Sample preservation methods for nitrous oxide concentration and isotope ratio measurements in aquatic environments

The nitrogen (N) and oxygen (O) stable isotope analysis of dissolved nitrous oxide (N₂O) can provide important constraints on the sources and cycling of N₂O in aquatic environments. The isotopic composition of aqueous N₂O, both in field (natural abundance) or experimental (¹⁵N-labeling) samples, however, may be altered by abiotic reactions involving nitrite (${\mathrm{NO}}_2^{-}$) or hydroxylamine (NH₂OH) and microbial activity during sample storage, if samples are not adequately preserved. Here we tested five different preservatives, mercuric chloride (HgCl₂), copper sulfate (CuSO₄), zinc chloride (ZnCl₂), hydrochloric acid (HCl) mixed with sulfamic acid (SFA), and sodium hydroxide (NaOH), for fixing natural water samples from an estuary and a lake with different ${\mathrm{NO}}_2^{-}$ concentrations over a range of different storage times for N₂O analyses. ZnCl₂ and CuSO₄ decreased the pH, and led to abiotic N₂O production from ${\hspace{0.17em}}^{15}N{O}_2^{-}$, shifting the N₂O isotopic composition significantly. Removal of ${\mathrm{NO}}_2^{-}$ with a mixture of SFA and HCl did not always prevent the alteration of the original N and O isotope composition of N₂O, confirming the requirement for complete ${\mathrm{NO}}_2^{-}$ removal, and underscoring the biasing effects of ${\mathrm{NO}}_2^{-}$ at very low pH, even at trace levels. At low ${\mathrm{NO}}_2^{-}$ background, HgCl₂ preservation led to robust and accurate results. However, in light of its toxicity and bioaccumulation potential in food webs, this fixative should be avoided. The addition of NaOH reduced abiotic side reactions involving ${\mathrm{NO}}_2^{-}$, and yielded the most reliable and reproducible results for N₂O concentration and isotope ratio measurements across tested settings and storage times.