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Engineered Biofiltration for the Removal of Disinfection By- Product Precursors, Genotoxicity and Emerging Contaminants by Michael James McKie A thesis submitted in conformity with the requirements for the degree of Masters of Applied Science Civil Engineering University of Toronto © Copyright by Michael James McKie 2015

Engineered Biofiltration for the Removal of Disinfection ... ENGINEERED BIOFILTRATION FOR THE REMOVAL OF DISINFECTION BY-PRODUCT PRECURSORS, GENOTOXICITY AND EMERGING CONTAMINANTS

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Engineered Biofiltration for the Removal of Disinfection By-

Product Precursors, Genotoxicity and Emerging Contaminants

by

Michael James McKie

A thesis submitted in conformity with the requirements

for the degree of Masters of Applied Science

Civil Engineering

University of Toronto

© Copyright by Michael James McKie 2015

ii

ENGINEERED BIOFILTRATION FOR THE REMOVAL OF

DISINFECTION BY-PRODUCT PRECURSORS, GENOTOXICITY AND

EMERGING CONTAMINANTS

Michael James McKie

Master of Applied Science, 2015

Graduate Department of Civil Engineering

University of Toronto

ABSTRACT

Biofilters have been operated in a passive manner without operator control, but the focus

has shifted to the potential for enhancement. This pilot study compared conventional treatment

(coagulation, flocculation, sedimentation, non-biological filtration) to passive and engineered

direct biofilters for the removal of disinfection by-product precursors, and genotoxic precursors

for two different source waters (Lake Ontario and Otonabee River). Additionally, 9

pharmaceuticals and 2 artificial sweeteners were spiked into the pilot to observe the reduction of

these emerging contaminants through the biofilters.

Conventional treatment provided superior performance compared to biofiltration for the

removal of DBP precursors, but performed equally well with respect to genotoxicity reduction in

Otonabee River samples. Lake Ontario water was best treated by filters receiving 0.8 mg/L

PACl. Engineered biofilters, enhanced with nutrients or hydrogen peroxide, were typically no

better than a passively operated biofilter. The removal of pharmaceuticals and sweeteners was

improved with a combination of biological and chemical treatments.

iii

ACKNOWLEDGEMENTS

This work was funded by the Natural Sciences and Engineering Research Council of

Canada (NSERC) Chair in Drinking Water Research at the University of Toronto, and the

Ontario Research Fund (ORF).

I would first like to thank my supervisors Dr. R.C. Andrews, and Dr. S. Andrews. Their

continued support, guidance and encouragement were critical to my success and the project. I

would also like to thank Dr. R. Hofmann for reviewing this thesis and his guidance throughout

my time at the DWRG. Thank you to Liz Taylor-Edmonds for all of your help, and friendship.

Without your daily support we wouldn’t have been able to accomplish everything we did with

this project.

I cannot possibly thank all of the members of the DWRG who were a part of this project

enough. Jim Wang, Isabelle Netto, Jules Carlson, John Gibson, Nicolas Peleato, Joshua Elliott,

Ewalina Chojecka, Patrick King, and Yuxuan Cao, thank you for all of your contributions.

Thank you also to John Armour, Kevan Light, Renee Gagnon and everyone else at the

Peterborough Water Treatment Plant. As well, thank you to Dave Scott, Liza Ballantyne, Ahbay

Tadwalker and all of the staff at the Toronto Water Harris Water Treatment Plant. Without the

efforts and knowledge of everyone involved with the pilots this project would not have been

possible.

Thank you to my family for their constant support, encouragement and love. I wouldn’t

be able to achieve all that I can without you. Finally, thank you to my wife Leslea for your

editing help, thesis writing guidance and infinite patience through this process. It was a journey I

could not have completed without you beside me.

iv

TABLE OF CONTENTS

ABSTRACT .................................................................................................................................... ii

ACKNOWLEDGEMENTS ........................................................................................................... iii

TABLE OF CONTENTS ............................................................................................................... iv

LIST OF TABLES ........................................................................................................................ vii

LIST OF FIGURES ....................................................................................................................... ix

NOMENCLATURE ...................................................................................................................... xi

1. Introduction ................................................................................................................................ 1

1.1 Background ......................................................................................................................... 1

1.2 Objectives ........................................................................................................................... 2

1.3 Description of Chapters ...................................................................................................... 3

2. Literature Review ....................................................................................................................... 4

2.1 Biofiltration Overview and Performance ............................................................................ 4

2.1.1 Engineered Biofiltration .......................................................................................... 6

2.2 Biological Characterization ................................................................................................ 7

2.3 Regulated Disinfection By-Product Formation .................................................................. 9

2.4 Halogenated Furanones ..................................................................................................... 11

2.5 Genotoxicity ...................................................................................................................... 14

2.6 Anthropogenic Pollutants .................................................................................................. 15

2.6.1 Pharmaceutically Active Compounds and Endocrine Disrupting Compounds .... 15

2.6.2 Artificial Sweeteners ............................................................................................. 16

2.7 Knowledge Gaps ............................................................................................................... 17

3. Materials and Methods ............................................................................................................. 19

3.1 Experimental Protocol ...................................................................................................... 19

3.1.1 Pilot-Scale Biofilters ............................................................................................. 19

3.1.2 Spiking Procedure ................................................................................................. 22

3.1.3 Selection of Pharmaceuticals and Artificial Sweeteners ...................................... 23

3.1.4 Sampling Schedule ................................................................................................ 23

3.2 Analytical Methods ........................................................................................................... 25

3.2.1 Adenosine Triphosphate (ATP) Analysis ............................................................. 25

3.2.2 EPS Analysis ......................................................................................................... 25

v

3.2.3 Dissolved Organic Carbon .................................................................................... 26

3.2.4 Liquid Chromatography-Organic Carbon Detection (LC-OCD) .......................... 28

3.2.5 UV254 ..................................................................................................................... 29

3.2.6 Disinfection By-Product Formation Test .............................................................. 29

3.2.7 Trihalomethanes .................................................................................................... 29

3.2.8 Haloacetic Acids ................................................................................................... 31

3.2.9 Adsorbable Organic Halides (AOX) ..................................................................... 32

3.2.10 Halogenated Furanones ......................................................................................... 32

3.2.11 Genotoxicity with the SOS Chromotest ................................................................ 34

3.2.12 Pharmaceuticals and Endocrine Disruptors .......................................................... 35

3.2.13 Artificial Sweeteners ............................................................................................. 38

3.3 Statistical Analysis ............................................................................................................ 40

4. Engineered Biofiltration for the Removal of DBP Precursors, and Genotoxicity ................... 41

4.1 Introduction ....................................................................................................................... 41

4.2 Materials and Methods ...................................................................................................... 42

4.2.1 Source Waters ....................................................................................................... 42

4.2.2 Pilot Plant Configurations ..................................................................................... 42

4.2.3 Analytical Methods ............................................................................................... 43

4.2.4 Statistical Methods ................................................................................................ 45

4.3 Results and Discussion ..................................................................................................... 45

4.3.1 Biological Characterization of the Filter Media ................................................... 45

4.3.2 Removal of DBP Precursors Relative to DOC ..................................................... 51

4.3.3 Reduction of Genotoxicity .................................................................................... 59

4.4 Summary ........................................................................................................................... 62

5. Removal of Emerging Contaminants by Engineered Biofiltration .......................................... 64

5.1 Introduction ....................................................................................................................... 64

5.2 Materials and Methods ...................................................................................................... 66

5.2.1 Compounds of Interest .......................................................................................... 66

5.2.2 Source Waters ....................................................................................................... 67

5.2.3 Pilot Plant Configurations ..................................................................................... 67

5.2.4 Analytical Methods ............................................................................................... 70

5.3 Results and Discussion ..................................................................................................... 70

vi

5.3.1 Characterization of the Pilot-Scale Filters ............................................................ 70

5.3.2 Removal of Pharmaceutical Compounds by Biological Processes ....................... 71

5.3.3 Impact of Coagulation on Removal of Artificial Sweeteners ............................... 75

5.4 Summary ........................................................................................................................... 78

6. Summary, Conclusions, and Recommendations ...................................................................... 80

6.1 Summary ........................................................................................................................... 80

6.2 Conclusions ....................................................................................................................... 80

6.3 Recommendations ............................................................................................................. 81

7. References ................................................................................................................................ 82

8. Appendices ............................................................................................................................. 102

8.1 Standard Operating Procedure Outlines .......................................................................... 102

8.2 Raw Data ......................................................................................................................... 114

8.3 Sample Quality Assurance/Quality Control Charts ........................................................ 130

vii

LIST OF TABLES

Table 2-1: Summary of Biofiltration Operational Parameters ........................................................ 5

Table 2-2: Summary of DBP Precursor Removal by Various Treatment Methods ..................... 10

Table 2-3: Summary of MX Precursor Formation and Treatment ............................................... 13

Table 3-1: Summary of Pilot Plant Influent Water Quality .......................................................... 19

Table 3-2: Selected Pharmaceutically Active Compounds and Artificial Sweeteners ................. 24

Table 3-3: Sampling Schedule at the Otonabee River and Lake Ontario Pilot Plants .................. 24

Table 3-4: EPS Reagents .............................................................................................................. 25

Table 3-5: DOC Analyzer Conditions .......................................................................................... 27

Table 3-6: DOC Analysis Reagents .............................................................................................. 27

Table 3-7: THM Instrument Conditions ....................................................................................... 30

Table 3-8: THM Reagent Compounds .......................................................................................... 30

Table 3-9: HAA Instrument Conditions ....................................................................................... 31

Table 3-10: HAA Analysis Required Reagents ............................................................................ 31

Table 3-11: Operating Conditions for the Analysis of MX and MCA ......................................... 33

Table 3-12: Reagents Used for MX and MCA Analysis .............................................................. 34

Table 3-13: Preparation of Reagents for MX and MCA Analysis ................................................ 34

Table 3-14: LC-MS-MS Operating Parameters - PhAC and EDC Analysis ................................ 36

Table 3-15: Reagents Used in PhAC and EDC Analysis ............................................................. 37

Table 3-16: Preparation Steps for Reagents Used in PhAC Analysis .......................................... 37

Table 3-17: Surrogate and Internal Standards for PhAC Analysis .............................................. 38

Table 3-18: Operating Conditions for Artificial Sweetener Analysis .......................................... 39

Table 3-19: Reagents Used in Artificial Sweetener Analysis ....................................................... 39

Table 3-20: Internal Standards Used in Artificial Sweetener Analysis ........................................ 39

Table 3-21: Preparation of Reagents for Artificial Sweetener Analysis ....................................... 40

Table 4-1: Statistical Comparison of Treatment Processes using Paired T-tests – Otonabee River

....................................................................................................................................................... 48

Table 4-2: Statistical Comparison of Treatment Processes using Paired T-tests – Lake Ontario 50

Table 5-1: PhACs, EDCs, and Artificial Sweetener Details ......................................................... 66

Table 5-2: Pharmaceutical Removals Compared to Existing Literature ...................................... 75

Table 8-1: ATP Analysis Method ............................................................................................... 102

Table 8-2: EPS Analysis Method Outline ................................................................................... 103

viii

Table 8-3: DOC Method Outline ................................................................................................ 104

Table 8-4: THM Extraction and Analysis Procedure ................................................................. 104

Table 8-5: HAA Analysis Procedure .......................................................................................... 106

Table 8-6: MX and MCA Sample Preparation Details ............................................................... 108

Table 8-7: MX and MCA Solid Phase Extraction Details .......................................................... 108

Table 8-8: Genotoxicity Sample Preparation .............................................................................. 109

Table 8-9: PhAC Sample Preparation Method ........................................................................... 110

Table 8-10: PhAC and EDC Extraction Method ........................................................................ 111

Table 8-11: Artificial Sweetener Sample Preparation Process ................................................... 112

Table 8-12: Artificial Sweetener SPE Details ............................................................................ 112

Table 8-13 Raw Data - Otonabee River Pilot Plant .................................................................... 114

Table 8-14: Raw Data - Lake Ontario Pilot Plant ....................................................................... 123

Table 8-15: PhAC, EDC, and Artificial Sweetener Raw Data - Lake Ontario Pilot Plant ......... 128

ix

LIST OF FIGURES

Figure 3-1: Schematic of the Otonabee River Pilot Plant ............................................................. 20

Figure 3-2: Schematic of the Lake Ontario Pilot Plant ................................................................. 22

Figure 3-3: Sample Calibration Curve - Protein Analysis (March 2014) ..................................... 26

Figure 3-4: Sample Calibration Curve - Polysaccharides Analysis (March 2014) ....................... 26

Figure 3-5: Sample Calibration Curve - DOC (March 2014) ....................................................... 28

Figure 3-6: Sample Chloroform Calibration Curve (April 2014) ................................................. 30

Figure 3-7: Sample HAA Calibration Curve (April 2014) ........................................................... 32

Figure 4-1: Otonabee River Pilot-Scale Filter Media Biomass Characterization ......................... 46

Figure 4-2: Lake Ontario Pilot-Scale Filter Media Biomass Characterization ............................. 49

Figure 4-3: LC-OCD Fractionation as a % of Total DOC Through Various Treatments –

Otonabee River ............................................................................................................................. 52

Figure 4-4: DBP Precursor Removal – Otonabee River ............................................................... 54

Figure 4-5: DBP Precursor Removal – Lake Ontario .................................................................. 56

Figure 4-6: Seasonal Formation of MX as a Function of THM Formation – Otonabee River ..... 57

Figure 4-7: MX FP as a function of THM FP – Otonabee River ................................................. 58

Figure 4-8: MCA FP as a function of THM FP – Otonabee River ............................................... 59

Figure 4-9: Genotoxic Response with Respect to AOX FP – Otonabee River ............................ 60

Figure 4-10: Genotoxic Response with Respect to AOX FP – Lake Ontario .............................. 61

Figure 4-11: Genotoxic Response as a Function of THM FP – Otonabee River – Circled areas

represent biological and conventional treatment .......................................................................... 61

Figure 5-1: Schematic of the Otonabee River Pilot Plant ............................................................. 68

Figure 5-2: Schematic of the Lake Ontario Pilot Plant ................................................................. 69

Figure 5-3: Influent Water Spiking Results - Lake Ontario ......................................................... 72

Figure 5-4: Average Analyte Removal – Otonabee River ........................................................... 73

Figure 5-5: Average Analyte Removal – Lake Ontario Water ..................................................... 74

Figure 5-6: Removal of Acesulfame-K – Otonabee River ........................................................... 76

Figure 5-7: Sucralose Removal – Otonabee River ....................................................................... 77

Figure 5-8: Sweetener Removal as a Function of PACl Dose – Lake Ontario ............................. 78

Figure 8-1: Quality Control Chart – Chloroform ........................................................................ 130

Figure 8-2: Quality Control Chart - Monochloroacetic Acid ..................................................... 131

Figure 8-3: Quality Control Chart – MX .................................................................................... 131

x

Figure 8-4: Quality Control Chart – MCA ................................................................................. 132

Figure 8-5: Quality Control Chart – MBA ................................................................................. 132

Figure 8-6: Quality Control Chart- Acesulfame – K .................................................................. 133

Figure 8-7: Quality Control Chart - Sucralose ............................................................................ 133

xi

NOMENCLATURE

~ Approximate

% Percent

< Less than

> Greater than

°C Degrees Celsius

α Confidence level

± Plus/minus

µg/g Micrograms per gram

µg/L Micrograms per litre

µL Microlitres

4-NQO 4-Nitroquinoline 1-oxide

AC Activated carbon

Acesulfame-K Acesulfame-potassium

Al2(SO4)3 Aluminum sulphate (alum)

AOX Adsorbable organic halides

ATP Adenosine triphosphate

AWWA American Water Works Association

AWWARF American Water Works Association Research Foundation

BAC Biologically active carbon

BDOC Biodegradable organic carbon

Biofilter Rapid, biologically active filter

BOM Biodegradable organic matter

C Carbon

C Concentration

C0 Initial concentration

cm Centimeter(s)

C:N:P Carbon:nitrogen:phosphorus ratio

Conventional Treatment Coagulation, flocculation, settling, non-biological filtration

Cremoved Carbon removed by treatment

d Diameter

xii

DBP Disinfection by-product

DBPR Disinfection by-product rule

DNA Deoxyribonucleic Acid

DOC Dissolved organic carbon

E. coli Escherichia coli

EBCT Empty bed contact time

EDC Endocrine disrupting compound

EPS Extracellular polymeric substances

FP Formation Potential

g Gram(s)

GAC Granular activated carbon

GC Gas chromatography

GC-MS Gas chromatography – mass spectrometry

GC-ECD Gas chromatography – electron capture detection

H2O2 Hydrogen peroxide

H2SO4 Sulphuric Acid

HAA(s) Haloacetic acid

HLB Hydrophilic-lipophilic balance

HPLC High performance liquid chromatography

HWTP R.C. Harris Water Treatment Plant

IF Induction factor

KHP Potassium hydrogen phthalate

L Litre(s)

LC-OCD Liquid chromatography – organic carbon detection

LMW Low molecular weight

m metre(s)

MCA Mucochloric acid

MDL Method detection limit

mg/L Milligram(s) per litre

mg/mL Milligram(s) per millilitre

min Minute(s)

mL/hr millilitre(s) per hour

xiii

mL/min millilitre(s) per minute

mm Millimetre(s)

MTBE Methyl-tert-butyl-ether

MX (Mutagen X) 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone

N Nitrogen

ng/g nanogram(s)/gram

ng/L nanogram(s)/litre

nm nanometre(s)

Na2SO4 Sodium sulphate

NaOH Sodium hydroxide

NOM Natural organic matter

NTU Nephelometric TurbidityUnit

OCD Organic carbon detection

ON Ontario

P Phosphorus

PAC Powdered activated carbon

PACL Polyaluminum chloride

pH -log (hydrogen ion concentration)

ppb Parts per billion

PhAC Pharmaceutically active compounds

ppb Parts per billion

PPCP Pharmaceuticals and personal care products

ppm Parts per million

PWTP Peterborough Water Treatment Plant

QA/QC Quality assurance/quality control

R Pearson correlation coefficient

R2/r

2 Coefficient of determination

sec Second(s)

SOP Standard operating procedure

SOSIP SOS Inducing Potency

SPE Solid phase extraction

t Student T-test value or Student T probability distribution

xiv

THM(s) Trihalomethane(s)

TIC Total inorganic carbon

TOC Total organic carbon

TOX Total organic halogen

USEPA United States Environmental Protection Agency

UV254 UV absorbance at 254 nm

UVD Ultraviolet radiation detection

1

1. Introduction

1.1 Background

Drinking water treatment facilities are required to remove different classes of

contaminants, including microbiological, chemical (metals, organics and disinfection by-

products) and radiological. Most facilities use chemical (e.g. coagulation and chlorination) and

physical (e.g. flocculation, settling and filtration) processes to meet the existing regulations

(AWWA 2006 a,b). Recently, new classes of contaminants have been identified in source waters.

Emerging contaminants such as pharmaceutically active compounds (PhACs), endocrine

disrupting compounds (EDCs) and artificial sweeteners are environmentally stable,

anthropogenic compounds that are being found in increasing concentrations, but may not be

effectively removed by existing treatment processes (USEPA 2001). At the same time,

disinfection by-product regulations are becoming more strict and are forcing water providers to

optimize treatment to meet these new regulations. In an effort to improve the performance of

existing systems, biological treatment processes have been used to improve removal of organic

compounds, lower chlorine demands and improve biological stability in distribution networks

(Urfer & Huck, 1997). Biological treatment may also provide another barrier against emerging

contaminants.

Many different forms of biological treatment are employed at drinking water treatment

plants around the world. River bank and slow sand filtration are commonly used in Europe and

in small systems (Kuehn & Mueller, 2000; Ellis & Wood, 1985), but have not been applied to

municipal facilities in North America. Rapid biological filtration using sand, anthracite, or

granular activated carbon filter media is a commonly utilized method of incorporating biological

treatment in North America (Emelko et al., 2006). Biological filters are developed by

backwashing existing filters with unchlorinated water, and allowing microbial communities to

form on the filter media. Until recently, biological filters have been operated passively, without

control by plant operators, unlike other treatment processes. Due to the passive operation, issues

may arise, including the excessive formation of biomass leading to headloss, sloughing of

biological material due to nutrient deficiencies, and inconsistent performance due to changes in

influent water temperature and nutrient composition (Zhu et al., 2010). In an effort to improve

biofilter performance, Lauderdale et al. (2012) introduced the concept of engineered biofiltration

2

whereby they enhanced the biofilter influent with nutrients (phosphorus and nitrogen) to improve

biomass growth and consumption of organic compounds; and hydrogen peroxide was added to

minimize headloss formation as a result of excessive biofilm development.

Organic compounds are not inherently problematic to drinking water treatment facilities

or consumers, but when combined with disinfectants the organics transform into potentially

harmful disinfection by-products (Roberts et al., 2002). Trihalomethanes (THMs) and haloacetic

acids (HAAs) are regulated DBPs, but have been shown to have no genotoxic response (Hrudey,

2009). Genotoxicity is a measure of DNA damage in genetically engineered cells, and is an

indicator of potential risk to consumers (Richardson et al., 2007). Of greater genotoxic concern

are emerging DBPs, such as halogenated furanones, which can be highly genotoxic at very low

concentrations. Over 50% of genotoxicity in chlorinated water samples can be attributed to the

halogenated furanone 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (Mutagen X, MX)

at ng/L concentrations, whereas THMs and HAAs show no genotoxic response at concentrations

more than 1000 times higher (Zheng et al., 2014).

Emerging contaminants have been shown to have negative impacts on aquatic

ecosystems at low concentrations, but the impacts to human consumers are unknown (Safe,

2004). Biofiltration may provide an additional treatment barrier for the removal of these

contaminants. Many of the compounds of interest are recalcitrant to chemical and physical

treatment, but have been shown to be effectively removed by biological treatments (Zearly &

Summers, 2012). Biological treatment performance has been demonstrated in many different

wastewater applications, but less data is available for drinking water treatment, particularly at

pilot-scale.

1.2 Objectives

This study utilized pilot-scale biofilters to assess the following objectives:

1. Assess the performance of engineered biofilters which are enhanced with nutrients

(phosphorous and nitrogen), hydrogen peroxide, and low doses of coagulant for the

removal of organic carbon, DBP formation potential, and filter performance (headloss

and turbidity) to passively operated biofilters and conventional treatment (coagulation,

flocculation, sedimentation, non-biological filtration)

3

2. Compare engineered biofilters to passively operated biofilters and conventional treatment

for the removal of halogenated furanones (including MX) and the reduction of genotoxic

response

3. Compare engineered biofilters to passively operated biofilters and conventional treatment

for the removal of pharmaceutically active compounds, endocrine disrupting compounds

and artificial sweeteners that have been spiked into the pilot system

1.3 Description of Chapters

Chapter 2: provides background information about biofiltration, disinfection by-

products, genotoxicity, pharmaceuticals, and artificial sweeteners

Chapter 3: details the pilot systems, experimental design, sampling procedures,

analytical methods, and statistical analysis

Chapter 4: compares engineered biofiltration to passive biofiltration and conventional

filtration for the removal of specific organic carbon fractions identified by LC-OCD,

disinfection by-products, halogenated furanones, and genotoxicity

Chapter 5: provides experimental results detailing the removal of pharmaceutical

compounds, endocrine disrupting compounds and artificial sweeteners by biofiltration

and conventional filtration

Chapter 6: summarizes the results of the pilot studies completed

Chapter 7: contains a list of references utilized in this study

Chapter 8: appendices containing standard operating procedures, raw data and QA/QC

information

4

2. Literature Review

2.1 Biofiltration Overview and Performance

Conventional drinking water treatment facilities typically consist of coagulation,

flocculation, sedimentation, filtration and disinfection processes (AWWA, 2006a,b) and are

designed to prevent the proliferation of waterborne disease caused by microbial contamination

(Xagoraraki et al., 2004). However, disinfection by-product (DBP) precursors and emerging

contaminants may be poorly removed by traditional drinking water treatment practices (Kim et

al., 1997). In addition to the risk presented by chemical contaminants, treated water that is

biologically unstable can cause problems including microbial growth and regrowth, accelerated

corrosion of water mains, increased chlorine demands, and can cause taste and odour issues

(Rittmann & McCarty, 2001; Rittmann & Snoeyink, 1984). A common solution to this problem

is increasing chlorine residual at the treatment plant, but this can lead to the formation of DBPs

such as trihalomethanes (THMs) and haloacetic acids (HAAs) at levels exceeding local

regulations (Rittmann & McCarty, 2001).

Biological treatment can be used to overcome the potential risk associated with chemical

contaminants and biological instability by reducing the concentration of electron donors

including biodegradable organic matter (BOM) and nitrogenous compounds (Rittmann &

McCarty, 2001). These compounds serve as possible DBP precursors that should be removed or

reduced prior to disinfection to ensure that all regulatory requirements are met (Hua & Reckhow,

2007; Chen & Westerhoff, 2010).

One proposed method of incorporating biological processes into drinking water treatment

is biological filtration. A biologically active filter (biofilter) is defined as any filter media that

has attached biomass (Chaudhary et al., 2003). Common biofilter configurations include:

riverbank filtration (Kuehn & Mueller, 2000); slow sand filtration (McNair et al., 1987); rapid

biological filtration using sand or anthracite (Miltner et al., 1995; Emelko et al., 2006) and

granular activated carbon (Wang et al., 1995; Najm et al., 2005). Riverbank filtration is widely

used in Europe, but has not gained support in North America (Kuehn & Mueller, 2000;

Sontheimer, 1980; Weiss et al., 2003). However, rapid filtration is commonly used throughout

North American treatment plants and can be easily converted to operate biologically (Hozalski &

Bouwer, 1998; Emelko et al., 2006). Previous studies have shown backwashing, chlorine in

5

backwash water, backwashing frequency and water temperature have minimal impact on

biofiltration performance (Table 2-1).

Table 2-1: Summary of Biofiltration Operational Parameters

Operational

Parameter

Results Impact

Backwashing -Backwashing with or without

air scour does not impact BOM

removal or particle removal

(Emelko et al., 2006).

Routine backwashing based

on head loss or effluent

turbidity does not impact

biological filter performance.

Chlorinated

Backwash Water

-Backwashing with chlorinated

water impairs biofilm growth

(Wang et al., 1995)

-Chlorinated backwash reduces

biological activity by 22%, but

is recovered over filter run

(Miltner et al., 1995).

Efforts should be taken to

ensure that backwash water

is not chlorinated.

Prechlorination (a method

of controlling zebra mussel

growth) should be avoided

since it will limit biofilm

growth (Wang et al., 1995).

Media Type (GAC

versus anthracite and

sand)

-GAC based filters achieve

biological activity 3 to 8 times

that of sand and anthracite

filters (Wang et al., 1995).

-Biodegradable organic matter

(BOM) removal equal at high

temperatures but much better in

GAC filters at low temperatures

(Emelko et al., 2006).

Better performance may be

expected from GAC based

biofilters if the material is

available due to increased

biological activity and

better performance during

cold water periods (Emelko

et al., 2006).

Biological filters have been shown to significantly reduce the concentration of BOM

(Wang et al., 1995; Miltner et al., 1995; LeChevallier et al., 1992; Persson et al., 2006), DBP

formation potential (FP) (Wang et al., 1995; Onstad et al., 2008; Farré et al., 2011), and

biological instability (Emelko et al., 2006; Liu et al., 2001) when compared to non-biological

filtration. However, the biofilters used in these studies were not optimized to improve

performance, but were allowed to operate under naturally occurring conditions. As a result, it

may be possible to achieve better performance if the filters are artificially enhanced.

6

2.1.1 Engineered Biofiltration

Engineered biofiltration was a concept introduced by Lauderdale et al. (2012) to describe

a rapid biological filtration system that is designed, enhanced, and controlled to improve effluent

water quality and hydraulic performance. Heterotrophic bacteria found in biomass require

carbon, nitrogen, and phosphorus in a stoichiometric ratio of approximately 100:10:1 (C:N:P) to

effectively consume organic contaminants and reduce biological instability (LeChevallier et al.,

1991). However, biofilter influent, especially if coagulation occurs prior to filtration, is

considered to be oligotrophic, and the influent cannot provide the nutrients required for optimal

biomass growth and activity (Lauderdale et al., 2012). To achieve better reduction of electron

donors and promote biological activity within the filter, engineered biofiltration systems can be

enhanced by the addition of nitrogen and phosphorus (USEPA, 1991).

To ensure that carbon is the nutrient limiting substrate removal in the filter, phosphorus

must be present at a concentration >0.026 mg-P/mg-Cremoved and the nitrogen concentration must

exceed 0.117 mg-N/mg-Cremoved based on the stoichiometry presented above. Lauderdale et al.

(2012) reported DOC uptake across the control filter to be 0.4 mg/L and to ensure that carbon

was the limiting nutrient the filter influent was supplemented with 0.02 mg-P/L (200% required

dose from stoichiometry) and nitrogen was found in excess (>0.06 mg-N/L compared to 0.05

mg-N/L required). The authors also dosed the filter influent with hydrogen peroxide because it

was hypothesized to increase the available dissolved oxygen concentration and catalyze the

oxidation of organic contaminants.

The authors reported that the engineered biofilter, with carbon limiting conditions, was

able to decrease terminal head loss (head loss after 18 hr filter run) by 15% and deviation (head

loss range at the end of 18 hr filter run) by 50%; remove 75% more DOC (0.7 mg/L with nutrient

enhancement compared to 0.4 mg/L in the control); increase microbial activity by 30% as

measured by adenosine triphosphate (ATP); and developed approximately 50% less extracellular

polymeric substances (EPS) (~7.5 mg/L free + bound EPS in the enhanced filter versus ~15

mg/L free + bound EPS in the control) when compared to a passively operated control biofilter.

Hydrogen peroxide was found to reduce terminal head loss in the biofilter by over 60% while not

impacting the formation of biomass compared to the control (Lauderdale et al., 2012). The

authors also suggest that the additional contaminant removal and longer filter run times would

7

easily offset the chemical costs (~$4/ML) associated with engineered biofilters; potentially

making this a viable treatment option for water providers across North America.

