DETERMINATION OF PESTICIDE RESIDUES IN Brassica oleracea (KALES)

SPRAYED WITH CHEMICAL PESTICIDES.

EDNAH KATULA

BTMB/119J/2017

A Project report submitted to the Department of Pure and Applied Sciences in partial

fulfillment of the requirement for a Bachelor’s Degree in Industrial Microbiology and

Biotechnology at the Technical University of Mombasa

JANUARY,2023
 DECLARATION

This Project is my original work and has not been presented for any academic award in any

university.

Signature…………………………………………………Date………………………………

EDNAH KATULA

BTMB/119J/2017

This project has been submitted for examination with my approval as a Technical University

of Mombasa supervisor.

Signature………………………………… Date…………………………………………

DR. MAGWA RISPER

Department of pure and Applied Sciences

II
 ACKNOWLEDGEMENT

I would like to appreciate and give a special “thank you” to the following groups of people

for their continued support and cooperation in this noble course;

The Almighty God for the far He has brought me and the blessings he has showered me

throughout the journey of my academic achievements and the entire research work.

I also express my deep and sincere gratitude to my research supervisor Dr. Risper Magwa for

her guidance, advice and assistance in every step of this work.

My fellow colleagues for always being there to help when needed and giving moral support

when proceeding with the research work.

My parents and siblings for their understanding and readiness to offer any support when

needed.

III
 TABLE OF CONTENT

DECLARATION..........................................................................................................II
ACKNOWLEDGEMENT.........................................................................................III
TABLE OF CONTENT.............................................................................................IV
ABBREVIATIONS...................................................................................................VII
ABSTRACT.............................................................................................................VIII
CHAPTER ONE............................................................................................................9
1.1 Background information.......................................................................................9
1.2. Statement of the problem...................................................................................10
1.3 Justification.........................................................................................................11
1.4 Objectives............................................................................................................12
1.4.1 General Objective...............................................................................................................12
1.4.2 Specific Objectives.............................................................................................................12
1.5 Research Questions.............................................................................................13
CHAPTER TWO........................................................................................................15
LITERATURE REVIEW...........................................................................................15
2.1 Introduction.........................................................................................................15
2.1.1. Classification of pesticides.................................................................................................16
2.2. CONFIDOR WG70............................................................................................17
2.3 DUDUTHRIN....................................................................................................18
2.4 Pesticide Residues and Maximum Residual Limits for Pesticides.....................18
2.5 Effect of handling practices and environmental factors on concentration of
pesticide residues............................................................................................19
2.6 Human exposure to pesticides.............................................................................21
2.7 Pesticide Residues in Food..................................................................................22
8 High Performance Liquid Chromatography...........................................................24
2.9.1 Benefits of Kale..................................................................................................................25
2.9.2 Ecological requirements......................................................................................................26
2.9.7 Major pests and diseases.....................................................................................................27
2.9.7.1 Pests.............................................................................................................................27
2.9.7.2 Diseases.......................................................................................................................28
2.9.8 Harvesting and post-harvesting handling............................................................................29

IV
 CHAPTER THREE....................................................................................................31
MATERIALS AND METHODS...............................................................................31
3.1 Study Area...........................................................................................................31
3.2 Research design...................................................................................................31
3.3 Field operations.....................................................................................................................31
3.3.1 Land preparation.................................................................................................................31
3.3.2 Raising seedlings................................................................................................................31
3.3.3 Transplanting......................................................................................................................32
3.5 Sample preparation..............................................................................................32
3.6 Extraction of the samples....................................................................................32
3.7 Pesticide residue analysis....................................................................................33
3.8 Examination of Leaf Morphology.......................................................................34
3.8.1 leaf size, plant height and number of leaves per plant.....................................34
3.8.2 Determination of fresh and dry weight................................................................................35
3.10 Statistical Analysis............................................................................................35
CHAPTER FOUR.......................................................................................................36
RESULTS AND DISCUSSION...............................................................................36
4.0Introduction..........................................................................................................36
4.1pesticide residue analysis.....................................................................................36
4.2Concentration of pesticide residues in vegetables................................................37
4.3Graphical representation of the data.....................................................................40
4.4Statistical analysis using One Way Anova statistical Analysis............................41
4.4.1 Anova: Single Factor for confider wg70.............................................................................41
4.4.2 Anova: Single Factor for Duduthrin....................................................................................42
4.4.3Statistical explanation..........................................................................................................42
4.5Results for examination of leaf morphology........................................................43
4.5.1determination of leaf size( in centimetres)...........................................................................43
4.5.2number of leaves per plant...................................................................................................44
4.5.3 Plant height (in centimetres)...............................................................................................44
4.5.4Fresh weight( in grams).......................................................................................................44
CHAPTER FIVE.........................................................................................................47
CONCLUSION AND RECOMMENDATIONS......................................................47
5.1 CONCLUSION...................................................................................................47
5.2Recommendations................................................................................................48

V
REFERENCES............................................................................................................49
Appendices:..................................................................................................................54

VI
 ABBREVIATIONS

FAO Food and Agricultural Organization

GHS Globally Harmonized System

WHO World Health Organization

ISO International Organization For Standardization

EFSA European Food Safety Authority

DDT Dichlorodiphenyltrichloroethane

DAP Diammonium Phosphate

CAN Calcium Ammonium Nitrate

ALS Amyotrophic Lateral Slerosis

ASD Autism Spectrum Disorder

MRL Maximum Residue Limits

HPLC High Performance Liquid Chromatography

HCH Hexachlorocyclohexane

PSA Primary Secondary Amine

UNEP United Nations Environment Programme

AOAC Association of Official Analytical Chemists

VII
 ABSTRACT

There is an increasing public concern of human health risks associated with extensive use of

pesticides in agriculture. Regulation of pesticide maximum residue limits in food

commodities is established in many countries. For in Kenya, this regulation exists in law but

is not fully enforced. Therefore, pesticide residues in vegetables have not been well

monitored. This study was done to investigate the pesticide residues in kales, a commonly

eaten vegetable among Kenyans. Vegetables constitute an important part of the human diet as

a source of nutrients such as vitamins. The emerging lifestyle changes and need to live a

healthy life have led to increased consumption of vegetables. This study was done to

determine the levels of pesticide residues in kale leaves and roots and also the soil on which

the kales are grown .Determination of the pesticide residues was to enable evaluation of the

safety profile of kales sprayed with chemical pesticides and those nurtured organically

without use of chemicals by measuring the levels of pesticide residues .The kales vegetables

will be grown in two separate gardens where one will be nurtured with pesticides and the

other without use of any pesticides. The study used experimental study design. Sample

extraction was done using matured leaves and roots. Pesticides residue analysis and safety

profile was done using AOAC 2007.01 method. The results were keyed into Microsoft excel

and analysed using one way ANOVA. The data was then subjected to descriptive statistic and

presented in tables and graphs.

CHAPTER ONE

INTRODUCTION

1.1 Background information

Pesticides have different physicochemical characteristics, structures, modes of action, and

uses in agriculture. Depending on their molecular structure, pesticides are classified into
VIII
different groups, such as organochlorines, organophosphates, neonicotinoids, carbamates,

triazine, urines, phenoxyacids, pyrethroids, and triazoles . According to their use in

agriculture, pesticides are classified as insecticides, fungicides, nematicides, acaricides, and

herbicides .Chemical pesticides are widely used globally as they are perceived to be more

effective and relatively cheap. Kenya being a country that widely relies on agriculture, there

is widespread use of chemical pesticides to control plant diseases and pests. This is in effort

to improve yield and to produce blemish-free products. Emergence of new diseases and pests

has led to the need of better pesticides to counter this problem. Farmers are encouraged to

embrace new methods of controlling plant diseases and pests such as integrated pest

management to reduce reliance on chemical pesticides. Despite this effort, use of chemical

pesticides is still wide spread in the country. Bio-magnification also known as biological

magnification is the increase in concentration of chemicals in organisms as one moves from

lower to higher trophic level. Pesticides that can be bio-magnified are mostly lipophilic such

as DDT, carbaryl and proporxur (Bonita, 2015). Biomagnification of these pesticides occurs

because they either cannot be excreted from the body easily or their rate of degradation is

slower. For example, when DDT is sprayed on or carried by surface runoff to aquatic

ecosystem, it is fed on by it is taken in by phytoplanktons and zooplanktons which are

subsequently fed on by small fish then big fish. Therefore, any organisms including human

beings who feed on big fish take higher concentrations of DDT. The high concentration taken

can affect human health. Bioaccumulation also known as biological accumulation is the

build-up in concentration of chemicals in organs of an organism over a period of time.

Chemical pesticides can accumulate in the human body organs especially the lipophilic ones.

Human beings can be exposed to pesticides from different sources such as vegetables and

meat from animals that have feed on feeds containing pesticides. The chemicals from these
IX
different sources take longer in the human body resulting to their accumulation.

Accumulation of these chemicals can cause harm to human health over a period of time.

