EGU General Assembly 2020
© Author(s) 2020. This work is distributed under
the Creative Commons Attribution 4.0 License.

Analysis of plant water stable isotopes using the water-vapor equilibrium method

Michael Stockinger1, Sabrina Santos Pires1,2, and Christine Stumpp1
Michael Stockinger et al.
  • 1University of Natural Resources and Life Sciences, Institute for Soil Physics and Rural Water Management (SoPhy), Vienna, Austria (
  • 2University of Hohenheim, Institute of Soil Science and Land Evaluation, Stuttgart, Germany

Plant water stable isotopes (δ18O, δ2H) have been used in eco-hydrological, biogeochemical and hydrological studies to e.g., quantify terrestrial water fluxes or to determine plant water sources. Current plant water extraction methods for isotope measurements are either expensive, labor-intensive or can lead to isotopic fractionation. Recent studies employed a new, extraction-free measurement method that was originally developed for the analysis of isotopes in sediment pore water: the water-vapor equilibrium method. It still needs to be tested if this method can be reliably used for isotope analysis of plant samples and how to best prepare the samples. Therefore, we investigated the effects of various preparation steps when measuring the plant water stable isotopes using this new method. We chose tomato and strawberry plants and prepared roots, shoots, leaves and fruits by either grinding or cutting them into pieces. Further, the necessary sample amount and the effect of equilibration time was evaluated. We investigated the effect of the preparation steps on mean values, standard deviations and a measurement device-specific value (LWV) that indicates a negative impact of volatile organic compounds (VOC) on reported isotope values. Results showed that an equilibration time longer than 24 hours is not advisable as the relationship between δ18O and δ2H of all plant samples worsened with R² declining from 0.97 to a minimum of 0.16. Additionally, the LVW indicated the influence of VOC with progressing equilibration time. Optimum amounts of plant material for roots were 3 g while for all other plant parts 5 g was necessary. In contrast to cut samples, kinetic fractionation effects were observed for grinded samples which could also be apparent fractionation effects because of the observed changes in LWV indicative of VOC interferences. For both plants the successive enrichment of the irrigation water from roots to leaves was observed. Fruits showed differences in their isotopic composition of the water stored inside the fruit compared to the water in the skin, with the inside water closer to the applied irrigation water. The intersection of the dual-isotope plot of all measured plant samples with the local meteoric water line was close to the applied irrigation water, making it theoretically possible to acquire information about the plant source water and enrichment factors in future studies when using the water-vapor equilibration method. From the findings of this study protocols can be established for sample preparation and plant water stable isotope analysis using the water-vapor equilibrium method.

How to cite: Stockinger, M., Santos Pires, S., and Stumpp, C.: Analysis of plant water stable isotopes using the water-vapor equilibrium method, EGU General Assembly 2020, Online, 4–8 May 2020, EGU2020-8859,, 2020

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  • CC1: Comment on EGU2020-8859, agnese aguzzoni, 06 May 2020

    Dear dr. Stockinger,

    the results of your test after water-vapor equilibrium are very interesting for their potential application to the study of plant water, overcoming the critical issues related to other water extraction techniques. Are you planning a comparison of these results with the results of other techniques? 

    In your opinion, combining this method to the isotope analysis, could it be possible to measure the isotope ratio of the mobile water only? 

    Finally, a question about your analyser: how is the CRDS equipped to directly measure the headspace isotope composition?

    many thanks,


    • AC1: Reply to CC1, Michael Stockinger, 06 May 2020

      Dear Agnese,

      thank you for your questions.

      We already planned to compare the results of the water-vapor equilibrium method with other techniques such as cryogenic extraction (keeping in mind which water pools each method accesses), but the current situation prevented us from the necessary laboratory work. We will do so in the near future.

      Regarding mobile water, I think the method has a good chance of aquiring mostly mobile water, keeping in mind the following: We finally used cut-up plant samples instead of grinding. I think grinding has a higher chance to access immobile (cell) water and the isotopes indicated that with much more enriched values compared to cutting. While cutting definitely also damages cells, it is my believe that this happens to a much lesser extent. In my view, the situation is further complicated by possible phloem water coming from the leaves and the potential of secondary evaporation from unsubarized stems.

      About setting up the CRDS Picarro: the Picarro has a line that continually sucks in carrier gas (atmosphere, or N2-gas, depending on lab setup). We used this line tubing and fitted it at the "atmosphere end" with a needle from medical supply. The Picarro continously measures the air coming in through this line, even if the autosampler/vaporizer are not operating. We now insert the needle into the headspace of our bags, and after about 4 minutes the ppm H2O and isotope water values stabilize. If the explanation was unclear, I could make a photo the next time in the lab (no garantuee when this will be).

