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Gas chromatography-massspectrometric determinationof polycyclic aromatichydrocarbons in five speciesof fish from three sites in theArabian GulfIman Al-Saleh a & Inaam Al-Doush aa Biological and Medical Research Department(MBC#03) , King Faisal Specialist Hospital andResearch Centre , P.O. Box: 3354, Riyadh, 11211,Saudi ArabiaPublished online: 21 Jul 2010.

To cite this article: Iman Al-Saleh & Inaam Al-Doush (2002) Gas chromatography-mass spectrometric determination of polycyclic aromatic hydrocarbons in fivespecies of fish from three sites in the Arabian Gulf, International Journal ofEnvironmental Health Research, 12:2, 193-200, DOI: 10.1080/096012022129373

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ISSN 0960-3123 printed/ISSN 1369-1619 online/02/020193-08 © 2002 Taylor & Francis LtdDOI: 10.1080/09603120220129373

International Journal ofEnvironmental Health Research 12, 193–200 (2002)

TECHNICAL NOTE

Gas chromatography–mass spectrometricdetermination of polycyclic aromatichydrocarbons in five species of fish from threesites in the Arabian GulfIMAN AL-SALEH and INAAM AL-DOUSH

Biological and Medical Research Department (MBC#03), King Faisal Specialist Hospital and Research Centre, P.O.Box: 3354, Riyadh 11211, Saudi Arabia

A gas chromatography–mass spectrmetroic (GC–MS) method was developed to measure six polycyclicaromatic hydrocarbons (PAHs) in 54 fish samples. Five fish species highly consumed by the localpopulation (shrimps, Emperors, Rabbitfish, Doublebar Bream and Greasy Grouper) were selected fromthree different sites on the Gulf coast of Saudi Arabia where agricultural, municipal and petroleum industryactivities take place. Variations in PAH levels among the three sites were not significant. Totalconcentrations of PAHs benzo(a)anthracene, chrysene, and benzo(b)fluoranthene ranged from non-detectable to 44.9 mg kg–1. In this study, concentrations of benzo(a)anthracene, chrysene, benzo(b-)fluoranthene and total PAHs greater than the acceptable tolerance limit (1 mg kg–1) were found in 68.5,40.7, 51.9 and 83.3% of the fish samples, respectively. PAH contents in fish vary considerably withspecies; Doublebar bream contain the highest while shrimps contain the lowest. This pilot study clearlyshows that the consumption of fish could be a source of exposure of the local population to PAHs. Sincethere is a consensus on the substantial contribution of PAHs to cancer in humans, it would be interestingto conduct further research in order to determine the magnitude of the problem along other coastal regionsof Saudi Arabia.

Keywords: Polycyclic aromatic hydrocarbons (PAH); fish; Arabian Gulf; GC–MS; Saudi Arabia.

Introduction

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants. Anumber of them are carcinogenic and/or mutagenic, which may have a significant contributionto human cancer (Mastranglelo et al. 1996). PAHs containing four fused rings, such asbenzo(a)anthracene and chrysene, are weakly carcinogenic. Five- or six-fused ring polycyclichydrocarbons include several very potent carcinogens, such as benzo(b)fluoranthene, benzo(a-)pyrene, and indo(1,2,3-cd)pyrene (Lijinsky 1991). These compounds are primarily activatedthrough an oxidative metabolic pathway to electrophilic intermediates capable of covalent

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194 Al-Saleh and Al-Doush

binding to cellular DNA to form DNA adducts. Such adducts, if they are not repaired ormisrepaired, may initiate gene mutations and lead to adverse health effects in humans (Peltonenand Dipple 1995; Kriek et al. 1998). Exposure to PAHs can be due to occupational,environmental and dietary sources. The general population is exposed to PAHs through pollutedair, drinking water and predominantly food. Contamination of food usually results frompreparation particularly smoking and cooking processes and by deposition of small airborneparticles containing PAHs on plants (Bartle 1991; Lijinsky 1991). The aquatic environment isknown to contain a variety of natural and anthropogenic compounds that are capable ofinteracting with the genetic material of aquatic organisms (De Flora et al. 1991). A number ofstudies revealed the contamination of the aquatic environment with PAHs (Akpan et al. 1994;Hernandez et al. 1995; Austin 1999). Sources of contamination can include oil spills and run-offfrom land and industrial effluent as well as deposition of atmospheric PAHs to the aquaticenvironment (Meador et al. 1995; Phillips 1999). PAHs become concentrated in marinesediments, especially in coastal waters, where bottom-feeding fish and filter-feedinginvertebrates are particularly prone to exposure and accumulation of such compounds (Phillips1999). Petroleum products contain considerable quantities of PAHs (Lijinsky 1991). There is amajor concern about the contamination of Arabian Gulf fishes with PAHs after the Gulf war in1991. Furthermore, the presence of major petroleum industries in the Eastern Province mightcontribute significantly to the contamination of the aquatic environment.

