1. 4 Application of Environmental Isotopes in Studies of Biodegradation of Organic Contaminants in Groundwater Ramon Aravena, Department of Earth Sciences, University of Waterloo, Waterloo, Canada Daniel Hunkeler, Centre for Hydrogeology, University of Neuchâtel, Switzerland Introduction Biodegradation can lead to transformation of organic contaminants in groundwater to non toxic products under natural conditions (natural attenuation) or as part of a engineered remediation strategy. However, it is often difficult to assess biodegradation at field sites because contaminant concentration vary also due to dilution and sorption or as function of varying water levels and groundwater flow directions. Analysis of stable isotope ratios is a possible way to trace biodegradation. A number of studies have shown that no significant isotope fractionation in organic contaminants occurs due to physical processes such as sorption and volatilization. This presentation summaries the current knowledge of isotope fractionation during biodegradation of organic compounds gained from laboratory and field studies. Biodegradation of methyl tert-butyl ether (MTBE) Acknowledgements This project was supported through grants from the National Sciences and Engineering Research Council of Canada, the Centre for Research in Earth and Space Technology and the University Consortium Solvents-in-Groundwater Research Program. Quantification of isotope fractionation in laboratory experiments Example: Reductive dechlorination of vinyl chloride (initial concentration 40 ppm) to ethene Case Study: Reductive dechlorination of PCE in a sandy aquifer in Toronto, Canada Quantification of carbon isotope fractionation using modified Rayleigh equation:  13 C=  13 C 0 +  ln C/C 0  13 C 0 initial isotope ratio C o initial concentration  13 C 0 initial isotope ratio at time t C concentration at time t  isotopic enrichment factor Kinetic isotope effect k 12 > k 13 Results: Isotope fractionation during biodegradation organic contaminants Part 1: Chlorinated hydrocarbons Part 2: Petroleum hydrocarbons 1 2 3 4 5 6 7 8 Carbon isotopic enrichment factors  ‰) Ref. 1 PCE →TCE -2 -2.7, -5.2, -5,5 1919 2 TCE → cisDCE -4, -2.5, -6.6, -7.1, -13.8 1, 2 9, 8 3 cis-DCE → VC -12, -14.1, -16.1, -20.4, -19.9 1, 2 9, 5 4 trans-DCE→ VC -30.35 5 1,1-DCE → VC -7.35 6 VC → Ethene -26.0, -21.5, -26.6, -22.4, -31.1 1, 2 9, 5 7 1,1,2-TCA → VC -2.05 8 1,2DCA→ Ethene -32.15 Carbon isotopic enrichment factors  ‰) Hydro. isotopic enrichment factors  ‰) Ref. 1 Benzene, Aerobic -1.5 -3.5 -12.8 -11.24 2 Toluene Aerobic, ring oxygenase Aerobic, methyl oxygenase Sulfate reducing -1.1 -0.4 -3.3 -2.5 -1.8 -16.0 -28.0 -905 -198.1 -726.0 7776677766 3 MTBE, Aerobic -1.7 -1.5 to –2.4 n.d. -29 to -66 3 10 4 TBA, Aerobic -4.2n.d.3 References 1)Hunkeler, D., Aravena, R. and Butler, B.J., 1999. Environmental Science and Technology, 33, 2733-2738. 2)Bloom, Y., Aravena, R., Hunkeler, D., Edwards, E., and Frape, S.K., 2000. Environmental Science and Technology, 34, 2768-2772. 3)Hunkeler, D., Butler, B.J., Aravena, R. and Barker, J.F., 2001. Environmental Science and Technology, 35, 676-681. 4)Hunkeler, D., Andersen, N., Aravena, R., Bernasconi, S.M. and Butler, B.J., 2001. Environmental Science and Technology, 35, 3462-3467. 5)Hunkeler, D., Aravena, R., and Cox E., 2002. Environmental Science and Technology, 36, 3378-3384. 6)Morasch, B., Richnow, H.H., Schink, B., and Meckenstock, R.U., 2001. Applied and Environmental Microbiology, 67, 4842-4849. 7)Morasch, B., Richnow, H.H., Schink, B., Vieth, A. and Meckenstock, R.U., 2002. Applied and Environmental Microbiology, 68, 5191-5194. 8)Sherwood Lollar, B, Slater G.F., Ahad, J. et al., 1999. Organic Geochemistry, 30, 813-820. 9)Slater, G.F., Sherwood Lollar, B., Sleep, B.E., and Edwards, E.A., 2001. Environmental Science and Technology, 35, 901-907. 10)Gray, J.R., Lacrampe-Couloume, G., Gandhi, D. et al., 2002. Environmental Science and Technology, 36, 1931-1938. Cis-DCE and VC become strongly enriched in 13 C during biodegradation confirming that strong carbon isotope fractionation also occurs under field conditions. Discussion The magnitude of carbon isotope fractionation tends to be larger for chlorinated hydrocarbons than for petroleum hydrocarbons. The difference is likely due to differences in the type of bonds that are broken and formed during the initial transformation step and due to the differences in size of molecules. The larger number of carbon atoms in petroleum hydrocarbons tends to « dilute » the isotope effect. For petroleum hydrocarbons, the isotopic enrichment factors are generally larger for hydrogen than for carbon, which can be explained by the larger relative mass difference between D and H than between 13 C and 12 C. A particularly larger H isotope effect occurs for pathways during which a C-H bond is broken in the initial transformation step (toluene degradation by methyl oxygenase and under sulfate reducing conditions). For several of the compounds, the carbon isotopic enrichment factor varies relatively litlle between microcosms from different sites or different cultures suggesting that carbon isotope ratios can be used to quantify biodegradation. Regarding hydrogen isotopes, the enrichment factors are larger but more variable suggesting that in some cases hydrogen isotopes could be a very sensitive qualitative parameter to confirm biodegradation. 1 2 3 4