1. Response of Juvenile Rainbow Trout to Pesticides in Surface Waters within Urban Streams in Western Washington Christie Shavey Sara Bogard, Jacki Bricker, Erin Lowery, Cara Menard University of Washington, School of Aquatic and Fishery Sciences FISH 455 / ESC 457 Cat Curran Martin Grassley Chris Grue University of Washington, Washington Cooperative Fish and Wildlife Research Unit

2. Pesticide Usage Pesticide Concentrations in Surface Waters Salmonids Acetylcholinesterase (AChE) Organophosphates Carbamates

3. Objective: To determine effects of a chemical cocktail representative of that reported in storm waters flows in Western Washington on brain acetylcholinesterase activity, behavior and survival of juvenile rainbow trout (Oncorhynchus mykiss) Endpoints: Mortality Behavior Changes in AChE activity Photo by M. Lorenzoni

4. Chemical Cocktail NameNominal Concentration (ppb) Atrazine0.02 2,4-D3.30 Dicamba0.09 Dichlobenil0.54 MCPA0.38 MCPP0.77 Prometon0.19 Simazine4.99 Triclopyr2.70 Carbaryl0.21 Diazinon0.43 Malathion0.22 Pentachlorophenol0.13 4-nitrophenol0.29 Maximum Concentration Values

5. Available Products at Retail Stores Photo by M. Grassley

6. Photos by M. Grassley

7. Testing Two Tests Conducted: Replicates of 10 / concentration Test 1: Control & 1x original chemical cocktail n = 20 Test 2: Control, 1x, 3.3x & 10x original chemical cocktail n = 10

8. Testing Followed U.S. EPA Protocols for Static 96-hour Toxicity Tests Fish Loading: 1.25 L / g Average Fish Length / Weight Test 1: Length 9.7 cm / Weight 10.38 g Test 2: Length 9.8 cm / Weight 10.62 g

9. Environmental Chamber Photos by M. Grassley

10. Methods Fish Behavior Water Quality Photos by M. Grassley

11. Actual Chemical Concentrations

12. Photos by M. Grassley Methods Brain Assay

13. TreatmentnTemp (°C)DO (mg/L)pH Conductivity (µS) Test 1 Control2012.7 ± 0.79.27 ± 0.56.75 ± 0.270.4 ± 4.0 1X2012.7 ± 0.69.00 ± 1.06.88 ± 0.171.0 ± 4.9 Test 2 Control1012.62 ± 1.69.96 ± 0.776.94 ± 0.7375.22 ± 4.6 1X1012.66 ± 2.010.07 ± 0.957.03 ± 0.675.68 ± 5.6 3.3X1013.10 ± 1.69.75 ± 1.727.00 ± 0.4474.68 ± 4.0 10X1012.69 ± 2.09.76 ± 2.666.97 ± 0.6875.77 ± 6.2 Water Quality Data Results Meets EPA requirements: Temp: 12 ± 2 o C DO: ≥5 mg/L pH: 6 - 8 O2O2 O2O2 O2O2

14. Brain Cholinesterase Results – Test 2 AChE Activity in Juvenile Rainbow Trout Exposed to Chemical Cocktail

15. Key: Active; Lethargic; Dead Fish Behavior Results – Test 2 Percent Responding

16. Interpretations Maximum Concentrations ↔ Concentrations in Streams Brain Cholinesterase Inhibition: 1x = No 3.3x & 10x = Yes Fish Behavioral Effects

17. 2,4-D Unfiltered Geometric Mean = 0.206 ppb 95% CI = 0.145 – 0.295 Maximum 3.30 ppb

18. Geometric Mean = 0.022 ppb 95% CI = 0.016 – 0.029 Prometon Filtered Maximum 0.19

19. Name1x ConcentrationLC50 (ppb) Atrazine 0.02 4,000 (18,000-32,000) 2,4-D3.30>100,000 Dicamba0.0928,000 Dichlobenil0.546,260 (4,680-8,370) MCPA*0.38>10,000 MCPP0.77124,800 Prometon0.1920,000 Simazine4.99>100,000 Triclopyr2.70>100,000 Carbaryl0.211,950 (1450-2630) Diazinon0.4390 Malathion0.22200 (160-240) Pentachlorophenol0.13115 (108-122) 4-nitrophenol0.293,800 LC 50 Values for Rainbow Trout * Indicates different species

20. If this is the case… Why did we see mortality?

21. Chemical Interactions 2.1 ppb Carbaryl 4.3 ppb Diazinon 2.2 ppb Malathion 84% Inhibition or Mortality Synergism &/or Potentiation

22. Summary No brain inhibition at 1x Brain inhibition of 23% at 3.3x & 84% at 10x Apparent change in behavior at 1x, 3.3x & 10x Potential chemical interactions resulting in brain cholinesterase & mortality Concentrations tested were 1-2 orders of magnitude greater than average field concentrations

23. Future Research Chemical interactions Survival & reproduction of adult coho

24. Acknowledgements We thank the USGS Cooperative Units Program, the School of Aquatic and Fishery Sciences and the Washington Cooperative Fish and Wildlife Research Unit. The unit is financially supported by the USGS, University of Washington, Washington State University and the Washington Departments of Ecology, Fish and Wildlife, and Natural Resources. We also thank Windy Madsen for help with the toxicity tests and cholinesterase assays.