ABSTRACT
The injudicious and indiscriminate use of pesticides is a threat to not only the target organisms but to non-target organisms. This study determined the haemato-toxicity and histopathology of selected agro-pesticides (basudyne and carbofuran) in albino rats. Haematological parameters were determined at once for each blood sample using an automated haematology analyser model. Alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase (ALP) and serum bilirubin were determined using commercial kits and following standard protocols prescribed by the producer, Randox laboratory limited, United Kingdom. The liver and stomach histology were determined based on international histological protocol. The experiment was laid out in a complete randomized design (CRD). Each group was assigned a particular oral dose level of the pesticides in the order 5000, 7000, 9000, 11000, 13000 and 15000mg/kg body weight. The control group was not exposed to the treatment. The rats were sacrificed within 24 hours after administration of the toxicants. Blood collection by cardiac puncture into ethylenediaminetetraacetic acid (k3 EDTA) and plane bottles for haematology and liver function tests. The liver and stomach were preserved in 10% formalin for histological examination. Results obtained showed a gradual decrease in the mean and standard deviation values of red blood cell count, pack cell volume, haemoglobin, mean corpuscular haemoglobin and mean corpuscular haemoglobin concentration at P≤0.05 as the pesticides concentration increases. A rise and fall in mean and standard deviation is seen in white blood cell count. Neutrophils, lymphocytes and monocytes compared with the mean and standard deviation of the control group in both organophosphate based pesticide (basudyne) and carbamate based pesticide (carbofuran). Liver dysfunction biomarkers aspartate amino transferase, alanine aminotransferase, alkaline phosphatase, total protein, albumin and globulin concentrations were significantly increased when compared to the control group as the dose increases. The elevations observed in aspartate aminotransferase and alanine aminotransferase values though differed from that of the control, still fall within safety range. No observable treatment related effect was seen in the liver and stomach histology. The use of pesticides should be in a controlled rate which does not affect the non-target organism.
TABLE OF CONTENTS
Title
Page i
Declaration ii
Certification iii
Dedication iv
Acknowledgements v
Table
of Contents vi
List
of Tables ix
List
of Figures x
List
of Plates xi
Abstract xiv
CHAPTER 1: INTRODUCTION
1.0 Background
to the Study 1
1.1 Statement
of Problem 3
1.2 Aim
of Study 4
1.3 Specific
Objectives of the Study 4
1.4 Justification
of the Study 5
1.5 Significance
of the Study 6
1.6 Scope
of the Study 6
1.7 Research
Questions 7
1.8 Test
of Hypotheses 7
CHAPTER
2: LITERATURE REVIEW
2.0 Pesticides 9
2.1 Pesticide
Toxicity 9
2.2 Classification
of Toxicity by Type of Exposure 11
2.3 Classification of Toxicity by Route of
Entry 12
2.4 Organophosphate
Pesticides 14
2.5 Carbamates 14
2.5.1 Carbofuran 15
2.6 Organochlorides 15
2.6.1 Biochemical toxicity of organochlorines 16
2.7 Pyrethroides 17
2.8 Phenylamides 17
2.9 Triazines 17
2.10 Pesticides and their Applications 17
2.11 Agricultural Pesticide Use and Food Safety 18
2.12 Pesticide Misuse and Abuse 19
2.13 Effect of Pesticides to Living Organisms 20
2.14 Toxic Effect of Pesticides in Farm Animals 20
2.15 Potential Impact of Pesticides on Human
Health 21
2.15.1 Acute intoxication 22
2.15.2 Chronic intoxication 22
2.15.3 Neurobehavioral effects 23
2.15.4 Developmental and reproductive effects 23
2.15.5 Carcinogenic effects 24
2.15.6 Immunological effects 24
2.16 The Blood 24
2.17 Histopathology 25
2.18 Liver Function Parameters as Markers of
Toxicity 26
2.19 Changes in Haematological Parameters as
Indicators of Toxicity 27
CHAPTER 3: MATERIALS AND METHOD
3.1 Materials and Their Uses 28
3.2 Research Design 29
3.3 Test Organisms 29
3.4 Assessment of Safety of the Preparation 29
3.4.1 Grouping of analysis and treatment for
animals administered with carbamate
based pesticide (Carbofuran) 30
3.4.2 Animals administered with organophosphate
based pesticides 30
