Abstract
Effect-directed analysis (EDA) that combines effect-based methods (EBMs) with high-performance thin-layer chromatography (HPTLC) is a useful technique for spatial, temporal, and process-related effect evaluation and may provide a link between effect testing and responsible substance identification. In this study, a yeast multi endocrine-effect screen (YMEES) for the detection of endocrine effects is combined with HPTLC. Simultaneous detection of estrogenic, androgenic, and gestagenic effects on the HPTLC plate is achieved by mixing different genetically modified Arxula adeninivorans yeast strains, which contain either the human estrogen, androgen, or progesterone receptor. Depending on the yeast strain, different fluorescent proteins are formed when an appropriate substance binds to the specific hormone receptor. This allows to measure hormonal effects at different wavelengths. Two yeast cell application approaches, immersion and spraying, are compared. The sensitivity and reproducibility of the method are shown by dose-response investigations for reference compounds. The spraying approach indicated similar sensitivities and higher precisions for the tested hormones compared to immersion. The EC10s for estrone (E1), 17β-estradiol (E2), 17α-ethinylestradiol (EE2), 5α-dihydrotestosterone (DHT), and progesterone (P4) were 95, 1.4, 10, 7.4, and 15 pg/spot, respectively. Recovery rates of E1, E2, EE2, DHT, and P4 between 88 and 120% show the usability of the general method in combination with sample enrichment by solid phase extraction (SPE). The simultaneous detection of estrogenic, androgenic, and gestagenic effects in wastewater and surface water samples demonstrates the successful application of the YMEES in such matrices. This promising method allows us to identify more than one endocrine effect on the same HPTLC plate, which saves time and material. The method could be used for comparison, evaluation, and monitoring of different river sites and wastewater treatment steps and should be tested in further studies.
Graphical abstract
Similar content being viewed by others
Data availability
The datasets generated during and analyzed during the current study are available from the corresponding author on reasonable request.
References
Rogowska J, Cieszynska-Semenowicz M, Ratajczyk W, Wolska L. Micropollutants in treated wastewater. Ambio. 2019;49(2):487–503. https://doi.org/10.1007/s13280-019-01219-5.
Henneberg A, Bender K, Blaha L, Giebner S, Kuch B, Kohler HR, et al. Are in vitro methods for the detection of endocrine potentials in the aquatic environment predictive for in vivo effects? Outcomes of the projects SchussenAktiv and SchussenAktivplus in the Lake Constance area, Germany. PLoS One. 2014;9(6):e98307. https://doi.org/10.1371/journal.pone.0098307.
Gehrmann L, Bielak H, Behr M, Itzel F, Lyko S, Simon A, et al. (Anti-)estrogenic and (anti-)androgenic effects in wastewater during advanced treatment: comparison of three in vitro bioassays. Environ Sci Pollut Res Int. 2018;25(5):4094–104. https://doi.org/10.1007/s11356-016-7165-4.
Harth FUR, Arras C, Brettschneider DJ, Misovic A, Oehlmann J, Schulte-Oehlmann U, et al. Small but with big impact? Ecotoxicological effects of a municipal wastewater effluent on a small creek. J Environ Sci Health A Tox Hazard Subst Environ Eng. 2018;53(13):1149–60. https://doi.org/10.1080/10934529.2018.1530328.
Itzel F, Jewell KS, Leonhardt J, Gehrmann L, Nielsen U, Ternes TA, et al. Comprehensive analysis of antagonistic endocrine activity during ozone treatment of hospital wastewater. Sci Total Environ. 2018;624:1443–54. https://doi.org/10.1016/j.scitotenv.2017.12.181.
Fent K. Progestins as endocrine disrupters in aquatic ecosystems: concentrations, effects and risk assessment. Environ Int. 2015;84:115–30. https://doi.org/10.1016/j.envint.2015.06.012.
Chang H, Wan Y, Wu S, Fan Z, Hu J. Occurrence of androgens and progestogens in wastewater treatment plants and receiving river waters: comparison to estrogens. Water Res. 2011;45(2):732–40. https://doi.org/10.1016/j.watres.2010.08.046.
