FACULTY OF BIOLOGICAL SCIENCE · I am ever grateful to Dr. C. S. Ubani, Dr. Parker Elijah Joshua...

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OKENYI, ANAYO DAVID

PG/M.Sc./12/62123

LEVELS OF POLYCYCLIC AROMATIC HYDROCARBONS IN FRESH WATER

FISH DRIED UNDER DIFFERENT DRYING REGIMES

DEPARTMENT OF BIOCHEMISTRY

FACULTY OF BIOLOGICAL SCIENCE

Godwin Valentine

Digitally Signed by: Content manager’s Name

DN : CN = Webmaster’s name

O= University of Nigeria, Nsukka

OU = Innovation Centre

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LEVELS OF POLYCYCLIC AROMATIC HYDROCARBONS IN FRESH WATER FISH DRIED UNDER DIFFERENT

DRYING REGIMES

BY

OKENYI, ANAYO DAVID

PG/M.Sc./12/62123

DEPARTMENT OF BIOCHEMISTRY

UNIVERSITY OF NIGERIA, NSUKKA

JUNE, 2014.

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TITLE PAGE

LEVELS OF POLYCYCLIC AROMATIC HYDROCARBON IN FRESH WATER FISH DRIED UNDER

DIFFERENT DRYING REGIMES

4

CERTIFICATION

Okenyi, Anayo David, a Postgraduate Student with Registration Number, PG/M.Sc./12/62123, in

the Department of Biochemistry has satisfactorily completed the requirements for course work

and research for the degree of Master of Science (M.Sc.) in Industrial Biochemistry. The work

embodied in this dissertation is original and has not been submitted in part or in full for any

other diploma or degree of this or any other University.

_______________________ ____________________

PROF. I. N. E. ONWURAH PROF. O.F.C. NWODO

(Supervisor) (Head of Department)

___________________________________

EXTERNAL EXAMINER

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DEDICATION

This work is dedicated to the loving memory of my late father, Mr. Okenyi, Ani Benjamin, and

my dearest mother, Mrs. Okenyi Oyibo Beatrice who have been a great motivation in my

academic pursuits.

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ACKNOWLEDGEMENT

I wholeheartedly give glory to God Almighty whose immeasurable grace and faithfulness has

guided me thus far in my pursuits in life. May only His Holy name be exalted for ever. Amen

My immense gratitude goes to the Head of Department, Prof O. F. C. Nwodo and my supervisor,

Prof. I. N. E. Onwurah and their families for their concern, understanding, encouragement and

guidance which enabled me to complete this work.

I am ever grateful to Dr. C. S. Ubani, Dr. Parker Elijah Joshua and Mr. Obinna Oje. I thank you for

all the technical assistance and your voluntary supervision. God bless you and your family

immensely.

I am especially indebted to Prof. E. C. Onyeneke, Dr. Victor Oguagua, Dr. Eric Ozougwu and Dr.

S. O. O. Eze whose various encouragements stabilized me during the entire programme. Indeed

you all are worthy associates and my God will reward you abundantly.

My unalloyed appreciation and thanks go to all my lecturers (Prof. L. U. S. Ezeanyika, Prof. F. C.

Chilaka, Prof. E. O. Alumona, Prof. O. U. Njoku, Prof. E. N. Uzoegwu, Prof. H. A. Onwubiko, Prof.

B. C. Nwanguma, Dr. O. C. Enechi, Dr (Mrs.) C. A. Anosike and Dr. (Mrs.) U. Njoku and Dr. P. A. C.

Egbuna in the Department of Biochemistry, University of Nigeria, Nsukka for their critical inputs

in making this study a worthy experience. Of special mention are my dear colleagues and

classmates White Alalibo, Dominic Ogbonna, Kingsley, Maximus, Kachi and the rest. Thank you

so much for your various encouragements.

Also my special thanks goes to my loving and beautiful wife Mrs. Okenyi Nkechi Loretta and my

wonderful children Kosi, Amarachi, Ebube and Ebuka for their concerns, worries, deprivations

and prayer that indeed strengthened me to accomplish this aim.

Finally to my dear siblings Ngozi, Uche, Chioma, Nkechi, Beatrice, Chinyere, Ego, Oby, Ani and

Ebere and my worthy in-laws Julius, Okechukwu, Marcel, Chimezie and Kelechi. I say thank you

all and remain blessed.

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ABSTRACT

Preservation of fish by drying over different types of heat regimes have been known. However,

there has not been a comprehensive comparison in terms of the possible contamination

associated with these drying regimes. This work was set to evaluate the levels of PAHs that are

likely to accumulate in the bodies of fresh water fishes dried under heat from charcoal, sun (sun

drying), electric oven and polythene augmented drying regimes (burning of used cellophone

materials). The levels of sixteen PAHs were determined in fish samples harvested from Otuocha

River in Anambra State, Nigeria. The fish samples were dried, pulverized and subjected to

soxhlet extraction using n-hexane at 600c for 8hrs. The water content of the eluants were

further removed with florisil clean-up before Gas chromatographic – mass spectrometric

analysis. Results obtained showed that sun-dried fish had PAHs concentration to be 35.7+

0.2µg/g; oven dried gave 47.7+ 0.2µg/g and charcoal dried 79.53+ 0.2µg/g, while drying with

firewood resulted in 188.1+ 0.2µg/g. Charcoal drying augmented with polythene resulted into

PAHs level of 166.2+ 0.1µg/g while fish dried under heat generated from burning firewood and

polythene material resulted into PAHs concentration of 696.3+0.2µg/g. Preliminary analysis of

the fresh water samples and the undried fish samples (control) revealed that the fresh water

contained total PAHs level of 2.86+ 0.1µg/ml, while the fresh fish 4.97+ 0.2µg/g. The

concentration of PAHs in all the dried fish under different drying agents were significantly

higher than the control. The result is more worrisome in that even the fishes dried under the

sun have PAHs significantly higher than that of the control (p<0.05). It is apparent that the

increase in PAHs must have come from the environmental PAHs (exposure) under which the

fishes were dried (under sun). For the other drying regimes, in which the levels of PAHs were

significantly higher than that of sun-dried, it can be concluded that the excessive PAHs in the

body of the dried fish were from the “burning” or drying agents. More significantly are the

observed very high increase in PAHs when drying was augmented with polythene, an agent

known to be a high source of PAHs when incinerated. Consumers of dried fish should therefore

beware of the dried fish they purchase from the local market.

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TABLE OF CONTENTS

Title Page - - - - - - - - - - i

Certification - - - - - - - - - - - ii

Acknowledgment- - - - - - - - - - iii

Abstract - - - - - - - - - - - iv

Table of Content - - - - - - - - - - v

List of Figures - - - - - - - - - - vi

List of Tables - - - - - - - - - - vii

List of Abbreviations - - - - - - - - - viii

CHAPTER ONE: INTRODUCTION

1.1. Introduction - - - - - - - - 1

1.2. Physical and Chemical Characteristics of PAHs - - - 2

1.3. Sources and Emission of PAHs - - - - - 5

1.3.1. Stationary Sources - - - - - - - - 5

1.3.1.1. Domestic Sources - - - - - - - 5

1.3.1.2. Industrial Sources - - - - - - - 6

1.3.2. Mobile Sources - - - - - - - 6

1.3.3. Agricultural Sources - - - - - - - 7

1.3.4. Natural Sources - - - - - - - 7

1.3.5 Uses of PAHs- - - - - - - - 8

1.4. Routes of Exposure for PAHs - - - - - - 8

1.4.1 Air - - - - - - - - - - 9

1.4.2 Water - - - - - - - - - 9

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1.4.3 Soil - - - - - - - - - 10

1.4.4. Foodstuffs - - - - - - - - - 10

1.4.5. Other Sources of Exposure - - - - - - 11

1.5. Individuals at Risk of Exposure - - - - - - 11

1.6. Standards and Regulation for PAH Exposure - - - 12

1.7. Metabolism of PAHs - - - - - - - 15

1.7.1. Fate of PAHs in Soil and Groundwater Environment - - 20

1.7.2. Fate of PAHs in Air and their Ecotoxicological consequences - 21

1.8 Human Health Effects - - - - - - 22

1.8.1 Acute or Short-Term Health Effects - - - - - 22

1.8.2. Chronic or Long-Term Health Effects - - - - 23

1.8.3 Carcinogenicity - - - - - - - 23

1.8.4. Teratogenicity - - - - - - - 24

1.8.5. Genotoxicity - - - - - - - - 25

1.8.6. Immunotoxicity - - - - - - - 25

1.8.7. effect of PAHs Pathogenic Change - - - - - 25

1.9. Fish - - - - - - - - - 28

1.9.1. Food Smoking - - - - - - - 29

1.10. Rationale of Study - - - - - - - 30

1.11. Aims and Objectives - - - - - - - 30

CHAPTER TWO: MATERIALS AND METHODS

2.0 Material and methods - - - - - - 32

2.1. Materials - - - - - - - - 32

2.1.1. Apparatus and Equipment - - - - - - 32

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2.1.2. Chemicals - - - - - - - - 32

2.1.3. Fish Samples - - - - - - - - 33

2.1.4. Study Site - - - - - - - - 33

2.2. Methods - - - - - - - - 35

2.2.1. Collection of Fish Samples and Drying - - - - 35

2.2.2. Sample Preparation for the Analysis of Dried Fishes - - - 36

2.2.3. Preparation of Florisil for clean-up - - - - - 37

2.2.4. Instrument Analysis - - - - - - - 38

CHAPTER THREE: RESULTS

Result - - - - - - - - - 41

CHAPTER FOUR

4.0. Discussion - - - - - - - - 80

Conclusion - - - - - - - - 84

Reference

Appendices

LIST OF TABLES

Table 1: Physical and Chemical Properties of PAHs - - - - 4

Table 2: levels of PAHs Exposures from Workplace - - - - 13

Table3: Carcinogenic Classification of Selected PAHs - - - 19

Table4: Temperature Condition of GC-MS - - - - 39

Table 5: Weight of Fish used in October, November and January 2014 - 41

Table 6: GC-MS result of fish samples in October 2013 - - - 43

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Table 7: GC-MS result of fish samples in November 2013 - - - 44

Table 8: GC-MS result of fish samples in January 201 - - - 45

Table 9: Statistical mean value of GC-Ms result of the three months - -

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LIST OF FIGURES

Figure 1: Mechanism of Activation of BaP by Cytochrome P450 and

Epoxide Hydroxilase - - - - - - - 16

Figure 2: Aryl hydrocarbon receptor (AhR) pathway activated by BaP - 17

Figure 3: Bay region of some PAHs - - - - - - 27

Figure 4: Map Showing Otuocha River in Anambra State - - - 34

Figure 5: Monthly distribution of Acenaphthylene in various treatments - - 49

Figure 6: Monthly distribution of Anthracene in various treatments - - 51

Figure 7: Monthly distribution of 1,2 Benzanthracene in various treatments - 53

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Figure 8: Monthly distribution of Benzo(pyrene) in various treatments - 55

Figure 9: Monthly distribution of Benzo(fluoranthene) in various treatments

57

Figure 10: Monthly distribution of Benzo(g,h,i)perylene in various treatments

59

Figure 11: Monthly distribution of Benzo(k)fluoranthene in various treatments 61

Figure 12: Monthly distribution of chrysene in various treatments - - 63

Figure 13: Monthly distribution of Dibenz(a,h)anthracene in various treatments 65

Figure 14: Monthly distribution of fluoranthene in various treatments -

67

Figure 15: Monthly distribution of fluorene in various treatments - - 69

Figure 16: Monthly distribution of indeno(1,2,3-cd)pyrene in various treatments 71

Figure 17: Monthly distribution of Naphthalene in various treatments - - 73

Figure18: Monthly distribution of Pyrene in various treatments - - 74

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LIST OF ABBREVIATIONS

PAHs – Polycyclic Aromatic Hydrocarbons

LMW – Low Molecular Weight

HMW – High Molecular Weight

ATSDR – Agency for Toxic Substances and Disease Registry

EPA – Environmental Protection Agency

POP - Persistent Organic Pollutants

WHO - World Health Organization

MCL - Maximum Contaminant

PPB - Parts Per Billion

IARC – International Agency for Research on Cancer

OSHA – Occupational Safety and Health Administration

Ctpv – Coal Tar Pitch Volatiles

PEL – Permissible Exposure Limit

NIOSH – National Institute for Occupational Safety and Health

TLV- Threshold Limit Value

TWA – Time Weighted Average

REL – Recommended Exposure Limit

FAO – Food and Agricultural Organization

FDA Food and Drug Administration

BAP – Benzo (a) Pyrene

CDC – Center for Disease Control and Prevention

BEI – Biological Exposure Index

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DNA – Deoxynbonucleic Acid

SPSS – Statistical Product and Solution Services

ANOVA – One Way Analysis of Variance

GC-MS – Gas Chromatography Mass Spectrometer

F/P – Ratio Flouranthene to Pyrene

KOW – Octanol-Water Partition Coefficients

KOC – Partition Coefficient for Organic Carbon

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CHAPTER ONE

1.1 INTRODUCTION

Polycyclic aromatic hydrocarbons (PAHs) are a group of organic compounds consisting of two

or more fused benzene rings (linear, cluster or angular arrangement), or compounds made up of

carbon and hydrogen atoms grouped into rings containing five or six carbon atoms. They are

called “PAH derivatives” when an alkyl or other radical is introduced to the ring, and

heterocyclic aromatic compounds (HACs) when one carbon atom in a ring is replaced by a

nitrogen, oxygen or sulphur atoms. PAHs originate mainly from anthropogenic processes

particularly from incomplete combustion of organic fuels. PAHs are distributed widely in the

atmosphere. Natural processes, such as volcanic eruptions and forest fires, also contribute to an

ambient existence of PAHs (Suchanova et al., 2008). PAHs can be present in both particulate

and gaseous phases, depending on their volatility. Low molecular weight PAHs (LMW PAHs)

that have two or three aromatic rings (molecular weight from 152 to 178g/mol) are emitted in the

gaseous phase, while high molecular weight PAHs (HMW PAHs), molecular weight ranging

from 228 to 278g/mol, with five or more rings, are emitted in the particulate phase, (ATSDR,

1995) . In the atmosphere, PAHs can undergo photo-degradation and react with other pollutants,

such as sulfur dioxide, nitrogen oxides, and ozone. Due to widespread sources and persistent

characteristics, PAHs disperse through atmospheric transport and exist almost everywhere. There

are hundreds of PAH compounds in the environment but in practice PAH analysis is restricted to

the determination of six (6) to sixteen (16) compounds. Human beings are exposed to PAH

mixtures in gaseous or particulate phases in ambient air. Long term exposure to high

concentration of PAHs is associated with adverse health problems. Since some PAHs are

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considered carcinogens, inhalation of PAHs in particulates is a potentially serious health risk

linked to lung cancer (Philips, 1999).

1.2. Physical and Chemical Characteristics of PAHs.

PAHs are a group of several hundred individual organic compounds which contain two or more

aromatic rings and generally occur as complex mixtures rather than single compounds. PAHs are

classified by their melting and boiling points, vapour pressure, and water solubility, depending

on their structure. Pure PAHs are usually coloured, crystalline solids at ambient temperature. The

physical properties of PAHs vary with their molecular weight and structure (Table1). Except for

naphthalene, they have very low to low water solubilities, and low to moderately high vapour

pressures. Their octanol-water partition coefficients (Kow) are relatively high, indicating a

relatively high potential for adsorption to suspended particles in the air and in water, and for

bioconcentration in organisms (Sloof et al., 1989). Table 1 shows physical and chemical

characteristics of few selected PAHs from the sixteen (16) priority PAHs, listed by the US EPA.

