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Effect of microwave hydrolysis on transformation of steroidal hormones during anaerobic digestion of… Hamid, Hanna 2013

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EFFECT OF MICROWAVE HYDROLYSIS ON TRANSFORMATION OF STEROIDAL HORMONES DURING ANAEROBIC DIGESTION OF MUNICIPAL SLUDGE CAKE  by  Hanna Hamid B.Sc., Bangladesh University of Engineering and Technology, 2009  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF APPLIED SCIENCE  in  The College of Graduate Studies (Civil Engineering)  THE UNIVERSITY OF BRITISH COLUMBIA (Okanagan) February 2013  © Hanna Hamid, 2013  Abstract This research evaluates the fate of 16 steroidal (estrogenic, androgenic and progestogenic) hormones during advanced anaerobic digestion of municipal sludge cake generated at the Kelowna’s wastewater treatment plant using microwave (MW) pretreatment. Effect of sludge pretreatment temperature (80, 120, 160oC), digester operating temperature (mesophilic at 35 ± 2oC, thermophilic at 55 ± 2oC) and sludge retention time (SRT: 20, 10, 5 days) were studied employing eight lab-scale semi-continuously fed digesters. To determine the potential effect of MW hydrolysis, hormones were quantified in sludge (total) and supernatant (soluble) phases of the digester influent and effluent streams. Seven of 16 hormones were above reporting limit (RL) in one or more of the samples. Hormone concentrations upto 1,640 ng/L and 2491 ng/g (for androstenedione, (Ad)), respectively, were detected in soluble and total phases of the influents. Microwave hydrolysis resulted in both release and attenuation of hormones in the soluble phase. High accumulations (upto 30 times for androstenedione (Ad) of the influent concentration) of hormones observed in the effluents of un-pretreated (control) digesters suggested that anaerobic digestion was inefficient to remove these compounds. Simultaneous accumulation and removal of 17β-estradiol (E2) and estrone (E1) as well as progesterone (Pr) and androstenedione (Ad) indicated possible transformations among the hormones. At a 20-day SRT, thermophilic digesters contained overall less concentration of steroidal hormones in their digestate supernatants to be recycled to the beginning of the treatment plant. However, mesophilic digesters seemed to perform better in terms of total decrease of hormones and would likely to have lower concentrations in their dewatered digestates to be disposed of via land application at same SRT (20 days). Microwave pretreatment increased total chemical oxygen demand (TCOD) volatile solids (VS) removals and methane production compared to un-pretreated (control) digesters. However, improvements were more prominent at the shortest SRT of 5 days.  ii  Preface Abridged version of chapter 2 was submitted to the journal Water Research on April 22, 2012 and was accepted for publication on August 2, 2012. The full citation of the article was “Hamid, Hanna and Eskicioglu, Cigdem, 2012. Fate of estrogenic hormones in wastewater and sludge treatment: A review of properties and analytical detection techniques in sludge matrix. Water Research 46(18), 5813-5833. http://dx.doi.org/10.1016/j.watres.2012.08.002.” Abstracts based on some results of chapter 3 were submitted for presentations in Water Environment Federation (WEF) Residuals and Biosolids 2013 Conference (May 5-8, 2013, Nashville, Tennessee) and International Water Association (IWA) 13th World Congress on Anaerobic Digestion (June 25-28, 2013, Santiago de Compostela, Spain). Also, an abridged version of chapter 3 has been submitted to Water Research as a journal paper and currently it is under review.  iii  Table of Contents Abstract .................................................................................................... ii Preface ..................................................................................................... iii Table of Contents ................................................................................... iv List of Tables ......................................................................................... vii List of Figures ....................................................................................... viii List of Abbreviations .............................................................................. x List of Symbols ....................................................................................... xi Acknowledgements ............................................................................... xii Dedication ............................................................................................. xiii Chapter 1 Introduction .......................................................................... 1 1.1 Background .................................................................................................... 1 1.2 Motivation for research ................................................................................. 2 1.3 Objectives........................................................................................................ 3 1.4 Thesis organization ........................................................................................ 4  Chapter 2 Literature Review ................................................................. 5 2.1 Introduction .................................................................................................... 5 2.2 Properties of hormones as endocrine disrupters ........................................ 6 2.3 Health effects of steroidal compounds ....................................................... 10 2.4 Screening program and regulatory aspects of EDC ................................. 11 2.5 Sources and pathways of hormones in environment ................................ 12 iv  2.6 Removal pathways for steroidal compounds ............................................ 15 2.6.1 Volatilization ............................................................................................................. 15 2.6.2 Biodegradation by sewage bacteria ......................................................................... 15 2.6.3 Adsorption ................................................................................................................. 20  2.7 Fate of steroidal compounds in engineered processes .............................. 22 2.7.1 Fate of steroidal compounds during conventional WWTPs ................................. 22 2.7.2 Fate of estrogenic compounds during sludge stabilization ................................... 29  2.8 Analytical techniques for steroidal compounds in sludge samples ......... 33 2.9 Summary ....................................................................................................... 39  Chapter 3 Effect of microwave hydrolysis on transformation of steroidal hormones during anaerobic digestion of municipal sludge cake ......................................................................................................... 40 3.1 Materials and methods ................................................................................ 41 3.1.1 Substrate and inocula ............................................................................................... 41 3.2.2 Microwave pretreatment .......................................................................................... 43 3.2.3 Digester studies.......................................................................................................... 45  3.3 Analytical techniques ................................................................................... 46 3.3.1 Characterization of influent and effluent ............................................................... 46 3.3.2 Hormone analysis ...................................................................................................... 47 3.3.3 Calculations ............................................................................................................... 48  3.4 Results and discussion ................................................................................. 52 3.4.1 Occurrence of steroids in anaerobic digester streams ........................................... 52 3.4.2 Effects of microwave pretreatment on steroids in digester feed........................... 57 3.4.3 Effects of microwave pretreatment on digester performance .............................. 58 v  3.4.3 Effects of pretreatment and anaerobic digester operating conditions on hormone concentrations .................................................................................................................... 63  3.5 Summary ....................................................................................................... 71  Chapter 4 Conclusions and perspectives ............................................ 72 4.1 Conclusions ................................................................................................... 72 4.2 Thesis contributions ..................................................................................... 74 4.3 Perspectives .................................................................................................. 74  References .............................................................................................. 76 Appendices ............................................................................................. 96 Appendix A: Calibration charts ....................................................................... 96 Appendix B: Analytical detection of hormones .............................................. 98 Appendix C: Analysis of variance .................................................................. 106  vi  List of Tables Table 2.1 Physical and chemical properties, relative binding affinity and surrogate standard of the hormones .............................................................................................................. 9 Table 2.2 Estimates of estrogen excretion by humans (per person) in µg/day ............................. 13 Table 2.3 Estimated total daily estrogen excretion of different livestock species ........................14 Table 2.4 Biodegradation kinetic constants for estrogens.. .......................................................... 19 Table 2.5 Occurrence and fate of some hormones in wastewater treatment plants (WWTPs) .... 23 Table 2.6 Analytic techniques for estrogenic compounds ............................................................ 38 Table 3.1 Characterization of raw (control) and pretreated digester feed after dilution ............... 45 Table 3.2 Method detection limits, reporting limits and quantification references for analytes, surrogate standards ........................................................................................ 50 Table 3.3 Concentration (ng/L) of steroidal hormones in soluble phase of digester influent and effluents at different SRTs ..................................................................................... 53 Table 3.4 Concentration (ng/g) of steroidal hormones in total phase of digester influent and effluents at different SRTs ........................................................................................... 55 Table 3.5 Biogas composition (%) at various sludge retention times (SRTs).............................. 63 Table 3.6 Analysis of Variance for removal efficiency of progesterone (Pr) from total phase .... 68 Table B.1 Quality control acceptance criteria ...............................................................................98 Table.B.2.Reporting limit (RL) of steroidal hormones in supernatant (ng/L) of influent and effluents at different SRT's ........................................................................................... 99 Table.B.3.Reporting limit (RL) of steroidal hormones in total phase (ng/g) of influent and effluents at different SRT's ......................................................................................... 101 Table B.4 Total solids (TS) concentrations (%, w/w) of the influent and effluent samples analyzed for hormones ..............................................................................................102 Table.C.1.Analysis of Variance for Removal efficiency of androstenedione (Ad) from soluble ........................................................................................................................ 106 Table.C.2.Analysis of Variance for Removal efficiency of estrone (E1) from soluble phase ... 106  vii  List of Figures Figure 2.1 Chemical structures of some natural and synthetic hormones ...................................... 8 Figure 2.2 Sources and pathways of steroidal hormones in environment .................................... 12 Figure 2.3 Proposed metabolic pathway of E2 by sewage bacteria .............................................. 17 Figure.2.4.Typical setup of a wastewater treatment plant showing wastewater sludge production and the split arrows indicate EDC release/transformation during various unit processes of treatment ............................................................................. 26 Figure 2.5 Steps of anaerobic digestion process ........................................................................... 30 Figure 3.1 Existing process flow diagram in City of Kelowna wastewater treatment plant ........ 42 Figure.3.2.a) Ethos microwave station (2.45 GHz, 0 - 1200 Watt, 25 - 300oC, 0 - 35 bars); b) heating and cooling profiles of the microwave unit.................................................... 44 Figure 3.3 One of the eight lab-scale semi-continuous anaerobic digesters ................................. 46 Figure 3.4 Experimental methodology showing sampling locations ............................................ 49 Figure.3.5.Flow chart of analytical determination of steroidal hormones in solid and liquid samples ........................................................................................................................ 51 Figure 3.6 Effect of microwave (MW) pretreatment on waste sludge cake solubilization .......... 57 Figure 3.7 Effect of microwave (MW) on hormone concentrations in the a) soluble (liquid) phase and b) total (solid) phase of the influent ........................................................... 59 Figure 3.8 Daily biogas productions (normalized to volatile solids (VS) content of the feed) of control (fed with unpretreated) and MW160 (fed with microwave pretreated sludge at 160oC) thermophilic (TH) and mesophilic (ME) digesters at STP (1 atm, 0oC) during the 20, 10 and 5 days of sluge retention time (SRT)....................... 60 Figure 3.9.Total chemical oxygen demand (TCOD) removal efficiencies at sludge retention times (SRTs) of 20, 10 and 5 days .............................................................................. 61 Figure 3.10 Volatile solids (VS) removal efficiencies at sludge retention times (SRTs) of 20, 10 and 5 days ............................................................................................................. 62 Figure 3.11 Average daily biogas and methane production rate at different sludge retention time (SRT) at STP ...................................................................................................... 62  viii  Figure 3.12 Concentrations of a) estrone (E1) and b) 17β-estradiol (E2) in soluble phases of the influent and effluents at different sludge retention times (SRTs) ........................ 64 Figure 3.13 Concentrations of androstenedione (Ad) in a) soluble phase and b) total phase of the influent and effluents at different sludge retention times (SRTs) ........................ 66 Figure 3.14 Concentrations of progesterone (Pr) in a) soluble phase and b) total phase of the influent and effluents at different sludge retention times (SRTs) .............................. 67 Figure.3.15.Total hormone concentrations (estrone (E1), 17β-estradiol (E2), androstenedione (Ad) and progesterone (Pr)) in the a) soluble phase (supernatant) and b) total phase (sludge) of the influent/digester feed and effluent at 20 days SRT.............................................................................................. 70 Figure A.1 Calibration curve for COD determination..................................................................96 Figure A.2 Calibration curve for NH3-N determination ..............................................................96 Figure A.3 Calibration curve for biogas measurement via manometer at STP (0oC, 1 atm) .......97 Figure.B.1.Concentrations of testosterone (Tr) in a) soluble phase and b) total phase of the influent and effluents at different sludge retention times (SRTs) .............................103 Figure.B.2.Concentrations of androsterone (An) in a) soluble phase and b) total phase of the influent and effluents at different sludge retention times (SRTs) .............................104 Figure.B.3.Concentrations of mestranol (Ms) in a) soluble phase and b) total phase of the influent and effluents at different sludge retention times (SRTs) .............................105 Figure.C.1.Normal probability plot for analysis of variance of removal efficiency of androstenedione (Ad) from soluble phase ................................................................107 Figure.C.2.Normal probability plot for analysis of variance of removal efficiency of estrone (E1) from soluble phase ............................................................................................107 Figure.C.3.Normal probability plot for analysis of variance of removal efficiency of progesterone (Pr) from total phase ............................................................................108  ix  List of Abbreviations Ad ADD An AOB APCI AS ASE CAS COD DHT E1 E2 E3 EE2 EDC Eq Eqn ESI GC GPC HRT HRTh KPCC LC MAE MDL MS Ms MW OLR Pr PS RL SCOD SDL SPE SRT TCOD Tr TS TVFA TWAS VFA WAS WWTP YES  Androstenedione Androstadienedione Androsterone Ammonia oxidizing bacteria Atmospheric pressure chemical ionization Activated sludge Accelerated solvent extraction Chemical abstract services Chemical oxygen demand 17β-hydroxy-androstanedieneone Estrone 17β-estradiol Estriol 17α-ethinylestradiol Endocrine disrupting compound Equilin Equilenin Electrospray ionization Gas chromatography Gel permeation chromatography Hydraulic retention time Hormone replacement therapy Kelowna Pollution Control Center Liquid chromatography Microwave assisted extraction Method detection limit Mass spectrometry Mestranol Microwave Organic loading rate Progesterone Primary sludge Reporting limit Soluble chemical oxygen demand Sample specific detection limit Solid phase extraction Sludge retention time Total chemical oxygen demand Testosterone Total solids Total volatile fatty acids Thickened waste activated sludge Volatile fatty acids Waste activated sludge Wastewater treatment plant Yeast estrogen screen  x  List of Symbols C Cs Cw k Kd KF KOM KOC Kow OC pKa TSS VSS  Total compound concentration Concentration in solids Concentration in water Reaction rate constant Distribution coefficient Freundlich parameter Distribution coefficient normalized to organic matter Distribution coefficient normalized to organic carbon content Octanol /water partition coefficient Percentage of organic carbon Acid dissociation constant Total suspended solids Volatile suspended solids  xi  Acknowledgements First of all, I would like to thank almighty Allah for giving me the strength and courage to complete this project. Next, my heartfelt gratitude goes to my supervisor, Dr. Cigdem Eskicioglu, for her enduring patience, guidance and motivation throughout the last two years of my graduate study. Whenever I needed her, she was always there for me. Our late afternoon meetings have always been a source of great inspiration for me. I would also like to extend deepest gratitude to my committee members, Dr. Rehan Sadiq and Dr. Bahman Naser for their valuable advice and time. In addition, I am very much grateful to the fellow students of my research team Neda Mehdizadeh, Piero Galvagno, Kafi Wahidunnabi and Mariel Barrantes Leiva. My especial thanks go to Shashank Gupta, summer intern in Dr. Eskicioglu’s research group. This research would not have been possible without their help and support. Last but not the least; I am thankful to my family members and friends for their unconditional love and encouragement throughout my entire life.  xii  Dedication  To my father and mother  xiii  Chapter 1 Introduction 1.1 Background In Canada, approximately 388,700 dry tones of biosolids are produced yearly; about 43% of which is land applied. This percentage is expected to increase as different municipalities are recognizing land application to be the most sustainable and environment friendly disposal option for biosolids, as opposed to landfilling and incineration (Apedaile, 2001). However, in addition to existing environmental and health concerns, presence of some natural hormones, pharmaceutical and personal care products in biosolids are causing public opposition against the beneficial reuse of biosolids (Citulski and Farahbakhsh, 2010). A number of these compounds are known to interfere with the natural functioning of endocrine system in human and animals and are called endocrine disrupting compounds (EDCs) (Hansen et al., 1998; Tyler et al., 2005). Among these, natural steroidal hormones have been characterized to be the most potent of all EDCs (Legler et al., 1999; Legler et al., 2002a; Legler et al., 2002b). Some studies show that although steroidal hormones have the potential to undergo transformations in agricultural settings, they are likely to persist long enough to impact the water quality of runoff (Yang et al., 2012). Furthermore, others indicate that hormone leaching and runoff can result in after land application of manure (Kjaer et al., 2007; Jenkins et al., 2009). Most commonly used processes for volatile content stabilization of biosolids include digestion, alkali stabilization, composting and heat drying (Tchobanoglous et al., 2002). Among these, anaerobic digestion is a biosolids treatment method that converts the organic waste into methane-enriched biogas and fertilizer by combined action of a mixed community of microorganisms (Botheju and Bakke, 2011). Stabilization of high-strength organic waste, production of heat or electricity by recovered methane, and reduced greenhouse gas emissions have made anaerobic digestion more favorable in today's world. Major drawbacks of conventional anaerobic digestion, such as longer digestion retention time requirement (20 – 50 days), low organic degradation efficiency (20  50%) are often thought to be associated with  hydrolysis, which is one of the three stages of anaerobic digestion (i.e., hydrolysis, acetogenesis and methanogenesis) (Tyagi and Lo, 2011). Recent developments in this field indicate that thermal (i.e., conventional and microwave (MW) heating) (Ge et al., 2010; Wett et al., 2010; Yu 1  et al., 2010), mechanical (e.g., high pressure homogenizer, sonication, stirred ball mills (Onyeche, 2006; Wood et al., 2009; Salsabil et al., 2010) and chemical (e.g., acidic and alkaline, ozonation.) (Lin et al., 2009; Kim and Youn, 2011) pretreatments applied to complex organic waste (i.e. industrial and municipal secondary sludge, manure) prior to anaerobic digestion can substantially accelerate the hydrolysis of biosolids and subsequent methane recovery in smaller digesters. Different processes, e.g., bulking and foaming, dewatering, conditioning, are also shown to improve after pretreatment (Muller, 2000; Tyagi and Lo, 2011). Due to the aforementioned advantages, a number of proprietary advanced hydrolysis processes are currently being applied at the full-scale, i.e., Cambi™, Microsludge™, Sonix™, OpenCEL™ (Jolly and Gillard, 2009; Pilli et al., 2011). Among these, thermal hydrolysis methods are reported to show improved pathogen destruction and dewaterability in addition to higher improvement in methane recovery compared to other pretreatment methods (Pino-Jelcic et al., 2006; Carballa et al., 2009; Yu et al., 2009).  