the Creative Commons Attribution 4.0 License.
the Creative Commons Attribution 4.0 License.
| 26 Jun 2026
Technical note: Evaluation of a new cryogenic airtight vapor extraction (CRAVE) method for soil and plant water
Abstract. Accurate extraction of soil and plant water for stable isotope analysis remains a methodological challenge in ecohydrology, particularly due to isotopic biases introduced by heating or selective pore-water extraction in conventional techniques. This study developed and evaluated a cryogenic airtight vapor extraction (CRAVE) method from soil and vegetation samples at ambient temperature within a recirculating vapor-liquid pathway. This approach avoids heating-induced non-equilibrium effects and reduces matrix-dependent artifacts and organic contamination, thereby facilitating direct comparison of isotopic compositions between soil and plant water. The results demonstrate that CRAVE-derived isotopic signatures align with both cryogenic vacuum distillation (CVD) and suction lysimeter (SL) benchmarks. However, systematic deviations were observed based on specific matrix properties. For xylem water, the d2H offset between CRAVE and CVD was strongly modulated by gravimetric water content (dry-weight basis), with CVD exhibiting greater hydrogen isotope depletion under low-moisture conditions (< 0.8 g·g-1). For soils, the isotopic divergence between CRAVE and CVD was driven primarily by soil texture, with offsets increasing as clay content and depth increased (r = 0.82–0.94), where CVD-extracted bulk water became depleted progressively in both δ2H and δ18O relative to the mobile-capillary pool captured by SL and CRAVE. The Rayleigh-based framework provides a physically grounded means to reconstruct source-water isotope values from condensate measurements; its potential use for mobile–immobile partitioning should, however, be treated as a future application pending targeted validation. Overall, CRAVE represents a promising ambient-temperature extraction method for tracing water partitioning and source-uptake dynamics within the soil–plant–atmosphere continuum.
- Preprint
(1447 KB) - Metadata XML
- BibTeX
- EndNote
Status: final response (author comments only)
-
RC1: 'Comment on egusphere-2026-3419', Anonymous Referee #1, 30 Jul 2026
-
AC1: 'Reply on RC1', Xiuqiang Liu, 06 Sep 2026
Title: Technical note: Evaluation of a new cryogenic airtight vapor extraction (CRAVE) method for soil and plant water
Author(s): Xiuqiang Liu, Hongxiu Wang, Xi Chen, Ying Zhao, Magali F. Nehemy, and Jeffrey J. McDonnell
MS No.: egusphere-2026-3419
MS type: Technical note
Reviewer 1:
Comments on egusphere-2026-3419
Water stable isotope biogeochemistry and, by extension, its domain of applications (e.g. isotope ecohydrology) is limited by how water is collected from mineral media and living tissues. Aside from technical issues potentially leading to incomplete recovery, questions recurrently arise about the representativeness of the extracted water as a pool or source mobilized by some process (e.g. plant root water uptake). In recent years, new methods and novel declinations of well-established ones have been published, aiming at simplifying procedures or at targeting water sources to address the aforementioned issues.
Xiuqiang Liu & colleagues’ method, namely the cryogenic airtight vapor extraction (CRAVE), is another relevant and potentially useful addition; it should, for instance, remove potential effects associated with the heating of the samples, their texture, and water availability.
I found the manuscript to be well-written, although would require streamlining in some sections (e.g in the introduction) and editing (isotope and physics terminology) – please see my specific comments below.
I have one general issue with the use of the Rayleigh distillation framework in the specific context of this study and urge the authors to address it. I simply do not think Eq. (1) applies here. Rayleigh distillation only applies during condensation of a water vapor reservoir into liquid water – with or without the removal of the condensate from the vapor-liquid system during “rain-out”. The authors’ case is rather different, they have instead:
- one the one hand a liquid water sample (supposedly) equilibrating with its own produced but also recirculated – and potentially fractionated water vapor (stemming from incomplete removal of the vapor in the “condensation” U-tube) – inside the sample vial headspace, and,
- on the other hand, a (partial) inverse sublimation of the vapor inside the “condensation” U-tube.
The authors should also provide a comparison between the isotopic composition values of the water collected in the “condensation” U-tube and the values after correction with Eq. (3). This is missing for now and would help the readers to assess the magnitude of these corrections.
My specific comments follow below:
Response: We thank the reviewer for identifying the need to distinguish the fractionation and collection steps. The intended Rayleigh reservoir is the source liquid water in the sample vial, not the vapor in the cold trap. Gat (1996, Sect. 2.2, pp. 229–231; Fig. 2) derives the Rayleigh relation for progressive fractionating removal from a mixed reservoir, including liquid-to-vapor transfer. We revised Sect. 2.2 to define the vapor-to-liquid fractionation factor, the removed-water fraction, and the quantitative cumulative-capture assumption. Instead of deriving the relationship between the isotopic value of the residual water and that of the initial source water, we considered the isotopic mass balance among the residual water, removed water, and initial source water to establish the relationship between the isotopic value of the removed water and that of the initial source water. The liquid-water benchmark evaluates the resulting correction separately from gravimetric recovery. Across 33 liquid-water samples, mean raw trapped-water values were −125.8‰ for δ2H and −17.5‰ for δ18O; corrected means were −53.3‰ and −8.1‰, compared with direct source-water means of −51.5‰ and −7.6‰. This improvement supports the correction under the tested conditions, without independently proving each model assumption. The detailed response to L160 below addresses recirculation, deposition, and the distinction between the >98% recovery criterion and isotope agreement.
Change in manuscript:
“A Rayleigh-type working correction estimates the initial liquid-water isotope composition from the measured cumulative trap-water value, the fraction of water removed from the source sample, and temperature-dependent liquid–vapor equilibrium fractionation (Majoube, 1971; Gat, 1996).”
“To evaluate the isotopic fidelity of the CRAVE method, we first conducted a water-to-water test using 33 liquid precipitation samples. These samples covered a broad isotopic range, providing a fundamental benchmark to assess the system's performance in the absence of complex matrix effects (Table A2). For each sample, source-water mass, trap-water mass, raw trap-water isotope values (δc), and Eq. (3)-transformed values (δI) were retained. Direct source-water analysis provided the comparator.”
“For the liquid-water benchmark (n = 33; Table A2), mean CRAVE-corrected values were −53.3‰ for δ2H and −8.1‰ for δ18O, compared with −51.5‰ and −7.6‰, respectively, for the direct measurements. Before correction, mean trapped-water values were −125.8‰ for δ2H and −17.5‰ for δ18O. Application of Eq. (3) shifted these means by +72.5‰ and +9.5‰, respectively.”
“The mean CRAVE values were lower than the direct measurements by 1.7‰ for δ2H and 0.4‰ for δ18O. The δ18O difference was systematic (Wilcoxon signed-rank test, p = 0.002), whereas no statistically significant δ2H difference was detected (p = 0.06). Both mean differences were smaller than the corresponding laboratory 2σ uncertainties (±2‰ for δ2H and ±0.8‰ for δ18O). The corresponding agreement statistics were R2 = 0.95–0.97 and ICC = 0.97 (Fig. 3).”
Title
I encourage the authors note to use the adjective “new”. The presented method will not indefinitely be “new” but the title will remain as is…
Response: Agreed. The adjective “new” was removed, and “airtight” was replaced by “recirculating airflow” to describe the system more precisely.
Change in manuscript:
Title. Technical note: Evaluation of a cryogenic recirculating airflow vapor extraction (CRAVE) method for soil and plant water
Abstract
Please harmonize and simplify the isotopic terminology (“composition” vs “signature”, “δ” instead of “d”).
L25. “greater hydrogen isotope depletion” -> “lower isotope composition”.
L26. “g water (g of dry soil)-1”. You may delete mention of “dry-weight basis”
L27. “depth” is not explained. Why would depth be a factor here? Information about the experimental design is missing.
L27. “where CVD-extracted bulk water δ2H and δ18O progressively decreased…”
L28. “The Rayleigh-based framework” is not evident and should be shortly explained before.
L31. This method does not qualify to “ambient-temperature extraction” as it operates with liquid Nitrogen, but the reader might think otherwise from reading the abstract alone.
Response: All of these points were addressed in the revised abstract. Isotope terminology was harmonized around “isotopic composition” and δ notation; the dry-weight-basis wording was removed from the abstract unit; the experimental design now states that 42 soil samples were collected from 5–150 cm depth; the depth result is linked explicitly to deeper, more clay-rich samples; the Rayleigh-type mass-balance correction is briefly defined before its result is presented; and the former “ambient-temperature extraction” wording was replaced by an explicit description in which the sample vial remains near room temperature while the trap is cooled with liquid nitrogen. The wording for the CVD depth trend was also revised as suggested.
Change in manuscript:
Abstract. “Reliable characterization of soil and plant water isotopic composition depends on how water is recovered from complex matrices. We evaluated cryogenic recirculating airflow vapor extraction (CRAVE), in which the sample vial remains near room temperature while recirculated vapor is deposited as ice in a liquid-nitrogen-cooled trap and subsequently thawed for analysis. CRAVE was tested with 33 liquid-water samples, 42 soil samples collected from 5–150 cm depth, and 37 xylem samples, and was compared with cryogenic vacuum distillation (CVD) and, for soil, suction lysimeters (SL). A Rayleigh-type mass-balance correction, based on temperature-dependent liquid–vapor fractionation and isotope-mass balance, was used as a working transformation to estimate the initial water isotopic composition from the trapped water. In the liquid-water benchmark, the correction shifted mean trapped-water values from −126‰ to −53‰ for δ2H and from −17.5‰ to −8.1‰ for δ18O, close to the directly measured source-water means of −52‰ and −7.6‰, respectively. Method-dependent differences remained in environmental matrices. For xylem water, the CRAVE–CVD δ2H difference varied with gravimetric water content, with lower CVD δ2H values at low moisture content (<0.8 g g−1). For soil, CVD-extracted bulk-water δ2H and δ18O progressively decreased relative to CRAVE and SL in deeper, more clay-rich samples. CRAVE avoids direct heating of the sample matrix, yields a storable liquid-water sample after thawing, and provides a common extraction workflow for soil and plant water isotope analysis under the tested conditions.”
Introduction
L40. Consider citing Ceperley et al.
Ceperley, N., Gimeno, T. E., Jacobs, S. R., Beyer, M., Dubbert, M., Fischer, B., Geris, J., Holko, L., Kuebert, A., Le Gall, S., Lehmann, M. M., Llorens, P., Millar, C., Penna, D., Prieto, I., Radolinski, J., Scandellari, F., Stockinger, M., Stumpp, C., Tetzlaff, D., van Meerveld, I., Werner, C., Yildiz, O., Zuecco, G., Barbeta, A., Orlowski, N., and Rothfuss, Y.: Toward a common methodological framework for the sampling, extraction, and isotopic analysis of water in the Critical Zone to study vegetation water use. Wiley Interdisciplinary Reviews-Water, 11(4), https://www.doi.org/10.1002/wat2.1727, 2024.
Response: Thank you. Ceperley et al. (2024) was added to the opening methodological context, where we now frame sampling, extraction, and isotope analysis as a linked process chain and explicitly consider the water fraction represented by each method.
Change in manuscript:
“Stable isotopes of hydrogen and oxygen (2H, 18O) have been used to trace water movement in hydrological and ecohydrological processes (West et al., 2006; Meißner et al., 2014; Tao et al., 2025). By analyzing the isotopic compositions of xylem water, soil water, precipitation, streamflow and groundwater, researchers can identify water sources and quantify key processes such as evaporation, infiltration, and plant water uptake (Sprenger et al., 2017; Liu et al., 2025; Tao et al., 2025). While liquid water can be analyzed directly for its isotopic composition using isotope ratio mass spectrometry (IRMS) or laser spectroscopy (LS), water in soil and plant samples must first be extracted or measured in situ (Beyer et al., 2020), which remains a technically challenging process (Orlowski et al., 2016; Kocum et al., 2025). A recent community framework therefore recommends evaluating sampling, extraction, and isotope analysis as one process chain, with explicit attention to the water fraction represented by each method (Ceperley et al., 2024).”
L47. I personally question any study extracting water at temperatures that high! This is contrary to standard recommendations (see study of Ceperley et al.). You want to achieve inverse-sublimation of water, not pyrolysis!
Response: We clarified this point. The 200 °C value does not describe the CRAVE sample temperature; it refers to heating-based CVD and, in our paired comparison, to the heating-device set point. CRAVE itself does not heat the sample. The revised text now uses “inverse sublimation (deposition)” for the vapor-to-ice phase change in the liquid-nitrogen trap and explicitly states that the sample vial is maintained near room temperature.
Change in manuscript:
“Multiple extraction and equilibration techniques are available, each with method-specific sources of isotope fractionation, incomplete water recovery, organic contamination, or matrix-dependent bias (see Table A1 for a summary). Cryogenic vacuum distillation (CVD) remains widely used but requires dedicated vacuum and cryogenic equipment (Orlowski et al., 2016). Heating-based extractions such as CVD seek to limit incomplete recovery and associated fractionation by releasing tightly bound water (Wang et al., 2024). Elevated extraction temperatures can also introduce sample-specific isotope effects. For soils, high temperatures increase the risk of releasing isotopically distinct structural water from hydrous minerals (Orlowski et al., 2018; Wen et al., 2023; Duvert et al., 2024). In plant tissues, heating can enhance wood-water hydrogen exchange and alter δ2H even when total water recovery is high (Younger et al., 2024). Heating can also increase co-extraction of volatile and semi-volatile organic compounds from soil and plant matrices; these compounds may cause spectral interference in laser-based isotope analysis (Millar et al., 2018; Bowen et al., 2025).”
“The CRAVE system recovers a small fraction of sample water by continuously recirculating headspace vapor between a sample vial maintained near room temperature and a cryogenic trapping U-tube (Fig. 1). Water evaporates from the sample into the vial headspace and is transported by controlled airflow to the U-tube immersed in liquid nitrogen, where it undergoes inverse sublimation (deposition) directly from vapor to ice. After the extraction, the trapped ice is subsequently thawed to obtain a liquid-water sample for isotope analysis. The sample itself is not heated or subjected to vacuum extraction.”
“CVD was performed using a capillary-and-vial system at 200 °C (heating-device set point) for 30 min for soil and 24 min for xylem samples”
L55. Suction cups are not an alternative. On the one hand you have a destructive method and on the other hand an invasive one. The true alternatives are presented in the next paragraph and table 1 (e.g. centrifugation, direct soil vapor equilibration…).
Response: Agreed. Suction lysimeters are no longer presented as an “alternative” to bulk-water extraction. They are now described as serving a different purpose by collecting a tension-accessible soil-water fraction in situ.
Change in manuscript:
“Suction lysimeters serve a different purpose from bulk-water extraction by collecting a tension-accessible fraction of soil water in situ. These instruments are restricted to high water content soils and often fail to collect a sample below ~100 cm H2O water tension (Sprenger et al., 2015; Sprenger et al., 2019).”
L59. Please cite properly, either by year or first author name – please check journal guidelines.
Response: The citations in this section were standardized to author–year form and retained as EndNote citation fields in the revised manuscript.
L60. “These approaches offer high throughput and, in some cases, in situ monitoring capability when coupled with LS.” That cannot be the case, in situ observation are made where the experiments are conducted.
Response: We rephrased this comparison. The revised text distinguishes laboratory DVE, which can support batch analysis, from in situ probes, which provide repeated measurements at the installed location when coupled with laser spectroscopy.
