Time-Resolved Method for Monitoring Hydroxyl Radical Formation Capacity in UVB/UVC Water Treatment Processes

• Time-resolved trac of HO • and H₂O₂ in UV-AOPs using a mirrored irradiation setup • Continuous ROS monitoring without perturbing pollutant degradation in the primary reactor • Accurate HO • quantification achieved despite the presence of fluorescent intermediates • Degradation intermediates shown to sustain HO • formation at later treatment stages • Platform readily expandable to multi-ROS detection for mechanistic AOP studies Monitoring the evolution of reactive oxygen species (ROS) during UV-based advanced oxidation processes (AOPs) is critical for understanding degradation mechanisms and assessing water treatment performance. However, the nanosecond lifetime of hydroxyl radicals (HO • ) prevents their direct measurement during irradiation, which limits insight into temporal variations in oxidative capacity as pollutants transform. This work introduces the MiMe (Mirror Irradiation Monitoring Experiment) method, a novel analytical approach enabling time-resolved quantification of HO • production capacity in UV-AOPs while simultaneously monitoring hydrogen peroxide (H₂O₂). The MiMe method relies on periodic sampling from a primary UV reactor, followed by brief secondary irradiation of each aliquot in the presence of terephthalic acid (TA). In this mirrored configuration, HO • precursors such as H₂O₂ and photogenerated intermediates are converted into HO • , which reacts with TA to form the fluorescent product 2-hydroxyterephthalic acid (TAOH). In parallel, H₂O₂ is quantified using the 4-hydroxyphenylacetic acid (HPAA)–horseradish peroxidase (HRP) assay. Both analytes are selectively quantified by HPLC with fluorescence detection (down to nM concentration range), ensuring reliable analysis even in complex matrices. Key experimental parameters, including probe concentration, irradiated volume, and photon flux, were optimised to ensure robust and reproducible quantification under realistic conditions (less than 5% variation across 3 independent secondary reactors). Application of the MiMe method to the UVC degradation of acetaminophen revealed that dimeric intermediates and humic-like substances continue to promote HO • generation even after substantial depletion of the parent compound. This previously unrecognised behaviour alters ROS dynamics during later treatment stages and highlights the mechanistic role of transformation products in sustaining oxidative capacity. Overall, the MiMe method provides an accessible and versatile tool for time-resolved ROS characterisation and offers strong potential for future multi-ROS monitoring and optimisation of UV-AOPs for wastewater treatment applications.