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9 2079 9510

This is to certify that the

dissertation entitled

Postharvest Treatment to Reduce or Remove
Ethylenebisdithiocarbamate (EBDC) Fungicides
from Apples and Apple Products & Elucidation

of Possible Degradation By—products and Pathways

presented by

Eun-Sun Hwang

has been accepted towards fulfillment
of the requirements for

Ph.D. . Food Science/
degree in

Environmental Toxicology

 

 

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ajor pro essor

Dae 12/06/99

MS U is an Affirmative Action/Equal Opportunity Institution 0- 12771

PLACE IN RETURN BOX to remove this checkout from your record.
TO AVOID FINE return on or before date due.
MAY BE RECALLED with earlier due date if requested.

 

DATE DUE l DATE DUE DATE DUE

 

@2015th

 

I-JCT 2 5 2005

 

6113 '13

 

 

 

 

 

 

 

 

 

 

 

 

11/00 mm.w5-p.14

 

 

POSTHA
REMOVE
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in

POSTHARVEST TREATMENT TO REDUCE OR
REMOVE ETHYLENEBISDITHIOCARBAMATE
(EBDC) FUNGICIDE RESIDUES FROM
APPLES & APPLE PRODUCTS AND
ELUCIDATION OF POSSIBLE DEGRADATION

BY—PRODUCTS & PATHWAYS

By

Eun—Sun Hwang

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Food Science & Human Nutrition
Institute for Environmental Toxicology

1999

 

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ABSTRACT

POSTHARVEST TREATMENT TO REDUCE OR REMOVE
ETHYLENEBISDITHIOCARBAMATE (EBDC) FUNGICIDE RESIDUES
FROM APPLES AND APPLE PRODUCTS & ELUCIDATION OF
POSSIBLE DEGRADATION BY—PRODUCTS AND PATHWAYS

By

Eun—Sun Hwang

The overall goal of this research was to reduce or eliminate
mancozeb residues in apples and apple products, determine the
effectiveness of different postharvest treatments and processing on the
reduction of mancozeb and ethylenethiourea (ETU) residues and
elucidate possible degradation products and pathways of this pesticide
when treated with various oxidation agents.

In the first part of the research, laboratory studies were
conducted using a model system to determine the effects of calcium
hypochlorite (50, 250 85 500 ppm), chlorine dioxide (5 8t. 10 ppm), ozone
(1 8r, 3 ppm) and hydrogen peroxyacetic acid (HPAA) (5 8B 50 ppm) at pH
4.6, 7.0, 10.7 and at 10°C and 21°C on the degradation of mancozeb in
solution over a 30 minute period. Rate of mancozeb degradation was
dependent on pH, with pH 7.0 being the most effective. Under controlled
conditions, ETU residue concentrations increased up to 15 minutes

reaction time and then decreased in all three pH ranges. Ozonation was

decree in 1::-
Ci‘t'r'ze dz):
concenraizon

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effective in the degradation of ETU residue in mancozeb solution.
Chlorine dioxide was an excellent degradation agent at low
concentration.

The second part of this study included laboratory whole fruit
studies. Mancozeb was spiked on the surface of apples at two different
concentrations and the effectiveness of each oxidizing agent was
determined on the reduction and degradation of mancozeb and ETU
residues on actual fruit as compared to the solution experiments. The
results showed similar patterns to the model system studies.

In the third part of this study, mancozeb was applied on
orchard apples throughout the growing season at the recommended rate.
Postharvest wash treatments were used, based on results of the model
system study: (1) no wash, (2) water wash, (3) calcium hypochlorite wash
@ 50 and 500 ppm (4) chlorine dioxide wash @ 10 ppm (5) ozone wash @
3 ppm and (6) HPAA wash @ 50 ppm. Wash treated apples were
processed as whole fruits, slices, sauce (peeled and unpeeled), juice and
pomace and frozen at —20°C until residue analysis. When wash
treatments were combined with processing, mancozeb and ETU were
reduced by 100% (i.e., below detectable limits).

The last part of this study involved investigation of degradation
products and possible pathways during chemical oxidation reaction.
Samples were detected by Time—of—Flight Mass Spectrometry (TOFMS)
with an electron ionization source. Several degradation by—products were

detected and identified.

Copyright©
by

Eun—Sun Hwang

1999

 

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I dedicate this dissertation to my beloved parents and God.
For my parents, whose unspoken love, expectations and understanding
have always been treasured by their daughter.
I thank God for my very good fortune for

I am living a very blessed life indeed.

1 WSh 1;. r-
in are Wd‘s‘ 0T ‘-

smdr. I have be.”

 

 

 

 

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ACKNOWLEDGEMENTS

I wish to express my sincere gratitude to all those people who have
in one way or another contributed to the successful completion of my
study. I have been blessed to meet numerous people with the knowledge
and patience necessary to aid me in my experience. In particular, I would
like to express very special thanks to my major advisor, Dr. Jerry N.
Cash, for his kind support, endless understanding, warm encouragement
and professional guidance. His support and trust in my ability to learn
gave me the determination to complete my Ph.D. program and this
dissertation.

I also would like to extend my sincere gratitude to my committee,
Dr. Matthew J. Zabik, Dr. Mark A. Uebersax, Dr. Gale Strasburg and Dr.
Alan L. Jones for their interest in my research, useful advice and
reviewing this manuscript. Especially, I am very grateful to Dr. Matthew
J. Zabik for his guidance and expertise with analytical instruments, as
well as many helpful suggestions with the direction and focus of my
research.

My thanks also go to Dr. Randy Beaudry who allowed me to use his
lab and mass spectrometry. I would like to acknowledge Gail Ehret for
providing the apple samples during last three years. Acknowledgment is
made to Michigan State University Agricultural Experimental Station and

Michigan Apple Committee for their support of this research.

vi

 

 

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I owe a great debt of thanks to my teachers, colleague and friends
who freely gave me a tremendous amount of technical and personal
support prior to and throughout this study. There are many people who
assisted, but unfortunately, they can not all be mentioned by name. Of
these people, I wish to give thanks to my lab members, Muhammad
Siddiq, Chris Vandervoort, Violet Morre and other fellow graduate
students for their help and friendship.

This undertaking would not have been possible without the
encouragement of my family across the Pacific. My parents, Byung—Eun
Hwang and Ki—Soon Kim, have been never ending sources of support and
encouragement. They always show me what is valuable in my life with
love. I also want to give special thanks to my beloved brother, Ki—Ho, and
my sister, Kyung—Sun. Their unlimited love, encouragement and prayers
have helped me to study abroad.

Finally, but most importantly, my deepest gratitude goes to my
father, almighty God, for spiritual guidance that I have received until
now and will be forever. He continues to remind me that all things are

possible while I am traveling against the wind.

vii

LIST OF TABLE
L'ST OF flG'L'F

LIST OF APPEX

 

 

LITERITLRE "'

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TABLE OF CONTENTS

Page
LIST OF TABLES .............................................................................. XIV
LIST OF FIGURES ........................................................................... XVI
LIST OF APPENDICES ..................................................................... XXI
LITERATURE REVIEW ..................................................................... 1
A. General Aspects of Pesticides .......................................... 1
B. The Fate of Pesticides in the Environment after Application 4
c_ General Aspects Of Fungicides ............................................. 8
D. EBDC Fungicides ............................................................... g
E. Toxicological PI‘OPCI‘IICS Of EBDCS ....................................... 14
F. Degradation Of EBDCS ...................................................... 16
G. Toxicological Properties Of ETU .......................................... 21
H. Formation of ETU during Heat Treatment ------------------------ 22
I. Degradation of Pesticide in Environment -------------------------- 25
J. Degradation of Pesticides in Solution ----------------------------- 28
(I) Hydrolysis .................................................................. 28
(II) Chemical Oxidation ................................................... 28
1) Chlorine ............................................................... 3O
2) Chlorine Dioxide ................................................... 34
3) Ozone .................................................................. 37
4) Hydrogen Peroxide ................................................ 40

K. Effect of Processing Operations on Pesticide Residues
in Foods ........................................................................... 41

L. Chemical By-Products and Degradation Pathways of

Pesticides ........................................................................... 46

CHAPTER I. STUDIES ON THE DEGRADATION OF PESTICIDES
IN A MODEL SYSTEM

INTRODUCTION .............................................................................. 48

MATERIALS AND METHODS ............................................................ 51

MATERIALS .................................................................................... 5 1
viii

 

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A. Reagents .............................................................................. 5 1

(I) Solvents ........................................................................ 51

(II) Chemicals ..................................................................... 51

B. Glassware .............................................................................. 52

METHODS ....................................................................................... 52

A. Sample preparation ............................................................ 53

(1) Calcium HypOChlorite ...................................................... 53

(11) Chlorine Dioxide ......................................................... 54

(III) Ozone ........................................................................ 55

(1V) Hydrogen Peroxyacetic Acid .......................................... 56

B. Pesticide Residue Analyses ................................................... 57

(I) Mancozeb ..................................................................... 57

(II) ETU .............................................................................. 58

C_ Chromatographic Analyses ................................................... 58

(I) Mancozeb ..................................................................... 58

(II) BTU .............................................................................. 59

D. Calculation of Pesticide Residue Concentration ------------------- 59

E. Statistical Analyses ............................................................ 60

RESULTS & DISCUSSION ............................................................... 62

A. Chromatographic Analysis ................................................... 62

(I) Mancozeb ........................................................................ 62

(II) ETU .............................................................................. 65

B, Degradation of Mancozeb in Solution .................................... 67

(I) Degradation of Mancozeb by Hydrolysis -------------------------- 67

(II) Degradation of Mancozeb by Calcium Hypochlorite ------------ 70

(III) Degradation of Mancozeb by Chlorine Dioxide ---------------- 77

(IV) Degradation Of Mancozeb by Ozone ................................. 83

(V) Degradation of Mancozeb by Hydrogen Peroxyacetic Acid "'89

C. Degradation of Mancozeb into ETU in Solution ----------------------- 95

(I) Degradation of BTU by Hydrolysis ................................. 95

(II) Degradation of ETU by Calcium Hypochlorite -------------- 97

(III) Degradation of ETU by Chlorine Dioxide ----------------------- 100

(IV) Degradation of ETU by 020116 ....................................... 103

(V) Degradation of ETU by Hydrogen Peroxyacetic Acid -------- 103

SUMMARY 85 CONCLUSIONS ......................................................... 109
ix

 

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CHAPTER II. STUDIES ON THE DEGRADATION OF PESTICIDES

IN SPIKED APPLES
INTRODUCTION ........................................................................... 11 1
MATERIALS AND METHODS ......................................................... 114
MATERIALS ................................................................................. 1 14
A. Apple Samples ..................................................................... 114
B. Reagents ........................................................................... 114
(I) SOIVCI‘IIS ........................................................................ 114
(11) Chemicals .................................................................. 1 15
C. Glassware ........................................................................ I 15
METHODS ................................................................................. I 15
A_ Sample Extraction ............................................................ 1 16
B. Pesticide Residue Analyses ............................................. 117
(I) Mancozeb ............................................................... 117
(II) ETU ........................................................................ 1 18
C. Chromatographic Analyses ............................................. 118
(I) Mancozeb ............................................................... 118
(III ETU ........................................................................ 119
D. Calculation of Pesticide Residue Concentration --------------- 119
E. Statistical AnaIYSes ................................................... 120
RESULTS 85 DISCUSSION ............................................................... 122
A. Recovery Stlldy ............................................................ 122
B. Degradation of Mancozeb in Spiked Apples --------------------- 123
C. Comparison of the Effects of Various Oxidizing Agents on the
Degradation of Mancozeb Residues ................................. 12 8
D. Degradation of Mancozeb into BTU in Spiked Apples -------- 136
SUMMARY & CONCLUSIONS ......................................................... 143

CHAPTER III. STUDIES ON THE DEGRADATION OF PESTICIDES
IN FRESH AND PROCESSED APPLES

INTRODUCTION ........................................................................... 145

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MATERIALS AND METHODS ......................................................... 148

MATERIALS ................................................................................. 148
A. Apple Samples .................................................................. 148
B. Reagents ........................................................................... 148

(I) Solvents ..................................................................... 148
(H) Chemicals ............................................................... 149
C. GIassware ........................................................................ 149

METHODS ................................................................................. 150
A. Pesticide Application and Spray Schedule ------------------------- 150
B_ Apple Sampling and Harvesting .......................................... 151
C. Apple Post Harvest Treatments .......................................... 155

(I) Wash Treatments ...................................................... 155

(II) SampIe processing ...................................................... 156

I. Slices ............................................................... 157

2. Sauce, peeled and Unpeeled ................................. 157

3. Juice .................................................................. 158

4. pomace ............................................................... 158

D. Pesticide Residue Analyses ................................................... 158
(I) Mancozeb .................................................................. 158

(II) E’I‘U ........................................................................ 159

(III) Recovery Study ......................................................... 160

E. Chromatographic Analyses ................................................... 160
(I) Mancozeb Residue Analyses ....................................... 160

(11) ETU Residue Analyses ................................................ 161

F. Calculation of Pesticide Residue Concentration ------------------- 161
G. Statistical Analysis ............................................................ 162

RESULTS 85 DISCUSSION

A. Pesticide Residues in Unprocessed Apples -------------------------- 164
(I) Effect of PHI on the Pesticide Residue Levels -------------------- 164
(II) Apple Processing 85 product Yield .................................... 167

B_ Recovery Study .................................................................. 173

C. Mancozeb Residue Study ...................................................... 174
(I) Comparison of Postharvest Wash Treatment on the Reduction

of Mancozeb Residues ...................................................... 174

(II) Comparison of Percent Reduction of Mancozeb Levels ----- 181
(III) Comparison of Mancozeb Residue Levels between

products ..................................................................... 189

xi

 

 

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INTRODC C T1 Q .‘~

.....
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D. ETU Residue Study ............................................................ 191
(I) Comparison of Postharvest Wash Treatment on the Reduction

of ETU Residues ............................................................ 191

(11) Comparison of Percent Reduction of ETU Levels ------------ 198

(III) Comparison of ETU Residue Levels between Products ------ 205
SUMMARY 85 CONCLUSIONS ......................................................... 206

CHAPTER IV. STUDIES OF THE DETERMINATION OF THE
DEGRADATION PRODUCTS & PATHWAYS

INTRODUCTION ........................................................................... 208
MATERIALS AND METHODS ......................................................... 213
MATERIALS ................................................................................. 2 13
A. Reagents ........................................................................... 213
(I) Solvents ..................................................................... 213
(II) Standard Chemicals ................................................... 213
B. Glassware ........................................................................ 214
METHODS
A_ Ozonation Procedure ...................................................... 214
B. Chlorination procedure ................................................... 215
C. Sample Extraction ............................................................ 216
D. GC/MS Analysis ............................................................ 219
RESULTS 81. DISCUSSION
A, By—Produets Formed from Hydrolysis .................................... 220
(I) Degradation of Mancozeb ................................................ 220
(II) Degradation of ETU ...................................................... 221
(III) Effect of pH on the Formation of Mancozeb Degradation
Product ..................................................................... 227
B. By-Products Formed from Ozonation -------------------------------- 232
(I) Degradation of Mancozeb ............................................. 232
(II) Degradation Of ETU ...................................................... 233
C. By-Products Formed from Chlorine Dioxide ----------------------- 236
(1) Degradation of Mancozeb ................................................ 236
(II) Degradation of ETU ...................................................... 241

xii

 

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SUMMARY & CONCLUSIONS ......................................................... 247
FUTURE WORK ........................................................................... 249
APPENDIX .................................................................................... 250
BIBLIOGRAPHY ........................................................................... 279

xiii

 

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Table 1.
Table 2.
Table 3.
Table 4.
Table 5.

Table 6.

Table 7.

Table 8.

Table 9.

LIST OF TABLES

Page
Chemical and physical properties of the Mancozeb -------------- 13
Chemical and physical properties of 'the ETU ------------------------ 19
Oxidation—reduction potentials of various compounds --------- 29

The effects of processing on pesticide residues in fruits and
vegetables ........................................................................... 42
Recovery (0/0) i SD (n=3) for the Mancozeb on apple

samples ........................................................................... 122
Effects of various oxidants on the Mancozeb residue
concentrations and % remaining of Mancozeb at 1 ppm
Mancozeb spiked level ...................................................... 132
Effects of various oxidants on the Mancozeb residue
concentrations and % remaining of Mancozeb at 10 ppm
Mancozeb spiked level ...................................................... 133
Effects of various oxidants on the ETU residue

concentrations and % remaining of ETU at 1 ppm Mancozeb
spiked level ..................................................................... 139
Effects of various oxidants on the ETU residue

concentrations and % remaining of ETU at 10 ppm Mancozeb
spiked ICVCI ..................................................................... 140

Table 10. 1997 Spray schedule for Cortland apples (77 day PHI) ------ 152

Table 11. 1998 Spray schedule for Golden Delicious apples

(4 day PHI) ..................................................................... 153
Table 12. 1999 Spray schedule for Golden Delicious apples ----------- 154
Table 13. 1998 Spray schedule for Cortland apples (Control) -------- 155

xiv

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Table 14.

Table 15.
Table 16.
Table 17.

Comparison of specific characteristics of Cortland and Golden

Delicious apples ............................................................ 165
Percent yields of processed apple products, 1997 ------------ 168
Percent yields of processed apple products, 1998 ---------- 170
Percent recovery of Mancozeb and ETU -------------------------- 174

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Figure 2.
Figure 3.
Figure 4.
Figure 5.
Figure 6.

Figure 7.
Figure 8.
Figure 9.

Figure 10.
Figure 11.

Figure 12.
Figure 13.
Figure 14.
Figure 15.
Figure 16.
Figure 17.
Figure 18.
Figure 19.

Figure 20.

LIST OF FIGURES

Page
Pesticide use of major crops, 1964-1994 ----------------------------- 3
The fate of pesticide in the environment after application ------ 5
Chemical SthtureS of EBDCS .......................................... 11
Degradation pathway of EBDCS ....................................... 18
Microbial degradation of some pesticides --------------------------- 27
Relative amounts of HOCl and OCl' formed at various pH
levels .............................................................................. 32
GC chromatogram of a Mancozeb standard ------------------------ 63
GC chromatogram of a Mancozeb sample ------------------------ 64
HPLC chromatogram of a ETU standard ----------------------- 66
HPLC chromatogram of a ETU sample .............................. 68
Effect of 50ppm Ca(OC1)2 on the degradation of 2 ppm
Mancozeb at 10 and 210C ............................................. 69
Effects of reaction time and temperature on the degradation
of Manoezeb at 50ppm Ca(OCl)2 .................................... '71
Effect of 250ppm Ca(OCl)2 on the degradation of 2 ppm
Mancozeb at 10 and 210C ............................................. 72
Effects of reaction time and temperature on the degradation
of Mancozeb at 250ppm Ca(OCl)2 .................................... '73
Effect of 500ppm Ca(OCl)2 on the degradation of 2 ppm
Mancozeb at 10 and 210C ................................................ 74
Effects of reaction time and temperature on the degradation
of Mancozeb at 500ppm Ca(OC1)2 .................................... 75
Effects of temperature and pH on the degradation of
Mancozeb in Ca(OCl)2 treatments .................................... 76
Effect of 5 ppm C102 on the degradation of 2 ppm Mancozeb
at 10 and 210C ............................................................ 78
Effects of reaction time and temperature on the degradation
of Mancozeb at 5 ppm C102 ............................................. 79
Effect of 10 ppm C102 on the degradation of 2 ppm Mancozeb

at 10 and 210C ............................................................ 8O

xvi

Figure 21.
Figure 22.
Figure 23.
Figure 24.
Figure 25.
Figure 26.
Figure 27.
Figure 28.
Figure 29.
Figure 30.
Figure 31.
Figure 32.
Figure 33.
Figure 34.
Figure 35.

Figure 36.
Figure 37.

Figure 38.
Figure 39.

Figure 40.

Figure 41.

Effects of reaction time and temperature on the degradation

of Mancozeb at 10 ppm C102 .......................................... 81
Effects of temperature and pH on the degradation of
Mancozeb in chlorine dioxide treatments ----------------------- 82

Effect of 1 ppm 03 on the degradation of 2 ppm Mancozeb

at 10 and 210C ............................................................ 84
Effects of reaction time and temperature on the degradation
of Mancozeb at 1 ppm 03 ................................................ 85
Effect of 3 ppm 03 on the degradation of 2 ppm Mancozeb

at 10 and 210C ............................................................ 86
Effects of reaction time and temperature on the degradation
of Mancozeb at 3 ppm 03 ................................................ 87
Effects of temperature and pH on the degradation of
Mancozeb in ozone treatments ....................................... 88
Effect of 5 ppm HPAA on the degradation of 2 ppm Mancozeb
at 10 and 210C ............................................................ 90
Effects of reaction time and temperature on the degradation

of Mancozeb at 5 ppm HPAA ............................................. 91
Effect of 50 ppm HPAA on the degradation of 2 ppm
Mancozeb at 10 and 21°C ................................................ 92
Effects of reaction time and temperature on the degradation
of Mancozeb at 50 ppm HPAA .......................................... 93

Effects of temperature and pH on the degradation of
Mancozeb in HPAA treatments ....................................... 94
Effect of pH and reaction time on the conversion of Mancozeb

into ETU in control ......................................................... 96
Effect of Ca(OCl)2 on the concentration of ETU with

time .............................................................................. 98
Effect of pH and reaction time on the conversion of Mancozeb
into ETU in Ca(OCl)2 treatments .................................... 99
Effect of C102 on the concentration of ETU with time ------ 101

Effect of pH and reaction time on the conversion of Mancozeb
into ETU in C102 treatments ....................................... 102
Effect of 03 on the concentration of ETU with time --------- 104
Effect of pH and reaction time on the conversion of Mancozeb

into ETU in ozone treatments ....................................... 105
Effect of HPAA on the concentration of ETU with
time ........................................................................... 106

Effect of pH and reaction time on the conversion of Mancozeb
into ETU in HPAA treatments ....................................... 107

xvii

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Figure 43.
Figure 44.
Figure 45.
Figure 46.
Figure 47.
Figure 48.
Figure 49.
Figure 50.
Figure 51.

Figure 52.
Figure 53.

Figure 54.
Figure 55.
Figure 56.
Figure 57.
Figure 58.
Figure 59.
Figure 60.

Figure 61.

Effect of Ca(0C1)2 on the degradation of Mancozeb in spike

apples ........................................................................ 124
Effect of C102 on the degradation of Mancozeb in spike
apples ........................................................................ 126
Effect of 03 on the degradation of Mancozeb in spike
apples ........................................................................ 127
Effect of HPAA on the degradation of Mancozeb in spike
apples ........................................................................ 129

Comparison of various oxidizing agents on the degradation of
1 ppm Mancozeb ...................................................... 130
Comparison of various oxidizing agents on the degradation of
10 ppm Mancozeb ...................................................... 131
Percent reduction of Mancozeb after various oxidizing agents

treatments ............................................................... 135
Comparison of various oxidizing agents on the conversion of

1 ppm Mancozeb into ETU .......................................... 137
Comparison of various oxidizing agents on the conversion of

10 ppm Mancozeb into ETU .......................................... 138
Percent reduction of ETU after various oxidizing agents

treatments .................................................................. 142
Effect of PHI on ETU residues in raw apples ----------------- 166
Concentration of Mancozeb residues in whole fruit after

postharvest wash treatment .......................................... 176
Concentration of Mancozeb residues in slices after

postharvest wash treatment .......................................... 177
Concentration of Mancozeb residues in unpeeled sauce

after postharvest wash treatment ................................. 178

Concentration of Mancozeb residues in peeled sauce after
postharvest wash treatment ....................................... 179

Concentration of Mancozeb residues in juice after postharvest
waSh treatment ......................................................... 180

Concentration of Mancozeb residues in juice pomace after

postharvest wash treatment .......................................... 182
Percent reduction of Mancozeb residues in whole fruit after
postharvest wash treatment .......................................... 183
Percent reduction of Mancozeb residues in slices after
postharvest wash treatment .......................................... 185
Percent reduction of Mancozeb residues in unpeeled sauce
after postharvest wash treatment ................................. 186

xviii

 

 

 

 

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Figure 62.
Figure 63.
Figure 64.
Figure 65.
Figure 66.
Figure 67.
Figure 68.
Figure 69.
Figure 70.
Figure 71.
Figure 72.
Figure 73.
Figure 74.
Figure 75.
Figure 76.
Figure 77.
Figure 78.
Figure 79.

Figure 80.
Figure 81.

Percent reduction of Mancozeb residues in peeled sauce after

postharvest wash treatment .......................................... 187
Percent reduction of Mancozeb residues in juice after
pOStharVCSt wash treatment .......................................... 188

Percent reduction of Mancozeb residues in juice pomace after

pOStharVeSt waSh treatment ....................................... 190
Concentration of ETU residues in whole fruit after
posthaI'VCSt waSh treatment .......................................... 192
Concentration of ETU residues in slices after postharvest
wash treatment ............................................................ 193
Concentration of ETU residues in unpeeled sauce after
postharvest wash treatment .......................................... 194
Concentration of ETU residues in peeled sauce after
postharvest waSh treatment .......................................... 195
Concentration of ETU residues in juice after postharvest
wash treatment ............................................................ 196
Concentration of ETU residues in juice pomace after
postharvest wash treatment .......................................... 197
Percent reduction of ETU residues in whole fruit after
pOStharveSt waSh treatment .......................................... 199
Percent reduction of ETU residues in slices after postharvest

wash treatment ......................................................... 200
Percent reduction of ETU residues in unpeeled sauce after

postharvest wash treatment .......................................... 201
Percent reduction of ETU residues in peeled sauce after
postharvest wash treatment ....................................... 202
Percent reduction of ETU residues in juice after postharvest
waSh treatment ............................................................ 203
Percent reduction of ETU residues in juice pomace after
postharvest wash treatment ....................................... 204
Overall mass analysis process in time—of—flight mass
Spectrometry ............................................................... 2 1 1
A typical spectrum of Mancozeb from (A) standard solution at
100 ppm in distilled water and (B) chloroform extract 222
The mass spectrum of Mancozeb obtained from library
seaI‘Ch ........................................................................ 223
Possible fragmentation of Mancozeb by hydrolysis --------- 224
Proposed degradation pathway of Mancozeb in aqueous
solution by hydrolysis ................................................... 225

xix

 

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Figure 82.
Figure 83.
Figure 84.
Figure 85.

Figure 86.

Figure 87.
Figure 88.
Figure 89.

Figure 90.
Figure 91.

Figure 92.

Figure 93.

A typical spectrum of ETU from (A) standard solution at

100 ppm in distilled water and (B) chloroform extract ----- 226
The mass spectrum of ETU obtained from library search-“228
The mass spectrum of ETU obtained from chloroform extract
at (A) 0 minute and (B) 60 minute reaction time in distilled
water ........................................................................... 229
Effect of ozone on time dependence of the GC/ MS response
on the formation of molecular ion (M+ 144) from CHC13 layer
(A) and CH2C12 layer (B) ................................................ 230
Effect of chlorine dioxide on time dependence of the GC/ MS
response on the formation of molecular Ion (M+ 144) from
CHC13 layer (A) and CHQCIQ layer (B) .............................. 231
Comparison of chromatogram of Mancozeb at (A) 0 minutes
and (B) 60 minutes in ozone treatment ---------------------- 234
Total ion current of ETU in ozone treatment at (A) 0 minutes
and (B) 60 minutes in chloroform extract --------------------- 235
The molecular ions found as ETU degradation products by
ozone ........................................................................ 237
Proposed degradation pathway of ETU in ozonation """"""" 240
Total ion current of ETU in chlorine dioxide treatment at

(A) 0 minutes and (B) 60 minutes in chloroform extract-”242
The molecular ions found as ETU degradation products by

Chlorine diOXidC ......................................................... 243
Proposed degradation pathway of ETU
by Chlorine dioxide ......................................................... 245

XX

LIST OF APPENDICES

Page
Appendix 1. A typical standard curve for Mancozeb standards -------- 250
Appendix 2. A typical standard curve for ETU standards ----------------- 251
Appendix 3. Raw data for Mancozeb residues (ppm) with 2 ppm spiked
Mancozeb in a model syStem ....................................... 252
Appendix 4. Raw data for ETU residues (ppb) with 2 ppm spiked
Mancozeb in a model system ....................................... 262
Appendix 5. Raw data for Mancozeb residues (ppm) in 1 and 10 ppm
Mancozeb Spiked apples ............................................. 267
Appendix 6. Raw data for Mancozeb residues (ppm) in apples and apple
products (1997) ......................................................... 270
Appendix 7. Raw data for ETU residues (ppm) in apples and apple
products (1997) ......................................................... 271
Appendix 8. Raw data for Mancozeb residues (ppm) in apples and apple
products (1998) ......................................................... 2'72
Appendix 9. Raw data for ETU residues (ppb) in apples and apple
products (1998) ......................................................... 2’75
Appendix 10 Field plot diagram ................................................... 278

xxi

A. General ASP

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LITERATURE REVIEW

A. General Aspects of Pesticides

Federal law defines a pesticide as “any substance or mixture of
substances intended for preventing, destroying, repelling, or mitigating
any pest” (CFR, 1988). Pesticides may also be described as any physical,
chemical or biological agent that will kill an undesirable plant or animal
pest (Ecobichon, 1996). Pesticide is a general term for many types of
products including insecticides, herbicides, fungicides and rodenticides.
Pesticides may be chemical or bacterial, natural or man—made. There are
approximately 320 active pesticide ingredients that are available in
several thousand different registered formulations (Hotchkiss, 1992). The
US. Environmental Protection Agency (EPA) reported over 811 million
pounds of pesticides, excluding wood preservatives and disinfectants,
used in US. agriculture in 1993, at a cost of $6.1 million (Schubert et
aL,1996)

Pesticide use in agriculture over the last several decades has
proven to be a great benefit to the production of food. Pesticides protect
crops by controlling insects, diseases, weeds, fungi (mold) and other

pests. They work because they are toxic to target organisms or otherwise

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disrupt natural processes necessary for the organisms’ survival. Pesticide
use has improved both the efficiency of growing crops and the quality of
food produced. Protecting crops from pests gives higher yields and better
quality, resulting in greater variety and availability of food at a low cost.
However, along with the benefits, there are the potential effects of trace
amounts of pesticide residues remaining on some commodities at the
time of harvest or sale to the general public. Pesticides are potentially
harmful to humans and can cause various health problems such as
cancer, birth defects, changes in genetic material that may be inherited
by the next generation (genetic mutations), and nerve damage, among
other debilitating or lethal effects.

Pesticides are applied directly to many crops, especially fresh
fruit and vegetables. Many factors can influence the nature and extent of
pesticide residues on a crop, such as sunlight, water, bacteria in the
soils and other physical factors. The resulting breakdown products may
be biologically inactive compounds or may be chemicals that are
themselves toxic (Cooley and Manning, 1995).

Figure 1 shows the pesticide use on major crops between 1964
and 1997. Pesticide use increased from 1964 to 1982 but decreased from
1982 through 1991. This is probably due to integrated pest management
(1PM) practices designed to maintain disease and pest control using

minimum levels of pesticide. After 1991, the overall pesticide use

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increased slightly. This indicates that consumers still demand good
sensory quality of products even though they have concerns about
pesticide residues. Food safety has received increased attention in recent
years as a major consumer concern. In several consumer surveys, 70—
80% of the respondents expressed concern about the health risks
associated with pesticide residues (Food Marketing Institute, 1992; Ott et
al., 1991). This has resulted in extensive research on the biological

efficacy and environmental fate of pesticides.

B. The Fate of Pesticides in the Environment after Application
Pesticides can be introduced directly into the environment in a
liquid phase, as a dispersion or solution, or in the solid phase, as a
powder, dust, microcapsule, or granule. The pesticides are exposed to
many agents capable of transforming them into various other forms.
After entering both target and non-target biota, pesticides are subjected
to attack by detoxification enzymes. However, the major proportion of an
applied pesticide does not immediately enter any organism, but remains
in soil, water or air where it is subjected to further transformation and
transport to different locations, as well as, uptake by organisms at that
site (Fuhr, 1982). Figure 2 is a simplified scheme illustrating the various
processes to which pesticides applied for plant protection are subjected

(direct application to soil and/ or to plant surface is the main route of

 

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Figure 2. The fate of pesticide in the environment after application

(Schubert et al., 1996).

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pesticide input to the environment). The fate of a pesticide in the
environment is governed by the retention, transformation, transport
processes and the interaction of these processes. Retention is the
consequence of interaction between the pesticide chemical and the soil
particle surface or soil components. The retention processes are
frequently described as adsorption or simply as sorption. Degradation
tends to decrease the chemical’s toxicity although occasionally the
metabolic products could be even more toxic than the parent compound.
Volatilization leads to the distribution of pesticides from the soil to the
atmosphere. Leaching leads to the movement of the pesticides toward the
ground waters and overland flows move the pesticides into surface
waters.

