Rain recharges soil water storages and either percolates downward into aquifers and streams or is returned to the atmosphere through evapotranspiration. Although it is commonly assumed that summer rainfall recharges plant-available water during the growing season, the seasonal origins of water used by plants have not been systematically explored. We characterize the seasonal origins of waters in soils and trees by comparing their midsummer isotopic signatures (delta H-2) to seasonal isotopic cycles in precipitation, using a new seasonal origin index. Across 182 Swiss forest sites, xylem water isotopic signatures show that summer rain was not the predominant water source for midsummer transpiration in any of the three sampled tree species. Beech and oak mostly used winter precipitation, whereas spruce used water of more diverse seasonal origins. Even in the same plots, beech consistently used more winter precipitation than spruce, demonstrating consistent niche partitioning in the rhizosphere. All three species' xylem water isotopes indicate that trees used more winter precipitation in drier regions, potentially mitigating their vulnerability to summer droughts. The widespread occurrence of winter isotopic signatures in midsummer xylem implies that growing-season rainfall may have minimally recharged the soil water storages that supply tree growth, even across diverse humid climates (690-2068 mm annual precipitation). These results challenge common assumptions concerning how water flows through soils and is accessed by trees. Beyond these ecological and hydrological implications, our findings also imply that stable isotopes of delta O-18 and delta H-2 in plant tissues, which are often used in climate reconstructions, may not reflect water from growing-season climates.

A growing number of field studies report isotopic offsets between stem water and its potential sources that prevent the unambiguous identification of plant water origin using water isotopes. We explored the causes of this isotopic offset by conducting a controlled experiment on the temperate tree species Fagus sylvatica. We measured δ H and δ O of soil and stem water from potted saplings growing on three soil substrates and subjected to two watering regimes. Regardless of substrate, soil and stem water δ H were similar only near permanent wilting point. Under moister conditions, stem water δ H was 11 ± 3‰ more negative than soil water δ H, coherent with field studies. Under drier conditions, stem water δ H became progressively more enriched than soil water δ H. Although stem water δ O broadly reflected that of soil water, soil-stem δ H and δ O differences were correlated (r = 0.76) and increased with transpiration rates indicated by proxies. Soil-stem isotopic offsets are more likely to be caused by water isotope heterogeneities within the soil pore and stem tissues, which would be masked under drier conditions as a result of evaporative enrichment, than by fractionation under root water uptake. Our results challenge our current understanding of isotopic signals in the soil-plant continuum.© 2020 The Authors. New Phytologist © 2020 New Phytologist Trust.

. The number of ecohydrological studies involving water stable isotope measurements has been increasing steadily due to technological (e.g., field-deployable laser spectroscopy and cheaper instruments) and methodological (i.e., tracer approaches or improvements in root water uptake models)\nadvances in recent years. This enables researchers from a broad scientific background to incorporate water-isotope-based methods into their studies. Several isotope effects are currently not fully understood but might be essential when investigating root water uptake depths of vegetation and\nseparating isotope processes in the soil–vegetation–atmosphere continuum. Different viewpoints exist on (i) extraction methods for soil and plant\nwater and methodological artifacts potentially introduced by them, (ii) the pools of water (mobile vs. immobile) measured with those methods, and (iii) spatial variability and temporal dynamics of the water isotope composition of different compartments in terrestrial ecosystems. In situ methods have been proposed as an innovative and necessary way to address these issues and are required in order to disentangle isotope\neffects and take them into account when studying root water uptake depths of plants and for studying soil–plant–atmosphere interaction based on\nwater stable isotopes. Herein, we review the current status of in situ measurements of water stable isotopes in soils and plants, point out\ncurrent issues and highlight the potential for future research. Moreover, we put a strong focus and incorporate practical aspects into this review in\norder to provide a guideline for researchers with limited previous experience with in situ methods. We also include a section on opportunities for\nincorporating data obtained with described in situ methods into existing isotope-enabled ecohydrological models and provide examples illustrating\npotential benefits of doing so. Finally, we propose an integrated methodology for measuring both soil and plant water isotopes in situ when\ncarrying out studies at the soil–vegetation–atmosphere continuum. Several authors have shown that reliable data can be generated in the field using\nin situ methods for measuring the soil water isotope composition. For transpiration, reliable methods also exist but are not common in\necohydrological field studies due to the required effort. Little attention has been paid to in situ xylem water isotope measurements. Research\nneeds to focus on improving and further developing those methods. There is a need for a consistent and combined (soils and plants) methodology for ecohydrological studies. Such systems should be designed and\nadapted to the environment to be studied. We further conclude that many studies currently might not rely on in situ methods extensively because of\nthe technical difficulty and existing methodological uncertainties. Future research needs to aim on developing a simplified approach that provides a\nreasonable trade-off between practicability and precision and accuracy.\n

. Measurements of the isotopic composition of separate and\npotentially interacting pools of soil water provide a powerful means to\nprecisely resolve plant water sources and quantify water residence time and\nconnectivity among soil water regions during recharge events. Here we\npresent an approach for quantifying the time-dependent isotopic mixing of\nwater recovered at separate suction pressures or tensions in soil over an\nentire moisture release curve. We wetted oven-dried, homogenized sandy loam\nsoil first with isotopically “light” water (δ2H =-130 ‰; δ18O =-17.6 ‰) to represent antecedent moisture held at high matric tension. We then\nbrought the soil to near saturation with “heavy” water (δ2H =-44 ‰; δ18O =-7.8 ‰) that represented new input water. Soil water samples\nwere subsequently sequentially extracted at three tensions (“low-tension”\ncentrifugation ≈0.016 MPa; “mid-tension” centrifugation ≈1.14 MPa; and “high-tension” cryogenic vacuum distillation at an\nestimated tension greater than 100 MPa) after variable\nequilibration periods of 0 h, 8 h, 1 d, 3 d, and 7 d. We assessed the differences\nin the isotopic composition of extracted water over the 7 d equilibration\nperiod with a MANOVA and a model quantifying the time-dependent isotopic mixing\nof water towards equilibrium via self-diffusion. The simplified and\nhomogenous soil structure and nearly saturated moisture conditions used in\nour experiment likely facilitated rapid isotope mixing and equilibration\namong antecedent and new input water. Despite this, the isotope composition\nof waters extracted at mid compared with high tension remained significantly\ndifferent for up to 1 d, and waters extracted at low compared with\nhigh tension remained significantly different for longer than 3 d.\nComplete mixing (assuming no fractionation) for the pool of water extracted\nat high tension occurred after approximately 4.33 d. Our combination\napproach involving the extraction of water over different domains of the\nmoisture release curve will be useful for assessing how soil texture and\nother physical and chemical properties influence isotope exchange and mixing\ntimes for studies aiming to properly characterize and interpret the isotopic\ncomposition of extracted soil and plant waters, especially under variably\nunsaturated conditions.\n

