Administrative Operations Staff (MD-49), Air Pollution Prevention and Control Division, National Risk Management Research Lab, US EPA, Research Triangle Park, NC 27711

A -- COOPERATIVE AGREEMENT ADDRESSING PFIESTERIA PISCICIDA IMPACT SOL NHEERL 99-ETD-01 DUE 062499 POC Margaret Mann (919) 541-4896 WEB: Not applicable, http://www.epa.gov/nheerl/organ.htm. E-MAIL: Not applicable, mann.margaret@epamail.epa.gov. The US EPA's National Health and Environmental Effects Research Laboratory (NHEERL), in cooperation with US EPA Region III Office, Philadelphia, PA, intends to conduct research to address various hypotheses concerning the potential impact of several agricultural runoff products on the diversity and populace of Pfiesteria piscicida, its major prey and predators, and on fish immunological functions via the establishment of an assistance agreement. Interested parties should have: 1) a Level 3 Biohazard Containment System custom designed for toxic Pfiesteria research, which is thought to be essential for the protection of the health of researchers working with toxic forms of Pfiesteria piscicida and; 2) an extensive history of research and publication on the toxic forms of Pfiesteria piscicida. The anticipated project period will be 2 years with anticipated funding at a level of $100,000 per year. The research will be conducted in two phases. In Phase One, begun during the first year of the project, subsamples of amoeba and zoospores of Pfiesteria piscicida (grown with live fish to induct toxicity) grown with several of Pfiesteria's major prey and predators will be used as replicates for batch culture experiments. In a series of experiments using concentration gradients of Dithane, Maneb, Zineb, copper, and zinc alone and in combination, Pfiesteria, its prey and predators will be characterized for species, growth, population density, and cell production using scanning electron microscopic, reporter gene, and other techniques. Split samples will be sent to NHEERL's Gulf Ecology Division (GED), Gulf Breeze, FL, for characterization of the diversity and dynamics of the microbial planktonic community using flow cytometry techniques. Samples will be fixed in 0.5% paraformaldehyde, frozen in liquid nitrogen, and shipped to GED, following protocols supplied by GED. Data collected during the experiments will be used to test certain hypotheses and to assess related issues. Using dose-response information from Phase Oneon effects of the chemicals on Pfiesteria amoeba and zoospore forms, Phase Two, begun by the second year of the project, will examine effects of chemical and Pfiesteria toxin(s) stressors on fish immunological functions. Pfiesteria at sublethal concentrations, its prey, and its predators will be raised with live fish in aquariums and treated with the chemicals alone and in combination at concentrations which do not adversely impact Pfiesteria's growth or toxicity. Standardized tests of fish immunological function (e.g., plaque forming cell assay, active immune assays including antibody production) will be administered to these fish, and the results will be compared to those from fish raised without exposure to the chemicals and/or Pfiesteria. Fish speciments may be required for shipment to EPA or a third party for immunological assays, according to aprotocol to be supplied. Data collected during the experiments will be used to test certain hypothesis and to assess related issues. In addition, live fish will be raised with Pfiesteria cysts formed during prey-deprived and prey-abundant conditions. Scanning electron microscopic or other techniques will be used to assess the proportions of Pfiesteria in toxic zoospore form. The fish bioassay will be used to assess Pfiesteria toxicity. Split samples will be sent to GED for characterization of the diversity and dynamics of the microbial planktonic community using flow cytometry techniques in order to verify findings from Phase One. Requests for this solicitation must be received at EPA, Attn: Margaret Mann, Mail Drop 85, Research Triangle Park, NC 27711, no later than May 24, 1999. Telephone and FAX requests will not be honored. Posted 05/07/99 (W-SN329064). (0127)

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