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 (H 2 O 2 ) in natural water due to its high sensitivity and specificity. However, the methodology developed for photochemistry may overestimate the H 2 O 2 content estimated in sediment pore water, where H 2 O 2 production results mainly from the oxidation of reduced species. Indeed, in previously published protocols, the measurement of H 2 O 2 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 H 2 O 2 . 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 H 2 O 2 concentration; II─the H 2 O 2 production rate; and III─the total H 2 O 2 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.