i

CYANIDE AND SOME METAL TOXICANTS IN FERMENTED CASSAVA FLOUR FROM

SOUTH-WEST, NIGERIA.

BY

ABIMBOLA OLUWATOYIN

PG/M.SC/09/50608

A RESEARCH PROJECT SUBMITTED IN PARTIAL FUFILMENT OF THE

REQUIREMENT FOR THE AWARD OF THE DEGREE OF MASTER OF SCIENCE IN

ANALYTICAL CHEMISTRY IN THE DEPARTMENT OF PURE AND INDUSTRIAL

CHEMISTRY, UNIVERSITY OF NIGERIA, NSUKKA

MARCH, 2012.

ii

APPROVAL PAGE

This research project has been approved for the Department of Pure and Industrial

Chemistry, Faculty of Physical Sciences, University of Nigeria, Nsukka.

BY

________________ ________________

DR. P. O OBUASI DR . C. O .B. OKOYE

HEAD OF DEPARTMENT PROJECT SUPERVISOR

DATE____________ DATE____________

_______________

EXTERNAL SUPERVISOR

DATE ____________

iii

CERTIFICATION

ABIMBOLA OLUWATOYIN, a post graduate student in Department of Pure and Industrial

Chemistry with the registration number PG/M.Sc/09/50608, has satisfactorily completed the

requirements for course and research work for the Degree of Master of Science in

Analytical Chemistry. The work embodied in this project is original and has not been

submitted in part or whole for any other diploma or degree of this or any other university.

__________________ __________________

DR. P.O OBUASI DR. C. O. B. OKOYE

HEAD OF DEPARTMENT PROJECT SUPERVISOR

DATE______________ DATE_______________

iv

DEDICATION

This project is dedicated to Almighty God for his provisions, inspiration and the

wisdom he bequeathed on me throughout the period of this research project and to my

beloved parents, Elder and Deaconess J.D Abimbola for their financial support, prayers and

encouragement.

v

ACKNOWLEDGEMENT

A research project like this would not have been successful without the effort of

some people who worked behind the scene, I am grateful to all of you.

First and foremost is my able and resourceful research supervisor, Dr C.O.B Okoye,

whose supervision and correction made this work, in no small way, a huge success. My

hearty thanks also goes to Dr Oluwole Akinnagbe of Agric Extension Department,

University of Nigeria, Nsukka whose immense contributions cannot be overlooked. I also

appreciate all my lecturers in the Department of Pure and Industrial Chemistry and also my

senior colleague, Emmanuel Okon, for his contributions.

I am not forgetting my lovely siblings, Tolu, Barrister Foluke, David and my

wonderful friends, Chidinma, Vivian, Ndidi, Jane, Temitayo, Barrister Kate, Grace, Lucy,

for their love and encouragement.

Worthy of remembrance are my colleagues, Ejike, Ifeoma Anigbogu, Chioma,

Emmanuel Onunze, Lady Okonkwo, Ify, Hillary, Christiana, Helen, Adika, Kalu, C.C, Walter,

Kenneth and Chijioke. I will miss all of you.

vi

ABSTRACT

Cassava flour, a processed product from cassava consumed in Nigeria as a staple food, is

a major source of carbohydrate. Analysis was carried out on the cassava flour to ascertain

the levels of some toxic contaminants like cyanide and metals introduced into the food

naturally or as a result of human activities such as industrial emission, effluent discharges,

solid waste disposal, usage of agrochemicals and sewage sludge in agricultural practices,

incineration, traffic pollution, atmospheric pollution, vehicular emission, combustion of

plastics, fossil fuels e.t.c, which are more prominent in urban areas where multitudes of

stationary and mobile sources release large quantities of these toxic contaminants into the

environment which are then taken up by man, soil, water, plant, food than in rural areas. At

selected sites, 20 (twenty) samples were collected, digested and analyzed. Cyanide in the

cassava flour was determined by UV-Visible spectrophotometer. Lead, cadmium and nickel

were determined using Atomic Absorption Spectrophotometer (AAS) while arsenic and

selenium were determined using Titrimetric method. The mean concentration levels in

mg/kg of cyanide, lead, cadmium, nickel, arsenic and selenium in the cassava flour from

the urban areas were 0.07±0.03, 0.13±0.14, 0.03±0.02, 0.60±0.18, 0.30±0.07 and

5.12±0.90. These were higher than the mean concentration levels, 0.03±0.02, 0.04±0.10,

0.02±0.02, 0.36±0.11, 0.20±0.03 and 4.50±0.80 respectively determined in the cassava

flour from the rural areas. However, all the determined levels of these elements from both

urban and rural areas are far below the WHO guideline values or permissible levels of

cyanide and metal in food. This implies that cyanide and the metal toxicants are present in

the cassava flour in such low concentrations that render the food non-toxic.

vii

LIST OF FIGURES Fig 2.1: Processing of cassava flour (lafu)- - - - - - 16

Fig 2.2: Operation of an Atomic Absorption spectrophotometer - - - 26

Fig 2.3: Calibration graph for AAS Determination - - - - - 26

Fig 2.4: Principle of uv/visible spectrophotometer- - - - - 29

Fig 2.5: EDTA, a chelating agent, binds a heavy metal, sequestering it - - 50

Fig 4.1: Graph of absorbance versus concentration for cyanide determination - 89

viii

LIST OF TABLES

Table 2.1: Concentration in mg/kg of cyanide in cassava and its processed

products - - - - - - - - - 11

Table 2.2: WHO recommended (guideline) concentration values of some heavy metals and

cyanide in food - - - - - - - - - 22

Table 2.3: U.S Recommended Dietary Allowances - - - - - 23

Table 4.1: Lead, cadmium and nickel concentration levels in the samples from

the urban areas of Southwest, Nigeria - - - - - 84

Table 4.2: Lead, cadmium and nickel concentration levels in the samples from the

rural areas of Southwest, Nigeria - - - - - 85

Table 4.3: Arsenic and selenium concentration levels in the samples from the urban

areas of Southwest, Nigeria - - - - - - 86

Table 4.4: Arsenic and selenium concentration levels in the samples from the rural

areas of Southwest, Nigeria - - - - - - 87

Table 4.5: Absorbances of various standard solutions of cyanide at 490nm - 88

Table 4.6: Cyanide concentration levels in the samples from the urban and rural

areas of Southwest, Nigeria - - - - - - - 90

ix

TABLE OF CONTENTS

Title page - - - - - - - - - - - i

Approval page - - - - - - - - - - ii

Certification - - - - - - - - - - - iii

Dedication - - - - - - - - - - iv

Acknowledgement - - - - - - - - - - v

Abstract - - - - - - - - - - - vi

List of Figures - - - - - - - - - - vii

List of Tables - - - - - - - - - - viii

Table of Contents - - - - - - - - - - ix

CHAPTER ONE

1.0 Introduction - - - - - - - - - - 1

1.1 Pollution - - - - - - - - - - - 1

1.2 Background of the Research project - - - - - - - 3

1.3 Aim and Objectives of the Research project - - - - - 4

CHAPTER TWO

2.0 Literature Review - - - - - - - - - 5

2.1 Cassava Plant (Manihot esculenta crantz) - - - - - 5

2.1.1 History of cassava plant - - - - - - - - 6

2.1.2 Production / Economic Impact of cassava - - - - - - 6

2.1.3 Importance of cassava processing - - - - - - - 8

2.1.4 Uses of cassava - - - - - - - - - 11

2.1.5 Cassava processed products (cassava flour) - - - - 15

2.1.6 Cassava toxicity - - - - - - - - - 17

2.2 Heavy metals - - - - - - - - - 18

2.2.1 Sources of Heavy metals - - - - - - - - 19

2.2.2 Heavy metals in food - - - - - - - - - 21

2.2.3 Recommended levels of cyanide and heavy metals in food - - 22

x

2.2.4 Analytical Techniques for cyanide and heavy metal determination

in food- - - - - - - - - - - 24

2.2.5 Atomic Absorption Spectrophotometry - - - - - - 25

2.2.5.1 Interferences - - - - - - - - 27

2.2.6 UV-Visible Spectrophotometry - - - - - - 28

2.2.7 Titrimetry methods - - - - - - - - - 30

2.2.7.1 Redox Titration - - - - - - - - - 31

2.2.7.2 Common Titrants in Redox Titration - - - - - - 32

2.2.7.3 Applications of Redox Titration - - - - - - - 34

2.3 Chemistry of cyanide and heavy metals - - - - - 34

2.3.1 Chemistry of cyanide - - - - - - - - 34

2.3.2 Chemistry of lead - - - - - - - - - 42

2.3.3 Chemistry of cadmium - - - - - - - - 50

2.3.4 Chemistry of Nickel - - - - - - - - - 56

2.3.5 Chemistry of Arsenic - - - - - - - - - 63

2.3.6 Chemistry of selenium - - - - - - - - 70

CHAPTER THREE

3.0 Experimentals - - - - - - - - - 80

3.1 Sample collection - - - - - - - - - 80

3.2 Sample preparation - - - - - - - - - 80

3.2.1Ashing procedure for analysis of sample for lead, cadmium and

nickel determination - - - - - - - 80

3.2.2 Digestion procedure for analysis of sample for arsenic and selenium

determination - - - - - - - - - 81

3.2.3 Extraction procedure of sample for cyanide determination- - - 81

3.3 Sample analysis - - - - - - - - 82

3.3.1 Determination of lead, cadmium and nickel using atomic

absorption spectrophotometry - - - - - - 82

xi

3.3.2 Determination of arsenic using titrimetric method - - - - - 82

3.3.3 Determination of selenium using titrimetric method - - - - 82

3.3.4 Preparation of stock solution of cyanide - - - - - 83

3.3.5 Calibration curve for cyanide determination - - - - - 83

3.3.6 Preparation of alkaline picrate solution - - - - - - 83

3.3.7Determination of cyanide using uv/visible spectrophotometer- - - 83

CHAPTER FOUR 4.0 Results and Discussion - - - - - - - - - - 84

4.1 Concentration levels of lead, cadmium and nickel in the samples from

urban and rural areas of Southwest, Nigeria - - - - - 84

4.2 Concentration levels of arsenic and selenium in the samples from urban

and rural areas of Southwest, Nigeria - - - - - - 85

4.3 Absorbances of various standard solutions of cyanide for the calibration

curve- - - - - - - - - - 88

4.4 Concentration levels of cyanide in the samples from urban and rural

areas of Southwest, Nigeria - - - - - - - - 90

CHAPTER FIVE

5.0 Conclusion - - - - - - - - - - 94

REFERENCES - - - - - - - - - - 95

1

CHAPTER ONE

1.0 INTRODUCTION

1.1 Pollution

Pollution is the introduction of contaminants into an environment, soil, food, water e.t.c

that causes instability, disorder, harm or discomfort to the ecosystem or living organisms[1].

Sources of pollution can be natural or man-made. The major sources of cyanide and metal

pollution in urban areas are anthropogenic (human activities) while contamination from natural

sources predominate in the rural areas[2]. High concentration of toxic contaminants like

cyanide and heavy metals are generally found in the urban areas with large population, high

traffic density and industries[2].

Consequently, their undue presence in the environment through industrial emission, effluent

discharges, solid waste disposal, usage of agrochemicals and sewage sludge in agricultural

practices, traffic pollution, atmospheric pollution, automobile activities, combustion of fossil

fuels, coal, steel, plastics e.t.c, are more prominent in urban areas where multitudes of

stationary and mobile sources release large quantities of toxic contaminants into the

atmosphere.

Cyanide, a toxic contaminant, occurs naturally in most plants but has high

concentration in cassava and bamboo shoot. It is released into the environment through

volcanoes and natural biogenic processes from higher plants, bacteria, algae and fungi[3],

biomass burning, discharges from industries, waste water treatment, tobacco smoke, wood

smoke, smoke from burning plastics, vehicular emission, inadequately processed cassava

products e.t.c. Exposure to small amounts of cyanide can be deadly regardless of the route of

2

exposure. Cyanide is very poisonous, it stops cellular respiration by inhibiting an enzyme in

mitochondria called cytochrome c. oxidase in the body.

The term heavy metals, is a group name for some metals and metalloids associated

with pollution and toxicity, but also includes some elements which are essential for living

organisms at low concentrations. Heavy metals like zinc, manganese, copper, chromium, iron,

cobalt, selenium, magnesium and calcium are essential trace elements for man, animal and

plants but become toxic if the homeostatic mechanisms maintaining their physiological limit are

disrupted or they become toxic if their concentration is very high in the body while lead,

cadmium, nickel, mercury, arsenic are potentially toxic at certain levels[4].

Despite the fact that some heavy metals are beneficial, essential and non-essential, they can

cause morphological abnormalities, reduced growth, increased human mortality rate and

mutagenic effect in human when present in excessive levels[5]. Also, excessive accumulation

of heavy metals in the human body system usually results from increased human exposure to

the metals and this may cause health problems such as cancer, anaemia, neurological

problems, renal dysfunction, damage to the hepatic, hematological, neuromuscular,

reproductive, renal and central nervous system [6,7].

Cassava flour, a processed product from cassava, popularly known as “lafu” in Yoruba

language is a staple food in Nigeria which contains essential and beneficial minerals needed

for the body morphological processes. It is a rich source of carbohydrate, often referred to as

the major fuel of the body tissues[8] that releases energy needed by the body to function

properly in its daily activities. Human activities (anthropogenic sources) could favour the

presence of toxic contaminants like cyanide and heavy metals in cassava flour which may

render it unfit for human consumption especially when they are in high concentration.

3

Research have it that particulate air pollution and vehicle emission are the main causes

of heavy metals contamination in the urban areas[9]. Ano et al reported that atmospheric

deposition of heavy metals on cassava and soils along express road is higher than on cassava

and soils in remote villages (non road side environment) because of low vehicle emission in

the remote villages[10]. Alloway reported that plants accumulate considerable amount of heavy

metals in root and leaves[11]. Amusan et al also reported that there is high concentration of

heavy metal in vegetables grown in waste dump soil[12]. Ugwu et al reported high

concentrations of lead, cadmium and nickel in cassava flour sundried along a major

highway[13].

Detailed studies of the fate and contents of these heavy metals in various food and

human being have become a major task in research and there is continuos challenge to

develop new methodology and optimize that already available.

1.2 Background of the Research Project

In food processing, it is often necessary to carry out trace element analysis to ensure

that harmful and non-essential elements are kept at low concentrations as much as possible.

Most of these ions are toxic to human beings by interfering with enzyme functions while some

may have stimulatory effects. When the metals intake is at low concentration, the body system

might not be able to remove it and it will remain in the body as impurities for a short time.

The increase of cyanide and some metal toxicants in cassava flour can cause toxic effects for

consumers. The gravity of toxic effect depends on the nature, quantity, chemical form on body

resistance and on synergetic or antagonistic effects of other chemical contaminants. Sometime

in 2008, it was widely reported about a family who was nearly wiped out after eating food

4

prepared from fermented cassava flour in Ondo state. Such food poisoning could result from

pollution by known and unknown toxicants.

1.3 Aim and Objectives of the Research Project

1. This research work focuses on the determination of cyanide, lead, cadmium, nickel, arsenic

and selenium concentration levels in fermented cassava flour from some urban areas and rural

areas of Ekiti, Oyo, Lagos, Ondo and Osun states, all in South-West, Nigeria.

2. To find out if there is any significant differences in the concentration levels of cyanide, lead,

cadmium, nickel, arsenic and selenium in fermented cassava flour sourced from urban and

rural areas of these states.

3. To examine the dietary exposure to cyanide, lead, cadmium, nickel, arsenic and selenium

through consumption of cassava flour and create public awareness. This will make people to

know the inherent dangers of consumption of cassava flour and other food products that may

contain cyanide and these metals above the W.H.O (World Health Organization) guideline or

permissible level.

5

CHAPTER TWO

2.0 LITERATURE REVIEW

2.1 Cassava Plant (Manihot esculenta crantz)

Cassava Plant (Manihot esculenta crantz) belong to the family of Euphorbiaceae

(Spurge family), native of Brazil and Paraguay[14] that is extensively cultivated as an annual

crop in tropical and subtropical regions for its edible starchy tuberous root, is a major source of

carbohydrates.

The cassava root is long and tapered with a firm homogenous flesh encased in a

detectable rind about 1mm thick, rough and brown on the outside. Commercial varieties can be

5 to 10 cm in diameter at the top and 50 to 80 cm long. A woody cordon runs along the root‟s

axis. The flesh tuber can be chalk-white or yellowish.

Cassava roots are very rich in starch and contain significant amount of calcium (50 mg/100g),

phosphorus (40 mg/100g) and vitamin C (25 mg/100g)[15], however they are poor in protein

and other nutrients. In contrast, cassava leaves are a good source of protein, it is

supplemented with the amino acid methionine despite containing cyanide[15].

Cassava root size and shape depends on varietal and environmental factors. Cassava is a

tropical root crop requiring at least 8 months of warm weather to produce a crop. It is

traditionally grown in a savanna climate but can be grown in extreme of rainfall. In most areas,

it does not tolerate flooding, in droughty areas, it losses its leaves to conserve moisture,

producing new leaves when rain resume. It takes 18 or more months to produce a crop under

adverse condition such as cool or dry weather. Cassava does not tolerate freezing conditions.

It tolerates a wide range of soil pH 4.0 to 8.0 and is most productive in full sun.

6

2.1.1 History of the Cassava Plant

The oldest direct evidence of cassava cultivation came from a 1,400 years old Maya

site in El Salvador[16], although the specie Manihot esculenta originated further in South Brazil

and Paraguay. Wild population of Manihot esculenta sub specie, Flebellifolia, is shown to be

the progenitor of domesticated cassava found in west central Brazil where it was likely first

domesticated not more than 10,000 years A.D. By 6,600 B.C, manioc pollen appears in the

gulf of Mexico lowlands at the San Andres archaeological site[17]. With its high food potential,

it had become a staple food of the native population of North and South America, South

Mesoamerica and the Caribbean by the time of the Spanish conquest and its cultivation was

continued by the Colonial Portuguese. Forms of the modern domesticated specie can be found

growing in the South of Brazil and there are several wild Manihot species, all varieties of

Manihot esculenta are cultigens.

2.1.2 Production / Economic Impact of Cassava

Cassava is one of the most staple food crops of more than 500 million people and is a

typical crop in developing countries[18]. Cassava is considered an important source of energy

in diets. Cassava is known to produce 250,000 calories/hectare/day compared to 200,000 for

maize, 176,600 for rice, 114,000 for sorghum and 110,000 for wheat. Cassava is the third

largest source of human food in the world, with Africa as its largest centre of production[19].

Cassava plays a major role in efforts to alleviate the African food crisis because of its efficient

production of food energy, year-round availability, tolerance to extreme stress conditions and

suitability to present farming and food systems in Africa[18].

7

World production of cassava root was estimated to be 184 million tonnes in 2002[15].

The majority of production is in Africa where 99.1 million tonnes were grown, 51.5 million

tonnes were grown in Asia and 33.2 million tonnes in Latin America and the Carribean[15].

Nigeria is the world‟s largest producer of cassava [15]. However, based on the statistics from

the Food and Agricultural Organization (FAO) of the United Nations, Thailand is the largest

world exporter of cassava in 2005. The second largest exporting country is Vietnam with

13.6%, and then increased to 55.8% and Costa Rica 2.1%[15]. World-wide cassava production

increased by 15.5% between 1988 and 1990[15]. Cassava plays a role in developing countries

farming especially in sub-saharan Africa because it thrives well on poor soil and low rainfall

and is a perennial crop that can be harvested as required. Its wide harvesting window allows it

to act as a famine reserve crop and is invaluable in managing labour schedules. Cassava is

regarded as subsistence or cash crop of low-income families or resource poor farmers[20].

A 1992 study revealed that about 42% of harvested cassava roots in West and East Africa are

processed into dried chips and flour [15]. In Ghana, cassava and yam occupy important

positions in the agricultural economy and contribute about 46% of agricultural gross domestic

product (GDP)[15]. Cassava accounts for a daily calorie intake of 3% in Ghana and it is grown

by every farming family. The importance of cassava to many Africans is epitomized in the Ewe

(a language spoken in Ghana, Togo and Benin) name for the plant, meaning “there is life”.

