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HEAVY METAL CONTAMINATION AND HEALTH RISK ASSESSMENT IN VEGETABLE GROWN AROUND BAMO RIVER

January 28, 2019 0 Comment

HEAVY METAL CONTAMINATION AND HEALTH RISK ASSESSMENT IN VEGETABLE GROWN AROUND BAMO RIVER ,GOBA WREDA SINJA KEBELE ,SOUTH EASTERN ETHIOPIA
SUBMITTED BY:
SEIFEDEN ABDULSEMED (BSC)
ADVISORS: M. SARASWATHI, (PHD)
CO ADVISOR: FTSUM GEBREYOHANNES (MSc.)
A RESEARCH PROPOSAL SUBMITTED TO:
COLLEGE OF NATURAL AND COMPUTATIONAL SCIENCE
MADDA WALABU UNIVERSITY IN PARTIAL FULFILLMENT OF
THE REQUIREMENTS FOR THE DEGREE OF MASTERS OF SCIENCE IN ENVIRONMENTAL SCIENCE
(STREAM: ENVIRONMENTAL POLLUTION AND SANITATION )
MADDAWALABU UNIVERSITY
January, 2018
Robe, Ethiopia

SUMMARY
Vegetables are very important in our everyday diet because of the presence of nutrients and minerals. Due to its anti-oxidative properties, prevents the diseases and keeps us healthy. The present study will be analysis and to assess heavy metal concentration in vegetables that are mostly locally available in Robe market in the period of four months, from may 2018 – October 2018 and the results will be compared with WHO&FAO limits .The vegetables that will be selected for this study is the vegetables that can be marketed locally in Robe market only like carrot, onion, potatoes and cabbages. In order to quantify the analyzed of heavy metal in vegetables the collected sample will be analyzed by using AAS. This study may provide the basic information related to heavy metal concentration and human health risks, which is consuming vegetables from Robe Market.
Key words: -vegetables, Health risk, Heavy metals, AAS.

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ABBREVIATIONS/ACRONYMS
FAO Food and Agricultural Organization
DNA Deoxyribo Nucleic Acid
AAS Atomic Absorption Spectrometry
LOD Limit Of Detection
SRM Standard Reference Material
SOM Soil Organic Matter
WHO World Health Organization

LIST OF TABLES
Table 1. work plan (time table )…………………………………………………………. 25
Table 2. personal and laboratory expense………………………………………………… 26
Table 3. Transport Expense and per diem………………………………………………… 26
Table 4. Storage and Stationery Material………………………………………………………27
Table 5. Budget Summery……………………………………………………………………27

LIST OF FIGURE
The study area of the map (robe town) ………………………………………………………22

Introduction
Background of the study
Environmental pollution by heavy metals can occur by many different ways, either directly or indirectly. Soils, water and plants are contaminated by material from the air or by direct deposition of pollutants. Heavy metals are introduced into the eco-system by the manufacturers and the use of materials containing heavy metals as well as the disposal of this waste. Heavy metals in air, soil, and water are global problems that are a growing threat to the environment. There are many sources of heavy metal pollution, including the coal, natural gas, paper, and industries (Alloway 1990).
The main routes to transfer metals throughout the environment are the atmosphere and flowing waters. Under normal conditions, the end results of migration are sediments, soil and underground waters. Heavy metals may enter the food chain as a result of their uptake by edible plants, thus, the determination of heavy metals in environmental samples is very important. The importance of interactions between metals and solid phases of soils, soil water, and air within and above soil depends on a variety of chemical factors. Absorption of metals from soil water to soil particles is the most important chemical determinant that limits mobility in soils (Bara?kiewicz and Siepak 1999).
The accumulation of these contaminants is aided by the capability of soil to bind them with clay minerals or organic substances. Heavy metals are natural components of soil. Most elements are only present in minimal, insignificant Eco toxicological concentrations in undisturbed locations. A few heavy metals are important as trace elements for physiological processes in plants and animals. Heavy metals contamination of soil is widespread due to metal processing industries, tannery, combustion of wood, coal and mineral oil, traffic, and plant protection (Margesin & Schinner 2005).
Heavy metals may reach and contaminate plants, vegetables, fruits and canned foods through air, water, and soil during cultivation (Husain et al., 1995; Ozores et al., 1997; Geert et al.,1989).
Inhalation and ingestion of heavy metals may cause various diseases such as anemia, neuropsychological effects, liver diseases, gastrointestinal pathologies, teratogenic implications (Needleman & Bellinger, 1991).
Moreover, it is known that the DNA-damaging effects of certain metals in humans can lead to induction of cancer and a decrease of fertility (Snow, 1992). In addition, heavy metals in soils may adversely affect soil ecology, agricultural production or products and water quality (Wang et al., 2001).
Some metals are essential for life, but if an individual’s intake exceeds a certain threshold, toxicity may develop. Metals and minerals in food and fodder are of great interest because of their potential effects on human and animal health. Some have no beneficial biological function but exposures in differs on deficiency may be harmful to health. For example, organic mercury compounds are neurotoxins, exposure to lead can be harmful to neurophysiological development; inorganic arsenic is a human carcinogen and cadmium can affect renal function. While some
elements, such as cobalt, iron and copper are essential to health, they may be toxic at high levels of exposure. Exposure to metals can be in a number of ways, including at work in certain industries, from drinking water and eating contaminated foods (Ministry of Agriculture, Fisheries and Food, UK, 1998a,b).
Heavy metal pollution is a rising environmental problem, which requires immediate attention. With current commercial remediation reagents failing to provide the needed requirements as safe and effective metal chelators, the need for new technology is critical. The emissions of sulfur per day, together with dust loaded heavy metals, both discharged from smelter and industries cause many environmental pollution. The annual mean of the global emissions into the atmosphere reach about 150 ton of sulfur and 3.5 ton of dust loaded with heavy metals (Lacatusu et al., 1999).
The risk to health from certain elements in food can be assessed by comparing estimates of dietary exposures with the Provisional Tolerable Weekly Intakes (PTWIs) and Provisional Maximum Tolerable Daily Intakes (PMTDIs) recommended by the Joint Expert Committee on Food Additives (JECFA) of Food and Agriculture Organization (FAO) and World Health Organization (WHO) programmes on chemical safety (WHO, 1982a,b, 1989a,b, 1993a,b).
Extreme accumulation of heavy metals in agricultural soils through waste water irrigation, may not only result in soil contamination, but also lead to elevated heavy metal uptake by crops, and thus affect food quality and safety. Heavy metal accumulation in soils and plants is of increasing concern because of the potential human health risks. This food chain contamination is one of the important pathways for the entry of these toxic pollutants into the body of the human. Heavy metal accumulation in plants depends on plant species, and the efficiency of different plants in absorbing metals is evaluated by either plant uptake or soil-to plant transfer factors of the metals. Vegetables cultivated in wastewater-irrigated soils take up heavy metals in large enough quantities to cause potential health risks to the consumers. In order to assess the health risks, it is necessary to identify the potential of a source to introduce risk agents into the environment, estimate the amount of risk agents that come into contact with the human-environment (Khan et al., 2008).
Anthropogenic activities (mining, ultimate disposal of treated and untreated waste) effluents containing toxic metals as well as metal chelates from different industries and also the indiscriminate use of heavy metal containing fertilizers and pesticides in agriculture resulted in deterioration of water quality rendering serious environmental problems posing threat on human beings. However some of the metals for example Cu, Fe, Mn, Ni and Zn are essential as micronutrients for life processes in plants and microorganisms, while many other metals like Cd, Cr and Pb have no known physiological activity, (Kar et al., 2007).
As a result, monitoring contamination of heavy metals is important for safety assessment of the environment and human health in particular. Regarding this background, it was therefore of necessity to determine contamination of heavy metals in water, soil and vegetables and assess health risk related to contamination of water, soil, vegetables Around Bamo River In Goba Sinja Kebeles .

