The Direct Effect of Metals on Ontogeny, Sexual Development, and Reproduction in the Northern Leopard Frog, Rana pipiens, of Great Lakes

EPA Grant Number: U916133
Title: The Direct Effect of Metals on Ontogeny, Sexual Development, and Reproduction in the Northern Leopard Frog, Rana pipiens, of Great Lakes
Investigators: Gross, Jason A.
Institution: University of Wisconsin - Madison
EPA Project Officer: Lee, Sonja
Project Period: January 1, 2003 through January 1, 2006
Project Amount: $108,000
RFA: STAR Graduate Fellowships (2003) Recipients Lists
Research Category: Fellowship - Toxicology , Academic Fellowships , Health Effects


During the last decade, there has been a rise in research on amphibians in response to public interest in declining amphibian populations, increased incidence of malformations, and the possible effects of endocrine-modulating substances. Some metals are putative endocrine disruptors, but the sensitivity of amphibians to these effects is practically unstudied. Because leopard frogs frequent transition zones between terrestrial and aquatic habitats, they may be key biological indicators for the evaluation of wetland communities affected by metal contaminants. Additionally, amphibian eggs, larvae, and adults serve as prey for many fish, birds, and mammals, therefore playing a role in contaminant transfer through the food web. The overall objective of this research project is to address amphibian reproductive toxicity through routes of exposures in different life-history stages (adult, embryo, and tadpole). The specific objective of this research project is to conduct laboratory studies to determine whether: (1) exposure to metals at ecologically relevant levels during development alters leopard frog sexual development and ontogeny; and (2) chronic exposure of male and female leopard frogs to metals negatively impacts their fitness. This research project will address the question of whether amphibians are adequately protected in both a terrestrial and aquatic ecosystem based on findings in other vertebrate species.


Clutches of eggs will be collected from in vitro fertilization. For tadpole exposures to cadmium (Cd), lead (Pb), mercury (Hg), copper, chromium, and arsenic, 30 eggs selected from each of 6 clutches are raised in 15.12 L of water (n = 4 tanks in each treatment) using a static renewal tank system with water changes occurring every other day. Doses will bracket the highest concentrations of metals that occur in the field and concentrations that have been shown to have effects in amphibians and other aquatic vertebrates. Tanks will be checked daily for mortality, and dead tadpoles will be removed and preserved in 10 percent formalin. Mass, snout vent length, stage, and malformations will be recorded every 2 weeks after posthatch day 30. Tadpoles will be sacrificed following complete tail resorption for analysis of metals, and gonadal morphology and sex ratio will be histologically evaluated. Adult male and female leopard frogs will be divided into high, low, and control exposure groups (n = 8 frogs in each treatment) for Cd, Pb, and Hg. Each animal will be housed in a separate aquarium and fed two crickets per day for 5 months. Twice per week, frogs will be fed two crickets into which a solution of the specific metal was injected. Following the dosing period, frogs will be hibernated and bred, and tadpole experiments will be conducted as described above. Frogs will be sacrificed, and the ovaries and entire body will be analyzed for metals.

Supplemental Keywords:

fellowship, metals, Northern Leopard Frog, Rana pipiens, amphibians, Great Lakes, amphibian populations, reproductive toxicology., RFA, Health, Scientific Discipline, Environmental Chemistry, Endocrine Disruptors - Environmental Exposure & Risk, endocrine disruptors, Environmental Microbiology, Biochemistry, Biology, Endocrine Disruptors - Human Health, aquatic ecosystem, microbial degradation, amphibian lifecycle testing, EDCs, endocrine disrupting chemicals, chlorpyrifos, sexual development, sex differences, estrogen response, reproductive processes, frog embryo study

Progress and Final Reports:

  • 2003
  • 2004
  • Final