MELANIE DOMENICA CEVALLOS NUNEZ · 2021. 3. 15. · Melanie Domenica Cevallos Nunez Department of...

PERACETIC ACID DISINFECTION SYNERGY WITH ALUM

AND DIRECT REACTION WITH CHLORINE

by

MELANIE DOMENICA CEVALLOS NUNEZ

A thesis submitted in conformity with the requirements

for the degree of Masters of Applied Science

Graduate Department of Civil Engineering

University of Toronto

© Copyright by Melanie Domenica Cevallos Nunez 2021

iii

PERACETIC ACID DISINFECTION SYNERGY WITH ALUM AND DIRECT

REACTION WITH CHLORINE

Melanie Domenica Cevallos Nunez

Department of Civil Engineering, University of Toronto

Degree of Masters of Applied Science

Convocation 2021

ABSTRACT

Peracetic acid (PAA) is an effective disinfectant during wastewater treatment. Anecdotal reports

suggest that its disinfection efficiency increases when combined with residual alum from upstream

coagulation. This research focused on determining if PAA and aluminum could initiate an

advanced oxidation process. Two probes were used during this study to test the presence of

hydroxyl radicals (OH•), and neither showed OH• formation. E. coli inactivation was also

assessed, with no significant increase in log reduction observed when alum was present during

PAA disinfection. This research also focused on the effect of PAA on chlorine disinfection when

used as a pretreatment for DBP and mussel control during drinking water treatment. A kinetic

model was developed to simulate the impact of PAA on chlorine consumption as a function of pH.

It was determined that chlorine decay was attributed mainly to the reaction with the slow formation

of H2O2 associated with PAA decomposition.

iv

ACKNOWLEDGMENTS

This research was funded by the Natural Science and Engineering Research Council of Canada

(NSERC) Chair in Drinking Water Research at the University of Toronto.

I would like to thank my supervisor, Dr. Ron Hofmann, it has been an absolute pleasure having

the opportunity to learn from you and I would like to express my sincere gratitude for your

unwavering support, guidance and mentorship. Thank you for pushing me to sharpen my skills to

become the professional I am today.

Furthermore, some other notable mentions include Liz Taylor-Edmonds for teaching me

everything I know about E. coli and to Chengjing Wang for sharing your wisdom and always

finding time to help me plan experiments. Thank you to all the members of the DWRG for their

support, in particular Meaghan Keon, Emily Bridgehouse, Alonso Hurtado, and Emily Curling,

thank you for the laughs, tears and immense joy.

Lastly, I would like to thank my family for their unconditional love and support. They have always

given me the ability to follow my dreams.

Domenica Cevallos

v

TABLE OF CONTENTS

ABSTRACT .................................................................................................................................. iii

ACKNOWLEDGMENTS ........................................................................................................... iv

TABLE OF CONTENTS ............................................................................................................. v

LIST OF TABLES ..................................................................................................................... viii

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

GLOSSARY................................................................................................................................... x

1 INTRODUCTION AND RESEARCH OBJECTIVES ..................................................... 1

1.1 Motivation ................................................................................................................... 1

1.2 Research Objectives .................................................................................................... 1

1.3 Description of Chapters .............................................................................................. 2

1.4 References ................................................................................................................... 2

2 PAA LITERATURE REVIEW ........................................................................................... 4

2.1 Overview/History ........................................................................................................ 4

2.2 Chemical and physical properties ............................................................................... 4

2.2.1 Decomposition .......................................................................................................... 6

2.2.2 Equilibrium: Generation and hydrolysis of PAA...................................................... 7

2.2.3 Oxidation Demand .................................................................................................... 8

2.3 PAA in the water industry ......................................................................................... 10

2.3.1 PAA synergy with other technologies/treatments................................................... 12

2.4 Disinfection kinetics ................................................................................................. 13

2.4.1 Mechanisms of inactivation .................................................................................... 13

2.4.2 Inactivation kinetics ................................................................................................ 14

2.4.3 Radical Formation ................................................................................................... 16

2.5 Water quality ............................................................................................................. 18

vi

2.5.1 Organic matter ........................................................................................................ 19

2.5.2 Suspended solids and particulate matter ................................................................. 19

2.6 PAA analysis ............................................................................................................. 20

2.7 Toxicity ..................................................................................................................... 21

2.8 Research needs .......................................................................................................... 22

2.9 References ................................................................................................................. 23

3 PERACETIC ACID REACTION WITH THIOSULPHATE AND WITH

CHLORINE ....................................................................................................................... 30

3.1 Introduction ............................................................................................................... 31

3.1.1 Neutralization of PAA/H2O2 solution ..................................................................... 31

3.1.1.1 Quenching of H2O2 .......................................................................................... 31

3.1.1.2 Quenching of PAA .......................................................................................... 32

3.1.2 PAA & Chlorine ..................................................................................................... 33

3.2 Objective ................................................................................................................... 35

3.3 Materials and Methods .............................................................................................. 36

3.3.1 Quenching experiments .......................................................................................... 36

3.3.1.1 Reagents and Equipment ................................................................................. 36

3.3.1.2 Experimental Protocols.................................................................................... 36

3.3.2 Kinetic model .......................................................................................................... 37

3.3.3 Direct PAA/OCl- reaction tests ............................................................................... 39

3.4 Results and Discussion .............................................................................................. 39

3.4.1 Quenching tests ....................................................................................................... 39

3.4.2 Kinetic model .......................................................................................................... 40

3.4.2.1 PAA/H2O2/AA system ..................................................................................... 40

3.4.2.2 PAA/H2O2/AA/OCl- system ............................................................................ 41

vii

3.4.3 Direct PAA/OCl- reaction ....................................................................................... 42

3.1 Summary and Conclusions ........................................................................................ 45

3.2 References ................................................................................................................. 46

4 PAA + ALUM EXPERIMENTS ....................................................................................... 49

4.1 Introduction ............................................................................................................... 49

4.2 Objective ................................................................................................................... 52

4.3 Materials and Method ............................................................................................... 52

4.3.1 Reagents and Equipment......................................................................................... 52

4.1.1 Experimental protocols ........................................................................................... 53

4.1.1.1 Radical formation analysis and PAA consumption ......................................... 53

4.1.1.2 Experimental QA/QC ...................................................................................... 54

4.1.1.3 Microbial analysis............................................................................................ 54

4.2 Results and Discussion .............................................................................................. 56

4.2.1 Radical quantification and PAA decay ................................................................... 56

4.1.1 Microbial analysis ................................................................................................... 58

4.2 Summary and Conclusions ........................................................................................ 59

4.3 References ................................................................................................................. 60

5 SUMMARY, CONCLUSIONS AND RECOMMENDATIONS .................................... 62

A. APPENDIX 1 EXPERIMENTAL DATA ......................................................................... 64

A.1 Experimental data for chapter 3 ................................................................................ 65

A.1.1 Analytical methods .......................................................................................................... 67

A.2 Experimental data for chapter 4 ................................................................................ 69

A.2.1 Radical formation analysis .............................................................................................. 69

A.2.2 Salicylic acid method for radical detection ..................................................................... 70

A.2.3 Microbial analysis ........................................................................................................... 74

viii

LIST OF TABLES

Table 2-1. PAA chemical and physical properties.......................................................................... 5

Table 2-2. Common values for k and D in wastewater following first-order kinetics ................... 9

Table 2-3. Model parameter ranges for commonly used inactivation models .............................. 14

Table 2-4. Wastewater disinfection models .................................................................................. 15

Table 3-1. Quenching of 5 and 10 mg/L PAA (pH 7 & 5 sec retention time) .............................. 40

Table A-1. Average PAA, H2O2, Cl2 concentration at pH 6......................................................... 65

Table A-2. Average PAA/H2O2/Cl2 concentration at pH 7 .......................................................... 65



Table A-5. Average MB absorbance in the presence of PAA/alum ............................................. 69

Table A-6. Average PAA and H2O2 concentration (mg/L) in the presence of alum .................... 70

Table A-7. Calculated molar absorptivity for SA method ............................................................ 70

Table A-8. Average SA molar concentration in SA/PAA/alum system ....................................... 72

Table A-9. SA molar concentration during control (SA/alum) .................................................... 72

Table A-10. SA molar concentration during second control (SA/PAA) ...................................... 73

Table A-11. Two-tail t-test values for MB decay ......................................................................... 73

Table A-12. Two-tail t-test values for SA decay .......................................................................... 73

Table A-13. Two-tail t-test values for PAA & H2O2 decay .......................................................... 74

Table A-14. E. coli only data ........................................................................................................ 77

Table A-15. Alum data ................................................................................................................. 78

Table A-16. PAA data................................................................................................................... 78

Table A-17. Alum + PAA data ..................................................................................................... 79

Table A-18. Sodium thiosulfate and catalase data ........................................................................ 79

Table A-19. Two-tail t-test values for alum control ..................................................................... 79

Table A-20. Two-tail t-test values for quenching agents as a control .......................................... 80

Table A-21. Two-tail t-test values for PAA vs alum + PAA (1 mg/L) ........................................ 80

Table A-22. Two-tail t-test values for PAA vs alum + PAA (5 mg/L) ........................................ 81

ix

LIST OF FIGURES

Figure 2-1. Peracetic acid molecule ................................................................................................ 4

Figure 2-2. Initial demand and decay rate of PAA ....................................................................... 10

Figure 2-3. Radical formation mechanism from PAA through UV light ..................................... 18

Figure 3-1. MATLAB function and script used to solve a system of 4 differential equations ..... 39

Figure 3-2. Simulation of PAA decay due to decomposition ....................................................... 41

Figure 3-3. Simulation of chlorine effect on PAA equilibrium trhough time at different pH

values and 296 K ....................................................................................................... 42

Figure 3-4. PAA, H2O2, and Cl2 concentration profiles, pH 6...................................................... 43




Figure 4-1. Microbial reduction in wastewater effluent flocculated by PIX and PAX, and

subsequent 3 mg/L PAA disinfection. ...................................................................... 50

Figure 4-2. Methylene blue as a chemical probe for the detection of radicals ............................. 57

Figure 4-3. Salicylic acid decay due to reaction with hydroxyl radical........................................ 57

Figure 4-4. PAA decay in the presence of alum ........................................................................... 58

Figure 4-5. Log reduction of E. coli ............................................................................................. 59

x

GLOSSARY

AA Acetic acid

AOP Advanced oxidation process

CFU Colony-forming unit

Cl2 Chlorine

COD Chemical oxygen demand

DBP Disinfection by-product

DPD N,N-diethyl-p-phenylelnediamine

EC50 Half maximal effective concentration

EDTA Ethylenediaminetetraacetic acid

FC Fecal coliform

MB Methylene blue

NOEC No-observed-effect concentration

ODE Ordinary differential equations

OD600 Optical density at 600 nm

OH• Hydroxyl radical

OCl- Hypochlorite ion

PAA Peracetic acid

ROS Reactive oxygen species

SA Salicylic acid

SS Suspended solids

TAED Tetraacetylenediamine

TC Total coliform

US EPA United States Environmental Protection Agency

UV Ultraviolet

WWTP Wastewater treatment plant

Domenica Cevallos

Department of Civil Engineering, University of Toronto 2021

1

1 INTRODUCTION AND RESEARCH OBJECTIVES

1.1 MOTIVATION

Peracetic acid (PAA) has become a suitable alternative to chlorine disinfection during wastewater

treatment. Its biggest advantage over chlorine-based disinfectants is attributed to the fact that PAA

does not form halogenated DBPs (Dell’Erba et al., 2007; Kitis, 2004). PAA has also proven to

have a synergetic affect when combined with metals such as iron thanks to the rupture of its

acyloxy bond to form reactive oxygen species (ROS) (Flores et al., 2014; Lubello et al., 2002).

The formation of free radicals using PAA is believed to take place in an analogous manner to a

Fenton and Fenton-like process, where iron acts as a catalyst in the reaction (Rokhina et al., 2010).

There is anecdotal evidence of an increase in PAA disinfection efficiency in the presence of alum

residual over iron-based coagulant from pilot-and full-scale. There is little research available that

assesses whether PAA and alum form radicals and that would provide an explanation of why PAA

disinfection efficiency increases in the presence of alum.

PAA has also been suggested as a pretreatment during drinking water treatment to reduced DBP

precursors and most recently as an alternative to mussel control (Griffin et al., 2018; Hurtado,

2020). In solution, peracetic acid exists as an equilibrium mixture with hydrogen peroxide and

acetic acid. The equilibrium of this mixture is pH dependent, with a faster PAA decomposition

into H2O2 and acetic acid (AA) as pH increases (Yuan et al., 1997a; Zhao et al., 2007). The effect

of H2O2 over chlorine consumption is well documented in the literature since chlorine can act as a

quenching agent for hydrogen peroxide. This reaction also depends on pH, with a higher reaction

rate coefficient at higher pH values (Held et al., 1978; Wang et al., 2019). However, there is no

available research on the effect of peracetic acid over chlorine.

1.2 RESEARCH OBJECTIVES

This research has two major goals. The first objective is to assess the potential synergy between

PAA and alum residual. The second objective is to assess the effect of peracetic acid residual on

choline disinfection. More specifically, the objectives are:

1. To determine the formation of hydroxyl radicals in a PAA/alum system using chemical

probes.

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2. To perform microbial analysis under a control environment to proof if there is a

synergetic effect between PAA and alum.

3. To assess the effect of PAA/H2O2 on chlorine.

a. Develop a kinetic model that would predict chlorine decay in the presence of

PAA and H2O2.

b. Obtain experimental data of concentration profiles in a PAA/H2O2 and chlorine

system.

1.3 DESCRIPTION OF CHAPTERS

• Chapter 2: Provides an in-depth literature review on peracetic acid and its role as a

disinfectant in the water industry.

• Chapter 3: Focusses on the effect of PAA as a solution in equilibrium on chlorine demand

and how pH impacts PAA decomposition into H2O2 and acetic acid. It also evaluates the

neutralization of peracetic acid and hydrogen peroxide during laboratory procedures.

• Chapter 4: Explores the potential synergy between PAA and alum by assessing hydroxyl

radical formation and microbial reduction.

• Chapter 5: Summarizes the findings of this research and recommends potential future work

1.4 REFERENCES

Dell’Erba, Falsanisi, D., Liberti, L., Notarnicola, M., Santoro, D., 2007. Disinfection by-products

formation during wastewater disinfection with peracetic acid. Desalination 215, 177–186.

Flores, M.J., Lescano, M.R., Brandi, R.J., Cassano, A.E., Labas, M.D., 2014. A novel approach to

explain the inactivation mechanism of Escherichia coli employing a commercially available

peracetic acid. Water Sci. Technol. 69, 358–363.

Griffin, A., DeWolfe, J., Kocak, S., Stoner, M., 2018. Peracetic acid as a pretreatment alternative

to chlorine in a DBP sensitive application. Water Qual. Technol. Conf.

Held, A.M., Halko, D.J., Hurst, J.K., 1978. Mechanisms of chlorine oxidation of hydrogen

peroxide. J. Am. Chem. Soc. 100, 5732–5740.

Hurtado, A., 2020. Evaluation of mussel control strategies in drinking water treatment plants:

Peracetic acid, Earthtec QZ, and prechlorination. University of Toronto.

Kitis, M., 2004. Disinfection of wastewater with peracetic acid: A review. Environ. Int. 30, 47–

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55.

Lubello, C., Caretti, C., Gori, R., 2002. Comparison between PAA / UV and H2O2 / UV

disinfection for wastewater reuse. Water Sci. Technol. Water Supply 205–212.

Rokhina, E. V., Makarova, K., Golovina, E.A., Van As, H., Virkutyte, J., 2010. Free radical

reaction pathway, thermochemistry of peracetic acid homolysis, and its application for phenol

degradation: Spectroscopic study and quantum chemistry calculations. Environ. Sci. Technol.

44, 6815–6821.

Wang, C., Hofmann, M., Safari, A., Viole, I., Andrews, S., Hofmann, R., 2019. Chlorine is

preferred over bisulfite for H2O2 quenching following UV-AOP drinking water treatment.

Water Res. 165, 115000.

Yuan, Z., Ni, Y., Van Heiningen, A.R.P., 1997. Kinetics of peracetic acid decompostition part I:

Spontaneous decomposition at typical pulp bleaching conditions. Canidian J. Chem. Eng. 75,

37–41.

Zhao, X., Zhang, T., Zhou, Y., Liu, D., 2007. Preparation of peracetic acid from hydrogen

peroxide. Part I: Kinetics for peracetic acid synthesis and hydrolysis. J. Mol. Catal. A Chem.

271, 246–252.

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2 PAA LITERATURE REVIEW

2.1 OVERVIEW/HISTORY

Peracetic acid (PAA) is a strong oxidant that was prepared for the first time in the early 1900s

(Swern, 1971). Its bactericidal, virucidal and sporicidal properties have been known since the

1960s (Dell’Erba et al., 2007). It has been used in the past in several industries including food

processing, beverage, medical and pharmaceutical, textile, and pulp and paper industries (Kitis,

2004). However, its use for wastewater disinfection only started around 1980 (Baldry et al., 1991;

Baldry and French, 1989).