1.2 Motivation for research To date, conventional performance parameters, such as organics removal, methane recovery, pathogen removal, dewaterability, have been the focus of almost all the studies dealing with thermal hydrolysis in lab- or full-scale advanced anaerobic digester operations to treat municipal biosolids. A large research gap exists regarding the effects of thermal hydrolysis on fate and removal of micropollutants, e.g., steroidal hormones, in municipal waste sludge streams during advanced anaerobic digestion. Only a few studies have reported fate and removal of estrogenic hormones (e.g. estrone (E1), 17β-estradiol (E2), estriol (E3) and 17α-ethinylestradiol (EE2)) from full-scale conventional anaerobic digesters (Andersen et al., 2003; Muller et al., 2010; Ifelebuegu, 2011). Furthermore, only one study has reported the loadings of androstenedione (Ad), testosterone (Tr), progesterone (Pr), mestranol (Ms) and androsterone (An) in full-scale sludge digesters (Furlong et al., 2010). Regarding the effect of thermal hydrolysis on hormones, a single study exits using conventional heating (Carballa et al., 2006), where removal efficiencies of E1, E2 and EE3 in pilot-scale mesophilic and thermophilic digesters utilizing mixed municipal waste sludge were reported. As the pretreatments disintegrate the complex polymeric network in waste sludge samples, it is likely that some hormones, initially encapsulated within the polymeric network, may be 2  released into the soluble phase and render themselves more or less biodegradable depending on the changes on their molecular structures at different pretreatment conditions (low or elevated temperatures, mechanical shear, or chemical dose). Furthermore, as sludge pretreatments solubilize organics (hormones), it could be postulated that there is a potential to shift the ultimate disposal route of residual hormones in digestate from a landfill or agricultural land to main wastewater stream. As wastewater treatment plants (WWTPs) incorporating thermal hydrolysis prior to anaerobic digestion may contain higher solubilized hormones in their digestates, there may be higher concentration of hormones recycled back to the head of the treatment train with the digester centrate.  1.3 Objectives The general objective of this work was to study the effect of MW hydrolysis on fate and removal of 16 steroidal hormones (synthetic and natural displayed in Table 2.1) during advanced anaerobic digestion of municipal sludge cake sampled from Kelowna’s WWTP. The effect of sludge cake pretreatment temperature (80, 120, 160oC), digester operating temperature (mesophilic at 35 ± 2oC, thermophilic at 55 ± 2oC) and sludge retention time (SRT: 20, 10, 5 days) were evaluated using eight lab-scale semi-continuously fed anaerobic digesters. The main goal set above was achieved by systematically investigating the following:   Biosolids particulate chemical oxygen demand (COD) solubilization following MW pretreatment at different temperatures    Concentration of the target steroidal hormones in control (un-pretreated) and pretreated digester feeds in sludge (total) and supernatant (soluble) phases    Mesophilic and thermophilic anaerobic digestion of un-pretreated and MW pretreated municipal sludge cake (at SRTs of 20, 10 and 5 days) to evaluate  Organic removal efficiency  Biogas and methane recovery  Concentration of target hormones in the effluent of control and pretreated digesters in sludge (total) and supernatant (soluble) phases  3  1.4 Thesis organization The thesis has been organized into four chapters. Chapter 1 provides a brief background and motivation for this research. General and specific objectives of this study are also outlined in this chapter. In chapter 2, literature involving physiochemical properties, source, pathway and fate of steroidal hormones in different units of WWTPs are reviewed. Furthermore, effects of advanced anaerobic digestion with MW pretreatment on different sludge (municipal and industrial) are also discussed here. Chapter 3 presents materials and methods along with the results addressing the objectives of the study. Final conclusions, thesis contributions and recommendations for future research are summarized in the chapter 4.  4  Chapter 2 Literature Review 2.1 Introduction Some natural and synthetic compounds are attracting attention due to their interferences with the usual functioning of the endocrine system in humans and animals. Collectively these are called endocrine disrupting compounds (EDCs). When present in environment above a certain (threshold) concentration, these compounds can cause adverse health effects on wildlife (Hansen et al., 1998; Tyler et al., 2005). EDCs mainly consist of natural and synthetic hormones and their metabolites. Several non-steroidal and synthetic compounds e.g., plasticizer, flame retardants, surfactants, and pesticides etc., as well as some pharmaceutical and personal care products also exhibit endocrine disruption potency (Caliman and Gavrilescu, 2009). Among these different classes of endocrine disrupters, human and animal waste born hormones, often known as endogenous steroidal hormones, has been characterized by very high endocrine disrupting potency. Compared to the exogenous endocrine disrupters, such as the organochlorine pesticides and industrial compounds, endogenous hormones has been found to be 102  107 times more  potent (Legler et al., 1999; Legler et al., 2002a; Legler et al., 2002b). Municipal wastewater is the main disposal pathway for the human waste born estrogenic and androgenic compounds. In addition, synthetic hormones widely used as oral contraceptives, hormone replacement therapy and anabolic steroids are ingested in humans and after excretion enter the wastewater stream. Optimizing the removal of micropollutants like steroidal hormones is not a design criterion for conventional wastewater and sludge treatment plants. Upon wastewater treatment, stabilized biosolids (treated sewage sludge) may act as source of these micropollutants due to incomplete removal from solid and/or liquid phase of wastewater. Studying the fate of these chemicals throughout different unit treatment processes is important to determine the removal and incoming load of steroidal hormones to the environment. The goal of this chapter is to provide a state-of the-art review of the current treatment techniques employed in wastewater and biosolids treatment and their performances regarding removal of these compounds. Furthermore, the current practices and the challenges involved in analytical determination of these compounds in biosolids samples are also highlighted. 5  2.2 Properties of hormones as endocrine disrupters Natural steroids in mammals are biosynthesized from cholesterol, a 27 carbon precursor. As these hormones are lipids, following their synthesis and transportation, they pass through cell wall and bind with respective receptors to bring changes within cells. Based on the binding receptors, steroids can be categorized into five groups: androgens, estrogens, progestagens, glucocorticoids and mineralocorticoids (Martin et al., 1998). Steroidal hormones share the same tetracyclic network consisting of three six carbon and one five carbon ring (Figure 2.1). All of them have oxygen at C3 and a varied substituent at C17. For various substituent, different compounds are formed with either α or β depending on where they are situated below or above the plane of the molecule (Considine and Considine, 1984). In addition to natural hormones, a number of synthetic hormones from humans and animals, as well as some estrogen mimicking compounds derived from plants also act as endocrine disruptors. Considering their origins, these compounds can be grouped as following:   Natural steroidal hormones: estrone (E1), 17β-estradiol (E2), testosterone etc.    Synthetic  hormones:  17α-ethinylestradiol  (EE2),  diethylstilbestrol,  19-  norethindrone etc.   Phyto- and mycoestrogens: daidzein, genistein, zearalenone etc.  Table 2.1 (Liu et al., 2009a) shows potencies of some synthetic and natural estrogenic hormones relative to E2. These values are obtained using a yeast-based in vitro bioassay (YES assay). YES utilizes a human estrogen receptor recombinant engineered with a betagalactosidase reporter (Routledge and Sumpter, 1996). The potencies vary depending on the type of bioassay used and the method of determination; however studies indicate EE2 and E2 to be of most potent estrogenic compounds, followed by E1 and estriol (E3) (Folmar et al., 2002). These natural and synthetic hormones are primarily of concern because very low concentration (ng/L range) has been proven to adversely affect a number of aquatic species (Panter et al., 1998; Irwin et al., 2001). In addition, relative binding affinity determined by estrogen receptor ligand competitive binding assay (ER-binding assay) or androgen receptor ligand competitive binding assay (AR-binding assay) for a number of hormones are also presented in Table 2.1. Physiochemical properties of these compounds (Table 2.1) play a significant role in determining their fate in natural and engineered environment. As indicated in Table 2.1, steroids are poorly 6  soluble in water (Liu et al., 2009a). The octanol/water partition coefficient (Kow), defined as the ratio of concentration of a compound in n-octanol and water under equilibrium condition at a specific temperature, are also shown in Table 2.1. Since the distribution of organic compounds between water and other natural solids are often considered as a partitioning process between the aqueous and organic phase, log Kow values are used to roughly predict the sorption (Carballa et al., 2004). Log Kow values for steroidal hormones vary between 2.5 to 4.0. As a result, they are often cited as moderately hydrophobic (Jones-Lepp and Stevens, 2007) and have a tendency to partition with solid phase. Majority of the total estrogenic and androgenic hormones produced by the body are excreted in conjugated form by urinary route (Adlercreutz et al., 1987). Prior to excretion via urine, conjugated estrogens are formed by glucuronide and/or sulfate groups at the position(s) of C3 and/or C17. These polar conjugates are biologically inactive and more soluble in water compared to their corresponding free or un-conjugated counterpart. Despite conjugated estrogens being the predominant form while leaving human body, studies (D'Ascenzo et al., 2003; Gomes et al., 2005; Reddy et al., 2005) indicate free estrogens and sulfate conjugates are dominant species in the influent and effluent of WWTP. This suggests deconjugation occurs somewhere between excretion and discharge of the effluent from WWTP. In fact, deconjugation has been shown to be a natural process and fecal bacteria such as Escherichia coli (E. coli) are able to deconjugate estrogens by synthesizing large quantities of the enzyme β-glucuronidase (Adlercreutz and Martin, 1980). The recalcitrant nature of sulphate conjugates along the wastewater treatment train can be explained by weaker arylsulfatase activity of E. coli compared to β-glucuronidase (Shackleton, 1986).  7  11 1 2  4  D  H  16  H  8 14 15  B 5  13 17  C  10 9  A 3  12  7  OH  6 Cholesterol  Steroid backbone O H H  H  H H  H  Estrone (E1) OH  OH 17 alpha-ethinylestradiol (EE2) O  O  H  H  H H  O  H  H  OH Androstenedione (Ad)  H  H  O H  H H  H O  Mestranol (Ms)  OH Androsterone (An)  OH  OH  O  H  H  O Testosterone (Tr)  H  H  OH 17 beta-estradiol (E2)  OH  H  OH  OH  H  H  H  H  H H  H  H  O Norethindrone  Progesterone (Pr)  Figure 2.1 Chemical structures of some natural and synthetic hormones  8  Table 2.1 Physical and chemical properties, relative binding affinity and surrogate standard of the hormones a (Liu et al., 2009a) Hormone Allyl trenbolone Norethindrone Norgestrel Progesterone (Pr) Androstenedione (Ad)  Category  CAS  Progestogen (S) Progestogen (S) Progestogen (S) Progestogen (N)  850-52-2 68-22-4 6533-00-2 57-83-0 63-05-8  Androgen (N) Androgen (N) Androgen (N) Estrogen (N) Estrogen (S) Estrogen (N) Estrogen (N) Estrogen (N) Estrogen (N) Estrogen (N) Estrogen (N)  Mol. wt. (g/mol) 310.43 298.4 312.45 314.5 286.4  Log Kow  Melt. pt. (oC)  H (Pa.m3 .mol-1)  RBA (ER)  RBA (AR)  YES potency  -d 2.97 3.87 -  120 203-204  -  1.5×10-03  0.146  -  205-207 128.5-131  -  -  0.0359  170-173  2.0×10-11  0.33  2.41e− 3 5.7e− 4 0.685 3.2e−4  -  Androsterone (An) 53-41-8 290.4 3.69 181-184 155 Testosterone (Tr) 58-22-0 288.4 3.32 .024c 282 Estriol (E3) 50-27-1 288.4 2.45 Mestranol (Ms) 72-33-3 310.43 150-151 17α-dihydroequilin 651-55-8 270.4 155-158 Equilenin 517-09-9 266.33 258.5 238-240 0.289 1.05e− 3 Equilin 474-86-2 268.4 3.35 c 216-219 0.801 8.85e−4 .3 , .075 17α-estradiol 57-91-0 272.4 4.01 -7 173-179 6.3×10 1 0.66 1 17ß-estradiol (E2) 50-28-2 274.4 4.01 -7 6.2×10 0.44 1.3e− 3 .38c, 1 Estrone (E1) 53-16-7 270.4 3.13 254.5-256 17α-ethinylestradiol (EE2) Estrogen (S) 57-63-6 296.4 3.67 182-184 3.8×10-7 1.4 4.28e− 3 1.19c, 1.5 a CAS: Chemical abstract services number, Kow: octanol-water partition coefficient, RBA (AR)/(ER): relative binding affinity determined by androgen receptor/ estrogen receptor ligand competitive binding assay, Mol. Wt.: molecular weight, Melt. Pt.: melting point, N: natural, S: synthetic, H: Henry’s law coefficient. b (IARC, 2007). c (Teske and Arnold, 2008) d not available  9  2.3 Health effects of steroidal compounds Studies revealed that the major health effects associated with exposure of different fish species to estrogenic compounds include altered sexual development, presence of intersex species, changed mating behavior. High incidence of intersexuality has been reported in wild roach (Rutilus rutilus) population and freshwater fish walleye (Sander vitreus) in the rivers receiving effluent from domestic and municipal WWTP that contains estrogenic hormones (Purdom et al., 1994; Jobling et al., 1998; Pollock et al., 2010). Elevated plasma vitellogenin levels in fathead minnows (Pimephales promelas) after 21 days exposure to E1 and E2 at environmentally relevant concentration accompanied by an inhibition of testicular growth has been observed (Panter et al., 1998). Chronic exposure of fathead minnows to environmentally relevant concentration of EE2 resulted in feminization of the males through vitellogenin production that led to a near collapse of the species in the experimental lake area near northwestern Ontario, Canada (Kidd et al., 2007). Early exposure to EE2 at a concentration of 9.86 ng/L resulted in diminished courting behavior of female zebrafish (Danio rerio) resulting in reduced female reproductive success (Coe et al., 2010). In a field study, elevated levels of vitellogenin in female painted turtle (Chrysemys picta) exposed to E2 has been reported (Irwin et al., 2001), which may have implication in terms of reproductive fitness and shifting energy allocation from other survival needs in turtles. Phytoestrogens present on a certain strain of clover caused severe infertility in sheep grazing on them (Adams, 1998). Effect of estrogenic hormones on plant growth has been studied to a much lesser extent compared to wildlife. Treatment with E1 and E2 influenced the root and shoot growth in potato plant and sunflower seedlings, as well as, altered the morphology and flowering patterns (Janeczko and Skoczowski, 2005; Brown, 2006). Although, compared to estrogens, adverse effects of other steroids (e.g., androgen, progestogen etc.) are studied to a lesser extent, studies reported exposure to pulp and paper mill effluent in river resulted in masculinization of fish (Jenkins et al., 2003; Fan et al., 2011). Despite the compelling evidence on wildlife, adverse health effects of estrogenic hormones on human are still a debatable issue. Some studies have reported lower sperm count, declining male reproductive health and breast cancer as an aftermath of increased exposure to endogenous and exogenous estrogenic compounds (Ahmed, 2000; Delbes et al., 2006; McLachlan et al., 2006) while others refuted this, suggesting other factors, e.g. geographic variation, cultural factors, as more important players in sperm count decline than these pollutants (Fisch and 10  Goluboff, 1996; Safe, 2000). Systematic investigation of health effects on human involves many challenges, e.g. lag time between exposure and manifestation of clinical disorder, age and duration of exposure (Lopez, 2010).  2.4 Screening program and regulatory aspects of EDC The issue of ubiquitous presence of EDCs in environment and their potential to harm human and animal health has forced the government of different countries to develop testing protocols and strategies. Programs establishing testing procedure and regulatory framework for risk assessment of EDCs include, a two-tier Endocrine Disruptor Screening Program of the U.S. Environmental Protection Agency (USEPA), the Strategic Programs on Endocrine Disruptors of the Japan Environment Agency, and the Joint Working Group on Endocrine Disrupters Testing and Assessment sponsored by the Organization for Economic Cooperation and Development (Hecker and Hollert, 2011). Endocrine Disruptor Screening Program, undertaken by USEPA, is currently developing in vitro and in vivo assays to identify/clarify substances relative to their potential interaction with endocrine systems (Tier 1) and then developing dose-response relationships in animal models (Tier 2) (U. S. Environment Protection Agency, 2011). Endocrine Disrupters Testing and Assessment developed a five-level conceptual framework for testing and assessment of potential EDCs, each level corresponding to a different level of biological complexity (Organization for Economic Co-operation and Development, 2002). Canadian government is supporting both institutional and academic research through international cooperation for developing standardized test method (Office of Auditor General of Canada, 2011). In 2010, Environment Canada has published a proposed Wastewater Systems Effluent Regulations in the Canada Gazette, Part I, which is expected to reduce the discharge of micropollutants associated with WWTP effluent employing secondary or equivalent treatment (Environment Canada, 2011). While there is some progress made in the testing procedure and chemical risk assessment, no efforts have been made in terms of environmental risk assessment of these compounds concerning their discharge with the effluent in relation to drinking water safety and agricultural practices. Furthermore, to date no regulatory values exists for these compounds (Hecker and Hollert, 2011).  