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In March/April 2018 during a cruise on R/V Sally Ride, SR1805, 15N-NH4+ incubations in 60mL glass serum bottles were performed to measure ammonium oxidation rates to nitrite and nitrous oxide in different depth at 3 different stations in the oxygen deficient zone (ODZ) of the Eastern Tropical North Pacific off the coast of Mexico. Water samples were collected from 30L Niskin bottles deployed with a conductivity-temperature-depth profiler (CTD, Seabird Electronics). The goal was to get a better understanding on the controls of nitrous oxide (N2O) production. The N2O production rate experiments were performed according to Bourbonnais et al. 2021 (https://doi.org/10.3389/fmars.2021.611937). Furthermore, ammonium (NH4+), nitrite (NO2-) and nitrate (NO3-) as well as N2O concentrations were determined using standard fluorometric (Holmes et al. 1999, https://doi.org/10.1139/f99-128), photometric (Strickland and Parsons 1972, hdl:10013/epic.46454.d001), chemiluminescent (Braman and Hendrix 1989, doi:10.1021/ac00199a007) and mass spectrometric techniques (McIlvin and Casciotti 2010, https://doi.org/10.4319/lom.2010.8.54), respectively. The N2O yield per nitrite produced was calculated. The archaeal ammonia monooxygenase gene subunit A (amoA) copy numbers/mL were determined using qPCR as described previously (Peng et al. 2015, https://doi.org/10.1002/2015GB005278). The data table contains the depth profiles of salinity, temperature (°C), oxygen concentration (µmol/L), ammonium (µmol/L), nitrate (µmol/L), nitrite (µmol/L) and nitrous oxide concentration (nmol/L) for three stations in the oxygen deficient zones (ODZ) in the Eastern Tropical North Pacific. It also contains the rate measurements of N2O production rate (nmol N2O/L*d) with standard error, ammonium oxidation rate (nmol N2O/L*d) to nitrite with standard error as well as N2O yield (%) with standard deviation. The % hybrid N2O production refers to the production of 45N2O, which requires incorporation of a 14N substrate other than NH4+. It is expressed in percent with respect to the total N2O produced from 15NH4+. Furthermore, the archaeal amoA copy numbers / mL and the standard deviation are given for the different depths.

Nitrous oxide (N2O) is a potent greenhouse gas and an ozone destroying substance. Yet, clear step-by-step protocols to measure N2O transformation rates in freshwater and marine environments are still lacking, challenging inter-comparability efforts. Here we present detailed protocols currently used by leading experts in the field to measure water-column N2O production and consumption rates in both marine and other aquatic environments. We present example 15N-tracer incubation experiments in marine environments as well as templates to calculate both N2O production and consumption rates. We discuss important considerations and recommendations regarding (1) precautions to prevent oxygen (O2) contamination during low-oxygen and anoxic incubations, (2) preferred bottles and stoppers, (3) procedures for 15N-tracer addition, and (4) the choice of a fixative. We finally discuss data reporting and archiving. We expect these protocols will make 15N-labeled N2O transformation rate measurements more accessible to the wider community and facilitate future inter-comparison between different laboratories.

AbstractMarine oxygen‐deficient zones represent a natural source of nitrous oxide (N2O), a potent greenhouse gas and ozone‐depleting agent. To investigate controls on N2O production, the responses of ammonia oxidation (AO) to nitrite () and N2O with respect to oxygen (O2), ammonium () and concentrations were evaluated using tracer incubations in the Eastern Tropical North Pacific. Within the oxycline, additions of and O2stimulated N2O production according to Michaelis–Menten kinetics, indicating that both substrates were limiting, and that N2O production, even if the exact mechanisms remain uncertain, is mediated by predictable kinetics. Low half‐saturation constants for (12–28 nM) and O2(460 ± 130 nM) during N2O production indicate that AO communities are well adapted to low concentrations of both substrates. Hybrid N2O formation (i.e., from one and one unlabeled nitrogen (N) source, e.g., , NO) accounted for ~ 90% of the N2O production from and was robust across the different O2, , and conditions. Lack of response to variable substrate concentrations implies that the unlabeled N source was not limiting for N2O production. Although both O2and were key modulators of N2O production rates, N2O yield (N2O produced per produced) seemed to be controlled solely by O2. The N2O yield increased when O2concentrations dropped below the half‐saturation concentration for AO to (< 1.4 μM), the range where production decreased faster than N2O production. Our study shows that O2control on N2O yield from AO is robust across stations and depths.