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The fate of emerging contaminants in wastewater treatment plants Simhon, Michal Vered 2013

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THE FATE OF EMERGING CONTAMINANTS  IN WASTEWATER TREATMENT PLANTS  by  Michal Vered Simhon  B.Sc. (Chemical Engineering), Queen?s University, 2007  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF APPLIED SCIENCE in The Faculty of Graduate and Postdoctoral Studies (Civil Engineering)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  December 2013  ? Michal Vered Simhon, 2013 ii  ABSTRACT Potential risk and toxicity of emerging contaminants (ECs) on the human population and the surrounding ecosystem have led to growing concern in the scientific world. This class of contaminants includes a variety of commonly used compounds such as pharmaceuticals and personal care products, as well as industry-based compounds such as surfactants and plasticizers. There are many challenges involved when studying these contaminants with the most apparent one relating to the lack of a standardized analytical method. As a result, this research investigated and optimized an analytical method for the determination of selected EC compounds in the soluble and particulate fractions of samples collected from the wastewater treatment pilot plant located at the University of British Columbia, Vancouver, Canada. Once the method was optimized, the effects of hydraulic retention time (HRT) and solids retention time (SRT) on the fate of ECs through the wastewater treatment process were studied using the UBC membrane biological nutrient removal pilot plant. The study involving the HRT failed to provide sufficient evidence to adequately assess the observed effects, since an inadequate quantity of each contaminant was spiked into the pilot plant. However, the spiking quantity was adjusted for the individual contaminants based on these observations, enabling the effect of the SRT to be studied. All analytes exhibited one of two trends during the mass balance analysis. For all of the EC compounds that were studied, sorption to the mixed liquor particulates and washout in the effluent were not the only removal mechanisms in the experimental system of the UBC pilot plant.    iii  PREFACE A condensed version of Chapter 2 and Chapter 3 were provided to the Canadian Water Network (CWN) to summarize the findings from my research, specifically relating to the analytical method development of emerging contaminants, and their individual responses to changes at the UBC pilot plant.   iv  TABLE OF CONTENTS ABSTRACT ................................................................................................................................. ii PREFACE .................................................................................................................................. iii TABLE OF CONTENTS ............................................................................................................. iv LIST OF TABLES ...................................................................................................................... vii LIST OF FIGURES .................................................................................................................... ix ACKNOWLEDGEMENTS .......................................................................................................... xi 1 INTRODUCTION ................................................................................................................ 1 1.1 Literature Review ......................................................................................................... 1 1.1.1 Background on Emerging Contaminants ............................................................... 1 1.1.2 Chemical Properties .............................................................................................. 2 1.1.3 Risks and Toxicity ................................................................................................. 5 1.1.4 Sampling and Analysis .......................................................................................... 6 1.1.5 Treatment Methods ............................................................................................... 9 1.1.6 Regulations and Policies in Canada .....................................................................14 1.1.7 Modeling and Design ...........................................................................................15 1.1.8 Selection of the Suite of Emerging Contaminants ................................................16 1.2 Motivation for the Research ........................................................................................22 1.3 Research Objectives ...................................................................................................23 2 MATERIALS AND EXPERIMENTAL METHODS ...............................................................24 2.1 Experimental Details ...................................................................................................24 2.2 Materials .....................................................................................................................27 2.2.1 Tracer Compound ................................................................................................27 2.2.2 Emerging Contaminants for Stock Solutions ........................................................27 2.2.3 Solvents ...............................................................................................................27 2.2.4 Derivatization Solvents ........................................................................................27 2.2.5 Bottles..................................................................................................................28 2.3 Analytical Instrumentation ...........................................................................................28 2.4 Extraction Methodology ..............................................................................................30 2.4.1 Optimized Extraction Method for Soluble Fraction:  INF-?, ML-?, and EFF ..........30 2.4.2 Optimized Extraction Method for Particulate Fraction: INF-?, ML-? ......................31 2.4.3 Optimized Analytical Method for Extracted Samples: All Samples .......................32 2.5 QA/QC ........................................................................................................................33 2.6 Measure for Hydrophobicity of ML ..............................................................................36 3 RESULTS AND DISCUSSIONS ........................................................................................38 3.1 Method Development ..................................................................................................38 v  3.1.1 Analytical Method Development for Soluble Fraction ...........................................38 3.1.1.1 SPE Method .................................................................................................38 3.1.1.2 LLE Method ..................................................................................................41 3.1.2 Analytical Method Development for Particulate Fraction ......................................45 3.1.3 Method Development for Analysis on the GC-MS .....................................................46 3.2 Tracer Study ...............................................................................................................49 3.3 Sampling Program Design ..........................................................................................52 3.4 Comparison of Steady versus Variable HRT ...............................................................53 3.4.1 Spiking Mass of Background Concentration for Each Analyte ..............................53 3.4.2 Concentration Profile ...........................................................................................57 3.5 Comparison of Long versus Short SRT .......................................................................65 3.5.1 Background Influent Concentrations ....................................................................67 3.5.2 Soluble and Particulate Mass Fraction .................................................................69 3.5.3 Concentration Profile ...........................................................................................74 3.5.4 QA/QC .................................................................................................................80 3.5.5 Methodology for Mass Balance ............................................................................82 3.5.6 Trends from the Mass Balance ............................................................................84 3.5.6.1 Hydrophobic Analytes ...................................................................................84 3.5.6.2 Hydrophilic and Other Analytes .....................................................................90 3.5.7 Interpreting the Unaccounted Mass .....................................................................94 4 CONCLUSIONS AND RECOMMENDATIONS...................................................................96 4.1 Conclusions ................................................................................................................96 4.2 Recommendations ......................................................................................................97 REFERENCES .........................................................................................................................98 APPENDIX A: ANALYTICAL METHOD................................................................................... 102 Appendix A.1: Liquid ? Liquid Extraction (LLE) for INF-?, ML-?, and EFF ........................... 102 Appendix A.2: Ultrasonic Extraction (USE) for INF-? and ML-? ........................................... 103 Appendix A.3: Optimization of Analytical Method ................................................................. 104 APPENDIX B: RAW CONCENTRATION DATA ...................................................................... 106 Appendix B.1: Data for Stage 1 (Comparison of Steady vs. Diurnal HRT) ........................... 106 Appendix B.2: Data for Stage 2 (Short vs. Long SRT) ......................................................... 118 APPENDIX C: MASS BALANCE RESULTS FOR REMAINDER OF ANALYTES .................... 143 Appendix C.1: Mass Balance ? Hydrophobic Analytes ........................................................ 143 Appendix C.2: Mass Balance ? Hydrophilic and Other Analytes .......................................... 152 APPENDIX D: SAMPLING SCHEDULE .................................................................................. 161 Appendix D.1: Sampling Schedule for Stage 1 .................................................................... 161 vi  Appendix D.2: Sampling Schedule for Stage 2 .................................................................... 163 Appendix D.3: Task Checklist for Stage 2 ............................................................................ 165 Day 1 Tasks ..................................................................................................................... 165 Day 2 Tasks ..................................................................................................................... 170 Day 3 Tasks ..................................................................................................................... 176 Day 4 Tasks ..................................................................................................................... 182 Day 5 Tasks ..................................................................................................................... 186 Day 6 Tasks ..................................................................................................................... 190 Day 7 Tasks ..................................................................................................................... 194 Day 8 Tasks ..................................................................................................................... 197 APPENDIX E: SUPPLEMENTARY INFORMATION FOR QA/QC ? STAGE 2 ........................ 209   vii  LIST OF TABLES Table 1: Indicative percent removals of organic chemicals during various stages of wastewater treatment (USEPA, 2012) .........................................................................................................13 Table 2: Examples of models to calculate properties required to predict the fate and transport of contaminants (Stuart et al., 2012) .............................................................................................16 Table 3: Properties of chosen emerging contaminants for research ..........................................17 Table 4: Full scale treatment systems average removal rates of emerging contaminants (Adapted from USEPA, 2010) ...................................................................................................21 Table 5: Average values for parameters from both sides of the UBC pilot plant ........................25 Table 6: Details of varying parameters at the UBC pilot plant for both experimental stages ......26 Table 7: Characteristics of emerging contaminants on the GC-MS ...........................................29 Table 8: Temperature program in the GC-MS ...........................................................................33 Table 9: IDL for suite of ECs on the GC-MS ..............................................................................35 Table 10: MDL for suite of ECs .................................................................................................36 Table 11: Summary of stock solution concentrations and volumes used for derivatization ........47 Table 12: Temperature program for the GC-MS ........................................................................49 Table 13: Summary of sampling program ..................................................................................53 Table 14: Average concentrations of ECs in wastewater influent ..............................................55 Table 15: Background concentrations .......................................................................................64 Table 16: Parameters at the UBC pilot plant ? Stage 2 .............................................................65 Table 17: Summary of analyte concentrations added to the UBC pilot plant .............................66 Table 18: Effluent and soluble ML data for EE2 and caffeine ....................................................80 Table 19: Average RSD values for analytes ..............................................................................81 Table 20: Average percent recoveries for analytes ...................................................................81 Table 21: Unaccounted mass for all analytes ............................................................................95  Table A - 1: Optimization parameters for analytical method .................................................... 104  Table B - 1: Raw concentration data for Stage 1 experiment................................................... 106 Table B - 2: Raw concentration data for Stage 2 experiment, divided according to particulate and soluble fraction ................................................................................................................. 118 Table B - 3: Raw total concentration data for Stage 2 experiment ........................................... 133   Table D - 1: Sampling schedule for Stage 1 ............................................................................ 161 Table D - 2: Sampling schedule for Stage 2 ............................................................................ 163  viii  Table E - 1: RSD and recovery percentages at time = 3 hours ................................................ 209 Table E - 2: RSD and recovery percentages at time = 24 hours .............................................. 210 Table E - 3: RSD and recovery percentages at time = 48 hours .............................................. 211   ix  LIST OF FIGURES Figure 1: Sources and pathways for emerging contaminants to reach various receptors (Stuart et al., 2012) ................................................................................................................................ 3 Figure 2: Limitations and complexities of environmental chemical analysis (Daughton, 2003) ... 7 Figure 3: Pilot plant configuration at UBC ..................................................................................24 Figure 4: LiCl tracer results at the pilot plant .............................................................................51 Figure 5: Particulate and soluble influent concentrations for all 10 analytes at the UBC pilot plant (Stage 1) ...................................................................................................................................56 Figure 6: Concentration profile for EE2 in the A-Side (steady HRT) ..........................................59 Figure 7: Concentration profile for EE2 in the B-Side (variable HRT) ........................................60 Figure 8: Concentration profile for caffeine in the A-Side (steady HRT) ....................................62 Figure 9: Concentration profile for caffeine in the B-Side (variable HRT)...................................63 Figure 10: Influent concentrations for all analytes at the UBC pilot plant (Stage 2) ....................68 Figure 11: Soluble and particulate fractions of target ECs at time zero in the INF .....................70 Figure 12: Soluble and particulate fractions of target ECs at time zero in the A-Side ML (SRT = 15 d) .........................................................................................................................................72 Figure 13: Soluble and particulate fractions of target ECs at time zero in the B-Side ML (SRT = 24 d) .........................................................................................................................................73 Figure 14: Concentration profile for EE2 in the A-Side (SRT = 15 days) ...................................75 Figure 15: Concentration profile for EE2 in the B-Side (SRT = 24 days) ...................................76 Figure 16: Concentration profile for caffeine in the A-Side (SRT = 15 days) ..............................77 Figure 17: Concentration profile for caffeine in the B-Side (SRT = 24 days) ..............................78 Figure 18: Mass Balance for nonylphenol in the A-Side (SRT = 15 d) .......................................85 Figure 19: Mass Balance for nonylphenol in the B-Side (SRT = 24 d) .......................................86 Figure 20: Mass Balance for Dibutyl Phthalate in the A-Side (SRT = 15d) ................................92 Figure 21: Mass Balance for Dibutyl Phthalate in the B-Side (SRT = 24d) ................................93  x  Figure C - 1: Mass Balance for Tonalide in the A-Side (SRT = 15d) ........................................ 144 Figure C - 2: Mass Balance for Tonalide in the B-Side (SRT = 24d) ........................................ 145 Figure C - 3: Mass Balance for Irgasan in the A-Side (SRT = 15d) ......................................... 146 Figure C - 4: Mass Balance for Irgasan in the B-Side (SRT = 24d) ......................................... 147 Figure C - 5: Mass Balance for EE2 in the A-Side (SRT = 15d)............................................... 148 Figure C - 6: Mass Balance for EE2 in the B-Side (SRT = 24d)............................................... 149 Figure C - 7: Mass Balance for Gemfibrozil in the A-Side (SRT = 15d) ................................... 150 Figure C - 8: Mass Balance for Gemfibrozil in the B-Side (SRT = 24d) ................................... 151 Figure C - 9: Mass Balance for Naproxen in the A-Side (SRT = 15d) ...................................... 153 Figure C - 10: Mass Balance for Naproxen in the B-Side (SRT = 24d) .................................... 154 Figure C - 11: Mass Balance for Estrone in the A-Side (SRT = 15d) ....................................... 155 Figure C - 12: Mass Balance for Estrone in the B-Side (SRT = 24d) ....................................... 156 Figure C - 13: Mass Balance for Caffeine in the A-Side (SRT = 15d) ...................................... 157 Figure C - 14: Mass Balance for Caffeine in the B-Side (SRT = 24d) ...................................... 158 Figure C - 15: Mass Balance for Ibuprofen in the A-Side (SRT = 15d) .................................... 159 Figure C - 16: Mass Balance for Ibuprofen in the B-Side (SRT = 24d) .................................... 160   xi  ACKNOWLEDGEMENTS I would like to thank my supervisor Dr. Eric Hall and Research Assistant Patricia Keen for providing me with assistance and support throughout the research. I would also like to thank Paula Parkinson and Timothy Ma for their guidance and direction in the laboratory, and Fred Koch for his advice in regards to the UBC pilot plant. In addition, I am grateful to my family and friends for their tremendous support and encouragement during my studies at UBC.   Finally, I am appreciative to the Canadian Water Network for funding my research, and allowing me to contribute to the collaborative efforts of Environment Canada, the Ontario Ministry of the Environment and the Minist?re de D?veloppement Durable, de l?Environnement et des Parcs du Qu?bec in an effort to minimize the knowledge gaps surrounding emerging contaminants in common Canadian wastewater treatment systems.     1  1 INTRODUCTION The purpose of this research was to investigate the fate of emerging contaminants (ECs) in wastewater treatment plants (WWTPs). These contaminants are gaining significant attention due to their potential risk to human health, and their presence is largely attributed to their release into the environment via wastewater effluents. Therefore, beyond the challenges in research and public health, ECs have now become a practical problem pertaining to civil engineering (specifically relating to wastewater treatment systems).   This research comprises a fraction of a broader study undertaken by the Canadian Water Network (CWN) in 2011. The intent of the CWN project was to decrease the knowledge gap surrounding ECs by conducting a multi-disciplinary nation-wide study examining process configuration and climate effects on the removal of ECs, the modelling of the fate of ECs throughout WWTP under various conditions, and the completion of chemical and toxicological assessments based on exposure to ECs.   1.1 Literature Review  1.1.1 Background on Emerging Contaminants Emerging contaminants are compounds that are either present in the environment for prolonged periods of time, but only recently detected by humans, or newly synthesized compounds currently being introduced and detected in the environment (Smital, 2008). These compounds have the potential of migrating into the environment via water sources, land, or the atmosphere.  The contaminants that fall under the EC category defined by the USEPA are unregulated substances that have been identified or reviewed recently in water sources which may pose a 2  risk to the aquatic environment. Furthermore, some of the compounds are considered to be ECs because the method of detection has only been developed recently (USEPA, 2008).   The types of ECs that exist are persistent organic pollutants (POPs), pharmaceuticals and personal care products (PPCPs), veterinary medicines such as antibiotics and antifungals, endocrine disrupting chemicals (EDCs) (either anthropogenic or naturally occurring), and nanomaterials such as carbon nanotubes (USEPA, 2008). These ECs are present at low concentrations in the environment (Kolpin et al., 2002). This review will focus on organic ECs typically found in wastewater, sludge, surface water, groundwater, soil, and sediment (Pal et al., 2010; Xu et al., 2009).  Researching ECs is particularly challenging for several reasons. First, the compounds occur at very low concentrations, making them difficult to detect. Second, the compounds have varying physicochemical properties, further limiting the use of a representative chemical species as an indicator. Finally, it is difficult to analyze sludge or wastewater samples due to the complexity of the matrices (Bolong et al., 2009). It is, therefore, crucial to reduce these challenges and improve the monitoring and surveillance systems of such contaminants.  1.1.2 Chemical Properties ECs are a type of hazardous material that can be classified based on persistence, bioaccumulation, and toxicity to an ecosystem and its organisms. Persistence refers to the stability or half-life of these contaminants. Some compounds may reach pseudo-steady state conditions due to continuous discharge into the environment. Bioaccumulation relates to the contaminant?s preferred environment and where it will end up in the food chain. Finally, toxicity refers to the impact of compounds as determined by either acute or chronic testing.  Currently 3  sub-lethal toxicity for many ECs is unknown due to a lack of chronic exposure evaluations, but it is recognized that some of these contaminants act as endocrine disrupting compounds (Smital, 2008).   The transfer of ECs in the environment is based on three key components using the source-pathway-receptor model (shown in Figure 1). First, there must be a source or discharge for the ECs. This may either be localized (referred to as a point source) or diffuse (referred to as a non-point source). Sources of ECs include WWTPs, industrial effluents, combined sewer overflows, and agricultural runoff. Second, the ECs travel from the source using a specific pathway. Finally, a receptor refers to a final location or action where the ECs are placed in contact with humans or an element of the environment. This could include contact through drinking tap water, surface waters, and groundwater (Stuart et al., 2012).  Figure 1: Sources and pathways for emerging contaminants to reach various receptors (Stuart et al., 2012) 4   The fate of compounds relates to the compound?s preferred environment or molecular structure.  Once ECs are discharged into the environment, the prevailing fate mechanisms include degradation (either chemically or biologically), conjugation, volatilization, dispersion, photolysis, biotransformation, or leaching (Pal et al., 2010; Xu et al., 2009). Regardless of the fate mechanism, the contaminant subsequently either remains in solution or sorbs onto the particulate fraction. Of the ECs, polar contaminants (containing acidic constituents) pose the greatest risk to the environment given their high water solubility and slow degradation rates (Petrovic et al., 2003).   Various physicochemical parameters govern the fate of ECs in the environment. Degradation is characterized by a compound?s stability, which is denoted as its half-life (t1/2). The octanol-water partition coefficient (Kow) describes the preference of a compound to be partitioned in water or in octanol. The solid-water partition coefficient (Kd) governs the preference of a compound to be associated with either the water or particulate  phases (Xu et al., 2009). In general, compounds with a high molecular weight and a log Kow above 5, are classified as hydrophobic, and these tend to sorb effectively onto particulates or sediment, resulting in effective coagulation. Furthermore, compounds with a log Kow below 2.5 are likely to be found in the water, rather than the particulate phase. For compounds falling in the log Kow range between 2.5 and 5, likely have a preference to either water or sediments, depending on the compound. Furthermore, compounds with a Kd value between 2.1 and 2.9 (such as estrogens) exhibit preferred sorption to sludge in a WWTP. In contrast, compounds with a Kd value between 0.2 and 1.9 (such as pharmaceuticals) would exhibit negligible removal by sorption on sludge (Pal et al., 2010).  5  1.1.3 Risks and Toxicity Although exposure to ECs from drinking water or non-potable water reuse poses a minimal public health risk (USEPA, 2012), evidence suggests that an environmental risk from both wastewater discharge or stormwater runoff does exist (USEPA, 2012).  Among the most prominent risks associated with exposure to ECs are reproductive defects in wildlife (Toppari et al., 1996), sterility (Jobling et al., 1998), and antimicrobial resistance (K?mmerer, 2004). ECs can pose a risk both to the aquatic environment and to humans, resulting in health threats such as cancer, immune system disturbances, development of antibiotic resistance, and endocrine disruption (Xu et al., 2009). Currently, the toxicity of ECs may be known, but cannot be regulated due to inadequate sub-lethal chronic toxicity exposure evaluations (Smital, 2008). Furthermore, toxicity testing alone would provide insufficient data, given the low background concentrations of the ECs in the environment, further straining the evaluation of potential risks (USEPA, 2012).   The aquatic life criterion (ALC) is a measure required for ECs that are both detected and believed to have an adverse effect on the environment. ALC can be sub-categorized into the criterion maximum concentration (CMC) and criterion continuous concentration (CCC). The first refers to the allowable concentration to prevent acute effects (following an exposure time of one hour). It is based on the median lethal concentration (LC50), or the median effect concentration (EC50). The CCC refers to the allowable concentration to prevent chronic (long term) effects following a four day exposure. This measure is based on the no observed effect concentration (NOEC), or the lowest observed effect concentration (LOEC). Examples of such effects include defects in reproduction, growth, survival, behaviour, etc. (USEPA, 2008).   