ABSTRACT
This study was aimed at comparatively evaluating the hepatotoxicity, haematological and antioxidant effects of differently prepared (parboiled and un-parboiled) beans treated with sniper (i.e. a dichlorvos insecticide) and fed to Wistar albino rats. Thirty (30) male Wistar albino rats of known body weight were assigned into six (6) groups of 5 rats each. The beans were treated with 0.7 ml (high dose) and 0.3 ml (low dose) of dichlorvos per one kg of beans. Group 1 and group 2 of un-parboiled beans compounded with standard feed stock, group 3 and group 4 were fed with high dose and low dose of parboiled beans compounded with feed stock, group 5 received beans only while group 6 received standard feed for a period of 30 days. The rats were euthanized, and blood samples were collected after the termination of the study. The increases in the activities of liver enzymes (ALT, AST and ALP) in the rats’ sera of different groups showed a hepatocellular damage as a result of dichlorvos toxicity which also significantly (P<0.05) decreased the activities of the antioxidant enzymes (GSH, GPx, SOD, CAT) in the rats of groups fed with un-parboiled beans unlike the groups fed with parboiled beans which were non-significantly decreased. There was significant increase in the malondialdehyde concentration of the rats of the groups fed with un-parboiled beans when compared to group 6 rats. Dose dependent variations were seen in the packed cell volume (PCV), white blood cell (WBC), haemoglobin (Hb) and platelet. However, a reduced concentration of red blood cell (RBC) count for the un-parboiled groups and an increase in the parboiled group were seen, although both were not significant (P>0.05). Consumption of un-parboiled beans exposes consumers to the risk of dichlorvos contamination and its harmful effects as seen in this study, hence there is need for the parboiling of beans which helps to reduce the dichlorvos residues deposited on beans.
TABLE OF
CONTENTS
Title Page i
Declaration ii
Dedication iii
Certification iv
Acknowledgements v
Table of Contents vi
List of Tables ix
List of Figures x
List of Plates xi
Abstract xii
CHAPTER 1: INTRODUCTION
1.1 Background of the Study 1
1.2 Prevalence
of Pesticides in Agricultural Products 2
1.3 Statement
of the Problem 3
1.4 Justification of the Study 3
1.5 Aim of the Study 4
1.6 Objectives of the Study 4
CHAPTER 2: LITERATURE REVIEW
2.1. Cowpea 5
2.1.1 Nutritional
composition of cowpea 6
2.1.2 Preservation
and storage of cowpea 7
2.2 Pesticides 9
2.2.1 Organochlorines
10
2.2.2 Carbamates 13
2.2.3 Pyrethroids 13
2.2.4 Organophosphorous 15
2.3 Metabolic Fate of Dichlorvos 16
CHAPTER 3:
MATERIALS AND METHODS
3.1 Materials 19
3.1.1 Dichlorvos 19
3.1.2 Beans 19
3.1.3 List of chemicals/reagents used 19
3.1.4 List of equipment used 20
3.1.5 Experimental animals 20
3.2 Method 20
3.2.1 Rats 20
3.2.2 Feed formulation 21
3.2.3 Experimental design and animal grouping 21
3.2.4 Serum preparation 22
3.2.5 Biochemical analyses 22
3.2.5.1 Assay
of serum aspartate amino transferase (AST) activity 22
3.2.5.2 Assay
of serum alanine aminotransferase (ALT) activity 23
3.2.5.3
Assay of serum alkaline
phosphatase (ALP) activity 24
3.2.5.4 Estimation
of serum reduced glutathione concentration 24
3.2.5.5 Assay of glutathione peroxidase
activity 25
3.2.5.6 Assay of catalase activity 26
3.2.5.7 Assay of superoxide dismutase
activity 27
3.2.5.8 Determination of serum
malondialdehyde concentration 28
3.2.5.9 Determination
