ABSTRACT
The presence of Organochlorine pesticide residues in nine (9) Liver samples collected from slaughter house in Zango, Zaria-Nigeria was determined. All samples were analyzed for the residual contents of α-Benzenehexachloride, β-Benzenehexachloride, Lindane(γ-Benzenehexachloride), endrin, Aldrin, chlorothalonil, δ-Benzenehexachloride, heptachlor, heptachlor epoxide, endosulfan, dieldrin, endosulfan II, p,p‟-Dichlorodiphenyldichloroethane, endosulfan sulfate, p.p‟-Dichlorodiphenyltrichloroethane, lambda cyhalothrin and permethrin by Gas Chromatography with Electron Capture Detector (GC-ECD). Sample extraction was carried out by warm soxhlet extraction followed by column clean-up with Florisil. The range of concentration was α-BHC 0.02 – 0.21 mg/kg, β-BHC 0.09 – 2.57 mg/kg, Lindane 0.06 – 0.24 mg/kg, chlorothalonil 0.05 – 0.32 mg/kg, δ-BHC 0.07 – 0.90 mg/kg, heptachlor 0.13 – 1.16 mg/kg, Aldrin 0.27 – 0.61 mg/kg, heptachlor epoxide 0.13 – 1.16 mg/kg, endosulfan 0.02 –3.79 mg/kg, dieldrin 0.12 – 1.72 mg/kg, endrin 0.04 – 7.88 mg/kg, endosulfan II 0.12 – 1.56 mg/kg, p,p‟-DDD 0.14 – 0.73 mg/kg, endosulfan sulfate 0.12 – 1.56 mg/kg, p.p‟-DDT 0.12 – 1.20 mg/kg, lambda cyhalothrin 0.07- 0.37 mg/kg and permethrin 0.02 – 0.28 mg/kg. All the samples analyzed were found to contain these OCPs with higher concentrations observed in cattle above 24 months. Higher concentration of DDT than DDD suggests a more recent usage of the pesticides and calls for strict and effective measures to stop the sale and usage of the pesticides. This study has provided the preliminary information on the concentration of OCPs in liver of cattle for the first time in Zaria. The result will help in a scientific assessment of the implications of these pesticide residues with regards to human risk in Zaria.
TABLE OF CONTENTS
Title page number Title page i Declaration ii Certification iii Dedication iv Acknowledgement v Abstract vi Table of content vii List of Figures xii List of Tables xiii List of Appendices xiv Abbreviations xv CHAPTER ONE 1 1.0 INTRODUCTION 1 1.1 Background of the Study 4 1.2 Statement of the Problem 6 1.3 Justification for the Study 7 1.4 Aim of the Study 9 1.5 Objectives of the Study 9 1.6 Scope of Study 9 1.7 Limitations of the Study 10
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CHAPTER TWO 11 2.0 LITERATURE REVIEW 11 2.1 Review of Previous Studies on OCPs in Fruits and Vegetable 11 2.2 Review of Previous Studies on OCPs in Water, Sediment and Fishes 14 2.3 Review of Previous Studies on OCPs in Dairy and Meat Products 19 2.4 Review of Previous Studies on OCPs in Soil 21 2.5 Chemistry of Organochlorine Pesticides 24 2.5.1 Toxicokinetics of organochlorine pesticides 24 2.5.2 Absorption 25 2.5.3 Distribution 25 2.5.4 Metabolism 26 2.5.5 Excretion 29 2.5.6 Toxicity of organochlorine pesticides 30 2.6 Mechanism of Toxicity of Organochlorine Pesticides 31 2.7 Brief Description of Some Organochlorines and their Metabolites 32 2.7.1 Aldrin 32 2.7.2 Chlordane 33 2.7.3 DDT 33 2.7.4 Dieldrin 35
2.7.5 Endrin 36
