CHAPTER ONE

1.0 Introduction

Polycyclic aromatic hydrocarbons (PAHs) are a class of toxic organic compounds composed of
two or more benzene rings connected in linear, angular or clustered fused rings.(Zhang et al.,
2015) Although PAHs are present in trace amounts in environmental media, the long–distance
migration ability of PAHs makes its widespread and long–lasting existence in the environment
possible (Liberti et al., 2006). It then enters the human body via various routes such as
respiratory, digestive and skin contact, accumulating and causing carcinogenic, teratogenic and
mutagenic effects (Liberti et al., 2006). As a result, it has attracted wide attention from all over
the world for a long time. The US Environmental Protection Agency and the European Union
have listed 16 PAHs as precedence–controlled pollutants which are; Naphalene Acenaphthene,
Acenaphthylene, Fluorene, Phenanthrene, Anthracene, Fluoranthene, Pyrane,
Benzo[a]anthracene, Chrysene, Benzo[b]fluoranthene, Benzo[k]fluoranthene, Benzo[a]pyrene,
Indeno[1,2,3-cd]pyrene, Dibenz[a,h]anthracene, Benzo[g,hi] perylene. These PAHs are typically
formed during the incomplete combustion of organic materials and can be found in substance
like fossil fuels, tobacco smoke, and grilled foods (Liberti et al., 2006). Their regulation is
essential due to their harmful environmental and health impacts.

Polycyclic aromatic hydrocarbons (PAHs) comprise over 200 organic compounds containing two
or more fused aromatic rings (Qin et al., 2014). According to the number of aromatic rings, they
can be classified as light (2–3 rings) or heavy (4–6 rings) compounds (Talanta. 2013).
Environmental PAHs can originate from natural sources, such as forest fires and volcanic
emissions, and from sources associated with human activity, such as coal burning, vehicles
exhaust emissions, engine lubricating oils, and cigarette smoke (Talanta. 2013). The pyrolytic
process that generates PAHs involves three fundamental factors—high temperatures, reduced
oxygen levels, and organic matter—resulting in incomplete combustion. This process generally
yields a complex mixture of PAHs, which in turn can accumulate in the environment, affecting
water, air, and soil, thereby entering the food chain (Qin et al., 2014).

Humans are exposed to polycyclic aromatic hydrocarbons (PAHs) through both dietary and non-
dietary sources, such as inhalation and skin contact. Among these, dietary intake is the

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predominant route of exposure. According to Rengarajan et al., over 70% of PAH exposure in
non-smokers is linked to food consumption. PAHs are particularly concerning due to their
harmful effects on human health, including their roles in carcinogenesis, mutagenesis, and
immunosuppression. While not all PAHs are classified as carcinogens, they can still pose health
risks through synergistic effects, particularly due to their propensity to act as free radicals and
accumulate in the body, leading to cellular damage. Recent epidemiological studies in both
humans and animals suggest that the rising prevalence of cancer may be partly attributed to PAH
exposure. Additionally, other studies have shown that a significant proportion of cancer cases
may be partially linked to dietary factors, including exposure to PAHs through food (Zhi et al.,
2015). Therefore, the presence of these organic compounds in food is a significant concern,
necessitating ongoing monitoring.

Polycyclic aromatic hydrocarbons (PAHs) are commonly found in various categories of food and
beverages, existing as complex mixtures of both light and heavy compounds. The European
Commission has identified four key PAHs (PAH4) in foods: benz[a]anthracene (BaA), chrysene
(Chr), benzo[b]fluoranthene (BbF), and benzo[a]pyrene (BaP). Food contamination by PAHs can
occur due to environmental pollution or during food preparation and processing. Environmental
contamination primarily involves gas-phase light PAHs in the atmosphere, which can adsorb
onto particulate matter. These, along with light and heavy PAHs in soil and water, can enter the
food chain through absorption by vegetation and plant materials. When such contaminated
vegetation is used as livestock feed, PAHs accumulate in animal products and their derivatives.
Additionally, PAHs can form during food processing methods like smoking or drying—
particularly when fuel combustion is incomplete—as well as during cooking methods involving
high temperatures or open flames, such as grilling, toasting, roasting, and frying. (Willis et at.,
2009).

The prevalence of industrial, natural, and domestic processes has heightened the general
population's exposure to carcinogenic substances, including PAHs. Among PAHs,
benzo[a]pyrene (BaP) has been extensively studied due to its genotoxic properties, which result
from its ability to intercalate directly into DNA, causing structural disruptions that lead to
mutations. The International Agency for Research on Cancer (IARC) classifies BaP as a Group 1
carcinogen, indicating it is carcinogenic to humans, while BaA, BbF, and Chr are classified as

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Group 2B carcinogens, meaning they are possibly carcinogenic to humans. In addition to IARC,
organizations such as the Joint Food and Agriculture Organization/World Health Organization
(FAO/WHO), the Scientific Committee on Food (SCF), and the European Food Safety Authority
(EFSA) also evaluate the carcinogenicity of PAHs based on their occurrence in food. These
organizations consistently classify BaA, Chr, BbF, and BaP as mutagenic, genotoxic, and
carcinogenic compounds.

The absorption of PAHs is facilitated by their increased solubility in fats, and their lipophilic
nature allows them to bind with the cell membrane. This binding causes structural changes,
interfering with normal functions of the cell (Qin eet al., 2018). Among PAHs, BaP is the most
easily solubilized by lipids. This compound may associate with lipid distribution molecules, such
as chylomicrons and other lipoproteins, thereby permeating several systems responsible for lipid
absorption and distribution and, consequently, promoting its bioaccumulation in tissues and
organs that actively participate in these processes, such as the liver and small intestine (Zhou et
al., 2014).

The toxic effects of PAHs depend on the duration and mode of exposure. Short-term effects
include skin and eye irritation, nausea, vomiting, and inflammation, while long-term effects are
associated with skin, lung, bladder, and gastrointestinal cancers; kidney, and liver damage; and
cataracts; as well as genetic mutation, cell damage, and cardiopulmonary-related mortality.
Furthermore, PAHs are metabolized through several pathways involving phase I and II enzymes,
including the cytochrome P450 peroxidase and aldo-keto reductase pathways, generating
metabolites such as diol-epoxides and radical cations. These metabolites can bind to proteins and
DNA, forming DNA adducts that lead to biochemical disruptions and cellular damage,
consequently causing carcinogenic, mutagenic, immunosuppressive, and teratogenic damage.
These genetic mutations are associated with the development of fetal malformations and tumors.
In addition, considering that PAHs commonly exist as a complex mixture of different structures,
they can exhibit synergistic effects, possibly increasing their toxicity.

