presence of aflatoxins in smoked-dried fish sold in abeokuta, ogun ...

PRESENCE OF AFLATOXINS IN SMOKED-DRIED FISH SOLD
IN ABEOKUTA, OGUN STATE, SOUTHWESTERN NIGERIA
ADEJOLA QUADRI ADETAYO
(2006/0767)
A PROJECT REPORT SUBMITTED TO THE DEPARTMENT
OF AQUACULTURE AND FISHERIES MANAGEMENT
COLLEGE OF ENVIRONMENTAL RESOURCES
MANAGEMENT
UNIVERSITY OF AGRICULTURE, ABEOKUTA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR
THE AWARD OF BACHELOR OF AQUACULTURE AND
FISHERIES MANAGEMENT.
JUNE, 2011.
i

CERTIFICATION
I certify that this project was carried out by Adejola Quadri Adetayo of the Department of
Aquaculture and Fisheries Management, College of Environmental Resources Management,
University of Agriculture Abeokuta, Ogun State Nigeria., and the report was prepared under my
supervision.
.................................... ...............................
Prof. G.N.O. Ezeri
Date
Supervisor
........................................... ..................................
Prof. Y. Akegbejo-Samsons
Date
Head of Department,
Aquaculture and Fisheries Management
ii

DEDICATION
I dedicate this work to Almighty Allah who willed every being to be orderly, well calculated,
created as He willed and the giver of knowledge. To my parents, Mr. and Mrs. R. A. Adejola.
Also, to Late Agoro Adeyinka, may Allah make him one of the dwellers of paradise.
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ACKNOWLEDGEMENT
All praises and gratitude goes to Almighty Allah who has given me the great privilege and
strength to have gone this far. It would never have been possible without Him.
I appreciate the great effort of my supervisor, Prof. G.N.O. Ezeri. Thank you sir for ensuring that
my work is well done despite your busy schedule. Also to other lecturers in the department who
have nurtured me till this moment; Prof. Akegbejo-Samsons, Prof. Otubusin, Dr. Idowu, Dr.
Agbon, Dr. Abdul, just a few to mention, I say thank you.
My successful journey in life has been accomplished with the moral, physical, financial, and
spiritual support of my Parents, Mr. and Mrs. R.A. Adejola. I am most grateful. May Allah
continue to shower His blessings on you and strengthen you.
My acknowledgement will be incomplete without appreciating my siblings, Yusuf, Abdul Afeez,
and Moshood, and to my cousins, Mrs. Aminah, Mrs Ogunbiyi (Nee Adejola), Abdul Rahman
and Ahmed Adejola.
I also commend the effort of my dear brothers Abdul Akeem, Ismael, Hasheem and Adeseye for
their moral support. To my very dear friends, AbdulQodri, Hamzah, Gophuroh, Abdul Afeez,
Necholas, khaleed, Idris Adenekan, Olabisi, Seun, Oriyomi, Michael, Asiwaju, Olanrewaju. I
appreciate you all.
iv

TABLE OF CONTENTS
Title Page
Certification
Dedication
Acknowledgement
Table of Contents
List of Tables
List of Plates
List of Figures
Abstract
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ii
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ix
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CHAPTER ONE
1.0 INTRODUCTION 1
1.1 Aflatoxin 2
1.1.1 Physical Characteristics 3
1.1.2 Chemical Properties 3
1.1.3 Biology of A. Flavus and A. Parasiticus 4
1.1.4 Effect on Human Health 6
1.1.5 Acute toxicity 7
1.1.6 Chronic Toxicity 7
1.1.7 Cellular Effects 8
1.2 Objectives 8
1.3 Justification 8
v

CHAPTER TWO
2.0 LITERATURE REVIEW 9
2.1 Fungi Producing Aflatoxins 10
2.2 Production and Reduction 12
2.3 Uses 13
2.4 Formation and Occurence 13
2.4.1 Prevalence of Toxigenic Species in Foods 13
2.4.2 Factors Affecting Formation of Aflatoxins in Foods 13
2.4.3 Occurrence 14
2.5 Presence in Human Biological Fluids 14
2.6 Absorption, Metabolism and Excretion in Humans 15
2.7 Toxic Effects in Humans 16
2.7.1 Immuno-suppression 17
2.7.2 Reproductive and Developmental effects in Humans 18
2.7.3 Genetic and Related Effects in Humans 20
2.8 Studies of Cancer in Humans 21
2.9 Human Biological Fluids 22
2.10 Cases–Control Studies 23
2.11 Analysis of Aflatoxin in Foods 23
2.12 Regulations and Guidelines 25
2.13 Outstanding Health Questions in Aflatoxin Management 27
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CHAPTER THREE
3.0 MATERIALS AND METHODS 28
3.1 Sample Collection 28
3.2 Assay Principles 28
3.3 Materials Provided 28
3.4 Sample Preparation and Extraction 29
3.5 Test Procedure 30
3.6 Statistical Analysis 31
CHAPTER FOUR
4.0 Result 32
4.1 Sample Analysis 32
CHAPTER FIVE
5.0 DISCUSSION, CONCLUSION AND RECOMMENDATIONS 38
5.1 Discussion 38
5.1 Conclusion 39
5.2 Recommendations 40
REFERENCES 41
APPENDIX 49
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LIST OF TABLES
Table 1: Melting-Points and Ultraviolet Absorption of Aflatoxins 5
Table 2: Analytical methods validated by AOAC International and the EU 24
Table 3: The FDA regulatory levels for Aflatoxins 26
Table 4: The identified smoked-dried fishes sampled from the two markets
(Itoku and Lafenwa) totaling fifty 32
Table 5: Result of Laboratory Analysis for the Aflatoxin Levels of the Smoked-dried
Fishes sampled from the two markets 35
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LIST OF PLATES
Plate 1: Ethmalosa fimbriata 33
Plate 2: Synodontis budgeti 33
Plate 4: Ilisha africana 33
Plate 5: Calamoichthys calabaricus 33
Plate 5: Clarias gariepinus 34
Plate 6: Schilbe uranoscopus 34
Plate 7: Chrysichthys nigrodigitatus 34
Plate 8: Gymnallabes typus 34
Plate 9: Cynoglossus browni 34
Plate 10: Alestes nurse 34
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LIST OF FIGURES
Figure 1: Aflatoxin concentrations in the different smoked-dried fish samples 36
x

ABSTRACT
This study was aimed to estimate the aflatoxin contamination of smoked-dried fish samples of
Sole, Catfish, Silverside, Silver catfish, West African shad, Mud Catfish, Bonga, Catfish, Rope
and Butter fish in Abeokuta, Ogun State, Southwestern Nigeria. Fifty smoked-dried fish samples
sold at two different markets in Abeokuta town; Lafenwa and Itoku in Abeokuta, Ogun State,
Nigeria were found to be lightly contaminated with aflatoxin, after testing for their aflatoxin
levels using Veratox quantitative aflatoxin test.
The Aflatoxin concentrations in the samples were between 0.030ppb-1.150ppb with a mean of
0.5980±0.1050 ppb. Rope fish had the lowest aflatoxin concentration while Mud catfish had the
highest aflatoxin concentration respectively. Aflatoxins are known to be carcinogenic (causing
hepatoma – cancer of the liver), acute hepatitis, reduced red blood cell and decreased immune
system in man. Prolonged intake of smoked fish with these metabolites may constitute potential
public health hazard.
In conclusion smoked-dried fishes stored for sale in Abeokuta markets were not heavily
contaminated with aflatoxins.
xi

CHAPTER ONE
1.0 INTRODUCTION
Fish is an important source of food and income to many people in the developing world. In
Africa, some 5 percent of the population, about 35 million people, depend wholly or partly on
the fisheries sector, mostly artisanal fisheries, for their livelihood.
Fish supplies a good balance of protein, vitamins and minerals. It has a relatively 10% calories
content hence its role in nutrition is recognized. Fish and fish products constitute more than 60%
of the total protein intake in adults especially in the rural areas. They are widely accepted on the
menu card and form a much-cherished delicacy that cuts across socio economic, age, religious
and educational barriers Fish flesh is one of the best sources of protein. Its flesh is tender due to
bundles of muscle fibers, which are held together by fibrous material when heated. It is better
digested than beef or other types of protein.
In Nigeria, fish is eaten fresh, preserved or processed. The percentage composition of the
different methods of fish disposed for consumption in the artisanal sector according to Tobor
(2004) are as follows live fish 7%, fresh fish 27%, smoke dried 45%, sun dried 20% salted and
sun dried 10%. Various traditional methods are employed to preserve and process fish for
consumption and storage. These include smoking, drying, salting, frying and fermenting and
various combinations of these. In Nigeria, smoking is the most widely practiced method.
Practically all species of fish available in the country can be smoked and it has been estimated
that 70-80 percent of the domestic marine and freshwater catch is consumed in smoked form.
Smoke drying methods used in Nigeria requires low capital, investment and it is conducted in
fishermen camps and fish processing centuries in traditional smoking kilns of clay, cement
blocks, drums or iron sheets This result in a very short shelf life and low market value as well as
inability to withstand handling and transportation by retailers (Eyo, 1992).
Smoked fish constitute a major source of animal protein for a vast majority of the population in
Nigeria, particularly the rural population. These products can be kept for 2-4 weeks in market
stalls with poor storage facilities. In a survey of aflatoxin contamination of common Nigerian
1

