Mechanism And Improvement Of Isotopic Bias On Cryogenic Vacuum Extracted Soil Water And Plant Water

Understanding the water relationship and interaction between soil and plant has always been a hot spot and difficulty in eco-hydrological research.Stable hydrogen and oxygen isotopes have become an important technique to solve soil and eco-hydrological scientific questions because of their accurate water-tracing function.However,the inaccuracy or mismatch of water isotopes,sampled from complex systems such as soil and plants,has become one of the main bottlenecks limiting the application of isotopes.Cryogenic vacuum extraction（CVE）,the most widely used mothed to extract water from soil and plant,can lead to different degrees of isotope deviation,but the fractionation phenomenon and mechanism in the CVE process are still poorly understood.Therefore,this study systematically explored the mechanism of soil and plant water isotope errors caused by CVE,then put forward corresponding improvements.The main conclusions are as follows:（1）Spiking tests were conducted on soil samples oven-dried at 105℃and 205℃,respectively.The recovery rate and isotopic composition of the extracted water were analyzed.Our results show that conventional ovendry（105℃）was not sufficient to liberate tightly bound water from soils but ovendry at temperature above 205℃was.Part of the tightly bound water in soil was extracted by CVE,because the extraction capacity of CVE（heating at 95℃）was equivalent to ovendrying at temperature between 105℃and 205℃.Moreover,the two pretreatments yielded markedly differences in water recovery rates.With increasing ratios of clay content to water content,the recovery rates of the pretreated soil at 105℃trended upward above 100%,but that of 205℃trended downward below 100%.Further,²H and ¹⁸O of the extracted water from the soil pretreated at 205℃were both depleted relative to reference water,but the pretreatment at 105℃showed enriched ¹⁸O and depleted ²H.This suggests that oxygen isotope exchange may have occurred between tightly bound water and soil minerals,resulting in enriched ¹⁸O in water for the 105℃pretreatments.For the 205℃pretreatment,soil water was completely removed,isotopic signatures of the extracted water followed equilibrium fractionation law due to incomplete extraction.Therefore,our results suggested that the biased isotopic signatures of water extracted by CVE may be partially due to incomplete extraction and mixture of added water with the residual tightly bound water that pre-exists before spiking.（2）Four soil samples with different textures were oven-dried at 205°C,wetted to varying water contents(0.05–0.30 g g^-1)with reference water of known isotope composition,and then subjected to CVE for water extraction.The isotopic differences（biases）between the reference and extracted water were related to the soil textures and water contents.Then,the relationship was used to assess how the cryogenic extraction biases potentially influence soil water isotope applications based on data from three previous studies.The results showed that the biases for84%CVE samples were more than twice the standard errors of currently available isotope analyzers,and biases increased significantly with increasing clay and decreasing water contents.Correction equations for CVE-obtainedδ²H andδ¹⁸O were established:R²=0.84,P<0.01,and R²=0.60,P<0.01,respectively.The|ME|and RMSE are less than the measurement accuracy of isotope analysis.When these corrections were applied to three different scenarios,they led to conclusions significantly different from those established with uncorrected values.This study provides a correction method for soil water vacuum extraction bias to improve the effectiveness of ²H and ¹⁸O applications in soil hydrology.This study suggests that cryogenic extraction biases should be considered seriously for quantitative analysis under low water contents and with a narrow spread of isotopic compositions among the potential end-members.（3）Rehydration experiments were performed on plant samples from two species and three organs with two distinct-isotopic spiking waters.Centrifugation and high-pressure mechanical squeezing were used to separate sap water and tissue water for stable isotope analyses.Plant waters extracted by CVE differed significantly from reference waters inδ²H,but not inδ¹⁸O.Theδ²H bias was linearly correlated to the xylem water content,and this relationship is affected significantly by plant organs/species and the isotopic signature of the spiking water.Moreover,theδ²H bias induced by CVE（-8.52±0.90‰）was significantly greater than theδ²H difference between the tissue and sap waters（-3.33±0.76‰）for apple stems possessing similar water contents.Thus,hydrogen exchange between plant organics and water,and isotopic heterogeneity within plants both contribute to the negativeδ²H bias,but the former is the dominant cause.The multiple factors governing the CVE-inducedδ²H bias,make it difficult to establish a unified bias correction equation.Our results question the usefulness of cryogenic extraction as a standard for plant water extraction.（4）Four motheds to extract plant water for isotope anslysis,such as gas pushing（GP）,centrifugation（CE）,squeezing（SQ）and CVE,were compared by sap water replacement and pot experiments.The results showed that CVE caused a significant ²H deviation of-8.21±0.67‰and-9.73±1.39‰in sap replacement and pot experiment,respectively.δ²H andδ¹⁸O of extracted water neither had significant difference form 0‰（P<0.05）among GP,CE and SQ.Isotopic accuracy of extracted water was:GP>CE>SQ;Meanwhile,the extraction success rate on potted plants:CE>SQ>GP.In many respects,CE can meet the requirements of accuracy and water volume for plant water isotopic analysis,thus be an alternative method to extract plant water.Based on CE extracted water from plant xylem,the ²H and ¹⁸O isotopes were not significantly different from those of labeled water or irrigation water（P>0.05）,indicating that there was no significant isotope fractionation in root water uptake and xylem water transport.In conclusion,the CVE-casued soil water isotopic bias can be corrected by indirect method.However,the deuterium deviation in plant water mainly resulted from the hydrogen exchange between organic matter and water,and was affected by multiple factors.Therefore,it is difficult to effectively correct the deuterium deviation for CVE plant water.Methods that can directly obtaining sap water in xylem（such as centrifugation）should be used as an alternative.These results could play a positive role in breaking through the main bottleneck of isotope technology in eco-hydrological research-the inaccuracy of sampling and analysis,and thus provide scientific basis and technical support for clarification of soil-plant water relationship.