The use of advanced oxidation processes in the degradation ...

The use of advanced oxidation processes in the degradation of the pesticide chlorpyrifos

and reduction of microbial contamination on apples

By

Jordan Ho

A Thesis

presented to

The University of Guelph

In partial fulfillment of requirements

For the degree of

Master of Science

In

Food Science

Guelph, Ontario, Canada

© Jordan Ho, December, 2019

ABSTRACT

The use of advanced oxidation processes in the degradation of the pesticide chlorpyrifos and

reduction of microbial contamination on apples

Jordan Ho Advisors:

University of Guelph, 2019 Dr. Keith Warriner

Dr. Ryan Prosser

A method based on Advanced Oxidation Process (AOP) was validated for the degradation of

chlorpyrifos on apples and inactivating Escherichia coli O157:H7, along with Aspergillus niger

spores. AOP generates free-radicals using UV-C light (at 254 nm), hydrogen peroxide, and

ozone. The use of gaseous ozone alone was ineffective in chlorpyrifos degradation and was

therefore excluded from AOP use. Response surface methodology (RSM) was used to find that

the degradation of chlorpyrifos was primarily dependent on UV-C dose and hydrogen peroxide

temperature but not hydrogen peroxide concentration. The maximal degradation of chlorpyrifos

on apples was achieved through an AOP treatment applying 68.4 kJ/m2 UV dose and 1.22% v/v

H2O2 at 66°C that resulted in a 47% reduction (92µg) of the pesticide with < 0.004 µg

accumulation of chlorpyrifos-oxon. The same treatment supported a >6.56 log CFU reduction in

E. coli O157:H7 and >6.56 log CFU reduction of A. niger spores.

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ACKNOWLEDGEMENTS

First, I’d like to thank my advisor Dr. Keith Warriner for taking me on as a graduate student, a

cautionary tale I’m sure he would not repeat. Thanks for these two years, I’ve learned a lot in the

field of food safety and enjoyed every minute of it. I also learned, more than I wanted, about how

the world of academia works which is why I would hate to pursue a PhD degree! Too much

politics and drama, although entertaining as a spectator, it doesn’t seem fun as a participant. I’d

also like to thank my committee members Dr. Ryan Prosser and Dr. David Lubitz for helping me

develop my presentation skills during my committee presentations as well as helping me edit this

thesis. I hope you both well wishes and I couldn’t have asked for a better committee.

Thank you everyone in my lab, especially Fan, Mahdiyeh, and Joanna for helping me out with my

experiments. Next, I’d like to thank my friends and family for their support, especially Emma,

Jesse, Nick, and Siva. Special thanks to Andrew Green for helping answer any questions I had

about microbiology and life too maybe. Also special thanks to Katherine Katie Kathy Petker, and

Dr. Shane Walker for not throwing a toaster at me for my constant annoyance. May WW continue

again someday even though I am no longer there.

Lastly, thank you OMAFRA Agri-Food and Innovation for funding this project along with Clean

Works Corp. for their donation of equipment and consultancy.

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TABLE OF CONTENTS

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

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

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

LIST OF TABLES ....................................................................................................................................... vi

LIST OF FIGURES ....................................................................................................................................viii

LIST OF ABBREVIATIONS ......................................................................................................................... xi

Chapter 1: Introduction and Literature Review ........................................................................................ 1

1.1 Introduction ................................................................................................................................... 1

1.2 Size of the Fresh Produce Sector .................................................................................................... 2

1.3 Climate change and fresh produce safety ....................................................................................... 3

1.4 Pesticides ....................................................................................................................................... 4

1.4.1 Chlorpyrifos .......................................................................................................................... 10

1.4.2 Chlorpyrifos-Oxon ................................................................................................................. 13

1.5 Microbial Hazards ........................................................................................................................ 15

1.5.1 Escherichia coli O157:H7 ....................................................................................................... 16

1.5.2 Aspergillus niger spores......................................................................................................... 19

1.6 Current Treatments to Decrease Pesticide Residues Found on Produce ....................................... 21

1.6.1 Ultraviolet Radiation ............................................................................................................. 24

1.6.2 Ozone ................................................................................................................................... 26

1.7 Advanced Oxidation Process ........................................................................................................ 29

1.7.1 UV-based Advanced Oxidation Process ................................................................................. 32

1.7.2 Ozone-based Advanced Oxidation Process ............................................................................ 34

1.8 Response Surface Methodology ................................................................................................... 36

Hypothesis and Objectives..................................................................................................................... 38

2. Materials and Methods...................................................................................................................... 39

2.1 Materials ..................................................................................................................................... 39

2.2 Advanced Oxidation Process Reactor ........................................................................................... 39


2.4 AOP Treatment of Apple skins Spiked with Chlorpyrifos ............................................................... 43

v

2.5 Validation Trial ............................................................................................................................. 45

2.6 UV Only Trial ................................................................................................................................ 45

2.7 Ozone Treatment of Apple Skins Spiked with Chlorpyrifos............................................................ 46

2.8 HPLC Analysis ............................................................................................................................... 48

2.9 Determination of Chlorpyrifos-Oxon Production by AOP Treatment using Q-ToF analysis ............ 48

2.10 Inactivation of Escherichia coli O157:H7 and Aspergillus niger .................................................... 50

2.11 Experimental plan and statistics ................................................................................................. 52

3. Results and Discussion ....................................................................................................................... 52

3.1 Optimization of Recovery Method and HPLC Analysis .................................................................. 52


3.3 Ultraviolet light mediated degradation of CPY .............................................................................. 63

3.4 CPY-O production from AOP exposure ......................................................................................... 65

3.5 Ozone Treatment ......................................................................................................................... 66

3.6 Microbial Inactivation .................................................................................................................. 68

4. Conclusions ....................................................................................................................................... 71

Future work ........................................................................................................................................... 72

References ............................................................................................................................................ 74

vi

LIST OF TABLES Table 1. Types of pesticides and their target organisms with examples that are currently used in

agriculture. .............................................................................................................................................. 4

Table 2. Different types of insecticides and their mechanism of action. Sourced from (NPIC, 2019). ...... 7

Table 3. Produce with the highest and lowest pesticide residues scores. Adapted from Environmental

Working Group. ...................................................................................................................................... 8

Table 4. Produce-linked foodborne outbreaks of Escherichia coli O157:H7 in North American between

1996 and 2018. Modified from (Green, 2018). ....................................................................................... 16

Table 5. Reduction of pesticide residues on produce by current washing treatments. .............................. 22

Table 6. Degradation of pesticides using AOPs and individual processes. .............................................. 29

Table 7. Oxidation potential of reactive species. Adapted from Parsons (2004). ..................................... 31

Table 8. Contamination and waste products treated with AOP technologies with pesticides and

microorganisms highlighted. Modified from Vogelpohl (2003). ............................................................. 32

Table 9. RSM trials (20) using various test parameters of UV-C dose (kJ/m2), temperature (°C) and

concentration (%) of hydrogen peroxide. ............................................................................................... 42

Table 10: Six center point replicate trials determining the replicability and precision of the experimental

method. Arranged from lowest to highest reduction corrected with the average (n=177) positive control

recovery of 89.4%. The standard deviation between reductions was 3.6 µg. ........................................... 43

Table 11. RSM trials with amount of CPY reduction (µg) observed using various test parameters of UV-C

dose (kJ/m2), temperature (°C) and concentration (%) of hydrogen peroxide. Reduction from each trial

was corrected using the average positive control recovery of 89.4% (n=177). ........................................ 58

Table 12: Six center point replicate trials determining the replicability and precision of the experimental

method. Arranged from lowest to highest reduction corrected with the average (n=177) positive control

recovery of 89.4%. The standard deviation between reductions was 3.6 µg. ........................................... 59

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Table 13. ANOVA analysis table of the linear model indicating that values lower than P < 0.05 are

significant. In this case, UV-C dose and H2O2 temperature are significant whereas H2O2 concentration

was not significant. * indicates P < 0.05................................................................................................. 59

Table 14. Q-ToF analysis of CPY-O on apple skins spiked with CPY. It can be observed that apple skin

samples treated with AOP produced the by-product CPY-O while samples spiked with CPY but not

treated with AOP had concentrations below the limit of detection. ......................................................... 66

Table 15. Log count reduction of Escherichia coli O157:H7 and Aspergillus niger spores inoculated onto

apple peel sections then treated with UV-C (at 254 nm), hydrogen peroxide (1.22% v/v at 66C) or a

combination of UV-C and hydrogen peroxide ........................................................................................ 70

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LIST OF FIGURES

Figure 1. Bioaccumulation of the pesticide DDT throughout the food chain. Modified from Penn State

Pesticide Education Manual. .................................................................................................................... 5

Figure 2. Mechanism of action of acetylcholinesterase breaking down acetylcholine into choline and

acetic acid. .............................................................................................................................................. 6

Figure 3. Fate of pesticides after application and factors affecting them. Adapted from Declour et al.

(2015). .................................................................................................................................................. 10

Figure 4. Structures of chlorpyrifos and its active form chlorpyrifos-oxon as well as the inactive form

TCP. Adapted from USEPA (n.d). ......................................................................................................... 14

Figure 5. Mechanism of action of chlorpyrifos-oxon. It can phosphorylate AChE to permanently

inactivate the enzyme or it can temporarily inactivate it. Adapted from Solomon (2014). ....................... 14

Figure 6. Mechanism of action of STEC in causing cell death. 1.) STEC adheres to intestinal lumen, 2.)

Shiga toxin (Stx) is produced, 3.) Stx adhering onto cell, 4.) Endocytosis of Stx into the cell, 5.) Stx in a

vesicle to be transported into the Golgi complex, 6.) Stx in the Golgi complex, 7.) Stx breaks apart and

the toxic A1 subunit is released, 8.) A1 subunit attaches to rRNA, 9.) RNA cannot be translated due to the

A1 subunit so protein synthesis is inhibited, 10.) Lack of protein synthesis leads to cell death. Modified

from V. Castro, Carvalho, Conte Junior, & Figueiredo (2017). ............................................................... 17

Figure 7. Transmission routes of STEC from farm cattle ingesting STEC (1) then contaminating the

environment with fecal shedding (2) leading to contamination of food and water (3 and 4) consumed by

humans. People can also transmit STEC from one to another (5). Adapted from Fairbrother and Nadeau

(2006). .................................................................................................................................................. 19

Figure 8. The UV light spectrum of the electromagnetic spectrum. Modified from Evoqua Water

Technologies, LLC. https://www.evoqua.com/en/brands/ETS_UV/Pages/how-ets-uv-works.aspx .......... 25

ix

Figure 9. Schematic of ozone production using a corona discharge ozone generator. Modified from

Oxidation Technologies, LLC. https://www.oxidationtech.com/ozone/ozone-production/corona-

discharge.html ....................................................................................................................................... 27

Figure 10. Absorbance spectra of hydrogen peroxide at different wavelengths in Angstroms (254 nm =

2540 Angstroms). Modified from USP technologies. http://www.h2o2.com/technical-library/physical-

chemical-properties/radiation-properties/default.aspx?pid=65&name=Ultraviolet-Absorption-Spectrum 33

Figure 11. Photolysis reaction of ozone when exposed to UV-C light at 254 nm. One oxygen molecule

and hydroxyl radical is produced. Modified from Spartan Environmental Technologies, LLC. ............... 35

Figure 12. Response surface methodology design with center points and star points. Modified from Stat-

Ease, Inc. ............................................................................................................................................... 37

Figure 13. Schematic diagram (A) and photograph (B) of the advanced oxidation process reactor used for

chlorpyrifos degradation and microbial inactivation. .............................................................................. 40

Figure 14. Schematic (A) and photograph (B) of ozone chamber treatment of CPY spiked apple skins. . 47

Figure 15. Calibration curve of chlorpyrifos-oxon standards ranging in concentrations from 0.01 – 0.31

µg/mL. Concentrations of 0.16 and 0.31 µg/mL were removed due to low accuracy. R2 value = 0.9993. 50

Figure 16. 3D surface graph of different interaction effects of an advanced oxidation process for

degrading CPY on apples. CPY was introduced onto apple peel and treated with various combinations of

hydrogen peroxide and UV dose with the decrease in pesticide levels being recorded. The plots illustrate

UV dose and H2O2 temperature (A), UV dose and H2O2 concentration (B) & H2O2 concentration and

H2O2 temperature (C). ........................................................................................................................... 62

Figure 17. Predicted vs actual reduction of CPY table and graph illustrating the accuracy of the RSM

model in predicting CPY degradation under known conditions. ............................................................. 63

Figure 18. Graph of CPY reduction using only UV radiation with a linear trend line (orange line).

Reductions were calculated using HPLC analysis. A R2 value of 0.982 was calculated using 5 data points

of varying UV doses (kJ/m2) and CPY reduction (µg). ........................................................................... 64

x

Figure 19. Log counts of Escherichia coli O157:H7 and Aspergillus niger spores. *<2.3 CFU/mL is the

limit of enumeration. All treatments are determined to be statistically significant for both organisms. .... 70

xi

LIST OF ABBREVIATIONS 2,4-D…………………………………………………...…………2,4-Dichlorophenoxyacetic acid

ACh……………………………………………………………………..…………...Acetylcholine

AChE…………………………………………………………………..……..Acetylcholinesterase

ANOVA………………………………………………………..…………..…Analysis of variance

AOP……………………………………………….………………….Advanced oxidation process

BBD…………………………………………………………………………..Box-Behnkin design

CCD……………………………………………………………….……..Central composite design

CFU……………………………………………………………………...……Colony forming unit

CPY………………………………………………………………………………..….Chlorpyrifos

CPY-O……………………………………………………………………….….Chlorpyrifos-oxon

DDT………………………………………………………..…….Dichlorodiphenyltrichloroethane

EHEC……………………………………………………………………Enterohemorrhagic E.coli

EWG…………………………………………………..…………. Environmental Working Group

FDA………………………………………………………………..Food and Drug Administration

GRAS……………………………………………………...………....Generally recognized as safe

HPLC…………………………...………………………High performance liquid chromatography

HUS………………………………………………..…………………Hemolytic uremic syndrome

LOD……………………………………………………………………………..Limit of detection

LOQ………………………………………………………………….……..Limit of quantification

LPM……………………………………………………………..………….Low pressure mercury

MDL………………………………………………………………..………Method detection limit

MPM…………………………………………………………...……….Medium pressure mercury

MRL……………………………………………………………………….Maximum residue level

OP………………………………………………………………………..………Organophosphate

PDA………………………………………………………………………..….Potato dextrose agar

PON1………………………………………………………………………………..Paraoxonase 1

Q-ToF…………………………………………………………...……..Quadrupole Time of Flight

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RSM……………………………………………………………….Response surface methodology

Stx………………………………………………………………………………………Shiga toxin

STEC……………………………………………...………………….Shiga toxin producing E.coli

R2….……………………………………………………….………….coefficient of determination

TCP…………………………………………………….……………..3,5,6 – trichloro-2-pyridinol

TSB………………………………………………………………..………………Trypic soy broth

USE…………………………………………………………………..Ultrasonic solvent extraction

USEPA…………………………………….…….United States Environmental Protection Agency

UV light……………………………………………………………………………Ultraviolet light

1

Chapter 1: Introduction and Literature Review

1.1 Introduction

Pesticides are an important part of modern day farming and are necessary in the growth and

production of fresh fruits and vegetables due to their ability to eliminate pests (Randall, Hock,

Crow, Hudak-Wise, & Kasai, 2014). Despite their necessity in agriculture, they have been

associated with numerous human health concerns as well as damage to the environment

(Stoytcheva, 2011). One of the most controversial pesticides is chlorpyrifos (CPY), which could

cause adverse effects on the nervous system of children (M. Bouchard, F. et al., 2011). Humans

can be exposed to pesticides through a number of different routes, one of them being through

ingestion of contaminated produce (Giesy et al., 2014).

The most common current approach to remaining CPY on produce is through washing, however,

this process results in a redistribution of not only pesticides but also pathogenic microorganisms

between produce batches and further dissemination within the environment via wastewater

disposal (Burkul, Ranade, & Pangarkar, 2015; Köck-Schulmeyer et al., 2013). A more effective

approach is to use water-free technologies to decontaminate fruits and vegetables thereby

reducing pesticide levels entering down-stream washing processes. One potential approach is

based on the advanced oxidation process (AOP) that has previously been shown to inactivate

Listeria monocytogenes on apples (Murray et al., 2018). AOP involves generating hydroxyl

radicals from the degradation of ozone and/or hydrogen peroxide. The generation of free radicals

can be achieved through the interaction between ozone and hydrogen peroxide, or more rapidly

via UV-C (254nm) mediated decomposition of the oxidants (Abramovic, Banic, & Sojic, 2010;

Femia, Mariani, Zalazar, & Tiscornia, 2013; Marican & Durán-Lara, 2018).

2

In the following study the AOP originally developed to decontaminate apples was applied for

CPY degradation as well as inactivate the pathogen Escherichia coli O157:H7 due to the

previous links to foodborne illness outbreaks linked to apples (Kenney, Burnett, & Beuchat,

2001). Aspergillus niger was also selected as a test microbe due to its inherent resistance to UV

in the spore form, in addition to acting as a surrogate for potential mycotoxin producing molds

(Taylor-Edmonds, Lichi, Rotstein-Mayer, & Mamane, 2015). A key part of the AOP is

optimizing the hydrogen peroxide concentration and temperature along with UV-C dose. In the

current study response surface methodology (RSM) was used to determine which factors and

their settings affect the degradation of CPY.

1.2 Size of the Fresh Produce Sector

Fresh produce is important to Canadians, with about 28.6% of Canadians aged 12 and over

(approximately 8.3 million people) reported to consume 5 or more fruits and vegetables per day

(Statistics Canada, 2017). Fresh produce is also important to the Canadian economy, grossing

approximately $2.24 billion in 2017 (Agriculture and Agri-Food Canada, 2017a, 2017b;

Statistics Canada, 2017). The fruit industry makes up about $1.04 billion annually while

vegetables make up the rest (Agriculture and Agri-Food Canada, 2017a). Of this value, apples

have been the highest grossing fruit since 2013, making approximately $224.6 million in 2017

(Agriculture and Agri-Food Canada, 2017a). Apples are one of the most widely grown fruit

species in the world with approximately 5.2 million hectares in production worldwide in 2016

(Agriculture and Agri-Food Canada, 2017a). According to Statistics Canada, apples are the most

widely produced fruit in Canada with an estimated 424,709 tons being produced in 2018. In

comparison, the second most widely produced fruit in Canada is cranberries with only 195,196

tons (Statistics Canada, 2018).

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1.3 Climate change and fresh produce safety

Climate change is believed to be caused by an accumulation of various greenhouse gases, mostly

carbon dioxide, which in effect is increasing a trend of warm and humid weather (Butler, 2018).

Climate change has been shown to increase the prevalence of pests such as insects, pathogenic

microorganisms, and weeds (Delcour, Spanoghe, & Uyttendaele, 2015; Gregory, Johnson,

Newton, & Ingram, 2009; Roos, Hopkins, Kvarnheden, & Dixelius, 2011; Ziska, Teasdale, &

Bunce, 1999). Although some crops are capable of growing faster at higher temperatures, they

may be subjected to an increase in pests and pathogens that also benefit from higher

temperatures and humidity (Gregory et al., 2009). Increased temperatures can be associated with

an enhanced metabolic rate in various insects which would increase both their rate of food

consumption and rate of reproduction (Delcour et al., 2015; Deutsch et al., 2018). Due to this, it

has been predicted that yields of grain such as maize, rice and wheat will decrease by 10 to 25%

per degree of global mean surface warming (Deutsch et al., 2018). This prediction is based on

various models that show a 2 °C increase in global temperature is capable of causing an average

yield loss of 46, 19, and 31% for wheat, rice and maize, respectively (Deutsch et al., 2018).

These predictions differ from previous models which showed a rice yield reduction of 5% per °C

rise above 32 °C (Walker, Steffen, Canadell, & Ingram, 1999) and a reduction of 10% maize

yield (Jones & Thornton, 2003).