The air, water and soil in rural farming areas may be
contaminated with pesticides or their degradation products. Pesticides
also contaminate ecosystems and may produce harmful effects in
wildlife. At the same time, the vast majority of adverse effects due to
pesticides are largely unknown. Pesticide products to which we are
exposed are a combination of chemical ingredients that include the active
ingredients disclosed on the product label, which attack the target pest,
and “inert” ingredients. However, the active ingredients are usually the
smallest percentage of total ingredients, which are principally the

undisclosed or secret “inert” ingredients. This part of the formulation can

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be biologically and chemically active and even more toxic than the
actives, but are protected as trade secrets (Schubert et al., 1996). Beyond
these components of a formulation, a pesticide product also contains
contaminants and breakdown products or metabolites. These, too, can
be the most toxic part of the pesticide product (Schubert et al., 1996).

Of all forms of pesticide pollution, groundwater degradation is
especially serious, because groundwater is the source of public drinking
water. Once groundwater contamination is discovered, clean—up is often
neither technically nor economically feasible. The contamination of
groundwater by pesticides is quite extensive. In a 1988 report, EPA
documented the presence of 74 different pesticides in the groundwater of
32 states. In particular, EPA discovered widespread contamination by the
pesticides aldicarb, atrazine and alachlor. A more extensive EPA study
released in November 1990 found further evidence of contamination.
Based on sampling results, EPA estimated that 10.4 percent of
community water system wells and 4.2 percent of rural domestic wells in
the US. contaminated at least one pesticide or pesticide degradation
product. EPA’s survey reveals that at a minimum, over 1.3 million people
are drinking water contaminated with one or more pesticide from private

wells.

C, General As
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C. General Aspects of Fungicides

Fungicides haveithe longest history of the three main groups of
crop protection agents (insecticides, herbicides and fungicides) (Uesugi,
1998). They are derived from a variety of structures ranging from simple
inorganic compounds, such as sulfur and copper sulfate, through the
aryl- and alkyl-mercurial compounds and chlorinated phenols to metal-
containing derivatives of thiocarbamic acid (Ecobichon, I996).
Fungicides may be described as protective, curative or eradicative
according to their mode of action. Protective fungicides, applied to the
plant before the appearance of any phytopathic fungi, prevent infection
by either sporicidal activity or by changing the physiological environment
on the leaf surface. Curative fungicides are used when an infestation has
already begun to invade the plant, and these chemicals function by
penetrating the plant cuticle and destroying the young fungal mycelium
growing in the epidermis of the plant, preventing further development.
Eradicative fungicides control fungal development following the
appearance of symptoms, usually after sporulation, by killing both the
new spores and the mycelium and by penetrating the cuticle of the plant
to the subdermal level (Kramer, 1983).

To be an effective fungicide, a chemical must posses the
following pr0perties: (1) low toxicity to the plant but high toxicity to the

particular fungus; (2) active or capable of conversion (by plant or fungal

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enzymes) into a toxic intermediate; (3) the ability to penetrate fungal
spores or the developing mycelium to reach a site of action; and (4) forms
a protective, tenacious deposit on the plant surface that will be resistant
to weathering by sunlight, rain and wind (Cremlyn, 1978). This list of
properties is never fulfilled entirely by any single fungicides and all
commercially available compounds show some phytotoxicity, lack of
persistence due to environmental degradation and so forth. Thus, the
timing of the application is critical in terms of the development of the
plant as well as the fungus.

The topic of fungicidal toxicity has been extensively reviewed by
Hayes (1982) and Edwards et al. (1991). With a few exceptions, most of
these chemicals have a low toxicity to mammals. However, all fungicides
are cytotoxic and most produce positive results in the usual in vitro
microbial mutagenicity test systems. Public concern has been focused on
the positive mutagenicity test obtained with many fungicides and the

predictive possibility of both teratogenic and carcinogenic potential.

D. EBDC Fungicides

Ethylene bisdithiocarbamates (EBDCs) are one of the oldest and
most widely used classes of organic fungicides in the world. They were
first introduced during the 1940s and are widely used nonsystemic

fungicides with low water solubility, which results in the pesticide

 

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remaining as superficial deposits on the surface of treated crops. This
allows it to be partly removed by water, especially on non-waxy crops
such as strawberries (Federal Register, 1989). EBDCs have been used to
control some 400 pathogens on more than 70 crops worldwide and
approximately one—third of all fruits and vegetables in the United States
are treated with EBDCs (Banrc, 1987). The major crops are apples,
tomatoes, potatoes, grapes, bananas, corn and wheat. (EPA, 1989). The
EBDCs registered for food uses in the US. are mancozeb, maneb,
metiram, nabam and zineb (Lentza—Rizos, 1990). Figure 3 shows
chemical structures of major EBDCs. These organic fungicides are
usually more effective than inorganic fungicides because organic
molecules tend to be more compatible with fungal cells which are
surrounded by walls and membranes in which a lipid layer is important
in exchanging substances through the layer. EBDCs act on various sites
in fungal physiolgy. These types of multiple-site inhibiting fungicides,
which are also called multisite inhibitors, are liable to act on organisms
other than their targets. EBDCs are applied as their manganese and zinc
complex form (maneb or mancozeb). The solubility, activity, and stability
of the EBDCs are dependent of the metal ion form (Lentza—Rizos, 1990).
EBDCs fit well into integrated pest management (1PM) practices
designed to maintain disease and pest control using minimum levels of

pesticide. One of the most important assets of EBDC fungicides is that,

10

 

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S S
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S S
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Figure 3. Chemical structures of EBDCs.

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in all their years of use, no known disease resistance to them has
developed, as is the case with many systemic fungicides (DuPont, 1992).
Because EBDCs act in a preventive mode, the pathogen does not have
the opportunity to infect the crop. EBDCs are also valuable in IPM
programs because they are not harmful to beneficial insects. This helps
reduce use of potentially more toxic pesticides. EBDCs are contact
fungicides, which remain on the surface of the plant. A synergistic effect
occurs when EBDCs are used with copper (DuPont, 1992).

Mancozeb (Dithane 75 DF®) is registered as a general use
pesticide by the US. Environmental Protection Agency (EPA). It is a
polymeric complex of ethylene bisdithiocarbamate manganese and zinc
salt. It contains 75% of ethylene bisdithiocarbamate in which the
ingredients are 15% of manganese, 1.87% of zinc and 58.13% of ethylene
bisdithiocarbamate ion (C4H6N2S4) and 25.00% of inert ingredients. It is
one of the most widely used EBDC fungicides to protect many fruits,
vegetables, nuts and field crops against a wide spectrum of diseases,
including potato blight, leaf spot, scab on apples and pears and rust on
roses (DuPont, 1992). It is also used for seed treatment of cotton,
potatoes, corn, safflower, sorghum, peanuts, tomatoes, flax and cereal
grains (Hayes and Laws, 1990; Meister, 1992). It is a grayish powder,

practically insoluble in water and in most organic solvents. Mancozeb is

12

Table 1- Che!
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Table 1. Chemical and physical properties of the Mancozeb
(Rohm & Haas Co., 1997)

 

 

Structure:

S S

|| ||
[—MnSCNHCHgCHgNHCS—]x [Zn2+]y

Common Name: Mancozeb

CAS Register No.2 8018—01—7

Trade Name: Dithane

Molecular Weight: ?

Manufacturer: Rohm 8r, Haas Company

Physical Form: Yellow powdered solid

Odor Characteristic : Musty odor

Melting Point : 192 to 204 °C / 378 to 399°F

Vapor Pressure: Negligible

Specific Gravity (Water = 1) 0.35 to 0.50 g. /cc. Bulk Density

Stability Media: Stable; However, keep away from moisture,
heat or flame.

Solubility in Water : Dispersible

Percent Volatility : 1% Water

 

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available as dusts, liquids, water—dispersible granules, as wettable

powders and as ready—to—use formulations (Meister, 1992).

E. Toxicological Properties of EBDCs

The EBDCs, which include mancozeb, are generally considered
to have low short-term toxicity to mammals. No toxicological effects were
observed in a long term study with rats fed doses of 5 mg/ kg (Hayes and
Laws, 1990). The major routes of exposure to mancozeb are through the
skin or from inhalation (US. EPA, 1987). In spray or dust forms, the
EBDCs are moderately irritating to the skin and respiratory mucous
membranes. Symptoms of poisoning from this class of chemicals include
itching, scratchy throat, sneezing, coughing, inflammation of the nose or
throat and bronchitis (Morgan, 1982; OHS, 1991). There is no evidence of
‘neurotoxicity’, nerve tissue destruction or behavior change, from the
EBDCs (Morgan, 1982). However, dithiocarbamates are partially
chemically broken down or metabolized to carbon disulfide, a neurotoxin
capable of damaging nerve tissue (Hallenbeck and Cunningham—Burns,
1985). The oral LDso for mancozeb ranges from 4,500 to 11,200 mg/ kg in
rats. When applied to the skin of rabbits, its dermal LDso is 5,000 to
15,000 mg/kg (Berg, 1988; US. EPA, 1987; Hayes and Laws, 1990;
Meister, 1992). It is a mild skin irritant and sensitizer and a mild to
moderate eye irritant in rabbits (DuPont, 1983). Agricultural workers

14

“*4‘uv‘ 1'.
haluAAAlg CTC:
rasnes (Hajve'
. c ‘ O A.
lflllCch’G 31a.

emflNOELifi

TE: In '11le 1'.

did La\\‘s
0 7“- fl‘
:uxTaugn Tc

handling crops treated with mancozeb have developed sensitization
rashes (Hayes and Laws, 1990). A two—year feeding study on rats
indicated that 6.25 mg/ kg of maneb in the diet is the no observable effect
level (NOEL) for rats. However, the next and highest level that was fed to
rats in this two—year study did produce signs of poisoning. A one—year
feeding study in dogs concluded that 20 mg/kg/day is a NOEL for dogs.
Toxic effects were seen in the dogs at daily doses of 75 mg/ kg and 250
mg/ kg (DuPont, 1983).

In a three—generation rat study with mancozeb at a dietary level
of 50 mg/ kg there was reduced fertility but no indication of embryo toxic
or teratogenic effects. In another study in which pregnant rats were
exposed to mancozeb by inhalation, toxic effects on the embryos were
observed only at doses (55 mg/ m3) that were also toxic to mothers (Hayes
and Laws, 1990). No teratogenic effects were observed in a three-
generation rat study with mancozeb at a dietary level of 50 mg/ kg (Hayes
and Laws, 1990). Specific developmental abnormalities of the body wall,
central nervous system, eye, ear and musculoskeletal system were
observed in experimental rats which were given 1,320 mg/ kg of
mancozeb on the 11th day of pregnancy (NIOSH, 1986). When it was
inhaled at concentrations of 0.017 mg/ L, mancozeb was not teratogenic
to pregnant rats (DuPont, 1983). Teratogenic activity was found in mice

given 1,320 mg/ kg of maneb (Shepard, 1989).

15

N3:
in chronic fee-
Mgneozeb pit.
tzrnes per wee.
tumors were :
shear: rapzd r-
fie. goizer). .‘.f
uptake was r:

maneb. anome

F‘ Degradatim

oxygen. and ir‘.

:egraded in If”.

lined,

C .41 [he en\.lr(
“N
t. l ,

L ETL has
50.

 

Non—tumorigenicity was reported for maneb, zineb and nabam
in chronic feeding studies on three strains of mice (Lentza—Rizos, 1990).
Mancozeb produced skin tumors in mice at 100 mg/ kg body weight, 3
times per week for 31 weeks. Historical examination revealed that these
tumors were mostly benign (Shukla et al., 1990). Several studies have
shown rapid reduction in the uptake of iodine and swelling of the thyroid
(i.e. goiter). Morgan (1982) found that a marked reduction of iodine
uptake was measured 24—hours after administration of a large dose of

maneb, another EBDC fungicide.

F. Degradation of EBDCs

The EBDCs are generally unstable in the presence of moisture,
oxygen, and in biological systems (US EPA, 1992). They are easily
degraded in these conditions and several degradation products are
formed, including ethylenethiourea (imidazolidine—2—thione, ETU)
(Lentza—Rizos, 1990). This rapid degradation lowers the need for concern
about the environmental fate of EBDCs and focuses such concern on
ETU. ETU has been identified as an impurity in commercial EBDC
formulations (Clarke et al., 1951; Bontoyan et al., 1972). Most
commercial EBDC formulations contain 0.02—5% of ETU (Bontoyan et al.,
1977). It has been reported that ETU occurs as a result of metabolic

(Engst and Schnaak, 1974) and chemical (Fishbein and Fawkes, 1965;

16

Ercst and SC?
1115 been ic‘er‘.
sprayed “1:3:
Newsome. l9

1'

it the Iowan;

“ W“
i'J1l"
mt dtz‘fi‘
SEC);-

Engst and Schnaak, 1974) alterations of the commercial fungicides. ETU
has been identified on a number of different crops which had been field-
sprayed with a commercial formulation of EBDC (Yip et al., 1971;
Newsome, 197 2). Cooking of foods containing EBDC residues also results
in the formulation of ETU (Newsome and Laver, 1973; Watts et al., 1974).

Engst and Schnaak (1974) suggested a possible degradation
scheme for metabolic derivatives of the ethylenebisdithiocarbamate
(Figure 4), speculating that ethylenebisdithiocarbamic acid readily forms
ETU under highly alkaline conditions (pH 10.5) and that ETU obtained
under these conditions may be formed from ethylenethiuram
monosulphide (ETM) by the loss of a molecule of carbon disulfide.

ETU has been known to be a possible degradation product of
EBDC fungicides for over 40 years (Clarke et al., 1951; Fishbein and
Fawkes, 1965; Bontoyan et al., 1972). It may be formed during
manufacture or storage of the EBDCs (Fishbein and Fawkes, 1965) on
plants following application of EBDC formulations, or in food containing
EBDC residues during cooking and processing procedures (Watts et al.,
1974). Pesticide degradation during storage results mainly from
hydrolysis and oxidation (Egli, 1982). Photolysis may not be an
important degradative reaction during storage since samples are usually
stored in the dark at -20°C. Oxidation, especially, is an important

reaction for readily oxidizable thio compounds. ETU is degraded from

17

‘1'

 

' "‘6:

“gm 4. DeS

 

 

 

 

 

 

 

 

 

CHI-N—C’
ca, 8
\/\ /
N S
[e
s +
I
on 411-6-qu -NH cu —NH
Oxidation ’ . 1 2 a a
Wz-NH-f-SH cm-uu-g-sa eta-nu,
8 s
[-st [-1133
i i
3-NHjI-6CH3-NH—‘Ks—CH1-NH-C-5H a—Nflz
. 4:,
g-NH-c-s ”Irma-1.4.; 332-".c.s CH 1"" -¢ .6
8
ETO" m0 .4135
CHz-N'C's
3-N'C'5
4:33
.3
ETU
a
-s ‘—
h car-NH
"In“ Oxidation

“la-\c cure?
............ 1...!“ 1.

Figure 4. Degradation pathway of EBDCs; + = polymeric products.

18

Table 2. Ch
al.

1 .
er- ,‘Y""
:4 \suA.<

A .......

53016011151:
Mm‘eféct;
Phl‘Sical F.
Odor Char

Xifi‘l‘wa

““1118 Po

Table 2. Chemical and physical properties of the ETU (VVindholz et
al., 1983; U.S. EPA 1986)

 

 

Structure:

(3201

/\
H—N N-H

H2C

 

CH2

Common Name: Ethylenethiourea

CAS Register No.: 9645—7

Chemical Name: imidazolidine—2—thione
Molecular Weight: 102.2
Manufacturer: Aldrich Company
Physical Form: White Crystals

Odor Characteristic : Musty odor
Melting Point : 203 °C /400°F

Vapor Pressure: —

Specific Gravity (Water = 1) 0.35 to 0.50 g./ cc. Bulk Density
Stability Media: Stable

Solubility in Water (30°C): 20g/ L

Percent Volatility : 1% Water

19

 

EBDC fun?

O
x

I.-
I

Nash. 19

1!‘ ’1 D~-v
18.01. ...

crops (Cni-

Marshall.

mancozeb. 1
at elevated

during cool;
The rate of :

uv,‘

EBDC fungicides in crops, rice (Rhodes, 1977; Ripley and Cox, 1978;
Nash, 1976), aqueous media (Marshall, 1977) and by heat (Newsome,
1976). During storage, ETU has been found to be unstable in certain
crops (Uno et al., 1978) and tomato sauce and paste (Ankumah and
Marshall, 1984). ETU is soluble in water and readily absorbed and
metabolized by plants (Engst and Schnaak, 1974; Newsome and Laver,
1973). It is a common contaminant in technical grade fungicides such as
mancozeb, maneb, zineb, and nabam. It may also be formed from EBDC
at elevated temperatures, high humidity, environmental degradation or
during cooking of food containing EBDC residues (Meneguz et al., 1987).
The rate of degradation of EBDC’s to ETU is influenced by temperature,
available oxygen and pH of the system. (Marshall, 1977).

Several workers have reported the instability of ETU.
Cruickshank and Jarrow (1975) reported that ultraviolet light can
degrade ETU on a solid substrate such as silica gel to produce 2—
imidazolidone as the major product. ETU degradation was especially
rapid in the presence of photosensitizers such as acetonaphthone,
naphthaldehyde, methylene blue, benzophenone, and crystal violet. Ross
and Crosby (1973) found that dissolved oxygen and sensitizers such as
acetone or riboflavin degrade ETU in the presence of light. Marshall
(1979) reported the oxidative degradation of ETU by hydrogen peroxide

and hypochlorite.

20

G. Toxicol

fr3:1 ETL’.

EBDCS. No

cancer in e:

mung SOme

." .~
11 .
a,‘

"1 demo:

rats, CD~1 In

G. Toxicological Properties of ETU

A major toxicological concern surrounding the EBDCs comes
from ETU, an industrial contaminant and a breakdown product of
EBDCs. No suitable information was found in the available literature on
the health effects of ETU in humans. In animal studies, the acute oral
LDso for ETU was 1,832 mg/kg in rats (U.S. EPA, 1982). ETU has caused
cancer in experimental animals and has been classified as a Group B2
probable human carcinogen based on sufficient evidence from animal
studies by the EPA (US EPA, 1992). Because of the report of their
carcinogenic (IARC, 1974), mutagenic (Teramoto et al., 1977), goitrogenic
(Graham et al., 1975) and teratogenic (Teramoto et al., 1980) effects in
laboratory animals, ETU has become a major human health concern
among some consumer groups (Lentza—Rizos, 1990). Chernoff et al.
(1979) demonstrated the teratogenic effects of ETU in Sprague—Dawley
rats, CD—1 mice and golden hamsters. Based on the results of this study,
the no observable adverse effect levels (NOAELs) for maternal and
developmental toxicity were 40 mg/ kg/ day in the rat, 200 mg/ kg/ day in
the mouse and 300mg/ kg/ day in the hamster. A 90—day study of the
effects of ETU revealed a NOEL of 5 ppm (0.25 mg/kg/day) (Morgan,
1982; Hayes and Laws, 1990; US EPA 1992). Seiler (1973) described ETU
as exhibiting weak but significant mutagenic activity in Salmonella

typhimurium. A 2.5—fold increase in mutation frequencies was seen at

21

  
   
 
   

irzemediate ‘
coneennano:
fire test color.
(12.151975) re;
and female C:

GEEK? l‘S‘Ce'fi

intermediate concentrations (100 or 1,000 ppm/ plate), but at higher
concentrations (10,000 and 25,000 ppm), ETU was somewhat lethal to
the test colonies resulting in lower relative mutagenic indices. Graham et
al. (1975) reported that ETU was a follicular thyroid carcinogen in male
and female Charles River rats that were fed the compound for 2 years at
dietary levels of 250 and 500 ppm (approximately 12.5 and 25
mg/kg/day)-

The thyroid appears to be the primary target organ for ETU
toxicity in long-term exposure studies. Ulland et al. (1972) reported a
dose related increased incidence of hyperplastic goiter in male and
female rats fed ETU at 175 and 350 ppm (approximately 8.75 and 17.5
mg/ kg/ day) in their diet for 18 months. An increased incidence of simple
goiter was also reported in all treatment groups. Arnold et al. (1983)
showed that the thyroid effects of ETU administered in the diet for 7
weeks to male and female Sprague—Dawley rats were reversible when

ETU was removed from the diet.

H. Formation of ETU During Heat Treatment

The nonbiological degradation of EBDCs to ETU is accelerated
by heat treatment and EBDC residues are known to be converted to ETU
during normal industrial processing of field-treated produce (Newsome

and Laver, 1973; Watts et al., 1974; Marshall, 1977; Phillips et al., 197 7).

22

Tne comers

maimed CC?
1:116 and an;
pracessed pr.
and the ETL‘
tempered to :3
beax‘een higher

:16 same $8111

 

 

Ride rage of co!
S“4739165 shower

25()
a. 1.08 mg kg

The conversion of these surface residues to ETU during cooking,
blanching or other processing has been demonstrated on snap beans,
tomatoes (Newsome et al., 1975), carrots, spinach (Phillips et al., 1977)
and grapes (Ripley et al., 1978).

Ripley and Cox (1978) processed field—treated tomatoes, using
simulated commercial methods, into whole pack tomatoes and tomato
juice and analyzed these products for EBDC and ETU residues. In the
processed products, the EBDC concentration was reduced by 50—75%
and the ETU concentration was about the same or slightly elevated
compared to the unprocessed fruit levels. They found a good correlation
between higher EBDC concentrations and higher ETU concentrations in
the same sample. However, the variability of their results indicated a
wide rage of conversion due to processing. It should be noted that some
samples showed no detectable EBDC residue, but had ETU levels as high
as 0.08 mg/kg.

The fate of ETU in the sterile environment of a processed food is
controversial. It has been reported that ETU, during a 4—week storage (at
1.0 or 0.1 ppm), decreased to 1% of the initial amount in pickles, 1—5%
in apple sauce, 0.1—0.2% in tomato sauce, and 9-12% in spinach (Han,
1977). In contrast, Uno et al. (1978) have reported that ETU in tomato
Puree was stable for up to 200 days. Efficient decontamination

Procedures are available for the removal of EBDC surface residues from

23

 

9..-]: " I
climax h-‘C

n
{a
‘g I
'1)
53.
£1“
{/1
m

as ms 03’
Ross
mecczeb 33‘

EBDC residliL

 

 

reatrnent. AU

ITS;

r1.

ties. but

pesticide treat:
was prepared

149‘C for 15 h
prodUctionl. Tl
mancozeb 1'14.
L‘1e heat treat
TfSpeCtively). TL
331‘? Domace.

rem -
016d befo rr-

tomatoes and green beans prior to processing (Marshall and Jarvis,
1979; Marshall, 1982). A four—minute preprocessing wash with dilute
alkaline hydrochlorite followed by a 30—second dip in dilute sodium
sulfite was demonstrated to reduce field residues of EBDC and ETU to
the limits of analytical significance.

Ross et al. (1978) found apples field-treated nine times with
mancozeb and metiram contained, respectively, 0.17 and 0.50 mg/kg
EBDC residue and 0.01 and 0.03 mg/ kg ETU 42 days after the last
treatment. Apple juice made from this produce did not contain EBDC
residues, but 0.05 mg/kg ETU was present in samples from both
pesticide treatments. Dried pomace, which is used as a feed for livestock,
was prepared in a laboratory scale experiment by drying the apples at
149°C for 15 hours (a more severe treatment than in commercial pomace
production). This dried pomace contained surprisingly high levels of both
mancozeb (14.9 mg/ kg) and metiram (3.3 mg/ kg) residues considering
the heat treatment, and high levels of ETU (0.17 and 0.15 mg/kg,
respectively). These levels were attributed to the apple peel concentration
in the pomace. Apple sauce prepared from apples with the peel and cores
removed before grinding and cooking contained residues of EBDC and
ETU at the 0.09 and 0.05 mg/kg level, respectively, in the case of

mancozeb and 0.09 and 0.04 mg/kg in the case of metiram.

24

 

 

:g kg. ETL
tomatoes m
; . I
mashmg. True

canned whoit
1:18 juice the

““3113.

 

 

residues from

ally-tints (0.1:;

Von Stryk and Jarvis (1978) analyzed tomatoes sprayed with
maneb and mancozeb and found EBDC levels between 0.03 and 0.80
mg/kg. ETU was detected only in one sample at 0.03 mg/kg. The
tomatoes were processed into juice and canned whole fruits, after
washing. The juice contained more fungicide and ETU residues than the
canned whole fruits. This was attributed to the fact that in preparation of
the juice the skins were not removed, whereas for whole tomatoes they
were.

Cabras et al. (1987) reviewed the fate of EBDC and ETU
residues from vine to wine. According to the data given, most EBDC
residues are absorbed by scums and ETU residues may remain in
amounts <0.01 mg/ kg. However, Kakalikova et al. (1988) showed that
the amount of ETU varies in relation to the amount of EBDC residues
present on harvested grapes. Must and wine produced from grapes
treated with mancozeb 14 or 28 days before harvest contained detectable
ETU residues, whereas those made from grapes harvested 42 days after

treatment did not.

1. Degradation of Pesticide in the Environment
The principal degradation pathways for pesticides in
environment can be classified as physical, chemical, and biological

factors (Coats, 1991). Under field conditions, a combination of these

25

,
t.- fi’fi'

1&1.“-

.

—Ov'.'

fr‘ 1
‘ 1145‘

-~v\fl.v"

~..~"J.-
‘

hr», a
Hi J]; 65
.

’ 9
guW-C‘
\-
"sun“
-

!7_
.LCUD
A

A

‘ .
1'31‘

...__;Cf

P A
U, cahl'
K I

factors usually influences the breakdown of a pesticides and their
relative importance depends on the chemical, physical properties of
pesticides and their chemical structures. Environmental factors such as
moisture, temperature and various management practices also play an
important role in degradation of pesticides (Coats, 1991).

The two primary physical agents involved in the degradation
process are light and heat. Photolysis of pesticide residues is extremely
significant on vegetation, on the soil surface, in water and atmosphere
(Zepp, 1991). Direct photo reactions account for only a part of sunlight-
induced reactions. Other photochemical reactions which produce
reactive transients such as hydroxyl, hydroperoxyl/superoxide,
organoperoxyl and other radicals as well as singlet molecular oxygen may
influence the fate of pesticides in the environment. Thermal
decomposition of the chemicals often occurs. Cold, especially freezing
temperatures, can also contribute occasionally to pesticide degradation
(Coats, 1991).

Chemical degradation occurs as a result of the various reactive
agents in the formulations, tank mixes and in the environment. Water is
responsible for considerable breakdown of pesticides in solution,
especially in conjunction with extremes of pH. Even slight variance from
a neutral pH can cause rapid decomposition of pH-sensitive compounds.

Molecular oxygen and its several more reactive forms (e.g., ozone,

26

superoxide, peroxides) are capable of reacting with many chemicals to
generate oxidation products. Chemical oxidations as well as reductions
can progress in the presence of inorganic, mostly metallic reagents (Zepp,
1991)

Microorganisms such as bacteria and fungi represent the most
important group of pesticide degraders in soil and water (Racke and
Coats, 1990). Pesticides can be utilized as a nutrient or energy source by
microorganisms, mainly bacteria that have adapted (following repeated
exposure) to utilize the pesticide molecule as a source of carbon or
nitrogen. This requires an initial hydrolysis of the pesticide, followed by
the utilization of at least one metabolite as a nutrient (Figure 5). Plants,
invertebrates and vertebrates are further degradation agents. The latter
group possesses the most sophisticated enzymatic system capable of
biodegrading xenobiotics (Moffat and Whittle, 1999). These systems are
most effective in birds and mammals; the spectrum of transformation
reactions is very broad and the rates of detoxification and elimination are

typically high (Moffat and Whittle, 1999).

Pesticide ED Degradation CI) Nutrient
Hydrolysis Product Catabolism

Figure 5. Microbial degradation of some pesticides.

27

J. Degradation of Pesticides in Solution
(1) Hydrolysis

For many pesticide molecules, hydrolysis is a primary route of
degradation. Laboratory studies on the effect of pH and temperature on
the breakdown of pesticides in aqueous solution have been conduced to
provide information on their relative persistence. Many types of esters are
hydrolytically cleaved, yielding two fragments with little or no pesticidal
activity. Hydrolysis of esters can occur by hydrolytic decomposition of
some esters, while acid—activated hydrolysis typically is induced only by

strongly acidic solutions (e.g., pH 3—4).

(II) Chemical Oxidation

Chlorine, chlorine dioxide, potassium permanganate and ozone
have been employed historically for the oxidation of organic compounds
at water treatment plants and were consequently investigated for their
capacity to degrade organic pesticides (Gomma and Faust, 1974; Cash et
al, 1997).

The capability of one substance to oxidize another is measured
by its oxidation potential, normally expressed in volts of electrical energy.
The oxidation potential is a measure of the relative ease by an atom, ion,

molecule or compound to lose electrons, thereby being converted to a

28

 

 

 

 

 

 

 

 

 

 

 

 

7..
na- 0‘”

higher state of oxidation. In general, the higher the oxidation potential,
the stronger it is as an oxidant. As indicated in Table 3, HOCl is a
stronger oxidizing agent (1.49V) than is free chlorine (1.36V), so that
HOCl is actually more desirable when using chlorine as an oxidant in

aqueous solution.

Table 3. Oxidation—reduction potentials of various compounds

 

 

Reactions Potential In Volts (E°) 25°C
F2 + 2e -—> 2F" 2.88
03 + 2H+ + 2c -> 02 + H2O 2.07
H202 + 2H“ + 2e —> 2H2O (acid) 1.76
Mn04’ + 4H" + 3e —> MnO2 + 2H2O 1.68
HClO2 + 3I-I+ + 4e —> C1' + 21-120 1.57
MnO4‘ + 8H+ + 5e —-> Mn2+ + 4H2O 1.49
HOC1+ H" + 2c —> C1" + H20 1.49
C12 + 2e —> 2Cl’ 1.36
HOBr + H+ + 2e —> Br‘ + H2O 1.33
03 + H2O + 2e —> 02 + 2OH‘ 1.24
C102 (gas) + e —> ClO2' 1.15
Br2 + 2e —> 2Br’ 1.07
HOI + 11* + 2e —> I” + H20 0.99
C102 (aq) + e —> ClO2' 0.95
ClO‘ + 2H2O + 2e —-> C1" + 20H” 0.90
H202 + 2H3O+ + 26 —-) 41-120 (basic) 0.87
C102“ + 2H2O + 4e —) C1’ + 40H“ 0.78
OBr‘ + H20 + 2e —> Br‘ + 4011‘ 0.70
I2 + 2e —> 21‘ 0.54
13 + 3e —> 31' 0.53
01' + H2O + 2e —> I‘ + 2OH‘ 0.49
02 + 2H2O + 4e —> 40H“ 0.40

 

Handbook of Chemistry 85 Physics, 56th Ed. (1975—76)

29

1) Ch

1.1.—

'h'OCI)
2:123:13 a
“171, Ht

11:? RE M

m

Cl.

. +

1) Chlorine
Chlorine Chemistry

Chlorine is presently used as a sanitizer in the food industry for
utensils and food-contact surfaces as well as for the treatment of public
water supplies. This is used either as gaseous or liquid chlorine or as
hypochlorite ion to generate nascent oxygen atoms by the reaction C12 +
H20 —> 2HCl + 0. This approach finds broad, international use for
disinfection of drinking water and as the final treatment for wastewater.
Because of its safety requirements, the use of gaseous or liquid chlorine
is usually limited to large facilities with the hypochlorite route being
more common at smaller sites. In either case, serious questions have
arisen concerning the possible generation of more hazardous chlorinated
by-products during the treatment.

Chlorine in water is hydrolyzed very easily to form hypochlorous
(HOCl) and hydrochloric acid (HCl). For normal conditions of
chlorination, the hydrolysis is essentially completed at pH values >6. In
turn, HOCl dissociates with a dissociation constant ranging from 1.6 x

10'8 M at 0°C to 3.2 x 10“8 M at 25°C (Morris, 1966).

C12 + H20 —> HOCl + H+ + Cl‘ Equationl

3O

This reaction is essentially complete within a few seconds. In
dilute solution and at pH levels above 4, the equilibrium shown in
Equation 1 is displaced to the right and very little Cl exists in solution
(Laubusch, 1962). Hypochlorous acid is a weak acid (Equation 2) with a
dissociation constant at 0°C to 25°C of 1.6 to 3.2 x 10‘2 M and a pKa of

7.8 to 7.5 (Morris, 1966).

HOCl —) H+ + OCl’ (pKa = 7.5) Equation2

As a result, the chlorine species present in the pH range 3.0—8.0
(the range for most foods) would be HOCl and the hypochlorite ion (0Cl’).
At pH 5.0, the species distribution would be 99.7% HOCl vs. 0.03% 0C1"
for a 10'2 M chlorine solution at 20°C. At pH 8.0, species distribution
shifts to 23.2% HOCl vs. 76.8% OCI’ for the same 10’2 M solution (Figure
6). At pH 7 .5, approximately equimolar concentrations of HOC1 and 0Cl‘
are present. Generally, HOCI plays a main role in bactericidal and
disinfecting function. The bactericidal efficiency of HOC1 is nearly 80
times higher than 0C1". The higher the pH, the lower the concentration of
HOCl and hence weaker activity and poorer disinfection (Morris, 1966).