More recently, Azzeh et al. (2014) examined engineered biofiltration at the Peterborough

Water Treatment Plant in Peterborough, Ontario, Canada. This study examined the impact of

biofilter enhancement using nutrients (phosphorus and nitrogen), hydrogen peroxide, and low

doses of alum (<0.5 mg/L) added to filter influent without flocculation. This study found that

DOC was poorly removed (5±2%) from Otonabee River water and enhancing the filters was

ineffective at improving these removals. Biopolymers were reduced by approximately 20%, but

biofiltration did not remove humic substances.

This study also examined the removal of disinfection by-product (DBP) precursors.

THMs and HAAs were found to be reduced by biofiltration by approximately 19% and 13%,

respectively. Adsorbable organic halides (AOX) were also found to be removed by an average of

6 to 11%. Enhancements were unable to consistently improve the removal of DBP precursors in

this study.

2.2 Biological Characterization

An important aspect of biofiltration is determining when a filter is biologically active,

quantifying how biologically active the filter media is, and determining how much biomass

exists on the filter media. Many different methods have been used to quantify biological activity

including phospholipid analysis (Wang et al., 1995), epifluorescence microscopic total cell count

(Dewaters and Digiano, 1990), heterotrophic plate counts (Stewart et al., 1990), biomass

respiration potential (Urfer & Huck, 2001), and adenosine tri-phosphate (ATP) analysis (Magic-

Knezev & van der Kooij, 2004). Since there are so many possible methods of quantifying

biomass, method selection is crucial. Methods utilizing heterotrophic plate counts or

phospholipid analysis can be used for measuring biomass, but may not include bacteria that are

not culturable, or be able to differentiate between live and dead cells. Respiration or consumption

based tests can be excellent at determining cellular activity, but does not measure all types of

cells, or determine the quantity of cells.

ATP analysis on the other hand, is an excellent measure of cellular activity and can be

used to approximate the quantity of live cells on filter media. This test is particularly beneficial

because it requires very little equipment for analysis, and can be completed easily in the field as

8

opposed to in a laboratory setting (Pharand et al., 2014). The test consists of an extraction step

(chemical, physical or enzymatic) to remove biomass from the filter media, the addition of a

luciferase-luciferin complex to react with ATP and produce light, and determining the light

intensity using a luminometer (Hammes et al., 2010).

This is a particularly interesting test method because it allows for the direct comparison

of filter media from different source waters. ATP on GAC filter media from a plant treating Lake

Ontario water was measured to be 11 ng/cm3 during cold water conditions (3-14°C), but 230

ng/cm3 during warm water conditions (10-19°C) (Pharand et al., 2014). In contrast, GAC media

from Lake Simcoe, Ontario was found to have ATP measurements between 328-270 ng/cm3

(Taylor-Edmonds et al., 2013), possibly due to higher influent DOC (2 mg/L in Lake Ontario,

compared to 4 mg/L in Lake Simcoe). Finally, an analysis of Grand River and Saugeen River

filter media showed ATP concentrations of 1,268 ng/cm3 and 163 ng/cm

3, respectively, despite

having the same DOC (~4 mg/L) (Pharand et al., 2013; Rahman, 2013).

Further characterization of biofilter media can be completed by quantifying

extrapolymeric substances (EPS). EPS is a gel like substance that holds microbes to the filter

media (Flemming & Wingender, 2001). In addition to providing the structural components

needed for the success of the biofilm, EPS protects microbial communities from high or low pH,

oxidants, and shear stresses (Wang et al., 2008; Sutherland, 2001). EPS is composed of

polysaccharides, proteins, and other polymeric substances (Flemming & Wingender, 2001).

Proteins are thought of as the structural components that hold microbial communities together,

while the polysaccharides are the waste products that are emitted from the cells. It is important to

quantify these compounds because they may be the primary contributor to biofilter clogging by

filling the voids between filter media, or binding media together (Mauclaire et al., 2004).

Although few direct studies of biofilms have been completed, Lauderdale et al. (2012)

examined the impact of nutrient and hydrogen peroxide enhancement on EPS formation in

drinking water biofilters. Nutrient enhancement, with nitrogen and phosphorus addition, reduced

free EPS by 43% and bound EPS by 55%. Although numerical results were not provided from

hydrogen peroxide analysis it was shown to reduce headloss by more than 60% with no change

in organics removal (Lauderdale et al., 2012), and it is believed that the peroxide was able to

oxidize inactive organisms and EPS.

9

2.3 Regulated Disinfection By-Product Formation

Trihalomethanes (THMs) are a group of four chemical compounds including:

chloroform, bromodichloromethane, dibromochloromethane, and bromoform (Health Canada,

2006). Chloroform was initially believed to be a carcinogenic compound (National Cancer

Institute, 1976), but was later shown to not be a carcinogen at the concentrations present in

drinking water (Hrudey, 2009). A potential causal link still exists between chlorinated DBPs and

bladder cancer, and until DBPs can definitively be shown to be non-cancerous they must be

controlled (Hrudey, 2009). As a result, THMs are regulated in Canada at a maximum acceptable

concentration of 100 μg/L (Health Canada, 2009), and in the United States at 80 μg/L as part of

the disinfection by-product rule (DBPR) stage 1 (USEPA, 2012a). The regulated group of

haloacetic acids (HAA5) is comprised of: monochloroacetic acid, dichloroacetic acid,

trichloroacetic acid, monobromoacetic acid and dibromoacetic acid (Health Canada, 2008).

These five compounds have a total maximum acceptable concentration of 80 μg/L in Canada

(Health Canada, 2008) and 60 μg/L in the United States as part of the DBPR stage 1 (USEPA,

2012a). Although being the major classes of regulated DBPs, THMs and HAAs are responsible

for a small percentage (<30%) of total organic halogen (TOX) detected in treated water samples

(Krasner et al., 2006).

Humic substances, typically composed of a combination of humic and fulvic acids, are a

component of natural organic matter (NOM) found in all surface waters (Narkis & Rebhun,

1977). When humic substances are chlorinated they serve as the primary precursors to the

formation of THMs and HAAs (Joyce et al., 1984; Amy et al., 1990; Hubel & Edzwald, 1987;

Roberts et al., 2002). Much of the existing literature has examined the removal of precursors, by

measuring DBP formation potential, as opposed to the removal of the DBPs themselves.

Coagulation (Bolto et al., 2002; Bell-Ajy et al., 2000), adsorption (Babi et al., 2007; Chiu et al.,

2012) and ozonation (Chang and Singer, 1991; Bose & Reckhow, 2007; Chiang et al., 2009)

were tested for their viability as DBP precursor removal processes. These processes are not

entirely effective at precursor removal, and some processes may transform the NOM into more

reactive degradation products (Reungoat et al., 2011) or leave more reactive fractions to be

disinfected (Bolto et al., 2002). A summary of the removal of DBP precursors by conventional

treatment methods can be found in Table 2-2.

10

Table 2-2: Summary of DBP Precursor Removal by Various Treatment Methods

Treatment

Process

Treatment Conditions Results Impact

Coagulation Various coagulants

tested including: alum

up to 125 mg/L,

polyaluminum chloride

(PACl) up to 50 mg/L,

and ferric chloride up to

70 mg/L (Volk et al.,

2000; Bell-Ajy et al.,

2000).

Alum removed between

12% and 59% TOC and

4% to 56% DOC. PACl

removed 10 to 52%

TOC and 5 to 55%

DOC. Ferric chloride

removed 0 to 72% TOC

and 12 to 58% DOC.

Coagulation is not

completely efficient at

removing organic

carbon from source

water. Many potential

DBP precursors remain

that could be utilized

as a carbon source in

biofiltration.

Activated Carbon

(AC)

May be either granular

(GAC) or powdered

(PAC). Used as either

filter-adsorber bed or as

suspended particles

which are later removed

by settling or filtration

(Calgon Carbon, 2013).

Pilot-scale studies see

DOC removals

approach 100% before

decreasing to <50%

(Babi et al., 2007). Full

scale treatment

averages 53% TOC

removal (Matilainen et

al., 2006).

AC can effectively

remove DBP

precursors, but its

treatment capacity is

diminished over time.

Conversion to

biologically active

carbon may be

beneficial in reducing

DBP formation.

Ozonation Ozone can be used as a

pre-oxidation,

intermediate oxidation

or final disinfection

process to degrade

compounds or enhance

removal efficiency by

other processes (Camel

& Bermond, 1998).

Preozonation (up to 3

mg/L) able to reduce

THM formation

potential by 32 to 35%

(Kleiser & Frimmel,

2000; Chiaket et al.,

2002) and HAA

formation potential by

43% (Chaiket et al.,

2002).

Ozonation degrades

NOM that can act as

DBP precursors and

creates biodegradable

by-products amenable

to biofiltration

(Siddiqui et al., 1997).

Biological

Filtration

Biofilters may be GAC

based, anthracite based,

sand based, or a

combination of media.

Often preceded by

ozonation in the

literature (Chaiket et al.,

2002; Nishijima et al.,

2003).

THM FP changed from

-16 to 17% while HAA

FP reduction ranged

12% - 100%. TOC was

removed by 12-17%

(Chaiket et al., 2002;

Onstad et al., 2008).

Increased reduction of

DBP expected from the

implementation of

biofiltration.

11

Biofiltration may be a viable alternative to improve on the DBP precursor removal

provided by traditional treatment processes. The primary precursors of THMs and HAAs are

NOM, and it has been shown in passive biofilters that the NOM fractions responsible for DBP

formation may also be used by biofilms as an electron donor. Hozalski et al. (1995) found that

biofiltration was able to remove up to an additional 12% of total organic carbon (TOC) and

eliminate almost 100% of the biodegradable fraction of TOC. From these results it is

hypothesized that engineered biofiltration will provide additional removal of NOM, when

compared to passive systems, and result in reduced formation of THMs and HAAs.

2.4 Halogenated Furanones

Halogenated furanones were first observed as unknown, highly mutagenic compounds in

the effluent from 4 pulp mills in Canada in the early 1980’s (Holmbom et al., 1984). The primary

compound known as Mutagen X, or MX, was isolated and identified as 3-chloro-4-

(dichloromethyl)-5-hydroxy-2(5H)-furanone (Holmbom et al., 1984). MX and its geometric

isomer (E)-2-chloro-3-(dichloromethyl)-4-oxobutenoic acid (EMX) were later discovered in

chlorinated humic waters and drinking waters (Kronberg et al., 1988). Further research

discovered a total of 12 analogues to MX (Onstad et al., 2008).

Halogenated furanones are most commonly found in waters treated with free chlorine as

opposed to chloramination or chlorine dioxide (Onstad et al., 2008). The formation of MX and

its analogues are usually a result of chlorine interaction with humic acids in raw water (Huixian

et al., 1999). MX and its 12 analogues are speciated based on pH. Ring forms (MX) dominate at

low pH and as pH rises the ring opens (ZMX) then tautomerizes (EMX) before degrading at

higher pH levels (>8) (USEPA, 2008).

As mentioned previously, natural organic matter (NOM), specifically humic and fulvic

acids, is generally accepted as the primary precursors for MX formation (Huixian et al., 1999).

Smeds et al. (1997) showed that MX formation was a factor of Cl2:TOC ratio and the total

chlorine demand. This indicates that as more chlorine is added, due to high levels of TOC or

high demand, the mutagenicity of the treated water increases as well.

Mutagenicity is a measure of a chemical’s ability to alter DNA (Noot et al., 1989). A

common method of measuring mutagenicity is the Ames test (Meier & Daniel, 1990). This test

detects changes in DNA by observing the alteration of Salmonella bacteria from being histidine-

12

requiring to histidine-independent (Noot et al., 1989; Meier & Daniel, 1990). MX and its isomers

have been found to contribute between 11 and 71% of total mutagenicity in drinking water

samples, making the removal of MX precursors very important (Smeds et al., 1997).

Huixian et al. (1999) tested 21 different suspected MX precursors to determine which

compounds were major contributors to MX formation. This study found that 8 had a significant

yield of MX including: syringaldehyde, tryptophan, tyrosine, 3-ethoxy-4-hydroxybenzaldehyde,

acetosyringone, 3,4,5-trimethoxy-benzaldehyde, ferulic acid, and vanillin. Although vanillin is

not a high yield precursor (~11 ng MX/μg vanillin) it can be found in wastewater effluent at

concentrations approaching 0.5 μg/L (Agus et al., 2012). Some of the other compounds are found

naturally in source waters and others are found as oxidation byproducts of organic matter in

source water. Syringaldehyde was found to produce less MX when more chlorine disinfectant

was added while the other 7 compounds were found to form more MX when more chlorine was

added (Huixian et al., 1999). A summary of selected MX precursors can be found in Table 2-3.

Similarly to THMs and HAAs, the precursors to MX formation may be easily reduced by

biological processes. As the precursors are again organic compounds, it is conceivable that a

fraction of the precursors would be consumed as substrate by micro-organisms. Onstad et al.

(2008) showed that MX was removed to below detection limits (<20 ng/L) using GAC based

biofiltration, while MX analogues were removed by as much as 87%. These results indicate that

biofiltration may be an appropriate process to control MX concentrations in treated waters.

However, enhanced biofiltration using non-adsorbent materials must be examined to further

evaluate the removal of MX precursors by biological filtration.

Another form of MX that is commonly found in drinking waters is mucochloric acid

(MCA). Jansson et al. (1995) found that MCA was much less mutagenic than MX when

examined in Salmonella typhimurium strain TA100, but equally mutagenic in Chinese hamster

ovary cells. These results are of particular concern because MCA is commonly found in treated

drinking water at concentrations up to 300 ng/L (Weinberg et al., 2002). MCA is rarely analyzed

in municipal drinking water treatment plants because it is an expensive analyte to test for, and it

is not currently regulated. However, the formation of MX and MCA has been found to be

correlated to the formation of THMs and HAAs which indicates that improvements in the

removal of the regulated DBPs will likely lead to improved removals of halogenated furanones.

13

Table 2-3: Summary of MX Precursor Formation and Treatment

Precursor Effect of Disinfectant Effect of Conventional

Treatment

Effect of

Biofiltration

Humic

substances

-Chlorination creates 1.4

to 8.3 ng MX/mg TOC

-Fulvic acids contribute

up to 80% of MX formed

(Xu et al., 1997)

-Alum coagulation able to

remove up to 59% TOC

(Volk et al., 2000; Bell-

Ajy et al., 2000)

-Activated carbon

removes >50% TOC

(Matilainen et al., 2006)

-Up to 35% removal

of humic substances

(Basu & Huck, 2004).

-Potential for

increased removal by

enhanced biofiltration

Syringaldehyde -Conversion up to .2 ng

MX / μg syringaldehyde

(Chengyong et al., 2000).

-Conversion decreases as

chlorine dose increases

(Huixian et al., 1999).

-No specific literature

focussed on the removal

of this compound by

conventional methods.

-Expected to be removed

similarly to other organic

compounds such as humic

substances

-Anaerobically

biodegraded (~100%)

to form methane from

lignin residues

(Barakat et al., 2012;

Healy & Young,

1979)

Vanillin -Chlorination may yield

up to 11 ng MX/ μg

Vanillin (Huixian et al.,

2000).

-Present in wastewater

effluent at 150 – 470

ng/L (Trenholm et al.,

2008).

-No specific literature

focussed on the removal

of this compound by

conventional methods.

-Expected to be removed

similarly to other organic

compounds such as humic

substances

-Well biodegraded

(>75%) in anerobic

conditions (Barakat et

al., 2012; Healy &

Young, 1979)

-Potential for aerobic

degradation must be

tested

14

2.5 Genotoxicity

The precursors and formation mechanisms of many different classes of disinfection by-

products have been discussed, but the overall impact of these compounds to human health is

difficult to quantify. One method of objectively comparing treatment processes is to use

biotesting to determine the resulting genotoxicity or mutagenicity. The term mutagenicity refers

to compounds that change cellular DNA sequences (either genes or chromosomal mutations),

and genotoxicity encompasses mutagenicity and DNA damage (DNA adducts or strand breaks)

(Richardson et al., 2007). Simply, these parameters measure the impact of contaminants in water

on cells without having to identify or quantify the chemicals causing the contamination.

Many different cell-based assays have been used to measure genotoxicity including:

salmonella (DeMarini et al., 1997; Kundu et al., 2004), Escherichia coli (E. coli) (Araki et al.,

2004; Giller et al., 1995; Liu et al., 2004), Chinese hamster ovaries (Cemeli et al., 2006; Plewa et

al., 2004), and bioluminescent bacteria Vibrio fisheri (Parkinson et al., 2001) among others. The

benefit of these in vitro tests is that they are sensitive, cost effective, and simple methods that can

provide a more robust evaluation of water quality (Zegura et al., 2009). These tests are also able

to quantify the synergistic or antagonistic effects that occur when combining a large number of

complex DBPs (Simmons et al., 2002).

Of particular interest is the SOS Chromotest which is used for the determination of

genotoxicity within a water sample. The SOS repair system is activated in E. coli in response to

DNA damage. The SOS Chromotest is a colourimetric assay used to detect the activation of the

DNA repair mechanism due to lesions in genetic material. In this test, the β-galactosidase (β-gal)

gene (lacZ) is fused to the bacterial sfiA SOS operon in a genetically engineered strain of E. coli

PQ37. This means that the SOS repair system of PQ37 is engineered such that SOS promoter

induces lacZ expression and subsequent synthesis of β-gal. The amount of β-gal formed can be

quantified photometrically using a chromogenic substrate to form a blue colour, and is

representative of bacterial genotoxicity (Kocak et al., 2010). Wang et al. (2011) used the SOS

Chromotest to assess the genotoxic potential of seven chlorinated drinking waters in China. This

study found that the chlorinated, finished waters were more likely to have a genotoxic response

(induction factor (IF) > 2.0) than the raw water samples. These results are indicative of the

potential issues surrounding DBPs, and further emphasize the need to remove DBPs efficiently.

15

2.6 Anthropogenic Pollutants

Anthropogenic contaminants are a class of compounds typically found in source waters

as a result of de facto water reuse. The compounds of interest are typically synthetic (USEPA,

2001), and can be useful indicators of wastewater contamination of source waters as they are

poorly treated in wastewater treatment facilities (Ternes et al., 2004; Westerhoff et al., 2005).

This section will focus on two groups of anthropogenic contaminants: endocrine disrupting

compounds (EDCs) and pharmaceutically active compounds (PhACs); and artificial sweeteners.

2.6.1 Pharmaceutically Active Compounds and Endocrine Disrupting Compounds

EDCs are chemicals which interfere with the synthesis, secretion, transport, action or

degradation of natural hormones by mimicking or blocking natural hormones (USEPA, 2001).

PhACs are used to diagnose, treat and prevent illness; and for the growth and health of livestock

and agricultural crops (USEPA, 2012b). There is concern that these compounds may have

negative impacts to the natural environment, including the feminization of male fish from

exposure to estrogen compounds (Larsson et al., 1999; Jobling et al., 1998; Purdom et al., 1994),

and salmonid reproductive and developmental problems from the introduction of toxic

compounds to Great Lakes waters (Leatherland, 1993). In addition, some EDCs and PhACs have

the ability to induce spawning in zebra mussels in laboratory settings (Fong, 1998). It is also

hypothesized that EDCs and PhACs may be responsible for increased rates of cancer in humans,

particularly breast and testicular, but there is not yet a definitive study on this topic and more

research is needed to identify the risks posed to human consumers by trace levels of these

compounds (Safe, 2004; Schwab et al., 2005; Brody and Rudel, 2003; Weber et al,, 2002).

Although consuming water with ng/L concentrations of individual EDCs or PhACs is not

expected to have significant negative health impacts, the impact of consuming a combination of

many compounds is uncertain (Webb et al., 2003; Servos et al., 2007). To reduce this

uncertainty, the removal of these compounds from drinking water and wastewater has been the

focus of many different studies (Westerhoff et al., 2005; Lishman et al., 2006; Reungoat et al.,

2011; Nakada et al., 2007; Carballa et al., 2007). Ozonation is a popular treatment method

(Nakada et al., 2007; Lee et al., 2012; Esplugas et al., 2007); however, ozonation does not

16

remove the compounds from water. Instead, ozonation creates degradation products that pose

unknown risks to the consumer (Westerhoff et al., 2005; Dodd et al., 2009).

Biofiltration has also been shown to remove these compounds from water. Reungoat et

al. (2011) looked at biofiltration in a wastewater treatment facility with quantifiable levels of 37

different EDCs and PPCPs in the filter influent. This study found that sand based biofiltration

was able to remove up to 85% of individual compounds, but the average removal was typically

much lower (<20%). GAC based biofiltration was found to be much more effective with average

removal of the 37 compounds above 90%. However, it is unclear whether these improved

removals are a factor of GAC adsorption or biodegradation by well adapted micro-organisms.

Research by Halle (2009a) showed anthracite and sand based biofiltration to achieve near 100%

removal of biodegradable PhACs and EDCs from Grand River water with low adsorption to the

media. These results indicate that biological filtration is a viable option for the removal of these

compounds.

2.6.2 Artificial Sweeteners

Artificial sweeteners are anthropogenic chemicals used to sweeten foods and beverages

(Scheurer et al., 2010). Four artificial sweeteners have been approved for consumption in Canada

including: acesulfame-potassium (acesulfame-k), aspartame, neotame, and sucralose (Health

Canada, 2008), and the United States also allows the use of Saccharin (FDA, 2010). In the

European Union neohesperidin dihydrochalcone (NHDC) and cyclamate have been approved for

use in foodstuffs (EU 1994, 2003).

Although these compounds are widely consumed there is growing concern because the

long-term health impacts are presently unknown (Mawhinney et al., 2011). They are also poorly

removed by traditional wastewater treatment facilities which allows for entry into the natural

environment and wastewater impacted source waters (Torres et al., 2011). Artificial sweeteners

have been detected in water in Canada (Van Stempoort et al., 2011), the United States

(Mawhinney et al., 2011), and 27 European countries (Loos et al., 2009). Of the seven possible

sweeteners, four have been detected in the environment at concentrations in the μg/L range:

acesulfame-k, cyclamate, saccharin, and sucralose (Lange et al., 2012). Since these compounds

are primarily introduced to the environment through wastewater effluent discharge, many studies

have looked into their viability as indicator compounds for wastewater contamination of source

17

waters (Oppenheimer et al., 2011; Mawhinney et al., 2011; Robertson et al., 2013).

Oppenheimer et al. (2011) found sucralose to be an excellent indicator compound in surface

waters impacted by wastewater because it is only found in waters with known wastewater

discharges and it is consistently present in wastewater discharges. Robertson et al. (2013) used

acesulfame-k as an indicator for septic system impacts on groundwater because it is persistent in

the environment and found at concentrations 1000 times greater in wastewater plumes than

background concentrations.

Since these compounds are impacting source waters, drinking water treatment options for

the removal of artificial sweeteners must be explored. Scheurer et al. (2010) found that saccharin

and cyclamate are both completely removed by biologically active treatment units such as

riverbank filtration. Acesulfame-k and sucralose are not easily biodegradable allowing them to

be effective indicator compounds (Scheurer et al., 2010). Rapid biological filtration was not

tested in this study and it would be beneficial to confirm the biodegradability of saccharin and

cyclamate and test the performance with respect to the other compounds. Mawhinney et al.

(2011) examined sucralose removal across 19 drinking water treatment plants throughout the

United States. Sucralose concentrations were reduced by 12% on average, but systems with GAC

and sand biofiltration saw removals of 50 – 58%; indicating a possible removal mechanism.

However, since GAC was used at those locations it is important to distinguish between

biological removal and adsorption since GAC has been shown to be a viable sucralose treatment

option (Scheurer et al., 2010).

2.7 Knowledge Gaps

Although biofiltration has been extensively tested for the removal or reduction of many

compounds, there is limited data available regarding engineered biofilters that are enhanced with

the addition of nutrients. Results reported by Lauderdale et al. (2012) and Azzeh et al. (2014)

provide a background for the theory and implementation of engineered biofiltration; however

their studies did not examine the longer-term impacts of nutrient enhancement, or potential

benefits with respect to the removal of emerging contaminants.

Lauderdale et al. (2012) reported a 75% increase in DOC removal when using engineered

biofilters, and Azzeh et al. (2014) found reductions in the formation potential of THMs (19%),

HAAs (13%), and AOX (11%). However, these were shorter-term studies with biofilter

18

enhancements occurring for a few days or weeks before examining a different enhancement

strategy. These studies also failed to examine the formation of halogenated furanones, or

quantify the reduction of genotoxicity that may occur by implementing biofiltration.

The removal of anthropogenic compounds from drinking water by engineered

biofiltration should also be examined. Many studies have used wastewater treatment facilities

(Reungoat et al., 2011; Nakada et al., 2007; Farré et al., 2011), but limited literature exists

exploring biofiltration of drinking water for the removal of these compounds (Halle, 2009a),

especially at pilot-scale. It is predicted that this knowledge gap exists because the concentration

of anthropogenic compounds in drinking water sources is typically orders of magnitude lower

than in wastewater (Metcalfe et al., 2003; Servos et al., 2007). Additionally, the results of many

of the studies are confounded by the use of adsorbent filter materials (Reungoat et al., 2011;

Farré et al., 2011) or ozonation of the filter influent (Nakada et al., 2007; Reungoat et al., 2011;

Lee et al., 2012). A representative group of PPCPs and EDCs should be selected and monitored

in an engineered biofiltration system to determine the impact of biofiltration on the removal of

these compounds without ozonation or adsorption of the analytes of interest.

Finally, the removal of artificial sweeteners by engineered biofiltration should be

examined to determine whether these compounds are recalcitrant to biological degradation

(Mawhinney et al., 2011; Scheurer et al., 2010). Previous studies have reported that biological

processes such as river bank filtration or groundwater recharge did not remove acesulfame or

sucralose (Scheurer et al., 2010), and increased levels of sucralose were reported from biological

filters in full scale treatment plants in the United States. However, no study to this point has

looked at engineered biofiltration as a treatment method, and until the treatment efficiency of this

process is isolated and tested, this class of compounds should not be considered to be entirely

recalcitrant to treatment.

19

3. Materials and Methods

3.1 Experimental Protocol

3.1.1 Pilot-Scale Biofilters

Biofiltration pilot plants were located at the Peterborough Water Treatment Plant

(PWTP) which treats Otonabee River water in Peterborough, Ontario, and the R.C. Harris Water

Treatment Plant which treats Lake Ontario water in Toronto, Ontario. A summary of pilot plant

influent water quality is provided in Table 3-1.

Table 3-1: Summary of Pilot Plant Influent Water Quality

Raw Water Source Otonabee River Lake Ontario

Temperature (⁰C) 0 – 28 0 – 15

pH 7.3 – 8.6 7.7 – 8.2

Turbidity (NTU) 0.3 – 2.4 0.2 – 1.0

DOC (mg/L) 4 - 6 1.5 – 2.5

Raw water at the Otonabee River pilot plant was treated by seven parallel filter trains

(Figure 3-1). Of the seven filters, six were operated biologically by backwashing them with their

unchlorinated effluent to prevent enhancement chemicals from transferring between filters. All

of the biofilters were operated with an empty bed contact time (EBCT) of 10 minutes while the

conventional filter was operated at 15 minutes to match full-scale operation. Five biofilters and

the conventional filter contained 50 cm of anthracite over 50 cm of sand. The sixth biofilter

contained 50 cm of exhausted GAC (Filtrasorb300, >8 years of service), obtained from another

drinking water treatment facility in southern Ontario, over 50 cm of sand. Raw water was

prechlorinated to achieve a free chlorine residual of approximately 0.2 mg/L when water

temperatures were above 12°C. The chlorine residual was quenched using sodium thiosulphate at

a molar ratio of 3:1 (sodium thiosulphate:free chlorine) prior to entering the constant head tank.

20

Raw Water

Rapid

Mix

Tapered

Flocculation

Parallel

Plate

Settlers

GAC

Media

EBCT

10 min

Mature

Control

EBCT:

10 min

0.2 mg/L

Alum

EBCT:

10 min

0.2 mg/L

H2O2

EBCT:

10 min

New

Control

EBCT:

10 min

0.5 mg/L

P & N

EBCT

10 min

Figure 3-1: Schematic of the Otonabee River Pilot Plant

Two of the biofilters were operated passively, without chemical enhancement, and served

as experimental controls. One of the filters without chemical enhancement was in operation for

four months longer than the other, and was referred to as the “mature control.” The other filter

without pre-treatment is designated the “new control.” In addition to the passively operated

filters, biofilters were enhanced with nutrients (phosphorus and nitrogen), hydrogen peroxide,

and in-line alum. Based on previous studies at the Otonabee River pilot plant (Azzeh et al.,

2014), phosphorus (as phosphoric acid) and nitrogen (as ammonium chloride) were added to one

of the filters to achieve an influent concentration of 0.5 mg/L. Based on the source water

chemistry, this nutrient dose ensures that carbon limiting conditions exist, and the biological

conditions are suitable for carbon uptake. Hydrogen peroxide was added to another of the

enhanced biofilters to limit the formation of extrapolymeric substances (EPS), and reduce the

formation of headloss. Hydrogen peroxide was dosed at 0.2 mg/L to minimize headloss

formation, but not increase disinfection by-product (DBP) precursor concentrations as reported

by Azzeh et al. (2014). Another biofilter was enhanced with 0.2 mg/L of alum to improve

biopolymer removal for ultrafilter pre-treatment, but not increase headloss in the filter.

21

The conventional treatment train consisted of alum coagulation dosed at approximately

40 mg/L to match full-scale treatment, tapered flocculation and parallel plate sedimentation. The

conventional pilot plant was compared to the full-scale plant effluent in terms of pH, turbidity

and dissolved organic carbon (DOC) concentration to ensure that the two systems were operating

similarly. All of the biofilters were backwashed with their own unchlorinated effluent three times

per week, or when they were unable to achieve the desired flow rate (i.e. critical headloss). The

conventional filter was backwashed using chlorinated water from the full-scale plant to limit the

growth of microorganisms on the filter media.