Along with a change in temperature, changes in precipitation, light exposure, and humidity can

also affect the growth of pests and pathogens (Delcour et al., 2015; Holland & Sinclair, 2004;

Keikotlhaile, 2011). Higher moisture is also associated with various plant diseases since

microorganisms are capable of thriving in wet conditions, promoting the growth of mold and

bacteria as well as spore germination (Roos et al., 2011).

4

1.4 Pesticides

Pesticides are substances, either chemical or biological, used by humans for controlling pests that

are capable of harming human health and crop yield (Randall et al., 2014). Pests can be weeds,

insects, animals, and even pathogenic microorganisms that can cause harm to both humans and

plants (Randall et al., 2014). Chemical pesticides were used as early as 2500 B.C. when sulphur

compounds were used for insect control by ancient Sumerians (Randall et al., 2014). Nowadays,

there are multiple types of pesticides including fungicides, herbicides, insecticides, repellents,

and disinfectants which control fungi, plants, insects/invertebrates, animals, and pathogenic

microorganisms respectively (Randall et al., 2014) (Table 1).

Table 1. Types of pesticides and their target organisms with examples that are currently used in

agriculture.

Type of Pesticide Target Organism Examples

Bactericides Bacteria Alcohols, disinfectants

Fungicides Fungi Tea tree oil, Bacillus subtilis

Herbicides Plants Glyphosate, 2-4 D

Insecticides Insects Chlorpyrifos, imidacloprid

Larvicides Larvae Methoprene, temephos

Rodenticides Rodents Anticoagulants, metal

phosphides

Many classes of synthetic chemical pesticides used in the modern era were originally produced

researched for use in warfare during World War II, with some of these early synthetic pesticides

still being used today for agricultural purposes (Sanborn, Cole, Helena Sanin, & Bassil, 2004).

The most notable of those pesticides being 2,4-Dichlorophenoxyacetic acid (2,4-D), a phenoxy

5

herbicide that kills weeds by causing uncontrolled and unsustainable growth (Randall et al.,

2014; Sanborn et al., 2004). Another noteworthy pesticide is dichlorodiphenyltrichloroethane

(DDT), an insecticide which was used in war to prevent malaria and typhus (Beard, 2006). In the

United States, it was approved for use in agriculture and households from 1945 until 1972 when

it was eventually banned in 1972 (Beard, 2006). This ban was due to the fact that DDT was

shown to be very persistent in the environment while maintaining its residual effects and it can

biomagnify in the food chain (Figure 1) (Beard, 2006; Randall et al., 2014). To this day, DDT

still persists in the environment and it is believed that every living organism on Earth has been

exposed to DDT resulting in body fat storage (Nicolopoulou-Stamati, Maipas, Kotampasi,

Stamatis, & Hens, 2016).

After DDT was banned from being used in the United States, farmers started to use more

organophosphate (OP) insecticides which have a relatively high mammalian toxicity but do not

persist in the environment and are effective for a wide spectrum of insects compared to other

insecticides (Davis, 2014).

Figure 1. Bioaccumulation of the pesticide DDT throughout the food chain. Modified from Penn

State Pesticide Education Manual.

6

OPs are effective at controlling insects and mammalian pests through their ability to inactivate

acetylcholinesterase (AChE), an enzyme required for proper nervous system function

(Williamson, Terry, & Bartlett, 2006). AChE breaks down acetylcholine (ACh), a

neurotransmitter that stimulates nerve fibres and muscles, into choline and acetic acid (Figure 2)

(Fukuto, 1990). If AChE is inhibited, ACh will continue to stimulate nerve fibres and muscles

uncontrollably which in effect will cause muscle spasms and involuntary movement (Fukuto,

1990). Besides mammals and insects, AChE is also present in birds, fish and reptiles (Fukuto,

1990). Exposure to high enough concentrations of OPs can lead to unintentional deaths of these

animals.

Figure 2. Mechanism of action of acetylcholinesterase breaking down acetylcholine into choline and

acetic acid.

Although pesticides are crucial in food production, they are capable of causing harm to human

health, especially insecticides (Ansari, Moraiet, & Ahmad, 2014). There are multiple types of

insecticides that have mechanism of actions, that target the nervous system of insects (Casida &

Durkin, 2013) (Table 2). Like DDT, residues of these insecticides can remain on produce after

being harvested from the fields (Bajwa & Sandhu, 2014) and may also be found in water, soil,

and/or air following field application (Ansari et al., 2014). It has been estimated that every year,

over 26 million people, especially those working in the agriculture industry, are poisoned by

7

pesticides, resulting in over 220,000 deaths (Ansari et al., 2014). One of the major human health

concerns associated with insecticides is neurotoxicity (Casida & Durkin, 2013).

Table 2. Different types of insecticides and their mechanism of action. Sourced from (NPIC, 2019).

Type of Insecticide Mechanism of Action Example

Carbamates Acetylcholinesterase inhibitor Carbaryl

Organochlorine Opens nerve cell sodium

channels DDT

Organophosphates

Acetylcholinesterase inhibitor

more toxic and longer lasting

than carbamates

Chlorpyrifos

Phenylpyrazoles Blocks glutamate-acitivated

chloride channels Fipronil

Pyrethroids Modulates sodium channels Cypermethrin

Neonicotinoids Nicotinic Acetylcholine

receptor agonist Imidacloprid

Ryanoids Binds to calcium channels to

block nerve transmission Chlorantraniliprole

Consumers are concerned about pesticides used in agriculture and in their food (Consumer

Reports, 2015; Koch, Astrid Epp, Mark Lohmann, & Gaby-Fleur Böl, 2017). A survey by

Consumer Reports in 2015 showed that of 1050 Americans surveyed, approximately 85% were

concerned about pesticides in their food (Consumer Reports, 2015). The Environmental Working

Group (EWG) states that in 2019, the top five fruits and vegetables linked to the highest end-user

pesticide exposure are: strawberries, spinach, kale, nectarines, and apples (Table 3). The EWG

also state that two or more pesticide residues were detected in about 90% of strawberries, apples,

cherries, spinach, nectarines, and kale samples tested (EWG, 2019).

8

Table 3. Produce with the highest and lowest pesticide residues scores. Adapted from

Environmental Working Group.

The CDC states that approximately 29 pesticides can be found in trace amounts in the average

human body in the United States. When exposed to the same concentration of pesticides,

children are more susceptible to danger than adults due to the difference in body sizes (Eskenazi,

Bradman, & Castorina, 1999). Children also have a different metabolism than adults so their

bodies are unable to remove toxins as quickly (Eskenazi et al., 1999). Due to these factors,

maximum residue limits (MRLs) are put in place to ensure that pesticide levels are safe for

children and infants. Studies have shown a correlation between pesticides and human health risks

such as lower IQ in children, Alzheimer’s disease, Parkinson’s disease, and ADHD to name a

few (M. Bouchard, F. et al., 2011; M. F. Bouchard, Bellinger, Wright, & Weisskopf, 2010;

Brown, Rumsby, Capleton, Rushton, & Levy, 2006; Eskenazi et al., 1999; Kim, Lee, Lee,

Jacobs, & Lee, 2015; Kuehn, 2010). Cancer development due to pesticide exposure is also a

great concern for both children and adults. Multiple studies have shown a link between pesticide

exposure and tumour growth (Bassil et al., 2007). Due to this, it has been suggested that a

reduction of pesticide use, especially for non-commercial purposes, should be considered (Bassil

9

et al., 2007). As a result of these concerns, organophosphate insecticide use in the United States

has decreased from 70 million lbs in 2000 to 20 million lbs in 2012 (Atwood & Paisley-Jones,

2017). The use of all insecticides have also decreased from 99 million lbs in 2000 to 60 million

lbs in 2012 (Atwood & Paisley-Jones, 2017). Although the use of insecticides has decreased, the

sale and usage of all pesticides combined are increasing. In 2008, about 4850 million lbs of

pesticides were being used but in 2012, an estimated 5821 million lbs were used (Atwood &

Paisley-Jones, 2017). Despite the current effort to reduce the use of pesticides, the need for them

may soon increase due to climate change (Delcour et al., 2015).

Increased temperature, humidity and CO2 can induce a higher growth rate of plants which in

effect will lead to a dilution of pesticide concentrations thus reducing residue levels which will

require more frequent applications as the plant matures (Holland & Sinclair, 2004). Higher levels

of precipitation and higher moisture levels can cause pesticides to dissipate faster which will also

lead to reduced residue concentrations found on plants and require more applications (Delcour et

al., 2015). Higher CO2 levels have also been shown to increase weed tolerance to herbicides

which may lead to the use of more potent herbicides (Ziska et al., 1999). Due to these factors

decreasing the amount of pesticide residues found on crops, the frequency of pesticide

application would increase which in effect leads to an increase in the risk of pesticide residue

carriage on produce (Delcour et al., 2015). When pesticides are applied, they can be transported

to different components of the environment, including: air, soil, ground water, and surface water

(Figure 3).

10

Figure 3. Fate of pesticides after application and factors affecting them. Adapted from Declour et

al. (2015).

1.4.1 Chlorpyrifos

One pesticide of great interest is chlorpyrifos (O,O-diethyl-O-3,5,6-trichloro-2-

pyridylphosphorothioate), a broad spectrum OP insecticide that is widely used in the agricultural

industry (Mladenova & Shtereva, 2009; K. R. Solomon et al., 2014; USEPA, 2006). In 2012,

CPY was ranked as the 14th most used pesticide and the most widely used insecticide in the

United States by the United States Department of Agriculture – National Agricultural Statistics

Service (Atwood & Paisley-Jones, 2017). CPY is capable of controlling insects that commonly

consume maize, fruit still on trees or vines, and root vegetables (Gomez, 2009; K. R. Solomon et

al., 2014). It has been estimated that CPY is used to treat over 50 different crops (Gomez, 2009)

11

and fruits grown on trees such as apples account for approximately 10 percent of CPY usage

(Eaton et al., 2008).

CPY has been shown to be a neurotoxin which can affect the mental development of children

(Trasande, 2017). Longitudinal studies have shown that prenatal exposure to OPs is associated

with neurological deficits in children (M. Bouchard, F. et al., 2011; Rauh et al., 2012). Bouchard

et al. (2011) conducted a 7-year birth cohort study (n = 329) and reported that children exposed

to higher levels of OPs had an average of 7 lower IQ points than children exposed to lower levels

of OPs.

CPY exposure can occur in a number of ways including ingestion through food or water, skin

contact, and inhalation through air (Giesy & Solomon, 2014). CPY can be used on crops

throughout the year and its use is determined by the amount of pests present at a given time. It

can be used in the winter season on tree crops and used in the summer season on field crops

(Giesy & Solomon, 2014). The United States National Institutes of Health (NIH) has stated that

the half-life of CPY is approximately 4.2 days in the summer and 9.7 days in the winter (Toxnet,

2014). CPY is capable of absorbing light in the UV range with a maximum absorbance of 290

nm (Zalat, Elsayed, Fayed, & Abd El Megid, 2014). The lower half-life in the summer is due to

photolysis by sunlight because of this absorption (Toxnet, 2014). Photolysis requires the

presence of water to occur (Eto, 1974). Under dry conditions, only an estimated 2%

decomposition of CPY after 1200 hours of irradiation is predicted (Eto, 1974).

Some jurisdictions have decided the risk of CPY to human health and the environment is too

great to allow continued use in agriculture. For example, the State of Hawaii has recently banned

the use of CPY starting in 2022, being the first State to do so in the United States of America

12

(State of Hawaii, 2017). New York has also proposed to ban all use of CPY by 2021 ("Assembly

Bill A2477B," 2019). Although Hawaii and New York may have banned the use of CPY, other

jurisdictions have not followed the same course of action. In 2017, a petition to ban the use of

CPY was denied by the U.S. Environmental Protection Agency (EPA) stating that “the pesticide

is crucial to U.S. agriculture.” (Environmental Protection Agency, 2017, March 29). As CPY

continues to be used in agriculture, it is necessary to limit exposure to the public. Currently, the

maximum residue limit (MRL) for CPY on apples in Canada is set at 0.01 µg/g (Health Canada,

2012, October 1). In the United States, the limit on apples is also set at 0.01 µg/g and due to its

possible detrimental effects, produce such as spinach, squash and carrots cannot be treated using

CPY as they are commonly fed to children (USEPA, 2006). If residue is found on these crops,

they are considered to be adulterated which allows the FDA to consider enforcement actions

(FDA, 1995).

If humans are exposed to high enough concentrations of CPY (LD50 in rats was determined to

be 60 mg/kg bw (EXTOXNET, 1993), adverse effects including nausea, dizziness, respiratory

paralysis and even death can occur (USEPA, 2006). It has also been shown recently that CPY

exposure can promote the growth of mammary tumors and may be a risk factor for breast cancer

development (Ventura et al., 2019). Various diseases that affect the nervous system such as

Alzheimer’s and Parkinson’s disease have also been associated with, exposure to, insecticides

but more research is required (Brown et al., 2006; Casida & Durkin, 2013; Kim et al., 2015). In

response to this, it is important to have post-harvest techniques and treatments in place to

minimize the amount of pesticide residue on produce to protect consumers.

13

1.4.2 Chlorpyrifos-Oxon

CPY can be degraded into two different metabolites, chlorpyrifos-oxon (CPY-O) and 3,5,6 –

trichloro-2-pyridinol (TCP). CPY-O is the active form of CPY and is capable of irreversibly

binding to AChE through phosphorylation (Giesy et al., 2014; Williamson et al., 2006).

Structurally, CPY-O and CPY are similar but CPY-O contains a phosphorus-oxygen double

bond (P=O) group whereas CPY contains a phosphothionate (P=S) group (Figure 4). Due to this

structural difference, CPY-O is more efficient at binding to AChE than CPY (Giesy et al., 2014).

The mechanism of action of CPY is to be transformed into CPY-O which prevents AChE action

(Figure 5). This transformation has been shown to occur in the environment due to oxidative

desulfonation which can be accelerated by photolysis (USEPA, n.d.-a) and in animals through

multifunction oxidase (MFO) (Giesy et al., 2014). El-Merhibi et al (2004) showed in aquatic

organisms, when MFO is inhibited, the toxicity of CPY can be reduced by up to six-fold (El-

Merhibi, Kumar, & Smeaton, 2004; Giesy et al., 2014). Acute CPY poisoning will show

symptoms when over 70% of AChE molecules have been bound (Connors et al., 2008). It has

also been suggested by the USEPA that CPY-O can exert the same or greater levels of toxicity

than CPY (Slotkin, Seidler, Wu, MacKillop, & Linden, 2009).They reported that CPY-O is 41

more times toxic than CPY when tested on aquatic animals (USEPA, n.d.-a). CPY-O can be

detoxified in human bodies through hydrolysis facilitated by the enzyme paraoxonase 1 (PON1)

(Costa, Giordano, Cole, Marsillach, & Furlong, 2013). When hydrolyzed, CPY-O will turn into

the less harmful TCP metabolite and be excreted through urine (Costa et al., 2013).

14

Figure 4. Structures of chlorpyrifos and its active form chlorpyrifos-oxon as well as the inactive

form TCP. Adapted from USEPA (n.d).

Figure 5. Mechanism of action of chlorpyrifos-oxon. It can phosphorylate AChE to permanently

inactivate the enzyme or it can temporarily inactivate it. Adapted from Solomon (2014).

15

TCP is the inactive form of CPY and is filtered by the kidney without toxic effects. However, It

has been reported in one study by Kashanian et al. (2012) that TCP has DNA binding effects

implying that TCP may exert mutagenic effects (Kashanian, Shariati, Roshanfekr, & Ghobadi,

2012). The USEPA concluded however that TCP is significantly less toxic than CPY and is

considered to be toxicologically insignificant (USEPA, n.d.-a). An animal study conducted by

Hanley et al. (2000), reported a LD50 oral dose of 800 mg/kg for TCP. They concluded that

because TCP lacks the phosphate group seen in CPY it cannot inhibit AChE and would therefore

be less toxic than its parent compound (Hanley, Carney, & Johnson, 2000). They also stated that

CPY is quickly broken down into TCP in mammalian systems through hydrolysis which would

effectively detoxify CPY (Hanley et al., 2000).

1.5 Microbial Hazards

Besides chemical contamination via pesticides, microbial contamination can also be present on

produce and some pathogens are capable of causing severe illness and even death for consumers

(Rangel, Sparling, Crowe, Griffin, & Swerdlow, 2005). One pathogen of concern is Escherichia

coli O157:H7 which has been implicated in numerous produce-linked foodborne outbreaks

(Table 4). A recent outbreak occurred in Canada in 2018 when romaine lettuce imported from

California was contaminated with E.coli O157:H7 which affected 29 individuals, 2 of which

developed hemolytic uremic syndrome (Canada, 2019).

16

Table 4. Produce-linked foodborne outbreaks of Escherichia coli O157:H7 in North American

between 1996 and 2018. Modified from (Green, 2018).

Year Source Area(s) affected Total cases

2019 Romaine lettuce 23 states in the USA 102

2018 Romaine lettuce British Columbia,

Alberta,

Saskatchewan,

Ontario, Quebec; 36

states in the USA

8 (Canada) + 210

(USA)

2018 Leafy greens Ontario, Quebec,

New Brunswick,

Nova Scotia, and

Newfoundland and

Labrador; 15 states

in the USA

67

2018 Alfalfa sprouts Minnesota,

Wisconsin (USA)

11

2017 Leafy greens 15 States in the USA 25

2015 Leafy greens Alberta,

Saskatchewan,

Ontario,

Newfoundland and

Labrador (Canada)

13

2013 Shredded lettuce Ontario, New

Brunswick, Nova

Scotia (Canada)

30

2013 Ready to eat salads Washington,

California, Arizona,

Texas (USA)

33

2012 Organic spinach and

spring mix

Ney York,

Pennsylvania,

Virginia, Maine,

Connecticut (USA)

33

2012 Romaine lettuce 9 states in the USA 58

2006 Spinach

26 states in the USA

199

1.5.1 Escherichia coli O157:H7

E. coli O157:H7 is classified as an enterohemorrhagic E.coli (EHEC) which can cause

gastroenteritis, enterocolitis, and bloody diarrhea in infected humans due to the production of

shiga toxins (Stx), one of the most potent biological poisons (Lim, Yoon, & Hovde, 2010;

17

Melton-Celsa, 2014). EHEC is a subgroup of Stx producing E.coli (STEC) that can induce

apoptosis in cells (Figure 6) which causes bloody diarrhea and in worse cases, hemolytic-uremic

syndrome (HUS), a condition involving kidney failure, haemolytic anemia, and

thrombocytopenia (a reduction in blood platelets that help in blood clot formation) (Melton-

Celsa, 2014). An estimated 8% of STEC infections leads to HUS (D. E. Thomas & Elliott, 2013).

Figure 6. Mechanism of action of STEC in causing cell death. 1.) STEC adheres to intestinal lumen,

2.) Shiga toxin (Stx) is produced, 3.) Stx adhering onto cell, 4.) Endocytosis of Stx into the cell, 5.)

Stx in a vesicle to be transported into the Golgi complex, 6.) Stx in the Golgi complex, 7.) Stx breaks

apart and the toxic A1 subunit is released, 8.) A1 subunit attaches to rRNA, 9.) RNA cannot be

translated due to the A1 subunit so protein synthesis is inhibited, 10.) Lack of protein synthesis

leads to cell death. Modified from V. Castro, Carvalho, Conte Junior, & Figueiredo (2017).

18

STEC can be transferred from person-to-person and contaminated food and drinking water

(Figure 7) (Fairbrother & Nadeau, 2006; Lim et al., 2010; Salvadori et al., 2009). The most

common route of STEC transmission is through consumption of contaminated food and

beverages, commonly linked to undercooked ground beef and raw milk (CDC, 2016; Gally &

Stevens, 2017; Lim et al., 2010). STEC has often been linked back to cattle manure runoff;

approximately 30% of feed cattle shed E.coli O157:H7 in their manure (D. E. Thomas & Elliott,

2013). It has been estimated that less than 100 cells of E.coli O157:H7 can cause disease in

susceptible hosts (Leach, Stroot, & Lim, 2010). The products most associated with human

exposure are: ground meat, leafy greens, raw milk, and apple juice/cider (Lim et al., 2010).