6  Methods for testing the toxicity of compounds employ the use of freshwater invertebrates such as daphnia (most sensitive), fish, algae (least sensitive), and mussels. Using such methods, toxicity analysis for compounds generally requires the determination of the lowest predicted no-effect concentration (PNEC), which is calculated using the NOEC value for the most sensitive species and a certain safety factor. If the NOEC value is unavailable, the value may be substituted using the minimum inhibitory concentration (MIC) or LOEC. Many ECs have been detected in the environment at concentrations greater than their respective PNEC value (Pal et al., 2010) and therefore, improving our understanding of the fate of ECs is critical to protection of the overall health of the ecosystem.   1.1.4 Sampling and Analysis As previously mentioned, no standardized method exists for the analysis of ECs. This is attributed to the required low detection limits, the complexity of the samples, and the struggle of compound separation within samples. Figure 2 (Daughton, 2003) illustrates the complexity in the analyses of samples containing environmentally-relevant concentrations of contaminants. It indicates that when a matrix (such as water or air) is analyzed, the samples must first be prepared by an extraction method (discussed in detail below). However, not all compounds can be extracted, depending on the sample medium or the extraction procedure used. Of the compounds that may be successfully extracted, separation will only be achieved on a portion of them. Following the physical processes, the samples are analyzed using an analytical instrument (also detailed below), and successful detection of native compounds as well as artifacts (referring to compounds that are not naturally present) occurs. Finally, of the compounds successfully detected, only a portion can accurately be identified. Therefore, many contaminants are overlooked and neglected, implying that there will continue to be newly identified or detected ECs.  7    Figure 2: Limitations and complexities of environmental chemical analysis (Daughton, 2003)  Derivatization is often employed with ECs, as this ensures that the analytes exhibit better chromatography (through sharper and smoother peaks) on a gas chromatograph ? mass spectrometer (GC-MS) by making the analytes more volatile, less polar, and more stable. This step is often employed in the separation process shown in Figure 2. A derivatization agent, such as N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) + 1% trimethylchlorosilane (TMCS) may be 8  used to add a trimethyl silyl (TMS) group to the compound in place of a hydrogen molecule (Farr? et al., 2007).   Of the various analytical methods that exist to measure such compounds, the most common ones are further addressed in this section. The first is the coupling of liquid chromatography with mass spectrometry (LC/MS) (Richardson & Ternes, 2011).  Using this method, three types of instrumentation exist including the triple quadrapole, the ion trap, and the quadrapole time of flight (Q-TOF) (Snow et al., 2009). Q-TOF is most commonly used for the identification of known and unknown polar compounds. It may also be coupled with atmospheric pressure photoionization (APPI) for non-polar compounds (Richardson & Ternes, 2011).  Another analytical method commonly used for steroidal hormones, involves the pairing of gas chromatography with mass spectrometry (GC-MS) (Snow et al., 2009). Furthermore, isotopic standards, as well as liquid chromatography tandem mass spectrometry (LC/MS/MS) may also be applied as an analytical method. The latter may require the use of nuclear magnetic resonance (NMR) spectroscopy to confirm results (Richardson & Ternes, 2011).   It should be noted that the analysis of ECs has primarily focussed on surface or sewage water and not sludge. Sludges are typically analyzed using GC or LC coupled with MS or MS/MS (tandem). However, sludge is extremely non-homogenous and therefore, difficult to analyse. The analysis may also be influenced by the presence of additional compounds such as wastewater treatment aids (D?az-Cruz et al., 2009).  As Figure 2 illustrates, typically analysis focuses on target compounds where standards are created, analytical methods are optimized, and specific ions are monitored. However, throughout the acquisition of results, it may appear that the analysis should be conducted on 9  non-target compounds, which may include unknown transformation products of target analytes. In this case, no standards are available, and compounds are identified using a full scan approach (rather than specific ions). For these purposes, technologies that are able to provide a sensitive full scan range in addition to a large database on the library are deemed to be most effective. LC/MS, high-resolution mass spectrometry (HRMS), and Q-TOF have all shown promise (Ag?era et al., 2013).   Prior to analysing ECs, extraction methods must be selected. Solid phase extraction (SPE) and liquid-liquid extraction (LLE) are the methods most commonly implemented. However, some solvent-less extraction methods exist, including solid phase micro-extraction (SPME), single-drop micro-extraction (SDME), stir bar sorptive extraction, or hollow-fibre membrane micro-extraction. In addition, molecularly imprinted polymers (MIPs) are commonly used for select extraction methods of pharmaceuticals, EDCs, and pesticides. Nanomaterials and microsensor arrays imprinted onto compact discs (CD?s) have also been used to facilitate the detection of some compounds (Richardson & Ternes, 2011).   1.1.5 Treatment Methods The EC removal achieved in WWTPs depends on the solids retention time (SRT), hydraulic retention time (HRT), and temperature. For example, with warmer temperatures that occur during the summer, kinetic rates for removal of ECs are increased. Furthermore, it is believed that a longer SRT (in the order of months) is required for the most effective removal of these compounds. However, this condition is not feasible because of complications that would arise from operational maintenance  (Knepper, 2008).   10  Conventional treatment processes in WWTPs are incapable of entirely removing trace ECs (USEPA, 2012).  Nevertheless, sorption to biosolids may occur if the ECs are lipophilic (i.e. hydrophobic compounds) (Knepper, 2008).   Information regarding treatment technologies in WWTPs specified for the degradation of ECs is scarce. Coagulation and flocculation, as well as biological treatment have proven to be ineffective for the complete removal of EDCs and PPCPs. Advanced treatment methods such as UV photolysis and ion exchange have been deemed successful but not feasible for WWTPs because of increased disinfectant dosage requirements (Bolong et al., 2009). In addition, activated carbon adsorption has illustrated removal of various ECs excluding polar water soluble contaminants (which comprise a significant quantity of ECs) (USEPA, 2012).   However, it has been shown that oxidation processes (such as ozonation, advanced oxidation processes (AOPs), or ferrate) have successfully broken down ECs such as pharmaceuticals (Petrovic et al., 2003). Interestingly, ozonation has been demonstrated to degrade ibuprofen and naproxen by converting the compounds to biologically active components (Pal et al., 2010). Nevertheless, caution must be taken with these daughter products, as they may pose a greater risk than the parent compound (Richardson & Ternes, 2011). Although AOPs may lead to a slight increase in EC removal, they are associated with a high operating cost (USEPA, 2012). This corresponds to high electricity requirements, large quantities of oxidants required, slow kinetics, and required pH adjustments (Klamerth et al., 2010).  An alternative to the use of AOPs for the breakdown of ECs is the use of photo-Fenton AOP processes. This technology utilizes energy from the sun which significantly reduces the operating costs. In this process, the ferrous ion (produced photolytically) is oxidized with hydrogen peroxide to yield oxidants. However, the conventional photo-Fenton process cannot 11  be utilized to remove high concentrations of compounds (such as those present in industrial waste) because excessive iron concentrations would accumulate in the effluent. Results have further demonstrated that most of the EC degradation occurs in the Fenton process, while the photo process is required for the purpose of reducing the ECs to levels below the limit of detection (LOD). Alternatively, the Fenton process utilizes hydroxide radicals produced from hydrogen peroxide and ferrous ion (Klamerth et al., 2010).   Other promising technologies that have been demonstrated for the removal of compounds in WWTPs include membrane bioreactors (MBRs) or membrane filtration exclusively. Once again, the available literature on the effectiveness for ECs is scarce. Nevertheless, microfiltration (MF) and ultrafiltration (UF) have been deemed not viable due to their large pore sizes. The only potential removal mechanism for ECs in these systems is based on hydrophobic interactions between the contaminants and the membranes (USEPA, 2012).   Other filtration methods such as nanofiltration (NF), and reverse osmosis (RO) have shown promise in removing ECs (Petrovic et al., 2003). The removal mechanisms for both NF and RO systems are based on size of the pores (for organic solutes), zeta potential (for inorganic ionic solutes), and the interaction between solute, solvent, and membrane (for uncharged organic solutes), where the latter mechanism relies on polarity. Nevertheless, studies using wastewater samples with NF/RO systems with multiple ECs yielded ineffective results. No clear explanation could be conveyed for the retention mechanisms of the ECs (Kunst, 2008).   Given the immense complexity of the interactions among various ECs found in the water system, it is impractical to use solely a single treatment method for these contaminants. Table 1 (adapted from USEPA, 2012) illustrates the removal efficiencies of numerous organic pollutants 12  using a select number of wastewater treatment technologies, clearly demonstrating that removal efficiencies for a given technology are contaminant specific.   A multi-step approach can be incorporated whereby numerous technologies are placed in series to maximize EC removal. For instance, literature has demonstrated that the combination of biological and chemical processes with activated carbon adsorption is an effective method. Also, physical separation (such as reverse osmosis (RO)) in conjunction with chemical oxidation has been demonstrated to be an effective means of EC removal. Finally, the use of natural processes with chemical oxidation or activated carbon adsorption may also be beneficial for EC removal (USEPA, 2012).  13  Table 1: Indicative percent removals of organic chemicals during various stages of wastewater treatment (USEPA, 2012) Treatment Percent Removal B(a)p Antibiotics1 Pharmaceuticals Hormones Fragrance NDMA DZP CBZ DCF IBP PCT Steroid2 Anabolic3 Secondary (activated sludge) nd 10-50 nd - 10-50 >90 nd >90 nd 50-90 - Soil aquifer treatment nd nd nd 25-50 >90 >90 >90 >90 nd >90 >90 Aquifer storage nd 50-90 10-50 -      - - Microfiltration nd <20 <20 <20 <20 <20 <20 <20 nd <20  Ultrafiltration/ powdered activated carbon (PAC) nd >90 >90 >90 >90 >90 nd >90 nd >90 >90 Nanofiltration >80 50-80 50-80 50-80 50-80 50-80 50-80 50-80 50-80 50-80  Reverse osmosis >80 >95 >95 >95 >95 >95 >95 >95 >95 >95 25-50 PAC >80 20->80 50-80 50-80 20-50 <20 50-80 50-80 50-80 50-80  Granulated activated carbon  >90 >90 >90 >90 >90  >90  >90 >90 Ozonation >80 >95 50-80 50-80 >95 50-80 >95 >95 >80 50-90 50-90 Advanced oxidation  50-80 50-80 >80 >80 >80 >80 >80 >80 50-80 >90 High-level ultraviolet  20->80 <20 20-50 >80 20-50 >80 >80 20-50 nd >90 Chlorination >80 >80 20-50 -<20 >80 <20 >80 >80 <20 20->80 - Chloramination 50-80 <20 <20 <20 50-80 <20 >80 >80 <20 <20   B(a)p = benz(a)pyrene; DZP = diazepam; CBZ = carbamazepine; DCF = diclofenac; IBP = ibuprofen; PCT = paracetamol; NDMA = N-nitosodimethylamine; nd = no data; PAC = powdered activated carbon 1 erythromycin, sulfamethoxazole, triclosan, trimethoprim 2 ethynylestradiol, estrone, estradiol, estroil 3 progesterone, testosterone     14  1.1.6 Regulations and Policies in Canada Although no current regulations have been put into place in regards to removing ECs in WWTPs, government agencies have been using past research and studies to ban or restrict harmful contaminants at the source. For instance, Canada has completely eliminated the use of several pesticides (including DDT), polychlorinated biphenyl (PCBs) used for insulation, and select EDCs such as dioxin (Holtz, 2006).  The 2004 convention on persistent organic pollutants and the Rotterdam Convention for the treaty on Prior Informed Consent led to the banning of, or increased restrictions on, 32 chemicals thought to be harmful. Nevertheless, there exist differences in opinion on select contaminants between different countries. For example, recombinant Bovine Growth Hormone (rBGH) is no longer sanctioned in the European Union and Canada, but remains unregulated for use in the United States (Holtz, 2006).  Canada has yet to create regulations to control the use or release of antibiotics into the environment, which is of particular concern given the potential for development of antimicrobial resistance in microbial communities or human pathogens. Historically, regulatory agencies have focused on the presence of such drugs in meat or milk, where now the attention must be shifted to address antimicrobial resistance. However, this issue has been a growing concern, with attention received from governmental health, food, and drug agencies. Since 2002, the Canadian Public Health Agency has conducted the Canadian Integrated Program for Antimicrobial Resistance Surveillance (CIPARS) to monitor trends and effects of antibiotic resistance in various organisms (Holtz, 2006) with increasing concern for the consequences of antimicrobial resistance in the environment.   15  The regulation of EDCs is far more challenging given the lack of agreement for contaminants that should fall under this category. Nevertheless, there is a consensus on common contaminants. The international Organisation for Economic Co-operation and Development (OECD) and the USEPA are in the process of creating a procedure for testing and monitoring of such contaminants; Canada will likely adhere to such protocols once created (Holtz, 2006).    Although government agencies are responsible for establishing regulation, social implications must be addressed. It is crucial to educate the public on decreased use and proper disposal of ECs (Holtz, 2006). Since many of these contaminants exhibit pseudo-persistence in the environment due to continuous flushing into the sewer system, it is up to the public to control the source and mitigate the release of such contaminants.  1.1.7 Modeling and Design To most accurately examine the fate of ECs, it is vital to emulate the real-life conditions of a full-scale WWTP. Assuming steady state behaviour for these systems is unrealistic. Instead, the dynamic behaviour must be understood and incorporated into the study. Monitoring of the dynamic behaviour of a WWTP must detect the state of the process and its response to disturbances, correlate cause and effect relationships (such as the way in which the influent affects the effluent), and control the disturbances. Specifically, within the WWTP, dynamic behaviour in sludge is affected by an increase in temperature, and contact between sludge and the treated liquid. However, dynamic behaviour in the clarifiers is governed by the recycled sludge liquor (Lessard & Lava, 1991).   To date, a select number of models exists to estimate the properties of various ECs (adapted in Table 2 from Stuart et al., 2012). However, the complex fate of these contaminants in the 16  various matrices has created a challenging setting for behavioural prediction (Stuart et al., 2012).    Table 2: Examples of models to calculate properties required to predict the fate and transport of contaminants (Stuart et al., 2012) Model Description Parameters Predicted EPI ? Suite Fragment constant Kow Solubility Hydrolysis rate KNN Atom-centered constant Indirect photolysis Biodegradation Hydrolysis SPARC Fundamental chemical structure theory (LFER and PMO) Thermodynamic properties Physicochemical properties CATABOL Degradation simulator using hierarchy of abiotic and enzymatic reactions Biotransformation pathways and metabolites SAR/QSAR type Molecular connectivity Structural activity relationship Physical and chemical properties Environmental fate Ecological effects Health effects of organic  1.1.8 Selection of the Suite of Emerging Contaminants The following section describes in further detail the ECs that were considered for investigation in this research. To incorporate a wide range of ECs, contaminants typically found in wastewater influent were selected. These included contaminants with acidic (ibuprofen naproxen, gemfibrozil) or basic (estrone, EE2, nonylphenol, caffeine) characteristics. In addition, these ECs have varying polarities based on their chemical structure, specifically the location of oxygen, hydroxyl, or nitrogen chemical groups as well as the location of single or double bonds. Furthermore, the log Kow values are provided as a reference to the sorption capability of each compound. As previously stated in Section 1.1.2, a log Kow value of less than 2.5 indicates that the analyte will remain in soluble form rather than sorb onto particulate matter. The greater the value, the greater the potential for sorption onto particulates (Miao et al., 2005). Table 3 17  provides a detailed summary of the considered ECs including molecular weight, log Kow, chemical and physical properties, and observed degradation products in the environment.   Table 3: Properties of chosen emerging contaminants for research Emerging Contaminant Molecular Weight (g/mol) Log KOW Properties Degradation Product  Ibuprofen 206.29 3.97 ? Most commonly used in analgesic drugs ? Classified as a Non-Steroidal Anti-Inflammatory Drugs (NSAIDs) ? Readily metabolized in the human body and photodegrades in river water  ? (Ontario Ministry of the Environment, 2006) ? Hydroxy-ibuprofen  ? 2-hydroxy-ibuprofen ? Carboxy ibuprofen ? (Buser et al., 1999; Quintana et al., 2005) Nonylphenol 220.35 5.76 ? A non-ionic surfactant ? Degradation of nonylphenolic compounds in conventional activated sludge (CAS) processes is low, with the majority ending up in the sludge ? Some of its metabolites are more persistent than the parent compounds ? (Knepper, 2008) ? No Data Caffeine 194.19 -0.07 ? One of the most commonly ingested compounds, found in various food types and beverages ? Also be used for cardiac, cerebral, and respiratory stimulant and as a diuretic ? Commonly used as an indicator for wastewater contamination in water bodies  ? (Buerge et al., 2003) ? 1,7-dimethylxanthine ? (Stackelberg et al., 2004) Tonalide 289.54 5.80 ? An anthropogenic fragrance with a polycyclic structure used to add scent ? (Richardson & Ternes, 2011) ? No Data Gemfibrozil 250.33 4.77 ? A highly common lipid regulator ? Has demonstrated reduced production of testosterone in select species ? Persists in the aquatic environment  ? (Mimeault et al., 2005) ? 5-(2-methyl-5-carboxyphenoxy)-2 ? 2-dimethylpentanoicacid ? (Winker et al., 2008) 18  Emerging Contaminant Molecular Weight (g/mol) Log KOW Properties Degradation Product  Dibutyl Phthalate 278.34 4.45 ? Commonly used in the production of plasticizers and additives in adhesives or paints ? Persists in the aquatic environment as a teratogen ? Typically does not degrade easily ? (Bajt et al., 2001) ? No Data Naproxen 230.26 3.18 ? A highly popular anti-inflammatory drug taken for pain relief or disease ? Has been found in varying concentrations in both anthropogenic and natural water bodies ? Removed by biodegradation, phototransformation, disinfection, and adsorption  ? (Boyd et al., 2005) ? O-Desmethyl-naproxen ? (Quintana et al., 2005) Irgasan (Triclosan) 289.54 4.76 ? An antiseptic (antimicrobial) used in liquid hand soap that has been commonly detected in breast milk and urine ? Demonstrated effect on animals includes disruptions to androgens, thyroid gland, and dermal irradiation ? No evidence to suggest triclosan as being a carcinogen or tetratogen ? May possibly enhance antibiotic resistance if used frequently  ? (Smital, 2008) ? 2,7/2,8-dibenzodichloro-p-dioxin (DCDD) by photodegradation ? (Mezcua et al., 2004)  Estrone 270.37 3.13 ? A naturally occurring estrogenic compound, classified as an endocrine disrupting compound (EDC) ? A transformation product of other estrogenic compounds  ? (Nghiem et al., 2004) ? No Data 17-? Ethinyl Estradiol (EE2) 296.43 3.67 ? An anthropogenic compound used as a contraceptive ? Exposure effects have been observed at very low concentrations, including changes in the female to male ratio, as well as reduced egg fertilization ? (Richardson & Ternes, 2011) ? Estrone ? Estriol ? 16?-hydroxyestrone ? 2-methoxyestradiol ? 2-methocyestrone  ? (Lee & Liu, 2002) Triclocarban* 315.58 4.90 ? An antiseptic commonly added to personal care products such as antimicrobial soaps, and cosmetics ? Has a high sorption potential  ? (Heidler et al., 2006) ? 4-chloroaniline ? 3,4-dichloroaniline ? (Heidler et al., 2006) 19  Emerging Contaminant Molecular Weight (g/mol) Log KOW Properties Degradation Product  4 ? nonylphenol* 220.35 5.76 ? A non-ionic surfactant ? Degradation of nonylphenolic compounds in conventional activated sludge (CAS) processes is low, with the majority ending up in the sludge ? Some of its metabolites are more persistent than the parent compounds ? (Knepper, 2008) ? No Data Bis (2EH) ? Phthalate* 390.56 7.5 ? An industrial plasticizer commonly found in human blood and urine ? Exposure methods include cosmetics, consumer products, ingestion of food-wrapped products, and inhalation of contaminated air ? Effects of exposure to lab animals include developmental problems (in the male reproductive system), destruction of reproductive organs, adrenal, liver, and kidney, as well as lower sperm motility for males  ? (Smital, 2008) ? No Data Sulfamethoxazole* 253.28 0.89 ? A commonly used type of sulfonamide, utilized as a synthetic antimicrobial ? Quite persistent in the environment due to its high usage rate  ? (Garoma et al., 2010) ? N4-Acetylsulfamethoxazole ? (P?rez & Barcel?, 2007) Carbamazepine* 236.27 2.25 ? A widely used antiepileptic drug ? Commonly found in aquatic environments.  ? Has been considered as an anthropogenic marker  ? (Zhang et al., 2008) ? 10,11-dihydro-10,11-epoxycarbamazepine (CBZ-EP) ? 11-dihydro-10,11-epoxycarbamazepine (CBZ-DiOH) ? 2-hydroxycarbamazepine ? (CBZ-2OH) ? 3-hydroxycarbamazepine (CBZ-3OH) ? 10,11-dihydro-10-hydroxycarbamazepine (CBZ-10OH)  ? (Miao et al., 2005) Ciprofloxacin* 331.34 0.28 ? A commonly prescribed chemotherapeutic antibiotic within the fluoroquinolone class ? Commonly detected in hospital effluents  ? (Githinji et al., 2010) ? No Data * Compounds that were initially selected for analysis, but were removed from further consideration during the preliminary stages   20  Several of the candidate compounds were eliminated from the EC study due to the occurrence of ambiguous chromatograms on the GC-MS during preliminary testing (triclocarban, sulfamethoxazole, carbamazepine, and ciprofloxacin), cost (4-nonylphenol), or ease of contamination (bis (2EH) ? phthalate). This will be discussed in further detail in Chapter 2.   Table 4 illustrates the reported average removal efficiencies for the chosen suite of ten emerging contaminants in various full scale treatment systems, adapted from USEPA (2010).  21  Table 4: Full scale treatment systems average removal rates of emerging contaminants (Adapted from USEPA, 2010) Emerging Contaminants Activated Sludge Treatment Fixed Film Biological Treatment Phosphorus Removal (Biological) Phosphorus Removal (Chemical) Denitrification Ibuprofen 90% NR 92% 98% 91% Nonylphenol 90% 100% NR 90% 83% Caffeine 94% NR 100% 100% 94% Tonalide 67% NR 70% 67% 84% Gemfibrozil 77% NR 68% 83% 64% Dibutyl Phthalate 88% NR NR 88% 88% Naproxen 85% NR 88% 95% 85% Irgasan (Triclosan) 89% 87% 69% 94% 96% Estrone 77% 76% 97% 85% 74% 17-? Ethinyl Estradiol 66% 46% 86% 66% 75% Emerging Contaminants Nitrification Granular Activated Carbon Adsoprtion Chlorine Disinfection UV Disinfection Ozone Disinfection Reverse Osmosis (RO) Ibuprofen 92% NR 78% 90% 95% 72% Nonylphenol 83% NR NR NR 85% NR Caffeine 94% NR 98% 97% NR 96% Tonalide 84% NR 79% 52% NR NR Gemfibrozil NR NR 83% 90% 90% 90% Dibutyl Phthalate 88% NR NR NR NR NR Naproxen NR NR 93% 97% 84% 90% Irgasan (Triclosan) 96& NR 83% 90% 99% 67% Estrone 74% 100% 37% 74% 94% 84% 17-? Ethinyl Estradiol 86% NR 42% 0.77% NR NR *NR = Not reported 22  1.2 Motivation for the Research  Although a moderate amount of information is currently available about the fate of ECs in the environment, various knowledge gaps exist in this line of research, specifically relating to the fate mechanisms of ECs in the environment, the biological responses from exposure to ECs, and the potential correlation between biological and chemical responses. Policy makers and regulators desire evidence upon which decisions to control exposure to these contaminants to avoid human health risks can be based; such regulations can only materialize once the ECs are comprehensively understood and a standardized method for the analysis of the ECs is defined.  Given the lack of understanding of ECs in WWTPs, this research project attempted to investigate the fate of ECs under dynamic conditions at the University of British Columbia (UBC) wastewater treatment pilot plant by the independent manipulation of the SRT and HRT. Results from these experiments were compared to lab scale experiments previously conducted in 2011, in which steady state conditions were simulated using synthetic wastewater. Although significant work has been conducted on the quantification of the ECs at varying locations along WWTPs, very little research has focussed on emulating real-life conditions (i.e. dynamic behaviour), illustrating the importance and necessity for this research topic. Furthermore, no research has been conducted on two parallel WWTPs running under identical conditions, excluding the parameters that were manipulated independently (i.e. the SRT or the HRT). Finally, the goal of this research was to conduct a complete mass balance analysis, wherein both the particulate and soluble portions of ECs were investigated, a vital component that is deficient in a considerable amount of the published research.   23  1.3 Research Objectives Based on the literature and current state of knowledge, the primary hypothesis for the present research was that an increased removal efficiency of ECs would be observed at a longer SRT. However, it was expected that the respective fate of the ECs would be dependent on the individual characteristics of each compound.  It was also expected that diurnal changes in the HRT, in comparison to a pseudo-steady state HRT, would result in changes in the removal efficiencies of the emerging contaminants, however to a lesser extent than the effect of SRT. To explore these hypotheses, the research objective involved studying the individual effects of HRT and SRT on the removal of ECs at the UBC pilot plant, and accounting for the selected contaminants using a mass balance approach.    In order to address these hypotheses, two peripheral goals needed to be achieved. The first required the monitoring of the background concentrations for the chosen suite of ECs at the UBC pilot plant. The second required a more thorough investigation into the fate and overall trends in concentrations of the ECs following spiking into the pilot plant.   Within the scope of the nation-wide multi-disciplinary project directed by the CWN, the objectives for the research to be conducted by UBC were two-fold. The first was to examine the impacts of dynamic operating conditions on the removal of ECs at the UBC pilot plant. This was to be conducted by studying the independent influences of the SRT and HRT. The experiments were to be run under pseudo-steady state conditions using a stimulus response spiking approach. The second objective was to analyze synthetic bench-scale wastewater effluent both chemically and biologically to explore a possible correlation between the two. The research described in this thesis focuses solely on the experimental methods designed to meet the first objective. 24  2 MATERIALS AND EXPERIMENTAL METHODS 2.1 Experimental Details The wastewater treatment pilot plant at UBC is comprised of two parallel trains operating as a membrane enhanced biological phosphorus removal process (MEBPR) utilizing a University of Cape Town (UCT) design with three zones: anaerobic zone, anoxic zone, and aerobic zone (where the membrane is located) (See Figure 3). In this configuration, the recycle flows are all at a 1:1 ratio.  Figure 3: Pilot plant configuration at UBC  Table 5 details several of the parameters measured at the UBC pilot plant on a regular basis. These values were averaged during the year of 2012. The wastewater is comprised of municipal and institutional (university) wastewater, with no contributions from industrial sources. The population demographic is largely comprised of students from UBC and non-student residents who live on the UBC campus. As a result, the wastewater influent is expected to have pharmaceuticals and other ECs present.  