Probing the Microheterogeneous Distribution of Photochemically Produced Hydroxyl Radicals in Dissolved Organic Matter

Dissolved organic matter (DOM) is a major photosensitizer in sunlit surface waters that generates hydroxyl radicals (^•OH). While ^•OH is believed to form within hydrophobic DOM microdomains, its spatial distribution and phase-specific reactivity remain poorly characterized among DOM from diverse environments. In this study, we employed testosterone as a probe to quantify DOM-phase ^•OH concentration ([^•OH]_DOM) via hydrophobic partitioning. Across eight DOM isolates, [^•OH]_DOM was found to be 20-150 times higher than aqueous phase concentration ([^•OH]_aq). To explore the underlying drivers of this microheterogeneity, we evaluated [^•OH]_DOM/[^•OH]_aq in relation to DOM composition. We report, for the first time, a negative correlation between [^•OH]_DOM/[^•OH]_aq and aromaticity and a positive correlation with aliphaticity. Testosterone was further employed to quantify [^•OH]_DOM/[^•OH]_aq for Suwannee River natural organic matter and humic acid isolates size fractionated with a 3 kDa ultrafiltration membrane. As expected, the <3 kDa showed little evidence of ^•OH microheterogeneity. In contrast, the >3 kDa fraction showed lower [^•OH]_DOM/[^•OH]_aq than the bulk fraction, suggesting that both high- and low-molecular weight components are important for the formation of microheterogeneous ^•OH. Overall, these results establish the ubiquity of microheterogeneous ^•OH formed during DOM photolysis and suggest that the abundance of aliphatic and aromatic carbon are important structural features governing this microheterogeneity.