Leafy greens such as lettuce can be contaminated through a number of ways including: human

transfer, manure runoff, and irrigation water (E. B. Solomon, Yaron, & Matthews, 2002). It was

demonstrated by Solomon, Yaron, and Matthews (2002) that E.coli O157:H7 can enter the root

system of mature lettuce and be found in the edible leafy portions. They concluded that due to

this route of entry, E.coli O157:H7 can avoid complete disinfection through surface sanitation of

lettuce. In the 1990s, seven cases of E. coli O157:H7 outbreaks from contaminated apple

products such as apple juice and apple cider were reported (Rangel et al., 2005). Due to being

able to tolerate low pH levels, below 2, E.coli O157:H7 can grow in apple products without

difficulty which is why nowadays, apple juices and ciders require pasteurization (Miller &

Kaspar, 1994). One case occurring in 1996 determined that apples were contaminated in the

orchard by contacting manure left by wildlife carrying E.coli O157:H7 as well as the use of

rotten apples (Cody et al., 1999). From these events, it was suggested that washing apples could

reduce the risk of transmission (Besser et al., 1993).

19

Figure 7. Transmission routes of STEC from farm cattle ingesting STEC (1) then contaminating the

environment with fecal shedding (2) leading to contamination of food and water (3 and 4) consumed

by humans. People can also transmit STEC from one to another (5). Adapted from Fairbrother and

Nadeau (2006).

1.5.2 Aspergillus niger spores

Another organism of interest is Aspergillus niger (A. niger) which is commonly implicated in

spoilage of produce. Aspergillus niger is a saprophytic filamentous fungus with a brown to dark

brown colour that is commonly found in soil, compost and decaying plants (Schuster, Dunn-

Coleman, Frisvad, & van Dijck, 2002). It is capable of growing at temperatures ranging from 25

– 40 °C, a pH range of 1.4 – 9.8 and a water activity down to 0.88 (Gautam, Sharma, Avasthi, &

Bhadauria, 2011; Ladaniya, 2008). These conditions mean warm and humid environments are

ideal for supporting growth on fruits (Gautam et al., 2011). Aspergillus niger reproduces

asexually, resulting in the growth of vegetative mycelium and conidiophores that leads to spore

formation (Krijgsheld et al., 2013). Once spores are formed they are able to survive in

20

environments outside of its usual growth parameters and then germinate once proper growth

conditions occur (Krijgsheld et al., 2013).

Aspergillus niger is labelled as “generally recognized as safe (GRAS)” by the FDA for use in

citric acid and enzyme production (R. Sharma, 2012) however multiple Aspergillus species are

capable of causing harm to human health (Al-hindi, Alnajada, & Mohamed, 2011). For example,

Aspergillus fumigatus is capable of causing allergic reactions as well as life threatening

infections in humans (Paulussen et al., 2017). Aspergillus fumigatus is responsible for 90% of

aspergillus related infections followed by Aspergillus flavus and A. niger (Dagenais & Keller,

2009; Paulussen et al., 2017). Aspergillus niger has been found to be an opportunistic infectious

organism to humans (R. Sharma, 2012). There have been rare cases of A. niger spores infecting

patients with severe immunodeficiency (Schuster et al., 2002). One such case involved a patient

with invasive pulmonary aspergillosis, a lung infection caused by aspergillus species which leads

to necrosis of lung tissue (Person, Chudgar, Norton, Tong, & Stout, 2010). Various strains of A.

niger are also capable of producing mycotoxins: aflatoxin, ochratoxin, and fumonisin B2

(Frisvad, Smedsgaard, Samson, Larsen, & Thrane, 2007; Gautam et al., 2011; Schuster et al.,

2002). These mycotoxins are dangerous when consumed by humans and are capable of inducing

hepatotoxicity, nephrotoxicity, immunotoxicity, and possible carcinogenicity (Gautam et al.,

2011; Schuster et al., 2002).

Aspergillus niger is widely used in industry due to its ability to produce citric acid in copious

amounts while also being economical (Gautam et al., 2011; Show et al., 2015). Aspergillus niger

can also produce various other organic acids as well as food enzymes such as pectinase,

proteases, and glucoamylase (Schuster et al., 2002). Due to its ability to produce organic acids

and enzymes, A. niger can cause decay in nuts, legumes, cereals, and fresh produce such as

21

apples (Gautam et al., 2011). Aspergillus niger is one of the most prevalent spoilage

microorganisms. One studied showed that A. niger was the most common fungi found in infected

fruits such as pineapples, watermelon, and oranges with a 38% occurrence frequency (Mailafia,

Okoh, Olabode, & Osanupin, 2017).

1.6 Current Treatments to Decrease Pesticide Residues Found on Produce

The most common current technology used to remove pesticide residues from produce is through

post-harvest washing with clean, potable water (Table 5). Rawn and colleagues showed that

washing and rubbing apples under running de-ionized water could effectively remove pesticide

residues by approximately 50% (Rawn et al., 2008). Washing was shown to be effective,

reducing CPY levels on fruits and vegetables by 30-50% (Mi-Gyung Lee, 2001; Rawn et al.,

2008). It was also shown that washing removed approximately 60% of CPY on rice grains (Lee,

Mourer, & Shibamoto, 1991). Another study showed that when shaken in water, red peppers

that were artificially spiked with CPY had a 30-40% reduction after 5 minutes (Mi-Gyung Lee,

2001). Washing produce with different low concentration solutions of acetic acid, citric acid and

potassium permanganate have also been shown to reduce pesticide concentrations in date fruits

(Osman, Al-Humaid, Al-Redhaiman, & El-Mergawi, 2014). The problem with batch washing

produce is that pesticides are removed from the surface of the product which in effect generates

wastewater containing pesticides that can lead to cross contamination between batches (Burkul et

al., 2015; Kayla Murray, Wu, Shi, Jun Xue, & Warriner, 2017). Although washing can reduce

pesticide concentrations on produce, it does not degrade pesticides but rather redistributes it

between batches and into the environment via the disposal of spent wash water (Burkul et al.,

2015; Köck-Schulmeyer et al., 2013).

22

Table 5. Reduction of pesticide residues on produce by current washing treatments.

Treatment Reduction Reference

Washing apples using

deionized water 50% Reduction of Captan (Rawn et al., 2008)

Washing of red peppers using

tap water 30-40% Reduction of CPY (Mi-Gyung Lee, 2001)


tap water then hot-air dried

for 26 hrs

67% Reduction of CPY

(Chun & Lee, 2006)


tap water then hot-air dried

and UV treated for 26 hrs



20% hydrogen peroxide

solution


Washing of rice using tap

water 60% Reduction of CPY (Lee et al., 1991)

Pesticides are also able to adhere onto soft skins and some may even be absorbed into the plant

(Yang et al., 2017). Yang and colleagues showed that washing with a baking soda solution was

more effective than with water or hypochlorite. They also found that the systemic pesticide

thiabendazole is capable of penetrating the skin of apples which makes it harder to be removed

through washing alone (Yang et al., 2017).

23

Approximately 6% of consumers do not wash their produce or choose to wash produce only

when deemed necessary (Parnell & Harris, 2003). In 2016, the FDA conducted a survey asking

consumers if they washed their produce before consumption. Only 54% replied “yes” when

asked if they wash their lettuce before consumption and 56% washed their avocadoes (Lando,

Verrill, Liu, & Smith, 2016). They also noted that when asked if they washed their strawberries

or tomatoes before consumption, close to 100% responded “yes”. Washing has also been shown

to be effective at removing microorganisms off the skin of produce (Parnell & Harris, 2003).

Parnell and Harris showed, in the lab, that a 3.2 log reduction of Salmonella on apples can be

achieved through rubbing and rinsing with 200 mL of water for 5 seconds in combination with

drying using a sterile paper towel. They also demonstrated that washing with a 5% vinegar or

200 µg/mL chlorine solution were better at reducing Salmonella than water with reductions

ranging from 5.2 – 6.2 logs (Parnell & Harris, 2003). Another study also showed that chlorine

bleach wash was more effective than running water at reducing Salmonella as well as E.coli

O157:H7 (Fishburn, Tang, & Frank, 2012). Fishburn and colleagues (2012) showed that when

washing lettuce under running water, log reductions of 1.58 and 1.69 can be observed for

Salmonella and E.coli O157:H7 respectively while chlorine bleach reduced 2.05 and 2.34 logs.

In commercial testing, it was shown that washing only resulted in a 1 – 2 log reduction of E.coli

(Murray et al., 2017, Barrera, 2012).

In-kitchen techniques and the application of heat (cooking and boiling) have also shown to be

effective in reducing pesticide residues (Bajwa & Sandhu, 2014). It was also reported by Sharma

and his colleagues in 2001, peeling in combination with cooking methods (boiling, steaming,

etc.) to be the most effective method of removing the pesticide mancozeb from the surface of

apples. They showed that washing alone reduced 20-52% of residues and when combined with

24

cooking led to 53-73% reduction (I. D. Sharma, Patyal, Nath, & Joshi, 2001). Cooking alone has

been shown to not completely remove all pesticides in tea leaves and crops such as spinach,

strawberries (made into jam), and oranges (Nagayama, 1996). Washing was also shown to be

ineffective at reducing levels of CPY to below the maximum residue limit (MRL) (Nagesh &

Verma, 1997). Lee and his colleagues also showed that approximately 30% of CPY residue

remained on rice after cooking (Lee et al., 1991). Singh et al. (2017) conducted heat treatments

such as boiling, pasteurization, and sterilization on CPY and found that after boiling for 5

minutes, 44% of CPY was degraded whereas sterilization at 121 °C for 15 minutes degraded

68% CPY (S. Singh, Krishnaiah, Rao, Pushpa, & Reddy, 2017). One possible explanation for

CPY not being fully degraded may be because CPY decomposes at 160 °C, above the boiling

point of water. These results suggests that heat may be a significant factor in CPY reduction.

1.6.1 Ultraviolet Radiation

The use of ultraviolet (UV) light has gained popularity over the years for food decontamination

because it is cost effective, easy to use and monitor, and residue-free as no chemicals are

required (Keklik, Krishnamurthy, & Demirci, 2012; National Environmental Services Center

[NESC], 2000). The UV light spectrum ranges from 100-400 nm and can be generated by low or

medium pressure mercury lamps (Keklik et al., 2012). This spectrum is categorized into four

sections: UV-A (315-400 nm), UV-B (280-315 nm), UV-C (200-280 nm) and Vacuum UV (100-

200 nm) (Figure 8). The region of interest is UV-C light at 254 nm which has been shown to be

the most effective at inactivating microorganisms (Keklik et al., 2012). UV-C light can induce a

germicidal effect on microorganisms through the absorption of UV light by living cells which

damages DNA (Keklik et al., 2012). Due to this damage, bacterial cells are incapable of

replicating DNA thus causing the cell to become inactivated (Keklik et al., 2012).

25

Low pressure mercury (LPM) lamps are capable of emitting this wavelength of 254 nm

monochromatically whereas medium pressure mercury (MPM) lamps emit polychromatic light at

wavelengths between 185-600 nm (Bohrerova, Shemer, Lantis, Impellitteri, & Linden, 2008).

These lamps operate by using electricity to excite mercury vapor within a silica or quartz tube

which then generates UV radiation when the mercury vapor returns to a lower energy level

(Bohrerova et al., 2008; Keklik et al., 2012). Exact wavelengths emitted depend on temperature

as well as the vapor pressure within the tube which is why LPM lamps produce a more

monochromatic wavelength compared to MPM lamps (Keklik et al., 2012). Low pressure lamps

have an operating temperature between 30 - 50 °C with vapor pressure between 0.1-10 Pa

whereas medium pressure lamps operate at temperatures between 600 – 900 °C with a vapor

pressure from 50 – 300 kPa (Keklik et al., 2012).

Figure 8. The UV light spectrum of the electromagnetic spectrum. Modified from Evoqua Water

Technologies, LLC. https://www.evoqua.com/en/brands/ETS_UV/Pages/how-ets-uv-

works.aspx

https://www.evoqua.com/en/brands/ETS_UV/Pages/how-ets-uv-works.aspx

https://www.evoqua.com/en/brands/ETS_UV/Pages/how-ets-uv-works.aspx

26

1.6.2 Ozone

The use of ozone for drinking water disinfection and oxidation has been around since the early

1900s in Europe (Langlais, Reckhow, & R. Brink, 1991). Nowadays it is not only employed in

water treatment but also in the food industry for increasing produce shelf life and ensuring

sanitation of surfaces (O'Donnell, Brijesh kumar, Cullen, & G. Rice, 2012; Wang, Wang, Wang,

Li, & Wu, 2019). It has been shown that ozone treatment is capable of reducing cellular

respiration and ethylene production which delays fruit ripening hence prolonging shelf life

(Wang et al., 2019). For ozone to be generated, a diatomic oxygen molecule needs to be broken

to create free radical oxygen which reacts with another diatomic oxygen thus forming a triatomic

ozone molecule (O'Donnell et al., 2012). There are multiple ways to generate ozone, including

the use of ultraviolet light at 188 nm which is capable of breaking the diatomic oxygen bond

(O'Donnell et al., 2012). Another method to produce ozone is corona discharge, in which

oxygen-containing gas is passed between two electrodes (Figure 9). These electrodes have a

voltage applied to them which produces electrons that dissociate the oxygen molecules to form

ozone (O'Donnell et al., 2012). It is estimated that if air is passed through the electrodes, only 1-

3% ozone is produced but if high-purity oxygen is passed through, production can be as high as

up to 16% (O'Donnell et al., 2012). In a contained environment, ozone concentration will reach

equilibrium with its surroundings when the generation and degradation rate is the same. Once

this point is reached, ozone concentrations cannot be increased any further (O'Donnell et al.,

2012).

27

Figure 9. Schematic of ozone production using a corona discharge ozone generator. Modified from

Oxidation Technologies, LLC. https://www.oxidationtech.com/ozone/ozone-

production/corona-discharge.html

Ozone has multiple uses in the food industry including plant sanitation, pest reduction,

packaging material disinfection as well as direct contact with food products to reduce

microorganisms that negatively impact food quality and safety (Jegadeeshwar, Shankar, Kumar,

& thottiam Vasudevan, 2017). In 2001, the FDA recognized ozone as an antimicrobial food

additive and labelled it as GRAS (O'Donnell et al., 2012). The FDA states that ozone can be used

as an additive in the “treatment, storage, and processing of foods” and currently has no limits set

(Regulations, 2018). However, the FDA also states that any devices that generate ozone at a level

over 0.05 ppm or accumulate ozone at a level over 0.05 ppm will be considered “adulterated

and/or mislabelled” in animal products (FDA, 2018). This is because ozone can harm human

health. It is capable of causing damage to the lungs, chest pain, coughing, shortness of breath and

even throat irritations (USEPA, n.d). The National Institute of Occupational Safety and Health

(NIOSH) recommends that ozone concentrations should not exceed 0.1 ppm at any time and the

Occupational Safety and Health Administration (OSHA) states that workers should not be

https://www.oxidationtech.com/ozone/ozone-production/corona-discharge.html

https://www.oxidationtech.com/ozone/ozone-production/corona-discharge.html

28

exposed to an average concentration of more than 0.1 ppm for 8 hours (USEPA, n.d). Ozone

must also be able to produce a minimum 5 log reduction for microorganisms commonly found in

juices in order to comply with 21 CFR 101.17(g)(7) (FDA, 1977). Ozone can be applied in either

an aqueous or gaseous form on produce to reduce the amount of pathogenic and spoilage

microorganisms by direct oxidation (O'Donnell et al., 2012). Ozone disperses rapidly and

decomposes into oxygen which leaves zero residues on food products (O'Donnell et al., 2012).

Since ozone does not leave residues on products and surfaces it has grown in popularity in its use

in the food industry (Jegadeeshwar et al., 2017). However, like UV treatments, ozone can

degrade pesticides into more dangerous by-products than its parent compound such as the

formation of oxons (Wang et al., 2019).

In the fruit and vegetable industry, ozone is generally used to inactivate pathogenic and spoilage-

causing microorganisms as well as to reduce the amount of post-harvest pesticide and chemical

residues (O'Donnell et al., 2012). Ozone is more effective when added to water due to increased

contact between the surface being decontaminated leading to the formation of hydroxyl radicals

which have a higher reaction rate and oxidizing power than ozone (Krishnan, Rawindran,

Sinnathambi, & Lim, 2017). Ozone is capable of decomposing into hydroxyl radicals faster in

water than air due to having a shorter half-life in water (LennTech, 2019). When in 20°C air,

ozone has a half-life of 3 days whereas in 20°C water (pH = 7), the half-life is 20 minutes

(LennTech, 2019). Achen and Yousef (2001) showed that apples treated with ozone being added

during water washing inactivated Escherichia coli O157:H7 better than apples placed in pre-

ozonated water with log reductions of 3.7 and 2.6, respectively. The time needed to generate

hydroxyl radicals can also be shortened through increased temperature, higher pH, decreased

concentrations of organic material, and increased dose of UV light (LennTech, 2019).

29

Maximizing the formation of hydroxyl radicals through optimizing the listed factors is known as

an advanced oxidation process (Abramovic et al., 2010; LennTech, 2019).

1.7 Advanced Oxidation Process

A potential method to decrease the public’s exposure to CPY is the use of advanced oxidation

processes (AOPs) combining UV-C light, hydrogen peroxide (H2O2), and ozone to degrade

various organic contaminants (Abramovic et al., 2010; Femia et al., 2013; Marican & Durán-

Lara, 2018). AOP has long been used in the treatment of wastewater (Andreozzi, Caprio, Insola,

& Marotta, 1999; Gottschalk, Saupe, & Libra, 2010; Oppenländer, 2003) and has been shown to

be effective in pesticide residue removal (Table 6) (Abramovic et al., 2010; Ikehata & Gamal El-

Din, 2005).

Table 6. Degradation of pesticides using AOPs and individual processes.

Target Pesticide Treatment Degradation Reference

UV/H2O2 exposure

for 120 minutes. 97% Degradation

(Abramovic et al.,

2010) Thiacloprid

UV exposure for 120

minutes No significant

degradation

H2O2 exposure for

120 minutes

Chlorpyrifos UV/H2O2 exposure

for 20 minutes

93% Degradation (14

mg/L) (Femia et al., 2013)

Chlorpyrifos UV exposure for 300

minutes

100% Degradation (5

mg/L) (Muhamad, 2010)

Chlorpyrifos Ozone exposure No significant

degradation

(Ikehata & Gamal El-

Din, 2005)

30

Target Pesticide Treatment Degradation Reference

Chlorpyrifos

Lychee fruit –

Gaseous ozone

exposure

45% Degradation (Whangchai,

Uthaibutra,

Phiyanalinmat,

Pengphol, & Nomura,

2011) Chlorpyrifos

Lychee fruit –

Aqueous ozone

exposure

10% Degradation

Chlorpyrifos

Apples – Aqueous

ozone exposure for 15

minute

69% Degradation

(0.36 µg/g)

(Swami et al., 2016) Apples – Aqueous

ozone exposure for 30

minute

95% Degradation

(0.06 µg/g)

AOPs are chemical treatments that produce hydroxyl radicals (·OH) in copious amounts to

oxidize organic and inorganic material in water and wastewater (Abramovic et al., 2010;

Andreozzi et al., 1999; Femia et al., 2013; Glaze, Kang, & H. Chapin, 1987; Marican & Durán-

Lara, 2018). Hydroxyl radicals are extremely reactive with little selectivity and a short life span

of less than a second (Deng & Zhao, 2015). According to Parsons, the oxidation potential of

hydroxyl radicals is +2.80 volts (V) which is almost as high as fluorine with a V of +3.06 (Table

7). The rate of oxidation is dependent on a number of factors including the concentrations of

radicals, oxygen, and pollutants (Parsons, 2004). One of the main goals when utilizing an AOP

is to elevate the production of hydroxyl radicals to increase oxidation of organic and inorganic

micropollutants (Katsoyiannis, Canonica, & von Gunten, 2011). It should be noted however that

due to the reactivity of hydroxyl radicals, a large number of reactions can occur and it would be

difficult to predict every product generated through oxidation (Parsons, 2004). Also, due to

hydroxyl reactivity, an equilibrium will be reached between the generation and dissipation of

hydroxyl radicals (Parsons, 2004).