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2.7.6 Heptachlor 36 2.7.7 Mirex 37 2.7.8 Toxaphene 38 2.7.9 Hexachlorobenzene (HCB) or Hexachlorohexane or Benzenehexachloride 39 2.8 Effects of Organochlorine Pesticides on Human Health 42 2.8.1 Cancer 43 2.8.2 Effect on reproductive system 44 2.8.3 Endocrine disruption 45 2.8.4 Immune system dysfunction 46 2.8.5 Parkinson‟s disease 47 2.8.6 Cytotoxic defects 47 2.8.7 Childhood developmental disorders 48 2.8.8 Diabetes 49 2.9 Extraction Methods and Clean-up of OCPs 50 2.9.1 Clean-up methods 50 2.10 Detection Techniques of OCPs 52 CHAPTER THREE 54
3.0 MATERIALS AND METHODS 54
3.1 Materials 54
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3.2 Reagents 55 3.3 Study Area 55 3.4 Sampling 58 3.5 Sample Preparation 58 3.5.1 Pre-treatment of Sodium sulphate 58 3.6 Sample Extraction 59 3.7 Column Clean-up 59 3.7.1 Preparation of stock solution 60 3.8. Proximate Composition 60 3.8.1 Determination of moisture content 60 3.8.2 Determination of ash content 60 3.8.3 Determination of lipid content 61 3.9 Determination of Pesticide Residue 62 3.10 Statistical Analysis 62 CHAPTER FOUR 63 4.0 RESULT 63 4.1 Proximate Parameters of Liver Samples 63 4.1.1 Percentage lipid content 63 4.1.2 Percentage ash content 63
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4.1.3 Percentage moisture content 64 4.2 Results for the Concentration of OCPs using GC-ECD 65 4.3 Mean Concentration of the OCPs in the Liver Samples 67 4.4 Correlation Matrix of Different Parameters Associated with OCP Determination 69 CHAPTER FIVE 70 5.0 DISCUSSION 70 5.1 Discussion 70 5.2 Incidence of Contamination with the Different Organochlorines 72 5.2.1 Benzenehexachlorides (BHCs) or Hexachlorohexanes (HCHs) 73 5.2.2 Chlorinated cyclodienes 74 5.2.3 Diphenyl-aliphatics (DDT and its metabolites) 74 5.3 Proximate Parameters and OCPs 75 CHAPTER SIX 76 6.0 SUMMARY, CONCLUSION AND RECOMMENDATION 76 6.1 Summary 76 6.2 Conclusion 77 6.3 Recommendations 77 REFERENCES 79
APPENDICES 89
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CHAPTER ONE
1.0 INTRODUCTION Organochlorine pesticides (OCPs) are synthetic organochlorines which are lipophilic and hydrophobic. Their lipophilicity, hydrophobicity, stability to oxidation, low vapour pressure and low chemical and biological degradation rates have led to their accumulation in biological tissues and the subsequent magnification of concentrations in organisms, progressing through to the food chain (Helberg et al., 2005). They can be recycled through food chains and produce a significant magnification of the original concentration at the end of the chain (Doong et al., 2002). They are resistant to natural breakdown processes and are extremely stable and persistent, highly toxic and bioaccumulate in the fatty tissues of animals and humans (Forget et al., 2001). Bioaccumulation is the ability of a pollutant to accumulate in living tissues at levels higher than those in the surrounding environment. When chemical pollutant enters into the body of an organism and is not excreted but rather collected in the organism‟s tissues, bioaccumulation sets in (Zweig et al., 1999).
