Abstract. The extracellular concentration of H₂O₂ in surface aquatic environments is controlled by a balance between photochemical production and the microbial synthesis of catalase and peroxidase enzymes to remove H₂O₂ from solution. In any kind of incubation experiment, the formation rates and equilibrium concentrations of ROS may be sensitive to both the experiment design (particularly to the regulation of incident light) and the abundance of different microbial groups (as both cellular H₂O₂ production and catalase/peroxidase enzyme production rates differ between species). Whilst there are extensive measurements of photochemical H₂O₂ formation rates and the distribution of H₂O₂ in the marine environment, it is poorly constrained how different microbial groups affect extracellular H₂O₂ concentrations, how comparable extracellular H₂O₂ concentrations within large scale incubation experiments are to those observed in the surface-mixed layer, and to what extent a miss-match with environmentally relevant concentrations of ROS in incubations could influence biological processes differently to what would be observed in nature. Here we show that both experiment design and bacterial abundance consistently exert control on extracellular H₂O₂ concentrations across a range of incubation experiments in diverse marine environments.

During 4 large scale (> 1000 L) mesocosm experiments (in Gran Canaria, the Mediteranean, Patagonia and Svalbard) most experimental factors appeared to exert only minor, or no, direct effect on H₂O₂ concentrations. For example, in 3 of 4 experiments where pH was manipulated (to 0.4–0.5 below ambient pH) no significant change was evident in extracellular H₂O₂ concentrations relative to controls. An influence was sometimes inferred from zooplankton density, but not consistently between different incubation experiments and no change in H₂O₂ was evident in controlled experiments using different densities of the copepod Calanus finmarchichus grazing on the diatom Skeletonema costatum (< 1 % change in [H₂O₂] comparing copepod densities from 1–10 L⁻¹). Instead, the changes in H₂O₂ concentration contrasting high/low zooplankton incubations appeared to arise from the resulting changes in bacterial activity. The correlation between bacterial abundance and extracellular H₂O₂ was stronger in some incubations than others (R² range 0.09 to 0.55), yet high bacterial densities were consistently associated with low H₂O₂. Nonetheless, the main control on H₂O₂ concentrations during incubation experiments relative to those in ambient, unenclosed waters was the regulation of incident light. In an open (lidless) mesocosm experiment in Gran Canaria, H₂O₂ was persistently elevated (2–6 fold) above ambient concentrations; whereas using closed high density polyethylene mesocosms in Crete, Svalbard and Patagonia H₂O₂ within incubations was always reduced (median 10–90 %) relative to ambient waters.