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1.1 Aim of the study

1.2 OBJECTIVES OF THE STUDY

The specific objective of this study were;

 To measure the concentration of PAHs within Abakpa using advanced analytical

techniques and compare these levels against established safety guidelines.
 To evaluate the levels, sources, and potential health risks associated with polycyclic
aromatic hydrocarbons (PAHs) in Abakpa
 To investigate the effectiveness of various mitigation strategies for reducing PAH
exposure.
 To determine the major sources of PAH contamination in the selected samples,
differentiating between environmental pollution, industrial processes, and food
preparation methods.
i. To Determine PAH(-) Concentration in Snail.
ii. To Determine PAH(-) Concentration in Carfish
iii. To Determine PAH(-) Concentration in Periwinkle

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 CHAPTER TWO
Literature Review
2.0 Section

Polyaromatic hydrocarbons are a class of organic compounds composed of multiple fused

aromatic rings (Kundu & Das, 2016). These compounds have been the subject of extensive
research and study due to their wide range of applications and environmental concerns (Gaffney
& Marley, 2018). In the field of environmental science, polyaromatic hydrocarbons are of
particular interest due to their ubiquitous presence in the environment and their potential adverse
effects on human health and the ecosystem (Wever et al., 2018).

Numerous studies have focused on the sources, distribution, fate, and toxicity of polyaromatic
hydrocarbons in various environmental compartments such as air, water, soil, and sediments.
One study conducted by Park et al. investigated the sources and distribution of polyaromatic
hydrocarbons in the atmosphere over a major urban area. The study found that vehicular
emissions were the primary source of polyaromatic hydrocarbons in the air, with higher
concentrations observed in areas with heavy traffic. (Broyde et al., 2011)

Polycyclic aromatic hydrocarbons (PAHs) can be formed both during biological processes or as
products of incomplete combustion from either natural combustion sources (forest and brush
fires) or man-made combustion sources (automobile emissions and cigarette smoke). As a result,
PAHs are commonly detected in air, soil, and water and, as such, they are considered ubiquitous
in the environment. (Wang et al., 2015)

Even in pristine areas, surficial soils can contain detectable levels of PAHs as a result of both
atmospheric deposition of particulates containing PAHs and natural processes, such as vegetative
decay. Thus, because of natural and human processes, soils often contain detectable amounts of
PAHs which can be considered “background” levels. Since there are so many large and small
sources which contribute PAHs to the environment, it is impossible to determine all of the
original sources of low “background” levels of PAHs found in surface and near surface soils.
Nevertheless, distinguishing between these “background” concentrations of PAHs and PAHs
from slightly contaminated media has become increasingly important as regulatory requirements

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or guidance levels, such as risk-based concentrations (RBCs), become more stringent. (Li et al.,
2012)

Generally, sample pre-treatment comprises:

 Solvent extraction of PAHs from filters and sorbents

 Extract concentration
 Some form of liquid, or solid, chromatography clean-up
 Eluent concentration
 Injection into a gas chromatography (GC) for analysis.

PAHs in air (Szulejko, Kim et al., 2014),water (Zhou and Gao, 2014), food, soil and waste
sludge are usually extracted into suitable conventional organic solvent before their introduction
into separating or analytical equipments. Analysis by GC, high performance thin layer
chromatography (HPTLC) and Fourier transform infrared spectroscopy (FTIR) (Mukherjee et
al., 2008) showed that the biosurfactant could effectively entrap and solubilize PAH. Water is an
alternative solvent and reaction medium to many conventional organic solvents. Other alternative
extraction agents that have been studied, including surfactants, biosurfactants, microemulsions,
natural surfactants, cyclodextrins, vegetable oil and solution with solid phase particles. These
extraction agents have been found to remove PAHs from soil at percentages ranging from 47 to
100% for various PAHs (Gan et al., 2014).

Hewitt et al., (2014) extracted sixteen environmental protection agency-polycyclic aromatic

hydrocarbons via solid-phase nanoextraction-gas chromatography/mass spectrometry. (Xu, Zhao
et al. 2014) determined PAHs in environmental water based on magnetite
nanoparticles/polypyrrole magnetic solid-phase extraction.

Zhang, (Zang et al., 2014) used polydimethylsiloxane/metal-organic frameworks coated fibre for
solid-phase micro extraction of polycyclic aromatic hydrocarbons in river and lake water
samples. Corrotea, Sánchez et al. (Sánchez et al., 2014) extracted PAH from water samples into a
rotating-disk microextractor and the subsequent determination by gas chromatography-mass
spectrometry.

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Gholivand et al. (2015) prepared and evaluated a novel solid-phase microextraction fibre based
on functionalized nanoporous silica coating for extraction of PAHs from water samples followed
by GC–MS detection. Messer and Taylordeveloped a method for the quantitation of trace PAHs
from water via solid-phase extraction with supercritical fluid elution. Concentrated PAHs in
water to sodium dodecyl sulphate-[gamma]-alumina admicelle., Desbands et al.,2002; Zhou and
Gao 2014) determined PAHs in water samples by temperature-controlled ionic liquid dispersive
liquid-liquid microextraction combined with high performance liquid chromatography (HPLC).

(Hewitt et al., 2014) extracted sixteen environmental protection agency-polycyclic aromatic

hydrocarbons via solid-phase nanoextraction-gas chromatography/mass spectrometry. (Zhao et
al., 2014) determined PAHs in environmental water based on magnetite
nanoparticles/polypyrrole magnetic solid-phase extraction.

