Melissa Churchel 1 , Jim Hanula 2 , Wayne Berisford 1 and Jim Vose 2 1 Department of Entomology University of Georgi

1. Environmental Fate of Imidacloprid Melissa Churchel1, Jim Hanula2, Wayne Berisford1 and Jim Vose2 1 Department of Entomology University of Georgia 2 USDA Forest Service Southern Research Station University of Georgia

2. Chemical Control Options Stem Injection with Imidacloprid Horticultural Oil Soil Injection with Imidacloprid

3. What’s the Forest Service Doing? • National Forests in the south are trying to save a remnant population of hemlocks throughout their forest

4. Saving a Remnant Population Trying to save 60 trees in selected areas using soil injection of imidacloprid

5. Guidelines for Soil Injection on National Forests in GA and NC • Sample soil to determine presence of highly permeable soils (sands or gravel) • Scout area for presence of surface water (springs, creeks, ponds, bogs, etc.) • Eliminate any trees with a direct vegetative connection to surface water from soil injection. • Treat 60 trees per selected area

6. Imidacloprid • PROBLEM: • Highly mobile but soil absorption increases with increasing organic matter content • Does imidacloprid make its way through the soil after injected and enter nearby streams?

7. Imidacloprid Toxicity • Toxic to aquatic organisms • LC50 values range from 10.5 to 10440 ppb for aquatic insects

8. Our Objectives • Determine if imidacloprid entered the streams using the guidelines • Determine if the treatments outlined by the national forests were affecting stream insects • Determine if the treatments were effectively controlling HWA

9. Methods – Study Sites

10. Methods • Selected 4 small streams with moderate flow rates and sufficient hemlocks • 2 treatment methods used • Holcomb Tributary, Addie Branch, and Billingsley Creek: • Treated 60 trees around each stream using Kioritz soil injectorto inject imidacloprid (Merit 75 WSP) • 1 g ai per inch diam in 10 ml H2O • Injected 2 inches deep, 1 injection/inch diameter in a ring 12 inches from tree bole. • ~ 670g ai. applied per site • Treated November 1, 2005

11. Holcomb Tributary

12. Addie Branch

13. Billingsley Creek

14. Methods • Dryman Fork • 2000 ft treatment area • Treated all trees within 50 ft on either side of stream with at least 10 inch diameter at base • 88 trees next to stream treated with Mauget II Generation Tree Injector • 109 trees treated with soil injections • Treated May 17, 18, and 19 2006 • Adjacent watershed used as reference condition

15. Insect Sampling • 4 riffles sampled in each stream using a Surber sampler with fixed area of 1 m2 • Collected all contents with sampler down to 5 cm • Large cobble was scrubbed to remove insects • Samples preserved in 95% alcohol • Large samples were subsampled as needed • All larger insects were identified to genus or lowest taxonomic level possible

16. Surber Sampler

17. Samplingfor Larger Insects and Their Relatives Post-treatment Sampling • Started 1 week after insecticide application • Sampled bi-weekly for first 4 months • Sampled monthly for rest of study (2 years)

18. Water Sampling Post-treatment Sampling • Grab samples taken downstream of treatment area using 1000 ml glass bottle • Samples stored in cold room until analysis • Analysis conducted by the University of Georgia, Pesticide and Hazardous Waste Laboratory

19. Extraction and Analysis of Imidacloprid from Water 1 2 3 Stopper funnel; shake; allow layers to separate. 500 ml sample into 1-L Separatory Funnel Add 75 mL methylene chloride 4 8 Analyze on HPLC Drain the MeCl2 layer; repeat Step 2-3 twice more; combine all drained layers. Concentrate the extract under nitrogen 7 6 5 Filter the extract (removes sodium sulfate) Add sodium sulfate to flask & swirl (removes excess water)

20. Data Analysis • Number of taxa • Number of mayflies, stoneflies and caddis flies collectively (EPT) • Abundance • North Carolina Biotic Index (NCBI) for each stream and sample date • Index specific to SE U.S. • Indicator of general health of stream biotic community

21. Data Analysis North Carolina Biotic Index (NCBI) NCBI = ∑ TVi Ni Total N Where: TVi = tolerance value of the ith taxa Ni = abundance of the ith taxa Total N = number of individuals in the sample Tolerance values range from 0 to 10 Abundance values are transformed into Rare (1-2 per sample), Common (3-9 per sample), or Abundant (≥10 per sample

22. Data Analysis • Data for each stream pooled by season • Compared each stream to reference • If results were significantly lower than in the reference, we analyzed seasonal variability within that stream • Determine if a significant reduction in the macroinvertebrate community occurred

23. Results • Collected 217,587 insects and relatives • 83 taxa from 18 orders and 66 families • Trichoptera and Diptera most diverse

24. Average Number of Taxa

25. Mayflies, Stoneflies and Caddis Flies

26. Average Abundance

27. NCBI Scores Mountain Ecoregion: Water Quality Class < 4.18 Excellent 4.17 - 5.09 Good 5.10 - 5.91 Good-Fair 5.92 - 7.05 Fair > 7.05 Poor

28. Water Samples • A water sample was collected each time we sampled insects • Holcomb Tributary - Oct. 22, 2007 • < 1.0 ppb • No evidence of an impact on aquatic macroinvertebrates

29. HWA Control • ????

30. Insect Sampling Summary • Avg. Number of Taxa • Addie Branch – Winter 2006/07 significantly lower than reference stream • But not lower than Fall ’06 • Due to seasonal variation & smaller community of invertebrates overall than in reference

31. Insect Sampling Summary • EPT Taxa • Addie Branch – Summer 2006 significantly lower than reference stream • Also significantly lower than Spring 2006 • Follows same pattern of seasonal variation due to emergence of adults as other streams • More pronounced due to smaller community

32. Insect Sampling Summary • Abundance • Addie Branch – Fall 06 & Winter 06/07 • Dryman Fork – Fall 07 • Significantly lower than reference stream • Not significantly different from previous season • NCBI • None with significantly lower scores than reference stream

33. Conclusion • Good news – treatments had no effect on macroinvertebrate communities in mountain streams • A small amount of imidacloprid was detected in Holcomb Tributary, but had no effect on invertebrates

34. Implications • Soil injections can safely be used in the southern Appalachians • Only a trace amount of imidacloprid entered the streams over a 2 year period • Not enough to significantly impact the aquatic organisms