31

Table 7. Oxidation potential of reactive species. Adapted from Parsons (2004).

When AOPs were first introduced for industrial purposes, they were mainly used in the treatment

of wastewater and were later utilized in the 1980s in the treatment of potable water (Deng &

Zhao, 2015). These early uses of AOP technologies used combinations of ozone, hydrogen

peroxide, and ultraviolet light to generate hydroxyl radicals (Glaze et al., 1987). Ozone and

hydrogen peroxide are the most commonly used chemicals to generate hydroxyl radicals with

ultraviolet light acting as a catalyst to start the reaction (Abramovic et al., 2010). Another early

use of AOPs was in the treatment of wastewater due to its strong oxidation potential to remove

both organic and inorganic pollutants such as benzene and iron (II) (Deng & Zhao, 2015).

Nowadays, AOP technologies are used to treat a number of different contaminants and wastes

(Table 8) (Vogelpohl, 2003). AOPs are useful in treating wastewater due to their ability to

reduce pathogens and pesticide concentrations (Abramovic et al., 2010; E. L. Thomas, Milligan,

Joyner, & Jefferson, 1994).

32

Table 8. Contamination and waste products reduced by AOP technology treatments. Modified from

Vogelpohl (2003).

Amino acids Parasites

Antibiotics Pesticide wastewater

Arsenic pesticides

Coliforms Phenolic wastewater

Cryptosprodium Natural organic matter

Escherichia coli Municipal wastewater

Hospital wastewater Taste and odour causing compounds

Insecticide Trinitrotoluene (TNT)

1.7.1 UV-based Advanced Oxidation Process

When using UV light by itself to destroy contaminants, photons from UV light must be able to

be absorbed and undergo degradation from their excited state (Parsons, 2004). This limits the

application of direct UV photolysis in industry. In an AOP system however, UV light is

generally used to initiate the production of hydroxyl radicals through decomposing ozone and

hydrogen peroxide (Abramovic et al., 2010). Hydrogen peroxide and oxygen radicals are

produced when UV light reacts with ozone and it is then cleaved into two hydroxyl radicals

(equation 1 and 2) (Deng & Zhao, 2015). This is possible due to hydrogen peroxide being able to

absorb the UV wavelength of 254 nm (Figure 10) (Gottschalk et al., 2010).

O3 + H2O + hv -> H2O2 + O2 (Equation 1)

H2O2 + hv-> 2OH (Equation 2)

33

Figure 10. Absorbance spectra of hydrogen peroxide at different wavelengths in Angstroms (254

nm = 2540 Angstroms). Modified from USP technologies. http://www.h2o2.com/technical-

library/physical-chemical-properties/radiation-

properties/default.aspx?pid=65&name=Ultraviolet-Absorption-Spectrum

One study using thiacloprid, a neonicotinoid insecticide, found that this pesticide can be

photodegraded when exposed to UV radiation between 200 – 280 nm with its maximum

absorbance wavelength being 242 nm (Abramovic et al., 2010). This study also showed that

when exposed to higher wavelengths, outside of the absorbance range (200 – 280 nm), pesticide

degradation was minimal. When UV is combined with hydrogen peroxide however, a synergistic

effect is observed due to the production of hydroxyl radicals (Abramovic et al., 2010;

Krzemińska, Neczaj, & Borowski, 2015).

http://www.h2o2.com/technical-library/physical-chemical-properties/radiation-properties/default.aspx?pid=65&name=Ultraviolet-Absorption-Spectrum



34

It has been shown that CPY can be degraded into its oxon form when exposed to UV-C light

(Figure 4) (Savić et al., 2019). Other OPs have been observed to degrade under UV-C light

(Bustos, Cruz-Alcalde, Iriel, Fernández Cirelli, & Sans, 2019).

1.7.2 Ozone-based Advanced Oxidation Process

Since ozone degrades into hydroxyl radicals, it is necessary for AOPs to optimize the factors

listed in Section 1.6.2 affecting ozone degradation (Gottschalk et al., 2010). These processes can

involve the use of hydrogen peroxide and/or UV light as stated previously to increase ozone

degradation and hydroxyl radical formation (Deng & Zhao, 2015; EPA, 1999; O'Donnell et al.,

2012).

When ozone reacts with H2O2, which is commonly referred to as the peroxone process, hydroxyl

radicals are produced along with oxygen (Merényi, Lind, Naumov, & Sonntag, 2010). By adding

hydrogen peroxide into ozonated water, the decomposition of ozone is increased, in effect

elevating the concentration of hydroxyl radicals (Deng & Zhao, 2015; EPA, 1999).

H2O2 -> HO2- + H+ (Equation 3)

HO2- + O3 -> OH. + O2

- + O2 (Equation 4)

This process of generating hydroxyl radicals was optimized by Duguet et al. (1985) who

concluded that hydrogen peroxide should be added in a controlled amount after the oxidation of

highly reactive substances by ozone (P. Duguet, Brodard, Dussert, & Mallevialle, 1985). This

mechanism is driven by hydrogen peroxide decomposing into hydroperoxide anions which react

with ozone to produce hydroxyl radicals (Deng & Zhao, 2015). Due to improved oxidation

through elevated concentrations of hydroxyl radicals, the peroxone process is considered

superior to ozone on its own when degrading organic material and it has been shown to be able

35

to degrade difficult-to-treat organics such as geosmin, an organic compound with an earthy taste

(EPA, 1999). However, peroxone is less effective at oxidizing inorganic material such as iron

and manganese compared to ozone.

Peroxone is generally used for oxidizing taste and odor compounds, synthetic organic

compounds, pathogens and pesticide degradation for examples, geosmin, 1,1 – dichloropropene,

poliovirus, and atrazine (EPA, 1999). When dealing with pathogens and viruses in a peroxone

system, ozone has been shown to be the main cause for reductions due to the combination of

direct ozone contact and reactivity of hydroxyl radicals in the oxidation of pathogens (EPA,

1999).

When exposed to UV-C light, ozone undergoes a photolysis reaction (Figure 11) and hydroxyl

radicals are formed. This synergistic reaction can help reduce dissolved organic matter better

than UV light or ozone alone (USEPA, n.d.-b). It is estimated that the initial ozonation rate of

organic substances can be increased up to 10,000 times when combined with UV irradiation

which can help speed up the removal of total organic matter (USEPA, n.d.-b).

Figure 11. Photolysis reaction of ozone when exposed to UV-C light at 254 nm. One oxygen

molecule and hydroxyl radical is produced. Modified from Spartan Environmental Technologies,

LLC.

36

1.8 Response Surface Methodology

When determining whether a new technology such as AOP is capable of degrading pesticides, it

is necessary to observe which operational factors are significant and if they can be optimized to

achieve a maximal degradation. This study aimed to determine the significance of the following

factors: temperature of hydrogen peroxide, concentration of hydrogen peroxide, ozone

concentration, and UV dose. One way of determining the significance of these factors and their

optimized settings for pesticide degradation is by using response surface methodology (RSM), a

multivariable statistic technique that utilizes a set of mathematical and statistical techniques to

explore the relationship between multiple variables and their response variables (Bezerra,

Santelli, Oliveira, Villar, & Escaleira, 2008). In this study, the multiple variables (temperature of

hydrogen peroxide, concentration of hydrogen peroxide, ozone concentration, and UV dose) are

set at different parameters (high, medium, and low) and the response variable is the amount of

CPY degraded in micrograms (µg). By comparing these factors at different settings, RSM can

determine their significance in CPY degradation and predict an optimized setting for each factor

to achieve maximal response (Bezerra et al., 2008).

There are multiple types of RSM design, with central composite design (CCD) and Box-Behnkin

design (BBD) being the most widely used (Pennsylvania State University, 2018). CCD consists

of multiple parameter settings; a central point representing the medium setting of each factor,

several axial (or “star”) points (Figure 12) which represent the highest or lowest settings, and a

factorial design, either full or fractional, that observes several combinations of different factor

settings (Bezerra et al., 2008). Full factorial design experiments consists of multiple factors,

usually two or more, occurring in every possible combination with one another (Bezerra et al.,

2008). Fractional factorial designs are similar to full factorial designs but only have a select few

37

combinations to test (Bezerra et al., 2008). BBDs are similar to CCDs but avoid testing star

points and consists of fewer experiments which makes it more time efficient and less costly

(Bezerra et al., 2008). In analytical chemistry, CCDs are used far more often than BBD as they

provide more information and tests for star points. By testing for star points, a more complete

data set is given which will provide more accurate predictive values (NIST, 2013).

Figure 12. Response surface methodology design with center points and star points. Modified from

Stat-Ease, Inc.

The use of RSM for determining the optimal treatment for chemical degradation using AOPs has

been shown to be effective (Ahmadi, Mohammadi, Igwegbe, Rahdar, & Banach, 2018; Behin &

Farhadian, 2017). Ahmadi and colleagues found that reactive blue 19 dye removal using AOP

can be optimized through using RSM and the experimental degradation values were close to the

predicted values suggested by RSM. Behin and Farhadian (2017) found that the pesticide

trifluralin can be fully degraded, into chemically inactive byproducts, by using optimal treatment

values of: ozone flow rate, UV light exposure time, and hydrogen peroxide solution pH. These

values were determined through a full factorial central composite design (Behin & Farhadian,

2017).

38

Hypothesis and Objectives

It is expected that an optimized AOP treatment that applies ozone, UV radiation and hydrogen

peroxide in combination will degrade the pesticide CPY and inactivate microorganisms on the

surface of apples without leaving toxic by-product residues such as CPY-O. The objectives of

this study were to:

1.) Develop an analytical method to analyze CPY on apple skin.

2.) Develop a recovery method of CPY from apple skins that have been treated or not treated

in the AOP reactor.

3.) Use RSM to model the degradation of CPY by AOP treatments with the following

factors: Ozone concentration, UV dose, temperature of hydrogen peroxide, and

concentration of hydrogen peroxide.

4.) Validate the model, determine which factors are significant, and optimize the AOP

factors for maximal CPY degradation.

5.) Determine whether AOP treatment will degrade CPY into its more toxic by-product

chlorpyrifos-oxon.

6.) Determine if the optimized parameters can inactivate the microorganisms Escherichia

coli O157:H7 and Aspergillus niger.

39

2. Materials and Methods

2.1 Materials

Chlorpyrifos analytical standards (>99% purity, PESTANAL®), hydrogen peroxide solution

(30% v/v), trifluoroacetic acid (>99% purity) and nalidixic acid sodium salt were obtained from

Sigma Aldrich (St. Louis, MO, USA). Crispin apples were provided by Moyer’s Apple Products

Ltd. (Beamville, ON, Canada). Acetonitrile (Optima™, HPLC grade) was purchased from

Thermo Fisher Scientific (Waltham, MA, USA). Chlorpyrifos-oxon standards (>99% purity)

were purchased from Toronto Research Chemicals (North York, ON, Canada). Trypic soy broth

(TSB) and potato dextrose agar (PDA) were purchased from Oxoid (Hampshire, UK).

MacConkey agar with sorbitol, cefixime, and tellurite (CT-SMac) was purchased from Thermo-

Fisher Scientific.

2.2 Advanced Oxidation Process Reactor

The advanced oxidative process (AOP) reactor (Clean Works Corporation, Beamsville, ON, CA)

(Figure 13) was constructed from stainless steel with frame dimensions of 122 cm × 38 cm × 20

cm (length × width × height). The reactor was fitted with a lightbox (61 × 35 × 15 cm) housing 4

× 25 W UV-C lamps (60 cm length) emitting at 254 nm (Sani-Ray, Hauppauge, NY, USA). The

UV intensity was varied through altering the distance of the lamps from the samples from 10 cm

– 16 cm). A radiometer (Trojan Technologies Inc, London, ON, CA) was placed in the middle of

reactor and used to measure the UV-intensity with treatment times being used to deliver a

defined UV-dose. The initial UV intensity (start of AOP treatment) and final UV intensity (end

of AOP treatment) were recorded and the average was used along with treatment time to

calculate the total UV dose. Prior to introducing the apple samples into the UV reactor, the

40

hydrogen peroxide (0.2 mL) of defined concentration (1-6% v/v) and temperature (23 – 70°C)

was dispensed onto the sample.

Figure 13. Schematic diagram (A) and photograph (B) of the advanced oxidation process reactor

used for chlorpyrifos degradation and microbial inactivation.

A

B

Light box housing 4 x 254 nm UV lamps

Height adjustable rack

Samples on a tray

10 or 16 cm

Entrance

Distance between light box and sample surface

61 cm

122 cm

Radiometer

41


RSM was used in the experimental design to determine the effectiveness of AOP in degrading

the pesticide CPY. Design Expert® Software 11.1.0 (Stat-Ease Inc, Minneapolis, MN, USA) was

used to produce a fractional factorial central composite design investigating the effect of UV

dose (kJ/m2), temperature of hydrogen peroxide (°C), and concentration of hydrogen peroxide

(% v/v) on the degradation of CPY (Table 9). The experimental design of this study included a

total of 20 runs with six replicate center points (Table 10). Runs were performed in triplicate

involving the three previously mentioned factors. The response variable can be expressed as a

function (Eqn. 5) of the independent process variables according to the following response

surface quadratic model.

3 3 2 32

0

1 1 1 1

i i ii ij i j

i i i j i

Y a a x a x a x x

(Equation 5)

where Y is the predicted response, xi and xj are factors influencing the predicted response, αi, αii,

and αij are the coefficients of the linear, quadratic, and interaction terms respectively. Lastly, ε is

a random error.

Regression analysis for the quadratic equation model was determined using Design Expert®

11.1.0 software. First, the model fit was tested to determine the quality of the model using the

coefficient of determination (R2). ANOVA analysis was conducted to determine whether the test

variables listed are significant or not. This was done using F-test and P-values with 95%

confidence level.

42

Table 9. RSM trials (20) using various test parameters of UV-C dose (kJ/m2), temperature (°C) and

concentration (%) of hydrogen peroxide.

Trial UV dose

(kJ/m2)(X1)

Temperature of

H2O2 (°C) (X2) H2O2 (%) (X3)

1 34.8 46.5 3.5

2 22.5 32.5 5.0

3 54.0 32.5 5.0

4 37.0 46.5 1.0

5 22.4 60.5 5.0

6 38.3 70.0 3.5

7 39.4 46.5 3.5

8 39.4 46.5 3.5

9 11.7 46.5 3.5

10 38.9 46.5 3.5

11 56.1 60.5 2.0

12 50.3 32.5 2.0

13 37.9 46.5 6.0

14 21.8 32.5 2.0

15 39.3 46.5 3.5

16 37.2 46.5 3.5

17 62.3 46.5 3.5

18 38.5 23.0 3.5

19 22.3 60.5 2.0

20 53.3 60.5 5.0

43

Table 10: Six center point replicate trials determining the replicability and precision of the

experimental method. Arranged from lowest to highest reduction corrected with the average

(n=177) positive control recovery of 89.4%. The standard deviation between reductions was 3.6 µg.

Trial #

Temperature of

H2O2 (˚C)

Actual

Temperature

(˚C)

Percent H2O2

(% v/v)

UV-C

Dose

(kJ/m2)

1 46.5 45 3.5 34.8

15 46.5 46 3.5 39.3

8 46.5 46 3.5 39.4

7 46.5 47 3.5 39.4

10 46.5 45 3.5 38.9

16 46.5 47 3.5 37.2

2.4 AOP Treatment of Apple skins Spiked with Chlorpyrifos

Crispin apples were stored in a cold storage room at 4°C until required. Apples were taken out of

cold storage when needed then washed under running municipal tap water followed with towel

drying. The washed apples were then inspected for damage including spots, bumps, bruises and

discolouration. Apples were then peeled vertically from the bottom to the top with a vegetable

peeler. Peeled apple skins were then cut into two 2.5 cm × 3 cm pieces per fruit. The peel

sections were then placed on individually labelled aluminum weigh dishes. Aliquots (0.2 mL) of

a working 1000 µg/mL CPY stock solution, prepared by dissolving 10 mg of CPY standard into

10 mL of acetonitrile, were introduced onto the surface of the apple peel samples and allowed to

44

dry at room temperature (approximately 25 °C) in the dark until dried. Thus, 200 µg of CPY was

deposited onto each piece of apple skin. One sample was treated with the AOP and the other was

left in the dark, untreated (positive control). Each treatment involved three pieces of apple skin

containing CPY that were treated with the AOP, three pieces of apple skin containing CPY that

were not treated with the AOP (positive controls), and two negative controls. One piece of apple

skin that did not contain CPY and was treated with the AOP (negative control 1) and one piece

of apple skin that did not contain CPY and was not treated with the AOP (negative control 2).

The spiked samples along with a negative control were lined up lengthwise on a tray with

dimensions 31 x 23.5 cm (length x width) and set 5 cm apart from one another lengthwise.

Treatment with 1 of the 20 listed AOP treatment parameters were performed (Table 9), the tray

was introduced into the reactor through its entrance and placed on top of an adjustable rack set so

that samples were 16 cm away from the UV light source (Figure 13A).

After treatment, the tray containing samples was removed through the reactor entrance and all

apple skins (treated, positive controls, and negative controls) were then placed inside

individually labelled 50-mL centrifuge tubes. A 20-mL aliquot of acetonitrile was used to rinse

the aluminum dish and was then placed inside the centrifuge tube containing the corresponding

apple skin. Assuming no degradation, a final concentration of 10 µg/mL would be present in the

rinsate. All centrifuge tubes with an apple skin and rinsate were sonicated (Branson 2510,

Crystal Electronics, Newmarket, ON, CA) for 20 minutes at 40 kHz. Due to heat being generated

during sonication (up to 30C), water was replaced with cool tap water to keep sonicater water

temperature constant at approximately 23C ± 2C. After sonication, 1 mL of rinsate was

removed from each tube and placed into individually labelled high performance liquid

chromatography (HPLC) vials. This process was repeated two more times to produce a triplicate

45

set of samples for each treatment (n = 3). A total of 24 samples were produced per trial (9 spiked

and treated, 9 spiked and untreated (positive control), and 6 not spiked/untreated (negative

controls). Sample vials were stored at 4 C for a maximum period of 24 h before quantification

using HPLC analysis. It should be noted in the current study, the spiked concentration of CPY,

10 µg/mL is 1000 times higher than the MRL of CPY on apples in Canada (0.01 µg/mL) (Health

Canada, 2008).

2.5 Validation Trial

Once RSM was completed, results were analyzed using Design Expert® Software. A validation

trial was suggested by the software in order to confirm the accuracy and validity of the model.

The given parameters for this trial were as follows: 69 kJ/m2 UV dose and 1.22% v/v H2O2 at

66°C. The predicted reduction for this trial was 92 µg (46% reduction of spiked chlorpyrifos).

Samples were prepared and treated using the methodology as described above in Section 2.4.

2.6 UV Only Trial

Treatments with UV light only were conducted using the same sample preparation and HPLC

analysis methods describe above in Section 2.4. Five trials at different UV doses (11, 23, 41, 53,

and 66 kJ/m2) were tested. After these trials were completed, the UV lights were brought closer

to the samples to be treated. By reducing the distance between UV lights and samples, UV dose

per minute would be increased thus reducing treatment time. The trial 53 kJ/m2 was re-conducted

at this higher UV dose to compare the difference in treatment times between same UV doses.

The positive controls between the higher and lower UV intensities were averaged and were used

as the positive control corrections.

46

2.7 Ozone Treatment of Apple Skins Spiked with Chlorpyrifos

Crispin apples were prepared the same way as described above in Section 2.4. Nine apple skin

sections were spiked with 0.2 mL of 1000 µg/mL CPY solution (200 µg) and dried in the dark

before treatment. Twelve apple skins were left blank (no spike) to act as a negative control.

Treatment occurred in a 30-L plastic container containing 2 holes, 1 for tubing connected to an

ozone monitor (linear dynamic range of 0-100 mg/L) (Model 106-L, 2B Technologies, Colorado,

USA) the other for tubing connected to a 2 g/h corona discharge ozone generator provided by Dr.

Thomas Graham (University of Guelph, School of Environmental Science) (Figure 14). Three

spiked skins along with 1 negative control was placed into the container with the lid closed.

