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

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

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

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 Pentachlorophenol nitrophenol0.29 Maximum Concentration Values

Available Products at Retail Stores Photo by M. Grassley

Photos by M. Grassley

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

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 g Test 2: Length 9.8 cm / Weight g

Environmental Chamber Photos by M. Grassley

Methods Fish Behavior Water Quality Photos by M. Grassley

Actual Chemical Concentrations

Photos by M. Grassley Methods Brain Assay

TreatmentnTemp (°C)DO (mg/L)pH Conductivity (µS) Test 1 Control ± ± ± ± 4.0 1X ± ± ± ± 4.9 Test 2 Control ± ± ± ± 4.6 1X ± ± ± ± X ± ± ± ± X ± ± ± ± 6.2 Water Quality Data Results Meets EPA requirements: Temp: 12 ± 2 o C DO: ≥5 mg/L pH: O2O2 O2O2 O2O2

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

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

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

2,4-D Unfiltered Geometric Mean = ppb 95% CI = – Maximum 3.30 ppb

Geometric Mean = ppb 95% CI = – Prometon Filtered Maximum 0.19

Name1x ConcentrationLC50 (ppb) Atrazine ,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 MCPP ,800 Prometon0.1920,000 Simazine4.99>100,000 Triclopyr2.70>100,000 Carbaryl0.211,950 ( ) Diazinon Malathion ( ) Pentachlorophenol ( ) 4-nitrophenol0.293,800 LC 50 Values for Rainbow Trout * Indicates different species

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

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

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

Future Research Chemical interactions Survival & reproduction of adult coho

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.