(see appendix). Most PAHs, especially as molecular weight increases, are soluble in non-polar

organic solvents and are barely soluble in water (ATSDR, 1995).

Most PAHs are persistent organic pollutants (POPs) in the environment. Many of them are

chemically inert. However, PAHs can be photochemically decomposed under strong ultraviolet

light or sunlight, and thus some PAHs can be lost during atmospheric sampling. Also, PAHs can

react with ozone, hydroxyl radicals, nitrogen and sulfur oxides, and nitric and sulfuric acids

which affect the environmental fate or conditions of PAHs (Dennis et al., 1984; Simko, 1991).

PAHs possess very characteristic UV absorbance spectra. Each ring structure has a unique UV

spectrum, thus each isomer has a different UV absorbance spectrum. This is especially useful in

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the identification of PAHs. Most PAHs are also fluorescent, emitting characteristic wavelengths

of light when they are excited (when the molecules absorb light). Generally, PAHs only weakly

absorb light of infrared wavelengths between 7 and 14µm, the wavelength usually absorbed by

chemical involved in global warning (Ramanathan, 1985).

Polycyclic aromatic hydrocarbons are present in the environment as complex mixtures that are

difficult to characterize and measure. They are generally analyzed using gas chromatography

coupled with mass spectrometry (GC-MS) or by using high pressure liquid chromatography

(HPLC) with ultraviolet (UV) and fluorescence dectetors (Slooff et al., 1989)

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Table 1 Physical and Chemical Characteristics of Some Popular PAHs

s/n Names of PAHs Chemical structure/formula Mol

weight

Vapour pressure Partition

coefficient

(kow)

1 Naphthalene

C10H8

128.17

0.087mmHg

3.29

2 Fluorine

C13H10

166.2

3.2x10-4mmHg

4.18

3 Fluoranthene

C16H10

202.26

5.0 x10-6mmHg

4.90

4 Pyrene

C16H10

202.3

2.5 x10-6mmHg

4.88

5 Benzo(a)anthracene

C20H12

228.29

2.5 x10-6mmHg

5.61

6 Benzo(k)fluoranthene

C20H12

252.3

9.59x10-11mmHg

6.06

7 Benzo(a)pyrene

C20H12

252.3

5.6x10-9mmHg

6.06

8 Indeno(1,2,3-c,d)pyrene

C22H12

276.3

10x10-16mmHG

6.58

Sources: (ATSDR, 1995)

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Source and Emission of PAHs

PAHs are mainly derived from anthropogenic activities related to pyrolysis and incomplete

combustion of organic matter. Sources of PAHs affect their characterization and distribution, as

well as their toxicity. Major sources of PAH emissions may be divided into four classes:

stationary sources (including domestic and industrial sources), mobile emission, agriculture

activities, and natural sources (Wania et al, 1996).

1.3. Stationary Sources

Some PAHs are emitted from point sources and this is hardly shifted (moved) for a long period

of time. Stationary sources are further subdivided into two main sources: domestic and industrial.

1.3.1. Domestic Sources

Heating and cooking are dominant domestic sources of PAHs. The burning and pyrolysis of coal,

oil, gas, garbage, wood, or other substances are the main domestic sources. Domestic sources are

important contributors to the total PAHs emitted into the environment. Difference in climate

patterns and domestic heating systems produce large geographic variations in domestic emission.

PAH emissions from these sources may be a major health concern because of their prevalence in

indoor environments (Ravindra et al., 2008). According to a recent World Health Organization

(WHO) report, more than 75% of people in China, India, and South East Asia and 50-75% of

people in parts of South America and Africa use combustion of solid fuel, such as wood,

charcoal for daily cooking.

Main indoor PAH sources are cooking and heating and infiltration from outdoors. PAH

emissions from cooking account for 32.8% of total indoor PAHs (Zhu et al., 2009). LMW PAHs

which originate from indoor sources are the predominant proportion of the total PAHs identified

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in residential non-smoking air. Toxicity of PAH mixtures from indoor sources is lower than

mixtures which contain large amounts of high molecular weight PAHs. Cigarette smoke is also a

dominant sources of PAHs in indoor environments. In many studies, PAHs in the indoor air of

smoking residences tend to be higher than those of non-smoking residences.

1.3.2. Industrial Sources

Sources of PAHs include emission from industrial activities, such as primary aluminum and coke

production, petrochemical industries, rubber tire and cement manufacturing, bitumen and asphalt

industries, wood preservation, commercial heat and power generation, and waste incineration

(Fabbri and Vassura , 2006).

1.3.3. Mobile Sources

Mobile sources are major causes of PAHs emissions in urban areas. PAHs are mainly emitted

from exhaust fumes of vehicles, including automobile, railways, ships, aircrafts, and other motor

vehicles. PAHs emissions from mobile sources are associated with use of diesel, coal, gasoline,

oils, and lubricant oil. Exhaust emissions of PAHs from motor vehicles are formed by three

mechanisms: (1) synthesis from smaller molecules and aromatic compounds in fuel; (2) storage

in engine deposits and in fuel; (3) pyrolysis of lubricants (Baek et al., 1991). One of the major

influences on the production of PAHs from gasoline automobiles is the air-to-fuel ratio. It has

been reported that the amount of PAHs in engine exhaust decreases with leaner mixtures

(Ravindra et al., 2006b). A main contribution to PAH concentrations in road dust as well as

urban areas is vehicle exhaust. Abrantes et al., (2009) reported that the total emissions and

toxicities of PAHs released from light-duty vehicles using ethanol fuels are less than those using

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gasohol. Low molecular weight PAHs are the dominant PAHs emitted from light duty vehicles

and helicopter engines.

1.3.4 Agricultural Sources

Open burning of bush wood, straw, moorland heather, and stubble are agricultural sources of

PAHs. All of those activities involve burning organic materials under suboptimum combustion

conditions. Thus it is expected that a significant amount of PAHs are produced from the open

burning of biomass. PAH concentrations released from wood combustion depend on wood type,

kiln type, and combustion temperature. Between 80 – 90% of PAHs emitted from biomass

burning are low molecular weight PAHs, including naphthalene acenaphthylene, phenanthene,

fluoranthene and pyrene. Lu et al., (2009) reported that PAHs emitted from the open burning of

rice and bean straw are influenced by combustion parameters. Total emissions of 16 PAHs from

the burning of rice and bean straw varied from 9.29 to 23.6µg/g and from 3.13 to 49.9µg/g

respectively. PAH emissions increased with increasing temperature from 200 to 7000c.

Maximum emissions of PAHs were observed at 40% O2 content in supplied air. However,

emission of PAHs released from the open burning of rice straw negatively correlate with the

moisture content in the straw (Lu et al., 2009).

1.3.5. Natural Sources

Accidental burning of forests, woodland, and moorland due to lightning strikes are natural

sources of PAHs. Furthermore, volcanic eruptions and decaying organic matter are also

important natural sources, contributing to the levels of PAHs in the atmosphere. The degree of

PAH production depends on meteorological conditions such as wind, temperature, humidity, and

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fuel characteristics and type; such as moisture content, green wood, and seasonal wood (Wild

and Jones, 1995).

1.3.6 Uses of PAHs

PAHs are not synthesized chemically for industrial purposes. Rather than industrial sources, the

major source of PAH is the incomplete combustion of organic material such as coal, oil, and

wood. However, there are a few commercial uses for many PAHs. They are mostly used as

intermediaries in pharmaceuticals, agricultural products, photographic products, thermosetting

plastics, lubricating materials, and other chemical industries. Acenaphthene, Anthracene,

Fluoranthene, Fluorene, Phenanthrene and Pyrene are used in the manufacture of dyes, plastics,

pigments, pharmaceutical and agrochemicals such as pesticides, wood preservatives resins and

so on.

Other PAHs may be contained in asphalt used for the construction of roads, as well as roofing

tar. Precise PAHs, specific refined products, are used also in the field of electronics, functional

plastics, and liquid crystals. (Katarina, 2011).

1.4 Routes of Exposure for PAHs

PAH exposure through air, water, soil, and food sources occurs on a regular basis. The routes of

exposure include ingestion, inhalation, and dermal contact in both occupational and non-

occupational settings. Some exposure may involve more than one route simultaneously,

affecting the total absorbed dose (such as dermal and inhalation exposure from contaminated

air). All non-workplace source of exposure such as diet, smoking, and burning of coal and wood

should be taken into consideration (ATSDR, 1995).

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1.4.1 Air

PAHs concentrations in air can vary from less than 5 to 200,000 (ng/m3) (Cherng et al., 1996;

Georgiadis and Kyrtopoulos, 1999). Although environmental air levels are lower than those

associated with specific occupational exposure, they are of public health concern when spread

over large urban populations (Zmirou et al., 2000).

The background levels of the Agency for Toxic Substances and Disease Registry’s toxicological

priority for PAHs in ambient air have been reported to be 0.02 – 1.2 ng/m3 in rural areas and

0.15 – 19.3 ng/m3 in urban areas (ATSDR, 1995).

Cigarette smoking and environmental tobacco are other sources of air exposure. Smoking one

cigarette can yield an intake of 20-40ng of benzo (a) pyrene (Philips, 1996; O’Neill et al.,

1997). Smoking one pack of unfiltered cigarette per day yields 0.7µg/day benzo (a) pyrene

exposure. Smoking a pack of filtered cigarette per day yields 0.4 µg/day (Sullivan and Krieger

2001).

Environmental tobacco smoke contains a variety of PAHs, such as benzo (a) pyrene, and more

than 40 known or suspected human carcinogens. Side-stream smoke (smoke emitted from a

burning cigarette between puffs) contains PAHs and other cytotoxic substances in quantities

much higher than those found in mainstream smoke (exhaled smoke of smoker) (Jinot and

Bayard, 1996; Nelson, 2001).

1.4.2. Water

PAHs can leach from soil into ground water. Water contamination also occurs from industrial

effluents and accidental spills during oil shipment at sea. Concentrations of benzo (a) pyrene in

24

drinking water are generally lower than those in untreated water and about 100 fold lower than

the US Environmental Protection Agency’s (EPA) drinking water standard. (EPA’s maximum

contaminant level (MCL) for benzo (a) pyrene in drinking water is 0.2 parts per billion

{ppb}(US EPA, 1995).

1.4.3 Soil

Soil contains measurable amounts of PAHs primarily from airborne fallout. Documented level

of PAHs in soil near oil refineries have been as high as 200,000 micrograms per kilogram

(µg/kg) of dried soil. Levels in soil samples obtained near cities and areas with heavy traffic

were typically less than 2,000 µg/kg (IARC, 1973).

1.4.4 Food Stuffs

In non-occupational settings, up to 70% of PAH exposure for non-smoking person can be

associated with diet (Skupinska et al., 2004). PAH concentrations in foodstuffs vary. Charring

meat or barbecuing food over a charcoal, wood, or other type of fire greatly increase the

concentration of PAHs. For example, the PAH level for charring meat can be as high as 10-20

µg/kg (Philips, 1999). Charbroiled and smoked meats and fish contain more PAHs than do

uncooked products, with up to 2.0 µg/kg of benzo (a) pyrene detected in smoked fish. Tea,

roasted peanuts, coffee, refined vegetable oil, cereals, spinach, and many other foodstuffs

contain PAHs. Some crops such as wheat, rye and lentils, may synthesize PAHs or absorb them

via water, air, or soil (Grimmer, 1968; Shabad and Cohan 1972; IARC, 1973).

25

1.4.5 Other Sources of Exposure

PAHs are found in prescription and non-prescription coal tar products used to treat

dermatologic disorders such as psoriasis and dandruff (Van Schooten, 1996). PAHs and their

metabolites are excreted in breastmilk, and they readily cross the placenta.

Antracene laxative use has been associated with melanosis of the colon and rectum (Badiali et

al., 1985).

1.5 Individuals at Risk of Exposure

Workers in industries or trades using or producing coal or coal products are at highest risk for

PAHs exposure. Those workers include, but are not limited to Aluminum workers, Asphalt

workers, Carbon black workers, Chimney sweeps, Coal-gas workers, Fishermen (coal tar on

nets), Graphite electrode workers, Machinists, Mechanics (auto and disel engine), Printers,

Road (pavement) workers, Roofers, Steel foundry workers, Tire and rubber manufacturing

workers, and Workers exposed to creosote, such as Carpenters, Farmers, railroad workers,

Tunnel construction workers, and Utility workers

Exposure is almost always to mixtures that pose a challenge in developing conclusions (Samet,

1995). Fetuses may be at risk for PAH exposure. PAH and its metabolites have been shown to

cross the placenta in various animal studies (ATSDR, 1995). Because PAH are excreted in breast

milk, nursing infants of exposed mothers can be easily exposed.

26

1.6 Standard and Regulations of PAHs Exposure.

The United States Government Agencies have established standards that are relevant to PAHs

exposure in the workplace and the environment. There is a standard relating to PAHs in the

workplace, and also a standard for PAHs in drinking water.

Occupational safety and health administrations (OSHA) have not established a substance-

specific standard for occupational exposure to PAHs. Exposures are regulated under OSHA’s Air

contaminants standard for substances termed coal tar pitch volatiles (CTPVs) and coke oven

emission. Employees exposed to CTPVs in the coke oven industry are covered by the coke oven

emissions standard.

The OSHA coke oven emission standard required employers to control employee exposure to

coke oven emissions by the use of engineering controls and work practices.

Whenever the engineering and work practices control that can be instituted are not sufficient to

reduce employee exposure to or below the permissible exposure limit (PEL), the employer shall

nonetheless use them to reduce exposure to the lowest level achievable by these controls and

shall supplement them by the use of respiratory protection. The OSHA standards also include

elements of medical surveillance for workers exposed to coke oven emissions (ATSDR, 1995).

Air

The OSHA PEL for PAHs in the workplace is 0.2miligram/cubic meter (mg/m3). The OSHA –

mandated PAH workroom air standard is an 8-hour time-weighted average (TWA) permissible

exposure limit (PEL) of 0.2 mg/m3, measured as the benzene-solube fraction of coal tar pitch

volatiles. The OSHA standard for coke oven emissions is 0.15 mg/m3. The National Institute for

27

Occupational Safety and Health (NIOSH) has recommended that the workplace exposure limit

for PAHs be set at the lowest detectable concentration which was 0.1 mg/m3 for coal tar pitch

volatile agents at the time of the recommendation (ATSDR, 1995).

Table 2: Levels of PAHs Exposures from Workplace

Agency Focus Level Comments

American conference

of governmental

industrial hygienists

Air workplace 0.2 mg/m3 for

benzene – soluble

coal tar pitch fraction

Advisory: TLV (8 –

hours TWA)

National institute for

occupational safety

and health

Air: workplace 0.1 mg/m3 for coal tar

pitch volatile agents

REL (8 – hour TWA)

Occupational safety

and health

administration.

Air: workplace 0.2mg/m3 for

benzene-soluble coal

tar pitch fraction

Regulation: (benzene

soluble fraction of

coal tar volatiles) PEL

8 – hour workday.

U.S. environmental

protection agency

Water 0.0001miligrams per

litre (mg/l)

MCL for benz (a)

anthracene

0.0002mg/l

MCL for benzo (a)

pyrene, benzo (b)

fluoranthene, benzo

(k) fluoranthene,

chrysene.

0.0003mg/l

MCL for dibenz (a,h)

anthracene

0.0004mg/l MCL for indeno

(1,2,3-c,d) pyrene

(ATSDR, 1995).

28

• TLV: threshold limit value.

• TWA (time – weighted average), concentration for a normal 8-hour workday and a 40-hour

workweek to which nearly all workers may be repeatedly exposed.

• REL (recommended exposure limit): recommended airborne exposure limit for coal pitch

volatiles (cyclohexane – extractable fraction) averaged over a 10 – hour work shift.