Other species besides HOC1 includes the hypochlorous

hydronium ion, H20Cl+ , the chloronium ion, Cl+ and C13+ which may be

31

present

  
 

IfaCilVlili‘

it may 5;

displacerrx.

 

reactions c
and Span- ‘1
decrease

[Emperam

reactivity

present in very low concentrations and/or have very low specific
reactivities (Laubusch, 1962).

The tendency for chlorine to acquire electrons is so strong that
it may split from the molecule and form the reduced chloride ion by
displacement (Wei et al., 1985). This is the basis for the oxidation
reactions of HOCl with organic compounds. The antibacterial efficiency
and sporicidal effectiveness of chlorine solution has been shown to
decrease with increasing pH (Dychdala, 1991). An increase in
temperature will decrease the percent of HOCl, and consequently its

reactivity with organic compounds (Wei et al., 1985).

 

Joe—T , °
\\

 

 

 

 

 

V. HOCI
3}
pi"
wffm—vd
2
Y- OCI”

 

 

 

 

 

 

 

 

 

 

 

 

 

8 9 10

Figure 6. Relative amounts of HOCl and OCl‘ formed at
various pH levels (Fair et al., 1948).

32

 

been used 5
and disinfez.

and use of C

 

1119911.

Aq‘.
Séti‘ize foot
to rinse ray.
Canned 100:
IT. the fish“

Aurerjds‘ 19 .

Uses of Chlorine

Chlorine as sodium, potassium, or calcium hypochlorite has
been used for many years by the food industry as the principal sanitizing
and disinfecting agent (Reina et al,. 1995). The history of the discovery
and use of chlorine in the food industry has been reviewed by Dychdala
(1991}

Aqueous chlorine is used extensively in the food industry to
sanitize food processing equipment and food containers (100—200 ppm),
to rinse raw fruits and vegetables (1—5 ppm), and to cool heat-sterilized
canned foods (1-2 ppm) (Foegeding, 1983). Chlorine is also widely used
in the fishing industry (Lane, 1974); in washing nutmeats (Smith and
Arends, 1976); and in processing seafood (Moody, 1976), poultry (Ranken
et al., 1965), and red meats (Kotula et al., 1974). Chlorine gas is used in
the flour industry as an oxidizing and bleaching agent to improve the
quality of flours (Johnston et al., 1980).

Chlorine, in gaseous form and derivatives such as calcium and
sodium hypochlorite, has been used widely in the United States for
disinfection of public water supplies and general sanitation. They are
powerful disinfectants which are active against a wide spectrum of
organisms, and are non—toxic to humans at low concentrations
(Dychdala, 1991). Many organic compounds present in water and foods

treated with chlorine are subject to chlorination reactions. When chlorine

33

is appize
met ased

increases

 

ht'WX'ma

unquestior...
01' foods. Her.
use of chit-1r
activity sue?
1013\‘alttate

needed cog
Praeess. 1h

e“i'el‘BS‘nre le'

A
;r

'JHCQP v Y‘ ,_ ‘
9'?“

,.
5 t
TEN
‘t‘ "fi
J, ‘ne ‘

is applied to organic molecules, they are changed to molecules with an
increased hydrophobicity or lipophilic nature. This in turn often
increases the toxicity and bioaccumulation of these compounds
(Kopperman et al., 1978). The use of chlorine in food processing is
unquestionable in preventing food spoilage and prolonging the shelf life
of foods. However, there are potential health hazards connected with the
use of chlorine because reaction products are formed that have toxic
activity such as mutagenicity, teratogenicity or carcinogenicity. In order
to evaluate the possible hazard to human health, more information is
needed concerning the level and reactivity of chlorine used in each
process, the identification and toxicity of the by-products, and the

exposure levels of the population to these compounds.

2) Chlorine Dioxide
Chemistry of Chlorine Dioxide

Chlorine dioxide is a gas that is soluble in water. At low
concentrations, the color of the solution is yellow-green, changing to
orange-brown at higher concentrations. The odor is similar to chlorine
but more pungent. The solubility of chlorine dioxide gas in water is 2.9
g/ L at room temperature and 30 mm partial pressure (Latshaw, 1994).
Chlorine dioxide is virtually pH independent and is effective at pH 4—10

(Latshaw, 1994). Gaseous chlorine dioxide is sensitive to pressure and

34

temperatur
on site.

Ti:
Miler e: a

charges in ti

 

 

 

 

Cl

 

temperature, so it is impossible to ship in bulk and must be generated
on site.

The amount of chlorine in chlorine dioxide is 52.6% by weight
(Miller et al., 1978). Since the chlorine atom undergoes five valence

charges in the process of oxidation to the chloride ion:

C102 + 5e‘ = C1’ + 20‘2

the equivalent available chlorine content is 52.6 x 5 = 263%. In effect,
this indicates that chlorine dioxide theoretically has about 2.5 times the
oxidizing power of chlorine (Miller et al., 1978). Chlorine dioxide achieved
faster kill of microorganisms at lower concentrations than did other
chlorine-based sanitizers (Aieta et al., 1980). Chlorine dioxide is of equal
bactericidal activity to sodium hypochlorite at one-seventh the
concentration of hypochlorite, when used for sanitation of poultry
processing water (Lillard, 1979). It is shown that oxidation capacity of
chlorine dioxide depends upon the acidity and basicity of the solution.
The stronger the acidity of solution, the higher the oxidation capacity of
chlorine dioxide.
Uses of Chlorine dioxide

Chlorine dioxide offers many advantages over chlorine as a

biocide in water systems. Chorine reacts with organic materials to form

35

chloroform
reaet “1'11
chloroform
lasted as s-
tater by tit.-

Chi

 

 

 

 

is 11110ng
addressed :1:
pepalations

‘iillard. l9T€
and an effee
ind 0dor~ce
tr

2”mm-gallis.

331er8 (B.

chloroform and trihalomethanes. In contrast, chlorine dioxide does not
react with organics, such as ammonia or nitrogenous compounds, so no
chloroform or other trihalomethanes are formed. Trihalomethanes are
listed as suspected carcinogens and are limited to 10 ppb in drinking
water by the U.S., Environmental Protection Agency (Latshaw, 1994).
Chlorine dioxide is used to disinfect public water supplies and
is finding application in the food industry. Several reports have
addressed the use of chlorine dioxide as a bactericide to reduce bacterial
populations both in poultry chiller water and on poultry carcasses
(Lillard, 1979; Lillard, 1980). This has proved to be an excellent biocide
and an effective oxidant in drinking water, cooling water, waste water,
and odor-control applications. This also achieved faster kill of
microorganisms at lower concentrations than did other chlorine-based
sanitizers (Bohner and Bradley, 1991). Chlorine dioxide has been used as
a drinking water treatment agent since 1944. (Aieta and Berg, 1986). In
treating drinking water, chlorine dioxide is used for taste and odor
control, color removal, iron and manganese oxidation, oxidation of
organics, disinfection and for providing a lasting residual in distribution
systems. Average dosages of chlorine dioxide can range from 0.1 to 1.5
mg/ L, depending on whether the oxidant is used for final treatment

(disinfection) or for pretreatment (removal of algae, Fe, Mn, etc).

36

as 11 fond ;‘

1d Bradle;

3) Ozone
Ct‘ristrt'

021; f

 

 

 

Tiiiseratec tt
. ,1
”All “limero;

1" ,
W ,
MSthn

er tr.

'5'»
”WE-tare c

DTUL’Iinem bei:

The, f.

03
H0
0.1

0H

Considerable quantities of chlorine dioxide are used daily for
bleaching in the pulp and paper industry. It is also used in large
amounts in the textile industry for bleaching and dye stripping, as well
as in food processing for bleaching of flour, fats, oils and waxes (Bohner

and Bradley, 1991).

3) Ozone
Chemistry of Ozone Technology

Ozone (CAS No. 10028—15—6) is a gas at ambient and
refrigerated temperatures. It is a very powerful oxidant that can react
with numerous organic chemicals. The oxidation potential of ozone (2.07
V) is higher than HOCl and free chlorine (Table 3). It is a partially soluble
in water and, like most gases, increases in solubility as the water
temperature decreases (Graham, 1997). It has the unique property of
autodecomposition, producing numerous free radical species, the most
prominent being the hydroxyl free radical (OH).

The following reaction mechanism was suggested by Alder and

Hill (1950).

03 + H20 ——> HOa“ + OH_ (1)
HOa“ + OH- ——) 2H02’ (2)
03 + HOz’ —-) HO' + 202 (3)
0H + H02 -+ H20 + 02 (4)

(1.
farting :11

to form 02: ‘

 

The orera'i 1-

Dee

maifi‘ ions.

 

n:1at:0n step

 

”.2835 before
'e. .l .
“CIECUlaI’ 020:
“:43.“

"“1de are t};

L'wL D
”m H Y;
- , CORC‘:

391115 directlr '

”l l .

“75761555, 5

Uch

Ozone is made by rupturing the stable oxygen molecule,
forming two oxygen fragments, which can combine with oxygen molecule

to form ozone:

02 . <—> 2 [O]
2[0] + 202 <—) 203

The overall equation as follows:

203 —-> 302 (5)

Decomposition of ozone can be initiated by hydroxide ions,
formate ions, or a variety of other species (Glaze, 1987). A single
initiation step can cause the decomposition of hundreds of molecules of
ozone before the chain ends. The electrophilic direct ozonolyses by
molecular ozone of double or triple bonds and the reactions with CH-
radicals are the two most important steps (Stockinger et al., 1994). At
high pH condition, the formation of hydroxyl radicals increases and this
lowers directly the rate of ozonolyses and vice versa at low pH. As the pH
of solutions containing dissolved ozone increases, the rate of
decomposition of molecular ozone to produce hydroxyl free radicals also

increases, such that at a pH 10 ozone decomposes instantaneously.

38

 

1
cliorine for
the most r

recognzzed .

1995). 0213?

 

sanitizer in l

The

[m f;
“mine. With
GOG p?-

Uses of Ozone

Ozone has been shown to be a more powerful disinfectant than
chlorine for deactivation of a very large number of organisms, including
the most recalcitrant. Ozonation is approved in the U.S. as generally
recognized as safe (GRAS) for treatment of bottled drinking water (FDA,
1995). Ozone has certain characteristics that make it attractive as a
sanitizer in food processing, and it is safer than other sanitizer systems.
Many applications appear in the food industry. These include the use of
gaseous ozone for increasing storage life and dissolved ozone in water for
sanitizing surfaces of vegetables, fruits, and other agricultural products.
Also, ozone has been used for washing food equipment, food and
packaging materials.

The U.S. Fish and Wildlife Service’s Coleman Fish Hatchery
uses ozonation to inactivate viruses, bacteria, and parasites for
protection of spawning salmon (Jennings, 1996). FDA has accepted the
use of gaseous ozone up to 0.1 ppm in meat—aging coolers (Ronk, 1975).
Ozone decontamination of beef carcasses is also being used in the U.S.
(Reagan et al., 1996). Ozone does not remain in water for a very long
period of time, thus its use is considered as a process rather than a food
additive, with no safety concerns about consumption of residual ozone in

food products (Graham, 1997).

39

 

products (

hear of ta

4) Hydrog

H
S~ tGRis

fixing 8

1.31.1338
fife

366 s»
Specifies U

«Tina .

Ozone has been applied in the food industry in Europe for
decades, especially in France and Germany, where ozone has been the
primary sanitizer for public water system (Graham, 1997). In other
European countries, ozone has long been used for various applications,
including air purification, storage of meat, fruit, cheese and other
products (Easton, 1951). Israel uses ozonation to control postharvest

decay of table grapes (Sarig et al., 1996).

4) Hydrogen Peroxide

Hydrogen peroxide (H202) is classified as generally recognized as
safe (GRAS) for use in food products as a bleaching agent, oxidizing and
reducing agent, and antimicrobial agent (Sapers and Simmons, 1998).
Three antimicrobial applications are approved by the Food and Drug
Administration (Sapers and Simmons, 1998): treatment of milk for use in
cheese, preparation of modified whey, and preparation of thermophile
free starch. For these and other food applications, the FDA regulation
specifies use levels and requires that residual hydrogen peroxide be
removed by appropriate physical and chemical means during processing.

Various experimental antimicrobial applications of hydrogen
peroxide for foods have been described, including preservation of fresh
vegetables and fruits (Honnay, 1988), control of postharvest decay in

table grapes (Forney et al., 1991; Rij and Forney, 1995), washing of fresh

4O

mushroo

‘3‘
Ft}
( I "'1

sala

peroxide
equipmer

Hl‘_\kv- ‘
. . H
rdk‘ cgaad

\.

FL Effeo

1f

91 “8,9, “‘-

ab‘(
“0m fOod
“6 Specifr

FA
Jr“

“align

5C1 -
CW pr:

{Fea~
~. ‘41:}
Ed 11“]

mushrooms (McConnell, 1991; Sapers et al., 1995) and preservation of
salad vegetables, berries, and fresh cut melons (Sapers et al., 1995).

The antimicrobial properties of hydrogen peroxide have long
been recognized (Block, 1991). Dilute hydrogen peroxide is used as a
topical disinfectant and is available as a consumer product. Hydrogen
peroxide vapor shows promise as a sterilizing agent for medical
equipment and supplies (Klapes and Vesley, 1990) and for aseptic

packaging systems and packaging materials (Wang and Toledo, 1986).

K. Effect of Processing Operations on Pesticide Residues in Foods
Various processing operations on foods give a reduction in the
level of pesticide residue (Cash et al., 1997; Siler, 1998). Residues which
are loosely held on the surface are removed by washing and blanching,
but residues which penetrate the tissues are more difficult to remove
from the food. Table 4 presents the effects of processing on the reduction
of pesticide residues in fruits and vegetables. The removal of residues
from foods depends upon numerous factors, including the type of food,
the specific characteristic of pesticides and the severity of the processing
operation. Certain residues such as the chlorinated hydrocarbons are
located primarily in the lipid materials of animal products and tend to be

retained with the lipids during processing (Geisman, 197 5).

41

Table 4.

 

Food

 

Apple

a? at: O
/7/7
a) n j— r —’

Table 4. The effects of processing on pesticide residues in fruits
and vegetables (Cash et al., 1997; Fahey et al., 1971; Farrow
et al., 1969; Ong et al., 1996; Siler, 1998; Tafuri et al., 1970)

 

 

 

 

 

 

 

Food Residue Process % Reduction
Apple Captan, Carzol, Chlorine wash (50 and 500 ppm) 80—100
and Guthion Ozone wash (0.25 ppm) 29—42
Captan and Chlorine wash (500 ppm) 87—100
Azinphosmethyl Detergent wash (2% SDS) 50-80
Peeling, steaming
Propargite Ozone wash (1, 5, 10 ppm) 30—100
Peeling, steaming
Broccoli Carbaryl Washing (detergent ) 77
Blanching, washing 99
Parathion Water wash None
Detergnet wash 30—33
Blanching, washing 10
Hand washing None
Washing, blanching, freezing 10
Malathion Washing, cooking 7—34
Storage (6 months frozen) 45—77
Grapes Chlorcholine Wine making None
chloride
Orange Guthion Washing 30
Peaches Gordona Lye peeling 99
Pears Gordona Canning and peeling 98

 

42

 

Table 1 11

food

Potatoes

Tr.
“‘J‘T‘dmes

Table 4 (Cont’d)

 

 

 

 

Food Residue Process % Reduction
Potatoes DDT Peeling (home) 91
Washing (5% lye) and peeling 94
Washing (15% lye) 90
Washing, blanching, canning 96
Washing, commercial 20
Spinach DDT Detergent washing 48
Blanching, washing 60
Washing, blanching, canning 91
Carbaryl Washing, blanching, canning 99
Detergent washing 87
Diazinon Blanching, washing 60
Water (detergent) washing None
Parathion Blanching, washing 71
Water washing 9
Detergent washing 24
Hand washing (home) 39
Washing, blanching, canning 66
Tomatoes Azodrin Cold wash 36—77
Hot lye peel 93
Carbaryl Detergent washing 97
Storage (55°F; 7 days) 30
Cooking 69
Home canning 92

 

43

Table «l (1

Food

 

 

Table 4 (Cont’d)

 

Food Residue

Process

% Reduction

 

Tomatoes Malathion

Water washing
Detergent wash

Cooking

36—79
90-95
90

 

Tomato Carbaryl

Juice

Home canning

67

 

 

44

unit ope:
pr: "act.
include

. . l
pasteun :r‘

 

 

 

 

 

110‘

Most commodities for processing are subjected to a number of
unit operations depending on both the commodity and the finished
product. Specific unit operations that may affect pesticide residues
include inspecting, washing, blanching, peeling and retorting or
pasteurizing. Inspection of the raw product with subsequent removal of
damaged material could reduce residue. Washing including rinsing is the
one of common unit operation to the preparation of nearly all fruits and
vegetables for processing. Various physical and chemical parameters of
the operation are important in reducing pesticide residues. The physical
aspects included soaking time, soaking temperature, agitation during
soaking, rotation of commodities under spray rinse, number and type of
nozzles, spray rinse pressure and volume. The chemical aspects of
washing are wetting agent type and chemical concentration. Blanching is
a mild heat treatment or partial cooking usually employed with
vegetables. The blanching operation is usually accomplished either in
steam or hot water. This operation may also accomplish some washing of
the product. Peeling, when applicable, would remove any surface
Contaminants. The main disadvantage is that not all commodities can be
Peeled. Peeling may be done by hand, mechanically or chemically. Most
PCSticides which appear to be heat unstable may be degraded when
hefitted in the presence of food products. Any unit operation which

employs heat offers potential in reducing residues. Home preparation

45

 

 

lO CHT'EFOI‘.

public def

L‘p
‘Q Y-s
.e {C
’1.
K161-

and cooking of many products also aids in reducing or removing residues

(Geisman, 1975).

L. Chemical By-Products and Degradation Pathways of Pesticides

Knowledge of the fate of pesticides in the environment is critical
to environmental risk assessments and management decisions. The
public demands a safe environment relatively free from toxic chemicals
and pesticides used in agricultural industries. In particular, there is a
need for understanding the fate and pathways of chemicals in the
environment to assess the exposure to humans and animals.

The fate of pesticide through processing of raw agricultural
commodities to finished foods is poorly understood and very few
published studies address this issue. Chlorination of drinking water is
known to produce some chemicals that cause cancer in laboratory
animals. These are chloroform, bromodidichloromethane, and MX [3—
chloro—4—(dichloromethyl)-5-hydroxy—2(5H)—furanone] (Richardson,
1998). Because of these concerns, alternative disinfectants are being
explored as disinfectants for food processing and drinking water.

Use of ozone, chlorine dioxide and chloramine as alternatives to
chlorine for treatment of drinking water is increasing, mainly because
they produce fewer chlorinated disinfection by—products (DBPs). Because

the alternative disinfectants do not form appreciable levels of these

46

 

DBPs. t1";
whether 1
these pro.
iden‘afr B
treat foods
that than;

reaetables

b

 

 

DBPs, they are gaining in popularity and use. However, it is unknown
whether they produce compounds as harmful or more harmful than
those produced by chlorine. No research is currently being conducted to
identify DBPs formed when these alternative disinfectants are used to
treat foods. Because of the similarity in precursor material, it is possible
that many of the by—products formed in the processing of fruits and

vegetables will be similar to those formed in drinking water treatment.

47

C HAPT

 

CHAPTER I. STUDIES ON THE DEGRADATION
OF PESTICIDES IN A MODEL
SYSTEM

 

trons in

 

potential 11
residues in
residues in
that of the

probable 0

Pesticides

1;.
“'33" are

I:
.51 p," ‘ .
kslt} IS her
6:5

INTRODUCTION

No one can doubt the efficacy of pesticides for the protection of
crops in the field, thereby providing us with abundant, inexpensive,
wholesome and attractive fruits and vegetables. However, the widespread
use and misuse of the toxic pesticides created an awareness of the
potential health hazards and of the need to protect the consumer from
residues in foods. Today, the total health risks presented by pesticide
residues in our food supply remain unknown. Experimental data indicate
that of the 300 pesticides used on food, as many as 71 are known,
probable or possible human carcinogens (Hajslova, 1999). Other
pesticides in food have been shown to cause neurotoxicity or
reproductive toxicity. Children may be uniquely vulnerable because their
food intake is a larger percentage of their body weight than adults.

EBDC compounds have been employed as fungicides and they
are widely used on a large variety of small fruits and vegetables. The
nomenclature of these agents comes from the metal cations with which
they are associated. The EBDCs registered for food uses in the U.S. are
mancozeb, maneb, penncozeb, ferbam and polyram. Although their
toxicity is negligible in animal feeding studies even at high doses, EBDCs

are subject to decomposition, and yield ethylenethiourea (ETU) as one of

48

their deg
goiteroge i

laboratorf

' “ Q
Th" 39h-
HAdL (11-4..

lipophilic

 

 

 

iECOUlCh-flr
TL

peSUCldes 1

69 CO

indUSm.‘ a

pesticide re

their degradation products. ETU is toxicologically significant because of
goiterogenic, oncogenic and teratogenic effects after being applied to
laboratory animals (Lentza—Rizos, 1990). ETU is present in nearly all
commercial formulations of EBDCs (Bontoyan et al., 1977). Some
evidence might point to hazards from other breakdown products of
EBDCs, such as carbon disulfide, as a neurotoxicant. It is also known
that dithiocarbamates can bind various divalent metal to form more
lipophilic complexes capable of entering the central nerve system
(Ecobichon, 1994).

The present study was focused on the use of chlorine, chlorine
dioxide, ozone and hydrogen peroxyacetic acid in the degradation of
pesticides in a model system solution. Calcium hypochlorite and chlorine
dioxide, common disinfecting and bleaching chemicals used in the food
industry, are potent oxidizing and chlorinating agents. Ozone has been
shown to be a more powerful disinfectant than the most commonly used
chlorine for deactivation of a very large number of microorganisms and
pesticide residues (Ong et al., 1996). Hydrogen peroxide has been shown
to have bleaching, oxidizing and antimicrobial properties (Sapers and
Simmnos, 1998). Hydrogen peroxide is unstable in solution but
combined with acetic acid, it forms peroxyacetic acid or hydrogen

peroxyacetic acid (HPAA), which is a fairly stable compound.

49

 

 

different
an aque.
diorde,

9~ _'
deter"...1::

 

The objective of this study was to determine the effectiveness of
different chemical oxidants on the degradation of mancozeb and ETU in
an aqueous solution model system, using calcium hypochlorite, chlorine
dioxide, ozone and HPAA treatment. The optimum parameters
determined in the laboratory studies were then applied to apples

and apple products.

50

 

l
MATER

11 Reagt

1D Solver

Uh Chen

\l.
0“

MATERIALS AND METHODS

MATERIALS

A. Reagents

(I) Solvents

All organic solvents used for preparation of stock solutions, in
sample extraction and high performance liquid chromatography (HPLC)
were distilled-in—glass grade. Acetone and methylene chloride were

obtained from J. T. Baker, Co. (Phillipsburg, NJ).

(11) Chemicals

Mancozeb standard was obtained from Rohm 85 Haas
(Philadelphia, PA). ETU standard was obtained from Aldrich Co.
(Milwaukee, WI). The stock solutions of mancozeb and ETU were
prepared in distilled water at concentration of 100 rig/100 ml. The
standards were protected from light and stored in refrigerator at 4°C.
Chlorine solutions were prepared from calcium hypochlorite (Milwaukee,
WI). Sodium thiosulfate, sodium sulfate, potassium iodide, potassium

indigo trisulfonate were all reagent grade.

51

B. Glassware
All glassware was thoroughly washed with detergent and warm
water, then rinsed with distilled water. The glassware was then rinsed

with acetone and placed in an oven at 400°C overnight before use.

METHODS

Solution studies were conducted in a model system to determine
the effect of (i) calcium hypochlorite at three concentrations (50, 250 85
500 ppm), chlorine dioxide at two concentrations (5 8t. 10 ppm), ozone at
two concentrations (1 85 3 ppm), and hydrogen peroxyacetic acid at two
concentrations (5 8t. 50 ppm) (ii) three pH’s: 4.6, 7.0, and 10.7 (pH 4.6,
0.2 M citrate—phosphate; pH 7.0, 0.2 M sodium—phosphate; and pH 10.7,
0.2 M carbonate—bicarbonate) (iii) two temperatures: low (10°C) and
ambient temperature (21°C). Degradation of the mancozeb was studied
over a 30—minute period because the typical water contact time in a
commercial plant is about 10—15 minutes and under normal conditions
would rarely exceed 30 minutes. There were three replications per
treatment. Samples were taken at appropriate intervals for analysis of

mancozeb and ETU residues.

52

A.Sal

(D Ca

"fif

{oqu

COCCE‘T

1116831.;

Sample

TESpeC'

Where 1

A. Sample Preparation
(I) Calcium Hypochlorite

For the chlorine source, calcium hypochlorite stock solution
(5000 ppm) was added to each pH solution to bring the final chlorine
concentration to 50, 250 or 500 ppm. Each pH solution was spiked with
the mancozeb stock solution to give a final concentration of 2 ppm. Total
available chlorine was determined by total residual chlorine and
measured using the iodometric method (Standard Methods for
Examination of Water and Wastewater, 1987). 10 ml, 20 m1 and 100 ml
samples from the 500, 250 and 50 ppm chlorine sample solution,
respectively, were pipetted to Erlenmeyer flasks containing 5ml acetic
acid and 1 g potassium iodide. The stirred samples were titrated with
0.01N sodium thiosulfate, Na2S203, until the endpoints were achieved.

The total residual C12 was determined using the formula :

mg C12 / L = [(A i B) x N x 35450] / ml of sample

where A is the amount Na2S203 titrated for the sample (in ml), B is the

amount Na28203 titrated for the blank (in ml) and N is the normality of

Na28203 (0.01 N).

53

(II) Chlorine Dioxide

Chlorine dioxide (C102) was generated in the laboratory using
the manufacturer’s (S.C. Johnson Professional, WI.) instructions as
follows: 100 mls of the stock 2% Oxine FP solution were added to a 200
ml French square screw capped bottle. Twenty five mls of 75% w/w food
grade phosphoric acid were added, sealed and allowed to generate
chlorine dioxide for 5 minutes with a magnetic stirrer to ensure though
mixing. After 5 minutes, the concentrated chlorine dioxide was
transferred into 5 gallons of each pI-I solution in a closed container to
serve as a stock solution. For 5 or 10 ppm of chlorine dioxide, 2 or 4
liters of stock solution, respectively, were diluted to 10 gallons with each
pH buffer solution.

The final concentration of chlorine dioxide was determined
using the HACH chlorine colorimeter (Model CN-66, Cat. No. 2231—01,
HACH Co., Loveland, CO.) before and after each sampling run. A 1:2000
dilution of unactivated Oxine FP solution was used as a control blank.
Ten mls of the control stock solution or test solutions were transferred
into test solution vials. Two or three drops of Hach Glycine reagent and
one “free chlorine DPD” were added into the vials and then mixed gently.

After 1 minute, the blank solution was read using the colorimeter and

54

then t1".

nonpr-

(HI) 021

appronrt:

appropna

 

then the test solution was read. The reading on the colorimeter was

multiplied by 1.9 to achieve final concentration of chlorine dioxide.

(III) Ozone

Ozone was bubbled through a glass sparger (i.e. bubbles of
approximately 10 mm i.d.) into 990 m1 of distilled water at the
appropriate temperature adjusted by a circulating water bath and pH
adjusted by the addition of standard buffer solutions under 25 psi at 15
SCFH of oxygen until the desired ozone concentration (1 or 3 ppm) was
attained. One hundred ml of ozonated water was spiked with mancozeb
to give a final concentration of 2 ppm.

Ozone detection and monitoring were performed using the
indigo colorimetric method as described in Standard Methods for the
Examination of Water and Wastewater (1987). All reagents were prepared
just prior to use. The ozone concentration was monitored before and after
each sampling run. The ozonated water was collected into a 100 m1
volumetric flask containing 10 ml of the indigo reagent to minimize loss
of ozone. A separate volumetric flask was filled with distilled water
containing 10 ml indigo reagent to serve as a blank. The solutions were
mixed thoroughly and the absorbance of each solution was immediately

measured at 600 nm in a 1 cm cell.

55

formula

Where A
sdunon,

mil. and f

 

 

f!

V

.1

f

T0335 0f p}
11‘:
Q‘CaIOr \i-

n

:18 dddfi‘d

dig
Ds,untn

The concentration of ozone, in mg/ L, was calculated using the

formula:

mgOalL=(1000xA)/(fxbe)

where A is the difference in absorbance between sample and blank
solution, b is the path length (1 cm), V is the volume of the sample (90

ml), and f is a constant of 0.42.

(IV) Hydrogen Peroxyacetic Acid Study

An appropriate amount of peroxyacetic acid stock solution was
added to each pH solution to bring the final peroxyacetic acid
concentration to 5 or 50 ppm. Each pH solution was spiked with
mancozeb stock solution to give a final concentration of 2 ppm.

Total residual peroxyacetic acid was measured using the POAA
test kit (Ecolab Inc., 1997). The procedure is as follows;

Each solution was less than 90°F prior to testing. Vials were
rinsed with solution to be tested then filled with 10 m1 of the test
solution. Five drops of potassium iodide were added and mixed. Five
drops of phosphoric acid were added, mixed and then five drops of starch
indicator were added with vigorous mixing. Sodium thiosulfate (N/ 200)
was added, one drop at a time, counting drops and mixing between

drops, until blue color just disappeared.

56

residua.

Tor

B. Pestit

(I) Manc-

gas liquid

 

Calculation: Each drop of thiosulfate N / 200 equals lppm

residual peroxyacetic acid (POAA).

Total residual POAA

= (Drops for solution-drops for blank) x 1 ppm residual POAA

B. Pesticide Residue Analyses
(I) Mancozeb

Mancozeb residues were analyzed as carbon disulfide (CS2) by
gas liquid chromatographic headspace analysis (Ahmad et al, 1995).
Twenty mls of sample were transferred at 0, 5, 15, and 30 minutes
interval into sample bottles. A 0.5% 0.1 M sodium thiosulfate solution
was added to the samples at the appropriate time to quench the reaction.
Forty mls of 1.5% stannous chloride in 5 M HCl were added and
immediately sealed with a crimped septum. Fifty uls of a 1 mg/ml
thiophene solution were injected into each bottle and incubated at 70—
80°C in a water bath for 15 minutes. Bottles were removed and agitated
for 2 minutes by hand. Bottles were replaced in the water bath with
1‘ epeated shaking for 1 hour. A 100 pl sample was removed with a gas

tight syringe from the bottle headspace, and injected into the GC.

57

 

(11) er

inleCtt‘d 1::

C' Chmmz

(I) Manet

 

 

 

(II) ETU

ETU residues were determined using a modification of the HPLC
method published by Ahmad et al. (1995). Twenty mls of sample were
weighed into an Erlenmeyer flask, then 8 g of potassium fluoride and 0.6
g of ammonium chloride were added. This mixture was extracted with 50
ml methylene chloride 2 times. The methylene chloride layer was passed
through a bed of 25 g anhydrous sodium sulfate (120°C for at least 12
hr), collected in a Zymark Turbovap tube and evaporated to dryness on
an automated Zymark Turbovap evaporator (Zymark Inc., Hopkin, MA) at
40°C. The residue was dissolved in 3 ml distilled water and 50 pls were

injected into an HPLC column.

C. Chromatographic Analyses

(I) Mancozeb

Mancozeb residues were detected and quantified using a
Hewlett Packard Series II 5890 gas chromatograph (GC) equipped with a
flame photometric detector (FPD) in the sulfur mode. The GC was
equipped with a Supel—Q—Plot fused silica capillary column (30 m long x
0.53 mm ID) with a film thickness of 0.25 pm (Supelco Inc., Bellefonte,
FA). The oven temperature was 80°C, while the injector and detector
temperatures were 230°C and 300°C, respectively. Helium and nitrogen

Were used as the GC carrier gas and makeup gas, respectively. Carrier

58

(ll) ET

:1
(J I
T

.53
:3
U!
1:
1

 

D‘ CalCul

4..
;AG

Ledcmated

gas flow through the column was 20 ml /min. Integration was carried out

with HP Chemstation software interfaced to the GC.

(11) ETU

ETU residues were detected and quantified using a Waters
liquid chromatograph with a Hypersil BDS C18 column (250 mm x 4.6
mm, 5 pm particles), a Hypersil BDS C18 guard column (10 mm x 4.6
mm, 5 pm particles) and UV detector set at 240 nm. The mobile phase
was 0.72% butylamine in distilled water at pH 3.0—3.2. A M—45 Waters
HPLC pump (Waters Associates, Inc., Milford, MA.) was used for solvent
delivery at a flow rate of 0.5ml / minutes. After the system was stabilized
(about 1 hour from initial warm—up), 75 pl samples were injected via
Rheodyne syringe loop injector (50 pl loop) for analysis. Integration was

carried out using 3390 A Hewlett Packard integrator.

D. Calculation of Pesticide Residue Concentration

Mancozeb and ETU residue concentrations in solution were
calculated based on the area of the integrated peaks of the samples
compared with known concentrations of analytical standard of the
respective pesticides. Standard curves of the mancozeb and ETU were
plotted and least square linear regression was obtained using a Microsoft

Excel (Microsoft Corporation, Redmond, WA) software. Appendix 1 and 2

59

 

 

‘ Av
Sffim

' " ‘.1
luau

~31?

show examples of standard curves for mancozeb and ETU standard of
known concentrations.

