Víctor Matamoros

Wastewater treatment plants (WWTPs) often fail to fully remove hazardous waterborne contaminants, allowing antibiotic resistance genes (ARGs), pathogenic microorganisms, and persistent microcontaminants to reach receiving aquatic environments. Here, we assessed the seasonal performance of a demonstration-scale zooplankton-based quaternary treatment system combining Daphnia magna with lamella-supported biofilm developed within the reactor, treating up to 80 m³/day of secondary wastewater at a hydraulic retention time of 2 days. Across seasons, the system substantially reduced the abundance of potentially pathogenic (e.g., Aeromonas, Mycobacterium) and WWTP-associated bacterial genera (e.g., Thiothrix, Brachymonas, Arcobacter, and Comamonas), achieved consistently high ARG removal (91–96%), and enriched freshwater-associated taxa (hgcl_clade, Rhodoluna) in the reclaimed water. The lamella-supported biofilm exhibited low ARG prevalence and harboured taxa with biodegradation potential (e.g., Alphaproteobacteria, Rhodobacter), highlighting its role as a degradation interface within the system. However, removal of 25 pharmaceuticals and industrial compounds ranged from negligible to 78%, indicating constrained degradation mechanisms for microcontaminants in the system. Overall, this system represents an effective polishing step to improve wastewater ecological quality by mitigating microbiological hazards, reducing antimicrobial resistance, and promoting the renaturalisation of wastewater microbiomes prior to discharge into aquatic ecosystems.

This study provides a comprehensive assessment of emerging contaminants (ECs), antibiotic resistance genes (ARGs), and heavy metals simultaneously in a complete soil-plant-grain system following biosolids application. Using durum wheat (Triticum aestivum subsp. L.) under controlled greenhouse conditions, we evaluated targeted mitigation strategies, composting and biochar amendment, across contrasting soil textures to understand their differential effects on multi-contaminant fate. Fifty-eight ECs, ten ARGs/horizontal gene transfer markers, and seven heavy metals were monitored over 140 days in a sandy soil and silty clay loam (SCL). Biosolids contained ECs up to 793 ng g−1, dominated by fluoroquinolones and macrolides, alongside all target ARGs. We demonstrate that soil texture acts as a master variable controlling contaminant persistence, with sandy soil promoting EC and ARG retention while SCL soil enhanced temporal attenuation. Critically, mitigation strategies showed soil-texture-dependent efficacy: composting reduced initial contaminant loads but paradoxically increased persistence in SCL soil, whereas 10% biochar selectively reduced both EC persistence and β-lactam ARGs in the same soil. Most importantly, despite detectable soil contamination, no ECs or ARGs transferred to wheat grain, indicating negligible direct food safety risks under single-application scenarios. This multi-contaminant approach reveals previously unknown soil-amendment interactions and establishes that effective biosolids management requires soil-specific strategies rather than universal approaches.

The new European (EU) regulation on water reuse explicitly incorporates antimicrobial resistance (AMR) into routine monitoring and risk management, creating an urgent need to define target antibiotic resistance genes (ARGs) for reclaimed irrigation water and agricultural sludge. However, existing global data largely focus on secondary effluents, providing little actionable evidence for reuse-oriented systems. Here, we present the first integrated framework combining targeted antibiotic residue analysis with shotgun metagenomics of the resistome, mobilome, and microbiome across full-scale reuse-oriented wastewater treatment plants (WWTPs) in Southern Europe to identify sentinel antibiotic resistance families for monitoring. Reclaimed effluents reflected low AMR exposure levels (<0.5 ARGs/cell), but mobile genetic element (MGE) levels comparable to secondary effluents (1-2 MGEs/cell). Effluent communities differed by WWTP configuration: membrane bioreactor combined with ultrafiltration favored nutrient-removal/oxidative-stress taxa and reduced transferable MGEs, whereas plants relying on physical separation (sand filtration or reverse osmosis) retained fecal-associated taxa and MGEs. Specific clinically relevant ARGs persisted after treatments, including aadA and aph(3'')-Ibs (resistance to aminoglycosides), ermB and mphA (resistance to macrolides), and blaOXA-129 (resistance to beta-lactams), which we identify as sentinel markers for monitoring reclaimed water and sludge. We advance a generalizable two-step framework, metagenomic discovery to identify sentinel markers, followed by targeted assays for streamlined surveillance, that provides the first operational blueprint for integrating AMR into water reuse management under the EU regulation.