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 nitrogen and oxygen isotope analysis of nitrate (NO₃⁻) and nitrite (NO₂⁻), but concurrent reduction of both species (NO₃⁻ and NO₂⁻) to N₂O obscures their individual isotopic signals. This study developed an integrated ion-exchange (AG1-X8 resin) and denitrification for the methods, achieving quantitative NO₃⁻/NO₂⁻ separation (0.25 mol per L KCl for NO₂⁻ and 0.5 mol per L HCl for NO₃⁻ at 0.5–3 mL min⁻¹) and high-precision isotopic analysis (δ¹⁵N: SD ≤ 0.2‰ and δ¹⁸O: SD ≤ 0.3‰ for both NO₂⁻ and NO₃⁻ at ≥0.054 mmol L⁻¹). This method provides a safe, stable, and high-accuracy approach for separation of NO₃⁻/NO₂⁻ in high-NO₂⁻ freshwater systems. In addition, to effectively address the instability of NO₂⁻ during storage, samples can be preserved immediately upon collection by adsorption onto resin. In conclusion, this study has established an efficient and accurate pretreatment method for separating NO₂⁻ and NO₃⁻ and determining their individual N and O isotopes in natural freshwater. This method provides a reliable analytical technique for in-depth investigation of nitrogen transformation mechanisms, especially in high NO₂⁻ environments, using NO₂⁻ isotopic signatures as an indicator.