The residue concentrations were calculated based on the
following formula:
(a) Mancozeb residue in pg/ml

ppm = ng Mancozeb

 

mg sample injected

where, ng Mancozeb was derived from standard curve

mg sample injected =

20g

 

headspace volume sample — containing reaction vial x pL injected

where, headspace volume of sample — containing reaction vial = 40 mL

(h) ETU residue in pg/ ml =

Conc. of ETU in sample extract based on std. curve(pg/g) x Vol. final extract (3 ml)

Weight of sample analyzed (20 g)

E. Statistical Analyses

All determinations were replicated three times. Mean standard
deviations, mean square errors, two factor ANOVA, correlation and
interaction of main effects were calculated using Sigmastat computer

software 1.0 (Jandel Corp., San Rafael, CA). Appropriate comparisons

60

111376

CD?“
.pa

were made using Student—Newman—Keuls Method for multiple

comparisons. A p<0.05 was considered statistically significant.

61

RESULTS & DISCUSSION

A. Chromatographic Analyses
(I) Mancozeb

A variety of analytical methods have been developed for
mancozeb analysis. As for many other dithiocarbamate pesticides, one of
the most widely used procedures for determining EBDC residues is the
headspace gas—liquid chromatographic (GLC) method. The matrix was
heated with hot acid and degraded the active ingredient to carbon
disulfide (CS2). Released carbon disulfide was detected and measured
directly by GLC headspace analysis linked to flame photometric detector
(FPD). To improve its sensitivity and resolution, thiophene was
incorporated as an internal standard. In the GLC analysis, carbon
disulfide appeared as a single sharp peak at a retention time of 5.1
minutes. Figure 7 shows a typical chromatogram of mancozeb standard
at a concentration of 1 ppm in distilled water, while Figure 8 shows an
example of a chromatogram of a sample in a 3 ppm ozonated water at pH
7.0 solution, room temperature and sampled at 5 minutes. The standard
curve shown in Appendix 1 was representative of the standard curves
used to calculated mancozeb concentration in the sample solutions. The

correlation coefficients (R2) for linear regression of the standard curves

62

Relative Intensity

.(De:€¥

.(De3€¥

.(De:€¥

.(D¢3€¥

 

 

L 1

 

 

ll

 

 

 

C)

.j
£3

Time (min)

Figure 7. GC chromatogram of a Mancozeb standard

1) 1.0 ppm standard in distilled water

2) Rt = 5.1 minutes

63

£5.C)e:€%

L

IL
:11

vcv

Relative Intensity

5.141

12.()ee€¥

1.(De=€fi

*kl J

 

 

 

 

 

 

Time (min)

Figure 8. GO chromatogram of a Mancozeb sample
1) 3ppm 03 in pH 7.0 at ambient temp. ; reaction time = 5 minutes
2) Rt = 5.1 minutes

64

 

lH) l

,‘ r'v-rn

0.1,.

1“.“
,1h,‘..
L‘d
51:
l
'3"—

were between 0.91 and 0.99, showing that the response was linear over

the concentration range of 1 to 500 pg.

(II) ETU

GLC methods have been the widely used for the determination
of ETU, because of its high sensitivity and specificity obtained by the use
of a number of different detectors. It must be pointed out, however, that
many workers have encountered difficulties with direct analysis of ETU
at low residue levels and some have demonstrated that the results
obtained using GLC must be treated with caution because of the
possibility of breakdown of any EBDCs and intermediate breakdown
products present under the conditions used for gas chromatography.
Comparisons of the results obtained on analysis of formulations using
both GLC and HPLC have shown that GLC may give abnormal results
(Bottomley et al., 1985). HPLC gives a better estimate of the ETU content
because of the lower operating temperatures as compared to the high
temperatures involved in GLC which may give rise to the degradation of
co-extractives on the column to form ETU. Consistently higher results
were obtained using GLC than by HPLC.

ETU was detected using liquid chromatography linked to a
ultraviolet (UV) spectrophotometric detector. ETU appeared as a peak

with a retention time of 10.4 minutes. Figure 9 shows a typical

65

 

'\

I) a"

10.40

 

Relative Intensity

 

 

Time (min)

Figure 9. HPLC chromatogram of a ETU standard
1) 1.0 ppm standard in distilled water
2) Rt = 10.4 minutes

66

 

wait
sen

m‘n\
ailaal\

B. D.

(U D

l v
(‘9‘

hurt

chromatogram of ETU standard at a concentration of 1 ppm in distilled
water, while Figure 10 shows an example of a chromatogram of a control
sample in pH 7 .0 solution, ambient temperature and sampled at 5
minutes. Standard curves for ETU standards were plotted, and a typical
curve is shown in Appendix 2. The correlation coefficients (R2) for the

linear regression of the curves were between 0.94 and 1.00.

B. Degradation of Mancozeb in Solution
(I) Degradation of Mancozeb by Hydrolysis

Mancozeb was stable at pH 7.0 at both 10°C and 21°C with very
little degradation due to hydrolysis. Between 95—99% (10°C) and 95—97%
(21°C) residual mancozeb remained after 30 minutes. Mancozeb was
relatively less stable at pH 4.6 and 10.7, with about 78 and 80%
remaining, respectively after 30 minutes at ambient temperature (Figure
11). This indicates mancozeb is less stable under basic and acidic
conditions than neutral condition. Appendix 3 shows raw data for
mancozeb residues in a model system under various oxidizing agents,

temperature and pH conditions.

67

Relative Intensity

 

1816 E

718
8A0

' 659

Time (min)

Figure 10. HPLC chromatogram of a ETU sample
1) Control in pH 7.0 at ambient temp. ; reaction time = 5 minutes
2) Rt = 10.46 minutes

68

 

 

100 0

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

\ 10°C + Control, pH 4.6
. —v— Control. pH 7.0
'3 80 ~ , -I— Control, pH 10.7
3 —<>— 50mm Ca(OCI)2. pH 4.6
g + 50ppm Ca(OCI)2, pH 7,0
I! I .
E 60 _ —o— 50ppm Ca(OC )2, pH 10 7
a.
O
a
.E
.E 40 ~
0
E ..
" L
K ..
c\" 20 ~
7 J.
0 l l V I r j
0 5 10 15 20 25 30
Time (min)
100 o ——— i a
21°C 4. + Control, pH 4.6
—v— Control, pH 7.0
.3 30 - o + fl + Control, pH 10.7
3 . —<>— 50ppm Ca(OCl)2, pH 4.6
g g + 50ppm Ca(OCI)2. pH 7.0
Q
E 60 _ p 4’ —o— 50ppm Ca(OCl), pH 10.7
h
0
D
.5
,5 40 a
«I
E
0
It ..
32 20 ~ ‘5
0 vi 1 O l I Q‘L
0 5 10 15 20 25 30

Time (min)

Figure 11. Effect of 50 ppm Ca(OCI)2 on the Degradation of 2 ppm Mancozeb
at 10 and 21°C.

69

 

‘1‘]

’9
“I

6}“;

(II) Degradation of Mancozeb by Calcium Hypochlorite

Degradation of mancozeb by calcium hypochlorite solution was
greatest at pH 4.6 and decreased with increasing pH (Figures 11—17).
The chlorine treatment at pH 10.7 was the least effective at both 10°C
and 21°C. Its degradation was only about 27 and 40% after 5 minutes at
50 ppm calcium hypochlorite (Figure 11). In 50 ppm calcium
hypochlorite solution, mancozeb was completely degraded at pH 4.6 after
5 minutes at ambient temperature (Figure 12). The 50 ppm chlorine
treatment at pH 10.7 was the least effective, with degradation only about
20% and 36% after 5 and 30 minutes, respectively. Low temperature
decreased the degradation of mancozeb at all pH ranges during the entire
sampling period (Figures 11—12). Chlorination at 50, 250 and 500 ppm
significantly (p<0.05) increased the rate of degradation of mancozeb in all
three pH treatments and at both temperatures. No mancozeb remained
with 250 and 500 ppm calcium hypochlorite treatments at pH 4.6 and
ambient temperature after 5 minutes (Figures 13—16). At pH 10.7, 50%
and 30% mancozeb residues remained after 5 minutes at 250 and 500
ppm calcium hypochlorite, respectively at ambient temperature (Figures
13, 15). The effects of pH on the degradation of mancozeb in solution are
illustrated in Figure 17. Again, the most effective pH for the degradation
of mancozeb with chlorination was pH 4.6, while pH 10.7 was the least

effective treatment.

70

 

2.00
1.50
1.00 4

(“9’91

0.50 + a

Mancozeb residue

 

a
r:-=—-I

b

 

 

0.00
5 min

30 min

Reaction time

pH 7.0

 

2.00
1.50
1.00 +

(ugly)

0.50 «»

Mancozeb residue

 

 

 

0.00

 

5 min

b

 

I

b

 

30 min

Reaction time

pH 10.7

 

(unis)
.0 .-* .-‘ .~
8 8 8’ 8

Mancozeb residue

 

 

 

 

 

0.00

 

5 min

 

 

30 min

Reaction time

(0100
@219 »

' 10VC;
210‘ :4

D l

r

1

li

Figure 12. Effects of reaction time and temperature on the
degradation of Mancozeb at 50 ppm Ca(OCI)2.
"’ Values with same letters are not significantly different (p <0.05).

71

 

 

100

 

 

 

 

 

 

 

 

 

 

10°C + Control, pH 4.6
n -v— Control, pH 7.0
Q 80 l < + Control, pH 10.7
3 —<>— 250po Ca(OCI)2. pH 4.6
8 + 250ppm Ca(OCI)2, pH 70
g 50 i . —o— 250ppm Ca(OCl)2, pH 10.7
h
o O
O
IE
.5 4o « -
w 1 g
E
0
r:
.\° 20 l
l
O J}
O f 1 Y I I Q
0 5 10 '15 20 25 30
Time (min)

 

 

 

1000 —-—-r l 4

 

 

 

 

 

 

 

 

 

O
21 C ‘” + Control, pH 4.6
Q —v— Control, pH 7.0
“g 80 1 T? + Control, pH 10.7
0 + 250ppm Ca(OCl)2, pH 4.6
g + 250ppm Ca(OCI)2, pH 7.0
g 60 ~ --0— 250ppm Ca(OCI)2, pH 10.7
h
0 .
O
C o
.E 40 ‘ L
a .
g .e
a:
32 20 .
0 \F l <> 1 i
0 5 1O 15 20 25 30

Time (min)

Figure ‘13. Effect of 250 ppm Ca(OCl)2 on the Degradation of 2 ppm Mancozeb
at 10 and 21°C.

72

Fi

pH 4.6

 

2.00

1.50 ~ .1- i - ,
100 10100
' i $819,?
0.50 4. ,

I a a a
0.00 +

5 min 30 min

Mancozeb residue
(ugly)

 

 

 

2.00 “~-

 

 

1.501> ,,,,
lEl10C,.

”’01 a .l21C}

'_"—J b
0.50 . C
0.00 * I ‘-

5 min 30 min

0

 

Mancozeb residue
lug/9)

 

 

 

Reaction time

pH 10.7

 

,u1oc
£219?

 

Mancozeb residue
(us/9)
8

 

 

 

 

 

 

 

 

5 min

Reaction time

Figure 14. Effects of reaction time and temperature on the
degradation of Mancozeb at 250 ppm Ca(OCI)2.
* Values with same letters are not significantly different (p <0.05).

73

100 C

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

\ 10°C + Control, pH 4.6
—v— Control, pH 7.0
g 80 - ., + Control, pH 10.7
g —<>— 5009er Ca(OCI)2, pH 4.6
g + 500ppm Ca(OCI)2, pH 70
g 60 l —o— 500ppm Ca(OCl)2, pH 10.7
I.—
O
a
.E
,E 40 —
g '.
O
a:
°\° 20 r o
0 Y 1 c l l 3
0 5 10 15 20 25 30
Time (min)
100 ~—-: ¥
0
21 C 1’ + Control, pH 4.6
—v— Control, pH 7.0
'8 30 ‘ i 4 + Control, pH 10.7
g _<>_ 500ppm Ca(OCI)2, pH 4.6
g + 500ppm Ca(OC|)2, pH 7.0
‘5' 60 .l —o— 500ppm Ca(OCl)2, pH 10.7
h
0
O
.E
,E 40 A
N
E .
g 0
.\° 20 — '
0 V T V I l
0 5 10 15 20 25 30

Time (min)

Figure 15. Effect of 500 ppm Ca(OCl)2 on the Degradation of 2 ppm Mancozeb
at 10 and 21°C.

74

 

pH 4.6 l

 

 

 

 

 

 

 

 

 

 

 

 

w 2.0
5
- 1.51 i, 1-, ,‘
is 10 jl1oc
$3: - "‘ hI21C‘
g 0.5 r
(I N. D. N. D.
5 0.0
5min 30 min
Reaction time
pH 7.0 _
a, 2.0 ,
3 2
'3 1.5 .____ ~§
fig 10 time)
3 a - ' (.210 .
g ,2, H .-.
g b
5min 30 min
Reaction time
,, s s s s - ;
s l ,
o- l ,___..__,,. ’
, § 1 lmocli
. .0 = l . l
l 3 l ll2‘l C«
l 8 b b r we. .2 -1
, c ‘
fl
, 2 l:—
j 5min 30min

Reaction time

Figure 16. Effects of reaction time and temperature on the
degradation of Mancozeb at 500 ppm Ca(0C1)2.
* Values with same letters are not significantly different (p <0.05).
* N. D. = None detected.

75

 

2.00

 

 

 

 

 

 

   

 

 

 

 

 

 

 

 

 

 

.1 g 150 13611376 ’,
g g 100 leH 7.0 ‘
N v l
§ 0 50 LE] 8WD].
II
5 0.00 «
10C
3 Temperature ;
l l
,1 i,_iii ,,,-i hit
250 ppm Ca(0Cl): l
7 ,, 2.00 3
l 3 _~ A w“ l
l '3 1501 c {IpH 4.6 3
3 8 § 1.00 * ° (IpH 7.0 If
N V , ‘
§ 0.50 b b . lEipH10.7‘
a a a
5 0.00 3 ,
100 21 0
Temperature
500 ppm Ca(OCI)2
. o 2.00 ;
l 3 i- _ i ,, A,
J E- 1'50” 'IpH4.6 ‘j
3 figi-OW 3IpH7.01‘.
l N a. 3 ,
, g: 0.50 .. b b LEIPHJQW 3
a a a l—T—l a a I I
5 0.00 «
100 21 C
Temperature

Figure 17. Effects of temperature and pH on the degradation
of Mancozeb in Ca(OCI)2 treatments at 15 minute
reaction time.

* Values with same letters are not significantly different (p <0.05).

76

 

(III) Degradation of Mancozeb by Chlorine Dioxide

Degradation of mancozeb by chlorine dioxide showed a pattern
similar to calcium hypochlorite treatment (Figures 18—22). As can be
seen in Figure 18 and 20, when chlorine dioxide and liquid chlorine were
used to degrade mancozeb residues, the required amount of chlorine
dioxide was lower than that of chlorine. Maximum degradation of
mancozeb by chlorine dioxide was observed at pH 4.6. For the 5 ppm
chlorine dioxide treatment, between 62 and 78% of mancozeb remained
after 5 minutes at both 10 and 21°C (Figure 18). Chlorine dioxide at 10
ppm significantly (p<0.05) increased the rate of degradation of mancozeb
at pH 4.6 at both temperatures. However, there was no significant
(p<0.05) difference in the degradation of mancozeb between 5 and 10
ppm chlorine dioxide at pH 7.0 and 10.7 at either temperatures. The
effects of pH on the degradation of mancozeb in solution are illustrated
in Figure 22. The most effective pH for the degradation of mancozeb with
chlorine dioxide was in pH 4.6, while pH 10.7 was the least effective
treatment.

The mechanism of chlorination and oxidation of organic
compounds by chlorine dioxide are not known. Chlorination in aqueous
solutions may occur indirectly through a progressive reduction of

chlorine dioxide, which passes through the HOCl stage.

77

 

 

DONOUC'E ho 33.9..Irtnvul c\:

 

 

 

 

 

% Remaining of Mancozeb

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

4o - l
O
D.
20 1
0 I i I j I
o 5 1o 15 20 25 30
Time (min)
100 l
21°C 4-
: sol + a
N
o
0
C
a
5 60 .
*-
° .l
a
.E
.E 40 —
(B
E
o
I!
.\° 20 -
0 I I T T I l
o 5 1o 15 20 25 30
Time (min)

 

 

+ Control, pH 4.6
-v— Control, pH 7.0
+ Control, pH 10.7
—<>— 5ppm CIOZ, pH 4.6
+ 5ppm CIOZ, pH 7_o

—o— 5ppm CIOZ, pH 10.7

 

 

 

 

—0— Control, pH 4.6
-V- Control, pH 7.0
+ Control, pH 10.7
—<>— 5ppm CIOZ, pH 4.6
+ 5ppm CIOZ, pH 7.0

—o— 5ppm CIOZ, pH 10.7

 

Figure 18. Effect of 5 ppm 0102 on the Degradation of 2 ppm Mancozeb

at 10 and 21°C.

78

 

A

Mancozeb residue (uglg

8

Mancozeb residue (uglg)

Mancozeb residue (uglg)

9.0

pH 4.6

2.00

 

5%

3- b

5 min

 

 

 

 

 

Reaction time

pH 7.0

2.00 ~ WW

 

b

30 min

1

 

 

1.50 +

-im

 

1.00 1-
0.50 ..

 

 

 

 

 

 

0.00
5 min

Reaction time

pH 10.7

30 min

 

 

 

1.50 4)

 

d

 

 

 

 

 

 

 

88

5 min
Reaction time

30 min

 

Enoc
521C

moc
.!219

moo
__I21C

Figure 19. Effects of reaction time and temperature on the
degradation of Mancozeb at 5 ppm ClOz.
* Values with same letters are not significantly different (p <0.05).

79

 

 

 

 

+ Control, pH 4.6
—v— Control, pH 7.0
—l— Control, pH 10.7
—<>— 10mm 0'02, pH 4.6
+ 10ppm Cl02, pH 70

—o— 10ppm Cl02. pH 10.7

 

 

 

 

 

 

 

% Remaining of Mancozeb

20‘

‘

 

 

 

0 I I I I I
0 5 10 15 20 25 30

 

Time (min)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

100 . i I
T O
‘ 21 C l + Control, pH 4.6
—v— Control pH 7.0
D . + .
a 80 a + Control, pH 10.7
8 —<>— 10ppm ClOz, pH 4.6
g + 10ppm ClOz, pH 7.0
5 60 - e —o— 10ppm ClOz, pH 10.7
“-
O ' .t
O)
.5
IE 40 ~
(I
E
0
r:
°\° 20 ~
~fi’}
o f I I l I 1
0 5 10 15 20 25 30
Time (min)

Figure 20. Effect of 10 ppm CIO2 on the Degradation of 2 ppm Mancozeb
at 10 and 21°C.

80

Mancozeb residue (uglg) Mancozeb Mid“ (09/0)

Mancozeb residue

pH 4.6

 

8

1.50 3 , .
U1OC

1.00 '21 C

a
0.50 b a a
0.00 I i [ lu—-—._____ ..

5 min 30 min

 

 

 

Reaction time

8

 

150+ a a lEl1OC"

a l ;

5 min 30 min

1.00 ..
0.50

 

 

 

 

 

0.00

 

 

Reaction time

 

2.00

1.50.3 -W W
b b (0100.

1.00 “r [.21 C

(0W9)

0.50 .-

 

 

 

 

 

 

0.00

 

 

5 min 30 min

Reaction time

Figure 21. Effects of reaction time and temperature on the

degradation of Mancozeb at 10 ppm CIOz.
* Values with same letters are not significantly different (p <0.05).

81

5 ppm ClOz

 

 

 

 

 

 

 

 

 

 

 

2.00
’6
3° 1.50
q, - _ i _
.3 lpH46
E 1.00 . TlpH 7.0
g EipH 10 7
N ,
§ 0.50 3»
(I
2
0.00 -
100
Temperature
10 ppm CID:
2.00
1:2?
31.50 .
8 c ' '
, 2 IpH 4.6
l g 1.00‘ ‘IpH 7-0 ‘
l a {09610-7
1
. 5 0.50 ‘-
‘ E
0.00 -

 

 

 

 

 

 

 

 

10 C
Temperature

Figure 22. Effects of temperature and pH on the degradation
of Mancozeb in 0102 treatments at 15 minute
reaction time.
* Values with same letters are not significantly different (p <0.05).

82

(IV) Degradation of Mancozeb by Ozone

Degradation of mancozeb by ozone was greatest at pH 7.0 and
decreased with increasing pH (Figures 23—27). The ozone treatment at pH
10.7 was the least effective at both 10°C and 21°C. Its degradation was
only 10% and 18% after 5 and 30 minutes, respectively at 21°C (Figures
23, 25). For the 1 ppm ozone treatment, almost 96% of the initial amount
of mancozeb was degraded after 30 minutes at pH 7.0 and ambient
temperature (Figure 23). Ozonation at 3 ppm significantly (p<0.05)
increased the rate of degradation of mancozeb in pH 4.6 and pH 7.0
treatments at ambient temperature. Only about 1% of mancozeb
remained at pH 7.0 after 30 minutes at 21°C. At pH 7.0, almost 65% of
the initial amount of mancozeb was degraded after only 5 minutes in a 3
ppm ozone concentration (Figure 25). Ozone degraded the majority of the
mancozeb residues within the first 5 minutes. This has important
implications from a practical situation, since the time required to lower
the concentration of any pesticide will affect cost. Again, the most
effective treatment was ozonation at 3 ppm in the pH 7.0 solution, while
pH 10.7 was the least effective treatment (Figure 27).

Many factors govern the solubility of ozone in water, one being
temperature. Ozone is partially soluble in water and, like most gases,

increases in solubility as the water temperature decreases. Dissolved

83

% Remaining of Mancozeb

% Remaining of Mancozeb

 

 

 

 

 

 

 

 

 

\.

\k 10°C
.

80 "

60 1

4o 3
.E

20 r

O I I I r I
0 5 10 15 20 25 30

Time (min)

 

 

 

 

20—

 

0 I I I I

 

 

 

O 5 10 15 20

Time (min)

25

30

 

 

—e— Control, pH 4.6
—v-— Control, pH 7.0
-I- Control, pH 10.7
—(>— 1 ppm 0,, pH 4.6
+ 1 ppm 0,, pH 7.0

—0— 1 ppm 0,, pH 10.7

 

 

 

 

+ Control, pH 4.6
—v— Control, pH 7.0
+ Control, pH 10.7
—<>— 1 ppm 03. pH 4.6
+ 1 ppm 03, pH 7.0

—o— 1 ppm 03, pH 10.7

 

Figure 23. Effect of1 ppm 03 on the degradation of 2 ppm Mancozeb

at 10 and 21°C.

84

 

pH 4.6

 

nfoc
I21 0

 

 

 

 

 

Mancozeb residue
(ugly)
8

 

 

 

 

5 min 30 min

Reaction time

pH 7.0

 

2.00

‘D‘lOC
a (.21 C

+LL_

5 min 30 min

 

 

Mancozeb residue
tools)
0 .3 d
8 8 8

 

 

 

 

 

0.00

Reaction time

pH 10.7

 

 

2.00

1.50 .
13100

(.21 C
0.50 ,

 

 

Mancozeb residue
(vein)
8

 

 

 

 

 

 

0.00
5 min 30 min

Reaction time

Figure 24. Effects of reaction time and temperature on the
degradation of Mancozeb at 1 ppm 0:.
* Values with same letters are not significantly different (p <0.05).

85

DGNOOCGS— ho Griz—59:0”; e\o CQNOUCQE 3° huts-s‘eeehsem 6A.

% Remaining of Mancozeb

% Remaining of Mancozeb

100°

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

\ 10°C
80 " o
O
60 . ’
40 ~
20 - f
0 I I I m I
o 5 1o 15 20 25 30
Time (min)
100 . T‘ q
T 21°C .-
80 1 + a
so - N
>
0
40 ~
0
20 ~ ..
>
4+
0 I I I I T
o 5 1o 15 20 25 30
Time (min)

 

 

+ Control, pH 4.6
—v— Control, pH 7.0
+ Control, pH 10.7
—o-— 3 ppm 03, pH 4.6
+ 3 ppm 0,, pH 7.0

—o— 3 ppm 03, pH 10.7

 

 

 

+ Control, pH 4.6
—v— Control, pH 7.0
+ Control, pH 10.7

—<>— 3 ppm 03, pH 4.6
+ 3 ppm 03, pH 7.0

-o— 3 ppm 03. pH 10.7

 

Figure 25. Effect of 3 ppm 03 on the degradation of 2 ppm Mancozeb
at 10 and 21°C.

86

 

 

pH 4.6

 

2.00
a
1.50 T a

 

E1100

1.00 3210

0.50 1)

 

 

 

 

 

 

 

Mancozeb residue (uglg)

8

5 min 30 min

Reaction time

2.00

 

1.50 4
100 a 1:100

u21 C x
0.50 b
h b
0.00 ,

——im

 

 

 

Mancozeb residue
lug/9)

 

 

 

 

 

5 min 30 min

 

0.50 1

 

,, 2.00

8
l '3 1.50*f 5 2,.1
l 58100 1D100
; fig {.2103
1 8

fl

2

 

 

 

 

0.00

 

 

5 min 30 min

Reaction time

Figure 26. Effects of reaction time and temperature on the
degradation of Mancozeb at 3 ppm 03.
* Values with same letters are not significantly different (p <0.05).

87

1 ppm 03

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

2.00
3 a
l S 1.50
g r _ r _ ,
2 lpH 4.6 ‘
, £1.00, IpH7.0 l'
‘ § (03519.7
§ 0.50 . 3
(I
s
0.00 . 1
100
Temperature
3 ppm 03
2.00
a.
E 1.50 « a
a T _. __ _,
:3 1.pH4.6 .
3 100 ~ leH 7.0 ,
1 , l
I g LquH 10:7?
! § 0.50 .- ~
l a
2
0.00 .

 

 

 

 

 

 

 

 

10 C
Temperature

Figure 27. Effects of temperature and pH on the degradation
of Mancozeb in 03 treatments at 15 minute
reaction time.
* Values with same letters are not significantly different (p <0.05).

88

ozone residuals also decrease with increasing temperature, due to
thermal decomposition (Hewes and Davison, 1971), which could
adversely effect the overall degradation process. In this study, two
temperatures, 10°C and 21°C, were used.

Ozone has the unique property of autodecomposition, producing
numerous free radical species, the most prominent being the hydroxyl
free radical (OH-). As the pH of solutions containing dissolved ozone
increases, the rate of decomposition of molecular ozone to produce
hydroxyl free radicals also increases, such that at a pH of about 10,
ozone decomposes instantaneously (Graham, 1997). Kearney et. al.
(1988) found that ozonation at high pH was less effective, due to the
instability of ozone in solution as the pH increases. This is due to the
catalytic effect of hydroxyl ions on the ozone decomposition process. As
the hydroxide ion is a promoter of ozone decomposition, the half-life of
ozone is very short under alkaline conditions. At pH 10, the half-life for
ozone in pure water is approximately 30 seconds (Masten et al., 1994).
Therefore, pH increases reduced the effect of ozone on the degradation of

mancozeb, while the effect of hydrolysis increased slightly.

(V) Degradation of Mancozeb by Hydrogen Peroxyacetic Acid
Maximum degradation of mancozeb by HPAA was observed at

pH 7 .0 (Figures 28—32). For the 5 ppm HPAA treatment, between 50 and

89

 

 

100

 

 

 

 

 

 

 

 

 

 

\ o
\, 1° C + Control, pH 4.6
a -V— Control, pH 7.0
o 80 _ ° 0 + Control, pH 10.7
3 ’ —<>— 5 ppm HPAA, pH 45
g e + 5 ppm HPAA, pH 7.0
N
E 60 4 —O— 5 ppm HPAA, pH 10.7
“-
O
a
.E
,5 4o 3
N
E
0
a:
.\° 20 1 IE

0 I T I fi T

0 5 10 15 20 25 30

Time (min)

 

 

 

 

+ Control, pH 4.6

—v— Control, pH 7.0

+ Control, pH 10.7
-<>— 5 ppm HPAA, pH 4.6
+ 5 ppm HPAA, pH 7.0
—O— 5 ppm HPAA, pH 10.7

 

 

 

 

 

 

% Remaining of Mancozeb

 

 

 

0 5 10 15 20 25 30

Time (min)

Figure 28. Effect of 5 ppm HPAA on the degradation of 2 ppm Mancozeb
at 10 and 21°C.

90

pH 4.6

 

2.00 ‘ a

1.50 1W2. WW ,
100 *El1OC,
‘ * iwc

0.50 1

 

Mancozeb residue
(uglg)

 

 

 

 

 

 

 

0.00

 

5 min 30 min

Reaction time

pH 7.0

 

2.00

._£; ‘
1.50 a W _W 1
100 (E1100
' ” @310
0.50 + b b :
0.00 m—

5 min 30 min

 

 

 

 

 

Mancozeb residue (uglg)

Reaction time

pH 10.7

 

2.00

1.50 -3 , g - j
( ‘1
321 C 3
0.50 —»

 

 

   

 

 

 

 

 

Mancozeb residue (uglg)
p A
8 8

5 min 30 min

Reaction time

Figure 29. Effects of reaction time and temperature on the
degradation of Mancozeb at 5 ppm HPAA.
* Values with same letters are not significantly different (p <0.05).

91

 

 

100 fi‘

 

 

 

 

 

 

 

 

 

 

 

\— o
10 C + Control, pH 4.6
.n 30 l e —v— Control, pH 7.0
a l + Control, pH 10.7
8 —<>— 50 ppm HPAA, pH 45
E + 50 ppm HPAA. pH 7.0
g 60 . ° —0— 50 ppm HPAA, pH 10.7
h
0
a r-
.E >
,E 40 1
N ‘o
E
0
0:
.\° 20 -
O
0 I I T j I
0 5 1O 15 20 25 30
Time (min)

 

 

 

 

 

 

 

 

 

 

 

100 — l
O
21 C 3" + Control, pH 4.6
.n 30 - —§ 5 —v— Control, pH 7.0
a —I— Control, pH 10.7
0 —<>— 50 ppm HPAA, pH 4.6
g + 50 ppm HPAA, pH 7.0
«I
2 60 1 —O— 50 ppm HPAA, pH 10.7
I-
O O
a
.E
,E 40 -
N
E
w e
K e
°\° 20 —
0 1 I ' W I I
0 5 10 15 20 25 30

Time (min)

Figure 30. Effect of 50 ppm HPAA on the degradation of 2 ppm Mancozeb
at 10 and 21°C.

92

 

2.00 1
1.50 «

 

D10C
£210

 

1.00
0.50

 

Mancozeb residue
(uglg)

 

 

   

0.00

 

5 min 30 min

Reaction time

pH 7.0

 

us (0919)
7.; N
OI O
O O

[1100
(um

_s
o
O

8 '8
l

 

 

 

Mancozeb resid

5 min 30 min

Reaction time

pH 10.7

 

2.00

150+ , .g j
100 D10C
' ‘I21C

0.50 ,

 

Mancozeb residue
(uglg)

 

 

 

 

0.00

 

5 min 30 min

Reaction time

Figure 31. Effects of reaction time and temperature on the
degradation of Mancozeb at 50 ppm HPAA.

* Values with same letters are not significantly different (p <0.05).

93

 

 

 

 

 

 

 

 

 

 

 

 

2.00
a
31.50.
.5; 7 .pH 11.6 *
g 1.00 IpH 7.0
g an10.7
8
c 0.50
I!
2
000
10c
Temperature
50 ppm HPAA
2.00 WW,
8
g 1.50
. 8 IpH 4.5
‘ § 100* *IpH 7.0 ~
1 3
r g. TUPH10-7
8
c 0.50 1.
ll
2 C
0.00

 

 

 

 

Temperature

Figure 32. Effects of temperature and pH on the degradation
of Mancozeb in HPAA treatments at 15 minute
reaction time.

* Values with same letters are not significantly different (p <0.05).

94

70% of mancozeb remained after 5 minutes at both 10 and 21°C at pH
7.0. Treatments at pH 4.6 and pH 10.7 were less effective than pH 7.0.
Degradation of mancozeb at pH 7.0 at both 10 and 21°C was significantly
(p<0.05) different than at pH 4.6 (Figures 28—29). The HPAA treatment at
pH 4.6 was the least effective at both 10 and 21°C with 45—75%
degradation after 30 minutes. The 50 ppm HPAA treatment for the
degradation of mancozeb was much more effective than 5 ppm HPAA for
all three pH treatments and at both temperatures. Increased temperature
completely degraded mancozeb after 15 minutes in 50 ppm HPAA at
21°C (Figures 30—31). HPAA treatment at neutral pH was more effective
than alkaline or acidic conditions (Figure 32). This relates to the stability

of HPAA at various pH ranges.

