Thin-layer chromatography bioassay coupled detection of organic micro-pollutant induced effects in the environment

• Detektion von mikroverunreinigungs-induzierten Effekten in der Umwelt mittels Dünnschichtchromatographie-Bioassay-Kopplung

Riegraf, Carolin Elena; Hollert, Henner (Thesis advisor); Schäffer, Andreas (Thesis advisor)

Aachen : RWTH Aachen University (2021)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2021

Abstract

A vast number of different micro-pollutants (MPs) continuously enters the aquatic environment via diverse pathways and is ubiquitously detected in surface and ground waters. Hence, aquatic ecosystems are exposed to a mixture of MPs including those potentially causing adverse effects in the environment. Endocrine disrupting compounds (EDCs) are MPs of special concern, because of their potential impact on different components of the complex endocrine system and its essential functions like reproduction and development. The major target of EDCs are different hormone receptors, e.g., estrogen, androgen or thyroid hormone receptor. Another important group of MPs are genotoxic compounds, which can irreversibly affect the genome, potentially leading to effects on the population level. Further, herbicides applied to impede weeds’ growth, e.g., by inhibiting the photosystem II (PSII), can leach into adjacent water bodies and thus adversely affect the aquatic environment. Prospective and retrospective risk assessments, in e.g., the European Union, were installed to achieve a good chemical and ecological status of freshwater systems. The focus of the current European Water Framework Directive (WFD) to determine the chemical status is mainly compound-based and relies on the detection and monitoring of a defined list of potential adverse compounds, so called priority substances. However, the detection of biologically active compounds, including unknown transformation products, cannot be guaranteed, nor possible combinatory effects of compounds’ mixtures can be captured. Furthermore, the enormous number of compounds present in the aquatic environment poses a challenge for the assessment of risk to the aquatic environment and hampers comprehensive chemical characterization. Effect-based methods (EBMs), capturing all substances contributing to an observed effect, are therefore discussed as complementary elements to chemical analysis in line with the WFD. However, the exclusive use of EBMs to directly identify the sample components responsible for the detected biological activity is not possible, especially in environmental samples constituted of a mixture of diverse components. Thus, a combination of chemical analysis of a sample, along an assaying of the adverse biological responses of its constituents, is required. Effect-directed analysis (EDA) combines both, ecotoxicological and analytical methods, to directly link potential adverse biological responses of separated fractions detected by bioassays with the characterization of contaminants by chemical analysis. As an alternative to high-performance liquid chromatography, high-performance thin-layer chromatography (HPTLC) can be used for sample fractionation prior to the assessment of effects using bioassays performed directly on the surface of the HPTLC plate. In this context, the present thesis aims to broaden and further improve the spectrum of specific bioassays combined with HPTLC for an efficient and robust effect screening of environmental samples to guide a subsequent in-depth EDA. The main objectives of the present thesis are to (i) combine HPTLC with bioassays for the detection of dioxin-like activity and hormone-like effects associated with endocrine disruption, (ii) develop an approach to detect direct genotoxicity in combination with HPTLC, (iii) investigate the possibility to perform the PSII-inhibition test directly on a HPTLC plate surface, (iv) identify effect-inducing compounds by hyphenation of the planar bioassays with analytical techniques and (v) assess the potential of the developed methods by investigating complex environmental samples. A stepwise adaptation of existing methods, optimization of procedures as well as adjustment of test conditions resulted in the successful combination of HPTLC with bioassays for the detection of androgenicity (p-YAS), dioxin-like activity (p-YDS), effects on the vitamin D receptor (p-YVS), genotoxicity (p-recA) and PSII-inhibition (p-PSII). Also, bioassays for the detection of thyroidogenicity (p-YTS) as well as of effects on the retinoic acid receptor (p-YRaS) could be successfully performed on the surface of a HPTLC plate. The developed planar test systems were thoroughly characterized in terms of sensitivity, repeatability and robustness using several model compounds tested individually as well as in mixture. The limit of quantification (LOQ) with chromatographic development was 37 pg for testosterone (p-YAS), 0.48 ng for beta-naphthoflavone (p-YDS) and 1.0 ng for calcipotriol hydrate (p-YVS). In the p-PSII, LOQs of 1.9 ng and 99 pg were established for atrazine and diuron, respectively. A LOQ without chromatographic development of 10 pg for 3,3’,5-triiodothyroacetic acid (p-YTS), 0.41 pg for tamibarotene (p-YRaS) and 0.86 ng for mitomycin C (p-recA) was determined. The versatile area of application of the developed methodologies was shown by investigating matrix-rich influent and effluent samples from wastewater treatment plants (WWTPs), landfill leachates and surface waters. The establishment of activity profiles allowed the performance comparison of different WWTPs especially regarding fraction specific removal efficiencies of agonistic effects. Observed specific effects were examined qualitatively based on visualized activity profiles and quantitatively by calculating biological equivalence concentrations for bioactive fractions. The investigated WWTPs showed high effect-based removal efficiencies of estrogenicity, androgenicity and partly of PSII-inhibition. However, differences among treatments and wastewater profiles could be observed. In contrast to the classic well-based approach, the established activity profiles of a sample revealed fraction specific information of potential effects. Thus, a comparison of the WWTP samples showed that although the overall activity decreased in most tests from influent to effluent, fractions showing increasing activities could be determined and characterized. Furthermore, the formation or the activation of compounds possibly due to metabolization processes during the wastewater treatment caused an increased genotoxicity. In landfill leachates, estrogenicity, dioxin-like effects as well as PSII-inhibition were detected with the developed methodologies. Bisphenol A was identified as a main driver of estrogenicity in landfill leachates. This finding was verified by hyphenation of the planar bioassay with liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry. Surface water extracts collected by passive sampling devices were successfully investigated regarding PSII-inhibition. The results obtained by the p-PSII correlated well with results from LC-MS, but also revealed unknown PSII-inhibiting activities. The presented approaches can be used in the context of the WFD as fast and robust screening tools for environmental samples. The effect-based approach allows to directly focus on the detection of environmentally relevant compounds eliciting specific effects. Compounds that might pose a risk for the environment can be captured based on their effects, even if the chemical structure of the respective environmental contamination might be yet unknown. The screening tools could be applied to track pressures and sources of chemical contaminations, to analyze emission pathways of biologically active substances or to support the control and optimization of processes in such as wastewater treatment. The characterization of effect profiles with the presented approach enables the compound-based assessment of the chemical status by including selected priority compounds as reference compounds. Moreover, it captures untargeted MPs exhibiting the same mode of action, which makes it a suitable screening tool for a possible future effect-based assessment of the status of freshwater bodies like rivers and lakes using effect-based trigger values.

Institutions

• Department of Biology [160000]
• Department of Ecosystem Analysis [162420]
• Chair of Environmental Biology and Chemodynamics [162710]