11  2.5 Sources and pathways of hormones in environment As previously mentioned, human and animal excreta is cited to be the main source of steroidal hormones in aquatic environment (de Mes et al., 2005; Jobling et al., 2006). The plantprocessing industry, including bio-ethanol and bio-diesel production, also significantly contributes towards the presence of phytoestrogens in surface water bodies (Lundgren and Novak, 2009). As shown in Figure 2.2, after excretion, the natural and synthetic hormones and their metabolites eventually reach WWTP. The treated solid and liquid fraction of the wastewater act as potential entry routes of these compounds in the environment. Hormones  Animal Growth promoter  Natural  Urine, faeces  Manure  Ground water  Human  Industry  Surface water  Plant processing  Natural  Wastewater stream  Dietary intake (phytoestrogens)  HRTh  Urine, faeces  WWTP  Land application, landfilling of sludge  Figure 2.2 Sources and pathways of steroidal hormones in environment (HRTh: hormone replacement therapy, WWTP: wastewater treatment plant) Among the 18 natural estrogens excreted by human urine, E1, E2 and E3 are shown to account for 66  82% estrogenicity of the urine in different population group (e.g, male,  menstruating female, menopausal female etc.). Average daily excretion rates of these three estrogenic and major androgenic hormones are given in Table 2.2. It is evident from Table 2.2 that the average excretion rate of estrogenic hormones for a menstruating female is about twice compared to a male, whereas, excretion rate of androgenic hormones are more than double in males compared to a female. However, excretion rate of estrogenic hormones can be very high 12  (upto 6 mg/day) during pregnancy in women. In addition to the natural estrogenic compounds, synthetic estrogens used in pharmaceuticals are also ingested and reach the WWTP via human waste. As a result, hormone concentrations measured in the wastewater is affected greatly by demographics of that particular community. Widely used synthetic estrogen in contraceptive pill, EE2, is considered as a major contributor to the total estrogenicity of sewage effluent (Cargouet et al., 2004; Kidd et al., 2007). EE2 is engineered from E2 by adding an ethinyl group at C17 (Figure 2.1) position resulting in a compound that is much more resistant to biodegradation compared to parent natural hormone (Clouzot et al., 2008). Johnson and Williams (2004) developed a Pharmaceutical Assessment and Transport Evaluation model integrating excretion rate data of different population groups to predict input concentrations of EE2 in WWTPs. Starting with a dose of 26 µg/day, 43% was predicted to be metabolized within the body, 27% excreted as conjugated molecules, and 30% as free form. Considering transformation and biodegradation in the pathway towards WWTP, the model estimates 40% of the total EE2, about 10.5 µg/day, reaches the sewage influent. Table 2.2 Estimates of estrogen excretion by humans (per person) in µg/day* (Johnson et al., 2000; Liu et al., 2009b) E1  E2  E3  Tr  An  Ad  Males  1.6  3.9  1.5  56.65  3340  3.4  Menstruating females  3.5  8  4.8  6.78 1570 N.A 2.3 4 1 Pregnant women 259 600 6000 *E1: estrogen, E2: 17β-estradiol, E3: estriol, Tr: testosterone, An: androsterone, Ad: androstenedione, N.A: not available. Menopausal females  Hormone replacement therapy (HRTh) involving oral intake of estrogens, progesterone and sometimes testosterone, can contribute to the total estrogenicity of municipal wastewater. HRTh is available in various forms, the most common being the conjugated equine estrogens (brand names Premarin® and Prempro®), a mixture of estrogens derived from the urine of pregnant mares. This mixture contains estrone sulfate that naturally occurs in women and metabolites of the B-ring unsaturated estrogens, equilin (Eq) and equilenin (Eqn) that are specific to horses (Tyler et al., 2009). Although there is a declining trend towards the use of HRTh since 2002, a recent study by Kim et al. (2007) showed that in the USA, approximately 17% of women aged 50 and greater (and up to 25% of women aged 50  59) remain on HRTh. Only one study has 13  been found evaluating the effect of conjugated equine estrogens from HRTh in four WWTPs in UK and reported their estrogenic potencies (Tyler et al., 2009). The same study also reported equine estrogen Eqn, and its metabolite 17β-dihydroequilenin (17β-Eqn) to be present in all the influent and 83% of the effluent samples at concentrations upto 26 ng/L. When applied to in vitro estrogen receptor in fish, their potency varied between 2.4 to 3490% of that of E2. Also 21 days exposure of Eqn and 17β-Eqn induced estrogenic responses including hepatic growth and vitellogenin production at concentrations as low as 0.6 – 4.2 ng/L. Recent studies indicated that the amount of steroidal hormones excreted by livestock is on the same order or often more than human excretion rate (Liu et al., 2012b). The yearly production of manure in the USA is 133 million tons (dry weight basis). That is 13-fold more solid waste compared to the human sanitary waste production (Burkholder et al., 2007). Different estrogens are common to different species of animals. 17α-E2, E1, E3 are found in excreta of cattle, whereas, other animal species (poultry and swine) mainly excrete E2, E1, and E3 (Wise et al., 2011). Table 2.3 shows daily excretion rate for estrogenic compounds for different livestock. Organic contaminants from animal waste can enter the environment through seepage from poorly constructed manure lagoon, overflow of lagoons in care of extreme rainfall events or runoff from recent application to agricultural fields (Burkholder et al., 2007). Several studies have reported or estimated the contribution of livestock to total estrogenicity of waterways. In UK, Johnson et al. (2006) estimated about 1% of the estrogens excreted by animals reaches water assuming the estrogenic compounds behaves like herbicides, which is about 15% of all estrogens in water. Table 2.3 Estimated total daily estrogen excretion of different livestock species (µg/day) (Wise et al., 2011) Species  Type  Total estrogen in urine (μg/day)  Total estrogen in feces (μg/day)  Total estrogen (μg/day)  Calves 15 30 45 Cycling cows 99 200 299 Cattle Pregnantb 320-104,320 256-7,300 56-111,620 Cycling sow 82 21 103 Pigb Pregnant 700-17,000 61 Cycling ewes 3 20 23 a Sheep Rams 3 22 25 a Data are estimated as total of estrone (E1), 17β-estradiol (E2), 17α- estradiol (E2), estriol (E3) excretions and include hormones from veterinary treatment (Lange et al., 2002) b Pig and pregnant cattle data are from (Johnson et al., 2006) a  14  2.6 Removal pathways for steroidal compounds The possible removal pathways of the hormones from different unit treatment processes include volatilization, biological degradation, abiotic degradation and adsorption onto solids. In the following section, these pathways are discussed in light of previous lab-scale studies. 2.6.1 Volatilization The extent of volatilization of estrogenic compounds can be predicted by the Henry’s law constant which is a ratio of the fractions of these compounds dissolved in water and those in air. Compared to volatile organic pollutant (e.g. chlorinated hydrocarbons, aromatics etc.) with value a in the range of 103 (Mackay and Shiu, 1981), estrogenic hormones have a small Henry’s law constant (Table 2.1); which makes these compounds less susceptible to volatilization under normal pressure and temperature (Khanal et al., 2006; Estrada-Arriaga and Mijaylova, 2010). 2.6.2 Biodegradation by sewage bacteria A number of bacteria for example many actinobacteria species including Arthrobacter, Mycobacterium, Nocardia, and Rhodococcus have been shown to degrade steroidal hormones (Yoshimoto et al., 2004; Donova, 2007; Yang et al., 2011). Different studies investigating biodegradation of estrogens suggest that removal is achieved by the direct use as electron donors by heterotrophs or by co-metabolism (i.e. organic compounds are only modified by microorganisms but not used for growth) by ammonia oxidizing bacteria (AOB) (Vader et al., 2000; Fang et al., 2003; Ren et al., 2007; Estrada-Arriaga and Mijaylova, 2010). Although most of these studies described degradation by first-order reaction kinetics (Shi et al., 2004; Li et al., 2005; Ren et al., 2007; Zeng et al., 2009b) as shown in eq. (2.1), few also suggest pseudo firstorder kinetics (Joss et al., 2004; McAdam et al., 2010) as in eq. (2.2). (2.1)  (2.2) Where, C is the total compound concentrations (ng/L), S is the soluble compound concentration (ng/L), t is the time (d), k is the reaction rate constant (d-1, for eq. (2.1) and L/g VSS d for eq. (2.2)), and VSS is the volatile suspended solids concentration (g/L). 15  A batch test study (Ternes et al., 1999a) using spiked levels of estrogens in diluted slurry of activated sludge (AS) collected from a full scale WWTP demonstrated that natural estrogens and their glucuronides are readily biodegradable by the microorganisms present in AS under aerobic condition. In this study, E2 spiked in ng/mL and µg/mL range was quantitatively oxidized to E1 and no further degradation products of E1 were identified before its elimination. Also, major portion of the spiked E2 glucuronides were cleaved after coming into contact with AS. In contrast, EE2 appeared to be much more persistent to biodegradation. This finding is supported by Layton et al. (2000), who investigated the removal of estrogenic compounds with laboratory mineralization assays using  14  C-labeled estrogens with biosolids from four municipal treatment  plants and one industrial system. After 24 hrs, the mineralization for and  14  14  C-17α-ethinylestradiol  C-17β-estradiol were 20% and 75%, respectively. The difference in mineralization (84%  versus 4%) of 14C-17β-estradiol using municipal and industrial biosolids in the same study also indicates the importance of adapted microbial population in biodegradation. Fuji et al. (2002) and Yoshimoto et al. (2004) isolated Rhodococcus zopfii and Rhodococcus equi from waste AS, that were able to degrade 100 mg/L of E2 to 1 mg/L after 24 hr. After investigating the fate of E2 and its 5 metabolites in a slurry of AS, Lee and Liu (2002) confirmed the biodegradation of these compounds under both aerobic and anaerobic conditions, aerobic being the more favorable with higher degradation rates. This study also affirms the previous finding of E1 being the major metabolite of E2 and identified a new metabolite X1 at the early stage of degradation. According to proposed pathway (Figure 2.3), degradation of E2 by sewage bacteria appeared to initiate at C17 at D ring, leading to the formation of E1. E1 is further oxidized into a labile metabolite with lactone structure (X1) and finally to carbon dioxide through a tricarboxylic acid cycle. Previous studies indicate Tr to be easily degraded by manure-borne bacteria under various environmental condition with mainly three degradation products (i.e., androstenedione (Ad), androstadienedione (ADD), 17β-hydroxy-androstanedieneone (DHT)). For steroids with 3-oxo4-ene structures such as Tr and Ad, degradation usually occurs through B ring cleavage involving 9β-hydroxylation and 1(2)-dehydrogenation (Donova, 2007; Yang et al., 2011). In a recent study, swine manure-borne culture has been shown to completely mineralize Tr (initial concentration 3 mg/L) to CO2 within 29 hrs, following a lag phase of 22 hrs under aerobic condition (Yang et al., 2011). In addition to the three above mentioned products, 9αhydroxytestosterone, 9α-hydroxy-ADD, 9α-hydroxy-DHT were also identified. 16  OH O  C A OH  D  B E2  OH E1  O OH  O  OH O OH OH X1 OH COOH OH  Tricarboxylic Acid Cycle Ring Cleavage  Figure 2.3 Proposed metabolic pathway of E2 by sewage bacteria (Lee and Liu, 2002) Several studies have reported that pure AOB cultures (Shi et al., 2004; Skotnicka-Pitak et al., 2009), enriched ammonia oxidizing cultures (Vader et al., 2000; Yi and Harper, 2007; Forrez et al., 2009) and nitrifying activated sludge (Ren et al., 2007; De Gusseme et al., 2009) are able to bio-transform comparatively recalcitrant synthetic estrogen EE2 at elevated concentration (> 50 µg/L), which may explain the augmented removal of estrogens in WWTP employing nitrification. The authors suggested that co-metabolism by the ammonium mono-oxygenase enzyme in presence of other organic substance was responsible for EE2 degradation. On the contrary, another study (Gaulke et al., 2008) claimed that pure cultures of AOB, such as N. europae and Nitrosospira (Ns.) multiformis, were not able to degrade EE2 at low concentrations typical for AS. The apparent degradation of EE2 reported in the previous studies was in fact abiotic transformation and heterotrophs are mainly responsible for EE2 removal in a full-scale 17  plant. However, a more recent study (Khunjar et al., 2011) demonstrated that both AOB and heterotrophic cultures are able to biodegrade EE2 (feed concentration 500 ng/L). Although, AOB biodegraded EE2 at a faster rate, heterotrophs were able to mineralize the compound as well as some AOB produced metabolites of EE2 suggesting both of these cultures may function cooperatively to enhance removal of these compounds. The effect of redox condition (i.e. aerobic, anoxic and anaerobic) on biodegradation has been also studied by Joss et al. (2004). Their study revealed that both E1 and E2 are biodegraded under all the three redox conditions, with aerobic being the most favorable for biodegradation. In contrast, substantial EE2 degradation is observed only under aerobic condition. A change in condition from anaerobic to anoxic and anoxic to aerobic corresponds to an increase by a factor 3 to 5 in E1 degradation rate, while in case of E2, this is less than 3. The biodegradation kinetic constants reported by several studies under different experimental conditions are shown in Table 2.4. Another bench-scale study under anaerobic-anoxic-oxic AS system by Li et al. (2011) substantiated E2 being biodegradable in all three conditions and EE2 accumulating under anaerobic condition. Elevated temperature has been shown to favor the degradation of estrogens (Li et al., 2005; Zeng et al., 2009b). Li et al. (2005) found a similar temperature co-efficient to that of biochemical oxygen demand, which varied between 1.026 to 1.09, for a temperature range of 5oC – 35oC during an aerobic biodegradation study of E2.  18  Table 2.4 Biodegradation kinetic constants for estrogens under different experiment condition* Hormones  E1, E2, EE2  E1, E2, E3, EE2  Agent/matrix type  Conventional activated sludge  Nitrosomonas europaea from nitrifying activated sludge  E2, EE2  Cultures established from lake water and sediments  E2  Acclimated aerobic activated sludge  Rate constants Pseudo first order rate constant, kE1: 162 ± 25 L/g SSb. d, kE2: 350 ± 42 L/g SS. d, kEE2: 8 ± 2 L/g SS. d kE1: 30 ± 10 L/g SS. d, kE2: 460 ± 60 L/g SS. d, kEE2: 1.2 ± 0.3 L/g SS. d kE1: 10 ± 1 L/g SS. d, kE1.reda: 52 ± 2 L/g SS. d, kE2: 175 ± 10 L/g SS. d, kEE2: 1.2 ± 0.3 L/g SS. d  Experimental condition  First order reaction rate constant, kE1: 0.056 h-1, kE2: 1.3 h-1, kE3: 0.030 h-1, kEE2: 0.035 h-1  Batch studies with initial concentration of 1 mg/L  Shi et al. (2004)  Initial concentration 5 mg/L, methanogenic, sulphate-, iron-, and nitrate-reducing anaerobic conditions  Czajka and Londry (2006)  Initial concentration varied 5 – 15 µg/L at temperature range 10-30oC.  Zeng et al. (2009b)  No anaerobic degradation of EE2 over long incubation period (over three years), E2 transformation rate of E2 to E1 under all four anaerobic conditions varied between 99 – 176 μgL−1day−1 First order reaction rate constant, kE2: 3.54 – 3.47 h-1 (did not change in investigated temperature change)  References  Batch studies, aerobic condition Batch studies, anoxic condition  Joss et al. (2004)  Batch studies, anaerobic condition  Nitrifying activated Pseudo first order rate constant, kE1: 38.400 ± Estrada-Arriaga sludge from Initial concentration varied E1, E2, EE2 3.601 L/gvss. d, kE2: 47.040 ± 3.924 L/gvss. d, and Mijaylova a membrane between 126 – 170 ng/L kEE2: 6.720 ± 4.101 L/gvss. d (2010) bioreactor Culture enriched First-order mineralization rate constant, Aerobic condition, 3 mg/L Yang et al. Tr from swine manure k: 0.005 to 0.072 h-1 initial concentration at 22oC (2011) * SS: suspended solids, VSS: volatile suspended solids, E1: estrone, E2: 17β-estradiol, E3: estriol, EE2: 17α-ethinylestradiol, Tr: testosterone a E1 is reduced to E2 under anaerobic condition  19  2.6.3 Adsorption Adsorption to sewage sludge is important because it represents a removal pathway with the excess sludge and often cited as the first stage of biodegradation. Batch experiments of adsorption at multiple concentrations usually involves construction of sorption isotherms and determining a characteristics value (Clara et al., 2004). Distribution coefficient (Kd) is the most commonly used characteristics value, defined as a ratio of equilibrium concentration of a dissolved adsorbate in a two-phase system consisting of sewage sludge and water as shown in eq. (2.3):  (2.3) Where, CS is the concentration in the solids in ng/kg and CW is the concentration in water in ng/l. Often Freundlich sorption isotherm is used to define partition of a compound between two phases as described by following: (2.4) KF and n are called Freundlich parameters and in case of linear isotherms (n = 1), KF corresponds to Kd value. In some studies, the Kd value is normalized to organic matter (KOM) and/ organic carbon content (KOC) of the sorbent (eqs. (2.5) and (2.6)). (2.5)  (2.6) Where, VSS and TSS are volatile and total suspended solid concentrations respectively. OC represents the percentage of organic carbon present in the sewage sludge. Clara et al. (2004) and Andersen et al. (2005) investigated sorption of E1, E2, E3 and EE2 onto activated sewage sludge and developed Freundlich sorption isotherms. From the fitted data, the Freundlich parameter, n, is found to be near unity, suggesting that the sorption of estrogenic compounds can be described by linear adsorption. The calculated distribution coefficient (log Kd) for the estrogens varied from 2.60 to 2.84 and is well within the range where sorption is a 20  relevant removal process. Despite the high initial concentration, the batch experiments did not reach saturation indicating very high adsorption potential of sewage for estrogenic hormones (Clara et al., 2004). In another study, Ren et al. (2007) described sorption of E1, E2, E3, EE2 and equol onto sewage sludge deactivated by heat treatment. Although batch experimental data fitted well to Freundlich isotherm, in contrast to previous studies, they observed sorption behavior independent of the Kow values of respective compounds. Thermodynamic analysis suggested that sorption of estrogens can be regarded as an exothermic, reversible and physical process, whereas, sorption of equol was an endothermic, irreversible, chemical process. Zheng et al. (2009b) studied sorption and biodegradation of E2 simultaneously, under aerobic conditions in both activated and inactivated sludge. The experimental data agreed well with both Freundlich and linear model, conforming to the previous studies. Concentration profiles of E2 in aqueous, solid and mixed liquor revealed that removal was achieved by sorption onto solid and subsequent biodegradation by the microorganisms. No significant difference has been found between the sorption onto activated and inactivated sludge (Clara et al., 2004; Zeng et al., 2009a; StevensGarmon et al., 2011). Measured sorption (expressed in terms of Kd and log Koc) of estrogenic and androgenic hormones were reported to be comparable between the primary sludge (PS) and WAS in a WWTP and between nitrifying activated sludge of two different WWTPs (StevensGarmon et al., 2011). For E1, E2 and EE2, Kd varied from 533 to 1550 L/kg SS, which is higher compared to the Kd (130 to 170 L/kg SS) of androgenic hormones (e.g., Tr and Ad). The effect of redox condition on adsorption has been studied by Li et al. (2011), who demonstrated that compared to anoxic and aerobic, anaerobic condition favored the sorption. Thus, recalcitrant estrogens like EE2 are prone to accumulation in sewage sludge under anaerobic condition. Zeng et al. (2009b) observed that sorption of E2 decreased with increasing temperature suggesting the reactions of exothermic nature. For a reduction of 10oC temperature, they observed 20% increases in both Kd and KF. Besides the temperature effect, Clara et al. (2004) studied sorption behavior of estrogens at high pH and found no detectable change up to pH 9. However, 30 to 50% of the initially adsorbed estrogens were desorbed between pH 9 to 10, which corresponds to the range of pKa values of the compounds studied. This is of special interest during sludge digestion which sometimes involves application of lime to elevate the pH of the sludge. Although readily biodegradable nature of most of the estrogenic hormones suggest that biodegradation plays a more significant role than sorption (Andersen et al., 2005; Li et al., 21  2005), sorption is likely to be important in the case of persistent compounds and under anoxic or anaerobic conditions. Under anaerobic condition Kd values of estrogenic compounds (E1, E2 and EE2) in digested sludge are shown to be in the same range (log Kd: 2.1  2.9) of that of in PS  and WAS. Despite the changes in operating condition of the digesters (e.g. SRT, temperature, pretreatment etc.), the sorption properties remained unaffected (Carballa et al., 2008).  2.7 Fate of steroidal compounds in engineered processes Although current WWTPs are designed on the basis of nitrogen, carbon and phosphorous removal, partial removal of EDCs have been attained in many cases. Table 2.5 presents a wide variation in removal performances of existing WWTP in different countries. These variations emphasize the importance of site-specific parameters on the removal of these compounds. The data in Table 2.5 also show the presence of these compounds in the effluents in varying concentration, which is considered to be the most significant pathway through which steroidal compounds enter the environment. This indicates the need for optimizing the existing facilities in terms of their operational parameters such as SRT, and hydraulic retention time (HRT) etc. Typical municipal WWTPs consist of preliminary, primary and secondary treatment units; in addition, tertiary treatment is often included in the case of discharging the effluent in surface water bodies. Although each unit contributes more or less to the overall removal efficiency of the plant, secondary treatment, namely biological treatment, plays the most important role in removing steroidal hormones (Andersen et al., 2003; Koh et al., 2008). In the following sections, the fates of these compounds are discussed along the treatment train in a conventional AS system. 2.7.1 Fate of steroidal compounds during conventional WWTPs 2.7.1 Preliminary treatment As preliminary treatment removes very little or no organic substances, it is unlikely to impart a significant effect of preliminary treatment on the incoming hormone loading to a WWTP (Khanal et al., 2006; Teske and Arnold, 2008). Figure 2.4 shows a conventional WWTP with the possible release or transformation of the EDCs in various unit processes indicated by dotted arrows. In addition to the PS and WAS, the liquid phase is also a potential source of EDCs. 22  Table 2.5 Occurrence and fate of some hormones in wastewater treatment plants (WWTPs) (concentration expressed in ng/L)* Target hormone  Influent  Effluent  Removal (%)  Treatment process  Sample type  References  83  ASS  Ternes et al. (1999b)  −22 to 95  ASS (12 – 14 hr)b  Flow proportional composite Flow proportional composite Time proportional composite Flow proportional composite Flow proportional composite Grab  Estrogens d  40 25 – 132  Estrone (E1)  17β-Estradiol (E2)  2.5  8.2  44  17  61 (mean)  ASS (12 – 14 hr)  29 – 670  N.Dc – 72  20 – 130  < 0.3 – 11  − 111 to 100 –  57.8 – 83.3  6.3 – 49.1  41 – 89  ASS, ASS+N, P removal, MBR ASS + N, P (chemical) removal TF, ASS  64.5 – 116.0  22.4  81 – 86  8.6  ASS, ASS+N removal, RBC, Oxidation ditch ASS  20.2  10.2  21  –  49.5 (mean) 99.9  4.0 – 25  0.35 – 3.5  59 – 98  ASS (12 – 14 hr)  11  1.6  85 (mean)  ASS (12 – 14 hr)  35 – 125  N.D – 30  44 – 100  2.4 – 26  0.2 – 14.7  17 – 150  < 0.8  − 18.5 to 98.8 –  15.67 – 82.55  0.9 – 3.9  83 – 97  ASS, ASS+N, P removal, MBR ASS, ASS+N removal, Lagoon (3 to > 150 hr) ASS + N, P (chemical) removal ASS, ASS+N removal, RBC, Oxidation ditch  ASS  Flow proportional composite Time proportional composite Time proportional composite Flow proportional composite Time proportional composite Flow proportional composite Flow proportional composite Flow proportional composite Flow proportional composite  Baronti et al. (2000) D'Ascenzo et al. (2003) Clara et al. (2005) Vethaak et al. (2005) Chimchirian et al. (2007) Ifelebuegu (2011) Zhang et al. (2011) Ternes et al. (1999b) Baronti et al.