In the manuscript “Triple oxygen isotope evidence for the pathway of nitrous oxide production in a forested soil with increased emission on rainy days” by Ding et al., the authors present results from nitrous oxide Δ¹⁷O measurements at a forested site. Additionally, they conducted tracer addition experiments, including nitrate enriched in Δ¹⁷O, to complement the in situ measurements. The results show that nitrous oxide produced under high and low soil moisture conditions can have different Δ¹⁷O values within the limits of analytical precision. These variations fall within the range defined by the Δ¹⁷O of soil O₂, H₂O, and NO₂⁻, suggesting that N₂O production from nitrification and denitrification could be inferred by comparing the Δ¹⁷O values of N₂O and potential oxygen sources.

Overall, I find this manuscript well written, with the results clearly presented. I am particularly impressed by the analytical rigor demonstrated in this study, which is critical given the typically subtle variations in Δ¹⁷O. However, I do have several specific questions and comments that I hope the authors can address in their revision:

[1] Section 2.3: While extraction with 2 M KCl is standard for measuring extractable soil nitrate and ammonium, significant loss of soil nitrite can occur during the extraction. This issue is well recognized in the soil nitrogen cycling community (e.g., Homyak et al., 2015). Given that nitrite concentration and Δ¹⁷O measurements are critical to this study, could the authors discuss how potential nitrite loss and associated isotopic fractionation might impact their analysis?

[2] Section 2.4: The manuscript uses a β (triple oxygen proportionality factor) range of 0.525 to 0.5305 to quantify the potential impact on Δ¹⁷O. Although several references are cited, it is unclear why this specific range was chosen. Please clarify. In particular, earlier studies (e.g., Matsuhisa et al., 1978; summarized by Miller, 2002) reported lower β values (e.g., ~0.5164). How would using lower β values affect the results?

[3] Lines 362–363 and throughout the analysis: A constant Δ¹⁷O value was assumed for O₂ (-0.44‰) and soil H₂O (0.03‰). Please clarify whether these values could vary due to hydrological and biogeochemical cycling. For instance, could O₂ diffusion and heterotrophic consumption affect O₂ Δ¹⁷O, or could evaporation significantly alter soil H₂O Δ¹⁷O?

[4] Lines 378–397: The characterization of δ¹⁸O offsets between O₂ and N₂O, and between NO₂⁻ and N₂O, does not necessarily represent true isotope effects between N₂O and its oxygen precursors because field-measured N₂O is a mixture of multiple sources. For example, in Fig. 6a, the actual δ¹⁸O difference between NO₂⁻ and N₂O may be larger than calculated if O₂-derived N₂O has δ¹⁸O values similar to that of O₂. Similarly, the O₂-N₂O difference may be smaller than estimated. This mixing effect could confound the use of δ¹⁸O differences to estimate Δ¹⁷O variations and warrants further clarification.

[5] Fig. 7 and related discussion: I commend the authors for conducting a sensitivity analysis to assess how much Δ¹⁷O variation may stem from biogeochemical processes versus purely geochemical processes (i.e., β variability). However, applying the β range to the net δ¹⁸O difference between N₂O and oxygen sources treats the N₂O-producing processes as a single step. In reality, processes like nitrite reduction involve multiple sub-steps (e.g., NO₂⁻ to NO, NO to N₂O, isotope exchange with H₂O), each potentially associated with different β values. This could lead to larger Δ¹⁷O variations than those estimated from a single-step approach. This limitation should be discussed.

[6] Lines 434–438: It is unclear how the 24% contribution of soil H₂O was derived. Additionally, Fig. 6b shows that the Δ¹⁷O of N₂O in the CS plot was significantly lower than that of NO₂⁻ six days after tracer addition. This suggests that soil H₂O may have played a significant role during nitrite reduction to N₂O.

[7] Lines 444–449 and Fig. 6b: Apparent differences in Δ¹⁷O between soil H₂O and N₂O cannot be used to conclusively rule out H₂O contributions during N₂O production. In the NF and U plots, the Δ¹⁷O of soil H₂O lies between that of NO₂⁻ and N₂O, and both soil H₂O and NO₂⁻ have higher Δ¹⁷O than O₂. Could significant H₂O exchange during N₂O production explain these observations, leading to a mixed Δ¹⁷O signal from both H₂O- and O₂-derived N₂O?