Volatile organic compounds (VOCs) such as methanol and ethanol in water extracted from plants cause spectral interference in isotope ratio infrared spectroscopy (IRIS). This contamination degrades the accuracy of measurements, limiting the use of IRIS. In response, this study presents a new decontamination method of VOCs for enhanced IRIS measurements.The isotopic compositions of water from laboratory-made and field-collected plant samples pre- and post-treatment were analyzed using IRIS. Traditional treatment methods of activated charcoal and commercial pre-combustion systems (MCM) were compared with our new treatment method that implements solid-phase extraction (SPE). The absolute concentrations of contaminants pre- and post-treatment were determined using (1)H and (13)C nuclear magnetic resonance to assess the effectiveness of the different treatments.SPE removes an average of 86.7% and 78.8% ethanol and methanol, respectively, significantly reducing spectral interference. SPE reduces errors to within instrumental noise for both ethanol and methanol at concentrations found in nature (<3.0% and 0.08%, respectively). Activated charcoal minimally affected alcohol concentrations. MCM significantly worsened ethanol-contaminated water isotope measurements by producing primary alcohol oxidation products such as formic acid, another compound that interferes with IRIS absorption.SPE is an effective, low-cost method for eliminating errors in ethanol-contaminated samples. For samples where methanol is prevalent, combining SPE and MCM is more effective than the use of SPE alone. Hence, SPE treatment alone or in conjunction with MCM is recommended as an effective pre-analysis purification method for water extracted from plants.Copyright © 2016 John Wiley & Sons, Ltd.

. Hydrophilic surfaces influence the structure of water close to them and may thus affect the isotope composition of water. Such an effect should be relevant and detectable for materials with large surface areas and low water contents. The relationship between the volumetric solid : water ratio and the isotopic fractionation between adsorbed water and unconfined water was investigated for the materials silage, hay, organic soil (litter), filter paper, cotton, casein and flour. Each of these materials was equilibrated via the gas phase with unconfined water of known isotopic composition to quantify the isotopic difference between adsorbed water and unconfined water. Across all materials, isotopic fractionation was significant (p&lt;0.05) and negative (on average −0.91 ± 0.22 ‰ for 18∕16O and −20.6 ± 2.4 ‰ for 2∕1H at an average solid : water ratio of 0.9). The observed isotopic fractionation was not caused by solutes, volatiles or old water because the fractionation did not disappear for washed or oven-dried silage, the isotopic fractionation was also found in filter paper and cotton, and the fractionation was independent of the isotopic composition of the unconfined water. Isotopic fractionation became linearly more negative with increasing volumetric solid : water ratio and even exceeded −4 ‰ for 18∕16O and −44 ‰ for 2∕1H. This fractionation behaviour could be modelled by assuming two water layers: a thin layer that is in direct contact and influenced by the surface of the solid and a second layer of varying thickness depending on the total moisture content that is in equilibrium with the surrounding vapour. When we applied the model to soil water under grassland, the soil water extracted from 7 and 20 cm depth was significantly closer to local meteoric water than without correction for the surface effect. This study has major implications for the interpretation of the isotopic composition of water extracted from organic matter, especially when the volumetric solid : water ratio is larger than 0.5 or for processes occurring at the solid–water interface.\n

The hydrogen isotope ratio of water cryogenically extracted from plant stem samples (δH) is routinely used to aid isotope applications that span hydrological, ecological, and paleoclimatological research. However, an increasing number of studies have shown that a key assumption of these applications-that δH is equal to the δH of plant source water (δH)-is not necessarily met in plants from various habitats. To examine this assumption, we purposedly designed an experimental system to allow independent measurements of δH, δH, and δH of water transported in xylem conduits (δH) under controlled conditions. Our measurements performed on nine woody plant species from diverse habitats revealed a consistent and significant depletion in δH compared with both δH and δH Meanwhile, no significant discrepancy was observed between δH and δH in any of the plants investigated. These results cast significant doubt on the long-standing view that deuterium fractionation occurs during root water uptake and, alternatively, suggest that measurement bias inherent in the cryogenic extraction method is the root cause of δH depletion. We used a rehydration experiment to show that the stem water cryogenic extraction error could originate from a dynamic exchange between organically bound deuterium and liquid water during water extraction. In light of our finding, we suggest caution when partitioning plant water sources and reconstructing past climates using hydrogen isotopes, and carefully propose that the paradigm-shifting phenomenon of ecohydrological separation ("two water worlds") is underpinned by an extraction artifact.

