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Exposure-Related Effects of Formulated Pseudomonas Fluorescens Strain CL145A to Glochidia from Seven Unionid Mussel Species

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Exposure-Related Effects of Formulated Pseudomonas fluorescens Strain CL145A to Glochidia from Seven Unionid Mussel Species By James A. Luoma, Kerry L. Weber, Todd J. Severson, Theresa M. Schreier, Denise A. Mayer, Douglas B. Aloisi and Nathan L. Eckert Open-File Report 2015–1094 U.S. Department of the Interior U.S. Geological Survey U.S. Department of the Interior SALLY JEWELL, Secretary U.S. Geological Survey Suzette M. Kimball, Acting Director U.S. Geological Survey, Reston, Virginia: 2015 For more information on the USGS—the Federal source for science about the Earth, its natural and living resources, natural hazards, and the environment—visit http://www.usgs.gov or call 1–888–ASK–USGS (1–888–275–8747) For an overview of USGS information products, including maps, imagery, and publications, visit http://www.usgs.gov/pubprod Any use of trade, firm, or product names is for descriptive purposes only and does not imply endorsement by the U.S. Government. Although this information product, for the most part, is in the public domain, it also may contain copyrighted materials as noted in the text. Permission to reproduce copyrighted items must be secured from the copyright owner. Suggested citation: Luoma, J.A., Weber, K.L., Severson, T.J, Schreier ,T.M., Mayer, D.A., Aloisi, D.B, and Eckert N.L., 2015, Exposure-related effects of formulated Pseudomonas fluorescens strain CL145A to glochidia from seven unionid mussel species: U.S. Geological Survey Open-File Report 2015–1094, 474 p., http://dx.doi.org/10.3133/ofr20151094. ISSN 2331-1258 (online) Acknowledgments This study was funded through a grant from the U.S. Environmental Protection Agency Great Lakes Restoration Initiative awarded to the New York State Education Department and U.S. Geological Survey appropriated funds. The authors thank Mark P. Gaikowski of the Upper Midwest Environmental Sciences Center for assisting with study design and statistical analysis as well as Jeremy K. Wise, Samuel M. Stafslien, Pajtshiab L. Moua, and Susan M. Schleis for technical assistance and data collection. iii Contents Acknowledgments .......................................................................................................................................... iii Abstract.......................................................................................................................................................... 1 Introduction .................................................................................................................................................... 1 Materials and Methods .................................................................................................................................... 3 Experimental Design .................................................................................................................................... 3 Test Article .............................................................................................................................................. 3 Test system ............................................................................................................................................. 4 Animal Collection and Distribution .............................................................................................................. 7 Test Article Preparation and Application ..................................................................................................... 8 Water Chemistry ...................................................................................................................................... 9 Glochidia Viability Assessments................................................................................................................. 9 Data Analysis................................................................................................................................................ 10 Results and Discussion.................................................................................................................................. 10 Conclusions.................................................................................................................................................. 17 References Cited .......................................................................................................................................... 17 Appendix 1. Study Protocol, Amendments, and Datasheets .............................................................................. 19 Appendix 2. Deviations from the Study Protocol ............................................................................................. 113 Appendix 3. Randomization Assignments ...................................................................................................... 135 Appendix 4. Test Article Information.............................................................................................................. 237 Appendix 5. Test Animal Information............................................................................................................. 282 Appendix 6. Water Quality ........................................................................................................................... 325 Appendix 7. Glochidia Viability ..................................................................................................................... 353 v Figures Figure 1. Photograph showing glochidia test system with individual test chambers......................................... 6 Figure 2. Photographs showing A, the extraction of glochidia from a gravid female mussel and, B, extracted glochidia. ................................................................................................................................... 7 Figure 3. Photomicrographs showing A, glochidia before a drop of sodium chloride solution was added to induce valve closure in viable animals and, B, glochidia one minute after addition of the sodium chloride solution. ........................................................................................................................ 8 Figure 4. Photograph showing the distribution of glochidia to test chambers. ................................................. 8 Tables Table 1. Data for test animals, test articles and, exposure dates for studies of selected native freshwater mussel glochidia exposed to two formulations of Pseudomonas fluorescens, strain CL145A. ............ 4 Table 2. Data for initial glochidia viability by individual female mussel for each species. ................................ 5 Table 3. Estimated number of glochidia distributed to test chambers. .......................................................... 6 Table 4. Mean temperature, alkalinity, hardness, and conductivity of pre-exposure source water and pooled- exposure termination-water samples. ......................................................................................... 12 Table 5. Mean dissolved oxygen, mean temperature, and pH range of exposure water. .............................. 12 Table 6. Mean total ammonia nitrogen of exposure water by treatment group at 24 hours .......................... 14 Table 7. Mean un-adjusted glochidia viability from selected native freshwater mussels at 6, 12 and 24 hours............................................................................... 15 Table 8. Mean adjusted glochidia viability from selected native freshwater mussels at 6, 12 and 24 hours. ... 16 vi Conversion Factors International System of Units to Inch/Pound Multiply By To obtain Length centimeter (cm) 0.3937 inch (in.) micrometer (µm) 3.937x10-5 inch (in.) millimeter (mm) 0.03937 inch (in.) Volume liter (L) 1.057 quart (qt) microliter (µL) 0.000033814 ounce, fluid (fl. oz) milliliter (mL) 0.03382 ounce, fluid (fl. oz) Flow rate milliliter per minute (mL/min) 0.0002642 gallon per minute (gal/min) Mass gram (g) 0.03527 ounce, avoirdupois (oz) milligra m (mg) 3.527 x10-5 ounce, avoirdupois (oz) Temperature in degrees Celsius (°C) may be converted to degrees Fahrenheit (°F) as °F = (1.8 × °C) + 32. Conductivity is given in microsiemens per centimeter at 25 degrees Celsius (µS/cm at 25 °C). Concentrations of chemical constituents in water are given in milligrams per liter (mg/L). Abbreviations AI active ingredient α alpha CaCO3 calcium carbonate DO dissolved oxygen FDP freeze-dried powder HD heat deactivated p p-value SDP spray-dried powder TAN total ammonia nitrogen USGS U.S. Geological Survey vii Exposure-Related Effects of Formulated Pseudomonas fluorescens Strain CL145A to Glochidia from Seven Unionid Mussel Species By James A. Luoma1, Kerry L. Weber1, Todd J. Severson1, Theresa M. Schreier1, Denise A. Mayer2, Douglas B. Aloisi3, and Nathan L. Eckert3 Abstract The study was completed to evaluate the exposure-related effects of a biopesticide for dreissenid mussel (Dreissena polymorpha, zebra mussel and Dreissena rostriformis bugensis, quagga mussel) control on glochidia from unionid mussels endemic to the Great Lakes and Upper Mississippi River Basins. The commercially prepared biopesticide was either a spray-dried powder (SDP) or freeze-dried powder (FDP) formulation of Pseudomonas fluorescens, strain CL145A. Glochidia of the unionid mussel species Lampsilis cardium, Lampsilis siliquoidea, Lampsilis higginsii, Ligumia recta, Obovaria olivaria, and Actinonaias ligamentina were exposed to SDP-formulated P. fluorescens and Lampsilis cardium and Megalonaias nervosa were exposed to FDP-formulated
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