(Zang et al., 2014) used polydimethylsiloxane/metal-organic frameworks coated fibre for solid-
phase micro extraction of polycyclic aromatic hydrocarbons in river and lake water samples.
(Sánchez et al., 2014) extracted PAH from water samples into a rotating-disk microextractor and
the subsequent determination by gas chromatography-mass spectrometry. (Shamsipur,
Gholivand et al., 2015) prepared and evaluated a novel solid-phase microextraction fibre based
on functionalized nanoporous silica coating for extraction of PAHs from water samples followed
by GC–MS detection.( Messer and Taylor 1995) developed a method for the quantitation of trace
PAHs from water via solid-phase extraction with supercritical fluid elution. (Saitoh, Matsushima
et al. 2005) Concentrated PAHs in water to sodium dodecyl sulphate-[gamma]-alumina
admicelle. ( Desbands et al.2002; Zhou and Gao Kronholm 2010; Zhou and Gao 2014)
determined PAHs in water samples by temperature-controlled ionic liquid dispersive liquid-
liquid microextraction combined with high performance liquid chromatography (HPLC).

2.1 Analytical Methods

Samples are usually extracted and “cleaned up” with organic solvents and analysed for PAHs
content by chromatographic techniques coupled with variety of sensitive detectors. Ultraviolet
absorption and fluorescence spectroscopy are the most frequently used detectors in HPLC
systems, while flame ionization, electron capture and nitrogen detectors and mass spectrometers
have been coupled to GLC instruments. Some of these measurement methods are very sensitive

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and can identify and quantify individual PAH compounds, however, information on exposure is
usually available only several days after sampling, instrumentation is very expensive and sample
workup is manpower-intensive and therefore very costly. Although this approach offers both
good sensitivity and good quality data, the results can rarely be used to provide rapid response to
potentially harmful exposures. For PAHs, there is currently an unsatisfied requirement for
methods that can accurately estimate exposures in real-time or at least within minutes of an
exposure. Indeed, this has been the thrust of some research in the past 10 years and has led to the
development of some new techniques of direct importance to the monitoring of toxic compounds
in the working environment. The most promising of the new techniques use the principle of
synchronous luminescence, room temperature phosphorescence, and ultraviolet absorption.
These instrumental techniques are very sensitive, ensuring detection of very low concentrations
and, in addition, may provide information on sample PAHs content.(Zhou and Gao 2014)

2.2 PAH in Air

PAHs in the ambient air exist in vapour phase or adsorb into airborne particulate matter
depending on the atmospheric conditions (ambient temperature, relative humidity, etc.), the
nature (i.e., origin and properties) of the aerosol, and the properties of the individual PAH (Sokhi
et al., 2008).

PAHs are complex hazardous organic compounds that are introduced into the atmosphere as by-
products of partial combustion processes. Atmospheric emissions of airborne PAHs can originate
from heat and power generation (e.g. coal, gas, wood, oil, charcoals), industrial processes (e.g.,
coke production) (Notarnicola et al., 2006, ). Refuse burning and vehicle emissions. Human
exposure to airborne PAHs can result from emissions from other sources, such as cooking,
smoking(Bradshaw et al., 2016), incense, mosquito coil (Verma, Patel et al., 2015) and
materials containing PAH (e.g., petroleum products, synthetic fuels). Urban road dusts are
carriers of polycyclic aromatic compounds (PACs) and are therefore considered to be a major
source of contamination of other environmental compartments and a source of exposure to PACs
for urban populations. Street dust is an indicator of PAHs pollution status of a city area
( Bandowe and Nkansah 2016; Rahman et al. 2015). PAH exposure from solid fuel burning
represents an important public health issue for the majority of the global population. Yet,
understanding of individual-level exposures remains limited. Indoor air pollution from use of

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indoor stoves and/or fireplaces is an important source of ambient PAH exposure. Vehicular traffic
is a major source of outdoor air pollution such as PAHs in urban areas, and studies have shown
that air pollution is worse during hours of commuting to and from work and school. However, it
is unclear to what extent different commuting behaviors are a source of air pollution compared to
non-commuters, and if air pollution exposure actually differs by the mode of commuting
(Kameda et al., 2013) . Parkıng Garages is another source of PAHs emission in urban area(Kara
and Aydin 2015) . Kitchen is also a main source of PAHs emission and urinary PAH metabolites
in kitchen workers can be associated with higher concentrations of PAHs in kitchen indoor air
(Robert et al., 2015).

PAHs are detected in air and monitoring of atmospheric organic pollutants, in other words they
are usually conducted by means of active or passive air samplers .

2.4. PAH in Water

PAHs, one of the major groups of anthropogenic environmental pollutants, were firstly identified
and measured in coastal waters, native mussels and fish of an industrialized South American
estuary.Zhi et al., (2015) studied the fate of PAHs in the water columns from Poyang Lake. The
predominance PAHs are of lower molecular weight congeners demonstrated that petroleum and
the combustion products of fuel oil, as well as other pyrogenic sources, contributed to the main
input of PAHs in the Poyang region. The vehicle emissions were mostly from diesel engines. Qin
et al. ( He et al., 2014) used GC-MS to measure the residual levels of PAHs in the water,
suspended particular matter (SPM) and sediment from Lake Chaohu .The spatial–temporal
distributions and the SPM–water partition of PAHs and their influencing factors were
investigated. The potential sources and contributions of PAHs in the sediment were estimated by
positive matrix factorization (PMF) and probabilistic stable isotopic analysis (PSIA). The results
showed that the average residual levels of total PAHs (PAH16) in the water, SPM and sediment
were 170.7 ± 70.8 ng/L, 210.7 ± 160.7 ng/L and 908.5 ± 1878.1 ng/g dry weights, respectively.
The same spatial distribution trend of PAH16 in the water, SPM and sediment was found from
high to low: river inflows; western lake; eastern lake; water source area. There was an obvious
seasonal trend of PAH16 in the water, while no obvious seasonal trend was found in the SPM.
The residues and distributions of PAHs in the water, SPM and sediment relied heavily on carbon
content. Significant Pearson correlations were found between LogKoc and LogKow as well as