Total fishery production of the Kingdom of Saudi Arabia in 1997 was 53,170 metric tons,whereas the production in the Arabian Gulf was 22,875 metric tons (Fisheries Statistics of SaudiArabia 1997). The 10 major species fish groups caught in 1997 for the Arabian Gulf wereshrimps, Emperors, Kingfishes, Seabream, Groupers, Scads-Jacks-Trevallies, Rabbitfish,Snappers-Jobfishes, marine crabs and tuna.

In this study, a rapid method using gas chromatography –mass spectrophotometry wasdeveloped for the separation, extraction and quantitation of PAHs in fish samples. Sixcarcinogenic PAHs were selected and determined in five different fish species consumed in largequantities by populations from the Arabian Gulf at three different sites where there areagricultural, municipal and petroleum industrial activities. These PAHs include benzo(a)an-thracene (BaA), chrysene (Ch), benzo(b)fluoranthene (BaF), benzo(a)pyrene (BaP),indo(1,2,3,cd)pyrene (IcdP) and dibenzo(a,h)anthracene (DbahA), with benzo(b)chrysene(BbCh) and b,b-binaphthyl as internal standards (IS). The presence of these compounds in fishwould serve as an indicator of the extent of pollution in the Arabian Gulf.

Materials and methods

Fresh samples of five species of fish commonly consumed by the local population in SaudiArabia were selected for this study from the coastal Arabian Gulf waters of the Eastern province.The studied fish species were shrimps (Penaeus semisulcatus), Emperors (Lethrinus miniatus),Greasy Grouper (Epinephelus tauvina), Rabbitfish (Siganus canaliculatus), and DoublebarBream (Acanthoparagus bifasciatus). Three selected sites were sampled. Dammam and SharqDareen affected mainly by agricultural activities and municipal wastes (an approximate distanceof 26 km between the two sites). Industrial production is the source of pollution of Munifah site.The latter was located 160 km from Dammam and Sharq Dareen sites. A total of 54 fishbelonging to five species were analyzed for PAHs. A composite sample for each species wasprepared and homongenized from the whole tissue of 15 specimens in a food processor andstored at –20°C. Frozen fish aliquots (25 g) were thawed to room temperature and spiked with

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PAHs in fish in the Arabian Gulf 195

IS and extracted. Reference PAHs standards were purchased from ULTRA Scientific (250 SmithStreet, North Kingstoun, RI 02852, USA). Bbch and b,b-binaphthyl were used as internalstandards and obtained from AccuStandard Inc. Standards Solutions (AccuStandard, 25 SciencePark, New Haven, CT 06511, USA) were stored at 4°C in amber glass vials wrapped withaluminium foil to avoid possible light degradation of some PAHs.

For extraction and clean up, we used a modified method described by Hong et al. (1993).Bbch and b,b-binaphyl (0.4 mg g–1) were added to each fish sample, working standards andspiked samples as internal standards. Analysis of selected PAHs in fish samples were performedusing a Hewlett-Packard gas chromatograph Model 5890 series II with automatic injector anda 7673 autosampler, coupled to a Hewlett-Packard Model 5972 quadrupole mass spectrometerdetector (MSD). A Hewlett-Packard Vectra 466/33N and Hewlett-Packard MS Chemstationsoftware controlled the system. A cross-linked methyl silicone HP5-MS capillary column(30 m 0.25 mm i.d. 0.25 mm film thickness) was used with a column head pressure of 11 p.s.i.in splitless injection mode. High purity helium (99.99%) was used as a carrier gas. The GC oventemperature program was as follows: initial oven temperature was 45°C held for 1 min,increased by 30°C/min to 190°C, and held for 4 min. Then, heated to 310°C at 5°C/min, heldfor 1 min. Total run time was 31.8 min. Injector temperature was 280°C and injection volumewas 1 ml. The MSD was operated at 70 eV in the electron impact (EI) ionization mode.Quantification was based on selected ion monitoring (SIM) as follows: BaA m/z 228, 114, 101;Ch m/z 228, 113, 101; BbF and BaP m/z 252, 126, 113; IcdP m/z 276, 138; DbahA and BbChm/z 278, 139; b,b-binaphthyl m/z 254, 126, 113; and m/z 278, 139.

