Molecular oxygen (O₂) is one of the most important electron acceptors for enzymatic and abiotic redox reactions in the environment. Enzymes that utilize O₂ as the terminal electron acceptor catalyze a large number of biological reactions involved in processes of metabolic activity, cellular detoxification, and biosynthesis. Many of these reactions are essential for life, but some of them also contribute to the metabolic and co-metabolic removal of anthropogenic organic contaminants in natural aquatic environments and during wastewater treatment. Abiotic reduction of O₂ is important for the biogeochemical cycling of elements, such as iron or other trace metals, as well as the photomineralization of organic compounds in sunlit surface water. Measuring systematic changes in the stable isotopic composition (i.e., isotope fractionation) of dissolved O₂ during enzymatic reactions has been shown to enable the identification of underlying O₂ reduction mechanisms. Consequently, measuring isotope fractionation of O₂ is a promising tool for investigating environmentally relevant redox reactions. However, to ultimately apply this approach to reactions with unknown O₂ reduction mechanisms, it is important to systematically extend the current knowledge of reaction-specific ¹⁸O-kinetic isotope effects (¹⁸O-KIEs), which ultimately determine observable isotope fractionation of enzymatic and abiotic reactions of O₂ in aquatic environments.The overall objective of this project is to experimentally determine reaction-specific ¹⁸O-KIEs associated with selected enzymatic and abiotic O₂ reduction reactions and, in turn, to evaluate the potential of O₂ isotope analysis as a tool to study environmental redox reactions. The central part of this project will entail the systematic investigation of well-known enzymatic reactions in laboratory experiments to determine the dependence of O₂ consumption-associated ¹⁸O-KIEs on the type of enzyme, substrate, and catalyzed reaction. Different O₂ reduction reactions will be studied in headspace-free reactors containing an oxidase or oxygenase enzyme, native or non-native substrates, and required cofactors and/or co-substrates. Reaction-specific ¹⁸O-KIEs will be determined in replicate experiments, where O₂ concentrations will be monitored continuously, and the isotopic composition of dissolved O₂ will be measured at different time-points. Comparison of these ¹⁸O-KIEs with isotope fractionation of O₂ during enzymatic oxidations of anthropogenic contaminants with unknown reaction mechanisms will represent the first step in our efforts to evaluate the suitability of this approach to study O₂ reduction mechanisms in anthropogenically impacted ecosystems. Similarly, ¹⁸O-KIEs will be studied for photochemical reactions involving O₂ , including singlet oxygen and superoxide radical formation. The gas chromatography isotope ratio mass spectrometry (GC/IRMS) method used for these experiments will be optimized regarding sample size and the O₂ extraction procedure at the beginning of the proposed project.It is expected that results from this project will enable the routine use of isotope analysis of O₂ to study environmentally relevant redox reactions in laboratory-scale experiments and provide valuable mechanistic insights into specific enzymatic and abiotic redox reactions. The comprehensive set of ¹⁸O-KIEs determined in this work will establish benchmarks for various abiotic and microbial O₂ consumption processes in the aquatic environment, which can be used in future studies applying isotope analysis of dissolved O₂ , or of organic products containing O-atoms derived from O₂, in natural systems.