Change in manuscript:
“Vapor-based methods include laboratory direct vapor equilibration (DVE), such as CO2 or H2 equilibration and liquid–vapor equilibration coupled to laser spectroscopy, as well as in situ equilibration probes (Wassenaar et al., 2008; Oerter et al., 2019; Marshall et al., 2020; Vadibeler et al., 2022; Pyschik et al., 2025). Laboratory DVE can support batch analysis, whereas in situ probes provide repeated measurements at the installed location when coupled with LS. Bag- or vial-based vapor sampling depends on vapor containment, storage duration, sampling conditions, and correction of organic-vapor interference in laser spectrometers (Mattei et al., 2019; Nehemy et al., 2019; Gralher et al., 2021; Millar et al., 2021; Iraheta et al., 2026). In situ equilibration probes reduce sample handling and can resolve high-frequency isotope dynamics, but they still require field-deployable laser systems and remain susceptible to interference from plant VOCs, especially in xylem applications (Herbstritt et al., 2023). Magh et al. (2022) proposed a discrete vapor-sampling technique that flushes dry air through samples to collect equilibrated vapor without requiring in situ instrumentation. In the reported tests, vapor storage duration affected the isotope values, particularly δ18O after several days. These approaches address different spatial and temporal scales. A complementary laboratory workflow would retain near-room-temperature vapor equilibration while producing a storable liquid-water sample for subsequent analysis.”
L63. You cannot compare in situ high frequency approaches with in labo low frequency ones: the latter measure repeatedly from the same sample, the latter includes spatial heterogeneities in isotopic composition.
Response: Agreed. We no longer treat laboratory batch methods and in situ high-frequency measurements as directly comparable. The revised introduction describes their different sampling modes and explicitly states that these approaches address different spatial and temporal scales.
Change in manuscript:
“Vapor-based methods include laboratory direct vapor equilibration (DVE), such as CO2, H2, N2, and dry air equilibration, as well as in situ equilibration probes, coupled with LS(Wassenaar et al., 2008; Oerter et al., 2019; Marshall et al., 2020; Vadibeler et al., 2022; Pyschik et al., 2025). Laboratory DVE can support batch analysis, whereas in situ probes provide repeated measurements at the installed location when coupled with LS. Bag- or vial-based vapor sampling depends on vapor containment, storage duration, sampling conditions, and correction of organic-vapor interference in laser spectrometers (Mattei et al., 2019; Nehemy et al., 2019; Gralher et al., 2021; Millar et al., 2021; Iraheta et al., 2026). In situ equilibration probes reduce sample handling and can resolve high-frequency isotope dynamics, but they still require field-deployable laser systems and remain susceptible to interference from plant VOCs, especially in xylem applications (Herbstritt et al., 2023). Magh et al. (2022) proposed a discrete vapor-sampling technique that flushes dry air through samples to collect equilibrated vapor without requiring in situ instrumentation. In the reported tests, vapor storage duration affected the isotope values, particularly δ18O after several days. These approaches address different spatial and temporal scales. A complementary laboratory workflow would retain near-room-temperature vapor equilibration while producing a storable liquid-water sample for subsequent analysis.”
Table 1 should be streamlined, e.g. do not write sentences in full, rather use bullet points. Waasenaar et al. DVE method is not to be pooled together with the rest…The rest of the “equilibration methods”, the in situ one, are not 1:1 comparable to the rest – please see my comment above – and should be treaded differently. I would actually not even bother to cite them here and in the intro at all.
Response: We streamlined the method overview and moved the broad comparison table to Appendix A. In the text, laboratory DVE, in situ equilibration probes, and discrete vapor-sampling approaches are now described separately rather than pooled as equivalent methods.
Change in manuscript:
“Table A1. Comparison of different water extraction methods for soil and plant isotope analysis”
Azeotropic distillation
Solvent-assisted distillation
Soil
Low extraction temperature; high recovery for moist samples
Organic/metal reagents; complex handling; contamination risk
Allison et al. (1983); Revesz and Woods (1990); Thorburn et al. (1993); Kelln et al. (2001); Figueroa-Johnson et al. (2007); Zhu et al. (2014)
Mechanical squeezing or centrifugation
Pressure or centrifugal force
Soil / plant
No heating; rapid recovery of readily mobile water
Incomplete recovery in dry or clay-rich matrices; operationally selective
Eichler (1966); Edmunds and Bath (1976); Jusserand (1980); Kelln et al. (2001); Moncur et al. (2013); Millar et al. (2018); Adams et al. (2020); Wen et al. (2023); Nehemy et al. (2025)
Cavitron extraction
Centrifugal tension applied to xylem conduits
Plant
Rapid; no heating; targets functional xylem water
Plant-specific; geometry and vessel continuity constrain yield; specialized equipment
Cochard (2002); Cochard et al. (2005); Barbeta et al. (2022); He et al. (2023); Duvert et al. (2024); Wang et al. (2024)
Microwave water extraction
Microwave heating and vapor recovery
Plant
Rapid; comparatively simple apparatus
Heating must be controlled; uneven heating or incomplete recovery can bias results
Munksgaard et al. (2014); Orlowski et al. (2016); Millar et al. (2018)
Cryogenic vacuum distillation
Vacuum heating followed by cryogenic trapping
Soil / plant
Broad matrix applicability; potentially high recovery
Equipment-intensive; heat-, mineral-, and wood-related exchange; co-extracted organics
West et al. (2006); Koeniger et al. (2011); Orlowski et al. (2013); Jones et al. (2017); Millar et al. (2018); Chen et al. (2020); Wen et al. (2021); Wu et al. (2021); Zeng et al. (2021); Diao et al. (2022); Liu et al. (2022); Wang et al. (2024); Cai et al. (2025); Liu et al. (2025); Nehemy et al. (2025)
Laboratory direct vapor equilibration (DVE)
Liquid–vapor equilibration followed by vapor measurement
Soil / plant
No liquid extraction; compatible with batch laboratory analysis
Requires equilibration control; sensitive to leakage, storage, humidity, and VOC interference
Wassenaar et al. (2008); Rothfuss et al. (2013); Volkmann and Weiler (2014); Hendry et al. (2015); Orlowski et al. (2016); Gaj and McDonnell (2019); Mattei et al. (2019); Wang et al. (2020); (Gralher et al., 2021); Seeger and Weiler (2021); Kühnhammer et al. (2022); Magh et al. (2022); Vadibeler et al. (2022); (Ring et al., 2024); Pyschik et al. (2025); Sprenger et al. (2025); Vadibeler et al. (2025)
Suction / tension lysimeter
Applied suction through a porous interface
Soil
In situ liquid sample; no heating; repeated sampling possible
Samples an operationally tension-accessible fraction; recovery depends on texture and wetness
Anderson et al. (1997); Kosugi and Katsuyama (2004); Yano et al. (2006); Kabeya et al. (2007); Li et al. (2007); Weihermüller et al. (2007); Sprenger et al. (2015); Floriancic et al. (2024); Costanza et al. (2025); Liu et al. (2025); Xu et al. (2025); Zhang et al. (2025)
Method positioning and Methods
L74-104. Text is not well structured despite the attempt to list the features of the presented method. Please streamline this and hierarchize the information in order of importance.
Response: The entire method-positioning passage was reorganized into a short hierarchy: what CRAVE recovers, how the vapor is trapped, how the Rayleigh-type correction is used, how collection efficiency functions as QA/QC, and how the method is evaluated. The previous (i)–(iii) list was removed.
Change in manuscript:
“Here, we evaluate cryogenic recirculating airflow vapor extraction (CRAVE), which recovers a measured fraction of sample water by depositing headspace vapor as ice in a liquid-nitrogen-cooled trap and subsequently thawing it for stable isotope analysis. CRAVE shares with vapor-equilibration methods the use of liquid–vapor isotope relationships while the sample vial remains near room temperature (Wassenaar et al., 2008; Volkmann and Weiler, 2014; Seeger and Weiler, 2021). Its distinguishing operational feature is that a measured fraction of vapor is trapped as ice and recovered as a storable liquid-water sample for subsequent isotope analysis.
CRAVE circulates headspace vapor through a liquid-nitrogen-cooled trap, where water vapor is deposited as ice and subsequently thawed. The thawed trap water can then be analyzed by IRMS or laser spectroscopy. A desiccant-buffered vent limits pressure excursions and reduces atmospheric water-vapor ingress (Millar et al., 2021; Herbstritt et al., 2024). The loop is pressure-buffered rather than fully airtight, and its temperature and humidity conditions must be controlled and documented as in other vapor-based isotope measurements (Bailey et al., 2015; Beyer et al., 2020). The resulting liquid sample can be transferred to sealed glass vials for storage, repeated analysis, or measurement by either IRMS or LS.
A Rayleigh-type working correction estimates the initial liquid-water isotope composition from the measured cumulative trap-water value, the fraction of water removed from the source sample, and temperature-dependent liquid–vapor equilibrium fractionation (Majoube, 1971; Gat, 1996).
Overall, CRAVE is designed to recover a measured fraction of water from soil or plant samples. The initial isotope composition is estimated from the trap-water value, removed-water fraction, and equilibrium-fractionation term described above. The evaluation therefore includes sample-vial temperature monitoring and gravimetric checks of cumulative water recovery (Mattei et al., 2019; Gralher et al., 2021). We evaluated CRAVE with liquid-water standards and paired soil and xylem samples, compared corrected and uncorrected values with direct standards, CVD, and, for soil, suction-lysimeter water, and examined whether method offsets varied with depth, texture, and water content.”
L75, L82. “sublimating”, not “condensing”
Response: Corrected. The revised manuscript uses “deposition” or “inverse sublimation” for the vapor-to-ice process and reserves “thawing” for recovery of the liquid sample.
L74-79. Not clear at all what “quantified” and “measured” mean here.
Response: We clarified the wording. “Measured fraction” now refers to the fraction of source water removed from the sample vial and recovered through the trapping workflow, with the removed-water fraction quantified from water-mass accounting rather than used as an undefined descriptor.
Change in manuscript:
“Here, we evaluate cryogenic recirculating airflow vapor extraction (CRAVE), which recovers a measured fraction of sample water by depositing headspace vapor as ice in a liquid-nitrogen-cooled trap and subsequently thawing it for stable isotope analysis. CRAVE shares with vapor-equilibration methods the use of liquid–vapor isotope relationships while the sample vial remains near room temperature (Wassenaar et al., 2008; Volkmann and Weiler, 2014; Seeger and Weiler, 2021). Its distinguishing operational feature is that a measured fraction of vapor is trapped as ice and recovered as a storable liquid-water sample for subsequent isotope analysis.”
“Overall, CRAVE is designed to recover a measured fraction of water from soil or plant samples. The initial isotope composition is estimated from the trap-water value, removed-water fraction, and equilibrium-fractionation term described above. The evaluation therefore includes sample-vial temperature monitoring and gravimetric checks of cumulative water recovery (Mattei et al., 2019; Gralher et al., 2021). We evaluated CRAVE with liquid-water standards and paired soil and xylem samples, compared corrected and uncorrected values with direct standards, CVD, and, for soil, suction-lysimeter water, and examined whether method offsets varied with depth, texture, and water content.”
“The sample vial containing the residual material is reweighed (Gssv′), and the mass of water removed from the sample is calculated from the sample-vial mass loss as Gloss = Gssv − Gssv′. Gravimetric collection efficiency (η) is calculated as η = Gc/Gloss × 100%. A collection-efficiency threshold of >98% is used as a quality assurance/quality control (QA/QC) criterion. Lower values indicate possible leakage or incomplete cryogenic trapping, whereas values >100% indicate possible ingress of atmospheric moisture or weighing uncertainty.
L78. “However” is not correct. Also the three items (i)-(iii) do not add structuration and do not relate as is to the text preceding them. Please streamline this.
Response: Agreed. The “However” transition and the three-item (i)–(iii) structure were removed. The method is now presented as a continuous sequence of operational features and evaluation steps.
L83. A desiccant cannot trap VOCs. A solid phase extractor may do the job well, depending on the nature of the VOC (e.g. methanol, ethanol).
Response: Agreed. The desiccant is no longer described as trapping VOCs. Its role is limited to pressure buffering and minimizing ingress of atmospheric water vapor.
Change in manuscript:
“CRAVE circulates headspace vapor through a liquid-nitrogen-cooled trap, where water vapor is deposited as ice and subsequently thawed. The thawed trap water can then be analyzed by IRMS or laser spectroscopy. A desiccant-buffered vent limits pressure excursions and reduces atmospheric water-vapor ingress (Millar et al., 2021; Herbstritt et al., 2024). The loop is pressure-buffered rather than fully airtight, and its temperature and humidity conditions must be controlled and documented as in other vapor-based isotope measurements (Bailey et al., 2015; Beyer et al., 2020). The resulting liquid sample can be transferred to sealed glass vials for storage, repeated analysis, or measurement by either IRMS or LS.”
L85. Again, you are comparing apples with oranges and the statement is rather unfair to the in situ methods: condensation is not inherent to the in situ family of methods after the collection point and can be largely avoided. Condensation is, on the other hand, a major problem in water vapor continuous analysis.
Response: We revised this comparison to avoid implying that condensation is intrinsic to in situ equilibration methods. The introduction now distinguishes laboratory DVE, in situ probes, and discrete vapor sampling by their sampling mode and scale, while Sect. 4.2 focuses on the specific operational difference that CRAVE recovers a storable liquid-water endpoint.
Change in manuscript:
“Vapor-based methods include laboratory direct vapor equilibration (DVE), such as CO2 or H2 equilibration and liquid–vapor equilibration coupled to laser spectroscopy, as well as in situ equilibration probes (Wassenaar et al., 2008; Oerter et al., 2019; Marshall et al., 2020; Vadibeler et al., 2022; Pyschik et al., 2025). Laboratory DVE can support batch analysis, whereas in situ probes provide repeated measurements at the installed location when coupled with LS. Bag- or vial-based vapor sampling depends on vapor containment, storage duration, sampling conditions, and correction of organic-vapor interference in laser spectrometers (Mattei et al., 2019; Nehemy et al., 2019; Gralher et al., 2021; Millar et al., 2021; Iraheta et al., 2026). In situ equilibration probes reduce sample handling and can resolve high-frequency isotope dynamics, but they still require field-deployable laser systems and remain susceptible to interference from plant VOCs, especially in xylem applications (Herbstritt et al., 2023). Magh et al. (2022) proposed a discrete vapor-sampling technique that flushes dry air through samples to collect equilibrated vapor without requiring in situ instrumentation. In the reported tests, vapor storage duration affected the isotope values, particularly δ18O after several days. These approaches address different spatial and temporal scales. A complementary laboratory workflow would retain near-room-temperature vapor equilibration while producing a storable liquid-water sample for subsequent analysis.”
“CRAVE also separates water recovery from isotope measurement. Direct vapor equilibration infers liquid water isotope composition from equilibrated vapor measured by laser spectroscopy (Wassenaar et al., 2008; Gralher et al., 2021). In discrete vapor sampling, the isotopic stability of stored vapor depends on sampling and storage conditions (Iraheta et al., 2026). CRAVE instead deposits vapor as ice in the cryogenic trap, recovers it as liquid water after thawing, and records the recovered mass. The recovered liquid water can be stored, remeasured, or analyzed by either laser spectroscopy or IRMS, while the recovered water mass enters the working Rayleigh-type correction. This liquid endpoint removes the need to keep the extraction apparatus coupled directly to a laser analyzer and permits repeated measurements from the same recovered sample.”
L93. Which method uses “empirical post hoc corrections”? Also CRAVE uses a rayleigh distillation framework, for which α is determined empirically and a posteriori. So I don’t think the statement is valid.
Response: The statement contrasting CRAVE with “empirical post hoc corrections” was removed. Sect. 2.2 now explicitly defines α as the vapor-to-liquid equilibrium isotope ratio, Rv/Rl, calculated from the sample-vial temperature using the published equilibrium relationship of Majoube (1971). This is distinct from assigning a separate fitted fractionation factor to the cryogenic deposition step. The correction is evaluated against direct liquid-water measurements.
Change in manuscript:
“A Rayleigh-type working correction estimates the initial liquid-water isotope composition from the measured cumulative trap-water value, the fraction of water removed from the source sample, and temperature-dependent liquid–vapor equilibrium fractionation (Majoube, 1971; Gat, 1996).”
L95. “QA/QC” is not defined.
Response: Corrected. QA/QC is now expanded as quality assurance/quality control in Sect. 2.1.
Change in manuscript:
“A collection-efficiency threshold of >98% is used as a quality assurance/quality control (QA/QC) criterion.”
