ABSTRACT
The impact of sub-lethal concentrations of glyphosate (an active ingredient in Roundup® herbicides) on growth, haematology and histology of Clarias gariepinus were evaluated. Uncontrolled discharge of glyphosate from agricultural farm land to aquaculture facilities necessitated the study. Sub-lethal concentrations (0.30mg/L, 0.50mg/L, 0.70mg/L and 1.40mg/L) examined were established after series of range finding tests. Weight in (g) and length measurement (cm) were taken on biweekly basis to monitor growth. Blood parameters analysed were Red Blood Cell (RBC), Haemoglobin content (Hb), Packed Cell Volume (PCV), White Blood Cell (WBC), Platelet (PLT), Mean Corpuscular Volume (MCV), Mean Corpuscular Haemoglobin (MCH) and Mean Corpuscular Haemoglobin Concentration (MCHC). Automated haematology analyser was used to determine the blood parameters. Histology of fish liver was studied among treatments. The liver was examined for lesions, oedema, lytic necrosis, focal necrosis, cloudy swelling, marked congestion and haemorrhages. The physico-chemical parameters monitored were temperature, pH, Dissolved Oxygen and Electrical Conductivity. Thermometer, pH meter, dissolved oxygen meter and Electrical conductivity meter respectively were used. There was significant difference (P<0.05) in growth among the treatments. The best growth was recorded in 0.30mg/L while poorest growth was recorded in 1.40mg/L. There was significant difference in haematological parameters among the treatments (P<0.05). The concentration of 0.30mg/L showed lesions and vacuolization, 0.50mg/L multifocal necrosis and shrunk hepatic cells, 0.70 mg/L lytic necrosis and moderate haemorrhages and 1.4mg/L showed marked congestion, haemorrhages and necrosis of the hepatocyte while the control (0.00mg/L) showed normal central vein and the unaffected sinusoids. The findings in this study showed that 0.30mg/L to 1.40mg/L of the toxicants were harmful to Clarias gariepinus. The report therefore recommended examination of lower concentrations (<0.30mg/L) in future study to establish the limit beyond which glyphosate should not be allowed in aquaculture system.
TABLE
OF CONTENTS
Title page i
Declaration ii
Certification iii
Dedication iv
Acknowledgements v
Table of Contents vi
List of Tables x
List of Figures xi
List of Plates xii
Abstract xiii
CHAPTER 1: INTRODUCTION
1.1 Background of the Study 1
1.2 Statement of
problem 4
1.3 Aims and Objectives 5
1.4 Justification 5
CHAPTER 2: LITERATURE
REVIEW
2.1 The African Catfish – Clarias gariepinus 7
2.1.1
Description of Clarias gariepinus 7
2.1.2
Taxonomic classification of the African
Catfish
(Clarias gariepinus) 8
2.1.3
Ecology of Clarias gariepinus 9
2.2
Glyphosate herbicide 10
2.2.1 Physical and chemical
properties of glyphosate 11
2.2.2 Mode of
action 13
2.2.3
Metals in glyphosate-based herbicide 13
2.2.4 Toxicity
of Co-formulants in glyphosate 14
2.2.5 Glyphosate in aquatic environment 14
2.2.6 Effect on aquatic life (Fish) 16
2.3 Effect
of Pesticides on Ecosystem 17
2.4 Susceptibility
of fish to toxicants 18
2.5 LC50 18
2.6 Sub-lethal
and Acute Toxicity test 19
2.6.1
Sub-lethal test 19
2.6.2 Acute toxicity test 20
2.7 Effect of herbicides
on the Biology and Physiology of Fish 21
2.7.1 Alterations
in blood biochemical parameters 21
2.7.2 Tissue
and organ damage 22
2.7.3 Reproductive
dysfunction 23
2.7.4 Development
disorders 25
2.7.5 Neurotoxicity 26
2.7.6 Behavioral
alterations 27
2.7.7 Genotoxicity 28
2.7.8 Immuno
suppression 29
2.8 Fish Haematology 30
2.9 Histological Studies 31
2.9.1 Liver 32
2.9.2 Gill 33
2.10 Liver function
parameters as markers of toxicity 33
2.11 Water Quality 34
2.11.1 Dissolved Oxygen 35
2.11.2 Temperature 36
2.11.3 pH 36
2.11.4 Ammonia (NH3) 36
2.12 Fish Behavioural Indices 37
CHAPTER
3: MATERIALS AND METHODS
3.1 Experimental Site 41
3.2 Collection
and Acclimation of Experimental Fish 41
3.3 Glyphosate
Source 42
3.3.1 Range finding test (LC50) 42
3.3.2 Preparation
of test solution 43
3.3.3 Sub-lethal
toxicity bioassay 43
3.4
Behavioural Studies 44
3.5 Experimental
Design 45
3.6 Haematological
Analysis 45
3.7 Histological
Analysis 48
3.8 Analysis
of fish growth 49
3.8.1 Weight Gain
(WG) 49
3.8.2
Percentage Weight Gain (WG %) 50
3.8.3 Specific
growth rate (SGR) 50
3.9
Water Quality Analysis 50
3.9.1
Dissolved Oxygen 51
3.9.2
pH 51
3.9.3
Temperature 51
3.9.4
Electrical conductivity 51
3.10
Test Disposal Organism 52
3.11 Data Analysis 52
CHAPTER 4: RESULTS AND
DISCUSSION
