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U.S. Environmental Protection Agency
Office of Research and Development
National Health and Environmental Effects Research Laboratory
Mid-Continent Ecology Division


Investigations Into the Causes of Amphibian Malformations in the Lake Champlain Basin of New England

Solicitation Number: DUL-99-1231

Opening Date: February 14, 2000
Closing Date: April 14, 2000

Requirements for Proposals
Proposal Evaluation Factors
Solicitation Due Date and Cooperative Agreement Schedule
Address for Mailing Proposals
Eligible Applicants


The U.S. Environmental Protection Agency is issuing a request for applications (RFA) under the Regional Applied Research Effort (RARE) program to conduct investigations into the possible causes of amphibian malformations in the Lake Champlain basin of New England. Recent studies in the Lake Champlain basin have documented the occurrence of malformed Rana pipiens. The typical malformation observed in these anurans is terminal truncations of the hindlimbs, similar to observations in Quebec (Ouellet et al 1997). Ongoing studies suggest that the elevated prevalence of malformations is real (Hoppe 1998) and that there may be several possible causes of malformations, including parasitic (trematode) infestations, early developmental exposure to xenobiotic chemicals, such as pesticides and/or their metabolites, or naturally occurring chemicals; ultraviolet (UV) radiation; and disease (Tietge et al 1996).

The role of parasites as causative factors in limb malformations was proposed by Sessions and Ruth (1990). In a recently conducted laboratory study, infection of Hyla regilla with the cercariae of a trematode, Ribeiroia sp., resulted in an array of hindlimb malformations including missing and extra limbs (Johnson et al 1999). However, field investigations have been inconclusive as malformed anurans in some sites are apparently not infected. This observation is consistent with the possibility that other environmental factors may cause limb malformations.

The role of xenobiotic chemicals as a possible cause has been under investigation by several laboratories. Chemicals which interact with the retinoid pathway are plausible causes of limb malformations, as natural retinoids are known to be important to the development of several tissues and organs, including limbs, via a nuclear receptor mediated pathway. Laboratory exposures to exogenous retinoids have been shown to induce limb malformations in anurans (Scadding and Maden, 1986). In a recent analysis of phenotypes of malformed anurans from the field, the dysmorphologies of some of the supernumerary limbs were similar to those induced by laboratory exposure to retinoids (Gardiner and Hoppe, 1999), suggesting that retinoid-like chemicals might be involved. Although several investigations into a possible chemical etiology are being conducted, no link has yet been established between chemical exposure and limb malformations in the field.

The role of UV light as a possible cause of limb malformations was advanced by recent studies which demonstrated the ability of laboratory UV light alone to induce terminal truncations of hindlimbs in Rana pipiens (Ankley et al 1998). Further studies have concluded that ambient solar UV intensities are indeed sufficient to result in similar limb truncations. Furthermore, UV filtration of full spectrum solar light was shown to eliminate the limb effects. Exposure of anurans to UV in the field is likely to be increasing due to reductions in stratospheric ozone (Kerr and McElroy, 1993; Herman et al 1996) and reductions in the attenuating characteristics of surface waters (Schindler, 1998). However, adequate dose-response data and in-situ dosimetry estimates for anurans are lacking.

Requirements for Proposals

The proposals must contain two standard federal forms, a work plan with budget, and appendices, as described below. Please follow instructions and do not submit additional items.

Application for Federal Assistance (SF-424) and Budget Information (SF-424A): The SF-424 and SF-424A must be submitted as part of your proposal. Only finalists will be asked to submit additional federal forms needed to process their proposal. EPA will make copies of your proposal for use by assistance reviewers. Funds will be provided for Fiscal Year (FY) 2000 and FY 2001, with a final report submitted by September 30, 2001.

Work plan and appendices: A work plan describes your proposed research project, and your appendices establish your timeline, your qualifications, and your partnerships with other organizations, where applicable.

(a) Timeline-Include a "time line" to link your activities to a clear project schedule and indicate at what point over the months of your budget period each action, event, product, development, etc. occurs.

(b) Key Personnel- Attach a one page resume for the key personnel conducting the project (maximum of five resumes please).

(c) Letters of Commitment- If there are partners, include one page letters of commitment from the partners explaining their role in the proposed project.

The scope of the proposal developed in response to this solicitation must focus on conducting studies which address one or more of the possible causes listed above or an alternative hypothesis, as defined by the investigator. The proposal should include studies designed to test the hypothesis that a specific factor results in developmental abnormalities, with an emphasis on limb development. These studies may utilize laboratory or field methods to test single or multiple factors. The scope may also include descriptive studies which document the type(s) of malformation, the prevalence of malformations, or the temporal and spatial changes in prevalence of malformations in native anuran populations of the Lake Champlain basin.

Each proposal must identify the experience of the principal investigator(s) with this or related research issues, the proposed field and laboratory analytical methods to be used, the available laboratory facilities, other related activities in this research area with other cooperating agencies, institutions and individuals, and the possible linkage of this work to related investigations by the same or different research groups.

Proposals and resultant work generated under this solicitation will be subject to peer review, at EPA's expense, and must comply with all EPA quality assurance requirements. Instructions for preparing a Quality Assurance Project Plan, can be found in EPA QA/G-5, Guidance for Quality Assurance Project Plans, which is on INTERNET site, http://www.epa.gov/quality/qa_docs.html.

