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The Use of a Multiparameter Bacterial Aquatic Toxicity Test

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The Use of a Multiparameter Bacterial Aquatic Toxicity Test The use of a multiparameter bacterial aquatic toxicity test Item Type Thesis-Reproduction (electronic); text Authors Pill, Kenneth Goodman,1961- Publisher The University of Arizona. Rights Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. Download date 24/09/2021 19:59:45 Link to Item http://hdl.handle.net/10150/192019 1 THE USE OF A MULTIPARAMETER BACTERIAL AQUATIC TOXICITY TEST by Kenneth Goodman Pill A Thesis Submitted to the Falculty of the DEPARTMENT OF HYDROLOGY AND WATER RESOURCES In Partial Fulfillment of the Requirements For the Degree of MASTER OF SCIENCE WITH A MAJOR IN HYDROLOGY In the Graduate College THE UNIVERSITY OF ARIZONA 1989 2 STATEMENT BY AUTHOR This thesis has been submitted in partial fulfillment of requirements for an advanced degree at The University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library. Brief quotations from this thesis are allowable without special permission, provided that accurate acknowledgment of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his or her judgement the proposed use of the material is in the interests of scholarship. In all other instances, however, permission must be obtained from the author. SIGNED: /4."' 6,e) APPROVAL BY THESIS DIRECTORS This thesis has been approved on the date shown below: 6-+-e-cpc 4 +1,0 t Robert G. Arnold Date Professor of Civil Engineering (7/Odk, re/- 1 /27 Roger C. Bales Date Professor of Hydrology 3 ACKNOWLEDGEMENTS My family played a large part in the completion of this thesis. My parents, Griz and Lil' J, provided much needed support throughout my stay here, and it is to them that I dedicate this work. My grandmother, Anne Krotter, Chuck and Barbara all supported my pursuit of my goals, and I love them all very much. I am also very grateful to Bob Arnold. His sense of humor, guidance and patience made tough times easier to handle. He taught me many things outside of this project that will certainly help me down the road, and I feel fortunate to have had him as my advisor. He also pitches a mean batting practice. Roger Bales and Martha Conklin provided much needed advise, and I appreciate their help and patience during the final stages of this work. Special thanks to some special friends that I've met along the way; Gerald Vogt, Bruce Smith, Kristen McClellan and Steve Price. They will remain friends for life, and riding just won't be the same without them. There are many others that have helped me along the way. Thanks to Greg Kupillas for getting me started on this project and his continued input along the way. Cathy Vogel made lab work more tolerable, along with Willie Odem and David Graham. Bruce Alleman always took the time to answer my sometimes stupid questions, and always made me pay for it in the Friday afternoon basketball games. Liz Kostiuk provided some much needed typing when things looked bleak. Thanks to all of you. I also appreciate the financial support provided by the National Science Foundation and Weyerhaeuser Corporation. 4 TABLE OF CONTENTS Page List of Figures 7 List of Tables 12 Abstract 14 Chapter 1 - Introduction 15 Chapter 2 - Background 2.1 Fish Bioassays 17 2.2 Bacterial Assays 18 2.2.1 Microtox Test 19 2.2.2 SpirtIlum volutans Test 21 2.2.3 Two-organism Test of Tchan 22 2.2.4 Cell Multiplication Inhibition Test 23 2.2.5 Microcalorimetry 24 2.3 Analysis of Microbial Assays 25 2.3.1 Comparisons 25 2.3.2 Summary 28 2.4 Procedure Proposed by Kupillas 30 2.5 Bacteriology 31 2.5.1 Introduction 31 2.5.2 Metabolic Pathway 33 2.5.3 Formation of ATP 37 2.5.4 Oxidative Phosphorylation 37 2.5.5 Summary of Metabolic Pathway 39 2.6 Compounds Investigated 41 2.6.1 Inhibitors 41 2.6.2 Chloramphenicol 41 2.6.3 Cyanide 43 2.6.4 DCCD 43 2.6.5 2,4-DNP 44 2.6.6 Summary of Inhibitors 44 2.6.7 Chlorinated Phenols, Ethanes and Metals. 