IODINATED X-RAY CONTRAST MEDIA (ICM) AS PRECURSORS TO
DISINFECTION BYPRODUCT (DBP) FORMATION AND ENHANCED TOXICITY
AS A FUNCTION OF pH AND CHLORINATED OXIDANTS
A Thesis
Presented to
The Graduate Faculty of The University of Akron
In Partial Fulfilment
Of the Requirements for the Degree
Master of Science
Alexis Helen Killinger
May, 2016
IODINATED X-RAY CONTRAST MEDIA (ICM) AS PRECURSORS TO
DISINFECTION BYPRODUCT (DBP) FORMATION AND ENHANCED TOXICITY
AS A FUNCTION OF pH AND CHLORINATED OXIDANTS
Alexis Helen Killinger
Thesis
Approved: Accepted:
______Advisor Interim Dean of the College Dr. Stephen Duirk Dr. Eric J. Amis
______Faculty Reader Dean of the Graduate School Dr. Teresa Cutright Dr. Chand K. Midha
______Faculty Reader Date Dr. Lan Zhang
______Department Chair Dr. Wieslaw Binienda
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ABSTRACT
The objective of this study was to investigate the formation of iodinated
disinfection by-products (iodo-DBPs) and mammalian cell cyto/genotoxicity in the presence of iodinated x-ray contrast media (ICM), chlorinated oxidants, and natural organic matter (NOM). The ICM chosen as the source of iodide for these experiments were iopamidol and iohexol. The experiments were conducted with aqueous chlorine or monochloramine as the chlorinated oxidant. The experiments were conducted at pH 6.5 -
9.0. The NOM concentration was constant [DOC] = 5.57 mg/L. Chloroform formation in the presence of iopamidol, a regulated DBP, was highest at pH 9.0 with aqueous chlorine
(up to 4014 nM) and pH 6.5 with monochloramine (up to 56 nM). Additionally, dichloroiodomethane followed a similar trend in the presence of iopamidol with concentrations in the chlorine experiments forming up to 650 nM and the monochloramine experiments forming up to 52 nM. Trichloroacetic acid (TCAA) formation generally decreased as pH increased; however, iopamidol significantly contributes to TCAA formation at pH 7.5 disrupting the normally observed TCAA formation trend in the presence of NOM. Toxicity of the iopamidol transformation in the presence of chlorinated oxidants and NOM was also investigated. The cytotoxicity index
(CTI) values were highest for chlorine (i.e., 21.0) and monochloramine (i.e., 16.5) when compared to control experiments without iopamidol. Genotoxicity index (GTI) values were highest for chlorine (i.e., 7.1) and monochloramine (i.e., 3.5) when compared to control experiments. Iohexol DBP experiments resulted in an enhanced formation of iii chloroform, while very little dichloroiodomethane formed, and the TCAA formation observed was comparable to the chlorinated source water controls. With iohexol as the iodide source, the CTI and GIT values were highest with aqueous chlorine at 21.6 and
3.8, respectively, when compared to control experiments without iohexol present.
Iopamidol acts as a precursor to DBP formation, while also enhancing mammalian cell cytotoxicity and genotoxicity after chlorine or chloramine disinfection. While iohexol formed regulated and unregulated DBPs, it also enhanced mammalian cell cytotoxicity and genotoxicity, though to a lesser extent than iopamidol.
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ACKNOWLEDGEMENTS
I would like to express my sincere appreciations to my advisor, Dr. Stephen
Duirk, for his support, guidance, and advice throughout my master’s studies. I would also
like to thank Dr. Teresa Cutright for her teachings and direction over the past few years.
Thank you to my committee, as well, for their support and advice: Dr. Stephen Duirk, Dr.
Teresa Cutright, and Dr. Lan Zhang. Additionally, thank you to all of my laboratory
associates, and friends, for all of the lessons and collaboration during this research:
Elizabeth Crafton, Edward Machek, and Nana Ackerson. Finally, my deepest gratitude is felt for my parents, Dave and Cathy Killinger, my sister, Sydney Killinger, and my
boyfriend, Sean Edwards, for all of their unconditional love, support, and advice.
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TABLE OF CONTENTS
Page
LIST OF TABLES ...... ix
LIST OF FIGURES ...... xiv
CHAPTER
I INTRODUCTION ...... 1
1.1 Background ...... 1
1.2 Problem Statement ...... 4
1.3 Specific Objectives ...... 5
II LITERATURE REVIEW ...... 7
2.1 Iodinated X-Ray Contrast Media ...... 7
2.2 Occurrence of ICM in Water and Wastewater ...... 8
2.3 Disinfection and Disinfection By-Products (DBPs)...... 10
2.3.1 Chemical Oxidants Used in Drinking Water Treatment ...... 10
2.3.2 Disinfection By-Product (DBP) Formation with Natural Organic Matter (NOM) ...... 11
2.3.3 Aqueous Chlorine Disinfection and DBP Formation with Micropollutants .... 13
2.3.4 Chloramine Disinfection and DBP Formation ...... 15
2.4 ICM Transformation and Iodo-DBP Formation...... 17
2.4.1 ICM Transformation ...... 17
2.4.2 Microbial Transformation of ICM ...... 17
2.4.3 Chemical Transformation of ICM ...... 18
vi
2.5 Toxicity of DBPs ...... 20
2.5.1 TOX Toxicity ...... 22
III MATERIALS AND METHODS ...... 25
3.1 Chemicals and Reagents...... 25
3.2 Source Water Characterization...... 26
3.3 Experimental Methods ...... 31
3.3.1 Disinfection By-Product Experiments with Source Water ...... 31
3.3.2 Cytotoxicity and Genotoxicity Experiments with Source Water ...... 34
3.3.3 Disinfection By-Product Analytical Methods ...... 37
3.4 Analysis of DBPs ...... 39
IV RESULTS AND DISCUSSION ...... 56
4.1 Introduction ...... 56
4.2 DBP Formation in the Presence of ICM and Chlorinated Oxidants ...... 57
4.2.1 DBP Formation in the presence of Aqueous Chlorine and Iopamidol ...... 57
4.2.2 Akron Kinetics Experiments in the presence of Aqueous Chlorine and Iopamidol ...... 63
4.2.3 DBP Formation in the presence of Monochloramine and Iopamidol ...... 69
4.2.4 Akron Kinetics Experiments in the presence of Monochloramine and Iopamidol ...... 74
4.2.5 DBP Formation in the presence of Aqueous Chlorine and Iohexol ...... 79
4.3 Chronic Cytotoxicity and Acute Genotoxicity Analysis for ICM in the Presence of Chlorinated Oxidants and Akron Source Water ...... 83
4.3.1 Cyto/Genotoxicity of Iopamidol in the presence of Chlorinated Oxidants ...... 84
4.3.2 Cyto/Genotoxicity of Iohexol in the presence of Aqueous Chlorine ...... 95
V CONCLUSIONS AND RECOMMENDATIONS ...... 104
5.1 Introduction ...... 104
vii
5.2 Conclusions ...... 104
5.3 Recommendations ...... 106
BIBLIOGRAPHY ...... 107
APPENDIX ...... 124
viii
LIST OF TABLES
Table Page
3.1: Source water characteristics for Akron water ...... 27
3.2: Fluorescence EEM regions proposed by Chen et al. (2003)...... 29
3.3: Fluorescence regions for Akron source water for 1 mg/L C ...... 29
3.4: Aqueous conditions for reactors used for toxicity experiments ...... 35
3.5: Oven temperature programming for THMs and HANs analysis on GC/μECD ...... 40
3.6: Oven temperature programming for HAAs analysis on GC/μECD ...... 40
3.7: Limit of quantification for the detection of DBPs ...... 55
A.1: Observed DBP formation at 72 hours as a function of pH, [NOM] = 5.57 mg/L-C, [iopamidol] = 5.0 μM, [Cl2]T = 100 μM, [Buffer] = 4 mM, temperature = 25 °C, ...... 124
A.2: Observed DBP formation at 72 hours as a function of pH, [NOM] = 5.57 mg/L-C, [ICM] = 0.0 μM, [Cl2]T = 100 μM, [Buffer] = 4 mM, temperature = 25 °C,...... 125
A.3: Observed DBP formation at 72 hours as a function of pH, [NOM] = 5.57 mg/L-C, [iopamidol] = 5.0 μM, [NH2Cl]T = 100 μM, [Buffer] = 4 mM, temperature = 25 °C ... 129
A.4: Observed DBP formation at 72 hours as a function of pH, [NOM] = 5.57 mg/L-C, [iopamidol] = 0.0 μM, [NH2Cl]T = 100 μM, [Buffer] = 4 mM, temperature = 25 °C ... 130
A.5: Observed CHO Cell Chronic Cytotoxicity as a function of Mean Cell Density for sample AOHTOX-SW. Concentration LC50 value = 161.3; pH = 7.5, [NOM] = 5.57 mg/L-C, [ICM] = 0.0 μM, [Cl2]T = 0 μM, [Buffer] = 4 mM, temperature = 25 °C ...... 134
A.6: ANOVA Test Cytotoxicity Statistical Analysis for AOHTOX-SW...... 134
A.7: Cytotoxicity Statistical Analysis Multiple Comparisons versus Control Group for AOHTOX-SW using Holm-Sidak Method. Overall significance level = 0.05 ...... 134
A.8: Observed CHO Cell Chronic Cytotoxicity as a function of Mean Cell Density for sample AOHTOX-CL. Concentration LC50 value = 53.0; pH = 7.5, [NOM] = 5.57 mg/L- C, [ICM] = 0.0 μM, [Cl2]T = 100 μM, [Buffer] = 4 mM, temperature = 25 °C ...... 135
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A.9: ANOVA Test Cytotoxicity Statistical Analysis for AOHTOX-CL ...... 135
A.10: Cytotoxicity Statistical Analysis Multiple Comparisons versus Control Group for AOHTOX-CL using Holm-Sidak Method. Overall significance level = 0.05 ...... 135
A.11: Observed CHO Cell Chronic Cytotoxicity as a function of Mean Cell Density for sample AOHTOX-IDOL. Concentration LC50 value = 123.7; pH = 7.5, [NOM] = 5.57 mg/L-C, [iopamidol] = 5.0 μM, [Cl2]T = 0 μM, [Buffer] = 4 mM, temperature = 25 °C ...... 136
A.12: ANOVA Test Cytotoxicity Statistical Analysis for AOHTOX-IDOL ...... 136
A.13: Cytotoxicity Statistical Analysis Multiple Comparisons versus Control Group for AOHTOX-IDOL using Holm-Sidak Method. Overall significance level = 0.05 ...... 136
A.14: Observed CHO Cell Chronic Cytotoxicity as a function of Mean Cell Density for sample AOHTOX-CL-IDOL. Concentration LC50 value = 47.6; pH = 7.5, [NOM] = 5.57 mg/L-C, [iopamidol] = 5.0 μM, [Cl2]T = 100 μM, [Buffer] = 4 mM, temperature = 25 °C ...... 137
A.15: ANOVA Test Cytotoxicity Statistical Analysis for AOHTOX-CL-IDOL...... 137
A.16: Cytotoxicity Statistical Analysis Multiple Comparisons versus Control Group for AOHTOX-CL-IDOL using Holm-Sidak Method. Overall significance level = 0.05 .... 137
A.17: Comparative CHO cell chronic cytotoxicity of X-ray contrast agent iopamidol in Akron OH source water samples disinfected with and without Cl and NH2Cl ...... 138
A.18: Observed CHO Cell Chronic Cytotoxicity as a function of Mean Cell Density for sample AOHTOX-NH2CL. Concentration LC50 value = 129.3; pH = 7.5, [NOM] = 5.57 mg/L-C, [iopamidol] = 0.0 μM, [NH2Cl]T = 100 μM, [Buffer] = 4 mM, temperature = 25 °C ...... 143
A.19: ANOVA Test Cytotoxicity Statistical Analysis for AOHTOX- NH2CL ...... 143
A.20: Cytotoxicity Statistical Analysis Multiple Comparisons versus Control Group for AOHTOX- NH2CL using Holm-Sidak Method. Overall significance level = 0.05 ...... 143
A.21: Observed CHO Cell Chronic Cytotoxicity as a function of Mean Cell Density for sample AOHTOX-NH2CL-IDOL. Concentration LC50 value = 60.3; pH = 7.5, [NOM] = 5.57 mg/L-C, [iopamidol] = 5.0 μM, [NH2Cl]T = 100 μM, [Buffer] = 4 mM, temperature = 25 °C ...... 144
A.22: ANOVA Test Cytotoxicity Statistical Analysis for AOHTOX- NH2CL-IDOL .. 144
A.23: Cytotoxicity Statistical Analysis Multiple Comparisons versus Control Group for AOHTOX- NH2CL-IDOL using Holm-Sidak Method. Overall significance level = 0.05 ...... 144
x
A.24: Observed CHO Cell Chronic Genotoxicity as a function of Genomic DNA Damage for sample AOHTOX-SW. Concentration factor SCGE 50% Tail DNA value = 680.0; pH = 7.5, [NOM] = 5.57 mg/L-C, [iopamidol] = 0.0 μM, [Cl2]T = 0.0 μM, [Buffer] = 4 mM, temperature = 25 °C ...... 147
A.25: ANOVA Test Genotoxicity Statistical Analysis for AOHTOX- SW ...... 147
A.26: Genotoxicity Statistical Analysis Multiple Comparisons versus Control Group for AOHTOX- SW using Holm-Sidak Method. Overall significance level = 0.05 ...... 148
A.27: Observed CHO Cell Chronic Genotoxicity as a function of Genomic DNA Damage for sample AOHTOX-CL. Concentration factor SCGE 50% Tail DNA value = 244.3; pH = 7.5, [NOM] = 5.57 mg/L-C, [iopamidol] = 0.0 μM, [Cl2]T = 100.0 μM, [Buffer] = 4 mM, temperature = 25 °C ...... 149
A.28: ANOVA Test Genotoxicity Statistical Analysis for AOHTOX- CL...... 149
A.29: Genotoxicity Statistical Analysis Multiple Comparisons versus Control Group for AOHTOX-CL using Holm-Sidak Method. Overall significance level = 0.05 ...... 149
A.30: Observed CHO Cell Chronic Genotoxicity as a function of Genomic DNA Damage for sample AOHTOX-IDOL. Concentration factor SCGE 50% Tail DNA value = 722.9; pH = 7.5, [NOM] = 5.57 mg/L-C, [iopamidol] = 5.0 μM, [Cl2]T = 0.0 μM, [Buffer] = 4 mM, temperature = 25 °C ...... 150
A.31: ANOVA Test Genotoxicity Statistical Analysis for AOHTOX-IDOL ...... 150
A.32: Genotoxicity Statistical Analysis Multiple Comparisons versus Control Group for AOHTOX-IDOL using Holm-Sidak Method. Overall significance level = 0.05 ...... 150
A.33: Observed CHO Cell Chronic Genotoxicity as a function of Genomic DNA Damage for sample AOHTOX-CL-IDOL. Concentration factor SCGE 50% Tail DNA value = 140.1; pH = 7.5, [NOM] = 5.57 mg/L-C, [iopamidol] = 5.0 μM, [Cl2]T = 100.0 μM, [Buffer] = 4 mM, temperature = 25 °C ...... 151
A.34: ANOVA Test Genotoxicity Statistical Analysis for AOHTOX-CL-IDOL ...... 151
A.35: Genotoxicity Statistical Analysis Multiple Comparisons versus Control Group for AOHTOX-CL-IDOL using Holm-Sidak Method. Overall significance level = 0.05 .... 151
A.36: Comparative CHO cell chronic genotoxicity of X-ray contrast agent iopamidol in Akron OH water samples disinfected with and without Cl and NH2Cl ...... 152
A.37: Observed CHO Cell Chronic Cytotoxicity as a function of Mean Cell Density for sample AOHTOX-NH2CL. Concentration LC50 value = 161.3; pH = 7.5, [NOM] = 5.57 mg/L-C, [iopamidol] = 0.0 μM, [NH2Cl]T = 100.0 μM, [Buffer] = 4 mM, temperature = 25 °C ...... 157
xi
A.38: ANOVA Test Genotoxicity Statistical Analysis for AOHTOX-NH2CL ...... 157
A.39: Genotoxicity Statistical Analysis Multiple Comparisons versus Control Group for AOHTOX-NHCL using Holm-Sidak Method. Overall significance level = 0.05 ...... 158
A.40: Observed CHO Cell Chronic Cytotoxicity as a function of Mean Cell Density for sample AOHTOX-NH2CL-IDOL. Concentration LC50 value = 287.9; pH = 7.5, [NOM] = 5.57 mg/L-C, [iopamidol] = 5.0 μM, [NH2Cl]T = 100.0 μM, [Buffer] = 4 mM, temperature = 25 °C ...... 159
A.41: ANOVA Genotoxicity Statistical Analysis for AOHTOX-NH2CL-IDOL ...... 159
A.42: Genotoxicity Statistical Analysis Multiple Comparisons vs. Control Group AOHTOX-NHCL-IDOL using Holm-Sidak Method. Overall significance level = 0.05 ...... 159
A.43: Observed DBP formation at 72 hours as a function of pH, [NOM] = 5.57 mg/L-C, [iohexol] = 5.0 μM, [Cl2]T = 100 μM, [Buffer] = 4 mM, temperature = 25 °C ...... 162
A.44: Observed DBP formation at 72 hours as a function of pH, [NOM] = 5.57 mg/L-C, [ICM] = 0.0 μM, [Cl2]T = 100 μM, [Buffer] = 4 mM, temperature = 25 °C...... 163
A.45: Observed CHO Cell Chronic Cytotoxicity as a function of Mean Cell Density for sample AOHTOX-SW. Concentration LC50 value = 107.1; pH = 7.5, [NOM] = 5.57 mg/L-C, [iohexol] = 0.0 μM, [Cl2]T = 0 μM, [Buffer] = 4 mM, temperature = 25 °C .. 164
A.46: ANOVA Test Cytotoxicity Statistical Analysis for AOHTOX-SW...... 164
A.47: Cytotoxicity Statistical Analysis Multiple Comparisons versus Control Group for AOHTOX-SW using Holm-Sidak Method. Overall significance level = 0.05 ...... 165
A.48: Observed CHO Cell Chronic Cytotoxicity as a function of Mean Cell Density for sample AOHTOX-CL. Concentration LC50 value = 55.9; pH = 7.5, [NOM] = 5.57 mg/L- C, [iohexol] = 0.0 μM, [Cl2]T = 100.0 μM, [Buffer] = 4 mM, temp. = 25 °C...... 166
A.49: ANOVA Test Cytotoxicity Statistical Analysis for AOHTOX-CL ...... 166
A.50: Cytotoxicity Statistical Analysis Multiple Comparisons versus Control Group for AOHTOX-CL using Holm-Sidak Method. Overall significance level = 0.05 ...... 166
A.51: Observed CHO Cell Chronic Cytotoxicity as a function of Mean Cell Density for sample AOHTOX-CL-IHXL. Concentration LC50 value = 45.5; pH = 7.5, [NOM] = 5.57 mg/L-C, [iohexol] = 5.0 μM, [Cl2]T = 100.0 μM, [Buffer] = 4 mM, temperature = 25 °C ...... 166
A.52: ANOVA Test Cytotoxicity Statistical Analysis for AOHTOX-CL-IHXL...... 167
xii
A.53: Cytotoxicity Statistical Analysis Multiple Comparisons vs. Control Group for AOHTOX-CL-IHXL using Holm-Sidak Method. Overall significance level = 0.05 .... 168
A.54: Comparative CHO cell chronic cytotoxicity of X-ray contrast agent iohexol in Akron OH water samples with and without Cl2 disinfection ...... 168
A.55: Observed CHO Cell Chronic Genotoxicity as a function of Genomic DNA Damage for sample AOHTOX-SW. Concentration factor SCGE 50% Tail DNA value = 400.4; pH = 7.5, [NOM] = 5.57 mg/L-C, [iohexol] = 0.0 μM, [Cl2]T = 0.0 μM, [Buffer] = 4 mM, temperature = 25 °C ...... 172
A.56: ANOVA Test Genotoxicity Statistical Analysis for AOHTOX- SW ...... 172
A.57: Genotoxicity Statistical Analysis Multiple Comparisons versus Control Group for AOHTOX-SW using Holm-Sidak Method. Overall significance level = 0.05 ...... 172
A.58: Observed CHO Cell Chronic Genotoxicity as a function of Genomic DNA Damage for sample AOHTOX-CL. Concentration factor SCGE 50% Tail DNA value = 362.4; pH = 7.5, [NOM] = 5.57 mg/L-C, [iohexol] = 0.0 μM, [Cl2]T = 100.0 μM, [Buffer] = 4 mM, temperature = 25 °C ...... 173
A.59: ANOVA Test Genotoxicity Statistical Analysis for AOHTOX- CL...... 173
A.60: Genotoxicity Statistical Analysis Multiple Comparisons versus Control Group for AOHTOX-CL using Holm-Sidak Method. Overall significance level = 0.05 ...... 173
A.61: Observed CHO Cell Chronic Genotoxicity as a function of Genomic DNA Damage for sample AOHTOX-CL-IXHL. Concentration factor SCGE 50% Tail DNA value = 258.4; pH = 7.5, [NOM] = 5.57 mg/L-C, [iohexol] = 5.0 μM, [Cl2]T = 100.0 μM, [Buffer] = 4 mM, temperature = 25 °C ...... 174
A.62: ANOVA Test Genotoxicity Statistical Analysis for AOHTOX- CL-IHXL ...... 174
A.63: Genotoxicity Statistical Analysis Multiple Comparisons versus Control Group for AOHTOX-CL-IHXL using Holm-Sidak Method. Overall significance level = 0.05 .... 174
A.64: Comparative CHO cell chronic genotoxicity of X-ray contrast agent iohexol in Akron OH water samples with and without Cl2 disinfection ...... 175
xiii
LIST OF FIGURES
Figure Page
2.1: The chemical structures of ICM commonly used within the medical field ...... 8
2.2: Iodo-DBP formation pathway due to iopamidol reacting with chlorinated oxidants in the presence of NOM (Duirk et al., 2011) ...... 20
3.1: Fluorescence excitation-emission spectrum of Akron source water. [DOC] = 5.57 mg/L, SUVA254 = 2.27 L/mg-m ...... 30
3.2: Calibration curve for CHCl3using chloroform. [CHCl3] = 0 – 1000 nM ...... 42
3.3: Calibration curve for CHCl2Br using bromodichloromethane. [CHCl2Br] = 0 – 400 nM ...... 42
3.4: Calibration curve for CHBr2Cl using dibromochloromethane. [CHBr2Cl] = 0 – 300 nM ...... 43
3.5: Calibration curve for CHCl2I using dichloroiodomethane. [CHCl2I] = 0 – 500 nM 43
3.6: Calibration curve for CHBrClI using bromochloroiodomethane. [CHBrClI] = 0 – 250 nM ...... 44
3.7: Calibration curve for CHBr3 using bromoform. [CHBr3] = 0 – 500 nM ...... 44
3.8: Calibration curve for CHBr2I using dibromoiodomethane. [CHBr2I] = 0 – 250 nM 45
3.9: Calibration curve for CHClI2 using chlorodiiodomethane. [CHClI2] = 0 – 250 nM 45
3.10: Calibration curve for CHBrI2 using bromodiiodomethane. [CHBrI2] = 0 – 125 nM ...... 46
3.11: Calibration curve for CHI3 using iodoform. [CHI3] = 0 – 50 nM ...... 46
3.12: Calibration curve for CAN using chloroacetonitrile. [CAN] = 0 – 500 nM ...... 47
3.13: Calibration curve for TCAN using trichloroacetonitrile. [TCAN] = 0 – 125 nM ... 47
3.14: Calibration curve for DCAN using dichloroacetonitrile. [DCAN] = 0 – 500 nM ... 48
xiv
3.15: Calibration curve for BAN using bromoacetonitrile. [BAN] = 0 – 125 nM ...... 48
3.16: Calibration curve for BCAN using bromochloroacetonitrile. [BCAN]=0–250 nM 49
3.17: Calibration curve for DBAN using dibromoacetonitrile. [DBAN] = 0 – 250 nM... 49
3.18: Calibration curve for IAN using iodoacetonitrile. [IAN] = 0 – 31 nM ...... 50
3.19: Calibration curve for CAA using chloroacetic acid. [CAA] = 0 – 250 nM ...... 50
3.20: Calibration curve for BAA using bromoacetic acid. [BAA] = 0 – 1000 nM ...... 51
3.21: Calibration curve for DCAA using dichloroacetic acid. [DCAA] = 0 – 500 nM .... 51
3.22: Calibration curve for TCAA using trichloroacetic acid. [TCAA] = 0 – 250 nM .... 52
3.23: Calibration curve for IAA using iodoacetic acid. [IAA] = 0 – 125 nM ...... 52
3.24: Calibration curve for BCAA using bromochloroacetic acid. [BCAA]=0–250 nM . 53
3.25: Calibration curve for BDCAA using bromodichloroacetic acid. [BDCAA] = 0 –250 nM ...... 53
3.26: Calibration curve for DBAA using dibromoacetic acid. [DBAA] = 0 – 500 nM.... 54
4.1: Observed chloroform formation at 72 hours as a function of pH. [NOM] = 5.57 mg/L-C, [iopamidol] = 5.0 μM, [Cl2]T = 100 μM, [Buffer] = 4 mM, temperature = 25°C ...... 58
4.2: Observed dichloroiodomethane formation at 72 hours as a function of pH, [NOM] = 5.57 mg/L-C, [iopamidol] = 5.0 μM, [Cl2]T = 100 μM, [Buffer] = 4 mM, temperature = 25°C ...... 60
4.3: Observed trichloroacetic acid formation at 72 hours as a function of pH, [NOM] = 5.57 mg/L-C, [iopamidol] = 5.0 μM, [Cl2]T = 100 μM, [Buffer] = 4 mM, temperature = 25°C ...... 62
4.4: Observed DBP formation at pH 6.5 as a function of time. [NOM] = 5.57 mg/L-C, [iopamidol] = 2.5 μM, [Cl2]T = 100 μM, [Buffer] = 4 mM, temperature = 25°C ...... 65
4.5: Observed DBP formation at pH 7.5 as a function of time, [NOM] = 5.57 mg/L-C, [iopamidol] = 2.5 μM, [Cl2]T = 100 μM, [Buffer] = 4 mM, temperature = 25°C ...... 66
4.6: Observed DBP formation at pH 8.5 as a function of time, [NOM] = 5.57 mg/L-C, [iopamidol] = 2.5 μM, [Cl2]T = 100 μM, [Buffer] = 4 mM, temperature = 25°C ...... 68
4.7: Observed DBP formation at pH 9.0 as a function of time, [NOM] = 5.57 mg/L-C, [iopamidol] = 2.5 μM, [Cl2]T = 100 μM, [Buffer] = 4 mM, temperature = 25°C ...... 69
xv
4.8: Observed chloroform formation at 72 hours as a function of pH, [NOM] = 5.57 mg/L-C, [iopamidol] = 5.0 μM, [NH2Cl]T = 100 μM, [Buffer] = 4 mM, temperature = 25°C ...... 71
4.9: Observed dichloroiodomethane formation at 72 hours as a function of pH, [NOM] = 5.57 mg/L-C, [iopamidol] = 5.0 μM, [NH2Cl]T = 100 μM, [Buffer] = 4 mM, temperature = 25°C ...... 72
4.10: Observed trichloroacetic acid formation at 72 hours as a function of pH, [NOM] = 5.57 mg/L-C, [iopamidol] = 5.0 μM, [NH2Cl]T = 100 μM, [Buffer] = 4 mM, temperature = 25°C ...... 73
4.11: Observed DBP formation at pH 6.5 as a function of time, [NOM] = 5.57 mg/L-C, [iopamidol] = 2.5 μM, [NH2Cl]T = 100 μM, [Buffer] = 4 mM, temperature = 25°C ...... 76
4.12: Observed DBP formation at pH 7.5 as a function of time, [NOM] = 5.57 mg/L-C, [iopamidol] = 2.5 μM, [NH2Cl]T = 100 μM, [Buffer] = 4 mM, temperature = 25°C ...... 77
4.13: Observed DBP formation at pH 8.5 as a function of time, [NOM] = 5.57 mg/L-C, [iopamidol] = 2.5 μM, [NH2Cl]T = 100 μM, [Buffer] = 4 mM, temperature = 25°C ...... 78
4.14: Observed DBP formation at pH 9.0 as a function of time, [NOM] = 5.57 mg/L-C, [iopamidol] = 2.5 μM, [NH2Cl]T = 100 μM, [Buffer] = 4 mM, temperature = 25°C ...... 79
4.15: Observed chloroform formation at 72 hours as a function of pH, [NOM] = 5.57 mg/L-C, [iohexol] = 5.0 μM, [Cl2]T = 100 μM, [Buffer] = 4 mM, temperature = 25°C . 81
4.16: Observed dichloroiodomethane formation at 72 hours as a function of pH, [NOM] = 5.57 mg/L-C, [iohexol] = 5.0 μM, [Cl2]T = 100 μM, [Buffer] = 4 mM, temperature = 25°C ...... 82
4.17: Observed trichloroacetic acid formation at 72 hours as a function of pH, [NOM] = 5.57 mg/L-C, [iohexol] = 5.0 μM, [Cl2]T = 100 μM, [Buffer] = 4 mM, temperature = 25°C ...... 83
4.18: Comparison of observed CHO Cell Chronic Cytotoxicity as a function of Mean Cell Density for samples AOHTOX-SW, AOHTOX-CL, and AOHTOX-CL-IDOL...... 86
4.19: Comparison of observed CHO Cell Chronic Cytotoxicity as a function of Mean Cell Density for samples AOHTOX-SW, AOHTOX-NH2CL, and AOHTOX-NH2CL-IDOL...... 88
-1 3 4.20: All CHO Cytotoxicity Index Values calculated as (LC50) x 10 ...... 89
4.21: Comparison of observed CHO Cell Acute Genotoxicity as a function of Genomic DNA Damage for samples AOHTOX-SW, AOHTOX-CL, and AOHTOX-CL-IDOL. . 91
xvi
4.22: Comparison of observed CHO Cell Acute Genotoxicity as a function of Genomic DNA Damage for samples AOHTOX-SW, AOHTOX-NH2CL, and AOHTOX-NH2CL- IDOL...... 93
4.23: All CHO Genotoxicity Index Values calculated as (50% Tail DNA)-1 x 103...... 94
4.24: Comparison of observed CHO Cell Chronic Cytotoxicity as a function of Mean Cell Density for samples AOHTOX-SW, AOHTOX-CL, and AOHTOX-CL-IHXL...... 97
-1 3 4.25: All CHO Cytotoxicity Index Values calculated as (LC50) x 10 ...... 98
4.26: Comparison of observed CHO Cell Acute Genotoxicity as a function of Genomic DNA Damage for samples AOHTOX-SW, AOHTOX-CL, and AOHTOX-CL-IHXL. 101
4.27: All CHO Genotoxicity Index Values calculated as (50% Tail DNA)-1 x 103...... 102
