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TOPICS IN PESTICIDE RESIDUE ANALYSIS: ASSESSMENT OF
CONTAMINATION OF SURFACE WATER AND FISH FROM COTE D'IVOIRE
AND THE EVALUATION OF IMMUNOASSAYS FOR DETECTION OF

PESTICIDES IN PLANT AND FISH SAMPLES

by

Eboua Narcisse Wandan

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Department of Entomology and the Program of Environmental Toxicology

1996

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ABSTRACT

TOPICS IN PESTICIDE RESIDUE ANALYSIS: ASSESSMENT OF
CONTAMINATION OF SURFACE WATER AND FISH FROM COTE D'IVOIRE
AND THE EVALUATION OF IMMUNOASSAY FOR DETECTION OF
PESTICIDES IN PLANT AND FISH SAMPLES

BY

Eboua Narcisse Wandan

The developing world, has focused on producing more food to feed the growing
population. To do so, farmers must not only rely on improved seeds and mechanization
but also on fertilizer and pesticides. As in the developed world, the assessment of
contamination of the environment by these chemicals should be of great concern. For this
reason, water and fish samples were collected from selected rivers and lagoons from C6te
d'Ivoire and were brought to the USA to be analyzed. The physico-chemical
characteristics values indicate that these water are suitable for drinking. Only two metals,
zinc and copper were detected at very low levels (range and values). The levels of
organochlorine pesticides detected in water and fish samples were below the extraneous
residue limits (ERL) and the acceptable daily intake set by the FAG/WHO codex
alimentaruis commission. The levels were higher in the south of the country where
agriculture is more intensified and in urban areas compared to the north and rural areas.
These results indicate that agricultural and industrial activities are the most important

source of surface water contamination by xenobiotics. Three commercially available

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immunoassay kits from different manufacturers were evaluated for the determination of
pesticides, as an alternative to gas chromatographic (GC) methods used for the
determination of organochlorine pesticides. Interference due to fish and com leaf
coextracts was corrected with dilution using distillate water. The resulting assays showed
good reproducibility and accuracy and had an estimated limit of pesticide detection of
0.25 ppb in fish and plant material. The results of the study indicated that the two types
of immunoassay kits gave similar results in the detection of alachlor, atrazine, and
carbofuran in corn leaf and fish fillet but the Ohmicron RaPID Assays® kit was more
accurate and sensitive and less expensive compared to the Millipore Envirogardm kit. The
analysis of incurred corn leaf and fish samples show that the ELISA compares favorably
with GC measurements. The ELISA was found to be less expensive and easy to use
compared to gas chromatography and could be a good analytical tool for developing

countries where financial resources are scarce.

This dissertation is dedicated to the memories of my mother in law Ekora and my sister
Andeh who passed away while I was here. May they rest in peace.

iv

ACKNOWLEDGMENTS

I would first like to express my gratitude and my sincere appreciation to Dr. Matt
Zabik, my major professor for the opportunity to work in his laboratory and for his
guidance during the past six years. I would like also to thank Drs. R. Leavitt, P. Hart,
Geo Bird, and specially Don Penner for his help in all the aspects involving this work.

I would like also to express my heartfelt appreciation to my lab mates: Gamal,
Glenn, Melvin, Matt. and the team of 1R4.

I would like to deeply thank the government of Cote d’Ivoire and the College of
Agriculture (ENSA), the African-American Institute (AF GRAD), together with Michigan
State University for allowing me to follow this program and providing me with their
financial support. Grateful acknowledgment is extended to Ms. Elizabeth Ward,
Academic advisor at the AAI, for her counseling and support during my stay here.

Special thanks to everyone of my family, particularly my father and mother for
providing me with their love and support and for teaching me that success comes with

hard work and perseverance.

Finally and most importantly, I would like to thank my lovely wife Brigitte and

my two sons Miessan-Aka and Kadjo Wandan, Jr. for their love and support

 

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TABLE OF CONTENTS

PART I
ASSESSMENT OF CONTAMINATION OF SURFACE WATER AND FISH FROM
COTE D'IVOIRE

, Page
LIST OF TABLES ............................................... xi

LIST OF FIGURES .............................................. xv
A. INTRODUCTION ............................................ 1
B. OVERVIEW OF THE COUNTRY ............................... 15
1. GENERAL DESCRIPTION ............................... 15
2. SURFACE WATER ..................................... l7
3. ICULTURE .......................................... 22

4. PEST PROBLEMS ..................................... 25
a CASH CROPS ................................... 26
b. FOCD CROPS ................................... 27
c. MAIN CEREALS ................................. 27
d. £1111 AND VEGETASLES .......................... 28
e. PCSTHARVEST PESTS ............................ 28
f. INSECTS VECTOR CF DISEASES .................... 29
s. PEST CONTROL MEASURES AND PRESENT USE OF PESTICIDE 30
a AGRICULTURE .................................. 30
b. HUMAN HEALTH ................................ 32
c. EXPERIMENTAL PROCEDURE ................................ 34
1. STUDY DESIGN AND SAMPLE COLLECTION ............... 34
a SAIQLINC SITES ................................ 34
b. SAMPLE COLLECTION A12 PRCCESSINC ............ 37
2. MATERIALS ......................................... 37
a Sow .................................... 37
b. A N ............. 39

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3. ANALYTICAL METHODS ............................... 40
a. ETERMINATI NOF PHY - HEMI AL PR PERTIES . 40

b. A METAL DETERMINATI N IN WATER ........ 40
c. R ANO PE TI IDES DETERMINATI N IN
FISH ............................................ 41
(1. HR MATO RAPHI ETERMINATI N .............. 42
D. RESULTS AND DISCUSSION ................................. 44
l. PHYSICOCHEMICAL PROPERTIES ........................ 44
2. HEAVY METALS CONCENTRATION IN WATER SAMPLES ..... 46
3. ORGANOCHLORINE PESTICIDES IN WATER ................ 47
4. ORGANOCHLORINE PESTICIDES IN FISH .................. 51
E. CONCLUSIONS ............................................ 54

APPENDIX A: LIST OF THE PESTICIDES DISTRIBUTED IN COTE D'IVOIRE 59
APPENDIX B: CHROMATOGRAMS OF THE ANALYSIS ............... 65

LIST OF REFERENCES ............................................. 70

PART II

THE EVALUATION OF IMMUNOASSAY FOR DETECTION OF PESTICIDES IN
PLANT AND FISH SAMPLES

Page

A. INTRODUCTION ........................................... 76
B. ENZYME IMMUNOASSAYS AND ITS APPLICATIONS .............. 80
1. THE IMMUNE RESPONSE ........... ' .................... 80

2. ANTIBODY STRUCTURE AND FUNCTION .................. 82

3. ANTIGEN STRUCTURE ................................. 82

Vii

4. PRODUCTION OF ANTIBODIES FOR LABORATORY USE ...... 85

a MONOCLONAL ANTIBODIES ....................... 85

b. ANTIBODY-ANTIGEN INTERACTIONS ............... 87

5. IMMUNOASSAYS ..................................... 88
a ENZYME IMMUNOASSAYS ........................ 88

b. TYPE OF ENZYME IMMUNOASSAYS ................ 90

6. APPLICATION OF IMMUNOASSAYS ...................... 91
a HEALTH AND AND CLINICAL MEDECINE ............ 91

b. AGRICULTURAL USES ........................... 92

c. TOXINS AND CONTAMINANTS SCREENING IN FOOD . . . 93

d. ENVIRONMENTAL ANALYSIS ..................... 93

C. CHARACTERISTICS OF THE PESTICIDES ....................... 95
1. ALACHLOR .......................................... 95
a. USES .......................................... 95

b. BEHAVIOR IN PLANTS ........................... 97

c. BEHAVIOR OF ALACHLOR IN SOIL ................. 97

d. BEHAVIOR IN AQUATIC ENVIRONMENT ............ 97

e. TOXICOLOGICAL PROPERTIES ................. 98

2. ATRAZINE .......................................... 99
a. USES ........................................... 99

b. BEHAVIOR IN PLANTS ........................... 101

c. BEHAVIOR IN SOIL .............................. 101

d. BEHAVIOR IN THE AQUATIC ENVIRONMENT ......... 102

e. TOXICOLOGICAL PROPERTIES ..................... 103

3. CARBOFURAN ....................................... 105
a. USES .......................................... 105

b. BEHAVIOR IN PLANTS ........................... 107

c. BEHAVIOR IN SOILS ............................. 107

d. BEHAVIOR IN AQUATIC ENVIRONMENT ............. 108

e. TOXICOLOGICAL PROPERTIES ..................... 108

D. MATERIALS AND METHODS ................................. 112
1. STUDY DESIGN AND SAMPLE COLLECTION ............... 112
a. FISH REARING .................................. 112

b. CORN RAISING ................................. 113

 

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a. REAGENTS ..................................... 114

b EQUIPMENT .................................... 114

a1. ELISA .................................. 114

ll. CHROMATOGRAPHY ....................... 115
3.ANALYTICALMETHODS 116
a. EXTRACTION OF FISH FOR ELISA DETECTION ........ 116

b. EXTRACTION OF CORN LEAF FOR ELISA DETECTION . . 116
c. EXTRACTION OF CORN LEAF FOR CHROMATOGRAPHIC

d. ANALYSIS ..................................... 117
e. EXTRACTION OF FISH FOR CHROMATOGRAPHY ........ .119
f. DETECTION AND QUANTIFICATION ................. 119
f1. W ................. 119
D. W ........... 120

f3. W
f4. W ......................... 121
f5. 90W ........................... 122
E. RESULTS AND DISCUSSION ...................... . ........... 123
1. ALACHLOR .......................................... 123
a ACCURACY .................................... 125
a1. w .............................. 125
12. CCRNLEAF ............................... 125
b. REPRODUCIBILITY .............................. 128
c. CROSS-REACTIVITY .............................. 130
d. SENSITIVITY ................................... 132
e. INCURRED SAMPLES ............................. 133
el. W .......... ' .................... 133

e2. W

2. ATRAZINE .......................................... 136
a. ACCURACY .................................... 137
:1. W .............................. 137
I2. W .............................. 140
b. REPRODUCIBILITY .............................. 141
c. CROSS-REACTIVITY .............................. 142
d. SENSITIVITY ................................... 145
e. INCURRED SAMPLES ............................. 146

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e1. CQE LEAF .............................. 146

62. W ............................ 147

3. CARBOFURAN ............................................ 149
a ACCURACY .................................... 150

a1. FISH FILLET .............................. 150

82. CCRN LEAF .............................. 153

b. REAPITABILITY ................................. 154

c. SENSITIVITY ................................... 156

d. CROSS-REACTIVITY ............................. 157

e. INCURRED SAMPLES ............................. 159

el. CORN LEAF .............................. 159

82. FISH FILLET .............................. 161

F. CONCLUSIONS ............................................ 163
APPENDIX A: RESULTS OF ALACHLOR DETERMINATION ............ 178
APPENDIX B: RESULTS OF ATRAZINE DETERMINATION ............ 179
APPENDIX C: RESULTS OF CARBOFURAN DETERMINATION ......... 187
REFERENCES CITED . . . .' ...................................... 199

 

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LIST OF TABLES

Inblsl'ills Ease

Al.

A2.

A3.

A4.

Organochlorine pesticides commonly encountered in natural waters. Extracted
from Environmental Chemistry; 4th ed. Stanley E. Manahan, Lewis '

Publishers. 1991. .......................................... 7
Occurrence and significance of trace elements in natural waters. Adapted

from Environmental chemistry. Stanley E. Manahand 5th Ed, 1991. . . . . 10
Major crops grown in C6te d'Ivoire ............................ 23

Physico—chemical characteristics of water samples from the collection sites . . . . 45

Heavy metals contaminant levels (pg/L) in water samples. Mean from
five determinations ....................................... 46

Mean recovery (Va) :1: RSD (n = 3) for the pesticides in fish-and detection
limits (pg/kg).

Mean (n = 5) pesticide residue levels (mg/kg) in fillet of Tilapia from the
nine collection sites ....................................... 52

List of the insecticides used in crop protection in Céte d'Ivoire.
Source: Rapport annual sur la vente des pesticides pour utilisation
agricole. UNIPHYTO (1991). ............................... 60

List of the fungicides used in crop protection in Cote d'Ivoire
Source: Rapport annual sur la vente des pesticides pour utilisation
agricole. UNIPHYTO (1991). ............................... 61

List of herbicides used in crop protection in Cote d'Ivoire.
Source: Rapport annual sur la vente des pesticides pour utilisation
agricole, UNIPHYTO (1991) ................................ 62

List of nematicides and miscellaneous pesticides used in crop protection in
COte d'Ivoire. Source: Rapport annual sur la vente des pesticides pour

xi

 

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ll.

12.

14.

15.

16.

17.

utilisation agricole, UNIPHYTO (1991). ........................ 63

PART II
IN! Ease
Chemical and physical properties of the technical alachlor ............ 96
Chemical and physical properties of the pure atrazine .............. 100
Chromatographic conditions for the determinations of the pesticide ..... 121

Accuracy of alachlor determination in spiked corn leaf
(2 replications per assay). A: Ohmicron kit; B; Millipore kit. ......... 127

Assay reproducibility (%CV) for the EIA for alachlor in corn leaf extract . . . . 129

Method determination limit (WL) calculated from standard deviation (0) of
6 replicate assays of corn leaf (A) and fish tissue (B) spiked with alachlor. 132

Alachlor concentration in incurred fish fillet samples by ELISA. 2 assays
per samples. ........................................... 136

Accuracy of Ohmicron (A) and Millipore (B) test kits for the determination
alachlor in spiked fish fillets (2 replications per assay). .............. 140

Assay reproducibility for of the EIA for alachlor in corn leaf extract . . . . 141

Alachlor concentration in incurred fish fillet samples by ELISA. 2 assays
per samples. ............................................ 148

Accuracy of Ohmicron (A) and Millipore (B) test kits for the
determination carbofuran in spiked fish fillets (2 replications per assay). . 152

Accuracy of Ohmicron (A) and Millipore (B) test kits for the
determination carbofuran in spiked fish fillets (2 replications per assay). . . 154

Assay reproducibility for of the EIA for carbofuran in com leaf extract . . 155

xii

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A2.

A3.

A5.

A6.

A7.

A8.

B1.

B3.

B4.

BS.

B6.

B7.

BS.

C1.

C4.

C5.

Method determination limit (MDL) calculated from standard deviation (0) of

6 replicates assays of the samples spiked with carbofuran ................ 156
Carbofuran concentration in incurred corn leaf samples by ELISA. 2 assays

per samples. ............................................ 161
Standard Curve Data ................................... . . . 170
Recovery Study Data for Fish Samples ......................... 172
Recovery Study Data for Corn Leaf data ........................ 173
Sensitivity Study Data ..................................... 175
Cross-Reactivity Study Data ................................. 176

Incurred Leaf Study Datal77

Incurred Fish Study Data ................................... 178
Standard Curve Data ........................... i ........... 179
Recovery Study Data for Corn Leaf data . . . .. .................... 181
Repeatability Study Data ................................... 182
Sensitivity Study Data ..................................... 183
Cross-Reactivity Study Data ................................. 184
Incurred Leaf Study Data ................................... 185
Incurred Fish Study Data ................................... 186
Standard Curve Data ...................................... 187
Repeatability Study Data ................................... 190
Sensitivity Study Data ..................................... 191

xiii

C6.

C7.

C8.

D1.

D2.

D3.

Cross-Reactivity Study Data ................................. 192

Incurred Leaf Study Data ................................... 193
Incurred Fish Study Data ................................... 194
Tank Water Characteristics for Alachlor ........................ 196
Tank Water Characteristics for Atrazine ..... ' .................... 197
Tank Water Characteristics for Carbofuran ....................... 198

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LIST OF FIGURES

PART I

Figure Tith Page
1. Processes influencing the behavior and fate of xenobiotics in the environment

(1973) ................................................. 3
2. Map of physical characteristics of COte d'Ivoire ................... l6
3. Map of the water systems illustrating the lagoons and rivers in Cote d'Ivoire.19
4. Structure of pesticide market in Cote d’Ivoire ..................... 32
5. Map of Céte D’Ivoire illustrating the sample collection sites .......... 36

Bl. Chromatogram of 00.1 ppm of standard solution of organochlorine

pesticide mixture ........................................ 65
BZ. Chromatogram of water sample from .......................... 66
B3. Chromatogram of water sample from .......................... 67
B4. Chromatogram of fish sample from ............................ 68
BS. Chromatogram of fish sample from ............................ 69

PART II

Elam Ifle has
1 Cellular events leading to antibody production following B-cells activation

by antigen molecules. ...................................... 81
2. The Structure of an Antibody Molecule. Four protein chains combine to form

an antibody molecule. ..................................... 82

10.

ll.

12.

13.

14.

Illustration of the binding of the antibodies to the antigen specific binding
sites called epitopes. Each epitope can bind to a different antibody containing
a specific antigen-binding site. ........... ' .................... 84

Illustration of a synthetic antigen indicating the hapten (small organic
chemical) covalently attached to immunogenic carrier molecule. ....... 85

Polyclonal antiserum containing a mixture of antibodies produced by multiple
B cells. ............................................... 86

Illustration of Monoclonal Antibodies Production. One B cell is fused in
the laboratory with a tumor cell. The resulting hybridoma produces
multiple copies of specific type of antibody (monoclonal antibody). ..... 87

Enzyme conjugate. Enzyme are physically linked to antibodies or antigens to

form an indicator system. The enzyme, the antibody, and the antigen must
retain their activities and binding capacities to be useful. ............. 89

Color reaction catalyzed enzymes used as indicators or tags in immunoassay. 89
Plot of the standard curves Of the Ohmicron and Millipore test kits
(average of 4 determinations). %Bo = % (absorbance of the

sample/absorbance of thezerocontrol). .......................... 124

Response of the ELISA kits to alachlor and metolachlor. (D) alachlor;
(I) metolachlor; (O ) mixture. ............................... 131

Alachlor concentration in incurred corn leaf samples by ELISA. 2 assays
per samples. . . ......................................... 134

Correlation of alachlor concentrations as determined by ELISA and gc
methods, n=4, r = 0.996 , y = 1.017 X + 0.0005. ................. 136

Confirmation of the ELISA results by GC/MS ..................... 136

Plot of the standard curves of the two test kits (average of 4 determinations).
%B0 = % (absorbance of the sample/absorbance of the zero control) .........

xvi

15.

16.

17.

18.

19.

20.

21.

22.

23.

Response of the ELISA kits to alachlor and metolachlor. (D) alachlor;

(I) metolachlor; (0 ) mixture. .............................. 143
Alachlor concentration in incurred corn leaf samples by ELISA.

2 assays per samples. ..................................... 146
Correlation of alachlor concentrations as determined by ELISA and go

methods, n = 4, r = 0.996 , y = 1.017 X + 0.0005. ................ 147
Confirmation of the ELISA results by GC/MS ..................... 149

Plot of the standard curves of the two test kits (average of 4 determinations).
%Bo = % (absorbance of the sample/absorbance of the zero control). . . . 150

Response of the ELISA kits to carbofuran; 3-ketocarbofuran and mixture . . . . 158

Carbofuran concentration in incurred corn leaf samples by Ohmicron EIA kit.
2 assays per samples. ...................................... 160

Correlation of carbofuran concentrations as determined by ELISA and go
methods, n = 4, r = 0.996 , y = 1.017 X + 0.0005. ................ 160

Mass spectra of GC peaks of incurred corn leaf and fish fillet.

(A) carbofuran standard solution; (B) non treated corn leaf, (C) treated corn
leaf; (D) non-treated fish fillet, (E) treated fish fillet. ............... 163

xvii

PART I
ASSESSMENT OF CONTAMINATION OF SURFACE WATER AND FISH FROM

COTE D'IVOIRE

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CHAPTER I

INTRODUCTION

In 1988, the world population amounted to 5 billion, and it is estimated to reach
6.5 billion in the year 2000. While the increase has stabilized in the developed world (0.6
% annual growth rate), it is increasing in the developing world at the rate of 1.6% per
year (IBRD, 1989). As result of this steep increase in population there is insufficient food
supply leading to malnourishment and a shorter life span. To feed this growing
population, the world food production must increase. This has been the case in the
developed world and in some underdeveloped countries. But many of the nations of sub-
Saharan Africa still suffer from low productivity in part due to insects and other
destructive pests. In all of these countries pesticides use will help prevent loss due to
pests. The use of pesticides, together with other means like irrigation, fertilization, and
mechanization would help reduce damage to crops and maintain adequate food supplies
in order to feed the growing population.
Most of the developing countries are located in areas where endemic diseases are still
prevalent. Reports of the World Health Organization (1985, 1987) indicate that a third
of the total population is threatened by vector-Dom diseases. Drugs and vaccinations have

been used to reduced the impact of these diseases. Although some success have been

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achieved by these means, the most efficient way to reduce the impact of these diseases
has been the control of the insect vectors by continuous application of insecticides and
molluscides.

From the economic standpoint as well as human and animal health, pesticides use
in Africa is Vital in the production of food and for the protection of man an animal. But
pesticides applied to croplands or in localized areas have been shown to move through
the environment affecting not only non-target organisms but also contaminate soil, surface
and groundwater, and air creating a great concern among general public. It is therefore
imperative to ascertain the extent of environmental contamination.

Pesticides applied in the lithosphere for pest control are transported to the aquatic
environment through atmospheric transport, soil runoff, erosion, and leaching (Figure 1).
The atmosphere is a mobile medium and serve as major transport route to move pesticides
to aquatic environments. The atmosphere becomes contaminated with pesticides by drift
during application, volatilization, and wind erosion. Drift is the portion that is moved
away from the target area by wind. Aerial application contributes to more drift but in
Céte d'Ivoire this method is used only in banana production because of the size of the
banana plant and for insect vectors control; all others crops receive ground application.
The extent of pesticide dispersal in the air due to drifi is governed primary by prevailing
conditions, formulation of the pesticides, and the method of application. Drift losses
reduce application efficiency, necessitating more frequent applications, increases costs,
and create hazards to non-target organisms and the environment. The quantity and extent

of drift can be reduced by considering the appropriate spray formulation, the spray

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equipment used such as ultra-low volume (ULV) and, weather conditions. Volatilization
has been recognized as a major pathway for loss of pesticides from soil, plant, and water
surfaces (Spencer et al., 1973).

Volatilization from the soil is evident for both surface-applied and soil
incorporated pesticides. The vaporization rate of a pesticide is function of its vapor
pressure, but once it is in contact with the soil its vapor pressure is modified by
environmental variables. Climatic and soil factors regulating volatilization rate include,
air movement, temperature, relative humidity, soil moisture content, soil organic matter
content, and pesticide concentration in the soil. Field measurements indicate that
significant volatilization loss may occur if pesticides are not incorporated in the soil.
Losses have been observed for DDT (Spencer and Cliath, 1972) and related compounds
such as lindane (Spencer and Cliath, 1970). To prevent excessive loss of more volatile
pesticides, it is necessary to incorporate them into the soil immediately after application.
However, under normal farming practices, pesticides incorporation is not always possible
because many of the insecticides and fungicides are applied on soil and plant surfaces.
Pesticides dispersed in the atmosphere may be associated with airborne particulate matter
and transported. Wind erosion may provide an important atmospheric source of pesticides
whereby they are redisposed on aquatic ecosystems. Data indicates that DDT distribution
in coastal and oceanic waters results from fallout of airborne particulate material
(Risebrough, 1969). This implies that a vast area of water surface, such as lagoons may
receive significant inputs of pesticides from the atmosphere.

Pesticide contamination of groundwater and surface water can occur through

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leaching. This downward movement is controlled by soil, pesticides, and climatic factors.
The leachability of a compound depends primarily to the degree to which it is adsorbed
to soil colloids. Pesticides are leached more readily in coarse-textured soils than in fine-
textured soils as the latter contain more clay and organic matter. The solubility of
pesticides plays an important role in their movement in the soil since solubility limits the
concentration of the compound in the soil-water phase. The transport of pesticides
through the soil is conditioned by the amount, intensity, and frequency of percolating
water. Adsorption of non-ionic pesticides, which includes the organochlorine and
organophosphate insecticides, is correlated primarily with soil organic matter content
(Bailey and White, 1970) and to a lesser extent with clay content. Retention of acidic and
basic compounds is affected markedly by soil pH (Donaldson and Foy, 1965). Soil pH
controls the overall charge of the molecule an hence its adsorptivity to clay and organic
colloids. The organic cations; diquat and paraquat, are held strongly by clay minerals and
are often adsorbed irreversibly (Weed and Weber, 1969). Weakly adsorbed water-soluble
compounds are desorbed readily by water and hence pose a greater potential for leaching.
Studies have Shown that the organochlorine (OC) insecticides, which have limited water
solubility are the least mobile, followed by the organophosphate insecticides. The water-
soluble acidic herbicides are most mobile. Most of the pesticides, such as the triazines,
phenylureas and carbarnates, have intermediate mobility. Organochlorine insecticides are,
in general, non leachable. Field trials have shown that they are retained largely in the
upper 15-20 cm layer of most agricultural soils (Cliath and Spencer, 1971.). Any

movement to lower depths and subsequently to water tables may be attributed to physical

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transport of adsorbed compounds through vertical cracks formed during dry periods

(Willis and Hamilton, 1973).

Pesticides present on agricultural land may be transported through surface runoff
either in solution and/or as adsorbed on soil particles. Since surface soils are susceptible
to erosion, pesticides retained in subsurface layer are potentially transportable by surface
drainage. The degree of pesticide loss by runoff depends principally on soil properties,
nature of the pesticide, and climatic factors (Bailey , 1966). Studies revealed that losses
of most OC insecticides relative to the amount applied are low even for surface-applied
insecticides. Once DC are present in the soil, they may persist for a long period of time
and are capable of being carried from one season to the next (Hindin and Bennett, 1970).
Pesticides associated with soil may be subjected to continual runoff, thereby providing a
steady, low level residue to aquatic systems. Since aquatic ecosystems may serve as
reservoirs or sinks for vaIiOUS chemicals, the indigenous species of animals, plants, and
microorganisms immersed in the water medium may incorporated these chemicals into
the food chain and pass them along to the highest predators in the food chain which may
result in higher concentration. Therefore, aquatic animals consumed as foodstuff may
represent a potential source of human exposure to toxic chemicals, including carcinogens
and mutagens.

Organochlorine pesticides such as DDT, aldrin, lindane, dieldrin, and heptachlor
(Table 1) were introduced in the early 40’s and were very successful for the control of
pests. They have a long persistence in soils and provide a greater potential than the other

insecticides for contaminating aquatic systems. This may result in their deposition in

Table 1.0rganochorine pesticides commonly encountered in natural waters. Extracted from
Environmental Chemistry, 4th ed. Stanley E. Manahan, Lewis Publishers. 1991.

 

Pesticide Formula

Aldrin-Dieldrin

DDT

Endosulfan

Endrin

Heptachlor

Lindane

‘Fresh-water quality Uses and characteritics

criteria

0.003 ug/L

0.00 jig/L

0.003 pg/L

0.004 jig/L

0.001 pg/L

0.01 pg/L

Persistent and stable in soil,
effective against insect in soil.
Organisms convert aldrin to
dieldrin, known to be
carcinogenic to mice. Banned
in the USA for most uses in
1975.

Low acute toxicity to mammals;
persistent; accumulate in food
chain. Some evidence of
carcinogenicity, use banned in
the US. in 1972.

Used in fruit to control aphids,
beetles, and caterpillars. Lower
chronic toxicity to mammals

Special precautions must be
used to avoid skin contact
during application; readily
photolyzes to non-toxic ketone
form.

Used to control pests in soil;
insecticide in feed. Change to
more the more toxic epoxide
which persists for a long time
in the soil; use restricted in
US. in 1978.

Used to control insects, plant
pests, animal parasites; widely
manufactures because of
convenience, lack of odor,
minimal residue

‘ Public Health Service Drinking Water Standard, (1.8. Public Health Service, 1962

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aquatic organisms, such as fish resulting in their accumulation in the food chain. These
compounds may be also detrimental to fish by interfering with their metabolism and/or
reduce egg hatchability. Because of their adverse environmental impacts, the OC have
generated considerable public concern and were banned in most industrial world.

OC were introduced in Céte d'Ivoire in the 50’s for the control of pests in
commercial agriculture (coffee, cocoa, cotton, etc.) and to protect human health against
vectors of diseases. Although some of them were commercially banned, many are still
in use because of their efficiency (e.g. lindane, endosulfan) or through their illegal entry
in the country.

A number of studies have been conducted on the presence of OC in water system
in Africa. In Sudan, fish collected from the Gwydir River in the cotton growing area
were found to contain endosulfan residues. In the same country, fish from Gezira (El
ZorghaG.A., 1980), Lake Nubia (Novak and N. Ahmad, 1989), were found to contain
total residues concentration of endosulfan ranging from 0.27 to 16 mg/kg and from 2 to
184 mg/kg respectively.

Kenya is the only country in Africa where inland waters and fish have been
intensively investigated. The first studies done by Koeman et a1. (1972) in the Rift Valley
lake found very low to undetectable residue of DDE in Tilapia and high levels of DDE
in bottom-feeding fish. Lincer et a1. (1981) reported extremely low levels (below 0.007
mg/kg) of dieldrin, p,p'-DDT
and undetectable to low levels of DDE in fish from lake Nakuru and lake Naivasha

respectively. Studies done in the 1980's reported levels of DDT ranging from 0.004 to

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0.367 mg/kg in fish from Lake Victoria (Kenja, 1989; Mitema and Gitau, 1989). More

recent studies reported finding DDT in all fish samples. Mugachia et al. (1992) found
that 73% of estuarine fish from the Athi River were positive for one or more of the OC.
Mitema and Gitau (1990) reported mean DDT levels Of 0.45 mg/kg in fresh Nile perch
fillet.

In Céte d'Ivoire, only the Ebrié lagoon has been investigated for the presence of
organochlorine pesticides (OC). The results indicated levels of DDT and its metabolites
ranging from 60-200 mg/kg in the sediment (Marchand and Martin, 1985). In analyzing
for residues in fresh water fish from Main lake in Nigeria, Koffi Kobenan (1986) found
residues of OC in all the samples at concentration ranging from trace to 0.593 ppm. He
also found traces of some organophosphates such as malaoxon (0.220 ppm).

Heavy metal contamination of aquatic environment is a continuing global concern
and seems to be more pronounced in countries where environmental regulation and
monitoring is not routine practice. The most problematic heavy metals are lead, mercury,
arsenic cadmium, tin, chromium, zinc, and copper. These metals are widely used in
industry, particularly in metal-working or metal plating-shops and in such products as
batteries and electronic (Table 2). Because heavy metals have brilliant color; they are
used in paint pigments, glazes, inks, and dyes. They are also used in some pesticides and
medicines. Thus heavy metals may enter the environment, wherever any of these
products are produced, used, and ultimately discarded. Heavy metals are extremely toxic

(Table 2) because, as ions or in certain compounds, they are soluble in water and may be

readily absorbed into the body, Where they tend to combine with and inhibit the

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functioning of particular vital enzymes. Very small amounts can have severe
physiological or neurological consequences.

Because of their greater solubility and volatility, the mercury find their way into
water. In Sweden, fresh water fish such as perch and pike were found to contain 0.5-3.5
ppm Hg in their axial muscle. In Japan, the consumption of fish and shellfish
contaminated with mercury wastes from chloroalkali plants caused an epidemic of
paralysis among human populations at Minamata (Goldwater, L.J., 1972).

Information on the presence of heavy metals in African waters is scattered and
scarce. Recently several monitoring programs have been initiated at various universities
and scientific institutions but most of them deal with marine pollution. Concerning fresh
waters, studies done in Northern Africa have been concentrating on Egyptian inland
waters and coastal zones, particularly on the River Nile and the delta lagoons. Toga et
a1. (1981) found high concentrations of heavy metals in the western part of the Nile.
They attributed these findings to contaminated drainage waters. Saad et al. (19810)
investigating Lake Mariut, found the levels of metals (Zn, Cu, Fe, Mn, and Cd) to be
much higher in fish than in water.

In western Africa, the occurrence of heavy metals in fresh water have been investigated
in Nigeria, Ghana, and Céte d'Ivoire. In Nigeria, Okoye et al.(l991) reported heavy metal
enrichment of Cd, Co, Cu, Cr, Fe, Mn, Ni, Pb, and Zn in the Lagos lagoon. He
implicated land base urban and industrial waste as sources of this contamination. In the
same country, the studies of pollution in 26 rivers (Ayayi and Osibanjo, 1981), in the

rivers in the Niger delta (Kakulu and Osibanjo, 1992), in the cocoa growing area of Ondo

11

Table 2. Sources and significance of heavy metals in natural waters. Extracted fiom
Environmental Chemistry, 4th ed. Stanley E. Manahan, Lewis Publishers. 1991.

 

 

Element Sources Efl'ecas and Significances ‘U.S. Public health
Service Limit, rug/L
Arsenic Mining try-product, Toxic, possible carcinogenic 0.05
pesticides, chemical waste
Cadnium Industrial discharge, Replaces zinc biochemically, 0.01
mining waste, metal causeshigh blood pressureand
- plsating, water pipes kidney damage, destroy
testicular tissue and red blood
cells, toxic to aquatic biota
Copper Metal plating, industrial Essential trace element, not 1.0
and domestic wastes, very toxic to animals, toxic to
mining, mineral leaching plants and algae at moderate
levels
Lead Industry, mining, Toxicity (anemia, kidney 0.05
plumbing, coal, gasoline disease, nervous system),
wildlife destruction
Chromium Metal plating, cooling- Essential trace element 0.05
tower, water additive (glucose tolerance factor),
(chromate), normally found possibly carcinogenic as
as Cr(VI) in polluted water Cr(VI)
Mercury Industrial waste, mining, Acute and chronic toxicity Not given
pesticides, coal
Zinc Industrial waste, metal Essential element in many 5.0
. plating, plumbing metallo-enzymes, component
of sewage sludge
Selenium Natural, geological sources, Essential at low levels, toxic at 0.01

sulfur,coal

high levels, possible
carcinogen

‘ Public Health Service Drinking Water Standard, (1.5. Public Health Service, 1962

12

(1991b) demonstrated that with the exception of iron, the concentrations of most trace
metals in the surface waters are generally lower than global average levels for surface
waters and the international drinking water standards. In Ghana, Amasse (1975) found
concentrations of AS above normal values in the area of Obuasi gold mining. A survey
conducted on fish and sediments from the river Wiwi in Kumasi demonstrated higher
levels of Cd and Hg in fish compared to sediment. In COte d'Ivoire, Marchand and
Martin (1985), and Kouadio and Tefry (1987) have studied the sediment of the Ebrie
lagoon and reported metal concentrations in excess of background levels; they attributed
their findings to the disposal of untreated sewage and industrial effluents. A comparative
study by Metongo (1991) of Cd, Cu, Hg, and Zn in samples of oysters (Crassostrea
gasar) from urban and rural lagoons of COte d'Ivoire revealed higher but background
levels of the metals in the urban area.

In Eastern Africa, most of the studies of heavy metals contamination of fresh
waters have been done in Kenya. In 1972, Koeman et a1, concluded that metals (As, Cu,
Zn, Cd, and Hg) concentrations did not constitute a hazard to the biota of Lake Nakuru.
Six years later, Greichus, found slightly elevated levels of the same metals in the same
lake. In lake Victoria, earlier studies (Ochieng, 1987) indicated no Significant heavy
metal pollution. However, more recent studies in the same area revealed increased lead
levels largely due to increased shipping traffic and associated problems from car washing,
and discharge from local industries (Onyari and Wandiga, 1989).

In southern Africa, higher levels of heavy metals were found in birds compared

to fish and sediment in Hartbeesport dam (South Africa) (Greichus, 1977). Greichus et

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13

al (1978b) investigating metals in lake Mclwaine (Harare, Zimbabwe) and found
intermediate levels of heavy metals than those found in South Africa

Surface water in C6te d'Ivoire is composed of rivers and lagoons. Pesticides
contamination of surface water may come from agricultural, industrial activities.
Agrochemicals (fertilizers and pesticides) sprayed on farmland or in the cities for insect
vectors control may be transported into the rivers and lagoons. Industrial activities
generate wastewater that is discharged untreated or partially treated into the waters. Thus,
chemical plants particularly those formulating pesticides may discharge pesticides into the
aquatic ecosystem. Finally fishermen have been found to use pesticides to kill fish
resulting in possible contamination of surface water.

Heavy metals contamination of surface wateIS‘in COte d'Ivoire many come, as
illustrated in Table 2, from chemical and metallurgical plants and a number of small
factories (food , mechanic, tanneries, building, woodpulp, plastic, etc.) that have been
built in cities. Many of these cities are located along side rivers and lagoons and waste
waters from these factories and from the cities which may contain heavy metals are
dumped without any treatment, into the lagoons or rejected into the rivers which flow into
the lagoons. Heavy metals may also originate from agricultural activities; mainly from
pesticides. The mercurials used as foliar fungicides or seed protectant (ethylmercuric
chloride) can be source of general contamination. Until the 70’s the timber industry in
COte d’Ivoire was the third exportation product after coffee and cocoa. Thus
organomercuric compounds may have been used in the country as fungicides in pulp

industries and as slimicide or mold retardant in paper industry. Finally transportation and

l4

tourism may also generate heavy metals in the water systems.

Apart from the few studies reported above, no comprehensive study of the
contamination of the aquatic system by pesticides and heavy metals have been conducted
in C6te d’Ivoire. Moreover, rivers and lagoons represent an important source of protein
in form of fish, water supplies for both human needs and agricultural activities, and
waterway for transportation. Therefore to protect global health and productivity of the
population, it is important that this fragile ecosystem be analyzed for the presence of
pollutants.

