ARB&ARGs during subsoil passages from surface water to groundwater; ♦development of models for the fate of ARB&ARGs in soil/ground/surface waters; ♦determination of taxonomic changes of endophytic bacteria; ♦ development of WW technologies for minimizing A&ARB&ARGs; ♦evaluation of the potential of TPs to induce biological effects; ♦development of an automated procedure for prioritisation of A/TPs based on their hazards; ♦determination of guidelines for the establishment of ELVs for A/TPs.

ANSWER resulted in various scientific/technological/socioeconomic impacts, including expansion of the NORMAN database, contribution to the initiative of the EC to develop a Legislative Instrument for minimum quality criteria for WW reuse, impact on the institutions/organisations and the ESRs, development of a children's book and an educational program.