Statement of the Problem
During the past few years, many death causes of human have been reported by the Goba health office in the Oromia regional state Bale zone Goba wereda Sinja kebele including infectious and noninfectious diseases. In addition, some human diseases which could be related to diet or drinking water may be correlated to heavy metals. However, there were some human fatalities for which the potential cause of death could not be agreed upon and affected human showed no specific symptoms before death. An atmosphere, waters, soils and vagatable contaminated by heavy metals via washing of car near areas of the farm and the use of fertilizers and insecticide in the farms. To date, no comprehensive study has been conducted to estimate the levels of contamination of these metals in the water, soil and vagatable in the Goba wereda. Therefore I will interested to interest to conduct our study to estimate the levels of these contamination heavy metals in water, soil and vegetable and it will conducted to assess health risk related to heavy metal of around Bamo river Goba Wereda Sinja Kebele.
1.3. Objectives
1.3.1. General objectives
To assess heavy metal contamination and health risk assessment in vegetable grown around bamo river in Sinja Kebele Goba Woreda
1.3.2. specific objective
To determine the levels of some selected heavy metals in locally available vegetable, soil and waters
To compare the results according to WHO and FAO guidelines
To establish some possible recommendations on utilization of vegetables in order to ensure significant improvement in food safety
To find a correlation between the concentrations of heavy metals in soil, well water and plant

1.4 Significance of Study
The study will provide the baseline data of the levels of cadmium (Cd), copper (Cu), , iron (Fe), lead (Pb), manganese (Mn), in well water, soil and vegetables of selected regions and will help to make a basis for further studies/ monitoring of their concentrations in soil, water and vegetables of The Sinja kebeles. Moreover, it will help in forecasting the potential threats caused by their excess or deficiency to human population of Sinja kebeles. The outcomes of this study will be of great interest to the environmental agency, Goba health office and agricultural societies particularly in Goba wereda Sinja kebeles.
1.5 Scope of Study
The scope of the present study will be encompasses the analysis of five heavy metals, namely as cadmium (Cd), copper (Cu), , iron (Fe), lead (Pb), manganese (Mn),. Environmental samples analyzed included water, soil and vagatables. Heavy metals are analyzed will be by using inductively coupled plasmaoptical emission spectrometry (ICP-OES) and FAAS.