(2000) D'Ascenzo et al. (2003) Clara et al. (2005) Servos et al. (2005) Vethaak et al. (2005) Ifelebuegu (2011) Continued on next page  23  Table 2.5 Continued from previous page Target hormone  Influent  Effluent  Removal (%)  17α-E2  <0.7 – 15  < 0.4  –d  24 – 188  0.43 – 18  72 23 – 660  Estriol (E3)  17αethinylestradiol (EE2)  16αhydroxyestrone 17αdihydroequilin Androgens Androstenedione (Ad)  Treatment process  Sample type  77 – 99  ASS + N, P (chemical) removal ASS (12 – 14 hr)  2.3  97 (mean)  ASS (12 – 14 hr)  18 – 100  138 – 381  N.Dc – 275 <1 – 4.9  0.40 – 13  N.D. – 1.7  52 – 100  ASS, ASS+N, P removal, MBR ASS, ASS+N, P removal, MBR (1.2 – 13.7 hr)b ASS (12 14 hr)  < 0.3 – 5.9  < 0.3 – 2.6  –  3 – 70  N.D – 5  33.3 – 100  <0.7 – 14.4  <0.7 – 4.1  71 – 93  6.3  N.D  55  0.5 – 1.54  0.3 – 0.5  41 – 58  ASS, ASS+N removal, RBC, Oxidation ditch  1.0  -  68  ASS  Flow proportional composite Flow proportional composite Time proportional composite Flow proportional composite Flow proportional composite Flow proportional composite Flow proportional composite Flow proportional composite Flow proportional composite Time proportional composite Flow proportional composite Flow proportional composite  23.8 ± 3.2  23.6 ± 0.6  1.0  TF, ASS  Grab sample  802 ± 162  18.9 ± 5.9 8 5.5 ± 0.3  97  ASS with N and P removal  82  ASS with N and P removal  24 hr composite samples 24 hr composite samples  31.1 ± 5.1  >7  ASS + N, P (chemical) removal ASS, ASS+N, P removal, MBR ASS, ASS+N, P removal, MBR (1.2 – 13.7 hr) ASS  References Vethaak et al. (2005) Baronti et al. (2000) D'Ascenzo et al. (2003) Clara et al. (2005) Drewes et al. (2005) Baronti et al. (2000) Vethaak et al. (2005) Clara et al. (2005) Drewes et al. (2005) Zhang et al. (2011) Ifelebuegu (2011) Ternes et al. (1999b) Chimchirian et al. (2007) Fan et al. (2011) Liu et al. (2012a) Continued on next page  24  Table 2.5 Continued from previous page Treatment process  Sample type  17.1 ± 8.0  Removal (%) 99  ASS with N and P removal  Fan et al. (2011)  229 ± 10.6  N. D  100  ASS with N and P removal  62.7 ± 7.3  1.2 ± 0.2  98  ASS with N and P removal  8.9 ± 1.0  1.5 ± 0.2  83  ASS with N and P removal  24 hr composite samples 24 hr composite samples 24 hr composite samples 24 hr composite samples  33.1 ± 8.0  5.0 ± 1.1  84  ASS with N and P removal  24 hr composite samples 12.2 ± 1.9 1.1 ± 0.1 90 ASS with N and P removal 24 hr composite samples * ASS: Activated sludge system, MBR: Membrane bioreactor, RBC: rotating biological contactor, TF: trickling filter b HRT is reported in parenthesis c Not detected d Not available  Fan et al. (2011)  Target hormone Androsterone (An)  Testosterone (Tr)  Influent  Effluent  2264 ± 492  References  Liu et al. (2012a) Fan et al. (2011) Liu et al. (2012a)  Progestagens Progesterone (Pr)  Liu et al. (2012a)  25  Bar screen  Grit removal  Primary sedimentation  Secondary treatment  Secondary clarifier  Tertiary treatment Effluent  Wastewater  E-3  E-2  EDC’s  EDC’s  PS WAS  EDC’s  EDC’s  TWAS  Centrate Digester  Sludge thickener  E-7  EDC’s  E-6  Dewatering EDC’s  Sludge cake to landfill/ land application  Figure 2.4 Typical setup of a wastewater treatment plant showing wastewater sludge production and the split arrows indicate EDC release/transformation during various unit processes of treatment (Barnabe et al., 2009) (EDC’s: endocrine disrupting compounds, PS: primary sludge, WAS: waste activated sludge, TWAS: thickened WAS) 2.7.1.2 Primary treatment Possible removal pathways of steroids during primary treatment is by partitioning with fat, oil and grease and/or sorption onto PS and subsequent removal by flotation or sedimentation (Khanal et al., 2006). The degree of target compound removed is largely determined by physiochemical properties of compound, e.g. hydrophobicity, suspended solid content of the wastewater and their settling characteristics, and retention time in the settling tank etc. (Koh et al., 2008). Khanal et al. (2006) described a model to predict the removal of moderately hydrophobic estrogenic compounds (Table 2.1) by sludge sorption and sedimentation. Based on this model, mass of PS, partition coefficient of estrogens and HRT are the three governing factors for estrogen removal during primary treatment. Studies have described the fate of estrogenic compounds during primary treatment with very little (Carballa et al., 2004; Zhang et al., 2011) or no removal at all (Servos et al., 2005; Ying et al., 2008). Interestingly, some of these studies (Carballa et al., 2004; Ifelebuegu, 2011; Liu et al., 2012a) identified an increasing trend of a number hormones (e.g., E1, Ad, An, Pr, Tr) concentration after primary treatment. Deconjugation of the steroid conjugates in the aqueous phase mediated by fecal microbes (e.g, E. coli) can attribute to this phenomenon. In addition, transformation of one hormone to another 26  (e.g., E3 and E2 transformed to E1) can also contribute to this (Fan et al., 2011; Liu et al., 2012a). 2.7.1.3 Secondary treatment Secondary or biological treatment plays the most significant role in removing micropollutants like steroidal compounds in a WWTP. During biological treatment, these pollutants are adsorbed onto the micro-flocs and subsequently biodegraded by the microbial growth (Khanal et al., 2006; Zeng et al., 2009b). Therefore, both adsorption and biodegradation determine the fate of hormones in this step. However, the type of process, i.e. trickling filter, AS, bio-contactors etc., also influence the extent of biodegradation by controlling the population dynamics of the microbes. Conventional AS is the most widely used secondary treatment for municipal and industrial wastewater treatment and has been reported to show better attenuation of estrogenic hormones compared to other treatment options. Baronti et al. (2000) reported average removal efficiencies for E1, E2, E3 and EE2 to be 61%, 87%, 95% and 85%, respectively, in six Roman municipal WWTPs with AS process. Similar type of studies (Komori et al., 2004; Chimchirian et al., 2007; Tan et al., 2007; Furlong et al., 2010) suggested efficient removals for E2 and E3, ranging between 90 – 99%. On the other hand, a variable removal has been observed in case of E1; often a net increase in E1 concentration in the effluent has been reported (Baronti et al., 2000; Carballa et al., 2004; Servos et al., 2005; Hashimoto et al., 2007). Oxidation of E2 to E1 by the microorganisms under aerobic condition and stability of the estrone conjugates, especially sulfate conjugates, are presumably the main reason behind the lower removal of E1 compared to other natural estrogens. Despite being present in smaller concentration than natural estrogens, EE2 persists in the effluent (Table 2.5) because of its recalcitrant nature. Comparative studies (Ternes et al., 1999b; Servos et al., 2005; Chimchirian et al., 2007; Fernandez et al., 2007) revealed that trickling filter is less effective at removing estrogenic compounds from wastewater than AS system. In an attempt to understand the fate of estrogenic compounds, Servos et al. (2005) studied 18 municipal WWTPs in Canada and found greater concentration of E1 and E2 and increased estrogenicity in the secondary effluent of a trickling filter. Similar observations were made by Svenson et al. (2003) and Fernandez et al. (2007) while investigating the change of estrogenicity in a trickling filter using in-vitro bioassay. In another study (Jiang et al., 2005), an extensive two-stage trickling filter with two-stage post 27  sedimentation showed comparable removal with AS system. In addition to AS and trickling filter system, some other biological treatment options (e.g. bio-contactors, oxidation ditch, stabilization pond etc.) mainly used for small, rural areas have been studied for their hormone removal efficiency in several studies (Svenson et al., 2003; Hashimoto et al., 2007; Froehner et al., 2011; Ifelebuegu, 2011). These studies show that, apart from AS system, oxidation ditch is capable of attenuating different classes of steroids successfully (Ifelebuegu, 2011; Liu et al., 2012a) because of the aerobic nature of the treatment and longer SRT/HRT compared to the other processes (Hashimoto et al., 2007). 2.7.1.4 Nutrient removal processes Conventional AS system with nutrient removal process has been reported to show augmented (ranging between 80 and 100%, Table 2.5) steroidal hormone removal (Andersen et al., 2003; Fan et al., 2011; Liu et al., 2012a). Natural estrogens are shown to be degraded in both denitrifying and nitrifying conditions; nitrifying being more efficient at removing both natural and synthetic hormones (e.g., EE2) (Andersen et al., 2003). This is in agreement with the findings of Vader et al. (2000), who showed that AS enriched with nitrifying bacteria strains is capable of biodegrading EE2. In contrast to estrogens, androgens and progestagens have shown better removal in anoxic and anaerobic tanks during AS process (Fan et al., 2011; Liu et al., 2012a). A recent study (McAdam et al., 2010) reported biodegradation efficiencies for total estrogens to be 51%, 80% and 91% for carbonaceous, nitrification/denitrification and nitrification  processes,  respectively.  Despite  the  lowest  degradation  efficiency and  underdeveloped autotrophic community leading incomplete nitrification, carbonaceous AS actually showed that the highest biomass activity (µg of estrogen degraded kg-1 of biomass day1  ). This implies the importance of heterotrophic community for effective biodegradation; also  suggests that process conditions (hormone loading, SRT, HRT etc.) may play a more significant role than nitrification solely. Sludge retention time and HRT have been postulated to be important parameters for removal of steroidal compounds during biological treatment. Although, no statistical correlation exists between SRT/HRT and removal during biological treatment, an apparent higher removal is associated with longer SRT and HRT (Andersen et al., 2003; Kreuzinger et al., 2004; Servos et al., 2005; Hashimoto et al., 2007). Kreuzinger et al. (2004) hypothesized that higher SRT allows 28  the enrichment of slow growing bacteria and provides a more diverse bio-consortium, enabling increased biodegradation of micropollutants compared to biological processes with lower SRT. The resultant relationship from biodegradation data of McAdam et al. (2010) and Koh et al. (2009) suggests that > 70% biodegradation is achievable employing an SRT > 10 days and more than 80% biodegradation is possible for SRT over 20 days. Similar findings by Clara et al. (2005) points out a ‘critical SRT’ of 10 days at a temperature 10oC, below which low concentration of micropollutants cannot be achieved. In addition to SRT and HRT, Joss et al. (2004) hypothesized the importance of estrogen loading by considering a potential for limited biodegradation caused by competitive substrate inhibition. A moderate positive correlation (r2 = 0.55) between total estrogen loading and average biomass activity suggests that it is possible to observe concentration dependent removal by applying appropriate loading in a full-scale WWTP (McAdam et al., 2010). 2.7.2 Fate of estrogenic compounds during sludge stabilization Several studies (Joss et al., 2004; Andersen et al., 2005; Khanal et al., 2006) have also pointed out the tendency of steroidal hormones to partition with sludge flocs during biological wastewater treatment because of their moderately high to low log Kow (ranging between 2.5 – 4.0) value. Depending on the process utilized for volatile content stabilization, i.e. microbial degradation, chemical oxidation, application of heat, most commonly used digestion processes are anaerobic digestion, aerobic digestion, alkali stabilization, composting and heat drying (Tchobanoglous et al., 2002). The physical, chemical, biological and mechanical changes occurring during biosolids treatment train can influence the adsorption, degradation and to a smaller extent volatilization and photolysis of micropollutants (Barnabe et al., 2009). Due to the complex composition of sludge and associated matrix interferences during quantitative analysis, the number of studies describing fate of EDCs during sludge stabilization process is smaller compared to the fate studies along the liquid stream treatment train. In the following sections, fate of steroids, namely estrogenic compounds, is described during different sludge stabilization processes. 2.7.2.1 Anaerobic digestion Anaerobic digestion is a biosolids treatment method that converts the organic waste into methane-enriched biogas and fertilizer by combined action of a mixed community of 29  microorganisms. Stabilization of high-strength organic waste, production of heat or electricity by recovered methane, and reduced greenhouse gas emissions have made anaerobic digestion more favorable in today's world. As shown in Figure 2.5, anaerobic digestion is mainly a three step process: hydrolysis, acetogenesis and methanogenesis.  Suspended organics in waste samples  Fermentative microorganisms 20%  4%  Hydrolysis  76% Alcohols, carboxylic acids (except acetate) 24%  52%  Acetogenesis Acetogens CO2, H2  Methanogenesis  Acetate  Hydrogenophilic 28% methanogens  72%  Acetophilic methanogens  CH4, CO2  Figure 2.5 Steps of anaerobic digestion process (Droste, 1997) During hydrolysis, high molecular weight organic substances (e.g., protein, carbohydrates, lipids etc.) are converted to low molecular weight soluble organics (e.g., amino acids and fatty acids) by the action of exocellular enzymes. The hydrolyzed organics then splits up into alcohols and higher organic acids. These compounds are converted into simpler compounds like volatile fatty acids (VFA) (e.g., acetic, propionic, butyric, valeric), CO2 and H2 during the acetogenesis  30  phase. Finally, the methane former microorganisms produces CH4 and CO2 utilizing the products of acetogenesis phase (Botheju and Bakke, 2011). Major drawbacks of conventional anaerobic digestion, such as longer retention time requirement (20  50 days), low organic degradation efficiency (20  50%) are often thought to  be associated with hydrolysis (Tyagi and Lo, 2011). Recent developments in this field indicate that thermal (i.e., conventional and microwave heating) (Ge et al., 2010; Wett et al., 2010; Yu et al., 2010), mechanical (e.g., high pressure homogenizer, sonication, stirred ball mills etc.) (Onyeche, 2006; Wood et al., 2009; Salsabil et al., 2010) and chemical (e.g., acidic and alkaline, ozonation etc.) (Lin et al., 2009; Kim and Youn, 2011) pretreatments applied to complex organic waste (i.e. industrial and municipal sludge, manure) prior to anaerobic digestion can substantially accelerate the hydrolysis of biosolids and subsequent methane recovery in digesters with smaller volume/foot print. Different processes, e.g., bulking and foaming, dewatering, conditioning, are also shown to improve after pretreatment (Muller, 2000; Tyagi and Lo, 2011). Due to the aforementioned advantages, there are a number of proprietary advanced hydrolysis processes currently being applied at the full-scale, i.e., Cambi™, Microsludge™, Sonix™, OpenCEL™ (Jolly and Gillard, 2009; Pilli et al., 2011). Among different pretreatment techniques, thermal hydrolysis is one of the most commonly used techniques in full-scale application. Thermal energy disrupts the gel like structure of sludge, releases the lined water and improves anaerobic digester performance (Skiadas et al., 2005). In addition to increased methane recovery, improved pathogen destruction and dewaterability has also been observed after thermal pretreatment (Pino-Jelcic et al., 2006; Carballa et al., 2009; Yu et al., 2009). Although conventional heating involving high temperature and pressure has been used so far in the full-scale, a growing interest can be identified towards application of thermal hydrolysis by microwave (MW) irradiation in lab-scale digesters. This is mostly because of its volumetric and uniform heating with rapid processing time (Mudhoo and Sharma, 2011). Initially, MW irradiation (below boiling point of water) at 2.45 GHz using kitchen type MW oven has shown to effectively break the complex polymeric network of WAS and disintegrate the organic macromolecules (e.g., protein, polysaccharides and nucleic acids). Later, pretreatment of thickened WAS (TWAS) with bench-scale MW unit and pressure sealed vessel resulted in 31% higher biogas production and 75% improvement in dewaterability at a 31  temperature of 175oC (Eskicioglu et al., 2009). More recent studies have suggested that in addition to municipal sludge, degradation and methane yield of industrial sludge (e.g., pulp and paper mill, food wastes etc.) can also be enhanced using MW pretreatment before anaerobic digestion (Beszedes et al., 2011; Saha et al., 2011). 2.7.2.2 Fate of steroidal hormones during anaerobic and aerobic digestion One of the earliest studies in this field by Holbrook et al. (2002) investigated the changes in total estrogenicity using YES assay in two full-scale sludge treatment facilities employing a doublestage mesophilic anaerobic digestion (35 ± 2oC) and a combined three-stage thermophilic (55 ± 2oC) followed by two-stage mesophilic aerobic digestion. The study revealed an increasing trend of estrogenicity during both mesophilic anaerobic and thermophilic aerobic stages; only mesophilic aerobic treatment showed a slight decrease in estrogenicity. Also, the recycled liquid stream of aerobic digestion showed more estrogenicity compared with that of the anaerobic digestion and is likely to contribute to the influent estrogenicity of the WWTP. About 5 to 10% of the total influent estrogenicity was present in the processed biosolids. This is similar to the 9% of E1, 1% of E2, and 6% of total E1+E2 being removed with WAS in another study (Braga et al., 2005). In contrast to high removal rate (75% – 85%) reported in pilot-scale study (Carballa et al., 2006), Muller et al. (2010) found no significant (around 30% – 40%) removal of three natural (E1+E2+E3) hormones in a full-scale anaerobic digester with similar feed (65:35, v/v, PS:WAS). Also, EE2 concentration increased from below detection limit in the mixed sludge (feed) to > 2 ng.g-1 dw in the digested sludge indicating dewatering can result in increase of the estrogen levels in dehydrated sludge, especially in the case of the most hydrophobic estrogens, E2 and EE2 suggesting that the thermal-pressurized treatment applied for stabilization was responsible to increase the extractability of the most tightly bound estrogens. So far, the most comprehensive study in this field by Furlong et al. (2010) suggested aerobic digestion removed most of the natural and synthetic estrogenic compounds successfully and corresponding to 18% reduction of estrogenicity observed in the YES bioassay. In contrast, thermophilic and mesophilic anaerobic digestion resulted a net increase in estrogenicity as measured by YES bioassay and the contribution of estrogenic hormones to total estrogenicity more than doubled after anaerobic digestion. The well documented transformation of E2 to E1 under different redox conditions (Ternes et al., 1999a; Joss et al., 2004) could not account for high levels of E1 observed in some digested sludge samples. Therefore, the high levels of E3 led to a proposed transformation 32  pathway of E3 to E1. If E3 is a potential source of E1 formation, some sludge digestion processes may actually increase the estrogenicity while decreasing estrogen concentration, because of higher estrogenic potency of E1 than E3. Ifelebuegu (2011) analyzed mixed liquor sludge and anaerobically digested sludge in two WWTPs in UK and found a removal efficiency varying between 10% – 32% for E1, E2 and EE2. Regarding the effect of pretreatments on hormone removal, Carballa et al. (2006) and Carballa et al. (2007) studied the behavior of 13 micropollutants including estrogens during pilot-scale anaerobic digestion of 70:30, v/v, PS:WAS, preceded by different pre-treatment options, i.e. thermal, alkaline and ozonation. Despite higher biogas production and organic removal from pretreated digesters, little or no effect of operational parameters (temperature, SRT of digesters) and pretreatment condition was observed on the removal efficiencies (> 75%) of these compounds. 2.7.2.3 Chemical sludge stabilization Literature describing the fate of estrogenic compounds during sludge stabilization processes, other than aerobic and anaerobic digestion is scarce. Ivashechkin et al. (2004) suggested that alkali stabilization may actually result in removal of estrogenic compounds from the sludge phase to the supernatant due to desorption induced by high pH. In two sampling campaigns in a full-scale lime stabilization process, the lime stabilization appeared to be more effective during winter than in the summer (Furlong et al., 2010). The authors observed most of the monitored estrogenic compounds decreased in the samples; however, the total estrogenicity activity measured by YES bioassay increased approximately four fold, suggesting the need for further research.  