Proposal Evaluation Factors

1) The ability of the proposal to systematically address possible causal mechanisms, spatial prevalence, temporal changes, or morphological characterization of amphibian malformations using sound methodologies and hypothesis testing. (40%)

2) Relevance of the proposed work to malformations in the Lake Champlain basin. (20%)

3) Experience of the principal investigators in the proposed research area. (20%)

4) Suitability of personnel, facilities, and methods to achieve the stated goals of the proposal. (10%)

5) Willingness and ability of the principal investigators to work cooperatively with EPA.

6) Institutional cost-share is not required. However, the degree of recipient cost sharing incorporated into the proposal, such as in-kind contributions, including: facilities, equipment, materials, professional services, or volunteer staff provided by non-Federal public agencies, organizations, or individuals, will be considered.

7) Results of proposal peer review.

The total number of points possible for each proposal is 100%. The maximum number of points each proposal may receive for the seven rating factors is 90. Reviewers will be given the flexibility to provide up to 10 bonus points for exceptional projects based on the overall quality of the proposal, evidence that the research will be advanced by the project, and that it will have a good return for the investment. The maximum number of points for each proposal is 100.

Solicitation Due Date and Cooperative Agreement Schedule

An original proposal signed by an authorized representative plus three copies, must be mailed to EPA postmarked no later than April 14, 2000. Proposals postmarked after that date will not be considered for funding. Proposal budgets must total $100,000 or less (exclusive of cost sharing provisions), including all overhead and indirect costs at approved federal government rates. If the cooperative agreement application is under $100,000, then the small grant policy applies, which is the original and one copy (in conformance with EPA Order 5700.2, entitled, "Implementation of Streamline Small Grants"). EPA expects to announce the successful recipient of one cooperative agreement during Spring of 2000. Applicants should anticipate project start dates for the Summer of 2000, and for planning purposes may use July 1, 2000, as the earliest project start date. Please remember, however, that EPA's Grants Administration Division must issue an assistance award before EPA's offer for assistance is official. Applicants that were not selected for award will be notified at the time the selection decision has been tentatively approved by the award official.

Address for Mailing Proposals

Proposals must be mailed to: U.S. EPA, NHEERL\MED, 6201 Congdon Boulevard, Duluth, Minnesota 55804. Information: Craig L. Johnson, (218) 529-5016.

Eligible Applicants

Any State water pollution control agencies, interstate agencies, other public or non-profit private agencies, institutions, organizations and individuals. Research will be funded under the statutory authority of the Clean Water Act (P.L.92-500, as amended) Section 104(a)(1). Interested applicants must be eligible to receive Federal assistance under this Act and the Code of Federal Regulations (CFR) Part 30 - General Regulation for Assistance, Part 31- Uniform Administrative Requirements for Grants and Cooperative Agreements to State and Local Government, Part 34- New Restrictions on Lobbying, and Part 40 - Research and Demonstration Grants. The purpose is to commence research projects relating to the causes, effects, extent, prevention, reduction and elimination of water pollution.


Joseph E. Tietge: Project Officer, 218-529-5176
USEPA Mid-Continent Ecology Division,
National Health and Environmental Effects Research Laboratory,
Office of Research and Development,
6201 Congdon Boulevard,
Duluth, MN 55804


Ankley GT, Tietge JE, DeFoe DL, Jensen KM, Holcombe GW, Durhan EJ, Diamond SA. 1998. Effects of ultraviolet light and methoprene on survival and development of Rana pipiens. Environ Toxicol Chem, 17:2530-2542.

Gardiner D , Hoppe DM. 1999. Environmentally induced limb malformations in mink frogs (Rana septentrionalis). J Exp Zool, 284:207-16

Herman JR, Bhartia PK, Ziemke J, Ahmad Z, Larko D. 1996 UV-B increases (1979-1992) from decreases in total ozone. Geophys Res Lett, 23:2117-2120.

Hoppe DM. 1998. History of Minnesota frog abnormalities: do recent findings represent a new phenomenon? Proceedings of the Midwest Declining Amphibians Conference, Milwaukee, WI, (abstract).

Johnson PTJ, Lunde KB, Ritchie EG, Launer AE. 1999. The effect of trematode infection on amphibian limb development and survivorship. Science, 284:802-804.

Kerr JB, McElroy CT. 1993. Evidence for large upward trends of ultraviolet-B radiation linked to ozone depletion. Science, 262:1032-1034.

Ouellet M, Bonin J, Rodrigue J, DesGranges JL, Lair S. 1997. Hindlimb deformities (ectromelia, ectrodactyly) in free-living anurans from agricultural habitats. J Wildl Dis, 33:95-104.

Scadding SR, Maden M. 1986. The effects of local application of retinoic acid on limb development and regeneration in tadpoles of Xenopus laevis. J Embryol exp Morph, 91:55-63.

Schindler DW. 1998. A dim future for boreal waters and landscapes. Bioscience 48:157-164.

Sessions SK, Ruth SB. 1990. Explanation for naturally occurring supernumerary limbs in amphibians. J of Exp Zool, 254:38-47

Tietge JE, Lannoo M, Beasley V. 1996. North American Amphibian Monitoring Program III - Deformed Frogs. Discussion of findings relative to meeting objectives. NAAMP III Online Paper. http://www.im.nbs.gov.naamp3/papers/60df.html.

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