Several cases on effects of pesticides have been reported, for example; in 1958, all members

of a local chief’s family in Nigeria who were cocoa farmers got hospitalized after feeding on

a leaf vegetable that was undergrowth after spraying the cocoa with lindane. In another

incident in 2004, residues of carbofuran were found on noodles manufactured in Nigeria and

they were reported to have caused twenty three cases of vomiting and one death (Monosson,

2011). Apart from these specific cases, the WHO estimates that there are three million cases

of pesticide poisoning and two twenty thousand deaths each year (WHO, 2008). Some

pesticides are banned both internationally and locally. Locally, for example DDT and

dimethoate have been banned for use in vegetables. However, a study done in lake Naivasha

basin showed that there is still use of banned pesticides such as endosulfan trading in

different names (Njogu, 2014). Another study done among tomato farmers in Kathiani

showed that there is use of dimethoate (Mutuku et al., 2014). Dimethoate was banned in 2012

by the government of Kenya (MoA, 2012). Continuous use of banned pesticides in the

country can be attributed to weak regulatory framework on pesticides and inadequate

capacity to carry out surveillance on pesticides being imported and used in the country. The

lack of food safety standards, traceability, good agricultural practices, and enforcement of

pesticide residues can be a source of chemical and biological risks to the health of consumers.

The aim of this study is to analyse pesticide residues and safety profile of kales sprayed with

CONFIDOR WG70 and DUDUTHRIN.

1.2. Statement of the problem

Farmers use pesticides to control pests and disease to improve yields and quality of their

products. New plant diseases have been experienced in different parts of the country as a
X
result of climate change. In addition, there is increased demand of vegetables as people seek

to maintain healthy lifestyles. At the same time, consumers also demand for products that are

of good quality and blemish free. To respond to the expanding market demand, vegetable

growers have drastically changed their farming practices to fulfil the needs of their customers

with not just the best but with consistent quality and quantity. This has resulted in

indiscriminate use of pesticides to manage pests and diseases. Most pesticides have a pre-

harvest period after application but most farmers do not observe this period hence the

likelihood of harvesting vegetables that have pesticide residues. Consumers are therefore at a

risk of being exposed to high pesticides levels in their vegetables. Various studies have

indicated the link between pesticide residues to negative health effects in human beings such

as cancer (Berrada et al., 2010). Consumption of such vegetables with pesticide residues can

expose people and increase their risk to such health effects. This study was therefore done to

assess the pesticide residues and safety profile of pesticides on kales grown and sprayed with

chemical pesticides. The research findings are aimed at creating awareness to the general

public and the relevant authorities on the need to monitor pesticide residues in locally

consumed products for the protection of public health.

1.3 Justification

In Kenya, pesticide residues levels in vegetables are commonly monitored for export

products. However, monitoring of pesticide residue levels in vegetables consumed locally is

insufficiently done. This could be attributed to lack of adequate resources, weak regulatory

framework and ignorance from consumers. There is inadequate documented data on pesticide

residue levels for locally consumed horticultural products. Kales being a commonly

consumed vegetable thus its of great importance to study them to know their quality as sold

locally to protect the consumer. Analyzing for pesticide residue levels in the kales gave a
XI
good indication on the risks surrounding the consumer. Even though pesticide residues in

foods is not directly mentioned in sustainable development goals, it is an element of safe food

in the first target of goal two which states that “By 2030, end hunger and ensure access by all

people, in particular the poor and people in vulnerable situations, including infants, to safe,

nutritious and sufficient food all year round (UNDP, 2015). Data from this study will help in

providing information for future development of monitoring strategy of pesticide residue

levels for locally consume vegetables for the protection of human health. It can also be used

by entities concerned with food safety such as the Department of public health and Kenya

bureau of standards.

1.4 Objectives

1.4.1 General Objective

To determine the concentration of pesticide residues in kales sprayed with chemical

pesticides, (CONFIDOR WG70 and DUDUTHRIN).

1.4.2 Specific Objectives

1. To determine the concentration levels of CONFIDOR WG70 in leaves, roots of

kales.

2. To determine the concentration levels of DUDUTHRIN in leaves, roots of kales

3. To determine the concentration of CONFIDOR WG70 and DUDUTHRIN in soil.

4. To compare the morphology of kales sprayed with pesticides with the non-sprayed

kales

XII
1.5 Research Questions

1. What is the concentration of CONFIDOR WG70 in roots and leaves of kales?

2. What is the concentration of DUDUTHRIN in roots and leaves of kales?

3. What is the concentration of CONFIDOR WG70 and DUDUTHRIN in soil?

4. Do the pesticides affect the growth of kales?

1.6 Hypothesis

There is no significance difference in the concentration of the two pesticides in the leaves,

roots, and soil.

1.7 Scope of study and limitations

This study was done in order to determine the concentration of pesticide residues in kales

nurtured using chemical pesticides for the purpose of evaluating their safety profile. The

limitation is that there are several chemical pesticides in the market being used on kales and

thus a hard task to select on the ones to be used in the study.

XIII
XIV
 CHAPTER TWO

LITERATURE REVIEW

2.1 Introduction

A pesticide is any substance or mixture of substances intended for preventing, destroying, or

controlling any pest including vectors of human or animal diseases, unwanted species of

plants or animals causing harm during, or otherwise interfering with, the production,

processing, storage, or marketing of food, agricultural commodities, wood and wood

products, or animal feedstuff, or which may be administered to animals for the control of

arachnids or other pesticide in or on their bodies. UNEP and FAO (1990), defined pesticide

as chemicals that are designed to combat attacks of pests and vectors on crops, domestic

animals and human beings. Since pesticides are used to control noxious and unwanted living

species (Baxter et al., 2010), they find their application in agriculture, in industry to protect

machineries and products from biological degradation, in public health for controlling vector

borne disease and other activities such as gardening (Collotta et al., 2013). Consumption of

vegetables is wide spread among most people in the world. According to WHO (2003), fruits

and vegetables consist an average of 30% (based on mass) of food consumption and are the

most frequently consumed food group. Consumption of vegetables differs across groups of

people depending on locality, availability and cultural practices. The World Health

Organisation recommends a minimum intake of 400g of vegetables per person per day or

146kg/person/year. However, a survey done in 21 countries in 2003 showed that only three

countries that is, Israel, Italy and Spain had reach the minimum recommended level (IARC,

2003). Vegetable consumption is projected to improve over the years as more people become

aware of the importance of consuming vegetables. Consumption of vegetables in Kenya in

XV
2005 was estimated to be 88.3kg/person/year which is way below the minimum

recommended intake (Ruel et al., 2005) There is inadequate information on consumption

rates of exotic vegetables in Kenya. According to Hamilton et al., (2004), food consumption

data can be obtained by conducting food supply surveys, household inventories, household

food use and individual food intake. There are various methods of dietary assessment

methods which include; food frequency, dietary records, diet history and dietary recall

(EFSA, 2009). Food consumption data can be collected using surveys based on food recall

method (Qaim, 2014, Agudo, 2006) Vegetables provide nutrients that are vital for human

health. They contain Vitamin A and C, potassium, dietary fibre, antioxidants and folate.

Vegetables rich in potassium help to maintain blood pressure reduce the risk of kidney stones

and decreases bone loss. Dietary fibre is important as it helps in reducing blood cholesterol

levels, lower the risk of heart disease, obesity and type II diabetes. Fibre is also important for

proper functioning of bowel, reduces constipation and diverticulosis. Vitamin A helps in

keeping eyes and skin healthy and protects them against infections. On the other hand,

Vitamin C helps in healing cuts and wounds and keeps gums and teeth healthy. It also helps

in iron absorption in the body. Folate helps in the formation of red bloods cells in the body. It

is also important to expectant women for reducing risk of birth defects such as neural tube

defects, spina bifida and anencephaly as the foetus develops (USDA, 2015). Vegetables

contain a group of antioxidants that are important in fighting cellular damage and helps in

preventing cancer, heart disease, Parkinson disease, atherosclerosis and Alzheimer,s disease

(Hyland-Tassava, 2013).

2.1.1. Classification of pesticides

Globally harmonized system of classification and labeling of chemicals (GHS) provides

guidelines on classification of chemicals and their labeling. The system classifies chemical in
XVI
terms of physical hazards, effect on human health and effects on environment (United

Nations, 2011). According to Louis (1994) and Buchel (1983), pesticides can be classified

according to chemical composition as organochlorines, organophosphates, carbamates and

pyrethroids or pyrethrins. Organochlorines are organic compounds with five chlorines atoms

within its structure such as lindane, endosulfan, aldrin and chlordane. Organophosphates on

the other hand contain a phosphate group within its structure. Unlike organochlorines,

Organophosphates degrade easily through chemical and biological actions in the environment

hence they are not persistent. According to WHO (2009) pesticides can be classified based on

toxicity as extremely hazardous (IA), highly hazardous (IB), moderately hazardous (II),

slightly hazardous (III), unlikely to 10 present acute hazard (U). Pesticides can also be

classified as insecticides, biopesticides, fungicides and others (Kodandaram et al., 2013).

