1. Tara Duffy and Stephen McCormick Conte Anadromous Fish Research Lab, Turners Falls, MA Differential life-stage response to common endocrine disruptors in two endangered species, Atlantic salmon and shortnose sturgeon

2. Tara Duffy and Stephen McCormick Conte Anadromous Fish Research Lab, Turners Falls, MA Differential life-stage response to common endocrine disruptors in two endangered species, Atlantic salmon and shortnose sturgeon Funding

3. Salmon and sturgeon conservation Anadromous Atlantic salmon and shortnose sturgeon are listed as endangered under the ESA Conservation efforts traditionally do not consider potential impacts of EDCs Habitat use and life strategies suggest these species may be particularly sensitive to degraded water quality

4. EPA WET testing and surrogate species WET: Whole Effluent Testing uses a single life-stage 4-day exposure, mortality is primary target Long-term, sublethal impacts are not routinely monitored Surrogate species Daphnia, brook trout, fathead minnow, silversides Sturgeon and salmon are very sensitive to common endocrine disruptors and effluent Dwyer et al. 2005, Arch. Environ. Toxicology and Contamination

5. Juveniles Embryo Life-stage and life-history will influence contaminant response Gonad differentiation or other factor Larvae Numerous life-stage sensitivity examples At what life stage will this chemical do the most damage?

6. Objectives Three year study: 1.Assess which life-stage may be most responsive to common feminizing EDCs 2.Assess vitellogenin (Vtg) as an appropriate biomarker for measuring contaminant sensitivity 3. Carry out chronic exposures (21-day) and assess impacts to fitness (growth, gonad histology, development, salinity tolerance)

7. Methodology 4-day exposures on early life-stages 1. Embryos 2. Yolk-sac larvae 3. Feeding fry 4. Juveniles/smolts Compounds used 1. α-ethinylestradiol (EE2) 2. 17 β-estradiol (E2) 3. 4-nonylphenol (NP) 15 o C target temperature for all life-stages (12 o C for salmon embryos)

8. Concentrations used represent: 1.Positive control dose 2.Concentration common to wastewater effluent 3.Low dose similar to that found in an urbanized river/estuary 4.Solvent control (<0.001%) Flow through system, 2 to 7 volumes d -1 Vtg gene expression (qPCR) and development of a sensitive EIA for Vtg protein concentration (from Peck et al. 2011) Whole body mRNA for early life-stages, liver in juveniles Mortality data Methodology

9. Vitellogenin as an indicator of life-stage sensitivity Common biomarker of estrogenic contaminant exposure Indicative of potential long-term reproductive impacts Could be used in the field in early life-stage fish

10. Atlantic salmon vitellogenin mRNA α-ethinylestradiol (EE2) 17 β-estradiol (E2) Nonylphenol (NP) control EE2 and E2 show significant treatment response, but this trend is not dose dependent (2-way ANOVA). NP is not significantly different from control. Relative transcription (normalized to EF-1α) embryos

11. Atlantic salmon vitellogenin mRNA α-ethinylestradiol (EE2) 17 β-estradiol (E2) Nonylphenol (NP) control Significant treatment effects, but dose-dependent response is less clear. Relative transcription (normalized to EF-1α) embryos * * Yolk-sac larvae * * * * *

12. All compounds show significant treatment effect, but suggestive dose-dependent response is non-significant. Vtg response is 200 to 500 times Vtg control transcription. α-ethinylestradiol (EE2) 17 β-estradiol (E2) Nonylphenol (NP) control Atlantic salmon vitellogenin mRNA Dose (nM) Relative transcription (normalized to EF-1α) Feeding fry * * * * *

13. EE2 and E2 elicit significant, dose-dependent response. Vtg mRNA is highest in this life-stage at 500-1500 times control transcription. α-ethinylestradiol (EE2) 17 β-estradiol (E2) Nonylphenol (NP) control Atlantic salmon vitellogenin mRNA Relative transcription (normalized to EF-1α) Dose (nM) Smolts * * * * 50

14. Summary E2 and EE2 elicit robust response in Vtg mRNA. Feeding fry show highest Vtg upregulation in response to NP. Overall expression is highest in salmon smolts (liver vs. whole animal differences?).

15. Dose (nM) Smolt plasma vitellogenin α-ethinylestradiol 17 β-estradiol Nonylphenol control Significant overall treatment effect and dose- dependent response Response to NP is muted compared to estrogens EIA shows a clearer response than qPCR data Plasma Vtg (μg/ml) 400 * * * * * * * * *

16. Sturgeon Vtg mRNA Relative Vtg transcription Dose (nM) Preliminary data Vtg response less robust than in salmon Full analysi Full analysis will demonstrate if Vtg is an appropriate biomarker in early life-stage sturgeon Feeding fry

17. Conclusions E2 and EE2 elicited nearly identical responses, and exposure to these compounds elicited strong Vtg response. Plasma Vtg may be the most appropriate biomarker for exogenous estrogen exposure. Vtg protein demonstrated a clear pattern of dose- dependent response, but not applicable to early life stages. Sensitivity vs. responsiveness of each life stage? Development of appropriate biomarker for sturgeon.

18. Future directions What are the implications of long-term exposure? Which species is most sensitive? Comparison to surrogate species

19. Funding and acknowledgements McCormick lab Amy Regish Amy Regish Arne Christensen Arne Christensen Michael O’Dea Michael O’Dea Joe Chadwick Joe Chadwick Barbara Arter Bill Kier-Kier Associates