Individual PAHs were quantified from a four-point calibration curve. Linear calibrationcurves were generated with linear correlation coefficients between 0.998 to 0.99999. PAHs wereanalyzed in four fish samples of each species. Concentrations were expressed as mg kg–1 wetweight of fresh fish. Detection limits for the individual PAHs in fish samples of 25 g using thismethod were 0.414 mg l–1 for BaA, 0.158 mg l–1 for Ch, 0.314 mg l–1 for BbF and 0.366 mg l–1

for BaP. Below detection limit was termed ND (not determined). The analytical recovery forPAHs at the various concentrations tested (0.5–4.0 mg l–1) was 80.6–109.5%, which wasthought to be satisfactory. These spiked fish samples were run with the test samples and blanksusing the same analytical procedure.

Non-parametric statistical analyses were employed including Wilcoxon matched-pairs testand Kruskal–Wallis one-way analysis of variance using Statgraphics Software (1994).

Results and discussion

All species of fish from three sites had undetectable levels of IcdP and DbahA. Unfortunately,we were not able to quantify BaP in the studied fish samples accurately due to degradation.Kruskal–Wallis one-way analyses of variance were carried out to test whether there is avariation in the concentration of individual PAHs among the different species within eachsampling site. No significant variations in the levels of BaA, Ch, BbF and SPAHs was notedamong the three sites, possibly reflecting similar patterns of pollution in waters of the ArabianGulf. Therefore, it was decided to pool the results of individual PAHs and SPAHs from the threesites. Summary statistics for individual PAHs and SPAHs concentrations among the five studiedfish species, shrimps (n = 12), Emperors (n = 10), Rabbitfish (n = 12), Greasy Grouper (n = 12)and Doublebar Bream (n = 8) as well as in all fish species (n = 54) collected from three sites atthe Arabian Gulf are presented in Table 1. Further statistical analyses were carried out to testwhether there is a variation in the concentrations of BaA, Ch, BbF and SPAHs among the five

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196 Al-Saleh and Al-Doush

Tabl

e 1.

Ari

thm

etic

mea

ns±

SD (

mg

kg–

1)

and

rang

es o

f PA

Hs

in t

he f

ive

sele

cted

fis

h sp

ecie

s of

the

Ara

bian

Gul

f

PAH

sSh

rim

ps( n

=12

)E

mpe

rors

( n=

10)

Rab

bitf

ish

( n=

12)

Dou

bleb

ar b

ream

( n=

8)G

reas

y G

roup

er( n

=12

)A

ll f

ish

spec

ies

( n=

54)

BaA

ND

6.63

8±

3.26

54( 2

.6–1

2.4)

8.31

08±

11.3

541

( 0–

40.0

)7.

2624

±3.

3307

( 1.0

09–1

1.6)

3.89

42±

1.96

42( 0

–7.0

)5.

017

±6.

366

( 0–

40.0

)

Ch

ND

1.10

01±

2.03

00( 0

–6.

4)0.

1808

±0.

2684

( 0–

0.6)

4.99

5±

2.34

56( 1

.8–

9.0)

1.82

08±

0.06

3( 1

.74–

1.96

)1.

389

± 2.

057

( 0–

9.0)

BbF

1.06

25±

1.76

13( 0

–6.

0)6.

142

±8.

9061

( 0–

23.3

)2.

7058

±1.

9717

( 0–

4.9)

7.84

±7.

4332

( 1.3

4–25

.0)

0.55

83±

1.93

41( 0

–6.

7)3.

260

±5.

527

( 0–

25.0

)

IcdP

ND

ND

ND

ND

ND

ND

Dba

hAN

DN

DN

DN

DN

DN

D

SPA

Hs

1.06

25±

1.76

13( 0

–6.

0)13

.880

±13

.702

( 4.4

–42

.1)

11.1

98±

12.5

01( 1

.8–1

3.16

)20

.097

±8.

488

( 9.5

4–37

.62)

6.27

3±

2.91

6( 0

.57–

44.9

)9.