A.1: Observed chloroform formation at pH 6.5, 7.5, 8.5 and 9.0 as a function of time, [NOM] = 5.57 mg/L-C, [iopamidol] = 2.5 μM, [Cl2]T = 100 μM, [Buffer] = 4 mM, temperature = 25°C ...... 126
A.2: Observed dichloroiodomethane formation at pH 6.5, 7.5, 8.5 and 9.0 as a function of time, [NOM] = 5.57 mg/L-C, [iopamidol] = 2.5 μM, [Cl2]T = 100 μM, [Buffer] = 4 mM, temperature = 25°C ...... 127
A.3: Observed trichloroacetic acid formation at pH 6.5, 7.5, 8.5 and 9.0 as a function of time, [NOM] = 5.57 mg/L-C, [iopamidol] = 2.5 μM, [Cl2]T = 100 μM, [Buffer] = 4 mM, temperature = 25°C ...... 128
A.4: Observed chloroform formation at pH 6.5, 7.5, 8.5 and 9.0 as a function of time, [NOM] = 5.57 mg/L-C, [iopamidol] = 2.5 μM, [NH2Cl]T = 100 μM, [Buffer] = 4 mM, temperature = 25°C ...... 131
A.5: Observed dichloroiodomethane formation at pH 6.5, 7.5, 8.5 and 9.0 as a function of time, [NOM] = 5.57 mg/L-C, [iopamidol] = 2.5 μM, [NH2Cl]T = 100 μM, [Buffer] = 4 mM, temperature = 25°C ...... 132
A.6: Observed TCAA formation at pH 6.5, 7.5, 8.5 and 9.0 as a function of time, [NOM] = 5.57 mg/L-C, [iopamidol] = 2.5 μM, [NH2Cl]T = 100 μM, [Buffer] = 4 mM, temperature = 25°C ...... 133
A.7: Observed CHO Cell Chronic Cytotoxicity as a function of Mean Cell Density for sample AOHTOX-SW. Concentration LC50 value = 161.3; pH = 7.5, [NOM] = 5.57 mg/L-C, [iopamidol] = 0.0 μM, [Cl2]T = 0.0 μM, [Buffer] = 4 mM, temperature = 25 °C ...... 139
A.8: Observed CHO Cell Chronic Cytotoxicity as a function of Mean Cell Density for sample AOHTOX-CL. Concentration LC50 value = 53.0; pH = 7.5, [NOM] = 5.57 mg/L- C, [iopamidol] = 0.0 μM, [Cl2]T = 100 μM, [Buffer] = 4 mM, temperature = 25 °C .... 140
xvii
A.9: Observed CHO Cell Chronic Cytotoxicity as a function of Mean Cell Density for sample AOHTOX-IDOL. Concentration LC50 value = 123.7; pH = 7.5, [NOM] = 5.57 mg/L-C, [iopamidol] = 5.0 μM, [Cl2]T = 0.0 μM, [Buffer] = 4 mM, temperature = 25 °C ...... 141
A.10: Observed CHO Cell Chronic Cytotoxicity as a function of Mean Cell Density for sample AOHTOX-CL-IDOL. Concentration LC50 value = 47.6; pH = 7.5, [NOM] = 5.57 mg/L-C, [iopamidol] = 5.0 μM, [Cl2]T = 100.0 μM, [Buffer] = 4 mM, temperature = 25 °C ...... 142
A.11: Observed CHO Cell Chronic Cytotoxicity as a function of Mean Cell Density for sample AOHTOX-NH2CL. Concentration LC50 value = 129.3; pH = 7.5, [NOM] = 5.57 mg/L-C, [iopamidol] = 0.0 μM, [NH2Cl]T = 100 μM, [Buffer] = 4 mM, temperature = 25 °C ...... 145
A.12: Observed CHO Cell Chronic Cytotoxicity as a function of Mean Cell Density for sample AOHTOX-NH2CL-IDOL. Concentration LC50 value = 60.3; pH = 7.5, [NOM] = 5.57 mg/L-C, [iopamidol] = 5.0 μM, [NH2Cl]T = 100 μM, [Buffer] = 4 mM, temperature = 25 °C ...... 146
A.13: Observed CHO Cell Acute Genotoxicity as a function of Genomic DNA Damage for sample AOHTOX-SW. Concentration factor SCGE 50% Tail DNA value = 680.0; pH = 7.5, [NOM] = 5.57 mg/L-C, [iopamidol] = 0.0 μM, [Cl2]T = 0.0 μM, [Buffer] = 4 mM, temperature = 25 °C ...... 153
A.14: Observed CHO Cell Acute Genotoxicity as a function of Genomic DNA Damage for sample AOHTOX-CL. Concentration factor SCGE 50% Tail DNA value = 244.3; pH = 7.5, [NOM] = 5.57 mg/L-C, [iopamidol] = 0.0 μM, [Cl2]T = 100.0 μM, [Buffer] = 4 mM, temperature = 25 °C ...... 154
A.15: Observed CHO Cell Acute Genotoxicity as a function of Genomic DNA Damage for sample AOHTOX-IDOL. Concentration factor SCGE 50% Tail DNA value = 722.9; pH = 7.5, [NOM] = 5.57 mg/L-C, [iopamidol] = 5.0 μM, [Cl2]T = 0.0 μM, [Buffer] = 4 mM, temperature = 25 °C ...... 155
A.16: Observed CHO Cell Acute Genotoxicity as a function of Genomic DNA Damage for sample AOHTOX-CL-IDOL. Concentration factor SCGE 50% Tail DNA value = 140.1; pH = 7.5, [NOM] = 5.57 mg/L-C, [iopamidol] = 5.0 μM, [Cl2]T = 100.0 μM, [Buffer] = 4 mM, temperature = 25 °C ...... 156
A.17: Observed CHO Cell Acute Genotoxicity as a function of Genomic DNA Damage for sample AOHTOX-NH2CL. Concentration factor SCGE 50% Tail DNA value = 565.5; pH = 7.5, [NOM] = 5.57 mg/L-C, [iopamidol] = 0.0 μM, [NH2Cl]T = 100.0 μM, [Buffer] = 4 mM, temperature = 25 °C ...... 160
A.18: Observed CHO Cell Acute Genotoxicity as a function of Genomic DNA Damage for sample AOHTOX-NH2CL-IDOL. Concentration factor SCGE 50% Tail DNA value
xviii
= 287.9; pH = 7.5, [NOM] = 5.57 mg/L-C, [iopamidol] = 5.0 μM, [NH2Cl]T = 100.0 μM, [Buffer] = 4 mM, temperature = 25 °C ...... 161
A.19: Observed CHO Cell Chronic Cytotoxicity as a function of Mean Cell Density for sample AOHTOX-SW. Concentration LC50 value = 107.1; pH = 7.5, [NOM] = 5.57 mg/L-C, [iohexol] = 0.0 μM, [Cl2]T = 0.0 μM, [Buffer] = 4 mM, temperature = 25 °C 169
A.20: Observed CHO Cell Chronic Cytotoxicity as a function of Mean Cell Density for sample AOHTOX-CL. Concentration LC50 value = 55.9; pH = 7.5, [NOM] = 5.57 mg/L- C, [iohexol] = 0.0 μM, [Cl2]T = 100.0 μM, [Buffer] = 4 mM, temperature = 25 °C ..... 170
A.21: Observed CHO Cell Chronic Cytotoxicity as a function of Mean Cell Density for sample AOHTOX-CL-IHXL. Concentration LC50 value = 45.5; pH = 7.5, [NOM] = 5.57 mg/L-C, [iohexol] = 5.0 μM, [Cl2]T = 100.0 μM, [Buffer] = 4 mM, temperature = 25 °C ...... 171
A.22: Observed CHO Cell Acute Genotoxicity as a function of Genomic DNA Damage for sample AOHTOX-SW. Concentration factor SCGE 50% Tail DNA value = 400.4; pH = 7.5, [NOM] = 5.57 mg/L-C, [iohexol] = 0.0 μM, [Cl2]T = 0.0 μM, [Buffer] = 4 mM, temperature = 25 °C ...... 176
A.23: Observed CHO Cell Acute Genotoxicity as a function of Genomic DNA Damage for sample AOHTOX-CL. Concentration factor SCGE 50% Tail DNA value = 362.4; pH = 7.5, [NOM] = 5.57 mg/L-C, [iohexol] = 0.0 μM, [Cl2]T = 100.0 μM, [Buffer] = 4 mM, temperature = 25 °C ...... 177
A.24: Observed CHO Cell Acute Genotoxicity as a function of Genomic DNA Damage for sample AOHTOX-CL-IHXL. Concentration factor SCGE 50% Tail DNA value = 258.4; pH = 7.5, [NOM] = 5.57 mg/L-C, [iohexol] = 5.0 μM, [Cl2]T = 100.0 μM, [Buffer] = 4 mM, temperature = 25 °C ...... 178
xix
CHAPTER I
INTRODUCTION
1.1 Background
Clean drinking water is a staple of modern society and a necessity for public health and safety. Often times, however, drinking water sources become contaminated with anthropogenic point and non-point sources. Chemical and biological contaminants in drinking water sources can cause undesirable aesthetic effects or threaten public health
(Post et al. 2011). Potable water requires certain levels of treatment to be safe for human consumption. The first water treatment procedures focused on removing elements of taste, odor, and turbidity. From the late 19th to early 20th century, disease causing microbes were targeted for removal to enhance drinking water quality (US EPA, 2000).
These microbial contaminants were removed through the addition of disinfectant
(Zweiner and Richardson, 2005). The United States has observed a significant decline in water borne illnesses, such as typhoid fever and cholera, throughout the employment of drinking water disinfection (McGuire, 2006).
Drinking water disinfection is now a widely accepted practice utilizing disinfectants such as chlorine, chlorine dioxide, chloramines, ozone, and ultraviolet radiation. These chemical oxidants can also be used to oxidize taste and odor compounds,
1
remove micropollutants, and improve surface water coagulation (Bruchet and Duguet,
2004; Hoff and Gelderich, 1981; Hoigne, 1998; Morris, 1986; von Gunten, 2003; Wolfe
et al., 1984). While disinfection has contributed largely to the substantial decrease of
water borne illnesses, they have also unfortunately lead to the formation of disinfection
by-products (DBPs) (Richardson, 1998). DBPs are chemical compounds formed as a result of the reaction between disinfectants and micropollutant or natural organic matter
(NOM) precursors (Krasner et al., 2006; Plewa et al., 2004; Richardson et al., 2002;
Simmons et al., 2002).
A specific class of DBPs, known as trihalomethanes (THMs), was discovered upon chlorination of treated drinking water in the 1970s (Bellar et al., 1974; Bryant et al.,
1992; Rook, 1974). Shortly after, the U.S. Environmental Protection Agency (US EPA)
passed the Safe Drinking Water Act (SDWA) in 1974 and the Total Trihalomethane
(TTHM) Rule in 1979 in response to a report detailing the formation of chloroform and
other haloforms from the reaction between NOM and chlorinated oxidants (Roberson,
2008). There are currently 4 regulated THMs with a maximum contamination level of 80
μg/L and 5 regulated HAAs with a maximum contamination level of 60 μg/L.
Additionally, two inorganic DBPs (chlorite and bromate) are also regulated (US EPA,
2005). There are currently over 600 known DBPs, of which some are significantly more toxic than their currently regulated counterparts (Richardson, 2011; Plewa et al., 2004;
Plewa et al., 2013; Plewa et al., 2015). Iodinated DBPs, such as iodoacetic acid, have been found to be far more toxic than their chlorinated or brominated counterparts (Plewa et al., 2004).
2
Iodo-DBPs are more likely to form in source waters containing the iodo-DBP
precursor, iodine. There are varying species of iodine that may be found in source waters,
each dependent on different mobility, bioavailability, and environmental chemical
behavior. The iodine species commonly encountered are iodide, iodate, and organo-
iodine (Gilfedder et al., 2007; Hansen et al., 2011). Organic and inorganic iodine species
exhibit different hydrophilic and biophilic properties (Hua et al., 2006). Iodide has been
found to naturally occur in seawater at an average concentration of 30 μg/L (Yokota et
al., 2004). Iodide has also been found to exist in freshwater and rain water (Gilfedder, et al., 2009; Schwehr and Santschi, 2003). Additionally, iodide has been identified to occur within U.S., Canadian, and European rivers at concentrations ranging from 0.5 μg/L to
212 μg/L (Moran et al., 2002).
Iodo-DBPs are able to form during drinking water disinfection when the iodide is
oxidized to hypoiodous acid (HOI) by the disinfectant (Krasner et al., 2006; Richardson
- et al., 2008). HOI is then oxidized further to form iodate (IO3 ) by chlorine, chloramines,
or ozone (Bichsel and von Gunten, 1999; Garland et al., 1980; Kumar et al., 1986; Nagy
et al., 1988). Additionally, HOI can also react with natural organic matter (NOM) to form
iodo-DBPs (Krasner et al., 2006; Richardson et al., 2008). Iodo-DBP formation is
expected in areas where iodide occurs naturally; however, iodo-DBPs have also,
remarkably, been detected in areas where iodide levels are below detection limits
(Richardson et al., 2008). Alternative sources of iodide may have been present in these
source waters; thus began the investigation into iodinated contrast media (ICM) as a
potential iodine source.
3
Iodinated x-ray contrast media (ICM) are triiodobenzoic acid pharmaceuticals
used for medical imaging of soft tissues, internal organs, and blood vessels. ICM are
large molecules with masses around 600 daltons and can be administered to humans in
doses up to 200 g per diagnostic session (Perez and Barcelo, 2007). ICMs are highly
hydrophilic, thus they pass through the human body un-metabolized and are excreted
through feces and urine within 24 hours (Perez and Barcelo, 2007; Steger-Hartmann et
al., 2000; Weissbrodt et al., 2009). Consequently, ICM have been detected at high
concentrations within domestic and clinical wastewaters, surface waters, groundwaters,
and drinking water sources (Busetti et al., 2008; Hirsch et al., 2000; Putschew et al.,
2006; Sacher et al., 2001; Schulz et al., 2008; Seitz et al., 2006b; Ternes et al., 2007;
Ternes and Hirsch, 2000). ICM are not removed during conventional wastewater treatment. Additionally, advanced oxidation and ozonation processes have also shown only partial transformation of ICM (Bahr et al., 2007; Putschew et al., 2006; Seitz et al.,
2006a; Ternes et al., 2003). Conversely, Activated carbon filtration has achieved ICM removal from medical wastewater (Carballa et al., 2007; Seitz et al., 2006a). However, most wastewater treatment processes have been found to be unable to remove ICM as well as other micropollutants resulting in source water contamination.
1.2 Problem Statement
ICMs are known to be a potential source of iodo-DBP formation within
chlorinated and chloraminated waters (Duirk et al., 2011). These compounds, coming mainly from medical wastes, pass through the wastewater treatment systems and enter into surrounding source waters. From the surrounding source waters, these ICMs then 4
pass through drinking water treatment systems and are transformed during disinfection.
The ICM compounds, specifically iopamidol, release iodide from their aromatic rings in the presence of chlorinated oxidants; the released iodide is then oxidized to HOI and reacts with NOM to form iodo-DBPs (Duirk et al., 2011). This report investigates DBP formation and toxicity analysis within Akron source water.
1.3 Specific Objectives
In this research, the following specific objectives were considered:
1. Investigate the formation of regulated DBPs and unregulated iodo-DBPs with
iopamidol as the iodide source, with respect to pH (6.5, 7.5, 8.5, and 9.0) in the
presence of Akron source water and chlorinated oxidants (aqueous chlorine and
monochloramine). Iopamidol concentrations were 5 μM for 72 hour experiments and
2.5 μM for kinetic experiments. Chlorinated oxidant concentrations were kept
constant at 100 μM initial concentrations. Source water control experiments were
performed at full NOM concentration for direct experimental comparisons in order to
analyze the ICM effects on both regulated and unregulated DBP formation.
2. Investigate the formation of regulated DBPs and unregulated iodo-DBPs with iohexol
as the iodide source, with respect to pH (6.5, 7.5, 8.5, and 9.0) in the presence of
Akron source water and chlorinated oxidants (aqueous chlorine and
monochloramine). Iohexol concentration was kept constant at 5 μM for 72 hour
experiments. Aqueous chlorine concentration was kept constant at 100 μM. Source
5
water control experiments were performed at full NOM concentration for direct
experimental comparisons in order to analyze the ICM effects on DBP formation.
3. Investigate the cytotoxicity and genotoxicity with both ICM, iopamidol and iohexol,
as varying sources of iodide in the presence of chlorinated oxidants (aqueous chlorine
and monochlormaine). ICM concentrations were kept constant at 5 μM. Chlorinated
oxidant concentrations were kept constant at 100 μM. Source water control
experiments were performed at full NOM concentration for direct experimental
comparisons in order to analyze the ICM effects on toxicity.
6
CHAPTER II
LITERATURE REVIEW
2.1 Iodinated X-Ray Contrast Media
Iodinated X-Ray Contrast Media (ICM) are a type of radiocontrast agent used to improve the visibility of soft tissue within the body during medical imaging procedures such as CT scans. ICM are introduced to the body intravenously in doses within the range of 100-200 grams for each examination (Perez et al., 2007, Speck et al., 1999). Due to the chemical and biological stability of ICM, ICM are excreted from the body through urine and feces after 24 hours (Perez et al., 2006; Steger-Hartmann et al., 2000). These compounds are commonly used within the medical field throughout the United States and across the world with documented annual compound consumption at 1.33 x 106 kg/year and 3.5 x 106 kg/year, respectively (Steger-Hartmann et al., 2000).
These ICM are derivatives of 2,4,6-triiodobenzoic acid and often have side chains of hydroxyl, carboxyl, and amide moieties (Figure 2.1) that increase the molecular solubility (Krause and Schneider, 2002; Seitz et al., 2006b). There are over 35 ICM currently in use and are categorized into four different groups: water soluble, nephrotropic, high osmolar; water soluble, nephrotropic, low osmolar; water soluble, hepatotropic; or non-water soluble (http://www.whocc.no, 2015). Iopamidol and Iohexol
7
are examples of water soluble, nephrotropic, and low osmolar compounds; whereas,
diatrizoate is an example of a water soluble, nephrotropic and high osmolar compound
(http://www.whocc.no, 2015).
Figure 2.1: The chemical structures of ICM commonly used within the medical field
2.2 Occurrence of ICM in Water and Wastewater
ICM have been found to be resistant to conventional water and wastewater treatment methods; as a result, numerous studies have been performed to determine treatment efficiencies and residual ICM concentrations. Within the medical field, the highly soluble ICM compounds have been detected in the effluent of medical imaging facilities (Ziegler et al., 1997; Gartiser et al., 1996). McArdell et al. (2010) performed a
8
separate study to investigate 69 common pharmaceutical agents expected to be present
within hospital wastewater; of the 69 compounds tested, 52 pharmaceutical agents were
detected with iopamidol present as the highest concentration in the mg/L range. McArdell
et al. (2010) then proceeded to treat the medical facility effluent to reduce ICM
concentrations through biologically activated membranes and powder activated carbon
(PAC). When treated with the membrane bioreactors, the results showed less than 20%
removal of pharmaceuticals including ICMs (i.e. iopamidol, diatrizoate, ioxitalamic acid,
iomeprol, and iopromide). When treated with the PAC, 70% removal efficiency was
achieved for ICMs yielding a residual iopamidol concentration of 900 μg/L. Low removal
efficiency of iopamidol can be attributed to ICMs high polarity (Steger-Hartmann et al.,
1999).
ICM have been detected within wastewater treatment plant effluent, and as a
result, the surrounding rivers and creeks, due to low ICM removal efficiency. The ICM
removal efficiency has been found to be as low as 10% during wastewater treatment
yielding a concentration greater than 1 μg/L within surrounding aqueous environments
(Drews et al., 2001; Ternes and Hirsch, 2000; Hirsch et al., 2000; Putschew et at., 2000).
Iopamidol, specifically, has also been detected at a median concentration of 0.49 μg/L within natural waters (Ternes and Hirsch, 2000; Putschew et al., 2001). Additionally, in a study performed by Duirk et al. (2011), source waters from 10 different cities were
examined for the presence of commonly used ICM; four ICM were detected in the form
of iopaimdol, iopromide, iohexol, and diatrizoate. Of these ICM detected, iopamidol was
the most frequently detected at concentrations up to 2700 ng/L in 6 of the 10 plants.
9
2.3 Disinfection and Disinfection By-Products (DBPs)
Chemical oxidation is a common practice to disinfect drinking water during the
treatment process. Disinfection by-products (DBPs) are unwanted and potentially toxic compounds that form as a result of the chemical oxidation process.
2.3.1 Chemical Oxidants Used in Drinking Water Treatment
The drinking water treatment process includes many physical and chemical unit operations to ensure that the final product is safe for human consumption, the last of which is disinfection. Chemical oxidants are used as disinfecting agents to deactivate pathogenic microorganisms and oxidize anthropogenic and naturally occuring organic micropollutants (Nriagu and Simmons, 1994). This process can oxidizes pollutants down to their terminal end products (CO2 and H2O) or to less toxic intermediate product
counterparts (Nriagu and Simmons, 1994). Chemical oxidation may also be utilized to
oxidize soluble metals to precipitate them out (Crittenden et al., 2012). The most
common oxidants used in drinking water treatment are chlorine, chlorine dioxide, ozone,
hydrogen peroxide, potassium permanganate and chloramines (mono-, di-, and tri-
chloramine ) (Crittenden et al., 2012; Nriagu and Simmons, 1994). Disinfection is a
necessary treatment process step; however, this must be monitored closely due to the
potential for chemical oxidants to form harmful DBPs (Krasner et al., 2006; Simmons et
al., 2002; Bichsel and von Guten, 2000).
10
2.3.2 Disinfection By-Product (DBP) Formation with Natural Organic Matter (NOM)
Chlorination of drinking water was first employed as a method to inactivate
pathogenic microorganisms for public safety, thus resulting in a large reduction of
waterborne illnesses (Akin et al., 1982). The formation and occurrence of DBPs was undetected until the 1970s when chloroform was first reported in chlorinated drinking water (Bryant et al., 1992; Beller et al., 1974; Rook, 1974). There are now more than 600 known DBPs that have been identified within chlorinated drinking water (Richardson et al., 2007). Nonetheless, a large portion of halogenated DBPs have yet to be identified
(Richardson et al., 2002; Weinberg, 1999).
Initially, DBPs were defined as the by-products of the reaction between natural organic matter (NOM) and oxidants during drinking water treatment. This definition, however, was reexamined due to documented reactions between the oxidants and anthropogenic contaminants (such as personal care products, pharmaceuticals, and pesticides) that also resulted in the formation of harmful DBPs (Duirk and Collette,
2006). DBPs are now considered to be any product formed from the reaction of disinfectant and any organic/inorganic material. DBP formation is influenced by many
factors such as: disinfectant type and concentration, pH, temperature, source water
characteristics, inorganic precursors, and treatment processes. Source water
characteristics that have been found to effect DBP formation are SUVA254 (discussed in
Chapter III), TOC concentration, solubule microbial products, regional fluorescence excitation-emission matrix (EEM) spectra assessment, and halogen concentraitons
(Krasner, 2009; Richardson et al., 2007; Sanchez et al., 2013; Ueno et al., 1996).
Utilizing source water characterizations and oxidant types, DBP research has developed 11
numerous models that are able to describe formation pathways (Amy et al., 1987; Clark et al., 2001; Cowman et al., 1996; Duirk et al., 2002; Duirk et al., 2005; von Guten et al.,
2003).
Currently, the US EPA regulates two groups of DBPs, trihalomethanes (THMs) and haloacetic acid (HAAs); together they total 9 individual DBP compound. The 4 US
EPA regulated THM compounds are chloroform, bromodichloromethane, dibromochloromethane, and bormoform (US EPA, 2015). The 5 US EPA regulated HAA compounds are chloroacetic acid, dichloroacetic acid, trichloroacetic acid, bromoacetic acid, and dibromoacetic acid (US EPA, 2015). Additionally, the US EPA regulates bromate and chlorite within the DBP category (US EPA, 2015). The US EPA has set the maximum contamination levels (MCL) of the aforementioned compounds at 80 μg/L for
THMS, 10 μg/L for HAAs, 10μg/L for bromate, and 1.0 mg/L for chlorite (US EPA,
2015). The most genotoxic and cytotoxic DBP, however, have yet to be officially regulated (Richardson et al., 2008, Plewa et al., 2002).
The oxidation of natural and anthropogenic micropollutants may result in final transformation products that are potentially more toxic than the original parent compounds (Duirk et al., 2011). These final transformation products may increase DBP concentrations that can be classified as regulated DBPs, known unregulated DBPs, or unknown unregulated DBPs. An example of DBPs that are now considered within the above definition are iodo-DBPs formed as a result of ICM degradation (Duirk et al.,
2011). Iodo-DBPs were originally considered to form from iodide present in source water; iodide oxidizes to HOI from HOCl, and the HOI is incorporated in organic matter
(Bischell and von Guten, 1999a; Duirk et al., 2011; Hansen et al., 2011; Gilfedder et al., 12
2009). Iodide (I-) has been detected in seawater at concentrations of 30 μg/L and can then
be present in freshwater through groundwater intrusion (Gilfedder et al., 2009; Schwehr
and Santschi, 2003; Yokota et al., 2004). Iodine (I-) has been found within the United
States, Canada, and European rivers at concentrations up to 212 μg/L (Moran et al.,
2002). Iodide within source water can oxidize by HOCl; however, other oxidants such as ozone and chloramines can also oxidize iodide to HOI (Garland et al., 1980; Nagy et al.,
- 1988; Kumar et al., 1986). Once formed, HOI can be oxidize to iodate (IO3 ) by chlorine or ozone (Bichsel and von Gunten, 1999a). Finally, HOI not consumed within the reaction can potentially incorporate with organic matter and form total organic iodide
(TOI) as well as iodo-DBPs (Krasner et al., 2006; Richardson et al., 2008).