These samples were treated with ozone gas for 30 minutes then placed into 50-mL centrifuge

tubes and filled with 20 mL of acetonitrile (ACN). These tubes were then sonicated (Branson

2510, Crystal Electronics, Newmarket, ON, CA) at 40 Hz for 20 minutes. After sonication, 1 mL

of sample was removed from the tube and placed into a 2-mL HPLC vial. This was repeated for

each sample and the extracts were then analyzed using an Agilent 1100 Series HPLC (Agilent,

Fair Lawn, CA, USA).

47

Figure 14. Schematic (A) and photograph (B) of ozone chamber treatment of CPY spiked apple

skins.

A

A

B

48

2.8 HPLC Analysis

High performance liquid chromatography was performed with an Agilent 1100 Series HPLC

instrument equipped with a photodiode array detector (Agilent, Fair Lawn, CA, USA). An

Agilent Zorbax Eclipse Plus C18 analytical column (4.6 x 150 mm, 5.0 µm) (Agilent, Santa

Clara, CA, USA) equipped with an Agilent Zorbax Eclipse Plus C18 analytical guard column

(4.6 x 12.5 mm, 5- micron, Agilent, Santa Clara, CA, USA) was used as a stationary phase with

a flow rate of 1.0 mL/min and the column temperature was uncontrolled with an average room

temperature of 23°C. The injection volume for samples and standards was 40 µL. The mobile

phase was in isocratic mode with a composition of 85:15 (%, v/v) acetonitrile/ water with 0.1%

trifluoroacetic acid (Millipore Sigma, St Louis, MO, USA). The detection wavelength was set at

290 nm. The concentration of CPY samples were quantified by the use of an x-point calibration

curve. Calibration standards for CPY were prepared in acetonitrile at concentrations of: 2.5, 5,

10, 20, and 40 µg/mL. The method detection limit (MDL) was determined by spiking apple skin

pieces with 200 µg of CPY (resultant concentration of rinsate 10 µg/mL) and extracting the CPY

to get a measure of the methods accuracy and precision (n = 7) (Shrivastava & Gupta, 2011). The

average recovery was 81.1%. The LOD and LOQ were 0.5 µg/mL and 1.7 µg/mL, respectively.

Replicate trials from the RSM table conducted on separate days were also compared to determine

the replicability of trials.

2.9 Determination of Chlorpyrifos-Oxon Production by AOP Treatment using Q-

ToF analysis

Apple skin samples were prepared as per Section 2.4 and were treated under the optimized

conditions described in Section 2.5. Untreated spiked samples and treated samples prepared using

the optimal treatment along with analytical standards of CPY, CPY-O and TCP were submitted to

49

the Mass Spectrometry Facility of the Advanced Analysis Centre at the University of Guelph for

Quadrupole Time of Flight (Q-ToF) mass spectrometry analysis to determine the presence or

absence of these compounds.

Liquid chromatography–mass spectrometry analyses was performed on an Agilent 1200 HPLC

liquid chromatograph (Agilent, Fair Lawn, CA, USA) interfaced with an Agilent UHD 6530 Q-

Tof mass spectrometer (Agilent, Fair Lawn, CA, USA). An Agilent Poroshell 120 C18 column (

EC-C18 50 mm x 3.0 mm 2.7 µm) was used for chromatographic separation with the following

solvents water with 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B). The mobile

phase gradient was as follows: initial conditions were 10% B hold for 1 min then increasing to

100% B in 29 min followed by column wash at 100% B for 5 min and 20 min re-equilibration.

The flow rate was maintained at 0.4 mL/min and sample injection volume was 10 µL. The mass

spectrometer electrospray capillary voltage was maintained at 4.0 kV and the drying gas

temperature at 250 °C with a flow rate of 8 L/min. Nebulizer pressure was 30 psi and the

fragmentor was set to 160 °C. Nitrogen was used as both nebulizing and drying gas. The mass-to-

charge ratio was scanned across the m/z range of 50-1500 m/z in 4GHz (extended dynamic range)

positive and negative ion modes. The acquisition rate was set at 2 spectra/s. The instrument was

externally calibrated with the ESI TuneMix (Agilent, Santa Clara, CA, USA). Mass spectrometer

control, data acquisition and data analysis were performed with MassHunter® Workstation

software (B.04.00).

The concentration of CPY-O samples were quantified using an x-point calibration curve.

Calibration standards for CPY-O were prepared in acetonitrile at concentrations of 0.01, 0.02,

0.04, 0.08, 0.16, and 0.31 µg/mL. Concentrations of 0.16 and 0.31 µg/mL were removed due to

low accuracy of the result which did not conform to the linearity of the lower concentrations below

50

0.16 µg/mL. The R2 value of the remaining 4 standards was 0.9993 (Figure 15) and the LOD was

below 0.01 µg/mL.

Figure 15. Calibration curve of chlorpyrifos-oxon standards ranging in concentrations from 0.01 –

0.31 µg/mL. Concentrations of 0.16 and 0.31 µg/mL were removed due to low accuracy. R2 value =

0.9993.

2.10 Inactivation of Escherichia coli O157:H7 and Aspergillus niger

Escherichia coli O157:H7 PH1 (stx2, eae, nalidixic acid resistant) originally isolated from a

clinical case was provided by the Public Health Agency of Canada (Guelph. Ontario, Canada).

Escherichia coli O157:H7 was cultivated in TSB at 37C for 16 h. The cells were harvested by

centrifugation (Sorvall ST8, Thermo Fisher Scientific, Waltham, MA, USA) at 4000 rpm for 10

minutes and resuspended in saline and then optical density at 600 nm was measured and adjusted

51

to 0.2 to obtain approximately 8 log CFU/mL. The suspension was held at 4 C until required for

testing.

Aspergillus niger spores NU320 previously isolated from environmental sources was used in this

study. Aspergillus niger spores were produced by inoculating the mold onto PDA and incubating

for 14 days at 25 C. The spores were harvested by flooding the plate with sterile distilled water

and scraping with a spreader. The spore suspension was then harvested by centrifugation for 15

minutes at 4000 rpm followed by the pellet being resuspended in sterile distilled water prior to

storing at 4 C until required for testing.

Apple skin sections were inoculated with either E. coli O157:H7 or A. niger spores to give a final

cell density of 8 and 7 log CFU/sample, respectively. The inoculated apple samples were then

placed within a biosafety hood and allowed to dry at room temperature for 3.5 h. Three skin

samples were treated with UV and 0.2 mL of 1.2 % v/v hydrogen peroxide (diluted from 30 %

v/v hydrogen peroxide with milli-Q water; Sigma-Aldrich, Oakville, Ontario, Canada), three

other samples were treated with only UV with a dose of 69 kJ/m2 and another three samples were

treated with hydrogen peroxide only (1.2 % v/v). Optimal treatment parameters were used to

treat another three samples (1.2 % hydrogen peroxide heated to 69 °C and UV dose of 69 kJ/m2).

The treated samples and non-treated controls were placed in 20 mL of saline and vortexed for 10

seconds to release microbes from the apple skin samples. A dilution series was prepared in saline

and plated onto agar. Escherichia coli O157:H7 was plated onto TSA supplemented with 50

µg/mL nalidixic acid that was subsequently incubated at 37 C for 24 h. Colonies grown on

plates were then swabbed onto CT-SMac agar and incubated for another 24 h at 37 C to confirm

52

identification of E.coli 0157:H7. Aspergillus niger was enumerated on PDA plates incubated at

25 C for 5 days.

2.11 Experimental plan and statistics

For RSM, trials were conducted with varying parameters of UV dose (kJ/m2), temperature (°C)

and concentration (% v/v) of hydrogen peroxide. Each trial was conducted in triplicates with

three samples per replicate. A total of 9 samples were produced per trial. Six trials were

conducted around the central points with 54 samples produced under the same conditions. For

the additional testing conditions (UV only, ozone fumigation), trials were performed at least

twice. Analysis of variance (ANOVA) was performed on the data using Design Expert®

Software 11.1.0. Statistical significance was determined at P > 0.05. Calculations of means and

standard deviations were performed using Microsoft Excel.

For microbial trials, the log count reduction calculation was performed using Microsoft Excel

2013 and determined by comparing the log transformed CFU/sample of treated and positive

control (inoculated but not treated) samples. Statistical significance between log CFUs or log

count reductions were determined using a Welch’s t-test in Microsoft Excel 2013 at P > 0.05.

3. Results and Discussion

3.1 Optimization of Recovery Method and HPLC Analysis

The method of recovering CPY from apples skins involved several steps in order to achieve a

maximal recovery of ~89%. One step in the recovery method was the use of a sonicator which

utilizes acoustic cavitation to generate bubbles which help in extracting deposited CPY from the

apple skin thus facilitating higher recoveries of CPY. Without sonication, recovery rates of CPY

were less than 80% but when sonication was utilized, recovery rates were consistently greater

53

than 85%. Other studies have shown that ultrasonic solvent extraction (USE) is effective at

improving recoveries of pesticides from produce (Pan, Xia, & Liang, 2008; Ramos, Rial-Otero,

Ramos, & Capelo, 2008). Pan et al. (2008) showed that recoveries of the pesticides

monocrotophos, dimethoate, carbendazim, carbaryl, imidacloprid and simazine could be

optimized to over 90% when sonicated for 35 minutes at 28 kHz. In comparison to this result,

they demonstrated that recovery rates were lower with decreased sonication time, for example,

imidacloprid sonicated for 25 minutes at 28 kHz had a recovery of 71% (Pan et al., 2008). They

also observed that increasing the volume of solvent was positively correlated with pesticide

recovery with 40 mL per gram of sample proving optimal. For example, when comparing

different volumes of ethyl acetate at 25 minutes of sonication with 5 g of pesticide-spiked

spinach sample, a recovery of 40% of monocrotophos was observed using 10 mL of solvent

whereas a recovery of 90% could be seen when using 40 mL (Pan et al., 2008). These factors

were optimized in the current study and it was observed that a sonication time of 20 minutes and

20 mL of solvent (ACN) would support a sufficient and consistent recovery. Higher sonication

times and solvent volume did not increase recovery rates above 85%.

Apple skin constituents had a negligible effect on the HPLC analysis of CPY with no major

interfering peaks. One unknown peak derived from apple skins eluted around 3.9 min while CPY

had an elution time of 2.3 min when using a 1 mL/min flow rate. The CPY peak had a tailing

effect after elution resulting in it partially co-eluting with the peak associated with constituents of

the apple skin. Water and hydrogen peroxide also initially reduced peak resolution with an

elution time around 1.8 min. In order to separate these peaks, the HPLC column was changed

from an initial 5 micron pore size stationary phase to 3.5 micron size. The mobile phase

concentration ratios were also changed from 90:10 ACN/water with 0.1% TFA to 85:15. This

54

allowed for better resolution between peaks with no overlap due to an increase in CPY elution

time to 4.6 min. This longer elution time also removed tailing due to eluting after the unknown

apple skin peak. Because resolution was well established between peaks, there was no need for a

clean-up procedure of extracts which is commonly employed when working with produce

(Harshit, Charmy, & Nrupesh, 2017).


The average recovery (n = 177) of CPY from apple peels by using the applied methodology from

Section 2.4 was 89.4% 3.8%. In comparison, Osmann et al. (2012) had recoveries of CPY

between 87 – 92% from date fruits (n = 10) using GC-MS. Their method of extraction however

used acetone as the extraction solvent rather than acetonitrile (Osman et al., 2014). Whangchai et

al. (2011) also employed GC rather than HPLC and used acetone as the extraction solvent for

removing CPY from lychee fruit but did not include % recovery in their study. Rawn et al.

(2008) extracted captan, a fungicide, from apples and had an average recovery (n = 31) of 81%

using GC-MS as well. Their recovery method may be lower due to involving a clean-up step

which subjected the samples to gel permeation chromatography and then a Florisil column

(Rawn et al., 2008).

The mean recovery was used as a recovery correction factor for AOP treated samples. The

percent degradation of CPY across the various AOP treatments ranged from 28 % to 92 % (Table

11). The extent of CPY degradation was analyzed using Design Expert® 11.1.0 software and

was determined to fit a linear model (Table 13). ANOVA analysis showed the linear terms X1

(UV dose) and X2 (H2O2 temperature) were significant (P < 0.05) in CPY degradation whereas

X3 (H2O2 concentration) was not significant (Table 13).

55

The RSM approach demonstrated that AOP could degrade CPY via a process that was primarily

dependent on the UV-C dose and temperature of hydrogen peroxide. It has been reported that

CPY in aqueous solution is relatively stable to UV-C light with the addition of hydrogen

peroxide or iron (Fe3+) required to support degradation of the pesticide (Gandhi, Lari, Tripathi, &

Kanade, 2016). Further studies have confirmed the finding that UV-C degradation enhances the

degradation of CPY when applied as part of an advanced oxidation process (Gandhi et al., 2016;

Savic et al., 2019; Thind, Kumari, & John, 2018; Utzig et al., 2019). Thind et al. (2018) reported

that hydrogen peroxide within the range of 0.5 – 1.5 % v/v supported a 42 % decrease in CPY

levels but could be increased to 53 % reduction when used in combination with UV-C. A further

increase in CPY degradation up to 74 % was obtained by using a combination of hydrogen

peroxide, titanium dioxide and UV-C therefore suggesting a synergistic effect. Oliveria et al.

(2014) also showed that low concentrations of hydrogen peroxide between concentrations of

0.025 % to 0.2 % had an effect on the degradation of CPY ranging from 49.2% to 73.9%

reduction. In contrast to Oliveria et al. (2014), this study used concentrations ranging from 1.0 –

6.0%. Another study concluded that 0.16 mg of chlorpyrifos was degraded per mg of H2O2

consumed and this relationship was observed up to a maximum concentration 0.09% H2O2

(Femia et al., 2013). Utzig et al (2019) reported the degradation of CPY in acetonitrile with UV

supporting complete degradation of 200 µg/L within 120 min treatment and the process was

accelerated to 30 minutes with the inclusion of 12 mg/L hydrogen peroxide. The authors

determined that at a UV-C radiation intensity of 0.75 mW/cm2 caused the half- life of CPY

utilizing UV-C radiation alone would be 12.9 min whereas when combined with hydrogen

peroxide, the half-life decreased to 5.8 min.

56

Previous reports on the AOP mediated degradation of CPY have mainly focused on aqueous or

ethanolic solutions. In the current study, where the AOP process was performed on the surface of

apples, it was evident that hydrogen peroxide had a minor role on the overall degradation process

compared to when applied in an aqueous phase. The presence of increasing levels of hydrogen

peroxide in the AOP reduced the degree of CPY degradation by UV-C light. It has been stated

that higher concentrations of hydrogen peroxide may form water and a hydroperoxyl radical

(HO2) which is capable of interfering with chlorpyrifos degradation (Oliveira et al., 2014).

Another issue with using hydrogen peroxide and UV light is that hydrogen peroxide does not

absorb 254 nm light well due to its low molecular extinction coefficient of 19 L/ molcm (Figure

10) (Gottschalk et al., 2010) so OH radical production is low which will require longer UV

exposure times and a surplus of hydrogen peroxide (Gottschalk et al., 2010).

There could be several reasons underlying the differences observed in the degradation of CPY on

the surface of apples compared to in aqueous solution. For example, it is known that the

concentration of hydrogen peroxide has to be balanced with generating sufficient free-radicals to

react with CPY and an excess peroxide concentration can impede CPY degradation (Oliveira et

al., 2014). In the case of aqueous solutions, diffusion of hydrogen peroxide and generation of

hydroxyl radicals can prevent the reaction from being impeded. It is also conceivable that in air

the UV-C transmission to the surface of apples would be greater than in water, especially if

absorbing contaminants are present. Therefore, in aqueous solutions the hydrogen peroxide

would constitute the primary element of the AOP given the absorption of UV-C by the water

constituents. By directly illuminating the surface of apples, the generation of radicals can occur

but CPY degradation would be primarily due to UV-C photon exposure rather than hydrogen

peroxide. Additional possibilities are the aqueous phase forms a relative constant matrix to

57

support the reaction of radicals with CPY. For example, high alkaline pH and presence of Fe can

promote the CPY degradation but which could be less controlled on the apple surface (Behin &

Farhadian, 2017). Regardless of this fact, the results indicate that AOP studies performed in the

aqueous phase do not always translate to treating whole fruit in air.

In the current study the degradation of CPY was positively correlated to temperature which is in

agreement with others (Cengiz, Catal, Erler, & Bilgin, 2015; Oliveira et al., 2014). A study

conducted by Oliveria et al. (2014) investigating the degradation of CPY in an aqueous system

using AOP also showed that an increase in temperature slightly influenced CPY degradation.

They suggested that higher temperatures used in combination with UV radiation supported

higher degradation compared to elevated temperatures used in combination with AOP involving

UV exposure for 8 hours (no dose or intensity given) and hydrogen peroxide (0.1%) (Oliveira et

al., 2014). The same result was observed in this study, with AOP treatments producing a lower

reduction than UV used alone at the same dose. This may be due to hydrogen peroxide

scavenging UV-C radiation, which may be more significant in CPY degradation, to generate

minimal amounts of hydroxyl radicals (Gottschalk et al., 2010). In a different study conducted by

Cengiz et al. (2015), it was observed that the reduction of chlorpyrifos-ethyl could be as high as

0.56 µg/kg when treated for 60 minutes at 90°C whereas a lower heat treatment of 60°C at the

same time produced a reduction of 0.37 µg/kg (Cengiz et al., 2015). The mechanism of thermal

decomposition of CPY involves a series of stepwise elimination reactions that cleaves ethylene

residues to leave 3,5,6,-trichloro-2-pyridinol as the main degradation product (Kennedy &

Mackie, 2018). Although greater temperatures have been shown to increase degradation of CPY,

the temperature sensitivity of fruits would make higher temperatures non relevant in the context

of commercial food processing.

58

Table 11. RSM trials with amount of CPY reduction (µg) observed using various test parameters of

UV-C dose (kJ/m2), temperature (°C) and concentration (%) of hydrogen peroxide. Reduction from

each trial was corrected using the average positive control recovery of 89.4% (n=177).

Trial UV dose

(kJ/m2)(X1)

Temperature of

H2O2 (°C) (X2) H2O2 (%) (X3)

CPY

Reduction

(µg)(Y)

1 34.8 46.5 3.5 50

2 22.5 32.5 5.0 32

3 54.0 32.5 5.0 52

4 37.0 46.5 1.0 60

5 22.4 60.5 5.0 54

6 38.3 70.0 3.5 58

7 39.4 46.5 3.5 52

8 39.4 46.5 3.5 54

9 11.7 46.5 3.5 28

10 38.9 46.5 3.5 56

11 56.1 60.5 2.0 88

12 50.3 32.5 2.0 74

13 37.9 46.5 6.0 54

14 21.8 32.5 2.0 30

15 39.3 46.5 3.5 52

16 37.2 46.5 3.5 62

17 62.3 46.5 3.5 92

18 38.5 23.0 3.5 58

19 22.3 60.5 2.0 52

20 53.3 60.5 5.0 74

Max 62.3 70.0 6.0 92

Min 11.7 23.0 1.0 28

59

Table 12: Six center point replicate trials determining the replicability and precision of the

experimental method. Arranged from lowest to highest reduction corrected with the average

(n=177) positive control recovery of 89.4%. The standard deviation between reductions was 3.6 µg.

Trial # Temperature of

H2O2 (˚C)

Actual

Temperature

(˚C)

Percent H2O2

(%)

UV-C

Dose

(kJ/m2)

Correction using

Average Pos

Control (µg)

1 46.5 45 3.5 34.8 50

15 46.5 46 3.5 39.3 52

8 46.5 46 3.5 39.4 54

7 46.5 47 3.5 39.4 52

10 46.5 45 3.5 38.9 56

16 46.5 47 3.5 37.2 62

Table 13. ANOVA analysis table of the linear model indicating that values lower than P < 0.05 are

significant. In this case, UV-C dose and H2O2 temperature are significant whereas H2O2

concentration was not significant. * indicates P < 0.05.