3.4.3 Acute toxicity test (LD50) 31
3.4.4 Dermination of haematological parameters 31
3.4.5 Determination of some biochemical parameters
31
3.4.6 Liver and stomach histology 33
3.5 Data Analysis and Statistical Procedures 34
CHAPTER 4: RESULTS AND DISCUSSION
4.1 Data Presentation 35
4.2 Data Analysis /Test of Hypotheses 49
4.2.1 Effect of
acute organophosphate based pesticide (basudyne) treatment on the
liver
histology of rats
4.2.2 Effect of acute organophosphate
based pesticide (basudyne) treatment on the
stomach histology of rats
4.2.3 Effect of acute carbamate based
pesticide (carbofuran) treatment on the liver histology of rats
4.2.4 Effect of acute carbamate based
pesticide (carbofuran) treatment on the stomach histology of rats
4.3. Discussion
4.3.1 Acute toxicity (LD50)
4.3.2 Effect of carbamate based pesticide (carbofuran) and
organophosphate based
pesticide (basudyne) in liver function biomaker parameters
4.3.3 Effect of carbofuran and basudyne on haematological
parameters
4.3.4 Effect of organophosphate based pesticide (basudyne) in
liver histology
4.3.5 Effect of carbamate based pesticide (carbofuran) in liver
histology
4.3.6 Effect of organophosphate based pesticide (basudyne)
in stomach histology
4.3.7 Effect of carbamate based pesticide (carbofuran) in stomach
histology
CHAPTER
5: CONCLUSION AND RECOMMENDATIONS
5.1 Conclusion 84
5.2 Recommendations 85
References 87
LIST OF TABLES
2.1 Types of toxicity based on the extent
of exposure to a pesticide 12
4.1.1 Haematological parameters for organophosphate
based pesticides 35
4.1.2 Haematological
parameters for organophosphate based pesticides 37
4.1.3 Haematological
parameters for carbamate based pesticides 39
4.1.4 Haematological
parameters for carbamate based pesticides (Carbofuran) 41
4.1.5 Liver dysfunction biomarkers test for
organophosphate based pesticides (Basudyne) 43
4.1.6 Liver dysfunction biomarkers test for
carbamate based pesticides
(Carbofuran) 45
4.1.7 Acute
toxicity study of carbamate based pesticides (Carbofuran) 47
4.1.8 Acute
toxicity study of organophosphate based pesticides (Basudyne) 47
4.2.1 Effect
of acute organophosphate based pesticide (basudyne) treatment
on RBC parameters 49
4.2.2 Test
of hypothesis for pack cell volume 50
4.2.3 Effect
of acute carbamate based pesticide (carbofuran) treatment
on RBC parameters 51
4.2.4 Test
of hypothesis for pack cell volume 52
4.2.5 Effect of acute organophosphate based
pesticides (Basudyne)
treatment on WBC and
differential WBC counts 53
4.2.6 Test
of hypothesis for total leucocyte count 54
4.2.7 Effect
of acute carbamate based pesticides (Carbofuran) treatment
on
WBC and differential WBC counts 55
4.2.8 Test
of hypothesis for total leucocyte count 56
4.2.9 Effect
of acute organophosphate based pesticide (basudyne) treatment
on liver dysfunction
biomarker 57
4.2.10 Test of hypothesis for liver dysfunction biomarkers 59
4.2.11 Effect
of acute carbamate based pesticide (Carbofuran) treatment on
liver dysfunction biomarkers 60
4.2.12 Test
of hypothesis for liver dysfunction biomarkers 64
LIST OF FIGURE
E
1 Main routes of pesticides entry
in the human organism 13
LIST OF PLATES
1
A cross section of a
normal rat liver showing the portal triad,
central vein, blood vessel and bile duct, H&E ×
100 (Control group) 63
2 A cross section of a rat liver given (5000mg/kg body
weight) of the
pesticide,
showing the portal triad, central vein, blood vessel and bile
duct,
H&E × 100 64
3 A cross section of a rat liver given (7000mg/kg body
weight) of the
pesticide,
showing the portal triad, central vein, blood vessel and bile
duct,
H&E × 100 64
4 A cross section of a rat liver given (9000mg/kg body
weight) of the
pesticide,
showing the portal triad, central vein, blood vessel and bile
duct,
H&E × 100 65
5 A cross section of a rat liver given
(11000mg/kg body weight)
of the pesticide, showing the portal triad, central
vein, blood vessel
and bile duct, H&E × 100 65
6 A cross section of a
rat liver given (13000mg/kg body weight) of the
pesticide,
showing the portal triad, central vein, blood vessel and
bile
duct, H&E × 100 66
7 A cross section of a
rat liver given (15000mg/kg body weight) of the
pesticide,
showing the portal triad, central vein, blood vessel and bile
duct.
