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

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

Compound-Specific Chlorine Isotope Analysis of the Herbicides Atrazine, Acetochlor, and Metolachlor

A gas chromatography-single quadrupole mass spectrometry method was developed and validated for compound-specific chlorine isotope analysis (Cl-CSIA) of three chlorinated herbicides, atrazine, acetochlor, and metolachlor, which are widespread contaminants in the environment. For each compound, the two most abundant ions containing chlorine (202/200 for atrazine, 225/223 for acetochlor, and 240/238 for metolachlor) and a dwell time of 30 ms were determined as optimized MS parameters. A limit of precise isotope analysis for ethyl acetate solutions of 10 mg/L atrazine, 10 mg/L acetochlor, and 5 mg/L metolachlor could be reached with an associated uncertainty between 0.5 and 1 . To this end, samples were measured 10-fold and bracketed with two calibration standards that covered a wide range of δ37Cl values and for which amplitudes matched those of the samples within 20% tolerance. The method was applied to investigate chlorine isotope fractionation during alkaline hydrolysis of metolachlor, which showed a shift in δ37Cl of +46 after 98% degradation, demonstrating that chlorine isotope fractionation could be a sensitive indicator of transformation processes even when limited degradation occurs. This method, combined with large-volume solid-phase extraction (SPE), allowed application of Cl-CSIA to environmentally relevant concentrations of widespread herbicides (i.e., 0.5-5 μg/L in water before extraction). Therefore, the combination of large-volume SPE and Cl-CSIA is a promising tool for assessing the transformation processes of these pollutants in the environment

Solid-phase extraction method for stable isotope analysis of pesticides from large volume environmental water samples

Compound-specific isotope analysis (CSIA) is a valuable tool for assessing the fate of organic pollutants in the environment. However, the requirement of sufficient analyte mass for precise isotope ratio mass spectrometry combined with prevailing low environmental concentrations currently limits comprehensive applications to many micropollutants. Here, we evaluate the upscaling of solid-phase extraction (SPE) approaches for routine CSIA of herbicides. To cover a wide range of polarity, a SPE method with two sorbents (a hydrophobic hypercrosslinked sorbent and a hydrophilic sorbent) was developed. Extraction conditions, including the nature and volume of the elution solvent, the amount of sorbent and the solution pH, were optimized. Extractions of up to 10 L of agricultural drainage water (corresponding to up to 200 000-fold pre-concentration) were successfully performed for precise and sensitive carbon and nitrogen CSIA of the target herbicides atrazine, acetochlor, metolachlor and chloridazon, and metabolites desethylatrazine, desphenylchloridazon and 2,6-dichlorobenzamide in the sub-μg L−1-range. 13C/12C and 15N/14N ratios were measured by gas chromatography-isotope ratio mass spectrometry (GC/IRMS), except for desphenylchloridazon, for which liquid chromatography (LC/IRMS) and derivatization-GC/IRMS were used, respectively. The method validated in this study is an important step towards analyzing isotope ratios of pesticide mixtures in aquatic systems and holds great potential for multi-element CSIA applications to trace pesticide degradation in complex environments

Compound-specific chlorine isotope fractionation in biodegradation of atrazine

Atrazine is a frequently detected groundwater contaminant. It can be microbially degraded by oxidative dealkylation or by hydrolytic dechlorination. Compound-specific isotope analysis is a powerful tool to assess its transformation. In previous work, carbon and nitrogen isotope effects were found to reflect these different transformation pathways. However, chlorine isotope fractionation could be a particularly sensitive indicator of natural transformation since chlorine isotope effects are fully represented in the molecular average while carbon and nitrogen isotope effects are diluted by non-reacting atoms. Therefore, this study explored chlorine isotope effects during atrazine hydrolysis with Arthrobacter aurescens TC1 and oxidative dealkylation with Rhodococcus sp. NI86/21. Dual element isotope slopes of chlorine vs. carbon isotope fractionation (ΛArthroCl/C = 1.7 ± 0.9 vs. ΛRhodoCl/C = 0.6 ± 0.1) and chlorine vs. nitrogen isotope fractionation (ΛArthroCl/N = −1.2 ± 0.7 vs. ΛRhodoCl/N = 0.4 ± 0.2) provided reliable indicators of different pathways. Observed chlorine isotope effects in oxidative dealkylation (εCl = −4.3 ± 1.8 ) were surprisingly large, whereas in hydrolysis (εCl = −1.4 ± 0.6 ) they were small, indicating that C-Cl bond cleavage was not the rate-determining step. This demonstrates the importance of constraining expected isotope effects of new elements before using the approach in the field. Overall, the triple element isotope information brought forward here enables a more reliable identification of atrazine sources and degradation pathways

Adsorbing vs. nonadsorbing tracers for assessing pesticide transport in arable soils

The suitability of two different tracers to mimic the behavior of pesticides in agricultural soils and to evidence the potential for preferential flow was evaluated in outdoor lysimeter experiments. The herbicide atrazine [6‐chloro‐N‐ethyl‐N′‐(1‐methylethyl)‐1,3,5‐triazine‐2,4‐diamine] was used as a model compound. Two tracers were used: a nonadsorbing tracer (bromide) and a weakly adsorbing dye tracer (uranine). Two soils that are expected to show a different extent of macropore preferential flow were used: a well‐drained sandy‐loamy Cambisol (gravel soil) and a poorly drained loamy Cambisol (moraine soil). Conditions for preferential flow were promoted by applying heavy simulated rainfall shortly after pesticide application. In some of the experiments, preferential flow was also artificially simulated by injecting the solutes through a narrow tube below the root zone. With depth injection, preferential leaching of atrazine occurred shortly after application in both soil types, whereas with surface application, it occurred only in the moraine soil. Thereafter, atrazine transport was mainly through the porous soil matrix, although contributions of preferential flow were also observed. For all the application approaches and soil types, after 900 d, <3% of the applied amount of atrazine was recovered in the drainage water. Only uranine realistically illustrated the early atrazine breakthrough by transport through preferential flow. Uranine broke through during the first intense irrigation at the same time as atrazine. Bromide, however, appeared earlier than atrazine in some cases. The use of dye tracers as pesticide surrogates might assist in making sustainable decisions with respect to pesticide application timing relative to rainfall or soil potential for preferential flow

On the possibility of an Eley-Rideal mechanism for ammonia synthesis on Mn6N5+x (x=1)-(111) surfaces

Recently we reported an Eley–Rideal/Mars–van Krevelen mechanism for ammonia synthesis on cobalt molybdenum nitride (Co3Mo3N). In this mechanism hydrogenation of activated dinitrogen occurs directly from the gas phase in a low barrier step forming a hydrazinylidene intermediate [double bond, length as m-dash]NNH2. In this paper we study whether such a mechanism of ammonia synthesis could occur on the (111) surface of another metal nitride, Mn6N5+x (x = 1), as this would explain the low-T ammonia synthesis activity of Co3Mo3N. We find that although N2 adsorbs more strongly than H2 on the (111) surface, having also examined the (110) and the (100) surface, N2 is not significantly activated when adsorbed in an end-on configuration. The hydrogenation reactions via an Eley–Rideal mechanism are all high barrier processes (>182 kJ mol−1) and therefore an Eley–Rideal mechanism for ammonia synthesis is predicted to not occur on this material unless there are high temperatures. Our study indicates that the fact that an Eley–Rideal/Mars–van Krevelen mechanism occurs on Co3Mo3N is a result of the stronger activation of dinitrogen at nitrogen vacancies when dinitrogen is adsorbed in an end-on configuration