We propose in the present study, to assess the contamination of surface water and
fish from COte d'Ivoire. The first objective was to identify and quantify the levels of
organochlorine pesticide residues in surface water and fish samples from COte d'Ivoire
and to assess the extend of environmental contamination by these pesticides and also to
discuss the toxicological significance of the findings to the health of people. The second
objective was to assess the physico-chemical characteristics of the surface water as well

as their contamination by heavy metals.

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CHAPTER II

OVERVIEW OF THE COUNTRY

GENERAL DESCRIPTION

COte d‘Ivoire is located on the south side of the West African bulge. It covers an
area of 322,462 km2 (124,503 sq miles) (Figure 2). Except the west where the altitude
reaches above 1,300 In, the land is almost flat and does not exceed 800 m. The country
has three main types of vegetation; in the south reigns the tropical closed forest (humid
evergreen and semideciduous forest), and then there is a transition zone (forest-savannah
mosaic) in the center. The north is covered with vast woodlands or savannah.

The climate in the southern forest is tropical with two rainy seasons. The dry
seasons are from December to April and from August to September. The rainy seasons
are from May to July and from October to November. the rainfall may reach 1,250 to
2,400 mm annually. The temperatures are generally constant throughout the year (22 °C
to 33 °C) with high humidity. Toward the north, the rainfall diminishes to one rainy
season (May to October) and one dry season (November-April).

In 1960, the population was about 3.8 million. The 1975 census recorded a
population of 6.67 million. The population was estimated to be 13.03 millions as of mid
1991, more than tripling in three decades. The high population growth rate is attributed

to immigration, high fertility rate (7.4 births per woman), and improvements in healthcare.

16

 

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Ote d’Ivoire

Figure 2. Map of physical characteristics of C

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Life expectancy at birth, rose from 44 years in 1965 to 52 years in 1987 (Economic
Intelligent Unit, 1991).

An increased proportion of the population live in urban areas, for example, in 1960
the urban population as a proportion of the total population was estimated to be about
19.3 percent. It has increased to 32.2 percent by 1975, and by 1990 it was estimated to
about 46.6 percent. Between 1960 and 1990, the urban population grew at an annual
average of 7.2 percent, whereas the rural population grew at only 2.7 percent (FAO year

book,1993)

SURFACE WATER

The lagoon systems are composed of three main lagoons which borders the eastern
equatorial Atlantic Ocean. They are Situated along the north coast of the gulf of Guinea,
between 2°50' W and 5°25' W (Figure 3). The lagoons cover a total surface area of 1200
km2 with a climate similar to the equatorial climate; the annual rainfall is about 2000
mm. These lagoons, initially separated, were connected by the construction of canals that
allow transportation across all of them. Each of the three main lagoons has a different
hydrological system which is influenced by continental fresh water from rivers and an
exchange with the marine environment. The lagoons are the reservoirs of the in-flow
water from the rivers; they are also subjected to intense transportation as well as tourism
activities. Fishing is the most important activity for people living around these lagoons

and its provides protein to these people as well as those living in cities. Among the

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ichthyologic fauna, there are many types of biological cycles depending on the various
types of salinity tolerances and on the conditions of the reproduction cycle.

The lagoon stocks are exploited by means of various types of artisanal fishing gear
(individual or collective). The estimation of catches for 1977 was about 7000 tons and
may have triple Since the introduction of commercial fishing. Seventy percent of the total
catch is made by purse seines and beach seines. For the three lagoons, the production
should probably be 15-20000 metric tons.

The Aby lagoon located in the east of the coast is surrounded by some of the most
productive farmland of the country; coffee, cocoa, palm trees and coconut trees are some
of the crops grown in this area. It covers a surface area Of 424 km2 and has an exchange
with the atlantic ocean by a natural opening. One big city, Adiake' and many Villages
inhabited by farmers and fishermen are located alongside this lagoon. The lagoon is
crossed by many boats for public as well as tourists transportation.

The Ebrie lagoon system is the largest of the three main lagoons that form the
Ivorian lagoonal system. This lagoon, located in the east-central part of the coastal zone,
has a length of 120 km, is 5 to 10 km wide and covers an area of 550 km2 (Dura and
Skubish, 1982; Dufour, 1982). It is composed of the main lagoon "Ebrie lagoon', 523
km2 and the lagoons of Aghien and Potou, 43 km2. Many creeks and rivers empty into
the lagoon. The Ebrie' Lagoon exchanges water with the Atlantic Ocean via the man-
made Vridi canal (300 m wide and 20 m deep) dug in 1950 for the construction of the
sea port of Abidjan. Five cities: Abidjan, Dabou, Bingerville and Bassam and many

villages where farmers and fisherman live are located around the lagoon.

 

 

 
    
   

 

 

 

 

 

 

 

 

 

 

 

  
  
 
 
  

 

 

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Due to the presence of the international airport and the sea port, Abidjan contains more
than 75% of the industrial and commercial activities (Dufour, 1982). Besides that, it
shelters about 3 millions inhabitants. Agricultural activity is also important in the land
around the lagoon; rubber trees, pineapple, vegetable, and floriculture are among the crops
grown in this area. The Grand Lahou lagoon is located in the midwest of the coastal
zone. It is the smallest and shallowest of the lagoons. The city of Grand Lahou as well
as small villages inhabited by fishermen are located alongside this lagoon. Palm oil and
rubber trees are among the crops grown in this area.

There is a pressure on the lagoons resulting from the increased demography of the
cities around them. For example, since 1960, the city of Abidjan located on the Ebrie
lagoon has had an annual population increase of 11%. Its population was 1,625,000 in
1980 and was estimated to have reached 3,000,000 by 1995. The development of cities
has resulted in the rejection of wastewater and untreated septic tank contents into the
lagoons. Second, the creation of industries, in many of the cities has resulted in the
generation of industrial wastewater that is dumped into the lagoons. Transportation is
also a source of pollution. Washoff of soil and floods of rivers during the rainy season
in May-June carries pesticides and fertilizers sprayed on farmland around the lagoons or
pesticide Sprayed for the control of vectors of diseases. Although officially banned,
certain chemicals such as pesticides are used as ichthyotoxic for fishing purposes.

The river system is composed of three large rivers and 6 small rivers (Figure 3).
Among these rivers, seven outflow in the lagoons. The Bandaman and the Boubo outflow

in the Grand Lahou lagoon, the Rivers Agneby, M6 and Comoé into the Ebrie lagoon and

IN.

 

21

finely the Rivers Bia and Tanoé into the Aby lagoon. Besides the Bandaman River and
Comoé that flow from the north of the country to the south, the others rivers are mostly
located in the southern part of the country in the forest land.

The river Comoé, the second largest rivers, flows from the savannah of the
neighboring country (Burkina Fasso), crosses through the northeast and east of the
country and outflows into the Ebrié lagoon in the south. In the northeast the River
Comoe’ crosses the cotton land around Bouna and Bondoukou. In the east it cross the
Indenié where coffee and cocoa are grown. In the south before the River Comoé reaches
the Ebrie lagoon, it crosses pineapple, rubber trees and banana plantations.

The Bandaman River is the largest and longest river in the country. It flows from
the north of the country, goes through the center of the country in the transition zone
between forest and savannah, and reaches the Ebrié lagoon in the south. Different parts
of the river have been enlarged for the construction of electrical as well as irrigation
dams. The bed of the river is surrounded by the cotton land in the north and center. In
the south the river cross rubber tree, palm tree, cocoa and coffee farms before reaching
the Grand Lahou lagoon.

The River Agneby is a small river flowing in the coastal zone. It is surrounded
by banana, pineapple, palm tree and rubber tree farms. It reaches the Ebrie' lagoon around
the city of Dabou.

The river Bia is a little longer than the Agneby River and flows along the eastern
part of the country. Two hydroelectrical dams have been constructed on this river in the

area of the city of Ayame'. This river crosses farm land composed of cocoa, coffee,

22

banana, pineapple, palm and coconut trees before reaching the Aby lagoon.
Agrochemicals (fertilizers and pesticides) used in agriculture may be carried by
erosion and reach many of these rivers. Besides, many cities are located alongside these
rivers and their waste may be discharged into these rivers. Finally, within the lagoons
pesticides may be illegally used for killing fish in these rivers. For all these reasons, the
investigation of these chemicals in surface water and fish is necessary to evaluate the

health threat to consumers as well as to ascertain the pollution status of these ecosystems.
AGRICULTURE

Agriculture is the keystone of the COte d'Ivoire economy, with a consistent annual
growth of nearly 7% from 1960 to 1980. It contributes about 33 percent of the gross
domestic product (GDP), provides between 50 to 75 percent of the nation's total export
earnings, and employs an estimated 79 percent of the labor force (Simeon K. Ehui).

In 1990, there were 1,240,000 ha of cropland; 13,000,000 ha of permanent pasture;
7,630,000 ha of forest and woodland; and 7,510,000 ha of other land including urban and
built-up areas (Agricultural Production Yearbook, 1991).

A wide variety of crops are grown in the country (Table 3) including small grains,
tubers and roots, cash crops, vegetables and fruits. Estimates indicate that crop
production apart from rice and wheat is sufficient to supply the food requirement of the
population. Although there are no statistics, one can assume that over 45% of cultivated

cropland is devoted to cash crops, 20% to small grain, 25% to tubers and roots, and the

23

remaining to others.

Table 3. Major crops grown in COte d'Ivoire

Source: Sustainable Agriculture and the Environment in the Humid Tropics (NRC, 1993)

. Table 1. Major crops grown in COte d’Ivoire

 

 

 

Principal crops 1988 1989 1990
Maize ..................................... 460 480 484
Millet. .................................... 43 45 44
Sorghum ................................ 24 25 24
Rice (paddy) ........................... 610 635 687
Potatoes" ................................ 24 24 24
Sweet potatoes‘ ...................... 12 18 18
Cassava (maniac) .................... 1,400 1460 1,393
Yams ...................................... 2,500 2,600 2,528
Taro (Coco yam) .................... 290 302 282
Pulses" .................................. 8 8 8
Tree nuts‘ ............................... 11 11 11
Sugar cane" ............................ 1,500 1,500 1,550
Palm kernels ........................... 35.8 20.2 36.8
Groundnuts (in shell) .............. 121 126 134
Cottonseed ............................. 136 148 134
Coconuts“ .............................. 470 470 470
Copra‘ ................................... 75 75 75
Tomatoes ................................ 20 21 22
Aubergines (Eggplants) .......... 25 27 30
Chilies, peppers“ .................... 23 23 23
Other vegetables" ................... 329 368 372
Oranges‘ ............................... 28 28 28
Other citus fruit“ ................... 30 30 30
Bananas ................................. 133 133 97
Plantains ................................ 1,100 1,145 1,087
Mangoes“ .............................. 14 14 14
Pineapples .............................. 196 209 136
Other fruit"I ............................ 12 12 13
Cofl‘ee (green) ........................ 187 239 219
Cocoa beans ........................... 849 725 700
Tobacco (leaves)“ ................... 2 2 2
Cotton lint) ............................. 114 128 108
Natural rubber (dry weight)... 61 60 74
‘ FAO estimate(s).

Source: FAO, Production Yearbook (1991).

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24

Cash crops consist of cultivated plants that are usually grown in monocroping plantations
for export or for use in local manufacturing industries. The major cash crops are coffee
(Coflea arabica), of which the country is the world's third largest producer; cocoa
(Theobroma cacao), of which the country is the world largest producer; and cotton for
which the country is becoming the second largest producer in Africa. Together these
commodities account for more than 60 percent of the area under cultivation. Since the
80's, the country has diversified its agriculture and today agricultural commodities have
expended to banana (Musa Sp.), palm tree (Elaeis guineensis), coconut (Cocos nucifera),
pineapple (Ananas chemises), rubber tree (Hevea brasiliensis), and sugarcane (Saccharum
Sp.).

Food crops are divided in two categories: (1) roots and tubers represent 76% in
value and 60% of the bulk of staple food output (4.5 million tons/year); and (2) cereals.
Cassava (Manihot esculenta Crantz), yams (Dioscorea spp,), cocoyam (Xanthosoma
sagittrfolium (L.) Schott) are the main root and tubers consumed by the population. The
swollen tubers (storage roots in the case of cassava) and the leaves (except yarns) are
commonly consumed in a wide variety of fresh and processed forms. The tubers are rich
in carbohydrates, while the leaves contain proteins, vitamins, and minerals.

Farming activities are distributed all over the country according to the climate and
vegetation. Most of the cash crops are located in the south of the country, only cotton,
sugarcane and tobacco are grown in the north. With food crops, small grains are
predominantly grown in the north while roots and tubers are grown in the south

Traditionally, land cultivation was done by Shifting (slash-and-bum). The creation

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25

of a farm was done by cutting and burning the forest or woodland (slash and burn). The
cleared area was cultivated for a few years (1 to 2 years). After that the land was
abandoned and allowed to return to forest or bush (fallow) for a period of 4 to 20 years.
Soils in thetropics have low nutrient content; thus clear and burn techniques make
available to the soil the nutrients in living plants in form of nutrient-rich ash fertilizer.
From biological point of View, annual food crops such as rice, maize, cassava, and
yams demand substantial quantities of nutrients for satisfactory yields, but many of the
soils in the tropics are dystrophic. Improved adapted varieties and cultural practices that
include minimum amounts of agricultural inputs (mainly fertilizers and herbicides) are

needed to improve agronomic sustainability.

PEST PROBLEMS

As in most of tTOpical Africa, climatic conditions in COte d'Ivoire are conducive
to the rapid multiplication of insects, many of them are vectors of diseases. For example,
407 insects species of major importance and 778 species of minor importance are listed
in Africa (Hill, 1975). Besides climatic conditions, recent changes in farming have
increased pest pressure. For example, enlargement and aggregation of fields in case of
cash crops has favored the rapid Spread of pests and hampered natural enemies of these
pests. Genotype uniformity has created extreme vulnerability to pests. Specialization
in case of corporate or state own plantations has helped increase pest pressure. Finally,

free international exchange and transboundry transfer of infected or infested plant

44.

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26

materials has greatly contributed to pest invasion.

AH RP

Cotton is attacked by many pests but the most important are the bollworms
(Heliothis armigera, Pectinophera Goddipiella (Saund), and Earias insulina (Boisd.));
thrips (Ihrips tobaci); aphids (Aphis gossypii Glov.); cotton leaf worm (Spodoptera
Iirtoralis (Boisd.); and jassids (Empoasca beica (De Berg)).

Coffee is also attacked by many pests; among them, the antestia bug (Antestiopsis
orbitalis (Westw.)), leaf miners (Leucoptera meyricki Ghesq.), mealybugs, looper
caterpillars (Epicampoptera strandi glauca Hmps. and E. ivoirensis), stem borer
(Xyleborus morstatti Hagdn.), berry borer (Hypothenemus hampei (F err.)), and the green
scale (Coccus viridis (Green)).

The major pest problems of cocoa are the mirids, Sahlbergella singularis Hagland
and Distantiella theobroma (Dist). Mirid feeding lesions on cocoa stems are invaded by
the weakly pathogenic fungus Calonectria rigidiuscula, resulting in extensive die-back of
branches and canopy degeneration. Earias biplaga Wlk. attacks the apical buds of young
cocoa plants and adversely affects establishment. Caterpillars of Earias also eat the
pericarp of green cocoa pods, Bothcoelr'a thalassina H.&S. The shield bug, is also
important on cocoa. Mealybugs, especially Planococcoides njalensis (Laing), are

notorious in spreading the cocoa swollen shot Virus.

 

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27
F DCR P

It is estimated that more than 30% of cassava is lost annually (Herren and Bennett,
1984). This loss is caused by the combined actions of cassava green mite and the cassava
mealybug, phenaoccus manihoti. The variegated grasshopper, Zonocerus variegatus "I.
defoliates the cassava plant, strips the bark, and sometimes eats the stems almost to
ground level. The whitefly, Bemissia tabaci (Genn.) is responsible for transmitting the
cassava mosaic virus disease (CMVD), which causes malformations in cassava leaves.
Preharvest damage to yam by insects pests (heteroligus spp.), nematodes, and pathogens

are responsible for 15-20% crop loss .

MAIN CEREALS

Cereals comprise paddy rice (Oryza spp.), maize (Zea mays L.), sorghum
(Sorghum bicolor L.), and millet (Pennisetum glaucum L.). The production of cereals is
estimated at 1 million tons per year. The increase of these food crops is bellow the
increase in population rate resulting in the importation of rice and bread. In 1983,
imports of rice and wheat amounted to 590,000 tons representing nearly 40% of the
national cereal consumption.

The most important pests are Lepidopterous borers: Eldana saccharina Wlk.,
dipterous: Diopsis thoracica Westw. Generally, stem borers cause damage to cereals by
feeding on the leaves and in the leaf whorls and boring into the stems and fruit heads.
Estimates of grain yield losses caused by stem borers damage in Africa vary considerably.

About 14% of the rice cultivated in Africa is lost to insect pests. In COte d'Ivoire. it is

 

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28

estimated that insect pests damage to rice results in loss of up to 1 ton of paddy/ha.

FR ANDVE TAB E

Fruits and vegetables are important components of farming in Africa. They
provide essential vitamins and minerals in the diet. Many subsistence farmers intercrop
fruit and leafy vegetables with roots and tubers. The principal fruits grown in COte
d'Ivoire are citrus, papaya, guava, mango, banana, pineapple, cashew, passion fruit.
Among the fruits, pineapple, banana, citrus represent cash crops. Vegetables include
tomato, onions, okra, cabbage, cucurbits, chili, eggplant, and a wide variety of leafy
vegetables.

Citrus are attacked by a variety of red scale that attack the young citrus seedling
and affect establishment. Citrus are also attacked by fruit flies that pierce the citrus fruit
and can cause severe crop loss. The most important damage on mango is done by
mealybugs. Bananas (fruit and plantain) are attacked by the weevil Cosmopolites
sordidus (Germ) and pineapple suffer the pineapple mealybug, Dysmicoccus brevipes
(Ckll.).

In general, vegetable crops are attacked by a wide range of insects pests including
heliothis amigera (Hb.) on cucurbits, Bemissia tabaci, Heliothis Sp. and Agrotis Sp. on
tomato and cabbage, Thrips tabaci Lind, on onions, Dysdercus spp. (F), and the leaf roller

Sylepta derogata (F .) on okra.

29
P THARVEST PEST

Stored grain and other food items are also attacked and damaged by a large
number of insect, causing serious losses at a time when the production system cannot
compensate for such loses. In Africa, where farm storage systems at the subsistence
farmer level are poor, average loses of stored grain have been estimated to exceed 30%
(Ezueh, 1983) and may be estimated at millions of dollars annually.

The major storage pests of grain include Sitophilus zeamais Motschulsky,
T ribolium castaneum (Herbst), Sitophilus oryzae (L.), Sitroga cerealella (Olivier),
T rogoderma granarium Everts., Callosobruchus maculatue (F .), and the larger borer,
Prostephanus truncatus (Horn). Some of these pests actually infest the crops in the field
and are subsequently carried into storage, where they develop under favorable conditions.

The economic impact of these storage loses extends well beyond reduction in grain
weights. Grain damaged by storage pests is very much reduced in market value and
consumer acceptance, especially in urban communities. Grain lose their viability,
resulting in low germination potential and thus reducing the availability of planting

materials for subsequent crop production.

IN T T R ISEA ES

To the above losses should be added the economic damage caused by insects
which act asivectors of debiliting diseases of man and animals. Some representative of
man vector-bom diseases in C6te d'Ivoire are malaria (Anopheles spp), filariasis (cuIex

spp, mansonia spp, anopheles spp), onchocerciasis (Simulium spp), and Schistosomiasis

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30
(shellfish). Southwood (1977), pointed out that about one in six of mankind is suffering

from insect-born diseases. The full costs borne by individuals and families are largely

unknown and the cost in term of loss in productivity from these disease are enormous.

PEST CONTROL MEASURES AND PRESENT USE OF PESTICIDE

AGRICULILJE

Traditionally, farmers relied on their own knowledge and understanding of the
ecosystem and made decisions relating to farm practice independently of government
control. They relied on a variety of management practices to deal with pest problems.
Farmers used two main strategies for the control of pests. The first consists of direct,
non-chemical methods (i.e., cultural, mechanical, physical and biological practices). The
second one consists of a built-in pest control mechanisms inherent to the biotic and
structural diversity of complex farming system. The farmers also use a variety of other
management practices that, although targeted for other farm purposes, significantly impact
pest dynamics.

With the introduction of modern agriculture, the accent has been placed on
integrated pest control when possible. The drawback is the fact that integrated schemes
of pest control are most easily implemented on large plantation like corporate owned or
state owned plantations of rubber trees, pineapple, banana, palm and coconut trees.
Because of traditional and hereditary land fragmentation, individual farmer holdings tend

to be small (often only around 2 ha). Thus, they may be less easily amenable to the

31

integrated approach. The best avenue to this problem has been via farmer cooperatives.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

PRIvATE cOMPANIES
IMPORTED
BASE EXPORT
MATERIALS RETAIL
STORES — FARMERS
Puauc
HEALTH
LocAL
FORMULATION SNOW
COTE D'IVOIRE C'DT
GOVERNMENT ; DEVELOPMENT
COMPANIES
\ PALM.
INDUSTRIE
IMPORTED v DPV
PasncIDES

 

 

 

 

 

 

 

 

 

 

 

 

Figure 4. Structure of pesticide market in Cote d'Ivoire

32

Besides the fact that farmers are able to purchase inputs and application equipments
together, it is also easy to allocate them an extension agent.

Over the past few decades, the use of pesticides has shown an upward trend which
is a logical consequence of the changes in land use and agricultural practice in general.
A number of pesticides are used in COte d'Ivoire, including insecticides, fungicides and
herbicides as listed in appendix 1. Pesticides used in the country are in part imported
already formulated or formulated by local companies from imported base materials. The
Structure of pesticide market is shown in figure 2. Pesticides are either supplied to the
farmers through state own companies or are purchased directly from retail stores by
farmers. Farmers producing cotton or palm/coconut Oil producers are supplied through
two developmental companies, CIDT (cotton) and PALMINDUSTRIE (palm and coconut
oil) which assist farmers through extension service and also by buying their products.
The state is reimbursed for the supply of the equipment and materials, including seed,
fertilizers and pesticides after the sale of their production.

Insecticides account for most of the pesticides applied to the cultures (Figure 3),
they represent 60% of the total pesticides; followed by herbicides (27%) (Anonymous,
1095). Herbicide use is increasing because of the departure of young people to the cities
and decreasing foreign labor. Among crops, cash crops receive the major treatment of
pesticides, with cotton receiving 46%, followed by cocoa (15%) and banana (14%).

Recently, pesticide use in food crops and vegetables has increased.

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33
NHEATHPR TE TI N

In many of the developing countries, the amount of pesticides used in public
health programs may currently exceed the amount used for the control of agricultural
pests and diseases. In C6te d'Ivoire one may think of the control of mosquitoes (Malaria,
yellow fever, Simulium larvae (Onchocerciasis), tsetse flies (T rypanosomiasis), and snails
(Schistosomiasis). Apart from the Simulium larvae control program, accurate quantitative
data concerning the type of pesticide used and the amount have not yet been summarized
in the literature. Onchocerciasis is endemic to the savannah area of the Volta River basin
in west Africa covering part of the center-north of COte d'Ivoire. In 1974, a control
program was initiated in the region by WHO. The Objective of the program was to
eliminate onchocerciasis as a disease of public health and socioeconomic importance
throughout the area covered by the program and to ensure that there is no outbreak of the
disease in the future. Abate, temephos, and chlorphoxim were the first insecticides used
in the program. Bt (H-l4), permethrin, and carbosulfan were later added. From an
environmental point of view it is important to stress the fact that chemical control of these
vectors takes place in more or less natural habitats. Thus, non-target organisms are more
liable to get exposed to the pesticides than is the case in various agricultural applications.
The same applies to other uses such as in forestry and livestock protection. The use of
pesticides in forestry is not widely practiced in the country; however this may change

with the recent intensive forest management program financed by the World bank.

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34
CHAPTER IV

EXPERIMENTAL PROCEDURE

STUDY DESIGN AND SAMPLE COLLECTION

AMPIN ITE

Nine collection sites located in areas of intense agricultural practice were selected
for the study; three sites were selected on lagoons and 7 on rivers (Figure 5). The
lagoons samples were:

1. The lagoon Aby located in the southeast of the country. This lagoon is surrounded
by one of the most productive farmland of the country; coffee, cocoa palm tree and
coconut tree are some of the crops grown in this area.

2. The lagoon Ebrié border the capital city, Abidjan. Due to the presence of the
intematio'nal airport and the port, most of the industrial and commercial activities are
located in this area The agricultural activity is also important; Hevea, pineapple,
vegetable, and floriculture are among the crops grown in this area.

3. The lagoon of Azagny is in the south midwest of the country. Palm oil, rubber tree
are among the crops grown in this area

All these lagoons are the reservoir of the in-flow water from the rivers. They are
also subjected to intense transportation as well as tourism activities. Fishing is the most

important activity for people living around these lagoons which provides protein to these

I.

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35

people as well as those living in cities.

Seven collection sites were selected on rivers. Two were located on the River Comoé,

one in the south near the city of Moossou and the second in the east in the village of

Aniassué.

1.

The river Comoé flows from the savannah of the north of the country (cotton) through
the east of the country (coffee, cocoa) to the lagoon Ebrié in the south (pineapple,
rubber tree, banana).

The river Agneby flows in the central part of the country from the center to the
south. Itis surrounded by banana, pineapple, palm oil production. The sampling site
was located near the city of Dabou where the river reaches the Ebrié lagoon.

The river Bandaman flows from the north of the country through the center to the
south into the lagoon of Dabou. Two sampling were chosen on this river; the first
site was located at five kilometers from the city of Ferkéssédougou and the second in
the Lake Kossou. In the north, the Bandaman river is surrounded by the cotton land.
In the center, the river is enlarged (Lake Kossou) for the building of the third

electrical dam of the country.

SAMPLE CQECIIQN AND PROCESSING

The samples for analysis were collected from October to November 1994. Water

samples. were collected midstream at depths of 15-20 cm by dipping the glass containers

into the river or lagoon from a row boat. Five samples were collected from each

sampling site for the measurement of the physicochemical characteristics (temperature,

36

  
 

  

Site 1
Ferk
. o
Kor . go
Boua~:
I
ite 2
D aloa
0 ,Yamous ~kro Ab ngourou
~ Site6 3
J
1 Site 7
Site 9 T - I
Abidjan

ATLANTIC OCEAN

Figure 5. Map of COte d’Ivoire illustrating the sampling collection sites

fir.

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37

pH, color, total hardness, alkalinity, suspended solid, dissolved oxygen (DO), and
chemical oxygen demand (COD), heavy metals (Cr, Se, As, Zn, Cd, Cu, Hg, and Pb), and
organochlorine pesticides.

Tilapia (Oreochromis niloticus) was the fish selected for this study because they
are found in most warm water (rivers and lagoons). Tilapia are resistant to disease, very
hardy, and tolerant to low levels of dissolved oxygen allowing them to overcome
overcrowded conditions. Because of their high yield potential and mild flavor, they are
appreciated by the population. They were caught either by gillnets or by line and were
transported from the fishing sites to the laboratory in a freezer. All the fish were
processed within 24 hours after harvest. All the fish were processed as followed; each
fish was scaled, deheaded, deguted and cleaned. The fish were then filleted and samples
from each fish were wrapped in aluminum foil and packed with labelled plastic bags and

stored at -20 °C until transportation to the USA.

MATERIALS

E PMENT
- pH meter (Beckman PHY 72)
0 cm x 1 cm i.d. chromatographic column fitted with a 200 ml reservoir.
0 250 m1 round-bottom flask
0 500 ml separatory flask

0 Graduate cylinders 25-1000 ml

38

0 Column for resin (Econo pack, Millipore)

0 Rotary evaporator (Buchler Instruments)

0 Zymark Turbo-Vap® evaporator

0 HPLC pump (Waters model 510)

0 Programmable HPLC pump (waters 590)

0 Automatic injector for HPLC (Waters WISP 712)

0 Fraction collector (Waters)

0 Ultrastyragel 500 A resin column (Waters, Millipore) cat No 20574, 19 x 300
mm

0 Gas chromatograph (Hewlett-Packard 5890 Series II) equipped with 8 “Ni
electron capture detector (ECD).

0 Automatic injector for GC (HP 7673)

0 Hewlett-Packard computer and Laserjet IIIp printer for GC data handling

0 GLC column. J&W fused Silica capillary column (Durabond); ID #122-5042;
Liquid phase: DB-S (non-extractable bonded phase); Film thickness, 0.25 mm;
Column dimensions, (30 M x 0.333 mm id).

0 Glass chromatographic column: 1.2 x 22 cm (1.0 cm i.d.), topped with a 3 x
10 cm reservoir and fitted a teflon stopcock with a 1.5 cm delivery tip.

All glassware (separatory funnels, beakers, funnels, teflon seals, and

chromatographic columns) were thoroughly washed in hot water with detergent, rinsed
with tap water, and distillate water, then with acetone, and finally with hexane when

necessary. The cleaned glassware was dried in an oven.

39

NT REAENT ND LUTINS

SPE C" cartridges (Alltech Associates)
Chelex 100 resin (hydrogen form, 200-400 mesh, 3 nm pore diameter) from
BioRad Laboratories,

Florisil; 60-80 mesh. Activate the florisil by placing in an oven ~130 °C for 16

. hours. Cool before using

Glass fiber filter paper (Whatman Gf 0.5 mm).

Silane treated glass wool (Anspec; Ann Arbor, MI)

Hexane (96% n-hexane), distilled in glass IT baker analyzed HPLC

Ethyl acetate, distilled in glass Malinckrodt ChroAR HPLC

Methanol, distilled in glass IT baker analyzed HPLC

Dichloromethane, distilled in glass JT baker analyzed HPLC

Sodium sulfate (Na2SO,)-granular anhydrous

Sulfuric acid 95-98%, ABCS reagent (BASE, C)

phenolphthalein , ABCS reagent (BASF, C)

bromocresol green /methyl red indicator (Aldrich).

EDTA (BASE, C)

Eriochrome black T indicator (BASE, C).

Nitric acid 90% ABCS reagent (BASE, C)

Ethyl acetate in hexane (v/v)was prepared by measuring 2,100 mls hexane into
a suitable container (empty solvent jug), add to the hexane 900 mls ethyl

acetate, shake well to ensure complete mixing.

40

ANALYTICAL METHODS

QEIERMINATIQN QF PHYSICQ-CHEMICAL PRQPERTIES

The physicochemical properties of water samples were determined according to
the Standard Methods for the Examination of Water and Wastewater (Clesceri et al.,
1989). DO was determined by the azide modification of the Winkler's method on dilute
samples (Hanson 1973). Suspended solids were determined by filtering a known volume
of water through a glass fiber membrane filter (GF/C, 0.25 mm), drying and weighing
pH was determined with a direct reading pH meter (Beckman PHY 72), standardized with
acetate and phosphate buffers at pH 4.0 and 9.2 respectively. Alkalinity was determined
by titration of 50 ml sample with 0.01 M H2804 using phenolphthalein and a mixed
indicator bromocresol green-methyl red. Total hardness was determined by means of

EDTA titration using eriochrome black T indicator.

HEAVY T DE RMINATI N IN WATER

One hundred ml of the water sample was filtered through a glass fiber (Whatman
Gf 0.5 mm). The filtrate was acid digested and passed through a column (Econo pack,
Millipore) filled with Chelex 100 resin (hydrogen form, 200-400 mesh, 3 nm pore
diameter) obtained from BioRad Laboratories, CA. The resin columns were kept frozen
until their transportation to the USA. In the USA, the heavy metals retained in the
columns were eluted with strong nitric acid and analyzed by ICP (Termo Iarrell Ash,

Polyscan 61E).

41
QRQANQCHLQENE PESTICIDES DETERMINATION IN WATER

The solid phase extraction (SPE) procedure was adapted from the method provided
by Alltech Associates, Inc (Anonymous). The solvents used were "pesticide residue
grade”. Sop-Pack cartridges (Alltech Associates, INC) containing 1000 mg of packing
material (C,,—bounded silica) were used for sample collection. The cartridges were
coupled to a vacuum glass. The cartridges were washed with 10 ml hexane followed by
10 ml ethyl acetate. The cartridges were dried briefly, under vacuum to remove excess
solvent and were conditioned with 10 ml methanol (Meow) then 10 ml deionized water.
Five hundred ml separatory funnel containing 250 ml of the water sample was connected
to the cartridge and water was passed through the column by hand suction at a rate of
approximately 15 ml/min. After passing the samples, the cartridge were stored at 4 °C
until transportation to the USA.

In the USA, the cartridges were inserted into a vacuum manifold and dried at a
pressure of 500 mbar. The columns were then washed with 10 ml deionized water
followed by 10 ml Meowzdeionized water (20:80 v/V). The absorbed pesticides were
eluted with a solution of hexanezethyl acetate (70:30) at a flow rate of approximately 2-3

ml/min until 4 ml were collected.

R AN HL RI ESTICIDE DETERMINATION IN FISH
Ten g of fish tissue was homogenized with 40 g anhydrous Na2SO4. The dried
mixture was ground to a fine powder and packed into a 30 cm x 1 cm i.d.

chromatographic column fitted with a 200 ml reservoir. The samples were extracted with

42

200 ml of dichloromethane at a flow rate of 34 mL/min. The lipid extracts were collected
in a 250 ml round bottomed flask, and the solvent was reduced to approximately 1 ml by
rotary evaporation. The concentrated extracts were then diluted to 10 ml with hexane and
used for gel permeation (GPC) fractionation .

Automated GPC consisting of a 60 g bed Ultrastyragel 500 A resin (Waters,
Millipore) attached to waters programmable HPLC pump (Waters 590) and waters fraction
collector was used to separate the OC from lipids and other pesticides. An aliquot of the
lipid extract (200 ml) was injected into the GPC column. The first 100 ml of eluate were
dumped and the next 50 ml containing the OC was collected in a flask and turbo-vaped
to approximately 5 ml.

The concentrated lipid extract was transferred into a column filled with a small
plug of glass wool at the base followed by 7 grams of florisil and topped with 1-2 grams
of anhydrous sodium sulfate. After addition of the GPC concentrate, the column was
eluted with 40 ml of elution solvent (dichloromethane). The eluate was concentrated by
turbo-evaporation (Zymark) to 0.5 ml, and then redissolved with 2 ml hexane prior to

GC/ECD analysis.

MAT P ETERMINATI N

The detection and quantification of the pesticides in water and fish samples was
performed by using a Hewlett-Packard 5890 Series II gas chromatograph equipped with
a “Ni electron capture detector (ECD). The conditions of analysis were the following:

0 Column: DB-5 fused capillary column (30 m x 0.333 mm id) with

Oven:
Injector :
Detector:
Carrier gas :

Make-up gas:

43

0.25 pm phase thickness.
isothermal temperature 275 OC
temperature 270 °C

temperature 250 OC.

Helium at the pressure Of 150 Kpa

Nitrogen

The acquisition of the data was done using a Hewlett-Packard Bell computer equipped

with HPCHEM software (Hewlett-Packard, Palo Alto, CA).

 

 

 

$1

131

44
CHAPTER V

RESULTS AND DISCUSSION

PHYSICOCHEMICAL PROPERTIES

Physicochemical properties for the selected rivers and lagoons are given in Table
3. The temperature of water of the lagoons was higher than the temperature of water
from the rivers. The lagoons are large bodies of water with more evapotranspiration than
rivers. The mean pH of 7.18 was within the range of the WHO recommendation of 7.0 -
8.5 for drinking water. The high value observed at Site 9 may be attributed to the
industrial activities surrounding the area.
Total hardness ranged between 21.2 and 41.5. Most of the values were below 40 mg/L
indicating that these waters are not too soft. As the color index indicated, most of the
water samples were turbid and colored. It did rain at the collection sites 1, 3, and 6 the
morning before the collection of the samples. the transport of organic matter, clay and
surface litter to the rivers is origin of this brownish color observed. The turbidity as well
as the brown color observed at sites 5 and 9 may be attributed to agricultural and
industrial activities surrounding these sites. The COD observed increased from the north
to the south of the country, principally in the area around the capital city. The increase
in COD may be attributed to the increase of organic matter due to agriculture (pesticides,

fertilizers), urbanization (waste water) and industrialization (oil refineries, food processing

plants, etc..).