2.8 Analytical techniques for steroidal compounds in sludge samples So far, a number of studies developed methods on analytic determination of steroids in water matrices and review of these studies can be found elsewhere (Briciu et al., 2009; KozlowskaTylingo et al., 2010). In this section only the studies reporting concentration of steroids in sludge samples has been reviewed due to the relevance to this research. Inadequacy of the studies addressing fate of the micropollutants during sludge digestion can be attributed to the difficulties associated with quantification of these compounds in sludge phase. Inherent complexity associated with the composition of sludge and very low concentration of these pollutants make it challenging to measure the trace organic contaminants, e.g., estrogens, androgens etc. with high 33  accuracy and confidence (Citulski and Farahbakhsh, 2010). Detection and measurement of different natural and synthetic hormones in any sludge sample involves sample stabilization, extraction of the target compounds from sludge matrix, cleaning up the sample and finally quantification. Although sample preparation and adequate clean up steps are of immense importance to minimize the matrix interference during the final quantification step, extensive extraction and clean up may as well result in loss of analytes (Gomes et al., 2004). After collection, immediate stabilization of the sludge samples is necessary to minimize the microbial activity. Otherwise, degradation of the target compounds, e.g., conversion of E2 to E1, would cause inaccurate measurements (Gomes et al., 2004). Previous literature suggests use of lyophilisation or freeze drying (Ternes et al., 2002; Muller et al., 2008; Nieto et al., 2008; Chiu et al., 2009), autoclaving (Braga et al., 2005) and addition of formaldehyde (Muller et al., 2010) as options for sample preservation techniques. Quantitative analysis of steroidal compounds in biosolids requires effective as well as selective extraction of target analytes from the matrix constituents. Traditional extraction techniques such as the well-known Soxhlet extraction, sonication (Ternes et al., 2002; Braga et al., 2005; Fan et al., 2011; Liu et al., 2011) and rotary shaking (Gomes et al., 2004; Chiu et al., 2009) have previously been used to extract these compounds from sludge samples. In terms of solvent selection, non-polar and polar aprotic organic solvents such as hexane, acetone, ethyl acetate, are usually used during solvent extraction because of the moderate hydrophobic nature of the target compounds (Citulski and Farahbakhsh, 2010). The major drawbacks of the traditional extraction techniques include longer extraction time and very high solvent consumption. However, a more recent technique called accelerated solvent extraction (ASE) is now being increasingly used for extracting estrogens from sludge samples (Muller et al., 2008; Nieto et al., 2008; Fernandez et al., 2009). In ASE, pressure is applied in the extraction cells to achieve high temperature. By decreasing viscosity and increasing diffusion coefficient of the solvent, high temperature disrupts the analyte-matrix interaction. Also, the high pressure provides better penetration of the solvent in the matrix, which in turn increases the extraction efficiency (Camel, 2001). Microwave assisted extraction (MAE) is another technique that has been used in several studies to extract estrogenic compounds from river and marine sediments (Liu et al., 2004; 34  Labadie and Budzinski, 2005; Hibberd et al., 2009). Greater than 80% recovery in sediment samples (Matejicek et al., 2007; Hibberd et al., 2009) promotes MAE as a good candidate to extract estrogenic compounds from sludge samples as well. Compared to traditional methods, MAE has the advantage of smaller solvent requirements, reduced extraction time, and multiple sample extractions (Hibberd et al., 2009). Although, the recommended solvents for Soxhlet and sonication can be used for ASE (Camel, 2001), special attention has to be given to the microwave absorbing properties of solvents in MAE. The selected solvent should be able to absorb microwaves but should not over-heat the sample, which may lead to degradation of the analytes. Polar solvents (e.g. methanol, water) have been reported to perform efficiently in MAE (Liu et al., 2004; Labadie and Budzinski, 2005; Matejicek et al., 2007). Due to the reasons explained above, ASE and MAE are advantageous over traditional techniques given that operating parameters, e.g, temperature, pressure, and solvent volume, are optimized considering the nature of sludge matrix that needs to be analyzed. Mass of the sample used evinced as an important factor to affect recovery of these micropollutants from sludge samples according to related previous studies. Literature suggests extraction of sample amount in a range of 0.1 g to 1 g (Table 2.6). Chiu et al. (2009) studied the effect of sample size (0.1 g, 0.2 g and 0.5 g) on the recovery of estrogens from PS samples and observed that 0.1 g sludge sample provided > 84% recovery with excellent repeatability. During quantification of micropollutants, an increase in sample mass is usually associated with increased co-extracted compounds from the matrix and results in matrix interferences during detection techniques (Citulski and Farahbakhsh, 2010). To minimize the effect of co-extracted compounds on quantification, extensive cleanup steps are necessary. As shown in Table 2.6, column chromatography with alumina, silica and florisil as the stationary single layer or in combination has been previously used. Gel permeation chromatography (GPC) is another widely used cleanup technique. In contrast to column chromatography, where separation is achieved by polarity of the compounds, GPC separates the analytes based on their molecular size and is often known as size exclusion chromatography. However most widely used cleanup techniques include solid phase extraction (SPE) alone or in combination with chromatographic techniques. During SPE, the solid phase acts as a selective adsorbent media and separates the analytes from liquid sample. After washing the solid phase to remove any unwanted compounds that has been adsorbed with the target analytes, elution with specific organic solvent is done (Chang et al., 2009). Several 35  studies (Ternes et al., 2002; Gomes et al., 2004; Braga et al., 2005) have used octadecyl (C18)bonded silica adsorbent cartridge which is common for reverse phase extraction of non-polar to moderately polar compounds. In addition, another widely used (Muller et al., 2010; Salgado et al., 2010) sorbent is a copolymer of divinylbenzene and vinylpyrrolidone, which has been commercialized under the trade name of OasisTM-HLB by Waters (Buchberger, 2007). The main reason behind the widespread use of Oasis-HLB is the simultaneous extraction of a wide range of compounds (i.e. acidic, neutral and basic) at neutral pH (Weigel et al., 2004) by a single SPE adsorbent. In addition to the aforementioned solid adsorbents, few studies reported use of aminopropyl bonded silica (LC-NH2) or anion exchange SPE to cleanup the samples after extraction. The selection of eluting solvent to clean and concentrate the analytes must be done carefully. Due to the variation in polarity of steroidal hormones, more polar compounds such as E3 and estrogen conjugates strongly binds them with the stationary phase and needs polar solvents for full recovery (Chiu et al., 2009). Upon extraction and cleaning, quantification of estrogenic compounds in solid matrix has been achieved by using gas chromatography (GC) or liquid chromatography (LC) followed by mass spectrometry (MS) (Table 2.6). Usually GC/MS(/MS) or LC/MS(/MS) can measure environmental concentration of these micropollutants down to lower ng/L level if efficient extraction and adequate cleanup of the sample are followed (Richardson, 2008). Use of GC/MS in quantifying different EDCs including estrogens is well established. The usual mode of ionization, such as electron impact or chemical ionization, are less susceptible to matrix interferences compared to LC/MS (Buchberger, 2007). However, non-volatile nature of steroidal compounds call for derivatization to give sufficient volatility prior to the injection in GC. For estrogens, silylation is the most commonly used derivatization technique. Among a large number of available silylation reagents, N-(trimethylsilyl) trifluoroacetamide alone or in combination with catalysts, for example trifluoroacetamide/trimethylsilylimidazole/dithioerytrol, is frequently used (Ternes et al., 2002). As shown by Ternes et al. (1999b), use of single GC/MS has a larger possibility of overestimating the environmental concentration, whereas a tandem MS provides better selectivity and requires smaller amount of sample thus decreasing the chances of matrix interferences. Despite the GC being a well established analytical technique, a recent trend towards widespread use of LC/MS (/MS) can be identified. The main advantage of LC is no derivatization, therefore avoids the chances of errors due to side reactions during derivatization 36  (Buchberger, 2007). The major drawback is that LC/MS suffers from matrix effects (ion suppression) due to the co-eluting residual matrix constituents, where electrospray ionization (ESI) interface is used and not properly accounted for (Villagrasa et al., 2007). In a recent study, Chiu et al. (2009) has shown that more polar estrogens (E3, E1) exhibited the least signal suppression (8 – 18% versus 10 – 28%) compared to more hydrophobic estrogens such as E2, EE2. Nonetheless, literature proves that extensive cleaning of the samples can actually reduce ion suppression and reliable quantification in the ng/L range can be achieved (Nieto et al., 2008; Chiu et al., 2009). Recent development in this field includes use of ultrafast method such as laser diode thermal desorption-atmospheric pressure chemical ionization (LDTD-APCI) coupled to tandem mass spectrometry (MS/MS) (Viglino et al., 2011) to analyze estrogens in different solid matrices including biosolids. This technique uses infrared laser diode to desorb the analyte thermally under atmospheric pressure condition followed by ionization using APCI and introduce the ions directly to the MS thus eliminates the requirement and limitations of liquid mobile phase (Segura et al., 2010). This type of high throughput method can be used for faster and effective quantitative analysis as it is capable of reducing the 5 to 30 min runs of LC/MS/MS to 10 to 30 sec with good method performance (Viglino et al., 2011).  37  Table 2.6 Analytic techniques for estrogenic compoundsª Sample DS and WAS  Sample dry weight (g) 0.5  Extraction  Cleaning  Analytical technique GC-MS/MS  Method recovery ratio (%) > 70  References  Solvent extraction GPC followed by Ternes et al. (2002) with sonication silica gel clean up DS 0.2 Accelerated solvent 5% deactivated LC-MS/MS >80 Fernandez et al. (2009) extraction florisil cleanup WAS 0.5 Solvent extraction GPC followed by LC/MS or >75 Gomes et al. (2004) with rotary shaker SPE GC/MS (twice) PS 0.1 Solvent extraction SPE, GPC, anionLC-MS/MS >84 Chiu et al. (2009) with rotary shaker exchange SPE PS and WAS Accelerated solvent 2 step SPE GC-MS >90 Muller et al. (2008) extraction Raw sewage 50 mL sludge Solvent extraction SPE GC-MS 75-95 Braga et al. (2005) sample with sonication Sludge 1.0 Accelerated solvent Microfiltration LC-MS/MS >81 Nieto et al. (2008) extraction ªDS: digested sludge; WAS: waste activated sludge; PS: primary sludge; GC: gas chromatography; MS: Mass spectrometry; LC: liquid chromatography; SPE: solid phase extraction; GPC: gel permeation chromatography  38  2.9 Summary This chapter presented a review of the sources and occurrence of steroidal compounds as well as their fate during the entire wastewater treatment train. Different batch- and full-scale plant studies indicated biodegradation and sorption of estrogenic and androgenic compounds onto the sludge as relevant removal pathways in WWTPs. Although numerous studies investigating fate of steroidal hormones exist in liquid treatment train of the WWTPs, a research gap has been identified regarding the fate studies during sludge stabilization. This may have been caused mostly because of the difficulties associated with quantifying these micropollutants in sludge at very low level concentrations. Very few studies available in this field investigated the removal and change in estrogenicity during conventional aerobic and anaerobic digestion only. Although, studies indicated inefficient removal from solid phase especially in case of anaerobic digestion, effects of operating parameters, e.g. temperature, SRT, on the attenuation process are not well understood. Also, fate and removal of steroidal hormones are yet to be studied during emerging advanced sludge digestion process for example anaerobic digestion preceded by thermal, mechanical or chemical pretreatments; that are applied at the full-scale for enhanced biodegradation, pathogen removal and sludge dewaterability. Moreover, data on androgenic and progestogenic hormones are scarce. Considering these, the intent of this research was to study the fate and removal of steroidal hormones during anaerobic digestion of municipal waste sludge followed by MW pretreatment (hydrolysis) at a range of pretreatment temperature and digester temperature and SRTs.  39  Chapter 3 Effect of microwave hydrolysis on transformation of steroidal hormones during anaerobic digestion of municipal sludge cake This chapter aims at quantification of steroidal hormones in Kelowna’s municipal sludge cake before and after conventional and advanced digestion coupled with a thermal hydrolysis for enhanced biodegradation and improved digestate (remaining material after digestion utilized as soil amendment) quality. The City of Kelowna operates a sewage treatment plant called Kelowna Pollution Prevention Centre (KPPC). The solid by-products (waste sludge) from the KPPC, produced at an average rate of 2.5 truckloads a day, are dewatered, and then hauled to a composting facility located near the City of Vernon. The stabilized compost material is marketed under the name of Ogogrow as a soil amendment. In recent years, with the alignment of the BC’s Energy Plan for reducing greenhouse emissions and for the goal of obtaining energy selfsufficiency by 2020, the City has begun to consider the implementation of an anaerobic digester, an alternative biosolids treatment method to composting. Anaerobic digester converts organic waste, such as biosolids, into a methane rich biogas and fertilizer by a group of microorganisms. Under this scenario, methane recovered from the biosolids during anaerobic digestion can be utilized as part of the landfill biogas system for electricity generation. The anaerobic digester would achieve around 50% volume reduction of biosolids (experimentally determined in this chapter) and the remaining materials part can be utilized as fertilizer or hauled to the composting facility for further treatment depending on the quality of soil amendment desired. The benefits of implementing an anaerobic digester versus maintaining the status quo are unclear from the point of removal of contaminants of emerging concern, i.e. hormones, personal care products, pharmaceuticals. The strategic decisions concerning the waste management options require laboratory testing for quantification of target compounds (main goal of this chapter) before and after different digester scenarios. Therefore, the objective of this work was to study the effect of microwave (MW) hydrolysis on fate and removal of 16 steroidal hormones (synthetic and natural) during advanced anaerobic digestion of municipal sludge cake (biosolids). The effect of sludge cake pretreatment temperature (80, 120, 160oC), digester operating temperature (mesophilic at 35 ± 2oC, thermophilic at 55 ± 2oC) and digester sludge retention 40  time (SRT; 20, 10, 5 days) were evaluated using eight lab-scale semi-continuously fed anaerobic digesters. To determine the potential effect of MW hydrolysis, hormones were quantified in sludge (total) and supernatant (soluble) phase of the digester influent and effluent streams. The results showed that, 7 out of 16 hormones were above reporting limit (RL) in one or more of the samples. Hormone concentrations upto 1,640 ng/L and 2491 ng/g (for androstenedione, (Ad)), respectively, were detected in soluble and total phases of the influents. Microwave hydrolysis resulted in both release and attenuation of hormones in the soluble phase. High accumulations (upto 30 times for androstenedione (Ad) of the influent concentration) of hormones observed in the effluents of un-pretreated (control) digesters suggested that anaerobic digestion was inefficient to remove these compounds. This agrees with the previous studies showing anaerobic digestion resulted in increased estrogenicity of digested sludge. Simultaneous accumulation and removal of 17β-estradiol (E2) and estrone (E1) as well as progesterone (Pr) and androstenedione (Ad) indicated possible transformation among the hormones. MW pretreatment also enhanced volatile solids (VS) removal and methane production compared to unpretreated (control) digesters; however, improvements were more prominent at the shortest SRT (5 days).  3.1 Materials and methods 3.1.1 Substrate and inocula Sewage sludge cake was collected bi-weekly from Kelowna Pollution Control Center (KPCC) (British Columbia (BC), Canada), currently serving more than 94,000 population. In this WWTP (Figure 3.1) after preliminary treatment and primary sedimentation, wastewater flows into a modified Bardenpho unit designed to remove C, P and N. The secondary effluent then undergoes UV disinfection before discharging into the Okanagan Lake. Fermented and gravity thickened primary sludge (PS) and waste activated sludge (WAS), thickened by a dissolved air flotation unit, are pumped separately and mixed (40:60, v/v) before dewatering by centrifugation. The dewatered sludge cake with a total solids (TS) content of 17.5 ± 1% (w/w) is used for MW pretreatment to minimize the input energy requirement per dry weight. MW pretreated and unpretreated (for controls) sludge cakes were mixed with leachate (< 1% TS, w/w) collected from Glenmore landfill (BC, Canada) and tap water (3:5 in liquid mixture) to attain a typical feed 41  concentration of 3.4% TS (w/w) for anaerobic digesters. Due to restrictions on land availability and limitations on expansion at the KPCC, a potential digester may be located at the local landfill (~ 15 km from the KPCC). Therefore, in future excess leachate of the landfill can be utilized to dilute the sludge cake stream into the digester.  RS pump house  RS  Primary sedimentation  Pretreatment  RS  Modified bardenpho unit  Secondary Filtration and clarifier UV disinfection Effluent (to Okanagan lake)  SRS RAS  Centrate PS WAS  GTOF Gravity thickener fermenter  TWAS  TOF Dissolved air flotation  Centrifuge  Sludge cake to landfill  Sampling point  Figure 3.1 Existing process flow diagram in City of Kelowna wastewater treatment plant (GTOF: gravity thickener overflow, PS: primary sludge, RAS: return activated sludge, RS: raw sewage, SRS: screened raw sewage, TOF: thickener overflow, TWAS: thickened waste activated sludge, WAS: waste activated sludge) For the bench-scale digester studies, the mesophilic and thermophilic inocula were collected from the full-scale anaerobic digesters located at Penticton and Annacis Island WWTPs (BC, Canada), respectively. Prior to actual digester experiments, the inocula were acclimatized to MW pretreated sludge at a temperature of 175oC to avoid any possible acute inhibition at elevated hydrolysis temperatures. During acclimation, the initial organic loading rate (OLR) of the digesters was 1.58 ± 0.27 g chemical oxygen demand (COD)/L/d at a safe SRT of 20 d. This loading was doubled gradually with a feed concentration ranging between 1.71 ± 0.15% to 3.65 ± 0.30% TS (w/w). Once the digesters reached steady state (> 10% variation in biogas 42  production) at the final feed concentration, they were maintained at the same condition until the experimentation began. This whole preparation period lasted for a total of 7 months. 3.2.2 Microwave pretreatment A closed-vessel microwave digestion system (ETHOS-EZ, Milestone Inc., Connecticut, USA) operating at 2.45 GHz with maximum power, temperature and pressure of 1200 watts, 300oC and 35 bars, respectively was used. ETHOS-EZ is equipped with ATC-400-CE (thermocouple) temperature probe housed in a reference vessel. The unit has 12 pressure sealed vessels (100 ml each) rotating in a carousal. ETHOS-EZ is easily programmable to heat the sample at different ramping rates and holding times, which allows for optimization of thermal hydrolysis (Figure 3.2). During hydrolysis, municipal sludge cake was placed in 12 Teflon vessels (45 g each) and irradiated to final desired temperatures of 80, 120 and 160oC. This range of temperature was selected to investigate the thermal effect both above and below boiling point. In order to make the heating profile comparable, a uniform rate of ramping (7.5oC/min), a constant holding time (1 min) and venting time (25 min) were applied in all three temperatures. After venting, the vessels were cooled down to room temperature (20  22oC) before opening to avoid any loss of  organics by evaporation. The MW irradiated sludge was stored at 4oC for digester feed preparation. A previous study investigating the effects of pretreatment temperature (110, 150 and 175oC) and ramping rate (3.75 and 7.5oC/min) demonstrated for concentrated TWAS (11.85% TS) that the ramping rate was not a significant factor in consideration to increased solubilization (Toreci et al., 2010). In accordance with this, the higher ramping rate (7.5oC/min) was chosen, which could also minimize the energy input/loss during heating. Table 3.1 displays the characterization of digester feed streams before and after MW pretreatment and dilution.  