Identification of Reactive Oxygen Species Generated by Irradiation of Aqueous Humic Acid Solution

Abstract

Electron spin resonance (ESR) spectroscopy coupled with the spin-trapping or chemicalquenching method was applied to identify the reactive oxygen species generated by irradiation of aqueous humic acid solution. Upon xenon lamp irradiation of aqueous humic acid in the presence of spin trap 5, 5-dimethyl-1-pyrroline-N-oxide (DMPO), a spin adduct (DMPO-OH) of hydroxyl radical with DMPO was detected. Formation of the DMPO-OH was sensitive to superoxide dismutase, dioxygen and hydroxyl radical scavengers, indicating the formation of superoxide anion and hydroxyl radicals. The irradiation of aqueous humic acid solution in the presence of 2, 2, 6, 6-tetramethyl-4-piperidinol resulted in the formation of 2, 2, 6, 6-tetramethylpiperidine-1-oxyl-4-ol (TEMPOL) nitroxide radical. The ESR signal intensity of the TEMPOL radical was enhanced in deuterium oxide, and reduced in the presence of sodium azide and 1, 4-diazabicyclo-[2.2.2]octane (DABCO), indicating the formation of singlet molecular oxygen. The generation of hydrogen peroxide was confirmed spectrophotometrically based on the peroxidase-catalyzed oxidation of leuco crystal violet.