ABSTRACT CHRISTINE A. LA ROCCA. Effects of Diet on the Survival, Reproduction and Sensitivity of Ceriodaphnia dubia. (Under the direction of DR. DONALD E. FRANCISCO) The effect of diet on the health and robustness of Ceriodaphnia dubia was investigated. Cj. dubia were raised on three diets for 19 generations to evaluate survival and reproduction. The three diets used to culture C^ dubia were the green alga Selenastrum capricornutum plus a mixture of yeast, Cerophyl^, and trout chow (YCT), the green alga Chlamydomonas reinhardtii plus YCT, and a combination of the two species of algae plus YCT. C^ dubia were also subjected to various copper concentrations to evaluate the relative sensitivity to toxicants of animals raised on different diets. The levels of survival and reproduction of C^ dubia raised on all three diets satisfied the North Carolina Division of Environmental Management Mini-chronic Pass/Fail Ceriodaphnia Effluent Toxicity Test minimum standards for control animals used in a chronic toxicity test. Survival was not significantly different in any diet tested. Reproduction was higher in the S^ capricornutum/C. reinhardtii/YCT diet than in the other diets. C^. dubia raised on the S_^ capricornutum/C. reinhardtii/YCT diet were also less sensitive to copper than animals raised on the single algae diets. The results suggest that a diet that includes multiple species of algae is nutritionally superior to one that includes a single species of algae. TABLE OF CONTENTS I. Introduction...........................................1 Background.........................................1 Literature Review.................................10 Ob j actives........................................17 II. Materials and Methods.................................18 General Culture Maintenance.......................18 Algal Cultures....................................23 Long-term Culture Experiment......................26 Copper Toxicant Experiments.......................29 III. Results...............................................32 Long-term Culture Experiment......................32 Copper Toxicant Experiment........................60 IV. Discussion............................................83 V. Conclusions and Recommendations.......................91 VI. Bibliography..........................................93 VII. Appendix A............................................97 VIII. Appendix B............................................98 IX. Appendix C............................................99 X. Appendix D...........................................102 XI. Appendix E...........................................105 XII. Appendix F...........................................141 XIII. Appendix G...........................................142 XIV. Appendix H...........................................143 LIST OF FIGURES 1. Diagram of the long-term culture experiment.............. 28 2. Average 7-day survival of C^ dubia raised on three different diets during the long-term culture experiment..........................................33 3. Survival of adult C^^ dubia raised on Selenastrum/YCT......34 4. Survival of adult C^. dubia raised on Chlamydomonas/YCT. .35 5. Survival of adult C^. dubia raised on Selenastrum/ Chlamydomonas/YCT....................................36 6. Average 3-brood reproduction during the long-term culture experiment...........................................39 7. Average reproduction during the 19 generations of the long-term culture experiment.........................40 8. Average reproduction of C^. dubia raised on Selenastrum/ YCT (+ and - one standard deviation).................42 9. Average reproduction of C^ dubia raised on Ch1amydomonas/ YCT (+ and - one standard deviation).................43 10. Average reproduction of C^ dubia raised on Selenastrum/ Chlamydomonas/YCT (+ and - one standard deviation)...44 11. Reproduction in Culture Line A............................45 12 . Reproduction in Culture Line B............................46 13 . Reproduction in Culture Line C............................47 14. Reproduction in Culture Line D............................48 15. Reproduction in Culture Line E............................49 16. Reproduction in Culture Line F............................50 17. Reproduction in Culture Line G............................51 18. Reproduction in Culture Line H............................52 19. Reproduction in Culture Line 1............................53 20. Reproduction in Culture Line J............................54 ii. 21. Reproduction in Culture Line K............................55 22. Reproduction in Culture Line L............................56 23. Reproduction in each Culture Line during the long-term culture experiment...................................59 24. Average