REVIEW OF LETRATURE
Heavy Metal
A metal of relatively high density (specific gravity greater than about 5) or of high relative atomic weight is defined as a heavy metal. The term “Heavy metals” is used to describe more than a dozen elements that are metals or metalloids e.g. chromium, arsenic, cadmium, lead, mercury, manganese, etc (Vogel’s; 1989);
Heavy metals are natural constituents of the Earth’s crust. Because they cannot be degraded or destroyed, heavy metals are persistent in all parts of the environment. In small amounts, they enter the human body via food, drinking water and air. Living organisms require varying amounts of heavy metals. Iron, cobalt, copper, manganese, molybdenum, and zinc are required by humans. Excessive levels can be damaging to the organisms. Therefore, heavy metals can be described as any metallic element that has a relatively high density and is toxic or poisonous at low concentration (Vogel’s; 1989).
The Occurrence of Heavy Metals in Nature
Metals in the environment may be present in the solid, liquid or gaseous state. They may be present as individual elements, and as organic and inorganic compounds. The movement of metals between environmental reservoirs may or may not involve changes of state. The geosphere is the original source of all metals (except those that enter the atmosphere from space in the form of meteorites and cosmic dust). Within the geosphere, metals may be present in minerals, glasses, and melts. In the hydrosphere, metals occur as dissolved ions and complexes, colloids, and suspended solids. In the atmosphere, metals may be present as gaseous elements and compounds and as particulates and aerosols (Nriagu; 1989).
Gaseous and particulate metals may be inhaled and solid and liquid (aqueous-phase) metals may be ingested or absorbed, thereby entering the biosphere. In addition to being the original source of all terrestrial metals, the geosphere may represent a sink for metals. The atmosphere and hydrosphere also constitute sinks for metals; however, from a geological perspective, they are more likely to be considered as agents of transport. The movement of metals from one site to another depends on the linear and temporal scales of observation. For example, the oceans are a vast reservoir for a variety of chemical elements. They also serve as a conduit for elements derived from weathering of rocks to return to the geosphere through sedimentation. A reservoir may act as a catalyst for changes of state of metals and metal compounds, without actually having incorporated those metals, as in the case of some biologically mediated reactions (Adrienne et.al.2008).
Behavior of heavy metals in the Environment
The main source for metal input to plants and soils is atmospheric deposition. Volatile metalloids such as As, Hg, Se, and Sb can be transported over long distances in gaseous forms or enriched in particles, while trace metals such as Cu, Pb, and Zn are transported in particulate phases. In terrestrial ecosystems, soils are the major recipient of metal contaminants, while in aquatic systems sediments are the major sink for metals. Freshwater systems are contaminated due to runoff and drainage via sediments or disposal, while groundwater is impacted through leaching or transport via mobile colloids. A number of biogeochemical processes take place at the heterogeneous interface between the rock, soil, water, air and living organisms. These processes or interactions in turn control the solubility, mobility, bioavailability and toxicity of metals. Metals are found in soil solution as free ions or complexes to inorganic or organic ligands. Both die free ions and the metal-ligand complexes can be
taken up by plants,
retained on mineral surfaces, natural organic matter, and microbes,
transported through the soil profile into groundwater via leaching or by colloid-facilitated transport,
precipitated as solid phases, and
diffused in porous media such as soils.
Microorganisms can transform metals such as Hg, Se, Sn, As and Cr by means of oxidation-reduction and methylation (the process of replacing an atom, usually a H atom, with a methyl group) mechanisms and dimethylazion reactions. These processes affect transport or mobility and solubility or toxicity of metals. For example, methylated (organic) forms of Hg are more toxic than inorganic forms of the element and they bio accumulate in organisms. Methylation is favoured in environments characterized by low oxygen levels, low pH, and high soil organic matter (SOM) contents(Sparks, 2005, Adriano, 2001; and Adriano et al. 2005).
Heavy metal contamination of soil is a far more serious problem than air or water pollution because heavy metals are usually tightly bound by the organic components in the surface layers of the soil. Consequently, the soil is an important geochemical sink which accumulates heavy metals quickly and usually depletes them very slowly by leaching into groundwater aquifers or bio accumulating into plants (Infotox, 2000).
Uses of Heavy Metals
Heavy metals are important components of building materials, vehicles, appliances, tools and computers; and are essential in the infrastructure including highways, bridges, railroads, airports, electrical utilities and food production and distribution (Sparks, 2005).
Natural and consumer products contain small concentrations of different heavy metals. For example, cadmium is mainly used in batteries, plastics and it is also found in cigarette smoke, in shellfish and vegetables (ATSDR, 1999).
Toxicity of heavy metals
Human activities affect the natural geological and biological distribution of heavy metals through pollution of air, water, and soil. Humans are also responsible for altering the chemical forms of heavy metals released to the environment. Such alterations often affect a heavy metal’s toxicity by allowing it to bio accumulate in plants and animals, bio concentrate in the food chain, or attack specific organs of the body. Bioaccumulation refers to an increase in the concentration of a metal in a biological organism over time, compared to the normal concentration in the environment Many metals and other chemicals accumulate in living things any time they are taken up and stored faster than they are broken down’ (metabolized) or excreted Some heavy metals such as mercury and lead are toxic metals that have no known vital or beneficial effect on organisms, and their accumulation over time in the bodies of animals can cause serious illness. Certain elements that are normally toxic are, for certain organisms or under certain conditions, may be beneficial. Examples include vanadium, tungsten, and even cadmium. Heavy metals are stable and persistent environmental contaminants since they cannot be degraded or destroyed. Therefore, they tend to accumulate in the soil, seawater, freshwater, and sediments. In small quantities, certain heavy metals are nutritionally essential for a healthy life (e.g., iron, copper, manganese, and zinc). Some of these are referred to as the trace elements. These elements, or some form of them, are commonly found naturally in foodstuffs, in fruits and vegetables, and in commercially available multivitamin products (Nriagu; 1989).
