Effects of Diflubenzuron On Nontarget Organisms In Broadleaf Forested Watersheds In The Northeast Richard C. Reardon Handbook Coordinator FHM-NC-05-95 Acknowledgments This handbook was prepared as part of the USDA Forest Service’s program to improve aerial application of insecticides, specifically, to apply insecticides more effectively and efficiently to eastern broadleaf forests while minimizing environmental impacts. This program is supported through the efforts of the Northeast Forest Aerial Application Technology Group and the USDA Forest Service’s National Center of Forest Health Management. Richard C. Reardon thanks the USDA Forest Service, Northeastern Area State and Private Forestry, Appalachian Integrated Pest Management Gypsy Moth Project for funding this effort; Duphar B.V. and Uniroyal Chemical Company for technical advice and technical grade diflubenzuron; the staff of the USDA Forest Service’s Fernow Experimental Forest -- the main study site; and other staff of the West Virginia University Experimental Forest -- the preliminary study site on diflubenzuron persistence; Roberta Burzynski for editing; Lisa Cress and Linda Kent for typing the final draft; and the contributors for their time and efforts in preparing the individual sections in this handbook. Contributors Edward M. Barrows Tara Dubey Georgetown University P.O. Box 411 Department of Biology Elizabeth, IL 61028 Washington, DC 20057 Pamela J. Edwards Denise Biion USDA Forest Service Forest Health Protection P.O. Box 404 - Nursery Bottom NA State and Private Parsons, WV 26287 Forestry USDA Forest Service Patricia Gundrum 180 Canfield Street Forest Health Protection Morgantown, WV 26505 NA State and Private Forestry USDA Forest Service Linda Butler 180 Canfield Street West Virginia University Morgantown, WV 26505 P.O. Box 6108 Morgantown, WV 26506- Felton Hastings 6108 North Carolina State University Department of Entomology Matt Cochran Box 7626 WV Dept. of Agriculture Raleigh, NC 27695 State Capitol Building Charleston, WV 25305 Alan Iskra Forest Health Management Dan Downey NA State and Private Forestry James Madison University USDA Forest Service Department of Chemistry 180 Canfield Street Harrisonburg, VA 22807 Morgantown, WV 26505 V John C. Landolt Richard C. Reardon Shepherd College National Center of Forest Health Mgmt. Division of Science and Math NA State and Private Forestry Shepherdstown, WV 25443 USDA Forest Service 180 Canfield Street Morgantown, WV 26505 Thomas K. Pauley Marshall University Alan Sexstone Dept. of Biological Sciences West Virginia University Huntington, WV 25701 Plant and Soil Sciences P.O. Box 6057 Morgantown, WV 26506-6057 William B. Peny West Virginia University Steven L. Stephenson P.O. Box 6125 Fairmont State College Morgantown, WV 26506-6 125 Department of Biology Fainnont, WV 26554 Sue A. Perry Mary J. Wimmer National Biological Service West Virginia University Coop Fish and Wildlife Research Department of Biochemistry Unit Health Sciences Center West Virginia University P.O. Box 9142 P.O. Box 6125 Morgantown, WV 26506-9 142 Morgantown, WV 26506-6 125 He Thong North Carolina State University Paul Poling Department of Entomology WV Department of Agriculture Box 7626 Route 1 Box 46 Raleigh, NC 27695 Hambleton, WV 26269 Mention of trade, firm, or corporation names is for information only, and does not constitute endorsement by the authors or the U.S. Department of Agriculture. Information about insecticides appears in this handbook. Publication of this information does not constitute endorsement or recommendation by the U.S. Department of Agriculture, nor does it imply that all uses discussed have been registered. Use of most insecticides is regulated by State and Federal law. Applicable regulations must be obtained from appropriate regulatory agencies. CAUTION: Insecticides can be injurious to humans, domestic animals, plants, fish, or other wildlife if not handled or applied properly. Use all insecticides selectively and carefully. Follow recommended practices given on the label for use and disposal of insecticides and insecticide containers. vi Table of Contents Acknowledgments … v Contributors … v Part A. Project Description … 1 Chapter 1. Introduction … 2 Introduction … 3 Diflubenzuron … 3 The Nontarget Project … 4 Objectives … 4 Study Location … 4 Study Design … 5 References … 5 Chapter 2. Site Characteristics … 6 Climate … 7 Study Watersheds … 7 References … 9 Part B. Aquatic Organisms … 13 Chapter 3. Aquatic Salamanders … 14 Introduction … 15 Laboratory Study Methods and Costs … 15 Field Study Methods and Costs … 16 Advantages and Disadvantages … 16 Results … 17 Laboratory Study … 17 Field Study … 17 Discussion … 17 Recommendations for Future Studies … 18 References … 18 Chapter 4. Macroinvertebrates … 