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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