Human Exposure and Health Hazards associated with Heavy Metals
Humans are always exposed to the natural levels of trace elements. Under normal circumstances; the body is able to control some of these amounts. However, continuous exposure to elevated levels of metals could cause serious illness or death. Increased exposure may occur through inhalation of airborne particles or through ingestion of contaminated soil by children or by absorption through the skin ( Okonkwo, 2005; Rollin et al., 2005; Moja, 2007 and WHO, 1981).
Metals and their compounds can accumulate in the body’s tissues, such as bones or nerves. They can cross the placenta and harm an unborn child in pregnant women. Children are the most susceptible to health problems caused by heavy metals, because their bodies are smaller and still developing The health hazards presented by heavy metals depend on the level and the length of exposure. In some cases, the health effects are immediately apparent; in others, the effects are delayed. High levels of toxic metals deposited in body tissues and subsequently in the brain, may cause significant developmental and neurological damage, including depression, increased irritability, anxiety, insomnia, hallucination, memory loss, aggression and many other disorders(Thomson, 2005, Fergusson, 1991; Thomson, 2007).
Cadmium (cd)
Cadmium is present as an impurity in several products, including phosphate fertilizers, detergents and re?ned petroleum products. In addition, acid rain and the resulting acidi?cation of soils and surface waters have increased the geochemical mobility of Cd, and as a result its surface-water concentrations tend to increase as lake water pH decreases. Cadmium is produced as an inevitable byproduct of Zn and occasionally lead re?ning. The application of agricultural inputs such as fertilizers, pesticides, and bio solids (sewage sludge), the disposal of industrial wastes or the deposition of atmospheric contaminants increases the total concentration of Cd in soils (Raymond et al., 2011).
Cadmium in the body is known to a?ect several enzymes. It is believed that the renal damage that results in proteinuria is the result of Cd adversely a?ecting enzymes responsible for reabsorption of proteins in kidney tubules. And also reduces the activity of alcohol dehydrogenase, and lipo amide dehydrogenase, whereas it enhances the activity of pyruvate dehydrogenase, and pyruvate decarboxylase (Manahan, 2003).
Chromium (cr)
Chromium is one of the known environmental toxic pollutants in the world. The main sources of chromium contamination are tanneries, steel industries and sewage sludge application and fly ash. Besides these, chromium plating and alloys in motor vehicles are considered to be a more probable sources of chromium at an elevated concentration it could be toxic for plants and animals. Concentrations between 5-30mg/kg are considered critical for plants and could cause yield reductions (McGrath and Smith, 1990 and Shaheen, 1975).
The problems that are associated with chromium exposure are skin rashes, upset stomach, ulcers, respiratory problems, weakened immune systems, kidney and liver damage, alteration of genetic material, lung cancer and ultimately death (McGrath and Smith 1990 and Pendias et al., 1984).
Among the different heavy metals, Chromium is a common and very toxic pollutant introduced into natural waters from a variety of industrial wastewaters. The two main forms of Chromium, (chromate and dichromate) pose significantly higher levels of toxicity than the other valence states be low toxic limit balances blood sugar levels, regulates hunger, reduces cravings, protect DNA and RNA improves heart function, helps control fat and cholesterol levels in the blood (Krejpcio, 2001; Agaje, 2007; and Qaiser et al., 2009). Chromium (Cr)).
Copper (cu)
Copper is an essential micronutrient required in the growth of both plants and animals. In humans, it helps in the production of blood hemoglobin. In plants, Cu is especially important in seed production, disease resistance, and regulation of water. Copper is critical for energy production in the cells. It is also involved in nerve conduction, connective tissue, the cardiovascular system and the immune system. Copper is closely related to estrogen metabolism, and is required for women’s fertility and to maintain pregnancy. Normal Values of Cu in Serum = 12 – 26 µmol/Land Urine = 0.05 – 0.55 µmol/da Deficiency of copper effect upon thyroid function caused Vascular lesions Central nervous system disorder and convulsion, Hair abnormalities0ther over intake of copper caused Decreased hemoglobin and erythrocyte levels, Death and Cancer. Copper is indeed essential, but in high doses it can cause anemia, liver and kidney damage, and stomach and intestinal irritation (Raymond et al., 2011,Tsugutoshi Aoki 2004 and David L. Watts 1989 ).
Iron (fe)
Iron trace mineral because your body only require very small amounts. Every tissue in the body and numerous cellular processes require iron is a part of hemoglobin found in red blood cells. They play important role as. Oxygen transport and storage Cognitive development. Detoxification, neurotransmitters and hormone synthesis. Energy metabolism and Immune functioning the body require for all age groups of men and post-menopausal women is 8 mg/day, and the RDA for iron for pre-menopausal women is 18mg/day. The difference in values between the two groups is primarily related to the need to replace iron losses due to menstruation (Laura Rowe 2004).
Lead (pb)
Lead is regarded as highly hazardous for plants, animals and particularly for microorganisms. The main sources of lead pollution in agriculture and plants are lead mines, fuel combustion, sewage sludge applications and farm yard manure. The maximum acceptable concentration for lead in food stuffs is around 1 mg/kg. Long-term exposure to lead can result in a buildup of lead in the body and severe symptoms. These include anemia, pale skin, a decrease handgrip strength, abdominal pain, nausea, vomiting and paralysis of the wrist joint. Prolonged exposure may also result in kidney damage. If the nervous system is affected, usually due to very high exposure, the resulting effects include severe headache, coma, delirium and death. Continued exposure can lead to decreased fertility and/or increased chance of miscarriage or birth defects (Dobrzanski. et al., 2005).

