the Creative Commons Attribution 4.0 License.
the Creative Commons Attribution 4.0 License.
| 21 May 2026
Technical note: Method effects on isotope-based inference of apple tree water sources
Abstract. Stable hydrogen and oxygen isotopes are widely used to trace plant water sources, but extraction and analytical choices can bias interpretation. Using an apple orchard on the Shandong Peninsula as a field example, we compared cryogenic vacuum extraction (CVE) with centrifugation, laser spectroscopy with isotope-ratio mass spectrometry (IRMS), and tested how apparent xylem δ²H offsets affect MixSIAR source apportionment. Across plant organs, water became progressively enriched from roots and branches to leaves and fruit. In paired branch samples measured by IRMS, centrifugation yielded δ²H values about 10‰ higher than CVE, whereas δ¹⁸O differences were small (≈1–2‰). Laser spectroscopy produced systematically higher δ¹⁸O than IRMS for plant extracts (typically 1–3‰), while non-plant source waters agreed closely between instruments. A δ²H offset correction shifted inferred uptake from shallow to deeper soil water. These results show that methodological choices can alter isotope-based inference of plant water sources and should be explicitly evaluated and reported.
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RC1: 'Comment on egusphere-2026-2258', Christina Hackmann, 18 Jun 2026
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The comment was uploaded in the form of a supplement: https://egusphere.copernicus.org/preprints/2026/egusphere-2026-2258/egusphere-2026-2258-RC1-supplement.pdfReplyCitation: https://doi.org/
10.5194/egusphere-2026-2258-RC1 -
RC2: 'Comment on egusphere-2026-2258', Anonymous Referee #2, 24 Jul 2026
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General comments:
This Technical Note examines a practical methodological problem in isotope-based studies of plant water sources. Using an apple orchard as a case study, the authors compare water-extraction and isotope-measurement methods and then ask whether the resulting differences affect source attribution in a mixing model. This is a suitable topic for a Technical Note in HESS. I also appreciate that the manuscript does not present either CVE or centrifugation as universally superior.
My main concern is not that the experiment needs to be repeated, but that the existing experiment and model analysis are not yet reported in enough detail. It is difficult to judge whether the method effects are consistent among paired samples, and the information provided is insufficient to reproduce Fig. 6. The sensitivity analysis suggests that a δ²H shift of roughly this magnitude can substantially change the model output, but it does not show that the adjusted scenario represents the true uptake depth of the trees. The paper could make a useful contribution if the authors provide fuller reporting of the paired comparisons, analytical quality control, and MixSIAR analysis, and narrow the conclusions accordingly. I therefore recommend major revision rather than additional field sampling.
Specific comments:
1. The approximately 10‰ difference in branch-water δ²H reported in Sect. 3.1 is one of the central empirical results, but the figures and text do not yet show how stable this difference is. Please report the actual number of paired samples and explain how material from the same branch was divided between CVE and centrifugation. It would help to show the difference for each pair and report a paired effect estimate with an uncertainty interval. This would be more informative than regression lines or pooled group means alone. If the samples span different plots, trees, or dates, please also indicate whether the method difference is broadly consistent among these groups.
2. The laser spectroscopy-IRMS comparison needs some additional methodological information. Please report how many samples of each plant material were measured with both instruments and whether the measurements used aliquots of the same extract or separately extracted samples. The Methods mention spectral screening for potential organic interference, but no screening results are presented, such as the number of flagged samples or whether any correction or exclusion was applied. The manuscript already uses the appropriately cautious phrase "consistent with sample-dependent spectral interference." Without further quality-control evidence, I would not take the interpretation beyond this wording.
3. The source classification shown in Fig. 6 is not clearly explained in the Methods. The actual soil-water sampling intervals were 0–20, 20–40, 40–60, 60–100, and 100–140 cm. Section 2.4 then defines the MixSIAR sources as 0–40, 40–80, and 80–140 cm soil water, together with precipitation, irrigation water, and groundwater, whereas Fig. 6 shows only <60 cm, >60 cm, and groundwater. Please explain the complete mapping from sampling intervals to MixSIAR sources and then to the categories displayed in Fig. 6. The 60 cm boundary needs particular attention because it is not a model-source boundary and it divides the stated 40–80 cm source. Please also explain how precipitation and irrigation water were treated and whether Fig. 6 aggregates posterior estimates or represents a separate model run with a different source structure. The supplement should provide the model inputs, sample sizes and means/variances for each source; Fig. 6 should also include credible intervals. The manuscript already commits to making the isotope data and MixSIAR input files public upon acceptance. Because these materials are needed to assess the central result, the data and analysis files or scripts should be made available during revision.
Technical correction:
Figure 1 defines LS and MS, whereas the main text generally uses IRMS, so the abbreviations are inconsistent between the text and the figure captions. Please use LS and MS/IRMS consistently in the text and figure captions and define them clearly at first use. The sample labels in Figs. 1–3 also mix plant material and extraction method, for example "Xylem," "Root," and "Branch-Centrifugal." A consistent naming scheme that identifies both plant material and extraction method would be clearer.
Citation: https://doi.org/10.5194/egusphere-2026-2258-RC2
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