8

2.1.3 Importance of Cassava Processing

Raw cassava roots must be used immediately, processed or preserved in order to

prevent decomposition since they cannot be stored for long because they rot within 3 - 4 days

of harvest. Thus processing helps to remove or reduce the level of toxic cyanogenic glucosides

and other toxic contaminants present in the cassava root as well as altering the available

energy of the cassava, increasing the shelf life, improving the palatability and conversion into

stable product [21].

Cassava processing varies according to the form in which the cassava is to be

consumed from simple sun-drying to complex methods involving fermentation to complicated

procedures like processing into gari[21]. Some of these processes reduce cyanide more

effectively than others. The specific effects of various processing techniques on the cyanide

content of cassava are discussed below.

Peeling: Many methods of cassava processing starts with the peeling of the tubers. Generally,

the cassava peel contains higher cyanide content than the pulp. Removal of the peels reduces

the cyanogenic glucoside content considerably to at least 50% in cassava tubers. In studies

carried out by Tewe, the peel of the “bitter” cassava variety was shown to contain an average

of 650 ppm and the pulp to contain 310 ppm total cyanide[22].

Grating: This process takes place after peeling and is sometimes applied to whole tubers.

Grating of the whole tuber ensures the even distribution of the cyanide in the product and will

also make the nutrients contained in the peel available for use. In the grated product, the

concentration of cyanide depends on the time during which the glucoside and the glucosidase

interact in an aqueous medium. Grating also, obviously, provides a greater surface area for

fermentation to take place.

9

Soaking: It provides a suitably larger medium for fermentation and allows for greater

extraction of the soluble cyanide into the soaking water. The process removes about 20% of

the free cyanide in fresh root chips after four hours, although bound cyanide is only negligibly

reduced. Bound cyanide begins to decrease only after the onset of fermentation[23]. A very

significant reduction in total cyanide is achieved if the soaking water is routinely changed over

a period of 3-5 days. A stimulation of the technique, followed by sun drying showed a reduction

of cyanide of about 98.6% of the initial content in the roots[23].

Fermentation: Microbial fermentation have traditionally played important roles in food

processing for thousands of years. Most marketed cassava products like “gari”, “fufu”, “lafu”,

“pupuru” etc. in Africa are obtained through fermentation. The importance of fermentation in

cassava processing is based on its ability to reduce the cyanogenic glucosides to relatively

insignificant level. Some cyanidrophilic/cyanide tolerant microorganisms effect breakdown of

the cyanogenic glucoside. Also, the longer the fermentation process, the lower the residual

cyanide content and generally, fermented cassava products store better and are often low in

residual cyanide content. Onabowale developed a combined acid hydrolysis and fermentation

process at FIIRO (Federal Institute for Industrial Research, Oshodi, Nigeria) and achieved 98%

reduction in total cyanide after dehydration of the cassava flour for use in the feeding of

chickens[24].

For “lafu” (cassava flour) production in Nigeria, peeled or unpeeled cassava tubers are

traditionally immersed either in a running stream, or in stationary water (near a stream) or in an

earthen ware vessel and fermented until the roots become soft. The peel and central fibres of

the fermented roots are manually removed and the recovered pulp is hand mashed or

pounded. The microorganisms involved in “lafu” (cassava flour) production include four yeasts:

10

Pichia onychis, Candida tropicalis, Geotrichum candida and Rhodotorula sp. ; two molds:

Aspergillus niger and penicillium sp. and two bacteria: leuconostoc sp. and corynebacterium

sp[25]. Moisture, pH and temperature conditions are critical for the growth of these

microorganisms in roots and thus for fermentation.

Drying: Drying is the simplest method of processing cassava. Drying reduces moisture,

volume and cyanide content of roots, thereby prolonging product shelf life and facilitates the

continuation of the fermentation process. Total cyanide content of cassava chips could be

decreased by only 10 - 30% through fast air drying. Slow sun-drying, however, produces

greater loss of cyanide. Drying may be in sun or over a fire. The former is more common

because it is simple and does not require fuel wood[22]. Gomez et al indicated that more than

86% of cyanide present in cassava was lost during sun drying[26].

Boiling/Cooking: As with soaking, the free cyanide of cassava chips is rapidly lost in boiling

water. About 90% of free cyanide is removed within 15 minutes of boiling fresh cassava chips,

compared to a 55% reduction in bound cyanide after 25 minutes. Cooking destroys the

enzyme linamarase at about 72 0C thus leaving a considerable portion of the glucoside intact.

Milling: The dried root pieces and fermented / dried pulp are milled into flour by pounding in

mortar or using hammer mills. Milling with hammer mills done at village level may also reduce

cyanide. The dried cassava roots (both fermented and unfermented) are often mixed in a ratio

of 2 to 3 parts cassava with one part of sorghum, millet and or maize and milled into a

composite flour. Mixing cassava with cereals increases food protein and enhances palatability

by improving consistency.

11

Table 2.1 shows the concentrations in mg/kg of cyanide in cassava and its processed

products.

Table 2.1 Concentrations in mg/kg of cyanide in cassava and its processed products

Unprocessed cassava and cassava

processed products

Concentration of cyanide in mg/kg[3]

1. Cassava (bitter) dried root cortex 2360

2 Cassava (bitter) leaves 300

3. Cassava (bitter) whole tubers 380

4. Cassava (sweet) leaves 451

5. Cassava (sweet) whole tubers 445

6. Cassava flour (lafu) 0.1[27]

7. Fufu 0.25[27]

8. Gari 0.19[27]

2.1.4 Uses of Cassava

Animal Feed

Cassava is widely used in most tropical areas for feeding pigs, cattle, sheep and poultry. Dried

peels of cassava roots are fed to sheep and goats and raw or boiled roots are mixed into a

mash with protein concentrates such as maize, sorghum, groundnut or oil-palm kernel meals

and mineral salts for livestock feeding. In Brazil and many parts of Southeast Asia, large

quantities of cassava roots, stems and leaves are chopped and mixed into a silage for the

feeding of cattle and pigs. However, cassava cannot be used as the sole feed stuff because of

12

its deficiency in protein and vitamins, but must be supplemented with other feeds that are rich

in these elements.

Fermented Products

Cassava Alcohol

Cassava is one of the richest fermentable substances for the production of alcohol. The fresh

roots contain about 30% starch and 5% sugars and the dried roots contain about 80%

fermentable substances which are equivalent to rice as a source of alcohol.

When cassava is used, the roots are washed, crushed into a thin pulp and then screened.

Saccharification is carried out by adding sulphuric acid to the pulp in pressure cookers until

total sugars reach 15% - 17% of the contents. The pH value is adjusted by using sodium

carbonate, and then yeast fermentation is allowed for 3 - 4days at a suitable temperature for

the production of alcohol, carbondioxide and small amounts of other substances from sugar. It

is usually utilized for industrial purposes, as in cosmetics, solvents and pharmaceutical

products. If the production is required for human consumption, special care should be taken in

handling the roots to rid them of hydrocyanic acid.

Dried Yeast

Microbial protein is attracting growing interest owning to the enormous protein requirements of

the world. Cassava starch and cassava roots are being used in Malaysia and some other

countries for the production of yeasts for animal feed, the human diet and for bakery yeast.

The starch is hydrolyzed into simple sugars (predominantly glucose) by means of mineral acid

or by enzymes. Certain yeasts are then propagated which assimilate the simple sugars and

produce microbial cellular substances.

13

Human Food and Food Industries

The food industries are one of the largest consumers of cassava and cassava product

like starch. Cassava can be cooked in various ways, cassava soft boiled root can replace

boiled potatoes in many uses as an accomplishment for meat dishes or made into purees,

dumpling, soups, stew, gravies e.t.c. Other traditional culinary preparation of cassava in Africa

are fufu, lafu, eba, chickwangue, abacha, etc. Tapioca is a flavourless starchy ingredient or

tecula produced from treated and dried cassava root and used in cooking. It is similar to sago

and is commonly used to make milky pudding cassava flour also called tapioca flour or tapioca

starch and also replace wheat flour and is used by some people with wheat allergies or celiac

disease. Boba tapioca pearls are made from cassava root. It is also used in cereals for which

several tribes in South America have used it extensively[15].The juice of the bitter cassava

boiled to the consistence of thick syrup and flavoured with spices is called cassareep. It is used

as a basis for various sauces and as a culinary flavouring principally in tropical countries. It is

also used in bubble drink in East Africa. The leaves can be pounded to fine chaff(washed

thoroughly to remove bitterness) and cooked in a palaver sauce(with meat and fish) in Sierra

Leone, usually with palm oil or vegetable oil.

Also in bakery products, cassava starch is the major constituent of flours which is used

in bread baking and biscuit making to increase volume and crispness and in confectioneries to

manufacture gums, paste and sweets in order to prevent them from sticking together.

Non- Food Uses

Non-food uses of cassava starch such as coating, sizing and adhesives account for about 75%

of the output of the commercial starch industry. The application of cassava in adhesives

continues to be one of the most important end uses of the product. It is used in the

14

manufacture of glue used in wood furniture and remoistening gums for stamps, envelope flaps,

used in corrugated cardboard industry for manufacture of cartons, boxes and other packing

materials. In foundry, starch is used as an adhesive for coating the sand grains and binding

them together in making cores which are placed in moulds in the manufacture of castings for

metals. Cassava starch is used as tubs size and beater size in the manufacture of paper and in

textile industry for cloth printing, producing certain design in various colours on the smooth

surface of a finished fabric and warp sizing (application of a protective coating to prevent the

single yarns from disintegrating during weaving).

Particle board from cassava stalks

As cassava cultivation increases, more stalks will become available for disposal. The Tropical

Product Institute, London, has been working on the utilization of the cassava plant. Particle

boards could be made from cassava stalks by cutting them into small sections and mixing

them with certain resins. The strength of the board can be varied by altering the resin content

or the density.

Biofuel

In many counties, significant research has begun to evaluate the use of cassava as an ethanol

biofuel. Under the Development Plan for Renewable Energy in the 11th five-year plan in China,

the target is to increase the application of ethanol fuel by non-grain feed stock to 2 million

tonnes and that of bio-diesel to 200,000 tonnes by 2010[28].This will be equivalent to a

substitute of 10 million tonnes of petroleum. As a result, cassava (tapioca) chips have

gradually become a major source for ethanol production. On December 22, 2007, the largest

cassava ethanol fuel production facility was completed in Beihai with annual output of 200,000

tons, which would need an average of one and half million tons of cassava[15]. In November

15

2008, China-based Hainan Yedao Group reportedly invested $51.5m (£31.8m) in a new biofuel

facility that is expected to produce 33 million gallons a year of bio-ethanol from cassava

plants[28].

Ethno medicine

The bitter variety of cassava is used to treat diarrhea and malaria. Cubans commonly use

cassava irritable bowel syndrome, the paste is eaten in excess during treatment. As cassava is

a gluten-free natural starch, there have been increasing incidences of its appearance in

western cuisine as wheat alternative for sufferers or celiac disease[15].

2.1.5 Cassava Processed Product (Cassava flour)

Cassava have been variously used in the production of different types of food most

especially in Africa[29] apart from non food uses. They are referred to as cassava processed

product and they are garri, fufu, cassava flour, wet pulp, smoked cassava ball (kumkum),

fermented and dried cassava pulp starch, chickwague (Zaire, Congo, Sudan, Gabon, Angola,

Cameroon and Central Africa Republic), abacha, tapioca etc.

The main focus of this research is cassava flour which is also known as “lafu” in Nigeria. Figure

2.1 shows the steps in processing of cassava into flour. Cassava flour (lafu) is a fibrous

powdery form of cassava similar to “fufu” in Nigeria. The method of production of “lafu” is

different from “fufu” and is given below. Unlike fufu, the fibres in the retted root for lafu are

dried along with the mash and later sieved out. Thus, lafu is coarser than fufu in texture. The

flour is made into dough with boiling water before consumption. When properly stored, it has a

shelf life of six months or more.

16

Fig 2.1 Processing of cassava flour (lafu)

Harvest/sorting of cassava

Peeling

Peel by hand and remove woody tips

Select fresh mature cassava roots without rot

Size reduction (optional)

Reduce size of cassava pieces by cutting

Washing

Wash in clean water to remove pieces of peel, sand etc

Steeping

Soak inside water in bowl for 3-4 days at room temperature

Crushing/pulping

By hand

Dewatering

Fermented paste is filled into hessin or polypropylene sacks and placed

in a hydraulic jerk press

Drying

On rocks or block inclined surfaces at ambient temperature

Milling/sieving

To obtain powder and remove fibre

Packing

In polythene bags

Storing

In a cool, dry place

17

2.1.6 Cassava Toxicity

Cassava roots and leaves cannot be consumed raw because they contain two

cyanogenic glucosides, linamarin and lotaustralin. These are decomposed by linamarase, a

naturally occurring enzyme in cassava, liberating hydrogen cyanide (HCN)[30]. Cassava

varieties are often categorized as either “sweet” or “bitter”, signifying the absence or presence

of toxic levels of cyanogenic glucosides. The so called “sweet” (actually not bitter) cultivars can

produce as little as 20 mg/kg of cyanide of fresh roots, whereas “bitter” ones may produce

more than 50 times as much (1 g/kg) of cyanide. Cassava grown during drought is high in

these toxins, cyanide[31]. A dose 40 mg of pure cassava cyanogenic glycoside is sufficient to

kill a cow. In case of human malnutrition, where the diet lacks protein and iodine processed

roots of high hydrogen cyanide cultivars may result in serious health problems.

Apart from cyanide which is part of cassava plant and a contaminant that causes

cassava poisoning when not properly processed, heavy metals are also contaminants in food

like cassava according to Shroeder (1973)[32]. Contaminants refer to undesirable materials

which have been added inadvertently before, during or after processing of food. Food plants,

vegetable or other farm products may be contaminated by agricultural sprays and heavy

metals may get incorporated into the food due to attack on the processing plant or container.

Although it is difficult to legislate specially against all possible forms of contamination,

maximum limits have been recommended for heavy metals[32].

Shroeder also reported that heavy metal refers to the inorganic elements which may be trace

metals which may be present in foods in amounts usually well below 50 ppm and have some

toxicology or nutritional significance[32] broadly classified trace metals according to their effect

on life and placed them into three classes which are;

18

i. The essential nutritive element e.g Co, Cu, Fe, Mn, Zn.

ii. The non-nutritive, non-toxic elements e.g Al, B, Cr, Ni, Sn which are not known to

have produced harmful effects when present in quantities not exceeding 100 ppm

and

iii. The non-nutritive, toxic elements e.g. As, Sb, Cd, F, Pb, Hg, Se which are known to

have deterious effect even when the diet contains less than 100 ppm.

Shroeder reported that a complicating factor, however is that heavy metals such as

copper and zinc, although essentials for life processes, when present in traces have an emetic

action when ingested in higher amounts. Cumulative poisoning due to ingestion of food

containing element such as lead or arsenic over a long period is probably rare. Consequently,

they tend to reach the environment from a vast array of anthropogenic sources as well as

natural geochemical processes[32].

2.2 Heavy Metals

Heavy metals are ill defined subset of element that exhibit metallic properties which

include the transition metals, some metalloids, lanthanides and actinides. Although there is no

clear definition of what a heavy metal is, density is most cases taken to be the defining factor.

Heavy metals are thus commonly defined as those having a specific density of more than

5 g/cm3.

It is very difficult to demarcate toxic metals from essential ones. In fact any metal could

become toxic if ingested in sufficiently large amounts.

19

2.2.1 Sources of Heavy Metals

The sources of heavy metal can be grouped into two broad headings namely: natural

sources and anthropogenic sources.

Natural Sources: This was the primary sources of these metals before population explosion

and industrialization. The origin of these metals is traced from nature. Their occurrence and

distribution is solely due to phenomena like geological weathering, volcanic eruption, erosion

and sometimes forest fire. Almost all metals which occur naturally are found to exist in the

earth crust where parent rocks constitute the reserves and primary sources of metals.

Generally, the levels of heavy metals emitted by natural processes are exceedingly low, they

are found to be distributed in trace amount and hence not harmful to organisms. Accumulation

at a particular point may come from weathering of rocks.

Anthropogenic Sources: This is a dominant source of trace metals in the environment. These

sources has been traced to the daily human activities such as industrial production, agricultural

activities, urban life and commerce. Approximately 100,000 different chemicals are produced

and used throughout the world, their production and usage are concentrated in industrialized

countries[33]. These chemical and even larger number of byproducts find their way into the

environment by different pathways. These include atmospheric distribution by which the metals

are carried as aerosols e.g from motor vehicles. The use of leaded petrol has been responsible

for lead emission through exhaust pipes which is carried along by air and distribution to the

nearby surroundings. The combustion of fossil fuel also result in the dispersion of many

elements in the air over large area. Agricultural fertilizer, slaps from iron manufacture,

pesticides and herbicides contain various combination of heavy metals either as impurities or

active constituents and serve as sources of heavy metal contamination.

20

The disposal of urban and industrial waste can lead to soil pollution. The deposition of particles

emitted during incineration of metal containing material can lead to soil pollution. The careless

dumping or disposal of metal containing dry cell batteries, abandoned vehicles and vehicle

components, for example lead acid batteries can give rise to small areas of very high metal

concentration in the soil or environment.

Metallurgical industries also contribute to soil pollution by emission of fumes and dusts

containing metals which are transported in the air and eventually deposited on soils and

vegetation. Industrial effluent play important role in polluting the environment when they are

allowed to flow freely or during rainfall, they are carried as run off flood. The mining and

smelting of metal ores also lead to pollution of mine areas.

It is generally believed that anthropogenic release of lead into the environment in the

modern era can be traced to the use of lead alkyl in leaded gasoline as an antiknock agent to

increase octane number[34]. Due to the non-biodegradability and cumulative tendency of lead,

the prevalent use of lead alkyl has been left behind footprints of lead contamination in the

ecosystem through air, soil and water[35]. Zinc is emitted into the environment as a result of

wear from tyres of automobiles, combustion of automobile tyres and motor vehicle exhaust as

a result of zinc containing compounds in lubricating oils. Copper and iron on the other hand are

essential components of motor vehicle parts[36]. They are emitted into the environment as a

result of wear of vehicle parts, whereas manganese is naturally associated with crude oils,

which are the sources of energy for motor vehicles, most of the manganese in the air is due to

the burning of fuels[37]. Cadmium is emitted into the environment as a result of combustion of

fossil fuels, iron and steel production, non-ferrous metal production and municipal solid waste

combustion. Combustion of fossil fuel, coal, municipal incineration releases nickel into the

21

environment[38]. Arsenic is emitted into the environment as a result of by-product of the

smelting of copper, lead and zinc sulphide ores[39], also, smelting of sulphide ores releases

selenium into the environment.

2.2.2 Heavy Metals in Food

Heavy metals gets into food plant through the soil. Heavy metals occur naturally in the

soil and soil is ultimately the primary natural source of trace metal but rarely at toxic level[40].

These amount can be changed because of many industrial processes such as smelting,

burning of fossil fuels, electroplating operation, pesticides, petroleum spill, fertilizer application

and mining.

The presence of heavy metals in food is highly significant for they are capable of causing

serious health problem depending on the nature of the heavy metal. They do this through

interfering with the normal biological functioning of the animal health.

