Citation:

LAZORCHAK, J. M., R. W. FLICK, K. KIDD, V. PALACE, AND R. EVANS. FATHEAD MINNOW AND PEARL DACE PILOT AT CANADIAN EXPERIMENTAL LAKES AREA. 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:

There is increasing concern about the potential impact of EDCs on aquatic organisms. Among the EDCs found in aquatic habitats are synthetic estrogens, which are used in contraceptives and other pharmaceuticals. These chemicals enter waterways through sewage treatment plants and septic systems and are also found in surface waters. EE2 is one of the most commonly used synthetic estrogens. Several studies have found EE2 in waters downstream of sewage treatment plants. In these rivers, concentrations of EE2 in the low ng/L levels are common, with some sites having concentrations as high as 40-60 ng/L.

A whole-lake endocrine-disruption experiment was conducted by Fisheries and Oceans Canada at the Experimental Lakes Area (ELA) in northwestern Ontario for three years beginning in 2001. This experiment examined population, organismal, biochemical, and cellular-level effects in lake trout, white sucker, fathead minnow, and pearl dace exposed to environmentally relevant (46 ng/L) concentrations of the synthetic estrogen, EE2. The U.S. Environmental Protection Agency (U.S EPA) collaborated in this study by evaluating VTG gene expression in (1) indigenous fathead minnows and pearl dace collected from the dosed and control lakes in 2001 through 2003, before and after dosing; (2) indigenous minnows collected in 2001from the control lake and deployed for 1, 3, 7, and 13 days in the dosed and control lakes; and (3) Cincinnati-cultured minnows exposed to water collected in 2001 through 2004 from ELA lakes and shipped to Cincinnati. In addition to water exposures, embryolarval fish and adult male fathead minnows were exposed to control- and dosed-lake whole sediment and sediment elutriate samples.

Methods developed to measure VTG gene expression in fathead minnows were validated for use in another cyprinid, pearl dace. Indigenous male fathead minnows and pearl dace collected at all time intervals from the dosed lake showed a constant level of elevation in VTG gene expression. Gene expression in the 2001 fathead minnow deployment study was detected within 24 hours after deployment of control fish into the treated lake and stayed elevated for the entire 13-day study. Highly variable gene expression was found in fathead minnow fry exposed to dosed-lake sediment elutriates, but no significant gene expression was found in fry exposed to control-lake sediment elutriates. Male adult fathead minnows exposed to sediment elutriates from sediments collected in 2004 in the previously dosed lake showed significant VTG gene expression. Results indicate that Real Time Polymerase Chain Reaction (RT-PCR) analyses of total RNA can be used to provide a rapid and timely estimate of exposure to estrogenic substances to indigenous organisms and in laboratory animals exposed to water and sediment samples. Furthermore, gene expression results, in combination with biochemical, histological, and chemical information, show that indigenous fathead minnows and pearl dace continuously exposed to 5 ng/L of EE2 will have adverse exposures.

Impacts and Outcomes

An EDC multiyear plan, "Annual Performance Measure," has been prepared and will be distributed to the Office of Water and regional offices to support the development of environmental policy and water quality criteria and standards development. A manuscript will also be prepared and submitted. Overall, this pilot research activity extended the capabilities of the U.S. EPA/ORD EDC monitoring efforts to a successful collaboration on an international whole-lake ecosystem study at an extremely remote location.

Record Details: