Increase in organic carbon stocks in subsoil layers after two decades of organic amendment application cannot be explained by the estimated contribution of multiple C inputs

• Accrual in SOC stocks in the subsoil after 24 years of organic amendments application. • Limited contribution of DOC leaching, bioturbation and illuviation. • Main contribution of belowground crop C inputs. • Discrepancies in our understanding of the mechanisms influencing subsoil SOC. Soil organic carbon (SOC) is important for both soil health and climate change mitigation. Organic amendment (OA) applications are known to increase SOC stocks. However, most existing studies have focused on surface soil layers, whereas the subsoil is an important SOC stock. In this study, we quantified SOC stock accrual over 0–90 cm in the QualiAgro long-term experiment after 24 years of OA application. While surface SOC stocks increased only with OA application, they increased significantly in the subsoil both with and without OA application. Compared to the control, OA application increased subsoil SOC stocks: from 5 % to 10 % and from 3 % to 13 % of the total SOC stocks accrual over the whole soil profile occurred in the 35–50 cm and 50–90 cm soil layers, respectively. C inputs to the subsoil layers were estimated: belowground crop C inputs using crop yields and allometric coefficients, dissolved organic carbon (DOC) leaching by direct measurement (lysimeter), illuviation and bioturbation by image analysis of soil thin sections. Potential C inputs did not fully explain the subsoil SOC stocks accrual. The contribution of belowground crop C ranged from 32 % to 54 % of the SOC stocks accrual. DOC leaching showed a limited contribution (1 to 5 %). The contribution of bioturbation and illuviation was very limited (≤3%). Our study suggests discrepancies in our current understanding of the mechanisms influencing subsoil SOC, and highlights the importance of considering the subsoil when monitoring the effect of OA application on SOC stocks.