Heavy metals impose contamination either during manufacture, processing and storage or

may be added directly as essential nutrient supplementation. Commercial foods like beans,

yam, cassava, rice, meat, garri e.t.c. may be focused on as they are the main sources of most

consumers. But reports has also been given of food that consumers collect from the wild, some

fungi can accumulate contaminants that are present in the environment. The principal factors

influencing the accumulation of contaminants by fungi are not only environmental factor such

as metal concentration in the soil and pH but also factors such as fungal structure[41]. Studies

have shown that the fruiting bodies (e.g mushrooms) of many fungal species can accumulate

mercury, cadmium and lead with some very high lead concentration being reported in food

growing in the vicinity of high ways or other source of lead[41]. It is obvious that food grown or

dried along the expresssway are liable to contamination with both manganese, lead, titanium,

22

cadmium, arsenic e.t.c. Other metals such as sodium, potassium, magnesium and calcium are

needed at comparatively greater amount and are regards as micronutrient. Their deficiency in

food must be supplemental or else the animal suffers from both biological imbalance and

physiological ill-condition. They play most important role in body functioning of an organism.

However, when in excess intake, they will also subject the body to some ill conditions.

2.2.3 Recommended levels of cyanide and heavy metals in food

In cognizance of the effects of heavy metals and other pollutants on human, animals

and plant life, various bodies, both national and global have recommended levels at which the

pollutants are permissible. This is part of efforts towards making the environment a healthy

place to live in by controlling natural and man-made activities, ending to make it otherwise[42].

Below are the various values recommended by the World Health Organization (WHO)

as the maximum permissible levels for some heavy metals and cyanide in food materials for

human consumption.

Table 2.2 WHO recommended (guideline) concentration values of some heavy metals

and cyanide (mg/kg) in food

Some heavy metals and cyanide WHO guideline values in mg/kg[42]

Lead 0.1

Cadmium 3

Nickel 1.037

Arsenic 2

23

Selenium 0.1-10

Copper 1.5-3.0

Zinc 15

Manganese 2.0-5.0

Iron 10

Cyanide 10 [43]

Table 2.3 US Recommended Dietary Allowances[44][8]

Trace

metals and

cyanide

Recommended Dietary Range Adequate

Level (mg/day)

Tolerable Upper Intake

Level (mg/day)

Lead 0.00 0.00

Arsenic 0.00 0.00

Cadmium 0.00 0.00

Zinc 12-15 10-40

Chromium 0.1-1 2-3

Copper 2-2 4-10

Cobalt 3-5 6-10

Iron 6-15 40-50

Nickel 0.40-0.70 1.00[45]

Selenium 0.055 0.40[45]

Cyanide 0.00 0.00

24

2.2.4 Analytical techniques for determination of cyanide and heavy metals in food

samples

Over the years, several analytical methods have been devised for quantitative

determination of cyanide and heavy metals in food. These include X-ray fluorescence

spectrophotometry, Atomic fluorescence spectrometry, Inductively coupled plasma atomic

emission spectroscopy (ICP/AES), polarography, chromatography, mass spectrometry,

voltammetry, titrimetry, flame atomic absorption spectrophotometry, ultraviolet / visible

spectrophotometry etc[46]. Some factors such as initial cost of instrument, technical know-

how, consumable and costly maintenance of technique, lack of specificity or sensitivity restrict

the wide applicability of these techniques, particularly in laboratories with limited budget in

developing countries and for field work.

The most common method of determination of heavy metals in food is Atomic

Absorption Spectrophotometry (AAS) and is the most widely used because it is sensitive,

specific, accurate, precise, reliable, simple and effective but this method has limits such as

requiring skilled personnel, time consuming, high capital and maintenance cost[47].

The application of ultraviolet/visible spectrophotometry in determination of metals in

food, biological samples, is still popular in many laboratories. The technique provides easy

determination of many metals from low to high concentration at affordable cost[48]. It is very

simple, rapid and less expensive but its limit is problem of selectivity and some of its reagents

are expensive.

Titrimetry method is the cheapest, simplest of all analytical techniques. A wide range of

analytes can be conveniently determined by this technique. Titrimetric methods of analysis are

25

capable of rapid and convenient analyte determinations with high accuracy and precision but

its limit is the inconsistent stiochiometry and instability of titrant and analyte solutions[49].

2.2.5 Atomic Absorption Spectrophotometry (AAS)

The technique was introduced in 1955 by Walsh in Austria and by Alkemade and Milatz

in Holland. The first commercial atomic absorption spectrophotometer was introduced in 1959

and it grew explosively after that[50].

Atomic Absorption Spectroscopy can be simply defined as the absorption of radiant

energy by atoms. The production of atoms from a chemical compound requires the absorption

of energy, the energy is usually supplied in form of heat from a flame. The compound

introduced into flame after vapourisation is partially or wholly dissociated into its elements in a

gaseous form and some of these atoms absorb radiant energy of a characteristics wavelength

and become excited to a higher energy state.

Atomic absorption spectroscopy makes use of the fact that free atoms of an element absorbs

light at wavelength characteristic of that element and determined by its outer electronic

structure. It has high specificity making it possible for elements to be determined in the

presence of each other. The extent of absorption is the measure of the number of atoms in the

light path. An illustration below shows the sequence through which atomic absorption

spectroscopy operates.

26

Fig 2.2 Operation of an Atomic Absorption Spectrophotometer

An energy source such as flame decomposes the sample into its constituent atoms that is

flame atomizer , the monochromator isolates the required wavelength which is characteristics

of the element, the photomultiplier detects and the readout devices is used to read out the

absorbance.

The linear relationship between the measured absorbance and the concentration of

analyte atoms in the flame which is proportional to the concentration of atoms in the sample

solution is expressed using the Beer-Lambert graph.

Beer – Lambert Law A = KC

Fig 2.3 A Calibration graph for AAS Determination.

A

K

C (PPM)

27

A = Absorbance of sample solution.

K = Slope of the curve.

C = Concentration of the metal in the same solution.

According to Beer-Lambert‟s law, absorbance is directly proportional to the

concentration of the absorbing specie C and to the path length L of the absorbing medium as

expressed by the equation below.

Where A is the absorbance, no unit because it is calculated as log A = Log10 Io I E is the molar absorbtivity or molar extinction coefficient (dm3/mol/cm). It is a proportionality

constant.

C is the concentration of the compound in the solution (mol/dm3)

L is the pathlenght of light in sample (cm)

The molar absorbtivity must have units that cancel out (L) and (C) which make absorbance unit

less. Then the above equation can also be represented as follows: A = ELC.

2.2.5.1 Interferences

Since the concentration of the analyte element is considered to be the ground state, any factor

that affects the ground state population of the analyte element can be classified as

interferences. Factors that may affect the ability of the instrument to read this parameter can

also be classified as interference. The following are the most common interferences.

i. Spectral interferences due to radiation overlapping of the light sources.

ii. Formation of compounds that do not dissociate in the flame.

A = Log10 Io = ELC

I

28

iii. Ionization of the analyte reduces the signal.

iv. Matrix interferences due to differences between surface tension and viscosity of test

solutions and standards.

v. Broadening of a spectral line, which can occur due to a number of factors.

2.2.6 UV/Visible Spectrophotometry

UV/Visible spectrophotometry refers to absorption spectroscopy in the ultraviolet-visible

spectral region which uses light in the visible and adjacent (near-uv and near-infrared) ranges.

The absorption in the visible range directly affects the perceived colour of the chemicals

involved. In this region, molecules undergo electronic transitions from ground state to excited

state[51] because of its ability to absorb colour of solution in the wavelength limits 185-769nm.

It is used for both qualitative and quantitative investigations of samples.

The instrument uv/visible spectrophotometer measures the intensity of light passing

through a sample (I) and compares it to the intensity of light before it passes through the

sample (Io). The basic parts are a light source, a holder for the sample, a diffraction grating in a

monochromator or a prism to separate different wavelength of light and a detector (typically a

photomultiplier tube or photodiode). Single photodiode detectors and photomultiplier tubes are

used with scanning monochromators which filter the light so that only light of a single wave

length reaches the detector at one time. The scanning monochromator moves the diffraction

grating to “step through” each wavelength so that its intensity may be measured as a function

of wavelength.

29

Fig 2.4 Principle of uv/vis spectrophotometer[53]

UV/Visible spectrophotometer also observe Beer-Lambert‟s law like atomic absorption

spectrophotometer in that the wavelength at the maximum of the absorption band will give

information about the structure of the molecule or ion and the extent of the absorption is

proportional with the amount of the species absorbing the light which is based on Beer-

Lambert‟s law. Instead of molar absorbance, a specific molar absorbance is used whose

concentration unit is applied. Molar absorbance is a function of wavelength, so Beer‟s law is

applied at one (or several) specific wavelength values.

Determination of food, biological samples e.t.c by uv/visible spectrophotometer could

be influenced by the following factors[52].

i. Selectivity Problems: The absorbance of other substance or substances being present

might be added to the absorbance of the compound under investigation. The effect of this

30

phenomenon could be eliminated either by increasing the selectivity by chemical reaction or by

preliminary separation using either extraction or chromatography.

ii. Deviations from the Beer’s Law: If using a “not properly monochromator” light beam, the

high absorbance are decreased compared to the real values. The association of molecules in

the sample (which depends on the concentration) also could cause deviations.

The analysis of cyanide in food, water, soil, plant has been conveniently and

successfully determined by uv/visible spectrophotometer. Research have it that a procedure

for the quantitative determination of hydrogen cyanide released by plants has been developed

based on the uv-vis spectrum of the sodium pictrate-cyanide complex[54]. Miguel etal reported

that uv/visible spectrophotometry and HPLC were used in sample analyses of assessment and

degradation study of total carotenoid and β-carotene in bitter yellow cassava varieties[55]. It

has also been used in cyanogen content of cassava mash and pre fufu mash (unprocessed

product), fufu and gari (processed product)[56].

2.2.7 Titrimetry Method

Titrimetry also known as “titration”[57] comes from the latin word titulus, meaning

inscription or title. The French word “titre” also from this origin means rank. Titration is a

common laboratory method of quantitative chemical analysis that is used to determine

unknown concentrations of a known analyte, because volume measurements play a key role in

titrating, it is also known as volumetric analysis. Using a calibrated burette or pipetting syringe

to add the titrant, it is possible to determine the exact amount that has been consumed when

the endpoint is reached. The end point is the point at which titration is complete as determined

by an indicator.

31

Titrimetic methods of analysis are capable of rapid and convenient analyte

determinations with high accuracy and precision[58]. There are various types of titrimetric

methods (titration) which include, acid base titrations, redox titrations, complexometric titrations

(EDTA titration), precipitation titrations etc. The most common types are the acid base titrations

and redox titrations.

2.2.7.1 Redox Titration

Redox titrations are the most diverse of all types of titration and a wide variation of

analytes can be conveniently determined by redox titrations[58]. They are based on a redox

reaction between an oxidizing agent and a reducing agent. Kleinmann and Pangritz regard

coloumetric methods of Gutzeit type as inherently unreliable and consider the titrimetric

methods (redox titration) the technique of Billister who dissolves the marsh mirror in iodine and

back titrates with thiosulphate is the most accurate[59]. Pretreatment of the analyte with an

oxidizing or reducing agent is often needed in redox titrations.

The procedure for carrying out redox titrations is similar to that required in acid-base

titration[57]. A potentiometer or a redox indicator is used to determine the end point of redox

titration[57].When one of the constituents of the titration is oxidizing agent, potassium

dichromate, the colour change of the solution from orange to green is not definite, an indicator

such as sodium diphenylamine is used. Report have it that the analysis of wine for their

sulphurdioxide content requires the use of iodine as an oxidizing agent, starch is used as an

indicator, a blue starch iodine complex is formed once an excess of iodine is present thus

signaling the end point of titration[57]. On the other hand, some redox titrations do not require

an indicators due to the intense colour of some of the constituents. For instance, in a titration

32

where the oxidizing agent, potassium permanganate is present, a slight faint persisting pink

colour signals the end point of the titration and no particular indicator is therefore required.

2.2.7.2 Common Titrants in Redox Titration

Reducing Agents

Reducing agents are not stable in air (undergo air oxidation) and so are not often used[58].

The two most common reducing titrants are ferrous ammonium sulphate (FAS) and sodium

thiosulphate. They are capable of determining the concentration analytes that are (at least)

moderately strong oxidizing agent[58].

Sodium Thiosulphate Na2S2O3

i. Thiosulphate is a moderately strong reducing agent:

ii. Thiosulphate is not suitable for the direct analysis of most oxidizing agents, since reactions

with thiosulphate tends to produce sulphite and sulphate. However, it is widely used in back

titrations of iodine that is produced by the reactions of oxidizing agents with iodide, another

reducing agent (this procedure is called iodometry)[58].

iii. Thiosulphate solutions are standardized with iodine which has been prepared by acidifying

primary standard potassium iodate in the presence of a slight excess of potassium iodide:

The titration reaction between iodine and thiosulphate is fairly straight forward:

iv. Alkaline solutions of sodium thiosulphate are fairly stable.

2S2O32- S4O6

2- + 2e- E0=0.09V

I2 + 2S2 O32- 2I- + S4O6

2-

S4O623H2O -

IO3- + 5I- + 6H+ 3I2(aq) + 3H2O acidic solution

33

Oxidizing Agents

Oxidizing agents are used for the analysis of reducing agents. Pre-reduction of analyte is

common, analyte is often unstable in reduced form and care must be taken in sample handling.

They are Potassium Permanganate, Ceric Sulphate, Potassium Dichromate and Iodine[58].

Iodine I2

i. It is used to analyze moderately strong oxidants or reductants. Advantage of its moderate

strength as a redox reagent is that titrations involving iodine are more selective than those

involving more powerful redox reagents[58].

ii. The standard reduction of iodine is

iii. There are two classes of titrations involving iodine, iodine is a moderate oxidizing agent and

iodide is a moderate reducing agents.

a. Iodimetry: This is based on the reaction between the analyte and iodine. Since iodine is an

oxidizing agent, iodimetry is used for the analysis of reducing agents (reductants).

b. Iodometry: This is based on the reaction between the analyte and an unmeasured excess

of iodide to produce iodine, followed by back titration with thiosulphate (i.e. measured by

titration with thiosulphate). The amount of iodine produced by this reaction is stoichiometrically

related to the amount of analyte originally present in the solution. Since iodide is a reducing

agent, iodometry is used for the analysis of oxidizing agent (oxidants).

Under acidic conditions, iodide is slowly air oxidized to produce iodine. In alkaline

solution, iodine will disproportionate to produce iodide and iodate.

I2(aq) + 2e- 2I- E=0.621v

3I2 + 3H2O IO3- + 5I- + 6H+

34

This reaction is quite reversible upon acidification, the reaction shifts to the left as iodate reacts

with iodide to form iodine.

Iodine solutions are generally most stable at neutral pH values and must be

standardized fairly frequently[58].

2.2.7.3 Applications of Redox Titrations

i. It is used in the analysis of iron ores by dichromate titration.

ii. It is used in the analysis of residual chlorine by iodometric titration.

iii. It is used in the analysis of ascorbic acid (vitamin C), hydrogen perioxide, bleach

e.t.c.

iv. It is used to determine dissolved oxygen (DO) by iodometric titration.

v. It is used to get the chemical oxygen demand (COD) by dichromate back titration

using Ferrous Ammonium Sulphate (FAS).

2.3 Chemistry of Cyanide and Heavy metals

2.3.1 Cyanide

Cyanide is a chemical compound that contains the cyano group -C≡N, which consists

of a carbon atom triple bonded to a nitrogen atom[60]. Cyanide could be a gas; hydrogen

cyanide or a solid; potassium cyanide or sodium cyanide and they are fast-acting poisons that

can be lethal.

35

History of cyanide

The word “cyanide” was extracted from “ferrocyanide”, a cyanide derivative of iron. The name

“ferrocyanide” was invented as meaning “blue substance with iron” as ferrocyanides were first

discovered as components of the intensely coloured dye prussian blue. Kyanos is Greek for

(dark) blue.

Cyanide didn‟t really become a widely used poison until the 18th century, beginning with some

experiments by a German painter, Heinrich Diesbach who in 1704 was only trying to improve

the colours on his palette. He spent hours at the laboratory of a Berlin Alchemist, trying to

create a low shade of red paint. He swirled together wilder and wider mixtures, eventually

mixing dried blood, potash (potassium carbonate) and green vitriol (iron sulfate) and then

stewing them over an open flame. Instead of the bloody crimson he expected, the flask yielded

a different brilliance, the deep violet-blue glow of a fading twilight. Diesbach called the vivid

pigment berlin blue, english chemist would later rename it prussian blue.

In the history of poisons, the story of cyanide is always coloured blue.

Chemistry of cyanide

Coordination chemistry: The cyanide anion is a potent ligand for many transition metals[61].

The very high affinities of metals for this anion can be attributed to its negative charge,

compactness and ability to engage in π-bonding. Well known complexes include:

Hexacyanides {M(CN)6}3- {M is Ti, V, Cr, Mn, Fe, Co} which are octahedral in shape.

Tetracyanides {M(CN)4}2- {M is Ni, Pd, Pt} which are square planar in their geometry.

Dicyanides {M(CN)2} - {M is Cu, Ag, Au} which are linear in geometry.

36

Certain enzymes, the hydrogenase contain cyanide ligands attached to iron in their active

sites. The biosynthesis of cyanide in the (NIFE)-hydrogenases proceeds from carbamoyl

phosphate which converts to cysteinyl thiocyanate, the CN-donor[62].

Organic derivatives: Because of the cyanide anion‟s high nucleophilicity, cyano groups are

readily introduced into organic molecules by displacement of a halide group (e.g the chloride

on methyl chloride). Organic cyanides are generally called nitriles. Thus, CH3CN can be called

methyl cyanide but more commonly referred to as acetonitrile. In organic synthesis, cyanide

can be used to lengthen a carbon chain by one, while retaining the ability to be functionalized.

RX + CN- RCN + X- (nucleophilic substitution) followed by

1) RCN + 2H20 RCOOH + NH3 (hydrolysis under reflux with mineral acid catalyst) or

2) 2RCN + Li AlH4 + (2nd step) 4H20 2RCH2NH2 + Li Al (OH)4 (under reflux in dry ether,

followed by addition of H20).

Chemical tests for cyanide

Prussian blue: Iron (II) sulphate is added to a solution suspected of containing cyanide such

as the filtrate from the sodium fusion test. The resulting mixture is acidified with mineral acid.

The formation of prussian blue is a positive result for cyanide.

Para-benzoquinone in DMSO: A solution of para-benzoquinone in DMSO react with inorganic

cyanide to form a cyanophenol which is fluorescent. Illumination with a uv light gives a

green/blue glow if the test is positive[63].

Copper and an aromatic amine: As used by fumigators to detect hydrogen cyanide, copper

(II) salt and an aromatic amine such as benzidine is added to the sample. A positive test gives

a blue colour. Copper (I) cyanide is poorly soluble. By sequestering the copper (I), the copper

37

(II) is rendered a stronger oxidant. The copper in a cyanide facilitated oxidation, converts the

amine into a coloured compound.

Pyridine-barbituric acid colorimetry: A sample containing inorganic cyanide is purged with

air from a boiling acid solution into a basic absorber solution. The cyanide salt absorbed in the

basic solution is buffered at pH 4.5 and then reacted with chlorine to form cyanogens chloride.

The cyanogen chloride formed, couples pyridine with barbituric acid to form a strongly coloured

red dye that is proportional to the cyanide concentration. This colorimetric method is used to

analyze cyanide in water, waste water and contaminated soils.

Gas diffusion flow injection analysis-(amperometry): Instead of distilling, the sample is

injected into an acidic stream where the HCN formed is passed under a hydrophobic gas

diffusion membrane that selectively allows only HCN to pass through. The HCN that passes

through membrane is absorbed into a basic carrier solution that transports the cyanide to an

amperometric detector that accurately measures cyanide concentration with high sensitivity.

Sources of cyanide

Cyanides are produced by certain bacteria, fungi and algae and are found in nearly 1,500

plants generally in form of sugars or lipids. Cyanides occur naturally in peach seeds, cherry

pits, tapioca apple seeds, green beans, bitter almonds, peas, apricots, cassava root, sweet

potato, flax seeds, lima beans and bamboo shoots which contain the highest amount of

cyanide sugar. Some millipedes, insects like burnet release hydrogen cyanide as a defence

mechanism. The combustion of any material containing carbon and nitrogen, fumes from

exhaust of vehicles, tobacco smoke, wood smoke and smoke from burning nitrogen containing

38

plastics (particularly acrylonitriles) predictably release clinically significant amount of cyanide

when burnt[64].