2.2 CHEMICAL AND PHYSICAL PROPERTIES

Peracetic acid (PAA, CH3COOOH), also known as peroxyacetic acid, is commercially available

as a solution in equilibrium with hydrogen peroxide (H2O2) and acetic acid (AA, CH3COOH), as

shown in Equation 1 (Gehr et al., 2003; Zhao et al., 2008). PAA is usually prepared by reacting

hydrogen peroxide and acetic acid and adding sulfuric acid to catalyze the synthesis (Swern, 1971).

The decomposition of PAA is exothermic and is promoted by high pH or temperature, or by the

reaction with a transition metal such as manganese, cooper, cobalt, and iron (Yuan et al., 1997b).

Industrial grade PAA is produced in concentrations ranging from 0.3 to 40% JACC, (2001), but

solutions above 15% tend to be more explosive, unstable, and reactive, so solutions ranging from

10-15% are used more often (Block, 2001; Kitis, 2004). The stability of peracetic acid also depends

on temperature. A PAA solution of 15 % has an approximate shelf life of 1 year at 30oC, but only

1 month at 43.3oC (PeroxyChem, 2014).

O

O

OH

H3C

Figure 2-1. Peracetic acid molecule

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CH3COOOH+ H2O ↔ CH3COOH+ H2O2 (1)

PAA is a colorless liquid which is very soluble in water and has a pungent odor. Due to its low

octanol-water partition coefficient (Kow) of 0.3 it is not bioaccumuable (JACC, 2001). Additional

PAA chemical and physical properties can be found in Table 2-1. It is important to note that some

properties change according to the concentration ratio of PAA with individual components. These

properties are listed for PAA concentrations of 5, 15, and 35%. Moreover, volatilisation of PAA

from aqueous solutions is fairly low, but is dependent on the partial vapor pressure (JACC, 2001).

PAA should be stored in original containers at cool temperatures. It can be kept in glass, pure

aluminum, stainless steel, tin-plated iron and some plastics (Kitis, 2004).

Table 2-1. PAA chemical and physical properties (JACC, 2001; Swern, 1971)

Property Value

Molecular weight (g/mol) 76.051

pKa at 20oC 8.2

Kow at 25oC 0.3

pH <1

Odor threshold (ppb) 50

Viscosity 1.5 (5%) or 2.89 (100%)

5% 15% 35%

Boiling point 99-105oC >100oC >105oC

Melting point -26 to -30oC -30 to -50oC -44oC

Vapor pressure at 20oC 21 to 27 hPa 25 hPa 17 hPa

Flash point (open cup) - >100oC -

Flash point (closed cup) 74 to 83oC 68 to 81oC 46 to 62oC

The redox potential of PAA under standard conditions (pH 7, 25oC, 1 atm) is 1.385 V, but it can

achieve a higher potential of up to 1.748 V in more acidic conditions (Zhang et al., 2018). This is

higher than the redox potential for hydrogen peroxide (1.349 V). The redox potential of PAA is

also higher than many other disinfectants such as chlorine based oxidizers, including Cl2, OCl-,

and HOCl, each having 1.358 V, 0.81 V, and 1.482 V, respectively (Vanýsek, 2018).

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2.2.1 Decomposition

Peracetic acid is typically consumed in three different decomposition reactions (Yuan et al.,

1997a). The first reaction is spontaneous decomposition where PAA degrades into acetic acid and

oxygen (Equation 2). This reaction drives decomposition for pH values between 5.5 and 8.2. It

follows a second-order kinetics with respect to the total peracetic acid concentration and with a

maximum rate at pH 8.2, which is the pKa value of PAA. The spontaneous decomposition rate

decreases with increasing pH, becoming negligible at pH 10.5.

2CH3COOOH → 2CH3COOH+ O2 (2)

The dissociation of PAA follows the equilibrium in Equation 3 . Yuan et al. (1997b) developed a

kinetic expression for the spontaneous decomposition of PAA in an aqueous system that takes into

account the relationship between the rate of decay and temperature through an Arrhenius plot. This

expression can be seen in Equation 4, where M is the ratio of proton concentration to the

dissociation constant (M=[H+]/ka).

CH3OOOH ↔ CH3COOO− + H+ (3)

−d[CH3CO3H]t

dt=

2M

(1 +M)2× 9.21x1013 × exp (−

11338.7

T) × [CH3CO3H]t

2 (4)

However, this expression only works for temperatures between 293 and 333 K and pH values

between 5.5 and 9. Zhao et al. (2008) took into consideration that during PAA synthesis there are

strong acidic conditions ([H+]>0.1 molL-1) due to the addition of sulfuric acid, and developed an

expression for the rate constant of spontaneous decomposition at temperatures ranging from 328

to 368 K (Equation 5).

𝑘 =2.72𝑥1019 exp (−

118529.37𝑅𝑇 ) [𝐻+]

{1 + 2.528𝑥106 exp (−30151.55𝑅𝑇 ) [𝐻+]}2

(5)

The second reaction responsible for the consumption of PAA is hydrolysis, where PAA reacts with

water to form acetic acid and hydrogen peroxide (Equation 6). This reaction is present at the same

Domenica Cevallos


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time as spontaneous decomposition at pH 8.2 but to a much lesser extent, and it is favored as the

pH increases, especially when the pH is 10.5 and higher. It was determined that this decomposition

follows a first-order decay with respect to total peracetic acid concentration (Yuan et al., 1997b).

CH3COOOH+H2O → CH3COOH+ H2O2 (6)

The third and final decomposition reaction is transition metal catalyzed decomposition where PAA

reacts with a transition metal to form acetic acid and oxygen as well as other products (Equation

7). This reaction is also favored at pH values higher than 10.5. When a chelating agent such as

diethylenetriaminepentaacetic (DTMPA) is present this decomposition becomes negligible (Yuan

et al., 1997a, 1997b). Consequently, it has been shown that at pH 10.5 when DTMPA is introduced,

not only spontaneous decomposition becomes negligible, but the metal catalyzed reaction does as

well leaving hydrolysis as the prominent decomposition mechanism. All decomposition products

have a much lesser oxidation potential than PAA; thus, when one of the three decomposition

mechanisms occur there is a loss of oxidation power.

CH3COOOH+M+ → CH3COOH+ O2 + other decompostion products (7)

2.2.2 Equilibrium: Generation and hydrolysis of PAA

When assessing the equilibrium reaction of PAA, synthesis (the forward reaction) and hydrolysis

(the reverse reaction) must be considered. As previously reported the rate at which equilibrium is

achieved can be catalyzed by adding a strong acid like sulfuric acid (JACC, 2001). It has been

reported that both synthesis and hydrolysis are first-order reactions with respect to reactant and H+

concentrations (Zhao et al., 2008, 2007). It was determined that at temperatures below 328 K the

spontaneous decomposition of PAA is negligible, leaving hydrolysis as the only decomposition

mechanism. Thus, at lower temperatures and acidic conditions the system follows Equation 8, and

the reaction rate constants in the temperature range of 293-323 K can be seen in Equation 9 - 10.

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CH3COOH+ H2O2 H2SO4, k1↔ CH3COOOH+ H2O (8)

k1 = [H+] × 6.83x108 exp (−

57846.15

RT) (9)

k2 = [H+] × 6.73x108 exp (−

60407.78

RT) (10)

2.2.3 Oxidation Demand

The loss of PAA through reaction with other constituents in typical waters is often observed to

occur in two phases: (i) an initial instantaneous decrease in concentration and (ii) a subsequent

slow decomposition of residual. It is not easy to distinguish between all the factors that could affect

the initial demand of PAA, such as particulates, iron, reduced organics, microorganisms, and

manganese (Luukkonen et al., 2014). Domínguez-Henao et al. (2018b) has shown that organic

matter can be responsible for the initial PAA demand, with a significant demand occurring within

the first 5 minutes after adding PAA. This initial demand was independent of PAA initial

concentration (2-10 mg/L PAA). Surrogates of compounds that represent the components of

wastewater secondary effluent were used and it was determined that proteins were a main

contributor to the instantaneous demand, whereas carbohydrates and lipids were not. The same

study observed that whereas organics controlled the instantaneous demand, the consumption of

PAA due to inorganics was slow, and was dependent on the initial PAA concentration and took

place throughout the entire experiment (60 min). It was shown that inorganics, specifically

transition metals such as reduced iron, affected the rate constant of PAA decay unless a chelating

agent was present. It was observed that after the rapid consumption of organics, after a few minutes

this reaction became almost nil. After the initial demand took place, the exponential decay due to

reaction with inorganics was 14 to 18 times higher than the decay kinetic rate constant for the

blank (kblank) for PAA initial concentrations of 2 and 10 mg/L respectively, whereas organics only

led to 1 to 3 times higher reaction rate constants than kblank. Modelled results showed that the higher

the initial concentration of PAA, the faster the interaction between PAA with iron and

orthophosphates.

Disinfection pilot studies have confirmed that the initial PAA demand is dependent on feed

characteristics such as suspended solids (SS), chemical oxygen demand (COD), pH, etc., and that

the residual PAA decreases at a very slow rate as seen in Error! Reference source not found..

k2

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Many authors have agreed that PAA decomposition follows first-order kinetics (Falsanisi et al.,

2006; Luukkonen et al., 2015, 2014). Luukkonen et al. (2015) found that the decomposition of

PAA fits first-order kinetics with an R2 value of 0.992 in wastewater.

The residual concentration of PAA can be determined using the modified first-order kinetic model

proposed by Haas and Finch (Equation 11) where Ct is the concentration of PAA at time t (min),

C0 is the initial concentration (mg/L), D is the instantaneous demand (mg/l), and k is the first order

decay coefficient (min-1). The values of D and k can be obtained from experimental data. Authors

have used Equation 11 and determined the D and k parameters in different ways (Falsanisi et al.,

2006). This is usually done by assessing the goodness of fit coefficients including R2, absolute

sum of squares [SS], and standard deviation of the vertical distances [Sy,x] and looking for the

lowest values of [SS] and [Sy,x] and the highest R2.

Falsanisi et al. (2006) has put emphasis on the importance of including the PAA initial demand

(D) into the PAA decay model, finding that when comparing the first-order model for D = 0 and

D ≠ 0, there is statistical improvement when using the D ≠ 0 hypothesis.

Ct = (Co −D) × e−kt (11)

Dell’Erba et al. (2004) observed that the initial demand of PAA (D) varied more than the first-

order decay coefficient for different types of water quality. However, Falsanisi et al. (2006) found

that both D and k values are higher when treating primary sedimentation effluent (PSE) rather than

secondary sedimentation (SSE). It was observed that the D value for PSE was 19.41 mg/L whereas

for SSE it was 0.44 mg/L (Table 2-2). The k value for PSE was almost ten times higher than SSE:

0.0028 min-1 versus 0.0396 min-1. When testing the PAA decay in tap water, which included metal

catalysts but very little organics compared to wastewater, it was observed that after 1 hour the

PAA had decreased by 25-30 % (Rossi et al., 2007).

Table 2-2. Common values for k and D in wastewater following first-order kinetics

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k Value D Value Reference

0.016 min-1 0.8 mg/L Dell’Erba et al. (2004)

0.004-0.005 min-1 0.925 mg/L Luukkonen et al. (2015)

0.0028-0.0396 min-1 0.44 mg/L Falsanisi et al. (2006)

0.007 min-1 0.415 mg/L Antonelli et al. (2013)

CPAA(t) = (C0(PAA) − D) × e−kt

Figure 2-2. Initial demand and decay rate of PAA (Domínguez-Henao et al., 2018b)

2.3 PAA IN THE WATER INDUSTRY

To prevent the spread of human pathogens present in wastewater effluents, disinfection must take

place. It is important to have an efficient inactivation of pathogenic bacteria, viruses, and protozoan

parasites from water and wastewater (Flores et al., 2014). Chlorine and chlorine-based compounds

are the most common agents used during water and wastewater disinfection. However, they can

have a disadvantage due to the formation of disinfection by-products (DBPs). These toxic and

mutagenic halogenated by-products are the result of the reaction of chlorine and organic material

(Monarca et al., 2000).

The recent popularity of PAA in the water industry is attributed to several factors, but the lack of

DBPs after its use is the most important advantage along with its ease of implementation without

the need of expensive capital investment, short contact time, small dependence on pH, and

effectiveness for primary and secondary effluent (Kitis, 2004). During a pilot-plant study using

effluent form secondary treatment it was observed that after adding PAA for disinfection, phenols

were detected in negligible concentrations. Brominated phenols were also detected but at

Glucose

Cellulose

Butyric acid

Oleic acid

Casein

Peptone

N-NH4+

N-NO2-

N-NO3-

Fe2+

P-PO43-

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unrealistic pH values, 3.8-4.2, and halogenated phenols were not present (Dell’Erba et al., 2007).

Thus, the conclusion was that PAA does not form significant concentration of disinfection by-

products. Booth and Lester (1994) looked into the potential increase of chlorinated and brominated

phenols due to the interaction between PAA with bromides, chlorides and organics and determined

that in the original effluents the chlorophenol concentrations were much less than 100 ng/L and

although when introducing PAA the concentration increased, it never surpassed 100 ng/L. A study

also showed that the main by-products in river waters treated with PAA were carboxylic acids

which are not mutagenic, and a few non-halogenated alcohols and carbonyl-containing compounds

were present (Monarca et al., 2002).

However, there are also some disadvantages to using PAA such as a potential increase in the

organic matter content in the effluent due to acetic acid, which can be used for food in microbial

regrowth. Another drawback of using PAA is the high cost due to the limited production, however

as the demand increases the prices may become as cost-effective as chlorine (Kitis, 2004).

In the United States, PAA has been adopted in several wastewater treatment plants temporarily

and also permanently. In November 2018, the Maynard C. Stiles Wastewater Treatment Facility

in Memphis started using PAA for disinfection, and it is expected that a second treatment plant,

also in Memphis, will start to use it by November 2020. At the moment in the US less that 1% of

the wastewater market is occupied by PAA, but it is expected that it will reach 10-15%

(Bettenhausen, 2020). The town of Mount Holly in New Jersey has been using PAA for about 3

years, and Denver’s biggest plant has had full-scale trials since 2018 and is hoping to permanently

change to PAA at the end of the year. In Canada, the Northwest Langley WWTP (NLWWTP) in

Vancouver, which treats 3.2 MGD of wastewater, tested at full-scale the use of PAA as an

alternative to sodium hypochlorite. During these tests it was concluded that PAA is just as effective

as sodium hypochlorite at meeting compliance discharge in log reduction of fecal coliforms,

making it a suitable alternative disinfectant technology. PAA was dosed at different dosages of

12% solution (1.5, 2.0, 2.5, and 3.0 mg/L). The dosages were kept under 3 mg/L due to an effluent

limit of 2.0 mg/L that was imposed (Nguyen et al., 2014).

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2.3.1 PAA synergy with other technologies/treatments

Since both peracetic acid and hydrogen peroxide are strong oxidants and coexist in equilibrium,

they may both contribute to disinfection from the mixture (Alasri et al., 1992). However, they do

not have equal effects. Some batch disinfection tests have been run to determine the dose needed

of each disinfectant to achieve a 2-3 log reduction of fecal coliforms. These test have shown that

to achieve this reduction only 0.6-1 mg/L of PAA were required, while on the other hand, when

using only hydrogen peroxide, 106-285 mg/L were necessary (Wagner et al., 2002). Thus, even

though hydrogen peroxide is also a disinfectant, it is desirable to have a higher concentration of

PAA than hydrogen peroxide because PAA requires a lower concentration and acts against a wide

spectrum of microorganisms by being an exceptional bactericide, fungicide, and sporicide (Baldry,

1983).

Primary and secondary treatment in wastewater can significantly reduce the number of pathogenic

microorganisms, but this is not enough. Thus, the use of coagulant followed by disinfection in

tertiary treatment has been studied. Pradhan et al. (2013) has shown that when using iron-based

coagulants (FeCl3, PIX) followed by PAA, disinfection results in a bigger reduction of E. coli than

when using an aluminum-based coagulant (AlCl3, PAX) followed by PAA. The authors showed

that PIX by itself did not reach the same level of microbial reduction than when using PIX+PAA

with 83.3 and >99% E. coli reduction, respectively. There were no results on the level that PAA

could achieve by itself without PIX or PAX as a pre-treatment. The PAX+PAA combination

resulted in a slightly higher reduction of somatic coliphage than PIX, PIX+PAA and PAX alone.

A synergy between PAA and UV has been reported in the literature. The efficacy of PAA in

concentrations of 2-8 mg/L and UV fluence at 100-300 mJ/cm2 has been investigated (Caretti and

Lubello, 2003). The authors found that when using only PAA (2 mg/L) or UV (165 mJ/cm2) a 2.52

and 3.50 log reduction of total coliforms (TC) was achieved, respectively. When using PAA

followed by UV (PAA+UV) a complete inactivation of all total coliforms was observed. The

combined treatments proved to be less effective when UV was followed by PAA (UV+PAA), with

a 4.57 log reduction of TC. The higher effectiveness found in adding PAA before UV and not vice

versa could be attributed to the formation of radicals. PAA could be photolyzed during UV

radiation leading to the formation of radicals including the hydroxyl radical.

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However, it is still unclear whether there is indeed a synergetic effect between UV and PAA. Even

though some authors have reported an increase in the effectiveness of disinfection when PAA is

added before UV as previously mentioned, there are also authors who claimed that no synergetic

effect was observed regardless of whether PAA was added before or after UV (González et al.,

2012). Others have suggested that water quality plays an important role in this potential synergy.

