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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 [email protected]
2 USGS., WRD, 520 N. Park Ave, Suite 221, Tucson, AZ 85719 [email protected]
3 USGS-Florida Caribbean Science Center, 7920 NW 71st St., Gainesville Florida, 32653 [email protected]
5 USGS, WRD, P.O. Box 1230, Iowa City, IA 52240 [email protected]
6 Delaware State University, 1200 N. DuPont Highway Dover, DE 19901 [email protected]
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?