Pesticides can be classified based on target pest species. The word usually has a suffix – cide

which means to kill. Such pesticide include: insecticides, herbicides, fungicides, miticides,

rodenticides, piscicides and nematicides among others. These pesticides are used to control

and kill insects, weeds, fungi, mites, rodents, fish and nematodes respectively. Pesticides can

also be classified or grouped on their mode of action. In this classification system, pesticides

are classified as contact pesticides, systemic pesticides, foliar pesticides, soil-applied

pesticides, fumigants, preplant herbicides, premergent herbicides, postmergent herbicides,

translocated herbicides, eradicant fungicides, protectant fungicides, selective pesticides, non-

selective pesticides, suffocating insecticides, residual pesticides and non-residual pesticides.

Other classifications include classification by mode of action, chemical structure, pesticide

formulations and activity spectrum that is either broad spectrum or narrow spectrum.

(Ministry of Agriculture, 2017). Pesticides can also be classified or grouped on their mode of

action. In this classification system, pesticides are classified as contact pesticides, systemic
XVII
pesticides, foliar pesticides, soil-applied pesticides, fumigants, preplant herbicides,

premergent herbicides, postmergent herbicides, translocated herbicides, eradicant fungicides,

protectant fungicides, selective pesticides, non-selective pesticides, suffocating insecticides,

residual pesticides and non-residual pesticides. Other classifications include classification by

mode of action, chemical structure, pesticide formulations and activity spectrum that is either

broad spectrum or narrow spectrum (Ministry of Agriculture, 2017).

2.2. CONFIDOR WG70

CONFIDOR WG70 is a systemic insecticide for foliar and soil treatment for control of a

wide range of sucking, chewing and soil pests in coffee, vegetables, ornamentals and other

crops. The target pests are thrips, aphids, whiteflies,red beetle and other leaf piercers.

CONFIDOR WG70 contains water dispersible granules (WG) and a chemical compound

known as imidacloprid which is a neonicoticoid. It works by blocking the activities of nerves

in the peripheral and central nervous system of insects.

2.3 DUDUTHRIN

DUDUTHRIN is a synthetic pyrethroid which acts on the nervous system of the insects and

disturbs the function of the neurons by interacting with sodium channel.

The active ingredient in this insecticide is the lambdacyhalothrin.

2.4 Pesticide Residues and Maximum Residual Limits for Pesticides

In the e nvironment, pesticides can breakdown through physical and chemical means to form

other products. This affects the original concentration of pesticides and results to presence of

metabolites. Pesticide metabolites are also used in determination of pesticide residue since

some are toxic to people and animals. In a study done in Ghana, pesticide residue of selected

pesticides on selected fruits and vegetable were found to exceed MRLs set by WHO (Bempah
XVIII
et al., 2011). It was also established that banned pesticides such as DDT are used in Ghana.

Pesticide residues in one-third of samples of major stable foods in Ethiopia were found to be

above MRLs (Mekonen et al., 2014). In another study conducted in Lebanon indicated that

55% of the samples collected and analysed were found to have one pesticide residue while

45% of the samples were found to have 2-4 residues. The pesticides that were detected

included chlopyrifos, procymidone, primiphos methyl, dimethoate and dieldrin (Nasreddine

et al., 2002). Residues of organochlorine pesticides such as hexachlorobenzene, DDT,

Chlordane compounds, hexachlorocyclohexane and 14 heptachlor were detected in sampled

foods in a study done in china. Even though they were detected, they did not exceed the

MRLs (Zhou et al., 2012). A study done in Pakistan by Aamir et al., (2018) on dietary

exposure to DDT and Hexachlorocyclohexane (HCH) found out that there were residues of

the pesticides in both food of animal origin and vegetable origin. The study also associated

the risk of cancer with consumption of foodstuffs contaminated with DDT and HCH.

Monitoring of Pesticide residues in Kenya on foods and agricultural products is done by

KEPHIS for exports (European Commission, 2014). According to KEPHIS (2013), pesticide

residue levels were analysed for export produce, locally consumed and industrial produce. It

was found that pesticide residues levels in some locally consumed products exceeded MRLs

standards. A study done by (Kithure et al., (2017) on fate of lambda-cyhalothrin in kales,

tomatoes and cabbage found out that the residues of the pesticide in the vegetables were

below the MRLs. In another study on deltamethrin residues in kales, cabbage and tomatoes,

found out that the residues of the pesticide were below the MRLs (Kithure et al., 2014).

However, in a study done by Inonda et al., (2015) found out that various pesticide residues in

kales and French beans exceeded the MRLs. Maximum Residual Levels refers to maximum

concentration of a residue that is legally permitted or recognized as acceptable in or on a food

XIX
or agricultural commodity or animal feedstuff (FAO, 2005). According to EFSA (2010),

Maximum residue levels are the highest levels of residues expected to be in/on food when

pesticide is used according to authorized good agricultural practices. Pesticide residue is

defined as any specified substances such as derivatives of a pesticide and metabolites in or on

food, agricultural commodities or animal feed resulting from use of pesticide (FAO, 2005). In

setting MRLs, marker residues are used. According to JMPR, a marker residue is a residue

whose concentration decreases in known relationship to the level of total residues in or on

foods. In doing this, a pesticide labelled with radioactive isotope is used and results of the

determinations of total residue (total radioactivity) are compared with the concentrations of

marker residue. These studies are carried by JMPR in selecting pesticides used on food and

residue(s) that are used in dietary risk assessment and on which MRLs are set by CAC

(FAO/WHO, 2006).

2.5 Effect of handling practices and environmental factors on concentration of pesticide

residues

Handling practices of food stuff before consumption affects the amount of pesticide residues

that one can be exposed. According to USFDA, (2016) vegetables have to be thoroughly

washed using clean running water. This is done to remove dirt and bacteria on the surface of

vegetables that might cause diseases. Handling practices such as washing with clean running

water have been found to reduce pesticide residue in vegetables. A study done by (Selim et

al., (2011) on the effect of processing vegetables in households on pesticide residues and

found out that washing sweet pepper with tap water reduced the concentration of

cypermethrin, dicofol, endosulfan, metalaxyl, pirimiphos-methyl, dimethoate, and methomyl

by 65%, 67%, 49%, 30%, 10%, 15% and 59% respectively. The same study also found out

that adding acetic acid to water used to wash vegetables reduced the residues further for
XX
methomyl (99.7%), dimethoate (34%), pirimiphos-methyl (89%), metalaxyl (61%),

endosulfan (90%), dicofol (100%), and cypermethrin (100%). In another study done by

Bonnechѐre et al.,( 2012) found out that residues of boscalid, chlorpyrifos, tebuconazole,

dimethoate, difenoconazole and linuron in carrots reduced by up to 90% after washing.

However, a study done by Chavarria et al., (2004) indicated that washing of asparagus did not

have a significant effect in reducing the residue levels of Chlopyrifos in them. Food

processing practices such as blanching, boiling, frying and roasting enhance volatilization

and hydrolysis of chemicals hence altering their concentration levels in food (Kiwango et al.,

2018). Cooking has been found to reduce deltamethrin residues in vegetables by up to 40%

(Tomer et al., 2013). Blanching has been found to reduce residue levels of fat-soluble

pesticides by up to 72% and water-soluble by 79% in cauliflower (Sheikh et al.,2013).

Residue levels of organophosphates in tomatoes, okra, capsicum, eggplant, beans and

cauliflower were found to reduce by 52-100% after being boiled (Satpathy et al.,2011). In the

same study, frying was found to reduce fat-soluble pesticide such as profenofos, endosulfan

and bifenthrin by 96.75%, 94.32% and 98.71% respectively. Studies done by Keikothaile et

al.,(2011), Yang et al., (2012), Bajwa et al., (2014) and Inonda et al.,(2015) showed that

washing, cooking and other handling/processing practices of vegetables reduce pesticide

residues upto 50%. Other methods such as blanching and washing vegetables with vinegar

were also found to reduce pesticide 16 residues (Wanwimolruk et al., 2015). However, in

another study, it was found that pre-heating, pulping, half-pasteurization and evaporation

increased the concentration levels of deltamethrin residues by 2.33% while reducing that of

endosulfan by 66.5% (Tomer et al.,2013). Various environmental factors such as

precipitation, humidity, Air movement (wind), temperature and radiation affect pesticide

residues in vegetables in various ways. Precipitation such as rain and hail wash off pesticides
XXI
from surfaces of vegetables hence reducing the amount of pesticide plants. Excess moisture

in plants is excreted through leaves and roots, this also helps in reducing pesticide residues.

High humidity in the atmosphere affects volatilization of pesticides by lessening it. Air

movement in form of wind physically removes pesticides on the surface of plants. It also

influences volatilization of pesticides on the plants. Radiation from sunlight enhances the

breakdown of pesticides on plants and increases volatilization of pesticides (Edwards, 1975).

High temperature increases the rate pesticide degradation on plant surfaces (Ebeling, 1963)

2.6 Human exposure to pesticides

2.6.1 Exposure routes

Pesticides get into human bodies through various exposure routes which include inhalation,

dermal contact, ingestion and ocular. Multiple exposures to pesticides can occur through the

various routes. For example dermal absorption can occur through accidental contact,

occupational exposure, residues on surfaces, contact with contaminated clothing and

medicinal use (WHO, 2008). Ingestion of pesticides can occur through accidental ingestion

and residues in foods. According to Juraske et al., (2009) exposure to pesticide residues

through dietary intake is estimated to be five times higher than other exposure routes.