C. Degradation of ETU in Solution
(I) Degradation of ETU by Hydrolysis

The degradation of mancozeb to ETU in solution due to
hydrolysis shown in Figure 33. It was found that the rate of
decomposition of mancozeb to ETU was influenced by pH. The total yield
of ETU was decreased when the pH was lowered from 7.0 or 10.7 to 4.6.
At pH 7.0, the initial ETU concentration was 17.3 ppb, which increased
to 21.9 ppb after 15 minutes and then decreased to 12.3 ppb after 60

minutes. In the case of pH 4.6, the initial ETU concentration was 11.9

95

Control

 

 

 

 

25.00
15 20.00
2’
3 15.00 ‘ IPH 4-6
m , :IpH 7.0
'5_ 10.00 EIpH1Q.7
8
O
0 5.00

0.00

0 min 15 min
Reaction time
Control

25.00
A 20.00 .
U!
Tc» ,
5' 15.00 « llpH 4-6
E ‘lpH 7.0 j .
'5_ 10.00 » 1'32”, 107.7%
2 .
O
0 5.00

0.00

 

 

 

30 min 60 min
Reaction time

Figure 33. Effects of pH and reaction time on the conversion
of Mancozeb into ETU in control.
" Values with same letters are not significantly different (p <0.05).

96

ppb, which increased to 14.3 ppb after 15 minutes and then decreased to
5.3 ppb after 60 minutes. This shows that acidic pH is much more
effective in reducing the conversion rate of mancozeb into ETU compared
with neutral or alkaline pH ranges. In processing, acidic treatments can
be used as a preventative method for ETU production. Engst and
Schnaak (197 4) reported that ethylenebisdithiocarbamic acid readily
forms ETU under highly alkaline conditions (pH 10.5). As shown in figure
34—41, conversion of mancozeb to ETU reached a maximum at 15 minute
reaction time and then decreased for all three pH ranges and all
treatments. Appendix 4 shows raw data for ETU residues from the

degradation of mancozeb in a model system.

(11) Degradation of ETU by Calcium Hypochlorite

Degradation of ETU by calcium hypochlorite solution was
greatest at pH 4.6 and decreased with increasing pH (Figures 34—35).
The chlorine treatment at pH 10.7 was the least effective at both 50 and
250 ppm. Its degradation was only about 89 and 75% after 5 and 15
minutes, respectively at 50 ppm calcium hypochlorite (Figure 34). In 50
ppm calcium hypochlorite solution, ETU was completely degraded at pH
4.6 after 5 minutes at ambient temperature. Longer reaction time and
higher chlorine concentration increased the degradation of ETU at both

50 and 250 ppm (Figure 35). Chlorination at 50 and 250 ppm

97

 

25

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

50 ppm Ca(OCI)2
+ Control, pH 4.6
—v— Control, pH 7.0
-I-— Control, pH 10.7
3 —<>— Ca(OCI)2, pH 4.6
a + Ca(OCI)2, pH 7.0
v —o— Ca(OCl)2, pH 10.7
D
.—
[Ll
- .
O -1
d 10 -1 o
1: ‘°
0
0 1' K \ 4
5 d o
0 V T \A/ 17 O 1 I ,
0 1O 20 3O 4O 50 60
Time (min)
25 .
250 m Ca OCI
' pp ( )2 + Control, pH 4.6
—v— Control, pH 7.0
20 v + Control, pH 10.7
A 0 —<>— Ca(OCI)2, pH 4.6
\\ V
'3 + Ca(OC|)2, pH 7.0
9; 15 l .0. Ca(OC|)2, pH 10.7
D k 41.
m .l_
3 ‘ ‘ ~11-
d 10 a o T
C o
8 o\
5 -‘ -1
0 1 4 1 t 1 r fi
0 10 20 30 4O 50 60
Time (min)

Figure 34. Effect of Ca(OCI)2 on the concentration of ETU with time.

50 ppm Ca(0Cl):

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

25.00
. A 20.00
( 5' 15.00. 5.9” 4'6 1
l E (IpH 7.0 M,
‘ '5. 10.00 ‘EipH10.7 ~
0 _ W - , ,
g b
o 5.00 L
a a
0.00 .
15min 60 min
Reaction time
250 ppm Ca(0Cl):
25.00
3’ 20.00 ,_
g ,
5' 15.00 IDH 4-6
E lIDH 7.0
3 10.00 ”L b ‘DpH10.7 ‘
2 J
O
o 5.00 . 1
a a a a ~
0.00 .
15min 60 min

Reaction time 1

Figure 35. Effects of pH and reaction time on the conversion
of Mancozeb into ETU in Ca(OCl)2 treatments.
* Values with same letters are not significantly different (p <0.05).

99

significantly (p<0.05) increased the rate of degradation of ETU. No ETU
was detected in either 50 or 250 ppm calcium hypochlorite treatment at
pH 4.6 and 7.0 after 60 minutes (Figure 35). However, in 250 ppm
chlorine solution, 51% of ETU residue still remained after 60 minutes.
Again, the most effective pH for the degradation of ETU was chlorination

in pH 4.6 solution, while pH 10.7 was the least effective treatment.

(111) Degradation of ETU by Chlorine Dioxide

Degradation of ETU by chlorine dioxide showed a pattern
similar to calcium hypochlorite treatment (Figures 36—37). As can be
seen in Figure 36 and 37, when chlorine dioxide and chlorine were used
to degrade ETU residues, the required amount of chlorine dioxide was
lower than that of chlorine. Maximum degradation of ETU by chlorine
dioxide was observed at pH 4.6. No ETU residues were detected at either
5 or 10 ppm chlorine dioxide at pH 4.6 after 5 minutes (Figure 36). The
effects of pH and reaction time on the degradation of ETU in solution are
illustrated in Figure 37. Chlorine dioxide at 10 ppm significantly (p<0.05)
increased the rate of degradation of ETU in all pH ranges. However, all
ETU residues were completely degraded at 10 ppm chlorine dioxide in
three pHs after 60 minutes so there was no significant (p<0.05) difference

at this point. The most effective pH on the degradation of ETU was

100

 

Conc. of ETU (ppb)

 

 

 

Time (min)

 

 

 

 

 

25 *
. 10 ppm CIO2
20 v
A 0
a ‘\ '
a. l
e 15
D .0.
F di-
l.l.l
~_ .
o 4
g 10 ~ .
O
U
5 + ‘
O
0 “ I fil> I V I I
O 10 20 30 40 50

Time (min)

60

 

 

--0— Control, pH 4.6
—v— Control, pH 7.0
+ Control, pH 10.7
—<>— ClOz. pH 4.6
+ ClOz, pH 7.0

—o— CIOZ, pH 10.7

 

 

 

+ Control, pH 4.6
—v— Control, pH 7.0
+ Control, pH 10.7

—o— CIOZ, pH 4.6
+ CIOZ, pH 7.0

—O— ClOz, pH 10.7

 

Figure 36. Effect of CIO2 on the concentration of ETU with time.

101

 

 

5 ppm CIOz

 

 

 

 

 

 

 

 

 

 

25.00
a 20.00
5' 15.00 ”IpH 4-6 ,
E IpH 7.0 _
'3_ 10.00 leH1o.7;
8 b i' ***** l
O b l
0 5.00
a
0.00 -
15min 60min
Reaction time
10 ppm ClOz
25.00
:6 20.00
g ,, W W .
315.00 :IpH4.6 ,
'13 llpH 7.0 ‘
3 10.00 ., lngjQJH
8 l
o l
0 5.00 .
a a a
0.00 4

 

 

 

 

 

60 min
Reaction time

Figure 37. Effects of pH and reaction time on the conversion
of Mancozeb into ETU in ClOz treatments.

* Values with same letters are not significantly different (p <0.05).

102

chlorine dioxide in pH 4.6 solution, while pH 10.7 was the least effective

treatment.

(IV) Degradation of ETU by Ozone

Degradation of ETU by ozone was greatest at pH 4.6 and 7.0.
(Figures 38—39). The ozone treatment at pH 10.7 was the least effective
with degradation of 62 and 49% after 5 and 60 minutes, respectively at 1
ppm concentration (Figure 38). At 1 ppm ozone treatments, no ETU was
detected after 30 minutes at either pH 4.6 or 7.0. Ozonation at 3 ppm
significantly (p<0.05) increased the rate of degradation of ETU in all three
pHs. Ozone showed the most powerful effects on the degradation of ETU
compared to the other agents, with complete degradation of all of ETU

within the first 15 minutes (Figure 39).

(V) Degradation of ETU by Hydrogen Peroxyacetic Acid

Maximum degradation of ETU by HPAA was observed at pH 4.6,
whereas 10.7 and pH 7.0 showed the least effectiveness (Figures 40-41)
5 and 50 ppm after 15 minutes. In 5 and 50 ppm HPAA treatments, no
ETU was detected at both pH 4.6 and 10.7 after only 5 minute reaction
time (Figure 40). In 5 ppm HPAA treatment, between 46 and 30% of
initial ETU remained after 5 and 30 minutes at pH 7.0. However,

increased reaction time (60 minutes) completely degraded all ETU

103

Conc. of ETU (ppb)

Conc. of ETU (ppb)

 

25*

1 ppm 03

 

 

 

 

0 V f A I r

 

 

 

 

 

0 1o 20 30 40 50 60
Time (min)
25
3 ppm 03
20 a
-l v
\ V
l
15 l
+
. 1,
10 ~
5 -« ° r
0 ‘ I V I G I I O
o 10 20 30 40 50 60
Time (min)

 

 

+ Control, pH 4.6
—v— Control, pH 7.0
+ Control, pH 10.7

—<>— 03, pH 4.6
+ 03, pH 7.0

—o-— 03, pH 10.7

 

 

 

+ Control, pH 4.6
—v— Control, pH 7.0
+ Control, pH 10.7

—o— 03, pH 4.6
+ 03, pH 7.0

—o— 03, pH 10.7

 

Figure 38. Effect of 03 on the concentration of ETU with time.

104

 

 

 

 

 

 

 

 

 

 

   

 

 

 

 

25.00
. 20.00
0
E: W W W W _ _
5" 15.00 « IpH 4.6
1: IpH 7 o
'3. 10-00 a DpH10.7
O T
I:
O
0 5.00
d d
0.00 .
60 min
Reaction time
3 ppm 03
25.00
1'7 20.00
g . __ __
~ g 15.00 » IpH 4-6
I E. J pH 7.0
l 9 10.00 :DpH1q.7
. 2
O
o 5.00
N. D. N. D.
0.00
15 min 60 min

Reaction time

Figure 39. Effects of pH and reaction time on the conversion
of Mancozeb into ETU in 0: treatments.
* Values with same letters are not significantly different (p <0.05).
* N. D. = None detected.

105

 

Conc. of ETU (ppb)

 

 

 

Time (min)

 

 

25

20

15

10‘

Conc. of ETU (ppb)

_‘ I

e
k

50 ppm HPAA

 

 

 

Time (min)

 

 

+ Control, pH 4.6
—v— Control, pH 7.0
—I— Control, pH 10.7
—<>— HPAA. pH 4.6
+ HPAA, pH 7.0
—o— HPAA, pH 10.7

 

 

 

+ Control, pH 4.6
—v— Control, pH 7.0
—I— Control, pH 10.7
—<>— HPAA, pH 4.6
+ HPAA, pH 7.0
—0— HPAA, pH 10.7

 

Figure 40. Effect of HPAA on the concentration of ETU with time.

106

 

 

5 ppm HPAA

 

 

 

 

 

 

 

 

 

 

 

25.00
a 20.00
2’ -
3 15.00 IpH 4.6
E (IpH 7.0
9 10.00 lElpH10.7 »
8
O
0 5.00

a a a a a
0.00
60 min
Reaction time
50 ppm HPAA

25.00
:6 20.00
2’
5' 15.00 IpH 4.6
E IpH 7.0
'5. 10.00 Ian1o.7
8
o
0 5.00

a a a a a
0.00 -
15 min 60 min

Reaction time

Figure 41. Effects of pH and reaction time on the conversion
of Mancozeb into ETU in HPAA treatments.
* Values with same letters are not significantly different (p <0.05).

107

residues at both concentration of HPAA (Figure 41). This indicate that
longer contact time with oxidizing agents play an important role in the
reduction of pesticide residues.

The rate of degradation of the EBDC to ETU is influenced by
temperature, reaction time and pH of the system (Marshall, 1977). A
model system study was developed which was shown to be effective in
monitoring the degradation or disappearance of mancozeb through the
use of various pH, temperature, chlorine and chlorine dioxide, ozone and
HPAA treatments. These treatments indicated the potential for a removal

of pesticide residues on the fruit and in processed products.

108

SUMMARY & CONCLUSION

The objective of this study was to determine the effectiveness of
chlorine and chlorine dioxide, ozone and HPAA treatment on the
dissipation of mancozeb and ETU in buffered solution. A model system
was developed, which was shown to be effective in monitoring the
degradation or disappearance of mancozeb through the use of various
pH, temperature, chlorine and chlorine dioxide treatments. Calcium
hypochlorite, chlorine dioxide, ozone and HPAA treatments were effective
in reducing mancozeb and ETU residues. The rate of degradation of
mancozeb by chlorine and chlorine dioxide was dependent on pH, with
pH 4.6 being the most effective. Mancozeb residues decreased 40—1000/0
with chlorine and chlorine dioxide treatments. Degradation of ETU by
calcium hypochlorite and chlorine dioxide was greatest at pH 4.6 and
lowest at pH 10.7. Chlorination at pH 4.6, yielded no ETU residues for
both calcium hypochlorite and chlorine dioxide. Chlorine dioxide gave
excellent degradation effects at lower concentrations than liquid chlorine.
Mancozeb residues in model system solutions decreased 56—1000/0 with
ozone treatment. At 3 ppm ozone treatment, no ETU residues were
detected at all three pH ranges after 15 minute reaction time. HPAA was

also effective in degrading the mancozeb residues. Degradation of ETU by

109

HPAA was greatest at pH 4.6 and no ETU residues remained after 5
minutes at both 5 and 50 ppm. ETU residues were quickly degraded
under acidic conditions with both ozone and HPAA treatments.

The results showed that all oxidizing agents used in this study
gave excellent degradation of pesticide residues depending on pH and
temperature. These experiments indicated the potential for the removal

of pesticide residues on fruit and in processed products.

CHAPTER II. STUDIES ON THE DEGRADATION
OF PESTICIDES IN SPIKED APPLES

INTRODUCTION

Pesticide use in agriculture over the last several decades has
proven to be a great benefit to the production of our food supply.
Pesticide use has improved both the efficiency of growing crops and the
quality of food produced. This has, in turn, lowered the cost of the
household food budget. However, along with the benefits emerged the
potential effect of trace amounts of pesticide residues remaining on some
commodities at the time of sale to the general public. There has recently
been concern by consumer groups demanding assurance from the
agricultural community that the food we eat is indeed safe.

The pesticide selected in this study was mancozeb (Dithane 75
DF®), which is an ethylenebisdithiocarbamate (EBDC). EBDCs are
fungicides which are frequently used for the control of fungal diseases in
a wide range of fruits and vegetables. EBDCs are highly active and
reliable nonsystemic fungicides and have gradually replaced the older
products, establishing higher levels of disease control (Uesugi, 1998).
Compared with nonsystemics, the systemic fungicides are approximately
twice as valuable in terms of effects. Their success relies as much on
their technical strength as on their low cost. In many cases, their use of

multi—site modes of action is essential in mixtures or program

111

applications with more sophisticated products in order to control
resistance (Uesugi, 1998). Concerns about the safety of mancozeb, for
example, have been rebutted, but if they had been accepted and
mancozeb withdrawn from the market, several crops would have been
devastated by fungal attack and many systemic fungicides exposed to
increased problems of resistance risk. However, EBDCs are subject to
decomposition at elevated temperatures and high humidity and yield
ethylenethiourea (ETU) as the principal metabolite in foods which
contain EBDC’s (Lenza—Rizos, 1990). BTU is also formed during the
dissipation of the EBDC fungicides and the conversion rate or
degradation of ETU is greater than its formation rate.

Chlorine, chlorine dioxide, ozone and hydrogen peroxyacetic
acid (HPAA) have been employed historically for the oxidation of organic
compounds at water treatment plants and were consequently
investigated for their capacity to degrade organic pesticides. Chlorine and
ozone treatments have shown to be effective on reduction of azinphos—
methyl, captan, formetanate—hydrochloride and propargite residues in
apples and apple products (Ong et al., 1996; Cash et al., 1997).

The previous solution laboratory studies were used to determine
the optimum parameters for the degradation of mancozeb and ETU.
These results were then used to determine the conditions that were

subsequently employed for these laboratory whole fruit studies. The

112

objective of this study was to reduce or eliminate mancozeb and ETU
residues in mancozeb spiked apples. The effectiveness of chlorine,
chlorine dioxide, ozone and HPAA on the reduction of mancozeb and ETU

residues were also examined based on previous model system studies.

MATERIALS AND METHODS

MATERIALS

A. Apple Samples

Mature Golden Delicious apples were obtained from a
commercial orchard in Onondaga, Michigan. These apples had not been
sprayed with mancozeb during growing seasons. The fruits were hand
picked randomly from various regions of the trees, thoroughly mixed and

stored at 4°C until they were prepared for residue analysis.

B. Reagents

(I) Solvents

All organic solvents used for the preparation of stock solutions,
extraction, gas chromatography (GC) and high performance liquid
chromatography (HPLC) were distilled—in-glass residue grade or better.
Acetone and methylene chloride were obtained from J. T. Baker, Co.

(Phillipsburg, NJ).

(11) Chemicals

Mancozeb standard was obtained from Rohm & Haas
(Philadelphia, PA). ETU standard was obtained from Aldrich Co.
(Milwaukee, WI). The stock solutions of mancozeb and ETU were
prepared in distilled water at concentration of 100 pg/IOO ml. The
standards were protected from light and stored in refrigerator at 4°C.
Chlorine solutions were prepared from calcium hypochlorite (Aldrich,
Milwaukee, WI) as a source of chlorine. Sodium thiosulfate, sodium
sulfate, potassium iodide, potassium indigo trisulfonate were all reagent

grade .

C. Glassware
All glassware was thoroughly washed with detergent and warm
water, then rinsed with distilled water. The glassware was then rinsed

with acetone and placed in an oven at 400°C overnight before use.

METHODS

The model system solution studies were used to determine the
optimum parameters for the degradation of mancozeb and ETU. These

results were then used to determine the conditions that were

subsequently employed for these laboratory whole fruit studies. Based on
model system studies, (i) calcium hypochlorite at two concentrations (50
and 500 ppm), chlorine dioxide at two concentrations (5 and 10 ppm),
ozone at two concentrations (1 and 3 ppm), and hydrogen peroxyacetic
acid at two concentrations (50 and 500 ppm) (ii) one ambient pH of 6.7
(distilled water) (iii) one ambient temperatures (21°C) were selected.
Degradation of the mancozeb was studied over a 30 minute period
because the typical water contact time in a commercial plant is about
10—15 minutes and under normal conditions would rarely exceed 30
minutes. There were three replications per treatment. Samples were
taken at appropriate intervals for analysis of mancozeb and ETU
residues.

Calcium hypochlorite stock solution (5000 ppm) and HPAA
stock solution were used as a chlorine and peroxyacetic acid sources.
Chlorine dioxide and ozone were generated in the laboratory. The
detailed preparation and determination methods are given in the Method

Section of Chapter I.

A. Sample Extraction
Apples were coated by carefully dipping 5 ml of water containing
1 ug/ ml and 10 ug/ m1 of mancozeb onto each individual apple surface.

The water was allowed to evaporate and then the apples (five at a time)

116

were placed in 500 m1 of distilled water, at room temperature and desired
concentration of solution of calcium hypochlorite (50 and 500 ppm),
chlorine dioxide (5 and 10 ppm), ozone (1 and 3 ppm) or hydrogen
peroxyacetic acid (50 and 500 ppm). At the predetermined reaction time
(0, 3, 15 and 30 min) the apples were removed, the surface extracted
with 20 ml of water and analyzed for mancozeb residues by GC. Dipping

solutions were also analyzed by GC for mancozeb residues.

B. Pesticide Residue Analyses
(I) Mancozeb

Mancozeb residues were analyzed as carbon disulfide (C82) by
gas liquid chromatographic headspace analysis (Ahmad et al, 1995).
Twenty mls of sample were transferred at 0, 5, 15, and 30 minutes
interval into sample bottles. A 0.5% 0.1 M sodium thiosulfate solution
was added to the samples at the appropriate time to quench the reaction.
Forty mls of 1.5% stannous chloride in 5 M HCl were added and
immediately sealed with a crimped septum. Fifty uls of a 1 mg/ ml
thiophene solution were injected into each bottle and incubated at 70—
80°C in a water bath for 15 minutes. Bottles were removed and agitated
for 2 minutes by hand. Bottles were replaced in the water bath with
repeated shaking for 1 hour. A 100 pl sample was removed with a gas

tight syringe from the bottle headspace, and injected into the GC.

117

(II) ETU

ETU residues were determined using a modification of the HPLC
method published by Ahmad et al. (1995). Twenty mls of sample were
weighed into an Erlenmeyer flask, then 8 g of potassium fluoride and 0.6
g of ammonium chloride were added. This mixture was extracted with 50
ml methylene chloride 2 times. The methylene chloride layer was passed
through a bed of 25 g anhydrous sodium sulfate collected in a Zymark
Turbovap tube and evaporated to dryness on an automated Zymark
Turbovap evaporator (Zymark Inc., Hopkin, MA) at 40°C. The residue was
dissolved in 3 ml distilled water and 50 1113 were injected into an HPLC

column.

C. Chromatographic Analyses
(I) Mancozeb

Mancozeb residues were detected and quantified using a
Hewlett Packard Series II 5890 gas chromatograph (GC) equipped with a
flame photometric detector (FPD) in the sulfur mode. The GC was
equipped with a Supel—Q-Plot fused silica capillary column (30 m long x
0.53 mm ID) with a film thickness of 0.25 um (Supelco Inc., Bellefonte,
PA). The oven temperature was 80°C, while the injector and detector

temperatures were 230°C and 300°C, respectively. Helium and nitrogen

118

were used as the GC carrier gas and makeup gas, respectively. Carrier
gas flow through the column was 20 ml /min. Integration was carried out

with HP Chemstation software interfaced to the GC.

(II) ETU

ETU residues were detected and quantified using a Waters
liquid chromatograph with a Hypersil BDS C18 column (250 mm x 4.6
mm, 5 pm particles), a Hypersil BDS C18 guard column (10 mm x 4.6
mm, 5 pm particles) and UV detector set at 240 nm. The mobile phase
was 0.72% butylamine in distilled water at pH 3.0—3.2. A M—45 Waters
HPLC pump (Waters Associates, Inc., Milford, MA.) was used for solvent
delivery at a flow rate of 0.5 m1 /minutes. After the system was stabilized
(about 1 hour from initial warm—up), 75 pl samples were injected via a
Rheodyne syringe loop injector (50 pl loop) for analysis. Integration was

carried out using 3390 A Hewlett Packard integrator.

D. Calculation of Pesticide Residue Concentration

Mancozeb and ET U residue concentrations in solution were
calculated based on the area of the integrated peaks of the samples
compared with known concentrations of analytical standard of the

respective pesticides. Standard curves of the mancozeb and ETU were

plotted and least square linear regression was obtained using a Microsoft
Excel (Microsoft Corporation, Redmond, WA) software.
The residue concentrations were calculated based on the

following formula:

(a) Mancozeb residue in pg/ml

ppm = ng Mancozeb

 

mg sample injected

where, ng Mancozeb was derived from standard curve

mg sample injected =

20g

 

headspace volume sample — containing reaction vial x pL injected

where, headspace volume of sample - containing reaction vial = 40 mL

(b) ETU residue in pg/ ml

Cone. of ETU in sample extract based on std. Curve(ug/ g) x Vol. final extract (3 m1)

 

Weight of sample analyzed (20 g)

E. Statistical Analyses
All determinations were replicated three times. Mean standard
deviations, mean square errors, two factor ANOVA, correlation and

interaction of main effects were calculated using Sigmastat computer

120

software 1.0 (Jandel Corp., San Rafael, CA). Appropriate comparisons
were made using Student—Newman-Keuls Method for multiple

comparisons. A p<0.05 was considered statistically significant.

121

RESULTS & DISCUSSION

A. Recovery Study

Based on model system studies, whole fruit studies were
conducted. To determine the extraction efficiency of the mancozeb on
apples by presented extraction techniques, five apples (about 700 g) were
fortified with mancozeb at two concentration levels (1 and 10 ug/ ml).
Table 5 gives the percent recoveries obtained from fortified apples. On
the basis of the regression equation, average recoveries of mancozeb were

84.0% at 1 ug/ml and 91.3% at 10 pg/ m1 spiked level.

Table 5. Recovery (%) i SD (n = 3) for the Mancozeb on apple
samples

 

Recovery %

 

0.01 ug/ m1 spiked 1 ug/ ml spiked 10 pg/ ml spiked

 

 

# 1 88.3 87.7 89.7
# 2 79.2 83.8 94.1
# 3 76.9 80.6 90.2
Mean 77.3 i 1.7 84.0 i 3.6 91.3 i 2.4

 

The method of detection limit (MDL) for mancozeb was
determined to be 0.01 ug/ ml. The percent recoveries at MDL are

presented in Table 5. Relatively high recoveries were obtained for all

122

three spiked levels. Recoveries appeared to decline when spiked at a
lower level. The lower recoveries may be a result of matrix effects on
extraction efficiency. Samples which contain low levels initially, are more

likely to show these discrepancies (Siler, 1998).

B. Degradation of Mancozeb in Spiked Apples

Based on model system study, ambient temperature (21°C) and
pH were used in this study. This experiment utilized five apples (about
700 g of apples) coated with l or 10 ppm mancozeb. The whole fruit
spiked with mancozeb gave results similar to those found in the model
system studies. Appendix 5 shows raw data for mancozeb residues in
spiked apples at various time and treatments. Control studies conducted
with mancozeb coated apples under the exact conditions with the treated
samples but exposed only to distilled water with no other treatments
showed only slight dissipation of mancozeb residues (Figure 42). This
indicates that mancozeb was relatively stable in distilled water, at least,
during 30 minute period. Figure 42 shows the rates of decline for
mancozeb on apples. At zero reaction time, spiked mancozeb
concentration was approximately 1 ppm. This decreased gradually to
about 0.11 ppm and 0.01 ppm at 50 and 500 ppm calcium hypochlorite,
respectively after 30 minute reaction time. In 50 ppm calcium

hypochlorite treatment, almost 94% and 75% of the initial amount of

123

% Remaining of Mancozeb

°/o Remaining of Mancozeb

 

100

    
 

 

 

 

 

1 ppm spiked Mancozeb
8O -
60 ~
40 ~
20 ~
0 I r ‘rL l r
o 5 10 15 20 25 30

Time (min)

 

 

 

 

 

100 I l

10 ppm spiked Mancozeb -~
80 -
60 ~
401
20 -

L

0 1 I I I I
0 5 1o 15 20 25 30

Time (min)

 

 

+ Control
+ 50ppm Ca(OCl)2

+ 500ppm Ca(OCl)2

 

 

 

+ Control
—I— 50ppm Ca(OCI)2

+ 500ppm Ca(OCl)2

 

Figure 42. Effect of Ca(OCI)2 on the degradation of Mancozeb

in spiked apples.

124

 

 

mancozeb was degraded after 30 minutes at 1 and 10 ppm spiked levels,
respectively. Chlorine at 500 ppm significantly (p<0.05) increased the
rate of degradation of mancozeb. Only about 0.01 % and 0.04% of
mancozeb remained at l and 10 ppm spiked levels after 30 minute
reaction time

Degradation of mancozeb residues by chlorine dioxide is shown
in Figure 43. At 1 ppm mancozeb spiked level, there was no significant
difference between 5 and 10 ppm chlorine dioxide treatment and the
effects were lower than calcium hypochlorite. In this case, between 34
and 32% of mancozeb remained after 5 minutes at both 5 and 10 ppm
chlorine dioxide treatment, respectively. After 15 minutes, degradation of
mancozeb increased up to 24 and 22%; however, there was no significant
difference with reaction time. In 10 ppm mancozeb spiked level, 64 and
16% of mancozeb remained after 5 minutes and 41 and 13 % of
mancozeb residues after 30 minutes at 5 and 10 ppm chlorine dioxide
treatment (Figure 43). It is anticipated that residue levels would be
reduced considerably by the chlorine dioxide treatment if the
concentration of chlorine dioxide is increased above the 10 ppm that was
used in this study.

Ozonation at 1 ppm and 3 ppm significantly (p<0.05) increased
the rate of degradation of mancozeb in 10 ppm mancozeb spiked level

(Figure 44). At 3 ppm ozone concentration, 3% of the mancozeb residue

125

 

100

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1 ppm spiked Mancozeb
+ Control
" + 5 m ClO
.0 80 - 1 PD 2
a + 10 ppm Cl02
o
0
C
a
s 60 -
1..
o
a:
DE
,5 40 J
N
E
0
n:
o\° 20 -
0 . , , W I
o 5 1o 15 20 25 30
Time (min)
100 .
10 ppm spiked Mancozeb» + Control
43, 80 « + 5 ppm 0'02
3 + 10 ppm CIOZ
0
t:
N
E 60 «
Q-
o
O
.E .l
.E 40 ~
I!
E
0
a:
o\° 20 —
0 I I I 1 I
0 5 1o 15 20 25 30
Time (min)

Figure 43. Effect of CIO2 on the degradation of Mancozeb

in spiked apples.

126

% Remaining of Mancozeb

% Remaining of Mancozeb

100

 

80‘

60*

40—

20‘

 

1 ppm spiked Mancozeb

+ Control
+ 1 ppm 03
+ 3 ppm 03

 

 

 

 

 

 

100

I I I l I

10 15 20 25 30

Time (min)

 

80-

60-

40«

20—

 

 

 

WI

10 ppm spiked Mancozeb .. + Control
+ 1 ppm 03

+ 3 ppm 03

 

 

 

 

 

I l l I l

10 15 20 25 30

Time (min)

Figure 44. Effect of 03 on the degradation of Mancozeb

in spiked apples.

127

remained after 30 minutes at the 10 ppm spiked level, with 16% of the
mancozeb residue remained at the 1 ppm spiked level. Ozone has shown
to be relatively stable at neutral pH range which is close to the pH of
distilled water, so this can be easily applied in commercial plants.
Degradation of mancozeb by HPAA was significantly increased
at higher HPAA concentration. In 50 ppm HPAA treatment, almost 83
and 66% of the initial amount of mancozeb was degraded after 30
minutes. HPAA treatments at 500 ppm showed greater effects than 50
ppm HPAA at 1 and 10 ppm mancozeb spiked level after 30 minutes,

with 99% and 98% degradation of mancozeb, respectively (Figure 45).

C. Comparison of the Effects of Various Oxidizing Agents on the
Degradation of Mancozeb Residues

The effects of various oxidizing agents on the degradation of
mancozeb are shown in Figures 46—47 and Tables 6—7. Mancozeb
residues in all the samples were significantly reduced compared to
control by exposure to various oxidizing agents. In 1 ppm mancozeb,
there were no significant differences among various treatments except
chlorine at 500 ppm at both 3 minute and 30 minute reaction time
(Figure 46). At the 10 ppm mancozeb spiked level, 10 ppm chlorine
dioxide treatment showed the best effect at 3 minute reaction time

(Figure 47). With longer reaction times of 30 minutes, chlorine at 500

128

% Remaining of Mancozeb

% Remaining of Mancozeb

 

100

 

 

   
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1 ppm spiked Mancozeb

+ Control
+ 50 ppm HPAA

80 q + 500 ppm HPAA

1-
60 .
4o -
.l.
20 — ..
0 I T I I l l
0 5 10 15 20 25 30
Time (min)
100 -
10 m s iked Mancozeb ._ + COMFO'

80 - pp p + 50 ppm HPAA
+ 500 ppm HPAA

60 -

40 —

20 —

o . , W I W
0 5 10 15 20 25 30
Time (min)

Figure 45. Effect of HPAA on the degradation of Mancozeb
in spiked apples.

129

1 ppm spiked Mancozeb @ 3 min

 

 

 

 

 

 

 

 

 

1.00 .
a
3 0.80
5
g 0.60 ~
g l
2" 0.40 - b b l
“a :
g 0.20 , *
8 c
0.00 4 1 i i 1 *r . l
1 2 3 4 5 6 7 8 9 '
Treatments .
1. Control 2. 50 ppm chlorine 3. 500 ppm chlorine
4. 5 ppm chlorine dioxide 5. 10 ppm chlorine dioxide 6. 1 ppm ozone
7. 3 ppm ozone 8. 50 ppm HPAA 9. 500 ppm HPAA
1 ppm spiked Mancozeb @ 30 min
. 1.00 ~
; 5
' a 0.80 .
~ 5
§ 0.60
l 5
; s 0.40 .
1 “<3
.‘ § 0.20
o
0.00 -

 

 

 

Treatments

Figure 46. Comparison of various oxidizing agents on the
degradation of 1 ppm Mancozeb.

