Effect-based monitoring of emerging organic micropollutant mixtures in conventional wastewater treatment plant effluents in Flanders, Belgium

Wastewater treatment plants are recognized as major sources of organic micropollutants (OMPs) for aquatic environments. Yet, chemical monitoring alone may underestimate the ecological risks posed by complex OMP mixtures. Here, we combined an effect-based monitoring approach with targeted chemical analysis to assess environmental risks of OMP mixtures in effluents from 16 wastewater treatment plants in Flanders, Belgium. Effluent sites were selected from a five-year regional monitoring dataset, prioritizing locations with high cumulative risk quotients. Bioassays using Microcystis aeruginosa (cyanobacteria growth inhibition) and Danio rerio (zebrafish larvae, dark-light locomotive assay) were conducted on effluent extracts. High-resolution mass spectrometry identified 130 compounds, with 26 OMPs quantified across classes, including pharmaceuticals, antibiotics, herbicides, and per- and polyfluoroalkyl substances. Median and 10% effective concentrations for cyanobacteria inhibition ranged in relative enrichment factors of 4.1 to 38 and 1.1 to 4.7, respectively. Iceberg modeling identified azithromycin and clarithromycin as the main drivers of cyanobacterial inhibition. Zebrafish behavioral responses were significantly affected in 8 of 16 samples (relative enrichment factors, 1.25-20), but these differences could not be explained by the available chemical data. Only some suspect compounds were identified, including antidepressants and pesticides; therefore, this remains an interesting aspect for future investigations. Risk characterization for receiving surface waters using chemical-based risk quotients, margin of safety, and effect-based trigger values revealed ecological risk (risk quotient >1) in 13 of 16 sites. This study highlights the added value of integrating effect-based monitoring with chemical monitoring to explain mixture effects, identify key toxicants, and support improved regulatory frameworks for environmental management.