Sources and Fate of Emerging Contaminants in Municipal Wastewater Treatment

By Daniel Shaver

A Thesis presented to The University of Guelph

In partial fulfillment of the requirements for the degree of Master of Applied Science In Engineering

Guelph, Ontario, Canada

Daniel Shaver, October 2011

ABSTRACT
Sources and Fate of Emerging Contaminants In Municipal Wastewater Treatment

Dan Shaver University of Guelph, 2011

Advisors: Professor H. Salsali Professor E. McBean

This thesis presents the results from a study investigating the sources and fate of emerging contaminants in wastewater treatment facilities for three municipalities in Ontario, Canada. As the potential environmental effects of emerging contaminants from wastewater treatment plants are being discovered, it is vital to understand their transport pathways and behaviour within the wastewater treatment system, to mitigate exposure and increase removal efficiencies. Through sampling the wastewater effluents of hospitals, funeral homes, slaughterhouses and residential neighbourhoods, each location type is shown to contribute to the loading of triclosan, triclocarban, beta-blockers, and antidepressants to the sewer system. Within the three different wastewater treatment plants monitored, the results show that conventional wastewater treatment processes remove less than 30% beta-blockers and that the disinfectants triclosan and triclocarban are primarily removed due to biological degradation.

ACKNOWLEDGEMENTS
I would like to thank my advisors Hamidreza Salsali and Edward McBean for their guidance and support throughout my studies as well as Joanne Ryks for her help in the laboratory. I would also like to thank the many operators and staff at the three wastewater treatment plants for obtaining the samples needed for this study.

iii

TABLE OF CONTENTS
INTRODUCTION ...................................................................................................................1!
Objectives................................................................................................................................... 3! Organization of Thesis ............................................................................................................... 3!

BACKGROUND, ORGANIZATION, AND LOGISTICS ............................................................4!

Background ................................................................................................................................ 4! Legislation and Regulations....................................................................................................... 5! Physical and Chemical Properties of Emerging Contaminants................................................ 11! Project Overview........................................................................................................................ 7! Logistics and Data Assembly..................................................................................................... 9! Emerging Contaminant Classes ............................................................................................... 10! Previous Research .................................................................................................................... 13! Personal Care Products ............................................................................................................ 14! Antidepressants ........................................................................................................................ 16! Pharmaceuticals (Acid/Basic/Neutral) ..................................................................................... 17! Musks ....................................................................................................................................... 19! PBDEs ...................................................................................................................................... 20! Antibiotics ................................................................................................................................ 21! Estrogenic Compounds (Natural/Industrial) ............................................................................ 22! Perfluorinated Compounds....................................................................................................... 24! References .................................................................................................................................. 1!

FATE OF PRION PROTEINS IN WASTEWATER: A REVIEW .............................................29! ABSTRACT .........................................................................................................................29! INTRODUCTION .................................................................................................................30!
!"#"$#%&'()"#*&+,.................................................................................................................. 31! -"./0"(1&'#/2%'/#%&'(3&%'#, ............................................................................................... 32!

PRIONS IN WASTE WATER TREATMENT PLANTS ...........................................................32!

4/#"(&5(36%&',(%'(7/'+(899:%$/#%&',(/'+(7/'+5%::%'0............................................................ 35! Risk .......................................................................................................................................... 36!

iv

CONCLUSIONS AND RECOMENDATIONS ..........................................................................38! REFERENCES .....................................................................................................................40! SOURCES OF !-BLOCKERS, ANTIDEPRESSANTS, AND DISINFECTANTS IN MUNICIPAL WASTEWATER ..................................................................................................................43! ABSTRACT .........................................................................................................................43! 1.! INTRODUCTION ..........................................................................................................44! 2.! METHODS ...................................................................................................................48!
Sampling .................................................................................................................................. 48! Sample Locations ..................................................................................................................... 48! Analysis.................................................................................................................................... 49!

3.! RESULTS .....................................................................................................................50! 4.! DISCUSSION ................................................................................................................53!

Source Concentrations ............................................................................................................. 53! Slaughter House and Funeral Homes....................................................................................... 55! Hospital Loadings .................................................................................................................... 55!

5.! CONCLUSION ..............................................................................................................57! 6.! ACKNOWLEDGMENTS ................................................................................................57! 7.! REFERENCES ..............................................................................................................58! FATE OF !-BLOCKERS, TRICLOSAN, AND TRICLOCARBAN IN MUNICIPAL WASTEWATER TREATMENT ............................................................................................61! ABSTRACT .........................................................................................................................61! 1.! INTRODUCTION ..........................................................................................................62! 2.! METHODS ...................................................................................................................68!
Sampling .................................................................................................................................. 68! Sample Locations ..................................................................................................................... 68! Analysis.................................................................................................................................... 69!

3.! RESULTS .....................................................................................................................71!

Removal Efficiencies ............................................................................................................... 71! Loadings................................................................................................................................... 72!

4.! DISCUSSION ................................................................................................................74!

Adsorption Coefficient............................................................................................................. 75! Fate........................................................................................................................................... 77! Environmental Risk.................................................................................................................. 79!

5.! CONCLUSION ..............................................................................................................81! 6.! ACKNOWLEDGMENTS ................................................................................................82! 7.! REFERENCES ..............................................................................................................83! SUMMARY, CONCLUSION AND RECOMMENDATIONS .....................................................86! APPENDIX ..........................................................................................................................93!
Beta Blocker Raw Data............................................................................................................ 93! Triclosan and Triclocarban ...................................................................................................... 95! Antidepressants ........................................................................................................................ 96!

vi

TABLE OF FIGURES
Figure 1: Conventional Wastewater Treatment Plant Diagram...................................................... 8! Figure 3: Dose-Response Curve for BSE in humans (Gale 2005)................................................ 37! Figure 4: Triclosan Concentration at Different Sample Locations (Funeral Home B= 70,000ng/l) ....................................................................................................................................................... 50! Figure 5: Triclocarban Concentration at Different Sample Locations.......................................... 51! Figure 6: Beta Blocker Concentrations at Different Sample Locations ....................................... 51! Figure 7: Venlafaxine and O-desmethyl venlafaxine Concentration at Different Sample Locations....................................................................................................................................... 52! Figure 8: Citalopram and desmethyl citalopram Concentration at Different Sample Locations.. 52! Figure 9: WWTP flow diagram with sample locations ................................................................ 69! Figure 10: TCC Loadings within treatment plant C ..................................................................... 72! Figure 11: TCS Loadings within treatment plant C (TCS plant influent: 71 g/day) .................... 72! Figure 12: Atenolol and Sotalol Loadings within treatment plant C ........................................... 73! Figure 13: Metoprolol and Propranolol Loadings within treatment plant C ............................... 73! Figure 14: Relative Fates by Loading within WWTP C (Sotalol was observed to have negative removal efficiency) ....................................................................................................................... 77!

vii

TABLE OF TABLES
Table 1: Emerging Contaminant Regulations Summary ................................................................ 6! Table 2: Typical Environmental Concentrations of Emerging Contaminants................................ 9! Table 3: Summary Table for Analytical Tests, Their Location and Status .................................. 11! Table 4: Physical and Chemical Properties of Selected Emerging Contaminants........................ 13! Table 5: Lab scale removals of emerging contaminants by selected treatment processes ........... 14! Table 6: Selected Literature Concentration Values for Personal Care Products in WWT ........... 15! Table 7: Selected Literature Concentration Values for Antidepressants ...................................... 17! Table 8: Selected Literature Concentration Values for Pharmaceuticals in WWTPs .................. 18! Table 9: Selected Literature Concentration Values for Musks in WWTPs .................................. 20! Table 10: Selected Literature Concentration Values for PBDEs in WWTPs............................... 21! Table 11: Selected Literature Concentration Values for Antibiotics in WWTPs......................... 22! Table 12: Selected Literature Concentration Values for Estrogens in WWTPs........................... 23! Table 13: Selected Literature Concentration Values for Perfluorinated Compounds in WWTPs 24! Table 10: PrPTSE Transport Parameters from Column Tests (Jacobsen et al., 2009) ................... 36! Table 11: Physical and Chemical Properties of Beta-Blockers, Antidepressants, Triclosan and Triclocarban .................................................................................................................................. 47! Table 12: Sample Locations.......................................................................................................... 49! Table 13: Hospital Loadings and Percentages .............................................................................. 56! Table 14: Physical and Chemical Properties of Beta Blockers, Triclosan and Triclocarban ....... 65! Table 15: Summary of Features of Treatment Plants ................................................................... 68! Table 16: Primary, Secondary, and Overall Removal Efficiency for TCC and TCS from the Aqueous Phase .............................................................................................................................. 71! Table 17: Primary, Secondary, and Overall Removal Efficiency for Beta-Blockers from the Aqueous Phase .............................................................................................................................. 72!

viii

CHAPTER 1
Introduction
Recently, scientists from North America and Europe have observed the presence of emerging contaminants such as pharmaceuticals, personal care products, bacteria, viruses, and pesticides in wastewater, groundwater, and surface waters. The search to find the sources of these compounds has routinely led to wastewater treatment plants (WWTPs) as an entry point of contaminants into the natural environment. The unknown effects of low concentrations of emerging contaminants in the aquatic ecosystem require scientists to study the occurrence, sources, fate, and transport of these compounds in wastewater treatment, to better understand and possibly identify mitigation opportunities. Reducing the contaminant levels in WWTP effluent helps minimize the contamination in lakes and rivers, which are both WWTP receiving waters as well as drinking water sources. Emerging contaminants end up in wastewater through several pathways including the disposal and use of consumer products, farm runoff, toxic spills, and excretion via the urine and feces of those consuming pharmaceuticals. The human body only metabolizes a percentage of each drug taken, expelling the rest into the municipal wastewater system. Another source is from consumer products such as soap, shampoo, disinfectant washes, and toothpaste which contain biologically active compounds that, when used, release these contaminants into the sewer system where they are then transported to a wastewater treatment plant (WWTP). Municipal wastewater treatment plants are not specifically designed to deal with the trace levels of emerging contaminates found in wastewater and many compounds pass through

conventional treatment systems without removal. From the WWTP effluent, emerging contaminants are discharged into surface waters where they may have measurable effects on aquatic life at low concentrations (Haider and Baqri, 2007). Once in surface waters,

pharmaceuticals have been shown to interrupt the natural biochemistry of many aquatic organisms including fish and algae (Hirsch et al., 1998; Michele et al., 2001). Many of the problems associated with the removal of emerging contaminants from municipal wastewater stem from their low concentrations and chemical diversity, which make detection and analysis difficult. Low concentrations require extremely sensitive analytical equipment while the wide range of distinct chemical compounds necessitates techniques to identify many chemicals at once. Only recently have scientists become aware of the presence of some emerging contaminants in wastewater because analytical techniques able to detect them at the ng/l have only recently been developed. As laboratory procedures are developed and emerging

contaminants can be accurately quantified, scientists are becoming increasingly able investigate the sources, removal pathways, and fate of pharmaceuticals in municipal wastewater. In addition to emerging contaminants, the potential entrance of prions into the wastewater system and their fate in wastewater treatment is an area of concern and a topic of interest in this study.

!"#$%&'($)*
Study of emerging contaminants a new topic of research whose largely unknown environmental effects require the study of their sources, fate, and transport within the wastewater treatment system in an attempt to understand whether the need exists to abate their release. This study has the following objectives: Review the fate of prion proteins in the wastewater treatment system Monitor emerging contaminant concentrations at potential source locations such as hospitals, funeral homes, slaughterhouses and residential areas. Monitor and compare emerging contaminant concentrations at multiple locations within the wastewater treatment plants of three municipalities to determine the fate and removal of these compounds in the wastewater treatment process.

!+,-.'/-&'!.*!0*&1$)')*
This thesis contains five chapters. Chapter 1 provides a global overview of the research, describes emerging contaminants, and outlines previous research investigating emerging contaminants in municipal wastewater. In Chapter 2, a literature review outlining the fate of Prions in municipal wastewater is presented. Chapter 3 investigates the sources of beta-blockers, antidepressants, and disinfectants in the municipal wastewater system and the role that hospitals, funeral homes, slaughterhouses, and residential areas play as point sources of these contaminants. In Chapter 4, the fate and transport of beta-blockers and disinfectants in

municipal wastewater treatment plants is investigated. Finally, Chapter 5 summarizes the results

of this study, comments on the ongoing sections of the project, and recommends directions of future research.

Background, Organization, and Logistics

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Emerging contaminants (ECs) such as pharmaceuticals, endocrine disruptors, pesticides, x-ray contrast media, and personal care products have been found in wastewater, groundwater, and surface waters (Barber et al., 2000; Bauman et al., 1990; Emmanuel et al., 2005). These compounds can enter the environment through leaky sewers and septic systems, which can allow contaminants to infiltrate into the groundwater, and pass through wastewater treatment plants to discharge contaminants into receiving waters. The fate and concentrations of many ECs in the environment are mostly unknown, making the design of treatment strategies difficult. One class of EC attracting particular attention is known as endocrine disrupting compounds (EDCs). These natural or synthetic compounds mimic biological hormones disrupting an organisms natural processes. EDCs have been shown to be biologically active down to concentrations as low as the ng/L level and have been linked to significant change in wildlife including the feminization of fish (Hirsch et al., 1998; Michele et al., 2001). Pharmaceuticals and personal care products (PPCPs) are another group of emerging contaminants gaining recent attention. These include x-ray media, analgesics, antiseptics, antibiotics etc. This class of contaminants often contains polar functional groups making the detection and removal process more difficult. 4

5$,')5-&'!.*-.4*+$,35-&'!.)
The lack of information on the fate, effects, and concentrations of emerging contaminants makes it difficult for governments to regulate their use as well as deal with the levels already existing in the environment. There are currently no laws or regulations outlining the maximum concentrations of emerging contaminants in wastewater effluent, drinking water or the environment. In the United States, the Environmental Protection Agency (EPA) maintains a list of compounds called the Contaminant Candidate List (CCL), which is a list of contaminants that are not subject to any drinking water regulations but are being monitored and may be included in future regulations. The most recent version, CCL3 was published in 2008 and includes several endocrine-disrupting compounds such as estrone, perfluoronated compounds such as PFOS, and some flame-retardants but no pharmaceuticals or personal care products (EPA CCL3, 2008). Regulations outlining maximum allowable concentrations of these CCL3 compounds may be implemented in the future but have yet to be put into place. In the aquatic environment, the Food and Drug Administration currently does not require ecological testing of pharmaceuticals unless the environmental concentration exceeds 1ug/L (Snyder et al., 2003). This is a much greater than the ng/L level at which pharmaceuticals have been shown to interact with the aquatic environment (Hirsch et al., 1998; Michele et al., 2001). In the Europe, the European Commission of the Environment called for the development of a comprehensive list of emerging contaminants in 2006 to comprehend the scope of contamination and develop steps to reduce contaminant levels (Brown et al., 1999; Hirsch et al., 1998). Similarly to the United States, the commission implemented a monitoring strategy and in August 2011 published document outlining the preliminary approach to reduce the interference of EDCs

on humans and wildlife (European Commission 2011). The document outlines a series of recommendations focusing on the reduction of endocrine disrupting compounds in consumer goods, food additives, and cosmetics but does not set any recommendations for maximum allowable concentrations in drinking water, wastewater, or the environment (European Commission 2011). Country or Region USA European Union EC Regulations or Status -None currently -Contaminant Candidate List for monitoring compound occurrence includes endocrine disrupting compounds -In progress -Monitoring EDCs -Recommends the reduction of EDC as additives in consumer goods -None

Canada

Table 1: Emerging Contaminant Regulations Summary In 2011, the World Health Organization published a report entitled Pharmaceuticals in Drinking-water which reviews the risks to human health associated with exposure to trace concentrations of pharmaceuticals in drinking-water. The report concludes that chlorination during the treatment of drinking water is sufficient to remove 50% of these compounds while advanced techniques such as ozonation and activated carbon can remove more than 99% of pharmaceutical molecules (WHO 2011). The authors suggest that pharmaceuticals in drinkingwater are unlikely to pose a risk to human health and that concern over pharmaceuticals should not divert resources away from bacterial, viral, and other chemical contaminants such as lead which are of a higher priority (WHO 2011). From the USA to the European Union, to the World Health Organization, the lack of knowledge regarding emerging contaminants is a major barrier for regulatory agencies responsible for drinking water and wastewater legislation. The burden of proof is on the 6

scientific community to show emerging contaminants negatively affect the environment without which there is no motivation for governments to limit their use and exposure. With a more detailed and thorough look at the sources, fate, and transport of emerging contaminants, decision-makers are better able to implement regulations and design removal and mitigation strategies limiting their release into the natural environment.

6+!#$%&*!($+('$7*
This study seeks to improve the knowledge of emerging contaminants fate within the municipal wastewater system. Through a partnership with three different Ontario municipalities, the source characteristics of hospitals, funeral homes, residential areas, and slaughterhouses were identified. By monitoring multiple locations within the wastewater treatment facilities of each municipality, the fate and transport of emerging contaminants inside WWTPs was examined. To assemble a greater understanding of the potential pathways of emerging contaminants when entering the wastewater system, source sample locations were chosen to monitor sites where ECs were hypothesized to be used. In each of the three municipalities, hospitals were monitored due to the high use of pharmaceuticals, antibiotics, x-ray contrast media, disinfectants, and other emerging contaminants in the health care field. Funeral homes were also monitored to see if the chemicals used in the embalming process leads to high discharge rates of emerging contaminants. Disinfectants and pharmaceuticals in the effluent from a slaughterhouse were monitored in order to determine the role of farming practices in the loading of ECs in wastewater. Finally, several residential neighborhoods were monitored to compare which contaminants come from general population use, and which ones are due to point sources.

Within each of the three wastewater treatment plants, multiple sample locations were selected to track the fate of each compound through the system. Samples were taken from both the aqueous and sludge streams in order to calculate the removal efficiencies of each stage. The following diagram shows a basic schematic of a conventional wastewater treatment plant.

Figure 1: Conventional Wastewater Treatment Plant Diagram Emerging contaminants enter the wastewater treatment plant at the Primary Clarifier where a portion physically adsorb to particulate matter and are settled out of suspension. The liquid discharge is sent to the aeration tank where aerobic bacteria work to break down the organic matter in the wastewater. After passing through another secondary clarifier, the aqueous phase is typically disinfected by chlorination prior to discharge to receiving waters. The sludge from both the primary and secondary clarifiers is sent to an anaerobic digester tank where bacteria further break down the sludge in the absence of oxygen. For each of the WWTPs in this study, samples were taken after each of the major steps in the treatment process as well as overall plant influents and effluents. 8

Location WWTP Biosolids Raw Sewage Treated Sewage Surface waters Drinking water

Concentration mg/kg or ppm ug/L or ppb ng/L or ppt Low ng/L Very low ng/L to non-detect

Table 2: Typical Environmental Concentrations of Emerging Contaminants The above table shows the typical concentrations ranges for emerging contaminants found in different locations. The lowest concentrations are found in drinking water with the greatest being observed in the biosolids from wastewater treatment plants.

