ABSTRACT
This study was carried out to determine sub-lethal effects of Atrazine herbicide in juveniles of Clarias gariepinus and Hybrid (Heterobranchus longifilis X Clarias gariepinus). Fish species were exposed to acute concentrations of 5mg/l, 10mg/l, 15mg/l, and 20mg/L of Atrazine, as well as sub-lethal doses of 0.6mg/l, 1.3mg/l and 2.5mg/L of Atrazine. Both experiments had a control each, which served as a reference point. The LC50 value of atrazine established from the study was 12.5mg/L for both species. A total number of one hundred and twenty (120) juveniles each of Clarias gariepinus and Hybrids of mean weight 7.60±0.10g and mean standard length 3.51±0.13cm, (regardless of sex) were exposed to sub-lethal atrazine concentration for 12 weeks. The effect of the sub-lethal exposure of both fish species to atrazine, was evaluated using standard methods that assessed fish behaviour, histology, haematological, biochemical, proximate composition and atrazine accumulation in the muscles. Atrazine induced hyperactivity and loss of equilibrium at acute levels of exposure. The physico-chemical parameters obtained before and after the experiment were within the tolerated limit. Histopathological analysis of the fish organs examined revealed varying degrees of pathological alterations to the gill and liver of both fish species. The gill showed alterations like fusion and shortening of primary and secondary lamella. The liver showed histopathological changes such as congestion of central vein and degeneration of hepatocytes amongst other alterations. Assessment of haematology and biochemical parameters revealed significant differences between exposed groups and control. Atrazine also has a dose dependent effect on the growth parameters, with both highest and lowest growth rate recorded in the control and exposed group of Hybrids respectively. Proximate composition of exposed groups was significantly different from the control of both species. Atrazine accumulation in the muscle was found highest in Hybrid juveniles exposed to highest Atrazine concentration (2.5mg/l). Results of the sub-lethal toxicity test showed that Atrazine was toxic to the both species in a dose-dependent manner. The toxicity assessment parameters were more pronounced in the exposed groups of Hybrids than Clarias gariepinus juveniles though not statistically different. All parameters for toxicity assessment showed significant differences in the exposed groups when compared to the control. The study revealed that Atrazine was moderately toxic to C. gariepinus and Hybrid (Heterobranchus longifilis X Clarias gariepinus) juveniles and affected the behaviour, haematology, biochemistry, histology, as well as growth of the fish. Hence, the use of the herbicide should be used with caution especially near water bodies.
TABLE
OF CONTENTS
Title page i
Certification ii
Declaration iii
Dedication iv
Acknowledgement v
Table of Contents vi
List of Tables x
List of Plates xii
List of Figures xiii
Abstract xiv
CHAPTER 1: INTRODUCTION
1.1 General Background 1
1.2 Statement of Problem 3
1.3 Justification 4
1.4 Objectives 6
CHAPTER 2: LITERATURE
REVIEW
2.1 General Scope of Pesticides 7
2.2 Effects of Pesticide on the Ecosystem 8
2.3 Atrazine as a Herbicide 10
2.3.1 Properties of atrazine at standard temperature
and pressure 11
2.4 Mode of Action 11
2.5 Environmental Fate of Atrazine 12
2.6 Toxicity of Atrazine 13
2.7 Atrazine Toxicity to other Animals 14
2.8 Effects of Atrazine on Humans 15
2.9 Atrazine Toxicity to Fish 17
2.9.1 Susceptibility of fish to toxicants 18
2.9.2 Pesticide toxicity and fish behavior 19
2.9.3 Pesticide
toxicity and fish histology 21
2.9.4 Pesticide
toxicity and fish haematology and biochemical
Properties 20
2.9.5 Pesticide toxicity and fish growth 27
[
CHAPTER 3: MATERIALS AND
METHODS
3.1. Experimental Fish Specimens 29
3.2 Preparation of Test Solution 29
3.3 Exposure of Fish Specimens to Atrazine 30
3.3.1 Range Finding test for the determination of atrazine
concentration 30
3.3.2 Acute toxicity of atrazine herbicide in
Clarias
gariepinus juveniles. 30
3.3.3 Acute toxicity of atrazine herbicide in hybrid juveniles. 31
3.4 Experimental Design 32
3.5 Experimental Procedure 34