Commission Implementing Decision (EU) 2018/840 of 5 June 2018 Establishing a watch list of substances for Union-wide monitoring in the field of water policy pursuant to Directive 2008/105/EC of the European Parliament and of the Council and repealing Commission Implementing Decision (EU) 2015/495. Official Journal of the European Union
Duft M, Schulte-Oehlmann U, Tillmann M, Markert B, Oehlmann J. Toxicity of triphenyltin and tributyltin to the freshwater mudsnail Potamopyrgus antipodarum in a new sediment biotest. Environ Toxicol Chem. 2003;22(1):145–52.
Jobling S, Casey D, Rodgers-Gray T, Oehlmann J, Schulte-Oehlmann U, Pawlowski S, et al. Comparative responses of molluscs and fish to environmental estrogens and an estrogenic effluent. Aquat Toxicol. 2004;66(2):207–22. https://doi.org/10.1016/j.aquatox.2004.01.002.
Kidd KA, Blanchfield PJ, Mills KH, Palace VP, Evans RE, Lazorchak JM, et al. Collapse of a fish population after exposure to a synthetic estrogen. PNAS. 2007;104(21):8897–901. https://doi.org/10.1073/pnas.0609568104.
Aris AZ, Shamsuddin AS, Praveena SM. Occurrence of 17alpha-ethynylestradiol (EE2) in the environment and effect on exposed biota: a review. Environ Int. 2014;69:104–19. https://doi.org/10.1016/j.envint.2014.04.011.
Orlando EF, Ellestad LE. Sources, concentrations, and exposure effects of environmental gestagens on fish and other aquatic wildlife, with an emphasis on reproduction. Gen Comp Endocrinol. 2014;203:241–9. https://doi.org/10.1016/j.ygcen.2014.03.038.
Örn S, Holbech H, Norrgren L. Sexual disruption in zebrafish (Danio rerio) exposed to mixtures of 17alpha-ethinylestradiol and 17beta-trenbolone. Environ Toxicol Pharmacol. 2016;41:225–31. https://doi.org/10.1016/j.etap.2015.12.010.
Rivero-Wendt CL, Oliveira R, Monteiro MS, Domingues I, Soares AM, Grisolia CK. Steroid androgen 17alpha-methyltestosterone induces malformations and biochemical alterations in zebrafish embryos. Environ Toxicol Pharmacol. 2016;44:107–13. https://doi.org/10.1016/j.etap.2016.04.014.
Kuckelkorn J, Redelstein R, Heide T, Kunze J, Maletz S, Waldmann P, et al. A hierarchical testing strategy for micropollutants in drinking water regarding their potential endocrine-disrupting effects-towards health-related indicator values. Environ Sci Pollut Res Int. 2018;25(5):4051–65. https://doi.org/10.1007/s11356-017-0155-3.
Ziková A, Lorenz C, Hoffmann F, Kleiner W, Lutz I, Stöck M, et al. Endocrine disruption by environmental gestagens in amphibians - a short review supported by new in vitro data using gonads of Xenopus laevis. Chemosphere. 2017;181:74–82. https://doi.org/10.1016/j.chemosphere.2017.04.021.
Knoop O, Hohrenk LL, Lutze HV, Schmidt TC. Ozonation of tamoxifen and toremifene: reaction kinetics and transformation products. Environ Sci Technol. 2018;52(21):12583–91. https://doi.org/10.1021/acs.est.8b00996.
Knoop O, Itzel F, Tuerk J, Lutze HV, Schmidt TC. Endocrine effects after ozonation of tamoxifen. Sci Total Environ. 2018;622–623:71–8. https://doi.org/10.1016/j.scitotenv.2017.11.286.
Brack W, Aissa SA, Backhaus T, Dulio V, Escher BI, Faust M, et al. Effect-based methods are key. The European Collaborative Project SOLUTIONS recommends integrating effect-based methods for diagnosis and monitoring of water quality. Environ Sci Eur. 2019;31(1). https://doi.org/10.1186/s12302-019-0192-2.
Schuetzle D, Lewtas J. Bioassay-directed chemical analysis in environmental research. Anal Chem. 1986;58(11):1060A–75A. https://doi.org/10.1021/ac00124a001.