foods, Nwokolo andOkonkwo (1998) found that improperly stored dried fish contained
aflatoxins.
1.1 Aflatoxins
Aflatoxin is a toxic compound produced by Aspergillus flavus and A. parasiticus.
The molds can grow in improperly stored feeds and feeds with inferior quality of ingredients.
Aflatoxins represent a serious source of contamination in foods and feeds in many parts of
the world. These toxins have been incriminated as the cause of high mortality in livestock
and in some cases of death in human beings. Aflatoxin B1 is known to be the most significant
form that causes serious risk to animals and human health (Murjani, 2003).
For long, fungi were regarded as causing only anesthetics spoilage of food. But during 1966,
when the famous “Turkey X” diseases killed 10,000 turkey poultry in Great Britain, Western
world became aware that common spoilage molds could produce significant of toxigenic fungi
and potentially toxic compounds have been discovered. Aflatoxins, a group of toxic metabolic
produced by certain Aspergillus species have been found to be carcinogenic tetratogenic and
mutagenic to several species of experiment animals. Aflatoxin occurs in a variety of crops and
animal product. The conditions that contribute to fungal growth and production of aflatoxins are
a hot and humid climate, moisture content of 16% and above favorable substrate characteristics
and factors that decrease the host’s immunity such as insect damage.
Aflatoxins have a high melting point i.e. 250°C. It has been proved that food items do carry
residue of the toxin. Thus, it’s certain that human beings are exposed to aflatoxins through
contaminated food items among which fish is an important component (Murjani, 2003).
2

1.1.1 Physical Characteristics
According to ICRI (2000), Aspergillus flavus and Aspergillus parasiticus are mostly the molds
that produce Aflatoxin which are potent toxic, carcinogenic, mutagenic, immunosuppressive
agents. These fungi can produce their toxic compounds on almost any food that will support
growth. Among 18 different types of aflatoxins identified, major members which are metabolites
produced by these fungi are named AFB1, AFB2, AFG1, and AFG2, all which occur naturally.
Of the four, AFB1 is found in highest concentrations followed by AFG1, AFB2 and AFG2.
Aspergillus flavus only produces AFB1 and AFB2 and Aspergillus parasiticus produces these
same metabolites along with G1 and G2. Aspergillus flavus typically produces AFB1 and AFB2
whereas A. parasiticus produce AFG1 and AFG2 as well as AFB1 and AFB2. Four other
aflatoxins M1, M2, B2A, G2A which may be produced in minute amount were subsequently
isolated from cultures of A. flavus and A. parasiticus. A member of closely related compounds
namely aflatoxin GM1, parasiticol are also produced by A. flavus. Aflatoxin M1 and M2 are
major metabolites of aflatoxin B1 and B2 respectively, found in milk of animals that have
consumed feed contaminated with aflatoxins.
Description: Colourless to pale-yellow crystals. Intensely fluorescent in ultraviolet light,
emitting blue (aflatoxins B1 and B2) or green (aflatoxin G1) and green–blue (aflatoxin
G2) fluorescence, from which the designations B and G were derived, or blue–violet
fluorescence (aflatoxin M1).
Solubility: Very slightly soluble in water (10–30 µg/ml); insoluble in non-polar solvents;
freely soluble in moderately polar organic solvents (e.g., chloroform and methanol) and
especially in dimethyl sulfoxide (Cole & Cox, 1981).
Melting-points: see Table 1.
Absorption spectroscopy: see Table 1
1.1.2 Chemical Properties
Stability: Unstable to ultraviolet light in the presence of oxygen, to extremes of pH (< 3,
> 10) and to oxidizing agents.
3

Reactivity: The lactone ring is susceptible to alkaline hydrolysis. Aflatoxins are also
degraded by reaction with ammonia or sodium hypochlorite. This hydrolysis appears to
be reversible since it has shown that recyclization occurs following acidification of a
basic solution containing aflatoxin. At higher temperatures (100 0 C) ring opening
followed by decarboxylation occurs and reaction may proceed further, leading to the loss
of the methoxy group from the aromatic ring. In the presence of mineral acids, aflatoxin
B1 and G1 are converted into aflatoxin B2A and G2A due to acid-catalyzed addition of
water across the double bond in the furan ring. In the presence of acetic anhydride and
hydrochloric acid, the reaction proceeds further to give the acetoxy derivative. Similar
adducts of aflatoxins B1 and G1 are formed with formic acid-thionyl chloride, acetic
acid-thionyl chloride and trifluoroacetic acid
Oxidizing agents: Many oxidizing agents such as Sodium hypochlorite, potassium
permanganate, chlorine hydrogen peroxide, ozone amd sodium perborate react with
aflatoxin and change the aflatoxin molecule in some way as indicated by the loss of
yielded aflatoxin RB1 and RB2 respectively. These arise as a result of opening of the
lactone ring followed by the reduction of the acid group and reduction of the keto group
in the cyclopentene ring (Marryann et al., 2001).
1.1.3 Biological Properties
The two fungi, Aspergillus. flavus and A. parasiticus are closely related and grow as saprophytes
on plant debris of many crops left on and in the soil. They are distributed worldwide, with a
tendency to be more common in countries with tropical climates that have extreme ranges of
rainfall, temperature and humidity. Members of the genus Aspergillus are characterized by the
production of non-septate conidiophores, which are quite distinct from hyphae and which are
swollen at the top to form a vesicle on which numerous specialized spore-producing cells, known
as phialides or sterigmata are borne either directly (uniseriate) or on short outgrowths known as
mutalae (biseriate). Colonies of A. flavus are green – yellow to yellow – green on zapek’s agar.
They usually have biseriate sterigmata; reddish – brown sclerotia are often present, conidia finely
roughened, variable in size and oval to spherical in shape. Aflatoxin are one of the most potent
toxic substances that occur naturally. These are a group of closely related mycotoxins produced
4

y fungi Aspergillus flavus and A. parasiticus. Aflatoxicosis is poisoning that result from the
ingestion of aflatoxin in contaminated foods or seeds (Sorenson et al., 1984).
Table 1. Melting-Points and Ultraviolet Absorption of Aflatoxins
Aflatoxin Melting-point ( 0 C) Ultraviolet absorption (ethanol)
λmax (nm) ε(L mol –1 cm –1 )
B1 268–269 (decomposition) 223 25 600
(crystals from chloroform) 265 13 400
362 21 800
B2 286–289 (decomposition) 265 11 700
(crystals from chloroform-pentane) 363 23 400
G1 244–246 (decomposition) 243 11 500
(crystals from chloroform-methane) 257 9 900
264 10 000
362 16 100
G2 237–240 (decomposition) 265 9 700
(crystals from ethyl acetate) 363 21 000
M1 299 (decomposition) 226 23 100
(crystals from methanol) 265 11 600
357 19 000
Source: O’Neil et al. (2001)
5

1.1.4 Effect on Human Health
According to ICRI (2000), Humans are exposed to aflatoxins by consuming foods contaminated
with products of fungal growth. Such exposure is difficult to avoid because fungal growth in
foods is not easy to prevent. Even though heavily contaminated food supplies are not permitted
in the market place in developed countries, concern still remains for the possible adverse effects
resulting from long-term exposure to low levels of aflatoxins in the food supply. Evidence of
acute aflatoxicosis in humans has been reported from many parts of the world. The syndrome is
characterized by vomiting, abdominal pain, pulmonary edema, convulsions, coma, and death
with cerebral oedema and fatty involvement of the liver, kidney, and heart. Conditions increasing
the likelihood of acute aflatoxicosis in humans include limited availability of food,
environmental conditions that favour fungal development in crops and commodities, and lack of
regulatory systems for aflatoxin monitoring and control.
The expression of aflatoxin related diseases in humans may be influenced by factors such as age,
sex, nutritional status, and/or concurrent exposure to other causative agents such as viral hepatitis
(HBV) or parasite infestation. Ingestion of aflatoxin, viral diseases, and hereditary factors have
been suggested as possible aetiological agents of childhood cirrhosis. There are evidences to
indicate that children exposed to aflatoxin breast milk and dietary items such as unrefined
groundnut oil, may develop cirrhosis. Malnourished children are also prone to childhood
cirrhosis on consumption of contaminated food. Several investigators have suggested aflatoxin as
an aetiological agent of Reye’s syndrome in children in Thailand, New Zealand etc. Though
there is no conclusive evidence as yet. Epidemiological studies have shown the involvement of
aflatoxins in Kwashiorkor mainly in malnourished children. The diagnostic features of
Kwashiorkor are edema, damage to liver etc. These outbreaks of aflatoxicosis in man have been
attributed to ingestion of contaminated food such as animal products, maize, groundnut etc.
Hence it is very important to reduce the dietary intake of aflatoxins by following the procedures
for monitoring levels of aflatoxins in foodstuffs.
There are differences in species with respect to their susceptibility to aflatoxins, but in general,
most animals, including humans, are affected in the same manner.
6