Primary Clarifier Storage Tank Influent 25  Table 5: Average values for parameters from both sides of the UBC pilot plant Parameter INF ANA ANX AER EFF Time Period T (?C) 18.71 April 2012 - July 2012 pH 7.1 7.1 7.1 7.0 7.0 April 2012 - July 2012 DO (mg/L) N/A N/A N/A 3.6 N/A April 2012 - July 2012 TSS (mg/L) 117 1500 2700 3690 0 April 2012 - July 2012 VSS (mg/L) 80 1390 2580 3600 0 April 2012 - July 2012 SVI (mL/g) N/A N/A N/A 198 N/A April 2012 - July 2012 NH3-N (mg/L) 36.5 23.0 9.5 1.1 0.2 April 2012 - July 2012 NOx-  (mg/L) 0.2 0.2 2.4 10.1 10.7 April 2012 - July 2012 TKN (mg/L) 430 N/A N/A N/A 13 April 2012 - July 2012 PO4-P (mg/L) 2.5 13.5 1.4 2.3 0.1 April 2012 - July 2012 TP (mg/L) 42.3 N/A N/A N/A 3.8 April 2012 - July 2012 VFA's: Acetic Acid (mg/L) 22.2 11.3 N/A N/A N/A April 2012 - July 2012 VFA's: Propionic Acid (mg/L) 5.5 0.9 N/A N/A N/A April 2012 - July 2012 VFA's: n-Butyric Acid (mg/L) 2.1 0.2 N/A N/A N/A April 2012 - July 2012 VFA's: n-Valeric Acid (mg/L) 0.3 0.2 N/A N/A N/A April 2012 - July 2012 COD (mg/L) 350 N/A N/A N/A 24 Feb 2012 - Jun 2012  For the purposes of examining the fate of ECs under varying conditions at the UBC pilot plant, two experimental stages were purposed. The first one was intended to examine the effects of diurnal versus constant HRT. In this case, the diurnal variation in the HRT was representative of realistic field conditions and was emulated at the pilot plant using a pump on a timer. For this stage, the SRT was held constant in both trains. However, in the second stage of the experiment, the HRT was the same for both sides while the SRT was compared in the two parallel trains. The parameters during these experimental stages are detailed in Table 6.   26  Table 6: Details of varying parameters at the UBC pilot plant for both experimental stages Stage Train Flowrate (L/min) HRT (h) SRT (d) Zone Volume  (L) 1  (November 2012) A-Side 3.5 Constant during the whole day 10.37 12 Anaerobic 229 Anoxic 616 Aerobic 1302 Holding Tank 31 B-Side 1.5  5.5 3.5 1 AM to 7 AM 7 AM to 1 PM 1 PM to 1 AM 24.19 10.37 6.60 12 Anaerobic 229 Anoxic 616 Aerobic 1302 Holding Tank 31 2  (January 2013) A-Side 3.5 Constant during the whole day 10.37 15 Anaerobic 229 Anoxic 616 Aerobic 1302 Holding Tank 31 B-Side 3.5 Constant during the whole day 10.37 25 Anaerobic 229 Anoxic 616 Aerobic 1302 Holding Tank 31  The sampling program commenced once the system was acclimatized to the operating conditions. In both scenarios, the acclimation period was equivalent to three times the longest SRT in that stage.   The acclimatization for the first stage was assumed to be complete after 36 days, while in the second stage, 75 days was allowed.  For the duration of the experiment, a slug (or impulse) of ECs was introduced to the system in the anaerobic zone of each train. Samples were obtained from the influent (after the primary clarifier), the recycle flow from the aerobic zone, and the effluent (or permeate of the membrane), over a period of 48 hours.  Examining the effects of HRT and SRT on the fate of ECs required the quantification of the chosen analytes in all streams (influent, effluent, and mixed liquor (ML)). Therefore, a robust, reliable, and reproducible method was required to analyze the contaminants in the various matrices. This required the development of an optimized analytical technique, detailed in the subsequent sections.  27  2.2 Materials 2.2.1 Tracer Compound Lithium chloride, utilized as the tracer throughout this research, was purchased from Fisher Scientific.  2.2.2 Emerging Contaminants for Stock Solutions Ibuprofen, irgasan (or triclosan), bis (2-ethylhexyl) phthalate, estrone, EE2, nonylphenol, caffeine, triclocarban, naproxen, sulfamethoxazole, gemfibrozil, carbamazepine, and ciprofloxacin were all purchased as analytical standards from Sigma Aldrich. Tonalide was purchased from Santa Cruz Biotechnology Inc. The surrogates used in this research, meclofenamic acid and 2,4-MCPB, 4-(4-chloro-o-tolyloxy)butyric acid (MCPB), as well as anthracene used as the internal standard were purchased from Sigma Aldrich.  2.2.3 Solvents Methanol, methyl tert-butyl ether (MTBE), toluene, acetone, acetonitrile, pyridine dichloromethane (DCM) and ethyl acetate were all utilized during the extraction and reconstitution methods, and were purchased from Sigma Aldrich. Stock solutions for all of the investigated ECs were prepared in methanol and stored in amber 40 mL bottles with rubber septa at -20?C.   2.2.4 Derivatization Solvents For the purposes of derivatization, N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) + 1% trimethylchlorosilane (TMCS) as well as N-methyl-N-tert-butyldimethylsilyltrifluoroacetamide (MTBSTFA) were purchased from Sigma Aldrich. 28  2.2.5 Bottles During sample collection, pre-cleaned 1 L and 500 mL amber bottles with rubber septa were purchased from Fisher Scientific. Also, 50 mL polypropylene bottles, utilized for centrifugation of the complex matrices (influent and ML), were purchased from Fisher Scientific.   2.3 Analytical Instrumentation Once analytical standards were created for the ECs of interest, all standards were loaded onto the gas chromatograph-mass spectrometer (GC-MS) to find the required operating conditions. A non-polar DB-5% (phenyl methyl silicone) column was installed in the GC-MS to allow for maximized transport and chromatography, while helium gas was used as the mobile phase. The GC-MS was comprised of an HP 6890 GC series in addition to a HP 5973 mass selective detector. Given that this research involved determination of contaminants at trace levels, the GC-MS was operated using splitless injection, allowing for both the mobile phase (helium gas) and the sample to go through the column.   Each compound has a unique retention time after which it will be ideally seen as one sharp and smooth peak on the chromatogram. The peak is comprised of a set of mass ranges or ions that are used in their respective ratios to identify the compound, while the area under the peak is used to quantify the compound. The GC-MS can operate either in full scan mode or selective ion monitoring (SIM) mode. In the full scan mode, the GC-MS monitors all mass ranges, and was initially used to find the retention times and specific ions for each compound. Following this step, the SIM mode was then used only on the previously established ions for a more rigorous and sensitive quantification of the compound.   29  Table 7 provides information on the base peak ions and quantifying ions for each analyte in the GC-MS that were obtained in the Scan Mode. The retention times for each analyte depended on the type of column used, such that the values in Table 7 indicate the retention time during the Stage 2 experimentation. The retention times for the last five compounds were not applicable since the compounds were removed from the target list prior to Stage 2. The ions used to identify each compound were based on ionic weight, and referred to as Q1, Q2, and Q3. Triclocarban yielded multiple peaks, of which only the last one was quantified for consistency purposes. Nonylphenol also yielded multiple peaks over an equal span of time but the analyte was quantified as the area under all of the peaks, given the close proximity of the multiple peaks.   Table 7: Characteristics of emerging contaminants on the GC-MS Compound Retention Time (min) Ionic Weight (g/mol) Q1 Q2 Q3 Ibuprofen 8.727 160.1 263.1 278.1 Irgasan (Triclosan) 13.124 347 345 200 Tonalide (AHTN) 10.904 243.2 258.1 201.1 Dibutyl Phthalate 11.737 149.0 223.0 205.0 Estrone 16.713 342.2 257.1 218.1 17?- Ethinylestradiol (EE2) 17.632 425.3 285.1 426.3 Caffeine 10.838 194.1 165 109 Naproxen 12.762 185.1 243.1 302.1 Nonylphenol 10.223 207.1 221.1  Gemfibrozil 11.507 179.0 201.1 202.1 MCPB (Surrogate) 11.314 159.0 199.0 141.0 Anthracene (Internal Std.) 10.510 178.0 179.0 152.0 Bis (2-EH) Phthalate N/A 149 167 279 Triclocarban N/A 161 163 165 Sulfamethoxazole N/A 357 358 359 Carbamazepine N/A 193 194 293 Meclofenamic Acid N/A 242 367 277  Throughout all of the experiments, the maintenance of the GC-MS involved replacement of the injection port and the septum when needed and regularly checking the condition of the auto-sampler syringe.  Following any changes that were conducted on the unit, the GC-MS was 30  baked out at 300?C and the mass spectrometer was re-tuned. Subsequent to this step, a standard was run to adjust the retention times and ensure that the peak intensities were behaving as expected by monitoring consistent peak amplitudes.    2.4 Extraction Methodology The samples obtained from the UBC pilot plant consisted of influent, effluent, and ML (mixed liquor) samples from both parallel trains. Given the high particulate concentration and complexity of the samples, the influent and ML samples needed to be analyzed separately for soluble (INF-?, ML-?) and particulate (INF-?, ML-?) fractions.  On the other hand, no particulates were present in the effluent (membrane permeate) samples, simplifying the analysis on the effluent samples. Influent and effluent samples were collected in 1 L amber bottles, while ML samples were collected in 500 mL amber bottles.   The methods in this section detail the optimized analyses that yielded the best reproducibility and recoveries. Section 3.1 details the optimization steps taken to arrive at the final method.    2.4.1 Optimized Extraction Method for Soluble Fraction:  INF-?, ML-?, and EFF An aliquot of 100 mL of each sample (influent, ML, or effluent) was measured using a graduated cylinder. The influent and ML samples were centrifuged at a relative centrifugal force (rcf) of 913 x g for 15 minutes using two 50 mL polypropylene bottles. The supernatant was then decanted into a beaker for analysis. The particulate fraction from the influent sample was then utilized for the method described in Section 2.4.2, while the particulate fraction from the ML samples was discarded.   31  All samples were acidified to a pH of 3 using a pipette to transfer 10-15 drops of 10% HCl solution. The pH was measured using litmus paper. Following acidification, the samples were transferred to a 250 mL separatory funnel (located in a fume hood), and spiked with the 75 ?L of the surrogate (MCPB ? 128 ng/?L solution in methanol) using a glass syringe. A volume of 25 mL of DCM was measured using a graduated cylinder, and transferred into the separatory funnel. The solution in the separatory funnel was shaken for 2 minutes with intermittent venting. The contents of the separatory funnel were then set aside for 10 minutes to allow for separation of the phases. Once the top of the separatory funnel was opened, the bottom (DCM) layer was discharged through a solvent-rinsed glass funnel full of anhydrous sodium sulfate supported on a Q8 Fisher Brand Filter Paper (Fisher Scientific ;12.5 cm diameter) and into a 100 mL round bottom flask. The addition and mixing of DCM was repeated two additional times, with the round bottom flask containing the combined volumes of the post-extraction DCM. The remaining aqueous phase was discarded from the separatory funnel, which was then washed with 5 mL DCM and emptied into the round bottom flask.   2.4.2 Optimized Extraction Method for Particulate Fraction: INF-?, ML-? For the ML samples, 5 mL was measured using a graduated cylinder, and transferred into a 10 mL glass HACH vial. The vial was centrifuged at an rcf of 913 x g for 5 minutes. The top (aqueous) phase was discarded using a Pasteur pipette, and the vials were capped tightly. The influent samples were obtained by transferring the particulate fraction following centrifugation (Section 2.4.1) into a 10 mL glass HACH vial, which was also capped tightly. All vials were subsequently placed in a freezer (-20?C) overnight. The following day, the caps were removed, and the10 mL glass HACH vials were placed in an ilShin Freeze Dryer overnight, to ensure complete removal of water from the samples.   32  Unlike the method for the soluble fraction, no pH adjustment was required for analysis of the particulates. As a result, the 10 mL glass HACH vials were spiked with 75 ?L of the surrogate (MCPB - 128 ng/?L solution in methanol) using a glass syringe. A mixture consisting of 4 mL of MTBE and 1 mL of methanol (both measured using a graduated cylinder) was added to the 10 mL glass HACH vials. Following the capping of the vials, the vials were given a quick shake and vented. The vials were subsequently placed in an ultrasonic water bath at 40?C for 15 minutes. The solvent was then transferred into a 100 mL round bottom flask using a Pasteur pipette. The addition and extraction with MTBE and methanol was repeated two times, with the solvent volumes added to the round bottom flask at the end of each round.   2.4.3 Optimized Analytical Method for Extracted Samples: All Samples Following the physical separation, the round bottom flask was placed on a Heldolph Rotary Evaporator, with a water bath set at 50?C. The contents in the round bottom flask were evaporated until an approximate final volume of 2 mL was reached. The remaining sample was transferred into a 10 mL glass HACH vial using a Pasteur pipette, and blown to complete dryness using nitrogen gas.   Following the drying of the sample, the derivatization step required the addition of 150 ?L of pure BSTFA (with 1% TMCS) and 50 ?L of pure pyridine into the 10 mL glass HACH vial using two separate glass syringes. The 10 mL glass HACH vial was vortexed for 10 seconds, and subsequently baked in an oven at 70?C for 1 hour. Once the 10 mL glass HACH vial was removed from the oven, the contents of the vial were once again blown down to dryness using nitrogen gas.   33  Finally, for the reconstitution step, 950 ?L of toluene (measured using a pipette) and 50 ?L of the internal standard (85.2 ng/?L anthracene in methanol ? measured using a glass syringe) were added to the 10 mL glass HACH vial. The vials were once again vortexed for 10 seconds. The contents of the vial were transferred into a GC vial using a Pasteur pipette and the vial was capped, and placed on the GC for analysis.   The temperature program for each run in the GC-MS is detailed in Table 8.   Table 8: Temperature program in the GC-MS Temperature Increase Rate (?C/min) Final Temperature (?C) Hold Time (min) Run Time (min)  95 1.00 1.00 12 290 2.75 20.00  300 5.00 25.00  The method development is detailed in Section 3.1, and a step-by-step detailed analytical method is provided in Appendix A.1 (soluble fraction) and Appendix A.2 (particulate fraction). Given that a standardized method for the analysis of ECs did not yet exist, the method is further summarized in Appendix A.3.   2.5 QA/QC For quality assurance and quality control, several steps were applied throughout the sampling plan. First, replicates and spikes were prepared using samples taken from each sample location once a day. The relative standard deviations (RSD) for the replicate runs and percent recoveries from spikes were determined. Unfortunately, obtaining a larger number of replicate samples was not possible due to time constraints.  A poor recovery of the spike was considered to be lower than 60%, and a poor RSD of replicate samples was considered to be greater than 40%.  34   Calibration curves were generated throughout each of the sample runs. Therefore, to gain a level of confidence in the analysis on the GC-MS, calibration standards were placed at the beginning of the run, after every 15 samples throughout the run, and once again at the end of the run. Furthermore, anthracene was used as an internal standard to account for any loss of sample volume. MCPB was also utilized as the surrogate throughout the analysis procedure.   The final portion of the QA/QC program entailed the determination of the method detection limit (MDL). This relates to the determination of the minimum detection concentration on the optimized extraction methods utilized during this research with a confidence that the result is greater than zero. This assessment posed numerous challenges. First, the determination of the MDL should utilize the same analytical procedure that is used for sample analysis. As a result, the matrix encountered during sample analysis should also be used during the determination of the MDL (USEPA, 1997). For the analysis of dissolved analytes, the possible matrices would include the pilot plant influent and effluent. In general, representative matrices that are analyte-free should be utilized and spiking into these matrices should be done at the expected MDL level. However, neither the influent nor the effluent matrices were analyte-free. Furthermore, the composition of samples could vary on a daily basis (depending on the sewage introduced into the system), increasing the complexity of the matrix. Therefore, for simplicity purposes, distilled water was used as the matrix of choice. The other challenge related to determining the MDL on the particulate matrix. Since the mixed liquor (ML) from the pilot plant similarly contained the examined analytes, and peat moss, used as a simulated blank (obtained from the nearby UBC farm) was not a true representative sample of the complex ML, it was decided to forgo the estimation of the MDL on the particulate fraction.   35  The MDL is typically determined using seven replicate spikes on a representative sample and is calculated as the product of the Student?s t value for 99% confidence level and the standard deviation of the replicated analyses. The level spiked for the determination of the MDL was based on the instrument detection limit (IDL). The IDLs for the analytes were determined using stock solutions of the ECs, where low calibration levels were spiked onto the GC column. The IDL corresponded to a concentration that would yield a noise to peak ratio of 3, which was below the level injected onto the GC column (Table 9).  Table 9: IDL for suite of ECs on the GC-MS Emerging Contaminant Allowable mass based on IDL (pg) Allowable minimum concentration in the vial (ng/mL) Ibuprofen 18.5 18.5 Nonylphenol 6.8 6.8 Caffeine 0.6 0.6 Tonalide (AHTN) 0.3 0.3 Gemfibrozil 3.5 3.5 Dibutyl phthalate 0.1 0.1 Naproxen 3.8 3.8 Irgasan (Triclosan) 0.3 0.3 Estrone 0.3 0.3 17?- Ethinylestradiol (EE2) 1.0 1.0  The resultant individual MDLs were determined using 6 spiked water samples, given that the remainder of the 8 spiked samples yielded unrealistic or unrepresentative results. It appeared that the MDL determinations for ibuprofen, gemfibrozil, naproxen, and nonylphenol were the most problematic, leading to lower confidence in the results for these compounds (Table 10). However, overall, the MDL provided a good understanding of the limits of the analytical method for the chosen ECs.   36  Table 10: MDL for suite of ECs Compounds Mean Standard Deviation Relative Standard Deviation (RSD) MDL on a mass basis (pg) MDL in a GC vial (ng/mL) Ibuprofen 134.31 114.45 85% 385 385 Nonylphenol 144.69 58.30 40% 196 196 Caffeine 236.89 49.89 21% 168 168 Tonalide 145.28 39.06 27% 131 131 Gemfibrozil 51.44 20.38 40% 69 69 Dibutyl Phthalate 424.96 83.41 20% 281 281 Naproxen 154.19 96.53 63% 325 325 Irgasan 85.91 17.52 20% 59 59 Estrone 32.94 6.28 19% 21 21 EE2 32.57 5.43 17% 18 18   2.6 Measure for Hydrophobicity of ML To explain some of the observed trends for the ECs, the hydrophobicity of the ML was of examined using a method modified from that of Rosenberg et al. (1980). It was postulated that the sorption of hydrophobic analytes decreased at the longer SRT (Section 3.5) as a result of a decrease in the hydrophobicity of the ML. In this method, 2 mL of ML was centrifuged at an rcf of 913 x g for 5 minutes. A PUM buffer solution was created by measuring 22.2 g of K2HPO4, 7.26g of KH2PO4, 1.8g of urea, 0.2g of MgSO4 . 7H2O and made into solution with 1000 mL of distilled water in a 1 L bottle. The supernatant was discarded and 2 mL of the PUM buffer solution was added using a pipette. The suspension was resuspended and centrifuged at an rcf of 913 x g for 5 minutes, and the supernatant was discarded again. This washing procedure was repeated one additional time with 10 mL of PUM buffer added (measured with a pipette). However, the samples were usually too turbid for analysis, requiring dilution. Therefore, after washing the sample with PUM buffer, 3 mL of the washed ML (measured using a pipette) was re-suspended with an additional 3 mL of PUM buffer in a separate vial.  The initial absorbance was measured using 0.5 mL of the washed and diluted ML in a cuvette at 400 nm using the 10 37  mL glass HACH Spectrophotometer DR 2800. The washed ML was then mixed with 1 mL of n-hexadecane, shaken for 2 minutes, and set aside for 15 minutes to allow for settling. An aliquot of 0.5 mL of the bottom (particulate) layer was then transferred to a cuvette using a pipette, with the final absorbance measured. The relative hydrophobicity was measured using Equation 1, where the PUM buffer was utilized as a blank solution:                         (  (                                ))           (1)    38  3 RESULTS AND DISCUSSIONS 3.1 Method Development 3.1.1 Analytical Method Development for Soluble Fraction Prior to the optimization of the analytical methods, stock solutions for each analyte were made in 100% methanol (held in a 40 mL amber bottle and stored in a freezer at -20?C). Compounds that initially were difficult to dissolve in methanol were either exposed to heat (40?C) in a water bath for a maximum of 5 minutes and/or sonicated for a maximum of 1 minute.   The following section details the parameters chosen in the optimization of the analytical method for the soluble fraction. They include the extraction method, the pH level, the temperature program, the solvents used, the derivatization agents used, filtration methods, chosen ECs, silanization of the glassware, and blowing down the solvents to dryness. The criteria for poor recoveries or reproducibility are detailed in the Section 2.5 ? QA/QC.   3.1.1.1 SPE Method The initial method utilized for the analysis of the selected ECs in the soluble phase was based on USEPA?s Method 1694 (USEPA, 2007). This method was intended for pharmaceuticals and personal care products in water, soil, sediment, and biosolids. Although most of the target ECs were present in the USEPA list, the USEPA method used a high performance liquid chromatography tandem mass spectrometer (HPLC/MS/MS) as the analytical instrument for analysis. Since the UBC laboratory did not have access to an HPLC/MS/MS, the USEPA method was modified using a GC-MS in place of an HPLC/MS/MS.  Initially the modified method was tested on influent samples obtained from the UBC pilot plant, spiked with a mixture of the 39  ECs (triclocarban, ibuprofen, nonylphenol, tonalide, caffeine, gemfibrozil, naproxen, irgasan, sulfamethoxazole, bis(2-EH) phthalate, and EE2) and the meclofenamic acid as the surrogate.   Prior to the extraction process of the soluble fraction, a pre-filtration step was required for the influent and ML samples. Using a vacuum and a 0.45 ?m Mixed Cellulose Easter HAWM filter, filtration was initially attempted on 1 L influent samples. This was also compared to centrifugation of the 1 L sample at an rcf of 2000 x g for 15 minutes. Since both results yielded comparable background concentrations of the ECs present in the influent samples, centrifugation was chosen as the separation method due to ease of use.   The method required the acidification of a 1 L soluble sample to a pH level of 2, followed by the addition of 500 mg of Na4EDTA. A C18 Supelco 6 mL SPE cartridge was then conditioned with 20 mL of methanol followed by 6 mL of distilled water. The conditioning fluid was discarded and the 1 L sample was loaded and allowed to flow through the cartridge. The cartridge was then washed with 10 mL of water (to remove the EDTA) and dried under vacuum for 5 minutes. The analytes were eluted with 12 mL of methanol using a vacuum followed by 6 mL of a 1:1 mixture of methanol:acetone. The combined 18 mL of the eluent was collected in two 10 mL glass HACH vial and then concentrated to dryness in the HACH vial with the rotary evaporator and nitrogen gas in a 50?C water bath. Using toluene, the sample was brought to a final volume of 1 mL. The sample was then derivatized with 50 ?L BSTFA + 1% TMCS at 70?C for one hour, vortexed, and placed in a GC vial. This method yielded unrealistic results (with negative recoveries for the analytes studied).  To try and gain better recoveries of the analytes, the elution of the samples was slightly modified, eluting the cartridges with 15 mL of acetone after the methanol, and washing the cartridge with 5 mL of acetone before methanol. The increase in the volume of acetone used 40  was postulated to improve the recoveries, as it would allow for the analytes that prefer acetone (such as triclocarban and irgasan) to be recovered more easily. Unfortunately, recoveries remained unrealistic (with negative recoveries once again). It was then supposed that the analytes were not recovering well since they remained on the physical cartridge. As a result, 10 mL of methylene chloride, a more potent solvent, was applied on the used cartridges. However, this did not yield improved results.   One of the key elements in a SPE relates to the type of cartridge used. Each cartridge is filled with media that has varying properties, and is chosen if it complements the analytes studied. For this study, C18 Supelco and Waters (6 mL) cartridges were compared to each other.  C18 cartridges retain analytes through a reverse retention mechanism, employing hydrophobic interactions between the sorbent and non-polar compounds. Given the poor results obtained using influent samples for both cartridges (negative recoveries and non-detects), and the polarity of some of the analytes, a different cartridge was chosen. The Supelco HLB cartridges were utilized as they are comprised of a hydrophilic modified styrene based polymer. The retention mechanisms in these cartridges include both reverse phase and ion exchange, thereby targeting polar analytes. Unfortunately, the recoveries for the influent samples did not improve.  The final component to be optimized in the SPE method entailed the drying out of the cartridge under a vacuum prior to elution. However, there was concern that this could cause physical damage of the media.  As a consequence the drying method was modified to employ gravity in place of a vacuum. Once again, recoveries for the analytes in the influent did not improve, and the SPE method was abandoned, and replaced by LLE (liquid-liquid extraction).    41  3.1.1.2 LLE Method The LLE method was based on previously used LLE methods in the UBC Environmental Engineering Laboratory, as well as modifications from the SPE method (such as pH adjustments). Initially, 100 mL of soluble influent or effluent samples were acidified to a pH level of 2 and then placed in a 250 mL separatory funnel along with 25 mL of DCM. The samples were shaken for 2 minutes with periodic venting. The contents were allowed to settle for 10 minutes ensuring full separation of the organic layer from the water layer. DCM was extracted through a solvent-rinsed glass funnel full of anhydrous sodium sulfate supported on a Q8 Fisher Brand Filter Paper (Fisher Scientific ;12.5 cm diameter) into a round bottom flask. The extraction step was repeated two additional times and the solvent layers were combined.   During the extraction of the influent samples, an emulsion formed on the lower (organic) phase. Initially, a glass stir rod was utilized to break apart the emulsion, but this failed to improve the separation of the two phases. Therefore, the emulsion phase was drained into a 125 mL separatory funnel and shaken again (with additional solvent added if required). The layers would then successfully separate, allowing for the draining of the organic layer into a round bottom flask.   Following the extraction, the same steps as detailed in the SPE method were utilized such that the samples were evaporated using a rotary evaporator followed by nitrogen gas. The samples were then derivatized and analyzed by GC-MS. Results were significantly better in comparison to the SPE method, yielding improved reproducibility (RSD less than 40%) and positive recoveries. Nevertheless, meclofenamic acid (utilized as the surrogate) was yielding both problematic calibration curves and recoveries.   42  To further improve the recoveries obtained in the extraction, the initially recommended adjustment of pH level to 2 was addressed. It is important for the pH value of the matrix to be below the lowest pKa value of the analytes in the matrix. This prevents the analyte from dissociating in the water solution, thereby making it less hydrophobic, and with a greater affinity for the organic solvent. Since the lowest pKa value of all the analytes in this study was 4.2 (for naproxen), the pH simply had to be below that, but not necessarily as low as 2. When pH levels of 3.5 and 3 were utilized, recoveries were improved, and the latter was the most effective.  A crucial step prior to performing the extraction method entailed the proper cleaning of the glassware utilized in these applications. Initially, all the glassware utilized was washed with soap and water (following complete solvent evaporation), and subsequently baked at 550?C for a minimum of one hour. However, to further improve the observed results, silanization of the glassware was attempted. This procedure prevents the adherence of the analytes onto the glassware by coating it with silanizing agents. The glassware was treated with 10% (v/v) dimethylchlorosilane in toluene for one hour. The glassware was then washed with 5 mL of toluene and 5 mL of methanol. The final step involved the drying of the glassware at 100?C for one hour (Favaro et al., 2008). Unfortunately, silanization did not improve the results of the LLE method as it yielded negative recoveries. It was then decided to spike the solutions with a concentration 100 times greater than the previous to ensure that substantial quantities of the ECs were present. Although there were excellent recoveries observed, the concentrations were unrealistic and non-representative of those likely to be found in the environment. Also, bis (2-EH) phthalate continued to be present in all samples (including the blanks), while carbamazepine and sulfamethoxazole continued to be problematic during analysis and calibrations, yielding complicated chromatograms with numerous sharp peaks instead of one single smooth peak. As a result, carbamazepine and sulfamethoxazole were removed from the list of analytes investigated in this research.  