3.5.1 Fish growth analysis 34
3.5.1.1
Mean weight gain 34
3.5.1.2
Specific growth rate 34
3.6 Haematological Analysis 34
3.6.1 Determination
of packed cell volume (PCV) 35
3.6.2 Determination of haemoglobin
concentration (Hb) 35
3.6.3 Red blood cell (RBC) count 36
3.6.4 Total
white blood cell (WBC) count 36
3.7 Determination
of Serum Biochemical Indices 37
3.7.1 Alanine amino transferase (ALT) 37
3.7.2 Aspartate amino transferase (AST) 38
3.7.3 Determination
of total protein 39
3.7.4 Determination
of cholesterol levels 40
3.7.5 Determination
of blood glucose 40
3.8 Determination of Atrazine Accumulation of
Fish Muscle of
Control and Exposed Groups. 40
3.9 Determination
of Proximate Composition of Experimental
Fish Muscle 41
3.9.1 Moisture
content 41
3.9.2 Ash
content 41
3.9.3 Crude
fibre 42
3.9.4 Crude
fat 43
3.9.5 Determination of
crude protein 43
3.9.6 Determination
of carbohydrate 44
3.10
Histopathological Examinations 44
3.11 Water Quality Determination 44
3.12 Data Analysis 45
3.13 Study Area 45
CHAPTER 4: RESULTS AND
DISCUSSION
4.1 Lc50 of Atrazine, Behavioural Responses, and
Physio Chemical
Parameters of Exposed Groups 46
4.2 Effect
of Atrazine on the Haematological Parameters of
C. gariepinus and Hybrid
Juveniles Exposed to Sub-lethal
Concentrations of Atrazine. 50
4.3 Effects of Atrazine on the Biochemical Parameters
of
Clarias
gariepinus and Hybrid Juveniles 56
4.4 Histopathology of Gills and Liver of Clarias gariepinus and Hybrid
Juveniles Exposed to Sub-lethal Concentrations
of Atrazine 62
4.4.1
Histopathology of gills of Clarias
gariepinus and hybrid
juveniles exposed to sub-lethal concentrations of atrazine 62
4.4.2
Histopathology of Liver of Clarias
gariepinus and hybrid
juveniles exposed to sub-lethal concentrations of atrazine 63
4.5 Effects of Atrazine on the Proximate
Composition of Muscles
of Clarias
gariepinus and Hybrid Juveniles 75
4.6 Atrazine Accumulation in the Muscle of Clarias gariepinus and
Hybrid Juveniles 78
4.7 Sub-lethal Effects of Atrazine on the
Growth of C.gariepinus and
Hybrid Juveniles 79
CHAPTER 5: CONCLUSION AND
RECOMMENDATION
5.1 Conclusion 88
5.2 Recommendation 89
References 90
LIST
OF TABLES
3.1: Acute Toxicity of Atrazine in Clarias gariepinus Juveniles. 31
3.2: Acute Toxicity of Atrazine in Hybrid Juveniles. 32
3.4.1:
Experimental setup Clarias gariepinus juveniles 33
3.4.2:
Experimental setup hybrid juveniles. 33
4.1: Effect of Atrazine on the Haematological
Parameters of
C. gariepinus and Hybrid Juveniles
Exposed to Varying
Concentrations
of Atrazine. 51
4.2: Effect of Atrazine on the Biochemical Parameters
of
C. gariepinus and Hybrid
Juveniles Exposed to Varying
Concentrations of Atrazine. 57
4.3: Proximate
Composition of C. gariepinus and
Hybrid Juvenile
Muscle Exposed to Varying Concentrations
of Atrazine. 76
4.4: Accumulation of Atrazine in Muscles of C. gariepinus
and Hybrid Juveniles
Exposed to Varying Concentrations
of Atrazine. 79
4.5: Sub-lethal Effects of Atrazine on the Growth
of C. gariepinus
and Hybrid
Juveniles. 85
LIST
OF PLATES
1A Histopathology of Gill of Clarias gariepinus Juvenile
Exposed to 0mg/l of Atrazine Showing
Normal Primary and
Secondary Lamellae and Afferent Artery 65
1B Histopathology of Gill of Hybrid Juveniles Exposed
to
0mg/l of Atrazine Normal Primary and
Secondary Lamellae
with Afferent Artery and Cartilaginous
Core 65
2A Histopathology of Gill of Clarias gariepinus Juvenile
Exposed to 0.6mg/l of Atrazine Showing
Congestion of
Primary Lamellae at the Extremities 66
2B Histopathology of Gill of Hybrid Juveniles Exposed
to
0.6mg/l of Atrazine Showing Congestion
of Primary Lamellae
at the Extremities and Mild Fusion of
Secondary Lamellae. 66
3A Histopathology of Gill of Clarias gariepinus Juvenile
Exposed to 1.3mg/l of Atrazine Showing
Disintegration of
Arterial Walls Fusion of Primary
Lamellae at the Extremities 67
3B Histopathology of Gill of Hybrid Juveniles
Exposed to
1.3mg/L of
Atrazine Showing Fusion of Primary Lamellae Shortening of Secondary Lamellae and
Mild Blood Vessels
Congestion. 67
4A Histopathology of Gill of Clarias Gariepinus Juvenile Exposed
to 2.5mg/L of Atrazine Showing Swelling
and Disruption of
both Primary and Secondary Lamellae at the Extremities.
