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Estrogene in Küstengewässern - Untersuchungen in der Ostsee mittels chemischer Analytik und einem in vitro-Biotest

Estrogens in coastal surface waters - Investigations in the Baltic Sea using chemical analysis and an in vitro-bio assay

  • In der vorliegenden Arbeit wurde ein spurenanalytisches Verfahren zur simultanen Identifizierung und Quantifizierung von Estrogenen in Küstengewässern entwickelt. Für die chemische „target“-Analyse wurden die natürlichen Steroide Estron, 17ß-Estradiol, Estriol, das synthetische Hormon 17α-Ethinylestradiol, die Phytoestrogene Daidzein und Genistein sowie mit Nonylphenol, 4-tert-Octylphenol und Bisphenol A drei weit verbreitete Xenoestrogene ausgewählt. Das Verfahren beinhaltet eine großvolumige Festphasenextraktion von durchschnittlich 49 Litern Oberflächen-wasser mit Hilfe des Copolymers Oasis HLB und eine sich anschließende Extraktaufreinigung an Kieselgel. Von 400 µL Extraktvolumen wurde ein 100 µL Aliquot für die chemisch-analytische Bestimmung der ausgewählten Analyte verwendet. Mit Hilfe der großvolumigen Anreicherung in Kombination mit leistungsstarker Messtechnik, Hochleistungs-Flüssigchromatographie in Kombination mit Elektrospray-Tandem-Massenspektrometrie, wurden Nachweisgrenzen im Bereich zwischen 0,02 (Estron) und 1 ng L-1 (Estriol) erreicht. Bei der Vermessung von Realproben auftretende Signalsuppressionen wurden für jede Probe ermittelt und die Ergebnisse diesbezüglich korrigiert. Die für die entwickelte Methode kalkulierten Wiederfindungen der Extraktion lagen zwischen 52 (4-tert-Octylphenol) and 91% (Nonylphenol). Die Ergebnisse der vorliegenden Arbeit zeigen, dass nicht nur Kläranlagenausläufe und Flüsse vom Eintrag anthropogener Estrogene betroffen sind, sondern auch die Küstenzonen mariner Ökosysteme. Das Vorkommen von Estrogenen in einem umweltrelevanten Konzentrationsbereich und das damit verbundene estrogene Wirkpotenzial im küstennahen Oberflächenwasser der Deutschen Ostsee stellen eine mögliche Erklärung für die in dieser Küstenzone bei Fischen festgestellten Reproduktionsstörungen dar. Vor diesem Hintergrund sollten auch Küstenzonen zunehmend im Fokus wissenschaftlicher Studien zum Thema Endokrine Disruptoren stehen.
  • An analytical method has been developed for the determination of different estrogenic substances in coastal waters. For the target-analyses three natural steroids (estradiol, estriol, estrone), two phytoestrogens (genistein, daidzein) and three phenolic xenoestrogens (nonylphenol, 4-tert-octylphenol, bisphenol A) were chosen. The procedure includes a solid-phase extraction of approx. 49 litres using the copolymer Oasis HLB followed by a clean-up on silica. 100 µL of the total extract volume of 400 µL were used for the identification of the target-analytes using high performance liquid chromatography coupled with electrospray-ionisation tandem mass spectrometry (HPLC-ESI-MS/MS). Method detection limits ranged from 0.02 (estrone) to 1 ng L-1 (estriol). Signal suppression occuring during electrospray ionisation was calculated for each field sample and quantitation results were corrected. For the method developed calculated extraction recoveries between 52 (4-tert-octylphenol) and 91% (nonylphenol) were obtained. During two sampling campaigns in July 2003 and 2004, the method was applied to determine environmental estrogens in coastal waters of the Baltic Sea. The analyses showed the presence of the steroids estrone and ethinylestradiol, and the xenoestrogens nonylphenol, 4-tert-octylphenol and bisphenol A, all in the lower ng per litre range. Due to their high relative estrogenic potency, the concentrations of estrone and ethinylestradiol are of concern. In inner coastal waters generally higher concentrations of analytes could be observed. The yeast estrogen screen (YES) was used for the subsequent biological analyses to assess the estrogenic activity in surface waters of a coastal region in the German Baltic Sea and was applied to 100 µL sample aliquots. Results were expressed as a percentage of the maximum response of the positive control 17ß-estradiol and on the basis of a 20% response measured estradiol equivalents (EEQs) were calculated. The occurrence of estrogens in coastal waters of the Baltic Sea was confirmed by the YES showing measured estrogenic activities up to 0.82 ng L-1 EEQs. From 29 samples taken during two campaigns (2003 and 2004) at five different stations 27 samples showed an estrogenic response higher than 10%. Samples from inner coastal waters generally showed higher responses than samples from the Outer Wismar Bay or Darss Peninsula. To determine the substances mainly responsible for the observed estrogenicity, the approach of the bioassay-directed chemical analysis was used. A reversed-phase fractionation method was developed and applied to 100 µL aliquots of estrogenic field samples. From seven individual fractions, three showed significant estrogenicity. Taking their relative estrogenic potencies into account, the chemical analysis identified the synthetic hormone 17α-ethinylestradiol, but particularly the natural steroids 17ß-estradiol and estrone as main contributors to the overall estrogenicities. The fractionation of the total extracts lead to fractions of higher purity with lower signal suppression during LC-MS/MS measurements. Hence, the detection of the female steroid 17ß-estradiol became possible. Thus, only steroidal hormones seem to be responsible for the observed estrogenicity in coastal surface waters of the German Baltic Sea. Based on concentration data and relative estrogenic potencies of analytes, calculated estradiol equivalents were determined for each individual fraction. Measured and calculated estradiol equivalents correlated well, when disregarding results for the second fractions, where high deviations occurred. The concentrations of 17α-ethinylestradiol determined in the second fractions didn’t match the comparatively low estrogenic activities. Possible reasons are the analytical measurement uncertainties in quantifying 17α-ethinylestradiol in field samples. Peak interferences during the HPLC-ESI-MS/MS measurements may lead to a significant overestimation of 17α-ethinylestradiol concentrations. Additionally, the presence of anti-estrogenic compounds could contribute to the observed lower activities. Results obtained within this work indicate, that not only sewage treatment plant effluents and river waters are significantly influenced by estrogenic substances, but also coastal areas of marine ecosystems. The occurrence of estrogens in the range of effect concentrations could be one possible explanation for reproductive disorders found in the German Baltic Sea. For this reason, coastal zones should be increasingly in focus of environmental research in the field of endocrine disruptors.

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Metadaten
Author: Iris-Constanze Beck
URN:urn:nbn:de:gbv:luen4-opus-3807
URL: https://pub-data.leuphana.de/frontdoor/index/index/docId/425
Advisor:W.-U. Palm (Dr.)
Document Type:Doctoral Thesis
Language:German
Year of Completion:2006
Date of Publication (online):2006/07/17
Publishing Institution:Leuphana Universität Lüneburg
Granting Institution:Leuphana Universität Lüneburg, Nachhaltigkeit
Date of final exam:2006/06/28
Release Date:2006/07/17
Tag:HPLC-ESI-MS/MS
Baltic Sea; Bioassay; Estrogens; HPLC-ESI-MS/MS
GND Keyword:Ostsee; Biotest; HPLC-MS; Östrogene
Institutes:Universität / Frühere Fachbereiche
Dewey Decimal Classification:5 Naturwissenschaften und Mathematik / 57 Biowissenschaften; Biologie / 570 Biowissenschaften; Biologie