the Creative Commons Attribution 4.0 License.
the Creative Commons Attribution 4.0 License.
| 21 May 2026
Identifying the underpinnings of δ2H discrepancies between plant stem and soil water: extraction-induced methodological artifacts versus biological fractionation effects
Abstract. Recent studies have reported widespread presence of hydrogen isotope offset (HIO) between cryogenically-extracted plant stem and soil water, challenging the long-standing assumption that the isotopic composition of stem xylem water reliably represents that of its source water. Despite intensive researches on this topic over the past decade, it remains debated as to whether and/or to what extent HIO originates from extraction-related artifacts or from in situ isotope mixing/fractionation during water transport from soil to plants. Here, we used cryogenic vacuum distillation (CVD) to extract stem and soil water from eight species (trees, shrubs, and grasses) grown under two humidity regimes. We quantified species-specific HIO, tested its associations with ecophysiological and environmental variables, and conducted immersion-based rehydration experiments to assess CVD-induced biases. Across species, HIO ranged from −7.2‰ to 3.2‰: trees were consistently negative, whereas shrubs and grasses were near-zero to slightly positive. Rehydration experiments revealed CVD-induced δ2H biases in stem (−4.5‰) and soil water (−2.5‰). When these extraction-related biases in both stem and soil water were simultaneously corrected, species-level HIO (mean = 0.2‰) was no longer different from zero, and showed no significant correlations with ecophysiological or environmental variables. These results suggest that apparent HIO is largely driven by CVD-induced artifacts rather than ecophysiological/environmental processes that cause isotopic fractionation during water transport along the soil-xylem continuum. We conclude that simultaneously correcting CVD-induced biases in both stem and soil water is critical to avoid spurious HIO signals and to improve isotope-based estimation of plant water sources.
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Status: final response (author comments only)
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RC1: 'Comment on egusphere-2026-2605', Anonymous Referee #1, 30 Jun 2026
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AC1: 'Reply on RC1', Xin Song, 01 Aug 2026
General comments
The authors of this manuscript conducted experiments with eight different plant species to evaluate the isotopic offsets between plant stem and soil water extracted by cryogenic vacuum distillation (CVD). The authors also investigated the possible main factors explaining these offsets, such as species, relative humidity, and soil and stem relative water contents, and they proposed corrections for the CVD-induced isotopic biases.
The experiments appear well designed, and the results and applied corrections may be potentially useful for developing standardized procedures for stem water extraction and the correction of isotopic biases.
Overall, the manuscript is well written and structured; however, I have major comments regarding the presentation of the dataset and the description of some data analyses. My main concerns relate to the lack of presentation of the 'original' isotopic data obtained for stem and soil water samples and for the reference water, and to the fact that some methodological steps are not entirely clear (i.e., how the corrections were implemented).
Response: Dear Reviewer, we sincerely thank you for the careful evaluation of our manuscript and for the insightful and constructive comments. We are encouraged that the reviewer found the manuscript to be well written and recognized the potential value of the proposed correction approach. We also fully agree that several aspects of the methodology and presentation of the results require further clarification. The reviewer's comments have helped us identify several important points that should be described more clearly and comprehensively in the revised manuscript. Below, we provide a detailed, point-by-point response to each comment and outline how we intend to revise the manuscript accordingly.
Specific comments
- Lines 109-118: This paragraph could be improved by stating the research hypotheses and by formulating clearly the specific objectives or the research questions.
Response: Thank you for this helpful suggestion. Our intention was to allow the experimental design itself to reveal the logic of the study. However, we recognize that explicitly stating the hypotheses would make the manuscript easier to follow.
In the revised manuscript, we will rewrite the final paragraph of the Introduction to explicitly state the central hypotheses and objectives. Specifically, we will clarify that this study was designed to test whether: (i) apparent HIO varies systematically among species and under different atmospheric humidity conditions; (ii) the observed HIO can be largely explained by CVD-induced extraction artifacts quantified through immersion-based rehydration experiments; and (iii) correcting CVD-induced biases in both stem water and soil water would substantially reduce the apparent HIO and weaken its dependence on ecophysiological and environmental variables.
- Line 112: Here the authors should clarify whether the two humidity regimes refer to soil moisture or to air humidity.
Response: The reviewer is correct that this statement is potentially ambiguous. The two humidity treatments refer exclusively to air relative humidity (RH). Soil water availability was intentionally maintained close to saturation throughout the experiment. We will revise the manuscript to explicitly state that the treatments refer to air RH.
- Line 130: I suggest providing an explanation for the choice of only two relative humidity levels and for these two specific values. What did the authors expect from using these two levels?
Response: This is an important point. The purpose of introducing two RH treatments was not to establish a quantitative response curve of HIO across atmospheric humidity, but rather to create two contrasting evaporative environments while minimizing variation in other environmental factors. During the experiment, the measured RH averaged 39.2 ± 5.2% (mean ± SD) under the low-humidity treatment and 68.3 ± 4.8% under the high-humidity treatment. These two levels were selected because they provide a substantial contrast in atmospheric evaporative demand while maintaining healthy plant growth and avoiding severe water stress conditions (Grantz et al., 1990; Kahmen et al., 2013).