43   As replicate runs were conducted to test the reproducibility of the method, it was observed that the effluent samples exhibited good recoveries (greater than 50%) and reproducibility (RSD less than 40%). However, the analysis of the influent samples did not perform as well, illustrating that the problem was probably associated with the complexity of the influent matrix and interferences from the particulates. To address this problem, whole influent samples (prior to separation of the particulate and soluble fractions) were extracted using MTBE and methanol. It was postulated that given the increase in polarity of MTBE, there may have been less interference from the particulates (this occurred prior to the investigation of filtration methods). Importantly, in this extraction method, the post-extraction organic phase was located on top of the water phase. Therefore, a difficulty that arose from this method was the presence of water in the organic phase, which was resolved using anhydrous sodium sulfate. Nevertheless, this method yielded inconsistent results for the influent samples with extremely high variability among replicates, and improvement only in a select number of analytes.   A combination MTBE and methanol (first extraction) with DCM (second and third extraction) were also tested to see if each organic solvent would be able to draw out the analytes that performed well in the earlier evaluations. The first extraction was then carried out with 50 mL of MTBE, followed by the second and third wash using 25 mL of DCM. There were no improvements in the recoveries with this modified method. Additionally, compounds that were previously improved solely with MTBE, did not improve during this combined extraction method of MTBE and DCM.   To further ensure that the problem stemmed from the method itself, and not from the performance of the GC-MS, derivatized analytes were spiked into already-extracted influent samples. Recoveries continued to be poor and calibration curves were not linear. Therefore, it 44  was postulated that a reaction may potentially have occurred within the GC vial post extraction or that the analytes were underivatized.   To address this problem, the subsequent trial did not involve blowing down the derivatization agents to dryness, post-extraction with DCM. This was tested using two possible scenarios. In the first, the volume of the derivatization agents was increased to 100 ?L and 30 ?L for BSTFA (+ 1% TMCS) and pyridine, respectively. In this scenario, the final sample was not to be reconstituted in toluene, and therefore was placed in an insert within the GC vial. The second scenario utilized 50 ?L and 15 ?L for BSTFA (+ 1% TMCS) and pyridine, respectively. Here, the final sample was reconstituted to 1 mL. Results indicated that omitting the blow down step of the derivatization agents indeed improved the recoveries of the analytes, and that of the two methods previously described, the samples lacking toluene produced the best results.   To minimize the complexity of the influent samples, an investigation of filtration methods was conducted for the influent samples prior to extraction with DCM. Two solids-liquid separation methods were examined: glass fibre filtration using a vacuum filtration apparatus and centrifugation at a relative centrifugal force (rcf) of 2060 x g for 20 minutes. Here, the volumes of the derivatization agents were increased slightly to 120 ?L and 30 ?L for BSTFA (+ 1% TMCS) and pyridine, respectively. Once again, the samples were not reconstituted in toluene. There appeared to be minimal differences between the two methods, however, due to the ease of centrifugation, this was chosen as the final method for separating the liquid and particulate phases. During further centrifugation runs, it was found that an rcf of 2060 x g broke the vials, and as a result, the speed was reduced to achieve an rcf of 913 x g. Results were promising, illustrating good recoveries and reproducibility, excluding triclocarban (which was a non-detect), bis (2-EH) phthalate (which had negative recoveries), and caffeine (which had >> 100% recoveries). As a result, triclocarban was removed from the target list of analytes, and bis (2-45  EH) phthalate was substituted by the dibutyl phthalate (another common analyte in the same chemical family). The same derivatization volumes were also utilized in replicates on the effluent (excluding centrifugation) with excellent recoveries.  3.1.2 Analytical Method Development for Particulate Fraction The initial extraction method for the particulate fraction was based on a method by Mahmood-Khan (2005) using ML samples obtained from the pilot plant. In this LLE method, 5 mL of ML was extracted using a mixed solution consisting of 4 mL of MTBE and 1 mL of methanol. The samples were shaken using a mechanical shaker for 20 minutes. Following that, samples were centrifuged at an rcf of 913 x g for 10 minutes. The top layer was then transferred through anhydrous sodium sulfate into a round bottom flask. The extraction was repeated three times, followed by derivatization.   It was important to evaluate whether a pH change was required. Decreasing the pH was detrimental to the detection of numerous analytes, indicating acidification in the extraction of the particulate fraction was unnecessary.   To further improve this extraction method, freeze drying of the ML prior to the extraction was employed. During this procedure, 5 mL of ML were centrifuged at an rcf of 913 x g for 5 minutes. The water layer was discarded and the pellet was frozen overnight at -20?C. The following day, the samples were freeze dried overnight. Following freeze drying overnight, the LLE method using MTBE and methanol was applied.   The final step taken to improve the extraction method for the particulate portion involved the use of ultrasonic solvent extraction (USE) in place of LLE, as described by Spongberg et al. (2008). 46  This method did not use pH adjustment and once again utilized 4 mL of MTBE and 1 mL of methanol following freeze drying. However, instead of shaking the samples on a mechanical shaker, the samples were placed in an ultrasonic bath at 40?C for 15 minutes. As in previous extraction methods, this step was repeated three times. Fortunately, this method yielded the best recoveries (recoveries greater than 30%) and reproducibility (RSD values less than 30%) for the particulate phases.  3.1.3 Method Development for Analysis on the GC-MS Derivatization of the analytes is required to ensure strong chromatography on the GC-MS, by making the analytes more volatile, less polar, and more stable. Given the wide variety of analytes in this investigation, it was essential to ensure that the derivatization step was complete using the respective standard solutions. As stated in Section 2.2, stock solutions for all ECs were made in methanol, held in 40 mL amber bottles, and stored in a freezer at -20?C. Using the individual EC stock solutions, the chosen volumes shown in Table 11 were transferred for each EC into an individual 10 mL glass HACH vial using a pipette. The methanol in the solution was evaporated using nitrogen gas. Once dryness of the 10 mL glass HACH vial was complete, the derivatization method could be optimized. Initially, 50 ?L of BSTFA + 1% TMCS and 15 ?L of pyridine were added to the dried 10 mL glass HACH vial, vortexed, and baked at 70?C for 20 minutes. However, when the baking time was extended to 60 minutes, the chromatography was dramatically improved, resulting in single, sharp peaks for a greater number of the analytes. This excluded ciprofloxacin, which remained the only non-detect and, carbamazepine which had multiple peaks (indicating incomplete derivatization).   47  Table 11: Summary of stock solution concentrations and volumes used for derivatization Analyte Stock Solution Concentration (ng/?L) Volume of Stock Solution used for Derivatization Method (?L) Ibuprofen 1069 50 Naproxen 2018 50 Irgasan 1954 50 Bis-(2EH) phthalate 2776 50 Estrone 2090 50 EE2 2266 50 Caffeine 1092 50 Triclocarban 2105 50 Tonalide 468 200 Gemfibrozil 1585 50 Carbamazepine 898 50 Sulfamethoxazole 412 200 Ciprofloxacin 338 200 Nonylphenol 1932 100  It was later decided to utilize N-methyl-N-tert-butyldimethylsilyltrifluoroacetamide (MTBSTFA) + 1% tert-Butyldimethylchlorosilane (TBDMS) as an alternative derivatization agent (Gunnar et al., 2004). However, this agent did not behave as effectively as the previous one, leading to complicated chromatograms and incomplete derivatization for over half the analytes.   The specific derivatization agents utilized in this step were also optimized. As previously mentioned, BSTFA + 1% TMCS was the first derivatization agent utilized on the chosen analytes. Since some of the analytes (carbamazepine, sulfamethoxazole, and ciprofloxacin) did not derivatize successfully or yield clear chromatograms, N-methyl-N-tert-butyldimethylsilyltrifluoroacetamide (MTBSTFA) + 1% tert-Butyldimethylchlorosilane (TBDMS) was used as an alternative derivatization agent (Gunnar et al. 2004). However, in comparison to BSTFA + 1% TMCS, this derivatization agent behaved poorly, resulting in complicated chromatograms and incomplete derivatization for most of the analytes. As a result, future work was completed with BSTFA + 1% TMCS, and ciprofloxacin was eliminated from the target list of analytes.  48   It was postulated that the blowing down of the derivatization agents with nitrogen gas post-derivatization may have hindered the derivatization reaction. As a result, the succeeding trial bypassed the blowing down, and utilized two scenarios of increased volumes of the derivatization agents.  The first incorporated 100 ?L and 30 ?L BSTFA + 1% TMCS and pyridine, respectively, as well as a bypassing of the reconstitution step. The second used 50 ?L and 15 ?L for BSTFA + 1% TMCS and pyridine, respectively, followed by reconstitution of the sample to a final volume of 1 mL using toluene. It appeared as though removing the blow down step improved the recoveries of the analytes, with increased improvement for the latter method. However, the presence of the derivatization agent in the GC vials obstructed the injector on the GC-MS unit, thereby reverting the derivatization method to include a blow down step. Nevertheless, the use of an increased volume of the derivatization agent 150 ?L and 50 ?L for BSTFA + 1% TMCS and pyridine, respectively improved the recoveries.   The final optimized parameter in this study related to the temperature program on the GC-MS. It is imperative for the temperature to be at a sufficiently high to be able to spread out the chromatographic peaks of interfering compounds present in the wastewater matrices. Two temperature programs were utilized, the first of which illustrated the greatest promise, given the lack of clear peaks on the latter approach (Table 12).  49  Table 12: Temperature program for the GC-MS Option Number Temperature Increase Rate (?C/min) Final Temperature (?C) Hold Time (min) Run Time (min) 1  95 1.00 1.00 12 290 2.75 20.00  300 5.00 25.00 2  95 1.00 1.00 15 170 0.50 6.50 8 290 5.00 26.50  300 5.00 31.50  To generate further confidence in the analytical method, anthracene was utilized as an internal standard in each sample. The internal standard was added post-extraction and prior to transferring of the sample to the GC vial. The use of anthracene on the GC-MS was first carried out on influent samples derivatized with 150 ?L and 50 ?L for BSTFA (+ 1% TMCS) and pyridine, respectively. Using the built-in library software of the GC-MS, it was possible to determine the quantifying ions and the retention time. This was conducted in triplicate influent samples to ensure consistency.   As an added measure to determine the efficacy of the derivatization procedure, MCPB was utilized as an additional internal standard to be added to the samples before derivatization.   3.2 Tracer Study Lithium chloride, a non-reactive compound was utilized as a soluble, conservative tracer to better understand the liquid phase residence time distribution at the UBC pilot plant. First, 26.6 g lithium chloride was dissolved in a 10 L bucket with 5 L of tap water. Using the A-Side of the pilot plant, the lithium chloride solution was added as an impulse to the anaerobic zone to yield a nominal 2 mg/L lithium concentration in the whole system. Using an automated ISCO Model 50  3700R refrigerated sequential sampler, 250 mL samples were obtained on an hourly basis for 72 hours from the permeate of the membrane (Figure 4).    Typically, a tracer curve for a continuous stirred tank reactor (CSTR) demonstrates an exponential decay from a maximum that occurs at time zero.  However, Figure 4 illustrates that the maximum was reached after a lag of approximately 3 hours, with completion of the washout after 48 hours. Therefore, from time zero to 3 hours, the pilot plant exhibited some plug flow-like behaviour, while in the remainder of the time, the pilot plant exhibited CSTR-like behaviour.   Consequently, the theoretical tracer curve (based on the volume and flowrates associated with the pilot plant) for a CSTR was overlaid on the same figure, beginning at the maximum time of 3 hours. The maximum for the LiCl curve (at the time of 3 hours) is lower than the theoretical maximum. This may be attributed to the loss of the tracer mass to the system. Nevertheless, following the maximum at 3 hours, the trends for the theoretical and measured tracer curves converge, displaying an exponential decay. This was attributed to the multiple recycle flows (Figure 3) which enhanced back-mixing in the pilot plant. Furthermore, the mean residence time of the tracer (equivalent to 12.2 hours) was in close accordance with the HRT of the pilot plant (equivalent to 10.4 hours).   This tracer test illustrated that following a slug addition of the analytes, a 48 hour sampling program would capture the movement of the analytes through the pilot plant.  In addition, the residence time distribution of the soluble tracer in the liquid was more representative of well-mixed conditions than of plug flow conditions.51    Figure 4: LiCl tracer results at the pilot plant 00.10.20.30.40.50.60.70.80.90 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80Concentration (mg/L) Time (h) Measured Tracer CurveTheoretical Tracer Curvet maxt pilot plantt tracer52  3.3 Sampling Program Design During the planning of the experimental portion of this research, it was important to define the method by which the ECs would be spiked into the UBC pilot plant. There were two possible options. The first entailed the addition of the analytes as a slug or impulse. This would be a one time, instantaneous addition to the influent or the process, that would lead to an observed decay behaviour measurable in the effluent and ML. The other option would involve a step-feed addition of the analytes. In this scenario, the analytes would be added at a continuous rate, with a constant concentration over a certain amount of time. The trend in the effluent and ML would then be investigated and samples would be taken from the influent as a composite sample to ensure there was no change in background concentrations. Since the overall trend (rather than the specific concentration) was the critical factor, samples could be spaced farther apart.   To determine which of the analyte addition methods would be most beneficial for this research, the cost for sustaining both options was estimated, where the second option involved a constant addition of the analytes over a period of one month. Given the large cost of the second option, it was decided to proceed with the research using a slug addition approach.    A sampling schedule for Stage 1 is detailed in Table D - 1. Note that at each time interval five samples were obtained (INF, EFF ? A, EFF ? B, ML ? A, and ML - B).   Similarly, Table D - 2 presents the sampling schedule for Stage 2 (comparison of short to long SRT), with a summary detailed in Table 13. However, in this stage, the influent and ML were each analyzed for both the soluble and particulate fractions (denoted as ? and ?, respectively). Therefore, at each time interval, 7 samples were obtained (INF-?, INF-?, EFF ? A, EFF ? B, ML ? ? A, ML ? ? A, ML ? ? B, ML ? - B). Given the increased complexity of the sample collection in 53  Stage 2 (comparison of short to long SRT), a checklist for each step from sample collection until analysis on the GC-MS was created (see Appendix D.3).   Table 13: Summary of sampling program Time Number of Samples Taken Location Type of Sample Day #1: 9 AM 1 Influent Grab 2 Effluent (A-Side & B-Side) Grab 2 ML (A-Side & B-Side) Grab Day #1: 12 PM 2 Effluent (A-Side & B-Side) Grab 2 ML (A-Side & B-Side) Grab Day #1: 9 PM 2 Effluent (A-Side & B-Side) Grab 2 ML (A-Side & B-Side) Grab TOTAL DAY 1: 13 Samples Day #2: 9 AM 1 Influent Grab 2 Effluent (A-Side & B-Side) Grab 2 ML (A-Side & B-Side) Grab Day #2: 9 PM 2 Effluent (A-Side & B-Side) Grab 2 ML (A-Side & B-Side) Grab TOTAL DAY 2: 9 Samples Day #3: 9 AM 1 Influent Grab 2 Effluent (A-Side & B-Side) Grab 2 ML (A-Side & B-Side) Grab TOTAL DAY 3: 5 Samples TOTAL (over 3 days): 27 Samples  The sample extraction was conducted within 48 hours of sample collection, based on the time that it took for the tracer to completely washout of the system. The remaining volume of each sample was refrigerated at 4?C for archival purposes.   3.4 Comparison of Steady versus Variable HRT 3.4.1 Spiking Mass of Background Concentration for Each Analyte As previously stated, the objective of this research was to determine the effects of selected UBC pilot plant operational parameters on the behaviour of the target suite of ECs and to observe possible trends. Stage 1 was intended to investigate the effects of steady and variable HRT on 54  the ECs in the system, as detailed in Table 6. This was to done by comparing samples from one pilot train operated a constant HRT of 10.37 hours on the A-Side to samples from a simulated diurnal HRT varying between 24.19 hours, 10.37 hours, and 6.6 hours on the B-Side. The overall average HRTs in both trains were the same. At this time, the SRT in both trains was 12 days.     The ECs in the ML measured only in the particulate fractions. An important assumption that was made for the quantification of the analytes was that the analyte concentrations in the soluble fraction of the ML would be equivalent to those in the effluent or permeate.  To explore whether a trend in analyte concentration could be observed in the different matrices (influent, effluent, and ML), a previously determined mass of each EC was added into the anaerobic zone of the UBC pilot plant as an instantaneous slug. Based on results from the calibrations, an equal quantity of each EC was prepared in a combined stock solution to be added for simplicity purposes  To emulate realistic conditions, the spiking concentration into the UBC pilot plant was to be representative of typical concentrations found in wastewater influent, based on literature. Given that the various ECs under investigation are present at various concentrations, a value within the range presented in Table 14 was chosen. The specific value of 3,750 ng/L (or 8.16 mg) was chosen since the equivalent spiking volume (75 ?L) from the stock solutions of the ECs had been used repeatedly during the development of the analytical method. To achieve the desired concentration, the mass of EC required was calculated assuming complete mixing across the entire volume of the UBC pilot plant of 2177 L.   55  Table 14: Average concentrations of ECs in wastewater influent  Emerging Contaminant Average Concentration of EC in Wastewater Influent from Literature (ng/L)  (Mohammadali, 2011) Ibuprofen 27,026 Triclosan (Irgasan) 3,000 Tonalide 1,000 Di (2-ethylhexyl) phthalate 40,010 Estrone 70 17?- Ethinylestradiol 6 Nonylphenol 21,580 Caffeine 45,072 Naproxen 15,026 Gemfibrozil 419  Throughout the 48 hour experiment, the influent background concentrations were measured at each sampling time to confirm that significant external disturbances did not occur within the system during the experimental period. These samples were taken from the influent stream prior to splitting off into the two trains at the UBC pilot plant. Figure 5 illustrates the varying range of the contaminants present in the influent, ranging from 0 ng/L (for EE2 and gemfibrozil) to 35,000 ng/L (for caffeine). Interestingly, these concentrations were in accordance with the levels found in literature (Table 14).  Furthermore, the concentrations appeared to remain relatively steady throughout the 48 hour sampling period, with a steady decline for ibuprofen and naproxen. However, the decrease may simply be attributed to random variability among the samples.     56    Figure 5: Particulate and soluble influent concentrations for all 10 analytes at the UBC pilot plant (Stage 1)   0500010000150002000025000300003500040000Concentration (ng/L) 031224303648Time (h) 57  3.4.2 Concentration Profile The following figures illustrate the challenges that arose from this experiment, with specific examples for EE2 (a hydrophobic compound) and caffeine (a hydrophilic compound). The raw data for the remainder of the analytes are archived in Appendix B.1.  To ensure accuracy and precision in the research, three replicate samples were collected and analyzed at times 3 hours, 24 hours, and 48 hours (as shown in Appendix B). Error bars are displayed for the replicate samples and these represent the maximum and minimum value for each EC. The plotted point represents the average of the three replicate concentrations.  Figure 6 illustrates the concentration profile for EE2 on the A-Side (steady HRT). The influent concentration remained at zero, since there was no EE2 in the wastewater. Therefore, at time zero, the concentrations in the system (ML A) and in the effluent were also zero. Furthermore, the mass of EE2 spiked into the system (8.16 mg) should have been a sufficient quantity to cause a measurable perturbation in the ML concentration. Following the addition of the spike, an increase was observed at time 3 hours both in the ML and in the effluent. As time progressed, the concentration at both sampling locations continued to decrease. This indicates that EE2 was either washed out or degraded during the time of the experiment.   Figure 7 on the other hand demonstrates a different concentration profile for the B-Side (variable HRT). Once again, the influent concentration remained at zero. For these samples, EE2 could not be detected in the (particulate) ML samples. Since only the particulate fraction of EE2 was measured, the soluble concentration in the ML was assumed to be equivalent in concentration to that present in the effluent. Given the non-detects in the particulate fraction, the effluent and the ML concentrations on the B-Side exhibited the same trend. At this point in the 58  experiment, it was unclear as to whether it was reasonable to have assumed that the effluent concentration was equal to the ML soluble concentration. However, this assumption could have resulted in a misrepresentation of the analyte concentration in the ML. Section 3.5.3 will address this assumption more closely and will illustrate the flaws in using this assumption.   Nevertheless, the absence of EE2 in the particulate phase of the ML, and the high variability in the effluent (and therefore soluble ML fraction) concentrations indicated inconsistencies with the developed analytical method, which appeared to be more pronounced on the B-Side (variable HRT).  59    Figure 6: Concentration profile for EE2 in the A-Side (steady HRT)  05000100001500020000250000 3 12 24 30 36 48Concentration (ng/L) Time (h) EFF AINFML A60    Figure 7: Concentration profile for EE2 in the B-Side (variable HRT) 0200400600800100012001400160018000 3 12 24 30 36 48Concentration (ng/L) Time (h) EFF BINFML B61  Figure 8 illustrates the concentration profile for the more hydrophilic caffeine on the A-Side (steady HRT). The measured influent concentration varied between 23,000 ng/L and 35,500 ng/L. More importantly, the concentrations of caffeine in the effluent and the ML were two orders of magnitude lower, at a maximum of 120 ng/L. This illustrates that the addition of 8.16 mg into the system was not a sufficient quantity to create a perturbation within the system itself. A possible explanation for the disappearance of the mass spiked may be attributed to a high degradation rate of caffeine within the system.   Recall that only the particulate fraction of the ML was measured, and that the soluble fraction was assumed to be equal to the effluent concentration. Furthermore, the measured concentrations shown in Figure 8 for the ML and the effluent are very similar to each other, given the absence of caffeine in the particulate fraction of the ML. This is an expected behaviour given caffeine?s hydrophilic and highly soluble nature.   Similar observations can be made from Figure 9, which illustrates the concentration profiles for caffeine on the B-Side (variable HRT). An inadequate mass of caffeine was spiked into the system to generate a perturbation in the system. However, a suspiciously high ML concentration (given the solubility and hydrophilic nature of caffeine) was observed at 36 hours. Unfortunately, no replicate samples were taken at this sampling time. Therefore, this could potentially demonstrate once again a limitation with the developed analytical method. Furthermore, this limitation may be constrained to the B-Side (variable HRT) of the pilot plant. 62   Figure 8: Concentration profile for caffeine in the A-Side (steady HRT)   05000100001500020000250003000035000400000204060801001201400 3 12 24 30 36 48Influent Concentration (ng/L) EFF and ML Concentration (ng/L) Time (h) EFF AML AINF63     Figure 9: Concentration profile for caffeine in the B-Side (variable HRT)  0500010000150002000025000300003500040000020040060080010001200140016000 3 12 24 30 36 48Influent Concentration (ng/L) EFF and ML Concentration (ng/L) Time (h) EFF BML BINF64  Based on the concentration profiles of the ECs, it was realized that adding 8.16 mg of each EC failed to impose a significant transient concentration on the system. Nevertheless, compounds that were known to be subject to sorption on particulates (including nonylphenol, tonalide, dibutyl phthalate, and irgasan) were detected in the ML at considerably higher levels than in the influent (Table 15) or effluent. Similar to the background influent sample, the background ML values were obtained from samples at time zero.   Table 15: Background concentrations Compounds Average Background Influent Concentrations (ng/L) Background ML Concentrations (ng/L) Ibuprofen 17,725 0 Nonylphenol 8,942 43,799 Caffeine 27,437 272 Tonalide 2,787 41,189 Gemfibrozil 0 0 Dibutyl Phthalate 7,991 81,341 Naproxen 13,935 43,734 Irgasan 5,200 29,871 Estrone 405 6,572 EE2 0 0  Although an insufficient quantity of ECs was added to the system to clearly elevate the concentrations of most of the analytes and thus, to permit observation of a response in the behaviour of the ECs, a mass balance calculation was attempted on the limited collected data.  However, based on the data analyzed, it was determined that equating the effluent concentration to the soluble ML concentration was likely incorrect, possibly due to the significance of the membrane in the performance of the system. For this reason, the subsequent Stage 2 experiment (comparison of short to long SRT) ensured that all fractions for the influent and ML were measured directly and not simply inferred. Furthermore, given the potential difficulties with the analytical method, instead of analysis of triplicate samples, the subsequent 65  experimental stage was to incorporate duplicate runs as well as a spike test to determine the percent recoveries. 3.5 Comparison of Long versus Short SRT The SRT controls the ML and more specifically the microbial community in a WWTP. In turn, the conditions at which the microbial community exists, control the effectiveness of the treatment system. As a result, the effects of SRT, a critical operating parameter in a WWTP, on the suite of ECs was of great interest. The A-Side was set at a lower SRT of 15 days, while the B-Side was increased to 24 days. During this time period, the HRT of 10.37 hours was held constant in both trains, without any attempt to simulate diurnal flow variation. The SRT governs the suspended solids concentration and Table 16 details the resulting TSS concentrations in each zone of the parallel trains.   Table 16: Parameters at the UBC pilot plant ? Stage 2 Zone Side of the pilot plant Volume (L) TSS (mg/L) Aerobic A ? Side 1300 4320 B ? Side 1300 6360 Anoxic A ? Side 600 1780 B ? Side 600 4820 Anaerobic A ? Side 230 1110 B ? Side 230 2570  Based on results from Section 3.4, it was determined that spiking the same amount for all analytes (3,750 ng/L) resulted in an insufficient quantity of material added for many of the analytes. Furthermore, highly sorptive compounds were already present in the ML matrix at greater concentrations than in the influent matrix. This was attributed to the accumulation effect exhibited in the ML phase. As a result, to ensure that a reasonable perturbation would be observed at the pilot plant, the quantity of each analyte added was calculated to produce an increase in  the nominal bioreactor concentrations that were equivalent to the maximum 66  background concentrations of each EC (from either the influent or in the ML). The nominal concentrations expected to arise from complete mixing of the spiked ECs in the parallel reactors are displayed in  Table 17.   