2.3.3 Aqueous Chlorine Disinfection and DBP Formation with Micropollutants
Chlorine is a very widely used and affordable oxidant that can be employed in
varying forms such as a solid (Ca(OCl)2), liquid (NaOCl), or gas (Cl2). Aqueous chlorine
can uniquely react with anthropogenic organic compounds resulting in the formation of toxic but stable transformation products (Arnold et al., 2008, Duirk and Collette, 2006;
Gallard and von Guten, 2002). It has been documented that there exist five main
pathways describing the reaction of chlorine with water: addition, substitution, oxidation,
light decomposition, and hydrolysis (Gang et al., 2003; Johnson and Jensen, 1986, Duirk
and Collette, 2006). Addition describes a chlorine incorporation into organic material;
likewise, the substitution pathway describes chlorine substituting into existing organic
13
matter. These pathways can then form chlorinated intermediate products that break down into known or identifiable DBPs (van Hoof, 1992).
The speciation of chlorine is dependent upon pH. Drinking water criteria recommend a pH between 6.5 and 8.5 be maintained at all times. When considering chlorine speciation coefficient is pKa of 7.54, the two chlorine species observed in that pH range are hypochlorous acid (HOCl) and hypochlorite (OCl-), as shown in Equation
2.1 (Deborde and von Gunten, 2008). Free chlorine, or total chlorine, can now be calculated by adding the conjugate acid and conjugate base. HOCl is a stronger oxidant than OCl-; thus, the loss of total chlorine is considered a function of aqueous pH (Duirk and Collette, 2006).
+ = 7.54 (2.1) − + 𝐻𝐻𝐻𝐻𝐻𝐻𝐻𝐻 ↔ 𝑂𝑂𝑂𝑂𝑂𝑂 𝐻𝐻 𝑝𝑝𝑝𝑝𝑎𝑎 The most common oxidation reaction between drinking water and HOCl is described below in Equation 2.2., where the organic/inorganic compound are represented by “X” (Deborde and von Gunten, 2008). This is assumed to be an elemental stoichiometric reaction and an overall second-order reaction, the reaction is first-order with respect to [HOCl]T and [X]T, and the rate expression for this reaction is shown in
Equation 2.3. The second-order rate coefficient is an apparent second-order due to the pH dependency of total chlorine speciation and X (Equation 2.4 and 2.5) (Deborde and von
Gunten, 2008).
+ (2.2)
𝐻𝐻𝐻𝐻𝐻𝐻𝐻𝐻 𝑋𝑋 → 𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃𝑃 [ ] = [ ] [ ] (2.3) 𝑑𝑑 𝑋𝑋 𝑇𝑇 𝑘𝑘𝑎𝑎𝑎𝑎𝑎𝑎 𝐻𝐻𝐻𝐻𝐻𝐻𝐻𝐻 𝑇𝑇 𝑋𝑋 𝑇𝑇 𝑑𝑑𝑑𝑑 14
=
𝑘𝑘𝑎𝑎𝑎𝑎𝑎𝑎 𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎𝑎 𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜 𝑟𝑟𝑟𝑟𝑟𝑟𝑟𝑟 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 [ ] = [ ] + [ ] (2.4) − 𝐻𝐻𝐻𝐻𝐻𝐻𝐻𝐻 𝑇𝑇 𝐻𝐻𝐻𝐻𝐻𝐻𝐻𝐻 𝑂𝑂𝑂𝑂𝑂𝑂 [ ] = [ ] + [ ] (2.5) − 𝑋𝑋 𝑇𝑇 𝐻𝐻𝐻𝐻 𝑋𝑋 The actual reactivity with respect to HOCl and OCl- has been documented to vary
depending on the compounds present (Abia et al., 1998; Armesto et al., 1994; Deborde et
al., 2004; Dodd et al., 2005; Gallard et al., 2004; Gallard and von Gunten, 2002). HOCl reacts with inorganic compounds via electrophilic attack, depending on the compounds nucleophilic properties (Deborde and von Gunten, 2008). HOCl is generally the dominant chlorine species reacting with organic compounds by means of oxidation, addition, and electrophilic substitution. These reactions result in the formation of more oxidized and chlorinated compounds (Dore, 1989). In the presence of chlorine, phenolic compounds undergo an electrophilic substitution reaction in which the attack occurs at the ortho- or para- ring position (Deborde and von Gunten, 2008; Roberts and Caserio, 1968). This reaction is governed by the electron donor properties and the aromatic ring charge density change (Deborde and von Gunten, 2008). Conversely to HOCl electrophilic reactions,
OCl- has been found to accelerate base-catalyzed hydrolytic reactions as a nucleophile
(Duirk and Collette, 2006).
2.3.4 Chloramine Disinfection and DBP Formation
Chloramines are often used as an oxidant in drinking water disinfection as an
alternative to chlorine. There are three species of chloramines: mono-, di-, and tri-
chloramine. This oxidant is a product of a substitution reaction between free chlorine and 15
ammonia (NH3). Monochloramine is the predominant species found within a typical
drinking water pH range of 6.5 – 8.5 (Vikesland et al., 1998). Though monochlormaine
and chlorine exhibit similar oxidizing capacities, monochloramine is a less effective
disinfectant (Wolfe et al., 1984). Additionally, chloramines are not very stable around the
drinking water pH range; they are known to auto-decompose, resulting in the oxidation of
ammonia and reduction of active chlorine (Jafvert and Valentine, 1992). These reactions
depend on the solution pH and chlorine to ammonia nitrogen ratios, as the oxidation of
ammonia increases with a larger ratio (Vikesland et al., 2000).
Chloramines are typically utilized when a treated source water chlorine residual
cannot be maintained throughout the distribution system or when excessive DBPs are
formed. . Monochloramine concentrations within drinking water distribution systems have been detected at 0.5 – 2.0 mg/L; in this case, monochloramine was the primary disinfectant or used to provide chlorine residual (Bull et al., 1991). Chloraminated treated waters have been found to still from known and regulated DBPs at lower concentrations.
Duirk et al. (2005) investigated the monochloramine autodecomposition and reaction pathways with NOM to further understand the formation of DBPs. Monochloarmine undergoes a biphasic reaction; the fast reaction involves a direct reaction between monochloramine and NOM, while the slow reaction accounts for oxidation loss (Duirk et al., 2005). In a study performed by Cowman and Singer (1996), they found that HAA formation did occur in chloraminated samples, but at concentrations 90—95% less than the chlorinated samples. DCAA was the predominant HAA formed in the chloraminated samples at concentrations of 9μg/L or less (Cowman and Singer, 1996). Monochloramine has also been found to react with the organic substance dimethylamine (NMA) to form
16
the detrimental compound N-nitrosodimethylamine (NDMA) (Choi and Valentine 2001;
Mitch and Sedlak, 2002). NDMA is extremely toxic and carcinogenic; however, it is only regulated in only a few States.
2.4 ICM Transformation and Iodo-DBP Formation
The transformations of ICM can occur various ways by means of chemical and/or biological agents; each case is very distinctive and subjective. Iodo-DBPs are generally formed by the chemical oxidation of ICMs.
2.4.1 ICM Transformation
Iodinated X-ray Contrast Media are susceptible to biological and chemical degradation processes. A common ICM chemical degradation pathway occurs during the drinking water disinfection process; ICM and transformation prodcuts are exposed to oxidants and may then form Iodo-DBPs. Current pharmaceutical removal treatment processes consist of partitioning (adsorption with activated carbon membrane separation) and transformation (oxidation via microbial degradation or chemical oxidation) (Adams,
2009; Grassi et al., 2012).
2.4.2 Microbial Transformation of ICM
ICM transformation has been detected within biological wastewater treatment; contrary to their inherently high water solubility and typical occurrence within drinking 17
water treatment. Iopromide, specifically, has been investigated within a soil/water
mixture for biological degradation assessment; as a result, 12 transformation products
were identified (Shulz et al., 2008). The researchers identified the end products through
high performance liquid chromatography coupled with ultraviolet (HPLC-UV) and liquid chromatography with tandem mass spectrometry (LC-MS) (Shulz et al., 2008).
Additionally, a separate German based research team also investigated ICM biological
degradation; they conducted experiments in batch reactors on soil/water mixtures and
river sediment/water mixtures (Kromos et al., 2010). This team utilized HPLC-US and
LC-MS for analysis and found that ionic ICMs (i.e. diatrizoate) were not susceptible to microbial transformations; however, non-ionic ICMs (i.e. iohexol, iomeprol and
iopamidol) were susceptible to microbial transformation (Kromos et al., 2010). It should
also be noted that in the latter study, the biological degradation of ICM experiments were
conducted at time intervals up to 159 days (Kromos et al., 2010).
2.4.3 Chemical Transformation of ICM
A chemical transformation of ICM occurs during the disinfection of drinking
water; often, this results in the formation of Iodo-DBPs. Natural iodide concentrations
within source waters have been documented at very low levels, often below detection
limits (Richardson et al., 2008). Despite this circumstance, there are disproportional
amounts of Iodo-DBPs forming; thus, suggesting that there are alternative sources of
iodide present in the source waters (Bischel and von Gunten, 2000; Richardson et al.,
2008).
18
Investigations by Duirk et al. (2011) have found that ICMs are an organic source
of iodide in the formation of Iodo-DBPs. During this study, the release of organically bound iodide from an aromatic compound ring in the presence of a chlorinated oxidant was demonstrated; likewise, this consequently integrated into the NOM structure resulting in the formation of Iodo-DBPs (Duirk et al., 2011). Interestingly though,
- experiments that were performed in the absence of NOM yielded only IO3 and trace
levels of DBP formations (Duirk et al., 2011). Based on these findings, it is suggested
that the chlorinated oxidants cleave the moieties of ICM changing the compound polarity
and releasing free iodine; the now released iodine is able to form Iodo-DBPs (Duirk et
al., 2011).
The hypochlorite ion is believed to be the primary chlorine species responsible for
iopamidol transformation in the presence of chlorinated oxidants (Wendel et al., 2014).
This claim is supported by observed pH dependency and lower Iodo-DBP formations in
monochloramine experiments (a result of monochloramine auto-decomposition)
compared to chlorine experiments (Duirk et al., 2011). This is also supported by Ye et al.
(2014), who investigated the formation of iodo-DBPs during chlorination and
chloramination and found that the ICM iopamidol formed the most THMs dependent on
pH. It has been determined that iopamidol is considered a precursor to Iodo-DBP
formation when coupled with a chlorinated oxidant (Duirk et al., 2011; Richardson et al.,
2008). Iopamidol, in the presence of chlorine, has been documented as forming up to 212
nM of dichloroiodomethane and 3.0 nM of iodoacetic acid (Duirk et al., 2011).
Additionally, it has been determined that iopamidol completely transforms itself at pH
8.5 after 24 hours; iodine is released and mainly oxidized to iodate, and only a small
19
percentage (<2% after 24 hours) of transformation products identified were Iodo-DBPs
(Wendel et al., 2014). The proposed reaction pathway for Iopamidol degradation is
presented below in Figure 2.2 (Duirk et al., 2011).
Figure 2.2: Iodo-DBP formation pathway due to iopamidol reacting with chlorinated oxidants in the presence of NOM (Duirk et al., 2011)
2.5 Toxicity of DBPs
As previously discussed, there are currently 9 DBPs regulated by the US EPA: 4
THMs and 5 HAAs (US EPA, 2015). It has been documented that these compounds are not just carcinogenic, but genotoxic and cytotoxic, as well (Richardson et al., 2007).
Chlorination of water has been connected to an elevated risk of cancer within epidemiologic studies; the specific forms of cancer include pancreas, stomach, kidney,
20
rectum, and Hodgkin’s and non-Hodgkin’s lymphoma (Bull et al., 1995; Koivusalo et al.,
1994; Morris et al., 1992). More health problems that have been connected to ingestion of chlorinated water are birth defects and spontaneous abortions (Nieuwenhuijsen et al.,
2000; Waller et al., 2001).
Additional investigations were performed with concentrated extracts from drinking water samples to show their toxicity through in vivo and in vitro bioassays
(Wilcox and Williamson, 1986). As a result, within the strains of Salmonella tested, brominated THMs stimulated genotoxicity along the glutathione S-transferase theta
(GSTT1-1) enzyme (Kogevinas et al., 2010; Kargalioglu, et al., 2002). In a separate
related study, Plewa et al. (2002) determined the cytotoxicity and genotoxicity of
brominated and chlorinated HAAs through experimentation with Chinese Hamster
Ovarian (CHO) mammalian cells. In this study, bromoacetic acid was determined as the
most cytotoxic and genotoxic; additionally, brominated HAAs were determined to be
more cytotoxic and genotoxic than their chlorinated countreparts (Plewa et al., 2002).
Again, another separate study performed had concluded that dichloroacetic acid and
trichloroacetic acid were mutagenic toward mouse lymphoma cells (Harrington-Brook et al., 1998).
Richardson et al. (2008) had conducted a study of chlorinated and chloraminated drinking waters across 23 cities within the United States and Canada to investigate five iodo-acids and two THMs. This study established that iodinated DBPs were highly cytotoxic and genotoxic within the drinking waters; specifically, iodoacetic acid was the most genotoxic formation within mammalian cells (Richardson et al., 2008). This study also determined that iodo-acids were more cytotoxic than their iodo-THM counterparts;
21
except iodoform, which showed a high cytotoxicity (Richardson et al., 2008). This is supported by the Plewa et al. (2004) study that determined iodoacetic acid to be more highly cytotoxic and genotoxic than bromoacetic acid within mammalian cells.
Duirk et al. (2011) also supported this by the experiments performed to determine the toxicity of source water (Athen-Clark County, Georgia) with iopamidol and oxidation by chlorine or monochloramine at pH 7.5. This study also exhibited increased cytotoxicity and genotoxicity with relation to experiments lacking ICM. Within chlorinated source water experiments, iodoacetic acid was the most cytotoxic, followed by in decreasing order chloroiodomethan, dichloroiodomethane, iodoform, and bromochloromethane. Within chloraminated source water experiments, iodoacetic acid was the most cytotoxic, followed by in decreasing order chlorodiiodomethane and dichloroiodomethane (Duirk et al., 2011). It was determined that iodo-DBPs exhibit greater genotoxicity than their brominated and chlorinated counterparts (Duirk et al.,
2011).
2.5.1 TOX Toxicity
Total organic halogen (TOX) is an analytically defined measurement applied to specially drinking waters. In comparison to detection methods forindividual DBPs, TOX is an inexpensive measurement to screen a large number of samples for halogenated organic components (Li et. al., 2002). TOX is measured with a view to estimating the total amount of organically bound chlorine, bromine and iodine in water samples. TOX is considered as a collective parameter and a toxicity indicator for all the halogenated
22
organic DBPs present in water sample (Li et al., 2002). Unknown fraction of TOX can
be estimated by comparing the TOX values with the halides attributed to known
quantifiable halogenated DBPs (Singer and Chang, 1989; Krasner et al. 2006; Hua and
Rekhow, 2006). Other than the volatile trihalomethanes, haloacetonitriles, haloketones
most of the total organic halogen (TOX) are non-volatile that show stronger mutagenic activity than the volatiles (Meire et al., 1983).Undoubtedly, major health concerns over disinfection by-products are concentrated not only to regulated or unregulated DBPs, but
should include all non-volatile TOX (Suzuki and Nakanishi, 1987). Johnson and Jensen
(1986) studied the TOX produced from the reaction of monochloramine and isolated
fulvic acid and found that TOX resulting from chloramination was more hydrophilic and
higher in molecular weight than TOX produced by chlorine. Zhang et al. (2000) studied
the TOX formation from the reactions of a fulvic acid and different chemical
disinfectants. For the chloramine treated sample, more than 80% of the TOX could not
be represented by the commonly known DBPs. A substantial amount of the halogenated
DBPs formed by chloramines is unknown. A larger unknown fraction of TOX is
produced from chloramination than from chlorination. And thus there remains a major
scope of further study to investigate the toxicity associated with the TOX. Savitz et al.
(2006) conducted a study in three US locations of varying DBP levels to evaluate the
pregnancy loss due to exposure to drinking water DBPs and found a possible association
for TOX, not addressed in any of the previous studies. Given that there are hundreds of
chemicals beyond the THMs and HAAs in chlorinated and chloraminated drinking water
(Krasner et al., 2006), possibly differing across the study sites, some harmful constituent
may be better reflected in the comprehensive measure, TOX, than in any of the other
23
DBP indices examined. Schenck et al. (2009) evaluated the relationship between
mutagenicity and water quality parameters. The study included information on treatment,
mutagenicity data, and water quality data for source waters, finished waters, and
distribution samples collected from five full-scale drinking water treatment plants, which used chlorine exclusively for disinfection. They found the highest correlation between mutagenicity and the total organic halide concentrations in the treated samples.
24
CHAPTER III
MATERIALS AND METHODS
3.1 Chemicals and Reagents
The iopamidol reference standard was purchased from U.S. Pharmacopeia
(Rockville, MD, USA). Potassium iodide (KI) (99.5%) was purchased from Fisher
Scientific (NJ, USA). Commercial (10-15%) sodium hypochlorite (NaOCl) containing equimolar amounts of OCl- and Cl- was purchased from Sigma Aldrich (St. Louis, MO,
USA). The standard reference solutions used for disinfection by-product analysis
included the following: iodoform and iodoacetic acid purchased from Sigma Aldrich (St.
Louis, MO, USA), haloacetic acid mix (containing monochloro-, monobromo-, dichloro-,
trichloro-, bromochloro-, dibromo-, bromodichloro-, chlorodibromo-, and tribromoacetic
acid mixed within MtBE) purchased from Restek (Bellefonte, PA, USA), trihalomethane
mix (containing chloroform, bromoform, bromodichloromethane, and
dibromochloromethane) purchased from Chem Service (West Chester, PA, USA), iodo-
trihalomethanes (dichloroiodo-, dibromoiodo-, bromochloroiodo-, chlorodiiodo-, and
bromodiiodomethane) purchased from CanSyn Chem Corporation (Toronto, ON,
Canada), chloro-, dichloro, and trichloroacetonitrile purchased from Chem Service (West
Chester, PA, USA), bromoacetonitrile and dibromoacetonictile purchased from Acros
Organics (Geel, Belguim), bromochloroacetonitrile purchased from Crescent Chemical 25
(Ward Hill, MA, USA), and iodoacetonitrile purchased from Alfa Aesar (Ward Hill, MA,
USA). All disinfection by-products standards were purchased at the highest available purities. All additional organic and inorganic chemicals used were certified American
Chemical Society reagent grade.
Deionized water used for experiments at 18.2 MΩ∙cm-1 was prepared with a
Barnstean ROPure Infinity/NANOPure system (Barnstead-Thermolyne Corp. Dubuque,
IA, USA). Experimental pH was measured with an Orion 5 Star pH Meter equipped with
Ross ultra-combination electrode (Thermo Fisher Scientific, Pittsburgh, PA, USA).
Experimental pH adjustments were performed with 1N H2SO4 and 1 NNaOH. Prior to experimental use, all glassware and polytetrafluoroethylene (PTFE) items were soaked in a chlorine bath for 24 hours, rinsed with deionized water, and thoroughly dried.
3.2 Source Water Characterization
Source water for the experiments was sampled from the intake of the Akron
Water Supply drinking water treatment plant. The source water characteristics for the
Akron sampled water are shown in Table 3.1. The total organic carbon (TOC) concentrations were measured using a Shimadzu TOC analyzer (Shimadzu Scientific,
Columbia, MD, USA) and calibrated according to the Standard Method 505A (APHA et al., 1992). The spectral characteristics and ultraviolet absorbance 254 nm (UV254) of the
NOM were measured using a Shimadzu UV 1601 UV Visible Spectrometer according to
Standard Method 5910B (APHA et al., 1995). The specific ultraviolet absorbance 254 nm
(SUVA254) was calculated from the following relation: SUVA254 = 100 x UV254/DOC.
26
DBP formation has been linked to water characteristics like SUVA254, bromide
concentrations and DOC concentrations (Njam et al., 1994).
Table 3.1: Source water characteristics for Akron water Characteristic Akron Source Water
DOC (mg/L C) 5.57
Bromide (μM) 1.6
Iodide (μM) <0.5
-1 UV254 (cm ) 0.121
SUVA254 (L/mg-m) 2.17
Next, the source water was characterized using fluorescence spectroscopy; this
yielded the excitation-emission matrix (EEM) spectra. Parlanti et al. (2000) used ratios of
fluorescence EEM peak intensities to track the NOM changes within natural waters. The
sample preparation for the fluorescence spectra detection followed the method developed
by Chen et al. (2003) with slight modification. The water samples were acidified with
sulfuric acid to lower pH to 2.75 – 3.25 to remove any organic carbon present. The
samples were then diluted to a final DOC of 1 mg/L with 0.01 M KCl to allow for direct
comparisons of the fluorescence intensities (Nguyen et al., 2005). The EEM fluorescence
spectra were obtained with an F-7000 FL Fluorescence Spectrometer (Hitachi Hi-Tech,
Tokyo, Japan); the spectrophotometer uses a xenon lamp as the light source. The excitation slit and emission slit were set to a band-pass of 10 nm. The spectra of the source water samples were then measured at successive emission spectra at 2 nm intervals across the range of 290 – 550 nm and using excitation wavelengths spaced at 5
27
nm from 204 – 404 nm. Next, the resulting spectra were merged into the EEM and combined within SigmaPlot 12.0 (SPSS Inc.) to generate contour maps of the fluorescence intensity along with regional integration, as seen in Figure 3.1.
Fluorescence regional integration (FRI) was proposed by Chen et al. (2003) to quantify multiple broad-shaped EEM peaks. FRI is a quantitative technique that integrates volume under the EEM region, as seen in Table 3.2. Also, the FRI method has been used to quantitatively analyze all wavelength-dependent fluorescence intensity data from EEM spectra (Marhuenda-Egea et al., 2007). The five distinctive fluorescence reasons as proposed by Chen et al. (2003) are listed in Table 3.2. The five regions found in the NOM EEM of the Akron source water are also shown in Table 3.3.
28
Table 3.2: Fluorescence EEM regions proposed by Chen et al. (2003) Excitation Emission
Regions Representation Range (nm) Range (nm)
I Aromatic 200 – 250 280 – 330
II Aromatic protein-like 200 – 250 330 – 380
III Fulvic acids 200 – 250 380 – 550
IV Soluble microbial by-products 250 – 400 280 – 380
V Humic acids 250 – 400 380 – 550
Table 3.3: Fluorescence regions for Akron source water for 1 mg/L C Fluorescence Regions Volume %
Aromatic (I) 1.9 14.7
Aromatic Protein-Like (II) 3.4 25.9
Fulvics (III) 5.1 38.5
Microbial (IV) 1.1 8.5
Humics (V) 1.6 12.3
Total 13.1 100
29
Figure 3.1: Fluorescence excitation-emission spectrum of Akron source water. [DOC] = 5.57 mg/L, SUVA254 = 2.27 L/mg-m
The EEM for the Akron source water seen in Figure 3.1 shows a large response in
Region III; this suggests that there is a large amount of fulvic acid present. A likely source for this high response in Region III is decaying leaf material, as this has been found to be a vital source of fulvic acid (Schlesinger, 1997). Humic acid is also present in the water as indicated by the large response in Region V. Humic acid results from the degradation of plant materials by biological and natural chemical processes (Hudson et al., 2007). Region II also shows a high response indicating the presence of aromatic
30
proteins. These aromatic proteins are likely of bacterial origin present as specific enzymes that a microbial community uses to break down leaf material (Allan and
Castillo, 2007; Benfield, 2006). It should be noted that there is a low response in Region
IV, indicating a low volume of soluble microbial by-products.
3.3 Experimental Methods
The experimental procedures were categorized as one experiment. This experimental plan investigated a constant level of the ICM iopamidol in the source water.
This experiment revealed the effects of ICM on regulated and unregulated DBP formation.
3.3.1 Disinfection By-Product Experiments with Source Water
Source water was collected from Lake Rockwell at the inlet of the Akron Water
Supply drinking water treatment plant. This raw source water was filtered through 5 μm and 0.45 μm Whatman nylon membrane filters (Whatman, West Chester, PA, USA) and stored in the fridge at 4°C before use. Chlorination and chloramination experiments were performed under a pseudo first-order condition over a pH range 6.5 – 9.0 using
[Oxidant]T:[Iopamidol] = 20:1, where [Iopamidol] = 5 μM and [Oxidant] = 100 μM.
Aqueous solutions for the source water at each pH (6.5, 7.5, 8.5 and 9.0) were prepared in batch reactors. Each batch reactor consisted of source water, iopamidol and buffer in a 500 mL Erlenmyer flask. Buffer was used to maintain the pH of the solutions;
31
4 mM phosphate buffer (for pH 6.5 and 7.5) and 4 mM borate buffer (for pH 8.5 and 9.0).
Under rapid mix conditions, using PTFE coated stir bar and magnetic stir plate, aqueous
chlorine was added to each aqueous solution reactor at the predetermined
[Cl2]T:[Iopamidol] ratio. The aqueous chlorine was added at a relatively high
concentration to ensure that excess chlorine would be present within the aqueous mixture
throughout the duration of the experiment. Before the addition of aqueous chlorine to each reactor, the chlorine concentration was verified using a Shimadzu UV-vis
Spectrometer. N’-diphenyl-p-phenylenediamine DPD was added to a test sample of the aqueous chlorine solution and DI water; the concentration was determined from a calibration curve derived in lab. The calibration curve for the UV-vis spectrometer was itself calibrated against the known method using ferrous ammonium sulfate (FAS)/N, N’- diphenyl-p-phenylenediamin (DPD) titration (APHA et al., 2005). Once the aqueous chlorine stock concentration was determined, an appropriate calculated amount was added to each reactor ensuring a reactor chlorine concentration of 100 μM. The experimental aqueous solution was stirred under rapid mix for 3 minutes to ensure homogeneity. Aliquots of the reactor solutions were then transferred to 128 mL amber bottles with PTFE septa caps, headspace free. Each reactor would yield 3 sub samples of
128 mL bottles; each of the amber bottles were then stored at 25±1°C in an incubator for a reaction time of 72 hours.
A similar experimental design as described above was also utilized by using monochloramine as the oxidant instead of chlorine. Pre-formed monochloramine was used to avoid the artefacts caused by reactions of excess free chlorine that may briefly exist when forming monochloramine in-situ (Duirk et al., 2005). A pre-formed
32
monochloramine solution was prepared by mixing 5.64 mM ammonium chloride with 3.7 mM hypochlorous acid to achieve a Cl/N molar ratio of 0.7 in a 4 mM borate buffer solution. The aqueous solution was rapidly mixed with a PTFE stir bar and magnetic stir plate at pH 8.5 and allowed to react and reach equilibrium for 30 minutes. A higher pH of
8.5 was used to minimize monochloramine decomposition and to ensure that monochloramine remains the active species in solution (Symons et al., 1998). Lastly, the monochloramine concentration was verified with the UV-vis spectrometer and FAS/DPD titration (APHA et al., 2005).
Experiments were preformed using the procedures described above with Akron source water using both oxidants (chlorine and monochloramine) and iopamidol concentrations (0 μM and 5 μM) at pH 6.5, 7.5, 8.5, and 9.0 for a reaction time of 72 hours. The 72 hour chlorine and monochloramine source water and iopamidol experiments were not performed in triplicate, but were performed with sub-sampling triplicates. Kinetic experiments were also performed using both oxidants with a lower concentration of iopamidol, 2.5 μM, at pH 6.5, 7.5, 8.5, and 9.0 at reaction times of 1, 6,
12, 24, 48 and 72 hours. A lower concentration of iopamidol was used in the kinetics experiments to ensure the oxidant was present in excess; the [Oxidant]T:[Iopamidol] ratio for kinetics experiments was 40:1. The kinetic experiments were performed in triplicate, as opposed to sub-sampling triplicates. At each reaction time, the samples were taken from the incubator and the residual chlorine was quenched with an aqueous sodium sulfite solution at 120% of the initial total oxidant concentration. Each sample was quenched to stop the oxidant reaction allowing for analysis of the DBP concentrations that were formed.