Cyanide may be found in water and soil near discharges from organic chemical industries, iron

and steel works, waste water treatment facilities,etc.

Production and synthesis of cyanide

The principal process used to manufacture cyanides is the Andrussow process invented by

Leonid Andrussow at IG Farben in which gaseous hydrogen cyanide is produced from

methane and ammonia in the presence of oxygen and a platinum catalyst[65].

CH4 + NH3 + 1.5O2 HCN + 3H20.

The energy needed for the reaction is provided by the partial oxidation of methane and

ammonia.

Applications and uses of cyanide

Mining (Gold cyanidation): Cyanide is mainly produced for the mining of gold and silver. It

helps dissolve these metals and their ores.

Cyanide is also used in electroplating.

Industrial organic chemistry: Some nitriles are produced on a large scale e.g. adiponitrile is

a precursor to nylon. Such compounds are often generated by combining hydrogen cyanide

and alkenes i.e. hydrocyanation.

RCH=CH2 RCH (CH3)

Metal catalysts are required for such reactions.

39

Medical uses: The cyanide compound, sodium nitroprusside is mainly used in clinical

chemistry to measure urine ketone bodies mainly as a follow-up to diabetic patients. On

occasion, it is used in emergency medical situations to produce a rapid decrease in blood

pressure in human. During world war I, a copper cyanide compound was briefly used by

Japanese physicians for the treatment of tuberculosis and leprosy[66].

Cyanide is also used in jewellery making and certain kinds of photography.

Cyanides are used as insectides for fumigating ships. Cyanide salts are used for

killing ants and used as rat poison.

Although usually thought to be toxic, cyanide and cyanohydrins have been

demonstrated to increase germination in various plant species[67].

Food additive: Due to high stability of their complexation with iron, ferrocyanides (sodium

ferrocyanide) E535, potassium ferrocyanide E536 and calcium ferrocyanide E538 do not

decompose to lethal level in the human body and are used in the food industry as an

anticaking agent in table salt.

Cyanide is used in the manufacture of paper, textiles and plastics.

The most extreme use of cyanide is as a chemical weapon, since high does can kill

large groups of people almost instantly. This application especially by terrorists has

become of increasing concern since the September 11, 2001 terrorist attacks on the

United states and the subsequent anthrax attacks.

40

Cyanide poisoning

Cyanide is poisonous because it stop cellular respiration by inhibiting an enzyme in

mitochondria called cytochrome c oxidase in the fourth complex of the election transport chain

(found in the membrane of the mitochondria of the eukaryotic cells), It attaches to the iron

within this protein. The binding of cyanide to this cytochrome prevent transport of electrons

from cytochrome c oxidase to oxygen. As a result, the electron transport chain is disrupted,

meaning that the cell can no longer aerobically produce ATP for energy. Tissues that mainly

depend on aerobic respiration such as the central nervous system and the heart are particulary

affected that is stop its action in respiration. Following absorption, cyanide is quickly and widely

distributed to all organs and tissues of the body. Ingestion leads to particularly high levels in

the liver when compared with inhalation exposure, but both routes lead to high concentrations

in plasma and enythrocytes and in the heart, lung and brain.

Effect of cyanide poisoning

Exposure to small amounts of cyanide can be deadly regardless of the route of exposure.

Inhalation of high concentrations of cyanide within a short time harms the brain and heart

which can cause a coma with seizures, aphea and cardiac arrest leading to death in a matter

of minutes. These effects can occur rapidly depending on the amount eaten or inhaled. At

lower dose, loss of consciousness may be preceded by general weakness, giddiness,

headache, vertigo, confusion and perceived difficulty in breathing, nausea, vomiting, sweating,

burning sensation in the mouth and throat etc. Chronic exposure to lower levels of cyanide

over a long period result in increased blood cyanide levels which can result in weakness,

permanent paralysis, nervous lesions[68], hypothyroidism and abortions, mild liver and kidney

41

damages, pulmonary edema and hypertension. Also children have been born with thyroid

disease because of the mother‟s exposure to cyanide and thiocyanate during pregnancy. Other

effects of cyanide include cyanide disease like Konzo (local name for irreversible paralysis of

the legs in children and women of child bearing age), tropical ataxic neuropathy(TAN), goitre

and cretinism and stunting of children.

Cyanide poisoning detection

After cyanide poisoning, increased levels of cyanide and thiocyanate are detectable in blood

and urine. Harmful effects can occur when blood levels of cyanides are higher than 0.05 ppm,

but some effects can occur at lower levels. Tissue levels of cyanide can be measured if

cyanide poisoning is suspected. However, cyanide and thiocyanate are cleared rapidly from

the body in urine or exhaled breath, for that reason, blood measurements are only useful for

detecting recent exposure. In general, if cyanide exposure is suspected, treatment should be

started immediately without waiting for the results of blood cyanide measurement. Blood

cyanide concentrations may be measured as a means of confirming the diagnosis in

hospitalized patients or to assist in the forensic investigation of a criminal poisoning.

Cyanide poisoning treatment

Symptomatic patients especially those with severe manifestations, may further benefit from

specific antidotal therapy or agent which are found in the standard cyanide antidote kit used by

the militaries of some countries which include amylnitrite, sodium nitrite and sodium

thiosulphate with high dose oxygen should be given as soon as possible. Other new antidotal

agent recommended by health agencies like International programme on chemical safety

42

U.S.A, UK Health and Safety Executive (HSE), Environmental Protection Agency (EPA),

Occupational Safety and Health Administration (OSHA), Food and Drug Administration (FDA)

are hydroxocobalamin which is available in a cyanokit antidote kits, 4-dimethylaminophenol,

dicobalt edetate, glucose and oxygen therapy[69].

Also one can reduce the exposure to cyanide by not breathing in tobacco smoke, in the

event of a building fire, one should evacuate the building immediately, smoke from burning

plastics contain cyanide (and carbon monoxide), smoke can lead to unconsciousness or death.

People should avoid eating pits and seeds to prevent accidental cyanide poisoning. Diets

containing adequate amounts of protein improve recovery from cyanide exposure incidents.

Also vitamin B12 reduce the negative effects of chronic exposure.

2.3.2 Lead

Lead is a main-group element with the symbol Pb (from latin word “Plumbum” for lead),

present below tin in group 14, belong to period 6 and p block of the periodic table with the

atomic number of 82 and atomic mass of 207. 2 gmol-l.

Generally, lead is the end product of a radioactive decay, hence it is harmful in nature. Like the

element, mercury another heavy metal, lead is a potent neurotoxin that accumulates both in

soft tissues and the bones.

History of lead

Lead was one of the earliest metal discovered by the human race and was in use by 3000 B.C.

The ancient Romans used lead for making water pipes and lining baths and the plumber who

join and mend pipes takes his name from the Latin word plumbum, meaning lead. Plumbum is

43

also the origin of the terms „plumb bob‟ and „plumb line‟ used in surveying and also the

chemical symbol for lead, Pb. In medieval times, most especially for the Roman Empire, lead

came to be used for roofing, coffins, cisterns, tanks, gutters, statues, ornaments and paint.

Also lead acetate or “sugar of lead” was put into wine and other foods to make them sweeter.

Lead touched many areas of Roman life. The Babylonians and the Assyrians used soldered

lead sheets to fasten bolts and construct buildings. The Chinese, Greeks and Romans used

lead to make coins 4,000 years ago. Early warriors made bullets out of it and gladiators

covered their fists with leaden knuckles.

Chemistry of lead

Various oxidized forms of lead are easily reduced to the metal. An example is heating

PbO with mild organic reducing agents such as glucose. A mixture of the oxide and the sulfide

heated together will also form the metal[70].

Metallic lead is attacked (oxidized) only superficially by air, forming a thin layer of lead

oxide that protects it from further oxidation. The metal is not attacked by sulfuric or hydrochloric

acids. It dissolves in nitric acid with the evolution of nitric oxide gas to form dissolved Pb(NO3)2.

Lead(II) oxide is also soluble in alkali hydroxide solutions to form the corresponding

plumbite salt[70].

Lead dioxide is representative of the +4 oxidation state, and is a powerful oxidizing

agent. The chloride of this oxidation state is formed only with difficulty and decomposes readily

2PbO + PbS 3Pb + SO2.

3Pb + 8H+

+ 8NO3- 3Pb

2+ + 6NO3

- + 2NO + 4H2O

PbO + 2OH- + H2O Pb(OH)4

2-

44

into lead(II) chloride and chlorine gas. The bromide and iodide of lead(IV) are not known to

exist[71]. Lead dioxide dissolves in alkali hydroxide solutions to form the corresponding

plumbates.

Lead readily forms an equimolar alloy with sodium metal that reacts with akyl halides to

form organometallic compounds of lead such as tetraethyllead[72].

Chloride complexes: Lead (II) forms a series of complexes with chloride, the formation of

which alters the corrosion chemistry of the lead. This will tend to limit the solubility of lead in

saline media.

Properties of lead

Lead is a dense, ductile metal with a low tensile strength, very soft, highly malleable, bluish-

white heavy metal that has poor electrical conductivity when compared to most other metals. It

has a lustrous silver blue appearance when freshly cut but rapidly tarnishes to a dull grayish

colour when exposed to air. Because lead is very malleable and resistant to corrosion, it is

extensively used in building construction, for example in the external coverings of roofing

joints. Lead has a face-centered cubic crystalline structure.

Lead can be toughened by addition of small amounts of antimony, or a small number of other

metals such as calcium. All isotopes of lead, except for lead-204, can be found in the end

products of the radioactive decay of the even heavier elements, uranium and thorium.

Powdered lead burns with a bluish white flame. As with many metals, finely divided powdered

lead exhibits pyrophoricity[73].

PbO2 + 2OH- + 2H2O Pb(OH)6

2-

45

Isotopes of lead

There are four natural isotopes of lead with three of them being stable. The four natural

isotopes of lead are 204Pb(1.48%), 206Pb(23.6%), 207Pb(22.6%), 208Pb(52.3%). 204Pb is

completely primordial lead and the stable isotopes 206, 207, 208 being formed probably from

the radioactive decay of two isotopes of uranium (U-235 and U-238) and one isotope of

thorium (Th-232).The one common radiogenic isotope of lead,202Pb, has a half life of about

53,000years.

Occurrences and sources of lead

Lead occur naturally in the environment. However, most lead concentrations that are found in

the environment are as a result of human activities. Metallic lead does occur in nature, but it is

rare. Lead is usually found in ore with zinc, silver and most abundantly, copper and is extracted

together with these metals. The primary lead mineral is galena (PbS) that contains 86.6% lead.

Some other common varieties of lead are anglesite (PbSO4) and cerussite (PbCOз).

Sources of lead are children‟s jewelry and toys, cosmetics, herbal medicines, industries where

lead is used in production of batteries, metal products, ammunition and devices, gasoline

products, pipe solder e.t.c. The most common cause of lead poisoning is dust and chips from

old paint. Lead seldom occurs naturally in water supplies like rivers, lakes, soil e.t.c. It gets into

drinking water primarily as a result of corrosion or wearing away of materials containing lead in

the water distribution systems, household or building plumbings or gets into the food through

the soil or as a result of food stored in ceramics, pottery, lead-glazed dishes e.t.c. Lead can

also be found in imported candies or food, batteries, radiators for cars and trucks and ink

colours.

46

Production and synthesis of lead

Sulfide ores are roasted producing primarily lead oxide and a mixture of sulfates and

silicates of lead and other metals contained in the ore[74]. Metallic lead that results from the

roasting and blast furnace processes still contain significant contaminants of arsenic, antimony,

bismuth, zinc, copper, silver, and gold. The melt is treated in a reverberatory furnace with air,

steam and sulfur, which oxidizes the contaminants except silver, gold, and bismuth. The

oxidized contaminants are removed by drossing, where they float to the top and are skimmed

off [74][75]. Very pure lead can be obtained by processing smelted lead electrolytically by

means of the Betts process. The process uses anodes of impure lead and cathodes of pure

lead in an electrolyte of silica fluoride[74][75].

Production and consumption of lead is increasing worldwide. Total annual production is

about 8 million tonnes, about half is produced from recycled scrap, the top lead producing

countries as of 2008, are Australia, China, USA, Peru, Canada, Mexico, Sweden, Morocco,

South Africa and North Korea[75]. Australia, China and the United States account for more

than half of primary production. 2008 mine production: 3,886,000 tonnes. 2008 metal

production: 8,725,000 tonnes. 2008 metal consumption: 8,706,000 tonnes.

Applications and uses of lead

1. Due to its half life of 22.2 years, the radioactive isotope 210Pb is used for dating material

from marine sediment cores by radiometric methods.

2. Lead bricks are commonly used as radiation shielding (like in x-ray rooms).

3. Because of its high density and resistance from corrosion, lead is used for the ballast

keel of sailboats. Its high density allows it to counterbalance the heeling effect of wind on the

47

sails while at the same time occupying a small volume and thus offering the least underwater

resistance.

4. The low melting point makes casting of lead easy, and therefore small arms

ammunition and shotgun pellets can be cast with minimal technical equipment. It is also

inexpensive and denser than other common metals[76].

5. Its corrosion resistance makes it suitable for outdoor applications when in contact with

water.

6. More than half of the worldwide lead production is used as electrodes in the lead-acid

battery, used extensively as a car battery.

7. Lead is the traditional base metal of organ pipes, mixed with varying amounts of tin to

control the tone of the pipe[77].

8. Lead is used in high voltage power cables as sheathing material to prevent water

diffusion into insulation. Lead is added to brass to reduce machine tool wear. Lead, in form of

strips, or tape, is used for the customization of tennis rackets.

9. Lead has many uses in the construction industry (e.g lead sheets are used as

architectural metals in roofing material, cladding, flashing, gutters and gutter joints, and on

roofs parapets). Detailed lead moldings are used as decorative motifs used to fix lead sheet.

Lead is still widely used in status and sculptures.

Lead poisoning

Lead poisoning also known as plumbisim, colica pictonium, saturnism, painter‟s colic is a

medical condition caused by increased levels of the heavy metal lead in the body. Lead

interferes with a variety of body processes and is toxic to many organs and tissues including

48

the heart, bone, intestines, kidneys, reproductive and nervous systems. The main target of

lead poisoning is nervous system both in adults and children and is therefore particularly toxic

to children causing potentially permanent learning and behaviour disorders.

Lead can enter the human body through uptake of food (65%), water (20%) and air (15%) but

may also occur after accidental ingestion of contaminated soil, dust or lead based paint[78].

Health and environmental effect of lead

Long term exposure to lead or its salt can cause nephropathy and colic-like abdominal pains. It

may also cause weakness in fingers, wrists or ankles, small increase in blood pressure,

particularly in middle-aged and older people and can cause anemia. It can damage nervous

connections (especially in young children), causes blood and brain disorders. Exposure to high

lead levels can severely damage the brain and kidneys in adults or children and ultimately

cause death. In pregnant women, it may cause miscarriage and reduce fertility in males

through sperm damage.

For environmental effect, lead can end up in water and soils through corrosion of leaded pipes

in a water transporting system and through corrosion of leaded paint. Soil functions are

disturbed by lead concentration especially near highways and farmland. Lead accumulates in

the bodies of water organisms and soil organisms and they experience effects from lead

poisoning. Health effects on shellfish can take place even when only very small concentration

of lead are present, also, body functions of phytoplankton can be disturbed when lead

interferes. Phytoplankton is an important source of oxygen production in seas and many larger

sea-animals eat it. That is why we now begin to wonder whether lead pollution can influence

global balances.

49

Lead poisoning detection

Elevated lead in the body can be detected by the presence of changes in blood cells visible

with a microscope and dense lines in the bones of children seen on X-ray. However the main

tool for diagnosis is measurement of the blood lead level. Analysis of lead in whole blood is the

most common and accurate method of assessing lead exposure in human. Enythrocyte

protoporphyrin (EP) tests can also be used to measure lead exposure, but are not sensitive at

low blood levels (< 0.2 mg/l). Lead in blood reflects recent exposure, bone lead measurements

are an indicator of cumulative exposure, they are not reliable.

Lead poisoning treatment

Treatment of organic lead poisoning involves removing the lead compound from the skin,

preventing further exposure, treating seizures and possibly chelation therapy (administration of

agents that bind lead so it can be excreted) for people with high blood lead concentrations. A

chelating agent is a molecule with at least two negatively charged groups that allow it to form

complexes with metal ions with multiple positive charges such as lead. The chelate formed is

non toxic[79] and can be excreted in the urine initially at up to 50times the normal rate. The

chelating agents used for the treatment of lead poisoning are edetate disodium calcium (CaNa2

EDTA), dimercaprol (BAL) which are injected and succimer and d-penicillamine which are

administered orally.

50

Fig 2.5 EDTA, a chelating agent, binds a heavy metal, sequestering it.

To reduce lead poisoning is to prevent exposure to lead[80]. Recommended steps to

reduce blood lead levels in adults and children include increasing frequently hand washing,

increase intake of calcium and iron, eliminating lead containing objects like blinds and jewellery

in the house[81]. In houses with lead pipes or plumbing solder, run water in the morning to

flush out the most contaminated water and to prevent corrosion of pipes. Lead testing kits

should be provided in the house to screen and test the blood of children for exposure. Using

cold water for drinking, cooking and for making baby formula can avoid lead exposure. Hot

water is more likely to contain higher amounts of lead than cold water.

2.3.3 Cadmium

Cadmium is a transition metal with the symbol Cd, atomic number of 48 and atomic

mass of 112.411 g/mol, belong to period 5 and block d of the periodic table. It is a soft, bluish

white metal which is chemically similar to the two other metals in group 12, zinc and mercury.

Similar to zinc, it prefers oxidation state +2 in most of its compounds and similar to mercury, it

shows a low melting point for a transition metal.

51

History of cadmium

Cadmium is from a Latin word “cadmia” and Greek word “Kadmeia” which are ancient names

for calamine(zinc carbonate). It was discovered in Germany in 1817 by Fredrick Strohmeyer, a

German chemist, as an impurity in zinc carbonate (calamine). When heated, he noticed that

some samples of calamine glowed with a yellow colour while other samples did not. After

further examination, he noticed that the calamine that changed colour when heated contained

trace amounts of a new element. There is only one mineral that contains significant amounts of

cadmium, greenockite (CdS), but it is not common enough to mine profitably. Fortunately,

small amounts of cadmium are found in zinc ores and most of the cadmium produced today is

obtained as a byproduct of mining zinc.

Chemistry of cadmium

Cadmium burns in air to form brown amorphous cadmium oxide (CdO).

The crystalline form of hydrochloric acid, sulphuric acid and nitric acid dissolve cadmium by

forming cadmium chloride (CdCl2 ), cadmium sulphate (CdSO4 ) or cadmium nitrate (Cd(NO3)2).

1. Cd + 2HCl CdCl2 + H2

2. Cd + H2SO4 CdSO4 + H2

3. Cd + 2HNO3 (Cd(NO3)2) + H2

The most common oxidation state of cadmium is +2, though rare, example of +1 can be found.

The oxidation state +1 can be reached by dissolving cadmium in a mixture of cadmium chloride

and aluminum chloride, forming Cd22+ which is similar to the Hg2

2+ in mercury (1) chloride.

Cd + CdCl + 2AlCl3 Cd2 (AlCl4)2

52

Isotopes of cadmium

Naturally occurring cadmium is composed of 8 isotopes in which 3 are stable. For two of them,

natural radioactivity was observed and the three others are predicted to be radioactive but their

decay is not observed due to extremely long half-life times.

