The popular sulfonamide antibiotics are one of many groups of micropollutants found widely spread in the environment, while comprehensive knowledge on their transformation products (TPs) and their biotransformation pathways remains lacking. Therefore, this Master thesis investigates the biotransformation of five selected sulfonamide antibiotics (sulfamethoxazole, sulfamethazine, sulfadiazine, sulfathiazole and sulfapyridine) and aims to elucidate their related biotransformation pathways in activated sludge. Batch reactor experiments with activated sludge over a time frame of 72h were performed using non-labeled and ¹⁴C-radiolabeled sulfonamides in parallel. By virtue of these two parallel experiments, formed TPs could be identified through a sensitive suspect screening with HPLC-HRMS/MS, whereas accurate mass balances could be achieved through the spiked radioactivity.
Biotransformation was shown to be the main removal process for the parent sulfonamides from the aqueous phase and abiotic transformation, mineralisation and sorption to the suspended solids remained low over the entire experimental period. Moreover, the evaluation of radioactivity mass balances revealed that all of the radioactivity was recovered after chromatographic separation of supernatant samples and that hence, no analytes were lost during analytical measurements. Throughout the experiments, all investigated sulfonamides displayed a similar biotransformation behaviour, likely because of their homologous structures. Particularly, similar TPs in a similar order of intensity were detected for all investigated antibiotics. The pterine-conjugate PtO-sulfonamide was formed for all sulfonamides as one of the major TPs. Additionally, for sulfamethazine, sulfathiazole and sulfapyridine, besides PtO-sulfonamide, other conjugated TPs corresponding to the mass shifts acetyl + hydroxy (AcOH-sulfonamide), N4-formyl-sulfonamide and the conjugation rest of PtO-sulfonamide were detected as important TPs. Notably, these TPs all resulted from the same major biotransformation pathway: the interference of the sulfonamide antibiotic with the folic acid synthesis pathway, which is the actual mode of action of the sulfonamide antibiotic. Along this pathway, sulfonamide antibiotics are transformed into pterine-conjugates which are thought to be further transformed through a series of hydrolysis, oxidation and decarboxylation reactions into the TPs AcOH-sulfonamide and subsequently N4-formyl-sulfonamide.
In contrast to humans, the vital folic acid is generally synthesized by all bacteria. It is therefore not surprising, that a partial removal of sulfonamides in activated sludge is observed ubiquitously. However, the majority of formed TPs was found to be conjugated sulfonamides, which still possess their original sulfonamide structure and thus, they possibly also retain antibiotic activity. Moreover, a backformation potential of the formed conjugated TPs cannot be excluded, as was shown for N4-acetyl-sulfonamide. As a result, the observed disappearance of sulfonamide antibiotics in activated sludge cannot be compared to the degradation of sulfonamide antibiotics and the related reduction in antibiotic activity.
Finally, although an estimation of mass balances indicated that not all TPs for the investigated sulfonamides have been found yet, the results of this study provide strong indications that the main TPs and their related biotransformation pathway have been elucidated. As a result, the findings of this Master thesis give a better understanding of sulfonamide removal in activated sludge and they help to understand previous observations related to sulfonamide degradation in activated sludge, that could not be interpreted so far.