Vegetables are consumed raw, steamed or cooked. They are hence more likely to contain

pesticide residues compared to other foodstuffs that undergo processing. Human beings

become exposed to pesticide residues through consumption of food. According to Jalalizand

et al., (2011), pesticide residues contamination levels in vegetables can be reduced by

washing. However, removing toxic components from inner tissues is difficult. Therefore,

people are at a risk of consuming them. The risk of exposure to pesticides in Kenya is not

studied adequately. This needs to be studied to provide information on the risk of exposure

through consumption of vegetables.

XXII
2.6.2 Effects of pesticides on human health

Various studies have been done on the potential negative impacts on health caused by

pesticide residues in food. These effects include carcinogenic and teratogenic (Criswell et al.,

2013). Pesticides have been associated with sarcomas, cancer of the prostrate, pancrease,

lungs, ovaries, breasts, kidneys and intestines. They have also been associated with multiple

myelomas and brain tumors (Zahm and Ward, 1998; Alavanja et al., 2004). Pesticides have

been found to cause negative effects on the nervous system such as impairing its development

which leads to lowered intelligence and behavioral abnormalities (Grandjean and Landrigan,

2006). A study done in California, United States by Roechr (2014) linked severe Autism

Spectrum Disorder (ASD) with organophosphates and Pyrethroids before conception and

during the third trimester. The study also linked carbamates with delayed development. Some

pesticides have also been found to increase the risk of fatal neurological disease known as

amyotrophic lateral sclerosis (ALS) which affects the brain and progressively inhibits it to

control functions such as movement, speech, eating and breathing. They have also been

linked to increased risk of Parkinson’s disease (Firestone et al. 2005, Hancock et al., 2008).

In addition, pesticides have also been found to cause negative reproductive effects such as

stillbirths, low birth weight, early neonatal deaths and spontaneous abortions. They have also

been associated with causing hormonal imbalances, reduced sperm count and sterility in

males (Goldman, 1997, Grandjean and Landrigan, 2006). Pesticides also weaken the immune

system especially in growing children which increases their risk to infectious diseases hence

high mortality rates especially in developing countries (Repetto and Baliga, 1996). They can

also promote autoimmune diseases such as diabetes, lupus and rheumatoid arthritis and

allergy sensitization reactions (Devon, 2016)

2.7 Pesticide Residues in Food

XXIII
Pesticide residues are very small amounts of pesticides that can remain in or on a crop after

harvesting or storage and make their way into the food chain (Food Standards Agency, 2010).

They can remain even when pesticides are applied in the right amount and at the right time.

They may need to be on the surface of foodstuffs to protect them from pest during storage

and some are applied after harvest for this purpose. The Codex definition of a pesticide

residue refers not only to the active ingredient but also to any derivatives of pesticide, such as

conversion products, metabolites, reaction products and impurities considered to be of

toxicological significance (Hamilton and Crossley, 2004).

Studies of the environmental fate, metabolism and processing of food provide basic

information for studying residue levels in food. Chronic and acute consumer intake estimates

compare dietary exposure with acceptable intakes derived from the toxicology of food.

Natural compounds, for proprietary reasons, have not usually been studied as thoroughly as

synthetic compounds and therefore the safety of these compounds is frequently less well

known (Hamilton and Crossley, 2004). The risk assessment of residues in food must be

acceptable at the international level to protect the consumer and to prevent disruption of the

international trade in food.

Most pesticide residues occur in food as a result of the direct application of a pesticide to a

crop or farm animal or the post-harvest treatments of food commodities such as grains to

prevent pest attack. Residues also occur in meat, milk and eggs from the consumption by

farm animals of feed from treated crops. However, residues can also occur in foods from

environmental contamination and spray drift at the time of application. In addition, transport

of residues and sediment, for example in storm water run-off or leaching through the soil to

ground water, may also contaminate drinking water sources (Hamilton and Crossley, 2004)

XXIV
 There has been increased public concern about the impact of organochlorine pesticides such

as dichlorodiphenyltrichloroethane (DDT) and dieldrin on the environment. These

compounds have both high environmental persistence and high fat solubility which

commonly lead to residues occurring in meat, milk and eggs. Most countries have now

withdrawn the registration of these persistent organochlorine pesticides. However, residues

are occasionally detected in food because of the environmental contamination that remains

from historical usage of the chemical. For example, animals grazing on contaminated land

readily consume residues, which can be detected in the fat. Grazing cattle may consume 1 kg

of soil per head per day and so will ingest the residue directly from the soil as well as residue

in the pasture or forage itself. Of the crops grown in soil contaminated with organochlorines,

root crops are the most likely to take up residues (Hamilton and Crossley, 2004).

8 High Performance Liquid Chromatography

High performance liquid chromatography is a chromatographic technique that is used to

seperate, identify and quantify components in a mixture. Liquid sample is injected into the

solvent which is the mobile phase flowing through a column packed with seperating medium

which is the stationary phase. A pump is used to control the flow of the mobile phase.( Meyer,

V.R and Wiley, 2010)

The seperation principle of HPLC is based on the distribution of the analyte (sample) between

the mobile phase (eluent) and the stationary phase (packing material of the column). The

mobile phase is usually a solvent such as Acetonitrile which carries the sample through the

column. The stationary phase is packed with beads such as silica beads and acts as the

seperating medium.( Blum F., 2014)

Depending on the chemical structure of the analyte, the molecules are retarded while passing

through the stationary phase. The specific intermolecular interactions between the molecules on
XXV
the sample determine retention on the column. Hence different samples are eluted at different

times these intermolecular interactions are physical in nataure such as hydrophobic, dipole-

dipole and ionic.

After the seperation of the sample ingredients is achieved , a detection unit recognises the

analytes after leaving the column and are converted recorded by the computer software as signal

peaks. The total amount of all peaks is called chromatogram. Each individual peak provides

qualitative and quantitative information of the analyte .(Dolan J.W, 2002)

Qualitative information is given by the peak itself( i.e shape, intensity of the signal time of

appearance on the chromatogram.the are of peak is proportional to the concentration of the

substance.

High performance liquid chromatography has a number of advantages; it has high resolution,

quick analysis and it can seperate both volatile and non-volatile components. However, it also

has disadvantages which include; high cost and its also relatively difficult to operate.

Kales

Kale, (Brassica oleracea, variety acephala), locally known as sukumawiki, is an edible plant

belongs to the Brassicas family. The loose-leafed plant is grown mainly for autumn and

winter harvest as cold improves its flavour and qualities since it hardens permitting harvest

for fresh greens after most fresh vegetables have become available. Kale can be eaten fresh or

as cooked and is a good source of vitamin A, vitamin C, calcium, Iron and vitamin B6.

The leaves of Kale produce a rosette to frilled margins. The leaves are green in color but can

also be purple, red or light green depending on the variety. The main stem can reach a height

of 60cm (inches) or more on a long growing season. It can be harvested by cutting the entire

rosette before the stem has elongated or progressively as the stem elongates. Kale is a

biennial plant though usually grown as an annual and produce yellow loose clusters in its
XXVI
 second year (Green life , CROP PROTECTION AFRICA).

2.9.1 Benefits of Kale

According to Green life, CROP PROTECTION AFRICA( CPA), the demand for kales is

usually very high because of their benefits, which include the following;

High in iron which is essential for good health, e.g. in formation of haemoglobin and

enzymes, cell growth, proper functioning of the liver, among others.

Rich in vitamin K which helps in protecting the body against various cancers and is also

necessary for a wide range of bodily functions.

High in fibre, low in calorie and zero fat

 Has anti-inflammatory properties which helps in fighting against asthma, arthritis and

autoimmune disorders

 Rich in antioxidants, e.g. flavonoids and carotenoids which protect the body against

various cancers.

 Rich in vitamin A and C

 Good source of calcium which helps in preventing osteoporosis, bone loss and maintaining

a healthy metabolism.

 Rich in zeaxanthin and lutein which are powerful nutrients which protect the eyes

2.9.2 Ecological requirements

Kale is grown in a wide range of climatic conditions provided water is available.

The listed below are the common ecological requirements;

 Soils- perform best in well drained soils which are rich in organic matter with a pH of 5.5-

7.5.

XXVII
 Altitude- does well at altitudes of 800-2200M above sea level

 Temperature- requires an optimum temperature range of 16-21o

 Rainfall- the crop requires sufficient amounts of moisture throughout the season. A well

distributed rainfall of 30-500mm is ideal for optimum yield. Irrigation is recommended if

rainfall is inadequate.

Some of the commonly grown varieties include the following;

 Thousand headed – has smaller leaves than Collard and is slow growing compared to other

varieties. It has long harvesting time and produces many heads thus requires frequent

pruning.

 Collards southern Georgia– it is a drought tolerant short variety that produces large, tender,

bluish green leaves which spread widely.