The two natural radioactive isotopes are 113Cd (beta decay, half-life is 7.7x1015 years) and

116Cd (two-neutrino double beta decay, half life is 2.9x1019 years). The other three are 106Cd,

108Cd (double electron capture) and 114Cd(double beta decay), only lower limits on their half life

times have been set. The three isotopes that are stable are 110Cd, 111Cd and 112Cd. Among the

isotopes absent in natural cadmium, the most long lived are 109Cd, with half life of 462.6 days

and 115Cd with half life of 53.4 hours. All of the remaining radioactive isotopes have half lives

that are less than 2.5 hours and the majority of these have half lives that are less than 5

minutes. This element also have 8 known metal states with the most stable being 113mCd (t1/2

14.1 years), 115mCd (t1/2 44.6 days) and 117mCd (t1/2 3.36 hours).

Occurrences and sources of cadmium

Cadmium can mainly be found in the earth crust. It always occur in combination with zinc. After

being applied, it enters the environment mainly through the ground because it is found in

manures and pesticides. Naturally, a very large amount of cadmium is released into the

environment about 25,000 tonnes a year through weathering of rocks and volcanoes and

through forest fires. The rest is released through human activities such as manufacturing[82].

Human uptake of cadmium takes place mainly through food. Food stuffs that are rich in

Cadmium can greatly increase the cadmium concentration in human bodies. Examples are

liver, mushrooms, shelfish, mussels, cocoa powder and dried seaweed.

53

An exposure to significantly higher cadmium levels occur when people smoke tobacco, smoke

transports cadmium into the lungs, blood will transport it through the rest of the body where it

can increase effects by potentiating cadmium that is already present from cadmium rich food.

Applications and uses of cadmium

About three-quarters of all the cadmium used are in batteries, predominantly in

rechargeable nickel cadmium batteries[82]. The remaining quarter is used mainly for cadmium

pigments, coatings and plating and as stabilizers for plastics.

Cadmium can also be applied in some of the lowest melting alloys like wood metal and

in bearing alloys due to a low coefficient of friction and very good fatigue resistance.

Cadmium is also used as a barrier to control neutrons in nuclear fission[83]. In

molecular biology, it is used to block voltage-dependent calcium channels from fluxing calcium

ions.

Biological application: A role of cadmium in biology has been recently discovered. A

cadmium dependent carbonic anhydrates has been found in marine diatoms. Cadmium does

the same job as zinc in other anhydrate, but the diatoms live in the environment with very low

zinc concentration, thus biology has taken cadmium rather than zinc and made it work. This

discovery was made using x-ray absorption fluorescence spectroscopy (XAFS) and cadmium

was characterized by noting the energy of the x-rays that were absorbed[84].

Cadmium poisoning

When people breathe in cadmium, it can severely damage the lungs and this may cause

death. Cadmium is first transported to the liver through the blood. Therefore, it is bonded to

54

protein to form complexes that are transported to the kidneys where it damages filtering

mechanisms. This causes the extraction of essential proteins and sugars from the body and

further kidney damage[82]. It takes a very long time before cadmium accumulated into the

kidney is excreted from human body.

Health and environmental effect of cadmium poisoning

Cadmium poisoning can cause diarrhea, stomach pains, severe vomiting, bone fracture,

reproductive failure and infertility, damage to the central nervous system, damage to immune

system, psychological disorders, possibly DNA damage or cancer development and eventually

lead to death.

Cadmium is strongly absorbed by organic matter in soils and when cadmium is present in soils

as a result of waste streams from industries like zinc production, phosphate ore, bio industrial

manure, burning of household waste and fossil fuel, it can be extremely dangerous as the

uptake through food will increase. Soils that are acidified enhance the cadmium uptake by

plants. This is potential danger to the animals like cow that are dependent upon the plants for

survival, cadmium can accumulate in their bodies and kidney especially when they eat multiple

plants and they can sometimes get high blood pressure, liver disease and brain damage.

Earthworms and other essential soil organisms are extremely susceptive to cadmium

poisoning. They can die at very low concentration and this has consequences for soil structure.

When cadmium concentrations in soil are high, they can influence soil processes of micro

organisms and threat the whole soil ecosystem.

In aquatic ecosystems, cadmium can bio accumulate in muscle oysters, shrimps, lobsters and

fish. The susceptibility to cadmium can vary greatly between aquatic organisms.

55

When cadmium is transported over a great distance, it is absorbed by sludge and cadmium

rich sludge can pollute surface water as well as soil.

Cadmium poisoning detection

Cadmium can be detected when about 10 milligrams of cadmium contents has been absorbed

either through the skin, inhalation or ingestion. The following signs can help to detect cadmium

level in the body[85]. Elevated levels of creatine in the blood and urine may confirm cadmium

poisoning generally. When a person or animal is exposed to cadmium over a long period of

time and in smaller doses, he or she starts noticing shortness of breath, tooth-staining and

weight loss resulting in damaged liver and kidney, sweet or metallic taste in the mouth,

increased amount of saliva, vomiting, choking, anemia, abdominal pains and spasm of

ingested cadmium, chest pain, wheezing, inflammation of the lungs, weakness in muscles and

leg pain.

Cadmium poisoning treatment

Although cadmium cannot be treated because of its stability and cannot be broken or

destroyed, but can be cured by preventing the body from exposure. The following steps will

prevent a case of cadmium poisoning[86].

1. Get a medical examination to diagnose cadmium poisoning. This is usually done

clinically by diagnosing the symptoms especially when cadmium exposure is already

suspected.

2. Provide gastric leverage or induce vomiting within one hour if cadmium salts have been

ingested. Get away from the cadmium exposure immediately and administer oxygen.

56

Chelating therapy is contradicted because it is generally toxic to kidney when combine

with cadmium.

3. Prevent future exposures to cadmium, stop smoking and check products in your home

for cadmium containing compounds, especially fungicides. Store any nickel-cadmium

out of reach of children. If you use a well, check the cadmium level in your water.

2.3.4 Nickel

Nickel is a hard slivery white transition metal with the symbol Ni, atomic number of 28

and atomic mass of 58. 71 g/mol, belongs to group 10, period 4 and the d block of the periodic

table. It is the 24th element in order of natural abundance in the earth crust. It is of the iron

group and it takes on a high polish.

History of nickel

In 1751, Baron Axel Fredrick Cronstedt was attempting to extract copper from kupfernickel and

obtained instead a white metal that he named after the spirit which had given its name to the

mineral, nickel[87]. In the modern German, kupternickel or kupfernickel designates the alloy

cupronickel. After its discovery, the only source for nickel was the rare kupfernickel, but from

1824 on, the nickel was obtained as by product of cobalt blue production. The first large scale

producer of nickel was Norway, which exploited nickel rick pyrrhotite from 1848 on.

57

Chemistry of nickel

Nickel is a relatively unreactive element. At room temperature, it does not combine with oxygen

or water or dissolve in most acids. At higher temperatures, it becomes more active. For

example,

i. Nickel burns in oxygen to form nickel oxide (NiO):

Ni + O NiO

ii. It also reacts with steams to give nickel oxide and hydrogen gas:

Ni + H2O NiO + H2

iii. Nickel metal dissolves slowly in dilute mineral acids. It does not dissolve in

concentrated nitric acid. Attack on the strongly oxidizing acid results in formation of

a protective oxide coat that stops further reaction.

Properties of nickel

1. Nickel is a fairly good conductor of heat and electricity. It is bivalent, although it assumes

other valences.

2. Nickel carbonyl (Ni(CO)4) is a volatile, colourless liquid with a boiling point of 43 0C, it

decomposes at temperature above 50 0C. In biological system, nickel form complex with

adenosine triphosphate, amino acids, peptides, proteins and deoxyribonucleic acid.

3. Nickel also form a number of complex compounds, most nickel compounds are blue or

green. Although it form compounds in several oxidation states, the divalent ion seems to be

the most important for both organic and inorganic substance but the trivalent form may be

generated by redox reaction in the cell[88]. Nickel compound that are practically insoluble

in water include carbonate, sulphides, the main form being amorphous or crystalline

58

monosulphides (NiS), subsulphide (Ni3S2) and oxides (NiO). Water - insoluble nickel

compounds may dissolve in biological fluids. Nickel and its compounds have no

characteristic odour or taste[89].

Isotopes of nickel

Naturally occurring nickel is composed of 5 stable isotopes, 58Ni, 60N, 61Ni, 62Ni and 64Ni with

58Ni being the most abundant (68.077% natural and abundance). 62Ni is the most stable known

nuclide of all the existing elements, even exceeding the stability of 56Fe[90]. 18 radioisotopes

have been characterized with the most stable being 59Ni with a half life of 76,000 years. 63Ni

with a half life of 100.1 years and 56Ni with a half life of 6.077 days. All of the remaining

radioactive isotopes have half lives that are less than 60 hours and the majority of these half

lives are less than 30 seconds. Nickel-78‟s half life was recently measured to be 110

milliseconds and is believed to be an important isotope involved in supernova nucleosynthesis

of elements heavier than iron .

Occurrences and sources of nickel

Nickel is widely distributed in nature forming about 0.008% of the earth crust. The core of the

earth contains 8.5% nickel, deep-sea nodules 1.5%, meteorites have found to contain 5-50%

nickel[91]. Natural sources of atmospheric nickel include dust from volcanic emissions and

weathering of rocks and soils. Global input of nickel into the human environment is

approximately 150,000 metric tonnes per year from natural sources and 180,000 metric tonnes

per year from anthropogenic sources, including emissions from fossil fuel consumption and

industrial production, use and disposal of nickel compounds and alloys[91].

59

Other sources of route of exposure to nickel include air, when air borne nickel is inhaled,

ingested and absorbed through the skin thereby entering the respiratory tract. Also in drinking

water, nickel may however be leached from nickel containing plumbing fittings and levels up to

500 mg/litre have been recorded in water left over night in such fittings[92].

High concentration of nickel have been found in aquatic plants, cocoa and chocolate, soya

beans, root and vegetables, oatmeal, almonds, bread and cereal food group, some legumes

and various nut. Nickel was found to be higher in the organs of wild ruminants than those of

domestic animals, because of the higher nickel content in their grazing areas[93].

Production and uses of nickel

There are two commercial classes of nickel ore, the sulphide ore (pentlandite and pyrrhotite)

and the silicate oxide. Most nickel is produced from the sulphide ores and the two largest

producers, Canada and the Russia account for 20-25% each of the total production which was

784.82 thousand tonnes in 1988[91].

Intermediate uses of nickel include 42% in steel production and 36% in the production of other

alloys. Electroplating in the form of nickel sulphate accounts for 18%. The most important end

uses are transportation 23%, chemical industry 15%, electrical equipment 12% and

construction 10%[94]. It is used in the automobile industry, electronic, nickel-cadmium batteries

and accumulators, many household products as well as in cheap jewelry. Many nickel

compounds are catalysts and pigment. Nickel is an important metal particularly in various

alloys, nickel alloys are often used in nails and prostheses[95].

60

Health and nutrition function of nickel

Since 1970, nickel has been known to be essential for proper functioning of human, animal,

organism and plants[96]. Food has been found to be the main sources of nickel intake by

man[89]. Nickel is one of the trace mineral or micro nutrient in our body since it is present in

very small amount in our body but it plays an important part in overall health of the human

body and in bodily processes. Nickel is found to be beneficial being an important cofactor to

various enzymes where it acts to accelerate the normal chemical reactions occurring in our

body[97]. This element has been shown to take part in reaction catalyzed by oxidoreductase

and hydrolyses (e.g urease). Nickel is in RNA and DNA of our body where it functions in

association with these nucleic acids. It probably has a role in stabilizing RNA structure. It is

found to be helpful in normal bone functioning and health, cell membrane and lipid also[97].

Also for plants, it is required for the enzyme urease to breakdown urea to liberate the nitrogen

into a usable form for plants. Nickel is required for iron absorption, seeds need nickel in order

to germinate. Plants grown without additional nickel will gradually reach a deficient level at

about the time they mature and begin reproductive growth. If nickel is deficient in plants, plants

may fail to produce viable seeds. Nickel produce red blood cells in the human body.

Effects of nickel deficiency

Effect of nickel deficiency include depressed growth, reproductive performance and plasma

glucose[98]. Nickel deficiency results in lower activities of different hydrogenases and

transaminases and above all, of alpha-amylase and particularly affects carbohydrate

metabolism. Nickel deficiency causes a significant triacylglycerol accumulation in liver with

greater concentration of saturated fatty acids and polyunsaturated fatty acids than nickel

61

adequate. Nickel deficiency also slightly compromised iron status, it has been suggested that

at least some of the observed alterations are due to moderate iron deficiency[99]. Nickel

deficiency also affects distribution and proper functioning of other nutrients including calcium,

iron, zinc and vitamin B12[98].

Nickel poisoning and effect

This metal is not a cumulative poisoning in animals or in human[89]. Human exposure to highly

nickel polluted environment such as those associated with nickel refining, electroplating and

welding has the potential to produce a variety of poisoning effects. Pathological alterations of

nickel metabolism are recognized in several human diseases. The diverse clinical

manifestations of nickel poisoning include; acute pneumonitis from inhalation of nickel

carbonyl, chronic rhinitis and sinusitis from inhalation of nickel aerosols, cancer of nasal

cavities and lungs in nickel workers, dermatitis and other hypersensitive reactions form

cutaneous and parenteral exposures to nickel alloys[100].

Almost all cases of acute nickel poisoning result from exposure to nickel carbonyl[89]. The

initial effects involve irritation of the respiratory tract and non specific symptoms. Patients with

severe poisoning develop intense pulmonary and gastrointestinal toxicity. The most common

harmful effects of nickel in human is allergic reaction. Most cases of nickel allergy can be

related to skin contact with nickel containing metallic items as buttons, suspender, ear

ornaments. Soluble nickel appear to increase respiratory cancer risks at lower exposure

concentration (>1 mgNi/m3 work-place dust)[101].

62

Nickel poisoning detection

Ni poisoning can be detected in the saliva, urine, water etc. The following signs can

help to detect nickel poisoning in the body[102].

i. Know that nickel poisoning will occur if nickel carbonyl is inhaled, contaminated food

or water taken or can be absorbed through the skin from contaminated soil, water or

by handling coins.

ii. Watch for common symptoms like insomina, frontal headaches, irritability, vertigo,

nausea, vomiting, difficulty in sleeping, pneumonia like chest pain, rapid heart rate,

dry cough, sweating and weakness.

iii. Look for allergic reaction to nickel, although rare, manifest in a rash of little red

bumps. Areas of the skin that encounter items such as belts or jewelry are generally

affected.

iv. Get tested. A urine test can measure the level of nickel in body to determine the

severity of acute nickel carbonyl poisoning.

Nickel poisoning treatment[103]

i. In case of outbreak of nickel poisoning, get out of the area immediately, remove any

contaminated clothes and see a doctor immediately, make sure there is plenty of

access to clean air or hospital may provide the oxygen.

ii. Chelation therapy can also be administered whereby Doctor gives drug that will

bond with the nickel, reduce its toxic level and move it out of the body through urine

or feaces. The drug is usually combined with antibiotics to the body to keep its

strength up.

63

iii. Recovering from nickel poisoning takes a long time, physical exercise can delay

recovery and cause other complications as well, so a lot of rest is needed.

2.3.5 Arsenic

Arsenic is a metalloid, a natural element that is not actually a metal but has some

properties of a metal. Arsenic is a chemical element that has the symbol As, atomic number 33

and atomic mass 74.92 g/mol. It belongs to group 15, period 4 and p block of the periodic

table. Some forms of arsenic are inorganic, they do not contain carbon and other forms contain

carbon and are classified as organic. Inorganic arsenic exists in four main chemical forms,

which are known as valency or oxidation.

History of arsenic

For over 2,400 years, arsenic was from the Greek word “arsenikon” and Latin word

“arsenicum” meaning “potent” which is the Greek name for the “pigment yellow orpiment”

which has been used both as a therapeutic agent and a poison[104].

Albert Magnus (Albert the Great, 1193-1280) was first to discover and isolate the element in

1250 by heating soap together with arsenic trisulphide[105]. In 1700, English inventor Thomas

Fowler developed a solution of arsenic trioxide in potassium bicarbonate that was used to treat

asthma, chorea, eczema, pemphigius, psoriasis, anemia, hodgkin‟s lymphoma and Leukaemia.

In 1878, the compound aptly named “Fowler‟s solution” was discovered to lower white blood

cell count in normal individual with a more significant decrease occurring in those with chronic

myelogenous Leukaemia treated for 10weeks. After this finding, Fowler‟s solution was used as

64

a main stay in the treatment of Leukaemia until it was succeeded by radiation in the 20 th

century.

During bronze age, arsenic was included in bronze which made the alloy harder so called

“arsenical bronze”[106] and in Victorian era, arsenic was rubbed on the faces and arms of

women to improve their complexion. The accidental use of arsenic in the adulteration of food

stuffs led to the Bradford sweet poisoning in 1858 which resulted in approximately 20 deaths.

Chemistry of arsenic

When heated in air, arsenic oxidizes to arsenic trioxide, the fumes from this reaction have an

odour resembling garlic. This odour can be detected on striking arsenide minerals such as

arsenopyrite with a hammer. Arsenic (and some arsenic compounds) sublimes upon heating at

atmospheric pressure, converting directly to a gaseous form without an intervening liquid

state[108].

Properties of arsenic

Like phosphorus, arsenic exhibits allotropy, the three common allotropes are metallic grey,

yellow and black arsenic[107]. The most common and important allotrope of arsenic under

normal condition is grey arsenic, which is the usual stable form. It is a semi conductor and a

very brittle semi metallic solid with high density of 5.73 g/cm3[108]. It is steel gray in colour,

crystalline, tarnishes readily in air and is rapidly oxidized to arsenous oxide, upon heating the

arsenous oxide exudes the odour of garlic. Yellow arsenic is soft and waxy, this unstable

allotrope, being molecular, is the most volatile, least dense and most toxic. Yellow arsenic is

65

produced by rapid cooling of arsenic vapour, for example with liquid nitrogen. It is rapidly

transformed into the grey arsenic by light. The yellow form has a density of 1.97 g/cm3[108].

Black arsenic is similar in structure to red phosphorus[108]. It is glassy, brittle and a poor semi

conductor. Three metalloidal forms of arsenic, each with a different structure are found free in

nature (the minerals arsenic sensu stricto and the much rarer arsenolamprite and

pararsenolamprite).

Isotopes of arsenic

Naturally occurring arsenic is composed of one stable isotope 75As[109]. Isotopes that are

lighter than the stable 75As tend to decay by β+ decay and those that are heavier tends to

decay by β- decay with some exceptions. 33 radioisotopes of arsenic have also been

synthesized, ranging in atomic mass from 60 to 92. The most stable of these is 73As with a

halflife of 80.3 days.

Sources of arsenic

Arsenic occurs in large quantities in the earth surface. Potential source of arsenic in our

environment is the major industrial processes from the mining and smelting of non-ferrous

metal like copper, burning of fossil fuels, industries that produces pesticides, agricultural

insecticide, wood preservative etc.

Naturally occurring pathway of exposure to arsenic include volcanic ash, weathering of the

arsenic containing mineral and ores as well as ground water.

Arsenic is also found in food (like seafood), water, soil, air, tobacco smoke, laundry, detergent,

beer, hair follicles, finger nails and skin, homeopathic preparations containing sulphur, Korean

66

herbal preparations used to treat hemorrhoids, kelp supplements and traditional Chinese

herbal balls.

Production and synthesis of arsenic

In 2005, China was the top producer of white arsenic with almost 50% world share followed by

Chile, Peru and Morocco. Arsenic is recovered mainly as a side product from the purification of

copper. It is also a part of the smelter dust from copper, gold and lead smelters[110].

On roasting in air of arsenopyrite, arsenic sublimes as arsenic (III) oxide leaving iron oxides,

while roasting without air results in the production of metallic arsenic. Further purification from

sulphur and other chalcogens is achieved by sublimation in vacuum or in a hydrogen

atmosphere or by distillation from molten lead-arsenic mixture[111].

Applications and uses of arsenic

Agricultural uses: Arsenic is used as wood preservatives and to treat and preserve lumber.

The toxicity of arsenic to insects, bacteria and fungi led to its use as a wood preservative[112].