The recent development of isotope ratio infrared spectroscopy (IRIS) was quickly followed by the addition of online extraction and analysis systems, making it faster and easier to measure soil and plant water isotopes. However, memory and sample size effects limit the efficiency and accuracy of these new setups. In response, this study presents a scheme dedicated to estimating and eliminating these two effects.Memory effect was determined by injecting two standard waters alternately. Each standard was injected nine times in a row and analyzed using induction module cavity ring-down spectroscopy (IM-CRDS). Memory coefficients were calculated using a new "multistage jump" algorithm. Sample size effects were evaluated by injecting water volumes ranging from 1 μL to 6 μL. Finally, the influence of cellulose filter paper on the isotopic measurements, the memory, and the sample size effect was evaluated by comparing it with glass filter paper.Memory effects were detected for both δ O and δ H values, with the latter being stronger. Isotopic differences between replicates of the same plant or soil sample showed a clear decrease after memory correction. A small water volume effect was found only when the injected water volume was larger than 3 μL. However, while the correction method performed well for laboratory-made samples, it did not for field samples, due to the heterogeneity of the isotopic composition of the samples. Stronger memory and water volume effects were found for cellulose filter paper.The memory coefficients and the water volume-isotope relationship improved the consistency and accuracy of both laboratory and field data. Our results indicate that cellulose filter paper may not be a suitable medium to measure standard waters and evaluate memory and water volume effects. Finally, a detailed correction and calibration protocol is suggested, along with notes on best practices to obtain good-quality IM-CRDS data. Copyright © 2017 John Wiley & Sons, Ltd.Copyright © 2017 John Wiley & Sons, Ltd.

. Stable isotopologues of water are widely used to derive relative root water uptake (RWU) profiles and average RWU depth in lignified plants. Uniform isotope composition of plant xylem water (δxyl) along the stem length of woody plants is a central assumption of the isotope tracing approach which has never been properly evaluated. Here we evaluate whether strong variation in δxyl within woody plants exists using empirical field observations from French Guiana, northwestern China, and Germany. In addition, supported by a mechanistic plant hydraulic model, we test hypotheses on how variation in δxyl can develop through the effects of diurnal variation in RWU, sap flux density, diffusion, and various other soil and plant parameters on the δxyl of woody plants. The hydrogen and oxygen isotope composition of plant xylem water shows strong temporal (i.e., sub-daily) and spatial (i.e., along the stem) variation ranging up to 25.2 ‰ and 6.8 ‰ for δ2H and δ18O, respectively, greatly exceeding the measurement error range in all evaluated datasets. Model explorations predict that significant δxyl variation could arise from diurnal RWU fluctuations and vertical soil water heterogeneity. Moreover, significant differences in δxyl emerge between individuals that differ only in sap flux densities or are monitored at different times or heights. This work shows a complex pattern of δxyl transport in the soil–root–xylem system which can be related to the dynamics of RWU by plants. These dynamics complicate the assessment of RWU when using stable water isotopologues but also open new opportunities to study drought responses to environmental drivers. We propose including the monitoring of sap flow and soil matric potential for more robust estimates of average RWU depth and expansion of attainable insights in plant drought strategies and responses.

Plants mediate water fluxes within the soil-vegetation-atmosphere continuum. This water transfer in soils, through plants, into the atmosphere can be effectively traced by stable isotopologues of water. However, rapid dynamic processes have only recently gained attention, such as adaptations in root water uptake depths (within hours to days) or the imprint of transpirational fluxes on atmospheric moisture, particularly promoted by the development of real-time in-situ water vapour stable isotope observation techniques. We focus on open questions and emerging insights at the soil-plant and plant-atmosphere interfaces, as we believe that these are the controlling factors for ecosystem water cycling. At both interfaces, complex pictures of interacting ecophysiological and hydrological processes emerge: root water uptake dynamics depend on both spatiotemporal variations in water availability and species-specific regulation of adaptive root conductivity within the rooting system by, for example, modulating soil-root conductivity in response to water and nutrient demands. Similarly, plant water transport and losses are a fine-tuned interplay between species-specific structural and functional strategies of water use and atmospheric processes. We propose that only by explicitly merging insights from distinct disciplines - for example, hydrology, plant physiology and atmospheric sciences - will we gain a holistic picture of the impact of vegetation on processes governing the soil-plant-atmosphere continuum.© 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.

This study demonstrates the application of Wavelength-Scanned Cavity Ring-Down Spectroscopy (WS-CRDS) technology which is used to measure the stable isotopic composition of water. This isotopic water analyzer incorporates an evaporator system that allows liquid water as well as water vapor to be measured with high precision. The analyzer can measure H2(18)O, H2(16)O and HD(16)O content of the water sample simultaneously. The results of a laboratory test and two field trials with this analyzer are described. The results of these trials show that the isotopic water analyzer gives precise, accurate measurements with little or no instrument drift for the two most common isotopologues of water. In the laboratory the analyzer has a precision of 0.5 per mil for deltaD and 0.1 per mil for delta(18)O which is similar to the precision obtained by laboratory-based isotope ratio mass spectrometers. In the field, when measuring vapor samples, the analyzer has a precision of 1.0 per mil for deltaD and 0.2 per mil for delta(18)O. These results demonstrate that the isotopic water analyzer is a powerful tool that is appropriate for use in a wide range of applications and environments.2009 John Wiley & Sons, Ltd.

. A method to measure the δ2H and δ18O composition of pore waters in saturated and unsaturated geologic core samples using direct vapour equilibration and laser spectrometry (DVE–LS) was first described in 2008, and has since been rapidly adopted. Here, we describe a number of important methodological improvements and limitations encountered in routine application of DVE–LS over several years. Generally, good comparative agreement, as well as accuracy, is obtained between core pore water isotopic data obtained using DVE–LS and that measured on water squeezed from the same core. In complex hydrogeologic settings, high-resolution DVE–LS depth profiles provide greater spatial resolution of isotopic profiles compared to long-screened or nested piezometers. When fluid is used during drilling and coring (e.g. water rotary or wet sonic drill methods), spiking the drill fluid with 2H can be conducted to identify core contamination. DVE–LS analyses yield accurate formational isotopic data for fine-textured core (e.g. clay, shale) samples, but are less effective for cores obtained from saturated permeable (e.g. sand, gravels) geologic media or on chip samples that are easily contaminated by wet rotary drilling fluid. Data obtained from DVE–LS analyses of core samples collected using wet (contamination by drill water) and dry sonic (water loss by heating) methods were also problematic. Accurate DVE–LS results can be obtained on core samples with gravimetric water contents &gt; 5 % by increasing the sample size tested. Inexpensive Ziploc™ gas-sampling bags were determined to be as good as, if not better than, other, more expensive specialty bags. Sample storage in sample bags provides acceptable results for up to 10 days of storage; however, measurable water loss, as well as evaporitic isotopic enrichment, occurs for samples stored for up to 6 months. With appropriate care taken during sample collection and storage, the DVE–LS approach for obtaining high-resolution pore water isotopic data is a promising alternative to study the hydrogeology of saturated and unsaturated sediments. Eliminating analytical interferences from volatile organics remains a challenge.\n