Weng et al. (2018) observed that even though there was an increase of at least 1 log of additive

reduction of MS2 when PAA+UV was used in contrast to using only PAA, this was only observed

when the experiments were performed in pure lab-grade water, and not in real municipal

wastewater, highlighting the importance of the water matrix and the presence of radical scavengers

in wastewater.

2.4 DISINFECTION KINETICS

2.4.1 Mechanisms of inactivation

PAA can dissociated to peracetate anion (PAA-) as a function of pH. However, the biocidal form

of peracetic acid is the undissociated acid (CH3COOOH), which is the primary molecule at pH

less than the pKa value of 8.2. The PAA oxidation mechanism can also be attributed to the

generation of oxidizing radicals such as the hydroxyl radical (Flores et al., 2014; Kitis, 2004). The

greater bacterial inactivation performance of PAA at lower pH values could also be the result of

PAA having a higher redox potential as the pH decreases, with 1.748 V at pH 0 and 1.005 V at pH

14 (Cai et al., 2017; Zhang et al., 2018). The disinfection mode of action of PAA is believed to be

analogous to other disinfectants by disrupting sulfhydryl (-SH), and disulphide (S-S) bonds in

enzymes and destroying key components of the membrane wall by oxidation (Flores et al., 2014;

Lefevre et al., 1992). The cell wall permeability increases due to the interruption of the lipoprotein

cytoplasmatic membrane chemi-osmotic function (Baldry and Fraser 1988). The sporicidal and

ovicide characteristics of PAA can be explained through its action as a protein denaturant (Block,

2001). An important benefit of using PAA is that it inactivates the catalase which is found in living

organisms and is identified to detoxify free hydroxyl radicals. The equilibrium solution of PAA

seems to act by having PAA alone attacking the cell first, followed by an attack of both PAA and

H2O2 (Flores et al., 2014).

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2.4.2 Inactivation kinetics

Inactivation models using PAA as a disinfectant can be used to determine the optimal

concentration and time required for effective disinfection. Some of the models that have been

tested include Chick-Watson, Hom, and the S-model (Luukkonen et al., 2015). However, it has

been established that the Chick-Watson model is inadequate to describe microbial inactivation

with tailing or shoulders, as a result more advanced models have been developed to take into

account these phenomena. The most common and validated inactivation models follow the

differential rate law seen in Equation 12. Falsanisi et al. (2006) called it the generalized

inactivation rate (GIR), where the special cases of each parameter lead to specific models (Table

2-3) such as Hom, Power law, Hom Power Law, Chick including Chick-Watson (Santoro et al.,

2007).

dN

dt= −k′CnmNxtm−1 (12)

where,

k’, n, m, x = model parameters (n, m, and x are exponents [dimensionless]; k’ takes the SI units

depending on the values of the n, m, and x exponents, to give the left term of the equation in

CFU/100 ml per minute).

N = microbial density (CFU/100 ml)

C = disinfection concentration (mg/L)

T = contact time (min)

Table 2-3. Model parameter ranges for commonly used inactivation models (Santoro et al., 2007)

Model Parameters Chick Chick-

Watson Hom Power law Hom PL

k’ k’ ≠ 0 k’≠ 0 k’≠ 0 k’≠ 0 k’≠ 0

N 0 n ≠ 0 n ≠ 0 n ≠ 0 n ≠ 0

m 1 1 m ≠ 1 1 m ≠ 1

x 1 1 1 x ≠ 1 x ≠ 1

Inactivation rate (dN/dt) -k’N -k’CnN -k’CnmNtm-1 -k’CnNx -k’CnmNxtm-1

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The Hom model can describe deviations from linearity in the survival curve (Table 2-4). Its

empirical constant, m, describes tailing and shoulders in microbial inactivation, with values of m

> 1 representing that the survival curve has an initial resistance to inactivation (shoulder) and m <

1 meaning that the survival curve has asymptotic inactivation (tailing-off) (Luukkonen et al.,

2015). The S-model includes the empirical constant h (mg min L-1) which means that the

inactivation kinetics has all three phases: shoulder, exponential inactivation and tailing-off.

Rossi et al. (2007) concluded that when comparing the Chick-Watson, Hom, Selleck, and S-model,

the first two were not appropriate to describe microorganism inactivation (R2<0.4), and only the

S-model, and Hom model were appropriate. It was concluded that the S-model fits best for PAA

dosages less than 5 mg/L, especially for E. coli since it takes into account inactivation lag where

initial PAA diffusion resistance through cellular membrane takes place and hinders the disinfection

process. Hom’s model fits better for PAA dosages above 5 mg/L where the diffusion resistance is

insignificant.

Table 2-4. Wastewater disinfection models

Chick-Watson Hom S-model

LogNtN0= −ΛCnt Log

NtN0= −kCntm Log

NtN0= −

kCn

1 + (hCt)

m

Including the disinfectant demand into a model is important because it represents a more realistic

view of the disinfection process since PAA consumption can have effects on disinfection efficacy

(Luukkonen and Pehkonen, 2017). To obtain a form of a formula where disinfectant consumption

and decay are accounted for, microorganism disinfection equations most be combined with PAA

decay terms. However, analytical solutions may not be available for all these combinations. For

example, when including a first-order disinfectant decay into the Hom’s model, the only analytical

solution includes the incomplete gamma function (Haas and Joffe, 1994). Santoro et al. (2007) has

concluded that the Power law or the Hom-power law are the most suitable when taking disinfectant

decay into account. Falsanisi et al. (2006) used the modified Hass and Joffe approximate

expression which fit the experimental data very well. Luukkonen et al. (2015) concluded that the

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inactivation of E. coli and enterococci by PAA fits best the S-model (R2=0.945 and R2=0.981,

respectively).

An inactivation kinetic model was developed by Santoro et al. (2015), known as the double-

exponential model, which uses a typical biphasic behavior where a rapid inactivation region is

followed by a tailing effect as observed in unfiltered secondary settled effluents (Equation 13).

This model directly relates the log inactivation to the integral of CT. There are two subgroups of

bacterial population associated with each decay coefficient: non-particle associated or free floating

(kd) which has a fast inactivation rate, and a particle associated subgroup (kp) which has a slow

inactivation rate. When the secondary effluent is filtered, there is no need for two subpopulation

decay coefficients, and only one needs to be used since the microbial population is more similar.

Nt = N0(1 − β)e−kdCTPAA +N0(β)e

−kpCTPAA (13)

where,

Nt = microorganism concentration at time t (CFU/100 mL)

N0 = initial microbial concentration (CFU/100 mL)

𝛽 = particle associated fraction of E. coli

Kp = inactivation rate constant of particle associated E. coli (L mg -1s-1)

Kd = inactivation rate constant of free E. coli (L mg -1s-1)

CTPAA = the integral CT (mg L-1 s)

2.4.3 Radical Formation

One mode of action of PAA during disinfection is the formation of radicals. Hydroxyl radicals are

very strong oxidizers with a redox potential of 2.8 V, higher than those of PAA and H2O2. Its

average lifespan is very short, about 10 s, and it reacts with most organics and several dissolved

inorganics (Hoigné, 1997). It not only attacks these compounds but it can also react with

microorganisms and other chemicals to form other radicals (Lubello et al., 2004).

Peracetic acid can form radicals through homolysis. This process takes place due to the rupture of

the O-O bond in PAA and requires the addition of a catalyst transition metal or in the presence of

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UV radiation (Rokhina et al., 2010). However, the pathway of this homolytic process is

complicated and not as straight forward as other radical generation reactions for advanced

oxidation processes (AOPs). The first step in the formation of radicals during PAA disinfection

can be seen in Equation 14 (Lubello et al., 2004). This reaction results in the formation of acyloxy

and hydroxyl radicals and has been determined to be the rate controlling step (Flores et al., 2014).

The reaction is followed by Equation 15-20.

CH3COOOH → CH3COO ∙ + HO ∙ (14)

CH3COOOH+HO ∙ → CH3CO ∙ + O2 + H2O (15)

CH3COOOH+ HO ∙ → CH3COOO ∙ +H2O (16)

CH3COO ∙ → ∙ CH3 + CO2 (17)

2CH3COO ∙ ↔ 2 ∙ CH3 + 2CO2 + O2 (18)

∙ CH3 + O2 → CH3COO ∙ (19)

CH3COO ∙ + HO ∙ → CH3COOOH (20)

Adding a transition metal as previously mentioned can catalyze the decomposition of PAA into

oxygen and other products (Equation 7). However, transition metals can also serve as a catalyst

for the formation of the hydroxyl radical. Equation 14 requires the presence of an eligible catalyst

like the ones responsible for Fenton-like reactions. Therefore, the radical generation with PAA can

be seen as analogous to the Fenton and Fenton-like reaction, where a metal catalyst is introduced

to disinfection with hydrogen peroxide to form hydroxyl radicals as seen in Equation 21 (Neyens

and Baeyens, 2003; Wang, 2008). It has been observed that intra- or extra-cellular Fe2+ could be

responsible for this reaction and only small amounts are needed for the reaction to take place

(Flores et al., 2014).

H2O2 + Fe2+ → Fe3+ + OH ∙ +OH− (21)

There are several AOPs that generate free radicals, and oxidize contaminants, with hydroxyl

radical being responsible for this oxidation. These processes include UV/H2O2, O3/H2O2, and

O3/UV. It has been reported that there exists a synergetic effect in the use of UV plus PAA. This

synergy was observed through the increase in microbial inactivation when PAA was added before

UV radiation, whereas no synergetic effect was observed when PAA was added after UV (Caretti

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and Lubello, 2003). Hence, it is suggested that UV radiation ruptures the O-O PAA bond through

photolysis and forms radicals (Figure 2-3), in a similar manner to the UV/H2O2 process. However,

it was suggested that the O-O bond of PAA is longer and weaker than the one for H2O2 which

makes it more efficient for radical formation through photocatalysis (da Silva et al., 2020).

Furthermore, the products of UV/H2O2 and UV/PAA are not the same, the latter having not only

hydroxyl radical but carbon-centered radicals as seen in Equation 22 (Chen et al., 2019).

CH3COOOH+ hv → CH3COO ∙ + HO ∙ (22)

2.5 WATER QUALITY

Water quality plays an important role in the efficacy of PAA as a disinfectant. The presence of

organic material, solids, transition metals, salinity, and water hardness can affect the

decomposition of peracids (Luukkonen and Pehkonen, 2017). It has been shown that treating

wastewater with high contents of TSS, COD, and enteric microbes like the ones coming out of

primary treatment reduces the efficacy of PAA (Koivunen and Heinonen-Tanski, 2005). Thus, to

achieve the same level of disinfection in the primary treatment as in secondary treatment a higher

dose of PAA must be applied due to the water quality.

O H CH C O

O ℎ𝑣

H . O

Figure 2-3. Radical formation mechanism from PAA through UV light

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2.5.1 Organic matter

The effect of organic matter on the consumption of PAA has been assessed by several authors.

Organic matter exerts a significant oxidation demand on PAA. However, not all organic matter

has been proven to be responsible for the initial PAA consumption. When looking into proteins,

carbohydrates, and lipids, only the former was responsible for the PAA initial demand

(Domínguez-Henao et al., 2018b). This agrees with Kerkaert et al. (2011), who found that PAA

can degrade cysteine, tryptophan, and methionine in dairy proteins. Pedersen et al. (2013) observed

that when using PAA dosages between 0 and 2 mg/L in fresh water with high concentration of

organic matter in the form of chemical oxidation demand (COD), up to 70.8 mg/L of COD resulted

in instantaneous PAA consumption above 0.2 mg/L. Liu et al. (2014) found a similar trend in the

increase of PAA oxidant demand as the dissolved organic carbon (DOC) increased (8-24 mg/L).

They concluded that DOC and salinity stimulated degradation of PAA. Other authors have found

that other proteins such as peptone (50 mg/L) have a negligible effect on the efficacy of PAA

(Harakeh, 1984).

2.5.2 Suspended solids and particulate matter

The presence of high concentrations of total suspended solids (TSS) can affect PAA disinfection

by increasing PAA decay rates, especially if the TSS concentration is above 40 mg/L. TSS can

also affect E. coli inactivation kinetics since it can act as a protective shield protecting bacteria

from PAA and lowering the bacteria inactivation. The protection given by TSS to E.coli depends

on the PAA concentration: the higher the concentration of PAA the higher the protective effect of

TSS (Domínguez-Henao et al., 2018a). This agrees with Dietrich et al. (2003), who stated that the

diffusion of disinfectant into wastewater particles involves a macro- and microporous network of

pathways within each particle, and incomplete penetration into the full network can result in

residual concentration of targeted organisms. Thus, wastewater particles can contain regions that

can completely shield bacteria from disinfection. McFadden et al. (2017) found that wastewater

particles with diameters between 10 µm and 100 µm had little effect on PAA disinfection, whereas

Falsanisi et al. (2008) found that TSS afforded different levels of protection depending on its

particle size. They reported that particles between 10-120 µm gave a 0.6 log protection to fecal

coliforms, and particles with >120 µm provided a 1.3 log protection.

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Lazarova et al. (1999) observed that the impact of suspended matter at concentrations between 10

and 40 mg/L on disinfection remained constant. Lefevre et al. (1992) found that at concentrations

of 5 to 10 mg/L TSS, there was no significant effect on PAA performance. Stampi et al. (2001)

observed that suspended solids with a mean value of 17.6 mg/L did not affect the action of the

disinfectant.

2.6 PAA ANALYSIS

The most common methods for analysis of PAA and H2O2 incorporate redox titration. However,

there are other techniques for PAA quantification such as the DPD calorimetric method which has

great sensitivity and good cost (Cavallini et al., 2013).

The colorimetric technique is based on the DPD (N,N-diethyl-p-phenylelnediamine) method,

which has been widely used to quantify other oxidants. The US Environmental Protection Agency

(EPA) has approved this method to quantify total chlorine (Method 330.5). It consists on having

the oxidant liberate iodine from potassium iodide which reacts with the DPD and forms a pink

color (EPA, 1978). For PAA specifically this process takes place when the total DPD reacts with

PAA to form the pink color species which are produced proportionally to the concentration of

peracetic acid (Domínguez-Henao et al., 2018c). Since PAA exists in equilibrium with H2O2, the

latter also must be quantified. Methods to measure both have been developed, including the HACH

10290 method which can measure PAA and H2O2 in the range of 0.1-10 mg/L and 0.05-5.00 mg/L,

respectively. Here, hydrogen peroxide does not interfere with the PAA quantification because

hydrogen peroxide analysis requires the addition of catalysts to react with DPD. Once PAA is

quantified, H2O2 can be analyzed by adding ammonium molybdate catalyst to the sample and

repeating the DPD method.

Iodometric titration of PAA is a redox titration that can be performed by first quenching hydrogen

peroxide with bovine liver catalase. The subsequent reactions are described according to Equations

24-25 where PAA oxidizes iodide (potassium iodide) into iodine and then the liberated iodine is

titrated with sodium thiosulfate using starch as an indicator. Iodometric titration can be used to

quantify PAA concentrations between 1-5 mg/L (Cavallini et al., 2013). The concentration of PAA

can be calculated using Equation Error! Reference source not found..

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CH3COOOH+ 2I− + 2H+ → CH3COOH+ I2 + H2O

(24)

I2 + 2S2O32− → 2I− + S4O6

2−

(25)

CPAA =VT × CS2O32−

VS × 2 (26)

where,

VT = volume of sodium thiosulfate used (mL)

VS = sample volume (mL)

CPAA = PAA concentration (mol/L)

CS2O32− = Sodium thiosulfate concentration (mol/L)

Other titration methods avoid neutralizing hydrogen peroxide by lowering the temperature to less

than 5oC, such that H2O2 reacts with iodide extremely slowly (Hatcher and W., 1927). After PAA

is determined, H2O2 can be quantified by adding ammonium molybdate as a catalyst to accelerate

the quantification reaction like in the DPD method (Sully and Williams, 1962).

2.7 TOXICITY

Peracetic acid does not persist in the environment due to its decomposition into other species such

as acetic acid, water, hydrogen peroxide, and oxygen as seen in previous sections. However, the

US EPA has approved a maximum wastewater disinfection effluent concentration of PAA of 1

mg/L. The aquatic toxicity of PAA can be assessed by looking into three main groups present in

freshwater: invertebrates, algae, and fish.

When assessing the toxicity of Daphnia magna it has been observed that the median concentration

expected to have an effect in 50 % of the test organisms (EC50) ranged between 0.5 and 1 mg/L

PAA after 48 hours of exposure (Douglas and Pell, 1986; Lamy et al., 1997). A no-observed-effect

concentration (NOEC) value as low as 0.15 mg/L PAA was determined. Daphnia magna is a good

indicator of toxicity since this micro-crustacean is reasonably sensitive to pollutants in comparison

to other invertebrates present in fresh water (Panouille, 2007). When comparing the toxicity of

PAA to invertebrates against chlorine toxicity, one study showed that PAA toxicity after 40 hours

of exposure is lower than that of sodium hypochlorite. The study determined an EC50 value of 0.73

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22

mg/L for PAA and 0.033 mg/L for sodium hypochlorite. The lower EC50 value for sodium

hypochlorite shows that, for chlorine, lower concentrations still represent a risk to aquatic life

(Gardner et al., 1996).

Peracetic acid toxicity to algae, especifically to Selenastrum capricornutum, has been evaluated.