Arsenic was also used in various agricultural insecticides, weed killers, termination and poison

in killing pests like rats and mice.

Arsenic is still added to animal food, particularly in the U.S as a method of disease prevention

and growth stimulation. One example is roxarsone which is used as broiler starter by about

70% of the broiler growers since 1995[113].

Medical uses: The U.S Food and Drug Administration in 2000 approved Arsenic trioxide

(As203) for the treatment of patients with cancer, acute promyclocytic leukaemia (APL) that is

resistant to ATRA[114]. It was also used as fowler‟s solution in psoriasis. Recently, new

67

research has been done in locating tumours using arsenic-74 (a positron emitter). The

advantages of using this isotope instead of the previously used iodine-124 is that the signal in

the PET scan is clearer as the body tends to transport iodine to the thyroid gland producing a

lot of noise[115].

Alloys and trace components of other various products: The main use of metallic arsenic

is for alloying with copper and especially lead. Lead components in automotive batteries are

strengthened by the presence of a few percent of arsenic. Gallium arsenide is an important

semi conductor material used in integrated circuits. It is fabricated by chemical vapour

deposition. Circuits made from gallium arsenide are much faster than those made in silicon.

Arsenic is used as a doping agent in solid state devices. Arsenic is added in small qualities to

alpha-brass to make it dezincification resistant. This grade of brass is used to make plumbing

fittings or other items which are in constant contact with water.

Arsenic is used in ammunition manufacturing, because it helps to create harder and rounder

bullets. Arsenic is used for taxonomic sample preservation.

Biological role: Under oxidative environmental conditions, some bacteria use arsenite, which

is oxidized to arsenate as fuel for their metabolism[116]. The enzymes involved are known as

arsenate reductases (Arr). Arsenic has been linked to epigenetic changes which are heritable

change in DNA sequence and include DNA methylation, histone modification and RNA

interference. Arsenic substitute for phosphorus as a building block of life in the molecule of the

bacteria cell including DNA and ATP[117].

68

Arsenic poisoning

Arsenic and many of its compound are potent poisons. Many water supply close to mines are

contaminated by these poisons. Arsenic disrupts ATP production through several mechanisms.

At the level of the critic acid cycle, arsenic inhibits lipoic acid which is a co factor for pyruvate

dehydrogenase and by competing with phosphate, it uncouples oxidative phosphorylation, thus

inhibiting energy-linked reduction of NAD, mitochondrial respiration and ATP synthesis.

Hydrogen peroxide production is also increased, which might form reactive oxygen species

and oxidative stress. These metabolic interferences lead to death from multi-system organ

failure, probably from necrotic cell death. A post mortem reveals brick red coloured mucosa,

owing to severe haemorrhage. Although arsenic causes toxicity, it can also play a protective

role[118].

Health and environmental effect of arsenic poisoning

Exposure to a toxic dose initially produces a dry burning sensation in the mouth and throat and

a constricted feeling in the throat. This is followed by severe abdominal pain, cramping,

diarrhea, and vomiting. Inhalation of toxic amounts of arsine gas results in headache, malaise,

weakness, dizziness and dyspnea accompanied by gastrointestinal distress. Chronic toxic

effects are fatigue, loss of energy, nasal septum perforation, ulceration in folds of skin,

increased pigmentation of skin, exfoliative dermatitis, rashes, muscular paralyses and atrophy,

sensory disturbances, visual disturbances and blindness, degeneration of liver (cirrhosis) and

kidneys, garlic odour breath, non cirrhotic portal hypertension and reproductive disorder.

Environmental exposure to well water containing inorganic arsenic can result in skin

hyperpigmentation or an eczematous dermatitis. Peripheral vascular involvement may occur

69

with acrocyanosis and distal neuropathy that presents like Guillain-Barre syndrome and

sideroblastic anaemia[119].

Arsenic poisoning detection

Since arsenic stays in the body only short time, test must be done soon after exposure.

However, the Agency for Toxic Substances and Disease Registry(ATSDR) states that long

term effects of arsenic exposure cannot be predicted. Blood, urine, hair and nails tested for

arsenic can measure exposure to high levels but not very useful for low exposure level[120].

Arsenic poisoning treatment

Arsenic effect can last for years after the exposure has been eliminated. The risk of developing

adverse effects may persist for many years after exposure has ceased, so taking folic acid

supplements, food that contains sulphur (egg, onion, beans, legumes and garlic), fibre (grains

and cereals, fruits and vegetables), folate (vitamin from leafy vegetables, citrus fruits, beans

and whole grain) each day can dramatically lower blood arsenic levels in individuals chronically

exposed to arsenic contaminated drinking water, food, etc[121].

Chelation therapy is a series of injections of ethylenediaminetetra acetic acid (EDTA) that is

done in the hospital and the procedure is safe. Chelation therapy can also be done at home by

taking chelation formulas made with alfalfa, garlic, fibre, tunin[121].Chronic arsenic poisoning

can be treated by haemodialysis and the use of BAL, British anti-lewisite (Dimercaprol)[121].

70

2.3.6 Selenium

Selenium is a metalloid represented by the chemical symbol Se, with the atomic

number 34 and atomic mass of 78.96 gmol-1. It belongs to Group 16, period 4 and p block of

the periodic table, located between sulphur and tellurium and resembles sulphur both in its

various form and compound[122].

Selenium is a trace element that is essential for good health in the body but required only in

small amount because when in large amount, it is toxic[123].

History of selenium

Selenium is from the Greek word selene meaning “moon”. It was discovered in 1817 by Jons

Jakob Berzelius[124] who found the element associated with tellurium (named for the Earth). It

was discovered as a byproduct of sulphuric acid production.

It came to medical notice later because of its toxicity to human working in industry. It was also

recognized as an important veterinary toxin seen in animals eating high selenium plants. In

1954, the first hints of specific biological functions of selenium were discovered in micro

organisms. Its essentiality for mammalian life was discovered in 1957. In the 1970s, it was

shown to be present in two independent sets of enzymes. This was followed by the discovery

of selenocysteine in proteins.

71

Chemistry of selenium

Chalogen Compound

I. Selenium forms two oxides: seleniumdioxide (SeO2) and selenium trioxide(SeO3)[125].

Seleniumdioxide is formed by the reaction, elemental selenium with oxygen:

II. Selenous acid, (H2SeO3) can also be made directly by oxidizing elemental selenium with

nitric acid:

Salts of selenous acid are called selenites. These include silver selenite (Ag2SeO3) and

sodium selenite (Na2SeO3).

III. Hydrogen sulphide reacts with aqueous selenous acid to produce selenium disulphide:

H2SeO3 + 2H2S SeS2 + 3H2O

.

IV. Unlike sulphur, which forms a stable trioxide, selenium trioxide is unstable and decomposes

to the dioxide above 185OC[125][126].

2SeO3 2Se O2 + O2 (ΔH = - 54 KJmol-1)

Selenium trioxide may be synthesized by dehydrating selenic acid, H2SeO4, which itself is

produced by the oxidation of selenium dioxide with hydrogen peroxide[127].

SeO2 + H2O2 H2SeO4

Hot concentrated selenic acid is capable of dissolving gold, forming gold (iii) selenate[128].

Se8+8O2 8SeO2

3Se + 4HNO3 + H2O 3H2SeO3 + 4NO 8SeO2

72

Halogen Compound

Selenium reacts with fluorine to form selenium hexafluoride:

Se8 + 24 F2 8SeF6

Other selenium halides include SeF4, Se2Cl2, SeCl4 and Se2Br2.

Selenides

Like oxygen and Sulphur, selenium forms selenides with metals. For example, reaction

with aluminium forms aluminum selenide[125]:

3Se8 + 16 Al 8Al2Se3

Other selenides include mercury selenide (HgSe), lead selenide (PbSe), magnesium selenide

(MgSe) and zinc selenide (ZnSe).

Other Compounds

Selenium reacts with cyanides to yield selenocyanates[125]:

8KCN +Se8 8KSeCN

Properties of selenium

Selenium occurs in a number of allotropic forms; amorphous form (powder form); black

(vitreous form) and crystalline form; deep red and grey metal like. The most popular are a red

amorphous powder, a red crystalline material and a grey crystalline metal like[129]. The most

stable is the grey crystalline metal like which conducts electricity better in the light than in the

dark and is used in photo cells.

73

Isotopes of selenium

Out of the known isotopes of selenium, selenium has only six naturally occurring isotopes, five

of which are stable: 74Se, 76Se, 77Se, 78Se and 80Se. The last three also occur as fission

products along with 79Se which has a half life of 327,000 years[130]. The final naturally

occurring isotope, 82Se has a very long half life (1020 years decaying via double beta decay to

82kr), which for practical purpose can be considered to be stable. 75Se is used commercially to

study the function of two organs in the body, the pancreas and the parathyroid gland. The

isotope gives off radiation when it reaches these organs which indicated whether the organs

are functioning properly by the amount and location of radiation given off.

Occurrence and sources of selenium

Selenium occurs naturally in the environment, it is released by both natural processes and

human activities. Well fertilized soil contain selenium since selenium is present in phosphate

fertilizer and is often added as a trace nutrient. Anthropogenic sources of selenium includes

coal burning, mining and smelting of sulphide ores, by product of refining copper e.t.c

Plant foods are the major dietary sources of selenium in most countries throughout the world.

The content of selenium in food depends on the selenium content of the soil where plants are

grown or animals are raised. Animals that eat grains or plants that are grown on selenium-rich

soil have higher levels of selenium in their muscle. Selenium is naturally present in grain,

cereal and meat and high levels of selenium can be found in nuts, meat, bread, brazil nuts,

beef, cheese, spaghetti, cod, noodles, egg, cottage, oatmeal, rice, wheat, mushroom, fish,

kidney, tuna, crab and lobster[131].

74

Production and synthesis of selenium

Selenium is most commonly produced from selenide in many sulphide ores such as

those of copper, silver or lead. It is obtained as a byproduct of the processing of these ores

and the mud from the lead chambers of sulphuric plants. These muds can be processed by

number of means to obtain free selenium. Industrial production of selenium often involves the

extraction of seleniumdioxide from residues obtained during the purification of copper.

Common production begins by oxidation with sodium carbonate to produce selenium dioxide.

Cu2Se + Na2CO3 + 2O2 2CuO + Na2SeO3 + CO2

The seleniumdioxide is then mixed with water and the solution is acidified to form selenous

acid (oxidation step). Selenous acid is bubbled with sulphurdioxide (reduction step) to give

elemental selenium.

H2SeO3 + 2SO2 + H2O Se + 2H2SO4

Selenium is present in the atmosphere as methyl derivatives. Uncombined selenium is

occasionally found and there are about 40 known selenium containing minerals, some of which

can have as much as 30% selenium but all are rare and generally they occur together with

sulphide of metals such as copper, zinc and lead. The main producing countries are Canada,

USA, Germany, Japan, Bolivia and Russia. Global industrial production of selenium is around

1,500 tonnes a year and about 150 tonnes of selenium are recycled from industrial waste and

reclaimed from old photocopiers.

Health and nutrition function of selenium

1. In human body, selenium is incorporated into proteins to make selenoproteins notably

glutathione peroxidase which are important antioxidant enzymes that help protect the cells in

75

the body from free radical cellular damage. Free radicals are natural by-products of oxygen

metabolism that may contribute to the development of chronic diseases such as cancer and

heart disease[132].

2. Although it is toxic in large doses, selenium is an essential micronutrient for animals. In

plants, it occurs as a bystander mineral, sometimes in toxic proportions in forage (some plants

may accumulate selenium as a defence against being eaten by animals, but other plants such

as locoweed require selenium and their growth indicates the presence of selenium in soil)[133].

3. Indicator plants: certain species of plants are considered indicators of high selenium content

of the soil, since they require high levels of selenium to thrive. The main selenium indicator

plants are Astragalus species (including some locoweeds), prince‟s plume (Stanleya sp.),

wood asters (Xylorhiza sp.) and false goldenweed (Oonopsis sp.)[134].

4. Selenium supplements like selenium yeast can be inform of selenomethionine. This was

used in large scale cancer prevention trial in 1983 which demonstrated that taking a daily

supplement about 200 microgram per day could lower the risk of developing prostrate lung and

colorectal cancer[135]. Also selenium supplements may be protective against goitre

(enlargement of the thyroid gland) and iodine deficiency[136].

5. Also, selenium primary function in the human body is to work in conjunction with vitamin E in

the preservation and elasticity of tissues, it is also necessary in the slowing down of the

process of aging and improving the blood and oxygen supply to the heart muscle. The

prostaglandins in the human body which protect against high blood pressure cannot be formed

without selenium. Prostaglandins aid in the prevention of abnormal blood clots thereby

preventing stroke, heart attack and also assist in the stimulation of uterine contraction in

pregnancy.

76

Effect of deficiency of selenium

1. Most cases of selenium depletion or deficiency are associated with severe

gastrointestinal problems such as crohn‟s disease or with surgical removal of part of the

stomach. This impair selenium absorption and other nutrients[137] and causes acute

severe illness which develop inflammation and widespread infection.

2. Selenium deficiency has also been seen in people who rely on total parenteral nutrition

(TPN) as their sole source of nutrition[138]. TPN is a method of feeding nutrients

through an intravenous(IV) line to people whose digestive systems do not function.

Forms of nutrients that do not require digestion are dissolved in liquid and infused

through the IV line. It is important for TPN solutions to provide selenium in order to

prevent a deficiency[138].

3. Selenium deficiency may also occur when a low selenium status is linked with an

additional stress, such as chemical exposure or increased oxidant stress due to vitamin

E deficiency[139].

4. There is evidence that selenium deficiency may contribute to development of a form of

heart disease, hypothyroidism, muscle problem and a weakened immune system[140].

Also people dependent on food grown on selenium deficient soil are at risk. There is

also evidence that selenium deficiency does not usually cause illness by itself, rather it

can make the body more susceptible to illnesses caused by other nutritional,

biochemical or infectious stresses[141]. Three specific diseases associated with

selenium deficiency are keshan disease (enlarged heart and poor heart function in

selenium deficient children), kashin-beck disease which result in osteoarthropathy and

myxedematous endemic cretinism which result in mental retardation.

77

Applications of selenium

Biological applications

1) Medical use: The substance loosely called selenium sulphide (SeS2) is the active ingredient

in some anti-dandruff shampoos. The selenium compound kills the scalp fungus Malassezia

which causes shedding of dry skin fragments. The ingredient is also used in body lotion to treat

Tinea versicolor due to infection by a different species of Malassezia fungus[142].

2) Nutrition: Selenium is used widely in vitamin preparations and other dietary supplements in

small does. Some livestock feeds are fortified with selenium as well.

Non-biological applications

1) Chemistry : Selenium is a catalyst in many chemical reactions and is widely used in

various industrial and laboratory syntheses, especially in organoselenium chemistry. It is also

widely used in structure determination of protein and nucleic acids by x-ray crystallography.

2) Manufacturing and materials use

i) The largest use of selenium worldwide is in glass and ceramic manufacturing, where it is

used to give a red colour to glasses, enamels and glazes as well as to remove colour from

glass by counteracting the green tint imparted by ferrous impurities.

ii) Selenium is used with bismuth in brasses to replace more toxic lead. It is also used to

improve abrasion resistance in vulcanized rubbers.

3) Electronics

i) Because of its photovoltaic and photoconductive properties, selenium is used in

photocopying, photocells, light meters and solar cells. Because it can convert AC electricity to

DC, it is widely used in rectifiers. These uses have mostly been replaced by silicon-based

devices. The most notable exception is in power DC surge protection, where the superior

78

energy capabilities of selenium suppressors make them more desirable than metal oxide

varistors.

ii) Sheets of amorphous selenium convert x-ray images to patterns of charge in

xeroradiography, solid state and flat-panel x-ray cameras.

4) Photography: Selenium is used in the toning of photographic prints and it is sold as a toner

by numerous photographic manufacturers including KODAK and FOTOSPEED. Its use

intensifies and extends the tonal rage of black and white photographic images as well as

improving the permanence of prints.

Selenium poisoning

Although selenium is an essential trace element, it is toxic if taken in excess. High blood levels

of selenium greater than the tolerable upper intake level of 400 microgram per day can lead to

a condition called selenosis[143].

Selenium poisoning is rare in most individual but the few reported cases have been associated

with industrial accidents and a manufacturing error that led to an excessively high dose of

selenium in a supplement[144] since selenium is active in only tiny amounts.

Health and environmental effect of selenium poisoning

When selenium uptake is too high, health effects will likely to come about. High blood levels of

selenium can result in a condition called selenosis. Symptoms of selenosis include

gastrointestinal disorder, garlic breath odour, hair loss, white blotchy nails, fatigue, irritability

and mild nerve damage[129]. Extreme cases of selenosis can result in cirrhosis of the liver,

pulmonary edema and death[145].

79

In environmental effect, there is evidence selenium can accumulate in the body tissues of

organisms and can then be passed up through the food chain. Usually this biomagnifications of

selenium starts when animals eat a lot of plants that have been absorbing large amounts of

selenium prior to digestion. Due to irrigation run-off, concentrations of selenium tend to be very

high in aquatic organisms and wetland birds which causes cogenital disorders. When animals

absorb or accumulate extremely high concentrations of selenium, it can cause reproductive

failure and birth defects[129][146].

Detection of selenium poisoning.

Selenium may be detected or measured in blood, plasma, serum or urine to monitor

excessive environmental or occupational exposure, confirm a diagnosis of poisoning in

hospitalized victims or to assist in forensic investigation in a case of fatal overdosage. Both

organic and inorganic forms of selenium are largely converted to monosaccharide conjugates

(seleno sugars) in the body prior to being eliminated in the urine. Cancer patients receiving

daily oral doses of selenothione may achieve very high plasma and urine selenium

concentrations[147].

Treatment of selenium poisoning

To treat patients suffering from selenium is usually aimed at treating symptoms. There is no

specific antidote or treatments for selenium poisoning. Anyone who has ingested mineral

supplements containing selenium and suffering from the symptoms of selenium poisoning

should stop taking these products immediately since most selenium poisoning comes from

high dose of selenium in a supplement[148].

80

CHAPTER THREE

3.0 EXPERIMENTALS

3.1 Sample Collection

20 samples of finely ground fermented cassava flour were purchased from different

markets in both rural and urban areas of Ekiti, Oyo, Lagos, Ondo and Osun States in

Southwest Nigeria. Two samples were purchased from two different urban markets and

another two samples from two different rural markets in each state as follows: In Ekiti state,

(Ado Ekiti-urban and Erio-rural); Oyo state (Ibadan-urban and Iseyin-rural); Lagos state

(Oshodi-urban and Aro-rural); Ondo state (Akure-urban and Epinmi-rural); and Osun state

(Oshogbo-urban and Ede-rural). This area was chosen for the case study because the South

west people (Yorubas) are the largest consumers of cassava flour.

To avoid further contamination during sampling, transporting and storage, the samples were

kept in air tight polyethylene bags that has been rinsed with dilute HCl and dried before use.

3.2 Sample Preparation

3.2.1 Ashing procedure for analysis of sample for lead, cadmium and nickel

determination[149]

2g of finely ground fermented cassava flour sample was weighed into a porcelain

crucible and 1ml conc HN03 was added and the sample was charred on an electric hot plate.

The charred sample was later heated in a controlled muffle furnace at a temperature of 450 0C

until there was no brown fumes generated and perfectly white ash was obtained. The ash

obtained was allowed to cool in the furnace and later 5 ml of IM HN03 solution and 5 ml of 30%

HCl were added and the solution was warmed on an electric hot plate. The solution was

81

allowed to cool and was decanted into 10 ml volumetric flask using funnel and rinsed with de-

ionized water. The solution was made up to the mark with de-ionized water. A blank solution

was also prepared using the same amounts of reagents and made up to the mark with de-

ionized water. The procedure was repeated for each sample and the resulting solutions were

poured into sample bottles for Atomic Absorption Spectrophotometry (AAS) analysis for lead,

cadmium and nickel.

