Ecological succession and functional alterations in stream periphyton exposed to episodic wastewater- derived pollutants

The ecological state of a peri-urban stream was assessed through a 10-week developmental analysis of periphyton microbial community structures on clay bead artificial substrates (CBAS), coupled with hydrological and chemical monitoring. Forty pharmaceuticals and 12 pesticides were quantified via POCIS samplers, and microbial source tracking (MST) assays of human and ruminant fecal contaminations. Rain events triggered sharp increases in contaminants and MST markers, revealing episodic wastewater inputs. Early-stage periphytons exposed to pollution showed reduced protein content but elevated respiratory activity, indicating energetic shifts. Bacterial densities reached ~108 16S rRNA gene copies/cm², with stable algal-to-bacterial ratios and significant fungal colonization. Metabarcoding analyses revealed distinct shifts in microbial diversity and composition between early and late biofilms. FEAST analysis showed increasing similarity to natural benthic periphytons over time, while planktonic signatures declined. Flavobacterium, Luteolibacter, and phototrophs dominated initial biofilms, progressively replaced by Acidobacteriota, Planctomycetota, and Nitrospirota—taxa involved in nitrogen and carbon cycling. These reassortments indicated a maturing microbial network, with enhanced ureolytic, nitrifying, and acetate-producing functions. The data point to substantial ecological disruption from sewer-derived pollutants, with measurable effects on microbial succession and periphyton functionality. CBAS periphytons emerge as sensitive indicators of wastewater-driven perturbation in urban stream ecosystems.