• PEL (permissible exposure limit): the legal airborne permissible exposure limit (PEL) for

coal tar pitch volatiles (Benezene soluble fraction) averaged over an 8 – hour work shift.

• MCL: maximum contaminant level. (ATSDR, 1995).

Water

The maximum contaminant level goal for benzo (a) pyrene in drinking water is 0.2 parts per

billions (ppb). In 1980, EPA developed ambient water quality criteria to protect human health

from the carcinogenic effects of PAH exposure. The recommendation was a goal of zero (non-

detectable level for carcinogenic PAHs in ambient water). EPA, as a regulatory agency, sets a

maximum contaminant level (MCL) for benzo (a) pyrene, the most carcinogenic PAH at

0.2ppb. EPA also sets MCLs for five other carcinogenic PAHs (see table 2) (ATSDR, 1995).

Food

The U.S. Food and Drug Administration has not established standard governing the PAH

content of foodstuffs but the Food and Agricultural Organization (FAO) and World Health

Organization (WHO) have set a maximum permissible level for total polycyclic aromatic

hydrocarbons and benzo (a) pyrene in certain foods. Recently the maximum permissible level of

health hazard dietary intake of the PAHs in cooked and processed food are not defined

accurately and varies from one country to another. Janoszka et al., (2004) reported that the

29

health hazard level of the PAHs daily ingested in diet was found to be 3.7µg/kg in Great Britain,

5.17µg/kg in Germany, 1.2 µg/kg in New Zealand and 3 µg/kg in Italy. Generally it is known

that the maximum permissible level (MPLs) of total PAHs and BaP are 10 and 1µg/kg wet

cooked or processed meat and fishery products respectively as reported by FAO/WHO and

Stolyhow and Sikorski (2005). The above and the Health hazard level of 5.7µg/day as reported

by Janoszka et al., (2004) are the accepted reference standards even in Nigeria.

1.7 Metabolism of PAHs

Once PAHs enter the body they are metabolized in a number of organs (including liver, kidney,

lungs), excreted in bile, urine or breast milk and stored to a limited degree in adipose tissue. The

principal routes of exposure are: inhalation, ingestion, and dermal contact. The lipophilicity of

PAHs enables them to readily penetrate cellular membranes (Yu, 2005). Subsequently

metabolism renders them more water-soluble making them easier for the body to remove.

However, PAHs can also be converted to more toxic or carcinogenic metabolites.

Phase I metabolism of PAHs

There are three main pathways for activation of PAHs: the formation of PAH radical cation in a

metabolic oxidation process involving cytochrome P450 peroxidase, the formation of PAH-o-

quinones by dihydrodiol dehydrogenase-catalysed oxidation and finally the creation of

dihydrodiol epoxides, catalysed by cytochome P450 (CYP) enzymes (Guengerich, 2000). The

most common mechanism of metabolic activation of PAHs, such as Benzo (a) pyrene (B(a)P), is

via the formation of bay-region dihydrodiol epoxides eg. Benzo (a)pyrene-7, 8-dihydrodiol-9,10-

epoxide (BPDE), via CYP450 and epoxide hydrolase (EH) as seen in figure 1 below.

30

Fig.1: Mechanism of activation of BaP by cytochrome P450 (CPY) and epoxide hydrolase (EH).

The most important enzymes in the metabolism of PAHs are CYPs IA1, IA2, IB1 and 3A4.

CYPIAI is highly inducible by PAHs such as B(a)P and some polyhalogenated hydrocarbons.

Recombinant human CYPIAI metabolizes compounds such as B(a)P, 2-acetylamino-fluorene

and 7,8-diol, 7-12-dimethylbenz (a) anthracene (Kim et al., 1998). CYPIA2 and CYPIB2 are

also inducible by the exposure to PAHs. These enzymes share the same mechanism with which

PAH molecules interact with the aryl hydrocarbon receptor (AHR). The AHR is present in the

cytoplasm as a complex with other proteins such as heat shock protein 90 (HSP 90), p23 and

AhR-interacting protein. After forming a complex with PAHs, the Hsp90 is released and the

AhR-PAH complex translocates to the nucleus as seen in Figure 2.

Benzo (a) pyrene

CYP450

Benzo (a)pyrene 7,8-diol HO

HO

CYP

HO

HO

O

Benzo (a)pyrene 7,8-diol 9,10 epoxide

O Benzo (a) pyrene

7,8-epoxide

Epoxide

Hydrolase

31

Fig. 2: Aryl hydrocarbon receptor (AhR) pathway activated by BaP induces expression of

cyp1A1 and cyp1B1

32

Here, it creates a heterodimer with a ARNT (Ah Receptor Nuclear Translocator) and afterwards

binds to DNA via the xenobiotic response element (XRE) situated in the promoter region of

CYPIA and CYPIB genes (Shimada et al., 2002).

Other phase I enzymes related to PAHs metabolism are the aldo-keto reductases. These enzymes

oxidize polycyclic aromatic (PAH) trans-dihydrodiols to reactive and redox-active O-quinones in

vitro (Quinn and Penning, 2006). Specifically, AKRIAI, and members of the AKRIC

dihydrodiol/hydroxysteroid dehydrogenase subfamily, AKRICI-AKRICA are involved in

metabolic activation of PAH trans-dihydrodiol. Production of O-quinone metabolites by these

enzymes has been shown in vitro and in cell lines to amplify ROS and oxidative damage to DNA

bases to form the highly mutagenic lesion 8-oxo-deoxyguanosine (8-oxo-Guo) and render

damaged and carcinogenic DNA (Quinn et al., 2008).

Phase II metabolism of PAHs

Phase II metabolism includes conjugation of metabolites from phase I with small molecules

catalysed by specific or glutathione S-transferases (GSTs). SULTs have been shown to activate

some metabolities of PAHs such as 7, 12-dimethylbenz(a)anthracene and its methyl-

hydroxylated derivatives, in different tissues (Chou et al., 1998). Polymorphisms of SULTIAI

have been associated with PAH-DNA adduct levels (Tong et al., 2003). Like sulfation,

glucuronidation produces polar conjugates that are readily excreted. Oxygenated benzo (a)

pyrene derivatives are common substrates of UDP-glucuronly-transferase (Bansal et al., 1981),

the resulting metabolites, I-hydroxypyrene glucuronide, and the parental I-hydroxypyrene are

used as biomarkers of PAH exposure (Strickland et al., 1994). Finally, GSTs are also involved in

conjugation of PAH derivatives. Glutathione conjugates are further metabolized to mercapturic

33

acids in the kidney and are excreted in the urine. On the other hand, polymorphisms of phase II

metabolism are associated with carcinogenesis and with DNA demage. For instance, there is an

important association between GSTMI gene polymorphism and the DNA adduct levels (Binkova

et al., 2007). The classification of some PAHs by some agencies and their carcinogenic

tendencies as shown in table 3.

Table 3: carcinogenic classification of selected PAHs by specific agencies

Agency PAH Compound (s) Carcinogenic Classification

U.S. Department of Health and

Human Services (HHs)

• Benz (a)anthracene

• Benz (b) flouranthene,

• Benzo (a) pyrene,

• Dibenz (a, h) antracene, and

• Indeno (1,2,3-cd)pyrene

Known animal carcinogens

International Agency for Research

on Cancer (IARC)

• Benz (a)anthracene

• Benzo (a) pyrene,

Probably carcinogenic to humans

• Benzo (b) fluoranthene,

• Benzo (k) fluoranthene, and

• Indeno (1,2,3-cd)pyrene.

Possible carcinogenic to humans

• Anthracene

• Benzo (g,h,i)perlyene,

• Benzo (e) pyrene

• Chrysene

• Fluoranthene,

• Fluorene

• Phenanthrene , and

• pyrene.

Not classifiable as to their

carcinogenicity to humans

• Benz (a) anthracene,

• Benzo (a) pyrene

• Benzo (b) fluoranthene

• Benzo (k) fluoranthene

• Chrysene,

• Dibenz (a,h) anthracene, and

• Indeno (1,2,3-cd)pyrene.

Probable human carcinogens

• Acenaphtylene,

• Anthracene

• Benzo (g,h,i) perylene

• Fluoranthene,

• Fluorene

• Phenanthrene, and pyrene

Not classifiable as to

human carcinogenicity

(ATSDR, 1995)

34

1.7.1. Fate of PAHs in Soil and Groundwater Environment

Low molecular weight (LMW) PAHs (two or three rings) are relatively volatile, soluble and

more degradable than are the higher molecular weight compounds. High molecular weights

(HMW) (four or more rings) sorb strongly to soils and sediments and are resistant to microbial

degradation (Sikkema et al., 1995).

Because of the very low water solubility and high Kow values, they will tend to be sorbed to the

organic matter in the soil instead of being solubilized in the infiltrating water and through this be

transported downwards to the groundwater reservoirs. The sorption process is therefore

counteractive to efficient biodegradation since it will decrease bioavailability (Zhang et al.,

1998). Bacterial strains that are able to degrade aromatic hydrocarbons have been repeatedly

isolated mainly from soil. These are usually gram negative bacteria (especially germs

Pseudomonas). It has been claimed that a slow sorption following the initial rapid and reversible

sorption lead to a chemical fraction that is very resistant to desorption. This phenomenon is

called aging, and the existence of such a desorption – resistant residues may increase with time

as the compound stay in the soil (Hatzinger and Alexander, 1995). PAHs have also been shown

to be partitioned or incorporated more or less reversibly into the humic substances of the soil

after partial degradation and thereby be even more immobilized in the soil (Kastner et al., 1999;

Ressler et al., 1999). They also show very low aerobic degradability depending on the

environmental conditions and the available concentration. Only two-and three-ringed

components have been shown to be degraded under anaerobic conditions with nitrate or sulphate

as the terminal electron acceptor (Mihelic and Luthy, 1988; Coates et al., 1996). Low

concentrations of bacteria have a strong influence on the biodegradation of such hydrophobic

compounds, and some studies have indicated that the process stops below a certain threshold

35

concentration (Alexander, 1985). The low mobility and persistence means that PAHs can stay in

the soil for decades, and even at sites with contamination dating at least fifty (50) years back

with 4- or 5- ringed PAHs found near the soil surface.

1.7.2. Fate of PAHs in Air and their Ecotoxicological consequences

PAHs are usually released into the air or they evaporate into the air when they are released to soil

or water. PAHs often adsorb to dust particles in the atmosphere, where they undergo photo

oxidation in the presence of sunlight, especially when they are adsorbed to particles. This

oxidation process can break down the chemicals over a period of days to weeks. Since PAHs are

generally insoluble in water, they are generally found adsorbed in particulates and precipitated in

the bottom of lakes and rivers or solubilized in any oily matter which may contaminate water,

sediments and soil. Mixed microbial populations in sediments/water systems may degrade some

PAHs over a period of weeks to months. The toxicity of PAHs to aquatic organisms is affected

by metabolism and photo-oxidation, and they are generally more toxic in the presence of

ultraviolet light. PAHs have moderate to high acute toxicity to aquatic life and birds. PAHs in

soil are unlikely to exert toxic effects on terrestrial invertebrates, except when the soil is highly

contaminated. Adverse effects on these organisms include tumors, adverse effects on

reproduction, development, and immunity. Mammals can absorb PAHs by various routes e.g.

inhalation, dermal contact, and ingestion (ATSDR, 1995).

Plants can absorb PAHs from soils through their roots and translocate them to other plant parts.

Uptake rates are generally governed by concentration, water solubility, and their

physicochemical state as well as soil type. PAH-induced phytotoxic effects are rare, however the

database on this is still limited. Certain plants contain substances that can protect against PAH

36

effects, whereas others can synthesize PAHs that act as growth hormones. PAHs are moderately

persistent in the environment and can bioacculate. The concentration of PAHs found in fish and

shellfish are expected to be much higher than in the environment from which they were taken.

Bioaccumulation has also been shown in terrestrial invertebrates, however PAH metabolism is

sufficient to prevent biomagnifications (Katarina, 2011).

1.8 Human Health Effects

1.8.1 Acute or Short-term Health Effects

The effect on human health will depend mainly on the length and route of exposure, the amount

or concentration of PAHs one is exposed to, and of course the innate toxicity of the PAHs (IPCS,

1998). A variety of other factors can also affect health impacts including subjective factors such

as pre-existing health status and age. The ability of PAHs to induce short-term health effects in

humans is not clear. Occupational exposure to high levels of pollutant mixtures containing PAHs

has resulted in symptoms such as eye irritation, nausea, vomiting, diarrhea and confusion (IPCS,

1998). However, it is not known which component of the mixture were responsible for these

effects and other compounds commonly found with PAHs may be the cause of these symptoms.

Mixtures of PAHs are also known to cause skin irritation and inflammation. Anthracene, benzo

(a) pyrene and naphthalene are direct skin irritants while anthracene and benzo (a) pyrene are

reported to be skin sensitizers (cause an allergic skin response in animals and human) (Rom,

1998). Some PAHs have low acute toxicity, other more acutely toxic agents probably cause the

acute symptoms attributed to PAHs. Hydrogen sulfide in roofing tars and sulfur dioxide in

foundries are examples of concomitant, acutely toxic contaminants. Naphthalene, the most

37

abundant constituent of coal tar, is a skin irritant, and its vapors may cause headache, nausea,

vomiting, diaphoresis (Rom, 1998).

1.8.2 Chronic or Long-term Health Effect

Health effects from chronic or long-term exposure to PAHs may include decreased immune

function, cataracts, kidney and liver damage (e.g. jaundice), and breathing problems, asthma –

like symptoms, and lung function abnormalities, whereas repeated contact with skin may induce

redness and skin inflammation (IPCS, 1998). Naphthalene, a specific PAH, can cause the

breakdown of red blood cells if inhaled or ingested in large amounts.

Many PAHs are only slightly mutagenic or even non-mutagenic in vitro. However, their

metabolites or derivatives can be potent mutagens (Gupta et al., 1991). Reported health effects

associated with chronic exposure to coal tar and its by-products (e.g. PAHs) are:

• Skin: erythema, burns, and warts on sun-exposed areas with progression to cancer. The

toxic effects of coal tar are enhanced by exposure to ultraviolet light.

• Eyes: irritation and photosensitivity

• Respiratory system: cough, bronchitis, and bronchogenic cancer.

• Gastrointestinal system: leukoplakia, buccal-pharyn-geal cancer and cancer of the lip.

• Hematopoietic system: leukemia (inconclusive) and lymphoma.

• Genitourinary system: hematuria and kidney and bladder cancers (Rom, 1998).

1.8.3 Carcinogenicity

The carcinogenicity of certain PAHs is well established in laboratory animals. Both the

International Agency for Research on Cancer (IARC, 1987) and US EPA (1994) classified a

38

number of PAHs as carcinogenic to animals and some PAH-rich mixtures as carcinogenic to

humans. The EPA has classified seven PAH compounds, as probable human carcinogens these

include, Benz (a) anthracene, Benzo (a) pyrene, Benzo (b) fluoranthene, Benzo (k) fluoranthene,

Chrysene, Dibenz (a, h) anthracene and Ideno (1,2,3-cd) pyrene.

Researchers have reported increased incidences of skin, lung, bladder, liver and stomach cancers,

as well as injection-site sarcomas, in animals (Blanton 1986, 1988). Animal studies show that

certain PAHs also can affect the hematopoietic and immune systems and can produce

reproductive, neurologic, and developmental effects (Dasgupta and Lahiri, 1992; Zhao, 1990). It

is difficult to ascribe observed health effects in epidemiological studies to specific PAHs because

most exposures are to PAH mixtures. Increased incidences of lung, skin, and bladder cancer are

associated with occupational exposure to PAHs. Epidemiologic reports of PAH-exposed workers

have noted increased incidences of skin, lung, bladder, and gastrointestinal cancer. These reports

however provide only qualitative evidence of the carcinogenic potential of PAHs in humans

because of the presence of multiple PAH compounds and other suspected carcinogens. Some of

these reports also indicate the lack of quantitative monitoring data (Hammond, et al., 1976;

Lloyd, 1971).

1.8.4 Teratogenicity

Embryotoxic effects of PAHs have been described in experimental animals exposed to PAHs

such as benzo (a) anthracene, benzo (a) pyrene, and naphthalene. The laboratory studies

conducted on mice have demonstrated that ingestion of high levels of benzo (a) pyrene during

pregnancy resulted in birth defects and decreased body weight in the offspring. It is not known

whether those effects can occur in humans. However, the centre for children’s environmental

39

health reports studies that demonstrate that exposure to PAH pollution during pregnancy is

related to adverse birth outcomes including low birth weight, premature delivery, and heart

malformations.

High prenatal exposure to PAH is also associated with lower 1Q at age three, increased

behaviourial problems at ages six and eight, and childhood asthma. Cord blood of exposed

babies shows DNA damage that has been linked to cancer. (IARC, 2010).

1.8.5 Genotoxicity

Genotoxic effects for some PAHs have been demonstrated both in rodents and in vitro tests using

mammalian (including human) cell lines. Most of the PAHs are not genotoxic by themselves and

they need to be metabolized to the diol epoxides which react with DNA, thus inducing genotoxic

damage. Genotoxicity plays important role in carcinogenicity process and may be in some forms

of developmental toxicity as well (IARC, 2010).

1.8.6 Immunotoxicity

PAHs have also been reported to suppress immune reaction in rodents. The precise mechanisms

of PAH-induced immunotoxicity are still not clear; however, it appears that immuno supression

may be involved in the mechanisms by which PAHs induce cancer (IARC, 2010).

1.8.7 Effect of PAHs on Pathogenic Change

A key factor in PAH toxicity is the formation of reactive metabolites. Not all the PAHs are of the

same toxicity because of differences in structure that affect metabolism.

Another factor to consider is the biologic effective dose, or the amount of toxics that actually

reaches the cells or target sites where interaction and adverse effects can occur. Because of solid

40

state, high molecular weight and hydrophobicity PAHs are very toxic to whole cells. CYPIAI,

the primary cytocrome P-450 isoenzyme that biologically activates benzo (a) pyrene, may be

induced by other substances (Kemena et al., 1988; Robinson et al., 1975).

The mechanism of PAH-induced carcinogenesis is believed to be via the binding of PAH

metabolites to deoxyribonucleic acid (DNA). Some parent PAHs are weak carcinogens that

require metabolism to become more potent carcinogens. Diol epoxides – PAH intermediate

metabolites – are mutagenic and affect normal cell replication when they react with DNA to

form adducts. A theory to explain the variability in the potency of different diol epoxides, “the

bay theory”, predicts that an epoxide will be highly reactive and mutagenic if it is in the “bay”

region of the PAH molecule (Jerina, 1976 and 1980; Weis, 1998). The bay region is as indicated

in Figure 3 below using the structure of Benzo(a) pyrene, Chrysene and Dibenz(a,h) anthracene

41

Figure 3: Bay region of some PAHs (The arrows indicate bay regions. The bay region is the

space between the aromatic rings of the PAH molecule).

PAH-induced carcinogenesis can result when a PAH-DNA adduct forms at a site critical to the

regulation of cell differentiation or growth. A mutation occurs during cell replication if the

aberration remains unrepaired. Cells affected most significantly by acute PAH exposure appear

to be those with rapid replicative turnover, such as those in bone marrow, skin, and lung tissue.

Tissues with slower turnover rates, such as liver tissue, are less susceptible.

12 1

11

10

9

8

7 6 5

4

3

2

Benzo (a) pyrene

12

1

11

10

9

8

7 6

5

4

3

2

13 14

Dibenz (a,h) anthracene

9

8

12 1

11

10

7 6

5

4

3

2

Chrysene

Bay Region

Bay Region

Bay Region

Bay Region

Bay Region

42

Benzo(a)pyrene diol epoxide adducts bind covalently to several guanine positions of the

bronchial epithelial cell DNA p53 gene, where cancers mutations are known to occur from

exposure to cigarette smoke. This is one possible genotoxic mechanism of cancer causation by

tobacco (Denissenko, 1996).

Persons with a high degree of CYPIAI inducibility may be more susceptible to PAH health risk.

Genetic variation in CYPIAI inducibility has been implicated as a determining factor for

susceptibility to lung and laryngeal cancer. Glutathione transferase deficiencies may result in

elevated cancer risk. Several studies have focused on breast cancer risk and metabolism of PAHs

(Ambrosone et al., 1995). Also several animal studies have implicated the ras oncogene in PAH

tumor induction (Chakravarti et al., 1995).

1.9 Fish

Fish is a rich source of lysine which is suitable for supplementing high carbohydrate diet. It is a

good source of thiamin, riboflavin, vitamins A and D, phosphorus, calcium and iron. It is high in

polyunsaturated fatty acids that are important in lowering blood cholesterol level (Al-Jediah et

al., 1999). In Nigeria, smoked fish products are the most readily form of fish product for

consumption. Out of the total of 194,000 metric tons of dry fish produced in Nigeria, about 61%

of it was smoked. One of the greatest problems affecting the fishing industry all over the world is

fish spoilage. In high ambient temperature of the tropics, fresh fish have the tendency to spoil

within 12 to 20h (Clucas, 1981). Attempt has been made to reduce fish spoilage to the minimum

through improved preservation techniques. At harvest time, fish are usually available in excess

of demand. This leads to lower market price and fish spoilage but if storage facilities are

provided, the surplus of the harvest could be stored and distributed during the off season.

43

Preservation and processing methods explore ways by which spoilage are stopped or slowed

down to give product a longer shelf life (Silva et al., 2011).

1.9.1 Food Smoking

Food smoking belongs to one of the oldest technologies of food preservation which mankind has

used in fish processing. Smoking has become a means of offering diversified, high value added

products as an additional marketing option for certain fish species where fresh consumption

becomes limited (Gomez-Estaca et al., 2009). Traditional smoking techniques involve treating of

presalted, whole or filleted fish with heat from charred wood in which smoke from incomplete

wood burning comes into direct contact with the product. This can lead to its contamination with

PAHs if the process is not adequately controlled or if very intense smoking procedures are

employed ((Gomez-Estaca et al., 2011). The smoke is produced by smouldering wood and

shavings or sawdust in the oven, directly below the hanging fish or fillets, laid out on mesh trays.

The actual level of PAHs in smoked foods depends on several variables in the heating process,

including type of smoke generator, combustion temperature and degree of smoking (Garcia-

Falcon and Simal-Gandara, 2005; Muthumbi et al., 2003). The combustion temperature during

the generation of smoke seems particularly critical and PAH is formed during incomplete

combustion processes, which occur in varying degree whenever wood, coal or oil is

burnt(Wrething et al., 2010). PAHs may be formed in three ways by high temperature (for

example, 700oc), pyrolysis of organic materials by low or moderate temperature (for example, 70

to 150 oc) and digenesis of organic material by microorganisms (Neff, 1985). The composition of

the smoke and the conditions of processing affect the sensory quality, shelf life, and

wholesomeness of the product. Potential health hazards associated with dried foods may be

44

caused by carcinogenic components of wood smoke; mainly PAHs, derivatives of PAHs such as

nitro-PAHs or oxygenated PAHs and to a lesser extent heterocyclic amines (Stolyhwo and

Sikorski, 2005). The smoke for ‘smoking of food’ develops due to the partial burning of wood,

predominantly hardwood, softwood and bagasse. Among PAHs, the Benzo (a) pyrene (BaP)

concentration has received particular attention due to its higher contribution to overall burden of

cancer in humans, being used as a marker for the occurrence and effect of carcinogenic PAHs in

food (Rey-Salguiero et al., 2009).

1.10. Rationale of the Study

Traditionally fish is smoked with firewood and charcoal to extend their shelf-life. In various

wealthy homes, the oven serves as a drying agent for preservation. From literature it is assumed

that direct exposure of fish to smoke brings about higher concentrations of polycyclic aromatic

hydrocarbons (PAHs) in the fish. This study aims at investigating the above fact against the

indirect drying methods of the sun and oven. Also whether the habit of augmenting smoke (from

firewood and charcoal) with polyethylene materials will significantly affect the levels of PAHs

deposited on the fish which is a large part of our usual daily diet. Therefore studying the various

types and levels of PAHs ingested by Nigerians from consuming smoked fish becomes

imperative.

1.11 Aim and objectives of Study

� This study is aimed at determing the levels of sixteen (16) PAHs (Acenaphthene,

Acenaphthylene, Anthracene, 1,2 Benzoanthracene, Benzo (a) Pyrene, Benzo (b)

Fluoranthene, Benzo (g,h,i) Perylene, Benzo (k) Fluoranthene, Chrysene, Dibenz (a, h)

45

anthracene, Fluoranthene, Fluorene Ideno (1,2,3-cd) Pyrene, Naphthalene, Phenantherene

and Pyrene) in fresh fish samples, dried under different heating regimes.

The specific objectives are to;

� (a) To determine the level of these PAHs in fresh water fish from Otuocha River.

� To determine the level of these PAHs in the river water sample.

� To determine the level of these PAHs in different smoking regimes:

(b) The sun

(c) The oven

(d) Charcoal

(e) Firewood

(f) Charcoal + 20g polythene material

(g) Firewood + 20g polythene material

Statistical Analysis

The data obtained from the laboratory experiment were subjected to one way analysis of variance

((ANOVA). Post Hoc test was used to separate and compare the means. Data obtained from the

groups were subjected to comparison across the different groups and differences were considered

significant at p<0.05. This analysis was estimated using computer software known as Statistical

Product and Solution Services (SPSS) version 18.

46

CHAPTER TWO

2.0. Material and Methods

2.1 Materials

2.1.1 Apparatus and Equipment

The following apparatus were used in this study which includes

Beakers Pyrex

Test-tube Pyrex

Soxhlet extractor Pyrex

Oven Samsumg

Grinder Locally Produced

Polythene Locally Produced

Charcoal Locally Available

Firewood Locally Available

Drums Locally Produced

GC-MS spectrophotometer GC-MS-QP2010 plus, Shimadzu Japan

2.1.2. Chemicals

All the chemicals used in this study were of analytical grade and in their pure forms

n-Hexane Sigma-aldrich

Dicholoromethane BDH, England

Sodium Sulphate Aldrich chemie Germany

Magnesium Silicate Aldrich chemie germany

Methanol 99.5+% BDH, England

47

Acetone 99.5+% BDH, England

PAHs solution catalog number z-013-17, LOT 213061049 Accustandard Inc, USA

200µg/ml Analyte

Acenaphthene

Acenaphthylene

Anthracene

1,2-Benzanthracene

Benzo (a) pyrene

Benzo (b) floranthene

Benzo (g,h,i)perylene

Benzo(k)fluoranthene

Chrsene

Dibenz (a,h) anthracene

Fluoranthene,

Fluorine

Indeno (1,2,3-cd)pyrene

Naphthalene

Phananthrene and

Pyrene

2.1.3 Fish Samples

The fishes used in this study were obtained from the Otuocha River in Anambra state within the

periods of October 2013, November 2013 and January 2014.

2.1.4 Study Site

Eastern Nigeria stretches from the Atlantic Ocean covering wide expanse of the forest region up

the lower boundary of the savannah forest belt. Otuocha in Anambra State is located between

48

longitude 6.85000 and latitude 6.33330. The region land mass falls within several communities

such as Ogurugu, Onitsha and Nsugbe all in Anambra State.

Fig.4: Map showing Otuocha River in Anambra State

49

2.2 Methods

2.2.1. Collection of fish Samples and Drying

The fish samples used in this study were collected during the dry season months of October to

January.

The fish samples, which was a collection of Tilapia spp., Arius heude loti and others are

predominant during the months of October to January when the dry season is in session, were

collected from Otuocha river, where relatively no explorative activity has taken place recently.

The choice of Otuocha River was to avoid all chances of pollution originating from explorative

activity (petroleum). There was no differentiation of the fish samples into different species since

the work centred on determing the level of PAHs deposited on the fish samples from the

different drying regimes. Nonetheless, the river water was collected for analysis to determine the

amount of PAHs in the river and possibly from other sources which could affect the results

obtained.

The fishes were divided into groups and the wet weight of each group was noted. These groups

are:

Group A: homonized fresh fish

Group B: Sundried fishes

Group C: Oven dried fishes

Group D: Charcoal smoked fishes

Group E: Firewood smoked fishes

50

Group F: Fishes smoked with charcoal augmented polythene material (20g)

Group G: Fishes smoked with firewood augmented of polythene material (20g)

The smoking of the fishes were for 3 days at 2 hours each day at high temperature of above

2500c.

The smoking process involved producing smoke from smouldering wood (hardwood) or charcoal

placed directly below the hanging fishes laid out on mesh trays. A piece of cardboard is placed

over the fishes as done locally to cover the fishes during the process. The piece of cardboard

traps the smoke to enable it act directly on the fish samples.

The group dried under the sun was done in Uwelu Ibeku Opi in Nsulla local Government Area

for 3 days.

The smoked and dried fishes were homogenized immediately using a very clean and dry grinder

and stored in a refrigerator at 40C prior to extraction and analysis. The extraction and analysis

followed immediately to avoid ageing.

2.2.2. Sample Preparation for the Analysis of Dried Fishes:

Soxhlet Extraction:

The sample of fishes were homogenized, minced into smaller fillets and blended using a grinder.

Twenty grams (20g) of the homogenized fish sample was thoroughly mixed with 60g of

anhydrous sodium sulphate in an agate mortar (Wang et al., 1999) to absorb moisture. The

homogenate was placed into an extraction cellulose thimble covered with a Whatman filter paper

(125mm diameter) and inserted into a soxhlet extraction chamber of the soxhlet extraction unit.

Extractions were then carried out with 200ml of n-hexane using EPA 3540C method (US EPA,

51

1994) for 8 hours. The crude extract obtained was carefully evaporated using Ribby RE 200B

rotary vacuum evaporator at 400C, just to dryness. The residue was redisolved in 5ml of n-

hexane and transferred onto a 10ml florisil column for clean up.

2.2.3. Preparation of Florisil for Clean-up:

This clean-up step to remove more polar substances was performed using activated florisil

(Magnessium silicate) and anhydrous Na2SO4. The florisil was heated in an oven at 1300c

overnight and transferred to a 250ml size beaker and placed in a desicator.

Anhydrous Na2SO4 (1.0g) was added to 2.0g of activated florisil (60-100mm mesh) on a 10ml

column which was plugged with glass wool. The packed column was filled with 5ml n-haxane

for conditioning.

The stopcock on the set-up was opened to allow the n-hexane run out until n-hexane just reached

the top of the sodium sulphate into a receiving vessel whilst taping gently the top of the column

till the florisil settled well in the column. The extract was then transferred onto the column with a

disposable Pasteur pipette from an evaporating flask. The crude extract was eluted on the column

with the wide opening of the stopcock. Each evaporating flask was immediately rinsed twice

with 1ml n-hexane and added to the column by the use of the Pasteur pipette. The eluate was

collected into an evaporating flask and rotary evaporated to dryness. The dry eluate was then

dissolved in 1ml n-hexane for Gas chromatographic analysis.

52

2.2.4. Instrumental Analysis

Gases used are Helium and Hydrogen gases. Hydrogen and Helium with a purity of 99.999%

were used as carrier gas at a constant flow of 30 and 300ml/min respectively. The determination

of PAHs was performed on the samples and standards using a Buck 901 GC-FID equipped with

a split /split-less injection port. 1.0g of extracted samples were dissolved in10ml n-hexane. Some

quantity were taken into 2ml chromatographic vial and made up to 2ml with toluene, injected

and separated on a Restek chrompack capillary column CP5860 with 95% methyl and 5%

phenylpolysiloxane phase, (oven max. temperature 3500C).