3.2.2 Digestion procedure for analysis of sample for arsenic and selenium[150][151]

2g of the finely ground fermented cassava flour was weighed into a Macro kjedahl flask.

20 ml of 1:1 perchloric acid, nitric acid solution were added into the sample and allowed to stay

overnight. The mixture was heated on a heating mantle in a fume cupboard until the brown

fumes clear off and the sample was completely digested into a nearly colourless solution. The

solution was allowed to cool and then filtered into a 100 ml volumetric flask using funnel and

whatman 44 filter paper and made up to the mark with distilled water.

3.2.3 Extraction procedure of sample for cyanide determination[152]

2g of finely ground fermented cassava flour was made into a paste and the paste was

dissolved with distilled water in a corked conical flask and allowed to stay overnight. The

mixture was filtered into 50 ml volumetric flask using funnel and whatman 44 filter paper and

made up to the mark with distilled water.

82

3.3 Sample Analysis

3.3.1 Determination of lead, cadmium and nickel using atomic absorption

spectrophotometry

The lead, cadmium and nickel content in the sample solutions were determined using

an atomic absorption spectrophotometer (GBC avanta version model 2.02) with air acetylene

flame at specific wavelength for each metal. The digested sample was passed into the burner

through a mixing chamber, the air met the fuel gas (C2H2), acetylene supplied to the burner at

a given pressure and this mixture were burnt, the radiations from the resulting flame were read.

3.3.2 Determination of arsenic using titrimetric method[150]

20 ml of the sample solution was put in a 250 ml conical flask, 10 ml of distilled water

was added, 1g of sodium bicarbonate crystal and 1 ml of 1% starch solution were also added

and swirled carefully until the crystal has dissolved. Then the solution was titrated slowly with

0.02N iodine solution contained in the burette until a permanent blue colour solution is formed

which is the end point.

3.3.3 Determination of selenium using titrimetric method[151]

40 ml of the sample solution was put in a 250 ml conical flask, 10 ml of 2% starch

solution and 6 ml of 1:1 hydrochloric acid were added. To expel oxygen, 0.4g of pure sodium

bicarbonate was added. 10 ml of 10% potassium iodide solution was also added in a thin

stream while swirling the solution. After 1 minute, the solution was titrated with 0.1N sodium

thiosulphate contained in the burette until the colour changes from blue through an

intermediate dirty brown to violet red.

83

3.3.4 Preparation of stock solution of cyanide[152]

0.40 mg/L stock solution of cyanide was prepared by dissolving 1g of KCN with distilled

water in 1000 ml volumetric flask and made up to the mark with distilled water.

3.3.5 Calibration curve for cyanide determination[152]

0.40 mg/L stock solution of cyanide was diluted to prepare 0.02 mg/L, 0.04 mg/L, 0.06

mg/L, 0.08 mg/L and 0.10 mg/L standard solutions of cyanide. A blank solution was

also prepared. The absorbance of each concentration was measured at 490nm using a

Novaspec model 4049 uv/vis spectrophotometer. The calibration curve for the cyanide

determination was obtained by plotting absorbances against concentrations of the

standard cyanide solutions. Then the graph factor obtained from the plot was used to

calculate the cyanide content of the samples.

3.3.6 Preparation of Alkaline picrate solution[152]

Alkaline picrate solution was prepared by dissolving 1g of picrate and 2g of sodium

carbonate in a volume of minimally warm water in 100 ml volumetric flask and made up to the

mark with distilled water.

3.3.7 Determination of cyanide using uv/visible spectrophotometer[152]

5 ml of the sample filtrate were put in a corked testube and 4 ml of the alkaline picrate

were added and the solution was incubated in a water bath for 5 minutes. After colour

development (reddish brown colour), the absorbance of the corked testube was read on a

Novaspec model 4049 uv/visible spectrophotometer at 490nm which is the wavelenght of

maximum absorption (λmax) of cyanide and this procedure was repeated for each sample. The

absorbance of a blank solution containing 1 ml distilled water and 4 ml alkaline picrate solution

was also read and extrapolated on the calibration graph.

84

CHAPTER FOUR

4.0 RESULTS AND DISCUSSION

4.1 The concentration levels of lead, cadmium and nickel in the fermented cassava flour

samples from the urban and rural areas of Southwest, Nigeria are given in Tables 4.1 and 4.2

respectively.

Table 4.1 Lead, cadmium and nickel concentration levels in the samples from the urban areas of Southwest, Nigeria

S/N Sample ID (urban areas)

concentration of lead in the samples (mg/kg)

concentration of cadmium in the samples (mg/kg)

concentration of nickel in the samples (mg/kg)

1. Ekiti 1 0.36 0.05 0.95

2. Ekiti 2 0.06 0.05 0.49

3. Oyo 1 0.06 0.04 0.69

4. Oyo 2 0.01 0.01 0.53

5. Lagos 1 0.06 0.01 0.43

6. Lagos 2 0.02 0.07 0.51

7. Ondo 1 0.03 0.01 0.32

8. Ondo 2 0.08 0.06 0.65

9. Osun 1 0.35 ND 0.78

10. Osun 2 0.30 0.04 0.69

Mean value±SD 0.13 ± 0.14 0.03 ± 0.02 0.60 ± 0.18

Range 0.01 – 0.36 ND – 0.07 0.32 – 0.95

ND = Not Detectable

85

Table 4.2 Lead, cadmium and nickel concentration levels in the samples from the rural

areas of Southwest, Nigeria

S/N Sample ID (rural areas)

concentration of lead in the samples (mg/kg)

concentration of cadmium in the samples (mg/kg)

concentration of nickel in the samples (mg/kg)

1. Ekiti 1 0.32 0.03 0.31

2. Ekiti 2 0.01 0.04 0.49

3. Oyo 1 ND 0.01 0.39

4. Oyo 2 ND 0.02 0.29

5. Lagos 1 ND ND 0.39

6. Lagos 2 0.01 0.05 0.20

7. Ondo 1 ND 0.01 0.27

8. Ondo 2 ND 0.02 0.46

9. Osun 1 ND ND 0.50

10. Osun 2 0.10 0.02 0.30

Mean value ± SD 0.04 ± 0.10 0.02 ± 0.16 0.36 ± 0.11

Range ND – 0.32 ND – 0.05 0.20 – 0.50

ND = Not Detectable

4.2 The concentration levels of arsenic and selenium in the fermented cassava flour

samples from the urban and rural areas of Southwest, Nigeria are given below in Tables 4.3

and 4.4 respectively.

86

Table 4.3 Arsenic and selenium concentration levels in the samples from the urban

areas of Southwest, Nigeria

S/N Sample ID (urban areas)

concentration of arsenic in the samples (mg/kg)

concentration of selenium in the samples (mg/kg)

1. Ekiti 1 0.38 3.94

2. Ekiti 2 0.23 3.94

3. Oyo 1 0.28 5.92

4. Oyo 2 0.28 5.52

5. Lagos 1 0.38 4.94

6. Lagos 2 0.30 5.01

7. Ondo 1 0.21 6.42

8. Ondo 2 0.21 5.52

9. Osun 1 0.19 4.94

10. Osun 2 0.21 5.01

Mean value ± SD 0.30 ± 0.07 5.12 ± 0.90

Range 0.19 – 0.38 3.92 – 6.42

87

Table 4.4 Arsenic and selenium concentration levels in the samples from the rural

areas of Southwest, Nigeria

S/N Sample ID (rural areas)

concentration of arsenic in the samples (mg/kg)

concentration of selenium in the sample (mg/kg)

1. Ekiti 1 0.19 3.94

2. Ekiti 2 0.19 3.94

3. Oyo 1 0.19 5.43

4. Oyo 2 0.19 4.95

5. Lagos 1 0.28 3.46

6. Lagos 2 0.21 3.46

7. Ondo 1 0.19 5.43

8. Ondo 2 0.21 5.37

9. Osun 1 0.19 4.44

10. Osun 2 0.18 4.46

Mean value ± SD 0.20 ± 0.03 4.50 ± 0.80

Range 0.18 – 0.28 3.46 – 5.43

88

4.3 Absorbances of various standard solutions of cyanide for the calibration curve are shown

in Table 4.5 while Fig 4.1 shows the calibration graph.

Table 4.5 Absorbances of various standard solutions of cyanide at 490nm

S/N Concentration in mg/L Absorbance at λ490nm

1. 0.02 0.120

2. 0.04 0.273

3. 0.06 0.400

4. 0.08 0.520

5. 0.10 0.650

89

Fig 4.1 Graph of absorbance versus concentration for cyanide determination at

490nm.

90

4.4 Concentration levels of cyanide in the fermented cassava flour samples from the urban

and rural areas of Southwest, Nigeria are given in Table 4.6.

Table 4.6 Cyanide concentration levels in the samples from the urban and rural areas

of Southwest, Nigeria

S/N Sample ID concentration of cyanide (mg/kg) in the samples (urban areas)

concentration of cyanide (mg/kg) in the samples (rural areas)

1. Ekiti 1 0.06 0.01

2. Ekiti 2 0.04 0.01

3. Oyo 1 0.11 0.04

4. Oyo 2 0.08 0.04

5. Lagos 1 0.05 0.01

6. Lagos 2 0.08 0.05

7. Ondo 1 0.08 0.08

8. Ondo 2 0.03 0.03

9. Osun 1 0.09 0.03

10. Osun 2 0.03 0.03

Mean value ± SD 0.07± 0.03 0.03 ± 0.02

Range 0.03 – 0.11 0.01 – 0.08

From the above results (Table 4.1 – 4.6), the mean values (mg/kg) of lead, cadmium,

nickel, arsenic, selenium and cyanide in the fermented cassava flour samples from the urban

areas are 0.13±0.14, 0.03±0.02, 0.60±0.18, 0.30±0.07, 5.12±0.90 and 0.07±0.03.These are

higher than the mean values: 0.04±0.10, 0.02±0.02, 0.36±0.11, 0.20±0.03, 4.50±0.80 and

91

0.03±0.02 respectively determined in the samples from the rural areas. The higher

concentration levels of lead, cadmium, nickel, arsenic, selenium and cyanide in fermented

cassava flour from the urban areas could be attributed to exposure of cassava flour to

atmospheric deposition of dust and traffic pollution in the urban markets places most especially

where there are heavy traffic of vehicles. Ugwu et al reported that air pollution may pose a

threat to cassava flour especially during the hazy and harmattan season and along

roadside[13]. Furthermore, as the vehicles plow through dusty roads, they cause pre-released

lead and other toxic metals like nickel, cadmium and arsenic to be wafted back up into the air.

Fumes from automobile exhaust would accumulate more on exposed food such as cassava

flour from high traffic density (go-slow) than in low traffic condition. Report have it that vehicle

contribute enormously to the level of metallic compound in food sample which depends on the

level of traffic in such location[153].

Also particulate air pollution from industries, motor vehicles, waste disposal activity,

incineration, chemical plants, metal production and PVC factories, oil refineries e.t.c contribute

to release of toxic contaminants in the urban areas. Sharma et al reported that particulate air

pollution and vehicles are the main causes of heavy metal contamination in urban areas[9].

Similar works show that traffic volume, industrial activities and intensity of human activities are

sources of soil contaminant in the urban areas[154]. Some works have it that rapid

urbanization, unorganized industrialization and increased use of automobile have contributed

to the elevated levels of heavy metals in the urban environments[155]. Akeredolu also reported

that the exceptional lead concentration is due to the heavy lead of contaminated dust in the air

of a very crowded city and fumes from automobile[153]. Dust mobilization due to automobiles

92

has been estimated to be 6.5g/vehicle km for paved roads and 61.5g/vehicle km for unpaved

roads in Nigeria compared to only 0.1g/vehicle km for streets in London, England[153].

The lower concentration levels of lead, cadmium, nickel, arsenic, selenium and cyanide

in fermented cassava flour from the rural areas where vehicular and industrial activities are

less could be attributed to background levels of these contaminants in the soil where the

cassava was planted.

The data were analyzed statistically to determine whether there was significant

differences between the concentration levels of lead, cadmium, nickel, arsenic, selenium and

cyanide in fermented cassava flour from the urban and rural areas. Mean concentration levels

of lead and cadmium in cassava flour from the urban and rural areas were not significantly

different (p > 0.05). Most of the lead and cadmium concentration levels in the fermented

cassava flour are from the soil where the cassava is planted since most of the cassava flour

sold in the urban areas are actually from the rural areas where industrial and vehicular

activities are less. This shows that urban deposition had not significant influence on the lead

and cadmium levels in cassava flour samples purchased from urban areas. This further

indicates that the flour samples purchased from the township have not been exposed to

atmospheric deposition since cassava flour is a fast selling commodity.

The mean concentration levels of nickel, arsenic, selenium and cyanide in fermented

cassava flour from the urban areas were significantly higher (p < 0.05) than the mean

concentration levels determined in the samples purchased from the rural areas. These

differences could be attributed to vehicles emission, particulate air pollution from industries,

traffic density, crowded areas which are the main sources of contamination in the urban areas

whereas the presence of these contaminants in the samples from the rural areas can be linked

93

to background levels in the soil where the cassava plants were grown. Report have it that the

content of selenium in food depends on the selenium content of the soil where plants are

grown[156]. Other works show that although nickel, arsenic and cyanide occur naturally in the

soil through volcanic emission, weathering of soil and rock, they can easily be released into the

air through burning of fossil fuels and plastics containing nitrogen particularly

acrylonitriles[64][91].

94

CHAPTER FIVE

5.0 CONCLUSION

The results obtained from the study shows that the mean concentration levels of lead,

cadmium, nickel, arsenic, selenium and cyanide in fermented cassava flour from both urban and

rural areas are far below the World Health Organization (WHO) guideline or permissible safe levels

of cyanide and metals in food. This shows that cyanide and metals are present in the cassava flour

in such low concentrations that render the food non-toxic and not to cause any harmful effect .

95

REFERENCES

1.Gari, L. (2002): “Arabic treatises on environmental pollution up to the end as the thirteenth

century,” Environmental and History 8(4), 75-488.

2.Hutchsinson,T.C and Meema, K.M. (1987): Lead, mercury, cadmium and arsenic in the

environment. p. 336-337.

3.Agency for Toxic Substances and Disease Registry (1997): Toxicological profile for cyanide.

Atlanta, G.A, US Department of Health and Human Services.

4.Kaur, Anuprat and Gupta, Usha. (2009): Preconcentration of zinc and manganese using 1-(-

2-pyridylazo)-2-naphthol anchored Si02 nano particles. Eurasian Journal of Analytical

Chemistry, 4(3): 234-244.

5.Khursid, S.R; and Qureshi, I.H. (1984): The role of inorganic elements in human body, the

nucleus, 21: 3-24.

6.Li,X.D; Poon,C.S and Liu,P.S. (2001): Heavy metal contamination of urban soils and street

dusts in Hong Kong. Applied Geochem, 16:1361-1368.

7.Agency for Toxic Substances and Disease Registry (1997): Draft toxicology profile for

cadmium. Public Health services, U.S Department of Health and Human services.

Altlanta, Georgia.

8.Robert,K.M; Daryl,K.G; Peter,A.M and Victor,W.R. (2000): Harpers Biochemistry. 25th

edition, Mc Graw Hill, New York, p. 658-670.

9.Sharma,R.K; Agrawal,M and Marshal, F.M. (2008): Heavy metal contamination of vegetables

in urban India: A Case Study in Vanasi Environ. Pollution. 154:234-263.

10.Ano,A.O; Odoemelam,S.A and Ekwueme,P.O. (2007): Lead and cadmium levels in soils

and cassava along Enugu-Port Harcourt expressway in Nigeria. Electronic. J. of

Environ. Agric and Food Chemistry, 6(5): 2024-2031.

11.Alloway, B.J. (1995): Heavy metals in soil (2nd edition). Chemical Principles of Enviromental

Pollution. Blackie and sons. Chap. 3, p. 156-246.

12.Amusan,A.A; Ige,P.V and Olawale, R. (1999): Preliminary investigation on the use of

municipal waste for farming. Paper Presentation on the Annual Conference of Soil

Science Society of Nigeria held Nov. 21-25, 1999, Benin City –Nigeria.

96

13.Ugwu,J.N; Okoye,C.O.B and Ibeto,C.N. (2011): Impacts of Vehicle Emissions and Ambient

Atmospheric Deposition in Nigeria on the Pb, Cd and Ni Content of Fermented Cassava

Flour Processed by Sun-Drying. Human and Ecological Risk Assessment,17:478 – 488.

14.Stephen, O.K. (1995): Tropical Research and Education Centre, Florida University Press.

15.http://en.wikipedia.org/wiki/cassava. Retrieved 17/12/2010.

16.University of Colorado at Boulder (2007): “Cu-Boulder Archaeology Team Discovers First

Ancient Manioc Field in Africa”. http://www.Colorado.edu/news/release/2007/305.html.

Press released, August 20, 2007, Accessed August 29, 2007.

17.Pope,K; Pohl,M.E.D; Jones,J.G; Lentz,D.L; Von,Nagy,C; Vega,F.J and Quitmyer,I.R.

(2001): “Origin and Environmental Setting of Ancient Agriculture in the low lands of

Meso America”, vol. 292 :1370-1373.

18.Hahn, S.K and Keyser, J. (1985): Cassava, a basic food of Africa. Outlook on Agriculture,

4: 95-100.

19.Philips, T.P. (1983): An Overview of Cassava Consumption and Production; International

Development Research Centre, Ottaura, Canada, p. 83-88.

20.Stone, Glenn, D. (2002): Fallacies in the genetic-modification wars. Implications for

developing countries and anthropological perspectives, Current Anthropology, 43(4):

611-630.

21.Wyllie,D; Mtui,M; Oloya,J.D and Kategile,J.A. (1984): The processing of cassava meal for

chicks. Nutrition Reports International 30(5): 1127-1136.

22.Tewe, O.O. (1983): Thyroid cassava toxicity in animals. Cassava toxicity and thyroid

research and public health issues. p. 114-118.

23.Cooke, R.D and Maduagwu, E.N. (1985): The effect of simple processing on the cyanide

content of cassava chips. Journal of Food Technology 13:299-306.

24.Onabowale,S.O.(1988): Processing of cassava for poultry feeds. In proceedings of national

workshop on alternative livestock feed formulations in Nigeria. November 21-25 (Ilorin),

p. 460-472.

25.Nwachukwu, S.U and Edwards, A.W. (1987): Microorganisms associated with cassava

fermentation for lafu production. Journal of Food and Agriculture,1:39-42.

97

26.Gomez,G; Valdivieso,M; Dela,Cuesta,D and Salcedo, T.S. (1984): Effect of variety and

plant age on the cyanide content of whole root cassava chips and its reduction by

sundrying. Animal Feed Science and Technology 11: 57-65.

27.Oke, O.L. (1968): Cassava as food in Nigeria. World Review of Nutrition and Dietetics. p.

227-250.

28.Renewable Energy Magazine Reports “China uses cassava for bioethanol/biodiesel”

www.biodieselinvesting.com/biodiesel. Retrieved 20/02/2011.

29.Okoro, C.C. (2007): Effect of process modification on the physio-chemical and sensory

quality of fufu-flour and dough. African Journal of Biotechnology, vol. 6(16), p. 1949.

30.Casereda, M.P and Matos, M.C.Y. (1996): Linamerin-The toxic compound of cassava.

Journal of Venomous Animals and Toxins, vol.2 (1). Centre of Tropical Root, Sao Paulo

State University, Botucatu (sp), Brazil.

31.Aregheore, E.M and Agunbiade, O.O. (1991): “The toxic effects of cassava diets on human,

a review.” Vet. Human Toxicology 33, p. 274-275.

32.Shroeder, H.A.(1973): The Trace elements and nutrition. Faber and Faber, London. p.261-

280.

33.Chlopecka,A; Bacon,J.R; Wilson,M.J and Kay,J. (1996): Heavy metals in the environment.

Forms of cadmium, lead and zinc in contaminated soils from southwest Poland. J.

Environmental Quality, 25:69-79.

34.Stanley, E.M. (1992): Toxicological Chemistry. Lewis Publishers. London. p.249-286.