4.1 Effect of sub-lethal concentrations of glyphosate
on Growth of
Clarias
gariepinus 53
4.2 Effect
of sub-lethal concentrations of glyphosate on blood
parameters
of Clarias gariepinus fingerlings 56
4.3 Histology
of the liver tissue of Clarias gariepinus
presented
after exposure to glyphosate 60
4.4 Effect of sub-lethal concentrations of glyphosate
on
physicochemical
parameters of water 65
4.5 Discussion 67
4.5.1 Growth
rate 67
4.5.2 Behavioural
response 68
4.5.3 Haematology 69
4.5.4 Histological
changes 71
4.5.5 Water
parameters 73
CHAPTER 5: CONCLUSION AND
RECOMMENDATIONS
5.1
Conclusion 74
5.2 Recommendations 75
References 76
LIST
OF TABLES
2.1 Physical
and chemical characteristics of Glyphosate 12
2.2 Terms
suitable for describing fish appearance and behavior 37
4.1 Mean weight of Clarias gariepinus exposed to sub-lethal
concentration
of Glyphosate 55
4.2 Mean of Sub-lethal concentration of
glyphosate on some
Haematological
Parameters of Clarias gariepinus 59
4.3 Mean Physico-chemical parameters of water
during exposure
of
fish to Glyphosate 66
LIST
OF FIGURES
2.1 African
Catfish (Clarias gariepinus, Burchell 1822) 10
2.2 Structure
of Glyphosate Isopropylamine salt 12
3.1 MINDRAY
BC 2800 Automated Haematology Analyser 47
LIST
OF PLATES
I. Photomicrograph of Liver cell without the
toxicants 60
II. Photomicrograph of the Liver cell exposed
to 0.30mg/L of
Glyphosate 61
III. Photomicrograph of the Liver cell exposed
to 0.50mg/L of
Glyphosate 62
IV. Photomicrograph of the Liver cell exposed
to 0.70mg/L of
Glyphosate 63
V. Photomicrograph of the Liver cell exposed
to 1.40mg/L of
Glyphosate 64
CHAPTER 1
INTRODUCTION
1.1 BACKGROUND OF THE STUDY
The non-stop discharge of contaminants from anthropogenic actions
into surface waters is of predominant subject globally as it portends
multiplied dangers of uptake and toxicity to humans and wildlife (Adeogun et al., 2013). As such the chemical
burden of the environment is related to the release of commercial waste water
and has the ability to have an effect on critical biological processes of
resident species which include reproductive success (Alquezar et al., 2006).
Drainage
and irrigation systems may become polluted through the application of
pesticides during agricultural and pest control activities, and this may
negatively impact the living and non-living members of the contaminated water
course (Mohamed et al., 2012). The persistence in the environment of
some pesticides may contaminate any of the terrestrial or aquatic species
(Bakry et al., 2011).
The poisonous results of chemicals in the water body are numerous
relying at the sort of compound which could either act alone or
synergistically, mainly in combinations of chemical substances (UKWIR, 2002).
Toxic pollutants often cause
characteristic responses in the affected organism, commonly known as
'toxicological endpoints' or 'biomarkers'. A biomarker, is "a biochemical, genetic, cellular, physiological
or behavioural variation that may be measured in tissue or body fluid samples
or at the extent of the entire organism (either individuals or populations),
that gives prove of exposure and/or results of one or greater chemical
pollution and/or radiation". Many research performed proved the mutagenic,
clastogenic and carcinogenic results of metals in mammals (Alimba et al., 2006; Bakare et al., 2007).
More recently, Wright et al.,
(2010) mentioned that environmental metallic exposures display prove of changes
in epigenetic marks which factors to a probable link among heritable changes in
gene expression and susceptibility to diseases. The mechanism of induction of
chromosomal aberrations in fish though, has not been properly elucidated.
Fish are important sources of protein and other nutrients in the
diets of man, it's far vital therefore, to understand whether or not
mutagenicity in fish can function as early warning of potential dangers both
for the fish and the human or non-human consumer of the contaminated fish or
whether the fish can act as sources of transmission of mutagenic chemical
substances to consumers in their tissues.
The choice of fish as a model in ecotoxicological research could
be valued as fish serves as a totally sensitive bio-indicator of aquatic
infection in tropical regions (Mdegela et
al., 2006). The potentiality of application of the findings from those
researches on humans and different environmental health problems has made fish
a greater appealing model organism in toxicology research (Govind, 2011).
