395

Journal homepage: www.fia.usv.ro/fiajournal

Journal of Faculty of Food Engineering,

Ştefan cel Mare University of Suceava, Romania

Volume XVII, Issue 4- 2018, pag. 395 - 403

DETERMINATION OF POLYCYCLIC AROMATIC HYDROCARBONS AND

HEAVY METAL CONTENTS OF BARBECUE BEEF, FISH AND CHICKEN

* Abel INOBEME
1
, Alexander Ikechukwu AJAI

2
,

Abdullahi MANN
2
, Yahaya Ahmed IYAKA

1Department of Chemistry, Edo University Iyamho, Edo State, Nigeria, inobeme.abel@edouniversity.edu.ng
2Department of Chemistry, Federal University of Technology, Minna, Niger State, Nigeria,

talk2alexajai@gmail.com

*Corresponding author
Received 16th October 2018, accepted 20th December 2018

Abstract: The purpose of this work is to investigate the polycyclic aromatic hydrocarbons (PAHs) and

heavy metal contents of barbecue fish, beef and chicken obtained from Minna, Niger state, using
standard procedures. Extraction of PAHs was done using sonification method. n-Hexane,

dichloromethane (DCM) and mixture of both (1:1) were used as extractants. PAHs content was

analysed using GC-MS while the heavy metal analysis was done using AAS. The results obtained from
the study showed that grilled chicken had the highest content of total PAHs (214.41µg/kg) while beef

had the least (25.71µg/kg). 45 to 98% of the fractions of PAHs observed in the samples were 4 and 5

member rings. Benzo (a) pyrene was detected in all the samples analysed, its concentrations varied
from 1.82 to 12.89 µg/kg. The concentrations of PAHs in the grilled samples were generally higher

than the control. The efficiency of the solvents from this study were in the order: n-hexane > (n-

hexane: DCM) > DCM. With the exception of Pb which had a concentration of 3.17 mg/kg to 4.68

mg/kg, other metals investigated were within safe limit based on international standard. The
concentrations of the metals in chicken and beef samples were in the general order: Fe>Pb>Cu>Mn.

There is need to continually check the contents of PAHs and heavy metals in barbecue food since they

have bio-accumulative tendencies and are deleterious to health.

Keywords: barbecue; heavy metals; polycyclic aromatic hydrocarbons

1. Introduction

The demand for fish, beef and chickens as

well as their products is increasing in most

countries, especially in view of their

significant nutritional values. They are

major components of human diets and

supply essential amino acids and minerals

[1]. However, some of these food

substances get contaminated during

processing either by heat treatment and

other exposure processes. These

contaminants such as polycyclic aromatic

hydrocarbons (PAHs), polycyclic amines

and heavy metals have lethal effects. This

has raised an increasing concern about the

quality and safety of foods in several parts

of the world [2]. Some of the contaminants

even when present in trace amount have

significant ecological impacts due to their

ability to enter the food chain and bio-

accumulate in the tissues of living

organisms [3]. PAHs are known to

constitute one of the prominent classes of

hazardous pollutants. Of primary concern

is the fact that some of them are

carcinogenic, teratogenic and mutagenic

[4]. PAHs are group of organic compounds

containing two or more fused aromatic

rings made up of carbon and hydrogen

atoms. Their formation occurs during

processing of coal, crude oil and natural

gas, as well as incomplete combustion of

coal and other organic substances

including food [5]. The presence of these

compounds in processed meat and other

http://www.fia.usv.ro/fiajournal
mailto:inobeme.abel@edouniversity.edu.ng
mailto:talk2alexajai@gmail.com

Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava

Volume XVII, Issue 4 – 2018

Abel INOBEME , Alexander I. AJAI , Abdullahi MANN , Yahaya A. IYAKA, Determination of polycyclic aromatic
hydrocarbons and heavy metal contents of barbecue beef, fish and chicken, Food and Environment Safety, Volume XVII,
Issue – 2018, pag. 395 – 403

