Deciphering Chlorohydrocarbon Transformation Mechanisms by Advancing δ13C/δ37Cl Compound-Specific Isotope Analysis

Abstract

Chlorinated organic compounds are ubiquitous in daily life. Chlorohydrocarbons are used as solvents in industry (e.g. chlorinated ethenes) and as herbicides in agriculture (e.g. atrazine). However, when present in ground- and surface water, they pose a threat to drinking water resources. Therefore, investigating and understanding their environmental fate is important to guarantee correct pesticide management and to develop successful (bio)remediation strategies at contaminated sites. When traditional concentration-based assessments fall short because mass balances cannot be closed, a promising approach for tracing the sources of contamination and studying the transformation pathways of such contaminants is compound-specific stable isotope analysis (CSIA). Analyzing changes in natural occurring isotope ratios (e.g. 13C/12C, 15N/14N, 37Cl/35Cl) during (bio)chemical transformations allows the detection and the assessment of degradation processes. Furthermore, isotopic information from more than one element enables the differentiation and even the identification of different (bio)chemical reaction mechanisms. The first part of this thesis focuses on advancing CSIA of chlorine. In the last years instrumental and methodical optimizations continually improved chlorine isotope analysis facilitating also the analysis of more complex organic compounds. For accurate chlorine isotope analysis, however, in-house referencing and substance-specific working standards are critically needed. Ideally two standards of each substance are required that display different isotope values to enable a two-point calibration. However, almost all international chlorine isotope reference materials have similar isotope values except one which is, therefore, very valuable and should not be used for routine analysis. Here, a synthesis route was identified resulting in a chloride salt which shows a pronounced negative chlorine isotope value. This chloride salt can be used as a second anchor for two-point calibration of in-house working standards in the future. Furthermore, it was demonstrated that substance-specific working standards of more complex organic chlorohydrocarbons (like the herbicides acetochlor and S-metolachlor) can be generated easily by using chemical reactions with pronounced chlorine isotope effects from organic chemistry. With these synthesis routes every laboratory has the opportunity to generate its own in-house standards leading to more accurate results in chlorine isotope analysis. The second part of the thesis tackles the question why bioremediation of the chlorinated ethenes tetrachloroethene (PCE) and trichloroethene (TCE) often stops at toxic cis-1,2-dichloroethene (cis-DCE) or vinyl chloride (VC). By studying dual element isotope plots of carbon and chlorine a model study recently identified two different chemical mechanisms which are at work during reductive dechlorination of PCE (addition-elimination) and cis-DCE (addition-protonation). For TCE dechlorination both mechanisms could be observed. In this thesis it was investigated whether the same mechanisms can also be observed during microbial reductive dechlorination with pure and mixed cultures. Dual element isotope trends of carbon and chlorine indeed indicated that bacteria dechlorinating cis-DCE or PCE followed the same mechanisms which were identified in the model study. Microbial TCE dechlorination followed the addition-protonation pathway if the cultures were already adapted to higher chlorinated substrates. If the bacteria were maintained on less chlorinated substrates before TCE dechlorination, they followed the addition-elimination pathway. Therefore, it was concluded that reductive dehalogenases (RDases, the enzymes catalyzing reductive dechlorination) are likely specialized in different chemical mechanisms. The fact that some RDases are specifically tailored to the dechlorination of PCE and TCE, but are not able to degrade cis-DCE or VC may offer an explanation for the question why bioremediation often stalls at cis-DCE or VC. Based on these results, a new classification system based on dual element isotope trends (C, Cl) and detected RDases could help to identify natural processes at contaminated field sites. The third part of this thesis studies chlorine, carbon and nitrogen isotope fractionation during microbial atrazine hydrolysis with the pure culture Arthrobacter aurescens TC1 and oxidative dealkylation with Rhodococcus sp. NI86/21. Carbon and nitrogen isotope effects confirmed that the bacteria followed the pathways which were proposed in previous studies. Dual element isotope plots of the measured elements (C/N, Cl/C, Cl/N) allowed a reliable distinction of the two pathways. In contrast to nitrogen and carbon isotope effects, chlorine isotope effects are not diluted by non-reacting atoms which could turn chlorine isotope fractionation into a sensitive indicator for transformation processes. During microbial hydrolysis of atrazine unexpected small chlorine isotope effects were observed indicating that the cleavage of the C-Cl bond is not the rate-limiting step in this reaction. On the other hand, oxidative dealkylation resulted in unexpected large chlorine isotope effects suggesting the involvement of enzymatic interactions. Regarding these unexpected results this study demonstrated that a complete understanding of chemical mechanisms is very important before applying this new approach to the field. Additionally, triple element isotope analysis, not only of atrazine, but also of other chlorohydrocarbons, will improve the source identification of contaminants and also the differentiation of degradation pathways.