3 Results and Discussion

3.1 SWRC

Figure 2 shows the modelled SWRCs for the measured means of the silty sand and clayey loam used in our study. The agreement between the fitted curves and the measured data was very good (R2=1.0 for the clayey loam and 0.98 for the silty sand, respectively). The silty sand’s SWRC was steeper and declined faster than the SWRC of the clayey loam. A sudden steepening of the slope indicates a distinct air-entry tension value, common for coarse soils (Wassar et al., 2016). The SWRC for the clayey loam showed a very high water retention at a suction head of 15,000 cm H2O. This water, held in the smallest pore spaces, is considered immobile or residual water. Soils with a high clay content appear to have a larger bound/residual water pool (Adams et al., 2019). The residual water content for the clayey loam was 26.4±1.1 Vol %, whereas the silty sand’s residual water content was 3.7±0.4 Vol %. Differences in SWRCs are dependent on various soil properties such as bulk density, organic carbon, soil texture and aggregate size (Lipiec et al., 2007). Sandy soils involve mainly capillary binding, and therefore release most of the water at higher potentials, while clayey soils, with adhesive and osmotic binding, release water at lower (more negative) potentials (Binkley and Fisher, 2012). Cryogenic water extraction following pF 3 resulted in a mean water content for the silty sand of 4.8±2.6 Vol % and for the clayey loam of 28.7±0.9 Vol % indicating that water extraction was not fully complete when compared to the values at the end of the pressure extraction. Cryogenic water extraction following pF 4.2 led to mean volumetric water contents of 3.8±1. Vol % and 26.1±1.3 %, respectively, which is comparable to the residual water content obtained via pressure plate extraction.