H&E × 100 66
8 A cross section of a normal rat stomach showing the
gastric gland,
muscularis
mucosa, muscularis propria and surface epithelium
H&E ×
100 67
9 A cross section of a
rat stomach given (5000mg/kg body weight)
of the
pesticide, showing the gastric gland, muscularis mucosa,
muscularis
propria and surface epithelium H&E × 100
67
10 A cross section of a rat stomach given (7000mg/kg body
weight)
of the
pesticide, showing the gastric gland, muscularis mucosa,
muscularis propria and surface
epithelium H&E × 100 68
11 A cross section of a rat stomach given (9000mg/kg body
weight)
of the
pesticide, showing the gastric gland, muscularis mucosa,
muscularis
propria and surface epithelium H&E × 100 68
12 A cross section of a rat stomach given (11000mg/kg body
weight)
of the
pesticide, showing the gastric gland, muscularis mucosa,
muscularis
propria and surface epithelium H&E × 100 69
13 A cross section of a rat stomach given (13000mg/kg body
weight)
of the
pesticide, showing the gastric gland, muscularis mucosa,
muscularis
propria and surface epithelium H&E × 100 69
14 A cross section of a rat stomach given (15000mg/kg body
weight)
of the
pesticide, showing the gastric gland, muscularis mucosa,
muscularis
propria and surface epithelium H&E × 100 70
15 A cross section of a normal rat liver showing the portal
triad,
central
vein, blood vessel and bile duct, H&E × 100 70
16 A cross section of a
rat liver given (5000mg/kg body weight) of the
pesticide,
showing the portal triad, central vein, blood vessel and
bile
duct, H&E × 100 71
17 A cross section of a rat liver given (7000mg/kg body weight)
of the
pesticide,
showing the portal triad, central vein, blood vessel and
bile duct,
H&E × 100 71
18 A cross section of a rat liver given (9000mg/kg body weight) of
the
pesticide,
showing the portal triad, central vein, blood vessel and bile
duct,
H&E × 100 72
19 A
cross section of a rat liver given (11000mg/kg body weight) of the
pesticide,
showing the portal triad, central vein, blood vessel and bile
duct,
H&E × 100 72
20 A
cross section of a rat liver given (13000mg/kg body weight) of the
pesticide,
showing the portal triad, central vein, blood vessel and bile
duct,
H&E × 100 73
21 A cross section of a rat liver given (15000mg/kg body weight)
of the
pesticide,
showing the portal triad, central vein, blood vessel and bile
duct,
H&E × 100 73
22 A cross section of a
normal rat stomach showing the gastric gland,
muscularis
mucosa, muscularis propria and surface epithelium
H&E ×
100 74
23 A
cross section of a rat stomach given (5000mg/kg body weight)
of the
pesticide, showing the gastric gland, muscularis mucosa,
muscularis
propria and surface epithelium H&E × 100 74
24 A
cross section of a rat stomach given (7000mg/kg body weight)
of the
pesticide, showing the gastric gland, muscularis mucosa,
muscularis
propria and surface epithelium H&E × 100 75
25 A
cross section of a rat stomach given (9000mg/kg body weight)
of the
pesticide, showing the gastric gland, muscularis mucosa,
muscularis
propria and surface epithelium H&E × 100 75
26 A
cross section of a rat stomach given (11000mg/kg body weight)
of the
pesticide, showing the gastric gland, muscularis mucosa,
muscularis
propria and surface epithelium H&E × 100 76
27 A
cross section of a rat stomach given (13000mg/kg body weight)
of the
pesticide, showing the gastric gland, muscularis mucosa,
muscularis
propria and surface epithelium H&E × 100 76
28 A cross section of a rat stomach given (15000mg/kg body
weight)
of the
pesticide, showing the gastric gland, muscularis mucosa,
muscularis
propria and surface epithelium. H&E × 100 77
CHAPTER 1
INTRODUCTION
1.1 BACKGROUND
TO THE STUDY
Pesticides
are said to be universal chemical substances and global statistics have shown
increasing use of these chemicals for
pest control (Zhang et al., 2011).
