ijst comparative study on the influence of grilling height ... · food processing or cooking steps...

International Journal of Science and Technology Volume 5 No. 9, September, 2016

IJST © 2016– IJST Publications UK. All rights reserved. 425

Comparative Study on the Influence of Grilling Height on the Concentration

of Polycyclic Aromatic Hydrocarbons (PAHS) and Some Toxic Metals in

Grilled Foods

Onwukeme, V. I., Obijiofor, O. C. and Tabugbo, I. B.

Pure and Industrial Chemistry Department, Nnamdi Azikiwe University, Awka, Anambra State, Nigeria.

ABSTRACT

A comparative study was carried out on the effect of grilling height on the extent of accumulation of the 16 US EPA priority PAHs

and some volatile (As and Hg) and non-volatile (Mn, Cu, Pb, Cr, Cd and Ni) heavy metals in two kinds of roasted foods; yam

(Dioscorea Rotundata ) and plantain (Musa Paradisiaca) commonly consumed in Nigeria. Yam and two variants of plantain (unripe

and ripe) were considered for this study. Two grilling heights (10 cm and 20 cm) were employed on each of the samples, amounting

to a total of nine samples with the raw samples of each serving as reference. The samples were simulated as obtainable in normal

practice for an expected duration, dried to constant weight, pulverised and homogenized. PAHs were extracted using soxhlet extraction

technique, prior to clean up by column chromatography and determined using High Performance Liquid Chromatography with Ultra

Violet (UV) Detector (HPLC-UVD), (ChemStation). Whilst, the heavy metals were determined using Atomic Absorption Spectrometer

(AAS), (Perkin Elmer, Aanalyst 200), after acid digestion with aqua regia. The total PAHs and heavy metals concentrations were

investigated to be higher in the 10 cm grilling height than in the 20 cm grilling height.

Keywords: Polycyclic aromatic hydrocarbons (PAHs), Heavy metals, grilling heights, HPLC and AAS.

1. INTRODUCTION

Roasting/grilling is a dry heat cooking process and is one of the

most popular methods used for cooking foods. The roasting

process tends to evaporate and reduce the moisture content of any

type of food, shrinking the fibre and making the food tough.

The safety issues related to the possibility of heavy metals and

some polycyclic aromatic hydrocarbons are of concern. The

subject of heavy metals and polycyclic aromatic hydrocarbons is

receiving increasing popularity in food industry due to increasing

incidents of contamination in agriculture and seafood sources.

Apart from the threat from polluted environment, smoked food

is subjected to heavy metals and polycyclic aromatic

hydrocarbons contamination during the smoking process. The

ingestion of food is an obvious means of exposure to these

toxicants as grilled/roasted foods are one of the delicacies

enjoyed by Nigerians [1].

The levels of polycyclic aromatic hydrocarbons and heavy

metals in our environment and foods have been the focus of

scientific investigations in the recent time because of the negative

health impacts of some of them. Their presence in foods has been

linked with the various methods of preparing our foods. Grilled

yam and plantain otherwise called roasted yam and plantain are

two widely consumed snacks in Nigeria. These snacks are

prepared by direct exposure of the fresh forms of these snacks to

heat from wood charcoal or coal. As part of efforts to quantify

the contribution of methods of cooking to the levels of PAHs and

some heavy metals in some of Nigerian foods, the levels of PAHs

and some toxic metals in grilled/roasted yam and plantain was

investigated in this study.

Food processing or cooking steps such as roasting, grilling,

barbecuing and smoking are known to generate and increase the

level of PAHs and heavy metals in the food being cooked [2, 3].

Some findings have shown that people who take diets which are

rich in roasted, barbecued or grilled and smoked foods are likely

to have significant intake of these toxicants [2]. Another major

source of PAHs exposure is cigarette smoking especially the

smokers [1]. As polycyclic aromatic hydrocarbons (PAHs) are a

group of chemical compounds composed of two or more fused

aromatic rings that are formed during the incomplete combustion

or high-temperature pyrolysis of coal, oil, gas, wood, fossil fuels,

garbage, or other substances, such as tobacco and charbroiled

meat [4].

