Nitrogen isotopic analysis of nitrate in aquatic environment using cadmium–hydroxylamine hydrochloride reduction

Rationale: The nitrogen isotopic ratio of nitrate (δ¹⁵N-NO₃ ⁻ value) is a critical parameter for understanding nitrogen biogeochemical cycling in aquatic systems. Current approaches to the determination of δ¹⁵N-NO₃ ⁻ values involve time-intensive handling procedures, the use of toxic chemicals and complicated microbial incubation. Methods: A chemical reduction method for measuring the δ¹⁵N-NO₃ ⁻ values of aquatic samples was established. Nitrate was first quantitatively reduced to nitrite in a column filled with copper-coated cadmium granules, and the produced nitrite further reduced to nitrous oxide gas with hydroxylamine hydrochloride. The nitrogen isotope ratio of the produced nitrous oxide was measured using a continuous-flow isotope ratio mass spectrometer coupled with a purge and cryogenic trap system. Results: The optimized experimental conditions were: solution acidity, H⁺ concentration of 0.46 M, pH = 0.34; dosage of hydroxylamine, molar ratio of NH₂OH to NO₂ ⁻ of 4; reaction temperature, 45°C; and reaction time, 14–16 h. No salt effect was found for this method. The reproducibility of the δ¹⁵N-NO₃ ⁻ value for the laboratory standard was better than 0.3‰ for long-term measurements (20 nmol NO₃ ⁻ requirement). Conclusions: This method provides a reliable approach for the determination of δ¹⁵N-NO₃ ⁻ values at natural abundance. It provides (1) high measurement accuracy, (2) ease of operation, (3) environmental-friendly procedure (less toxic regents used), and (4) suitability for both freshwater and saline water samples.