Hicks et al. (1996) found that during a 130-hour exposure duration the EC50 and NOEC values for

Selenastrum capricornutum were 0.18 mg/L and 0.13 mg/L PAA, respectively. Toxicity to fish

was lower: it was observed that for 12 different species exposed during 24 hours, the average LC50

values were between 2.8-9.3 mg/L PAA, and the no-observed-effect concentration (NOEC) was

between 1.9-5.8 mg/L PAA (Straus, 2018). The most tolerant specie to PAA exposure were tilapia,

and the most sensitive were fathead minnow. It was also observed that as the alkalinity/hardness

decreased the toxicity also decreased. This agrees with Marchand et al. (2013), who found that the

toxicity of PAA to zebrafish embryos was negatively correlated to water hardness.

Whole effluent toxicity (WET) refers to the aggregate toxic effect to aquatic organisms from all

pollutants contained in a wastewater facility effluent. These tests measure the effect of wastewater

on specific test organisms’ ability to survive, grow and reproduce (EPA, 2019). They are

performed by exposing an organism to dilute and undilute effluent samples under controlled

conditions (SETAC, 2004). During a disinfection pilot trial at Little Miami WWTP in Ohio, a

WET study was performed in the WWTP’s effluent. Ceriodaphnia dubia and Pimephales

promelas were tested for acute toxicity with PAA concentrations of 1 and 2 mg/L. This two-day

WET test resulted in a “passing” performance, where the values for acute toxicity were below

detection for both organisms. This demonstrated that PAA could be an environmentally friendly

disinfection process (EPA, 2017).

2.8 RESEARCH NEEDS

The literature has previously explored the reaction between PAA and transition metals such as

iron, and how their reaction can lead to the formation of reactive oxygen species that could enhance

the PAA oxidation process (Rokhina et al., 2010). There is anecdotal evidence from pilot-and full-

scale showing that the disinfection efficiency of PAA increases when using alum instead of iron-

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based coagulants during wastewater treatment. However, there is little available research on how

peracetic acid interacts with non-transition metals like Al3+ and the pathways that could lead to a

synergetic effect between the two. A study should be conducted in order to determine if aluminum

reacts with PAA in the same manner as iron producing free radicals as a consequence.

Moreover, as peracetic acid becomes more popular in the water industry its uses go beyond

wastewater disinfection. It has started to be considered as a pretreatment for DBP minimization

and mussel control during drinking water treatment (Griffin et al., 2018; Hurtado, 2020). Thus,

there is a need to understand the impacts of PAA addition to subsequent chlorine disinfection.

2.9 REFERENCES

Alasri, A., Roques, C., Michel, G., Cabassud, C., Aptel, P., 1992. Bactericidal properties of

peracetic acid and hydrogen peroxide, alone and in combination, and chlorine and

formaldehyde against bacterial water strains. Can. J. Microbiol. 38, 635–642.

Antonelli, M., Turolla, A., Mezzanotte, V., Nurizzo, C., 2013. Peracetic acid for secondary effluent

disinfection: A comprehensive performance assessment. Water Sci. Technol. 68, 2638–2644.

Baldry, M.G.C., 1983. The bactericidal, fungicidal and sporicidal properties of hydrogen peroxide

and peracetic acid. J. Appl. Bacteriol. 54, 417–423.

Baldry, M.G.C., French, M.S., 1989. Disinfection of sewage effluent with peracetic acid. Water

Sci. Technol. 21, 203–206.

Baldry, M.G.C., French, M.S., Slater, D., 1991. The activity of peracetic acid on sewage indicator

bacteria and viruses. Water Sci. Technol. 24, 353–357.

Bettenhausen, C.A., 2020. How peracetic acid is changing wastewater treatment. Chem. Eng.

News 98.

Block, S.S., 2001. Disinfection, Sterilization, and Preservation, Disinfection, Serilization & P.

Lippincott Williams & Wilkins.

Booth, R.A., Lester, J.N., 1994. The potential formation of halogenated by-products during

peracetic acid treatment of final sewage effluent. Wat. Res. 29, 1793–1801.

Cai, M., Sun, P., Zhang, L., Huang, C.H., 2017. UV/Peracetic acid for degradation of

pharmaceuticals and reactive species evaluation. Environ. Sci. Technol. 51, 14217–14224.

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Caretti, C., Lubello, C., 2003. Wastewater disinfection with PAA and UV combined treatment: A

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3 PERACETIC ACID REACTION WITH THIOSULPHATE

AND WITH CHLORINE

ABSTRACT

The reaction kinetics of peracetic acid (PAA) with sodium thiosulphate has not

been reported in the literature. This is important information in a laboratory context

when conducting experiments that require the PAA to be quenched quickly. In this

study, the reaction rate was examined using PAA concentrations of 5 and 10 mg/L

and their corresponding H2O2 concentrations (0.3 and 0.4 mg/L, respectively).

Sodium thiosulfate in a 1:1 stoichiometric ratio to PAA and H2O2 was enough to

immediately quench both species, eliminating the need to subsequently add catalase

to quench residual H2O2.

In drinking water treatment, peracetic acid may be added at the front of the

treatment train to destroy disinfection byproduct precursors, but residual PAA and

H2O2 may exert a downstream chlorine demand. A kinetic reaction model was

developed using MATLAB to predict this reaction. The model suggested that while

the initial H2O2 in the mixture quickly reacts with chlorine, the PAA and any H2O2

that is formed through PAA hydrolysis in an attempt to re-stablish equilibrium

would not exert any significant chlorine demand over a 20-hour time frame.

Experimental data suggests that there is no direct reaction between PAA and

chlorine over a 24-hour period and that chlorine decay is attributed to H2O2

formation only. The kinetic model also tested the PAA decay alone due to

hydrolysis and spontaneous decomposition. The PAA decay was proportional to

increase in temperature and pH. An initial concentration of 1 mg/L PAA decayed

to almost zero after 20 hours at 333 K and pH 10.

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3.1 INTRODUCTION

3.1.1 Neutralization of PAA/H2O2 solution

PAA is considered a promising alternative to chlorine-based disinfectants, especially for municipal

wastewater treatment (Rossi et al., 2007). As a disinfectant for secondary effluent in a wastewater

treatment plant, PAA may not require quenching because it does not leave a mutagenic or toxic

residual (Kitis, 2004). However, to assess its disinfection efficacy on microbial communities in a

laboratory setting, a quenching method is required not only for PAA but for its equilibrium

components since H2O2 is also a strong oxidant.

3.1.1.1 Quenching of H2O2

Hydrogen peroxide is one of the equilibrium components in a PAA solution, alongside acetic acid.

When PAA quenching is required, this process also includes neutralizing H2O2. Hydrogen

peroxide can act an oxidizer or as a reducing agent. It can be reduced by reagents such as sulfite,

thiosulfate, and bisulfite, or it can be oxidized by chemicals such as chlorine (Wang et al., 2019).

It has been found that the reaction between H2O2 and free chlorine is very rapid (Equation 1-3),

and proceeds at a faster rate than the reaction between H2O2 and catalase, which is effective to

quench hydrogen peroxide (Equation 4) (Keen et al., 2013). It has been stablished that the second-

order rate constant for the reaction between Cl2 and H2O2 will increase with pH, with a rate as fast

as 3.2x103 Ms-1 at pH 10 and after this it will decrease with higher pH values (Held et al., 1978).

Cl2 +H2O → HOCl + Cl− +H+ (1)

HOCl + H2O2 → H2O + Cl− + O2 +H

+ (2)

OCl− + H2O2 → H2O + Cl− +O2 (3)

H2O2Catalase→ O2 +H2O (4)

Keen et al. (2013) also suggested sodium thiosulfate (Na2S2O3) and sodium sulfite to quench H2O2

as alternatives to chlorine and catalase. The authors found that the reaction between sodium

thiosulfate and hydrogen peroxide (Equation 5) is slow compared to using free chlorine or sodium

sulfite as quenching agents. Using exact stoichiometric ratios to quench 1 mg/L of H2O2, would

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require 1.2 mg/L of sodium thiosulfate, but it takes hours to completely neutralize the hydrogen

peroxide.

2Na2S2O3 + H2O2 → Na2S4O6 + 2NaOH (5)

Sodium bisulfate has also been studied in the literature as a quenching agent for hydrogen peroxide.

It has been observed that the reaction between H2O2 and bisulfite is rather slow with a half-life in

the order of hours to days, which depends on the reagent concentration and pH value (Wang et al.,

2019). Thus, chlorine is much preferred for neutralizing H2O2, except at pH values <5.7 where

bisulfite becomes the faster reagent (Wang et al., 2019).

3.1.1.2 Quenching of PAA

When assessing the efficacy of PAA disinfection in the lab it is necessary to have a method for

PAA quenching that completely eliminates any disinfectant residual before microbial analysis.

Peracetic acid is an oxidant and, theoretically, can be quenched by many reductants such as

thiosulfate, metabisulfite, bisulfite, sulfite, and ascorbic acid, although the kinetics of these

quenching reactions remain to be defined (Corcoran and Whinston, 1991; Hilgren et al., 2011). On

the other hand, PAA decomposition can be accelerated by metals (e.g., iron, cobalt, manganese,

and copper (Yuan et al., 1997b)). Bases such as sodium carbonate and sodium hydroxide can also

be used to quench PAA when its concentrations are high (e.g., as a disinfectant in the health care

industry) (Corcoran and Whinston, 1991). Enzymes such as catalase, which are effective in

neutralizing hydrogen peroxide, has been proven ineffective in quenching PAA (Block 2001,

Rizzo et al. 2018). Therefore, the most feasible options to remove PAA is through the adoption of

the reductants.

In the water and wastewater industry, sodium thiosulfate (Na2S2O3) is the most commonly used

quenching agent for PAA (Antonelli et al., 2013; Dunkin et al., 2017; Lazarova et al., 1999; Manoli

et al., 2019; Rajala-Mustonen et al., 1997; Rossi et al., 2007; Shah et al., 2015; Weng et al., 2018).

The reaction between sodium thiosulfate and PAA follows Equation 6. Some technical reports

suggest to add sodium thiosulfate in a 1:1 stoichiometric ratio (Peroxychem, 2017), while others

suggest to add it in excess to compensate for the slow kinetics. It has been recommended that the

dosages to completely quench PAA should be 1X, 2X, and 5X molar excess for 1% PAA, and

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100X stoichiometric excess for 0.001% PAA (EPA, 2014). Shah et al. (2015) used sodium

thiosulfate at a ratio of 2:1 to the initial molar concertation of PAA of 2.0 mM.

CH3COOOH+ 2S2O32− + 2H+ → CH3COOH+H2O + S4O6

2− (6)

The sulfur(IV) reductants are another group of potential PAA quenching agents which include

sodium metabisulfite (SMBS, Na2S2O5), sodium bisulfite (SBS, NaHSO3) (EnviroTech, n.d.) and

sulfite (Wang and Zhao, 2018). These chemicals result in the same species, SO32- or HSO3

-

depending on the pH, after dissolving in the water. Equation 7 shows the reaction of sodium

bisulfite with PAA, with a molar ratio of 1:1. A similar or shorter reaction time is expected when

using sulfur(IV) reducing agents in comparison to using thiosulfate as the former is a strong

reducing agent. Hilgren et al. (2011) found that at a SMBS/PAA ratio of 1.75, 60 mg/L PAA can

be quenched to 20 mg/L within half an hour.

NaHSO3 + CH3COOOH → CH3COOH+ NaHSO4 (7)

3.1.2 PAA & Chlorine

Peracetic acid has also been considered as a pretreatment to reduce disinfection by-products

(DBPs) precursors during drinking water treatment (Griffin et al., 2018). When used for pre-

oxidation, residual PAA and its equilibrium components (H2O2 and acetic acid) could have an

impact on the subsequent treatment, specifically on chlorine disinfection

There are three reactions responsible for PAA concentration at a given time: formation, which is

the forward reaction during equilibrium and consists of the formation of PAA through acetic acid

and hydrogen peroxide (Equation 8); hydrolysis, which is the decomposition of PAA in water into

H2O2 and acetic acid (AA) (Equation 9); and spontaneous decomposition (Equation 10) (Yuan et

al., 1997a; Zhao et al., 2007). The presence of a transition metal can result in a third decomposition

reaction for PAA, but it will not be considered in this chapter.

CH3COOH+H2O2 𝑘1→ CH3COOOH+ H2O (8)

CH3COOOH+ H2O 𝑘2→ CH3COOH+ H2O2 (9)

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2CH3COOOH 𝑘3→ 2CH3COOH+ O2 (10)

Kinetic constants for these reactions have been developed in the literature, Yuan et al. (1997b,

1997a) developed expressions for the rate constants k2 and k3 in an aqueous system as seen in

Equation 11 and 12, respectively. The pH of the solution determines which reaction is favored,

with hydrolysis being more prominent at high pH values, particularly 10.5 and higher.

Spontaneous decomposition has a faster rate at lower pH than hydrolysis, with the highest rate at

the pka value of 8.2, and at any pH higher than this the spontaneous decomposition rate will

decrease. It has been determined that while hydrolysis is a first-order reaction with respect to PAA,

spontaneous decomposition is second-order (Koubek et al., 1963). Other authors have also

developed expressions for these rate constants, but they lack the aqueous system used in the kinetic

model for this chapter (Zhao et al., 2008, 2007).

k2 = 2.32 × 108 exp (−

7488.68

T)

kaka + [H+]

+ 1.19 × 109 exp (−5903.40

T)

[H+]

ka + [H+] (11)

k3 = 9.21 × 1013exp (−

11338.71

T)

2[H+]/ka(1 + [H+]/ka)2

(12)

where,

k2= PAA hydrolysis reaction rate coefficient (L/mol s)

k3= PAA spontaneous decomposition reaction rate coefficient (L/mol s)

ka= PAA dissociation constant

T= temperature (K)

Since there is not a reaction rate coefficient for PAA synthesis in aqueous phase, Janković and

Sinadinović-Fišer (2005) determined the chemical equilibrium constant (K) in the liquid phase

using an integrated form of the van’t Hoff equation, Equation 13. From here, using another form

of the chemical equilibrium constant for the water phase which includes the forward and reverse

reaction rate constants (Equation 14), the value for the formation rate constant can be calculated

(Rangarajan et al., 1995).

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K = exp (12.2324 ln(T) − 0.0229913T + 9.70452 × 10−6T2 +3045.76

T− 72.8758)

(13)

K =k1k2=CPAACH2O

CAACH2O2 (14)

As previously mentioned, H2O2 can be oxidized by chlorine, but the effect of chlorine on PAA and

vice versa is unknown (Wang et al., 2019). The rate coefficient of reaction between H2O2 and

chlorine is pH dependent and can be calculated using Equation 15 (Held et al., 1978).

k4 =3.4 × 103M−1S−1

1 +[H+]

2.9 × 10−8M

× 1 +2.2 × 10−12M

[H+] (15)

3.2 OBJECTIVE

The first objective of this chapter is to determine the most appropriate method for PAA and H2O2

quenching in a laboratory setting. The effectiveness of sodium thiosulfate to neutralize PAA and

catalase to neutralize hydrogen peroxide will be assessed. A second objective is to determine the

effect of pH in PAA decay in the PAA/H2O2/AA/OCl- system as a result of the chlorine-hydrogen

peroxide reaction by developing a kinetic model. The last objective is to demonstrate PAA

decomposition as a function of temperature and pH. More specifically the objectives of this chapter

are:

1. To quantify how much sodium thiosulfate and catalase are needed to completely quench

PAA and H2O2, respectively.

2. To determine how the chlorine-hydrogen peroxide reaction affects the decay of PAA, and

how the PAA solution in turn exerts a chlorine demand.

3. To predict how fast PAA decays due to spontaneous decomposition and hydrolysis.

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3.3 MATERIALS AND METHODS

3.3.1 Quenching experiments

3.3.1.1 Reagents and Equipment

Quenching experiments were performed to determine the sodium thiosulfate and catalase doses

required to quench 5 and 10 mg/L of PAA and their corresponding H2O2 concentrations (0.3 and

0.4 mg/L, respectively). A magnetic stirring plate was used to mix the quenching agents with the

PAA solution. Peracetic acid was made using the reagents detailed below and quantified using a

HACH DR 2700 spectrophotometer (DPD method).

PAA synthesis (100 mg/L)

• 0.19 g TAED granule

• 0.285 g Sodium percarbonate

• 0.05 g EDTA

• 0.26 g Citric acid

Quenching Agents

• Sodium thiosulfate (Na2S2O3)

• Catalase

3.3.1.2 Experimental Protocols

PAA was synthesized by adding the TAED, sodium percarbonate and EDTA into 1 L of Milli-Q

water with continuous stirring with a magnetic bar for 30 minutes. Citric acid is added almost at

the end of the mixing time to reduce the pH to 6. The resulting PAA had a concentration of 100

mg/L and 5.36 mg/L of H2O2 and was placed in a 1 L amber bottle. For the quenching tests the

stock PAA solution was diluted into 30 mL of 5 and 10 mg/L. The quenching process followed

procedures outlined in studies like Martin and Gehr (2007) and Wagner et al. (2002) who use

sodium thiosulfate to quench PAA subsequently followed by catalase to neutralize H2O2 since

thiosulfate only quenches hydrogen peroxide slowly.