45

Table 4. Physico-chemical characteristics of water samples from the nine collection sites

 

 

Collection Sites
Mean Values S.l S2 S3 S4 S5 S6 S7 S8 89
Temperature (C) 27.4 28.0 27.7 27.2 26.7 27.2 31.3 30.5 31.7
pH 1 7.6 7.2 7.1 6.8 6.5 6.2 6.9 7.5 8.9
Color ' 85.5 15.3 87.2 25.2 88.5 75.7 15.4 13.2 89.3
Total hardness (mg/L) 39.2 29.4 28.4 31.0 37.0 35.2 41.5 42.0 21.2
Total solid (mg/L) 45.2 15.2 50.0 22.5 32.1 44.4 20.3 15.2 19.6
DO“ 5.4 2.3 8.2 3.5 10.1 10.2 11.2 10.1 12.2
Total alkalinity 15.0 5.5 17.2 4.2 17.0 16.5 20.1 21.2 30.2
COD" 87.0 102.0 145.0 105.0 125.0 130.0 121.0 103.0 254.0

 

* Disolved oxygen
" Chanical oxygen demand

46
HEAVY METALS CONCENTRATION IN WATER SAMPLES

The average concentrations ”of metals in water samples are given in table 4. Cr,
Se, As, Cd, Hg, and Pb were not detected Copper was detected in waters from four
samfling sites ( 2, 3, 6, and 7) but the levels found was lower than the US. Public Health
Service limit for this metal in drinking water which is 1.0 mg/L (US Public Health
Service, 1962). Zinc is the only metal found at eight out of the nine sampling sites.
Marchand and Martin ( 1985), and Kouadio and Trefry (1987) found zinc in the sediment
of the Ebrie lagoon at levels 6 to 20 times higher than the background levels. Apart from

site 5 where the level found (1.73 jig/L) is higher, the levels in the other sites

Table 5. Heavy metals contaminant levels (pg/L) in water samples. Mean from five
determinations

 

 

Sampling Sites
Contaminants Sitel Site2 Site3 Site4 SiteS Site? Site6 Site8 Site9
Cr <10 <10 <10 <10 <10 <10 <10 <10 <10
Se <100 <100 <100 <100 <100 <100 <100 <100 <100
As <50 <50 <50 <50 <50 <50 <50 <50 60
Zn 45 58 70 42 1730 3 10 73 1 <5 32
Cd <5 <5 <5 <5 G <5 <5 6 6
Cu <5 7 7 <5 <5 <5 10 7 <5
Hg Q0 <50 <50 <50 <50 <50 <50 <50 <50
Pb Q0 Q0 Q0 <20 Q0 Q0 Q0 Q0 Q0

 

47

is lower than the limit of 5.0 mg/L set for this metal in drinking water (US. Public Health
Services, 1962). In general as indicated by Table 2, the sources of zinc is industrial
waste, metal plating or plumbing. Zinc inputs at sites: 5, 6, 7 where the levels are high,
are most likely related to effluent discharge. In Others area the presence of zinc might

be related to background.

ORGANOCHLORINE PESTICIDES IN WATER

For statistically evaluating the extraction efficiency of the targeted pesticides in
water by solid phase extraction (SPE) techniques, 500 ml of distillate water from our

laboratory was fortified with the compounds at three concentration levels (35.0, 55.0,

250.0 jig/L).

Table 6. Recovery (%) 2t RSD (n = 3) for the organochlorine pesticides in water samples
and detection limits (pg/L).

 

 

 

Spiking Levels (pg/L) Detection
Pesticide 35.0 55.0 250.0 Limits
Aldrin 41 :1: 1.5 45 i 5.6 50:1: 4.1 0.002
DDD 98 :1: 9.0 88 :t 7.5 102:1: 5.6 0.003
DDE 101 :l: 7.0 99 i 6.2 77:1: 4.7 0.002
DDT 97 :t 5.2 102 :1: 4.0 78:1: 3.3 0.003
Dieldt‘in 105 :1: 6.6 77 :1: 6.1 72:1: 4.4 0.004
Endosulfan 55 :1: 3.1 46 :1: 4.5 49:1: 6.1 0.003
Endrin ‘ 96 :1: 9.0 97 :1: 8.2 921: 6.5 0.005
Heptachlor 92 :1: 9.7 95 :1: 9.0 105:1: 8.7 0.002
Lindane 92 :l: 3.2 102 i 5.1 99:1: 4.5 0.001

 

 

 

 

0?

48

The average recoveries obtained were between 72 and 105% except for aldrin and
endosulfan with 41 and 50% (Table 6). The relative standard deviation was between 2
and 22% (in accord with residue analysis, Grove 1984).

Identification and quantitation of compounds in water and fish samples was
accomplished using reference solutions of a mixture containing the targeted pesticides.
One 111 of 0.01 to 0.08 ppm of the mixture solutions were injected into the GC and a
standard curve was determined and used to quantitate the solutes in the samples. Results
given in table 6 present the concentration levels of organochlorine pesticides in water
samples from the nine collection sites. Among the targeted pesticides, five were found
in the water samples. Dieldrin and lindane occurred in all the samples except in the
samples from site 1. p,p'-DDT and endosulfan were detected in samples from 6 Sites
whereas aldrin was found only at three locations. The mean concentration of aldrin, p,p'-
DDT and dieldrin were 0.3 mg/l, 0.5, and 0.4 mg/l respectively. The mean concentration
of endosulfan was 1.7 (range 1.3-1.9). The mean concentration of lindane was 2.6 (range
0.3-3.9).

The results of the study (Table 7) Show that among the targeted pesticides, five
were found in the water samples. Dieldrin and lindane occurred in all the samples except
in the sample from site 1 (north of the country). p,p'-DDT end endosulfan were detected
in samples from 6 sites whereas aldrin was found only at 3 locations. The metabolites
of DDT (DDD and DDD) were not found. The mean concentration of aldrin, p,p’-DDT
and dieldrin'were 0.3, 0.5, and 0.4 ug/L respectively. The mean concentrations and

ranges of endosulfan and lindane were 1.7 pg/L (1.3-l.9 ug/L) and 2.6 ug/L (0.3-3.9

49

rig/L) respectively.

Table 7. Mean (n = 5) pesticide residue levels (pg/L) in water samples from the nine
collection sites. nd = non detected at the detection limit.

 

 

Collection Sites
Compound SI S2 $3 8.4 8.5 8.6 S7 S8 S9
Aldrin nd nd nd nd nd nd 0.3 0.3 0.5

p,p'-DDE nd nd nd nd nd nd nd nd nd
p,p'-DDD nd nd nd nd nd nd nd nd nd
p,p'-DDT nd nd nd 0.4 0.5 0.3 0.5 0.5 0.6
Dieldrin 0.1 0.2 0.3 0.5 0.4 0.4 0.5 0.5 0.6
Endosulfan nd nd nd 1.3 1.7 1.7 1.9 1.9 1.9
Endrin nd nd nd nd nd nd nd nd nd
Heptachlor nd nd nd nd nd nd nd nd nd
Lindane nd 0.3 1.1 3.1 2.9 3.1 3.2 3.3 3.9

‘ nd = non detected atbthe detection limit

The results of the study Show that the levels of OC found in the rivers were higher
in the south than in the north of the country. For example the mean concentration of
lindane in the river Comoé was 1.1 mg/L at site 3 (upper Comoé) while in the lower

Comoé (site 5) it was 2.9 mg/L. The rivers flow from the north to the south of the

50

country and they carry with them run-off water from farmlands that may contain dissolved
pesticides and/or pesticides attached to soil particles. The findings can also be explained
by the difference in agricultural activities; in the north of the country only cotton, and
recently sugar cane are grown whereas most of the commercial crops are grown in the
south.

The results indicate also that the levels of OC in the lagoons were higher than
those found in the rivers. This finding can be explained by a biomagnification process
because the rivers flow from the north of the country, crossing all the farmlands and
outflow in the lagoons in the south. Thus any chemical (pesticide) transported by the
rivers reaches the lagoons.

The residue levels of lindane (range 0.3-3.9) and endosulfan (range 1.3-1.9) were
higher than those of the other compounds. This finding can be explained by the fact that
lindane and endosulfan are Still extensively used in the country and therefore are carried
by run-off to surface water. Endosulfan is used in crop protection as an insecticide
against termites and variegated grasshopper and as a nematicide in banana production.
Endosulfan is also used against vectors of diseases. Lindane is extensively used against
cocoa mirids and other pests (Anonymous). The level of lindane found in water samples
from the Ebrie lagoon (3.2-3.9) at the collection site 7 and 9 is higher than the levels
found in the'sediments from the same site (0.6-1.7 ppb) as reported by Marchand and
martin (1985). This finding is perhaps related to the possible use of lindane as
ichthyotoxic compound for the fishing activities in these areas (Colconap and Dufour,

1982). The fact that DDT was found in water samples but not its metabolites can be only

 

 

 

be

51

explained by recent use of this pesticide

The concentration of organochlorine insecticides in water samples were
considerably lower than that reported in River Nile by El-Dib and Badawy (1985) and
in several African lakes (Greichus, 1978). The residue levels of the studied compounds
were still low compared with the permissible levels for drinking waters (Train, 1979,

WHO, 1982).
ORGANOCHLORINE PESTICIDES IN FISH

To evaluate the performance of the extraction and cleanup procedures, fish samples
were fortified at three levels with known amounts of the pesticides (0.5, 1.5, 5.0 mg/kg)
and then analyzed. The recoveries for the selected pesticides were between 75-110%
(Table 8).

Table 9 shows the results of the fish analysis expressed as mg/kg wet weight.
The chromatograms showed no indication of polychlorinated biphenyl (PCB)
contamination. Out of the forty five samples Of fish tissue analyzed, 47% contain aldrin,
56% DDE, 11% endrin, 76% lindane, and 69% endosulfan. DDD, DDT, dieldrin and
heptachlor were not detected. The results show that the levels of OC found in fish tissue
were higher in fish from the south than from the north. This was in concordance with the
findings for water. DDT was found in water samples but not in fish samples while its
metabolite DDE was not found in water but was found in fish samples. Since DDT is

banned in the country, its presence in fish samples suggests a possible uptake from the

52

Table 8. Mean recovery (%) :1: RSD (n = 3) for the pesticides in fish and detection limits
(us/ks).

 

 

 

Spiking Levels Detection Limits

Pesticide 0. 5 1 .5 5 .0

Aldrin 75 :1: 4.4 80 :1: 6.0 82 :1: 6.1 0.002
DDD 110i 5.1 95: 5.0 100:1:7.2 0.003
DDE 90 i 6.6 89 i 6.5 104 :1: 4.0 0.002
DDT 86 d: 7.7 100 i 7.7 93 :1: 10.1 0.003
Dieldrin 84 :1: 7.3 87 :t 7.1 87 :1: 8.5 0.003
Endosulfan 67 :1: 6.6 65 a: 8.2 81 i 9.2 0.005
Endrin 102 a: 9.0 98 :1: 7.0 88 :1: 9.5 0.005
Heptachlor 101 :1: 5.5 97 i 3.6 93 :1: 6.3 0.002
Lindane 107 :1: 7.1 95 d: 5.6 92 :1: 7.3 0.001

 

water column. The OC adsorbed on sediment or plants, may be released in water and be
absorbed by fish. Fish are able to uptake DDT adsorbed on the surface of particles (silt),
and plants such as algae. The BHC isomers are relatively short-lived compounds and

have a bioconcentration factor (BCF) of 50-900 and therefore they should not normally

accumulate.

53

Table 9. Mean (11 = 5) pesticide residue levels (mg/kg) in fillet of Tilapia from the nine
collection sites. nd = non detected at the detection limit.

 

Collection Sites
Compound SI S2 8.3 8.4 8.5 S6 S7 S8 S9
Aldrin nd 0.018 0.005 0.004 0.029 0.026 0.015 0.010 0.027
p,p'-DDE 0.005 0.034 0.107 0.057 0.249 0.184 0.048 0.208 0.493
p,p'-DDD nd nd nd nd nd nd nd nd nd
p,p'-DDT nd nd nd nd nd nd nd nd nd
Dieldrin nd nd nd nd nd nd nd nd nd
Endosulfan nd 0.006 0.997 1.300 1.700 1 .700 l .900 1.900 1.900
Endrin nd nd nd nd 0.013 0.017 nd nd 0.061
Heptachlor nd nd nd nd nd nd nd nd nd

Lindane 0.045 0.080 0.086 0.073 0.155 0.142 0.056 0.114 0.220

The fact that lindane was found at high concentration compared to the others OC indicates
a build-up or a continuing input into the aquatic habitat. These findings can be explained
by the fact that lindane is still in use in the country. The presence of aldrin in the

majority of the samples may be explained by its relative high BCF's and long half-lives

54

in fish samples (Clark et al., 1983). Endosulfan presence in the samples can be explained
by the fact that it is still in used in the country.

The results show that none of the samples analyzed had residue levels above the
extraneous residue limits (ERL) and acceptable daily intake (ADI) for the respective
pesticides set by the FAO/WHO codex alimentarius commission (1986). This indicates
that the residue levels of OC in fish were within the acceptable limits for human
consumption.

The levels Of OC found in this study are lower than those reported in fish
elsewhere in Africa. El Zorgani (1980) reported a sum of DDT ranging from 0.38 to 1.31
mg/kg in Tilapia niloticus from Lake Nuba in Sudan; Mugachia and a1. (1992) reported

levels of DDT and lindane ranging from 0.102-l.185 to 0033-0295 respectively.

55
CHAPTER VI

CONCLUSIONS

The present study shows that the rivers and lagoons investigated do not appear,
at present, to have serious pesticide and heavy metal pollution problem. Physicochemical
characteristics of the rivers and lagoons sampled are in general in the normal range. The
organochlorine pesticides when found were at a very low levels. This was true for
metals although it is difficult to distinguish between naturally occurring metals
(background from the crust) and those due to human activities.

Recently, blooming of aquatic plants has been observed in many of the rivers and lagoons
indicating nutrient enrichment of the water systems. For example the lagoon Ebrié in the
Capitol city Abidjan has been periodically invaded in the last five years by aquatic plants.
This has caused serious difficulties for urban transportation by boat. Furthermore, the
Ebrie' lagoon is now less than appealing for swimming, boating, and sport fishing. Even
the lagoon has foul Odors and fish have been found dead. This eutrophication is seen in
other lagoons although very minor. The major sources of this eutrophication-causing
nutrient enrichment are: (l). agriculture (eutrophication from the croplands, leaching of
fertilizer applied to crops, runoff from animal feedlots, dairy barns); (2). urban/suburban
runoff; (3). sewage effluents (discharge from treated and untreated sewage, usage of
detergent containing phosphate, sewage from individual septic systems). Urbanization is

rapidly growing in the country, the population of Abidjan, the capitol city has triple in

56

less than ten years and the population of other cities is also increasing. In order to
prevent future environmental disaster, routine monitoring of the aquatic systems (water
and sediments) and stricter regulations in waste discharge must be invoked.

Concerning the organochlorine pesticides, their use in the country is expected to
increase although many of them have been replaced by less persistent pesticides such as
carbamates and organophosphates. Until now, the country has been able to feed its
population but the situation is changing because of the rapid growing of the population.
. Agriculture must produce more food but the young people able to farm have fled the
villages to the cities for an illusive better life. In order to produce more with less people,
agriculture must be intensified; one the components of this intensification is pesticides.
By the use of pesticides, farmers will be able to reduce the damage to their crops in the
field and in the storage rooms. Because of the reduction in manpower, more herbicides
will be needed for weed control.

COte d'Ivoire relies on importation of meat from neighboring countries but recent
drought in many of these countries have severely impaired animal husbandry. For this
reasons, programs have been implemented in the country to increase cattle and sheep
production in order to meet the demand in meat. The major constraint to cattle and sheep
rasing in Céte d’Ivoire has been diseases. For animal raising, there is a necessity to use
pesticides to control insect vectors such as trypanosomiasis.

Everything indicates that pesticides will be an important component of pest control
in C6te d'Ivoire. But the goal here should be to rationalize their use in order to reduce

ecological disruptions, which may threaten long-terrn sustainability, and to reduce

 

I?!

t)‘
‘4

 

 

57

environmental and health hazards. Pesticide usage must be rationalized, and means and
ways have to be found to attain this objective. This is not possible unless a groundtruth
data-base is available on pesticides at the grassroots level. It Should be mentioned that
proper and solid legislation with regards to pesticide sale, handling, storage and disposal,
as well as worker protection from occupational hazards does exist but its implementation
leaves much to be desired. One of my future studies will be to survey for pests,
pesticides, pesticide legislation and management in the country. This work will allow the
identification of the various factors necessary for good pesticide use practice SO that
necessary corrections may be made. Pesticide use on vegetables has increased recently
but there is no information regarding the use of pesticides by the small vegetable growers.
There is no extension service for these farmers, and therefore, such a study may help
understand where and how they get their advice and what kind of records they keep. My
second subject of concern will be to assess the impact of pesticide use on animal
husbandry. In rural areas, small scale farmers associate crop production with animal
husbandry, the latter feeding on fallen grains, insects and worms. In case of aerial spray,
poultry may ingest sprayed insects that may have ingested or adsorbed the insecticide.
Poultry may also ingested granular insecticides. No data exist in the codex alimantarus
on the ADI of pesticides in Africa because of the lack of diet determination. I would like
to conduct research in this area. Concerning the heavy metals, one possible study would
be to determine the sources of contamination by monitoring points of injection. This can
be done only if there is a strong support from the authorities. One feasible study would

be to monitor these chemicals in air due to the emission from the refineries by using

passive detection devices.

58

APPENDICES

APPENDIX A

LIST OF THE PESTICIDES DISTRIBUTED IN COTE D’IVOIRE

—..{\_ ~71 Q.

rrU C PL

~ 4

4
V

»L

.t

71.0

p\a

 

LIST OF THE PESTICIDES DISTRIBUTED IN COTE D’IVOIRE

59
APPENDIX A

Table A1. List of the insecticides used in crop protection in COte d’Ivoire.

Source: Rapport annual sur la vente des pesticides pour utilisation agricole.

UNIPHYTO (1991).

 

 

 

 

 

Commercial Speciality Active Ingredient Distributor
Typhon 50 EC 500 g/l Ethylparathion Sofaco
Systhoate 40 400 g/l Dirnethoate Sofaco
Dyfonate SG 5% Fonofos Sofaco
Gammatif 5 5% lindane Sofaco

Decis D5+150 ULV 5 g/l Deltamethrine Sofaco

Thioral 25/25 25% Thirame + 25% Sofaco
Malathion CE 50 500 g/l Malathion Shell

Ekalux Forte 480 g/l Quinalphos Shell

Basudine 600 600 g/l Diazinon Ciba-

Thiodan 50 EC 500 g/l Endosulfan Hoechst
Sumithion CE 60 600 g/l Fenitrothion shell

Sumicidin 100 CE 100 g/l Fenvalerate Shell-Chimie-CI
Undone 75 PM 75% Propoxur Bayer

Callifan 50 EC 50 g/l Endosulfan Callivoire
Marshall 25 ST 25% Carbosulfan Callivoire
Karate CE 50 g/l Lamdacyhalothrine Rhone-Poulenc
Nurelle D 12/100 ULV & ULV P 12 g/l Cyperm + 50 g/l STEPC
Dursban 100 ULV 100 g/l Chlorpyrophos ethyl Shell-Chimie-CI
Sevin 480 g/l Carbaryl Shell-Chimie-CI
Fastac 40 EC 400 g/l Alphacypennethrine Shell-Chimie-CI
Lindal 90 90 g/l Lindane Callivoire
Teknar 1 Kg/l unit Aedes Acgypti Sandoz

Caid Procida 2.5 g/l Chlorophacinone Sofaco
Folithion EC 500 500 g/l F cnitrothion Bayer

Solfac EC 050 50 y] Cyfluthrine Bayer

Marshall 2% PP 20 g/kg Carbosulfan Rhone-Poulenc
Thionex 50 EC 500 g/l Endosulfan Rhone -Poulenc

 

 

60

Table A2. List of the fungicides used in crop protection in Cote d’Ivoire
Source: Rapport annual sur la vente des pesticides pour utilisation agricole.
UNIPHYTO (1991).

 

 

 

 

Commercial Active Ingredient if ‘DIstutribor j.
Speciality

‘l' °domil 25 25 % Metalaxyl Ciba-Geigy/SOCHIM
Caocobre 50 % Oxyde of cupper Agro Business

: 'BS Procida 25 % Sulphate of cupper SOFACO 1
Difolatan 80 80 % captafol STEPC/Rhone-Poulenc
ou-Foura 1.6 % Thiabendazole/ 1.5 % Permethrine Callivoire

Alto 100 SL 100 g/l Cyproconazole Sandoz

Tilt 250 100 g/l Propiconazole Ciba-Geigy/SOCHIM
Sandofan 20 % Oxadixil Sandoz

Fungasil 100 100 g/l Imazalil SOFACO

manate 80 80 % Manebe SOFACO

Alliette 800 g/l Phosethyl-Al Rhone-poulenc

Benlate 50 % Benomyl Rhone-Poulenc

Punch 40 EC 400 g/l Flusilazol SOFACO

Manesan 80 % Manebe Rhone-Poulenc

 

 

) >—.1
F1:
:0

301;! C

 

 

61

Table A3. List of herbicides used in crop protection in Cote d'Ivoire.
Source: Rapport annual sur la vente des pesticides pour utilisation agricole,
UNIPHYTO (1991)

 

 

 

W Luv—WW" W
'Wfl'... WI 9111 ; I [noun 1.- .‘r ; ”Muffin. 1" gy 0' ‘11'
«can 500 500 OIL Fluometurnn Cibs-Ocigy/SOCHIM
elpar L 240 M Shell Chimio-Cl
Ronstar 25 CE 250 M Oxadiazon Rhone-Poulaic
'- .. 12L 120M0xadiazon Rhone-Poulcnc
' - ~ . PL 100 M Oxadiazon/300 M Propanil Rhone-Portions
azalon 80 PL 500 M Atrazine SOFACO
azelon 80 PM 80 % Atrazine SOFACO
Basagran 480 M Bentazone BASF
- -0k 200 M bananas/200 M Atrazine BASF
- . - 820 480 M Butrahne Ciba-Ooigy/SOCHIM
$0me 100 M Parquet/300 M Diuron SOFACO
facox 200 M Paraquat SOFACO
1 Procida 80 % Diuron 1 SOFACO
Herbazol 2,4—D 720 M SOFACO
urnn 100 M Parquet/300 M Diurnn SOFACO
t oxone 200 G/L Paraquat SOFACO
lifor 250 M Promethrine/250 M Fluometrion Callivoire
~ 200 200 M Flumxypyr Callivoire
Roundup 360 M Olyphosate Rhone-Poulenc
t lent 125 104 M Haloxyfop acid Callivoire
Bastas LS 200 M Olufosinate SOFACO
Hyvar XL 200 M bromacil Dupont/Rono-Poulenc
Hyvar X i80% Brornacil Rhone-Paulette
ordon 101 CE 64 M “chlorine/240 M 2.4-D Shell Chimio-Cl
ordon 225 E 120 Pichlorand120 M 2.4-D Shell Chirnio-Cl
onion 155 120 M “chlorine/480 M 2.4-0 Shell Chirnie-Cl
t - pax 500 500 M Antonino Ciba-Geigy/SOCHIM
t 3 pax Combi~500 250 M Arnetrind250 M Atrazine Ciba-Geigy/SOCHIM
« - 80 80 M Ametrine Ciba-Geigy/SOCHIM
Lasso 480 GIL Alachlore Rhone-Poulenc
Lasso OD 350 M Alachlore/ZOO M strazinc Rhone-Poulcnc
Prirnagram 250 M Metolachlort-J235 M Atrazine Ciba-Geigy/SOCHIM
c. . 4 161.5 v. Butoxy ethylic m ofTriclopyr SOFACO
~ pics 30 57 % Diana/23 % Brnrnacil SOFACO
tral 80 80 % Atrazine Callivoire
- tral 50 500 M Atrazine Callivoire
tnzine 4L 480 M Atrazine Callivoire
- etral 80 PM 80 Va Ametrync Callivoire
. oral 50 FW 500 M Ametryne Callivoire
netral Mine-PM 250 M Atrazine/40 % Ametryne Callivoire
..-- Mixto-L 250MAtnzine/250MAmetryne Callivoire
c ’ EF 72 M Triclopyr ester butoxy ouryl Callivoire
.. « TM 90 gm Olyphosate STEPC
‘ 4 ox 40 400 M Asularne Rhone-Poulcnc
= l 400 M MSMA/200 M Diumn SOFAOO
Roaster 38 FLO 380 M Oxadiazon Rhone-Poulcnc

 

Table A4. List of nematicides and miscellaneous pesticides used in crop protection in '

COte d’Ivoire.

Source: Rapport annual sur la vente des pesticides pour utilisation agricole,

UNIPHYTO (1991).

62

 

 

 

 

Commercial Speciality Active Ingredient Distributor
; emacure SG 5 % Phenamiphos Bayer
1
lMiral 10 G 10 % Isazophos Ciba-Geigy/SOCHIM
I emacure 400 EC 400 g/l Phenamiphos BAYER
Mocap 10 G 10 % Ethophos SOFACO
Ternik 10 10 % Aldicarbe Rhone-Poulenc
Furadan 4F 480 g/l Carbofuran STEPC
Furadan 5G 5 % Carbofiiran STEPC
Furadan 10 G 10 % Carbofuran STEPC
lTelone 11 EC 90 % Dichloropropene SOFACO
l Spic SG 5 % Metahaldehyde SOFACO
Carel 480 480 g/l Ethephon Callivoire
lEthrel Stirnulatex 480 g/l Etephon Ciba-Geigy/SOCHIM
threl Special ananas 480 g/l Etephon Ciba-Geigy/SOCHIM
1 erat Blocs 0.05 %Brodifacoum SOFACO
1Klerat Granules 0.05 % Brodacoum _ SOFACO

APPENDIX B

CHROMATOGRAMS OF THE ANALYSIS

63

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1 1:
SL‘
8000-
7000— 1?; 5 .§
‘ 3
‘ ‘T
O...
600 §
- 8 s.
s:
5000—
4000-
" .7 VLJL L/xl r
3000 -
l
0 50

Figure Bl. Chromatogram of 00.1 ppm of standard solution of organochlorine
pesticide mixture

64

7.504
33.506

1
7333

 

14.248

1:591

 

:l‘ ."‘" "

 

 

l
o 5 0

Figure BZ. Chromatogram of water sample from site 1 (Ferké)

65

 

 

 

Figure B3. Chromatogram of water sample from site 3 (Abengourou)

66

 

 

 

 

 

 

 

 

 

8
at.
2'?
N
3
‘3
if
n
#1;
I.
”r
5"» n
.n A '2 1h {'5-
- e. e 7 '7 ..
l , 4‘- v ,9;
.1 -- §.:: 1.. U"
:1 '
3300- r. :w
4 | 7. l1

1 I
‘=i____
‘lmmm
V‘L-IPW
W

 

 

1
O 5 0

Figure B4. Chromatogram of fish sample from site 5 (Moossou)

67

 

 

 

 

 

 

 

 

 

 

 

Figure BS. Chromatogram of fish sample from site 9 (Abidjan)

LIST OF REFERENCES

68
LIST OF REFERENCES

Abedi, Z.H. and DE. Turton. 1968. Note on the response of zebrafish larvae to folpet and
difolatan. ,1. Assee. Off. Anfl, Chem. 51:1108-1109.

Alexander, M. Microbial degradation of pesticides. In: F Matsumura, G.M. Boush, and
T. Misato (eds). Environmental Toxicology of Pesticides, Academic Press, New York,
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Anonymous. 1984. Codex Alimentarius. Ambrus A. and Grenhalgh, R. Eds. WHO/FAQ.
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Ayayi, 8.0. and Osibanjo, 0. Environ. 1981. Pollution Studies on Nigerian Rivers: II.
Water Quality of Some Nigerian Rivers. Pollut. (B) 2:87-95

 

Bailey G,W. ‘1966. Entry of biocides into water courses. Proc. Symp. Agr. Waste Waters.
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Cliath, M.M. and Spencer, W.F 1971. Movement and persistence of dieldrin and lindane
in soil as influenced by placement and irrigation. Soil Sei. Sec, Amer. Prec. 35:791-785.

Bailey G.W. and White, J.L. 1970. Factors influencing the adsorption and movement of
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D'Itri, F, 1986. Impact of Toxic Contaminants on Fisheries Management. In 'I‘oxic

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Donaldson, T.W. and Foy C.L., 1965. The phytotoxicity and persistence in soils of
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Durand, JR. and Skubich, M.1982 Les Lagunes Ivoiriennes, Ageecultere, 27. 211-250.

Clesceri, S.SL., Greenberg, A.E., and Trussell, RR. (1989). Stmdard Methods for the
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69

Crop protection Strategies for Subsistence Farmers. Edited by Miguel A. Altieri.
Westview Press, Inc., 1993.

El-Dib M.A. and Badawi M.I. (1985) Organochlorine insecticides and PCBs in River Nile
water, Egypt. Bull Environ Centm. Texicel 40:86-93.

Ehui, SK. 1993. Cote d'Ivoire i_n_ "Sustainable Agriculture and the Environemnt in the
Humid Tropics". National Academy Press, Washington, DC.

El Zorgani,G.A. (1980). Residues of organochlorine Pesticides in fish in Sudan. L
Envireg, 591 hegth, B15(6), 1091-1098.

Goldwater, L.J. 1972.Human toxicology of mercury. In Environmental Toxicology of
Pesticides. Eds. F .Matsumura, G.M.Boush, and T.Misato. Academic pp. 165-175.

Greve, RA. (1984). Good Laboratory practice in pesticide residue analysis. In "Pesticide
residue analysis” (Ambrus A. and Greenhalgh R., eds), WHO/FAQ, Rome, p.281.

Greichus Y.A., Greichus A., Aman B.D., and Hopcraft J. (1978). Insecticides,
polychorinated biphenyls and metals in African lake ecosystems III, Lake Nakuru, Kenya
Bell, Enviren Qentm Texieol 19:455.

Hindin,E. and Bennett RI. 1970. Transport of organic insecticides to the aquatic
environment. Intern. Water Pell, Res. Cenf., Pree. 5th. HI: 19/1-19/16. San Francisco,
Ca

Hill, D. (1975). Agricultural insect pests of the tropics and their control. Cambridge
University Press, London.516 pp.

I.ATA. Rapport of the Biological Control program center for Africa, 5-9 December 1988.
Cotonou, Benin.

Kenaga, BE, 1972. Guideline for environmental study of pesticides: Determination of
bioconcentration potentials. Residue Revs. 44:73-113.

Koeman J.H., Pennings J.H., De Goeij, J.J.M., Tjioe P.S., Olindo P.M., and Hopcraft, J.

(1972). A preliminary survey of the possible contamination of Lake Nakuru in Kenya with
some metals and Chlorinated hydrocarbons. 1, Appl, Eeel., 9:411.

Kouadio, I. and Trefry, J. H. 1987. Sediment Trace Metal Contamination in the Ivory
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Manahan, SE. 1991. Environmental Chemistry. 5th Ed. Lewis Publishers, Inc.

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Marchand, M. and Martin, J-L. 1985. Determination de la pollution chimiques

(hydrocarbures, organochlorés, métaux) dans les lagunes d'Abidjan (Cote d'Ivoire) par
l'étude des sediments. Qeégegr, Trop. 20:25-39.

Mugachia, J.C, Kanja, L, and Maitho, TE. (1992). Organochlorine Pesticides in estuarine
Fish from the Athi River. Kenya Bull. Environ. Centam. Toxieel., 49:199-206.

Okeye, B.C.0., Afolabi, O.A., and Ajao, EA. 1991. Heavy metals in the Lagos lagoon
sediments. kit, 1, Enviren. Stud. 37:35-41.

Risebrough, R.W. 1969. Chlorinated hydrocarbons inn marine ecosystems. In “Chemical
fallout: Current research on persistent pesticides'. Eds M.W Miller and 6.6, Berg.
Charles C. Thomas, Springfield, 111., pp.5-23.

Saad, M.H., Ezzat, A.A., El-Rayis, O.A., and Hafez, H. 1981c. Occurence and
Distribution of Chemical Pollutants in Lake Mariut, Egypt: 11. Heavy Metals. Water Air
Sei Pellet, 16:401-407

Sinha, A.P., Kishnan Singh, and AN. Mukhopadhyay. Soil Fungicides. Vol II. CRC
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Spencer W.F. and Cliath, M.M. 1970. Vapor density and apparent vapor pressure of
lindane. 1. Agr, Food Chem, 18:529-530.

Spencer W.F. and Cliath, M. 1972. Volatility of DDT and related compounds. 1. Agr.
Feed Chem. 20:645-649.

Spencer, W.F, Farmer, W.J., and Cliath, M.M. 1973. Pesticide volatilization. Residue
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Southwood, T.R.E. 1977. Entomology and mankind. Proc. XV. International Congr. Ent.
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Toma, S.A., Saad, M.A.H., Salama, MS, and Halim, Y.J. 1981. The distribution of some
absorbed elements on the Nile continental shelf sediments. 1 Etud. Pellut. CIESM.,

5:377-382.
UNIPHYTO (1991). Rapport annuel sur la vente des pesticides pour utilisation agricole.
U.S.E.P.A. 1976.Quality Criteria for water.Washington, D.C.

Weed, SB. and Weber, J.B. 1969. The effect of cation exchange capacity on the
retention of diquat2+ and paraquat2+ by three layers type clay minerals. 1. Adsorption and

71
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Willis, G.H. and Hamilton, RA. 1973. Agricultural chemicals in surface runoff,
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World Bank Technical paper N0. 142. Africa Technical Department Series. Integrated Pest
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Yaninek, IS. and HR. Herren. Biological control: A sustainable solution to crop pest
problems in Africa. Proceedings of the inaugural conference and workshop of the IITA

PART II

EVALUATION OF COMMERCIAL IMMUNOASSAY FOR THE DETECTION OF

PESTICIDES IN PLANT AND FISH

72
CHAPTER I

INTRODUCTION

Today's farm practices are being scrutinized for their contributions to water
pollution, water shortages and soil erosion. There is public perception that food is unsafe
because of the presence of pesticides and other chemical residues in food. There is also
a concern by farmers for their own health and for the quality of the environment. Nearly
half of the farmers in 1989 nationwide survey by Jefferson Davis Associates in Iowa were
worried that their use of chemicals poses a danger to themselves and to the environment
(Anonymous, 1990). As public concern about the pesticide issue increases, the pressure
to provide new information and guidelines on the fate of pesticides in the environment
has become important for regulatory agencies and governments. Many countries have not
only introduced rigid legislation requiring detailed examination of all aspects of the
potential hazard before a new chemical can be approved for specific usages but also
surveillance of the food supply for the presence pesticides. Furthermore research must
provide critical evaluations on the fate of pesticides in the soil, water, and food. At the
stage of inquiry and for purposes of implementing legislation, analytical methods are
required to locate and quantify contamination, to determine the risks that pollutants pose

to human and ecological health, and to actively remediate polluted sites when necessary.

73

This presents an imposing analytical challenge when one considers the total number of
analyses needed, the broad spectrum of analytes which must be determined, the multitude
of matrices in which theses analytes must be quantified, and the economic constraint in
carrying out these measurements. Various analytical methods can be used for the
determination of pesticides in different matrices but up today chromatographic techniques
(gas chromatography, high-performance liquid chromatography, and thin layer
chromatography) alone or coupled with mass spectrometry are the most commonly used.
However monitoring the supply or the environment for residue by these analytical
methods is expensive, time consuming, complicated, potentially unsafe, and require the
use of polluting solvents.

For example, the general procedure for the determination of most pesticides and
their metabolites in plant materials involves the following steps: 1. collection of sample
materials; 2. extraction of the sample with an organic solvent 3. decoloration with
activated carbon/Attaclay mixture; 4. partition with an organic solvent followed by a
second partition with a mixture of organic solvent; 5. column chromatography clean-up;
and 6. analysis by chromatography with a specific detector. This method may take
several hours to analyze a dozen samples and generates appreciable amount of hazardous
waste.

More recently enzyme-linked immunosorbent assays (ELISAs) have gained interest
for pesticide residue analysis. Several books, articles and reviews have discussed the
theory and applications of these techniques (Newsome, 1986; Van Emon and Mumma,

1990; Van Emmon et al., 1989; Van Emmon and Lopez-Avilila, 1992). Commercial

74

enzyme-linked immunosorbent assays (ELISA) are becoming increasingly available that
a section was totally dedicated to this technique during the ACS 211th meeting in New
Orleans. These techniques offer several advantages over chromatographic techniques;
relatively rapid analysis times, high samples throughput and sensitivity at a relatively low
cost. Thus these techniques are appealing for developing countries such as C6te d'Ivoire
where large scale of pesticide analysis is often difficult as instrumentations such as GC
or HPLC are often unavailable or when they are available, the cost associated with their
maintenance and the purchasing of solvent is often too high.

Most of the commercially available ELISA kits have been marketed for the
analysis of water samples because of the absence of matrix interference. For example,
ELISA kits have been used for the determination of atrazine (Bushway et al., 1988;
Schaleppi et al., 1989), alachlor (Feng et al., 1990; Rittenberg et al., 1991; Lawruk et al.,
1992), and carbofuran (Bushway et al., 1992) in water. Recently works have been done
to extend the use of these kits to more complicated matrix such as soil samples, food.
The present study investigate a broader use of commercial kits as rapid detection systems
for the screening of three commonly use pesticides in two complex matrices: corn leaves
and fish. The first objective is to verify the method precision and accuracy by comparing
it to established chromatographic methods and establish the method sensitivity by
determining its limit of quantitation (LOQ) and its limit of detection (LOD). LOQ is the
level above .which quantitative results may be obtained with a specified degree of
confidence while (LOD) represents the lowest concentration that can be determined to be

statistically different from blank.

75

Because of the binding of the pesticide to the matrix (soil) or its conjugation to
matrix components (plant or animal tissue), immunoassay may give different results with
environmental samples compared to fortified samples. Samples components (proteins, fat,
pigments), pH and ionic strength, viscosity, solubility of chemicals and extraction solvents
may interfere with the reading and give false results. The second objective of this study
was to analyze environmental plant material and fish samples and to determine the effect
of matrices on the accuracy and efficacy of the ELISA.

