Studien zum metabolischen Schicksal von 13C-Nonylphenol in Wasser und Sediment und entsprechenden mikrobiellen Systemen (UBC
Projektlaufzeit: 2004 - 2007
EU (Marie Curie Programm Aquabase)
The aim of this research project is to study the metabolic fate of 13C-labelled nonylphenol in water, sediment and related microbial model systems.
The main use of nonylphenol is the production of NP polyethoxilates. These nonionic surfactants have different application such as the production of industrial and household detergents. The most common route of nonylphenol to enter in the environment is through the wastewater. In fact NP polyethoxilates is most used in cleaners and for this reasons is discharged directly in the sewage system. Under anaerobic conditions NP polyetoxilates is degraded to NP. Laboratories studies on NP have demonstrated that NP can be classified as endocrine disrupter compound. Isomers highly branched in the alpha position of the nonyl chain show an higher estrogenic activity and that the para position is favorite as well for estrogenic activity.So far data on the metabolic fate of NP in water and soil are limitated in literature.
Several studies have been performed in the laboratories about the metabolic fate of EDC using 14C labelled compounds. But this lead to 2 disadvantages: 1) the complete identification of the metabolites was not possible using GC/MS and 2) problems related with the discharge of radioactive compounds. For the previous reasons isomers of NP (353-NP, 363-NP, 33-NP) will be synthesised and labelled with 13C on the aromatic ring. An equimolar mixture of the labelled compound with the corresponding non labelled compound will yields a characteristic double peak with approximately the same abundance in MS analyses. Taking advantages of this it will be able to follow the degradation of nonylphenol in the experiments. The metabolic fate of NP exposed to different conditions will be studied: exposed to UV light, degraded by a recombinant yeast and in aerobic and anaerobic conditions.
The first line of the project is to study the metabolic fate of NP exposed under condition of photo degradation. In fact NP can be exposed to sunlight when present in the aquatic environment. UV rays are of a short wavelength and have the energy to degrade products in sunlights. A lamp that had a ratio of UV-A and UV-B quite similar to the sun light was chosen. The NP degradation will be followed in a water/sediment system and in chlorinated water. The water sediment system and chlorinated water will be spiked with the nonylphenol, than the sample will be exposed to UV light at difference distance from the lamp and for different time. Than the metabolites will be extracted and analyzed via GC/MS.
The second line of the project is to study the metabolic fate of NP after degradation with a recombinant yeast. The host used will be the yeast Saccharomyces cerevisiae, because it is well known and the genome of this yeast has been completely sequenced. The yeast will be transformed with an expression vector containing one cytochrome P-450 and the yeast reductase. The cytochrome P-450 is a super family of heme-thiolate enzymes which are involved in the metabolism of several xenobiotics. A large number of cytochrome P450 are expressed in human liver. Only 2 cytochrome are able to degrade partially NP: CYP2B6 and CYP2C19. The yeast reductase is used because previous studies suggested that the limited amount of yeast NADPH-P450 reducatese is the main limiting factor for the activity of monooxygenase P450. Furthermore, the S. cerevisiae reductase was chosen for overproduction since mammalian P450 reductase has been reported to be poorly synthesized in yeast. These two transformed yeast will be used for studies on the metabolic fate of the isomers of NP in vivo. To verify the biodegradation rate of the isomers of NP labelled with 13C the yeast will be transformed with both genes. The transformation will be performed using the two plasmids cloned previously, having a different selection marker.
The third and final line of the research project will study the metabolic fate of NP in anaerobic and aerobic conditions. Data regarding the biodegradation of nonylphenol in water are few. A water/sediment system and chlorinated water will be spiked with labelled isomers to follow their metabolic fate in aerobic and anaerobic conditions.