Application of a superoxide (O₂^-) thermal source (SOTS-1) for the determination and calibration of O₂^- fluxes in seawater

Superoxide (O₂^-) is an important short lived transient reactive oxygen species (ROS) in seawater. The main source of O₂^- in the ocean is believed to be through photochemical reactions though biological processes may also be important. Sink terms for O₂^- include redox reactions with bioactive trace metals, including Cu and Fe, and to a lesser extent dissolved organic matter (DOM). Information on the source fluxes, sinks and concentration of superoxide in the open ocean are crucial to improving our understanding of the biogeochemical cycling of redox active species. As O₂^- is a highly reactive transient species present at low concentrations it is not a trivial task to make accurate and precise measurements in seawater. In this study we developed the appropriate numerical analysis tools and investigated a number of superoxide sources and methods for the purposes of calibrating O₂^- concentrations and/or fluxes specifically in seawater. We found the superoxide thermal source bis(4-carboxybenzyl)hyponitrite (SOTS)-1 easy to employ as a reliable source of O₂^- which could be successfully applied in seawater. The thermal decomposition of SOTS-1 in seawater was evaluated over a range of seawater temperatures using both a flux based detection scheme developed using two spectrophotometric methods: (i) 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) and (ii) ferricytochrome c (FC), or a concentration based detection scheme using a chemiluminescence flow injection method based on the Cypridina luciferin analog 2-methyl-6-(p-methoxyphenyl)3-7-dihydroimidazol[1,2-α]pyrazin-3-one (MCLA) as reagent. Our results suggest SOTS-1 is the best available O₂^- source for determining concentrations and fluxes, all detection systems tested have their pros and cons and the choice of which to use depends more on the duration and type of experiment that is required.