68
4B Histopathology
of Gill of Hybrid Juveniles Exposed to 2.5mg/l
of Atrazine Showing
Fusion/Shortening/Swelling of both
Primary and Secondary Lamellae and
Disruption of the
Cartilaginous core
68
5A Histopathology
of Liver of Clarias gariepinus Juvenile
Exposed to 0mg/l
of Atrazine showing Normal Central
Vein and Small
Blood Vessels Dispersed at Intervals 69
5B Histopathology of Liver of Hybrid Juveniles Exposed
to
0mg/l
of Atrazine Central Vein and Small Blood Vessels
Dispersed at Intervals 69
6A Histopathology of Liver of Clarias gariepinus Juvenile
Exposed
to 0.6mg/l of Atrazine Showing Perivascular Cuffing
around the Central Vein and
Congestion of Small Blood
Vessels 70
6B Histopathology of Liver of Hybrid Juveniles Exposed
to
0.6mg/l of Atrazine Congestion of
Central Vein a Mild
Congestion of Sinusoids 70
7A Histopathology of Liver of Clarias gariepinus Juvenile
Exposed to 1.3mg/l of Atrazine
showing Mild Congestion of
Central Vein and Congestion of Sinusoids. 71
7B Histopathology of Liver of Hybrid Juveniles Exposed
to 1.6mg/l
of Atrazine Showing Congestion around
the Central Vein and
Mononuclear Inflammatory Cells in the Liver Parenchyma. 71
8A Histopathology of Liver of Clarias gariepinus Juvenile
Exposed to 2.5mg/l of Atrazine Showing
Congested Central
Vein,
Congested Sinusoids and other Blood Vessels 72
8B Histopathology of Liver of Hybrid Juveniles
Exposed to 2.5mg/l
of Atrazine Showing Congested
Sinusoids, Areas of Fatty
Degeneration of Hepatocytes and
Inflammatory Cells Linning
the
Blood Vessels 72
LIST
OF FIGURES
1: Graphical Representation of Growth Rate of
Clarias gariepinus Juveniles. 81
2: Graphical Representation of Growth Rate
of Hybrid
Juveniles 81
4.3: Graphical Representation of Growth Rate of Clarias
gariepinus
and Hybrid Juveniles Exposed to 0.6 mg/l of
Atrazine. 82
4.4:
Graphical Representation of Growth Rate
of Clarias
gariepinus and Hybrid Juveniles
Exposed to 1.3 mg/l of
Atrazine. 83
4.5:
Graphical Representation of Growth Rate
of Clarias
gariepinus and Hybrid Juveniles
Exposed to 2.5 mg/l of
Atrazine. 84
CHAPTER 1
INTRODUCTION
1.1 GENERAL BACKGROUND
The use of herbicides to control weeds has been a
common practice in global agriculture, mainly with the objective to increase
agricultural production. According to Vasilescu and Medvedovici (2005),
herbicides are defined as any substance, individually or in mixtures, whose
function is to control, destroy, repel or mitigate the growth of weeds in a
crop. Herbicides are the most used chemical substances throughout the world (He
et al., 2012).
They are used extensively for agricultural purposes
and their use is necessary to control nuisance organisms and increase crop
yields to support the rising global population. However, when these chemicals are
used in an uncontrolled manner, they can cause impacts on non-target organisms,
especially on those that live in aquatic environments.
Major disadvantages of herbicides are that they are
not biodegradable and as a result can persist in the environment for a long
period of time, also they are mildly toxic, cause diseases and even accidental
death. They can even be carried into rivers by rainwater or be leached to
groundwater thereby causing pollution of the underground water reserve.
Some herbicides can accumulate in the food chain and
become toxic for animals, including man.
Some herbicides, when at low concentrations, cannot
cause immediate detectable effects in the organisms, but, in long term can reduce
their lifespan longevity. The contamination of aquatic environments by
herbicides has been characterized as a major world concern.
This aquatic contamination is due to the use of these
products in the control of aquatic plants, leachate and runoff of agricultural
areas. It is a growing public concern about the amount of herbicides that have
been introduced into the environment by leachate and runoff, not to mention
that the contaminations of the aquatic environments generally occur by a
mixture of these compounds and not by isolated substances (He et al., 2012)
Atrazine, a well-known herbicide was invented in 1958
in the Geigy
laboratories as the second of a series of 1, 3, 5-triazines (Wolfgang
Krämer 2007). The molecular weight of Atrazine is 215.69 and
its chemical formula is: (2–chloro-4-ethylamino-6-
isopropylamino-S-triazine). Like other triazine
herbicides, atrazine functions by binding to the plastoquinone-binding
protein
in photosystem
II, which animals lack.
