1. Melissa Churchel 1, Jim Hanula 2, Wayne Berisford 1 and Jim Vose 2 1 Department of Entomology University of Georgia 2 USDA Forest Service Southern Research Station Environmental Fate of Imidacloprid University of Georgia

2. Chemical Control Options Horticultural Oil Soil Injection with Imidacloprid Stem 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 LC 50 values range from 10.5 to 10440 ppb for aquatic insects

8. Our Objectives 1)Determine if imidacloprid entered the streams using the guidelines 2)Determine if the treatments outlined by the national forests were affecting stream insects 3)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 injector to inject imidacloprid (Merit 75 WSP) –1 g ai per inch diam in 10 ml H 2 O –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. 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 Methods

15. Insect Sampling 4 riffles sampled in each stream using a Surber sampler with fixed area of 1 m 2 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. Sampling for Larger Insects and Their Relatives Started 1 week after insecticide application Sampled bi-weekly for first 4 months Sampled monthly for rest of study (2 years) Post-treatment Sampling

18. Water 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 Post-treatment Sampling

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

22. 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 Data Analysis

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 < 4.18Excellent 4.17 - 5.09Good 5.10 - 5.91Good-Fair 5.92 - 7.05Fair > 7.05Poor

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. 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 Insect Sampling Summary

32. 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 Insect Sampling Summary

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