3.2 pF effects on isotopic composition

When now comparing the stable isotope values in dual isotope space from the sampling along the pF curve, most of the soil water isotope values plotted below and slightly to the right of the Global Meteoric Water Line (GMWL) (Fig. 3), whereas the introduced isotopic label (DIW) plotted on the GMWL. In general, the pF curve extracts from the silty sand and the clayey loam showed a similar isotopic composition and extraction behavior. With increasing pF values, the δ2H and δ18O composition tended to get heavier and moved up and slightly to the right of the GMWL (apart from pF 4.2). Only extracts from pF 4.2 showed a depletion in heavy isotopes for both soil types in comparison to the introduced label (DIW). This depletion was more pronounced for the silty sand extracts. When looking at the cryogenic extracts, the clayey loam cryo extracts taken after pF 4.2 did not show significant differences to the silty sand’s extracts at pF 4.2, the silty sand’s cryo extracts taken after pF 4.2 and the clayey loam cryo extracts taken after pF 3. However, they differed statistically significantly from all other pressure extracts. The silty sand’s cryo extracts taken after pF 3 showed the largest mean difference to the DIW in 18O-direction (3.3‰) and +12.4‰ in 2H-direction, whereas the cryo extracts taken after pF 4.2 and the pressure extracts from pF 4.2 differed from the DIW mainly in negative 2H-direction by-9.35‰ and -9.84‰, respectively. For the clayey loam, the pF 1.8 pressure extract showed the largest mean difference to the DIW (+1.19‰ for δ18O and +3.62‰ for δ2H). For both soil types, the extracts from pF 1.4 showed the smallest mean difference to the DIW in 18O-direction (-0.03‰ for the silty sand and +0.06‰ for the clayey loam). For δ2H, the mean difference to the DIW was within the range of the measurement accuracy of the isotope analysis (-0.12‰ for the silty sand and +0.40‰ for the clayey loam, respectively).
In general, the isotope values for the silty sand showed a much larger SD than the values of the clayey loam extracts (Fig. 3). For the silty sand, the cryo extracts from pF 3 were enriched in comparison to the DIW, whereas, the cryo extracts from pF 4.2 were depleted in heavy isotopes. Both differed significantly between each other (p=0.006). Such depletion could be an artifact of the cryogenic extraction and has been observed during water recovery tests of the same soil types in a dataset by Orlowski et al. (2013) and by others (e.g., Adams et al., 2019). However, in the study by Orlowski et al. (2013), the clayey loam cryo extracts showed a much larger depletion than the silty sand extracts, which was further dependent on water extraction times. The SDs of the δ18O values of the silty sand ranged between 0.02 and 0.73‰ and for the clayey loam between 0.07and 0.22‰ (pF 1.4–4.2). Whereas for δ2H, the SDs of the silty sand varied between 0.10 and 9.03 and for the clayey loam between 0.30 and 2.21. Except for the SD of 9.03 (pF 4.2, silty sand), the SDs of the δ2H and δ18O values were either within the range of the measurement accuracy for our isotope analyses (±0.2/±0.8‰ for δ18O/δ2H) or only slightly higher.
Lin et al. (2018) and Lin and Horita (2016) showed that the equilibrium fractionation factor changes if the vapor pressure controls the quantity of water adsorbed on a surface. These surface isotope effects are much stronger for 2H than for 18O (Chen et al., 2016; Lin et al., 2018). Gaj and McDonnell (2019) found out that soil tension affects the equilibrium fractionation factor for soil tensions above pF 3.1 (1,260 hPa). Their study included the exact same soil types as used in our study. In their study, the tension effect on the equilibrium fractionation factor increased linearly with increasing soil tension, which was independent on soil texture. The higher the soil tension, the farther away the isotope values plot from the introduced isotope label in their water recovery study using the water-vapor equilibrium method by Wassenaar et al. (2008). The authors hypothesized that adhesion is the cause of the additional fractionation on the water vapor isotopic composition. In our study, liquid water extracts were compared and not vapor samples. Nevertheless, our work showed that under dry conditions, soil tension is the main driver for isotope fractionation leading to isotopically enriched water extracts (see Fig. 3). According to Gaj and McDonnell (2019) immobile water at high soil tension would be depleted in the heavy isotopic species, which was only the case for the pF 4.2 extracts in our study but we did not take samples between pF 3 and 4.2, which could have further underlined this hypothesis. Nevertheless, this does not explain the isotopically enriched values of the silty sand’s cryo extracts taken after pF 3 (Fig. 3). We therefore suggest that future studies should consider testing soil tension effects for a variety of different soil types and at much higher resolution than in our study. However, if our findings are supported by others in future experiments, this would have important consequences for recent research on plant water uptake studies and interpreting the water isotopic composition of mobile and bulk water in soils (with respect to the tension water is held in the soil). We know that plants can apply high tensions to withdraw soil water, especially under dry conditions and that responses to water stress are species-specifically different (Fotelli et al., 2000). Given our findings, this would imply that isotopically more enriched water would be taken up by plants during dryer conditions (at higher tensions). However, soil properties and the soil’s water retention characteristics affect the isotopic composition of this water pool. Further, water uptake strategies of plants are highly species-specific and are not only influenced by soil water availability (Larcher, 2003). In contrast, Vargas et al. (2017) showed that avocado plants might preferentially take up 1H and 16O, leaving the remaining pool of water in the soil enriched. This discrimination was a function of the soil water loss and soil type. Barbeta et al. (2020) recently conducted a drought experiment with Fagus sylvaticawhere they compared the soil and stem water isotopic compositions. Under drier conditions, the authors observed soil-stem isotopic offsets. They hypothesized that adsorbed water dominates the fraction of bulk soil water under dry conditions. Depending on the balance between the isotopic enrichment caused by evaporation and the depletion caused by the higher fraction of adsorbed water, the isotopic composition of bulk soil water may therefore exhibit different trends with regard to its2H and 18O composition. This further affects the observed soil-stem isotopic offset (Barbeta et al., 2020). Lu (2016) demonstrated that the fraction of adsorbed water varies highly depending on the soil type and may range from 1.7% VWC in sandy soils to 12.8% VWC in silty clay soils. In the experiment by Barbeta et al. (2020), soil types showed a significant effect on the drying rate. Thus, exploring soil water retention characteristics with regard to plant water availabilities is important when comparing soil and plant water isotopic compositions and drawing assumptions with respect to plant water uptake depths, times and water stress responses.