The Lake Ontario pilot plant consisted of seven parallel filter trains preceded by

ozonation (Figure 3-2). Lake Ontario water was preozonated (dose: 1 mg/L, contact time: 8 min,

O3:DOC: 0.5) prior to filtration. All of the filters were operated in a biological manner. Three

filters were setup with the same configuration as the full-scale plant it was mimicking: 150 cm of

granular activated carbon (GAC) or anthracite over 15 cm of sand and gravel support media. The

other four filters contained 50 cm of GAC over 50 cm of sand. The pilot plant was operated to

examine the impact of hydrogen peroxide addition, nutrient addition, varying doses of PACl pre-

treatment, and different EBCTs.

The four filters containing 50 cm of GAC over 50 cm of sand operated with an EBCT of

16 minutes. These four filters included the control, nutrient enhancements, hydrogen peroxide

addition and in-line coagulant addition. The experimental control filter was operated with a 16

minute EBCT and without chemical enhancements. Similar to the Otonabee River pilot plant, the

filters were spiked with 0.5 mg/L of phosphorus and nitrogen; 0.2 mg/L of hydrogen peroxide;

and 0.2 mg/L PACl.

The three filters containing 150 cm of media over 15 cm of sand and gravel received

influent water dosed with 0.8 mg/L PACl to match full-scale operation. One of the GAC filters

was operated with a 16 minute EBCT while the other was operated at 26 minutes. The anthracite

filter was also operated with a 26 minute EBCT. An additional set of three filters were operated

in the same way, but controlled to achieve objectives determined by the Toronto Water staff.

These three filters were monitored, but not included in the study results presented in this

document. All of the filters were backwashed with unchlorinated effluent once a week or when

flow could not be maintained.

22

GAC

EBCT:

16 min

ANTH.

EBCT:

26 min

GAC

EBCT:

26 min

Control

EBCT:

16 min

Nutrient

Addition

0.5 mg/L

P & N

16 min

Peroxide

Addition

0.2 mg/L

16 min

In-line

PACL

0.2 mg/L

16 min

Figure 3-2: Schematic of the Lake Ontario Pilot Plant

3.1.2 Spiking Procedure

Two different classes of compounds were spiked into the Otonabee River and Lake

Ontario pilot plants: filter enhancements, and experimental analytes. The filter enhancements

included hydrogen peroxide, phosphoric acid, ammonium chloride, and coagulants.

Experimental analytes included PhACs, EDCs and artificial sweeteners.

To determine the dose of a particular filter enhancement, the influent flow rate to the

filter was measured manually by collecting water for a given period of time. After an accurate

flow rate was determined, the quantity of the compound required for the spiking solution was

calculated based on the dose of the particular enhancement. The highly concentrated stock

solutions were then diluted into 20 L of distilled water, and pumped into the respective filter at a

flow rate of 1 mL/min using a variable speed, digital drive Masterflex® peristaltic pump. The

diluted solutions were pumped into a graduated cylinder for a minimum of five minutes to ensure

that the dose was being applied correctly.

23

The experimental analytes were spiked into the raw water constant head tank at a flow

rate of 1 mL/hr using a Masterflex® C/L peristaltic tubing pump. Influent flow rates were

determined and based on the average flow. The correct mass of neat compounds was added to

240 mL of solvent (10 day spiking volume). After the pump was setup, it pumped the solutions

into a 10 mL graduated cylinder for at least five hours to confirm that the pump was dosing

accurately at a rate of 1 mL/hr.

3.1.3 Selection of Pharmaceuticals and Artificial Sweeteners

The pharmaceutically active compounds (PhAC) used for this experiment were chosen

based on two different criteria: occurrence in natural water bodies and diverse physical and

chemical properties. Based on these criteria the compounds chosen by Wray (2014) were

selected. Although many artificial sweeteners have been approved for use and can be found in

Canadian surface waters, only two have been shown to be recalcitrant to biological treatment.

These compounds, acesulfame-K and sucralose, were spiked along with the pharmaceuticals, and

their chemical properties are presented in Table 3-2.

3.1.4 Sampling Schedule

Sampling at each pilot plant was completed over a two week period. Samples for

dissolved organic carbon (DOC), UV254, pH, turbidity, disinfection by-product (DBP)

formation potential (FP), halogenated furanones and genotoxicity were collected during the first

week and DOC, UV254, pH, turbidity, pharmaceuticals, and artificial sweetener samples were

collected seven days after. Five sampling events were completed at the Otonabee River pilot

plant, but only three sets of samples were collected at the Lake Ontario pilot plant due to

operational issues. Samples were collected between March and September to incorporate a range

of water temperatures and influent conditions. The beginning of the sampling events is presented

in Table 3-3, and the second week of sampling occurred seven days after the date listed.

24

Table 3-2: Selected Pharmaceutically Active Compounds and Artificial Sweeteners

Compound Use CAS MW

(g/mol)

Water

Solubility

(g/L)

Log

Kow

pKa

Acetaminophen Analgesic 103-90-2 151.17 15 0.46 9.38

Bisphenol A Plasticizer 80-05-7 228.29 0.071 3.32 10.3

Carbamazepine Antiepileptic 298-46-4 236.28 0.022 2.45 13.9

Clofibric Acid Metabolite of lipid

regulator

882-09-7 214.65 100 2.57 3.37

Diclofenac Analgesic 15307-86-5 296.16 2.3 4.51 4.15

17β Estradiol Reproductive

Hormone

50-28-2 272.39 0.0036 4.01 10.4

Estriol Reproductive

Hormone

50-27-1 288.39 0.029 2.45 10.4

Estrone Reproductive

Hormone

53-16-7 270.37 0.147 3.13 10.4

Gemfibrozil Lipid Regulator 25812-30-0 250.34 11 4.77 4.42

Ketoprofen Analgesic and anti-

inflammatory

22071-15-4 254.29 5.8 3.12 4.45

Naproxen Anti-inflammatory 22204-53-1 230.26 0.115 3.18 4.15

Pentoxifylline Vasodilator 6493-05-6 278.31 9.2 0.29 0.28

Acesulfame-K Artificial sweetener 55589-62-3 201.24 270 -1.0 2

Sucralose Artificial sweetener 56038-13-2 397.64 283 -2.67 12.52

Table 3-3: Sampling Schedule at the Otonabee River and Lake Ontario Pilot Plants

Otonabee River Sampling Events Lake Ontario Sampling Events

March 4, 2014 April 8, 2014

May 20, 2014 July 7, 2014

June 23, 2014 August 11, 2014

July 28, 2014

September 22, 2014

25

3.2 Analytical Methods

3.2.1 Adenosine Triphosphate (ATP) Analysis

Adenosine triphosphate (ATP) analysis was completed using a Luminultra analysis kit

(DSA-100C). Media samples were obtained from the top of the biofilter and analyzed following

the manufacturer’s instructions provided in Appendix Table 8-1.

3.2.2 EPS Analysis

Extracellular polymeric substances (EPS) analysis was completed on filter media

throughout the study. Protein and polysaccharides were analyzed at the University of Toronto

laboratory (Toronto, ON) using a method adapted from Papineau et al. (2013) and DuBois et al.

(1956), respectively. The samples were extracted using a Tris-EDTA extraction method as

described by Liu and Fang (2002). A fresh calibration curve was prepared for each analysis

incorporating concentrations of 0, 3.125, 6.25, 12.5, 25, 50, and 100 mg/L of glucose and Bovine

Serum Albumin (BSA) for polysaccharides and proteins, respectively. The reagents used for EPS

analysis are presented in Table 3-4, and the method outlines are presented in Appendix Table

8-2. Sample calibration curves for proteins (Figure 3-3) and polysaccharides (Figure 3-4) are

presented below.

Table 3-4: EPS Reagents

Reagent Details

Ethylenediaminetetraacetic acid (EDTA) disodium salt

dehydrate

Sigma Aldrich, ACS Grade

Tris (Hydrohymethyl) Aminomethane, BioUltrapure BioShop, >99.9%

Bovine Serum Albumin (BSA) solution BioLabs, 10 mg/mL

D-glucose Anhydrous, reagent grade

Pierce™ BCA Reagent A + B Thermo Scientific

Phenol Alfa Aesar, ACS grade

Sulphuric Acid Anachemia, 98+%

26

Figure 3-3: Sample Calibration Curve - Protein Analysis (March 2014)

Figure 3-4: Sample Calibration Curve - Polysaccharides Analysis (March 2014)

3.2.3 Dissolved Organic Carbon

DOC was measured based on standard method 5310 D using an O-I Corporation Model

1010 Analytical TOC Analyzer with a Model 1051 Vial Multi-Sampler (APHA, 2005). Samples

were filtered into 40 mL amber vials through a 0.45 μm glass fiber filter, and acidified to pH ≤ 2

using concentrated sulfuric acid if samples were not analyzed immediately after preparation. The

vials were sealed with Teflon®-lined septum screw caps and stored at 4⁰C. All samples were

prepared on the day of collection and tested within 7 days. A summary of the instrument

conditions are presented in Table 3-5. DOC concentrations in water were quantified using

anhydrous potassium hydrogen phthalate (KHP) in Milli-Q® water as a calibration solution. The

calibration solutions were prepared at a concentration of 10 mg/L and diluted by the instrument

y = 0.0044x - 0.0023 R² = 0.9984

0.00

0.10

0.20

0.30

0.40

0.50

0 20 40 60 80 100 120

OD

56

2

BSA concentration in mg/L

y = 0.0121x + 0.0028 R² = 0.9979

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

0 20 40 60 80 100 120

OD

49

2

Glucose concentration in mg/L

27

to concentrations of 0, 0.625, 1.25, 2.5 and 5 mg/L for a 6 point calibration curve. A 10 mg/L

calibration sample was prepared, diluted and analyzed before each sample set. Check standards

(C = 2.5 mg/L) were tested after every 10 samples, and at the end of every sample set.

Additionally, a minimum of three blank samples were tested after calibration, and before every

check standard sample. The reagent list and a sample calibration curve are presented in Table 3-6

and Figure 3-5. Sample preparation and the method outline details are presented in Appendix

Table 8-3.

Table 3-5: DOC Analyzer Conditions

Parameter Description

Acid volume 200 μL of 5% phosphoric acid

Oxidant volume 1000 μL of 100 g/L sodium persulphate

Sample volume 15 mL

Rinses per sample 1

Volume per rinse 15 mL

Replicates per sample 3

Reaction time (min:sec) TIC 2:00; TOC 2:30

Detection time (min:sec) TIC 2:40; TOC 2:00

Purge gas Nitrogen

Loop size 5 mL

Table 3-6: DOC Analysis Reagents

Reagent Supplier and purity

Milli-Q® water Prepared in the laboratory

Sulphuric acid, H2SO4 VWR International, 98%+

Sodium persulphate, Na2(SO4) Sigma Aldrich, 98%+, anhydrous

Potassium hydrogen phthalate (KHP), C8H5KO4 Sigma Aldrich, 98%+

Phosphoric acid, H3PO4 Caledon, >85%

Nitrogen gas, N2 Praxair, Ultra high purity (UHP)

28

Figure 3-5: Sample Calibration Curve - DOC (March 2014)

3.2.4 Liquid Chromatography-Organic Carbon Detection (LC-OCD)

Liquid chromatography-organic carbon detection (LC-OCD) characterized the organic

compounds found in raw and treated water samples. LC-OCD identifies five different fractions

of DOC including: biopolymers, humic substances, building blocks, low molecular weight

neutrals and low molecular weight acids.

Water samples were filtered using a 0.45 µm glass fiber filter (Gelman Supor, Gelman

Sciences, Ann Arbor, MI) and collected in 40 mL amber glass vials with Teflon® lined silicon

septa and screw caps (VWR International, Mississauga, ON). The samples were stored at 4°C

until being shipped to the University of Waterloo (Waterloo, ON) for analysis. Based on a

method by Huber et al., (2011), the samples are first separated chromatographically using a weak

cation exchange column (250 mm x 20 mm, Tosoh, Japan). The mobile phase used was a

phosphate buffer exposed to UV irradiation in an annular UV reactor, delivered at a flow rate of

1.1 mL/min to an autosampler (MLE, Dresden, Germany, 1 mL injection volume).

Chromatographic separation was followed by UV254 detection (UVD), and organic carbon

detection (OCD). The solution was acidified to form carbonic acid from carbonates at the OCD

inlet. Total DOC for each sample was measured using a column bypass. Calibration was

completed using a potassium hydrogen phthalate (KHP) solution. Data processing and

y = 4131.1x + 991.56 R² = 0.9999

0

5000

10000

15000

20000

25000

30000

35000

40000

45000

0 2 4 6 8 10 12

Pe

ak A

rea

Co

un

t

DOC (mg/L)

29

acquisition was completed using a custom ChromCALC software package (Karlsruhe,

Germany).

3.2.5 UV254

UV254 is a measure of ultraviolet absorbance at wavelengths of 254 nm, and can be used

to approximate the concentration of organic materials in a water sample (Kitis et al., 2002).

Samples were placed in 1 cm quartz cell (Hewlett Packard, Mississagua) and measured using a

CE 3055 Single Beam Cecil UV/Visible Spectrophotometer (Cambridge, England). The device

was zeroed with Milli-Q® water. The cells were rinsed with Milli-Q® water twice between

sampling and rinsed again with the sample water before analysis to minimize contamination.

3.2.6 Disinfection By-Product Formation Test

To determine the correct disinfectant dose, the chlorine demand was determined by

adding 4 and 6 mg/L of chlorine to 500 mL of sample. The samples were disinfected in a

chlorine-demand-free amber bottle, and incubated at room temperature (20°C ± 1.0°C) for 24

hours. The difference between the initial disinfectant concentration and the final concentration

was determined to be the chlorine demand.

After the chlorine demand was determined, new samples were collected in chlorine-

demand-free amber jars. The samples were chlorinated to have a residual concentration of 1±0.4

mg/L after 24 hours. After 24 hours, the residual concentration was measured, and if it was

correct, the disinfectant in each sample was quenched with 20 mg of L-ascorbic acid. The bottles

were then acidified to pH 2 with H2SO4 and stored in the dark at 4°C until extraction.

3.2.7 Trihalomethanes

Trihalomethane analysis was conducted to quantify the concentration of four compounds:

chloroform (trichloromethane; TCM), bromodichloromethane (BDCM), dibromochloromethane

(DBCM), and bromoform (tribromomethane; TBM). A liquid-liquid extraction with gas

chromatography was performed based on Standard Method 6232 B (APHA, 2005). The analysis

was conducted at the University of Toronto drinking water research laboratory (Toronto,

Ontario) using a Hewlett Packard 5890 Series II Plus gas chromatograph (Mississauga, Ontario)

equipped with an electron capture detector (GC-ECD) and a DB 5.625 capillary column (Agilent

30

Technologies Canada Inc., Mississauga, Ontario). A THM stock solution (2000 μg/mL) was used

to create an intermediate solution (20 mg/L). This intermediate was used to generate calibration

standards of 0, 5, 10, 20, 40, 60, 100, 150, and 200 μg/L. The instrument conditions (Table 3-7)

and required reagents (Table 3-8) are presented below. The method outline is presented in

Appendix Table 8-4. A sample calibration curve is presented in Figure 3-6. An example QA/QC

chart for chloroform is presented in Appendix Figure 8-1.

Table 3-7: THM Instrument Conditions

Parameter Description

Injector Temperature 200⁰C

Detector Temperature 300⁰C

Temperature Program 40⁰C for 4.0 min

4⁰C/min temperature ramp to 95⁰C

60⁰C/min temperature ramp to 200⁰C

Carrier Gas Helium

Flow Rate 1.2 mL/min at 35⁰C

Table 3-8: THM Reagent Compounds

Reagent Source

Milli-Q® water Prepared in the laboratory

Concentrated trihalomethane stock for

calibration

Supelco, 2000 μg/mL in methanol

(48140-U)

Sodium sulphate [Na2SO4] Sigma Aldrich, ACS Grade

Methyl-tert-butyl-ether (MTBE) Fluka, >99.8%

Figure 3-6: Sample Chloroform Calibration Curve (April 2014)

y = 355.84x - 2.5139 R² = 0.9958

0

20

40

60

80

100

0 0.05 0.1 0.15 0.2 0.25

Ch

loro

form

(u

g/L)

Ratio Response

31

3.2.8 Haloacetic Acids

The analysis of haloacetic acids (HAAs) involved quantifying the following 9

compounds: monochloroacetic acid (MCAA), monobromoacetic acid (MBAA), dichloroacetic

acid (DCAA), trichloroacetic acid (TCAA), bromochloroacetic acid (BCAA), dibromoacetic acid

(DBAA), bromodichloroacetic acid (BDCAA), dibromochloroacetic acid (DBCAA), and

tribromoacetic acid (TBAA). The analysis was completed using a liquid-liquid extraction and a

gas chromatograph based on Standard Method 6251 B (APHA, 2005). The analyses were

completed at the University of Toronto drinking water research laboratory (Toronto, Ontario)

using a Hewlett Packard 5890 Series II Plus gas chromatograph (Mississauga, Ontario) paired

with an electron capture detector (GC-ECD) and a DB 5.625 capillary column (Agilent

Technologies Canada Inc., Mississauga, Ontario). The instrument conditions (Table 3-9),

required reagents (Table 3-10), and a calibration curve (Figure 3-6) are presented below. An

example quality control chart for monochloroacetic acid is provided in Appendix Figure 8-2.

Table 3-9: HAA Instrument Conditions

Parameter Description

Injector Temperature 200⁰C

Detector Temperature 300⁰C

Temperature Program 35⁰C for 10.0 min

2.5⁰C/min temperature ramp to 65⁰C

10⁰C/min temperature ramp to 85⁰C

20⁰C/min temperature ramp to 205⁰C, hold for 7 minutes

Carrier Gas Helium

Flow Rate 1.2 mL/min at 35⁰C

Table 3-10: HAA Analysis Required Reagents

Reagent Source

Diethyl ether [C2H5OCH2CH2OCH2CH2OH] Sigma Aldrich, 99+%

N-methyl-N-nitroso-p-toluene sulfonamide

(Diazald) [ CH3C6H4SO2N(CH3)NO]

Sigma Aldrich, 99+%

Potassium Hydroxide (KOH) BDH, 85.0+%, ACD Grade

Sulphuric acid [H2SO4] E.M. Science, 98+%

Haloacetic acids concentrated stock EPA 552.2 Acids Calibration Mix in MTBE

Sodium sulphate [Na2SO4] Sigma Aldrich, ACS Grade

Methyl-tert-butyl-ether (MTBE) Sigma Aldrich, >99.8%

32

Figure 3-7: Sample HAA Calibration Curve (April 2014)

3.2.9 Adsorbable Organic Halides (AOX)

Adsorbable organic halides (AOX) were analyzed using a Trace Elemental Instruments

Xplorer Instrument (TE Instruments, Delft, Netherlands). A 50 mg dose of activated carbon (Trace

Elemental Instruments CON100400, Delft, Netherlands) to adsorb organic halogens was added to

100 mL of chlorinated sample, and mixed with an orbital shaker for 1 hour. Mixed samples were

filtered through a quartz frit to recover activated carbon. Combustion followed by colourimetric

titration of the released organic halogens was used to determine AOX in each sample. All samples

were prepared in duplicate with replicate analytical samples.

3.2.10 Halogenated Furanones

Samples for 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (Mutagen X, MX)

and 2,3-dichloromalealdehydic acid (mucochloric acid, MCA) were collected in 1 L amber glass

bottles and chlorinated using the method described in Section 3.2.6. After 24 hours chlorine

residuals were measured and quenched with 20 mg of L-ascorbic acid if the residual was 1.0±0.4

mg/L. The samples were then acidified to pH 2 with H2SO4 and stored in the dark at 4°C until

extraction. A surrogate standard, 2,3-dibromoalealdehydic acid (mucobromic acid, MBA), was

spiked at 100 ng/L into all samples and calibration standards. Analysis was completed using a

Varian 3800 GC paired with a 4000 MS and equipped with a DB-1701 column (30 m x 0.25 mm

y = 575.54x + 0.228 R² = 0.9997

0

10

20

30

40

50

60

70

0 0.02 0.04 0.06 0.08 0.1 0.12

Mo

no

chlo

ro a

ceti

c ac

id (

ug/

L)

Ratio Response

33

x 0.25 µm). Instrument details are provided in Table 3-11. Solid phase extraction (SPE) was

completed using a tandem cartridge setup. A trifunctional C18 (tC18) cartridge (Sep Pak 6 cc,

Waters Corporation, Mississauga, ON) was used for humics removal prior to an Oasis HLB

cartridge (12 cc, Waters Corporation, Mississauga, ON). Using a vacuum manifold the samples

are loaded onto the cartridges, and the cartridges are dried under vacuum. The HLB cartridges

are eluted by gravity with 10 mL of acetone, and dried under nitrogen until approximately 0.5

mL remains. The remaining acetone is then transferred to GC vials, blown to dryness, and

reconstituted with 2% H2SO4 in methanol. The samples are then derivatized for one hour before

adding 0.75 mL of 2% NaHCO3 in Milli-Q® water to neutralize the acid. Extraction with 0.6 mL

hexane was completed twice before being concentrated to 0.1 mL under nitrogen. Detailed

sample preparation and extraction instructions are provided in Appendix Table 8-6 and Table

8-7, respectively. Reagents and reagent preparation information is provided in Table 3-12 and

Table 3-13; and the QA/QC charts are provided in Appendix Figure 8-3 and Figure 8-4.

Table 3-11: Operating Conditions for the Analysis of MX and MCA

Parameter Conditions

System Varian 3800 GC with 4000 MS and CombiPAL autosampler

Column DB-1701 (30m x 0.25mm OD x 0.25m ID, Agilent J & W)

Injection volume 8 L

Injection mode Initial splitless injection

Split on at 0.1 min at 5:1

Split off at 0.8 min

Split on at 6.0 min at 30:1

Injector temperature 50°C hold for 0.8 min

200°C/min to 200°C, hold for 20 min

Oven temperature 40°C, hold for 2 min

20°C/min to 100°C

6°C/min to 170°C

15°C/min to 260°C, hold for 3 min

Carrier gas Helium, constant flow at 1.2mL/min

Transfer line temperature 275°C

Ion source temperature 150 °C

Ionization mode Electron impact (EI)

Scan range 50-400 amu

MS scan mode MS/MS

34

Table 3-12: Reagents Used for MX and MCA Analysis

Reagent Supplier and Description

Acetone Sigma-Aldrich, LC Grade

Hexane Sigma-Aldrich, LC Grade

Sulfuric acid (H2SO4) Fluka, >95% Purity

Methanol (CH3OH) Sigma-Aldrich, LC Grade

Ethyl Acetate Sigma-Aldrich, Chromasolv Plus ®

Sodium bicarbonate Sigma-Aldrich, LC Grade

Table 3-13: Preparation of Reagents for MX and MCA Analysis

Solution Preparation Steps

MX and MCA working

solution

Add 100 µL of intermediate solution (25 mg/L in ethyl acetate) to

9.9 mL of ethyl acetate. Concentration of MX/MCA is 250 µg/L

MBA working solution Add 100 µL of intermediate solution (25 mg/L in ethyl acetate) to

9.9 mL of ethyl acetate. Concentration of MX/MCA is 250 µg/L

2% H2SO4 in methanol Add 2 mL of H2SO4 to a 100 mL volumetric flask and fill to the

line with methanol

2% NaHCO3 Add 2.00 g of NaHCO3 to a 100 mL volumetric flask and fill to

the line with Milli-Q® water

3.2.11 Genotoxicity with the SOS Chromotest

The SOS repair system is activated in Escherichia coli (E. coli) in response to DNA

damage. The SOS Chromotest is a colourimetric assay used to detect the activation of the DNA

repair mechanism due to lesions in genetic material. The SOS Chromotest test kit used in this

study was developed and supplied by Environmental Bio-Detection Products Inc. (EBPI,

Mississauga, ON).

In this test, the β-galactosidase (β-gal) gene (lacZ) is fused to the bacterial sfiA SOS

operon in a genetically engineered strain of E. coli PQ37. This means that the SOS repair system

of PQ37 is engineered such that SOS promoter induces lacZ¸ expression and synthesis of β-gal.

The amount of β-gal formed can be quantified photometrically using a chromogenic substrate to

form a blue colour, and is representative of bacterial genotoxicity (Kocak et al., 2010). 2 L

samples are collected in amber glass bottles and chlorinated as per section 3.2.6. Sample

concentration, extraction and analysis details can be found in Appendix Table 8-8.

35

The specific equations for the determination of β-gal specific activity (Rc), AP reduction

specific activity (R0) and the induction factor (IF) are given below:

(3.1)

(3.2)

(3.3)

A serial, step-wise dilution (six different concentrations for each sample) was used in this

test. The amount of sample in each dilution is quantified using a relative enrichment factor (REF)

defined by Escher & Leusch (2011):

(3.4)

Where the enrichment factor is the concentration achieved during sample extraction and the

dilution factor is the dilution amount in each step of the bioassay.

Using one dilution after an observed IF of 2.0 a slope of the linear portion of IF vs. REF

can be found. For the samples of the known carcinogen, 4-NQO, a known mass in pmol is used

instead of the REF. The slopes of the 4-NQO are corrected with the SOS induction potential

(SOSIP) factor to normalize out the mass unit. The SOSIP value for known carcinogens is

consistent and reported. Finally, the toxic equivalent concentration (TEQ4-NQO) is determined by

comparing the slope of the sample to the known carcinogen as follows:

(3.5)

The TEQ value represents the genotoxicity of each sample relative to the genotoxicity of the

carcinogen 4-NQO. For example, a sample as carcinogenic as 4-NQO would have a TEQ of 1.0.

3.2.12 Pharmaceuticals and Endocrine Disruptors

Pharmaceutically active compounds (PhACs) and endocrine disruptor compounds

(EDCs) (acetaminophen, carbamazepine, clofibric acid, gemfibrozil, ketoprofen, naproxen,

pentoxifylline, 17 β-estradiol, estriol, estrone, diethylstilbestrol, bisphenol A) were analyzed

using a method based on the Ontario Ministry of the Environment (MOE) method EOP-E3454,

36

version 2.0 (MOE, 2008). Analyses were conducted at the University of Toronto using an

Agilent 6460 triple quadrupole mass spectrometer with an Agilent 1200 series autosampler,

pump, and column heater (Agilent Technologies, Mississauga, Ontario). It was equipped with a

Poroshell EC-C18 guard column (5 mm x 2.1 mm x 2.7 µm) and a Poroshell EC-C18 column (50

mm x 2.1 mm x 2.7 µm) (Agilent Technologies, Mississauga, Ontario). Operating conditions are

presented in Table 3-14.

Table 3-14: LC-MS-MS Operating Parameters - PhAC and EDC Analysis

Parameter Conditions

System Agilent 6460 Triple Quad MS with an Agilent 1200 series

autosampler, pump, and column heater.

Column Agilent Poroshell EC-C18 column 50 mm x 2.1 mm x 2.7 um I.D with

a Poroshell EC-C18 5 mm x 2.1 mm x 2.7 um guard cartridge

Injection volume 10 uL (Positive Mode); 30 µL (Negative Mode)

Column temperature 30°C

Mobile phase A: 0.03 % HFBA in LC Grade Water

B: Acetonitrile Positive

Mode

A: 5 mM NH4OAc with NH4OH to pH 7.0

B: Acetonitrile

Negative

Mode

Gradient program 0 - 0.01 min 300 uL/min hold at 92:8 A:B

0.01 – 5.00 min 300 uL/min hold at 65:35 A:B

5.00 – 8.00 min 300 uL/min ramp to 2:98 A:B

8.01 – 11.00 min 500 uL/min hold at 92:8 A:B

11.00 – 14.00 min 300 uL/min hold at 92:8 A:B

Positive

Mode

0.00 min 300 uL/min hold at 80:20 A:B

0.01 – 9.00 min 300 uL/min ramp to 10:90 A:B

9.00 – 13.00 min 500 uL/min hold at 10:90 A:B

13.01 – 16.00 min 500 uL/min hold at 80:20 A:B

16.00 – 18.49 min 300 uL/min hold at 80:20 A:B

Negative

Mode

Total Run Time 14.01 minutes (Positive Mode)

18.50 minutes (Negative Mode)

Samples were collected in 500 mL amber glass bottles with Teflon ™ lined caps that had

not been cleaned with detergent to minimize interference (Systems Plus, Baden, ON). Surrogate

compounds were added to the samples prior to extraction. Samples (400 mL) were extracted with

6 cc Waters Oasis Hydrophilic-Lipophilic Balance (HLB) solid phase extraction (SPE) cartridges

37

(Waters, Mississauga, ON) and eluted with methanol. Methanol extracts were subsampled (1

mL), blown to dryness with nitrogen, and reconstituted with 200 µL of internal standard solution

prior to analysis by LC-MS-MS. Concentrations of analytes were determined by correlation with

an eight point calibration curve. Calibration standards were prepared and extracted in the same

manner as samples. Samples were stored at 4°C in the dark until analysis, extracted within 15

days, and analyzed within 50 days as per the MOE method (MOE, 2008). A detailed description

of the sample preparation and the SPE procedures are presented in Table 8-9 and Table 8-10.

Reagents are presented in Table 3-15, and the preparation steps for extraction reagents are shown

in Table 3-16. Surrogate and internal standard information is given in Table 3-17.