Our expectation was that, if biological fractionation during water transport contributes to HIO, the stronger transpiration associated with lower RH would enhance such effects. Conversely, if HIO primarily originates from extraction-related methodological artifacts, any differences between the two RH treatments should become negligible after correcting the extraction-related isotope biases. Thus, the two RH treatments were selected as contrasting conditions for hypothesis testing rather than to comprehensively characterize the response of HIO to atmospheric humidity.
We agree that this rationale was not sufficiently explained in the original manuscript. In the revised version, we will explicitly report the measured RH values for both treatments in the Methods section and clarify the reasoning behind the selection of these two humidity levels.
- Lines 132-133: It is not clear why the soil was maintained very wet or saturated throughout the plants' growth. Considering the analysis conducted for relative air humidity, I would have expected the application of different soil moisture levels as well.
Response: We fully understand the reviewer's expectation that soil moisture could have been incorporated as an additional treatment factor, particularly because soil water availability is known to influence plant water uptake and has frequently been discussed as a potential driver of isotope offsets (Vargas et al., 2017; Barbeta et al., 2020).
Our intention, however, was to isolate the potential influence of atmospheric evaporative demand on HIO while minimizing confounding effects associated with soil water availability. Had different soil moisture treatments also been imposed, several additional factors would have changed simultaneously, including plant water status, soil water isotopic heterogeneity, root water uptake patterns, stem and soil relative water contents (RWC), and potentially the magnitude of CVD-induced extraction bias itself. Under such conditions, it would have become considerably more difficult to distinguish whether differences in HIO originated from biological fractionation processes or from methodological artifacts associated with water extraction. Maintaining the soil close to saturation therefore served two purposes. First, it ensured that all species had unrestricted access to water, minimizing drought-induced physiological responses. Second, it reduced spatial heterogeneity in soil water isotopic composition, allowing the comparison between stem and soil water isotopes to be interpreted more directly.
We agree that this experimental rationale is insufficiently explained in the current manuscript. In the revised version, we will explicitly state that soil moisture was intentionally controlled rather than investigated, because the objective of this study was to isolate the effects of atmospheric humidity on HIO while minimizing other environmental sources of variation. We will also clarify that future studies incorporating both soil moisture and atmospheric humidity gradients would be valuable for evaluating whether the proposed correction approach remains applicable under drought conditions.
- Sections 2.1, 2.4 and 2.5: These sections lack key details in the descriptions of the methods, namely the isotopic compositions of the irrigation water (tap water) and of the reference water (is the same tap water used for irrigation?). Furthermore, does the reference/irrigation water change across samples, given that there may be temporal variability in the isotopic signature of tap water?
Response: Thank you for pointing out this omission. In the revised manuscript, we will provide the measured isotopic compositions of the irrigation water and reference water. For the low-humidity treatment, the irrigation water had δ2H and δ18O values of −27.0 ± 0.7‰ and −4.5 ± 0.1‰ (mean ± SD, n = 7), respectively, whereas those for the high-humidity treatment were −30.8 ± 0.6‰ and −4.9 ± 0.1‰ (n = 7), respectively. These values showed only minor variations during the 7-day experimental period. The reference water used for stem rehydration had δ2H and δ18O values of −35.4 ± 0.3‰ and −5.5 ± 0.1‰ (n = 4), respectively, while the reference water used for soil rehydration had values of −33.8 ± 0.1‰ and −5.3 ± 0.1‰ (n = 4), respectively. The isotopic compositions of the rehydration waters were relatively close to those of the irrigation waters.
- Section 2.3: Please add the number of samples and replicates taken for each sample type.
Response: Thank you for this suggestion. In the revised manuscript, we will report the number of biological replicates and sample sizes more clearly. Specifically, we will add the following sentence to Section 2.3: "For each species under each RH treatment, four stem samples and their corresponding soil samples (n = 4) were collected, resulting in a total of 64 stem samples and 64 corresponding soil samples across the eight species and two humidity treatments." We will also clarify the sample numbers used in the subsequent statistical analyses where appropriate.
- Lines 193-194: I find this description of the corrections insufficient, as the results (e.g., Figure 3) include an evaluation of stem, soil, and stem+soil corrections. Specifically, I recommend reporting the equations used, discussing the uncertainty associated with these equations (e.g., analysis of residuals, coefficient of determination, etc.), and describing any other methodological steps performed for — or prior to — the corrections.
Response: We sincerely thank the reviewer for this insightful comment, which we believe addresses one of the most important methodological aspects of the manuscript. Our original intention was to avoid unnecessarily repeating information, because the empirical correction equations and their goodness-of-fit (R2) are presented in the Results section where the correction models are developed. In retrospect, however, we agree that the description of the correction procedure in the Methods section was too brief, making it difficult for readers to understand exactly how the corrections were implemented.
In the revised manuscript, we will therefore substantially expand the description of the correction procedure. Specifically, before presenting the results, we will clearly describe the overall correction workflow, including (i) quantifying extraction-induced isotope biases using the rehydration experiments, (ii) establishing empirical relationships between isotope bias and sample water status, (iii) applying these empirical relationships independently to stem water and soil water, and (iv) recalculating HIO after correcting one or both water pools. This will allow readers to clearly understand how the three correction scenarios (stem-only, soil-only, and stem + soil correction) were generated.