43  a)  Temperature probe  Teflon vessel  Controller  180  b) Temperature (degree C)  160 140 120 100 MW 80  80  MW 120  60  MW 160  40 20 0 0  20  40  60  Time (min)  Figure 3.2 a) Ethos microwave station (2.45 GHz, 0  1200 Watt, 25  300oC, 0  35 bars); b)  heating and cooling profiles of the microwave unit (MW: microwave, 80, 120 and 160: ultimate pretreatment temperatures in oC unit)  44  Table 3.1 Characterization of raw (control) and pretreated digester feed after dilutiona  Parameter  Control  MW80  MW120  MW160  pH  6.66 (0.38, 12)b  6.71 (0.31, 12)  6.75 (0.02, 12)  6.56 (0.25, 12)  TS (%,w/w)  3.48 (0.31, 24)  3.38 (0.23, 24)  3.37 (0.28, 24)  3.42 (0.19, 24)  VS (%, w/w)  2.74 (0.22, 24)  2.84 (0.01, 24)  2.78 (0.2, 24)  2.85 (0.21, 24)  TCOD (mg/L)  34,743 (1677, 24)  33,149 (3495, 24)  37,611 (1589, 24)  33,786 (3327, 24)  SCOD (mg/L)  3,222 (67, 4)  5,342 (62, 4)  6,324 (17, 4)  7,084 (125, 4)  Alkalinity (mg CaCO3/L)  1636 (150, 3)  1199 (86, 3)  953 (46, 3)  968 (58, 3)  NH3-N (mg/L)  668 (21, 2)  640 (15, 2)  305 (46, 2)  430 (26, 2)  Acetic acid (mg/L)  795 (343, 3)  552 (316, 3)  426 (17, 3)  241 (80, 3)  Propionic acid (mg/L)  390 (301, 3)  187 (75, 3)  98 (45, 3)  69 (39, 3)  Volatile Fatty Acids  Butyric acid (mg/L)  38 21 10 8 (22, 3) (0, 3) (1, 3) (4, 3) a TS: Total solids, VS: volatile solids, TCOD: total chemical oxygen demand, SCOD: soluble chemical oxygen demand b arithmetic mean (standard deviation, number of data points)  3.2.3 Digester studies A total number of eight, side-armed erlenmeyer flasks (Figure 3.3) with total and liquid volumes of 1 and 0.5 L, respectively, were used as semi-continuous flow digesters (fed once a day). Erlenmeyer flasks were sealed with rubber stoppers and side arms were used to feed the reactors. One sampling port was bored into the rubber stopper to withdraw the sludge from the digester. The second sampling port was connected to 2 L Tedlar bag for biogas collection. The Tedlar bags were equipped with on/off valves and a septum fitting that was used for gas composition sampling. Volume of daily biogas collected was measured by a manometer. Four of the digesters were operated at the mesophilic temperature (35 ± 2oC) and the remaining four were kept at the thermophilic condition (55 ± 2oC) in temperature controlled shakers (90 rpm). One of the four digesters at each operating temperature was fed with un-pretreated sludge and was called control, while rests of the three were fed with pretreated sludge at 80, 120 and 160oC temperature. The 45  digesters were run for SRTs of 20, 10 and 5 days. At each operational stage, once the steady state (< 10% variation in biogas production rate) has been reached, the digesters were maintained under the identical loading for over a period of three SRTs. Effluents were collected during this time as 7-day composite samples in LDPE bottles and refrigerated at 4oC for hormone analysis. Chemical oxygen demand (COD), TS, volatile solids (VS), pH, alkalinity, ammonia, total volatile fatty acids (TVFAs) of effluent and feed along with biogas composition were monitored twice a week. Digester pH and biogas volumes were measured daily.  Figure 3.3 Two of the eight lab-scale semi-continuous anaerobic digesters  3.3 Analytical techniques 3.3.1 Characterization of influent and effluent Total solids (TS), VS, alkalinity and ammonia were analyzed according to Standard Methods procedure 2540B, 2540E, 2320B and 4500D, respectively (APHA, 2005). The closed reflux colorimetric method was used for COD measurement according to procedure 5250D (APHA, 2005) using Spectronic 20D+ (Thermo-Electron Corporation) spectrophotometer and 600 nm wavelength absorbencies. Sludge samples were centrifuged for 15 min at 8,000 rpm and the collected supernatants were used for both ammonia and alkalinity. However, for soluble COD (SCOD) determination, the supernatants were further filtered through 0.45 µm pore-sized membrane discs. 46  To measure the TVFAs (summation of acetic, propionic and butyric acids), the supernatants were first filtered through membrane discs (0.2 µm pore size) and then injected into an Agilent 7890A Gas Chromatograph (GC). The GC contains a capillary column (Agilent 19091F-112, HP-FFAP polyethylene glycol TPA column length x ID: 25 m × 320 µm) and a flame ionization detector (oven, inlet and outlet temperatures: 200, 220 and 300oC, respectively, carrier gas flow rate: 25 mL helium/min) and is equipped with an auto-sampler (Agilent 7693A). The method developed by Ackman (1972) used iso-butyric acid as an internal standard. An Agilent 7820A GC with a thermal conductivity detector (oven, inlet and outlet temperatures: 70, 100 and 150o C, respectively) and packed column (Agilent G3591-8003/80002) was used to determine the biogas composition (CH4, CO2, O2, N2 percentages) in the headspace of digesters using helium as the carrier gas (flow rate: 25 mL/min). The method was developed by Van Huyssteen (1967). 3.3.2 Hormone analysis Digester effluent and influent samples were centrifuged at 8,000 rpm for 20 min and the supernatants were collected and filtered through 1 µm filter papers. The filtrate (< 1 µm), which represents the soluble fraction of sample, along with collected influent and effluent samples were used for hormone analysis (Figure 3.4). Digester operation, sampling, centrifugation/filtration was done at the Environmental Engineering Laboratory in the School of Engineering (UBC Okanagan Campus) and samples were shipped in coolers packed with dry ice via overnight courier to a commercial laboratory (AXYS Analytical Services Ltd., Sidney, BC) for extraction/clean-up and subsequent hormone quantification by an LC-MS/MS. Currently, an LCMS/MS is not available at the School of Engineering. Firstly, samples were spiked with surrogates (Table 3.2) and adjusted to the required pH (2.0) to prevent deuterium-hydrogen exchange occurring between the sample and labeled surrogates. The sludge samples were extracted by sonication with aqueous buffered acetonitrile and with pure acetonitrile, concentrated by rotary evaporation, and diluted with ultra-pure water to 200 mL. Aqueous samples and the diluted extracts of sludge samples were cleaned-up using hydrophilic lipophilic balanced (HLB) solid phase extraction (SPE) cartridges. After addition of recovery standards, the extract was filtered and analyzed by Waters 2795 (or Waters 2695) High Pressure Liquid Chromatograph equipped with a Micromass Quattro Ultima Mass Spectometer and workstations 47  running QuanLynx/Masslynx software (LC-MS/MS). The instrument described required a total of two different LC runs in positive and negative ionization modes to analyze a total of 16 hormones displayed in Table 3.2. Instrument calibration was performed using a series of calibration solutions (6 points) covering the working concentration range of the instrument (0.2 ng/mL to 3,000 ng/mL) specific for the individual compounds of interest. The LC-MS/MS was run in multiple reaction monitoring mode and quantification was performed by recording the peak areas of the applicable daughter ion of specified parent ion/daughter transitions (AXYS, 2012). The detailed extraction and cleanup steps are provided in Figure 3.5. The concentrations obtained from the analysis were reported in ng.g-1 dry weight (total phase) and ng.L-1 (soluble phase) unit. Ongoing precision recovery (OPR) standards (spiked matrix of the analytes) and procedural blanks were used for quality control purpose. Laboratory performance in terms of OPR recovery and blank levels were compared to established performance criteria (Appendix B, Table B.1). Method detection limits (MDLs) of quantification (Table 3.2) were the levels, at which analytes could be detected in absence of interferences. However, due to complex nature of sludge matrix, a number of compounds other than target analytes present in the sample may affect detection performance. To account for the matrix interferences, sample specific detection limits (SDLs) of the target hormones were determined for each sample run. The SDLs varied mainly depending on the sample type and size and were calculated as the analyte concentration corresponding to a peak with 3:1 signal to noise ratio. The reporting limit (RL) for each target compound was defined as the lowest calibration standard which is higher than MDL, or the SDL, whichever was greater. 3.3.3 Calculations For more than duplicate data points, mean and standard deviation were reported. To determine the significant factors, multi-factor analysis (digester operating temperature, pretreatment temperature and SRT) of variance was used, with p ≤ 0.05 for hormones Ad, Pr and E1. Removal efficiency [based on eq. (3.1)] was considered as the response variable for these analyses. During removal efficiency calculations, below detection limits concentrations were assumed to be equal to the RL for that specific sample. Also, total phase concentrations of the analytes initially determined in ng/g were converted into ng/L unit using their TS concentrations (Appendix B, 48  Table B.4) for removal efficiency calculation. As the digester feed was prepared in the same manner, and feed characterization results in terms of typical parameters, i.e., TS/VS, CODs etc. showed variations less than ± 9% during the experimental period (January  May, 2012),  influent hormone concentrations were assumed to remain the same in the digester feed for the three SRTs studied. As previously mentioned, digester effluent samples were 7-day composite samples. Removal efficiency (%) =  (3.1)  Where, Ci is the concentration of hormone in total (ng/L) or soluble (ng/L) phase of the influent, Ce is the concentration of hormone in total (ng/L) or soluble (ng/L) phase of the effluent. Sewage sludge cake (17% TS, w/w)  MW pretreatment at 80, 120 and 160oC  Digester feed (3.5% TS, w/w) preparation with pretreated sludge  Feed/influent sampled (sludge or total phase)  Feeding semi-continuous anaerobic digesters  Collection of effluent from digesters  Centrifugation and filtration of supernatant (soluble phase)  Quantification of target hormones in total and soluble phases  Effluent sampled (sludge or total phase)  Centrifugation and filtration of supernatant (soluble phase)  Figure 3.4 Experimental methodology showing sampling locations (MW: microwave, TS: total solids)  49  Table 3.2 Method detection limits, reporting limits and quantification references for analytes, surrogate standards* Matrix Liquid (supernatant) Solid (sludge) ng/L (0.250 L), (4000 uL) ng/g (0.5 g), (4000 uL) Units (sample size), (extract volume) Quantified against Analyte (ESI positive) MDL RL MDL RL Allyl Trenbolone 2.64 3.2 1.32 1.6 d6-Norethindrone Androstenedione 1.76 0.88 4.0 d6-Norethindrone 8.0 Androsterone 30.3 15.1 40.0 d6-Norethindrone 80.0 Estriol 175 88 120.0 d6-Norethindrone 240 Mestranol 100 50 32.0 d6-Norethindrone 64.0 Norethindrone 2.58 1.29 40.0 d6-Norethindrone 80.0 Norgestrel 6.1 3.1 8.0 d6-Norgestrel 16.0 Progesterone 3.02 1.51 8.0 d9-Progesterone 16.0 Testosterone 1.57 0.79 1.6 d6-Norethindrone 3.2 Analyte (ESI negative) 17α-Dihydroequilin 8.3 4.1 d4-17β-Estradiol 16.0 8.0 Equilenin 2.64 1.32 d4-17β-Estradiol 3.2 1.6 Equilin 11.7 5.9 d4-17β-Estradiol 32.0 16.0 17α-Estradiol 6.6 16.0 3.3 8.0 d4-17β-Estradiol 16.0 8.0 17ß-Estradiol 14.9 7.4 d4-17β-Estradiol 16.0 8.0 Estrone 11.7 5.9 d4-17β-Estradiol 20.0 10.0 17α-Ethinylestradiol 38.5 19.25 d4-Ethinylestradiol Surrogate standard 13 d6-Norethindrone C3-Atrazine 13 d6-Norgestrel C3-Atrazine 13 d9-Progesterone C3-Atrazine 13 d4-17β-Estradiol C6-2,4,5-Trichlorophenoxyacetic acid 13 d4-Ethinylestradiol C6-2,4,5-Trichlorophenoxyacetic acid Recovery standard 13 C3-Atrazine External standard 13 C6-2,4,5-Trichlorophenoxyacetic acid External standard *MDL: method detection limit (estimated), RL: reporting limit (based on lowest calibration point), ESI: electrospray ionization, N. A: not applicable  50  Solid sample (≈ 5 g, wet)  Addition of 15 mL pH 2.0 phosphate buffer and labeled compounds, vortexing  Addition of 20 mL CH3CN, sonication of 30 min, centrifuge (approx. 5 min at 3000 rpm)  Addition of 15 mL pH 2.0 phosphate buffer and 20 mL CH3CN, vortexing, sonication, centrifugation  15 mL CH3CN, vortexing, sonication, centrifugation  Concentration of extracts by rotary evaporation to 20-30 mL at 50oC, dilution with 200 mL reagent water, addition of 500 mg Na4EDTA  Loading (5-10 mL/min) of solid extracts and liquid samples to SPE HLB conditioned with MeOH (20 mL), Mili-Q water (6 mL), acidified (pH 2.0) Mili-Q water (6 mL)  Washing cartridge with 10 mL water, drying for 5 min, eluting with 12 mL MeOH, 6mL acetone: MeOH (1:1), drying under N2 stream  Reconstituting in 3 mL MeOH, injection of internal standards, diluting to 4 mL with 0.1% formic acid buffer, vortexing  Analysis by LC/MS/MS in MRM mode under ESI+ and ESI- condition  Figure 3.5 Flow chart of analytical determination of steroidal hormones in solid and liquid samples (SPE: solid phase extraction, HLB: hydrophilic lipophilic balanced, MRM: multiple reaction monitoring, ESI: electrospray ionization) 51  3.4 Results and discussion 3.4.1 Occurrence of steroids in anaerobic digester streams Hormones detected in one or more influent and effluent samples from the lab-scale anaerobic digester utilizing Kelowna sludge cake are presented in Tables 3.3 and 3.4, for both liquid (soluble phase) and solid (total phase) matrixes. Although for most of effluent and influent samples the peak of mestranol (Ms) and androsterone (An) were detected, quantification criteria (Appendix B, Table B.1) were not met. For such cases, concentrations were reported as a 'K' flagged value and represented the estimated maximum possible concentration in the soluble phase or total phase matrixes. During analyses, five of the 16 target hormones were detected in total phases of one or more influent total phase samples. Among these, three were androgenic (Ad, An and Tr), one progestagen (Pr) and one estrogenic (Ms). The majority of the estrogens studied, i.e., E1, E2, E3 and EE2, were below the RL in all the influent samples. Based on the results presented in Table 3.4, concentrations of Tr (4.76 – 6.29 ng/g) and Pr (< 4.76 – 22.7 ng/g) were comparable to previously reported concentrations of hormones in dewatered sludge (Fan et al., 2011; Liu et al., 2011; Liu et al., 2012a); while concentration of Ad (< 11.9 – 75.5 ng/g) was slightly higher than the previous studies (6.7 – 12 ng/g). Regarding the occurrence of steroids in the soluble phases, six out of 16 hormones were detected in soluble phase of at least one of the influents (Table 3.3). Both E1 and E2 were detected and their concentrations varied in a range of 22.4 – 92.4 ng/L; while Pr fell below the RL (3.08 – 3.71 ng/L) in the soluble phase.  52  Table 3.3 Concentration (ng/L) of steroidal hormones in soluble phase of digester influent and effluents at different SRTsa Mesophilic Hormones  Androstenedione (Ad)  Androsterone (An)  Mestranol (Ms)  Progesterone (Pr)b  Testosterone (Tr)  Sample Influent Effluent (20 d) Effluent (10 d) Effluent (5 d) Influent Effluent (20 d) Effluent (10 d) Effluent (5 d) Influent Effluent (20 d) Effluent (10 d) Effluent (5 d) Influent Effluent (20 d) Effluent (10 d) Effluent (5 d) Influent Effluent (20 d) Effluent (10 d) Effluent (5 d)  Thermophilic  Control  MW80  MW120  MW160  Control  MW80  MW120  MW160  1640 732 < 18.2 1230 K 17700 < 30200 K 19500 < 890 K 86400 K 5170 K 2100 K 3700 < 3.08 317 N.A 359 < 3.08 < 39.3 < 6.12 < 62.8  480 327 < 20.8 1370 K 36800 < 27200 K 13400 < 807 K 9510 < 4190 K 2570 K 4280 < 3.12 218 N.A 179 139 136 46.7 < 46.2  < 45.9 246 < 21.4 762 K 22300 < 30600 K 13200 < 613 K 8970 < 4800 < 745 K 4210 < 3.16 207 N.A 89.5 < 3.16 828 < 18.2 < 12.0  < 50.3 156 < 22.2 687 K 42900 K 29900 K 17400 < 271 K 16500 < 4830 K 2270 K 2770 < 3.71 < 5.25 N.A 206 < 3.71 861 K 396 < 75.6  1640 104 1100 703 K 17700 K 31900 K 25000 < 1820 K 86400 < 7250 K 11700 K 37800 < 3.08 153 N.A 1540 < 3.08 405 K 794 < 61.4  480 68.8 1550 393 K 36800 K 45800 K 22700 < 360 K 9510 < 8500 K 5930 K 13700 < 3.12 225 N.A 315 139 588 K 1020 < 10.6  < 45.9 66.4 85.8 234 K 22300 K 30800 K 24300 < 1500 K 8970 < 3520 K 5670 K 12600 < 3.16 154 N.A 245 < 3.16 514 K 1990 < 48.2  < 50.3 89.1 89.2 413 K 42900 K 43200 K 20100 < 748 K 16500 < 5560 K 6520 K 13600 < 3.71 264 N.A 575 < 3.71 965 K 3220 < 82.9  Spiked matrix (% recovery) < 8.00 72.6 < 13.3 92.1 < 20.0 136 < 2.00 71.4 < 82.2 53.5 < 153 88.9 K 551 170 < 23.2 64.3 < 80.0 55.4 < 133 111 K 663 138 K45.9 73.7 < 3.20 80.3 < 5.33 167 N.A N.A < 0.80 86.4 < 3.20 72.9 < 5.33 90.4 < 8.00 162 < 0.80 70.7 (Continued on next page) Lab blank  53  Table 3.3 Continued from previous page Mesophilic Thermophilic Lab Spiked matrix blank (% recovery) Control MW80 MW120 MW160 Control MW80 MW120 MW160 Influent < 34.0 88.8 49.7 92.4 < 34.0 88.8 49.7 92.4 < 16.0 96.8 17 betaEffluent (20 d) < 27.9 < 28.0 < 27.5 < 26.2 < 28.4 < 30.6 < 30.2 < 28.5 < 26.7 106 estradiol Effluent (10 d) 27.1 65 52.2 33.3 59.3 62.9 46 99.2 < 40.0 97.2 (E2) Effluent (5 d) < 60.2 < 90.0 < 60.2 < 42.1 < 78.7 < 53.1 < 53.6 < 61.1 < 40.0 80.5 Influent 58.6 28.5 22.4 36.1 58.6 28.5 22.4 36.1 < 16.0 85.4 Effluent (20 d) 94.6 100 77.9 65.9 78 89.2 33 59.7 < 26.7 88.5 Estrone (E1) Effluent (10 d) 112 75 82.3 67.4 67.4 60.5 54.4 63 < 40.0 91.5 Effluent (5 d) < 60.2 < 90.0 < 60.2 < 42.1 < 78.7 < 53.1 < 53.6 < 61.1 < 40.0 84.8 a K: estimated maximum possible concentration, '< ': number following this symbol represents the reporting limit (RL), control: un-pretreated, MW: microwave, 80,120 and 160 are the final pretreatment temperatures, lab blank: an aliquot of reagent water that is treated exactly as a sample including exposure to all glassware, equipment, solvents, reagents, internal standards, and surrogates that are used with samples, spiked matrix: matrix containing known quantity of analytes b Non-quantifiable (NQ) in 10 day SRT effluent Hormone  Sample  54  Table 3.4 Concentration (ng/g of dry weight) of steroidal hormones in total phase of digester influent and effluents at different SRTsa Mesophilic Hormone  Sample  Influent Androstenedione Effluent (20 d) (Ad) Effluent (10 d) Effluent (5 d) Influent Androsterone Effluent (20 d) (An) Effluent (10 d) Effluent (5 d) Influent Effluent (20 d) Mestranol (Ms) Effluent (10 d) Effluent (5 d) Influent Progesterone Effluent (20 d) (Pr) Effluent (10 d) Effluent (5 d) Influent Testosterone Effluent (20 d) (Tr) Effluent (10 d) Effluent (5 d)  Thermophilic  Control  MW80  MW120  MW160  Control  MW80  MW120  75.5 125 46.7 62.3 K 923 < 556 < 526 < 124 K 599 < 437 K 467 K 586 < 4.76 < 16.6 23.1 51.3 < 4.76 31.9 K 47.2 7.88  < 11.9 46.7 26 < 11.8 K 589 < 349 < 377 < 205 K 471 < 350 K 403 K 317 18 8.12 < 12.2 < 4.74 < 4.77 K 63.0 K 55.0 < 4.74  < 12.1 26.1 K 17.1 < 12.4 K 733 < 321 < 556 < 127 K 852 K 578 < 629 K 401 22.7 < 9.89 < 10.2 5.82 6.29 K 84.9 K 183 < 4.96  16.2 22.3 < 16.5 < 12.5 K 799 < 338 K 1000 < 129 K 1640 < 215 K 690 K 466 12.9 < 9.25 < 22.2 < 5.02 < 4.47 K 108 K 193 < 5.02  75.5 < 20.9 197 26.7 K 923 < 419 < 473 < 140 K 599 < 209 2330 K 495 < 4.76 33.9 67.7 184 < 4.76 < 9.91 K 51.5 < 5.43  < 11.9 48.7 365 11.7 K 589 < 349 < 433 < 109 K 471 K 902 K 1130 K 293 18 67.7 19.2 9.61 < 4.77 K 36.5 K 120 < 4.23  < 12.1 27.6 < 18.2 < 15.8 K 733 < 380 < 356 < 163 K 852 K 1050 K 1130 K 573 22.7 34.6 48.1 < 6.34 6.29 K 21.8 K 24.7 K 20.3  Spiked matrix (% MW160 recovery) 16.2 33.7 93.6 < 23.3 < 20.0 80.3 < 15.5 < 20.0 80.3 < 12.1 < 13.3 78.6 K 799 < 139 79.5 < 727 < 284 76.7 < 408 < 284 76.7 < 125 < 145 79.4 K 1640 < 100 79.3 K 1230 < 200 89.9 2100 < 200 89.9 K 573 K 648 64.6 12.9 < 4.00 79.7 86.5 < 8.00 94.5 69.2 < 8.00 94.5 9.43 < 5.33 80.8 < 4.47 < 4.00 88 K 21.6 < 8.00 80 K 41.7 < 8.00 80 K 7.56 < 5.33 79.8 (Continued on next page) Lab blank  55  Table 3.4 Continued from previous page Spiked matrix (% Control MW80 MW120 MW160 Control MW80 MW120 MW160 recovery) Influent < 80.0 < 56.0 < 77.5 < 70.0 < 80.0 < 56.0 < 77.5 < 70.0 < 20.0 96.4 17 β-estradiol Effluent (20 d) < 37.1 < 35.0 < 34.5 < 43.1 < 41.8 < 35.4 < 39.6 < 46.7 < 40.0 70.7 (E2) Effluent (10 d) < 41.0 < 37.2 < 33.2 < 33.0 < 42.9 < 36.4 < 36.4 < 31.0 < 40.0 70.7 Effluent (5 d) < 24.2 < 23.7 < 24.8 < 25.1 < 27.2 < 21.1 < 31.7 < 24.2 < 40.0 81.6 Influent < 23.8 < 23.8 < 24.3 < 22.4 < 23.8 < 23.8 < 24.3 < 22.4 < 20.0 84.9 Effluent (20 d) < 37.1 < 35.0 < 34.5 < 43.1 < 41.8 < 35.4 < 39.6 < 46.7 < 40.0 80 Estrone (E1) Effluent (10 d) < 41.0 < 37.2 < 33.2 < 33.0 < 42.9 < 36.4 < 36.4 < 31.0 < 40.0 80 Effluent (5 d) < 24.2 < 23.7 < 24.8 < 25.1 < 27.2 < 21.1 < 31.7 < 24.2 < 40.0 87.5 a K: estimated maximum possible concentration, '< ': number following this symbol represents the reporting limit (RL), control: un-pretreated, MW: microwave (80,120 and 160 are the final pretreatment temperatures), lab blank: an aliquot of reagent water that is treated exactly as a sample including exposure to all glassware, equipment, solvents, reagents, internal standards, and surrogates that are used with samples, spiked matrix: matrix containing known quantity of analytes Mesophilic  Hormone  Sample  Thermophilic  Lab blank  56  3.4.2 Effects of microwave pretreatment on steroids in digester feed As expected, MW pretreatment resulted in a linear particulate COD solubilization with increased temperature as shown in Figure 3.6. However, there was no consistent pattern among different steroidal hormone concentrations with an increase in MW irradiation temperature in soluble (Figure 3.7a) or total phases (Figure 3.7b) of digester feeds. Compared to estrogenic (i.e. E1, E2, EE2), androgenic and progestogenic hormones have been shown to partition with aqueous phase mostly (Esperanza et al., 2007). This study indicated very high concentrations of Ad in the soluble phase of the control (not-pretreated) samples. However, MW pretreatment led to attenuation of these two hormones in both soluble and total phases of the digester influents with an increase in the MW temperature (Figure 3.7a). Similarly, E1 decreased in the soluble phase with an increase in the pretreatment temperature and was below the RL in the total phase.  