Citation:

LAZORCHAK, J. M., M. MILLS, AND G. SAYLES. NATIONAL WWTP EFFLUENT STUDY. Presented at 2005 EPA Science Forum, Washington, DC, May 16 - 18, 2005.

Impact/Purpose:

The indeterminate condition of exposure indicator research stands to change markedly with the ability to connect molecular biological technologies with cellular or tissue effects and outcomes. Three focal areas of ecological research aim to develop a sequence of approaches where "the earliest recognizable signatures of exposure" (i.e., unique patterns of up- and down-regulated genes and proteins) are identified for numerous stressors, demonstrable in case studies and incorporated into Agency, State and Regional studies supported by EMAP and other programs.

Area 1, Computational Toxicology Research: Exposure assessment has historically been based on use of chemical analysis data to generate exposure models. While biological activity of chemicals has been recognized to be important for exposure risk assessments, measurement of such activity has been limited to whole organism toxicity tests. Use of molecular approaches will:

improve extrapolation between components of source-to-outcome continuum (source , exposure , dose , effect , outcome)

Using a systems modeling approach, gene and protein expression data, in small fish models (fathead minnow and zebrafish), will be integrated with metabolomic and histopathological data. This will assist in prediction of environmental transformation and chemical effects based on structural characteristics, and enhance quantitative risk assessments, including areas of uncertainty such as a basis for extrapolation of effects of endocrine disrupting chemicals, interspecies extrapolation, complex chemical mixtures and dose-response assessment.

Area 2, Ecological Research-Environmental Diagnostics: Development of molecular diagnostic indicators contributes to several of the GPRA Diagnostic Research Goals. Methods will employ DNA microarray technology and expression proteomics, focusing on species of relevance to aquatic ecosystem risk assessment. Significantly, these diagnostic indicators will open the door to understanding subcellular interactions resulting from exposure to complex chemical mixtures.

define relationship between genetic disposition of populations and degree/specificity of stressor-specific gene transcriptional response in aquatic organisms (fish and invertebrates)

identify of chemical mixture induced transcriptional "patterns" using microarrays and hyperspectral scanning - via collaboration with DOE Sandia National Labs

apply molecular indicators to watershed level stressor study, including pilot studies with targeted pesticides and toxins indicators

develop molecular indicators of exposure for invertebrates (Daphnia, Lumbriculus, Chironomus)

Area 3, Exposure Research in Endocrine Disruptors:

Subobjective 1: Develop exposure methods, measurement protocols, and models for assessment of risk management practices of endocrine disrupting compounds. As risk management approaches are identified and developed, there will be a need to identify, adapt and develop bioassay screening tools and other analytical methods to assess their efficacy. Measurements research will be performed to define management needs. This effort will entail cross-lab participation from NRMRL, NERL and NHEERL.

Subobjective 2: Determine extent of environmental and human exposures to EDCs, characterize sources and factors influencing these exposures, develop and evaluate risk management strategies to reduce exposures. In order to develop effective risk management strategies, it is important to understand the extent of exposures to endocrine disrupting compounds and factors influencing source-to-exposure-to-dose relationships.

apply molecular indicators of exposure to estrogenic compounds in selected wastewater treatment plants located in ten USEPA Regions

identify differential gene expression following exposure of fathead minnows to environmental androgens and androgen-like compounds

apply molecular indicators of exposu

Description:

Reports of potential wildlife risk from exposure to environmental estrogens emphasize the need to better understand both estrogenic presence and persistence in treated wastewater effluents. In addition to wildlife exposure, human exposure should also be examined, especially in situations when estrogenic effluents may return to a drinking water supply. This potential has been examined in rivers and reservoirs in the United Kingdom, where they found reduced estrogenicity downstream from wastewater outfalls and no estrogenicity in reservoirs receiving these waters. In 2000, the NERL and the University of North Texas collaborated in a study in which the toxicity and estrogenicity of a final treated municipal effluent was examined. Male fathead minnows were deployed in the effluent for three weeks. VTG protein, Gonado-Somatic Index (GSI), Hepato-Somatic Index (HSI), and secondary sexual characteristics were biomarkers used in fish models to assess aqueous estrogenicity. VTG gene expression was also measured. There was a very good correspondence in the biomarker and gene expression results. This study provided evidence that the VTG gene expression assay could be used to assess estrogenic EDC exposures in effluents.

In 2002 and 2003, the NERL and NRMRL contacted our ten regional biologists, regional science liaisons, U.S. Environmental Protection Agency (U.S. EPA) inspectors, state U.S. EPA inspectors, and municipal plant operators to voluntarily collect up to 50 effluents and ship them to Cincinnati for male and female fathead minnow exposures. The objective of this study was to determine whether adult male or female fathead minnows exposed to a municipal WWTP effluent elicited a change in VTG gene expression above or below a lab water control. The goal was to use VTG gene expression results to assess whether certain types of domestic WWTPs or operations are more effective in removing estrogenic EDCs. Of the 50 effluents tested, 13 (26%) effluents upregulated VTG expression in male fathead minnows, two effluents downregulated VTG expression in female fathead minnows, and two effluents that were resampled and tested demonstrated upregulation of VTG in male fathead minnows. This study demonstrated the utility of using the U.S. EPA's male fathead minnow VTG gene expression assay as a tool for screening effluents for estrogenic EDCs. Additional assessment of individual operational information of those wastewater plants demonstrating estrogenicity and similar treatment facilities that did not show estrogenicity need to take place to determine whether certain types of treatment processes and/or operations are more efficient at removing estrogenic EDCs than others. Chemical analyses of effluent samples are currently underway. Results will be used to look for the relationships among the detected estrogens and androgens to gene expression results.

Impacts and Outcomes

This research study initiated the ORD's efforts to monitor EDCs on more than a local scale. Two ORD national laboratories collaborated to expand the scope of a local WWTP study to include samples from several states. The feasibility of sample collection, shipment, and analysis at this scale was shown. Positive findings are setting the research agenda for both future studies and development of additional indicators. Additional studies are currently being planned to look at relationships between the presence of EDCs in effluents and in-stream adverse exposures. An EDC multiyear plan, "Annual Performance Measure," has been generated on the gene expression results and is being distributed to the regions and the Office of Water.

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