We will also expand the Results section by providing additional information on the performance of the regression models, including analyses of the residuals and a discussion of the uncertainty associated with the empirical correction equations. We believe these revisions will substantially improve the transparency and reproducibility of the proposed correction approach.
- Section 3.1: Before presenting the isotopic offsets, I recommend showing a dual isotope plot including stem samples, soil samples, and reference water, and adding a table with descriptive statistics of the isotopic signatures. Please note that no isotopic values of the samples are presented in either the manuscript or the supplementary material. Showing the original isotopic values in a dual isotope plot and in a table would help the reader understand the magnitude of the offset.
Response: Following this comment and a similar suggestion from Reviewer #2, we will add a new dual isotope plot showing the measured isotopic compositions of stem water, soil water, and irrigation water. In addition, the corresponding isotopic data and descriptive statistics will be included in the Supplementary Table S1. As the reviewer pointed out, presenting the original isotopic measurements before discussing HIO will improve the clarity of the Results section and allow readers to better evaluate the magnitude of the isotopic offsets and the subsequent correction analyses.
- Table 1: Please add the sample size for each test and provide in the caption the name of the statistical test used to determine the differences between the isotopic compositions of stem and soil water.
Response: Although the statistical methods are described in the Methods section, we agree that the table should be understandable without requiring readers to refer back to previous sections.
In the revised manuscript, we will therefore add the sample size (n = 4) for each comparison and explicitly state in the one-sample t-tests were used to assess whether HIO and OIO differed significantly from zero.
- Figure 1: I suggest adding horizontal lines to represent the uncertainty in the isotopic analyses. Regarding HIO, there is an interesting pattern of increasing values; do the authors have any explanation for the positive HIO obtained for grass species?
Response: We agree that presenting the analytical uncertainty would help readers better evaluate the significance of relatively small isotopic offsets, particularly for species with HIO values close to zero. Therefore, we will revise Fig. 1 by adding horizontal lines representing the analytical uncertainty of the isotope measurements.
Regarding the slightly positive HIO values observed in grass species, we also find this pattern interesting. Based on our results, we hypothesize that the positive HIO values are mainly attributable to the larger CVD-induced δ2H bias in soil water compared with that in stem water. This may be related to the extremely high RWC of grass stems, which reduces the magnitude of CVD-induced δ2H bias in stem water. Consequently, the difference between stem water and soil water δ2H becomes slightly positive. This interpretation is further supported by Fig. 3g, h, where HIO values of the grass species become close to zero after correcting for the CVD-induced δ2H biases in both soil and stem waters. This possible explanation is discussed in Lines 347–359 of the manuscript. We will further expand this discussion in the revised manuscript to clarify the underlying mechanism and better explain the observed positive HIO values in grasses. We trust that this additional clarification addresses the reviewer's concern.
- Section 3.4: The reader cannot observe or evaluate the effect of the correction on the isotopic values, as no data are shown. I recommend adding either a dual isotope plot with corrected and uncorrected values or a scatter plot comparing corrected and uncorrected isotopic values.
Response: Thank you for this helpful suggestion. In the revised manuscript, we will add a new dual-isotope plot comparing the uncorrected and corrected isotopic compositions to the Supplementary Material. We believe that this presentation will allow readers to more directly visualize and evaluate the effect of the correction on the isotopic values.
- Figure 3: In some cases, HIO is smaller for the uncorrected data than for the corrected ones (e.g., GH). Do the authors have an explanation for this? In certain cases, the corrections may not be appropriate; is there a way to determine when corrections are needed and when they are not?
Response: We appreciate the reviewer’s concern, which likely arises from our insufficient description of how the corrections were applied in the original manuscript (as also mentioned in the previous comment). Here, we provide further clarification. According to Eqn 1, HIO is calculated as: HIO = δ2Hstem water − δ2Hsoil water. Because CVD induces negative δ2H biases in both stem water and soil water (with average offsets of −4.47‰ for stem water and −2.52‰ for soil water; Fig. S1), the direction and magnitude of the correction depend on which component is corrected. For example, when only the CVD-induced δ2H bias in stem water is corrected, HIO increases by 4.47‰ (using the mean bias as an example). In contrast, when only the soil water bias is corrected, HIO decreases by 2.52‰. When both stem water and soil water biases are corrected simultaneously, HIO increases by 4.47‰ and then decreases by 2.52‰, resulting in a net increase of 1.95‰. Therefore, the cases where corrected HIO values are larger than uncorrected values (e.g., Fig. 3) are expected when stem water corrections contribute more strongly than soil water corrections. This apparent pattern does not indicate that the correction is inappropriate, but rather reflects the different contributions of the two CVD-induced biases to the calculated HIO values. We acknowledge that the original manuscript did not sufficiently explain this correction procedure, and we will add a more detailed description in the revised manuscript, as mentioned above.
Regarding when corrections are necessary, we suggest that CVD-induced δ2H biases should be considered for correction when they are detectable and significant, especially when HIO is interpreted in terms of biological fractionation processes. Accounting for such methodological effects is essential to avoid misattributing extraction-related artifacts to biological mechanisms.