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some hydro-meteorological factors. Three major sources of PAHs including coal and biomass
combustions, and vehicle emissions were identified. Penezic et al. ( Gašparović et al., 2014)
developed a new sensing system for PAHs in waters. The system consists of a wafer-based
device with chip-based mercury on platinum microelectrode as a working electrode and a
platinum auxiliary electrode, incorporated into a flow cell system with an external reference
electrode. The Hg microelectrode was coated with a phospholipid-triglyceride mixed layer and
interactions between anthracene, phenanthrene, pyrene and fluoranthene and the layer were
monitored using rapid cyclic voltammetry. The layer proved sensitive to interactions with PAHs
in ‘organic matter free’ seawater, with respective detection limits of 0.33, 0.35, 0.15 and
0.32μgL-1 for phenanthrene, pyrene, anthracene and fluoranthene. Tested interferences, such as
sodium humate, dextran T-500 and bovine serum albumin, representing humic substances,
polysaccharides and proteins, did not have an influence on the layer response. The system was
also tested with a river water sample where concentrations of PAHs were determined using the
standard addition method and compared with the results obtained by using gas chromatography-
mass spectrometry (GC-MS). The concentration of total PAHs obtained by the standard addition
method is ∼80% lower compared with the results obtained by GC-MS analysis. The difference is
explained by the fact that the electrochemical method measures water-soluble and free PAHs
whereas the chromatographic method measures both dissolved and particulate-organic PAHs.
( Gašparović et al., 2014).

2.5 PAH in Soil and Sediment

Pollution pressures are always placed in the coastal and estuarine ecosystems of any developing and
developed countries because of the elevated pollutants discharged from various sources such as industries,
waste and human activities. PAHs in the environment were closely linked to human activities, which have
been intensively studied for their geochemical interest as markers. Riverine inputs (Boonyatumanond et
al., 2006) are the most important sources of PAHs in the coastal sediments and soil has been identified as
the primary reservoir for PAHs in the United Kingdom (Cave et al., 2016). The waste generated from
industrial processes and operations including domestic wastes when treated partially and disposed in soil–
water environment enter to lakes, streams, rivers, oceans and other water bodies. The pollutants get
dissolved or lie suspended in water or get deposited on soil sediment beds.

Wang et al. (Liu et al., 2014) collected surface water and sediments from eleven locations on the middle
region of Huaihe River to evaluate the environmental effects of urban and industrial activities. The

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concentrations of sixteen PAHs in water and sediments were between 1.2-5.1 μg/L and 72-139 ng/g,
respectively. High levels of PAHs in water were recorded in comparison to those of other countries, while
the concentrations in sediments were low to moderate levels. A weak positive correlation was observed
(R2 = 0.49, P = 0.13) between PAHs and total organic carbon in water. However, a significant positive
correlation (R2 = 0.79, P = 0.004) was found in sediments, which indicated that total organic carbon was
a factor in PAH levels in sediments but not in water. The parent ratios were studied by principal
component analysis that showed the PAHs from water were predominantly derived from coal combustion
(34%), oil spills (49%), and vehicular emissions (17%), while the major PAH sources to sediments were
coal combustion. Using the ecological toxic equivalency factor index, it was concluded that the PAHs
levels may provide a potential risk in water but not in sediments. (Li and Duan 2015) reviewed the status
of PAH contamination in China Sea and assessed the concentrations, sources, and fates of PAHs in
sediments of China Sea. PAH concentrations in China Sea sediments decreased from north to south due to
the higher emissions in North China. Atmosphere was probably the main carrier of PAHs in the north due
to the higher contents of atmospheric fine particles and higher wind speeds. However, riverine inputs
were probably the most important sources of PAHs in the coastal sediments of South China due to higher
rainfall.

2.6. PAH in Sewage Sludge

Waste and sewage sludge both industrial and domestic have recently increased as a result of rapid
industrialization and overpopulation. Waste water of these waste and sewage sludge is often use to irrigate
farmland and the application of sewage sludge as a fertilizer is a common method used to improve soil
properties. However, sewage sludge and waste water may contain various organic pollutants including
PAHs. The presence of contaminants considerably restricts the application of sewage sludge for the
fertilization and reclamation of soils. PAHs are ubiquitous in the environment and vary widely in sewage
sludge depending on the input of industrial effluents. PAHs may be present as contaminants in wastewater
sewage sludge. Due to their toxicity and persistence in the solid phase, information are gathered relating
to their presence in sewage sludge in order to determine their contamination risks after land application
(Higashi et al., 2010; Lin et al., 2014). Wastewater sewage sludge increased the removal of PAHs from
soils but the effect is controlled by the physical, chemical and microbial soil properties, the contaminant
and microorganisms in wastewater sludge (Yang et al., 2015; Fernández-Luqueño et al., 2016) .(Ning et
al., 2016) established an efficient oxidation process for the degradation of PAHs in textile dyeing sludge,
which increased the removal of PAHs in the sludge.

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Recent reports show that as much as 10 – 106 t of sewage sludge was produced in the member countries
of the European Union between 2003 and 2006; 8.7106 t in the EU-1 and additional 1.2 – 106 t for the 12
new Member States, and significant amounts of this total have been applied to land, 37% (DGEnv, 2010).

Aiemi et al.,( 2016) studied the fate of organic matter during anaerobic digestion of sewage sludge in
batch systems thanks to a sequential chemical fractionation of the particulate phase coupled to
fluorescence spectroscopy. Polycyclic Aromatic Hydrocarbons (PAHs) distribution within the organic
pools was characterized from their analysis in the residual fraction after each extraction. Both methods
were combined to understand the link between PAHs presence in organic pools and their spectral
characterization after extraction. Two batch systems (sludge and inoculum mixture) were set up to study
the impact of PAHs spiking on their fate and distribution. The sequential fractionation allowed us to
extract and characterize about 50% of total Chemical Oxygen Demand. Moreover, fluorescence
spectroscopy helped us to understand the organic pools evolution: the most easily extracted pools
composed of protein-like molecules were highly degraded meaning that chemical accessibility mimics the
bioaccessibility to degrading microorganisms. PAHs were present in all pools of organic matter but native
PAHs were mainly present in low accessible (hardly extractable) fractions and during anaerobic digestion,
they accumulated in the non-accessible (non extractable) fraction. Spiked PAHs were more dissipated
during anaerobic digestion since spiking made them present in more accessible fractions. (Cave et
al., 2016). During the anaerobic digestion, contrary to native PAHs, spiked ones relocated toward less
accessible organic fractions confirming the ageing phenomenon. PCA analysis showed that, in spiked
mixture, PAHs presence in organic pools is linked to both PAHs physical–chemical properties and
quality/quantity of the associated organic pools. (Li et al., 2016)