Water stress is regarded as a global challenge to forests. Unlike other water-limited areas, the water use strategies of rocky mountainous forests, which play an important ecohydrological role, have not received sufficient attention. To prove our hypothesis that species adopt different water use strategies to avoid competition of limited water resources, we used site abiotic monitoring, sap flow and stable isotope method to study the biophysiological responses and water use preferences of two commonly distributed forest species, (Pt) and (Qv). The results showed that Pt transpired higher than Qv. Pt was also prone to adopt isohydric water use strategy as it demonstrated sensitive stomatal control over water loss through transpiration. Qv developed cavitation which was reflected by the dropping in response to high vapor pressure deficit, concentrated peak sap flux density (), and enlarged hysteresis loop. Considering the average soil depth of 52.8 cm on the site, a common strategy shared by both species was the ability to tap water from deep soil layers (below 40 cm) when soil water was limited, and this contributed to the whole growing season transpiration. The contribution of surface layer water to plant water use increased and became the main water source for transpiration after rainfall. Qv was more efficient at using water from surface layer than Pt due to the developed surface root system when soil water content was not stressed. Our study proves that different water-using strategies of co-occurring species may be conducive to avoid competition of limited water resources to guarantee their survival. Knowledge of water stress-coping strategies of trees has implications for the understanding and prediction of vegetation composition in similar areas and can facilitate forest management criteria for plantations.

Our understanding of how temporal variations of atmospheric water vapour and its isotopic composition (δ O ) influence water and assimilates in plants remains limited, restricting our ability to use δ O as a tracer of ecophysiological processes. We exposed oak (Quercus robur) saplings under wet and dry soil moisture conditions to O-depleted water vapour (c. - 200‰) at high relative humidity (c. 93%) for 5 h, simulating a fog event. We then traced the step change in δ O into water and assimilates (e.g. sucrose, hexoses, quercitol and starch) in the leaf lamina, main veins and twigs over 24 h. The immediate δ O effect was highest for δ O of leaf lamina water, but 40% lower on δ O of main vein water. To a smaller extent, we also observed changes in δ O of twig xylem water. Depending on the individual assimilation rate of each plant, the O-label was partitioned among different assimilates, with highest changes in δ O of starch/sucrose and lowest in δ O of quercitol. Additionally, O-label partitioning and allocation towards leaf starch and twig phloem sugars was influenced by the plant water status. Our results have important implications for water isotope heterogeneity in plants and for our understanding of how the δ O signal is incorporated into biomarkers.© 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

Newly available gas analyzers based on off-axis integrated cavity output spectroscopy (OA-ICOS) lasers have been advocated as an alternative to conventional isotope-ratio mass spectrometers (IRMS) for the stable isotopic analysis of water samples. In the case of H2O, OA-ICOS is attractive because it has comparatively low capital and maintenance costs, the instrument is small and field laboratory portable, and provides simultaneous D/H and 16O/18O ratio measurements directly on H2O molecules with no conversion of H2O to H2, CO, or H2/CO2-water equilibration required. Here we present a detailed assessment of the performance of a liquid-water isotope analyzer, including instrument precision, estimates of sample memory and sample mass effects, and instrumental drift. We provide a recommended analysis procedure to achieve optimum results using OA-ICOS. Our results show that, by using a systematic sample analysis and data normalization procedure routine, measurement accuracies of +/-0.8 per thousand for deltaD and +/-0.1 per thousand delta18O are achievable on nanoliter water samples. This is equivalent or better than current IRMS-based methods and at a comparable sample throughput rate.

以往通常假设降水与土壤水完全混合后形成径流,而基于δD-δ¹⁸O关系研究表明,降水与土壤水混合存在生态水文分离现象,即土壤水可分为由土壤无效水和可供植物吸收的有效水构成的束缚水及自由移动形成径流的自由水,且两个水库间存在着部分混合即连接性.本研究系统阐述了“生态水文分离”的概念及其内涵,描述了降水与土壤水的混合过程以及两个水库δD和δ¹⁸O的特征与关系,总结了土壤水、束缚水及自由水δD和δ¹⁸O直接观测及替代观测方法的优缺点,并阐明了径流小区及流域尺度上基于直接及替代观测方法的土壤束缚水与自由水完全分离及连接性的定性研究进展,同时阐明了基于模型和控制试验的土壤束缚水与自由水完全分离及连接性的定量研究进展,并指出应加强生态水文分离过程的定性及定量方法以及对传统生态水文模型的影响和改进研究.

Determining the mechanisms of flux through protein channels requires a combination of structural data, permeability measurement, and molecular dynamics (MD) simulations. To further clarify the mechanism of flux through aquaporin 1 (AQP1), osmotic pf (cm3/s/pore) and diffusion pd (cm3/s/pore) permeability coefficients per pore of H2O and D2O in AQP1 were calculated using MD simulations. We then compared the simulation results with experimental measurements of the osmotic AQP1 permeabilities of H2O and D2O. In this manner we evaluated the ability of MD simulations to predict actual flux results. For the MD simulations, the force field parameters of the D2O model were reparameterized from the TIP3P water model to reproduce the experimentally observed difference in the bulk self diffusion constants of H2O vs. D2O. Two MD systems (one for each solvent) were constructed, each containing explicit palmitoyl-oleoyl-phosphatidyl-ethanolamine (POPE) phospholipid molecules, solvent, and AQP1. It was found that the calculated value of pf for D2O is ∼15% smaller than for H2O. Bovine AQP1 was reconstituted into palmitoyl-oleoyl-phosphatidylcholine (POPC) liposomes, and it was found that the measured macroscopic osmotic permeability coefficient Pf (cm/s) of D2O is ∼21% lower than for H2O. The combined computational and experimental results suggest that deuterium oxide permeability through AQP1 is similar to that of water. The slightly lower observed osmotic permeability of D2O compared to H2O in AQP1 is most likely due to the lower self diffusion constant of D2O.

