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Causes and Factors of Cryogenic Extraction Biases on Isotopes of Xylem Water

Cryogenic vacuum extraction (CVE) has been considered as the standard technique for the analysis of plant water stable isotopes in ecohydrological research. Recent studies reported that CVE can introduce significant bias in stable isotope analyses, yet the causes and influencing factors of the CVE‐induced deuterium offsets remain poorly understood. Here, we performed rehydration experiments 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 in δ2H, but not in δ18O. The δ2H 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, the δ2H bias induced by CVE (−8.52 ± 0.90‰) was significantly greater than the δ2H 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 negative δ2H bias, but the former is the dominant cause. The multiple factors governing the CVE‐induced δ2H 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.