1. The Ecological Effects of Endocrine Disruption Dr. David Walker University of Arizona David Walker 1, Nick Paretti 2, Gail Cordy 2, Timothy S. Gross 3, Edward T. Furlong 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. For Our Purposes… An endocrine disruptor is a synthetic (anthropogenic) chemical that when absorbed into the body mimics, blocks, or otherwise alters hormone level, function, or binding and subsequently disrupts normal bodily functions including behavioral and/or strictly physiologic responses.

4. Aquatic Ecology and Endocrine Disruption An individual 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.

5. Genetic conservation of traits is initiated, and sustained by, subtle behavioral cues for mating, spawning, aggression, territoriality, avoidance, etc. Any impairment of these behavioral cues or manifestation into physiological or morphological changes has the capability to stunt speciation by lowering fertility and fecundity.

6. Endocrine Disrupting Compounds FAR more than what can be included in this presentation. By the time breakdown products and metabolites are added to the mix, iterations become astronomical.

7. Just a Few Examples by Use Category Detergent Metabolites Fire/Flame Retardants Fragrances/Flavors Fuels/PAH’s Herbicides/Insecticides Household Wastewater Compounds Non-Prescription Drugs Plasticizers/Antioxidants Prescription Drugs Steroids

8. At the landscape scale, those compounds known to be “powerful” EDC’s, but are not environmentally-persistent, exert less of an effect than those persistent, but relatively weaker, compounds.

9. 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

10. Observational versus Controlled Studies True control and replication is not possible in the field. Laboratory studies with control and replication give up some ecological significance.

11. Exposure and Causation Several studies have examined the effect of one or a very few EDC’s on the physiological response of an organism. The vast magnitude of compounds in a matrix makes assumptions about individual compounds difficult to ascertain. Non-monotonic dose-response curves

12. With new and emerging contaminants found almost on a daily basis, making assumptions about exposure and physiologic response must always carry the caveat “of the compounds we analyzed for”…

13. Grab, Composite, or Integrated Samples? Problems associated with not knowing long-term exposure to fish or other organisms.

14. Passive Organic Chemical Integrative Sampler (POCIS) and/or Semi-Permeable Membrane Devices (SPMD’s aka “fatbags”)

15. Of all the tools at our disposal to study complex environmental issues in aquatic ecosystems, a sound understanding of ecological principles as they pertain to these ecosystems is the most essential.

16. Quantifying Endocrine Disruption in a Threatened and Endangered Fish Species

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

19. The Santa Cruz River Near Tucson, Arizona Flows from Mexico near Nogales, Sonora northward to Tucson, Arizona. The only sections with flowing water are those due to discharge from WWTP’s.

21. 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

22. Roger Road WWTP Tucson Santa Cruz River

24. 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.

25. Bonytail Chub (Gila elegans)

26. Treatment Control

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

30. 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

31. Results

34. 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

35. 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

38. 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.

39. 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

40. 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

41. Synergism, feedback mechanisms, and non-linearity of bio-markers makes data reduction necessary to determine trends. Ordination is a good statistical tool but still assumes some degree of linear correlation as would occur with a typical dose-response curve.

42. Eigenvalue2.49941.59831.35200.78430.43640.23060.0990 Percent35.705922.832819.314511.20386.23413.29501.4140 Eigenvectors Impairment0.032070.47871-0.599710.031980.52405-0.28832-0.22668 Det. Met-0.02386-0.69182-0.127190.137210.651810.205030.13755 Fire Ret.0.272060.146640.488350.742880.19262-0.10884-0.25528 Fragrances-0.453150.458510.068260.203180.170530.609720.37099 Herbicides-0.243540.130710.61232-0.518830.45492-0.26042-0.06864 HH Waste0.592040.168670.05748-0.082590.11987-0.159140.75579 Plasticizers0.556070.122270.05280-0.333150.114090.63094-0.38854 Males

43. Eigenvalue2.54241.37851.11610.94300.65820.27600.0858 Percent36.319919.695215.944013.47109.40303.94331.2262 Eigenvectors Impairment0.292450.18743-0.641530.257360.51884-0.35362-0.08546 Det. Met0.05636-0.564190.380590.434440.557230.180170.04430 Fire Ret.0.041970.511510.472400.59978-0.16230-0.31014-0.17655 Fragrances-0.434530.42877-0.210060.275960.176820.664760.18408 Herbicides-0.268750.341610.37217-0.519330.59522-0.22040-0.00263 HH Waste0.578080.218460.15670-0.069810.017240.100290.76047 Plasticizers0.560640.191470.11504-0.185900.090080.49575-0.58936 Females

44. Summary Significant hormonal impairment of both sexes, as compared to controls, at very low concentration of compounds. This impairment could never have been determined in a field study. Commonly-used parametric analyses are often inadequate in determining impairment.

45. Summary (cont) Determination of either hormonal impairment or endocrine disruption requires 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

46. Current and Future Research Fertility/fecundity and sex ratio/development of F2 generation. Behavior. Treatments using streambed sediment from affected EDW’s.

47. This study is highly representative of the biological effect of endocrine-disrupting compounds at the landscape scale.

48. Questions?