 Sukuma Siku Hybrid – produces curled leaves with soft texture and has good tolerance to

Diamond Back Moth.

 Collard Mfalme F1 – it is a hybrid variety with short internodes and many tender leaves per

internode hence more yield per unit area. It is tolerant to a wide range of diseases.

 Marrow stem – Produces dark green leaves and prefers cool climate with moderate to fairly

heavy and well-distributed rainfall.

2.9.7 Major pests and diseases

2.9.7.1 Pests

Cutworms– these are soft bodied, smooth caterpillars, brown to black in colour, which cut

seedlings or young plants near the soil level mainly during morning and evening hours. When

disturbed, they curl up tightly. Heavy infestations cause significant crop loss.
XXVIII
Drench soil with PROFILE® 440EC 60ml/20L or PENTAGON® 50EC 20ml/20L or

LOYALTY® 700WDG 10g/20L

Diamondback moth (DBM) – the larva is a green caterpillar which feeds on the leaf tissue

except the veins. Infestation causes significant losses if the pest is not controlled.

Spray ESCORT® 19EC 10ml/20L or BACIGUARD® 16WDG 15g/20L or LEXUS® 247SC

8ml/20L or OCCASION STAR® 200SC 3ml/20L

Aphids– these are tiny black and green soft –bodied sap sucking insects usually on the

undersides of the leaves or stems. They cause damage by sucking sap from leaves and stems

of plants causing leaf curling and distortion. In severe cases of infestation, wilting of the crop

occurs. They also excrete a sticky sugary substance called honeydew, which encourages the

growth of sooty mold.

Spray KINGCODE ELITE® 50EC 10ml/20L or PENTAGON® 50EC 10ml/20L or

LEXUS® 247SC 8ml/20L

To get rid of the sooty mold, spray JAMBO CLEAN® 100ml/20L.

Sawfly– the larvae feed on the leaves creating small regular holes and this leads to

production of poor quality leaves.

Spray ESCORT® 19EC 10ml/20L or BACIGUARD® 16WDG 15g/20L or LEXUS® 247SC

8ml/20L or OCCASION STAR® 200SC 3ml/20L or KINGCODE ELITE® 50EC 10ml/20L

Thrips– these are small, slender sap sucking insects with fringed wings. As they feed, they

damage the crop, causing distortion of leaves, stunted growth and sunken tissues on the

undersides of the leaves.

Spray ALONZE® 50EC 5ml/20L or BAZOOKA® 18EC 10ml/20L or DEFENDER® 25EC

40ml/20L

Whiteflies–These are small white insects which suck plant sap and excrete honeydew where
XXIX
molds grow, affecting plant’s growth and vigour. Their feeding causes crop damages and

reduces the crop’s vigour.

Spray TAURUS® 500SP 10g/20L or LEXUS® 247SC 8ml/20L or KINGCODE

ELITE® 50EC 10ml/20L

Spray the crop with JAMBO CLEAN® 100ml/20L to clean the sooty mold.

Root knot nematodes– these are microscopic parasites found in the soil and whose

infestation leads to formation of galls/swellings on the roots, which reduces plant vigour thus

causing stunting of the crop and eventual death.

Drench soil with ALONZE® 50EC 10ml/20L or mix basal fertilizer, 50kg with 2kg of

ADVENTURE® 0.5GR.

2.9.7.2 Diseases

Damping-off– this is a soilborne disease caused by Pythium species, Rhizoctonia species,

and Fusarium species which frequently occurs in the nurseries.  The diseased seeds do not

germinate while the emerged seedlings rot and eventually die. White cottony growth is seen

on the roots of the infected seedlings.

Drench the soil with PYRAMID® 700WP 100g/20L and spray the crop with

DOMAIN® 250EC 10ml/20L or GEARLOCK TURBO® 250WP 25g/20L

Black rot– this is a bacterial disease whose symptoms are seen as v-shaped yellow to brown

lesions which start forming from the leaf margin. In severe cases of infection, leaves drop and

subsequently the crop dies.

Spray GREENCOP® 500WP 50g/20L, a copper-based products which suppress the activity of

the pathogen.

Leaf spots – infection leads to development of small dark spots on leaves which enlarge

forming brown lesions, become brittle and eventually the necrotic centres crack. The lesions
XXX
can also develop on stems and petioles.

Spray RANSOM® 600WP 15g/20L or CHARIOT® 500SC 20ml/20L or ABSOLUTE® 375SC

10ml/20L

Powdery mildew -symptoms first develop as whitish talcum like powdery growth on upper

leaf surface and as infection progresses, the stems also get infected. Severely infected plant

parts become chlorotic and eventually wilt. Infection also causes stunted growth.

Spray RANSOM® 600WP 15g/20L or DOMAIN® 250EC 10ml/20L or ABSOLUTE® 375SC

10ml/20L

Downy mildew– infection leads to formation of yellow patches on the underside of the leaf.

These patches turn light brown and eventually fall off as disease progresses. Stunted growth

occurs.

Spray GEARLOCK TURBO® 250WP 25g/20L or FORTRESS GOLD ® 720WP 40g/20L or

TOWER® 720WP 50g/20L

2.9.8 Harvesting and post-harvesting handling

Most kale varieties are ready for harvesting within four weeks after transplanting. However,

harvesting can start as soon as the crop begins growing leaves.

Harvesting is done through hand picking the leaves.

The time frame for harvesting is usually a personal decision, based on various preferences.

For instance, if one requires a lighter side to kale’s taste, younger leaves are best, while for

those who like the more pungent and bold flavor of kale, the matured leaves are preferable.

The younger leaves are a tasty addition to some salads while the more mature ones become

more flavorful as they grow.

The harvested leaves should be handled with care in order to avoid bruises.
XXXI
If refrigerated, fresh harvested kale leaves can be stored for up to approximately (7-10) days

while once cooked, they can be frozen and stored for a longer period.( Green life ,CROP

PROTECTION AFRICA)

XXXII
 CHAPTER THREE

MATERIALS AND METHODS

3.1 Study Area

The research was conducted at Technical University of Mombasa (TUM) which is a public

university located in Tudor, Mombasa Kenya.  

3.2 Research design

The study was experimental design which includes field and laboratory works. This design

was also used to find out the concentration of pesticide residues in the kale leaves, roots and

the soil onto which the kales are grown.

3.3 Field operations

3.3.1 Land preparation

Land was prepared early in order to expose pests to sunlight and birds.

Ploughing was done 2 to 3 weeks in advance at about 7-9 inches deep, followed by

harrowing, 2 to 3 weeks after ploughing then preparation of the soil to a fine tilth.

3.3.2 Raising seedlings

Procedure

i. Nursery bed was prepared, about 1 metre wide against the required length.

ii. On the nursery drills were prepared across the bed at a spacing of 10 to 15 cm and a

depth of about 2cm.

iii. Seeds were sowed thinly and covered with soil lightly..

iv. The nursery bed was then covered with a thin layer of dry grass (mulch).

v. The nursery bed was then watered.

XXXIII
 3.3.3 Transplanting

The seedlings were ready for transplanting after 4-6 weeks, after attaining 3-4 leaves

Transplanting was on the plots prepared. Two plots where pesticides will be applied and the

other one plot where pesticides will not be used. The plot without pesticide will be prepared

in a seperate garden. The rate of irrigation was reduced a week before transplanting to harden

the seedlings.

Procedure

i. Raised or sunken beds were made on each plot. Raised beds were preferred for proper

drainage and root development.

ii. Soil was mixed with manure for efficient and improved nutrient uptake and stimulation of

root development.

iii. The beds were irrigated to allow easy planting.

iv. Seedlings were planted in the beds 30 by 30 centimetres apart and at a depth of 2cm.

v. Irrigation was done once daily.

vi. Weeding was also done to ensure they don’t compete with kales for water and nutrients.

3.4 Sample collection

After 6-8 weeks samples, which are leaves and roots of kales and soil were collected

randomly in triplicates from the biological garden where they are grown and transported to

laboratory for analysis.

3.5 Sample preparation

The leaves were thoroughly washed with running water to remove dirt and dust.

Kale samples were chopped and shredded using a blender to obtain a homogenized sample.

Each prepared sample was stored at-20 degrees centigrade awaiting analysis.

XXXIV
3.6 Extraction of the samples

i. 10 grams of the homogenized sample was weighed into 50mL Teflon centrifuge tube and

10mL Acetonitrile added.

ii. The sample was vigorously shaken using a vortex mixer at maximum speed for one

minute.

iii. 4 grams of anhydrous Magnesium Sulphate and 2g Sodium Chloride was added

immediately and mixed vigorously using a vortex mixer.40 mL of internal standard

solution was then added and mixed on a vortex mixer for 30 seconds

iv. The extract was then centrifuged for 5 minutes at 5000rpm.

v. 1mL aliquot of upper Acetonitrile layer was transferred to test tube containing 25mg

PSA sorbent and 150 mg anhydrous Magnesium Sulphate and capped.

vi. The mixture was then shaken with vortex mixer for 30 seconds and the extract

centrifuged for 1minute at 6000rpm to separate pellet from the supernatant.