- Lines 294-296: These findings may be expected, given that soil RWC is used for the correction. If environmental variables are used for the corrections, I do not understand what the authors' expectations are. Furthermore, to investigate the lack of significant correlations between the isotopic offsets of the corrected data and the various variables, the authors should better present the dataset and the methodologies, and likely expand the statistical analyses related to the regressions and residuals.
Response: Regarding the first point, we would like to clarify that environmental variables were not used for the correction itself. Instead, Lines 294-296 describe how the relationships between HIO and various ecophysiological variables change after simultaneously correcting the CVD-induced δ2H biases in both stem water and soil water. Specifically, we examined whether the previously observed associations between HIO and these variables persisted after correction.
Regarding the second point, we will follow the reviewer’s suggestion to improve presentation of the referred data and analytical methodologies, i.e., by adding scatter plots showing the relationships between corrected HIO values and the relevant variables, together with the corresponding regression analyses and statistical results, into the Supplementary Materials. We believe these additions will allow the reviewer and interested readers to more directly evaluate the regression patterns and residuals after correction.
Technical corrections
- Line 93: ‘etc.’ can be removed.
Response: We will make the correction in the revised manuscript. Thank you.
- Line 189: Please use here and in the following sections the term ‘generalized linear models’ instead of ‘general linear models’.
Response: Thank you for this comment. In our study, all response variables were continuous, and the analyses were performed using the General Linear Model procedure in SPSS (i.e., a factorial ANOVA with species and RH treated as fixed factors). We therefore believe that the term “general linear model” is appropriate and propose to retain this terminology in the revised manuscript. To avoid potential confusion, we will revise the Methods section to specify that a two-way general linear model wes used.
- Lines 234-235: Please specify these ecophysiological and environmental variables as they should differ from the species and relative humidity (RH).
Response: We will clarify these variables in the revised manuscript. Thank you.
- Table 2: I recommend adding an explanation of all acronyms in the caption.
Response: We agree with this suggestion and will include explanations of all acronyms in the caption of Table 2.
- Figure 3: In the legend, there is a missing ‘d’ in ‘uncorrected’.
Response: We will correct the word in the revised manuscript. Thank you for pointing this out.
References：
Barbeta, A., Gimeno, T. E., Clavé, L., Fréjaville, B., Jones, S. P., Delvigne, C., Wingate, L., and Ogée, J.: An explanation for the isotopic offset between soil and stem water in a temperate tree species, New Phytol., 227, 766–779, https://doi.org/10.1111/nph.16564, 2020.
Grantz, D. A.: Plant response to atmospheric humidity, Plant Cell & Environment, 13, 667–679, https://doi.org/10.1111/j.1365-3040.1990.tb01082.x, 1990.
Kahmen, A., Schefuß, E., and Sachse, D.: Leaf water deuterium enrichment shapes leaf wax n-alkane δD values of angiosperm plants I: Experimental evidence and mechanistic insights, Geochim. Cosmochim. Acta, 111, 39–49, https://doi.org/10.1016/j.gca.2012.09.003, 2013.
Vargas, A. I., Schaffer, B., Yuhong, L., and Sternberg, L. da S. L.: Testing plant use of mobile vs immobile soil water sources using stable isotope experiments, New Phytol., 215, 582–594, https://doi.org/10.1111/nph.14616, 2017.
Citation: https://doi.org/10.5194/egusphere-2026-2605-AC1
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AC1: 'Reply on RC1', Xin Song, 01 Aug 2026
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RC2: 'Comment on egusphere-2026-2605', Anonymous Referee #2, 16 Jul 2026
The manuscript investigates the recently heavily debated discrepancies between plant and soil water isotope values caused by extraction methods. The authors used cryogenic vacuum distillation (CVD) to extract stem and soil water from eight species (trees, shrubs, and grasses) grown under two humidity regimes. We quantified species-specific HIO, tested its associations with ecophysiological and environmental variables, and conducted immersion-based rehydration experiments to assess CVD-induced biases.
The manuscript is well-written and the experiment and developed correction measures potentially can be a useful contribution. However, the rehydration experiment in my opinion is faulty and the experiment itself introduces and isotopic bias. I would like to see the authors elaborate on that before a final decision. Here is a summary of my comments, with the rehydration experiment as main concern.
I believe this needs to be improved before a decision can be made. In my opinion it is crucial.
Nevertheless, I encourage the authors because potentially this is a very valuable contribution.
Main comments
- Section 3.1: Before presenting the isotopic offsets, I recommend showing a dual isotope plot as it provides a clear and understandable visualization of the overall isotopic space
- One thing that kept me thinking: Is the correction applicable generally? Or what is the practical use of the experiment? It would be tremendously laborious to carry out rehydration experiments for any given case.
- My main criticism:
2.4. For the rehydration experiment, samples were dried at 65°C to constant mass “to ensure complete removal of pre-existing water” according to the authors. But constant mass does not mean that all water is removed from the samples pre-rehydration. Let alone the fact that CVD was carried out at 195°C causes removal of additional water from the stems and soil. In other words, the drying at 65°C left less mobile water in soils and stems. When these are then spiked with the reference water, less mobile water plus spiked water will be contained in the sample. When extracting at 195°, however, both the less mobile and spiked water will be extracted. There are a number of papers examining this for soils (see below). This is a severe methodological error that questions the entire evaluation of the rehydration experiment and correction approach in my opinion. Likewise, a similar process could be relevant for stems, as Chen et al. also found. The fact that the CVD-induced bias was greater with lower RWC also supports my reasoning: with lower RWC, the relative contribution of “leftover”-water (caused by drying at 65°C only) is higher, whereas it diminishes with higher RWC.