of serum urea concentration 28
3.2.5.10 Determination of serum total protein concentration 29
3.2.5.11 Determination of serum
bilirubin concentration 30
3.2.5.12 Determination
of total white blood cell count by
haemocytometry 31
3.2.5.13 Determination
of platelet count 32
3.2.5.14 Packed
cell volume (PCV) estimation 33
3.2.5.15 Determination
of haemoglobin concentration 34
3.2.5.16 Determination
of erythrocyte count by haemocytometry 35
3.2.5.17 Determination of
mean corpuscular haemoglobin concentration
(MCHC) 36
3.2.5.18 Determination
of mean corpuscular volume (MCV) 36
3.2.5.19 Determination
of mean corpuscular hemoglobin (MCH) 37
3.2.6 Histopathological examination 37
3.2.6.1 Tissue
preparation 37
3.2.6.2 Slide
examination 37
3.2.7 Statistical
analysis 38
CHAPTER 4: RESULTS AND
DISCUSSIONS
4.1 Results 39
4.1.1 Effect of dichlorvos on aspartate
transaminase activity in Wistar rats 39
4.1.2 Effect of dichlorvos on alanine amino
transferase activity in Wistar
rats 40
4.1.3 Effect of dichlorvos on alkaline phosphatase
activity in Wistar rats 41
4.1.4 Effect of dichlorvos on serum glutathione
activity in Wistar rats 42
4.1.5 Effect of dichlorvos on serum glutathione peroxidase activity in
Wistar rats 43
4.1.6 Effect of dichlorvos on serum catalase
concentration in Wistar rats 44
4.1.7 Effect of dichlorvos on serum superoxide
dismutase activity in Wistar
rats 45
4.1.8 Effect of dichlorvos on serum
malondialdehyde concentration in Wistar
rats 46
4.1.9 Effect
of dichlorvos on serum urea concentration in Wistar rats 47
4.1.10 Effect of dichlorvos on serum total protein
concentration in Wistar rats 48
4.1.11 Effect
of dichlorvos on serum total bilirubin concentration (g/dl) in
Wistar rats 49
4.1.12 Effect of dichlorvos on serum direct bilirubin
concentration in Wistar
rats 50
4.1.13 Effect
of dichlorvos on some haematological
parameters 51
4.1.14 Histopathology 52
4.1.14.1 Histopathology
of the liver tissues of group 1 Wistar rats
exposed to dichlorvos 52
4.1.14.2 Histopathology
of the kidney tissue of group 1 Wistar rats
exposed to dichlorvos 53
4.1.14.3 Histopathology
of the liver tissues of group 2 Wistar rats
exposed to dichlorvos 54
4.1.14.4 Histopathology
of the kidney tissues of group 2 Wistar rats
exposed to dichlorvos 55
4.1.14.5 Histopathology
of the liver tissues of group 3 Wistar rats
exposed to dichlorvos 56
4.1.14.6 Histopathology
of the kidney tissues of group 3 Wistar rats
exposed to dichlorvos 57
4.1.14.7 Histopathology
of the liver tissues of group 4 Wistar rats
exposed to dichlorvos 58
14.1.14.8 Histopathology
of the kidney tissues of group 4 Wistar rats
exposed to dichlorvos 59
14.1.14.9 Histopathology
of the liver tissues of group 5 Wistar rats
exposed to dichlorvos 60
14.1.14.10 Histopathology of the kidney
tissues of group 5 Wistar rats
exposed to dichlorvos 61
14.1.14.11 Histopathology of the liver
tissues of group 6 Wistar rats
exposed to dichlorvos 62
14.1.14.12 Histopathology of the kidney
tissues of group 6 Wistar rats
exposed to dichlorvos 63
4.2 Discussions 64
CHAPTER 5: CONCLUSION AND
RECOMMENDATIONS
5.1 Conclusion 70
5.2 Recommendations 70
References 72
Appendix
LIST OF
TABLES
Page
3.1: List of
chemicals, reagents and their manufacturers. 19
3.2: List of equipment and their manufacturers. 20
4.1: Effect
of dichlorvos on some haematological parameters 51
LIST OF FIGURES
Page
1.1: Structure
of dichlorvos 1
2.1: Examples
and structures of organochlorine insecticide 12
2.2: Example
