Abstract. Recent studies have challenged the interpretation of plant water isotopes obtained through cryogenic vacuum distillation (CVD) based on observations of a large 2H fractionation. These studies have hypothesized the existence of an H-atom exchange between water and organic tissue during CVD extraction with the magnitude of H exchange related to relative water content of the sample; however, clear evidence is lacking. Here, we systematically tested the uncertainties in the isotopic composition of CVD-extracted water by conducting a series of incubation and rehydration experiments using isotopically depleted water, water at natural isotope abundance, woody materials with exchangeable H, and organic materials without exchangeable H (cellulose triacetate and caffeine). We show that the offsets between hydrogen and oxygen isotope ratios and expected reference values (Δ2H and Δ18O) have inversely proportional relationships with the absolute amount of water being extracted, i.e. the lower the water amount, the higher the Δ2H and Δ18O. However, neither Δ2H nor Δ18O values, were related to sample relative water content. The Δ2H pattern was more pronounced for materials with exchangeable H atoms than with non-exchangeable H atoms. This is caused by the combined effect of H exchange during the incubation of materials in water and isotopic enrichments during evaporation and sublimation that depend on absolute water amount. The H exchange during CVD extraction itself was negligible. Despite these technical issues, we observed that the water amount-dependent patterns were much less pronounced for samples at natural isotope abundance and particularly low when sufficiently high amounts of water were extracted (>600 µL). Our study provides new insights into the mechanisms causing isotope fractionation during CVD extraction of water. The methodological uncertainties can be controlled if large samples of natural isotope abundance are used in ecohydrological studies.

Carbon (C) isotope discrimination during photosynthetic CO2 assimilation has been extensively studied, but the whole process of fractionation from leaf to soil has been less well investigated. In the present study, we investigated the δ13C signature along the C transfer pathway from air to soil in a coniferous and broad-leaved mixed forest in northeast China and examined the relationship between δ13C of respiratory fluxes (leaf, trunk, soil, and the entire ecosystem) and environmental factors over a full growing season. This study found that the δ13C signal of CO2 from canopy air was strongly imprinted in the organic and respiratory pools throughout C transfer due to the effects of discrimination and isotopic mixing on C assimilation, allocation, and respiration processes. A significant difference in isotopic patterns was found between conifer and broadleaf species in terms of seasonal variations in leaf organic matter. This study also found that δ13C in trunk respiration, compared with that in leaf and soil respiration, was more sensitive to seasonal variations of environmental factors, especially soil temperature and soil moisture. Variation in the δ13C of ecosystem respiration was correlated with air temperature with no time lag and correlated with soil temperature and vapor pressure deficit with a lag time of 10 days, but this correlation was relatively weak, indicating a delayed linkage between above- and belowground processes. The isotopic linkage might be confounded by variations in atmospheric aerodynamic and soil diffusion conditions. These results will help with understanding species differences in isotopic patterns and promoting the incorporation of more influencing factors related to isotopic variation into process-based ecosystem models.