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ToxSci Advance Access published online on June 12, 2003
Toxicological Sciences, doi:10.1093/toxsci/kfg162
Toxicological Sciences © Society of Toxicology 2003; all rights reserved
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Received April 16, 2003; accepted May 27, 2003
© 2003 Society of Toxicology

Environmental Toxicology

Combined in Situ and in Vitro Assessment of the Estrogenic Activity of Sewage and Surface Water Samples

S. Pawlowski 1*, T. Ternes 2, M. Bonerz 2, T. Kluczka 3, B. van der Burg 4, H. Nau 3, L. Erdinger 5, and T. Braunbeck 1

1 Department of Zoology, University of Heidelberg, Im Neuenheimer Feld 230, D-69120 Heidelberg, Germany
2 ESWE-Institute for Water Research and Water Technology, Söhnleinstrasse 158, D-65201 Wiesbaden, Germany
3 Department of Food Toxicology, Center of Food Science, School of Veterinary Medicine Hannover, Bischofsholer Damm 15, D-30173 Hannover, Germany
4 Bio Detection Systems b.v., Kanaal Laboratorium, Badhuisweg 3, 1031 CM Amsterdam, The Netherlands
5 Department of Hygiene, University of Heidelberg, Im Neuenheimer Feld 354, D-69120 Heidelberg, Germany

* To whom correspondence should be addressed. E-mail: spawlows{at}ix.urz.uni-heidelberg.de.


  Abstract

In order to investigate the estrogenic activities of two municipal sewage treatment plant (STP; site A, B) effluents and of Rhine water sampled at Worms (site C; Rhine-Neckar triangle, Germany), data from in situ experiments measuring hepatic vitellogenin expression from caged rainbow trout (Oncorhynchus mykiss) were compared with data from in vitro bioassays (yeast estrogen screen, YES; ER luciferase assay with HEK 293 cells, HEK; primary rainbow trout hepatocytes, PH) and chemical analysis. Three sampling campaigns were carried out at each site between November 2000 and September 2001.

VTG-mRNA expression in male rainbow trout exposed for two weeks ranged from 3 ± 5 to 619 ± 188 and from 226 ± 38 to 3,373 ± 1,958 pg/µg total RNA at sites A and B, respectively. E2-Equivalents obtained from the in vitro bioassays gave values up to 0.21 ± 0.04 nM (57.3 ± 10.2 ng/L, PH), 0.07 ± 0.03 nM (20.2 ± 6.9 ng/L; YES) and 0.008 ± 0.002 nM (2.1 ± 0.7 ng/L; HEK). In contrast, in one year-old rainbow trout exposed at site C, no VTG-mRNA induction could be observed after two weeks of exposure. In vitro bioassays (YES, HEK, PH) indicated an estrogenic activity at site C, which, however, was lower than at the investigated STP effluents. Chemical analysis of representative water samples from site A identified steroidal estrogens up to 5.6 ng/L 17{beta}-estradiol (E2), 19 ng/L estrone as well as 1.5 ng/L 17{alpha}-ethinylestradiol. Furthermore, the sum of fecal- and phytosteroids, resorcyclic lactones, and flavonoids concentrations were 280 (A) and 1.200 ng/L (B). In addition, site C (river Rhine) contained 3.9 ng/L E2 and 250 ng/L of fecal- and phytosteroids, respectively. Thus, STP effluents and Rhine water contain biologically relevant concentrations of estrogenic compounds, the activity of which can be detected by means of various bioassays.

Key Words: Estrogenicity, STP effluent, surface water, Rhine, rainbow trout, primary hepatocytes, HEK 293 cells, yeast estrogen screen, solid phase extracted water samples, chemical analysis .


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