WCOT fused silica, 30m X 0.25mm id and 0.25µm film thickness with CP-sil 8 CB low

bleeds/MS coating. Carrier gas was helium 26cm sec. Temperature profile during the

chromatographic analysis was 500C for 3minutes, 80C/min to 3200C hold for 15 minutes and

detector at 3200C. Fixed setting: Generally the operator must adjust gas flows to the column, the

inlets, the detectors, and the split ratio. In addition, the injection and detector temperature must

be set. The detectors are generally held at the high end of the oven temperature range to

minimize the risk of analyte precipitation (Annual Book of ASTM standards, 2005). All of these

parameters should have been set to the correct values. A double check was done on all the

instrument: Agilent 6890 Gas chromatograph equipped with an on-column, automatic injector,

flame ionization detector, HP 88 capillary column (100m X 0.25 µm film thickness) CA, USA.

Detector Temp: 2500c

Injector Temp: 220c

Integrator Chart Speed: 2cm/min

53

Temperature Condition.

Table 4: Temperature condition of GC-MS

Initial Temp Hold Ramp Final Temp

700c 5min 10min 2200c

2200c 2min 5min 2800c

When the instrument is ready, the “NOT READY” light turns off, and the run begins. Then 1µL

sample was injected into column A using proper injector technique (US. EPA, 2003).

54

CHAPTER THREE

RESULTS

Fishes were caught with nets in October – November 2013 and in January 2014 at different

locations in Otuocha River (a strip of about 230 meters). The fishes were divided into six (6)

groups and then the control. Each group has about 5 or more fishes.

Table 5 shows the various groups and the corresponding weights before and after drying for the

three months of the study. The fishes were ascertained dry when a constant weight persists for

some period. The drying in each case was for 2hrs each day and lasted for 3 days

55

Table 5: weight of fishes used in this study in the months of October, November and January

Variables October November January

Wet

wt.

Dry

wt.

% water

loss

Mean +SD Wet

wt.

Dry

wt.

%

water

loss

Mean

+SD

Wet

wt.

Dry

wt.

%

water

loss

Mean +SD

Fresh fish 134 150 150

Firewood 245 53.10 78.30 78.30+0.01 300 61.00 79.60 79.65+0.1 270 57.5 78.7 78.71+0.01

Charcoal 250.30 55.00 78 78.05+0.1 270.50 58 78.50 78.55+0.1 240.2 50.8 78.9 78.82+0.1

Sunlight 211.90 47.60 77.50 77.55+0.10 230 50 78.30 78.35+0.1 235 50.7 78.4 78.42+0.02

Oven 200.30 45.30 77.40 77.55+0.10 220.40 47.20 78.60 78.65+0.1 240.2 51.2 78.8 78.60+0.01

Firewood + polythene

(20g)

233.70 50.60 78.30 78.35+0.10 260 56.20 77.60 77.65+0.1 240 50.8 78.8 78.81+0.01

Charcoal + polythene

(20g)

220.40 49.50 77.50 77.63+0.01 240.70 51.00 78.80 78.85+0.1 240.5 51.0 78.8 78.82+0.02

Total 77.90+0.3 78.61+0.6 78.69+0.2

56

Table5 shows the wet and dry weights of the fishes collected in the three months. In the month of

October 2013, the percentage of water removed varied a little with changes in the drying regime.

With the firewood dried, charcoal dried and firewood+20g polythene having the highest water

removal of 78.3%, 78.0% and 78.3% respectively. In November, percentage water loss was

lowest (77.6%) in the firewood + polythene (20g) fish sample while the highest percentage water

loss was observed in the fish sample dried with firewood.

In January 2014, the percentage water loss in the different drying regimes was fairly constant but

varied between 78.4 to 78.9. The average percentage water lost in the three months are 77.8% for

October, 78.6% for November and 78.7% for January.

From the statistical analysis, it was observed that p>0.05 which indicates that there is no

significant difference in the percentage water loss of the various drying regimes employed in

drying the fishes.

The water samples were taken from the same stretch of about 230 meters of the river (where the

fishes were caught) at 50meter intervals and at a depth of 5-10ft. The water samples were made

into a composite mixture of 400ml before analysis.

The GC-MS results of the levels of PAHs in the river water and the fish samples in the 3 months

are presented in tables 6, 7, 8 and 9.

57

Table 6: GC –MS Result of Fish Samples in October 2013 (µg/g)

Component Sundried Charcoal

dried

Firewood River Water

Sample

Oven dried Fresh fish Charcoal +20g

Polythene

Firewood+ 20g

Polythene

Acenaphthene ND ND ND ND ND ND ND ND

Acenaphthylene ND ND 0.5 ND ND ND ND ND

Anthracene ND ND ND ND ND ND ND 10.0

1,2 Benzanthracene ND ND 114.3 ND 38.0 ND ND 16.2

Benzo (a) pyrene 1.2 ND 64.4 ND ND ND 2.4 ND

Benzo (b)

fluoranthene

ND ND 2.6 ND ND ND ND 404.3

Benzo (g,h,i) pyrene ND ND 1.6 ND ND ND ND ND

Benzo (k)

flouranthene

27.0 40.4 ND 2.0 6.2 4.5 46.2 134.8

Chrysene ND ND 0.3 ND ND 0.65 ND ND

Dibenz (a,h)

anthracene

ND ND ND ND ND ND 9.0 ND

Fluoranthene ND ND ND ND ND ND ND 17.7

Indeno (1,2,3-cd)

pyrene

ND ND ND ND ND ND 68.0 4.4

Naphthalene 7.0 39.0 3.3 1.0 1.0 ND 39.1 2.3

Phananthrene ND ND ND ND ND ND ND ND

Pyrene ND ND ND ND ND ND ND 94.8

Fluorene ND ND ND ND ND ND ND 10.8

58

Table 7: GC –MS Result of Fish Samples in November 2013 (µg/g)


dried


Sample

Oven Dried Fresh fish Charcoal +20g

Polythene

Firewood+ 20g

Polythene






Benzo (b)

fluoranthene



Benzo (k)

flouranthene

27.2 40.3 ND 1.96 6.5 4.3 46.1 135.0


Dibenz (a,h)

anthracene




Indeno (1,2,3-cd)

pyrene


Naphthalene 7.1 39.1 3.5 0.93 1.2 ND 39.1 2.5



59

Table 8: GC –MS Result of Fish Samples in January 2014 (µg/g)


dried


Sample

Oven Dried Fresh fish Charcoal +20g

Polythene

Firewood+ 20g

Polythene






Benzo (b)

fluoranthene



Benzo (k)

flouranthene

27.4 40.5 ND 1.87 6.83 4.1 46.1 135.2


Dibenz (a,h)

anthracene




Indeno (1,2,3-cd)

pyrene


Naphthalene 7.3 39.3 3.7 0.84 1.4 ND 39.0 2.7



60

Table 9: statistical mean Values of GC-MS results of the three months

Component

Sundried

Sample

Charcoal

Dried

Firewood

Dried

Water (River)

Sample

Oven Dried Fresh Fish Charcoal Dried +

20g polythene

Firewood dried

+ 20g

polythene


Acenaphthylene ND ND 0.6+0.1 ND ND ND ND ND

Anthracene ND ND ND ND ND ND ND 9.9+0.1

1,2

Benzanthracene

ND ND 114.5+0.2 ND 40.0+0.2 ND ND 16.4+0.2

Benzo (a)

Pyrene

1.4+0.2

ND 64.6+0.2 ND ND ND 2.4+0.1 ND

Benzo (b)

Fluoranthene

ND ND 2.8+0.2 ND ND ND ND 404.5+0.2

Benzo (g,h,i)

Perylene

ND ND 1.8+0.2 ND ND ND ND ND

Benzo (k)

Fluoranthene

27.2+0.2 40.4+0.0001 ND 1.94+0.1 6.5+0.3 4.3+0.2 46.1+0.1 135.0+0.2

Chrysene ND ND 0.3+0.1 ND ND 0.67+0.1 ND ND

Dibenz (a,h)

Anthracene

ND ND ND ND ND ND 9.6+0.1 ND

Fluoranthene ND ND ND ND ND ND ND 17.5+0.2

Fluorene ND ND ND ND ND ND ND 11.0+0.2

Indeno (1,2,3, -

cd) Pyrene

ND ND ND ND ND ND 69.0+0.1 4.5+0.2

Naphthalene 7.1+0.2 39.13+0.2 3.5+0.4 0.92+0.1 1.2+0.2 ND 39.1+0.1 2.5+0.1


Pyrene ND ND ND ND ND ND ND 94.9+0.1

Total PAHs 35.7+0.2 79.53+0.2 188.1+0.2 2.86+0.1 47.7+0.2 4.97+0.2 166.2+0.1 696.3+0.2

ND = Not detected;

85

Table 9 above shows the statistical mean values of the PAH components of the three

months. The river water samples revealed the presence of Naphthalene (0.92+ 0.1

µg/g) and Benzo (k) fluoranthene (1.94+ 0.1 µg/g). The total PAH are 2.86+ 0.1 µg/g.

The other PAHs were not detected.

The fresh fish samples revealed the presence of Benzo (k) fluoranthene (4.3+ 0.2

µg/g) and Chrysene (0.67+ 0.1 µg/g) in the fresh fish samples in the months of

October, November and January. The total PAHs content of the Fresh fish samples

were (4.97µg/g). The other PAHs probably not in detectable levels.

The Sundried fish samples revealed the presence of Benzo (a) pyrene (1.4+ 0.2 µg/g)

Benzo (k) fluoranthene (27.2+ 0.2 µg/g) and Naphthalene (7.1+ 0.2 µg/g). The total

PAHs content is 35.7+ 0.2 µg/g. The other PAHs were not detected.

The oven dried fish samples revealed the presence of Naphthalene (1.2+ 0.2 µg/g),

1,2 Benzanthracene (40.0+ 0.2 µg/g) and Benzo (k) fluoranthene (6.5+ 0.3 µg/g). The

total PAH content is 47.7+ 0.2 µg/g. The other PAHs were not detected.

The charcoal dried fish samples revealed the presence of Naphthalene (39.13+

0.2µg/g) and Benzo (k) fluoranthene (40.4+ 0.1 µg/g) respectively. The total PAH

content is 79.53+ 0.2µg/g. The other PAHs were not detected.

The firewood dried fish samples shows the presence of naphthalene (3.5+ 0.2 µg/g),

acenaphthylene (0.6 + 0.1 µg/g), 1,2 benzanthracene (114.5.2 µg/g) chrysene (0.30 +

0.1 µg/g), benzo (b) fluoranthene (2.8+ 0.2 µg/g), benzo (a) pyrene (64.6+ 0.2 µg/g)

and benzo (g,h,i) perylene (1.8+ 0.2 µg/g) respectively. The total PAH content is

188.1+ 0.2 µg/g.. The other PAHs were not detected.

86

The charcoal + 20g polythene dried fish samples revealed the presence of naphthalene

(39.1+ 0.1µg/g), benzo (a) pyrene (2.4+ 0.1µg/g), benzo (k) fluoranthene (46.1+

0.1µg/g), dibenz (a,h) anthracene (9.6+ 0.1µg/g) and indeno (1,2,3-cd) pyrene (69.0+

0.1µg/g) respectively. The total PAH content is 166.2+ 0.1 µg/g. The other PAHs

were not detected.

The firewood + 20g polythene dried fish samples shows the presence of naphthalene

(2.5+ 0.1 µg/g), fluorene (11.0 + 0.2 µg/g), anthracene (9.9 + 0.1 µg/g) fluoranthene

(17.5 + 0.2 µg/g), Pyrene (94.9+ 0.1 µg/g), 1,2 benzanthracene (164+ 0.2 µg/g)

benzo (b) fluoranthene (404.5+ 0.2 µg/g), benzo (k) fluoranthene (135.0+ 0.2

µg/g)and indeno (1,2,3-cd) pyrene (4.5+ 0.2 µg/g) respectively. The total PAH

content is 696.3+ 0.1 µg/g. The other PAHs were not detected.

The GC-MS chromatograms are shown in appendix I-VIII.

87

0.000.100.200.300.400.500.60

µg/m

l

Treatment

AcenaphthyleneOct

0.000.100.200.300.400.500.600.70

µg/m

l

Treatment

Acenaphthylene

0.000.100.200.300.400.500.600.700.80

µg/m

l

Treatment

Acenaphthylene Jan

Fig 5: Monthly distribution of Acenaphthylene in various treatments

Nov

Monthly distribution of individual PAHS in various treatments

treatments

88

0.00

2.00

4.00

6.00

8.00

10.00

12.00µ

g/m

l

Treatment

Anthracene Oct

0.00

2.00

4.00

6.00

8.00

10.00

12.00

µg/m

l

Treatment

Anthracene Nov

Fig 5 is the distribution of Acenaphthylene in the months of October, November 2013

and January 2014. Acenaphthylene was detected in the fish sample dried with

firewood only. The component was not detected in the other types of treatment.

89

0.000

2.000

4.000

6.000

8.000

10.000

12.000

µg/m

l

Treatment

Anthracene

0.00

20.00

40.00

60.00

80.00

100.00

120.00

140.00

µg/m

l

Treatment

1,2 Benzanthracene oct

Fig 6: month

Fig 6: monthly distribution of Anthracene in the various treatments.

Fig 6 is the distribution of Anthracene in the months of October, November2013 and

January 2014. Anthracene was detected in the fish sample dried with firewood + 20g

polythene. The component was not detected in the other types of treatment.

Jan

90

0.0020.0040.0060.0080.00

100.00120.00140.00

µg/m

l

Treatment

1,2 Benzanthracene

0.0020.0040.0060.0080.00

100.00120.00140.00

µg/m

l

Treatment

1,2 Benzanthracene

Fig7: Monthly distribution of 1,2 Benzanthracene in the various treatments.

Fig7 is the distribution of 1,2 benzanthracene in the months of October, November

2013 and January 2014. 1,2 benzabthracene was detected in the fish sample dried

with firewood, oven and firewood +20g polythene. In the three months under

consideration, the concentration of 1,2 benzanthracene was highest in firewood dried

sample, followed by that dried with oven and least in the fish sample dried using

Nov

Jan

91

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

µg/m

l

Treatment

Benzo (a) pyrene Oct

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

µg/m

l

Treatment

Benzo (a) pyreneNov

firewood +20g polythene. The component was not detected in the other types of

treatment.

92

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

µg/m

l

Treatment

Benzo (a) pyreneJan

0.0050.00

100.00150.00200.00250.00300.00350.00400.00450.00

µg/m

l

Treatment

Benzo(b)fluorantheneOct

Fig8 represents the distribution of Benzo(a)pyrene in the months of October,

November2013 and January 2014. Benzo(a)pyrene was detected in fish samples dried

with firewood, charcoal+20gpolythene and the sun. in the three months considered,

the concentration of Benzo(a)pyrene was highest in firewood dried sample, followed

by the charcoal+20gpolythene sample and least in the sun dried sample. The

component was not detected in the other types of treatment.

Fig8: Monthly distribution of Benzo(a)pyrene in the various treatments.

