Compound specific isotope analysis to track sources and transformation processes of micropollutants – Investigations in engineered and natural systems with a special focus on diclofenac and chiral herbicides

Abstract

Pharmaceuticals and pesticides are among the most frequently detected micropollutants in natural waters because they enter the environment via sewage treatment plants or by application to fields. Hence, it is of great interest to know if they can be degraded in the environment and by which processes. However, the complexity of aquatic systems makes it challenging to assess the environmental fate of micropollutants by concentration measurements, because concentrations are not only affected by degradation, but also by dilution processes. In addition, concentration analysis cannot distinguish transformation processes and their underlying reaction mechanisms. To some part this can be achieved by the analysis of transformation products (TPs), but they are often not fully elucidated, can be further degraded and in some cases different reaction mechanisms yield the same transformation product. Compound specific isotope analysis (CSIA) is an elegant alternative that is independent of dilution and can even deliver insights into reaction mechanisms. This approach is based on the detection of stable isotopes of C (13C/12C) and N (15N/14N) which are an inherent trait of all organic substances. In the absence of transformation processes, isotope ratios are preserved and can be used to verify the origin of substances. If transformation takes place, isotope ratios of the substrate are shifted and the magnitude of this shift is determined by the reaction mechanism. Consequently, transformation reactions can be directly detected by isotope analysis of the substrate and the magnitude of fractionation can deliver insight into the underlying mechanism. Before this thesis was initiated the applicability of CSIA was limited to relatively high concentrations and isotope fractionation of pharmaceuticals during transformation reactions was unexplored territory. Hence, the present thesis developed for the first time compound specific isotope analysis (CSIA) for a pharmaceutical, using diclofenac as a model compound, which is one of the most frequently consumed pharmaceuticals, but also among the most frequently detected micropollutants in the environment. First, this method was used to demonstrate its applicability to the ng L-1 range, which makes it one of the most sensitive CSIA methods at all. Subsequently, CSIA was used to gain insights about its fate on three levels. (i) Since isotope ratios are determined by the resources used in synthesis and the synthesis pathway, the analysis of C and N isotope ratios could distinguish most of the tested commercial diclofenac products (tablets, gels). On the one hand this finding can be used to verify commercial products with a fraud-resistant method. On the other hand this approach can be extended to distinguish sources of diclofenac in the environment, such as sewage treatment plants or manure that originates from diclofenac treated cattle. (ii) CSIA could be established as a new line of evidence for transformation that is independent of TP analysis. Six transformation pathways were investigated and all of them could be tracked by CSIA. Moreover, the remarkable picture was obtained that all tested environmentally relevant transformation reactions can be separated into two groups that can be distinguished from each other by the analysis of C and N isotope ratios. Ozonation and photolysis cause inverse isotope fractionation, whereas biotransformation and MnO2 lead to normal isotope fractionation. This is of great interest for environmental investigations, since photolysis and biotransformation are discussed as the two most important transformation pathways in the environment and CSIA delivers a direct measure for their relative importance. But CSIA can also provide insights into engineered systems, because it can be used to investigate the efficiency of biotransformation and / or ozonation during (waste-) water treatment. (iii) Because isotope effects mirror transition states of transformation reactions, they deliver unique insights in reaction mechanisms that cannot be obtained by other methods under environmentally relevant conditions. The big potential of this approach became apparent when transformation by ABTS and MnO2 was compared. Both processes are supposed to mimic single electron oxidation (SET), which is proposed to be of big importance in enzymatic transformation reactions. CSIA revealed that ABTS is indeed a model for SET. In contrast, MnO2 showed a completely different isotope fractionation that was at the same time in perfect agreement with aerobic biotransformation. Moreover, it was strongly indicated that the observed isotope fractionation during MnO2 or biotransformation, points towards an overseen transformation pathway that can only be seen by CSIA at the moment. Furthermore, CSIA was applied for the first time to study transformation processes during ozonation. There, it provided evidence that ozone rather attacks at the aromatic ring of diclofenac than at the N-atom. (iv) Besides CSIA of diclofenac, the present thesis investigated also chiral herbicides. CSIA was combined with enantioselective separation to yield enantioselective an even more versatile approach – enantioselective stable isotope analysis (ESIA). ESIA was used for the first time to investigate the fate of the chiral herbicides and revealed that isotope ratios and enantiomeric ratios behave complementarily. In this thesis a case was studied were pronounced enantiomer fractionation, but no isotope fractionation was detected, while the opposite was found for an insecticide in another study. When ESIA was brought to the field results were even more fascinating, because isotope- and enantiomeric fractionation were observed simultaneously. The present thesis could demonstrate that CSIA and ESIA can improve our understanding of micropollutant transformation in the environment in several ways. There is great potential when the methodology of this dissertation will be applied to one of the other 10,000 pharmaceuticals that are on the market. But also the novel area of ESIA offers plenty of possibilities, when this approach will be transferred to other pesticides and processes. Even more exciting, ESIA of carbon can be combined with the analysis of nitrogen or hydrogen and this would increase the knowledge gain dramatically as shown for CSIA of diclofenac. Moreover, ESIA should not be restricted to pesticides, but this approach urges to be extended to chiral pharmaceuticals, such as ibuprofen or metoprolol.