L101. “leakage pathways”? “leakage” should be enough.
Response: Corrected. The wording was simplified to “leakage.”
L102. Because of the somewhat chaotic nature of the text L74-104, I don’t know what “finally” exactly refers to. What came first, then second etc. before “finally”?
Response: The ambiguous “finally” wording was removed together with the former list structure. The evaluation is now stated directly in the final paragraph of the method-positioning section.
Change in manuscript:
“Overall, CRAVE is designed to recover a measured fraction of water from soil or plant samples. The initial isotope composition is estimated from the trap-water value, removed-water fraction, and equilibrium-fractionation term described above. The evaluation therefore includes sample-vial temperature monitoring and gravimetric checks of cumulative water recovery (Mattei et al., 2019; Gralher et al., 2021). We evaluated CRAVE with liquid-water standards and paired soil and xylem samples, compared corrected and uncorrected values with direct standards, CVD, and, for soil, suction-lysimeter water, and examined whether method offsets varied with depth, texture, and water content.”
L110. Water does not condensate in liquid N, it inversely sublimates (sometimes referred as to “deposition”). This is an important terminology point.
Response: Corrected throughout Sect. 2.1. The phase change in the liquid-nitrogen trap is now described explicitly as inverse sublimation (deposition) from vapor to ice.
Change in manuscript:
“The CRAVE system recovers a small fraction of sample water by continuously recirculating headspace vapor between a sample vial maintained near room temperature and a cryogenic trapping U-tube (Fig. 1). Water evaporates from the sample into the vial headspace and is transported by controlled airflow to the U-tube immersed in liquid nitrogen, where it undergoes inverse sublimation (deposition) directly from vapor to ice. The trapped ice is subsequently thawed to obtain a liquid-water sample for isotope analysis. The sample itself is not heated or subjected to vacuum extraction.”
Figure 1. Schematic and experimental setup of the cryogenic recirculating airflow vapor extraction (CRAVE) system: (a) schematic diagram illustrating the recirculating airflow loop and cryogenic vapor-deposition pathway; (b) photograph of the laboratory-based setup for extracting pre-packaged soil and plant samples
L114. This is a “deposition U-tube”, not a “condensation U-tube”.
Response: Corrected. The component is now consistently termed a “deposition U-tube.”
Change in manuscript:
“The system consists of four main components: (1) a sample vial containing the soil, plant, or liquid-water sample; (2) a deposition U-tube placed in a liquid-nitrogen flask to trap water vapor as ice; (3) a recycling pump (GS150, Geesiniger, Germany) that drives continuous vapor circulation through the loop; and (4) a desiccant-buffered vent (drying tube) that provides pressure buffering while minimizing ingress of atmospheric water vapor. All tubing, needles, and connections are sealed with PTFE (Teflon) septa to minimize isotopic exchange with the atmosphere. A schematic of the CRAVE system and vapor-flow pathway is shown in Fig. 1.”
L120ff. “[deposition] vials (Gcv) [are] pre-weighed”. This is a general statement – did not happen once in the past. Same for “soil and plant materials [are] placed immediately” etc.
Response: Agreed. General protocol statements in Sect. 2.1 were converted to the present tense.
L129. What is meant by “pressure equilibrium”? Do you want to say “to avoid under/overpressure in the closed loop system due to changes in temperature overtime”? Either way, please add details in the text.
Response: We added the requested physical explanation. The drying tube provides a pressure-buffered connection to the atmosphere and prevents substantial under- or overpressure as gas temperature and vapor content change during pumping and cryogenic trapping; the desiccant minimizes atmospheric water-vapor ingress during this equalization.
Change in manuscript:
“The sample vial is connected to the deposition U-tube, which is immersed in liquid nitrogen so that the nitrogen level covers approximately one-third of the U-tube height. The opposite end of the U-tube is connected to the inlet of the recycling pump, and the pump outlet is connected back to the sample vial to drive recirculating airflow. The drying tube provides a pressure-buffered connection to the atmosphere, preventing substantial under- or overpressure as gas temperature and vapor content change during pumping and cryogenic trapping; the desiccant minimizes atmospheric water-vapor ingress during this pressure equalization.”
L131. “Cryogenic condensation” is “inverse sublimation”.
Response: Corrected. “Cryogenic condensation” was replaced by “cryogenic deposition.”
L132. How was the protocol “optimized”? Please add details.
Response: The word “optimized” was removed because no separate optimization experiment was performed. The revised protocol instead states the observed operational duration ranges and defines the termination criterion from the real-time mass-loss plateau.
Change in manuscript:
“Once the system is assembled, the recycling pump is activated to initiate vapor circulation and cryogenic deposition. Extraction duration is adjusted according to sample type and the real-time mass-loss response rather than a fixed optimized duration. Liquid-water standards typically require 0.5–1 h, whereas soil and plant samples require 0.5–3 h. The sample vial remains on a high-precision electronic balance throughout extraction, and the extraction is terminated when its mass reaches a stable plateau, defined as no further measurable mass loss for at least 15–20 min. This operational criterion allows extraction duration to vary with soil texture, vegetation type, and water content. The sample vial is maintained near room temperature (ca. 20–25 °C), and its internal temperature is monitored continuously using a high-precision thermistor (ON-403-PP, Omega Engineering, USA; accuracy ±0.1 °C) connected to a four-channel data logger (OM-CP-QUADTHERM-A1, Omega Engineering, USA; resolution 0.01 °C).”
L150. Water pools of what? Please explain / detail.
Response: Here, “water pools” referred to water held in different soil pores or plant tissue compartments. We have removed this broad term and clarified that collection efficiency measures the recovery of water removed from the sample vial. Gravimetric collection efficiency ( ) exceeded 98% for all samples. We therefore treated cumulative recovery of the removed water as approximately complete in the isotope mass-balance calculation.
Change in manuscript:
“The sample vial containing the residual material is reweighed (Gssv′), and the mass of water removed from the sample is calculated from the sample-vial mass loss as Gloss = Gssv − Gssv′. Gravimetric collection efficiency (η) is calculated as η = Gc/Gloss × 100%. A collection-efficiency threshold of >98% is used as a quality assurance/quality control (QA/QC) criterion. Lower values indicate possible leakage or incomplete cryogenic trapping, whereas values >100% indicate possible ingress of atmospheric moisture or weighing uncertainty.”
L160. I have a problem with using this equation in this particular context. Rayleigh distillation only applies during condensation of a water vapor reservoir into liquid water – with or without the removal of the condensate from the vapor-liquid system. Your case is rather different, you have:
- one the one hand a liquid water sample (supposedly) equilibrating with its own produced but also recirculated – and potentially fractionated water vapor (during incomplete removal of the vapor in the “consensation” U-tube) – inside the sample vial headspace, and
- On the other hand (partial) inverse sublimation of the vapor inside the “condensation” U-tube.
Therefore, “reconstruction” of the “isotopic compositions of soil and plant water derived from the condensed vapor” is not granted with the Rayleigh distillation framework.
On a side note, all parameters and variables in Eq. (1) (α, δ, f) are unitless, therefore the factor “1000” is unnecessary. It should just read “1”.
Response: We agree that the two phase-transfer steps must be distinguished. Gat (1996, pp. 229–231; Fig. 2) derives the Rayleigh relation for progressive fractionating removal from a mixed reservoir, including liquid-to-vapor transfer; it is not restricted to vapor condensation. Equation (1) describes the isotope evolution of the remaining source water, with α = Rv/Rl < 1 and f = Gloss/Gw,0 the fraction removed. Gat’s remaining fraction corresponds to 1 − f in our notation. These definitions are now explicit in Sect. 2.2.
Gat (1996)
The model assumes an effectively mixed source-water reservoir and vapor removal at a constant liquid–vapor equilibrium fractionation factor for each extraction, with negligible additional kinetic fractionation, external moisture exchange, and changes in loop vapor storage. The trapped water is removed from further exchange with the source. Recirculating carrier gas therefore does not by itself invalidate the Rayleigh reservoir description; returning water vapor matters if its amount or isotope composition materially affects the net removal process.
Equations (2) and (3) additionally assume that the pooled trap water represents the cumulative vapor removed. In the quantitative-capture limit, vapor-to-ice deposition followed by complete recovery and pooling of the ice conserves the isotope ratio of the total removed vapor. No additional ice–vapor equilibrium fractionation factor is required for that complete accumulated product. This is a mass-balance statement, not an assumption that the warm sample vial and liquid-nitrogen trap are in mutual isotopic equilibrium. We have made this distinction explicit around Eq. (2).
Cumulative gravimetric recovery, defined in Sect. 2.1, is a separate check and does not establish complete capture on every circulation pass. Likewise, agreement after isotope correction supports the combined model under the tested conditions but cannot identify each assumption separately. We report the before-and-after isotope comparison in Sect. 3.1 and Table A2, and address the residual corrected-value offset in our response to Fig. 3 below.
We did not change the factor 1000 to 1. The isotope values in the manuscript are expressed numerically in per mil (‰). By definition, R/Rstd = 1 + δ/1000; therefore, when the Rayleigh relationship is written directly in per-mil δ notation, the corresponding algebra contains δ + 1000. Replacing 1000 by 1 would only be correct if δ were expressed as an unscaled fractional deviation rather than in ‰. This convention is now stated immediately after Eq. (3).
Change in manuscript:
“A Rayleigh-type working correction estimates the initial liquid-water isotope composition from the measured cumulative trap-water value, the fraction of water removed from the source sample, and temperature-dependent liquid–vapor equilibrium fractionation (Majoube, 1971; Gat, 1996).”
“The initial sample-water isotopic composition is estimated using a Rayleigh-type model of progressive vapor removal from the source-water reservoir, following Gat (1996):”
“The model assumes an effectively mixed source-water reservoir, vapor removal at a constant liquid–vapor equilibrium fractionation factor for each extraction, irreversible removal of trapped water, and negligible external moisture exchange and changes in loop vapor storage. Additional kinetic or transport fractionation is neglected. The sample vial and cryogenic trap are not treated as a two-phase system in mutual isotopic equilibrium.”
“Under quantitative cumulative capture (Gc = Gloss), the pooled trap water represents all vapor removed from the source. The natural-abundance isotope mass balance between the initial water reservoir, the residual water, and the cumulatively trapped water is:”
“where δc is the isotopic composition of the total accumulated ice in the deposition U-tube after complete thawing. Under quantitative cumulative capture, it represents the mass-weighted mean of the removed vapor, not its instantaneous composition. An additional vapor-to-ice fractionation correction is not required in this limit because the complete accumulated product conserves the isotope ratio of the removed vapor.”
“All δ values in Eqs. (1)–(3) are numerical values in per mil (‰).”
“For the water-to-water validation experiment, the original liquid-water samples and the cryogenically trapped water were analyzed using a Los Gatos Research liquid-water Off-Axis Integrated-Cavity Output Spectroscopy (OA-ICOS) instrument and post-processed using LIMS for Lasers. A bracketed two-standard run template was used, with standards analyzed before and after every five samples, to normalize the data and minimize instrumental drift and memory effects following Wassenaar et al. (2014). Each sample and standard was measured using eight injections; the first three injections were discarded and the last five were averaged. An in-house calibrated control (δ2H = −135.9‰; δ18O = −17.0‰; δ17O = −8.8‰) was analyzed as a sample in each run to monitor accuracy and reproducibility. Two in-house standards spanning a broad natural-water isotope range were used for normalization: an enriched standard (δ2H = −0.6‰; δ18O = −0.5‰; δ17O = −2.0‰) and a depleted standard (δ2H = −209.6‰; δ18O = −27.3‰; δ17O = −14.1‰). The laboratory 2σ uncertainties were ±2‰ for δ2H, ±0.8‰ for δ18O, and ±0.5‰ for δ17O; corresponding laboratory reproducibilities were ±1.0‰ for δ2H and ±0.2‰ for δ18O and δ17O. All isotope values are reported in per mil (‰) relative to the VSMOW–SLAP scale.”
“To evaluate the isotopic fidelity of the CRAVE method, we first conducted a water-to-water test using 33 liquid precipitation samples. These samples covered a broad isotopic range, providing a fundamental benchmark to assess the system's performance in the absence of complex matrix effects (Table A2). For each sample, source-water mass, trap-water mass, raw trap-water isotope values (δc), and Eq. (3)-transformed values (δI) were retained. Direct source-water analysis provided the comparator.”
“For the liquid-water benchmark (n = 33; Table A2), mean CRAVE-corrected values were −53.3‰ for δ2H and −8.1‰ for δ18O, compared with −51.5‰ and −7.6‰, respectively, for the direct measurements. Before correction, mean trapped-water values were −125.8‰ for δ2H and −17.5‰ for δ18O. Application of Eq. (3) shifted these means by +72.5‰ and +9.5‰, respectively.”
L180. Please provide the definition of “immobile” versus “mobile” water pools in terms of matric potential.
Response: We added an operational definition tied to matric potential. “Mobile soil water” is the water collected by suction lysimeters under the site- and depth-specific applied suction limits derived from the water-retention curves (approximately 40–90 kPa in this dataset). Water retained beyond the corresponding lysimeter limit is termed the operationally less mobile fraction. We also distinguish CVD bulk-water recovery and the CRAVE vapor-exchange extract from this hydraulically defined SL fraction.
Change in manuscript:
“Extraction method and soil properties jointly determine the isotope composition recovered from soil samples (Adams et al., 2020). In this study, mobile soil water is defined operationally as water collected by suction lysimeters when the applied suction magnitude remained below the maximum value derived from the water retention curve for each site and depth. These limits ranged from approximately 40 to 90 kPa (Liu et al., 2026). Water retained beyond the corresponding lysimeter limit is termed the operationally less mobile fraction. This definition follows the hydraulic accessibility framework used to distinguish mobile and bulk soil water (Sprenger et al., 2018; Sprenger et al., 2019). CVD targets recoverable bulk water in the homogenized soil sample and therefore covers a broader range of retention states than SL. CRAVE samples a third operational domain. The sample vial remained at 20 to 25 °C, whereas the deposition U-tube was immersed in liquid nitrogen, creating a temperature difference greater than 200 °C. Water was transferred through headspace vapor exchange and cryogenic deposition without direct heating of the sample matrix or vacuum extraction. Because CRAVE imposes no defined matric suction, its product is classified here as a vapor exchange extract. Under the tested conditions, its mean soil water isotope composition was closer to SL water than to CVD bulk water (Fig. 2; Table A3).”
L193. Did you sample pairs in unicates (no replicates)?
Response: Yes. The comparison used one matched CRAVE–CVD subsample pair per field sample rather than technical replicate extractions of the same subsample. Soil samples were homogenized and divided into matched CRAVE and CVD subsamples; xylem material from the same branch segment was likewise divided into matched subsamples. The dataset therefore comprises 42 soil pairs and 37 xylem pairs.
Change in manuscript:
“Bulk soil and xylem samples were compared using matched subsamples. Each homogenized bulk-soil sample was divided into two subsamples for CRAVE and CVD, while SL-accessible water was collected from the corresponding soil. Xylem material from the same branch segment was likewise divided into matched CRAVE and CVD subsamples. The comparison comprised 42 soil pairs and 37 xylem pairs (79 pairs in total).”
L194. Again, 200 degC is much too high for soil already, but not acceptable for plant organs or tissues. Are you sure we are talking about the actual sample temperature or the set point temperature of the heating device (, which may significantly differ)??
Response: The 200 °C value is the heating-device set point used for the CVD protocol, not a direct measurement of the internal sample temperature. We revised the Methods to state this explicitly. The actual sample temperature during CVD was not measured independently in this experiment.
Change in manuscript:
“CVD was performed using a capillary-and-vial system at 200 °C (heating-device set point) for 30 min for soil and 24 min for xylem”
Results
L232ff. Parts of this section should be moved to the material and methods.
Response: Agreed. The liquid-water validation design and the description of what was retained for each sample were moved to Sect. 2.7, leaving Sect. 3.1 to report the results.