Stable isotopes are extensively used as tracers for the study of plant-water sources. Isotope-ratio infrared spectroscopy (IRIS) offers a cheaper alternative to isotope-ratio mass spectroscopy (IRMS), but its use in studying plant and soil water is limited by the spectral interference caused by organic contaminants. Here, we examine two approaches to cope with contaminated samples in IRIS: on-line oxidation of organic compounds (MCM) and post-processing correction. We assessed these methods compared to IRMS across 136 samples of xylem and soil water, and a set of ethanol- and methanol-water mixtures. A post-processing correction significantly improved IRIS accuracy in both natural samples and alcohol dilutions, being effective with concentrations up to 8% of ethanol and 0.4% of methanol. MCM outperformed the post-processing correction in removing methanol interference, but did not effectively remove interference for high concentrations of ethanol. By using both approaches, IRIS can overcome with reasonable accuracy the analytical uncertainties associated with most organic contaminants found in soil and xylem water. We recommend the post-processing correction as the first choice for analysis of samples of unknown contamination. Nevertheless, MCM can be more effective for evaluating samples containing contaminants responsible for strong spectral interferences at low concentrations, such as methanol. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

The stable isotope ratios of water (δ H and δ O values) have been widely used to trace water in plants in a variety of physiological, ecohydrological, biogeochemical and hydrological studies. In such work, the analyte must first be extracted from samples, prior to isotopic analysis. While cryogenic vacuum distillation is currently the most widely used method reported in the literature, a variety of extraction-collection-analysis methods exist. A formal inter-method comparison on plant tissues has yet to be carried out.We performed an inter-method comparison of six plant water extraction techniques: direct vapour equilibration, microwave extraction, two unique versions of cryogenic vacuum distillation, centrifugation, and high-pressure mechanical squeezing. These methods were applied to four isotopically unique plant portions (head, stem, leaf, and root crown) of spring wheat (Triticum aestivum L.). Extracted plant water was analyzed via spectrometric (OA-ICOS) and mass-based (IRMS) analysis systems when possible. Spring wheat was grown under controlled conditions with irrigation inputs of a known isotopic composition.The tested methods of extraction yielded markedly different isotopic signatures. Centrifugation, microwave extraction, direct vapour equilibration, and high-pressure mechanical squeezing produced water more enriched in H and O content. Both cryogenic vacuum distillation systems and the high-pressure mechanical squeezing method produced water more depleted in H and O content, depending upon the plant portion extracted. The various methods also produced differing concentrations of co-extracted organic compounds, depending on the mode of extraction. Overall, the direct vapor equilibration method outperformed all other methods.Despite its popularity, cryogenic vacuum distillation was outperformed by the direct vapor equilibration method in terms of limited co-extraction of volatile organic compounds, rapid sample throughput, and near instantaneous returned stable isotope results. More research is now needed with other plant species, especially woody plants, to see how far the findings from this study could be extended.Copyright © 2018 John Wiley & Sons, Ltd.

Stable isotopes are a powerful tool for ecologists, often used to assess contributions of different sources to a mixture (e.g. prey to a consumer). Mixing models use stable isotope data to estimate the contribution of sources to a mixture. Uncertainty associated with mixing models is often substantial, but has not yet been fully incorporated in models. We developed a Bayesian-mixing model that estimates probability distributions of source contributions to a mixture while explicitly accounting for uncertainty associated with multiple sources, fractionation and isotope signatures. This model also allows for optional incorporation of informative prior information in analyses. We demonstrate our model using a predator-prey case study. Accounting for uncertainty in mixing model inputs can change the variability, magnitude and rank order of estimates of prey (source) contributions to the predator (mixture). Isotope mixing models need to fully account for uncertainty in order to accurately estimate source contributions.

. For more than two decades, research groups in hydrology, ecology, soil science, and biogeochemistry have performed cryogenic water extractions (CWEs) for the analysis of δ2H and δ18O of soil water. Recent studies have shown that extraction conditions (time, temperature, and vacuum) along with physicochemical soil properties may affect extracted soil water isotope composition. Here we present results from the first worldwide round robin laboratory intercomparison. We test the null hypothesis that, with identical soils, standards, extraction protocols, and isotope analyses, cryogenic extractions across all laboratories are identical. Two standard soils with different physicochemical characteristics along with deionized (DI) reference water of known isotopic composition were shipped to 16 participating laboratories. Participants oven-dried and rewetted the soils to 8 and 20 % gravimetric water content (WC), using the deionized reference water. One batch of soil samples was extracted via predefined extraction conditions (time, temperature, and vacuum) identical to all laboratories; the second batch was extracted via conditions considered routine in the respective laboratory. All extracted water samples were analyzed for δ18O and δ2H by the lead laboratory (Global Institute for Water Security, GIWS, Saskatoon, Canada) using both a laser and an isotope ratio mass spectrometer (OA-ICOS and IRMS, respectively). We rejected the null hypothesis. Our results showed large differences in retrieved isotopic signatures among participating laboratories linked to soil type and soil water content with mean differences compared to the reference water ranging from +18.1 to −108.4 ‰ for δ2H and +11.8 to −14.9 ‰ for δ18O across all laboratories. In addition, differences were observed between OA-ICOS and IRMS isotope data. These were related to spectral interferences during OA-ICOS analysis that are especially problematic for the clayey loam soils used. While the types of cryogenic extraction lab construction varied from manifold systems to single chambers, no clear trends between system construction, applied extraction conditions, and extraction results were found. Rather, observed differences in the isotope data were influenced by interactions between multiple factors (soil type and properties, soil water content, system setup, extraction efficiency, extraction system leaks, and each lab's internal accuracy). Our results question the usefulness of cryogenic extraction as a standard for water extraction since results are not comparable across laboratories. This suggests that defining any sort of standard extraction procedure applicable across laboratories is challenging. Laboratories might have to establish calibration functions for their specific extraction system for each natural soil type, individually.\n

