The manuscript by Moonen et al. contributes to recent body of literature developing novel approaches to improve methods to partition net ET and NEE fluxes in its gross components. This is paramount to better understand the role of terrestrial ecosystems for regional water and carbon cycles and to allow better upscaling of these fluxes using remote sensing or modelling tools.

This partitioning is for various reasons not trivial for tropical forest and recently novel neural network-based methods have been developed that try to improve the “traditional” so-called daytime and nighttime methods. I think that besides the limitations, which are clearly recognized by the authors, adding isotopes exchanges to the ET and NEE partitioning “toolbox” disserves to be discussed in the scientific literature allowing for future experiments to improve also this method. Afterall, this isotope flux method It is simply a more direct and physical based method, i.e. we add one isotope flux equation to solve two unknowns.

This paper assesses this method on a very relevant site (ATTO, Brazil) and was made possible using novel eddy covariance measurements of isotopologues of H2O and CO2. The authors address in a very detailed and (often) clear way the followed methodology and potential caveats for an “average day” of a 13-day period in the dry season in the Amazonian tropical rainforest. Despite the limitations, but this is the case for all current existing partitioning methods, this paper disserves to be published to allow further refinement by other interested research groups, this is the only way how the potential of this method can be tested and further developed. Below I give one major comment mainly related to the steady state assumption made and several minor comments that are meant to further increase the clarity of this complex and integrated methodology. Finally, the paper also correctly advocates for detailed isotopic measurements of leaf and soil endmembers, which is paramount further refine and test this methodology.

Major comments

I have some concerns on what the authors describe as a midday steady state (line 68). This is mostly linked to the “isotopic composition of transpiration” (line 125). Can you really consider that mass of water that is entering the leave matches the mass of water evaporating from the leaf? During photosynthesis light splits H2O and is “consumed” to generate glucose. Furthermore, H2O is also used to incorporate H into leaf waxes (alkanes). These processes have a (large) biosynthetic fractionation factor and hence also isotopically enrich leaf water. Maybe there is a misunderstanding in the (too short) description of the steady state here? Furthermore, I agree, in general root water uptake is assumed not to be fractionating, though this is increasingly debated. In addition to that (and see comment on Table A1) root water uptake seems to come from deeper than 10 cm soil depth. This is also something that needs more attention in the text.

Minor comments

I believe also “non-isotope” experts would be highly interested in this methodology. Hence a consistent and unfirm terminology is important, i.e. what is a “delta flux” (line 7, 48, 90, etc.) or (H2O and CO2) isotopic fluxes (line 88, 68, etc.), explain the relation between delta and isotope ratio notation (e.g., Eq; 6, 7 vs. Eq. 4, as both are used in the various equations. 

Line 81: I don’t understand this sentence: … “describe the composition of a negative flux”?

Line 120, Eq. 6, 10: explain all parameters in the text. Note here you use isotope ratio’s (R) and not a delta notation (make clear to a non-expert public). Please check this also consistently for all other equations in the text.

Line 210: is it correct that “d18c” is calculated from Eq. 6?

Line 243: what is the meaning of 10^5 here?

Line 255: It is unclear how the outlier filtering was done.

Line 259: You are sure these uncertainties are correct?

Figure 1 and related text: please add a bit more technical details on the equipment and which isotope standards that have been used.

Line 269: Here you refer to the “steady state” (see major comment), we need a bit more explanation on this I believe.

Line 280: Explain why this background isotopic composition was unstable.

Line 301: “This finding was confirmed by,…” this “co-spectra” notation is not clear.

Fig. 2: improve legend and caption for a better understanding of the figure. For a) also add it is a “30 min water flux”.

Fig. 3: idem for legend in this figure.

Line 311-312: “stable conditions lead to horizontally heterogeneous conditions”; make clear please.

Line 322: correct terminology please: write (maybe) CO2-isotopologue flux,…

Line 343-345: this section is unclear to me. Can you try to rephrase?

Line 348: where we see this -15 per mill lower values?

Fig. 5: explain the y-axis titles well in the caption.

Line 379: Make clear in the captions that all data are for 14:00 hour and a 13-day composite diurnal flux - > independent reading of Tables and Figures.

Table 1: please not that Tair and Tleaf can be different. So not sure what the 33°C represents.

Line 394: “isotopic composition of the topsoil”. Please note root water uptake is not from the topsoil alone and, depending on tree species, distributed over different soil layers. Please make this and its complications clear here. This is also a bit misleading at the bottom of figure 6, as root water uptake is water with (for dD) a value between -25.8 and -14.4 per mil. Furthermore see also my comment for table A1 and in the major comment.