The commercial immunoassay kits may performed differently depending on the
type of solid phase by the manufacturer. The solid phase employed may be polystyrene
wells, balls or tubes, on which antibody or hapten-protein conjugate are passively
adsorbed or it can be magnetic particles coupled to the antibody. The third objective in
this study was to compare the sensitivity and precision of two types of these solid phases
for the analysis of sample extracts. The first kit Enviro-guard® (Millipore) has the
antibodies coated on the bottom of test tubes. The second type of kit is RAPD"
(Ohmicron corp.) has the antibodies adsorbed on fine magnetic particles suspended in
solution. The sensitivity and precision of these kits was compared. The last objective
of this study was to analyze the usefulness of ELISA compared to traditional techniques
by evaluating the cost of equipments and reagents; cost associated with the training of
technicians, cost associated with quality control, and the availability and stability of the

kits.

76
CHAPTER H

ENZYME IMMUNOASSAYS AND ITS APPLICATIONS

THE IMMUNE RESPONSE

The immune system protects animals from infectious organisms. It comprises
several different types of cells, each with a variety of functions. One group of white
blood cells, lymphocytes, secrete proteins that bind in a highly specific manner to foreign
molecules (Benjamin and Leskowitz, 1988). These proteins are called antibodies while
the foreign molecules are called antigens. Lymphocytes that produce antibodies are called
B lymphocytes or B cells (Benjamin and Leskowitz, 1988). B cells specifically bind to
a particular antigen (Figure 1). Once binding occurs, these cells are activated and divide,
producing identical copies of themselves (clones). Each new B cell secretes antibody
molecules that bind specifically to the antigen. B cells release antibodies which then
circulate throughout the body in the blood stream. When the antibody encounters their
specific antigen they bind to it, and the antigen is marked for destruction by other
components of the immune system such as the macrophages (scavengers cells that engulf
and destroy). Most of the antibody structure is relatively constant, except for the antigen-
binding site (variable region). There are at least two antigen binding sites per antibody

structure.

77

 

   

B Cells

/ \ Antigen Molecules
Antibodies

o ..

. .>I> ‘la 1’ \ ‘1 Q \ -jll" “

.- r” , ,‘u ,- ’ I
a

l
(

        

 

 

 

Figure 1. Cellular events leading to antibody production following B-cells activation
by antigen molecules. Source

78

 

 

Antigen Binding
Region ( Fab)

LIGHT CHAIN

 

Disulflde Bon . <

HEAVY CHAIN

 

 

 

 

 

Figure 2. The Structure of an Antibody Molecule. Four protein chains combine to form

an antibody molecule.

79
ANTIBODY STRUCTURE AND FUNCTION

Antibodies also called immunoglobulins (Ig) are glycoproteins (Goodman J.W.,
1991). They are grouped in five classes (IgA, IgD, IgE, IgG, IgM). Antibodies of
different class ranged from 150 to 900 Kilodaltons (Kd) in molecular weight and mediate
different immunological functions. Some other vertebrate animals produce fewer classes,
but most produce IgG and IgM.

An antibody molecule consists of two heavy chains and two light polypeptide
chains connected through disulfide bound (Fig 2). The Fab portion (light chain) of the
antibody contains the variable region that is responsible for the specificity of the
molecule. Variation of the polypeptide sequence of this region are complimentary to the
antigenic determinant, thus providing the basis for antigen/antibody binding. The Fc
region (heavy chain) of an antibody molecule is constant within a particular antibody
class. The Fc region mediates secondary immunological functions such as complement
fixation. Labels or "tags" which are used for visualization of immunoassays are generally
attached to the Fc region so the antibody retains the antibody/antigen capacity of the Fab

region.

ANTIGEN STRUCTURE

An antigen is any molecule which can bind to an antibody (Benjamin and
Leskowitz, 1988). Antigens can be biological molecules or synthetic compounds.

Immunogens are molecules or part of a molecule that stimulates a B cell to produce

80

antibodies. To be immunogenic a substance must:

(1) contain a region B cells recognize as foreign; (2) contain sufficient complexity; and
(3) have sufficient molecular weight (usually > 3,000 daltons). Large antigens may
contain a number of recognition sites called epitopes (Benjamin and Leskowitz, 1988);
each epitope activates a different B cell which in turn produces antibody with a unique
binding specificity. An immune response in which many different lymphocytes produces
antibodies to a complex immunogen is said to be polyclonal, and the resulting antibodies
are called polyclonal antibodies (Figure 3).

Some molecules are too small (MW < 1,000 daltons) to elicit an effective humoral
response. These molecules, defined as haptens, must be physically coupled to a larger
immunogenic molecule in order to elicit an antibody response to the hapten. The large
molecule, known as carrier, helps produce a necessary recognition signals for activation
of the immune response. This type of system is employed in immunoassay for analytical
purposes to produce antibodies which can then be bind to certain chemicals (e.g.
pesticides) and to small polypeptides (Van Emon and Lopez-Avila, 1992; Rittenburg et

al., 1989) (Figure 4).

81

 

 

Specific Binding Sites

  

Epitopes

  

Specific Antibody

 

 

Figure 3. Illustration of the binding of the antibodies to the antigen specific binding
sites called epitopes. Each epitope can bind to a different antibody containing a
specific antigen-binding site.

82

 

lmmunogenic
Carrier
Molecule

  

7'

Specific Binding Site

 

 

 

Figure 4. Illustration of a synthetic antigen indicating the hapten (small organic chemical)
covalently attached to immunogenic carrier molecule.

83
PRODUCTION OF ANTIBODIES FOR LABORATORY USE

The humoral response, which involves production of antibodies to foreign
substances (antigens), is the arm of the immune system which provides the basis for
immunoassay systems. Immunoassays are tests in which antibodies are used as analytical
chemistry reagents. Antibodies are produce for use in an immunoassay by exposing an
animal or specialized cells from an animal to a target substrate. For example, a laboratory
animal such a rabbit may be immunized with a preparation of the target substance (i.e.
pesticide) to stimulate the production of antibodies. Serum from the blood of the animal,
which contains the antibodies to a wide variety of substances, is then collected at the
appropriate time. This is called polyclonal serum (Figure 5). The antibodies are purified

and incorporated into a detection system.
MONOCLONAL ANTIBODIES

The production of monoclonal antibodies is one of the most useful recent scientific
advance applied to immunoassay technology. They have the ability to produce a single
type of antibody from the B-cell partner and the ability to survive and proliferate outside
the body of the animal for an extended period of time from the myeloma partner.

Monoclonal antibodies are produced in a series of steps as illustrated by figure 6.
It begins with the immunization of a mouse and then the removal of its spleen after an

appropriate period of time. Antibody-producing cells are isolated from the spleen and

84

 

B Cells

  

’73” ,1
i ‘4' ‘

 

 

 

Figure 5. Polyclonal antiserum containing a mixture of antibodies produced by multiple
B cells.

85

 

 

   

Specific Myeloma
B Cell Cell
Cells Fuse
Hybridoma / ”I ’ Monoclonal
Cell .57 ‘ , , Antibodies
FF ' ;/ I / \I i ' A \

e ”f .2_/ d I ,5‘\ \f \i': \ \..-

I I I! ’~\ A . l

 

 

Figure 6. Illustration of Monoclonal Antibodies Production. One B cell is fused in the
laboratory with a tumor cell. The resulting hybridoma produces multiple copies of one
specific type of antibody (monoclonal antibody).

86

fused with "immortal" myeloma cells from tissue culture through the use of polyethylene
glycol. Cells resulting from the fusion of a B-cell and myeloma cells are called
hybridomas. Through a series of manipulations in tissue culture, individual hybridomas
are isolated and allow to produce antibodies that are then tested for desirable antigen-

binding characteristics.
ANTIBODY-ANTIGEN INTERACTIONS

The binding of an antibody to an epitote on an antigen depends on non covalent
interaction such as electrostatic, hydrophobic, and Van der Waal forces (Benjamin, E. and
Leskowitz, S., 1988). The antigen-binding site and its epitope must be in close proximity
before optimal binding can occur. Hapten-carrier combinations can be designed with
various orientations, allowing an induction of an array of antibodies with different ability
to distinguish among closely related compounds. Relatively small changes in the structure
of the epitope itself or even its stereochemistry, can affect antibody binding.

Affinity is a measure of the strength of an individual antibody-antigen binding
interaction. Affinity is the most important consideration for the usefulness of a particular
antibody in an analytical assay. High affinity antibodies will bind larger amounts of
antigens in a shorter period of time than low affinity antibodies and produce a stable
complex. Monoclonal antibodies are homogeneous and their affinities can be determined
precisely. Polyclonal antibody mixtures have a variety of affinities so the overall binding

energy of the polyclonal mixture is referred to as avidity (Benjamin and Leskowitz, 1988).

87
IMMUNOASSAYS

Immunoassays are powerful techniques that rely on the specific interactions
between antibodies and antigen to detect and quantify a wide variety of substances
(microorganisms, environmental contaminants, etc). In a typical immunoassay either
antibodies or antigens are immobilized on a solid phase. Example of solid supports
include nitrocellulose or nylon membrane, test tubes, microscopic particles, or microtiter
plates (Van Emmon and Lopez-Avila, 1992).

The binding of the antigen to the antibody is detected by using markers. A
number of markers have been used in the detection system; among them radioactivity,
fluorescence, polarization of light, visible or ultraviolet absorbance, phosphorescence,
chemilunescence, bioluminescence or electron spin luminescence (Jung et al., 1989;
Kaufman et al. 1982; Anonymous 1990). Radioactive markers (tags) were used widely
for many years, but the inherent problems in handling radioactive compounds has driven
most immunoassays to employ non radioactive markers. Fluorescence tags are used in
many immunoassay procedures and can be detected with special instruments or

microscopes.

ENZYME IMMUNOASSAYS
The most widespread class of immunoassays employ enzymes as markers (Figure

7). These assays are called enzyme immunoassay (EIA) or Enzyme-linked
immunosorbent assay (ELISA). EIA produces a color reaction (Figure 8) that is

proportional to the level of the antigen being measured. The enzymes commonly use as

88

markers catalyze reactions that yield colored end-products. The enzyme must be stable,

 

 

Enzyme

    
  

    
 

Chemical

Attachment ’

Antibody

Antigen

 

 

Figure 7. Enzyme conjugate. Enzyme are physically linked to antibodies or antigens to
form an indicator system. The enzyme, the antibody, and the antigen must retain their
activities and binding capacities to be useful.

89

 

Enzyme

 

Colored

Colorless product

Substrate

 

 

 

 

 

 

 

Figure 8. Color reaction catalyzed enzymes used as indicators or tags in immunoassay.

90

operate under a wide range of conditions, and deliver a high reaction rate. The enzymes
most frequently used include horseradish peroxidase, alkaline phosphatase, galactosidase,
and urease.

The results of immunoassay are determined with instruments that measure the
amount of color, fluorescence or radioactivity of the assay. The level of signal detected

is either directly or inversely proportional to the concentration of the antigen of interest.

TYPE OF ENZYME IMMUNOASSAYS

Immunoassay product designs vary and certain formats may be appropriate for
specific applications. Immunoassays are categorized according to the way the antigen-
antibody complex forms. Competitive ELISA is the immunoassay format in which the
target analyte and an enzyme tagged with the target analyte compete to bind to an
antibody specific for the target. In this assay, the detecting reagent (antibody), is attached
to a solid support. Free antigen in the sample and a test antigen linked to an enzyme
compete to bind to the immobilized antibody. The ratio of free analyte to enzyme
conjugate determines the amount of enzyme conjugate that will bind to the immobilized
antibody. After the unreacted material is removed from the reaction solution, the enzyme
converts a substrate to a color product. The level of color is inversely proportional to the
amount of antigen in the sample since the free antigen prevents the enzyme-antigen
conjugate from binding. Competitive ELISAs are most often used to detect small organic
molecules such as pesticides or drugs and will be the format used in this study.

Double-antibody sandwich ELISA. Double-antibody sandwich assays are used to

91

determine the concentration of large antigens such as proteins, viruses, and bacteria.
Antibody is bound to a solid matrix, the sample antigen is allowed to bind, and unbound
antigens are removed by washing. Then a second, labelled antibody is added which binds
to the immobilized antibody-antigen complex. the assay is quantified by measuring the

color produced by the labeled second antibody.

APPLICATION OF IMMUNOASSAYS

HEALTH AND CLINICAL MEDICINE

Immunoassays are used routinely in a variety of applications in the medical field.
The first widespread applications were in biomedical research and human diagnostics.
Uses include the diagnosis of virus and bacterial infections, cancer screening, drug

monitoring, and pregnancy testing.

92

AGRICULTURAL USES
Immunoassays are used for the diagnosis of diseases and pregnancy in animals

(Miller, S. et al., 1988; Lankow, R.K. et al, 1987). More recently immunoassays have
been applied to the detection of crop diseases. They are simple, rapid, and sensitive
detection methods of pathogens in crops, seeds, bulbs, and soil. Traditionally, the
diagnosis of plant diseases was slow and inconsistent because plant pathogens are not
easily cultured. Besides conventional methods relied on specialized techniques such as
electron microscopy. In contrast detection by immunoassay methods do not require
specialized training or equipment. Once limited to the laboratory, immunoassays now
provide on-site testing by the growers, consultants and other agricultural professionals.
The ready availability of accurate information allows the farmer to make more timely and

informed management decisions regarding planting, pesticide use, and harvest timing.

93
TOXINS AND CONTAMINANTS SCREENING IN FOOD

Antibiotics can be found as residue in animal derived food if improperly used or
if withdrawal times have not been observed for treated animals. This can have potential
health hazard such as direct toxic effect on the consumer (sulfamethazine), transmission
of antibiotic resistance (salmonella), and development of allergy due to drugs that have
sensitized some individuals (penicillin). Mycotoxins are diverse family of poisonous
fungal metabolites. Aflatoxins, one these toxins can cause edema and necrosis of hepatic
and renal tissues. Cereals, bakery and oilseed products present high risk with regard to
aflatoxin contamination. Pesticides are applied at the farm levels for the protection of
crops against insects and diseases or on various foodstuffs after harvest for protection
against various types of pests during extending periods of storage. These chemicals may
be found at the levels above their tolerance levels in food and feed. Immunoassays
techniques can replace the standard chromatographic methods for the determination of

antibiotics, mycotoxins, and pesticides.

ENVIRONMENTAL ANALYSIS

During the past decade, immunoassays have been developed for the monitoring of
environmental contaminants. Monoclonal antibody technology combined with new
techniques to produce sensitive and specific assays have allow the detection and
quantification of pesticides, PCB's, petroleum and other contaminants. Today more than
50 commercial and experimental immunoassays have been described for the detection of

these types .of compounds. Immunoassays can be used during site assessment;

94

remediation and post remediation monitoring; RCRA testing.

95
CHAPTER III

CHARACTERISTICS OF THE PESTICIDES

ALACHLOR

USES

Alachlor [(chloro-N-(2,6-diethylphenyl)-N-(methoxymethyl) acetanilide], is a
selective systemic herbicide used for pre- and post-emergence control of most annual
grasses and broad-leaved weeds in such crops as corn (Zea mays L.), soybeans [Glycine
max (L.) Merr.], peanuts (Arachis hypogeae), cotton (Gossypium Spp.) and sugarcane
(Saccharum Spp.) (Anonymous, 1988). Lasso® which contains of alachlor as the active

ingredient is one of the most widely used herbicides in North America (Sun, 1986).

BEHAVIOR OF ALACHLOR IN PANTS

Alachlor is absorbed and translocated by tolerant and susceptible plants. The
basis for the selective toxicity is the rapidity of metabolic deactivation. In tolerant plants,
the herbicide is detoxified by rapid conjugation with glutathione (GSH) and/or
homoglutathione (hGSH) (Breaux et al., 1987). The GSH conjugate is subsequently
metabolized to malonylcysteine conjugate (W SSA Herbicide handbook, 7th edition, 1994).
The site of action of alachlor is unknown but it may as most of the chloroacetamides,

inhibit lipid and protein synthesis and interfere with respiration and photosynthesis and

 

Structure:

CAS No:
Common name:
Chemical name:
Trade name:

Chemical family:

Molecular formula:

Molecular weight:
Manufacturer:
Physical form:
Melting point:
Boiling point:

Vapor pressure:
Specific gravity:

Stability:

Solubility:

96

Table 1. Chemical and physical properties of the pure alachlor

 

15972-60-8

alachlor

2-chloro

Lasso

acetamide

CHHzoCl NO2

269.77

Monsanto

colorless to yellow crystals

39.5 - 41.5 °C

100 °C at 0.02 mm Hg or 135 °C at 0.3
mm Hg

2.9 mPa at 25 °C

1.133 at 25 °C

Hydrolyzed by strong acids and alkali.

Stable in UV light. Decomposes at 105C.

into water at 25 °C, 242 mg/L.
Soluble in diethylether, acetone,
chloroform, and ethyl acetate.

 

 

97
membrane phenomena (Ashton, FM. and Craft, AS, 1973. Mode of action of herbicides.

John Wiley & Sons, New York, pp 127-146.).

BEHAVIOR OF ALACHLOR IN SOIL

Alachlor undergoes chemical and microbial degradation in soil. Chemical
degradation of alachlor occurs under low humidity and high temperature conditions,
resulting in formation of 2-chloro-2',6'-diethyl acetanilide. This intermediate
decomposition product did not accumulate under natural soil conditions (Hargrove and
Merkle, 1971). Microbial degradation was found to be the major route of alachlor
degradation in soil with half-lives ranging between 2 to 14 days for several soils

(Beetstman and Deming, 1972).

BEHAVIOR IN AQUATIC ENVIRONMENT
Little information is available on the degradation of alachlor in aquatic systems.
Studies found mineralization in aquifer materials to be extremely slow (Novick et al.,

1986). Under flooded soil conditions, eight metabolites were detected (Lee, 1984).

TOXICOLOGICAL PROPERTIES

Metabolism of alachlor in domestic animals is poorly understood but it is similar
to that in plants. While plants retain metabolites, animal eliminate metabolites quickly
and almost entirely. In rat, alachlor was rapidly metabolized and the metabolites in urine

and feces were excreted as conjugates of mercapturic acid, glucuronic acid, sulfate and

98
products hydroxylated at the O-alkyl substituents (USEPA, 1984).

Alachlor has been classified in group B2 by the EPA, as a probable human
carcinogen (US. EPA, 1986). The USEPA has established residues tolerances for food
and feed expressed as DEA and HEEA. However, the proposed methods for tolerance
enforcement may not measure all metabolites of toxicological concern (Kovacs, 1986).
The wide use of alachlor suggests that the major pathways of human exposure are direct
contact during application, dietary exposures from ingested residue-containing foods, and
drinking contaminated water. Alachlor exhibits low mammalian acute toxicity, LD,0 =
0.93 (rat) and a systemic NOEL of 30 mg/kg in food (1.5 mg/kg/d) was determined
(USEPA, 1984).

Effects of alachlor on wildlife and aquatic organisms is of concern because the
herbicides may reach surface waters inadvertently through runoff from terrestrial treated
fields and spray drifts. Browsing animals may be exposed to residues that persist in
terrestrial plants. The herbicide has low avian toxicity, is slightly toxic to aquatic
invertebrates, and moderately toxic to fish. Dietary LCso values of alachlor for mallard
ducklings is >5,000 and the 96-hr LC,o for blue gill is 2.8 mg/L. Limited data show fish
are unlikely to accumulate alachlor because of rapid elimination. Call et al.(1984) found
that 85% of "C alachlor injected into rainbow trout is readily eliminated within 24 hr,
40% as metabolites. The same study showed that 14C-Alachlor was absorbed rapidly by
fathead minnows and the BCFs of total radioactivity from 1-21 d were 50 and 41 for
exposures at 0.66 and 9.95 mg/L respectively. However, only 13% of total 14C was

extracted as parent compound, for a mean BCF of 6.0 as alachlor. In all the studies,

99

tissue residues declined to low levels after depuration, probably due to metabolism and

excretion.

ATRAZINE

USES
Atrazine [2-chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine] is a triazine

herbicide marketed under the trade names of Gesaprim® and Aatrex®. Atrazine is the
second most widely used pesticide in the USA, mostly in corn production. It is also used
in sorghum, sugarcane and a variety of other crops. Current annual sales are

approximately 27.2 million kg (Regehr, 1992).

BEHAVIOR IN PLANTS

Atrazine is absorbed through roots from soil applications and translocates to the
shoots via the apoplast. It is also adsorbed into leaves from post applications. Tolerant
plants such as maize or millet possess efficient detoxification mechanisms. In these
plants, atrazine is rapidly detoxified by conjugation with Gluthatione (GSH).
Benzoxazinone-catalyzed hydrolysis and N-dealkylation of side chains contributed
significantly to the detoxification of atrazine (Anonymous, 1994).

Atrazine inhibits the photosynthetic transport of electrons and this inhibition affects
processes dependent on photosynthesis such as the opening of stomata, transpiration, ion
transport, which may lead to the disruption of overall metabolism including RNA,

enzyme, and protein synthesis (Ebert, E. and Dunford, S.W., 1976).

 

 

100

Table 2. Chemical and physical properties of the pure atrazine

Common name:
CAS register No:

Chemical name:

Trade name:
Geigy).

Chemical family:

Molecular formula:

Molecular weight:
Manufacture:
Physical form:
Melting point:
Vapor pressure:

Stability:

Solubility:

11 CL
1
N / N

H NCH3CHJ

 

Atrazine
1912-24-9

2-chloro-4-(ethylamino)-6-(isopropylamino)-s-
triazine

Gesaprim (Ciba-Geigy), Primatol (Ciba-
Aatrex (Ciba-Geigy)

Triazine

C,H,,Cl N,S
215.69
Ciba-geigy
colorless crystals
176 °C

0.04 mPa at 20 °C

stable in neutral, weekly acidic and weakly
alkaline media Hydrolyzed to the herbicidally-
inactive hydroxy derivative in strong acid and
alkalis, and at higher. temperature, in neutral
media.

in water at 20 °C, 28 mg/L; in dimethylsulfoxide 183,
chloroform 52, ethyl acetate 28. methanol 18, diethyl ether

 

 

 

101

In the environment atrazine is metabolized in three ways (Knuesli et al.,1969): (1).
By hydrolysis of the chlorine-carbon bonds yielding a non phytotoxic compound,
hydroxyatrazine, which is one of the main metabolites in both soil and aquatic systems;
(2). By N-dealkylation of carbon atom 4 and/or carbon atom 6. This gives rise to
deethylatrazine, deisopropylatrazine, and diaminochloro-s-triazine (Schiavon, 1988; ). (3).
By splitting of the triazine ring, usually caused by microorganisms (Wolf and
Marin,1975). The products resulting from decomposition are less toxic to plants and

animals than the original substances (Straton, 1984).

BEHAVIOR IN SOIL

Atrazine has come under close scrutiny due to its persistence in soil which can
causes injury to succeeding sensitive plants during crop rotation. Its concentration and
persistence in the soil depends on the amount applied, soil composition, and climate. The
data for the persistence of atrazine in the soil are extremely variable. The half-life range
from 20 to more than 385 days (Winkelmann and Klaine, 1991).

Atrazine is moderately adsorbed to soil and adsorption increases at lower pH.
Biological degradation contributes to a moderate extent to field dissipation of atrazine.
The products of biological degradation are N-deethylated atrazine and N-demethylated
atrazine. Soil hydrolysis of atrazine is slow at high pH (7.5-8), but it contributes to
degradation at lower pH (5.5-6.5) producing hydroxy atrazine.

Atrazine has recently been identify as a potential pollutant of both ground water

and surface water. Surface water contamination occurs primarily as the results of runoff

102

processes following precipitation or irrigation. Loss due to runoff may reach up to 18%
of the total volume of applied atrazine. The transport of atrazine in the soil is affected
by structure and composition of the soil and by climatic conditions (Premazzi and Steechi,
1990). Sandy soils allow considerably more rapid translocation than humus soils. Under
moderately moist conditions, over a period of one year, virtually no translocation occurs
beyond a depth of 30 cm (Frank. and Sirons, 1979). In general, atrazine may be

classified as a substance that is moderately mobile in soil.

BEHAVIOR IN THE AQUATIC ENVIRONMENT

In aquatic systems, the half-life of atrazine is reported to be between 3 and 300
days (Yoo and Solomon, 1981). Hydrolytic decomposition can be a factor if the medium
is slightly acidic. The rate of decomposition is affected by the chemical composition of
water. A higher saline content, such as may occur in the estuaries appears to accelerate
the decomposition in water (Jones et al., 1982).

A large number of reports have documented the occurrence of atrazine in surface
water. In the USA, atrazine concentration of 0-87 mg/L have been found, with the
majority of the findings below 10 mg/L (Eisler, 1989). A study by Hoennann et al.(1979)
investigating nine central European rivers found that 59% of the tested samples contained
<0.4 mg/L; only 2% exceeded the 10 mg/L limit. 11 the case of atrazine-contaminated
rivers, concentrations of between 1.4 and 95 mg/L were detected in the river silts
(Waldron, 974).

In standing freshwater (ponds, natural lakes, and reservoirs), the levels of atrazine

103

was found to be considerably lower. In the USA, concentrations up to 2 mg/L have been
recorded (Premazzi and Steechi, 1990).

Many studies have reported on groundwater contamination by atrazine but many
of these studies involved major uncertainty due to shortcomings such as non
representative sampling selection, non standardized sampling, and non conformity of
sampling container used. The EPA study " National Survey of Pesticides in Drinking
Water Wells" (U.S.E.P.A, 1990) which seems to be more rigorous reported atrazine
concentrations of between 0.18 and 1.04 mg/l with 50% of the tested wells showing

concentrations below 0.28 mg/L.

TOXICOLOGICAL PROPERTIES

For most of the organisms, atrazine is taken up from water by adsorption or via
the food chain. In fish, direct accumulation of atrazine from water takes place according
to simple saturation kinetics; the saturation point is reached after 6 h. The concentration
limit for atrazine in the case of whitefish ranges between two to five and does not change
significantly even in cases of long-terrn exposure (Gunkel, 1981). Investigations of
atrazine uptake via contaminated food show atrazine taken up from contaminated food is
assimilated rapidly. Only 70% of the atrazine taken up can still be detenninated 30 min
after the food has been ingested. After that the quantity of detectable atrazine in fish
declines rapidly within 12 h. Effective elimination mechanisms prevent residual
concentrations of atrazine in fish. (Gunkel, 1981).

Atrazine degradation is insignificant in most aquatic organisms. Elimination is the

104

most important pathway of decontamination. The rates of atrazine elimination vary
widely depending on the organism. In the algae it is within one minute, hours in case of
water fleas, molluscs, and leeches. By contrast, elimination periods reported for fish
range from 1.5 to several days (Gunkel, 1981).

In summary, atrazine is taken up more or less easily by most aquatic organisms.
However, a large portion of the adsorbed substance is eliminated quickly by the organisms
when they reach non contaminated water.

Atrazine has not been shown to present serious adverse effect in wildlife. In fish
there is some indications that atrazine has an impact on carp (Cyprinus carpio) at even
low concentrations (100-1000 mg/L) and short exposure period. At these concentrations,
the hydrocortisone and glucose levels increase which indicates a typical stress defense
reaction (Eisler, R., 1989).

The LC,o values based on 96 h of exposure are variable depending on the species.
Values of 19,000 mg/L have been reported for common carp (C. carpio) and 42, 000
mg/L for bluegill sunfish (Lepomis macrochirus) (Premazzi and Stecchi, 1990; Eisler,
1989)

Various studies have reported with NOEC or LOEC values. These values vary
between 100 and 2,100 mg/L in the case of trout (S. gairdneri) with an exposure time of
96 h. Values of 1,000 mg/L have been measured for Blue gill (Lepomr‘s macrochirus).
The NOEC values decline with longer exposure time (Premazzi and Stecchi, 1990; Eisler,

1989)

105
CARBOFURAN

USES
Carbofuran, (2,3-dihydro-2,2-dimethyl-7-benzofuranyl N-methylcarbarnate), is a

broadspectrum insecticide-nematicide used in a variety of crops (Cox, 1966; Tumipseed,
1967). It is effective as both a contact and systemic insecticides (Shorey and Hale, 1967).
It is used to control a wide range of agricultural pests (Caro et al., 1973). In the midwest
of the USA, carbofuran has provided effective control of the corn root worm. Carbofuran
is also used for the control of rot weevils and a moth larva in small fruits. These include:
the strawberry root weevil, Bracyrhinus ovatus (L.), and the bush weevil, Nemocestes
incauprus (Horn) in strawberry; the bud weevil, B. Singularis (L.) in raspberry; the black
vine weevil, B. sulcarus (Fab,), and in blueberry; the black-headed fireworm, Rhopobota
naevana (Hub.). In Africa, carbofuran has been increasingly used as the most effective
insecticide to control rice pests in paddy fields. Some of the pests controlled are the
green leafiiopper (Nephotem'x virescens), the brown planthopper (Nilaparvata lugens) and

the stem borers (Tryphorhiza mcertulas, chilo suppressalis).

BEHAVIOR IN PLANTS

Because of its systemic nature, carbofuran is taken up by the root system and
distributes throughout the entire plant and could remain in grains or plant materials after
harvesting. Carbofuran is metabolized by hydroxylation and hydrolysis in plants (Metcalf
et al., 1968). Carbofuran deteriorates rapidly on vegetation sprayed with flowable and

wettable powder formulation (e.g., half-life of less tan 7 days on alfalfa and Bermuda

 

 

 

 

Common name:
CAS register No.2
Chemical name:
Trade name:

Chemical family:

Molecular formula:

Molecular weight:
Manufacturer:
Physical form:

Melting point:

Vapor pressure:
Specific gravity:

Stability:
media

Solubility:

106

Table 3. Chemical and physical properties of the pure atrazine

Carbofuran

1563-66-2

2,3-dihydro-2,2-dimethyl-7-benzofi1ranyl N-methylcarbarnate
Furadan (FMC), Curater (Bayer), Bay 70143 (bayer)
carbamate

C,2H,,,NO3

221.25

FMC, Bayer

colorless crystals

153-154 °c (pure), 150-152 °C

2.7 mPa at 33 °C
118 at 20 °C
unstable in alkaline media, stable in acidic and neutral

150.
120,

in water, 700 mg/L. In acetone
acetonitrile 140, dichloromethane
cyclohexanone 90, benzene 40,

 

 

107

grass) (Leuck et al., 1968; Fahey et al., 1970). In furrow application of granular
formulation carbofuran is readily translocated through the roots and stems, with significant
insecticidal activity continuing in foliage for 2 to 4 months. An appreciable amount of
carbofuran and its major metabolite 3-hydroxycarbofuran remain in the leaves of corn at

silage stage as well as at harvest (Turner and Caro, 1973).

BEHAVIOR IN SOILS

Carbofuran's fate in soil is affected by the pesticide formulation, the rate and
method of application, soil type, pH, rainfall and irrigation, temperature, moisture content,
and microbial population (Kuhr and Borough, 1976). Carbofuran is stable at pH 5.5 but
decomposes rapidly in alkaline soil. The hydrolytic half-life in soil at pH 7 is about 35
days (Finlayson et al, 1979). Temperature and moisture content are positively correlated
with degradation; maximum degradation to hydrolytic metabolites occurs at 27 °C (Du et
al., 1982). The terrestrial dissipation half-life of carbofuran in irrigated soils is reported
to be 4 to 11 days in sandy loam, and less than 5 months in silty loam (US. EPA, 1991).
Carbofuran is mobile and likely to be found in streams, surface water, and runoff
sediments from treated watersheds (US. EPA, 1991). This suggests that carbofuran may

be quite stable in regions of significant acid precipitation

BEHAVIOR IN AQUATIC ENVIRONMENT
Carbofuran is soluble in water to 700 mg/l (25 °C) and in most organic solvents

to 30%. It is essentially stable in acidic medium (Baron, 1991). The fate of carbofuran

108

in water is predominantly function of the pH, but is also influenced by photolysis,
temperature, and trace impurities (Seiber et al., 1978). The half life of carbofuran in
distilled water at 25 °C and pH 5.5 is 16.4; and decreases when pH increases. At pH 9
the half-life is 6 h (Finlayson et al., 1979). The rate of hydrolysis is positivelycorrelated

with ambient temperature.

TOXICOLOGICAL PROPERTIES

Aquatic invertebrates and fish do not tend to bioaccumulate carbofuran when
studied in slightly alkaline model ecosystems (Isensee and Tayaputch, 1986). Crabs may
be the exception, as one species (Uca mimax) is reported to bioaccumulate carbofuran (Yu
et al., 1974).

Carbofuran is unstable in living animals and is readily excreted. Therefore
significant bioaccumulation is not expected from sublethal exposure of either invertebrates
or vertebrates (Finlayson et al., 1979). Nonetheless, a secondary hazard from carbofuran
occurs in predatory vertebrates that feed on dead and struggling insects, earthworms, and
small birds and mammals (US EPA, 1991). The source of poisoning is most likely to be
from unabsorbed carbofuran on the cuticle of arthropods or in the gut of worms and small
vertebrates.

Carbofuran is highly toxic to fish, but considerably less toxic to tubificid worms
and marine shellfish. The 96-h acute toxicity tests of with seven species of juvenile fresh
water fish indicate that LCsos varied from 147 mg of carbofuran per liter for yellow perch

(Perch flavescens) to 872 mg of carbofuran per liter for fathead minnow (pimephales

109

promelas) (Johnson and Finley, 1980). Two species of freshwater annelid worms and four
species of salt water bivalve molluscs gave 96-h LCso for carbofuran that varies from 3.75
to 125 mg/l (Eisler, 1985). The 96-h LC,o for the red crayfish (Procambarus clarki) is
2.26 mg/l (US. EPA, 1991). The 48-h EC,0 for Daphnia magna and Chimnomus n‘parr'us
is 48 and 56 mg of carbofuran per liter (Johnson, 1986). Few adverse non lethal effects
are reported for carbofuran in practical controlled studies of aquatic organisms. At current
registered application rates, carbofuran has not proven accumulative in aquatic systems
and poses little chronic hazard to fish and invertebrates. However, it has been observed
that at the application rates in variety of formulations, carbofuran has been held
responsible for sporadic fish kills (Flickinger et al., 1980).

Because carbofuran is sufficiently toxic to most aquatic organisms, a special
warning is required on all use labels against application of carbofuran to water either
directly or through drift or run-off from treated areas.

Carbofuran is highly toxic to most terrestrial animals and is non specific in its
action on non beneficial non targeted invertebrates species (Finlayson et al., 1979). At
recommended field application rates, losses of earthworms and springtails (Collembola)
have occurred. Similarly, predatory and parasitic soil insects and parasites and predators
of foliage pests are also vulnerable.

Birds and mammals are highly sensitive to acute or oral dosage of carbofuran and
usually die within a few minutes of exposure, or recover with little evidence of toxicity
within 0.5 to 2 h (Hill and camardese, 1984). The toxicity of carbofuran is a function of

the formulation; the flowable concentrate, Ferritin® 4F is about four times as toxic as

1 10
granular Ferritin® 15G to northern bobwhite and the wettable powder Ferritin® 75 WP

is nearly seven times as toxic as granular Ferritin® 10G to laboratory rats (Finlayson et
al, 1979).

Experimental and operational agricultural applications of both foliar and soil-
incorporated treatment with carbofuran in various formulations have consistently killed
large number of birds of many species. Field studies have shown avian mortality and
death of amphibians, reptiles, and mammals when carbofuran is applied in different
formulation on corn, rice, and pine seed orchard, and alfalfa (US. EPA, 1991).
Carbofuran is especially hazardous because of its extreme acute toxicity.

Carbofuran formulations and applications are highly toxic to most terrestrial
wildlife, but the acute action is short-lived in survivors (Hill, 1992), and it is non-
accumulative in most biological systems (Finlayson et al., 1979). Sublethal exposure to
carbofuran is not usually expected to pose an important direct hazard to birds and
mammals, but indirect effects due to reduced invertebrate food base and plant pollinators
may be significant (Eisler, 1985). Also there is evidence that inclement weather may
exarcebate the toxicity of low-grade exposure in young birds (Martin and Salmon, 1991).

Because of its high acute toxicity to fish and mammals (LD50 11 mg/Kg in rats),
the fate of its residues in terms of its persistence, mobility, and dissipation pathways is

of great concern.

lll
CHAPTERIV

MATERIALS AND METHODS

STUDY DESIGN AND SAMPLE COLLECTION

FISH REARING

The fish used in the experiment were bluegills (Lepomis macrochirus) size 4-5 inches,
purchased from a commercial hatchery in Dexter, Michigan. The fish were first
acclimated in the greenhouse by putting them in a big tank, and later they were
transferred in 40 ml aquarium and exposed to the pesticides. The rearing conditions are
summarized in table 1 (Appendix D). Stock solutions were prepared by diluting 1 ml of
the commercial formulation in distilled water to reach 2.4 g/L for carbofuran (Furadan)
and 1.2 g/L for atrazine (Aatrex) and alachlor (Lasso). For each pesticide the final
concentration in the tanks were made in consideration of the 96h LC,0 for bluegill. The
experiment consisted on a control (non treated) and two treatments as follow: for atrazine;
for alachlor 1.2 and 2.4 mg/L; for carbofuran 0.12 and 0.24 mg/L. To avoid ammonia
build-up in the tank, 25% of water in the tank was replaced each day. In order to keep
constant exposure, 25% of the initial amount of pesticide was added each day. Each
treatment was replicated and replication 1 was treated each day until the fourteen day by
which the fish were removed from the tanks,and killed. For replication 2, the treatments

were stopped 7 days before the fish were removed from the tanks. At the end of the

112

exposition, fish were removed from the tanks, killed, the scales removed, and effiletted.

The fillets were put on plastic bags , marked and kept in the freezer until analysis.