Raw foods should usually not contain high levels of PAH. In

areas remote from urban or industrial activities, the levels of

PAH found in unprocessed foods reflect the background

contamination, which originates from long distance airborne

transportation of contaminated particles and natural emissions

from volcanoes and forest fires. In the neighbourhood of

industrial areas or along highways, the contamination of

vegetation can be ten-fold higher than in rural areas [5].

Heavy metals are very harmful because of their non-

biodegradable nature, long biological half-lives and their

potential to accumulate in different body parts [6, 7]. Excessive

accumulation of heavy metals in agricultural soils through waste

water irrigation may not result in soil contamination, but also

affect food quality and safety [8].

International Journal of Science and Technology (IJST) – Volume 5 No. 9, September, 2016


PAHs are most often identified and quantified using either gas

chromatography (GC) with flame ionization detection (FID) or

coupled to mass spectrometry (MS) or high performance liquid

chromatography (HPLC) with ultraviolet or fluorescence

detection or coupled to MS. While the main analytical techniques

used to determine heavy metals in environmental matrices are:

Atomic Absorption Spectrometry (AAS), Inductively Coupled

Plasma Atomic Emission Spectrometry (ICP/AES), Inductively

Coupled Plasma Mass Spectrometry (ICP/MS), Neutron

Activation Analysis (NAA), X-ray fluorescence (XRF) and Ion

Chromatography (IC).

2. METHODOLOGY

The methodology in this research included sample collection,

sample preparation, sample homogenization, sample extraction,

sample pre-concentration, sample clean-up and the instrumental

analysis.

a. Sample Collection

The fresh samples, yam (Dioscorea Rotundata) and ripe and

unripe plantain (Musa Paradisiaca) used for this investigation

were gotten from a local market, Eke-Awka, in Anambra State,

South-Eastern Nigeria and subsequently identified in the

Department of Botany, Herbarium section, Nnamdi Azikiwe

University, Awka, Anambra State, Nigeria.

b. Sample Preparation

The method of preparation of these snacks involved direct

exposure of peeled fresh yam and the two variants of plantain to

charcoal heat. The fresh yam and plantain were placed on a grid

/wire mesh which was placed on charcoal contained in a charcoal

pot. The heights of the grid from the charcoal were varied with

measures of stacked stones to 10 cm and 20 cm. The lighted

charcoal was continuously fanned to supply heat to the material

until it was satisfactorily grilled.

The direct exposure of these snacks to heat (from wood charcoal)

during their preparation gave motivation for this work, because

direct exposure of food to heat and incomplete combustion of

charcoal have been linked with generation of PAHs [9]. Thus,

the aim of the present study is to compare the level of polycyclic

aromatic hydrocarbons and some heavy metals in grilled/roasted

yam and plantain which are highly consumed by Nigerians at

two employable grilling heights which are highly consumed by

Nigerians.

Raw and roasted food samples were ground, blended and stored

in the refrigerator at 4 ºC prior analysis

.

c. Sample Extraction

1. Polycyclic Aromatic Hydrocarbons (PAHs)

10 g of the blended sample was mixed with 10 g of anhydrous

sodium sulphate and was transferred into a soxhlet extractor

thimble. Approximately 200 mL of the extraction solvent

(dichloromethane) was measured into a 500-mL round bottom

flask containing two clean boiling chips. The flask was attached

to the extractor and the sample was extracted for 6 hours at 4 - 6

cycles/hour. The extract was allowed to cool after the extraction

was complete. The dichloromethane extract was made to dry up

on a water bath set at 50°C and exchanged with 4ml Cyclohexane

for clean-up [10, 11].

