Protocol for Ammonium Isotope Analysis of Low-Concentration Environmental Water Samples

In this study, ammonium was separated from water samples using a diffusion technique and oxidized to nitrate. Nitrate was then denitrified and analyzed for isotopic values using isotope ratio mass spectrometry. Each step was carefully examined, particularly oxidation, which was conducted under a near-neutral pH through the addition of a buffer. This precaution was critical because highly acidic or alkaline conditions can lead to nitrogen loss during high-temperature and high-pressure oxidation. The results indicate that the employed ammonium diffusion technique combined with the optimized oxidation process can yield precise and reproducible δ15N values for low-concentration NH4+ water samples. Notably, our method requires only 10 µg of NH4+ for both diffusion and isotope analyses, which is a notable improvement over conventional diffusion techniques, which typically require 50–150 µg. To evaluate potential matrix interferences, we conducted recovery tests by spiking ammonium standards into three different hot spring samples, which often contain low ammonium concentrations and high concentrations of other ions. The results of recovery tests showed robust performance of the proposed method, with recovery rates ranging from 87% to 102%. These results confirm that our method is unaffected by high ion concentrations in complex natural matrices. Additionally, comparison with a previously reported technique revealed no significant difference in the isotope results (paired t-test, p > 0.05). We analyzed several natural hot spring samples across Japan using the proposed method, with the obtained results clarifying the origin of ammonium in the natural hot springs at the investigated locations. Therefore, our analytical method is efficient and highly sensitive for the analysis of ammonium isotopes in environmental water samples with low ammonium concentrations; furthermore, it enables the processing of large numbers of samples in a short time. The δ15NH4+ results contributed to elucidating the NH4+ sources and transport mechanisms at the study locations.