Oerter, E., Finstad, K., Schaefer, J., Goldsmith, G. R., Dawson, T., & Amundson, R. (2014). Oxygen isotope fractionation effects in soil water via interaction with cations (Mg, Ca, K, Na) adsorbed to phyllosilicate clay minerals. Journal of Hydrology, 515, 1–9. https://doi.org/10.1016/J.JHYDROL.2014.04.029
Gaj, M., Kaufhold, S., Koeniger, P., Beyer, M., Weiler, M., & Himmelsbach, T. (2017). Mineral mediated isotope fractionation of soil water. Rapid Communications in Mass Spectrometry, 31(3). https://doi.org/10.1002/rcm.7787
Minor comments:
Abstract
Well written, but the information is missing that the observed bias was corrected and how the correction was done. The abstract only speaks about the results of the correction.
2.2.: Please add an introductory sentence to why these gas exchange measurements were conducted and necessary for the experiment. It currently comes out of nowhere
l.151: it is sometimes argued that plant samples should not be stored under such cold temperatures, because some cells will break and potentially cause organic contamination of the sample. Well, I guess that would be another cryogenic-introduced bias needing investigation…
l. 20: research
l.90 assess
l.115 approach
l.125: ca. instead of c. (also line 145, please check throughout the ms)
l. 174: 195°C is an unusually high extraction temperature. For plants, it has been stated previously that the risk of contamination with organic compound is higher with increasing extraction temperature. Please provide a reasoning for this high temperature used (even though measurement with IRMS should not be affected by organic contamination).
l. 175-178: after extraction, the samples are commonly put in the drying oven at the extraction temperature to assess extraction efficiency
l. 180-185: The definition used here for the isotopic offset is only valid under the assumption that there is no other potential water source for delta stem water. While this is clear for the experiments that were done, it should be clarified to avoid confusion or misuse of this definition.
Citation: https://doi.org/10.5194/egusphere-2026-2605-RC2 -
AC2: 'Reply on RC2', Xin Song, 01 Aug 2026
The manuscript investigates the recently heavily debated discrepancies between plant and soil water isotope values caused by extraction methods. The authors used cryogenic vacuum distillation (CVD) to extract stem and soil water from eight species (trees, shrubs, and grasses) grown under two humidity regimes. We quantified species-specific HIO, tested its associations with ecophysiological and environmental variables, and conducted immersion-based rehydration experiments to assess CVD-induced biases.
The manuscript is well-written and the experiment and developed correction measures potentially can be a useful contribution. However, the rehydration experiment in my opinion is faulty and the experiment itself introduces and isotopic bias. I would like to see the authors elaborate on that before a final decision. Here is a summary of my comments, with the rehydration experiment as main concern.
I believe this needs to be improved before a decision can be made. In my opinion it is crucial.
Nevertheless, I encourage the authors because potentially this is a very valuable contribution.
Response: Dear Reviewer, we sincerely thank you for your careful evaluation of our manuscript and for your insightful comments. We are encouraged by your recognition that the manuscript is well written and that the proposed correction approach has the potential to make a useful contribution. We particularly appreciate your detailed comments regarding the immersion-based rehydration experiment, as this experiment constitutes the methodological foundation of the present study. We agree that several assumptions underlying this approach require further clarification, and your comments have helped us identify important aspects that should be discussed more explicitly in the revised manuscript. Below, we provide point-by-point responses to your comments and describe how we will revise the manuscript accordingly.
Main comments
- Section 3.1: Before presenting the isotopic offsets, I recommend showing a dual isotope plot as it provides a clear and understandable visualization of the overall isotopic space
Response: Thank you for this helpful suggestion. We agree that presenting the original isotope dataset before discussing hydrogen isotope offset (HIO) would improve the clarity of the manuscript. Because HIO is calculated directly from the differences between stem-water and soil-water isotope compositions, showing the original isotope values first would help readers better understand the isotopic variability and the magnitude of the observed offsets.
Following this suggestion, and also considering a related comment from Reviewer #1, we propose to add a dual-isotope plot showing stem water, soil water, and irrigation water in the revised manuscript.
- One thing that kept me thinking: Is the correction applicable generally? Or what is the practical use of the experiment? It would be tremendously laborious to carry out rehydration experiments for any given case.
Response: Thank you for highlighting this important point. The reviewer’s comment provides an opportunity for us to further clarify the intended application and scope of the proposed correction approach. We would like to emphasize that our intention is not to suggest that immersion-based rehydration experiments should become a routine step before every CVD-based isotope analysis. Rather, the purpose of the rehydration experiment was to provide an independent approach to quantify extraction-induced isotope biases and to evaluate the extent to which apparent HIO may result from methodological artifacts. Accordingly, the correction relationships developed here should be considered as a proof-of-concept rather than universal correction equations applicable to all conditions. The magnitude of extraction-induced isotope biases may vary among laboratories and CVD systems due to differences in extraction procedures, instrument configurations, sample types, species, and water contents. Therefore, further inter-laboratory comparisons and broader validation experiments will be needed before generally applicable correction functions can be established. Nevertheless, we believe that the broader implication of this study extends beyond the specific empirical equations. Our results highlight that HIO should be interpreted by considering potential isotope biases associated with both stem-water and soil-water extraction, and provide a framework for distinguishing methodological artifacts from biological signals.
