Evidence for reactive oxygen species formation associated with bulk nanobubbles in pure water

• Bulk nanobubbles produced by ultrasonic irradiation show gas-dependent oxidative probe responses in water. • O 2 - and N 2 -derived nanobubbles exhibit increased and time-evolving H 2 O 2 -equivalent signals. • Oxidative probe responses are significantly reduced in CO 2 -derived nanobubble suspensions. • Interfacial electrostatic environments and CO 2 -related radical scavenging are suggested to influence observed oxidative signals. Bulk nanobubbles (NBs) have been reported to exhibit enhanced chemical reactivity in aqueous systems, yet their physicochemical origins remain unclear. Probe-based studies have suggested the possible involvement of reactive oxygen species (ROS), but processes at NB interfaces are poorly understood. In this study, bulk NB suspensions were prepared by ultrasonic irradiation of ultrapure water bubbled with O 2 , CO 2 , or N 2 , and temporal variations in their oxidative probe responses were investigated using H 2 O 2 -responsive absorption spectrophotometry, electron spin resonance spectroscopy with a spin-trapping agent, and aminophenyl fluorescein fluorescence measurements. O 2 - and N 2 -derived NBs exhibited increased H 2 O 2 -equivalent signals during ultrasonic irradiation, and these responses persisted and evolved during storage without further irradiation. Meanwhile, CO 2 -derived NBs showed markedly suppressed probe responses under similar conditions. These gas-dependent trends suggest that interfacial processes may influence oxidative probe responses. To account for these observations, a tentative mechanism was proposed, in which the negatively charged NB interface potentially created a locally enhanced electric field environment, promoting ROS-related probe responses. In CO 2 -containing systems, interfacial CO 2 dissolution and bicarbonate formation might contribute to suppression of oxidative signals through hydroxyl-radical scavenging. The proposed mechanism is presented as a working hypothesis to explain the observed probe responses in bulk NB systems.