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Sodium thiosulfate was added to the PAA dilutions under constant stirring at a ratio of 2 moles of

sodium thiosulfate per 1 mol of PAA. Catalase was added immediately following the sodium

thiosulfate addition to a final concentration of 13.2 µg/L. The final concentrations of PAA and

H2O2 were measured after approximately 5 seconds using the HACH DPD method.

3.3.2 Kinetic model

The PAA used for the kinetic model was regent grade PAA (32%) purchased from Sigma-Aldrich

where the equilibrium components are present in the following molar ratios: 0.3 mol H2O2/ 1 mol

PAA and 1.8 mol AA/ 1 mol PAA. It is assumed that there is no transition metal or organic matter

present for this simulation that could affect the consumption of PAA. The concentration of residual

PAA used in the PAA/H2O2/AA/OCl- system was 1 mg/L and the chlorine dose was 1.5 mg/L.

The rate constants used during this simulation are seen in Equations 11 and 12 for PAA hydrolysis

and spontaneous decomposition, respectively. The PAA formation rate constant is obtained

through Equations 13 and 14 since there is no available expression in the literature for an aqueous

system. The rate constant for the reaction between chlorine and H2O2 was determined using

Equation 15 at pH values ranging from 6-10.

The mathematical model used is given in Equations 16-19, as a system of differential equations

where the concentration of PAA, H2O2, AA, and chlorine can be modeled through time. This

system of differential equations was solved using MATLAB R2020a as a stiff first-order ODE.

dCPAAdt

= k1CAACH2O2 − k2CPAACOH− − k3CPAA2 (16)

dCH2O2dt

= −k1CAACH2O2 + k2CPAACOH− − k4CH2O2COCl− (17)

dCAAdt

= −k1CAACH2O2 + k2CPAACOH− + k3CPAA2 (18)

dCOCl−

dt= −k4CH2O2COCl− (19)

The live function used for the simulation is in Figure 3-1. The function was paired to a script which

includes the ode15s solver with an specific time span [t0 tf], time step of 0.01 hr, and initial

conditions y(0) for each component.

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function dydt = PAA&Cl(t, y)

T=296; %(K)

ka=10^(-8.2);

pH=4;

W=10^(-pH);%[H]

K=exp(12.2324*log(T)-0.0229913*T+(9.70452*10^-6)*T*T+(3045.76/T)-72.8758);

k2=(2.32*10^8)*exp(-7488.68/T)*(ka/(ka+W))+(1.19*10^9)*exp(-5903.4/T)*(W/(ka+W))*60*60;

%(L/mol*hr)

k1=K*k2; %(L/(mol*hr)

k3=(9.21*10^13)*exp(-11338.71/T)*(2*W/ka)/((1+W/ka)^2)*60*60;

k4=0.98*60*60; %(L/(mol*hr)

pOH=14-pH;

OH=10^(-pOH);%[OH]

%dPAA/dt=k1[PAA][H2O2]-k2[PAA][OH]-k3[PAA]^2

%dH2O2/dT=-k1[PAA][H2O2]+k2[PAA][OH]-k4[H2O2][OCl]

%dAA/dt=-k1[PAA][H2O2]+k2[PAA][OH]+k3[PAA]^2

%dOCl/dt=-k4[H2O2][OCl]

dydt=[k1*(y(3)*y(2))-k2*y(1)*OH-k3*(y(1)^2);-k1*(y(3)*y(2))+k2*y(1)*OH-k4*(y(2)*y(4));-

k1*(y(3)*y(2))+k2*y(1)*OH+k3*(y(1)^2);-k4*y(2)*y(4)];

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end

Figure 3-1. MATLAB function and script used to solve a system of 4 differential equations

3.3.3 Direct PAA/OCl- reaction tests

Chlorine was added to a PAA solution in high enough concentrations so the chlorine residual

(measured as Cl2) after reacting with H2O2 would be close to a 1:1 molar ratio with PAA. The

concentration profiles of PAA/H2O2, and Cl2 were measured throughout a 24-hour period using

the HACH DPD method. Strong phosphate buffers (0.5 M) were used in order maintain the pH at

6, 7, 8, and 9. The PAA used during the experiments was a dilution of the regent grade PAA (32%)

purchased from Sigma-Aldrich. The H2O2 concentration measured was about 10-12% wt. All

experiments were run in duplicate for quality control, and there were two controls consisting of

Cl2 only and PAA/H2O2 solution only. The interference of high concentrations of PAA on Cl2

readings is about 0.1 mg/L for which the final results have been adjusted. Furthermore, chlorine

also interferes with the PAA reading, it was found that 1 mg/L of pure Cl2 is displayed as 1 mg/L

PAA. Thus, PAA concentrations were obtained by subtracting the PAA reading, by the Cl2

measured previously. 100 mL amber bottles were used as reactors to avoid light exposure. Little

air space was left in the bottles in order to prevent volatilization of the components. Due to the

high concentrations of PAA and Cl2, the samples were diluted into an acidic medium before

analysis in order to stay within the range of the HACH spectrophotometer, 0.1 - 10mg/L for PAA

and 0.02 - 2.0 mg/L for free chlorine.

3.4 RESULTS AND DISCUSSION

3.4.1 Quenching tests

Quenching tests were performed to determine the required concentration of sodium thiosulfate and

catalase needed to completely neutralize PAA and H2O2. The tests were conducted at pH 7 and

using stoichiometric ratios according to Equation 6. As seen in Table 3-1 and 3-2, the 2:1 sodium

%PAA(0)=0.0000131 (1mg/L)

%H2O2(0)=0.00000394

%AA(0)=0.0000238

%OCl(0)=0.000029 (1.5mg/L)

[t,y]=ode15s(@PAA&Cl,[0:0.01:20],[0.0000131;0.00000394;0.0000238;0.000029]);

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thiosulfate/PAA ratio is enough to completely quench both PAA and H2O2 within seconds without

the need to add catalase. Moreover, the quenching agent only required a few seconds to fully

neutralize the PAA solution under constant mixing.

Table 3-1. Quenching of 5 and 10 mg/L PAA (pH 7 & 5 sec retention time)

Sodium thiosulfate

(mg/L)

Catalase

(µg/L)

5 mg/L PAA

PAA (mg/L) H2O2 (mg/L)

- - 4.8 0.3

20.8 - LDL LDL

20.8 13.2 LDL LDL

10 mg/L PAA

PAA (mg/L) H2O2 (mg/L)

- - 9.7 0.46

41.5 - LDL LDL

41.5 13.2 LDL LDL

LDL: Less than the minimum detection limit (0.1 mg/L)

3.4.2 Kinetic model

3.4.2.1 PAA/H2O2/AA system

In this study, the effect of pH on peracetic acid was assessed by modelling the decay of PAA due

to spontaneous decomposition and hydrolysis, assuming no transition metals, chlorine, and organic

matter are present. Figure 3-2 shows the PAA decay at different pH and temperature values. When

the pH values are low (below 8.2), the main decomposition mechanism of PAA is spontaneous

decomposition. However, at temperatures lower than 328 K and in the absence of transition metals

this mechanism is almost negligible. This agrees with the obtained results. It can be seen that at

pH 7.5 and temperature 296 K the decay of PAA is the slowest, but once the temperature increases

to 333 K the decay increases because spontaneous decomposition is more prominent. When the

pH increases hydrolysis becomes the dominant mechanism of decomposition and as can be seen

at pH 10 the decay of PAA is more significant. It is also shown that hydrolysis has a greater effect

on PAA decay than spontaneous decomposition. These results also show that hydrolysis takes

place at a faster rate when temperature increases.

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Figure 3-2. Simulation of PAA decay due to decomposition

3.4.2.2 PAA/H2O2/AA/OCl- system

In this section the effect of PAA on chlorine and vice versa was determined in order to understand

how peracetic acid residual can affect chlorine disinfection. This kinetic model uses the same

assumptions of the model used in the previous section, but it also includes the presence of chlorine

in the system. The effect of chlorine in the equilibrium of PAA can be observed in Figure 3-3. An

initial PAA concentration of 1 mg/L does not seem to drastically decay in the presence of chlorine.

This is because the model used in this study assumes PAA does not react with OCl- as seen in

previous work (Hurtado, 2020). On the other hand, a hydrogen peroxide initial concentration of

0.1 mg/L abruptly decays to almost zero within 2 minutes and occurs faster as the pH increases.

This is attributed to the chlorine-hydrogen peroxide reaction, whose rate increases proportionally

to the increase in pH.

0

0.2

0.4

0.6

0.8

1

1.2

0 5 10 15 20

Conce

ntr

atio

n (

mg/L

)

Time (hr)

PAA pH:7.5 T:296

PAA pH:7.5 T:333

PAA pH:10 T:296

PAA pH:10 T:333

H2O2 pH:7.5 T:296

H2O2 pH:7.5 T:333

H2O2 pH:10 T:296

H2O2 pH:10 T:333

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Figure 3-3. Simulation of chlorine effect on PAA equilibrium trhough time at different pH

values and 296 K

Figure 3-3 shows that even though the initial concentration of H2O2 is consumed almost

immediately, chlorine continues to decay throughout the entire 20 hours, which means that more

H2O2 is added to the system. This can be a result of PAA decomposing into hydrogen peroxide, as

pH increases and in the absence of transition metals, hydrolysis is favored over spontaneous

decomposition. Therefore, there is more production of H2O2 by PAA hydrolysis which in turn

decreases the PAA concentration throughout the simulation.

3.4.3 Direct PAA/OCl- reaction

To test whether PAA reacts directly with chlorine, the concentration profiles of a PAA/H2O2/Cl2

system were determined. Figures 3-4 to 3-7 show that PAA decay in the system is similar to that

in the control. This suggests that there is no PAA consumption attributed to a direct reaction with

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

0 5 10 15 20

Conce

ntr

atio

n (

mg/L

)

Time (hr)

pH 6 PAA

H2O2

AA

OCl-

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

0 5 10 15 20

Conce

ntr

atio

n (

mg/L

)

Time (hr)

pH 7.5PAA

H2O2

AA

OCl-

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

0 5 10 15 20

Conce

ntr

atio

n (

mg/L

)

Time (hr)

pH 8.3 PAA

H2O2

AA

OCl-

-0.2

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

0 5 10 15 20

Conce

ntr

atio

n (

mg/L

)

Time (hr)

pH 10PAA

H2O2

AA

OCl-

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Cl2. The experiential chlorine decay after Cl2 initial reaction with H2O2 follows the same trajectory

as the theoretical Cl2 decay due to H2O2 in the control. When comparing the experimental results

to those of the kinetic model in a PAA/H2O2/Cl2 system it can be seen that chlorine and PAA have

a greater decay in reality than they do in the simulation. This could be attributed to more hydrogen

peroxide being formed by PAA decomposition.

Figure 3-4. PAA, H2O2, and Cl2 concentration profiles, pH 6

0

20

40

60

80

100

120

140

160

0 5 10 15 20 25

Conc.

(m

g/L

)

Time (hr)

Cl2 (PAA/H2O2/Cl2 system) PAA (PAA/H2O2/Cl2 system) H2O2 (PAA/H2O2/Cl2 system)

Cl2 control PAA control (PAA/H2O2 system) H2O2 control (PAA/H2O2 system)

Cl2 model (PAA/H2O2/Cl2 system) PAA model (PAA/H2O2/Cl2 system) H2O2 model (PAA/H2O2/Cl2 system)

Cl2 based on H2O2 control

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0

20

40

60

80

100

120

140

160

0 5 10 15 20 25

Conc.

(m

g/L

)

Time (hr)





0

20

40

60

80

100

120

140

0 5 10 15 20 25

Conc.

(m

g/l

)

Time (hr)





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The figures above show that as pH increases, the Cl2 and PAA decay increases as well. This is

supported by the fact that spontaneous decomposition of PAA into H2O2 and AA is favored at

greater pH values. It can be concluded that the kinetic model might underestimate the decay of

chlorine and PAA. However, the experimental method has several limitations, including the

analysis of its components. Since PAA concentration is determined by subtracting the previously

analyzed Cl2 value from the PAA reading, the resulting PAA values might not be an accurate

representation of the true PAA value. A better analysis method should be developed in order to

quantify all the components simultaneously.

3.1 SUMMARY AND CONCLUSIONS

Quenching experiments were performed to determine the required doses of sodium thiosulfate and

catalase to completely quench PAA and H2O2, respectively. It was observed that when adding

sodium thiosulfate to PAA at the molar stoichiometric ratio of 2:1, it not only completely quenched

PAA within seconds but also neutralized H2O2. This eliminated the need to subsequently add an

extra quenching agent such as catalase to neutralize hydrogen peroxide. This will be useful in a

0

20

40

60

80

100

120

140

0 5 10 15 20 25

Conc.

(m

g/L

)

Time (hr)





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laboratory setting in future disinfection chapters where PAA and H2O2 must be quenched before

microbial enumeration in order to avoid further disinfection.

A kinetic model to assesses how the PAA profile changes in the presence of chlorine was

developed. The system was made of 4 differential equations, one for each component: PAA, H2O2,

AA, and chlorine. The kinetic model considered PAA formation, hydrolysis, and spontaneous

decomposition, and that the only component reacting with chlorine was H2O2. It was concluded

that chlorine will exert a higher demand of PAA as pH increases since chlorine reacts at a faster

rate with H2O2 with higher pH values. This in turn will shift the equilibrium reaction to form more

hydrogen peroxide and therefore consume more PAA. Experiments were conducted to test the

potential direct reaction between PAA and chlorine by evaluating the decay of each element

throughout time. It was concluded that in a PAA/H2O2/Cl2 system the chlorine decay is only

attributed to hydrogen peroxide formation as result of PAA decomposition. The effect of

decomposition on PAA alone (no chlorine present) was also determined. It was observed that the

PAA decay was pH and temperature dependent, just as suggested by the literature, with more

decay at higher pH and temperature values. It is recommended that for an aqueous system where

only PAA/H2O2/AA are present, a more appropriate formation constant k1 should be developed

since there is no available literature that provides one.

3.2 REFERENCES

Antonelli, M., Turolla, A., Mezzanotte, V., Nurizzo, C., 2013. Peracetic acid for secondary effluent

disinfection: A comprehensive performance assessment. Water Sci. Technol. 68, 2638–2644.

Corcoran, R., Whinston, J., 1991. Method of disinfection contact lenses with peracetic acid.

4986963.

Dunkin, N., Weng, S., Schwab, K.J., McQuarrie, J., Bell, K., Jacangelo, J.G., 2017. Comparative

inactivation of Murine norovirus and MS2 bacteriophage by peracetic acid and

monochloramine in municipal secondary wastewater effluent. Environ. Sci. Technol. 51,

2972–2981.

EnviroTech, n.d. Neutralization of Perasan® A and BioSide® HS 15% using sodium metabisulfite

and sodium bisulfite.

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47

EPA, 2014. Parametric testing of decontamination chemistries to guide decontaminant selection

I : Peracetic acid, Office of Research and Development’s National Homeland Security

Research Center.



Held, A.M., Halko, D.J., Hurst, J.K., 1978. Mechanisms of chlorine oxidation of hydrogen

peroxide. J. Am. Chem. Soc. 100, 5732–5740.

Hilgren, J., Lanting, J., Tippett, R.J.A., 2011. Method for processing peroxygen solutions.

20110217761.



Janković, M., Sinadinović-Fišer, S., 2005. Prediction of the chemical equilibrium constant for

peracetic acid formation by hydrogen peroxide. JAOCS, J. Am. Oil Chem. Soc. 82, 301–303.

Keen, O.S., Dotson, A.D., Linden, K.G., 2013. Evaluation of hydrogen peroxide chemical

quenching agents following an advanced oxidation process. J. Environ. Eng. (United States)

139, 137–140.


55.

Koubek, E., Haggett, M.L., Battaglia, C.J., Ibne-Rasa, K.M., Pyun, H.Y., Edwards, J.O., 1963.

Kinetics and mechanism of the spontaneous decompositions of some peroxoacids, hydrogen

peroxide and t-butyl hydroperoxide. J. Am. Chem. Soc. 85, 2263–2268.

Lazarova, V., Savoye, P., Janex, M.L., III, B., Pommepuy, E.R. and, 1999. Advanced wastewater

disinfection technologies: State of the art perspectives. Water Sci. Technol. 40, 203–213.

Manoli, K., Sarathy, S., Maffettone, R., Santoro, D., 2019. Detailed modeling and advanced

control for chemical disinfection of secondary effluent wastewater by peracetic acid. Water

Res. 153, 251–262.

Peroxychem, 2017. The Use of Peracetic Acid as a “Pre-Oxidant” for Drinking Water

Applications.

Rajala-Mustonen, R.L., Toivola, P.S., Heinonen-Tanski, H., 1997. Effects of peracetic acid and

UV irradiation on the inactivation of coliphages in wastewater. Water Sci. Technol. 35, 237–

241.

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Rangarajan, B., Havey, A., Grulke, E.A., Culnan, P.D., 1995. Kinetic parameters of a two-phase

model for in situ epoxidation of soybean oil. J. Am. Oil Chem. Soc. 72, 1161–1169.

Rossi, S., Antonelli, M., Mezzanotte, V., Nurizzo, C., 2007. Peracetic acid disinfection: A feasible

alternative to wastewater chlorination. Water Environ. Res. 79, 341–350.