Source Sum of

Squares df* Mean Square F-Value P-value

Model 7.70 3 2.57 23.72 < 0.0001*

A-UV Dose 6.48 1 6.48 59.93 < 0.0001*

B-H2O2 Temperature 0.8333 1 0.8333 7.70 0.0135*

C-H2O2 Concentration 0.3080 1 0.3080 2.85 0.1109

Residual 1.73 16 0.1082

Lack of Fit 1.73 15 0.1150 23.01 0.1623

Pure Error 0.0050 1 0.0050

*df is the degrees of freedom.

60

Surface response plots generated by Design Expert® software illustrates the effects of UV-C

dose, hydrogen peroxide concentration and temperature in degrading CPY (Figure 16). A

synergistic effect between hydrogen peroxide temperature and UV dose on the degradation of

CPY was observed (Figure 16A). In contrast, there was no significant (P > 0.05) interaction

between hydrogen peroxide concentration and the applied UV-C dose (Figure 16B). In a similar

manner, there was a relatively negligible interaction between the concentration and temperature

of hydrogen peroxide on CPY degradation (Figure 16C).

A

61

B

C

62

Figure 16. 3D surface graph of different interaction effects of an advanced oxidation process for

degrading CPY on apples. CPY was introduced onto apple peel and treated with various

combinations of hydrogen peroxide and UV dose with the decrease in pesticide levels being

recorded. The plots illustrate UV dose and H2O2 temperature (A), UV dose and H2O2 concentration

(B) & H2O2 concentration and H2O2 temperature (C).

From the interaction between the three parameters it was possible to generate a predictive

equation (Eqn. 6) used to describe the observed reduction of CPY. Reduction of CPY from the

six center points ranged from 50 – 62 µg (25 – 31% of initial concentration) with a standard

deviation of 3.6 µg, demonstrating reproducibility of the predictive model (Table 12). This

shows that the data is replicable with minimal variation between samples. In addition, through

verification, a 94 µg (47%) degradation of CPY was achieved using an optimal AOP treatment

of 68.4 kJ/m2 UV dose and 1.22% v/v H2O2 at 66°C. This reduction agreed with a predictive

value of 92 µg suggested by Design Expert software (Eqn. 6) and was within a ± 5% standard

deviation (87 - 97 µg). It can also be seen from the predicted vs actual degradation graph that the

model is capable of accurately predicting CPY degradation following specified parameters

(Figure 17).

𝑌 = 5.742 + 1.071 𝑋1 + 0.390 𝑋2 − 2.241 𝑋3 @ 36𝐽

𝑠 ∙ 𝑚2

(Equation 6)

63

Figure 17. Predicted vs actual reduction of CPY table and graph illustrating the accuracy of the

RSM model in predicting CPY degradation under known conditions.

3.3 Ultraviolet light mediated degradation of CPY

The average positive control samples recovery was 90.5 ± 1.8 % (n = 42) and was used as the

correction factor in subsequent trials. The UV-C mediated degradation of CPY followed linear

kinetics with (r2 = 0.982) with 66 kJ/m2 dose supported a 140 µg (70%) reduction of CPY

(Figure 18).

When the distance between the sample and the UV light was altered from 16 cm to 10 cm, the

average UV intensities changed from 36 J/sm2 to 43 J/sm2, respectively. At this higher intensity

(10 cm, 43 J/sm2), the average (n=17) positive control recovery of 87.7% ± 5.0% was used to

correct reduction from treatments. Treatments at higher and lower UV intensities were compared

64

with UV exposure times remaining the same which resulted in different UV doses. UV exposure

times of 25 minutes were compared. An average (n=15) positive control recovery of 87.8% ±

5.2% CPY was used for the reduction correction. After 25 minutes of UV exposure, the lower

UV intensity treatment (16 cm between UV light and sample) was subjected to a total UV dose

of 53 kJ/m2 which achieved a CPY reduction of 106 µg (53%). In comparison, the higher UV

intensity treatment (10 cm between UV light and sample) produced a total UV dose of 64 kJ/m2.

CPY reduction of 141 µg (71%) was observed at this higher UV dose. When comparing the same

UV dose of 53 kJ/m2 at different UV exposure treatment times of 20.5 and 25 minutes, no

significant difference was observed with both producing CPY reductions of 106 µg (53%). These

results showed that by increasing UV intensity and by decreasing the distance between the UV

source and sample, treatment times could be shortened.

Figure 18. Graph of CPY reduction using only UV radiation with a linear trend line (orange line).

Reductions were calculated using HPLC analysis. A R2 value of 0.982 was calculated using 5 data

points of varying UV doses (kJ/m2) and CPY reduction (µg).

65

3.4 CPY-O production from AOP exposure

The positive control apple skins (spiked with CPY but not treated) showed concentrations of

CPY-O below the limit of detection (0.001 µg). CYP-O was not detected in negative control

apple skins (not spiked with CPY) by Q-ToF analysis. Of the 9 treated apple skin samples spiked

with CPY, a single sample showed levels of CPY-O below the limit of detection. The other 8

samples showed concentrations ranging from 0.002 – 0.004 µg. These results indicate that CPY-

O was generated when treated with AOP technologies (Table 14 and Figure 4). This result

agreed with other studies that also observed the generation of CPY-O due to AOP exposure

(Bavcon, Franko, & Trebše, 2007; Savić et al., 2019; Utzig et al., 2019). CPY, like other OP

pesticides, contains a P=S group which has been known to be converted to its oxon derivate

group (P=O) by metabolic oxidation (Savić et al., 2019). Bavcon et al. (2007) demonstrated

using GC-MS that CPY-O was the main degradation product of CPY from photoexcitation via

xenon lamp emitting below 400 nm. They observed, after 60 minutes of exposure, CPY-O

generation did not exceed 1% of the initial CPY concentration (Bavcon et al., 2007). Savic et al.

(2019) concluded that UV-C irradiation of CPY resulted in the generation of CPY-O with

maximum generation of >1% of the initial CPY concentration after 80 minutes of exposure.

Utzig et al. (2019) reported that although CPY degradation could be observed when using UV

and hydrogen peroxide, the generation of CPY-O was also observed when the AOP was applied,

but not when UV-C was applied alone. These studies however did not state the UV dose but

rather included UV exposure time instead.

66

Table 14. Q-ToF analysis of CPY-O on apple skins spiked with CPY. It can be observed that apple

skin samples treated with AOP produced the by-product CPY-O while samples spiked with CPY

but not treated with AOP had concentrations below the limit of detection.

Positive Control (µg) Treated Sample (µg)

< 0.000 0.002

< 0.000 0.004

< 0.000 0.004

< 0.000 < 0.000

< 0.000 0.004

< 0.000 0.004

< 0.000 0.002

< 0.000 0.004

< 0.000 0.004

3.5 Ozone Treatment

In the first replicate, the average % recovery for positive controls (spiked, untreated) apple skins

was 66% (n=8), and the treated samples had a recovery of 65% (n=9). HPLC analysis showed

that ozone gas treatment for 30 minutes (~ 1 g of gaseous ozone) had no effect on CPY

degradation. The concentration of ozone was above the linear dynamic range of the ozone

monitor of 100 mg/L. This experiment was repeated but with a higher relative humidity (>70%

RH) and the % recovery for positive controls was 82% (n=6) compared to treated samples with

85% (n=6). Recovery may have been lower in the first experiment due to sample preparation and

storage errors. Another repeat of this experiment was conducted and showed an 82% (n=5)

recovery of positive controls and 85% (n=6) for treated samples. These results showed that the

average recoveries for both positive controls and treated samples were not significantly different

67

when compared using Welch’s t test (P <0.05). This suggests that the ozone treatment did not

have a measureable effect on CPY on the surface of apple skins.

In other studies, gaseous ozone was seen to be successful at degrading pesticides. Whangchai et

al. (2011) concluded that gaseous ozone fumigation for 1 hour at 240 mg/L could degrade 45%

(4.5 mg/L) of CPY on lychee fruit. This result however was not supported in the current study.

One possible reason for this may be due to the difference in the concentration of ozone generated

and exposure time. Karaca et al (2012) demonstrated that 3 out of 5 tested fungicides stored in

ozone enriched air degraded residues faster than storage in ambient air conditions (2°C and 95%

RH). They observed reductions of 34.7%, 51.6%, and 64.5% in cyprodinil, pyrimethanil, and

fenhexamid respectively (Karaca, Walse, & Smilanick, 2012). Research conducted on using

ozone in reducing pesticides dissolved in water has been shown to be effective (Ikehata & Gamal

El-Din, 2005; Swami et al., 2016; Wu, Luan, Lan, Lo, & Chan, 2007). Swami et al (2016)

demonstrated that CPY degradation of 95% (0.06 µg/g) could be achieved when aqueous

samples were treated for 30 minutes using a 200 mg/h ozone generator Wu et al. (2006) observed

different reductions of the insecticide cypermethrin on vegetables (Brassica rapa) such as bok

choy treated with differing concentrations of ozonated water and exposure times. When treated

for 15 minutes with tap water or ozonated water at 1.4 mg/L, and 2.0 mg/L, reductions of 26%,

33%, and 54% was observed, respectively (Wu et al., 2007). After 30 minutes of exposure,

reductions of 31%, 54%, and 61% were observed (Wu et al., 2007). They concluded that higher

concentrations and longer exposure times increased removal of cpyermethrin on vegetables.

They also saw the same results when using other pesticides including methyl-parathion,

parathion, and diazinon.

68

3.6 Microbial Inactivation

The initial loading of E .coli O157:H7 and A. niger were determined to be 8.59 ± 0.52 and 6.98 ±

0.35 log CFU/sample, respectively (Figure 19). The maximal CPY degradation reduction

treatment with a UV-C dose of 69 kJ/m2 and 1.22% v/v H2O2 at 66°C supported a >7 log CFU

reduction of E. coli O157:H7 and a >4.68 log CFU/sample reduction of A. niger (Table 15).

These reductions were determined to be statistically significant when assessed using Welch’s t-

test. When determining individual factors, 1.22% v/v hydrogen peroxide alone applied at 66C

for 29 minutes (approximate equivalent amount of time for UV-C dose to reach 69 kJ/m2) also

supported >8 log CFU reduction of E.coli but only supported a 1.65 log CFU/sample reduction

of A. niger spores (Table 15). According to Welch’s t-test, both of these reductions using heated

hydrogen peroxide were determined to be statistically significant (P < 0.05). It was suspected

that the use of heat alone could significantly reduce E.coli O157:H7, perhaps more than the

hydrogen peroxide itself. To test this theory, triplicate trials were conducted with sterile, room

temperature or heated (~66°C) milli-Q water. The initial load of E.coli O157:H7 was 5.92 ± 0.09

log CFU/sample. After treatments, the average log CFU/sample for the room temperature and

heated water treatments were 5.86 ± 0.06 and 5.81 ± 0.06 for room temperature and heat

treatment, respectively, and they were not significantly different from the initial load (P > 0.05).

Consequently, it can be assumed that heat alone played a minor role in E.coli O157:H7

reduction. This could be due to E.coli having the ability to survive in temperatures over 66°C for

an extended period of time (Droffner & Brinton, 1995; R. Singh, Kim, Shepherd, Luo, & Jiang,

2011). Droffner and Brinton (1995) showed that E.coli could survive in temperatures between 60

– 70 °C for 9 days under dry conditions. Singh et al. (2011) showed that E.coli O157:H7 could

survive in compost at 50% moisture for 1 day at 60 °C. In this study, heated solution was applied

69

onto room temperature apple skins which would in theory, begin cooling down the heated

solution when it comes in contact with surface of the apple. This may explain why a heated

solution did not be significantly reduce E.coli O157:H7. In comparison, hydrogen peroxide

treatments were re-conducted but with both room temperature and heated (~66°C) solutions and

an initial load of 5.84 ± 0.14 log CFU/sample of E.coli O157:H7. The resultant load of E.coli

O157:H7 was < 2.3 log CFU/sample for both room temperature and heated treatments. This

result showed that hydrogen peroxide could exert a germicidal effect on E.coli O157:H7 without

the addition of heat. A UV-C dose of 69 kJ/m2 alone was also determined to be statistically

significant, resulting in a >6.56 log CFU/sample decrease in E. coli O157:H7. In comparison, A.

niger spores with UV-C alone resulted in a >4.68 log CFU/sample reduction.

A UV-C process alone could be used to degrade CPY but for inactivation of E. coli O157:H7

there was a significant contribution from hydrogen peroxide exposure. Although hydrogen

peroxide does not have as large of a contribution to A. niger reduction relative to E. coli

O157:H7, it is still considered to be statistically significant.

The results are in agreement with other reports on the inactivation of microbes on fresh produce

and egg shell surfaces by AOP treatment (Hadjok, Mittal, & Warriner, 2008; K. Murray, Moyer,

Wu, Goyette, & Warriner, 2018; Rehkopf, Byrd, Coufal, & Duong, 2017; Xie, Hajdok, Mittal, &

Warriner, 2008). It is thought that the free radicals generated on the surface of the sample can

access microbes shielded from UV-C by surface structures. It is possible that the proportion of

free radicals required to inactive microbes is less than that needed for CPY oxidation. The results

suggest that hydrogen peroxide makes a greater contribution to antimicrobial action of AOP,

while photo-oxidation by UV-C makes a greater contribution to the degradation of CPY, and it

can be enhanced by temperature. In this respect, the optimum AOP treatment parameters

70

identified in this study can collectively act to reduce microbial and chemical hazards in fresh

produce.

Figure 19. Log counts of Escherichia coli O157:H7 and Aspergillus niger spores. *<2.3 CFU/mL is

the limit of enumeration. All treatments are determined to be statistically significant for both

organisms.

Table 15. Log count reduction of Escherichia coli O157:H7 and Aspergillus niger spores inoculated

onto apple peel sections then treated with UV-C (at 254 nm), hydrogen peroxide (1.22% v/v at

66C) or a combination of UV-C and hydrogen peroxide.

Microbe Treatment Log CFU/Sample

Initial

Loading

Post-Treatment LCR

Escherichia coli

O157:H7

Control

(Non-treated)

8.590.52

H2O2 (1.22% v/v) <2.30* >6.56a

UV-C (69 kJ/m2) 2.690.41 5.890.41b

71

UV-C + H2O2(69

kJ/m2 and 1.22%

v/v)

<2.30* >6.56a

Aspergillus

niger

Control

(Non-treated)

6.980.35

H2O2 (1.22% v/v) 5.320.40 1.650.40a

UV-C (69 kJ/m2) <2.30* >4.68b

UV-C + H2O2 (69

kJ/m2 and 1.22%

v/v)

<2.30* >4.68b

Mean values for each of the test microbes, followed by the same letter are not significantly

(P>0.05) different.

*Limit of enumeration = 2.30 log CFU/Sample

4. Conclusions

This study found that the use of AOPs involving UV radiation at 254 nm and hydrogen peroxide

were successful in degrading the pesticide chlorpyrifos (CPY) on the surface of apple skins. An

HPLC analysis method and recovery method for determining residual CPY levels on apples was

shown to be effective and accurate with an average positive control recovery of 89.4% 3.8%

(n=177). Through multiple ozone fumigation trials, it was shown that ozone gas was incapable of

degrading CPY on apple skins after 30 minutes of exposure in an enclosed space. A set of

experiments designed using RSM showed that hydrogen peroxide concentration is an

insignificant factor in degrading CPY while UV radiation and hydrogen peroxide temperature

were significant factors. When using the optimized treatment parameters of 68.4 kJ/m2 UV dose

and 1.22% v/v H2O2 at 66°C, an average degradation of 94 µg (47% of the initial concentration)

(n = 9) was observed. However, it was shown that UV radiation on its own was more effective

than when used in combination with hydrogen peroxide; a UV dose of 64 kJ/m2 alone produced

72

an average CPY degradation of 141 µg (n = 9). Although degradation of CPY was observed, Q-

ToF analysis showed that CPY degradation resulted in the production of chlorpyrifos-oxon

(CPY-O), the active and more toxic form of CPY. Treatment of 10 µg/mL CPY with optimized

degradation parameters resulted in the production of 0.004 µg (0.02 µg/mL) CPY-O. The spike

amount used in this study was 1000 times the MRL for CPY on apples in Canada of 0.01 µg/mL

which suggests that the production of CPY-O from treated apples with CPY concentrations near

the MRL would be minimal and not pose a significant risk to human health. Decreasing the

contact distance between the UV lamp source and apple skin surface resulted in the UV dose

being increased due to an increased UV intensity, thus reducing the treatment time required for

the same effect. AOP treatment was also successful in reducing microbial colonies of both the

pathogen E.coli O157:H7 and A. niger. Despite being insignificant in degrading CPY, the

addition of hydrogen peroxide is useful for treating microbial contamination, and was significant

in reducing E.coli O157:H7 to below the limit of enumeration (2.30 log CFU/sample).

Future work

This study focused only on the treatment of apples, which have a relatively smooth surface

compared to most other fruits and vegetables. Different types of produce should be tested with

the AOP to determine its effectiveness in reducing CPY and microbial contaminants. Other types

of insecticides besides CPY should be tested as well including: carbamates, organochlorides,

phenylpyrazole, pyrethroids, neonicotinoids, and ryanoids. Other types of pesticides can also be

tested such as fungicides, bactericides, and especially herbicides, which are the most widely used

type of pesticide in the world. Modern day pesticide analysis involves testing for multiple

pesticide residues at the same time. A known concentration pesticide solution containing

multiple pesticides should be spiked onto apple skin samples and analyzed using triple-quad LC-

73

MS-MS in order to determine the degradation of multiple pesticides at the same time rather than

using HPLC to determine the degradation of a single pesticide as performed in the current study.

Other future work of interest may be to use AOP to treat lower CPY spike concentrations, down

to 0.01 µg/mL (maximal residue limit on apples in Canada). It may also be worth investigating

how to obtain a higher UV dose in order to decrease treatment time, which will impact the rate of

food production.

Different microbial organisms can also be tested on apples or different types of produce

including: salmonella and if possible, viruses. UV-C radiation has been known to inactivate

viruses as well as bacteria and molds (Tseng & Li, 2007; Watanabe et al., 2010)

74

References

Abramovic, B. F., Banic, N. D., & Sojic, D. V. (2010). Degradation of thiacloprid in aqueous solution by UV and UV/H2O2 treatments. Chemosphere, 81(1), 114-119. doi: 10.1016/j.chemosphere.2010.07.016

Agriculture and Agri-Food Canada. (2017a). Statistical Overview of the Canadian Fruit Industry 2017.

Agriculture and Agri-Food Canada. (2017b). Statistical Overview of the Canadian Vegetable Industry 2017.

Ahmadi, S., Mohammadi, L., Igwegbe, C. A., Rahdar, S., & Banach, A. M. (2018). Application of response surface methodology in the degradation of Reactive Blue 19 using H2O2/MgO nanoparticles advanced oxidation process. International Journal of Industrial Chemistry, 9(3), 241-253. doi: 10.1007/s40090-018-0153-4

Al-hindi, R., Alnajada, A., & Mohamed, S. (2011). Isolation and identification of some fruit spoilage fungi: Screening of plant cell wall degrading enzymes. African Journal of Microbiology Research, 5, 443-448.

Andreozzi, R., Caprio, V., Insola, A., & Marotta, R. (1999). Advanced oxidation processes (AOP) for water purification and recovery. Catalysis Today, 53(1), 51-59. doi: https://doi.org/10.1016/S0920-5861(99)00102-9

Ansari, M. S., Moraiet, M. A., & Ahmad, S. (2014). Insecticides: Impact on the Environment and Human Health. In A. Malik, E. Grohmann & R. Akhtar (Eds.), Environmental Deterioration and Human Health: Natural and anthropogenic determinants (pp. 99-123). Dordrecht: Springer Netherlands.

Assembly Bill A2477B, (2019).

Atwood, D., & Paisley-Jones, C. (2017). Pesticide Industry Sales and Usage 2008 - 2012.

Bajwa, U., & Sandhu, K. S. (2014). Effect of handling and processing on pesticide residues in food- a review. Journal of Food Science and Technology, 51(2), 201-220. doi: 10.1007/s13197-011-0499-5

Bassil, K. L., Vakil, C., Sanborn, M., Cole, D. C., Kaur, J. S., & Kerr, K. J. (2007). Cancer health effects of pesticides: systematic review. Canadian family physician Medecin de famille canadien, 53(10), 1704-1711.