3.3 Isotopic variation over time

Figure 4 depicts the δ2H and δ18O variation over 7 days for the two soil types when permanently exposed to a pressure of 15 bar in the pressure extractor (experiment 2). In general, the δ2H and δ18O values showed a similar trend. The silty sand extracts’ isotope values started off within the range of the DIW values, approached LW values over time and then decreased again. More specifically, the δ-values started to decrease after 30 min of extraction. From 150 min to 7,200 min the δ-values increased until the final sample reached approximately the starting value of -58.7‰ for δ2H. For silty sand’s δ18O values, the last sample’s δ18O value was by 0.5‰ higher than the starting value of -8.5‰. For silty sand’s δ2H values, the point of greatest difference to the starting value was reached after 60 min and it seemed like a plateau was reached which remained constant (within the range of measurement inaccuracy) for the next five values. The greatest SDs for the silty sand isotope values were observed at 7,200 min extraction time for both isotopes.
For the clayey loam, the isotopic variation over time was generally smaller. Interestingly, the δ18O values remained close to the DIW value (-8.6±0.2‰) almost over the entire 7 day extraction period, which was not the case for the silty sand extracts. This is surprising since LW was used for spiking the soil samples and DIW for wetting the ceramic plates of the extractors. The δ2H values remained within the range of the starting value (-58.7‰) and the DIW (-58.4‰), respectively, until they slightly decreased from 60 min onwards. The difference to the LW was smallest after 150 min extraction time. Surprisingly, the δ-values never reached the LW isotope values used for spiking, neither for δ2H nor for δ18O. The last sample’s δ-values (at 10,080 min) were even more positive than the DIW signature and showed the greatest difference to both the DIW and LW.
Figure 5 shows the mean differences over time between the clayey loam and silty sand extracts compared to the DIW’s and LW’s δ-values. Statistically, mean δ2H and δ18O differences to the LW were significantly different from zero for both soil types (p=0.00). However, the mean δ2H and δ18O differences to the DIW were only significantly different for the silty sand (p=0.00 for δ18O and δ2H) but nor for the clayey loam (p>0.05 for both isotopes).
Mean differences for the clayey loam extracts to the DIW ranged from -2.7‰ to 0.1‰ for δ2H and from -0.4‰ to 0.1‰ for δ18O, respectively. Mean differences to the LW showed a larger variation: from 3.6‰ to 6.3‰ for δ2H and from 0.8‰ to 1.3‰ for δ18O. For the δ2H values of the silty sand extracts, mean differences to the DIW ranged from -5.6‰ to 0.0‰ and to the LW from 0.7‰ to 6.3‰. For δ18O, mean differences to the DIW ranged from -1.1‰ to 0.6‰ and to the LW from 0.1‰ to 1.8‰. For the δ18O values of the clayey loam extracts, largest differences occurred at extraction times of 80 min (to DIW) and 10,080 min (to LW). Generally, δ-value differences were smaller to DIW than to LW for the clayey loam. This was not the case for the silty sand extracts. Interestingly, silty sand extracts from 60 to 105 min showed the smallest isotopic difference to the introduced LW, which was used for spiking the soil samples.
Thus, our null hypothesis that soil water collected sequentially over a period of 7 days under a 15 bar pressure would not differ isotopically from the introduced isotopic label did not hold true. The time at which the water draining from the pressure extractor did play a crucial role for the recovery of the introduced isotopic label. We further observed that the clayey loam extracts seemed to have liberated water from the ceramic plates, which was imprinted in the extracted isotopic signature (Fig. 4 and 5). However, the spiked label for the ceramic plates could still be recovered via cryogenic vacuum extraction and did not differ statistically significantly from the DIW (see Fig. 5). Thus, not as expected, we observed a temporal change (over 7 days) of the isotopic composition of the water extracted at 15 bar. Our findings suggest that isotopically different fractions of water in the two soil types were released over time. Since the silty sand releases water much faster than the clayey loam (Fig. 2), the introduced isotopic label was visible in the extracts after 60 min. However, for the clayey loam, it is obscure that even under 15 bar pressure a different soil water fraction can remain almost constant over time (Fig. 4) and the introduced isotopic label could not be recovered even when considering the given SD. This somehow contradicts the findings of others (Sprenger et al., 2016; Vargas et al., 2017) that tightly bound soil water quickly exchanges with mobile water in soils. Barbeta et al. (2020) demonstrated in a drought experiment with Fagus sylvatica an opposite isotope trend for δ2H and δ18O in soil water when the permanent wilting point had been reached and water potentials fell below -1MPa. They argue that soil evaporative enrichment creates a stronger enrichment in 18O than in2H and surface isotope effects are much stronger for2H than for 18O (Chen et al., 2016; Lin et al., 2018). Given this, it is possible that soil water δ18O enriches while soil water δ2H becomes depleted, at least when the soil water balance is dominated by root water uptake.
Gaj and McDonnell (2019) hypothesized that the soil water and soil vapor fractionation at high soil tension are driven by the surface properties and the ionic strength of the remaining soil solution. This implies that e.g. the interlayer space of clay minerals and mineral surfaces impact the amount and strength at which water is held in the soil (Gaj et al., 2017; Oerter et al., 2014). Further, water retention and O and H interactions with the soil matrix are higher for clay soils than for sandy soils (Thielemann et al., 2019). Adams et al. (2019) showed that the retention increased with increasing clay and silt contents. Our silty sand consisted of 92.7% sand and only 4.8% silt but our clayey loam had a clay fraction of 41.9%, which is rich in Vermiculite (43.4 %) (see Orlowski et al., 2018a). This is a 2:1 clay with a medium shrink-swell capacity but high cation exchange capacity. This has been shown to affect mineral-water interactions and thus cause isotope fractionation effects (Gaj et al., 2017; Meißner et al., 2014; Oerter et al., 2014). This might explain why in our experiment 2, the clayey loam water extracts showed a different extraction behavior to the silty sand when exposed to the highest pressure level (Fig. 5 and 6) and the clayey loam soil water most likely interacted with the ceramic plate water. Nevertheless, our results showed an extraction time-dependent effect on soil water held at 15bar. This might have implications on how we sample and interpret plant available soil water. If at a certain point in time plants would apply a constant tension to take up soil water, the timing of sampling for studying plant water uptake patterns would be highly relevant; since given our findings at 15bar pressure, the soil water isotopic composition would change over time. We admit that this is highly speculative but underlines the need for more research on time-variant changes in soil water pools relevant for plant water supply by e.g. simultaneously applying high-resolution in-situ isotope measurements at the soil and plant level.

