Abstract
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.