33
3.3.2 Cytotoxicity and Genotoxicity Experiments with Source Water
Mammalian cell cytotoxicity measurements were conducted on organic extracts
from 20 L reactors of Akron source water with 5 µM ICM, 100 mM aqueous chlorine or
monochloramine, at pH 7.5 with 10 mM phosphate buffer and allowed to react for 72
hours in the dark prior to extraction. Table 3.4 shows the number of experiments
conducted including controls and the experimental conditions.
Treated waters were concentrated using XAD resins (30 mL XAD-8 over 30 mL
XAD-2), as described in a previously published procedure (Duirk et al, 2011). Briefly,
the 20 L reactors would acidify the water to a pH of 1-2 with concentrated sulfuric acid,
approximately 20-25 mL per liter of water. The acidified water samples were poured
over the XAD resin bed at 30 mL/min. Then, the column bed was rinsed with 200 mL of
ethyl acetate to elute the sorbed organic compounds and collected to be dried with
anhydrous sodium sulfate in a separatory funnel. The ethyl acetate extract was
concentrated to approximately 5.0 mL with a rotovap and then blowing the remainder
down with a gentle stream of nitrogen to 2 mL.
The extracted samples were shipped to Dr. Plewa’s laboratory for analysis. The ethyl acetate was removed over a stream of N2 gas and then solvent exchanged into
dimethylsulfoxide (DMSO). Cytotoxicity was measured as a reduction in cell density
after cell exposure to a water sample concentrate (WSC) for 72 hours (Plewa et al.,
2015). Highly concentrated WSCs were stored within DMSO inside a freezer. A dilution
series of 10 concentrations was prepared by diluting the WSCs into culture medium; this
was performed immediately prior to the experiment. Each dilution was transferred to the
34
Table 3.4: Aqueous conditions for reactors used for toxicity experiments
Experimental Code Conditions AOHTOX-DI: 20 L of DI water extracted/concentrated to 2 ml ethyl acetate
AOHTOX-SW 20 L of Akron source water (ASW) adjusted to pH 7.5 and extracted/concentrated to 2 ml ethyl acetate
AOHTOX-Cl 20 L chlorinated ([CL2]T = 100 M) AS extracted/concentrated to 2 ml ethyl acetate
AOHTOX-Cl-IDOL 20 L chlorinated ([CL2]T = 100 M M at pH 7.5 extracted/concentrated to 2 ml ethyl acetate
AOHTOX-IDOL 20 L of ASW with IDOL = 5 M at pH 7.5 extracted/concentrated to 2 ml ethyl acetate
AOHTOX-NH2Cl- 20 L chloraminated ([NH2Cl] = 100 IDOL 5 M
AOHTOX-NH2Cl 20 L chloraminated ([NH2Cl] = 100 at M)pH ASW7.5 extracted/concentrated to 2 ml ethyl acetate
AOHTOX-Iopromide 20 L of Akron source water (ASW) with Iopromide = 5 M at pH 7.5 extracted/concentrated to 2 ml ethyl acetate
AOHTOX-Cl-Iopromide 20 L chlorinated ([CL2]T = 100 mideM) =AS 5 M
AOHTOX-Iohexol 20 L of Akron source water (ASW) with Iohexol = 5 M pH 7.5 extracted/concentrated to 2 ml ethyl acetate
AOHTOX-Cl-Iohexol 20 L chlorinated ([CL2]T = 100 M) ASW with Iohexol = 5 M at pH 7.5 extracted/concentrated to 2 ml ethyl acetate
AOHTOX-Iomeprol 20 L of Akron source water (ASW) with Iomeprol = 5 pH 7.5 extracted/concentrated to 2 ml ethyl acetate
AOHTOX-Cl-Iomeprol 20 L chlorinated ([CL2]T = 100 M at pH 7.5 extracted/concentrated to 2 ml ethyl acetate
AOHTOX-Diatrizoate 20 L of Akron source water (ASW) with Diatrizoate = 5 at pH 7.5 extracted/concentrated to 2 ml ethyl acetate
AOHTOX-Cl- 20 L chlorinated ([CL2]T = 100 Diatrizoate 5 M
35
CHO cells in 96-well microplates and the positive and negative controls; all microplates
were then covered with AlumnaSeal to prevent any sample volatization. The samples
were then left to set for the exposure period of 72 hours. After the exposure period, the
cell density for each microplate was determined through histological staining and
absorbency using crystal violet and a microplate reader, respectively (Plewa et al., 2015).
A cytotoxic concentration-response curve for each WSC was developed from the data collected from the combined replicate experiments. A concentration factor associated with 50% reduction in cell density as compared to the negative controls was determined as LC50. Each LC50 was calculated for each WSC from the concentration-
response curve.
Genotoxicity was measured through CHO single cell gel electrophoresis; a
molecular genetic test to quantitatively measure the level of genomic DNA damage
induced by a test agent or water sample concentrate in individual nuclei of cells (Plewa et
al., 2015). This experiment focuses on the %Tail DNA value, the DNA amount that has
migrated from the nucleus to the microgel, as the indication for DNA damage induced
from the water samples. A microplate methodology similar to that described above for
cytotoxicity experiments was used. Again, the microplate were covered with AlumnaSeal
to prevent sample volatization; however, the exposure time was only 4 hours, in order to
maximize the ability to detect genomic damage while limiting the effect of DNA repair.
The concentration factor associated with 50% reduction in genomic DNA is noted as the
50% Tail DNA value. Each 50% Tail DNA value was calculated for each WSC from the
concentration response curve.
36
3.3.3 Disinfection By-Product Analytical Methods
THM, HAN and HAA analyses were carried out using a micro liquid-liquid extraction method with MtBE with an acidic pH. The predominant THMs that were repeatedly analyzed for include chloroform (CHCl3), bromodichloromethane (CHBrCl2), dibromochloromethane (CHBr2Cl), dichloroiodomethane (CHCl2I), bromochloroiodomethane (CHBrClI), bromoform (CHBr3), dibromoiodomethane
(CHBr2I), chlorodiiodomethane (CHClI2), bromodiiodomethane (CHBrI2), and iodoform (CHI3). The predominant HANs that were repeatedly analyzed for include chloroacetonitrile (CAN), trichloroacetonitrile (TCAN), dichloroacetonitrile (DCAN), bromoacetonitrile (BAN), bromochloroacetonitrile (BCAN), dibromoacetonitrile
(DBAN), and iodoacetonitrile (IAN).
THMs and HANs were extracted using the US EPA method 551.1 (Munch and
Hautman, 1998) with some modifications. At the designated reaction times, each sample was quenched with the aqueous sodium sulfite solution to end the oxidant reactions. The sample was acidified with 5 mL of 36 N sulfuric acid to drop the sample’s pH to around pH 2. Next, 3mL of MtBE and 10 μL of 123.9 mM of 1,2-dibromopropane internal standard were added to the sample to achieve nearly 12.4 μM internal standard concentration within the sample. MtBE was used to extract the non-dissociated acidic compounds (APHA AWWA and WEF, 1995). Next, 30g of anhydrous sodium sulfate salt (dried at 100°C) was added to decrease activity of inorganic compounds and increase activity of organic compounds – increasing partitioning of DBPs from aqueous phase to
MtBE (US EPA, 2013). The samples, still in the 128 mL amber bottles, were then capped with polyseal cone-lined caps, shaken by hand for one minute, and then shaken on the
37
wrist-action-shaker (Burrell Scientific, Pittsburgh, PA, USA) for 30 minutes. After the mechanical shake, the samples were left to settle for 3 minutes. Next, a disposable pasteur pipette was used to transfer roughly 1.5 mL of MtBE extract into a 2 mL CG autosampler vial. Before entering the vial, the MtBE extract was filtered through another pasteur pipette containing glass wool and dried anhydrous sodium sulfite salt, in order to remove any excess water from the extract. The extracted sample was then split; 0.5 mL being used for diazomethane derivatization for HAA analysis, and the remaining extract being used for THM and HAN analyses. All extracted samples were stored in the freezer until required for GC analysis.
HAAs were measured using US EPA method 552.1 (Hodgeson and Becker, 1992) with some modifications. This utilizes a liquid-liquid extraction with MtBE, derivatization with diazomethane, and analysis with a GC/MS. The predominant HAAs that were repeatedly analyzed for include chloroacetic acid (CAA), bromoacetic acid
(BAA), dichloroacetic acid (DCAA), trichloroacetic acid (TCAA), iodoacetic acid (IAA), bromochloroacetic acid (BCAA), bromodichloroacetic acid (BDCAA), and dibromoacetic acid (DBAA). An aliquot of the extracted sample was methylated with diazomethane for the production of methyl ester or other derivatives for gas chromatographic separation (APHA, AWWA, and WEF, 1995). The diazomethane was created by adding 0.367 g of diazald and 1 mL of carbitol (2-[2-ethoxyethoxy] ethanol) to the inner tube of the diazomethane generator. Next, 3 mL of MtBE was added to the outer tube of the diazomethane generator. The two parts of the diazomethane generator were assembled; the lower part of the outer tube was immersed in an ice bath to maintain isothermal conditions of 0ºC. After equilibriating to 0ºC, 1.5 mL of KOH (37%) was
38
injected (slowly, dropwise) into the inner tube of the diazomethane generator. The generator was then gently shaken by hand to ensure mixing of the inner tube reactants, without spilling into the outer tube. The outer tube MtBE solution may turn yellow, indicating an excess of diazomethane production. The diazomethane generator was left to set for 50 minutes for diazomethane production to occur. Next, the generator was opened to destroy any unreacted diazomethane with the addition of activated silica. Once the diazomethane had been prepared, 250 μL of diazomethane was added to the 0.5 mL of extracted sample in the GC autosampler vial. This was allowed to set for 30 minutes to allow adequate methylation of the HAAs. Lastly, 1 – 3 grains of activated silica were added to the GC autosampler vial to destroy any unreacted diazomethane.
3.4 Analysis of DBPs
After extraction and derivatization, the samples were analyzed on a 7890 GC system equipped with 63Ni microelectron capture detector (μECD) (Agilent
Technologies, Santa Clara, CA, USA) and a 7693 Autosampler (Agilent Technologies,
Santa Clara, CA, USA). A Restek 13638-127 GC column (Restek Corporation,
Bellefonte, PA, USA) was used in the GC connected from the injector port to the μECD to achieve separation of the analytes. The column parameters consisted of a length of 30 m, internal diameter of 0.25 mm, film thickness of 0.5 μm, and a flow rate of 1 mL/min.
Splitless injection of the sample was achieved by injecting 1 μL into the column. The operating temperature of the μECD was held constant at 250°C while the column make- up gas consisted of ultrahigh purity nitrogen gas with a 19 mL/min column flow rate; the column carrier gas consisted of ultrahigh purity helium gas. There were two oven 39
temperature ramp programs used for the analysis of THMs and HANs (Table 3.5) and
HAAs (Table 3.6), respectively.
Table 3.5: Oven temperature programming for THMs and HANs analysis on GC/μECD Rate (°C/min) Temperature (°C) Hold Time (min) Run Time (min)
Initial N/A 50 10 10
Ramp 1 2.5 65 0 16
Ramp 2 5 85 0 20
Ramp 3 7.5 205 0 36
Ramp 4 10 280 0 43.6
Table 3.6: Oven temperature programming for HAAs analysis on GC/μECD Rate (°C/min) Temperature (°C) Hold Time (min) Run Time (min)
Initial N/A 50 10 10
Ramp 1 0.25 50.5 5 17
Ramp 2 0.25 52 5 28
Ramp 3 0.25 62.5 0 70
Ramp 4 35 280 0 76.214
The standard solutions for THMs, HANs, and HAAs were prepared with deionized water. Known concentrations of the THMs and HANs standards were extracted using the procedure previously described along with 10 μL of 123.9 mM 1,2- dibromopropane internal standard, resulting in 12.4 μM internal standard sample concentration. Known concentrations of the HAAs standard were extracted using the procedure previously described along with the same volume and concentration of 1,2- 40
dibromopropane as the THMs and HANs standards; additionally, the HAAs standards were derivatized with diazomethane after the described extraction. The standards were then run and analyzed on the 7890 GC system with μECD; the THMs and HANs samples ran on the method as depicted in Table 3.5, while the HAAs samples ran on the method as depicted in Table 3.6. Calibration curves were created for all standards as shown in
Figures 3.2 – 3.26. The response ratio vs. compound concentration were plotted and the line of best fit was applied with the respective equation; this equation was used to calculate the corresponding DBP concentration for each sample tested.
The relevant DBP limits of quantification (LOQ) for this project are shown in
Table 3.7. The actual LOQs for each compound were much lower than the table values presented; however, the table values were preferred due to the estimated excess noise detected within the actual source water samples. It should also be noted that throughout the length of the project, there was constant maintenance being performed on the 7890
GC to ensure proper analyses. General maintenance consisted of changing the columns, changing the inlet liners, changing the inlet septa, changing the inlet gold seal, and baking out the μECD. Each time that maintenance was performed on the 7890 GC, all standards would be run again as a check on the GC performance. The check standard results showed similarly consistent response ratios, allowing for continued 7890 GC sample analysis.
41
1200
1000
800 ](nM)
3 600 y = 1471.4x R² = 0.9785 [CHCl 400
200
0 0.0 0.2 0.4 0.6 0.8 1.0
Response ratio Figure 3.2: Calibration curve for CHCl3using chloroform. [CHCl3] = 0 – 1000 nM
400
300
Br](nM) 200 2 y = 158.05x R² = 0.9965 [CHCl
100
0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Response Ratio Figure 3.3: Calibration curve for CHCl2Br using bromodichloromethane. [CHCl2Br] = 0 – 400 nM
42
350
300
250
200 Cl](nM) 2 150 y = 109.38x
[CHBr R² = 0.9974 100
50
0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Response ratio Figure 3.4: Calibration curve for CHBr2Cl using dibromochloromethane. [CHBr2Cl] = 0 – 300 nM
600
500
400
y = 2013.1x I] (nM) I]
2 300 R² = 0.9985 [CHCl 200
100
0 0.00 0.05 0.10 0.15 0.20 0.25 0.30 Response ratio Figure 3.5: Calibration curve for CHCl2I using dichloroiodomethane. [CHCl2I] = 0 – 500 nM 43
300
250
200
150 y = 1595.3x R² = 0.998 [CHBrClI](nM) 100
50
0 0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 0.18 Response ratio Figure 3.6: Calibration curve for CHBrClI using bromochloroiodomethane. [CHBrClI] = 0 – 250 nM
600
500
400 ](nM)
3 300 y = 204.28x R² = 0.9968 [CHBr 200
100
0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Response ratio Figure 3.7: Calibration curve for CHBr3 using bromoform. [CHBr3] = 0 – 500 nM
44
300
250
200 I] (nM) I]
2 150 y = 1152.7x
[CHBr R² = 0.9941 100
50
0 0.00 0.05 0.10 0.15 0.20 0.25 Response ratio Figure 3.8: Calibration curve for CHBr2I using dibromoiodomethane. [CHBr2I] = 0 – 250 nM
300
250
200 ](nM) 2 150 y = 266.06x
[CHClI R² = 0.9993 100
50
0 0.0 0.2 0.4 0.6 0.8 1.0 Response ratio Figure 3.9: Calibration curve for CHClI2 using chlorodiiodomethane. [CHClI2] = 0 – 250 nM
45
160
140
120
100 ](nM) 2 80
[CHBrI 60 y = 135.95x R² = 0.9925 40
20
0 0.0 0.2 0.4 0.6 0.8 1.0 Response ratio Figure 3.10: Calibration curve for CHBrI2 using bromodiiodomethane. [CHBrI2] = 0 – 125 nM
60
50
40
y = 68.338x ](nM) 3 30 R² = 0.9933 [CHI 20
10
0 0.0 0.2 0.4 0.6 0.8 1.0 Response ratio Figure 3.11: Calibration curve for CHI3 using iodoform. [CHI3] = 0 – 50 nM
46
600
500
400
300
[CAN](nM) y = 478.7x 200 R² = 0.9988
100
0 0.0 0.2 0.4 0.6 0.8 1.0 Response ratio Figure 3.12: Calibration curve for CAN using chloroacetonitrile. [CAN] = 0 – 500 nM
140
120
100
80
60
[TCAN](nM) y = 160.4x R² = 0.9631 40
20
0 0.0 0.2 0.4 0.6 0.8 1.0 Response ratio Figure 3.13: Calibration curve for TCAN using trichloroacetonitrile. [TCAN] = 0 – 125 nM
47
600
500
400
300 y = 2852.9x [DCAN](nM) 200 R² = 0.9991
100
0 0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 0.18 0.20 Response ratio Figure 3.14: Calibration curve for DCAN using dichloroacetonitrile. [DCAN] = 0 – 500 nM
140
120
100
80
y = 129.6x 60 R² = 0.9987 [BAN](nM)
40
20
0 0.0 0.2 0.4 0.6 0.8 1.0 1.2 Response ratio Figure 3.15: Calibration curve for BAN using bromoacetonitrile. [BAN] = 0 – 125 nM
48
300
250
200
150 y = 372.69x R² = 0.9931 [BCAN](nM) 100
50
0 0.0 0.2 0.4 0.6 0.8 Response ratio Figure 3.16: Calibration curve for BCAN using bromochloroacetonitrile. [BCAN]=0–250 nM
300
250
200
150 y = 346.63x R² = 0.9977 [DBAN](nM) 100
50
0 0.0 0.2 0.4 0.6 0.8 Response ratio Figure 3.17: Calibration curve for DBAN using dibromoacetonitrile. [DBAN] = 0 – 250 nM
49
35
30
25
20
y = 33.062x 15
[IAN](nM) R² = 0.9946
10
5
0 0.0 0.2 0.4 0.6 0.8 1.0 1.2
Response ratio Figure 3.18: Calibration curve for IAN using iodoacetonitrile. [IAN] = 0 – 31 nM
300
250
200
150 y = 773.91x
[CAA](nM) R² = 0.9997 100
50
0 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 Response ratio Figure 3.19: Calibration curve for CAA using chloroacetic acid. [CAA] = 0 – 250 nM
50
1200
1000
800
600 y = 1378.1x [BAA](nM) R² = 0.991 400
200
0 0.0 0.2 0.4 0.6 0.8 Response ratio Figure 3.20: Calibration curve for BAA using bromoacetic acid. [BAA] = 0 – 1000 nM
600
500
400
300
y = 376.03x [DCAA](nM) 200 R² = 0.9693
100
0 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 Response ratio Figure 3.21: Calibration curve for DCAA using dichloroacetic acid. [DCAA] = 0 – 500 nM
51
300
250
200
150
[TCAA](nM) y = 100.41x 100 R² = 0.9986
50
0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Response ratio Figure 3.22: Calibration curve for TCAA using trichloroacetic acid. [TCAA] = 0 – 250 nM
140
120
100
80
y = 83.588x 60
[IAA](nM) R² = 0.9896
40
20
0 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 Response ratio Figure 3.23: Calibration curve for IAA using iodoacetic acid. [IAA] = 0 – 125 nM
52
300
250
200
150 y = 105.74x
[BCAA](nM) R² = 0.9969 100
50
0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Response ratio Figure 3.24: Calibration curve for BCAA using bromochloroacetic acid. [BCAA]=0–250 nM
300
250
200
150 y = 85.951x R² = 0.9982 [BDCAA](nM) 100
50
0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 Response ratio Figure 3.25: Calibration curve for BDCAA using bromodichloroacetic acid. [BDCAA] = 0 –250 nM
53
600
500
400
300
[DBAA](nM) y = 139.48x 200 R² = 0.9949
100
0 0 1 2 3 4 Response ratio Figure 3.26: Calibration curve for DBAA using dibromoacetic acid. [DBAA] = 0 – 500 nM
54
Table 3.7: Limit of quantification for the detection of DBPs DBPs Limit of Quantification (nM) CHCl3 1.0 CHBrCl2 1.0 CHBr2Cl 1.0 CHClBrI 1.0 CHCl2I 0.2 CHClI2 0.2 CHBr3 1.0 CHBr2I 0.2 CHBrI2 0.2 CHI3 0.2 CAN 1.0 TCAN 1.0 DCAN 1.0 BAN 1.0 BCAN 1.0 DBAN 1.0 IAN 0.2 CAA 1.0 BAA 1.0 DCAA 1.0 TCAA 1.0 IAA 0.2 BCAA 1.0 BDCAA 1.0 DBAA 1.0
55
CHAPTER IV
RESULTS AND DISCUSSION
4.1 Introduction
This chapter evaluates the data collected through experimental investigations of
ICM transformation in the presence of chlorinated oxidants. The experiments explored factors that influence regulated and unregulated DBP formation, as well as enhanced cell cytotoxicity and genotoxicity, due to the degradation of iodinated pharmaceuticals in the presence of chlorinated oxidants and NOM. The factors investigated were pH, chlorinated oxidant type (chlorine and monochloramine), and ICM type (iopamidol and iohexol). This work also incorporates the collaborative toxicity research previously performed by Dr. Michael Plewa et al. (2013, 2015).
This chapter will be discussed in three main sections. The first section will discuss the DBP formation in the presence of iopamidol and iohexol. This will include 72 hour batch experiments and DBP formation kinetics. The second section will discuss toxicity analysis of iopamidol transformation in the chlorinated oxidants and Akron source water.
Finally, the last section will discuss the toxicity analysis of iohexol in the presence of aqueous chlorine and Akron source water.
56
4.2 DBP Formation in the Presence of ICM and Chlorinated Oxidants
The next sections will discusss the DBP formation detected in experiments
performed in the presence of ICM (iopamidol and iohexol) and chlorinated oxidants
(chlorine and monochloramine)
4.2.1 DBP Formation in the presence of Aqueous Chlorine and Iopamidol
In the aqueous chlorine and monochloramine experiments, the DBPs most
frequently detected were chloroform (CHCl3), dichloroiodomethane (CHCl2I), and
trichloroacetic acid (C2HCl3O2). In all cases, chloroform formed in the highest
concentrations. Dichloroiodomethane was the iodo-DBP that formed in the highest
concentrations. Trichloroacetic acid also formed in high concentrations. There were no
iodo-acids detected in these experiments. There were additional DBPs that were detected
and can be found in Section A1 of the appendix; however, these will not be discussed
here due to the low quantities and difficulty of interpretation.
The first 72 hour DBP formation experiments were conducted over the pH range of 6.5 – 9.0 with and without the presence of iopamidol [NOM] = 5.57 mg/L and [Cl2]T
= 100 μM,. The results from this can be seen in the following Figures (4.1 – 4.3) shown
as the differences between the source water controls and the iopamidol experiments.
Figure 4.1 shows the chloroform formation with chlorine as the oxidant. As we can see,
the concentration increases from 1564 nM at pH 6.5 to 4014 nM at pH 9.0 in the
iodinated sample. This formation is expected, as this follows the trend of THM formation
increasing as pH increases. The greatest chloroform concentration increase in the
iodinated sample is seen from 1856 nM at pH 8.5 to 4014 nM at pH 9.0, while this
57
increase was not seen in the controls. Over the pH range of 6.5-8.5, chloroform concentrations were found not to be statistically significantly different from the experiments with or without iopamidol. At pH 9 however, there was a significant increase in chloroform concentration for the experiments with iopamidol present. This suggests that iopamidol is highly reactive with aqueous chlorine inferring, and that iopamidol is a prominent chloroform precursor above pH 8.5. These results support previous findings in which it was suggested that iopamidol and its transformation products have the potential to form regulated DBPs, such as chloroform (Ackerson, 2014;
Crafton, 2014; Machek, 2015).
5000 5.0 µM IP 0.0 µM IP 4000
3000 ](nM) 3
2000 [CHCl
1000
0 6.5 7.5 8.5 9.0 pH
Figure 4.1: Observed chloroform formation at 72 hours as a function of pH. [NOM] = 5.57 mg/L-C, [iopamidol] = 5.0 μM, [Cl2]T = 100 μM, [Buffer] = 4 mM, temperature = 25°C
58
As shown below in Figure 4.2, dichloroiodomethane was formed in the source
water experiments at low concentrations from 1.36 nM at pH 6.5 to 3.06 nM at pH 9.0.
From pH 6.5-9.0, the iopamidol experiments formed dichloroiodomethane at higher concentrations from 215 to 650 This can be attributed to the transformation of iopamidol
resulting in a release of an active iodinated oxidant and reacting with NOM to form iodo-
DBPs (Duirk et al, 2011; Ye et al., 2014). Dichloroiodomethane was also found to share the same pH formation trend as chloroformTHM formation as the pH increases (Duirk et
al., 2011; Ye et al., 2014). The experiments containing iopamidol formed much larger concentration of the iodo-DBP dichloroiodomethane; whereas, the experiments without iopamidol barely formed any dichloroiodomethane. This suggests that the iopamidol is degraded in the presence of aqueous chlorine – the iodine attached to the central benzene ring is removed and transformed into iodo-DBP precursors in the NOM structure (Duirk et al., 2011).
59
1000 5.0 µM IP 0.0 µM IP 800
600 I] (nM) I] 2
400 [CHCl
200
0 6.5 7.5 8.5 9.0
pH
Figure 4.2: Observed dichloroiodomethane formation at 72 hours as a function of pH, [NOM] = 5.57 mg/L-C, [iopamidol] = 5.0 μM, [Cl2]T = 100 μM, [Buffer] = 4 mM, temperature = 25°C
It has been proposed that the hypochlorite ion (OCl-) may be the primary initiator
in iopamidol transformation (Duirk et al., 2011). OCl- is a strong nucleophile that is thought to initiate attacks on one of the amide side chains of the partially positive compound, iopamidol. This proposed nucleophile attack on the amide side chain of iopamidol would result in a primary amine transformation product (Wendel et al., 2015).
Additionally, after the side chain is removed from the reaction with OCl-, the iodine
remaining on the aromatic ring now becomes more susceptible to reaction with
hypochlorous acid (HOCl). The iodine, an electronegative nucleophile, is then more
60
likely to donate an electron pair and react with the hypochlorous acid, an electrophile.
This reaction then will rapidly oxidize the iodine from the aromatic ring forming
hypoiodous acid (HOI) (Duirk et al., 2011).
Next to be discussed will be haloacetic acid (HAA) formations. As shown in
Figure 4.3, the source water experiments and iopamidol experiments are graphed next to one another. Trichloroacetic acid (TCAA) was the focus since iopamidol is a known
TCAA precursor (Ackerson, 2014). As seen in Figure 4.3, with respect to the source water experiments, TCAA concentrations range from 825 nM at pH 6.5 down to 231 nM at pH 9.0. Additionally, with respect to the iopamidol experiments, the TCAA concentrations range from 651 nM at pH 6.5 down to 232 nM at pH 9.0. This follows the expected trend of TCAA formation decreasing as pH increases (AWWA, 2008). Upon inspection, it appears as though the iopamidol actually inhibits TCAA formation. This could be due to competitive demand for aqueous chlorine between the source water NOM and iopamidol.
61
1000 5.0 µM IP 0.0 µM IP 800
600
400 [TCAA](nM)
200
0 6.5 7.5 8.5 9.0
pH
Figure 4.3: Observed trichloroacetic acid formation at 72 hours as a function of pH, [NOM] = 5.57 mg/L-C, [iopamidol] = 5.0 μM, [Cl2]T = 100 μM, [Buffer] = 4 mM, temperature = 25°C
This study only performed DBP experiments at one NOM concentration, 5.57
mg/L; however, it should be noted that NOM concentration does have an effect on DBP
formation. Crafton (2014) found that lower levels of NOM resulted in higher formation
of iodo-DBPs, whereas Machek (2015) found that lower levels of NOM resulted in lower levels of iodo-DBPs. Ackerson (2014) also found that iopamidol forms negligible concentrations of iodo-DBPs in experiments without NOM. The formation of iodo-DBPs is seemingly dependent on the varying source of organic material, as described by
Machek (2015). Akron source water, as used for this study, varies greatly from Cleveland
62
source water, as used by Crafton (2014), and Barberton source water, as used by Machek
(2015). Source water characteristics include TOC, SUVA, and percent compositions of
EEM regions. Source water characteristics, such as humic and fulvic content, play
important roles in the type and amounts of DBPs that are able to form (Duirk et al.,
2011). Fulvic acid may be more competitive with iopamidol with regards to DBP
transformation. DBP formation has been shown to be linked to SUVA254 and DOC
concentration (Njam et al., 1994).