of structures of carbamate insecticide (cabaryl) 13
2.3: Example of structures of pyrethroid insecticide 14
2.4: Examples and structures of some
organophosphate pesticides. 16
2.5: Proposed pathway for the breakdown of
dichlorvos in
soil
and water/sediment system, including abiotic hydrolysis and
microbial
degradation steps. 17
2.6: Mammalian pathway of metabolism of dichlorvos 18
4.1:
Activity of aspartate transaminase in
Wistar rats of different rat groups 39
4.2:
Activity of alanine amino transferase
in Wistar rats of different rat
groups 40
4.3:
Activity of alkaline phosphatase in Wistar rats of different rat groups 41
4.4:
Serum glutathione activity in Wistar rats of different rat groups 42
4.5:
Serum glutathione peroxidase activity
in Wistar rats of different rat
groups 43
4.6: Serum
catalase activity in Wistar rats of different
rat groups 44
4.7: Serum
superoxide dismutase activity in Wistar
rats of different rat
groups 45
4.8:
Serum total malondialdehyde
concentration in Wistar rats of different
rat
groups 46
4.9: Serum
urea concentration in Wistar rats of different
rat groups 47
4.10:
Serum total protein concentration in Wistar rats of different rat groups 48
4.11: Serum
total bilirubin concentration in Wistar
rats of different rat
groups 49
4.12:
Serum direct bilirubin concentration in
Wistar rats of different rat groups 50
LIST
OF PLATES
Plate Page
1a: Histology of the liver tissue of group 1 rats 52
1b: Histology of the kidney tissue of group 1 rats 53
2a: Histology of the liver tissue of group 2 rats 54
2b: Histology of the kidney tissue of group 2 rats 55
3a: Histology of the liver tissue of group 3 rats 56
3b: Histology of the kidney tissue of group 3 rats 57
4a: Histology of the liver tissue of group 4 rats 58
4b: Histology of the kidney tissue of group 4 rats 59
5a: Histology of the liver tissue of group 5 rats 60
5b: Histology of the kidney tissue of group 5 rats 61
6a: Histology of the liver tissue of group 6 rats 62
6b: Histology of the kidney tissue of group 6 rats 63
CHAPTER 1
INTRODUCTION
1.1
BACKGROUND OF THE STUDY
Dichlorvos
(2,2-dichlorovinyldimethylphosphate) was first introduced in 1961 (Mennear,
1998). It has a molecular formular, C4H7Cl2O4P
(Fig 1.1), and molecular weight of 220.98. It is one of the most commonly used
organophosphate pesticides in developing countries (Binukumar and Gill, 2010).
The WHO categorized it as Class IB, ‘highly hazardous chemical (WHO, 1992).
Fig. 1.1: Structure
of dichlorvos
|
Dichlorvos is a highly volatile organophosphate,
a pesticide which is used to control domestic pests, in environmental health, as
well as preserving stored agricultural produce from storage insects like
weevils.
Apart
from its usage in agriculture as an insecticide for stored products, it has
been reported that it is also used as an anti-helminthes (de-worming agent) for
dogs, swine, and horses, as a botacide; agent that kills fly larvae (USEPA,
1994; Carpenter, 2004). Organophosphates function mainly by the inhibition of
acetylcholinesterase – an enzyme that breaks down acetylcholine (Lewalter et al., 1986; Harlin et al., 1993; Yair et al., 2008). Being an acetylcholinesterase inhibitor, its
overdose symptoms include weakness, headache, and chest congestion, blurred
vision, salivation, sweating, nausea, vomiting, diarrhea, respiratory failure,
and abdominal cramps. It is mainly metabolized by esterase to dimethyl
phosphate and dichloroacetaldehyde in mammals (Loeffler et al., 1976; Wright et al.,
1979).