Table 17: Summary of analyte concentrations added to the UBC pilot plant Compounds Background Concentration from ML or INF (whichever is highest) (ng/L) Concentration resulting from mass actually added to the reactor - A-Side (ng/L) Concentration resulting from mass actually added to the reactor - B-Side (ng/L) Concentration expected in the reactor after spiking - A-Side (ng/L) Concentration expected in the reactor after spiking - B-Side (ng/L) Ibuprofen 17700 17500 17600 35200 35300 Nonylphenol 43800 45000 43800 88800 87600 Caffeine 27400 27400 27500 54800 55000 Tonalide 41200 40900 41000 82100 82200 Gemfibrozil 0 2100 1900 2100 1900 Dibutyl Phthalate 81300 81200 83000 162600 164300 Naproxen 13900 14000 13800 27900 27800 Irgasan 29900 30200 30400 60100 60300 Estrone 6600 6700 6700 13300 13300 EE2 0 1400 1500 1400 1500  To ensure accuracy and precision in the research, two replicate samples and one spike sample were prepared at time 3 hours, 24 hours, and 48 hours (as shown in Appendix B). Error bars are displayed for the replicate samples and these represent the maximum and minimum value for each EC. The recovery and reproducibility in each sample medium is detailed in the following section based on the EC analyzed. As stated in Section 2.5, an unsatisfactory recovery was considered to be lower than 60%, and an unsatisfactory RSD of replicate samples was considered to be greater than 40%. Although the surrogate recovery varied, it was the best result that could be obtained using the specific GC-MS methodology.  Rough trends were apparent and are described in detail below. 67  3.5.1 Background Influent Concentrations As in Stage 1, the influent concentrations were measured throughout the experiment to ensure that no external disturbances occurred in the system. Therefore, a stable concentration over the sampling period (with no trend or significant variation) would indicate reasonably steady input conditions. Figure 10 illustrates the influent concentrations observed during Stage 2. To minimize the analytical workload required in the laboratory, the concentrations in the influent were measured only once a day. Nevertheless, these measured concentrations were in close accordance with those measured during Stage 1 (see Section 3.4). Furthermore, Figure 10 illustrates the relatively stable concentrations for each analyte in the influent during the Stage 2 experiment.   68    Figure 10: Influent concentrations for all analytes at the UBC pilot plant (Stage 2)  050001000015000200002500030000Concentration (ng/L) 02448Time (h) 69  3.5.2 Soluble and Particulate Mass Fraction Compounds known to be highly sorptive to the ML typically exhibit those characteristics due to strong hydrophobic interactions between the analyte and the particulates in the ML. Analytes of this nature are typically characterized with a high log Kow value (see Table 3Table 3).  Figure 11 illustrates the portion of each analyte that was in either the particulate or soluble fraction in influent samples. This representation is taken from samples taken at time zero, prior to the addition of the spike to the system, such that a typical representation of the system can be seen. EE2 and gemfibrozil were not detected in the influent samples.   Given the low particulate content in influent samples (relative to the ML), it was expected that the largest portion of all analytes would be in the soluble phase. However, analytes known to have high log Kow values were expected to be largely present in the particulate phase. This sorptive behaviour was exhibited by dibutyl phthalate, irgasan, nonylphenol, and tonalide. Conversely, analytes known to be highly soluble (including caffeine and ibuprofen) were essentially 100% present in the soluble fraction of the influent.    70    Figure 11: Soluble and particulate fractions of target ECs at time zero in the INF0%10%20%30%40%50%60%70%80%90%100%Mass Fraction Particulate Fraction in INFSoluble Fraction in INF71  Given the significantly higher particulates concentrations in the ML (see Table 16 for the TSS concentrations), the analytes from the ML samples were expected to be present to a greater proportion in the particulate phase than in the soluble phase. Figure 12 and Figure 13 illustrate the proportions of each analyte in the ML liquor soluble and particulate phases in the A-Side and the B-Side, respectively.   As expected, EE2 and gemfibrozil were not detected in the ML samples prior to the spike addition, due to their absence in the influent samples.   Several unforeseen trends were observed. First, caffeine was not detected in the A-Side, either in the soluble or in the particulate fraction of the ML, as well as in the effluent. This may be attributed to high degradation rates within the system, causing the analyte to completely degrade to its metabolites. Second, ibuprofen behaved in the ML in a contrary way to the behaviour in the influent samples. Given its high solubility (and low log Kow) ibuprofen was expected to remain in the soluble fraction. In both the A-Side and the B-Side, ibuprofen was entirely present in the particulate fraction of the ML samples. This may be attributed to the soluble fraction of ibuprofen being degraded into its metabolites at a high rate. However, for the fraction of ibuprofen that sorbs onto the particulates, the degradation process may be delayed. Finally, estrone behaved irregularly in the B-Side of the ML, detected only in the soluble fraction. Given the high affinity of estrone to particulates, this behaviour was unforeseen.   Nevertheless, the highly sorptive compounds, including dibutyl phthalate, irgasan, naproxen, and nonylphenol all had slightly greater fractions of the analytes present in the particulate fraction on the B-Side (longer SRT of 24 days). Given the increased concentration of particulates in the B-Side (due to the greater SRT), it is reasonable that a greater fraction of these analytes would sorb onto the solids.  72   Figure 12: Soluble and particulate fractions of target ECs at time zero in the A-Side ML (SRT = 15 d)     0%10%20%30%40%50%60%70%80%90%100%Mass Fraction Particulate Fraction in MLSoluble Fraction in ML73    Figure 13: Soluble and particulate fractions of target ECs at time zero in the B-Side ML (SRT = 24 d) 0%10%20%30%40%50%60%70%80%90%100%Mass Fraction Particulate Fraction in MLSoluble Fraction in ML74  3.5.3 Concentration Profile Similarly to the approach taken in Stage 1 (where a steady HRT was compared to a variable HRT), the concentration was measured for each analyte in each sampling location: influent (A-Side and B-Side), effluent, and ML (A-Side and B-Side). In contrast to Stage 1, in this stage of the experiment (comparing short and long SRT), both the particulate and soluble phases were measured for the influent and ML samples.   The following figures once again detail the concentration profiles for EE2 and caffeine. The raw data for the remainder of the analytes are in Appendix B.2.  Figure 14 and Figure 15 illustrate the concentration profile for EE2 on the A-Side (SRT = 15 days) and the B-Side (SRT = 24 days), respectively. Once again, no EE2 was present in the influent. Furthermore, for both figures, a maximum was achieved both in the ML and in the effluent after the spike was added. Following the maximum, EE2 rapidly decreased in both phases, either due to rapid washout and/or degradation. Nevertheless, the error bars from the replicate runs illustrate that there may have continued to be limitations with the analytical method.  Figure 16 and Figure 17 illustrate the concentration profile for caffeine on the A-Side (SRT = 15 days) and the B-Side (SRT = 24 days), respectively.  Once again, the influent concentrations are in the range of 23,000 ng/L to 27,000 ng/L. However, the trend for caffeine in this stage of the experiment was more complex. Notwithstanding, it appears that the spike caused a substantial increase in the ML (more significantly in the A-Side), illustrating that an adequate mass of analyte was spiked into the system. It was expected for the caffeine concentrations in the effluent to be low, given the highly biodegradable nature of caffeine in WWTPs.  75   Figure 14: Concentration profile for EE2 in the A-Side (SRT = 15 days)   020004000600080001000012000140001600018000200000204060801001200 3 12 24 36 48ML Cpncentration (ng/L) Effluent Concentration (ng/L) Time (h) EFF AINFML A76   Figure 15: Concentration profile for EE2 in the B-Side (SRT = 24 days) 02000400060008000100001200014000160000501001502002503000 3 12 24 36 48Time (h) ML Concentration (ng/L) Effluent Concentration (ng/L) EFF BINFML B77   Figure 16: Concentration profile for caffeine in the A-Side (SRT = 15 days)   2000021000220002300024000250002600027000280000500100015002000250030003500400045000 3 12 24 36 48Influent Concentration EFF and ML Concentration (ng/L) Time (h) EFF AML AINF78   Figure 17: Concentration profile for caffeine in the B-Side (SRT = 24 days) 200002100022000230002400025000260002700028000050001000015000200002500030000350000 3 12 24 36 48Influent Concentration (ng/L) EFF and ML Concentration (ng/L) Time (h) EFF BML BINF79  The major limitations of the concentration data in the first stage of this study included an insufficient mass of analyte spiked into the system, a potentially incorrect assumption of the effluent concentration being equivalent to the soluble ML concentration, and challenges associated with the analytical method.  Both concentration profiles for EE2 and caffeine illustrated that an adequate mass of analyte was added in this stage. Furthermore, the variability in the analytical method (demonstrated with error bars) appeared to have decreased due to modifications that were made to the method. This may largely be the result of measuring of both the particulate and soluble fractions of the ML and the influent. This leads to the final limitation exhibited in Stage 1. Since both fractions of the ML and the influent were measured, the reliability of the assumption for the particulate phase of the ML could be examined.   Table 18 compares the effluent concentration to the soluble ML concentration for both EE2 and caffeine. In the case of EE2, the ML soluble concentration was consistently greater than the effluent concentration. The lower concentration in the effluent may be attributed to the membrane?s role in the fate of EE2. Interestingly, the opposite was observed for caffeine, where the ML concentrations were lower than the effluent concentrations. Nevertheless, it is clear that assuming the effluent concentration to be equivalent to the soluble ML concentration was not valid.   80  Table 18: Effluent and soluble ML data for EE2 and caffeine EE2 Time (h) EFF A Concentration (ng/L) ML A Soluble Concentration (ng/L) EFF B Concentration (ng/L) ML B Soluble Concentration (ng/L) 0 82 0 0 0 3 72 175 55 0 12 114 233 268 742 24 40 349 62 472 36 31 254 33 239 48 0 120 0 52  Caffeine Time (h) EFF A Concentration (ng/L) ML A Soluble Concentration (ng/L) EFF B Concentration (ng/L) ML B Soluble Concentration (ng/L) 0 2684 0 745 39 3 722 0 0 89 12 0 0 0 0 24 1201 0 278 77 36 117 0 69 58 48 60 40 68 37  3.5.4 QA/QC Section 2.5 illustrated the QA/QC measures taken in this experiment. Duplicate sampling and spike testing were conducted once a day for the concentration measurements previously addressed. From these data, RSD and percent recovery values were determined (Table 19 and Table 20, respectively). Recall the criterion for an unsatisfactory recovery of the spike was considered to be a value that was lower than 60%, and an unsatisfactory RSD for replicate samples was considered to be greater than 40%. Unfortunately, and inexplicably, a significant number of the samples generated unsatisfactory results (highlighted in the following tables). However, it is important to mention that during the analyses for this stage of the experiment, the GC-MS initially did not work properly. All samples were removed immediately, and placed in the freezer until it was confirmed that the instrument was working properly. Following the repair of 81  the instrument, all samples were re-applied onto the GC-MS for analysis. This indicates that the analytical method may have not been completely optimized for the analytes, and further work should be conducted to improve the analytical results.    Table 19: Average RSD values for analytes Compound Location of Sample EFF A EFF B A-Side B-Side ML - ? (soluble) ML - ? (particulate) ML - ? (soluble) ML - ? (particulate) Caffeine 22% 73% 1% 37% 31% 93% Dibutyl Phthalate 11% 25% 2% 20% 7169% 39% EE2 2% 80% 7% 57% 97% 44% Estrone Non detect Non detect 34% 16% 36% 74% Gemfibrozil 13% 13% 95% 77% 70% 30% Ibuprofen 33% Non detect 123% 27% 42% 61% Irgasan 2% 9% 43% 61% 45% 26% Naproxen 34% 98% 67% 78% 34% 72% Nonylphenol 19% 35% 29% 62% 18% 34% Tonalide 4% 5% 15% 13% 5% 13%  Table 20: Average percent recoveries for analytes Compound Location of Sample EFF A EFF B A-Side B-Side ML - ? (soluble) ML - ? (particulate) ML - ? (soluble) ML - ? (particulate) Caffeine 76% 69% 60% 85% 39% 85% Dibutyl Phthalate 87% 80% 66% 99% 44% 95% EE2 24% 15% 76% 99% 133% 126% Estrone 15% 12% 80% 79% 34% 83% Gemfibrozil 15% 14% 134% 155% 56% 171% Ibuprofen 4% 3% 38% 38% 11% 49% Irgasan 7% 5% 91% 42% 13% 61% Naproxen 134% 123% 104% 142% 70% 137% Nonylphenol Non detect 8% 108% 61% 77% 113% Tonalide Non detect 123% 104% 142% 70% 137%   82  3.5.5 Methodology for Mass Balance Using the concentration data for each analyte, a mass balance approach was utilized to examine the EC concentration data obtained during Stage 2 of the experiment. As previously mentioned, it was not possible to complete this step for Stage 1 due to insufficient data.    The first step entailed the calculation of the mass of each analyte in the system using concentration data from the ML. Using Equations 2 to 5, the mass of each analyte in the particulate fraction was calculated, at each time point. Since all ML samples were taken from the aerobic zone and since it was assumed that the ML particulates were reasonably well mixed across all three redox zones, it was also assumed that the analyte particulate phase concentrations or loadings (mg analyte/g of TSS) were the same in each zone. In addition, it was assumed that the concentration of each analyte per litre of ML had no spatial variation within each zone. Therefore, the particulate phase concentration of each analyte in each zone was estimated as a function of the relative suspended solids concentration in that zone (measured as the total suspended solids concentration (TSS), see Table 16).                                                                  (2)                                           (                                            )     (3)                                                 (                                                  )   (4)                                                                                         (5)  The total soluble mass in the system ML at each time point was calculated using Equation 6. As suggested by the lithium tracer study results, it was assumed that the liquid phase of the MEBPR was quite well mixed across all three redox zones. Once again, the soluble concentration was assumed to be constant throughout the system. 83                                                                                 (6)  Equation 7 was subsequently used to calculate the total mass in the system (particulate and soluble).                                                          (7)  To determine the cumulative mass added over two days to the system via the spike (at time zero) and the influent, Equations 8 and 9 were utilized.            (                             )                         (8) Where                        (9)  Similarly, the cumulative mass removed from the system over two days via the effluent or wasting was calculated using Equations 10 to 12. Sludge wasting for SRT control was performed once per day as a discrete event. It was, therefore, only applicable at time points 3 hours and 24 hours.               (        )                        (10)                (                                       )                (11)                                                       (12) Where                   (13)  The resultant mass that was unaccounted for, was therefore calculated using Equation 14.                                                                                  (14)  84  The following sections detail the observed mass balance results for the suite of emerging contaminants studied.  3.5.6 Trends from the Mass Balance 3.5.6.1 Hydrophobic Analytes As a group, the hydrophobic analytes (with high log Kow value (see Section 1.1.8)) were expected to behave similarly.   This group, which included nonylphenol, tonalide, irgasan, EE2, and gemfibrozil, presented similar trends, explained in the following section. The discussion uses the results observed for nonylphenol as an example of the trends observed in the mass balances for the hydrophobic group. The trends for the other hydrophobic analytes are detailed in Appendix C.1.     Figure 18 and Figure 19 illustrate the cumulative mass balance results for nonylphenol on the A-Side and B-Side of the pilot plant, respectively. Several observations should be considered. First, the cumulative mass inflow at time zero for both figures is at 98 mg. This is attributed to the addition of the spike of analyte at time zero. As expected, beyond t = 0, the mass inflow increases at a slow and steady rate due to the analyte being inputted into the system from the influent stream. At time zero, no mass has been removed from the system yet. However, once time zero had passed, mass was removed from the system in a similar fashion to the influent, due to the continuous outflow from the effluent and intermittent flow from the discrete wasting events.  85    Figure 18: Mass Balance for nonylphenol in the A-Side (SRT = 15 d) 0501001502002503000 10 20 30 40 50 60Mass (mg) Time (h) Accumulated Mass in SystemCumulative Mass InflowCumulative Mass Outflow86   Figure 19: Mass Balance for nonylphenol in the B-Side (SRT = 24 d) 0501001502002500 10 20 30 40 50 60Mass (mg) Time (h) Accumulated Mass in SystemCumulative Mass InflowCumulative Mass Outflow87  Furthermore, the figures illustrate the mass in the system (i.e. in the ML). At time zero, the concentration in the system is attributed to the concentration of the analytes already present in the ML: 39 mg for the A-Side and 29 mg for the B-Side. The concentration in the system then increases rapidly, since the system is bombarded with the analytes in the spike. Theoretically, the maximum should not be greater than 137 mg (98 mg from the spike + 39 mg from the mass present at time zero in the system) for the A-Side and 127 mg (98 mg from the spike + 29 mg from the mass present at time zero in the system) for the B-Side. These maxima will be addressed subsequently in further detail. Finally, once the spiking event completed (after 48 hours), the mass in the system decreases. If, however, the mass in the system after 48 hours is lower than the mass in the system at time zero (as seen in Figure 18 for nonylphenol), de-accumulation has occurred, indicating degradation of the analyte.  As previously mentioned, maxima for mass in the system were observed at 12 hours and 36 hours for the A-Side, and at 24 hours for the B-Side. An increase in the mass in the system after time zero should be exhibited, given the addition of the spike at t = 0. As the lithium tracer study illustrated, the maximum concentration was achieved only at 3 hours. However, the tracer study utilized a conservative tracer that would not sorb onto particulate matter. Therefore, the residence time distribution observed with lithium chloride is pertinent only to analytes that would remain in the soluble phase. If an analyte with sorbing characteristics is considered, it is reasonable to expect the particulate matter to have a greater residence time than for the soluble phase.    When the lithium tracer study was conducted, the results indicated that once the maximum was achieved, the liquid phase of the system behaved as a completely mixed reactor (as shown by the tracer curve). However, this trend may not be observed as easily in the system for the sorbing analytes. A possible cause for an increased residence time for the particulates may be 88  attributed to imperfect mixing, whereby a more substantial plug flow-like behaviour is exhibited. When the analytes were spiked into the anaerobic zone, the concentration of the analytes in this specific zone increased dramatically. However, as those particulates move from the anaerobic zone, through the anoxic zone, and into the aerobic zone the analytes may not be well mixed among all the particulates, resulting in clusters of extremely high concentrations and clusters of low concentrations of the analyte. As a result, assuming that the analyte concentration on the particulates in the aerobic zone (which is the location in which the ML samples were obtained) is consistent throughout the system may not be completely accurate.  Nevertheless, it is important to note the differences between the A-Side (SRT = 15 d) and the B-Side (SRT = 24 d). More specifically, in the A-side, where two maximums were observed, the reduced SRT may result in partial mixing of the analyte in the system, resulting in two plug-like concentrated zones moving throughout the plant. However, it would be difficult to confirm this observation, given the limited number of samples obtained. It is possible that the real maxima were missed from either one or both systems. As a result, comparing the time of the apparent maxima may not be conceivable.  The questionable peaks observed in the mass in the system may also be attributed to the QA/QC data (previously discussed in Section 3.5.4). RSD and recoveries for nonylphenol were not as promising as expected. It is likely that the analytical method developed in this research had weaknesses relating to the complexity of the systems (most notably seen in the ML samples).   As previously mentioned, similar patterns in the mass balance plots were observed for tonalide, irgasan, EE2, and gemfibrozil. These analytes (all of which are hydrophobic, like nonylphenol) 89  appeared to clearly exhibit two maxima on the A-Side of the pilot plant (SRT = 15 d), and one maxima in the B-Side of the pilot plant (SRT = 24 d).    Notwithstanding a number of differences between the observed mass balance plots for these analytes can be addressed. Nonylphenol, tonalide, and EE2 all appeared to exhibit maxima that were significantly greater than the associated cumulative mass inflow, which was previously explained. On the other hand, the data for irgasan and gemfibrozil indicated that the maxima mass in the system was either very close to, or less than the cumulative mass inflow. The latter observation indicates a more realistic result, whereby the mass of analyte in the system did not exceed the mass actually added to the system. This illustrates that the mass was therefore conserved.   Another important observation for this group of analytes is the pattern of the mass inflow into the system over the 48 hour experiment. Following the addition of the spike (at time zero), the accumulated mass inflow for EE2 and gemfibrozil was flat. This is attributed to the absence of these specific analytes from the wastewater influent (as seen in Figure 10). Conversely, mass inflow curves for nonylphenol, tonalide, and irgasan exhibited a steady increase, due to the presence of these ECs in the influent wastewater.    90  3.5.6.2 Hydrophilic and Other Analytes The remaining analytes were grouped together as they exhibited similar patterns in the mass balance data that were distinctly different from those discussed for the previously discussed hydrophobic contaminants. Although these analytes exhibited similar mass balance trends to each other, differences in their properties do exist. For instance, dibutyl phthalate, naproxen, and estrone are all hydrophobic analytes, while caffeine and ibuprofen are hydrophilic analytes. This section will display the mass data for dibutyl phthalate (Figure 20 for the A-Side and Figure 21 for the B-Side), with the remainder of the figures for naproxen, estrone, caffeine, and ibuprofen displayed in Appendix C.2.   In comparison to the results presented in the previous section, the patterns in the mass balance data observed for these analytes was more straightforward. Figure 20 and Figure 21 illustrate the mass balance trend for dibutyl phthalate for the A-Side (SRT=15 days) and B-Side (SRT=24 days), respectively. The mass inflow at time zero is 178 mg attributed to the mass of dibutyl phthalate spiked into the anaerobic zone of the system at time zero. Following time zero, the mass inflow into the system increased linearly and rapidly, given the high concentration of dibutyl phthalate present in the influent. The mass outflowing from the system exhibited a steadier trend, accounting for mass released either in the effluent or during intermittent wasting. Finally, the mass in the system at time zero was 61 mg for the A-Side and 32 mg for the B-Side. Following the 48 hour experiment, the final mass in the A-Side was 28 mg, suggesting desorption (and therefore biodegradation) of dibutyl phthalate in the system. Conversely, the mass in the B-Side was 58 mg, illustrating accumulation of dibutyl phthalate in the system. This was the only apparent difference between the A-Side and the B-Side.   91  Interestingly, the mass inflow was significantly greater than the sum of the mass outflow and the mass in the system. This behaviour indicates that a large quantity of the mass could not be accounted for, which provides an additional confirmation that biodegradation likely plays a key role for the presence dibutyl phthalate in the pilot plant.   The remainder of the analytes (estrone, naproxen, caffeine, and ibuprofen) exhibited similar trends. The mass inflow into the system increased linearly, given the presence of all analytes in the influent. Depending on the quantities present in the influent, the slopes of the cumulative mass inflow plots were either relatively large (i.e. dibutyl phthalate, caffeine, and ibuprofen), or moderate (i.e. naproxen and estrone).  In addition, the mass inflow was consistently greater than the mass outflow and the mass in the system. It is likely that naproxen, estrone, caffeine, and ibuprofen also experienced biodegradation within the wastewater treatment system. This possibility is discussed in further detail in the next section.  In particular for caffeine and ibuprofen, biodegradation in wastewater treatment systems is well documented in literature. Caffeine is known to degrade into its metabolites such as 1,7-dimethylxanthine (Stackelberg et al., 2004). Similarly, ibuprofen is also known be highly degradable in wastewater systems with hydroxyl-ibuprofen, 2-hydroxy-ibuprofen, and carboxy-ibuprofen as its metabolites (Buser et al., 1999; Quintana et al., 2005). Since the degradation products for these analytes were known, additional analytical work was performed. The ions of the metabolites were searched on the full Scan Mode on the GC-MS, without success. But once again, this may only be attributed to the analytical method being optimized for the chosen suite of ECs, and not for these specific metabolites.   92   Figure 20: Mass Balance for Dibutyl Phthalate in the A-Side (SRT = 15d)   0501001502002503003500 10 20 30 40 50 60Mass (mg) Time (h) Accumulated Mass in SystemCumulative Mass InflowCumulative Mass Outflow93   Figure 21: Mass Balance for Dibutyl Phthalate in the B-Side (SRT = 24d)0501001502002503003500 10 20 30 40 50 60Mass (mg) Time (h) Accumulated Mass in SystemCumulative Mass InflowCumulative Mass Outflow94  3.5.7 Interpreting the Unaccounted Mass As Section 3.5.6 detailed, using a mass balance approach, and accounting for all of the mass that entered the system, accumulated within the system, or exited the system, a fraction of mass for all analytes could not be accounted for. The previous section indicated the reason for the large quantity of mass that was unaccounted for dibutyl phthalate, naproxen, caffeine, and ibuprofen.  Interestingly, there appears to be a common difference for all analytes based on the SRT of the plant. All analytes were more easily accounted for on the B-Side of the pilot plant, which translates to an increased SRT of 24 days rather than 15 days. This observation may be attributed to the increased SRT resulting in a decreased degradation or transformation of the analytes within the system.   Nevertheless, the mass that is unaccounted for in both sides of the analytes (and for all analytes), likely exists due to degradation products that were not measured in this experiment (Table 21). In addition, some discrepancies may occur due to inaccuracies present in the analytical method, as seen in Section 3.5.4.   95  Table 21: Unaccounted mass for all analytes Analyte Unaccounted Mass (mg) A-Side  (SRT = 15d) B-Side (SRT = 24d) Nonylphenol 153 94 Tonalide 18 8 Irgasan 90 83 Naproxen 144 133 Dibutyl Phthalate 297 238 Estrone 15 12 EE2 8 0 Caffeine 522 498 Ibuprofen 353 246 Gemfibrozil 19 6     96  4 CONCLUSIONS AND RECOMMENDATIONS 4.1 Conclusions Emerging contaminants are a growing concern due to their potential for deleterious health effects on the human population. It is therefore, imperative to create a standardized analytical method such that proper regulation and control can be enforced. Specifically, these contaminants must be monitored in wastewater treatment settings due to their presence in the effluent and wasting streams. A large knowledge gap exists in the effects of these contaminants in wastewater streams, consequently requiring research on the fate of these contaminants.  