5!,')&'%)*-.4*4-&-*-))$8"59**
Over the course of this research, samples were obtained every two months for a period of twelve months. In each wastewater treatment plant, municipal staff obtained three liters of sample at each location using a 24-hour composite auto sampler to ensure that the sample contained an average representation of the locations contents. The three liters were split into two amber glass bottles and one HDPE plastic bottle, and stored at 4oC until a researcher from the University of Guelph picked them up. The composition of emerging contaminants can be altered by prolonged exposure to light and thus dark amber bottles are used to prevent the degradation of the samples. Similarly, for the source sampling within the sewer catchment areas, municipal staff set up 24-hour composite samplers in the sewage manholes outside of the target hospitals, slaughterhouses, and residential areas. Once the samples from all three municipalities had been collected at the University of Guelph, they were transported to the Worsfold Water Quality Center at Trent University where the majority of the analysis was undertaken. The Water Quality Center (WQC) is an internationally recognized laboratory specializing in environmental chemistry. Their advanced techniques and 9

specialized equipment allow the detection of emerging contaminants down to the ng/L level required to accurately study EC presence in municipal wastewater as seen in Table 1. Once the analytical work was completed, the raw data was then sent back to the University of Guelph for analysis and interpretation. As well as chemical tests at the WQC, samples were also shipped to Carleton University for toxicity and microbial testing. At Carleton, a laboratory led by Dr. Golshani is overseeing bacterial and toxicology testing.

$8$+,'.,*%!.&-8'.-.&*%5-))$)*
The following table outlines the different categories of emerging contaminants investigated in this study and their status.
Test Type Chemical Chemical Chemical Chemical Chemical Type Beta-Blockers Antidepressants Disinfectants Antibiotics Compounds Atenolol, Metoprolol, Propranolol, Sotolol Citalopram desmethylcitalopram, Venlafaxine, desmethyl-venlafaxine Triclosan Triclocarban Ciprofloxacin Erythromycin Sulfamethoxazole Sulfapyridine Trimethoprim !-estradiol !-ethinylestradiol Estrone Nonylphenol Octylphenol Bisphenol A Celestolide Phantolide Tonalide Lab Location WQC WQC WQC WQC Lab Status Complete Complete Complete Partially Complete

Chemical

Natural Estrogens

WQC

In Progress

Chemical

Industrial Estrogens

WQC

In Progress

Chemical

Musks

WQC

In Progress

10

Chemical

Perfluorinated Compounds PBDEs

Traseolide Cashmeran Galaxolide Musk ketone Musk xylene Perfluorooctanoate Perfluorooctane sulfonate PBDE 28, PBDE 33 PBDE 47, PBDE 99 PBDE 100, PBDE 153 PBDE 154, PBDE 183 PBDE 209 Acetaminophen Gemfibrozil Ibuprofen Naproxen Caffeine Carbamazapine

WQC

In Progress

Chemical

WQC

In Progress

Chemical

Acidic Pharmaceuticals

WQC

In Progress

Chemical Toxicity Tests Microbial Tests

Basic/Neutral Pharmaceuticals -

WQC Carleton University Carleton University

In Progress In Progress In Progress

Table 3: Summary Table for Analytical Tests, Their Location and Status As seen in the table above, many of the analytical tests in this study are still in progress. As more data are obtained, future researchers will conduct additional analyses.

619)'%-5*-.4*%1$8'%-5*6+!6$+&'$)*!0*$8$+,'.,*%!.&-8'.-.&)*
When examining the fate, pathways, and partitioning of emerging contaminants, it is important to consider the physical and chemical properties of each compound. After a chemical is created, the route that it takes between initial observation and final observations is referred to as a pathway. Common pathways include manufacture to initial use, initial used to disposal and initial use to release to the environment. The result of interactions between a chemical compound and its environment over a series of events and procedures is known as its fate. Thus the study of

11

a compounds fate and pathways seeks to understand where a particular compound goes and how it is affected by different situations. By studying the physical and chemical properties of chemical compounds it is possible to predict their fate in some situations. One commonly used class of physical properties is partitioning coefficients. Partitioning refers to the tendency of a chemical to concentrate in one phase of a two-phase mixture at equilibrium. The mixture can be two liquid phases, a liquid and a solid phase, a liquid and a gas phase, or a combination thereof. A partitioning coefficient is the dimensionless ratio of

concentrations present in the two different phases of a two-phase mixture. The octanol-water partitioning coefficient is a measure of the partitioning between octanol and water, which describes the hydrophobicity of a compound and is inversely related to the solubility of a compound in water. Compounds with a high kow have been shown to preferentially adsorb to soil and sediment particles in water (Karickhoff et al., 1979). Similarly, a sludge adsorption coefficient or kd, is a ratio of the amount of compound adsorbed to sludge compared to the amount present in aqueous solution under the specific conditions the measurement was taken. In water and wastewater treatment, the sludge adsorption coefficient is commonly used to predict the extent to which a compound can be removed by physical adsorption to sludge particles in a primary or secondary clarification unit. A commonly use chemical property is the acid dissociation constant of Ka. It is a measure of the strength of an acid in solution and is the concentration ratio of ionized to un-ionized species of a compound at equilibrium. The Ka of a compound enables the concentration of ionized or un-ionized versions of a chemical to be calculated for a given pH. Due to the large range in magnitudes of Kas, the logarithmic constant (pKa) is commonly used. 12

Compound or Class

Molecular Weight (g/mol)

Acidity Constant (pKa)

Octanol-water Partitioning Coefficient (log Kow)

Water Solubility (mg/L at 25oC)

Beta Blockers Sotalol Atenolol Metoprolol Propranolol Disinfectants Triclosan Triclocarban Antidepressants Venlafaxine O-desmethylvenlafaxine Citalopram desmethylcitalopram Musks Musk ketone Musk xylene Galaxolide Tonalide Antibiotics Erythromycin Pharmaceuticals Naproxen Carbamazepine Ibuprofen Estrogens Estrone Bisphenol A Perfluorinated Compounds Perfluorooctanoate PBDEs 272.4 266.3 267.4 259.3 289.5 315.6 277.41 263.38 324.4 310.37 294.3 297.3 258.4 258.4 733 230.3 206.3 270.4 228.3 9.5 9.2 9.7 9.45 7.9 12.7 9.24 9.74 9.5 10.5 8.9 4.2 13.9 4.9 10.7 10.3 0.24 0.16-0.46 1.69-1.88 3.48-3.03 4.8 4.3 0.43 0.74 3.74 4.3 4.9 5.9 5.7 3.06 3.18 2.25 3.5 3.13 2.2-3.4 137,000 13,300 4,780 70 2-5 0.6-1.5 1.9 0.49 2000 5.11 800 120

414.0 -

3.8 4-11

6.3 -

Table 4: Physical and Chemical Properties of Selected Emerging Contaminants

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Studies investigating the removal of emerging contaminants from wastewater have been conducted at the lab bench scale as well as full wastewater treatment plant scale. The following table outlines the removal of emerging contaminants by selected treatment processes during lab scale experiments. 13

EC Class

Ozonation (Wastewater) (Rosal et al., 2010)

Powdered Activated Carbon (5mg/L, 4h contact time) (Westerhoff et al., 2005) >90% >90% >90% >90% 40-60% -

Ultrafiltration (Drinking water) (Snyder et al., 2007)

MBR (Drinking water) (Snyder et al., 2007) Removed Removed Removed Not Removed

Beta Blockers Antidepressants Disinfectants Antibiotics Estrogens Perfluorinated Compounds PBDEs Pharmaceuticals Musks

Removed Removed Not removed Removed Removed Removed Not removed

Removed Not Removed Removed Not Removed Not Removed

Table 5: Lab scale removals of emerging contaminants by selected treatment processes Where not specified, removal in the above table refers to reduction to levels lower than the detection limit used in the experiment. Ozonation and powdered activated carbon show the potential to be effective removal methods for some classes of emerging contaminants however more data is needed at the pilot and WWTP scale. The following section expands on each EC type monitored in this research and provides removal efficiencies of full scale WWTPs as reported in current literature.

6$+)!.-5*%-+$*6+!43%&)*
Personal Care Products are a class of chemicals found in wide ranging consumer products from toothpaste to soap, to kitchen utensils. They are generally used for their antimicrobial and antifungal properties. In this study, wastewater samples were analysed for triclosan (TCS) and triclocarban (TCC) concentrations. TCS and TCC are both anti-bacterial and anti-fungal agents commonly used in consumer products such as soaps, disinfectants, toothpastes, body washes, and medical disinfectant of which these products contain between 0.1% and 2% of TCS or TCC by

14

weight (Chalew and Halden 2009). Approximately 96% of the triclosan is used in consumer goods, which are disposed of to the sewer system (Riess et al., 2002). They have both been used since the 1970s and have been detected in wastewater treatment plant effluent and surface waters (Singer et al., 2002, Heidler et al., 2006). TSC and TCC interact with bacteria by binding to the enoyl-acyl carrier protein reductase enzyme (ENR) found in their cell membrane inhibiting fatty acid synthesis (Heath et al., 1999). Bacteria need these fatty acids to build new cell membranes and without them they do not have enough material to build new cells and thus cannot reproduce. The following table outlines some triclosan and triclocarban concentrations found in other wastewater treatment plants.
Compound Triclosan Triclosan Triclosan Influent (ng/L) 1900 5200 2500 Effluent (ng/L) 114 260 625 Removal Efficiency 94% 95% 75% Location Ontario Columbus, Ohio United Kingdom Source Lishman et al., 2006 McAvoy et al., 2002 Thompson et al., 2005 Treatment Type Activated sludge process Activated sludge process Rotating biological contactor as secondary -

Triclocarban

6100

170

97%

Heidler et al., 2007

Table 6: Selected Literature Concentration Values for Personal Care Products in WWT The above papers report removal efficiency of 75 to 97% and suggest that an activated sludge process may remove more triclosan than a rotating biological contactor process though detailed comparisons are not possible without detailed operating conditions.

15

-.&'4$6+$))-.&)*
Antidepressants are a class of pharmaceuticals that affect neurotransmitters, the chemicals that nerves within the brain use to communicate with each other. Examples of neurotransmitters include serotonin, dopamine and norepinephrine. Antidepressants are believed to work by inhibiting the release or affecting the action of neurotransmitters. The antidepressant compounds in this study are velafaxine, O-desmethylvenlafaxine, citalopram, and desmethylcitalopram. Venlafaxine is a serotonin-norepinephrine reuptake inhibitor (SNRI) prescribed for the treatment of depression, depression with associated symptoms of anxiety, generalized anxiety disorder, social anxiety disorder and adult panic disorder. O-desmethylvenlafaxine, a major active metabolite of venlafaxine, also functions as an SNRI. It is also synthetically produced (desvenlafaxine) and was approved by Health Canada in 2009 for treatment of depression. Citalopram is a selective serotonin reuptake inhibitor (SSRI) prescribed for the management of depression as well as treating obsessive-compulsive disorder, panic disorder, premenstrual dysphoric disorder, anxiety disorder and posttraumatic stress disorder. Desmethylcitalopram, an active metabolite of citalopram, also functions as an SSRI. The following table outlines the concentrations of Citalopram and Venlafaxine found in other wastewater treatment plants.

16

Compound Citalopram Venlafaxine Venlafaxine

Influent (ng/L) 52 195 540

Effluent (ng/L) 46 175 300

Removal Efficiency 11% 10% 44%

Location Montreal Canada Montreal Canada Spain

Source Lajeunesse at el 2008 Lajeunesse at el 2008 Gracia-Lor et al 2010

Treatment Type Primary Treatment Only Primary Treatment Only Not specified

Table 7: Selected Literature Concentration Values for Antidepressants The removal rate for the wastewater treatment plant in Montreal, Canada was reported as 10 to 12% (Lajeunesse et al., 2008). This low removal efficiency suggests that antidepressants are difficult to remove, however, the wastewater treatment plant in Montreal, Canada is only equipped with primary treatment and municipalities with secondary or tertiary treatment would likely expect a higher removal efficiency. Gracia-Lor et al. (2010) does not specify the treatment type used in their study.

61-+8-%$3&'%-5)*:-%'4;"-)'%;.$3&+-5<*
Pharmaceuticals can be divided up into acidic, basic, or neutral compounds and can be used to treat pain, inflammation and a variety of other conditions. They range in prevalence from Naproxen, which is a prescription drug, to caffeine, which is found in coffee, tea, and chocolate among other goods. The following table outlines examples of the WWTP concentrations found in literature for some of the compounds in this study.

17

Compound Acetaminophen

Influent (ng/L) 23202

Effluent (ng/L) 100

Removal Efficiency 99%

Location Spain

Source Rosal et al., 2009

Treatment Type Biological Treatment with nitrificationdenitrification Biological Treatment with nitrificationdenitrification Activated Sludge and gravity filtration Biological Treatment with nitrificationdenitrification Biological Treatment with nitrificationdenitrification Activated Sludge and gravity filtration Activated Sludge Activated Sludge Activated Sludge

Ibuprofen

2687

135

94%

Spain

Rosal et al., 2009

Ibuprofen

9500

18

99%

Virginia

Thomas and Foster 2004 Rosal et al., 2009

Naproxen

2363

923

60%

Spain

Caffeine

22849

1176

94%

Spain

Rosal et al., 2009

Caffeine

43800

36

99%

Virginia

Thomas and Foster 2004 Santos et al., 2005 Santos et al., 2005 Santos et al., 2005

Caffeine Ibuprofen Naproxen

2700 83500 6000

560 6500 2560

79% 92% 57%

Spain Spain Spain

Table 8: Selected Literature Concentration Values for Pharmaceuticals in WWTPs The reported removal rates for these pharmaceuticals range from 79 to 99% with the exception of Naproxen in Rosal et al. and Santos et al. which reported 60% and 57% respectively. This highlights the differences between specific pharmaceuticals and the need to

18

study each individually. Overall, however, literature suggests that most pharmaceuticals except for naproxen are greater than 90% removed from wastewater.

83)2)*
Musks are a class of compound known for their fragrance. They have broad uses from cosmetics to detergents. They can be separated into three classes: aromatic nitro musks (musk ketone, musk xylene), polycyclic musks(tonalide, galaxolide, celestolide), macrocyclic musks. The following table outlines some musk concentrations found in municipal wastewater treatment plants.
Compound Galaxolide Galaxolide Tonalide Traseolide Tonaline Phantolide Celestolide Galaxolide Tonalide Influent (ng/L) 4000 1701 3000 131 687 22 34 1941 583 Effluent (ng/L) 80 876 150 47 298 20 695 212 Removal Efficiency 98% 48% 95% 64% 56% 41% 64% 63% Location USA Ontario USA Ontario Ontario Ontario Ontario Germany Germany Source Horii et al., 2007 Lishman et al., 2006 Horii et al., 2007 Lishman et al., 2006 Lishman et al., 2006 Lishman et al., 2006 Lishman et al., 2006 Bester 2004 Bester 2004 Treatment Type Activated Sludge Activated Sludge Activated Sludge Activated Sludge Activated Sludge Activated Sludge Activated Sludge Activated Sludge Activated Sludge

19

Musk ketone Musk xylene Tonalide Galaxolide

569 376 10700 13700

99 5 1180 1170

82% 98% 88% 91%

Ohio Ohio Ohio Ohio

Simonich et al., 2000 Simonich et al., 2000 Simonich et al., 2000 Simonich et al., 2000

Activated Sludge Activated Sludge Activated Sludge Activated Sludge

Table 9: Selected Literature Concentration Values for Musks in WWTPs The removal rates for musks in the wastewater treatment plants described above range from 41 to 99%. This high variability suggests that the removal of musks is inconsistent between wastewater treatment plants.

6"4$)*
Polybrominated diphenyl ethers or PBDEs are commonly used as flame-retardants in a variety of products including electronics, cars, textiles, and plastics. The PDBE family includes 209 individual compounds, which are generally used as a mixture rather then a pure compound. The following table outlines the concentrations of common PDBEs found at different municipal wastewater treatment plants.
Compound PBDE-47 PBDE-99 PBDE-100 PBDE-153 Influent (ng/L) 102 121 19 11 Effluent (ng/L) 14 14 2.8 1.6 Removal Efficiency 86% 88% 85% 85% Location Ontario Ontario Ontario Ontario Source Song et al., 2006 Song et al., 2006 Song et al., 2006 Song et al., 2006 Treatment Type Activated Sludge Activated Sludge Activated Sludge Activated Sludge

20

PBDE-154 PBDE-183 Total PBDE Total PBDE

7.6 1.7 265 -

1.1 36 29

85% 86% -

Ontario Ontario Ontario California

Song et al., 2006 Song et al., 2006 Song et al., 2006 North 2004

Activated Sludge Activated Sludge Activated Sludge Activate Sludge with gravity filter Anoxic, anaerobic, aerobic biological treatment (A2O) A2O

PBDE-47

3.4

0.1

97%

China

Peng et al., 2009

PBDE-99

3.4

0.09

97%

China

Peng et al., 2009

Table 10: Selected Literature Concentration Values for PBDEs in WWTPs All of the wastewater treatment plants documented above have a removal efficiency ranging from 85 to 97% with respect to PBDEs. This indicates that PBDEs are consistently at efficiencies higher than 80% by activated sludge processes.

-.&'"'!&'%)*
Antibiotics are a class of drug used to kill or slow the growth of bacteria. They are available by prescription from a doctor and are used to treat a variety of bacterial infections. The following table outlines concentrations of various antibiotics in municipal wastewater plants.
Compound Ciprofloxacin Influent (ng/L) 150 Effluent (ng/L) 60 Removal Efficiency 60% Location Wisconsin Source Karthikeyan and Meyer 2005 Karthikeyan and Meyer Treatment Type Activate Sludge Activate

Sulfamethoxazole

300

200

33%

Wisconsin

21

2005 Trimethoprim 330 170 48% Wisconsin Karthikeyan and Meyer 2005 Karthikeyan and Meyer 2005 Watkinson et al., 2007 Watkinson et al., 2007 Watkinson et al., 2007 Brown et al., 2006 Brown et al., 2006

Sludge Activate Sludge Activate Sludge Activate Sludge Activate Sludge Activate Sludge Activate Sludge Activate Sludge

Erythromycin

3900

1100

71%

Wisconsin

Ciprofloxacin Sulfamethoxazole Trimethoprim Sulfamethoxazole Trimethoprim

3800 360 340 390 590

640 270 50 310 180

83% 25% 85% 20% 69%

Australia Australia Australia Mexico Mexico

Table 11: Selected Literature Concentration Values for Antibiotics in WWTPs The papers above report similar removal rates for respective compounds across locations ranging from 21 to 33% for Sulfamethoxazole, 60 to 83% for Ciprofloxacin, and 48 to 85% for Trimethoprim.