4.2.2 Akron Kinetics Experiments in the presence of Aqueous Chlorine and Iopamidol
The next set of experiments conducted were kinetic experiments to observe DBP
formation over 72 hours. These experiments were conducted under the same
experimental conditions as the previously discussed 72 hour experiments; therefore,
direct comparisons can be made. Shown below in Figures 4.4 – 4.7 are the kinetics
experiments at each pH from 0-72 hours with chlorine as the oxidant. The iopamidol concentration used for these experiments was decreased to 2.5 μM, as opposed to 5 μM in the 72 hour experiments. This was done to ensure that there would be enough chlorine to react throughout the entire 72 hours and result in an excess residual chlorine concentration.
The kinetics experiment graphs are displayed such that each shows the formation concentration for three compounds at one pH, in an attempt to simplify the analysis.
Additional kinetics graphs displayed such that each shows one compound’s DBP formation at all four pHs tested can be found in Section A.1 of the appendix. The three
63
compounds chosen for kinetics analysis were chloroform, dichloroiodomethane, and trichloroacetic acid.
As seen in Figure 4.4, observed DBP formation as a function of time is graphed at pH 6.5. The compounds displayed are chloroform, dichloroiodomethane, and trichloroacetic acid. TCAA is the highest forming DBP at pH 6.5 with a final concentration of 1893 nM. Chloroform was the second highest DBP formation with a final concentration of 1749 nM. The lowest forming DBP at pH 6.5 was dichloroiodomethane with a final concentration of 160 nM. These values are similar to those from the 72 hour chlorine experiments and discussed earlier.
Next to be discussed will be DBP kinetics formation at pH 7.5, as seen in Figure
4.5. Chloroform was the DBP that formed in the highest concentration at 1942 nM. The next highest forming DBP was TCAA at 1333 nM; the lowest forming DBP was dichloroiodomethane at 157 nM. These results match those discussed earlier for the 72 hour chlorine experiments.
64
2500
CHCl3 CHCl I 2000 2 TCAA
1500
1000 Concentration(nM)
500
0 0 20 40 60 80
Time (hours) Figure 4.4: Observed DBP formation at pH 6.5 as a function of time. [NOM] = 5.57 mg/L-C, [iopamidol] = 2.5 μM, [Cl2]T = 100 μM, [Buffer] = 4 mM, temperature = 25°C
65
2500
CHCl3 CHCl I 2000 2 TCAA
1500
1000 Concentration(nM)
500
0 0 20 40 60 80
Time (hours) Figure 4.5: Observed DBP formation at pH 7.5 as a function of time, [NOM] = 5.57 mg/L-C, [iopamidol] = 2.5 μM, [Cl2]T = 100 μM, [Buffer] = 4 mM, temperature = 25°C
66
The DBP kinetics formation at pH 8.5 are shown in Figure 4.6. Again, chloroform formed in the highest concentration at 1887 nM. TCAA formation was second highest, at
637 nM; and dichloroiodomethane formation was the lowest, at 150 nM. These results coincide with those of the original 72 hour experiments, as discussed earlier.
Last to be discussed will be the DBP formation at pH 9.0, as seen in Figure 4.7.
Again, chloroform formation was the highest at 2349 nM. TCAA formation was second highest, at 502 nM; and dichloroiodomethane formation was lowest, at 187 nM. Again, these results match the earlier discussed results from the 72 hour chlorine experiments.
These kinetics experiments follow the expected trend for THM formation in which concentration increases as pH increases. Additionally, these graphs support the claim that most of the DBP formation occurs within the first 24 hours regardless of pH, as found in previous works (Ackerson, 2014; Crafton, 2014; Machek, 2015). A possible reason for this is that the oxidant and DBP precursor compounds are being consumed, resulting in slower reaction rates as they continue consumption reactions.
67
2500
CHCl3 2000 CHCl2I TCAA
1500
1000 Concentration(nM)
500
0 0 20 40 60 80
Time (hours) Figure 4.6: Observed DBP formation at pH 8.5 as a function of time, [NOM] = 5.57 mg/L-C, [iopamidol] = 2.5 μM, [Cl2]T = 100 μM, [Buffer] = 4 mM, temperature = 25°C
68
3000
CHCl3 2500 CHCl2I TCAA 2000
1500
Concentration(nM) 1000
500
0 0 20 40 60 80
Time (hours) Figure 4.7: Observed DBP formation at pH 9.0 as a function of time, [NOM] = 5.57 mg/L-C, [iopamidol] = 2.5 μM, [Cl2]T = 100 μM, [Buffer] = 4 mM, temperature = 25°C
4.2.3 DBP Formation in the presence of Monochloramine and Iopamidol
Experiments were conducted using monochloramine (NH2Cl) as the chlorinated
oxidant under the same conditions as the aqueous chlorine experiments. Similar to the
chlorine experiments, the DBPs most frequently detected were chloroform (CHCl3),
dichloroiodomethane (CHCl2I), and trichloroacetic acid (TCAA). The following
discussion focuses on the formation trends of these DBPs as a function of pH. Additional
information regarding monochloramine experiments can be found in Section A1 of the
appendix.
69
The 72 hour monochloramine DBP formation experiments were conducted over the pH range of 6.5 – 9.0 with and without the presence of iopamidol in the presence of
Akron source water. In figures (4.8 – 4.10), the differences between the source water controls and the iopamidol experiments are shown. Figure 4.8 shows the chloroform formation with monochloramine as the oxidant. The concentration of chloroform decreases from 56 nM at pH 6.5 to 13 nM at pH 9.0 in the iodinated sample. This follows the expected formation trend in the presence of monochloramine, chloroform formation decreases as pH increases. The greatest concentration decrease in the iodinated sample is seen from 56 nM at pH 6.5 to 31 nM at pH 7.5. The decrease in chloroform formation as the pH increases is due to complex reactions resulting in monochloramine decomposition which leaves aqueous chlorine more available to react with NOM near a neutral pH
(Duirk et al. 2011). The slightly higher concentrations of chloroform in the experiments with iopamidol than the control experiment is likely due to the fact that iopamidol is transformed and the transformation products continue to react with monochloramine resulting in the formation of chloroform.
70
70 5.0 µM IP 60 0.0 µM IP
50
40 ](nM) 3 30 [CHCl
20
10
0 6.5 7.5 8.5 9.0
pH
Figure 4.8: Observed chloroform formation at 72 hours as a function of pH, [NOM] = 5.57 mg/L-C, [iopamidol] = 5.0 μM, [NH2Cl]T = 100 μM, [Buffer] = 4 mM, temperature = 25°C
Dichloroiodomethane was the only iodo-DBP detected in monochloramine experiments at concentrations above detection limits of 1 nM. As shown in Figure 4.9, dichloroiodomethane formation in control experiments and iopamidol experiments were plotted as a function of pH. The formation of this iodo-DBP in the source water experiments suggests that there may be trace amounts of iodide within the Akron source water. This formation was not as evident in the aqueous chlorine experiments due to the fact that aqueous chlorine is a stronger oxidant and oxidized the residual iodide to iodate
(Bishell and von Gunten, 1999a). Dichloroiodomethane formation at pH 6.5 was 52 nM 71
in iopamidol experiments compared to 25 nM in control experiments. As pH increased
from 6.5-9.0, dichloroiodomethane decreased; this is due to the stability of monochloramine increasing as pH increases (Duirk et al., 2005). Dichloroiodomethane was detected at higher concentrations with the iopamidol than without it being present; this supports that iopmaidol does result in the formation of iodo-DPBs in the presence of
ICM and NOM (Duirk et al., 2011; Ye et al., 2014).
60 5.0 µM IP 0.0 µM IP 50
40 I] (nM) I]
2 30 [CHCl 20
10
0 6.5 7.5 8.5 9.0
pH
Figure 4.9: Observed dichloroiodomethane formation at 72 hours as a function of pH, [NOM] = 5.57 mg/L-C, [iopamidol] = 5.0 μM, [NH2Cl]T = 100 μM, [Buffer] = 4 mM, temperature = 25°C
72
Trichloroacetic acid formation in the presence of monochloramine with and
without iopamidol present was monitored as a function of pH. As shown below in Figure
4.10, TCAA formation was plotted from the control and iopamidol experiments. The observed concentrations of TCAA were extremely low regardless of pH. Therefore, it was difficult to determine any trend that was statistically significant. It appears to follow the trend of decreasing TCAA formation as the pH increases; however, it is difficult to definitively state due to the extremely low concentrations.
8 5.0 µM IP 0.0 µM IP
6
4 [TCAA](nM)
2
0 6.5 7.5 8.5 9.0
pH
Figure 4.10: Observed trichloroacetic acid formation at 72 hours as a function of pH, [NOM] = 5.57 mg/L-C, [iopamidol] = 5.0 μM, [NH2Cl]T = 100 μM, [Buffer] = 4 mM, temperature = 25°C
73
The experiments presented in this work did not vary the concentration of NOM.
However, in previous works by Crafton (2014) and Machek (2015) DBP formation in the
presence of monochloramine decreased as NOM levels decreased. Though the
monochloramine experiments still produced increased levels of DBPs compared to the
control experiments, the concentrations were significantly lower than those produced in
the aqueous chlorine experiments. This can be attributed to the differences in oxidants
used and the rate at which they react with organic material; monochloramine reacts at a
much slower rate. Monochloramine has been shown to react with iopamidol to form iodo-
DBPs in the presence of NOM (Duirk et al., 2011). The iodide on the aromatic ring of
iopamidol can be oxidized to HOI, similar to the chlorine reaction pathways (Bichsel and
von Gunten, 1999). HOI has been determined to be stable in the presence of
monochloramine, as supported by the lack of formation of iodate detected by Ackerson
(2014) (Bichsel and von Gunten, 1999a). This work supports the findings by Duirk et al.
(2011), in that iodo-DBP formation was greatest at a lower pH of 6.5 in the presence on
monochloramine.
4.2.4 Akron Kinetics Experiments in the presence of Monochloramine and Iopamidol
The next set of experiments conducted were monochloramine kinetic experiments to observe DBP formation over 72 hours. The iopamidol concentration used for these experiments was decreased to 2.5 μM, as opposed to 5 μM in the 72 hour experiments.
This was done to ensure that there would be enough active oxidant resulting in a higher detectable residual oxidant concentration throughout the entire 72 hours.
74
The kinetics experiments (Figure 4.11-4.14) are displayed such that each shows the formation concentration for three compounds at one pH, in an attempt to simplify the analysis. Additional kinetics graphs displayed such that each shows one compound’s
DBP formation at all four pH tested can be found in Section A.1 of the appendix. As seen in Figure 4.11, observed DBP formation was plotted as a function of time. The compounds displayed are chloroform, dichloroiodomethane, and trichloroacetic acid.
Chloroform was formed the most with a final concentration of 61 nM. TCAA is the second highest forming DBP at pH 6.5 with a final concentration of 14 nM. The lowest forming DBP at pH 6.5 was dichloroiodomethane with a final concentration of 11 nM.
Next to be discussed will be DBP kinetics formation at pH 7.5, as seen in Figure 4.12.
Chloroform was the DBP that formed in the highest concentration at 48 nM. The next highest forming DBP was TCAA at 8 nM; the lowest forming DBP was dichloroiodomethane at 7 nM. The DBP kinetics formation at pH 8.5 are shown in Figure
4.13. Again, chloroform formed in the highest concentration at 41 nM.
Dichloroiodomethane formation was second highest, at 6 nM; and TCAA formation was the lowest, at 5 nM. Last to be discussed will be the DBP formation at pH 9.0, as seen in
Figure 4.14. Again, chloroform formation was the highest at 38 nM.
Dichloroiodomethane formation was second highest, at 5 nM; and TCAA formation was lowest, at 4 nM. These kinetics experiments follow the expected trend that DBP formation concentrations decreases as pH increases. Additionally, this supports that most of the DBPs form within the first 24 hours regardless of pH, as found in previous works
(Ackerson, 2014; Crafton, 2014; Machek, 2015).
75
80
CHCl3 CHCl2I 60 TCAA
40 Concentration(nM) 20
0 0 20 40 60 80
Time (hours) Figure 4.11: Observed DBP formation at pH 6.5 as a function of time, [NOM] = 5.57 mg/L-C, [iopamidol] = 2.5 μM, [NH2Cl]T = 100 μM, [Buffer] = 4 mM, temperature = 25°C
76
60
CHCl3
50 CHCl2I TCAA
40
30
20 Concentration(nM)
10
0 0 20 40 60 80 Time (hours)
Figure 4.12: Observed DBP formation at pH 7.5 as a function of time, [NOM] = 5.57 mg/L-C, [iopamidol] = 2.5 μM, [NH2Cl]T = 100 μM, [Buffer] = 4 mM, temperature = 25°C
77
60
CHCl3 50 CHCl2I TCAA
40
30
Concentration(nM) 20
10
0 0 20 40 60 80
Time (hours) Figure 4.13: Observed DBP formation at pH 8.5 as a function of time, [NOM] = 5.57 mg/L-C, [iopamidol] = 2.5 μM, [NH2Cl]T = 100 μM, [Buffer] = 4 mM, temperature = 25°C
78
50 CHCl3 CHCl2I 40 TCAA
30
20 Concentration(nM)
10
0 0 20 40 60 80 Time (hours)
Figure 4.14: Observed DBP formation at pH 9.0 as a function of time, [NOM] = 5.57 mg/L-C, [iopamidol] = 2.5 μM, [NH2Cl]T = 100 μM, [Buffer] = 4 mM, temperature = 25°C
4.2.5 DBP Formation in the presence of Aqueous Chlorine and Iohexol
The iohexol experiments were conducted over 72 hours with the same chlorine and equivalent iohexol dose in Akron source water; thus, these experiments are directly comparable to the 72 hour iopamidol experiments over the pH range of 6.5 – 9.0. The
DBPs monitored were chloroform (CHCl3) dichloroiodomethane (CHCl2I), and trichloroacetic acid (TCAA). There were no iodo-acids detected in these experiments or the iopamidol experiments. Additional DBPs that were detected and can be found in
Section A2 of the appendix. 79
The results from the iohexol experiments can be seen in the following Figures
(4.15 – 4.17) shown as the differences between the source water controls and the iohexol
experiments. Figure 4.15 shows the chloroform formation concentration only slightly
increases from 2437 nM at pH 6.5 to 2613 nM at pH 9.0 in the iohexol source water
experiment, which is significantly different than both the control and in the iopamidol
experiments. While iohexol is know not to be reactive with aqueous chlorine (Wendel
2014), chlorination of the iohexol reagent standard was conducted over the pH range of
6.5-9.0 in the absence of NOM. As shown in Figure 4.15, chloroform formation was
found to be relatively constant as a function of pH in the iohexol DI experiment. This
chloroform formation in the iohexol DI experiments proves that there was an unidentified
product present that was reactive with aqueous chlorine and responsible for the additional
chloroform formation. This suggests that the elevated chloroform concentrations in the
iohexol source water experiments may be due to the unidentified product within the
iohexol standard; thus, the chloroform may not be formed by the reaction of iohexol with
chlorine.
Dichloroiodomethane formation was significantly low, but still observable. In
Figure 4.16, the source water experiments formed dichloroiodomethane at low concentrations from 1.36 nM to 3.06 nM as pH increased from 6.5-9.0. Whereas, the iohexol experiments formed dichloroiodomethane at slightly higher concentrations from
5 nM to 17 nM as pH increased from 6.5-9.0. This is somewhat unexpected, as it has been suggested that iohexol does not react with aqueous chlorine (Wendel et al., 2014).
The trend shown here dichloroiodomethane formation in the presence of iohexol is
similar to the iopamidol but at significantly lower concentrations (Ye et al., 2014).
80
However, the increase of dichloroiodomethane concentrations from the source water experiments to the iohexol experiments is quite a small amount, only 15 nM at pH 9. This could be due to an unidentified product in the purchased standard.
3500 5.0 µM IHX - SW 3000 0.0 µM IHX - SW 5.0 µM IHX - DI
2500
2000 ](nM) 3 1500 [CHCl
1000
500
0 6.5 7.5 8.5 9.0 pH
Figure 4.15: Observed chloroform formation at 72 hours as a function of pH, [NOM] = 5.57 mg/L-C, [iohexol] = 5.0 μM, [Cl2]T = 100 μM, [Buffer] = 4 mM, temperature = 25°C
81
20
18 5.0 µM IHX 0.0 µM IHX 16
14
12 I] (nM) I]
2 10
8 [CHCl 6
4
2
0 6.5 7.5 8.5 9.0
pH Figure 4.16: Observed dichloroiodomethane formation at 72 hours as a function of pH, [NOM] = 5.57 mg/L-C, [iohexol] = 5.0 μM, [Cl2]T = 100 μM, [Buffer] = 4 mM, temperature = 25°C
TCAA formation concentrations, shown in Figure 4.17, range from 825 nM at pH
6.5 down to 231 nM at pH 9.0 in the control experiments. Additionally, with respect to the iohexol experiments, the TCAA concentrations range from 847 nM at pH 6.5 down to
202 nM at pH 9.0. The TCAA concentrations detected for the control and iohexol experiments are at nearly the same levels. These results suggest that there was no additional TCAA formation within the source water in the presence of iohexol. This is expected, as iohexol has been suggested as being unreactive in the presence of aqueous chlorine (Wendel et al., 2014).
82
1000 5.0 µM IHX 0.0 µM IHX 800
600
400 [TCAA](nM)
200
0 6.5 7.5 8.5 9.0
pH Figure 4.17: Observed trichloroacetic acid formation at 72 hours as a function of pH, [NOM] = 5.57 mg/L-C, [iohexol] = 5.0 μM, [Cl2]T = 100 μM, [Buffer] = 4 mM, temperature = 25°C
4.3 Chronic Cytotoxicity and Acute Genotoxicity Analysis for ICM in the Presence of
Chlorinated Oxidants and Akron Source Water
The next sections will disucss the cytotoxicity and genotoxicity analysis for ICM
(iopamidol and iohexol) in the presence of chlorinated oxidants (chlorine and monochloramine). The iopamidol experiments were performed in the presence of both oxidants, while the iohexol experiments were only performed in the presence of chlorine.
83
4.3.1 Cyto/Genotoxicity of Iopamidol in the presence of Chlorinated Oxidants
Mammalian Chinese hamster ovarian (CHO) cells were utilized in the chronic
cytotoxicity and acute genotoxicity experiments with Akron source water. This section
discusses the experimental results from the analytical biological studies on the
cytotoxicity and genotoxicity of Akron water samples treated with chlorine, with and
without the presence of iopamidol. Twenty liters of Akron source water containing 5 μM
iopamidol was chlorinated with 100 μM chlorine at pH 7.5. The treated source water was
then concentrated using XAD resins, eluted with 400 mL of ethyl acetate, and was
concentrated to 1 mL, as described in Chapter III. The extracted samples were then
shipped to Dr. Plewa’s laboratory for toxicity analysis.
Cytotoxicity was measured as a reduction in mammalian cell density after cell
exposure to the water sample concentration (WSC) for 72 hours (Plewa et al., 2013;
Plewa et al., 2015). Highly concentrated WSCs were stored in the freezer in dimethyl sulfoxide (DMSO). For each experiment, a dilution series (generally 10 concentrations) was created by diluting the WSCs into the culture prior to the experiment. The dilutions were then transferred to the CHO cells in 96-well microplates, as well as the positive and negative controls, and allowed to set for 72 hours before histological staining to interpret the results. The dilution series constructed from the stock concentrate represents a range of concentration factors for the organics in the original water. The range in concentration factors is selected to span the range between no significant reduction in growth (similar to an untreated, negative control) and high concentration factors where no or very little growth is observed (Plewa et al., 2013; Plewa et al., 2015). A cytotoxicity concentration- response curve for each WSC was then generated from the summary data from combined 84
replicate experiments. The concentration factor associated with a 50% reduction in cell
density as compared to the concurrent negative controls (LC50) was calculated for each
WSC from a non-linear regression analysis of the concentration-response curve (Plewa et
al., 2013; Plewa et al., 2015). The LC50 values can then be used to quantify the relative cytotoxicity, by using cytotoxicity index values, of different water samples (Plewa et al.,
2013; Plewa et al., 2015). The variations of experiments performed can be seen in Table
3.4 of Chapter III. It should be noted that AOHTOX-Iopromide, AOHTOX-Cl-
Iopromide, AOHTOX-Iomeprol, AOHTOX-Cl-Iomeprol, AOHTOX-Diatrizoate, and
AOHTOX-Cl-Diatrizoate will not be discussed, as the results from these experiments showed no reactivity with chlorine.
The cytotoxicity response curves for the aqueous chlorine experiments, with and without the presence of iopamidol, can be seen in Figure 4.18. The cytotoxicity response curves are displayed for the Akron Source Water (ASW) control experiment, the aqueous chlorine experiment, and the aqueous chlorine experiment with iopamidol. The LC50
values, as well as additional individual concentration response curves can be found in
Section A1 of the appendix. Figure 4.18 shows that as we move from the control
experiment, to the chlorination control, to the chlorine and iopamidol experiment – the
LC50 values decrease from 161.3, to 53.0, to 47.6, respectively. Cytotoxicity was the
greatest when Akron source water was chlorinated with iopamidol present.
85
ASW Control 100 ASW + Cl2 ASW + Cl2 + IDOL
80
60
40
20 asthe Percent of Negative Control (±SE) CHO Cell Cytotoxicity: Mean Cell Density Cell Mean Cytotoxicity: Cell CHO 0
0 50 100 150 200 250 Akron OH Source Water 2013 Samples (Concentration Factor) Figure 4.18: Comparison of observed CHO Cell Chronic Cytotoxicity as a function of Mean Cell Density for samples AOHTOX-SW, AOHTOX-CL, and AOHTOX-CL- IDOL.
The next section will discuss the cytotoxicity experimental results with monochloramine as the oxidant. The variations of experiments performed can be seen in
Table 3.4 of Chapter III. It should be noted that AOHTOX-Iopromide, AOHTOX-Cl-
Iopromide, AOHTOX-Iomeprol, AOHTOX-Cl-Iomeprol, AOHTOX-Diatrizoate, and
AOHTOX-Cl-Diatrizoate will not be discussed, as the results from these experiments showed no reactivity with monochloramine.
The cytotoxicity response curves for the monochloramine experiments, with and without the presence of iopamidol, can be seen below in Figure 4.19. The cytotoxicity 86
response curves are displayed for the Akron Source Water (ASW) control experiment,
the monochloramine experiment, and the monochloramine experiment with iopamidol.
The concentration factor associated with 50% reduction in cell density (LC50), as
compared to the negative controls, was calculated for each WSC from a non-linear
regression analysis of the concentration-response curve (Plewa et al., 2013; Plewa et al.,
2015). The LC50 values, as well as additional individual concentration response curves
can be found in Section A1 of the appendix.
Figure 4.19 shows that as we move from the control experiment, to the
monochloramine experiment, and finally, monochloramine and iopamidol – the LC50 values decrease from 161.3, to 129.3, to 60.3, respectively. As the point at which 50% reduction in cell density occurs decreases, the cytotoxicity increases. In other words, the cytotoxicity increases as the mean cell density decreases.
Additionally, Figure 4.20 displays the CHO cytotoxic index values for all experiments graphed next to one another. The cytotoxicity index (CTI) values were calculated using the LC50 values from each of the graphed cytotoxicity response curves.
-1 3 The CTI was calculated as (LC50) x 10 . The CTI values can be used to easily quantify cytotoxicity results as the greater the CTI value, the greater the cytotoxicity of the sample. From Figure 20, we can see that aqueous chlorine with iopamidol seems to induce a higher level of mammalian cell cytotoxicity than with disinfectant alone with values of 21.0 and 18.8, respectively. This is expected and supports the previous conclusions that iopamidol transforms in the presence of aqueous chlorine to form DBPs.
Additionally, we can see that monochloramine with iopamidol also appears to induce a higher level of mammalian cell cytotoxicity than monochloramine alone with values of 87
16.5 and 7.8, respectively, though not to the extent of the chlorine experiments. The chlorine experiments generated a 2.3x higher level of cytotoxicity as compared to the chloramine experiments. This is expected and supports the previous conclusions that iopamidol transforms in the presence of monochloramine to form DBPs at lower concentrations than with chlorine disinfection.
ASW Control 100 ASW + NH2Cl ASW + NH2Cl + IDOL 80
60
40
20 asthe Percent of Negative Control (±SE) CHO Cell Cytotoxicity: Mean Cell Density Cell Mean Cytotoxicity: Cell CHO 0
0 50 100 150 200 250 Akron OH Source Water 2013 Samples (Concentration Factor) Figure 4.19: Comparison of observed CHO Cell Chronic Cytotoxicity as a function of Mean Cell Density for samples AOHTOX-SW, AOHTOX-NH2CL, and AOHTOX- NH2CL-IDOL.
88
25
20
15
10 CHOCell SCGE Averages
Cytotoxicity Index Values (±SE) Values Index Cytotoxicity 5
0
AOHTOX-CL AOHTOX-SW AOHTOX-IDOL AOHTOX-CL-IDOLAOHTOX-NH2CL AOHTOX-NH2CL-IDOL
-1 3 Figure 4.20: All CHO Cytotoxicity Index Values calculated as (LC50) x 10
Next to be discussed will be the CHO single cell gel electrophoresis (SCGE)
assay genotoxicity analysis. More specifically, SCGE is a molecular genetic assay that
quantitatively measures the level of genomic DNA damage induced by a test agent or
WSC in individual nuclei of cells (Plewa et al., 2013; Plewa et al., 2015). Induced DNA lesions that lead to single and double strand DNA breaks allow the DNA to migrate out of the nucleus within a microgel subjected to electrophoresis (Plewa et al., 2013; Plewa et al., 2015). The SCGE metric for genomic DNA damage is the % Tail DNA value, i.e. the amount of DNA that has migrated from the nucleus into the microgel (Plewa et al., 2013; 89
Plewa et al., 2015). The microplate methodology for genotoxicity analysis was similar to
the cytotoxicity analysis, as previously discussed, however the length of exposure with
the WSC was only 4 hours, in order to maximize the ability to detect genomic damage
while limiting the effect of DNA repair. Again, the variations of experiments performed can be seen in Table 3.4 of Chapter III. Concentration response curves, such as those shown in Figure 4.21, were created and non-linear regression analysis was performed to fit each curve and determine the concentration factor that induces 50% of the genomic
DNA to migrate from the nucleus (50% Tail DNA Value).
The genotoxicity response curves for the aqueous chlorine experiments, with and without the presence of iopamidol are shown in Figure 4.21. The genotoxicity response curves are displayed for the Akron Source Water (ASW) control experiment, the aqueous chlorine experiment, and the aqueous chlorine experiment with iopamidol. The concentration factor associated with 50% of the genomic DNA migrating from the nucleus (50% Tail DNA Value), as compared to the negative controls, was calculated for each WSC from a non-linear regression analysis of the concentration-response curve
(Plewa et al., 2013; Plewa et al., 2015). The 50% Tail DNA Values, as well as additional individual concentration response curves can be found in Section A1 of the appendix.
Figure 4.21 shows that as we move from the control experiment, to the chlorine experiment, and finally, chlorine and iopamidol – the 50% Tail DNA Values decrease from 680.0, to 244.3, to 140.1, respectively. As the point at which 50% of the genomic
DNA migrating from the nucleus occurs decreases, the genotoxicity increases. In other words, the genotoxicity increases as the 50% Tail DNA Value decreases.
90
80 ASW Control ASW + Cl2 ASW + Cl2 + IDOL 60
40
20 MeanSCGE Tail% DNA Value(±SE) CHOCell Genoimc DNA Damage as the Average 0 0 200 400 600 Akron OH Source Water 2013 Samples (Concentration Factor) Figure 4.21: Comparison of observed CHO Cell Acute Genotoxicity as a function of Genomic DNA Damage for samples AOHTOX-SW, AOHTOX-CL, and AOHTOX-CL- IDOL.
The genotoxicity response curves for the monochloramine experiments, with and without the presence of iopamidol are shown in Figure 4.22. The genotoxicity response curves are displayed for the Akron Source Water (ASW) control experiment, the monochloramine experiment, and the monochloramine experiment with iopamidol. The concentration factor associated with 50% of the genomic DNA migrating from the nucleus (50% Tail DNA Value), as compared to the negative controls, was calculated for each WSC from a non-linear regression analysis of the concentration-response curve
91
(Plewa et al., 2013; Plewa et al., 2015). The 50% Tail DNA Values, as well as additional
individual concentration response curves can be found in Section A1 of the appendix.
Figure 4.22 shows that as we move from the control experiment, to the
chloramine experiment, and finally, chloramine and iopamidol – the 50% Tail DNA
Values decrease from 680.0, to 565.5, to 287.9, respectively. As the point at which 50%
of the genomic DNA migrating from the nucleus occurs decreases, the genotoxicity increases. In other words, the genotoxicity increases as the 50% Tail DNA Value decreases.
