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FINAL REPORT Characterization of Aquatic Nonindigenous Species for Department of Defense Vessels: Bacteria, Algae, and Small Microfauna (< 80 μm) in Ballast Water SERDP Project SI-1244 July 2005 JoAnn Burkholder Center for Applied Aquatic Ecology, North Carolina State University, Raleigh, NC Gregory Melia Department of Environment & Natural Resources, Ecosystem Enhancement Program, Raleigh, NC Andrew Cohen San Francisco Estuary Institute, Oakland, CA Michael Mallin Center for Marine Science, University of North Carolina at Wilmington Matthew Parrow Center for Applied Aquatic Ecology, North Carolina State University, Raleigh, NC Gustaaf Hallegraeff School of Plant Science, University of Tasmania, David Oldach Holly Bowers Institute of Human Virology Institute of Human Virology, School of Medicine, University of Maryland Approved for public release This report was prepared under contract to the Department of Defense Strategic Environmental Research and Development Program (SERDP). The publication of this report does not indicate endorsement by the Department of Defense, nor should the contents be construed as reflecting the official policy or position of the Department of Defense. Reference herein to any specific commercial product, process, or service by trade name, trademark, manufacturer, or otherwise, does not necessarily constitute or imply its endorsement, recommendation, or favoring by the Department of Defense. iii Contents 28 July 2005 Final Characterization of Aquatic Nonindigenous Species for Department of Defense Vessels: Bacteria, Algae, and Small Microfauna (< 80 µm) in Ballast Water SI-1244 JoAnn Burkholder, Gregory Melia, Andrew Cohen, Michael Mallin, Matthew Parrow, Gustaaf Hallegraeff, David Oldach Center for Applied Aquatic Ecology North Carolina State University 620 Hutton Street, Suite 104 Raleigh, NC 27606 Strategic Environmental Research and Development Program SERDP Sustainable Infrastructure 901 North Stuart Street, Suite 303 Arlington, VA 22203 For general distribution after the draft report is finalized, pending SERDP review. This study characterized the bacteria, phytoplankton, and small microzooplankton (maximum dimension < 80 µm) in ballast tanks of DoD vessels operating from harbors on the U.S. East and West Coasts. From Sept. 2002- July 2004, 62 ballast tanks were sampled aboard 28 ships operated by the MSC and MARAD. A high percentage of the tanks (94%) had adequate records to determine the source locations and age of the ballast water, and 90% had had ballast exchange with open-ocean waters. Sources of variability in microbiota abundances were evaluated within and among ships that used different ballasting practices; an electronic Atlas of Phytoplankton Species was created; a database was designed to assist in risk analysis of microflora and microfauna species introductions by DoD ships; and laboratory-bench-scale experiments were conducted to examine the effectiveness of heat treatment on survival of selected species. The data were used to make recommendations to improve ballast water management practices, and to further advance understanding about the potential for introductions of aquatic invasive microbiota by DoD ships. Nonindigenous species, ballast water, bacteria, phytoplankton, microzooplankton JoAnn M. Burkholder UNCLASSIFIED UNCLASSIFIED UNCLASSIFIED SAR ix+71, and Appendices (919) 515-2726 ii Contents Page Cover Page ………………………………………………………………………………. i Report Documentation Page (Form OMB 0704-0188) ………………………………… ii Contents ………………………………………………………………………………….. iii Tables ……………………………………………………………………………………. v Figures …………………………………………………………………………………… vi Administrative Information ……………………………………………………………… ix Acknowledgments ……………………………………………………………………….. ix 1. Executive Summary …………………………………………………………………… 1 2. Introduction …………………………………………………………………………… 5 2a. Background ………………………………………………………………………. 5 2b. Regulatory Drivers ……………………………………………………………….. 6 2c. Previous Data on Species Transport by Ballast Water of DoD Vessels …………. 8 2d. Project Goals and Technical Objectives …………………………………………. 8 3. Materials and Methods ……………………………………………………………….. 10 3a. Operational Definitions ………………………………………………………….. 10 3b. Sampling Design and Methods …………………………………………………. 11 3b.1. Ballast Tank Sampling ………………………………………………….... 11 3b.2. Water Quality Analyses ………………………………………………….. 14 3b.3. Phytoplankton Analyses ………………………………………………….. 15 3b.4. Atlas of Phytoplankton Species ………………………………………….. 17 3b.5. Microzooplankton Analyses ……………………………………………... 17 3b.6. Bacterial Analyses ……………………………………………………….. 17 3b.7. Molecular Techniques: Ligation, Cloning, and Sequence Analyses for Selected Dinoflagellates and Bacteria ……………………... 20 3b.8. Heat Treatment Experiments …………………………………………….. 