$)&+!,$.'%*%!86!3.4)*:.-&3+-5;'.43)&+'-5<*
Estrogenic compounds are substances that interact with the human bodys hormone system. They include natural compounds such as estrogen and synthetic compounds such as Bisphenol A and nonylphenol. The following table outlines the concentrations of some estrogenic compounds found in municipal wastewater treatment plants.

22

Compound Esterone Estradiol Estrone

Influent (ng/L) 30 8 54.8

Effluent (ng/L) 13 8.1

Removal Efficiency 56% 85%

Location Ontario Ontario Australia

Source Lishman et al., 2006 Lishman et al., 2006 Braga et al., 2005

Treatment Type Activated Sludge Activated Sludge Activated Sludge with Reverse osmosis Activated Sludge with Reverse osmosis Not specified Anaerobic/ aerobic with activated sludge Anaerobic/ aerobic with activated sludge Anaerobic/ aerobic with activated sludge

a-estradiol

22

0.95

95%

Australia

Braga et al., 2005

Bisphenol A Bisphenol A

1600 140

900 86

43% 38%

Greece Australia

Gatidou et al., 2006 Tan et al., 2007

Nonylphenol

3070

335

89%

Australia

Tan et al., 2007

Octylphenol

229

23

89%

Australia

Tan et al., 2007

Table 12: Selected Literature Concentration Values for Estrogens in WWTPs The wastewater treatment plants as reported above have estrogenic compound removal efficiencies ranging from 40 to 90% with the highest removal efficiencies found in locations with tertiary, reverse osmosis treatment systems.

23

6$+053!+'.-&$4*%!86!3.4)*
Perfluorinated compounds are organofluorine compounds with hydrogens replaced with fluorine on the carbon chain. They are used in a large variety of products for properties such as water, oil, and stain resistance. PFOA is used, for example, to make Teflon while PFOS is used in the semiconductor industry,
Compound PFOA PFOA PFOS PFOS PFOS Influent (ng/L) 100 50 16 7.8 20 Effluent (ng/L) 122 53 13 9.3 24 Removal Efficiency 0% 0% 18% 0% 0% Location Kentucky Georgia Kentucky Georgia Oregon Source Loganathan et., al 2007 Loganathan et al., 2007 Loganathan et al., 2007 Loganathan et al., 2007 Schultz et al., 2006 Schultz et al., 2006 Yu et al., 2009 Treatment Type Activated Sludge Activated Sludge Activated Sludge Activated Sludge Trickling filter and activated sludge Trickling filter and activated sludge Activated Sludge and Membrane Bioreactor Activated Sludge and Membrane Bioreactor

PFOA

15

11

26%

Oregon

PFOA

16.3

24.3

0%

Singapore

PFOS

13.9

12.6

10%

Singapore

Yu et al., 2009

Table 13: Selected Literature Concentration Values for Perfluorinated Compounds in WWTPs

24

The removal efficiencies reported in the table above range from -49% to 20%. These low rates are reported across all of the papers and locations indicating perfluorinated compounds are not effectively removed from wastewater streams by activated sludge, membrane bioreactor, or trickling filter systems.

25

+$0$+$.%$)*
Barber, L. B.; Brown, G. K.; Zaugg, S. D. (2000) In Analysis of Environmental Endocrine Disruptors; Keith, L. H., Jones Lepp, T. L., Needham, L. L., Eds.; ACS Symposium Series 747; American Chemical Society: Washington, DC, 2000; pp 97 123. Bauman, L.C. ; Stenstrom, M.K. (1990) Removal of organohalogens and organohalogen precursors in reclaimed wastewater. Water Research, v 24, n 8, p 949 955. Braga O., G. Smythe, A. Schafer, A. Feitz, Fate of Steroid Estrogens in Australian Inland and Coastal Wastewater Treatment Plants, Environ. Sci. Technol. 2005, 39, 3351-3358 Brown K., Jerzy Kulis, Bruce Thomson, Timothy H. Chapman, Douglas B. Mawhinney, Occurrence of antibiotics in hospital, residential, and dairy effluent, municipal wastewater, and the Rio Grande in New Mexico, Science of the Total Environment 366 (2006) 772783 Brown, Gregory K.; Zaugg, Steven D.; Barber, Larry B. (1999) Wastewater analysis by gas chromatography/mass spectrometry. Water Resour. Invest. Rep. U.S. Geol Surv.No. 99 4018B, pp 431 435. Bester, K. Retention characteristics and balance assessment for two polycyclic musk fragrances (HHCB and AHTN) in a typical German sewage treatment plant, Chemosphere 57 (2004) 863870 Emmanuel E, Hanna K, Bazin C, Keck G, Clement B, Perrodin Y. (2005) Fate of glutaraldehyde in hospital wastewater and combined effects of glutaraldehyde and surfactants on aquatic organisms. Environment International, 31(3):399 406. Environmental Protection Agency. Fact Sheet: Final Third Drinking Water Contaminant Candidate List (CCL 3). 2008. www.epa.gov/safwater European Commission. Commission Staff Working Paper: Report on the implementation of the Community Strategy for Endocrine Disrupters a range of substances suspected of interfering with the hormone systems of humans and wildlife. Brussels, 2011 Gatidou G, Nikolaos S. Thomaidis, Athanasios S. Stasinakis, Themistokles D. Lekkas, Simultaneous determination of the endocrine disrupting compounds nonylphenol, nonylphenol ethoxylates, triclosan and bisphenol A in wastewater and sewage sludge by gas chromatographymass spectrometry, Journal of Chromatography A, 1138 (2007) 3241 Gracia-Lor E., Juan V. Sancho, Flix Hernndez, Simultaneous determination of acidic, neutral and basic pharmaceuticals in urban wastewater by ultra high-pressure liquid chromatographytandem mass spectrometry. Journal of Chromatography A, 1217 (2010) 622-633 Haider S.; Baqri S.S.R., 2000. beta-Adrenoceptor antagonists reinitiate meiotic maturation in Clarias batrachus oocytes. Comp Biochem Physiol Mol Integr Physiol , 126: 51725. 26

Hirsch R, Ternes TA, Haberer K, Mehlich A, Ballwanz F, Kratz KL. (1998) Determination of antibiotics in different water compartments via liquid chromatography-electrospray tandem mass spectrometry. J Chromatogr A. 31;815(2):213-23. Karickhoff S.; Brown D.; Scott T.; Sorption of hydrophobic pollutants on natural sediments. Water Research. 1979. 13:241-248. Karthikeyan K., M. Meyer, Occurrence of antibiotics in wastewater treatment facilities in Wisconsin, USA, Science of the Total Environment 361 (2006) 196207 Lajeunese, A. C. Gagnon, S. Sauve. Determination of Basic Antidepressants and Their NDesmethyl Metabolites in Raw Sewage and Wastewater Using Solid-Phase Extraction and Liquid Chromatography-Tandem Mass Spectrometry, Anal. Chem. Lenz, Katharina; Hann, Stephan; Koellensperger, Gunda; Stefanka, Zsolt; Stingeder, Gerhard; Weissenbacher, Norbert; Mahnik, Susanne N.; Fuerhacker, Maria. (2005) Presence of cancerostatic platinum compounds in hospital wastewater and possible elimination by adsorption to activated sludge. Science of the Total Environment, v 345, n 1 3, p 141 152. Lishman L., Shirley Anne Smyth, Kurtis Sarafin, Sonya Kleywegt, John Toito, Thomas Peart Bill Lee, Mark Servos, Michel Beland, Peter Seto, Occurrence and reductions of pharmaceuticals and personal care products and estrogens by municipal wastewater treatment plants in Ontario, Canada, Science of the Total Environment 367 (2006) 544558 Loganathan, B., Kenneth S. Sajwan, Ewan Sinclair, Kurunthachalam Senthil Kumar, Kurunthachalam Kannan, Perfluoroalkyl sulfonates and perfluorocarboxylates in two wastewater treatment facilities in Kentucky and Georgia, Water Research 41 (2007) 4611 4620 Michele E. Lindsey, Michael Meyer, and E. M. Thurman. (2001)Analysis of Trace Levels of Sulfonamide and Tetracycline Antimicrobials in Groundwater and Surface Water Using Solid-Phase Extraction and Liquid Chromatography/Mass Spectrometry. Anal. Chem.; 73(19) pp 4640 4646. North, K. Tracking Polybrominated Diphenyl Ether Releases in a Wastewater Treatment Plant Effluent, Palo Alto, California. Environ. Sci. Technol. 2004, 38, 4484-4488 Reinthaler, F.F. ; Posch, J.; Feierl, G.; Wust, G.; Haas, D.; Ruckenbauer, G.; Mascher, F.; Marth. (2003). Antibiotic resistance of E. coli in sewage and sludge : Water Research, v 37, n 8, p 1685 1690. Rosal, R, Antonio Rodrguez, Jose Antonio Perdigo !n-Melon, Alice Petrea, Eloy Garca-Calvo, Mar !a Jose ! Go !mez, Ana Aguera, Amadeo R. Ferna !ndez-Alba. Occurrence of emerging pollutants in urban wastewater and their removal through biological treatment followed by ozonation. Water Research 44(2010) 578-588 Salako Q, DeNardo SJ.(1997) Analysis of long lived radionuclidic impurities in short lived radiopharmaceutical waste using gamma spectrometry. Health Phys. 1997 Jan;72(1):56 9. 27

Santos, J. I. Aparicio, E. Alonso, M. Callejn. Simultaneous determination of pharmaceutically active compounds in wastewater samples by solid phase extraction and high-performance liquid chromatography with diode array and fluorescence detectors. Analytica Chimica Acta 550 (2005) 116122 Schultz, M., C. Higgens, C. Huset, R. Luthy, D. Barofsky, J. Field, Fluorochemical Mass Flows in a Municipal Wastewater Treatment Facility, Environ. Sci. Technol. 2006, 40, 7350-7357 Simonich, S. W. Begley, G. Debaere, W. Eckhoff. Trace Analysis of Fragrance Materials in Wastewater and Treated Wastewater, Environ. Sci. Technol. 2000, 34, 959-965 Song, M. S. Chu, R. Letcher, R. Seth, Fate, Partitioning, and Mass Loading of Polybrominated Diphenyl Ethers (PBDEs) during the Treatment Processing of Municipal Sewage, Environ. Sci. Technol. (2006) 40, 6241-6246 Snyder S.; Adham S.; Redding A.; Cannon F.; DeCarolis J.; Oppenheimer J.; Wert E.; Yoon Y. Role of membranes and activated carbon in removal of endocrine disruptors and pharmaceuticals. Desalination (2007) 202,156-181 Snyder S. ,Vanderford, B. R. Pearson, O. Quinones and Y. Yoon, Analytical methods used to measure endocrine disrupting compounds in water, Practice Periodical of Hazardous, Toxic and Radioactive Waste Management ASCE, 7(4) (2003) 224234 Synder S, Westerhoff P, Yoon Y, Sedlak D. Pharmaceuticals, Personal Care Products, and Endocrine Disruptors in Water: Implications for the Water Industry. Environmental Engineering Science,. (2003) 20:5:449-469 Tan B, Darryl W. Hawker, Jochen F. Mu ller, Fre !de !ric D.L. Leusch, Louis A. Tremblay, Heather F. Chapman, Modelling of the fate of selected endocrine disruptors in a municipal wastewater treatment plant in South East Queensland, Australia, Chemosphere 69 (2007) 644654 Thomas, P. G. Foster, Tracking Acidic Pharmaceuticals, Caffeine and Troclosan through the Wastewater Treatment Process. Environmental Toxicology and Chemistry 24(2005) 25-30 Watkinson, A., S. Costanzo. Removal of antibiotics in conventional and advanced wastewater treatment: Implications for environmental discharge and wastewater recycling, Water Research 41 (2007) 4164 4176 World Health Organization. WHO/HSE/WSH/11.5 Pharmaceuticals in Drinking-water. Geniva. 2011

Yu, J., J. Hu, S. Tanaka, S. Fujii, Perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) in sewage treatment plants, Water Research 43 (2009) 23992408

28

CHAPTER 2
Fate of Prion Proteins in Wastewater: A Review
Daniel P. Shaver*a, Hamid Salsalia, Edward A. McBeana , M. Alamgirb, A. Golshanib
a

School of Engineering, University of Guelph, Thornbrough Building, N1G 2W1 Guelph,

Ontario, Canada
b

Department of Biology, Carleton University, Ottawa, Ontario, Canada

*dshaver@uoguelph.ca

Abstract
Since the 1980s, scientists have been aware of abnormally-folded proteins that cause a group of neurodegenerative diseases such as Transmissible Spongiform Encephalopathies (TSEs) in humans, Bovine Spongiform Encephalophathy (BSE) and Scrapie in animals, collectively called proteinaceous infectious particles, or prions. Although numerous investigators have tried to establish an understanding of the details of their impacts not only in animals, but also in the environment, little is known about their fate, function and detection in wastewater treatment. Evidence suggests that these abnormally-folded proteins are resistant to extreme heat and chemical treatment and may remain viable in the environment for years. This review provides an overview of the fate of prions in wastewater treatment, which indicates that adsorption leads to partitioning into the wastewater sludge, and that prions are not destroyed during wastewater treatment.

29

Introduction
Neurodegenerative diseases are caused by death or loss of the functionality of neuron cells due to the mutation of genes (Thompson, 2008) or the accumulation of toxic proteins (Rubinsztein, 2006) that form "-sheet rich amyloidic deposits in the central nervous system(CNS) (Song et al., 2008). One such class of neurodegenerative diseases is known as Transmissible Spongiform Encephalopathy (TSEs). These TSEs are caused by a natural prion protein in a misfolded form. The normal PrPC protein is a cellular glycoprotein found in the cell membrane. It has been suggested that PrPC is involved in the function of long-term memory (Shorter and Lindquist, 2005) or stem cell renewal (Maglio et al., 2004). Studies suggest that the infectious agents of TSEs are an abnormally folded version of PrPC, called PrPSC (Wiggins, 2009). Although the amino acid sequences of normal cellular PrPC and abnormally folded PrPSc are the same, these proteins differ in their conformation, i.e. spatial arrangement of the molecules, due to post-translational modification (Herms et al., 1999). Although very little is known about the propagation of prion proteins within cells, it is thought that the PrPSc protein may cause the normal form of the protein, PrPc, to fold improperly, resulting in propagation of the PrPSC protein and a cascade of harmful effects (Wiggins, 2009). TSEs affect many different mammals such as cattle (bovine spongiform encephalopathy BSE) sheep and goats (scrapie), deer (chronic wasting disease CWD), and humans (Creutzfeld-Jakob Desease CJD). Although horizontal transmission in sheep and deer is well documented, very little data are available to explain how the human variants of Prion diseases, such as CJD and Fatal Familial Insomnia are transmitted. Human exposure to prions is very rare and has historically been limited to consuming meats that are contaminated with infectious prions (Genovesi et al., 2007).

30

Since little is known about their transmission, it is important to understand their fate in the environment and the possible ways by which humans can be exposed to them, to facilitate identifying how to adequately protect the public. This review will outline current research investigating the fate of prions from various sources such as slaughterhouses, through the wastewater treatment process, to their eventual destruction or release back into the environment. 4$&$%&'!.*8$&1!4)* In order to investigate the fate of prions in wastewater treatment, reliable methods of detection must be applied. There are various techniques available for the identification of prions, such as immunoassays (Reuter et al., 2009; Paspaltsis et al., 2009; Genovesi et al., 2007; Xiong et al., 2009), and neuronal cell assays (Klohn et al., 2003). Immunoassays utilize a specially chosen compound that specifically binds to the prion protein, which can then be detected by chemiluminescence or other detection techniques. These techniques are generally very costly and have low sensitivity, making it difficult to detect target proteins in low concentrations. A relatively new detection method for prions is called Protein Misfolding Cyclic Amplification (PMCA). Conceptually similar to polymerase chain reaction, PMCA adds normal PrPC proteins to the sample containing trace amounts of misfolded PrPSC proteins and through a series of cycles, the PrPC proteins are converted to PrPSC amplifying the amount of prions to more easily detectable levels by traditional immunoassays (Castilla et al. 2006, Soto et al. 2002). The primary advantage of this method is the very low concentrations of prions that can be detected such as those found in wastewater though the technique has not yet been found practical in for environmental analysis. Of the several detection methods, a highly sensitive, accurate, and cost effective technique has not be found and continues to be a topic of research.

31

*)$7-,$*%!.&-8'.-&'!.*6!'.&)*

There are several ways that prions can be introduced into the wastewater system. In animals, it has been show that CWD and Scrapie can be shed from the hosts urine, feces, and blood (Seeger et al., 2005; Gonzales-Romero et al., 2008; Maluquer de Motes et al., 2008; Saunders 2010). If a slaughterhouse or other animal processing facility is connected to the main sewer system, it is possible for contaminated blood or urine to enter the wastewater system. It is also possible that human hosts of CJD may excrete prions into the wastewater system through their urine though there have been no reports of this process. Another potential source of prions into the wastewater system is through slaughterhouse waste. It is known that prion proteins are primarily found in the brain and spinal tissues of infected animals. If an infected animal is processed in a slaughterhouse facility, there is the possibility of prions being released into the wastewater system.

