Accurate prediction of the response of freshwater fish to a mixture of estrogenic chemicals

Environ Health Perspect. 2005 Jun;113(6):721-8. doi: 10.1289/ehp.7598.


Existing environmental risk assessment procedures are limited in their ability to evaluate the combined effects of chemical mixtures. We investigated the implications of this by analyzing the combined effects of a multicomponent mixture of five estrogenic chemicals using vitellogenin induction in male fathead minnows as an end point. The mixture consisted of estradiol, ethynylestradiol, nonylphenol, octylphenol, and bisphenol A. We determined concentration-response curves for each of the chemicals individually. The chemicals were then combined at equipotent concentrations and the mixture tested using fixed-ratio design. The effects of the mixture were compared with those predicted by the model of concentration addition using biomathematical methods, which revealed that there was no deviation between the observed and predicted effects of the mixture. These findings demonstrate that estrogenic chemicals have the capacity to act together in an additive manner and that their combined effects can be accurately predicted by concentration addition. We also explored the potential for mixture effects at low concentrations by exposing the fish to each chemical at one-fifth of its median effective concentration (EC50). Individually, the chemicals did not induce a significant response, although their combined effects were consistent with the predictions of concentration addition. This demonstrates the potential for estrogenic chemicals to act additively at environmentally relevant concentrations. These findings highlight the potential for existing environmental risk assessment procedures to underestimate the hazard posed by mixtures of chemicals that act via a similar mode of action, thereby leading to erroneous conclusions of absence of risk.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Benzhydryl Compounds
  • Cyprinidae
  • Drug Synergism
  • Estradiol / toxicity*
  • Estrogens / toxicity
  • Estrogens, Non-Steroidal / toxicity
  • Ethinyl Estradiol / toxicity*
  • Female
  • Forecasting
  • Fresh Water
  • Male
  • Phenols / toxicity*
  • Vitellogenins / biosynthesis*
  • Vitellogenins / blood
  • Water Pollutants, Chemical / toxicity


  • Benzhydryl Compounds
  • Estrogens
  • Estrogens, Non-Steroidal
  • Phenols
  • Vitellogenins
  • Water Pollutants, Chemical
  • Ethinyl Estradiol
  • Estradiol
  • 4-octylphenol
  • 4-nonylphenol
  • bisphenol A