Why are amphibians good models? Gonadal development in Rana pipiens tadpoles and the influence of Aatrex (Atrazine) and Roundup (Glyphosate) exposure.

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1 Gonadal development in Rana pipiens tadpoles and the influence of Aatrex (Atrazine) and Roundup (Glyphosate) exposure. Amphibians as a model species for detecting endocrine disrupting effects of environmental contaminants CHRISTINA M. FRIDGEN 1, M. BERRILL 2, K. DOE 3, P. JACKMAN 3, AND B.D. PAULI 4 1 Watershed Ecosystems Graduate Program, Trent University Peterborough, ON 2 Department of Biology, Trent University, Peterborough, ON 3 Environment Canada, Moncton, NB 4 Environment Canada, Carleton University, Ottawa, ON. Natural variation in ual differentiation and metamorphosis Effects of Roundup and Atrazine on amphibian development Canadian Wildlife Service Comparing research within and between laboratories Why are amphibians good models? High risk habitat External development Global status sensitivity» Not to mention, their convenient abundance on campus Environmental/Chemical Plasticity Thyroid mediated Metamorphosis Sex Steroid mediated Sexual Development 1

2 Glyphosate Herbicide Use Conifer forest clear cuts (Vision ) Thyroid Hormone TRβ relative expression Tail tips Genetically Engineered Crops Heads Shoreline Weed Control (Rodeo ) Roundup (Glyphosate) induced Abnormal Gonadal Development Sexual differentiation New Glyphosate Formulations Gly-Flo Rodeo Glyfos (regular, Envision, Kernel, Aquatic, Custom, Roundup (Original, Zacca, Forza) Original MAX, Transorb, Glyphomax (regular, Ultra, UltraDry, Plus) UltraMAX, WeatherMAX, Glyphosate Pro (I, II) to name a few) Mirage Sequence Ranger Touchdown (CF, IQ, Extreme HiTech, Total FieldMaster Vision ReadyMaster ATZ Vantage to name a few. 2

3 Morphometrics Roundup Original and R. WeatherMax affected rate of metamorphosis and increased length Alternative Thyroid Endpoints tail tip cultures in vitro thyroid gland histology -Dr. Carolyn Kapron, Trent University Atrazine Affects Morphometric Endpoints Gosner Stage SNV Length

4 Atrazine Affects Time to Metamorphosis 5-Sep-05 Treatment; LS Means Current effect: F(5, 177)=2.8868, p= Ef f ectiv e hy pothesis decomposition Vertical bars denote 0.95 conf idence interv als Ovary Size 31-Aug-05 Days to Metamorphosis (dd-mos-yy) 26-Aug Aug Aug Aug-05 6-Aug-05 1-Aug-05 Testis Size Jul Atrazine [ppb] Aatrax (Atrazine) induced Abnormal Gonadal Development CONTROL 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% Control 0.1 Atrazine ATRAZINE (0.1 and 1.8 µg/l) Atrazine Abnormal Male Female H&E Stain PAS Stain Differences between and within labs -Time to Metamorphosis B A C A D CDE CD F (32) (16) (20) (114) (21) (22) (32) (26) 4

5 Differences between and within labs SNV Length (mm) AC AB AB D GOSNER STAGE 26 AB Male ABC Timing of Exposure Dependence of gonadal development on AGE ABC Female E (32) (21) (16) (22) (20) (32) (114) Week 3 Week 6 Week 12 (26) Timing of Exposure Semi-differentiated Males Dependence of gonadal development on STAGE Male AGE WEEK 11 Female vs. INTERSEX Males Stage 26 Stage 30 Stage 36 5

6 Oocyte diameters of control tadpoles (females and semi-differentiated males) vs pesticide exposed tadpoles (inter) Box Plot (oocyte_diameter.sta 10v324c) Length (cm) Least Squares Means (some means not estimable) Current effect: F(1, 201)=20.768, p= Effective hypothesis decomposition Vertical bars denote 0.95 confidence intervals Statistical analysis and hidden interactive effects Diameter VAR3 (µm) control control female inter male VAR1 Non-Outlier Max Non-Outlier Min 75% 25% Median Outliers m f inter SNV Length (mm) TreatmentSEX; LS Means Current eff ect: F(1, 36)=.00012, p= Ef f ectiv e hypothesis decomposition Vertical bars denote 0.95 conf idence intervals RC 2000 RC 2001 Treatment SEX M SEX F Histological ing may actually be an important endpoint for determining 30 morphometric Least Squares Means (some means not estimable) effects!!! Current effect: F(24, 201)=1.0817, p= p<0.05 from control Effective hypothesis decomposition p<0.05 from control Vertical bars denote 0.95 confidence intervals and betw een es Length (cm) CON00 GLY00 POEA100 POEA200 RO100 RO200 RT100 RT200 CON01 CHA101 CHA201 AU101 AU201 DWN101 DWN301 BCT101 BCT201 m f inter 6

7 Acknowledgements Supervisor: Michael Berrill, Department of Biology, Trent University, Peterborough, ON Collaboration and Funding: Ken Doe, Paula Jackman and summer students Environment Canada, Moncton, NB Natacha Hogan CAREG, University of Ottawa The Project Coordination and Funding: Bruce Pauli Canadian Wildlife Service Environment Canada, Carleton University, Ottawa, ON. Rana pipiens 7