High-Throughput Effect-Directed Analysis: a novel platform for rapid and sensitive identification of toxic compounds in the aquatic environment
Start date: 15 Dec 2012
End date: 1 April 2017
Funding: STW Open Technology Programme
Introduction to the research project
All kinds of emerging contaminants, including pharmaceuticals, endocrine disrupting compounds, and perfluorinated compounds, are present in the aquatic environment, wastewater and drinking water sources. In addition, numerous bioassay surveys have demonstrated the presence of not yet identified compounds with toxic activities in the environment, which is evidently of much concern. For example, genotoxic and endocrine disrupting compounds may lead to DNA damage, to disruption of development and reproduction and/or to cancer.
At this moment, identification of toxic compounds is done by effect-directed analysis (EDA). In this approach, complex environmental samples showing toxic activity are fractionated with liquid chromatography into a limited number of fractions. Each fraction is tested for toxic activity in a bioassay. Ultimately, the “hot fractions” are analyzed with mass spectrometry to identify the responsible compounds. EDA studies often fail to identify the responsible toxic compounds, because, even after repeated fractionation, biologically active fractions remain too complex for chemical identification. A second problem is that current EDA studies are labor-intensive and as such not suitable for routine monitoring.
Drinking water companies, water boards, and other government agencies surveying water quality for toxic contaminants will directly benefit from the HT-EDA project. They will be equipped with a tool to efficiently identify compounds with biological activities that might threat environmental and human health. The methodology means to solve a major problem they currently are confronted with: identification of unknown toxic compounds and the unlimited and costly growth of target analyses of emerging substances within their monitoring programs. Implementation of the methodology will enable replacement of parts of monitoring programs that are currently performed separately and enables replacement of expensive targeted analyses saving time and money. In addition, the methodology developed will be of interest to analytical instrumentation companies.
The project will be performed in close cooperation with end-users, which is emphasized by the secondment of the two PhD students and implementation of the platform at an end-users’ laboratory. This will provide end-users already during the project with knowledge on the identity of toxic compounds possibly present in their samples and will guarantee that the platform developed is suitable for application to the samples they work with. The implementation of the platform is thus an integral part of the project.
End-users recognize the proposed model types of biological activity (endocrine disruption and genotoxicity) as very relevant to be investigated, first because of the importance for human health. In addition, these bioactivities are detected in surface waters and during water treatment. The platform can be adapted to other bioassays in a straightforward fashion. Finally, instrument manufacturers involved will profit by commercialization of the platform developed.
- Development of a platform centralized around a spotter technology for nanofractionation onto high density microtiter plates (e.g. 1536 wells). The spotter technology provides high-resolution fractionation that maintains chromatographic separation efficiency. By integrating mammalian and other cellular bioassay responses and mass spectrometry, biological activity-to-identity correlation is greatly improved.
- Miniaturization of mammalian cellular bioassays for high density microtiter well plates and high-resolution analysis formats for analysis of toxic activities in mixtures (e.g. water samples).
- Applying this platform to water samples containing unidentified toxicants. Final end goal is to provide a functional platform suitable for valorisation and subsequent distribution to end-users (e.g. surface water, wastewater and drinking water laboratories).
The HT-EDA project comprises the development of a robust, high-resolution effect-directed analysis platform for environmental and (drinking) water samples, allowing the rapid identification of (unknown) toxicants. Compounds in a sample are separated with liquid chromatography. Subsequently, the eluent is split. By means of an innovative spotting technology, one part is transferred to a high-resolution (mammalian or yeast-based cellular) bioassay format to test the biological activity of eluting compounds. The other part is transferred to a mass spectrometer. Peak shapes from ‘reconstructed bioassay chromatograms’ using the individually collected and bioassayed fractions are efficiently correlated to compound identity. The research is carried out by two PhD students.
PhD student 1, embedded in the BioAnalytical Chemistry Group of the VU, focuses on the spotting technology. PhD student 2 (department Enviroment and Health) works on miniaturization of cellular bioassays for endocrine disruption and genotoxicity, and will implement sample clean-up and pre-concentration procedures. In phase II of the project, both PhD students have worked synergistically on application of the platform in collaboration with end-users. In phase II the platform has been implemented at Het Waterlaboratorium (HWL), Haarlem The Netherlands.
- Zwart, N., Hamers, T., Jonker, W., Houtman, C.J., Somsens, G., De Boer, J., Kool, J., Lamoree, M. Application of miniaturized cell-based bioassays for high-throughput effect-directed analysis of passive sampler extracts using microfractionation. Presented at the 26th SETAC Europe Annual Meeting, 22-26 May 2016, Nantes, France.
- Zwart, N., Hamers, T., Jonker, W., Houtman, C.J., De Boer, J., Kool, J., Lamoree, M. Development of a miniaturized AMES assay for high throughput effect-directed analysis of water samples using microfractionation. Presented at the 25th SETAC Europe Annual Meeting, 5 May 2015, Barcelona, Spain.
- Jonker, W., Ballesteros-Gómez, A., Hamers, T., Somsen, G.W., Lamoree, M.H. and Kool, J. Highly selective screening of estrogenic compounds in consumer-electronics plastics by liquid chromatography in parallel combined with nanofractionation-bioactivity detection and mass spectrometry. Environmental Science & Technology, 50 (2016) 12385−12393
- Jonker, W., Lamoree, M.H., Houtman, C.J., Hamers, T., Somsen, G.W., Kool, J. Rapid activity-directed screening of estrogens by parallel coupling of liquid chromatography with a functional gene reporter assay and mass spectrometry. Journal of Chromatography A, 1406 (2015) 165-174.
This project is carried out in close collaboration with the BioAnalytical Chemistry group of the Faculty of Earth and Life Sciences of the Vrije Universiteit, contact person dr. Jeroen Kool, and with dr. Corine Houtman of Het Waterlaboratorium, Haarlem, The Netherlands.