Amphibian Metamorphosis: A Sensitive Model for Examining the Developmental Effects of Ammonium Perchlorate.

1. Amphibian Metamorphosis: A Sensitive Model for Examining the Developmental Effects of Ammonium Perchlorate. James A. Carr, Ph.D. Department of Biological Sciences1 Texas Tech University, Lubbock, TX 79409 Co-performers: W.L.Goleman1*, L.J. Urquidi1, C.S. McMurry2,3, T.A. Anderson3, and R.J. Kendall3. 2Texas Coop. Fish and Wildlife Res. Unit, 3TIEHH, TTU

2. Ammonium Perchlorate (AP) • AP is the oxidizer and primary component in solid propellant for rocket motors. • ClO4- is themost potent known competitive inhibitor of thyroidal iodide uptake; Ki for the mammalian thyroidal Na+/I- symporter is 1.8 mM • AP has been detected in surface and ground waters near aerospace and munitions facilities.

3. TG iodination TG uptake I- I- I- TG-R ? TG I- I- I- lysosomes T4, T3 I- Na+ Na+ I- K+ TSH-R I- Na+ Na+ Na+ Blood I- • ClO4: • blocks I- transport • blocks T4, T3 synthesis • causes enlarged thyroid ClO4

4. From Shi, 2000

5. Objectives • Sub-lethal effects of AP on thyroid-sensitive indices of development and metamorphosis in Xenopuslaevis. • Effects of environmentally-relevant concentrations of AP on development and thyroid function in in Xenopus. • To apply the EDSTAC Tier I tail resorption assay for Xenopus laevis to environmentally relevant concentrations of AP. • Effects of water from AP contaminated and reference sites on tail resorption using EDSTAC Tier I criteria. • Aspects of development and growth in tadpoles from contaminated and reference sites.

6. Sub-lethal effects of AP on thyroid-sensitive indices of development and metamorphosis in Xenopuslaevis. • Xenopus eggs and larvae were exposed to a range of AP concentrations to determine the concentration-dependent effects of AP on metamorphosis. • Thyroid-sensitive indices of development: • Fore-limb emergence • Hind limb length • Tail resorption

7. 5 Day Post-hatch Mortality After AP Exposure in XenopusExperiment 1-2 LC50 = 460 mg/L % Mortality AP Concentration (ppm)

8. Effect of AP on Fore-limb Emergence After 70 d Exposure

9. Effect of AP on Hind Limb Length After 70 d Exposure

10. Effect of AP on Tail Resorption After 70 d Exposure

11. Objectives • Sub-lethal effects of AP on thyroid-sensitive indices of development and metamorphosis in Xenopuslaevis. • Effects of environmentally-relevant concentrations of AP on development and thyroid function in in Xenopus. • To apply the EDSTAC Tier I tail resorption assay for Xenopus laevis to environmentally relevant concentrations of AP. • Effects of water from AP contaminated and reference sites on tail resorption using EDSTAC Tier I criteria. • Aspects of development and growth in tadpoles from contaminated and reference sites.

12. Effects of environmentally-relevant concentrations of AP on development and thyroid function in in Xenopus. • To examine the effects of environmentally-relevant concentrations of AP on metamorphosis, eggs and larvae were exposed to 0, 38 ppb, or 14,040 ppb for 10 wks, followed by removal from AP and maintenance in control media for an additional 4 wks. 2 d 42 d 70 d 98 d AP Exposure FETAX

13. Long-Horn Army Ammunition Plant (LHAAP) • LHAAP, located in Karnack, TX, manufactured various military weapons and rocket motors from 1942 until the mid-1990s and presently awaits decommissioning. • The AP concentrations used in sub-lethal and EDSTAC experiments represent high and low concentrations reported by CLS Laboratory in April 1999 for effluent from the burning ground No. 3 ground water treatment plant (GWTP) and treated effluent from Harrison Bayou which drains into Caddo Lake, the only natural lake in Texas.

14. Tail Resorption Fore-limb Emergence

15. “Reversible” effects of AP on Hind-limb Length

18. Objectives • Sub-lethal effects of AP on thyroid-sensitive indices of development and metamorphosis in Xenopuslaevis. • Effects of environmentally-relevant concentrations of AP on development and thyroid function in in Xenopus. • To apply the EDSTAC Tier I tail resorption assay for Xenopus laevis to environmentally relevant concentrations of AP. • Effects of water from AP contaminated and reference sites on tail resorption using EDSTAC Tier I criteria. • Aspects of development and growth in tadpoles from contaminated and reference sites.

19. EDSTAC Tier I Test • USEPA -Endocrine Disruptor Screening and Testing Advisory Committee- formed 1996 • NF Stage 60 larvae were exposed to control media and 1.2E-4 M AP for 14 d for the EDSTAC Tier I assay. • NF Stage 60 larvae were exposed to surface water collected from contaminated and reference sites at LHAAP.

20. From Shi, 2000

21. AP Effects in EDSTAC Tier I Assay for Thyroid • Hormone Disruption • Day 0, stage 60 Xenopus • 14 day effects on tail resorption

22. 14-d EDSTAC Tail Resorption, Stage 60

23. From Shi, 2000

24. Objectives • Sub-lethal effects of AP on thyroid-sensitive indices of development and metamorphosis in Xenopuslaevis. • Effects of environmentally-relevant concentrations of AP on development and thyroid function in in Xenopus. • To apply the EDSTAC Tier I tail resorption assay for Xenopus laevis to environmentally relevant concentrations of AP. • Effects of water from AP contaminated and reference sites on tail resorption using EDSTAC Tier I criteria. • Aspects of development and growth in tadpoles from contaminated and reference sites.

25. EDSTAC Tier I Test • Bullfrog tadpoles (Rana catesbeiana) were collected from a reference site and nearby site contaminated with ppm AP. • Tadpoles were staged, weighed, and a number of morphological features (tail length, hind limb length, SVL) measured. Histological analysis is currently underway.

27. Contaminated Site Reference Site Hindlimb Hindlimb

28. Contaminated site Reference site

29. Hind-limb Length is Significantly Reduced in Bullfrog Tadpoles Collected From an AP contaminated Site Compared with Tadpoles Collected from a Reference site

30. Summary and Conclusions • The 5- and 70-day LC50s were 460 mg/L to 498 mg/L and 290 mg/L to 293 mg/L, respectively. • Others (Dumont, J.N and Harvey, G.J.) have shown that that AP is significantly more toxic than other perchlorate salts in the 98 hr FETAX assay. • AP inhibits several thyroid-hormone sensitive indices of development (HLL, FLE, tail resorption) at ppb concentrations in FETAX-medium lab experiments. • Environmentally-relevant concentrations of AP reduce whole body T4 content, cause enlargement of the thyroid gland, and inhibit tail resorption in the 14 d EDSTAC tail resorption test. • Pond water contaminated with AP has little or no effect on tail resorption in the 14-d EDSTAC test. • Tadpoles collected from an AP contaminated site have stunted hind limb development consistent with AP exposure.

31. Acknowledgements TTU: • Lou Densmore • Ellen Roots • Jeremy Unruh • Elisha Hatfield • Ryan Bounds • Catherine Bens Brooks AFB: • Elizabeth Maull Supported by the U.S. Department of Defense, through the Strategic Environmental Research and Development Program (SERDP) under a Cooperative Agreement with the USAF, Institute for Environment, Safety, and Occupational Health, Brooks AFB, TX.

32. Caddo Lake N TX LA Creeks, Bayous Roads LHAAP Boundary Open Water Sampling Sites