L238. The reader cannot verify the statement “These metrics indicate that the Rayleigh-based equilibrium correction (Eq. 3) accurately reconstructs the source water composition by neutralizing effectively fractionation during vapor circulation and condensation.”) himself without the authors providing a comparison with and without “reconstruction” with Eq. 3. Please provide the corresponding data in the form of a table or figure.
Response: Sect. 3.1 and Table A2 now provide the requested before-and-after comparison: raw cumulative trap-water values, Eq. (3)-corrected values, and direct source-water measurements. We also revised the Conclusion to state that the correction substantially reduced the source-water offset.
Change in manuscript:
“To evaluate the isotopic fidelity of the CRAVE method, we first conducted a water-to-water test using 33 liquid precipitation samples. These samples covered a broad isotopic range, providing a fundamental benchmark to assess the system's performance in the absence of complex matrix effects (Table A2). For each sample, source-water mass, trap-water mass, raw trap-water isotope values (δc), and Eq. (3)-transformed values (δI) were retained. Direct source-water analysis provided the comparator.”
“For the liquid-water benchmark (n = 33; Table A2), mean CRAVE-corrected values were −53.3‰ for δ2H and −8.1‰ for δ18O, compared with −51.5‰ and −7.6‰, respectively, for the direct measurements. Before correction, mean trapped-water values were −125.8‰ for δ2H and −17.5‰ for δ18O. Application of Eq. (3) shifted these means by +72.5‰ and +9.5‰, respectively.”
“The mean CRAVE values were lower than the direct measurements by 1.7‰ for δ2H and 0.4‰ for δ18O. The δ18O difference was systematic (Wilcoxon signed-rank test, p = 0.002), whereas no statistically significant δ2H difference was detected (p = 0.06). Both mean differences were smaller than the corresponding laboratory 2σ uncertainties (±2‰ for δ2H and ±0.8‰ for δ18O). The corresponding agreement statistics were R2 = 0.95–0.97 and ICC = 0.97 (Fig. 3).”
“Table A2. Sample information and isotope values (δ2H and δ18O) of liquid water and cryogenic recirculating airflow vapor extraction (CRAVE) samples.”
W1
6.81
0.27
-19.11
-4.32
-96.80
-14.59
-22.24
-5.16
W2
13.14
0.15
-38.44
-6.45
-113.88
-15.10
-40.32
-5.61
W3
7.88
0.63
-30.95
-4.85
-99.23
-13.92
-27.53
-4.81
W4
10.96
0.35
-20.20
-4.00
-94.64
-13.67
-19.81
-4.21
W5
6.79
0.37
-17.53
-3.33
-89.89
-13.21
-16.44
-3.96
W6
7.08
0.23
-27.56
-5.01
-103.07
-14.00
-29.23
-4.57
W7
10.02
0.22
-16.05
-3.87
-96.41
-13.57
-22.58
-4.22
W8
8.25
0.80
-76.03
-10.50
-141.56
-20.17
-72.40
-11.02
W9
8.43
0.68
-18.88
-4.31
-90.19
-14.19
-15.99
-4.88
W10
11.68
0.50
-47.45
-7.30
-119.17
-16.63
-46.81
-7.27
W11
16.16
0.40
-39.63
-6.58
-113.14
-15.62
-38.89
-6.07
W12
16.04
0.49
-46.91
-5.32
-119.36
-17.49
-45.99
-8.01
W13
12.66
0.30
-65.40
-9.41
-139.40
-19.39
-68.09
-9.96
W14
11.56
0.16
-73.56
-10.20
-145.72
-19.50
-74.61
-10.02
W15
15.46
0.15
-6.01
-3.16
-101.81
-12.83
-27.09
-3.30
W16
18.57
0.43
-100.94
-12.24
-154.10
-21.01
-84.12
-11.60
W17
20.21
0.58
-58.68
-8.55
-133.41
-18.82
-61.79
-9.40
W18
20.60
0.40
-83.22
-12.14
-157.51
-22.93
-87.43
-13.49
W19
22.04
0.18
-17.65
-3.42
-96.98
-13.30
-21.95
-3.78
W20
25.97
0.40
-69.46
-9.70
-138.16
-19.16
-65.69
-9.60
W21
24.03
0.54
-39.44
-6.23
-114.06
-16.17
-39.92
-6.63
W22
24.02
0.42
-32.59
-5.68
-109.01
-15.22
-34.32
-5.66
W23
20.20
0.21
-92.06
-12.22
-165.38
-21.82
-94.90
-12.26
W24
18.30
0.16
-68.35
-9.66
-142.27
-18.86
-70.17
-9.31
W25
18.86
0.54
-35.35
-5.43
-110.09
-15.62
-36.23
-6.16
W26
12.21
0.44
-65.71
-9.34
-140.73
-20.49
-69.37
-11.07
W27
18.58
0.49
-69.19
-9.78
-142.17
-19.74
-70.59
-10.26
W28
19.50
0.13
-71.29
-10.13
-165.31
-21.52
-95.18
-12.00
W29
23.03
0.25
-55.64
-7.09
-129.64
-17.43
-56.41
-7.87
W30
17.93
0.36
-51.83
-7.21
-121.38
-17.17
-47.69
-7.63
W31
18.70
0.15
-45.28
-7.24
-122.24
-17.72
-48.21
-8.13
W32
17.83
0.39
-65.71
-9.34
-143.08
-20.11
-71.31
-10.61
W33
20.18
0.22
-134.67
-17.97
-201.85
-27.46
-134.63
-17.97
Mean
15.87
0.36
-51.54
-7.63
-125.81
-17.53
-53.27
-8.08
L256. Providing two digits after the decimal point for δ2H and δ18O when in fact the precision is 10 or 100 times more (±1.0-2.0‰ for δ2H and ±0.2-0.8‰ for δ18O) is not correct.
Response: We agree that inferential interpretation should reflect analytical precision. Summary values in the revised Results are therefore reported primarily to one decimal place. The Appendix tables retain the stored instrumental values at two decimals for data traceability and recalculation; those extra digits are not interpreted as analytical accuracy. The analytical uncertainty and reproducibility of the measurements are stated explicitly in Sect. 2.5.
Figures
Fig. 3. Nice figure! One can see from the spread in CVD versus CRAVE reference water extractions that CRAVE is generally producing more depleted water that CVD, which points out to possible incomplete water deposition (inverse sublimation) and/or non-equilibrium between water vapor and liquid water in the sample vial.
Response: Figure 3 compares corrected CRAVE values with direct measurements of the liquid-water samples; the comparator is not a CVD extraction. The corrected values remain lower on average by 1.7‰ for δ2H and 0.4‰ for δ18O. As reported in Sect. 3.1, the δ18O offset is systematic (p = 0.002), whereas no statistically significant δ2H offset was detected (p = 0.06). These residual differences must be distinguished from the much larger depletion of the raw trapped water, which is expected from liquid–vapor fractionation. The residual offset alone cannot distinguish incomplete deposition from non-equilibrium exchange or transport fractionation. We therefore report the residual bias alongside the improvement after correction and use the separate gravimetric recovery criterion in Sect. 2.1 to assess water-mass recovery. The Conclusion has been narrowed accordingly.
Fig. 5. Apparently, only CVD shows a depletion in both isotopes in deeper (>1 m) water. CRAVE and SL see less of a vertical isotopic composition gradient.
Response: We agree with this interpretation and revised the Results accordingly. The text now states explicitly that the deeper depletion is mainly associated with CVD, whereas CRAVE and SL show a much weaker vertical isotope gradient.
Change in manuscript:
“Fig. 5 shows the isotopic profiles (δ2H and δ18O) of soil water extracted by CRAVE, CVD, and SL methods at different depths, while Fig. B1 presents the depth-dependent variation of the mean signed relative error (Eref) of CRAVE relative to CVD. The three methods yielded comparable isotopic compositions in the upper soil layers, but their differences became more pronounced below 30 cm. In deeper layers, the δ2H and δ18O values obtained by CVD were systematically more depleted than those derived from CRAVE and SL, and the deviation increased with depth. This pattern is consistent with the positive correlation between Eref and soil depth (r = 0.77 for δ18O, r = 0.66 for δ2H; Fig. B2), indicating that CRAVE-derived isotope values became enriched increasingly relative to CVD with increasing depth.”
Fig. 6. Only the relationships between Eref and two of the three soil texture classes is needed since the 3rd relationship is a function of the first two. In other words, the displayed data is partly redundant.
Response: Agreed. The redundant texture relationships were removed. Figure 6 now focuses on clay content, which showed the strongest relationship with Eref, for δ18O and δ2H.
Change in manuscript:
“Figure 6. Relationships between clay content and the mean signed relative error (Eref) of cryogenic recirculating airflow vapor extraction (CRAVE) relative to cryogenic vacuum distillation (CVD) for δ18O (left) and δ2H (right).”
Fig. 7. Providing the R2 for linear regressions that have a y-intercept different than zero makes little sense if you are aiming to evaluate how two procedures plot with respect to the 1:1 line.
Response: We retained the regression line and R² in Fig. 7 as descriptive information on linear association, but we no longer use R² as the primary measure of 1:1 agreement. The revised Results evaluate agreement using ICC(A,1), the mean paired difference, and the Wilcoxon signed-rank test, and interpret the direction of the paired offsets directly.
Change in manuscript:
“Figure 7 compares 37 matched xylem water samples analyzed after CRAVE and CVD extraction. Agreement was higher for δ18O (ICC = 0.90) than for δ2H (ICC = 0.83). The mean paired difference, calculated as CVD minus CRAVE, was −0.3‰ for δ18O and −4.0‰ for δ2H. The Wilcoxon test of paired differences gave p = 0.20 for δ18O and p = 0.005 for δ2H. Paired δ18O values showed no directional shift, whereas CVD produced lower δ2H values across the xylem sample set.”
Fig. 8. See previous comment.
Response: The same treatment was applied to Fig. 8. Regression information is retained descriptively in the figure, while the revised text focuses on the paired platform differences and their statistical significance rather than using R² as an agreement criterion.
Change in manuscript:
“A subset of xylem water samples extracted by CVD was measured by both OA-ICOS laser spectroscopy and isotope ratio mass spectrometry (Fig. 8). Mean laser values were −2.3‰ for δ18O and −29.8‰ for δ2H, compared with −3.2‰ and −33.9‰ by isotope ratio mass spectrometry. The mean paired difference, calculated as laser spectroscopy minus isotope ratio mass spectrometry, was +0.9‰ for δ18O and +4.2‰ for δ2H; both differences had p < 0.001. OA-ICOS produced systematically higher values than isotope ratio mass spectrometry for these CVD extracts, with a larger mean difference for δ2H (+4.2‰) than for δ18O (+0.9‰).”
Fig. 9. 1- Do you assume that the water content is the same between samples of each xylem pair? 2- How do you determine the xylem water content of the CRAVE sample? 3- I see that there are, for example, less samples between 1.0 and 1.5 g/g for δ18O than δ2H, how come?
Response: The xylem samples were paired by dividing the same branch segment into matched CRAVE and CVD subsamples. One dry-weight gravimetric water-content value was therefore assigned to both members of each pair. Gravimetric water content was determined from the original fresh mass and the oven-dried mass after drying at 105 °C. Water mass was calculated as the difference between fresh and dry mass, and gravimetric water content was calculated as (w = (Gfresh-Gdry)/Gdry). All 37 pairs were included for both isotopes. The apparent lower number of δ18O points between 1.0 and 1.5 g g-1 is a plotting-range effect: two δ18O values fall below the displayed lower limit because the corresponding CVD isotope values are close to zero.
Change in manuscript:
“Each xylem pair came from one branch segment divided into matched subsamples, so one dry weight gravimetric water content was assigned to both extraction methods. The relation between Eref and water content was negative for δ2H (r = −0.52, p = 0.001) and weaker for δ18O (r = −0.30, p = 0.07; Fig. 9). The fitted δ2H relation crossed zero at approximately 0.81 g water (g dry matter)−1; below this value, CVD generally produced more negative δ2H values than CRAVE. All 37 pairs were included for both isotopes.”
“Figure 9. Dry weight gravimetric water content and signed relative error (Eref) between cryogenic recirculating airflow vapor extraction (CRAVE) and cryogenic vacuum distillation (CVD) for matched xylem samples (n = 37). One water content value was assigned to each pair because both subsamples came from the same branch segment. Two δ18O values lie below the displayed lower limit because the corresponding CVD isotope values approached zero.”
Discussion
L366. A clear definition of mobile/immobile water in the context of the present study is still missing at this point.
Response: A study-specific operational definition is now provided at the beginning of the Discussion, including the suction limits used to define SL-accessible “mobile” water and the corresponding operationally less mobile fraction.
Change in manuscript:
“Extraction method and soil properties jointly determine the isotope composition recovered from soil samples (Adams et al., 2020). In this study, mobile soil water is defined operationally as water collected by suction lysimeters when the applied suction magnitude remained below the maximum value derived from the water retention curve for each site and depth. These limits ranged from approximately 40 to 90 kPa (Liu et al., 2026). Water retained beyond the corresponding lysimeter limit is termed the operationally less mobile fraction. This definition follows the hydraulic accessibility framework used to distinguish mobile and bulk soil water (Sprenger et al., 2018; Sprenger et al., 2019). CVD targets recoverable bulk water in the homogenized soil sample and therefore covers a broader range of retention states than SL. CRAVE samples a third operational domain. The sample vial remained at 20 to 25 °C, whereas the deposition U-tube was immersed in liquid nitrogen, creating a temperature difference greater than 200 °C. Water was transferred through headspace vapor exchange and cryogenic deposition without direct heating of the sample matrix or vacuum extraction. Because CRAVE imposes no defined matric suction, its product is classified here as a vapor exchange extract. Under the tested conditions, its mean soil water isotope composition was closer to SL water than to CVD bulk water (Fig. 2; Table A3).”
L371. CRAVE does not run isothermally, there is roughly a 200 degC difference between the sample and the trap.
Response: Agreed. We removed the description of CRAVE as isothermal. The revised text distinguishes the sample-vial temperature (20–25 °C) from the liquid-nitrogen-cooled deposition U-tube and explicitly states that the temperature difference exceeds 200 °C.
Change in manuscript:
“The sample vial remained at 20 to 25 °C, whereas the deposition U-tube was immersed in liquid nitrogen, creating a temperature difference greater than 200 °C.”
406ff. The advantages of the CRAVE method, especially in terms of ease of use, number of samples to be extracted simultaneously etc. should be discussed here as well.
Response: We added a dedicated discussion of operational advantages. The revised Sect. 4.2 describes the simple modular hardware, absence of a sample oven/vacuum pump/central vacuum manifold, the common soil–xylem workflow, one sample per independent loop during a 0.5–3 h extraction, the possibility of running additional loops in parallel, and the ability to store and remeasure the recovered liquid water.
Change in manuscript:
“The operational benefit of CRAVE lies in its simple, modular extraction pathway. Samples remain in the same sealed glass vials used for collection, and each vial is connected directly to a compact recirculating pump and a liquid-nitrogen-cooled deposition U-tube. The operating sequence consists of weighing the sample vial and U-tube, connecting the recirculating loop, maintaining the liquid nitrogen level during extraction, and thawing the deposited ice for isotope analysis. No sample oven, vacuum pump, or central vacuum manifold is required. The same hardware and workflow are used for both soil and xylem samples. Each independent loop processes one sample during the 0.5–3 h extraction interval. The design is modular because additional pump–U-tube loops can in principle be operated in parallel, with one sample in each independent flow path.
CRAVE also separates water recovery from isotope measurement. Direct vapor equilibration infers liquid water isotope composition from equilibrated vapor measured by laser spectroscopy (Wassenaar et al., 2008; Gralher et al., 2021). In discrete vapor sampling, the isotopic stability of stored vapor depends on sampling and storage conditions (Iraheta et al., 2026). CRAVE instead deposits vapor as ice in the cryogenic trap, recovers it as liquid water after thawing, and records the recovered mass. The recovered liquid water can be stored, remeasured, or analyzed by either laser spectroscopy or IRMS, while the recovered water mass enters the working Rayleigh-type correction. This liquid endpoint removes the need to keep the extraction apparatus coupled directly to a laser analyzer and permits repeated measurements from the same recovered sample.