3.7 Pesticide residue analysis

Pesticide residue analysis was done using AOAC 2007.01 method. It was conducted to

obtain data on concentration of pesticide residues in the collected kale samples. Handling of

samples that is, sampling, packaging, reception and storage to avoid contamination was also

done according to the same guidelines. Samples were stored at a temperature range of

between 1°C to 5°C away from direct sunlight waiting for analysis. Quick, Easy, Cheap,

Effective, Rugged and Safe Method commonly known as the QuEChERS was used in the

analysis of pesticide residues (Anastassiades et al., 2003). This method is also registered as

AOAC 2007.01 official method for pesticide residue analysis.

XXXV
 i. 0.5 mL of extract was transferred to HPLC analysis.

ii. High performance liquid chromatography machine Waters 600 Controller model was

used for analysis.

iii. The machine was connected to a detector Waters 484 Tunable Absorbance Detector

model. Acetonitrile and water was used as mobile phase in a ratio of 80:20 v/v

respectively.

iv. The 31 column (LiChrospher® 100 Rp-18, 5µm) was set at room temperature while the

flow rate was 1ml/min.

v. The wavelength of the detector was set at 205nm with a sensitivity of 0.5. 20µL of extract

was injected into the HPLC machine for analysis using a micro-syringe.

vi. Pure standards of CONFIDOR WG70 and DUDUTHRIN was ran in the HPLC machine

and the retention time of each determined.

vii. This was done to identify peaks of the two pesticides in the samples. Concentration of

pesticide residues for each pesticide tested will be determined by calculating the area of

the peaks. Hence; Concentration of pesticide residue = Area of peak.

3.8 Examination of Leaf Morphology

3.8.1 leaf size, plant height and number of leaves per plant

 After 4-6 weeks the size of the leaf samples from the two biological gardens was

determined by measuring the width and length using a ruler.

 The measurements were recorded and comparison done between the leaves from the two

biological gardens.
XXXVI
 Measurements were in centimetres.

 Number of leaves per plant was also counted and compared from the two biological

gardens.

 The kale plants were uprooted and the height determined by measuring using a ruler.

Comparison was done based on the measurements obtained

3.8.2 Determination of fresh and dry weight

Fresh leaves of kales from the two biological gardens were weighed using the analytical

balance. The same leaves were then dried in an oven after which their weight were

determined using the analytical balance. The results were compared

3.9 Data management.

All activities related to the project were recorded in laboratory notebook. Raw and processed

data was entered routinely in the project database. Daily reports were used to determine the

progress of this project.

3.10 Statistical Analysis

Collected data was analyzed using Descriptive statistics. Descriptive statistics for

concentration of pesticide residue (area of peaks) was calculated using Motic Images plus

2.0. One-way Analysis of Variance was used to determine if there is significant difference in

the concentration of the two pesticides in the samples used. The data was then presented in

tables and graphs.

Statistical significance where P values of <0.05 or equal to 0.05 were considered statistically

significant.

XXXVII
XXXVIII
 CHAPTER FOUR

RESULTS AND DISCUSSION

4.0Introduction
Discussions were made in relation to the results obtained after pesticide residue analysis

in the leaves, roots and the soil using HPLC. Also the examination of leaf morphology ,

the size of leaves, no of leaves per plant , the height of the plant and fresh and dry weight

was determined.

4.1pesticide residue analysis

HPLC was conducted to determine the concentration of CONFIDOR WG70 and

DUDUTHRIN residues in the leaves, roots and the soil on which kales were planted.

Plot A(sprayed Roots Leaves Soil concentration

with CONFIDOR concentration concentration (mg/kg)

WG70) (mg/kg) (mg/kg)

1 0.188 0.508 0.097

2 0.182 0.519 0.099

3 0.185 0.515 0.099

Mean 0.185 0.514 0.295

standard deviation 0.003 0.005567764 0.001154701

Plot B(sprayed Roots Leaves Soil concentration

with concentration concentration

DUDUTHRIN)

1 0.083 0.257 0.034

2 0.083 0.259 0.038

3 0.087 0.255 0.031

XXXIX
Mean 0.084333333 0.257 0.034333333

standard deviation 0.002309401 0.002 0.003511885

Plot 3(Not      

sprayed by any

pesticide)-control

1 BLD BLD BLD

2 BLD BLD BLD

3 BLD BLD BLD

Standard 0 0 0

deviation

mean 0 0 0

BDL- Below Detection Limit.

Table 4.1 .1Concentration of CONFIDOR WG70 and DUDUTHRIN

4.2Concentration of pesticide residues in vegetables

Pure standard samples of CONFIDOR WG70, DUDUTHRIN were dissolved

in Acetonitrile and ran individually in the HPLC to determine their

concentration. The standards were then mixed to check if the HPLC machine

was capable of separating the peaks. Dilution of the standards of 0.0001,

0.001 and 0.01mg/l were used to make calibration curves. Concentration of

pesticide residues were determined by calculating the area of peaks that were

plotted. This was done using a software known as Motic Images Plus 2.0.

XL
Analysis of concentration of pesticide residues in the vegetables leaves, roots and the soils

was done in plot A, B and D..In plot A confider wg70 was the pesticide sprayed. In Plot B

duduthrin was sprayed and in plot C was used as a negative control and hence was not

sprayed with the pesticides. The concentration of the pesticide residues for CONFIDOR

WG70, DUDUTHRIN are as shown in above.

Generally, the results indicated that the concentration of confider W70 residues was higher

than those of duduthrin in all the tested samples with an exception of the plot c(negative control )

that had BDL- Below Detection Limit residues . The concentration of confider in all three samples

of roots,leaves and soil ranged between 0.519 and 0.097 mg/kg with a mean of 0.185,0.514 and

0.295 mg/kg in roots,leaves and soil samples respectively and with a standard deviation of

0.003,0.006,001 mg/kg in roots ,leaves and soil samples respectively.

On the other hand, The concentration of Duduthrin in all three samples of roots,leaves and soil

ranged between 0.259 and 0.031 mg/kg with a mean of 0.084,0.257 and 0.034 mg/kg in

roots,leaves and soil samples respectively and a standard deviation of 0.002,0.002,004 mg/kg in

roots ,leaves and soil samples respectively.

The low residue concentration of duduthrin can be attributed to its shorter half-life

compared to confider wg70 that have a longer half life.It was also noted that leaves had

the highest concentration of both pesticide residues followed by roots and lastly a low

concentration was observed in the soil. The leaves had the highest concentration since is

the part that was directly sprayed and the leaves of kales are the ones that store food thus

the pesticides were also translocated along with the food components. Roots had a medium

concentration since they absorb water and mineral salts and in turn get to absorb the

pesticide residues in the soil. The soil had the least pesticide residues which might have

been attributed by the exposure of soil to air and other soil microbes that might have

utilized the pesticides in one way or another. A study done to determine the concentration
41
of Duduthrin and confider wg70 in tomatoes and kales ,it indicated residue concentration

ranged between 0.035 and 0.135mg/kg (Malhat et al., 2016).

Kales are smooth and shiny surface which make the leaves more exposed to the sun hence

photodegradation of pesticides occurs gradually.. The characteristics of Kales leaves also

make it easy for pesticides to be absorbed and retained in them e.g their broadness and

flat surface . On the other hand, roots are hidden in the soil particle, making it difficult for

the pesticides reaching them.i.e they are not exposed .

The maximum residue levels for confider wg70 and duduthrin and in Kales are 0.45 and

0.3 mg/kg respectively (FAO/WHO, 2014). Kales from plot A sprayed by confider wg70

exceeded the recommended level while those from Plot B and C didn’t exceed the

recommended levels . A study done in Baghladesh indicated that duduthrin was below the

recommended level in kales (Hossain et al., 2016). The results in this study indicates that

consumers who get the commodities that exceed the recommended residue limits from the

respective markets or gardens are likely to suffer negative health effects such as

uncoordinated movements, body tremors and other neurological effects. . This shows that

consumers are likely to suffer negative chronic health effects due to exposure to the

pesticide In addition, consumers who buy vegetables that are not properly cleaned and do

not clean and wash them well are also likely to be exposed to the residues. However, if

they are washed with clean running water and processed as required, exposure to the

pesticide residues can be reduced.

42
4.3Graphical representation of the data

Plot A with CONFIDOR WG70

0.6
0.5
0.4
pesticide concentration

0.3
0.2 result 1
0.1 result 2
0
n n n result 3
tio tio tio
rt a rt a rt a
en en en
onc onc onc
s c c il c
ot es So
Ro eav
L
Sample results

Figure.4.3.1 Plot A sprayed with confider wg70 pesticide

Plot B with DUDUTHRIN

0.3
0.25
0.2
pesticide conventration

0.15
0.1 Result 1
0.05 result 2
0
n n n result 3
tio tio tio
rt a rt a rt a
en en en
onc onc onc
sc es
c il c
ot So
Ro eav
L
sample results

Figure 4.3.2 Plot B sprayed with duduthrin pesticide

In figure4.3.1 it represent confider wg70 pesticide that was used in plot A.vertically are the

pestcide concentration levels in mg/kg of the the samples of the roots,leaves and soil.The highest

concentration was 0.519 mg/kg and the lowest 0.097 mg/kg.Horizontally ,are the samples reults

per run that were obtned after running the HPLC machine.Three samples of each roots,leaves and

soil samples were runned.The blue color represent the results of the first running of the HPLC ,the

43
 dark and green ones represent the second and thirn running results obtained from HPLC

In figure4.3.2 it represent duduthrin pesticide that was used in plot A.vertically are the pestcide

concentration levels in mg/kg of the the samples of the roots,leaves and soil.The highest

concentration was 0.259 mg/kg and the lowest 0.031 mg/kg.Horizontally ,are the samples reults

per run that were obtned after running the HPLC machine.Three samples of each roots,leaves and

soil samples were runned.The blue color represent the results of the first running of the HPLC ,the

dark and green ones represent the second and third running results obtained from HPLC.