Additionally, Figure 4.23 displays the CHO genotoxic index values for all experiments graphed next to one another. The genotoxicity index (GTI) values were calculated using the 50% Tail DNA values from each of the graphed genotoxicity response curves. The GTI was calculated as (50% Tail DNA)-1 x 103. The GTI values can
be used to easily quantify genotoxicity results as the greater the GTI value, the greater the
genotoxicity of the sample. From Figure 4.23, we can see that chlorinated water with
iopamidol induced a higher level of genotoxicity that chlorinated water alone at values of
7.1 and 4.0, respectively. From Figure4.23, we can also see that monochloraminated
water with iopamidol induced a higher level of genotoxicity that monochloraminated
water alone at values of 3.5 and 1.7, respectively. Although iopamidol itself was not
genotoxic, it enhanced the genotoxicity of source water after chlorine and
monochloramine disinfection; however the chlorine disinfected waters were still more
genotoxic than chloraminated waters. This is expected and supports the previous
conclusions that iopamidol transforms in the presence of aqueous chlorine and
monochloramine to form DBPs. It can be concluded that iopamidol, when added to 92
source water, is generating additional DBPs (especially iodo-DBPs) after disinfection
with chlorine and chloramines.
ASW Control ASW + NH2Cl 80 ASW + NH2Cl + IDOL
60
40
20 MeanSCGE Tail% DNA Value (±SE) CHOCell Genoimc DNA Damage as the Average 0 0 200 400 600 Akron OH Source Water 2013 Samples (Concentration Factor) Figure 4.22: Comparison of observed CHO Cell Acute Genotoxicity as a function of Genomic DNA Damage for samples AOHTOX-SW, AOHTOX-NH2CL, and AOHTOX- NH2CL-IDOL.
93
8
6
4
CHOCell SCGE Averages 2 Genotoxicity Index Values (±SE) Index Genotoxicity
0
AOHTOX-CL AOHTOX-SW AOHTOX-IDOL AOHTOX-CL-IDOLAOHTOX-NH2CL AOHTOX-NH2CL-IDOL
Figure 4.23: All CHO Genotoxicity Index Values calculated as (50% Tail DNA)-1 x 103
A statistical analysis was also performed for the iopamidol cytotoxic and genotoxic results, more details and the results from this can be seen in Section A1 of the appendix. Precision analyses were conducted on each set of data for each bioassay. The process follows the generation of the concentration-response curve from combined replicate experiments with a test for significance using a one-way analysis of variance
(ANOVA) test (Plewa et al., 2013; Plewa et al., 2015). If a significant F value (P ≤ 0.05) was obtained, a Holm-Sidak multiple comparison test versus the control group was
94
conducted to identify the lowest toxic concentration factor (Plewa et al., 2015). After the
non-linear regression analysis for cytotoxic or genotoxic assays, a bootstrap statistic was
conducted for the results of each bioassay dataset and a mean toxicity index value was
calculated (Plewa et al., 2013; Plewa, et al., 2015). Using the toxicity index values, an
ANOVA test was conducted for each WSC and significant differences were identified.
4.3.2 Cyto/Genotoxicity of Iohexol in the presence of Aqueous Chlorine
Cytotoxicity was measured as a reduction in cell density after cell exposure to the
water sample concentration (WSC) for 72 hours (Plewa et al., 2015). For each
experiment, a dilution series (generally 10 concentrations) was created by diluting the
WSCs into the culture prior to the experiment. The dilutions were then transferred to the
CHO cells in 96-well microplates, as well as the positive and negative controls, and
allowed to set for 72 hours before histological staining to interpret the results, similar to the iopamidol toxicity experiments. The variations of experiments performed can be seen in Table 3.4 of Chapter III.
The cytotoxicity response curves for the aqueous chlorine experiments, with and without the presence of iohexol, can be seen in Figure 4.24. The concentration factor associated with 50% reduction in cell density (LC50), as compared to the negative
controls, was calculated for each WSC from a non-linear regression analysis of the concentration-response curve (Plewa et al., 2013; Plewa et al., 2015). The LC50 values,
as well as additional individual concentration response curves can be found in Section A1
of the appendix. Figure 4.24 shows that as we move from the control experiment, to the
95
chlorine experiment, and finally, chlorine and iohexol – the LC50 values decrease from
107.1, to 55.9, to 45.5, respectively. As the point at which 50% reduction in cell density occurs decreases, the cytotoxicity increases. In other words, the cytotoxicity increases as the mean cell density decreases.
The cytotoxicity index (CTI) values were calculated using the LC50 values from
-1 each of the graphed cytotoxicity response curves. The CTI was calculated as (LC50) x
103. The CTI values can be used to easily quantify cytotoxicity results as the greater the
CTI value, the greater the cytotoxicity of the sample. Figure 4.25 displays the CHO
cytotoxic index values for all experiments graphed next to one another; chlorine with
iohexol exhibits greater cytotoxicity than chlorine alone, with values of 21.6 and 18.0,
respectively. From Figures 4.24 and 4.25, we can see that iohexol with aqueous chlorine
appears to induce a higher level of mammalian cell cytotoxicity than with disinfectant
alone. It should be noted that the increased cytotoxicity within the iohexol results may be
due to the chloroform contaminant detected within the iohexol analytical standard, as
previously discussed. Regardless, iohexol appeared to enhance the cytotoxicity of the
Akron water, though to a lesser extent than iopamidol.
96
120 ASW Control ASW + Cl2 100 ASW + Cl2 + IHXL
80
60
40
20 CHO Cell Toxicity: Mean Cell Density Cell Mean Toxicity: Cell CHO asthe Percent of Negative Control (±SE)
0
0 20 40 60 80 100 120 140 160 Akron OH Source Water 2015 Samples (Concentration Factor) Figure 4.24: Comparison of observed CHO Cell Chronic Cytotoxicity as a function of Mean Cell Density for samples AOHTOX-SW, AOHTOX-CL, and AOHTOX-CL- IHXL.
97
25
20
15
10 CHOCell SCGE Averages
Cytotoxicity Index Values (±SE) Values Index Cytotoxicity 5
0
ASW + Cl2
ASW Negative Control ASW + Cl2 + Iohexol
-1 3 Figure 4.25: All CHO Cytotoxicity Index Values calculated as (LC50) x 10
CHO single cell gel electrophoresis (SCGE) assay genotoxicity analysis was
performed on the cytotoxicity samples. Genotoxicity was measured as the level of
genomic DNA damage induced by the WSC in individual nuclei of cells (Plewa et al.,
2013; Plewa et al., 2015). The SCGE metric for genomic DNA damage is the % Tail
DNA value, i.e. the amount of DNA that has migrated from the nucleus into the microgel
(Plewa et al., 2013; Plewa et al., 2015). The microplate methodology for genotoxicity
analysis was similar to the cytotoxicity analysis, as previously discussed, however the
length of exposure with the WSC was only 4 hours. Again, the variations of experiments
98
performed can be seen in Table 3.4 of Chapter III. Concentration response curves, such as those shown above in Figure 4.26, were created and non-linear regression analysis was performed to fit each curve and determine the concentration factor that induces 50% of the genomic DNA to migrate from the nucleus (50% Tail DNA Value).
The genotoxicity response curves for the aqueous chlorine experiments, with and without the presence of iohexol are shown in Figure 4.26. The genotoxicity response curves are displayed for the Akron Source Water (ASW) control experiment, the aqueous chlorine experiment, and the aqueous chlorine experiment with iohexol. The concentration factor associated with 50% of the genomic DNA migrating from the nucleus (50% Tail DNA Value), as compared to the negative controls, was calculated for each WSC from a non-linear regression analysis of the concentration-response curve
(Plewa et al., 2013; Plewa et al., 2015). The 50% Tail DNA Values, as well as additional individual concentration response curves can be found in Section A1 of the appendix.
Figure 4.26 shows that as we move from the control experiment, to the chlorine experiment, and finally, chlorine and iohexol – the 50% Tail DNA Values decrease from
400.4, to 362.4, to 258.4, respectively. As the point at which 50% of the genomic DNA migrating from the nucleus occurs decreases, the genotoxicity increases. In other words, the genotoxicity increases as the 50% Tail DNA Value decreases.
Additionally, Figure 4.27 displays the CHO genotoxic index values for all experiments graphed next to one another. The genotoxicity index (GTI) values were calculated using the 50% Tail DNA values from each of the graphed genotoxicity response curves. The GTI was calculated as (50% Tail DNA)-1 x 103. The GTI values can be used to easily quantify genotoxicity results as the greater the GTI value, the greater the 99
genotoxicity of the sample. From Figure 4.27, we can see that chlorinated water with iohexol induced a higher level of genotoxicity that chlorinated water alone at values of
3.8 and 2.8, respectively. From Figures 4.26 and 4.27, we can see that chlorinated water with iohexol induced a higher level of genotoxicity that chlorinated water alone. It should be noted that the increased genotoxicity within the iohexol results may be due to the chloroform contaminant detected within the iohexol analytical standard, as previously discussed. Although iohexol itself was not genotoxic, it enhanced the genotoxicity of source water after chlorine disinfection. It can be concluded that iohexol enhances the genotoxicity of Akron water.
100
100 ASW Control ASW + Cl2 80 ASW + Cl2 + IHXL
60
40
20 MeanSCGE Tail% DNA Value (±SE) CHOCell Genomic DNA Damage as the Average 0 0 100 200 300 400 500 Akron OH Source Water 2015 Samples (Concentration Factor) Figure 4.26: Comparison of observed CHO Cell Acute Genotoxicity as a function of Genomic DNA Damage for samples AOHTOX-SW, AOHTOX-CL, and AOHTOX-CL- IHXL.
101
5
4
3
2 CHOCell SCGE Averages
Genotoxicity Index Values (±SE) Index Genotoxicity 1
0
ASW + Cl2
ASW Negative Control ASW + Cl2 + Iohexol
Figure 4.27: All CHO Genotoxicity Index Values calculated as (50% Tail DNA)-1 x 103
A statistical analysis was also performed for the iohexol cytotoxic and genotoxic results, more details and the results from this can be seen in Section A1 of the appendix.
Precision analyses were conducted on each set of data for each bioassay. The process follows the generation of the concentration-response curve from combined replicate experiments with a test for significance using a one-way analysis of variance (ANOVA) test (Plewa et al., 2013; Plewa et al., 2015). If a significant F value (P ≤ 0.05) was obtained, a Holm-Sidak multiple comparison test versus the control group was conducted to identify the lowest toxic concentration factor (Plewa et al., 2013; Plewa et al., 2015). 102
After the non-linear regression analysis for cytotoxic or genotoxic assays, a bootstrap statistic was conducted for the results of each bioassay dataset and a mean toxicity index value was calculated (Plewa et al., 2013; Plewa, et al., 2015). Using the toxicity index values, an ANOVA test was conducted for each WSC and significant differences were identified.
103
CHAPTER V
CONCLUSIONS AND RECOMMENDATIONS
5.1 Introduction
This study investigated DBP formation and mammalian cell toxicity in Akron source water in the presence of ICM and chlorinated oxidants (chlorine and monochloramine). Iopamidol DBP formation and toxicity was investigated in the presence of chlorine and monochloramine. Iohexol DBP formation and toxicity was investigated in the presence of chlorine. DBP formation was investigated as a function of pH (6.5 – 9.0) and time (0 – 72 hours). NOM concentrations were held constant for the
DBP and toxicity experiments at full [NOM] = 5.57 mg/L. ICM concentrations were 5
μM for the 72 hour experiments and 2.5 μM for the kinetics experiments. ICM concentrations were held constant at 5 μM for all toxicity experiments.
5.2 Conclusions
1. Iopamidol formed regulated and unregulated DBPs in the presence of chlorinated
oxidants and NOM. Iopamidol acted as an indirect precursor to iodo-DBP formation
(i.e. dichloroiodomethane) and a direct precursor in the formation of chloroform and
TCAA. Additionally, chloroform formation proved to be dependent on the presence 104
of iopamidol and NOM. Iopamidol DBP formation was greatest at pH 9.0 for
chlorinated experiments and 6.5 for monochloraminated experiments.
2. Iopamidol appeared to enhance mammalian cell cytotoxicity and did enhance
genotoxicity in the experiments conducted with chlorine or monochloroamine. The
transformation of iopamidol seems to have enhanced toxicity in the presence of
chlorinated oxidants and NOM. The transformation of iopamidol results in the
formation of an active iodine oxidant generating additional DBPs (i.e., iodo-DBPs on
total organic iodide) in the presence of chlorinated oxidants.
3. Iohexol increased the formation of chloroform under the experimental conditions in
this study. Though it should be noted that the contaminant within the iohexol
analytical standard may have affected the increase in chloroform. Iohexol also formed
dichloroiodomethane at very low concentrations over the pH range of 6.5 – 9.0,
greater than the source water controls, with a maximum concentration of
apporiamtely 18 nM at pH 9. While iohexol does not appear to be a significant
precursor to iodo-DBP formation in the presence of chlorinated oxidants, it did result
in the enhanced formation of regulated DBPs.
4. Iohexol appears to enhance mammalian cell cytotoxicity and did enhance
genotoxicity in the experiments conducted after chlorine disinfection. Though it
should be noted that the contaminant within the iohexol analytical standard may have
influenced the enhanced toxicity results; regardless, iohexol does appear to enhance
toxicity in the presence of aqueous chlorine.
105
5.3 Recommendations
1. Additional work should be conducted to investigate DBP formation and possible
reaction mechanisms regarding ICM degradation over a higher pH range (i.e. 10.0 or
11.0).
2. Additional experiments should be conducted, similar to those performed in this work,
to verify the DBP formation within the iohexol experiments. Monochloramine should
also be investigated as an oxidant within iohexol experiments, in order to directly
compare the results to the iopamidol DBP experiments presented in this study.
3. A statistical model should be created comparing the results presented in this research
to the results presented in previous works by Crafton (2014) and Machek (2015). This
statistical model can directly compare the three data sets and prove useful for
identifying DBP precursors within source waters.
106
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APPENDIX
A.1 Iopamidol Experiments
A.1.1 DBP Formation in Aqueous Chlorine Experiments
Table A.1: Observed DBP formation at 72 hours as a function of pH, [NOM] = 5.57 mg/L-C, [iopamidol] = 5.0 μM, [Cl2]T = 100 μM, [Buffer] = 4 mM, temperature = 25 °C, pH 6.5 7.5 8.5 9.0 DBPs THMs CHCl3 1564.35 ± 258.83 1745.35 ± 254.10 1855.99 ± 264.77 4014.31 ± 221.62 CHCl2Br < 1.0 < 1.0 < 1.0 < 1.0 CHClBr2 < 1.0 < 1.0 < 1.0 < 1.0 CHBr3 < 1.0 < 1.0 < 1.0 < 1.0 CHCl2I 215.51 ± 46.18 262.92 ± 27.76 337.73 ± 34.89 650.67 ± 130.82 CHClI2 5.53 ± 0.95 10.69 ± 1.68 6.96 ± 0.69 5.86 ± 1.39 CHBrClI < 1.0 < 1.0 < 1.0 < 1.0 CHBr2I < 0.2 < 0.2 < 0.2 < 0.2 CHI3 < 0.2 < 0.2 < 0.2 < 0.2 HANs CAN 2.78 ± 0.37 4.32 ± 0.02 2.86 ± 0.25 1.47 ± 0.05 DCAN < 1.0 < 1.0 < 1.0 < 1.0 TCAN < 1.0 < 1.0 < 1.0 < 1.0 BAN < 1.0 < 1.0 < 1.0 < 1.0 DBAN < 1.0 < 1.0 < 1.0 < 1.0 BCAN < 1.0 < 1.0 < 1.0 < 1.0 IAN < 0.2 <0.2 < 0.2 < 0.2 HAAs CAA < 1.0 < 1.0 < 1.0 < 1.0 DCAA < 1.0 < 1.0 < 1.0 < 1.0 TCAA 651.77 ± 42.22 539.94 ± 60.54 287.84 ± 26.57 232.60 ± 98.94 BAA < 1.0 < 1.0 < 1.0 < 1.0 DBAA < 1.0 < 1.0 < 1.0 < 1.0 BCAA < 1.0 < 1.0 < 1.0 < 1.0 BDCAA < 1.0 < 1.0 < 1.0 < 1.0 IAA < 0.2 < 0.2 < 0.2 < 0.2
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Table A.2: Observed DBP formation at 72 hours as a function of pH, [NOM] = 5.57 mg/L-C, [ICM] = 0.0 μM, [Cl2]T = 100 μM, [Buffer] = 4 mM, temperature = 25 °C, pH 6.5 7.5 8.5 9.0 DBPs THMs CHCl3 1215.21 ± 70.23 1413.89 ± 182.0 1758.90 ± 150.33 1756.71 ± 207.89 CHCl2Br < 1.0 < 1.0 < 1.0 < 1.0 CHClBr2 < 1.0 < 1.0 < 1.0 < 1.0 CHBr3 < 1.0 < 1.0 < 1.0 < 1.0 CHCl2I 1.36 ± 0.93 2.06 ± 0.90 2.67 ± 1.36 3.06 ± 1.56 CHClI2 0.65 ± 0.58 0.52 ± 0.16 0.48 ± 0.21 0.28 ± 0.19 CHBrClI < 1.0 < 1.0 < 1.0 < 1.0 CHBr2I < 0.2 < 0.2 < 0.2 < 0.2 CHI3 < 0.2 < 0.2 < 0.2 < 0.2 HANs CAN < 1.0 < 1.0 < 1.0 < 1.0 DCAN < 1.0 < 1.0 < 1.0 < 1.0 TCAN < 1.0 < 1.0 < 1.0 < 1.0 BAN < 1.0 < 1.0 < 1.0 < 1.0 DBAN < 1.0 < 1.0 < 1.0 < 1.0 BCAN < 1.0 < 1.0 < 1.0 < 1.0 IAN < 0.2 <0.2 < 0.2 < 0.2 HAAs CAA < 1.0 < 1.0 < 1.0 < 1.0 DCAA < 1.0 < 1.0 < 1.0 < 1.0 TCAA 825.29 ± 28.06 650.0 ± 45.0 283.88 ± 14.60 231.05 ± 8.56 BAA < 1.0 < 1.0 < 1.0 < 1.0 DBAA < 1.0 < 1.0 < 1.0 < 1.0 BCAA < 1.0 < 1.0 < 1.0 < 1.0 BDCAA < 1.0 < 1.0 < 1.0 < 1.0 IAA < 0.2 < 0.2 < 0.2 < 0.2
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3000 pH 6.5 2500 pH 7.5 pH 8.5 pH 9.0 2000 (nM)
3 1500 CHCl 1000
500
0 0 20 40 60 80
Time (hours) Figure A.1: Observed chloroform formation at pH 6.5, 7.5, 8.5 and 9.0 as a function of time, [NOM] = 5.57 mg/L-C, [iopamidol] = 2.5 μM, [Cl2]T = 100 μM, [Buffer] = 4 mM, temperature = 25°C
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250
200
150 I (nM) I 2
CHCl 100
pH 6.5 50 pH 7.5 pH 8.5 pH 9.0 0 0 20 40 60 80 Time (hours)
Figure A.2: Observed dichloroiodomethane formation at pH 6.5, 7.5, 8.5 and 9.0 as a function of time, [NOM] = 5.57 mg/L-C, [iopamidol] = 2.5 μM, [Cl2]T = 100 μM, [Buffer] = 4 mM, temperature = 25°C
127
2500 pH 6.5 pH 7.5 2000 pH 8.5 pH 9.0
1500
TCAA(nM) 1000
500
0 0 20 40 60 80
Time (hours) Figure A.3: Observed trichloroacetic acid formation at pH 6.5, 7.5, 8.5 and 9.0 as a function of time, [NOM] = 5.57 mg/L-C, [iopamidol] = 2.5 μM, [Cl2]T = 100 μM, [Buffer] = 4 mM, temperature = 25°C
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A.1.2 DBP Formation in Monochloramine Experiments
Table A.3: Observed DBP formation at 72 hours as a function of pH, [NOM] = 5.57 mg/L-C, [iopamidol] = 5.0 μM, [NH2Cl]T = 100 μM, [Buffer] = 4 mM, temperature = 25 °C pH 6.5 7.5 8.5 9.0 DBPs THMs CHCl3 56.43 ± 2.19 31.39 ± 1.22 20.69 ± 1.56 13.43 ± 0.42 CHCl2Br < 1.0 < 1.0 < 1.0 < 1.0 CHClBr2 < 1.0 < 1.0 < 1.0 < 1.0 CHBr3 < 1.0 < 1.0 < 1.0 < 1.0 CHCl2I 52.17 ± 4.37 8.98 ± 0.35 1.05 ± 0.17 1.51 ± 0.07 CHClI2 < 0.2 < 0.2 < 0.2 < 0.2 CHBrClI < 1.0 < 1.0 < 1.0 < 1.0 CHBr2I < 0.2 < 0.2 < 0.2 < 0.2 CHI3 < 0.2 < 0.2 < 0.2 < 0.2 HAN CAN < 1.0 < 1.0 < 1.0 < 1.0 DCAN < 1.0 < 1.0 < 1.0 < 1.0 TCAN < 1.0 < 1.0 < 1.0 < 1.0 BAN < 1.0 < 1.0 < 1.0 < 1.0 DBAN < 1.0 < 1.0 < 1.0 < 1.0 BCAN < 1.0 < 1.0 < 1.0 < 1.0 IAN < 0.2 <0.2 < 0.2 < 0.2 HAAS CAA < 1.0 < 1.0 < 1.0 < 1.0 DCAA < 1.0 < 1.0 < 1.0 < 1.0 TCAA 5.34 ± 2.06 3.20 ± 0.34 2.20 ± 0.30 2.60 ± 0.37 BAA < 1.0 < 1.0 < 1.0 < 1.0 DBAA < 1.0 < 1.0 < 1.0 < 1.0 BCAA < 1.0 < 1.0 < 1.0 < 1.0 BDCAA < 1.0 < 1.0 < 1.0 < 1.0 IAA < 0.2 < 0.2 < 0.2 < 0.2
129
Table A.4: Observed DBP formation at 72 hours as a function of pH, [NOM] = 5.57 mg/L-C, [iopamidol] = 0.0 μM, [NH2Cl]T = 100 μM, [Buffer] = 4 mM, temperature = 25 °C pH 6.5 7.5 8.5 9.0 DBPs THMs CHCl3 44.51 ± 4.50 28.01 ± 2.65 14.66 ± 1.92 12.16 ± 1.33 CHCl2Br < 1.0 < 1.0 < 1.0 < 1.0 CHClBr2 < 1.0 < 1.0 < 1.0 < 1.0 CHBr3 < 1.0 < 1.0 < 1.0 < 1.0 CHCl2I 25.43 ± 4.92 4.97 ± 0.29 1.05 ± 0.11 0.65 ± 0.11 CHClI2 < 0.2 < 0.2 < 0.2 < 0.2 CHBrClI < 1.0 < 1.0 < 1.0 < 1.0 CHBr2I < 0.2 < 0.2 < 0.2 < 0.2 CHI3 < 0.2 < 0.2 < 0.2 < 0.2 HAN CAN < 1.0 < 1.0 < 1.0 < 1.0 DCAN < 1.0 < 1.0 < 1.0 < 1.0 TCAN < 1.0 < 1.0 < 1.0 < 1.0 BAN < 1.0 < 1.0 < 1.0 < 1.0 DBAN < 1.0 < 1.0 < 1.0 < 1.0 BCAN < 1.0 < 1.0 < 1.0 < 1.0 IAN < 0.2 <0.2 < 0.2 < 0.2 HAAS CAA < 1.0 < 1.0 < 1.0 < 1.0 DCAA < 1.0 < 1.0 < 1.0 < 1.0 TCAA 4.51 ± 1.68 2.68 ± 0.13 2.37 ± 0.18 1.66 ± 0.45 BAA < 1.0 < 1.0 < 1.0 < 1.0 DBAA < 1.0 < 1.0 < 1.0 < 1.0 BCAA < 1.0 < 1.0 < 1.0 < 1.0 BDCAA < 1.0 < 1.0 < 1.0 < 1.0 IAA < 0.2 < 0.2 < 0.2 < 0.2
130
80
pH 6.5 pH 7.5 pH 8.5 60 pH 9.0 (nM)
3 40 CHCl
20
0 0 20 40 60 80
Time (hours) Figure A.4: Observed chloroform formation at pH 6.5, 7.5, 8.5 and 9.0 as a function of time, [NOM] = 5.57 mg/L-C, [iopamidol] = 2.5 μM, [NH2Cl]T = 100 μM, [Buffer] = 4 mM, temperature = 25°C
131
18
16 pH 6.5 pH 7.5 14 pH 8.5 pH 9.0 12
10 I (nM) I 2 8 CHCl 6
4
2
0 0 20 40 60 80
Time (hours) Figure A.5: Observed dichloroiodomethane formation at pH 6.5, 7.5, 8.5 and 9.0 as a function of time, [NOM] = 5.57 mg/L-C, [iopamidol] = 2.5 μM, [NH2Cl]T = 100 μM, [Buffer] = 4 mM, temperature = 25°C