Worldwide, about 2.5 million tons of pesticides are used per annum in
agriculture (Zhang et al., 2011). Newest
evidence on pesticide worldwide clearly shows an increase from around US$7
billion to US$12 billion from 2000 to 2012 with a related trend across the
world (Plumer, 2013). About one-third of agricultural yields are achieved by using pesticides
and without pesticide usage, the damage of fruits, vegetables, and cereals as a
result of pest damage would reach 78, 54, and 32%, respectively (Zhang
et al., 2011).
There
have been many studies on determining the cruel effects of pesticide contact (McCauley
et al., 2006).
The United Nation (UN) Environment Programme and the World Health Organization evaluated
that each year, 3million farm workers in the developing world experience stark
pesticide poisoning of which about 18,000 were fatal (Miller, 2004). Facts from
India, disclosed that about 51% of the food material is polluted with residues
in contrast to 21% worldwide, of which 20% were above minimal risk level agreed
by Food and Agricultural Organisation (FAO) standards (ANON, 1999). The
contaminated food is usually not thrown away in the developing countries, but
enters the food chain out of ignorance, innocence and also importantly out of
lack of affordability by the consumers. Absence of awareness of the degree of
pesticide contaminated food might remain one of the explanations for increased
occurrences of cancers in developing world. In addition to the damages to human
health, an indiscriminate use of chemical pesticides harmfully disturbs the
natural bio-diversity that results in the decrease of natural competitors (Ranga
Rao et al., 2005).
Food safety in sub-Saharan Africa
mostly rely on improved food productivity through the use of sustainable
agricultural practices and the decrease of post-harvest losses instigated by pests
and diseases. According to Okori et al. (2004), without pesticides food
would be more costly; because production would entail more labour and more
intensive knowledgeable management. For decades, the pest control policy in
developing countries has been dependent upon the use of synthetic pesticides
(Ogendo et al., 2004). Pesticides are lethal in nature and do not distinguish
between target and non-target species of plants and animals, and hence should be
subjected to safe and cautious use. Due to injudicious and indiscriminate use
of pesticides, too many accidents have happened in diverse parts of the world,
and incidences of pesticides in foods, fruits, vegetables, and environment and
even in mother’s milk is a matter of great worry (FAO, 2005). Of all the
pesticides released into the environment every year by humans, persistent
pesticides are amid the most dangerous (FAO, 2005). They are extremely toxic,
causing an array of adverse effects, especially death, diseases and birth
defects among humans and animals. Insecticides are the quickest and most
pragmatic means of combating an infestation, but their uses have been
restricted in many parts of Africa because of cost and their capacity to
contaminate the environment, leaving damaging residue in produce and induce
resistance in pest species (Lale, 2002).