Persistent organic pollutants (POPs) are ubiquitous contaminants and have been detected far from their sources of origin because of long-range transport stemming from atmospheric exchange, water currents, animal migration and other pathways (Zhang et al., 2007). Efforts at minimizing and eventually phasing out POPs globally gave rise to the Stockholm Convention in 2001. OCPs namely aldrin, dieldrin, endrin, chlordane, dichlorodiphenyltrichloroethane (DDT), heptachlor, mirex, toxaphene, hexachlorobenzene (HCB) and industrial chemicals and byproducts, including polychlorinated biphenyls (PCBs), dioxins and furans, constitute the twelve chemical substances called the “dirty dozen” and defined under the Stockholm Convention. However, at its fourth meeting held in 2009, the Conference of the Parties (COP) adopted the amendments to annexes A (elimination), B (restriction) and C (unintentional
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production) of the Stockholm Convention to list nine additional chemicals as persistent organic pollutants, resulting in the “dirty twenty one”. The nine additional chemicals include chlordecone, alpha hexachlorocyclohexane, beta hexachlorocyclohexane, lindane, pentachlorobenzene, octabromodiphenyl ether, pentabromodiphenyl ether, perfluorooctane sulfonic acid and perfluorooctane sulfonyl fluoride. Residues and metabolites of many POPs are very stable, with long half-lives in the environment (UNEP, 2002). The manufacture and use of chlorinated pesticides have been banned or restricted in developed countries. Although these bans and restrictions were enacted during the 1970s and 1980s, some developing countries are still using OCPs for agricultural and public health purposes because of their low cost and versatility in controlling various pests (Xue et al., 2006). Again, they are being used in most developing countries, including Nigeria, due to a lack of appropriate regulatory control and management of the production, trade and use of these chemicals (Darko and Acquaah, 2007). There is also a paucity of data on the use of pesticides in the country, a reflection of the lack of a mechanism and planning programme in place for chemicals management as well as a low level of understanding of the environmental and public health hazards of pesticide use (Osibanjo et al., 2002).
Pesticides are chemicals used to kill or control pests. They are classified according to their chemical class or intended use (function). According to functions, we have acaricides, algicides, antifeedants, avicides, bactericides, repellants (fumigants), fungicides, chemosterilants, herbicides, insecticides, molluscides, nematicides, plant activators, growth regulators, Rodenticides and virucides (Nick, 2012). Chemical classification may vary from organochlorinates to organophosphates, polybiphenyls, carbamates, benzophenones, bipyridilium, carbazates, dicarboximides, fluorine rich pesticides etc. OCPs are basically insecticides composed primarily of carbon, hydrogen and chlorine and are classified thus;
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Hexachlorobenzenes (which include alpha, beta, delta and gamma(Lindane) HCBs), Cyclodienes (which include Aldrin, Eldrin, Endrin, cis-Chlordane, trans-Chlordane, Heptachlor etc.), Endosulfan ( alpha- and beta-endosulfan), Diphenyl-Aliphatics ( o,p‟-DDT, p,p‟-DDT, o,p‟-DDD, p,p‟-DDD, o,p‟-DDE, p,p‟-DDE) etc. They are found in the environment as a result of human activities. Insecticides act by poisoning the nervous system of target harmful insects. The use of OCPs takes many forms, ranging from pellet application in field crops to sprays for seed coating and grain storage. OCP residues enter vegetative environments through effluent release, discharges of domestic sewage and industrial wastewater, atmospheric deposition, runoff from agricultural fields, leaching, equipment washing, and disposal of empty containers and direct dumping of wastes into the vegetation systems (Yang et al., 2005). The distribution of various chlorinated contaminants in the land and vegetative environment depends on the physicochemical properties of the ecosystem as well as the partition coefficients of individual chlorinated hydrocarbons (Sarkar et al., 1997). OCPs could distribute among the components of the ecosystem, such as water and sediment, and accumulate in the biota. As a result of their persistence, OCPs in water can be transferred into the food chain and accumulate in aquatic organisms like plankton. Different pesticides pose varying degrees and types of risk to water quality. It is reported that approximately three million people are poisoned and two hundred thousand die each year around the world from pesticide poisoning, the majority of them from the developing countries (FAO, 2000). It is also believed that the incidence of pesticide poisoning in developing countries may be greater than reported due to under-reporting, lack of data and misdiagnosis (Cocco et al., 1997). Some of the symptoms of pesticides poisoning include irritation, dizziness, tremor, tonic and chronic convulsion (Winter, 1992). OCPs have been linked to human breast and liver cancers, testicular tumours and lower sperm counts in humans (Davis and Bradlow, 1995; Cocco et al., 1997).