Prions in Waste Water Treatment Plants

Partitioning refers to the process of separation of particles, compounds or contaminants into different phases of a system. Understanding partitioning is an important step in establishing the fate of any contaminants such as prions in wastewater treatment processes. When sewage from a municipal sewer system enters a wastewater treatment plant, it is treated in multi-step processes to remove or destroy different contaminants in the wastewater. Generally, microorganisms decompose the biodegradable materials of the sewage before the treated wastewater is discharged back into the environment. The particles and contaminants, which have been removed from the water phase, are then disposed of to a landfill or utilized for land application for its nutrient value. However, the potential exists that these particles may be a 32

source of contamination for fruits and vegetables if wastewater derived biosolids are used as fertilizer. As a result, researchers are trying to determine the percentage of active infectious materials which end up in wastewater, sludge, or in the biosolids following the wastewater treatment process. Due to the pathogenicity of prions, researchers may adopt use of an alternative to prions in their research. Depending on the type of research, one may choose to use either actual prions obtained from infected animals or proteins that behave similarly to prions. In some studies, researchers used non-pathogenic amyloid-like fibrils that have structural features similar to prions (Morales-Belpaire and Gerin, 2008). Amyloid fibrils are a type of extracellular protein that behave similarly to prions in terms of transport and partitioning situations and hence can be used in various model systems (Morales-Belpaire and Gerin, 2008). In addition, protein particles from infected hamsters (PsPTSE) may also be an alternative choice. For this approach, hamsters are infected with a TSE agent and then the brains are homogenized to isolate the PsPTSE. Hamster proteins are also used to test the transmissibility of the prions. In a study by Hinckley et al. (2008), the researchers spiked activated sludge from a wastewater treatment plant with infectious Prions PrPTSE and then subjected this sludge to simulated conditions similar to a typical activated sludge and anaerobic digestion treatment process. Once the process was completed, different fractions were collected and analyzed by using immunoblotting techniques. They found >99% of the PrPTSE partitioned into the sludge floc. This finding suggests that the majority of prions entering activated sludge remain active even after traditional treatment systems such as anaerobic digestion and remain un-degraded in the digested sludge (Hinckley, 2008). In addition, they showed that a large fraction of PrPTSE survived in the sludge even after 20-days of anaerobic digestion (Hinckley, 2008). This result 33

suggests that most of the PrPTSE would pass through the wastewater treatment processes followed by mesophilic anaerobic digestion, ending up in the digested sludge rather in the plants liquid effluent (Hinckley, 2008). Because Prions from infected animals are difficult to obtain and work with, recent work has been limited to hamster Prions or amyloid fibrils in simulated wastewater treatment processes. Morales-Belpaire and Gerin (2008) spiked activated sludge/supernatant mixtures obtained from a local WWTP with amyloid fibrils. After centrifuging at 6700g for ten minutes, they used fluorescence techniques to determine whether the fibrils partitioned into the sludge or into the supernatant. Their findings indicated that the fibrils were adsorbed to the sludge flocs and were not accumulating in the supernatant (Morales-Belpaire and Gerin, 2008). Diverse approaches have been taken by investigators to reduce the activity of prion proteins. Under appropriate conditions, Brown et al. (2004) show that it is possible to reduce the activity of prions from Scrapie-infected hamster brains by incineration at 10000C for 15 minutes, although a small amount of infectivity survived even at 600C. Recently, Paspaltsis et al. (2009) used titanium dioxide (TiO2) for the photocatalytic degradation of prions, which reduces the infectivity significantly. Although it has been suggested that prions remain infectious after passing though anaerobic digesters in wastewater treatment processes, it has been shown that UV-ozone treatment can potentially reduce the amount of prions present. Johnson et al. (2006) used prions from hamsters and adsorbed them on to silicon wafers, clay, and quartz based soil. They exposed them to UVOzone treatment for 1 week and tested for the presence of prions using immunoblot techniques. It was found that the UV-ozone treatment greatly reduced the amount of detectable prions in 34

each sample (Johnson et al., 2009). Currently, UV-ozone is not common method for treating wastewater in Ontario nor is incineration at 10000C although ongoing research may yield other techniques that are more applicable in a wastewater treatment context. 0-&$*!0*6+'!.)*'.*5-.4*-665'%-&'!.)*-.4*5-.40'55'.,* Previous studies show that a significant portion of proteins are transported from sewage to the treatment facility by adsorption to solid particulates. However, little is known about the binding mechanisms of prions to solid particles. It was found that different soil particles (e.g. clay, quartz, etc.) bind to the prion proteins in different manners. This is likely due to the presence of different surface characteristics of the soil minerals (Johnson et al., 2006). Jacobson et al. (2009) explain the transportation process of prion proteins through landfill refuse. They used a variety of soil types including sand, natural soil, green waste residual (composted organic matter or GWR) and municipal solid waste (MSW) to determine the migration of PrPSC protein in the refuse, using the colloid transport equation (Jacobson et al., 2009; Bradford et al., 2003):

where " is volumetric water content, C is the PrPSC concentration in the aqueous phase, t is time, z is vertical distance, v is seepage velocity, D is the dispersion coefficient, q is the Darcy velocity, #d is dry density of the medium in the column, Kd is the equilibrium distribution coefficient, katt is a first-order attachment coefficient, and kdet is a first order detachment coefficient. By using this equation, Jacobson was able to estimate the effects of Kd, katt, and kde

35

on PrPSC transport and found that Kd and kdet had to be extremely small to account for his results in the quartz sand and natural soil columns.
Porous medium Quartz Sand Boardman Silt Bluestem Clay Fresh MSW Aged MSW GWR Percent of Loaded PrPTSE eluted (%) 0 0 0 28 0.3 2 Kd (L/kg) 0.15 0.05 4.5 0.2 0.0 1.2 katt (h-1) >2.9 >2.6 >3.3 0.55 0.04 6.6 0.3 1.9 1.8 kdet (h-1) 0.003 0.003 0.001 0.002 0.01 0.38

Table 14: PrPTSE Transport Parameters from Column Tests (Jacobsen et al., 2009) This research by Jacobson et al. suggests that some soil types such as quartz sand may be able to retain prions and stop their release outside of the landfill site by having the prion adsorb strongly to the soil surface. This has implications both for the landfilling of municipal solid waste and the burying of culled livestock infected with TSEs. If it can be shown in future research that some soils form a barrier to prion transport, with the potential to be used as part of landfill liners designed in such a way to minimize the potential for prions to escape. It is also possible that the PrPSC could run off into surface water or could adsorb to soil particles from contaminated locations and may survive for three years in the soil (Brown and Gajdusek, 1991).

+')2
A risk assessment is a way for scientists to quantify the likelihood of people contracting prion diseases. Several studies have been done in England where outbreaks of BSE have led to mass cattle culls and raised concerns of increased TSE cases in humans. Due to the unknown concentration of TSE agents entering the WWTPs, risk calculations must be based on estimates of prions entering the sewage system from the processing of cattle infected with BSE. In a risk assessment by Gale and Stanfield (2001), it was concluded that prions in the biosolids from

36

WWTPs were not significant enough to explain a BSE outbreak in cows when spread on agricultural land. The risk to humans exposed to prions by consuming vegetables grown on soil and biosolids from WWTPs was also found to be acceptably low. Gale and Stanfield (2001) reports the annual risk of CJD infection is 1.32 x 10-7 person-1 year-1 assuming that the vegetables are not washed. In drinking water, it has been estimated that the risk to someone living near a rendering plant processing infected cattle is 1.5 x 10-8 person-1 year-1 (Gale et al., 1998). One of the primary objectives of a risk assessment is to construct a dose response curve indicating the risk of infection verses the dose exposed.

Figure 2: Dose-Response Curve for BSE in humans (Gale 2005) The dose-response curve above from Gale (2005) shows the median of this curve indicating the ID50 of humans consuming infected cow brain is about 24g while the arithmetic mean ID50 is 2.67g (Gale 2005). This lower estimate of 2.67g is high enough that the chances of someone being exposed to an ID50 through drinking water or crops grown on soil treated with sewage sludge is quite low (Gale 2005). The cumulative effects of multiple, low doses of prion proteins are unknown and a topic of continuing research. 37

Current assessments conclude that the risk of human infection from drinking water or biosolids use to be very low despite the lack of knowledge concerning prion concentrations entering WWTPs.

Conclusions and Recommendations

Although infectious particles can enter the wastewater stream from different sources and make their way to treatment plants during the wastewater treatment process, they tend to partition into the sludge fraction. It is still not clear to what degree strains remain infectious after rigorous wastewater treatment though it has been shown that prions do survive the anaerobic digestion of wastewater sludge. If infected biosolid wastes are sent to a landfill following treatment, different soil types such as quartz sand may potentially immobilize the prion proteins and serve as a reservoir for the proteins. This reservoir could provide a potential route for the introduction of infectious agents into the environment at a later stage if disturbed. Further research is needed to address and investigate the fate of prions in biomass from wastewater treatment and when the sludge is applied as a fertilizer for crops and vegetables. As emerging and trace contaminants draw more attention from the scientific community, prion research will continue to grow. In this situation, more advanced detection methods are required which will greatly facilitate the research. Currently, it is very difficult to detect prions quantitatively because of the low concentrations of prions found in the environment and in wastewater treatment plants. Most of the current detection strategies are based on immunochemical techniques that are limited in terms of the scope of applicability. The risk associated with prions entering WWTPs has been estimated to be quite low. Though there is limited data, plants grown on land with biosolids application are expected to have minimal risk

38

of TSE infection, as is drinking water. It may be useful to identify the risk factors by analyzing and assessing the factors associated with the environmental spread of prion diseases. These questions will help to further elucidate the impact on the environment and the fundamentals of the fate of prions in wastewater. As discussed, significant progress has been made so far, for the identification and fate of prions in wastewater, but since prions seem to be quite resilient in conventional treatment processes, the development of modern techniques will allow some key questions regarding the fate of Prions in wastewater treatment plants to be resolved.

39

References
Bradford, S. A., Simunek, J., Bettahar, M., van Genuchten, M. T., & Yates, S. R. (2003), Modeling colloid attachment, straining, and exclusion in saturated porous media. Environ Sci Technol. 37(10), 2242-2250. Brown, P., Rau, E. H., Lemieux, P., Johnson, B. K., Bacote, A. E., & Gajdusek, C. (2004), Infectivity studies of both ash and air emissions from simulated incineration of scrapiecontaminated tissues. Environ Sc. Technol., 38(22), 61556160.. Brown, P., & Gajdusek, D. C. (1991), Survival of scrapie virus after 3 years interment. Lancet, 337(8736), 269-270. Castilla J., Paula Sa, Rodrigo Morales, Karim Abid, Kinsey Maundrell and Claudio Soto. Protein Misfolding Cyclic Amplification for Diagnosis and Prion Propagation Studies. Methods in Enzymology. (2006) 412:3-21 Gale, P. (2006), BSE risk assessments in the UK: A risk tradeoff? J Appl Microbiol., 100(3), 417-427. Gale, P., & Stanfield, G. (2001), Towards a quantitative risk assessment for BSE in sewage sludge. J Appl Microbiol, 91(3), 563-569. Gale, P. (1998), Quantitative BSE risk assessment: relating exposures to risk. Letters in Applied Microbiology., 27: 239-242. Genovesi, S., Leita, L., Sequi, P., Andrighetto, I., Sorgato, M. C., & Bertoli, A. (2007), Direct detection of soil-bound Prions. PloS One, 2(10): e1069. Gonzalez-Romero D, Barria MA, Leon P, Morales R, Soto C. Detection of infectious prions in urine. FEBS Lett. (2008) 582(21-22):3161-6. Herms, J., Tings, T., Gall, S., Madlung, A., Giese, A., Siebert, H., Schrmann, P., Windl, O., Brose, N., & Kretzschmar, H. (1999), Evidence of presynaptic location and function of the Prion protein. J Neurosci., 19(20), 8866-8875. Hinckley, G. T., Johnson, C. J., Jacobson, K. H., Bartholomay, C., McMahon, K. D., McKenzie, D., Aiken, J. M., & Pedersen, J. A. (2008), Persistence of pathogenic Prion protein during simulated wastewater treatment processes. Environ Sci Technol., 42(14), 5254-5259. Jacobson, K. H., Lee, S., McKenzie, D., Benson, C. H., & Pedersen, J. A. (2009), Transport of the pathogenic Prion protein through landfill materials. Environ Sci Technol., 43(6), 20222028. Johnson, C. J., Gilbert, P. U., McKenzie, D., Pedersen, J. A., & Aiken, J. M. (2009), Ultravioletozone treatment reduces levels of disease-associated Prion protein and Prion infectivity. BMC Res Notes, 2:121. 40

Johnson, C. J., Phillips, K. E., Schramm, P. T., McKenzie, D., Aiken, J. M., & Pedersen, J. A. (2006), Prions adhere to soil minerals and remain infectious. PLoS Pathog., 2(4): e32. Klohn, P.C., Stoltze, L., Flechsig, E., Enari, M., & Weissmann, C. (2003), A quantitative, highly sensitive cell-based infectivity assay for mouse scrapie Prions, Proc Nat Acad Sci U. S. A., 100(20) ,1166611671. Maglio LE, Perez MF, Martins VR, Brentani RR, Ramirez OA., Hippocampal synaptic plasticity in mice devoid of cellular prion protein. Molecular Brain Research. 2004. 131 (1-2): 5864. Maluquer de Motes C, Grassi J, Simon S, Herva ME, Torres JM, Pumarola M, Girones R.Excretion of BSE and scrapie prions in stools from murine models. Vet Microbiol. (2008) 131(1-2):205-11 Maluquer de Motes, C., Cano, M. J., Torres, J. M., Pumarola, M., & Girones, R. (2008), Detection and survival of Prion agents in aquatic environments. Water Res., 42(10-11), 24652472. Maluquer de Motes, C., Simon, S., Grassi, J., Torres, J. M., Pumarola, M., & Girones, R. (2008), Assessing the presence of BSE and scrapie in slaughterhouse wastewater. J Appl Microbiol., 105(5), 1649-1657. Morales-Belpaire, I., & Gerin, P. A. (2008), Fate of amyloid fibrils introduced in wastewater sludge. Water Res., 42(17), 4449-4456. Paspaltsis, I., Berberidou, C., Poulios, I., & Sklaviadis, T. (2009), Photocatalytic degradation of Prions using the photo-Fenton reagent. J Hos Infect., 71(2), 149-156. Reuter,T., Gilroyed B. H., Alexander, T. W., Mitchell, G., Balachandran, A., Czub, S., & McAllister, T. A. (2009). Prion protein detection via direct immuno-quantitative real-time PCR. J Microbiol Methods., 78(3), 307-311. Rubinsztein, D. C. (2006), The roles of intracellular protein-degradation pathways in neurodegeneration. Nature 443(7113): 780786. Saunders, S. E., Bartz, J. C., Vercauteren, K. C., & Bartelt-Hunt, S. L. (2010), Enzymatic digestion of chronic wasting disease Prions bound to soil. Environmental Science & Technology, 44(11), 4129-4135. Seeger H, Heikenwalder M, Zeller N, Kranich J, Schwarz P, Gaspert A, Seifert B, Miele G, Aguzzi A., Coincident scrapie infection and nephritis lead to urinary prion excretion. Science. (2005) 310:324-6. Shorter J, Lindquist S., Prions as adaptive conduits of memory and inheritance. Nature Reviews. Genetics (2005). 6 (6): 43550

41

Soto C., Gabriela P. Saborio, Laurence Anderes. Cyclic amplification of protein misfolding: application to prion-related disorders and beyond. Trends in Neurosciences (2002) 25:8: 390394 Thompson, L. M. (2008), Neurodegeneration: A question of balance. Nature 452(7188), 707708. Taylor, D. M. (2000), Inactivation of transmissible degenerative encephalopathy agents: A review. Vet. J., 159 (1), 1017. Wiggins, R. C. (2009), Prion stability and infectivity in the environment. Neurochem Res., 34(1), 158-168. Xiong, X., Ouyang, J., Xiong, X., Zhai, S., & Baeyens, W. R. G. (2009), Chemiluminescencebased detection technologies for biomolecules, mainly in gel electrophoresis. TrAC Trends in Analytical Chemistry, 28 (8), 961-972.

42

CHAPTER 3
Sources of "-Blockers, Antidepressants, and Disinfectants in Municipal Wastewater
Daniel P. Shaver*a, Hamid Salsalia, Edward A. McBeana

School of Engineering, University of Guelph, Thornbrough Building, N1G 2W1 Guelph,

Ontario, Canada *dshaver@uoguelph.ca

Abstract
Emerging contaminants are a growing concern in wastewater treatment. Low concentrations of previously undetected chemicals are being identified in surface waters and wastewater treatment effluents with unknown effects on aquatic ecosystems. To effectively design a removal strategy, the source pathways of these compounds into the wastewater system must be classified. Effluents from hospitals, funeral homes, residential neighborhoods, and a slaughter house in three different cities are used to identify the sources of beta-blockers, antidepressants, and disinfectants in municipal wastewater treatment plant (WWTP). All of the compounds studied were found in each sample location and it was determined that each location type contributed to the overall loading to the WWTP. When hospital effluents were compared to wastewater treatment plant influents, it was found that hospitals contribute less than 1% of the total loading in each city for each compound studied.

