Using water-stable isotopes to track plant water uptake or soil water processes has become an invaluable tool in ecohydrology and physiological ecology. Recent studies have shown that laser absorption spectroscopy can measure equilibrated water vapour well enough to support inference of liquid-stable isotope composition of plant or soil water, on-site and in real-time. However, current in situ systems require the presence of an instrument in the field. Here we tested, first in the lab and then in the field, a method for equilibrating, collecting, storing, and finally
analysing water vapour for its isotopic composition that does not require an instrument in the field. We developed a vapour storage vial system (VSVS) that relies on in situ sampling into crimp neck vials with a double-coated cap using a pump and a flow metre powered through a small battery and measuring the samples in a laboratory. All components are inexpensive and commercially available. We tested the system's ability to store the isotopic composition of its contents by sampling a range of water vapour of known isotopic compositions (from
Since the introduction of isotope-ratio infrared spectrometers (IRIS), the analysis of water-stable isotope samples has become much more popular in many fields, e.g. in hydrogeological, watershed, oceanographic eco(hydro)logical studies (Tweed et al., 2019; Oerter and Bowen, 2017; Oerter et al., 2019; Beyer et al., 2020; Quade et al., 2019; Volkmann and Weiler, 2014; Volkmann et al., 2016b). This has led to an increased utility of water stable isotopes in applications, where interest in inferring plant water uptake depths and/or patterns and water movements through the soil matrix has grown tremendously (Eggemeyer et al., 2008; Liu et al., 2010; Beyer et al., 2016; Magh et al., 2020).
Until recently, however, samples of soil matrix- or plant tissue-bound water needed to be obtained destructively to extract the water samples. A method that is frequently used is cryogenic vacuum extraction, where a sample undergoes heating under vacuum, with the bound water evaporating in the process and subsequently being captured in a cryogenic trap (Ingraham and Shadel, 1992; Koeniger et al., 2011; Orlowski et al., 2013, 2016). This method was preferred because the assumed completeness of the water extraction was thought to eliminate fractionation. However, it has recently been heavily criticised for introducing biases due to artefacts coming from an exchangeable organic hydrogen pool in the plant biomass (Chen et al., 2020; Allen and Kirchner, 2022) and representing mainly the tightly bound water in the soil (Orlowski et al., 2016; Zhao et al., 2013).
A recently developed method based on direct vapour equilibration reduces the co-extraction of organic compounds and increases sample throughput (Millar et al., 2018; Wassenaar et al., 2008). One of the biggest advantages of in situ equilibration techniques is that water from plants and soils can be sampled at high temporal resolution without altering their physiology or physical properties (Kühnhammer et al., 2021). This is particularly noticeable when repeatedly sampling the same tree for cores, as water transport is repeatedly disrupted, whereas when using the in situ approach this only happens once. In the soil, the recurrence of drilling eventually alters the water flow of the entire plot as it opens many preferential flow channels in the same vicinity. Therefore, in situ measurements of water stable isotopes have gained popularity and have been proposed a way forward to disentangle isotopic processes in the critical zone or the soil–vegetation–atmosphere continuum (Rothfuss and Javaux, 2017; Beyer et al., 2020).
In situ measurement systems are based on direct inferences of liquid water isotopic composition from equilibrated water vapour from the soil or the plant (for a detailed review see Beyer et al., 2020). The vapour is collected using a gas-permeable membrane (the utility of which was proven by Herbstritt et al. (2012) buried in the soil (Rothfuss et al., 2013; Volkmann et al., 2016b; Volkmann and Weiler, 2014; Kübert et al., 2020), or in the xylem of woody species (Volkmann et al., 2016a, b; Seeger and Weiler, 2021), or drawing equilibrated water vapour from a borehole in the xylem directly (Marshall et al., 2020; Kühnhammer et al., 2021). Additionally, it is possible to measure the isotopic composition of plant transpiration and evapotranspiration in situ, using gas exchange chambers in the lab (Simonin et al., 2013; Dubbert et al., 2017), as well as in the field (Kübert et al., 2019; Dubbert et al., 2013; Wang et al., 2013).
The biggest advantage of these in situ systems is their ability to monitor real-time changes in water uptake and subsequent transport in plants and/or in soils and produce immediate data. The biggest disadvantage is the need for an IRIS at the site of measurement, which requires shelter, protection against vandals, and most importantly, access to a continuous power source. Additionally, the in situ setup in practice is limited in spatial resolution, as it requires tubing at the length of the distance from the sampling place to the IRIS, which is advisably kept short as increased tubing length increases the possibility of condensation (Beyer et al., 2020; Kühnhammer et al., 2021). These factors limit the utility of in situ measurement systems to field sites in the vicinity of the civil infrastructure, which potentially leads to research sites chosen because of their proximity to power rather than suitability as a research location, and therefore, location biases (e.g. monitoring wildlife in the vicinity of universities; Piccolo et al., 2020, or the location of protected areas worldwide; Joppa and Pfaff, 2009). Additionally, remote areas tend to lie in regions with less wealth, leading to an underrepresentation of research requiring cost-intensive equipment.
