amphibian metamorphosis: a sensitive model for examining the developmental effects of ammonium...

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)

• AP is the oxidizer and primary component in solid propellant for rocket motors.

• ClO4- is the most potent known competitive

inhibitor of thyroidal iodide uptake; Ki 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.

Na+

Na+

I-

I-

I-

I-

Na+

K+

TG

I-

I-

Na+

Na+ TSH-R

TG iodination

TG-R ?I-

I-

I-

I-

TG uptake

T4, T3

Blood

lysosomes

ClO4

ClO4:•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

0

10

20

30

40

50

60

70

80

90

100

0 0.001 0.01 0.1 1 11.8 118 351 585 1175

AP Concentration (ppm)

% M

orta

lity

LC50 = 460 mg/L

AP

0 1 12 118 1 12 118 1175

% F

LE

0

10

20

30

40

50

60

70

a

b

b

cc c c c

ppb ppm

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

c c c c

AP

0 1 12 118 1 12 118 1175

Hin

d Li

mb

Leng

th (

mm

)

0

2

4

6

8

10

12

14

16

18

a

b,c a,b

c c b cb

ppb ppm

Effect of AP on Hind Limb Length After 70 d Exposure

AP

0 1 12 118 1 12 118 1175

% T

ail R

esor

ptio

n

0

10

20

30

40

50

a

a,b

b,c

c c c c c

ppb ppm

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 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 98 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.

AP (ppb)

0 38 14040

% T

ail R

esor

ptio

n

0

20

40

60

80

100

70 d

AP (ppb)

0 38 14040

% T

ail R

esor

ptio

n

0

20

40

60

80

10098 d

a

b

b

ba,b

a

AP (ppb)

0 38 14040

%F

LE

0

20

40

60

80

100

70 d

AP (ppb)

0 38 14040

%F

LE

0

20

40

60

80

10098 d

a,b b

a

a

bb

Tail Resorption Fore-limb Emergence

Days Post-hatch16 21 26 31 36 40 46 51 56 61 66 68 79 86 90 96

Hin

d-lim

b Le

ngth

(m

m)

0

5

10

15

20

250 ppb AP38 ppb AP14,040 ppb AP

* *

* **

*

*

Remove from AP

“Reversible”effectsof AP onHind-limb Length

Ammonium Perchlorate (ppb)

0 38 14040

T4 (pg/g)

0

300

600

900

1200

1500

0 38 14040

T4 (pg)

0

300

600

900

1200

1500

70-d AP exposure-T4

*

Ammonium Perchlorate (ppb)

0 38 14040

70-d

0.000

0.002

0.004

0.006

0.008

0.010

0.012

0 38 14040

42-d

0.000

0.002

0.004

0.006

0.008

0.010

0.012

THYROID FOLLICLE CELL HEIGHT

**

Objectives• Sub-lethal effects of AP on thyroid-sensitive indices of development and

metamorphosis in Xenopus laevis.





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

Trial 2

FETAX 14 ppm AP

Per

cent

age

Tai

l Re

sorp

tion

0

20

40

60

80

100

120

CompleteIncomplete

29%

86%

Trial 3

FETAX 14 ppm APP

erce

ntag

e T

ail R

eso

rptio

n

0

20

40

60

80

100

120

12%

100%

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

INF pond

0 2 4 6 8 10 12 14 16

% T

ail R

esor

ptio

n

0

20

40

60

80

100

120

FETAXPond Water

Star Ranch

0 2 4 6 8 10 12 14 16

0

20

40

60

80

100

120

Bayou

DAYS

0 2 4 6 8 10 12 14 16

0

20

40

60

80

100

120Ditch

0 2 4 6 8 10 12 14 16

0

20

40

60

80

100

120

April 2000

AP = 1937 ppb

AP =0 ppbAP =0 ppb

AP =0 ppb

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.





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

ReferenceSite

HindlimbHindlimb

Contaminated site

Reference site

Ref INF

Hin

d-lim

b le

ngth

(m

m)

0

5

10

15

20

25

30

35P<0.0001

Ref INF

SV

L (m

m)

0

5

10

15

20

25

30

Ref INF

AP

(pp

b)

0

500

1000

1500

2000

2500

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 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.

AcknowledgementsTTU:• 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.

TexasTexas

2 0 2 Kilometers

N

Goo

se P

rair

ie C

reek

Har

riso

n B

ayou

Building 25C

Harrison Bayou Ponded Area

INF Pond

Harrison Bayou Discharge

Goo

se P

rair

ie C

reek

Har

riso

n B

ayou

Building 25C

Harrison Bayou Ponded Area

INF Pond

Harrison Bayou Discharge

Open Water

Sampling Sites

Creeks, Bayous RoadsLHAAP Boundary

Caddo Lake

TX LA

Caddo Lake

N

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