43

Keywords: emerging contaminants, municipal wastewater, hospitals, funeral homes, slaughterhouse

1. Introduction
Emerging contaminants such as pharmaceuticals and personal care products are receiving increased attention as researchers study their occurrence in the environment and their impacts on living organisms. In a search to find the point of entry for these contaminants into the natural environment, scientists are routinely led to municipal wastewater effluent. In order to understand the fate and transport pathways of these compounds within the greater wastewater system, the potential upstream sources of emerging contaminants into the wastewater catchment system must be studied. The three classes of emerging contaminants monitored in this study are beta-blockers, antidepressants, and disinfectants. Beta-blockers and antidepressants are both medications prescribed by medical professionals to treat a variety of conditions. The two disinfectants in this study are triclosan (TCS) and triclocarban (TCC), which are found in consumer products. Beta-blockers, or beta-adrenergic blocking agents, are a class of drugs first developed in the late 1950s. One of the most commonly used beta-blockers is propranolol, which revolutionized the treatment of angina (van der Vring et al., 1999). Beta-blockers are designed to inhibit the action of beta-adrenergic receptors that are part of the central nervous system. When these receptors are hindered, the result is a dilation of blood vessels, slowing of the heart rate, and opening of bronchi in the lungs (Manger and Gifford, 2001). Beta-blockers are therefore known to have an anti-hypertensive effect and are used to treat individuals with high blood pressure as

44

well as to treat patients after heart attacks. The four beta-blockers in this study are atenolol, sotalol, metoprolol, and propranolol. Antidepressants are a class of pharmaceuticals that affect neurotransmitters, the chemicals that nerves within the brain use to communicate with each other. Examples of neurotransmitters include serotonin, dopamine and norepinephrine. It is thought that an imbalance in these neurotransmitters is the cause of depression and also may play a role in anxiety. Antidepressants are believed to work by inhibiting the release or affecting the action of neurotransmitters. The antidepressant compounds in this study are velafaxine, O-desmethylvenlafaxine, citalopram, and desmethylcitalopram. Venlafaxine is a serotonin-norepinephrine reuptake inhibitor (SNRI) prescribed for the treatment of depression, depression with associated symptoms of anxiety, generalized anxiety disorder, social anxiety disorder and adult panic disorder. Odesmethylvenlafaxine, a major active metabolite of venlafaxine, also functions as an SNRI. It is also synthetically produced (desvenlafaxine) and was approved by Health Canada in 2009 for treatment of depression. Citalopram is a selective serotonin reuptake inhibitor (SSRI) prescribed for the management of depression as well as treating obsessive-compulsive disorder, panic disorder, premenstrual dysphoric disorder, anxiety disorder and posttraumatic stress disorder. Desmethylcitalopram, an active metabolite of citalopram, also functions as an SSRI. TCS and TCC are both anti-bacterial and anti-fungal agents commonly used in consumer products such as soaps, disinfectants, toothpastes, body washes, and medical disinfectant of which these products contain between 0.1% and 2% of TCS or TCC by weight (Chalew and Halden, 2009). Approximately 96% of the triclosan is used in consumer goods, which are disposed of to the sewer system (Riess et al., 2002). They have both been used since the 1970s and have been detected in wastewater treatment plant effluent and surface waters (Singer et al., 45

2002; Heidler et al., 2006). TSC and TCC interact with bacteria by binding to the enoyl-acyl carrier protein reductase enzyme (ENR) found in their cell membrane inhibiting fatty acid synthesis (Heath et al., 1999). Bacteria need these fatty acids to build cell membranes and thus cannot reproduce. Once in the natural environment, these compounds interact with aquatic organisms. Betablockers and antidepressants have been found in the tissues of fish while triclosan and triclocarban have been observed to disrupt the natural hormone levels in bullfrogs and rats (Haider and Baqri, 2007; Veldhoen et al., 2006; Zorrilla et al., 2008). All three of these emerging contaminant types have been found in wastewater treatment influents around the world (Miege et al., 2009; Wick et al., 2009; Ying and Kookana, 2006) although there have been few studies attempting to classify their sources. Of the work that has been done to classify the sources of pharmaceutical contaminants, hospitals have been the primary focus. Ort et al. (2010) reports that hospitals do not account for a disproportionate amount of total mass loading for the majority of pharmaceutical compounds found in municipal wastewater. Atenolol and metoprolol from hospital effluents were reported to account for 0.6% and 2.3% of the total loading in the WWTP influent respectively. Similarly. Citalopram,

venlafaxine and triclosan from hospitals were found to be 1.6%, 2%, and 6% respectively. This research investigates the possible sources of beta-blockers, antidepressants, triclosan and triclocarban into the sewer system by monitoring the wastewater effluents of hospitals, funeral homes, residential neighborhoods, and a slaughterhouse in order to determine the primary route of entry for these emerging contaminants into the wastewater treatment system. Three different municipalities were involved and will be referred to as cities A, B, and C. 46

The following table outlines the compounds in this study as well as their physical and chemical properties
Compound Molecular Acidity Weight constant (g/mol) (pKa) Octanolwater partition coefficient (logkow) Liposomewater distribution ratio at pH 7 (logDlipw) Sorption Coefficient dD (L/gss) Water Solubility (mg/L at 25oC) Structure

Beta Blockers Sotalol

272.4

9.5

0.24

n.d.

0.037

137,000

Atenolol

266.3

9.2

0.16-0.46

0.51-1.0

0.038

13,300

Metoprolol

267.4

9.7

1.69-1.88

1.43

0.001

4,780

Propranolol

259.3

9.45

3.48-3.03

2.5-3.0

0.317

70

Personal Care Triclosan Triclocarban Antidepressants Venlafaxine 277.41 9.24 0.43 289.5 315.6 7.9 12.7 4.8 4.3 2-5 0.6-1.5

Odesmethylvenlafaxine Citalopram

263.38

9.74

0.74

324.4

9.5

3.74

desmethylcitalopram

310.37

10.5

Table 15: Physical and Chemical Properties of Beta-Blockers, Antidepressants, Triclosan and Triclocarban

47

2. Methods
)-865'.,*
The sample locations were selected to best study the possible sources of emerging contaminants into municipal wastewater systems. Hospitals, and residential neighborhoods were chosen as locations with high disinfectant and consumer product use and therefore potential sources of TCC and TCS into the wastewater network as well as locations where beta-blocker and antidepressant use is expected. Funeral homes and slaughterhouses were chosen as possible entry points due to the use of disinfectant and sterilization solutions in the embalming and meat processing methods. Most of the samples were 24-hour composites obtained using an autosampler; where this was not possible, such as for the funeral homes, grab samples were used. Samples were taken every two months for four sampling events per location.

)-865$*5!%-&'!.)*
Samples were taken from manholes directly downstream of hospitals, funeral homes, residential neighborhoods, and a slaughterhouse by municipal staff. Due to the batch process of embalmining a body, grab samples were used for both funeral homes and coordinated with the funeral home staff to ensure that the sample was taken during a time when there was an embalming occurring. At each location, three liters of sample was collected and split into two amber glass bottles and one HDPE bottle before they were stored at 4oC until analysis. Analytical work was completed at the Worsfold Water Quality Centre at Trent University. The following table outlines the sampling locations within each city.

48

City A B C

Sampling location Residential Area, Hospital Two Residential Areas, Hospital, Funeral Home Hospital, Funeral Home, Slaughterhouse

Table 16: Sample Locations

-.-59)')*
The samples were filtered, acidified, and then extracted using Waters Oasis MCX solid phase extraction cartridges. The extracts were subsequently preconditioned with acetone, methanol, and dilute sulphuric acid before being eluted from the cartridge with ammonium hydroxide in methanol. They were then evaporated to almost dryness and reconstituted in methanol. The target compounds were analyzed by Micromass Quattro LC triple-quadrupole mass spectrometer. The target compounds were analyzed in positive ion mode. Multiple reaction monitoring (MRM) was employed for analyte quantisation. Chromatographic separation was conducted on a Waters model 2695 HPLC system with a Genesis C18 column (150 # 2.1 mm i.d., 4$m). The mobile phase A and B consisted of acetonitrile and aqueous ammonium acetate. Prior to extraction, samples were spiked with stable-isotope labeled standards. Concentrations of the analytes in the sample were determined by comparing the relative ratio of the response of the target analyte to the response of the labeled standard with external standards. The methodology for triclosan and triclocarban is an adaptation of a procedure from Sabourin et al. (2009) and can be found in more detail therein. The beta-blocker procedure followed the method described in Scheurer et al. (2009) For Quality Analysis and Quality Control, a laboratory blank for each sample batch and at least one real sample duplicate from each sample set from the same sampling location was conducted.

49

Also, surrogate standards were added into each sample to monitor and correct for any potential loss during the sample analysis.

3. Results
At each sampling location, the concentrations of beta-blockers, antidepressants, triclosan and triclocarban were characterized. The following graphs show the concentrations of each compound at the various source locations. Error bars are +/- one standard deviation

Figure 3: Triclosan Concentration at Different Sample Locations (Funeral Home B= 70,000ng/l)

50

Figure 4: Triclocarban Concentration at Different Sample Locations

Figure 5: Beta Blocker Concentrations at Different Sample Locations

51

Figure 6: Venlafaxine and O-desmethyl venlafaxine Concentration at Different Sample Locations

Figure 7: Citalopram and desmethyl citalopram Concentration at Different Sample Locations

52

4. Discussion
)!3+%$*%!.%$.&+-&'!.)*
The source sampling of triclosan as seen in Figure 1 shows high concentrations in the wastewater streams of hospital B and funeral home C. A composite sample of funeral home B was not feasible and hence a grab sample was used; this concentration of 70,000 ng/L may not be indicative of an average funeral home concentration. This large spike is likely due to the embalming and disinfecting batch processes used when preparing a body for burial rather than a baseline effluent concentration for the funeral home itself. Hospital B also exhibits a high triclosan effluent concentration though the large error bars highlight the variability of the data. Since Hospital B was sampled with a 24h auto-sampler over many months, the results are unlikely due to a small number of events but rather the continual, but inconsistent use of triclosan for tasks such as hand washing and equipment disinfecting. Figure 2 outlines the source concentrations of triclocarban and various locations. Residential areas A and B had slightly elevated levels of TCC while hospitals A and C had some of the lowest levels. Similarly to Figure 2, Hospital B had the highest levels of the three hospitals sampled and also had the greatest variability. Slaughterhouse C was found to have greater concentrations of triclocarban than two of the hospitals and was similar to the residential neighborhoods. This is possibly due to the use of disinfectants and antibacterial agents in the cleaning of slaughterhouse equipment.

53

Despite the increased levels of triclosan in Hospital B and triclocarban in Residential Area B, the variability of the data suggests that each class of potential source, both residential and hospital, contribute to the overall loading of triclosan and triclocarban in municipal wastewater. The beta-blocker concentrations as seen in Figures 3 to 6 also show similar variability to the triclosan and triclocarban samples. The metoprolol, atenolol, and sotalol concentrations are similar across the range of sample locations suggesting that none of the locations are distinct point sources of beta-blockers to the wastewater treatment system. Due to the long-term nature of beta-blocker prescriptions for conditions such as hypertension, individuals are likely to consume a large percentage of beta-blockers at home leading to high concentrations in residential effluent. Of the anti-depressants studied, venlafaxine and O-desmethyl venlafaxine show the highest concentrations at Hospital A. Residential area A shows the second highest concentrations with locations in city B and C being much lower. This may be due to different levels of antidepressant prescription between cities or differences between neighborhood types. Citalopram and

desmethyl citalopram exhibit more consistent concentrations across the range of sample locations with Hospital A and Funeral Home C recording the highest levels. Overall, none of the location types in this study discharge much greater levels of ECs than other sample locations. The high variability is spread over hospitals, residential areas, and funeral homes indicating that no sample location type contributes more contaminant per litre than the others.

54

)5-3,1&$+*1!3)$*-.4*03.$+-5*1!8$)*
Funeral homes and a slaughterhouse were included in this study to investigate their potential as a source of emerging contaminants into the wastewater treatment system. When not in the process of embalming a body, funeral home wastewater resembles a residential dwelling and therefore the sampling schedule was arranged to retrieve samples during the embalming process in order to assess the role of preserving a body on the funeral home effluent. Due to this, the results obtained from the funeral homes are not indicative of average effluent concentrations over long periods of time but rather give snapshots of the loadings when funeral homes are operational. Triclosan was the only compound, which was found at a funeral home in greater concentrations than the rest of the sample locations though this was expected as triclosan is a component of many disinfectants and antibacterial washes which are likely used in the embalming process. The slaughterhouse monitored in this study exhibited high concentrations of the disinfectant triclocarban likely used to clean surfaces and equipment within the facility. The slaughterhouse effluent contained measureable amounts of beta-blockers and antidepressants at similar levels found in the residential neighborhoods studied. This may be due beta-blockers and antidepressants being taken by slaughterhouse employees though further studies should be undertaken to rule out contribution by the slaughtered animals themselves.

1!)6'&-5*5!-4'.,)*
By estimating the total loading of each compound within hospital effluents, it is possible to assess the percentage that originates from the hospitals in this study. Using an average

55

wastewater per hospital bed value of 350L/bed/day (Salem et al., 2007), the hospital loading were calculated and compared to loading levels at the WWTP influents.
Compound Hospital B Effluent (g/day) 1.14 0.006 0.207 0.027 0.081 0.004 0.041 0.120 0.044 0.027 WWTP B Influent (g/day) 259.2 19.5 289.9 166.3 115.5 12.69 177.95 418.01 100.30 80.67 Percent from Hospital, City B (%) 0.44 0.032 0.07 0.02 0.07 0.04 0.023 0.029 0.044 0.033 Hospital C Effluent (g/day) 0.069 0.0017 0.0708 0.0072 0.031 0.0006 0.062 0.0036 0.022 0.0005 WWTP C Influent (g/day) 71.6 6.5 74.7 26.9 27.9 2.2 55.91 731.82 25.81 20.34 Percent from Hospital, City C (%) 0.098 0.027 0.095 0.027 0.113 0.029 0.112 0.0005 0.087 0.003

Triclosan Triclocarban Atenolol Sotalol Metoprolol Propranolol Venlafaxine O-desmethyl venlafaxine Citalopram Desmethyl citalopram

Table 17: Hospital Loadings and Percentages

As seen in Table 12, contaminant loading from hospitals is responsible for a very small percentage of the overall loading at the wastewater treatment plant influent. All of the individual hospitals contributed less than 0.5% of the overall loading per compound and most were under 0.1%. In a municipality such as City B with multiple hospitals, the loading per bed as calculated for Hospital B was extrapolated over the 1200 hospital beds throughout the city to estimate the loading percentages due to hospitals in general. This yielded values of 2.3% for triclosan while all other compounds were below 0.36%. Despite the prevailing thought that hospitals would be a significant source of pharmaceuticals to the wastewater system, this is not evident for the three classes of contaminants in this study. The current trend in the healthcare sector towards short hospital stays and early discharge times means patients are taking pharmaceuticals at home where they are excreted into the wastewater system through residential neighborhoods. 56

5. Conclusion
Through monitoring the wastewater effluents of hospitals, funeral homes, residential neighborhoods and a slaughterhouse for beta-blockers, antidepressants, and disinfectants, the sources of emerging contaminants into the wastewater treatment system were analyzed. It was found that all location types within this study contributed to the overall loading of pharmaceuticals in wastewater. When the contribution due to hospitals was compared to the total loading for each compound at the wastewater treatment influent, it was calculated that hospitals account for <1% of the mass loading, indicating that health care centers are not point sources for antidepressants and betablockers into the wastewater treatment system. Further studies into the causes of emerging contaminants from slaughterhouses would help explain why both antidepressants and beta-blockers were found in slaughterhouse effluent.

6. Acknowledgments
We thank the three municipalities for their funding and cooperation in the completion of this study.

57

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Andersen, H.; Siegrist, H.; Halling-Srensen, B.; Ternes, T.A., 2003. Fate of estrogens in a municipal sewage treatment plant. Environ. Sci. Technol. 37, 40214026. Bright, D.A; Healey, N. 2003. Contaminant risks from biosolids land application: Contemporary organic contaminant levels in digested sewage sludge from five treatment plants in Greater Vancouver, British Columbia. Environmental Pollution, 126 (1), 39-49. Chalew, T.; Halden, R.; 2009. Environmental Exposure of Aquatic and Terrestrial Biota to Triclosan and Triclocarban. JAWRA. 45(1):4-13 Chen, X.; Nielsen, J.; Furgal, K.; Lui, Y.; Lolas, I.; Bester, K. Biodegradation of triclosan and formation of methyl-triclosan in activated sludge under aerobic conditions. 2011. Chemosphere. 84(4):452-456. Christensen A. M.; Markussen B.; Baun A.; Halling-Sorensen B., 2009. Probabilistic environmental risk characterization of pharmaceuticals in sewage treatment plant discharges. Chemosphere , 77:351358. Cleuvers, M., 2005. Initial risk assessment for thee B-blockers found in the aquatic environment. Chemosphere. 50:199-205 Drillia, P.; Stametelatou, K.; Lyberatos, G., 2006. Fate and mobility of pharmaceutical is solid matrices. Chemosphere. 60:1034-1044 Fair, P.; Lee, H.; Adams, J.; Darling, C.; Pacepavicius, G.; Alaee, M.; Bossart, G.; Henry, H.; Muir, D. 2009. Occurrence of triclosan in plasma of wild Atlantic bottlenose dolphins (Tursiops truncatus) and in their environment. Environmental Pollution. 157(8):2248-2254. Federle, T.; Kaiser, S.; Nuck, B. 2002. Fate and Effects of Triclosan in Activated Sludge. Environmental Toxicology and Chemistry. 21(7)1330-1337. Gabet-Giraud, V.; Miege, C.; Choubert, J.M.; Martin Ruel, S.; Coquery, M., 2010. Occurrence and removal of estrogens and beta blockers by various processes in wastewater treatment plants. Science of the Total Enviromnent. 408:4257-4296. Haider S.; Baqri S.S.R., 2000. beta-Adrenoceptor antagonists reinitiate meiotic maturation in Clarias batrachus oocytes. Comp Biochem Physiol Mol Integr Physiol , 126: 51725. Halden, R.; Paull, D. 2005. Co-Occurrence of Triclocarban and Triclosan in U.S. Water Resources, Environ. Sci. Technol. 39:1420-1426. Heath, RJ.; Rubin, JR.; Holland, DR.; Zhang, E.; Snow, ME.; Rock, CO.; 1999. Mechanism of triclosan inhibition of bacterial fatty acid synthesis. J. Biol. Chem. 274 (16): 111104.

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Heidler, J.; Sapkota, A.; Halden, R. 2006. Partitioning, Persistence, and Accumulation in Digested Sludge of the Topical Antiseptic Tricloscarban during Wastewater Treatment. Environ. Sci. Technol. 40:3634-3639. Jones, O.; Voulvoulis, N.; Lester, J.N., 2002. Aquatic environmental assessment of the top 25 English prescription pharmaceuticals. Water Research. 36:5013-5022 Kinney, C.A.; Furlong, E.T.; Zaugg, S.D.; Burkhardt, M.R.; Werner, S.L.; Cahill, J.D.; Jorgensen, G.R.; 2006. Survey of Organic Wastewater Contaminants in Biosolids Destined for Land Application. Environmental Science & Technology, 40 (23):7207-7215. Lindstrom, A.; Buerge, I.; Poiger, T.; Bergqvist, P.; Muller, M.; Buser, H. 2002. Occurrence and Environmental Behavior of the Bactericide Triclosan and Its Methyl Derivative in Surface Waters and in Wastewater. Environ. Sci. Technol. 36:2322-2329. Lin, A.; Lin, A.; Tung, H.; Chary, N., 2010. Potential for biodegradation and sorption of acetaminophen, caffeine, propranolol and acebutolol in lab-scale aqueous environments. Journal of Hazardous Materials. 183:242-250 Manger, W. M.; Gifford, R. W., 2001. 100 Questions and Answers about Hypertension. Blackwell Science. . Maurer M.; Escher B.I.; Richle P.; Schaffner C.; Alder A.C. ,2007. Elimination of B-blockers in sweage treatment plants. Water Research. . 41:1614-1622. McAvoy, D.; Schatowitz, B.; Jacob, M.; Hauk A.; Eckhoff, W.S. 2002. Measurement of triclosan in wastewater treatment systems. Environ. Toxicol. Chem. 21 (7):1323-1329 Miege C.; Choubert J.M.; Ribeiro L.; Eusebe M.; Coquery M., 2009. Fate of pharmaceuticals and personal care products in wastewater treatment plants conception of a database and first results. Environ Pollut .157:17216. Quintana, J.; Reemtsma, T. 2004. Sensitive determination of acidic drugs and triclosan in surface and wastewater by ion-pair reverse-phase liquid chromatography/tandem mass spectrometry. Rapic Commun. Mas Spectrom. 18:765-774. Radjenovic, J.; Petrovic, M.; Barcelo, D., 2008. Fate and distribution of pharmaceuticals in wastewater and sewage slugde on the conventional activated sludge (CAS) and advanced membrane bioreactor (MBR) treatment. Water Research. 43, 831-841. Ramil, M.; Aref, T.; Fink, G.; Scheurer, M.; Ternes, T., 2009. Fate of Beta Blockers in AquaticSediment Systems: Sorption and Biotransformation. Environ. Sci. Technol. 44:962-970 Reiss, R.; Mackay, N.; Habig, C.; Griffin, J., 2002. Ecological Risk Assessment for Triclosan in Lotic Systems Following Discharge from Wastewater Treatment Plants in the United States. Environ. Toxicol. Chem. 21: 2483-2492.