Bavcon, M., Franko, M., & Trebše, P. (2007). Photodegradation of organophosphorus insecticides – Investigations of products and their toxicity using gas chromatography–mass spectrometry and AChE-thermal lens spectrometric bioassay. Chemosphere, 67(1), 99-107. doi: https://doi.org/10.1016/j.chemosphere.2006.09.039

Beard, J. (2006). DDT and human health. Science of The Total Environment, 355(1), 78-89. doi: https://doi.org/10.1016/j.scitotenv.2005.02.022

75

Behin, J., & Farhadian, N. (2017). Response surface methodology for ozonation of trifluralin using advanced oxidation processes in an airlift photoreactor. Applied Water Science, 7(6), 3103-3112. doi: 10.1007/s13201-016-0443-y

Besser, R. E., Lett, S. M., Weber, J. T., Doyle, M. P., Barrett, T. J., Wells, J. G., & Griffin, P. M. (1993). An outbreak of diarrhea and hemolytic uremic syndrome from Escherichia coli O157: H7 in fresh-pressed apple cider. JAMA, 269(17), 2217-2220.

Bezerra, M. A., Santelli, R. E., Oliveira, E. P., Villar, L. S., & Escaleira, L. A. (2008). Response surface methodology (RSM) as a tool for optimization in analytical chemistry. Talanta, 76(5), 965-977. doi: 10.1016/j.talanta.2008.05.019

Bohrerova, Z., Shemer, H., Lantis, R., Impellitteri, C. A., & Linden, K. G. (2008). Comparative disinfection efficiency of pulsed and continuous-wave UV irradiation technologies. Water Research, 42(12), 2975-2982. doi: https://doi.org/10.1016/j.watres.2008.04.001

Bouchard, M., F., Chevrier, J., Harley Kim, G., Kogut, K., Vedar, M., Calderon, N., . . . Eskenazi, B. (2011). Prenatal Exposure to Organophosphate Pesticides and IQ in 7-Year-Old Children. Environmental Health Perspectives, 119(8), 1189-1195. doi: 10.1289/ehp.1003185

Bouchard, M. F., Bellinger, D. C., Wright, R. O., & Weisskopf, M. G. (2010). Attention-deficit/hyperactivity disorder and urinary metabolites of organophosphate pesticides. Pediatrics, 125(6), e1270-e1277. doi: 10.1542/peds.2009-3058

Brown, T. P., Rumsby, P. C., Capleton, A. C., Rushton, L., & Levy, L. S. (2006). Pesticides and Parkinson's disease--is there a link? Environ Health Perspect, 114(2), 156-164. doi: 10.1289/ehp.8095

Burkul, R. M., Ranade, S. V., & Pangarkar, B. L. (2015). Removal of Pesticides by Using Various Treatment Method: Review. International Journal of Emerging Trend in Engineering and Basic Sciences, 2(2), 88-91.

Bustos, N., Cruz-Alcalde, A., Iriel, A., Fernández Cirelli, A., & Sans, C. (2019). Sunlight and UVC-254 irradiation induced photodegradation of organophosphorus pesticide dichlorvos in aqueous matrices. Science of The Total Environment, 649, 592-600. doi: https://doi.org/10.1016/j.scitotenv.2018.08.254

Butler, C. D. (2018). Climate Change, Health and Existential Risks to Civilization: A Comprehensive Review (1989⁻2013). International journal of environmental research and public health, 15(10), 2266. doi: 10.3390/ijerph15102266

Canada, G. o. (2019). Public Health Notice - Outbreak of E. coli infections linked to romaine lettuce. Retrieved from https://www.canada.ca/en/public-health/services/public-health-notices/2018/outbreak-ecoli-infections-linked-romaine-lettuce.html.

Casida, J. E., & Durkin, K. A. (2013). Neuroactive Insecticides: Targets, Selectivity, Resistance, and Secondary Effects. Annual Review of Entomology, 58(1), 99-117. doi: 10.1146/annurev-ento-120811-153645

CDC. (2016). Escherichia coli (E. coli) Factsheet

http://www.canada.ca/en/public-health/services/public-health-notices/2018/outbreak-ecoli-infections-linked-romaine-lettuce.html

http://www.canada.ca/en/public-health/services/public-health-notices/2018/outbreak-ecoli-infections-linked-romaine-lettuce.html

76

Cengiz, M. F., Catal, M., Erler, F., & Bilgin, K. (2015). The effects of heat treatment on the degradation of the organophosphate pesticide chlorpyrifos-ethyl in tomato homogenate. Quality Assurance and Safety of Crops & Foods, 7(4), 537-544. doi: 10.3920/Qas2013.0301

Chun, M. H., & Lee, M. C. (2006). Reduction of pesticide residues in the production of red pepper powder. Food Science and Biotechnology, 15, 57-62.

Cody, S. H., Glynn, M. K., Farrar, J. A., Cairns, K. L., Griffin, P. M., Kobayashi, J., . . . Vugia, D. J. (1999). An outbreak of Escherichia coli O157:H7 infection from unpasteurized commercial apple juice. Ann Intern Med, 130(3), 202-209. doi: 10.7326/0003-4819-130-3-199902020-00005

Connors, S. L., Levitt, P., Matthews, S. G., Slotkin, T. A., Johnston, M. V., Kinney, H. C., . . . Zimmerman, A. W. (2008). Fetal mechanisms in neurodevelopmental disorders. Pediatr Neurol, 38(3), 163-176. doi: 10.1016/j.pediatrneurol.2007.10.009

Consumer Reports. (2015). Pesticides in Produce. Retrieved June 6, 2019, from https://www.consumerreports.org/cro/health/natural-health/pesticides/index.htm

Costa, L. G., Giordano, G., Cole, T. B., Marsillach, J., & Furlong, C. E. (2013). Paraoxonase 1 (PON1) as a genetic determinant of susceptibility to organophosphate toxicity. Toxicology, 307, 115-122. doi: 10.1016/j.tox.2012.07.011

Dagenais, T. R. T., & Keller, N. P. (2009). Pathogenesis of Aspergillus fumigatus in Invasive Aspergillosis. Clinical microbiology reviews, 22(3), 447-465. doi: 10.1128/CMR.00055-08

Davis, F. R. (2014). Banned : A History of Pesticides and the Science of Toxicology. New Haven: Yale University Press.

Delcour, I., Spanoghe, P., & Uyttendaele, M. (2015). Literature review: Impact of climate change on pesticide use. Food Research International, 68, 7-15. doi: https://doi.org/10.1016/j.foodres.2014.09.030

Deng, Y., & Zhao, R. Z. (2015). Advanced Oxidation Processes (AOPs) in Wastewater Treatment. Current Pollution Reports, 1(3), 167-176. doi: 10.1007/s40726-015-0015-z

Deutsch, C. A., Tewksbury, J. J., Tigchelaar, M., Battisti, D. S., Merrill, S. C., Huey, R. B., & Naylor, R. L. (2018). Increase in crop losses to insect pests in a warming climate. Science, 361(6405), 916-919. doi: 10.1126/science.aat3466

Droffner, M. L., & Brinton, W. F. (1995). Survival of E. coli and Salmonella populations in aerobic thermophilic composts as measured with DNA gene probes. International journal of hygiene and environmental medicine, 197(5), 387-397.

Eaton, D. L., Daroff, R. B., Autrup, H., Bridges, J., Buffler, P., Costa, L. G., . . . Spencer, P. S. (2008). Review of the Toxicology of Chlorpyrifos With an Emphasis on Human Exposure and Neurodevelopment. Critical Reviews in Toxicology, 38(sup2), 1-125. doi: 10.1080/10408440802272158

http://www.consumerreports.org/cro/health/natural-health/pesticides/index.htm

77

El-Merhibi, A., Kumar, A., & Smeaton, T. (2004). Role of piperonyl butoxide in the toxicity of chlorpyrifos to Ceriodaphnia dubia and Xenopus laevis. Ecotoxicol Environ Saf, 57(2), 202-212. doi: 10.1016/S0147-6513(03)00082-4

Environmental Protection Agency. (2017, March 29). EPA Administrator Pruitt Denies Petition to Ban Widely Used Pesticide. from https://www.epa.gov/newsreleases/epa-administrator-pruitt-denies-petition-ban-widely-used-pesticide-0

EPA. (1999). Alternative Disinfectants and Oxidants Guidance Manual. Retrieved from https://nepis.epa.gov/Exe/ZyPDF.cgi/2000229L.PDF?Dockey=2000229L.PDF.

Eskenazi, B., Bradman, A., & Castorina, R. (1999). Exposures of children to organophosphate pesticides and their potential adverse health effects. Environ Health Perspect, 107 Suppl 3, 409-419. doi: 10.1289/ehp.99107s3409

Eto, M. (1974). Organophosphorus pesticides; organic and biological chemistry. Cleveland,: CRC Press.

EWG. (2019). EWG's 2019 Shopper's Guide to Pesticides in Produce™. Retrieved June 6, 2019, from https://www.ewg.org/foodnews/summary.php#dirty-dozen

EXTOXNET. (1993). Chlorpyrifos. Retrieved September 23, 2019, from http://pmep.cce.cornell.edu/profiles/extoxnet/carbaryl-dicrotophos/chlorpyrifos-ext.html

Fairbrother, J., & Nadeau, E. (2006). Escherichia coli: on-farm contamination of animals. Rev Sci Tech, 25(2), 555-569.

101.17 Food labeling warning, notice, and safe handling statements. (1977).

FDA. (1995). CPG Sec. 575.100 Pesticide Residues in Food and Feed - Enforcement Criteria.

Sec. 801.415 Maximum acceptable level of ozone. (2018).

Femia, J., Mariani, M., Zalazar, C., & Tiscornia, I. (2013). Photodegradation of chlorpyrifos in water by UV/H2O2 treatment: toxicity evaluation. Water Sci Technol, 68(10), 2279-2286. doi: 10.2166/wst.2013.493

Fishburn, J. D., Tang, Y., & Frank, J. F. (2012). Efficacy of various consumer-friendly produce washing technologies in reducing pathogens on fresh produce. Food Protection Trends, 32(8), 456-466.

Frisvad, J. C., Smedsgaard, J., Samson, R. A., Larsen, T. O., & Thrane, U. (2007). Fumonisin B2 Production by Aspergillus niger. Journal of Agricultural and Food Chemistry, 55(23), 9727-9732. doi: 10.1021/jf0718906

Fukuto, T. R. (1990). Mechanism of action of organophosphorus and carbamate insecticides. Environmental Health Perspectives, 87, 245-254. doi: 10.1289/ehp.9087245

Gally, D. L., & Stevens, M. P. (2017). Microbe Profile: Escherichia coli O157 : H7 – notorious relative of the microbiologist’s workhorse. Microbiology, 163(1), 1-3. doi: https://doi.org/10.1099/mic.0.000387

http://www.epa.gov/newsreleases/epa-administrator-pruitt-denies-petition-ban-widely-used-pesticide-0

http://www.epa.gov/newsreleases/epa-administrator-pruitt-denies-petition-ban-widely-used-pesticide-0

http://www.ewg.org/foodnews/summary.php#dirty-dozen

http://pmep.cce.cornell.edu/profiles/extoxnet/carbaryl-dicrotophos/chlorpyrifos-ext.html

78

Gandhi, K., Lari, S., Tripathi, D., & Kanade, G. (2016). Advanced oxidation processes for the treatment of chlorpyrifos, dimethoate and phorate in aqueous solution. Journal of Water Reuse and Desalination, 6(1), 195-203. doi: 10.2166/wrd.2015.062

Gautam, A. K., Sharma, S., Avasthi, S., & Bhadauria, R. (2011). Diversity, pathogenicity and toxicology of A. niger: an important spoilage fungi. Research Journal of Microbiology, 6(3), 270-280.

Giesy, J. P., & Solomon, K. R. (2014). Ecological Risk Assessment for Chlorpyrifos in Terrestrial and Aquatic Systems in the United States Special Foreword. Ecological Risk Assessment for Chlorpyrifos in Terrestrial and Aquatic Systems in the United States, 231, V-Vii. doi: Book_Doi 10.1007/978-3-319-03865-0

Giesy, J. P., Solomon, K. R., Cutler, G. C., Giddings, J. M., Mackay, D., Moore, D. R., . . . Williams, W. M. (2014). Ecological risk assessment of the uses of the organophosphorus insecticide chlorpyrifos, in the United States. Rev Environ Contam Toxicol, 231, 1-11. doi: 10.1007/978-3-319-03865-0_1

Glaze, W., Kang, J.-W., & H. Chapin, D. (1987). The Chemistry of Water Treatment Processes Involving Ozone, Hydrogen Peroxide and Ultraviolet Radiation (Vol. 9).

Gomez, L. E. (2009). Use and benefits of chlorpyrifos in US agriculture. Dow AgroSciences LLC.

Gottschalk, C., Saupe, A., & Libra, J. A. (2010). Ozonation of water and waste water: Wiley-VCH.

Green, A. (2018). UV light-based interventions to inactivate pathogens on fresh produce through the use of UV-LEDs and water-assisted processing. (Masters), University of Guelph.

Gregory, P. J., Johnson, S. N., Newton, A. C., & Ingram, J. S. I. (2009). Integrating pests and pathogens into the climate change/food security debate. Journal of Experimental Botany, 60(10), 2827-2838. doi: 10.1093/jxb/erp080

Hadjok, C., Mittal, G. S., & Warriner, K. (2008). Inactivation of human pathogens and spoilage bacteria on the surface and internalized within fresh produce by using a combination of ultraviolet light and hydrogen peroxide. Journal of applied microbiology, 104, 1014-1024.

Hanley, T. R., Jr., Carney, E. W., & Johnson, E. M. (2000). Developmental Toxicity Studies in Rats and Rabbits with 3,5,6-Trichloro-2-pyridinol, the Major Metabolite of Chlorpyrifos. Toxicological Sciences, 53(1), 100-108. doi: 10.1093/toxsci/53.1.100

Harshit, D., Charmy, K., & Nrupesh, P. (2017). Organophosphorus pesticides determination by novel HPLC and spectrophotometric method. Food Chemistry, 230, 448-453.

Health Canada. (2008). MRL Database - Chlorpyrifos on Apples. Canada Gazette II.

Health Canada. (2012, October 1). Maximum Residue Limits for Pesticides from http://pr-rp.hc-sc.gc.ca/mrl-lrm/index-eng.php

Holland, J., & Sinclair, P. (2004). Environmental Fate of Pesticides and the Consequences for Residues in Food and Drinking Water (pp. 27-62).

http://pr-rp.hc-sc.gc.ca/mrl-lrm/index-eng.php

http://pr-rp.hc-sc.gc.ca/mrl-lrm/index-eng.php

79

Ikehata, K., & Gamal El-Din, M. (2005). Aqueous Pesticide Degradation by Ozonation and Ozone-Based Advanced Oxidation Processes: A Review (Part I). Ozone: Science & Engineering, 27(2), 83-114. doi: 10.1080/01919510590925220

Jegadeeshwar, H., Shankar, V., Kumar, P., & thottiam Vasudevan, R. (2017). A Review on Utilization of Ozone in Food preservation.

Jones, P. G., & Thornton, P. K. (2003). The potential impacts of climate change on maize production in Africa and Latin America in 2055. Global environmental change, 13(1), 51-59.

Karaca, H., Walse, S. S., & Smilanick, J. L. (2012). Effect of continuous 0.3μL/L gaseous ozone exposure on fungicide residues on table grape berries. Postharvest Biology and Technology, 64(1), 154-159. doi: https://doi.org/10.1016/j.postharvbio.2011.07.004

Kashanian, S., Shariati, Z., Roshanfekr, H., & Ghobadi, S. (2012). DNA Binding Studies of 3, 5, 6-Trichloro-2-Pyridinol Pesticide Metabolite (Vol. 31).

Katsoyiannis, I. A., Canonica, S., & von Gunten, U. (2011). Efficiency and energy requirements for the transformation of organic micropollutants by ozone, O3/H2O2 and UV/H2O2. Water Research, 45(13), 3811-3822. doi: https://doi.org/10.1016/j.watres.2011.04.038

Keikotlhaile, B. M. (2011). Influence of the processing factors on pesticide residues in fruits and vegetables and its application in consumer risk assessment. Ghent University.

Keklik, N. M., Krishnamurthy, K., & Demirci, A. (2012). 12 - Microbial decontamination of food by ultraviolet (UV) and pulsed UV light. In A. Demirci & M. O. Ngadi (Eds.), Microbial Decontamination in the Food Industry (pp. 344-369): Woodhead Publishing.

Kennedy, E. M., & Mackie, J. C. (2018). Mechanism of the Thermal Decomposition of Chlorpyrifos and Formation of the Dioxin Analog, 2,3,7,8-Tetrachloro-1,4-dioxino-dipyridine (TCDDpy). Environmental Science & Technology, 52(13), 7327-7333. doi: 10.1021/acs.est.8b01626

Kenney, S. J., Burnett, S. L., & Beuchat, L. R. (2001). Location of Escherichia coli O157:H7 on and in apples as affected by bruising, washing, and rubbing. J Food Prot, 64(9), 1328-1333.

Kim, K.-S., Lee, Y.-M., Lee, H.-W., Jacobs, D. R., & Lee, D.-H. (2015). Associations between organochlorine pesticides and cognition in U.S. elders: National Health and Nutrition Examination Survey 1999–2002. Environment International, 75, 87-92. doi: https://doi.org/10.1016/j.envint.2014.11.003

Koch, S., Astrid Epp, Mark Lohmann, & Gaby-Fleur Böl. (2017). Pesticide Residues in Food: Attitudes, Beliefs, and Misconceptions among Conventional and Organic Consumers. J Food Prot, 80(12), 2083-2089. doi: 10.4315/0362-028x.jfp-17-104

Köck-Schulmeyer, M., Villagrasa, M., López de Alda, M., Céspedes-Sánchez, R., Ventura, F., & Barceló, D. (2013). Occurrence and behavior of pesticides in wastewater treatment plants and their environmental impact. Science of The Total Environment, 458-460, 466-476. doi: https://doi.org/10.1016/j.scitotenv.2013.04.010

80

Krijgsheld, P., Bleichrodt, R., van Veluw, G. J., Wang, F., Müller, W. H., Dijksterhuis, J., & Wösten, H. A. B. (2013). Development in Aspergillus. Studies in mycology, 74(1), 1-29. doi: 10.3114/sim0006

Krishnan, S., Rawindran, H., Sinnathambi, C. M., & Lim, J. W. (2017). Comparison of various advanced oxidation processes used in remediation of industrial wastewater laden with recalcitrant pollutants. 29th Symposium of Malaysian Chemical Engineers (Somche) 2016, 206. doi: Unsp 012089

10.1088/1757-899x/206/1/012089

Krzemińska, D., Neczaj, E., & Borowski, G. (2015). Advanced oxidation processes for food industrial wastewater decontamination (Vol. 16).

Kuehn, B. M. (2010). Increased Risk of ADHD Associated With Early Exposure to Pesticides, PCBs. JAMA, 304(1), 27-28. doi: 10.1001/jama.2010.860

Ladaniya, M. S. (2008). 16 - POSTHARVEST DISEASES AND THEIR MANAGEMENT. In M. S. Ladaniya (Ed.), Citrus Fruit (pp. 417-XIX). San Diego: Academic Press.

Lando, A., Verrill, L., Liu, S., & Smith, E. (2016). 2016 Food Safety Survey.

Langlais, B., Reckhow, D., & R. Brink, D. (1991). Ozone in Water Treatment.

Leach, K. M., Stroot, J. M., & Lim, D. V. (2010). Same-Day Detection of Escherichia coli O157:H7 from Spinach by Using Electrochemiluminescent and Cytometric Bead Array Biosensors. Applied and environmental microbiology, 76(24), 8044. doi: 10.1128/AEM.01990-10

Lee, S. R., Mourer, C. R., & Shibamoto, T. (1991). Analysis before and after Cooking Processes of a Trace Chlorpyrifos Spiked in Polished Rice. Journal of Agricultural and Food Chemistry, 39(5), 906-908. doi: Doi 10.1021/Jf00005a020

LennTech. (2019). Ozone Decomposition. Retrieved October 8, 2019, from https://www.lenntech.com/library/ozone/decomposition/ozone-decomposition.htm

Lim, J. Y., Yoon, J., & Hovde, C. J. (2010). A brief overview of Escherichia coli O157:H7 and its plasmid O157. Journal of microbiology and biotechnology, 20(1), 5-14.