3. SAMPLE CLEAN-UP

A column chromatographic technique was employed for the

clean-up. 10g of previously activated 100/200 mesh silica gel at

130°C for 16 hours was weighed into a 50ml beaker with

sufficient volume of methylene chloride, stirred with a glass

stirring rod until an even slurry was made. The slurry was

transferred into a previously cleaned and oven dried 10mm ID

chromatographic column. The column was tapped to settle the

silica gel and eluted the dichloromethane. 1-2 cm of anhydrous

sodium sulphate was added to the top of the silica gel. The

column was pre-eluted with 40 mL of pentane. The rate for all

elution was about 2 mL/min.; the eluate was discarded and just

prior to exposure of the sodium sulphate layer to the air, 2 mL

cyclohexane sample extract was transferred onto the column

using an additional 2 mL cyclohexane to complete the transfer.

Just prior to exposure of the sodium sulphate layer to the air, 25

mL of pentane was added and the elution of the column

continued. Pentane eluate was discarded. Next, the column was

eluted with 25 mL of methylene chloride/pentane (2:3) (V/V)

into a 50 mL K-D flask equipped with a 10 mL concentrator tube

[11, 12]. The collected fraction was further concentrated to

dryness and finally reconstituted in 5 mL acetonitrile for

HPLC/UVD analysis [13].

2. STOCK STANDARD SOLUTIONS

A stock standard solution previously prepared at a concentration

of 1000 μg /mL by dissolving 0.0100 g of assayed reference

material in acetonitrile and diluting to volume in a 10-mL

volumetric flask. The stock standard solution was transferred

into Teflon-sealed screw cap bottle. Store at 4°C and protected

from light.

3. CALIBRATION STANDARDS

PREPARATION

Calibration standards: Calibration standards of five

concentration levels were prepared through dilution of the stock

standards with acetonitrile. One of the concentration levels was

at a concentration near, but above, the method detection limit.

The remaining concentration levels corresponded to the

expected range of concentrations found in real samples or as

defined by the working range of the HPLC/UV detector.

1. HEAVY METALS

a.Digestion of Sample

2 g of dried and pulverized sample was taken into a 100 ml

kjeldahl flask, which was previously cleaned by boiling in a

diluted (2-fold) aqua regia for 8 hours. A 20 ml of aqua regia was

added to the samples and they were heated on a heating mantle

for 2 hours. The digests were allowed to cool and were filtered



into 50 ml volumetric flasks and were made up to mark with

distilled water. The resulting sample solutions were subjected to

the atomic absorption spectroscopic (AAS) measurements after

prior calibration of the Atomic Absorption Spectrometer for all

the metals by running different concentrations of standard

solutions, with average values of three replicates for each

determination [13]. The results were given as μg/g dry weight.

Data obtained from the experiment were statistically treated with

IBM SPSS statistics (version 21).

4. RESULTS AND DISCUSSION

Table I: Table showing the concentrations in μg/g of the 16 PAHs in the samples.

ND- not detected (*): IARC Group 2a: probably carcinogenic to human [14]. (**): IARC Group 2b: possibly carcinogenic to

human [14]. (* and **): classified as carcinogenic to human [15, 16, 17].

Raw

yam

Roasted

yam,

10cm

high

Roasted

yam,

20cm

high

Raw

unripe

plantain

Roasted

unripe

plantain,

10cm

high

Roasted

unripe

plantain,

20cm

high

Raw ripe

plantain

Roasted

ripe

plantain,

10cm

high

Roasted

ripe

plantain,

20cm

high

Naphthalene

(NAP)

2.1531 0.7046 0.3881 ND 2.0633 0.6721 0.6372 0.5040 0.3854

Acenaphthylene

(ACY)

34.8901 1.4238 0.7466 3.9036 14.2063 4.6480 ND 6.5291 4.9843

Acenaphthene

(ACP)

8.6268 0.7226 0.7733 ND 0.7014 0.6390 0.9271 0.7011 0.6249

Fluorene (FLR)) 5.7477 0.5362 0.4510 0.5856 0.6860 4.3529 0.6841 2.8651 5.6953

Phenanthrene

(PHE)