Plot A that was sprayed with confider wg70 showed higher concentrations of the pesticide

residues in comparison to Plot B and C in all the samples that were runned in HPLC..

4.4Statistical analysis using One Way Anova statistical Analysis

4.4.1 Anova: Single Factor

for confider wg70

SUMMARY

Averag Varian

Groups Count Sum e ce

0.55 6.33E-

Column 1 3 5 0.185 06

1.54 3.3333

Column 2 3 2 0.514 33

0.29 8.8574

Column 3 3 5 0.295 07

ANOVA

Source of SS df MS F P-value F crit

44
Variation

Between 59049. 29524. 1.0000 0.4218 5.1432

Groups 61 2 8 04 74 53

Within 177148 29524.

Groups .1 6 69

236197

Total .7 8        

4.4.2 Anova: Single Factor

for Duduthrin

SUMMARY

Averag Varian

Groups Count Sum e ce

0.25 0.0843 4.33E-

Column 1 3 3 3 06

0.77 0.257 1.63E-

Column 2 3 1 05

0.10 0.0343 1.23E-

Column 3 3 3 3 05

ANOVA

45
Source of

Variation SS df MS F P-value F crit

Between 0.0002 0.0001 10.373 0.0112 5.1432

Groups 28 2 14 74 88 53

Within 6.6E- 0.0000

Groups 05 6 11

0.0002

Total 94 8        

4.4.3Statistical explanation
One way anova was used to analyze the results as shown above P_-value of confider wg 70

was 0.042 and for duduthrin was 0.011.A significance level of 0.005 was used. When P

less than 0.05 ,it indicates that the null hypothesis is false and thus should be rejected but

instead alternative hypothesis is hence true as by Aires et al.,2009.the One-way ANOVA

test indicated that there was no significant difference in the concentration of duduthrin and

confider wg70 in the three plots of kales grown (p<0.05). Their significant levels does not

proves the results to be rejected .This thus proves to nullify the null hypothesis.

Note: There were no pesticide residues found in samples from plot C hence no relevance in

discussion statistically.

4.5Results for examination of leaf morphology

4.5.1determination of leaf size( in centimetres)
Table 4.5.1.1 plot A ( sprayed with CONFIDOR)

Length in Cm

Plant 1 14

46
Plant 2 18.

Plant 3 15

Table 4.5.1.2 Plot B (Sprayed with DUDUTHRIN)

Length in Cm

Plant 1 20

Plant 2 18.

Plant 3 21

Table 4.5.1.3Plot C ( No spray used )

Length in Cm

Plant 1 23

Plant 2 25.

Plant 3 21

4.5.2number of leaves per plant

Plot A Plot B Plot C
Plant 1 4 9 9
Plant 2 7 8 12
Plant 3 5 7 10

4.5.3 Plant height (in centimetres)

Plot A Plot B Plot C

Plant 1 18.5 24 32.5

Plant 2 19 22 29

Plant 3 17 20 26

47
4.5.4Fresh weight( in grams)
Plot A:
Weight in grams

Plant 1 6

Plant 2 5

Plant 3 4

Plot B sprayed with duduthrin

Weight in grams

Plant 1 6

Plant 2 9

Plant 3 7

Plot C( control)

Weight in grams

Plant 1 9.5

Plant 2 9

Plant 3 7

The length of leaves was measured from the apex to the petiole b placing a ruler along the midrib. one

leave per plant was pricked out and three plants per plot were measured .This was repeated in the three

plots and the average done. The mean length per plot was 15.67 cm ,19.67 cm ,and 23 cm in plot A,B, and

C respectively. this indicate that plot c that was not sprayed with the pesticides had the longest length

followed by plot B that was sprayed by duduthrin and plot A sprayed with confider wg70 had the shortest
48
length .this implies that the chemical pesticides affects the growth and development of leaves as evident in

plot Cthat had no pesticides sprayed, in plot B that had low concentration of chemical residues and in plot

A that had high residues concentration thus hinders growth

The number of leaves was determined by counting 3 plants per plot and doing thner average. Plot A had a

mean of 5 leaves ,plot B 6 and plot C had 10n leaves .This numbers were directly proportional to the

height of the plants where the heights were measured using a rule .three plants per plot were measured and

an average done . the mean heights were as follows :18.17 cm,22 cm and 29.17 cm in plot A, B and C

respectively. this indicate that plot c that was not sprayed with the pesticides had the longest heights and

number of leaves followed by plot B that was sprayed by duduthrin and plot A sprayed with confider

wg70 had the shortest height and number of leaves .this implies that the chemical pesticides affects the

growth and development of leaves and height of kales as evident in plot C that had no pesticides sprayed,

in plot B that had low concentration of chemical residues and in plot A that had high residues

concentration thus a retarded growth.

The weight of fresh leaves was measured from using a weighing balance in the .Three leaves from

different plants were measured per plot .The mean weights in grams recorded :5 g, 7.3 g and 8.5 g in

plots A, B and C respectively . this indicate that plot c that was not sprayed with the pesticides had the

heaviest leaves followed by plot B that was sprayed by duduthrin and plot A sprayed with confider wg70

had the shortest length .this implies that the chemical pesticides affects the growth and development of

leaves as evident in plot C that had no pesticides sprayed, in plot B that had low concentration of

chemical residues and in plot A that had high residues concentration thus affecting their growth. This

weights were mostly dictated by the leaf sizes that differed per plot.

49
 CHAPTER FIVE

CONCLUSION AND RECOMMENDATIONS

5.1 CONCLUSION
1. Kales sprayed with pesticides retain some pesticide residues in them at different

concentrations.

2. Not all pesticides are fit to be used by farmers .some exceed the agreed standards as

required by WHO guidelines. Confider wg70 exceeded the recommended maximum

residues while the duduthrin was below the recommended maximum residue

concentrations.

3. Pesticides affects morphological properties of kales as evident in the measure

weight,height,number of leaves and length of leaves in relation to the concentrations o

the pesticide residues.

4. According to the results gotten from the concentration of the two pesticides, consumers

of kales were at a high risk of exposure to the pesticides’ residues.

5. The two most commonly used pesticides were duduthrin and confider wg70. The

pesticides are classified as moderately hazardous by WHO and are used in kales.

6. Hazard quotient of kales sprayed with confider wg 70 for average and high consumers

were above the recommended value of ≤4.5 mg/kg except for consumers of kales

sprayed with duduthrin whose hazard quotient were below the recommended value of

≤0.3 mg/kg

7. The kales to be ingested as recommended by WHO that do not exceed the maximum

residue should also be thoroughly cleaned before cooking to lower health risks by the

small concentrations in the kales.

8. Inorganic farming of kales can give better yield that those organic farming that use

50
 chemical pesticides,

5.2Recommendations
1. Farmers should use duduthrin pesticide other than confider wg70 that exceed the

recommended maximum residues.

2. Consumers should be encouraged to eat kales that have the maximum residues that

are below the required levels as recommended quantity by WHO.

3. Consumers should handle kales as required such as washing them with clean

running water and other recommended practices to reduce exposure to pesticide

residues.

4. The concerned authorities such as Ministry of agriculture livestock and fisheries,

KEPHIS and KEBS should set and monitor regularly maximum residue levels of

pesticides in locally consumed commodities to protect the consumers. The relevant

authorities should pay attention to use of confider wg70 since smaller amounts of

the pesticide’s residue results to great significance to human health.

5. Consumers should be encouraged to do inorganic farming that avoids the use of

chemical pesticides to reduce chemical pesticide risky .