IRMS provides a distinct analytical route that does not rely on optical absorption spectra. Laser spectrometers determine isotope ratios from absorption features that can be distorted by overlapping VOC spectra (West et al., 2010; Millar et al., 2021; Herbstritt et al., 2024). IRMS instead determines isotope ratios after high-temperature conversion and gas separation and therefore does not require optical spectral correction (Gehre et al., 2015). In the paired CVD xylem extracts, OA-ICOS values exceeded IRMS values by 0.9‰ for δ18O and 4.2‰ for δ2H; the offset was larger for δ2H (Fig. 8). Overall, the CRAVE–IRMS workflow combines extraction in a sample vial maintained at 20–25 °C, recovery of storable liquid water, independent flow paths that can in principle be operated in parallel, and isotope measurement without laser spectral fitting.”
References
Adams, R. E., Hyodo, A., SantaMaria, T., Wright, C. L., Boutton, T. W., and West, J. B.: Bound and mobile soil water isotope ratios are affected by soil texture and mineralogy, whereas extraction method influences their measurement, Hydrological Processes, 34, 991-1003, 10.1002/hyp.13633, 2020.
Bailey, A., Noone, D., Berkelhammer, M., Steen-Larsen, H. C., and Sato, P.: The stability and calibration of water vapor isotope ratio measurements during long-term deployments, Atmos. Meas. Tech., 8, 4521-4538, 10.5194/amt-8-4521-2015, 2015.
Beyer, M., Kühnhammer, K., and Dubbert, M.: In situ measurements of soil and plant water isotopes: a review of approaches, practical considerations and a vision for the future, Hydrol. Earth Syst. Sci., 24, 4413-4440, 10.5194/hess-24-4413-2020, 2020.
Bowen, G. J., Banerjee, S., and Chakraborty, S.: Technical note: Spectral correction for cavity ring-down isotope analysis of plant and soil waters, Hydrol. Earth Syst. Sci., 29, 3907-3915, 10.5194/hess-29-3907-2025, 2025.
Ceperley, N., Gimeno, T. E., Jacobs, S. R., Beyer, M., Dubbert, M., Fischer, B., Geris, J., Holko, L., Kübert, A., Le Gall, S., Lehmann, M. M., Llorens, P., Millar, C., Penna, D., Prieto, I., Radolinski, J., Scandellari, F., Stockinger, M., Stumpp, C., Tetzlaff, D., van Meerveld, I., Werner, C., Yildiz, O., Zuecco, G., Barbeta, A., Orlowski, N., and Rothfuss, Y.: Toward a common methodological framework for the sampling, extraction, and isotopic analysis of water in the Critical Zone to study vegetation water use, WIREs Water, 11, e1727, 10.1002/wat2.1727, 2024.
Duvert, C., Barbeta, A., Hutley, L. B., Rodriguez, L., Irvine, D. J., and Taylor, A. R.: Cavitron extraction of xylem water suggests cryogenic extraction biases vary across species but are independent of tree water stress, Hydrological Processes, 38, e15099, https://doi.org/10.1002/hyp.15099, 2024.
Gat, J. R.: Oxygen and hydrogen isotopes in the hydrologic cycle, Annual Review of Earth and Planetary Sciences, 24, 225-262, https://doi.org/10.1146/annurev.earth.24.1.225, 1996.
Gehre, M., Renpenning, J., Gilevska, T., Qi, H., Coplen, T. B., Meijer, H. A. J., Brand, W. A., and Schimmelmann, A.: On-Line Hydrogen-Isotope Measurements of Organic Samples Using Elemental Chromium: An Extension for High Temperature Elemental-Analyzer Techniques, Analytical Chemistry, 87, 5198-5205, 10.1021/acs.analchem.5b00085, 2015.
Gralher, B., Herbstritt, B., and Weiler, M.: Technical note: Unresolved aspects of the direct vapor equilibration method for stable isotope analysis (δ18O, δ2H) of matrix-bound water: unifying protocols through empirical and mathematical scrutiny, Hydrology and Earth System Sciences, 25, 5219-5235, 10.5194/hess-25-5219-2021, 2021.
Herbstritt, B., Wengeler, L., and Orlowski, N.: Coping with spectral interferences when measuring water stable isotopes of vegetables, Rapid Communications in Mass Spectrometry, 38, e9907, https://doi.org/10.1002/rcm.9907, 2024.
Herbstritt, B., Gralher, B., Seeger, S., Rinderer, M., and Weiler, M.: Technical note: Discrete in situ vapor sampling for subsequent lab-based water stable isotope analysis, Hydrol. Earth Syst. Sci., 27, 3701-3718, 10.5194/hess-27-3701-2023, 2023.
Iraheta, A., Malsch-Fröhlich, E., Gerchow, M., and Beyer, M.: Technical note: Water vapor sampling for stable isotope analysis: systematic testing of sampling conditions and development of a robust protocol, Hydrol. Earth Syst. Sci., 30, 5005-5021, 10.5194/hess-30-5005-2026, 2026.
Kocum, J., Haidl, J., Gebousky, O., Falatkova, K., Sipek, V., Sanda, M., Orlowski, N., and Vlcek, L.: Technical note: A new laboratory approach to extract soil water for stable isotope analysis from large soil samples, Hydrol. Earth Syst. Sci., 29, 2863-2880, 10.5194/hess-29-2863-2025, 2025.
Liu, X., Chen, X., Zhang, Z., Liu, W., Peng, T., and McDonnell, J. J.: The role of rock fractures on tree water use of water stored in bedrock: Mixing and residence times, EGUsphere, 2025, 1-43, 10.5194/egusphere-2025-3937, 2025.
Liu, X., Chen, X., Zhang, Z., Liu, W., Peng, T., and McDonnell, J. J.: Rock fracture characteristics regulate water storage and seasonal tree water uptake in karst, Hydrology and Earth System Sciences, 30, 3061-3094, 10.5194/hess-30-3061-2026, 2026.
Magh, R. K., Gralher, B., Herbstritt, B., Kübert, A., Lim, H., Lundmark, T., and Marshall, J.: Technical note: Conservative storage of water vapour – practical in situ sampling of stable isotopes in tree stems, Hydrol. Earth Syst. Sci., 26, 3573-3587, 10.5194/hess-26-3573-2022, 2022.
Majoube, M.: Fractionnement en oxygène 18 et en deutérium entre l'eau et sa vapeur, Journal de Chimie Physique, 68, 1423-1436, 10.1051/jcp/1971681423, 1971.
Marshall, J. D., Cuntz, M., Beyer, M., Dubbert, M., and Kuehnhammer, K.: Borehole Equilibration: Testing a New Method to Monitor the Isotopic Composition of Tree Xylem Water in situ, Frontiers in Plant Science, Volume 11 - 2020, 10.3389/fpls.2020.00358, 2020.
Mattei, A., Barbecot, F., Guillon, S., Goblet, P., Hélie, J.-F., and Meyzonnat, G.: Improved accuracy and precision of water stable isotope measurements using the direct vapour equilibration method, Rapid Communications in Mass Spectrometry, 33, 1613-1622, https://doi.org/10.1002/rcm.8494, 2019.
Meißner, M., Köhler, M., Schwendenmann, L., Hölscher, D., and Dyckmans, J.: Soil water uptake by trees using water stable isotopes (δ2H and δ18O)−a method test regarding soil moisture, texture and carbonate, Plant and Soil, 376, 327-335, 10.1007/s11104-013-1970-z, 2014.
Millar, C., Pratt, D., Schneider, D. J., and McDonnell, J. J.: A comparison of extraction systems for plant water stable isotope analysis, Rapid Communications in Mass Spectrometry, 32, 1031-1044, https://doi.org/10.1002/rcm.8136, 2018.
Millar, C., Janzen, K., Nehemy, M. F., Koehler, G., Hervé-Fernández, P., and McDonnell, J. J.: Organic contamination detection for isotopic analysis of water by laser spectroscopy, Rapid Communications in Mass Spectrometry, 35, e9118, https://doi.org/10.1002/rcm.9118, 2021.
Nehemy, M. F., Millar, C., Janzen, K., Gaj, M., Pratt, D. L., Laroque, C. P., and McDonnell, J. J.: 17O-excess as a detector for co-extracted organics in vapor analyses of plant isotope signatures, Rapid Communications in Mass Spectrometry, 33, 1301-1310, https://doi.org/10.1002/rcm.8470, 2019.
Oerter, E. J., Siebert, G., Bowling, D. R., and Bowen, G.: Soil water vapour isotopes identify missing water source for streamside trees, Ecohydrology, 12, e2083, https://doi.org/10.1002/eco.2083, 2019.
Orlowski, N., Pratt, D. L., and McDonnell, J. J.: Intercomparison of soil pore water extraction methods for stable isotope analysis, Hydrological Processes, 30, 3434-3449, https://doi.org/10.1002/hyp.10870, 2016.
Orlowski, N., Breuer, L., Angeli, N., Boeckx, P., Brumbt, C., Cook, C. S., Dubbert, M., Dyckmans, J., Gallagher, B., Gralher, B., Herbstritt, B., Hervé-Fernández, P., Hissler, C., Koeniger, P., Legout, A., Macdonald, C. J., Oyarzún, C., Redelstein, R., Seidler, C., Siegwolf, R., Stumpp, C., Thomsen, S., Weiler, M., Werner, C., and McDonnell, J. J.: Inter-laboratory comparison of cryogenic water extraction systems for stable isotope analysis of soil water, Hydrol. Earth Syst. Sci., 22, 3619-3637, 10.5194/hess-22-3619-2018, 2018.
Pyschik, J., Seeger, S., Herbstritt, B., and Weiler, M.: Technical note: A fast and reproducible autosampler for direct vapor equilibration isotope measurements, Hydrol. Earth Syst. Sci., 29, 525-534, 10.5194/hess-29-525-2025, 2025.
Seeger, S. and Weiler, M.: Temporal dynamics of tree xylem water isotopes: in situ monitoring and modeling, Biogeosciences, 18, 4603-4627, 10.5194/bg-18-4603-2021, 2021.
Sprenger, M., Herbstritt, B., and Weiler, M.: Established methods and new opportunities for pore water stable isotope analysis, Hydrological Processes, 29, 5174-5192, https://doi.org/10.1002/hyp.10643, 2015.
Sprenger, M., Tetzlaff, D., and Soulsby, C.: Soil water stable isotopes reveal evaporation dynamics at the soil–plant–atmosphere interface of the critical zone, Hydrol. Earth Syst. Sci., 21, 3839-3858, 10.5194/hess-21-3839-2017, 2017.
Sprenger, M., Llorens, P., Cayuela, C., Gallart, F., and Latron, J.: Mechanisms of consistently disjunct soil water pools over (pore) space and time, Hydrology and Earth System Sciences, 23, 2751-2762, https://doi.org/10.5194/hess-23-2751-2019, 2019.
Sprenger, M., Tetzlaff, D., Buttle, J., Laudon, H., Leistert, H., Mitchell, C. P. J., Snelgrove, J., Weiler, M., and Soulsby, C.: Measuring and Modeling Stable Isotopes of Mobile and Bulk Soil Water, Vadose Zone Journal, 17, 170149, 10.2136/vzj2017.08.0149, 2018.
Tao, S., Xia, J., Xia, J., Chen, X., Lv, W., Gui, H., and Xu, J.: Stable water isotopes in soil–plant systems reveals ecohydrological dynamics in a typical flood wetland in the subtropical monsoon region, China, Ecological Indicators, 178, 114021, https://doi.org/10.1016/j.ecolind.2025.114021, 2025.
Vadibeler, D., Stockinger, M. P., Wassenaar, L. I., and Stumpp, C.: Influence of equilibration time, soil texture, and saturation on the accuracy of porewater water isotope assays using the direct H2O(liquid)–H2O(vapor) equilibration method, Journal of Hydrology, 607, 127560, https://doi.org/10.1016/j.jhydrol.2022.127560, 2022.
Volkmann, T. H. M. and Weiler, M.: Continual in situ monitoring of pore water stable isotopes in the subsurface, Hydrol. Earth Syst. Sci., 18, 1819-1833, 10.5194/hess-18-1819-2014, 2014.
Wang, H., Yu, H., He, D., Li, M., Si, B., McDonnell, J. J., and Nehemy, M. F.: Cryogenic vacuum distillation vs Cavitron methods in ecohydrology: Extraction protocol effects on plant water isotopic values, Journal of Hydrology, 642, 131853, https://doi.org/10.1016/j.jhydrol.2024.131853, 2024.
Wassenaar, L. I., Hendry, M. J., Chostner, V. L., and Lis, G. P.: High Resolution Pore Water δ2H and δ18O Measurements by H2O(liquid)−H2O(vapor) Equilibration Laser Spectroscopy, Environmental Science & Technology, 42, 9262-9267, 10.1021/es802065s, 2008.
Wen, M., Zhao, X., Si, B., He, D., Li, M., Gao, X., Cai, Y., Lu, Y., and Wang, Y.: Inter-comparison of extraction methods for plant water isotope analysis and its indicative significance, Journal of Hydrology, 625, 130015, https://doi.org/10.1016/j.jhydrol.2023.130015, 2023.
West, A. G., Patrickson, S. J., and Ehleringer, J. R.: Water extraction times for plant and soil materials used in stable isotope analysis, Rapid Communications in Mass Spectrometry, 20, 1317-1321, https://doi.org/10.1002/rcm.2456, 2006.
West, A. G., Goldsmith, G. R., Brooks, P. D., and Dawson, T. E.: Discrepancies between isotope ratio infrared spectroscopy and isotope ratio mass spectrometry for the stable isotope analysis of plant and soil waters, Rapid Communications in Mass Spectrometry, 24, 1948-1954, https://doi.org/10.1002/rcm.4597, 2010.
Younger, S. E., Blake, J., Jackson, C. R., and Aubrey, D. P.: δ2H isotopic offsets in xylem water measurements under cryogenic vacuum distillation: Quantifying and correcting wood-water hydrogen exchange influences, Ecohydrology, 17, e2640, https://doi.org/10.1002/eco.2640, 2024.
-
AC1: 'Reply on RC1', Xiuqiang Liu, 06 Sep 2026
-
RC2: 'Comment on egusphere-2026-3419', Anonymous Referee #2, 10 Aug 2026
General comments:
The authors provide a new method, similar to the established cryogenic vacuum distillation (CVD) method, for the extraction of matrix bound water (soil, xylem) and subsequent water isotope analysis with OA-ICOS and IRMS. The difference to CVD is, that sample temperature is ambient temperature, i.e. no heating is necessary and thus, no heating-induced effects occur; further, due to the smaller temperature gradient applied, the method is more preserving for the sample material.
I agree with and support most of the comments of Reviewer 1, but I have some additional concerns.
In general, the manuscript is well structured but parts could be rephrased to be more specific. Especially the introduction is a bit long. The table, comparing the existing methods gives a good but very broad overview; it could perhaps go to the appendix.As stated in the study, data of both methods, CRAVE and CVD show strong consistency (Fig. 7). Why should one use CRAVE instead of CVD, when the data is highly comparable?
In Figure 8 it doesn’t seem to improve the CVD-extracted xylem water data very much when analyzing with IRMS compared to OA-ICOS. I would assume that especially the d18-O data of the laser based instrument are more affected by organics and that there is a larger offset, compared to the d2H-data.
Your hypothesis is that CVD co-extracts organics to a higher extend than CRAVE, but there is not much evidence in your data. You state that the method reduces organic contamination which is correct, based on the mechanistic theory, but I am missing data that emphasize this. Did you check for any organics-indicating parameter? There are specific parameters which are recorded together with the isotope data by the OA-ICOS instruments anyway. It would be great to show these data in order to support your hypothesis.
Specific Comments:
I agree with Reviewer 1, don’t use “new” in the title.