[8] Section 4.5: Early in the manuscript, the authors argue that bulk isotopic and SP-based techniques for N₂O source apportionment are limited due to isotopic fractionations during cycling (lines 56-61), whereas Δ¹⁷O measurements may be more robust. After presenting the results, I would encourage the authors to revisit this point with more specificity. Given potential complications such as H₂O exchange and multiple contributing sources (H₂O, O₂, NO₂⁻), can Δ¹⁷O measurements realistically achieve quantitative source apportionment? If so, what would the total uncertainty be, considering analytical precision, β variability, and uncertainties from the Keeling approach? Under what conditions would Δ¹⁷O approaches be preferable to conventional methods, and when might they be less effective?

References:

Homyak, P.M., Vasquez, K.T., Sickman, J.O., Parker, D.R. and Schimel, J.P., 2015. Improving nitrite analysis in soils: Drawbacks of the conventional 2 M KCl extraction. Soil Science Society of America Journal, 79(4), pp.1237-1242.  

Miller, M.F., 2002. Isotopic fractionation and the quantification of 17O anomalies in the oxygen three-isotope system: an appraisal and geochemical significance. Geochimica et Cosmochimica Acta, 66(11), pp.1881-1889.

Matsuhisa, Y., Goldsmith, J.R. and Clayton, R.N., 1978. Mechanisms of hydrothermal crystallization of quartz at 250 C and 15 kbar. Geochimica et Cosmochimica Acta, 42(2), pp.173-182.

This study investigates the pathways of N₂O production in forest soils using Δ¹⁷O signatures of emitted N₂O and soil nitrite (NO₂⁻) and O2 (−0.44 ‰). The authors report higher N₂O fluxes (38.8 ± 28.0 vs. 3.8 ± 3.1 μg N m⁻² h⁻¹) and Δ¹⁷O values (+0.12 ± 0.13‰ vs. −0.30 ± 0.09‰) on rainy versus dry days. They attribute rainy-day emissions to denitrification (aligned with NO₂⁻ Δ¹⁷O: +0.23 ± 0.12‰) and dry-day emissions to nitrification (aligned with O₂ Δ¹⁷O: −0.44‰), concluding that Δ¹⁷O effectively identifies production pathways. This approach provides complementary insights to traditional δ¹⁵N, δ¹⁸O, or site preference methods.

Major comments:

1. The study site is very local. The study site’s soil type, vegetation, and climate should be contextualized relative to other ecosystems to assess broader applicability. In addition, the sample numbers of N2O gas samples are very limited in this study. For instance, only five data and 18 data are illustrated in Figure 3 and 4. The confidence degree of the line fitting and the representative of the results should be further clarified. The novelty of the findings and this study should be further highlighted by comparing with prior soil Δ¹⁷O studies.
2. The sample information is missing. The definition of “fine days” and “rainy days” (e.g., precipitation threshold, duration) must be clarified to ensure reproducibility. In addition to weather conditions (fine or rainy), the other influencing factors are not considered and discussed, for instance, soil physical, chemical and microbiological properties, wind speed, air temperature. These factors may affect the implications of the results. For instance, in different seasons, the soil properties, especially soil microorganisms, may largely change and lead to the variation in N2O emission regardless of rainy or fine days. Soil moisture, temperature, and redox data are critical to substantiate the claim that rain-induced anoxia drives denitrification. Their absence weakens causal inferences. Variability in N₂O fluxes (e.g., ±28.0 μg N m⁻² h⁻¹ on rainy days) warrants discussion (e.g., soil heterogeneity, rain intensity).
3. The Δ¹⁷O of N₂O on rainy days (+0.12‰) is lower than that of NO₂⁻ (+0.23‰). The authors should address whether this reflects mixing of oxygen sources (e.g., H₂O, O₂) or kinetic fractionation during denitrification. On fine days, the Δ¹⁷O of N₂O (−0.30‰) differs from O₂ (−0.44‰). Potential contributions from H₂O (Δ¹⁷O ≈ 0‰) during nitrification should be discussed.
4. A more in depth comparison with complementary isotopic compositions (e.g., δ¹⁵N, δ18O) would strengthen pathway discrimination.

Specific comments:

1. Line 56, 59, spell out “SP” for its first appearance
2. Line 150, which kind of autoanalyzer
3. Line 205, spell out “VSMOW” for its first appearance
4. Line 357, Identification of pathways of N2O production in forested soil using Δ17O signature, the subhead can be changed to “Identification of N2O production pathways in forested soil using Δ17O signature.
5. Section 4.2, this section only reports experimental results and has no discussion.
6. The figures appear to be crudely constructed, seemingly pieced together, with text added afterward. Subfigures are misaligned, and axis labels are inconsistently positioned. It is recommended to use professional illustration tools to improve the clarity and precision of the figures.