3.4 Isotopic variation over time in dual isotope space

While testing whether soil water collected sequentially over a period of 7 days at 15 bar, we observed different effects on 2H and 18O when compared to the cryogenically extracted samples and the introduced labels (to the soils and ceramic plates). For δ18O, the DIW showed no statistically significant differences to the clayey loam extracts and the cryogenically extracted silty sand. However, for δ2H, DIW was significantly different to all tested subgroups (Fig. 6). Interestingly, the LW was statistically similar to the cryogenically extracted water from the ceramic plates for both isotopes. Additionally for δ2H there were no significant differences to the cryogenically extracted silty sand samples (p=0.41). The clayey loam extracts were statistically similar to the cryogenically extracted silty sand samples for δ18O, which was not true for δ2H (p=0.001). For δ18O, the cryogenically extracted clayey loam samples showed no significant differences to the silty sand samples from the pressure extractor but were significantly different to the clayey loam samples (p=0.006). The cryogenically extracted silty sand samples on the other hand did not differ significantly from the silty sand samples from the pressure extractor. This did not hold true for δ2H. But the cryogenically extracted silty sand samples were statistically similar to the cryogenically extracted ceramic plates and the cryogenically extracted clayey loam samples for δ2H. Given the statistical differences between the LW (used for spiking the soil samples) and the pressure plate extracts, we had to reject our null hypothesis. Soil water collected sequentially over 7 days at 15 bar did not show the same isotopic composition as the water used for spiking the samples. The water draining from the clayey loam surprisingly showed statistical similarities to the δ18O of the DIW (p=0.67) used for rewetting the ceramic plates of the extractor. This was not the case for δ2H. However, over time the LW must have exchanged the DIW of the ceramic plates, since we did not find statistical differences between the ceramic plate water and the LW for both isotopes. This leads to the conclusion that there was an isotopic exchange between the ceramic plate water and the water to be extracted from the saturated soil samples. Surprisingly, the exchange did not occur in experiment 1, which leads us to the conclusion that it is pressure level dependent. If it would have occurred, the extracted water isotopic composition would have plotted closer to the spiked isotopic label.
Through the cryogenic extraction of the silty sand, the isotopic composition of the water changed in a way that the δ2H values became more negative and the δ18O values became more positive in comparison to the water extracted from the pressure plates. Interestingly, the cryogenically extracted water from the clayey loam was more depleted in both heavy isotopes (2H and18O) than the clayey loam water extracted via the pressure plate but also the cryogenically extracted water from the silty sand was more depleted than the silty sand water from the pressure plate extraction. For a spiking experiment with the same soil types, Orlowski et al. (2013) observed that the cryogenically extracted silty sand water was more enriched in heavy isotopes and showed a smaller deviation from the spike water than the clayey loam, which plotted furthest away from the spike water. Here we saw a similar behavior with the cryogenically extracted clayey loam samples being more depleted but only the hydrogen isotopic composition of the silty sand extracts did not differ significantly from the introduced LW. Thus, we observed a deviation from the water used for spiking for both soil types.
When plotting the data in dual isotope space, we found statistically significant linear regressions for the samples of the different subgroups. The cryogenically extracted silty sand samples showed a much higher correlation among each other (R2=0.97) than the cryogenically extracted clayey loam samples (R2=0.62). The same was true for the clayey loam (R2=0.65) and silty sand samples (R2=0.96) from the pressure extractor (Fig. 6). The slopes of the different regression lines were very similar: The silty sand’s line had a slope of 4.01, the clayey loam’s of 4.50 and the cryo silty sand’s of 4.14; only the cryo clayey loam’s slope of 5.81 was slightly higher than the others. The intercepts of the regression lines from the cryogenically extracted samples and the pressure plate extraction for both soil types showed a difference of approximately 5. In the study by Gaj and McDonnell (2019), which included the same soil types, the slope of the regression lines decreased with decreasing grain size. Their sandy soils plotted on a regression line with a slope of 3 and the clayey soils’ regression line had a slope of 2. Such strong grain size dependency was not reflected in the slopes of the regression lines in our study. Much rather was there a cryogenic extraction induced effect on the clayey loam as previously observed by Orlowski et al. (2013).
In summary, the soil water sampled sequentially over 7 days at 15 bar deviated from the spiking water. The observed deviations changed over the time of the experiment and were larger for the clayey loam than for the silty sand and also different for the two isotopes (2H and 18O).