Shah, A.D., Liu, Z.Q., Salhi, E., Höfer, T., Von Gunten, U., 2015. Peracetic acid oxidation of

saline waters in the absence and presence of H2O2: Secondary oxidant and disinfection

byproduct formation. Environ. Sci. Technol. 49, 1698–1705.

Wang, C., Hofmann, M., Safari, A., Viole, I., Andrews, S., Hofmann, R., 2019. Chlorine is

preferred over bisulfite for H2O2 quenching following UV-AOP drinking water treatment.

Water Res. 165, 115000.

Weng, S.C., Dunkin, N., Schwab, K.J., McQuarrie, J., Bell, K., Jacangelo, J.G., 2018. Infectivity

reduction efficacy of UV irradiation and peracetic acid-UV combined treatment on MS2

bacteriophage and murine norovirus in secondary wastewater effluent. J. Environ. Manage.

221, 1–9.

Yuan, Z., Ni, Y., Van Heiningen, A.R.P., 1997a. Kinetics of peracetic acid decomposition part II:

PH effect and alkaline hydrolysis. Can. J. Chem. Eng. 75, 42–47.

Yuan, Z., Ni, Y., Van Heiningen, A.R.P., 1997b. Kinetics of peracetic acid decompostition part I:


37–41.

Zhao, X., Cheng, K., Hao, J., Liu, D., 2008. Preparation of peracetic acid from hydrogen peroxide,

part II: Kinetics for spontaneous decomposition of peracetic acid in the liquid phase. J. Mol.

Catal. A Chem. 284, 58–68.



271, 246–252.

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4 PAA + ALUM EXPERIMENTS

ABSTRACT

Peracetic acid is known to be an effective disinfectant during wastewater treatment.

There is anecdotal evidence that PAA disinfection efficiency increases when

combined with residual alum coagulant. The potential formation of hydroxyl

radicals due to a reaction between peracetic acid and alum was evaluated. Salicylic

acid and methylene blue were used as probes to quantify the presence of these

radicals. No decay on methylene blue absorbance was observed during a 60-minute

period and salicylic acid concentrations were also constant throughout these tests.

The lack of changes in concentration in both compounds suggests that there were

no hydroxyl radicals being formed as a consequence of a reaction between PAA

and aluminum. The PAA microbial inactivation was compared to that of alum +

PAA. Using contact times of 10 and 20 minutes, and PAA doses of 1 and 5 mg/L,

the E. coli log reduction was determined. Alum was used in small doses (0.1 mg/L

aluminum), representative of residual concentrations in wastewater effluent. It was

found that there was no significant increase in log reduction when alum was present

during PAA disinfection of E. coli.

4.1 INTRODUCTION

Peracetic acid is a promising alternative for wastewater disinfection due to its strong oxidation

properties, its ease of implementation, and its lack of persistent residuals or disinfection by-

products (Kitis, 2004; Koivunen and Heinonen-Tanski, 2005). In wastewater treatment,

physiochemical processes like ferric chloride (FeCl3) and alum coagulation can be used to assist

the removal of suspended solids and phosphorous (Gehr et al., 2003). There is some anecdotal

evidence from pilot-and full-scale that when combined with residual alum coagulant from

upstream, PAA efficacy increases. In the literature there is some evidence of enhanced disinfection

when using alum-based versus iron-based coagulants. Gehr et al. (2003) evaluated disinfection

alternatives to chlorine such as ozone at the City of Montreal Wastewater treatment plant. The

authors found that when using ferric chloride as the upstream coagulant the ozone dose required

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to meet effluent criteria of 2 log reduction of fecal coliforms was 17 mg/l, whereas using alum

required only 10 mg/L of ozone. Other authors have investigated the same phenomenon and found

that replacing FeCl3 for alum coagulation reduces the required dose of ozone by 35% to reach the

same 5,000 CFU/100 mL target level (Gher and Nicell, 1996). The oxidation mechanism of ozone

works through the formation of free radicals, and the authors suggested that the increase in

disinfection efficiency observed with alum over ferric chloride can be attributed to iron acting as

a scavenger to the free radicals formed through ozonation.

A different study focused on microbial inactivation as a result of coagulation with aluminum- or

ferric-chloride combined with PAA disinfection. It was observed that using ferric chloride (FeCl3,

PIX) coagulation followed by PAA disinfection (PIX-PAA) had a greater reduction in E. coli

(P=0.34) and enterococci (P=0.05) compared to aluminum chloride (AlCl3, PAX) coagulation

followed by PAA (PAX-PAA) as seen in Figure 4-1 (Pradhan et al., 2013).

Figure 4-1. Microbial reduction in wastewater effluent flocculated by PIX and PAX, and

subsequent 3 mg/L PAA disinfection. The letters a and b above the same microbe indicate a

statistical significantly different result (Pradhan et al., 2013)

Transition metals, especially reduced metal ions such as iron, manganese, cobalt, and copper are

responsible for PAA catalyzed decomposition, where PAA decomposes into acetic acid, oxygen,

and other products (Equation 1) (Yuan et al., 1997a; Zhao et al., 2007). This, along with all other

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decomposition reactions, reduce the oxidation power of PAA because its products are not as strong

oxidants as peracetic acid. Chelating agents such as diethylenetriaminepentaacetic acid (DTPA)

and diethylenetriaminepentamethylenephosphonic acid (DTMPA) can be used to avoid the

transition metal catalyzed decomposition.

CH3COOOH+ M+ → CH3COOH+ O2 + other decomposition products (1)

However, transition metals can also enhance the PAA disinfection efficiency. Similar to ozone,

the mode of action of PAA can be through the generation of radical species. The homolytic PAA

reaction consists on the rupture of its peroxy bond into acyloxy and hydroxyl radicals, referred to

as the initiation step (Equation 2) (Flores et al., 2014; Lubello et al., 2002). There are subsequent

reactive oxygen species (ROS) formed after the initiation reaction (Equation 3-8) that could

contribute to oxidation of target pollutants and disinfection of microorganisms, but it is mainly the

hydroxyl (OH•), acetyl (CH3COO•), and the methyl radical (CH3•) that are responsible for the

oxidation (Bianchini et al., 2002; Flores et al., 2014; Zhou et al., 2015). The activation of PAA to

form radicals can be catalyzed in the presence of a transition metal in an analogous manner to the

Fenton and Fenton-like reactions (Rokhina et al., 2010). Cavallini et al. (2015) found that just as

H2O2, organic peroxides such as PAA could also be decomposed by iron to form CH3COO• and

CH3COOO•, but that it does not form OH•. Luna-Pabello et al. (2009) reported a reduction in

required contact time for Ag+/PAA and Ag+/Cu2+/PAA systems during the removal of helminth

eggs (HE) and fecal coliforms in raw wastewater. The authors attribute the effectiveness of the

Ag+/Cu2+/PAA system to the oxidation of Ag and Cu in the presence of PAA to highly reactive

compounds like Ag2O, Ag2O2, and Cu2O.

CH3COOOH → CH3COO ∙ + HO ∙ (2)

CH3COOOH+HO ∙ → CH3CO ∙ + O2 + H2O (3)

CH3COOOH+ HO ∙ → CH3COOO ∙ +H2O (4)

CH3COO ∙ → ∙ CH3 + CO2 (5)

2CH3COO ∙ ↔ 2 ∙ CH3 + 2CO2 + O2 (6)

∙ CH3 + O2 → CH3COO ∙ (7)

CH3COO ∙ + HO ∙ → CH3COOOH (8)

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Researchers have further investigated the reaction between PAA and other transition metals. Popov

et al. (2005) assessed the decomposition of PAA by Mn2+ and determined that it takes place

through multiple redox reactions involving different manganese oxidation states. The authors also

determined that a radical pathway decomposition induced by Mn was not likely in the presence of

a chelating agent like DTPA. Other studies have explored the decomposition of PAA catalyzed by

Co2+, suggesting that although it does not produce OH• radical, it does generate CH3COO• and

CH3COOO• radicals (Zhang et al., 1998).

It is not clear whether alum reacts with PAA to form any ROS or other type of highly reactive

compounds, as do the transition metals. Non-ferrous Fenton catalysts involving elements such as

aluminum to activate H2O2 have been investigated. However, the only available state of aluminum

in aqueous solution is Al3+, where an electron transfer between Al3+ and H2O2 is not feasible

(Bokare and Choi, 2014). Thus, a similar phenomenon is unlikely to occur in a PAA and Al3+

system.

4.2 OBJECTIVE

The objective of this chapter is to assess whether the efficiency of PAA disinfection of E. coli is

affected by the presence of alum in a controlled environment. This involves assessing whether

PAA and alum react by determining if radicals are being formed as products, and by evaluating

PAA consumption in the system as a consequence of reaction with alum.

4.3 MATERIALS AND METHOD

4.3.1 Reagents and Equipment

Radical analysis was performed using two different probes: methylene blue (MB) and salicylic

acid (SA, C7H6O3), both from Sigma-Aldrich. Both methods employed an Agilent 8453 UV-Vis

spectrophotometer (Agilent Technologies Canada Inc.) with a 1 cm quartz crystal cuvette to

measure absorbance. Peracetic acid was synthesized in situ using tetraacetylethylenediamine

(TAED) (Warwick Chemicals, United Kingdom), sodium percarbonate (OCI Peroxygens, LLC,

Alabama), (EDTA) (BioShop, Canada), ethylenediaminetetraacetic acid (EDTA) (BioShop,

Canada), and citric acid (Sigma-Aldrich, Canada). The reactants were added to 1 L of Milli-Q

water and mixed for 30 minutes. Both peracetic acid and hydrogen peroxide were quantified using

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a HACH DR2700 spectrophotometer (DPD method 10290). All experiments were performed

using a 0.1 M buffer solution made of dipotassium and monopotassium phosphate (K2HPO4 &

KH2PO4) (Sigma-Aldrich) in Milli-Q water to avoid alum affecting the pH. Aluminum sulfate

hydrate (alum, Al2(SO4)3•xH2O) from Sigma-Aldrich was used for the experiments.

4.1.1 Experimental protocols

4.1.1.1 Radical formation analysis and PAA consumption

Radicals can contribute to the degradation of methylene blue (Shimizu et al., 2007). To quantify

the formation of hydroxyl radicals in a PAA/alum system, the decay in MB absorbance is used as

an indicator. In 500 mL of a 0.1 M buffer solution, 1mL of methylene blue was added and

completely mixed with a stirring rod. The solution was completely wrapped in aluminum foil to

minimize light exposure since MB might decompose slowly in light. PAA stock solution was

added to the solution to a final concentration of 10 mg/L. At time zero, 45 mg/L of alum (3.64

mg/L Al3+) were introduced into the mixture and stirred for 60 minutes. The MB absorbance was

taken every 10 minutes using a UV-Vis spectrophotometer at a 664 nm wavelength.

Salicylic acid as a chemical probe for hydroxyl radical followed the analytical method developed

by Peralta et al. (2014). The hydroxyl radical reacts with SA to form 2,3- and 2,5- dihidroxybenzoic

acid (DHBA) (Equation 9). A mixture containing 45 mg/L alum (3.64 mg/L Al3+) and salicylic

acid (0.000141 M) in a 0.1 M buffer solution was prepared. The diluted PAA was added (time 0)

to the mixture and stirring took place for 60 minutes. Salicylic acid absorbance was quantified

every 10 minutes using the UV-Vis spectrophotometer and converted into molar concentrations

using Equation 10. Assuming the concentration of SA is in large excess compared to that of the

hydroxyl radical, a pseudo-first order reaction can be assumed and the OH• concentration can be

calculated using Equation 11. The k value used in Equation 11 is the rate constant for the reaction

between SA and the hydroxyl radical reported in the literature, 5x109 M-1s-1 (Villamena, 2017).

SA + OH · → 2,3 − DHBA + 2,5 − DHBA (9)

[𝑆𝐴] = (8.69x10−4)A297nm − (7.87x10−4)A307nm + (1.80x10

−4)A321nm (10)

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[OH ·] = −ln (𝑆𝐴𝑡𝑆𝐴0

)

t ∗ k

(11)

To determine if there is a chemical reaction between Al3+ and PAA, the peracetic acid decay profile

in the presence of alum was developed. PAA stock was diluted into a 0.1 M buffer solution to a

final concentration of 10 mg/L. At time 0, 855.37 mg/L of alum (69.28 mg/L Al3+) were added to

the mixture and stirred for 60 minutes. PAA was quantified every 10 minutes using the HACH

DR2700 spectrophotometer (DPD method).

4.1.1.2 Experimental QA/QC

All experiments to quantify radical formation as well as PAA decay were replicated twice. The

vertical bars plotted in the results section show the standard deviation of the average values. During

the methylene blue experiments, a control (only MB) was used for quality control. The

experiments using salicylic acid used controls made of SA+alum and SA+PAA. Furthermore, the

MDL for hydroxyl radical quantification through SA analysis was calculated to be 1.39 E-12 M.

The literature reports a low concentration of OH• to be around 1.0E-12 M (Li et al., 2017). Thus,

the SA method can accurately determine the presence of even low hydroxyl radical concentrations.

The PAA decay experiments also used a control (PAA only) to test the decay of PAA in the

presence of alum. All experiments performed with the UV-vis spectrophotometer used Milli-Q

water as a blank.

4.1.1.3 Microbial analysis

The microbial enumeration method took place based on a previously developed standard operating

procedure (SOP), see Appendix A.2.3. The procedure used E. coli (ATCC ® 23631™) stock stored

at -80oC, the evening prior to the test a portion of the stock was reanimated by touching the frozen

stock with a pipette tip and depositing it in 120 mL of LB broth contained in a 500 mL Erlenmeyer

flask. The solution was then incubated overnight at 37oC while shaking at 1000 rpm.

The morning of the test the optical density (OD600) of the solution was measured using a UV-vis

spectrophotometer and diluted to an OD600 of ~0.5 in LB broth. 30 mL of this solution were

transferred into a 50 mL centrifuge tube and spined for 15 minutes at 4000 rpm. The supernatant

was discharged and the pallet rinse using 30 mL of ¼ Ringer’s solution. This process was repeated

2 more times in order to avoid any LB broth residual in the E. coli pallet. Then, 30 mL of Ringer’s

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solution was added and vortexed to ensure even distribution of bacteria. The OD600 of the solution

was measured and used as the stock solution concentration.

Five different reactors consisting of 100 mL Erlenmeyer flasks were used during each experiment.

All reactors contained enough E. coli stock in 50 mL of Ringer’s solution for a final OD600 of 0.05.

Reactor 1 contained E. coli only as a control. The second reactor consisted of E. coli and alum (0.1

mg/L aluminum) as a control to determine if alum alone had any effect on microbial inactivation.

The third reactor was made of E. coli, sodium thiosulfate (600 mg/L), and catalase (2.5 mg/L). The

purpose of it was to assess if the quenching agents used during the experiment would have an

effect in the microbial community. The fourth and fifth reactors were the actual treatments used to

assess E. coli reduction. The first treatment was PAA (1 mg/L for the first experiment and 5 mg/L

for the second), and the second treatment was PAA in the previously mentioned doses and alum

(0.1 mg/L aluminum).

After a retention time of 10 and 20 minutes, 1 mL from each reactor containing a control was

removed and added directly into a polypropylene tube containing 9 mL of 0.9% NaCl solution.

For the treatments, 20 mL were first removed and quenched using 600 mg/L of sodium thiosulfate

to neutralize PAA, and 2.5 mg/L to neutralize the H2O2 in the PAA mixture. Then, 1 mL of each

solution was removed and added into the polypropylene tubes. Eight serial dilutions were prepared

for each solution in polypropylene tubes, being vortexed to assure proper distribution of E. coli.

All dilutions were run in duplicate for quality control purposes. 100 µL of each dilution were

pipetted in the middle of a pre-poured agar plate and spread using a sterile spreader until the

dilution was completely absorbed into the agar. The plates were incubated overnight upside down

in a 37oC incubator. The next day the number of E. coli colonies formed in the agar was counted

and recorded. The observed colony-forming units (CFU) was calculated using Equation 12. Then

the log reduction of E. coli was calculated using Equation 13, based on the treatment and the E.

coli only control.

CFU

mL=

colonies counted

(mL of sample plated) x (dilution factor) (12)

log reduction = C0Cf (13)

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where,

C0= The concentration of E. coli in the control at time t (CFU/mL)

Cf= The concentration of E. coli in the treatment at time t (CFU/mL)

4.2 RESULTS AND DISCUSSION

4.2.1 Radical quantification and PAA decay

Based on the results seen in Figure 4-2, methylene blue absorbance did not significantly decay in

any of the four systems analyzed (MB, MB+PAA, MB+alum, and MB+PAA+alum). During the

60-minute experiment, the absorbance in the MB/PAA/alum system went from 0.20 to 0.18.

However, all scenarios had a similar decrease by the end of the reaction time. This suggests that

no hydroxyl radicals are being formed as a result of a PAA/alum reaction. A two-tail t-test was

conducted to determine if the slope in the MB absorbance for a system containing PAA and alum

is statistically different than 0. It was concluded that the decay in MB is not statistically significant

(p value > 0.05).

The salicylic acid molar concentration in the SA+PAA+alum system seemed to increase in the

first 10 minutes of the test and then remained constant for the rest of the experiment (Figure 4-3).

