The assessment of microbial biomass C in subsoil samples using fumigation-extraction is negligibly affected by residual chloroform

The potential overestimation of soil microbial biolmass C, using the fumigation-extraction method, arising from the contribution of residual (i.e., non-evacuated) chloroform (CHCl₃) to the fumigation flush of C, was recently shown to be minor for agricultural topsoil samples. However, we hypothesized larger flush proportions of residual CHCl₃–C, and hence greater biomass overestimations in low-biomass, subsoil samples due to the characteristically lower fumigation flushes of C in subsoils. The depth stratification (0–100 cm) of fumigation-extractable C and CHCl₃–C was determined in six soil profiles (three loesses ranging from 6 to 20% clay and three vertisols ranging from 46 to 69% clay). Residual chloroform levels correlated positively with soil organic matter content but not with clay content and decreased down the profiles. Residual chloroform contributed ∼2.0% to biomass-C overestimation, independent of soil type or depth, thus refuting the study hypothesis and supporting the safe use of the fumigation-extraction method also with subsoil samples.