Quantifying Endocrine Disruption in a Threatened and Endangered Fish Species Dr. David Walker...

Quantifying Endocrine Disruption in a Threatened and Endangered Fish Species

Dr. David WalkerUniversity of Arizona

David Walker1, Nick Paretti2, Gail Cordy2, Timothy S. Gross3,

Dana W. Kolpin5, and Dennis McIntosh6

1 University of Arizona, Environmental Research Laboratory, 2601 E. Airport Dr., Tucson, AZ 85706 dwalker@ag.arizona.edu

2 USGS., WRD, 520 N. Park Ave, Suite 221, Tucson, AZ 85719 nvp@email.arizona.edu

3 USGS-Florida Caribbean Science Center, 7920 NW 71st St., Gainesville Florida, 32653 tim_s_gross@usgs.gov

5 USGS, WRD, P.O. Box 1230, Iowa City, IA 52240 dwkolpin@usgs.gov

6 Delaware State University, 1200 N. DuPont Highway Dover, DE 19901 dmcintosh@desu.edu

Quantification and Research Design Issues; One Size Does Not Fit All

Mech

anistic U

nd

erstand

ing

Ecological Significance

Genetic

Biochemical

Physiological

Behavioral

Reproductive

Assemblages

Histopathological

Immunological

Bioenergetic

Populations

Unlike semi-arid or north-temperate regions, effluent-dependent water’s (EDW’s) in arid regions usually contain 100% effluent year-round.

Roger Road WWTP

• Built in 1951.• Treated effluent is discharged into the

Santa Cruz River or diverted into the city’s reclaimed water system.

• Treats the wastewater generated by a population of about 419,000.

• A capacity of 41 mgd and treated an average of 38 mgd from 2004 to 2005.

• Produces secondarily-treated wastewater

Roger Road WWTP

Tucson

Santa Cruz River

This Study

• Laboratory study with controls, replicates, and randomization.

• Use fish native to the region (largely pollution-tolerant).

• Framework or foundation for refinement of future studies.

• Varying doses of effluent (“treatments”).• Concentrate on long-term, persistent

compounds.

Bonytail Chub (Gila elegans)

Treatment Treatment

Control

Control

Water temperature maintained between 25-29o C.

Photoperiod was maintained at 12 hours of light and dark

Treatment/Dosages

• Fish in raceways exposed for 3 months per treatment

• 1st treatment = 1/3 by volume treated ww and 2/3 water treated by RO

• 2nd treatment = 2/3 by volume treated ww and 1/3 water treated by RO

• 3rd treatment = full strength treated ww

Experimental Design• Fish are PIT tagged • Fish randomly assigned to tanks• Blood collected from 10 randomly selected

individuals within each raceway• Blood drawn from the same individual fish

contiguously over the extent of experiment• 17β-estradiol, 11-ketotestosterone, and

vitellogenin

0

0.2

0.4

0.6

0.8

1

1.2

1.4

Dose 2Dose1 Dose 3

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

Dose 1 Dose 2 Dose 3

Detergent Metabolites

Fire/Flame Retardants

Fragrances/flavors

Fuels/PAHs

Plasticizers/antioxidants

Herbicides/insecticides

Non-prescription drugs

Prescription Drugs SteroidsHousehold Wastewater Compounds

Males - Overall

17β-Estradiol

Control (n = 6): 217.3

Treatment (n = 13): = 547.4

11-ketotestosterone

Control (n = 6): = 820.8

Treatment (n = 13): = 473.5

Vitellogenin

Control (n = 6): = 0.09

Treatment (n = 13): = 0.32

17-B

ETA

(pg/

ml)

100

200

300

400

500

600

700

800

900

1000

1100

Control Treatment

T/C

11-K

ETO

(pg/

ml)

0

200

400

600

800

1000

1200

1400

Control Treatment

T/C

VIT

ELLO

(pg/

ml)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

Control Treatment

T/C

Females - Overall

17β-Estradiol

Control (n = 54): 568.2

Treatment (n = 47): 403.7

11-ketotestosterone

Control (n = 54): 591.3

Treatment (n = 47): 530.4

Vitellogenin

Control (n = 54): 0.18

Treatment (n = 47): 0.18

17-B

ETA

(pg/

ml)

0

200

400

600

800

1000

1200

Control Treatment

T/C

Missing Rows 25

Oneway Anova

Oneway Analysis of 17-BETA (pg/ml) By T/C

11-K

ETO

(pg/

ml)

100200300400500600700800900

1000110012001300

Control Treatment

T/C

Missing Rows 25

Oneway Anova

Oneway Analysis of 11-KETO (pg/ml) By T/C

VIT

ELLO

(pg/

ml)

-0.1

0.1

0.3

0.5

0.7

0.9

1.1

Control Treatment

T/C

Missing Rows 25

Oneway Anova

Oneway Analysis of VITELLO (pg/ml) By T/C

CONTROL FEMALE

TREATMENT MALE

CONTROL FEMALE

TREATMENT MALE

CONTROL FEMALE

TREATMENT MALE1/

32/

33/

3

Trea

tmen

t S

ex/C

ontr

ol S

ex w

ithin

DO

SE

0 200 400 600 800

Mean(17-BETA (pg/ml))

CONTROL FEMALE

TREATMENT MALE

CONTROL FEMALE

TREATMENT MALE

CONTROL FEMALE

TREATMENT MALE1/

32/

33/

3

Trea

tmen

t S

ex/C

ontr

ol S

ex w

ithin

DO

SE

.0 .1 .2 .3 .4 .5

Mean(VITELLO (pg/ml))

Synergistic Effects

• Ratios of primary male and female sex hormones, in “undisturbed” populations would be expected to have an inverse relationship i.e. as one increased, the other would decrease.