93

0.0050.00

100.00150.00200.00250.00300.00350.00400.00450.00

µg/m

l

Treatment

Benzo(b)fluoranthene

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

µg/m

l

Treatment

Benzo(b)fluoranthene

Fig9 is the distribution of benzo(b)fluoranthene in the months of October, November

2013 and January 2014. Benzo(b)fluoranthene was detected only in the fish samples

Nov

Jan

Fig9: monthly distribution of Benzo(b)fluoranthene in the various treatments

94

0.000.200.400.600.801.001.201.401.601.80

µg/m

l

Treatment

Benzo (g,h,i) perylene Oct

0.000.200.400.600.801.001.201.401.601.802.00

µg/m

l

Treatment

Benzo (g,h,i) perylene Nov

dried with firewood+20gpolythene. The component was not detected in the other

treatments

95

0.00

0.50

1.00

1.50

2.00

2.50

µg/m

l

Treatment

Benzo (g,h,i) peryleneJan

0.0020.0040.0060.0080.00

100.00120.00140.00160.00

µg/m

l

Treatment

Benzo(k) flourantheneOct

Fig10 represents the distribution of Benzo(g,h,i)perylene in the months of October,

November 2013 and January 2014. Benzo(g,h,i)perylene was detected only in the fish

samples dried with firewood. The component was not detected in the other

treatments.

Fig10: monthly distribution of Benzo(g,h,i)perylene in the various treatments.

96

0.0020.0040.0060.0080.00

100.00120.00140.00160.00

µg/m

l

Treatment

Benzo(k) flourantheneNov

0.0020.0040.0060.0080.00

100.00120.00140.00160.00

µg/m

l;

Treatment

Benzo(k) flourantheneJan

Fig11 represents the distribution of Benzo(k)fluoranthene in the months of October,

November 2013 and January 2014. Benzo(k)fluoranthene was detected in all the

various treatments except in the fishes dried with firewood. While the concentration

of Benzo(k)fluoranthene was highest in the fish dried with firewood+20g polythene,

it was least in the river water sample.

Fig11: monthly distribution of Benzo(k)fluoranthene in the various treatments.

97

0.000.100.200.300.400.500.600.700.80

µg/m

l

Treatment

Chrysene Oct

0.000.100.200.300.400.500.600.700.80

µg/m

l

Treatment

ChryseneNov

98

0.000.100.200.300.400.500.600.700.80

µg/m

l

Treatment

Chrysene Jan

0.001.002.003.004.005.006.007.008.009.00

10.00

µg/m

l

Treatment

Dibenz(a,h)anthracene Oct

Fig12 represents the distribution of chrysene in the months of October, November

2013 and January 2014. Chrysene was detected in fresh fish and fishes dried using

firewood. The concentration of chrysene was higher in the fresh fish than in the

firewood dried fish sample. The component was not detected in the other treatments.

Fig12: Monthly distribution of chrysene in the various treatments.

99

0.00

0.20

0.40

0.60

0.80

1.00

1.20

µg/m

l

Treatment

Dibenz(a,h)anthracene Nov

0.00

2.00

4.00

6.00

8.00

10.00

12.00

µg/m

l

Treatment

Dibenz(a,h)anthraceneJan

Fig13 represents the distribution of Dibenz(a,h)anthracene in the months of October,

November 2013 and January 2014. Dibenz(a,h)anthracene was detected in fish dried

with charcoal+20g polythene only. The component was not detected in other

treatments.

Fig13: Monthly distribution of Dibenz(a,h)anthracene in the various treatments.

100

0.002.004.006.008.00

10.0012.0014.0016.0018.0020.00

µg/m

l

Treatment

FluorantheneOct

0.002.004.006.008.00

10.0012.0014.0016.0018.0020.00

µg/m

l

Treatment

Fluoranthene Nov

101

0.002.004.006.008.00

10.0012.0014.0016.0018.0020.00

Axµ

g/m

l

Treatment

FluorantheneJan

0.00

2.00

4.00

6.00

8.00

10.00

12.00

µg/m

l

Treatment

FloureneOct

Fig14 is the distribution of Fluoranthene in the months of October, November 2013

and January 2014. Fluoranthene was detected in fish dried with firewood +20g

polythene only. The component was not detected in other treatments.

Fig14: Monthly distribution of Fluoranthene in the various treatments.

102

0.00

2.00

4.00

6.00

8.00

10.00

12.00

µg/m

l

Treatment

FluoreneNov

0.00002.00004.00006.00008.0000

10.000012.000014.000016.000018.0000

µg/m

l

Treatment

Fluorene

Fig15 represents the distribution of fluorine in the months of October, November

2013 and January 2014. Fluorine was detected in the fish dried with firewood +20g

polythene only. The components was not detected in other treatments.

Fig15: Monthly distribution of Fluorene in the various treatments.

103

0.0010.0020.0030.0040.0050.0060.0070.0080.00

µg/m

l

Treatment

Indeno(1,2,3-cd)pyreneOct

0.0010.0020.0030.0040.0050.0060.0070.0080.00

µg/m

l

Treatment

Indeno(1,2,3-cd)pyrene

Nov

104

0.0010.0020.0030.0040.0050.0060.0070.0080.00

µg/m

l

Treatment

Indeno(1,2,3-cd)pyreneJan

Fig16 represents the distribution of Indeno(1,2,3-cd)pyrene in the months of October,

November 2013 and January 2014. Indeno(1,2,3-cd)pyrene was detected in the fish

dried with charcoal +20g polythene and that of firewood +20g polythene. The

concentration of Indeno(1,2,3-cd)pyrene was higher in the charcoal +20g polythene

dried fish. The component was not detected in other treatments.

Fig16: Monthly distribution of Indeno(1,2,3-cd)pyrene in the various treatments.

105

0.005.00

10.0015.0020.0025.0030.0035.0040.0045.00

µg/m

l

Treatment

NaphthaleneOct

0.005.00

10.0015.0020.0025.0030.0035.0040.0045.00

µg/m

l

Treatment

NaphthaleneNov

0.005.00

10.0015.0020.0025.0030.0035.0040.0045.00

µg/m

l

Treatment

Naphthalene

Fig17: Monthly distribution of Naphthalene in the various treatments.

106

0.0010.0020.0030.0040.0050.0060.0070.0080.0090.00

100.00

µg/m

l

Treatment

pyreneOct

0.0010.0020.0030.0040.0050.0060.0070.0080.0090.00

100.00

µg/m

l

Treatment

PyreneNov

Fig17 represents the distribution of Naphthalene in the months of October, November

2013 and January 2014. Naphthalene was detected in all the treatments except that of

fresh fish. While the concentration of Naphthalene detected in the fish dried with

charcoal +20g polythene was highest, that detected in the river water sample was the

lowest.

107

0.0010.0020.0030.0040.0050.0060.0070.0080.0090.00

100.00

µg/m

l

Treatment

PyreneJan

Fig18 is the distribution of Pyrene in the months of October, November 2013 and

January 2014. Pyrene was detected only in the fish sample dried with firewood +20g

polythene. The component was not detected in other treatments.

From statistical analysis PAH component in October Acenaphthylene correlated

negatively with anthracene, benzo(b)fluoranthene, benzo(k)fluoranthene,

dibenz(a,h)anthracene, fluoranthene, fluorine, indeno(1,2,3-cd)pyrene, naphthalene

and pyrene while it correlated positively with 1,2-benzanthracene, benzo(a)pyrene,

benzo(g,h,i)perylene and chrysene. This pattern of correlation occurred similarly in

November but differed slightly in January 2014 because Acenaphthylene correlated

negatively with chrysene and positively with benzo(b)fluoranthene.

Fig18: monthly distribution of Pyrene in the various treatments.

108

PAH component, anthracene constantly correlated positively with

benzo(k)fluoranthene, fluoranthene and pyrene but varied between negative and

positive for benzo(b)fluoranthene, fluorine and naphthalene for the three months in

consideration. For the other components anthracene correlated negatively in the three

months.

PAH component, 1,2 benzothracene correlated positively with Acenaphthylene,

benzo(a)pyrene, benzo(g,h,i)perylene and chrysene in the months of october and

November 2013 but varied slightly in January 2014 when chrysene became negative

and benzo(b)fluoranthene correlated positively. The other PAH components

correlated negatively in the three months under consideration.

PAH component, benzo(a)pyrene in the three months considered correlated positively

with Acenaphthylene, 1,2 benzanthracene and benzo(g,h,i) perylene while chrysene

which was positively in October and November 2013 correlated negatively in January

2014. When benzo(b)fluoranthene became positive. The other PAH components

correlated negatively in the three months.

PAH component, benzo(b)fluoranthene in October correlated positively with

anthracene, benzo(k)fluoranthene, fluoranthene, naphthalene and pyrene, in

November. It positively correlated with anthracene, benzo(k)fluoranthene,

fluoranthene, flourene and pyrene while in January it positively correlated with

Acenaphthylene, 1,2 benzanthracene, benzo(a)pyrene and benzo(g,h,I,)perylene. The

other PAH components correlated negatively.

109

PAH component, benzo(g,h,i)perylene in the three months correlated positively with

Acenaphthylene, 1,2 benzanthracene, benzo(a)pyrene and benzo(b)fluoranthene

(January only), and negatively with the rest of the PAH components.

PAH component, benzo(k)fluoranthene in the three months considered correlated

negatively with Acenaphthylene, 1,2 benzanthracene, benzo(a)pyren,

benzo(g,h,i)perylene, chrysene and dibenz(a,h)anthracene and indeno(1,2,3-cd)pyrene

(in November only) while it correlated positively with the PAH components.

PAH component, chrysene in the three months correlated positively with

Acenaphthylene, 1,2 benzanthracene, benzo(a)pyrene and benzo(g,h,i)perylene for

the months of october and November and naphthalene only in January. The remaining

PAH components correlated negatively with chrysene.

PAH component, dibenz(a,h)anthracene correlated positively in the three months as

follows – benzo(k)fluoranthene, fluorine and indeno (1,2,3-CD)pyrene for October,

naphthalene and indeno (1,2,3-cd)pyrene and naphthalene was positive for January

2014. The remaining PAHs correlated negatively.

PAH component, fluoranthene in the three months correlated positivelt with

antracene, benzo(b)fluoranthene and pyrene for October and November. Fluorine

(November and january), pyrene, anthracene and benzo(k) fluoranthene correlated

positively in January. The remaining PAHs have negative correlation with

fluoranthene.

PAH component, fluorine correlated positively in the three months as follows:

Benzo(k)fluoranthene, dibenz(a,h)anthracene and indeno (1,2,3-cd)pyrene in October,

110

anthracene, benzo(b)fluoranthene, benzo(k)fluoranthene, pyrene and fluoranthene in

November and anthracene, benzo(k)fluoranthene, fluorine there and pyrene in

January 2014. The other PAH components were negatively correlated.

PAH component, indeno (1,2,3-cd)pyrene correlated positively on the three months

as follows: Benzo(k)fluoranthene, dibenz(a,h)anthracene and fluorine in October,

dibenz(a,h)anthracene and naphthalene in November and benzo(k)fluoranthene,

dibenz(a,h)anthracerne and naphthalene in January 2014. The other PAHs correlated

negatively.

PAH component, Naphthalene correlated positively in the three months as follows:

anthracene, benzo(b)fluoranthene, benzo(k)fluoranthene, flouranthene and pyrene in

October, benzo(k)fluoranthene, dibenz(a,h)anthracene and indeno(1,2,3-cd)pyrene in

November and benzo(k)fluoranthene, chrysene, dibenz(a,h)anthracene and

indeno(1,2,3-cd)pyrene in January. The other PAH components were negatively

correlated.

PAH components, pyrene correlated positively in the three months as follows:

anthracene, benzo(b)fluoranthene, benzo(k)fluoranthene, fluoranthene and

naphthalene in October, anthracene, benzo(b)fluoranthene, benzo(k)fluoranthene,

fluoranthene and fluorine in November and anthracene, benzo(k)fluoranthene,

fluoranthene and fluorine in january. The other PAH components correlated

negatively with pyrene.

111

CHAPTER FOUR

4.0. DISCUSSION

The emphasis in this work is to determine the level of polycyclic aromatic

hydrocarbons (PAHs) in fresh water fish dried under different drying agents. After

the drying an average 78% body weight of the fishes were lost as water during the

various regimes used in drying the fish samples.

PAHs were detected in the river water sample from Otuocha in Anambra State. The

concentration of these PAHs is well above the US EPA maximum contaminant level

(0.1-0.4 µg/ml) in water and appeared consistently throughout the period of the work

indicating that the source of contamination was the water or sediment. The increased

total PAHs content of the fresh fish sample (the control) could have been due to

bioaccumulation in the fishes from the river. This level is below the maximum

permissible level of 10µg/kg for total PAHs, but should be checked immediately

since it could also rise to dangerous proportions and affect the health of the fishes and

its consumers. The review of Katarina, (2011) reports that the concentration of PAHs

found in fish and shellfish are expected to be much higher than in the environment

from which they were taken thus confirming the above difference in total PAH

concentration. Chrysene detected in the fresh fish but not in the river suggest there

could be another source or that the fishes could have migrated from any nearby water

body containing chrysene.

The Sun and Oven drying methods are the two non-smoking regimes used in this

work. The total concentration in the sun dried fish sample was higher than that of the

112

fresh fish sample (positive control) thus suggesting that the difference in PAH

concentration could have come from the environment where the fishes were sundried.

The oven dried fish samples had more PAHs than the fresh fish sample (control)

possibly due to the water lost during the use of the oven which could have

concentrated the PAHs in the fish relative to the weight (dry weight). Therefore since

more intense heat was involved in the oven, more PAHs were present. This confirms

the work of Lu et al., (2009) which reported that PAH emissions increased with

increasing temperature from 200 to 7000c. The above two (2) regimes has more total

PAHs concentrations than the control (fresh fish) which is in line with the work of

Agerstad and Skog (2005) which reported that cooking and food processing at high

temperature have been shown to generate various kinds of genotoxic substances, or

cooking toxicants, including PAHs. Naphthalene and Benzo(a)pyrene detected in the

sundried fish but was absent in the control sample could have come from the air

where the fishes were sundried. This confirms that certain PAHs especially the low

molecular weight PAHs are air-borne and that PAHs can be present in both

particulate and gaseous phases, depending on their volatility.

The charcoal and firewood are the most ancient and prevalent methods of both

cooking and drying foods in the African traditional setting. They involve the use of

smoke from the charcoal and firewood to cook and dry the foods. In this work the

charcoal and firewood are from the local hardwood called oil bean tree (Pentaclethra

macrophylla). Pentaclethra macrophylla wood is a multipurpose tree from Africa

with potential for agroforestry in the tropics. It is highly suitable for fuel wood and

charcoal making (Ladipo et al., 1993). The total PAHs concentration from the

113

charcoal dried fish samples are much lower than that of the firewood dried fish

samples. Thus suggesting that the smoke from the firewood has much more PAH

components than that of charcoal. This confirms the report of Peter et al., (2003) that

plants can absorb PAHs through their roots and translocate them to other plant parts.

These are released onto food as they are burnt in the fire. The charcoal having

undergone burning may have lost most of the PAHs originally in it, thus the

difference in PAH concentration between the two methods. From the result two (2)

PAH components were detected from the charcoal dried fish sample whereas seven

(7) were detected from the firewood dried sample. This could only be from the wood

smoke which has more PAH component than the smoke from charcoal. To further

confirm this the work of Silva et al., (2011) showed that smoked fish samples

processed by charcoal gave the lowest level of Total PAHs, followed by firewood

method, while the sawdust method gave the highest level of total PAHs in the smoked

fishes.

