Bakterielle Genotoxizitätstests im Rahmen des gefährdungsbasierten Risikomanagements für Trinkwasser relevante Spurenstoffe

• Bacterial genotoxicity tests within the framework of hazard-based risk management for drinking water relevant trace substances

Buchner, Eva-Maria; Hollert, Henner (Thesis advisor); Schäffer, Andreas (Thesis advisor)

Aachen (2020)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2020

Abstract

Objectives: With regard to the entire water cycle, a reliable assessment of questionable single substances and real water samples of the drinking water must be ensured. A corresponding end-point-related test system for the evaluation of corresponding substances or samples for their toxicological hazard potential is an important step to guarantee the toxicological evaluation process. It is especially for the rapid and safe exclusion of a human-toxicologically relevant genotoxic potential. The main task of the dissertation was to contribute to a holistic toxicological assessment of anthropogenic trace substances, for single substances as well as for transformation products and mixtures of substances. Within this dissertation, the use of the two bacterial genotoxicity tests, Ames- and umu-Test was further developed with regard to the evaluation of drinking water samples, supplemented with genetically modified additional tester strains and put to the test. Current state of scienceThe GOW concept (GOW = health orientation value) as a single substance-related evaluation concept requires good data base for toxicological assessment. If there are large gaps in the existing data base and the individual substance cannot be isolated from the water sample in a sufficient quantity for the toxicological testing, the evaluation of the substances is difficult. This applies above all to trace substance metabolites and transformation products (e.g. due to lack of stability), which can already occur in raw water, but also can occur during the water treatment (e.g. chlorination, UV treatment). The concentration gap between the effective threshold of the biological tests and the real substance concentrations in the water can also cause problems for the assessment of the samples. In order to fill this gap, various enrichment methods are required to concentrate the trace substances and transformation products. Although some of these methods have already been standardized, there is a great need to check their compatibility with biological test methods with regard to testing, development and standardization. For example, the physiological boundary conditions and the effective threshold of the test organisms must be taken into account. Method applied, method optimization and findings: With the standard versions of the Ames-fluctuationtest and the umu-Test, 31 water-relevant single substances were tested. Eight of them resulted positive in the bacterial tests, but only three of them were mutagenic in both test systems. This can probably be explained by the different endpoints. When tested with the respective additional tester strains expressing additional mammalian enzymes, selected substances for which the expressed enzymes play a role in substance metabolism showed a significantly more sensitive genotoxic reaction. The two test systems and the corresponding additional tester strains are suitable for analysis after successful establishment in the laboratory. The fact that these are fast, simple and well standardised test procedures opens up the possibility that water supply companies will actively participate in the investigation of drinking water-relevant substances in their own laboratories in the future and thus contribute significantly to the evaluation process on the basis of the GOW concept. Furthermore, it could be shown by way of example that by the supplementary use of genetically modified, metabolism-competent additional tester strains, the verifiable spectrum of human-relevant genotoxins can be extended and important information on the mechanisms of action of the tested substances can be obtained. However, before using these additional strains it was necessary to establish the additional strains in the laboratory with partial modifications of the standard protocol (e.g. bacterial density, incubation time and establishment of suitable positive controls).The investigations of the water samples within the scope of this dissertation revealed problems with the enrichment processes and the stability of the transformation products. Therefore, different water concentrate samples were investigated. During the investigation, the great importance of a blank sample was shown in order to be able to sort out false positive results. Compliance with the physiological limits of the test organisms also had to be adapted to the test system and tested. Another important examination parameter for real water samples is the effective threshold. The effectiveness threshold of the known toxicological test methods is usually several orders of magnitude above the concentration range in drinking water relevant for the evaluation of potential microcontaminants and thus these microcontaminants had to be concentrated using suitable enrichment methods. It also had to be ensured that the laboratory tests for the evaluation of the extracts could be transferred to reality. Laboratory-scale investigations may cause conditions to deviate from those in the real environment and lead to results that may not be relevant to reality. If all these basic conditions (suitable blank sample, compliance with physiological tolerances, effective threshold and transfer from laboratory scale to real environmental conditions) are observed, the general suitability of the enrichment methods for the evaluation is ensured and the effective threshold of the test system is reached. This is of great importance for the evaluation of the parameter genotoxicity in order to obtain reliable statements on the toxicological potential of the concentrate. Résumé: These results from the dissertation project contributed to the establishment of an assessment strategy for the quick detection of human genotoxins. The two bacterial genotoxicity tests could be recommended together with the micronucleus test as components of a basic test battery in the sense of a first evaluation stage. All in all, these tests showed how important but at the same time how complex an evaluation of substances or concentrates relevant for drinking water is. In order to be able to make a reliable statement about the genotoxic potential of the samples, the general conditions of the test systems must be observed. The two investigated biological genotoxicity tests, umu-Test and Ames-Test, allow a quick classification of substances not yet evaluated. By extending testing with metabolism-competent tester strains, it is possible to gain information on possible mechanisms of action. Therefore, a safe drinking water supply can be guaranteed and a contribution for the evaluation can also be made directly by smaller laboratories of the water suppliers.