We therefore propose to adapt the above presented in situ measurement systems to mixed systems, where sample equilibration occurs in situ but analysis at a central laboratory. This should be useful where in situ measurements are impossible, owing to a lack of power supply and safe storage of equipment, or when large numbers of samples or simultaneous observation are required.
Here, we introduce an adapted sampling method based on a vacuum pump powered by a 12 V battery (derived from the borehole method by Marshall et al., 2020) and a commercially available storage container (adapted from the SWIS System introduced by Havranek et al. (2020), making the presence of an IRIS in the field redundant. We tested our VSVS (Vapour Storage Vial System) using water sources of known isotopic composition in an extensive lab trial and added data from a field trial carried out in a boreal forest in northern Sweden, where we could test the suitability of the proposed method and identify possible limitations. We include a section “preceding work” in the Results section to give the reader a chance to avoid repeating our failures if attempting to improve this methodology.
We conducted a laboratory test with water of known isotopic composition
(i.e. standards). The liquid standards (50 mL) were stored in 250 mL
Duran® bottles (DWK Life Sciences, Staffordshire, UK) closed with a rubber stopper allowing repeated sampling. The sampling vials were 50 mL crimp neck vials (VWR1548-2092, VWR International AB, Stockholm, Sweden). The vials were dried in the oven at 65
Setup of sampling in the lab experiment. The CRDS creates suction from the headspace of a water source of known isotopic signature (dark blue) into the crimped vials (turquoise). Pressure deficit is compensated for by air from a desiccant (Drierite®). The right side of the figure includes the same setup, this time as a schematic plan to improve readability. The isotopic composition and the water vapour concentration are monitored for 10 min before the vial is disconnected from the flow and stored for later analysis.
The lids ensured that the sample was in contact with only glass or PTFE (inner surface of the lid). PTFE is a diffusion-tight material, which is hydrophobic and chemically inert. It is recommended by the Picarro, Inc. (Santa Barbara, CA, USA) to use PTFE-coated lids to store liquid samples, and is therefore, to date, the most suitable material for storing water (vapour) samples if glass and stainless steel are unavailable.
The outer seal made from butyl ensured air-tight re-sealing after sampling via a 0.7 mm needle. Subsequently, the vials were flushed with air containing equilibrated water vapour of known isotopic composition (hereafter referred to as “Source”) for 10 min (see Fig. 1 for the setup) using the suction created by the cavity ring down spectrometer (CRDS, L2130-i; Picarro Inc., Santa Clara, CA, USA). Dry air was pulled from a laboratory gas drying unit (Drierite®, Fisher Scientific, UK), which dried the air down to 250–800 ppmV depending on room temperature. The dry air supply was connected using a silicone tube forced over PTFE tubing (
We monitored the water vapour concentration and isotopic composition as we
flushed the sample to be able to detect the time when the water vapour
concentration stabilised, which was after 8 min. After stabilisation, we
flushed the samples for 2 more minutes to allow for one more complete
exchange of the sample volume, leading to a total flushing time of 10 min
and six complete turnovers. As the flow rate created by the CRDS can vary
between instruments (ours was
Means and standard deviation (SD) for the five sources observed
immediately after the vials were filled (“0 d” samples), as well as
values for
Exemplified measurement data for the “heavy” source over a time
period of 5 min. Vapour concentration
We selected five sources of water with different isotopic composition to
test this method, not only for natural abundance applications but also for
examining the applicability for labelling studies, where water enriched in
Replicated vials were stored for 0, 1, 3, 4, 7, and 14 d, where storage of 0 d means the samples were analysed on the same day on which they were collected (“0 d” samples). Samples were kept in racks at room temperature in the
lab. Each source and each storage time consisted of at least 10 (five for
the sources “heavy” and “very heavy”) replicates. Before analysis, the
racks with the samples were placed on a heating plate at 40
For sample analysis, the dry air supply and the CRDS were directly connected
to the vial. We let the CRDS pull the sample vapour from the vial at the
same time as dry air replaced the now missing volume in the vial (at
We excluded the initial isotope purge by calculating the slope of the vapour
concentration over time. We filtered out all data before
To show that the cleaning protocol and diffusive exchange into or out of the sample vials was negligible, we sampled dry air from the desiccant tower into three sampling vials and stored those for 14 d. We then measured the vials using the same setup as with the “real” samples. The analysis of these vials was done as described in the Supplement and the data are summarised in Table S1 in the Supplement.