• We could therefore assume that major deviations from this inverse relationship between male and female primary sex hormones, could be attributed to impairment.

5

5.5

6

6.5

7

6

6.25

6.5

6.75

7

7.25

0.1

0.3

0.5

0.7

0.9

ln 17-beta

5 5.5 6 6.5 7

ln 11-keto

6 6.25 6.5 6.75 7 7.25

sqrtvitello

.1 .2 .3 .4 .5 .6 .7 .8 .9 1

5.5

6

6.5

7

5.5

6

6.5

7

0.1

0.2

0.3

0.4

0.5

0.6

0.7

ln 17ß

5.5 6 6.5 7

ln 11Kt

5.5 6 6.5 7

ln vtg

.1 .2 .3 .4 .5 .6 .7

ln 17β ln 11-keto

ln Vtg

ln 17β 1.00 -0.89 0.74

ln 11-keto

-0.89 1.00 -0.73

ln Vtg 0.74 -0.73 1.00

Control Males Treatment Malesln 17β ln 11-

ketoln Vtg

ln 17β 1.00 -0.50 0.69

ln 11-keto

-0.50 1.00 -0.28

ln Vtg 0.69 -0.28 1.00

5

5.5

6

6.5

7

5

5.5

6

6.5

7

0.1

0.3

0.5

0.7

0.9

ln 17ß

5 5.5 6 6.5 7

ln 11Kt

5 5.5 6 6.5 7

ln vtg

.1 .2 .3 .4 .5 .6 .7 .8 .9 1

5

5.5

6

6.5

7

5

5.5

6

6.5

7

0.1

0.3

0.5

0.7

0.9

ln 17ß

5 5.5 6 6.5 7

ln 11Kt

5 5.5 6 6.5 7

ln vtg

.1 .2 .3 .4 .5 .6 .7 .8 .9 1

ln 17β ln 11-keto

ln Vtg

ln 17β 1.00 -0.67 0.52

ln 11-keto

-0.67 1.00 -0.70

ln Vtg 0.52 -0.70 1.00

Control Females Treatment Females

ln 17β ln 11-keto

ln Vtg

ln 17β 1.00 0.11 0.28

ln 11-keto

0.11 1.00 -0.36

ln Vtg 0.28 -0.36 1.00

Eigenvalue 2.4994 1.5983 1.3520 0.7843 0.4364 0.2306 0.0990

Percent 35.7059 22.8328 19.3145 11.2038 6.2341 3.2950 1.4140

Eigenvectors

Impairment 0.03207 0.47871 -0.59971 0.03198 0.52405 -0.28832 -0.22668

Det. Met -0.02386 -0.69182 -0.12719 0.13721 0.65181 0.20503 0.13755

Fire Ret. 0.27206 0.14664 0.48835 0.74288 0.19262 -0.10884 -0.25528

Fragrances -0.45315 0.45851 0.06826 0.20318 0.17053 0.60972 0.37099

Herbicides -0.24354 0.13071 0.61232 -0.51883 0.45492 -0.26042 -0.06864

HH Waste 0.59204 0.16867 0.05748 -0.08259 0.11987 -0.15914 0.75579

Plasticizers 0.55607 0.12227 0.05280 -0.33315 0.11409 0.63094 -0.38854

Males

17ß-11KT Impair

Detergent Met.

Fire/Flame Ret.

Fragrances

Herbicides

Household Waste

Plasticizers

x

y

z

Eigenvalue 2.5424 1.3785 1.1161 0.9430 0.6582 0.2760 0.0858

Percent 36.3199 19.6952 15.9440 13.4710 9.4030 3.9433 1.2262

Eigenvectors

Impairment 0.29245 0.18743 -0.64153 0.25736 0.51884 -0.35362 -0.08546

Det. Met 0.05636 -0.56419 0.38059 0.43444 0.55723 0.18017 0.04430

Fire Ret. 0.04197 0.51151 0.47240 0.59978 -0.16230 -0.31014 -0.17655

Fragrances -0.43453 0.42877 -0.21006 0.27596 0.17682 0.66476 0.18408

Herbicides -0.26875 0.34161 0.37217 -0.51933 0.59522 -0.22040 -0.00263

HH Waste 0.57808 0.21846 0.15670 -0.06981 0.01724 0.10029 0.76047

Plasticizers 0.56064 0.19147 0.11504 -0.18590 0.09008 0.49575 -0.58936

17ß-11KT Impair

Detergent Met.

Fire/Flame Ret.

FragrancesHerbicides

Household Waste

Plasticizers

x

y

z

Females

Summary• Significant hormonal impairment of

both sexes, as compared to controls, at very low concentration of compounds.

• This impairment could have never been determined in a field study.

• Commonly-used parametric analyses are often inadequate in determining impairment.

Summary (cont)

• Determination of impairment using phased biomarkers.– Phase 1: Aromatase/GnRH– Phase 2: GtH I, GtH II– Phase 3: Sex hormones– Phase 4: Protein development (vtg,

oocyte, spermiation)– Phase 5: Intersex/Morphological change

This study is highly representative of the biological effect of endocrine-disrupting

compounds at the landscape scale.

Current and Future Research

• Fertility/fecundity and sex ratio/development of F2 generation (funded and initiated).

• Behavior.• Treatments using streambed sediment

from affected EDW’s. • Assemblage Effects

Questions?