CORN RAISING

Field corns (Great Lakes Signature hybrid GL 420) were grown in greenhouse and
treated with the three pesticides. Atrazine (Aatrex 4L) and alachlor (Lasso 4EC) were
applied by spray after corn plants have emerged. he experiment consisted on three
treatments and one control. he treatments consist on half of the recommended rate (1 lb
a.i/A); the recommended rate (2 lb ai./A), and twice the recommended rate (4 lb a.i.lA).
These treatment correspond to 2.5 ml, 5.0, 10.0 of ai. in 250 ml of water respectively.
The characteristic of the sprayer were the following: 8001E/100 mesh screen, 10 inches
high, 14 inches band width, and 30 gpa.

Because of its high vapor pressure that may cause intoxication, carbofuran was
incorporated to the soil. The pots were first filled with soil and topped with 1 cm of the
treated. The experiment consists on three treatment and a control (non treated).
Treatment one was the recommended concentration (1 1b a.i.lA), treatment two was twice
the recommended rate (2 lb a.i.lA) and treatment three was three time the recommended
rate (3 lb aii/A).

Corn leaves were sampled five time during the experiment as follow: one day
before corn plant were treated, the day of treatment, five, seven, and eleven days after the
treatment. The leaves were chopped using a food chopper and after thorough mixing, the

samples were stored in refrigerator in deep freeze for further analysis.

1 13
MATERIALS

REAGENTS

o Solvents: methanol, acetone, and acetonitrile (Burdick and Jackson, Muskegon, MI)
were pesticide grade (ChromAR-HPLC).

0 Analytical Reference: Analytical reference of carbofuran (99.5%) and its metabolites
3-hydroxycarbofuran were provided free by FCC Corp., Agricultural Chemical Group
(Princeton, NJ). Analytical standard stock solution of carbofuran was prepared in
HPLC-grade methanol and stored in refrigerator. Formulated carbofuran (4F) used for
fish exposure was provided by FCC Ag (Princeton, NJ). Atrazine and its metabolites
were provided free by Ciba Geigy (Greensboro, NC). Formulated atrazine (Aatrex
4L) used for fish exposure and plant treatment was provided free by Ciba-Geigy.
Alachlor and its analogs acetolachlor, metolachlor, and propachlor were purchased
from AccuStandard (New Haven, CT). he formulated alachlor (Lasso) used for fish

exposure and plant treatment was provided by Monsanto.

EQUIPMENT

ELISA
The RaPID" Assays kits for alachlor, carbofuran, and atrazine; the magnetic separation
rack; and the RAPID Photometric Analyzer" were purchased from Ohmicron (Newton,

PA). The kit contained: anti-pesticide antibody coupled to magnetic particles; pesticide

114

coupled to peroxidase; standard solutions of the pesticides in water; enzyme substrate
solution; color generating products (peroxidase solution and chromogen solution); stopping
solution (2M sulfuric acid solution); buffer saline diluent; washing solution; and test
tubes.

EnviroGardTM assay kits for alachlor, carbofuran, and atrazine were purchased from
Millipore (Bedford, MA). he kits contain antibody-coated test tubes; standard solutions
of the pesticides; pesticide enzyme conjugate; enzyme substrate solution; Chromogen

solution

CHROMATOGRAPHY

0 Hewlett-Packard Model 5890 Series H GC (Palo Alto, CA) equipped with a
“Ni electron capture detector (ECD) and a nitrogen specific detector (NPD).

0 HP Model 7673 automatic injector

0 J&W fused silica capillary column (Durabond); ID #122-5042; Liquid phase:
DB-5 (non-extractable bonded phase); film thickness, 0.25 pm; column
dimensions: 30 M x 0.333 mm id.

0 J&W fused silica capillary column (Durabond); ID #122-5042; Liquid phase:
DB-5 non-extractable bonded phase); film thickness, 0.25 pm; column

dimensions: 15 x 0.333 mm id.

1 15
ANALYTICAL METHODS

EXTRACTION OF FISH FOR ELISA DETECTION

Five g of chopped fish filet was spiked with standard solution of atrazine and let
stand for 2 hr at room temperature (25 °C). The sample was grounded with twice its
weight with Na,SO, in a mortar and placed into an erlenmeyer. The grounded sample
was homogenized for 10 min with 50 ml acetonitrile using a mechanical homogenizer.
he homogenate let stand alone for 3-5 min, then an aliquot of the supernatant was
collected and analyzed after dilution with the supplied buffered saline diluent (Ohmicron
kits) or distillate water (Millipore kits). For carbofuran, the samples were also extracted
with water and the results compared with acetonitrile. For water extraction, fish fillet was
chopped in small pieces, grounded with 5 grams of Na,SO4 and blended in Sorvall Omni-
Mixer with 50 ml distillate. The extract was analyzed according to the same procedure

described above.

EXTRACTION OF CORN LEAF FOR ELISA DETECTION

Atrazine. 15 g of chopped corn leaves was blended for 2 min with 50 ml methanol
and filtered with celite 455 under vacuum using whatrnan No. 1 filter.

Carbofuran. 15 gram chopped leave samples were placed into a round bottom
flask. Seventy five ml of 0.25N HCl solution was added to the flask and heated for 1 hr
(stirring). After cooling, the slurry was filtered under vacuum using Whatrnan No 1 filter

paper and analyzed after dilution. hen decoloration was needed, the slurry was mixed

116

with 2 grams of charcoal and celite 455 and filtered under vacuum using Whatman No
1 filter paper.

Alachlor. 15 grams of chopped leaves were placed into an erlenmeyer, 50 ml
solution of 10% water in acetonitrile was added and homogenized and filtered under

vacuum using whatrnan No 1 filter paper and analyzed after dilution.

EXTRACTION OF CORN LEAF FOR CHROMATOGRAPHIC ANALYSIS

Atrazine. 50 g of sample was blended with 100 ml of methanol for 2 min. The
homogenate was filtered with celite 455 under vacuum using whatrnan No 1 filter paper.
he filter cake was washed with 50 ml and an aliquot of 50 m1 of extract was placed into
a separatory funnel and extracted three times with 50 ml dichloromethane (DCM). The
DCM extract was evaporated to dryness under vacuum, and diluted with 2 ml hexane and
submitted toclean-up. For the clean-up, the glass column was filled with 10 g basic
alumina topped with 2-3 g NaQSO,. The column was washed with 40 ml 20% (v/v) ethyl
ether (EE)/hexane. After transferring the extract into the column, the column was eluted
with 40 ml 20% (v/v) EE/hexane which was discarded, then with 120 ml 20% EE/hexane
which was collected and evaporated near dryness, redissolved in 2 ml hexane and injected
into the GC.

Carbofuran. 25 g of sample of chopped sample was placed into a round bottom
flask. ne hundred fifty ml of 0.25N HCl solution was added to the flask and refluxed for
1 hr (stirring). After cooling,, the homogenate was filtered under vacuum using Whatman

No 1 filter paper and the filter cake rinsed with 50 ml methylene chloride followed by 50

117

ml of 0.25N HCL. Each filtrate was transferred to a separatory funnel and after shaking,
the methylene chloride layer was removed. Then the aqueous fraction was further
extracted with 3 x 50 ml methylene chloride. The combined methylene layer was dried
over anhydrous Naso, and decolonized by adding 2 g of charcoal and filtered later by
gravity. The filtrate was evaporated to dryness, then redissolved in 5 ml of 5% (v/v)
acetone in hexane and subjected to a clean-up.
For the clean-up, the column was filled with 7 g of silica gel and topped with 2-3 g of
anhydrous Na2804. The column was washed with 50 ml hexane and the 5 ml extract was
loaded into the column. Initially the column was eluted with 50 ml 5% (v/v) acetone in
hexane, which was discarded. It was further eluted with 250 ml of 10% acetone in
hexane, which was then evaporated to nearly dryness and redissolved in 5 ml hexane and
analyzed by NPD-GC.

Alachlor. Twenty five grams of chopped leaves was blended for 3 minutes with
250 mL 10% acetone in water. The suspension was filtered under vacuum and the filtrate
reduced to ~30 ml using a rotary evaporator. The evaporate residue was transferred into
a separatory funnel with 2 x 50 m1 5% sodium sulfate solution and partitioned twice with
100 ml hexane. The combined hexane layers was dried over anhydrous Na,SO, and
evaporated to dryness using a rotary evaporator. The residue was taken to 10 ml with
hexane and subjected to clean-up. For the clean-up, the column was filled with 5.5 grams
of alumina/activated florisil and topped with 3-4 grams of anhydrous Na2804. The
column was pro-washed successively with 50 ml of 10% ethyl acetate in hexane and 50

ml of hexane. Then the extract was loaded into the column. Initially the column was

118

eluted with 50 ml 2% (v/v) ethyl acetate in hexane followed by 20 ml 5% ethyl acetate
in hexane, which was discarded. It was further eluted with 50 ml 10% ethyl acetate in
hexane, which was then evaporated to nearly dryness and redissolved in 5 ml hexane and

analyzed by ECD-GC.

EXTRACTION OF FISH FOR CHROMATOGRAPHIC ANALYSIS

For all the three pesticides, 10 gram of sample were extracted according to the
method of Martin and al. (12). Basically, the sample mixed with 50 gram Na,SO,
anhydrous, were grounded with a mortar and homogenized with acetonitrile. The
homogenate was filtered, first partitioned with hexane, and second with 10% NaCl water

and finely cleanup on silica gel column and analyzed by GC.

DETECTION AND QUANT'IFICAT'ION
ELISA ASSAY PRQCEDURES

The assay, as applied to corn leaves and fish extract was carried out according to
the procedure for water samples specified in the kit by the manufacturer, except all
determinations were done in duplicate. Briefly, for Millipore kit, the samples and the
enzyme conjugate were added into the test tubes and were allowed to incubate for a 20
minutes at room temperature. After washing the tubes four times with tap water, the
substrate was added to the tubes followed by the chromogen and the tubes were allowed
to incubate for 10 minutes. After that, the stop solution was added to the tubes.

Absorbance readings were made at 450 nm with a portable tube photometer. For

119

Ohmicron, the standard, control or samples, carbofuran-enzyme conjugate and anti-
carbofuran antibody coupled to magnetic particles were incubated in polystyrene tube held
in a rack at room temperature for 30 minutes. After the incubation, a magnetic base was
coupled to the rack, the tubes were rinsed twice with the washing solution. After having
remove the rack from the separator, a freshly prepared chromogen solution was added to
each tube and the tubes were incubated. After 20 minutes, an enzyme inhibitor (stop
solution) was added to the tubes to stop the color development. The absorbance of the
color in the sample and standard tubes was read at 450 nm using a portable tube
photometer at 450 nm and the amount of the pesticides determined by reference to the
standard curve

The concentration of the pesticide in fish or corn leaf extract is calculated by using

the above equation:

3 I Extract vol.(mL) vol. wracKmL) wot. dihunxml.)
Y readt(ppb) x sample welghdg) x wlmacronl)

HR MAT PHI PROCED S
A Hewlett packard 5890 Series 11 gas chromatograph equipped with a 63Ni electron
capture detector (ECD) and a nitrogen specific detector (NPD) was used for the detection
of the pesticides in fish and corn leaf samples. The injection of the samples into the GC
were done automatically using a HP Model 7673 automatic injector. A Hewlett packard

computer and Laserjet IIIp printer for GC was used for data handling and processing.

120

Table 4. Chromatographic conditions for the determinations of the pesticide

_
Compound Detector Column Type Temperature 'C

 

Oven Injector Detector

 

Alachlor ECD DB-5 fused capillary
60 M x 25 mm id. 180 250 250

0.25 um phase thickness.
J7W # 122-5042

DB-5 fused capillary
Atrazine NPD 30 M x 0.333 mm id. 200 250 250
0.25 pm phase thickness
17W 3 122-5043

DB-5 fused capillary
Carbofuran NPD 30 M x 0.333 mm id. 200 250 250
0.25 um phase thickness
NW 3 122-5043

AL AT‘I N F THE PESTI IDE ONCENTRATION
Quantification of pesticides were based on peak areas. A series of pesticide
standard solutions were injected and a standard curve was traced. calculation of the
concentration levels for each pesticide in a sample on the wet weight of fish or volume

of water was accomplished by the use of the above equations.

(1)Standard curve: y =ax+c ~ 1: =!:_; x =g of pesticide, y=peak area
a

121

ng of pesticide x 1000 ”um,

(2)Pesticide on final extract, rig/ml = . ,
111 injectron volume

(3 ) ppm =ng/ml (extract Vol.) x final Vol . x (1 / sample Wt) x 1 ug/ 1000 ng

CONFIRMATI N
Confirmation of the pesticide confirmation were performed on a tandem Hewlett-

Packard Model 5890A GC (Palo Alto, CA) and a 5970A mass selective detector.
Operating conditions were as followed: ionization voltage, 70 eV; ion source temperature
260 °C; electron multiplier 300 V above autotune; direct capillary interface at 300 °C. The
filament and multiplier were not turn on until 5 min into the run. DB-5 30 M x 0.333
mm id fused capillary column, 0.25 um phase thickness was used. The detection was
made by using electron capture/negative ionization (ECNCI) in the scan mode. Initial
column temperature was set at 80 °C for 5 min and programmed at 15 °C/min for 10 min.

Confirmation was based upon presence of the molecular ion and two confirming ions.

APPENDIX C

RESULTS OF CARBOFURAN DETERMINATION

122
CHAPTER V

RESULTS AND DISCUSSION

The absorbance readings from ELISA are inversely proportional to the
concentration of analytes in the sample extracts. The percentage control (total binding)
was calculated as the absorbance of the sample (B) at 450 nm divided by the absorbance
of the negative control (extract blank, Bo) at 450 nm and times 100. In this study B/Bo
was referred as Bo, which plotted versus the log concentration is linear with a negative
slope. These plots were used to approximate concentrations of the samples. Cross-
reactivities (%CR) were calculated by dividing the metabolite concentration which
produced 50% B0 by the alachlor concentration that gave 50% B0. The least detectable

dose (LDD) was the calculated analyte concentration yielding 90% Bo.

ALACHLOR

While performing the ELISA for the present study, blanks and calibration
standards were always assayed with the samples. The results are summarized in appendix
A. A compilation of alachlor calibration curves generated with the two different test kits
shows a linearity in the range of 0.1 ppb-5 ppb and 0.5-10 ppb for the Ohmicron and
Millipore test kits respectively (Figure 9). The RaPIDT" kit generated an equation with

R2 = 0.99 while the EnvriroGardO (Millipore) kits gave an equation with R2 = 0.97.

123

% Bo

1001

90—
80*
70~

60~

      
 

 

50‘ V, Ohmicrorj

40 _ 5 8M0 Millipore

 

 

3o—
20-
10M] - 1W] . 1W]
1E-01 _ .1 10
Concentration (ppb)

Figure 9. Plot of the standard curves of the Ohmicron and Millipore test kits (average of
4 determinations). %Bo = % (absorbance of the sample/absorbance of the zero control).

124
The Ohmicron kit has the lowest least detectable dose (LDD) of 0.07 ppb; the LDD for

the Millipore kit was determined to be 1.3 ppb.

ACCURACY

The accuracy of the ELISA method was tested using fortified corn leaves and
fish samples. Fish samples were spiked at the levels ranging from 5.5 ppb to 880 ppb
and corn leaves samples were spiked at the levels ranging from 5.5 to 2200 ppb and
assayed using the two ELISA kits.

A limiting factor in the use of ELISA kits for pesticide residue analysis is their
requirement for water miscible sample extract. It was therefore necessary to use water
miscible solvents for extraction. Acetonitrile and acetone were chosen because of their
ability to efficiently extract alachlor from fish and corn leaves respectively. During this
study the effects of corn leaves and fish coextracts as well extraction solvent on the

determination by the kits were assessed.

FISH FILLET

Non-diluted acetonitrile (ACN) extract was found to give a positive response to
the kit; the recovery was between 135 to 167% and 141 to 177% for the RaPle and
EnvriroGard° kits respectively (Table 5). This response enhancement must have been due
to either sample matrix or ACN interference. To eliminate this enhancement of the

readings, the samples were diluted 10 times. The recovery found ranged from 103 to

115% for the RaPIDTM kit and 91 to 116% for EnvriroGard’ kit (Table 5). When the

125

Table 5. Accuracy of alachlor determination in spiked fish fillets (2 replications per
assay). A: Ohmicron kit; B: Millipore kit.

 

Spiking Levels (ng/g) *Assay 1 "Assay 2 "*Assay 3

 

 

 

5.5 127 106 nd

11 122 120 108

'22 126 113 98

44 113 102 92

110 nd 103 103

154 nd 116 95

Mean 112.75 97.25 99.20
SDV 7.68 4.35 6.40
%CV 7 4 6

 

 

Spiking Levels (Lg/g) *Assay 1 "Assay 2 l""""Assay 3

 

 

 

22 137 91 nd

55 128 111 nd

82.5 121 114 nd

.165 119 86 101

275 129 111 96

550 114 102 106

880 112 115 90

1100 139 nd nd

2200 89 nd nd

Mean 120.89 99.40 98.25
SDV 15.20 5.46 6.80
%CV 13 5 7

 

*Dilution 1:10; no decoloration; provided standard
"Dilution 1:10; decoloration; provided standard
"*Dilution 1:50; decoloration; provided standard

stande used in the assay was prepared in 10% ACN in water, the recovery found was

in the range of 90.7-100.7% for The RaPID‘" kit and 91 - 108% for EnvriroGard® kit.

126

Thus diluting the extract reduced the effect of matrix without eliminating the positive
response due to the organic solvent. The solvent effect was eliminated by using a

standard prepared in 10% ACN in water.

C RNLEAF

The assay of the undiluted sample extract gave a recovery between 137-187%.
As in the previous case (Fish), this high enhancement of the response must be explained
by sample matrices or solvent interference. Diluting the extract 1:10 with water fairly
reduced the enhancement effect yielding a recovery of 118-127% as reported in Table 6.
When the extract was decolonized by mixing the extract with decolonizing agent and
diluted to times, the enhancement previously observed was markedly reduced (recovery

106-120%).

When the extract was diluted 1:50 after decoloration, the response enhancement was
eliminated and the recovery was in the normal range (95-108%). The results showed the
positive response due to the pigments (carotenoids and xanthophiles) which was
eliminated by decolonizing the extract.

The recovery results ranged from 98 to 110% (fish) and 92 to 108% (com leaves)
for the Ohmicron kit and 91 to 117% (fish) and 90 to 106% (com leaves) for Millipore
kit (Table 6). In both fish sample and corn leaves extraction, the recoveries obtained are
good. They seemed to be higher for fish samples compared to corn leaves sample. This
enhancement may come from the fish matrix since the fish extract was diluted only 10x

while corn leaves extracts were diluted 50x.

127

Table 6. Accuracy of alachlor determination in spiked corn leaf (2 replications per assay).
A: Ohmicron kit; B: Millipore kit.

 

 

 

 

 

 

 

 

Ohmicron
Spiked Levels (nflg) I"Assay 1 "Assay 2 "*Assay 3
5.5 127 106 nd
1 l 122 120 108
22 126 1 13 98
44 1 18 102 92
l 10 nd 108 103
154 nd 1 16 95
Mean 123.25 1 10.83 99.20
SDV 4.1 6.7 6.4
% CV 3% 6% 6%
* Dilution 1:10; No decoloration; Standard provided
" Dilution 1:10; Decoloration Standard provided
"* Dilution 1:50; Decoloration; Standard provided
Millipore
Spiked Levels (ng/gL l"Assay l “Assay 2 "*Assay 3
22 137 91 nd
55 128 1 1 1 nd
82.5 121 1 14 nd
165 1 19 86 101
275 129 1 1 1 96
550 1 14 1021 106
880 1 12 l 15 9O
1 100 139 nd nd
2200 89 nd nd
Mean 120.89 235.57 98.25
SDV 15.2 346.5 6.8
% CV 13% 147% 7%

 

" Dilution 1:10; No decoloration; Standard provided
" Dilution 1:10; Decoloration Standard provided
"" Dilution 1:50; Decoloration; Standard provided

128

Table 7. Assay reproducibility (%CV) for of the EIA for alachlor in corn leaf extract.

 

 

 

 

 

 

 

 

 

 

Sample ‘Intra-Assay " ‘Inter-Assay
Standard solutions (ppb)

0.0 4.3 2.9

2.0 5.1 5.9

10.0 6.9 7.9

20.0 6.1 9.3

100.0 9.5 8.9

Mean 6.38 6.98

Spikes (Hg/g)

55.0 1 1.1 9.4

165.0 5.1 10.7

550.0 ' 4.7 12.1

Mean 7.00 10.70

Sample ‘Intra-Assay "Inter-Assay
Standard solutions (ppb)

0.0 3.0 2.9

0.1 7.9 5.9

1.0 6.4 7.9

5.0 5.9 9.3

Mean 5.8 6.98

Spikes (118/8)

5.5 3.0 6.7

11.0 7.9 7.9

44.0 6.4 9.0

110.0 . 5.9 6.7

154.0 5.7 5.6

Mean 5.80 7.20

%
‘6 assays Within 1 day

"'1 assayperdayduring6days

129

As with any analytical technique, precision within and between days is
important. The reproducibility of the extraction technique as well as the ELISA test were
determined by performing 3 and 6 replicates assays were performed respectively on
standard solutions provided with both ELISA kits and on spiked corn leaves samples.
Within-day coefficient variation of 4.3-9.5% (standards) and 4.7-ll.1 (spiked samples)
were obtained for the Millipore kit and 3.0-7.9% (standards) and 3.0-7.9 (spiked samples)
for Ohmicron kit (Table 7). In general the coefficient of variation of Millipore kit are
higher than those of Ohmicron kit.

Between-day assays were performed at 3 days and 6 days successively for
Millipore and Ohmicron kits respectively. The results showed a coefficient of variation
of 2.9-12% for Millipore kit and 4.1-9.0% for Ohmicron kit (Table 7). As the results
indicated there is no big difference between the two types of kits. Both kits showed
slightly higher variation with inter-days tests compared to between-day assays, but in
general the coefficients of variation were the ranges recommended by the manufacturers

(%CV < 12%).

CROSS-REACTIVITY

Alachlor belongs to a family of structurally similar chloroacetanilide herbicides.
Specificity of the antibodies for alachlor was therefore crucial for the successful
application of this assay to residue analysis. Studies were conducted to determine the

specificity of the antibodies for alachlor. Both alachlor kits rely on antibodies which are

130

Figure 10. Response of the ELISA kits to alachlor and metolachlor. (D) alachlor; (I)
metolachlor; (0 ) mixture.

A. Ohmicron test kit

 

% Bo

 

l 10 100 1000

Concentration (ppb)

 

 

 

B. Millipore test kit

 

% Bo

 

Concentration (ppb)

 

 

 

131

specific to alachlor. In order to measure cross reactivity between alachlor and its related
compounds, metolachlor, acetolachlor, and propachlor, corn leaf extracts were spiked at
various levels of alachlor, acetolachlor, and propachlor separately. The preliminary results
showed no detection of propachlor at reasonable levels. With both EIA test kits, the
response of metolachlor and acetolachlor were significantly lower than that of alachlor
(Figure 10). The presence of metolachlor and acetolachlor produced only a small
enhancement to alachlor response. The response of metolachlor and acetolachlor were
found to be respectively 7% and 1.7% of that of alachlor with Ohmicron kit, and 18%
and 7.3 % with Millipore kit. Feng et al. (1990) have found cross reactivities lower than
those found in this study but their study used antibodies developed by themselves which
may differ in affinity. The cross-reactivities of the test kit for chemical analogs may due
to how a chemical is conjugated to the IgG in the immunization antigen. The exact
structure of the alachlor- immunogen used to produce the polyclonal antibodies used in
this kit is proprietary, however, it must be similar in structure to those used by other
investigators performing ELISA quantitation of alachlor residues (Feng et al., 1994).
Only metolachlor, acetolachlor and propachlor were investigated in this study because
their are the most widely used chloroacetanilide herbicides

Other chloroacetanilides, mainly those containing a thiolether functional group may
have greater cross-reactivity with the assay because of the thioether linkages in the
immunogen antigen. These compounds were not investigated because they are not widely
used. No metabolite was investigated in this study because they are not sold

commercially and we were not able to obtain them from the manufacturer.

1 3 2
SENSITIVITY

Table 8. Method determination limit (MDL) calculated from standard deviation (0) of 6
replicate assays of corn leaf (A) and fish tissue (B) spiked with alachlor.

 

 

 

 

 

 

 

 

 

A. Corn leaf
Ollnicrontoltkit | . Mimic-tut

Assay# Abst Asz Mean %Bo wept] All! A1332 m %Bo Concentration”)
Control 1.700 1.612 1.656 1.675 1.625 1.650

1 1.040 1.117 1.079 65 0.530 1.432 1.412 1.422 36 1.700

2 0.995 1.126 1.061 64 0.563 1343 1.377 1.360 32 2150

3 1.107 1.034 1.071 65 0.544 1.266 1.234 1.250 76 3.240

4 1.130 0.939 1.035 65 0.51 1.332 1.355 1.369 33 2690

5 1.223 1.040 1.132 63 0.443 1.523 1.544 1.534 93 1.120

6 0.936 1.050 1.013 61 0.592 1.243 1.320 1.232 73 2330

Mean 65 1M... 33 2.297

Stdv 222 0.05 $th 6.17 0.79

3@ 6.66 0.152 39 13.50 2.330

B. Fish fillet

Mounts-tut I 1131111151..th

Assayfl A1131 m2 Mm %Bo WWI Abtl A1332 Meat %Bo Concentration”)
Control 1.412 1.377 1.395 1.745 1.721 1.733

1 0.376 0.356 0.366 62 0.54 1.465 1.444 1.455 34 2330

2 0.912 0.397 0.905 65 0. 1.432 1.337 1.410 31 2.730

3 0.354 0.366 0.360 62 0.55 1.435 1.476 1.456 34 2.320

4 0.343 0.900 0.372 62 0.53 1.373 1.427 1.403 31 4.040

5 0.335 0.921 0.373 63 0.515 1.437 1.466 1.477 35 2.150

6 0.900 0.372 0.336 64 0.493 1.366 1.354 1.360 73 3.260

Mean 63 0.51 32 2.305

Stdv 1.15 0.03 250 0.72

E 3.44 0.103 Q 7.51 2175

 

Method detection limit for each kit was evaluated from standard deviation (6) of

6 replicates of fish and corn leaves samples spiked with alachlor standard solution. With

133
both test kits, the minimum detectable levels (MDL) (Table 8) obtained were slightly

higher than the lower limit of quantification (LOQ) which were 2 ppb and 0.1 ppb for

EnviroGard° kit and RAPID" kit respectively.
INCURRED SAWLES

Fish fillets and corn leaves coming form fish and corn raised in the greenhouse
and treated with alachlor were analyzed using the two ELISA kits and gas
chromatography. Because of inhibition due to the extraction solvents and matrices
interference, the extracts were diluted 1:10 and 1:50 for fish and corn leaves respectively

prior to their analysis.

W

No alachlor was found in the non-treated samples as well as the samples collected
the day before the herbicide application (Figure 11). Both kits detected alachlor at similar
levels (0.02 ug/g - 0.6 ug/g) in the samples and the levels found were higher in the
samples collected the day of treatment and decreased on subsequent days. The decrease
in the amount found may be explained by the metabolism of alachlor in corn.

Because some of the metabolites of alachlor may cross react with the detection of
alachlor by the ELISA kits, GC and GC/MS were used to the evaluate results obtained
by the kits. Gas chromatography equipped with electron capture detector (ECD) was used

to analyze the samples. The conditions of analysis are described in the method section.

134

Figure 11. Alachlor concentration in incurred corn leaf samples by ELISA. 2 assays per
samples.

DBT = Day before treatment; DOT = Day of treatment; DAT = Day of treatment

NT = Non treated; Trt = treatment

Ohm = Ohmicron; Mill = Millipore

 

0.5

   
  
  
         

% 0.4
5 T11 33 Mill
E 0 3 . Tn #3 on...
c ’ Tn #2 Mill
In
g 0 2 Tn 32 Ohm
Trt 31 Mill
0,] Trt #1 Ohm

. NT Ohm
1 DBT DOT . -
5 DAT 7 DAT

11 DAT

Samplig Day

 

 

 

135

Figure 12 illustrates the correlation obtained when plotting the results of the ELISA tests
against GC determination. The results obtained showed a good correlation between GC
and the two kits with Ohmicron kit giving a slightbetter correlation coefficient (R2 =
0.997) compared to the Ohmicron kit (R2 = 0.996).

In plants, alachlor is first conjugated with glutathione (GSH) and/or
homoglutathione (hGSH) and later metabolized to malonylcysteine conjugate (Breaux et
al., 1987) Therefore, there are no metabolites structurally close to alachlor. The
chromatographs obtained with GC analysis contained different peaks beside alachlor peak
which might correspond to coextracts from plant materials. The results obtained from the
ELISA test kits correlated very well with the Ge determination. From this I can assume
that there is no cross reactivity at the levels obtained from any metabolite or any plant
component and therefore the ELISA kits can be successfully used for the quantification
of alachlor in plant material.

Confirmation of the incurred corn leaves was accomplished using GC/MS. As
illustrated in Figure 13 the mass spectrum of cut 2 (Treatment #3) collected at 7 days
after alachlor application yielded the molecular ion at 269 m/e and the fragments at 237,
146, and 160 m/e which were seen in the standard. The non treated sample did not yield
the molecular ion nor the fragments seen in the standard. These results confirmed that
alachlor was present in the samples for which the ELISA kits as well as GC

determinations were positive.

136

 

 

 
     

 

 

 

   

 

 

 

 

 

 

 

 

 

0.3
’03 = -
h 0.6 .. y 1.0175x 0.0005
5 R = 0.9966
8 0.4 ..
>3
.4:
E 0.2 --
0 . . ; i .
0 0.1 0.2 0.3 0.4 0.5 0.6
Level by Ohmicron Kit
(Ila/a)
0.6
y = 1.01275: + 0.0083
0.5 -- ,
a R - 0.9956
E
5
>.
.e
i
O t 2 t t .
0 0.1 0.2 0.3 0.4 ' 0.5 0.6
Level by Millipore Kit (rig/g)

 

 

 

Figure 12. Correlation of alachlor concentrations as determined by ELISA and go
methods, n=4, r = 0.996 , y = 1.017 X + 0.0005.

137

 

 

..
0.. .U
R. x. .. .U
«H. / . ...J B
./ . g
L n.
—./r . in”
...J ./..I 4. w
.3 a
S
.U a“
l. .U in...
C7.
1.. /.
/Il|l.|.|.
nut-rum
Q‘-
C.”. 4.4
(.1..- 4. t..ll.
0 Ag
1.18
S.
\\ 4.

 

GU Co 4. n:

 

\R...

\ 4. ...
\ ........... .-

 

....w

 

'lli’llll'l'lllll

:1.
E
Q

a

...u

to Ag
33 S
.. Go
“.3
a
in.”
P...
/..
3
a” C
.. ..w ..M
oJl— Mr
..U
I

....mucwwcjac.. ..J

 

 

Figure 13. Confirmation of the ELISA results by GC/MS.

FISH FILLET

138

Apart from one sample (fish #3 of treatment #2-1), no alachlor was found in any other

sample by both ELISA kits. The GC determinations showed no alachlor indicating that

there is no cross reactivity to the ELISA kits from any metabolite that might be present

in the samples nor any fish component. Because of its low detection limit, the Ohmicron

kit was able to detect alachlor at the level of 0.007 ppm in one fish.

Table 9. Alachlor concentration in incurred fish fillet samples by ELISA. 2 assays per

 

 

 

 

 

 

 

 

  
  
   
    

 

 

  

 

 

 

              

samples.
Sample Non Treated #1 Treatment #1-1 Treatment #2-1
Ohm Mill GC Ohm Mill GC Ohm Mill GJ
Fish 1 nd nd nd nd nd nd nd nd nd
Fish 2 nd nd nd nd nd nd 0.006 nd nd
Fish 3 nd nd nd nd nd nd nd nd nd
Fish 4 nd nd nd nd nd nd nd nd nd
Fish 5 nd nd nd nd nd nd nd nd
ID Non Treated #2 Treatmentjifz Treatment #2-2
Ohm Mill GC Ohm Mill GC Ohm Mill GC

Fish 1 nd nd nd nd nd nd nd nd nd
Fish 2 nd nd nd nd nd nd nd nd nd
Fish 3 nd nd nd nd nd nd nd nd nd

' nd nd nd nd nd nd nd nd nd

     

       

 

nd

 

nd nd

 

 

 

nd

 

nd

      

nd

  

 

 

     

 

nd nd nd

139
ATRAZINE

Blanks and calibration standard assays results are summarized in Appendix B. The
linearity range of the Millipore and Ohmicron kits were 0.05-5 ppb and 0.25-10 ppb
respectively (Figure 14). These results showed that Ohmicron kit was slightly more
sensitive than Millipore kit. These results indicated also that the Ohmicron assay can
detect very low concentrations (~ 0.05 ppb), while the Millipore kit can detect higher

concentrations (~ 15 ppb).

ACCURACY

Corn leaves and fish fillets were spiked with standard solutions, extracted and
analyzed with the ELISA kits. The accuracy of the determinations were assessed by
calculating the percent recoveries. The results are summarized in Appendix B. Strong
matrices and solvent interferences were observed with the assays detections during the
recovery study.

CORN LEAF

In the first two assays, the standard provided by the manufacturer was used.
When the extract was diluted 1:10, the recoveries were very high indicating inhibition
due to either coextracts or the extraction solvent. Diluting the extract 1:50 slightly
reduced the enhancement observed in the first assay. A standard prepared in 10%

methanol in water was used in the third assay.

140

Figure 14. Plot of the standard curves of the two test kits (average of 4 determinations).

%Bo = % (absorbance of the sample/absorbance of the zero control).

 

100.00

0

1

0

1

0.

.01

0

on (ppb)

Concentrati

141

As the results (Table 10) indicated, the inhibition due to methanol as extraction solvent
was reduced but still the recoveries were above the normal range indicating interferences

due to sample matrix.

Table 10. Accuracy of Ohmicron (A) and Millipore (B) test kits for the determination
atrazine in spiked fish fillets (2 replications per assay).

 

 

 

A. Millipore
Spiking Levels (nng) *Assay l "Assay 2 "*Assay 3 ""Assay 4
2.5 176 173 nd nd
25.0 141 132 121 102
50.0 154 122 118 96
100.0 138 116 133 98
Mean 152.3 135.8 124.0 98.7
SDV 17.29 25.70 7.94 3.06
%CV 11% 19% 6% 3%

 

* Dilution 1:10; Standard provided

" Dilution 1:50; Standard provided

‘" Dilution 1:50; Standard in 10% Methanol in water
“” Dilution 1:50; Standard in corn leaf extract

 

 

 

B. Ohmicron

Spiking Levels (ngjg) ‘Assay 1 "Assay 2 "*Assay 3 I""“'”"Assay 4
0.7 123 108 98 104
1.0 125 119 91 91
1.5 151 109 116 99
2.0 136 129 120 93
4.0 117 98 100 97
7.0 122 123 123 89

Mean 129.0 114.3 108.0 95.5

SDV 12.47 11.38 13.31 5.58

%CV 10% 10% 12% 6%

 

’ Dilution 1:10; Standard provided

" Dilution 1:50; Standard provided

“* Dilution 1:50; Standard in 10% Methanol in water
"” Dilution 1:50; Standard in corn leaf extract

142

In the fourth assay, 5 grams of untreated corn leaves were extracted with methanol. The
extract diluted 100 times was kept in the refrigerator and used to prepare the standard
solutions. The calibration standard solutions were prepared by adding the appropriate
amount of atrazine standard solution solutions (10% methanol in water) to an aliquot of

this extract. With this standard, the inhibition due to matrix interferences was eliminated.

FISH FILLET
The preliminary results indicated that when the extracts were diluted 1:10 and
analyzed using the standards provided by the manufacturer, recoveries above 100% were

obtained (Table 10).

Table 11. Accuracy of Ohmicron (A) and Millipore (B) test kits for the determination
atrazine in spiked fish fillets (2 replications per assay).

 

 

 

 

 

Millipore Ohmicron
Spiking Levels (“fl *Assay 1 "Assay 2 *Assay 1 “Assay 2
0.7 nd nd 122 104
1.0 129 100 124 97
1.5 1 1 l 109 118 100
2.0 115 94 106 99
4.0 119 96 110 95
7.0 122 105 134 94
Mean 119.2 100.8 119.0 98.2
SDV 6.87 6.22 10.10 3.66
%CV 6% 6% 8% 4%

 

 

‘ Dilution 1:10; Standard provided
“ Dilution 1:10; Standard in 10% ACN in water

143

When the standard used was prepared in 10% ACN in water, the mean recoveries were
101% and 98% for Millipore and Ohmicron respectively. These results suggested that
acetonitrile can be used as extraction solvent for the ELISA determination but the
standard used in the assay should contain a portion of acetonitrile in order to eliminate
any inhibition of the detection which may result in false positives.

The overall results showed that in general more matrices interferences occurred
in the assay with the Millipore kit compared to the Ohmicron kit. These results showed
good recoveries for both kits ranging from 94-109% to 94-104% for the Millipore and
Ohmicron kits respectively. The results indicate that there is no significant difference

between the two kits.

REPRODUCIBILITY

The results of reproducibility studies are summarized in Appendix B. In this study,
6 replicate assays were performed in one day, on standard solutions provided with the kits
for the determination of within-day coefficient of variation. For the determination of the
between-day coefficient of variation, duplicate assays were performed on 6 successive
days on the same standard solutions. The same assays were performed on corn leaf
extracts spiked with standard solutions. Both within and between-assay coefficient of
variation were good (Table 12). There was no difference between the two types of kit.
The results indicated between-assay coefficient of variation slightly higher than within-

assay coefficient of variation.