27.5519 1.0744 0.9045 0.6438 1.9096 1.8278 0.4253 2.5347 2.4680

Anthracene

(ANT)

ND 1.2297 1.4309 0.9270 1.9592 1.5065 1.6882 1.8599 1.4804

Fluoranthrene

(FLT)

ND ND ND 0.5222 9.5031 9.2770 2.0002 5.3686 5.2245

Pyrene (PYR) ND 47.1786 41.9955 47.3839 27.7411 55.6250 33.0089 47.5313 41.4598

Chrysene

(CHR)**

ND 0.5909 0.6735 3.3147 1.2905 1.9353 2.4940 2.4380 2.5975

Benzo

[a]anthracene

(BaA)*

ND 1.6494 1.6202 1.0390 2.1920 2.1264 2.1958 1.8043 1.8873

Benzo

[b]fluoranthene

(BbF)**

ND 311.0290 289.6232 173.2246 372.4130 328.4420 64.1304 ND 68.2971

Benzo

[k]fluoranthene

(BkF)**

ND 438.2500 347.4643 97.8393 742.7857 583.1786 571.2143 801.2500 756.8571

Benzo[a]pyrene

(BaP)*

ND 1.9527 1.8059 ND 6.5865 3.9970 3.4658 ND ND

Dibenzo[a,h]ant

hracene (DhA)*

ND 135.2000 100.5125 233.5833 249.0000 238.7083 411.0417 697.0833 622.0417

Benzo[g,h,i]pery

lene (BghiP)

ND 2.1712 ND 15.6753 5.7383 0.6372 ND 4.6684 ND

Indeno[1,2, 3 -

cd]pyrene

(IcdP)**

ND 4.1179 2.7136 1.8974 1.6766 0.3589 1.5726 1.1704 0.4434

Total PAHs 78.9696 947.8310 791.1031 580.5397 1440.4530 1237.9320 1095.4860 1576.3080 1514.4470

Total

carcinogenic

PAHs

0.0000 892.7899 744.4132 510.8983 1375.9440 1158.7470 1056.1150 1503.7460 1452.1240



Figure I: A comparative chart of the concentration of 16 PAHs in the raw sample and samples of the 10 cm and 20 cm grilling

heights in yam.

Figure II: A comparative chart of the concentration of 16 PAHs in the raw sample and samples of the 10 cm and 20 cm grilling

heights unripe plantain.

Figure III: A comparative chart of the concentration of 16 PAHs in the raw sample and samples of the 10 cm and 20 cm grilling

heights in ripe plantain.

The results of the levels of polycyclic aromatic hydrocarbons

(PAHs) in the samples are shown in Table I. Figures I, II and III

represent the comparative charts of the concentration of the 16

PAHs in the raw sample and samples of the 10 cm and 20 cm

grilling/roasting heights of each of the study samples.

The table comprises of the raw and two varied roasting heights

of each of the samples of Yam (Dioscorea Rotundata), unripe

0

500

NAP ACY ACP FLR PHE ANT FLT PYR CHR BaA BbF BkF BaP DhA BghiP IcdP

Co

nce

ntr

atio

n (μg/g

)Raw Yam Roasted Yam, 10cm high Roasted Yam, 20cm high

0

500

1000

Cn

cen

trat

ion

(μg/g

)

Raw Unripe Plantain Roasted Unripe Plantain, 10 cm high Roasted Unripe Plantain, 20cm high

0

500

1000

con

cen

trat

ion

(μg/g

)

Raw Ripe Plantain Roasted Ripe Plantain, 10 cm high Roasted Ripe Plantain, 20 cm high



and ripe plantain (Musa Paradisiaca), giving a total of 9 (nine)

samples.