. In this commentary, we summarize and build upon discussions that\nemerged during the workshop “Isotope-based studies of water partitioning and\nplant–soil interactions in forested and agricultural environments” held in\nSan Casciano in Val di Pesa, Italy, in September 2017. Quantifying and\nunderstanding how water cycles through the Earth's critical zone is important\nto provide society and policymakers with the scientific background to manage\nwater resources sustainably, especially considering the ever-increasing\nworldwide concern about water scarcity. Stable isotopes of hydrogen and\noxygen in water have proven to be a powerful tool for tracking water fluxes in\nthe critical zone. However, both mechanistic complexities (e.g. mixing and\nfractionation processes, heterogeneity of natural systems) and methodological\nissues (e.g. lack of standard protocols to sample specific compartments,\nsuch as soil water and xylem water) limit the application of stable water\nisotopes in critical-zone\nscience. In this commentary, we examine some of the\nopportunities and critical challenges of isotope-based ecohydrological\napplications and outline new perspectives focused on interdisciplinary\nresearch opportunities for this important tool in water and environmental\nscience.\n

. This study evaluated between-sample memory in isotopic measurements of δ2H and δ18O in water samples by laser spectroscopy. Ten isotopically depleted water samples spanning a broad range of oxygen and hydrogen isotopic compositions were measured by three generations of off-axis integrated cavity output spectroscopy and cavity ring-down spectroscopy instruments. The analysis procedure encompassed small (less than 2‰ for δ2H and 1‰ for δ18O) and large (up to 201‰ for δ2H and 25‰ for δ18O) differences in isotopic compositions between adjacent sample vials. Samples were injected 18 times each, and the between-sample memory effect was quantified for each analysis run. Results showed that samples adversely affected by between-sample isotopic differences stabilised after seven–eight injections. The between-sample memory effect ranged from 14% and 9% for δ2H and δ18O measurements, respectively, but declined to negligible carryover (between 0.1% and 0.3% for both isotopes) when the first ten injections of each sample were discarded. The measurement variability (range and standard deviation) was strongly dependent on the isotopic difference between adjacent vials. Standard deviations were up to 7.5‰ for δ2H and 0.54‰ for δ18O when all injections were retained in the computation of the reportable δ-value, but a significant increase in measurement precision (standard deviation in the range 0.1‰–1.0‰ for δ2H and 0.05‰–0.17‰ for δ18O) was obtained when the first eight injections were discarded. In conclusion, this study provided a practical solution to mitigate between-sample memory effects in the isotopic analysis of water samples by laser spectroscopy.\n

Stable isotopes are increasingly being used as tracers in environmental studies. One application is to use isotopic ratios to quantitatively determine the proportional contribution of several sources to a mixture, such as the proportion of various pollution sources in a waste stream. In general, the proportional contributions of n+1 different sources can be uniquely determined by the use of n different isotope system tracers (e.g., delta13C, delta15N, delta18O) with linear mixing models based on mass balance equations. Often, however, the number of potential sources exceeds n+1, which prevents finding a unique solution of source proportions. What can be done in these situations? While no definitive solution exists, we propose a method that is informative in determining bounds for the contributions of each source. In this method, all possible combinations of each source contribution (0-100%) are examined in small increments (e.g., 1%). Combinations that sum to the observed mixture isotopic signatures within a small tolerance (e.g., +/-0.1 per thousand ) are considered to be feasible solutions, from which the frequency and range of potential source contributions can be determined. To avoid misrepresenting the results, users of this procedure should report the distribution of feasible solutions rather than focusing on a single value such as the mean. We applied this method to a variety of environmental studies in which stable isotope tracers were used to quantify the relative magnitude of multiple sources, including (1) plant water use, (2) geochemistry, (3) air pollution, and (4) dietary analysis. This method gives the range of isotopically determined source contributions; additional non-isotopic constraints specific to each study may be used to further restrict this range. The breadth of the isotopically determined ranges depends on the geometry of the mixing space and the similarity of source and mixture isotopic signatures. A sensitivity analysis indicated that the estimated ranges vary only modestly with different choices of source increment and mass balance tolerance parameter values. A computer program (IsoSource) to perform these calculations for user-specified data is available at http://www.epa.gov/wed/pages/models.htm.

. Plant root water uptake (RWU) has been documented for the past five decades from water stable isotopic analysis. By comparing the (hydrogen or oxygen) stable isotopic compositions of plant xylem water to those of potential contributive water sources (e.g., water from different soil layers, groundwater, water from recent precipitation or from a nearby stream), studies were able to determine the relative contributions of these water sources to RWU. In this paper, the different methods used for locating/quantifying relative contributions of water sources to RWU (i.e., graphical inference, statistical (e.g., Bayesian) multi-source linear mixing models) are reviewed with emphasis on their respective advantages and drawbacks. The graphical and statistical methods are tested against a physically based analytical RWU model during a series of virtual experiments differing in the depth of the groundwater table, the soil surface water status, and the plant transpiration rate value. The benchmarking of these methods illustrates the limitations of the graphical and statistical methods while it underlines the performance of one Bayesian mixing model. The simplest two-end-member mixing model is also successfully tested when all possible sources in the soil can be identified to define the two end-members and compute their isotopic compositions. Finally, the authors call for a development of approaches coupling physically based RWU models with controlled condition experimental setups.\n