The initial increase in SA concentration was also observed in a control test using SA+alum but not

in a SA+PAA system, attributing this increase to the presence of alum in the system. When testing

the presence of a slope on SA concentration through time using a two-tail t-test, it was found that

no statistically significant decay took place (p value > 0.05). The steady concentration of SA

throughout time shows the lack of OH• being formed, suggesting once again that PAA does not

react with Al3+ to form hydroxyl radicals. However, aluminum and PAA might be reacting to form

free radicals other than OH•.

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Figure 4-2. Methylene blue as a chemical probe for the detection of radicals

Figure 4-3. Salicylic acid decay due to reaction with hydroxyl radical

When measuring the PAA and H2O2 concentrations in the presence of alum, no statistically

significant change was observed over 60 minutes (p value > 0.05) (Figure 4-4). These results agree

with the previous evidence of a lack of radical formation as indicated by the MB and SA probes.

It can be concluded that neither peracetic acid, nor hydrogen peroxide, are being consumed by a

reaction with Al3+, and no hydroxyl radicals that would react with MB or SA are being formed.

0.00

0.05

0.10

0.15

0.20

0.25

0 10 20 30 40 50 60

MB

abso

rban

ce

Time (min)

MB

MB + PAA

MB + alum

MB + PAA + alum

0

0.00002

0.00004

0.00006

0.00008

0.0001

0.00012

0.00014

0.00016

0.00018

0 10 20 30 40 50 60

[SA

]

Time (min)

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Figure 4-4. PAA decay in the presence of alum

4.1.1 Microbial analysis

E. coli inactivation was assessed in order to determine if residual alum enhances the disinfection

efficiency of PAA. Figure 4-5 shows how the log reduction of E. coli increased with contact time.

PAA (1 mg/L) + alum (0.1 mg/L aluminum) had a 0.44 and 4.81 log reduction at 10- and 20-

minutes contact time, respectively. When the PAA dose increased from 1 to 5 mg/L while still

using 0.1 mg/L aluminum, the log reduction increased from 0.44 to 3.2. When 5 mg/L PAA was

used for more than 10 minutes, no colony-forming units were observed after the treatment. It can

be seen that the microbial reduction using PAA + alum was slightly higher than the one observed

using PAA alone. However, by performing a two-tail t-test it was found that the increase in

inactivation was not statically significant (p > 0.05). This suggests that small doses of alum do not

significantly increase PAA disinfection efficiency during 10 and 20 minutes contact time. These

results agree with the lack of radical formation previously observed.

0

2

4

6

8

10

0 10 20 30 40 50 60

Conc.

(m

g/L

)

Time (min)

Control PAA + alum

PAA

PAA

PAA

PAA

PAA

PAA

PAA

PAA

PAA

PAA

PAA

PAA

PAA

PAA

PAA

PAA

H2O2

H2O2

H2O2

H2O2

H2O2

H2O2

H2O2

H2O2

H2O2

H2O2

H2O2

H2O2

H2O2

H2O2

H2O2

H2O2

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Figure 4-5. Log reduction of E. coli

4.2 SUMMARY AND CONCLUSIONS

Experiments were performed in order to determine if alum reacts with PAA to enhance the

disinfection process during wastewater treatment. Anecdotal evidence suggests that when plant

operators use alum as a coagulant for enhanced phosphorous removal, they have reported enhanced

disinfection performance, meaning that a lower dose of PAA was required in order to meet

bacterial compliance. One of the theories that would justify an increase in disinfection performance

was the potential formation of free radicals which could take place in an analogous manner to

Fenton and Fenton-like reactions. In this chapter it was determined that no hydroxyl radicals were

formed in a PAA/aluminum system, as demonstrated using two chemical probes, salicylic acid and

methylene blue. The potential reaction between Al3+ and PAA in a different manner to OH•

formation was also tested. It was concluded that PAA and H2O2 do not significantly decay in the

presence of alum, which once again suggest that no reaction is taking place, and if it is, it takes

place at a very slow rate. Finally, the microbial inactivation of PAA + alum was determined using

nonpathogenic E. coli. It was found that the presence of alum does not significantly increase the

disinfection performance of PAA at two different doses (1 and 5 mg/L PAA) and contact times

(10 and 20 minutes). Therefore, it can be concluded that introducing alum to the PAA disinfection

process using a clean water matrix does not have a significant impact on E. coli reduction. The

0

1

2

3

4

5

6

0 2 4 6 8 10 12 14 16 18 20

Log-r

educt

ion

Time (min)

PAA (1 mg/L)

alum + PAA (1 mg/L)

PAA (5 mg/L)

alum + PAA (5 mg/L)

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60

water matrix might play an important role in this process and experiments using wastewater

samples should be conducted in the future.

4.3 REFERENCES

Bianchini, R., Calucci, L., Caretti, C., Lubello, C., Pinzino, C., Piscicelli, M., 2002. An EPR study

on wastewater disinfection by peracetic acid, hydrogen peroxide and UV irradiation. Ann.

Chim. 92, 783—793.

Bokare, A.D., Choi, W., 2014. Review of iron-free Fenton-like systems for activating H2O2 in

advanced oxidation processes. J. Hazard. Mater. 275, 121–135.

Cavallini, G.S., Vidal, C.M., Souza, J.B., Campos, S.X., 2015. Fenton coagulation/oxidation using

Fe2+ and Fe3+ íons and peracetic acid for the treatment of wastewater. Orbital - Electron. J.

Chem. 7.




Gehr, R., Wagner, M., Veerasubramanian, P., Payment, P., 2003. Disinfection efficiency of

peracetic acid, UV and ozone after enhanced primary treatment of municipal wastewater.

Water Res. 37, 4573–4586.

Gher, R., Nicell, J., 1996. Pilot studies and assessment of downstream effects of UV and ozone

disinfection of a physicochemical wastewater. Water Qual. Res. J. Canada 31, 263–281.


55.

Koivunen, J., Heinonen-Tanski, H., 2005. Peracetic acid (PAA) disinfection of primary, secondary

and tertiary treated municipal wastewaters. Water Res. 39, 4445–4453.

Li, W., Jain, T., Ishida, K., Liu, H., 2017. A mechanistic understanding of the degradation of trace

organic contaminants by UV/hydrogen peroxide, UV/persulfate and UV/free chlorine for

water reuse. Environ. Sci. Water Res. Technol. 3, 128–138.

https://doi.org/10.1039/c6ew00242k

Lubello, C., Caretti, C., Gori, R., 2002. Comparison between PAA / UV and H2O2 / UV

disinfection for wastewater reuse. Water Sci. Technol. Water Supply 205–212.

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61

Luna-Pabello, V.M., Ríos, M.M., Jiménez, B., Orta De Velasquez, M.T., 2009. Effectiveness of

the use of Ag, Cu and PAA to disinfect municipal wastewater. Environ. Technol. 30, 129–

139.

Peralta, E., Roa, G., Hernandez-Servin, J.A., Romero, R., Balderas, P., Natividad, R., 2014.

Hydroxyl radicals quantification by UV spectrophotometry. Electrochim. Acta 129, 137–141.

Popov, E., Eloranta, J., Hietapelto, V., Vuorenpalo, V.M., Aksela, R., Jäkärä, J., 2005. Mechanism

of decomposition of peracetic acid by manganese ions and diethylenetriaminepentaacetic acid

(DTPA). Holzforschung 59, 507–513.

Pradhan, S.K., Kauppinen, A., Martikainen, K., Pitkan̈en, T., Kusnetsov, J., Miettinen, I.T., Pessi,

M., Poutiainen, H., Heinonen-Tanski, H., 2013. Microbial reduction in wastewater treatment

using Fe3+and Al3+ coagulants and PAA disinfectant. J. Water Health 11, 581–589.




44, 6815–6821.

Shimizu, N., Ogino, C., Dadjour, M.F., Murata, T., 2007. Sonocatalytic degradation of methylene

blue with TiO2 pellets in water. Ultrason. Sonochem. 14, 184–190.

Villamena, F.A., 2017. Chapter 6 - UV–Vis absorption and chemiluminescence techniques, in:

Villamena, F.A.B.T.-R.S.D. in B. (Ed.), . Elsevier, Boston, pp. 203–251.

Yuan, Z., Ni, Y., Van Heiningen, A.R.P., 1997. Kinetics of peracetic acid decompostition part I:


37–41.

Zhang, X.Z., Francis, R.C., Dutton, D.B., Hill, R.T., 1998. Decomposition of peracetic acid

catalyzed by cobalt(III) and vanadium(V). Can. J. Chem. 76, 1064–1069.



271, 246–252.

Zhou, F., Lu, C., Yao, Y., Sun, L., Gong, F., Li, D., Pei, K., Lu, W., Chen, W., 2015. Activated

carbon fibers as an effective metal-free catalyst for peracetic acid activation: Implications for

the removal of organic pollutants. Chem. Eng. J. 281, 953–960.

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5 SUMMARY, CONCLUSIONS AND RECOMMENDATIONS

The potential synergy between alum residual and peracetic acid during disinfection was

determined. PAA can react with some metals to form reactive oxygen species that would enhance

the disinfection process. Thus, the formation of hydroxyl radicals was assessed using two different

chemical probes, salicylic acid (SA) and methylene blue (MB). The probes would react with the

OH• formed resulting in a decay in SA and MB concentration. Each experiment was run during a

60-minute period, with the PAA/SA molar ratio at about 1:1. The results showed a lack in SA and

MB decay throughout time, suggesting that no hydroxyl radicals were being formed. However,

aluminum and PAA could be forming ROS other than the hydroxyl radical. The concentration

profiles of PAA and H2O2 in the presence of alum were also evaluated, in order to understand if

the PAA solution reacts in any other form with aluminum. It was found that peracetic acid and

hydrogen peroxide were not significantly consumed in the presence of alum, which indicates that

no reaction was taking place during the 60-minute test. The effect of alum and peracetic acid on

E. coli inactivation was also tested. The experiments took place using two different PAA doses of

1 and 5 mg/L, and two different contact times, 10 and 20 minutes. The alum dose (0.1 mg/L

aluminum) was constant for all tests. There was no statistically significant change in microbial

inactivation in the presence of alum, suggesting that alum residual does not enhance the PAA

disinfection process. These tests were conducted using a clean water matrix. Future experiments

could be run using wastewater samples that would better mimic the water matrix were the

anecdotal synergy between PAA and alum was observed.

The effect of peracetic acid as a pretreatment on downstream chlorine disinfection during drinking

water treatment was assessed. A kinetic model was developed in order to predict how PAA as a

solution in equilibrium with H2O2 and acetic acid can exert a chlorine demand and vice versa. The

model assumed that hydrogen peroxide was the only component reacting with Cl2, and that no

organic matter or transition metals were present in the system. It also took into consideration PAA

formation, hydrolysis, and spontaneous decomposition which are pH dependent, so four different

pH values were simulated (6, 7.5, 8.3, and 10). It was found that as pH increased, chlorine exerted

a higher demand on PAA during a 24-hour period. This is attributed to the fact that hydrolysis of

PAA into H2O2 is favored at higher pH values. Thus, as pH increases more hydrogen peroxide is

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being formed and therefore more chlorine is being consumed. The kinetic model used reaction

constants previously reported in the literature. However, a PAA formation constant (k1) has not

being previously developed and had to be estimated using the equilibrium and decomposition k

values. This suggests that a more accurate value for k1 should be found. The kinetic model results

were supported with experimental data. These tests assessed the concentration profiles of each

component in a PAA/H2O2/Cl2 system during a 20-hour period at four different pH values (6, 7,

8, and 9). It was found that chlorine consumption is mainly attributed to the reaction with H2O2

and that after the initial Cl2 demand exerted by H2O2 present in the PAA solution, the continuous

decay in chlorine concentration was due to the formation of hydrogen peroxide through PAA

hydrolysis as seen in the kinetic model results. Nevertheless, the experimental results showed a

higher decrease in chlorine than the kinetic model predicted, suggesting that the model might

underestimate the rate of PAA hydrolysis (k1). The analytical method used during these tests to

quantify PAA and H2O2 was not completely accurate, as currently there is no quantification

method to accurately determine PAA in the presence of chlorine, since Cl2 interferes with the DPD

method used for PAA/H2O2. Thus, a more accurate method for the simultaneous analysis of all

three components could be further developed as part of future work.

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A. APPENDIX 1 EXPERIMENTAL DATA

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A.1 EXPERIMENTAL DATA FOR CHAPTER 3

Table A-1. Average PAA, H2O2, Cl2 concentration at pH 6

Time (hr) Average Cl2

(mg/L) SD Time (hr)

Average

PAA (mg/L) SD

0 147.5 0 0 99.8 0

0.05 130 0 0.11 93.5 6.36

0.53 127.5 0.70 0.58 92 2.82

1.5 123.5 0.70 1.53 89.5 2.12

2.5 120 2.82 2.53 84.5 2.12

3.5 117.5 2.12 3.53 82 4.24

4.5 116 4.24 4.53 78 4.24

17.5 102 1.41 17.53 70.5 3.53

19.5 101.5 2.12 19.53 65 3.53

22 100 2.82 22.03 69 7.07

23.2 98 2.82 23.31 62.5 3.53

Table A-2. Average PAA/H2O2/Cl2 concentration at pH 7


(mg/L) SD Time (hr)

Average

PAA (mg/L) SD

0 137.8 0 0 99.81 0

0.05 107 - 0.1 84 -

0.68 91 2.82 0.73 77 5.65

1.73 81 0 1.78 67.5 6.36

2.73 78 5.65 2.78 57.5 2.12

3.43 74.5 4.94 3.53 54.5 3.53

4.4 73.5 4.94 4.4 47.5 2.12

5.06 71 2.82 5.16 45.5 3.53

17.6 52.5 3.53 17.8 20.5 4.94

19 48.5 2.12 19.1 19.5 3.53

21.6 48 1.41 21.6 18 4.24

22.9 46.5 3.53 22.9 18 5.65

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24.61 44 1.41 24.66 16.5 3.53



(mg/L) SD Time (hr)

Average

PAA (mg/L) SD

0 137.8 0 0 99.81 0

0.05 107 - 0.1 84 -

0.68 91 2.82 0.73 77 5.65

1.73 81 0 1.78 67.5 6.36

2.73 78 5.65 2.78 57.5 2.12

3.48 74.5 4.94 3.53 54.5 3.53

4.4 73.5 4.94 4.45 47.5 2.12

5.06 71 2.82 5.11 45.5 3.53

17.68 52.5 3.53 17.83 20.5 4.94

19.05 48.5 2.12 19.1 19.5 3.53

21.6 48 1.41 21.65 18 4.24

22.9 46.5 3.53 22.95 18 5.65

24.61 44 1.41 24.66 16.5 3.53



(mg/L) SD Time (hr)

Average

PAA (mg/L) SD

0 135 0 0 99.81 0

0.05 102.5 2.12 0.08 91 4.24

0.88 87.5 3.53 0.91 66 2.82

1.71 79.5 0.70 1.78 61.5 13.43

2.55 76.5 3.53 2.58 54.5 0.70

4.36 67 5.65 4.4 46 2.82

5.58 66 4.24 5.65 39.5 3.53

17.28 44 2.82 17.33 15.5 2.12

19.4 47 - 19.46 17 2.82

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20.51 43 2.82 20.56 12.5 2.12

21.23 39.5 0.70 21.26 12.5 0.70

22.28 38 1.41 22.31 9.5 2.12

A.1.1 ANALYTICAL METHODS

Standard Operating Procedure (SOP)

Quantification of Peracetic Acid in water (PAA)

Prepared: July 2019, by Alonso Hurtado and Domenica Cevallos

Principle

The quantification of PAA is performed according to the method 10290 of the Hach Company,

which outlines the application of N-N-diethyl-p-phenylendiamine (DPD) for the measurement of

PAA. The spectrophotometer used for this analysis is the DR2700- program 790. The measurement

range is 0.10 mg/L - 10 mg/L PAA, and the measurement wavelength is 530 nm. Total DPD reacts

with PAA to form a pink color, hydrogen peroxide does not interfere with the PAA reaction since

the peroxide reaction requires the addition of catalyst and longer reaction time.

Samples must be analyzed immediately at room temperature after collecting them and bright light

conditions must be avoided by using the protective cover during the measurements. They cannot

be preserved for later analysis. The only reagent required is the DPD Total Chlorine Reagent

Powder Pillows, 25 ml.

Safety notes

Nitrile gloves, safety glasses, and a lab coat must be worn at all times.

Biosafety Certificate is not required

Operational Concerns

Samples must be collected or prepared in containers that do not have residual chlorine or other

oxidants on them. Tap water must be avoided to clean any laboratory glassware or the square cell

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used for this method. Additionally, optical interference stemming from sample background color

and turbidity will be problematic.

Acidity and alkalinity of the samples cannot be more than 150 mg/L and 250 mg/L CaCO3,

respectively. In case the values are higher, adjust the pH to 6-7 with 1 N sodium hydroxide for

acidity and with 1 N sulfuric acid for alkalinity. The test result must be corrected for the dilution

form the volume addition.

Equipment and Materials

· Square glass 10 ml matched sample cell. Do not use plastic cells!