The samples of raw yam, roasted yam at 10cm and 20 cm

simulated roasting heights were found to have a total levels of

PAHs as 78.9696 μg/g, 947.8310 μg/g and 791.1030 μg/g

respectively, and total carcinogenic PAHs as 0.0000 μg/g,

892.7899 μg/g and 744.4132 μg/g respectively. The raw yam

sample had only Naphthalene (2.1531 μg/g), Acenaphthylene

(34.5901 μg/g), Acenaphthene (8.6268 μg/g), Fluorene (5.7477

μg/g) and Phenanthrene (27.5519 μg/g) of the 16 PAHs under

investigation.

Sample of the roasted yam at 10 cm height had all of the PAHs

detected, but Fluoranthrene. Whereas at 20 cm roasting height,

all but Fluoranthrene and Benzo[g,h,i]perylene were not

detected. There was also a notice of distinct lower concentrations

of the PAHs at the 20 cm roasting height. Naphthalene,

Acenaphthene and Benzo[a]pyrene were not detected in the raw

unripe plantain sample. The concentrations of all the PAHs were

noticed to be reduced at the higher roasting height of 20 cm

except for Fluorene, Pyrene and Chrysene.

The unripe plantain samples had total PAHs concentrations of

580.5397 μg/g, 1440.4530 μg/g and 1237.9320 μg/g and total

carcinogenic PAHs of 510.8983 μg/g, 1375.9440 μg/g and

1158.7470 μg/g for raw unripe plantain and 10 and 20 cm grilling

heights roasted unripe plantain samples respectively.

The concentrations of Chrysene, Benzo [a] anthracene and Benzo

[b] fluoranthene was found higher in the roasted ripe plantain at

20 cm roasting height than in the 10 cm roasting height.

Acenaphthylene and Benzo [g,h,i] perylene were not detected in

the raw ripe plantain sample. Benzo [a] pyrene was found absent

in the roasted ripe plantain samples (10 cm and 20 cm heights).

Benzo [b] fluoranthene was not detected in the 10 cm roasting

height. More also, Benzo [g,h,i] perylene was found absent in the

roasted ripe plantain sample of 20 cm roasting height. On the

overall, the total PAHs and total carcinogenic PAHs for the raw

ripe plantain sample and roasted ripe plantain samples of roasting

heights 10 cm and 20 cm were as follows; 1095.4860 μg/g,

1576.3080 μg/g and 1514.4470 μg/g and 1056.1150 μg/g,

1503.7460 μg/g and 1452.1240 μg/g respectively.

Table II: Table showing the mean concentration in μg/g and standard deviation of heavy metal content in the

samples.

Raw yam Roasted

yam, 10cm

high

Roasted

yam, 20cm

high

Raw

Unripe

Plantain

Roasted

unripe

Plantain,

10cm high

Roasted

unripe

Plantain,

20cm high

Raw Ripe

Plantain

Roasted

ripe

plantain,

10cm high

Roasted

ripe

plantain,

20cm high

M

n

0.032

±0.002

0.039±0.01

1

0.033±0.01

4

0.046±0.02

3

0.062±0.02

2

0.046±0.04

5

0.032±0.00

7

0.072±0.02

3

0.047

±0.012

Pb ND ND ND ND 0.002±0.00

2

ND ND ND ND

Cu 0.131±0.01

4

0.432±0.03

2

0.321±0.01

4

0.511±0.00

3

0.456±0.00

0

0.422±0.03

3

0.123±0.12

4

0.942±0.01

4

0.342±0.00

4

Ni 0.010±0.00

1

ND ND ND ND ND 0.004±0.02

0

0.014±0.00

2

0.012±0.00

0

Cr 0.007±0.00

0

ND ND 0.005±0.00

0

ND ND 0.001±0.00

0

0.021±0.00

3

0.012±0.00

8

Cd 0.002±0.00

0

ND ND ND ND ND 0.001±0.00

1

0.006±0.00

3

0.006±0.00

2

As ND ND ND ND ND ND ND ND ND

Hg ND ND ND ND ND ND ND ND ND

Values are Mean±SD, ND: Not detected

Figure IV: A comparative chart of the concentration of eight heavy metals in the raw sample and samples of the 10 cm and 20

cm grilling heights in yam.

