RESUMO
This work describes the use of different complementing methods (mass balance, polymerase chain reaction assays and compound-specific stable isotope analysis) to demonstrate the existence and effectiveness of biodegradation of chlorinated solvents in an alluvial aquifer. The solvent-contaminated site is an old chemical factory located in an alluvial plain in France. As most of the chlorinated contaminants currently found in the groundwater at this site were produced by local industries at various times in the past, it is not enough to analyze chlorinated solvent concentrations along a flow path to convincingly demonstrate biodegradation. Moreover, only a few data were initially available to characterize the geochemical conditions at this site, which were apparently complex at the source zone due to (i) the presence of a steady oxygen supply to the groundwater by irrigation canal losses and river infiltration and (ii) an alkaline pH higher than 10 due to former underground lime disposal. A demonstration of the existence of biodegradation processes was however required by the regulatory authority within a timeframe that did not allow a full geochemical characterization of such a complex site. Thus a combination of different fast methods was used to obtain a proof of the biodegradation occurrence. First, a mass balance analysis was performed which revealed the existence of a strong natural attenuation process (biodegradation, volatilization or dilution), despite the huge uncertainty on these calculations. Second, a good agreement was found between carbon isotopic measurements and PCR assays (based on 16S RNA gene sequences and functional genes), which clearly indicated reductive dechlorination of different hydrocarbons (Tetrachloroethene--PCE-, Trichloroethene--TCE-, 1,2-cisDichloroethene--cis-1,2-DCE-, 1,2-transDichloroethene-trans--1,2-DCE-, 1,1-Dichloroethene--1,1-DCE-, and Vinyl Chloride--VC) to ethene. According to these carbon isotope measurements, although TCE biodegradation seems to occur only in the upgradient part of the studied zone, DCE and VC dechlorination (originating from the initial TCE dechlorination) occurs along the entire flowpath. TCE reductase was not detected among the Dehalococcoides bacteria identified by quantitative PCR (qPCR), while DCE and VC reductases were present in the majority of the population. Reverse transcriptase PCR assays (rt-PCR) also indicated that bacteria and their DCE and VC reductases were active. Mass balance calculations showed moreover that 1,1-DCE was the predominant DCE isomer produced by TCE dechlorination in the upgradient part of the site. Consequently, coupling rt-PCR assays with isotope measurements removes the uncertainties inherent in a simple mass balance approach, so that when the three methods are used jointly, they allow the identification and quantification of natural biodegradation, even under apparently complex geochemical and hydraulic conditions.