. Disentangling ecosystem evapotranspiration (ET) into evaporation (E) and transpiration (T) is of high relevance for a wide range of applications, from land surface modelling to policymaking. Identifying and analysing the determinants of the ratio of T to ET (T/ET) for various land covers and uses, especially in view of climate change with an increased frequency of extreme events (e.g. heatwaves and floods), is prerequisite for forecasting the hydroclimate of the future and tackling present issues, such as agricultural and irrigation practices. One partitioning method consists of determining the water stable isotopic compositions of ET, E, and T (δET, δE, and δE, respectively) from the water retrieved from the atmosphere, the soil, and the plant vascular tissues. The present work emphasizes the challenges this particular method faces (e.g. the spatial and temporal representativeness of the T/ET estimates, the limitations of the models used, and the sensitivities to their driving parameters) and the progress that needs to be made in light of the recent methodological developments. As our review is intended for a broader audience beyond the isotopic ecohydrological and micrometeorological communities, it also attempts to provide a thorough review of the ensemble of techniques used for determining δET, δE, and δE and solving the partitioning equation for T/ET. From the current state of research, we conclude that the most promising way forward to ET partitioning and capturing the subdaily dynamics of T/ET is by making use of non-destructive online monitoring techniques of the stable isotopic composition of soil and xylem water. Effort should continue towards the application of the eddy covariance technique for high-frequency determination of δET at the field scale as well as the concomitant determination of δET, δE, and δE at high vertical resolution with field-deployable lift systems.

Concern exists about the suitability of laser spectroscopic instruments for the measurement of the (18)O/(16)O and (2)H/(1)H values of liquid samples other than pure water. It is possible to derive erroneous isotope values due to optical interference by certain organic compounds, including some commonly present in ecosystem-derived samples such as leaf or soil waters. Here we investigated the reliability of wavelength-scanned cavity ring-down spectroscopy (CRDS) (18)O/(16)O and (2)H/(1)H measurements from a range of ecosystem-derived waters, through comparison with isotope ratio mass spectrometry (IRMS). We tested the residual of the spectral fit S(r) calculated by the CRDS instrument as a means to quantify the difference between the CRDS and IRMS δ-values. There was very good overall agreement between the CRDS and IRMS values for both isotopes, but differences of up to 2.3‰ (δ(18)O values) and 23‰ (δ(2)H values) were observed in leaf water extracts from Citrus limon and Alnus cordata. The S(r) statistic successfully detected contaminated samples. Treatment of Citrus leaf water with activated charcoal reduced, but did not eliminate, δ(2)H(CRDS) - δ(2)H(IRMS) linearly for the tested range of 0-20% charcoal. The effect of distillation temperature on the degree of contamination was large, particularly for δ(2)H values but variable, resulting in positive, negative or no correlation with distillation temperature. S(r) and δ(CRDS) - δ(IRMS) were highly correlated, in particular for δ(2)H values, across the range of samples that we tested, indicating the potential to use this relationship to correct the δ-values of contaminated plant water extracts. We also examined the sensitivity of the CRDS system to changes in the temperature of its operating environment. We found that temperature changes ≥4 °C for δ(18)O values and ≥10 °C for δ(2)H values resulted in errors larger than the CRDS precision for the respective isotopes and advise the use of such instruments only in sufficiently temperature-stabilised environments.Copyright © 2011 John Wiley & Sons, Ltd.

. We developed a setup for a fully automated, high-frequency in situ monitoring system of the stable water isotope deuterium and 18O in soil water and tree xylem. The setup was tested for 12 weeks within an isotopic labeling experiment during a large artificial sprinkling experiment including three mature European beech (Fagus sylvatica) trees. Our setup allowed for one measurement every 12–20 min, enabling us to obtain about seven measurements per day for each of our 15 in situ probes in the soil and tree xylem. While the labeling induced an abrupt step pulse in the soil water isotopic signature, it took 7 to 10 d until the isotopic signatures at the trees' stem bases reached their peak label concentrations and it took about 14 d until the isotopic signatures at 8 m stem height leveled off around the same values. During the experiment, we observed the effects of several rain events and dry periods on the xylem water isotopic signatures, which fluctuated between the measured isotopic signatures observed in the upper and lower soil horizons. In order to explain our observations, we combined an already existing root water uptake (RWU) model with a newly developed approach to simulate the propagation of isotopic signatures from the root tips to the stem base and further up along the stem. The key to a proper simulation of the observed short-term dynamics of xylem water isotopes was accounting for sap flow velocities and the flow path length distribution within the root and stem xylem. Our modeling framework allowed us to identify parameter values that relate to root depth, horizontal root distribution and wilting point. The insights gained from this study can help to improve the representation of stable water isotopes in trees within ecohydrological models and the prediction of transit time distribution and water age of transpiration fluxes.

The two-pool and Péclet effect models represent two theories describing mechanistic controls underlying leaf water oxygen isotope composition at the whole-leaf level (δ(18) OL ). To test these models, we used a laser spectrometer coupled to a gas-exchange cuvette to make online measurements of δ(18) O of transpiration (δ(18) Otrans ) and transpiration rate (E) in 61 cotton (Gossypium hirsutum) leaves. δ(18) Otrans measurements permitted direct calculation of δ(18) O at the sites of evaporation (δ(18) Oe ) which, combined with values of δ(18) OL from the same leaves, allowed unbiased estimation of the proportional deviation of enrichment of δ(18) OL from that of δ(18) Oe (f) under both steady-state (SS) and non-steady-state (NSS) conditions. Among all leaves measured, f expressed relative to both δ(18) O of transpired water (ftrans ) and source water (fsw ) remained relatively constant with a mean ± SD of 0.11 ± 0.05 and 0.13 ± 0.05, respectively, regardless of variation in E spanning 0.8-9.1 mmol m(-2)  s(-1). Neither ftrans nor fsw exhibited a significant difference between the SS and NSS leaves at the P < 0.05 level. Our results suggest that the simpler two-pool model is adequate for predicting cotton leaf water enrichment at the whole-leaf level. We discuss the implications of adopting a two-pool concept for isotopic applications in ecological studies. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