Plant death results from starvation and oxidative
damage caused by breakdown in the electron transport
process. Oxidative damage is accelerated at high light intensity (Appleby
et al., 2001).
Atrazine is one of the most widely used pesticides in
the world. Approximately 80 million pounds are applied annually in the United
States alone, and atrazine is the most common pesticide contaminant of ground
and surface water (Solomon et al.,
2013).
Atrazine has been proven to covalently bind to a large
number of mammalian proteins (Dooley et al., 2008). Atrazine has been
known to have a persistent effect on the environment. Due to Atrazine's
persistence in the environment and its readiness to enter water systems via
runoff, drinking
water contamination
is a major public health issue in areas of heavy application (ATSDR, 2003)
Fish are often used as sentinel organisms for
ecotoxicological studies because they play a number of roles in the trophic
web, accumulate toxic substances and respond to low concentrations of mutagens
(Cavas and Ergene-Gözükara, 2005). To access environmental quality, fish are
usually used as bio indicators. They have the ability to detect the presence of
chemicals even at sub-lethal levels.
Toxicity testing in fish is of great concern due to
their potential adverse effects on human health after consumption, thus
toxicity studies are essential to determine sensitivity of animals to toxicants
and also useful for evaluating the degree of damage to target organs and the
consequent physiological, biochemical and behavioral disorders (Omoniyi 2018).
1.2 STATEMENT OF
PROBLEM
Worldwide use of pesticides in 2007 was
estimated at 2.4 billion kg of which the largest proportion, 40% or 950 million
kg was herbicides (U.S EPA, 2012a). The
intense use of herbicides on agricultural fields to control diseases and
increase food production has contributed to environmental pollution in both terrestrial
and aquatic ecosystems.
The presence of pesticides in the environment
has caused significant social and scientific development anxiety worldwide, as
their all over the world extensive usage can create potential risks to the
environment and human health, as they can easily pollute bodies of water,
resulting in extensive damage to non-target species, including fish (Moreno et
al., 2010).
Atrazine can be transported more than 1,000 km from
the point of application via rainfall and, as a result, contaminates otherwise
pristine habitats, even in remote areas where it is not used (Thurman and
Cromwell, 2000; Mast et al., 2007).
Fish serves as bio-indicators of environmental
pollution and can play significant roles in assessing potential risk associated
with contamination in aquatic environment since they are directly exposed to
chemicals resulting from agricultural production via surface run-off or
indirectly through food chain of ecosystem (Lakra and Nagpure, 2009).
1.3 JUSTIFICATION
The issue of food security in Nigeria has raised
questions on how to feed the teeming population that is in excess of 213
million inhabitants. Food security in Nigeria has been a topical issue of
discourse in government circle since the initiation of the Millennium
Development Goals (MGDs) at the onset of the twenty first century (Agbon et al., 2014).
Herbicides
are used to control weeds and are usually targeted to processes and target
sites that are specific to plants but in most cases these chemicals reaches
non-target areas and exerts both acute and chronic hazards to non- target
organisms. Environmental
threats to freshwater ecosystems are increasing at faster rates as industrialization,
urbanization and agricultural activities intensify (Amaeze et al., 2020).
Billions of kilograms of industrial
chemicals find their way into fresh water bodies around the world annually
including 140 billion kilograms of pesticides (Mensah et al., 2014). Over the last decades,
the extensive agricultural and non-agricultural use of pesticides has elicited
extensive research on their effects on non-target organisms.
Despite the existence of several toxicological studies
carried out with herbicides in different organisms to quantify the impacts of
these pollutants and know their mechanisms of action, there is a great need to
expand even more the knowledge about the effects of herbicides in aquatic and
terrestrial ecosystems.
Environmental contamination due to excessive use of
pesticides has become a great concern to the public and environmental
regulatory authorities.
In Nigeria, agrochemicals that
contain pesticides especially chlorinated hydrocarbons and the
organophosphates, are routinely employed as part of the integrated farming
practice to protect crops and animals from insects, weeds and diseases (Fafioye et al., 2001; Ezike, 2017), as a result of this, there is need to study the effect of
these chemicals on aquatic organisms which are to a greater extent, the
non-targeted organisms.
1.4 OBJECTIVES
The specific objectives of the study are to determine:
i.
Effect of sub-lethal
concentrations of Atrazine on growth of Clarias
gariepinus and Hybrid Juveniles.
ii.
Effect of sub-lethal
concentrations of Atrazine on the histology of the gills and liver of Clarias gariepinus and Hybrid Juveniles.
iii.
Sub-lethal effect of Atrazine
on haematological and biochemical parameters of Clarias gariepinus and Hybrid Juveniles.
iv.
Effect of sub-lethal
concentrations of Atrazine on proximate composition of muscle of Clarias gariepinus and Hybrid Juveniles.
v.
Level of Atrazine accumulation in the muscle of test organisms
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