During pyrolysis of sewage a great number of carcinogenic PAHs are emitted into the haracteri and there
are some dissipation of PAHs in soil amended with sewage sludge compost. (Zielińska et al., 2014)
identified the effect of various biochars on the content of freely dissolved (Cfree) PAHs in sewage sludge.
Apart from the evaluation of biochars obtained from various materials, the study also included the
determination of the effects of biochar particle sizes and biochar production temperature on their ability to
bind PAHs in sewage sludge. Increase in biochar dose caused a gradual reduction of C free PAHs content,
but only up to the biochar dose of 5%. Depending on the kind of initial material from which the biochar
was produced, the reduction of C free PAHs content in sewage sludge varied from 17.4% to 58.0%. Both
the temperature and the particle size of biochar had an effect on PAH free concentration reduction.
Biochars haracterized by a low polarity index (O/C or (O + N)/C) reduced the level of C free PAHs better
than biochars with a higher polarity index value.

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2.7. PAHs in Food

Food consumption is an important pathway for human exposure to environmental contaminants such as
PAHs. The exposure to PAHs from food sources is ubiquitous and dietary intake is one of the major
exposure pathways of PAHs (Shen et al., 2016) .

The occurrence of PAHs in food is due to either food preparation or processing techniques or entry into
the food chain when either crops or plants are grown in contaminated soil or marine life or fish live in
contaminated water. Dietary intake of PAHs is the major route of human exposure (Duan et al., 2010).
Food can be contaminated by PAHs that are present in air, soil, or water, or during food processing and
cooking. The presence of PAHs in food implies a potential risk to human health. Their presence in the
environment is reflected in their presence at detectable levels in many types of uncooked food. In
addition, cooking processes can generate PAHs in food. PAHs can also be formed during the curing and
processing of raw food prior to cooking. Several studies have been carried out to determine the levels of
exposure to PAHs from representative human diets, and the proportion of the overall burden of
environmental exposure to PAHs that is attributable to the diet. In most cases, it is concluded that diet is
the major source of human exposure to PAHs. The major dietary sources of PAHs are cereals and
vegetables, rather than meat, except where there is high consumption of meat cooked over an open flame.
Cereals and cereal products were identified as a major contributor to the intake of PAHs, owing to their
high consumption. PAHs might occur in fats and oils such as sunflower, olive, soybean, palm, and
rapeseed oils. Vegetable fats and oils have higher concentration of PAHs in their food group. The
concentrations of PAHs are generally greater on the plant surface (outer leaves, peel) than in internal
tissue. Seafood and fish can be exposed to PAHs present in water and sediments due to atmospheric
pollution or oil spills. PAHs are also formed as a result of some domestic food preparation methods.
When food, particularly meat and fish, are cooked over an open flame, PAHs are formed. If the grilled
food is in direct contact with the flame, pyrolysis of the drippings from meat or fish generates PAHs that
can be deposited on its surface (Kim et al., 2016). Even if not in direct contact, fat dripping onto the flame
or hot coals generates these compounds that are carried back onto the surface of the food (Lijinsky and
Shubik 1965). PAH formation during charcoal grilling was shown to be dependent upon the fat content of
the meat, duration of cooking and the temperature used.

Smoked and grilled food may contribute significantly to the intake of PAHs if such foods are a
large part of the usual diet. Simple practices such as selecting preferentially lean meat and fish
and avoiding contact of foods with flames for barbecuing, using less fat for grilling, and cooking

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at lower temperature forlonger time, results in a significant reduction of food contamination by
PAHs (Lijinsky and Ross, 1967).

Roasting and drying of coffee beans, cocoa beans and tea leaves may increase the PAH content
(Schummer et al., 2014) mainly of those which undergo an inappropriate drying process. Rose et
al. (Holland et al., 2015) investigated the effects of frying, grilling, barbecuing, toasting and
roasting on the formation of 27 different PAHs in foods. A total of 256 samples from in-house
cooking experiments were produced. There was little evidence of PAH formation during the
grilling, frying, roasting and toasting experiments. Comparison with the raw materials used in the
experiments showed little or no increase in PAH concentrations for all of the sample types,
regardless of distances from the heat source, cooking mediums and intensity of cooking
conditions. Barbecuing with charcoal plus wood chips however resulted in the formation of
benzo[a]pyrene in most foods; for beef burgers only, barbecuing over charcoal (without the use
of wood chips) gave the highest levels. In general PAH levels increased when the food was
barbequed closer to the heat source. For sausages cooked over briquettes, and for beef burgers,
beef and salmon cooked over charcoal, the concentration of PAHs was lower when the food was
closer to the heat source. Cooking time may result in a moderate increase of PAHs in some
foods, although concentrations in beef burgers appeared to fall when cooking time was extended
by 50–100%. (Wu et al. 2016) determined the concentration of PAH in Youtiao, or oil stick.
Youtiao is typical, traditional and widely-consumed fried food in China. The concentration of
PAHs in youtiao from different origins was determined. The dietary exposure and cancer risk
associated with benzo[a]pyrene equivalents from youtiao consumption were estimated using
Monte Carlo simulation. Analysis of 16 PAHs in youtiao was completed by GC-MS.
Concentrations of the sum of 16 PAHs were between 9.90 and 89.97μg/kg. The sum
concentrations of PAH4, including benzo[a]anthracene (BaA), chrysene (Chr), benzo[.
B]fluoranthene (BbF) and benzo[. A]pyrene (BaP), ranged from 1.41 to 26.56μg/kg. The median
dietary exposure of BaPeq concentrations from youtiao for children, adolescents, adults and
seniors in China, were 0.0147, 0.0101, 0.0561 and 0.0106ng/(kg·day), respectively. Health risk
estimates expressed as the 95th percentile incremental lifetime cancer risks (ILCRs) with respect
to PAHs indicated a slight potential carcinogenic risk for children in northern China and adults in
both the north and south.

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2.8. PAH in Coal and Petroleum Products

The ever-growing energy demand for industrial development in an eco-friendly manner

necessitates the evaluation of coal and its combustion residues. In management of coal
combustion and its residues on sustainable basis, characterization of PAHs is imperative and
essential. A high amount of PAHs are emitted from processing coal, during incomplete
combustion of organic matter (Achten and Hofmann 2009). Coke oven battery is one of the main
sources of fugitive total suspend particles (TSP) and PAHs in coke plants(Kozielska and
Konieczyński, 2015). The petroleum oil contamination has damaged the environment according
to the different decomposed level of compounds which exist in diverse oil pollutants (Lindén
and Pålsson 2013).