· DR 2700 Spectrophotometer

· Chronometer

· DPD Total Chlorine Reagent Powder Pillow, 25

· Sealing Film, Parafilm M

· Waste Beaker

· Kimwipes

· Pasteur Pipettes

Method

Blank preparation

a) Start program 790 PAA. It can be found in the favourite programs list.

b) Rinse the square cell with the samples at least two or three times.

c) Fill the cell to the 10 mL mark with the sample. If required used a Pasteur pipette to adjust

to the 10 mL mark.

d) Clean the cell with the wipes and insert the blank into the cell holder

e) Push zero to measure the blank. The display shows 0.0 mg/L PAA

Sample preparation and quantification

a) Rinse the cell with the solution at least two or three times

b) Fill the second sample cell to the 10 ml mark with samples

c) Peel the sealing film and be ready to cover the cell quickly with it. Ideally a glass stopper

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will be used to seal the cell

d) Add the content of one DP Total Chlorine Powder pillow 25 ml to the sample. The sample

will turn pink, the intensity of the color depends on the amount of PAA

e) Start the instrument timer immediately after adding the total content of the pillow

f) Cover the cell with the sealing film or the stopper

g) Shake the sample cell until the chronometer displays 40 s to dissolve the reagent. Check

for no solid particles or bubbles in the cell.

h) Clean the sample cell with the wipes, and insert it into the cell holder

i) At 55 s push Read. The results are shown in mg/L PAA

Waste Disposal and clean up

The blank and sample with the DPD can be flushed down the sink. After the analysis clean up

the square cell with deionized or Milli Q water.

A.2 EXPERIMENTAL DATA FOR CHAPTER 4

A.2.1 RADICAL FORMATION ANALYSIS

Table A-5. Average MB absorbance in the presence of PAA/alum MB

PAA

PAA + Alum

Alum

Time

(min) Abs/cm SD Abs/cm SD Abs/cm SD Abs/cm SD

0 0.20 0 0.17 0 0.20 0 0.22 0

10 0.22 0.001 0.17 0.006 0.19 0 0.21 0

20 0.19 0.004 0.16 0.009 0.17 0.001 0.21 0.009

30 0.19 0.0009 0.17 0.012 0.18 0.007 0.20 0.01

40 0.21 0.0068 0.15 0.003 0.15 0.001 0.19 0.01

50 0.19 0.0044 0.16 0.015 0.16 2.83E-05 0.20 0.008

60 0.18 0.008 0.15 0.008 0.15 0.01 0.20 0.006

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Table A-6. Average PAA and H2O2 concentration (mg/L) in the presence of alum

Control (PAA and H2O2 only) Trial 1 & 2 (alum present)

PAA HP PAA HP

Time Average SD Average SD Average SD Average SD

0 8.9 0.02 1.47 0.34 8.77 0.24 1.44 0.23

10 8.85 0.06 1.56 0.17 8.62 0.10 1.71 0.12

20 8.94 0.23 1.62 0.15 8.51 0.13 1.64 0.04

30 8.96 0.19 1.61 0.14 8.69 0.23 1.72 0.007

40 8.57 0.18 1.47 0.11 8.37 0.21 1.74 0.06

50 8.82 0.19 1.54 0.21 8.6 0.18 1.77 0.28

60 8.82 0.14 1.69 0.05 8.47 0.06 1.79 0.04

A.2.2 SALICYLIC ACID METHOD FOR RADICAL DETECTION

The concentration of hydroxyl radicals follows Equation 1. It was previously observed that salicylic

acid (SA), 2,3-DHBA, and 2,5-DHBA have their maximum absorbance at 297 nm, 307 nm, and 321

nm respectively.

SA + ∙𝑂𝐻 → 2,3−𝐷𝐻𝐵𝐴+ 2,5−𝐷𝐻𝐵𝐴 (1)

When taking into consideration that the peaks of each analyte overlap, concentrations can be

determine using Beer Lambert Law (Equation 2) and linear algebra. Thus, molar absorptivity is

calculated using Equation 2, where A is absorbance, 𝜀 is the molar absorptivity (M-1 cm-1), c is molar

concentration (M), and l is the optical path length (1 cm).

𝐴=𝜀𝑐l (2)

Table A-7. Calculated molar absorptivity for SA method

Analyte λ (nm) Absorbance Conc. (M) 𝜀 (M-1 cm-1)

SA 297 0.31 1E-4 3114

2,3-DHBA 297 0.25 1E-4 2521

2,5-DHBA 297 0.12 8E-5 1528

SA 307 0.22 1E-4 2282

2,3-DHBA 307 0.31 1E-4 3100

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2,5-DHBA 307 0.20 8E-5 2522

SA 321 0.12 1E-4 1220

2,3-DHBA 321 0.21 1E-4 2181

2,5-DHBA 321 0.29 8E-5 3630

Therefore,

(𝐴1𝐴2𝐴3

) = 𝑏 (ε𝑋1 ε𝑌1 ε𝑍1ε𝑋2 ε𝑌2 ε𝑍2ε𝑋3 ε𝑌3 ε𝑍3

)(

[𝑋][𝑌][𝑍])

Where 1, 2, 3 correspond to each wavelength, and can be solved as:

(

[𝑆𝐴][2,3 − 𝐷𝐻𝐵𝐴][2,5 − 𝐷𝐻𝐵𝐴]

) = 1−1 (3114 2282 12202521 3100 21811528 2522 3630

)

−1

(𝐴297𝑛𝑚𝐴307𝑛𝑚𝐴321𝑛𝑚

)

Results:

[SA]= (8.69E-4) A297 nm - (7.87E-4) A307 nm + (1.80E-4) A321 nm

[2,3-DHBA]= - (8.79E-4) A297 nm + (1.42E-3) A307 nm - (5.61E-4) A321 nm

[2,5-DHBA]= (2.45E-4) A297 nm - (6.60E-4) A307 nm + (5.89E-4) A321 nm

Method detection limit (MDL):

Prepared 8 salicylic acid (SA) standard solutions at 10X the lowest molar concentration in the SA

calibration curve range and measured the absorbance at 297nm. Using the calibration curve

previously developed, the molar concentration for each absorbance was calculated based on the

absorbance per cm.

MDL=[t(n-1, =0.01)]x[S]

where,

S= standard deviation of the replicate analyses

t(n-1, =0.01)= value appropriate for a 99% confidence level (CIV 1319 table)

Calibration Curve:

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Linear Range [SA] Regression Equation R2

3.0E-6 – 1.0E-4 Abs297nm=3456.4[SA]-0.0056 0.99

MDL:

n-1=7

t(n-1, =0.01)]=2.998

S=7.27E-7 (Calculated with Excel)

𝑀𝐷𝐿 = 2.998𝑥7.27𝐸 − 7 = 2.18𝐸 − 6 𝑀

Hydroxyl radical analysis:

SA_initial:1.41E-4 M

SA_t: 2.18E-6 M (This is the previously calculated MDL)

k: 5.0E9 M-1s-1

t:10 min

[𝑂𝐻𝑠𝑠] =ln (2.18𝐸 − 6 𝑀1.41𝐸 − 4 𝑀)

10 𝑚𝑖𝑛 ∗ 60 ∗ (5.0𝐸9 𝑀−1𝑠−1)= 1.39𝐸 − 12

Table A-8. Average SA molar concentration in SA/PAA/alum system

Time (min) [SA ave.] SD [OH ave.] SD

0 0.00014 0 0 0

10 0.00016 5.78E-06 -3.04E-12 7.03E-13

20 0.00016 5.43E-06 -1.53E-12 3.30E-13

30 0.00016 5.28E-06 -1.02E-12 2.14E-13

40 0.00016 7.50E-06 -7.27E-13 2.30E-13

50 0.00016 5.18E-06 -6.15E-13 1.26E-13

60 0.00016 6.45E-06 -5.17E-13 1.30E-13

Table A-9. SA molar concentration during control (SA/alum)

Time (min) Abs297 Abs307 Abs321 [SA] [OH]

0 0.42 0.30 6.01E-2 0.00014 0

10 0.50 0.38 0.14547 0.00016 -2.87E-12

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60 0.50 0.38 0.14624 0.00016 -4.77E-13

Table A-10. SA molar concentration during second control (SA/PAA)

Time (min) Abs297 Abs307 Abs321 [SA] [OH]

0 0.42 0.30 6.01E-2 0.00014 0

10 0.39 0.28 5.76E-2 0.00013 1.29E-12

60 0.39 0.28 5.80E-2 0.00013 2.096E-13

Table A-11. Two-tail t-test values for MB decay

Variable 1 Variable 2

Mean -0.00047 0

Variance 3.69E-07 0

Observations 2 2

Hypothesized Mean

Difference 0

df 1

t Stat -1.09

P(T<=t) one-tail 0.23

t Critical one-tail 6.31

P(T<=t) two-tail 0.47

t Critical two-tail 12.7

Table A-12. Two-tail t-test values for SA decay


Mean 5.25E-08 0

Variance 4.51E-15 0

Observations 2 2

Hypothesized Mean

Difference 0

df 1

t Stat 1.10


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One or more steps in this SOP (see below for specific directions) must be performed in (circle):

Fume hood biosafety cabinet

Other:__ Cold room______________

Safety Equipment Required: acid-resistant gloves nitrile gloves

safety glasses UV-filtering safety glasses

face-shield lab coat

other____________________________

Biosafety Certificate Required: yes no




Table A-13. Two-tail t-test values for PAA & H2O2 decay

A.2.3 MICROBIAL ANALYSIS

Standard Operating Procedure (SOP)

Spread Plating Method for E. coli Enumeration

Prepared by Sarah Larlee November 2017

Revised by Domenica Cevallos, November 2019

Safety Notes:

PAA H2O2

Variable 1 Variable 2 Variable 1 Variable 2

Mean -0.0038 0 0.004 0

Variance 0.00001 0 2.2E-06 0

Observations 2 2 2 2

Hypothesized Mean

Difference 0

0

df 1

1

t Stat -1.35

4.33


0.07


6.31


0.14

t Critical two-tail 12.7 12.70

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Reagents

• Agar

• LB broth

• E. coli stock culture (ATCC ® 23631)

• For making Ringer’s solution

o Calcium Chloride (CaCl2)

o Sodium Chloride (NaCl)

o Potassium Chloride (KCl)

o Sodium Bicarbonate (NaHCO3)

• 0.9 % Sodium Chloride (NaCl)

Equipment and Materials

• Biosafety cabinet

• Serological pipettes

• Sterile pipette tips

• Automatic pipettor

• 70% ethanol spray

• Sterile spreader

• 500 mL Erlenmeyer flask

• Shaker

• Incubator at 37°C

• Low temperature freezer at -80°C

• UV-vis spectrophotometer

• 50 mL centrifuge tubes

• Centrifuge

Methods

Agar preparation and plate pouring

i. Fill a 500 mL bottle to approximately half with distilled water

ii. Weigh 10 g of powdered LB and 7.5 g of agar and add to bottle

iii. Fill bottle to the 500 mL mark, close, and shake vigorously

iv. Autoclave at 121°C making sure cap is loose

v. Allow solution to cool slightly in the biosafety cabinet, but not enough so that it

solidifies

vi. Pour agar into 100 mm petri dishes so that the dishes are ¾ full. Let the agar solidify

and turn the petri dishes upside down to avoid condensation in the agar and leave them

overnight in the biosafety cabinet

vii. The plates can be put in a sealed bag and left in the 4°C fridge for up to 2 weeks.

Ensure that petri dishes are stored upside down.

LB broth preparation

Domenica Cevallos


76


ii. Weigh 10 g of powdered LB and add to bottle



v. Allow solution to cool slightly in the biosafety cabinet, and store in the 4°C fridge.

Ringer’s solution preparation


ii. Weight out the following ingredients into the bottle:

a. Calcium chloride: 0.125 g

b. Potassium chloride: 0.21 g

c. Sodium bicarbonate: 0.1 g

d. Sodium chloride: 3.25 g


iv. Dilute to ¼ strength by removing 125 mL of the solution and adding to another 500 mL

bottle and filling to the 500 mL mark

v. Autoclave at 121°C making sure cap is loose

vi. Allow solution to cool slightly in the biosafety cabinet, and store in the 4°C fridge

0.9% Sodium Chloride preparation


ii. Weigh 4.5 g of NaCl and add to bottle



v. Allow solution to cool slightly in the biosafety cabinet, and store in the 4°C fridge.

E. coli culture from frozen stock preparation

i. The evening prior to testing, reanimate a portion of the E. coli stock by touching a

sterile pipette tip to the frozen culture then immerse the tip in 120 mL of LB broth in a

500 mL Erlenmeyer flask. Incubate the solution overnight at 37°C while shaking at 100

rpm.

ii. The day of the experiment measure the OD of the solution and diluted to an OD ~0.5 in

LB broth.

iii. Transfer 30 mL of the solution to a sterile, autoclaved 50 mL centrifuge tube and spin

for 15 minutes at 4000 rpm. Pour off the supernatant growth media and add 30 mL of ¼

strength Ringer’s solution. Gently rinse the pellet with the ¼ Ringer’s solution.

iv. Spin down the solution for 15 minutes at 4000 rpm and pour off the supernatant. Add

30 mL of ¼ strength Ringer’s solution and rinse the pellet.

v. Repeat step iv)

vi. Once the pellet has been rinsed for the third time, pour off the supernatant and add 30

mL of Ringer’s solution. Rinse the pellet and vortex to ensure the even distribution of

the bacteria.

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vii. Check the OD of the solution and use as stock

Plating

i. The Remove agar plates from fridge at the beginning of the experiments to allow them

to reach room temperature

ii. Prepare polypropylene tubes with 9 mL of 0.9% NaCl for each dilution.

iii. Immediately prior to experimentation, add enough volume of the E. coli stock to 50 mL

of Ringer’s solution to achieve a starting OD of 0.05 in each reactor.

iv. Remove 1 mL from each reactor add directly to the first prepared polypropylene tube.

v. Make eight serial dilutions in the prepared polypropylene tubes vortex each dilution to

ensure proper mixing.

vi. Pipette 100 µL of each dilution in the middle of a pre-poured agar plate and manually

spread using a sterile spreader until the entire sample is absorbed on to the agar.

vii. Incubate the plates upside down overnight in a 37°C incubator.

viii. Count the CFU formed on the plates and calculate the concentration in the samples

using the dilution factors and the following formula:

CFU

mL=

colonies counted

(mL of sample plated) x (dilution factor)

ix. Put plates in an autoclave bag, wrap with tape, spray alcohol and discard in regular

waste after 12 hours of contact with the alcohol.

Table A-14. E. coli only data

1 mg/L PAA & 0.1 mg/L aluminum

Time (min) Colonies Dilution factor CFU/mL Average

10 28 0.00001 2.80E+07 2.25E+07

10 17 0.00001 1.70E+07

20 29 0.00001 2.90E+07 2.65E+07

20 24 0.00001 2.40E+07



10 21 0.00001 2.10E+07 1.95E+07

10 18 0.00001 1.80E+07

20 24 0.00001 2.40E+07 2.15E+07

20 19 0.00001 1.90E+07

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Table A-15. Alum data



10 23 0.00001 2.30E+07 2.40E+07

10 25 0.00001 2.50E+07

20 20 0.00001 2.00E+07 1.80E+07

20 16 0.00001 1.60E+07



10 22 0.00001 2.20E+07 2.00E+07

10 18 0.00001 1.80E+07

20 19 0.00001 1.90E+07 2.10E+07

20 23 0.00001 2.30E+07

Table A-16. PAA data



10 9 0.00001 9.00E+06 1.00E+07

10 11 0.00001 1.10E+07

20 6 0.1 6.00E+02 9.50E+02

20 13 0.1 1.30E+03



10 15 0.01 1.50E+04 1.25E+04

10 10 0.01 1.00E+04

20 - - - -

20 - - -

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Table A-17. Alum + PAA data



10 7 0.00001 7.00E+06 8.00E+06

10 9 0.00001 9.00E+06

20 2 0.1 2.00E+02 5.00E+02

20 8 0.1 8.00E+02



10 4 0.01 4.00E+03 5.50E+03

10 7 0.01 7.00E+03

20 - - - -

20 - - -

Table A-18. Sodium thiosulfate and catalase data



20 31 0.00001 3.10E+07 2.70E+07

20 23 0.00001 2.30E+07

Table A-19. Two-tail t-test values for alum control

Time 10 min Time 20 min


Mean 22.5 24 26.5 18

Variance 60.5 2 12.5 8


Hypothesized Mean

Difference 0 0

df 1 2

t Stat -0.26 2.65

P(T<=t) one-tail 0.41 0.05

t Critical one-tail 6.31 2.91

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Table A-20. Two-tail t-test values for quenching agents as a control


Mean 26.5 27

Variance 12.5 32

Observations 2 2

Hypothesized Mean

Difference 0

df 2

t Stat -0.10





Table A-21. Two-tail t-test values for PAA vs alum + PAA (1 mg/L)

Time 10 minutes Time 20 minutes


Mean 1.0E+7 8.0E+6 950 500

Variance 2E+12 2E+12 2.45E+5 1.8E+5


Hypothesized Mean

Difference 0

0

df 2

2

t Stat 1.41

0.97


0.21


2.91


0.43

P(T<=t) two-tail 0.83 0.11

t Critical two-tail 12.70 4.30

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4.30

Table A-22. Two-tail t-test values for PAA vs alum + PAA (5 mg/L)

Time 10 minutes


Mean 5.5E+5 1.25E+5

Variance 4.5E+8 1.25E+9

Observations 2 2

Hypothesized Mean

Difference 0

df 2

t Stat -2.40





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