Zinc (zn)
Zn is an essential micronutrient that can be found in all tissues of the body and is essential for cell growth, differentiation, healthy immune system and DNA synthesis). Zinc occurs naturally in soil about 70mgkg?1 in crustal rocks. Most Zn is added during industrial activities, such as mining, coal, and waste combustion and steel processing. But water-soluble zinc that is located in soils can contaminate groundwater (Davies and Jones, 1988 and Sand stead, 1991).
Zn can interrupt the activity in soils, as it negatively in?uences the activity of microorganisms and earthworms, thus retarding the breakdown of organic matter. Zinc toxicity and gastric distress can occur from moderately high intakes of Zn greater than 150 mg per day over long period of time (Raymond et al., 2011 and Samman, 2002).
Heavy Metals in plant
Until 1920 it was believed that the total nutrient requirement of plants were fully satisfied by ten essential element : the seven inorganic elements (N, S, P, k, Ca, Na, and Fe) supplied by the cultural solution as salts plus carbon (C) from carbon dioxide and hydrogen (H) and oxygen (O) from water. Recent knowledge has revealed that plants require at least seven other elements in trace amounts (B, Cu, Cl, Mn, Mo, Na, and Zn). The ultimate source of trace elements in the soil (Hala Ehamed 2012).

MATERIAL AND METHODS
3.1 Description of the Study Area
The study will be carried out at around in Ethiopia, Oromia Regional, Bale Zone, Goba Woreda, Sinja Kebeles Around Bamo River, located 430 km to the south east of Addis Ababa, The topographic elevation of the town ranges from 2270 meters up to 2690 meters above sea level with mean annual temperature of 15oc which experienced cool temperature and with a mean annual rainfall of 1100mm.It covers a total area of land of 80,240 km2 (Robe town land administration and development plan 2011).
The study area of the map (Robe Town )

our study center and sample collection will be from this center and the vegetables, soil, water samples will be collected.