If we assume that the tubes began the 2-week test at
For further testing whether diffusive exchange was affecting the isotopic composition of the stored vials, we measured the isotopic composition of the atmosphere on several days during the lab experiment. We expected diffusive exchange with the atmosphere to lead to altered isotopic compositions of the samples in the direction towards said atmospheric composition.
We conducted our field test opportunistically during an ongoing tracer
pulse-chase experiment. The pulse chase involved the addition of
Comparison of in situ field setup
Using the VSVS, samples were collected by connecting the “inlet” side of
the borehole (in the original in situ system this side was exposed to the atmosphere) to a gas-drying unit (Drierite®, Fisher Scientific, UK) and using a vacuum pump (no name, 24 V,
We set the flow rate of the MFC to 110 mL min
Standards (i.e. the sources “light”, “heavy”, and “very heavy” from the lab test) were prepared in the same way as in the lab test, with modification of the higher flow rate and use of the pump in the field. All standards and samples were assigned to a storage group (i.e. 0, 1, 3, 7, 14 d). All samples were stored in the lab until analysis, except for the “0 d” samples, which were measured directly in the field 3 h after sampling.
Measurements were conducted as previously described in Sect. 2.1, with the
modification of measuring each sample for only 3.5 min. This was done because on the day of the field trial the inside of the borehole was colder
than the lab during the lab trial. The sampled air was therefore less moist,
leading to lower water vapour mixing ratios (wvmr, in ppmV) in the vials.
This meant that mixing with the dry air led to lower wvmr values more quickly
than for the samples in the lab test, reducing the time period when wvmr values
were within the target range between
We switched from the battery-driven pump sampling to the in situ system every 4 h. Because the schedule of the in situ setup measured this pine tree every 4 h, we were able to obtain two in situ measurements during the VSVS sampling day (one at 10:00 LT and again at 14:00 LT
Calculations as well as graphical representations were conducted using the
“tidyverse” packages in R (Wickham et al., 2019; R Core Team, 2020). To assess the VSVS's suitability to reliably store collected water vapour (assessing the “storage effect”), we calculated the change in isotopic composition (see Eq. 2 for either
We used the same model coefficients determined from the lab data to correct the field data samples. We additionally calculated the mean for each storage group (by source) and conducted pairwise Wilcoxon tests between the “0 d” samples and every other storage group, to disentangle effects introduced by the sampling method from storage. A Wilcoxon test is a non-parametric approach to detecting differences between two groups of data that are not normally distributed. Wilcoxon tests were conducted using the “compare_means” function of the “ggpubr” package in R (Kassambara, 2020).
To relate measurements to the liquid true values we used a linear regression
model for each storage group using the “lme4” package (Bates et al., 2015). We used three-point calibration for both
The first tests for this method originate from a field trial in a boreal forest, where some of the authors attempted to trace an enriched water pulse through 120 trees simultaneously. Briefly, a hole was drilled through the entire diameter of a tree stem, equipped with brass fittings (Ahlsell AB, Sweden), and sealed from the atmosphere using chlorol-butyl septa (Exetainer; Labco, Lampeter, UK). Syringes (Henke Sass Wolf, Tuttlingen, Germany) were used to draw out 20 mL of equilibrated xylem sap vapour and the isotopic composition was subsequently measured on a CRDS via injection into a dry air stream (Magh et al., 2021). The time between sampling and measurement varied between 20 min and up to 5 h.
We noticed that the water concentration and isotopic composition of the vapour in the syringes were altered within hours after sampling. The test revealed suitability for heavy label detection studies where, for example, response times revealed by isotope dynamics rather than absolute values may be of prime interest. However, we do not recommend using plastic syringes for long-term storage or for natural-abundance studies.
When developing the presented method further, we also tested crimp neck vials of 20 mL volumes, which would be even easier to transport and handle. However, after the first rounds of testing, we discovered that the volume was not large enough to give a stable 2 min isotope plateau when measuring, so we discarded the idea of using vials smaller than 50 mL.
Table 1 shows the mean and the variation occurring immediately after the vials were filled (“0 d” samples). These data give an overview of the minimum possible variation (method precision) during the sampling procedure and compare it with the expected values defined through the measurement of the liquid source on the CRDS. Results depended on the source sampled (see SD values in Table 1), indicating that the vapour sampling procedure introduces greater variation than the liquid phase measurements (Table 1).
We observed two different patterns between
Linear regressions for the change (
Looking at
The global meteoric water line reveals a tight relation between
We then analysed the uncertainty of the stored vapour samples based on their
true liquid isotopic composition. We used linear regression models for three
natural abundance sources (“light”, “medium”, “heavy”) and for three enriched sources (“heavy”, “very heavy”, “crazy heavy”) for
Linear regression through
The calibrated and uncalibrated data can be derived from Table S3 and are plotted in Fig. 7, showing that storage-effect correction and calibration reduce the variability between the storage groups, moving the samples close to their true liquid value.
