The effects of progesterone and synthetic derivatives on Fathead Minnow (Pimphales promelas) embryos.

JA Stine and DB Huggett, Ph.D.Department of Biological Sciences

Ronald E. McNair Post Baccalaureate Achievement ProgramUniversity of North Texas

Denton, Texas

Introduction:

• Pharmaceuticals and personal care products are being routinely detected in surface waters throughout the United States.

• Little information is available on their fate and potential effects in aquatic systems.

• This project attempts to quantify the effect of progesterone and its synthetic derivatives on aquatic organisms.

Progesterone Background• Progesterone and its synthetic forms are

commonly used for the purpose of oral contraception and hormone replacement therapy in humans.

• In fish, progestins (e.g. 17α,20β-dihydroxy-4-pregnen-3-one) are extremely important for many physiological processes, including oocyte maturation and embryo morphogenesis.

• Given the importance of progestins in mammals and fish, it is essential to determine the developmental consequences of progesterone and synthetic derivatives in the environment.

Fathead Minnow

• The organism used in this study is the fathead minnow (Pimephales promelas).

• Vast amount of data available concerning all aspects of growth, life cycle, and development.• Known “sentinel species” for aquatic toxicity.• Widely available and easily cultured.

Objectives of Study:

• To determine the developmental toxicity of progesterone, norethindrone, and medroxyprogesterone to the fathead minnow.

• This objective will be achieved by:1.Exposing fathead minnow embryos for 96 hr to each

of the test chemicals2.microscopically staging and investigating

developmental abnormalities3.assessing survival after the 96 hr exposure.

Materials and Methods:

• P. promelas were cultured and held in standard systems in the Aquatic Toxicology Lab at the University of North Texas.

• < 24 hr old eggs collected and sorted prior to exposure.

• P. promelas eggs were placed in exposure jars with differing concentrations of progesterone or synthetic derivatives.

• 10 eggs were placed in each of three replicate jars for a total of 30 eggs exposed per concentration.

Materials and Methods, Cont’d.• The eggs were staged and observed every 24

hr until the exposure was terminated (96 hr). • Abnormalities or death of embryos were

noted and recorded.• Exposure solutions were renewed every 24 hr

(static renewal).• Images of abnormal embryos were taken on

the last day of the study using a Ziess dissecting scope, digital camera, and Axiovision software.

Figure 1. Lethality of Compounds

Progesterone Medroxyprogesterone Norethindrone0

5

10

15

20

25

30

35

Lethality of Progesterone Compunds 96 Hr. post Exposure

ControlSolvent Control0.625 mg/L1.25 mg/L2.50 mg/L5.0 mg/L

Exposure Compound

Num

ber S

urvi

ving

Figure 2: Number Of Abnormalities

Progesterone Medroxyprogesterone Norethindrone0

5

10

15

20

25

30

Number of Abnormalities 96 hr. Post Exposure

.078 mg/L

.15625 mg/L

.3125 mg/L0.625 mg/L21.25 mg/L22.50 mg/L25.0 mg/L2

Exposure Compound

Num

ber o

f Def

orm

ities

Figure 3: Bradycardia per Concentration

Control Solvent Control

0.78 mg/L .15625 mg/L

.3125 mg/L 0.625 mg/L 1.25 mg/L 2.50 mg/L 5.00 mg/L0

20

40

60

80

100

120

140

160

Bradycardia Per Exposure Concentration

ProgesteroneMedroxyprogesterone

Concentration of Exposure Compound

Hear

trat

e in

Bea

ts P

er M

inut

e (b

pm)

Discussion of Findings:

• The rank order potency in this experiment was: progesterone > medroxyprogesterone > norethindrone

• Progesterone significantly reduced survival at concentrations ≥ 1.25 mg/L.

• All compounds tested produced cardiovascular abnormalities.

Discussion of Findings, Cont’d.

• An increased prevalence of cardiac edema, ventricular enlargement and elongation were observed at 0.625 mg/L.

• Bradycardia was observed beginning at the lowest dose concentration, 0.078 mg/L, and increased proportionally per exposure concentration.

• Mammalian data demonstrate that progesterone does alter normal cardiovascular physiology.

• In the 2.5 and 5 mg/L medroxyprogesterone exposed fish, blood accumulated in the heads of the fish.

Figure 4. Representative Embryos following 96 Hr. Exposure to Progesterone

Directions for Future Research:

• Further study is needed to determine the mechanism(s) of malformations in the fathead minnow embryos following progestin exposure.

• Are amphibian embryos affected in the same way as piscine embryos?

• What are the Lowest Observable Effect Level (LOEL) for progesterone and synthetics or both fish and amphibians?

• What is the LC50 for progesterone and synthetics?

• Is the Nip3a pathway affected by presence of exogenous progesterone?

• Perform field studies to investigate the impacts of progesterone on fish and amphibians

• Cattle dominated watersheds

Acknowledgements:

UNT McNair Scholars ProgramDr. David HalaDr. Lene PetersenMr. David Baxter UNT Department of Biological Sciences

Literature Cited:

Brammer, JD; Puyear, R; et al; Prehatching development of the Fathead Minnow, Pimephales promelas Rafinesque.; July 1996. EPA 600-R-96-079. Office of Research and Development, U.S. Environmental Protection Agency. 49 pages