Sample collection
Our sample will be collected from four different areas for each water, soil and vegetables from Sinja Kebeles Around Bamo river, for each waters, soil and vegetables I will analysis five types of metals namely iron , manganese, zinc, copper and cadmium the procedure that require for analysis of heavy metal listed below
3.2.1. Water Sample
Sampling will be carried out for physico-chemical parameters of the Bamo River from upstream to the downstream in the different four sampling sites using standard sampling techniques from june 2018 to july, 2018. Samples will be collected in pre-cleaned plastic containers, which are cleaned by detergent and de-ionized water and then rinsed using 10% HNO3 appropriately. During sampling, sample bottles were rinsed with sampled water three times and then filled to the brim from each of four sampling sites. The samples will be labelled and transported to the laboratory using icebox and stored in a refrigerator at about 4 ºC prior to analysis.
3.2.2. Soil Samples
About 1 kg of soil samples will be collected from each four sampling sites of vegetable garden using plastic spade vertically from 0-15 cm borehole once at each sampling day after the removal of grassy part. The collected samples will be thoroughly mixed to give a representative samples and will be dried to constant weight, in an oven to remove excess moisture, then grounded and sieved with 200 mesh (75 µm) sieve. The dried samples will be kept packed till analysis.
3.2.3. Vegetable Samples
Potato vegetable sample which is mostly grown around Bamo River will be collected from the selected sites and kept in plastic containers. The samples will be washed with distilled water to remove dust particles and air dried. The dry vegetable samples will be crushed using mortar and pestle. Finally, the powder was packed for analysis of heavy metals.
3.2.4. Analytical Procedures
3.2.4.1. Physico-chemical analysis
The physico-chemical parameters will be analysed using the standard analytical methods . The pH, temperature, dissolved oxygen (DO) were measured in-situ during sampling; total dissolved solids (TDS), total solids (TS), and total suspended solids (TSS) of the samples will be determined using gravimetric method. Determination of total alkalinity, total hardness, calcium, magnesium and chloride were carried out by titration methods. Ammonium (NH4+-N), nitrite (NO2–N), total nitrogen (TN), total phosphorus (TP), soluble phosphate (PO43-) and chemical oxygen demand (COD) will be analysed using HACH DR/2800 spectrophotometer. Nitrate (NO3–N) was determined using UV spectroscopy. Suphate (SO42-) will determined using Nephelometric turbidity meter.
3.2.4.2. Heavy Metal Analysis from Water
Triplicate of 50 ml water sample was transferred in to three digestive tubes and 10 ml of conc. HNO3 to the tubes containing water sample which allowed to evaporate at temperature of 80 oC in a fume hood tile to half of its original volume on the digestive stove. The mixture was cooled and then filtered in to 100 ml volumetric flask using what man number 41 filter paper and the flask was filled up to the mark by using distilled water. Reagent blank also prepared to check the contamination. Finally heavy metals Cd, Pb, Zn, Cu, Cr and Fe will be determined by AAS.
3.2.4.3. Heavy Metal Analysis from Soil
A triplicate sample of 0.5 g of air-dried ground soil will be transferred to the digestive tube; in addition to that 5 ml of concentrated H2SO4 will added following the addition of 25 ml of concentrated HNO3 and 5 ml of concentrated HCl. The mixture of three acids with the sample has heated at 200 °C for one hour in a fuming hood and then cooled to room temperature. After that 20 ml distilled water added and then filtered. The process of digestion was completed. Finally, the mixture will be transferred to a 50 ml volumetric flask, filled to the mark and let to settle for at least 15 hours. The filtrates will be analyzed for total Cr, Fe, Cu, Pb, Zn and Cd by using FAAS.
3.2.4.4. Heavy Metal Analysis from Spinach Vegetable
A triplicate of approximately 0.5 g of the spinach vegetable powder put in to the digestive tube followed by 5 ml of concentrated HNO3 and 3 ml of concentrated H2O2. The mixture will heated at 160 OC for 1 hour in a fuming hood and then cooled to room temperature. About 20 ml of distilled water will be added and the mixtures were filtered. Finally, the mixture will be transferred to a 50 ml volumetric flask, filled to the mark and let to settle for at least 15 hours. The filtrates will be analyzed for total Cr, Fe, Cu, Pb, Zn and Cd by FAAS.
3.5. Health Risk Assessment
3.5.1. Transfer factor (TF)
Transfer factor is the relative tendency of a metal to be accumulated by a particular species of plant. It expresses by the bioavailability of a metal at a particular position on a species of plant. It is the ratio of the concentration of heavy metal in a plant to the concentration of heavy metal in soil, which is described as.
TF=(concentration of metal in edible part of vegetable)/(concentration of metal in soil)
3.5.2. Daily Intake of Metal (DIM)
The daily intake of metals (DIM) was calculated to averagely estimated the daily metal loading into the body system of a specified body weight of a consumer which informs the relative phyto-availability of metal. The daily intake metals (DIM) has determined as follows:
DIM=(Cmetal×Cfactor×Dfood intake)/BW
Where:
C metal= heavy metal concentrations in plants (mg/kg),
C factor = conversion factor (i.e.to convert fresh vegetable weight to dry weight)
D food intake = daily intake of vegetables
BW = average body weight (65 kg) and conversion factor of 0.085 is to convert fresh vegetable weight to dry weight
3.5.3. Health Risk Index (HRI)
The health risk index (HRI) was determined as described as follows:
HRI=DIM/RfDo
Where: RfDo = reference oral dose for each trace elements
NB: Human are considered to be safe if HRI < 1.
3.6. Statistical Analysis
All the data formulated will be analyzed statistically using SAS 9.1. Variation of each parameter between sites has analysed using one way ANOVA at 95% confidence level.
3.7. Sample preparation
The collected samples will be washed with distilled water to remove the dust particles. Then samples will cut into small pieces using clean steel knife. Different parts (roots, stems and leaves) of fruits will be dried in an oven at 100 0C. After drying the samples will be grinded into a fine powder using a commercial blender and stored in polyethylene bags, until using for acid digestion.
Acid digestion and metals analysis
Tri-acid mixture (15 ml, 70% high purity HNO3, 65% HClO4 and 70% H2SO4; 5:1:1) will be added to the beaker containing 1 g dry fruit sample , The mixture will be then digested at 800C till the transparent solution will be achieved. After cooling, the digested samples will be filtered using what man No. 42 filter paper and the filtrate will be dilute to 50 ml deionized water. Lastly of the selected heavy metals in the filtrate of fruits and atmospheric deposits will be achieved by Flam atomic absorption spectrophotometer (Allen et al., 2003).
Quality assurance