The principal target organ for aflatoxins is the liver. After the invasion of aflatoxins into the
liver, lipids infiltrate hepatocytes and leads to necrosis or liver cell death. The main reason for
this is that aflatoxin metabolites react negatively with different cell proteins, which leads to
inhibition of carbohydrate and lipid metabolism and protein synthesis. In correlation with the
decrease in liver function, there is a derangement of the blood clotting mechanism, icterus
(jaundice), and a decrease in essential serum proteins synthesized by the liver. Other general
signs of aflatoxicosis are edema of the lower extremities, abdominal pain, and vomiting.
1.1.5 Acute toxicity
ICRI (2000) also acute toxicity is less likely than chronic toxicity. The principal target organ for
aflatoxins is the liver. After the invasion of aflatoxins into the liver, lipids infiltrate hepatocytes
and leads to necrosis or liver cell death. The main reason for this is that aflatoxin metabolites
react negatively with different cell proteins, which leads to inhibition of carbohydrate and lipid
metabolism and protein synthesis. In correlation with the decrease in liver function, there is a
derangement of the blood clotting mechanism, icterus (jaundice), and a decrease in essential
serum proteins synthesized by the liver. Other general signs of aflatoxicosis are edema of the
lower extremities, abdominal pain, and vomiting.
1.1.6 Chronic Toxicity
Animals which consume sub-lethal quantities of aflatoxin for several days or weeks develop a
sub acute toxicity syndrome which commonly includes moderate to severe liver damage. Even
with low levels of aflatoxins in the diet, there will be a decrease in growth rate, lowered milk or
egg production, and immunosuppression. There is some observed carcinogenicity, mainly related
to aflatoxin B1. Liver damage is apparent due to the yellow color that is characteristic of
jaundice, and the gall bladder will become swollen. Immunosuppression is due to the reactivity
of aflatoxins with T-cells, decrease in Vitamin K activities, and a decrease in phagocytic activity
in macrophages (ICRI, 2000).
7

1.1.7 Cellular Effects
Aflatoxins are inhibitors of nucleic acid synthesis because they have a high affinity for nucleic
acids and polynucleotides. They attach to guanine residues and for nucleic acid adducts.
Aflatoxins also have been shown to decrease protein synthesis, lipid metabolism, and
mitochondrial respiration. They also cause an accumulation of lipids in the liver, causing a fatty
liver. This is due to impaired transport of lipids out of the liver after they are synthesized. This
leads to high fecal fat content. Carcinogenisis has been observed in rats, ducks, mice, trout, and
subhuman primates, and it is rarely seen in poultry or ruminants. Trout are the most susceptible.
In fact, 1ppb of aflatoxin B1 will cause liver cancer in trout. Carcinogenisis occurs due to the
formation of –8, 9-epoxide, which binds to DNA and alters gene expression. There is a
correlation with the presence of aflatoxins and increased liver cancer in individuals that are
hepatitis B carriers (ICRI, 2000).
1.2 JUSTIFICATION
Smoked-dried fish constitute a major source of animal protein for a vast majority of the
population in Nigeria, particularly the rural areas.
It is therefore important from toxicological points of view to investigate the effect of
smoke on aflatoxin production.
1.3 OBJECTIVES
The study was undertaken to determine the quantities of aflatoxins produced on smoke
dried fish.
To determine the implication of high level consumption of smoked dried fish infested
with aflatoxins on the health of human beings.
8

CHAPTER TWO
2.0 LITERATURE REVIEW
Aflatoxins are metabolites of Aspergillus flavus and A. parasiticus (Speare, 2005). The
compounds are both toxic and carcinogenic to a wide range of animals. A review by Ciegler
(1999), complete with bibliography, deals extensively with chemical properties, production
conditions, and biological effects of the aflatoxins. A number of investigators have studied the
formation of aflatoxins on human food. Marth (2000) observed aflatoxin production on Cheddar
cheese and casein, which had been inoculated with A. flavus and A. parasiticus. Frank (2000)
studied the production and diffusion of aflatoxins in apple juice, rye and wheat breads, and
cheese by using a strain of A. flavus isolated from food. Wildman et al. (1992) inoculated a large
number of sterilized and nonsterilized solid foods and fruit juices with A. flavus and obtained
aflatoxins. Sterilized beef infusion and beef pieces supported yields of 15 and 11 jug of aflatoxin
per g of meat, respectively, but no aflatoxins were produced on raw beef because of bacterial
overgrowth. Frank (2000) obtained aflatoxins from A. flavus grown on a large number of foods
including smoked fish, condensed and powdered milk, and egg noodles.
Molds capable of producing aflatoxins are occasional contaminants of foods. Van Walbeek et al.
(2005) found that 16 of 128 fungi isolated from74 food samples produced toxins when cultured
on complex media and on shredded wheat. Molds often grow on meats, fish especially cured
during storage or aging. Some of these molds have toxinogenic potential.
Results obtained from a study carried out on the level of aflatoxins in smoked-dried fish in three
markets in Uyo town, Akwa-Ibom State showed that Aspergillus flavus, Aspergillus tereus,
A.fumigatus, Absidia sp., Rhizopus sp., Aspergillus niger, Mucor sp. Cladosporum sp.,
Penicillium italicum, Penicilium viridatus, Candida tropicalis and Fusarium moniliformis were
found to be associated with smoked dried fishes sold in different market in Uyo. Aspergillus
flavus and Aspergillus tereus, A. fumigates were the dominant mycoflora in decreasing sequential
9

order. Adebayo et al. (2006) reported similar result in marketed bush mango seeds (Irvingia
spp.) stored for sale in Uyo. Penicillium viridatus, Candida tropicalis and Fusarium
moniliformis occurred less frequently. The presence of A. flavus in the samples might probably
make its consumption hazardous to health.
According to Akande and Tobor, 1992 in artisanal fishery, freshly caught fish are covered with
damp sacks and at times, they are mixed with wet grass or water weeds to reduce the
temperature. Fish treated this way is prone to contamination with microorganisms such as
bacteria and fungi. This indicates that spoilage of fish starts right from the aquatic ecosystem.
Handling fishes are also prone to microbial attack especially in artisanal fishery due to
unhygienic methods of reducing temperature. During the smoke drying period, smoking kilns
used in artisanal fishery and the overloading of the fishes on the trays leads to improper
processing which in turn encourages fungal attack (Eyo, 1992). During storage of smoked dried
fish products, good storage practices are not adhering by wholesaler hence stores are not well
ventilated and pest can easily gain access into the stores. The environment in which fishes are
displayed in the market is not always hygienic and this is another avenue for microbial
contamination. Very often, retailers display the smoked-dried fish samples in open trays beside
the gutter on refuse heaps, this also encourages fungi attack and subsequent production of toxins.
This is in agreement with the report of Akande and Tobor, 1992.
2.1 Fungi Producing Aflatoxins
Aflatoxins are produced by the common fungi Aspergillus flavus and the closely related species
A. parasiticus. These are well defined species: A. flavus produces only B aflatoxins and
sometimes the mycotoxin cyclopiazonic acid (CPA), while A. parasiticus produces both B and G
aflatoxins, but never CPA (Pitt, 1993). This simple situation, of just two aflatoxigenic species,
has been complicated by more recent taxonomic findings. Kurtzman et al. (1987) described A.
nomius, a species closely related to A. flavus but which produces small bullet-shaped sclerotia, as
distinct from the large spherical sclerotia produced by many A. flavus isolates. This species is
also distinguished from A. flavus by the production of both B and G aflatoxins (Saito et al.,
2003).
10

A second new species, closely related to A. nomius, was described by Peterson et al. (2001) and
named A. bombycis. These two species were distinguished from each other by differences in
DNA, and also by differences in growth rates at 37 °C. Like A. nomius, A. bombycis produces
both B and G aflatoxins. The species A. ochraceoroseus described by Bartoli et al. (1978) was
recently shown to be another aflatoxin producer. It also produces sterigmatocystin (Klich et al.,
2000). This same isolate was reported by Stubblefield et al. (1970) to produce B but not G
aflatoxins, in line with those assessments. Moreover, Geiser et al. (2000) showed that the
production of small versus large sclerotia does not have taxonomic significance within A. flavus.
Two aflatoxin-producing isolates from Japan, originally classified as aberrant A. tamari (Goto et
al., 1996), were recently described as A. pseudotamarii. Like A. flavus, this species produces B
aflatoxins and CPA, but differs from A. flavus by the production of orange-brown conidia (Ito et
al., 2001).
In studying population genetics of A. flavus, Geiser et al. (2000) showed that A. flavus from an
Australian peanut field comprised two distinct subgroups, which they termed Group I and Group
II, and suggested that Group II differed from Group I sufficiently to be raised to species level.
Further studies by Geiser et al. (2000) and independent observations have confirmed that A.
flavus Group II comprises a distinct species, which will be described as ‘Aspergillus australis’.
Unlike any other known species, A. australis produces both B and G aflatoxins and also CPA.
It appears to occur almost exclusively in the southern hemisphere, where it has been found in
Argentina, Australia, Indonesia and South Africa.
The evidence indicates that A. flavus and A. parasiticus are responsible for the overwhelming
proportion of aflatoxins found in foodstuffs throughout the world. Of the other species, only A.
australis, which appears to be widespread in the southern hemisphere and is common in
Australian peanut soils, may also be an important source of aflatoxins in a few countries.
Results obtained from a study carried out on the level of aflatoxins in smoked-dried fish in three
markets in Uyo town, Akwa-Ibom State showed that Aspergillus flavus, Aspergillus tereus,
A.fumigatus, Absidia sp., Rhizopus sp., Aspergillus niger, Mucor sp. Cladosporum sp.,
Penicillium italicum, Penicilium viridatus, Candida tropicalis and Fusarium moniliformis
were found to be associated with smoked dried fishes sold in different market in Uyo.
11