A pseudo-steady state stimulus response approach was undertaken to study the effects of HRT and SRT on the fate of such contaminants. To do so, an optimized analytical method was developed for a chosen suite of ten emerging contaminants. This method involved the use of liquid liquid extraction with DCM (for the soluble fraction) or ultrasonic extraction with MTBE and methanol (for the particulate fraction), derivatization with BSTFA, and quantitation on the GC-MS unit.  The dynamic experiments based on the HRT studies (and set out by the CWN) were not successful. The only non-steady state aspects of the study were due to uncontrolled dynamics in the influent composition, while all other parameters were maintained at steady state conditions.  Following a number of modifications to the analytical and experimental method, a detailed analysis was conducted for the comparison of short to long SRT. Using the concentration measurements, a mass balance analysis was conducted on the individual ECs. Both parallel sides at the UBC pilot plant (with a greater proportion observed at a shorter SRT) demonstrated that the greatest fraction of the mass fed into the system was unaccounted for, signifying that 97  sorption and washout were not the only removal mechanisms for these contaminants in this particular experimental system. Degradation and other removal mechanisms beyond the scope of this research were likely responsible for the removal of these contaminants, demonstrating a great need for further research in this area.   4.2 Recommendations Working with the chosen suite of ECs proved to be challenging given the lack of information on many aspects relating to the role of wastewater treatment on the fate of these contaminants. Nevertheless, the research conducted at UBC further demonstrates the need for the study of degradation products in wastewater treatment plants, as well as other removal mechanisms for the ECs. In addition, confirmation of the observed results using an analysis using LC/MS/MS in a facility such as a certified laboratory would be required. However, due to monetary constraints, this option was not viable for this research. Furthermore, the membrane?s role, the role of the three zones (i.e. anaerobic, anoxic, and aerobic), and the foam in the aerobic zone should all be investigated in regards to removal mechanisms. Finally, a more comprehensive study examining the behaviour of the solids in the ML should be conducted (potentially using a sorbing-type tracer).   On a larger scope, the effects of the ECs on a microbial level must be investigated including testing for changes in gene expression or uptake effects. In addition, the public must be educated on proper disposal of these contaminants given that inadequate disposal is a probable cause of high concentrations released into the environment. Finally, and most importantly, a standardized method for analyzing these contaminants is imperative. Only once this step is complete, can these analytes be regulated and monitored properly on a full scale level.       98  REFERENCES Ag?era, A., Mart?nez Bueno, M. J., & Fern?ndez-Alba, A. R. (2013). New trends in the analytical determination of emerging contaminants and their transformation products in environmental waters. Environmental Science and Pollution Research International, 1?20. Bajt, O., Mailhot, G., & Bolte, M. (2001). Degradation of dibutyl phthalate by homogeneous photocatalysis with Fe(III) in aqueous solution. Applied Catalysis B: Environmental, 33(3), 239?248. Bolong, N., Ismail, A. F., Salim, M. R., & Matsuura, T. (2009). 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For INF and ML, centrifuge at an rcf of 913*g for 15 minutes and decant the supernatant 3. For INF, obtain the particulate fraction and proceed to Appendix C.2 for freeze drying (A smaller sample size will be used for ML) 4. Acidify sample using 10% HCl solution (approximately 10-15 drops) 5. Transfer to separatory funnel and spike with surrogate (MCPB) 6. Add 25 mL of DCM 7. Shake and vent for 2 minutes 8. Allow the contents of the separatory funnel to sit for 10 minutes and allow for separation 9. Open the tap on the separatory funnel and discharge the bottom (DCM layer) through anhydrous sodium sulfate to a round bottom flask 10. Repeat Steps 6 to 9 two more times 11. Empty the contents of the separatory funnel (aqueous phase) and wash with DCM 12. Place the round bottom flask on a rotary evaporator and allow the solvent to evaporate to an approximate final volume of 2 mL 13. Transfer the contents from each round bottom flask into a 10 mL glass HACH vial 14. Blow down to dryness using nitrogen gas 15. Add 150 ?L of BSTFA (with 1% TMCS) and 50 ?L of pyridine for derivatization 16. Vortex each vial for 10 seconds 17. Bake in an oven at 70?C for 1 hour 18. Remove from oven and blow down to dryness with nitrogen gas 19. Add 1 mL of toluene (spiked with internal standard) 20. Vortex each vial for 10 seconds 21. Transfer contents of the vial to a GC-MS vial using a pasteur pipette 22. Place vial on the GC-MS for analysis      103  Appendix A.2: Ultrasonic Extraction (USE) for INF-? and ML-? Freeze drying of ML-? 1. Measure 5mL of sludge into a 10 mL glass HACH vial 2. Centrifuge at an rcf of 913*g for 5 minutes 3. Pipette out all the water (should be the top phase) 4. Cap the 10 mL glass HACH vials tightly 5. Freeze the vials in a freezer (-20?C) overnight 6. Take off the caps from the 10 mL glass HACH vials 7. Place in freeze dryer overnight (this sucks out all the water remaining in the samples)  Freeze drying of INF-? 1. Freeze the vials from (Step 3 in Appendix C.1) in a freezer (-20?C) overnight 2. Take off the caps from the 10 mL glass HACH vials 3. Place in freeze dryer overnight (this sucks out all the water remaining in the samples)  Ultrasonic extraction of INF-? and ML-? 1. No pH adjustment is required 2. Spike the samples with the surrogate 3. Add 4mL of MTBE and 1mL of methanol to 10 mL glass HACH vial  4. Cap the vials 5. Give a quick shake and vent to the vials 6. Place the vials in an ultrasonic water bath at 40?C for 15 minutes 7. Pipette out the solvent (top phase) into a round bottom flask 8. Repeat steps 3 to 7 an additional two times 9. Blow down the solvent in the round bottom flask to ~2 mL using the Rotary Evaporator 10. Transfer to clean 10 mL glass HACH vial using a pasteur pipette 11. Rinse the round bottom flask with ~2 mL MTBE and methanol and transfer to 10 mL glass HACH vial 12. Blow down solvent in 10 mL glass HACH vial to dryness using nitrogen gas 13. Add 150 ?L BSTFA (+1% TMCS) and 50?L pyridine (derivatization agents) 14. Cap the vials and vortex for 10 seconds 15. Bake in oven at 70?C for 1 hour 16. Blow down to dryness using nitrogen gas 17. Add 1mL of toluene 18. Cap the vials and vortex for 10 seconds 19. Transfer the sample to a GC vial using a pipette 20. Place vial on the GC-MS for analysis      104  Appendix A.3: Optimization of Analytical Method  Table A - 1: Optimization parameters for analytical method Parameter Optimized Use of Parameter Details Used in final Method (Yes or No) Suite of ECs Compounds that couldn?t be analyzed on the GC-MS Ciprofloxacin  No Sulfamethoxazole No Carbamezapine No Triclocarban No Compounds with too much interference Bis(2-ethylhexyl) phthalate  No (replaced by dibutyl phthalate) Surrogate Meclofenamic acid  No MCPB [2,4-4-(4-chloro-o-tolyloxy)butyric acid] used Yes (but still not successful) Measuring INF concentrations Separation processes Filter soluble fraction with 0.45 ?m filter No Centrifuge soluble fraction Yes Contamination  Silanization of glassware Glass treated with 10% dimethylchlorosilane in toluene No pH level Prior to extraction 2 No 3 Yes Extraction method for soluble fraction   Solid phase extraction (SPE) cartridge types Discovery Supelco  No Waters No Supelco HLB No Solid phase extraction (SPE) elution solvents Acetone  No Methanol No Acetonitrile No Liquid-liquid extraction (LLE) solvents used MTBE  No DCM + MTBE No DCM Yes Derivatization Baking time 20 minutes No 1 hour Yes Derivatization agents BSTFA (50 ?L) + pyridine (15 ?L) No MTBSTFA (100 ?L) + acetonitrile (100 ?L) No BSTFA (150 ?L) + pyridine (5o ?L) Yes Blowing down of derivatization agents after baking Skip this step No With nitrogen gas Yes GC-MS vials Reconstitution of sample in solvent Derivatization agents (no solvent) No Ethyl Acetate No Toluene Yes GC-MS Operation Holding time after each sample 0 minutes No 5 minutes Yes 105  Parameter Optimized Use of Parameter Details Used in final Method (Yes or No) Temperature program Increase at 15?C/min to 170?C and hold for 0.5 minutes, then increase at 8?C/min until 290?C and hold for 5 minutes, then hold at 300?C for 5 minutes  (Total run time = 31.5 minutes) No Increase at 12?C/min to 290?C and hold for 5 minutes, then hold at 300?C for 5 minutes  (Total run time = 27.27 minutes) Yes Analyte quantification Full scan No Selected ion monitoring (SIM) Yes    106  APPENDIX B: RAW CONCENTRATION DATA  Appendix B.1: Data for Stage 1 (Comparison of Steady vs. Diurnal HRT) Table B - 1: Raw concentration data for Stage 1 experiment Time (h) Sample Number Sample Location at the Pilot Plant Analyte Average Concentration in Pilot Plant (ng/L) Minimum concentration in the pilot plant (ng/L)  Based on replicate runs only Maximum concentration in the pilot plant (ng/L)  Based on replicate runs only 0 1 INF Ibuprofen 24904 N/A N/A 0 1 INF Nonylphenol 9512 N/A N/A 0 1 INF Caffeine 23729 N/A N/A 0 1 INF Tonalide 2754 N/A N/A 0 1 INF MCPB 20917 N/A N/A 0 1 INF Gemfibrozil 0 N/A N/A 0 1 INF Dibutyl Phthalate 9206 N/A N/A 0 1 INF Naproxen 23692 N/A N/A 0 1 INF Irgasan 6140 N/A N/A 0 1 INF Estrone 749 N/A N/A 0 1 INF EE2 0 N/A N/A 0 2 EFF A Ibuprofen 0 N/A N/A 0 2 EFF A Nonylphenol 4135 N/A N/A 0 2 EFF A Caffeine 0 N/A N/A 0 2 EFF A Tonalide 1121 N/A N/A 0 2 EFF A MCPB 14392 N/A N/A 0 2 EFF A Gemfibrozil 0 N/A N/A 0 2 EFF A Dibutyl Phthalate 11476 N/A N/A 0 2 EFF A Naproxen 1278 N/A N/A 0 2 EFF A Irgasan 339 N/A N/A 0 2 EFF A Estrone 0 N/A N/A 0 2 EFF A EE2 0 N/A N/A 0 3 EFF B Ibuprofen 0 N/A N/A 0 3 EFF B Nonylphenol 3347 N/A N/A 0 3 EFF B Caffeine 272 N/A N/A 0 3 EFF B Tonalide 1155 N/A N/A 0 3 EFF B MCPB 10565 N/A N/A 0 3 EFF B Gemfibrozil 0 N/A N/A 107  Time (h) Sample Number Sample Location at the Pilot Plant Analyte Average Concentration in Pilot Plant (ng/L) Minimum concentration in the pilot plant (ng/L)  Based on replicate runs only Maximum concentration in the pilot plant (ng/L)  Based on replicate runs only 0 3 EFF B Dibutyl Phthalate 6985 N/A N/A 0 3 EFF B Naproxen 1592 N/A N/A 0 3 EFF B Irgasan 385 N/A N/A 0 3 EFF B Estrone 0 N/A N/A 0 3 EFF B EE2 0 N/A N/A 0 4 ML A Ibuprofen 0 N/A N/A 0 4 ML A Nonylphenol 44999 N/A N/A 0 4 ML A Caffeine 0 N/A N/A 0 4 ML A Tonalide 33718 N/A N/A 0 4 ML A MCPB 151815 N/A N/A 0 4 ML A Gemfibrozil 0 N/A N/A 0 4 ML A Dibutyl Phthalate 72178 N/A N/A 0 4 ML A Naproxen 72649 N/A N/A 0 4 ML A Irgasan 33645 N/A N/A 0 4 ML A Estrone 6694 N/A N/A 0 4 ML A EE2 0 N/A N/A 0 5 ML B Ibuprofen 0 N/A N/A 0 5 ML B Nonylphenol 43791 N/A N/A 0 5 ML B Caffeine 272 N/A N/A 0 5 ML B Tonalide 41186 N/A N/A 0 5 ML B MCPB 306529 N/A N/A 0 5 ML B Gemfibrozil 0 N/A N/A 0 5 ML B Dibutyl Phthalate 81323 N/A N/A 0 5 ML B Naproxen 51509 N/A N/A 0 5 ML B Irgasan 29870 N/A N/A 0 5 ML B Estrone 6572 N/A N/A 0 5 ML B EE2 0 N/A N/A 3 6-8 INF Ibuprofen 21849 19910 24801 3 6-8 INF Nonylphenol 8467 7644 9169 3 6-8 INF Caffeine 26797 25874 27700 3 6-8 INF Tonalide 2865 2528 3041 3 6-8 INF MCPB 19459 17922 21545 3 6-8 INF Gemfibrozil 0 0 0 3 6-8 INF Dibutyl 9453 8954 10004 108  Time (h) Sample Number Sample Location at the Pilot Plant Analyte Average Concentration in Pilot Plant (ng/L) Minimum concentration in the pilot plant (ng/L)  Based on replicate runs only Maximum concentration in the pilot plant (ng/L)  Based on replicate runs only Phthalate 3 6-8 INF Naproxen 19362 17199 22300 3 6-8 INF Irgasan 5837 5608 6117 3 6-8 INF Estrone 498 379 636 3 6-8 INF EE2 0 0 0 3 9-10 EFF A Ibuprofen 1178 1116 1241 3 9-10 EFF A Nonylphenol 5393 4202 6584 3 9-10 EFF A Caffeine 106 100 112 3 9-10 EFF A Tonalide 1094 1019 1169 3 9-10 EFF A MCPB 18356 18322 18391 3 9-10 EFF A Gemfibrozil 2847 2800 2894 3 9-10 EFF A Dibutyl Phthalate 7270 6563 7978 3 9-10 EFF A Naproxen 3511 3433 3589 3 9-10 EFF A Irgasan 439 427 451 3 9-10 EFF A Estrone 0 0 0 3 9-10 EFF A EE2 1590 504 2676 3 12-14 EFF B Ibuprofen 768 0 947 3 12-14 EFF B Nonylphenol 3446 2659 4232 3 12-14 EFF B Caffeine 275 267 288 3 12-14 EFF B Tonalide 890 835 935 3 12-14 EFF B MCPB 15605 6911 17195 3 12-14 EFF B Gemfibrozil 2260 1372 2301 3 12-14 EFF B Dibutyl Phthalate 5649 3759 7539 3 12-14 EFF B Naproxen 2582 1069 2991 3 12-14 EFF B Irgasan 334 295 363 3 12-14 EFF B Estrone 0 0 0 3 12-14 EFF B EE2 1141 0 1547 3 15-17 ML A Ibuprofen 1175 1113 1238 3 15-17 ML A Nonylphenol 53932 48055 61842 3 15-17 ML A Caffeine 106 100 112 3 15-17 ML A Tonalide 47434 44409 51020 3 15-17 ML A MCPB 311200 271225 334393 3 15-17 ML A Gemfibrozil 2840 2793 2887 3 15-17 ML A Dibutyl Phthalate 69262 58015 78171 109  Time (h) Sample Number Sample Location at the Pilot Plant Analyte Average Concentration in Pilot Plant (ng/L) Minimum concentration in the pilot plant (ng/L)  Based on replicate runs only Maximum concentration in the pilot plant (ng/L)  Based on replicate runs only 3 15-17 ML A Naproxen 3503 3425 3580 3 15-17 ML A Irgasan 33077 30724 35506 3 15-17 ML A Estrone 0 0 0 3 15-17 ML A EE2 18870 502 20525 3 18-20 ML B Ibuprofen 766 0 944 3 18-20 ML B Nonylphenol 55576 42145 76226 3 18-20 ML B Caffeine 274 266 287 3 18-20 ML B Tonalide 52392 49409 54427 3 18-20 ML B MCPB 363045 226556 543558 3 18-20 ML B Gemfibrozil 2254 1368 2295 3 18-20 ML B Dibutyl Phthalate 58404 53330 63090 3 18-20 ML B Naproxen 2575 1066 2984 3 18-20 ML B Irgasan 29134 22764 39310 3 18-20 ML B Estrone 0 0 0 3 18-20 ML B EE2 1139 0 1544 12 21 INF Ibuprofen 15159 N/A N/A 12 21 INF Nonylphenol 8854 N/A N/A 12 21 INF Caffeine 32109 N/A N/A 12 21 INF Tonalide 3004 N/A N/A 12 21 INF MCPB 17527 N/A N/A 12 21 INF Gemfibrozil 0 N/A N/A 12 21 INF Dibutyl Phthalate 6083 N/A N/A 12 21 INF Naproxen 16294 N/A N/A 12 21 INF Irgasan 5480 N/A N/A 12 21 INF Estrone 0 N/A N/A 12 21 INF EE2 0 N/A N/A 12 22 EFF A Ibuprofen 447 N/A N/A 12 22 EFF A Nonylphenol 2629 N/A N/A 12 22 EFF A Caffeine 125 N/A N/A 12 22 EFF A Tonalide 760 N/A N/A 12 22 EFF A MCPB 12708 N/A N/A 12 22 EFF A Gemfibrozil 0 N/A N/A 12 22 EFF A Dibutyl Phthalate 2635 N/A N/A 12 22 EFF A Naproxen 1532 N/A N/A 110  Time (h) Sample Number Sample Location at the Pilot Plant Analyte Average Concentration in Pilot Plant (ng/L) Minimum concentration in the pilot plant (ng/L)  Based on replicate runs only Maximum concentration in the pilot plant (ng/L)  Based on replicate runs only 12 22 EFF A Irgasan 403 N/A N/A 12 22 EFF A Estrone 0 N/A N/A 12 22 EFF A EE2 902 N/A N/A 12 23 EFF B Ibuprofen 399 N/A N/A 12 23 EFF B Nonylphenol 2852 N/A N/A 12 23 EFF B Caffeine 239 N/A N/A 12 23 EFF B Tonalide 1089 N/A N/A 12 23 EFF B MCPB 6970 N/A N/A 12 23 EFF B Gemfibrozil 0 N/A N/A 12 23 EFF B Dibutyl Phthalate 4823 N/A N/A 12 23 EFF B Naproxen 630 N/A N/A 12 23 EFF B Irgasan 171 N/A N/A 12 23 EFF B Estrone 0 N/A N/A 12 23 EFF B EE2 930 N/A N/A 12 24 ML A Ibuprofen 5402 N/A N/A 12 24 ML A Nonylphenol 45782 N/A N/A 12 24 ML A Caffeine 124 N/A N/A 12 24 ML A Tonalide 55514 N/A N/A 12 24 ML A MCPB 340319 N/A N/A 12 24 ML A Gemfibrozil 0 N/A N/A 12 24 ML A Dibutyl Phthalate 98733 N/A N/A 12 24 ML A Naproxen 1528 N/A N/A 12 24 ML A Irgasan 32938 N/A N/A 12 24 ML A Estrone 0 N/A N/A 12 24 ML A EE2 900 N/A N/A 12 25 ML B Ibuprofen 398 N/A N/A 12 25 ML B Nonylphenol 2845 N/A N/A 12 25 ML B Caffeine 238 N/A N/A 12 25 ML B Tonalide 30500 N/A N/A 12 25 ML B MCPB 253467 N/A N/A 12 25 ML B Gemfibrozil 0 N/A N/A 12 25 ML B Dibutyl Phthalate 102067 N/A N/A 12 25 ML B Naproxen 628 N/A N/A 12 25 ML B Irgasan 13792 N/A N/A 111  Time (h) Sample Number Sample Location at the Pilot Plant Analyte Average Concentration in Pilot Plant (ng/L) Minimum concentration in the pilot plant (ng/L)  Based on replicate runs only Maximum concentration in the pilot plant (ng/L)  Based on replicate runs only 12 25 ML B Estrone 0 N/A N/A 12 25 ML B EE2 927 N/A N/A 24 26-27 INF Ibuprofen 19128 17191 21065 24 26-27 INF Nonylphenol 10468 9880 11057 24 26-27 INF Caffeine 22213 20934 23491 24 26-27 INF Tonalide 2463 2445 2482 24 26-27 INF MCPB 17638 16289 18988 24 26-27 INF Gemfibrozil 0 0 0 24 26-27 INF Dibutyl Phthalate 5118 4701 5534 24 26-27 INF Naproxen 18099 16531 19667 24 26-27 INF Irgasan 5004 4500 5509 24 26-27 INF Estrone 560 474 645 24 26-27 INF EE2 0 0 0 24 29-31 EFF A Ibuprofen 0 0 0 24 29-31 EFF A Nonylphenol 2444 2351 2545 24 29-31 EFF A Caffeine 0 0 0 24 29-31 EFF A Tonalide 910 833 1010 24 29-31 EFF A MCPB 9292 6696 13161 24 29-31 EFF A Gemfibrozil 0 0 0 24 29-31 EFF A Dibutyl Phthalate 3327 3155 3534 24 29-31 EFF A Naproxen 950 0 1206 24 29-31 EFF A Irgasan 448 377 565 24 29-31 EFF A Estrone 0 0 0 24 29-31 EFF A EE2 206 154 236 24 32-34 EFF B Ibuprofen 0 0 0 24 32-34 EFF B Nonylphenol 2499 1678 2974 24 32-34 EFF B Caffeine 226 192 256 24 32-34 EFF B Tonalide 973 777 1100 24 32-34 EFF B MCPB 8145 5203 11689 24 32-34 EFF B Gemfibrozil 0 0 0 24 32-34 EFF B Dibutyl Phthalate 3483 2443 4203 24 32-34 EFF B Naproxen 758 576 1026 24 32-34 EFF B Irgasan 384 315 506 24 32-34 EFF B Estrone 0 0 0 112  Time (h) Sample Number Sample Location at the Pilot Plant Analyte Average Concentration in Pilot Plant (ng/L) Minimum concentration in the pilot plant (ng/L)  Based on replicate runs only Maximum concentration in the pilot plant (ng/L)  Based on replicate runs only 24 32-34 EFF B EE2 405 335 496 24 35-37 ML A Ibuprofen 0 0 0 24 35-37 ML A Nonylphenol 39966 34929 48621 24 35-37 ML A Caffeine 0 0 0 24 35-37 ML A Tonalide 48120 45959 49355 24 35-37 ML A MCPB 338531 308847 365413 24 35-37 ML A Gemfibrozil 0 0 0 24 35-37 ML A Dibutyl Phthalate 103307 73181 153769 24 35-37 ML A Naproxen 948 0 1203 24 35-37 ML A Irgasan 29072 26482 30612 24 35-37 ML A Estrone 0 0 0 24 35-37 ML A EE2 205 154 235 24 38-40 ML B Ibuprofen 0 0 0 24 38-40 ML B Nonylphenol 41672 37052 48474 24 38-40 ML B Caffeine 225 191 255 24 38-40 ML B Tonalide 51841 50413 52950 24 38-40 ML B MCPB 336057 257614 427574 24 38-40 ML B Gemfibrozil 0 0 0 24 38-40 ML B Dibutyl Phthalate 75916 70585 79378 24 38-40 ML B Naproxen 756 574 1023 24 38-40 ML B Irgasan 24121 22178 27123 24 38-40 ML B Estrone 0 0 0 24 38-40 ML B EE2 404 334 495 30 41 INF Ibuprofen 14943 N/A N/A 30 41 INF Nonylphenol 8843 N/A N/A 30 41 INF Caffeine 26123 N/A N/A 30 41 INF Tonalide 2750 N/A N/A 30 41 INF MCPB 16273 N/A N/A 30 41 INF Gemfibrozil 0 N/A N/A 30 41 INF Dibutyl Phthalate 9839 N/A N/A 30 41 INF Naproxen 10426 N/A N/A 30 41 INF Irgasan 5247 N/A N/A 30 41 INF Estrone 270 N/A N/A 30 41 INF EE2 0 N/A N/A 113  Time (h) Sample Number Sample Location at the Pilot Plant Analyte Average Concentration in Pilot Plant (ng/L) Minimum concentration in the pilot plant (ng/L)  Based on replicate runs only Maximum concentration in the pilot plant (ng/L)  Based on replicate runs only 30 42 EFF A Ibuprofen 570 N/A N/A 30 42 EFF A Nonylphenol 2752 N/A N/A 30 42 EFF A Caffeine 0 N/A N/A 30 42 EFF A Tonalide 1119 N/A N/A 30 42 EFF A MCPB 16990 N/A N/A 30 42 EFF A Gemfibrozil 0 N/A N/A 30 42 EFF A Dibutyl Phthalate 6385 N/A N/A 30 42 EFF A Naproxen 1653 N/A N/A 30 42 EFF A Irgasan 369 N/A N/A 30 42 EFF A Estrone 0 N/A N/A 30 42 EFF A EE2 253 N/A N/A 30 43 EFF B Ibuprofen 0 N/A N/A 30 43 EFF B Nonylphenol 2818 N/A N/A 30 43 EFF B Caffeine 237 N/A N/A 30 43 EFF B Tonalide 1170 N/A N/A 30 43 EFF B MCPB 7424 N/A N/A 30 43 EFF B Gemfibrozil 0 N/A N/A 30 43 EFF B Dibutyl Phthalate 3827 N/A N/A 30 43 EFF B Naproxen 516 N/A N/A 30 43 EFF B Irgasan 338 N/A N/A 30 43 EFF B Estrone 0 N/A N/A 30 43 EFF B EE2 395 N/A N/A 30 44 ML A Ibuprofen 569 N/A N/A 30 44 ML A Nonylphenol 35493 N/A N/A 30 44 ML A Caffeine 0 N/A N/A 30 44 ML A Tonalide 48764 N/A N/A 30 44 ML A MCPB 317401 N/A N/A 30 44 ML A Gemfibrozil 0 N/A N/A 30 44 ML A Dibutyl Phthalate 106895 N/A N/A 30 44 ML A Naproxen 1649 N/A N/A 30 44 ML A Irgasan 22036 N/A N/A 30 44 ML A Estrone 0 N/A N/A 30 44 ML A EE2 253 N/A N/A 30 45 ML B Ibuprofen 0 N/A N/A 114  Time (h) Sample Number Sample Location at the Pilot Plant Analyte Average Concentration in Pilot Plant (ng/L) Minimum concentration in the pilot plant (ng/L)  Based on replicate runs only Maximum concentration in the pilot plant (ng/L)  Based on replicate runs only 30 45 ML B Nonylphenol 41633 N/A N/A 30 45 ML B Caffeine 1382 N/A N/A 30 45 ML B Tonalide 48945 N/A N/A 30 45 ML B MCPB 287060 N/A N/A 30 45 ML B Gemfibrozil 0 N/A N/A 30 45 ML B Dibutyl Phthalate 92863 N/A N/A 30 45 ML B Naproxen 515 N/A N/A 30 45 ML B Irgasan 18647 N/A N/A 30 45 ML B Estrone 0 N/A N/A 30 45 ML B EE2 394 N/A N/A 36 46 INF Ibuprofen 16398 N/A N/A 36 46 INF Nonylphenol 7618 N/A N/A 36 46 INF Caffeine 35372 N/A N/A 36 46 INF Tonalide 3335 N/A N/A 36 46 INF MCPB 15873 N/A N/A 36 46 INF Gemfibrozil 0 N/A N/A 36 46 INF Dibutyl Phthalate 9116 N/A N/A 36 46 INF Naproxen 5011 N/A N/A 36 46 INF Irgasan 4607 N/A N/A 36 46 INF Estrone 384 N/A N/A 36 46 INF EE2 0 N/A N/A 36 47 EFF A Ibuprofen 0 N/A N/A 36 47 EFF A Nonylphenol 6346 N/A N/A 36 47 EFF A Caffeine 0 N/A N/A 36 47 EFF A Tonalide 1372 N/A N/A 36 47 EFF A MCPB 3620 N/A N/A 36 47 EFF A Gemfibrozil 0 N/A N/A 36 47 EFF A Dibutyl Phthalate 14355 N/A N/A 36 47 EFF A Naproxen 0 N/A N/A 36 47 EFF A Irgasan 179 N/A N/A 36 47 EFF A Estrone 0 N/A N/A 36 47 EFF A EE2 0 N/A N/A 36 48 EFF B Ibuprofen 0 N/A N/A 36 48 EFF B Nonylphenol 1125 N/A N/A 115  Time (h) Sample Number Sample Location at the Pilot Plant Analyte Average Concentration in Pilot Plant (ng/L) Minimum concentration in the pilot plant (ng/L)  Based on replicate runs only Maximum concentration in the pilot plant (ng/L)  Based on replicate runs only 36 48 EFF B Caffeine 0 N/A N/A 36 48 EFF B Tonalide 609 N/A N/A 36 48 EFF B MCPB 2480 N/A N/A 36 48 EFF B Gemfibrozil 0 N/A N/A 36 48 EFF B Dibutyl Phthalate 3944 N/A N/A 36 48 EFF B Naproxen 0 N/A N/A 36 48 EFF B Irgasan 0 N/A N/A 36 48 EFF B Estrone 0 N/A N/A 36 48 EFF B EE2 0 N/A N/A 36 49 ML A Ibuprofen 0 N/A N/A 36 49 ML A Nonylphenol 35688 N/A N/A 36 49 ML A Caffeine 0 N/A N/A 36 49 ML A Tonalide 44636 N/A N/A 36 49 ML A MCPB 298939 N/A N/A 36 49 ML A Gemfibrozil 0 N/A N/A 36 49 ML A Dibutyl Phthalate 127799 N/A N/A 36 49 ML A Naproxen 0 N/A N/A 36 49 ML A Irgasan 25301 N/A N/A 36 49 ML A Estrone 0 N/A N/A 36 49 ML A EE2 0 N/A N/A 36 50 ML B Ibuprofen 0 N/A N/A 36 50 ML B Nonylphenol 38444 N/A N/A 36 50 ML B Caffeine 0 N/A N/A 36 50 ML B Tonalide 75827 N/A N/A 36 50 ML B MCPB 311548 N/A N/A 36 50 ML B Gemfibrozil 0 N/A N/A 36 50 ML B Dibutyl Phthalate 68970 N/A N/A 36 50 ML B Naproxen 0 N/A N/A 36 50 ML B Irgasan 24410 N/A N/A 36 50 ML B Estrone 0 N/A N/A 36 50 ML B EE2 0 N/A N/A 48 51-53 INF Ibuprofen 11693 10390 12461 48 51-53 INF Nonylphenol 8829 8660 8950 48 51-53 INF Caffeine 25716 23019 27336 116  Time (h) Sample Number Sample Location at the Pilot Plant Analyte Average Concentration in Pilot Plant (ng/L) Minimum concentration in the pilot plant (ng/L)  Based on replicate runs only Maximum concentration in the pilot plant (ng/L)  Based on replicate runs only 48 51-53 INF Tonalide 2339 2117 2683 48 51-53 INF MCPB 12360 10705 13432 48 51-53 INF Gemfibrozil 0 0 0 48 51-53 INF Dibutyl Phthalate 7122 6824 7272 48 51-53 INF Naproxen 4660 4220 5074 48 51-53 INF Irgasan 4087 3584 4629 48 51-53 INF Estrone 372 283 422 48 51-53 INF EE2 0 0 0 48 54-56 EFF A Ibuprofen 0 0 0 48 54-56 EFF A Nonylphenol 1270 1188 3045 48 54-56 EFF A Caffeine 0 0 43 48 54-56 EFF A Tonalide 301 280 778 48 54-56 EFF A MCPB 2621 1109 11659 48 54-56 EFF A Gemfibrozil 0 0 0 48 54-56 EFF A Dibutyl Phthalate 2428 2007 6951 48 54-56 EFF A Naproxen 167 0 1176 48 54-56 EFF A Irgasan 67 24 68 48 54-56 EFF A Estrone 0 0 0 48 54-56 EFF A EE2 0 0 0 48 57-59 EFF B Ibuprofen 0 0 0 48 57-59 EFF B Nonylphenol 3519 2760 3662 48 57-59 EFF B Caffeine 0 0 0 48 57-59 EFF B Tonalide 696 530 862 48 57-59 EFF B MCPB 5226 4768 13990 48 57-59 EFF B Gemfibrozil 0 0 0 48 57-59 EFF B Dibutyl Phthalate 8136 6789 9482 48 57-59 EFF B Naproxen 0 0 1419 48 57-59 EFF B Irgasan 208 143 286 48 57-59 EFF B Estrone 0 0 0 48 57-59 EFF B EE2 0 0 0 48 60-62 ML A Ibuprofen 0 0 0 48 60-62 ML A Nonylphenol 29660 26573 36048 48 60-62 ML A Caffeine 0 0 42 48 60-62 ML A Tonalide 43869 39905 46598 117  Time (h) Sample Number Sample Location at the Pilot Plant Analyte Average Concentration in Pilot Plant (ng/L) Minimum concentration in the pilot plant (ng/L)  Based on replicate runs only Maximum concentration in the pilot plant (ng/L)  Based on replicate runs only 48 60-62 ML A MCPB 282729 260022 305935 48 60-62 ML A Gemfibrozil 0 0 0 48 60-62 ML A Dibutyl Phthalate 60857 59516 66478 48 60-62 ML A Naproxen 167 0 1173 48 60-62 ML A Irgasan 15423 14130 16134 48 60-62 ML A Estrone 0 0 0 48 60-62 ML A EE2 0 0 0 48 63-65 ML B Ibuprofen 0 0 0 48 63-65 ML B Nonylphenol 35734 28149 43526 48 63-65 ML B Caffeine 0 0 0 48 63-65 ML B Tonalide 55204 50892 60496 48 63-65 ML B MCPB 269436 210302 322304 48 63-65 ML B Gemfibrozil 0 0 0 48 63-65 ML B Dibutyl Phthalate 67810 60165 80263 48 63-65 ML B Naproxen 0 0 1415 48 63-65 ML B Irgasan 16226 11476 21520 48 63-65 ML B Estrone 0 0 0 48 63-65 ML B EE2 0 0 0    118  Appendix B.2: Data for Stage 2 (Short vs. Long SRT) Table B - 2: Raw concentration data for Stage 2 experiment, divided according to particulate and soluble fraction Time (h) Sample Location at the Pilot Plant Sample Number Fraction  (a = soluble, b = particulate) Analyte Average Sample Concentration (ng/L) Difference between maximum or minimum and the average (ng/L)  Only for replicate runs 0 INF 1 a Ibuprofen 19470 N/A 0 INF 1 a Nonylphenol 3898 N/A 0 INF 1 a Caffeine 22989 N/A 0 INF 1 a Tonalide 1435 N/A 0 INF 1 a MCPB 7097 N/A 0 INF 1 a Gemfibrozil 0 N/A 0 INF 1 a Dibutyl Phthalate 11878 N/A 0 INF 1 a Naproxen 915 N/A 0 INF 1 a Irgasan 404 N/A 0 INF 1 a Estrone 24 N/A 0 INF 1 a EE2 0 N/A 0 INF 1 b Ibuprofen 123 N/A 0 INF 1 b Nonylphenol 2429 N/A 0 INF 1 b Caffeine 0 N/A 0 INF 1 b Tonalide 297 N/A 0 INF 1 b MCPB 5746 N/A 0 INF 1 b Gemfibrozil 0 N/A 0 INF 1 b Dibutyl Phthalate 1223 N/A 0 INF 1 b Naproxen 0 N/A 0 INF 1 b Irgasan 241 N/A 0 INF 1 b Estrone 0 N/A 0 INF 1 b EE2 0 N/A 0 EFF A 2 a Ibuprofen 44 N/A 0 EFF A 2 a Nonylphenol 477 N/A 0 EFF A 2 a Caffeine 2684 N/A 0 EFF A 2 a Tonalide 338 N/A 0 EFF A 2 a MCPB 1054 N/A 0 EFF A 2 a Gemfibrozil 0 N/A 119  Time (h) Sample Location at the Pilot Plant Sample Number Fraction  (a = soluble, b = particulate) Analyte Average Sample Concentration (ng/L) Difference between maximum or minimum and the average (ng/L)  Only for replicate runs 0 EFF A 2 a Dibutyl Phthalate 9709 N/A 0 EFF A 2 a Naproxen 174 N/A 0 EFF A 2 a Irgasan 15 N/A 0 EFF A 2 a Estrone 15 N/A 0 EFF A 2 a EE2 82 N/A 0 EFF B 3 a Ibuprofen 0 N/A 0 EFF B 3 a Nonylphenol 324 N/A 0 EFF B 3 a Caffeine 745 N/A 0 EFF B 3 a Tonalide 299 N/A 0 EFF B 3 a MCPB 908 N/A 0 EFF B 3 a Gemfibrozil 0 N/A 0 EFF B 3 a Dibutyl Phthalate 7731 N/A 0 EFF B 3 a Naproxen 64 N/A 0 EFF B 3 a Irgasan 9 N/A 0 EFF B 3 a Estrone 0 N/A 0 EFF B 3 a EE2 0 N/A 0 ML A 4 a Ibuprofen 0 N/A 0 ML A 4 a Nonylphenol 810 N/A 0 ML A 4 a Caffeine 0 N/A 0 ML A 4 a Tonalide 238 N/A 0 ML A 4 a MCPB 1504 N/A 0 ML A 4 a Gemfibrozil 0 N/A 0 ML A 4 a Dibutyl Phthalate 1484 N/A 0 ML A 4 a Naproxen 100 N/A 0 ML A 4 a Irgasan 30 N/A 0 ML A 4 a Estrone 20 N/A 0 ML A 4 a EE2 0 N/A 0 ML A 4 b Ibuprofen 4138 N/A 0 ML A 4 b Nonylphenol 23582 N/A 0 ML A 4 b Caffeine 0 N/A 0 ML A 4 b Tonalide 23368 N/A 0 ML A 4 b MCPB 223334 N/A 120  Time (h) Sample Location at the Pilot Plant Sample Number Fraction  (a = soluble, b = particulate) Analyte Average Sample Concentration (ng/L) Difference between maximum or minimum and the average (ng/L)  Only for replicate runs 0 ML A 4 b Gemfibrozil 0 N/A 0 ML A 4 b Dibutyl Phthalate 35790 N/A 0 ML A 4 b Naproxen 3582 N/A 0 ML A 4 b Irgasan 4260 N/A 0 ML A 4 b Estrone 218 N/A 0 ML A 4 b EE2 0 N/A 0 ML B 5 a Ibuprofen 0 N/A 0 ML B 5 a Nonylphenol 835 N/A 0 ML B 5 a Caffeine 39 N/A 0 ML B 5 a Tonalide 483 N/A 0 ML B 5 a MCPB 2661 N/A 0 ML B 5 a Gemfibrozil 0 N/A 0 ML B 5 a Dibutyl Phthalate 1467 N/A 0 ML B 5 a Naproxen 50 N/A 0 ML B 5 a Irgasan 79 N/A 0 ML B 5 a Estrone 20 N/A 0 ML B 5 a EE2 0 N/A 0 ML B 5 b Ibuprofen 2748 N/A 0 ML B 5 b Nonylphenol 14828 N/A 0 ML B 5 b Caffeine 0 N/A 0 ML B 5 b Tonalide 19718 N/A 0 ML B 5 b MCPB 226290 N/A 0 ML B 5 b Gemfibrozil 0 N/A 0 ML B 5 b Dibutyl Phthalate 15674 N/A 0 ML B 5 b Naproxen 2042 N/A 0 ML B 5 b Irgasan 6918 N/A 0 ML B 5 b Estrone 0 N/A 0 ML B 5 b EE2 0 N/A 3 EFF A 6-7 a Ibuprofen 145 186 3 EFF A 6-7 a Nonylphenol 230 496 3 EFF A 6-7 a Caffeine 722 1160 3 EFF A 6-7 a Tonalide 222 109 121  Time (h) Sample Location at the Pilot Plant Sample Number Fraction  (a = soluble, b = particulate) Analyte Average Sample Concentration (ng/L) Difference between maximum or minimum and the average (ng/L)  Only for replicate runs 3 EFF A 6-7 a MCPB 1507 1543 3 EFF A 6-7 a Gemfibrozil 421 265 3 EFF A 6-7 a Dibutyl Phthalate 11142 12630 3 EFF A 6-7 a Naproxen 5164 3715 3 EFF A 6-7 a Irgasan 0 0 3 EFF A 6-7 a Estrone 0 0 3 EFF A 6-7 a EE2 72 1 3 EFF B 9-10 a Ibuprofen 0 0 3 EFF B 9-10 a Nonylphenol 414 2121 3 EFF B 9-10 a Caffeine 0 0 3 EFF B 9-10 a Tonalide 237 44 3 EFF B 9-10 a MCPB 850 1051 3 EFF B 9-10 a Gemfibrozil 71 82 3 EFF B 9-10 a Dibutyl Phthalate 12235 24302 3 EFF B 9-10 a Naproxen 218 258 3 EFF B 9-10 a Irgasan 0 0 3 EFF B 9-10 a Estrone 0 0 3 EFF B 9-10 a EE2 55 692 3 ML A 12-13 a Ibuprofen 415 3210 3 ML A 12-13 a Nonylphenol 1738 2773 3 ML A 12-13 a Caffeine 0 0 3 