Toxicity
testing of chemicals on animals has been used for a long time to detect the
potential hazards posed by chemicals to environment and human (OECD, 2021). Aquatic
Bioassays are necessary in water pollution control to determine whether a
potential toxicant is dangerous to aquatic life and if so, to find the
relationship between the toxicant concentration and its effect on aquatic animals
(USEPA, 2008).
The use of herbicides to check weeds has been identified as part
of agricultural practices throughout the world. Unfortunately, the
indiscriminate use of those herbicides includes chemical substances which
enhance agricultural manufacturing and yield can also have impacts on
non-target organisms, mainly aquatic existence forms (fish kill) and their
environment. As such, it may become hazardous to the health of man
when these aquatic organisms are harvested and consumed (Williams, 2011). Herbicides are normally carried out in dry season or early wet
season, which regularly coincide with the breeding season of many fish species.
Some of those fishes breed in aquatic habitats receiving the runoff drained
from the cultivation fields.
Glyphosate (N–phosphonomethyl glycine) is the active ingredient in
“Roundup” broad-spectrum,
post-emergence, non-selective herbicides
used for controlling annual and perennial grasses, broad-based leafed weeds,
trees and other species (Okayi et al., 2010).
It is one of the established herbicide used worldwide and it is a major
pollutant of rivers and surface water. Furthermore, it is perhaps the most
important herbicide ever developed because of its low persistence. Glyphosate
is considered a probable human carcinogen based on the scientific based
evaluation of cancer reported in humans and other laboratory animals (Portier,
2016).
In
this study, glyphosate a common herbicide used by Nigerian farmers for the
control of weeds in crop land areas especially in irrigated canals, rice fields
etc. is evaluated for its impact on Clarias gariepinus.
1.2 STATEMENT
OF PROBLEM
The contamination of water body by herbicides originating from
agricultural runoff has posed a serious threat globally (Grzegorz et al., 2012). The application of
herbicides as a method of controlling weed is at the boom and contributes significantly
to aquatic pollution which end up to low water quality.
Sub-lethal concentrations of toxicants in the environment will not
necessarily bring about outright mortality of aquatic organisms. Uren Webster et al., (2014) reported that
they have significant effects that could bring about numerous physiological
dysfunctions in the fish when the concentration is high. The increase in
numbers and volumes of chemical substances, both natural and synthetic which
have been released in the environment during the last fifty years has been
immense. These chemicals are used for the production of pesticides and could be
harmful to the health of the ecosystem (Saravanan
et al., 2011).
In 1942, most effective 600 thousand (600,000) chemical substances
had been regarded. This number has now multiplied to nearly eleven million
(11,000,000) (Sveltana et al., 2004)
and most of these compounds are genotoxic, neurotoxic, nephrotoxic,
hepatotoxic, carcinogenic and non-biodegradable. Pollution from agricultural
runoff like the use of herbicide including “Roundup” which contains glyphosate
remains a major threat to the aquatic ecosystem.
1.3 AIM AND
OBJECTIVES OF THE STUDY
The aim
of the study is to access the impact of sub-lethal concentrations of Glyphosate
on Clarias gariepinus.
The
objectives of the study are:
1. To determine the effect of sub-lethal dosage of
glyphosate on the growth of Clarias
gariepinus.
2. To determine the effect of sub-lethal dosage of
glyphosate on the blood parameter of Clarias
gariepinus.
3. To determine the effect of sub-lethal dosage of
glyphosate on histology of Clarias
gariepinus.
1.4
JUSTIFICATION
Aquaculture is gaining attention all around the world today as a method
of enhancing world fish production. This ability can be hampered by means of
the frequent use of pesticides and herbicide on farmland which finally pollutes
the water body through run off and ultimately disrupts the physiological processes
of fish and different aquatic organism. This necessitates research on impact of
glyphosate on the growth, haematology and histological changes of fish.
Clarias
gariepinus is an important fish in
aquaculture and essential supply of protein in Nigeria. Fish are extensively
used to determine the health of aquatic ecosystems, any biochemical changes
observed in fish serve as very sensitive bio-indicator of aquatic contamination
(Mdegela et al., 2006). Generally,
the indiscriminate use of glyphosate-based herbicides including “Round up”
related to careless handling, unintended spillage or discharge of untreated
effluents into natural waterways can promote long-term biological effects yet
to be discovered (Gallardo et al.,
2016; Moustafa et al., 2016).
Studies carried out have characterized the effects of individual
glyphosate-based herbicide formulations in a wide variety of aquatic organisms
including fish using standard toxicity bioassays (Glusczak et al., 2011; Hued et al.,
2012; Menezes et al., 2011; Modesto
and Martinez, 2010a)
Since
the liver is one of the target and detoxifying organ, it is important to check
the histopathological indices of the fish. The findings from this study will
provide information that will be relevant in setting out environmental limits
as regards use of glyphosate.
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