Minimizing H 2 O 2 Overestimation in Pore Waters via High-Frequency Amplex Red Measurements

The Amplex Red (AR) assay is a widely used method for the quantification of hydrogen peroxide (H2O2) in natural water due to its high sensitivity and specificity. However, the methodology developed for photochemistry may overestimate the H2O2 content estimated in sediment pore water, where H2O2 production results mainly from the oxidation of reduced species. Indeed, in previously published protocols, the measurement of H2O2 is not conducted a few minutes after the sample’s mixing with the probe. We demonstrate that this delay is frequently sufficient for the reduced species present in pore water to produce a significant amount of H2O2. This study refines the AR technique by implementing high-frequency (HF, 10 Hz) measurements following probe addition. Moreover, the HF AR method gives access to three key parameters: I─the initial H2O2 concentration; II─the H2O2 production rate; and III─the total H2O2 production capacity of the sample upon oxidation. The accuracy and robustness of the method were demonstrated through a series of controlled experiments based on iron oxidation in the presence of citrate ligands. The efficiency of the method was demonstrated through its application in the field of sulfur- and iron-rich pore water. The findings represent a substantial enhancement in comparison to the prevailing protocols.