Investigations into the Causes of Amphibian Malformations in the Lake Champlain Basin of New England Prepared By: Richard Levey Vermont Department of Environmental Conservation R.A.LaRosa Environmental Laboratory 103 S. Main St. Waterbury, VT 05671 In Collaboration with: Nathaniel Shambaugh – VT Department of Agricultural, Food & Markets Douglas Fort – Fort Environmental Laboratories James Andrews – Middlebury College Final Report May 2003 Investigations into the Causes of Amphibian Malformations in the Lake Champlain Basin of New England Prepared By: Richard Levey Vermont Department of Environmental Conservation R.A.LaRosa Environmental Laboratory 103 S. Main St. Waterbury, VT 05671 In Collaboration with: Nathaniel Shambaugh – VT Department of Agricultural, Food & Markets Douglas Fort – Fort Environmental Laboratories James Andrews – Middlebury College Final Report May 2003 ACKNOWLEDGMENTS This study would not have been possible without the generous assistance of many individuals. For aid with field work, data collection and management we thank Peter Steckler whose enthusiasm and dedication to the study were unrelenting. Special thanks to Dr. Michael J. Lannoo, for providing radiographic images of Vermont specimens, interpretations of digital and radiographic images and for editorial comments. Dr. Carol Meteyer for providing interpretations of digital and radiographic images and editorial comments. Laura Eaton-Poole for assisting with field surveys and sharing survey results. Douglas Burnham VTDEC project manager, for providing technical and management oversight on the project. The Vermont Department of Environmental Conservation’s RA LaRosa Environmental Laboratory provided chemical and analytical services. Peer review comments from Judy Helgen, Carol Meteyer, Martin Ouellet and William Smith were greatly appreciated. We thank Joseph Tietge from EPA’s Mid- Continent Division in Duluth, MN for providing technical and management oversight on the project. Greg Hellyer from EPA-New England’s Regional Lab provided support for the project and peer review. This project was made possible with funding by the US EPA, Regional Applied Research Effort (RARE). A peer review of the project was conducted in accordance with the US EPA peer review policy and procedures. However, it has not been subject to the US EPA’s policy review, and therefore, does not necessarily reflect the views of that Agency, and thus no official endorsement should be inferred. The Vermont Agency of Natural Resources, Department of Environmental Conservation is an equal opportunity agency and offers all persons the benefits of participating in each of their programs and competing in all areas of employment regardless of race, color, religion, sex, national origin, age, disability, sexual orientation, or other non-merit factors. Persons with a TDD may call 1-800-253-0191 (Vermont Relay Information Service) to communicate requests to offices that do not have a TDD. Disclaimer The mention of trade names or commercial products within this document does not constitute endorsement or recommendations by the State of Vermont, Agency of Natural Resources. TABLE OF CONTENTS List of Figures..........................................................................................................................................i List of Tables ...........................................................................................................................................vi Project Description: Background..........................................................................................................1 Objectives .........................................................................................................................3 Methods ....................................................................................................................................................5 Sampling Design Rationale...............................................................................................5 Site Selection ....................................................................................................................5 Site Characterization.........................................................................................................7 Northern Leopard Frog (R. pipiens) Survey .....................................................................7 Tadpole Surveys................................................................................................................8 Drift-Fence Monitoring at Ward Marsh 1998-2001 .........................................................8 Limb Bud Assays..............................................................................................................13 Water Quality Measurements ...........................................................................................14 Water and Sediment Collection for Pesticide Analysis and Limb Bud Assays 14 Pesticide Analysis .............................................................................................................16 Results ......................................................................................................................................................21 Site Characterizations .......................................................................................................21 Northern Leopard Frog (R. pipiens) Metamorph Surveys................................................27 Categories of Metamorph Abnormalities..........................................................................35 Age and Growth – Metamorphs........................................................................................46 Tadpole Surveys – Rana pipiens.......................................................................................52 Growth – Tadpoles............................................................................................................60 Drift-Fence Monitoring at Ward Marsh 1998-2001 .........................................................63 Limb Bud Assays..............................................................................................................95 Water Quality Measurements – Field ...............................................................................120 Water Quality Measurements – Laboratory......................................................................124 Pesticides...........................................................................................................................131 Discussion.................................................................................................................................................136 References................................................................................................................................................146 Appendices: Rana pipiens Metamorph and Tadpole Data .........................................................................Appendix A Photo Atlas: Radiographs of Rana pipiens ............................................................................Appendix B Photo Atlas: Rana pipiens Metamorph..................................................................................Appendix C Photo Atlas: Rana pipiens Larvae..........................................................................................Appendix D Raw Data-Limb Bud Assays –Water Chemistry ...................................................................Appendix E Photo Atlas: Rana pipiens and Xenopus laevis......................................................................Appendix F Definitions..............................................................................................................................Appendix G LIST OF FIGURES Figure 1. Lake Champlain Basin Map ......................................................................................................4 Figure 2. Sampling Sites...........................................................................................................................6 Figure 3. Location of Seven Drift-Fences at Ward Marsh in West Haven, VT .......................................9 Figure 4. Diagram of the Design of the Drift-Fences ...............................................................................10 Figure 5. Drift-fence at Ward Marsh in West Haven, VT ........................................................................11 Figure 6. Otter Creek WMA .....................................................................................................................21 Figure 6a. Otter Creek Wetland................................................................................................................21 Figure 7. Mud Creek WMA......................................................................................................................22 Figure 7a. Mud Creek Marsh....................................................................................................................22 Figure 8. Alburg Dune State Park.............................................................................................................23 Figure 9. Arial Photograph of Alburg.......................................................................................................24 Figure 9a. North Hero Floodplain.............................................................................................................24 Figure 10. North Hero Study Site .............................................................................................................25