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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
2
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.
3
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
5
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.
6
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.
7
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.
8
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
9
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
10
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
11
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 - -
46
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
12
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
13
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
14
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
15
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
16
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
17
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)
18
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)
19
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
20
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
21
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
22
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).
23
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)
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 0.6 ND ND ND ND ND
Anthracene ND ND ND ND ND ND ND 9.8
1,2 Benzanthracene ND ND 114.5 ND 40.0 ND ND 16.6
Benzo (a) pyrene 1.4 ND 64.6 ND ND ND 2.6 ND
Benzo (b)
fluoranthene
ND ND 2.8 ND ND ND ND 404.5
Benzo (g,h,i) pyrene ND ND 1.8 ND ND ND ND ND
Benzo (k)
flouranthene
27.2 40.3 ND 1.96 6.5 4.3 46.1 135.0
Chrysene ND ND 0.2 ND ND 0.67 ND ND
Dibenz (a,h)
anthracene
ND ND ND ND ND ND 10.0 ND
Fluoranthene ND ND ND ND ND ND ND 17.5
Fluorene ND ND ND ND ND ND ND 11.2
Indeno (1,2,3-cd)
pyrene
ND ND ND ND ND ND 69.0 4.6
Naphthalene 7.1 39.1 3.5 0.93 1.2 ND 39.1 2.5
Phananthrene ND ND ND ND ND ND ND ND
Pyrene ND ND ND ND ND ND ND 95.0
59
Table 8: GC –MS Result of Fish Samples in January 2014 (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 0.7 ND ND ND ND ND
Anthracene ND ND ND ND ND ND ND 9.9
1,2 Benzanthracene ND ND 114.7 ND 42.0 ND ND 16.4
Benzo (a) pyrene 1.6 ND 64.8 ND ND ND 2.2 ND
Benzo (b)
fluoranthene
ND ND 3.0 ND ND ND ND 404.7
Benzo (g,h,i) pyrene ND ND 2.0 ND ND ND ND ND
Benzo (k)
flouranthene
27.4 40.5 ND 1.87 6.83 4.1 46.1 135.2
Chrysene ND ND 0.4 ND ND 0.69 ND ND
Dibenz (a,h)
anthracene
ND ND ND ND ND ND 10.0 ND
Fluoranthene ND ND ND ND ND ND ND 17.5
Fluorene ND ND ND ND ND ND ND 11.0
Indeno (1,2,3-cd)
pyrene
ND ND ND ND ND ND 69.0 4.6
Naphthalene 7.3 39.3 3.7 0.84 1.4 ND 39.0 2.7
Phananthrene ND ND ND ND ND ND ND ND
Pyrene ND ND ND ND ND ND ND 94.9
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
Acenaphthene ND ND ND ND ND ND ND ND
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
Phananthrene ND ND ND ND ND ND ND ND
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 (k) fluoranthene 27.2 + 0.2µg/g
Benzo (a) pyrene 1.4 + 0.2µg/g
Total PAHs 35.7 + 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
Benzo (k) fluoranthene 6.5 + 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
Total PAHs 79.5+ 0.2µg/g
131
Appendix VI
PAHs Composition of Charcoal + 20g Polythene Dried Fish Sample
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
Indenol (1,2,3-cd)pyrene 69.0 + 0.1µg/g
Naphthalene 39.1+ 0.1µg/g
Total PAHs 166.2 + 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
Total PAHs 188.1+ 0.2µg/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
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
Fire wood + 20g poly-ethene
2 77.650000 .0707107 .0500000 77.014690 78.285310 77.6000 77.7000
Chacaol + 20g poly-ethene
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
Fire wood + 20g poly-ethene
2 78.805000 .0070711 .0050000 78.741469 78.868531 78.8000 78.8100
Chacaol + 20g poly-ethene
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
Chacaol + 20g poly-ethene .4200000* .0580948 .000 .277847 .562153
sun light Fire wood -.7550000* .0580948 .000 -.897153 -.612847
144
Charcoal -.5000000* .0580948 .000 -.642153 -.357847
Oven 0.0000000 .0580948 1.000 -.142153 .142153
Fire wood + 20g poly-ethene -.8000000* .0580948 .000 -.942153 -.657847
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 + 20g poly-ethene -.8000000* .0580948 .000 -.942153 -.657847
Chacaol + 20g poly-ethene -.0800000 .0580948 .218 -.222153 .062153
Fire wood + 20g poly-ethene
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
Chacaol + 20g poly-ethene .7200000* .0580948 .000 .577847 .862153
Chacaol + 20g poly-ethene
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
Fire wood + 20g poly-ethene -.7200000* .0580948 .000 -.862153 -.577847
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
Chacaol + 20g poly-ethene .8000000* .0707107 .000 .626977 .973023
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
Chacaol + 20g poly-ethene -.5000000* .0707107 .000 -.673023 -.326977
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
Chacaol + 20g poly-ethene -.2000000* .0707107 .030 -.373023 -.026977
Fire wood + 20g poly-ethene
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 #######
Chacaol + 20g poly-ethene
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
Chacaol + 20g poly-ethene -.1050000 .0510718 .086 -.229968 .019968
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
Fire wood + 20g poly-ethene -.3900000* .0510718 .000 -.514968 -.265032
Chacaol + 20g poly-ethene -.4000000* .0510718 .000 -.524968 -.275032
Oven Fire wood -.1050000 .0510718 .086 -.229968 .019968
Charcoal -.2100000* .0510718 .006 -.334968 -.085032
sun light .1900000* .0510718 .010 .065032 .314968
Fire wood + 20g poly-ethene -.2000000* .0510718 .008 -.324968 -.075032
Chacaol + 20g poly-ethene -.2100000* .0510718 .006 -.334968 -.085032
147
Fire wood + 20g poly-ethene
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
Chacaol + 20g poly-ethene -.0100000 .0510718 .851 -.134968 .114968
Chacaol + 20g poly-ethene
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.