The act of augmenting fire is an age long habit in Africa and is seen in the use of

various chaffs (e.g Palm kernel chaffs, coconut and rice huscs and so on) and other

light materials (paper, sawdust, cellophone and plastics) to ignite fire especially

during rainy seasons when wood or charcoal is wet or damp. The use of 20g of

polythene material (pure water sachets) to augment the charcoal and firewood used in

drying the fish samples in this work significantly increased the total PAH

concentrations and the Total carcinogenic PAH concentrations. These significant

increases could only be due to the augmentations with polythene materials during the

drying regimes. There were not only increases in the individual PAHs as a result of

114

the augmentation with polythene but the introduction of other PAHs onto the fish

samples which could only have come from the polythene materials. The effect of

augmentation as seen from the results is to generally increase the concentrations of

Total PAHs in the fish sample. Some of the PAHs increased or introduced by

argumentation with polythene (or tire and plastics) are classified by US EPA as

probable human carcinogens (Benzoanthracene, Benzo(a)pyrene,

Benzo(k)fluoranthene, ideno(1,2,3-cd)pyrene). This unwholesome practice is

widespread as it is not only limited to fishes but can also be seen practiced by food

vendors who use polythene and so on to augment firewood or charcoal while roasting

corn, yams, plantain or meat (suya) that we consume daily. Added to the above route

of exposure which is by ingestion are other routes such as inhalation and dermal

contacts which some workers (mechanics, printers, carpenters, farmers, roofers,

aluminum workers and so on) are daily exposed to. These long term (chronic)

exposure to PAHs may lead to decreased immune function, skin inflammations,

cataracts, kidney and liver damage, breathing problems and lung function

abnormalities in these individuals. Though the human body (liver, kidney and lungs)

by metabolism renders PAHs more water soluble and removes them via the bile and

urine, accumulation of PAHs in the body (adipose tissue) could lead to biological

effective dose that could cause pathogenic changes (carcinogenicity, genotoxicity and

so on) in the individuals.

Based on the results from the different drying regimes used in this study, the safest

method of drying fish is the Oven method. But due to cost and maintenance, the Oven

may not be affordable for the average African home. Thus the charcoal method

115

because it is next in safety is recommended and moreso it is cheap and affordable.

The act of augmentation of charcoal or firewood smoke with polythene or plastic

materials should be discouraged out rightly since from the results obtained in this

work, these PAHs could build-up to very dangerous proportions (above the maximum

permissible level of 10 and 1µg/kg for total PAHS and BaP) in the human body.

Conclusion

Polycyclic aromatic hydrocarbon (PAH) components detected in Otuocha river,

though of low concentration, indicate a regular source of pollution entering into the

river which if not stopped immediately could increase to dangerous proportions

especially in the fishes and this could affect the people consuming these fishes from

the river.

For the drying regimes, in which the levels of PAHs were significantly higher than

that of sun-dried, it can be concluded that the excessive PAHs in the body of the dried

fish were from the “burning” or drying agents. More significantly are the observed

very high increase in PAHs when drying was augmented with polythene, an agent

known to be a high source of PAHs when incinerated. These proportions may not

only lead to various forms of cancerous growths but could affect the eyes (irritation

116

and photosensitivity), respiratory system (Bronchitis), Gastrointestinal system

(Leukoplakia), Hematopoietic system (Leukemia) and hematuria in the Genitourinary

system. Consumers of dried fish should therefore beware of the dried fish they

purchase from the local market.

Further research is indeed needful regarding:

1. The mutagenic and carcinogenic effects from chronic exposure to PAHs and

metabolites.

2. Proper classification of the PAH compounds which are pathogenic and those

which are not.

117

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126

Appendix I

PAH Composition of River Water Samples in µg/ml

Naphthalene 0.92µg/ml + 0.1

Benzo (k) flouranthene 1.94µg/ml + 0.1

Total PAHs 2.86µg/ml + 0.1

127

Appendix II

PAH Composition of Fresh Fish Samples in µg/g

Chrysene 0.67 + 0.1µg/g

Benzo (k) fluoranthene 4.3 + 0.2µg/g

Total PAHs 4.97 + 0.1µg/g

128

Appendix III

PAHs Composition of Sundried Fish Samples (µg/g)

Naphthalene 7.1 + 0.2µg/g


Benzo (a) pyrene 1.4 + 0.2µg/g


129

Appendix IV

PAHs composition of Oven Dried Fish Samples

Naphthalene 1.2 + 0.2µg/g

1,2 Benzathracene 40.0 + 0.2µg/g


Total PAHs 47.7+ 0.2µg/g

130

Appendix V

PAHs Composition of Charcoal Dried Fish Sample

Naphthalene 39.13+ 0.2µg/g

Benzo (k) fluoranthene 40.4+ 0.1µg/g


131

Appendix VI

PAHs Composition of Charcoal + 20g Polythene Dried Fish Sample

Benzo (a) pyrene 2.4 + 0.1µg/g


Dibenz (a,h) anthracene 9.6 + 0.1µg/g

Indenol (1,2,3-cd)pyrene 69.0 + 0.1µg/g

Naphthalene 39.1+ 0.1µg/g


132

Appendix VII

PAHs Composition of Firewood Dried Fish Sample

Naphethalene 3.5 + 0.4µg/g

Acenaphthylene 0.6+ 0.1µg/g

1,2 benzantharacene 114.5 + 0.2µg/g

Chrysene 0.3 + 0.1µg/g

Benzo (b) fluoranthanthene 2.8 + 0.2µg/g

Benzo (a) pyrene 64.6+ 0.2µg/g

Benzo (g,h,i) perylene 1.8 + 0.1µg/g


133

Appendix VIII

PAHs Composition of Firewood + 20g Polythene Dried Fish Sample

FIREWOOD DRIED SAMPLE + 20G POLYTHENE

PAH COMPONENTS ARE

Naphthalene 2.5µg/ g + 0.1

fluorene 11.0 µg/ g + 0.2

Anthracene 9.9 µg/ g + 0.1

Fluoranthene 17.5 µg/ g + 0.2

Pyrene 94.9 µg/ g + 0.1

1,2 Benzanthracene 16.4 µg/ g + 0.2

Benzo (b) Fluoranthene 404.5µg/ g + 0.2

Benzo (k) Fluoranthene 135.0/µg/ g + 0.2

Indeno (1,2,3 -cd) Pyrene 4.5µg/ g + 0.2

Total PAHs 696.3µg/g+ 0

135

Acenaph

thylene

Anthrac

ene

1,2

Benzant

hracene

Benzo

(a)

pyrene

Benzo

(b)

fluoranth

ene

Benzo

(g,h,i)

perylene

Benzo

(k)

fluorant

hene

Chrysene Dibenz

(a,h)

anthrace

ne

Fluoran

thene

Fluorene Indeno

(1,2,3-

cd)

pyrene

Naphth

alene

Pyrene

Acenaphthylene

Pearson

Sig. (2-tailed)

N

1

24

-.142

.507

24

.938**

.000

24

.996**

.000

24

-.136

.526

24

.996**

.000

24

-.292

.167

24

.331

.114

24

-.142

.507

24

-.142

.507

24

-.153

.476

24

-.195

.361

24

-.142

.507

24

-.142

.507

24

Anthracene

Pearson

Sig. (2-tailed)

N

-.142

.507

24

1

24

-.048

.823

24

-.152

.478

24

1.000**

.000

24

-.143

.505

24

.916**

.000

24

-.202

.343

24

-.143

.505

24

1.000**

.000

24

-.079

.715

24

-.219

.305

24

1.000**

.000

24

1.000**

.000

24

1,2

Benzanthracene

Pearson

Sig. (2-tailed)

N

.938**

.000

24

-.048

.823

24

1

24

.936**

.000

24

-.042

.844

24

.941**

.000

24

-.243

.253

24

.234

.271

24

-.213

.318

24

-.048

.823

24

-.217

.308

24

-.313

.136

24

-.048

.823

24

-.048

.823

24

Benzo (a) pyrene

Pearson

Sig. (2-t ailed)

N

.996**

.000

24

-.152

.478

24

.936**

.000

24

1

24

-.146

.497

24

.999**

.000

24

-.291

.168

24

.319

.129

24

-.109

.614

24

-.152

.478

24

-.119

.579

24

-.175

.414

24

-.152

.478

24

-.152

.478

24

Benzo(b)fluoranth

ene

Pearson

Sig. (2-tailed)

N

-.136

.526

24

1.000**

.000

24

-.042

.844

24

-.146

.497

24

1

24

-.137

.525

24

.915**

.000

24

-.200

.348

24

-.144

.502

24

1.000**

.000

24

-.080

.711

24

-.220

.301

24

1.000**

.000

24

1.000**

.000

24

Benzo (g,h,i)

pyrene

Pearson

Sig. (2-tailed)

N

.996**

.000

24

-.143

.505

24

.941**

.000

24

.999**

.000

24

-.137

.525

24

1

24

-.293

.165

24

.327

.118

24

-.143

.505

24

-.143

.505

24

-.153

.475

24

-.196

.359

24

-.143

.505

24

-.143

.505

24

Benzo(k)

flouranthene

Pearson

Sig. (2-tailed)

N

-.292

.167

24

.916

.000

24

-.243

.253

24

-.291

.168

24

.915**

.000

24

-.293

.165

24

1

24

-.377

.069

24

.121

.574

24

.916

.000

24

.181

.396

24

.161

.452

24

.916**

.000

24

.916**

.000

24

Chrysene

Pearson

Sig. (2-tailed)

N

.331

.114

24

-.202

.343

24

.234

.271

24

.319

.129

24

-.200

.348

24

.327

.118

24

-.377

.069

24

1

24

-.202

.343

24

-.202

.343

24

-.217

.308

24

-.352

.091

24

-.202

.343

24

-.202

.343

24

Dibenz(a,h)anthra

cene

Pearson

Sig. (2-tailed)

N

-.142

.507

24

-.143

.505

24

-.213

.318

24

-.109

.614

24

-.144

.502

24

-.143

.505

24

.121

.574

24

-.202

.343

24

1

24

-.143

.505

24

.998**

.000

24

.651**

.001

24

-.143

.505

24

-.143

.505

24

Fluoranthene

Pearson

Sig. (2-tailed)

N

-.142

.507

24

1.000**

.000

24

-.048

.823

24

-.152

.478

24

1.000**

.000

24

-.143

.505

24

.916**

.000

24

-.202

.343

24

-.143

.505

24

1

24

-.079

.715

24

-.219

.305

24

1.000**

.000

24

1.000**

.000

24

Fluorene

Pearson

Sig. (2-tailed)

N

-.153

.476

24

-.079

.715

24

-.217

.308

24

-.119

.579

24

-.080

.711

24

-.153

.475

24

.181

.396

24

-.217

.308

24

.998**

.000

24

-.079

.715

24

1

24

.642**

.001

24

-.079

.715

24

-.079

.715

24

Indeno (1,2,3-

cd)pyrene

Pearson

Sig. (2-tailed)

N

-.195

.361

24

-.219

.305

24

-.313

.136

24

-.175

.414

24

-.220

.301

24

-.196

.359

24

.161

.452

24

-.352

.091

24

.651**

.001

24

-.219

.305

24

622**

.001

24

1

24

-.219

.305

24

-.219

.305

24

Naphthalene

Pearson

Sig. (2-tailed)

N

-.142

.507

24

1.000**

.000

24

-.048

.823

24

-.152

.478

24

1000**

.000

24

-.143

.505

24

.916**

.000

24

-.202

.343

24

-.143

.505

24

1.000**

.000

24

-.079

.715

24

-.219

.305

24

1

24

1.000**

.000

24

Appendix IX

CORRELATIONS OCTOBER

2014

136

=**. Correlation is significant at the 0.01 level (2-tailed)

Pyrene

Pearson

Sig. (2-tailed)

N

-.142

.507

24

1.000**

.000

24

-.048

.823

24

-.152

.478

24

1.000**

.000

24

-.143

.505

24

916**

.000

24

-.202

.343

24

-.143

.505

24

1.000**

.000

24

-.079

.715

24

-.219

.305

24

1.000**

.000

24

1

24

137

APPENDIX X

CORRELATIONS FOR NOVEMBER 2014.

138

Acenaph

thylene

Anthrac

ene

1,2

Benzant

hracene

Benzo

(a)

pyrene

Benzo

(b)

fluoranth

ene

Benzo

(g,h,i)

perylene

Benzo

(k)

fluorant

hene

Chrysene Dibenz

(a,h)

anthrace

ne

Fluorant

hene

Fluoren

e

Indeno

(1,2,3-

cd)

pyrene

Naphth

alene

Pyrene

Acenaphthylene

Pearson

Sig. (2-tailed)

N

1

24

-.143

.505

24

.935**

.000

24

.999**

.000

24

-.136

.526

24

1.000**

.000

24

-.259

.221

24

.154

.473

24

-.143

.506

24

-.143

.505

24

-.143

.506

24

-.154

.473

24

-.193

.365

24

-.143

.505

24

Anthracene

Pearson

Sig. (2-tailed)

N

-.143

.505

24

1

24

-.048

.825

24

-.153

.476

24

1.000**

.000

24

-.143

.505

24

.951**

.000

24

-.185

.387

24

-.143

.506

24

1.000**

.000

24

1.000**

.000

24

-.075

.727

24

-.217

.307

24

1.000**

.000

24

1,2

Benzanthracene

Pearson

Sig. (2-tailed)

N

.935**

.000

24

-.048

.825

24

1

24

.929**

.000

24

-.041

.849

24

.935**

.000

24

-.193

.367

24

.063

.771

24

-.215

.313

24

-.048

.825

24

-.048

.825

24

-.220

302

24

-.314

.135

24

-.048

.825

24

Benzo (a) pyrene Pearson

Sig. (2-t ailed)

N

.999**

.000

24

-.153

.476

24

.929**

.000

24

1

24

-.146

.496

24

.999**

.000

24

-.266

.208

24

.144

.503

24

-.106

.620

24

-.153

.476

24

-.153

.476

24

-.118

583

24

-.171

.424

24

-.153

.476

24

Benzo(b)fluoranth

ene

Pearson

Sig. (2-tailed)

N

-.135

.526

24

1.000**

.000

24

-.041

.849

24

-.146

.496

24

1

24

-.136

.526

24

.950**

.000

24

-.184

.389

24

-.144

.502

24

1.000**

.000

24

1.000**

.000

24

-.076

.723

24

-.219

.304

24

1.000**

.000

24

Benzo (g,h,i)

pyrene

Pearson

Sig. (2-tailed)

N

1.000**

.000

24

-.143

.505

24

.935**

.000

24

.999**

.000

24

-.136

.526

24

1

24

-.259

.221

24

.154

.473

24

-.143

.506

24

-.143

.505

24

-.143

.506

24

-.154

.473

24

-.193

.365

24

-.143

.505

24

Benzo(k)

flouranthene

Pearson

Sig. (2-tailed)

N

-.259

.221

24

.951**

.000

24

-.193

.367

24

-.266

.208

24

.950**

.000

24

-.259

.221

24

1

24

-.297

.158

24

-.116

.590

24

.951**

.000

24

.951**

.000

24

-.051

.812

24

.007

.972

24

.951**

.000

24

Chrysene

Pearson

Sig. (2-tailed)

N

.154

.473

24

-.185

.387

24

.063

.771

24

.144

.503

24

-.184

.389

24

.154

473

24

-.297

.158

24

1

24

-.185

.387

24

-.185

.387

24

-.185

.387

24

-.199

.351

24

-.333

.111

24

-.185

.387

24

Dibenz(a,h)anthra

cene

Pearson

Sig. (2-tailed)

N

-.143

.506

24

-.143

.506

24

-.215

.313

24

-.106

.620

24

-.144

.502

24

-.143

.506

24

-.116

.590

24

-.185

.387

24

1

24

-.143

.506

24

-.143

.506

24

.997**

.000

24

.650**

.001

24

-.143

.506

24

Fluoranthene

Pearson

Sig. (2-tailed)

N

-.143

.505

24

1.000**

.000

24

-.048

.825

24

-.153

.476

24

1.000**

.000

24

-.143

.505

24

.951**

.000

24

-.185

.387

24

-.143

.506

24

1

24

1.000**

.000

24

-.075

.727

24

-.217

.307

24

1.000**

.000

24

Fluorene

Pearson

Sig. (2-tailed)

