2006 Progress Report: Development of Receptor- to Population-Level Analytical Tools for Assessing Endocrine Disruptor Exposure in Wastewater-Impacted Estuarine SystemsEPA Grant Number: R832737
Title: Development of Receptor- to Population-Level Analytical Tools for Assessing Endocrine Disruptor Exposure in Wastewater-Impacted Estuarine Systems
Investigators: Ferguson, P. Lee , Chandler, G. Thomas
Institution: University of South Carolina at Columbia
EPA Project Officer: McOliver, Cynthia
Project Period: January 1, 2006 through December 31, 2009 (Extended to December 31, 2010)
Project Period Covered by this Report: January 1, 2006 through December 31, 2007
Project Amount: $526,028
RFA: Exposure Measurement Tools for Endocrine Disrupting Chemicals in Mixtures (2005) RFA Text | Recipients Lists
Research Category: Endocrine Disruptors , Health , Safer Chemicals
The general objectives of the proposed research are to:
- Develop nuclear hormone receptor-affinity extraction techniques as tools for isolating endocrine disrupting chemicals (EDCs) from complex wastewater mixtures.
- Apply these methods in combination with high performance mass spectrometry for activity-directed analysis of EDCs in wastewater and estuarine receiving waters on the SC coast.
- Utilize sensitive vertebrate (zebrafish) and invertebrate (copepod) EDC-exposure laboratory bioassays to link exposure measurements (above) to biological effects.
- Apply novel biomolecular endpoints to assess EDC exposure in field populations of sensitive meiobenthic invertebrates in wastewater-impacted estuarine environments.
Initial work on specific objective 1 of this project has focused on the cloning, expression, and purification of human estrogen receptor ligand binding domain (ER-LBD) and human androgen receptor ligand binding domain (AR-LBD) in bacterial vectors for use in developing receptor-affinity extraction methods suitable for isolation of receptor-active xeno-ligands from complex wastewater environments. To date, we have successfully expressed and purified active ER-LBD (in fusion with his6 and thioredoxin) in~10 mg L-1 yield and > 95% purity. The AR-LBD purification is currently being optimized to increase soluble protein expression level. We have successfully applied the ER-LBD to isolation of ER ligands from treated wastewater by receptor-affinity extraction methods. In combination with mass spectrometric (HPLC-MS/MS and HPLC-QTOF-MS) methods, natural estrogens (including estrone and 17β-estradiol) have been identified and quantified at ng L-1 concentrations in ER-affinity extracts of several wastewater samples obtained from coastal and freshwater discharge regimes within South Carolina. In support of this work we have adapted highly specific HPLC-MS/MS methods for quantitative analysis of a diverse range of xenoestrogens (including natural and synthetic hormones, bisphenol A, and alkylphenol ethoxylates) in wastewater samples. Current work focuses on validation of the ER-LBD receptor affinity extraction method for xenoestrogen isolation from wastewater and its combination with highly sensitive mass spectrometric methods for analysis of these compounds in wastewater-impacted receiving waters on the SC coast (addressing specific objective 2 above).
In parallel, we have applied sensitive, EDC-specific molecular bioassays to determine the effects of ecdysteroid-receptor active compound exposure on the harpacticoid copepod Amphiascus tenuiremus, in support of objectives 3 and 4 above. Specifically, we have performed microplate-based, full life-cycle screening assays (ASTM E 2317-04) using the insect growth-regulating pesticide tebufenozide and the fungicide fenarimol. No biologically significant effects were observed on A. tenuiremis growth, development, and reproduction following exposure to 0.005, 0.05, and 0.5 mg/L fenarimol, however exposure did cause a significant decrease in copepod ecdysteroid titers for both male and female copepods at all doses, suggesting that fenarimol either inhibits ecdysteroid biosynthesis or stimulates an acceleration of hormone metabolism. Tebufenozide exposure accelerated naupliar development, increased male abundance, and reduced female offspring production at 0.05, 0.5, and 2-mg/l exposure, respectively; however, no biologically-significant alterations of ecdysteroid titers were measured in copepods under laboratory exposure of copepods to tebufenozide.
Future work will focus on extending the receptor-affinity extraction methodology to include the human androgen receptor (expression and purification in progress) and the mysid ecdysone-receptor/ultraspiracle heterodimer (expression plasmid received). Method validation activities will include standard-addition experiments with known or suspected EDCs in wastewater extracts as well as targeted quantitative analysis of these compounds in wastewater and receiving waters on the SC coast. Identification of novel receptor-active EDCs will be facilitated by the application of high-resolution HPLC-QTOF-MS to receptor-affinity extracts from these samples. In addition, we are extending our suite of EDC bioassays to include a sensitive vertebrate xenoestrogen assay (vitellogenesis and sex-ratios in medaka fish embryos) for testing of EDCs identified in wastewater using the receptor-affinity extraction approach outlined above.