51
 REFERENCES

Aamir, M., Khan, S. & Li, G. (2018). Dietary exposure to HCH and DDT congeners and their associated

cancer risk based on Pakistani food consumption. Environ Sci Pollut Res Int 25:846. Retrieved

from https://doi.org/10.1007/s11356-017-1129-1

Agudo, A., (2006). Measuring intake of fruits and vegetables. World Health Organization. Retrieved

www.who.int/dietohysicalactivity/publications/f&v_intake_measure.pdf

Alavanja, M.C., Hoppin, J.A., & Kamel, F. (2004). Health Effects of Chronic Pesticide Exposure- Cancer

and Neurotoxicity, Annual Review of Public Health, 25: 155-197. Retrieved

https://www.annualreviews.org/doi/10.1146/annurev.publhealth.25.101802.123020

Bempah, K. C., Buah-kwofie, A., Denutsu, D., Asomaning, J. & Tutu, O. A. (2011). Monitoring of

Pesticide Residues in Fruits and vegetables and Related Health Risk Assessment in Kumasi

Metropolis, Ghana. Journal of Environmental and Earth Sciences 3(6): 761-771. Retrived from

http://maxwellsci.com/print/rjees/v3-761- 771.pdf

Bonita, C., (2015). The Effects of Biomagnification in Plants on Non-Target Species. Retrieved from

https://prezi.com/m/okpey94eavug/the-effects-of-biomagnification-and- pesticides-on-no-target/

Buchel, K. H. (1983). Chemistry of pesticides. JohnbWiley-Blackwell & Sons, Inc. New York, USA.

Chen, C., Qian, Y., Chen, Q., Tao, C., Li, C. & Li Y., (2011). Evaluation of pesticide residues in fruits and

vegetables from Xiamen, China. Food Control, 22(2011):1114-1120. Retrieved from

https://www.researchgate.net/229411226_Evaluation_of_pesticide_residues_in_fruits_

and_vegetables_from_Xiamen_China/

Collotta, M., Bertazzi, P.A., & Bollati, V. (2013). Epigenetics and pesticides. 307(2013), 35-

41.Retrieved from http://www.sciencedirect.com/science/article/pii/S0300483X1300022X.

Criswell J.T., Shelton K. & Luper C. (2013) Pesticide Applicator Certification Series: Toxicity of

pesticides. Retrieved from

http://dasnr22.dasnr.okstate.edu/docushare/dsweb/Get/Version-11545/EPP- 7457web.pdf
52
Devon L. J. (2016). Pesticides found to cause transgenerational mental disorders and obesity. Harmful

traits are inherited for Three generations, Natural News, Thursday, March 17,

2016.Retrievedfromhttp://www.naturalnews.com/053340_pesticides_genetic_expression_transgener

ational

Edwards C. A., (1975). Factors that affect the persistence of pesticides in plants and soils. Pure and

applied Chemistry, 42(1-2): 39-56. Retrieved fromhttps://doi.org/10.1351/pac197542010039

European commission, (2014). Final report on an Audit carried out in Kenya. Retrieved from

http://ec.europa.eu

FAO&WHO, (2006). Chapter 8; Updating the Principles and Methods of Risk Assessment: Maximum

Residue Levels (MRLs) for pesticides and Veterinary drugs. Retrieved from

www.who.int/foodsafety/chem/residue_limits.pdf

Firestone J.A., Smith-Weller T., Frankline G., Swanson A., & Checkowary H. (2005). Pesticides and risk

of Parkinson disease: A population-based case control study. Archives neurology,62(1):91-

95.Retrieved from https://www.ncbi.nlm.nih.gov/pubmed/15642854

Fishel, F. M., (2013). Management and pesticides: A historical perspective. University of Florida.

Retrieved from https://edis.ifas.ufl.edu/pi007

Fishel, F.M., (2014). Pesticides Characteristics. Gainesville. University of Florida Institute of Food and

Agriculture. Retrieved from https://edis.ifas.ufl.edu/pi202

Goldman L.R., (1997). New approaches for assessing the etiology and risks of developmental

abnormalities from chemical exposure. Reproduction Toxicology.

Grandjean P., & Landrigan P. J., (2006). Developmental Neurotoxicity of Industrial

Chemical.Lancet,368(9553).

Hamilton D., Ambrus A., Dieterle R., Felsot A., Harris C., Petersen B., Racke K., Wong S-S., Gonzalez

R., Tanaka K., et al., (2004). Pesticide residues in food – acute dietary exposure. Pest Manag Sci.

60(4): 311-339. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/15119595

53
Hancock D.B., Martin E.R., mayhew G.M., Jewett R., Stacy M.A., Scott B.L., Vance J.M. & Scott W.K.,

(2008). Pesticide exposure and risk of Parkinson’s Disease: A Family- based Case-control study.

Hyland-Tassava S., (2013). Why are vegetables Important to the Human Body?. Retrieved from

http://www.livestrong.com/article/505412-why-are-vegetables-important-to-the- human-body/

Inonda R., Njage E., Ngeranwa J. & Mutai C., (2015). Determination of Pesticide Residue in Locally

Consumed Vegetables in Kenya. African Journal of Pharmacology and Therapeutics, 4(1), 1-6.

Retrieved from http://journals.uonbi.ac.ke/ajpt

International Agency for Research on Cancer, (2003). Fruits and Vegetables. Handbooks of Cancer

Prevention, Vol. 8. IARC press, Lyon, France.

Kithure J.G.N., Murungi J.I., Tum P.K., Wanjau R.N. & Thoruwa C. L., ( 2017). Fate of Lambda-

Cyhalothrin in Kales, Tomatoes and Cabbage from Rural setting in Kenya. International

Journal of Science and Innovative Technology, 4(2). Retrieved from

https://profiles.uonbi.ac.ke/patricktum/publication/fate-lambda-cyhalothrin-kales- tomatoes-and-

cabbage-rural-setting-kenya

Kithure J.G.N., Murungi J.I., Wanjau R.N. & Thoruwa C. L., (2014). Analysis of Deltamethrin Residue

Amounts Using HPLC in Some Vegetables Consumed in a Rural Area Makuyu, Kenya. The

International Journal of Science and technology, 2(12). Retrieved from

https://profiles.uonbi.ac.ke/jkithure/publications/analysis-deltamethrin-residue-amounts-using-hplc-

some-vegetables-consumed-rura-3.

Ministry of Agriculture, British Columbia, (2017). About Pesticides: types, Names and Formulations.

Retrieved from https://www2.gov.bc.ca>plant-health>about- pesticides.pdf

Mutuku, M., Njogu, P. & Nyagah G., (2014). Assessment of Pesticide use and Application Practices in

Tomato based Agrosystem in Kaliluni Sub-located, Kathiani District, Kenya. Journal of

Agriculture, Science and Technology, 16(2). Retrieved from

http://www.ajol.info/index.php/jagst/article/view/112901
54
Nasreddine L, & Parent-Massin D., (2002). Food contamination by metals and pesticides in the European

Union. Should we worry? Toxicology Letters, 127(1): 29–41.

Njogu, P., (2011). Assessment of pollution and prediction of Environmental risks of organochlorine

pesticide residues on aquatic communities in Lake Naivasha, Kenya.

Retrievedhttp://www.oceandocs.org/bitstream/handle/1834/6850/ktf0225.pdf?sequence=Nordby

K.C., Andersen A. Irgens L.M. & Kristensen P., (2005). Indicators of mancozebexposure in

relation to thyroid cancer and neural tube defects in farmers’ families. Scandinavian Journal of

Work, Environment and Health, 31(2): 89-96. Retrieved from

https://www.jstor.org/stable/40967474?seq=1#page_scan_tab_contents

Qaim, M., (2014). Evaluating Nutritional and Health Impacts of Agricultural Innovations. George-

August-University of Goettingen. Retrieved from http://www.uni- goettingen.de/globalfood

Repetto R. & Baliga S., (1996). Pesticides and the immune system. The public health risks. World

Resources Institute: Washington, DC. Retrieved from http://www.wri.org/publication/pesticides-

and-immune-system

Ruel, M. T., Minot, N. & Smith, L., (2005). Patterns and determinants of fruit and vegetable consumption

in sub-sahara Africa: a multicountry comparison. International Food

USFDA, (2016). Produce: Selecting and Serving it safely. Retrieved from www.fda.gov

Wanwimolruk S., Kanchanamayoon O., Phopin K. & Prachayasittikul V., (2015). Food safety in

Thailand 2: pesticide residues found in Chinese kale ( Brassica oleracea), a commonly

consumed vegetable in Asian countries. Science of the total Environment, 447-445.

Retrieved

WHO, (2003). Fruit and Vegetable promotion initiative. A meeting Report/ 25-27/08/03.

WHO, (2008). Chapter 8: Maximum Residue Limits for pesticides and Veterinary drugs, Principles and

methods for the risk Assessment of chemicals in Food. Retrieved from

http://www.who.int/foodsafety/chem/residuelimits.pdf
55
WHO, (2009). The WHO Recommended Classification of Pesticides by Hazard and Guidelines to

Classification 2009.

Zahm S.H. & Ward M.H., (1998). Pesticides and childhood cancer. Environ Health Perspect 106(3):

893-908.

Zhang Z., Zhao X. & Qin X., (2017). Potential genotoxic and cytotoxicity of emamectin benzoate in

human normal liver cells. Oncotarget, 8(47): 82185-82195, doi:10.18632/oncotarget.18988. .

Zhou P., Zhao Y., Li J., Wu G., Zhang L., Liu Q., Fan S., Yang X., Li. X., & Wu Y., (2012) Dietary

exposure to persistent organochlorine pesticides in 2007 Chinese total diet study. Journal of

Environment International, 42:152-159. Retrieved from

https://doi.org/10.1016/j.envint.2011.05.018

56
Appendices:

57