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Studies have also suggested that OCPs may affect the normal function of the endocrine system of humans and wildlife (Xue et al., 2006). OCPs are among the most commonly detected pesticides around the world. Although most of them were banned in the 1970s and 1980s, they can still be found in the environment in several matrices such as water, soil, marine sediments and thus animal tissues (Sarkar et al., 2008; Guan et al., 2009; Essumang et al., 2009). Pesticides can be bioconcentrated through biogeochemical processes and can be scavenged from the water through sorption onto suspended material before they get deposited to the bottom substrate (Xue et al., 2006). The sediment component of aquatic ecosystem deposits pesticides. Sediment is one of the principal reservoirs of environmental pesticides, representing a source from which residues can be released to the atmosphere, groundwater and living organisms (Xue et al., 2006). Persistence of these organic pollutants in sediment is possible due to their low solubilities and tendency to associate with suspended particulate matter. As a result of their low water solubility, OCPs have a strong affinity for particulate matter. They are hydrophobic compounds that tend to adsorb to suspended particulate matter and benthic sediments in aquatic ecosystems (Ademoroti, 1996). Sediments serve as ultimate sinks for them. Indirect exposure to contaminated sediments takes place when fishes feed on benthic invertebrates that are ingesting particulate matter. Direct exposure through the sediment takes place by release of contaminated particulate matter into the water column by both natural and anthropogenic processes. 1.1 Background of the Study
The contamination of the environment, food and animal feed by chlorinated organic pesticides has become a topical issue of considerable concern in many parts of the world, and has led many researchers to investigate their occurrence, distribution and concentrations in several ecosystems (Sankar et al., 2006; Yang et al., 2007; Poolpak et al., 2008). The toxicity of pesticides varies greatly with their intrinsic properties, the species being studied and factors in
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the environment. Important factors that influence the impact of a pesticide on the aquatic environment are its persistence, the partitioning of the pesticide between the particulate and aqueous phases, toxicity to aquatic organisms and their tendency to bioaccumulate. All pesticides are toxic to some forms of life. Many modern pesticides are developed to be as selective against target organisms as possible, but it is rarely possible to achieve perfect control of one organism without the wider environment being exposed and susceptible non-target species being affected. Anthropogenic activities provide the primary point source of chlorinated hydrocarbon input into the aquatic environment (Malik et al., 2008). OCPs enter the environment by deliberate application or by accident. These substances are sometimes applied directly to animals, vegetation or water bodies to control pests. OCPs have been extensively used for agriculture and vector control purposes in Nigeria. The pesticides applied on land eventually find their way to the aquatic environment, thus contaminating it. Being lipophilic, OCPs can be concentrated to harmful levels in the environment through bioaccumulation and bio magnification (Toft et al., 2003). Consequently, aquatic organisms that are commercially exploited for human food may pose a risk to man. It has also been recognized that the persistence and bioaccumulative tendency of these substances, their metabolites and residues in the environment make them not to remain where they are applied but to be partitioned between the major environmental compartments in accordance with their physicochemical properties. Such environmental distributions may lead to the exposure of living organisms, including man, that are far removed from intended targets.
