The increasing prevalence of nitrate contamination in surface waters, groundwater, and ocean waters, represents a critical environmental challenge, particularly in regions with intensive agriculture and aquaculture. Denitrification, the microbial reduction of nitrate to dinitrogen gas, plays a pivotal role in mitigating this contamination and regulating the global nitrogen cycle. Stable isotope analysis provides critical insights into nitrate transformation pathways, distinguishing denitrification from anaerobic ammonium oxidation (anammox), another N-loss process, or internal recycling processes such as dissimilatory nitrate reduction to ammonium (DNRA).

This review highlights the importance of isotopic tools for assessing nitrate attenuation in natural and anthropogenic-impacted systems and explores the use of nitrogen (δ¹⁵N) and oxygen (δ¹⁸O) isotopic fractionation to trace denitrification and to quantify its extent in diverse aquatic environments. The nitrogen (N) and oxygen (O) isotopic fractionation during denitrification is evaluated at organism and ecosystem levels. Also, environmental factors modulating isotopic composition of N compounds in groundwater, rivers, lakes, riparian zones, coastal wetlands and oxygen-deficient marine regions are explored.

Advances in isotope biogeochemistry and analytical techniques improve our ability to assess the transport and fate of nitrate, integrating isotopic data with hydrological and biogeochemical models. A precise characterization of N and O isotopic enrichment factors for denitrification supports improved predictions of nitrogen cycling dynamics under changing environmental conditions. These approaches enhance understanding of nitrogen removal processes and help refine estimates of nitrogen fluxes at local, regional and global scales. By providing a quantitative framework for evaluating denitrification and related processes, this review contributes to developing more effective strategies for managing nitrogen pollution and mitigating its impacts on aquatic ecosystems.