Aspergillus flavus and Aspergillus tereus, A. fumigates were the dominant mycoflora in
decreasing sequential order. Adebayo et al. (2006) reported similar result in marketed bush
mango seeds (Irvingia spp.) stored for sale in Uyo. Penicillium viridatus, Candida tropicalis and
Fusarium moniliformis occurred less frequently. The presence of A. flavus in the samples might
probably make its consumption hazardous to health.
According to Akande and Tobor, 1992 in artisanal fishery, freshly caught fish are covered with
damp sacks and at times, they are mixed with wet grass or water weeds to reduce the
temperature. Fish treated this way is prone to contamination with microorganisms such as
bacteria and fungi. This indicates that spoilage of fish starts right from the aquatic ecosystem.
Handling fishes are also prone to microbial attack especially in artisanal fishery due to
unhygienic methods of reducing temperature. During the smoke drying period, smoking kilns
used in artisanal fishery and the overloading of the fishes on the trays leads to improper
processing which in turn encourages fungal attack (Eyo, 1992). During storage of smoked dried
fish products, good storage practices are not adhering by wholesaler hence stores are not well
ventilated and pest can easily gain access into the stores. The environment in which fishes are
displayed in the market is not always hygienic and this is another avenue for microbial
contamination. Very often, retailers display the smoked-dried fish samples in open trays beside
the gutter on refuse heaps, this also encourages fungi attack and subsequent production of toxins
(Akande and Tobor, 1992).
2.2 Production and Reduction
Apart from natural formation, aflatoxins are produced only in small quantities for research
purposes, by A. flavus or A. parasiticus fermentations on solid substrates or media in the
laboratory. Aflatoxins are extracted by solvents and purified by chromatography. Total annual
production is less than 100g (IARC, 1993).
Aflatoxins occurring naturally in foods and feeds may be reduced by a variety of procedures.
Improved farm management practices, more rapid drying and controlled storage are now defined
within GAP (Good Agricultural Practice) or HACCP (Hazard Analysis and Critical Control
Point) (FAO, 2005). By segregation of contaminated lots after aflatoxin analyses and by sorting
12

out contaminated nuts or grains by electronic sorters, contaminated lots of peanuts or maize can
be cleaned up to produce food-grade products.
Decontamination by ammoniation or other chemical procedures can be used for rendering highly
contaminated commodities suitable as animal feeds.
2.3 Uses
Aflatoxins are not used commercially, only for research.
2.4 FORMATION AND OCCURENCE
2.4.1 Prevalence of Toxigenic Species in Foods
Because of the importance of aflatoxins, A. flavus has become the most widely reported
foodborne fungus—even with the provision that A. parasiticus is sometimes not differentiated
from A. flavus in general mycological studies. A. flavus is especially abundant in the tropics.
Levels of A. flavus in warm temperate climates such as in the USA and Australia are generally
much lower, while the occurrence of A. flavus is uncommon in cool temperate climates except in
foods and feeds imported from tropical countries.
The major hosts of A. flavus among food and feed commodities are peanuts, maize and
cottonseed; in animals, they include fish, meat. In addition, various spices sometimes contain
aflatoxins, while tree nuts are contaminated less frequently. Low levels may be found in a wide
range of other foods (Pitt et al., 1997).
It seems probable that although A. parasiticus has the same geographical range as A. flavus, it is
less widely distributed. In particular, it has been found only rarely in southeast Asia. The food
related hosts of A. parasiticus are similar to those of A. flavus, except that A. parasiticus is very
uncommon in maize (Pitt et al., 1994).
2.4.2 Factors Affecting Formation of Aflatoxins in Foods
A fundamental distinction must be made between aflatoxin formation in crops and animals
before (or immediately after) harvest, and that occurring in stored commodities or foods.
13

Peanuts, maize and cottonseed are associated with A. flavus, and in the case of peanuts, also with
A. parasiticus, so that invasion of plants and developing seed or nut may occur before harvest.
This close association results in the potential for high levels of aflatoxins in these commodities
and is the reason for the continuing difficulty in eliminating aflatoxins from these products.
In contrast, A. flavus lacks this affinity for other crops and animals, so it is not normally present
at harvest. Prevention of the formation of aflatoxins therefore relies mainly on avoidance of
contamination after harvest, using rapid drying and good storage practice.
2.4.3 Occurrence
Aflatoxins have been found in a variety of agricultural commodities, but the most pronounced
contamination has been encountered in maize, peanuts, cottonseed and tree nuts. Aflatoxins were
first identified in 1961 in animal feed responsible for the deaths of 100, 000 turkeys in the United
Kingdom (Sargeant et al., 1991).
2.5 Presence in Human Biological Fluids
Covalent binding of aflatoxin to albumin in peripheral blood has been measured in a number of
studies (Montesano et al., 1997). The levels of these adducts are assumed to reflect exposure to
aflatoxin over the previous 2–3 months, based on the half-life of albumin. Experimental data
have also shown that this biomarker reflects the formation of the reactive metabolite of aflatoxin
B1 and the level of DNA damage occurring in the livers of rats treated with aflatoxin B1.
Maxwell (2008) has discussed the presence of aflatoxins in human body fluids and tissues in
relation to child health in the tropics. In Ghana, Kenya, Nigeria and SierraLeone, 25% of cord
blood samples contained aflatoxins, primarily M1 and M2, as well as others in variable amounts
(range: 1 µg aflatoxin M1/l to 64 973 µg aflatoxin B1/l).
Of 35 cord serum samples from Thailand, 48% contained aflatoxins at concentrations of 0.064–
13.6 nmol/ml (mean, 3.1 nmol/ml). By comparison, only two of 35 maternal sera obtained
immediately after birth contained aflatoxin (mean, 0.62 nmol/ml). These results show that
transplacental transfer and concentration of aflatoxin by the fetoplacental unit occur (Denning et
al., 1990).
14

Analyses of breast milk in Ghana, Nigeria, Sierra Leone and Sudan showed primarily aflatoxin
M1, aflatoxin M2 and aflatoxicol. Aflatoxin exposure pre- or post-natally at levels ≥ 100 µg/l
was very often associated with illness in the child (Maxwell, 2008). Exposure of infants to
aflatoxin M1 from mothers’ breast milk in the United Arab Emirates has been measured by Saad
et al. (1995). Among 445 donors of breast milk, 99.5% of samples contained aflatoxin M1 at
concentrations ranging from 2–3 µg/l. The mothers were of a wide range of nationalities, ages
and health status; no correlation was observed between these factors and aflatoxin M1 content of
the milk.
2.6 Absorption, Metabolism and Excretion in Humans
Rigorous quantitative comparisons of dietary intakes and aflatoxin metabolites in body fluids
following absorption and distribution are lacking, aflatoxin M1 concentrations in urine and
human milk have been correlated with dietary aflatoxin intake. However, studies of human
exposure have yielded quantitatively very different correlations between aflatoxin concentrations
in foods and either aflatoxin–protein or aflatoxin–DNA adducts in urine and sera (Hall et al.,,
1994). Hudson et al. (1992) very carefully measured aflatoxin intake based on plate foods in a
village in The Gambia. They found intakes less than those estimated from aflatoxin–serum and
urinary adduct levels in the same individuals. In humans, as with other species, the DNA binding
and carcinogenicity of aflatoxin B1 result from its conversion to the 8,9-epoxide by cytochrome
P450 (CYP) enzymes (Essigman et al., 2002). There is individual variability in the rate of
activation of aflatoxin, including between children and adults, which may be material to the
pharmacokinetics (Wild et al., 2001). The pharmacokinetics of aflatoxins in humans are still not
clearly known.
Factors that explain variation in response to aflatoxin between individual humans, animal species
and strains include the proportion of aflatoxin metabolized to the 8,9- epoxide (mainly by CYP
enzymes) relative to the other much less toxic metabolites and the prevalence of pathways
forming non-toxic conjugates with reduced mutagenicity and cytotoxicity. The metabolism of
aflatoxins in humans has been extensively studied and the major CYP enzymes involved have
been identified as CYP1A2 and CYP3A4 (Gallagher et al., 1998). CYP3A4 mediates formation
of the exo-epoxide and aflatoxin Q1 while CYP1A2 can generate some exo-epoxide but also a
15