Table 3-15: Reagents Used in PhAC and EDC Analysis

Reagent Supplier and Description

Acetonitrile (CH3CN) Sigma-Aldrich, LC Grade

Ammonium acetate (C2H7NO2) Fluka, LC Grade

Ammonium hydroxide (NH4OH) Fluka, LC Grade

Heptafluorobutyric acid (HFBA) (C4HF7O2) Fluka, Ion Chromatography Grade

Methanol (CH3OH) Sigma-Aldrich, LC Grade

Sodium hydroxide (NaOH) Sigma-Aldrich, 99% Purity

Sulfuric acid (H2SO4) Fluka, >95% Purity

Water Sigma-Aldrich, LC Grade

Table 3-16: Preparation Steps for Reagents Used in PhAC Analysis

Solution Preparation Steps

10 & 250 mM NH4Ac

(ammonium acetate)

Dissolve 0.385 5 or 19.27 g of ammonium acetate powder in 1000

mL of Milli-Q® water

5, 10, 25, 50% NaOH

(sodium hydroxide)

Add 5, 10, 25, or 50 mL of NaOH to a 100 mL volumetric flask and

fill to the line with Milli-Q® water

10% H2SO4 (sulfuric acid) Add 5 mL of H2SO4 to a 50 mL volumetric flask and fill to the line

with Milli-Q® water

5% CH3OH (methanol) Add 25 mL of methanol to a 500 mL volumetric flask and fill to the

line with Milli-Q® water

10 mM NH4Ac

(ammonium acetate)

Dissolve 0.385 g NH4Ac (ammonium acetate) in 1 L of HPLC

grade water.

0.03% C4HF7O2 (HFBA,

Heptafluorobutyric Acid)

Dissolve 300 µL of HFBA in 1 L of HPLC grade water. Filter with

Nalgene 0.2 µm filter unit (Thermo Scientific, Rochester, NY).

LC-MS-MS wash solution Combine equal parts in HPLC grade water and acetonitrile. Filter

with Nalgene 115 mL 0.2 µm filter unit

38

Table 3-17: Surrogate and Internal Standards for PhAC Analysis (Obtained from CDN Isotopes,

Pointe-Claire, QC)

Compound

Surrogates

D4-Acetaminophen

D10-Carbamazepine

D4-Clofibric Acid

D4-Diclofenac

D6-Gemfibrozil

D3-Naproxen

Internal Standards

D16-Bisphenol A (negative mode) 13

C6-Sulfamethazine (positive mode)

3.2.13 Artificial Sweeteners

Artificial sweeteners (acesulfame-K and sucralose) were analyzed using a method

developed at Trent University in Peterborough, Ontario. Analyses were conducted at the

University of Toronto using an Agilent 6460 triple quadrupole mass spectrometer with an

Agilent 1200 series autosampler, pump, and column heater (Agilent Technologies, Mississauga,

Ontario). It was equipped with a Poroshell EC-C18 guard column ( 5 mm x 2.1 mm x 2.7 µm)

and a Poroshell EC-C18 column (50 mm x 2.1 mm x 2.7 µm) (Agilent Technologies,

Mississauaga, Ontario). Operating conditions are presented in Table 3-18.

Samples were collected in 500 mL amber glass bottles with Teflon ™ lined caps that had

not been cleaned with detergent to minimize interference (Systems Plus, Baden, ON). Surrogate

compounds were added to the samples prior to extraction. Samples (200 mL) were extracted with

6 cc Oasis MCX solid phase extraction (SPE) cartridges (Waters, Mississauga, ON) and eluted

with a 5% solution of ammonium hydroxide in methanol. Ammonium hydroxide solutions were

evaporated until near dryness with nitrogen and then reconstituted with a solution of 0.1% acetic

acid in HPLC grade water. Concentrations of analytes were determined by correlation with an

eight point calibration curve. Calibration standards were prepared and extracted in the same

manner as samples. Reagent details, internal standard specifications and reagent preparation

steps are shown in Table 3-19, Table 3-20, and Table 3-21, respectively. Details for sample

39

preparation (Table 8-11) and extraction (Table 8-12) are presented in the Appendix. Quality

control charts for acesulfame-K (Figure 8-6) and sucralose (Figure 8-7) are also available in the

Appendix.

Table 3-18: Operating Conditions for Artificial Sweetener Analysis

Parameter Conditions

System Agilent 6460 Triple Quad MS with an Agilent 1200 series

autosampler, pump, and column heater.

Column Agilent Poroshell EC-C18 column 50 mm x 2.1 mm x 2.7 um I.D

with a Poroshell EC-C18 5 mm x 2.1 mm x 2.7 um guard

cartridge

Injection volume 20 uL

Column temperature 30°C

Mobile phase A: 0.1 % Acetic Acid in LC Grade Water

B: 0.1 % Acetic Acid in LC Grade Acetonitrile

Gradient programme 0-1.5 min 300 uL/min hold at 93:7 A:B

1.5-6 min 300 uL/min ramp to 2:98 A:B

6-8 min 300 uL/min hold at 2:98 A:B

8-9 min 300 uL/min hold at 2:98 A:B

9-11 min 800 uL/min hold at 93:7 A:B

11-15 min 300 uL/min hold at 93:7 A:B

Table 3-19: Reagents Used in Artificial Sweetener Analysis

Reagent Supplier and Description

Acetonitrile Sigma-Aldrich, LC Grade

Methanol Sigma-Aldrich, LC Grade

Water Sigma-Aldrich, LC Grade

Acetic Acid Sigma-Aldrich, LC Grade

Sulphuric Acid Fluka, >95% Purity

Ammonium Hydroxide Fluka, >25%, LC Grade

Table 3-20: Internal Standards Used in Artificial Sweetener Analysis

Compound

Internal Standards

D4-Acesulfame-K

D6-Sucralose

40

Table 3-21: Preparation of Reagents for Artificial Sweetener Analysis

Solution Preparation Steps

pH 1.5 Water Add drops of sulphuric acid (~50) to 500 mL

of LC grade water until pH reaches 1.5.

5% NH4OH in methanol (ammonium

hydroxide)

Add 20 mL of NaOH to 380 mL of LC grade

methanol

0.1% Acetic acid in acetonitrile (Organic

mobile phase)

Add 0.5 mL of acetic acid to a 500 mL

volumetric flask and fill to the line with

acetonitrile

0.1% Acetic acid in LC grade water (Aqueous

mobile phase)

Add 0.5 mL of acetic acid to a 500 mL

volumetric flask and fill to the line with LC

grade water

Reconstitution solution Same as the aqueous mobile phase, and can be

sampled from the prepared solution

3.3 Statistical Analysis

A Student’s paired t-test (equation 3.6) was used to compare the effectiveness of different

treatments to the untreated influent water, and to compare different treatment to each other.

(3.6)

Where t is the paired t-test value, ∑D is the sum of the difference between data pairs, (∑D2) is

the sum of the square difference between pairs, (∑D)2 is the square of the sum of the differences

between pairs, and n is the number of observations (pairs).

A two-tailed test (α/2) was used when the direction (positive or negative) of change

between the control and a treatment or enhancement was unknown and important (e.g.

differences in ATP measurements between the control and a treatment). A one-tailed test was

used (α) when testing to determine if a treatment was better than no treatment, or when an

enhancement was better than the control (e.g. removal of DOC).

41

4. Engineered Biofiltration for the Removal of DBP Precursors,

and Genotoxicity

4.1 Introduction

Natural organic matter (NOM) represents a mixture of chemicals found in water systems.

Although NOM itself is not regarded as a health hazard, when combined with chlorine used for

disinfection during drinking water treatment a wide range of disinfection by-products (DBPs) are

formed. These DBPs include commonly regulated compounds such as trihalomethanes (THMs),

and haloacetic acids (HAAs), as well as less common and unregulated compounds of health

concern such as the halogenated furanones: 3-chloro-4(dichloromethyl)-2(5H)-furanone (MX)

and mucochloric acid (MCA) (Krasner et al., 2006). The US EPA has set regulatory limits for

THMs (80 μg/L) HAAs (60 μg/L) while Health Canada has set guidelines for the concentration

of total THMs and the five most common HAAs at 100 μg/L and 80 μg/L, respectively (USEPA,

2012; Health Canada, 2008 & 2009).When present in drinking water these compounds may

create a genotoxic or carcinogenic response (Richardson et al., 2007). As such, it is important to

minimize NOM to limit DBP formation. Biologically active filtration (biofiltration) represents an

important water treatment option because of its ability to reduce biodegradable organic matter

(LeChevallier et al., 1992), and disinfection by-product precursors (Onstad et al., 2008), while

achieving acceptable filtration performance with respect to particle removal (Emelko et al.,

2006). However, until recently biofiltration has been operated in a passive manner without

anthropogenic influences.

Lauderdale et al. (2012) introduced the concept of engineered biofiltration whereby

phosphorus and nitrogen were added to increase biofilm growth and activity (increase reduction

of DOC by 0.3 mg/L), while hydrogen peroxide was introduced to limit headloss resulting from

extrapolymeric substance (EPS) formation by up to 66% without impacting DOC removal.

Azzeh et al. (2014) furthered this type of research by examining the removal of specific organic

fractions and DBP precursors. However, previous studies did not address longer-term impacts of

a consistent enhancement strategy, the removal of emerging DBPs such as MX and MCA, or the

reduction in genotoxic response.

This study compared conventional water treatment (coagulation, flocculation, settling,

dual media non-biological filtration) to passive and engineered biofiltration at pilot-scale for the

42

removal of NOM (measured as DOC and LC-OCD), DBP precursors (including THMs, HAAs,

adsorbable organic halides (AOX), MX, MCA), and genotoxicity (SOS Chromotest) in trials

employing a variety of continuous chemical enhancements (nutrient addition, hydrogen peroxide

addition, and in-line coagulant addition).

4.2 Materials and Methods

4.2.1 Source Waters

Pilot-scale biofiltration studies were conducted using water from the Otonabee River in

Peterborough, Ontario; and Lake Ontario water in Toronto, Ontario, Canada. Typical pilot plant

influent water quality is shown in Table 3-1.

4.2.2 Pilot Plant Configurations

The Otonabee River pilot-scale treatment system consisted of seven parallel trains

(Figure 3-1). All biofilters were operated with an empty bed contact time (EBCT) of 10 minutes,

while the conventional filter was operated at 15 minutes to match full-scale operation. Five of the

biofilters as well as the conventional filter contained 50 cm of anthracite (effective size d10 =

0.85 mm, uniformity coefficient UC = 1.8) over 50 cm of sand (d10 = 0.5 mm, UC = 1.8). The

sixth biofilter contained 50 cm of exhausted GAC (Filtrasorb 300, >8 years of service), obtained

from another drinking water treatment plant in southern Ontario, over 50 cm of sand.

Two of the biological filters and the conventional filter were installed and in operation for

four months before the starting of the others. As such, the original filter without pre-treatment

served as the “mature control”, while a new filter without pre-treatment was designated as the

“new control”. Based on previous work by Azzeh et al. (2014), phosphorus (as phosphoric acid),

and nitrogen (as ammonium chloride) were each added to one of the biological filters at a

concentration of 0.5 mg/L to test for biological impacts on DBP precursors when nutrient

limiting conditions were not present. Hydrogen peroxide was added to a different filter at 0.2

mg/L to determine if its effect on reducing headloss (Lauderdale et al., 2012) also impacted DBP

precursors. In-line alum was added to another filter at 0.2 mg/L as Al3+

to maximize organic

removal without significantly increasing headloss (Azzeh et al., 2014). The conventional filter

43

was preceded by alum coagulation (~40 mg/L), tapered flocculation, and parallel plate

sedimentation to match full-scale conditions. All biofilters were backwashed with their own

unchlorinated effluent three times per week, whereas the conventional filter was backwashed

using chlorinated water from the full-scale plant.

The Lake Ontario pilot-scale treatment system consisted of seven filters preceded by

ozonation (dose = 1 mg/L, contact time = 8 min, O3:DOC = 1:2) as shown in Figure 3-2. Three

filters received water coagulated with 0.8 mg/L polyaluminum hydroxychloride (PACl). Two of

the these filters were comprised of 150 cm of GAC over 15 cm of sand and gravel support

media, and operated at two different EBCTs (16 and 26 min EBCT). The third filter contained

150 cm of anthracite over 15 cm of sand and gravel support media (26 min EBCT). The four

remaining filters contained 50 cm of GAC over 50 cm of sand, and were operated at a 16 min

EBCT. One filter was operated as a control (no chemical additives), while the other three

received 0.2 mg/L hydrogen peroxide, 0.2 mg/L in-line PACl, and 0.5 mg/L of phosphorus and

nitrogen, respectively, to allow for comparison with the corresponding biofilters at the Otonabee

River pilot plant. All filters were backwashed with their own unchlorinated effluent at terminal

headloss, maximum once a week.

4.2.3 Analytical Methods

Biological activity on the filter media was characterized by adenosine triphosphate (ATP)

while proteins and polysaccharides were quantified to determine the concentration of

extracellular polymeric substances (EPS). ATP concentrations were assayed with a LuminUltra

Deposit Surface Analysis kit (DSA-100C, Fredricton, NB) following the manufacturer’s

instructions as presented in Section 3.2.1. EPS was extracted in a Tris-EDTA buffer (10mM

Tris, 10mM EDTA), where 10 mL was added to 2 g of filter media, shaken for 4h at 4⁰C and

centrifuged (Liu and Fang, 2002). The supernatant was then extracted and filtered through a 0.45

μm filter and stored at -20⁰C. Proteins were quantified with the Pierce BCA (Thermo Scientific),

and polysaccharides were quantified by the methods described in DuBois et al. (1956). A CE

3055 Single Beam Cecil UV/Visible Spectrophotometer (Cambridge, England) was used for

protein quantification, while a Hach Oyssey DR/2500 Scanning Spectrophotometer

44

(Mississauga, ON) was used for polysaccharide analysis. Complete details are presented in

Section 3.2.2.

DOC was measured using a persulfate wet oxidation method as described in Section

3.2.3. Recent advancements have allowed for the further characterization of organic compounds

using liquid chromatography – organic carbon detection (LC-OCD). This technology classifies

organic compounds into five fractions including: biopolymers (proteins and polysaccharides),

humic substances, building blocks, low molecular weight (LMW) acids, and LMW neutrals

(Huber et al., 2011). LC-OCD analyses were conducted at the University of Waterloo (Waterloo,

ON) according to a method described by Huber et al. (2011) and presented in Section 3.2.4.

Prior to DBP analyses, samples were chlorinated to achieve a residual of 1.0±0.4 mg/L

after 24±2 hours at 20⁰C, without pH correction (APHA, 2005). Free chlorine residuals were

measured as per Standard Method 4500-Cl G (APHA, 2005) and quenched with L-ascorbic acid.

THMs (chloroform, bromodichloromethane, dibromochloromethane, and bromoform) and nine

HAAs (monochloroacetic acid, monobromoacetic acid, dichloroacetic acid, trichloroacetic acid,

bromochloroacetic acid, dibromoacetic acid, bromodichloroacetic acid, dibromochloroacetic

acid, and tribromoacetic acid) were analyzed using liquid-liquid extraction and gas

chromatography based on Standard Methods 6232 B and 6251 (APHA, 2005), respectively. A

Hewlett Packard 5890 Series II Plus Gas Chromatograph (Mississauga, ON) equipped with an

electron capture detector (GC-ECD) and a DB 5.625 capillary column (Agilent Technologies

Canada Inc., Mississauga, ON) was used for both THM and HAA analysis. Complete details

pertaining to the quantification of THMs and HAAs can be found in Section 3.2.7 and Section

3.2.8, respectively. Adsorbable organic halides (AOX) were analyzed using a titration method

based on Standard Method 5320 (APHA, 2005) with a Trace Element Instruments Xplorer

Organic Halogens Analyzer (Delft, Netherlands) as described in Section 3.2.9.

MX and MCA samples were extracted using tandem solid phase extraction cartridges

with a trifunctional C18 (tC18) cartridge (Sep Pak 6 cc, 1 g, Waters Corporation, Mississauga,

ON) for humics removal in-line with an Oasis HLB cartridge (12 cc, 500 mg, Water

Corporation). Analyses were conducted using a Varian 3800 GC, 4000 MS with a CombiPAL

autosampler (Mississauga, ON) equipped with a J&W Scientific DB-1701 column (30 m, 0.25

mm I.D., 0.25 μm film thickness). The GC/MS method was adapted from one described by

45

Zwiener & Kronberg (2001). The specifics of the GC/MS method and sample extraction is

presented in Section 3.2.10.

Genotoxicity was quantified using the SOS Chromotest assay, following the

manufacturer’s instructions presented in Section 3.2.11 (Environmental Bio-Detection Products

Inc., Mississauga, ON). Results were corrected to the relative toxic equivalent concentration

(TEQ) of the known carcinogen 4-nitroquinoline 1-oxide (4-NQO).

4.2.4 Statistical Methods

All comparisons between treatments were conducted using a paired Student t-test with a

significance level of 95%. For comparisons where the objective was to determine if a difference

existed (e.g. comparison of two Otonabee River control biofilters) a two-tailed test was utilized.

To assess whether an enhancement strategy performed better than another strategy or the control,

or if a particular treatment removed a significant amount of a specific compound when compared

to raw water, a one-tailed test was utilized. Reported ρ-values indicate the level at which a

particular relationship would show a significant difference (i.e. the higher the value the more

likely they are to be the same).

4.3 Results and Discussion

4.3.1 Biological Characterization of the Filter Media

Biological activity within the filters was quantified using adenosine triphosphate (ATP)

whereas the presence of extrapolymeric substances (EPS) were measured by determining the

concentration of proteins and polysaccharides on the surface of the filter media. ATP

concentrations on media treating Otonabee River water were roughly ten times higher when

compared to media treating Lake Ontario water, likely due to higher levels of biodegradable

organic carbon (Pharand et al., 2014). A qualitative comparison of the source waters shows a

difference in DOC (5 mg/L to 2 mg/L), and nitrogen (0.3 mg/L to <0.1 mg/L) for the Otonabee

River and Lake Ontario, respectively. Protein concentrations on the Otonabee River biofilter

media were observed to be two to ten times those found on the Lake Ontario biofilters, although

polysaccharide concentrations were within the same range (0 – 100 μg/g media). Since

46

heterotrophic bacteria in biomass require carbon, nitrogen and phosphorus to effectively

consume organic contaminants and develop more biomass, the observed differences in ATP and

EPS (especially proteins) at the two locations confirm that the low carbon and nutrient

concentrations in Lake Ontario water limit possible biological activity compared to other sources

(LeChevallier et al., 1991).

ATP testing at the Otonabee River location confirmed that biological activity in the

conventional filter backwashed with chlorinated water was much less (10.5 – 129 ng/g media)

than the mature control biofilter (1,080 – 3,083 ng/g media) (Figure 4-1). These results are

similar to those reported by Pharand et al., (2013) for biofilter media treating Grand River Water

(705 - 2037 ng ATP/g media, EBCT 38 min), but much higher than by Rahman (2013) on media

treating Saugeen River water with similar biofilter conditions (73 – 294 ng ATP/g media, EBCT

10 min). None of the chemical enhancements (nutrients, hydrogen peroxide, or in-line alum)

were observed to have an impact on ATP. Temperature did not affect ATP concentration which

is consistent with Evans et al., (2013).

Figure 4-1: Otonabee River Pilot-Scale Filter Media Biomass Characterization (Conventional

Filter sample was not collected in May)

47

Table 4-1 summarizes the level of significance for differences in specific parameters

associated with various filter pre-treatments where values below 0.05 are deemed significant.

The conventional filter (72±58 ng/g) and new control (565±78 μg /g) were much lower than the

mature control biofilter (734±76 μg /g); in-line alum pre-treatment (432±64 μg/g) suppressed

protein formation when compared to the new control. It was expected that the conventional filter

would have a lower concentration of proteins than the biofilters since the overall biological

activity was less (Miltner et al., 1995). The mature control likely has a higher concentration of

proteins than the new control since it is has been in operation for four months longer and had a

more established biological community. In-line alum may suppress protein formation by

removing some of the organics required for EPS production prior to degradation by the biofilm.

Table 4-1 shows no significant differences between any of the pilot filters at the Otonabee River

site with respect to polysaccharide concentrations including the comparison of conventional to

biological filtration.

The control filter (92 – 290 ng ATP/g media) had similar ATP concentrations to other

biofilters on Lake Ontario (54 – 506 ng ATP/g media, EBCT 4 – 17 min) with preozonation

(Pharand et al., 2014). None of the enhancements (0.2 mg/L peroxide, 0.2 mg/L PACl, 0.5 mg/L

P & N) were observed to significantly change biological activity within the Lake Ontario pilot-

scale filters (Figure 4-2, Table 4-2). PACl has been shown to remove >50% of phosphorus from

water (Zheng et al., 2012), and nutrient deficient conditions can cause an increase in

polysaccharide formation (Vu et al., 2009). Additionally, nutrient deficient conditions can limit

the formation of biofilm, which will lower the measureable ATP in the filter. Filters which

received 0.8 mg/L PACl pre-treatment were found to have a significantly lower ATP than the

three that did not. The impact of PACl dose was confirmed by increasing its dose from 0.2 mg/L

to 0.8 mg/L. ATP decreased by 50% following this decrease. Higher doses of PACl influenced

the relationship between ATP and temperature. Addition of 0.8 mg/L PACl prevented an

increase in ATP which was observed in August (temp = 12.5⁰C). GAC filters were found to have

a significantly higher ATP than the anthracite filter consistent with Dussert and Tramposch

(1997) and Liu et al. (2001).

48

Table 4-1: Statistical Comparison of Treatment Processes using Paired T-tests – Otonabee River

Treatments Compared

AT

P

EP

S –

Pro

tein

EP

S -

Poly

sacc

hari

des

DO

C (

mg/L

)

Bio

poly

mer

s

Hu

mic

Su

bst

an

ces

Bu

ild

ing B

lock

LW

M N

eutr

als

TH

M

Pre

curs

ors

HA

A P

recu

rso

rs

AO

X P

recu

rso

rs

MX

Pre

curs

ors

MC

A

Pre

curs

ors

Gen

oto

xic

ity

#1 #2

Raw Settled Water NA NA NA 0.001 0.001 0.001 0.093 0.227 0.001 0.001 0.001 0.001 0.001 0.005

Raw Conventional

Treatment NA NA NA 0.001 0.001 0.001 0.047 0.003 0.001 0.001 0.001 0.001 0.001 0.002

Raw Mature Control NA NA NA 0.002 0.009 0.039 0.115 0.046 0.003 0.043 0.060 0.040 0.290 0.015

Raw New Control NA NA NA 0.002 0.019 0.001 0.032 0.002 0.044 0.062 0.010 NS NS NS

Raw Nutrient Addition NA NA NA 0.001 0.008 0.008 0.180 0.449 0.003 0.169 0.042 NS NS NS

Raw Peroxide Addition NA NA NA 0.001 0.025 0.003 0.013 0.029 0.020 0.198 0.022 NS NS NS

Raw In-line Alum NA NA NA 0.001 0.007 0.001 0.374 0.001 0.014 0.131 0.058 0.027 0.068 0.041

Raw GAC Media NA NA NA 0.001 0.001 0.031 0.063 0.449 0.011 0.149 0.002 NS NS NS

Mature New Control 0.040 0.008 0.095 0.421 0.425 0.167 0.471 0.148 0.425 0.986 0.367 NS NS NS

Control Nutrient Addition 0.477 0.994 0.846 0.449 0.390 0.406 0.167 0.128 0.366 0.662 0.694 NS NS NS

Control Peroxide Addition 0.385 0.175 0.776 0.480 0.429 0.173 0.105 0.157 0.989 0.502 0.910 NS NS NS

Control In-line Alum 0.569 0.020 0.542 0.361 0.372 0.119 0.336 0.180 0.619 0.847 0.650 0.312 0.236 0.056

Control GAC Media 0.360 0.579 0.426 0.047 0.297 0.199 0.139 0.238 0.502 0.845 0.350 NS NS NS

Control Settled Water NA NA NA 0.001 0.004 0.001 0.135 0.330 0.001 0.001 0.001 0.004 0.014 0.338

Control Non-Biological

Filtration 0.009 0.001 0.035 0.395 0.013 0.279 0.325 0.440 0.046 0.230 0.059 0.215 0.215 0.024

Settled Non-Biological

Filtration NA NA NA 0.369 0.001 0.001 0.459 0.383 0.001 0.001 0.001 0.005 0.040 0.018

p-values representing the level of significance of the difference between two treatments. (α=0.05)

Bold indicates a significant difference between two sampling points. Italics indicates Treatment #2 is lower than #1

NA = Not Applicable NS = Not Sampled <DL = Parameter below detection level

LMW Acids were not detected, and T-tests were not completed.

49

Figure 4-2: Lake Ontario Pilot-Scale Filter Media Biomass Characterization

When considering the impact of media type at the Lake Ontario facility, anthracite was

found to have significantly less protein and polysaccharide formation than the GAC filters

receiving the same pre-treatment, which also corresponds to lower ATP on the anthracite. Empty

bed contact time, filter age, and filter enhancements did not have a significant impact of protein

formation (Table 4-2). GAC filters that received 0.8 mg/L PACl were observed to have

significantly higher concentrations of polysaccharides (56.3±21 μg/g) than GAC filters that did

not receive PACl (18.7±6 μg/g).

50

Table 4-2: Statistical Comparison of Treatment Processes using Paired T-tests – Lake Ontario

Treatments Compared

AT

P

EP

S –

Pro

tein

EP

S -

Poly

sacc

hari

des

DO

C

Bio

poly

mer

s

Hu

mic

Su

bst

an

ces

Bu

ild

ing B

lock

LM

W A

cid

s

LW

M N

eutr

als

TH

M P

recu

rsors

HA

A P

recu

rso

rs

AO

X P

recu

rso

rs

MX

Pre

curs

ors

MC

A P

rec

urs

ors

Gen

oto

xic

ity

#1 #2

Raw Ozone NA NA NA 0.617 0.021 0.140 0.129 <DL 0.275 0.157 0.306 0.028 <DL NA 0.121

Ozone GAC 16 NA NA NA 0.016 0.002 0.030 0.040 <DL 0.272 0.010 0.001 0.001 <DL NA 0.008

Ozone GAC 26 NA NA NA 0.009 0.021 0.007 0.033 <DL 0.092 0.007 0.001 0.001 NS NS NS

Ozone Anthracite 26 NA NA NA 0.007 0.003 0.042 0.156 <DL 0.225 0.103 0.019 0.002 NS NS NS

Ozone Control NA NA NA 0.076 0.187 0.325 0.032 <DL 0.230 0.037 0.007 0.052 <DL NA 0.267

Ozone Nutrient Addition NA NA NA 0.038 0.420 0.175 0.002 <DL 0.113 0.126 0.115 0.043 NS NS NS

Ozone Peroxide Addition NA NA NA 0.229 0.236 0.486 0.141 <DL 0.171 0.059 0.027 0.001 NS NS NS

Ozone 0.2 mg/L PACl NA NA NA 0.280 0.151 0.057 0.093 <DL 0.076 0.047 0.030 0.007 <DL NA 0.034

Control GAC 16 0.225 0.083 0.098 0.006 0.003 0.053 0.190 <DL 0.498 0.056 0.008 0.072 <DL NA 0.159

Control GAC 26 0.165 0.956 0.028 0.003 0.018 0.066 0.162 <DL 0.207 0.058 0.003 0.014 NS NS NS

Control Anthracite 26 0.103 0.055 0.048 0.002 0.002 0.091 0.398 <DL 0.143 0.410 0.308 0.051 NS NS NS

Control Nutrient Addition 0.776 0.941 0.621 0.218 0.336 0.213 0.105 <DL 0.146 0.366 0.329 0.051 NS NS NS

Control Peroxide Addition 0.711 0.518 0.366 0.299 0.328 0.413 0.352 <DL 0.465 0.411 0.368 0.106 NS NS NS

Control 0.2 mg/L PACl 0.327 0.383 0.590 0.130 0.209 0.153 0.295 <DL 0.420 0.467 0.410 0.332 <DL NA 0.091

GAC 16 GAC 26 0.430 0.102 0.377 0.477 0.242 0.345 0.481 <DL 0.232 0.426 0.003 0.001 NS NS NS

GAC 26 Anthracite 26 0.133 0.001 0.008 0.253 0.330 0.402 0.182 <DL 0.067 0.098 0.027 0.002 NS NS NS

p-values representing the level of significance of the difference between two treatments. (α=0.05)

Bold indicates a significant difference between two sampling points. Italics indicates Treatment #2 is lower than #1

NA = Not Applicable NS = Not Sampled <DL = Parameter below detection level

LMW Acids were not detected, and T-tests were not completed.

51

4.3.2 Removal of DBP Precursors Relative to DOC

Changes in DOC concentrations as a result of biofiltration were typically small (<10-

15%) in this study, regardless of the water source, whereas changes in DBP formation were

greater (up to 21%). Fractionation of the DOC using LC-OCD showed that while the humic

fraction correlated with the formation of regulated DBPs such as THMs and HAAs, as

previously reported (Zheng et al., 2014), the biopolymer fraction was correlated with MX, an

emerging DBP.

Biofiltration Impacts on Dissolved Organic Carbon

The impact of biofiltration on DOC concentrations was generally small. Biofilters

without pretreatment at the Otonabee River plant were able to consume DOC (anthracite: 6%;

GAC: 11% reduction), but did not match the performance of coagulation for DOC reduction

(45%). None of the pretreatments applied (hydrogen peroxide, nutrients, or in-line alum) were

observed to improve the performance of biofiltration for the removal of DOC relative to the

control. DOC removal from Lake Ontario water was reliant on PACl addition. GAC filters

receiving 0.8 mg/L PACl reduced DOC by 12-14% while the anthracite filter removed 9%. No

DOC was consumed by the control biofilter, or the filters being enhanced with hydrogen

peroxide, 0.2 mg/L in-line PACl, or nutrients.

A typical set of LC-OCD data showing changes in the NOM fractions through treatment

of Otonabee River water is illustrated in Figure 4-3. The proportions of the different fractions in

the two raw waters were similar despite Lake Ontario having much less DOC (2mg/L) than the

Otonabee River (5mg/L).