Btw, CVD-extraction lines are also airtight.
Moreover, CRAVE is not perfectly airtight, there is a pressure release…IRMS can be less sensitive to VOC-induced spectral interference but this depends on the peripheral that was combined to the IRMS. Did you use an H-Device, which could be assumed from L 415 ff. or which one was used? Please add this information to the method section and discuss in the discussion section accordingly.
Technical corrections:
Figure 3: Nice figure, but values are hard to read. Please always add blanks after the values for d2H.
Figure 5: Legend is missing.Citation: https://doi.org/10.5194/egusphere-2026-3419-RC2 -
AC2: 'Reply on RC2', Xiuqiang Liu, 06 Sep 2026
Title: Technical note: Evaluation of a new cryogenic airtight vapor extraction (CRAVE) method for soil and plant water
Author(s): Xiuqiang Liu, Hongxiu Wang, Xi Chen, Ying Zhao, Magali F. Nehemy, and Jeffrey J. McDonnell
MS No.: egusphere-2026-3419
MS type: Technical note
Reviewer 2:
Comments on egusphere-2026-3419
Response: We thank the reviewer for the constructive comments. We revised the title and terminology, shortened and reorganized the introduction, moved the broad method-comparison table to the Appendix, clarified the pressure-buffered rather than fully airtight configuration, expanded the IRMS method description, and strengthened the discussion of CRAVE’s operational rationale and the limitations of the organic-interference comparison.
Title
I agree with Reviewer 1, don’t use ‘new’ in the title.
Response: Agreed. The adjective “new” was removed from the title.
Change in manuscript:
Title. “Technical note: Evaluation of a cryogenic recirculating airflow vapor extraction (CRAVE) method for soil and plant water”
Btw, CVD-extraction lines are also airtight.
Response: Agreed. “Airtight” was removed from the method name because it is not a unique feature relative to CVD and because the CRAVE loop includes a pressure-buffering vent. The revised name is “cryogenic recirculating airflow vapor extraction,” and the text now states explicitly that the loop is pressure-buffered rather than fully airtight.
Change in manuscript:
“CRAVE circulates headspace vapor through a liquid-nitrogen-cooled trap, where water vapor is deposited as ice and subsequently thawed. The thawed trap water can then be analyzed by IRMS or laser spectroscopy. A desiccant-buffered vent limits pressure excursions and reduces atmospheric water-vapor ingress (Millar et al., 2021; Herbstritt et al., 2024). The loop is pressure-buffered rather than fully airtight, and its temperature and humidity conditions must be controlled and documented as in other vapor-based isotope measurements (Bailey et al., 2015; Beyer et al., 2020). The resulting liquid sample can be transferred to sealed glass vials for storage, repeated analysis, or measurement by either IRMS or LS.”
“The system consists of four main components: (1) a sample vial containing the soil, plant, or liquid-water sample; (2) a deposition U-tube placed in a liquid-nitrogen flask to trap water vapor as ice; (3) a recycling pump (GS150, Geesiniger, Germany) that drives continuous vapor circulation through the loop; and (4) a desiccant-buffered vent (drying tube) that provides pressure buffering while minimizing ingress of atmospheric water vapor. All tubing, needles, and connections are sealed with PTFE (Teflon) septa to minimize isotopic exchange with the atmosphere. A schematic of the CRAVE system and vapor-flow pathway is shown in Fig. 1.
The sample vial is connected to the deposition U-tube, which is immersed in liquid nitrogen so that the nitrogen level covers approximately one-third of the U-tube height. The opposite end of the U-tube is connected to the inlet of the recycling pump, and the pump outlet is connected back to the sample vial to drive recirculating airflow. The drying tube provides a pressure-buffered connection to the atmosphere, preventing substantial under- or overpressure as gas temperature and vapor content change during pumping and cryogenic trapping; the desiccant minimizes atmospheric water-vapor ingress during this pressure equalization.”
Abstract and Introduction
Your hypothesis is that CVD co-extracts organics to a higher extend than CRAVE, but there is not much evidence in your data. You state that the method reduces organic contamination which is correct, based on the mechanistic theory, but I am missing data that emphasize this. Did you check for any organics-indicating parameter? There are specific parameters which are recorded together with the isotope data by the OA-ICOS instruments anyway. It would be great to show these data in order to support your hypothesis.
Response: We agree that the present dataset does not provide a direct chemical quantification of organic contamination. No spectral contamination indicator or direct chemical VOC measurement was included in this experiment. We therefore removed the earlier claim from the abstract that CRAVE “reduces organic contamination” and revised the Discussion so that the OA-ICOS–IRMS comparison is treated as an analytical-platform offset rather than direct proof that CRAVE eliminates organic interference. The manuscript now states explicitly what additional data would be needed to attribute the offset to VOCs.
Change in manuscript:
Abstract. “Reliable characterization of soil and plant water isotopic composition depends on how water is recovered from complex matrices. We evaluated cryogenic recirculating airflow vapor extraction (CRAVE), in which the sample vial remains near room temperature while recirculated vapor is deposited as ice in a liquid-nitrogen-cooled trap and subsequently thawed for analysis. CRAVE was tested with 33 liquid-water samples, 42 soil samples collected from 5–150 cm depth, and 37 xylem samples, and was compared with cryogenic vacuum distillation (CVD) and, for soil, suction lysimeters (SL). A Rayleigh-type mass-balance correction, based on temperature-dependent liquid–vapor fractionation and water-mass balance, was used as a working transformation to estimate the initial water isotopic composition from the trapped water. In the liquid-water benchmark, the correction shifted mean trapped-water values from −126‰ to −53‰ for δ2H and from −17.5‰ to −8.1‰ for δ18O, close to the directly measured source-water means of −52‰ and −7.6‰, respectively. Method-dependent differences remained in environmental matrices. For xylem water, the CRAVE–CVD δ2H difference varied with gravimetric water content, with lower CVD δ2H values at low moisture (<0.8 g water (g dry matter)−1). For soil, CVD-extracted bulk-water δ2H and δ18O progressively decreased relative to CRAVE and SL in deeper, more clay-rich samples. CRAVE avoids direct heating of the sample matrix, yields a storable liquid-water sample after thawing, and provides a common extraction workflow for soil and plant water isotope analysis under the tested conditions.”
“Volatile organic compounds represent a separate analytical source of variation. Heating can co-extract volatile compounds from plant tissue, and these compounds can interfere with laser spectroscopic isotope measurements (Martín-Gómez et al., 2015; Herbstritt et al., 2024). For the tested CVD extracts, OA-ICOS produced values that were higher than IRMS by 0.91‰ for δ18O and 4.15‰ for δ2H (Fig. 8). These paired measurements establish an analytical platform offset. Identifying the contribution of VOC interference would require spectral contamination indicators or direct chemical measurements, neither of which was included in this experiment.”
In general, the manuscript is well structured but parts could be rephrased to be more specific. Especially the introduction is a bit long.
Response: The introduction was shortened and made more specific. The revised version now moves directly from the general methodological problem to the distinction among bulk-water extraction, suction lysimeters, laboratory DVE, in situ probes, and discrete vapor sampling, followed by a concise five-paragraph definition and evaluation framework for CRAVE.
Change in manuscript:
“Stable isotopes of hydrogen and oxygen (2H, 18O) have been used to trace water movement in hydrological and ecohydrological processes (West et al., 2006; Meißner et al., 2014; Tao et al., 2025). By analyzing the isotopic compositions of xylem water, soil water, precipitation, streamflow and groundwater, researchers can identify water sources and quantify key processes such as evaporation, infiltration, and plant water uptake (Sprenger et al., 2017; Liu et al., 2025; Tao et al., 2025). While liquid water can be analyzed directly for its isotopic composition using isotope ratio mass spectrometry (IRMS) or laser spectroscopy (LS), water in soil and plant samples must first be extracted or measured in situ (Beyer et al., 2020), which remains a technically challenging process (Orlowski et al., 2016; Kocum et al., 2025). A recent community framework therefore recommends evaluating sampling, extraction, and isotope analysis as one process chain, with explicit attention to the water fraction represented by each method (Ceperley et al., 2024).
Multiple extraction and equilibration techniques are available, each with method-specific sources of isotope fractionation, incomplete water recovery, organic contamination, or matrix-dependent bias (see Table A1 for a summary). Cryogenic vacuum distillation (CVD) remains widely used but requires dedicated vacuum and cryogenic equipment (Orlowski et al., 2016). Heating-based extractions such as CVD seek to limit incomplete recovery and associated fractionation by releasing tightly bound water (Wang et al., 2024). Elevated extraction temperatures can also introduce sample-specific isotope effects. For soils, high temperatures increase the risk of releasing isotopically distinct structural water from hydrous minerals (Orlowski et al., 2018; Wen et al., 2023; Duvert et al., 2024). In plant tissues, heating can enhance wood-water hydrogen exchange and alter δ2H even when total water recovery is high (Younger et al., 2024). Heating can also increase co-extraction of volatile and semi-volatile organic compounds from soil and plant matrices; these compounds may cause spectral interference in laser-based isotope analysis (Millar et al., 2018; Bowen et al., 2025).
Suction lysimeters serve a different purpose from bulk-water extraction by collecting a tension-accessible fraction of soil water in situ. These instruments are restricted to high water content soils and often fail to collect a sample below ~100 cm H2O water tension (Sprenger et al., 2015; Sprenger et al., 2019).
Vapor-based methods include laboratory direct vapor equilibration (DVE), such as CO2 or H2 equilibration and liquid–vapor equilibration coupled to laser spectroscopy, as well as in situ equilibration probes (Wassenaar et al., 2008; Oerter et al., 2019; Marshall et al., 2020; Vadibeler et al., 2022; Pyschik et al., 2025). Laboratory DVE can support batch analysis, whereas in situ probes provide repeated measurements at the installed location when coupled with LS. Bag- or vial-based vapor sampling depends on vapor containment, storage duration, sampling conditions, and correction of organic-vapor interference in laser spectrometers (Mattei et al., 2019; Nehemy et al., 2019; Gralher et al., 2021; Millar et al., 2021; Iraheta et al., 2026). In situ equilibration probes reduce sample handling and can resolve high-frequency isotope dynamics, but they still require field-deployable laser systems and remain susceptible to interference from plant VOCs, especially in xylem applications (Herbstritt et al., 2023). Magh et al. (2022) proposed a discrete vapor-sampling technique that flushes dry air through samples to collect equilibrated vapor without requiring in situ instrumentation. In the reported tests, vapor storage duration affected the isotope values, particularly δ18O after several days. These approaches address different spatial and temporal scales. A complementary laboratory workflow would retain near-room-temperature vapor equilibration while producing a storable liquid-water sample for subsequent analysis.
Here, we evaluate cryogenic recirculating airflow vapor extraction (CRAVE), which recovers a measured fraction of sample water by depositing headspace vapor as ice in a liquid-nitrogen-cooled trap and subsequently thawing it for stable isotope analysis. CRAVE shares with vapor-equilibration methods the use of liquid–vapor isotope relationships while the sample vial remains near room temperature (Wassenaar et al., 2008; Volkmann and Weiler, 2014; Seeger and Weiler, 2021). Its distinguishing operational feature is that a measured fraction of vapor is trapped as ice and recovered as a storable liquid-water sample for subsequent isotope analysis. The revised workflow is described in the following paragraphs:
CRAVE circulates headspace vapor through a liquid-nitrogen-cooled trap, where water vapor is deposited as ice and subsequently thawed. The thawed trap water can then be analyzed by IRMS or laser spectroscopy. A desiccant-buffered vent limits pressure excursions and reduces atmospheric water-vapor ingress (Millar et al., 2021; Herbstritt et al., 2024). The loop is pressure-buffered rather than fully airtight, and its temperature and humidity conditions must be controlled and documented as in other vapor-based isotope measurements (Bailey et al., 2015; Beyer et al., 2020). The resulting liquid sample can be transferred to sealed glass vials for storage, repeated analysis, or measurement by either IRMS or LS.
A Rayleigh-type working correction estimates the initial liquid-water isotope composition from the measured cumulative trap-water value, the fraction of water removed from the source sample, and temperature-dependent liquid–vapor equilibrium fractionation (Majoube, 1971; Gat, 1996).
Overall, CRAVE is designed to recover a measured fraction of water from soil or plant samples. The initial isotope composition is estimated from the trap-water value, removed-water fraction, and equilibrium-fractionation term described above. The evaluation therefore includes sample-vial temperature monitoring and gravimetric checks of cumulative water recovery (Mattei et al., 2019; Gralher et al., 2021). We evaluated CRAVE with liquid-water standards and paired soil and xylem samples, compared corrected and uncorrected values with direct standards, CVD, and, for soil, suction-lysimeter water, and examined whether method offsets varied with depth, texture, and water content.”
The table, comparing the existing methods gives a good but very broad overview; it could perhaps go to the appendix.
Response: Agreed. The broad comparison table was moved from the main text to Appendix A as Table A1.
Methods
Moreover, CRAVE is not perfectly airtight, there is a pressure release…
Response: Correct. The revised manuscript no longer describes the system as perfectly airtight. It now states that the loop is pressure-buffered rather than fully airtight and explains the function of the desiccant-buffered vent in preventing substantial under- or overpressure while minimizing atmospheric water-vapor ingress.
Change in manuscript:
“CRAVE circulates headspace vapor through a liquid-nitrogen-cooled trap, where water vapor is deposited as ice and subsequently thawed. The thawed trap water can then be analyzed by IRMS or laser spectroscopy. A desiccant-buffered vent limits pressure excursions and reduces atmospheric water-vapor ingress (Millar et al., 2021; Herbstritt et al., 2024). The loop is pressure-buffered rather than fully airtight, and its temperature and humidity conditions must be controlled and documented as in other vapor-based isotope measurements (Bailey et al., 2015; Beyer et al., 2020). The resulting liquid sample can be transferred to sealed glass vials for storage, repeated analysis, or measurement by either IRMS or LS.
The system consists of four main components: (1) a sample vial containing the soil, plant, or liquid-water sample; (2) a deposition U-tube placed in a liquid-nitrogen flask to trap water vapor as ice; (3) a recycling pump (GS150, Geesiniger, Germany) that drives continuous vapor circulation through the loop; and (4) a desiccant-buffered vent (drying tube) that provides pressure buffering while minimizing ingress of atmospheric water vapor. All tubing, needles, and connections are sealed with PTFE (Teflon) septa to minimize isotopic exchange with the atmosphere. A schematic of the CRAVE system and vapor-flow pathway is shown in Fig. 1.
The sample vial is connected to the deposition U-tube, which is immersed in liquid nitrogen so that the nitrogen level covers approximately one-third of the U-tube height. The opposite end of the U-tube is connected to the inlet of the recycling pump, and the pump outlet is connected back to the sample vial to drive recirculating airflow. The drying tube provides a pressure-buffered connection to the atmosphere, preventing substantial under- or overpressure as gas temperature and vapor content change during pumping and cryogenic trapping; the desiccant minimizes atmospheric water-vapor ingress during this pressure equalization.”
IRMS can be less sensitive to VOC-induced spectral interference but this depends on the peripheral that was combined to the IRMS. Did you use an H-Device, which could be assumed from L 415 ff. or which one was used? Please add this information to the method section and discuss in the discussion section accordingly.
Response: We added the requested instrumental information. The system was not an H-Device. Water extracted from soil and plant samples was analyzed with a vario PYRO cube elemental analyzer coupled to a precisION IRMS (Elementar), using high-temperature pyrolysis over glassy carbon chips at 1450 °C. The Discussion was also revised to distinguish IRMS from laser spectroscopy on the basis of the measurement pathway rather than making a generic claim that all IRMS configurations are equally insensitive to organic compounds.