132
20
18 pH 6.5 pH 7.5 16 pH 8.5 pH 9.0 14
12
10
TCAA(nM) 8
6
4
2
0 0 20 40 60 80
Time (hours) Figure A.6: Observed TCAA formation at pH 6.5, 7.5, 8.5 and 9.0 as a function of time, [NOM] = 5.57 mg/L-C, [iopamidol] = 2.5 μM, [NH2Cl]T = 100 μM, [Buffer] = 4 mM, temperature = 25°C
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A.1.3 CHO Cell Chronic Cytotoxicity Analysis in Aqueous Chlorine Experiments
Table A.5: Observed CHO Cell Chronic Cytotoxicity as a function of Mean Cell Density for sample AOHTOX-SW. Concentration LC50 value = 161.3; pH = 7.5, [NOM] = 5.57 mg/L-C, [ICM] = 0.0 μM, [Cl2]T = 0 μM, [Buffer] = 4 mM, temperature = 25 °C Group Name N Mean Std Dev SW0 16 100.047 8.079 SW75 5 83.365 13.669 SW100 5 70.798 10.559 SW125 5 61.504 8.658 SW130 4 31.487 6.370 SW140 4 59.863 4.895 SW150 5 54.358 8.021 SW160 4 52.901 8.339 SW180 4 43.724 4.873 SW200 5 41.688 13.305 SW250 4 13.871 3.768
Table A.6: ANOVA Test Cytotoxicity Statistical Analysis for AOHTOX-SW Source of Variation DF SS MS F P Between Groups 10 42302.63 4230.264 53.495 <0.00 9 1 Residual 50 3953.883 79.078 Total 60 46256.52 3
Table A.7: Cytotoxicity Statistical Analysis Multiple Comparisons versus Control Group for AOHTOX-SW using Holm-Sidak Method. Overall significance level = 0.05 Comparison Diff of t Unadjusted Critical Significant? Means P Level SW0 vs. SW250 86.176 17.335 <0.001 0.005 Yes SW0 vs. SW130 68.560 13.792 <0.001 0.006 Yes SW0 vs. SW200 58.359 12.809 <0.001 0.006 Yes SW0 vs. SW180 56.342 11.330 <0.001 0.007 Yes SW0 vs. SW150 45.689 10.028 <0.001 0.009 Yes SW0 vs. SW160 47.689 9.484 <0.001 0.010 Yes SW0 vs. SW125 38.543 8.460 <0.001 0.013 Yes SW0 vs. SW140 40.184 8.084 <0.001 0.017 Yes SW0 vs. SW100 29.250 6.420 <0.001 0.025 Yes SW0 vs. SW75 16.682 3.661 <0.001 0.050 Yes
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Table A.8: Observed CHO Cell Chronic Cytotoxicity as a function of Mean Cell Density for sample AOHTOX-CL. Concentration LC50 value = 53.0; pH = 7.5, [NOM] = 5.57 mg/L-C, [ICM] = 0.0 μM, [Cl2]T = 100 μM, [Buffer] = 4 mM, temperature = 25 °C Group Name N Mean Std Dev CL0 16 100.047 8.079 CL25 4 79.694 7.250 CL30 4 75.316 6.284 CL40 4 81.593 7.382 CL50 4 48.418 9.576 CL60 4 20.886 7.646 CL70 4 20.622 6.968 CL75 4 22.310 8.658 CL80 4 25.158 5.246 CL100 4 11.762 3.211 CL125 4 1.899 4.036
Table A.9: ANOVA Test Cytotoxicity Statistical Analysis for AOHTOX-CL Source of Variation DF SS MS F P Between Groups 10 75445.356 7544.536 141.332 <0.001 Residual 45 2402.167 53.381 Total 55 77847.523
Table A.10: Cytotoxicity Statistical Analysis Multiple Comparisons versus Control Group for AOHTOX-CL using Holm-Sidak Method. Overall significance level = 0.05 Comparison Diff of t Unadjusted Critical Significant? Means P Level CL0 vs. CL125 98.148 24.031 <0.001 0.005 Yes CL0 vs. CL100 88.286 21.616 <0.001 0.006 Yes CL0 vs. CL70 79.425 19.446 <0.001 0.006 Yes CL0 vs. CL60 79.161 19.382 <0.001 0.007 Yes CL0 vs. CL75 77.737 19.033 <0.001 0.009 Yes CL0 vs. CL80 74.889 18.336 <0.001 0.010 Yes CL0 vs. CL50 51.629 12.641 <0.001 0.013 Yes CL0 vs. CL30 24.731 6.055 <0.001 0.017 Yes CL0 vs. CL25 20.353 4.983 <0.001 0.025 Yes CL0 vs. CL40 18.454 4.518 <0.001 0.050 Yes
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Table A.11: Observed CHO Cell Chronic Cytotoxicity as a function of Mean Cell Density for sample AOHTOX-IDOL. Concentration LC50 value = 123.7; pH = 7.5, [NOM] = 5.57 mg/L-C, [iopamidol] = 5.0 μM, [Cl2]T = 0 μM, [Buffer] = 4 mM, temperature = 25 °C
Group Name N Mean Std Dev IDOL0 16 100.000 7.022 IDOL50 5 100.504 7.481 IDOL75 5 79.449 7.983 IDOL100 5 74.999 9.883 IDOL110 4 67.080 8.874 IDOL125 5 47.285 6.644 IDOL140 5 33.517 4.992 IDOL150 4 9.917 4.610 IDOL175 4 15.909 5.348 IDOL125 5 3.313 4.983
Table A.12: ANOVA Test Cytotoxicity Statistical Analysis for AOHTOX-IDOL Source of Variation DF SS MS F P Between Groups 9 74607.851 8289.761 167.860 <0.001 Residual 48 2370.474 49.385 Total 57 76978.325
Table A.13: Cytotoxicity Statistical Analysis Multiple Comparisons versus Control Group for AOHTOX-IDOL using Holm-Sidak Method. Overall significance level = 0.05 Comparison Diff of t Unadjusted Critical Significant? Means P Level IDOL0 vs. IDOL125 96.687 26.854 <0.001 0.006 Yes IDOL0 vs. IDOL150 90.082 22.931 <0.001 0.006 Yes IDOL0 vs. IDOL175 84.090 21.405 <0.001 0.007 Yes IDOL0 vs. IDOL140 66.483 18.465 <0.001 0.009 Yes IDOL0 vs. IDOL125 52.714 14.641 <0.001 0.010 Yes IDOL0 vs. IDOL110 32.920 8.380 <0.001 0.013 Yes IDOL0 vs. IDOL100 25.000 6.944 <0.001 0.017 Yes IDOL0 vs. IDOL75 20.551 5.708 <0.001 0.025 Yes IDOL0 vs. IDOL50 0.504 0.104 0.889 0.050 No
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Table A.14: Observed CHO Cell Chronic Cytotoxicity as a function of Mean Cell Density for sample AOHTOX-CL-IDOL. Concentration LC50 value = 47.6; pH = 7.5, [NOM] = 5.57 mg/L-C, [iopamidol] = 5.0 μM, [Cl2]T = 100 μM, [Buffer] = 4 mM, temperature = 25 °C
Group Name N Mean Std Dev CL-IDOL0 24 100.01 7.383 CL-IDOL20 8 96.222 4.784 CL-IDOL25 9 88.897 11.597 CL-IDOL30 12 89.415 13.066 CL-IDOL35 8 72.328 8.974 CL-IDOL40 12 62.407 7.210 CL-IDOL45 8 49.017 8.781 CL-IDOL50 13 43.104 17.25 CL-IDOL55 12 30.527 10.535 CL-IDOL60 12 21.475 6.838 CL-IDOL70 12 1.235 9.585 CL-IDOL75 4 -0.413 2.438
Table A.15: ANOVA Test Cytotoxicity Statistical Analysis for AOHTOX-CL-IDOL Source of Variation DF SS MS F P Between Groups 11 157000.923 14272.811 140.682 <0.001 Residual 122 12377.400 101.454 Total 133 169378.324
Table A.16: Cytotoxicity Statistical Analysis Multiple Comparisons versus Control Group for AOHTOX-CL-IDOL using Holm-Sidak Method. Overall significance level = 0.05 Comparison Diff of t Unadjusted Critical Significant? Means P Level CL-IDOL0 vs. CL-IDOL70 98.775 27.737 <0.001 0.005 Yes CL-IDOL0 vs. CL-IDOL60 78.535 22.053 <0.001 0.005 Yes CL-IDOL0 vs. CL-IDOL55 69.483 19.511 <0.001 0.006 Yes CL-IDOL0 vs. CL-IDOL75 100.423 18.461 <0.001 0.006 Yes CL-IDOL0 vs. CL-IDOL50 56.906 16.406 <0.001 0.007 Yes CL-IDOL0 vs. CL-IDOL45 50.993 12.401 <0.001 0.009 Yes CL-IDOL0 vs. CL-IDOL40 37.603 10.559 <0.001 0.010 Yes CL-IDOL0 vs. CL-IDOL35 27.682 6.732 <0.001 0.013 Yes CL-IDOL0 vs. CL-IDOL30 10.595 2.975 0.004 0.017 No CL-IDOL0 vs. CL-IDOL25 11.113 2.823 0.006 0.025 No CL-IDOL0 vs. CL-IDOL20 3.788 0.921 0.359 0.050 No
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Table A.17: Comparative CHO cell chronic cytotoxicity of X-ray contrast agent iopamidol in Akron OH source water samples disinfected with and without Cl and NH2Cl 2 Sample Lowest LC50 r AONVA Test Statistic Cytotoxic (CF) Conc. Factor AOHTOX-SW 75 161 0.94 F10,50 = 53.5; P ≥ 0.001 AOHTOX-CL 25 53.0 0.94 F10,40 = 141; P ≥ 0.001 AOHTOX-IDOL 75 123 0.96 F9,48 = 167; P ≥ 0.001 AOHTOX-CL-IDOL 25 47.6 0.97 F11,122 = 140; P ≥ 0.001 AOHTOX-NH2CL 100 129 0.94 F10,50 =72.3; P ≥ 0.001 AOHTOX-NH2CL-IDOL 25 60.3 0.99 F10,125 =154; P ≥ 0.001
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100
80
60
40
20 asthe Percent of Negative Control (±SE) CHO Cell Cytotoxicity: Mean Cell Density Cell Mean Cytotoxicity: Cell CHO
0 0 50 100 150 200 250 Water Sample AOHTOX-SW (Concentration Factor) Figure A.7: Observed CHO Cell Chronic Cytotoxicity as a function of Mean Cell Density for sample AOHTOX-SW. Concentration LC50 value = 161.3; pH = 7.5, [NOM] = 5.57 mg/L-C, [iopamidol] = 0.0 μM, [Cl2]T = 0.0 μM, [Buffer] = 4 mM, temperature = 25 °C
139
100
80
60
40
20 asthe Percent of Negative Control (±SE) CHO Cell Cytotoxicity: Mean Cell Density Cell Mean Cytotoxicity: Cell CHO 0
0 20 40 60 80 100 120 Water Sample AOHTOX-CL (Concentration Factor) Figure A.8: Observed CHO Cell Chronic Cytotoxicity as a function of Mean Cell Density for sample AOHTOX-CL. Concentration LC50 value = 53.0; pH = 7.5, [NOM] = 5.57 mg/L-C, [iopamidol] = 0.0 μM, [Cl2]T = 100 μM, [Buffer] = 4 mM, temperature = 25 °C
140
100
80
60
40
20 asthe Percent of Negative Control (±SE) CHO Cell Cytotoxicity: Mean Cell Density Cell Mean Cytotoxicity: Cell CHO 0
0 50 100 150 200 Water Sample AOHTOX-IDOL (Concentration Factor) Figure A.9: Observed CHO Cell Chronic Cytotoxicity as a function of Mean Cell Density for sample AOHTOX-IDOL. Concentration LC50 value = 123.7; pH = 7.5, [NOM] = 5.57 mg/L-C, [iopamidol] = 5.0 μM, [Cl2]T = 0.0 μM, [Buffer] = 4 mM, temperature = 25 °C
141
100
80
60
40
20 asthe Percent of Negative Control (±SE) CHO Cell Cytotoxicity: Mean Cell Density Cell Mean Cytotoxicity: Cell CHO 0
0 20 40 60 80 Water Sample AOHTOX-CL-IDOL (Concentration Factor) Figure A.10: Observed CHO Cell Chronic Cytotoxicity as a function of Mean Cell Density for sample AOHTOX-CL-IDOL. Concentration LC50 value = 47.6; pH = 7.5, [NOM] = 5.57 mg/L-C, [iopamidol] = 5.0 μM, [Cl2]T = 100.0 μM, [Buffer] = 4 mM, temperature = 25 °C
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A.1.4 Cytotoxicity Analysis in Aqueous Monochloramine Experiments
Table A.18: Observed CHO Cell Chronic Cytotoxicity as a function of Mean Cell Density for sample AOHTOX-NH2CL. Concentration LC50 value = 129.3; pH = 7.5, [NOM] = 5.57 mg/L-C, [iopamidol] = 0.0 μM, [NH2Cl]T = 100 μM, [Buffer] = 4 mM, temperature = 25 °C
Group Name N Mean Std Dev NH2CL0 16 100.047 9.975 NH2CL50 5 90.011 6.753 NH2CL75 5 91.693 14.752 NH2CL100 5 75.756 10.632 NH2CL125 5 50.868 13.607 NH2CL130 5 39.746 9.653 NH2CL140 5 43.288 2.179 NH2CL150 5 29.826 3.824 NH2CL160 5 21.564 6.639 NH2CL170 5 22.622 1.867 NH2CL200 5 12.993 9.355
Table A.19: ANOVA Test Cytotoxicity Statistical Analysis for AOHTOX- NH2CL Source of Variation DF SS MS F P Between Groups 10 65581.673 6558.167 72.277 <0.001 Residual 50 4536.828 90.737 Total 60 70118.501
Table A.20: Cytotoxicity Statistical Analysis Multiple Comparisons versus Control Group for AOHTOX- NH2CL using Holm-Sidak Method. Overall significance level = 0.05 Comparison Diff of t Unadjuste Critical Significant? Means d P Level NH2CL0 vs. NH2CL200 87.054 17.837 <0.001 0.005 Yes NH2CL0 vs. NH2CL160 78.483 14.739 <0.001 0.006 Yes NH2CL0 vs. NH2CL170 77.426 14.540 <0.001 0.006 Yes NH2CL0 vs. NH2CL150 70.221 14.388 <0.001 0.007 Yes NH2CL0 vs. NH2CL130 60.301 11.324 <0.001 0.009 Yes NH2CL0 vs. NH2CL140 56.760 10.659 <0.001 0.010 Yes NH2CL0 vs. NH2CL125 49.179 10.077 <0.001 0.013 Yes NH2CL0 vs. NH2CL100 24.291 4.977 <0.001 0.017 Yes NH2CL0 vs. NH2CL50 10.037 1.885 0.065 0.025 No NH2CL0 vs. NH2CL75 8.354 1.712 0.093 0.050 No
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Table A.21: Observed CHO Cell Chronic Cytotoxicity as a function of Mean Cell Density for sample AOHTOX-NH2CL-IDOL. Concentration LC50 value = 60.3; pH = 7.5, [NOM] = 5.57 mg/L-C, [iopamidol] = 5.0 μM, [NH2Cl]T = 100 μM, [Buffer] = 4 mM, temperature = 25 °C
Group Name N Mean Std Dev NH2CLIDOL0 24 100.057 9.947 NH2CLIDOL10 9 98.522 10.065 NH2CLIDOL20 8 102.348 5.146 NH2CLIDOL25 13 85.328 21.392 NH2CLIDOL40 8 81.995 5.048 NH2CLIDOL50 13 63.277 13.875 NH2CLIDOL75 13 34.971 15.779 NH2CLIDOL80 12 16.764 7.245 NH2CLIDOL85 12 13.277 8.365 NH2CLIDOL90 12 10.568 3.782 NH2CLIDOL100 12 3.479 7.145
Table A.22: ANOVA Test Cytotoxicity Statistical Analysis for AOHTOX- NH2CL- IDOL Source of Variation DF SS MS F P Between Groups 10 201285.895 20128.589 154.309 <0.001 Residual 125 16305.389 130.443 Total 135 217591.284
Table A.23: Cytotoxicity Statistical Analysis Multiple Comparisons versus Control Group for AOHTOX- NH2CL-IDOL using Holm-Sidak Method. Overall significance level = 0.05 Comparison Diff of t Unadjusted Critical Significant Means P Level ? NH2CLIDL0 v. NH2CLIDL100 96.578 23.917 <0.001 0.005 Yes NH2CLIDL0 v. NH2CLIDL90 89.488 22.162 <0.001 0.006 Yes NH2CLIDL0 v. NH2CLIDL85 86.780 21.491 <0.001 0.006 Yes NH2CLIDL0 v. NH2CLIDL80 83.293 20.627 <0.001 0.007 Yes NH2CLIDL0 v. NH2CLIDL75 65.086 16.548 <0.001 0.009 Yes NH2CLIDL0 v. NH2CLIDL50 36.780 9.351 <0.001 0.010 Yes NH2CLIDL0 v. NH2CLIDL40 18.062 3.874 <0.001 0.013 Yes NH2CLIDL0 v. NH2CLIDL25 14.729 3.745 <0.001 0.017 Yes NH2CLIDL0 v. NH2CLIDL20 2.292 0.492 0.624 0.025 No NH2CLIDL0 v. NH2CLIDL10 1.535 0.344 0.732 0.050 No
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100
80
60
40
20 asthe Percent of Negative Control (±SE) CHO Cell Cytotoxicity: Mean Cell Density Cell Mean Cytotoxicity: Cell CHO
0 0 50 100 150 200 Water Sample AOHTOX-NH2CL (Concentration Factor) Figure A.11: Observed CHO Cell Chronic Cytotoxicity as a function of Mean Cell Density for sample AOHTOX-NH2CL. Concentration LC50 value = 129.3; pH = 7.5, [NOM] = 5.57 mg/L-C, [iopamidol] = 0.0 μM, [NH2Cl]T = 100 μM, [Buffer] = 4 mM, temperature = 25 °C
145
100
80
60
40
20 asthe Percent of Negative Control (±SE) CHO Cell Cytotoxicity: Mean Cell Density Cell Mean Cytotoxicity: Cell CHO 0
0 20 40 60 80 100 Water Sample AOHTOX-NH2CL-IDOL (Concentration Factor) Figure A.12: Observed CHO Cell Chronic Cytotoxicity as a function of Mean Cell Density for sample AOHTOX-NH2CL-IDOL. Concentration LC50 value = 60.3; pH = 7.5, [NOM] = 5.57 mg/L-C, [iopamidol] = 5.0 μM, [NH2Cl]T = 100 μM, [Buffer] = 4 mM, temperature = 25 °C
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A.1.5 CHO Cell Chronic Genotoxicity Analysis in Aqueous Chlorine Experiments
Table A.24: Observed CHO Cell Chronic Genotoxicity as a function of Genomic DNA Damage for sample AOHTOX-SW. Concentration factor SCGE 50% Tail DNA value = 680.0; pH = 7.5, [NOM] = 5.57 mg/L-C, [iopamidol] = 0.0 μM, [Cl2]T = 0.0 μM, [Buffer] = 4 mM, temperature = 25 °C
Group Name N Mean Std Dev SW0 6 1.721 0.868 SW25 2 1.963 0.247 SW50 2 1.506 0.234 SW10 2 1.596 0.783 SW150 2 0.710 0.192 SW200 2 1.186 0.910 SW250 2 1.629 0.435 SW300 2 2.303 0.139 SW350 2 1.401 0.425 SW400 2 0.930 0.399 SW450 4 2.432 0.690 SW500 6 2.585 1.041 SW550 6 3.437 2.064 SW600 6 5.595 3.635 SW650 6 21.418 8.522
Table A.25: ANOVA Test Genotoxicity Statistical Analysis for AOHTOX- SW Source of Variation DF SS MS F P Between Groups 14 1983.789 141.699 11.318 <0.001 Residual 37 463.225 12.520 Total 51 2447.014
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Table A.26: Genotoxicity Statistical Analysis Multiple Comparisons versus Control Group for AOHTOX- SW using Holm-Sidak Method. Overall significance level = 0.05 Comparison Diff of t Unadjuste Critical Significant? Means d P Level SW0 vs. SW650 19.698 9.624 <0.001 0.004 Yes SW0 vs. SW600 3.874 1.897 0.066 0.004 No SW0 vs. SW550 1.716 0.840 0.406 0.004 No SW0 vs. SW500 0.864 0.423 0.675 0.005 No SW0 vs. SW150 1.010 0.350 0.729 0.005 No SW0 vs. SW450 0.712 0.312 0.757 0.006 No SW0 vs. SW400 0.790 0.274 0.786 0.006 No SW0 vs. SW300 0.582 0.201 0.841 0.007 No SW0 vs. SW200 0.535 0.185 0.854 0.009 No SW0 vs. SW350 0.320 0.111 0.912 0.010 No SW0 vs. SW25 0.243 0.0840 0.934 0.013 No SW0 vs. SW50 0.214 0.0741 0.941 0.017 No SW0 vs. SW10 0.124 0.0430 0.966 0.025 No SW0 vs. SW250 0.0916 0.0317 0.975 0.050 No
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Table A.27: Observed CHO Cell Chronic Genotoxicity as a function of Genomic DNA Damage for sample AOHTOX-CL. Concentration factor SCGE 50% Tail DNA value = 244.3; pH = 7.5, [NOM] = 5.57 mg/L-C, [iopamidol] = 0.0 μM, [Cl2]T = 100.0 μM, [Buffer] = 4 mM, temperature = 25 °C
Group Name N Mean Std Dev CL0 6 1.699 0.596 CL25 2 1.388 0.000493 CL50 2 1.496 0.691 CL100 2 1.233 0.0416 CL150 6 9.881 5.402 CL175 8 29.688 8.895 CL200 8 30.049 13.571 CL225 8 58.937 19.264 CL250 6 53.728 13.713 CL300 2 69.57 0.000
Table A.28: ANOVA Test Genotoxicity Statistical Analysis for AOHTOX- CL Source of Variation DF SS MS F P Between Groups 9 25358.117 2817.569 20.383 <0.001 Residual 40 5529.119 138.228 Total 49 30887.236
Table A.29: Genotoxicity Statistical Analysis Multiple Comparisons versus Control Group for AOHTOX-CL using Holm-Sidak Method. Overall significance level = 0.05 Comparison Diff of t Unadjusted Critical Significant? Means P Level CL0 vs. CL225 57.238 9.014 <0.001 0.006 Yes CL0 vs. CL250 52.029 7.665 <0.001 0.006 Yes CL0 vs. CL300 67.871 7.070 <0.001 0.007 Yes CL0 vs. CL200 28.350 4.465 <0.001 0.009 Yes CL0 vs. CL175 27.989 4.408 <0.001 0.010 Yes CL0 vs. CL150 8.182 1.205 0.235 0.013 No CL0 vs. CL100 0.466 0.0486 0.961 0.017 No CL0 vs. CL25 0.311 0.0324 0.974 0.025 No CL0 vs. CL50 0.203 0.0211 0.983 0.050 No
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Table A.30: Observed CHO Cell Chronic Genotoxicity as a function of Genomic DNA Damage for sample AOHTOX-IDOL. Concentration factor SCGE 50% Tail DNA value = 722.9; pH = 7.5, [NOM] = 5.57 mg/L-C, [iopamidol] = 5.0 μM, [Cl2]T = 0.0 μM, [Buffer] = 4 mM, temperature = 25 °C
Group Name N Mean Std Dev IDOL0 6 1.736 1.018 IDOL25 2 1.507 0.318 IDOL50 2 1.360 0.069 IDOL100 2 2.310 1.407 IDOL150 6 1.905 0.883 IDOL160 6 2.384 1.328 IDOL180 6 1.648 0.689 IDOL200 6 1.995 0.623 IDOL220 8 3.108 1.264 IDOL250 8 6.560 4.643
Table A.31: ANOVA Test Genotoxicity Statistical Analysis for AOHTOX-IDOL Source of Variation DF SS MS F P Between Groups 9 146.293 16.255 3.663 <0.001 Residual 42 186.397 4.438 Total 51 332.690
Table A.32: Genotoxicity Statistical Analysis Multiple Comparisons versus Control Group for AOHTOX-IDOL using Holm-Sidak Method. Overall significance level = 0.05 Comparison Diff of t Unadjusted Critical Significant? Means P Level IDOL0 vs. IDOL250 4.823 4.240 <0.001 0.006 Yes IDOL0 vs. IDOL220 1.371 1.205 0.235 0.006 No IDOL0 vs. IDOL160 0.648 0.533 0.597 0.007 No IDOL0 vs. IDOL100 0.574 0.334 0.740 0.009 No IDOL0 vs. IDOL50 0.376 0.219 0.828 0.010 No IDOL0 vs. IDOL200 0.258 0.213 0.833 0.013 No IDOL0 vs. IDOL150 0.168 0.138 0.891 0.017 No IDOL0 vs. IDOL25 0.229 0.133 0.895 0.025 No IDOL0 vs. IDOL180 0.0877 0.0721 0.943 0.050 No
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Table A.33: Observed CHO Cell Chronic Genotoxicity as a function of Genomic DNA Damage for sample AOHTOX-CL-IDOL. Concentration factor SCGE 50% Tail DNA value = 140.1; pH = 7.5, [NOM] = 5.57 mg/L-C, [iopamidol] = 5.0 μM, [Cl2]T = 100.0 μM, [Buffer] = 4 mM, temperature = 25 °C
Group Name N Mean Std Dev CL-IDOL0 6 2.357 1.002 CL-IDOL25 2 1.639 0.343 CL-IDOL50 4 3.995 2.894 CL-IDOL75 4 3.675 0.844 CL-IDOL100 6 14.597 9.298 CL-IDOL125 4 38.22 12.143 CL-IDOL150 2 61.683 6.029
Table A.34: ANOVA Test Genotoxicity Statistical Analysis for AOHTOX-CL-IDOL Source of Variation DF SS MS F P Between Groups 6 8827.499 1471.250 32.752 <0.001 Residual 21 943.339 44.921 Total 27 9770.838
Table A.35: Genotoxicity Statistical Analysis Multiple Comparisons versus Control Group for AOHTOX-CL-IDOL using Holm-Sidak Method. Overall significance level = 0.05 Comparison Diff of t Unadjusted Critical Significant? Means P Level CL-IDOL0 vs. CL-IDOL150 59.327 10.841 <0.001 0.009 Yes CL-IDOL0 vs. CL-IDOL125 35.863 8.289 <0.001 0.010 Yes CL-IDOL0 vs. CL-IDOL100 12.241 3.163 0.005 0.013 No CL-IDOL0 vs. CL-IDOL50 1.639 0.379 0.709 0.017 No CL-IDOL0 vs. CL-IDOL75 1.319 0.305 0.763 0.025 No CL-IDOL0 vs. CL-IDOL25 0.717 0.131 0.897 0.050 No
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Table A.36: Comparative CHO cell chronic genotoxicity of X-ray contrast agent iopamidol in Akron OH water samples disinfected with and without Cl and NH2Cl Sample Lowest SCGE 50% r2 AONVA Test Statistic Genotoxic Tail DNA Conc. Value (CF) Factor AOHTOX-SW 650 680 0.99 F14,37 = 11.3; P ≥ 0.001 AOHTOX-CL 175 244 0.92 F9,40 = 20.4; P ≥ 0.001 AOHTOX-IDOL 250 723 0.48 F9,42 = 3.66; P ≥ 0.001 AOHTOX-CL-IDOL 100 140 0.99 F6,21 = 32.7; P ≥ 0.001 AOHTOX-NH2CL 350 565 0.98 F14,27 = 64.6; P ≥ 0.001 AOHTOX-NH2CL-IDOL 250 288 0.93 F12,29 = 60.5; P ≥ 0.001
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5 MeanSCGE Tail% DNA Value (±SE) CHOCell Genoimc DNA Damage as the Average 0 0 100 200 300 400 500 600 Water Sample AOHTOX-SW (Concentration Factor) Figure A.13: Observed CHO Cell Acute Genotoxicity as a function of Genomic DNA Damage for sample AOHTOX-SW. Concentration factor SCGE 50% Tail DNA value = 680.0; pH = 7.5, [NOM] = 5.57 mg/L-C, [iopamidol] = 0.0 μM, [Cl2]T = 0.0 μM, [Buffer] = 4 mM, temperature = 25 °C
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20 MeanSCGE Tail% DNA Value (±SE)
CHOCell Genoimc DNA Damage as the Average 0 0 50 100 150 200 250 300 Water Sample AOHTOX-CL (Concentration Factor) Figure A.14: Observed CHO Cell Acute Genotoxicity as a function of Genomic DNA Damage for sample AOHTOX-CL. Concentration factor SCGE 50% Tail DNA value = 244.3; pH = 7.5, [NOM] = 5.57 mg/L-C, [iopamidol] = 0.0 μM, [Cl2]T = 100.0 μM, [Buffer] = 4 mM, temperature = 25 °C
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5 MeanSCGE Tail% DNA Value (±SE) CHOCell Genoimc DNA Damage as the Average 0 0 50 100 150 200 250 Water Sample AOHTOX-IDOL (Concentration Factor) Figure A.15: Observed CHO Cell Acute Genotoxicity as a function of Genomic DNA Damage for sample AOHTOX-IDOL. Concentration factor SCGE 50% Tail DNA value = 722.9; pH = 7.5, [NOM] = 5.57 mg/L-C, [iopamidol] = 5.0 μM, [Cl2]T = 0.0 μM, [Buffer] = 4 mM, temperature = 25 °C
