Assessment of a Daphnia–Biofilm Quaternary Water Treatment for Water Renaturalisation: Microbial and Chemical Changes

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.