130

10 ppm spiked Mancozeb @ 3 min

 

 

   

 

 

 

 

 

 

 

 

 

 

10.00 a

5 800

:3; ' ab

0

g 6.00 , b

5 p b

E 4.00 b b

‘6 b

g 2.00 I C

o

0.00 . T ; A; : 1 '
1 2 3 4 5 6 7 8 9
Treatments
1. Control 2. 50 ppm chlorine 3. 500 ppm chlorine
4. 5 ppm chlorine dioxide 5. 10 ppm chlorine dioxide 6. 1 ppm ozone
7. 3 ppm ozone 8. 50 ppm HPAA 9. 500 ppm HPAA
10 ppm spiked Mancozeb @ 30 min
10.00 F+

~ 3
l 3 8.00 -A

8

§ 6.00 ~

g b

s 4.00 .. b 4

'52 be 1

§ 2.00 ~ c I ‘

o C c

0.00 ~ +
1 2 3 4 5 6 7 8 9
Treatments

Figure 47. Comparison of various oxidizing agents on the
degradation of 10 ppm Mancozeb.

131

Table 6. Effects of Various Oxidants on the Mancozeb Residue

Concentrations and % Remaining of Mancozeb at 1 ppm
Mancozeb Spiked Level

 

Mancozeb Residue Conc. (ppm)

% Remaining

 

 

3 min 30 min 3 min 30 min

Control 0.93 i 0.038 0.83:0.028 100 100

50 ppm Ca(0C1)2 0.41 .1: 0.11b 0.11 i003b 44.09 13.25
500 ppm Ca(0C1)2 0.06 : 0.02c 0.01 i000b 6.45 1.20
5 ppm C102 0.32 i 0.05 b 0.20i 0.04b 34.40 24.10
10 ppm C102 0.30 i 0.05 b 0.18i 0.01b 32.26 21.69
1 ppm 03 0.41 1:0.02C 0.19 i 0.04b 44.09 22.89
3 ppm 03 0.34 : 0.04C 0.13 : 0.01b 36.56 15.66
5 ppm HPAA 0.49 : 0.14b 0.14i 0.03b 52.69 16.87
50 ppm HPAA 0.24 i 0.00b 0.01i 0.00b 25.81 1.20

 

Note: 1. Values are the means of triplicate determinations.

2. Values with same letters are not significantly different (p<0.05).

132

 

Table 7. Effects of Various Oxidants on the Mancozeb Residue
Concentrations and % Remaining of Mancozeb at 10 ppm
Mancozeb Spiked Level

 

Mancozeb Residue Conc. (ppm) % Remaining

 

 

3 min 30 min 3 min 30 min

Control 9.60 i 0.59"11 9.32 i 0.79%1 100 100

50 ppm Ca(0C1)2 3.91 i 0.06a 2.33 i 0.67b 40.73 25.00
500 ppm Ca(0C1)2 2.40 i 0.19b 0.58 i 0.01c 25.00 6.22
5 ppm C102 6.12 i 0.48a 3.78 i 0.55b 63.75 40.56
10 ppm C102 1.56 i 0.04 b 1.17 i 0.08C 16.25 12.55
1 ppm 03 4.03 i 0.06b 2.43 i 0.20b 41.98 26.07
3 ppm 03 3.39 i 0.19C 0.31 i 0.05C 35.31 3.33
5 ppm HPAA 5.37 i 0.383 3.13 i 0.10b 55.94 33.58
50 ppm HPAA 3.21 i 0.22b 0.13 : 0.02C 33.44 1.39

 

Note: 1. Values are the means of triplicate determinations.

2. Values with same letters are not significantly different (p<0.05).

133

ppm, chlorine dioxide at 10 ppm, ozone at 3 ppm and HPAA at 500 ppm
showed greater effects than other treatments. Five hundred ppm calcium
hypochlorite and 500 ppm HPAA treatments showed the greatest effects
with both 1 and 10 ppm mancozeb after 30 minutes. Figure 48 shows
the percent reduction in mancozeb residues in spiked apples at both 3
and 30 minutes. To determine the percent reduction all samples were
compared to the control which was exposed only to distilled water. For
the 50 ppm chlorine treatment, mancozeb residues were reduced about
56% and 59% at 3 minute and 87% and 75% after 30 minute dipping
time in 1 ppm and 10 ppm mancozeb, respectively. For the 500 ppm
chlorine and 500 ppm HPAA experiments, most residues were degraded
up to 99% in both 1 ppm and 10 ppm mancozeb levels. Ozone at 3 ppm
also showed effectiveness in reducing mancozeb levels at 10 ppm level
after 30 minutes. Chlorine dioxide at both 5 and 10 ppm showed less
effectiveness compared to other treatments. Generally, increased reaction
time (30 minutes) reduced mancozeb levels compared to 3 minute
reaction time. The overall reaction was much slower and less effective
than observed from the solution studies. Degradation of mancozeb at the
high concentration (10 ppm) was less effective than at the low

concentration (1 ppm).

134

1 ppm spiked Mancozeb

 

 

9499

 

 

 

 

 

 

 

 

 

 

0
0
1

0
9

momm

7

O
4

4

3

52253. x.

<<dI
Eaaoom

<<QI

Eanom

mo Edam

m0 E9:

No.0
Eager

No.0
Edam

~_o
Eaaoom

N6
Eaaom

.9250

1:13 min I30 min,

Treatments

10 ppm spiked Mancozeb

 

 

 

 

 

 

 

 

o
o

 

 

0
7

0
6

0
5

0
4

000
321

5:853. s

0

 

.9250

l

1

1:13 min I30 mini

l

Treatments

Figure 48. Percent reduction of Mancozeb after various oxidizing

agents treatments.

135

D. Degradation of Mancozeb into ETU in Spiked Apples

The next part of this work was the determination of conversion
of mancozeb to ETU in spiked apples. Figures 49—50 and Tables 8—9
shows the ETU residues formed from the 1 and 10 ppm mancozeb spiked
apples at 3 and 30 minute reaction times. Mancozeb produced significant
quantities of ETU. At the 1 ppm mancozeb, ETU after 3 minutes was
14.13 ppb and slowly increased to 15.12 ppb after 30 minutes reaction
time for control which was treated with only distilled water (Figure 49
and Table 8). Various oxidizing agents significantly reduced ETU residue
levels compared to the control. For the 1 ppm mancozeb experiments,
500 ppm calcium hypochlorite and 1 and 3 ppm ozone treatments
completely inhibited the conversion of mancozeb to ETU (Figure 49). At 3
minutes, chlorine dioxide and HPAA showed powerful effects in reducing
ETU levels compared to the control; however, there was no statistical
(p<0.05) difference between 5 and 10 ppm chlorine dioxide and 50 and
500 ppm HPAA. After 30 minutes, all ETU residues were degraded at
high concentrations of the oxidizing agents, small amounts of ETU were
still determined at lower concentration of oxidizing agents.

At the 10 ppm mancozeb, the conversion rate of mancozeb into
ETU was higher and the oxidizing agent treatments showed less effect
than at the 1 ppm level (Figure 50 and Table 9). In this case, increased

reaction time and higher concentration of oxidizing agents played an

136

1 ppm spiked Mancozeb @ 3 min

 

 

 

 

 

 

 

 

 

 

 

 

18.00
A 15.00 -
2’
E 12.00 ~
3
11': 9.00
'5. b
g 6.00 ~ b b b
o 3.00 ~ .
C C C ‘
0.00 . 4 4 l i I
1 2 3 4 5 6 7 8 9 l
Treatments 1
1. Control 2. 50 ppm chlorine 3. 500 ppm chlorine
4. 5 ppm chlorine dioxide 5. 10 ppm chlorine dioxide 6. 1 ppm ozone
7. 3 ppm ozone 8. 50 ppm HPAA 9. 500 ppm HPAA
1 ppm spiked Mancozeb @ 30 min
18.00 *+
A 15.00 W
U)
l E 12.00
C D
l E 9.00
"6
g 6.00 ~ 0 b
8
3.00 _ i C l
c c c c
0.00 - r + ; 'T— r . 5 ‘
3 4 5

Treatments

Figure 49. Comparison of various oxidizing agents on the
conversion of 1 ppm Mancozeb into ETU.

137

10 ppm spiked Mancozeb @ 3 min

40.00 a
35.00
30.00 5
25.00
20.00 ,
15.00

a
b
10.00
i b b
5‘00 I]
C
0.00 1 ‘ '1 ‘. ‘r
2 3 4 5 6 7

Treatments

 

Conc. of ETU (nglg)
m

 

 

 

1. Control 2. 50 ppm chlorine 3. 500 ppm chlorine
4. 5 ppm chlorine dioxide 5. 10 ppm chlorine dioxide 6. 1 ppm ozone
7. 3 ppm ozone 8. 50 ppm HPAA 9. 500 ppm HPAA

10 ppm spiked Mancozeb @ 30 min

45.00
40.00 ,.
35.00 —
30.00
25.00 pr
20.00 3
15.00 *5
10.00 1
5.00 1
0.00 ~

 

Conc. of ETU (nglg)

 

 

 

 

c
C c
i l l . . i
3 4 5 6 7 8 9
Treatments

Figure 50. Comparison of various oxidizing agents on the
conversion of 10 ppm Mancozeb into ETU.

138

Table 8. Effects of Various Oxidants on the ETU Residue

Concentrations and % Remaining of ETU at 1 ppm

 

 

 

Mancozeb Spiked Level
ETU Conc. (ppb) % Remaining

3 min 30 min 3 min 30 min
Control 14.13 i 1.938 15.12i6.93‘al 100 100
50 ppm Ca(0C1)2 7.02 i 2.57b 6.04 i314b 49.68 39.95
500 ppm Ca(OCl) 2 N.D.C N.Db 0.00 0.00
5 ppm C102 4.45 i 0.53 b 3.41i 1.42b 31.49 2.55
10 ppm C102 3.90 i 1.63 b 0.22: 1.19b 27.60 1.46
1ppm 03 N.D.C N.D.b 0.00 0.00
3 ppm 03 N.D.C N.D.b 0.00 0.00
5 ppm HPAA 4.06 i 1.27b 3.55i 1.00b 28.73 23.48
50 ppm HPAA 4.00 i 0.52b N.D.b 28.31 0.00

 

Note: 1. Values are the means of triplicate determinations.
2. Values with same letters are not significantly different (p<0.05).
3. N. D. = None Detected

139

Table 9. Effects of Various Oxidants on the ETU Residue

Concentrations and % Remaining of ETU at 10 ppm

Mancozeb Spiked Level

 

ETU Conc. (ppb) % Remaining

 

 

3 min 30 min 3 min 30 min

Control 30.25 i 5.018 40.88 i 3.078 100 100

50 ppm Ca(0C1)2 26.17 i 12.17a 24.82 i 5.28b 86.51 60.71
500 ppm Ca(0C1)2 6.23 : 4.64b 4.86 i 3.02C 20.60 11.89
5 ppm C102 16.91 i 0.448l 6.15 i 0.21C 55.90 15.04
10 ppm C102 6.09 i 1.32 b 4.57 i 0.23c 20.13 11.18
1 ppm 03 4.84 i 1.17b N.D.c 16.00 0.00
3 ppm 03 N.D.C N.D.c 0.00 0.00
5 ppm HPAA 19.20 i 1.1061 16.69 i 1.7001 63.47 40.83
50 ppm HPAA 10.34 i 4.48b 4.92 i: 2.35c 34.18 12.04

 

Note: 1. Values are the means of triplicate determinations.

2. Values with same letters are not significantly different (p<0.05).

3. N. D. = None Detected

140

important role in the reduction of ETU residues. Ozone at 3 ppm still
showed a powerful effect in reducing ETU levels. Ozone was also very
effective in the degradation of mancozeb as compared to other oxidants
at low concentration. This is probably due to the high oxidation potential
of ozone (2.07 V).

Figure 51 shows the percent reduction of ETU under various
oxidizing agent treatments. At 50 ppm calcium hypochlorite, ETU residue
decreased up to 50% of initial concentration in 1 ppm spiked mancozeb
after 3 minutes. However, at 10 ppm mancozeb level, very little reduction
of ETU occurred with only 13 and 39% reduction at 3 and 30 minutes,
respectively. This is due to the low concentration of chlorine and high
concentration of mancozeb. In 1 and 3 ppm ozone, ETU residues were
completely degraded at both 1 ppm and 10 ppm mancozeb. At low
mancozeb levels of 1 ppm, chlorine at 500 ppm and HPAA at 500 ppm
showed the best effects. However, increased mancozeb concentration
decreased the degrade time effects.

These findings indicate that EBDC content of a food should be
of concern in addition to ETU residues on the raw agricultural

commodities for any realistic evaluation of ETU exposure.

141

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142

Figure 51. Percent reduction of ETU after various oxidizing
agents treatments.

SUMMARY & CONCLUSION

The objective of this study was to determine the effectiveness
of chlorine, chlorine dioxide, ozone and HPAA treatment on the
degradation of mancozeb and ETU in spiked apples. This study was
developed based on model system experiments at ambient pH and
temperature. Two different levels of mancozeb (1 and 10 ug/ ml) were
added to non—mancozeb treated apples. Various oxidizing treatments
were effective in reducing or removing ETU residues as well as mancozeb
on spiked apples. Mancozeb residues decreased 56—99% with chlorine
and 36—87% with chlorine dioxide treatments. In this study, chlorine
dioxide showed less effectiveness in mancozeb degradation compared to
model study. This was due to low concentration of chlorine dioxide
compared to high mancozeb residue. The residue levels would be reduced
considerably if the concentration of chlorine dioxide is increased above
the 10 ppm that was used in this study. ETU was completely degraded
by 500 ppm calcium hypochlorite and 10 ppm chlorine dioxide at 1 ppm
spiked level. However, at 10 ppm spiked level, the effectiveness of ETU
degradation was lower than observed in 1 ppm spiked level. Mancozeb
residues decreased 56—97% with ozone treatment. At 1 and 3 ppm ozone

treatment, no ETU residue was detected at 1 ppm spiked mancozeb after

143

both 3 and 30 minutes. Again, at 10 ppm mancozeb spiked level, the rate
of degradation was low. HPAA was also effective in degrading the
mancozeb residues with 44—99% reduction at different time and
concentrations. ETU was completely degraded at 500 ppm HPAA after 30
minute reaction time.

Studies on whole fruit spiked with mancozeb gave results
similar to those found in the model system studies. However, the
reaction was much slower than observed for the model systems. These
treatments indicated good potential for the removal of pesticide residues

on fruit and in processed products.

144

 

CHAPTER III. STUDIES ON THE DEGRADATION
OF PESTICIDES IN FRESH AND
PROCESSED APPLE PRODUCTS

INTRODUCTION

Pesticides are used worldwide to protect crops by controlling
insects, diseases, fungi and other pests. Protecting crops from pests gives
higher yields, resulting in greater variety and availability of food at a low
cost. The demand by consumers for produce with good sensory quality
has continued to sustain the use of pesticides for control of insects and
diseases in fruits and vegetables. However, along with the benefits there
are potential effects of trace amounts of residues remaining on some
fruits and vegetables. Consumer groups have expressed concerns about
food safety, especially, pesticide residues on (or in) produce at harvest.
As a result, there is a need to develop methods for removing or reducing
the pesticide residues on fresh and processed fruits and vegetables after
harvest. Such methods could alleviate concerns about the hazard of
pesticide residues to humans and environment. Postharvest treatments,
such as the postharvest water wash and scrub that have been
traditionally employed to remove debris and dirt, have been shown to
reduce pesticide residues (E1 Hadidi, 1993). The use of postharvest
chlorine dips and ozonated water dips shows potential effect in the
removal of pesticide residues (Hendrix, 1991; Ong, 1996). Chlorination
and ozonation which are the principal processes of water purification,

145

may produce by-products as a result of reaction between chlorine or
ozone and pesticides in raw material, and it has been reported that for
some organophosphate pesticides, the degradation by—products have
higher toxicity than the original pesticides themselves (Kobayashi et al.,
1990). Chlorine dioxide has been used since 1944 by the food industry
as a sanitizing and disinfecting agent and for oxidizing organic
compounds at water treatment plants. It has a greater oxidizing capacity
than chlorine and does not react with ammonia or nitrogenous
compounds like chlorine (Bohner and Bradley, 1991). The use of a
mixture of acetic acid, hydrogen peroxide and peroxyacetic acid has also
been considered as a postharvest treatment method.

Apples (Malus X domestica Borkh.) are considered to be a major
agricultural product in Michigan (Downing, 1989). Michigan is one of the
nation’s most important apple producing states, with approximately 9%
of the total U.S. production (Ricks and Hull, 1992). As a result of its high
economic value as well as the large number of plant disease, insects, and
mites that infest apples during their growing seasons, significant
quantities of pesticides are often necessary for the protection of this crop.
This leads to residues on (or in) the fruit at harvest. The most widespread
apple disease, accounting for much of the apple pesticide use worldwide,
is apple scab, caused by the fungus Venturia inaequalis (Merwin et al.,

1994)

146

The pesticide selected in this study was mancozeb (dithane 75
DF®), which is an ethylenebisdithiocarbamate (EBDC). EBDCs are
fungicides which are frequently used for the control of fungal diseases in
a wide range of fruits and vegetables. These substances are not stable in
the presence of moisture, oxygen, and in biological systems. During their
degradation, several products are formed, but ethylenethiourea (ETU) is
one of the major by—products. Many researchers are interested in ETU
because this compound has been found to be carcinogenic and
teratogenic for laboratory animals and is considered dangerous to
human health (Fishbein, 1976).

The previous solution laboratory studies and the whole fruit
studies were used to determine the optimum parameters for this orchard
study. The objectives of this study are 1) reduce or eliminate mancozeb
and ETU residues in apples and apple products; 2) determine the
effectiveness of different post harvest treatments and processing on the
reduction of mancozeb and ETU residues when treated with chlorine,

chlorine dioxide, ozone or hydrogen peroxyacetic acid.

147

MATERIALS AND METHODS

MATERIALS

A. Apple Samples

Mature Cortland apples (1997) and Golden Delicious apples
(1998 and 1999) were harvested from the Botany Research Field
Laboratory at Michigan State University, East Lansing, at various
preharvest intervals. The fruits were hand picked randomly from various
regions of the treated trees, thoroughly mixed and stored at 4°C until

they were processed for residue analysis.

B. Reagents

(I) Solvents

All organic solvents used for preparation of stock solution, in
sample extraction and high performance liquid chromatography (HPLC)
were distilled-in-glass grade. Acetone and methylene chloride were

obtained from J. T. Baker, Co. (Phillipsburg, NJ).

148

(11) Chemicals

Mancozeb standard was obtained from Rohm 8r, Haas
(Philadelphia, PA). ETU standard was obtained from Aldrich Co.
(Milwaukee, WI). The standard stock solutions of mancozeb and ETU
were prepared in distilled water at concentration of 100 ug/ 100 ml. The
standards were protected from light and stored in refrigerator. Chlorine
solutions were prepared from calcium hypochlorite (Aldrich, Milwaukee,
WI) as a source of chlorine. Chlorine dioxide was generated in the
laboratory using the manufacturer’s (S.C. Johnson Professional, WI.)
instructions. Hydrogen peroxyacetic acid stock solution (Ecolab Inc.)
were used as a source of hydrogen peroxide. Sodium thiosulfate, sodium
sulfate, potassium iodide, potassium indigo trisulfonate were all reagent

grade .

C. Glassware
All glassware was thoroughly washed with detergent and warm
water then rinsed with distilled water. The glassware was then rinsed

with acetone and placed in an oven at 400°C overnight before use.

149

METHODS

The solution studies and the whole fruit studies were used to
determine the optimum parameters for this orchard study. The orchard
study was set up to investigate the effect of various postharvest wash
treatments and the effects of processing on the reduction of mancozeb

and ETU residues under actual field conditions.

A. Pesticide Application and Spray Schedule

The orchard study was conducted during three years (1997—
1999) and two different varieties were used; i) Cortland (1997) and ii)
Golden Delicious (1998 and 1999). Apples were grown at the Botany
Research Field Laboratory at Michigan State University in East Lansing.
The field diagrams are shown in Appendix 10. Maintenance pesticides
were applied throughout the growing season to provide the necessary
insect, mite and disease control. All pesticides were applied as a foliar
spray with an FMC airblast sprayer at 80 gallons/ acre and 300 psi.

Seventy seven day preharvest intervals (PHI) for 1997 studies
and 4—day PHI for 1998 studies were used. In the case of 1997 studies,
the mancozeb residues in some apple products were below detectable
limits (BDL), so, 4—day PHI was used in the 1998 studies to ensure the

measurable amount of residue on the fruits. For the final application,

150

mancozeb was tank—mixed and applied as a single application. A PHI
study was conducted in 1999. The apples were harvested at of PHI 0, l,
4, 7, 14 and 77 day along with a control. Control samples did not receive
a final application. Tables 10—12 show the records for the 1997—1999

spray applications.

B. Apple Sampling and Harvesting

At optimum harvest maturity the apples were randomly hand
picked from all regions of the tree except fruit within one foot of the
ground which was excluded. Gloves were worn during harvest and
changed between treatments and control to prevent cross contamination.
Twelve crates (53—60 lbs/ crate) of fruit were collected from each of the
areas. Immediately following harvest, the samples were transported to
refrigerated cubicles (2—4°C) for storage at the MSU Food Science and
Human Nutrition building until postharvest treatment and processing.

Control samples produced in 1998 were found to contain
residues of mancozeb. This was due to the cross contamination during
spraying. Thus control samples were obtained from a commercial
orchard which had not been sprayed with mancozeb in 1998. Refer to
Table 13 for records of the spray schedule of the sample. Control apples
received the same treatments and processes as described for the treated

samples.

151

Table 10.

1997 Spray Schedule for Cortland Apples (7 7 day PHI)

 

 

Dates Chemicals Rate Purpose of Application Temp.

April 21 Dithane 75DF 3 lb / A treatment spray 40—45°F
Nova 40W 5 02/ A apple scab

April 28 Dithane 75DF 3 lb / A treatment spray 40°F
Nova 40W 5 02/ A apple scab

May 7 Dithane 75DF 3 lb/ A treatment spray 60°F
Nova 40W 5 oz / A apple scab

May 16 Dithane 75DF 3 lb / A treatment spray 35°F
Nova 40W 5 oz / A apple scab

May 27 Dithane 75DF 3 lb / A treatment spray 40°F
Nova 40W 5 oz / A apple scab

June 6 Dithane 75DF 3 lb/ A treatment spray 60°F
Nova 40W 5 oz/ A apple scab

June 16 Dithane 75DF 3 lb / A treatment spray 74°F
Nova 40W 5 02/ A apple scab

June 30 Dithane 75DF 3 lb/ A treatment spray 75°F
Nova 40W 5 oz / A apple scab

July 14 Guthion 50W 1.5 lb / A curculio, aphids 75°F

July 24 Guthion 50W 1 lb / A curculio, aphids 70°F

 

Note — 1. Dithane 75 DF: Trade name of Mancozeb
2. 3 lb/ acre is equivalent to 3.36 kg/ ha.
2. Control spray: Nova 40W + Captan 50W

3. Harvest date: September 15, 1997 (1:30-2:40 PM)

152

Table 11. 1998 Spray Schedule for Golden Delicious Apples

 

 

(4 day PHI)

Dates Chemicals Rate Purpose of Application Temp.

April 1 Dithane 80DF 3 lb / A treatment spray 50—55°F
Nova 40W 5 oz / A apple scab

April 7 Dithane 80DF 3 lb/ A treatment spray 35—40°F
Nova 40W 5 oz / A apple scab

April 14 Dithane 80DF 3 lb/ A treatment spray 45—50°F
Nova 40W 5 oz / A apple scab

April 21 Dithane 80DF 3 lb / A treatment spray 60°F
Nova 40W 5 oz / A apple scab

April 28 Dithane 80DF 3 lb/ A treatment spray 35—40°F
Nova 40W 5 oz/ A apple scab

May 6 Dithane 80DF 3 1b / A treatment spray 50°F
Nova 40W 5 oz / A apple scab

May 15 Dithane 80DF 3 lb / A treatment spray 70°F
Nova 40W 5 02/ A apple scab

June 1 Dithane 80DF 3 lb / A treatment spray 65°F
Nova 40W 5 oz / A apple scab
Guthion 50W 1 lb / A curculio, aphids

June 15 Dithane 80DF 3 lb / A treatment spray 50—60°F
Nova 40W 5 oz / A apple scab
Guthion 50W 1 lb / A curculio, aphids

June 29 Dithane 80DF 3 lb / A treatment spray 80°F
Nova 40W 5 oz / A apple scab
Guthion 50W 1 lb / A curculio, aphids
Pyramite 4 .4oz/ A

July 16 Dithane 80DF 3 lb / A treatment spray 70—75°F
Nova 40W 5 02/ A apple scab
Guthion 50W 1 lb / A curculio, aphids

Sep. 28 Dithane 75 DF 3 lb / A treatment spray 60°F

 

Note — 1. Dithane 80DF and Dithane 75 DF: Trade name of Mancozeb
2. 3 lb/ acre is equivalent to 3.36 kg/ ha.

3. Control spray: Nova 40W + Captan 50W

4. Harvest date: October 2, 1998 (8:00-9:30 AM)

153

Table 12. 1999 Spray Schedule for Golden Delicious Apples

 

 

Dates Chemicals Rate Purpose of Application Temp.

April 13 Dithane 7 5DF 3 lb / A treatment spray 32°F
Nova 40W 5 oz / A apple scab

April 20 Dithane 75DF 3 lb / A treatment spray 40—45°F
Nova 40W 5 02/ A apple scab

April 26 Dithane 75DF 3 lb/ A treatment spray 40°F
Nova 40W 5 oz / A apple scab

May 5 Dithane 75DF 3 lb/ A treatment spray 50°F
Nova 40W 5 oz / A apple scab

May 11 Dithane 75DF 3 lb/ A treatment spray 55—60°F
Nova 40W 5 oz / A apple scab

May 28 Dithane 75DF 3 lb/ A treatment spray 60°F
Nova 40W 5 oz / A apple scab

June 14 Dithane 75DF 3 lb / A treatment spray 60°F
Nova 40W 5 oz / A apple scab

June 28 Dithane 75DF 3 lb / A treatment spray 70°F
Nova 40W 5 oz / A apple scab

July 12 Dithane 75DF 3 lb / A treatment spray 65°F
Nova 40W 5 oz / A apple scab

July 22 Dithane 75DF 3 lb/ A treatment spray 75°F
Nova 40W 5 oz / A apple scab

August 9 Dithane 75DF 3 lb / A treatment spray 60—65°F
Nova 40W 5 oz /A apple scab

Sep. 17 Dithane 75DF 3 lb/ A treatment spray 55—55°F
Nova 40W 5 oz / A apple scab

 

Note — 1. Dithane 75 DF: Trade name of Mancozeb

2. 3 lb / acre is equivalent to 3.36 kg/ha.

3. Control spray: Nova 40W + Captan 50W

4. Sampling date: Sep.17 (0 day PHI) at 10 A.M.
Sep. 18 (1 day PHI) at 10 A.M.
Sep. 21 (4 day PHI) at 10 A.M.
Sep. 24 (7 day PHI) at 10 A.M.
Oct. 1 (14 day PHI) at 10 A.M.
Oct. 25 (77 day PHI) at 10 A.M.

154

Table 13. 1998 Spray Schedule for Golden Delicious Apples (Control)

 

 

Dates Chemical Purpose
April 27 Syllit Scab
May 8 Syllit Scab
May 10 Captan Scab

 

* After May 10, spraying was aborted due to hail damage.

C. Fruit Postharvest Treatments

All postharvest treatment and processing were conducted at
the Fruit and Vegetable Processing Laboratory, Department of Food
Science and Human Nutrition, Michigan State University, East Lansing,
MI. Batches of apples (1.5—2 lbs) from each of the sample groups were
subjected to postharvest treatments, processed and then analyzed for
mancozeb and ETU residues in whole apples, peeled and cored slices,
peeled and cored sauce, unpeeled and uncored sauce, juice and wet

pomace.

(I) Wash Treatments
All wash treatments were prepared in 20—1iter containers with 7
liters of wash solution to allow for complete submersion of the apple

samples. 1.5—2 lbs apples were used per replication (3 replications per

155

treatment) and the use of mesh bags allowed samples to be easily
removed after 15 minute wash time. The three treatments in 1997 were:
(1) No wash, (2) Water wash and (3) Calcium hypochlorite wash @ 50 and
500 ppm. The six treatments in 1998 were: (1) No wash, (2) Water wash,
(3) Calcium hypochlorite wash @ 50 and 500 ppm, (4) Chlorine dioxide
wash @ 10 ppm, (5) Ozone wash @ 3 ppm and (6) HPAA wash @ 50 ppm.
The temperature, pH, Chorine, chlorine dioxide, ozone and HPAA
concentration were monitored before and after each wash treatment. A
calibrated pH meter, model 601A (Corning Glass Works, Medfield, MA),

was used to determine the pH of the wash treatments.

(II) Sample Processing

Fruit from all plots, replication and treatments were processed
into peeled and cored slices, peeled and cored apple sauce, unpeeled and
uncored apple sauce, juice and pomace. The methods used were
Standard Operating Procedures developed by the Department of Food
Science and Human Nutrition, Michigan State University. Apples were
postharvest—treated and/ or processed within one week of harvest. All
samples were weighed before and after processing in order to obtain
percent yield. The controls were treated and processed before the

mancozeb treated samples. After each sample was processed, the

156

processing equipment was thoroughly cleaned and pressure washed to

prevent cross contamination of samples with mancozeb residues.

1. Slices

Apples for slices were weighed, then peeled and cored on a
Leader apple peeler. The peeled and cored apples were sliced with a
Sunkist slicer. Samples for slices were weighed and placed into labeled
plastic ziplock bags then sealed and frozen at —20°C storage immediately

to await residue analysis.

2. Sauce, Peeled and Unpeeled

Two—lb samples of apples were processed into apple sauce
(peeled-cored and also unpeeled—uncored). The apples were first sliced
with a Sunkist slicer and subsequently blanched in a Dixie steam
blancher for 10 minutes at approximately 110°C to adequately soften the
fruit and inactivate enzymes. After steaming, the apples were cooled in
air for 1 to 2 minutes and then were passed through a Langsencamp
pilot plant finisher with a 0033—0045 inch screen to remove coarse
fibers, seeds, stems, and peel particles. The apple sauce samples were
transferred into plastic ziplock bags immediately after finishing, weighed

and stored at —20°C for residue analysis.

157

3. Juice

One batch of apples was processed into juice. Weighed,
unpeeled apples were sliced with a Sunkist slicer. The apple slices were
macerated in an Acme Juicerator equipped with stainless steel blades.
The grinder/ centrifuge basket was lined with one layer of Kimwipe
tissue. Apple slices were introduced into the juicerator and were
automatically ground, juiced and filtered. The volume of juice was
measured, filled into French square glass bottles and frozen at —20°C

immediately to await residue analysis.

4. Pomace
Pomace is the by—product of juice processing. After juice
processing, the pomace was collected, placed in labeled plastic ziplock

bags, and immediately frozen (—20°C) until analysis.

D. Pesticide Residue Analyses
(I) Mancozeb

Mancozeb residues were analyzed as carbon disulfide (CS2) by
gas liquid chromatographic headspace analysis (Ahmad et al, 1995).
Twenty mls of sample were transferred at 0, 5, 15, and 30 minute
intervals into sample bottles. A 0.5% 0.1 M sodium thiosulfate solution

was added to the samples at the appropriate time to quench the reaction.

158

Forty mls of 1.5% stannous chloride in 5 M HCl were added and
immediately sealed with a crimped septum. Fifty pls of a lmg/ ml
thiophene solution were injected into each bottle and incubated at 70—
80°C in a water bath for 15 minutes. Bottles were removed and agitated
for 2 minutes by hand. Bottles were replaced in the water bath with
repeated shaking for 1 hour. A 100 pl sample was removed with a gas

tight syringe from the bottle headspace and injected into the GC.

(II) ETU

ETU residues were determined using a modification of the HPLC
method published by Ahmad et al. (1995). Twenty mls of sample were
weighed into blender cup and added 160 ml of methanol + water (3:1,
V / V) blended with a homogenizer for 3 minutes at high speed. The slurry
was filtered under vacuum through a buchner funnel with a #4
Whatman filter paper. The blender cup and solids on filter were mixed
with 30 ml of solvent mixture. These were combined with initial filtrate
into Turbovap tube and concentrated extract at 60°C to ca. 20 ml on a
Zymark Turbovap evaporator (Zymark Inc., Hopkin, MA). The extracts
were quantitatively transferred to an Erlenmeyer flask by rinsing tubes
with 5 ml distilled water, and then 8 g of potassium fluoride and 0.6 g of
ammonium chloride were added. This mixture was extracted with 50 ml

methylene chloride two times. The methylene chloride layer was passed

159

through a bed of 25 g anhydrous sodium sulfate, collected in a Zymark
Turbovap tube and evaporated to dryness on an automated Zymark
Turbovap evaporator at 40°C. The residue was dissolved in 3 ml distilled

water and 75 pls were injected into an HPLC column.

