THE EFFECTS OF SELECTED ANTIBIOTICS ON NITROGEN UPTAKE BY Spirodela punctata By Cory M. Jones A Thesis Presented to The Faculty of Humboldt State University In Partial Fulfillment Of the Requirements for the Degree Master of Science In Natural Resources: Wastewater Utilization Program February, 2010 ABSTRACT The Effects of Selected Antibiotics on Nitrogen Uptake by Spirodela punctata Cory M. Jones The purpose of this study was to determine the effects of nitrogenous compound removal by the aquatic macrophyte, Spirodela punctata, when exposed to three selected antibiotics. Recent research has shown that certain antibiotics target the chloroplasts of aquatic species such as Lemna and Myriophyllum. Studies have demonstrated antibiotic toxicity to Lemna gibba at concentrations as low as 10 µg/L. Meanwhile, antibiotic concentrations in domestic wastewater lie in the nanogram to microgram range with an average of approximately 50 µg/L. In this study, Spirodela punctata was grown in a mineral salts medium containing the antibiotics chlortetracycline, lomefloxacin, and sulfamethoxazole in concentrations ranging from 10 µg/L to 300 µg/L. Fronds were allowed to grow in the medium for seven, fourteen, and twenty-one day periods. Following the growth periods, the medium was analyzed for nitrate and total nitrogen concentrations. Dry weights of fronds were taken and the dried plant material was analyzed for Total Kjeldahl Nitrogen (TKN) content. Effective concentrations (EC25 and EC50) that impacted total nitrogen and nitrate removal from the growth medium as well as dry weights and Total Kjeldahl Nitrogen of the plant tissue were calculated. Of the antibiotics tested, chlortetracycline had the most iii significant responses. The antibiotic reduced nitrate and total nitrogen removal from the medium and decreased plant biomass. For nitrate removal chlortetracycline had an EC25 of 32 µg/L after three weeks of exposure. Dry weight EC25 for chlortetracycline was 73 µg/L after seven days of exposure to the antibiotic. Control treatments containing the antibiotic and no plant material had similar results indicating that the compounds added nitrogen. Conversely, other compounds tested resulted in very few significant responses. Lomefloxacin only showed significance for nitrate removal during week two and total nitrogen for week one. Surprisingly, the antibiotic seemed to stimulate the ability of Spirodela punctata to remove total nitrogen during the first week of exposure. Sulfamethoxazole had only one significant response during the test period. The combination of the three antibiotics resulted in only a reduction in nitrate removal during the first week after which there was no significant effect. Overall, selected antibiotics had little direct effect on ability of Spirodela punctata to remove nitrogenous compounds. However, with respect to chlortetracycline, the reduction in the production of biomass resulted in the reduction in nitrate and total nitrogen removal. This reduction in biomass is perhaps the greatest impact on S. punctata’s ability to remove nitrogenous compounds from wastewater. iv ACKNOWLEDGEMENTS First and foremost, I would like to express my sincere appreciation towards Dr. Kristine Brenneman for the commitment, support, and expertise that she lent me throughout my graduate school experience. Dr. Brenneman was extremely patient with me through multiple thesis revisions and motivated me to keep working. I could not have done it without her. Secondly, I would like to thank Dr. Bill Bigg and his wife, Donna, for their help with statistics, experimental design, and the Total Kjeldahl Nitrogen analysis. They will be missed at Humboldt State University. Thanks to Ryan Faria-Cecil for all of his help during those long hours in the laboratory. I would also like to express my gratitude towards the city of Arcata for funding my research. My committee members, Dr. Frank Shaughnessy and Dr. Margaret Lang are greatly appreciated for their efforts providing insight regarding my project and for time spent reviewing my thesis. Thanks to the biology department for lending me laboratory materials without which I wouldn’t have been able to perform my research. Finally, I would like to thank my family for all of the support, financially and otherwise, that allowed me to achieve my goal. I am especially indebted to my beautiful wife, Lynette, who always supported and believed in me throughout this entire project. v TABLE OF CONTENTS Page ABSTRACT.......................................................................................................................iii ACKNOWLEDGEMENTS.................................................................................................v TABLE OF CONTENTS...................................................................................................vi LIST OF TABLES.............................................................................................................vii LIST OF FIGURES..........................................................................................................viii INTRODUCTION...............................................................................................................1 MATERIALS AND METHODS.........................................................................................5 Response Variables and Statistics............................................................................7 RESULTS............................................................................................................................8 DISCUSSION....................................................................................................................21 LITERATURE CITED......................................................................................................26 PERSONAL COMMUNICATION CITED......................................................................29 APPENDIX A....................................................................................................................30 Antibiotic Molecular Structures.............................................................................30 vi LIST OF TABLES Table Page 1 The p-values for significant responses for indicated assays of growth media treated with antibiotics as determined using analysis of variance (ANOVA) in the general linear model...................................9 2 The p-values and lowest-observable-effect concentrations (LOECs) for indicated assays of growth media and plant tissue of Spirodela punctata treated with antibiotics as determined using analysis of variance (ANOVA) in the general linear model and Dunnett’s two-sided test for treatment-control comparison............................11 3 Effective concentrations that cause a 25% change from the control for the indicated assays of growth media and plant tissue of Spirodela punctata treated with antibiotics as determined using curve fitting modeling techniques at the 95% confidence interval...................................................................................................................12 4 Effective concentrations that cause a 50% impact on the indicated assays of growth media and plant tissue of Spirodela punctata treated with antibiotics as determined using curve fitting modeling techniques at the 95% confidence interval............................................13 vii LIST OF FIGURES Figure Page 1 Significant (p<0.05) effects of the antibiotic chlortetracycline on nitrate removal by Spirodela punctata.............................................................14 2 Significant (p<0.05) effects of the antibiotic chlortetracycline on total nitrogen removal by Spirodela punctata..................................................15 3 Significant (p<0.05) effects of the antibiotic chlortetracycline on frond dry weight and Total Kjeldahl Nitrogen percentage of Spirodela punctata.................................................................................................................16 4 Significant (p<0.05) effects of the antibiotic lomefloxacin on nitrate removal and total nitrogen removal by Spirodela punctata.......................18 5 The significant (p<0.05) effect of the sulfonamide antibiotic sulfamethoxazole on total nitrogen removal by Spirodela punctata.....................19 6 The significant (p<0.05) effect of the combination of three antibiotics (chlortetracycline, lomefloxacin, and sulfamethoxazole) on nitrate removal by Spirodela punctata..............................................................20 viii INTRODUCTION Recent studies have provided evidence that antibiotics have phytotoxic effects on vascular plants including Lemna and Myriophyllum species (Brain et al. 2003, Brain et al. 2004, Pomati et al. 2004). The primary focus of these studies has been on the morphological effects that antibiotics impose including reduction of chlorophyll and biomass. To my knowledge, nothing is known about the effect antibiotics have on nutrient removal by such aquatic species. Lemnaceae have long been shown to be effective in wastewater treatment (Harvey and Fox 1973, Sutton and Ornes 1975, Hillman and Culley 1978). Lemnaceae are efficient in reducing nitrogen and phosphorus levels from various polluted water sources including animal wastes and municipal wastewater. A study by Tripathi (20003) showed that Lemna minor removed 62.5% of total nitrogen and 58.8% of phosphorus from secondarily treated dairy effluent in eight-weeks. Furthermore, Cheng et al. (2001) reported that within a 20-day