Line 399-400. Here is the concept of mass conservation again. See my comments above where I argue that is not really the case. Can this be adjusted?

Line 420: explain clearly to which “reservoir” you refer.

Fig. 6: I can’t recall from the text how/where the “canopy” values were derived? Maybe this can be further clarified?

Line 423, 433 (and elsewhere): I think you need to better clarify in the text (e.g. M&M) what you mean with kinetic, physical, apparent and equilibrium fractionation. This is now used a bit randomly/confusing. Please clarify this throughout the text.

Line 439-443: unclear please revise.

Line 447-448  this equality (for dE) needs to be better explained.

Line 458: can you better explain that equation?

Line 462: “Uptake fractionation”? See my earlier comments on fractionation

Line 498: you might consider discussing the possibility to derive integrated ci/ca ratios via 13C analyses in cellulose of leaves.

Line 507: the ci/ca of 0.7 seems to me on the high end? I would expect values of 0.5-0.6. Can you back this up with literature data?

Line 522, 542-544: if the isotope steady state is so crucial, I think you need to guide/explain the (new) reader a bit better on this here and in the Materials and Methods.

Line 560 and beyond: I agree you need better description of the diurnal dynamics of the isotope reservoirs. But I think to use this isotope-based partitioning method on a longer time scales than just days we need also seasonal (e.g. dry-wet season) information on the isotope reservoirs. Second, it would be good, if feasible, to compare your isotope-based partitioning with other (debated) flux partitioning methods such as the (modified day-time method) or more recent neural network-based approaches.

Figure A1: check the title of the y-axis. Is this notation correct?

Table A1/A2: mention in the caption this data is at 14h00 and 13-day composite average. Make sure your parameters have all the same notation as in the text (e.g. for the fractionation factors this seems different. If I look at the dD and d18O data for topsoil and xyleme water, it is clear (assuming no fractionation) that trees take up water from below 10 cm depth. I have added an earlier comment on root water uptake depth. You might need to (re)consider this aspect in the text.

Moonen et al. report on a very innovative measurement campaign using stable isotopes in CO₂ and H2O in eddy covariance flux measurements with the aim to partition net fluxes into its components. At the moment, there are very few of such using stable-isotope enabled eddy covariance measurements. Carrying out these measurements in a tropical environment is even more novel. In my view, this study is a tremendous measurement effort with a very interesting ecosystem flux analysis.

In my view, the manuscript is in general well written. There are, however, a number of smaller aspects that could help to improve clarity. At some places, details are missing to really understand what and how it done and also what were the limits of their analysis. Some parts that I was expecting in the Methods section only come later in the results, which makes the reading unnecessarily complicated.

Specific comments

Introduction

Line 54: Here the authors describe, what they do, but do not provide clear objectives or hypotheses. In my view, it is helpful for the reader to have clear objectives and hypotheses were possible.

Theory

Line 102: The section on measuring eddy covariance fluxes for isotopes could be strengthen by providing an overview of previous work on isotope EC.

Line 105: I would stay with the term “eddy covariance” to be consistent with the rest of the manuscript.

Line 130: Here assumptions about steady state are made. In my view, it would be help to have a stronger overview of literature here on this assumption and its limitations. I am aware that this is discussed later, but maybe one sentence with 1-2 references would help here.

Line 153/Eq 8: how are rs, rb, and ra calculated? Are they coming from Table 1 (but check that there the R are in capital)? If yes, please mention already here. What about ra?

Lines 180: Peclet effect: why assuming an effective path length on 8 mm? In the text it refers to Barbour and Farquhar 2004, which however shows a wider range. Barbour and Farquhar 2004 refer to other studies with an L=8 mm, but this is for winter wheat, not a tree species. Why were not own measurements done? Alternatively, Lai et al. 2008 Oecologia estimate a wide range of values for L from 0.01 to 0.57 m for seven tree species in the Amazon. This could help with a sensitivity analysis and/or error propagation.

Eq 9: How is transpiration (T) estimated here? Is this obtained from the ET partitioning or somehow assumed.

Line 206/eq. 11: please check the use of symbols. Here T stands for temperature, where as in eq. 9 it stands for transpiration. Maybe good to use a subscript somewhere.

Eq. 12 and 13: How is ci calculated? Is this also coming from Table 1. If yes, please mention already here.  Why is mesophyll conductance not considered here (please check Langendörfer et al. 2002).