high proportion of endo-epoxide and aflatoxin M1. In-vitro evidence that both CYP3A4 and 1A2
are responsible for aflatoxin metabolism in humans has been substantiated by biomarker studies.
Aflatoxins M1 and Q1, produced by CYP1A2 and 3A4, respectively, are present in the urine of
individuals exposed to aflatoxin in human fetal liver, which has the capacity to activate aflatoxin
B1 to the 8,9-epoxide (Kitada et al., 2000). This is consistent with the detection of aflatoxin–
albumin adducts in the cord blood of newborns whose mothers were exposed to dietary aflatoxin
in The Gambia (Wild et al., 2001).
2.7 Toxic Effects in Humans
There are data suggesting that children are more vulnerable than adults to acute hepatotoxicity
resulting from ingestion of aflatoxin. In 1988, 13 Chinese children died of acute hepatic
encephalopathy in Perak, Malaysia (Lye et al., 1995). Common symptoms included vomiting,
haematemesis and seizures; jaundice was detected in seven cases and all children had liver
dysfuntion with elevated serum concentrations of hepatic enzymes. The deaths occurred 1–7
days after hospital admission and were associated with consumption of Chinese rice noodles
shortly before the outbreak. Aflatoxins were found in blood and organs from the children (Chao
et al., 1991). Pesticides, carbon tetrachloride and mushroom poisons were not found. The flour
used to make the noodles was found to contain aflatoxin. Adults who presumably consumed the
same contaminated food were not reported to have been affected (Lye et al., 1995).
Children suffering from protein-energy malnutrition in developing countries may also be
exposed to aflatoxin. In a study conducted in South Africa, aflatoxin concentrations in serum
were higher in 74 children with protein-energy malnutrition than in 35 age-matched control
children. The control group, however, had a higher concentration of aflatoxins in urine (Ramjee
et al., 1992). [Possible explanations for this result are that aflatoxin metabolism is affected in
children with protein-energy malnutrition or that malnourished children are more highly
exposed]. A second study compared children with protein-energy malnutrition with high (n = 21)
and undetectable (n = 15) aflatoxin concentrations in serum and urine. The aflatoxin-positive
group of children with protein-energy malnutrition showed a significantly lower haemoglobin
level (p = 0.02), longer duration of oedema (p = 0.05), an increased number of infections (p =
0.03) and a longer duration of hospital stay (p = 0.008) than the aflatoxin-negative group
16

(Adhikari et al., 2004). This finding confirmed result of an earlier study which suggested that
malarial infections were increased in children exposed to aflatoxin, as determined on the basis of
the amounts of aflatoxin–albumin adducts (Allen et al., 1992). However, a similar study from the
Philippines gave inconclusive results (Denning et al., 1995).
2.7.1 Immuno-suppression
Studies on the immunosuppressive effects of aflatoxins published before 1993 were reviewed in
the previous monograph (IARC, 1993). Aflatoxins modulate the immune system in domestic and
laboratory animals after dietary intake of up to several milligrams per kg feed (Hall and Wild,
1994; Bondy and Pestka, 2000). The major effects involve suppression of cell-mediated
immunity, most notably impairment of delayed-type hypersensitivity, which has been a
consistent observation at low dose levels in various species (Bondy and Pestka, 2000). Other
notable effects include suppression of non-specific humoral substances, reduced antibody
formation, suppression of allograft rejection, decreased phagocytic activity and decreased
blastogenic response to mitogens (WHO, 1990). Strong modification of cytokine secretion and
interleukin gene expression has also been observed in vitro with mycotoxins, including
aflatoxins (Han et al., 1999). The immune system of developing pigs was affected by maternal
dietary exposure to aflatoxin B1 or aflatoxin G1 during gestation and lactation. Motility and
chemotaxis of neutrophils were inhibited in piglets from aflatoxin-treated sows (Silvotti et al.,
1997). In a further study, thymic cortical lymphocytes were depleted and thymus weight was
reduced in piglets from sows exposed to aflatoxin B1 (800 ppb in diet) from day 60 of gestation
up to day 28 of lactation (Mocchegiani et al., 1998).
The effects of aflatoxin B1 on growing rats have been shown to be similar to those in adult
animals. Weanling rats [strain unspecified] were given oral doses of 60, 300 or 600 µg/kg body
weight aflatoxin B1 in corn oil every other day for four weeks. Aflatoxin B1 selectively
suppressed cell-mediated immunity, assessed by measuring the delayed-type hypersensitivity
response, at the 300- and 600-µg/kg body weight doses (Raisuddin et al., 1993).
In order to determine the effect of aflatoxin B1 on the activation of toxoplasmosis, CF1 mice
were injected with the cyst-forming parasite Toxoplasma gondii one month before aflatoxin B1
was given by gavage daily for 50 days at 100 µg/kg body weight. Cysts developed in the brains
17

of all mice, but the lesions were judged to be more severe in the aflatoxin B1-treated animals
(Venturini et al., 2006).
2.7.2 Reproductive and Developmental effects in Humans
Several studies have reported high levels of free aflatoxins in maternal and umbilical cord blood
in humans living in areas where consumption of large amounts of food highly contaminated with
aflatoxins is suspected or has been demonstrated in previous studies.
However, the chemical analysis in each study relied on a single method and the results were not
confirmed by other means. A number of studies have reported effects in infants, but in most
studies, various confounders were not controlled for and exposure levels were not investigated.
Aflatoxins have been reported to occur in up to 40% of samples of breast milk collected from
women in tropical Africa (Hendrickse, 1997).
Maxwell (2008) reviewed the presence of aflatoxins in human body fluids and tissues in relation
to child health in the tropics. In Ghana, Kenya, Nigeria and Sierra Leone, 25% of cord blood
samples contained aflatoxins, primarily M1 and M2, in variable amounts (range for aflatoxin
M1: 7 µg/l–65 µg/l).
Of 35 cord serum samples from Thailand, 17 (48%) contained aflatoxin concentrations of 0.064–
13.6 nmol/ml (mean, 3.1 nmol/ml). By comparison, only two (6%) of 35 maternal sera obtained
immediately after birth of the child contained aflatoxin (mean, 0.62 nmol/ml). These results
demonstrate transplacental transfer and indicate that aflatoxin is concentrated by the feto
placental unit (Denning et al., 1990).
A study of 480 children (aged 1–5 years) in Benin and Togo examined aflatoxin exposure in
relation to growth parameters. Mean concentrations of aflatoxin–albumin adducts in the blood
were 2.5-fold higher in fully weaned children than in those who were still partially breast-fed.
There was a strong negative correlation between aflatoxin– albumin adduct levels in the blood
and both height-for-age (stunting) and weight for- age (being underweight) compared with WHO
reference population data after adjustment for age, sex, weaning status, socioeconomic status and
18

geographical location. These data suggest that aflatoxin may inhibit growth in West African
children (Gong et al., 2002).
In a small study of the presence of aflatoxin in cord blood in Ibadan, Nigeria, a significant
reduction in birth weight was found in jaundiced neonates, who had significantly higher serum
aflatoxin concentrations compared with babies without jaundice (Abulu et al., 1998).
In a study to investigate whether aflatoxins contribute to the occurrence of jaundice in Ibadan,
blood samples were obtained from 327 jaundiced neonates and 60 non-jaundiced controls.
Aflatoxins were detected in 24.7% of jaundiced neonates and in 16.6% of controls. Analysis of
the data indicated that either glucose-6-phosphate dehydrogenase deficiency or serum aflatoxin
are risk factors for neonatal jaundice; odds ratios were significantly increased: 3.0 (95% CI, 1.3–
6.7) and 2.7 (95% CI, 1.2–6.1), respectively (Sodeinde et al., 2005).
Aflatoxins were detected in 14 of 64 (37.8%) cord blood samples from jaundiced neonates
compared with 9 of 60 (22.5%) samples from non-jaundiced control babies in another study in
Nigeria, but the difference was not statistically significant (Ahmed et al., 1995).
Aflatoxins were detected in 37% of cord blood samples in a study of 125 pregnancies in rural
Kenya, with 53% of maternal blood samples being aflatoxin-positive. There was no correlation
between aflatoxins in maternal and cord blood. A significantly lower mean birth weight of
infants born to aflatoxin-positive mothers was recorded for female babies, but not for males (De
Vries et al., 1989).
In cord blood collected from 625 babies in Nigeria, aflatoxins were detected in 14.6% of the
samples. There was no significant difference in birth weight between the groups positive or
negative for aflatoxins (Maxwell et al., 2004).
In a study of the presence of the imidazole ring-opened form of aflatoxin B1–DNA adducts in
placenta and cord blood, 69 of 120 (57.5%) placentas contained the adduct at 0.6–6.3 µmol/mol
DNA and 5 of 56 (8.9%) cord blood samples contained the adduct at 1.4–2.7 µmol/mol DNA.
The results indicate that transplacental transfer of aflatoxin B1 and its metabolites to the progeny
is possible (Hsieh, 2003).
A random sampling of semen from adult men, comprising 50 samples collected from infertile
men and 50 samples from fertile men from the same community in Nigeria, revealed the
presence of aflatoxin B1 in 40% of samples from infertile men compared with 8% in fertile men.
19