Change in manuscript:
“Water extracted from soil and plant samples was analyzed at the Soil Science Isotope Laboratory, Department of Soil Science, University of Saskatchewan, Canada, using a vario PYRO cube elemental analyzer coupled to a precisION isotope ratio mass spectrometer (IRMS; Elementar). Hydrogen and oxygen isotope analyses were performed by high-temperature pyrolysis over glassy carbon chips at 1450 °C. To minimize between-sample memory effects, multiple injections were performed for each sample and standard; the first 1–4 injections were excluded as required and the final 3–4 injections were averaged. Measured isotope values were normalized to the Vienna Standard Mean Ocean Water–Standard Light Antarctic Precipitation (VSMOW–SLAP) scale using two calibrated laboratory standards. Natural-abundance controls and, when required, enriched controls were analyzed with every batch. Analytical uncertainty for the IRMS measurements was expressed as 2σ and calculated conservatively as twice the standard deviation of the corresponding control measurements.”
“IRMS provides a distinct analytical route that does not rely on optical absorption spectra. Laser spectrometers determine isotope ratios from absorption features that can be distorted by overlapping VOC spectra (West et al., 2010; Millar et al., 2021; Herbstritt et al., 2024). IRMS instead determines isotope ratios after high-temperature conversion and gas separation and therefore does not require optical spectral correction (Gehre et al., 2015). In the paired CVD xylem extracts, OA-ICOS values exceeded IRMS values by 0.9‰ for δ18O and 4.2‰ for δ2H; the offset was larger for δ2H (Fig. 8). Overall, the CRAVE–IRMS workflow combines extraction in a sample vial maintained at 20–25 °C, recovery of storable liquid water, independent flow paths that can in principle be operated in parallel, and isotope measurement without laser spectral fitting.”
Figures and Results
Figure 3: Nice figure, but values are hard to read. Please always add blanks after the values for d2H.
Response: Thank you. This is a figure-formatting issue rather than a data issue. We will standardize the spacing between numerical values and the per mil symbol in the δ2H annotation in the final production-quality Fig. 3.
Change in manuscript:
“Figure 2. Relationships between δ2H and δ18O for water extracted by different methods (cryogenic recirculating airflow vapor extraction (CRAVE), cryogenic vacuum distillation (CVD), and suction lysimeter (SL)).”
Figure 5: Legend is missing.
Response: Corrected. A legend identifying CRAVE, CVD, and SL is now included in Fig. 5.
Change in manuscript:
“Figure 5. Isotopic variations (δ2H and δ18O) of soil water extracted by cryogenic recirculating airflow vapor extraction (CRAVE), cryogenic vacuum distillation (CVD), and suction lysimeter (SL) methods at different soil depths.”
As stated in the study, data of both methods, CRAVE and CVD show strong consistency (Fig. 7). Why should one use CRAVE instead of CVD, when the data is highly comparable?
Response: We expanded the Discussion to make the rationale explicit. CRAVE is not presented as superior to CVD simply because the isotope values are comparable. Its practical distinction is the no-direct-heating sample workflow, simple modular hardware, use of the same extraction configuration for soil and xylem, recovery of a storable liquid-water sample, separation of water recovery from isotope measurement, and the possibility of operating independent loops in parallel. These features define use cases where CRAVE can be advantageous even when its isotope values agree closely with CVD.
Change in manuscript:
“The operational benefit of CRAVE lies in its simple, modular extraction pathway. Samples remain in the same sealed glass vials used for collection, and each vial is connected directly to a compact recirculating pump and a liquid-nitrogen-cooled deposition U-tube. The operating sequence consists of weighing the sample vial and U-tube, connecting the recirculating loop, maintaining the liquid nitrogen level during extraction, and thawing the deposited ice for isotope analysis. No sample oven, vacuum pump, or central vacuum manifold is required. The same hardware and workflow are used for both soil and xylem samples. Each independent loop processes one sample during the 0.5–3 h extraction interval. The design is modular because additional pump–U-tube loops can in principle be operated in parallel, with one sample in each independent flow path.
CRAVE also separates water recovery from isotope measurement. Direct vapor equilibration infers liquid water isotope composition from equilibrated vapor measured by laser spectroscopy (Wassenaar et al., 2008; Gralher et al., 2021). In discrete vapor sampling, the isotopic stability of stored vapor depends on sampling and storage conditions (Iraheta et al., 2026). CRAVE instead deposits vapor as ice in the cryogenic trap, recovers it as liquid water after thawing, and records the recovered mass. The recovered liquid water can be stored, remeasured, or analyzed by either laser spectroscopy or IRMS, while the recovered water mass enters the working Rayleigh-type correction. This liquid endpoint removes the need to keep the extraction apparatus coupled directly to a laser analyzer and permits repeated measurements from the same recovered sample.
IRMS provides a distinct analytical route that does not rely on optical absorption spectra. Laser spectrometers determine isotope ratios from absorption features that can be distorted by overlapping VOC spectra (West et al., 2010; Millar et al., 2021; Herbstritt et al., 2024). IRMS instead determines isotope ratios after high-temperature conversion and gas separation and therefore does not require optical spectral correction (Gehre et al., 2015). In the paired CVD xylem extracts, OA-ICOS values exceeded IRMS values by 0.9‰ for δ18O and 4.2‰ for δ2H; the offset was larger for δ2H (Fig. 8). Overall, the CRAVE–IRMS workflow combines extraction in a sample vial maintained at 20–25 °C, recovery of storable liquid water, independent flow paths that can in principle be operated in parallel, and isotope measurement without laser spectral fitting.”
“As shown in Tables A3 and A4, the isotopic compositions of plant and soil water extracted by the same method are generally consistent, whereas slight but systematic differences exist among different extraction techniques. Xylem water extracted by CVD shows slightly lower δ2H values than that obtained via CRAVE, while for soil water CVD produces slightly lower δ2H and δ18O values than CRAVE and SL. These results indicate that small isotopic offsets introduced during extraction are method-dependent. For studies that compare isotopic compositions between plant and soil water, particularly those using mixing models for source attribution, applying the same extraction method can help minimize methodological bias and improve data comparability. However, this does not exclude the use of different methods when justified by sample properties or practical limitations. For example, the Cavitron centrifuge has been shown to extract xylem water effectively with minimal thermal and organic interference (Barbeta et al., 2022), even though it cannot be used for soil samples. In such cases, calibration procedures, correction factors, or dual-method validation may be necessary to ensure consistency between plant and soil water isotopic measurements (Chen et al., 2020; Allen and Kirchner, 2022; He et al., 2023). In contrast, the CRAVE method evaluated in this study allows both soil and plant water to be extracted using a single, unified protocol. This feature supports internally consistent comparisons and reduces the need for cross-method correction.”
In Figure 8 it doesn’t seem to improve the CVD-extracted xylem water data very much when analyzing with IRMS compared to OA-ICOS. I would assume that especially the d18-O data of the laser based instrument are more affected by organics and that there is a larger offset, compared to the d2H-data.
Response: The observed data indeed do not show a larger δ18O offset than δ2H. We therefore do not interpret IRMS as automatically “improving” the CVD extract, nor do we use this comparison as proof of VOC interference. The revised Results report the empirical offsets directly (+0.9‰ for δ18O and +4.2‰ for δ2H, OA-ICOS minus IRMS), while the Discussion states that identifying the contribution of VOC interference would require spectral contamination indicators or direct chemical measurements, which were not included. The comparison is therefore presented as an analytical-platform offset, with the larger observed offset occurring for δ2H in this dataset.
Change in manuscript:
“A subset of xylem water samples extracted by CVD was measured by both OA-ICOS laser spectroscopy and isotope ratio mass spectrometry (Fig. 8). Mean laser values were −2.3‰ for δ18O and −29.8‰ for δ2H, compared with −3.2‰ and −33.9‰ by isotope ratio mass spectrometry. The mean paired difference, calculated as laser spectroscopy minus isotope ratio mass spectrometry, was +0.9‰ for δ18O and +4.2‰ for δ2H; both differences had p < 0.001. OA-ICOS produced systematically higher values than isotope ratio mass spectrometry for these CVD extracts, with a larger mean difference for δ2H (+4.2‰) than for δ18O (+0.9‰).”
“Volatile organic compounds represent a separate analytical source of variation. Heating can co-extract volatile compounds from plant tissue, and these compounds can interfere with laser spectroscopic isotope measurements (Martín-Gómez et al., 2015; Herbstritt et al., 2024). For the tested CVD extracts, OA-ICOS produced values that were higher than IRMS by 0.91‰ for δ18O and 4.15‰ for δ2H (Fig. 8). These paired measurements establish an analytical platform offset. Identifying the contribution of VOC interference would require spectral contamination indicators or direct chemical measurements, neither of which was included in this experiment.”
“IRMS provides a distinct analytical route that does not rely on optical absorption spectra. Laser spectrometers determine isotope ratios from absorption features that can be distorted by overlapping VOC spectra (West et al., 2010; Millar et al., 2021; Herbstritt et al., 2024). IRMS instead determines isotope ratios after high-temperature conversion and gas separation and therefore does not require optical spectral correction (Gehre et al., 2015). In the paired CVD xylem extracts, OA-ICOS values exceeded IRMS values by 0.9‰ for δ18O and 4.2‰ for δ2H; the offset was larger for δ2H (Fig. 8). Overall, the CRAVE–IRMS workflow combines extraction in a sample vial maintained at 20–25 °C, recovery of storable liquid water, independent flow paths that can in principle be operated in parallel, and isotope measurement without laser spectral fitting.”
References
Allen, S. T. and Kirchner, J. W.: Potential effects of cryogenic extraction biases on plant water source partitioning inferred from xylem-water isotope ratios, Hydrological Processes, 36, e14483, https://doi.org/10.1002/hyp.14483, 2022.
Bailey, A., Noone, D., Berkelhammer, M., Steen-Larsen, H. C., and Sato, P.: The stability and calibration of water vapor isotope ratio measurements during long-term deployments, Atmos. Meas. Tech., 8, 4521-4538, 10.5194/amt-8-4521-2015, 2015.
Barbeta, A., Burlett, R., Martín-Gómez, P., Fréjaville, B., Devert, N., Wingate, L., Domec, J.-C., and Ogée, J.: Evidence for distinct isotopic compositions of sap and tissue water in tree stems: consequences for plant water source identification, New Phytologist, 233, 1121-1132, https://doi.org/10.1111/nph.17857, 2022.
Beyer, M., Kühnhammer, K., and Dubbert, M.: In situ measurements of soil and plant water isotopes: a review of approaches, practical considerations and a vision for the future, Hydrol. Earth Syst. Sci., 24, 4413-4440, 10.5194/hess-24-4413-2020, 2020.
Bowen, G. J., Banerjee, S., and Chakraborty, S.: Technical note: Spectral correction for cavity ring-down isotope analysis of plant and soil waters, Hydrol. Earth Syst. Sci., 29, 3907-3915, 10.5194/hess-29-3907-2025, 2025.
Ceperley, N., Gimeno, T. E., Jacobs, S. R., Beyer, M., Dubbert, M., Fischer, B., Geris, J., Holko, L., Kübert, A., Le Gall, S., Lehmann, M. M., Llorens, P., Millar, C., Penna, D., Prieto, I., Radolinski, J., Scandellari, F., Stockinger, M., Stumpp, C., Tetzlaff, D., van Meerveld, I., Werner, C., Yildiz, O., Zuecco, G., Barbeta, A., Orlowski, N., and Rothfuss, Y.: Toward a common methodological framework for the sampling, extraction, and isotopic analysis of water in the Critical Zone to study vegetation water use, WIREs Water, 11, e1727, 10.1002/wat2.1727, 2024.
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, 10.1073/pnas.2014422117, 2020.
Duvert, C., Barbeta, A., Hutley, L. B., Rodriguez, L., Irvine, D. J., and Taylor, A. R.: Cavitron extraction of xylem water suggests cryogenic extraction biases vary across species but are independent of tree water stress, Hydrological Processes, 38, e15099, https://doi.org/10.1002/hyp.15099, 2024.
Gat, J. R.: Oxygen and hydrogen isotopes in the hydrologic cycle, Annual Review of Earth and Planetary Sciences, 24, 225-262, https://doi.org/10.1146/annurev.earth.24.1.225, 1996.
Gehre, M., Renpenning, J., Gilevska, T., Qi, H., Coplen, T. B., Meijer, H. A. J., Brand, W. A., and Schimmelmann, A.: On-Line Hydrogen-Isotope Measurements of Organic Samples Using Elemental Chromium: An Extension for High Temperature Elemental-Analyzer Techniques, Analytical Chemistry, 87, 5198-5205, 10.1021/acs.analchem.5b00085, 2015.
Gralher, B., Herbstritt, B., and Weiler, M.: Technical note: Unresolved aspects of the direct vapor equilibration method for stable isotope analysis (δ18O, δ2H) of matrix-bound water: unifying protocols through empirical and mathematical scrutiny, Hydrology and Earth System Sciences, 25, 5219-5235, 10.5194/hess-25-5219-2021, 2021.
He, D., Wen, M., Wang, Y., Du, G., Zhang, C., He, H., Jin, J., Li, M., and Si, B.: Xylem water cryogenic vacuum extraction: Testing correction methods with CaviTron-based apple twig sampling, Journal of Hydrology, 621, 129572, https://doi.org/10.1016/j.jhydrol.2023.129572, 2023.
Herbstritt, B., Wengeler, L., and Orlowski, N.: Coping with spectral interferences when measuring water stable isotopes of vegetables, Rapid Communications in Mass Spectrometry, 38, e9907, https://doi.org/10.1002/rcm.9907, 2024.
Herbstritt, B., Gralher, B., Seeger, S., Rinderer, M., and Weiler, M.: Technical note: Discrete in situ vapor sampling for subsequent lab-based water stable isotope analysis, Hydrol. Earth Syst. Sci., 27, 3701-3718, 10.5194/hess-27-3701-2023, 2023.
Iraheta, A., Malsch-Fröhlich, E., Gerchow, M., and Beyer, M.: Technical note: Water vapor sampling for stable isotope analysis: systematic testing of sampling conditions and development of a robust protocol, Hydrol. Earth Syst. Sci., 30, 5005-5021, 10.5194/hess-30-5005-2026, 2026.
Kocum, J., Haidl, J., Gebousky, O., Falatkova, K., Sipek, V., Sanda, M., Orlowski, N., and Vlcek, L.: Technical note: A new laboratory approach to extract soil water for stable isotope analysis from large soil samples, Hydrol. Earth Syst. Sci., 29, 2863-2880, 10.5194/hess-29-2863-2025, 2025.
Liu, X., Chen, X., Zhang, Z., Liu, W., Peng, T., and McDonnell, J. J.: The role of rock fractures on tree water use of water stored in bedrock: Mixing and residence times, EGUsphere, 2025, 1-43, 10.5194/egusphere-2025-3937, 2025.
Magh, R. K., Gralher, B., Herbstritt, B., Kübert, A., Lim, H., Lundmark, T., and Marshall, J.: Technical note: Conservative storage of water vapour – practical in situ sampling of stable isotopes in tree stems, Hydrol. Earth Syst. Sci., 26, 3573-3587, 10.5194/hess-26-3573-2022, 2022.
Majoube, M.: Fractionnement en oxygène 18 et en deutérium entre l'eau et sa vapeur, Journal de Chimie Physique, 68, 1423-1436, 10.1051/jcp/1971681423, 1971.
Marshall, J. D., Cuntz, M., Beyer, M., Dubbert, M., and Kuehnhammer, K.: Borehole Equilibration: Testing a New Method to Monitor the Isotopic Composition of Tree Xylem Water in situ, Frontiers in Plant Science, Volume 11 - 2020, 10.3389/fpls.2020.00358, 2020.
Martín-Gómez, P., Barbeta, A., Voltas, J., Peñuelas, J., Dennis, K., Palacio, S., Dawson, T. E., and Ferrio, J. P.: Isotope-ratio infrared spectroscopy: a reliable tool for the investigation of plant-water sources?, New Phytologist, 207, 914-927, 10.1111/nph.13376, 2015.
Mattei, A., Barbecot, F., Guillon, S., Goblet, P., Hélie, J.-F., and Meyzonnat, G.: Improved accuracy and precision of water stable isotope measurements using the direct vapour equilibration method, Rapid Communications in Mass Spectrometry, 33, 1613-1622, https://doi.org/10.1002/rcm.8494, 2019.