ML A 12-13 a Tonalide 1412 4112 3 ML A 12-13 a MCPB 2220 12690 3 ML A 12-13 a Gemfibrozil 609 2679 3 ML A 12-13 a Dibutyl Phthalate 1068 405 3 ML A 12-13 a Naproxen 5972 30973 3 ML A 12-13 a Irgasan 97 491 3 ML A 12-13 a Estrone 38 42 3 ML A 12-13 a EE2 175 345 3 ML A 12-13 b Ibuprofen 4726 11715 3 ML A 12-13 b Nonylphenol 80833 290701 3 ML A 12-13 b Caffeine 0 0 122  Time (h) Sample Location at the Pilot Plant Sample Number Fraction  (a = soluble, b = particulate) Analyte Average Sample Concentration (ng/L) Difference between maximum or minimum and the average (ng/L)  Only for replicate runs 3 ML A 12-13 b Tonalide 77210 86807 3 ML A 12-13 b MCPB 241912 215265 3 ML A 12-13 b Gemfibrozil 7732 8259 3 ML A 12-13 b Dibutyl Phthalate 18557 12134 3 ML A 12-13 b Naproxen 18634 69959 3 ML A 12-13 b Irgasan 20378 37102 3 ML A 12-13 b Estrone 280 712 3 ML A 12-13 b EE2 10546 9606 3 ML B 15-16 a Ibuprofen 111 619 3 ML B 15-16 a Nonylphenol 714 1261 3 ML B 15-16 a Caffeine 89 674 3 ML B 15-16 a Tonalide 475 238 3 ML B 15-16 a MCPB 3451 11735 3 ML B 15-16 a Gemfibrozil 178 745 3 ML B 15-16 a Dibutyl Phthalate 1022 0 3 ML B 15-16 a Naproxen 605 3255 3 ML B 15-16 a Irgasan 59 299 3 ML B 15-16 a Estrone 11 41 3 ML B 15-16 a EE2 0 0 3 ML B 15-16 b Ibuprofen 4512 12833 3 ML B 15-16 b Nonylphenol 14500 48054 3 ML B 15-16 b Caffeine 1078 661 3 ML B 15-16 b Tonalide 38946 25590 3 ML B 15-16 b MCPB 218541 103846 3 ML B 15-16 b Gemfibrozil 0 0 3 ML B 15-16 b Dibutyl Phthalate 22790 49528 3 ML B 15-16 b Naproxen 5120 30774 3 ML B 15-16 b Irgasan 6603 10432 3 ML B 15-16 b Estrone 0 0 3 ML B 15-16 b EE2 2958 7674 12 EFF A 18 a Ibuprofen 57 N/A 12 EFF A 18 a Nonylphenol 104 N/A 123  Time (h) Sample Location at the Pilot Plant Sample Number Fraction  (a = soluble, b = particulate) Analyte Average Sample Concentration (ng/L) Difference between maximum or minimum and the average (ng/L)  Only for replicate runs 12 EFF A 18 a Caffeine 0 N/A 12 EFF A 18 a Tonalide 148 N/A 12 EFF A 18 a MCPB 1679 N/A 12 EFF A 18 a Gemfibrozil 28 N/A 12 EFF A 18 a Dibutyl Phthalate 1103 N/A 12 EFF A 18 a Naproxen 632 N/A 12 EFF A 18 a Irgasan 7 N/A 12 EFF A 18 a Estrone 0 N/A 12 EFF A 18 a EE2 114 N/A 12 EFF B 19 a Ibuprofen 31 N/A 12 EFF B 19 a Nonylphenol 352 N/A 12 EFF B 19 a Caffeine 0 N/A 12 EFF B 19 a Tonalide 241 N/A 12 EFF B 19 a MCPB 1431 N/A 12 EFF B 19 a Gemfibrozil 0 N/A 12 EFF B 19 a Dibutyl Phthalate 3926 N/A 12 EFF B 19 a Naproxen 91 N/A 12 EFF B 19 a Irgasan 0 N/A 12 EFF B 19 a Estrone 0 N/A 12 EFF B 19 a EE2 268 N/A 12 ML A 20 a Ibuprofen 70 N/A 12 ML A 20 a Nonylphenol 2280 N/A 12 ML A 20 a Caffeine 0 N/A 12 ML A 20 a Tonalide 1606 N/A 12 ML A 20 a MCPB 2471 N/A 12 ML A 20 a Gemfibrozil 237 N/A 12 ML A 20 a Dibutyl Phthalate 1234 N/A 12 ML A 20 a Naproxen 945 N/A 12 ML A 20 a Irgasan 125 N/A 12 ML A 20 a Estrone 33 N/A 12 ML A 20 a EE2 233 N/A 12 ML A 20 b Ibuprofen 4994 N/A 124  Time (h) Sample Location at the Pilot Plant Sample Number Fraction  (a = soluble, b = particulate) Analyte Average Sample Concentration (ng/L) Difference between maximum or minimum and the average (ng/L)  Only for replicate runs 12 ML A 20 b Nonylphenol 174118 N/A 12 ML A 20 b Caffeine 1556 N/A 12 ML A 20 b Tonalide 201982 N/A 12 ML A 20 b MCPB 265356 N/A 12 ML A 20 b Gemfibrozil 0 N/A 12 ML A 20 b Dibutyl Phthalate 12158 N/A 12 ML A 20 b Naproxen 10424 N/A 12 ML A 20 b Irgasan 46782 N/A 12 ML A 20 b Estrone 338 N/A 12 ML A 20 b EE2 18594 N/A 12 ML B 21 a Ibuprofen 42 N/A 12 ML B 21 a Nonylphenol 1647 N/A 12 ML B 21 a Caffeine 0 N/A 12 ML B 21 a Tonalide 1313 N/A 12 ML B 21 a MCPB 1921 N/A 12 ML B 21 a Gemfibrozil 181 N/A 12 ML B 21 a Dibutyl Phthalate 1165 N/A 12 ML B 21 a Naproxen 76 N/A 12 ML B 21 a Irgasan 83 N/A 12 ML B 21 a Estrone 12 N/A 12 ML B 21 a EE2 742 N/A 12 ML B 21 b Ibuprofen 3950 N/A 12 ML B 21 b Nonylphenol 61746 N/A 12 ML B 21 b Caffeine 1274 N/A 12 ML B 21 b Tonalide 68576 N/A 12 ML B 21 b MCPB 282454 N/A 12 ML B 21 b Gemfibrozil 3698 N/A 12 ML B 21 b Dibutyl Phthalate 15434 N/A 12 ML B 21 b Naproxen 3472 N/A 12 ML B 21 b Irgasan 19572 N/A 12 ML B 21 b Estrone 280 N/A 12 ML B 21 b EE2 10678 N/A 125  Time (h) Sample Location at the Pilot Plant Sample Number Fraction  (a = soluble, b = particulate) Analyte Average Sample Concentration (ng/L) Difference between maximum or minimum and the average (ng/L)  Only for replicate runs 24 INF 22-23 a Ibuprofen 16992 5634 24 INF 22-23 a Nonylphenol 2444 6579 24 INF 22-23 a Caffeine 23653 6161 24 INF 22-23 a Tonalide 757 968 24 INF 22-23 a MCPB 7704 5822 24 INF 22-23 a Gemfibrozil 0 0 24 INF 22-23 a Dibutyl Phthalate 11833 20514 24 INF 22-23 a Naproxen 677 86 24 INF 22-23 a Irgasan 944 4545 24 INF 22-23 a Estrone 21 44 24 INF 22-23 a EE2 0 0 24 INF 22-23 b Ibuprofen 0 0 24 INF 22-23 b Nonylphenol 267 12388 24 INF 22-23 b Caffeine 106 6264 24 INF 22-23 b Tonalide 53 2452 24 INF 22-23 b MCPB 3375 12033 24 INF 22-23 b Gemfibrozil 0 0 24 INF 22-23 b Dibutyl Phthalate 683 7115 24 INF 22-23 b Naproxen 0 0 24 INF 22-23 b Irgasan 49 2312 24 INF 22-23 b Estrone 1 318 24 INF 22-23 b EE2 0 0 24 EFF A 25-26 a Ibuprofen 58 271 24 EFF A 25-26 a Nonylphenol 112 256 24 EFF A 25-26 a Caffeine 121 461 24 EFF A 25-26 a Tonalide 261 125 24 EFF A 25-26 a MCPB 1444 974 24 EFF A 25-26 a Gemfibrozil 40 64 24 EFF A 25-26 a Dibutyl Phthalate 859 1147 24 EFF A 25-26 a Naproxen 129 685 24 EFF A 25-26 a Irgasan 8 1 24 EFF A 25-26 a Estrone 0 0 126  Time (h) Sample Location at the Pilot Plant Sample Number Fraction  (a = soluble, b = particulate) Analyte Average Sample Concentration (ng/L) Difference between maximum or minimum and the average (ng/L)  Only for replicate runs 24 EFF A 25-26 a EE2 40 16 24 EFF B 28-29 a Ibuprofen 0 0 24 EFF B 28-29 a Nonylphenol 190 378 24 EFF B 28-29 a Caffeine 278 2796 24 EFF B 28-29 a Tonalide 248 227 24 EFF B 28-29 a MCPB 1207 1365 24 EFF B 28-29 a Gemfibrozil 0 0 24 EFF B 28-29 a Dibutyl Phthalate 1365 4685 24 EFF B 28-29 a Naproxen 13 167 24 EFF B 28-29 a Irgasan 4 4 24 EFF B 28-29 a Estrone 0 0 24 EFF B 28-29 a EE2 62 107 24 ML A 31-32 a Ibuprofen 34 426 24 ML A 31-32 a Nonylphenol 1845 4699 24 ML A 31-32 a Caffeine 0 0 24 ML A 31-32 a Tonalide 2622 1097 24 ML A 31-32 a MCPB 2877 6870 24 ML A 31-32 a Gemfibrozil 0 0 24 ML A 31-32 a Dibutyl Phthalate 1816 0 24 ML A 31-32 a Naproxen 109 33 24 ML A 31-32 a Irgasan 51 227 24 ML A 31-32 a Estrone 31 167 24 ML A 31-32 a EE2 349 548 24 ML A 31-32 b Ibuprofen 3786 8513 24 ML A 31-32 b Nonylphenol 28425 133400 24 ML A 31-32 b Caffeine 1067 1156 24 ML A 31-32 b Tonalide 80485 16302 24 ML A 31-32 b MCPB 223747 168367 24 ML A 31-32 b Gemfibrozil 2010 25539 24 ML A 31-32 b Dibutyl Phthalate 13681 40265 24 ML A 31-32 b Naproxen 3535 15819 24 ML A 31-32 b Irgasan 8774 16162 127  Time (h) Sample Location at the Pilot Plant Sample Number Fraction  (a = soluble, b = particulate) Analyte Average Sample Concentration (ng/L) Difference between maximum or minimum and the average (ng/L)  Only for replicate runs 24 ML A 31-32 b Estrone 279 89 24 ML A 31-32 b EE2 5207 9059 24 ML B 34-35 a Ibuprofen 218 433 24 ML B 34-35 a Nonylphenol 2082 1731 24 ML B 34-35 a Caffeine 77 2 24 ML B 34-35 a Tonalide 1639 461 24 ML B 34-35 a MCPB 4264 7396 24 ML B 34-35 a Gemfibrozil 459 975 24 ML B 34-35 a Dibutyl Phthalate 1101 1275 24 ML B 34-35 a Naproxen 325 192 24 ML B 34-35 a Irgasan 113 352 24 ML B 34-35 a Estrone 18 22 24 ML B 34-35 a EE2 472 323 24 ML B 34-35 b Ibuprofen 8389 13913 24 ML B 34-35 b Nonylphenol 112882 163831 24 ML B 34-35 b Caffeine 1820 23125 24 ML B 34-35 b Tonalide 85331 95663 24 ML B 34-35 b MCPB 291865 281273 24 ML B 34-35 b Gemfibrozil 4268 7776 24 ML B 34-35 b Dibutyl Phthalate 17622 41574 24 ML B 34-35 b Naproxen 6316 22591 24 ML B 34-35 b Irgasan 23754 33950 24 ML B 34-35 b Estrone 545 343 24 ML B 34-35 b EE2 11204 24091 36 EFF A 37 a Ibuprofen 52 N/A 36 EFF A 37 a Nonylphenol 126 N/A 36 EFF A 37 a Caffeine 117 N/A 36 EFF A 37 a Tonalide 388 N/A 36 EFF A 37 a MCPB 1484 N/A 36 EFF A 37 a Gemfibrozil 0 N/A 36 EFF A 37 a Dibutyl Phthalate 1720 N/A 36 EFF A 37 a Naproxen 103 N/A 128  Time (h) Sample Location at the Pilot Plant Sample Number Fraction  (a = soluble, b = particulate) Analyte Average Sample Concentration (ng/L) Difference between maximum or minimum and the average (ng/L)  Only for replicate runs 36 EFF A 37 a Irgasan 6 N/A 36 EFF A 37 a Estrone 0 N/A 36 EFF A 37 a EE2 31 N/A 36 EFF B 38 a Ibuprofen 0 N/A 36 EFF B 38 a Nonylphenol 79 N/A 36 EFF B 38 a Caffeine 69 N/A 36 EFF B 38 a Tonalide 279 N/A 36 EFF B 38 a MCPB 920 N/A 36 EFF B 38 a Gemfibrozil 0 N/A 36 EFF B 38 a Dibutyl Phthalate 820 N/A 36 EFF B 38 a Naproxen 0 N/A 36 EFF B 38 a Irgasan 5 N/A 36 EFF B 38 a Estrone 0 N/A 36 EFF B 38 a EE2 33 N/A 36 ML A 39 a Ibuprofen 52 N/A 36 ML A 39 a Nonylphenol 1250 N/A 36 ML A 39 a Caffeine 0 N/A 36 ML A 39 a Tonalide 1954 N/A 36 ML A 39 a MCPB 2534 N/A 36 ML A 39 a Gemfibrozil 0 N/A 36 ML A 39 a Dibutyl Phthalate 1075 N/A 36 ML A 39 a Naproxen 51 N/A 36 ML A 39 a Irgasan 53 N/A 36 ML A 39 a Estrone 38 N/A 36 ML A 39 a EE2 254 N/A 36 ML A 39 b Ibuprofen 11252 N/A 36 ML A 39 b Nonylphenol 53170 N/A 36 ML A 39 b Caffeine 3902 N/A 36 ML A 39 b Tonalide 85920 N/A 36 ML A 39 b MCPB 287740 N/A 36 ML A 39 b Gemfibrozil 4248 N/A 36 ML A 39 b Dibutyl Phthalate 24986 N/A 129  Time (h) Sample Location at the Pilot Plant Sample Number Fraction  (a = soluble, b = particulate) Analyte Average Sample Concentration (ng/L) Difference between maximum or minimum and the average (ng/L)  Only for replicate runs 36 ML A 39 b Naproxen 6096 N/A 36 ML A 39 b Irgasan 11614 N/A 36 ML A 39 b Estrone 766 N/A 36 ML A 39 b EE2 5894 N/A 36 ML B 40 a Ibuprofen 94 N/A 36 ML B 40 a Nonylphenol 857 N/A 36 ML B 40 a Caffeine 58 N/A 36 ML B 40 a Tonalide 2123 N/A 36 ML B 40 a MCPB 4134 N/A 36 ML B 40 a Gemfibrozil 158 N/A 36 ML B 40 a Dibutyl Phthalate 1230 N/A 36 ML B 40 a Naproxen 20 N/A 36 ML B 40 a Irgasan 50 N/A 36 ML B 40 a Estrone 21 N/A 36 ML B 40 a EE2 239 N/A 36 ML B 40 b Ibuprofen 0 N/A 36 ML B 40 b Nonylphenol 0 N/A 36 ML B 40 b Caffeine 0 N/A 36 ML B 40 b Tonalide 0 N/A 36 ML B 40 b MCPB 0 N/A 36 ML B 40 b Gemfibrozil 0 N/A 36 ML B 40 b Dibutyl Phthalate 0 N/A 36 ML B 40 b Naproxen 0 N/A 36 ML B 40 b Irgasan 0 N/A 36 ML B 40 b Estrone 0 N/A 36 ML B 40 b EE2 0 N/A 48 INF 41-42 a Ibuprofen 15469 32078 48 INF 41-42 a Nonylphenol 2078 327 48 INF 41-42 a Caffeine 26122 4151 48 INF 41-42 a Tonalide 1121 396 48 INF 41-42 a MCPB 5579 14153 48 INF 41-42 a Gemfibrozil 0 0 130  Time (h) Sample Location at the Pilot Plant Sample Number Fraction  (a = soluble, b = particulate) Analyte Average Sample Concentration (ng/L) Difference between maximum or minimum and the average (ng/L)  Only for replicate runs 48 INF 41-42 a Dibutyl Phthalate 4335 10266 48 INF 41-42 a Naproxen 405 356 48 INF 41-42 a Irgasan 552 1123 48 INF 41-42 a Estrone 12 41 48 INF 41-42 a EE2 0 0 48 INF 41-42 b Ibuprofen 441 32 48 INF 41-42 b Nonylphenol 5877 9331 48 INF 41-42 b Caffeine 489 112 48 INF 41-42 b Tonalide 2797 15153 48 INF 41-42 b MCPB 8488 10194 48 INF 41-42 b Gemfibrozil 0 0 48 INF 41-42 b Dibutyl Phthalate 3450 10256 48 INF 41-42 b Naproxen 0 0 48 INF 41-42 b Irgasan 4243 4131 48 INF 41-42 b Estrone 7 87 48 INF 41-42 b EE2 0 0 48 EFF A 44-45 a Ibuprofen 0 0 48 EFF A 44-45 a Nonylphenol 144 101 48 EFF A 44-45 a Caffeine 60 22 48 EFF A 44-45 a Tonalide 459 140 48 EFF A 44-45 a MCPB 385 398 48 EFF A 44-45 a Gemfibrozil 0 0 48 EFF A 44-45 a Dibutyl Phthalate 991 352 48 EFF A 44-45 a Naproxen 0 0 48 EFF A 44-45 a Irgasan 0 0 48 EFF A 44-45 a Estrone 0 0 48 EFF A 44-45 a EE2 0 0 48 EFF B 47-48 a Ibuprofen 0 0 48 EFF B 47-48 a Nonylphenol 126 294 48 EFF B 47-48 a Caffeine 68 203 48 EFF B 47-48 a Tonalide 374 146 48 EFF B 47-48 a MCPB 481 315 131  Time (h) Sample Location at the Pilot Plant Sample Number Fraction  (a = soluble, b = particulate) Analyte Average Sample Concentration (ng/L) Difference between maximum or minimum and the average (ng/L)  Only for replicate runs 48 EFF B 47-48 a Gemfibrozil 0 0 48 EFF B 47-48 a Dibutyl Phthalate 964 1376 48 EFF B 47-48 a Naproxen 10 121 48 EFF B 47-48 a Irgasan 8 4 48 EFF B 47-48 a Estrone 0 0 48 EFF B 47-48 a EE2 0 0 48 ML A 50-51 a Ibuprofen 37 466 48 ML A 50-51 a Nonylphenol 1803 6489 48 ML A 50-51 a Caffeine 40 2 48 ML A 50-51 a Tonalide 2595 1975 48 ML A 50-51 a MCPB 2142 2744 48 ML A 50-51 a Gemfibrozil 59 753 48 ML A 50-51 a Dibutyl Phthalate 1532 318 48 ML A 50-51 a Naproxen 19 244 48 ML A 50-51 a Irgasan 88 180 48 ML A 50-51 a Estrone 52 37 48 ML A 50-51 a EE2 120 57 48 ML A 50-51 b Ibuprofen 0 0 48 ML A 50-51 b Nonylphenol 12469 105777 48 ML A 50-51 b Caffeine 1398 7649 48 ML A 50-51 b Tonalide 65003 136729 48 ML A 50-51 b MCPB 120141 1360419 48 ML A 50-51 b Gemfibrozil 0 0 48 ML A 50-51 b Dibutyl Phthalate 15493 26949 48 ML A 50-51 b Naproxen 747 9491 48 ML A 50-51 b Irgasan 2167 27534 48 ML A 50-51 b Estrone 0 0 48 ML A 50-51 b EE2 2143 4200 48 ML B 53-54 a Ibuprofen 0 0 48 ML B 53-54 a Nonylphenol 743 1667 48 ML B 53-54 a Caffeine 37 31 48 ML B 53-54 a Tonalide 1051 672 132  Time (h) Sample Location at the Pilot Plant Sample Number Fraction  (a = soluble, b = particulate) Analyte Average Sample Concentration (ng/L) Difference between maximum or minimum and the average (ng/L)  Only for replicate runs 48 ML B 53-54 a MCPB 1347 12061 48 ML B 53-54 a Gemfibrozil 136 1722 48 ML B 53-54 a Dibutyl Phthalate 1503 6148 48 ML B 53-54 a Naproxen 0 0 48 ML B 53-54 a Irgasan 49 189 48 ML B 53-54 a Estrone 20 103 48 ML B 53-54 a EE2 52 658 48 ML B 53-54 b Ibuprofen 57837 689847 48 ML B 53-54 b Nonylphenol 37865 170476 48 ML B 53-54 b Caffeine 15079 177033 48 ML B 53-54 b Tonalide 67418 120580 48 ML B 53-54 b MCPB 357860 1560271 48 ML B 53-54 b Gemfibrozil 6837 24713 48 ML B 53-54 b Dibutyl Phthalate 29827 180006 48 ML B 53-54 b Naproxen 8111 78993 48 ML B 53-54 b Irgasan 9414 38474 48 ML B 53-54 b Estrone 1419 18030 48 ML B 53-54 b EE2 6762 47114    133  Table B - 3: Raw total concentration data for Stage 2 experiment Time (h) Sample Location at the Pilot Plant Sample Number Analyte Average Sample Concentration (ng/L) Difference between maximum or minimum and the average (ng/L)  Only for replicate runs 0 INF 1 Ibuprofen 19592 N/A 0 INF 1 Nonylphenol 6327 N/A 0 INF 1 Caffeine 22989 N/A 0 INF 1 Tonalide 1732 N/A 0 INF 1 MCPB 12843 N/A 0 INF 1 Gemfibrozil 0 N/A 0 INF 1 Dibutyl Phthalate 13101 N/A 0 INF 1 Naproxen 915 N/A 0 INF 1 Irgasan 644 N/A 0 INF 1 Estrone 24 N/A 0 INF 1 EE2 0 N/A 0 EFF A 2 Ibuprofen 44 N/A 0 EFF A 2 Nonylphenol 477 N/A 0 EFF A 2 Caffeine 2684 N/A 0 EFF A 2 Tonalide 338 N/A 0 EFF A 2 MCPB 1054 N/A 0 EFF A 2 Gemfibrozil 0 N/A 0 EFF A 2 Dibutyl Phthalate 9709 N/A 0 EFF A 2 Naproxen 174 N/A 0 EFF A 2 Irgasan 15 N/A 0 EFF A 2 Estrone 15 N/A 0 EFF A 2 EE2 82 N/A 0 EFF B 3 Ibuprofen 0 N/A 0 EFF B 3 Nonylphenol 324 N/A 0 EFF B 3 Caffeine 745 N/A 0 EFF B 3 Tonalide 299 N/A 0 EFF B 3 MCPB 908 N/A 0 EFF B 3 Gemfibrozil 0 N/A 0 EFF B 3 Dibutyl Phthalate 7731 N/A 0 EFF B 3 Naproxen 64 N/A 0 EFF B 3 Irgasan 9 N/A 0 EFF B 3 Estrone 0 N/A 0 EFF B 3 EE2 0 N/A 0 ML A 4 Ibuprofen 4138 N/A 134  Time (h) Sample Location at the Pilot Plant Sample Number Analyte Average Sample Concentration (ng/L) Difference between maximum or minimum and the average (ng/L)  Only for replicate runs 0 ML A 4 Nonylphenol 24392 N/A 0 ML A 4 Caffeine 0 N/A 0 ML A 4 Tonalide 23606 N/A 0 ML A 4 MCPB 224838 N/A 0 ML A 4 Gemfibrozil 0 N/A 0 ML A 4 Dibutyl Phthalate 37274 N/A 0 ML A 4 Naproxen 3682 N/A 0 ML A 4 Irgasan 4290 N/A 0 ML A 4 Estrone 238 N/A 0 ML A 4 EE2 0 N/A 0 ML B 5 Ibuprofen 2748 N/A 0 ML B 5 Nonylphenol 15663 N/A 0 ML B 5 Caffeine 39 N/A 0 ML B 5 Tonalide 20201 N/A 0 ML B 5 MCPB 228951 N/A 0 ML B 5 Gemfibrozil 0 N/A 0 ML B 5 Dibutyl Phthalate 17141 N/A 0 ML B 5 Naproxen 2092 N/A 0 ML B 5 Irgasan 6997 N/A 0 ML B 5 Estrone 20 N/A 0 ML B 5 EE2 0 N/A 3 EFF A 6-7 Ibuprofen 145 15 3 EFF A 6-7 Nonylphenol 230 39 3 EFF A 6-7 Caffeine 722 91 3 EFF A 6-7 Tonalide 222 9 3 EFF A 6-7 MCPB 1507 121 3 EFF A 6-7 Gemfibrozil 421 21 3 EFF A 6-7 Dibutyl Phthalate 11142 994 3 EFF A 6-7 Naproxen 5164 292 3 EFF A 6-7 Irgasan 0 0 3 EFF A 6-7 Estrone 0 0 3 EFF A 6-7 EE2 72 0 3 EFF B 9-10 Ibuprofen 0 0 3 EFF B 9-10 Nonylphenol 414 167 3 EFF B 9-10 Caffeine 0 0 135  Time (h) Sample Location at the Pilot Plant Sample Number Analyte Average Sample Concentration (ng/L) Difference between maximum or minimum and the average (ng/L)  Only for replicate runs 3 EFF B 9-10 Tonalide 237 3 3 EFF B 9-10 MCPB 850 83 3 EFF B 9-10 Gemfibrozil 71 6 3 EFF B 9-10 Dibutyl Phthalate 12235 1913 3 EFF B 9-10 Naproxen 218 20 3 EFF B 9-10 Irgasan 0 0 3 EFF B 9-10 Estrone 0 0 3 EFF B 9-10 EE2 55 55 3 ML A 12-13 Ibuprofen 230 1175 3 ML A 12-13 Nonylphenol 5141 23097 3 ML A 12-13 Caffeine 82571 0 3 ML A 12-13 Tonalide 0 7156 3 ML A 12-13 MCPB 78622 17941 3 ML A 12-13 Gemfibrozil 244132 861 3 ML A 12-13 Dibutyl Phthalate 8341 987 3 ML A 12-13 Naproxen 19625 7944 3 ML A 12-13 Irgasan 24606 2959 3 ML A 12-13 Estrone 20475 59 3 ML A 12-13 EE2 318 783 3 ML B 15-16 Ibuprofen 10546 1059 3 ML B 15-16 Nonylphenol 4623 3881 3 ML B 15-16 Caffeine 15214 105 3 ML B 15-16 Tonalide 1167 2033 3 ML B 15-16 MCPB 39421 9097 3 ML B 15-16 Gemfibrozil 221992 59 3 ML B 15-16 Dibutyl Phthalate 178 3898 3 ML B 15-16 Naproxen 23812 2678 3 ML B 15-16 Irgasan 5725 845 3 ML B 15-16 Estrone 6662 3 3 ML B 15-16 EE2 11 604 12 EFF A 18 Ibuprofen 57 N/A 12 EFF A 18 Nonylphenol 104 N/A 12 EFF A 18 Caffeine 0 N/A 12 EFF A 18 Tonalide 148 N/A 12 EFF A 18 MCPB 1679 N/A 136  Time (h) Sample Location at the Pilot Plant Sample Number Analyte Average Sample Concentration (ng/L) Difference between maximum or minimum and the average (ng/L)  Only for replicate runs 12 EFF A 18 Gemfibrozil 28 N/A 12 EFF A 18 Dibutyl Phthalate 1103 N/A 12 EFF A 18 Naproxen 632 N/A 12 EFF A 18 Irgasan 7 N/A 12 EFF A 18 Estrone 0 N/A 12 EFF A 18 EE2 114 N/A 12 EFF B 19 Ibuprofen 31 N/A 12 EFF B 19 Nonylphenol 352 N/A 12 EFF B 19 Caffeine 0 N/A 12 EFF B 19 Tonalide 241 N/A 12 EFF B 19 MCPB 1431 N/A 12 EFF B 19 Gemfibrozil 0 N/A 12 EFF B 19 Dibutyl Phthalate 3926 N/A 12 EFF B 19 Naproxen 91 N/A 12 EFF B 19 Irgasan 0 N/A 12 EFF B 19 Estrone 0 N/A 12 EFF B 19 EE2 268 N/A 12 ML A 20 Ibuprofen 5064 N/A 12 ML A 20 Nonylphenol 176398 N/A 12 ML A 20 Caffeine 1556 N/A 12 ML A 20 Tonalide 203588 N/A 12 ML A 20 MCPB 267827 N/A 12 ML A 20 Gemfibrozil 237 N/A 12 ML A 20 Dibutyl Phthalate 13392 N/A 12 ML A 20 Naproxen 11369 N/A 12 ML A 20 Irgasan 46907 N/A 12 ML A 20 Estrone 371 N/A 12 ML A 20 EE2 18827 N/A 12 ML B 21 Ibuprofen 3992 N/A 12 ML B 21 Nonylphenol 63393 N/A 12 ML B 21 Caffeine 1274 N/A 12 ML B 21 Tonalide 69889 N/A 12 ML B 21 MCPB 284375 N/A 12 ML B 21 Gemfibrozil 3879 N/A 12 ML B 21 Dibutyl Phthalate 16599 N/A 137  Time (h) Sample Location at the Pilot Plant Sample Number Analyte Average Sample Concentration (ng/L) Difference between maximum or minimum and the average (ng/L)  Only for replicate runs 12 ML B 21 Naproxen 3548 N/A 12 ML B 21 Irgasan 19655 N/A 12 ML B 21 Estrone 292 N/A 12 ML B 21 EE2 11420 N/A 24 INF 22-23 Ibuprofen 16992 443 24 INF 22-23 Nonylphenol 2711 1493 24 INF 22-23 Caffeine 23759 978 24 INF 22-23 Tonalide 809 269 24 INF 22-23 MCPB 11079 1405 24 INF 22-23 Gemfibrozil 0 0 24 INF 22-23 Dibutyl Phthalate 12515 2175 24 INF 22-23 Naproxen 677 7 24 INF 22-23 Irgasan 994 540 24 INF 22-23 Estrone 22 29 24 INF 22-23 EE2 0 0 24 EFF A 25-26 Ibuprofen 58 21 24 EFF A 25-26 Nonylphenol 112 20 24 EFF A 25-26 Caffeine 121 36 24 EFF A 25-26 Tonalide 261 10 24 EFF A 25-26 MCPB 1444 77 24 EFF A 25-26 Gemfibrozil 40 5 24 EFF A 25-26 Dibutyl Phthalate 859 90 24 EFF A 25-26 Naproxen 129 54 24 EFF A 25-26 Irgasan 8 0 24 EFF A 25-26 Estrone 0 0 24 EFF A 25-26 EE2 40 1 24 EFF B 28-29 Ibuprofen 0 0 24 EFF B 28-29 Nonylphenol 190 30 24 EFF B 28-29 Caffeine 278 220 24 EFF B 28-29 Tonalide 248 18 24 EFF B 28-29 MCPB 1207 107 24 EFF B 28-29 Gemfibrozil 0 0 24 EFF B 28-29 Dibutyl Phthalate 1365 369 24 EFF B 28-29 Naproxen 13 13 24 EFF B 28-29 Irgasan 4 0 138  Time (h) Sample Location at the Pilot Plant Sample Number Analyte Average Sample Concentration (ng/L) Difference between maximum or minimum and the average (ng/L)  Only for replicate runs 24 EFF B 28-29 Estrone 0 0 24 EFF B 28-29 EE2 62 8 24 ML A 31-32 Ibuprofen 3820 704 24 ML A 31-32 Nonylphenol 30270 10869 24 ML A 31-32 Caffeine 1067 91 24 ML A 31-32 Tonalide 83107 1369 24 ML A 31-32 MCPB 226624 13792 24 ML A 31-32 Gemfibrozil 2010 2010 24 ML A 31-32 Dibutyl Phthalate 15497 3172 24 ML A 31-32 Naproxen 3644 1248 24 ML A 31-32 Irgasan 8825 1290 24 ML A 31-32 Estrone 310 20 24 ML A 31-32 EE2 5556 756 24 ML B 34-35 Ibuprofen 8607 1129 24 ML B 34-35 Nonylphenol 114964 13030 24 ML B 34-35 Caffeine 1897 1820 24 ML B 34-35 Tonalide 86970 7565 24 ML B 34-35 MCPB 296129 22719 24 ML B 34-35 Gemfibrozil 4727 689 24 ML B 34-35 Dibutyl Phthalate 18723 3372 24 ML B 34-35 Naproxen 6641 1793 24 ML B 34-35 Irgasan 23867 2700 24 ML B 34-35 Estrone 563 29 24 ML B 34-35 EE2 11676 1921 36 EFF A 37 Ibuprofen 52 N/A 36 EFF A 37 Nonylphenol 126 N/A 36 EFF A 37 Caffeine 117 N/A 36 EFF A 37 Tonalide 388 N/A 36 EFF A 37 MCPB 1484 N/A 36 EFF A 37 Gemfibrozil 0 N/A 36 EFF A 37 Dibutyl Phthalate 1720 N/A 36 EFF A 37 Naproxen 103 N/A 36 EFF A 37 Irgasan 6 N/A 36 EFF A 37 Estrone 0 N/A 36 EFF A 37 EE2 31 N/A 139  Time (h) Sample Location at the Pilot Plant Sample Number Analyte Average Sample Concentration (ng/L) Difference between maximum or minimum and the average (ng/L)  Only for replicate runs 36 EFF B 38 Ibuprofen 0 N/A 36 EFF B 38 Nonylphenol 79 N/A 36 EFF B 38 Caffeine 69 N/A 36 EFF B 38 Tonalide 279 N/A 36 EFF B 38 MCPB 920 N/A 36 EFF B 38 Gemfibrozil 0 N/A 36 EFF B 38 Dibutyl Phthalate 820 N/A 36 EFF B 38 Naproxen 0 N/A 36 EFF B 38 Irgasan 5 N/A 36 EFF B 38 Estrone 0 N/A 36 EFF B 38 EE2 33 N/A 36 ML A 39 Ibuprofen 11304 N/A 36 ML A 39 Nonylphenol 54420 N/A 36 ML A 39 Caffeine 3902 N/A 36 ML A 39 Tonalide 87874 N/A 36 ML A 39 MCPB 290274 N/A 36 ML A 39 Gemfibrozil 4248 N/A 36 ML A 39 Dibutyl Phthalate 26061 N/A 36 ML A 39 Naproxen 6147 N/A 36 ML A 39 Irgasan 11667 N/A 36 ML A 39 Estrone 804 N/A 36 ML A 39 EE2 6148 N/A 36 ML B 40 Ibuprofen 94 N/A 36 ML B 40 Nonylphenol 857 N/A 36 ML B 40 Caffeine 58 N/A 36 ML B 40 Tonalide 2123 N/A 36 ML B 40 MCPB 4134 N/A 36 ML B 40 Gemfibrozil 158 N/A 36 ML B 40 Dibutyl Phthalate 1230 N/A 36 ML B 40 Naproxen 20 N/A 36 ML B 40 Irgasan 50 N/A 36 ML B 40 Estrone 21 N/A 36 ML B 40 EE2 239 N/A 48 INF 41-42 Ibuprofen 15909 2527 48 INF 41-42 Nonylphenol 7954 760 140  Time (h) Sample Location at the Pilot Plant Sample Number Analyte Average Sample Concentration (ng/L) Difference between maximum or minimum and the average (ng/L)  Only for replicate runs 48 INF 41-42 Caffeine 26611 336 48 INF 41-42 Tonalide 3918 1224 48 INF 41-42 MCPB 14067 1916 48 INF 41-42 Gemfibrozil 0 0 48 INF 41-42 Dibutyl Phthalate 7785 1615 48 INF 41-42 Naproxen 405 28 48 INF 41-42 Irgasan 4795 414 48 INF 41-42 Estrone 18 10 48 INF 41-42 EE2 0 0 48 EFF A 44-45 Ibuprofen 0 0 48 EFF A 44-45 Nonylphenol 144 8 48 EFF A 44-45 Caffeine 60 2 48 EFF A 44-45 Tonalide 459 11 48 EFF A 44-45 MCPB 385 31 48 EFF A 44-45 Gemfibrozil 0 0 48 EFF A 44-45 Dibutyl Phthalate 991 28 48 EFF A 44-45 Naproxen 0 0 48 EFF A 44-45 Irgasan 0 0 48 EFF A 44-45 Estrone 0 0 48 EFF A 44-45 EE2 0 0 48 EFF B 47-48 Ibuprofen 0 0 48 EFF B 47-48 Nonylphenol 126 23 48 EFF B 47-48 Caffeine 68 16 48 EFF B 47-48 Tonalide 374 12 48 EFF B 47-48 MCPB 481 25 48 EFF B 47-48 Gemfibrozil 0 0 48 EFF B 47-48 Dibutyl Phthalate 964 108 48 EFF B 47-48 Naproxen 10 10 48 EFF B 47-48 Irgasan 8 0 48 EFF B 47-48 Estrone 0 0 48 EFF B 47-48 EE2 0 0 48 ML A 50-51 Ibuprofen 37 37 48 ML A 50-51 Nonylphenol 14272 8836 48 ML A 50-51 Caffeine 1438 602 48 ML A 50-51 Tonalide 67598 10916 141  Time (h) Sample Location at the Pilot Plant Sample Number Analyte Average Sample Concentration (ng/L) Difference between maximum or minimum and the average (ng/L)  Only for replicate runs 48 ML A 50-51 MCPB 122283 107285 48 ML A 50-51 Gemfibrozil 59 59 48 ML A 50-51 Dibutyl Phthalate 17025 2146 48 ML A 50-51 Naproxen 766 766 48 ML A 50-51 Irgasan 2255 2181 48 ML A 50-51 Estrone 52 3 48 ML A 50-51 EE2 2263 2147 48 ML B 53-54 Ibuprofen 57837 54293 48 ML B 53-54 Nonylphenol 38608 13548 48 ML B 53-54 Caffeine 15116 13935 48 ML B 53-54 Tonalide 68469 9543 48 ML B 53-54 MCPB 359207 123747 48 ML B 53-54 Gemfibrozil 6973 2081 48 ML B 53-54 Dibutyl Phthalate 31330 14651 48 ML B 53-54 Naproxen 8111 6217 48 ML B 53-54 Irgasan 9463 3043 48 ML B 53-54 Estrone 1439 1427 48 ML B 53-54 EE2 6814 3760 0 Spike N/A Ibuprofen N/A N/A 0 Spike N/A Nonylphenol N/A N/A 0 Spike N/A Caffeine N/A N/A 0 Spike N/A Tonalide N/A N/A 0 Spike N/A Gemfibrozil N/A N/A 0 Spike N/A Dibutyl Phthalate N/A N/A 0 Spike N/A Naproxen N/A N/A 0 Spike N/A Irgasan N/A N/A 0 Spike N/A Estrone N/A N/A 0 Spike N/A EE2 N/A N/A 0 Spike N/A Ibuprofen N/A N/A 0 Spike N/A Nonylphenol N/A N/A 0 Spike N/A Caffeine N/A N/A 0 Spike N/A Tonalide N/A N/A 0 Spike N/A Gemfibrozil N/A N/A 0 Spike N/A Dibutyl Phthalate N/A N/A 0 Spike N/A Naproxen N/A N/A 142  Time (h) Sample Location at the Pilot Plant Sample Number Analyte Average Sample Concentration (ng/L) Difference between maximum or minimum and the average (ng/L)  Only for replicate runs 0 Spike N/A Irgasan N/A N/A 0 Spike N/A Estrone N/A N/A 0 Spike N/A EE2 N/A N/A   143  APPENDIX C: MASS BALANCE RESULTS FOR REMAINDER OF ANALYTES Appendix C.1: Mass Balance ? Hydrophobic Analytes The following section illustrates the mass balance results for tonalide, irgasan, EE2, and gemfibrozil discussed is Section 3.5.6.1.  144   Figure C - 1: Mass Balance for Tonalide in the A-Side (SRT = 15d)  0501001502002503003500 10 20 30 40 50 60Mass (mg) Time (h) Accumulated Mass in SystemCumulative Mass InflowCumulative Mass Outflow145    Figure C - 2: Mass Balance for Tonalide in the B-Side (SRT = 24d)   0204060801001201401601800 10 20 30 40 50 60Mass (mg) Time (h) Accumulated Mass in SystemCumulative Mass InflowCumulative Mass Outflow146   Figure C - 3: Mass Balance for Irgasan in the A-Side (SRT = 15d)  01020304050607080901000 10 20 30 40 50 60Mass (mg) Time (h) Accumulated Mass in SystemCumulative Mass InflowCumulative Mass Outflow147   Figure C - 4: Mass Balance for Irgasan in the B-Side (SRT = 24d)  01020304050607080901000 10 20 30 40 50 60Mass (mg) Time (h) Accumulated Mass in SystemCumulative Mass InflowCumulative Mass Outflow148   Figure C - 5: Mass Balance for EE2 in the A-Side (SRT = 15d)  051015202530350 10 20 30 40 50 60Mass (mg) Time (h) Accumulated Mass in SystemCumulative Mass InflowCumulative Mass Outflow149   Figure C - 6: Mass Balance for EE2 in the B-Side (SRT = 24d)   0510152025300 10 20 30 40 50 60Mass (mg) Time (h) Accumulated Mass in SystemCumulative Mass InflowCumulative Mass Outflow150   Figure C - 7: Mass Balance for Gemfibrozil in the A-Side (SRT = 15d)   05101520250 10 20 30 40 50 60Mass (mg) Time (h) Accumulated Mass in SystemCumulative Mass InflowCumulative Mass Outflow151   Figure C - 8: Mass Balance for Gemfibrozil in the B-Side (SRT = 24d) 05101520250 10 20 30 40 50 60Mass (mg) Time (h) Accumulated Mass in SystemCumulative Mass InflowCumulative Mass Outflow152  Appendix C.2: Mass Balance ? Hydrophilic and Other Analytes The following section illustrates the mass balance results for naproxen, estrone, caffeine, and ibuprofen discussed is Section 3.5.6.2.  153   Figure C - 9: Mass Balance for Naproxen in the A-Side (SRT = 15d)   0204060801001201401600 10 20 30 40 50 60Mass (mg) Time (h) Accumulated Mass in SystemCumulative Mass InflowCumulative Mass Outflow154   Figure C - 10: Mass Balance for Naproxen in the B-Side (SRT = 24d)   0204060801001201401600 10 20 30 40 50 60Mass (mg) Time (h) Accumulated Mass in SystemCumulative Mass InflowCumulative Mass Outflow155   Figure C - 11: Mass Balance for Estrone in the A-Side (SRT = 15d)   02468101214160 10 20 30 40 50 60Mass (mg) Time (h) Accumulated Mass in SystemCumulative Mass InflowCumulative Mass Outflow156   Figure C - 12: Mass Balance for Estrone in the B-Side (SRT = 24d)  02468101214160 10 20 30 40 50 60Mass (mg) Time (h) Accumulated Mass in SystemCumulative Mass InflowCumulative Mass Outflow157   Figure C - 13: Mass Balance for Caffeine in the A-Side (SRT = 15d)   01002003004005006000 10 20 30 40 50 60Mass (mg) Time (h) Accumulated Mass in SystemCumulative Mass InflowCumulative Mass Outflow158   Figure C - 14: Mass Balance for Caffeine in the B-Side (SRT = 24d)   01002003004005006000 10 20 30 40 50 60Mass (mg) Time (h) Accumulated Mass in SystemCumulative Mass InflowCumulative Mass Outflow159   Figure C - 15: Mass Balance for Ibuprofen in the A-Side (SRT = 15d)  0501001502002503003504000 10 20 30 40 50 60Mass (mg) Time (h) Accumulated Mass in SystemCumulative Mass InflowCumulative Mass Outflow160   Figure C - 16: Mass Balance for Ibuprofen in the B-Side (SRT = 24d)  0501001502002503003504000 10 20 30 40 50 60Mass (mg) Time (h) Accumulated Mass in SystemCumulative Mass InflowCumulative Mass Outflow161  APPENDIX D: SAMPLING SCHEDULE Appendix D.1 and Appendix D.2 provide the sampling schedule for the 48 hour experiment at the pilot plant during Stage 1 and Stage 2, respectively.  Appendix D.1: Sampling Schedule for Stage 1  Table D - 1: Sampling schedule for Stage 1 Date Time Location Number of Locations to Collect Sample From Total Replicate Samples Sample Number Bottle Number Monday Nov 12 9:00 AM (Spiked at this time) INF 1 0 1 A EFF 2 (A-Side & B-Side) 0 2 B 3 C Sludge 2 (A-Side & B-Side) 0 4 a 5 b 12:00 PM INF 1 3 6 D 7 8 EFF 2 (A-Side & B-Side) 6 9 E 10 11 12 F 13 14 Sludge 2 (A-Side & B-Side) 6 15 c 16 17 18 d 19 20 9:00 PM INF 1 0 21 G EFF 2 (A-Side & B-Side) 0 22 H 23 I Sludge 2 (A-Side & B-Side) 0 24 e 25 f Tuesday Nov 13 9:00 AM INF 1 3 26 J 27 28 EFF 2 (A-Side & 6 29 K 162  Date Time Location Number of Locations to Collect Sample From Total Replicate Samples Sample Number Bottle Number B-Side) 30 31 32 L 33 34 Sludge 2 (A-Side & B-Side) 6 35 g 36 37 38 h 39 40 3:00 PM INF 1 0 41 M EFF 2 (A-Side & B-Side) 0 42 N 43 O Sludge 2 (A-Side & B-Side) 0 44 i 45 j 9:00 PM INF 1 3 46 P EFF 2 (A-Side & B-Side) 6 47 Q 48 R Sludge 2 6 49 k 50 l Wednesday Nov 14 9:00 AM INF 1 3 51 S 52 53 EFF 2 (A-Side & B-Side) 6 54 T 55 56 57 U 58 59 Sludge 2 (A-Side & B-Side) 6 60 m 61 62 63 n 64 65  163  Appendix D.2: Sampling Schedule for Stage 2  Table D - 2: Sampling schedule for Stage 2 Day Time of Day Time of Experiment (h) Location Number of Locations to Collect Sample From Total Replicate Samples Bottle Number Sample Number Number of phases (N/A = 1, ? & ? = 2) Monday Jan 28 9:00 AM (Spiked at this time) 0 INF 1 0 A 1 ? and ? EFF 2 0 B 2 N/A C 3 N/A ML 2 0 D 4 ? and ? E 5 ? and ? 