The mean concentration of aflatoxins in semen of the infertile men was significantly higher than
that in semen of fertile men. Infertile men with aflatoxin in their semen showed a higher
percentage of spermatozoal abnormalities (50%) than the fertile men (10–15%) (Ibeh et al.,
1994).
2.7.3 Genetic and Related Effects in Humans
DNA and protein adducts of aflatoxin have been detected in many studies of human liver tissues
and body fluids (IARC, 1993). Some studies related the level of adducts detected to
polymorphisms in metabolizing enzymes, in order to investigate interindividual susceptibility to
aflatoxin.
Wild et al. (2001) measured serum aflatoxin–albumin adducts in 117 Gambian children in
relation to GSTM1 genotype and found no difference in adduct levels by genotype.
In a larger study of 357 adults in the same population, aflatoxin–albumin adduct levels were
examined in relation to genetic polymorphisms in the GSTM1, GSTT1, GSTP1 and epoxide
hydrolase genes. Only the GSTM1-null genotype was associated with a modest increase in
aflatoxin–albumin adduct levels and this effect was restricted to non-HBV-infected individuals.
CYP3A4 phenotype, as judged by urinary cortisol metabolite ratios, was also not associated with
adduct level. The main factors affecting the level of aflatoxin–albumin adducts were place of
residence (rural areas higher than urban areas) and season of blood sample collection (dry season
higher than wet season) (Wild et al., 2000). Kensler et al. (1998) also found no association
between aflatoxin– albumin adducts and GSTM1 genotype in 234 adults from Qidong County,
China.
Studies of the types of genetic alteration associated with exposure to aflatoxin in vivo have been
less extensive. In human subjects from Qidong County, China, aflatoxin exposure was
determined as high or low (dichotomized around the population mean) by aflatoxin– albumin
adduct level in serum and compared with the HPRT mutation frequency in lymphocytes. A
raised HPRT mutant frequency was observed in subjects with high compared with low aflatoxin
exposure (OR, 19; 95% CI, 2.0–183) (Wang et al., 1999).
The levels of chromosomal aberrations, micronuclei and sister chromatid exchange were studied
in 35 Gambian adults, 32 of whom had measurable concentrations of aflatoxin– albumin adducts.
20

There was no correlation within this group between the cytogenetic alterations and aflatoxin–
albumin adducts in peripheral blood at the individual level. In a further study, blood samples of
29 individuals of the same Gambian group were tested for DNA damage in the single-cell gel
electrophoresis (comet) assay but no correlation was observed with aflatoxin–albumin adducts or
GSTM1 genotype (Anderson et al., 1999).
2.8 Studies of Cancer in Humans
Beginning in the 1960s and throughout the 1980s, a large number of ecological correlation
studies were carried out to look for a possible correlation between dietary intake of aflatoxins
and risk of primary liver cancer (IARC, 1993). Most of these studies were carried out in
developing countries of sub-Saharan Africa or Asia, where liver cancer is common. With some
notable exceptions, and despite the methodological limitations of these studies, they tended to
show that areas with the highest presumed aflatoxin intake also had the highest liver cancer rates.
However, the limitations of these studies, including questionable diagnosis and registration of
liver cancer in the areas studied, questionable assessment of aflatoxin intake at the individual
level, non-existent or questionable control for the effect of hepatitis virus and the usual problem
of making inferences for individuals from observations on units at the ecological level, led to
increasing recognition of the need for studies based on individuals as units of observation.
In the 1980s, some case–control studies were carried out in high-risk areas, generally based on
reasonably reliable diagnostic criteria for liver cancer (IARC, 1993). The comparability of cases
and controls was limited in some of these studies. Exposure to aflatoxins was sometimes
assessed via dietary questionnaires and sometimes via biomarker measurements. As both of these
were collected after disease onset, their relevance to past lifetime intake of aflatoxins was
uncertain. Beginning in the mid 1980s, some prospective cohort studies were undertaken which
avoided many of the methodological limitations of earlier studies. Among the major advantages
of this new generation of studies were the following: new improved biomarkers of aflatoxin
exposure, improved ability to measure hepatitis infection, better comparability of cases and
controls within a well defined cohort, and control of the temporal sequence by measuring
exposure before disease onset.
In 1992, an IARC Working Group described all relevant human studies that had been
21

eported and concluded that there was sufficient evidence in humans for carcinogenicity
of aflatoxin B1 and of naturally-occurring mixtures of aflatoxins. The outcome investigated in
most studies was liver cancer. Different studies used different sources (e.g., death certificates,
hospital registries, medical examinations) and different criteria (clinical, cytological) for
definition of liver cancer. Different terms, such as liver cancer, primary liver cancer or
hepatocellular carcinoma (HCC) were used.
2.9 Human Biological Fluids
Covalent binding of aflatoxin to albumin in peripheral blood has been measured in a number of
studies (Montesano et al., 1997). The levels of these adducts are assumed to reflect exposure to
aflatoxin over the previous 2–3 months, based on the half-life of albumin. Experimental data
have also shown that this biomarker reflects the formation of the reactive metabolite of aflatoxin
B1 and the level of DNA damage occurring in the livers of rats treated with aflatoxin B1.
Maxwell (2008) has discussed the presence of aflatoxins in human body fluids and tissues in
relation to child health in the tropics. In Ghana, Kenya, Nigeria and Sierra Leone, 25% of cord
blood samples contained aflatoxins, primarily M1 and M2, as well as others in variable amounts
(range: 1 µg aflatoxin M1/l to 64 973 µg aflatoxin B1/l).
Of 35 cord serum samples from Thailand, 48% contained aflatoxins at concentrations of 0.064–
13.6 nmol/ml (mean, 3.1 nmol/ml). By comparison, only two of 35 maternal sera obtained
immediately after birth contained aflatoxin (mean, 0.62 nmol/ml). These results show that
transplacental transfer and concentration of aflatoxin by the fetoplacental unit occur (Denning et
al., 1990).
Analyses of breast milk in Ghana, Nigeria, Sierra Leone and Sudan showed primarily aflatoxin
M1, aflatoxin M2 and aflatoxicol. Aflatoxin exposure pre- or post-natally at levels ≥ 100 ng/L
was very often associated with illness in the child (Maxwell, 2008). Exposure of infants to
aflatoxin M1 from mothers’ breast milk in the United Arab Emirates has been measured by Saad
et al. (1995). Among 445 donors of breast milk, 99.5% of samples contained aflatoxin M1 at
concentrations ranging from 2–3 µg/L. The mothers were of a wide range of nationalities, ages
and health status; no correlation was observed between these factors and aflatoxin M1 content of
the milk.
22

2.10 Cases–Control Studies
Olubuyide et al. (1993) carried out a small case–control study in Nigeria to assess the role of
HBV and aflatoxins in primary hepatocellular carcinoma. Cases were 22 patients at a university
hospital in Ibadan in 1988. Controls were 22 patients from the gastroenterology ward of the same
hospital with acid peptic disease unrelated to liver diseases and matched to cases for sex and age.
Blood samples were collected after subjects were on hospital diet for one week and were
analysed for HBsAg and a number of aflatoxins (B1, B2, M1, M2, G1, G2) and aflatoxicol.
HBsAg was detected in 16 cases and 8 controls. Elevated levels of aflatoxins were detected in
five (23%) cases and one (5%) control, the difference being significant (p < 0.05).
2.11 Analysis of Aflatoxin in Foods
Methods for determining aflatoxins in agricultural commodities and food products have been
verified by AOAC International (Stroka et al., 2001) and by various international committees
(ISO, 2001). The methods have greatly improved in recent years with the commercial
availability of multifunctional columns and immunoaffinity columns, which are simple and rapid
to use, and with reduction in the use of toxic solvents for extraction and clean-up.
A number of approaches have been used to analyse aflatoxins and their metabolites in human
tissues and body fluids. These include immunoaffinity purification, immunoassay (Wild et al.,
1987), high performance liquid chromatography (HPLC) with fluorescence or ultraviolet
detection and synchronous fluorescence spectroscopy (Groopman et al., 1991). Molecular
biomarkers, such as urinary markers, metabolites in milk and parent compounds in blood, are
used for determining exposure to aflatoxins (Groopman, 1993).
23

Table2: Analytical methods validated by AOAC International and the EU
Method No. Aflatoxin Food Method Detection limit
(µg/kg)
975.36 All Food and feeds MC 5–15
(Screening)
979.18 All Maize and peanuts MC 10
(Screening)
990.31 All Maize and peanuts IC 10
(Screening)
994.08 B1, B2, Maize, Almond MFC/HPLC 5
G1, G2 (Mycosep)
999. All, B1 Peanut butter, IC/HPLC NG
(Screening)
989.06 B1 Cottonseed products ELISA 15
(Screening)
990.32 B1 Maize and roasted peanuts ELISA 20
24