Dual isotope plots of the raw mean (grey dots) and corrected and calibrated mean (coloured dots) of the lab trial storage data, corrected for the “storage effect” and calibrated using the linear regression models of each storage time. Sources are depicted by colour and the liquid true value is indicated by black stars. Panel
We recorded the atmosphere's isotopic composition during sampling and the measurement days to check for admixture of the atmosphere into the vial during storage (dashed blue lines in Fig. 7). We were thus able to rule out diffusion of atmosphere into the vials as all three standard sources would have been altered towards the atmospheric composition. This would have led to depletion rather than enrichment of heavy isotopes with increased storage periods, which was not the case (Figs. 4, 7 and Table S2).
The values of the VSVS samples were generally similar to the mean of the
in situ samples. The in situ data revealed stable
Dual isotope plot for a comparison of in situ and corrected VSVS data by storage time for the field trial. The in situ value is indicated by a star including the standard deviation represented as a grey bar. The uncorrected VSVS data are represented by grey dots, while the corrected data is indicated by color for the respective storage time.
We performed a lab trial of a water-vapour storage method using water sources covering stable isotope ratios in the natural abundance range and well beyond it into the range highly enriched in
We show that our adaptation of the in situ method (Marshall et al., 2020) can simplify the analysis while reliably reproducing the isotopic composition of natural abundance samples when measured on the same day. The method is robust and cost efficient as it uses only a battery-powered pump and a flow controller to collect the samples; then, the water vapour is stored in commercially available crimp vials, which can be re-used.
The reproducibility of measurements lies within the range reported for other
in situ approaches (e.g. Volkmann et al., 2016a). For example, the
median reproducibility was 2.8 ‰ for
Although we were unable to reproduce the standard value within the above
range for the samples highly enriched in
However, we point out that our sampling method did not reliably reproduce
In the crimp neck vials we observed a significant change in isotopic composition over time. The direction of change for
Cost [EUR] and time [
Changes in
In addition to the smaller sensitivity of
Given the potentially significant yet systematic shift in VSVS data over time, we strongly recommend preparing standards within the “equal treatment” framework, as emphasised in, for example, Gralher et al. (2021). This means that the standards are sampled on the same day as the samples, stored under the same conditions, and for the same period of time. One can then presume that any systematic, storage time-related isotopic shift in the samples is matched by the standards. Using this approach, we gained greater precision and accuracy for both lab- and field-based data.
For the field data set we emphasise the potential for additional variation
owing to the trees' water use and transport. As the sampled tree was constantly transpiring water throughout the sampling process and the sampling took roughly 50 min per storage group (for each sample, 10 min
To be able to make an informed decision about costs and time effort regarding the VSVS, we compare the VSVS with an in situ system and destructive sampling and subsequent extraction via a cryogenic extraction line after Koeniger et al. (2011) (Table 2). The data for the latter two have been obtained from Kübert et al. (2020). Each method has its own advantages and disadvantages. In terms of equipment costs, the VSVS is the cheapest, even when including the running costs involved in repeatedly buying new lids and needles. In terms of time effort, the in situ system and the VSVS are more efficient than obtaining and analysing samples for the cryogenic extraction line. Overall, the VSVS combines cost and time efficiency when compared with the two alternatives.
We introduced and tested a simple and cost-efficient approach to sampling and
storing water vapour to enable plant or soil water isotope measurements that
does not require access to line power. We proved the suitability of the sampling method within an extended precision range for natural abundance and
samples heavily enriched in
Code and data are available from the corresponding author upon request.
The supplement related to this article is available online at:
RKM and JM designed the experiments. RKM and AK conducted the experiments. RKM and HL performed the data analysis. RKM, BG, BH, and JM conceived the theoretical parts. RKM wrote the first draft of the manuscript and implemented the revisions together with JM. All authors contributed with advice, and prepared and reviewed the manuscript.
The contact author has declared that none of the authors has any competing interests.
Publisher's note: Copernicus Publications remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
We would like to thank Jose Gutierrez Lopez and Hjalmar Laudon for support during the field campaign. Special thanks also to Jonas Lundholm for providing advice and support in the lab.
Ruth-Kristina Magh and John Marshall were supported through grants from the Knut and Alice Wallenberg foundation: KAW 2018.0259 (Ruth-Kristina Magh and John Marshall) and KAW 2015.0047 (John Marshall). Angelika Kübert was funded by a “Short Term Scientific Mission (STSM)” grant provided by the COST Action (CA19120) WATer isotopeS in the critical zONe (WATSON).
This paper was edited by Miriam Coenders-Gerrits and reviewed by Rachel Havranek and one anonymous referee.