The
exposure of human-beings to harmful chemicals directly in the fields and
indirectly via contaminated intake, leads in the expression of residues of
organochlorines in human blood (3.3–6.3 mg per L) and milk (3.2–4.6 mg per L)
samples from lactating women. Increased levels of pesticide residues (15–605
times) were spotted in blood samples of cotton farmers from four villages in
Punjab (ANON, 2005). The possible risks to pesticides applicator or farm worker
occupationally exposed to pesticides are higher than the risks to someone in
the overall population exposed only to traces of pesticides in food and /or
water (Amer, 2002). Haematological and biochemical parameters
have been related with health indices and are of diagnostic significance in the
day by day evaluation of the state of health; hence the analysis of these
parameters is important to risk evaluations of alterations of the
haematological system in humans and other animals (Saliu et al., 2012). Exposure to low level of pesticides is known to
produce diverse biochemical changes, some of which may be responsible for the
adverse biological effects in humans (Elhalwagy et al., 2009; Ibrahim et al.,
2011). In some cases, some biochemical alterations may not necessarily lead to
clinically recognizable symptoms, though all the biochemical reactions can be
used as indicators of exposure or effect. Haematological records are used for
diagnosis of diseases in mammals and is a prodigious help in veterinary and
human medicine and could be used to decide the health status of fishes (Saliu
et al., 2012).
1.2 STATEMENT OF PROBLEM
There have been many studies
on determining the ill effects of pesticide exposure (McCauley et al., 2006). Annually
there are dozens of million cases of pesticide poisonings worldwide. The World Health Organization and the UN Environment
Programme estimate that each year, 3 million farm workers in the developing
world experience severe pesticide poisoning of whom about 18,000 were fatal
(Miller, 2004). Exposure to pesticides both occupationally and
environmentally causes a range of human health problems. It is estimated that
nearly 10,000 deaths annually are linked to use of chemical pesticide
worldwide, with about three-fourths of these occurring in developing countries
(Horrigan
et al., 2002).
Exposure to pesticides results in acute and chronic health problems (Yassi
et al., 2001).
Excessive and non-judicious use
of insecticides have led to the degradation of environmental quality, pest
resistance, pest resurgence and the contamination of agricultural products and
natural resources. Most of the studies on pesticides conducted in Asia reflect
the presence of pesticide residues in significant amounts in food and
agricultural commodities, and pesticide pollution does exist in the country;
and is a cause of concern for public health (Kumari et al., 2006). Pesticides applied to the soil or that eventually
end in the soil in agricultural areas can contribute to the contamination of
surface and ground waters (Gilliom et al.,
2006).
Moreover, it is now better agreed that
pesticides have significant chronic health effects, including cancer,
neurological effects, diabetes, respiratory diseases, fetal diseases, and
genetic disorders. Various inappropriate practices in
the use of pesticides cause possible poisoning symptoms generally among farmers
who do not wear protective clothing (Ntow et
al., 2006). It is well known now that a significant fraction of
pesticides are carcinogenic. It is also well known that pesticide residues
persist for long periods of time, leading to complex environmental
implications. Also, pesticides destroy domestic animals, fishes and bees.
Furthermore, their use results in the development and evolution of pesticide
resistance in insects, weeds and plant pathogens.
Unfortunately,
there is a research gap on the haematotoxicity and selected organ changes as a
result of exposures to some selected pesticides using albino rats as
experimental units.
1.2 AIM OF STUDY
Thus,
this study aims at determining the haemato-toxicity and histopathology of selected
agro-pesticides in Albino rats.
1.3 SPECIFIC
OBJECTIVES OF THE STUDY
The
specific objectives of this study includes the following;
·
To determine the packed cell volume (PCV) of albino
rat orally administered with organophospate
based pesticide (basudyne)
·
To determine the packed cell volume (PCV) of albino
rat orally administered with carbamate based pesticide (carbofuran).
·
To determine the total leucocyte count (TLC) of
albino rat after administration of organophosphate based pesticide (basudyne).
·
To determine the total leucocyte count (TLC) of
albino rat after administration of carbamate based
pesticide (carbofuran).
·
Determination
of the effect of orally administered organophosphate based pesticide (basudyne)
on the liver function biomarkers of albino rats.
·
Determination of the effect of orally administered
carbamate based pesticide (carbofuran) on the liver function biomarkers of
albino rats.
·
To ascertain the
histopathology of orally administered carbamate based pesticide (carbofuran) in
the liver and stomach of albino rats.
·
To ascertain the
histopathology of orally administered organophophate based pesticide (basudyne)
in the liver and stomach of albino rats.