OCPs are among the first set of pesticides to be used in Nigeria (Amakwe, 1984). They are still in use in Nigeria. Despite the difficulties associated with the analysis of organochlorine compounds, especially at the low levels normally found in tissue samples, there is evidence that they are major long-term contaminants of the environment (Yang et al., 2005; Malik et al.,
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2008). Concern about exposure to organochlorines among humans has arisen mainly because of the proven carcinogenicity of these chemicals in experiments with animals and their ubiquity, bioconcentration and persistence in human tissues (Skaare et al., 2000). Many POPs, which pollute the environment, become incorporated into food webs. Humans, being the final links in the food chain, are the most affected. Consequently, the general public has become increasingly concerned about the potential risk to human health from the consumption of such polluted biota. The ill effects of pesticides may arise from short- or long-term and low- or high-level exposure through dermal absorption, inhalation and oral ingestion. The toxicity of pesticides could be acute and chronic. There is growing evidence of cancer, neurological damage, endocrine disruption and birth defects arising from exposure (IARC, 2001). In view of the negative health effects of OCPs on humans and the nonexistence of a government policy on the regulation and monitoring of OCP levels in fishes in Nigeria, it is very important to evaluate the levels of chlorinated pollutants in commercially available cattle liver. Persistent organic pollutant levels are usually monitored in inorganic ecosystem compartments such as water, air and sediment or in biota. Monitoring in inorganic compartments has the advantage of producing an immediate, geographically localized measure of contamination, while biological monitoring provides information on the extent of biotransformation and bioaccumulation processes that the contaminants have undergone during their passage through biological systems. Biological monitoring, therefore, provides a more realistic view of the contaminant distribution in the environment site (Rissato et al., 2006). 1.2 Statement of the Problem
The occurrence of organochlorine pesticides in biological species even at trace levels is not desirable as they have toxic effects. Cattle meat and liver as good source of protein are at the top of the food chain and do bioaccumulate these OCP residues, leading to many diseases like
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Cancer, Endocrine disruption, Immune system dysfunction, Parkinson‟s disease, Cytotoxic defects, Child development disorder and Diabetes among others in man. 1.3 Justification for the Study Pesticides are known to be toxic to man (Ademoroti, 1996). The use of pesticides introduces some risks to the environment, the degree of risk depending upon the pesticide persistence, mobility, non-target toxicity and volume of use. The toxicity level of a pesticide also depends on the deadliness of the chemical, the length of exposure, the health status of the recipient and the route of entry into the body. OCPs contribute to many acute and chronic health effects, including cancer, neurological damage, birth defects, tremors, headache, dermal irritation, respiratory problems and dizziness (Garry, 1996; Mathur et al., 2002; Toppari, 2002). Animal studies by Garry (1996) have also shown the potential for reproductive and developmental effects and disruption of normal hormone function. Long-term exposure to sub-lethal levels of OCPs and their metabolites through various pathways in the aquatic environment may cause far reaching ecological damage and health problems to man. Residues of these toxic chemicals found in water, sediments and aquatic biota pose a risk to aquatic organisms, predators and humans.
Cattle meat and liver are suitable indicators for environmental pollution monitoring because they concentrate pollutants in their tissues directly from their diet, thus enabling the assessment and transfer of pollutants through the trophic web (Fisk et al., 2001; Lanfranchi et al., 2006). The low activity of the mono-oxygenase enzymes in cattle liver and tissue limits their ability to metabolize organochlorines (Dearthand Hites, 1991). Hence, animal tissues generally reflect the levels of organochlorine pollution in their diet (Muir et al., 1990). This offers the opportunity to study the influence of environmental and biological factors on the bioaccumulation of pollutants (Porte and Albaiges, 1993; Sarkar et al., 2008). Besides analyzing
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OCPs in the muscle tissues of cattle, it is vital to investigate their distribution in organs which could provide more information about the pathways along which bioaccumulation occurs, and thus reflect the environmental conditions. This is because they do possess pronounced ability to concentrate persistent organic pollutants from their diet (Yang et al., 2006; Zhou et al., 2008). It has been reported that the consumption of contaminated liver, meat and fishes is one of the important pathways of human exposure to OCPs (Mwevura et al., 2002; Zhou et al., 2007; Muralidharan et al., 2008). Indeed, studies have related the presence of organochlorine residues in breast milk to the consumption of contaminated fishes and cattle parts (Fitzgerald et al., 2001). Data on the presence and distribution of OCPs in cattle liver are, therefore, important from the ecological and human health perspectives. The transport, dispersion and the ultimate effects of pesticides in marine systems depend upon their persistence, bioaccumulation and biodegradation. OCPs could be associated with organic components of soils, biological tissues and dissolved organic carbon in living systems (Xue et al., 2006; Vagi et al., 2007; Imo et al., 2007; Zhou et al., 2007). The indiscriminate use of pesticides in Nigeria has resulted in the occurrence of the residues in biota and other abiotic compartments (Osibanjo and Bamgbose, 1990; Ize-Iyamu et al., 2007; Adeyemi et al., 2008). It is necessary to ascertain the distribution, behaviour and fate of these compounds in various animal compartments. The determination of POPs existing in biota could indicate the extent of contamination and the accumulation characteristics.