Appropriate quality assurance procedures and precautions will be taken to ensure the reliability of the results. Samples will be carefully handle to avoid cross contamination. Analytical grade chemicals will be used in present study. Deionize water will be used throughout the study. Reagent blank determinations will be used to apply corrections to the instrument readings. For validation of the analytical procedure, repeated analyses of the samples against internationally certified plant standard reference material (SRM) of the National Institute of Standard and Technology were used, and the results were found to lie within ±1% of the certified values.
Statistical Data analyses
Data will be analyzed statistically using the SPSS software version 17.0 by one way ANOVA. one-way analysis of variance will be conducted along with Duncan’s multiple range tests (at P = 0.01) to compare the means of each heavy metal across the five food items; and for each heavy metal, the mean for each food item was compared with the WHO/FAO permissible level as test value using a one-sample student-t test at 99% confidence interval of the difference (Sobukola et al., 2010).

EXPECTED OUTCOMES
From this research, the vegetable quality of the town will be evaluated in terms of its heavy metal concentration. This research work will be utilized as a decision making tool for future to reduce contamination of vegetable by heavy metals. It will be helpful to the society by means of indicating whether the vegetable is safe for appropriate purpose or not, with respect to health.
DISSEMINATION
The end results of this study will be presented to the Madda Walabu University. Finally the work will be published in high impact factor journals.
ENVIRONMENTAL CONSIDERATION
No environmental damage is expected in this investigation, rather than analysis and assessing risk of heavy metal that can be presented in the vegetables.

WORK PLAN ( TIME TABLE )

No
Activities feb March Appril may june july august sept october
1 Select the topic
2 Gathering the literature
3 Set the objective
4 Writing proposal
5 Submission and correction of proposal
6 Proposal defenses
7 Data collection
8 Data sorting and analysis
9 Interpretation of the data
10 Writing the thesis
11 Thesis defense
Table 1. work plan (time table )

BUDGET BREAK DOWN
the estimated cost earned in the process of accomplishing the study will be estimated as follow

No

Activities
Total cost
1 For laboratory analysis , for purchase chemical and vegetable sample 25000
2 Contingency 5000
Sub total 30000
Table 2. personal and laboratory expense

No

Person
Total Cost
1 Student 3000
2 Advisor 4000
Sub Total 7000
Table 3. Transport Expense and per diem

No
Item
Total cost

1 Paper ,pen , hard disk,

1000

Sub total
1000
Table 4 . storage and stationery material

No
Description

Total cost
1 Personal Expense 30000
2 Transport Expense And Per Diem 7000
3 Storage And Stationery Material 1000
Sub Total 38000
Table 5. Budget Summery

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