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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 79409

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slide1

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

ammonium perchlorate ap
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.
slide3

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

objectives
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.
slide6
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
5 day post hatch mortality after ap exposure in xenopus experiment 1 2
5 Day Post-hatch Mortality After AP Exposure in XenopusExperiment 1-2

LC50 = 460 mg/L

% Mortality

AP Concentration (ppm)

objectives11
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.
slide12
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

long horn army ammunition plant lhaap
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.
slide14

Tail Resorption

Fore-limb Emergence

slide15

“Reversible”

effects

of AP on

Hind-limb Length

objectives18
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.
edstac tier i test
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.
slide21

AP Effects in EDSTAC Tier I Assay for Thyroid

  • Hormone Disruption
  • Day 0, stage 60 Xenopus
  • 14 day effects on tail resorption
objectives24
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.
edstac tier i test25
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.
slide27

Contaminated

Site

Reference

Site

Hindlimb

Hindlimb

slide28

Contaminated site

Reference site

slide29

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
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.
acknowledgements
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.

slide32

Caddo Lake

N

TX LA

Creeks, Bayous

Roads

LHAAP Boundary

Open Water

Sampling Sites