Verma, Masto et al., (2015) evaluated the feed coal, fly ash, and bottom ashes from a power
plant (Unchahar, India). Feed coal is low grade high volatile sub-bituminous. Quartz and
kaolinite are the predominant minerals in the coal, whereas quartz, mullite, and anhydrite in the
ashes. The total PAHs content was much higher in coal (4542 μg kg −1) than fly ash (32.4 μg kg −1)
and bottom ash (10.1 μg kg−1). High molecular weight PAHs were predominant in coal and fly
ash, low molecular weight PAHs in bottom ash. (Kozielska and Konieczyński, 2015) studied the
content of selected PAHs associated with the TSP in the immediate vicinity of the coke oven
battery Radlin (Poland) was investigated. TSP collection places were located on battery wall,
battery roof, pusher machine and coal transfer car. Revealed TSP concentrations were within the
range of 0.50–5.15 mg/m3, the total content of PAHs within wide range of 216.6–28018.9 μg/g.
Regardless of the concentration level of PAHs connected with the TSP in the coke oven battery
surrounding, it was found that four rings PAHs are the main fraction (50–70%). The high average
concentrations of benzo[a]pyrene (BaP) and Toxicity Equivalent BaP (BaPeq) reaching 1.29 and
2.63 μg/m3 respectively in the immediate vicinity of coke oven battery, could pose a serious
threat to the health of a coking plant workers. Calculated diagnostic ratios BaA/ (BaA + Ch), Fl/
(Fl + Py), BaP/ (BaP + Ch), BbF/BkF, BaP/BghiP, BaA/Ch are characteristic for the coking
process (Chen, Zhi et al., 2015) found that both China and UNEP recommend replacing raw coal
chunks with coal briquettes in household sector as clean coal technology (CCT), which has been
confirmed by the decreased emissions of particulate matter and black carbon. However, the clean
effect has never been systematically checked by other pollutants like PAHs. In this study, 5 coals

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with different geological maturities were processed as both chunks and briquettes and burned in
3 typical coal stoves for the measurement of emission factors (Efs) of particle-bound PAHs. It
was found that the Efs of 16 parent PAHs, 26 nitrated PAHs, 6 oxygenated PAHs, and 8 alkylated
PAHs for coal briquettes were 6.90 ± 7.89, 0.04 ± 0.03, 0.65 ± 0.40, and 72.78 ± 18.23 mg/kg,
respectively, which were approximately 3.1, 3.7, 1.9, and 171 times those for coal chunks,
respectively. Such significant increases in PAH emissions increased human health risk and
challenged the policy of CCT,

Apicella et al.,(2015) found that the different thermal behaviors and solubilities of large and
structurally different PAH mixtures featuring coal tar pitch (CP) and naphthalene synthetic pitch
(NP) samples could be read in light of their different molecular weight (MW) distribution and
spectroscopic features. The number-average MW obtained by mass spectrometry for CP (417
Da) and NP (691 Da) resulted to be lower in comparison to the values evaluated by size-
exclusion chromatography (SEC) (796 and 824 Da for CP and NP, respectively) because of the
different response of the detector of mass spectrometry to low- and high-MW components.
Hence, SEC showed to be more suitable for the analysis of PAH mixtures overlapping and
covering a higher mass range in comparison to mass spectrometry. Insights into structural PAH
features were given by means of spectroscopic analysis [infrared (IR), ultraviolet-visible (UV-
vis), and fluorescence], allowing for the discrimination between different families of PAHs as
ortho-fused PAHs and rylenes interspersed with aliphatic (mainly naphthenic) groups, mainly
featuring CP and NP, respectively. Besides showing the different aromaticity and
aliphatic/aromatic hydrogen distribution, the improvement of Fourier transform infrared (FTIR)
and UV-vis absorption analysis put also in evidence the contribution of carbon-rich particle
impurities and PAH aggregates in CP and NP, respectively. (Relić et al., 2014) investigated the
threat PAHs from deposited coal ash, obtained by coal combustion in this power plant, can
represent for the surrounding environment, samples of coal ash were submitted to extraction with
river water used for transport of coal ash to the dump, as well as with water of different ionic
strength and acidity. It was found that, out of 16 EPA priority PAHs, only naphthalene,
acenaphthylene, fluorene, phenantrene, fluoranthene, and pyrene were found in measurable
concentrations in the different extracts. Their combined concentration was around 0.1 μg/L, so
they do not, in terms of leached concentrations, represent serious danger for the surrounding
environment. In all cases of established (and leached) PAH compounds, changes of ionic

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strength, acidity, or the presence of organic compounds in river water may to some extent
influence the leached concentrations. However, under the examined conditions, similar to those
present in the environment, leached concentrations were not more than 50% greater than the
concentrations leached by distilled water. Therefore, water desorption is likely the most
important mechanism responsible for leaching of PAH compounds from filter coal ash.

2.8.1. Toxicity, Carcinogenicity and Mutagenicity of PAHS

The growing rate of industrialization is gradually leading to contamination and deterioration of

the environment and pollution is likely to reach disturbing levels in the years ahead. PAHs are
major pollutant and public concern over the deleterious effects of PAHs has grown rapidly due to
recognition of their toxicity, carcinogenicity, and teratogenicity. Benzo(a)pyrene (BaP) possesses
the greatest carcinogenic potential among the various PAHs. PAHs are widespread in various
ecosystems and are pollutants of great concern due to their potential toxicity, mutagenicity and
carcinogenicity Twelve, likely carcinogenic, PAHs (i.e., phenanthrene (Phe), anthracene (An),
fluoranthene (Fluo), pyrene (Pyr), benz[a]anthracene (B[a]A), chrysene (Chry),
benzo[b]fluoranthene (B[b]F), benzo[k]fluoranthene (B[k]F), benzo[a]pyrene (B[a]P),
indeno[1,2,3-cd]pyrene (Ind), dibenz[a,h]anthracene (D[a,h]A), and benzo[g,h,i]perylene
(B[g,h]P) (Tipmanee et al. 2013).