mortality of animals in 2 ug-Cu/L.................61 25. Average mortality of animals in 4 ug-Cu/L.................62 26. Average mortality of animals in 8 ug-Cu/L.................63 27. Average reproduction of animals in 2 ug-Cu/L..............65 28. Average reproduction of animals in 4 ug-Cu/L..............66 29. Average reproduction of animals in 8 ug-Cu/L..............67 30. Average reproduction in all copper concentrations.........68 31. Reproduction in each Culture Line raised on Selenastrum/ YCT in 2 ug-Cu/L.....................................69 32. Reproduction in each Culture Line raised on Chlamydomonas/ YCT in 2 ug-Cu/L.....................................70 33. Reproduction in each Culture Line raised on Selenastrum/ Chlamydomonas/YCT in 2 ug-Cu/L.......................71 34. Reproduction in each Culture Line raised on Selenastrum/ YCT in 4 ug-Cu/L.....................................72 35. Reproduction in each Culture Line raised on Chlamydomonas/ YCT in 4 ug-Cu/L.....................................73 36. Reproduction in each Culture Line raised on Selenastrum/ Chlamydomonas/YCT in 4 ug-Cu/L.......................74 37. Reproduction in each Culture Line raised on Selenastrum/ YCT in 8 ug-Cu/L.....................................75 38. Reproduction in each Culture Line raised on Chlamydomonas/ YCT in 8 ug-Cu/L.....................................76 39. Reproduction in each Culture Line raised on Selenastrum/ Chlamydomonas/YCT in 8 ug-Cu/L.......................77 40. Difference in reproduction between controls and 2 ug-Cu/L in each Culture Line.................................79 ill. 41. Difference in reproduction between controls and 4 ug-Cu/L in each Culture Line.................................80 42. Difference in reproduction between controls and 8 ug-Cu/L in each Culture Line.................................81 iv. ACKNOWLEDGEMENTS I would like to offer my grateful appreciation to Dr. Donald E. Francisco for his advice, assistance and encouragement during the preparation of this document. I would also like to thank Dr. Michael J. Symons and Dr. Francis A. DiGiano for reading this report and offering many helpful suggestions. Many thanks also to my lab partner Michael C. Elias for his friendship, cooperation and company during many long hours at the Wastewater Research Center. Lastly, I must recognize the invaluable assistance of Marilyn J. Maerker. Her commitment to excellence was an inspiration to all of the students that spent time in her laboratory. INTRODUCTION Background Early in the history of U.S. environmental policy, state agencies bore the primary responsibility for the formulation, implementation and enforcement of water pollution control laws and regulations, with the federal role limited to matters of interstate pollution (Lamb, 1985). Recognizing the inability of the states to effectively control water pollution. Congress passed a series of laws designed to shift the responsibility of cleaning the nation's surface waters from the state governments to the federal government. The passage of the Water Quality Act of 1965 (P.L. 87-234) increased federal involvement in water pollution control, although the principal responsibility for establishing and enforcing standards remained with the states. One important provision of the act was the classification of streams according to their anticipated maximum beneficial use, which theoretically, forces states to reduce discharges into streams and prevents the further degradation of surface waters (Vesilind, Peirce and Weiner, 1990). 2. Through the Federal Water Pollution Control Act of 1972 (P.L. 92-500) Congress attempted to increase federal responsibility for establishing, implementing, and enforcing standards. A system was developed whereby minimum effluent standards are set universally for all dischargers, and these standards are modified based on the actual effect the discharge will have on the receiving water (Vesilind, Peirce and Weiner, 1990). This policy is implemented through the National Pollutant Discharge Elimination System (NPDES) which prohibits the discharge of pollutants into any public waterway unless authorized by a permit. NPDES permits contain effluent limits that require baseline use of treatment technologies regardless of the impact on receiving waters. The interim goal of the 1972 Federal Water Pollution Control Act was to provide for "...the protection and propagation of fish, shellfish, and wildlife, and...for recreation in and on the waters..." (Macek, 1985), with the ultimate goal being the attainment of zero discharge of wastes into streams by 1985 (Lamb, 1985). Also included in the legislation was the statement that "... it is the national policy that the discharge of toxic pollutants in toxic amounts be prohibited."