Improved Isotopic Analysis of Low-Concentration Freshwater Nitrite by Anion-Exchange Resin Enrichment and Azide Reduction

The isotopic composition (δ¹⁵N and δ¹⁸O) of nitrite (NO₂^-) provides valuable insights into nitrogen cycling in aquatic environments. However, its typically low concentrations as a transient redox intermediate in natural waters often limit isotopic analysis. In this study, a method for δ¹⁵N and δ¹⁸O analysis of low-level NO₂^- in freshwater has been developed by coupling anion-exchange resin preconcentration with the azide reduction method. This approach enables the measurement of δ¹⁵N and δ¹⁸O with low-concentrations (0.02 μmol L^-1), which significantly expands the applicability of NO₂^- isotope analysis to low-nutrient environments. Experimental evaluation of the azide reduction step demonstrated that oxygen exchange between NO₂^- and water was consistently suppressed to below 10% when the NaCl concentration exceeded 0.41 mol L^-1, and a 1:1 mixture of 90% acetic acid and 4 mol L^-1 sodium azide was added at dosages greater than 28 μL mL^-1. Field application in Lake Biwa yielded reasonable and interpretable NO₂^- concentration and isotope profiles, with nearly uniform vertical distributions during full mixing and clear contrasts across the thermocline during stratification. Our isotope data further suggest that NO₂^- derived from partial nitrate assimilation likely makes a non-negligible contribution to the NO₂^- pool in the upper layer of Lake Biwa. The robustness of this method in capturing NO₂^- dynamics in oligotrophic systems, as demonstrated here, adds a valuable tool to the toolbox for studying nitrogen cycling in freshwater systems.