Biopolymers were removed by the control biofilters from Otonabee River water (28-

30%) over the study period; however enhancements were unable to improve performance. In

contrast, biopolymers were only reduced from Lake Ontario water in filters receiving 0.8 mg/L

PACl (31-37%); but media type and EBCT were not significant factors. The filter dosed with 0.2

mg/L PACl reduced biopolymer concentration by 17%, whereas the filters not receiving PACl

removed less than 5% (~0.01 mg/L).

52

Figure 4-3: LC-OCD Fractionation as a % of Total DOC Through Various Treatments –

Otonabee River

Humic substances are comprised of humic and fulvic acids and serve as DBP precursors

when chlorinated (Hubel and Edzwald, 1987). Otonabee River biofiltration reduced humic

substances by 4-9%, and coagulation removed 55±6%. Biofilter enhancements did not improve

performance relative to the control. PACl was also required to reduce humic substances from

Lake Ontario water, but unlike biopolymers, 0.2 mg/L PACl (8±5%) performed as well as 0.8

mg/L PACl (14±6% reduction). The control, hydrogen peroxide enhanced, and nutrient addition

filters removed less than 2% of humic substances from Lake Ontario Water.

53

Biofiltration Impacts on Regulated Disinfection By-Products and AOX

Disinfection by-product formation potentials (DBP FP) relating to adsorbable organic

halides (AOX), trihalomethanes (THMs), and haloacetic acids (HAAs) were measured after

incubating at 20⁰C for 24 hours and achieving a free chlorine residual of 1.0 ± 0.4 mg/L. The

reduction of DBP FP from Otonabee River water is presented in Figure 4-4, while a summary of

the significance of the differences in results as compared to DBP FP in the raw water or control

filters are presented in Table 4-1.

Biofiltration of Otonabee River water was able to effectively reduce the concentration of

DBP precursors at a rate exceeding their ability to remove bulk DOC, but none of the

enhancements were able to improve performance with respect to precursor removal (Figure 4-4).

Precursors to the formation of THMs (8.5-17%), HAAs (4.3-10%), and AOX (9.2-21%) were

removed 1.3-2.6, 0.7-1.5, and 1.4-2.6 times more efficiently than DOC, respectively. The

relatively low removal of DBP precursors and DOC through the biofilters was likely due to the

absence of any pre-oxidation process such as ozonation (Chaiket et al., 2002; Siddiqui et al.,

1997). Consistent with previous literature, humic substances, as measured using LC-OCD, were

observed to be the primary precursors to DBP formation in Otonabee river water (Hubel and

Edzwald, 1987; Pomes et al., 1999). Humic substance concentrations were also strongly

correlated to the formation potential of AOX (R=0.95), THMs (R=0.96) and HAAs (R=0.96),

whereas other organic fractions were not correlated to DBP formation (R<0.7).

Ozonation of Lake Ontario water prior to biofiltration reduced AOX precursors

(8.2±3.5%), increased THM precursors (8.5±11%), and did not impact HAA precursors

(2.1±8.9%). DBP removal is presented in Figure 4-5. Significant differences in DBP formation

between different treatments are presented in Table 4-2. Biofiltration reduced AOX precursors

by 20 to 57%; however only filters dosed with 0.8 mg/L PACl improved performance compared

to the control. Increasing EBCT in filters receiving 0.8 mg/L PACl from 16 to 26 minutes

improved AOX removal from 36±2.3% to 57±3%. GAC filters operated with an EBCT of 26

min performed better than anthracite filters (39±3.9) with the same EBCT, consistent with

existing literature (Wang et al., 1995). THM FP was lowered by 20-42%, but none of the

enhancements were significantly better than the control (23±11%). HAA FP reductions of 22-

54% occurred through the biofilters. GAC filters dosed with 0.8 mg/L PACl were able to

54

remove 45±3% (16 min EBCT) and 54±1.3% (26 min EBCT), respectively. None of the other

enhancements improve performance compared to the control (26±5.2%).

Figure 4-4: DBP Precursor Removal – Otonabee River (No bar indicates no precursor removal)

55

Similar to the Otonabee River biofilters, DBP precursors were preferentially removed

from Lake Ontario water when compared to DOC. Filters not receiving PACl removed an

average of 20-28%, 22–34%, and 20–41% of the THM, HAA, and AOX precursors,

respectively, despite their inability to reduce DOC. Filters receiving 0.8 mg/L PACl removed 20-

42%, 28-52%, and 36-57% of the THM, HAA, and AOX precursors, respectively while only

removing 9-14% of the DOC.

It is also interesting to note that the biological parameters (ATP, proteins,

polysaccharides) were not correlated to the formation or removal of any of the organic fractions

or DBPs. This indicates that biological monitoring alone is not sufficient to properly quantify the

effectiveness and performance of a biological filter. However, combining biological monitoring

with routine monitoring of organic removal and DBP formation can provide data essential to

optimizing performance of biological treatment processes.

Reduction of Emerging DBP Formation

3-Chloro-4(dichloromethyl)-2(5H)-furanone, also known as Mutagen-X (MX), is a

highly mutagenic halogenated furanone that has been shown to have much more potential to be

harmful to human health than the regulated DBPs (Onstad & Weinberg, 2005). MX and MCA

(mucochloric acid, a much less mutagenic isomer of MX) are currently unregulated compounds

due to the cost and complexity of analysis, as well as the low concentrations found in most

treated water (Zheng et al., 2014). However, the formation of these compounds has been reported

to be correlated to compounds routinely monitored including THMs (Onstad et al., 2008). Five

sampling points were used to monitor MX and MCA formation potential at each of the Otonabee

River pilot plant (Raw, Settled, Conventional Filter Effluent, Mature Biofilter Effluent and

Biofiltration with 0.2 mg/L alum) and the Lake Ontario pilot plant (Raw, Post Ozone, GAC filter

with 16 min EBCT, Control Biofilter, and Biofilter with 0.2 mg/L PACl dose).

MX was consistently detected in the chlorinated Otonabee River water, but not in the

chlorinated Lake Ontario water. In Lake Ontario water, the low DOC and the use of ozone

resulted in only one post ozone sample forming MX (6 ng/L) above the detection limit (5 ng/L)

while all of the biofilter effluent samples were below detection. However, the MX formation

56

potential in Otonabee River water ranged from 12 – 22 ng/L. Formation was not observed to be

correlated with water temperature (r2=0.45) or influent water DOC (r

2=0.43).

Figure 4-5: DBP Precursor Removal – Lake Ontario (No bar indicates no precursor removal)

57

Figure 4-6 illustrates the range of observed MX FP levels as well as their relationship to

THM FP for each of the five sampling points in the study. Although there was a large variation

in the presence of precursors throughout the study, a strong, linear relationship existed between

THMs and MX for each specific sampling date (R-values ranging from 0.83 to 0.95). Therefore,

although the specific concentrations may change, reducing THM FP will also very likely reduce

MX FP.

Figure 4-6: Seasonal Formation of MX as a Function of THM Formation – Otonabee River

In addition, Figure 4-7 shows that raw water and the two biofilters had higher DBP

formation potential, and that raw water DBP formation and biofilter precursor removal was

highly variable throughout the study. Conventional treatment, including coagulation and non-

biological filtration, was able to reduce the concentration of DBP precursors consistently despite

changes in the raw water. MX precursors were significantly removed by conventional treatment

(61±7%), biofiltration without pre-treatment (20±19%), and biofiltration with 0.2 mg/L alum

(27±22%). (See Table 4-1 for a statistical comparison of treatments for the removal of MX.)

Average formation potentials of MX, MCA, AOX, THM, and HAA were normalized to their

values in influent water and then compared to each other at each sampling point. Correlations

between the different DBP FPs were very strong at the Otonabee River pilot with each R-value

higher than 0.96. This agrees with previous results presented by Zheng et al., (2014). One of the

relationships, that of THM formation potential to MX formation potential in Otonabee River

0

5

10

15

20

25

30

0 50 100 150 200 250 300

MX

FP

(n

g/L)

THM FP (ug/L)

March May

June July

September

58

water, is shown in Figure 4-7. These results are beneficial to water treatment plant operation

because the analysis of regulated DBPs (THMs and HAAs) provides important information

regarding the formation of the other compounds even if the precursors or final products cannot

be routinely determined.

Figure 4-7: MX FP as a function of THM FP – Otonabee River

MCA formation potentials were also monitored at the Otonabee River and Lake Ontario

pilot plants and the results were similar to those for MX. Influent Otonabee River water MCA

formation potential ranged from 285 – 705 ng/L, and was not observed to be correlated with

water temperature (r2= 0.15) or influent water DOC (r

2=0.13).

Similar to MX, the average formation of MCA was strongly correlated to THM FP

(R=0.96, Figure 4-8), HAA FP (R=0.98) and AOX FP (R=0.98) in Otonabee River water. In

Lake Ontario water the relationship between normalized AOX FP, THM FP and HAA FP was

strong (R>0.96), but the relationship between MCA formation and THM FP (R=0.71), HAA FP

(0.79) and AOX FP (0.90) varied. Despite the somewhat weaker relationships in Lake Ontario

water, the results still suggest that monitoring and control of regulated DBPs such as THMs will

likely correlate to emerging DBPs.

Influent Water

Settled Water

Conventional

Filter

Control

Biofilter

0.2 mg/L

Alum

MX FP = 0.98(THM FP) - 0.05

R = 0.96

0

0.2

0.4

0.6

0.8

1

1.2

0 0.2 0.4 0.6 0.8 1 1.2

Ave

rage

MX

FP

(C

/Co

)

Average THM FP (C/Co)

59

Figure 4-8: MCA FP as a function of THM FP – Otonabee River

MCA precursors were removed from Otonabee River water by coagulation (59±8.5%),

but not by biological filtration (<9%). In Lake Ontario water, MCA was only quantified above

detection (15 ng/L) in July and August with raw water formation potentials ranging from 65–76

ng/L. Ozonation removed 37% of precursors (5 – 70%), the control biofilter removed 35-41%,

0.2 and 0.8 mg/L PACl removed 15-50% and 11-58%, respectively.

MX and MCA formation in Otonabee River water were related to biopolymer

concentration (r2=0.95, and r

2=0.88, respectively) in contrast to AOX, THMs, and HAAs which

were correlated to humic substances. MCA formation in Lake Ontario water was correlated to

humic substances (r2=0.90), possibly because of different precursor compounds or the effect of

pre-ozonation.

4.3.3 Reduction of Genotoxicity

A cell-based assay (SOS Chromotest) was performed to determine the genotoxic

response of chlorinated water samples. A ratio between the SOS Inducing Potency (SOSIP) of

each sample and a known carcinogen, 4-nitroquinoline 1-oxide (4-NQO,) was calculated with a

result of 1 indicating a sample as carcinogenic as 4-NQO.

As observed for MX, MCA and the regulated DBPs, genotoxic responses from Lake

Ontario were lower than those for the Otonabee River. Genotoxic responses in Otonabee River

MCA FP = 1.09(THM FP) - 0.10

R = 0.96

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0 0.2 0.4 0.6 0.8 1 1.2

MC

A F

P (

C/C

o)

THM FP (C/Co)

Influent

Water

Control

Biofilter

0.2 mg/L

alum Settled

Water

Conventional

Filter

60

influent water ranged from 0.192 to 0.895. Biofiltration was unable to perform as well as

coagulation and conventional filtration with respect to DBP precursor removal; however the

control biofilter (26±14%) reduced genotoxic response similar to coagulation (30±10%), and

better than biofiltration including 0.2 mg/L alum (11±8%) from Otonabee River water. The

genotoxic response of Lake Ontario influent water ranged from 0.06 to 0.14. Ozonation

increased genotoxicity by 5% in April while reducing responses in July (35%) while

enhancement with 0.2 and 0.8 mg/L PACl reduced it by 27±13% and 43±9%, respectively.

Examining the formation of genotoxicity and DBPs through each treatment over the

study showed that genotoxicity was well correlated to AOX FP(R=0.86; 0.99), THM FP

(R=0.89; 0.90), and HAA FP (R=0.84; 0.94) in Otonabee River and Lake Ontario water,

respectively. Since genotoxicity may be used to estimate overall risk associated with chlorinated

organics, its correlation with AOX, a measure of all halogenated DBPs, was examined first.

Results for the Otonabee River (Figure 4-9) and Lake Ontario (Figure 4-10) are presented below.

Figure 4-9: Genotoxic Response with Respect to AOX FP – Otonabee River

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 200 400 600 800

SOSI

P C

orr

ect

ed

GTX

AOX FP (ug Cl/L)

March

May

June

July

September

Raw Water and Biofilter

Effluent

Conventional Treatment

61

Figure 4-10: Genotoxic Response with Respect to AOX FP – Lake Ontario

The relationship between THM FP and genotoxic response in Otonabee River water as a

function of various treatments is shown in Figure 4-11. The correlation between genotoxic

response and THM FP is very strong (r2=0.97) for the biofilter samples, but not when

considering conventional treatment (r2=0.64). The slope is much steeper when considering

biofiltration suggesting that it may preferentially remove precursors that lead to the formation of

a genotoxic response as opposed to those responsible for other DBPs.

Figure 4-11: Genotoxic Response as a Function of THM FP – Otonabee River – Circled areas

represent biological and conventional treatment

0

0.02

0.04

0.06

0.08

0.1

0.12

0.14

0.16

0 20 40 60 80 100 120

SOSI

P C

orr

ecte

d G

TX

AOX FP (ug Cl/L)

April

July

August Raw Water

Raw

Settled Water

Conventional Filter

Control

0.2 mg/L In-line alum

0

0.2

0.4

0.6

0.8

1

1.2

0 0.2 0.4 0.6 0.8 1 1.2

Ave

rage

SO

SIP

(C

/Co

)

Average THM FP (C/Co)

Biofilter genotoxicity = 1.6(THM FP(C/Co)) - 0.56

R² = 0.97

62

In contrast, MX FP did not show a strong correlation with genotoxic response in

Otonabee River water (R=0.56). Previous literature has shown that MX accounts for up to 67%

of the genotoxicity or carcinogenicity of disinfected waters (Zheng et al., 2014; McDonald and

Komulainen, 2005). When compared to total AOX FP, MX comprised less than 0.01%, but was

responsible for 17-93% (average = 47%) of the genotoxic response. Unknown AOX, comprised

32-63% of all compounds excluding THMs or HAAs, and was associated with the remaining 7–

83% (average = 53%) of the genotoxic response.

4.4 Summary

Conventional treatment, including coagulation and non-biological filtration, was

observed to better remove organic compounds, DBP precursors, and halogenated furanone

precursors when compared to passive biofiltration alone, while biofiltration was better at

removing precursors to genotoxicity. This study showed that source water chemistry and organic

characteristics will determine the effectiveness of biofiltration. While DOC, DBP precursors, and

genotoxicity precursors were effectively removed by passive biofiltration of Otonabee River

water, pre-treatment with PACl was required to achieve similar results for Lake Ontario water.

Except in a few instances, the addition of nutrients, hydrogen peroxide, and low doses of in-line

coagulant did not improve treatment through biofiltration. Utilizing GAC media, as opposed to

anthracite, did not show widespread benefits at either location, illustrating the need of pilot-scale

testing prior to conversion to biofiltration.

This study also showed that DBP precursors could be preferentially removed by

biofiltration, indicating that the most reactive compounds are easily removed compared to DOC

as a whole. Specific organic fraction removal is also very important as Otonabee River

biopolymers were related to MX and MCA formation whereas humic substances were highly

correlated to AOX, THM, and HAA formation. Lake Ontario MCA formation was related to

humic substances, while no relationship was observed between specific organic fractions and

AOX, THMs, or HAAs.

Regulated DBPs can be excellent surrogates for parameters such as halogenated

furanones and genotoxicity. Strong correlations were found for Otonabee River water between

MX formation and THM, HAA, and AOX FP. This relationship may reduce the need of complex

63

and expensive monitoring of more mutagenic compounds through the existing analysis of

regulated DBPs. Genotoxic response behaves in a similar manner, and is strongly correlated at

both locations to AOX, THM, and HAA formation.

The potential exists for biofiltration to reduce genotoxic risk as effectively as

conventional treatment. Otonabee River genotoxic response was equal in the control biofilter and

the convention filter which indicates that biofiltration may be able to remove the most reactive

DBP precursors, and effectively limit the formation of potentially genotoxic compounds.

Finally, a comprehensive monitoring program is required to be able to fully quantify the

performance of a biofiltration system. No individual parameter can be used to effectively

determine overall performance of a system.

64

5. Removal of Emerging Contaminants by Engineered

Biofiltration

5.1 Introduction

As discussed in Chapter 4, source water chemistry and organic characteristics will

determine the effectiveness of chemical and biological treatment in a given location.

Conventional treatment at the Otonabee River pilot plant included coagulation, flocculation,

sedimentation and non-biological filtration, and was generally superior to biofiltration for the

reduction of chemical contaminants. Despite poor performance with respect to DOC reduction

(<15%), biofiltration with or without chemical enhancement was able to preferentially remove

disinfection by-product precursors, and lowered genotoxic response more efficiently than

conventional treatment. At the Lake Ontario pilot plant, PACl addition was required to achieve

removal of DOC, DBP precursors and genotoxicity. Chemical enhancements such as nutrient

addition and hydrogen peroxide addition were generally no more effective than passive

biofiltration, and GAC media was not significantly better than anthracite. These results indicate

that biological degradation of organic contaminants is occurring within the filter which could be

useful for the reduction of emerging contaminants.

Anthropogenic contaminants are synthetic compounds that may be designed to be stable

in the environment (USEPA, 2001). Due to their design, they are poorly removed by wastewater

treatment facilities making them potential indicators of wastewater contamination (Ternes et al.,

2004). Included in the category of anthropogenic contaminants are pharmaceutically active

compounds (PhACs), endocrine disrupting compounds (EDCs), and artificial sweeteners. They

are of interest to drinking water professionals because of the potential risk that PhACs and EDCs

pose to consumers, or for artificial sweeteners ability to indicate wastewater contamination.

EDCs interfere with the synthesis, secretion, transport, action or degradation of natural

hormones by mimicking or blocking natural hormones (USEPA, 2001). PhACs are used to

diagnose, treat, alter, or prevent illness (USEPA, 2012). These compounds are of interest because

of the potential risk they pose to the natural environment, including feminization of male fish

from exposure to estrogen compounds (Larsson et al., 1999), and reproductive issues in Great

Lakes salmonids (Leatherland, 1993). Additionally, there is an unknown risk to human

65

consumers when they consume trace levels of these compounds for long periods of time (Safe,

2004; Schwab et al., 2005).

Artificial sweeteners are synthetic, anthropogenic contaminants used to sweeten foods

and beverages (Scheurer et al., 2010). Although these compounds have been approved for

consumption by government health organizations worldwide there is concern because long-term

health impacts are presently unknown (Mawhinney et al., 2011). In addition, these compounds

are poorly removed by traditional wastewater treatment facilities allowing for their entry into the

natural environment (Torres et al., 2011). Artificial sweeteners have been proposed as a tracer

for wastewater in drinking water sources due to the fact that they are poorly removed by

traditional waste and drinking water treatment processes (Oppenheimer et al., 2011).

EDCs, PhACs, and artificial sweeteners have been detected in source waters around the

world, and many studies have examined their removal with different water treatment processes.

Ozonation is an effective method of reducing the concentration of EDCs and PhACs, but

ozonation may create many unknown degradation products (Westerhoff et al., 2005; Dodd et al.,

2009). Coagulation has also been examined for the removal of PhACs and EDCs, but removals

are typically low (<30%) (Diemert & Andrews, 2013).

Biologically active filters (biofilters) are filters backwashed with non-chlorinated water to

allow a biofilm to form on the surface of the media. This treatment method is becoming more

popular in drinking water treatment facilities because of its ability to degrade organic compounds

while providing effective physical removal (LeChevallier et al., 1992). In addition to passive

operation, biofilters can be enhanced with low doses of in-line coagulant to improve removal of

large organic compounds without increasing headloss (Azzeh et al., 2014).

Biofiltration is most commonly used in drinking water treatment to remove organic

carbon and disinfection by-product precursors (Onstad et al., 2008). Recently, biofiltration has

been shown to remove EDCs, PhACs (Reungoat et al., 2011), and artificial sweeteners

(Mawhinney et al., 2011) from municipal wastewater and drinking water (Zearly and Summers,

2012).

This study examined the removal of 9 different EDCs and PhACs, and the artificial

sweeteners sucralose and acesulfame potassium (acesulfame-K), through pilot-scale

conventional treatment (coagulation, flocculation, settling, non-biological filtration), and pilot-

scale biofiltration (with or without coagulant enhancement). The objective of the study was to

quantify the removal of these compounds through typical chemical and biological drinking water

66

treatment processes, and determine which processes are most effective at removing these

compounds from drinking water.

5.2 Materials and Methods

5.2.1 Compounds of Interest

Compounds were selected due to their occurrence in the natural environment and range

of physical and chemical properties including: hydrophobicity, solubility, molecular weight, and

acidity (Table 5-1). Notably absent from this group of analytes are antibiotics due to the negative

impact they may have on biological growth within the filter media.

Table 5-1: PhACs, EDCs, and Artificial Sweetener Details

Compound Use CAS MW

(g/mol)

Water

Solubility

(g/L)

Log

Kow

pKa

Acetaminophen Analgesic 103-90-2 151.17 15 0.46 9.38

Carbamazepine Antiepileptic 298-46-4 236.28 0.022 2.45 13.9

Clofibric Acid Metabolite of lipid

regulator

882-09-7 214.65 100 2.57 3.37

Diclofenac Analgesic 15307-86-5 296.16 2.3 4.51 4.15

Estrone Reproductive

Hormone

53-16-7 270.37 0.147 3.13 10.4

Gemfibrozil Lipid Regulator 25812-30-0 250.34 11 4.77 4.42

Ketoprofen Analgesic and

anti-inflammatory

22071-15-4 254.29 5.8 3.12 4.45

Naproxen Anti-inflammatory 22204-53-1 230.26 0.115 3.18 4.15

Pentoxifylline Vasodilator 6493-05-6 278.31 9.2 0.29 0.28

Acesulfame-K Artificial

sweetener

55589-62-3 201.24 270 -1.0 2

Sucralose Artificial

sweetener

56038-13-2 397.64 283 -2.67 12.52

67

The EDCs and PhACs were dissolved in acetonitrile as per Diemert and Andrews (2013),

and spiked into the biofilter influent water to achieve a concentration of 500 ng/L. Analytical

detection of the artificial sweeteners was less sensitive than the pharmaceuticals. They were

dissolved in Milli-Q® water and spiked to 1000 ng/L to aid in quantification. Spiking began 7

days prior to sample collection to ensure consistent operation and feed concentrations. A

Masterflex C/L tubing peristaltic pump was used to dose the two sets of samples at a flow rate of

0.017 mL/min (1 mL/hr). The pump was calibrated in the lab, and flow rates were confirmed at

the pilot plants by pumping to a graduated cylinder for a minimum of four hours before spiking

the influent water. This flow rate was chosen to minimize the amount of solution that had to be

transported. Water samples were collected in 500 mL amber glass bottles and stored for a

maximum of 7 days prior to extraction.

5.2.2 Source Waters

Pilot-scale studies were completed at the Peterborough Water Treatment Plant located on

the Otonabee River in Peterborough, Ontario, and the Harris Water Treatment Plant located on

Lake Ontario in Toronto, Ontario. Pilot plant influent water quality is shown in Table 3-1. Both

pilot plants were spiked with the same group of analytes shown in Table 5-1.

5.2.3 Pilot Plant Configurations

Two biofilter pilot plants were used in this study. One pilot plant at the Otonabee River

was configured to examine biofiltration with or without in-line alum addition (0.2 mg/L) and

conventional filtration (Figure 5-1). The second pilot plant, utilizing ozonated Lake Ontario

water as an influent source, focussed on the impact of PACl dose on biofiltration.

The two Otonabee River biofilters were operated with an empty bed contact time (EBCT)

of 10 minutes, while the conventional filter was operated at 15 minutes to match full-scale

operation. The filters contained 50 cm of anthracite (effective size d10 = 0.85 mm, uniformity

coefficient UC = 1.8) over 50 cm of sand (d10 = 0.5 mm, UC = 1.8). One of the biofilters was

operated without chemical addition and is referred to as a control. The other biofilter was

enhanced with 0.2 mg/L of alum added in-line prior to filtration. The conventional train

consisted of alum coagulation (~40 mg/L), three-stage tapered flocculation, parallel plate

settling, and non-biological filtration. The biofilters were backwashed with their own effluent,

68

and the conventional filter was backwashed with chlorinated water from the full-scale treatment

plant. All filters were backwashed three times per week. The analytes of interest were spiked

directly into the constant head tank and completely mixed prior to treatment.

A second pilot-scale treatment system was fed raw Lake Ontario water and consisted of

three filters preceded by ozonation (dose = 1 mg/L, contact time = 8 min, O3:DOC = 1:2). One

filter influent was dosed with 0.8 mg/L polyaluminum hydroxychloride (PACl). One of the other

two filters acted as a control and the other received 0.2 mg/L of PACl (Figure 5-2).

Raw Water

Rapid

Mix

Tapered

Flocculation

Parallel

Plate

Settlers

Control

Biofilter

EBCT:

10 min

0.2 mg/L

Alum

EBCT:

10 min

Analyte Spike

Figure 5-1: Schematic of the Otonabee River Pilot Plant

All of the biofilters were operated with a 16 min EBCT and consisted of 50 – 150 cm

GAC over 15 – 50 cm of sand. All media had been in operation for at least 4 years prior to

sampling and was considered to be exhausted in terms of adsorption capacity. All filters were

backwashed with their own effluent at terminal headloss, maximum once a week.

69

All of the compounds were dosed into the pilot plant after ozonation to isolate the

biological processes occurring within the filters and to eliminate the impact of oxidation on

removal. Due to the configuration of the HWTP pilot, analytes were spiked into a constant head

tank prior to the filter receiving 0.8 mg/L PACl and in a separate constant head tank prior to the

two smaller filters. Samples were collected from the effluent of each filter and from each of the

mixing tanks to account for variations in the influent concentrations.

GAC

EBCT:

16 min

Control

EBCT:

16 min

In-line

PACl

0.2 mg/L

16 min

Figure 5-2: Schematic of the Lake Ontario Pilot Plant

70

The pilot systems were also tested to determine the amount of adsorption occurring to the

materials in the pilot plant. A filter column parallel to the three being tested at the Otonabee

River plant was operated without media, and samples were collected alongside the treated

samples. Results showed that adsorption to material within the pilot plant was generally small

(<12%), and likely did not influence the results of the study.

5.2.4 Analytical Methods

The EDCs and PhACs were analyzed by LC/MS/MS based on a method derived from

Ontario Ministry of the Environment (MOE) EOP-E3454, version 2.0 (MOE, 2008). Analysis

was completed at the University of Toronto using an Agilent 1200 series pump, Aglient

Poroshell EC-C18 column (5 cm x 2.1 cm x 2.7 µm particle size), and Agilent 6460 triple

quadrupole. Neat standards were purchased from Sigma-Aldrich Inc. (Oakville, ON) while

surrogates (d4acetaminophen, d10carbamazepine, d3naproxen, d4diclofenac, d6gemfibrozil,

and d4clofibric acid) and internal standards (13

C6sulfamethazine phenyl for positive mode and

d14bisphenol A for negative mode) were purchased from CDN Isotopes (Pointe-Claire, QC).

The artificial sweeteners were analyzed using the same LC/MS/MS equipment as the

pharmaceutical analysis and a method developed by the Water Quality Centre at Trent

University (Hoque et al., 2014). Neat standards (sucralose and acesulfame-K) were purchased

from Sigma-Aldrich Inc. (Oakville, ON) and internal standards (d6sucralose and d4acesulfame-

K) were purchased from Toronto Research Chemicals (Toronto, ON).

Adenosine triphosphate (ATP) was quantified with a LuminUltra Deposit Surface

Analysis kit (DSA-100C, Fredricton, NB) following the manufacturer’s instructions. DOC was

measured using a persulfate wet oxidation method as described in Standard Method 5310 D

(APHA, 2005) with an O-I Corporation Model 1010 TOC Analyzer (College Station, Texas,

USA). All other parameters were measured as per Standard Methods (APHA, 2005).

5.3 Results and Discussion

5.3.1 Characterization of the Pilot-Scale Filters

Biological activity within the biofilters was quantified by measuring adenosine

triphosphate (ATP) on the filter media and the removal of dissolved organic carbon (DOC)

71

through the filter. The ATP measured approximately ten times higher at the Otonabee River pilit

plant than the Lake Ontario pilot plant which was in line with the differences in their

biodegradable organic carbon (Pharand et al., 2014) and nutrients in the two water sources. The

source waters were very different with respect to DOC (5 mg/L to 2 mg/L) and nitrogen (0.3

mg/L to <0.1 mg/L) at the Otonabee River and Lake Ontario plants, respectively.

Filters operated biologically had more ATP than the conventional filter backwashed with

chlorinated water. The Otonabee River control biofilter had an average ATP of 1800 ng/g media,

while the biofilter receiving 0.2 mg/L alum had an average ATP of 1080 ng/g media. These are

both much higher than the conventional, non-biological filter which had an average ATP of 53

ng/g media. The control biofilter at the Lake Ontario pilot plant, in contrast, has an average ATP

of 190 ng/g media. The filter receiving 0.2 mg/L PACl measured 117 ng ATP/g media, and the

filter receiving 0.8 mg/L PACl resulted in an average ATP measurement of 93 ng/g media. These

were all higher than the 53 ng/g ATP measured in the Otonabee River conventional filter.

DOC removal was low through all of the filters. There was more removal of DOC

through the biofilters than the conventional filter, as expected. The control biofilter treating

Otonabee River water consumed 6.5% of the DOC, and the filter dosed with 0.2 mg/L alum

removed 6.7%. This indicates that biological processes in the filter are likely responsible for this

removal, and not chemical processes. The filters treating Lake Ontario water and receiving 0,

0.2, and 0.8 mg/L PACl removed 0, 2, and 13% of the DOC over the course of the study,

respectively. This indicates that chemical processes are likely responsible for DOC removal from

Lake Ontario water instead of biological processes. The difference between the removal

mechanisms at each location may be a factor of biological activity within the filter since

Otonabee River filters had ATP concentrations ten times higher than the Lake Ontario filters.

