1. Ecological Effects of Endocrine Disruption: Quantifying Generational Effects David Walker, PhD University of Arizona David Walker 1, Nick Paretti 2, Gail Cordy 2, Timothy S. Gross 3, Edward T. Furlong 4, Steven D. Zaugg 4 Dana W. Kolpin 5, and Dennis McIntosh 6 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 71 st St., Gainesville Florida, 32653 tim_s_gross@usgs.gov 4 USGS, National Water Quality Laboratory, Denver Federal Center, P.O. Box 25046, MS 407, Lakewood, CO 80225-0046 efurlong@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

2. Aquatic Ecology and Endocrine Disruption An organisms ability to better-exploit a resource (or group of resources) in the face of environmental stress and inter- specific competition, coupled with conservation of the genetic material enabling this exploitation, is what drives speciation.

3. Genetic conservation of traits is initiated, and sustained by, subtle behavioral cues for mating, spawning, aggression, territoriality, avoidance, etc.

4. Quantification and Research Design Issues; One Size Does Not Fit All Mechanistic Understanding Ecological Significance Genetic Biochemical Physiological Behavioral Reproductive Assemblages Histopathological Immunological Bioenergetic Populations

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

6. Roger Road WWTP Tucson Santa Cruz River

8. This Study Laboratory study with control, replication, and randomization. Use fish native to the region (largely pollution-tolerant). Concentrate on long-term, persistent compounds.

9. Morphology epitomizes adaptations to living in turbid, flashy rivers with extremes in environmental conditions. Pollution-tolerant Omnivorous, adults grow to 2 feet in length. Large enough to survive multiple venipuncture events. Readily available supply from USFWS Deter National Fish Hatchery and Technology Center

10. Treatment Control

11. Water temperature maintained between 25-29 o C. Photoperiod was maintained at 12 hours of light and dark

12. Treatment/Dosages Fish in raceways exposed for 3 months per treatment 1 st treatment = 1/3 by volume treated ww and 2/3 water treated by RO 2 nd treatment = 2/3 by volume treated ww and 1/3 water treated by RO 3 rd treatment = full strength treated ww

14. Results in µg/L

15. Males 17β-estradiol (pg/mL) Control (n = 6): 217.3 Treatment (n = 13): = 547.4 11-ketotestosterone (pg/mL) Control (n = 6): = 820.8 Treatment (n = 13): = 473.5 Vitellogenin (mg/mL Control (n = 6): = 0.09 Treatment (n = 13): = 0.32 P = 0.0016 P = 0.021 P = 0.036

16. Females 17β-estradiol (pg/mL) Control (n = 54): 568.2 Treatment (n = 47): 403.7 11-ketotestosterone (pg/mL) Control (n = 54): 591.3 Treatment (n = 47): 530.4 Vitellogenin (mg/mL) Control (n = 54): 0.18 Treatment (n = 47): 0.18 P = 0.001 P = 0.286 P = 0.986

17. ln 17βln 11- keto ln Vtg ln 17β 1.00-0.890.74 ln 11- keto -0.891.00-0.73 ln Vtg 0.74-0.731.00 Control MalesTreatment Males ln 17βln 11- keto ln Vtg ln 17β 1.00-0.500.69 ln 11- keto -0.501.00-0.28 ln Vtg 0.69-0.281.00

18. ln 17βln 11- keto ln Vtg ln 17β 1.00-0.670.52 ln 11- keto -0.671.00-0.70 ln Vtg 0.52-0.701.00 Control Females Treatment Females ln 17βln 11- keto ln Vtg ln 17β 1.000.110.28 ln 11- keto 0.111.00-0.36 ln Vtg 0.28-0.361.00

19. We believe this study is highly representative of the biological effect of endocrine-disrupting compounds on aquatic organisms at the landscape scale.

20. This study quantified hormonal impairment; but not fertility or fecundity

21. New (Ongoing) Research Repeat design. Quantify impairment following a 4 month exposure. Aromatase (?),GnRh, GtH I and II, steroidal hormones, protein development, GSI, HSI, and histology. Provide environmental stimuli for spawning. Collect eggs and quantify fertility and fecundity of parent generation.

23. F1 generation grown in effluent for one year for the determination of sex ratio and hormonal/morphological change. Compounds identified using POCIS and SPMD (David Alvarez/USGS-CERC). Sperm quality (Jill Jenkins/USGS)

24. Summary Biomarkers infer reproductive impairment, they don’t quantify it. Physiological impairment can occur without morphological change. Bio-indicator species selection should be ecosystem and objective-specific.

25. Acknowledgements USFWS Dexter National Fish Hatchery and Technology Center. USGS Toxics and Hydrology Program This work was supported by State of Arizona funding, through the Technology and Research Initiative..