SCOD/TCOD ratio  SCOD/TCOD ratio (%)  30  20  10  0 Control  MW80  MW120  MW160  Figure 3.6 Effect of microwave (MW) pretreatment on waste sludge cake solubilization (SCOD: soluble chemical oxygen demand, TCOD: total chemical oxygen demand; 80, 120 and 160: ultimate pretreatment temperatures in oC; data represent arithmetic mean of duplicates and error bars represent absolute difference between mean and duplicate measurements) The elevated melting points of these three hormones (155 – 256oC; Table 2.1) suggest that it is unlikely that the attenuation of these compounds were due to evaporations from the soluble phase during MW irradiation of digester feed in a range of 80  160oC. The attenuations could  have been caused by abiotic transformation of the hormones. Autoxidation of cholesterol and plant sterols by heating is well documented in the literature (Osada et al., 1993; Baggio and 57  Bragagnolo, 2006; Otaegui-Arrazola et al., 2010) and could explain the results obtained in the current study involving steroids with similar ring structures to sterols. This warrants further investigation to indentify the products of transformation. In the soluble phase of the digester feed, E2 was the only hormone showing some release with increasing temperature and was below the RL in the total phase. Based on these results, it can be postulated that both transformation (i.e. autoxidation, denaturation) and release of hormones from the complex polymeric floc structure occurred during the MW pretreatment. It is possible to postulate that for more hydrophobic hormones, such as E2, the release was higher. Furthermore, Pr was not detected in the soluble phase of the influent; however, it increased in the total phase. Based on the results in the literature, as the high MW pretreatment temperatures used could be inhibitory to any enzymatic activity in the digester feed (Daniel et al., 1996), transformation of one or more hormones into Pr was unlikely. However, this could be due to additional (to sonication described in section 3.3.2) hormone extraction step provided by the MW irradiation for the pretreated sludge samples. Literature shows that microwave assisted extraction has been used to extract hormones from solid samples (e.g., soil, sediment etc.) as an attractive alternative method to traditional extraction methods such as soxhlet extraction and sonication (Liu et al., 2004; Labadie and Budzinski, 2005; Hibberd et al., 2009). This could mean that the MW pretreated digester feed samples could be advantaged in terms of hormone extraction efficiency and subsequent detection sensitivity in the total phases due to hybrid (MW pretreatment of sludge followed by sonication as part of the analytical method for detection) extraction applied. 3.4.3 Effects of microwave pretreatment on digester performance Except for the control digesters at the 5-day SRT, all control and pretreated digesters achieved steady state at all three SRTs, corresponding to volumetric OLRs of 1.74 to 6.96 g COD/L/d (Figure 3.8). At the SRT of 5 days, both mesophilic and thermophilic controls stopped producing biogas after 20 days of operation with TVFA concentrations exceeding 1,818 mg/L at pH < 5.64 for mesophilic and 2,853 mg/L at pH < 7.02 for thermophilic controls while the pretreated digesters continued producing biogas.  58  Concentrations in soluble phase (ng/L)  a)  Androstenedione (Ad)  17 beta-estradiol (E2)  Estrone (E1)  Testosterone (Tr)  1000  100  10  1 Control  b)  MW80  Androstenedione (Ad)  MW120  MW160  Progesterone (Pr)  Testosterone (Tr)  Concentrations in total phase (ng/L)  10000  1000  100  10  Control  MW80  MW120  MW160  Figure 3.7 Effect of microwave (MW) on hormone concentrations in the a) soluble (supernatant) phase and b) total (sludge) phase of the influent (control: un-pretreated, 80, 120 and 160: ultimate MW pretreatment temperatures in oC; single data point concentrations; black filled data points represents concentrations below reporting limit (RL) which varied as Tr: 3.08 and 157  160 ng/L, Ad: 45.9  50 ng/L and 392  3.71 ng/L  399 ng/L, in soluble and total phase,  respectively, Pr: 157 ng/L in total phase, E2: 34 ng/L in soluble phase) 59  ME Control  TH Control  ME MW160  TH MW160  800  Biogas volume (mL/g VS per day)  700  600  500  400  300  200  100  SRT = 20 d  SRT = 10 d  23-May-12 25-May-12  19-May-12  15-May-12  11-May-12  7-May-12  3-May-12  29-Apr-12  19-Apr-12  15-Apr-12  11-Apr-12  7-Apr-12  3-Apr-12  30-Mar-12  26-Mar-12  22-Mar-12  18-Mar-12  14-Mar-12  8-Mar-12  4-Mar-12  29-Feb-12  25-Feb-12  21-Feb-12  17-Feb-12  13-Feb-12  9-Feb-12  5-Feb-12  1-Feb-12  28-Jan-12  24-Jan-12  20-Jan-12  16-Jan-12  12-Jan-12  7-Jan-12  0  SRT = 5 d  Figure 3.8 Daily biogas productions (normalized to volatile solids (VS) content of the feed) of control (fed with un-pretreated) and MW160 (fed with microwave pretreated sludge at 160oC) thermophilic (TH) and mesophilic (ME) digesters at STP (1 atm, 0oC) during the 20, 10 and 5 days of sludge retention times (SRTs)  60  Regarding the effect of pretreatments on digester performance, TCOD removal (Figure 3.9), VS removal (Figure 3.10) and daily methane productions (Figure 3.11) at standard temperature and pressures (STP; 0oC, 1 atm) improved for the pretreated digesters at both mesophilic and thermophilic digester temperatures. The pretreatment effects were more prominent at the shortest SRT of 5 days, which was in agreement with the previous studies reported on mesophilic semicontinuous digestion of municipal sludge (WAS and WAS+PS) and after MW irradiation below boiling point (< 96oC) temperatures (Eskicioglu et al., 2007; Park, 2011). During the 5-day SRT, pretreated digesters showed TCOD removal improvements with respect to control, in the range of 54  97% and 88  120% for mesophilic and thermophilic digesters, respectively. The  relative (to control) improvement for VS removal efficiencies was respectively in the range of 124  163% and 98  117% for mesophilic and thermophilic digesters, at the same SRT of 5  days. Similarly, pretreated digesters produced 101  121% and 57  81% higher methane,  respectively for mesophilic and thermophilic digesters, over controls at SRT of 5 days. Biogas composition (% of CH4, N2, O2, N2) at various SRTs as presented in Table 3.5 show that all of the digesters had 60% or higher methane content, except the controls at 5 days SRT.  20 days  70  10 days  5 days  Total COD removal (%)  60 50 40 30 20 10 0 Control  MW80  Mesophilic  MW120  MW160  Control  MW80  MW120  MW160  Thermophilic  Figure 3.9 Total chemical oxygen demand (TCOD) removal efficiencies at sludge retention times (SRTs) of 20, 10 and 5 days (80, 120 and 160: ultimate microwave pretreatment temperatures in oC; arithmetic means and standard deviations of 14 data points are shown)  61  70  20 days  10 days  5 days  60  VS removal (%)  50 40  30 20 10 0 Control  MW80  MW120  MW160  Control  Mesophilic  MW80  MW120  MW160  Thermophilic  Figure 3.10 Volatile solids (VS) removal efficiencies at sludge retention times (SRTs) of 20, 10 and 5 days (80, 120 and 160: ultimate microwave pretreatment temperatures in oC unit; arithmetic mean and standard deviation of 14 data points are shown) 3.5  Volume (L/L of digester/day)  CH4  CO2 + N2  3 2.5  2 1.5 1 0.5  Control  MW80  Mesophilic  MW120  MW160  Control  MW80  MW120  20 day 10 day 5 day  20 day 10 day 5 day  20 day 10 day 5 day  20 day 10 day 5 day  20 day 10 day 5 day  20 day 10 day 5 day  20 d 10 d 5d  20 day 10 day 5 day  0  MW160  Thermophilic  Figure 3.11 Average daily biogas (CH4, CO2+N2, assuming O2 is negligible) production rate at various sludge retention times (SRTs) at STP (80, 120 and 160: ultimate microwave pretreatment temperatures in oC; STP: 0oC, 1 atm; arithmatic mean and standard deviation of 60, 36 and 26 data points for SRT of 20, 10 and 5 days, respectively) 62  Table 3.5 Biogas composition (%) at various sludge retention times (SRTs) Control SRT = 20 days  Mesophilic MW80 MW120  MW160  Control  Thermophilic MW80 MW120  MW160  66  65  65  66  66  65  66  65  CH4  (2.0,4)a  (0,4)  (1,4)  (2,4)  (2,4)  (0,4)  (1,4)  (2,4)  CO2  29  29  29  30  29  29  29  29  N2  4 1  5 1  5 1  3 1  4 1  5 1  4 1  5 1  65  66  67  66  64  65  67  (2,4)  (2,4)  (2,4)  (1,4)  (3,4)  (1,4)  (2,4)  O2  SRT = 10 days 64 CH4 (0.5,4) CO2  32  32  31  30  30  31  31  30  N2  3  2  2  2  3  4  3  2  O2  1  1  1  1  1  1  1  1  43  63  64  64  58  63  63  61  (13, 9)  (1,9)  (1,9)  (1,9)  (4,9)  (1,9)  (1,9)  (1,9)  48  33  33  33  34  33  32  32  (11, 11)  (1, 6)  (1, 7)  (1.5, 5)  (2.5, 10)  (1, 5)  (0.6, 5)  (0.2, 3)  8  3  2  2  6  4  4  6  (4, 11)  (1, 6)  (1, 7)  (1, 5)  (3, 10)  (0.5, 5)  (2, 5)  (2, 3)  1  1  1  1  2  1  1  1  (0.6, 11)  (0.2, 6)  (0.6, 7)  (1, 5)  (0.5, 10)  (0.1, 5)  (0.5, 5)  (0.1, 3)  SRT = 5 days CH4 CO2 N2 O2 a  (standard deviation, number of data points)  3.4.3 Effects of pretreatment and anaerobic digester operating conditions on hormone concentrations The hormone concentrations before and after anaerobic digestion for different pretreatment temperatures (from both total and soluble phases) are displayed in Figures 3.12 to 3.14. Total phase concentrations of Ad and Pr were converted to ng/L by multiplying with the concentrations in ng/g by the TS (%) content (Appendix B, Table B.4) of respective samples in order to make them comparable to soluble phase concentrations in ng/L. Matrix interferences were observed in the cases of Tr, An and Ms and their detection in most of the samples did not meet quantification criteria specified in Table B. 1. Concentration profiles for these three hormones in soluble and total phases are presented in Appendix B (Figures B. 1 to B. 3). It 63  should be noted that hormone concentration in total phase can change after digestion as a result of biodegradation, abiotic transformation. However, soluble phase concentrations are affected by sorption and desorption to sludge in addition to the aforementioned processes. a)  Thermophilic  120.0 100.0 80.0 60.0 40.0  Control  MW80  b)  Effluent (20 d)  Effluent (10 d)  Effluent (5 d)  Influent  Effluent (20 d)  MW120  Mesophilic  MW160  Thermophilic  100 80 60 40  Control  MW80  MW120  Effluent (20 d)  Effluent (10 d)  Effluent (5 d)  Influent  Effluent (20 d)  Effluent (10 d)  Effluent (5 d)  Influent  Effluent (20 d)  Effluent (10 d)  Effluent (5 d)  Influent  Effluent (20 d)  Effluent (5 d)  0  Effluent (10 d)  20  Influent  E2 in soluble phase (ng/L)  Effluent (10 d)  Effluent (5 d)  Influent  Effluent (20 d)  Effluent (10 d)  Effluent (5 d)  Influent  Effluent (5 d)  Effluent (10 d)  0.0  Effluent (20 d)  20.0 Influent  E1 in soluble phase (ng/L)  Mesophilic  MW160  Figure 3.12 Concentrations of a) estrone (E1) and b) 17β-estradiol (E2) in soluble phases of the influent and effluents at various sludge retention times (SRTs) (control: un-pretreated; 80, 120 and 160 are ultimate microwave pretreatment temperatures in oC unit; black filled data points were below reporting limit (RL). RL varied as E1: 32  90 ng/L, E2: 26.2  90 ng/L) 64  Despite the known inefficiency of conventional anaerobic digesters to biodegrade the hormones (Andersen et al., 2003; Esperanza et al., 2007; Holmes et al., 2010; Ifelebuegu, 2011), below RL concentrations of estrogenic hormones (i.e. E1, E2, E3 and EE2) reported in Table 3.4 made it difficult to estimate their removal from the total phase. Due to same reason, calculation of degradation coefficient was also not possible. However, the soluble phase concentrations indicated accumulation of E1 (Figure 3.11) and removal of E2 (Figure 3.12) in both controls and pretreated digesters. Previous studies indicated that, E1 is an intermediate compound of E2 degradation (Czajka and Londry, 2006; Furlong et al., 2010; Holmes et al., 2010; Zheng et al., 2012), which may be responsible for the observed accumulation of E1. Another possible reason could be cleaving of glucuronide and sulfate conjugates present in the primary sludge during anaerobic digestion (Furlong et al., 2010). Although, there is not much information that exists regarding the biodegradation and fate of androgenic and progesteronic hormones under anaerobic conditions (Esperanza et al., 2007), this study demonstrated an overall accumulation of Ad and Pr in most of the digesters (Figures 3.13 and 3.14). Despite the lower concentrations of Ad in the pretreated influents than the controls, the effluent concentrations revealed accumulation in both phases in most of the pretreated digesters. Also, the longest SRT (20 days) seemed to worsen the situation in case of presence of Ad (both thermophilic and mesophilic digesters) and Pr (thermophilic digesters). Possible explanation of such accumulation could be microbial assisted transformation of hormones. For example, Ad has been the starting compound in synthesis of androgenic and anabolic drugs for a long time and many microorganisms have been isolated from environment that are able to produce Ad by ring cleavage of plant sterols (Malaviya and Gomes, 2008). Such bioconversion of phytosterols had been reported to cause accumulation of androgens (e.g., androstenedione (Ad)) in anoxic river sediments receiving pulp and paper mill effluent (Jenkins et al., 2004). Recently, another study reported Ad to be a metabolite of Pr degradation using soil mold Penicillium aurantiogriseum (Eshrat and Aroona, 2011). However, the majority of these studies deal with aerobic microorganisms and unfortunately not much is known regarding biotransformation under anoxic or anaerobic conditions. It is possible that some other steroidal hormones, not monitored in this study, could have acted as precursors of the target hormones resulting in their accumulation.  65  Mesophilic  Thermophilic  2000 1500 1000  Control  MW80  b)  Effluent (20 d)  Effluent (10 d)  Effluent (5 d)  Influent  Effluent (20 d)  MW120  Mesophilic Ad in total phase (ng/L)  Effluent (10 d)  Effluent (5 d)  Influent  Effluent (20 d)  Effluent (10 d)  Effluent (5 d)  Influent  Effluent (10 d)  Effluent (5 d)  0  Effluent (20 d)  500  Influent  Ad in soluble phase (ng/L)  a)  MW160  Thermophilic  8000 6000 4000 2000  Control  MW80  MW120  Effluent (20 d)  Effluent (10 d)  Effluent (5 d)  Influent  Effluent (20 d)  Effluent (10 d)  Effluent (5 d)  Influent  Effluent (20 d)  Effluent (10 d)  Effluent (5 d)  Influent  Effluent (20 d)  Effluent (10 d)  Effluent (5 d)  Influent  0  MW160  Figure 3.13 Concentrations of androstenedione (Ad) in a) soluble phase and b) total phase of the influent and effluents at various sludge retention times (SRTs) (control: un-pretreated; 80, 120 and 160 are ultimate microwave pretreatment temperatures in oC unit; black filled data points were below reporting limit (RL). RL varied as 18.2  50.3 ng/L and 362  412 ng/L in soluble  and total phases, respectively)  66  Mesophilic  a)  Thermophilic  1500 1000  Control  MW80  b)  Effluent (20 d)  Effluent (5 d)  Influent  Effluent (20 d)  MW120  Mesophilic  MW160  Thermophilic  6000 4000 2000  Control  MW80  MW120  Effluent (20 d)  Effluent (10 d)  Effluent (5 d)  Influent  Effluent (20 d)  Effluent (10 d)  Effluent (5 d)  Influent  Effluent (20 d)  Effluent (10 d)  Effluent (5 d)  Influent  Effluent (20 d)  Effluent (10 d)  Effluent (5 d)  0 Influent  Pr in total phase (ng/L)  Effluent (5 d)  Influent  Effluent (5 d)  Influent  Effluent (5 d)  Effluent (20 d)  0  Effluent (20 d)  500  Influent  Pr in soluble phase (ng/L)  2000  MW160  Figure 3.14 Concentrations of progesterone (Pr) in a) soluble phase and b) total phase of the influent and effluents at various sludge retention times (SRTs) (Pr was not quantifiable in soluble phase of the effluent of SRT 10 days; control: un-pretreated; 80, 120 and 160 are ultimate microwave pretreatment temperatures in oC unit; black filled data points were below reporting limit (RL). RL varied as 3.08  5.75 ng/L and 146  555 ng/L in soluble and total phases,  respectively)  67  Compared to control digesters, MW pretreated digesters, especially at elevated temperatures (120, 160oC), contained lower concentrations of Ad, Pr and E1 in their effluents. This trend was more apparent in the total phases of effluents for both Ad (Figure 3.13b) and Pr (Figure 3.14b) and soluble phase of E1 (Figure 3.12a) under both mesophilic and thermophilic digester temperatures. These results were confirmed by statistical analyses. Multi-factor analysis of variance performed on hormone removal efficiency [calculated from eq. (3.1)] of Pr (total phase) suggested that MW pretreatment was a statistically significant factor (P < 0.05) at 95% confidence level (Table 3.6). Similar statistical analysis of removal efficiency on Ad (soluble phase) revealed both SRT and pretreatment temperatures to be significant factors at the same (95%) confidence level. For the estrogenic hormones, although removal of E1 from soluble phase was significantly (P < 0.05) affected by digester operating and pretreatment temperatures; effect of SRT was found to be statistically insignificant. The statistical analysis for the hormones of Ad and E1 are provided in Tables C. 1 and C. 2 in Appendix C, along with the normality plots. On the other hand, removal of Pr (soluble phase) and Ad (total phase) were found to be not significantly affected by the studied factors. Table 3.6 Analysis of Variance for removal efficiency of progesterone (Pr) from total phasea Factor Digester (operating) temperature Sludge retention time (SRT) Microwave temperature (MW)  Unit o C days o C  Levels 2 3 4  Values 35 ± 2 (mesophilic), 55 ± 2 (thermophilic) 20, 10, 5 Control (un-pretreated), 80, 120, 160  Source DF SS MS F P Digester temperature 1 688224 688224 3.59 0.107 SRT 2 789332 394666 2.06 0.209 MW 3 3989744 1329915 6.94 0.022 Digester temperature × SRT 2 198784 99392 0.52 0.620 Digester temperature × MW 3 917019 305673 1.59 0.286 SRT × MW 6 3649407 608235 3.17 0.093 Error 6 1150023 191671 Total 23 11382534 a DF: degrees of freedom; SS: sum of square; MS: mean of square; F: observed F value, P: probability value  In contrast to these findings, previously, it had been reported that thermal pretreatment of a mixture of WAS and PS (30:70, v/v) with an autoclave (160oC, 60 min) followed by anaerobic digestion showed similar removal of estrogenic compounds (E1, E2 and EE2) relative to the control digesters for different SRTs (20 and 10 days for mesophilic, 10 and 6 days for 68  thermophilic) (Carballa et al., 2006). However, in the above mentioned study only a single pretreatment temperature (130oC for 60 min) was evaluated. Therefore, the current study, employing a wider range of pretreatment temperatures and three different digester SRTs suggested, for the first time, that pretreatment by MW can be a influencing factor in fate of steroidal hormones. In addition to overall lower concentrations of hormones (Ad, E1) detected in the digester effluents at elevated MW temperatures, thermophilic digesters consistently contained higher Pr in the effluents compared to those of mesophilic (Figure 3.14b). The sum of concentrations of four hormones (E1, E2, Ad and Pr) in the supernatant (soluble) and sludge (total) phases of effluent and influent at the SRT of 20 days are presented in Figure 3.15. These hormones were considered since they were above RL in most of the samples. Also, when these hormones were not detected at RL in some of the samples, their concentrations were assumed to be equal to the RL. Concentration profiles in the soluble phase (Figure 3.15a) revealed that both mesophilic and thermophilic effluent supernatants showed higher concentrations at the elevated pretreatment temperatures (120 and 160oC) compared to influents. This may indicate that pretreatments may have facilitated the release of hormones from the particulate phase to soluble phase during both mesophilic and thermophilic digesters. Furthermore, compared to control digesters, MW pretreatment was able to lower the hormone loads in mesophilic digester centrates recycled back to the main WWTP. For example, at the SRT of 20 days, mesophilic supernatants contained 42%, 52%, 78% less hormones in their digestate supernatants at MW pretreatment temperatures of 80, 120 and 160oC, compared to controls (Figure 3.15a). On the other hand, the thermophilic control contained more or less same concentrations of hormones in its digestate supernatant as the pretreated digesters. In fact, the thermophilic digester fed with pretreated sludge at 160oC showed 21% increase in total hormone concentrations compared to the control. Furthermore, in terms of the comparisons in the soluble phase, all of the thermophilic digesters, except MW 160, had smaller hormone concentrations compared to mesophilic digesters. However, the total phase concentration profiles (Figure 3.15b) suggested that thermophilic digesters actually accumulated more hormones with respect to both mesophilic digesters and influent. This in turn indicates that thermophilic anaerobic digesters may introduce higher amount of steroidal hormones to the environment in case of land application of dewatered digested sludge (biosolids).  69  a)  Influent (ME & TH)  Effluent (ME)  Effluent (TH)  Soluble phase (ng/L)  2000  1500  1000  500  0 Control  b)  MW80  Influent (ME & TH)  MW120  Effluent (ME)  MW160  Effluent (TH)  Total phase (ng/L)  6000  4000  2000  0 Control  MW80  MW120  MW160  Figure 3.15 Total hormone concentrations (∑ of estrone (E1), 17β-estradiol (E2), androstenedione (Ad) and progesterone (Pr)) in the a) soluble phase (supernatant) and b) total phase (sludge) of the influent and effluent at 20 days SRT (80, 120 and 160: ultimate microwave pretreatment temperatures in oC unit) The concentrations of estrogenic (E1+E2), androgenic (Ad) and progestogenic (Pr) hormones ranged between 30 and 140 ng/L, 66.4 and 732 ng/L and < 5.5 and 317 ng/L, respectively, in the soluble phase of control and pretreated digester effluents in the same SRT (20 70  days). These concentrations are comparable to the concentrations of hormones in the wastewater entering the WWTP (Chang et al., 2011; Liu et al., 2012a).  3.5 Summary In summary, transformation and fate 16 steroidal hormones were studied during anaerobic digestion of control (unpretreated) and MW pretreated municipal sludge cake using semicontinuous bench-scale digesters. Only five of the 16 hormones were detected in the total and/or soluble phases of digester influents. Among these five, only E2 fell below the detection limit and the rest were detected in the effluents. MW pretreatment at three different temperatures (i.e. 80, 120 and 160oC) showed release of comparatively more hydrophobic hormones (i.e. E2) in the soluble phase and attenuation of some other hormones (i.e. E1, Ad, Tr). Low biodegradation efficiency of steroidal hormones in anaerobic digesters resulted in accumulation of Ad and E1 in both soluble and total phases. However, compared to aforementioned hormones, Pr showed some removal from total phase of the mesophilic digesters. The simultaneous accumulation and removal can be attributed to microbial biotransformation of the steroidal hormones; however, this needs further investigation. At 20 days SRT, thermophilic digesters contained overall less concentration of steroidal hormones in the effluent supernatants. However, mesophilic digesters seemed to perform better in terms of total attenuation of hormones at the same SRT (20 days) and would likely to have lower concentrations in the dewatered effluents. MW pretreatment also showed increased organic removal and methane production rate, especially at shorter SRTs (5 days).  . 71  Chapter 4 Conclusions and perspectives 4.1 Conclusions This study investigated fate of 16 natural and synthetic hormones during advanced anaerobic digestion of municipal sludge cake using MW pretreatment prior to digestion. The factors studied here included pretreatment temperature (80, 120 and 160oC), SRT (20, 10 and 5 days) and operating temperature (mesophilic, 35 ± 2oC; thermophilic, 35 ± 2oC). To determine the potential effect of MW hydrolysis, hormones were quantified in sludge (total) and supernatant (soluble) phase of the digester influent and effluent streams. Following the collection and analysis of data obtained from eight semi-continuously fed lab-scale digesters, the following conclusions were drawn:   Seven (E1, E2, Ad, Pr, Tr, An and Ms) out of 16 hormones were above RL in one or more of the influent and/or effluent streams. For three of these hormones (Tr, An and Ms), despite the peaks being detected in mass spectrometry, most of the sample showed presence matrix interference.    The most potent estrogens, i.e., E1, E2, E3 and EE2, were below the RL in the sludge phase of all the influent samples; while both E1 and E2 were detected in the range of 22.4 – 92.4 ng/L. Concentrations of Tr (< 4.76 – 6.29 ng/g) and Pr (< 4.76 – 22.7 ng/g) were comparable to previously reported concentrations of hormones in dewatered sludge. However, while concentration of Ad (< 11.9 – 75.5 ng/g) was slightly higher than the previous studies (6.7 – 12 ng/g).    Microwave pretreatment of the digester feed (influent) led to attenuation of Ad and Tr in both soluble and total phases with an increase in the MW temperature. Similar trend was observed with E1 in the soluble phase. Furthermore, more hydrophobic hormones, such as E2, showed release to soluble phase from total phase. These suggest that both transformation (i.e. autoxidation, denaturation) and release of hormones from the complex polymeric floc structure occurred during the MW pretreatment.    Pretreated digesters performed better in terms of TCOD removal, VS removal and biogas production rate. This improvement was especially prominent during the shortest SRT 72  (highest organic loading rate) of operation (5 days). In fact, both mesophilic and thermophilic control (fed with unpretreated sludge) digesters stopped producing biogas after 20 days of operation as a result of VFA accumulation, while the pretreated digesters continued producing biogas.   Despite the attenuation of E2, most potent natural estrogen, anaerobic digestion showed overall increase in most of the hormones (E1, Ad, Pr) in effluent relative to influent concentrations. This supports the previous finding that these micropollutants often exhibit resistance to anaerobic degradation.    Although, below RL concentrations of E1, E2 in the sludge phase made it difficult to speculate their degradation, soluble phase concentrations of the influent and effluent stream from all the digesters revealed accumulation of E1 and attenuation of E2. Therefore, it is possible that E1 was an intermediate compound of E2 degradation, as suggested by previous literature.    Statistical analysis revealed MW pretreatment temperature was in fact a significant factor (P ≤ 0.05) in removal of Pr from sludge phase. Similarly, removal of Ad and E1 from soluble phase of digester effluent were significantly (P ≤ 0.05) different at studied pretreatment temperatures. Furthermore, SRT and operating temperatures influenced removal of Ad and E1 from soluble phase, respectively.    Total hormone concentration (E1 + E2 + Ad + Pr) in the supernatant (soluble phase) of mesophilic digesters linearly decreased (relative to control) with increasing MW temperature at 20 days SRT. This indicates that pretreated digester has the potential to introduce smaller amount of target compounds to the WWTP with the recycled supernatant of the digestate in the mesophilic temperature range. In contrast, at same SRT, thermophilic control performed in the same range as pretreated thermophilic digesters. In fact, effluent supernatant of the thermophilic digester fed with pretreated sludge at 160oC, had slightly higher total hormone concentration relative to control.    Despite thermophilic digesters showing lower hormone concentrations (E1 + E2 + Ad + Pr) in supernatants relative to mesophilic at 20 days SRT, total phase concentrations suggested more accumulation in thermophilic digesters. This may result in higher  73  steroidal hormone concentrations in dewatered digested sludge, eventually ending up in the agricultural land with biosolids.  4.2 Thesis contributions For the first time, this study investigated the fate of steroidal hormones in sewage sludge during anaerobic digestion coupled with MW pretreatment. Although, initial hypothesis was that pretreatment would release hormones from sludge floc to soluble phase of the sludge, the results indicated both release and transformation of hormones occurred during pretreatment. Overall hormone concentrations decreased in the influents of digesters following MW pretreatment. This has never been reported before. In addition, this research also affirms the previous findings that anaerobic digestion was mostly inefficient in degrading steroidal hormone concentrations. However, in contrast to previous studies, various factors (e.g., pretreatment temperatures, digestion temperatures, SRTs) were found to be significantly affecting fate of these compounds. This suggests that, it is possible to optimize the digestion system in terms of steroidal hormone removal.  4.3 Perspectives The conclusions presented here can be verified and expanded based on the following recommended research:   Microwave pretreated digester feeds showed reduction of some hormone concentrations. Exact mechanism of this attenuation is yet to be studied.    Anaerobic digesters showed high accumulation of some hormones, suggesting possibility of microbial assisted conversion of one hormone to another. Although, a number of reports showed microorganisms isolated from environment are able to perform such transformation, these are mostly under aerobic condition. Degradation of steroidal hormones under anaerobic conditions needs further studies.    Estrogenic and androgenic potency of the hormones varies over a wide range. Instead of quantifying each hormone separately, another approach could be to use bioassays. 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Environmental Science & Technology 46(10), 5471-5478.  95  Appendices Appendix A: Calibration charts  Concentration (mg/L)  800 y = 1516.6x R² = 0.9993  600 400 200  0 0  0.1  0.2 0.3 Absorbance at 600 nm  0.4  0.5  Concentration of NH3 -N (mg/L)  Figure A.1 Calibration curve for COD determination  1000 y = 0.6668e-0.038x R² = 0.9985 100  10  1 0  -50  -100  -150  -200  -250  NH3 probe reading (mv) Figure A.2 Calibration curve for NH3-N determination 96  Injected air volume (mL)  600 y = 1.4443x R² = 0.9987 400  200  0 0  100 200 300 Air volume in manometer (mL)  400  Figure A.3 Calibration curve for biogas measurement via manometer at STP (0oC, 1 atm)  97  Appendix B: Analytical detection of hormones Table B.1 Quality control acceptance criteria* IPR (%) Analytes Mestranol Estrone 17α-Dihydroequilin 17α-Ethinylestradiol 17α-Estradiol 17ß-Estradiol Norethindrone Equilin Norgestrel Testosterone Progesterone Androstenedione Estriol Allyl Trenbolone Androsterone Equilenin  OPR recovery  Sample recovery  Blank level  Recovery  RSD  (%)  (%)  (ng)  50 – 125 30 – 140 35 – 160 70 – 125 50 – 125 25 – 150 75 – 125 35 – 135 70 – 125 75 – 125 75 – 125 75 – 125 75 – 130 45 – 125 65 – 125 45 – 210  30 40 50 30 30 40 30 30 30 30 30 30 30 30 30 50  45 – 130 25 – 150 30 – 170 70 – 130 45 – 130 20 – 160 70 – 130 30 – 140 65 – 130 70 – 130 70 – 130 70 – 130 70 – 135 40 – 130 65 – 130 40 – 220  -  ≤ 40 ≤4 ≤4 ≤ 20 ≤4 ≤4 ≤4 ≤8 ≤4 ≤ 0.8 ≤ 0.8 ≤2 ≤ 48 ≤ 0.8 ≤ 20 ≤ 0.8  Surrogate Standard d4-17ß-Estradiol 25 – 180 40 20 – 190 20 – 190a d6-Norethindrone 75 – 130 30 70 – 130 70 – 130a d4-17α-Ethinylestradiol 50 – 190 30 40 – 200 40 – 200a d9-Progesterone 75 – 130 30 70 – 130 70 – 130a a d6-Norgestrel 75 – 130 30 70 – 135 70 – 135 *IPR: initial precision recovery, OPR: Ongoing precision recovery, RSD: relative standard deviation a Recoveries outside limits may be accepted based on application and professional judgment  98  Table B.2 Reporting limit (RL) of steroidal hormones in supernatant (ng/L) of influent and effluents at different SRT's* Hormones Allyl Trenbolone  Estriol (E3)  Norgestrel  17αdihydroequilin  Equilenin  Equilin  Sample Influent Effluent (20 d) Effluent (10 d) Effluent (5 d) Influent Effluent (20 d) Effluent (10 d) Effluent (5 d) Influent Effluent (20 d) Effluent (10 d) Effluent (5 d) Influent Effluent (20 d) Effluent (10 d) Effluent (5 d) Influent Effluent (20 d) Effluent (10 d) Effluent (5 d) Influent Effluent (20 d) Effluent (10 d) Effluent (5 d)  Control  Mesophilic MW80 MW120 MW160 Control  Thermophilic MW80 MW120 MW160  < 9.18 < 17.9 < 5.55 < 23.2 < 123 < 112 < 4120 < 984 < 230 < 95.8 < 27.1 < 60.2 < 50.3 < 31.2 < 27.1 < 60.2 < 3.31 < 5.59 < 5.41 < 12.0 < 30.8 < 55.9 < 54.1 < 120  < 12.4 < 24.1 < 5.59 < 33.6 < 125 < 112 < 554 < 4890 < 68.8 < 81.8 < 57.7 < 90.0 < 29.8 < 39.5 < 27.3 < 90.0 < 3.12 < 5.60 < 5.45 < 18.0 < 31.2 < 56.0 < 54.5 < 180  < 12.4 < 15.1 < 7.62 < 40.8 < 125 < 122 < 5840 < 5290 < 68.8 < 79.4 < 51.7 < 80.1 < 29.8 < 124 < 29.3 < 53.1 < 3.12 < 6.11 < 5.86 < 10.6 < 31.2 < 61.1 < 58.6 < 106  < 39.9 < 16.5 < 7.06 < 24.0 < 126 < 110 < 4070 < 4420 < 196 < 90.3 < 50.1 < 60.2 < 30.0 < 34.1 < 27.3 < 60.2 < 3.16 < 5.50 < 5.45 < 12.0 < 31.6 < 55.0 < 54.5 < 120  < 22.1 < 27.0 < 8.68 42.3 < 149 < 105 < 5890 < 8250 < 266 < 92.3 < 52.4 < 42.1 < 26.7 < 53.1 < 25.7 < 42.1 < 3.71 < 5.25 < 5.13 < 8.41 < 37.1 < 52.5 < 51.3 < 84.1  < 9.18 < 20.6 < 13.6 < 32.7 < 123 < 114 < 5440 < 8750 < 230 < 140 < 50.6 < 78.7 < 50.3 < 60.9 < 29.3 < 78.7 < 3.31 < 5.69 < 5.86 < 15.7 < 30.8 < 56.9 < 58.6 < 157  < 39.9 < 20.4 < 13.3 < 35.6 < 126 < 121 < 7260 < 5460 < 196 < 83.7 < 66.0 < 53.6 < 30.0 < 149 < 26.4 < 53.6 < 3.16 < 6.04 < 5.28 < 10.7 < 31.6 < 60.4 < 52.8 < 107  < 22.1 < 15.8 < 18.0 < 50.6 < 149 < 114 < 5030 < 8560 < 266 < 115 < 83.5 < 408 < 26.7 < 57.0 < 25.1 < 61.1 < 3.71 < 5.69 < 7.55 < 12.2 < 37.1 < 56.9 < 50.2 < 122  Lab blank  Spiked matrix (% recovery)  < 3.28 32.7 < 5.47 80.7 < 8.47 122 < 0.820 42.5 < 128 105 < 107 114 < 737 267 < 16.0 82.4 < 16.0 74 < 26.7 89.2 < 69.8 131 < 4.00 87.1 < 16.0 72.3 < 26.7 100 < 40.0 96.2 < 40.0 84.2 < 3.20 87.9 < 5.33 70.8 < 8.00 81.1 < 8.00 98.9 < 32.0 81 < 53.3 83 < 80.0 83.7 < 80.0 83.7 (Continued on next page)  99  Table B.2 Continued from previous page Mesophilic Thermophilic Lab Spiked matrix blank (% recovery) Control MW80 MW120 MW160 Control MW80 MW120 MW160 Influent < 15.4 < 15.6 < 15.8 < 18.6 < 15.4 < 15.6 < 15.8 < 18.6 < 16.0 92.4 Effluent (20 d) < 27.9 < 28.0 < 27.5 < 26.2 < 28.4 < 30.6 < 30.2 < 50.0 < 26.7 88 17α-estradiol Effluent (10 d) < 27.1 < 27.3 < 27.3 < 25.7 < 29.3 < 29.3 < 26.4 < 25.1 < 40.0 86.5 Effluent (5 d) < 60.2 < 90.0 < 60.2 < 42.1 < 78.7 < 53.1 < 53.6 < 61.1 < 40.0 81.1 Influent < 29.9 < 19.5 < 19.8 < 23.2 < 29.9 < 19.5 < 19.8 < 23.2 < 20.0 84.4 17αEffluent (20 d) < 34.9 < 35.0 < 34.4 < 32.8 < 35.5 < 38.2 < 37.8 < 35.6 < 33.3 99.9 ethinylestradiol Effluent (10 d) < 33.8 < 34.1 < 34.1 < 32.1 < 36.6 < 36.7 < 33.0 < 31.4 < 50.0 84.5 (EE2) Effluent (5 d) < 75.2 < 112 < 75.3 < 52.6 < 98.3 < 66.3 < 67.0 < 76.4 < 50.0 79.3 Influent < 44.2 < 44.8 < 92.4 < 108 < 44.2 < 44.8 < 92.4 < 108 < 16.0 68.3 Norethindrone Effluent (20 d) < 63.4 < 67.5 < 65.7 < 65.5 < 75.3 < 91.0 < 77.5 < 81.3 < 26.7 95.1 (Nr) Effluent (10 d) < 33.6 < 32.3 < 53.3 < 59.2 < 51.5 < 69.5 < 58.2 < 53.8 88.7 141 Effluent (5 d) 124 < 90.0 < 78.4 < 131 < 131 < 53.1 < 94.3 < 169 < 4.00 81.4 * <: number following this symbol represents the reporting limit (RL), control: un-pretreated, MW: microwave (80,120 and 160 are the final pretreatment temperatures), lab blank: an aliquot of reagent water that is treated exactly as a sample including exposure to all glassware, equipment, solvents, reagents, internal standards, and surrogates that are used with samples, spiked matrix: matrix containing known quantity of analytes Hormones  Sample  100  Table B.3 Reporting limit (RL) of steroidal hormones in total phase (ng/g of dry weight) of influent and effluents at different SRT's* Hormones  Allyl trenbolone  Estriol (E3)  Norgestrel  Equilenin  17αdihydroequilin  Equilin  Sample Influent Effluent (20 d) Effluent (10 d) Effluent (5 d) Influent Effluent (20 d) Effluent (10 d) Effluent (5 d) Influent Effluent (20 d) Effluent (10 d) Effluent (5 d) Influent Effluent (20 d) Effluent (10 d) Effluent (5 d) Influent Effluent (20 d) Effluent (10 d) Effluent (5 d) Influent Effluent (20 d) Effluent (10 d) Effluent (5 d)  Control < 6.87 < 8.05 < 11.0 < 4.96 < 1010 < 148 < 513 < 96.9 < 36.5 < 37.1 < 41.0 < 24.2 < 4.76 < 7.42 < 8.21 < 4.84 < 91.4 < 37.1 < 41.0 < 24.2 < 59.0 < 74.2 < 82.1 < 48.4  Mesophilic MW80 MW120 MW160 < 4.89 < 5.20 < 4.87 < 7.18 < 7.07 < 8.83 < 9.01 < 7.96 < 9.44 < 4.85 < 5.08 < 5.14 < 917 < 833 < 1610 < 487 < 138 < 172 < 149 < 394 < 615 < 94.7 < 99.2 < 100 < 40.9 < 46.1 < 42.4 < 35.0 < 34.5 < 43.1 < 37.2 < 33.2 < 33.0 < 23.7 < 24.8 < 25.1 < 4.77 < 4.86 < 4.47 < 7.01 < 6.90 < 8.61 < 7.44 < 6.64 < 6.61 < 4.74 < 4.96 < 5.02 < 52.7 < 48.1 < 39.2 < 35.0 < 34.5 < 43.1 < 37.2 < 33.2 < 33.0 < 23.7 < 24.8 < 25.1 < 51.7 < 48.6 < 49.1 < 70.1 < 69.0 < 86.1 < 74.4 < 66.4 < 66.1 < 47.4 < 49.6 < 50.2  Control < 6.87 < 9.35 < 11.9 < 5.57 < 1010 < 626 < 171 < 109 < 36.5 < 41.8 < 42.9 < 71.2 < 4.76 < 8.36 < 8.57 < 5.43 < 91.4 < 41.8 < 42.9 < 27.2 < 59.0 < 83.6 < 85.7 < 54.3  Thermophilic MW80 MW120 < 4.89 < 5.20 < 9.62 < 8.91 < 8.53 < 9.40 < 4.33 < 6.50 < 917 < 833 < 141 < 158 < 146 < 145 < 84.5 < 127 < 40.9 < 46.1 < 35.4 < 39.6 < 36.4 < 36.4 < 21.1 < 31.7 < 4.77 < 4.86 < 7.07 < 7.91 < 7.28 < 7.27 < 4.23 < 6.34 < 52.7 < 48.1 < 35.4 < 39.6 < 36.4 < 36.4 < 21.1 < 31.7 < 51.7 < 48.6 < 70.7 < 79.1 < 72.8 < 72.7 < 42.3 < 63.4  MW160 < 4.87 < 14.0 < 8.27 < 5.73 < 1610 < 187 < 124 < 97.0 < 42.4 < 46.7 < 31.0 < 24.2 < 4.47 < 9.33 < 6.21 < 4.85 < 39.2 < 46.7 < 31.0 < 24.2 < 49.1 < 93.3 < 62.1 < 48.5  Lab blank  Spiked matrix (% recovery)  < 4.10 53.3 < 8.20 63.2 < 8.20 63.2 < 5.47 49.3 < 268 132 < 160 76 < 160 76 < 107 85.2 < 20.0 71.2 < 40.0 98.7 < 40.0 98.7 < 26.7 89.1 < 4.00 87.8 < 8.00 124 < 8.00 124 < 8.00 114 < 20.0 82.3 < 40.0 71.7 < 40.0 71.7 < 40.0 75.8 < 40.0 69.6 < 80.0 73.1 < 80.0 73.1 < 80.0 84.1 (Continued on next page)  101  Table B.3 Continued from previous page Mesophilic Thermophilic Lab Spiked matrix blank (% recovery) Hormones Sample Control MW80 MW120 MW160 Control MW80 MW120 MW160 Influent < 29.7 < 29.8 < 30.4 < 27.9 < 29.7 < 29.8 < 30.4 < 27.9 < 25.0 97.3 17αEffluent (20 d) < 46.4 < 43.8 < 43.1 < 73.9 < 52.2 < 44.2 < 72.3 < 58.3 < 50.0 87.6 ethinylestradiol Effluent (10 d) < 51.3 < 46.5 < 41.5 < 41.3 < 53.6 < 45.5 < 45.5 < 38.8 < 50.0 87.6 (EE2) Effluent (5 d) < 30.3 < 29.6 < 31.0 < 31.3 < 33.9 < 26.4 < 39.6 < 30.3 < 50.0 89.1 Influent < 23.8 < 23.8 < 24.3 < 22.4 < 23.8 < 23.8 < 24.3 < 22.4 < 20.0 98.8 Effluent (20 d) < 37.1 < 35.0 < 34.5 < 43.1 < 41.8 < 35.4 < 39.6 < 46.7 < 40.0 82.1 17α-estradiol Effluent (10 d) < 41.0 < 37.2 < 33.2 < 33.0 < 42.9 < 36.4 < 36.4 < 31.0 < 40.0 82.1 Effluent (5 d) < 24.2 < 23.7 < 24.8 < 25.1 < 27.2 < 21.1 < 31.7 < 24.2 < 40.0 91 Influent < 23.8 < 23.8 < 24.3 < 22.4 < 23.8 < 23.8 < 24.3 < 22.4 < 20.0 73.9 Norethindrone Effluent (20 d) < 37.1 < 35.0 < 34.5 < 43.1 < 41.8 < 35.4 < 39.6 < 46.7 < 40.0 90.7 (Nr) Effluent (10 d) < 41.0 < 37.2 < 33.2 < 33.0 < 42.9 < 36.4 < 36.4 < 31.0 < 40.0 90.7 Effluent (5 d) < 24.2 < 23.7 < 24.8 < 25.1 < 27.2 < 21.1 < 31.7 < 24.2 < 26.7 85.1 * <: number following this symbol represents the reporting limit (RL), control: un-pretreated, MW: microwave (80,120 and 160 are the final pretreatment temperatures), lab blank: an aliquot of reagent water that is treated exactly as a sample including exposure to all glassware, equipment, solvents, reagents, internal standards, and surrogates that are used with samples, spiked matrix: matrix containing known quantity of analytes  Table B.4 Total solids (TS) concentrations (%, w/w) of the influent and effluent samples analyzed for hormones Sample Influent Effluent (20 d) Effluent (10 d) Effluent (5 d)  Control 3.3 2.1 2 3.3  Mesophilic MW80 MW120 3.3 3.3 2.2 2.2 2.1 2.4 3.1 3.2  MW160 3.6 1.9 2.5 3  Control 3.3 1.9 1.9 2.9  Thermophilic MW80 MW120 3.3 3.3 2.3 2.1 2.1 2.2 3.7 2.4  MW160 3.6 1.8 2.4 3.2  102  Mesophilic  Thermophilic  3000  K  2000  K K  1000  Control  MW80  b)  MW120  Mesophilic  MW160  Thermophilic  5000  K  K  4000 3000  K  2000  K  K  1000  K  K  K  K K  K  K  K  K  Control  MW80  MW120  Effluent (20 d)  Effluent (5 d)  Influent  Effluent (20 d)  Effluent (10 d)  Effluent (5 d)  Influent  Effluent (20 d)  Effluent (10 d)  Effluent (5 d)  Influent  Effluent (20 d)  Effluent (10 d)  Effluent (5 d)  0  Effluent (10 d)  K  Influent  Tr in total phase (ng/L)  Effluent (20 d)  Effluent (10 d)  K  Effluent (5 d)  Influent  Effluent (20 d)  Effluent (10 d)  Effluent (5 d)  Influent  Effluent (20 d)  Effluent (10 d)  Effluent (5 d)  Influent  Effluent (5 d)  Effluent (10 d)  0  Effluent (20 d)  K  Influent  Tr in soluble phase (ng/L)  a)  MW160  Figure B.1 Concentrations of testosterone (Tr) in a) soluble phase and b) total phase of the influent and effluents at different sludge retention times (SRTs) (80, 120 and 160 are ultimate pretreatment temperatures in oC unit; black filled data points were below reporting limit (RL); RL varied as 3.08  82.9 ng/L in soluble phases; K flagged data points represent estimated maximum concentration) 103  Mesophilic  a)  Thermophilic  An in soluble phase (µg/L)  60 K 40  K  K K K 20  K  K  K  K  K  K  K  K  K  K  K K  K  K  Control  b)  MW80  MW120  Mesophilic  Effluent (20 d)  Effluent (10 d)  Effluent (5 d)  Influent  Effluent (20 d)  Effluent (10 d)  Effluent (5 d)  Influent  Effluent (20 d)  Effluent (10 d)  Effluent (5 d)  Influent  Effluent (20 d)  Effluent (10 d)  Influent  Effluent (5 d)  0  MW160  Thermophilic  An in total phase (µg/L)  40 K  30  K K  K  20  K  10  Control  MW80  MW120  Effluent (20 d)  Effluent (10 d)  Effluent (5 d)  Influent  Effluent (20 d)  Effluent (10 d)  Effluent (5 d)  Influent  Effluent (20 d)  Effluent (10 d)  Effluent (5 d)  Influent  Effluent (20 d)  Effluent (10 d)  Effluent (5 d)  Influent  0  MW160  Figure B.2 Concentrations of androsterone (An) in a) soluble phase and b) total phase of the influent and effluents (µg/L) at different sludge retention times (SRTs) (80, 120 and 160 are ultimate pretreatment temperatures in oC unit; black filled data points were below reporting limit (RL); RL varied as 0.271 – 31.9 µg/L in soluble phases; K flagged data points represent estimated maximum concentration) 104  Mesophilic  a)  Thermophilic  K  80 60  40  K K  20 K  K  K  K  K  K  Control  MW80  Effluent (20 d)  Influent  Effluent (20 d)  MW120  Mesophilic  b)  Effluent (10 d)  Effluent (5 d)  Influent  Effluent (20 d)  Effluent (10 d)  Effluent (5 d)  Influent  Effluent (20 d)  Effluent (10 d)  Effluent (5 d)  0 Influent  K  K Effluent (10 d)  K  K  K  K  K  Effluent (5 d)  Ms in soluble phase (µg/L)  100  MW160  Thermophilic  K  60  40  Control  MW80  MW120  Effluent (10 d)  Effluent (5 d)  Influent  Effluent (20 d)  K Effluent (10 d)  Effluent (5 d)  Influent  Effluent (20 d)  Effluent (10 d)  Effluent (5 d)  0  K K  K  K Influent  K  K  K Effluent (20 d)  K  K  K  Effluent (5 d)  K  K  Effluent (20 d)  K  Effluent (10 d)  20  K  K  K K  Influent  Ms in total phase (µg/L)  80  MW160  Figure B.3 Concentrations of mestranol (Ms) in a) soluble phase and b) total phase of the influent and effluents (µg/L) at different sludge retention times (SRTs) (80, 120 and 160 are ultimate pretreatment temperatures in oC unit; black filled data points were below reporting limit (RL); RL varied as 0.745 – 8.5 µg/L in soluble phases; K flagged data points represent estimated maximum concentration) 105  Appendix C: Analysis of variance Table C.1 Analysis of variance for removal efficiency of androstenedione (Ad) from soluble phasea Factor Digester (operating) temperature Sludge retention time (SRT) Microwave temperature (MW)  Unit o C days o C  Levels 2 3 4  Values 35 ± 2 (mesophilic), 55 ± 2 (thermophilic) 20, 10, 5 Control (un-pretreated), 80, 120, 160  Source DF SS MS F P Digester temperature 1 148626 148626 4.59 0.076 SRT 2 1194801 597400 18.46 0.003 MW 3 1054529 351510 10.87 0.008 Digester temperature × SRT 2 418639 209320 6.47 0.032 Digester temperature × MW 3 228627 76209 2.35 0.171 SRT × MW 6 995421 165903 5.13 0.034 Error 6 194213 32369 Total 23 4235240 a DF: degrees of freedom; SS: sum of square; MS: adjusted mean of square; F: observed F value, P: probability value  Table C.2 Analysis of variance for removal efficiency of estrone (E1) from soluble phasea Factor Digester (operating) temperature Sludge retention time (SRT) Microwave temperature (MW)  Unit o C days o C  Levels 2 3 4  Values 35 ± 2 (mesophilic), 55 ± 2 (thermophilic) 20, 10, 5 Control (un-pretreated), 80, 120, 160  Source DF SS MS F P Digester temperature 1 16136 16136 8.89 0.025 SRT 2 5115 2558 1.41 0.315 MW 3 86389 28796 15.86 0.003 Digester temperature × SRT 2 3327 1664 0.92 0.450 Digester temperature × MW 3 13718 4573 2.52 0.155 SRT × MW 6 12249 2041 1.12 0.445 Error 6 10895 1816 Total 23 147830 a DF: degrees of freedom; SS: sum of square; MS: adjusted mean of square; F: observed F value, P: probability value  106  Normal Probability Plot 99  95 90 80  Percent  70 60 50 40 30 20 10 5  1  -200  -100  0 Residual  100  200  Figure C.1 Normal probability plot for analysis of variance of removal efficiency of androstenedione (Ad) from soluble phase Normal Probability Plot 99  95 90 80  Percent  70 60 50 40 30 20 10 5  1  -50  -25  0 Residual  25  50  Figure C.2 Normal probability plot for analysis of variance of removal efficiency of estrone (E1) from soluble phase 107  Normal Probability Plot 99  95 90 80  Percent  70 60 50 40 30 20 10 5  1  -750  -500  -250  0 Residual  250  500  Figure C.3 Normal probability plot for analysis of variance of removal efficiency of progesterone (Pr) from total phase  108  

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