Identifying the underpinnings of &delta;<sup>2</sup>H discrepancies between plant stem and soil water: extraction-induced methodological artifacts versus biological fractionation effects

Wen, Wei; Yue, Chao; Chen, Bingming; Tang, Xianhui; Wang, Yabo; Khattak, Wajid Ali; Song, Xin

doi:10.5194/egusphere-2026-2605

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https://doi.org/10.5194/egusphere-2026-2605

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21 May 2026

| 21 May 2026

Identifying the underpinnings of δ²H discrepancies between plant stem and soil water: extraction-induced methodological artifacts versus biological fractionation effects

Wei Wen, Chao Yue, Bingming Chen, Xianhui Tang, Yabo Wang, Wajid Ali Khattak, and Xin Song

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.

Received: 06 May 2026 – Discussion started: 21 May 2026

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Wei Wen, Chao Yue, Bingming Chen, Xianhui Tang, Yabo Wang, Wajid Ali Khattak, and Xin Song

Status: final response (author comments only)

RC1:
'Comment on egusphere-2026-2605', Anonymous Referee #1, 30 Jun 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).

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.

Line 112: Here the authors should clarify whether the two humidity regimes refer to soil moisture or to air humidity.

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?

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.

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?

Section 2.3: Please add the number of samples and replicates taken for each sample type.

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.

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.

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.

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?

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.

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?

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.

Technical corrections
Line 93: ‘etc.’ can be removed.

Line 189: Please use here and in the following sections the term ‘generalized linear models’ instead of ‘general linear models’.

Lines 234-235: Please specify these ecophysiological and environmental variables as they should differ from the species and relative humidity (RH).

Table 2: I recommend adding an explanation of all acronyms in the caption.

Figure 3: In the legend, there is a missing ‘d’ in ‘uncorrected’.
Citation: https://doi.org/10.5194/egusphere-2026-2605-RC1
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

Wei Wen, Chao Yue, Bingming Chen, Xianhui Tang, Yabo Wang, Wajid Ali Khattak, and Xin Song

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Wei Wen, Chao Yue, Bingming Chen, Xianhui Tang, Yabo Wang, Wajid Ali Khattak, and Xin Song

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Short summary

Hydrogen isotope discrepancies between plant stem and soil water extracted using cryogenic vacuum distillation challenge the assumption that plant stem water represents its source water. Using controlled experiments with multiple plant types, we tested whether this mismatch reflects biological processes or extraction biases. We found that the extraction method affects both stem and soil water. Correcting these biases removes the mismatch and improves the reliability of isotope-based methods.


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Identifying the underpinnings of δ2H discrepancies between plant stem and soil water: extraction-induced methodological artifacts versus biological fractionation effects

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Identifying the underpinnings of δ²H discrepancies between plant stem and soil water: extraction-induced methodological artifacts versus biological fractionation effects