Identification of ROS Produced by Nanobubbles and Their Positive and Negative Effects on Vegetable Seed Germination

Exogenous reactive oxygen species (ROS) produced by nanobubble (NB) water offer a reasonable explanation for NBs' physiological promotion and oxidation effects. To develop and exploit the NB technology, we have performed further research to identify the specific ROS produced by NBs. Using a fluorescent reagent APF, a Fenton reaction, a dismutation reaction of superoxide dismutase and DMSO, we distinguished four types of ROS (superoxide anion radical (O₂·^-), hydrogen peroxide (H₂O₂), hydroxyl radical (·OH), and singlet oxygen (¹O₂)). ·OH was confirmed to be the specific ROS produced by NB water. The role of ·OH produced by NB water in physiological processes depends on its concentration. The amount of exogenous ·OH has a positive correlation with the NB number density in the water. Here, spinach and carrot seed germination tests were repeatedly performed with three seed groups submerged in distilled water, high-number density NB water, and low-number density NB water under similar dissolved oxygen concentrations. The final germination rates of spinach seeds in distilled water, low-number density NB water, and high-number density NB water were 54%, 65%, and 69%, respectively. NBs can also promote sprout growth. The sprout lengths of spinach seeds dipped in NB water were longer than those in the distilled water. For carrot seeds, the amount of exogenous ·OH in high-number density NB water was beyond their toxic threshold, and negative effects were shown on hypocotyl elongation and chlorophyll formation. The presented results allow us to obtain a deeper understanding of the physiological promotion effects of NBs.