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Ricart, M.; Guasch, H.; Alberch, M. 2010. Triclosan persistence through wastewater treatment plants and its potential toxic effects on river biofilms. Aquat. Toxicol. 100 (4): 34653 Sabourin, L.; Beck, A.; Duenk, P,; Kleywegt, S.; Lapen, D.; Hongzxia, L.; Metcalfe, C.; Payne, M.; Topp, A. 2009, Sci Total Envion, 407(16): 4596-4604. Scheurer M.; Ramil M.; Metcalfe C.; Groh S.; Ternes T., 2009. The challenge of analyzing betablocker drugs in sludge and wastewater. Anal Bioanal Chem. 396:845-856 Sedlak, D.L.; Pinkston, K.E., 2001. Factors affecting the concentrations of pharmaceuticals released to the aquatic environment. Water Resource. 120, 5664. Singer, H.; Muller, S.; Tixier, C.; Pillonel, L. 2002. Triclosan: occurrence and fate of a widely used biocide in the aquatic environment: field measurements in wastewater treatment plants, surface waters, and lake sediments.. Environ Sci Technol. 36: 49985004 Ternes T.A.; Herrmann, N.; Bonerz, M.; Knacker, T.; Seigrist, H.; Joss, A., 2004. A rapid method to measure the solid-water distribution coefficient (Kd) for pharmaceutical and musk fragrances in sewage sludge. Water Research. 38:4075-4084. Thompson, A.; Griffin, P.; Stuetz, R.; Cartmell, E. 2005. The Fate and Removal of Triclosan during Wastewater Treatment. Water Environ. Res., 77:63-67 Tohren, C.; Kibbey, G.; Paruchuri, R.; Sabatini, D.; Chen, L. ,2007. Adsorption of Beta Blockers to Environmental Surfaces. Environ. Sci. Technol. 41:5349-5356 Veldhoen, V.; Skirrow, R.; Osachoff, H.; Wigmore, H.; Clapson, D.; Gunderson, M.; Van Aggelen, G.; Helbing, C.; 2006. The bactericidal agent triclosan modulates thyroid hormoneassociated gene expression and disrupts postembryonic anuran development. Aquatic Toxicology 80 (3): 217227. Wick A.; Fink G.; Joss A.; Seigrist H.; Ternes T., 2008. Fate of beta blockers and psycho-active drugs in conventional wastewater treamtment. Water Research. 43:1060-1074. van der Vring JA.; Danils M.C.; Holwerda N.J.,1999 Combination of calcium channel blockers and beta blockers for patients with exercise-induced angina pectoris: a double-blind parallelgroup comparison of different classes of calcium channel blockers. The Netherlands Working Group on Cardiovascular Research. 50, 44754. Zhao, JL.; Ying, GG.; Liu, YS.; Chen, F.; Yang, JF.; Wang, L. 2010. Occurrence and risks of triclosan and triclocarban in the Pear River system, South China: From source to the receiving environment. Journal of Hazardous Materials. 179:215-222. Zorrilla, L.; Gibson, E.; Jeffay, S.; Crofton, K.; Setzer, W.; Cooper, R.; Stoker, T. 2009. The Effects of Triclosan on Puberty and Thyroid Hormones in Male Wistar Rates. Toxicol. Sci. 107(1):56-64.

60

CHAPTER 4
Fate of "-Blockers, Triclosan, and Triclocarban in Municipal Wastewater Treatment
Daniel P. Shaver*a, Hamid Salsalia, Edward A. McBeana

Department of Engineering, University of Guelph, Thornbrough Building, N1G 2W1

Guelph, Ontario, Canada *dshaver@uoguelph.ca

Abstract
Emerging contaminants are a growing concern in wastewater treatment. Low concentrations of previously undetected chemicals are being found in surface waters and wastewater treatment effluents with unknown effects on aquatic ecosystems. The fate of six compounds within three different municipal wastewater treatment plants are assessed to determine their removal efficiencies and pathways. The adsorption coefficient (kd) values for each compound, as estimated from the data, indicate that the compounds partition into the sludge phase, although, sludge flow rates are so much smaller than the aqueous phase that removal due to physical adsorption is minimal. Biological degradation is shown to be the primary removal pathway for the compounds studied. The average overall removal efficiency for beta-blockers in the three WWTPs studied was 28%, 0%, 24%, and 25% for atenolol, sotalol, metoprolol, and propranolol,

61

respectively. Average triclosan and triclocarban removal efficiencies were 75% and 63% respectively.

1. Introduction
Emerging contaminants and pharmaceuticals have recently received increasing attention as scientists become aware of their presence in the natural environment. The search to find the sources of these compounds has routinely led to wastewater treatment plants (WWTP) as an entry point of contaminants into the natural environment. The unknown effects of low

concentrations of emerging contaminants in the ecosystem require scientists to study the occurrence, fate, and transport of these compounds in wastewater treatment in an attempt to better understand and mitigate their effects as pertinent. Beta-blockers, or beta-adrenergic blocking agents, are a class of drugs first developed in the late 1950s. One of the most commonly used beta-blockers was propranolol, which revolutionized the treatment of angina (van der Vring et al., 1999). Beta-blockers are designed to inhibit the action of beta-adrenergic receptors that are part of the central nervous system. When these receptors are hindered, the result is a dilation of blood vessels, slowing of the heart rate, and opening of bronchi in the lungs (Manger and Gifford, 2001). Beta-blockers are therefore known to have an antihypertensive effect and are used to treat individuals with high blood pressure as well as to treat patients after heart attacks. Triclosan (TCS) and Triclocarban (TCC) are both anti-bacterial and anti-fungal agents commonly used in consumer products such as soaps, disinfectants, toothpastes, body washes, and medical disinfectant of which these products contain between 0.1% and 2% of TCS or TCC by

62

weight (Chalew and Halden 2009). Approximately 96% of the triclosan is used in consumer goods, which are disposed to the sewer system (Riess et al., 2002). They have both been used since the 1970s and have been detected in wastewater treatment plant effluent and surface waters (Singer et al., 2002; Heidler et al., 2006). TSC and TCC interact with bacteria by binding to the enoyl-acyl carrier protein reductase enzyme (ENR) found in their cell membrane inhibiting fatty acid synthesis (Heath et al., 1999). Bacteria need these fatty acids to build cell membranes and thus cannot reproduce. As well as anti-bacterial affects, triclosan has also been shown to interfere with other aquatic organisms in surface water systems. Studies show that low doses of TCS may disturb the natural hormones in bullfrogs and rats (Veldhoen et al., 2006; Zorrilla et al., 2009). Triclosan also inhibits the photosynthesis of aquatic algae found in WWTP receiving waters (Ricart et al., 2010) and has been found in the blood plasma of wild Bottle Nosed Dolphins (Fair et al., 2009). Since there is evidence of triclosan and triclocarban in the natural environment and indications of toxicity to aquatic life, the discharge of TCC and TCS may need to be reduced. The effective removal of triclosan and triclocarban from wastewater effluent requires an understanding of the fate of these two compounds within the wastewater treatment system. Most studies monitoring the influent and effluent concentrations of WWTPs for triclosan and triclocarban report a high level of removal from the aqueous phase. Removals of 90% to 99% have been reported and attributed to biological degradation in activated sludge processes (Federle et al., 2002; McAvoy et al., 2001). Studies that have monitored other types of treatment systems detail similar findings. Thompson et al., (2005) studied treatment plants using Rotating Biological Contactors and Trickling Filters as secondary treatment processes and report removals of 86 to 97% for trickling filters and 58 to 96% for rotating biological contactors. 63

For the fraction of TCS and TCC that enters the aeration basin, biological degradation becomes a removal path. In laboratory-scale reactors, triclosan was found to be reduced by 75% under aerobic conditions after 150 hours (Chen et al., 2011). Activated sludge was taken from a local WWTP and spiked with triclosan and then analyzed by gas chromatography at various time intervals. Chen et al. (2011) concluded that below 20ug/L, triclosan exhibited first-order biological degradation kinetics with a half-life between 54 and 86h. The measured rate degradation rate constant (kbio) was estimated at 0.0081s-1 for an influent concentration of 20ug/L. The same study also reported very little measured degradation under anaerobic and anoxic conditions (Chen et al., 2011). This suggests that the primary location of biological degradation of triclosan is in the aeration basin of the WWTP and not it the anaerobic digesters. Treated sludge (biosolids) is regularly applied to agricultural land as a cheap fertilizer and to reduce landfilling (Kinney et al., 2006). Metal and pathogen content of land- applied sludge is regulated by most European and North American governments though there are no parameters on organic contaminants (Bright and Healey 2003). It is therefore vital to understand the fate of TCC and TCS in both the aquatic and sludge effluents of municipal wastewater treatment plants.

64

Compound

Molecular Acidity Weight constant (g/mol) (pKa)

Octanolwater partition coefficient (logkow) 0.24

Liposomewater distribution ratio at pH 7 (logDlipw) n.d.

Sorption Coefficient dD (L/gss) 0.037

Water Solubility (mg/L at 25oC) 137,000

Structure

Beta Blockers Sotalol

272.4

9.5

Atenolol

266.3

9.2

0.16-0.46

0.51-1.0

0.038

13,300

Metoprolol

267.4

9.7

1.69-1.88

1.43

0.001

4,780

Propranolol

259.3

9.45

3.48-3.03

2.5-3.0

0.317

70

Personal Care Triclosan Triclocarban 289.5 315.6 7.9 12.7 4.8 4.3 n.d. n.d. n.d. n.d. 2-5 0.6-1.5

Table 18: Physical and Chemical Properties of Beta Blockers, Triclosan and Triclocarban Recently, beta-blockers have been found in aquatic environments as well as in the tissues of fish (Christensen et al., 2009; Haider and Baqri 2007). It has been shown that wastewater treatment plants are one of the main points of entry for drugs into streams and lakes and thus there is a need to better understand the fate of pharmaceuticals in various wastewater treatment processes (Miege et al., 2009). The present body of research documenting beta-blockers in wastewater treatment is largely focused on influent and effluent concentrations of hormones (Miege et al., 2009) with fate and transport within the wastewater treatment plant receiving only recent attention. There have been studies, however, investigating the transport and partitioning of beta-blockers in aquatic systems and sediment, which provide insight into how beta-blockers may behave in the wastewater treatment system.

65

It has been shown that adsorption to soils and sediments correlates highly with hydrophobicity of the molecule with a high hydrophobicity leading to stronger adsorption (Tohren et al. 2007). In experiments with beta-blockers, Tohren et al. (2007) found that propranol, with a higher Kow, and thus higher hydrophobicity, adsorbed better to soils than metoprolol with a lower Kow. Mobility in a soil system depends on the soil type and aqueous flows but can be predicted by the compounds adsorption tendencies (Tohren et al., 2007). Compounds such as propranolol, which have high hydrophobicity, tend to adsorb strongly to sediment and not be very mobile in soils (Drillia et al., 2006). This trend of hydrophobic betablockers adsorbing to river sediment suggests that adsorption to particulate matter may play a role in their removal by wastewater treatment processes. The breakdown of beta-blockers by natural organisms has also been the subject of study. In a lab-scale experiment, Ramil et al. (2009) took contaminated river sediment and measured how long biotransformation of the beta-blockers took to occur. They found that it took 7-10, 25-27, 13-28, and 6-11 days for atenolol, sotalol, metoprolol, and propranolol respectively to reach 90% disappearance in a batch of river water under simulated conditions (Ramil et al., 2009). When the same experiment was completed in a river water/sediment matrix, the time needed to reach 90% disappearance increased to 37-100, 26-96, and 32-100 days for sotalol, metoprolol, and propranolol respectively, suggesting that the molecules are more difficult to biotransform when sorbed to sediments. In a similar experiment, Lin et al. (2010) concluded that for river water, adsorption is the more significant method of removal relative to biodegradation for water contaminated with propranolol. These studies jointly show that biodegradation of beta-blockers is much more difficult when sediments and particulate matter are present such as in an activated

66

sludge process and that for a wastewater treatment plant, removal by adsorption and sedimentation should be investigated as a removal path. In the research that has been completed for investigation of the partitioning and adsorption of beta-blockers in wastewater sludge, the adsorption coefficients reported range from 0.001 to 0.4 L/g suggesting that beta-blockers do not generally partition to the solids fraction (Maurer et al., 2007; Scheurer et al., 2009). These report that since the adsorption to sludge is low, any removal must be primarily due to biological degradation (Maurer et al., 2007; Scheurer et al., 2009). A partial goal of this study is to further investigate the role of physical adsorption in beta-blocker removal. As well as providing an overall picture of beta-blocker, triclosan, and triclocarban removal efficiency, this study improves the understanding of the fate and transport within the individual treatment systems by interpreting sampling results at various points within the treatment sequence. This paper describes the results as derived from monitoring at three wastewater treatment plants and assesses the removal effectiveness of various wastewater treatment components. Four different beta-blocker compounds are examined: atenolol, sotalol, metoprolol, and propranolol as well as triclosan and triclocarban. Some of the properties of these compounds are listed in Table 1. This study has sampled from three different municipalities each with different sizes and operating conditions. Table 2 outlines the characteristics of the three different plants.

67

Plant A

Primary and Secondary Treatment

Chemical Addition

Major Tertiary Treatment

B C

Sedimentation and ferrous chloride None Aeration with activated sludge Sedimentation and ferrous chloride None Aeration with activated sludge Sedimentation and ferrous chloride Rotating Aeration with activated Biological sludge Contactors + Sand Filter

City Population Treatment Plant (2006) Capacity (m3/day) 800,000 545,000

Measured Flow Rate (m3/day) 420,000

500,000 120,000

614,000 64,000

409,000 55,800

Table 19: Summary of Features of Treatment Plants

2. Methods
)-865'.,*
Sampling locations were selected to assess the fate of beta-blockers, TCC and TCS in response to different components of a WWTP. These include the plant influent and effluent as well as primary effluent, aeration basin, secondary effluent, waste activated sludge, raw sludge, digester effluent, and biosolids. Most of the samples were 24-hour composites obtained using an auto-sampler taken every two months over a six-month period for four sample events per location; where this was not possible, such as for the biosolids cake, grab-samples were used.

)-865$*5!%-&'!.)*
At each treatment plant, municipal staff used auto-samplers to take twenty-four hour composite samples totaling 3L at each location. These three litres were split into two amber glass bottles and one HDPE bottle before they were stored at 4oC until analysis. Common to all three plants were sample locations of Plant Influent, Raw Sludge, Mixed Liquor, Thickened Waste Activated Sludge (TWAS), Digester Effluent, Biosolids Cake, and Final Effluent. Treatment

68

Plant A also sampled Primary Effluent, Secondary Effluent, and Biosolids Cake. Analytical work was completed at the Worsfold Water Quality Centre at Trent University.

Figure 8: WWTP flow diagram with sample locations

-.-59)')*
Wastewater samples were filtered, acidified, and then extracted using Waters Oasis MCX solid phase extraction cartridges. The extracts were subsequently preconditioned with acetone, methanol, and dilute sulphuric acid before being eluted from the cartridge with ammonium hydroxide in methanol. They were then evaporated to almost dryness and reconstituted in methanol. Triclosan and triclocarban where analyzed by Micromass Quattro LC triple-quadrupole mass spectrometer. The target compounds were analyzed in positive ion mode. Multiple reaction monitoring (MRM) was employed for analyte quantisation. Chromatographic separation was conducted on a Waters model 2695 HPLC system with a Genesis C18 column (150 # 2.1 mm

69

i.d., 4$m). The mobile phase A and B consisted of acetonitrile and aqueous ammonium acetate. Prior to extraction, samples were spiked with stable-isotope labeled standards (Triclosan-13C12 and Triclorcarban-13C12). Concentrations of the analytes in the sample were determined by comparing the relative ratio of the response of the target analyte to the response of the labeled standard with external standards. The methodology is an adaptation of a procedure from Sabourin et al. (2009) and can be found in more detail therein. Beta-blocker pharmaceuticals were analyzed by Micromass Quattro LC triple-quadrupole mass spectrometer. The target compounds were analyzed in positive ion mode. Multiple reaction monitoring (MRM) was employed for analyte quantitation. Chromatographic separation was conducted on a Waters model 2695 HPLC system with a Genesis C18 column (150 # 2.1 mm i.d., 4$m) at a flow rate of 0.2-0.4 mL/min. The mobile phase A and B consisted of acetonitrile and 20 mM aqueous ammonium acetate, respectively. The applied gradient elution was as follows: mobile phase A was increased from 30% to 55% within 3 min, increased from 55% to 95% in 2 min, held for 2 min, and then decreased from 95% to 30% within 0.2 min and held for 8 min. The column was kept at room temperature and the injection volume was 20 $L. The procedure followed the method described in Scheurer et al. (2009) For Quality Analysis and Quality Control, a laboratory blank for each sample batch and at least one real sample duplicate from each sample set from the same sampling location was conducted. Also, surrogate standards were added into each sample to monitor and correct for any potential loss during the sample analysis.

70

3. Results
+$8!(-5*$00'%'$.%'$)*
For each compound, and each wastewater treatment plant, removal efficiencies were calculated for each treatment process step, as well as the overall removal efficiency by comparing the final drug concentration with the original concentration. Positive removal efficiencies indicate the compound is being removed. Negative removal efficiencies can occur due to changes in the extent of shielding by suspended solids or analytical uncertainty. Table 3 shows the primary, secondary, and overall removal efficiencies from the aqueous phase of the three wastewater treatment plants based on TCC and TCS concentrations.
Compound Wastewater Plant A Triclosan Triclocarban Wastewater Plant B Triclosan Triclocarban Wastewater Plant C Triclosan Triclocarban Primary Removal Efficiency 36% 35% 68% 8% 72% 64% Secondary Removal Efficiency 82% 42% -89% 38% 67% 56% Overall Efficiency 88% 63% 40% 43% 98% 84%

Table 20: Primary, Secondary, and Overall Removal Efficiency for TCC and TCS from the Aqueous Phase

As seen in Table 3, there is a range in the TCC and TCS removal efficiencies of the three WWTPs. Treatment plant B has the lowest removal with 40% overall efficiency while Treatment plant C removes 98% and 84% of triclosan and triclocarban respectively.
Compound Wastewater Plant A Atenolol Sotalol Metoprolol Propranolol Wastewater Plant B Atenolol Sotalol Metoprolol Primary Removal Efficiency 8% 21% -95% 18% 71% 18% 47% Secondary Removal Efficiency -19% -75% 34% -21% -253% -11% -83% Overall Efficiency 17% -21% -3% 25% -1% 10% 3%

71

Propranolol Wastewater Plant C Atenolol Sotalol Metoprolol Propranolol

48% 91% 34% 73% 34%

-78% -320% -83% -56% 15%

8% 62% -21% 58% 44%

Table 21: Primary, Secondary, and Overall Removal Efficiency for Beta-Blockers from the Aqueous Phase

5!-4'.,)**
Mass loadings for each compound were also calculated for the different points within wastewater treatment plant C.