396

food substances has become a subject of

much concern in the recent years [6]. The

concentration of PAHs determined in a

food matrix could be affected by

processing method, extraction techniques

as well as the efficiency of the solvent

employed. Various studies have reported

the presence of PAHs in processed food

substances in different parts of the world

[7]; but much has not been done on

barbecue fish, beef and chicken obtained

from Minna and its environs in Niger

State, Nigeria. Thus the aim of this study is

to determine the PAHs and heavy metals

content of barbecue fish, chicken and meat

obtained from Minna in Niger State.

2. Materials and methods

2.1 Sampling method

A total of six (6) samples were used for the

purpose of this study. At each of the

sampling point, five (5) subsamples were

collected and homogenised so as to ensure

a representative sampling. The samples

collected from each area include beef,

chicken (Agric. type) and fish (Catfish-

Clarias gariepinus) processed using

charcoal grilling. Fresh samples were also

collected and used as control. The samples

were packed in aluminium foil, placed in

polyethylene bags and then transported to

the laboratory for pre-treatment and

subsequent analyses [8].

2.2 Sample pre-treatment

The samples were dried in an oven at low

temperature of 105°C for eight hours. They

were then homogenised using a mortar and

pestle and stored in a refrigerator at 4°C

prior to extraction and other analyses.

2.3 Preparation of PAHs standard and

calibration curve

A mixture of 16 PAHs reference standards

was purchased from Supleco Inc., USA.

Five standard solutions each containing

1.0, 2.0, 5.0, 10.0 and 20.0 cm3 of 500

mg/L of each standard PAH was made up

to 100cm3 with dichloromethane. These

were transferred into a capped and sealed

vial until ready for analysis. Calibration

curve of PAHs was obtained by running

standards of 5, 10, 25, 50 and 100 mg/L. A

calibration curve was obtained by

analysing each of the standard PAHs

solutions prepared with GC/MS. The

concentration of each PAH in the sample

was determined in line with procedure

reported by [9].

2.4 Determination of selected

physicochemical properties

Some physicochemical parameters (ash,

moisture and fat contents) were determined

in line with [10].

2.5 Ultrasonic assisted extraction of

samples

The ultrasonic extraction was carried out in

line with the method reported by [9] with

slight modifications. The extract was then

filtered and concentrated using a rotary

evaporator under controlled vacuum.

2.6 Silica gel column clean-up

The clean-up was performed in line with

the method reported by [11] using

activated florisil (Magnesium silicate) and

anhydrous Na2SO4. The eluate was

collected into an evaporating flask and

rotary evaporated to dryness. The clean-up

procedure was repeated using the other

solvents (n-hexane and DCM) as eluents.

2.7 Determination of heavy metal

concentration

The determination of selected heavy

metals (Mn, Zn, Cu, Pb, Cr and Cd) was

done using the method reported by [12].

Analysis of variance (ANOVA) was

performed on PAH concentration data

using SPSS software. The significant

statistical level was set at P<0.05.

Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava

Volume XVII, Issue 4 – 2018

397

3. Results and discussion

3.1 Physico-chemical properties

Table 1

Physico-chemical properties of analysed Samples

Fish Chicken Beef

Fat content (%) 16.40±0.70 27.30±0.20 20.81±1.01

Moisture content (%) 25.15±1.21 15.30±1.90 29.40±0.99

Ash content (%) 2. 20±0.93 1.62±0.12 1.41±1.20

Results expressed as mean±SD for triplicate determinations.

Table 1 shows the physico-chemical

parameters of the samples analysed. The

fat contents were 16.40±0.70, 27.30±0.20

and 20.81±1.01% for fish, chicken and

beef respectively. Barbecue beef had the

highest moisture content (29.40±0.99%﴿

while chicken had the least (15.30±1.90%).