35.Coby,S.C.W and Xiang, D.L. (2004): Lead contamination and isotopic combination of urban

soils in HongKong. The Science of the Total Environment. 319, p.185-195.

36.Sutherland,R.A; Tolosa,C.A, Tack,F.M.G. (2000): Characterization of selected element

concentrations and enrichment ratios in background and anthropogenical impacted

road sides areas. Arch. Environ. Contox, 38, p.428-438.

37.Kleeman,M.J, Schauer,J.J; Cass,G.R. (2000): Size and composition distribution of fine

particulate matter emitted from motor vehicles. Environmental Science and Tech. 34

(7), p.1132-1142.

38.Schmidt and Andren. (1980): Overview on nickel, potential for human exposure. p.205.

98

39.Blot,W.J and Fraumeni,J.F Jnr. (1975): Arsenical air pollution and lung cancer. Lancet.

p.142.

40.Drobiora,H.F; Al-Awadhi,H.A and Obekwi,C. (2004): An investigation into the inhibitory

effects of lead and nickel in mineralization of hydrocarbon soil. The annual international

conference on contaminated soils, sediment and water. Faculty of science, Kuwait

University.

41.Ademoroti, C.M.A. (1996): Pollution by heavy metals. Environmental Chemistry and

Toxicology, Foluder press limited, Ibadan, p.56-58.

42.Friberg, L. (1997): Handbook of Toxicology of Metals. Lagos academic press.

43.FAO/WHO (1991): Joint FAO/WHO Standards Programme Codex Alimentarius

Commission XII, Supplement 4, FAO, Rome, Italy.

44.NAS (1998): Dietary Reference Intakes, National Academy of Sciences. Assessed 18/3/06.

http://www.nal.usda.gov/etex/0000/05html

45.Food and Nutrition Board Institute of Medicine, National Academics (2004): Dietary

Reference Intakes (DRI):Recommended Intakes for Individuals. Assessed 09/06/2009.

46.Abdulllahi,H; Shariat,P.M and Khoshayand,R.M. (2003): Simultaneous spectrophotometric

determination of iron, cobalt and copper by partial least square calibration method in

miscellular medium. Iranian Journal of Pharmaceutical Research. p.207.

47.RaO, A.N. (2005): Trace element estimation methods and clinical context. J.Health Allied

Sciences, 4:1-9.

48.Panali,A.P; Karami,M; Moniri,E; and Soudi, H. (2008): Development for Determination of

Copper. African Journal of Pure and Applied Chemistry, vol 2(16): 096-099.

49.https://facultystaff.richmond.edu/rdominey/titrimetrymethods. Retrieved 21/10/11.

50.Skoog,D.A; West,D.M; Holler,F.J and Crouch,S.R. (2007): Fundamentals of Analytical

Chemistry. Baba Barkha Nath Printers, Haryana, India. 8th edition. p.858-867.

51.Skoog, etal. (2007): Principles of Instrument Analysis. 6th edition. Thomas Brooks / Cole.

p.169-173.

52.http://www.cem.msu.edu/reusch/virtualTest/spectrpy/uv-vis/uvspec.htm#uv1. Retrieved

21/10/11.

53. http://www.shu.ac.uk/schools/sci/chem/tutorials/molspec/uvvisabl.htm

99

http://www.cem.msu.edu/reusch/virtualTest/spectrpy/uv-vis/spectrum.htm. Retrieved 21/10/11.

54.Alicides,R.G; Lucia De Carvalho,M.J; Marilia Nutti,R; Jose De Carvalho,L.V and Wania,

Goncalves,Fukuda. (2010): Assessment and degradation study of total carotenoid and

β-carotene in bitter yellow cassava varieties. African Journal of Food Science. vol 4(4),

p.148- 155.

55.Miguel,E; Alonso,Amelot and Alberto,Oliveros. (2000): A method for the qualification and

kinetic evaluation of cyanogenesis in plant material. Phytochem. Anal. 11:309-316.

56.Ajaelu,J.C; Bamgbose,J.T; Atolaiye,B.O and Adeloye,A.A. (2008): The use of

methemoglobin complex in estimating cyanogens potential of cassava and cassava

products. African Journal of Biotech.vol 7(10) p.1585-1587.

57.Titrimetry, n.OED 2nd edition, 1989, Online version Nov.2010. Retrieved. 6/9/2011.

58. http://facultystaff.richmond.edu/rdominey/Titrimetrymethods Retrieved 21/10/11.

59.Billister, (1923): Helv. Chim. Acta. 6, 771.

60.http://en.wikipedia.org/wiki/cyanide. Retrieved 19/10/2011.

61.Sharpe, A.G (1976): The chemistry of cyano complexes of the transition metals; academic

press. London.

62.Reissman,Stefanie; Elisabeth,Hochleitner; Haofan,Wang; Athanasios,Paschos; Friedrich,

Lottspeich; Richard,S.Glass and August, Bock.(2003): “Taming of a Poison”:

Biosynthesis of the NIFE-hydrogenase cyanide ligands. Science 299(5609) : 1067-

1070. Assessed 28/6/08.

63.Ganjeloo,A; Isom,G.E; Morgan,R.L and Way,J.L. (1980): “Fluorometric determination of

cyanide in biological fluids with p-benzoquinone1”. Toxicology and Applied

Pharmacology 55(1): 103-7.

64.Anon,(2004): “Facts about cyanide: where cyanide is found and how it is used” Center for

Disease Control and Prevention. Retrieved 13/04/10.

65.Leonid, Andrussow. (1935): “The catalytic oxidation of ammonia-methane-mixtures to

hydrogen cyanide” 48(37):593-595.

66.Takano, R. (1916): “The treatment leprosy with cyano cuprol”. The Journal of Experimental

Medicine, 24(2):207-211.

100

67.Taylorson,R and Hendricks,S.B. (1973):˝Promotion of seed germination by cyanide” Plant

Physiology. 52(1):23-27.

68.Soto-Blanco,B; Maiorka,P.C and Gorniak,S.L. (2002): “Effects of long term low-dose

cyanide administration to rats”. Ecotoxicology and Environmental Safety. 53(1):37-41.

69.Toxicity, cyanide – overview at e medicine. Retrieved 18/10/2011.

70.Linus, Pauling. (1947): General Chemistry. W.H. Freeman.

71.Brady, James. E and Holum, John R. (1996): Descriptive chemistry of the elements. John

wiley and sons.

72.Windholz, Martha.(1976): Merk Index of Chemicals and Drugs. 9th edition, Monograph

8393. Merck.

73.Charles,J; Kopf,P.W and Toby,S. (1966): „‟The reaction of pyrophoric lead with oxygen”.

Journal of Physical Chemistry 70:1478.

74.Samans, Carl. H. (1949): Engineering metals and their alloys. Macmillian.

75.Primary Lead Refining Technical Notes. (2007): LDA International. Archived from the

original on 22/03/07. Assessed 7/4/2007.

76.Rooney, Corinne. (2007): “Contamination at shooting ranges” (pdf). The Lead Group,

Incorporated. Assessed 7/4/07.

77.Audsley, George. Ashdown. (1988): The art of organ building 2. p.250-251.

78.Agency for Toxic Substances and Disease Registry / Division of Toxicology and

Environmental Medicine (2006): “tox FAQs:CABS / Chemical agent briefing sheet:

lead”.

79.Light foot, T.L and Yeager, J.M. (2008): “Pet bird toxicity and related environmental

concerns. “The veterinary clinics of North America. Exotic animal practice 11(2):229-59.

80.Rossi, E. (2008): “Low level environmental lead exposure – a continuing challenge” “The

Clinical Biochemist. Reviews / Australian Association of Clinical Biochemists 29(2):63-

70.

81.Payne, M. (2008): “Lead in drinking water”. Canadian Medical Association Journal. 179 (3):

253-4.

82. http://en.wikipedia.org/wiki/cadmium. Retrieved 20/5/2011.

101

83.Scoullos,M.J; Vonkeman,G.H; Thorton,L and Makuch,Z. (2001): Mercury, cadmium, lead,

Handbook for sustainable heavy metals policy and regulation. Spinger.

84.Lane,T.W; Saito,M.A; George,G.N and Pickering,I.J. (2005): “A cadmium enzyme from a

marine diatom”. Nature 435 (42).

85. http://www.ehow.com/howdetect-cadmium-poisoning. Retrieved 8/8/2011.

86. http://www.ehow.com/howtreat-cadmium-poisoning.html. Retrieved 8/8/2011.

87.Weeks, Mary. Elvia. (1932): “The discovering of the element III, some eighteenth century

metals”. Journal of Chemical Education. 9. 22.

88.Huang, X. etal. (1993): Nickel induces increased oxidant in cultured mammalian cell as

detected by dichlorofluorescein fluorescence. Toxicology and Applied Pharmacology

12: 29-36.

89.Barceloux, D.G. (1999): Nickel Toxicological Clinic. Toxicology 37: 239-258.

90. “The most tightly bound nuclei”.

http://hyperphysics.phyastr.gsu.edu/hbase/nucene/nucbin2.html#cl. Retrieved 19/11/10

91.International Agency for Research on Cancer (1990): IARC Monographs on the evaluation

of carcinogenic risks to human. Chromium, nickel and welding vol. 49; lyon: IARC

scientific publication, lion france. p.257- 445.

92.Andersen, K.E etal. (1983): Nickel in tapwater contact dermatitis, 9:140-143.

93.Alloway, B.J. (1999): Heavy metals in soil. Blackie and Son Ltd. Bishop Briggs Glasgrow.

p.43-56.

94.Nickel, Geneva, World Health Organization (1991): Environmental Health Criteria, No 108.

95.Grandjean, P. (1984): Human Exposure to Nickel. IARC Science Publication, 53: 469-485.

96.Spears, J.W. (1984): Nickel is a “never trace element” in the nutrition of domestic animals.

J. Annual Science 59:823-835.

97.Minerals:Nickel micronutrient. Nickel mineral/amino-acids-supplement.com contain 4:61-71.

98.Nielsen, F.H. (1991): Nutritional requirement for boron, silicon, vanadium, nickel and

arsenic. Current Knowledge and Speculation. FASEB 5:2661-2667.

99.Stangl, G.I and Kirchagessner, I.M. (1996): Nickel deficiency alters liver metabolism in rats.

J.Nutrition 126:2466-2473.

102

100.Sunderman, F.W. (1977): A Review of the Metabolism and Toxicology of Nickel. Ann. Clin.

Lab. Sci. 7:377-398.

101.Oller, A.R. (2002): Respiratory Carcinogenicity Assessment of Soluble Nickel Compounds.

Environ. Health. Persp. 110 (supp.5) 841-844.

102. http://www.ehow.com/howtospot-nickel-poisoning Retrieved 22/9/2011.

103. http://www.livestrong.com/treat-nickel-poisoning Retrieved 22/9/2011.

104.Online Etymology Dictionary."arsenic‟‟ http://www.etymonline.com/indexphy?term=arsenic.

Retrieved 15/5/2010.

105.Emsley, John. (2001): Nature‟s Building Blocks: An A-Z guide to the elements. Oxford

University Press. p. 43, 513, 529.

106.Turner, Allan. (1999): “View point, the story so far: An overview of developments in UK

food regulation and associated advisory committees”. British Food Journal 101 (4):274-

283.

107.Norman, Nicholas.C. (1998): Chemistry of Arsenic, Antimony and Bismuth. Springer. p.

50.

108.Holleman,Arnold.F; Wiberg,Egon and Wilberg,Nils. (1985): “Arsen” (in German) Walter de

Gruyter. p.675-681.

109.“The NUBASE Evaluation of Nuclear and Decay Properties” Nuclear Physics A (Atomic

Mass Data Centre) 729:3-128.

110.Brooks, William. E. (2007) “Minerals Yearbook : Arsenic”. United States Geological

Survey. http://minerals.cr.usgs.gov/minerals/commodity/arsenic/mvbl-2007-arsenpdf.

Assessed 8/11/08.

111.Whenlan,J.M; Struthers,J.D and Ditzenberger,J.A. (1960): “Separation of sulphur,

selenium and tellurium from arsenic”. Journal of Electrochemical Society 107(2): 982-

985.

112.Rahman,F.A; Allan,D.L; Rosen,C.J and Sadowskv,M.J. (2004): “Arsenic availability from

chromate copper arsenate(CCA)-treated wood”. Journal of Environmental Quality

33(1):173-80.

113.Jones, F.T. (2007): “A Broad View of Arsenic”. Poultry Science 86(1):2-14.

103

114.Antman,Karen.H. (2007): “The history of arsenic trioxide in cancer therapy”. The

Onocologist 6 (supp. 2):1-2.

115.Jennewein,Marc; Lewis,M.A; Zhao,D; Tsyganor,E; Slavine,N; He,J; Watkins,L;

kodibagakar,V.D etal. (2008): “Vascular imaging of solid tumors in rats with a

radioactive arsenic-labelled antimony that binds exposed phosphatidylserine”. Journal

of Clinical Cancer 14(5):1377-1385.

116.Stolz,John,F; Basu,Partha; Santini,Joanne,M and Oremland,Ronald,S. (2006): “Arsenic

and selenium in microbial metabolism”. Annual Review of Microbiology 60: 107.

117.Wolfe-Simon,Felisa; Suitzer,Blum,Jodis and Kulp,Thomas R. (2010): “A Bacterium that

can grow by using arsenic instead of phosphorus”. Science (47).

118.Klaassen,Curtis and Watkins,John. (2003): Casaretta and Doull‟s Essentials Toxicology.

McGraw-Hill. p. 512.

119.Frances, M. Dyro. (2010): New York Medical College, Westchester Medical centre,

Arsenic Contributor Information and Disclosures. Updated Jan 19, 2010.

120.The Agency for Toxic Substances and Diseases Registry. (2009): Retrieved at

http://www.atsdr.cdc.gov.

121.Akron, R. Bourne.(2007): “Folic acid lowers arsenic levels in blood, study reveals”

contributor on associated content in health and wellness. p. 38-39

122. http://en.wikpedia.org/wiki/selenium Retrieved 2/12/2011.

123.Thomas, C.D. (2004): Assessment of requirements for selenium and adequacy of

selenium status: A review. Eur. J. Clin. Nutr. 58: 391 - 402.

124.Berzelius, J.J. (1818): (Letter from Mr. Berzelius to Mr Bertholet on two new metals) series

2 vol. 7 p.199-206.

125.James, E. House. (2008): Inorganic Chemistry. Academic press, p.524.

126.Egon Wiberg; Nils Wilberg; Arnold Fredrick Holleman. (2001): Inorganic chemistry, san

diego, academic press, p. 583.

127.Seppelt, K; Desmarteau, Darryl. D. (1980): “Selenoyl difluoride”. Inorganic synthesis

20:36-38.

128.Lenher, V. (1902): “Action of selenic acid on gold”. Journal of the American Chemical

Society 24 (4) 36-38.

104

129.http://www.lentech.com/periodictable/elements/chemicalpropeties,healthandenviromental

effects of selenium. Retrieved 2/12/2011.

130.Jrg,Gerhard; Bhnemann,Rolf,Hollas; Kivel,Niko; Kossert,Karsten; Van Winckel,Stefaan;

Gostomski, Christoph and Lierse V. (2010): “Preparation of radiochemically pure 79Se

and highly precise determination of its halflife”. Applied Radiation and Isotopes 68(12):

2339-51.

131.Barclay, Magaret N.I; Allan Macpherson and James Dion. (1995): “Selenium content of

range of UK food”. Journal of food composition and analysis 8(4): 307-318.

132.Combs, G.F Jr and Gray, W.P. (1998): Chemopreventive Agents: Selenium Pharmacol

Ther 79: 179-92.

133.Ruyle, George. (2009): “Poisonous Plants on Arizona Rangelands” (pdf). The University of

Arizona. Assessed 5/12/08.

134. Zone Davis, T. (2008): “Selenium in plants” p.8 Retrieved 5/12/08.

135.Clark,L.C; Combs Jr,G.F; Turnbull,B.W; Slate,E.H; Chalker,D; Chow,J; Davis,L.S; Glover,

R.A; Graham,G.F; Gross,E.G; Krongrad,A; Lesher,J.L; Park,H.K; Sanders;B.B; Smith,

C.L and Taylor,J.R. (1996): Effects of selenium supplementation for cancer prevention

in patients with carcinoma of the skin. A randomized controlled trial. J. Am. Med. Ass.

276: 1957-63.

136.Derumeaux,H; Valeix,P; Castetbon,K; Besimon,M; Boutron-Rualt,M.C; Arnaud,J and

Herceberg, S.(2003): Association of Selenium with Thyroid Volume and Echo Structure

in 35 to 60 years old French Adults. Eur. J. Endocrinol. 148(3):309-15.

137.Kuroki,F; Matsumoto,T and Lida,M. (2003): Selenium is depleted in crohn‟s disease on

enteral nutrition. Digestive diseases 21: 266-70.

138.Abrams,C.K; Siram,S.M; Galsim,C; Johnson-Hamilton,H; Munford,F.L and Mezghebe,H.

(1992): Selenium deficiency in long-term total parenteral nutrition. Nutr. Clin. Pract. 7:

175-8.

139.Mann,Jim; Truswel,A and Stewart. (2002): Essentials of Human Nutrition (2nd edition).

Oxford university press.

140.Combs, G.F. (2002): Food system-based approaches to improving micronutrient nutrition:

the case for selenium. Bio factors 12:39-43.

105

141.Beck,M.A; Levander,O and Handy,J. (2003): Selenium deficiency and viral infection. J.

Nutr. 133: 1463S-67S.

142.“Selenium Sulphide”. Derm Net NZ. Assessed 6/1/2009.

143.National Institutes of Health (2009): Office of the Dietary Supplements, “Dietary

Supplement Fact sheet: Selenium”. Assessed 5/1/2009.

144.Hathcock, J. (1997): Vitamins and Minerals: Efficacy and Safety. Am. J. Clin. Nutr. 66:

427-37.

145.Agency for Toxic Substance and Disease Registry (2005): “Public Health Statement;

Health effects” (pdf).

146.Ohlendorf, H.M. (2003): “Ecotoxicology of Selenium”. Hand book of Ecotoxicology Boca

Raton, lewis publishers p. 466-491.

147.Baselt, R. (2008): Disposition of Toxic Drugs and Chemicals in Man, 8th edition Biomedical

Publications, Fostercity, C.A, 2008. p.1416-1420.

148.Selenium FAQs. Virgina Department of Health. Updated 7/3/2011.

149.International Institute of Tropical Agriculture (IITA), Ibadan (1979): Selected method for oil

and plant analysis. Manual series (I), p. 52.

150.Vogel, A.I. (1965): Quantitative Inorganic Analysis, 3rd edition, p. 355-356.

151.Vogel, A.I. (1965): Quantitative Inorganic Analysis, 3rd edition, p. 302-303.

152.Onwuka, G.I. (2005): Food Analysis and Instrumentation Theory and Practice, napthale

prints, Lagos, Nigeria. p. 142-143.

153.Akeredolu, F. (1989): Atmospheric Environmental Pollution, 23: 783-792.

154.Zheng,Y.M; Chen,T.B; Wu,H.T; Chen,H; Zhou,J.L; Luo,J.F and Huang,Z.C. (2003): The

Spatial Structure and Contents Distribution of Nickel in Soils of Beijing Suburb. Acta

Geographical Sinica, 58: 470-476.

155.Wong,C.S.C; X.D,Li; G. Zhang; S.H, Qi and X.Z Peng. (2003): Atmospheric Deposition of

Heavy metals in the Pearl River Delta, China Atmos. Eviron. 37:767-776.

156.Longnecker,M.P; Taylor,P.R; Levander,O.A; Howe,M; Veillon,C; McAdam,P.A;

Patterson,K.Y; Holden,J.M; Stamper,M.J; Morris,J.S and Willett,W.C. (1991): Selenium

in diet, blood and toe nails in relation to human health in a seleniferous area. Am. J.

Clin. Nutr. 53: 1288-94.