Nitrate (NO₃^-) leaching from fertilized soils is a major driver of water quality degradation, contributing to eutrophication and posing risks to human health. Understanding nitrate sources and transformations is crucial for mitigation, as these processes depend on farming practices, climate, and subsurface dynamics. Stable isotopes of nitrate (δ¹⁵N-NO₃^- and δ¹⁸O-NO₃^-) are powerful tools for tracing these dynamics and revealing biogeochemical processes such as nitrification and denitrification. Changes in nitrate isotope composition result from isotope effects, differences in reaction kinetics or equilibrium partitioning due to the tendency of heavier isotopes to react more slowly or fractionate differently than lighter ones. Although isotope effects are typically derived from controlled laboratory experiments, natural systems, marked by variable redox conditions, hydrology, and substrate heterogeneity, can substantially modify them.

To address this gap, we estimated isotope effects in situ through high-resolution monitoring of an agricultural soil profile, combining nitrate, redox, and isotopic measurements with numerical modeling and HYDRUS-based particle tracking. Evidence of nitrification was found in the upper soil horizons, supported by nitrate concentrations above 2.75 mM and δ¹⁵N values (δ¹⁵N = 4.2‰ ± 0.9‰) indicative of a soil nitrogen source, likely derived from immobilized fertilizer. A clear signature of denitrification is observed mainly in the capillary fringe, where nitrate levels drop to 0.004 mM and isotopic shifts follow a linear Δδ¹⁸O:Δδ¹⁵N slope of 0.79. The isotopic effects of nitrification were not well constrained, reflecting the limited ability to resolve parameters under the available data and model structure. In contrast, denitrification shows a median ¹⁵ε_NAR and ¹⁸ε_NAR of 7.77‰ and 5.78‰, respectively. These isotope effects show corresponding variations with redox conditions and display negative tendencies with both temperature change and pH. Lower redox potentials lead to reduced isotope effects, consistent with faster reactions under more reducing conditions. Similarly, within the observed range, higher pH and positive temperature changes correspond to enhanced microbial activity and smaller isotope effects.

Overall, integrating isotopic data with process-based modeling improved the understanding of nitrate transformations and highlighted the environmental controls governing isotopic fractionation in agricultural soils.