Moisture content relates to the freshness

and stability of food for long time storage

[13]. Food samples with very low moisture

content are less prone to microbial attack

hence less perishable [14].

The ash content of the samples in this

study was generally low (1.41 to 2.20%﴿

which relates to the mineral compositions

of the sample. Ash is a measure of the

inorganic components in food sample after

removal of water and organic matter. Fish

had the highest ash content (2.20±0.93%﴿

while beef had the least.

Fat content determination is basic to PAHs

analysis, since the dripping of fat into the

flame is one of the mechanisms of PAHs

formation. Chicken had the highest fat

content (27.30±0.20%﴿ while fish had the

least (16.40 ±0.70%﴿. The low value of fat

observed in some of the samples may be

attributed to the fact that the samples were

not analysed the same day of collection

thereby resulting to the oxidation of fat.

The difference in some of these parameters

with findings from other studies can be

attributed to geographical areas, sex,

amongst others [15].

3.2 Polycyclic Aromatic Hydrocarbon

Contents of the Samples

Most of the lower members PAHs were

not detected in the fish samples (table 2).

This observation may be connected with

the higher volatility of PAHs of lower

molecular weight. The highest amount of

benzo (a) pyrene was obtained from n-

hexane (12.89 µg/kg) and the mixture of

the two solvents (12.79 µg/kg). This

implies that n-hexane is a better extraction

solvent for B(a)P using sonification

method. n-Hexane extract gave a higher

total PAHs content for fish sample

(99.27µg/kg) than DCM (33.05µg/kg).

The total PAHs obtained from fish using n-

hexane was about thrice that of DCM. This

shows that n-hexane is more efficient for

PAHs extraction. The concentration of

B(a)P ranged from 0.99±0.31 to

12.89±0.13 µg/kg. The values for n-hexane

and combined solvent extracts are higher

than 0.63±0.57µg/kg reported by [16] for

B(a)P in a related study. The total PAHs

obtained is higher than a maximum of

14.95mg/kg documented by [17] in

charcoal broiled beef burger. The

concentration of B(a)P in DCM extract is

however lower than 5.0µg/kg based on

[17] standard.

Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava

Volume XVII, Issue 4 – 2018

398

Table 2

PAHs Content of Grilled Fish (µg/kg) using Different Extractants

PAHs DCM n-hexane DCM: n-hexane (1:1) Control

1. Napthalene ND ND ND ND
2. Napthalene, 2-methyl

ND ND ND

3. Biphenylene ND ND ND ND
4. Acenapthene ND ND ND ND

5. Fluorene ND 16.01±0.13b 1.59±0.09a ND
6. Phenanthrene 1.16±0.18a 7.01±0.91b 7.01±0.10b ND
7. Fluoranthene 1.06±0.12a 1.12±0.08a 1.17±0.11a ND
8. Pyrene 1.19±0.80a 4.18±0.10b 4.18±0.14b 0.98±0.91c
9. Benz [a] anthra 1.51±0.12a 11.69±0.16b 11.73±0.04b 3.10±0.32a
10. Triphenylene 1.74±0.10a 1.21±0.09a 1.21±0.21a 2.01±0.19a
11. Benz[b] flu.

7.95±0.18b 12.39±0.11c 12.39±0.09c
5.98±0.12a

12. Benzo [a] pyrene 1.92±0.13a 12.89±0.13b 12.79±0.11b 0.99±0.31a
13. Indeno [1,2,3]pyrene

5.12±1.07a 10.11±0.30b 10.11±0.08b

2.98±0.08a

14. Dibenz [a,h] anthrh 6.29±0.10a 17.19±0.05b 17.11±0.10b ND
15. Benzo [ghi] peryle 5.11±0.11a 5.49±0.81a 5.49±0.59a ND

Ʃ PAH 33.05 99.27 84.78 16.04
Results are expressed as mean ± SD. Values with same superscript on same row do not differ significantly at p< 0.05. ND:
Not detected.

