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P513 A RAPID APPROACH FOR DIAGNOSING CERIODAPHNIA DUBIA REPRODUCTIVE FAILURE
Citation:
Lazorchak, J M., M E. Smith, M Bennett, H Haring, M. W. Tucker, P. L. Andrews, AND H. J. Allen. P513 A RAPID APPROACH FOR DIAGNOSING CERIODAPHNIA DUBIA REPRODUCTIVE FAILURE. Presented at Society of Environmental Toxicology and Chemistry, Salt Lake City, UT, November 17-21, 2002.
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:
Sources of failure in C. dubia culture and testing can be difficult to determine without outside assistance. Investigation of these failures can be time consuming. In the current study, USEPA Region 7 laboratory suffered a drop in reproduction in the C. dubia cultures, with weekly averages less than the required 15 young/female minimum. Repeated attempts to restart the cultures with animals from an outside source failed. USEPA, NERL, Cincinnati, OH agreed to participate in an inter-lab study to determine the cause of the culture failure. Preliminary analysis of the culture failure pointed to a problem related to the food or water being used by Region 7. Specifically, the cereal leaves Region 7 used to prepare Yeast/Cereal Leaves/Trout Chow (YCT) were outdated. Region 7 supplied NERL with culture water, YCT and algae. NERL supplied Region 7 with culture water, YCT and algae, along with animals from a successful culture. Eight experimental combinations of food and water were developed, such that the performance of each individual component could be determined. After a 7-day culture period, it was determined that the culture failure in Region 7 was due to the YCT. C. dubia reproduction in the eight cultures (four in each laboratory) that used Region 7 HCT ranged from 4.2 young/female to 17.8 young/female. Reproduction in the eight cultures that used NERL YCT ranged from17.4 young/female to 30.9 young/female. Region 7 replaced cereal leaves with fresh alfalfa and digested trout chow with digested flake food in the preparation of YCT. Subsequent success of the Region 7 cultures (weekly averages in excess of 20 young/female) confirmed that the problem was resolved. Systematic round-robin testing is an effective tool that can rapidly diagnose and resolve problems related to C. dubia culturing.