Assessing the within‐site variability and apparent reproducibility of soil organic carbon fractionation methods in subsoil

Abstract

Soil organic carbon (SOC) fractionation methods are widely used to assess different carbon (C) pools, but their reproducibility, particularly with depth, remains unclear. This study evaluated the within‐site variability of chemical and physical fractionation methods for SOC at 0‐ to 30‐cm and 30‐ to 60‐cm depths across four apple ( Malus domestica ) orchards in northern Italy. All measured fractions: total organic carbon (TOC), dissolved organic carbon (DOC), hot water extractable carbon, permanganate oxidizable carbon (POXC), fulvic acids, humic acids, particulate organic matter carbon (POMC), particulate organic carbon (POC), and mineral‐associated organic carbon (MAOC) decreased significantly with depth; subsoils contained less than half the C of topsoils. Reproducibility declined in subsoil, with higher coefficient of variation, especially for POXC and MAOC (∼80%). Heterogeneity in subsoils was attributed to lower organic matter concentrations, greater C heterogeneity, texture differences, and more complex soil structure. POC and DOC exhibited lower variability in subsoil than topsoil, suggesting a reduced sensitivity to short‐term changes and therefore more stable indicators of subsoil C dynamics. The combined measurement of POC and MAOC, alongside DOC, reliably represented TOC at both depths, with POC + MAOC values closely aligning with measured TOC. Future studies should work toward improving fractionation protocols and normalization techniques to enhance both accuracy and comparability, particularly in the complex conditions of subsoil. This research adds valuable insight into C dynamics at depth and offers practical recommendations for choosing appropriate fractionation methods. It also underscores the need for careful consideration when comparing C fractions reported in different studies.