To better reflect this point, we propose to revise the Discussion by clarifying the intended application of the correction approach and emphasizing that the broader methodological insights, rather than the empirical equations alone, represent the main contribution of this study.
- My main criticism:
2.4. For the rehydration experiment, samples were dried at 65°C to constant mass “to ensure complete removal of pre-existing water” according to the authors. But constant mass does not mean that all water is removed from the samples pre-rehydration. Let alone the fact that CVD was carried out at 195°C causes removal of additional water from the stems and soil. In other words, the drying at 65°C left less mobile water in soils and stems. When these are then spiked with the reference water, less mobile water plus spiked water will be contained in the sample. When extracting at 195°, however, both the less mobile and spiked water will be extracted. There are a number of papers examining this for soils (see below). This is a severe methodological error that questions the entire evaluation of the rehydration experiment and correction approach in my opinion. Likewise, a similar process could be relevant for stems, as Chen et al. also found. The fact that the CVD-induced bias was greater with lower RWC also supports my reasoning: with lower RWC, the relative contribution of “leftover”-water (caused by drying at 65°C only) is higher, whereas it diminishes with higher RWC.
Oerter, E., Finstad, K., Schaefer, J., Goldsmith, G. R., Dawson, T., & Amundson, R. (2014). Oxygen isotope fractionation effects in soil water via interaction with cations (Mg, Ca, K, Na) adsorbed to phyllosilicate clay minerals. Journal of Hydrology, 515, 1–9. https://doi.org/10.1016/J.JHYDROL.2014.04.029
Gaj, M., Kaufhold, S., Koeniger, P., Beyer, M., Weiler, M., & Himmelsbach, T. (2017). Mineral mediated isotope fractionation of soil water. Rapid Communications in Mass Spectrometry, 31(3). https://doi.org/10.1002/rcm.7787
Response: Thank you very much for raising this important methodological concern. We agree that drying samples at 65°C to constant mass does not necessarily guarantee the complete removal of all pre-existing water, particularly water that is tightly bound to plant tissues, organic matter, or soil mineral surfaces. We appreciate the reviewer for highlighting this potential issue and agree that it deserves further clarification in the revised manuscript.
First, we would like to emphasize that although reaching constant mass does not prove the complete removal of all water, the drying conditions and duration used in this study are expected to remove the vast majority of mobile water from both stem and soil samples. Therefore, if a small amount of tightly bound water remained after drying, its absolute quantity − and consequently its contribution to the isotopic composition of the rehydrated samples − would likely be relatively small.
In addition, the existence of mobile and tightly bound water pools, as well as the extent of isotopic exchange between them, has been widely discussed in isotope-based ecohydrology, particularly in the context of the "two water worlds" hypothesis. However, whether such isotopically distinct water pools are ubiquitous, and to what extent isotope exchange occurs between them, remains an active topic of debate (Brooks et al., 2010; McDonnell et al., 2014; Evaristo et al., 2015; Chen et al., 2020; Finkenbiner et al., 2022). More recent studies have further suggested that substantial isotopic exchange (potentially as high as 75–95%) may occur even between water pools previously considered to be relatively isolated (e.g., Vargas et al., 2017). Consequently, if the residual water remaining after drying is exchangeable, its isotopic composition would be expected to approach that of the reference water after 48 h of equilibration with a large volume of reference water, thereby minimizing its influence on the rehydration experiment.
On the other hand, we also considered the alternative scenario proposed by the reviewer, namely that a small amount of genuinely non-exchangeable tightly bound water remains in the sample after drying. In this case, the water recovered during subsequent CVD extraction at 195 °C would indeed consist of both the added reference water and the residual tightly bound water. However, we do not believe that this possibility invalidates either the rehydration experiment or the proposed correction approach. It is important to emphasize that the objective of the rehydration experiment was not to isolate the contribution of each individual water pool or physical process to the observed isotope offset. Rather, its purpose was to quantify the net extraction-related isotopic bias introduced by the analytical workflow. If residual tightly bound water is indeed extracted during CVD and contributes to the measured isotope composition, then this influence is also inherently present during the extraction of the original stem and soil samples analyzed in this study. Specifically, both the rehydrated samples used to quantify extraction-related bias and the experimental stem and soil samples underwent exactly the same CVD extraction procedure, including identical extraction temperature, vacuum conditions, and extraction duration. Therefore, if residual tightly bound water contributes to isotope offsets during extraction, its effect is expected to be represented similarly in both the rehydrated samples and the original experimental samples. Consequently, any isotope offset arising from residual tightly bound water would be incorporated into the experimentally determined extraction-related bias and therefore accounted for during the correction procedure. From this perspective, even if a small amount of non-exchangeable tightly bound water remains after drying, the isotope offset quantified by the rehydration experiment still represents the integrated methodological bias associated with the complete extraction workflow, rather than the effect of any single physical process. Ultimately, for plant water source studies, the corrected soil water and stem water correspond to the water pools that are of primary ecohydrological interest − namely, the mobile water flowing through the soil-plant-atmosphere continuum, including mobile soil water available for root uptake and mobile xylem water transported within plant stems.