. The storage and release of water in soils is critical for\nsustaining plant transpiration and groundwater recharge. However, how much\nsubsurface mixing of water occurs, and how much of the water is available for plants or otherwise percolates to streams and the groundwater is not yet\nunderstood. Based on stable isotope (2H and\n18O) data, some studies have found that water infiltrating into soils can bypass older pore\nwater. However, the mechanisms leading to the separation of water routed\nto the streams and water held tightly in smaller pores are still unclear.\nHere, we address the current limitations of the understanding of subsurface\nmixing and their consequences regarding the application of stable isotopes in\necohydrological studies. We present an extensive data set, for which we\nsampled the isotopic composition of mobile and bulk soil water\nin parallel with groundwater at a fortnightly temporal resolution and stream water and rainfall at a much higher resolution in a Mediterranean\nlong-term research catchment, in Vallcebre, Spain. The data reveal that the\nmobile and tightly bound water of a silty loam soil in a Scots pine forest\ndo not mix well; however, they constitute two disjunct subsurface water pools\nwith little exchange, despite intense rainfall events leading to high soil\nwetness. We show that the isotopic compartmentalization results from the rewetting\nof small soil pores by isotopically depleted winter/spring rain. Thus,\nstable isotopes, and, in turn, water residence times, do not only vary\nacross soil depth, but also across soil pores. Our findings have important\nimplications for stable isotope applications in ecohydrological studies\nassessing the water uptake by plants or the process realism of hydrological models,\nas the observed processes are currently rarely implemented in the simulation\nof water partitioning into evapotranspiration and recharge in the critical\nzone.\n

水分是生态系统的重要因子, 水同位素自然示踪和人工标记是研究生态系统水循环过程的重要方法, 利用水同位素所具有的示踪、整合和指示等功能特征, 通过测量和分析生态系统中不同组分所含水分的氢氧同位素比值的变化情况, 可实现生态系统蒸散发的拆分、植物水分来源判定和叶片水同位素富集机理研究, 是研究生态系统水循环过程机理和生态学效应不可或缺的技术手段。该文首先简要回顾了生态系统水同位素发展和应用的历史, 在此基础上阐述了水同位素技术和方法在生态学研究热点领域应用的基本原理, 概述了水同位素在植物水分来源判定、蒸散发拆分、露水来源拆分、降水的水汽来源拆分以及 ¹⁷O-excess的研究进展, 并介绍了植物叶片水富集机理及基于稳定同位素的碳水耦合研究。最后, 指出了水同位素研究亟待解决的问题, 展望了水同位素应用的前沿方向, 旨在利用水同位素分析加深对生态系统的水分动态、植被格局和生理过程的理解。

We tested for isotope exchange between bound (immobile) and mobile soil water, and whether there is isotope fractionation during plant water uptake. These are critical assumptions to the formulation of the 'two water worlds' hypothesis based on isotope profiles of soil water. In two different soil types, soil-bound water in two sets of 19-l pots, each with a 2-yr-old avocado plant (Persea americana), were identically labeled with tap water. After which, one set received isotopically enriched water whereas the other set received tap water as the mobile phase water. After a dry down period, we analyzed plant stem water as a proxy for soil-bound water as well as total soil water by cryogenic distillation. Seventy-five to 95% of the bound water isotopically exchanged with the mobile water phase. In addition, plants discriminated against O and H during water uptake, and this discrimination is a function of the soil water loss and soil type. The present experiment shows that the assumptions for the 'two water worlds' hypothesis are not supported. We propose a novel explanation for the discrepancy between isotope ratios of the soil water profile and other water compartments in the hydrological cycle.© 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

. Stable isotope signatures provide an integral fingerprint of origin, flow paths, transport processes, and residence times of water in the environment. However, the full potential of stable isotopes to quantitatively characterize subsurface water dynamics is yet unfolded due to the difficulty in obtaining extensive, detailed, and repeated measurements of pore water in the unsaturated and saturated zone. This paper presents a functional and cost-efficient system for non-destructive continual in situ monitoring of pore water stable isotope signatures with high resolution. Automatic controllable valve arrays are used to continuously extract diluted water vapor in soil air via a branching network of small microporous probes into a commercial laser-based isotope analyzer. Normalized liquid-phase isotope signatures are then obtained based on a specific on-site calibration approach along with basic corrections for instrument bias and temperature dependent isotopic fractionation. The system was applied to sample depth profiles on three experimental plots with varied vegetation cover in southwest Germany. Two methods (i.e., based on advective versus diffusive vapor extraction) and two modes of sampling (i.e., using multiple permanently installed probes versus a single repeatedly inserted probe) were tested and compared. The results show that the isotope distribution along natural profiles could be resolved with sufficiently high accuracy and precision at sampling intervals of less than four minutes. The presented in situ approaches may thereby be used interchangeably with each other and with concurrent laboratory-based direct equilibration measurements of destructively collected samples. It is thus found that the introduced sampling techniques provide powerful tools towards a detailed quantitative understanding of dynamic and heterogeneous shallow subsurface and vadose zone processes.\n

土壤-植被-大气连续体(SPAC)是陆地水文学、生态学和全球变化领域的重要研究对象,其水碳循环过程及耦合机制是前沿性问题.稳定同位素技术示踪、整合和指示的特征有助于评估分析生态系统固碳和耗水情况.本文在简述稳定同位素应用原理和技术的基础上,重点阐释了基于稳定同位素光学技术的SPAC系统水碳交换研究进展,包括:在净碳通量中拆分光合与呼吸量,在蒸散通量中拆分蒸腾与蒸发量,以及在系统尺度上的水碳耦合研究.新兴的技术和方法实现了生态系统尺度上长期高频的同位素观测,但在测量精准度、生态系统呼吸拆分、非稳态模型适应性、尺度转换和水碳耦合机制等方面存在挑战.本文探讨了现有主要研究成果、局限性以及未来研究展望,以期对稳定同位素生态学领域的新研究和技术发展有所帮助.