144

Table 12. Assay reproducibility for of the EIA for atrazine in corn leaf extract

 

 

 

 

 

A. Millipore
Sample I"Intra-Assay "Inter-Assay
Standars Solutions (ppb)
0.0 4.1 7.6
0.5 5.1 ' 5.9
2.0 8.0 8.6
10.0 83L 7.7
Mean 6.5 7.5
Spikes (ng/g)
0.5 5.9 12.9
1.0 9.2 21.0
2.5 8.9 12.0
5.0 7.6 13.1
10.0 10.4 13.5
Mean 8rd 13.3

 

* 6 assays within 1 day
"”" 1 assay per day during 6 days

 

 

 

 

 

 

B. Ohmicron
Sample *Intra-Assay MInter-Assay
Standars Solutions (ppb)
0.0 4.8 5.6
0.1 3.5 6.1
1.0 4.1 5.2
5.0 8.3 9.9
Mean 5.175 6.7
Spikes (ng/g)
6.56 7.7 13.3
32.80 4.2 8.3
65.60 3.0 10.4
31.20 6.1 16.7
Mean fl 12.2

 

* 6 assays within 1 day
** 1 assay per day during 6 days

145

CROSS-REACTIVITY

Atrazine, molecular weight 269, is too small to be immunogenic in its own right.
For this reason, atrazine hapten was obtained by derivatizing atrazine at the 2-chloro
position which was later conjugated to bovine gamma globulin through a modified
carbodiimide by cross-linking procedure (Bushway et al., 1988). The procedures used to
prepare the kits used in this assay was not known for proprietary reasons but they must
follow the same scheme. Because of the way the hapten is synthesized, the antibodies
produced may cross react with compounds closely related to atrazine or metabolites of
atrazine.

To assess the cross-reactivity of metabolite and related compound to the kits,
assays of atrazine, simazine and hydroxyatrazine were carried out. The results are
reported in Appendix B. The response of simazine and hydroxy-atrazine were
significantly lower than the atrazine response (Figure 15). The results of Millipore kits
were based on 4 assays of 3-point calibration curves, whereas the Ohmicron results were
from 4 separate experiments. The reason that the LDD does not correlate with the cross-
reactivates is due to the less steep slopes of the metabolite hydroxy-atrazine versus the
greater atrazine sensitivity. The fact that hydroxy-atrazine concentration at 50% Bo was
more removed from atrazine response than 90% Bo.

The results indicate that simazine as well as hydroxy-atrazine cross reacted more
with Millipore kit than with Ohmicron kits; and the LDD's for Ohmicron kits are lower

than those of Millipore kits. The overall results indicate the possibility of simazine to

146

Figure 15. Response of the ELISA kits to atrazine (D), hydroxyatrazine (I), and simazine
(0 ).

%Bo
100

 

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cross react with both kits; this might be a drawback for the kits when samples to be
assayed contained simazine which is another s-triazine herbicide used extensively in
agriculture. Using Ohmicron kits reduced to a certain extent the possibility of the cross-
reactivity of simazine. Simazine as a related compound and hydroxy-atrazine as a
metabolite represent a few of compounds which might cross-react with the test kits, but
many more related compound (cyanazine, propazine, etc.) are used as herbicides sometime
in the same crops and other metabolites (DEA, etc.) occur in plant which may cross-react

with the test kit.

SENSITIVITY

Determination of the method detection limit (MDL) was accomplished by spiking
fish fillet and corn leave samples with the lowest concentration used in the recovery
studies. Eight replicates of these spiked extract were assayed; the results are summarized
in Appendix B. The MDL for Ohmicron kits were 0.27 ng/g and 0.20 ng/g for corn
leaves and fish fillet samples respectively, whereas the MDL for the Millipore kits were
1.97 ng/g and 0.55 ng/g respectively (Table 13). The MDL for the Ohmicron kits were
lower than the MDL for Millipore kits and therefore more sensitive. This can be
explained by the fact that com extract interfered more with the kits detection compared

to fish extracts.

148

INCURRED SAMPLES

LEAF

Corn leaves samples obtained from corn plants grown in the greenhouse and
sprayed with atrazine were assayed with both kits. The two test kits detect similar
amounts of atrazine, but only the Ohmicron kits were able to detect very low levels (0.04-
.06 ng/g) (Figure 16). This can be explained by the fact that Ohmicron kits (MDL =
0.27 ng/g) compared to Millipore kits (1.97 ng/g).

In order to verify the accuracy of the determination by both ELISA kits, corn
leaves samples were extracted according to the procedure described in the method section,
and analyzed by GC/NPD. The results are reported in Appendix B. The results showed
that there is good correlation (R2 = 99) between the ELISA kits and the GC
determinations (Figure 17). These results indicate that the ELISA kits can be used for

qualitative as well as quantitative determination of atrazine in plant materials.

FISH SAMPLES

Incurred fish samples were also analyzed by the ELISA kits. The results are
reported in appendix B. No atrazine was found in any of the extracts by either the
Millipore kit nor GC; only the Ohmicron kits, because of their high sensitivity, detected

atrazine at the levels ranging from 0042-0118 ug/g in two fish (Table 14).

149

 

Level Found (ng/g)

 

Sampling Day

 

 

 

Figure 16. Atrazine concentration in incurred corn leaf samples by ELISA. 2 assays per
samples.

150

 

 

 

 

 

 

 

 

 

 

 

 

y - 0.94671: + 0.0442
- 11’ -0.9901
1.50

A 1.20 ~-

E

e.
3
D 0.90 ~-
0

>.
-° 0.60 4»
.2

3

0
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0.00 4 ; . t
0.00 0.30 0.60 0.90 1.20 1.50
Levels by Millipore kit (ppm)
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13’ - 0.993
1.60

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3'

U

a 0.30 -

.o

733

:3 0.40 i

0.00 : 4
0.00 0.50 1.00 1.50
Levels by Ohmicron kit (ppm)

 

 

 

Figure 17. Correlation of atrazine concentrations as determined by ELISA and go
methods, 11 = 4, r = 0.996 , y = 1.017 X + 0.0005.

151

Table 14. Atrazine concentration in incurred fish fillet samples by ELISA. 2 assays per
samples.

ID Non Treated #1 Treatment #1-1 Treatment #2-1
Ohm Mill GC Ohm Mill GC Ohm Mill GC

 

 

 

Fish 1 nd nd nd nd nd nd nd nd nd
Fish 2 nd nd nd nd nd nd nd nd ndl

Fish 3 nd nd nd nd nd nd nd nd nd
Fish 4 nd nd ’
Fish 5 nd nd __ _ u ‘

 

 

 

 

 

 

 

 

 

 

 

 

 

ID Non Treated #2 Treatment #1-2 Treatment #2-2
*Ohm "Mill "*GC Ohm Mill GC Ohm Mill GC
Fish 1 nd nd nd nd nd nd nd nd nd
Fish 2 nd nd nd nd nd nd nd nd nd
Fish 3 nd nd nd nd nd nd nd nd nd
Fish 4 l nd nd nd nd nd nd nd nd nd
Fish 5 nd nd nd nd nd nd nd nd nd
" Ohmicron test kit
" Millipore test kit

"* GC determination

152

. Samples and standard solution of atrazine were run on GC/MS in order to confirm
the results obtained by the ELISA kits. The mass spectrum of atrazine standard yielded
the molecular ion at 215 m/e of atrazine and the fragmentation at 43, 93, and 200
characteristic of atrazine (S-triazine) (Figure 17). The same patterns were seen with the
following samples: cut 2 (Trt #3) and F2 (Trt #2-1) which were found to be positive for
atrazine by the kits and GC. The non-treated samples did not present the same pattern.

These results confirmed that the ELISA kits can successfully used for the
determination of atrazine in weathered environmental samples and food provided that

interference due to matrices and solvent inhibition be eliminated.

153

 

 

Hbundance

 

 

 

 

 

 

n:

p.“
O

 

HhUndance

a... L__ m n It_ L g g I

 

 

a I‘L

 

Figure 18. Confirmation of the ELISA results by GC/MS.

154
CARBOFURAN

Figure 19. Plot of the standard curves of the two test kits (average of 4 determinations).
%Bo = % (absorbance of the sample/absorbance of the zero control).

 

%Bo

o Ohmicronl

 

 

a Millipore

 

 

0.010 0.100 1.000 10.000

Concentration (ppb)

155

Standard solutions prepared in 10% methanol in water were assayed during the
study. The concentrations ranged from 0.07 ppb to 6.24 ppb. Two replicate assays were
performed (in duplicate) for the Millipore kits whereas 3 replicate assays (in duplicate)
were performed with the Ohmicron kits. The results are reported in Appendix C. Both
ELISA kits present a linear range between 0.312 ppb and 4 ppb (Figure 19). In this
range, the slope of the Millipore kits was slightly steeper than the slope of the Ohmicron
kits but R2 is the same for both kits. The least detectable dose (LDD) calculated as 90%

were similar and equal to 0.3 ppb.

ACCURACY

FISH FILLET

Because of the solubility of carbofuran in water, the extraction of the fish samples
was accomplished with water and acetonitrile for comparison purpose. For water
extraction, 5 grams of fish fillet were grounded with 10 grams of anhydrous sodium
sulfate; the powder obtained was homogenized with 50 ml water. After 3 to 5 minutes.
an aliquot of the supernatant was collected for the assay. In case of acetonitrile
extraction, the sample was grounded with 10 grams of anhydrous sodium sulfate and
homogenized with 50 ml acetonitrile. After decantation, an aliquot of the extract was

collected for the assays.

The preliminaries assays, without dilution, gave very high recoveries (> 177%)

156

for both extraction solvents. This enhancement observed with the kits indicated

Table 15. Accuracy of Ohmicron (A) and Millipore (B) test kits for the determination
carbofuran in spiked fish fillets (2 replications per assay).

% Recovery

 

3.28
5.65
16.4 32.8

Spiking Levels (ng/g)

III—
II‘

 

— ACN extraction Assay #2
ACN extraction Assay #1
Water extraction Assay #2

Water extraction Assay #1

157

interference due to matrices and/or extraction solvent inhibition. Subsequently, the
extracts were diluted for the assays. The results are reported in appendix C. As the
results indicate (Table 9), when the extracts obtained were diluted 10 times with distillate
water, the enhancement observed was markedly reduced for water extracts (112-122%
recovery) compared to acetonitrile extract (104-152% recovery). Diluting the extracts 50
times eliminate the enhancement in both extracts but the recoveries with water extracts
were low (70-90%) compared to acetonitrile extracts (104-107%).

These results can be explained by the fact that when the samples are extracted
with water, fat and fish components which make the extracts cloudy, were allowed to
settle out thereby reducing their interference with the assay. For this reason a 10x
dilution resulted in a marked decrease of the enhancement seen when the sample was not
diluted. The fact that 50 times dilution resulted in poor recoveries indicates possible loss
of the pesticide during extraction. Water as polar solvent cannot successfully extract
pesticides which tend to partition in the fat. Acetonitrile extracts have a high fat load
which causes the high enhancement observed with 10 times dilution which was eliminated

by diluting the extract 50 times.

W

Corn leaf samples free of any pesticide were spiked with a series of standard
solution of carbofuran according to the procedure described in the method section. The
extract were assayed directly using the Ohmicron test kits; the reading yielded recoveries

in the rage of 170-180 indicating interference due to coextracts. The results are

158

summarized in appendix C.

To eliminate the enhancement observed in the preliminary assay, the extracts were
diluted 50x with distilled water and assayed. The recoveries obtained ranged from 115-
137% (Table 16). This dilution markedly reduced the enhancement observed in the
preliminary assay but did totally eliminate the interference observed.

In the following two assays, the extract were decelerate to remove the green color
observed and diluted 50x and 100x respectively. When the discolored extract was diluted
50 times, the enhancement was reduced but the recoveries were still high (105-124%).
The enhancement was completely eliminated when the discolorized extracts were diluted
100x; the recoveries obtained ranged from 90 to 108%.

The results of this study indicate that plant coextract (pigments and others)
interfered with the ELISA detection. In order to eliminate this interference one must only
discolor the extracts but dilute the extract 100 times. The extractions were done using
0.25N HCl solution which may have inhibited to a certain degree the detection
contributing to the high enhancement observed during the preliminary assays. These
results showed that the Ohmicron kits can quantitatively be used to determine carbofuran
in plant material provide that matrices interference’s and/or extraction solvent inhibition
is eliminated.

The Millipore kits were not tested in this study because the kits were provided
with only two standard solutions: 0.2 and 5 ppb. No 2-point calibration curve can be

used for qualitative determination.

159

Table 16. Accuracy of Ohmicron (A) and Millipore (B) test kits for the determination
carbofuran in corn leaf (2 replications per assay).

 

 

I Assa y #1
IAssay #2
DAssay #3

% Recovery

 

 

 

 

 

 

15.62 39.00 117.00 195.00 234.20
Spiking Levels (ng/g)

160

REAPITABILITY

Table 17. Assay reproducibility for of the EIA for carbofuran in corn leaf extract

A. Millipore test kit

 

 

 

 

Sample *Intra-Assay "Inter-Assay
Standard Solutions (ppb)
0.0 5.0 5.7
0.2 4.2 5.9
5.0 8.9 9.5
Mean 6.0 7.0
Spikes (ng/g)
32.8 3.9 7.6
65.6 7.4 7.9
312.0 10.8 11.0
Mean 7.4 8.8

 

 

B. Ohmicron test kit

 

 

 

 

 

Sample *Intra-Assay "Inter-Assay
Standars Solutions (ppb)
0.0 1.3 5.0
0.1 3.1 4.0
1.0 4.0 6.0
5.0 7 .6 6.6
Mean 4 5.4
Spikes (03/3)
6.56 7.7 8.5
32.80 4.2 6.8
65.60 3.0 8.1
31.20 6.1 9.9
Mean 5.0 7.7

 

* 6 assays within 1 day
** 1 assay per day during 6 days

161

Standard solutions of carbofuran and spiked samples were assayed in order to
determine how much variation are introduced in the determination from assay to assay or
from day to day. For both test kits, 6 assays were conducted under the same conditions
to determine the within-day variation, and one assay was conducted for six consecutive
days for between-day variation determination. The results are reported in Appendix C.

The recoveries ranged from 3.9-10.8 (within-day) to 5.7-11% (between-day) for
the Millipore test kits and 1.3-7.7% (within-day) to 4.0-9.9% (between-day) for the
Ohmicron kits (Table 17). Within-day total coefficient of variation were determined to
be 6.65 and 4.6% for the Millipore and Ohmicron kits respectively, whereas they were
determined to be 8.1 and 6.9% respectively. The results showed also that variations were
more pronounced with the spiked samples compared to the standard solutions. This can
be explained by variations introduced during the different steps of extraction and
principally in dilution steps.

The overall results showed coefficient of variation for the assays of less than 12%

which is very good for qualitative determination.

SENSITIVITY

Sensitivity of the ELISA kits was assessed using Ohmicron test kits. Corn as well
as fish samples were spiked at 3.28 ng/g and 15.62 ng/g respectively, extracted and
assayed after dilution. Six replicate assays were done for both kits; the results of the

study are reported in Appendix C. The method detection limit (MDL) of the test kits

162

Table 18. Method determination limit (MDL) calculated from standard deviation (0) of
6 replicates assays of corn leaf (A) and fish tissue (B) spiked with carbofuran.

 

 

 

 

 

 

 

A. Corn leaf
Assay # Abs 1 %Bo Concentration (ppb)
Control 1.336

1 1.321 99 0.067
2 1.242 93 0.108
3 1.117 84 0.176
4 0.967 72 ' 0.317
5 1.105 83 0.185
6 0.845 63 0.512

Mean 82 0.228

Stdv 13.05 0.163.

3@ 39.16 0.5

B. Fish fillet

Assay # Abs 1 %Bo Concentration (ppb)

Control 1.346

1 1.212 90 0.121
2 1.180 88 0.138
3 0.997 74 ' 0.282
4 1.210 90 0.122
5 0.955 71 0.333
6 1.275 95 0.095

Mean 85 0.182

Stdv 9.66 0.100

3@ 23.99 0.30

163
were 0.5 ng/g and 0.3 ng/g for corn leaves and fish sample respectively (Table 15).

These values fall in the range of linearity of the standard solutions provided with the test

4kits and are well above the lowest standard solution (0.1 ppb).

CROSS-REACTIVITY

Carbofuran is metabolized by hydroxylation and hydrolysis in plants and the
major metabolites are 3-hydroxy-carbofuran and 3-keto-carbofuran (Metcalf et al. 1968).
These metabolites may cross react with the detection by the ELISA kits. For this reason,
fish samples were spiked separately and in a mixture with standard solutions of
carbofuran, 3-keto, and 3-hydroxy-carbofuran. The results of the assays a summarized
in Appendix C. The response of the metabolites (3-keto-carbofuran and 3-hydroxy-
carbofuran) were found to be significantly lower than the response for the parent
compound carbofuran (Figure 20). With both kits, the presence of 3-keto and 3-hydroxy-
carbofuran produced a slight enhancement to carbofuran responses with the 3-keto
metabolite producing twice the enhancement due to the 3-hydroxy metabolite.

The overall results indicated that the metabolites particularly the 3-keto exhibit
cross-reactivity with the Millipore kits (26%) whereas the cross reactivity was non
significant with the Ohmicron kits. Practically 3-hydroxy-carbofuran was undetectable

by the Ohmicron kits at the range of determination.

1 64
INCURRED SAMPLES

W

Ten g of sample were extracted according to the procedure described for the
extraction of carbofuran in corn leaf. The extracts were diluted 1:100 in order to eliminate
any interference or inhibition due to the matrix and the acid, and were assayed in
replicate. Carbofuran was not detected by the Ohmicron EIA kit in the non-treated
samples but was found in some of the treated samples (Figure 16). The concentrations
found were higher in samples treated with high rate of carbofuran compared to samples
treated with low rate carbofuran. The results indicate also that the concentrations found
were high in samples collected the same day after the treatment and decreased with time.

The same samples were also subjected to gas chromatographic determination. The
results are reported in Appendix C. All samples positive for carbofuran using Ohmicron
kits were also positive by GC determinations. Plotting the ELISA results against GC
results gave a regression line of 0.95 slope (R2 = 0.997) (Figure 21). These results
showed a good correlation between the ELISA and gas chromatography indicating that
the Ohmicron kits can be successively used for quantitative determination of carbofuran
in plant materials.

The Millipore kits were also used in this study, but because the kits contain only
two standard solutions it was not possible to quantitatively determine the concentration
of carbofuran in the samples. The samples positive for carbofuran by the Ohmicron kits

were found to be positive with the Millipore kits. For the Millipore kits the results were

165

A. Ohmicron test kit

 

%Bo
on
O
O

20.0 f

 

0.1 1.0 10.0 100.0
Concentration (ppb)

 

 

 

B. Millipore test kit

 

  
    

  

 

 

 

 

100.0 "5
3 ,0, fiflititihfléitiél
.\- 6,, an ‘S'lllffih 33131
40.0
20.0 .......
0.0 i
0.1 1.0 10.0

 

Concentration (ppb)

 

 

Figure 20. Response of the ELISA kits to carbofuran; 3-ketocarbofuran; and (0 ) mixture.

166

expressed as contain carbofuran at a level above 0.2 ppb. This kind of kit can only be
used for qualitative determination where one want to know yes or no as to whether the

samples contain a certain pesticide.

 

 

 

R

30

5 15

,3 .

E

=

O

‘i l

.6 Tn33

5 4
0‘5 Tnz

  

DOT

 
   

5 .
DAT 7 DAT

Sampling Day

11 DAT

 

 

 

Figure 21. Carbofuran concentration in incurred com leaf samples by Ohmicron EIA kit.
2 assays per samples.

167

 

2.5

Level by CC (us/8)

 

 

 

 

0 0:5 1 1:5 2 2.5
Level by ELSA ( nglg)
Page 1

Figure 22. Correlation of carbofuran concentrations as determined by ELISA and go
methods, 11 = 4, r = 0.996 , y = 1.017 X + 0.0005.

168
FI H FILLET

Fish samples were extracted with acetonitrile and assayed after 1:50 dilution.
Carbofuran was detected in only 2 fish (Treatment 2-1) by the Ohmicron kits. The levels
found using the Ohmicron kits ranged from 0.327 to 0.51 ng/g. The same fish were
found to contained carbofuran at the levels above 0.2 ng/g by the Millipore test kits
(Table 17). In order to assess the accuracy of the assays, the same samples were
extracted according to the procedures described in the methods and analyzed by GC. The
samples found to be positive for carbofuran by both kits were found to contain carbofuran
by GC. The GC results were found to be 93-98% in concordance with the Ohmicron
results.

Confirmation of the presence of carbofuran in corn leaf and fish fillet samples was
performed using GC/MS on selected samples. In case of corn leaf samples, cut 1 and 2
(Treatment #3) samples and non treated sample were run. Cut 3 (Treatment #3) contained
a molecular ion at 221 m/e in the negative ion mode with fragment ions at 43, 131, 148,
and 164 which were expected for carbofuran (N-methylcarbamate ). The control sample
(non treated) did not show either the molecular ion at 221 m/e nor the fragments observed
for the standard. With fish samples, the control sample did not also present the molecular
ion at 221 m/e; the samples positive for carbofuran by the ELISA showed the molecular
ion at 221 m/e and the fragments at 43, 131, 148, and 164. These results confirm the

results obtained with the ELISA kits and the GC determinations.

169

Table 17. Carbofuran concentration in fish fillet by ELISA. 2 assays per samples.

 

 

 

 

 

 

 

 

 

 

ID Non Treated #1 Treatment #1-1 Treatment #2-1 .
Ohm Mill GC Ohm Mill GC Ohm GC
Fish 1 nd nd nd nd nd nd nd 11 _
Fish 2 nd nd nd nd nd nd 0.510 > 0.2 ppb 0.47 4
Fish 3 nd nd nd nd nd nd 0.327 > 0.2 ppb 0.3201
Fish 4 nd nd nd nd nd nd nd r1
Fish 5 nd llnd nd nd i d nd
ID Non Treated #2 Treatment #1-2 Treatment #22 ,
Ohm Mill GC Ohm Mill GC Ohm Mill GC ,1
Fish 1 nd nd nd nd nd nd nd nd nd I
Fish 2 nd nd nd nd nd nd nd nd nd 1
Fish 3 nd nd nd nd nd nd nd nd nd '
Fish 4 nd nd nd nd nd nd nd nd nd ,
Fish 5 - nd m1 nd nd nd _ 11d __ nd lid __ 11d |

 

 

 

170

 

 

 
 

 

 

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standard solution; (B) non treated com leaf, (C) treated com leaf; (D) non-treated fish

fillet, (E) treated fish f1

llet.

171
CHAPTER VI

CONCLUSIONS

Commercial enzyme immunoassay kits (EIA) kits were successfully used to
determine carbofuran (insecticides), and alachlor and atrazine (herbicide) in plant and fish
samples. The comparison of the results showed good agreement between the
immunoassays results and gas chromatographic determinations. No positive sample were
found but significant matrices effects on the EIA assays were observed.

Most of the enzyme immunoassay kits were developed for the detection of
pesticides in water samples and for this reason, the interferences from commonly found
groundwater components were minimal. Our study showed that coextracts from fish (fat
and proteins) and com leaf (pigments, proteins, and cellulose) strongly interfered with the
assay detection. Dilution of the samples with distillate water was used to reduce this
interference but on the basis of recoveries, dilution was shown to be productive and
counterproductive. In case of corn extracts, it helped reduce the effects of coextracts and
the inhibition due to extraction solvents enabling the detection of the pesticides. But
when the range of detection is of the test kit is very low 0.1-5 ppb such as the Ohmicron
kits RaPID Assays® kit, the attempt to decrease the effects of background interference
by diluting may be counterproductive because the kit can no longer detect the analytes
at these level of dilution. In any case, as long as these interferences are relatively

constant from one sample to another of the same matrix, the EIA can be applied for

172

screening analysis. It is therefore invalid to make quantitative assessments of the
presence of a pesticide in matrices (e.g., fish) from calibration in another (e.g., corn leaf
extract). For this reason, EIA appears more suitable as a qualitative technique in complex
matrices rather than as quantitative method and will suited for screening purpose. Using
standard prepared in matrix extract helped counteract this problem and in this case EIA
could be use for qualitative purpose.

The results of the study indicated that the two types of kits gave similar results
in the detection of alachlor, atrazine, and carbofuran in corn leaf and fish fillet. The
comparison study of the ELISA kits from the two manufacturers showed each to have
separate advantages and disadvantages. The Millipore Envirogard"I kits showed more
variability compared to the Ohmicron kits which seems to be more precise and
reproducible. The solid phase employed in manufacturing the Millipore Envirogardm kit
is polystyrene tube on which antibodies are passively adsorbed. Studies have shown that
the desorption or leaching off of the antibodies which have been passively adsorbed are
majors factors that adversely affect assay precision and reproducibility (Howell et al.,
1981; Engvall, 1980; Lehtonen and Viljanen, 1980). The solid phase used in the
manufacturing of the Ohmicron RaPID Assays® kits are small magnetic particles on
which the antibodies are covalently bound. The dispersion of particles throughout the
reaction mixture allows precise addition of antibody. The Ohmicron RaPID Assays® kit
was more sensitive and had in general, a lower LDD compared to the Millipore
Envirogard" kits. This is explained by the fact that the lower limits of quantitation of the

RaPID Assays® kit is always lower than the lower limit of quantification of the

173
Envirogardn‘ kit. For this reason, the Ohmicron RaPID Assays® kit is suited for the

analysis of low level residue analysis while the Millipore EnvirogardT" kit is suited for
high level residue analysis. In many assays, samples are diluted in order to reduce
interferences due to matrices; the dilution may lower considerably the levels of the
pesticides to be detected. In such cases, the Ohmicron RaPID Assays® kit is the most
suited for the assay. In case where there is no dilution to be done such as water samples
both assays can be used but the Millipore EnvirogardTM kit had a clear advantage on the
Ohmicron RaPID Assays® kit because of the shorter time of this assay (30 min of
incubation) compared to the Ohmicron RaPID Assays® kit (50 min incubation time).
When considering the cost of the assay, the Ohmicron RaPID Assays® kit has an
advantage over the Millipore Envirogardm kit. The Millipore kit is sold in box of 40
tubes allowing the analysis of 32 samples which cost $10 per sample. The Ohmicron
RaPID Assays® kits is sold by batch of 30 or 100 test tubes. The cost per sample is
$8.63 and $4.72 for the 30 tubes and 100 tubes batch respectively. With the Ohmicron
RaPID Assays® kits (100 tubes), the samples can be analyzed in duplicate, and it will
cost $9.45 which is less than the cost of singlicate analysis by the Millipore assay
($10.18). Besides its low cost, the Ohmicron RaPID Assays® kits has the edge over the
Millipore Envirogard" kits by the fact that up to 46 samples can be analyzed in duplicate
or 96 samples in singlate with one batch whereas only up to 32 samples can be analyzed
using the Millipore EnvirogardTM kits.

The results of the study have shown that the ELISA compares favorably to GC

determinations but the ELISA present several advantages over classical techniques. The

174

length of analysis is shorten with the ELISA compared to chromatographic techniques.
The determination of the three pesticides in fish or corn leaf involved the extraction step,
followed by two clean-up steps (liquid-liquid partition and liquid-solid partition whereas
with the ELISA, the samples are assayed directly after the extraction. Besides, up to 90
samples can be assayed in less than one hour with the ELISA while the detection of the
pesticide in one sample alone by GC takes 20 min. In ELISA, not only low volume of
solvent were used for the extraction (‘ 50 ml) but small size sample were also used for the
assay whereas large volume of organic solvent (250-500 ml) and large sample size (15-25
g) were used in GC determination. Using low volume of solvent helps reduce the cost
of analysis the cost related to the disposal of the organic solvents. In analysis where the
size of the sample is a limiting factor, the ELISA has an edge over chromatographic
techniques.

A cost analysis was done, the ELISA costs approximately $9.7 to $10.4 per sample
whereas the GC determination costs $13.2 to $16.8. The cost included labor, depreciation
of instrumentation, disposables (which include pipette tips, vials, test tubes), and reagents.
The real advantage of the ELISA compared to chromatographic techniques for developing
counties such as my country is the cost associated with instrumentation; the GC
instrumentation used in our study is composed of a Hewlett-Packard Model 5890 Series
II with two detector (ECD and NPD) coupled with a HP Model 7673 automatic injector.
This system costs more than $ 35,000. Starting-up an EIA requires a photometer and
different types of pipeters; the total costs less than $3,000 (including the RaPID

analyzer®). If one want to use a more sophisticated photometer such as the

175

EnviroQuant" which can process the data, it costs $3,675.00. For developing country
one of the factor that might favor the ELISA over GC is the cost associated with
installation, maintenance, and training associated with the GC. It is far less expensive to
have a technician to install, maintain and train workers here in the USA than sending
somebody in my country. Usually when a GC or HPLC is not working in my country,
it takes six months, one to two years to be fixed. Because of the cost associated with
sending somebody to fix the instrument, the company such as Hewlett-packard will wait
to until it has several instrument to service before sending a technician over there to fix
the problem.

Besides its low cost, the ELISA determination can be done in the field using a
portable photometer. Example of on-site uses are remediation sites, in field after
pesticide application for reentry checking purpose or to check the level of a given
pesticide on vegetables before harvesting.

Using the EIA kits, it was possible of detecting 0.15 to 100 ppb of pesticide in
complex matrices such as corn leaf and fish tissue. The disadvantage of these commercial
kits include slow equilibration time and irreversible binding which prevent their re-use or
continuous application. This has been solved by the development of immunosensors
(biosensors) which may be regenerated several times.

As immunoassay for residue analysis becomes widely accepted and applied, new
challenges involving more complex chemicals in more difficult matrix arise. The
integration of traditional analytical methods of detection with immunoassay can provide

new approaches useful in the field of trace analysis. Such approach will be to use thin

176

layer chromatography (TLC) in tandem with EIA. Compounds may be separated by TLC
and the EIA used for determination. EIA can also be coupled with liquid chromatography
(LC). In such a case, EIA may be used as immunoaffinity chromatography or for the

quantitation of LC fractions.

APPENDICES

APPENDIX A

RESULTS OF ALACHLOR DETERMINATION

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179

Table A2. Recovety Study Data for Fish Samples

A. Ohmicron test kit assay

 

 

 

 

 

 

 

 

 

 

Standard
Concentration @9113) Abs 1 Abs 2 Mean % Bo
0.0 1.344 1.351 1.348
0.1 1.212 1.231 1.222 90.6
1.0 0.786 0.736 0.761 56.5
5.0 0.400 0.400 0.400 29.7
Spiking Levels (”HQ ‘Abs 1 Abs 2 Mean %Bo LogConc Cone %Rec
0.0 1.542 1.54 1.541
5.5 0.957 0.987 0.972 63 -0.22 6.08 110.5
11.0 0.812 0.832 0.822 53 0.06 11.38 103.4
22.0 0.635 0.623 0.629 41 0.41 25.48 115.8
44.0 0.466 0.476 0.471 31 0.69 49.31 112.1
110.0 0.213 0.234 0.224 15 nd nd nd
Standard
Concentration (Ppb) Abs 1 Abs 2 Mean % Bo
0.0 1.422 1.325 1.374
0.1 1.244 1.231 1.238 90.1
1.0 0.634 0.664 0.649 47.3
5.0 0.404 0.385 0.386 28.1
82% ' Levels (Egg) Abs 1 Abs 2 Mean %Bo logCon Cone %Rec
0.0 1.422 1.325 1.374
5.5 0.865 0.821 0.843 61 -0.27 5.38 97.9
11.0 0.712 0.656 0.684 50 0.04 11.08 100.7
22.0 0.577 0.532 0.555 40 0.30 19.95 90.7
44.0 0.389 0.377 0.383 28 0.64 43.46 98.8
110.0 0.211 0.178 0.195 nd nd nd nd

 

B. Millipore test kit assay

180

 

 

 

 

 

 

 

 

 

 

 

Standard
tamarind gnu) Abs 1 Abs T‘M—m
0.0 1.734 1.645 1.690
2.0 1.455 1.432 1.444
10.0 0.976 0.956 0.966
100.0 0.210 0.214 0.212
“spacinguveis (118/8) Absl Abs F Mean Lat-m one .
—_——0.0 1.737 1.645 1,690
22.0 1.487 1.455 1.471 0.89 24.32 110.6
55.0 1.168 1.178 1.173 1.78 59.52 108.2
82.5 0.954 1.101 1.028 2.22 92.14 111.7
165.0 0.823 0.855 0.839 2.79 162.29 98.4
275.0 0.666 0.611 0.639 3.39 296.33 107.8
550.0 0.375 0.388 0.382 4.16 641.14 116.6
880.0 0.310 0.303 0.307 4.39 803.09 91.3
Standard
toncentranfi (ppb) Abs 1 Abs 2 Mean
0.0 1.542 1.540 1.541
2.0 1.367 1.410 1.389
10.0 0.978 1.070 1.024
100.0 0.213 0.225 0.219
m“ Levels (ngg) Absl Abs2 Mean Lam
0.0 1.542 1.54 1.541
22.0 1.423 1.412 1.418 0.75 21.10 95.9
55.0 1.156 1.135 1.146 1.65 51.93 94.4
82.5 0.976 0.988 0.982 2.19 89.24 108.2
165.0 0.797 0.791 0.794 2.81 166.31 100.8
275.0 0.642 0.657 0.650 3.29 268.36 97.6
550.0 0.466 0.460 0.463 3.91 497.64 90.5
880.0 0.277 0.297 0.287 4.49 891.27 101.3

 

181

Table A3. Recovery Study for Corn Leaf

A. Ohmicron test kit
Standard

        
 
 
 
   

%Bo

0.1 1.120 1.213 1.167 87.8389
1.0 0.723 0.724 0.724 54.4804
21.9503

 

 

 

SM ' Levels! Egg) Absl Abs 2 Mean %Bo £59011 Conc %Rec
0.0 1.310 1.346 1.328
5.5 0.736 0.776 0.756 56.93 (0.16) 6.97 127
11.0 0.610 0.610 0.610 45.93 0.13 13.47 122
22.0 0.424 0.477 0.451 33.92 0.44 27.67 126
44.0 0.321 0.300 0.311 23.38 0.72 52.05 118
110.0 0.170 0.170 0.170 12.80 nd nd nd
154.0 0.167 0.161 0.164 12.35 nd nd nd
Standard

      

Absl Asz
0.0 1.455 1.51 1.483

0.1 1.258 1.289 1.274 85.9022
1.0 0.817 0.788 0.803 54.1315
0.331 0.322

  
 
 
 
  

   

 

 

 

SM ' Levels ( BEE) Abs 1 Abs 2 Mean %Bo LogCon Conc %Rec
0.0 1.310 1.346 1.328
5.5 1.154 1.102 1.128 85 -0.93 5.85 106
11.0 0.976 0.932 0.954 72 -0.58 13.17 120
22.0 0.823 0.811 0.817 62 -0.30 24.94 113
44.0 0.676 0.705 0.691 52 -0.05 44.98 102
110.0 0.477 0.486 0.482 36 0.38 119.16 108
154.0 0.401 0.387 0.394 30 0.55 179.17 116
Standard

     

%Bo

  
  
  
 
  

   

Abs 1 Abs 2 Mean
0.0 1.367 1.342 1.355

0.1 1.223 1.201 1.212 89.4795
1.0 0.767 0.753 0.760 56.1093
5.0 0.367 0.381 0.374 27.6117

    

    

   

 

 

§9Leve1s( ppb) Abs 1 Abs 2 Mean %Bo 1.9ng Cone %Rec
0.0 1.310 1.346 1.328

5.5 1.365 1.324 1.345 101 nd nd nd

11.0 0.987 1.050 1.019 77 -0.62 11.88 108

22.0 0.900 0.887 0.894 67 -0.36 21.62 98

44.0 0.750 0.775 0.763 57 .009 40.49 92

110.0 0.564 0.532 0.548 . 41 0.35 113.11 103

154.0 0.487 0.501 0.494 37 0.47 146.49 95

182

 

 

 

 

 

 

 

 

 

Assay #1
Standard
Concentration Eb) Abs 1 Abs 2 Mean
0.0 1.936 1.930 1.933
2.0 1.486 1.502 1.494
10.0 1.030 1.112 1.071
100.0 0.216 0.189 0.203
Spiking Levels ( Egg) Abs 1 Abs 2 Mean LnConc ‘Conc ' ‘%Rec
0.0 1.936 1.930 1.9?
22.0 1.40 1.400 1.400 1.10 30.10 137
55.0 1.11 1.122 1.116 1.95 70.64 128
82.5 0.977 1.024 1.001 . 2.30 99.92 121
165.0 0.767 0.782 0.775 2.98 196.97 119
275.0 0.583 0.575 0.579 3.57 354.30 129
550.0 0.377 0.400 0.389 4.14 627.80 114
880.0 0.245 0.233 0.239 4.59 983.56 112
1100.0 0.097 0.087 0.092 5.03 1529.38 139
2200.0 0.007 0.012 0.010 5.28 1959.34 89
" concentration
" Percent recovery
Assay #2
Standard
W Abs 1 Abm
0.0 1.755 1.682 1.719
2.0 1.464 1.453 1.459
10.0 1.030 1.117 1.074
100.0 0.210 0.231 0.221
Seiking Levels (£5) Abs 1 Abs 2 Mean LnCon Cone %Rec
0.0 1.755 1.682 1.719
22.0 1.512 1.503 1.508 0.69 19.98 91
55.0 1.178 1.124 1.151 1.81 60.88 111
82.5 0.997 1.025 1.011 2.24 94.29 114
165.0 0.876 0.883 0.880 2.65 142.21 86
275.0 0.645 0.623 0.634 3.42 306.27 1 1 1
550.0 0.432 0.455 0.444 4.02 555.45 101
880.0 0.267 0.235 0.251 4.62 1013.67 115
1100.0 0.210 0.187 0.199 nd nd nd
2200.0 0.191 0.169 0.180 nd nd nd

 

183

B. Millipore test kit assay. con't.

Assay #3
Standard
oncentranon p s ean
0.0 1.712 1.735 1.724
2.0 1.513 1.486 1.500
10.0 1.121 1.155 1.138
100.0 0.232 0.239 0.236

 

 

 

Spiking Levels (ng/g) AbsI Abs 2 Mean LnC—_C—_—%RW one
0 o

1.755 1.682 1.719

82.5 1.578 1.582 1.580 nd nd nd
165.0 1.388 1.400 1.394 . 1.20 166.2 101
275.0 1.277 1.210 1.244 1.66 263.0 96
550.0 0.945 1.020 0.983 2.46 582.8 106
880.0 0.897 0.865 0.881 2.77 794.1 90
1100.0 0.203 0.211 0.207 nd nd nd
2200.0 0.103 0.111 0.107 nd nd nd

 

184

 

 

 

 

 

 

 

  

 

 

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186

 

 

 

 

 

 

 

 

 

 

 

 

  
    
  
     
       
    
    

 

 

Absorbance
Standard Assay #1 Assay #2 Assay #3 Mean Stdv %CV
0 1.725 1.761 1.621 1.698 0.073 4.3
2 1.464 1.356 1.332 1.382 0.070 5.1
10 0.932 0.845 0.967 0.910 0.063 6.9
20 0.612 0.66 0.69 0.650 0.039 6.1
100 0.234 0.243 0.202 0.227 0.022 9.5
Spike (rig/s)
55 1.156 1.23 0.997 1.070 0.119 11.1
165 0.857 0.925 0.842 0.870 0.044 5.1
550 0.375 0.342 0.365 0.357 0.017 4.7
1 Absorbance I
JStandard Assay #1 Assay #2 Assay #3 Mean Stdv %cvll
' 0 1.656 1.684 1.756 1.778 0.052 2.9
2 1.276 1.367 1.456 1.532 0.090 5.9
10 0.765 0.945 0.912 1.210 0.096 7.9
20 0.545 0.633 0.66 0.650 0.060 9.3
0.225 0.24 0.2 0.227 0.020 8.9
1.21 1.17 1.02 1.070 0.100 9.4
0.812 0.956 0.987 0.870 0.093 10.7
0.36 0.357 0.043 12.1)

 

 

 

 

 

 

 

Table A5. Sensitivity Study Data.