666

±10

.766

( 0–

44.9

)

S-P

AH

s, B

aA+

Ch

+B

bF.

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PAHs in fish in the Arabian Gulf 197

Tabl

e 2.

Kru

skal

–W

alli

s on

e-w

ay a

naly

sis

of v

aria

nce

betw

een

five

fis

h sp

ecie

s co

llec

ted

from

the

Ara

bian

Gul

f w

ith

resp

ect

to P

AH

s ex

posu

re

Spec

ies

BaA

aver

age

rank

s

H( p

)C

hav

erag

era

nks

H( p

)B

bFav

erag

era

nks

H( p

)S

PAH

sav

erag

era

nks

H( p

)

Dou

ble-

brea

m (

n=

8)40

.75

28.4

06( 0

·000

01)

48.6

2539

.754

( 4·8

66 ´

10–

8)

43.3

1318

.975

( 7·9

5 ´

10–

2)

45.2

529

.654

( 5·7

´10

–4)

Em

pero

rs (

n=

10)

37.8

526

.029

.25

32.2

5G

reas

y G

roup

er (

n=

12)

25.0

4239

.083

16.3

3326

.917

Rab

bitf

ish

( n=

12)

31.0

18.0

8332

.25

31.4

17S

hrim

ps (

n=

12)

9.0

12.5

21.9

178.

375

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198 Al-Saleh and Al-Doush

fish species, as illustrated in Table 2. Kruskal–Wallis one-way analysis of variance showed thatthe highest average ranks for BaA, Ch, BbF and SPAHs was found in Doublebar Bream,whereas shrimps had the lowest average ranks. Variations in the contamination level of fishmight be related to a number of factors such as species, feeding habits, bioavalibility ofchemicals in food and water and physiochemical parameters of the aquatic environment (Pietersand Geuke 1994; Joiris et al. 1997; Svobodova et al. 1999). In Finland and Germany, a tolerancelimit of 1 mg kg–1 for BaP in smoked food, meat and cheese was established (Hietaniemi andKumpulainen 1996). In this study, concentrations of BaA, Ch, BbF and SPAHs greater than1 mg kg–1 were found in 68.5, 40.7, 51.9 and 83.3% of the fish samples, respectively. TheSPAHs concentrations in five fish species were in the following order: Doublebar Bream20.097mg kg–1) > Emperors (13.880 mg kg–1) > Rabbitfish (11.198 mg kg–1) > GreasyGrouper (6.273 mg kg–1) > shrimps (1.063 mg kg–1). Our data are higher than the sum of BaA,Ch and BbF reported by Lodovici et al. (1995). in trout (1.18 mg kg–1) and those measured byGomaa et al. (1993) in smoked fish with the range of 0 in shrimps to 12.4 mg kg–1 inoysters.

When we calculated the daily intake of SPAHs, expressed as mg day–1, obtained bymultiplying the average quantity of fish consumed per Saudi family per day (9.967 g) by theconcentration of SPAHs in studied fish (Al-Nozha et al. 1991), it would correspond to dailySPAHs intake of 0.096 mg in the range of 0–0.448 mg. Thomson et al. (1996) and Gomaa et al.(1993). reported figures of 1.2 and 3.0 mg daily total dietary intake of PAHs from New Zealandand Italian diets, respectively.

Conclusion

Although, the present study was limited to small area within the Eastern coastal region ofSaudi Arabia, it seems that the evidence of PAH contamination in the studied fish species issufficient to raise our concern about their consumption by the local population. Additionalfactors of contamination, such as mode of cooking, food processing and preserving (Lijinsky1991; Phillips 1999) should be taken into account. Concerns have been raised about thecarcinogenic properties of PAHs in experimental animals and humans (Lee et al. 1998; Arcaroet al. 1999; Roberts-Thomson et al. 1999; Ross and Nesnow 1999). On this basis, furtherinvestigation is needed to assess the magnitude of the problem along other coastal regions ofSaudi Arabia.

Acknowledgements

Authors would like to acknowledge gratefully the following contributors from Ministry ofAgriculture and Water: Mr. Abdulaziz Al-Yahya, the General Director of Marine Fisheries; Mr.Fahad Al-Jamea, the General Director of Fishery Research Centre in Qatief; and Mr. Nabil Fita,the Fishery Specialist for their help in selecting sampling sites, species of fish and providing fishsamples.

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