Fig.1 presents the sum total fraction of

PAHs from the grilled fish. Medium

molecular weight (MMW) PAHs

(4 and 5 ring members) constitute majority

(61.09 to 71.46%) of the total PAHs.

Fig.1 The fractions of total PAHs in grilled fish using different extractants LMW:

low molecular weight (2 and 3 rings Pahs); MMW: medium molecular weight (4 and 5 rings PAHs);

HMW: high molecular weight (6 rings).

Studies on carcinogenicity of PAHs have

shown that medium and higher molecular

weight PAHs (4 to 6) rings are more

carcinogenic when compared to lower

molecular weight members [18]. Also

DCM showed better efficiency in the

extraction of six ring PAHs (30.95%)

when compared to n-hexane (15.25%).

Table 3 gives the PAHs content of the

barbecue chicken using various

extractants. The total PAHs concentration

in chicken ranged from 165.63 to

214.41µg/kg. The highest for total PAHs

was observed in the extract of the

combined solvents. n-Hexane gave a

higher yield of total PAHs (185.63µg/kg)

DCM n-hexane DCM:n-hexane

LMW

MMW

HMW

Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava

Volume XVII, Issue 4 – 2018

399

when compared to DCM (165.63µg/kg).

While individual solvents did not extract

naphthalene and 2-methylnaphthalene, but

with a combination of the extracting

solvents, concentrations of 1.08±0.23 to

2.16±0.10 µg/kg were obtained. The least

amount was in the control (1.01µg/kg) The

PAHs contents obtained here are

comparable to the findings of [19] in their

study on smoked fish from southern

Nigeria. The concentrations of PAHs

obtained is lower than 0.12mg/kg reported

by [20] for B(a)P in their study on sardine.

Table 3

PAHs Content of Grilled Chicken (µg/kg) using Different Extractants

PAHs DCM n-hexane DCM: n-

hexane (1:1)

Control

1. Napthalene ND ND 1.08±0.23a ND

2. Napthalene, 2-
methyl ND ND 2.16±0.10a

3. Biphenylene 1.26±0.09a ND 16.11±0.06b ND

4. Acenapthene 2.66±0.01a 13.39±0.89b 13.41±0.12b ND

5. Fluorene 3.57±0.11a 11.89±1.03b 11.85±0.09b ND

6. Phenanthrene 4.94±0.01a 6.91±0.10a 5.89±0.92a ND

7. Fluoranthene 1.19±0.08a 3.73±0.04b 3.77±0.10b ND

8. Pyrene 50.91±0.01b 76.41±0.33c 76.39±2.07c 1.01±0.01
a

9. Benz [a] anthracene 8.19±0.15c 3.09±0.09b 3.09±0.02b 0.99±0.21
a

10. Triphenylene 1.78±0.91a 4.01±0.02b 6.74±0.03c 0.04±0.03
a

11. Benzo [b]
fluoranthene 10.39±0.01b 15.01±0.12c 11.51±0.10bc

2.97±0.01a

12. Benzo [a] pyrene 16.11±0.10c 11.01±0.07b 10.61±0.07b 3.02±0.10
a

13. Indeno[1,2,3,-cd]
pyre 9.04±1.09b 8.02±1.01b 11.59±0.11c

4.98±0.01a

14. Dibenz [a,h]
anthracene 24.79±0.09c 19.11±0.10b 28.11±0.11c

7.01±0.10a

15. Benzo [ghi]
perylene 30.81±0.12c 13.11±0.09b 12.11±0.09b

5.99±0.03a

Ʃ Pah 165.63 185.68 214.41 26.01

Results are expressed as mean ± SD. Values with same superscript on same row do not differ significantly at p< 0.05. ND;
Not detected

Fig.2 shows the distribution of PAHs

extracted from chicken in terms of the

number of rings present. n-Hexane was

more effective in extracting 4- 5 ring

PAHs (71.28%) compared to DCM

(68.43%), mixture of both solvents gave

the least (62.14%). DCM however gave the

highest yield for 6 ring members

(dibenz(a,h)anthracene and

benzo[ghi]perylene).