A. Ohmicron test kit assay

187

 

 

 

 

Standard (ppb) I"Abs 1 Abs 2 Mean %Bo

0.0 1.412 1.377 1.395

0.1 1.232 1.210 1.221 88

1 .0 0.766 0.782 0.774 56

5 .0 0.312 0.302 0.307 22
Absorbance at 450 nm

y- -38.129x+ 51.2
Standard Curve R2 _ 0.9869

 

 

 

 

 

 

 

 

 

 

 

 

LogColeentI-aflol

Assay # *Abs 1 Abs 2 Mean %Bo LogConc "Cone
Blk 1.412 1.377 1.395

1 0.876 0.856 0.866 61 -0.266 0.542

2 0.912 0.897 0.905 64 -0.337 0.460

3 0.854 0.866 0.860 61 -0.255 0.556

4 0.843 0.900 0.872 62 -0.276 0.530

5 0.835 0.921 0.878 62 -0.288 0.515

6 0.900 0.872 0.886 63 -0.303 0.498
Mean 0.878 62.158 -0.287 0.517
Stdv 0.016 1.131 0.030 0.035
3@ 0.048 3.394 0.089 0.104
‘Absorbance at 450 nm

"Concentration

A. Ohmicron test kit assay. con't

188

 

 

 

 

 

 

 
    

 

 

 

 

 

 

 

Standard (ppb) ‘Abs 1 Abs 2 Mean %Bo
0.0 1.343 1.400 1.372
0.1 1.240 1.265 1.253 91
1.0 0.754 0.764 0.759 55
5.0 0.312 0.304 0.308 22
" Absorbance at 450 nm
Standard Curve
100
:3 0 y - 40.2211: + 52.338
70 .. R’ - 0.9942
:3 i3 :1
0 40 ..
30 4
20 ..
10 +1
0 t .
-1 «0.5 0.5 1
Lotto-cannula.
Assay # Abs 1 Abs 2 Mean %Bo LogConc Conc Conc
Blk 1.700 1.612 1.656
1 1.040 1.117 1.079 63 -0.276 0.5296 5.296
2 0.995 1.126 1.061 62 -0.250 0.5627 5.627
3 1.107 1.034 1.071 63 -0.264 0.5441 5.441
4 1.180 0.989 1.085 64 -0.285 0.5190 5.190
5 1.223 1.040 1.132 67 -0.354 0.4430 4.430
6 0.986 1.105 1.046 62 -0.228 0.5918 5.918
Mean 1.079 63.4 -0.276 0.532 5.317
Stdv 0.029 1.7 0.043 0.051 0.505
3@ 0.088 5.2 0.129 0.152 1.515

 

B. Millipore test kit

189

 

 

 

 

 

 

 

 

 

 

 

 

 

Standard (ppb) ‘Abs 1 Abs 2 Mean
0.0 1.745 1.721 1.733
2.0 1.523 1.468 1.496
10.0 1.120 0.978 1.049
100.0 0.386 0.410 0.398
" Absorbance at 450 nm
Standard Curve y - 028071: + 1.692
111-1
1.500
1.000 4r
3 0.500
0.000 :
0 1 4 5
111 Concentration
Assay # 1"Abs 1 Ab?) Mean Ln Con "IT-on
Blk 1.745 1.721 1.733
1 1.465 1.444 1.455 0.8 2.33
2 1.432 1.387 1.410 1.0 2.73
3 1.435 1.476 1.456 0.8 2.32
4 1.378 1.427 1.300 1.4 4.04
5 1.487 1.466 1.477 0.8 2.15
6 1.366 1.354 1.360 1.2 3.26
Mean 1.427 1.426 1.409 1.0 2.80
Stdv 0.047 0.047 0.068 0.2 0.72
3@ 0.142 0.142 0.204 0.7 2.17

L

 

 

* Absorption at 450 nm
1" Concentration (ppb)

B. Millipore test kit assay; con't.

 

 

 

190

 

 

 

 

 

 

 

 

 

Standard Absl Asz Mean
0.0 1.675 1.625 1.650
2.0 1.423 1.375 1.399
10.0 0.912 0.921 0.917
100.0 0.347 0.361 0.354
Standard Curve y - 0.26568 + 1.5627
113-0.9967
1.5
1.2..
i 0.9 «r
3 0.6»
<
0.3.»
0 : 2 3 :
0 1 2 3 4 5
new
Assay # ‘Abs 1 Abs 2 Mean anon Con Conc
Blk 1.675 1.625 1.650
1 1.432 1.412 1.422 0.5 1.70 84.95
2 1.343 1.377 1.360 0.8 2.15 107.25
3 1.266 1.234 1.250 1.2 3.24 162.18
4 1.382 1.355 1.300 1.0 2.69 134.39
5 1.523 1.544 1.534 0.1 1:12 55.86
6 1.243 1.32 1.282 1.1 2.88 144.07
Mean 1.365 1.374 1.358 0.8 2.3 114.78
Stdv 0.105 0.103 0.106 0.4 0.8 39.77
_3_@ 0.314 0.310 0.317 1.2 2.4 119.3

 

‘Absorbanceat450nm

 

191

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.32 NE 88 caste—.0 .<

25 ....am £883.-..ec .2 ...:

B. Millipore test kit assay

192

 

 

 

 

Absorption
Spiking Levels ( ppb) Ala Meto Mixt
0 1.645
165 1.278 nd 1.167
880 0.734 1.456 0.510
1100 0.489 1.277 0.311
2200 0.210 0.931 0.127

 

Table A7. Incurred Corn Leaf Study

A. Gas Chromatographic determination

193

 

 

 

 

 

 

 

 

 

 

 

 

 

Concentration (ppb) Area 1 Area 2 Mean
0.0944 1421 1432 1427
0.1888 1943 1956 1950
0.2360 3123 3131 3127
0.4720 4254 4261 4258
0.9440 5532 5521 5527

y-1050.8x+105
R’s-0.9833
6000
g 4000..
< 2000.-
0 t f Y Y
o 1 2 3 4 5
Concentration (ppm)

Spiking Levels (ppb) Area] Area 2 Area 3 Mean Conc I"Concf "%Rec
0.236 146 158 168 157 0.050 0.249 105.5
0.472 195 203 205 201 0.091 0.457 96.8
0.944 288 300 291 293 0.179 0.895 94.8
1.180 367 354 336 352 0.235 1.177 99.7
2.360 523 573 555 550 0.424 2.119 89.8

194

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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:00 SD :82 N 82 _ 8?. :00 :00 :82 N «0?. _ 82 a
Na. “CUEBOHH Mfi «EDP—30.5.
v: v: a: a: a: v: v: u: a: 9.. m :6
v: v: a: a: a: u: v: v: a: a: v So
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E E 9. a: a: 89° :2 oNoN NEN mNoN N .8
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H ”new“ 502 N 8.2. _ 32 3&3 scant—50:00

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5.:— + .23: u N

 

 

..zou EcuaEEBNov 02985038950 95

195

Table A7. Incurred Corn Leaf Study

B. Ohmicron test kit assay data

Standard

 

Conc Abs1 Asz Mean %Bo
0.0 1.395 1.415 1.405
0.1 1.247 1.237 1.242 88
1.0 0.825 0.835 0.83 59
5.0 0.367 0.388 0.378 27
Conc = concentration (ppb)

 

y = 45.742: + 54.527
R‘ - 0.9835

 

%Bo

 

 

 

 

-1.0 -o.5 0.0 0.5 1.0
Log Concentration (ppb)

 

 

 

6
9
1

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325.3... 883 .95 .

 

 

 

 

 

 

 

 

 

 

 

 

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B. Millipore test kit assay

197

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Standard 1
Commioo (ppb) Absl Absl Mean 1
0.0 1.656 1.651 1.654 15 0
2.0 1.477 1.500 1.489
10.0 1.110 1.123 1.117 ‘ "
100.0 0.224 0.227 0.226 05 1'
o 4
0 l 2 3 4 5
Non Treated j I Tm #1
11) ABS LnCon Con Conf IABS Ln Can Can Conf
control 1.756 1.756
[DBT 1.654 0.35 1.42 1.577 0.58 1.79 ‘nd
DOT 1.560 0.64 1.89 1.540 0.70 2.01 66.25
SDAT 1.670 0.30 1.35 1.567 0.62 1.85 at!
7DAT 1.575 0.59 1.81 1.557 0.65 1.91 121!
1 lDAT 1.61 1 0.48 1.62 1.555 0.65 1.92 .21
Non dacunined
Treatment #2 ] Tm #3
11) was 14. Can “Can “ConflABS Ln Can Can Conf
control
1081‘ nd
DOT
SDAT
7DAT
llDAT
‘Comtmion

"Fin! «Inoculation

198

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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APPENDIX B

RESULTS OF ATRAZINE DETERMINATION

204

 

 

 

 

 

 

 

 

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205

 

 

 

 

 

 

 

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206

Table B2. Recovery Study Data for Fish Samples

A. Ohmicron test kit assay

 

 

 

 

 

Standard

Concentration (ppb) Absl Abs 2 Mean % Bo
0.0 0.905 1.11 1.008
0.1 0.783 0.791 0.787 78
1.0 0.427 0.445 0.436 43
5.0 0.185 0.2 0.193 19

ASSAY #1: Dilution 1:10; Standard provided

Spiking Levels w Absl Abs 2 Mean %Bo LoLCon Conc %Rec

control 0.905 0.915 0.910

0.7 0.727 0.738 0.733 80 -1.070 0.85 122
1.0 0.687 0.676 0.682 75 -0.908 1.24 124
1.5 0.637 0.627 0.632 , 69 -0.752 1.77 118
2.0 0.612 0.603 0.608 67 -0.674 2.12 106
4.0 0.502 0.512 0.507 56 -0.356 4.40 110
7.0 0.402 0.405 0.404 44 -0.029 9.36 134

 

ASSAY #2: Dilution 1:50; Stande prepared in 10% ACN in water

 

 

5711ng Levels Absl Abs 2 Mean T’/o Bo Logaon Eonc a/oRcc
control 0.925 0.91 5 0.920
0.7 0.757 0.768 0.763 83 -l.l4 0.73 103.93
1.0 0.720 0.725 0.723 79 -1.01 0.97 97.04
1.5 0.667 0.657 0.662 72 -0.82 1.50 100.04
2.0 0.620 0.628 0.624 68 -0.70 1.97 98.66
4.0 0.533 0.532 0.533 58 -0.42 3.81 95.37
7.0 0.450 0.465 0.458 50 -0. 18 6.55 93.54

 

B. Millipore test kit assay

207

 

 

 

 

 

 

 

 

 

 

 

Concentration (ppb) Abs 2 Abs 2 Mean
0.0 1.512 1.500 1.506
0.5 1.240 1.238 1.239
2.0 0.875 0.865 0.870
10.0 0.397 0.386 0.392
y - 41.28331: + 1.0509
113-0.999
1.3
1 .
0.5
0 . r t ; .
.1 o 1 2 3
ASSAY #1 Dilution 1:10, Standard provided
Spiking Levels (ppb) Absl LoLCon Conc %Rec
control 1.457
0.7 1.210 nd nd nd
1.0 0.978 0.2541 1.29 128.94
1.5 0.905 0.5118 1.67 111.22
2.0 0.813 0.8366 2.31 115.42
4.0 0.608 1.5602 4.76 118.99
7.0 0.442 2.1461 8.55 122.17

 

Absl LQLCon Conc %Rec
control 1.457

0.7 1.220 nd nd nd
1.0 1.050 0.000 1.00 100.00
1.5 0.910 0.494 1.64 109.28
2.0 0.870 0.635 1.89 94.39
4.0 0.670 1.341 3.82 95.60
7.0 0.485 1.994 7.35 104.96

ASSAY #2: Dilution 1:10; Standard prepared in 10%‘ACN in water
SM ' Levels (9!!)

 

 

208

Table B3. Recovery Study Data for Corn Leaf

A. Ohmicron test kit assay

 

 

Standard
Concentration (ppb) Abs 2 Abs 2 Mean % Bo
Blank 0.912 0.923 0.918
0.1 0.782 0.791 0.787 85.7
1.0 0.431 0.437 0.434 47 .3
5.0 0.147 0.152 0.150 16.3

 

 

y =- 40.696): + 45.689
R’ - 0.9984

 

 

 

 

 

 

 

 

ASSAY #1: Dilution 1:10; standard provided

 

 

Spiking Levels @818) Absl Abs 1 Mean % Bo LogECon Conc %Rec
control 0.942 0.95 1 0.947
0.7 0.830 0.856 0.843 89 -1.065 0.86 123
1.0 0.770 0.79 0.780 82 -0.902 1.25 125
1.5 0.677 0.684 0.681 72 -0.644 2.27 151
2.0 0.645 0.655 0.650 ~ 69 -0.564 2.73 136
4.0 0.550 0.568 0.559 59 -0.328 4.70 117
7.0 0.462 0.456 0.459 48 -0.069 8.54 122

 

209

Ohmicron test kit assay; con’t

ASSAY #2: Dilution 1:50

 

 

 

 

 

 

 

 

Sprlfl ' Levels (g5) Absl Abs 1 Mean % Bo LQLCon Conc %Rec
control 0.915 0.957 0.936 .
0.7 0.855 0.856 0.856 91 -l.123 0.75 107.66
1.0 0.771 0.79 0.781 83 -0.926 1.19 118.59
1.5 0.739 0.715 0.727 78 -0.786 1.64 109.24
2.0 0.649 0.655 0.652 70 -0.589 2.58 128.92
4.0 0.588 0.578 0.583 62 -0.408 3.91 97.82
7 .0 0.449 0.456 0.453 48 -0.065 8.61 123.01
ASSAY #3: Dilution 1:50; Standard prepared in 10% methanol in water
Spiking Levels Absl Abs 1 Mean °/o Bo LoLCon Conc %Rec
control 0.915 0.957 0.936
0.7 0.865 0.876 0.871 93 -1. 162 0.69 98.33
1.0 0.779 0.869 0.824 88 -1.040 0.91 91.17
1.5 0.729 0.705 0.717 77 -0.759 1.74 116.05
2.0 0.679 0.648 0.664 71 -0.619 2.41 120.26
4.0 0.566 0.594 0.580 62 -0.400 3.98 99.61
7.0 0.455 0.451 0.453 48 -0.066 8.58 122.64
ASSAY #3: Dilution 1:50; Standard prepared in corn leave extract
S ' ' Levels( ) Absl Abs 1 Mean % Bo LogCon Conc %Rec
control 0.915 0.957 0.936
0.7 0.877 0.844 0.861 92 -l.136 0.73 104.46
1.0 0.788 0.861 0.825 88 -1.041 0.91 90.89
1.5 0.736 0.749 0.743 79 -0.826 1.49 99.47
2.0 0.700 0.712 0.706 75 -0.730 1.86 93.02
4.0 0.576 0.594 0.585 63 -0.413 3.87 96.64
7.0 0.497 0.515 0.506 54 -0.205 6.23 89.02

 

B. Millipore test kit assay

210

 

 

Standard 1
Concentration (ppb) Abs1 Asz Mean
0.00 1 .460 1 .455 1 .458
0.50 1 .366 1 .378 1.372
2.00 0.881 0.877 0.879
10.00 0.388 0.395 0.392

 

 

1.5

 

1.2 V
0.9 0
0.6 0

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y - 03266:: +1.1315
R’ -= 0.9979

 

 

ASSAY #1: 1:10 Dilution; standard provided

 

 

 

 

 

Spiking Levels ‘an3} Abs1 ngConc 'Conc "%Rec
control 1.460
2.50 1.400 -0.8182 4.41 176.49
25.0 0.715 1.2576 35.17 140.68
50.0 0.456 2.0424 77.09 154.19
100.0 0.264 2.6242 137.94 137.94
'Concentration
"96 recovery
ASSAY #2 Dilution 1:50; standard provided
Faking Levels gum) Abs1 Log Con Conc %Rec' "
control 1.460
2.50 1 .407 -0.8394 21 .60 172.79
25.0 0.735 1.1970 165.50 132.40
50.0 0.533 1.8091 305.24 122.10
100.0 0.321 2.4515 580.30 116.06

 

‘ I - “'1“-

Millipore test kit assay con't

211

 

 

 

 

 

 

 

 

Standard 2
Concentration Jppb) Abs1 Asz Mean
0.0 1 .475 1 .521 1 .498
0.5 1 .263 1 .282 1.273
2.0 0.910 0.896 0.903
10.0 0.410 0.404 0.407
y - 0.289411 + 1.083
11’ - 0.9983
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Assay #3 Dilution 1:50; standard prepared in 10% water in methanol

 

 

 

 

 

Spiking Levels (M) Abs1 Log Con Conc %Rec
control 1.475
2.50 1.458 nd nd nd
25.0 0.765 1.1061 151.12 120.90
50.0 0.545 1.7727 294.34 117.74
100.0 0.275 2.5909 667.09 133.42
Assay #4: Dilution 1:50; Standard prepared with corn extract
Sg'kigg Levels (M) Abs1 Log Con Conc %Rec
control 1.458
2.50 1.462 nd nd nd
25.0 0.821 0.9364 127.53 102.03
50.0 0.612 1.5697 240.26 96.10
100.0 0.376 2.2848 98.24

 

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Table BS. Sensitivity study.

A. corn leaf with the Ohmicron test kit

 

 

 

 

 

 

 

 

 

 

 

 

 

Standard
Conc (ppb) Absl Asz Mean %Bo
0.0 1.212 1.243 1.228
0.1 1.121 1.135 1.128 92
1.0 0.555 0.564 0.560 46
5.0 0.211 0.207 0.209 17
y - -0.4422x + 0.4706
n’ - 0.9988
8
5
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8
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.1 .05 0 0.5 1
Log Concentration (ppb)
Assay # Abs %Bo Conf
control 1.228
1 0.279 0.23 0.407
2 0.222 0.18 0.512
3 0.105 0.09 0.727
4 0.200 0.16 ' 0.552
5 0.211 0.17 0.532
6 0.217 0.18 0.521
7 0.225 0.18 0.506
8 0.234 0.19 0.489
Mean 0.212 0.172 0.531
Stdv 0.049 0.040 0.090
3@ 0.147 0.120 0.271

 

 

217

B. Corn leaf with Millipore tests kit

 

 

 

 

 

 

 

 

 

 

 

 

Standard
Concentration (ppb) Ln Con Absl Abs2 Mean
0.0 1.317 1.325 1.321
0.5 -0.69 1.201 1.189 1.195
2.0 0.69 0.767 0.776 0.772
10.0 2.30 0.319 0.311 0.315
y - 02935: + 0.9858
n‘ - 09995
1.600
8 1.200 1.
5
f 0.800 ..
8
2 0.400 0
0.000 . . . 4.
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Log Concentration (ppb)
Assay # Abs Con Conf
Blk 1.321
1 0.535 1.53 4.637
2 0.501 1.65 5.205
3 0.521 1.58 4.863
4 0.498 1.66 5.259
5 0.426 1.90 6.718
6 0.512 1.61 5.014
7 0.504 1.64 5.152
8 0.531 1.55 4.700
Mean 0.504 1.641. 5.193
STDv 0.034 0.116 0.657
E 0.102 0.349 1.972

 

 

C. Fish fillet with Ohmicron tests kit

218

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Standard
—
Conc (ppb) Absl Asz Mean % Bo
0.0 1.130 1.121 1.126
0.1 0.975 0.985 0.980 87
1.0 0.510 0.521 0.516 46
5.0 0.275 0.260 0.268 24
1.00 y - 41.3754! '1' 0.4845
R’ - 0.9949
0.80 «-
8
g 0.60 0
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g 0.40 4»
<
0.20 ..
0.00
-1 -0.5 0 0.5 1
Ln Concentration (ppb)
Assay # Abs %Bo Conf
Blk 1.228 '
1 0.259 21% 0.729
2 0.212 17% 0.831
3 0.221 18% 0.81 1
4 0.207 17% 0.842
5 0.201 16% 0.855
6 0.147 12% 0.972
7 0.225 18% 0.803
8 0.217 18% 0.820
Mean 0.211 17% 0.833
STDv 0.031 3% 0.068
3@ 0.094 8% 0.204

219

D. Fish fillet with Millipore test kit

 

 

 

 

 

 

 

 

 

 

 

 

 

Standard
Conc (ppb) Absl Asz Mean
0.0 1.403 1.376 1.390
0.5 1.223 1.270 1.247
2.0 0.865 0.858 0.862
10.0 0.386 0.391 0.389
y - -0.4749x + 0.7572
1.20 1
R - 0.9997
0 0.90 .
U
5
f 0.60 -
S
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< 0.30 ..
0.00 . - ,
.0500 0.000 0.500 1.000
Ln Concentration (ppb)
Assay # Abs Ln Con Con
blank 1.390
1 0.543 0.451 1.72
2 0.600 0.331 1.82
3 0.621 0.287 1.86
4 0.498 0.546 1.65
5 0.342 0.874 1.41
6 0.410 0.731 1.51
7 0.375 0.805 1.45
8 0.612 0.306 1.84
Mean 0.500 0.541 1.658
Stdv 0.1 12 0.236 0.183
3@ 0.336 0.707 0.548

 

 

Table B6. Cross-Reactivity Study Data.

A. Ohmicron test kit assay

220

 

 

 

 

 

 

 

 

 

 

 

A. Atrazine
Assay #1
Concentation (ppb) Absl Asz Mean % Bo
0.0 0.925 0.915 0.920
0.1 0.786 0.802 0.794 86
1.0 0.457 0.427 0.442 48
5.0 0.132 0.140 0.136 15
Assay #2
Concentation gppb) Absl A1182 Mean % Bo
0.0 0.922 0.935 0.929
0.1 0.712 0.727 0.720 77
1.0 0.421 0.387 0.404 44
5.0 0.131 0.124 0.128 14
Assay #3
Concentation (ppb) absl 8sz Mean %Bo
0.0 0.916 0.907 0.9115
0.1 0.771 0.754 0.7625 84
1.0 0.407 0.412 0.4095 45
5.0 0.133 0.127 0.13 14
Assay #4
Concentation (ppb) absl asz ' Mean %Bo
0.0 0.912 0.925 0.919
0.1 0.717 0.731 0.724 79
1.0 0.417 0.391 0.404 44
5.0 0.133 0.131 0.132 14

 

Ohmicron test kit assay; con't

B. Hydroxy-atrazine

221

 

 

 

 

 

 

 

 

 

 

 

Assay #1
Concentation (ppm Absl Abs2 Mean % Bo
0.0 0.925 0.907 0.916
0.1 0.903 0.895 0.899 98
1.0 0.597 0.607 0.602 66
5.0 0.344 0.321 0.333 36
Assay #2
Concentation (ppb) absl asz Mean %Bo
0.0 0.916 0.907 0.912
0.1 0.901 0.897 0.899 99
1.0 0.612 0.600 0.606 66
5.0 0.311 0.318 0.315 35
Assay #3
Concentation (ppb) Absl Abs2 Mean % B0
0.0 0.922 0.935 0.929
0.1 0.867 0.897 0.882 95
1.0 0.587 0.596 0.592 64
5.0 0.312 0.319 0.316 34
Assay #4
Concentation (ppb) absl abs2 Mean %Bo
0.0 0.912 0.925 0.919
0.1 0.882 0.887 0.885 96
1.0 0.622 0.611 0.617 67
5.0 0.318 0.331 0.325 35

 

Ohmicron test kit assay; con't

222

 

 

 

 

 

 

 

 

 

 

 

c. Simazine
Assay #1
Concentation (ppb) Absl Ab82 Mean % Bo
0.0 0.925 0.915 - 0.920
0.1 0.912 0.902 0.907 99
1.0 0.800 0.821 0.811 88
5.0 0.645 0.655 0.650 71
Assay #2
Concentation (ppb) Absl Asz Mean % B0
0.0 0.922 0.935 0.929
0.1 0.922 0.931 0.927 100
1.0 0.845 0.856 0.851 92
5.0 0.677 0.687 0.682 73
Assay #3
Concentation (ppb) abs2 Mean %Bo
0.0 0.916 0.907 0.912
0.1 0.847 0.839 0.843 92
1.0 0.775 0.768 0.772 85
5.0 0.663 0.611 0.637 70
Assay #4
Concentation (ppb) absl abs2 Mean %Bo
0.0 0.912 0.925 0.919
0.1 0.835 0.830 0.833 91
1.0 0.811 0.789 0.800 87
5.0 0.664 0.678 0.671 73

 

B. Millipore test kit assay

223

 

 

 

 

 

 

 

 

A. Atrazrn' e

Concentation (ppb) Absl Ab82 Abs3 Abs4 Mean
0.0 1.510 1.477 1.510 1.477 1.494
0.5 1.100 1.230 1.179 1.227 1.203
2.0 0.927 0.901 0.956 0.946 0.951
10.0 0.470 0.450 0.558 0.555 0.558

B. Hydroxy-atrazine

Concentation (ppb) Abs 1 Abs 3 Abs 3 Abs 4 Mean
0.0 1.510 1.477 1.51 1.477 1.494
0.5 1.345 1.387 1.365 1.371 1.368
2.0 1.131 1.145 1.170 1.005 1.088
10.0 0.726 0.710 0.719 0.723 0.721

C. Simazine

Concentration (ppb) Absl Abs 2 Abs 3 Abs 4 Mean
0.0 1.510 1.545 1.510 1.561 1.536
0.5 1.512 1.524 1.487 1.494 1.491
2.0 1.237 1.224 1.217 1.203 1.210
10.0 0.767 0.800 0.826 0.819 0.823

 

 

224

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225

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Standard 1
EgoncentrafioM) A1181 Alisa Mean BIBo
0.0 1 .223 1 .275 1 .249
0.1 1.13 1.125 1.1275 90
1 .0 0.567 0.56 0.564 45
5.0 0201 0.205 0203 16
Control 8 non treated _
W ABA A882 Mean %Bo lgggonc cone Cont
control 1 .467 1 .455 1.461
1 DBT 1 .450 1 .420 1 .435 98.22 -1 .1 87 nd nd
DOT 1 .330 1 .310 1 .320 90.35 -1 .008 nd nd
SDAT 1 .367 1 .303 1 .335 91 .38 -1 .031 nd nd
7DAT 1 .312 1 .333 1 .323 90.52 -1 .012 nd nd
1 1 DAT 1.412 1.403 1.408 96.34 -1.144 nd nd
Treatment #1
W ABS1 AB§2 Mean %Bo log Cone cone cont
control 1 .323 1 .275 1 .299
1DBT 1 .127 1.357 1242 96 -1.128 nd nd
DOT 0.452 0.433 0.443 34 0.271 1 .87 37.35
SDAT 1.235 1.236 1.236 95 -1.116 nd nd
7DAT 1 .312 1.225 1.269 98 -1 .174 nd nd
1 1 DAT 1.212 1.307 1.260 97 -1.158 nd nd
1 Treatment #2
ID A381 A882 Mean %Bo flConc conc cont
control 1 .467 1 .455 1 .461
1DBT 1 .356 1 .362 1.359 93.02 -1 .069 nd nd
DOT 0.281 0.290 0.286 19.54 0.601 3.99 79.87
SDAT 0.407 0.384 0.396 27.07 0.430 2.69 53.86
7DAT 0.421 0.432 0.427 29.19 0.382 2.41 48.20
1 1 DAT 1 232 1.370 1.301 89.05 -0.978 nd nd
_ Treatment #3
10 °ABS1 A882 Mean %B—o log Cone conc “53117
control 1 .323 1 275 1 299 .
1DBT 1.312 1.310 1.311 101 -1.248 nd nd
DOT 0242 0235 0239 18 0.628 425 84.96
5DAT 0.304 0.31 1 0.308 24 0.507 3.22 64.34
7DAT 0.400 0.429 0.415 32 0.320 2.09 41 .81
11DAT 4 1.212 1250 1.231 95 -1.106 0.08 1.56
‘Absorbance at 450 nm

 

”Final concentration

226

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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228

 

 

 

 

 

 

 

 

 

 

 

 

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8:8 2.8 289-. 8... .8: ~22 .22 ..88 0:8 2.8.3 8... :82 ~22 .82 8.9.8
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. ...... .. n... .-
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2.2%

.983 ....— .3. 5.8.8.5 .m

229

 

 

 

 

 

 

 

 

 

 

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~-.2 2.258.. .-.... 2.258..

 

 

 

....8 $.88 .... .8. ..o..o_E._O

230

C. Millipore test kit data

 

 

Standard #1
Concentation (ppb) Absl Asz Mean
0.0 1.510 1.487 1.499
0.5 1.354 1.377 1.366
2.0 1.110 0.987 1.049
10.0 0.213 0.222 0.218

 

 

 

1.500

1.000 0

0.500 4»

0.000

y - -0.122x + 1.4137

R’ - 0.9m

 

 

 

 

 

2.0 4.0 6.0 8.0

 

 

 

 

 

 

 

0.0
Control 1

ID Absl Ln Conc Conc Concf (ppb)
BLK 1.645

Fishl 1.507 nd nd nd
Fish2 1.316 nd nd nd
Fish3 1.400 nd nd nd
Fish4 1 .585 nd nd nd
FishS 1.43 1 nd nd nd
Mean 1.448 nd nd nd

Control 2

ID Absl Ln Conc Conc Concf (ppb)
BLK 1.645

Fish] 1 .481 nd nd nd
Fish2 1.411 nd nd nd
Fish3 1 .302 nd nd nd
Fish4 1.382 nd ' nd nd
FishS 1 .520 nd nd nd
Mean 1.419 nd nd nd

 

231
Millipore test kit data; con't

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Standard #2
Concentation Absl Abs2 Mean
0.0 1.450 1.477 1.464
2.0 1.385 1.375 1.380
10.0 0.987 1.101 1.044
100.0 0.340 0.331 0.336
y - -0.2698x + LJ
3’ - 0.9898
1.600
1.400 J1
1.200 J+
31$ 1;
0.600 1*
0.400 0
0.200 2»
0.000 r . 4 .
0.00 1.00 2.00 3.00 4.00 5.00
TRT #14
1D Absl Ln Conc Conc Concf (gpb)
BLK 1.567
Fishl 1 .423 nd nd nd
Fish2 1.400 nd nd nd
Fish3 1.326 nd ' nd nd
Fish4 l .41 5 nd nd nd
FishS 2. 5 12 nd nd nd
Mean 1.615 nd nd nd
TRT #1-2
I'D Absl Ln Conc Conc Concf (ppb)
BLK 1.567
Fishl 1.325 nd nd nd
Fish2 1.317 nd nd nd
Fish3 1 .341 nd nd nd
Fish4 1.312 nd nd nd
Fish5 1.292 nd nd nd
Mean 1.3 17 nd nd nd

 

232
Millipore test kit data; con't

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Standard #3
Concentation (ppb) Absl Asz Mean
0.0 1.567 1.582 1.575
2.0 1.145 1.140 1.143
10.0 0.856 0.862 0.859
100.0 0.345 0.340 0.343
y - 0.2059: + 1.3029
n1 - 0.9958
1.2
0
E 0.8 ..
'E
g 0.4 <-
0 .1 . . .
0.00 1.00 2.00 3.00 4.00 5.00
Ln Concentration (ppb)
TRT #2-1
ID Absl LnConc Conc Concf (ppb)
blank 1.760
Fishl 1 .245 nd nd nd
F ish2 1.340 nd nd nd
Fish3 1.300 nd nd nd
Fish4 1.410 nd nd nd
F ish5 1 .430 nd nd nd
Mean
'I'RT #2-2
ID Absl Ln Conc Conc Concf (ppb)
BLK 1.760
Fishl 1.400 nd nd nd
Fish2 1.275 nd nd nd
Fish3 1.327 nd nd nd
Fish4 1.315 nd nd nd
Fish5 1.265 nd nd nd
Mean 1.316 nd nd nd

 

233

APPENDIX C

RESULTS OF CARBOFURAN DETERMINATION

Table Cl. Standard Curve Data.

A. Ohmicron test kit assay

 

 

 

 

 

 

 

 

 

ASSAY #1
Concentration (ppb) Abs 1 Abs 2 Mean sdv %CV %Bo
0.000 1.364 1.397 1.38r 0.023 1.7
0.078 1.315 1.322 1.319 0.005 0.4 96
0.156 1.312 1.307 1.310 0.004 0.3 95
0.312 1.187 1.201 1.194 0.010 0.8 86
0.624 0.877 0.881 0.879 0.003 0.3 64
1.248 0.545 0.540 0.543 0.004 0.7 39
2.496 0.400 0.378 0.389 0.016 4.0 28
4.368 0.302 0.285 0.294 0.012 4.1 21
6.240 0.277 0.295 0.286 0.013 4.5 21
ASSAY #2
Wucentration (ppb) Abs 1 Abs T Mean sdv %CV “7.8?
0.000 1.346 1.361 1.354 0.011 0.8
0.078 1.315 1.310 1.313 0.004 0.3 97
0.156 1.302 1.287 1.295 0.011 0.8 96
0.312 1.221 1.211 1.216 0.007 0.6 90
0.624 0.882 0.867 0.875 0.011 1.2 65
1.248 0.535 0.547 0.541 0.008 1.6 40
2.496 0.386 0.377 0.382 0.006 1.7 28
4.368 0.311 0.306 0.309 0.004 1.1 23
6.240 0.287 0.288 0.288 0.001 0.2 21
ASSAY #3
(Encentration (ppb) Abs 1 Abs 2 Mm sdv M
0.000 1.337 1.340 1.339 0.002 0.?
0.078 1.322 1.312 1.317 0.007 0.5 98
0.156 1.297 1.296 1.297 0.001 0.1 97
0.312 1.233 1.213 1.223 0.014 1.2 91
0.624 0.866 0.856 0.861 0.007 0.8 64
1.248 0.530 0.532 0.531 0.001 0.3 40
2.496 0.367 0.371 0.369 0.003 0.8 28
4.368 0.301 0.321 0.311 0.014 4.5 23
6.240 0.300 0.311 0.306 0.008 2.5 23

 

234

B. Millipore test kit assay

 

 

 

 

 

ASSAY #1
Concentration (ppb) Abs 1 Abs 2 Mean Stdv %CV
0.000 1.377 1.330 1.354 0.033 2.5
0.156 1.277 1.300 1.289 0.016 1.3
0.312 1.230 1.223 1.227 0.005 0.4
0.624 0.850 0.846 0.848 0.003 0.3
1.248 0.523 0.542 0.533 0.013 2.5
4.368 0.278 0.267 0.273 0.008 2.9
6.240 0.256 0.267 ' 0.262 0.008 3.0
ASSAY #2
Concentration (ppb) Abs 1 Abs 2 Mean Stdv %CV
0.000 1.400 1.356 1.378 0.031 2.3
0.156 1.323 1.367 1.345 0.031 2.3
0.312 1.220 1.267 1.244 0.033 2.7
0.624 0.853 0.861 0.857 0.006 0.7
1.248 0.523 0.542 0.533 0.013 2.5
4.368 0.288 0.255 0.272 0.023 8.6
6.240 0.266 0.247 0.257 0.013 5.2

 

Table C2. Recovery Study Data for Fish Samples.