20 3c. Construction of a Regional Database ……………………………………………. 21 3d. Data Analysis ……………………………………………………………………. 23 4. Results ………………………………………………………………………………... 24 4a. Vessels Sampled and Ballast Water Management Data …………………………. 24 4b. Physical and Chemical Data ……………………………………………………... 33 4c. Phytoplankton Assemblages ……………………………………………………... 35 4c.1. Taxonomic Composition, Including Cultures …………………………….. 35 4c.2. Phytoplankton Abundance ………………………………………………... 35 4c.3. Phytoplankton Diversity …………………………………………………... 44 iii Contents (cont’d.) Page 4d. Microzooplankton ………………………………………………………………… 47 4e. Bacteria …………………………………………………………………………… 51 4f. Comparative Analyses of the Microbiota ……………………………………… 53 4f.1. Abundances of Phytoplankton, Microzooplankton, and Bacteria ……… 53 4f.2. Variance in Biota Within vs. Across Ships …………………………….. 53 4f.3. Considerations Based on Proposed / Established Standards for Ballast Water ………………………………………………………... 56 4g. Heat Treatment Experiments ………………………………………………….. 58 5. Summary and Conclusions ………………………………………………………… 59 6. Recommendations …………………………………………………………………. 62 7. References …………………………………………………………………………. 63 Appendix 1 – Sampling Methods and Flow Charts Appendix 2 – Atlas of Phytoplankton Species (electronic copy also included) Appendix 3 – Database Structure, Contents, and Documentation (and electronic copy of the Database) iv Tables Page Table 1. Harmful phytoplankton effects on humans and beneficial aquatic life ………….. 10 Table 2. Age distribution of ballast water, for tanks that contained coastal sources ……... 11 Table 3. Ballast tank sampling decision tree ……………………………………………… 12 Table 4. Examples of the designation system for voyages and tanks …………………… 12 Table 5. Molecular techniques used to screen for potentially harmful eubacteria and cyanobacteria ……………………………………………………………..... 19 Table 6. Vessels included in the study ………………………………………………….... 24 Table 7. Ballast management summary for the tanks examined ……………………….... 25 Table 8. Salinity and temperature of the ballast water …………………………………... 33 Table 9. Nutrient concentrations and N:P ratios in the ballast waters ………………….... 34 Table 10. Phytoplankton taxa found in ballast water samples, indicating potential harmful traits ………………………………………………………….. 36 Table 11. Phytoplankton tax cultured from ballast water, and harmful status ……………. 41 Table 12. Microzooplankton taxa in the ballast tanks, indicating size and known distribution ……………………………………………………………… 49 Table 13. Molecular screening for pathogenic eubacteria ………………………………… 54 Table 14. Restricted maximum likelihood variance components for phytoplankton, microzooplankton, and bacterial abundances ………………………………….. 55 Table 15. Proposed regulatory discharge limits for viable phytoplankton and zooplankton, in comparison to the data from this study ……………………….. 58 Table 16. Heat treatment effects on selected phytoplankton and microzooplankton species ……………………………………………………… 59 v Figures Page Figure 1. Example of vessels sampled: an oiler operated by the Military Sealift Command (MSC)…………………………………………. 9 Figure 2. Labline whole-water composite sampler …………………………………... 13 Figure 3. Overview of the sampling structure ……………………………………….. 14 Figure 4. Dinophysis caudata, an example of a toxigenic dinoflagellate found in ballast tanks ………………………………………………………. 15 Figure 5. Demonstration of flow cytometric analysis, using a toxic algal strain ……. 21 Figure 6. Example of a GIS map for a DoD ship track, which can be produced, if desired, using the designed database ………………………... 22 Figure 7. Tank distribution based on ballast water management status …………….. 26 Figure 8. Distribution of tanks with coastal source water(s) ………………………... 27 Figure 9. Examples of mapped coastal ballasting activities ………………………… 28 Figure 10. Mean temperature and salinity in each ballast tank, considered by major source region …………………………………………………….. 33 Figure 11. Mean pH and dissolved oxygen in each tank, considered by major source region …………………………………………………….. 34 Figure 12. Example of depth profiles of temperature, pH, salinity, dissolved oxygen, turbidity, and chlorophyll a (Voyage 1) ………………. 34 Figure 13. Sample page from the Phytoplankton Atlas ………………………………. 40 Figure 14. Relative abundance of viable phytoplankton as general groupings ………. 42 Figure 15. Total and viable phytoplankton abundance for each tank, arranged by source region …………………………………………….….. 43 Figure 16. The relationship between total and viable phytoplankton abundance (log-transformed data) and ballast water age ……………………………… 43 Figure 17. Total and viable phytoplankton abundance by season ……………………. 44 vi Figures (cont’d.) Page Figure 18. Species richness of total phytoplankton and harmful taxa in each tank, considered by major source region, water age, and status coastal, open ocean)