Fig. 2. The fractions of total PAHs in grilled chicken using different extractants

DCM n-hexane DCM: n-hexane

LMW

MMW

HMW

Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava

Volume XVII, Issue 4 – 2018

400

The PAHs content in barbecued beef is

presented in table 4. The first five

members were not detected in the beef

extract. Indeno [1,2,3-cd] a six ring PAH

was present in highest concentration

among other PAHs (4.88 ±0.06 to

8.79±0.13µg/kg). The concentration of

B(a)P in the grilled samples ranged from

1.48±0.11 to 5.62±0.11µg/kg. Benzo (a)

pyrene (B(a)P) is considered as a

biomarker hence it presence is used in

assessing other PAHs in the environment

[19]. The concentration of

dibenzo(a,h)anthracene and fluorene

observed here are higher than

0.0099mg/kg to 0.104 mg/kg reported by

[21] in roasted meat. They are however

lower when compared to 1.34 to 5.56

µg/kg reported by [22] in a similar study at

Port Harcourt. The presence of pyrene and

benzo(a) pyrene in all the extract is in

agreement with findings of [23].

Table.4

PAHs Content of Grilled Beef (µg/kg) using Different Extractants

PAHs DCM n-hexane DCM: n-
hexane (1:1)

Control

1. Napthalene ND ND ND ND
2. Napthalene, 2-methyl ND ND ND ND
3. Biphenylene ND ND ND ND
4. Acenapthene ND ND ND ND
5. Fluorene ND ND ND ND
6. Phenanthrene 1.08±0.71a 1.11±0.09a ND 0.49±0.01a
7. Fluoranthene ND 1.07±0.55a ND 1.01±0.03a
8. Pyrene 7.01±0.011c 1.01±0.06a ND 0.01±0.21b
9. Benz [a] anthracene 1.29±0.07a 2.54±0.10a 1.59±0.01a 0.98±0.11a
10. Triphenylene 2.36±0.02a 2.66±0.09a 2.49±0.10a 1.02±0.15b
11. Benzo [b] fluoranthene 1.65±0.06a 4.31±0.05b 1.88±0.07a 1.03±0.31a
12. Benzo [a] pyren 1.48±0.11a 1.82±0.10a 5.62±0.11b 1.01±0.23a
13. Indeno [1,2,3, -cd] p 4.88±0.06a 7.89±0.07b 8.79±0.13b ND
14. Dibenz [a,h] anthrace 5.95±0.11a 6.96±0.12a 6.06±0.09a 2.03±0.01a
15. Benzo [ghi] perylene ND 5.49±1.08a ND ND

Ʃ PAH 25.71 34.86 26.52 7.58

Fig. 3. Fractions of total PAHs in grilled beef using different extractants

The fractions of PAHs extracted from beef

in terms of molecular weight and ring

number is shown in fig. 3 The fraction of

lower molecular weight PAHs in all the

100

DCM n-hexane DCM: n-hexane

LMW

MMW

HMW

Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava

Volume XVII, Issue 4 – 2018

401

beef extracts where insignificant (0.00 to

4.20%). 4 and 5 rings PAHs constituted the

major components of the total PAHs.

These groups appear to be of more concern

due to their carcinogenic risk. DCM gave

76.81% of the 4-5 rings compared to

58.43% from n-hexane. Presence of 4-5

member rings above 50% of total PAHs

shows dominance of combustion.

3.3 Heavy metal concentration

Table 5 shows the concentrations of the

metals in the samples. Chromium was not

detected in all the samples analysed. [24]

reported the concentration of chromium as

0.43 to 1.22mg/kg in his study on

barbecued food. The concentration of Fe

ranged from 2.52±0.036 to

3.52±0.100mg/kg. Barbecued beef had the

highest amount while the least was in fish.