1.4 JUSTIFICATION
OF THE STUDY
The
risk of pesticides to human health has been of public concern since the 1970s.
However, hundreds of pesticides are used worldwide, and some pesticides are
used in some countries but not in others. For instance, the leading pesticide
which is used in corn production in the US is atrazine, but this pesticide has
been banned in the European Union countries because of its toxicity since 2004
(European Union, 2004).
The
use of chemicals in modern agriculture has significantly improved productivity.
But it has significantly increased the concentration of pesticides in food and
in our environment, with associated negative impacts on human health (Horrigan
et al., 2002). Globally, there is an
increasing pressure on the agricultural sector to produce more food to meet
increased demand of the growing populations all around the world (Horrigan
et al., 2002). This has increased the
need for intensive plant protection with increased use of pesticides. Despite
decades of research about pesticides, there is still high uncertainty about
health effects of pesticides. These health effects are diverse depending on the
degree, and the type of exposure. Perceptions by
farmers of pesticide efficacy were found to play a major role in farmers’
behaviour towards the use of pesticides and the adoption of alternative methods
of pest control such as integrated pest management (IPM), (Hashemi and Damalas 2010).
For example, pesticide used on any crop depends on the farmer’s discretion
whether to enhance the productivity to meet the market demands in search of
enhanced income or subsistence farming for livelihood. The necessity of
pesticide residue analysis in various agro-based commodities has become more
relevant.
1.5 SIGNIFICANCE
OF THE STUDY
The study examined the haemato-toxicity and vital
organ changes of albino rats exposed to some selected pesticides. The findings
of the study will help ensure that the significant portion of
food samples will not exceed
the maximum residue limits set by regulators. In addition, the finding will
also add to the understanding of the health effects of pesticides exposure.
Also, farmers will be enlightened on the health implications of using various
concentrations of pesticides during food storage.
1.6 SCOPE
OF THE STUDY
The study
was a laboratory based experiment set up. The routine haematological variables
were used to establish potential toxic effects of pesticides in albino rats.
The histopathology of the pesticides on vital organ of albino rats were equally
explored.
1.7 RESEARCH
QUESTIONS
This
research answered the following questions:
·
Does the oral administration of organophosphate
based pesticide (basudyne) affect the pack cell volume (PCV) of albino rats?
·
Does the oral administration of carbamate based
pesticide (carbofuran) affect the pack cell volume (PCV) of albino rats?
·
Does the oral administration of organophosphate
based pesticide (basudyne) affect the total leucocyte count (TLC) of albino rats?
·
Does the oral administration of carbamate based
pesticide (carbofuran) affect the total leucocyte count (TLC) of albino rats?
·
Does the oral administration of organophosphate
based pesticide (basudyne) affect the liver function biomarkers of albino rats?
·
Does the oral administration of carbamate based
pesticide (carbofuran) affect the liver function biomarkers of albino rats?
·
Does the oral administration of organophosphate
based pesticide (basudyne) affect the liver histology of albino rats?
·
Does the oral administration of carbamate based
pesticide (carbofuran) affect the stomach histology of albino rats?
1.8
TEST
OF HYPOTHESES
This research work tested
the following null hypotheses (H0):
· There
is no significant difference in the pack cell volume (PCV) of albino
rats orally administered with organophosphate based pesticide (basudyne) at p≤0.05
confidence interval.
· There
is no significant difference in the pack cell volume (PCV) of albino
rats orally administered with carbamate based pesticide (carbofuran) at p≤0.05
confidence interval
· There
is no significant difference in the total leucocyte count (TLC) of
albino rats orally administered with organophosphate based pesticide (basudyne)
at p≤0.05 confidence interval.
· There
is no significant difference in the total leucocyte count (TLC) of
albino rats orally administered with carbamate based pesticide (carbofuran) at
p≤0.05 confidence interval.
· There
is no significant difference in the liver function biomarkers of
albino rats orally administered with organophosphate based pesticide (basudyne)
at p≤0.05 confidence interval.
· There
is no significant difference in the liver function biomarkers of
albino rats orally administered with carbamate based pesticide (carbofuran) at
p≤0.05 confidence interval.
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