Ecohydrological isotope based field research is often constrained by a lack of temporally explicit soil water data, usually related to the choice of destructive sampling in the field and subsequent analysis in the laboratory. New techniques based on gas permeable membranes allow to sample soil water vapor in situ and infer soil liquid water isotopic signatures. Here, a membrane-based in situ soil water vapor sampling method was tested at a grassland site in Freiburg, Germany. It was further compared with two commonly used destructive sampling approaches for determination of soil liquid water isotopic signatures: cryogenic vacuum extraction and centrifugation. All methods were tested under semi-controlled field conditions, conducting an experiment with dry-wet cycling and two isotopically different labeling irrigation waters. We found mean absolute differences between cryogenic vacuum extraction and in situ vapor measurements of 0.3-14.2 (δ 18 O) and 0.4-152.2 (δ 2 H) for soil liquid water. The smallest differences were found under natural abundance conditions of 2 H and 18 O, the strongest differences were observed after irrigation with labeled waters. Labeling strongly increased the isotopic variation in soil water: Mean soil water isotopic signatures derived by cryogenic vacuum extraction were-11.6 ± 10.9 (δ 18 O) and +61.9 ± 266.3 (δ 2 H). The in situ soil water vapor method showed isotopic signatures of-12.5 ± 9.4 (δ 18 O) and +169.3 ± 261.5 (δ 2 H). Centrifugation was unsuccessful for soil samples due to low water recovery rates. It is therefore not recommended. Our study highlights that the in situ soil water vapor method captures the temporal dynamics in the isotopic signature of soil water well while the destructive approach also includes the natural lateral isotopic heterogeneity. The different advantages and limitations of the three methods regarding

Rationale: New methods to measure stable isotopes of soil and tree water directly in the field enable us to increase the temporal resolution of obtained data and advance our knowledge on the dynamics of soil and plant water fluxes. Only few field applications exist. However, these are needed to further improve novel methods and hence exploit their full potential. Methods:We tested the borehole equilibration method in the field and collected in situ and destructive samples of stable isotopes of soil, trunk and root xylem water over a 2.5-month experiment in a tropical dry forest under natural abundance conditions and following labelled irrigation. Water from destructive samples was extracted using cryogenic vacuum extraction. Isotope ratios were determined with IRIS instruments using cavity ring-down spectroscopy both in the field and in the laboratory.Results: In general, timelines of both methods agreed well for both soil and xylem samples. Irrigation labelled with heavy hydrogen isotopes clearly impacted the isotope composition of soil water and one of the two studied tree species. Intermethod deviations increased in consequence of labelling, which revealed their different capabilities to cover spatial and temporal heterogeneities. Conclusions:We applied the novel borehole equilibration method in a remote field location. Our experiment reinforced the potential of this in situ method for measuring xylem water isotopes in both tree trunks and roots and confirmed the reliability of gas permeable soil probes. However, in situ xylem measurements should be further developed to reduce the uncertainty within the range of natural abundance and hence enable their full potential. | INTRODUCTIONPlant transpiration fuels the hydrological cycle by returning 35% to 90% of water from land surfaces to the atmosphere. 1,2 Therefore, transpiration greatly influences our climate and impacts water availability in consequence of land use and climate change. 3,4 However, why is the quantification of this essential water flux so uncertain? This comes down to knowledge gaps in the mechanistic functioning of root water uptake (RWU) as well as plant rooting depth that persist despite its crucial role in predicting the future of one of our most important resources. 5 A major constraint is the practical difficulty in observing below-ground processes. Moreover, the magnitude and location of RWU are the results of multiple influencing factors, such as extent of the root system and its hydraulic properties,

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