Decoding the stable isotope signature of the non-exchangeable oxygen fraction of bulk soil organic matter: methodological prerequisites

Oxygen (O), the most abundant element in the Earth's crust, has an underexplored isotope system in plant and soil sciences compared to carbon and nitrogen, despite its strong potential to serve as a robust proxy for climate, ecohydrology and biogeochemical studies. The stable isotope ratio of O (δ¹⁸O) in bulk soil organic matter (SOM) might reflect the isotope composition of soil water during SOM formation. However, this signal is blurred by the presence of O from inorganic minerals and a dynamic exchangeable O fraction that can quickly equilibrate with ambient water. To address these challenges, the O in SOM must be isolated from interfering O-containing inorganic compounds in plant OM and minerals. Moreover, the exchangeable O fraction must be accounted for. Although we hypothesise that the exchangeable O fraction in SOM is smaller than that of H, it can likely not be ignored.

We evaluated two alternative methods to separate organic and inorganic O from the soil: demineralisation (i.e., removal of inorganic compounds using HF and HCl) and removal of the organic compounds by muffling combined with a KCl treatment to remove oxyanions. After isolating the organic fraction, we applied a steam equilibration procedure, in which we equilibrated the samples with different water vapours of known O-isotopic composition to determine the δ¹⁸O value of the nonexchangeable O fraction, as has already been similarly established for H. We used standard materials like ethylene glycol, p-Nitro aniline, and Aldrich humic acid (AHA) for the demineralisation method and two O-containing minerals (Goethite and Apatite), both pure and mixed with AHA as model substances for the organic matter removal method and also ¹⁸O-spiked chemicals to select the procedure with no (or minimal) alterations of the original O isotope ratios. Our preliminary data reveal an exchangeable O fraction of 1-1.5% in AHA and excluding its effect by using mass balance calculation, the resulting δ¹⁸O value of the nonexchangeable fraction of AHA was ~15.2‰, which is significantly depleted relative to the humic acid extracted from natural soil (18.4-24.6‰), a discrepancy attributable to the absence of microbial decomposition and associated isotopic fractionation in our synthetic model compound (AHA). Thus, by quantifying the exchangeable O fraction and assessing the stability of the non-exchangeable O fraction against our treatments, this study provides a methodological prerequisite for the accurate determination of oxygen isotope ratios of the nonexchangeable O fraction in plant and soil science.