There is need for continuous monitoring to identify the occurrence and the levels of OCPs in fishes, water and sediment of aquatic ecosystems in Nigeria as literature data on the concentrations of OCP residues in the Nigerian environment are inadequate. Most institutions and researchers in Nigeria lack the analytical facilities/equipment for the detection and quantitative analysis of OCPs in environmental samples. Lack of adequately trained experts in trace organic analysis, as well as funds for the purchase of chemicals, are some of the main
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limiting factors for conducting research and monitoring OCP residues in biota and other media in the environment. Data gaps have thus been identified. There are gaps in evaluating the accumulation of OCPs in the organs of cattle and the daily intake of pesticide residues which the present study aims to bridge. This research serves to provide data on the prevailing levels of these persistent pollutants in cattle liver obtained in Zaria metropolis. 1.4 Aim of the Study This research was aimed at investigating the occurrence, concentration and distribution of organochlorine pesticides in cattle liver obtained in Zaria, Kaduna state with a view to assessing the current state of contamination and exposure to these toxicants. 1.5 Objectives of the Study The research was designed to achieve the following objectives:
i. Determination of the physicochemical properties of liver of cattle.
ii. Determination of organochlorine pesticide residues in the liver of cattle.
iii. Comparison of bioaccumulation levels of the organochlorine pesticide residues in the male cattle using age and the physicochemical parameters determined as a factor of categorization. Bioaccumulation of the OCPs are expected to increase as the duration of exposure increases hence, higher concentration of OCPs are expected as the age of the cattle increases.
iv. Evaluation of the intake of OCP residues through cattle liver to human beings per kilogram consumed.
v. To compare results obtained to Maximum Residual Limits as set by FAO (Food and Agriculture Organization ) and WHO (World Health Organization)
1.6 Scope of Study The scope of this work included
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i. The determination of proximate parameters such as moisture content, ash content and Lipid content of the liver samples.
ii. The qualitative and quantitative determination of Endosulfan, DDT and metabolites, Heptachlor and isomers, Lindane, Hexachlorohexane and isomers, Chlorothalonil, Aldrin, Dieldrin, Cyhalothrin, Permethrin and Endrin. using GC-ECD.
iii. The analysis of results obtained using the Tukey range test (ANOVA), correlation of results obtained with some proximate parameters using Pearson‟s correlation and comparison with MRLs as established by FAO/WHO.
1.7 Limitations of the Study
i. The research work was limited to Zaria, Nigeria. All samples were taken from Zaria.
ii. Only the liver organs of the cattle were analyzed.
iii. A Single specie of cattle was used for the analysis (the N‟dama). There are other cattle types that have not been analyzed in the present study.
iv. Cattle liver has been reported as a bioindicator.
v. Only male cattle liver was used in the research. This is because, OCP level in female animals have been known to fluctuate due to lactation.
vi. Not all the OCPs were determined. The OCPs of interest were those commonly used by farmers in Zaria and these are Endosulfan, DDT and metabolites, Heptachlor and isomers, Lindane, Hexachlorohexane and isomers, Chlorothalonil, Aldrin, Dieldrin, Cyhalothrin, Permethrin and Endrin.
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