Accurate Isotopic Analysis of Nitrate and Nitrite in Freshwater: Integration of Ion Exchange and Bacterial Denitrification Methods

Abstract

The bacterial denitrification method (Pseudomonas aureofaciens) is a widely used pretreatment technique for nitrate (NO₃⁻) and nitrite (NO₂⁻) nitrogen and oxygen isotope analysis, but concurrent reduction of both species to N₂O obscures their individual isotopic signals. This study developed an integrated ion-exchange (AG1-X8 resin) and denitrification method, achieving quantitative NO₃⁻/NO₂⁻ separation (0.25 mol/L KCl for NO₂⁻, 0.5 mol/L HCl for NO₃⁻ at 0.5–3 mL/min) and high-precision isotopic analysis (δ¹⁵N: SD ≤0.2‰ and δ¹⁸O: SD ≤0.5‰ for both NO₂⁻ and NO₃⁻ at ≥0.054 mmol/L). This method provides a safe, stable, and high-accuracy approach for single-run isotopic discrimination in high-NO₂⁻ freshwater systems. To effectively address the instability of NO₂⁻ during storage, samples can be preserved immediately upon collection by absorbing them onto AG1-X8 resin. Thereby, this method overcomes the limitation that the δ¹⁸O_NO₂⁻ fraction is prone to occur during storage due to O of NO₂⁻ exchange with H₂O. Furthermore, it overcomes major methodological constraints that mixed NO₃⁻ and NO₂⁻ isotope signals by Pseudomonas aureofaciens inherent to complex matrices.