Identification of putative steroid receptor antagonists in bottled water: combining bioassays and high-resolution mass spectrometry

PLoS One. 2013 Aug 28;8(8):e72472. doi: 10.1371/journal.pone.0072472. eCollection 2013.


Endocrine disrupting chemicals (EDCs) are man-made compounds interfering with hormone signaling and thereby adversely affecting human health. Recent reports provide evidence for the presence of EDCs in commercially available bottled water, including steroid receptor agonists and antagonists. However, since these findings are based on biological data the causative chemicals remain unidentified and, therefore, inaccessible for toxicological evaluation. Thus, the aim of this study is to assess the antiestrogenic and antiandrogenic activity of bottled water and to identify the causative steroid receptor antagonists. We evaluated the antiestrogenic and antiandrogenic activity of 18 bottled water products in reporter gene assays for human estrogen receptor alpha and androgen receptor. Using nontarget high-resolution mass spectrometry (LTQ-Orbitrap Velos), we acquired corresponding analytical data. We combined the biological and chemical information to determine the exact mass of the tentative steroid receptor antagonist. Further MS(n) experiments elucidated the molecule's structure and enabled its identification. We detected significant antiestrogenicity in 13 of 18 products. 16 samples were antiandrogenic inhibiting the androgen receptor by up to 90%. Nontarget chemical analysis revealed that out of 24520 candidates present in bottled water one was consistently correlated with the antagonistic activity. By combining experimental and in silico MS(n) data we identified this compound as di(2-ethylhexyl) fumarate (DEHF). We confirmed the identity and biological activity of DEHF and additional isomers of dioctyl fumarate and maleate using authentic standards. Since DEHF is antiestrogenic but not antiandrogenic we conclude that additional, yet unidentified EDCs must contribute to the antagonistic effect of bottled water. Applying a novel approach to combine biological and chemical analysis this is the first study to identify so far unknown EDCs in bottled water. Notably, dioctyl fumarates and maleates have been overlooked by science and regulation to date. This illustrates the need to identify novel toxicologically relevant compounds to establish a more holistic picture of the human exposome.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Androgen Receptor Antagonists / analysis*
  • Androgen Receptor Antagonists / isolation & purification
  • Androgen Receptor Antagonists / pharmacology
  • Biological Assay
  • Drinking Water / analysis*
  • Endocrine Disruptors / analysis*
  • Endocrine Disruptors / isolation & purification
  • Endocrine Disruptors / pharmacology
  • Estrogen Receptor alpha / antagonists & inhibitors
  • Estrogen Receptor alpha / metabolism
  • Fumarates / analysis
  • Fumarates / isolation & purification
  • Fumarates / pharmacology
  • Genes, Reporter
  • Humans
  • Inhibitory Concentration 50
  • Maleates / analysis
  • Maleates / isolation & purification
  • Maleates / pharmacology
  • Receptors, Androgen / metabolism
  • Solid Phase Extraction
  • Tandem Mass Spectrometry
  • Transcriptional Activation / drug effects
  • Yeasts


  • Androgen Receptor Antagonists
  • Drinking Water
  • ESR1 protein, human
  • Endocrine Disruptors
  • Estrogen Receptor alpha
  • Fumarates
  • Maleates
  • Receptors, Androgen

Grant support

This study was in part supported by the German Federal Environment Agency (, Research & Development Project 206 67 448/04). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. No additional external funding received for this study.