Amphibian Metamorphosis: A Sensitive Model for Examining the Developmental Effects of Ammonium Perchlorate. James A. Carr, Ph.D. Department of Biological Sciences 1 Texas Tech University, Lubbock, TX Co-performers: W.L.Goleman 1 *, L.J. Urquidi 1, C.S. McMurry 2,3, T.A. Anderson 3, and R.J. Kendall 3. 2 Texas Coop. Fish and Wildlife Res. Unit, 3 TIEHH, TTU

Ammonium Perchlorate (AP) AP is the oxidizer and primary component in solid propellant for rocket motors. ClO 4 - is the most potent known competitive inhibitor of thyroidal iodide uptake; K i for the mammalian thyroidal Na + /I - symporter is 1.8  M AP has been detected in surface and ground waters near aerospace and munitions facilities.

ClO 4 ClO 4 : blocks I- transport blocks T4, T3 synthesis causes enlarged thyroid

From Shi, 2000

Objectives Sub-lethal effects of AP on thyroid-sensitive indices of development and metamorphosis in Xenopus laevis. 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.

Sub-lethal effects of AP on thyroid- sensitive indices of development and metamorphosis in Xenopus laevis. 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

5 Day Post-hatch Mortality After AP Exposure in Xenopus Experiment 1-2 AP Concentration (ppm) % Mortality LC 50 = 460 mg/L

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

Effect of AP on Hind Limb Length After 70 d Exposure

Effect of AP on Tail Resorption After 70 d Exposure

Objectives Sub-lethal effects of AP on thyroid-sensitive indices of development and metamorphosis in Xenopus laevis. 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.

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 d98 d70 d42 d AP Exposure FETAX

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.

Tail ResorptionFore-limb Emergence

“Reversible” effects of AP on Hind-limb Length

Objectives Sub-lethal effects of AP on thyroid-sensitive indices of development and metamorphosis in Xenopus laevis. 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.

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.

From Shi, 2000

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

14-d EDSTAC Tail Resorption, Stage 60

From Shi, 2000

Objectives Sub-lethal effects of AP on thyroid-sensitive indices of development and metamorphosis in Xenopus laevis. 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.

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.

Contaminated Site Reference Site Hindlimb

Contaminated site Reference site

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

Summary and Conclusions The 5- and 70-day LC 50 s 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.

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.

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