Brack W. Effect-directed analysis: a promising tool for the identification of organic toxicants in complex mixtures? Anal Bioanal Chem. 2003;377(3):397–407. https://doi.org/10.1007/s00216-003-2139-z.
Brack W, editor. Effect-directed analysis of complex environmental contamination. The handbook of environmental chemistry. Berlin, Heidelberg: Springer; 2011.
Weller MG. A unifying review of bioassay-guided fractionation, effect-directed analysis and related techniques. Sensors (Basel). 2012;12(7):9181–209. https://doi.org/10.3390/s120709181.
Brack W, Ait-Aissa S, Burgess RM, Busch W, Creusot N, Di Paolo C, et al. Effect-directed analysis supporting monitoring of aquatic environments--an in-depth overview. Sci Total Environ. 2016;544:1073–118. https://doi.org/10.1016/j.scitotenv.2015.11.102.
Muschket M, Di Paolo C, Tindall AJ, Touak G, Phan A, Krauss M, et al. Identification of unknown antiandrogenic compounds in surface waters by effect-directed analysis (EDA) using a parallel fractionation approach. Environ Sci Technol. 2018;52(1):288–97. https://doi.org/10.1021/acs.est.7b04994.
Hashmi MAK, Krauss M, Escher BI, Teodorovic I, Brack W. Effect-directed analysis of progestogens and glucocorticoids at trace concentrations in river water. Environ Toxicol Chem. 2020;39(1):189–99. https://doi.org/10.1002/etc.4609.
Morlock G, Schwack W. Hyphenations in planar chromatography. J Chromatogr A. 2010;1217(43):6600–9. https://doi.org/10.1016/j.chroma.2010.04.058.
Buchinger S, Spira D, Bröder K, Schlüsener M, Ternes T, Reifferscheid G. Direct coupling of thin-layer chromatography with a bioassay for the detection of estrogenic compounds: applications for effect-directed analysis. Anal Chem. 2013;85(15):7248–56. https://doi.org/10.1021/ac4010925.
Weiss SC, Egetenmeyer N, Schulz W. Coupling of in vitro bioassays with planar chromatography in effect-directed analysis. Adv Biochem Eng Biotechnol. 2017;157:187–224. https://doi.org/10.1007/10_2016_16.
Chamas A, Pham HTM, Jähne M, Hettwer K, Gehrmann L, Tuerk J, et al. Separation and identification of hormone-active compounds using a combination of chromatographic separation and yeast-based reporter assay. Sci Total Environ. 2017;605–606:507–13. https://doi.org/10.1016/j.scitotenv.2017.06.077.
Stütz L, Weiss SC, Schulz W, Schwack W, Winzenbacher R. Selective two-dimensional effect-directed analysis with thin-layer chromatography. J Chromatogr A. 2017;1524:273–82. https://doi.org/10.1016/j.chroma.2017.10.009.
Stütz L, Schulz W, Winzenbacher R. Identification of acetylcholinesterase inhibitors in water by combining two-dimensional thin-layer chromatography and high-resolution mass spectrometry. J Chromatogr A. 2020;1624:461239. https://doi.org/10.1016/j.chroma.2020.461239.
Chamas A, Pham HTM, Jähne M, Hettwer K, Uhlig S, Simon K, et al. Simultaneous detection of three sex steroid hormone classes using a novel yeast-based biosensor. Biotechnol Bioeng. 2017;114(7):1539–49. https://doi.org/10.1002/bit.26249.
Schoenborn A, Schmid P, Bräm S, Reifferscheid G, Ohlig M, Buchinger S. Unprecedented sensitivity of the planar yeast estrogen screen by using a spray-on technology. J Chromatogr A. 2017;1530:185–91. https://doi.org/10.1016/j.chroma.2017.11.009.
Klingelhöfer I, Morlock GE. Sharp-bounded zones link to the effect in planar chromatography-bioassay-mass spectrometry. J Chromatogr A. 2014;1360:288–95. https://doi.org/10.1016/j.chroma.2014.07.083.
Grünebaum T, Jardin N, Lübken M, Marc W, Sven L, Rath L, et al. Untersuchung verschiedener Verfahren zur weitergehenden Spurenstoffelimination auf kommunalen Kläranlagen im großtechnischen Maßstab. Korrespondenz Abwasser. 2014;10:876–84.
