N2O isotope approaches for source partitioning of N2O production and estimation of N2O reduction – validation with the 15N gas-flux method in laboratory and field studies

The approaches based on natural abundance N2O stable isotopes are often applied for the estimation of mixing proportions between various N2O-producing pathways as well as for estimation of the extent of N2O reduction to N2. But such applications are associated with numerous uncertainties; hence, their limited accuracy needs to be considered. Here we present the first systematic validation of these methods for laboratory and field studies by applying the 15N gas-flux method as the reference approach. Besides applying dual-isotope plots for interpretation of N2O isotopic data, for the first time we propose a three dimensional N2O isotopocule model based on Bayesian statistics to estimate the N2O mixing proportions and reduction extent based simultaneously on three N2O isotopic signatures (δ15N, δ15NSP, and δ18O). Determination of the mixing proportions of individual pathways with N2O isotopic approaches often appears imprecise, mainly due to imperfect isotopic separation of the particular pathways. Nevertheless, the estimation of N2O reduction is much more robust, when applying an optimal calculation strategy, typically reaching an accuracy of N2O residual fraction determination of about 0.15.


Line 310: Why higher fungal DN compared to nitrification in this instance? The data in
can be used to briefly justify this?
We deal with rather high soil moisture, mostly over 65% WFPS, and also ammonium content was low, which rather favours fD than Ni. This explanation will be added.
Line 379: Some of the N2 fluxes are above the detection limit but the values written here are below the detection limit mentioned in line 373.
Sorry, this was a mistake, it is from 23 to 304 g N-N2. This will be corrected. Thank you for careful reading! Line 421 -423: Don't quite get what you mean here. Consider rephrasing. You mean corrected precursor ranges based on different fractionation factors?
It will be clarified. In this sentence one word was incorrectly used -precursors instead of endmembers. Sorry for this mistake.
Line 434: The minimum reduction line is not described in Fig. 1. The dotted lines and the mixing should be clearly described in the legend/caption. This explanation will be added to the Fig.1 caption: The soild lines (bD-fD mixing and mean reduction line) are main assumptions used in the calculation procedures for SP/O Map. The grey dashed line shows the alternative bD-Ni mixing line (calculations with this alternative scenario are also presented in the supplement Table S2). The red dashed line shows the minimum reduction line -for the case of minimal delta values of the bD endmember. And for Fig.3 caption: The dashed line shows the linear fit for all the points with its equation and statistics above.
Line 461: There was relatively large discrepancy between lab and field NO3-and NH4+ values. In fact, the d15N-NH4+ is very heavy and the possible factors driving these values should be discussed.
We comment this in the discussion, L680: This indicates that the ammonium pool was highly fractionated and nearly exhausted. This is most probably due to adsorption processes. But this is just a speculation so far. The discussion on this issue will be extended in the follow up paper, where we also include the 15N-NH4 treatment which was not presented here. This information will be added in the manuscript.

Line 490: The authors mentioned that the high d15N-NH4+ has shifted the location of the nD and Ni in the end member mixing plot. What is the author comparing the shift to?
Ccompared to cases when similar δ 15 N NH4 and δ 15 N NO3 values are determined or assumedthis will be clarified in the text.
Line 551: Amplitude for 3D1 model, case 1 is not always lower than the reference -at the start and towards the end of sampling, the amplitude is higher than the reference method. Any explanation on why this is the case?
I meant lower amplitude of the temporal changes, this will be clarified in the text. The uncertainty of each method mostly depend on the standard deviation of 4 repetitions of which each time sample consists.

Line 670: I agree with the authors that recalculation of the literature mixing endmember values is important but my question is what fractionation factors should be considered when correcting these values and how to evaluate that these corrected ranges are justified?
We can take the literature ranges for fractionation factors based on pure culture studies (we have presented the summarised values in Table S1, they are also summarised in the supplement to new perspective paper Yu et al., 2020 (https://onlinelibrary.wiley.com/doi/abs/10.1002/rcm.8858)) . These values can be also determined experimentally for the particular soil under study, at least for denitrification, but this is complex and time consuming. But importantly the literature fractionation factors for particular processes must be corrected with the substrate isotopic signatures, which should be determined for each soil study. The procedure of this correction is presented in Table S1. We have also extended the description of this correction and will move this whole paragraph to the methods section.
Hence, the endmember ranges represent the expected isotopic signatures of N 2 O originating from each mixing endmember for the particular case study characterised by specific precursor isotopic signatures. Such approach allows for presenting all data in the common isotopic scales without presumption on the dominating pathway and dominating precursor. Hence, this new approach presented here is actually a further development of Maps, since this allows for correcting both Ni and bD, fD and nD endmembers with relevant distinct precursors, in contrast to only correcting measured values with one common assumed precursor isotopic signature. We will also move the Table 1 into the main manuscript, since it contains important information for these corrections.

Line 686: Be specific of what shift is meant here? Temporal?
Yes, temporal shift, this will be added.
Line 820: This sentence is rather subjective. Is it possible to provide a more definitive range here? Can the authors make use of a sensitivity analysis to show the extent of substrate isotopic variations effects on the accuracy of the mapping approach?
This is quite a complex analysis -it has been done for SP/O Map (Wu et al., 2019 https://www.sciencedirect.com/science/article/abs/pii/S0013935119306036) but not yet for isotope Maps applying d15N. This is definitely the topic for the further work and it is planned to be done soon. Without a precise analysis it is not possible to provide a precise numbers here.