44 2.7 Wastewater 46 5 TABLE OF CONTENTS - Continued Chapter 3 - Materials and Methods 47 3.1 Tester Strain 47 3.2 General Procedure 48 3.2.1 Pure Compounds 48 3.2.2 Wastewater 53 3.2.3 Toxicants 53 3.3 Preparation of Samples 54 3.3.1 Pure Compounds 54 3.3.2 Wastewater 58 3.4 Procedure for ATP Experiments 58 3.5 Procedure for Respiration Experiments 60 3.6 Luminometer Calibrations 62 Chapter 4 - Results 64 4.1 Preliminary Work 64 4.1.1 Luminometer Calibration - Pure Compounds 64 4.1.2 Luminometer Calibration - Wastewater 64 4.2 Experiments Involving Pure Compounds 69 4.3 Experiments Involving Wastewater 74 4.4 Mechanism of Toxicity - Inhibitors 74 4.4.1 Introduction 74 4.4.2 Chloramphenicol 79 4.4.3 Cyanide 79 4.4.4 DCCD 80 4.4.5 2,4-DNP 80 4.4.6 Summary 81 4.5 Mechanism of Toxicity - Chlorinated Phenols 81 4.2.1 2-Chlorophenol 81 4.2.2 2,4-Dichlorophenol 84 4.2.3 2,4,6-Trichlorophenol 84 4.2.4 Pentachlorophenol 84 4.2.5 Summary of Phenolic Compounds 88 4.6 Mechanism of Toxicity - Chlorinated Ethanes 89 6 TABLE OF CONTENTS - Continued 4.7 Mechanism of Toxicity - Metals 89 4.8 Summary of Pure Compounds 94 4.9 Mechanism of Toxicity - Wastewater 98 4.10 Reproducibility 101 Chapter 5 - Discussion 5.1 Toxicity Assessment 104 5.1.1 Introduction 104 5.1.2 Importance of Undissociated Concentration 104 5.1.3 Toxicity - Pure Compounds 106 5.1.4 Toxicity - Wastewater 108 5.2 Octanol/Water Partition Coefficients 108 5.3 Correlations 110 5.3.1 Kow vs Undissociated Concentrations 110 5.3.2 Kow vs Toxicity 112 5.3.3 Tester Organism Sensitivity 112 5.3.4 Quantitative Structure-Activity Relationships (QSAR's) 115 5.3.5 S. aurantia Toxicity Data vs Other Bioassay Toxicity Data 117 Chapter 6 - Conclusions 125 6.1 Bioassay Procedure 125 6.2 Toxicants 125 6.3 Toxicity Assessment 127 APPENDIX A 129 APPENDIX B 142 REFERENCES CITED 147 7 LIST OF FIGURES Figure Page 2.1 Schematic diagram of overall microbial metabolic pathway 32 2.2 Glucose Translocation 34 2.3 Embden-Meyerhof-Parnas Pathway 35 2.4 The Tricarboxylic Acid (TCA) Cycle 36 2.5 Components of the Bacterial Respiratory Chain 38 2.6 Proton and electron movement along the Electron Transport Chain in E. cou 40 2.7 Active sites of inhibition for Chloramphenicol, Cyanide, DCCD and 2,4-DNP 42 3.1 Flow-chart of experimental procedures 49 3.2 Air-tight stoppers used to seal flasks when testing volatile compounds 51 3.3 Flow-chart of ATP analysis experimental procedure. 59 3.4 02 utilization experimental procedure 61 4.1 Luminometer ATP calibration curve 65 4.2 Wastewater ATP calibration curve, Abs = 0.234 66 4.3 Wastewater ATP calibration curve, Abs = 1.477 67 4.4 Specific inhibitor's effect on the growth of S. aurantia in time dependent plots. Absorbance was measured at A = 600 cm, 1 cm pathlength 70 4.5 Specific inhibitor's effect on ATP production of S. aurantia in time dependent plots 71 4.6 Specific inhibitor's effect on 02 utilization rate of S. aurantia in time dependent plots 72 4.7 S. aurantia' s response to Chloramphenicol as measured by specific growth rate, intracellular ATP concentration and 02 utilization rate in concentration dependent plots 75 8 LIST OF FIGURES - Continued 4.8 S. aurantia'S response to Cyanide as measured by specific growth rate, intracellular ATP concentration and 0 2 utilization rate in concentration dependent plots 76 4.9 S. aurantia'S response to DCCD as measured by specific growth rate, intracellular ATP concentration and 0 2 utilization rate in concentration dependent plots 77 4.10 S. aurantia' S response to 2,4-DNP as measured by specific growth rate, intracellular ATP concentration and 02 utilization rate in concentration dependent plots 78 4.11 S. aurantia'S response to 2-Chlorphenol as measured by specific growth rate, intracellular ATP concentration and 02 utilization rate in concentration dependent plots 83 4.12 S. aurantia'S response to 2,4-Dichlorophenol as measured by specific growth rate, intracellular ATP concentration and 0 2 utilization rate in concentration dependent plots 85 4 13 S. aurantia'S response to 2,4,6-Trichlorophenol as measured by specific growth rate, intracellular ATP concentration and 02 utilization rate in concentration dependent plots 86 4.14 S. aurantia'S response to Pentachlorophenol as measured by specific growth rate, intracellular ATP concentration and 0 2 utilization rate in concentration dependent plots 87 4.15 S. aurantia'S response to 1,2-Dichloroethane as measured by specific growth rate, intracellular ATP concentration and 0 2 utilization rate in concentration dependent plots 90 4.16 S. aurantia'S response to 1,1,1-Trichloroethane as measured by specific growth rate, intracellular ATP concentration and 02 utilization rate in concentration dependent plots 91 9 LIST OF FIGURES - Continued 4.17 S.
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