The conventional approach to evaluate biodegradation of organic contaminants in groundwater is to demonstrate an increase in the concentration of transformation products. This approach is problematic for MTBE from gasoline spills because the primary transformation product (TBA) can also be a component of gasoline. Compound-specific stable isotope analysis may provide a useful alternative to conventional practice. Changes in the delta13C and deltaD of MTBE during biodegradation of MTBE in an anaerobic enrichment culture were compared to the delta13C and deltaD of MTBE in groundwater at nine gasoline spill-sites. The stable isotopes of hydrogen and carbon were extensively fractionated during anaerobic biodegradation of MTBE. The stable isotope enrichment factor for carbon (epsilonC) in the enrichment cultures was -13 (-14.1 to -11.9 at 95% confidence level), and the hydrogen enrichment factor (epsilonH) was -16 (-21 to -11 at 95% confidence level). The isotope enrichment factors for carbon and hydrogen during anaerobic biodegradation indicate that the first reaction is enzymatic hydrolysis of the O-Cmethyl bond. The ratio of epsilonH to epsilonC was consistent between the enrichment culture and the field site that provided the inoculum, and with the other eight sites, suggesting a common degradation pathway. Compound-specific isotope evidence is discussed in terms of its utility for monitoring in situ biodegradation, in particular, for measuring how much MTBE was degraded. For the studied field sites, significant biodegradation of the original mass of MTBE is suggested, in some cases exceeding 90%.

Currently it is unclear if natural attenuation is an appropriate remedial approach for groundwater impacted by methyl tertiary butyl ether (MTBE). Site-characterization data at most gasoline release sites are adequate to evaluate attenuation in MTBE concentrations over time or distance. But, demonstrating natural biodegradation of MTBE requires laboratory microcosm studies, which could be expensive and time-consuming. Recently, compound-specific carbon isotope ratio analyses (13C/12C expressed in delta13C notation) have been used to demonstrate aerobic biodegradation of MTBE in laboratory incubations. This study explored the potential of this approach to distinguish MTBE biodegradation from other abiotic processes in an anaerobic groundwater plume that showed extensive decrease in MTBE concentrations. To our knowledge, this is the first study to use delta13C of MTBE data in groundwater and laboratory microcosms to demonstrate anaerobic biodegradation of MTBE. The delta13C of MTBE in monitoring wells increased by up to 31 per thousand (-25.5 per thousand to +5.5 per thousand) along with a 40-fold decrease in MTBE concentrations. Anaerobic incubations in laboratory microcosms indicated up to 20-fold reduction in MTBE concentrations with a corresponding increase in delta13C of MTBE of up to 33.4 per thousand (-28.7 per thousand to +4.7 per thousand) in live microcosms. Little enrichment was observed in autoclaved controls. These results demonstrate that anaerobic biodegradation was the dominant natural attenuation mechanism for MTBE at this site. The estimated isotopic enrichment factors (epsilon(field) = -8.10 per thousand and epsilon(lab) = -9.16 per thousand) were considerably larger than the range (-1.4 per thousand to -2.4 per thousand) previously reported for aerobic biodegradation of MTBE in laboratory incubations. These observations strongly suggest that delta13C of MTBE could be potentially useful as an "indicator" of in-situ MTBE biodegradation.

Contaminant attenuation studies utilizing CSIA (compound-specific isotope analysis) routinely assume that isotope effects (IEs) result only from degradation. Experimental results on MTBE behavior in diffusive volatilization and dynamic vapor extraction show measurable changes in the isotope ratios of the MTBE remaining in the aqueous or nonaqueous phase liquid (NAPL) matrix. A conceptual model for interpretation of those IEs is proposed, based on the physics of liquid-air partitioning. Normal or inverse IEs were observed for different volatilization scenarios. The range of carbon enrichment factors (epsilon) was from +0.7 per thousand (gasoline vapor extraction) to -1 per thousand (diffusive volatilization of MTBE from gasoline), the range of hydrogen epsilon was from +7 per thousand (gasoline vapor extraction) to -12 per thousand (air sparging of aqueous MTBE). The observed IEs are lower than those associated with MTBE degradation. However, under a realistic scenario for MTBE vapor removal, their magnitude is within the detection limits of CSIA. The potential for interference of those IEs is primarily in confusing the interpretation of samples with a small extent of fractionation and where only carbon CSIA data are available. The IEs resulting from volatilization and biodegradation, respectively, can be separated by combined carbon and hydrogen 2D-CSIA.

DNA was extracted from three fecal samples, more than 2,000 years old, from Hinds Cave, Texas. Amplification of human mtDNA sequences showed their affiliation with contemporary Native Americans, while sequences from pronghorn antelope, bighorn sheep, and cottontail rabbit allowed these animals to be identified as part of the diet of these individuals. Furthermore, amplification of chloroplast DNA sequences identified eight different plants as dietary elements. These archaic humans consumed 2-4 different animal species and 4 -8 different plant species during a short time period. The success rate for retrieval of DNA from paleofeces is in strong contrast to that from skeletal remains where the success rate is generally low. Thus, human paleofecal remains represent a source of ancient DNA that significantly complements and may in some cases be superior to that from skeletal tissue.