Seven PAH compounds have been classified as probable human carcinogens: benz(a)anthracene,
benzo(a)pyrene, benzo(b)fluoranthene, benzo(k) fluoranthene, chrysene, dibenz(ah)anthracene,
and indeno(1,2,3-cd) pyrene Table5. When PAHs enter agricultural soils through sewage sludge,
they pose an environmental risk to soil organisms, agricultural food grown in that soil and
humans that eats from the food grown on PAHs polluted soil. Industries such as petrochemical,
pharmaceutical, insecticides and fertilizers generates the hazardous waste comprising of
inorganic and organic compounds. Organic compounds mainly composed PAHs, are one of the
toxic environmental pollutant. Excessive exposure to PAHs often results in lung cancer, a disease
with the highest cancer mortality in the United States. After entry into the lung, PAHs induce
phase I metabolic enzymes such as cytochrome P450 (CYP) monooxygenases, i.e. CYP1A1/2
and 1B1, and phase II enzymes such as glutathione S-transferases, UDP glucuronyl transferases,

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NADPH quinone oxidoreductases (NQOs), aldo-keto reductases (AKRs), and epoxide
hydrolases (Ehs), via the aryl hydrocarbon receptor (AhR)-dependent and independent pathways.
PAHs are among the likely major causative agents for lung cancer in smokers. PAHs require
metabolic activation to exert their carcinogenic effects, and one important pathway proceeds
through a three-step sequence resulting in the formation of diol epoxides, which react with DNA
to produce adducts that can cause mutations and initiate the carcinogenic process. Humans can
also be exposed to PAHs through diet, via consumption of charcoal broiled foods. Metabolism of
PAHs through the CYP1A1/1B1/EH pathway, CYP peroxidase pathway, and AKR pathway leads
to the formation of the active carcinogens diol-epoxides, radical cations, and o-quinones(Chu et
al., 2015). Exposure to carcinogenic PAHs, such as benzoapyrene (BaP), may have a role in
increased esophageal cancer risk (Wei et al., 2013). With rapid urbanization, the contamination
of policy PAHs has become one of the major factors threatening urban soil quality and human
health.

PAHs are highly hazardous and can negatively affect health as well as the environment. These
health effects include carcinogenesis, localized skin effects, pulmonary and respiratory problems,
genetic reproductive and developmental effects, behavioral, neurotoxic, other organ system
effects. Human liver toxicity is associated with exposure to PAHs. PAHs are responsible for
more cancers, primarily lung cancers, than any other carcinogen (Hakura and Sonoda 2013).
PAHs in air caused asthma in children living in developed countries and the leading cause of
childhood hospitalization and school absenteeism(Karama, Peters et al. 2015). Exposures to
traffic-related air pollutants including PAH have been associated with the development and
exacerbation of asthma (Lovinsky-Desir et al,. 2015).The most studied PAHs are unsubstituted
multi-ring structures. These compounds are not DNA reactive, and must be modified to become
carcinogenic. PAHs are environmental carcinogens whose metabolites can react with DNA to
form bulky DNA adducts(Wang et al., 2011).

DNA damage is an important step in carcinogenesis. The Ames assay is a short-term screening of
carcinogens that induce DNA damage. Most carcinogens require enzymatic activation through
oxidation by cytochrome P450 (CYP450) in the presence of S9 mix. A combination of iron (Fe)
(III) porphyrin and an oxidant is also able to oxidize compounds as an alternative metabolic
pathway to CYP450. Previously it was reported that a chemical model containing a water-soluble

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5,10,15,20-tetrakis(1-methylpyridinium4-yl)porphyrinatoiron(III) chloride (4-Mpy) and tert-
butyl hydroperoxide (t-BuOOH) activated aromatic amines and amides(Inami, Ishikawa et al.
2010).

PAHs are considered to be important, dangerous and priority pollutants, which cause serious
health problems and/or genetic defects in humans, as well as harming the flora and fauna of
affected habitats. PAHs are ubiquitous environmental pollutants that are potent mutagens and
carcinogens. They are human carcinogens and mutagens; they are toxic to all living organisms.
Atmospheric PAHs may cause respiratory problems, impair pulmonary function and cause
bronchitis (Tsapakis and Stephanou 2005). PAHs are widely distributed in the environment and
human exposure to them is unavoidable. A number of them, such as benzo[a]pyrene, are
carcinogenic and mutagenic, and they are widely believed to make a substantial contribution to
the overall burden of cancer in humans. Due to their carcinogenic, mutagenic and teratogenic
potential, the removal of PAHs from aqueous environment using physical, biological and
chemical processes has been studied by several researchers. (Tishbee et al.1981).

2.9 Seafoods

Freshwater reservoirs play an important role in the livelihood of human populations. They are
used as a source of domestic water supply, irrigation, fishery development, hydropower
generation and flood control. Additional benefits of the reservoirs are tourist attraction and
opening up of new areas for development (Kitur, 2009). According to Dudgeon (2006) and Junk
(2002), freshwater ecosystems are vulnerable to human impacts hence, they are likely to be
influenced by reservoir catchment activities. This is because terrestrial ecosystems have linkages
with aquatic ecosystems (UNEP, 2000). Contamination of aquatic ecosystems with a wide range
of pollutants has become a matter of concern over the past few decades (Dirilgen, 2001;
Vutukuru, 2005; Yousafzai and Shakoori, 2008; Narayan and Vinodhini, 2008).