Method No. Aflatoxin Food Method Detection limit
(µg/kg)
2000.16 B1 Baby foods (infant formula) IC/HPLC 0.1
990.34 B1, B2, G1 Maize, cottonseed, peanuts ELISA 20–30
978.15 B1 Eggs TLC 0.1
982.24 B1 and M1 Liver TLC 0.1
974.17 M1 Dairy products TLC 0.1
986.16 M1 and M2 Fluid milk HPLC 0.1
6651 B1 Animal feeding stuff TLC/fluorescence 4
Source: IARC (1995); ISO (2001); Stroka et al. (2001)
2.12 Regulations and Guidelines
Efforts to reduce human and animal exposure to aflatoxins have resulted in the establishment of
regulatory limits and monitoring programme worldwide. The rationale for the establishment of
specific regulations varies widely; however, most regulations are based on some form of risk
analysis including the availability of toxicological data, information on susceptible commodities,
sampling and analytical capabilities, and the effect on the availability of an adequate food supply
(Stoloff et al., 1991). In 1995, among countries with more than five million inhabitants, 77 had
known regulations for mycotoxins (all of which included aflatoxins) and 13 reported the absence
25

of regulations. Data were not available for 40 countries (FAO, 2005). The regulation ranges for
aflatoxin B1 and total aflatoxins (B1, B2, G1, G2) were ‘none detectable’ to 30 or 50 µg/kg,
respectively. Seventeen countries had regulations for aflatoxin M1 in milk. The regulatory range
for aflatoxin M1 in milk was ‘none detectable’ to 1.0 µg/kg. New minimum EU regulations to
which all EU countries must adhere were provided in 1998 (European Commission, 1998).
These regulations apply to all aflatoxins (B1, B2, G1, G2) in raw commodities and processed
foods and to aflatoxin M1 in milk. Regulations for other commodities include infant foods
(European Commission, 2001) and selected spices (European Commission, 2002).
However, the US food and Drug Administration has set maximum allowable levels of aflatoxin
in food and feed. Therefore, accurate determination of the presence of the toxin is of major
importance to those monitoring the quality of food and feed in which aflatoxin may occur.
Testing these commodities for the toxin requires careful sampling, chemical extraction,
sanitation and quantitative analysis.
Table: 3 The FDA regulatory levels for aflatoxins
For Level Commodities
Humans 20 ppb All foods such as meat, fish,
except milk
All animal species 20 ppb All feeds (exceptions below)
Exceptions:
Breeding cattle
Breeding swine
Mature poultry
100 ppb Corn
Finishing swine
200 ppb Corn
(>100lbs.)
Finishing beef cattle 300 ppb Corn
Finishing beef cattle,
swine, poultry
300 ppb Cottonseed meal
26

2.13 Outstanding Health Questions in Aflatoxin Management
Increased liver cancer incidence associated with aflatoxins occurs in areas of the world with
chronic high levels of toxins, frequently exceeding the regulatory levels under consideration by
the Codex Alimentarius Commission (2001) by large amounts, sometimes factors of 10 or 100,
and endemic infection with hepatitis B or C viruses (HBV or HCV). Some basis exists for
quantifying the effects of aflatoxin exposure on liver cancer risk, and for the greater impact of
aflatoxins in areas of high HBV or HCV incidence (JECFA, 2001). However, uncertainty
remains regarding the health effects of occasional high exposures occurring due to unusual
weather patterns, in comparison with those due to normal chronic exposure; the effect of
combinations of mycotoxins, e.g. aflatoxins and fumonisins, on cancer risk; and the broader
health effects of aflatoxin exposure. Under the latter consideration, the most important aspects
are the greater sensitivity of children to acute toxicity of aflatoxins as compared with adults; the
in-utero effects of aflatoxins, known to cross the placenta; the immunosuppressive effects of
aflatoxins, which may influence susceptibility to infectious disease; the suppressive effects of
aflatoxins on growth; and the interactions between HCV, HBV and aflatoxins in liver cancer
development in various populations in the world.
The areas mentioned are important ones for future research, to provide information of direct
relevance to public health decisions regarding aflatoxin exposure.
27

CHAPTER THREE
3.0 MATERIALS AND METHODS
3.1 Sample Collection
Smoke dried fishes were randomly sampled and purchased from two different marketing sites,
Itoku and Lafenwa in Abeokuta town, Ogun State, Nigeria. Five samples of related species were
grouped together to make ten composite samples totaling fifty samples in all. They were
subsequently packed in sterile polyethylene bags and tagged accordingly, and taken to Zartech
laboratory for analyses.
3.2 Assay Principles
Veratox for Aflatoxin is a direct competitive ELISA in a microwell format which allows the user
to obtain exact concentrations in part per billion (ppb). Free aflatoxin in the samples and controls
are allowed to compete with enzyme-labelled aflatoxin (conjugate) for the antibody binding
sites. After a wash step, substrate is added, which reacts with the bound conjugate to produce
blue colour. More blue colour means less aflatoxin. The test is read in a microwell reader to yield
optical densities of the controls from the standard curve, and the sample optical densities are
plotted against the curve to calculate the exact concentration of aflatoxin.
3.3 Materials Provided
1. 48 antibody-coated microwells.
2. 48 red-marked mixing wells.
3. 4 yellow-labelled bottles of 0, 5, 15 and 50 ppb.
4. 1 blue-labelled bottle of aflatoxin-HRP conjugate solution.
5. 1 green-labelled bottle of K-Blue Substrate solution.
6. 1 red-labelled bottle of Red Stop solution.
7. Extraction materials (items c through e available in kit form from Neogen):
a. 70% ACS Grade methanol
28

. 250ml graduated cylinder
c. Container with 125ml capacity.
d. Neogen filter syringes. Whatman #1 filter paper
e. Sample collection tubes.
8. High-speed blender.
9. Agri-Grind grinder or blender.
10. Scale capable of weighing 5-50grams
11. Microwell reader with a 650nm filter.
12. Pipettor, 12-channel.
13. Pipettor, 100µl.
14. Pipette tips for 100µl and 12-channel pipettors.
15. Towel as an absorbent material.
16. Plastic bucket for use as a waste receptacle.
17. Microwell holder.
18. Timer.
19. Waterproof marker.
20. Wash bottle.
21. 2 reagents boats for 12-channel pipettor.
22. Good quality grade distilled water.
3.4 Sample Preparation and Extraction
1. The collected samples (smoked-dried fish) were ground into powdery form with the use
of high-speed blender, thoroughly mixed together and made into composite, followed by
weighing on an electronic scale. 5 grams of the representative sample was put into an
extraction cup.
2. 25ml of 70% methanol was added, the extraction cup was covered and shaked vigorously
using hand for 3 minutes, then the mixture was allowed to settle down.
3. The extract was filtered by pouring 5ml through a Whatman #1 filter syringe, and filtrate
was collected as a sample.
4. The sample is now ready for testing.
29

3.5 Test Procedure
1. The kit (containing coated well, mixing well, conjugate, subscript and stop solution) were
set at room temperature (20 0 C).
2. A well holder was obtained and 1 red-marked mixing well for each sample tested plus 4
red-marked wells for controls, and placed in the well holder.
3. An equal number of antibody-coated wells were removed. One end of strip was marked
with a “1”, and strip was placed in the well holder with the marked end on the left.
Marking the inside or bottom of the wells was avoided.
4. Each reagent was mixed swirling the reagent bottle prior to use.
5. 100µl of conjugate from the blue-labelled bottle was placed in each red-marked mixing
well.
6. Using a new pipette tip for each, 100µl of controls and samples were transferred to the
red-marked mixing wells as described below:
0 5 15 50 S1 S2 S3 S4 S5 S6 S7 S8 Strip 1
S9 S10 S11 S12 S13 S14 S15 S16 S17 S18 S19 S20 Strip2
7. Using a 12-channel pipettor, mix the liquid in the well and swirled up and down for 3
times. 100µl was transferred to the anti-body coated wells, then the red-marked mixing
wells.
8. The timer was set for 2 minutes, mixing the wells by forward pipetting and set at room
temperature for incubations by sliding the mixing wells back and forth on a flat surface
without splashing the wells.
9. The content of the antibody wells was shaked, filled with good quality distilled water and
dumped out. Then wells were turned upside-down and tap-dried with towel until the
remaining water has been removed.
10. With new tips on the 12-channel pipettor, 100µl of substrate was added into the wells.
11. The timer was set for 3 minutes, the wells were mixed by sliding back and forth on a flat
surface. The mixture was then discarded and the reagent boat was rinsed with distilled
water.
30

12. Red Stop solution was poured from the red-labelled bottle into the labelled reagent boat.
13. Excess substrate from the 12-channel pipette tips was ejected, and 100µl of Red Stop
solution was pipetted into each well, mixed back and forth on a flat surface and
discarded.
14. The bottom of the microwells was wiped with a dry towel such that there was no fluid
remaining, the plate was taking into the microwell reader using a 650nm filter. Air
bubbles were eliminated, as they could affect analytical results.
3.6 Statistical Analysis
The result was read and calculated using Neogen’s Veratox software, while T-test was used to
test for the significant level of the means.
31