0

0.2

0.4

0.6

Mn Pb Cu

Ni Cr

Cd As

Hg

Mea

n C

on

cen

trat

ion

(μg/g

)

Raw Yam Roasted Yam, 10 cm High Roasted Yam, 20 cm high



Figure V: A comparative chart of the concentration of eight heavy metals in the raw sample and samples of the 10 cm and

20 cm grilling heights in unripe plantain.

Figure VI: A comparative chart of the concentrations of eight heavy metals in the raw sample and samples of the 10 cm and 20

cm grilling heights in ripe plantain.

The results of the mean concentration (μg/g dry weight) and

standard deviation are shown in Table II. Figures IV, V and VI

represent the comparative charts of the concentrations of eight

heavy metals (Mn, Pb, Cu, Ni, Cr, Cd, As and Hg) in the raw

sample and samples of the 10 cm and 20 cm grilling/roasting

heights of each of the study samples.

After measuring the heavy metals concentrations by the process

of Atomic Absorption Spectrometry, data were collected and

their standard deviation was tabulated as in Table II.

Lead (Pb) was only detected in roasted unripe plantain

(0.002±0.002μg/g ) at 10 cm grilling height. Ni, Cr and Cd were

not detected in the roasted forms of yam and unripe plantain, but

in their raw yam 0.010±0.001, 0.007±0.000 and

0.002±0.000 μg/g respectively and only Cr was detected in raw

unripe plantain as 0.005±0.000 μg/g. Ripe plantain had all of

Ni, Cr and Cd detected but recorded a steep higher concentration

in the sample of 10 cm roasting height than of the 20 cm roasting

height. Concentrations of Mn and Cu varied from 0.032 to 0.042

μg/g and 0.123 to 0.511 μg/g in the raw samples and are slightly

higher in the roasted samples except for unripe plantain which

had a higher concentration of Cu in the raw sample

(0.511±0.003μg/g ) than in the roasted samples

(0.422±0.033 and 0.123±0.124μg/g for the 10 and 20 cm

roasting heights respectively). Roasting at 10 cm height appeared

to have greater accumulation of these metals than at 20 cm height.

No variation was recorded for the volatile Arsenic and Mercury

as they were not found present in any of the study samples.

5. CONCLUSION AND

RECOMMENDATION

PAHs production by cooking over charcoal (barbecued, grilled)

is a function of both the fat content of the food and the proximity

of the food to the heat source [18, 19]. Processing procedures,

such as smoking and drying, and cooking of food is commonly

thought to be the major source of contamination by PAHs.

Depending on a number of parameters: time, fuel used, distance

0

0.2

0.4

0.6

Mn Pb Cu Ni

Cr Cd

As Hg

Mea

n C

on

cen

trat

ion

(μg/g

)

Raw Unripe Plantain Roasted Unripe Plantain, 10 cm high Roasted Unripe Plantain, 20 cm high

0

0.5

1

Mn Pb Cu Ni

Cr Cd

As Hg

Mea

n C

on

cen

trat

ion

(μg/g

)

Raw Ripe Plantain Roasted Ripe Plantain, 10 cm high Roasted Ripe Plantain, 20 cm high



from the heat source and drainage of fat, type (grilling, frying,

roasting), cooking results in the production in the food of a

number of compounds including PAHs. Although not precisely

known, it is likely that there are several mechanisms of formation

of PAH such as melted fat that undergoes pyrolysis when

dripping onto the heat and pyrolysis of the meat due to the high

temperature [20, 21]. Conclusively, as gathered from this study,

distance from the heat source has a direct effect on the levels of

PAHs and some heavy metals on grilled or roasted foods. As a

product of this research, grilling/roasting of foods is hereby

recommended at farther distance from the heat source.

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