Chemical Mixture Toxicity and Metagenomic Profiles Temporally Covary in Wastewater from a Municipal Sewer

Municipal wastewater contains dynamic mixtures of bioactive chemicals and microbial communities that fluctuate on subdaily time scales, yet conventional monitoring obscures short-lived toxicity pulses and microbial shifts. This study investigates whether the known diurnal swing in wastewater chemical composition can be reflected using bioanalytical tools, and specifically whether subdaily variability alters interpretations of mixture toxicity, microbial community structure, and functional potential. We applied an integrated framework combining bulk chemical indicators, multispecies bacterial growth inhibition assays ( Escherichia coli, Pseudomonas putida, and Bacillus subtilis ), and long-read metagenomics to hourly composite samples collected across early morning, midday, and evening flow regimes at a single site within a sanitary-separated sewer. Water chemistry varied over the 16 h period, including shifts in dissolved organic carbon, ionic strength, and optical surrogate signals. Growth inhibition assays revealed organism-specific responses aligned with these transitions, while long-read metagenomics showed coordinated restructuring of taxonomic and functional profiles, including shifts in genes encoding oxidoreductases, transferases, and xenobiotic-responsive enzymes. These results show that diurnal chemical variability is directly reflected in mixture toxicity and microbial functional potential. This demonstrates that bioanalytical tools can resolve short-lived hazard dynamics that conventional sampling obscures.