N

-.143

.506

24

1.000**

.000

24

-.048

.825

24

-.153

.476

24

1.000**

.000

24

-.143

.506

24

.951**

.000

24

-.185

.387

24

-.143

.506

24

1.000**

.000

24

1

24

-.075

.727

24

-.217

.308

24

1.000**

.000

24

Indeno (1,2,3-

cd)pyrene

Pearson

Sig. (2-tailed)

N

-.143

.505

24

1.000**

.000

24

-.048

.825

24

-.153

.476

24

1.000**

.000

24

-.143

.505

24

.951**

.000

24

-.185

.387

24

-.143

.506

24

1.000**

.000

24

1.000**

.000

24

-.075

.727

24

-.217

.307

24

1

24

Naphthalene

Pearson

Sig. (2-tailed)

N

-.193

.365

24

-.217

.307

24

-.314

.135

24

-.171

.424

24

-.219

.304

24

-.193

.365

24

.007

.972

24

-.333

.111

24

.650**

.001

24

-.217

.307

24

-217

.308

24

.640**

.001

24

1

24

-.217

.308

24

139

**. Correlation is significant at the 0.01 level (2-tailed)

Pyrene

Pearson

Sig. (2-tailed)

N

-.154

.473

24

-.075

.727

24

-.220

.302

24

-.118

.583

24

-.076

.723

24

-.154

.473

24

-.051

.812

24

-.199

.351

24

.997**

.000

24

-.075

.727

24

-.075

.727

24

1

24

.640**

.001

24

-.075

.727

24

140

Acenaph

thylene

Anthrac

ene

1,2

Benzant

hracene

Benzo

(a)

pyrene

Benzo

(b)

fluoranth

ene

Benzo

(g,h,i)

perylene

Benzo

(k)

fluorant

hene

Chrysene Dibenz

(a,h)

anthrace

ne

Fluorant

hene

Fluoren

e

Indeno

(1,2,3-

cd)

pyrene

Naphth

alene

Pyrene

Acenaphthylene

Pearson

Sig. (2-tailed)

N

1

24

-.143

.506

24

.930**

.000

24

.999**

.000

24

.999**

.000

24

1.000**

.000

24

-.293

.165

24

-.210

.325

24

-.143

.506

24

-.143

.506

24

-.138

.519

24

-.153

.474

24

-.192

.369

24

-.143

.506

24

Anthracene

Pearson

Sig. (2-tailed)

N

-.143

.506

24

1

24

-.052

.809

24

-.152

.477

24

-.143

.506

24

-.143

.506

24

.916**

.000

24

-.210

.325

24

-.143

.505

24

1.000**

.000

24

.970**

.000

24

-.077

.721

24

-.215

.312

24

1.000**

.000

24

1,2

Benzanthracene

Pearson

Sig. (2-tailed)

N

.930**

.000

24

-.052

.809

24

1

24

.925**

.000

24

.930**

.000

24

.930**

.000

24

-.247

.244

24

-.318

.131

24

-.216

.310

24

-.052

.809

24

-.051

.814

24

-.221

.299

24

-.313

.136

24

-.052

.809

24

Benzo (a) pyrene Pearson

Sig. (2-t ailed)

N

.999**

.000

24

-.152

.477

24

.925**

.000

24

1

24

.999**

.000

24

.999**

.000

24

-.292

.166

24

-.224

.293

24

-.113

.599

24

-.152

.477

24

-.148

.490

24

-.124

.564

24

-.174

.417

24

-.152

.477

24

Benzo(b)fluoranth

ene

Pearson

Sig. (2-tailed)

N

.999**

.000

24

-.143

.505

24

.930**

.000

24

.999**

.000

24

1.000

.000

24

1.000**

.000

24

-.293

.165

24

-.210

.325

24

-.143

.505

24

-.143

.506

24

-.139

.518

24

-.153

.474

24

-.192

.369

24

-.143

.505

24

Benzo (g,h,i)

pyrene

Pearson

Sig. (2-tailed)

N

1.000**

.000

24

-.143

.506

24

.930**

.000

24

.999**

.000

24

1

24

1

24

-.293

.165

24

-.210

.325

24

-.143

.506

24

-.143

.506

24

-.139

.519

24

-.153

.474

24

-.192

.369

24

-.143

.506

24

Benzo(k)

flouranthene

Pearson

Sig. (2-tailed)

N

-.293

.165

24

.916**

.000

24

-.247

.244

24

-.292

.166

24

-.293

.165

24

-.293

.165

24

1

24

-.195

.362

24

.119

.579

24

.916**

.000

24

.889**

.000

24

.182

.396

24

.163

.446

24

.916**

.000

24

Chrysene

Pearson

Sig. (2-tailed)

N

-.210

325

24

-.210

.325

24

-.318

.131

24

-.224

.293

24

-.210

.325

24

-.210

.325

24

-.195

.265

24

1

24

-.210

.325

24

-.210

.325

24

-.203

.340

24

-.225

.290

24

.109

.612

24

-.210

.325

24

Dibenz(a,h)anthra

cene

Pearson

Sig. (2-tailed)

N

-.143

.506

24

-.143

.505

24

-.216

.310

24

-.113

.599

24

-.143

.505

24

-.143

.506

24

.119

.579

24

-.210

.325

24

1

24

-.143

.506

24

-.139

.518

24

.998**

.000

24

.645**

.001

24

-.143

.505

24

Fluoranthene

Pearson

Sig. (2-tailed)

N

-.143

.506

24

1.000**

.000

24

-.052

.809

24

-.152

.477

24

-.143

506

24

-.143

.506

24

.916**

.000

24

-.210

.325

24

-.143

.506

24

1

24

.972**

.000

24

-.077

.721

24

-.215

.312

24

1.000**.

000

24

Fluorene

Pearson

Sig. (2-tailed)

N

-.138

.519

24

.970**

.000

24

-.051

.814

24

-.148

.490

24

-.139

.516

24

-.139

.159

24

.889**

.000

24

-.203

.340

24

-.139

.518

24

.972**

.000

24

1

24

-.075

.729

24

-.209

.327

24

.970**

.000

24

Indeno (1,2,3-

cd)pyrene

Pearson

Sig. (2-tailed)

N

-.153

.474

24

-.077

.721

24

-.221

.299

24

-.124

564

24

-.153

.474

24

-.153

.474

24

.182

.396

24

-.225

.290

24

.998**

.000

24

-.077

.721

24

-.075

.729

24

1

24

.638**

.001

24

-.077

.721

24

Naphthalene

Pearson

Sig. (2-tailed)

N

-.192

.369

24

-.215

.312

24

-.313

.136

24

-.174

.417

24

-.192

.369

24

-.192

.369

24

.163

.446

24

.109

.612

24

.645

.001

24

-.215

.312

24

-.209

.327

24

.636**

.001

24

1

24

-.215

.312

24

Appendix XI

CORRELATIONS FOR JANUARY 2015.

141

**. Correlation is significant at the 0.01 level (2-tailed)

Pyrene

Pearson

Sig. (2-tailed)

N

-.143

.506

24

1.000**

.000

24

-.052

.809

24

-.152

.477

24

-.143

.505

24

-.143

.506

24

.916**

.000

24

-.210

.325

24.

-.143

.505

24

1.000**

.000

24

.970**

.000

24

-.077

.721

24

-.215

.312

24

1

24

142

Appendix XII

Statistical analysis of the percentage water loss in the different drying regimes

Descriptives

N Mean Std. Deviation Std. Error

95% Confidence Interval for Mean

Minimum Maximum Lower Bound Upper Bound

October Fire wood 2 78.305000 .0070711 .0050000 78.241469 78.368531 78.3000 78.3100

Charcoal 2 78.050000 .0707107 .0500000 77.414690 78.685310 78.0000 78.1000

sun light 2 77.550000 .0707107 .0500000 76.914690 78.185310 77.5000 77.6000

Oven 2 77.550000 .0707107 .0500000 76.914690 78.185310 77.5000 77.6000

Fire wood + 20g poly-ethene

2 78.350000 .0707107 .0500000 77.714690 78.985310 78.3000 78.4000

Chacaol + 20g poly-ethene

2 77.630000 .0141421 .0100000 77.502938 77.757062 77.6200 77.6400

Total 12 77.905833 .3610202 .1042176 77.676452 78.135215 77.5000 78.4000

November Fire wood 2 79.650000 .0707107 .0500000 79.014690 80.285310 79.6000 79.7000

Charcoal 2 78.550000 .0707107 .0500000 77.914690 79.185310 78.5000 78.6000

sun light 2 78.350000 .0707107 .0500000 77.714690 78.985310 78.3000 78.4000

Oven 2 78.650000 .0707107 .0500000 78.014690 79.285310 78.6000 78.7000


2 77.650000 .0707107 .0500000 77.014690 78.285310 77.6000 77.7000


2 78.850000 .0707107 .0500000 78.214690 79.485310 78.8000 78.9000

Total 12 78.616667 .6249848 .1804176 78.219570 79.013763 77.6000 79.7000

Jenuary Fire wood 2 78.710000 .0141421 .0100000 78.582938 78.837062 78.7000 78.7200

Charcoal 2 78.815000 .1202082 .0850000 77.734973 79.895027 78.7300 78.9000

143

sun light 2 78.415000 .0212132 .0150000 78.224407 78.605593 78.4000 78.4300

Oven 2 78.605000 .0070711 .0050000 78.541469 78.668531 78.6000 78.6100


2 78.805000 .0070711 .0050000 78.741469 78.868531 78.8000 78.8100


2 78.815000 .0212132 .0150000 78.624407 79.005593 78.8000 78.8300

Total 12 78.694167 .1569284 .0453013 78.594459 78.793874 78.4000 78.9000

Multiple Comparisons

Dependent Variable Mean

Difference (I-J) Std. Error Sig.

95% Confidence Interval

Lower Bound Upper Bound

October LSD Fire wood Charcoal .2550000* .0580948 .005 .112847 .397153

sun light .7550000* .0580948 .000 .612847 .897153

Oven .7550000* .0580948 .000 .612847 .897153

Fire wood + 20g poly-ethene -.0450000 .0580948 .468 -.187153 .097153

Chacaol + 20g poly-ethene .6750000* .0580948 .000 .532847 .817153

Charcoal Fire wood -.2550000* .0580948 .005 -.397153 -.112847

sun light .5000000* .0580948 .000 .357847 .642153

Oven .5000000* .0580948 .000 .357847 .642153

Fire wood + 20g poly-ethene -.3000000* .0580948 .002 -.442153 -.157847


sun light Fire wood -.7550000* .0580948 .000 -.897153 -.612847

144

Charcoal -.5000000* .0580948 .000 -.642153 -.357847

Oven 0.0000000 .0580948 1.000 -.142153 .142153


Chacaol + 20g poly-ethene -.0800000 .0580948 .218 -.222153 .062153

Oven Fire wood -.7550000* .0580948 .000 -.897153 -.612847

Charcoal -.5000000* .0580948 .000 -.642153 -.357847

sun light 0.0000000 .0580948 1.000 -.142153 .142153




Fire wood .0450000 .0580948 .468 -.097153 .187153

Charcoal .3000000* .0580948 .002 .157847 .442153

sun light .8000000* .0580948 .000 .657847 .942153

Oven .8000000* .0580948 .000 .657847 .942153



Fire wood -.6750000* .0580948 .000 -.817153 -.532847

Charcoal -.4200000* .0580948 .000 -.562153 -.277847

sun light .0800000 .0580948 .218 -.062153 .222153

Oven .0800000 .0580948 .218 -.062153 .222153


November LSD Fire wood Charcoal 1.1000000* .0707107 .000 .926977 1.273023

sun light 1.3000000* .0707107 .000 1.126977 1.473023

Oven 1.0000000* .0707107 .000 .826977 1.173023

Fire wood + 20g poly-ethene 2.0000000* .0707107 .000 1.826977 2.173023


145

Charcoal Fire wood -1.1000000* .0707107 .000 -1.273023 -.926977

sun light .2000000* .0707107 .030 .026977 .373023

Oven -.1000000 .0707107 .207 -.273023 .073023

Fire wood + 20g poly-ethene .9000000* .0707107 .000 .726977 1.073023

Chacaol + 20g poly-ethene -.3000000* .0707107 .005 -.473023 -.126977

sun light Fire wood -1.3000000* .0707107 .000 -1.473023 #######

Charcoal -.2000000* .0707107 .030 -.373023 -.026977

Oven -.3000000* .0707107 .005 -.473023 -.126977

Fire wood + 20g poly-ethene .7000000* .0707107 .000 .526977 .873023


Oven Fire wood -1.0000000* .0707107 .000 -1.173023 -.826977

Charcoal .1000000 .0707107 .207 -.073023 .273023

sun light .3000000* .0707107 .005 .126977 .473023

Fire wood + 20g poly-ethene 1.0000000* .0707107 .000 .826977 1.173023



Fire wood -2.0000000* .0707107 .000 -2.173023 #######

Charcoal -.9000000* .0707107 .000 -1.073023 -.726977

sun light -.7000000* .0707107 .000 -.873023 -.526977

Oven -1.0000000* .0707107 .000 -1.173023 -.826977

Chacaol + 20g poly-ethene -1.2000000* .0707107 .000 -1.373023 #######


Fire wood -.8000000* .0707107 .000 -.973023 -.626977

Charcoal .3000000* .0707107 .005 .126977 .473023

sun light .5000000* .0707107 .000 .326977 .673023

Oven .2000000* .0707107 .030 .026977 .373023

146

Fire wood + 20g poly-ethene 1.2000000* .0707107 .000 1.026977 1.373023

Jenuary LSD Fire wood Charcoal -.1050000 .0510718 .086 -.229968 .019968

sun light .2950000* .0510718 .001 .170032 .419968

Oven .1050000 .0510718 .086 -.019968 .229968

Fire wood + 20g poly-ethene -.0950000 .0510718 .112 -.219968 .029968


Charcoal Fire wood .1050000 .0510718 .086 -.019968 .229968

sun light .4000000* .0510718 .000 .275032 .524968

Oven .2100000* .0510718 .006 .085032 .334968

Fire wood + 20g poly-ethene .0100000 .0510718 .851 -.114968 .134968

Chacaol + 20g poly-ethene 0.0000000 .0510718 1.000 -.124968 .124968

sun light Fire wood -.2950000* .0510718 .001 -.419968 -.170032

Charcoal -.4000000* .0510718 .000 -.524968 -.275032

Oven -.1900000* .0510718 .010 -.314968 -.065032



Oven Fire wood -.1050000 .0510718 .086 -.229968 .019968

Charcoal -.2100000* .0510718 .006 -.334968 -.085032

sun light .1900000* .0510718 .010 .065032 .314968



147


Fire wood .0950000 .0510718 .112 -.029968 .219968

Charcoal -.0100000 .0510718 .851 -.134968 .114968

sun light .3900000* .0510718 .000 .265032 .514968

Oven .2000000* .0510718 .008 .075032 .324968



Fire wood .1050000 .0510718 .086 -.019968 .229968

Charcoal 0.0000000 .0510718 1.000 -.124968 .124968

sun light .4000000* .0510718 .000 .275032 .524968

Oven .2100000* .0510718 .006 .085032 .334968

Fire wood + 20g poly-ethene .0100000 .0510718 .851 -.114968 .134968

*. The mean difference is significant at the 0.05 level.

FACULTY OF BIOLOGICAL SCIENCE · I am ever grateful to Dr. C. S. Ubani, Dr. Parker Elijah Joshua...

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Transcript of FACULTY OF BIOLOGICAL SCIENCE · I am ever grateful to Dr. C. S. Ubani, Dr. Parker Elijah Joshua...