Seafood refers to any aquatic animal that humans eat both of freshwater and seawater origin
(Rogers, 2021). The harvesting and consumption of seafood is a long-standing practice that dates
back to the upper Palaeolithic period in history (Wikipedia 2021). Seafood, which contains
between 75 to 80 percent water and a good source of high-quality proteins, is crucial to global
food security (Gaynor et al 2018; Tahergorabi et al 2011). After cereals, fish and other seafood

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may be humanity’s most significant food, providing around 15% of the world’s protein intake
with individual consumption of fish and shellfish estimated to be over 13 kg per year depending
on geography and personal preferences (Rogers, 2021). Seafood is not just a source of nutrients
in human diet but also of environmental contaminants and unregulated consumption could pose a
risk to consumer’s health (Barchiesi F et al, 2020). The low fat content of many seafood species,
as well as the protective effects of n-3 polyunsaturated fatty acids found in oily (pelagic) fish on
coronary heart disease, are critical for health-conscious people, especially in countries where
cardiovascular disease is common (Burger et al, 2002). The seafood industry has evolved in
recent years, with significant expansion in aquaculture, geographic shifts in trade, and rising
commoditization and vertical integration. Seafoods such as – catfish
(Chrysichthysnigrodigitatus), periwinkles (Tympanotonusfuscatus), snail are favoured seafood
in Ogoni Land (Opuogulaya et al,2022) Silver catfish (Chrysichthysnigrodigitatus) is demersal;
it is omnivorous, feeding on seeds, insects, bivalves, detritus, decapods and fish. According to
Asuquo et al.,(include year)Chrysichthysnigrodigitatus forages on a variety of benthic food
sources in the tropical estuaries of Nigeria.

African periwinkle (Tympanotonusfuscatus) is mostly found in the sea, in the upper and middle
intertidal zones, and it can also be found in muddy environments like estuaries . Periwinkles are
omnivorous, feeding on algae and tiny organic debris such as barnacle larvae, and may survive
out of water for several days (Sankar et al 2007). African periwinkle (Tympanotonusfuscatus) is
mostly found in the sea, in the upper and middle intertidal zones, and it can also be found in
muddy environments like estuaries. Periwinkles are omnivorous, feeding on algae and tiny
organic debris such as barnacle larvae, and may survive out of water for several days. Heavy
metal comprises a number of elements which are manganese, chromium lead, cadmium, etc. .
(Mazvila, 2001), but some of these metals are also the most important source of necessary for
living organisms: that is, iron, zinc, and copper (Chovanec et al., 2003; Popek et al., 2008).

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 CHAPTER THREE

3.0 MATERIALS AND METHODS

3.1 Materials

i. Analytical weighing valance(OHAUS, USA)

ii. High performance liquid chromatography
iii. Beaker
iv. Burette
v. Reagent bottle
vi. Volumetric flask

3.2 Chemical and Reagents

The chemical and reagents used in the study are;

i. Acetonitrile (Air Liquid Port Harcourt)

ii. Distilled Water (Springboard Laboratory Awka)
iii. Trioxonitrate (v) acid (HNO3) (May and Beaker, England)
iv. Concentrated tetraoxusulphate (v) acid (H2SO4) (BDH England)
v. Copper Stock solution (BDH England)
vi. Mercury Stock solution (Qualikems, England)
vii. Aqua regiextraction acid (Qualikem England)
viii. Alcohol lead stock solution (Qualikem England)
ix. Zinc stock solution (BDH England)

3.3 Methodology

3.3. 1 Sample Collection

The seafoods (catfish and Periwinkle) were obtained from the different location Abakpa. It was
placed into a bowel and then transported to the Department of Biochemistry laboratory at Enugu
State University of Science and Technology.

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3.3.2 Description of Sample Collection area

Samples were also collected form Abakpa, which is suited in Nike, with a geographical trail of
(latitude: 6.4636ON, longitude: 7.4845OE). Samples where collected form Ogbete Market, which
is suited in Achara, Enugu South, with a geographical trail of (latitude: 6.4667 ON, longitude:
7.4845OE)., and lastly Samples were also collected form Garriki, which is suited in Enugu
South, with a geographical trail of (latitude: 6.4667 ON, longitude: 7.4833OE) all located in
Enugu State, Nigeria.

3.3.2. Determination of Polyaromatic Hydrocarbon Concentration

The sample was weighed (1-2 grams) and placed into a clean, dry container. PAHs were
extracted from the sample by adding 10ml. of acetonitrile, followed by ultrasonication for
30minutes. The extract was then filtered through a 0.45 µm PTFE filter and collected in an
HPLC vial. A 20µL aliquot of the filtered extract was injected into the HPLC system. The
separation was carried out using a C18 column with a mobile phase consisting of acetonitrile
and water in an 80:20 ratio, at a flow rate of 1mL/min. PAHs were detected using a fluorescence
detector set to excitation and emission wavelengths of 250 nm and 400nm, respectively. The
quantification of PAHs was performed by comparing the peak areas of the sample with those of
know standard.

3.3.3 Collection of sample

Heavy metal analysis was conducted using FS240AA Atomic Absorption Spectrophometer
according to the method of APHA 1995 (American Public Health Association)

3.3.4 Preparation of reference solution

A series of standard metal solution in the optimum concentration range are prepared, the
reference solution were prepared daily by diluting the sing stock element with water containing
1.5ml, concentrated nitric acid/liter. A Calibrated blank was prepared using all the reagents
except for the metal stock solutions.

Calibration curve for each metal was prepared by plotting absorbance of standards versus their
concentration

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3.3.5 Sample Preparation

The part of the seafood needed for the analysis was separated from the part not needed, the
sample was then dried, 2g of the dried sample was weighed out and was put into a digestion flask
and 20/ml of the acid mixture was added (650/ml Conc HNO3; 80/ml perchloric acid; 20/ml
Conc H2So4) the flask was then heated until a clear digest was observed. The digest was diluted
with 100/ml of H2O

3.3.6 Working Principle

Atomic absorption spectrometer’s working principle is based on the sample being aspirated into
flame and atomized when the AAS’s light beam is directed through the flame into the
monochromator, and onto the detector that the amount of light absorbed by the atomized element
in the flame. Since metals shave their own characteristic absorption wavelength, a source lamp
composed of that element is used, making the method relatively free from spectral interference.
The amount of energy of the characteristic wavelength absorbed in the flame is proportional to
the concentration of the element in the sample

3.5 For Liquid

Procedure; the sample is thoroughly mixed by shaking, and 100ml of it is transferred into a
glass beaker of 250ml volume, to which 5ml of Conc, nitric acid was added and heated to boil till
the volume is reduced to 1bout 15-20ml, by adding Conc. Nitric acid in increment of 5ml till the
residue is completely dissolved. The mixture is cooled, transferred an made up to 100ml using
metal free distilled water. The sample is aspirated into the oxidizing air-acetylene flame. When
the aqueous sample is aspirated the sensitivity for 1% abortion is observed.

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