12:00 PM 3 EFF 2 6 F 6 N/A 7 N/A 8 N/A G 9 N/A 10 N/A 11 N/A ML 2 6 H 12 ? and ? 13 ? and ? 14 ? and ? I 15 ? and ? 16 ? and ? 17 ? and ? 9:00 PM 12 EFF 2 0 J 18 N/A K 19 N/A ML 2 0 L 20 ? and ? M 21 ? and ? Tuesday Jan 29 9:00 AM 24 INF 1 3 N 22 ? and ? 23 ? and ? 24 ? and ? EFF 2 6 O 25 N/A 26 N/A 27 N/A P 28 N/A 29 N/A 30 N/A ML 2 6 Q 31 ? and ? 164  Day Time of Day Time of Experiment (h) Location Number of Locations to Collect Sample From Total Replicate Samples Bottle Number Sample Number Number of phases (N/A = 1, ? & ? = 2) 32 ? and ? 33 ? and ? R 34 ? and ? 35 ? and ? 36 ? and ? 9:00 PM 36 EFF 2 6 S 37 N/A T 38 N/A ML 2 6 U 39 ? and ? V 40 ? and ? Wednesday Jan 30 9:00 AM 48 INF 1 3 W 41 ? and ? 42 ? and ? 43 ? and ? EFF 2 6 X 44 N/A 45 N/A 46 N/A Y 47 N/A 48 N/A 49 N/A ML 2 6 Z 50 ? and ? 51 ? and ? 52 ? and ? AA 53 ? and ? 54 ? and ? 55 ? and ?    165  Appendix D.3: Task Checklist for Stage 2 Day 1 Tasks Task 1: Collect Samples         Monday Jan. 28 Bottle Number Location Volume (mL) Time of day Task Completed? A INF 500 8:00 AM   B EFF A 500 8:00 AM   C EFF B 500 8:00 AM   D ML A 500 8:00 AM   E ML B 500 8:00 AM   BB Field Blk (tap) 500 8:00 AM   F EFF A 500 11:00 AM   G EFF B 500 11:00 AM   H ML A 500 11:00 AM   I ML B 500 11:00 AM   J EFF A 500 8:00 PM   K EFF B 500 8:00 PM   L ML A 500 8:00 PM   M ML B 500 8:00 PM        Task 2: Transport back to the lab        Monday Jan. 28 Bottle Number Location Volume (mL) Time of day Task Completed? A INF 500 11 AM - 8 PM   B EFF A 500   C EFF B 500   D ML A 500   E ML B 500   F EFF A 500   G EFF B 500   H ML A 500   I ML B 500   BB Field Blk (tap) 500           166  Task 3: Measure 5mL, centrifuge, and keep solid phase in  HACH vial       Monday Jan. 28 Bottle Number Location Sample # Time of day Task Completed? D ML A 4-? 11 AM - 8 PM   E ML B 5-?   H ML A 12-?   13-?   14-?   I ML B 15-?   16-?   17-?   N/A ML Blk 62-?        Task 4: Centrifuge at an rcf of 913*g for 10 min        Monday Jan. 28 Bottle Number Location Sample # Time of day Task Completed? A INF 1 11 AM - 8 PM        Task 5: Freeze solid phase overnight        Monday Jan. 28 Bottle Number Location Sample # Time of day Task Completed? A INF 1-? 11 AM - 8 PM   D ML A 4-?   E ML B 5-?   H ML A 12-?   13-?   14-?   I ML B 15-?   16-?   17-?   N/A ML Blk 62-?               167  Task 6: Measure 100mL in beaker         Monday Jan. 28 Bottle Number Location Sample # Time of day Task Completed? A INF 1-? 11 AM - 8 PM   B EFF A 2   C EFF B 3   F EFF A 6   7   8   G EFF B 9   10   11   BB Field Blk (tap) 56   N/A Water Blk 59        Task 7: Acidify          Monday Jan. 28 Bottle Number Location Sample # Time of day HCl / Task Completed? A INF 1-? 11 AM - 8 PM 15 B EFF A 2 10 C EFF B 3 10 F EFF A 6 10 7 10 8 10 G EFF B 9 10 10 10 11 10 BB Field Blk (tap) 56 10 N/A Water Blk 59 6             168  Task 8: Transfer to separatory funnel and add surrogate (200 uL)       Monday Jan. 28 Bottle Number Location Sample # Time of day Task Completed? A INF 1-? 11 AM - 8 PM   B EFF A 2   C EFF B 3   F EFF A 6   7   8   G EFF B 9   10   11   BB Field Blk (tap) 56   N/A Water Blk 59        Task 9: LLE          Monday Jan. 28 Bottle Number Location Sample # Time of day Task Completed? A INF 1-? 11 AM - 8 PM   B EFF A 2   C EFF B 3   F EFF A 6   7   8   G EFF B 9   10   11   BB Field Blk (tap) 56   N/A Water Blk 59               169  Task 10: Rotavap and transfer to HACH vial        Monday Jan. 28 Bottle Number Location Sample # Time of day Task Completed? A INF 1-? 11 AM - 8 PM   B EFF A 2   C EFF B 3   F EFF A 6   7   8   G EFF B 9   10   11   BB Field Blk (tap) 56   N/A Water Blk 59        Task 11: Blow down to dryness and store in freezer        Monday Jan. 28 Bottle Number Location Sample # Time of day Task Completed? A INF 1-? 11 AM - 8 PM   B EFF A 2   C EFF B 3   F EFF A 6   7   8   G EFF B 9   10   11   BB Field Blk (tap) 56   N/A Water Blk 59               170  Task 12: Clean and bake-out         Monday Jan. 28  Equipment Purpose Quantity Task Completed?  500 mL beaker Measuring 13    100 mL glass bottle Centrifugation 3    pH paper Acidification 13    125 mL sep funnels LLE Extraction 3    250 mL sep funnels 13    Funnel 13    Roundbottom flsk Rotavap 13      Day 2 Tasks Task 1: Collect Samples         Tuesday Jan. 29 Bottle Number Location Volume (mL) Time of day Task Completed? N INF 500 8:00 AM   O EFF A 500 8:00 AM   P EFF B 500 8:00 AM   Q ML A 500 8:00 AM   R ML B 500 8:00 AM   CC Field Blk (tap) 500 8:00 AM   S EFF A 500 8:00 PM   T EFF B 500 8:00 PM   U ML A 500 8:00 PM   V ML B 500 8:00 PM               171  Task 2: Transport back to the lab         Tuesday Jan. 29 Bottle Number Location Volume (mL) Time of day Task Completed? J EFF A 500 8:00 AM   K EFF B 500   L ML A 500   M ML B 500   N INF 500   O EFF A 500   P EFF B 500   Q ML A 500   R ML B 500   CC Field Blk (tap) 500        Task 3: Freeze dry solid phase overnight        Tuesday Jan. 29 Bottle Number Location Sample # Time of day Task Completed? A INF 1-? 8 AM - 8 PM   D ML A 4-?   E ML B 5-?   H ML A 12-?   13-?   14-?   I ML B 15-?   16-?   17-?   N/A ML Blk 62-?          172  Task 4: Measure 5mL, centrifuge, and keep solid phase in HACH vial       Tuesday Jan. 29 Bottle Number Location Sample # Time of day Task Completed? L ML A 20-? 8 AM - 8 PM   M ML B 21-?   Q ML A 31-?   32-?   33-?   R ML B 34-?   35-?   36-?   N/A ML Blk 63-?        Task 5: Centrifuge at an rcf of 913*g for 10 min        Monday Jan. 28 Bottle Number Location Sample # Time of day Task Completed? N INF 22 8 AM - 8 PM   23   24        Task 6: Freeze solid phase overnight         Tuesday Jan. 29 Bottle Number Location Sample # Time of day Task Completed? N INF 22-? 8 AM - 8 PM   23-?   24-?   L ML A 20-?   M ML B 21-?   Q ML A 31-?   32-?   33-?   R ML B 34-?   35-?   36-?   N/A ML Blk 63-?          173  Task 7: Measure 100mL in beaker         Tuesday Jan. 29 Bottle Number Location Sample # Time of day Task Completed? J EFF A 18 8 AM - 8 PM   K EFF B 19   N INF 22-?   23-?   24-?   O EFF A 25   26   27   P EFF B 28   29   30   CC Field Blk (tap) 57   N/A Water Blk 60        Task 8: Acidify          Tuesday Jan. 29 Bottle Number Location Sample # Time of day HCl / Task Completed? J EFF A 18 8 AM - 8 PM   K EFF B 19   N INF 22-?   23-?   24-?   O EFF A 25   26   27   P EFF B 28   29   30   CC Field Blk (tap) 57   N/A Water Blk 60          174  Task 9: Transfer to separatory funnel and add surrogate (200 uL)       Tuesday Jan. 29 Bottle Number Location Sample # Time of day Task Completed? J EFF A 18 8 AM - 8 PM   K EFF B 19   N INF 22-?   23-?   24-?   O EFF A 25   26   27   P EFF B 28   29   30   CC Field Blk (tap) 57   N/A Water Blk 60        Task 10: LLE          Tuesday Jan. 29 Bottle Number Location Sample # Time of day Task Completed? J EFF A 18 8 AM - 8 PM   K EFF B 19   N INF 22-?   23-?   24-?   O EFF A 25   26   27   P EFF B 28   29   30   CC Field Blk (tap) 57   N/A Water Blk 60                                      175  Task 11 Rotavap and transfer to HACH vial        Tuesday Jan. 29 Bottle Number Location Sample # Time of day Task Completed? J EFF A 18 8 AM - 8 PM   K EFF B 19   N INF 22-?   23-?   24-?   O EFF A 25   26   27   P EFF B 28   29   30   CC Field Blk (tap) 57   N/A Water Blk 60        Task 12: Blow down to dryness and store in freezer        Tuesday Jan. 29 Bottle Number Location Sample # Time of day Task Completed? J EFF A 18 8 AM - 8 PM   K EFF B 19   N INF 22-?   23-?   24-?   O EFF A 25   26   27   P EFF B 28   29   30   CC Field Blk (tap) 57   N/A Water Blk 60                                 176       Task 13: Clean and bake-out          Tuesday Jan. 29        Equipment Purpose Quantity Task Completed?  500 mL beaker Measuring 13    100 mL glass bottle Centrifugation 3    pH paper Acidification 13    125 mL sep funnels LLE Extraction 3    250 mL sep funnels 13    Funnel 13    Roundbottom flsk Rotavap 13     Day 3 Tasks Task 1: Collect Samples         Wednesday Jan. 30 Bottle Number Location Volume (mL) Time of day Task Completed? W INF 500 8:00 AM   X EFF A 500 8:00 AM   Y EFF B 500 8:00 AM   Z ML A 500 8:00 AM   AA ML B 500 8:00 AM   DD Field Blk (tap) 500 8:00 AM        Task 2: Transport back to the lab         Wednesday Jan. 30 Bottle Number Location Volume (mL) Time of day Task Completed? S EFF A 500 8 AM - 8 PM   T EFF B 500   U ML A 500   V ML B 500   W INF 500   X EFF A 500   Y EFF B 500   Z ML A 500   AA ML B 500   DD Field Blk (tap) 500   177       Task 3: Freeze dry solid phase overnight        Wednesday Jan. 30 Bottle Number Location Sample # Time of day Task Completed? N INF 22-? 8 AM - 8 PM   23-?   24-?   L ML A 20-?   M ML B 21-?   Q ML A 31-?   32-?   33-?   R ML B 34-?   35-?   36-?   N/A ML Blk 63-?        Task 4: Measure 5mL, centrifuge, pipette out water, and place in HACH vial      Wednesday Jan. 30 Bottle Number Location Sample # Time of day Task Completed? U ML A 39-? 8 AM - 8 PM   V ML B 40-?   Z ML A 50-?   51-?   52-?   AA ML B 53-?   54-?   55-?   N/A ML Blk 64-?        Task 5: Centrifuge at an rcf of 913*g for 10 min        Wednesday Jan. 30 Bottle Number Location Sample # Time of day Task Completed? W INF 41 8 AM - 8 PM   42   43          178  Task 6: Freeze solid phase overnight         Wednesday Jan. 30 Bottle Number Location Sample # Time of day Task Completed? W INF 41-? 8 AM - 8 PM   42-?   43-?   U ML A 39-?   V ML B 40-?   Z ML A 50-?   51-?   52-?   AA ML B 53-?   54-?   55-?   N/A ML Blk 64-?        Task 7: Measure 100mL in beaker         Wednesday Jan. 30 Bottle Number Location Sample # Time of day Task Completed? S EFF A 37 8 AM - 8 PM   T EFF B 38   W INF 41-?   42-?   43-?   X EFF A 44   45   46   Y EFF B 47   48   49   DD Field Blk (tap) 58   N/A Water Blk 61               179  Task 8: Acidify          Wednesday Jan. 30 Bottle Number Location Sample # Time of day HCl / Task Completed? S EFF A 37 8 AM - 8 PM   T EFF B 38   W INF 41-?   42-?   43-?   X EFF A 44   45   46   Y EFF B 47   48   49   DD Field Blk (tap) 58   N/A Water Blk 61        Task 9: Transfer to separatory funnel and add surrogate (200 uL)       Wednesday Jan. 30 Bottle Number Location Sample # Time of day Task Completed? S EFF A 37 8 AM - 8 PM   T EFF B 38   W INF 41-?   42-?   43-?   X EFF A 44   45   46   Y EFF B 47   48   49   DD Field Blk (tap) 58   N/A Water Blk 61               180  Task 10: LLE          Wednesday Jan. 30 Bottle Number Location Sample # Time of day Task Completed? S EFF A 37 8 AM - 8 PM   T EFF B 38   W INF 41-?   42-?   43-?   X EFF A 44   45   46   Y EFF B 47   48   49   DD Field Blk (tap) 58   N/A Water Blk 61        Task 11: Rotavap and transfer to HACH vial        Wednesday Jan. 30 Bottle Number Location Sample # Time of day Task Completed? S EFF A 37 8 AM - 8 PM   T EFF B 38   W INF 41-?   42-?   43-?   X EFF A 44   45   46   Y EFF B 47   48   49   DD Field Blk (tap) 58   N/A Water Blk 61               181  Task 12: Blow down to dryness and store in freezer        Wednesday Jan. 30 Bottle Number Location Sample # Time of day Task Completed? S EFF A 37 8 AM - 8 PM   T EFF B 38   W INF 41-?   42-?   43-?   X EFF A 44   45   46   Y EFF B 47   48   49   DD Field Blk (tap) 58   N/A Water Blk 61        Task 13: Clean and bake-out         Wednesday Jan. 30  Equipment Purpose Quantity Task Completed?  500 mL beaker Measuring 13    100 mL glass bottle Centrifugation 3    pH paper Acidification 13    125 mL sep funnels LLE Extraction 3    250 mL sep funnels 13    Funnel 13    Roundbottom flsk Rotavap 13    HACH vials ML LLE 14       182  Day 4 Tasks Task 1: Freeze dry overnight         Thursday Jan. 31 Bottle Number Location Sample # Time of day Task Completed? W INF 41-? 8 AM - 8 PM   42-?   43-?   U ML A 39-?   V ML B 40-?   Z ML A 50-?   51-?   52-?   AA ML B 53-?   54-?   55-?   N/A ML Blk 64-?        Task 2: Measure 2 x 50mL ML into polypropylene bottles and centrifuge for 15min at an rcf of 2060*g      Thursday Jan. 31 Bottle Number Location Sample # Time of day Task Completed? D ML A 4-? 8 AM - 8 PM   E ML B 5-?   H ML A 12-?   13-?   14-?   I ML B 15-?   16-?   17-?   N/A ML Blk 62-?   L ML A 20-?   M ML B 21-?   Q ML A 31-?   32-?   33-?                            183  Task 3: Measure 85mL into beaker         Thursday Jan. 31 Bottle Number Location Sample # Time of day Task Completed? D ML A 4-? 8 AM - 8 PM   E ML B 5-?   H ML A 12-?   13-?   14-?   I ML B 15-?   16-?   17-?   N/A ML Blk 62-?   L ML A 20-?   M ML B 21-?   Q ML A 31-?   32-?   33-?        Task 4: Acidify          Thursday Jan. 31 Bottle Number Location Sample # Time of day Task Completed? D ML A 4-? 8 AM - 8 PM   E ML B 5-?   H ML A 12-?   13-?   14-?   I ML B 15-?   16-?   17-?   N/A ML Blk 62-?   L ML A 20-?   M ML B 21-?   Q ML A 31-?   32-?   33-?                       184       Task 5: Transfer to separatory funnel and add surrogate (200 uL)       Thursday Jan. 31 Bottle Number Location Sample # Time of day Task Completed? D ML A 4-? 8 AM - 8 PM   E ML B 5-?   H ML A 12-?   13-?   14-?   I ML B 15-?   16-?   17-?   N/A ML Blk 62-?   L ML A 20-?   M ML B 21-?   Q ML A 31-?   32-?   33-?        Task 6: LLE          Thursday Jan. 31 Bottle Number Location Sample # Time of day Task Completed? D ML A 4-? 8 AM - 8 PM   E ML B 5-?   H ML A 12-?   13-?   14-?   I ML B 15-?   16-?   17-?   N/A ML Blk 62-?   L ML A 20-?   M ML B 21-?   Q ML A 31-?   32-?   33-?                       185       Task 7: Rotavap and transfer to HACH vial        Thursday Jan. 31 Bottle Number Location Sample # Time of day Task Completed? D ML A 4-? 8 AM - 8 PM   E ML B 5-?   H ML A 12-?   13-?   14-?   I ML B 15-?   16-?   17-?   N/A ML Blk 62-?   L ML A 20-?   M ML B 21-?   Q ML A 31-?   32-?   33-?        Task 8: Blow down to dryness and store in freezer        Thursday Jan. 31 Bottle Number Location Sample # Time of day Task Completed? D ML A 4-? 8 AM - 8 PM   E ML B 5-?   H ML A 12-?   13-?   14-?   I ML B 15-?   16-?   17-?   N/A ML Blk 62-?   L ML A 20-?   M ML B 21-?   Q ML A 31-?   32-?   33-?                       186       Task 9: Clean and bake-out         Thursday Jan. 31  Equipment Purpose Quantity Task Completed?  500 mL beaker Measuring 14    50 mL plastic bottle Centrifugation 28    pH paper Acidification 14    125 mL sep funnels LLE Extraction 14    250 mL sep funnels 14    Funnel 14    Roundbottom flsk Rotavap 14    HACH vials ML LLE 14     Day 5 Tasks Task 1: Measure 2 x 50mL ML into polypropylene bottles and centrifuge for 15min at an rcf of 2060*g      Friday Feb. 1 Bottle Number Location Sample # Time of day Task Completed? R ML B 34-? 8 AM - 8 PM   35-?   36-?   N/A ML Blk 63-?   U ML A 39-?   V ML B 40-?   Z ML A 50-?   51-?   52-?   AA ML B 53-?   54-?   55-?   N/A ML Blk 64-?               187  Task 2: Measure 85mL into beaker         Friday Feb. 1 Bottle Number Location Sample # Time of day Task Completed? R ML B 34-? 8 AM - 8 PM   35-?   36-?   N/A ML Blk 63-?   U ML A 39-?   V ML B 40-?   Z ML A 50-?   51-?   52-?   AA ML B 53-?   54-?   55-?   N/A ML Blk 64-?        Task 3: Acidify          Friday Feb. 1 Bottle Number Location Sample # Time of day Task Completed? R ML B 34-? 8 AM - 8 PM   35-?   36-?   N/A ML Blk 63-?   U ML A 39-?   V ML B 40-?   Z ML A 50-?   51-?   52-?   AA ML B 53-?   54-?   55-?   N/A ML Blk 64-?               188  Task 4: Transfer to separatory funnel and add surrogate (200 uL)       Friday Feb. 1 Bottle Number Location Sample # Time of day Task Completed? R ML B 34-? 8 AM - 8 PM   35-?   36-?   N/A ML Blk 63-?   U ML A 39-?   V ML B 40-?   Z ML A 50-?   51-?   52-?   AA ML B 53-?   54-?   55-?   N/A ML Blk 64-?        Task 5: LLE          Friday Feb. 1 Bottle Number Location Sample # Time of day Task Completed? R ML B 34-? 8 AM - 8 PM   35-?   36-?   N/A ML Blk 63-?   U ML A 39-?   V ML B 40-?   Z ML A 50-?   51-?   52-?   AA ML B 53-?   54-?   55-?   N/A ML Blk 64-?               189  Task 6: Rotavap and transfer to HACH vial        Friday Feb. 1 Bottle Number Location Sample # Time of day Task Completed? R ML B 34-? 8 AM - 8 PM   35-?   36-?   N/A ML Blk 63-?   U ML A 39-?   V ML B 40-?   Z ML A 50-?   51-?   52-?   AA ML B 53-?   54-?   55-?   N/A ML Blk 64-?        Task 7: Blow down to dryness and store in freezer        Friday Feb. 1 Bottle Number Location Sample # Time of day Task Completed? R ML B 34-? 8 AM - 8 PM   35-?   36-?   N/A ML Blk 63-?   U ML A 39-?   V ML B 40-?   Z ML A 50-?   51-?   52-?   AA ML B 53-?   54-?   55-?   N/A ML Blk 64-?               190  Task 8: Clean and bake-out         Friday Feb. 1  Equipment Purpose Quantity Task Completed?  500 mL beaker Measuring 13    50 mL plastic bottle Centrifugation 26    pH paper Acidification 13    125 mL sep funnels LLE Extraction 13    250 mL sep funnels 13    Funnel 13    Roundbottom flsk Rotavap 13    HACH vials ML LLE 13     Day 6 Tasks Task 1: Add surrogate (200 uL)         Monday Feb. 4 Bottle Number Location Sample # Time of day Task Completed? A INF 1-? 8 AM - 8 PM   D ML A 4-?   E ML B 5-?   H ML A 12-?   13-?   14-?   I ML B 15-?   16-?   17-?   N/A ML Blk 62-?   N INF 22-?   23-?   24-?   L ML A 20-?   M ML B 21-?   Q ML A 31-?   32-?   33-?               191  Task 2: USE           Monday Feb. 4 Bottle Number Location Sample # Time of day Task Completed? A INF 1-? 8 AM - 8 PM   D ML A 4-?   E ML B 5-?   H ML A 12-?   13-?   14-?   I ML B 15-?   16-?   17-?   N/A ML Blk 62-?   N INF 22-?   23-?   24-?   L ML A 20-?   M ML B 21-?   Q ML A 31-?   32-?   33-?          192  Task 3: Rotavap and transfer to HACH vial        Monday Feb. 4 Bottle Number Location Sample # Time of day Task Completed? A INF 1-? 8 AM - 8 PM   D ML A 4-?   E ML B 5-?   H ML A 12-?   13-?   14-?   I ML B 15-?   16-?   17-?   N/A ML Blk 62-?   N INF 22-?   23-?   24-?   L ML A 20-?   M ML B 21-?   Q ML A 31-?   32-?   33-?               193  Task 4: Blow down to dryness and store in freezer        Monday Feb. 4 Bottle Number Location Sample # Time of day Task Completed? A INF 1-? 8 AM - 8 PM   D ML A 4-?   E ML B 5-?   H ML A 12-?   13-?   14-?   I ML B 15-?   16-?   17-?   N/A ML Blk 62-?   N INF 22-?   23-?   24-?   L ML A 20-?   M ML B 21-?   Q ML A 31-?   32-?   33-?        Task 5: Clean and bake-out         Friday Feb. 1  Equipment Purpose Quantity Task Completed?  HACH vials USE 18    Roundbottom flsk Rotavap 18       194  Day 7 Tasks Task 1: Add surrogate (200 uL)         Tuesday Feb. 5 Bottle Number Location Sample # Time of day Task Completed? R ML B 34-? 8 AM - 8 PM   35-?   36-?   N/A ML Blk 63-?   W INF 41-?   42-?   43-?   U ML A 39-?   V ML B 40-?   Z ML A 50-?   51-?   52-?   AA ML B 53-?   54-?   55-?   N/A ML Blk 64-?          195  Task 2: USE          Tuesday Feb. 5 Bottle Number Location Sample # Time of day Task Completed? R ML B 34-? 8 AM - 8 PM   35-?   36-?   N/A ML Blk 63-?   W INF 41-?   42-?   43-?   U ML A 39-?   V ML B 40-?   Z ML A 50-?   51-?   52-?   AA ML B 53-?   54-?   55-?   N/A ML Blk 64-?        Task 3: Rotavap and transfer to HACH vial        Tuesday Feb. 5 Bottle Number Location Sample # Time of day Task Completed? R ML B 34-? 8 AM - 8 PM   35-?   36-?   N/A ML Blk 63-?   W INF 41-?   42-?   43-?   U ML A 39-?   V ML B 40-?   Z ML A 50-?   51-?   52-?   AA ML B 53-?   54-?   55-?   N/A ML Blk 64-?   196  Task 4: Blow down to dryness and store in freezer       Tuesday Feb. 5 Bottle Number Location Sample # Time of day Task Completed? R ML B 34-? 8 AM - 8 PM   35-?   36-?   N/A ML Blk 63-?   W INF 41-?   42-?   43-?   U ML A 39-?   V ML B 40-?   Z ML A 50-?   51-?   52-?   AA ML B 53-?   54-?   55-?   N/A ML Blk 64-?       Task 5: Clean and bake-out         Friday Feb. 1  Equipment Purpose Quantity Task Completed?  HACH vials USE 16    Roundbottom flsk Rotavap 16         Task 6: Measure and blow down to dryness        Tuesday Feb. 5        Bottle Number Location Sample # Task Completed?  N/A N/A Calibration 1    N/A N/A Calibration 2    N/A N/A Calibration 3    N/A N/A Calibration 4    N/A N/A Calibration 5    197   Day 8 Tasks Task 1: Derivatize (50 uL pyridine + 150 uL BSTFA)       Wednesday Feb. 6 Bottle Number Location Sample Number Task Completed? A INF 1 ?   ?   B EFF 2 N/A   C 3 N/A   D ML 4 ?   ?   E 5 ?   ?   F EFF 6 N/A   7 N/A   8 N/A   G 9 N/A   10 N/A   11 N/A   H ML 12 ?   ?   13 ?   ?   14 ?   ?   I 15 ?   ?   16 ?   ?   17 ?   ?   J EFF 18 N/A   K 19 N/A   L ML 20 ?   ?   M 21 ?   ?      198  Wednesday Feb. 6 Bottle Number Location Sample Number Task Completed? N INF 22 ?   ?   23 ?   ?   24 ?   ?   O EFF 25 N/A   26 N/A   27 N/A   P 28 N/A   29 N/A   30 N/A   Q ML 31 ?   ?   32 ?   ?   33 ?   ?   R 34 ?   ?   35 ?   ?   36 ?   ?   S EFF 37 N/A   T 38 N/A   U ML 39 ?   ?   V 40 ?   ?   W INF 41 ?   ?   42 ?   ?   43 ?   ?      199  Wednesday Feb. 6 Bottle Number Location Sample Number Task Completed? X EFF 44 N/A   45 N/A   46 N/A   Y 47 N/A   48 N/A   49 N/A   Z ML 50 ?   ?   51 ?   ?   52 ?   ?   AA 53 ?   ?   54 ?   ?   55 ?   ?   BB Field blk 1 56 N/A   CC 57 N/A   DD 58 N/A   N/A Water blk 59 N/A   60 N/A   61 N/A   N/A ML blk 62 ?   ?   63 ?   ?   64 ?   ?   N/A N/A Calibration 1 N/A   N/A N/A Calibration 2 N/A   N/A N/A Calibration 3 N/A   N/A N/A Calibration 4 N/A   N/A N/A Calibration 5 N/A   N/A N/A Deriv. Agents Blk N/A      200  Task 2: Blow down to dryness         Wednesday Feb. 6 Bottle Number Location Sample Number Task Completed? A INF 1 ?   ?   B EFF 2 N/A   C 3 N/A   D ML 4 ?   ?   E 5 ?   ?   F EFF 6 N/A   7 N/A   8 N/A   G 9 N/A   10 N/A   11 N/A   H ML 12 ?   ?   13 ?   ?   14 ?   ?   I 15 ?   ?   16 ?   ?   17 ?   ?   J EFF 18 N/A   K 19 N/A   L ML 20 ?   ?   M 21 ?   ?      201  Wednesday Feb. 6 Bottle Number Location Sample Number Task Completed? N INF 22 ?   ?   23 ?   ?   24 ?   ?   O EFF 25 N/A   26 N/A   27 N/A   P 28 N/A   29 N/A   30 N/A   Q ML 31 ?   ?   32 ?   ?   33 ?   ?   R 34 ?   ?   35 ?   ?   36 ?   ?   S EFF 37 N/A   T 38 N/A   U ML 39 ?   ?   V 40 ?   ?   W INF 41 ?   ?   42 ?   ?   43 ?   ?      202  Wednesday Feb. 6 Bottle Number Location Sample Number Task Completed? X EFF 44 N/A   45 N/A   46 N/A   Y 47 N/A   48 N/A   49 N/A   Z ML 50 ?   ?   51 ?   ?   52 ?   ?   AA 53 ?   ?   54 ?   ?   55 ?   ?   BB Field blk 1 56 N/A   CC 57 N/A   DD 58 N/A   N/A Water blk 59 N/A   60 N/A   61 N/A   N/A ML blk 62 ?   ?   63 ?   ?   64 ?   ?   N/A N/A Calibration 1 N/A   N/A N/A Calibration 2 N/A   N/A N/A Calibration 3 N/A   N/A N/A Calibration 4 N/A   N/A N/A Calibration 5 N/A   N/A N/A Deriv. Agents Blk N/A      203  Task 3: Add internal std (50 uL) and toluene (950 uL)       Wednesday Feb. 6 Bottle Number Location Sample Number Task Completed? A INF 1 ?   ?   B EFF 2 N/A   C 3 N/A   D ML 4 ?   ?   E 5 ?   ?   F EFF 6 N/A   7 N/A   8 N/A   G 9 N/A   10 N/A   11 N/A   H ML 12 ?   ?   13 ?   ?   14 ?   ?   I 15 ?   ?   16 ?   ?   17 ?   ?   J EFF 18 N/A   K 19 N/A   L ML 20 ?   ?   M 21 ?   ?      204  Wednesday Feb. 6 Bottle Number Location Sample Number Task Completed? N INF 22 ?   ?   23 ?   ?   24 ?   ?   O EFF 25 N/A   26 N/A   27 N/A   P 28 N/A   29 N/A   30 N/A   Q ML 31 ?   ?   32 ?   ?   33 ?   ?   R 34 ?   ?   35 ?   ?   36 ?   ?   S EFF 37 N/A   T 38 N/A   U ML 39 ?   ?   V 40 ?   ?   W INF 41 ?   ?   42 ?   ?   43 ?   ?      205  Wednesday Feb. 6 Bottle Number Location Sample Number Task Completed? X EFF 44 N/A   45 N/A   46 N/A   Y 47 N/A   48 N/A   49 N/A   Z ML 50 ?   ?   51 ?   ?   52 ?   ?   AA 53 ?   ?   54 ?   ?   55 ?   ?   BB Field blk 1 56 N/A   CC 57 N/A   DD 58 N/A   N/A Water blk 59 N/A   60 N/A   61 N/A   N/A ML blk 62 ?   ?   63 ?   ?   64 ?   ?   N/A N/A Calibration 1 N/A   N/A N/A Calibration 2 N/A   N/A N/A Calibration 3 N/A   N/A N/A Calibration 4 N/A   N/A N/A Calibration 5 N/A   N/A N/A Deriv. Agents Blk N/A      206  Task 4: Transfer to GC vial and load on GC-MS        Wednesday Feb. 6 Bottle Number Location Sample Number Task Completed? A INF 1 ?   ?   B EFF 2 N/A   C 3 N/A   D ML 4 ?   ?   E 5 ?   ?   F EFF 6 N/A   7 N/A   8 N/A   G 9 N/A   10 N/A   11 N/A   H ML 12 ?   ?   13 ?   ?   14 ?   ?   I 15 ?   ?   16 ?   ?   17 ?   ?   J EFF 18 N/A   K 19 N/A   L ML 20 ?   ?   M 21 ?   ?      207  Wednesday Feb. 6 Bottle Number Location Sample Number Task Completed? N INF 22 ?   ?   23 ?   ?   24 ?   ?   O EFF 25 N/A   26 N/A   27 N/A   P 28 N/A   29 N/A   30 N/A   Q ML 31 ?   ?   32 ?   ?   33 ?   ?   R 34 ?   ?   35 ?   ?   36 ?   ?   S EFF 37 N/A   T 38 N/A   U ML 39 ?   ?   V 40 ?   ?   W INF 41 ?   ?   42 ?   ?   43 ?   ?      208  Wednesday Feb. 6 Bottle Number Location Sample Number Task Completed? X EFF 44 N/A   45 N/A   46 N/A   Y 47 N/A   48 N/A   49 N/A   Z ML 50 ?   ?   51 ?   ?   52 ?   ?   AA 53 ?   ?   54 ?   ?   55 ?   ?   BB Field blk 1 56 N/A   CC 57 N/A   DD 58 N/A   N/A Water blk 59 N/A   60 N/A   61 N/A   N/A ML blk 62 ?   ?   63 ?   ?   64 ?   ?   N/A N/A Calibration 1 N/A   N/A N/A Calibration 2 N/A   N/A N/A Calibration 3 N/A   N/A N/A Calibration 4 N/A   N/A N/A Calibration 5 N/A   N/A N/A Deriv. Agents Blk N/A      209  APPENDIX E: SUPPLEMENTARY INFORMATION FOR QA/QC ? STAGE 2  Table E - 1, Table E - 2, and Table E - 3 illustrate the relative standard deviation (RSD) and % recoveries from spikes at 3 hours, 24 hours, and 48 hours, respectively. Note that N/A (non-applicable) refers to non-detect value.  Table E - 1: RSD and recovery percentages at time = 3 hours t = 3 hours EFF A EFF B Compounds RSD (%) % Recovery RSD (%) % Recovery Ibuprofen 14% 7% N/A 7% Nonylphenol 24% 7% 57% 7% Caffeine 18% 70% N/A 70% Tonalide 5% 125% 2% 125% MCPB 11% 3% 14% 3% Gemfibrozil 7% 12% 13% 12% Dibutyl Phthalate 13% 91% 22% 91% Naproxen 8% 8% 13% 8% Irgasan N/A 2% N/A 2% Estrone N/A 5% N/A 5% EE2 0% 15% 141% 15% t = 3 hours ML A - ? ML B - ? Compounds RSD (%) % Recovery RSD (%) % Recovery Ibuprofen 86% 314% 62% 6% Nonylphenol 18% 186% 20% 9% Caffeine N/A 66% 84% 6% Tonalide 32% 116% 6% 11% MCPB 64% 311% 38% -29% Gemfibrozil 49% 183% 46% 1% Dibutyl Phthalate 4% 71% 21449% 7% Naproxen 58% 246% 60% 1% Irgasan 57% 86% 57% 3% Estrone N/A 5% N/A 5% EE2 0% 15% 141% 15%    210  t = 3 hours ML A - ? ML B - ? Compounds RSD (%) % Recovery RSD (%) % Recovery Ibuprofen 28% 241% 32% 238% Nonylphenol 40% 91% 37% 174% Caffeine N/A 87% 7% 79% Tonalide 13% 146% 7% 135% MCPB 10% 0% 5% 5% Gemfibrozil 12% 125% N/A 118% Dibutyl Phthalate 7% 104% 24% 92% Naproxen 42% 67% 67% 91% Irgasan 20% 63% 18% 71% Estrone 28% 67% N/A 94% EE2 10% 149% 29% 194%  Table E - 2: RSD and recovery percentages at time = 24 hours t = 24 hours INF - ? INF - ? Compounds RSD (%) % Recovery RSD (%) % Recovery Ibuprofen 4% 249% N/A 27% Nonylphenol 30% 209% 26% 63% Caffeine 3% 171% 33% 69% Tonalide 14% 308% 26% 118% MCPB 8% -37% 2% -20% Gemfibrozil N/A 191% N/A 24% Dibutyl Phthalate 19% 167% 6% 88% Naproxen 1% 30% N/A 10% Irgasan 54% 23% 26% 5% Estrone 24% 60% 141% 43% EE2 N/A 125% N/A 97% t = 24 hours EFF A EFF B Compounds RSD (%) % Recovery RSD (%) % Recovery Ibuprofen 52% 25% N/A 22% Nonylphenol 26% 4% 22% 5% Caffeine 43% 85% 112% 70% Tonalide 5% 149% 10% 133% MCPB 8% 5% 13% -5% Gemfibrozil 18% 16% N/A 13% Dibutyl Phthalate 15% 92% 38% 76% Naproxen 59% 12% 141% 7% Irgasan 2% 4% 12% 3% Estrone N/A 6% N/A 5% EE2 4% 12% 19% 9% 211  t = 24 hours ML A - ? ML B - ? Compounds RSD (%) % Recovery RSD (%) % Recovery Ibuprofen 141% 87% 22% 105% Nonylphenol 28% 149% 9% 121% Caffeine N/A 66% 0% 47% Tonalide 5% 115% 3% 97% MCPB 27% 22% 19% 17% Gemfibrozil N/A 59% 24% 53% Dibutyl Phthalate 0% 68% 13% 57% Naproxen 3% 27% 7% 29% Irgasan 50% 29% 35% 19% Estrone 59% 75% 14% 74% EE2 17% 221% 8% 187% t = 24 hours ML A - ? ML B - ? Compounds RSD (%) % Recovery RSD (%) % Recovery Ibuprofen 25% 206% 18% 15% Nonylphenol 52% 91% 16% -14% Caffeine 12% 74% 141% 92% Tonalide 2% 132% 12% 136% MCPB 8% -8% 11% -143% Gemfibrozil 141% 107% 20% 1% Dibutyl Phthalate 33% 90% 26% 96% Naproxen 50% 60% 40% 1% Irgasan 21% 49% 16% -4% Estrone 4% 66% 7% 3% EE2 19% 149% 24% -12%  Table E - 3: RSD and recovery percentages at time = 48 hours t = 48 hours INF - ? INF - ? Compounds RSD (%) % Recovery RSD (%) % Recovery Ibuprofen 23% 283% 1% 234% Nonylphenol 2% 317% 18% 194% Caffeine 2% 174% 3% 95% Tonalide 4% 332% 60% 156% MCPB 28% 13% 13% 41% Gemfibrozil N/A 179% N/A 134% Dibutyl Phthalate 26% 209% 33% 120% Naproxen 10% 30% N/A 9% Irgasan 23% 51% 11% 106% Estrone 39% 49% 141% 22% EE2 N/A 136% N/A 53% 212  t = 48 hours EFF A EFF B Compounds RSD (%) % Recovery RSD (%) % Recovery Ibuprofen N/A 14% N/A 13% Nonylphenol 8% 21% 26% 11% Caffeine 4% 73% 33% 68% Tonalide 3% 127% 4% 111% MCPB 11% 1% 7% -5% Gemfibrozil N/A 18% N/A 12% Dibutyl Phthalate 4% 79% 16% 73% Naproxen N/A 1% 141% 1% Irgasan N/A 6% 6% 4% Estrone N/A 9% N/A 6% EE2 N/A 46% N/A 20% t = 48 hours ML A - ? ML B - ? Compounds RSD (%) % Recovery RSD (%) % Recovery Ibuprofen 141% 0% N/A 57% Nonylphenol 40% -12% 25% 102% Caffeine 1% 49% 9% 64% Tonalide 8% 82% 7% 101% MCPB 14% -57% 100% 33% Gemfibrozil 141% -2% 141% 48% Dibutyl Phthalate 2% 59% 46% 67% Naproxen 141% 0% N/A 10% Irgasan 23% -1% 43% 11% Estrone 8% -3% 58% 66% EE2 5% -7% 141% 197% t = 48 hours ML A - ? ML B - ? Compounds RSD (%) % Recovery RSD (%) % Recovery Ibuprofen N/A 17% 133% 260% Nonylphenol 94% 0% 50% 178% Caffeine 61% 95% 131% 84% Tonalide 23% 148% 20% 140% MCPB 126% -60% 49% -40% Gemfibrozil N/A 5% 40% 130% Dibutyl Phthalate 19% 102% 67% 97% Naproxen 141% 0% 108% 91% Irgasan 141% 1% 45% 80% Estrone N/A 2% 141% 102% EE2 141% 0% 78% 197%   

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