The 18O signature of biogenic nitrous oxide is determined by O exchange with water.

To effectively mitigate emissions of the greenhouse gas nitrous oxide (N(2)O) it is essential to understand the biochemical pathways by which it is produced. The (18)O signature of N(2)O is increasingly used to characterize these processes. However, assumptions on the origin of the O atom and resultant isotopic composition of N(2)O that are based on reaction stoichiometry may be questioned. In particular, our deficient knowledge on O exchange between H(2)O and nitrogen oxides during N(2)O production complicates the interpretation of the (18)O signature of N(2)O.Here we studied O exchange during N(2)O formation in soil, using a novel combination of (18)O and (15)N tracing. Twelve soils were studied, covering soil and land-use variability across Europe. All soils demonstrated the significant presence of O exchange, as incorporation of O from (18)O-enriched H(2)O into N(2)O exceeded their maxima achievable through reaction stoichiometry. Based on the retention of the enrichment ratio of (18)O and (15)N of NO(3)(-) into N(2)O, we quantified O exchange during denitrification. Up to 97% (median 85%) of the N(2)O-O originated from H(2)O instead of from the denitrification substrate NO(3)(-).We conclude that in soil, the main source of atmospheric N(2)O, the (18)O signature of N(2)O is mainly determined by H(2)O due to O exchange between nitrogen oxides and H(2)O. This also challenges the assumption that the O of N(2)O originates from O(2) and NO(3)(-), in ratios reflecting reaction stoichiometry.