5.3.2 Removal of Pharmaceutical Compounds by Biological Processes

Five sampling events occurred at the Otonabee River pilot and three samples were

collected from the Lake Ontario pilot between March and September 2014. Although a nominal

concentration of 500 ng/L was generally achieved for most of the PhACs and EDCs, the

resulting raw water concentrations varied throughout the study due to changes in influent flow

rate during the spiking. Influent concentrations from the Lake Ontario pilot plant are given in

Figure 5-3. These variations did not affect the calculations of removals of the pharmaceuticals.

72

Figure 5-3: Influent Water Spiking Results - Lake Ontario

At the Otonabee River pilot plant biological treatment and chemical treatment

(coagulation and settling), either alone or in combination, could provide an effective treatment

strategy for the removal of EDCs and PhACs (Figure 5-4). Coagulation (settled water) removed

an average of 31% (18-56%) and non-biological filtration removed an additional 8% (0-28%)

from the settled water. Biofiltration with or without 0.2 mg/L alum removed an average of 44%

(0-78%) of the analytes. Five of the nine EDCs and PhACs were removed by more than 50%

through biofiltration and biofiltration with 0.2 mg/L (acetaminophen, estrone, gemfibrozil,

ketoprofen, and pentoxifylline); two of the remaining compounds were removed by more than

50% by conventional coagulation and non-biological filtration (clofibric acid and naproxen).

Diclofenac and carbamazepine were the two least treatable compounds, removed by a maximum

of 37% by conventional treatment.

73

Figure 5-4: Average Analyte Removal – Otonabee River (Error bars represent mean ± 1

standard deviation)

Still, taken together, the combination of biological and chemical treatment processes was

shown to effectively reduce the concentration of a wide range of PhACs and EDCs. All of the

compounds were removed by at least 37% by one of the two treatment mechanisms.

Biofilter enhancement through the addition of 0.2 mg/L alum provided benefit for the

removal of a few specific compounds. In particular, diclofenac and carbamazepine were not

removed by the control biofilter, but achieved 22% and 8% average removal when treated with

0.2 mg/L alum prior to biofiltration, respectively.

Testing at the Lake Ontario pilot plant focused on the addition of different coagulant

doses prior to biofiltration, as opposed to the comparison of chemical to biological treatment.

Three polyaluminum hydroxychloride (PACl) doses were tested: 0 mg/L, 0.2 mg/L, and 0.8

mg/L. Average results from three testing events indicate that increasing PACl concentrations

improve the removal of emerging contaminants (Figure 5-5).

The control biofilter, with no PACl added, was able to remove two of the ten compounds

by more than 50%. The average removal through the control biofilter was 39% with a range of 7-

86%. When treated with 0.2 mg/L PACl the filter reduced EDCs and PhACs by 12-87% with an

average of 45%. Increasing the dose to 0.8 mg/L PACl improved the performance such that

reductions averaged 70% and nine of the ten compounds were reduced by 50% or more (39-

91%).

0

20

40

60

80

100

120

Ave

rage

% R

em

ova

l

Settled Water

Conventional Treatment

Control Biofilter

Biofilter + 0.2 mg/L Alum

74

Figure 5-5: Average Analyte Removal – Lake Ontario Water

Despite significant differences between Otonabee River and Lake Ontario composition,

control biofilter operational details, and biological activities at the two pilot plants, the results

was similar. The control filter at the Otonabee River pilot plant average removal of EDCs and

PhACs was 44% (0-78%) while the control filter at the Lake Ontario pilot plant averaged 39%

removal (7-85%). This was not expected based on the biological activity measured in each filter,

and may suggest that these compounds are more easily biodegraded than bulk DOC. These

results also indicate that any filter backwashed without chlorine, and therefore allowed to operate

in a biological manner, may reduce the risk that low doses of EDCs and PhACs pose to the end

user.

The reported literature validates the results observed at the pilot plants. There are a few

notable comparisons to the existing literature as summarized in Table 5-2. First, reported

removals from chemical treatment were typically less than what was observed at the Otonabee

River pilot plant. This is likely because the literature values only examined coagulation at bench-

scale (jar tests), and not at pilot-scale in combination with filtration. Secondly, the results from

the Otonabee River control biofilter closely matched the results from existing literature except

for estrone and pentoxifylline. More research into these compounds may be required to

determine the metabolic pathways leading to improved removal in this test compared to previous

results. Finally, many of the compounds were removed from Lake Ontario more efficiently than

0

10

20

30

40

50

60

70

80

90

100

No PACl 0.2 mg/L PACl 0.8 mg/L PACl

Ave

rage

% R

em

ova

l

Acetaminophen

Clofibric Acid

Diclofenac

Estrone

Gemfibrozil

Carbamazepine

Ketoprofen

Naproxen

Pentoxifylline

75

reported in the literature.. These improved removals may be a factor of adsorption to the GAC

media used in the pilot plant, but was expected to be a small factor since the media was in use for

more than four years prior to testing.

Table 5-2: Pharmaceutical Removals Compared to Existing Literature

Otonabee River Lake Ontario Literature Results

Compound

Con

ven

tion

al

Fil

tra

tion

Con

trol

Bio

filt

rati

on

No P

AC

l

0.2

mg/L

PA

Cl

0.8

mg/L

PA

Cl

Biological

Filtration

Processes

Chemical

Treatment

Acetaminophen 46% 68% 82% 86% 91% 59±11%1 <20%

4

Carbamazepine 35% 5.2% 39% 25% 80% 0.5±1.1%1 32%

5

Clofibric Acid 52% 36% 14% NA 39% 35±6%1 <20%

6

Diclofenac 36% 8.7% 12% 27% 74% 21±1%1 <20%

4,6

Estrone 33% 60% 86% 87% 77% <1%2 <20%

4

Gemfibrozil 29% 73% 6.8% NA 58% 70±7%1 31-37%

5

Ketoprofen 38% 78% 29% 28% 63% Not Available <20%6

Naproxen 52% 29% 33% 12% 72% 68-75%3 <20%

4,6

Pentoxifylline 24% 74% 45% 60% 77% 13%2 <20%

4

NA = Not Available; 1 Zearly & Summers, 2012;

2 Snyder et al., 2007;

3 Halle, 2009b;

4 Westerhoff et al., 2005;

5 Diemert & Andrews, 2013;

6 Simizaki et al., 2008

5.3.3 Impact of Coagulation on Removal of Artificial Sweeteners

This study examined the removal of two artificial sweeteners, acesulfame-K and

sucralose, by chemical and biological processes. Acesulfame-K was best removed from

Otonabee River water by treatment processes that included alum addition. Coagulation (19%)

and biofiltration combined with 0.2 mg/L alum (8.1%) removed an average of 70 and 30 ng/L,

respectively (Figure 5-6). Conventional, non-biological filtration, and the control biofilter

76

reduced acesulfame-K concentrations by only 3% (9 ng/L). However, acesulfame-K removal

was quite variable over the study period, and that variability within the data is a more likely

explanation for the removals through biofiltration than a systematic biological removal process.

Coagulation was really the only process that showed consistent removals throughout the study.

Figure 5-6: Removal of Acesulfame-K – Otonabee River

Sucralose was not well removed from Otonabee River water by any of the treatment

processes examined. Coagulation (7%), conventional filtration (7%), biofiltration (7%), and

biofiltration with 0.2 mg/L alum (6%) all removed small concentrations of sucralose (~30 ng/L),

but none of the treatments provided consistent results (Figure 5-7). The results from the

Otonabee River pilot plant show that sucralose removal was highly variable with higher removal

events and samples with little to no removal. This also impacts that average removal since it is

impossible to determine if any of these results are outliers from the remaining data set. However,

it is expected that there will be low removal of sucralose through these treatments (Scheurer et

al., 2010).

0

5

10

15

20

25

30

Settled Water Conventional Filter Control Biofilter Biofilter - 0.2 mg/L alum

Ace

sulf

ame

-K (

% R

em

ova

l)

May

June

July

September

77

Figure 5-7: Sucralose Removal – Otonabee River

Further study into the removal of sweeteners via coagulation and biofiltration using the

Lake Ontario pilot plant shows that removals were dependent on PACl dose and confirmed the

compounds to be more amenable to treatment when PACl use was included. The control biofilter

removed an average of 15% acesulfame-K (0-38%) and 18% sucralose (0-36%) over three

sampling dates. The biofilter receiving 0.8 mg/L PACl removed 33% acesulfame-K (14-50%)

and 39% sucralose (26-58%). Due to pilot plant operational issues, only 1 reliable sample was

captured for the filter receiving 0.2 mg/L PACl which showed 14% and 23% removals of

acesulfame-K and sucralose, respectively. Figure 5-8 shows a strong relationship between the

removal of artificial sweeteners and the in-line PACl dose added to the filters as indicated by the

r2 values of 0.92 and 0.99 for acesulfame-K and sucralose, respectively. Although this was a very

limited range of coagulant doses, it was representative of the doses utilized at the full-scale plant

which the pilot mimics, and shows the potential for removal of sweeteners with optimized

coagulant doses.

0

5

10

15

20

25

Settled Water Conventional Filter Control Biofilter Biofilter - 0.2 mg/L alum

Sucr

alo

se (

% R

em

ova

l)

May

June

July

September

78

Figure 5-8: Sweetener Removal as a Function of PACl Dose – Lake Ontario

Previous studies have shown acesulfame-K and sucralose to be highly resistant to

removal or transformation by chemical or biological treatment, including oxidation with ozone

(Scheurer et al., 2010). However, GAC filtration was shown to provide partial to complete

removal of the sweeteners if adsorption sites on the filter media were not completely exhausted.

These results are confirmed by studies with Otonabee River water, but removals were much

higher than expected in Lake Ontario water. The filters in the Lake Ontario pilot plant likely

have adsorptive capacity remaining which has removed more of the sweeteners than expected.

5.4 Summary

This study involved spiking 9 EDCs and PhACs, and 2 artificial sweeteners into a pilot-

scale treatment system treating Otonabee River water, and another system treating Lake Ontario

water. Generally, biofiltration with or without in-line coagulant addition was able to lower

concentrations of PhACs and EDCs in treated water. At the Otonabee River pilot plant a

combination of conventional and biological treatment was able to reduce the concentration of 7

of the 9 EDCs and PhACs by more than 50%. At the Lake Ontario pilot plant, biofiltration

Acesulfame-K Removal = 23.865(PACL Dose) + 12.612 R² = 0.923

Sucralose Removal = 26.647(PACL Dose) + 18.11 R² = 0.999

0

5

10

15

20

25

30

35

40

45

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9

Swe

ete

ne

r %

Re

mo

val

PACL Dose (mg Al/L)

Acesulfame

Sucralose

79

without PACl reduced the concentration of 9 different compounds by 39%, but adding PACl up

to 0.8 mg/L increased the average removal of these compounds to 70%.

The artificial sweeteners acesulfame-K and sucralose were also spiked into each pilot

system. In Otonabee River water, the results were highly variable. Coagulation provided the

most reliable removals, and averaged 19% over the study. Sweetener removal in Lake Ontario

water was proportional to PACl dose with higher doses performing better. Overall, artificial

sweetener removal was low regardless of treatment method, confirming that these compounds

may be useful as indicators for wastewater impacts in drinking water sources.

80

6. Summary, Conclusions, and Recommendations

6.1 Summary

Pilot plants were operated to compare passive biofiltration to engineered biofiltration and

conventional treatment for the removal of NOM (DOC, UV254, and LC-OCD), DBP precursors

(THMs, HAAs, AOX), halogenated furanone precursors (MX and MCA), and genotoxicity.

Additional tests were completed to determine how efficiently pharmaceutically active

compounds (PhACs), endocrine disruptors (EDCs), and artificial sweeteners that had been

spiked into the pilot plant influent were removed. The study utilized pilot plants that treated

Otonabee River and Lake Ontario water. Parallel filter trains were operated to test passively

operated biofilters (experimental controls), nutrient enhancement with phosphorus and nitrogen,

hydrogen peroxide addition, and in line coagulant using alum (Otonabee River) and PACl (Lake

Ontario) in addition to conventional treatment (coagulation, flocculation, sedimentation and non-

biological filtration) at the Otonabee River pilot.

6.2 Conclusions

Results from the Otonabee River pilot plant indicate that biological filtration can

effectively remove NOM, DBP precursors and MX, but it is not as effective as conventional

treatment. Biofiltration was as effective as conventional treatment with respect to the reduction

of genotoxicity which may indicate that biofiltration is able to remove the most highly reactive

compounds. Chemical enhancements to the biofilters did not show any significant improvements

for the removal of these parameters. A combination of conventional treatment, and biological

filtration appeared to be an effective method of removing PhACs and EDCs by removing 9 of the

10 compounds by more than 50%. Artificial sweeteners were not reliably removed by any of the

processes examined at the Otonabee River location.

Passively operated biofilters were unable to remove DOC from ozonated Lake Ontario

water, but were able to reduce the formation of THMs and HAAs. These results indicate that

biofilters may preferentially remove DBP precursor compounds. Chemical enhancement with 0.8

mg/L PACl was superior to the control biofilters and was required to remove DOC, DBP

precursors, and genotoxicity. PhACs and EDCs were removed more effectively with 0.8 mg/L

than other treatments, and 9 of the 10 compounds were removed by >50%. Artificial sweetener

81

removal was strongly correlated to PACl dose, and 30-40% removals can be expected by dosing

0.8 mg/L.

6.3 Recommendations

The results of this work calls for further investigation into the following areas:

1. This work effectively examined the performance of direct biofiltration, but does not

assess the potential benefits of combining biofiltration with conventional treatment.

Further NOM and DBP precursor removal may be experienced by adding biological

filtration after coagulation, flocculation, and settling.

2. Further work should be completed to characterize microbial communities on the filter

media. If certain microbes can be attributed with contaminant removal the possibility of

spiking the filter with beneficial microbes may be able to improve performance.

3. The impacts of PACl on biofiltration should be explored in more detail. PACl was shown

to limit biofilm activity (as measured by ATP) on the Lake Ontario filters at 0.8 mg/L,

but improved the removal of organic compounds, DBP precursors and emerging

contaminants compared to alum addition at the Otonabee River plant.

4. The benefits of ozonation should be explored at both pilots. DOC removals from

Otonabee River water were lower than expected given the DOC, but may improve

dramatically with ozonation. Pre-ozonation was utilized at the Lake Ontario site, but

higher ozone doses may make the organics more amenable to removal without chemical

enhancements.

5. Additional studies should be completed into the preferential removal of DBP precursors

and genotoxic response by biofiltration. Conventional filtration exceeded the

performance of biofiltration for every parameter, but the combined effect measured by

genotoxicity was similar. The preferential removal of genotoxic precursors by

biofiltration should be further examined to determine what factors contribute to the

reduction and how this reduction can be optimized.

82

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102

8. Appendices

8.1 Standard Operating Procedure Outlines

The following tables describe the basic procedure that was used for sample preparation

and analysis throughout the study. Information for the analysis of ATP (Table 8-1), EPS (Table

8-2), DOC (Table 8-3), trihalomethanes (THMs) (Table 8-4), haloacetic acids (HAAs) (Table

8-5), adsorbable organic halides (AOX) (table), halogenated furanones (Table 8-6 and Table

8-7), genotoxicity (Table 8-8), pharmaceuticals and endocrine disruptors (Table 8-9 and Table

8-10), and artificial sweeteners (Table 8-11 and Table 8-12) is presented below.

Table 8-1: ATP Analysis Method

Calibration

1. Add 100 µL of enzyme reagent (Luminase) and 100 µL of ATP standard (Ultracheck 1)

in a 12 x 55 mm test tube

2. Measure the relative light units (RLU) using the luminometer (RLUstandard)

Sample Analysis

1. Weigh 1 g of sample and transfer to a 5 mL (pre-packaged) UltraLyse™7 tube and mix

2. Wait for 5 minutes

3. Transfer 1 mL of the UltraLyse™7 solution to a 9 mL (pre-packaged) UltraLute vial

4. After mixing the UltraLute solution, pipette 100 µL to a new 12 x 55 mm test tube.

5. Add 100 µL of Luminase and gently swirl 5 times

6. Place the test tube into the luminometer and measure the RLU (RLUsample)

7. Determine ATP concentration using the following equation:

103

Table 8-2: EPS Analysis Method Outline

Sample Storage and Preparation for Analysis

1. Collect 2 g samples in Falcon tubes and store in freezer (-11°C) for up to 14 days

2. Prior to extraction, remove samples from freezer and bring to room temperature (~20°C)

Sample Extraction

1. Prepare Tris-EDTA buffer from concentrate in a 100 mL graduated cylinder by adding 10

mL of concentrated Tris-EDTA and 90 mL of Milli-Q® water

2. Add a magnetic stir bar and stir for 30 min or until reagents are fully dissolved

3. Sterilize in the autoclave at 120°C for 20 minutes and keep at room temperature (~20°C)

4. Add 5 mL of Tris-EDTA buffer solution to each 2 g sample and incubate at 4°C on a

shaker plate for 4 hours

5. Centrifuge samples at 12,000 g at 4°C for 15 minutes

6. Pipette the supernatant while avoiding any surface residue from the media

7. Filter samples into a sterile Falcon tube using a 0.45 µm syringe filter

Protein Analysis

1. Place the hot water bath in the fume hood and heat to 60°C

2. Prepare Pierce™ BCA working reagent by adding 50 parts reagent A to 1 part reagent

3. Prepare 1.5 mL of working reagent for each sample, blank, standard and duplicate

4. Add 75 µL of each concentration of the BCA standard into microcentrifuge tubes

5. Add 75 µL of sample extract into the microcentrifuge tube (with duplicates for each)

6. Add 75 µL of Milli-Q® water into a microcentrifuge tube as a blank

7. In the fume hood, add 1.5 mL of Pierce™ BCA working reagent to the blank, the BSA

serial dilution and the samples

8. Incubate in water bath for 30 min

9. Cool to room temperature and read absorbance at 562 nm (OD562)

Polysaccharide Analysis

1. Place the hot water bath in the fume hood and heat to 100°C

104

Table 8-2: EPS Analysis Method Outline (Cont.)

2. Add 1 mL of each concentration of the glucose standard to the designated glass vials with

matching acid-resistant lids

3. Add 1 mL of sample extracts (in triplicate) and a Milli-Q® blank into the glass vials

4. Add 1 mL of 5% phenol solution into each vial

5. Add 5 mL of 98% sulfuric acid into each vial. Loosely close the vials

6. Using a low speed, vortex the samples in the fume hood

7. Place vials in the water bath for 5 minutes

8. Remove each sample from the water bath and vortex

9. Cover samples with aluminum foil, and incubate for 10 minutes before vortexing

10. Re-cover samples for 30 minutes and vortex

11. Read absorbance at 492 nm (OD492) using a spectrophotometer

Table 8-3: DOC Method Outline

Blanks

Use 40 mL of Milli-Q®

Stock Solution

Mix 2.13 g potassium hydrogen phthalate in 1 L Milli-Q® water. Store at pH ≤ 2 by acidifying

with H2SO4.

Calibration Solution (10 mg/L)

Add 1 mL of stock solution to 99 mL of Milli-Q® water.

Check Standard (2.5 mg/L)

Add 250 μL of stock solution to 99.75 mL Milli-Q® water.

Samples

Follow SOP for TOC analyzer.

Table 8-4: THM Extraction and Analysis Procedure

1. Collect samples in 20 mL amber vials and quench with 0.020 g of ascorbic acid

2. Store samples in the dark at 4⁰C for up to 7 days before analysis

105

Table 8-4: THM Extraction and Analysis Procedure (Cont.)

3. Remove from refrigerator and bring to room temperature before preparation

4. Blank preparation: Transfer 20 mL of Milli-Q® water into 40 mL vial and prepare with

samples.

5. Intermediate solution: Prepare 20 mg/L THM solution by adding 10 mL of stock solution

(2000 μg/mL) to 990 mL of Milli-Q® water.

6.

7. Calibration solutions: Prepare by adding the corresponding amount of intermediate

solution to Milli-Q® water to achieve concentrations of 0, 5, 10, 20, 40, 60, 100, 150, and

200 μg/L

8. **Wipe the syringe tip with a Kimwipe before measuring out of the THM stock or

intermediate solutions, and before adding to intermediate and calibration solutions.

Running Standards (40 μg/L)

1. Add 40 μL of intermediate solution to 20 mL of Milli-Q® water in a 40 mL vial and

process with other samples. Salt and MTBE should be added after adding intermediate

solution.

2. Analyze a blank and running standard after every tenth sample.

Extraction

1. Transfer the contents of each sample vial into a clean 40 mL vial.

2. Add 2 scoops of sodium sulphate (Na2SO4) to increase extraction efficiency. Add 4 mL

of MTBE extraction solvent and cap with a Teflon®-lined silicon septum and cap.

3. Shake vial vigorously for 30 seconds and place vial on its side. Repeat for all samples,

blanks and standards. Place samples upright in a rack and shake for 2 minutes. Let stand

for 10 minutes.

4. Extract 2 mL from the organic layer using a Pasteur pipette and place in a 1.8 mL GC

vial filled with 2 small scoops of Na2SO4 (no water should remain in the vial). Use a

clean pipette for each sample. Fill the vial to the top and cap immediately, ensuring there

is no headspace. To ensure the MTBE phase was the only one taken, freeze the vials for

2 hours and observe that only 1 phase is visible.

5. If not analyzing immediately, freeze samples for up to 21 days.

6. Analyze using a GC-ECD.

106

Table 8-5: HAA Analysis Procedure

1. Collect samples in 40 mL amber vials. Ensure that samples are free of headspace.

2. Store samples in the dark at 4⁰C for up to 9 days.

3. To begin sample preparation, remove from refrigerator and bring to room temperature.

Blanks

1. Transfer 40 mL of Milli-Q® water into 40 mL vials and process with other samples.

Calibration Solutions

1. Collect 20 mL of Milli-Q® in 40 mL vial

2. Using a 50 μL syringe and the “sandwich” technique, add 0, 5, 10, 20, 40, 60, 100, and

150 μL of HAA stock (2000 μg/mL each)

**Wipe the syringe tip with a Kimwipe before measuring out of the HAA stock and

before adding the stock to other solutions.

Running Standards

1. Add 50 μL of working solution to 20 mL of Milli-Q® water, process alongside samples.

2. Include blanks and running standards after every tenth sample.

Diazomethane Generation

1. Set up MNNG diazomethane generation apparatus on ice using a beaker filled with

crushed ice and water.

2. Add 2.5 mL of MTBE to outer tube of generator, and cover with tin foil before placing in

ice bath.

3. Add ~ 1.25 cm of diazald to inner tube of generator using large end of Pasteur pipette.

Add ~0.5 mL of methanol to cover diazald by 3 mm and secure cap and septum.

4. Place O-ring in glass joint. Position inside tube firmly on top and secure clamp. Ensure

that the vapour exit hole is located on the opposite side of the tube from the clamp and

place clamp on the spout of the beaker. Ensure the seal is tight to maximize CH2N2

generation and recovery.

5. Let cool on the ice bath for 10 min.

107

Table 8-5: HAA Extraction Procedure (Cont.)

6. Add 600 μL of 20% NaOH solution dropwise to inner tube with a gas tight syringe (1

drop/5 sec). Aim drops directly into the diazald in the bottom of the inner tube. Leave the

syringe in place after all of the NaOH is added to avoid a hole in the septum from which

CH2N2 may exit.

7. Allow CH2N2 to form for 30 to 45 minutes on the ice bath.

8. Transfer CH2N2 in MTBE (which is now yellow) to 4 mL vials using a specially flamed

Pasteur pipette and store vials in an explosion-proof freezer (use within 2-4 weeks).

9. Rinse inner tube and syringe several times with Milli-Q® water. Rinse inner and outer

tubes with methanol and MTBE until glassware is clean and dry at 100⁰C.

Extraction

1. Transfer 20 mL of sample into a clean 40 mL vial.

2. Add 3 mL of sulphuric acid (H2SO4). Add 6 g of sodium sulphate (Na2SO4). Add 5 mL of

MTBE extraction solvent and cap with a Teflon®-lined silicon septa and screw cap.

3. Shake sample for ~30 seconds and place vial on its side. Repeat for all blanks, calibration

solutions and samples.

4. Place samples upright in a rack and shake for 5 minutes. Let samples stand for 15

minutes.

5. Extract 1.5 mL from the organic layer and place into a 1.8 mL GC vial using a clean

pipette for each sample. Add 150 μL of diazomethane to the GC vial (submerging the

syringe tip before injection) and seal immediately.

6. If not analyzing immediately, store the samples in a freezer (-20⁰C) for up to 21 days.

7. Analyze using a GC-ECD.

108

Table 8-6: MX and MCA Sample Preparation Details

1. 1 L water samples in amber bottles

a. Calibration and check standards are prepared in matrix-matched 1L samples

(spike standards into 1L 0.45um filtered, acidified raw water samples)

2. Acidify to pH 2 with 20 drops of concentrated H2SO4

3. Keep samples in the dark at 4°C until ready for extraction

4. Just prior to extraction, take sample out of fridge and spike with 100 ng/L of MBA

Table 8-7: MX and MCA Solid Phase Extraction Details

1. Solid phase extraction (SPE) is completed using a Visiprep vacuum manifold and a

tandem SPE set-up, with a tC18 column (for humics removal) followed by an Oasis HLB

column for analyte retention

2. Condition the SPE columns under gravity (collect acetone and methanol in spare falcon

tubes for proper disposal). Ensure that the cartridges are not exposed to air after the

acetone step (Important! Maintain ~1mm of solvent/aqueous layer on top of SPE media

at all times)

a. tC18 (5mL acetone – preconditioning, 5mL methanol – conditioning, 5mL Milli-

Q® water – aqueous buffer)

b. Oasis HLB (10mL acetone – preconditioning, 10mL methanol – conditioning,

10mL Milli-Q® water – aqueous buffer)

3. Connect SPE sampling tube to tC18 cartridge, and connect tC18 column on top of HLB

column with fitting, place unit on appropriate port on the Visiprep SPE vacuum manifold

4. Loading of 1 L samples, under light vacuum at ~ 20 mL/min

5. After each sample is loaded, ~ 100 mL of acidified Milli-Q® water (pH 2) added to each

sample vessel to transfer any remaining sample

6. Discard t18 cartridge, HLB cartridges are then dried under vacuum for about 1 hour or

until colour change is noticed

7. Elution by gravity with two 5 mL aliquots of acetone, collected in falcon tubes

8. Evaporate acetone under light flow of nitrogen, until ~ 0.5 mL remaining

9. Transfer the 0.5 mL acetone samples into respective GC vials, then rinse the walls of

falcon tubes with fresh acetone (~ 0.5 mL) and add to GC vial

109

Table 8-7: MX and MCA Solid Phase Extraction Details (Cont.)

10. Evaporate GC vial samples to completion under nitrogen

11. Add 300 uL of 2% H2SO4 in methanol (v/v) for derivatization in the oven at 70ºC for 1 hr

12. Remove samples and allow them to cool to room temperature

13. Add 750 uL of 2% NaHCO3 in Milli-Q® water (w/v) to neutralize the samples

14. Add 600 uL of hexane, shake for 2 minutes, and transfer hexane layer to new GC vial

15. Repeat with another 600 uL of hexane

16. Hexane extract should be ~1 mL

17. Concentrate under nitrogen to a volume of ~0.1 mL

18. Transfer to glass inserts, place insert into GC vial

19. Samples are ready for GC-MS analysis

Table 8-8: Genotoxicity Sample Preparation

1. Solid phase extraction (SPE) is completed using a Visiprep vacuum manifold and an

Oasis HLB column for analyte retention

2. Condition the SPE columns under gravity with 10 mL of acetone for preconditioning,

10 mL of methanol for conditioning and 10 mL of Milli-Q® water as an aqueous buffer

(collect acetone and methanol in spare falcon tubes for proper disposal). Ensure that the

cartridges are not exposed to air after the acetone step (Important! Maintain ~1mm of

solvent/aqueous layer on top of SPE media at all times)

3. Connect SPE sampling tube to the top of HLB column with fitting, place unit on

appropriate port on the Visiprep SPE vacuum manifold

4. Loading of 2L samples, under light vacuum at ~ 20 mL/min

5. After each sample is loaded, ~ 100 mL of acidified Milli-Q® water (pH 2) added to

each sample vessel to transfer any remaining sample

6. HLB cartridges then dried under vacuum for about 1 hour or until colour change is

noticed

7. Elution by gravity with two 4.5 mL aliquots of acetone, collected in falcon tubes

110

Table 8-8: Genotoxicity Sample Preparation (Cont.)

8. Evaporate acetone under light flow of nitrogen

9. Reconstitute samples by adding 30 µL of dimethyl sulfoxide (DMSO).

10. Add 1 µL of the reconstituted sample to 4 µL of 10% DMSO solution into one section of

a 96 well microplate

11. Dilute each sample two-fold into the five respective wells, for a total of six test

concentrations for each sampl

12. Add varying concentrations of the known carcinogen 4-NQO to wells alongside the

diluted samples

13. Add 100 µL of diluted bacterial suspension (prepared overnight and diluted to 0.5 optical

density at 600 nm, OD600, prior to use) to each well

14. Incubate at 37°C for 2 hours

15. After incubation, add 100 µL of chromogen for β-gal and alkaline phophatase (AP)

activity determination and incubate for 1 hour at 37°C

16. Put plate on microplate reader to read the activity of β-gal (OD605) and AP (OD420)

Table 8-9: PhAC Sample Preparation Method

1. 400 mL water samples in amber bottles

a. Make sure samples are filtered through 0.45 um PTFE filter

b. Calibration and check standards are prepared in matrix-matched 400 mL samples

(also 0.45 um filtered)

2. Add ~ 2 g of Na2 EDTA to each sample, blank, and standard. Place on a shaker for 20

minutes or more to completely dissolve the EDTA.