Taken together, although extraction at 195 °C may recover additional residual water from both stem and soil samples, this extraction condition was applied consistently to both the rehydrated samples and the original experimental samples. The scenarios discussed above therefore indicate that the rehydration-based calibration captures the integrated methodological effect of the complete extraction procedure under the specific conditions used in this study. Within this methodological context, we believe that the proposed rehydration approach and the resulting correction scheme remain appropriate and ecologically meaningful for interpreting plant–soil water isotope data.
At the same time, we agree that the reviewer's hypothesis provides an important alternative interpretation of our observations. In particular, the dependence of the measured isotope bias on relative water content (RWC) could indeed be explained by an increasing proportional contribution of residual tightly bound water at lower water contents. However, we also note that the same experimental pattern is consistent with previously proposed mechanisms. For example, Chen et al. (2020) interpreted this dependence as resulting from dynamic hydrogen isotope exchange between water and exchangeable organic hydrogen during CVD extraction, an interpretation that was further supported by the recent study of Wen et al. (2026). Therefore, although our results are consistent with the reviewer's hypothesis, they are not unique to that explanation, and the present data do not allow us to distinguish among these alternative mechanisms. Based on the currently available evidence, we consider hydrogen isotope exchange during CVD extraction to be a likely major contributor to the observed extraction-related isotope bias, while at the same time acknowledging that a contribution from residual tightly bound water cannot be excluded.
We greatly appreciate the reviewer for drawing our attention to this important issue. In the revised manuscript, we will substantially expand the Discussion to clarify these assumptions, explicitly acknowledge this limitation, and better define the scope and interpretation of the proposed correction approach. We will also revise the terminology where appropriate to clarify that the isotope bias quantified by the rehydration experiment may include the potential influence of residual tightly bound water remaining after drying, rather than attributing the observed bias exclusively to the CVD extraction step.
Minor comments:
Abstract
Well written, but the information is missing that the observed bias was corrected and how the correction was done. The abstract only speaks about the results of the correction.
Response: Thank you for the suggestion. In the revised manuscript, we will revise the Abstract to briefly introduce the correction procedure.
2.2.: Please add an introductory sentence to why these gas exchange measurements were conducted and necessary for the experiment. It currently comes out of nowhere
Response: Thank you. We agree that the introduction to the gas-exchange measurements is currently rather abrupt. The purpose of these measurements was to evaluate whether apparent HIO was related to plant physiological activity, thereby providing an independent test of the hypothesis that biological processes contribute to the observed HIO. Specifically, if isotope fractionation during water transport were responsible for HIO, one might expect HIO to covary with transpiration-related physiological variables such as stomatal conductance or transpiration rate. Conversely, if HIO primarily results from extraction-related methodological artifacts, such relationships should weaken or disappear after correcting the extraction bias. We agree that this rationale should be introduced before describing the measurements themselves.
l.151: it is sometimes argued that plant samples should not be stored under such cold temperatures, because some cells will break and potentially cause organic contamination of the sample. Well, I guess that would be another cryogenic-introduced bias needing investigation…
Response: This is an interesting point. We agree that potential cell rupture during frozen storage and the resulting release of organic compounds represent an important methodological issue that deserves further investigation. In the present study, however, water isotope analyses were performed using isotope ratio mass spectrometry (IRMS), which is generally much less susceptible to interference from organic contaminants than isotope ratio infrared spectroscopy (IRIS). Therefore, we expect that any potential influence of organic contamination on the isotope measurements in our study is likely to be minimal.
- 20: research
Response: We will make the correction in the revised manuscript. Thank you.
l.90 assess
Response: Thank you for pointing this out. We will correct the wording in the revised manuscript.
l.115 approach
Response: We will correct the word in the revised manuscript.
l.125: ca. instead of c. (also line 145, please check throughout the ms)
Response: Thank you for pointing this out. We will replace “c.” with “ca.” throughout the manuscript and carefully check for consistency wherever this abbreviation is used.
- 174: 195°C is an unusually high extraction temperature. For plants, it has been stated previously that the risk of contamination with organic compound is higher with increasing extraction temperature. Please provide a reasoning for this high temperature used (even though measurement with IRMS should not be affected by organic contamination).
Response: Thank you for raising this important point. We agree that 195°C is relatively high compared with conventional CVD protocols and that higher extraction temperatures may increase the potential risk of co-extracting of organic compounds. In this study, we used an automated CVD system (LI-2100, LICA, Beijing, China), which follows the same basic principle as the classic CVD system described by West et al. (2006), but avoids the need for liquid nitrogen and intensive manual operation. Owing to its operational convenience, this type of automated system has been increasingly used for plant and soil water isotope analyses (e.g., Yang et al., 2023; Wang et al., 2025; Zhang et al., 2026). The extraction temperature of 195°C is the standard operating temperature recommended for this system and is commonly used to ensure efficient water recovery from samples with different physical properties, including plant tissues and soils. We therefore followed this standard protocol to maintain consistency and comparability with previous studies using the same extraction system.