Contamination by Fe and other metals may

occur during processing of meat and other

foods through contact with processing

materials and penetration through surfaces

exposed to surrounding environmental

contaminants [25]. The concentration of Fe

from this study is in consonance with 2.13

to 4.65mg/kg reported by [24] in a similar

study. Mn had the least concentration

among the elements analysed. The least

content was in barbecued chicken. Earlier

studies have attributed the presence of

some toxic metals to some of the additives

and spices which some barbecued meat

sellers add prior to processing [26].The

highest amount of copper was in beef

(2.97±0.35mg/kg), which was significantly

higher than those of fish and chicken. The

high content of copper may be related to

environmental contamination due to

exposure in the course of heating alongside

background content. [21] in a related study

on heavy metals in meat reported a

significant increase in the concentration of

metals after grilling, which he attributed to

the method employed in cooking. [25] in a

related study highlighted the content of

copper as 0.15 to 0.35mg/kg. Pb had the

highest concentration which ranged from

1.17±0.100 to 2.68±0.037mg/kg. Pb

content varied significantly among the

samples studied. Fish sample had the

highest content of Pb. The concentration of

Pb in all samples from the present study is

higher than the permissible limit of

0.5mg/kg in meat [8]. The value from the

present study is also higher than

0.02mg/kg reported by [27] in roasted

meat and 0.125 mg/kg documented by [26]

in a related study.

Table 5

Heavy Metals Content of analysed Samples (mg/kg﴿

Metals Fish Chicken Beef Permissible Limit

Cr ND ND ND 1.0 (USDA, 2006)
Fe 2.52±0.036a 2.58±0.047a 3.52±0.100b 43 (WHO, 2011)

Mn 0.51±0.015b 0.28±0.019a 0.48±0.015b 5.5 (WHO, 2011)

Cu 0.94±0.045a 0.95±0.050a 2.97±0.350b 10 (WHO, 2011)

Pb 2.68±0.037c 1.57±0.011b 1.17±0.100a 0.10(WHO, 2011)

Result expressed as mean ± SD for triplicate determinations. Values on same row with same superscript do not

differ significantly at p<0.05.

3.4 Inter-elemental correlation

coefficient

There was a strong positive association

between Fe and Cu (0.99) using Spearman

coefficient (table 6). Fe correlated

negatively with Pb. A weak positive

association (0.35 to 0.38) was observed

among most of the other metals

considered. The positive association

among some of the metals shows their

common origin.

Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava

Volume XVII, Issue 4 – 2018

402

Table 6

Interelemental correlation coefficients in the samples

Cr Fe Mn Cu Pb

Cr 1 - - - -

Fe - 1 0.35 0.99** -0.73

Mn - 0.35 1 0.38 0.37

Cu - 0.99** 0.38 1 -0.72

Pb - -0.73 0.37 -0.72 1

**. Correlation is significant at the 0.01 level (2-tailed).

4. Conclusion

Findings from this study have reveal that 4

to 5-membered ring PAHs made up major

fraction (60 to 80%) of the total PAHs

from the barbecued fish, chicken and beef.

With a few exceptions, the efficiency of

solvents in extraction of total PAHs from

the present study could best be arranged in

the order: n-hexane> mixture (1:1) >DCM.

The mean concentrations of PAHs in the

sample were in the following order:

chicken>fish>beef. The concentration of

B(a)P in some of the samples analysed

exceeded the maximum acceptable limit of

5µg/kg (EFSA, 2008). For heavy metal

analysis, Fe had the highest concentration

in chicken and beef while Mn had the least

in all the samples analysed.

5. Acknowledgments

The authors are sincerely grateful to the

Laboratory technologists of the

Department of Chemistry, Federal

University of Technology, Minna and the

staff of Central Research Laboratory,

University of Lagos, Nigeria.

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1. Introduction
4. Conclusion