3. Add 10 mL of 0.25 M ammonium acetate solution to each sample, blank, and standard.

4. Make sure the pH meter is calibrated with standards of pH 4, 7, 10.

5. Using the pH meter, slowly adjust the pH of each sample, blank, and standard to 6.95 ±

0.05 using 50 % NaOH, 10 % NaOH, and 10 % H2SO4 solutions.

6. Keep samples in the dark at 4°C until ready for extraction.

7. Samples and standards must be spiked with surrogate standards prior to SPE.

111

Table 8-10: PhAC and EDC Extraction Method

1. Solid phase extraction (SPE) is completed using a Visiprep vacuum manifold and a

tandem SPE set-up, with OASIS HLB 6 cc cartridges.

2. Condition the SPE columns under gravity (collect acetone and methanol in spare falcon

tubes for proper disposal). Important! Maintain ~1mm of solvent/aqueous layer on top of

SPE media at all times.

Oasis HLB (6 mL methanol – preconditioning, 6 mL Milli-Q® water –

preconditioning)

3. Refill the cartridges with 6 mL Milli-Q® water and leave until samples are introduced.

4. Load 400 mL samples under light vacuum at ~ 5 mL/min (to maintain a linear velocity of

0.2 cm/s). Make sure all sample bottles and cartridges are labelled.

5. After samples are passed through, dry cartridges under vacuum until they appear dry (at

least 30 minutes). Make sure to dry the cartridges with Kimwipes and dry the inside of

the SPE manifold prior to applying vacuum to speed up drying.

6. Dry the cartridges for under high vacuum until they appear dry.

7. Either store cartridges in the freezer until elution, or proceed with the elution.

8. Clean Teflon guides with methanol and replace on the bottom side of the manifold.

9. Add 5 mL of methanol to each cartridge and let it stand for 3 minutes.

10. Elute samples by gravity at 0.5 mL/min.

11. Mix the eluent in the centrifuge tubes by placing each sample on the vortex.

12. Using a cleaned syringe, transfer 1 mL of each final extract for each analysis into a 2 mL

amber vial.

13. Evaporate GC vial samples to dryness with nitrogen.

14. Bring volume of sample up to 300 uL using reconstitution solution (see solutions) to add

the internal standards.

15. Using the 0.5 mL luer lock syringe and a new disposable needle for each sample, draw

each sample into the syringe.

16. Place a 4 mm 0.2 um PTFE syringe filter on the end between the syringe and the needle.

112

Table 8-10: PhAC and EDC Extraction Method (Cont.)

17. Expel the sample out of the syringe into a glass insert. Make sure you leave some

headspace at the top or the autosampler will not be able to draw the sample properly.

18. Between each sample, use a dedicated disposable needle (this way it will not need to be

cleaned) for drawing LC water into the syringe to clean the syringe.

19. Samples are ready for LC-MS analysis

20. Samples best analyzed immediately after processing or store at 4°C until analysis.

Table 8-11: Artificial Sweetener Sample Preparation Process

1. 200 mL water samples in amber bottles

a. Make sure samples are filtered through 0.45 um PTFE filter

b. Calibration and check standards are prepared in matrix-matched 200 mL

samples (also 0.45 um filtered)

2. Acidify samples and standards to pH 1.5 with 15 drops of concentrated H2SO4

3. Keep samples in the dark at 4°C until ready for extraction

4. Samples and standards must be spiked with 1 ppm acesulfame K-d4 and 10 ppm

sucralose-d6 in acetonitrile prior to SPE

Table 8-12: Artificial Sweetener SPE Details

1. Solid phase extraction (SPE) is completed using a Visiprep vacuum manifold and a

tandem SPE set-up, with OASIS MCX 6 cc cartridges.

2. Condition the SPE columns under gravity (collect acetone and methanol in spare falcon

tubes for proper disposal). Important! Maintain ~1mm of solvent/aqueous layer on top of

SPE media at all times.

Oasis MCX (6 mL acetone – preconditioning, 6 mL methanol – preconditioning,

6 mL Milli-Q® water acidified to pH 1.5 using H2SO4 – preconditioning)

3. Refill the cartridges with 6 mL Milli-Q® water acidified to pH 1.5 and leave until

samples are introduced.

113

Table 8-12: Artificial Sweetener SPE Details (Cont.)

4. Load 200 mL samples under light vacuum at ~ 3 mL/min (to maintain a linear velocity of

0.1 cm/s). Make sure all sample bottles and cartridges are labeled.

5. After samples are passed through, dry cartridges under vacuum until they appear dry (at

least 30 minutes). Make sure to dry the cartridges with Kimwipes and dry the inside of

the SPE manifold prior to applying vacuum to speed up drying.

6. Clean Teflon guides with methanol and replace on the bottom side of the manifold.

Place glass 15 mL glass centrifuge tubes under each Teflon guide in the manifold.

7. Add 3 mL of 5 % ammonium hydroxide in methanol to each cartridge, wet the stationary

phase by pulling a little bit of solvent (~0.1-0.2 mL) by vacuum, and let it stand for 6

minutes.

8. Elute samples by gravity at 0.5 mL Do not let it go dry. Re-add 3 mL of 5 %

ammonium hydroxide in methanol to each cartridge and let it stand again for 6 minutes.

9. Elute one more time, and this time the sample can go dry.

10. Dry the cartridges for 2 minutes under high vacuum.

11. Either store eluent at 4oC overnight, or evaporate methanol under light flow of nitrogen,

until ~ 1-2 mL remaining.

12. Add 1-2 mL of methanol, vortex, and replace to continue evaporating under nitrogen.

13. Evaporate until the volume is about 0.5 mL, then transfer the 0.5 mL methanol samples

into respective GC vials, then rinse the walls of glass centrifuge tubes with fresh

methanol (~ 0.5 mL) and add to GC vial.

14. Evaporate GC vial samples to near dryness (~40 uL) with nitrogen.

15. Bring volume of sample up to 400 uL (with ~360 uL) using reconstitution solution to

bring to 90:10 water:acetonitrile with 0.1% acetic acid.

16. Using the 0.5 mL luer lock syringe and a new disposable needle for each sample, draw

each sample into the syringe.

17. Place a 4 mm 0.2 um PTFE syringe filter on the end between the syringe and the needle.

18. Expel the sample out of the syringe into a glass insert. Make sure you leave some

headspace at the top or the autosampler will not be able to draw the sample properly.

19. Between each sample, use a dedicated disposable needle (this way it will not need to be

cleaned) for drawing LC water into the syringe to clean the syringe.

8.2 Raw Data

Analytical results for the Otonabee River pilot plant are presented in Table 8-13. This table includes sample results for media

characterization (ATP, proteins, polysaccharides), general water quality (pH, turbidity, UV254, and DOC), LC-OCD, DPB FP,

genotoxicity, and emerging contaminant concentrations. Results for the Lake Ontario pilot plant are presented in Table 8-14 and Table

8-15. Biological characterization, general water quality, DBP FP, and genotoxicity is presented in Table 8-14, while emerging

contaminant data is presented in Table 8-15.

Table 8-13: Raw Data - Otonabee River Pilot Plant

Date Raw Water Settled Conv. Control Nutrients Control Peroxide Alum GAC

Media Type N/A N/A N/A Anthracite and Sand

Anthracite and Sand

Anthracite and Sand

Anthracite and Sand

Anthracite and Sand

Anthracite and Sand

GAC and Sand

Filter Type N/A N/A N/A Conv. Bio Bio Bio Bio Bio Bio

Influent Conditions

N/A Raw Water Settled Water

Alum Coag Control 0.5 mg/L PO4, NH4

Control 0.2 mg/L

H2O2 0.2 mg/L

Alum Control

Column Diameter N/A N/A N/A 15.24 cm 15.24 cm 15.24 cm 7.62 cm 7.62 cm 7.62 cm 7.62 cm

Flow Rate N/A N/A N/A 1.6 L/min 1.6 L/min 1.6 L/min 0.4 L/min 0.4 L/min 0.4 L/min 0.4 L/min

EBCT N/A N/A N/A 11 mins 11 mins 11 mins 11 mins 11 mins 11 mins 11 mins

ATP (pg/g media)

04-Mar-14

Not Sampled

Not Sampled

10526.5 1080434 1235653 818657 851704 880827 1006921

20-May-14 No Sample 1137000 1554000 829950 1041500 771850 875450

23-Jun-14 33810 1690000 1911000 421600 1102000 910700 2322000

28-Jul-14 129025 3083100 4795820 1320910 1845790 1263455 1064080

22-Sep-14 37820 2012814 2270995 1326613 1000582 1561928 1013532

TABLE 8-13: Raw Data – Otonabee River Pilot Plant (Cont.)

115

Date Raw Water Settled Conv. Control Nutrients Control Peroxide Alum GAC

Proteins (ug/g media)

04-Mar-14

Not Sampled

Not Sampled

156.6 745.2 662.3 482.2 382.2 389.0 595.8

20-May-14 No Sample 836.8 940.6 659.2 513.7 531.7 688.7

23-Jun-14 59.5 727.7 716.8 506.9 460.0 373.0 576.1

28-Jul-14 38.5 741.0 748.5 634.4 581.9 456.9 615.2

22-Sep-14 33.1 623.0 602.7 540.6 523.7 410.1 485.9

Polysacharides (ug/g media)

04-Mar-14

Not Sampled

Not Sampled

67.85 115.75 109.8 74.25 68.9 62.7 83.55

20-May-14 No Sample 244.57 214.23 85.17 62.94 133.67 107.60

23-Jun-14 6.16 124.78 117.58 28.38 45.18 46.58 52.78

28-Jul-14 1.65 53.55 57.6 39.05 35.95 36.2 31.7

22-Sep-14 33.55 89.4 87.05 50.3 45.95 62.7 72.05

pH

04-Mar-14 8.17 7.46 7.50 8.11 7.81 7.90 7.96 7.69 7.91

20-May-14 7.68 6.88 7.14 7.59 7.52 7.61 7.73 7.66 7.58

23-Jun-14 7.93 7.36 7.37 7.82 7.60 7.73 7.69 7.98 7.64

28-Jul-14 7.77 6.34 6.38 7.09 6.70 7.08 7.02 7.25 7.06

22-Sep-14 8.10 6.95 6.90 7.75 7.61 7.72 7.73 7.76 7.71

Turbidity (NTU)

04-Mar-14 0.32 1.16 0.088 0.18 0.187 0.165 0.174 0.182 0.165

20-May-14 0.442 0.366 0.074 0.19 0.154 0.138 0.14 0.155 0.164

23-Jun-14 0.858 0.232 0.098 0.188 0.23 0.2 0.238 0.214 0.212

28-Jul-14 0.575 0.227 0.102 0.374 0.365 0.347 0.234 0.206 0.297

22-Sep-14 0.61 0.217 0.079 0.133 0.706 0.207 0.158 0.195 0.202

UV254

04-Mar-14 0.168 0.062 0.059 0.158 0.167 0.166 0.166 0.166 0.165

20-May-14 0.144 0.059 0.055 0.137 0.170 0.139 0.139 0.141 0.136

23-Jun-14 0.128 0.052 0.053 0.120 0.114 0.121 0.116 0.122 0.090

28-Jul-14 0.127 0.056 0.054 0.103 0.108 0.104 0.103 0.106 0.098

22-Sep-14 0.112 0.053 0.047 0.109 0.111 0.111 0.111 0.112 0.106

TABLE 8-13: Raw Data – Otonabee River Pilot Plant (Cont.)

116

Date Raw Water Settled Conv. Control Nutrients Control Peroxide Alum GAC

DOC (mg/L)

04-Mar-14 5.17 2.67 2.67 5.46 5.01 4.94 5.00 4.92 5.03

20-May-14 4.70 2.59 2.55 4.46 6.47 4.23 4.26 4.21 4.25

23-Jun-14 4.84 2.62 2.76 4.34 4.29 4.38 4.26 4.45 3.37

28-Jul-14 4.96 2.77 2.73 4.62 4.89 4.82 4.60 4.84 4.62

22-Sep-14 4.95 2.72 2.61 4.55 4.64 4.52 4.61 4.68 4.63

Total THM (ug/L)

04-Mar-14 234.8 84.8 67.1 182.2 184.1 190.2 188.2 214.6 189.1

20-May-14 204.4 78.3 78.6 182.9 174.4 151.8 160.4 168.0 181.3

23-Jun-14 110.5 59.0 63.6 97.9 101.4 107.9 105.4 104.4 81.2

28-Jul-14 125.4 53.5 47.1 91.9 116.3 128.7 115.0 122.3 116.4

22-Sep-14 104.3 50.2 49.5 91.6 92.3 91.8 101.7 96.3 93.1

TCM (ug/L) (Avg. , (Std. Dev.))

04-Mar-14 204(2.31) 71.2 (0.94) 55.6 (0.33) 158.0 (0.98) 157.1 (1.33) 164.7 (3.42) 163.4 (1.48) 186.5 (4.22) 164.0 (3.69)

20-May-14 177(4.30) 65.3 (8.51) 65.5 (1.23) 158.2 (5.11) 152.0 (3.03) 132.5 (7.49) 140.5 (5.85) 145.7 (9.94) 158.2 (3.73)

23-Jun-14 92.5 (3.03) 47.9 (3.76) 51.4 (2.37) 83.0 (3.45) 86.5 (2.39) 92.6 (2.70) 90.7 (4.33) 88.4 (4.23) 63.4 (0.51)

28-Jul-14 103 (4.70) 42.5 (2.43) 37.3 (0.90) 75.4 (6.78) 96.1 (1.96) 106.8 (7.09) 95.1 (2.44) 101.0 (2.84) 96.4 (6.32)

22-Sep-14 75.8 (4.72) 35.6 (1.89) 37.0 (2.15) 68.5 (1.16) 73.0 (2.06) 72.5 (1.95) 80.1 (0.41) 74.5 (3.66) 73.4 (1.35)

BDCM (ug/L) (Avg., (Std. Dev.))

04-Mar-14 31.3 (1.69) 13.5 (0.69) 11.5 (0.24) 24.2 (0.71) 27.0 (0.35) 25.5 (0.71) 24.8 (0.01) 28.1 (3.08) 25.1 (0.46)

20-May-14 27.9 (1.11) 13.1 (0.93) 13.2 (0.38) 24.7 (0.97) 22.5 (0.24) 19.3 (1.23) 19.9 (0.80) 22.3 (1.47) 3.73 (0.67)

23-Jun-14 18.0 (0.38) 11.1 (0.69) 12.2 (0.20) 14.9 (0.64) 14.9 (0.71) 15.26 (0.45) 14.7 (0.43) 16.1 (0.87) 17.8 (0.23)

28-Jul-14 22.4 (1.38) 11.0 (0.45) 9.80 (0.29) 16.6 (1.48) 20.3 (0.48) 21.8 (1.45) 19.9 (0.63) 21.3 (0.67) 19.9 (1.26)

22-Sep-14 28.6 (5.89) 14.6 (3.54) 12.5 (0.95) 23.0 (6.07) 19.3 (0.56) 19.3 (0.59) 21.6 (0.22) 21.8 (0.75) 19.8 (0.41)

Total HAA (ug/L)

04-Mar-14 96.8 34.5 29.5 81.4 90.3 92.6 94.5 79.1 95.3

20-May-14 81.2 46.6 43.1 77.1 78.5 73.4 73.0 73.4 73.6

23-Jun-14 83.7 52.6 47.4 72.0 67.2 67.3 74.0 74.6 64.0

28-Jul-14 60.2 30.6 29.1 61.3 65.3 62.2 66.2 66.8 65.0

22-Sep-14 102.2 60.0 45.9 96.6 97.2 91.2 96.9 95.6 96.4

TABLE 8-13: Raw Data – Otonabee River Pilot Plant (Cont.)

117

Date Raw Water Settled Conv. Control Nutrients Control Peroxide Alum GAC

MCAA (ug/L) (Avg., (Std. Dev.))

04-Mar-14 1.86 (0.35) 1.38 (0.26) 0.82 (0.24) 1.35 (0.20) 2.74 (0.05) 2.58 (0.02) 2.71 (0.09) 2.53 (0.17) 2.66 (0.05)

20-May-14 3.08 (0.34) 3.67 (0.41) 3.77 (0.33) 3.76 (0.50) 3.83 (0.65) 3.90 (0.36) 3.65 (0.59) 3.60 (0.20) 3.67 (0.42)

23-Jun-14 1.69 (0.02) 0.94 (0.15) 0.84 (0.05) 0.96 (0.07) 0.91 (0.26) 0.90 (0.07) 0.90 (0.07) 1.17 (0.11) 0.59 (0.03)

28-Jul-14 1.75 (0.10) 1.26 (0.04) 1.21 (0.12) 2.07 (0.17) 2.16 (0.05) 2.15 (0.12) 2.06 (0.22) 2.61 (0.57) 2.05 (0.45)

22-Sep-14 5.21 (1.38) 3.19 (0.45) 2.50 (0.74) 4.43 (1.87) 6.61 (0.25) 6.58 (0.44) 4.40 (0.53) 4.08 (1.29) 5.78 (0.23)

DCAA (ug/L) (Avg., (Std. Dev.))

04-Mar-14 40.4 (0.83) 11.7 (0.03) 10.0 (0.84) 31.6 (0.67) 35.7 (0.89) 36.9 (0.53) 37.2 (0.59) 30.1 (0.57) 36.8 (0.21)

20-May-14 41.4 (1.17) 22.2 (0.14) 21.4 (0.57) 38.0 (0.73) 38.5 (0.42) 36.3 (1.51) 35.0 (0.64) 36.6 (0.61) 36.0 (0.49)

23-Jun-14 37.8 (1.21) 21.9 (0.25) 21.4 (0.19) 28.9 (0.14) 26.6 (3.98) 27.0 (0.86) 28.9 (0.91) 30.5 (0.24) 23.1 (0.48)

28-Jul-14 26.3 (0.47) 13.8 (1.88) 15.4 (0.43) 26.9 (0.83) 29.7 (0.70) 30.0 (0.43) 30.8 (0.39) 31.2 (0.37) 29.8 (0.46)

22-Sep-14 55.4 (1.89) 35.4 (1.86) 25.6 (2.44) 51.0 (7.28) 48.1 (0.09) 51.9 (3.50) 50.6 (1.41) 49.7 (3.40) 48.6 (1.52)

TCAA (ug/L) (Avg., (Std. Dev.))

04-Mar-14 54.6 (1.58) 21.4 (0.28) 18.8 (1.38) 48.5 (0.53) 51.9 (3.30) 53.1 (0.93) 54.6 (0.20) 46.5 (0.78) 55.8 (0.59)

20-May-14 36.7 (0.99) 20.7 (0.15) 18.0 (0.18) 35.3 (0.76) 36.2 (0.65) 33.2 (1.32) 34.3 (0.46) 33.2 (1.05) 33.9 (0.98)

23-Jun-14 44.2 (1.51) 29.7 (0.25) 25.2 (0.07) 42.2 (0.10) 39.7 (5.58) 39.4 (1.51) 44.1 (1.07) 42.9 (0.51) 40.4 (0.42)

28-Jul-14 32.2 (0.49) 15.5 (0.44) 12.5 (0.15) 32.3 (0.92) 33.4 (0.41) 30.0 (0.25) 33.3 (0.87) 33.0 (0.65) 33.1 (1.68)

22-Sep-14 41.6 (2.47) 21.5 (2.76) 17.8 (1.39) 41.3 (3.66) 42.5 (0.87) 32.7 (22.0) 42.0 (1.44) 41.8 (1.97) 42.0 (0.95)

Total AOX (ug/L) (Avg., (Std. Dev.))

04-Mar-14 691.9 (30.1) 269.3 (6.84) 262.1 (3.60) 668.7 (13.5) 634.7 (9.98) 581.1 (2.34) 621.3 (14.9) 702.6 (13.8) 577.4 (11.3)

20-May-14 597.8 (11.4) 185.0 (7.98) 200.2 (32.0) 432.8 (10.7) 429.3 (55.7) 435.3 (14.1) 411.8 (37.5) 400.2 (22.7) 474.4 (41.4)

23-Jun-14 454.5 (17.8) 206.7 (16.9) 241.8 (7.18) 438.6 (3.17) 432.6 (22.9) 416.0 (10.6) 420.8 (5.98) 405.3 (18.1) 323.9 (18.3)

28-Jul-14 433.8 (10.1) 219.4 (22.6) 209.0 (9.23) 417.4 (31.3) 438.2 (11.9) 416.0 (4.99) 416.1 (54.7) 417.4 (23.2) 391.6 (32.6)

22-Sep-14 438.3 (96.1) 231.1 (6.13) 215.1 (8.51) 403.5 (21.1) 349.2 (27.8) 349.9 (9.07) 319.3 (32.8) 382.3 (9.74) 319.8 (43.3)

Bio-polymers (mg/L)

04-Mar-14 0.274 0.151 0.141 0.275 0.245 0.298 0.307 0.266 0.388

20-May-14 0.352 0.128 0.142 0.242 0.265 0.190 0.197 0.215 0.200

23-Jun-14 0.479 0.219 0.190 0.294 0.273 0.287 0.241 0.322 0.292

28-Jul-14 0.587 0.180 0.161 0.377 0.510 0.397 0.260 0.441 0.391

22-Sep-14 0.404 0.139 0.139 0.261 0.261 0.231 0.284 0.27 0.281

TABLE 8-13: Raw Data – Otonabee River Pilot Plant (Cont.)

118

Date Raw Water Settled Conv. Control Nutrients Control Peroxide Alum GAC

Humics (mg/L)

04-Mar-14 3.97 1.87 1.48 4.01 3.80 3.70 3.84 3.74 3.78

20-May-14 3.49 1.64 1.58 3.17 3.47 3.20 3.21 3.30 3.17

23-Jun-14 3.31 1.43 1.72 3.18 3.08 3.10 3.16 3.23 2.54

28-Jul-14 3.38 1.25 1.36 3.22 3.19 3.25 3.23 3.20 3.18

22-Sep-14 3.347 1.745 1.717 3.261 3.191 3.214 3.243 3.216 3.235

Building Blocks (mg/L)

04-Mar-14 0.844 0.447 0.815 0.794 0.874 0.838 0.757 0.934 0.689

20-May-14 0.657 0.482 0.487 0.693 2.810 0.627 0.662 0.604 0.650

23-Jun-14 0.716 0.780 0.500 0.713 0.735 0.681 0.639 0.588 0.482

28-Jul-14 0.771 0.853 0.761 0.695 0.822 0.765 0.712 0.803 0.791

22-Sep-14 0.913 0.348 0.317 0.812 0.876 0.826 0.805 0.936 0.809

LWM Acids (mg/L)

04-Mar-14 n.q. n.q. n.q. 0.048 0.026 0.024 0.030 0.029 0.034

20-May-14 0.010 n.q. n.q. 0.011 n.q. 0.007 0.009 0.005 0.015

23-Jun-14 0.031 n.q. n.q. 0.002 0.006 0.013 n.q. 0.002 n.q.

28-Jul-14 0.003 n.q. n.q. 0.012 0.01 0.001 0.005 0.009 0.013

22-Sep-14 0.005 n.q. n.q. n.q. n.q. 0.003 n.q. 0.001 0.007

LMW Neutrals (mg/L)

04-Mar-14 0.554 0.583 0.513 0.580 0.533 0.492 0.560 0.516 0.574

20-May-14 0.448 0.482 0.422 0.384 0.499 0.401 0.394 0.402 0.357

23-Jun-14 0.553 0.538 0.464 0.496 0.457 0.468 0.463 0.487 0.337

28-Jul-14 0.466 0.359 0.389 0.407 0.536 0.437 0.421 0.413 0.638

22-Sep-14 0.484 0.435 0.418 0.457 0.477 0.4 0.465 0.438 0.532

MX (ng/L)

04-Mar-14 22.2 8.45 9.7 24.5

Not Sampled Not Sampled Not Sampled

23.6

Not Sampled

20-May-14 15.5 5 5 11 9

23-Jun-14 12 6 5 9 8

28-Jul-14 19 7 8 16 16

22-Sep-14 12.5 5 <MDL 7.5 6.5

TABLE 8-13: Raw Data – Otonabee River Pilot Plant (Cont.)

119

Date Raw Water Settled Conv. Control Nutrients Control Peroxide Alum GAC

MCA (ng/L)

04-Mar-14 705 230 215 855

Not Sampled Not Sampled Not Sampled

685

Not Sampled

20-May-14 Not Analyzed NA

23-Jun-14 630 330 240 540 440

28-Jul-14 535 225 220 545 505

22-Sep-14 285 105 40 165 150

Genotoxicity (SOSIP Corrected)

04-Mar-14 0.895 0.576 0.67 0.716

Not Sampled Not Sampled Not Sampled

0.829

Not Sampled

20-May-14 0.35 0.328 0.358 0.486 0.439

23-Jun-14 0.45 0.269 0.371 0.24 0.359

28-Jul-14 0.1925 0.142 0.1455 0.153 0.165

22-Sep-14 0.192 0.158 0.129 0.158 0.190

Sucralose (ng/L)

11-Mar-14 1500 1100 1050 1100

Not Sampled Not Sampled Not Sampled

700

Not Sampled

26-May-14 395 410 323 304 340

02-Jul-14 519 513 562 512 507.5

04-Aug-14 501 428 540 494 473

29-Sep-14 361 313 291 350 358

Acesulfame Potassium (ng/L)

11-Mar-14 2070 2400 2525 1980

Not Sampled Not Sampled Not Sampled

2220

Not Sampled

26-May-14 238.5 179.5 179 242.5 230

02-Jul-14 407 315 310.5 306 313.5

04-Aug-14 334 302 290 343 347

29-Sep-14 412 332 309 445 371

Acetaminophen

11-Mar-14 57 70.5 <MDL 98.5

Not Sampled Not Sampled Not Sampled

82

Not Sampled

26-May-14 240 49 0 26 95

02-Jul-14 79 51 49 <MDL <MDL

04-Aug-14 111 138 126 <MDL <MDL

29-Sep-14 149 124 91 <MDL <MDL

TABLE 8-13: Raw Data – Otonabee River Pilot Plant (Cont.)

120

Date Raw Water Settled Conv. Control Nutrients Control Peroxide Alum GAC

Bisphenol-A

11-Mar-14 20.5 32 79.4 50.5

Not Sampled Not Sampled Not Sampled

41.5

Not Sampled

26-May-14 <MDL <MDL <MDL <MDL <MDL

02-Jul-14 <MDL <MDL <MDL <MDL <MDL

04-Aug-14 <MDL <MDL <MDL <MDL <MDL

29-Sep-14 <MDL <MDL <MDL <MDL <MDL

Clofibric Acid

11-Mar-14 362.5 144.5 227 104

Not Sampled Not Sampled Not Sampled

192

Not Sampled

26-May-14 <MDL <MDL <MDL <MDL <MDL

02-Jul-14 <MDL <MDL <MDL <MDL <MDL

04-Aug-14 <MDL <MDL <MDL <MDL <MDL

29-Sep-14 173 136 122 142 152

Diclofenac

11-Mar-14 254 176 242 196

Not Sampled Not Sampled Not Sampled

153

Not Sampled

26-May-14 340.5 104 112.5 <MDL <MDL

02-Jul-14 264.5 196.5 196.5 437.5 153.5

04-Aug-14 174 150 144 157 164

29-Sep-14 173 126 104 175 154

Diethylstilbestrol

11-Mar-14 <MDL <MDL <MDL <MDL

Not Sampled Not Sampled Not Sampled

<MDL

Not Sampled

26-May-14 <MDL <MDL <MDL <MDL <MDL

02-Jul-14 <MDL <MDL <MDL <MDL <MDL

04-Aug-14 <MDL <MDL <MDL <MDL <MDL

29-Sep-14 <MDL <MDL <MDL <MDL <MDL

Estriol

11-Mar-14 <MDL <MDL <MDL <MDL

Not Sampled Not Sampled Not Sampled

<MDL

Not Sampled

26-May-14 <MDL <MDL <MDL <MDL <MDL

02-Jul-14 168.5 122 116 <MDL 191

04-Aug-14 <MDL <MDL <MDL <MDL <MDL

29-Sep-14 <MDL <MDL <MDL <MDL <MDL

TABLE 8-13: Raw Data – Otonabee River Pilot Plant (Cont.)

121

Date Raw Water Settled Conv. Control Nutrients Control Peroxide Alum GAC

Estrone

11-Mar-14 <MDL <MDL 68.1 <MDL

Not Sampled Not Sampled Not Sampled

<MDL

Not Sampled

26-May-14 <MDL <MDL <MDL <MDL <MDL

02-Jul-14 134 87 54.5 <MDL <MDL

04-Aug-14 34 35 46 <MDL <MDL

29-Sep-14 43 29 <MDL <MDL <MDL

Gemfibrozil

11-Mar-14 210.5 188 214.5 144.5

Not Sampled Not Sampled Not Sampled

178.5

Not Sampled

26-May-14 <MDL <MDL <MDL <MDL <MDL

02-Jul-14 195 147 150 54.5 <MDL

04-Aug-14 140 128 124 <MDL 78

29-Sep-14 160 129 117 28 75

17 β estradiol

11-Mar-14 <MDL <MDL <MDL <MDL

Not Sampled Not Sampled Not Sampled

<MDL

Not Sampled

26-May-14 <MDL <MDL <MDL <MDL <MDL

02-Jul-14 <MDL <MDL <MDL <MDL <MDL

04-Aug-14 <MDL <MDL <MDL <MDL <MDL

29-Sep-14 <MDL <MDL <MDL <MDL <MDL

Carbamazepine

11-Mar-14 227 155.5 209 177

Not Sampled Not Sampled Not Sampled

147

Not Sampled

26-May-14 185 0 <MDL 189.5 248.5

02-Jul-14 258 154 160 383 171

04-Aug-14 156 130 128 150 149

29-Sep-14 149 121 111 149 146

Ketoprofen

11-Mar-14 236 149 <MDL 158

Not Sampled Not Sampled Not Sampled

155

Not Sampled

26-May-14 <MDL <MDL <MDL <MDL <MDL

02-Jul-14 513 354 453.5 32 <MDL

04-Aug-14 132 147 133 <MDL 62

29-Sep-14 141 171 141 <MDL 68

TABLE 8-13: Raw Data – Otonabee River Pilot Plant (Cont.)