We acknowledge that higher extraction temperatures may increase the risk of organic contamination, particularly for IRIS. However, as noted by the reviewer, all isotope analyses in this study were conducted using IRMS, for which the influence of organic contamination is generally much smaller than that in laser spectroscopy. We will further clarify the rationale for using this extraction temperature in the revised manuscript.
- 175-178: after extraction, the samples are commonly put in the drying oven at the extraction temperature to assess extraction efficiency
Response: Thank you for pointing this out. In fact, we further dried the samples at the extraction temperature to verify the completeness of water extraction, as we did in our previous studies (Chen et al., 2020; Wen et al., 2026). We will clarify this procedure in the revised manuscript.
- 180-185: The definition used here for the isotopic offset is only valid under the assumption that there is no other potential water source for delta stem water. While this is clear for the experiments that were done, it should be clarified to avoid confusion or misuse of this definition.
Response: We agree and will clarify this assumption in the revised manuscript to avoid potential misunderstanding or inappropriate application of this definition. Thank you.
References：
Brooks, J. R., Barnard, H. R., Coulombe, R., and McDonnell, J. J.: Ecohydrologic separation of water between trees and streams in a Mediterranean climate, Nature Geosci, 3, 100–104, https://doi.org/10.1038/ngeo722, 2010.
Chen, Y., Helliker, B. R., Tang, X., Li, F., Zhou, Y., and Song, X.: Stem water cryogenic extraction biases estimation in deuterium isotope composition of plant source water, Proc. Natl. Acad. Sci. U.S.A., 117, 33345–33350, https://doi.org/10.1073/pnas.2014422117, 2020.
Evaristo, J., Jasechko, S., and McDonnell, J. J.: Global separation of plant transpiration from groundwater and streamflow, Nature, 525, 91–94, https://doi.org/10.1038/nature14983, 2015.
Finkenbiner, C. E., Good, S. P., Renée Brooks, J., Allen, S. T., and Sasidharan, S.: The extent to which soil hydraulics can explain ecohydrological separation, Nat Commun, 13, 6492, https://doi.org/10.1038/s41467-022-34215-7, 2022.
McDonnell, J.: The two water worlds hypothesis: Ecohydrological separation of water between streams and trees?, WIREs WATER, 1, https://doi.org/10.1002/wat2.1027, 2014.
Vargas, A. I., Schaffer, B., Yuhong, L., and Sternberg, L. da S. L.: Testing plant use of mobile vs immobile soil water sources using stable isotope experiments, New Phytol., 215, 582–594, https://doi.org/10.1111/nph.14616, 2017.
Wang, S., Yang, M., Gao, X., Si, B., and Zhao, X.: Climate-Dependent Hydrogen Isotopic Offset of Stem Water and Its Effect on Quantification of Plant Water Sources, Geophys. Res. Lett., 52, e2025GL115559, https://doi.org/10.1029/2025GL115559, 2025.
Wen, W., Tang, X., Lin, W., Chen, Y., Zhou, L., and Song, X.: Cryogenic vacuum distillation-induced deuterium isotope biases in leaf water and their ecophysiological implications, New Phytol., 249, 2776–2786, https://doi.org/10.1111/nph.70857, 2026.
West, A. G., Patrickson, S. J., and Ehleringer, J. R.: Water extraction times for plant and soil materials used in stable isotope analysis, Rapid Commun. Mass Spectrom., 20, 1317–1321, https://doi.org/10.1002/rcm.2456, 2006.
Yang, B., Dossa, G. G. O., Hu, Y., Liu, L., Meng, X., Du, Y., Li, J., Zhu, X., Zhang, Y., Singh, A. K., Yuan, X., Wu, J., Zakari, S., Liu, W., and Song, L.: Uncorrected soil water isotopes through cryogenic vacuum distillation may lead to a false estimation on plant water sources, Methods Ecol Evol, 14, 1443–1456, https://doi.org/10.1111/2041-210X.14107, 2023.
Zhang, Q., Su, Y., Feng, Q., Jian, C., and Yu, T.: Environmental drivers and correction methods optimization of isotope offsets in water source analysis of desert riparian Populus euphratica, J. Hydrol., 664, 134482, https://doi.org/10.1016/j.jhydrol.2025.134482, 2026.
Citation: https://doi.org/10.5194/egusphere-2026-2605-AC2
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General comments
The authors of this manuscript conducted experiments with eight different plant species to evaluate the isotopic offsets between plant stem and soil water extracted by cryogenic vacuum distillation (CVD). The authors also investigated the possible main factors explaining these offsets, such as species, relative humidity, and soil and stem relative water contents, and they proposed corrections for the CVD-induced isotopic biases.
The experiments appear well designed, and the results and applied corrections may be potentially useful for developing standardized procedures for stem water extraction and the correction of isotopic biases.
Overall, the manuscript is well written and structured; however, I have major comments regarding the presentation of the dataset and the description of some data analyses. My main concerns relate to the lack of presentation of the 'original' isotopic data obtained for stem and soil water samples and for the reference water, and to the fact that some methodological steps are not entirely clear (i.e., how the corrections were implemented).
Specific comments
Technical corrections