23 Introduction … 24 Laboratory Study Methods … 24 Field Study Methods … 25 Results … 26 Laboratory Study … 26 Field Study … 26 Discussion of the Laboratory Study … 27 Recommendations for Future Studies … 27 References … 28 Chapter 5. Aquatic Fungi … 31 Introduction … 3 Field Study Methods … 32 Results … 33 Pretreatment 1991 … 33 Treatment 1992 … 33 Discussion … 34 Recommendations For Future Studies … 35 References … 36 Part C. Terrestrial Organisms … 41 Chapter 6. Terrestrial Salamanders … 42 Introduction … 43 Laboratory Study Methods … 43 Field Study Methods and Costs … 43 Advantages and Disadvantages … 44 Results … 44 Laboratory Study … 44 Field Study … 45 Discussion … 45 Recommendations for Future Studies … 46 References … 46 Chapter 7. Canopy Arthropods … 53 Introduction … 54 Laboratory Study Methods … 56 Field Study Methods and Costs … 56 Advantages and Disadvantages … 57 Results … 58 Discussion … 59 Recommendations for Future Studies … 59 References … 60 Chapter 8. Pollinating Insects – Native Species … 66 Introduction … 67 Field Study Methods … 67 Results … 69 Discussion … 70 Recommendations for Future Studies … 71 References … 71 Chapter 9. Pollinating Insects – Honey Bees … 77 Introduction … 78 Field Study Methods … 78 Methods for Diflubenzuron Extraction and Analysis … 79 Results … 80 References … 80 Chapter 10. Invertebrates in Leaf Litter and Soil … 81 Introduction … 82 Field Study Methods … 82 Results … 85 Core Samples … 85 Bag Samples … 85 Discussion and Recommendations … 86 References … 86 Chapter 11. Soil Bacteria and Fungi … 93 Introduction … 94 Laboratory Study Methods … 94 Field Study Methods … 94 Results … 94 Laboratory Study … 95 Field Study … 95 Discussion … 96 References … 96 Chapter 12. Decomposer and Ectomycorrhizal Fungi … 106 Introduction … 107 Field Study Methods … 107 Results and Discussion … 108 Conclusions … 110 References … 111 Chapter 13. Terrestrial Fungi In Leaf Litter … 121 Introduction … 122 Laboratory Study Methods … 122 Results … 124 Discussion … 125 References … 126 Chapter 14. Microorganisms In Soil … 130 Introduction … 131 Laboratory Study Methods … 131 Results … 132 Discussion … 133 References … 133 Part D. Residue Levels and Persistence … 140 Chapter 15. Aquatic Environment … 141 Introduction … 142 Laboratory and Field Study Methods … 142 Results … 143 Discussion … 144 References … 145 Chapter 16. Terrestrial Environment … 146 Introduction … 147 Methods … 148 Reporting of Diflubenzuron Analysis Data … 151 Results … 151 References … 155 Chapter 17. Breakdown Products of Diflubenzuron … 161 Introduction … 162 Methods … 162 Results and Discussion … 163 Conclusions … 164 References … 164 Part E. Summary … 168 Introduction … 169 Aquatic Organisms … 169 Terrestrial Organisms … 170 Residue Levels and Persistence … 172 Conclusion … 174 Part A. Project Description Chapter 1. Introduction Richard C. Reardon Introduction Aerial application of insecticides is one of the control methods used for the gypsy moth, Lymantria dispar (L.) (Lepidoptera: Lymantriidae), an important defoliating pest in broadleaf forests. The synthetic arthropod growth regulator diflubenzuron (DFB) is one of the insecticides used in the Federal and State cooperative gypsy moth program. The others are the bacterium Bacillus thuringiensis (Bt) and the nucleopolyhedrosis virus product Gypchek®. From 1975 when this program began, to 1994, approximately 4 million acres infested with the gypsy moth were treated with DFB (NOTE: DFB wasn’t registered until 1976). From 1991 through 1994, approximately 1.1 million acres were treated with DFB, 2.1 million acres with B. t., and 26,000 acres with Gypchek (Machesky 1995). DFB is also used to control a wide variety of other insect pests on agricultural crops (cotton and soybeans), ornamentals in nurseries, and conifers and hardwoods in forests. Numerous studies, conducted before and after the registration of DEB by the U.S. Environmental Protection Agency (EPA) in 1976, have examined the potential impacts on selected nontarget species (Onken 1995, USDA 1995). One data gap identified by the Appalachian Integrated Pest Management (AIPM) Gypsy Moth Project, however, was potential impacts on nontarget organisms when DFB is applied to an entire broadleaf forested watershed, as can happen when spraying for the gypsy moth. An entire watershed can encompass stream headwaters and adjacent major streams. Diflubenzuron DFB, N-[[(4-chlorophenyl)amino]carbonyl]2,6-difluorobenzamide] was discovered by scientists at Philip Duphar B.V., Weesp, Holland, and was developed by the Thompson-Hayward Chemical Company for marketing in the United States. It is distributed in the United States by Uniroyal Chemical Company. DFB breaks down into either 2,6-difluorobenzoic acid (DFBA) or 4- chlorophenylurea