(III) Recovery Study

To evaluate performance of the foregoing analytical procedures,
known amounts of different concentration levels of mancozeb and ETU
were added to samples. Recoveries (% recovery) were determined at
fortification levels ranging from 0.01 to 2 ppm for mancozeb and 0.005
to 2 ppm for ETU and the fortified samples were then carried through the
above procedures. Percent recovery was derived from the equation:

% Recovery = Amount of pesticide obtained

 

x 100
Amount of pesticide added

E. Chromatographic Analyses
(I) Mancozeb Residue Analyses

Mancozeb residues were detected and quantified using a
Hewlett Packard Series II 5890 gas chromatograph (GC) equipped with a
flame photometric detector (FPD) in the sulfur mode. The GC was
equipped with a Supel—Q—Plot fused silica capillary column (30 m long x
0.53 mm ID) with a film thickness of 0.25 pm (Supelco Inc., Bellefonte,

PA). The oven temperature was 80°C, while the injector and detector

160

temperatures were 230°C and 300°C, respectively. Helium and nitrogen
were used as the GC carrier gas and makeup gas, respectively. Carrier
gas flow through the column was 20ml/ min. Integration was carried out

with HP Chemstation software interfaced to the GC.

(II) ETU Residue Analyses

A liquid chromatograph with a Hypersil BDS C18 column (250
mm x 4.6 mm, 5 pm particles), a Hypersil BDS C18 guard column (10 mm
x 4.6 mm, 5 pm particles) UV detector set at 240 nm were used. The
mobile phase was 0.72% butylamine in distilled water at pH 3.0—3.2. A
M—45 Waters HPLC pump (Waters Associates, Inc., Milford, MA.) was
used for solvent delivery at a flow rate of 0.5 ml/minutes. After the
system was stabilized (about 1 hour from initial warm—up), 75 p1 samples
were injected via Rheodyne syringe loop injector (50 pl loop) for analysis.

Integration was carried out using 3390 A Hewlett Packard integrator.

F. Calculation of Pesticide Residue Concentration

Mancozeb and ETU residue concentrations in fresh’or processed
apples were calculated based on the area of the integrated peaks of the
samples compared with known concentrations of analytical standard of
the respective pesticides. Standard curves of the mancozeb and ETU

were plotted and least square linear regression were obtained using

161

Microsoft Excel (Microsoft Corporation, Redmond, WA) software.
Examples of standard curves for mancozeb and ETU standard of known
concentrations are provided in the appendix.

The residue concentrations were calculated based on the
following formula:
(a) Mancozeb residue in pg/ ml

ppm = ng Mancozeb

 

mg sample injected

where, ng Mancozeb was derived from standard curve

mg sample injected =

20g

 

headspace volume sample — containing reaction vial x pL injected

where, headspace volume of sample - containing reaction vial = 40 mL

(b) ETU residue in pg/ml

Cone. of ETU in sample extract based on std. curve(pg/g) x Vol. final extract (3 ml)

 

Weight of sample analyzed (20 g)

G. Statistical Analysis
All determinations were replicated three times. Mean standard

deviations, mean square errors, two factor ANOVA, correlation and

162

interaction of main effects were calculated using Sigmastat computer
software 1.0 (Jandel Corp., San Rafael, CA). Appropriate comparisons
were made using Student—Newman—Keuls Method for multiple

comparisons. A p<0.05 was considered statistically significant.

163

 

RESULTS & DISCUSSION

A. Pesticide Residues in Unprocessed Apples

Two different varieties of apples were used in this study. Table
14 shows the comparison of specific characteristics of Cortland and
Golden Delicious apples. These are dual—purpose apple cultivars, for
fresh eating and processing (Downing, 1989). The reason for using two
different varieties of apples was to determine the relationship between
surface waxes and pesticide degradation patterns. However, no specific
relationships were noted for these characteristics between the two

cultivars from these experiments.

(I) Effect of PHI on the Pesticide Residue Levels

Preharvest interval (PHI), which is the time interval between the
last spray application and harvest, has been shown to affect pesticide
residue levels. Figure 52 shows the rate of decline for ETU on apples
according to PHI. The points on the curves represent an average of three
determinations. ETU residue concentration on the day of application was
approximately 2.47 ppm. The residue slightly increased after 1 day PHI

up to 2.50 ppm. However, this decreased gradually to about 1.89 ppm,

164

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ETU Concentration (ppm)

3i)

 

 

 

 

 

 

I I I I I I _I I

21 28 35 42 49 56 63 7O

Preharvest Interval (days)

Figure 52. Effect of PHI on ETU residues in raw apples.

166

77

 

0.84 ppm, 0.07 ppm after 4, 7 and 14 days, respectively. There was no
indication that ETU accumulated in the apples at 77 days. There were
several heavy rains between the 3 and 14 day. This seemed to cause
major changes in ETU residue concentrations.

Various studies indicate that longer PHI’s help to lower
pesticide residue levels. El—Zamity (1988) and Rashid et al. (1987) both
reported lower residue levels for captan, with an increase in PHI, on
tomatoes and apples, respectively. Similar studies by El—Hadidi (1993)
and Belanger et al. (1991) found residue levels to decrease with increased
PHI. Pesticide manufacturers often recommend a specific PHI in
combination with a particular crop and pesticide. Such
recommendations help to reduce the chance of residues exceeding
federal tolerances. The recommended PHI for mancozeb (Dithane 75 DF®)
is 77 days for apple (Code of Federal Regulations, 1996). For the first
year (1997) studies, 77 days PHI was applied. In this case, there was no
evidence of ETU residue in apples and apple products. Analysis of these
samples indicated ETU residues to be absent or below the method of

detection limit.

(II) Apple Processing & Product Yield
The apples were processed into slices, unpeeled sauce, peeled

sauce, juice and wet pomace. Tables 15—16 show yield data for two years

167

 

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of processing. The yield data shows various differences in final products.
This may be a result of a number of factors including condition of the
apples (firmness or juiciness), size, shape, and the efficiency of the
processing method. From an economic standpoint maximum recovery is
desirable. In juice processing, as the season progresses and apples are
less firm, the yield decreases even though the amount of press aid and
pressing time is increased (Downing, 1989). The low yield was a direct
result of processing, coarse fibers, seeds, stems, and peel particles.
Peeled apple sauce and pomace showed lower yield than other products
because of the number of unit operations necessary to obtain this

product.

B. Recovery Study

Recovery studies for mancozeb and ETU were carried out in
triplicate using the previously described analytical methods. The recovery
samples were prepared by adding a known amount of each pesticide to a
20 g sample of apple products and taking each sample through the entire
analytical method. Samples were spiked at a level of 0.2 and 2.0 pg/ g for
both mancozeb and ETU. Table 17 gives percent recoveries of mancozeb
and ETU added to apple products prepared with apples not treated with
mancozeb. Recovery percents ranging from 76% to 99.8% are indication

of good and dependable analytical procedures. Recoveries in this study

173

were higher than 80% for all levels except for the lowest ETU addition.

The method of detection limit (MDL) for mancozeb and ETU was

determined to be 0.01 ug/ g and 0.005 ug/ g, respectively.

Table 17. Percent Recovery of Mancozeb and ETU

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Apple products 0.01 pglg * 0.2 pg/g 2.0itglg
Mancozeb Slices 80.4 i 5.1 89.6 i- 2.9 85.3 i 4.2
Unpeeled sauce 77.5 i 4.6 90.4 i 4.8 93.4 i 5.3
Peeled sauce 82.8 i 7.6 85.2 i 5.0 90.5 i 5.8
Juice 76.4 i- 4.3 93.4 i 6.1 87.9 :L' 3.2
Pomace 78.4 i 6.7 88.5 i 5.7 92.5 i 4.7
Apple products 0.005 pg/g * 0.2 pg/g 2.0 pg/g
ETU Slices 80.5 i 5.3 95.0 i 5.6 96.3 i 2.8
Unpeeled sauce 82.4 i 2.8 87.7 i- 2.9 99.8 i- 3.0
Peeled sauce 79.9 i 6.9 93.5 i 5.5 96.7 i 7.2
Juice 75.7 i 5.6 88.6 i 7.1 98.1 i 5.3
Pomace 80.4 i 5.2 85.3 i 5.2 90.5 i 8.2

 

 

 

Note: 1. * Method detection limit (MDL) for mancozeb and ETU.
2. ** Values are the means of triplicate determinations.

C. Mancozeb Residue Study

 

(I) Comparison of Postharvest Wash Treatment on the Reduction of

Mancozeb Residues
Control samples were subjected to the same postharvest

treatment parameters except that they were not exposed to the mancozeb

174

during the growing seasons. The data illustrated in Figures 53—58, show
the effects of the various wash treatments on reduction of mancozeb
residues in /on apple and apple products. The total amount of residue on
the unwashed apple was determined to be 1.99 ppm. The established
tolerance level for mancozeb as published in the Code of Federal
Regulation, USA (1996) is 3.0 ppm. There were no statistical differences
between water wash and 50 ppm chlorine wash treatments (Figure 53).
For 500 ppm chlorine and 3 ppm ozone treatments, no mancozeb
residues were detected. Chlorine dioxide and HPAA were shown to be
effective treatments compared to no—wash, water-wash or 50 ppm
chlorine—wash; however, less effective than 500 ppm chlorine or ozone
treatments. For slices, no significant differences were found among no—
wash, water—wash and 50 ppm chlorine—wash treatments (Figure 54).
This indicates that water wash only is insufficient in removing pesticide
levels compared to no wash. In unpeeled sauce and peeled sauce, no
mancozeb residues were detected in all wash—treated samples (Figures
5556). In juice, there were no significant differences among no—wash,
water—wash. and 50 ppm chlorine—wash (Figure 57). High mancozeb
residues were detected in 50 ppm chlorine—wash than water—wash.
However, there was no significant difference between these two
treatments. Five hundred ppm chlorine wash still showed a powerful

effect among all treatments. In the case of pomace, relatively high levels

175

1997 Residue data, PHI = 77 days

 

 

 

 

2.50
‘6
3 2.00 -
3
g 1.50 -
0
c
a
a 1.00.
"6
‘g’ 0.50
o a b b b
000 j A
1 2 3 4
Wash treatments
1. No wash 2. Water wash 3. 50 ppm chlorine

4. 500 ppm chlorine 5. 10 ppm chlorine dioxide 6. 3 ppm ozone
7. 50 ppm HPAA

1998 Residue data, PHI = 4 days

 

 

 

   

 

 

a
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‘5
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3
3
3 1.50i
0
r:
e
E 1.00 -. b
“6
§ 050+
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1 2 3 4 5 6 7

Wash treatments

Figure 53. Concentration of Mancozeb residues in whole fruit
after postharvest wash treatments.
* Values with same letters are not significantly different (p<0.05).

176

1997 Residue data, PHI = 77 days

 

 

 

 

2.50
‘5
B: 2.00
a
fi
3 1.50 None detected
2
i 1.00 »
'6
2
o 0.50
o

0.00 .

1 2 3 4
Wash treatments
1. No wash 2. Water wash 3. 50 ppm chlorine

4. 500 ppm chlorine 5. 10 ppm chlorine dioxide 6. 3 ppm ozone
7. 50 ppm HPAA

1998 Residue data, PHI = 4 days

 

 

 

 

 

2.50
g 2.00
P.
3
3 1.50
0
5
a 1.00
"6
8 0.50
0
0 b b b b
0.00
1 2 3 4 5 6 7
Wash treatments

Figure 54. Concentration of Mancozeb residues in slices
after postharvest wash treatments.

* Values with same letters are not significantly different (p<0.05).

177

1997 Residue data, PHI = 77 days

 

 

 

 

2.50
a
a. 2.00
a
‘3
3 150 None detectOd
8
a
E 1.00
“‘2
§ 0.50
o

0.00 .

1 2 3 4
Wash treatments
1. No wash 2. Water wash 3. 50 ppm chlorine

4. 500 ppm chlorine 5. 10 ppm chlorine dioxide 6. 3 ppm ozone
7. 50 ppm HPAA

1998 Residue data, PHI = 4 days

 

 

 

 

 

 

2.50
:6
3 2.00
l .n
0
l N _
1 8 1.50
C
Cl
2 1.00 -«
"6 a a
‘3’ 0.50 ,.
0 b b b b b
0.00 7 7 r 7 ? TL
1 2 3 4 5 6 7
Wash treatments

Figure 55. Concentration of Mancozeb residues in unpeeled
sauce after postharvest treatments.
* Values with same letters are not significantly different (p<0.05).

178

1997 Residue data, PHI = 77 days

 

 

 

 

2.50
‘6
a 2.00
a
.0
g 150 None detected
8
fl
2 1.00
'6
2
o 0.50
o

0.00 -

1 2 3 4
Wash treatments
1. No wash 2. Water wash 3. 50 ppm chlorine

4. 500 ppm chlorine 5. 10 ppm chlorine dioxide 6. 3 ppm ozone
7. 50 ppm HPAA

1998 Residue data, PHI = 4 days

 

 

 

 

 

2.50
3
E 2.00
.D
g 1.50
0
C
N
s 1.00
'6
g 0.50
° b b b b b b
0.00 .
1 2 3 4 5 6 7
Wash treatments

Figure 56. Concentration of Mancozeb residues in peeled
sauce after postharvest treatments.
* Values with same letters are not significantly different (p<0.05).

179

1997 Residue data, PHI = 77 days

 

 

 

 

2.50
a
a 2.00
a
.n
g 1.50
0
C
(I
E 1.00
“6
§ 0.50
o a a b b
000 .___—____._____ , .
1 2 3 4
Wash treatments
1. No wash 2. Water wash 3. 50 ppm chlorine

4. 500 ppm chlorine 5. 10 ppm chlorine dioxide 6. 3 ppm ozone
7. 50 ppm HPAA

1998 Residue data, PHI = 4 days

 

2.50 r

8

1.50 1

§

Cone. of Mancozeb (uglg)
.0
8

 

 

 

 

0.00

Wash treatments

Figure 57. Concentration of Mancozeb residues in juice
after postharvest wash treatments.
* Values with same letters are not significantly different (p<0.05).

180

of mancozeb were detected in all wash—treated samples (Figure 58). In
the no—wash sample, the total concentration of mancozeb in pomace was
13.31 ppm which is much higher than the established tolerance level.
Various wash treatments reduced mancozeb levels but were not effective
compared to other products. In water—wash, 50 ppm chlorine and 10
ppm chlorine dioxide—wash, there were still high levels of mancozeb
detected. Five hundred ppm chlorine and 3 ppm ozone treatments
significantly (p<0.05) reduced mancozeb levels compared to other
washes. Pomace may be used as feeds for livestock so much more

concerns are needed.

(11) Comparison of Percent Reduction of Mancozeb Levels

The percent reduction in mancozeb residues in whole fruit,
apple slices, unpeeled apple sauce, peeled apple sauce, juice and pomace
are presented in Figures 59-64. To determine the percent residue
reduction, each wash treated sample was compared to residues from the
no wash treatment. The percent reduction in mancozeb was shown to
decrease in relation to higher initial residue levels. The mancozeb
residues in 1997 samples were lower than 1998 samples so, the overall
percent reductions were high in 1997 studies.

Almost 50% of mancozeb residue was removed from the fruit

with the water wash only in 1998 studies (Figure 59). In whole fruit,

181

1997 Residue data, PHI = 77 days

 

 

 

 

2.50
a
a. 2.00
a
.0
g 1.50 .
0
C
a
s 1.00 -
"6
§ 0.50
o a a a b
0 00 M -
1 2 3 4
Wash treatments
1. No wash 2. Water wash 3. 50 ppm chlorine

4. 500 ppm chlorine 5. 10 ppm chlorine dioxide 6. 3 ppm ozone
7. 50 ppm HPAA

1998 Residue data, PHI = 4 days

b

 

15.00
12.00 “t
9.00 a
6.00 1+

bc
3.00 «r

Conc. of Mancozeb (uglg)

 

 

   

0.00 1

 

1 2 3 4 5 6 7
Wash treatments

Figure 58. Concentration of Mancozeb residues in pomace
after postharvest wash treatments.
* Values with same letters are not significantly different (p<0.05).

182

 

     

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100

Figure 59. Percent reduction of Mancozeb residues in whole

fruit after postharvest wash treatments.

183

chlorine wash at 50 and 500 ppm removed about 53% and 100%
mancozeb residue, respectively in 1998 studies. Apples dipped in HPAA
and ozonated water reduced residue levels by about 82% and 100%,
respectively. While water alone reduced levels by 45%, the various other
treatments reduced mancozeb by 50—100%. No statistical difference
appeared between water—wash and 50 ppm chlorine—wash at the 5%
level. Also, there was no significant (p<0.05) difference between HPAA—
and chlorine dioxide—washes. The 500 ppm chlorine— and ozone—washes
were the most effective treatments for every apple product. Apple slices
and apple juice showed percent reduction of 45-100% and 47-100%,
respectively (Figure 60). No mancozeb residues were detected in 500 ppm
chlorine, 10 ppm chlorine dioxide, 3 ppm ozone, and 50 ppm HPAA
washes. Unwashed apples that were processed into unpeeled and peeled
sauce showed 70% and 100% reduction, respectively in residue level
compared to no washed whole fruit (Figures 61—62). Apples from the
various treatments that were subsequently processed into peeled and
unpeeled sauces showed mancozeb residue reductions of 100%. Percent
reduction in juice is shown in Figure 63. Water—wash and chlorine—wash
at 50 ppm removed about 47% and 56% mancozeb residue, respectively
in 1998 studies. In HPAA and ozonated water, mancozeb was reduced
about 63% and 82%, respectively. Chlorine at 500 ppm gave the best

effect on the reduction of mancozeb residue. Percent reduction of

184

1997, PHI = 77 days

 

 

 

 

4 l
i
g 3 None detected
a »
E 2
ill
2 1
o 20 4o 60 80 100
% Reduction of Mancozeb
1. No wash 2. Water wash 3. 50 ppm chlorine

4. 500 ppm chlorine 5. 10 ppm chlorine dioxide 6. 3 ppm ozone
7. 50 ppm HPAA

1998, PHI = 4 days

 

 

 

 

 

             

     

 

 

 

Wash treatments
A

 

 

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0 20 40 60 80 100

% Reduction of Mancozeb

Figure 60. Percent reduction of Mancozeb residues in slices
after Postharvest Wash Treatments.

185

1997, PHI = 77 days

 

3 None detected

Wash treatments

 

 

 

O 20 40 60 80 100
% Reduction of Mancozeb

1. No wash 2. Water wash 3. 50 ppm chlorine
4. 500 ppm chlorine 5. 10 ppm chlorine dioxide 6. 3 ppm ozone
7. 50 ppm HPAA

1998, PHI = 4 days

 

 

 

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0 20 40 60 80 100
% Reduction of Mancozeb

Figure 61. Percent reduction of Mancozeb residues in unpeeled
sauce after postharvest wash treatments.

186

1997, PHI = 77 days

 

3 None detected

Wash treatments

 

 

 

O 20 40 60 80 100
°/o Reduction of Mancozeb

1. No wash 2. Water wash 3. 50 ppm chlorine
4. 500 ppm chlorine 5. 10 ppm chlorine dioxide 6. 3 ppm ozone
7. 50 ppm HPAA

 

 

 

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Wash treatments

 

 

 

O 20 40 60 80 100
% Reduction of Mancozeb

Figure 62. Percent reduction of Mancozeb residues in peeled
sauce after postharvest wash treatments.

187

 

 

 

 

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4. 500 ppm chlorine 5. 10 ppm chlorine dioxide 6. 3 ppm ozone
7. 50 ppm HPAA

1. No wash

 

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Wash treatments
.5

 

 

 

0 20 40 60 80 100
% Reduction of Mancozeb

Figure 63. Percent reduction of Mancozeb residues in juice
after Postharvest Wash Treatments.

188

mancozeb in pomace showed patterns similar to other products;
however, the rate of reduction was lower (Figure 64). Water—wash seemed
to be more effective than chlorine wash at 50 ppm but there was no
statistically significant (p<0.05) difference. The patterns of overall percent

reduction was found to be consistent in all treatments.

(III) Comparison of Mancozeb Residue Levels between Products

The differences observed in the residue levels between products
is a direct result of the different processing methods used. In no—wash
and water—wash treatments, the differences in residues observed
between the two apple sauces is a result of peeling. When removing the
peel we also remove any residue which may bound on the surface of
skin. Processing into peeled apple sauce completely eliminated the
mancozeb in all wash-treatments. Mancozeb is a contact fungicide which
resides on the surface of the fruits so it would be assumed that removing
the peel would give a high reduction of mancozeb residues. Processing
the apples into slices, unpeeled sauce or peeled sauce significantly
reduced all residues in all the treatments. When apples were pressed for
juice the residues in the resulting pomace were concentrated and the
percent reduction of mancozeb in pomace by various washes was less
than in the other products. This was not unexpected, since a given

weight of pomace represents 4 to 7 times its weight of raw product before

189

1997, PHI = 77 days

 

 

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T

0 20 40 60 80 100
% Reduction of Mancozeb

1. No wash 2. Water wash 3. 50 ppm chlorine

4. 500 ppm chlorine 5. 10 ppm chlorine dioxide 6. 3 ppm ozone
7. 50 ppm HPAA

1998, PHI = 4 days

 

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Wash treatments

 

 

 

0 20 40 60 80 100 120
% Reduction of Mancozeb

Figure 64. Percent reduction of Mancozeb residues in pomace
after Postharvest Wash Treatments.

190

crushing and processing. Even so, the reductions by 500 ppm chlorine
and ozone were still significant. Apple pomace is the principal solid waste
generated in the apple processing industry. This is converted to various
marketable by-products such as animal feed, pectin or natural fiber
extract (Downing, 1989).

Overall, high reductions in mancozeb residues were observed in
all products from the combination of postharvest wash treatments and
processing. These findings are in agreement with previous studies by
Ong et al.(1996) and Siler (1998) on the ability of washing and processing
to significantly reduce pesticide residues in apples as well as other fruits
and vegetables. In the study by El—Hadidi (1993), processing of apples
into apple products such as apple slices, sauce and juice were
significantly effective in reducing the pesticide residue levels to non-

detectable amounts.

D. ETU Residue Study

(1) Comparison of Postharvest Wash Treatment on the Reduction of
ETU Residues

The data presented Figures 65—70, show the effects of the
various wash treatments on reduction of ETU residues in both 1997 and
1998 studies. The total amount of residue on the unwashed apples was

determined to be 0.02 ppm. ETU is a possible human carcinogen so its

191

1997 Residue data, PHI = 77 days

 

 

 

 

 

 

 

 

 

 

20.00
a
a 16.00
3
3
g 12.00 None detected
a 8.00
it
§ 4.00
o
0.00 . .
1 2 3 4
Wash treatments
1. No wash 2. Water wash 3. 50 ppm chlorine
4. 500 ppm chlorine 5. 10 ppm chlorine dioxide 6. 3 ppm ozone
7. 50 ppm HPAA
1998 Residue data. PHI = 4 days
20.00 a
a 16.00 -
a.
E.
.—
Ill
'53 8.00 ~
2
O
0 4.00 l
C C C C
0.00 .
1 2 3 4 5 6 7
Wash treatments
Figure 65. Concentration of ETU residues in whole fruit after

postharvest wash treatments.

* Values with same letters are not significantly different (p<0.05).

192

 

1997 Residue data, PHI = 77 days

 

 

 

 

20.00
g 16.00 ,
a
3
g 12.00 None detected
8
a
2 8.00
“6
2'
o 4.00»
o

0.00 e .~

1 2 3 4
Wash treatments
1. No wash 2. Water wash 3. 50 ppm chlorine

4. 500 ppm chlorine 5. 10 ppm chlorine dioxide 6. 3 ppm ozone
7. 50 ppm HPAA

1998 Residue data, PHI = 4 days

 

 

 

 

20.00
A1600
a
2°
312.00 None detected
I m
I Q-
; 9 8.00
. 0
' C
O
0 400 ~
0.00 - . 0+
1 2 3 4 5 6 7
Wash treatments

Figure 66. Concentration of ETU residues in slices after
postharvest wash treatments.
* Values with same letters are not significantly different (p<0.05).

193

1997 Residue data, PHI = 77 days

 

 

 

 

20.00

3

a 16.00

a.

8

g 12.00 None detected

8

a

2 8.00

"6

2

O 4.00

o

0.00 *
1 2 3 4
Wash treatments
1. No wash 2. Water wash 3. 50 ppm chlorine

4. 500 ppm chlorine 5. 10 ppm chlorine dioxide 6. 3 ppm ozone
7. 50 ppm HPAA

1998 Residue data, PHI = 4 days

 

 

 

 

20.00
a 16.00
2’
‘5 12.00 None detected
in
.-
9 8.00
0
C
o
0 4.00

0.00 .

1 2 3 4 5 6 7
Wash treatments

Figure 67. Concentration of ETU residues in unpeeled sauce after
postharvest wash treatments.
* Values with same letters are not significantly different (p<0.05).

194

1997 Residue data, PHI = 77 days ,

 

 

 

 

20.00

a
B 16.00
a
3
g 12.00 None detected
8
II
2 8.00
"6
2
o 4.00
o

0.00

1 2 3 4
Wash treatments
1. No wash 2. Water wash 3. 50 ppm chlorine

4. 500 ppm chlorine 5. 10 ppm chlorine dioxide 6. 3 ppm ozone
7. 50 ppm HPAA

1998 Residue data, PHI = 4 days

 

 

 

 

20.00 -----....._.,______
a 16.00
2
‘5 1200 None detected
8
Q-
9 8.00
0
c
O
0 4.00
0.00 . . ’
Wash treatments

Figure 68. Concentration of ETU residues in peeled sauce after
postharvest wash treatments.
* Values with same letters are not significantly different (p<0.05).

195

1997 Residue data, PHI = 77 days

 

 

 

 

 

 

 

 

 

 

20.00
a
a 16.00 ,
a
'8
3 12.00 None detected
2 8.00
'5.
§ 4.00
O
0.00 z
1 2 3
Wash treatments
1. No wash 2. Water wash 3. 50 ppm chlorine
4. 500 ppm chlorine 5. 10 ppm chlorine dioxide 6. 3 ppm ozone
7. 50 ppm HPAA
1998 Residue data, PHI = 4 days
20.00
“ g 16.00
1 5
'—
‘ ill
'53 8.00
8
O
o 4.00
b b b b b
0.00
1 2 3 4 5 6
Wash treatments

Figure 69. Concentration of ETU residues in juice after
postharvest wash treatments.

* Values with same letters are not significantly different (p<0.05).

196

1997 Residue data, PHI = 77 days

 

 

 

 

20.00 .
a 16.00
a
E.
3 12.00
.—
I.“
9 8.00
0
c
O
0 4.00
a a a a
0.00 .
1 2 3 4
Wash treatments .
1. No wash 2. Water wash 3. 50 ppm chlorine

4. 500 ppm chlorine 5. 10 ppm chlorine dioxide 6. 3 ppm ozone
7. 50 ppm HPAA

1998 Residue data, PHI = 4 days

 

 

100.00

cl

80.00

60.00 1

40.00 -

Conc. of ETU (nglg)

20.00 .

 

 

 

 

0.00 J

1 2 3 4 5 6 7
Wash treatments

Figure 70. Concentration of ETU residues in pomace after
postharvest wash treatments.
* Values with same letters are not significantly different (p<0.05).

197

presence is undesirable. In Figure 65, 500 ppm chlorine, chlorine dioxide
ozone and HPAA treatments reduced ETU levels to non-detection limits
in whole fruit. In slices, unpeeled sauce and peeled sauce, no ETU was
found (Figures 66—68). In pomace, relatively high levels of ETU were
detected in all wash treated samples (Figure 70). For the no—wash
sample, the total concentration of ETU was 0.07 ppm. Various wash
treatments reduced ETU concentration. For water—wash, 50 ppm
chlorine and 10 ppm chlorine dioxide wash, high levels of ETU were
detected. 500 ppm chlorine and 3 ppm ozone treatments significantly

(p<0.05) reduced ETU levels compared to other washes.

(II) Comparison of Percent Reduction of ETU Levels

The percent reduction of ETU residues in whole fruit, apple
slices, unpeeled apple sauce, peeled apple sauce, juice and pomace are
presented in Figures 71—76. To calculate the percent ETU residue
reduction, each wash—treated samples were compared to residues in
each of the no wash treatments. The ETU residues in 1997 all samples
and unpeeled and peeled sauce in 1998 samples were below the
detection limit in all wash treatments. So these samples were excluded in
the determination of percent reduction.

Almost 38% of ETU residue was removed from the fruit with the

water wash only in 1998 studies (Figure 71). In whole fruit, chlorine

198

1997, PHI = 77 days

 

3 None detected *‘

Wash treatments

 

 

 

0 20 40 60 80 100
% Reduction of Mancozeb

1. No wash 2. Water wash 3. 50 ppm chlorine

4. 500 ppm chlorine 5. 10 ppm chlorine dioxide 6. 3 ppm ozone
7. 50 ppm HPAA

 

 

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0 20 40 60 80 1 00 1 20
% Reduction of ETU

Figure 71. Percent reduction of ETU residues in whole fruit
after Postharvest Wash Treatments.

199

1997, PHI = 77 days I

 

3 None detected

Wash treatments

 

 

 

r I

0 20 40 60 80 1 00

% Reduction of Mancozeb l
I

1. No wash 2. Water wash 3. 50 ppm chlorine
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1 0.0

 

 

0 20 40 60 80 100
% Reduction of ETU

Figure 72. Percent reduction of ETU residues in slices after
postharvest wash treatments.

200

1997, PHI = 77 days

 

 

 

 

l
l
l

s 4 ,

g .

g 3 None detected

(U

E 2 1

3

g 1

0 20 40 60 80 100
% Reduction of Mancozeb
1. No wash 2. Water wash 3. 50 ppm chlorine

4. 500 ppm chlorine 5. 10 ppm chlorine dioxide 6. 3 ppm ozone
7. 50 ppm HPAA

1998, PHI = 4 days *

 

5 None detected

Wash treatments
.5

 

 

 

s l

0 20 40 60 80 100
% Reduction of ETU .

Figure 73. Percent reduction of ETU residues in unpeeled sauce
after Postharvest Wash Treatments.

201

1997, PHI = 77 days

 

 

 

 

8 4
g s»
g 3 None detected
a l
E 2
3
3 1
0 20 40 6O 80 100
% Reduction of Mancozeb
1. No wash 2. Water wash 3. 50 ppm chlorine

4. 500 ppm chlorine 5. 10 ppm chlorine dioxide 6. 3 ppm ozone
7. 50 ppm HPAA

1998, PHI = 4 days

 

5 None detected

Wash treatments
¢

 

 

 

O 20 40 60 80 100
% Reduction of ETU

Figure 74. Percent reduction of ETU residues in peeled sauce
after Postharvest Wash Treatments.

202

1997, PHI = 77 days

 

3 None detected

Wash treatments

 

 

 

40 6O 80
% Reduction of Mancozeb

2. Water wash
5. 10 ppm chlorine dioxide

1. N0 wash
4. 500 ppm chlorine
7. 50 ppm HPAA

1998, PHI = 4 days

100

3. 50 ppm chlorine
6. 3 ppm ozone

 

 

 

 

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Wash treatments
A

 

 

40 60 80

% Reduction of ETU

100

Figure 75. Percent reduction of ETU residues in juice after
postharvest wash treatments.

203

 

120

1997, PHI = 77 days

 

 

 

 

8 4

8

g 3 None detected

t!

E 2

3

g 1

0 20 40 60 80 100
% Reduction of Mancozeb

1. No wash 2. Water wash 3. 50 ppm chlorine
4. 500 ppm chlorine 5. 10 ppm chlorine dioxide 6. 3 ppm ozone
7. 50 ppm HPAA

 

 

 

 

 

 

 

 

 

 

 

7 I
I
I
6 76.1 ‘
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1 0.0
0 20 40 60 80 100
7. Reduction of ETU 7

Figure 76. Percent reduction of ETU residues in pomace after
postharvest wash treatments.

204

wash at 50 and 500 ppm removed about 56% and 100% ETU residue,
respectively. Apples dipped in chlorine dioxide and HPAA treated water
reduced residue levels by about 97%, both. For unpeeled and peeled
apple sauce, ETU residue was below the detection limit (Figures 73—74)
in 1998 samples. Apple slices and apple juice showed percent reduction
of 7 5—7 9% and 88—90%, respectively, in water wash only (Figures 72 and
75). In pomace, relatively low levels of percent reduction in ETU residues
were detected for all wash—treated samples (Figure 76). This is due to the
high concentration of ETU in pomace compared to other products. Even
chlorine at 500 ppm and ozone at 3 ppm, gave low percent of reductions

with approximately 78 and 76%, respectively.

(111) Comparison of ETU Residue Levels between Products

Reduction of ETU residues by various oxidizing agents showed a
pattern similar to mancozeb. High amounts of mancozeb residues
resulted in high ETU residues. In unpeeled and peeled apple sauce, no
mancozeb was detected. Again, pomace contained high levels of ETU
compared to other products for all wash treatments. This indicates that
certain processing procedure such as peeling or steaming can play an

important role in reducing pesticide residue levels.

205

SUMMARY & CONCLUSIONS

The objective of the present study was to determine the effects
of various wash treatments on the reduction of mancozeb and ETU
residues in apples and apple products and determine the effectiveness of
different post harvest treatments and processing procedures on the
reduction of mancozeb and ETU residues.