Bergmann AJ, Simon E, Schifferli A, Schönborn A, Vermeirssen ELM. Estrogenic activity of food contact materials-evaluation of 20 chemicals using a yeast estrogen screen on HPTLC or 96-well plates. Anal Bioanal Chem. 2020;412(19):4527–36. https://doi.org/10.1007/s00216-020-02701-w.
Van den Belt K, Berckmans P, Vangenechten C, Verheyen R, Witters H. Comparative study on the in vitro/in vivo estrogenic potencies of 17beta-estradiol, estrone, 17alpha-ethynylestradiol and nonylphenol. Aquat Toxicol. 2004;66(2):183–95. https://doi.org/10.1016/j.aquatox.2003.09.004.
Riegraf C, Reifferscheid G, Belkin S, Moscovici L, Shakibai D, Hollert H, et al. Combination of yeast-based in vitro screens with high-performance thin-layer chromatography as a novel tool for the detection of hormonal and dioxin-like compounds. Anal Chim Acta. 2019;1081:218–30. https://doi.org/10.1016/j.aca.2019.07.018.
Klingelhöfer I, Morlock GE. Bioprofiling of surface/wastewater and bioquantitation of discovered endocrine-active compounds by streamlined direct bioautography. Anal Chem. 2015;87(21):11098–104. https://doi.org/10.1021/acs.analchem.5b03233.
Günter A, Balsaa P, Werres F, Schmidt TC. Influence of the drying step within disk-based solid-phase extraction both on the recovery and the limit of quantification of organochlorine pesticides in surface waters including suspended particulate matter. J Chromatogr A. 2016;1450:1–8. https://doi.org/10.1016/j.chroma.2016.03.073.
Cotlet M, Goodwin PM, Waldo GS, Werner JH. A comparison of the fluorescence dynamics of single molecules of a green fluorescent protein: one- versus two-photon excitation. Chemphyschem. 2006;7(1):250–60. https://doi.org/10.1002/cphc.200500247.
Schick D, Schwack W. Planar yeast estrogen screen with resorufin-beta-d-galactopyranoside as substrate. J Chromatogr A. 2017;1497:155–63. https://doi.org/10.1016/j.chroma.2017.03.047.
Nakada N, Shinohara H, Murata A, Kiri K, Managaki S, Sato N, et al. Removal of selected pharmaceuticals and personal care products (PPCPs) and endocrine-disrupting chemicals (EDCs) during sand filtration and ozonation at a municipal sewage treatment plant. Water Res. 2007;41(19):4373–82. https://doi.org/10.1016/j.watres.2007.06.038.
Itzel F, Baetz N, Hohrenk LL, Gehrmann L, Antakyali D, Schmidt TC, et al. Evaluation of a biological post-treatment after full-scale ozonation at a municipal wastewater treatment plant. Water Res. 2020;170:115316. https://doi.org/10.1016/j.watres.2019.115316.
Acknowledgments
Financial support for the graduate program Future Water by the Ministry of Culture and Science of the Federal State of North Rhine-Westphalia (MKW NRW, Düsseldorf, Germany) is gratefully acknowledged. Gratefully acknowledged is also the support of the Ruhrverband (Essen, Germany) especially Prof. Dr. Grünebaum and the team from the WWTP Schwerte. Special thanks also go to new_diagnostics (Berlin, Germany) and especially Martin Jähne for providing the yeast strains and fruitful discussions. Further thanks also go to the reviewers of this work, who really helped to improve the manuscript and conclusions with their comments.
Funding
This study received funding from the graduate program Future Water by the Ministry of Culture and Science of the Federal State of North Rhine-Westphalia (MKW NRW, Düsseldorf, Germany).
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Ethics approval
Not applicable
Code availability
Not applicable
Additional information
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary information
ESM 1
(PDF 1146 kb)
Rights and permissions
About this article
Cite this article
Baetz, N., Rothe, L., Wirzberger, V. et al. High-performance thin-layer chromatography in combination with a yeast-based multi-effect bioassay to determine endocrine effects in environmental samples. Anal Bioanal Chem 413, 1321–1335 (2021). https://doi.org/10.1007/s00216-020-03095-5
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00216-020-03095-5