1.2
PREVALENCE OF PESTICIDES IN AGRICULTURAL PRODUCTS
Pesticides are ubiquitous and global statistics have revealed
increasing use of these chemicals for the control of pests (Zhang and Jiang, 2011). Pesticide, a damage control
input to safeguard from insects and other pests, is considered to improve
nutrition in food, and its use is assumed an economic, labour-saving, and
efficient tool for pest management (Damalas and Eleftherohorinos, 2011). They are poisons; however, they are
produced because they are toxic to one pest or the other (Banjo et al., 2010).
Although
pesticides play great roles in the management of agricultural enterprise, where
they potentiate yields and protect crops against insects at post-harvest and storage
stages, which have given modern agriculture a solid foundation because of its
unquantifiable benefits, including enhancement of shelf life of stored
agricultural products (Olabode et al.,
2011). It is worthy of note that insects and pests are getting immune to
the commercial pesticides due to over usage, hence leading to the development
of multiple pesticides with different active ingredients targeted at multiple
species (Speck-Planche et al., 2012).
Furthermore, the introduction of
pesticides in agriculture has improved the sector by giving it a competitive
advantage where agricultural products are consumed more than synthetic products
(Delcour et al., 2015). Hence pesticide
use is deemed essential for retaining current production and yield levels, as
well as maintaining a high-quality standard of life (Delcour et al., 2015).
The
World Health Organization (WHO) defined a pesticide as a chemical compound that
is used to kill pests, including insects, rodents, fungi and unwanted plants
(weeds) (WHO, 2017). Through some environmental processes such as leaching,
rainfall, etc, pesticides move from treated agricultural
areas into the broader environment, thereby affecting non-target organisms (Osman et al., 2010). This implies that pesticide usage may yield undesirable
results as a result of pesticide residues deposited as contaminants in trace
amounts on food, the environment and living tissues.
1.3
STATEMENT OF PROBLEM
Significant
contamination of dried beans has been reported in Nigeria. For example, Hassan (2018) in the Nation newspaper on Sunday, April 5 2015, it
was reported that 42 food items produced in the country were rejected by United
Kingdom (UK) for quality defects. Again, in the Editorial newspaper Thursday,
July 30 2015, European Union (EU) suspended the importation of dried beans
originating from Nigeria as a result of pesticide residues in them (Hassan et al., 2018). Recently,
retailers of beans in Nigeria in an attempt to protect the stored beans from
the attack of pests, they mix the beans with sniper – 2,2-dichlorovinyl
dimethyl phosphate (DDVP). And according to punch newspaper on 23rd
March, 2017, three (3) sellers of beans were arrested for mixing sniper with
beans (Afeez, 2017). This means that some of the beans consumed in Nigeria
are always preserved with organophosphates which indirectly poison the
consumers of beans.
1.4
JUSTIFICATION
Sequel to the
incident of poisoned beans, beans consumers are
advised to extensively parboil their beans before consumption. There has been
the argument that washing and parboiling beans would eliminate the pesticide.
Considering the regular pattern of beans preservation, there is need to
ascertain the safe level of different delicacies prepared with beans, either
parboiled or not (Harrison,
et al., 2018).
1.5
AIM
OF THE STUDY
This study was
aimed at evaluating the effect of different concentrations of
2,2-dichlorovinyldimethyl phosphate (DDVP) in both parboiled and un-parboiled
beans on some biochemical, antioxidant and haematological in albino Wistar rats.
1.6
OBJECTIVES
The objectives
of this study were to:
a)
determine liver toxicity
biomarkers (AST and ALT, ALP) of the various blended feed in the serum and
liver homogenate.
b) determine serum reduced glutathione concentration.
c) determine the levels
of the serum antioxidant enzymes (CAT, SOD, GPx).
d) determine the
extent of lipid peroxidation using malondialdehyde as the biomarker.
e) determine the
concentration of urea, total protein, direct and total bilirubin.
f) determine the effect of 2,2-dichlorovinyldimethyl
phosphate on haematological parameters and
g) assess the histopathological implications of frequent consumption of
dichlorvos-contaminated beans in the liver and kidney tissues.
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