Figure 9: TCC Loadings within treatment plant C (Raw Sludge = 0.03g/day)

Figure 10: TCS Loadings within treatment plant C (TCS plant influent: 71 g/day)

72

Figure 11: Atenolol and Sotalol Loadings within treatment plant C

Figure 12: Metoprolol and Propranolol Loadings within treatment plant C

Figures 2 to 4 shows the mass loadings of the different compounds in g/day for various points within wastewater treatment plant C. The highest loadings are found in the plant influent with lower loadings observed in the final effluent for most compounds.

73

4. Discussion
The high variability in beta-blocker concentrations found within the wastewater treatment plants make accurate tracing of the fate of these compounds through the system difficult. Neither Plant A nor B removed any of the beta-blockers to statistically significant values (95% confidence). Plant C removed a significant amount of atenolol (62%), metoprolol (58%) and propranolol (44%) but failed to remove sotalol. Of the four beta-blockers, sotalol is most difficult to remove, with Plants A and C showing an increase in concentration in the effluent while Plant B shows minimal change. All three plants show low concentrations exiting the aeration tank in the mixed liquor but then the concentrations rise dramatically in the secondary clarifier discharge. Since it is unlikely that the beta-blockers reform after being degraded in the aeration basin, it is considered that the observations may be caused by shielding of the compounds by particles or by the cleavage of conjugates as suggested by Wick et al. (2008). Microbial cleavage conjugates have been shown to skew wastewater mass balances (Andersen et al., 2003; Sedlak and Pinkston 2001). The tertiary treatment system in place at Plant C may also assist in the removal of betablockers. Plants B and C have similar concentrations in the Mixed Liquor, but Plant C achieves a better removal of three beta-blockers although not for sotalol. This may be due to biological degradation from the rotating biological contactors. Triclosan and Triclocarban were removed with greater efficiency than the beta-blockers studied with removal efficiencies ranging from 40% in plant B to 98% in plant C. With the exception of TCS in plant B, no increase in concentration after the secondary clarifier was observed as was the case with the beta-blockers. 74

-4)!+6&'!.*%!$00'%'$.&*
By considering the chemical and physical properties of each compound, one can predict the fate in wastewater treatment. A kd of less than 0.5 L/g suggests that physical removal by adsorption plays a limited role in elimination of organic compounds in wastewater treatment plants (Scheurer et al., 2009; Ternes et al., 2004). Using the following equation and the experimental data from this experiment, the sorption coefficient for each compound was estimated for Treatment Plant C kd(Lg-1COD) = Ssorbed/(Xbiomas * Sdissolved) (1)

Where Ssorbed is the concentration of sorbed substance (ng/L), Sdissolved is the concentration of dissolved substance, Xbiomass an equivalent for biomass in the system (g/L) (Maurer et al., 2007). For the estimation, Primary Raw Sludge concentration was used as Ssorbed while Sdissolved was the concentration in the Primary Effluent. A raw sewage biomass concentration of 1g/L was used for the estimate, which is on the high end of the spectrum for primary clarifier biomass concentration. Xbiomass Estimated Literature kd Literature kd (g/L) kd (L/gCOD)1 (L/gCOD)2 (L/g) Atenolol 2670 1232 1 2.16 0.001 0.033 Sotalol 1380 382 1 3.61 0.08 0.035 Metoprolol 333 501 1 0.66 0.01 0.023 Propranolol 32 32 1 18.2 0.4 0.058 Triclosan 932 462 1 0.94 Triclocarban 75 65 1 1.16 Table 5: Estimated Sorption Coefficient For Primary Clarifier of Plant C 1(Scheurer et al., 2009) 2(Maurer et al., 2007) As seen in Table 5, the sludge adsorption estimated from this study is much higher than those reported in current literature for beta-blockers even with a high estimated biomass concentration 75 Compound Ssorbed (ng/L) Sdissolved (ng/L)

(Xbiomass). The higher kd values indicate that a higher proportion of the beta-blockers are being removed by physical separation in the primary clarifier than would be predicted using the kd values found in Scheurer et al. (2009) and Maurer et al. (2007) This may be due to the measurement location; Scheurer et al. (2009) determined the kd values in activated sludge and digested sludge while Maurer et al. (2007) used primary clarifier effluent instead of testing for the kd within the primary clarifier. Ternes et al. (2004) demonstrated that kd values in the primary sludge and secondary sludge are significantly different. The adsorption coefficients for TCC and TCS as estimated in Table 5 indicate kd values around 1 L/g which denotes an approximately equal distribution of triclosan and triclocarban in the sludge and effluent from the primary clarifier. The adsorption coefficients of TCC and TCS indicate why there is a measurable concentration of both TCC and TCS in the raw sludge. Although the sludge adsorption values of all the compounds studied suggest that physical adsorption should play a role in their removal from the aqueous wastewater stream, the difference in flow rates between the aqueous and sludge phases from the primary clarifier mean that very little of each compound is removed in this manner. The mass loadings as seen in Figures 2 to 4 show that the Raw Sludge stream contains very little of any of the compounds in this study suggesting that physical adsorption in the primary clarifier is not a significant removal path for beta-blockers, TCC or TCS from municipal wastewater.

76

0-&$*
The distribution of each compound relative to the total plant influent loadings was then calculated to determine the comparative fates of each compound.

Figure 13: Relative Fates by Loading within WWTP C (Sotalol was observed to have negative removal efficiency)

Both triclosan and triclocarban appear to be primarily removed within the aeration basin where loading changes were attributed to biological degradation. A small portion of TCC and

77

TCS is removed in the anaerobic digester with the remainder exiting the plant in either the aqueous effluent or the sludge and biosolids. These observations support conclusions by Chen et al. (2011) suggesting that biological degradation under aerobic conditions is the primary method of triclosan removal in wastewater treatment. The beta-blockers atenolol and metoprolol were observed to be primarily removed in the aeration basin with 69% and 59% respectively while the remainder exited in the plant effluent without being removed. Propranolol was found to be removed in both the aeration basin and the anaerobic digester. This is likely due to the high proportion of propranolol in the raw sludge and TWAS streams feeding the anaerobic digester as suggested by the high sludge adsorption coefficient. Scheurer et al. (2009) reported that beta-blockers have low sorption coefficients with sludge, which indicates the primary removal mechanism is microbial degradation. This is consistent with the findings of this study and would explain why Plant C with the tertiary Rotating Biological Contactors has better overall removal efficiencies than the other two plants, which have no tertiary treatment. Although few other studies have sampled after each process in the wastewater treatment system, some overall removal efficiencies have been reported. Radjenovic et al. (2008) reported that Activated Sludge systems remove 61.218.6% atenolol, 21.431.5% sotalol, 24.744.9% metoprolol, and 58.824.5 % propranolol. The high variability reported by Radjenovic et al. (2008) is consistent with the findings reported herein, and include the possibility of negative removal efficiencies of the concentrating of beta-blockers in plant effluent.

78

Maurer et al. (2007) reported removals of 7917% Atenolol, 267% Sotalol, 3111% Metoprolol, and 282 % Propranolol. Maurer et al. (2007) also concluded that beta-blocker elimination increases with an increase in hydraulic retention time. Gabet-Giraud et al. (2010) have also studied the removal of beta-blockers in wastewater treatment plants. Their results are also highly variable with multiple reports of negative removal efficiencies. They attribute these negative overall removal efficiencies to analytical uncertainty.

$.('+!.8$.&-5*+')2*
The environmental risk associated with triclosan and triclocarban in surface waters has been studied on algae and fish at various concentrations. By studying the acute and toxic effects of triclosan and triclocarban on multiple forms of aquatic life, the most sensitive species can be identified and the toxicological data from it may be used as a worst case estimate for the risk associated with triclosan and triclocarban in surface waters. Riess et al. (2002) tested eight different aquatic species finding that the most sensitive to TCS had a No Observed Effect Concentration (NOEC) of 690 ng/L suggesting that any concentration below this level is unlikely to lead to negative effects on aquatic life. Similarly, Chalew and Halden (2009) surveyed multiple studies and found the lowest NOEC values to be 200ng/L and 60ng/L for triclosan and triclocarban respectively. These values are comparable to the Predicted No Effect Concentrations (PNEC) found by Zhao et al. (2010) of 50ng/L for triclosan and 58ng/L for triclocarban. Estimated surface water concentrations were calculated for City C in which river flow data was available. During the summer months of low flow the receiving water for WWTP C has an average flow of 146,880m3/day (Qsw). At an average effluent concentration of 18ng/L of

79

triclocarban and 28ng/L of triclosan (Ceff) and flow of 55800m3/day (Qeff), the estimated surface water concentration (Csw) was estimated as Csw = Ceff * (Qeff/Qeff + Qsw) (2)

The estimated surface water concentration was calculated to be 7ng/L for triclocarban and 11ng/L for triclosan. These values fall within the range of surface water concentrations reported in literature (Lindstrom et al., 2002; Quintana and Reemtsma 2004; Halden and Paull 2005). Using the conservative PNEC from Zhao et al. (2010), a risk quotient (RQ) for each compound was calculated using the previously estimated triclosan and triclocarban concentrations in the receiving waters of WWTP C. MEC/PNEC = RQ (3)

where MEC is the measured environmental concentration. The values yield risk quotients of RQ=0.12 for TCC and RQ=0.21 for TCS. Since both risk quotients are less than 1 when using conservative estimates for both the NOEC and MEC values, the environmental risk is considered to be low. Despite these results triclosan and triclocarban are often found together in the aquatic environment, and further risk studies should investigate the possible additive effects of multiple compounds in the receiving waters of municipal wastewater treatment plants. The risk associated with beta-blockers in the aquatic environment has been studied and the topological effects measured. When toxicity of propranolol, metoprolol, and atenol was measured in the water flea Daphnia magna, propranolol was found to be most toxic with an

80

EC50 of 7.7mg/L while metoprolol and atenolol were observed at 438mg/L and 313 mg/L respectively (Cleuvers 2004). In environmental risk assessments, the PEC/PNEC ratio is

commonly used as an indicator of risk where PEC is the predicted environmental concentration and PNEC is the predicted no effect concentration. If the PEC is less than the PNEC and thus PEC/PNEC < 1, no adverse effects are expected and the substance is not thought to pose an acute toxicity risk to the environment. Cleuvers (2004) reports a PEC/PNEC ratio of 0.81, 0.28, and 7.7x10-4 for propranolol, metoprolol, and atenolol respectively suggesting that none of these beta-blockers pose a risk to aquatic life at concentrations found in the environment. In a British study, atenolol was reported to have a PEC/PNEC ratio of 0.01 further suggesting that there is minimal acute risk to the environment (Jones et al., 2002). Despite these findings, there is a lack of chronic toxicity data and there may be an environmental risk associated with long-term exposure of aquatic life to beta-blockers in surface waters.

5. Conclusion
The results of this study reveal a large variation in beta-blocker, TCS and TCC concentrations after each major step in the wastewater treatment system. By estimating the adsorption coefficient for each compound in the primary clarifier, it was found that the kd values reported in this paper are much higher than current literature values suggesting that the compound prefer to partition into the solid sludge phase. When the flow rates and loadings are considered, however, the removal due to physical adsorption is minimal compared to biological degradation. When investigating the changes in concentrations of beta-blockers through the WWTPs, there were several instances where the concentrations rose substantially from one unit process to the next. This is thought to be due to analytical error as a result of shielding of the pharmaceuticals 81

by particulate matter in the wastewater. The average overall removal efficiency for the three treatment plants studied was 28%, 0%, 24%, and 25% for atenolol, sotalol, metoprolol, and propranolol respectively. These values are lower than values reported by other researchers, which highlights the variability of current data and the need for continuing research. The TCC and TCS aqueous removal efficiencies for the three treatment plants were 88% and 63% for Plant A, 40% and 43% for Plant B, and 98% and 84% for Plant C with regards to triclosan and triclocarban respectively. Treatment plant C provides tertiary treatment in the form of rotating biological contactors and was expected to have the highest removal efficiency. The risk to aquatic life from beta-blockers, triclosan and triclocarban loading in surface waters appears to be minimal however further study into additive effects of multiple compounds in the aquatic matrix as well as chronic affects should be undertaken.

6. Acknowledgments
We thank the three municipalities for their funding and cooperation in the completion of this study.

82

7. References
Andersen, H.; Siegrist, H.; Halling-Srensen, B.; Ternes, T.A., 2003. Fate of estrogens in a municipal sewage treatment plant. Environ. Sci. Technol. 37, 40214026. Bright, D.A; Healey, N. 2003. Contaminant risks from biosolids land application: Contemporary organic contaminant levels in digested sewage sludge from five treatment plants in Greater Vancouver, British Columbia. Environmental Pollution, 126 (1), 39-49. Chalew, T.; Halden, R.; 2009. Environmental Exposure of Aquatic and Terrestrial Biota to Triclosan and Triclocarban. JAWRA. 45(1):4-13 Chen, X.; Nielsen, J.; Furgal, K.; Lui, Y.; Lolas, I.; Bester, K. Biodegradation of triclosan and formation of methyl-triclosan in activated sludge under aerobic conditions. 2011. Chemosphere. 84(4):452-456. Christensen A. M.; Markussen B.; Baun A.; Halling-Sorensen B., 2009. Probabilistic environmental risk characterization of pharmaceuticals in sewage treatment plant discharges. Chemosphere , 77:351358. Cleuvers, M., 2005. Initial risk assessment for thee B-blockers found in the aquatic environment. Chemosphere. 50:199-205 Drillia, P.; Stametelatou, K.; Lyberatos, G., 2006. Fate and mobility of pharmaceutical is solid matrices. Chemosphere. 60:1034-1044 Fair, P.; Lee, H.; Adams, J.; Darling, C.; Pacepavicius, G.; Alaee, M.; Bossart, G.; Henry, H.; Muir, D. 2009. Occurrence of triclosan in plasma of wild Atlantic bottlenose dolphins (Tursiops truncatus) and in their environment. Environmental Pollution. 157(8):2248-2254. Federle, T.; Kaiser, S.; Nuck, B. 2002. Fate and Effects of Triclosan in Activated Sludge. Environmental Toxicology and Chemistry. 21(7)1330-1337. Gabet-Giraud, V.; Miege, C.; Choubert, J.M.; Martin Ruel, S.; Coquery, M., 2010. Occurrence and removal of estrogens and beta blockers by various processes in wastewater treatment plants. Science of the Total Enviromnent. 408:4257-4296. Haider S.; Baqri S.S.R., 2000. beta-Adrenoceptor antagonists reinitiate meiotic maturation in Clarias batrachus oocytes. Comp Biochem Physiol Mol Integr Physiol , 126: 51725. Halden, R.; Paull, D. 2005. Co-Occurrence of Triclocarban and Triclosan in U.S. Water Resources, Environ. Sci. Technol. 39:1420-1426. Heath, RJ.; Rubin, JR.; Holland, DR.; Zhang, E.; Snow, ME.; Rock, CO.; 1999. Mechanism of triclosan inhibition of bacterial fatty acid synthesis. J. Biol. Chem. 274 (16): 111104.

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Heidler, J.; Sapkota, A.; Halden, R. 2006. Partitioning, Persistence, and Accumulation in Digested Sludge of the Topical Antiseptic Tricloscarban during Wastewater Treatment. Environ. Sci. Technol. 40:3634-3639. Jones, O.; Voulvoulis, N.; Lester, J.N., 2002. Aquatic environmental assessment of the top 25 English prescription pharmaceuticals. Water Research. 36:5013-5022 Kinney, C.A.; Furlong, E.T.; Zaugg, S.D.; Burkhardt, M.R.; Werner, S.L.; Cahill, J.D.; Jorgensen, G.R.; 2006. Survey of Organic Wastewater Contaminants in Biosolids Destined for Land Application. Environmental Science & Technology, 40 (23):7207-7215. Lindstrom, A.; Buerge, I.; Poiger, T.; Bergqvist, P.; Muller, M.; Buser, H. 2002. Occurrence and Environmental Behavior of the Bactericide Triclosan and Its Methyl Derivative in Surface Waters and in Wastewater. Environ. Sci. Technol. 36:2322-2329. Lin, A.; Lin, A.; Tung, H.; Chary, N., 2010. Potential for biodegradation and sorption of acetaminophen, caffeine, propranolol and acebutolol in lab-scale aqueous environments. Journal of Hazardous Materials. 183:242-250 Manger, W. M.; Gifford, R. W., 2001. 100 Questions and Answers about Hypertension. Blackwell Science. . Maurer M.; Escher B.I.; Richle P.; Schaffner C.; Alder A.C. ,2007. Elimination of B-blockers in sweage treatment plants. Water Research. . 41:1614-1622. McAvoy, D.; Schatowitz, B.; Jacob, M.; Hauk A.; Eckhoff, W.S. 2002. Measurement of triclosan in wastewater treatment systems. Environ. Toxicol. Chem. 21 (7):1323-1329 Miege C.; Choubert J.M.; Ribeiro L.; Eusebe M.; Coquery M., 2009. Fate of pharmaceuticals and personal care products in wastewater treatment plants conception of a database and first results. Environ Pollut .157:17216. Quintana, J.; Reemtsma, T. 2004. Sensitive determination of acidic drugs and triclosan in surface and wastewater by ion-pair reverse-phase liquid chromatography/tandem mass spectrometry. Rapic Commun. Mas Spectrom. 18:765-774. Radjenovic, J.; Petrovic, M.; Barcelo, D., 2008. Fate and distribution of pharmaceuticals in wastewater and sewage slugde on the conventional activated sludge (CAS) and advanced membrane bioreactor (MBR) treatment. Water Research. 43, 831-841. Ramil, M.; Aref, T.; Fink, G.; Scheurer, M.; Ternes, T., 2009. Fate of Beta Blockers in AquaticSediment Systems: Sorption and Biotransformation. Environ. Sci. Technol. 44:962-970 Reiss, R.; Mackay, N.; Habig, C.; Griffin, J., 2002. Ecological Risk Assessment for Triclosan in Lotic Systems Following Discharge from Wastewater Treatment Plants in the United States. Environ. Toxicol. Chem. 21: 2483-2492.