Mailafia, S., Okoh, G. s. R., Olabode, H. O. K., & Osanupin, R. (2017). Isolation and identification of fungi associated with spoilt fruits vended in Gwagwalada market, Abuja, Nigeria. Veterinary world, 10(4), 393-397. doi: 10.14202/vetworld.2017.393-397

Marican, A., & Durán-Lara, E. F. (2018). A review on pesticide removal through different processes. Environmental Science and Pollution Research, 25(3), 2051-2064. doi: 10.1007/s11356-017-0796-2

Melton-Celsa, A. R. (2014). Shiga Toxin (Stx) Classification, Structure, and Function. Microbiology spectrum, 2(4), 10.1128/microbiolspec.EHEC-0024-2013-2013. doi: 10.1128/microbiolspec.EHEC-0024-2013

http://www.lenntech.com/library/ozone/decomposition/ozone-decomposition.htm

81

Merényi, G., Lind, J., Naumov, S., & Sonntag, C. v. (2010). Reaction of Ozone with Hydrogen Peroxide (Peroxone Process): A Revision of Current Mechanistic Concepts Based on Thermokinetic and Quantum-Chemical Considerations. Environmental Science & Technology, 44(9), 3505-3507. doi: 10.1021/es100277d

Mi-Gyung Lee. (2001). Reduction of Chlorpyrifos and Fenitrothion Residues in Red Pepper Peel by Washing and Drying. Food Science and Biotechnology, 10(4), 429-432.

Miller, L. G., & Kaspar, C. W. (1994). Escherichia coli O157: H7 acid tolerance and survival in apple cider. J Food Prot, 57(6), 460-464.

Mladenova, R., & Shtereva, D. (2009). Pesticide residues in apples grown under a conventional and integrated pest management system. Food Addit Contam Part A Chem Anal Control Expo Risk Assess, 26(6), 854-858. doi: 10.1080/02652030902726060

Muhamad, S. G. (2010). Kinetic studies of catalytic photodegradation of chlorpyrifos insecticide in various natural waters. Arabian Journal of Chemistry, 3(2), 127-133. doi: https://doi.org/10.1016/j.arabjc.2010.02.009

Murray, K., Moyer, P., Wu, F., Goyette, J. B., & Warriner, K. (2018). Inactivation of Listeria monocytogenes on and within Apples Destined for Caramel Apple Production by Using Sequential Forced Air Ozone Gas Followed by a Continuous Advanced Oxidative Process Treatment. J Food Prot, 81(3), 357-364. doi: 10.4315/0362-028X.JFP-17-306

Murray, K., Wu, F., Shi, J., Jun Xue, S., & Warriner, K. (2017). Challenges in the microbiological food safety of fresh produce: Limitations of post-harvest washing and the need for alternative interventions. Food Quality and Safety, 1(4), 289-301. doi: 10.1093/fqsafe/fyx027

Nagayama, T. (1996). Behavior of Residual Organophosphorus Pesticides in Foodstuffs during Leaching or Cooking. Journal of Agricultural and Food Chemistry, 44(8), 2388-2393. doi: 10.1021/jf950549v

Nagesh, M., & Verma, S. (1997). Decontamination of cabbage treated with chlorpyrifos and quinalphos. Indian Journal of Entomology, 59(4), 404-410.

National Environmental Services Center [NESC]. (2000). Ultraviolet Disinfection: University of West Virginia.

Nicolopoulou-Stamati, P., Maipas, S., Kotampasi, C., Stamatis, P., & Hens, L. (2016). Chemical Pesticides and Human Health: The Urgent Need for a New Concept in Agriculture. Frontiers in public health, 4, 148-148. doi: 10.3389/fpubh.2016.00148

NIST. (2013). 5.3.3.6.3. Comparisons of response surface designs. from https://www.itl.nist.gov/div898/handbook/pri/section3/pri3363.htm

NPIC. (2019, August 08, 2019). National Pesticide Information Center. Retrieved October 8, 2019, from http://npic.orst.edu/ingred/ptype/index.html

O'Donnell, C., Brijesh kumar, T., Cullen, P. J., & G. Rice, R. (2012). Ozone in Food Processing.

http://www.itl.nist.gov/div898/handbook/pri/section3/pri3363.htm

http://npic.orst.edu/ingred/ptype/index.html

82

Oliveira, A., Pereira Ribeiro, J., Oliveira, J., De Keukeleire, D., Saldanha Duarte, M., & Nascimento, R. (2014). Degradation of the Pesticide Chlorpyrifos in Aqueous Solutions with UV/H2O2: Optimization and Effect of Interfering Anions (Vol. 17).

Oppenländer, T. (2003). Photochemical Purification of Water and Air. Weinheim, Germany: Wiley-VCH Verlag,.

Osman, K. A., Al-Humaid, A. I., Al-Redhaiman, K. N., & El-Mergawi, R. A. (2014). Safety methods for chlorpyrifos removal from date fruits and its relation with sugars, phenolics and antioxidant capacity of fruits. Journal of Food Science and Technology, 51(9), 1762-1772. doi: 10.1007/s13197-012-0693-0

P. Duguet, J., Brodard, E., Dussert, B., & Mallevialle, J. (1985). Improvement in the Effectiveness of Ozonation of Drinking Water Through the Use of Hydrogen Peroxide (Vol. 7).

Pan, J., Xia, X.-X., & Liang, J. (2008). Analysis of pesticide multi-residues in leafy vegetables by ultrasonic solvent extraction and liquid chromatography-tandem mass spectrometry. Ultrasonics Sonochemistry, 15(1), 25-32. doi: https://doi.org/10.1016/j.ultsonch.2007.06.005

Parnell, T. L., & Harris, L. J. (2003). Reducing Salmonella on apples with wash practices commonly used by consumers. J Food Prot, 66(5), 741-747. doi: Doi 10.4315/0362-028x-66.5.741

Parsons, S. (2004). Advanced Oxidation Processes for Water and Wastewater Treatment: IWA Publishing.

Paulussen, C., Hallsworth, J. E., Álvarez-Pérez, S., Nierman, W. C., Hamill, P. G., Blain, D., . . . Lievens, B. (2017). Ecology of aspergillosis: insights into the pathogenic potency of Aspergillus fumigatus and some other Aspergillus species. Microbial biotechnology, 10(2), 296-322. doi: 10.1111/1751-7915.12367

Pennsylvania State University. (2018). Lession 11: Response Surface Methods and Designs. from https://newonlinecourses.science.psu.edu/stat503/node/57/

Person, A. K., Chudgar, S. M., Norton, B. L., Tong, B. C., & Stout, J. E. (2010). Aspergillus niger: an unusual cause of invasive pulmonary aspergillosis. Journal of medical microbiology, 59(Pt 7), 834-838. doi: 10.1099/jmm.0.018309-0

Ramos, J. J., Rial-Otero, R., Ramos, L., & Capelo, J. L. (2008). Ultrasonic-assisted matrix solid-phase dispersion as an improved methodology for the determination of pesticides in fruits. Journal of Chromatography A, 1212(1), 145-149. doi: https://doi.org/10.1016/j.chroma.2008.10.028

Randall, C., Hock, W., Crow, E., Hudak-Wise, C., & Kasai, J. (2014). National Pesticide Applicator Certification Core Manual (2nd ed.). Washington: National Association of State Departments of Agriculture Research.

Rangel, J. M., Sparling, P. H., Crowe, C., Griffin, P. M., & Swerdlow, D. L. (2005). Epidemiology of Escherichia coli O157:H7 outbreaks, United States, 1982-2002. Emerging infectious diseases, 11(4), 603-609. doi: 10.3201/eid1104.040739

83

Rauh, V. A., Perera, F. P., Horton, M. K., Whyatt, R. M., Bansal, R., Hao, X., . . . Peterson, B. S. (2012). Brain anomalies in children exposed prenatally to a common organophosphate pesticide. Proceedings of the National Academy of Sciences, 109(20), 7871-7876. doi: 10.1073/pnas.1203396109

Rawn, D. F. K., Quade, S. C., Sun, W.-F., Fouguet, A., Bélanger, A., & Smith, M. (2008). Captan residue reduction in apples as a result of rinsing and peeling. Food Chemistry, 109(4), 790-796. doi: https://doi.org/10.1016/j.foodchem.2008.01.061

Regulations, C. o. F. (2018). PART 173 -- SECONDARY DIRECT FOOD ADDITIVES PERMITTED IN FOOD FOR HUMAN CONSUMPTION.

Rehkopf, A. C., Byrd, J. A., Coufal, C. D., & Duong, T. (2017). Advanced Oxidation Process sanitization of hatching eggs reduces Salmonella in broiler chicks. Poultry Science, 96(10), 3709-3716. doi: 10.3382/ps/pex166

Roos, J., Hopkins, R., Kvarnheden, A., & Dixelius, C. (2011). The impact of global warming on plant diseases and insect vectors in Sweden. European Journal of Plant Pathology, 129(1), 9-19. doi: 10.1007/s10658-010-9692-z

Salvadori, M. I., Sontrop, J. M., Garg, A. X., Moist, L. M., Suri, R. S., & Clark, W. F. (2009). Factors that led to the Walkerton tragedy. Kidney International, 75, S33-S34. doi: https://doi.org/10.1038/ki.2008.616

Sanborn, M., Cole, D., Helena Sanin, L., & Bassil, K. (2004). Systematic Review of Pesticide Human Health Effects.

Savić, J. Z., Petrović, S. Ž., Leskovac, A. R., Lazarević Pašti, T. D., Nastasijević, B. J., Tanović, B. B., . . . Vasić, V. M. (2019). UV-C light irradiation enhances toxic effects of chlorpyrifos and its formulations. Food Chemistry, 271, 469-478. doi: https://doi.org/10.1016/j.foodchem.2018.07.207

Savic, J. Z., Petrovic, S. Z., Leskovac, A. R., Pasti, T. D. L., Nastasijevic, B. J., Tanovic, B. B., . . . Vasic, V. M. (2019). UV-C light irradiation enhances toxic effects of chlorpyrifos and its formulations. Food Chemistry, 271, 469-478. doi: 10.1016/j.foodchem.2018.07.2017

Schuster, E., Dunn-Coleman, N., Frisvad, J. C., & van Dijck, P. W. M. (2002). On the safety of Aspergillus niger - a review. Applied Microbiology and Biotechnology, 59(4-5), 426-435. doi: 10.1007/s00253-002-1032-6

Sharma, I. D., Patyal, S. K., Nath, A., & Joshi, V. K. (2001). Effect of processing on mancozeb residues in apple. Journal of Food Science and Technology-Mysore, 38(1), 23-25.

Sharma, R. (2012). Pathogenecity of Aspergillus niger in plants. Cibtech Journal of Microbiology, 1(1), 47-51.

Show, P. L., Oladele, K. O., Siew, Q. Y., Aziz Zakry, F. A., Lan, J. C.-W., & Ling, T. C. (2015). Overview of citric acid production from Aspergillus niger. Frontiers in Life Science, 8(3), 271-283. doi: 10.1080/21553769.2015.1033653

84

Shrivastava, A., & Gupta, V. (2011). Methods for the determination of limit of detection and limit of quantitation of the analytical methods. Chronicles of Young Scientists, 2(1), 21-25. doi: 10.4103/2229-5186.79345

Singh, R., Kim, J., Shepherd, M. W., Luo, F., & Jiang, X. (2011). Determining Thermal Inactivation of E.coli O157:H7 in Fresh Compost by Simulating Early Phases of the Composting Process. Applied and environmental microbiology, 77(12), 4126-4135. doi: 10.1128/aem.02873-10

Singh, S., Krishnaiah, N., Rao, T. M., Pushpa, R., & Reddy, G. (2017). Effect of heat processing on degradation of organophosphorus compounds in milk. The Pharma Innovation Journal, 6(3), 209-212.

Slotkin, T. A., Seidler, F. J., Wu, C., MacKillop, E. A., & Linden, K. G. (2009). Ultraviolet photolysis of chlorpyrifos: developmental neurotoxicity modeled in PC12 cells. Environmental Health Perspectives, 117(3), 338-343. doi: 10.1289/ehp.11592

Solomon, E. B., Yaron, S., & Matthews, K. R. (2002). Transmission of Escherichia coli O157:H7 from contaminated manure and irrigation water to lettuce plant tissue and its subsequent internalization. Applied and environmental microbiology, 68(1), 397-400. doi: 10.1128/AEM.68.1.397-400.2002

Solomon, K. R., Williams, W. M., Mackay, D., Purdy, J., Giddings, J. M., & Giesy, J. P. (2014). Properties and Uses of Chlorpyrifos in the United States. In J. P. Giesy & K. R. Solomon (Eds.), Ecological Risk Assessment for Chlorpyrifos in Terrestrial and Aquatic Systems in the United States (pp. 13-34). Cham: Springer International Publishing.

State of Hawaii. (2017). S.B. NO 844. Twenty-Ninth Legislature.

Statistics Canada. (2017). Health Fact Sheets - Fruit and vegetable consumption, 2017.

Statistics Canada. (2018). Table 32-10-0364-01 Estimates, production and farm gate value of fresh and processed fruits.

Stoytcheva, M. (2011). Pesticides in the Modern World: Risks and Benefits: BoD–Books on Demand.

Swami, S., Muzammil, R., Saha, S., Shabeer, A., Oulkar, D., Banerjee, K., & Singh, S. B. (2016). Evaluation of ozonation technique for pesticide residue removal and its effect on ascorbic acid, cyanidin-3-glucoside, and polyphenols in apple (Malus domesticus) fruits. Environmental monitoring and assessment, 188(5), 301.

Taylor-Edmonds, L., Lichi, T., Rotstein-Mayer, A., & Mamane, H. (2015). The impact of dose, irradiance and growth conditions on Aspergillus niger (renamed A. brasiliensis) spores low-pressure (LP) UV inactivation. Journal of Environmental Science and Health Part a-Toxic/Hazardous Substances & Environmental Engineering, 50(4), 341-347. doi: 10.1080/10934529.2015.987519

Thind, P. S., Kumari, D., & John, S. (2018). TiO2/H2O2 mediated UV photocatalysis of Chlorpyrifos: Optimization of process parameters using response surface methodology. Journal of Environmental Chemical Engineering, 6(3), 3602-3609. doi: 10.1016/j.jece.2017.05.031

85

Thomas, D. E., & Elliott, E. J. (2013). Interventions for preventing diarrhea-associated hemolytic uremic syndrome: systematic review. BMC public health, 13, 799-799. doi: 10.1186/1471-2458-13-799

Thomas, E. L., Milligan, T. W., Joyner, R. E., & Jefferson, M. M. (1994). Antibacterial activity of hydrogen peroxide and the lactoperoxidase-hydrogen peroxide-thiocyanate system against oral streptococci. Infect Immun, 62(2), 529-535.

Toxnet. (2014, September 9, 2014). Chlorpyrifos: Environmental Fate/Exposure Summary. 2019, from https://toxnet.nlm.nih.gov/cgi-bin/sis/search2/f?./temp/~1NEBSj:3

Trasande, L. (2017). When enough data are not enough to enact policy: The failure to ban chlorpyrifos. PLoS Biol, 15(12), e2003671. doi: 10.1371/journal.pbio.2003671

Tseng, C.-C., & Li, C.-S. (2007). Inactivation of Viruses on Surfaces by Ultraviolet Germicidal Irradiation. Journal of Occupational and Environmental Hygiene, 4(6), 400-405. doi: 10.1080/15459620701329012

USEPA. (2006). Reregistration Eligibility Decision (RED) for Chlorpyrifos. Washington, DC.

USEPA. (n.d). Ozone Generators that are Sold as Air Cleaners. Retrieved October 8, 2019, from https://www.epa.gov/indoor-air-quality-iaq/ozone-generators-are-sold-air-cleaners#harmful-ozone

USEPA. (n.d.-a). Appendix 1-9. Degradate Line of Evidence.

USEPA. (n.d.-b). Ultraviolet Irradiation + Ozone Drinking Water Treatability Database.

Utzig, L. M., Lima, R. M., Gomes, M. F., Ramsdorf, W. A., Martins, L. R. R., Liz, M. V., & Freitas, A. M. (2019). Ecotoxicity response of chlorpyrifos in Aedes aegypti larvae and Lactuca sativa seeds after UV/H2O2 and UVC oxidation. Ecotoxicology and Environmental Safety, 169, 449-456. doi: 10.1016/j.ecoenv.2018.11.003

Ventura, C., Zappia, C. D., Lasagna, M., Pavicic, W., Richard, S., Bolzan, A. D., . . . Cocca, C. (2019). Effects of the pesticide chlorpyrifos on breast cancer disease. Implication of epigenetic mechanisms. J Steroid Biochem Mol Biol, 186, 96-104. doi: 10.1016/j.jsbmb.2018.09.021

Vogelpohl, A. (2003, May 18-22). Proceedings of the 3rd Conference on Oxidation Technologies for Water and Wastewater Treatment, Goslar, Germany.

Walker, B., Steffen, W., Canadell, J., & Ingram, J. (1999). The terrestrial biosphere and global change: implications for natural and managed ecosystems (Vol. 4): Cambridge University Press.

Wang, S., Wang, J., Wang, T., Li, C., & Wu, Z. (2019). Effects of ozone treatment on pesticide residues in food: a review. International journal of food science & technology, 54(2), 301-312.

Watanabe, M., Masaki, H., Mori, T., Tsuchiya, T., Konuma, H., Hara-Kudo, Y., & Takatori, K. (2010). Inactivation Effects of UV Irradiation and Ozone Treatment on the Yeast and the Mold in Mineral Water. J Food Prot, 73(8), 1537-1542. doi: 10.4315/0362-028x-73.8.1537

http://www.epa.gov/indoor-air-quality-iaq/ozone-generators-are-sold-air-cleaners#harmful-ozone

http://www.epa.gov/indoor-air-quality-iaq/ozone-generators-are-sold-air-cleaners#harmful-ozone

86

Whangchai, K., Uthaibutra, J., Phiyanalinmat, S., Pengphol, S., & Nomura, N. (2011). Effect of Ozone Treatment on the Reduction of Chlorpyrifos Residues in Fresh Lychee Fruits. Ozone: Science & Engineering, 33(3), 232-235. doi: 10.1080/01919512.2011.554313

Williamson, L. N., Terry, A. V., Jr., & Bartlett, M. G. (2006). Determination of chlorpyrifos and its metabolites in rat brain tissue using coupled-column liquid chromatography/electrospray ionization tandem mass spectrometry. Rapid Commun Mass Spectrom, 20(18), 2689-2695. doi: 10.1002/rcm.2647

Wu, J. G., Luan, T. G., Lan, C. Y., Lo, W. H., & Chan, G. Y. S. (2007). Efficacy evaluation of low-concentration of ozonated water in removal of residual diazinon, parathion, methyl-parathion and cypermethrin on vegetable. Journal of Food Engineering, 79(3), 803-809. doi: https://doi.org/10.1016/j.jfoodeng.2006.02.044

Xie, Y., Hajdok, C., Mittal, G. S., & Warriner, K. (2008). Inactivation of MS2 F (+) coliphage on lettuce by a combination of UV light and hydrogen peroxide. Journal of food protection, 71, 903-907.

Yang, T., Doherty, J., Zhao, B., Kinchla, A. J., Clark, J. M., & He, L. (2017). Effectiveness of commercial and homemade washing agents in removing pesticide residues on and in apples. Journal of Agricultural and Food Chemistry, 65(44), 9744-9752.

Zalat, O. A., Elsayed, M. A., Fayed, M. S., & Abd El Megid, M. K. (2014). Validation of UV Spectrophotometric and HPLC Methods for Quantitative determination of chlorpyrifos. . International Letters of Chemistry, Physics and Astronomy(2), 58-63.

Ziska, L. H., Teasdale, J. R., & Bunce, J. A. (1999). Future atmospheric carbon dioxide may increase tolerance to glyphosate. Weed Science, 47(5), 608-615. doi: Doi 10.1017/S0043174500092341

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