122

Date Raw Water Settled Conv. Control Nutrients Control Peroxide Alum GAC

Naproxen

11-Mar-14 281.5 226 <MDL 122.5

Not Sampled Not Sampled Not Sampled

147

Not Sampled

26-May-14 354 144 <MDL 107 241.5

02-Jul-14 266 163.5 162.5 267 124.5

04-Aug-14 157 145 137 107 139

29-Sep-14 150 125 104 114 128

Pentoxifylline

11-Mar-14 261 185 209 163.5

Not Sampled Not Sampled Not Sampled

106

Not Sampled

26-May-14 <MDL <MDL <MDL <MDL <MDL

02-Jul-14 387 277.5 360.5 82.5 48

04-Aug-14 144 175 164 <MDL 69

29-Sep-14 146 163 162 <MDL 50

Table 8-14: Raw Data - Lake Ontario Pilot Plant

Date Raw

Water Ozonated

0.8 PACl GAC

16 min

0.8 PACl Anth.

26 min

0.8 PACl GAC

26 min

0.8 PACl GAC

16 min

0.8 PACl Anth.

26 min

0.8 PACl GAC

26 min

Control GAC

16 min

Peroxide GAC

16 min

0.2 PACl GAC

16 min

Nutrients GAC

16 min

Media Type N/A N/A N/A GAC and

Sand Anthracite and Sand

GAC and Sand

GAC and Sand

Anthracite and Sand

GAC and Sand

GAC and Sand

GAC and Sand

GAC and Sand

GAC and Sand

Filter Type N/A N/A N/A Bio Bio Bio Bio Bio Bio Bio Bio Bio Bio

Influent Conditions

N/A Raw

Water Ozonated

Water 0.8 mg/L

PACl 0.8 mg/L

PACl 0.8 mg/L

PACl 0.8 mg/L

PACl 0.8 mg/L

PACl 0.8 mg/L

PACl Control

0.2 mg/L H2O2

0.2 mg/L PACl

0.5 mg/L PO4, NH4

Column Diameter

N/A N/A N/A 15.24 cm

(6") 15.24 cm

(6") 15.24 cm

(6") 15.24 cm

(6") 15.24 cm

(6") 15.24 cm

(6") 7.62 cm

(3") 7.62 cm

(3") 7.62 cm

(3") 7.62 cm

(3")

Flow Rate N/A N/A N/A 2.0 L/min 1.2 L/min 1.2 L/min 2.0 L/min 1.2 L/min 1.2 L/min 0.3 L/min 0.3 L/min 0.3 L/min 0.3 L/min

EBCT N/A N/A N/A 16 mins 26 mins 26 mins 16 mins 26 mins 26 mins 16 mins 16 mins 16 mins 16 mins

ATP (pg/g media)

08-Apr-14 Not

Sampled Not

Sampled

79250 39580 77575 63710 25610 45210 189505 99765 127650 175815

07-Jul-14 70740 22600 90760 109605 20370 63470 92200 103030 100690 102745

11-Aug-14 89640 49110 114880 105820 39055 107740 289635 272355 123540 229945

Proteins (ug/g media)

08-Apr-14 Not

Sampled Not

Sampled

78.0 26.6 111.6 178.0 18.0 65.2 55.2 56.6 95.2 61.6

07-Jul-14 178.3 55.3 115.1 123.7 25.0 75.7 94.1 74.3 109.2 91.4

11-Aug-14 53.1 6.5 77.5 101.1 15.3 67.3 56.5 40.3 59.2 48.4

Polysacharides (ug/g media)

08-Apr-14 Not

Sampled Not

Sampled

44.7 5.1 45.3 97.1 9.7 44.5 23.2 15.5 23.4 18.1

07-Jul-14 45.7 15.7 41.8 50.2 11.0 38.6 14.0 7.1 16.2 19.8

11-Aug-14 55.5 12.7 55.5 48.8 0.0 58.9 21.9 21.1 27.0 27.7

pH

08-Apr-14 7.87 7.86 7.76 7.78 7.74 7.71 7.75 7.68 7.74 7.77 7.75 7.66

07-Jul-14 8.04 7.98 7.78 7.75 7.76 7.79 7.69 0.07 0.11 0.11 0.16 0.12

11-Aug-14 8.01 7.98 7.78 7.82 7.78 7.79 7.84 7.68 7.83 7.8 7.51 7.41

Turbidity (NTU)

08-Apr-14 0.67 0.36 0.06 0.05 0.05 0.07 0.06 0.06 0.16 0.19 0.13 0.17

07-Jul-14 0.19 0.15 0.10 0.09 0.09 0.08 0.08 0.07 0.11 0.11 0.16 0.12

11-Aug-14 0.36 0.29 0.11 0.12 0.12 0.06 0.19 0.16 0.21 0.34 0.13 0.21

Table 8-14: Raw Data - Lake Ontario Pilot Plant (Cont.)

124

Date Raw

Water Ozonated

0.8 PACl GAC

16 min

0.8 PACl Anth.

26 min

0.8 PACl GAC

26 min

0.8 PACl GAC

16 min

0.8 PACl Anth.

26 min

0.8 PACl GAC

26 min

Control GAC

16 min

Peroxide GAC

16 min

0.2 PACl GAC

16 min

Nutrients GAC

16 min

UV254

08-Apr-14 0.67 0.36 0.06 0.05 0.05 0.07 0.06 0.06 0.16 0.19 0.13 0.17

07-Jul-14 0.19 0.15 0.1 0.09 0.09 0.08 0.08 0.07 0.11 0.11 0.16 0.12

11-Aug-14 0.02 0.021 0.014 0.016 0.013 0.013 0.015 0.013 0.017 0.018 0.014 0.018

DOC (mg/L)

08-Apr-14 1.77 1.76 1.53 1.53 1.55 1.53 1.60 1.52 1.82 1.82 1.79 1.85

07-Jul-14 1.76 1.83 1.45 1.05 1.49 1.59 1.66 1.64 1.85 1.85 1.82 1.89

11-Aug-14 2.36 2.36 1.91 2.08 1.94 2.02 2.16 2.12 2.34 2.32 2.09 2.36

Total THM (ug/L)

08-Apr-14 39.1 46.1 22.3 30.4 20.7 21.7 26.8 23.3 29.4 29.8 28.8 27.2

07-Jul-14 41.1 39.6 29.4 27.9 27.2 26.9 36.3 26.5 34.1 36.3 35.7 32.9

11-Aug-14 31.0 34.6 20.5 21.4 20.9 20.6 31.0 21.3 27.7 28.1 25.9 23.6

TCM (ug/L) (Avg. , (Std.

Dev.))

08-Apr-14 17.7

(1.36) 17.8

(1.99) 8.86

(0.79) 13.9

(0.84) 8.08

(0.70) 8.66

(1.19) 11.8

(0.69) 9.26

(0.50) 13.12 (1.21)

13.5 (1.04)

12.6 (2.56)

10.5 (1.13)

07-Jul-14 16.5

(1.05) 14.6

(0.43) 11.1

(1.89) 10.3

(1.36) 9.78

(0.80) 10.1

(0.96) 14.7

(0.23) 9.48

(0.59) 13.3

(0.37) 14.3

(0.32) 14.0

(1.37) 12.5

(1.30)

11-Aug-14 13.3

(0.84) 12.7

(1.15) 7.09

(0.36) 8.43

(0.21) 7.20

(0.65) 7.26

(0.16) 12.8

(0.88) 7.39

(0.57) 10.8

(0.39) 10.9

(1.04) 9.88

(1.13) 7.77

(0.85)

BDCM (ug/L) (Avg., (Std.

Dev.))

08-Apr-14 16.1

(1.04) 22..9 (2.95)

8.97 (0.55)

11.6 (0.58)

8.44 (0.56)

8.73 (0.73)

10.6 (0.49)

9.17 (0.37)

11.7 (0.66)

11.8 (0.63)

11.6 (1.47)

11.2 (0.89)

07-Jul-14 18.2

(1.05) 14.6

(0.43) 11.1

(1.89) 10.3

(1.36) 9.78

(0.80) 10.1

(0.96) 14.7

(0.23) 9.48

(0.59) 13.3

(0.37) 14.3

(0.32) 14.0

(1.37) 12.5

(1.30)

11-Aug-14 13.8

(0.98) 17.7

(0.91) 8.70

(0.29) 9.18

(0.22) 8.78

(0.44) 8.75

(0.22) 12.8

(0.70) 8.87

(0.42) 11.8

(0.29) 12.2

(0.91) 11.0

(1.05) 11.0

(0.83)

CDBM (ug/L) (Avg., (Std.

Dev.))

08-Apr-14 5.28

(0.44) 5.33

(0.68) 4.48

(0.41) 4.89

(0.38) 4.15

(0.38) 4.27

(0.46) 4.42

(0.29) 4.87

(0.25) 4.53

(0.46) 4.51

(0.30) 4.58

(0.83) 5.53

(0.37)

07-Jul-14 6.41

(0.52) 5.14

(0.13) 5.63

(0.25) 5.32

(0.31) 5.62

(0.31) 5.29

(0.25) 6.01

(0.10) 5.60

(0.29) 6.30

(0.15) 6.31

(0.19) 6.34

(0.48) 5.82

(0.43)

11-Aug-14 3.87

(0.21) 4.10

(0.27) 4.67

(0.22) 3.80

(0.10) 4.91

(0.28) 4.56

(0.10) 5.44

(0.33) 5.08

(0.25) 5.09

(0.10) 5.05

(0.44) 5.04

(0.51) 4.82

(0.42)

Table 8-14: Raw Data - Lake Ontario Pilot Plant (Cont.)

125

Date Raw

Water Ozonated

0.8 PACl GAC

16 min

0.8 PACl Anth.

26 min

0.8 PACl GAC

26 min

0.8 PACl GAC

16 min

0.8 PACl Anth.

26 min

0.8 PACl GAC

26 min

Control GAC

16 min

Peroxide GAC

16 min

0.2 PACl GAC

16 min

Nutrients GAC

16 min

Total HAA (ug/L)

08-Apr-14 9.56 10.29 5.72 6.88 5.60 5.37 6.49 4.87 7.74 8.50 7.56 5.40

07-Jul-14 22.44 20.62 12.81 13.89 14.76 11.94 16.90 9.29 16.03 17.26 17.10 16.28

11-Aug-14 27.11 29.01 15.10 18.32 13.21 15.55 18.86 13.08 19.65 18.81 16.87 20.25

MCAA (ug/L) (Avg., (Std.

Dev.))

08-Apr-14 1.20

(0.14) 1.62

(0.03) 1.75

(0.12) 1.79

(0.05) 1.72

(0.04) 1.45

(0.05) 1.81

(0.15) 1.25

(0.10) 1.47

(0.06) 1.89

(0.15) 1.48

(0.08) 1.05

(0.16)

07-Jul-14 1.54

(0.12) 1.43

(0.30) 0.98

(0.11) 1.57

(0.67) <MDL

1.38 (0.16)

1.71 (0.68)

<MDL 1.55

(0.36) 2.30

(0.20) 1.88

(0.44) 1.68

(0.57)

11-Aug-14 3.04

(0.29) 4.51

(0.14) 3.80

(1.65) 4.25

(0.89) 2.15

(0.14) 3.36

(0.52) 4.51

(0.08) 3.48

(0.46) 3.12

(0.24) 4.81

(0.80) 2.78

(0.06) 1.69

(0.03)

MBAA (ug/L) (Avg., (Std.

Dev.))

08-Apr-14 0.65

(0.01) 0.62

(0.01) 0.20

(0.04) 0.19

(0.05) 0.26

(0.01) 0.48

(0.00) 0.17

(0.00) 0.21

(0.03) 0.63

(0.01) 0.22

(0.00) 0.60

(0.00) 0.31

(0.10)

07-Jul-14 3.51

(0.06) 3.06

(0.02) 2.73

(0.45) 2.73

(0.42) 2.43

(0.02) 2.56

(0.08) 3.27

(0.09) 2.33

(0.04) 2.87

(0.07) 3.01

(0.14) 3.11

(0.06) 3.24

(0.05)

11-Aug-14 3.83

(0.03) 3.74

(0.10) 2.82

(0.32) 3.11

(0.43) 2.98

(0.05) 3.12

(0.04) 2.72

(0.02) 2.04

(0.13) 3.55

(0.11) 2.20

(0.59) 3.26

(0.07) 4.27

(0.03)

DCAA (ug/L) (Avg., (Std.

Dev.))

08-Apr-14 3.45

(0.13) 3.66

(0.02) 1.82

(0.08) 2.42

(0.05) 1.72

(0.11) 1.36

(0.02) 2.07

(0.29) 1.67

(0.05) 2.34

(0.07) 3.05

(0.06) 2.27

(0.02) 1.54

(0.06)

07-Jul-14 8.26

(0.21) 7.29

(0.06) 3.42

(1.26) 3.65

(1.37) 2.72

(0.03) 3.20

(0.10) 5.15

(0.06) 2.76

(0.18) 5.47

(0.10) 5.01

(0.10) 5.66

(0.13) 5.05

(0.07)

11-Aug-14 9.58

(0.07) 10.0

(0.19) 3.69

(0.29) 5.25

(0.44) 3.55

(0.06) 4.12

(0.03) 5.63

(0.04) 3.43

(0.07) 6.08

(0.16) 5.44

(0.10) 5.27

(0.07) 6.15

(0.05)

TCAA (ug/L) (Avg., (Std.

Dev.))

08-Apr-14 2.21

(0.10) 1.74

(0.01) 0.60

(0.01) 1.05

(0.02) 0.63

(0.01) 0.75

(0.01) 0.99

(0.06) 0.50

(0.05) 1.61

(0.05) 1.60

(0.03) 1.51

(0.03) 1.03

(0.16)

07-Jul-14 1.80

(0.06) 1.03

(0.05) 0.48

(0.03) 0.69

(0.03) <MDL <MDL

0.80 (0.18)

<MDL 0.74

(0.03) 1.16

(0.33) 0.85

(0.03) 0.91

(0.11)

11-Aug-14 3.72

(0.08) 3.00

(0.11) 0.79

(0.13) 1.45

(0.12) 0.40

(0.16) 0.76

(0.06) 1.82

(0.04) 0.48

(0.05) 2.03

(0.08) 2.16

(0.06) 1.31

(0.06) 4.52

(0.05)

Table 8-14: Raw Data - Lake Ontario Pilot Plant (Cont.)

126

Date Raw

Water Ozonated

0.8 PACl GAC

16 min

0.8 PACl Anth.

26 min

0.8 PACl GAC

26 min

0.8 PACl GAC

16 min

0.8 PACl Anth.

26 min

0.8 PACl GAC

26 min

Control GAC

16 min

Peroxide GAC

16 min

0.2 PACl GAC

16 min

Nutrients GAC

16 min

BCAA (ug/L) (Avg., (Std.

Dev.))

08-Apr-14 1.26

(0.03) 1.24

(0.01) 0.64

(0.01) 0.73

(0.02) 0.64

(0.01) 0.64

(0.00) 0.71

(0.08) 0.61

(0.01) 0.90

(0.03) 0.89

(0.01) 0.92

(0.04) 0.71

(0.03)

07-Jul-14 3.34

(0.10) 2.92

(0.16) 2.01

(0.42) 2.04

(0.11) 6.59

(0.20) 1.67

(0.05) 2.50

(0.34) 1.41

(0.08) 2.14

(0.13) 2.32

(0.23) 2.15

(0.04) 2.05

(0.05)

11-Aug-14 3.54

(0.02) 3.09

(0.06) 1.63

(0.09) 1.80

(0.11) 1.58

(0.09) 1.60

(0.04) 1.81

(0.06) 1.42

(0.02) 2.06

(0.13) 1.92

(0.08) 1.77

(0.05) 2.41

(0.05)

DBAA (ug/L) (Avg., (Std.

Dev.))

08-Apr-14 0.79

(0.01) 1.41

(0.02) 0.71

(0.05) 0.70

(0.03) 0.63

(0.01) 0.69

(0.01) 0.74

(0.01) 0.63

(0.02) 0.79

(0.04) 0.85

(0.05) 0.78

(0.01) 0.76

(0.04)

07-Jul-14 3.99

(0.07) 4.89

(0.09) 3.20

(0.34) 3.21

(0.36) 3.02

(0.05) 3.13

(0.06) 3.47

(0.05) 2.79

(0.07) 3.25

(0.05) 3.46

(0.32) 3.45

(0.08) 3.36

(0.08)

11-Aug-14 3.39

(0.11) 4.66

(0.10) 2.37

(0.21) 2.46

(0.28) 2.55

(0.04) 2.59

(0.03) 2.37

(0.16) 2.24

(0.04) 2.81

(0.08) 2.28

(0.12) 2.47

(0.06) 1.22

(0.09)

Total AOX (ug/L)

08-Apr-14 91.5

(12.8) 80.4

(2.46) 38.9

(1.62) 49.8

(1.27) 39.7

(1.38) 53.3

(3.24) 45.5

(1.62) 34.5

(1.72) 69.6

(5.72) 51.8

(2.48) 65.7

(1.10) 42.7

(1.47)

07-Jul-14 78.9

(5.14) 73.2

(3.08) 48.0

(10.0) 47.8

(9.37) 42.7

(3.02) 45.2

(1.56) 44.7

(7.85) 34.0

(0.73) 48.1

(2.65) 49.7

(2.86) 53.7

(2.28) 47.1

(3.37)

11-Aug-14 96.6

(2.48) 91.5

(4.96) 61.7

(4.64) 66.5

(10.8) 55.2

(2.97) 57.5

(1.76) 58.8

(3.46) 36.1

(2.82) 79.8

(4.34) 63.4

(2.28) 66.8

(3.07) 114.5 (0.53)

Bio-polymers (mg/L)

08-Apr-14 0.224 0.21 0.108 0.116 0.105 0.132 0.126 0.124 0.187 0.202 0.18 0.185

07-Jul-14 0.256 0.221 0.129 0.078 0.128 0.133 0.151 0.148 0.227 0.227 0.227 0.238

11-Aug-14 0.403 0.359 0.238 0.22 0.226 0.242 0.246 0.292 0.34 0.339 0.214 0.36

Humics (mg/L)

08-Apr-14 0.99 0.95 0.864 0.816 0.87 0.857 0.825 0.853 0.947 0.875 0.87 0.957

07-Jul-14 1.185 0.916 0.855 0.538 0.852 0.818 0.876 0.78 0.952 1.101 0.888 0.965

11-Aug-14 1.093 1.07 1.02 1.003 0.896 0.846 0.902 0.95 0.96 0.951 0.921 1.07

Building Blocks (mg/L)

08-Apr-14 0.34 0.422 0.255 0.372 0.292 0.267 0.336 0.27 0.337 0.408 0.404 0.324

07-Jul-14 0.201 0.454 0.267 0.125 0.274 0.372 0.355 0.383 0.375 0.225 0.424 0.349

11-Aug-14 0.434 0.504 0.351 0.349 0.455 0.432 0.534 0.409 0.468 0.451 0.394 0.406

Table 8-14: Raw Data - Lake Ontario Pilot Plant (Cont.)

127

Date Raw

Water Ozonated

0.8 PACl GAC

16 min

0.8 PACl Anth.

26 min

0.8 PACl GAC

26 min

0.8 PACl GAC

16 min

0.8 PACl Anth.

26 min

0.8 PACl GAC

26 min

Control GAC

16 min

Peroxide GAC

16 min

0.2 PACl GAC

16 min

Nutrients GAC

16 min

LWM Acids (mg/L)

08-Apr-14 n.q. n.q. n.q. n.q. n.q. n.q. n.q. n.q. n.q. n.q. n.q. n.q.

07-Jul-14 n.q. n.q. n.q. n.q. n.q. n.q. n.q. n.q. n.q. n.q. n.q. n.q.

11-Aug-14 0.018 n.q. n.q. n.q. n.q. n.q. 0.013 0.045 n.q. n.q. n.q. n.q.

LMW Neutrals (mg/L)

08-Apr-14 0.383 0.236 0.097 0.234 0.182 0.248 0.237 0.182 0.243 0.244 0.221 0.247

07-Jul-14 0.225 0.207 0.181 0.243 0.184 0.203 0.26 0.179 0.202 0.188 0.204 0.215

11-Aug-14 0.223 0.254 0.202 0.282 0.184 0.208 0.25 0.251 0.215 0.226 0.226 0.253

MX (ng/L)

08-Apr-14 <MDL 5.5 Not

Sampled Not

Sampled Not

Sampled

<MDL Not

Sampled Not

Sampled

<MDL Not

Sampled

<MDL Not

Sampled 07-Jul-14 <MDL <MDL <MDL <MDL <MDL

11-Aug-14 <MDL <MDL <MDL <MDL <MDL

MCA (ng/L)

08-Apr-14 <MDL <MDL Not

Sampled Not

Sampled Not

Sampled

<MDL Not

Sampled Not

Sampled

<MDL Not

Sampled

<MDL Not

Sampled 07-Jul-14 76 23 20.5 15 19.5

11-Aug-14 64.5 61.5 25.5 36.5 31

Genotoxicity

08-Apr-14 0.14 0.147 Not

Sampled Not

Sampled Not

Sampled

0.082 Not

Sampled Not

Sampled

0.111 Not

Sampled

0.112 Not

Sampled 07-Jul-14 0.065 0.042 0.0205 0.04 0.0355

11-Aug-14 0.06 0.037 0.025 0.04 0.022

Table 8-15: PhAC, EDC, and Artificial Sweetener Raw Data - Lake Ontario Pilot Plant

Date Influent 0.8 PACl GAC

16 min Influent

Control GAC 16 min

0.2 PACl GAC 16 min

Media Type N/A N/A GAC and

Sand N/A

GAC and Sand

GAC and Sand

Filter Type N/A N/A Bio N/A Bio Bio

Influent Conditions

N/A N/A 0.8 mg/L

PACl N/A Control

0.2 mg/L PACl

Column Diameter N/A N/A 15.24 cm

(6") N/A 7.62 cm (3") 7.62 cm (3")

Flow Rate N/A N/A 2.0 L/min N/A 0.3 L/min 0.3 L/min

EBCT N/A N/A 16 mins N/A 16 mins 16 mins

ATP (pg/g media)

08-Apr-14 Not

Sampled

63710

Not Sampled

189505 127650

07-Jul-14 109605 92200 100690

11-Aug-14 105820 289635 123540

Proteins (ug/g media)

08-Apr-14 Not

Sampled

178.0

Not Sampled

55.2 95.2

07-Jul-14 123.7 94.1 109.2

11-Aug-14 101.1 56.5 59.2

Polysacharides (ug/g media)

08-Apr-14 Not

Sampled

97.1

Not Sampled

23.2 23.4

07-Jul-14 50.2 14.0 16.2

11-Aug-14 48.8 21.9 27.0

pH

28-Apr-14 Not

Sampled

7.9

Not Sampled

7.8 7.9

14-Jul-14 7.5 7.6 7.7

18-Aug-14 7.7 7.6 7.8

Turbidity (NTU)

28-Apr-14 Not

Sampled

0.054

Not Sampled

0.108 0.099

14-Jul-14 0.216 0.290 0.270

18-Aug-14 0.134 0.107 0.151

UV254

28-Apr-14 Not

Sampled

0.011

Not Sampled

0.015 0.016

14-Jul-14 0.015 0.019 0.019

18-Aug-14 0.017 0.022 0.022

DOC (mg/L)

28-Apr-14 Not

Sampled

1.440

Not Sampled

1.780 1.700

14-Jul-14 2.180 2.790 2.820

18-Aug-14 2.030

Sucralose (ng/L)

28-Apr-14 624 460 825 512 633

14-Jul-14 1458 614 1116.5 1445 1528

18-Aug-14 655.5 432 1904 1590 2465

Acesulfame Potassium (ng/L)

28-Apr-14 503 432 687 438 590

14-Jul-14 1264 632 974 1273 1370

18-Aug-14 624 417 1646 1500 2218

Table 8-15: PhAC, EDC, and Artificial Sweetener Raw Data - Lake Ontario Pilot Plant (Cont.)

129

Date Influent 0.8 PACl GAC

16 min Influent

Control GAC 16 min

0.2 PACl GAC 16 min

Acetaminophen

28-Apr-14 144 53 213.5 512 51

14-Jul-14 587.5 <MDL 757 27 29

18-Aug-14 728 <MDL 1786.5 <MDL 178

Bisphenol-A

28-Apr-14 423 <MDL 457.5 438 228

14-Jul-14 190.5 77 609.5 304 435

18-Aug-14 Not Detected Not Detected

Clofibric Acid

28-Apr-14 Not Detected Not Detected

14-Jul-14 Not Detected Not Detected

18-Aug-14 801.5 489.5 2233.5 1914.5 2739.5

Diclofenac

28-Apr-14 744 231.5 775 717.5 802.5

14-Jul-14 443 67 594.5 438.5 510.5

18-Aug-14 454 158.5 929 668.5 1165

Estriol

28-Apr-14

Not Detected Not Detected 14-Jul-14

18-Aug-14

Estrone

28-Apr-14 Not Detected Not Detected

14-Jul-14 179 <MDL 304 <MDL 40

18-Aug-14 64.5 <MDL 294.5 <MDL 140.5

Gemfibrozil

28-Apr-14 Not Detected Not Detected

14-Jul-14 481 228 660.5 528 607.5

18-Aug-14 354 175.5 922.5 707.5 1430

17 β estradiol

28-Apr-14

Not Detected Not Detected 14-Jul-14

18-Aug-14

Carbamazepine

28-Apr-14 647.5 78.5 851.5 584.5 650.5

14-Jul-14 436.5 105.5 628.5 368.5 460.5

18-Aug-14 332 80 871 498 1070

Ketoprofen

28-Apr-14 500.5 213 650 245 152.5

14-Jul-14 1075 291.5 1066.5 840 1283

18-Aug-14 279 113 680.5 652 971

Naproxen

28-Apr-14 709 167.5 629 470 612

14-Jul-14 514 142.5 752 484.5 586

18-Aug-14 343.5 114.5 839.5 513.5 1126

Pentoxifylline

28-Apr-14 810.5 30.3 1033.5 543.5 117

14-Jul-14 652.5 124.5 851 431.5 582

18-Aug-14 327 81 809 488 944.5

130

8.3 Sample Quality Assurance/Quality Control Charts

QA/QC charts were generated by analyzing 8 samples prepared at a concentration

equivalent to the expected concentrations in the samples being analyzed after each new

calibration. Check standards are prepared at the same concentration, and analyzed after every 10

samples. The check standards are compared to the mean and standard deviation calculated from

the original 8 samples as per Standard Method 1020 (APHA, 2005). The calibration is

considered unacceptable if:

- 2 consecutive measurements fall outside of the control limit (CL) of the mean ± 3 times

the standard deviation;

- 3 out of 4 consecutive measurements were outside the warning limits (WL) of the mean

± 2 times the standard deviation;

- 5 out of 6 consecutive measurements were outside of the mean ± the standard deviation;

- 5 out of 6 consecutive measurements exhibited an increasing or decreasing trend; or,

- 7 consecutive samples were greater, or less, than the mean

If any of these conditions were met a new calibration curve was generated and the samples were

reanalyzed. QA/QC charts are presented below for chloroform (Figure 8-1), monochloroacetic

acid (Figure 8-2), MX (Figure 8-3), MCA (Figure 8-4), MBA (Figure 8-5), acesulfame-K

(Figure 8-6), and sucralose (Figure 8-7).

Figure 8-1: Quality Control Chart – Chloroform

10

14

18

22

26

30

0 5 10 15 20 25 30

Concentr

ation u

g/L

Upper CL Upper WL Average Lower WL Lower CL

June 2013 Oct. 2013 Dec. 2013 Feb. 2014 April 2014

July 2013 Nov. 2013 Jan. 2014 Mar. 2014

131

Figure 8-2: Quality Control Chart - Monochloroacetic Acid

Figure 8-3: Quality Control Chart – MX

4

8

12

16

0 5 10 15 20 25 30

Concentr

ation u

g/L

Upper CL Upper WL Average Lower WL Lower CL

June 2013 Oct. 2013 Dec. 2013 Feb. 2014 April 2014

Jul. 2013 Nov. 2013 Jan. 2014 Mar. 2014

2000000

3000000

4000000

5000000

6000000

7000000

8000000

0 2 4 6 8 10

MX

Peak A

rea C

ount

Standard Sample Number

Upper CL Upper WL Average Lower WL Lower CL

132

Figure 8-4: Quality Control Chart – MCA

Figure 8-5: Quality Control Chart – MBA

1500000

1700000

1900000

2100000

2300000

2500000

2700000

2900000

3100000

0 2 4 6 8 10

MC

A P

eak A

rea C

ount

Standard Sample Number

Upper CL Upper WL Average Lower WL Lower CL

0

500000

1000000

1500000

2000000

2500000

3000000

3500000

4000000

4500000

0 2 4 6 8 10

MB

A P

eak A

rea C

ount

Standard Sample Number

Upper CL Upper WL Average Lower WL Lower CL

133

Figure 8-6: Quality Control Chart- Acesulfame – K

Figure 8-7: Quality Control Chart - Sucralose

80000

85000

90000

95000

100000

105000

110000

115000

120000

125000

0 2 4 6 8 10

Acesulfam

e-K

Peak A

rea C

ount Upper CL

Upper WL Average Lower WL Lower CL

3000

4000

5000

6000

7000

8000

9000

10000

0 2 4 6 8 10

Sucra

lose P

eak A

rea C

ount Upper CL

Upper WL Average Lower WL Lower CL