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Ricart, M.; Guasch, H.; Alberch, M. 2010. Triclosan persistence through wastewater treatment plants and its potential toxic effects on river biofilms. Aquat. Toxicol. 100 (4): 34653 Sabourin, L.; Beck, A.; Duenk, P,; Kleywegt, S.; Lapen, D.; Hongzxia, L.; Metcalfe, C.; Payne, M.; Topp, A. 2009, Sci Total Envion, 407(16): 4596-4604. Scheurer M.; Ramil M.; Metcalfe C.; Groh S.; Ternes T., 2009. The challenge of analyzing betablocker drugs in sludge and wastewater. Anal Bioanal Chem. 396:845-856 Sedlak, D.L.; Pinkston, K.E., 2001. Factors affecting the concentrations of pharmaceuticals released to the aquatic environment. Water Resource. 120, 5664. Singer, H.; Muller, S.; Tixier, C.; Pillonel, L. 2002. Triclosan: occurrence and fate of a widely used biocide in the aquatic environment: field measurements in wastewater treatment plants, surface waters, and lake sediments.. Environ Sci Technol. 36: 49985004 Ternes T.A.; Herrmann, N.; Bonerz, M.; Knacker, T.; Seigrist, H.; Joss, A., 2004. A rapid method to measure the solid-water distribution coefficient (Kd) for pharmaceutical and musk fragrances in sewage sludge. Water Research. 38:4075-4084. Thompson, A.; Griffin, P.; Stuetz, R.; Cartmell, E. 2005. The Fate and Removal of Triclosan during Wastewater Treatment. Water Environ. Res., 77:63-67 Tohren, C.; Kibbey, G.; Paruchuri, R.; Sabatini, D.; Chen, L. ,2007. Adsorption of Beta Blockers to Environmental Surfaces. Environ. Sci. Technol. 41:5349-5356 Veldhoen, V.; Skirrow, R.; Osachoff, H.; Wigmore, H.; Clapson, D.; Gunderson, M.; Van Aggelen, G.; Helbing, C.; 2006. The bactericidal agent triclosan modulates thyroid hormoneassociated gene expression and disrupts postembryonic anuran development. Aquatic Toxicology 80 (3): 217227. Wick A.; Fink G.; Joss A.; Seigrist H.; Ternes T., 2008. Fate of beta blockers and psycho-active drugs in conventional wastewater treamtment. Water Research. 43:1060-1074. van der Vring JA.; Danils M.C.; Holwerda N.J.,1999 Combination of calcium channel blockers and beta blockers for patients with exercise-induced angina pectoris: a double-blind parallelgroup comparison of different classes of calcium channel blockers. The Netherlands Working Group on Cardiovascular Research. 50, 44754. Zhao, JL.; Ying, GG.; Liu, YS.; Chen, F.; Yang, JF.; Wang, L. 2010. Occurrence and risks of triclosan and triclocarban in the Pear River system, South China: From source to the receiving environment. Journal of Hazardous Materials. 179:215-222. Zorrilla, L.; Gibson, E.; Jeffay, S.; Crofton, K.; Setzer, W.; Cooper, R.; Stoker, T. 2009. The Effects of Triclosan on Puberty and Thyroid Hormones in Male Wistar Rates. Toxicol. Sci. 107(1):56-64.

85

CHAPTER 5
Summary, Conclusion and Recommendations
Emerging contaminants have become a topic of increasing study in the scientific community as scientists become aware of their presence and environmental effects. A sampling program was implemented to monitor the sources and fate of emerging contaminants in three different municipal wastewater treatment systems. Laboratories tested for twelve different classes of emerging contaminants ranging from pharmaceuticals to flame-retardants. Hospitals, funeral homes, slaughterhouses and residential neighborhoods were monitored to determine possible point sources of contaminants into municipal sewer systems. Multiple locations within each wastewater treatment plant were monitored to trace the fate of each emerging contaminant class through the wastewater treatment process in an attempt to understand the fate removal pathways of each contaminant. This thesis reports on the first batch of data for three emerging contaminant classes: beta-blockers, antidepressants, and disinfectants. In Chapter 2, the following conclusions were made observing the current knowledge of prion proteins in wastewater treatment: Accurate detection and quantification of prions in a wastewater matrix is difficult with current analytical methods Prion proteins should partition into the solids phase during the wastewater treatment process where they likely survive anaerobic digestion and would be found in the biosolid effluent

86

Risks associated with prions entering the municipal wastewater system and being discharged into the environment are low

Chapter 3 discussed the sources of disinfectants, antidepressants, and beta-blockers into municipal sewer systems concluding that: Disinfectants, beta-blockers, and antidepressants were found in all locations monitored and it was observed that all source types contributed to the contaminant loading in municipal wastewater with no apparent point sources When hospital loadings were estimated and compared to total wastewater treatment plant influent loadings, it was calculated that hospitals account for <1% of the total disinfectant, beta-blocker, and antidepressant loading in a city. Further study is suggested to determine the source and cause of beta-blockers and antidepressants found in slaughterhouse effluent

In Chapter 4, the fate and removals of disinfectants and beta-blockers in municipal wastewater treatment plants was discussed: Large variations in contaminant concentrations were observed throughout the wastewater treatment plants. Beta-blockers, triclosan and triclocarban partition into the solids phase however the low flow rate of primary sludge dictates that physical removal of beta-blockers and disinfectants due to adsoprtion does not play a significant role.

87

Biological degradation was concluded to be the primary removal pathway with betablocker removal efficiencies from 0 to 28% and triclosan/triclocarban removal efficiencies from 40-98%

The risk associated with beta-blockers and disinfectants in the aquatic environment due to wastewater treatment effluent discharge was found to be low although the chronic affects and potential additive affects of multiple contaminants in the aquatic matrix are currently unknown.

The results discussed in Chapters 3 and 4 are only the first part of a larger study. As more data becomes available from the Worsfold Water Quality lab, the rest of the emerging contaminant classes described in Chapter 1 will need to be analyzed in a similar manner. Once a more complete picture describing the sources of emerging contaminants to municipal wastewater is obtained, reduction strategies can be investigated. If point sources for specific classes of emerging contaminants are identified, on-site pretreatment should be investigated. A small membrane bioreactor or similar on-site treatment system could remove contaminants from a concentrated effluent source such as a large hospital or slaughterhouse facility. On-site treatment may provide a viable option for contaminant removal if there are sources identified with high enough contaminant loadings to make practical and economical. If there are no specific point sources for the rest of the emerging contaminants studied, limiting the entry of these contaminants to the municipal wastewater system will be difficult. Removal strategies may have to be focused on enhancing the removal efficiencies of the wastewater treatment plants. Once the fate of the remaining emerging contaminant classes has been analyzed, methods to increase the removal capacity of treatment plants can be investigated. 88

For compounds such as beta-blockers and disinfectants where biological degradation is thought to be the primary removal mechanism, further research should investigate the differences in operating conditions between the three different wastewater treatment plants to determine why there was found to be differences in the removal efficiencies specifically around the aeration basin where aerobic degradation occurs. Increased removal capacity may also be added by constructing tertiary processes to existing facilities such as membrane bioreactors, sand filters, and rotating biological contactors. As scientists continue to investigate the prevalence of emerging contaminants in the environment and their effects on the aquatic ecosystem, there will be increased pressure to find solutions and methods in order to reduce the loading these contaminants into the natural world. By gaining a better understanding of the sources and fate of emerging contaminants in municipal wastewater, removal strategies can be developed mitigating the risk these compounds pose to both humans and the environment.

89

!"##$%&'()%*+),*-$.(//$),+&'()%*&(*0"&"1$*!&",$)&%* The following is a collection of suggestions and background information pertinent to students working on this or similar projects in the future. 21(3$.&*41#+)'5+&'()* Before sampling has commenced, it is recommended that the student meet in person with representatives from each municipality to develop a personal connection with the municipal staff to facilitate cooperation as the project is implemented. Without a clear contact person, communication with a municipal wastewater treatment plant is difficult. When choosing sample locations, glean as much information about the specific locations as possible before sampling starts. Useful information includes residential catchment area populations and specific hospital details. !+/67$*8(.+&'()%* The sample locations within each wastewater treatment plant were not consistent across the three municipalities. While they were similar enough to make some comparisons, identical locations would allow for more specific evaluations of the differences between WWTP performances. For a detailed description of WWTP sample locations, see the Appendix. Source sampling of hospitals was conducted at the sewage discharge point out side of each hospital. Some hospitals have multiple discharge points and thus sampling just one may give an inaccurate depiction of the overall hospital discharge especially if entire hospital units such as cancer or radiation suites are being released into an unmonitored discharge point. Since it is

90

difficult to determine how specific hospital discharge systems are laid out, this is another potential source of experimental error. For Municipality A, two discharge points from the same hospital were monitored and the values averaged. Funeral homes presented yet another challenge for sampling procedures. Partway through the project, it was discovered that some municipal staff in municipality C were presenting funeral home operators with bottles, which they then filled with the liquid discharge from the embalming process. In this way, data from Funeral Home C may not be indicative of an average funeral home effluent but rather describes funeral home discharge while an embalming is occurring. 9+&+*:)+7;%'%*+),*27+)&*9+&+* Since the laboratory analysis was completed at the Worsfold Water Quality Center, the data analysis timeline was dictated by the speed at which the water quality center returned the analytical results to the University of Guelph researchers. For the eleven classes of emerging contaminants being tested, only three classes of data are currently available. As more classes of contaminants are analyzed and returned to the University of Guelph, a more complete picture of emerging contaminants in municipal wastewater can be produced. When attempting to compare the three different wastewater treatment plants involved in this study, plant-operating data was requested from each of the municipalities. Municipality B was the only one to comply completely with this request while Municipality C provided some data and Municipality A neglecting to respond at all. With more details regarding the operating conditions of each plant, more comparisons could have been made between the differences in removal efficiencies seen in each wastewater treatment plant. All plant operating conditions made available by municipalities B and C can be found in the attached appendix. 91

**

92

Appendix
!"#$%!&'()"*%*$+%,$#$%
CITY A
Compound Plant Influent Avg Atenolol Sotalol Metoprolol Propranolol 999 457 415 59 StDev 407 115 188 38 Primary Effluent Avg 916 360 359 49 StDev 375 105 187 28 Digester Effluent Avg 849 1535 809 150 StDev 894 1270 703 139 Secondary Effluent Avg 1094 631 531 59 StDev 264 30 116 7 Final Effluent Avg 829 551 426 44 StDev 303 253 202 22 Residential Avg 1087 209 611 66 StDev 302 116 155 64 Hospital 1 Avg 406 367 368 9 StDev 147 262 128 6 Hospital 2 Avg 2155 306 2580 173 StDev 1619 214 1287 8

CITY B
Compound Atenolol Sotalol Metoprolol Propranolol Plant Influent Avg 990 568 394 43 StDev 221 93 35 2 Mixed Liquor Avg 348 464 209 22 StDev 183 255 145 11 Centrate Avg 649 579 256 59 StDev 435 74 12 25 Digester Effluent Avg 875 1475 493 123 StDev 1041 1399 448 151 Final Effluent Avg 951 513 384 40 StDev 261 71 68 5 Residential 1 Avg 577 449 603 127 StDev 77 257 420 70 Residential 2 Avg 1749 406 102 12 StDev 1009 122 328 21 Funeral Home Avg 21 98 590 1 StDev 5 28 936 1 Hospital Avg 2600 348 1020 56 StDev 877 330 309 91

93

City C
Compound Plant Influent Avg Atenolol Sotalol Metoprolol Propranolol 1340 484 501 40 StDev 141 136 118 5 Raw Sludge Avg 1157 1575 232 70 StDev 486 219 89 6 Mixed Liquer Avg 122 321 136 26 StDev 60 163 83 7 TWAS Avg 205 1536 336 149 StDev 52 798 91 47 Digester Effluent Avg 196 647 206 48 StDev 59 296 76 33 Final Effluent Avg 510 586 212 22 StDev 62 158 93 7 Slaughterhouse Avg 1190 400 357 31 StDev 566 #REF! 173 19 Funeral Home Avg 736 442 42 3 StDev 628 194 1017 0.4 Hospital Avg 1080 62 388 9 StDev 289 89 416 4

94

#*-(&'.$/%$/,%#*-(&'($*!$/%
CITY A
Compound Triclocarban Triclosan Plant Influent Avg 100 1547 Stdev 34 216 Digester Effluent Avg 422 1600 Stdev 139 552 Primary Effluent Avg 65 991 Stdev 38 424 Secondary Effluent Avg 31 215 Stdev 4 150 Final Effluent Avg 37 182 Stdev 9 23 Residential Avg 111 2035 Stdev 41 191 Hospital 1 Avg 13 432 Stdev 5 146 Hospital 2 Avg 61 401 Stdev 71 302

CITY B
Compound Plant Influent Avg Triclocarban Triclosan 67 885 Stdev 26 677 Mixed Liquer Avg 61 284 Stdev 45 178 Digester Effluent Avg 711 3807 Stdev 358 1180 Final Effluent Avg 38 534 Stdev 6 555 Residential 1 Avg 270 3453 Stdev 178 1156 Residential 2 Avg 115 1928 Stdev 90 1510 Funeral Home Avg 27 70000 Stdev 22 7355 Hospital Avg 77 14363 Stdev 127 19260

CITY C
Conpound Plant Influent Avg Triclocarban Triclosan 116 1284 Stdev 39 688 Raw Sludge Avg 75 932 Stdev 33 87 Mixed Liquor Avg 42 86 Stdev 43 34 TWAS Avg 187 312 Stdev 130 54 Digester Effluent Avg 214 727 Stdev 25 297 Final Effluent Avg 19 29 Stdev 15 6 Slaughterhouse Avg 113 226 Stdev 50 305 Funeral Home Avg 72 379 Stdev 80 337 Hospital Avg 30 1210 Stdev 25 200

95

$/#-,"0*"..$/#.%
CITY A
Compound Plant Influent Avg Venlafaxine Odesmethyl venalfaxine Citalopram Desmetyl citalpram 775 1803 301 234 StDev 184 404 36 44 Plant Influent Avg Venlafaxine O-desmethyl venalfaxine Citalopram Desmetyl citalpram 607 1427 342 275 StDev 89 364 27 65 Primary Effluent Avg 889 2317 340 295 StDev 142 136 85 95 Mixed Liquor Avg 263 825 156 395 StDev 176 354 12 406 Digester Effluent Avg 418 8633 340 430 StDev 140 1450 92 53 Secondary Effluent Avg 924 2140 361 224 StDev 249 354 69 12 Final Effluent Avg 823 1893 340 225 StDev 185 743 80 31 Residential Avg 3298 4350 354 275 StDev 3779 2560 95 132 Hospital 1 Avg 4307 1751 345 159 StDev 3439 943 135 47 Hospital 2 Avg 3535 11550 1775 990 Funeral Home Avg 21 98 1670 1 StDev 13 11 35 115 StDev 615 212 177 71

CITY B
Date Centrate Avg 371 1620 313 526 StDev 61 336 139 156 Digester Effluent Avg 738 4737 622 3687 StDev 773 5078 406 3855 Final Effluent Avg 648 1260 335 295 StDev 285 150 46 57 Residential 1 Avg 577 449 603 127 StDev 93 614 55 59 Residential 2 Avg 325 406 102 12 StDev 208 319 173 94 Hospital Avg 517 1500 552 337 StDev 343 668 106 91

CITY C
Compound Venlafaxine Odesmethyl venalfaxine Plant Influent Avg 1002 13115 StDev 153 16016 Raw Sludge Avg 2505 1660 StDev 233 396 Mixed Liquor Avg 507 1045 StDev 385 969 TWAS Avg 1077 1105 StDev 372 870 Digester Effluent Avg 458 3620 StDev 145 467 Final Effluent Avg 461 351 StDev 191 417 Slaughterhouse Avg 1190 400 StDev 319 809 Funeral Home Avg 736 442 StDev 16 89 Hospital Avg 1080 62 StDev 155 272

96

Citalopram Desmetyl citalpram

463 365

80 29

341 555

11 42

232 177

47 30

722 484

182 154

147 867

42 670

178 359

63 19

357 31

156 122

42 3

1306 958

388 9

36 103

.$10&"%&'($#-'/.%!2%+$.#"+$#"*%#*"$#1"/#%0&$/#%
WWTP A
Location Plant Influent Primary Clarifier Liquid Effluent Mixed Liquor TWAS Digester Effluent Secondary Clarifier Liquid Effluent Biosolids Final Effluent

Data Received from Worsfold Water Quality Center

Yes

Yes

No

No

Yes

Yes

No

Yes

WWTP B
Location Plant Influent Primary Sludge Mixed Liquor TWAS Digester Effluent Centrate from dewatering process Biosolids Final Effluent

Data Received

Yes

No

Yes

No

Yes

Yes

No

Yes

97

WWTP C
Location Plant Influent Primary Sludge Mixed Liquor TWAS Digester Effluent Final Effluent

Data Received

Yes

Yes

Yes

Yes

Yes

Yes

.$10&-/3%.(4",5&"%
Sample Date March 2010 (Municipality A only) Late June 2010 Late August 2010 Late October 2010 Mid January 2011 Early April 2011 Early June 2011

Data Returned from Worsfold Water Quality Center

Yes

Yes

Yes

Beta-Blockers Only

No

Beta-Blockers only

No

**Municipality A stopped participating after November 2010 and did not participate in the last three sampling events 98

0&$/#%'0"*$#-/3%,$#$%!2%+$.#"+$#"*%#*"$#1"/#%0&$/#%
WWTP B
Parameter Daily influent Flow Influent cBOD Influent TSS Influent COD Influent TP Primary Effluent TSS Primary Effluent cBOD Primary Effluent TP Aeration Retention Time SRT Aeration Retention Time HRT

Average Value

293 MLD

138 mg/L

195 mg/L

379mg/L

3.69 mg/L

82 mg/L

85 mg/L

2.05 mg/day

7.47 days

6.9 hours

Parameter

Final Effuent TSS

Final Effluent cBOD

Final Effluent TP

Ferric Sulfate dosage

Primary Clarifier HRT

Secondary Clarifier HRT

Total HRT

Average Value

11.59 mg/L

10.71 mg/L

0.493 mg/L

4.02 mg/L

2.99 hours

4.12 hours

14.02 hours

99