      All the best,


  • CC2: Comment on EGU2020-8859, agnese aguzzoni, 06 May 2020

    Dear Michael,

    thank you for the much informative answer. Another question: in your opinion, can sample be stored in freezer (-80°C) before the water-vapour equilibration or is it better to perform the equilibration as soon as the samples are collected?



    • AC2: Reply to CC2, Michael Stockinger, 06 May 2020

      I would measure them as soon as possible. In the freezer at -80°C I would worry if the forming ice crystals could break open the plant cells, only to release the cell water when unfrozen that might disturb the measurement.

  • CC3: Comment on EGU2020-8859, Grzegorz Skrzypek, 07 May 2020

    Hi Michael

    This looks very similar to the concept published by Wassenaar et al. 2008 Environ. Sci. Technol. 2008.


    How are you normalizing your data to VSMOW? Against liquid standards and recalculating water-vapor fractionation at a given temperature?


    Crushing fruits or green material may lead to the production of organic substances. Did you check potential interference from organic substances?


    Thanks a lot in advance

    Best regards



    • AC4: Reply to CC3, Michael Stockinger, 07 May 2020

      Dear Greg,

      please find my reply to your questinos below as AC3, that I accidentally posted.

      Thank you.

      • CC4: Correction function?, Matthias Sprenger, 07 May 2020

        Hi Michael,

        These are really interesting results you present!

        I wonder if you see a chance in correction for VOCs or similar by using a similar approach as Benjamin Gralher did for CO2 on the Picarro instrument.

        Also, the more enriched isotopes in plant water compared to the soil water that you shown in the dual-isotope plots, is that a result from evaporation(?) fractionation in the plant or probabaly due to interference of organics with the laser?

        P.S.: I made similar experiments with cut (not grinded) Scots Pine samples and found extremely highly enriched isotope values due to VOCs interference on a LGR. I could smell the VOCs in the bag though. For Willow, the results were better though (less VOC). How do you see the chances of transferring the approach to trees?


        • AC5: Reply to CC4, Michael Stockinger, 08 May 2020

          Dear Matthias,


          I have not read too much into VOC and possible correction methods yet and we still want to do our measurements about it, so my only guess right now is that it might be possible to correct it. This also connects with your next questions: as we have not made the VOC measurements yet, we only assumed the presence and interference. It could be a combination of evaporative enrichment and VOC.

          As for the transferability, I think it could be transfered to any plant (part) as long as no massive interfering VOC is involved and we can reasonable assume that we sample the water that we are interested in.

          All the best, Michael

  • AC3: Comment on EGU2020-8859, Michael Stockinger, 07 May 2020

    Dear Greg,

    our study is based on the method of Wassenaar et al. (2008) but instead of soil samples we decided to test it for plant samples.

    For VSMOW normalization, we use liquid standards inbetween samples and then a linear regression as a correction function. Using temperature and equilibrium fractionation factor would also be an option of course.

    I totally agree with the organic substances and we highly suspect interference. In a further step, we want to test the sample air for this substances but were not able to do it yet due to the lockdown.

    All the best, Michael

  • CC5: Comment on EGU2020-8859, Magali Nehemy, 08 May 2020

    Dear Michael,

    Thanks for sharing your results! They look very interesting!

    I have myself been interested in applying the vapor equilibrium method in plant materials, especially from trees. In our lab we have been using the LGR machines, and early work by a colleague showed promising results with winter wheat (Millar et al 2018). We then decided to apply the same method to three different species from our study sites. We carried out analysis similar to yours, chopped samples and 24 hours equilibrium for plants (and 48h soil). We found a large influence of possible VOCs (as we compare the plants with MeOH and EtOH) even with 24h equilibrium. However, some species more so than others. Because LGR does not have a quantitative way of identifying spectral contamination, we found that measuring 17O (17O-excess) helped to quantify (mainly for narrowband contamination) and agreed with a qualitative flagging system and tests done with known concentrations of organics. Because I’m not familiar with Picarro, I wonder if you have used any flagging software during vapor measurement, or any other measures.

    Thanks for your time!



    Our work can be found here:

    • AC6: Reply to CC5, Michael Stockinger, 08 May 2020

      Dear Magali,

      thanks for sharing your paper, I will definitely read it soon! The Picarro has a recorded variable that seems to react to VOC, but we are still learning about it, so I'm carefully phrasing this.

      All the best, and greetings to Jeff (I assume you currently work at GIWS in Saskatoon)


      • CC6: Reply to AC6, Magali Nehemy, 14 May 2020

        Thanks Michael for your reply! Glad to hear that there are tools being tested on vapor mode on the Picarro end. I look forward to see the outcomes! Yes, I'm in Saskatoon doing my PhD with Jeff.

        All the best,