Quantifying Endocrine Disruption in a Threatened and Endangered Fish Species Dr. David Walker University of Arizona David Walker 1, Nick Paretti 2, Gail Cordy 2, Timothy S. Gross 3, Dana W. Kolpin 5, and Dennis McIntosh 6 1 University of Arizona, Environmental Research Laboratory, 2601 E. Airport Dr., Tucson, AZ USGS., WRD, 520 N. Park Ave, Suite 221, Tucson, AZ USGS-Florida Caribbean Science Center, 7920 NW 71 st St., Gainesville Florida, USGS, WRD, P.O. Box 1230, Iowa City, IA Delaware State University, 1200 N. DuPont Highway Dover, DE

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

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 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 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 Control

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

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

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

Males - Overall 17β-Estradiol Control (n = 6): Treatment (n = 13): = ketotestosterone Control (n = 6): = Treatment (n = 13): = Vitellogenin Control (n = 6): = 0.09 Treatment (n = 13): = 0.32

Females - Overall 17β-Estradiol Control (n = 54): Treatment (n = 47): ketotestosterone Control (n = 54): Treatment (n = 47): Vitellogenin Control (n = 54): 0.18 Treatment (n = 47): 0.18

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.

ln 17βln 11- keto ln Vtg ln 17β ln 11- keto ln Vtg Control MalesTreatment Males ln 17βln 11- keto ln Vtg ln 17β ln 11- keto ln Vtg

ln 17βln 11- keto ln Vtg ln 17β ln 11- keto ln Vtg Control Females Treatment Females ln 17βln 11- keto ln Vtg ln 17β ln 11- keto ln Vtg

Eigenvalue Percent Eigenvectors Impairment Det. Met Fire Ret Fragrances Herbicides HH Waste Plasticizers Males

Eigenvalue Percent Eigenvectors Impairment Det. Met Fire Ret Fragrances Herbicides HH Waste Plasticizers 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?