235

A. Water extraction with Ohmicron test kit assay

 

Standard
Concentration (ppb) Abs 1 Abs 2 Mean %Bo
0.0 1.320 1.335 1.328
0.1 1.235 1.257 1.246 94
1.0 0.727 0.730 0.729 55
5.0 0.262 0.270 0.266 20

 

ASSAY #1: Dilution 1:10 Standard provided with the kit

 

 

 

 

 

SJ Levels (ppb) Abs 1 Abs 2 Mean %Bo Conc Conf * %Rec
0.00 1.402 1.377 1.390
3.28 1.105 1.010 1.0575 76 0.365 3.65 111
5.65 0.955 0.946 0.951 68 0.7 7 124
16.40 0.555 0.560 0.558 40 1.90 19 116
32.80 0.330 0.327 0.329 24 4.00 40 122
"' Percent recovery
Assay #2; Dilution 1:50; Standard provided
Sp Levels (ppb) Abs 1 Abs 2 Mean %Bo Conc Conf * %Rec
0.00 1.402 1.377 1.390 -
3.28 1.345 1.330 1.3375 96 nd nd nd
5.65 1.3 1.32 1.31 94 0.1 5 88.50
16.4 1.141 1.157 1.149 83 0.28 14 85.37
32.8 0.977 0.983 0.980 71 0.56 28 85.37

 

‘ percent recovery

236

B. Acetonitrile extraction with Ohmicron test kit

ACETONITRILE EXTRACTION

 

 

ASSAY #1; Dilution 1:10; Stande provided

 

 

%Bo

 

 

 

 

Sp Levels (ppb) Abs 1 Abs 2 Mean Conc Conf %Rec
0.00 1.440 1.435 1.438
3.28 1.120 1.115 1.1175 78 0.50 5.00 152.44
5.65 0.967 0.976 0.972 68 0.85 8.50 150.44
16.40 0.647 0.610 0.6285 44 2.20 22.00 134.15
32.80 0.395 0.371 0.383 27 3.40 34.00 103.66

Assay #2; Dilution 1:50; Standard provided

Sp Levels (ppb) Abs 1 Abs 2 Mean %Bo Conc Conf Rec
0.00 1.440 1.435 1.438
3.28 1.400 1.397 1.399 97 nd nd nd
5.65 1.347 1.354 1.351 94 0.12 6 106.19
16.4 1.129 1.300 1.215 A 84 0.340 17.00 103.66
32.8 1.000 1.102 1.051 73 0.700 35 106.71

 

237

C. Water extraction with Millipore test kit

ASSAY #1: Dilution 1:10 Standard provided with the kit

 

 

 

 

 

 

 

 

Standard
Concentration (ppb) Abs 1 Abs 2 Mean.
0.0 1.444 1.422 1.433
0.2 1.243 1.255 1.249
5.0 0.307 0.310 0.309
Sp Levels (ppb) Abs 1 Abs 2 Mean
0.00 1.444 1.422 1.433
3.28 1.005 1.105 1.055
5.65 0.522 0.946 0.734
16.40 0.343 0.341 0.342
32.80 0.122 0.1267 0.124
Assay #2; Dilution 1:50; Standard provided
Sp Levels (ppb) Abs 1 Abs 2 Mean
0.00 1.444 1.422 1.433
3.28 0.967 0.956 0.9615
5.65 0.723 0.712 0.7175
16.4 0.565 0.545 0.555
32.8 0.235 0.244 0.240

 

 

238

D. Acetonitrile extraction with Millipore test kit

Standard

 

Concentration (ppb) Absl Abs 2 Mean

 

0.0 1.41 1.425 1.418
0.2 1.215 1.244 1.230
5.0 0.288 0.285 0.287

 

ASSAY #1; Dilution 1:10; Standard provided

 

 

 

 

 

Sp Levels (ppb) Abs 1 Abs 2 Mean
0.00 1.41 1.425 1.418
3.28 0.967 0.955 0.961
5.65 0.775 0.77 0.773
16.40 0.435 0.427 0.431
32.80 0.118 0.105 0.1115
Assay #2; Dilution 1:50; Standard provided
Sp Levels (ppb) Abs 1 Abs 2 Mean
0.00 1.41 1.425 1.418
3.28 0.934 0.924 0.929
5.65 0.755 0.724 0.740
16.4 0.347 0.362 0.355
32.8 0.210 0.221 0.216

 

239
Table 3. Recovery Study for Corn Leaf

A. Ohmicron test kit assay

 

Standard 1
Concentration (ppb) Abs 1 Abs 2 Mean %Bo
0.0 1.311 1.318 1.3145
0.1 1.213 1.211 1.212 92
1.0 0.731 0.72 0.7255 55
5.0 0.264 0.261 0.2625 20

 

ASSAY #1: Dilution 1:50 Standard provided with the kit

 

 

 

 

 

 

 

Sp Levels ( ppb) Abs 1 Abs 2 Mean ‘ %Bo Conc Conf %Rec
Blank 1.455 1.477 1.466
15.62 0.98 0.975 0.978 67 0.43 21.5 138
39.00 0.735 0.732 0.734 50 0.95 47.5 122
117.00 0.405 0.41 0.408 28 2.8 140 120
195.00 0.297 0.285 0.291 20 4.5 225 115
234.20 0.21 0.201 0.206 14 5.6 280 120
Standard 2
Conc Abs 1 Abs 2 Mean %Bo
0.0 1.311 1.318 1.3145
0.1 1.213 1.211 1.212 92
1.0 0.731 0.72 0.7255 55
5.0 0.264 0.261 0.2625 20
ASSAY #2: Discoloration; Standard provided
Sp Levels (ppb) Abs 1 Abs 2 Mean %Bo Conc Conf %Rec
0.00 1.612 1.586 1.599
15.62 1.212 1.225 1.219 76 0.38 19 122
39.00 0.915 0.923 0.919 57 0.9 45 115
117.00 0.522 0.521 0.522 33 2.45 122.5 105
195.00 0.35 0.344 0.347 22 4.4 220 113
234.20 0.276 0.301 0.289 18 5.8 290 124

 

Ohmicron test kit assay; con't

240

 

Standard 3
Concentration (ppm Abs 1 Abs 2 Mean %Bo
0.0 1.311 1.318 1.3145
0.1 1.213 1.211 1.212 92
1.0 0.731 0.72 0.7255 55
5.0 0.264 0.261 0.2625 20

 

ASSAY #3: Dilution 1:100; Discoloration; Standard provided

 

 

Sp Levels ( ppb) Abs 1 Abs 2 Mean %Bo Conc Conf %Rec
Blank 1.522 1.517 1.520

15.62 1.355 1.357 1.356 89 0.14 14 90

39.00 1.145 1.138 1.142 75 0.4 40 103

117.00 0.797 0.799 0.798 53 1.05 105 90

195.00 0.565 0.545 0.555 37 2.1 210 108

234.20 0.53 0.525 0.528 35 2.2 220 94

 

. Millipore test kit assay

241

 

 

Standard

Concentration (ppb) Abs 1 Abs 2 Mean
0.0 1.412 1.455 1.434
0.2 1.245 1.236 1.241
5.0 0.277 0.286 0.282

 

ASSAY #1: Dilution 1:50 Standard provided with the kit

 

 

Sp Levels ( ppb) Abs 1 Abs 2 Mean Cone
0.00 1.412 1.455 1.434 ‘nd

15.62 0.756 0.743 0.750 nd

234.20 0.132 0.141 0.137 nd

 

"' Non-determined

ASSAY #2: Dilution 1:50; Discoloration; Standard provided

 

 

Sp Levels ( ppb) Abs 1 Abs 2 Mean Conc
0.00 1.412 1.455 1.434 nd

15.62 0.867 0.843 0.855 nd

234.20 0.104 0.107 0.106 nd

 

ASSAY #3: Dilution 1:100; Discoloration; Standard provided

 

 

Sp Levels ( ppb) Abs 1 Abs 2 Mean Cone
0 1.412 1.455 1.434 nd

15.62 1.115 1.102 1.109 nd

234.20 0.204 0.21 0.207 nd

 

242

 

 

 

 

 

...... ...... 8...... .8... ..8... .8... ...... 8.... .8... ..~...

.... ...... .~.... 8.... 8.... 8.... .... .~.... .~.... ......

.... ...... ...... .8... ~..... 8.... .8... .8... .8... 8.8

.... ...... ...... 3... ...... 8... 8... 8...... ...... ....
...... 8....

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.... 8.... ..~.. ..~.. ..~.. ~... .~.. ~.. .8. ....

.... ...... ~8. ~... .8. ..~.. .8. .8. 8... ....

>08 >.... 282 . 22 . 22 e 22 m 22 ~ 22 .22 3.... .888.

><wm<.¢m._.z.

... .~..... .8... .8... .8... ~..... .2... 8.... .8... ..~...

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...... 8....

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>08 >3. 58.2 . 22 m 22 v 22 ... 22 ~ 22 .22 3.2.. .588.

 

8.... 888-8...— .<

.... .8. 8.6.5.... .2

55.2.86 2.. .2... 8:38.83. .5 ......

 

243

 

 

 

 

 

 

c... an... ....o .o... mm... ~...o Na... ~...o ....o o~..n

... co... 2.... «o... ....o ....o me... -..o .o... o....

... «.... Hm... mm... m...o ....o Na... mm... mm... o..~n
.m\u:. 0...m

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m.. .2... ..~.. ..n.. H... ..~.. ..~.. o.~.~ ~.... ~..

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>0» >32 :80: . 2.2 m 22 .2 0.2 n 82 m 0.2 H 0.2 3...... .2323...
><mmcummazH

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.o\u:. 0....

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>0. >6». :80: . 222 m 022 ¢ 022 m 222 ~ n22 . 02.. .nmn. apnoea».

 

><mmdléBz H

.... .8. 96......2 .m

 

 

244

Table C5. Sensitivity Study Data

A. Corn Leaf with Ohmicron test kit

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Standard (ppb) Abs 1 Abs 2 Mean %Bo
0.0 1.400 1.338 1.369
0.1 1.210 1.245 1.228 90
1.0 0.710 0.721 0.716 52
5.0 0.227 0.210 0.219 16
y - 42.97: + 48.318
11’ - 0.9913
8
.\'
6.5 6 0.5 1
Log Concentration (ppb)
Assay# Abs %Bo Conc
control 1.336
1 1.321 98.877 0.067
2 1.242 92.964 0.108
3 1.1 17 83.608 0.176
4 0.967 72.380 0.317
5 1.105 82.710 0.185
6 0.845 63.249 0.512
Mean 1 . 100 82.298 0.228
Stdv 0.174 13.052 ° 0.164
g 0.523 39.156 0.491

 

 

245

B. Fish fillet with Ohmicron test kit

 

 

Standard

Levels (ppb) Abs 1 Abs 2 Mean %Bo
0.0 1.332 1.340 1.336
0.1 1.225 1.257 1.241 93
1.0 0.707 0.741 . 0.724 54
5.0 0.241 0.230 0.236 18

 

 

 

 

 

 

 

 

 

 

 

 

y = 43.913x + 50.496
112 = 0.9927
8
.\°
0 . . .
-1 «0.5 0 0.5 1
Log Concentration
Assay # Abs %Bo Concentration
control 1.336
1 1.212 90.7 0.121
2 1.180 88.3 0.138
3 0.997 74.6 0.282
4 1.210 90.6 0.122
5 0.955 71.5 0.333
6 1.275 95.4 0.095
Mean 1.138 85.2 0.182
Stdv 0.130 9.7 0.100

3@ 0.390 29.20 0.299

246

Table C6. Cross-Reactivity Study Data

A. Ohmicron test kit assay

fl

 

 

 

 

 

 

 

 

 

 

 

Sample Concentration (ppb Abs 1 Aha 2 Mean 1680
Non spiked 0.00 1.355 1.459 1.407
Carbofuran 6.56 1.13 1.137 1.134 80.6
Carbofuran 32.80 0.835 0.824 0.830 59.0
Carbofuran 65.60 0.612 0.6 0.606 43.1
Carbofuran 131.20 0.421 0.412 0.417 29.6
Carbofuran 196.80 0.321 0.315 0.318 22.6
‘3-Keto-Carb 60.0 1.315 1.321 1.318 93.7
3-Keto-Carb 120.0 1.05 1 .1 1 .075 76.4
3cKeto-Carb 300.0 0.917 0.915 0.916 65.1
3«Keto-Carb 600.0 0.535 0.546 0.541 36.4
”3-Hydroxy-Carb 600.0 1 .325 1.343 1 .334 94.8
3-Hydroxy—Carb 1280.0 1.156 1.164 1.160 82.4
3-l-lydroxy-Carb 1600.0 0.854 0.875 0.870 61.8
carb + 3-Keto-Carb 6.56 + 120 0.976 0.975 0.976 69.3
carb + 3-Keto-Carb 32.80 + 120 0.667 0.672 0.670 47.6
carb + 3-Keto-Carb 65.6 0 + 120 0.505 0.513 0.509 36.2
carb + 3-Keto-Carb 131.2 0 + 120 0.250 0.261 0.256 18.2
carb + 3.Keto-Carb 196.80 + 120 0.135 0.145 0.140 10.0
Carb + 3-Hydro-Carb 6.56 + 1280 1.100 1.050 1.075 76.4
Carb + 3-Hydro-Carb 32.80 + 1280 0.806 0.787 0.797 56.6
Carl) + 3-Hydro-Carb 65.6 0 + 1280 0.587 0.603 0.595 42.3
Carb + 3-Hydro-Carb 131.20 + 1280 0.365 0.376 0.371 26.3
Garb + 3-Hydro-Carb 196.8 0+ 1280 0.210 0.235 0.223 15.8
'3-keto-carbofuran

"3-hydroxy-carbofuran

bf.‘
M!

 

B. Millipore test kit assay

247

 

 

 

 

 

 

 

 

 

 

Sample Concentration (ppb Abs 1 Abs 2 Mean %Bo
Ns 0.00 1.421 1.455 1.438

Carb 6.56 1.310 1.300 1.305 90.8
Carb 32.80 0.932 0.921 0.927 64.4
Carb 65.60 0.733 0.725 0.729 50.7
Carb 131.20 0.482 0.477 0.480 33.3
Carb 196.80 0.300 0.307 0.304 21.1
3-Keto-Carb 60.0 1.376 1.345 1.361 94.6
3-Keto-Carb 120.0 1.157 1.205 1.181 82.1
3-Keto-Carb 300.0 0.923 0.932 0.928 64.5
3-Keto-Carb 600.0 0.712 0.734 0.723 50.3
3-Hydroxy-Carb 800.0 1.410 1.387 1.399 97.3
3-Hydroxy-Carb 1280.0 1.211 1.165 1.188 82.6
3-Hydroy-Carb 1600.0 0.977 1.105 1.041 72.4
carb + 3-Keto-Carb 32.80 + 120 0.976 0.985 0.981 68.2
carb + 3-Keto-Carb 65.6 0 + 120 0.810 0.787 0.799 55.5
carb + 3-Keto-Carb 131.2 0 + 120 0.396 0.403 0.400 27.8
carb + 3-Keto-Carb 196.80 + 120 0.231 0.241 0.236 16.4
Carb + 3-Hydro-Carb 32.80 + 1280 1.110 1.215 1.163 80.8
Carb + 3-Hydro-Carb 65.6 0 + 1280 0.800 0.800 0.800 55.6
Carb + 3-Hydro-Carb 131.20 + 1280 0.500 0.500 0.500 34.8
Carb + 3-hydro-carb 196.8 0+ 1280 0.300 0.300 0.300 20.9

 

Carb = carbofuran
3-keto-carb = 3-keto-carbofuran

3-hydro-c111b = 3-hydroxy-carbofuran

248

Table C7. Incurred Corn Leaf Study Data

A. Gas Chromatographic datermination

Standard

 

Conc (ppm) Area

 

 

 

 

 

 

 

 

 

 

 

 

 

 

0.137 3415
0.274 7550
0.548 15018
1.096 31005
1.507 41031
y - 2765831 . 99.588
a’=0.999
45000
30000
E 15000
0 i Y .
0 0.3 1 1.3 2
Concentration”)
*Sp Levels (ppm) Assay] Assay 2 Mean Conc "Concf %Rec
0.656 3575 3673 3624 0.135 0.673 103%
1.312 7120 6890 7005 0.257 1.284 98%
3.28 16322 16212 16267 0.592 2.959 90%
4.592 24130 24055 24093 0.875 4.373 95%
6.56 37024 37009 37017 1.342 6.710 102%
Mean 98%
‘SpikingLevels
”Finalconcentration

%Rec=%nccove1y

Gas chromatographic determination; con't

249

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Standard
Conc 1n Area
1.507 47562
2.192 65125
3.288 97500
5.460 162500
y- 29499x+1136.1
11’ - 0.9995
200000
130000 4
100000 ..
30000 ..
o 4 w 4 ¢
0 1 2 3 4 3 6
Non Treated
Sample Area 1 Area 2 Mean Cone Conf
Cut 1 np np np nd nd
Cut 2 np np np nd nd
Cut 3 up up up nd nd
Cut 4 np np np nd nd
Cut 5 np np np nd nd
Treatment #1
Sample Area 1 Area 2 Mean Conc Conf (ppm)
lDBT up up nd nd nd
DOT 210545 271235 240890 8.205 1.64
5DAT 40546 40215 40380.5 1.407 0.28
7DAT up up nd nd nd
llDAT np 11p nd nd nd

 

 

Gas Chromatographic determination; con't

250

 

 

 

 

 

Treatment #2
Sample Area 1 Area 2 Mean Conc Conf (ppm)
Cut 1 np np nd nd nd
Cut 2 275435 276745 276090 9.398 1.88
Cut 3 103250 103112 10318] 3.536 0.71
Cut 4 45745 45655 45700 1.588 0.32
Cut 5 np np nd nd 110

Treatment #3
Sample Area 1 Area 2 Mean Conc Conf (ppm)
Cut 1 np np nd nd nd
Cut 2 313750 312565 3131575 10.654 2.13
Cut 3 125455 124765 125110 4.280 0.86
Cut 4 48221 48210 48215.5 1.673 0.33
Cut 5 51212 52342 51777 1.794 0.36

 

251

B. Ohmicron test kit determination

Standard

 

Concentration (ppb) Abs] Abs 2 Mean %Bo

 

 

 

 

 

 

 

 

0.0 1.355 1.534 1.445

0.1 1.335 1.345 1.340 93

1.0 0.756 0.761 0.759 53

5.0 0.286 0.290 0.288 20

Non Treated
Sample Assay] Assay2 Mean %Bo Conc Conf (ppm)
control 1 .678 l .682 1.680
lDBT 1.600 1.587 1.594 95 nd nd
DOT 1.700 1.700 1.700 101 nd nd
5DAT 1.587 1.605 1.596 95 nd nd
7DAT 1.631 1.542 1.587 94 nd nd
11DAT 1.523 1.578 1.551 92 nd nd
Treatment #1

Sample Assay] Assay2 Mean %Bo Conc Concf (ppb)
control 1.712 1.734 1.723
lDBT 1.700 1.600 1.650 96 "nd nd
DOT 0.712 0.756 0.734 43 1.70 1700
5DAT 1.456 1.400 1.428 83 0.28 280
7DAT 1.552 1.534 1.543 90 nd nd
1 lDAT 1.623 1.63 5 1.629 95 nd nd
‘Final concentration

Non-detected

 

252

Ohmicron test kit determination; con't

 

 

 

 

 

Treatment #2
Sample Assay 1 Assay 2 Mean %Bo Con Conf (ppb)
control 1.512 1.534 1.523
lDBT 1.412 1.410 1.411 93 nd nd
DOT 0.580 0.565 0.573 38 2 2000
SDAT 0.947 0.962 0.955 63 0.750 750
7DAT 1.210 1.188 1.199 79 0.36 360
11DAT 1.451 1.431 1.441 95 nd nd
Treatment #3
Sample Area 1 Area 2 Mean %Bo Con Conf (ppb)
control 1.512 1.534 1.523 I
lDBT 1.478 1.51 1 1.495 98 nd nd
DOT 0.531 0.522 0.527 35 2.2 2200
5DAT 0.922 0.915 0.919 60 0.820 820
7DAT 1.210 1.173 1.192 78 0.38 380
11DAT 1.233 1.246 1.240 81 0.285 285

 

 

C. Millipore test kit determination

253

 

 

 

 

 

 

 

 

Standard 1

Concentration (ppb) Abs 1 Abs 2 Mean

0.0 1.650 1.531 1.591

0.2 1.434 1.41] 1.423

5.0 0.310 0.341 0.326

Non Treated
Sample Assay] Assay2 Mean Cone Conf (ppb)
control 1 .775 1 .734 1.755
1DBT 1.621 1.612 1.617 nd nd
DOT 1.735 1.756 1.746 nd nd
5DAT 1.610 1.602 1.606 nd nd
7DAT 1.567 1.612 1.590 nd nd
11DAT 1.665 1.728 1.697 . nd nd
Treatment #1

Sample Assay] Assay2 Mean Conc Conf
control 1.775 1.734 1.755
1DBT 1.675 1.710 1.693 nd nd
DOT 1.612 1.735 1.674 nd nd
5DAT 1.12] 1.105 1.113 >2ppb >2000 ppb
7DAT 1.465 1.421 1.443 nd nd
11DAT 1.524 1.561 1.543 nd nd

 

 

 

Millipore test kit determination; con't

254

 

 

 

 

 

 

 

 

Standard 2

Concentration (ppb) Abs 1 Abs 2 Mean

0.0 1.464 1.366 1.415

0.2 1.277 1.231 1.254

5.0 0.302 0.312 0.307 '

Treatment #2
Sample Area 1 Area 2 Mean Con Conf (ppb)
control 1 .677 1 .655 1.666
1DBT 1.566 1.545 1.556 nd nd
DOT 1 .267 1 .288 1.278 >2 >2000
5DAT 0.922 0.93 1 0.927 >2 >2000
7DAT 1.121 1.200 1.161 >2 >2000
11DAT 1.662 1.575 1.619 nd 110
Treatment #3

Sample Area 1 Area 2 Mean Con Conf (ppb)
control ] .677 1 .655 1.666
1DBT 1.632 1.621 1.627 nd nd
DOT 0.887 0.910 0.899 >2 >2000
5DAT 0.775 0.795 0.785 >2 >2000
7DAT 1.277 1.255 1.266 >2 >2000
11DAT 1.342 1.321 1.332 >2 >2000

 

 

255
Table C8. Incurred Fish Study Data

A. Gas chromatographic determination
Standard

Concentration Area 1 Area 2 Meat;
0. 137 322 3 12 317
0.274 812 795 803.5

 

0.548 1758 1784 1771

1.096 2981 2969 2975

1 .507 4048 4038 4043

 

 

4500

y - 962.3531 - 905.15
11’ - 0.9811

 

3000 4»

 

 

 

 

 

 

 

 

 

 

 

 

 

 

OMean
1500 3r
0 4 ;
o 1 2 3 4 5
Control 1

i) Area 1 Area 2 Mean (fine (ppm) Conc
Fishl *np up up nd ”nd
Fish2 np np np nd nd
Fish3 np np np nd nd
Fish4 up up up nd nd
Fish5 np np np nd nd
Mean np np np nd nd
__ Control 2

ID Area 1 Area 2 Mean Cone (ppm) Conc
Fish] np np np nd nd
Fish2 11p np np nd nd
F ish3 up up up nd nd
Fish4 up up up nd nd
F ish5 np np np nd nd
Mean 11p np np nd nd
*No peak

*Not detected

 

256

Gas chromatographic determination; con't

 

 

Treatment #1-1
II) Area 1 Area 2 Mean Cone (ppm) Conc
Fish] np np np nd nd
Fish2 np np np nd nd
Fish3 up up np nd nd
Fish4 np up up nd nd
Fish5 np np np nd nd
Mean up up up nd nd

 

Treatment #1-2

 

 

ID Area 1 Area 2 Mean Conc (ppm) Conc
BLK np np np nd nd
Fishl up up np nd nd
F ish2 np np np nd nd
Fish3 up up up nd nd
Fish4 np np np nd nd
Fish5 np np np nd nd
Mean up up up nd nd

 

Treatment #2-1

 

 

ID Area 1 Area 2 Mean Conc (ppm) Conc
Fishl 2092.0 2107.0 2099.5 0.003 0.54
Fish2 np np np nd nd
Fish3 up up up nd nd
Fish4 np np np nd nd
Fish5 np np np nd nd
Mean 2092 2107 2100 0.003 0.5382

 

Treatment #2-2

 

 

11) Area 1 Area 2 Mean Conc (ppm) Conc
Fish] nd nd nd nd nd
Fish2 nd nd nd nd nd
Fish3 nd nd nd nd nd
Fish4 nd nd nd nd nd
Fish5 nd nd nd up up

Mean nd nd nd nd nd

257

B. Ohmicron test kit determination

 

 

 

 

 

 

 

 

 

 

 

Standard 1

Concentration (ppb) Abs 1 Abs 2 Mean %Bo

0.0 1.337 1.342 1.340

0.1 1.213 1.207 1.210 90.3

1.0 0.725 0.722 0.724 54.0

5.0 0.254 0.274 0.264 19.7

Non Treated
Sample Assay 1 Assay 2 Mean %Bo .oLcon Con Conf
Blank 1.337 1.342 1.340
Fish 1 1.327 1.334 1.331 99.3 nd nd nd
Fish 2 1.289 1.275 1.282 95.7 nd nd nd
Fish 3 1.266 1.254 1.260 94.] nd nd nd
Fish 4 1.312 1.317 1.315 98.1 nd nd nd
Fish 5 1.227 1.266 1.247 94.8 nd nd nd
Treatment #1-1
Sample Assay 1 Assay 2 Mean %Bo Log con Con Conf
Blank 1.337 1.342 1.340
Fish 1 1.236 1.234 1.235 92.2 nd nd nd
Fish 2 1.227 1.228 1.228 91.6 nd nd nd
Fish 3 1.264 1.235 1.250 93.3 nd nd nd
Fish 4 1.312 1.322 1.317 98.3 nd nd nd
Fish 5 1.302 1.278 1.290 97.9 nd nd nd
Treatment #1-2

Sample Assay l Assay 2 Mean %Bo Log con Con Conf
Blank 1.337 1.342 1.340
Fish 1 1.275 1.270 1.273 95.0 nd nd nd
Fish 2 1.244 1.235 1.240 92.5 nd nd nd
Fish 3 1.255 1.251 1.253 93.5 nd nd nd
Fish 4 1.325 1.312 1.319 98.4 nd nd nd
Fish 5 1.321 1.332 1.327 100.6 nd nd nd

 

Ohmicron test kit determination; con't

258

 

 

 

 

 

 

 

 

Standard 2

Concentration (ppb) Abs 1 Abs 2 Mean %Bo

0.0 1.364 1.357 1.361

0.1 1.225 1.229 1.227 90.2

1.0 0.734 0.74] 0.738 54.2

5.0 0.270 0.282 0.276 20.3

Tmtment #2-1
Sample Assay l Assay 2 Mm %Bo logCon con conf
Blank 1.364 1.357 1.361
Fish 1 0.127 0.136 0.132 9.7 1.01 10.20 509.9
Fish 2 0.235 0.242 0.239 17.5 0.82 6.54 327.]
Fish 3 1.343 1.355 1.349 99.2 nd nd nd
Fish 4 1.267 1.270 1.269 93.2 nd nd nd
Fish 5 0.756 0.750 0.753 59.4 -0.21 0.62 30.8
Treatment #2-2

Sample Assay 1 Assay 2 Mean %Bo logCon con conf (ppb)
Blank 1.364 1.357 1.361
Fish 1 1.257 1.265 1.261 92.7 nd nd nd
Fish 2 1.332 1.338 1.335 98.] nd nd nd
Fish 3 1.244 1.251 1.248 91.7 nd nd nd
Fish 4 1.312 1.310 1.311 96.4 nd nd nd
Fish 5 1.255 1.250 1.253 95.5 nd nd nd

 

C. Millipore test kit determination

259

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Standard 1
Concentration (ppb) Abs 1 Abs 2 Mean %Bo
0.0 1.423 1.435 1.429
0.2 1.277 1.265 1.271 88.9
5.0 0.287 0.280 0.284 19.8
Non Trcated
Sample Assay 1 Assay 2 Mm %Bo Conc
Blank 1.423 1.435 1.429
Fish 1 1.423 1.420 1.422 99.5 nd
Fish 2 1.356 1.347 1.352 94.6 nd
Fish 3 1.322 1.231 1.277 89.3 nd
Fish4 1.412 1.421 1.417’ 99.1 nd
Fish 5 1.425 1.425 1.425 100.6 nd
Standard 2
Concentration (ppb) Abs 1 Abs 2 Mean %Bo
0.0 1.376 1.365 1.371
0.2 1.217 1.205 1.21] 88.4
5.0 0.301 0.275 0.288 21.0
Treatment #1-1
Sample Assay 1 Assay 2 Mm %Bo Conc
Blank 1.376 1.365 1.371
Fish 1 1.235 1.255 1.245 90.8 nd
Fish2 1.312 1.310 1.311 95.7 nd
Fish 3 1.300 1.287 1.294 94.4 nd
Fish 4 1.255 1.256 1.256 91.6 nd
Fish5 1.311 1.325 1.318. 105.0 nd
Standard 2
Concentration (ppb) Abs 1 Abs 2 Mean %Bo
0.0 1.376 1.365 1.371
0.2 1.217 1.205 1.211 88.4
5.0 0.301 0.275 0.288 21.0

 

 

APPENDIX D

TANK WATER CHARACTERISTICS IN FISH REARING EXPERIMENT

260

APPENDIX D

TANK WATER CHARACTERISTICS IN FISH REARING EXPERIMENT

Table D1. Tank Water Characteristics for Alachlor Study

ALACHLOR

Treatment date 03/05/93

 

 

Date
Treatment 5 6 8 9 10 11 12 13
Control #1 Toc 16.00 16.00 1 . 16.23 16.77 16.67 16.70 16.87 16.87

7
6 04
pl] 7.05 7.56 6 77 7.12 7.” 7.25 7.32 7.05 7.45
‘Control #2 101: 15.76 16.12 16.10 16.00 16.” 15.78 16.20 16.20 16.10
p11 7.00 7.00 7 12 7.23 7.45 8.00 8.00 7.77 7.45
Treatment #1 10C 15.30 16.00 15 67 15.67 16.12 16.10 15.87 15.45 16.15
p11 8.00 8.12 7 87 7.87 7.56 7.55 7.45 7.45 7.23
Treatment #1 101: 15.00 15.00 15.30 15.00 15.00 15.21 15.21 15.22 16.00
p11 7.12 8.00 7 78 8.00 7.56 7.67 8.12 8.12 7.77
Treatment #2 106 15.32 15.10 15.20 15.23 15.45 15.10 15.33 16.00 16.10
pl] 7.12 7.20 7.20 7.20 8.00 7.77 7.77 7.65 8.00
Treatment #2 Tot: 16.10 15.57 16 00 15.30 15.20 15.22 15.23 15.25 15.50
7 20

fl 8.w 7.40 . 7.23 7.30 7.25 7.15 8.00 8.10

Treatment 14 15 16 17 18 19 20 21
Control #1 Tot: 17.10 17.22 17.23 17.00 17.” 17.12 17.12 17.10
p11 8.04 8.04 8.12 8.21 7.78 8.35 8.25 8.12
Control #2 rec 16.77 16.05 16.03 16.12 17.01 17.00 17.21 17.20
pl] 7.55 8.00 8.10 8.12 8.00 7.45 7.77 7.23
Treatment #1 Tot: 17.00 16.77 17.20 16.75 16.40 16.4 17 15
pl] 7.45 8.00 8.00 8.10 8.30 8.35 8.15 8.13
Treatment #1 ToC 16.00 16.10 15.77 16.23 17.0 17 16.45 17.12
p11 8.23 8.23 8.23 8.12 7.76 7.45 7.56 8.12
Treatunt #2 Tot: 16.20 16.30 16.23 15.77 15.67 15.98 16.12 16.22
pl] 8.20 8.23 8.20 8.30 8.30 8.43 8.32 8.21

Treatmant#2‘l'oc 15.64 16.70 17.01 16.45 17.00 17.00 17.10 17.20
04

fl 8. 8.10 8.30 8.25 8.15 8.3 8.33 8.35

261

Table D2. Tank Water Characteristics for Atrazine Study

Treatment date 02/2/93

Treatment
Control #1 ToC
pfl
Control #2 ToC
pfl
Treatment #1 Tot:
pH
Treatment #1 ToC
pH
Treatment #2 ToC
on

Treatment #2 ToC

2
15.00
7.55
15.00
7.23
15.00
8.22
15.50

7.56

3
15.00
7.61
15.00
8.00
16.00
7.67
15.00

7.53
15.10
7.45
16.00

4
15.00
7.07
15.10
7.12
16.00
7.87
15.00

8.01
15.00
7.44
15.70

5
16.00
7.00
15.50
8.01
16.00
7.03
15.07

7.67
15.00
7.32

15.17

5
a: 7.10 7.10 7.54 7.45 8. 8.00 7.35 7.44 7.67

Date

6
15.00
7.35
15.20
7.88
16.02
8.00

7
16.00
7.12
15.10
7.45
15.30
7.45

15.26 16.00

7.69
15.45
7.02
1 .02

00

8.02
15.35
7.65
16.00

8
15.80
7.55
16.10
7.67
16.00
8.05
16.24

8.02
14.20
8.00
16.00

9
15.50
8.01
16.50
8.12
15.23
8.04
16.25

7.35
15.45
8.00
15.77

10

15.50
8.02
15.00
8.32
15.65

16.71
8.45
16.00
8.12
16.00

 

Treatment
Control #1 10C
pfl
Control #2 Tee
pH
Treatment #1 Toc
on
Treatment #1 Toc
on
Treatment #2 Toc
PH

Treatment #2 ToC

 

Date
11 12 13 14 15 16 17 18 19 20
15.00 14.00 16.00 16.00 16.30 16.00 15.60 15.50 16.05 16.20
7.87 7.45 7.56 8.20 8.23 8.12 7.53 7.01 8.15 8.25
15.00 16.05 16.03 15.00 15.00 16.00 16.30 16.50 17.00 15.30
7.65 7.02 7.05 8.03 7.45 7.55 7.55 7.35 7.44 7.67
15.45 16.00 16.00 16.00 16.02 16.05 16.11 15.87 15.81 16.05
8.20 7.65 8.56 8.54 8.67 8.11 7.87 8.87 8.71 7.45
16.02 16.00 16.45 16.44 16.2 15.77 16.87 16.05 16.12 16.33
8.01 7.68 7.65 8.21 8.20 7.68 7.45 8.02 8.25 8.77
16.00 16.00 15.01 15.23 16.00 16.11 15.64 16.22 16.05 16.12
8.07 8.12 8.12 8.23 8.51 8.31 7.05 7.75 7.77 7.81
16.05 16.23 15.77 15.43 16.02 16.32 16.01 15.78 16.00 16.24
8 56

262

Table D3. Tank Water Characteristics for Carbofuran Study

Treatment date 01107793

 

 

Treatment 7 8 9 10 11 12 13
Control #1 10¢ 15.0 15.5 15.5 15.0 15.0 15.5 15.8
p11 7.55 7.61 7.07 7.11 7.58 7.02 8.06
Control #2 ToC 15.0 15.0 15.5 15.0 15.0 15.5 15.8
p11 7.12 7.65 7.02 7.56 7.77 7.02 8.08
Treatment #1 ToC 15.0 15.0 15.5 15.0 15.0 15.5 15.5
p11 7.93 7.87 7.77 7.91 7.93 7.02 7.91
Treatment #1 100 15.5 15.5 15.5 15.0 15.0 15.5 15.5
p11 8.00 7.55 7.76 8.01 8.00 7.01 8.09
Treatment #2 ToC 15.0 7.0 15.0 7.3 15.0 15.5 15.5
p11 8.06 14.70 7.15 15.00 8.06 7.01 7.85

Treatment #2 ToC 15.0 8.1 15.0 15.0 15.0 15.5 15.5

a 8.20 15.00 8.20 8.16 8.20 7.02 8.31

 

Date
Treatment 16 15 16 17 13 19 20
Control #1 ToC 15.5 15.5 15.0 14.0 13.8 13.2 13.5
p11 7.96 7.50 7.73 3.50 3.19 3.12 8.16
control 32 161: 15.5 15.5 15.1 16.0 13.3 13.2 13.5
pll 3.03 3.02 7.33 3.51 3.33 8.36 3.22
Treatment 31 161: 15.5 15.5 15.1 16.0 13.4 13.6 13.3
p11 7.33 7.75 7.33 3.30 3.17 3.17 3.27
Treatment 31 to: 15.3 15.5 15.2 16.0 13.4 13.1 14.0
p11 3.23 7.39 7.93 3.25 3.12 3.16 3.05
Treatment 32 ToC 15.5 15.5 15.0 15.0 16.0 13.0 13.0
p11 7.30 7.32 3.36 3.11 8.06 3.11 3.09

Treatmt #2 ToC 15.5 15.0 15.5 14.1 14.0 13.4 13.1
p11 8.25 7.92 7.93 8.41 8.25 8.12 8.10

LITERATURE CITED

263
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