CHAPTER FOUR
4.0 RESULTS
4.1 Sample Analysis
The result of the total aflatoxin concentrations expressed in part per billion (ppb) obtained
from the sampling of ten smoked-dried fish species purchased from local markets in
Abeokuta are shown in table 5.
Table 4: Identified smoked-dried fishes sampled from the two markets (Itoku and
Lafenwa) totaling fifty
Scientific Names
Common Names
Alestes nurse
Synodontis budgeti
Ethmalosa fimbriata
Schilbe uranoscopus
Clarias gariepinus
Gymnallabes typus
Ilisha Africana
Chrysichthys nigrodigitatus
Cynoglossus browni
Calamoichthys calabaricus
Silverside fish
Catfish
Bonga fish
Butterfish
Mudcatfish
Catfish
West African Shad
Silver catfish
Sole
Rope fish
32

PLATES SHOWING THE SAMPLED SMOKED-DRIED FISH SPECIES
Plate 1: Ethmalosa fimbriata
Plate 2: Synodontis budgeti
Plate 5: Ilisha africana
Plate 4: Calamoichthys calabaricus
33

Plate 5: Clarias gariepinus
Plate 6: Schilbe uranoscopus
Plate 7: Chrysichthys nigrodigitatus
Plate 8: Gymnallabes typus
Plate 9: Cynoglossus browni
34
Plate 10: Alestes nurse

Table 5: Result of Laboratory Analysis for the Aflatoxin Levels of the Smoked-dried
fishes sampled from the two markets
Slope: -2.090 Correlation Co-efficient Units: ppb
Sample Description Optical Preliminary Dilution Final
Density Result Factor Result
1 0 ppb 1.220 0.0000
2 5 ppb 0.752 5.290
3 15 ppb 0.497 13.480
4 50 ppb 0.203 52.620
5 A 1.184 0.190 1.0 0.190 ± 0.105
6 B 1.121 0.620 1.0 0.620 ± 0.105
7 C 1.117 0.650 1.0 0.650 ± 0.105
8 D 1.127 0.570 1.0 0.570 ± 0.105
9 E 1.056 1.150 1.0 1.150 ± 0.105
10 F 1.096 0.810 1.0 0.810 ± 0.105
11 G 1.154 0.380 1.0 0.380 ± 0.105
12 H 1.083 0.910 1.0 0.910 ± 0.105
13 I 1.114 0.670 1.0 0.670 ± 0.105
14 J 1.214 0.030 1.0 0.030 ± 0.105
Keys:
A: Alestes nurse F: Gymnallabes typus
B: Synodontis budgeti G: Ilisha Africana
C: Ethmalosa fimbriata H: Chrysichthys nigrodigitatus
D: Schilbe uranoscopus I: Cynoglossus browni
E: Clarias gariepinus J: Calamoichthys calabaricus
Aflatoxin concentrations of the smoked-dried fish samples ranged from 0.030 ppb to 1.150 ppb
with a mean of 0.5980±0.1050 ppb.
35

Figure 1: Aflatoxin concentrations in the different smoked-dried fish samples
36

CHAPTER FIVE
5.0 DISCUSSION, CONCLUSION AND RECOMMENDATIONS
5.1 DISCUSSION
Results obtained from this study showed that aflatoxins were found to be associated with
smoked-dried fishes sold in different markets in Abeokuta, but in non-significant levels
(0.030ppb-1.150ppb) that may not be toxic to human health according to the regulatory levels for
aflatoxins issued by the Food and Administration (FDA) of the United States (The FDA
regulatory levels for aflatoxin intake for humans and all animal species is maximum of 20 ppb).
Adebayo-Tayo et al. (2006), reported different results in marketed smoked-dried fish stored for
sale in Uyo, Akwa-Ibom State, whereby the presence of aflatoxins were in higher concentrations
(The Aflatoxin concentrations in the samples were between 1.5ppb – 8.1ppb) in the samples
which might probably make their consumption hazardous to health. Such a higher difference in
aflatoxin concentrations might be as a result of higher relative humidity usually recorded in Uyo,
unlike Abeokuta town. This might favour the accumulation of aflatoxin due to high moisture
content when displayed for sale in the market. The processes of handling fishes are also prone to
aflatoxin contamination especially in artisanal fishery due to unhygienic methods of
preservation. According to Akande and Tobor (1992), in artisanal fishery, freshly caught fish are
covered with damp sacks and at times, they are mixed with wet grass or water weeds to reduce
the temperature. Fish treated this way is prone to contamination with microorganisms such as
bacteria and fungi. This indicates that spoilage of fish starts shortly after harvesting. During the
smoke-drying period, smoking kilns used in artisanal fishery and the overloading of the fishes on
the trays leads to improper processing which in turn encourages aflatoxin contamination (Eyo,
1992). During storage of smoked-dried fish products, good storage practices are not adhered to,
hence stores are not well ventilated and pests can easily gain access into the stores. The
environment in which fishes are displayed in the market is not always hygienic and this is
another avenue for aflatoxin contamination. Very often, retailers display the smoked-dried fishes
in open trays beside the gutter on refuse heaps, this also encourages fungal attack and subsequent
production of toxins. This is in agreement with the report of Akande and Tobor (1992). The
37

esult also revealed that Aflatoxins were detected in all of the samples. The aflatoxin
concentration ranged from 0.030ppb-1.150ppb (Table 4). Rope fish had the lowest aflatoxin
concentration while Mud catfish had the highest aflatoxin concentration as shown in Table 4.
The extracted smoked-dried fish samples produced bluish and greenish spots during laboratory
analysis. Sharma (2002) reported that the two major metabolites of Aspergillus sp. called
aflatoxins were designated B and G because they fluoresce blue (B ) and green (G ) when
exposed to long-wave ultraviolet light.
Aflatoxins are highly carcinogenic causing hepatoma (cancer of the liver) and have also been
associated with acute hepatitis in man, mostly the developing world (Eaton and Groopman,
2004). Aflatoxin have been reported in grapes in France (Sage et al., 2002), edible nuts and nut
products, milk and milk products (Prasad, 1992), bush mango seeds (Adebayo-Tayo et al.,
2006). The implication of this report is that, though in Abeokuta most of the populace feed on
smoked-dried fishes, it can be confirmed that most of the consumers would have been
consuming aflatoxins. Though, in relatively small amount, but, prolong intake of these aflatoxins
may constitute a health hazard. Goldbatt and Stolloff (1993) reported that aflatoxins occurred in
the human diet and this could pass from feed to milk. Since, improper smoking and drying of
fishes may lead to insect infestation, fungal attack, fragmentation and degradation of the product
(Eyo, 1992), it is therefore important that both the artisanal fishermen and the marketers should
adapt a better method of preservation. Better smoking kilns should be provided for artisanal
fishermen at subsidized rates and fish product should be well stored.
5.2 CONCLUSION
This work has dealt with a number of issues. Many areas have been highlighted which are
in need of much future research to provide information that is directly relevant not only
to public health, but also to availability of a wholesome food and feed supply.
Smoked-dried fishes samples stored for sale in Abeokuta markets were not heavily
contaminated with aflatoxins and they are still acceptable for consumption due to the
aflatoxin levels, which is less than the prescribed maximum concentration.
38

Increased liver cancer incidence associated with aflatoxin exposure occurs in areas where
there is intake of high levels of aflatoxins (often higher than the regulatory limits).
5.3 RECOMMENDATIONS
There is a need for further study of interaction between aflatoxins and hepatitis virus and
joint effects of aflatoxins and hepatitis virus on liver cancer.
Pharmacokinetic studies of ingested aflatoxins in humans (with and without liver
disease).
Populations with a low mean aflatoxin intake are likely to achieve a decrease in liver
cancer cases by introducing lower aflatoxin limits.
Surveillance should be targeted to staple foods.
HACCP (Hazard analysis and critical control point) principles can be used to highlight
the roles that fungal ecology play in aflatoxin contamination, prevention and control.
The stakeholders in the production chain, particularly farmers, fish processors should be
made aware of the importance of measures to reduce aflatoxin contamination.
Training programmes for the development of practical control and management strategies
should be developed and conducted in order to set up strong aflatoxin management
programmes.
Complicated aflatoxin analysis procedure should be replaced with commercial kits such
as Veratox Quantitative Aflatoxin Test used for this particular work, which is easy to run.
Health regulatory bodies such as NAFDAC should carry this out so that aflatoxin can be
easily detected and samples containing them in very high concentrations should be
discarded.
39

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48

APPENDIX I
One- Sample Statistics
FINAL
RESULT
N Mean Standard Standard Error
Deviation Mean
10 0.598000 0.3321245 0.1050270
The mean of the sample result obtained was 0.5980, while standard error was 0.3321.
The required maximum level of aflatoxin concentration in human and even all animal species is
20 ppb.
Thus,
H 0 : µ= 20
H 1 : µ≠20
Level of significant (α): 0.05
49

APPENDIX II
One –Sample T-test
Test Value =20
95% Confidence Interval of
the Difference
T df Sig. (2-tailed) Mean
Difference
Lower
Upper
FINAL
RESULT
-184.733 9 0.000 -19.4020000 -19.639588 -19.164412
P→Value = (0.000)
Since P Value is less than 0.05, which is the level of Significance used, we thereby reject Ho
(Null hypothesis) and accept H 1 (Alternative hypothesis).
However, from the analyses above, it shows that the level of aflatoxins in the smoked-fish
samples does not equal to 20. Since the average of the sample is less than 20, thus, it is not toxic
to human health.
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