PhD project

Title: Estrogenic and androgenic potential of municipal sewage in Australia and New Zealand.

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Abstract: Studies in Europe, Japan, and North America have reported that wild fish exposed to treated sewage effluents can exhibit significant physiological and reproductive abnormalities consistent with exposure to hormonally active chemicals. The main objective of this research project was to examine the estrogenic and androgenic activity in treated sewage to determine the risk associated with treated sewage discharges in Australia and New Zealand.

Several bioassays, including a sheep estrogen receptor and a rainbow trout androgen receptor binding assay, were set up and validated with model compounds. The assays were then used to measure the estrogenic and androgenic activity in sewage samples from 15 municipal sewage treatment plants (STP) utilizing a variety of treatment technologies. Raw sewage samples contained high levels of both estrogenic and androgenic activity, up to 185ng/L estradiol equivalents (EEq) and up to 9330ng/L testosterone equivalents (TEq), respectively. Secondary treatment processes such as activated sludge had the greatest impact on removal of biological activity from the wastewater. The estrogenic and androgenic activity in final treated effluents were <1 to 4.2ng/L EEq and <6.5 to 736ng/L TEq, respectively. Based on lowest observable effective concentrations reported in the literature, these levels are unlikely to induce biological effects in exposed fish in the short term.

To examine potential long-term effects, resident mosquitofish chronically exposed to undiluted treated sewage were sampled. Several morphological biomarkers indicative of endocrine disruption were measured and compared with mosquitofish captured at a reference site. Mosquitofish captured in a constructed wetland for tertiary treatment of secondary-treated sewage exhibited morphological differences such as elongated anal fins consistent with exposure to androgenic chemicals, although this effect was not measurable in fish collected at sites further downstream or at any of the other sites. Based on these results, it is unlikely that mosquitofish populations would be significantly affected by exposure to final treated sewage. A reverse transcription real-time polymerase chain reaction (RT-PCR) method to measure the production of a female-specific protein (vitellogenin) mRNA in adult male mosquitofish was developed, and this could be used as a rapid test to detect early changes in individuals exposed to estrogenic activity.

Keywords: activated sludge, androgen receptor (AR), biomarkers, estrogen receptor (ER), in vitro bioassay, mosquitofish, rainbow trout, receptor binding assays, sewage treatment, sheep, trickling filter, vitellogenin.

Collaborators on this project:

  • Dr Louis Tremblay, Landcare Research, Lincoln, New Zealand (PhD supervisor)
  • Dr Ravi Gooneratne, Lincoln University, Lincoln, New Zealand (PhD supervisor)
  • Dr Heather Chapman, Griffith University, Brisbane, QLD, Australia (PhD supervisor)
  • Dr Mike van den Heuvel, Forest Research, Rotorua, New Zealand
  • Dr Wolfgang Koerner, German EPA, Augsburg, Germany
  • Miss Katherine Trought, Landcare Research, Lincoln, New Zealand
  • Mr Benjamin Tan, Griffith University, Brisbane, Australia
  • Mr Sean Taylor, Forest Research, Rotorua, New Zealand
  • Miss Anna Eriksson, Landcare Research, Lincoln, New Zealand
  • Dr Andrew Laurie, Canterbury Health Laboratories, Christchurch, New Zealand

Peer-reviewed publications from this project:

  1. Leusch, F.D.L., H.F. Chapman, G.W. Kay, S.R. Gooneratne and L.A. Tremblay. 2006. Anal fin morphology and gonadal histopathology in the mosquitofish (Gambusia holbrooki) exposed to treated municipal sewage effluent. Arch. Environ. Contam. Toxicol. 50: 562-574.
  2. Leusch, F.D.L., H.F. Chapman, M.R. van den Heuvel, B.L.L. Tan, S.R. Gooneratne and L.A. Tremblay. 2006. Bioassay-derived androgenic and estrogenic activity in municipal sewage in Australia and New Zealand. Ecotox. Environ. Saf. 65: 403-411.
  3. Leusch, F.D.L., M.R. van den Heuvel, H.F. Chapman, S.R. Gooneratne, A.M.E. Eriksson and L.A. Tremblay. 2006. Development of methods for extraction and in vitro quantification of estrogenic and androgenic activity of wastewater samples.. Comp. Physiol. Biochem. Part C. 143: 117-126.
  4. Leusch, F.D.L., M.R. van den Heuvel, A.D. Laurie, H.F. Chapman, S.R. Gooneratne and L.A. Tremblay. 2005. Quantification of vitellogenin mRNA induction in mosquitofish (Gambusia affinis) by reverse transcription real-time polymerase chain reaction (RT-PCR). Biomarkers. 10(6): 429-438.
  5. Sarmah, A.K., G.L. Northcott, F.D.L. Leusch and L.A. Tremblay. 2005. A survey of endocrine disrupting chemicals (EDCs) in municipal sewage and animal waste effluents in the Waikato region of New Zealand. Sci. Total Environ. 335(1-3): 98-105.
  6. Leusch FDL, Chapman HF, Koerner W, Gooneratne SR, and Tremblay LA. 2005. Efficacy of an advanced sewage treatment plant in Southeast Queensland, Australia, to remove estrogenic chemicals. Environ. Sci. Technol. 39(15): 5781-5786.