We present an approach for characterizing in situ microbial degradation using the ¹³C/¹²C isotope fractionation of contaminants as an indicator of biodegradation. The ¹³C/¹²C isotope fractionation of aromatic hydrocarbons was studied in anoxic laboratory soil percolation columns with toluene or o-xylene as the sole carbon and electron source, and sulfate as electron acceptor. After approximately 2 months' of incubation, the soil microbial community degraded 32 mg toluene l⁻¹ and 44 mg o-xylene l⁻¹ to less than 0.05 mg l⁻¹, generating a stable concentration gradient in the column. The ¹³C/¹²C isotope ratio in the residual non-degraded fraction of toluene and o-xylene increased significantly, corresponding to isotope fractionation factors (αC) of 1.0015 and 1.0011, respectively. When the extent of biodegradation in the soil column was calculated based on the measured isotope ratios (R_t) and an isotope fractionation factor (αC=1.0017) obtained from a sulfate-reducing batch culture the theoretical residual substrate concentrations (C_t) matched the measured toluene concentrations in the column. This indicated that a calculation of biodegradation based on isotope fractionation could work in systems like soil columns.

In a field study, a polluted, anoxic aquifer was analyzed for BTEX and PAH contaminants. These compounds were found to exhibit a significant concentration gradient along an 800-m groundwater flow path downstream of the source of contamination. A distinct increase in the carbon isotope ratio (δ¹³C) was observed for the residual non-degraded toluene (7.2‰), o-xylene (8.1‰) and naphthalene fractions (1.2‰). Based on the isotope values and the laboratory-derived isotope fractionation factors for toluene and o-xylene, the extent to which the residual substrate fraction in the monitoring wells had been degraded by microorganisms was calculated. The results revealed significant biodegradation along the groundwater flow path. In the wells at the end of the plume, the bioavailable toluene and o-xylene fractions had been almost completely reduced by in situ microbial degradation.

Although indane and indene showed decreasing concentrations downstream of the groundwater flow path, suggesting microbial degradation, their carbon isotope ratios remained constant. As the physical properties of these compounds are similar to those of BTEX compounds, the constant isotope values of indane and indene indicated that microbial degradation did not lead to isotope fractionation of all aromatic hydrocarbons. In addition, physical interaction with the aquifer material during the groundwater passage did not significantly alter the carbon isotope composition of aromatic hydrocarbons.