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20 MeanSCGE Tail% DNA Value (±SE) CHOCell Genoimc DNA Damage as the Average 0 0 20 40 60 80 100 120 140 Water Sample AOHTOX-CL-IDOL (Concentration Factor) Figure A.16: Observed CHO Cell Acute Genotoxicity as a function of Genomic DNA Damage for sample AOHTOX-CL-IDOL. Concentration factor SCGE 50% Tail DNA value = 140.1; pH = 7.5, [NOM] = 5.57 mg/L-C, [iopamidol] = 5.0 μM, [Cl2]T = 100.0 μM, [Buffer] = 4 mM, temperature = 25 °C
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A.1.6 Genotoxicity Analysis in Aqueous Monochloramine Experiments
Table A.37: Observed CHO Cell Chronic Cytotoxicity as a function of Mean Cell Density for sample AOHTOX-NH2CL. Concentration LC50 value = 161.3; pH = 7.5, [NOM] = 5.57 mg/L-C, [iopamidol] = 0.0 μM, [NH2Cl]T = 100.0 μM, [Buffer] = 4 mM, temperature = 25 °C
Group Name N Mean Std Dev NH2CL0 6 1.462 0.373 NH2CL25 2 1.448 0.605 NH2CL50 2 1.338 0.255 NH2CL100 2 3.948 1.227 NH2CL150 2 4.237 3.627 NH2CL200 2 2.132 0.299 NH2CL250 4 2.101 0.580 NH2CL300 4 3.906 2.136 NH2CL350 4 10.413 7.609 NH2CL400 4 18.591 9.140 NH2CL450 2 24.849 4.133 NH2CL500 2 36.054 4.032 NH2CL550 2 51.63 6.600 NH2CL600 2 64.566 2.451 NH2CL650 2 68.169 2.649
Table A.38: ANOVA Test Genotoxicity Statistical Analysis for AOHTOX-NH2CL Source of Variation DF SS MS F P Between Groups 14 18424.525 1303.038 64.577 <0.001 Residual 27 544.809 20.178 Total 41 18787.334
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Table A.39: Genotoxicity Statistical Analysis Multiple Comparisons versus Control Group for AOHTOX-NHCL using Holm-Sidak Method. Overall significance level = 0.05 Comparison Diff of t Unadjusted Critical Significant? Means P Level NH2CL vs. NH2CL650 66.707 18.188 <0.001 0.004 Yes NH2CL vs. NH2CL600 63.104 17.205 <0.001 0.004 Yes NH2CL vs. NH2CL550 50.168 13.678 <0.001 0.004 Yes NH2CL vs. NH2CL500 34.593 9.432 <0.001 0.005 Yes NH2CL vs. NH2CL450 23.388 6.377 <0.001 0.005 Yes NH2CL vs. NH2CL400 17.129 5.907 <0.001 0.006 Yes NH2CL vs. NH2CL350 8.951 3.087 0.005 0.006 No NH2CL vs. NH2CL300 2.444 0.843 0.407 0.007 No NH2CL vs. NH2CL150 2.775 0.757 0.456 0.009 No NH2CL vs. NH2CL100 2.486 0.678 0.504 0.010 No NH2CL vs. NH2CL250 0.639 0.220 0.827 0.013 No NH2CL vs. NH2CL200 0.670 0.183 0.856 0.017 No NH2CL vs. NH2CL50 0.124 0.0338 0.973 0.025 No NH2CL vs. NH2CL25 0.0137 0.997 0.997 0.050 No
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Table A.40: Observed CHO Cell Chronic Cytotoxicity as a function of Mean Cell Density for sample AOHTOX-NH2CL-IDOL. Concentration LC50 value = 287.9; pH = 7.5, [NOM] = 5.57 mg/L-C, [iopamidol] = 5.0 μM, [NH2Cl]T = 100.0 μM, [Buffer] = 4 mM, temperature = 25 °C
Group Name N Mean Std Dev NH2CL-IDOL0 6 1.165 0.478 NH2CL-IDOL25 2 1.011 0.477 NH2CL-IDOL50 2 2.347 0.163 NH2CL-IDOL100 2 1.245 0.349 NH2CL-IDOL150 2 1.905 1.561 NH2CL-IDOL200 2 6.703 2.251 NH2CL-IDOL250 4 24.457 4.851 NH2CL-IDOL275 2 55.938 11.894 NH2CL-IDOL300 4 65.084 16.555 NH2CL-IDOL325 4 59.381 10.089 NH2CL-IDOL350 4 73.196 8.344 NH2CL-IDOL375 4 82.394 8.574 NH2CL-IDOL400 4 84.444 6.801
Table A.41: ANOVA Genotoxicity Statistical Analysis for AOHTOX-NH2CL-IDOL Source of Variation DF SS MS F P Between Groups 12 48029.995 4002.500 60.555 <0.001 Residual 29 1916.817 66.097 Total 41 49946.812
Table A.42: Genotoxicity Statistical Analysis Multiple Comparisons vs. Control Group AOHTOX-NHCL-IDOL using Holm-Sidak Method. Overall significance level = 0.05 Comparison Diff of t Unadjusted Critical Significant Means P Level ? NH2CLIDL v. NH2CLIDL400 83.279 15.869 <0.001 0.004 Yes NH2CLIDL v. NH2CLIDL375 81.229 15.478 <0.001 0.005 Yes NH2CLIDL v. NH2CLIDL350 72.031 13.726 <0.001 0.005 Yes NH2CLIDL v. NH2CLIDL300 63.919 12.180 <0.001 0.006 Yes NH2CLIDL v. NH2CLIDL325 58.216 11.093 <0.001 0.006 Yes NH2CLIDL v. NH2CLIDL275 54.773 8.251 <0.001 0.007 Yes NH2CLIDL v. NH2CLIDL250 23.292 4.438 <0.001 0.009 Yes NH2CLIDL v. NH2CLIDL200 5.538 0.834 0.411 0.010 No NH2CLIDL v. NH2CLIDL50 1.182 0.178 0.860 0.013 No NH2CLIDL v. NH2CLIDL150 0.740 0.111 0.912 0.017 No NH2CLIDL v. NH2CLIDL100 0.0798 0.012 0.990 0.050 No
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20 MeanSCGE Tail% DNA Value (±SE) CHOCell Genoimc DNA Damage as the Average 0 0 200 400 600 Water Sample AOHTOX-NH2CL (Concentration Factor) Figure A.17: Observed CHO Cell Acute Genotoxicity as a function of Genomic DNA Damage for sample AOHTOX-NH2CL. Concentration factor SCGE 50% Tail DNA value = 565.5; pH = 7.5, [NOM] = 5.57 mg/L-C, [iopamidol] = 0.0 μM, [NH2Cl]T = 100.0 μM, [Buffer] = 4 mM, temperature = 25 °C
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20 MeanSCGE Tail% DNA Value (±SE) CHOCell Genoimc DNA Damage as the Average 0 0 100 200 300 400 Water Sample AOHTOX-NH2CL -IDOL (Concentration Factor) Figure A.18: Observed CHO Cell Acute Genotoxicity as a function of Genomic DNA Damage for sample AOHTOX-NH2CL-IDOL. Concentration factor SCGE 50% Tail DNA value = 287.9; pH = 7.5, [NOM] = 5.57 mg/L-C, [iopamidol] = 5.0 μM, [NH2Cl]T = 100.0 μM, [Buffer] = 4 mM, temperature = 25 °C
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A.2 Iohexol Experiments
A.2.1 DBP Formation in Aqueous Chlorine Experiments
Table A.43: Observed DBP formation at 72 hours as a function of pH, [NOM] = 5.57 mg/L-C, [iohexol] = 5.0 μM, [Cl2]T = 100 μM, [Buffer] = 4 mM, temperature = 25 °C pH 6.5 7.5 8.5 9.0 DBPs THMs CHCl3 2436.66 ± 205.39 2469.20 ± 258.91 2650.43 ± 283.25 2613.53 ± 25.50 CHCl2Br < 1.0 < 1.0 < 1.0 < 1.0 CHClBr2 < 1.0 < 1.0 < 1.0 < 1.0 CHBr3 < 1.0 < 1.0 < 1.0 < 1.0 CHCl2I 5.41 ± 1.04 6.36 ± 0.62 11.69 ± 1.71 17.57 ± 0.32 CHClI2 2.94 ± 2.19 3.15 ± 0.32 2.91 ± 0.18 2.19 ± 0.35 CHBrClI < 1.0 < 1.0 < 1.0 < 1.0 CHBr2I < 0.2 < 0.2 < 0.2 < 0.2 CHI3 < 0.2 < 0.2 < 0.2 < 0.2 HAN CAN < 1.0 < 1.0 < 1.0 < 1.0 DCAN < 1.0 < 1.0 < 1.0 < 1.0 TCAN < 1.0 < 1.0 < 1.0 < 1.0 BAN < 1.0 < 1.0 < 1.0 < 1.0 DBAN < 1.0 < 1.0 < 1.0 < 1.0 BCAN < 1.0 < 1.0 < 1.0 < 1.0 IAN < 0.2 <0.2 < 0.2 < 0.2 HAAS CAA < 1.0 < 1.0 < 1.0 < 1.0 DCAA < 1.0 < 1.0 < 1.0 < 1.0 TCAA 846.78 ± 64.75 601.17 ± 64.40 307.33 ± 9.20 201.64 ± 4.67 BAA < 1.0 < 1.0 < 1.0 < 1.0 DBAA < 1.0 < 1.0 < 1.0 < 1.0 BCAA < 1.0 < 1.0 < 1.0 < 1.0 BDCAA < 1.0 < 1.0 < 1.0 < 1.0 IAA < 0.2 < 0.2 < 0.2 < 0.2
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Table A.44: Observed DBP formation at 72 hours as a function of pH, [NOM] = 5.57 mg/L-C, [ICM] = 0.0 μM, [Cl2]T = 100 μM, [Buffer] = 4 mM, temperature = 25 °C pH 6.5 7.5 8.5 9.0 DBPs THMs CHCl3 1215.21 ± 70.23 1413.89 ± 182.0 1758.90 ± 150.33 1756.71 ± 207.89 CHCl2Br < 1.0 < 1.0 < 1.0 < 1.0 CHClBr2 < 1.0 < 1.0 < 1.0 < 1.0 CHBr3 < 1.0 < 1.0 < 1.0 < 1.0 CHCl2I 1.36 ± 0.93 2.06 ± 0.90 2.67 ± 1.36 3.06 ± 1.56 CHClI2 0.65 ± 0.58 0.52 ± 0.16 0.48 ± 0.21 0.28 ± 0.19 CHBrClI < 1.0 < 1.0 < 1.0 < 1.0 CHBr2I < 0.2 < 0.2 < 0.2 < 0.2 CHI3 < 0.2 < 0.2 < 0.2 < 0.2 HAN CAN < 1.0 < 1.0 < 1.0 < 1.0 DCAN < 1.0 < 1.0 < 1.0 < 1.0 TCAN < 1.0 < 1.0 < 1.0 < 1.0 BAN < 1.0 < 1.0 < 1.0 < 1.0 DBAN < 1.0 < 1.0 < 1.0 < 1.0 BCAN < 1.0 < 1.0 < 1.0 < 1.0 IAN < 0.2 <0.2 < 0.2 < 0.2 HAAS CAA < 1.0 < 1.0 < 1.0 < 1.0 DCAA < 1.0 < 1.0 < 1.0 < 1.0 TCAA 825.29 ± 28.06 650.0 ± 45.0 283.88 ± 14.60 231.05 ± 8.56 BAA < 1.0 < 1.0 < 1.0 < 1.0 DBAA < 1.0 < 1.0 < 1.0 < 1.0 BCAA < 1.0 < 1.0 < 1.0 < 1.0 BDCAA < 1.0 < 1.0 < 1.0 < 1.0 IAA < 0.2 < 0.2 < 0.2 < 0.2
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A.2.2 CHO Cell Chronic Cytotoxicity Analysis in Aqueous Chlorine Experiments
Table A.45: Observed CHO Cell Chronic Cytotoxicity as a function of Mean Cell Density for sample AOHTOX-SW. Concentration LC50 value = 107.1; pH = 7.5, [NOM] = 5.57 mg/L-C, [iohexol] = 0.0 μM, [Cl2]T = 0 μM, [Buffer] = 4 mM, temperature = 25 °C
Group Name N Mean Std Dev SW0 16 99.935 7.858 SW10 4 99.725 6.597 SW20 4 101.654 7.399 SW30 4 96.794 7.828 SW40 4 89.225 3.251 SW50 8 89.14 5.054 SW55 4 83.693 2.288 SW60 4 80.19 6.837 SW65 4 74.167 8.369 SW70 4 77.114 3.841 SW75 4 74.922 2.943 SW80 8 70.58 5.462 SW85 4 72.304 5.87 SW90 8 61.165 9.214 SW95 4 61.353 4.093 SW100 8 53.067 5.558 SW105 4 55.056 4.009 SW110 8 46.532 6.769 SW115 8 42.171 6.668 SW120 8 40.439 2.712 SW130 8 32.733 4.794 SW140 4 27.721 1.131 SW160 4 19.972 3.839
Table A.46: ANOVA Test Cytotoxicity Statistical Analysis for AOHTOX-SW Source of Variation DF SS MS F P Between Groups 22 79638.347 3619.925 98.208 <0.001 Residual 113 4165.163 36.860 Total 135 83803.510
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Table A.47: Cytotoxicity Statistical Analysis Multiple Comparisons versus Control Group for AOHTOX-SW using Holm-Sidak Method. Overall significance level = 0.05 Comparison Diff of q’ P Significant? Means SW0 vs. SW160 79.963 23.561 <0.001 Yes SW0 vs. SW140 72.214 21.277 <0.001 Yes SW0 vs. SW130 67.202 25.563 <0.001 Yes SW0 vs. SW120 59.496 22.631 <0.001 Yes SW0 vs. SW115 57.764 21.973 <0.001 Yes SW0 vs. SW110 53.403 20.314 <0.001 Yes SW0 vs. SW100 46.868 17.828 <0.001 Yes SW0 vs. SW105 44.880 13.224 <0.001 Yes SW0 vs. SW90 38.771 14.748 <0.001 Yes SW0 vs. SW95 38.582 11.368 <0.001 Yes SW0 vs. SW80 29.355 11.166 <0.001 Yes SW0 vs. SW85 27.631 8.141 <0.001 Yes SW0 vs. SW65 25.768 7.593 <0.001 Yes SW0 vs. SW75 25.014 7.370 <0.001 Yes SW0 vs. SW70 22.821 6.724 <0.001 Yes SW0 vs. SW60 19.746 5.818 <0.001 Yes SW0 vs. SW55 16.242 4.786 <0.001 Yes SW0 vs. SW50 10.795 4.106 0.002 Yes SW0 vs. SW40 10.710 3.156 0.040 Yes SW0 vs. SW30 3.141 0.925 1.000 No SW0 vs. SW20 1.719 0.506 1.000 Do Not Test SW0 vs. SW10 0.210 0.062 1.000 Do Not Test
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Table A.48: Observed CHO Cell Chronic Cytotoxicity as a function of Mean Cell Density for sample AOHTOX-CL. Concentration LC50 value = 55.9; pH = 7.5, [NOM] = 5.57 mg/L-C, [iohexol] = 0.0 μM, [Cl2]T = 100.0 μM, [Buffer] = 4 mM, temp. = 25 °C Group Name N Mean Std Dev CL0 16 100.064 4.664 CL10 4 99.253 6.981 CL20 8 94.384 7.904 CL25 4 93.44 8.971 CL30 8 80.988 9.364 CL35 4 81.756 8.695 CL40 8 68.08 10.931 CL45 4 70.078 10.777 CL50 8 57.476 11.67 CL60 8 39.947 9.762 CL70 4 30.62 6.542 CL80 8 13.981 11.061 CL90 4 -1.375 1.775
Table A.49: ANOVA Test Cytotoxicity Statistical Analysis for AOHTOX-CL Source of Variation DF SS MS F P Between Groups 12 85976.769 7164.731 92.786 <0.001 Residual 75 5791.353 77.218 Total 87 91768.122
Table A.50: Cytotoxicity Statistical Analysis Multiple Comparisons versus Control Group for AOHTOX-CL using Holm-Sidak Method. Overall significance level = 0.05 Comparison Diff of q’ P Significant? Means CL0 vs. CL90 101.438 20.650 <0.001 Yes CL0 vs. CL80 86.083 22.623 <0.001 Yes CL0 vs. CL70 69.443 14.137 <0.001 Yes CL0 vs. CL60 60.117 15.799 <0.001 Yes CL0 vs. CL50 42.587 11.192 <0.001 Yes CL0 vs. CL40 31.984 8.406 <0.001 Yes CL0 vs. CL45 29.986 6.104 <0.001 Yes CL0 vs. CL30 19.076 5.013 <0.001 Yes CL0 vs. CL35 18.307 3.727 0.004 Yes CL0 vs. CL25 6.624 1.348 0.859 No CL0 vs. CL20 5.679 1.493 0.769 Do Not Test CL0 vs. CL10 0.811 0.165 1.000 Do Not Test Table A.51: Observed CHO Cell Chronic Cytotoxicity as a function of Mean Cell Density for sample AOHTOX-CL-IHXL. Concentration LC50 value = 45.5; pH = 7.5, 166
[NOM] = 5.57 mg/L-C, [iohexol] = 5.0 μM, [Cl2]T = 100.0 μM, [Buffer] = 4 mM, temperature = 25 °C
Group Name N Mean Std Dev CL-IHXL0 16 100.064 4.664 CL-IHXL10 4 99.545 2.778 CL-IHXL20 8 92.642 6.829 CL-IHXL25 4 94.653 6.362 CL-IHXL30 8 73.221 10.121 CL-IHXL35 4 81.501 5.614 CL-IHXL40 8 58.085 16.014 CL-IHXL45 4 63.932 10.418 CL-IHXL50 8 30.846 18.686 CL-IHXL60 8 17.935 12.319 CL-IHXL70 8 8.587 7.285 CL-IHXL80 8 7.413 6.407 CL-IHXL90 4 2.414 1.558 CL-IHXL100 4 0.687 0.888
Table A.52: ANOVA Test Cytotoxicity Statistical Analysis for AOHTOX-CL-IHXL Source of Variation DF SS MS F P Between Groups 12 85976.769 7164.731 92.786 <0.001 Residual 75 5791.353 77.218 Total 87 91768.122
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Table A.53: Cytotoxicity Statistical Analysis Multiple Comparisons vs. Control Group for AOHTOX-CL-IHXL using Holm-Sidak Method. Overall significance level = 0.05 Comparison Diff of q’ P Significant? Means CLIHXL0 v. CLIHXL1 99.376 18.106 <0.001 Yes CLIHXL0 v. CLIHXL90 97.650 17.791 <0.001 Yes CLIHXL0 v. CLIHXL80 92.650 21.793 <0.001 Yes CLIHXL0 v. CLIHXL70 91.476 21.517 <0.001 Yes CLIHXL0 v. CLIHXL60 82.129 19.318 <0.001 Yes CLIHXL0 v. CLIHXL50 69.218 16.281 <0.001 Yes CLIHXL0 v. CLIHXL40 41.979 9.874 <0.001 Yes CLIHXL0 v. CLIHXL45 36.131 6.583 <0.001 Yes CLIHXL0 v. CLIHXL30 26.843 6.314 <0.001 Yes CLIHXL0 v. CLIHXL35 18.563 3.382 0.013 Yes CLIHXL0 v. CLIHXL20 7.422 1.746 0.602 No CLIHXL0 v. CLIHXL25 5.410 0.986 0.986 Do Not Test CLIHXL0 v. CLIHXL10 0.519 0.095 1.000 Do Not Test
Table A.54: Comparative CHO cell chronic cytotoxicity of X-ray contrast agent iohexol in Akron OH water samples with and without Cl2 disinfection 2 Sample Lowest LC50 r AONVA Test Statistic Cytotoxic (CF) Conc. Factor AOHTOX-SW 40.0 107.1 0.99 F22,113 = 98.2; P ≥ 0.001 AOHTOX-CL 30.0 55.9 0.99 F12,75 = 92.8; P ≥ 0.001 AOHTOX-CL-IHXL 30.0 45.5 0.97 F13,82 = 109.9; P ≥ 0.001
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0 0 20 40 60 80 100 120 140 160 Water Sample AOHTOX-SW (Concentration Factor) Figure A.19: Observed CHO Cell Chronic Cytotoxicity as a function of Mean Cell Density for sample AOHTOX-SW. Concentration LC50 value = 107.1; pH = 7.5, [NOM] = 5.57 mg/L-C, [iohexol] = 0.0 μM, [Cl2]T = 0.0 μM, [Buffer] = 4 mM, temperature = 25 °C
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20 CHO Cell Cytotoxicity: Mean Cell Density Cell Mean Cytotoxicity: Cell CHO asthe Percent of the Negative Control (±SE) 0
0 20 40 60 80 100 Water Sample AOHTOX-CL (Concentration Factor) Figure A.20: Observed CHO Cell Chronic Cytotoxicity as a function of Mean Cell Density for sample AOHTOX-CL. Concentration LC50 value = 55.9; pH = 7.5, [NOM] = 5.57 mg/L-C, [iohexol] = 0.0 μM, [Cl2]T = 100.0 μM, [Buffer] = 4 mM, temperature = 25 °C
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20 CHO Cell Cytotoxicity: Mean Cell Density Cell Mean Cytotoxicity: Cell CHO asthe Percent of the Negative Control (±SE) 0
0 20 40 60 80 100 Water Sample AOHTOX-CL-IHXL (Concentration Factor) Figure A.21: Observed CHO Cell Chronic Cytotoxicity as a function of Mean Cell Density for sample AOHTOX-CL-IHXL. Concentration LC50 value = 45.5; pH = 7.5, [NOM] = 5.57 mg/L-C, [iohexol] = 5.0 μM, [Cl2]T = 100.0 μM, [Buffer] = 4 mM, temperature = 25 °C
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A.2.3 CHO Cell Chronic Genotoxicity Analysis in Aqueous Chlorine Experiments
Table A.55: Observed CHO Cell Chronic Genotoxicity as a function of Genomic DNA Damage for sample AOHTOX-SW. Concentration factor SCGE 50% Tail DNA value = 400.4; pH = 7.5, [NOM] = 5.57 mg/L-C, [iohexol] = 0.0 μM, [Cl2]T = 0.0 μM, [Buffer] = 4 mM, temperature = 25 °C
Group Name N Mean Std Dev SW0 6 0.981 0.490 SW50 2 0.752 0.528 SW100 2 0.603 0.070 SW200 6 1.024 0.703 SW250 4 1.382 0.395 SW300 6 2.610 1.304 SW325 4 11.425 9.699 SW350 4 13.195 8.502 SW375 4 29.812 15.208 SW400 6 60.202 15.273 SW425 4 66.442 12.465
Table A.56: ANOVA Test Genotoxicity Statistical Analysis for AOHTOX- SW Source of Variation DF SS MS F P Between Groups 10 28095.069 2809.507 36.916 <0.001 Residual 37 2815.938 76.106 Total 47 30911.007
Table A.57: Genotoxicity Statistical Analysis Multiple Comparisons versus Control Group for AOHTOX-SW using Holm-Sidak Method. Overall significance level = 0.05 Comparison Diff of q’ P Significant? Means SW0 vs. SW425 65.524 11.636 <0.001 Yes SW0 vs. SW400 59.284 11.770 <0.001 Yes SW0 vs. SW375 28.894 5.131 <0.001 Yes SW0 vs. SW350 12.277 2.180 0.234 No SW0 vs. SW325 10.534 1.871 0.399 Do Not Test SW0 vs. SW300 1.692 0.336 1.000 Do Not Test SW0 vs. SW250 0.464 0.0824 1.000 Do Not Test SW0 vs. SW100 0.315 0.0443 1.000 Do Not Test SW0 vs. SW50 0.166 0.0233 1.000 Do Not Test SW0 vs. SW200 0.106 0.0210 1.000 Do Not Test
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Table A.58: Observed CHO Cell Chronic Genotoxicity as a function of Genomic DNA Damage for sample AOHTOX-CL. Concentration factor SCGE 50% Tail DNA value = 362.4; pH = 7.5, [NOM] = 5.57 mg/L-C, [iohexol] = 0.0 μM, [Cl2]T = 100.0 μM, [Buffer] = 4 mM, temperature = 25 °C
Group Name N Mean Std Dev CL0 8 0.688 0.249 CL50 2 0.43 0.256 CL100 2 0.914 0.872 CL200 8 1.737 1.386 CL250 6 3.744 2.529 CL300 8 21.979 11.816 CL325 6 37.842 22.898 CL350 6 48.43 23.755 CL375 6 53.571 24.467 CL400 8 66.07 17.275 CL 450 6 80.903 11.054
Table A.59: ANOVA Test Genotoxicity Statistical Analysis for AOHTOX- CL Source of Variation DF SS MS F P Between Groups 10 52919.134 5291.913 23.935 <0.001 Residual 55 12160.294 221.096 Total 65 65079.428
Table A.60: Genotoxicity Statistical Analysis Multiple Comparisons versus Control Group for AOHTOX-CL using Holm-Sidak Method. Overall significance level = 0.05 Comparison Diff of q’ P Significant? Means CL0 vs. CL450 80.215 9.989 <0.001 Yes CL0 vs. CL400 65.382 8.794 <0.001 Yes CL0 vs. CL375 52.882 6.585 <0.001 Yes CL0 vs. CL350 47.742 5.945 <0.001 Yes CL0 vs. CL325 37.153 4.627 <0.001 Yes CL0 vs. CL300 21.290 2.864 0.048 Yes CL0 vs. CL250 3.055 0.380 1.000 No CL0 vs. CL200 1.048 0.141 1.000 Do Not Test CL0 vs. CL50 0.258 0.022 1.000 Do Not Test CL0 vs. CL100 0.226 0.019 1.000 Do Not Test
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Table A.61: Observed CHO Cell Chronic Genotoxicity as a function of Genomic DNA Damage for sample AOHTOX-CL-IXHL. Concentration factor SCGE 50% Tail DNA value = 258.4; pH = 7.5, [NOM] = 5.57 mg/L-C, [iohexol] = 5.0 μM, [Cl2]T = 100.0 μM, [Buffer] = 4 mM, temperature = 25 °C
Group Name N Mean Std Dev CL-IHXL0 8 0.839 0.285 CL-IHXL50 2 0.87 0.332 CL-IHXL100 8 1.077 0.468 CL-IHXL125 6 1.652 0.73 CL-IHXL150 6 2.297 1.088 CL-IHXL175 6 6.582 2.703 CL-IHXL200 8 20.996 9.541 CL-IHXL225 5 26.691 11.496 CL-IHXL250 6 47.225 14.182 CL-IHXL300 8 70.763 14.282 CL-IHXL350 6 89.732 4.316
Table A.62: ANOVA Test Genotoxicity Statistical Analysis for AOHTOX- CL-IHXL Source of Variation DF SS MS F P Between Groups 10 63784.295 6378.429 98.923 <0.001 Residual 58 3739.770 64.479 Total 68 67524.065
Table A.63: Genotoxicity Statistical Analysis Multiple Comparisons versus Control Group for AOHTOX-CL-IHXL using Holm-Sidak Method. Overall significance level = 0.05 Comparison Diff of q’ P Significant? Means CL-IHXL0 v. CL-IHXL350 88.893 20.498 <0.001 Yes CL-IHXL0 v. CL-IHXL300 69.924 17.416 <0.001 Yes CL-IHXL0 v. CL-IHXL250 46.386 10.969 <0.001 Yes CL-IHXL0 v. CL-IHXL225 25.852 5.647 <0.001 Yes CL-IHXL0 v. CL-IHXL200 20.157 5.020 <0.001 Yes CL-IHXL0 v. CL-IHXL175 5.743 1.324 0.771 No CL-IHXL0 v. CL-IHXL150 1.458 0.336 1.000 Do Not Test CL-IHXL0 v. CL-IHXL125 0.814 0.188 1.000 Do Not Test CL-IHXL0 v. CL-IHXL100 0.238 0.059 1.000 Do Not Test CL-IHXL0 v. CL-IHXL50 0.031 0.005 1.000 Do Not Test
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Table A.64: Comparative CHO cell chronic genotoxicity of X-ray contrast agent iohexol in Akron OH water samples with and without Cl2 disinfection Sample Lowest 50% Tail r2 AONVA Test Statistic Cytotoxic DNA (CF) Conc. Factor AOHTOX-SW 375 400.4 0.94 F10,37 = 36.9; P ≥ 0.001 AOHTOX-CL 300 362.4 0.99 F10,55 = 23.9; P ≥ 0.001 AOHTOX-CL-IHXL 200 258.4 0.99 F10,58 = 98.9; P ≥ 0.001
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CHOCell Genomic DNA Damage as the Average 0
0 100 200 300 400 500 Water Sample AOHTOX-SW (Concentration Factor) Figure A.22: Observed CHO Cell Acute Genotoxicity as a function of Genomic DNA Damage for sample AOHTOX-SW. Concentration factor SCGE 50% Tail DNA value = 400.4; pH = 7.5, [NOM] = 5.57 mg/L-C, [iohexol] = 0.0 μM, [Cl2]T = 0.0 μM, [Buffer] = 4 mM, temperature = 25 °C
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CHOCell Genomic DNA Damage as the Average 0
0 100 200 300 400 500 Water Sample AOHTOX-CL (Concentration Factor) Figure A.23: Observed CHO Cell Acute Genotoxicity as a function of Genomic DNA Damage for sample AOHTOX-CL. Concentration factor SCGE 50% Tail DNA value = 362.4; pH = 7.5, [NOM] = 5.57 mg/L-C, [iohexol] = 0.0 μM, [Cl2]T = 100.0 μM, [Buffer] = 4 mM, temperature = 25 °C
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20 MeanSCGE Tail% DNA Value (±SE)
CHOCell Genomic DNA Damage as the Average 0
0 100 200 300 Water Sample AOHTOX-CL-IHXL (Concentration Factor) Figure A.24: Observed CHO Cell Acute Genotoxicity as a function of Genomic DNA Damage for sample AOHTOX-CL-IHXL. Concentration factor SCGE 50% Tail DNA value = 258.4; pH = 7.5, [NOM] = 5.57 mg/L-C, [iohexol] = 5.0 μM, [Cl2]T = 100.0 μM, [Buffer] = 4 mM, temperature = 25 °C
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