Meißner, M., Köhler, M., Schwendenmann, L., Hölscher, D., and Dyckmans, J.: Soil water uptake by trees using water stable isotopes (δ2H and δ18O)−a method test regarding soil moisture, texture and carbonate, Plant and Soil, 376, 327-335, 10.1007/s11104-013-1970-z, 2014.
Millar, C., Pratt, D., Schneider, D. J., and McDonnell, J. J.: A comparison of extraction systems for plant water stable isotope analysis, Rapid Communications in Mass Spectrometry, 32, 1031-1044, https://doi.org/10.1002/rcm.8136, 2018.
Millar, C., Janzen, K., Nehemy, M. F., Koehler, G., Hervé-Fernández, P., and McDonnell, J. J.: Organic contamination detection for isotopic analysis of water by laser spectroscopy, Rapid Communications in Mass Spectrometry, 35, e9118, https://doi.org/10.1002/rcm.9118, 2021.
Nehemy, M. F., Millar, C., Janzen, K., Gaj, M., Pratt, D. L., Laroque, C. P., and McDonnell, J. J.: 17O-excess as a detector for co-extracted organics in vapor analyses of plant isotope signatures, Rapid Communications in Mass Spectrometry, 33, 1301-1310, https://doi.org/10.1002/rcm.8470, 2019.
Oerter, E. J., Siebert, G., Bowling, D. R., and Bowen, G.: Soil water vapour isotopes identify missing water source for streamside trees, Ecohydrology, 12, e2083, https://doi.org/10.1002/eco.2083, 2019.
Orlowski, N., Pratt, D. L., and McDonnell, J. J.: Intercomparison of soil pore water extraction methods for stable isotope analysis, Hydrological Processes, 30, 3434-3449, https://doi.org/10.1002/hyp.10870, 2016.
Orlowski, N., Breuer, L., Angeli, N., Boeckx, P., Brumbt, C., Cook, C. S., Dubbert, M., Dyckmans, J., Gallagher, B., Gralher, B., Herbstritt, B., Hervé-Fernández, P., Hissler, C., Koeniger, P., Legout, A., Macdonald, C. J., Oyarzún, C., Redelstein, R., Seidler, C., Siegwolf, R., Stumpp, C., Thomsen, S., Weiler, M., Werner, C., and McDonnell, J. J.: Inter-laboratory comparison of cryogenic water extraction systems for stable isotope analysis of soil water, Hydrol. Earth Syst. Sci., 22, 3619-3637, 10.5194/hess-22-3619-2018, 2018.
Pyschik, J., Seeger, S., Herbstritt, B., and Weiler, M.: Technical note: A fast and reproducible autosampler for direct vapor equilibration isotope measurements, Hydrol. Earth Syst. Sci., 29, 525-534, 10.5194/hess-29-525-2025, 2025.
Seeger, S. and Weiler, M.: Temporal dynamics of tree xylem water isotopes: in situ monitoring and modeling, Biogeosciences, 18, 4603-4627, 10.5194/bg-18-4603-2021, 2021.
Sprenger, M., Herbstritt, B., and Weiler, M.: Established methods and new opportunities for pore water stable isotope analysis, Hydrological Processes, 29, 5174-5192, https://doi.org/10.1002/hyp.10643, 2015.
Sprenger, M., Tetzlaff, D., and Soulsby, C.: Soil water stable isotopes reveal evaporation dynamics at the soil–plant–atmosphere interface of the critical zone, Hydrol. Earth Syst. Sci., 21, 3839-3858, 10.5194/hess-21-3839-2017, 2017.
Sprenger, M., Llorens, P., Cayuela, C., Gallart, F., and Latron, J.: Mechanisms of consistently disjunct soil water pools over (pore) space and time, Hydrology and Earth System Sciences, 23, 2751-2762, https://doi.org/10.5194/hess-23-2751-2019, 2019.
Tao, S., Xia, J., Xia, J., Chen, X., Lv, W., Gui, H., and Xu, J.: Stable water isotopes in soil–plant systems reveals ecohydrological dynamics in a typical flood wetland in the subtropical monsoon region, China, Ecological Indicators, 178, 114021, https://doi.org/10.1016/j.ecolind.2025.114021, 2025.
Vadibeler, D., Stockinger, M. P., Wassenaar, L. I., and Stumpp, C.: Influence of equilibration time, soil texture, and saturation on the accuracy of porewater water isotope assays using the direct H2O(liquid)–H2O(vapor) equilibration method, Journal of Hydrology, 607, 127560, https://doi.org/10.1016/j.jhydrol.2022.127560, 2022.
Volkmann, T. H. M. and Weiler, M.: Continual in situ monitoring of pore water stable isotopes in the subsurface, Hydrol. Earth Syst. Sci., 18, 1819-1833, 10.5194/hess-18-1819-2014, 2014.
Wang, H., Yu, H., He, D., Li, M., Si, B., McDonnell, J. J., and Nehemy, M. F.: Cryogenic vacuum distillation vs Cavitron methods in ecohydrology: Extraction protocol effects on plant water isotopic values, Journal of Hydrology, 642, 131853, https://doi.org/10.1016/j.jhydrol.2024.131853, 2024.
Wassenaar, L. I., Hendry, M. J., Chostner, V. L., and Lis, G. P.: High Resolution Pore Water δ2H and δ18O Measurements by H2O(liquid)−H2O(vapor) Equilibration Laser Spectroscopy, Environmental Science & Technology, 42, 9262-9267, 10.1021/es802065s, 2008.
Wen, M., Zhao, X., Si, B., He, D., Li, M., Gao, X., Cai, Y., Lu, Y., and Wang, Y.: Inter-comparison of extraction methods for plant water isotope analysis and its indicative significance, Journal of Hydrology, 625, 130015, https://doi.org/10.1016/j.jhydrol.2023.130015, 2023.
West, A. G., Patrickson, S. J., and Ehleringer, J. R.: Water extraction times for plant and soil materials used in stable isotope analysis, Rapid Communications in Mass Spectrometry, 20, 1317-1321, https://doi.org/10.1002/rcm.2456, 2006.
West, A. G., Goldsmith, G. R., Brooks, P. D., and Dawson, T. E.: Discrepancies between isotope ratio infrared spectroscopy and isotope ratio mass spectrometry for the stable isotope analysis of plant and soil waters, Rapid Communications in Mass Spectrometry, 24, 1948-1954, https://doi.org/10.1002/rcm.4597, 2010.
Younger, S. E., Blake, J., Jackson, C. R., and Aubrey, D. P.: δ2H isotopic offsets in xylem water measurements under cryogenic vacuum distillation: Quantifying and correcting wood-water hydrogen exchange influences, Ecohydrology, 17, e2640, https://doi.org/10.1002/eco.2640, 2024.
-
AC2: 'Reply on RC2', Xiuqiang Liu, 06 Sep 2026
Viewed
| HTML | XML | Total | BibTeX | EndNote | |
|---|---|---|---|---|---|
| 120 | 32 | 13 | 165 | 10 | 9 |
- HTML: 120
- PDF: 32
- XML: 13
- Total: 165
- BibTeX: 10
- EndNote: 9
Viewed (geographical distribution)
| Country | # | Views | % |
|---|
| Total: | 0 |
| HTML: | 0 |
| PDF: | 0 |
| XML: | 0 |
- 1
Water stable isotope biogeochemistry and, by extension, its domain of applications (e.g. isotope ecohydrology) is limited by how water is collected from mineral media and living tissues. Aside from technical issues potentially leading to incomplete recovery, questions recurrently arise about the representativeness of the extracted water as a pool or source mobilized by some process (e.g. plant root water uptake). In recent years, new methods and novel declinations of well-established ones have been published, aiming at simplifying procedures or at targeting water sources to address the aforementioned issues.
Xiuqiang Liu & colleagues’ method, namely the cryogenic airtight vapor extraction (CRAVE), is another relevant and potentially useful addition; it should, for instance, remove potential effects associated with the heating of the samples, their texture, and water availability.
I found the manuscript to be well-written, although would require streamlining in some sections (e.g in the introduction) and editing (isotope and physics terminology) – please see my specific comments below.
I have one general issue with the use of the Rayleigh distillation framework in the specific context of this study and urge the authors to address it. I simply do not think Eq. (1) applies here. Rayleigh distillation only applies during condensation of a water vapor reservoir into liquid water – with or without the removal of the condensate from the vapor-liquid system during “rain-out”. The authors’ case is rather different, they have instead:
The authors should also provide a comparison between the isotopic composition values of the water collected in the “condensation” U-tube and the values after correction with Eq. (3). This is missing for now and would help the readers to assess the magnitude of these corrections.
My specific comments follow below:
Title
I encourage the authors note to use the adjective “new”. The presented method will not indefinitely be “new” but the title will remain as is…
Abstract
Please harmonize and simplify the isotopic terminology (“composition” vs “signature”, “δ” instead of “d”).
L25. “greater hydrogen isotope depletion” -> “lower isotope composition”.
L26. “g water (g of dry soil)-1”. You may delete mention of “dry-weight basis”
L27. “depth” is not explained. Why would depth be a factor here? Information about the experimental design is missing.
L27. “where CVD-extracted bulk water δ2H and δ18O progressively decreased…”
L28. “The Rayleigh-based framework” is not evident and should be shortly explained before.
L31. This method does not qualify to “ambient-temperature extraction” as it operates with liquid Nitrogen, but the reader might think otherwise from reading the abstract alone.
Introduction
L40. Consider citing Ceperley et al.
Ceperley, N., Gimeno, T. E., Jacobs, S. R., Beyer, M., Dubbert, M., Fischer, B., Geris, J., Holko, L., Kuebert, A., Le Gall, S., Lehmann, M. M., Llorens, P., Millar, C., Penna, D., Prieto, I., Radolinski, J., Scandellari, F., Stockinger, M., Stumpp, C., Tetzlaff, D., van Meerveld, I., Werner, C., Yildiz, O., Zuecco, G., Barbeta, A., Orlowski, N., and Rothfuss, Y.: Toward a common methodological framework for the sampling, extraction, and isotopic analysis of water in the Critical Zone to study vegetation water use. Wiley Interdisciplinary Reviews-Water, 11(4), https://www.doi.org/10.1002/wat2.1727, 2024.
L47. I personally question any study extracting water at temperatures that high! This is contrary to standard recommendations (see study of Ceperley et al.). You want to achieve inverse-sublimation of water, not pyrolysis!
L55. Suction cups are not an alternative. On the one hand you have a destructive method and on the other hand an invasive one. The true alternatives are presented in the next paragraph and table 1 (e.g. centrifugation, direct soil vapor equilibration…).
L59. Please cite properly, either by year or first author name – please check journal guidelines.
L60. “These approaches offer high throughput and, in some cases, in situ monitoring capability when coupled with LS.” That cannot be the case, in situ observation are made where the experiments are conducted.
L63. You cannot compare in situ high frequency approaches with in labo low frequency ones: the latter measure repeatedly from the same sample, the latter includes spatial heterogeneities in isotopic composition.
Table 1 should be streamlined, e.g. do not write sentences in full, rather use bullet points. Waasenaar et al. DVE method is not to be pooled together with the rest…The rest of the “equilibration methods”, the in situ one, are not 1:1 comparable to the rest – please see my comment above – and should be treaded differently. I would actually not even bother to cite them here and in the intro at all.
L74-104. Text is not well structured despite the attempt to list the features of the presented method. Please streamline this and hierarchize the information in order of importance.
L75, L82. “sublimating”, not “condensing”
L74-79. Not clear at all what “quantified” and “measured” mean here.
L78. “However” is not correct. Also the three items (i)-(iii) do not add structuration and do not relate as is to the text preceding them. Please streamline this.
L83. A desiccant cannot trap VOCs. A solid phase extractor may do the job well, depending on the nature of the VOC (e.g. methanol, ethanol).
L85. Again, you are comparing apples with oranges and the statement is rather unfair to the in situ methods: condensation is not inherent to the in situ family of methods after the collection point and can be largely avoided. Condensation is, on the other hand, a major problem in water vapor continuous analysis.
L93. Which method uses “empirical post hoc corrections”? Also CRAVE uses a rayleigh distillation framework, for which α is determined empirically and a posteriori. So I don’t think the statement is valid.
L95. “QA/QC” is not defined.
L101. “leakage pathways”? “leakage” should be enough.
L102. Because of the somewhat chaotic nature of the text L74-104, I don’t know what “finally” exactly refers to. What came first, then second etc. before “finally”?
L110. Water does not condensate in liquid N, it inversely sublimates (sometimes referred as to “deposition”). This is an important terminology point.
L114. This is a “deposition U-tube”, not a “condensation U-tube”.
L120ff. “[deposition] vials (Gcv) [are] pre-weighed”. This is a general statement – did not happen once in the past. Same for “soil and plant materials [are] placed immediately” etc.
L129. What is meant by “pressure equilibrium”? Do you want to say “to avoid under/overpressure in the closed loop system due to changes in temperature overtime”? Either way, please add details in the text.
L131. “Cryogenic condensation” is “inverse sublimation”.
L132. How was the protocol “optimized”? Please add details.
L150. Water pools of what? Please explain / detail.
L160. I have a problem with using this equation in this particular context. Rayleigh distillation only applies during condensation of a water vapor reservoir into liquid water – with or without the removal of the condensate from the vapor-liquid system. Your case is rather different, you have:
Therefore, “reconstruction” of the “isotopic compositions of soil and plant water derived from the condensed vapor” is not granted with the Rayleigh distillation framework.
On a side note, all parameters and variables in Eq. (1) (α, δ, f) are unitless, therefore the factor “1000” is unnecessary. It should just read “1”.
L180. Please provide the definition of “immobile” versus “mobile” water pools in terms of matric potential.
L193. Did you sample pairs in unicates (no replicates)?
L194. Again, 200 degC is much too high for soil already, but not acceptable for plant organs or tissues. Are you sure we are talking about the actual sample temperature or the set point temperature of the heating device (, which may significantly differ)??
L232ff. Parts of this section should be moved to the material and methods.
L238. The reader cannot verify the statement “These metrics indicate that the Rayleigh-based equilibrium correction (Eq. 3) accurately reconstructs the source water composition by neutralizing effectively fractionation during vapor circulation and condensation.”) himself without the authors providing a comparison with and without “reconstruction” with Eq. 3. Please provide the corresponding data in the form of a table or figure.
L256. Providing two digits after the decimal point for δ2H and δ18O when in fact the precision is 10 or 100 times more (±1.0-2.0‰ for δ2H and ±0.2-0.8‰ for δ18O) is not correct.
Fig. 3. Nice figure! One can see from the spread in CVD versus CRAVE reference water extractions that CRAVE is generally producing more depleted water that CVD, which points out to possible incomplete water deposition (inverse sublimation) and/or non-equilibrium between water vapor and liquid water in the sample vial.
Fig. 5. Apparently, only CVD shows a depletion in both isotopes in deeper (>1 m) water. CRAVE and SL see less of a vertical isotopic composition gradient.
Fig. 6. Only the relationships between Eref and two of the three soil texture classes is needed since the 3rd relationship is a function of the first two. In other words, the displayed data is partly redundant.
Fig. 7. Providing the R2 for linear regressions that have a y-intercept different than zero makes little sense if you are aiming to evaluate how two procedures plot with respect to the 1:1 line.
Fig. 8. See previous comment.
Fig. 9. 1- Do you assume that the water content is the same between samples of each xylem pair? 2- How do you determine the xylem water content of the CRAVE sample? 3- I see that there are, for example, less samples between 1.0 and 1.5 g/g for δ18O than δ2H, how come?
L366. A clear definition of mobile/immobile water in the context of the present study is still missing at this point.
L371. CRAVE does not run isothermally, there is roughly a 200 degC difference between the sample and the trap.
406ff. The advantages of the CRAVE method, especially in terms of ease of use, number of samples to be extracted simultaneously etc. should be discussed here as well.