Apples sprayed with mancozeb were used to determine the
effectiveness of various wash treatments on the removal of the mancozeb
and ETU on and in fresh and processed apples. Two—lb apples were used
per replication (3 replications per treatment) and placed in a 20 L bucket
containing 7 L of water or each oxidizing agent solution. The three
treatments in 1997 were (1) No wash, (2) Water wash, (3) Calcium
hypochlorite wash @ 50 and 500 ppm and the six treatments in 1998
were (1) No wash, (2) Water wash, (3) Calcium hypochlorite wash @ 50
and 500 ppm, (4) Chlorine dioxide wash @ 10 ppm, (5) Ozone wash @ 3
ppm and (6) Hydrogen peroxyacetic acid wash @ 50 ppm. Mancozeb and
ETU residues were analyzed on and in the whole fruit and processed
apples using GLC and HPLC.

The amounts of mancozeb residue found on the unwashed

whole fruits were below the EPA tolerance level. However mancozeb

206

detected in pomace was more than EPA tolerance value (3 ppm).
Reduction in residual mancozeb was significantly (p<0.05) influenced by
the effect of various wash treatments as compared to the unwashed
samples. There was significantly higher residue in the water washed
apples than apples processed with other wash treatments. Chlorine wash
at 50 ppm was not especially effective due to its low concentration.
Chlorine wash at 500 ppm and ozone at 3 ppm were the most effective
treatments for mancozeb and ETU removal in all products. The addition
of chlorine, chlorine dioxide, hydrogen peroxyacetic acid, and ozone were
shown to be more effective in removing mancozeb residues than a water
wash alone. When various wash treatments were combined with
processing into apple sauce, mancozeb was reduced by 100% (ie. non—
detection levels). Between 48—100% of the mancozeb and 45—100% of the
ETU residues were removed after processing. This indicates that certain
processing procedure such as peeling or steaming play an important role

in reducing pesticide residue levels.

207

CHAPTER IV. STUDIES ON THE DETERMINATION
OF THE DEGRADATION PRODUCTS
AND PATHWAYS

INTRODUCTION

Ozone and chlorine dioxide have been widely used for treating
drinking water and food processing for many years in many countries.
The use of ozone is particularly attractive because it can be applied as a
gas or in water, and it dissipates quickly, so that no residue is left on
foods (Graham, 1997). Like ozone, chlorine dioxide is a good disinfectant
and can kill a large number of microorganisms, including some that are
resistant to treatment with chlorine (Richardson et al., 1994). Both these
compounds are also being explored for use in reducing pesticide residues
on fruits and vegetables and the results have shown them to be effective.
However, there is also concern over the presence of chemical by—
products that are formed when chlorine, ozone and chlorine dioxide are
used for reduction of pesticide residues. Chlorine treatment is known to
produce some chemicals that cause cancer in laboratory animals. Use of
ozone and chlorine dioxide as alternatives to chlorine for treatment of
drinking water and food processing is increasing, mainly because they
produce fewer disinfection by—products. Because the alternative
disinfectants do not form appreciable levels of these by-products, they
are gaining in popularity and use. However, it is unknown whether they

produce compounds as harmful or more harmful than those produced by

208

chlorine. EPA has therefore set out to identify all potentially harmful by—
products.

Gas chromatography (GC) is frequently interfaced with mass
spectrometry (MS) for confirmation and structural identification of
pesticides (Sherma, 1997). Chemical ionization mass spectrometry
(CI/MS) is frequently used to generate molecular ions. Electron
ionization (E1/ M8) is an indispensable tool for determining structures, as
it provides the necessary empirical formula information for the molecular
ion and fragments. It also helps to limit the number of possible
structures for each unknown by—product. GC / IR is useful for
determining the functional group (Richardson et al., 1998).

Mass spectrometry is an analytical technique used for the
detection of ions and the measurements of their masses, allowing for the
identification of the sample. The components become ionized, then travel
through a drift region. In the drift region the ions enter a reflectron. By
the time the ions reach the detector they are gathered into like—massed
groups. The ions hit the detector at the end of their drift. The output of
the recording device is a chromatogram. The mass spectrometry process
involves three steps: ionization of the sample, mass separation and
detection.

A Time—of—Flight Mass Spectrometer (TOFMS) is based on the

elapsed time the ion takes from the ion source to the detector. Ions

209

which have been accelerated to equal energies move with velocities
related to their mass—to—charge ratio; these characteristic velocities are
used for mass analysis in TOFMS. Ions simultaneously accelerated out of
an ion source separate into groups according to their velocities as they
travel through an evacuated, field—free tube, as shown in Figure 7 7. The
time elapsed between the extraction of an ion from the source and its
detection at the end of the tube is measured and used to calculate mass.
In a typical commercial TOFMS instrument, the energy applied for
extraction is sufficient to cause ions up to about m/z 1000 to arrive at
the detector within 100 us of the extraction pulse (Yefchak, 1990). The
instrument is therefore capable of producing a signal representing 104
complete mass spectra each second. This permits analysis of dozens of
compounds in 1—3 minutes (Song et al., 1997) due to the extremely rapid
spectral acquisition capacity (up to 500 spectra/ second) of the mass
spectrometer. The use of TOFMS for detection allows compression of
chromatography time by permitting significant overlap of eluting
compounds without loss of analytical capacity as long as the mass
spectra of overlapping compounds differ by a single m/z ratio. In
addition, compression of chromatography time results in an increase in
sensitivity in that the spectrometer response is concentrated over a
shorter time interval than by conventional chromatography. Thus,

sampling, chromatographic separation, detection and analysis potentially

210

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211

can be completed in minutes per sample with enhanced sensitivity (Song
et al, 1998).

Among various oxidizing agents used in Chapters I—III, ozone
and chlorine dioxide were selected for this study because they are known
to be relatively less toxic and would be good alternatives to chlorine
treatment. One objective of this investigation was to determine the by—
products of mancozeb and ETU when treated with ozone and chlorine
dioxide and elucidate possible degradation pathways of this pesticide. A
second objective was to compare our results to previous

findings.

212

MATERIALS AND METHODS

MATERIALS

A. Reagents

(I) Solvents

All organic solvents used for preparation of stock solution,
sample extraction and GC/ MS were distilled-in-glass grade. Hexane,
xylene, chloroform and methylene chloride were obtained from J. T.

Baker, Co. (Phillipsburg, NJ).

(11) Standard Chemicals

Mancozeb standard (79.8%) was obtained from Rohm 85 Hass
(Philadelphia, PA). Mancozeb is a complex polymeric, non-crystalline
organometallic solid that does not exist in pure form. Standard product
material is about 80% pure and contains some stabilizers and
formulation materials. Ethylenethiourea (ETU [2—imidazolidinethione],
CAS Registry No.96—45—7, 99.0%) and ethyleneurea (EU [2—
imidazolidineone], CAS Registry No. 120—93—4, chemical purity 96.0%)

standard were obtained from Aldrich Co. (Milwaukee, WI).

213

B. Glassware

All glassware was thoroughly washed with detergent and warm
water, then rinsed with distilled water. The glassware was then rinsed
with acetone before being placed in an oven at 400°C overnight before

1186.

METHODS

A. Ozonation Procedure

A laboratory research ozone generator (Allegheny Teledyne Inc.)
was used. Ozone (03) was bubbled through a glass sparger (produced
bubbles of approximately 10 mm i.d.) into 500 ml of distilled water at
ambient temperature and pH under 25 psi at 15 SCFH of oxygen until
the desired ozone concentration (3 ppm) was attained. Mancozeb or ETU
was spiked to give a final concentration of 100 ppm. After the addition of
the mancozeb or ETU, at the desired ozone concentration, the addition of
ozone was continued at 25 psi and 15 FCFN of oxygen. A 30 m1 sample
was transferred at l, 15, 30 and 60 minute intervals into an Erlenmeyer
flask. Two hundred pl of 0.5% 0.1 M sodium thiosulfate solution was

immediately added to the samples to quench the reaction.

214

All ozone concentrations were determined by the method
published in Standard Methods for the Examination of Water and

Wastewater, 17th Edition (4500—03 B Indigo Colorimetric Method, 1987).

B. Chlorination Procedure

Chlorine dioxide (C102) was generated in the laboratory using
the manufacturer’s (S.C. Johnson Professional, WI.) instructions as
follows. One hundred ml of the stock 2% Oxine FP solution were added
to a 200 ml volume French square screw—capped bottle. Twenty five mls
of 7 5% w/w food grade phosphoric acid were added, sealed and allowed
to generate chlorine dioxide for 5 minutes with a magnetic stirrer to
ensure though mixing. This served as a 100 ppm stock chlorine dioxide
solution to achieve the final test concentration. For 20 ppm of chlorine
dioxide, added 8 liters of stock solution and made to a total 10 gallon
with distilled water. Mancozeb or ETU was spiked to give a final
concentration of 100 ppm

All chlorine dioxide concentrations were determined using the
HACH chlorine colorimeter before and after each sampling run. The
detailed determination method is given in the methods section of Chapter

I.

215

C. Sample Extraction

Thirty i 0.1 mls prepared sample were weight in an Erlenmeyer
flask, with 8 g of potassium fluoride (KF) and 0.6 g of ammonium
chloride (NH4C1) and extracted in a 250 ml separatory funnel. In a
preliminary study, this mixture was extracted with 5 different solvents
according to their polarity. The solvents include hexane (polarity = 7.3),
xylene (polarity = 8.8), chloroform (polarity = 9.1), methylene chloride
(polarity = 9.6), and water (polarity = 21.0). Scheme 1 shows the diagram
of preliminary extraction procedure. From these results, mancozeb and
ETU residues were dissolved only in chloroform and methylene chloride
layer so these two solvents were used for further extraction procedure.
Scheme 2 shows the diagram of revised extraction procedure. The
mixture was extracted with 50 ml of chloroform and methylene chloride
two times. Then, the solvent layer was passed through a bed of 25 g
sodium sulfate (120°C for at least 12 hr) and collected in a Zymark
Turbovap tube. The extracted liquid was evaporated to 1 m1 at 40°C in a
Zymark Turbovap evaporator (Zymark Ind., Hopkin, MA) using nitrogen
gas. This reduced extract was determined by GC/ MS. By—products of
ozone or chlorine dioxide treatment and possible degradation pathways

were identified.

216

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218

D. GC/MS Analysis

GC / MS analyses was performed on mass spectrometer (LECO
Corp., 1997), equipped with a Hewlett Packard Model 6890 gas
chromatograph (Hewlett Packard Co., Wilmington, DE) and Pegasus II
Version 1.4 computer workstation (LECO Corp., 1997) was used.

Injections of 1 pl of the extract were introduced via a split
injector (split ratio=1z10) onto a J 85 W Scientific hp—S chromatographic
column (30 m, 0.25 mm i.d., 0.25 pm film thickness). Ultrapurified
helium (99.999%) was used as carrier gas at a flow rate of 1.5
ml/ minute. The GC temperature program consisted of an initial
temperature of 40°C, which was held for 1 minute, followed by an
increase at a rate of 55°C /minute to 300°C, which was held for 1 minute.
Transfer lines were held at 250°C, and the injection port was controlled
at 280°C. Sample detection was by Time—of—Flight Mass Spectrometry
(TOFMS) with an electron ionization source (FCD—650, LECO Corp, St.
Joseph, MI). Mass spectra were collected at a rate of 40 /s over the mass
range (m/z) 33—350. The electron ionization energy was 70 eV. The

temperature of the ion source was 200°C.

219

RESULTS & DISCUSSION

As a first step in this study, five different solvents were selected
according to their polarity. These include hexane, xylene, chloroform,
methylene chloride and water. In serial extraction, mancozeb and ETU
were found in chloroform and methylene chloride layer, so these solvents
were selected as a further extraction study. Then ozone and chlorine
dioxide treatments of the pesticide were performed and the by-products

were identified by GC/ MS.

A. By—Products Formed from Hydrolysis
(1) Degradation of Mancozeb

Identification of fragment ions was confirmed by comparison of
collected mass spectra with those of authenticated chemical standards
and to reference spectra in a mass spectral library (National Institute for
Standard Technology, Search Version 1.5, Gaithersburg, MD). A mass
spectrum is a graph of ion abundance versus mass to charge ratio. The
ions and their abundance serve to establish the molecular weight and
structure of the compound being analyzed. Since the ionization process
frequently breaks up or fragments the molecule, ions appear in the

spectrum at lower m/z values than that which corresponds to the

220

molecular mass of the molecule. Figure 78 (A) shows a typical spectrum
of mancozeb standard at a concentration of 100 ppm, while Figure 78
(B) shows the mass spectrum of the chloroform extract of mancozeb
obtained by GC/MS. These spectra corresponded to library search data
for mancozeb (Figure 79). In the mass spectrum of chloroform extract,
mancozeb has a strong molecular cluster at m/z 144, both with and
without computer background subtraction (Figure 78(B)). The average
retention time of this peak was approximately 181—189 seconds. This
corresponded to the ethylene bisdithiocarbamic acid compound minus
manganese and zinc ion (C4H4N282; S—Imidazoledithiocarboxylic acid)
(Figure 80). Metal ions in mancozeb structure are considered to be very
unstable and quickly lost when mancozeb is introduced into high
temperature condition. This compound can be present as linear or cyclic
form. The major peak with the highest intensity was m/z value 72 at
181—181 seconds and several other peaks which include m/z 60 and m/z
45 appeared. The ion at m/z 85 carried a smaller portion of the total ion
current. The fragment ions were used to determine molecular structure.

The proposed structures of the fragment ions are illustrated in Figure 81.

(II) Degradation of ETU
Figure 82 (A) shows a typical spectrum of ETU standard at a

concentration of 100 ppm, while Figure 82 (B) shows the mass spectrum

221

1000-

i§é

Relative Intensity
V
8

.§

8

g

§i§

.§

300-

Relative Intensity

—- m
8 8
' 1

O)
.8

‘3'

.§

§

(A) Mancozeb Standard Soln

 

 

1o 10 2 o 2 o 2 o 2'é6"'"5153"m§§3""'5416m
72 "V2

 

(B) Mancozeb; Chloroform Extract

117

 

50 1o ’ 1o 2 ww'vgafierfirfiifiiflaso
Z

/

m/

Figure 78. A typical spectrum of mancozeb from (A) standard
solution at 100 ppm in distilled water and (B)
chloroform extract.

222

Relative Intensity

 

 

 

144
72

8

 

 

 

 

., l I
ll.‘ . ! - i)

 

l
IIU'IIIIIIIIIIU'UIIVU'I'I'U'III'UII'I'III'VUUIU'I'U'UUIVIIUUI'UAY

60 80100 120 140 160 180 200 220 240260
m/z

Figure 79. The mass spectrum of Mancozeb obtained from library search.

223

s
CH2-NH-0-S
\(Zn. Mn)

CH2-NH-9-s
s

Mancozeb

 

l

s
CH2-NH-i'2-SH
CH2-NH-9-SH
s
M“ 212
/ or \
fi’
CH2-NH-C HCéN‘c=s
' l l
CH2-NH-9 HZC\N’C=S
S H
M+ 144 M‘ 144

5-lmidazoledithiocarboxylic acid

Figure 80. Possible fragmentation of Mancozeb by hydrolysis.

224

 

CH2-NH-g

HC¢N\c=s
°' Ht': c-s
CH2-NH? 2 \N/ -
S H
M+144
CH2-NH2 s-fi-N=CH2
CH2-NW m/z 72
m/z 60 l
S=fiyNH
m/260

Figure 81. Proposed degradation pathway of Mancozeb in aqueous

solution by hydrolysis.

225

Relative Intensity

Relative Intensity

10001 ‘02

i (A) ETU Standard Soln.
900.;
800
700—;

1
1
1
q
4
_
I
4

400.? 3°

I

1

.
.
300'
d

1

4

1

 

zoo-j

1oo-j

35.1.1.3:

' 60 """" i "20""1'10' 100 180 200220240260 260 300320340"
1000_ 102 m/z

 

 

 

  

 

mm? (B) ETU; Chloroform Extract
goo;
no.5
600-3
5.3..
400.3

4
1
4
<
l
—I
1
q
4
a

 

4
a

1

a

4

.1

4

4

a

4

1005 it
i 59
i ll 8?

 

 

 

Figure 82. A typical spectrum of ETU from (A) standard solution at
100 ppm in distilled water and (B) chloroform extract.

226

of chloroform extract of ETU obtained by GC/MS. These spectra
corresponded to library search data for ETU (Figure 83). After 60 minutes
reaction in distilled water, the spectrum showed similar patterns to that
of 0 minutes and still had a strong molecular cluster at m/z 102 (Figure
84). The M+ (102) corresponded to molecular weight of ETU. This
indicates that ETU was stable in distilled water and did not undergo
hydrolysis during 60 minutes. The average retention time of ETU was

approximately 210—230 seconds.

(111) Effect of pH on the Formation of Mancozeb Degradation
Product

The mass spectra of mancozeb in each pH solution were
collected and monitored for a period of sixty minutes at both chloroform
and methylene chloride layers. Chloroform layer showed more intensive
GC/MS response to the mancozeb degradation products than methylene
chloride layer. This was due to the effect of serial extraction. Most
mancozeb residues were extracted by chloroform and only small amounts
of mancozeb residues remained on the methylene chloride layer. In pure
mancozeb standard solution, the most abundant ion was m/z value 72.
In Figure 85—86, the time dependence of the GC/ MS response as the
peak area of the molecular ion (M+ 72) is shown. As time elapsed the

relative response of the ion currents at m/z values 72 increased in

227

 

Relative Intensity

100. 102

.8

 

 

 

 

UV'UUVII' 'UU'II'UU UV 1 I
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0 10 20 30 40 50 60 7 80 90 100
m/z

1:! 1 I!!! I“
0

Figure 83. The mass spectrum of ETU obtained from library search.

228

1 000
900
800
700 4
600 ~

500 .-

l
x . .

Relative Intensity
A
8

§ §

l-u44.... ... ..... .... ..

 

A. N (n (O
8 ° 8 8
o
l .i.........1.........;...-.....1.-... ..l..-.. . .

‘s’

Relative Intensity

A N
1. “1......1...

 

38

45

 

 

50

59

102

 

(A) 0 min

 

73

 

 

 

(B) 60 min

 

vvvvv

vavavavava

Figure 84. The mass spectrum of ETU obtained from chloroform extract at

(A) 0 minute and (B) 60 minute reaction time in distilled water.

229

GCIMS Response (Peak Area)

GCIMS Response (Peak Area)

 

2.5e+6
(A) oi-ICI3 layer
2.0e+6 ~

1.5e+6 ~

1.0e+6 «

 

l
I VT

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

5.0e+5 ‘ _ ’
' <-
‘f _
v ' L
0.0 I fl I I I I
0 1o 20 30 4o 50 60
Time (min)
5e+5
(B) CHZCI2 layer
4e+5 ~
3e+5 ~
0
2e+5 « L
O
I «)-
r
1’. A
1e+5 ~ "
I
‘ I
O I T i I I U
o 10 20 30 4o 50 60
Time (min)

 

 

+ Control, pH 4.6
—0— Control, pH 7.0
—v— Control, pH 10.7
—v— 03, pH 4.6
+ 03. pH 7.0
—-{:)— 03, pH 10.7

 

 

 

+ Control, pH 4.6
—0— Control, pH 7.0
+ Control, pH 10.7
—v— 03. pH 4.6
+ 03, pH 7.0
—{}—- 03, pH 10.7

 

Figure 85. Effect of ozone on time dependence of the GCIMS response
on the formation of molecular ion (MI 72) from (A) CHCI3 layer

and (B) CHZCIZ layer.

230

 

 

2.5e+6

 

2.0e+6 .

1.5e+6 4

GCIMS Response (Peak Area)

 

 

(A) CHC|3 layer

+ Control, pH 4.6
-O—— Control, pH 7.0
+ Control, pH 10.7
-v— CIOZ, pH 4.6

—Cl- CIOZ, pH 10.7

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1.0e+6 ~
5.0e+5 4
0.0
0
Time (min)
5e+5
B CH Cl la er
A ( ) 2 2 y + Control, pH 4.6
8 4e+5 « —0— Control, pH 7.0
2 + Control, pH 10.7
x ——v— C102, pH 4.6
8 + 010,. pH 7.0
E: 3e+5 ‘ —o— 0:0,, pH 10.7
3 .
C
o
o. L
m 2e+5 ~
&D 0 I
4:.
B 1e+5 0’. '
0 ' 7 4
..‘ .
“W
0 I T I I I I
o 10 20 3o 40 so 60
Time (min)
Figure 86. Effect of chlorine dioxide on time dependence of the GCIMS response

on the formation of molecular ion (M+ 72) from (A) CHCI3 layer and
(B) CHZCI2 layer.

231

control treatment at three pH ranges. The formation of m/z 72 was the
greatest at pH 7 .0 and decreased in pH 4.7 and pH 10.7. This result
suggest that the m/z 72 ion was stable at neutral pH and the formation
of this ion increased as time elapsed.

Ozone treatment at pH 4.6 showed preventive effect on the
formation of m/z 72 ion (Figure 85). The ozone treatment at pH 10.7 was
the least effective. No m/z 72 ion was detected at pH 4.6 or pH 7.0 after
60 minutes reaction time. This was due to the instability of ozone at
alkaline condition. These results corresponded to the model system
study. Chlorine dioxide also showed preventative effect on the formation
of m/z ion (Figure 86). pH 4.6 showed the most effectiveness and pH
10.7 was the least effective in both chloroform and methylene chloride
layer. However, the effect was lower than ozone treatment. m/z 72 ion

still remained at 20 ppm chlorine dioxide treatment after 60 minutes.

B. By—Products Formed from Ozonation
(1) Degradation of Mancozeb

Ozonation of mancozeb produced ETU, with a retention time of
206 seconds. When the reaction between mancozeb and ozone
continued, degradation of mancozeb occurred. At 30 minutes reaction
time, the total amount of m/z 144 ion decreased compared to 1 minute.

After 60 minutes ozone treatment, no m/z 144 was detected at 206

232

seconds (Figure 87). Oxidation due to ozonation or hydrolysis changes
the by—products into high polarity hydrophilic compounds, such as ETU
and others. Analysis of the aqueous ozonation of mancozeb and its
degradation products demonstrated that metal groups, such as
manganese and zinc, are the first Site of attack and the C82 or CS group
was removed.

Usually, reference standards are pure compounds, however the
sample extracts are not, so they can introduce interfering ions into the
mass spectrum, complicating the confirmation process. Mancozeb is a
complex polymeric, non-crystalline organometallic solid that does not
exist in pure form. Standard mancozeb is about 80% pure and contains
some stabilizers and formulation materials. So, determination of some

oxidation products was not possible because of matrix interference.

(II) Degradation of ETU

Treatment of ETU with ozone yielded several degradation
compounds. Figure 88 presents the total ion current (TIC) of ETU
obtained from chloroform layer with 3 ppm ozone treatment after 60
minutes. Prolonged ozonation (60 minutes) of ETU eventually gave rise to
EDA (ethylenediamine) and several degradation products but no
ethyleneurea (EU) was detected in this study. The mass spectrum of each

molecular ion (Mt) used to determine the possible degradation products

233

 

 

   

 

 

 

 

 

 

 

(A) 0 min

3

4.)
"-1

U)

c

0

4..)

C

H

0

>
-«-i

4.)

m
H

o

“ M+ 144

J.
Time (seconds)
(B) 60 min

>.

4.)

-H

(.0

a

m

4.)

c

H

o

>

"-1

4.)

w

H

o

m

No M+ 144
J.

 

 

 

 

Time (seconds)

Figure 87. Comparison of chromatogram of Mancozeb at (A) 0 minutes and

(B) 60 minutes in ozone treatment.

234

Relative Intensity

Relative Intensity

(A) 0 min

 

4-_A_
V———-—
g-

 

 

 

 

 

 

 

 

ETU

 

 

Time (seconds)

 

 

 

 

 

 

 

Wl (B) 60 min

 

 

Time (seconds)

Figure 88. Total ion current (TIC) of ETU in ozone treatment at

(A) 0 minutes and (B) 60 minutes in chloroform extract.

235

are shown in Figure 89. The molecular ions found as ETU degradation
products by ozone treatment were M+ 60 at 42.77 seconds, M“ 84 at
47.87 seconds, M+ 163 at 61.37 seconds, M+ 117 at 62.47 seconds and
M+ 267 at 131.57 seconds. The proposed structures of the degradation
products are illustrated in Figure 90. The degradation by-products were
confirmed with previous findings (Aizawa, 1991). The results suggest that
ozonation increases the removal of ETU and produce several degradation
products. These results, however, do not reveal the underlying
mechanism(s) or toxicity. Hence, more detailed studies are required in
order to identify these mechanisms and subsequently, optimize the

combined treatment process. Toxicity tests are also required.

C. By—Products Formed from Chlorine Dioxide
(1) Degradation of Mancozeb

Mancozeb with chlorine dioxide treatment produced ETU, with a
retention time of 206—218 seconds. When the reaction between
mancozeb and chlorine dioxide continued, the degradation of mancozeb
occurred. At 30 minutes reaction time, the total amount of m/z 144 ion
decreased as compared to 0 minutes. After 60 minutes chlorine dioxide
treatment, small peak of m/z 144 was still detected. This indicates that
mancozeb residue did not completely degrade into other by products but

still remained. This was probably due to the high concentration of

236

 

Relative Intensity

Relative Intensity

1000 102
(A) ETU (Parent Compd.)

at 208,99 seconds

mm
ami
mm
600 Q
500 1
«mg
an?

i 73
200 45

1003
i 59

 

 

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mm; (B) m/z 60 at42.77 seconds

4
.
4
-<
4
4
4
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4

4

4

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4

4

4

1

.
.

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&M‘
-

.

1

.

400;
uni
200-

100

 

 

 

Figure 89. The molecular ions found as ETU degradation products
by Ozone (Cont’d).

237

 

Relative Intensity

Relative Intensity

1000 49

(C) m/z 84 at 47.87 seconds
900
800
700
600
500
400
300 ~ 35
200 —:

100~

 

 

 

 

 

 

 

VVVVVV

 

 

m/z

1000 35

(D) m/z 163 at 61.37 seconds
mm!
700‘

mm:
. 117

4
4

4

4

4

..4

4

4

4

mm. 1

 

 

 

 

 

 

 

Figure 89. The molecular ions found as ETU degradation products
by Ozone.

238

Relative Intensity

Relative Intensity

1000
900
800
700
600 .
500
400 S
300
200

umé

 

900 ~
4
4
800 -:
4
4
4
4
700~
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4
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4
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4
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43

36

 

 

 

117

 

 

(E) m/z ll7at 62.47 seconds

 

73

 

A
vvvvvvv

(F) m/z 267 at 131.57 seconds

267

 

Figure 89. The molecular ions found as ETU degradation products

by Ozone.

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mancozeb (100 ppm) compared to low chlorine dioxide concentration. It
is anticipated that m/z 144 peak would completely disappear with
chlorine dioxide treatment if the concentration of chlorine dioxide is

increased above the 20 ppm that was used in this study.

(II) Degradation of ETU

Treatment of ETU with chlorine dioxide yielded several
degradation compounds. Figure 91 presents the total ion current (TIC) of
ETU obtained from chloroform layer with 20 ppm chlorine treatment
after 60 minutes. At prolonged ozonation (60 minutes), ETU was oxidized
to ethyleneurea (EU) at a retention time of 162-180 seconds. However,
ETU was still detected at 209—221 seconds in the spectra. This mean
that ETU did not completely degrade into other by products but still
remained in the reaction mixture. This was probably due to the high
concentration of ETU (100 ppm) compared to low chlorine dioxide
concentration. The mass spectrum of each molecular ion (M+) used to
determine the possible degradation products of chlorine dioxide
treatment are shown in Figure 92. The molecular ions found as ETU
degradation products were M” 117 at 62.72 seconds, M” 86 at 160.12
seconds and M+ 163 at 61.37 seconds. Several unknown products are
also present. The proposed structures of the degradation products are

illustrated in Figure 93. Chlorine dioxide showed less effectiveness in

241

 

 

 

 

 

 

 

 

 

(A) 0 min
w
4.)
H
(D
c
m
4J
c
H
m
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'° l
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m
m
Time (seconds)
H ' (B) 60 min
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m
c
m
4.)
c
H
m
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m
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Time (seconds)

Figure 91. Total ion current (TIC) of ETU in chlorine dioxide treatment at

(A) 0 minutes and (8)60 minutes in chloroform extract.

242

Relative Intensity

Relative Intensity

l
l
|

1000 ) ‘02

(A) ETU (Parent Compd.)
“mi 3 at 210.67 seconds

800 j
700

600 3
500
400
3.3m

73
200 «1 45

 

 

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mm? i (B) m/z 117 at 62.72 seconds

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800

700

600 .

500 -

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m/z

 

 

Figure 92. The molecular ions found as ETU degradation products
by Chlorine Dioxide (Cont’d).

243

 

Relative Intensity

Relative Intensity

1000
900 ~
800
mm
mm.
500 ;
«m{
an.
200

um:

 

1000

.
.
4
800‘
-

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200 ‘
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100 -.

 

 

 

 

43

 

59

83

 

 

74

 

 

(C) m/z 163 at 73.19 seconds

86 m/z

 

 

(D) m/z 86 at 160.12 seconds

 

Figure 92. The molecular ions found as ETU degradation products
by chlorine dioxide.

244

 

 

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degradation of ETU compared to ozone treatment. ETU was produced
less degradation products compared to ozonation. This is probably due to
the fact that ETU was not completely degraded by chlorine dioxide.

The results suggest that low dose chlorine dioxide treatment
does not significantly remove mancozeb and ETU. However, the effect of
chlorine treatment may be expected to depend on the applied chlorine
dioxide dosage, contact time, as well as the concentration of mancozeb
present in solution. Consequently, further studies are required in order
to assess these effects.

Overall, many by—products were identified, several of which
have never been reported previously. Many of the compounds were not
present in any spectral library (NIST or Wiley), and many of the ones that
were in the libraries did not give conclusive library matches (Richardson
et al., 1998). For many of the compounds, little information was provided
by the mass spectra, because of the absence of molecular ions, which

provide molecular weight information.

246

SUMMARY & CONCLUSIONS

The objective of the present study was to determine the
degradation products of mancozeb and ETU and elucidate the possible
degradation pathways in solutions as a result of chemical oxidation
using ozone and chlorine dioxide. This study was developed in a solution
at 100 ppm mancozeb and ETU concentration during 60 minutes. Two
different oxidizing agents used in this study were (1) Ozone @ 3 ppm and
(2) Chlorine dioxide @ 20 ppm. Ozone was continuously provided
throughout the course of the reaction. Degradation products were
detected with high resolution GC/ MS. The total analysis time was 4
minutes per sample combined with rapid gas chromatographic
separation and time—of—flight mass spectrometry (TOFMS).

Mancozeb lead to m/z 144 ion fragmentation, which is 5—
Imidazoledithiocarboxylic acid, as a major degradation product. ETU
showed M+ l02 which corresponds to its mass, was stable in distilled
water and did not undergo hydrolysis during 60 minutes. The average
retention time of mancozeb and ETU was approximately 181—189 and
210—230 seconds, respectively. Ozonation of mancozeb produced ETU as
a major product. Treatment of ETU with ozone produced several

degradation compounds. From prolonged ozonation, the C82 or CS group

247

Was removed. Overall, several by-products identified were M+ 60, M+ 84,
M+ 163, M+ 117 and M+ 267 by ozone and M+ 117, M+ 86 and M“ 163 by
chlorine dioxide treatment. Several of these have been reported but some
of those never been reported previously. Identification of fragment ions in
this study was not conducted for unknown compounds but confirmed by
comparison of published structural data with those of Degradation of
Pesticides (1991). Although mancozeb and ETU were degraded by
chlorine dioxide, this oxidant was less effective than ozone at the
concentration used in this study. However, it is anticipated that
mancozeb and ETU would be completely degraded by the chlorine dioxide
treatment if the concentration of chlorine dioxide is increased above the

20 ppm that was used in this study.

248

 

   

FUTURE WORK

Possible future research efforts include:

1. This study determined that the various wash treatments and
processing methods were effective in the degradation/ removal of
pesticide residues on apples at the pilot plant level. Future work should
focus on the possibility of scaling up to a commercial size operation.
More research should be carried out to set up the proper concentrations

which may be used to maximized reductions of pesticide residue levels.

2. This study elucidated some degradation products and pathways after
ozone and chlorine dioxide treatments in a model system. Future work
should include determination of possible products as a results of
chemical oxidation in processed apple products. Other analytical
equipment, such as GC/MS, LC /MS, IR, NMR and UV, should be used to
confirm the structure and pathways. Assessment of toxicity should also

be carried out on the degradation products.

249

 

 

APPENDICES

Peak Area

Appendix 1. A typical standard curve for Mancozeb standards.

1e+8

8e+7 ~

6e+7 ~

4e+7 .

2e+7 j

 

 

O

 

 

100

I l

200 300

Concentration (ug)

25C)

400

500

 

Peak Area

Appendix 2. A typical standard curve for ETU standards.

 

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400 600

Concentration (ppb)

251

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