Methods

Line 244: Which anemomenter was used? What is the OPGA? The Li-7500? Which CO₂ isotope analyzer and which H2O isotope analyzer was used? Please provide details.

Line 252: Regarding the calibration procedure it is referred to Moonen et al. 2025. This is fine, but nevertheless, it would be nice to have some information here on the instrument performance, e.g. on precision for 18O and D

Results

Line: 290: here the term “moisture flux” is used. I assume H2O flux is meant here as written earlier. Please use only one term for one thing.

Line 295: the terms “delta flux” is used whereas in the caption of Fig. 2 the term “isotope flux” is used. Pleas use also delta flux in the figure caption as delta flux was well defined in eq. 3 (with proper unit) whereas isotope flux is more a general term and not really defined. Please also check Fig. 4.

Line 304: where can we see the cospectra? How did you quantify that the highest frequency had negative contributions? Please add this to an appendix or show here in the discussion.

Line 315/Fig. 3 and line 339/Fig. 5: Why is deltaET-delta_atmo or deltaNEE-delta_atmo shown and not just deltaET or deltaNEE? In my view, it would make it easier to interpret Fig. 3 and Fig. 5.

Line 339/Fig. 5: The diel cycle of delta18O of NEE looks very similar to the work to Wehr et al. 2013, Sturm et al. 2012 and Griffis et al. 2008. Please check these references and refer to if meaningful.

Line 378: the term ABL is not defined before.

Line 455: here the authors calculate a respiration value of 44% of NEE. It would be interesting to compare this to the classical eddy covariance flux partitioning approaches (Reichstein et al. 2005 and Lasslop et al. 2010.

Line 464, Fig. 6: please add to the caption that the values shown here are for average conditions at 14:00.

Line 469: I don’t understand why it was not possible to determine uncertainties for the partitioning results.

Line 513 to 520: Here a fix value for ci/ca is used. It is good to see some discussion here on the sensitivity of the partitioning to this value. Nevertheless, I am wondering why the authors did not adapt an approach similar to Bowling et al. 2001 or Wehr et al. 2015 where they estimated canopy conductance based on the water flux to calculate ci/ca. I am aware this introduces some circularity, but might still provide a meaningful constraint.

References

Barbour, M. M. and Farquhar, G. D.: Do pathways of water movement and leaf anatomical dimensions allow development of gradients in H218O between veins and the sites of evaporation within leaves?, Plant, Cell and Environment, 27, 107–121, https://doi.org/10.1046/j.0016-8025.2003.01132.x, 2004.

Bowling, David R., Pieter P. Tans, and Russell K. Monson. "Partitioning net ecosystem carbon exchange with isotopic fluxes of CO2." Global Change Biology 7.2 (2001): 127-145.

Griffis, T. J., et al. "Direct measurement of biosphere‐atmosphere isotopic CO2 exchange using the eddy covariance technique." Journal of Geophysical Research: Atmospheres 113.D8 (2008).

Lai, Chun-Ta, et al. "Life form-specific variations in leaf water oxygen-18 enrichment in Amazonian vegetation." Oecologia 157.2 (2008): 197-210.

Langendörfer, Uwe, et al. "Modelling of biospheric CO2 gross fluxes via oxygen isotopes in a spruce forest canopy: a 222Rn calibrated box model approach." Tellus B: Chemical and Physical Meteorology 54.5 (2002): 476-496.

Lasslop, Gitta, et al. "Separation of net ecosystem exchange into assimilation and respiration using a light response curve approach: critical issues and global evaluation." Global change biology 16.1 (2010): 187-208.

Reichstein, Markus, et al. "On the separation of net ecosystem exchange into assimilation and ecosystem respiration: review and improved algorithm." Global change biology 11.9 (2005): 1424-1439.

Sturm, Patrick, Werner Eugster, and Alexander Knohl. "Eddy covariance measurements of CO2 isotopologues with a quantum cascade laser absorption spectrometer." Agricultural and Forest Meteorology 152 (2012): 73-82.

Wehr, R., et al. "Long-term eddy covariance measurements of the isotopic composition of the ecosystem–atmosphere exchange of CO2 in a temperate forest." Agricultural and forest meteorology 181 (2013): 69-84.

Wehr, R., and S. R. Saleska. "An improved isotopic method for partitioning net ecosystem–atmosphere CO2 exchange." Agricultural and Forest Meteorology 214 (2015): 515-531.