Food Chemistry 131 (2012) 469476

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Food Chemistry
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Contents of cadmium, copper, mercury and lead in sh from the Neretva river
(Bosnia and Herzegovina) determined by inductively coupled plasma mass
spectrometry (ICP-MS)
Jasmina Djedjibegovic a,, Thorjrn Larssen b,c, Armin Skrbo a, Aleksandra Marjanovic a, Miroslav Sober a
a
b
c

Faculty of Pharmacy, University of Sarajevo, Cekalusa 90, 71000 Sarajevo, Bosnia and Herzegovina
Norwegian Institute for Water Research, Norwegian Institute for Water Research (NIVA), Gaustadallen 21, NO-0349, Oslo, Norway
Department of Chemistry, University of Oslo, P.O. Box 1033, Blindern, N-0315 Oslo, Norway

a r t i c l e

i n f o

Article history:
Received 30 August 2009
Received in revised form 24 June 2011
Accepted 6 September 2011
Available online 10 September 2011
Keywords:
Heavy metals
Bioaccumulation
Food safety

a b s t r a c t
The concentrations of cadmium, copper, mercury and lead in sh from the Neretva river have been investigated in order to assess safety for consumers and the level of contamination. Samples of muscle and
liver from six sh species (brown trout, common carp, prussian carp, chub, rudd and gray mullet) were
collected at six locations upstream and downstream from the three largest towns along the Neretva river.
Metal concentrations were determined by inductively coupled plasma mass spectrometry after microwave-assisted acid mineralisation. Accumulation of metals in sh tissues was species-dependent. The
average content of metals in sh muscle samples was 0.0130.055, 0.06816.059, 0.0500.401 and
0.0550.703 mg/kg for Cd, Cu, Hg and Pb, respectively. Health effects due to sh consumption could
not be excluded. Further investigation is recommended, including a survey of sh consumption frequency
among the local inhabitants and sports shermen.
2011 Elsevier Ltd. All rights reserved.

1. Introduction
Fish is an important part of the human diet, but also a good indicator of trace metal pollution in the aquatic ecosystem (Rashed,
2001; Rayment & Barry, 2000). High toxicity, bioaccumulation and
environmental persistence make many metals dangerous health
hazards for organisms. Cadmium (Cd), mercury (Hg), copper (Cu)
and lead (Pb) are of particular interest for sheries (Svobodov,
Lloyd, Mchov, & Vykusov, 1993) and as a potential health risk
for humans. Mercury is of most widespread concern in connection
with sh consumption and advisories related to sh consumption
are issued by health authorities in many countries (UNEP, 2002).
However, many other metals are also of concern, although available
information and guidelines related to consumption and toxicity for
humans and predator wildlife are more scattered, but highly relevant (Burger & Gochfeld, 2005). Metals in waters may be of natural
origin from the rocks and soil or from human activities, including
industry, domestic wastewater, agricultural discharge, mine runoff,
solid waste disposal and atmospheric deposition.
Metals of major concern in rural waters include cadmium and
mercury. Practically all metals could be found in waters in urban/industrial regions, depending on type of industry (USEPA,
2000).
Corresponding author.
E-mail address: djedjibegovicj@gmail.com (J. Djedjibegovic).
0308-8146/$ - see front matter 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2011.09.009

The Neretva river, with its tributaries, makes up a great part of the
total water resource of Bosnia and Herzegovina. It is widely used for
sheries (wild sh and sh farming), sports and recreation, for irrigation and as a source of hydroelectric power. Very little recent
information is available regarding the contamination with metals
in the Neretva river catchment, and that is mostly from the lower
part of the Neretva delta in Croatia (Bogut & Bukvic, 2003; HasSchn, Bogut, & Strelec, 2006; Has-Schn et al., 2008). The war activities, uncontrolled sewage disposal and inadequate control by
authorities in the postwar period are reasons to fear serious environmental contamination. This work is the rst screening of metals in
the entire ow of the Neretva river in the post-war period.
Heavy metals and essential elements are routinely determined
in food products. Inductively coupled plasma mass spectrometry
(ICP-MS) is becoming a common analytical technique for such
determinations. The advantages of ICP-MS over other instrumental
techniques, such as ame atomic absorption spectrometry (FAAS),
graphite furnace atomic absorption spectrometry (GF AAS) and
inductively coupled plasma atomic emission spectrometry (ICPAES), have been reported by others (Nardi et al., 2009). Briey,
ICP-MS is characterised by capability of simultaneous multielemental measurement, low detection limits, wider linear dynamic
range, simpler spectral interpretation compared to ICP-AES and
less matrix or spectral interferences. For the above-mentioned reasons, ICP-MS was the technique of choice in the present work.

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J. Djedjibegovic et al. / Food Chemistry 131 (2012) 469476

The Cyprinidae, Salmonidae and Mugilidae sh species inhabiting

Neretva river, that are extensively consumed by the local population, were selected for the analysis. The selection of the sh tissue
to be analysed depends on the aim of the study. When the goal is to
evaluate the safety of sh for human consumption, edible muscle
aklovica, & Saracevic,
tissue is usually analysed (Alic, Milanovic, C
2004; Dalman, Ahmet, & Ahmet, 2006; Hinojosa Reyes, Mizanur
Rahman, & Skip Kingston, 2009; Tapia et al., 2009; Uluozlu, Tuzen,
& Medil, 2007). Evaluation of the bioaccumulation of the metals
(and relative level of the environmental contamination) typically
involves two or more sh tissues and organs, such as liver, gonads
and kidney (Qiao-quiao, Guang-wei, & Langdon, 2007; de Mora,
Fowler, Wyse, & Azemard, 2004; Marcovecchio, 2004; Mansour &
Sidky, 2002). In the present work we analysed metal contents in
the sh muscle and liver, the two organs for which many literature
data exist, in order to:
(a) assess the suitability of analysed sh species for human consumption in respect to concentrations of some heavy metals
(Cd, Cu, Hg and Pb) found in edible muscle tissue;
(b) evaluate the level of contamination of the Neretva river by
comparison of our results with those from other studies.
2. Materials and methods
2.1. Sampling sites
Samples were collected at six locations along the Neretva river.
Sampling sites were located upstream and downstream of three
major settlements (Konjic, Mostar and Metkovic) along the river
(Fig. 1).
The rst sampling site (L1) was approximately 2 km upstream
of Konjic, the rst urban area along the Neretva river. Upstream
of Konjic, the river runs through a mountainous area with very
sparse population, no industry or other major point sources of pollution. The main human activities are a few small sh ponds and
some tourism (especially rafting). The second sampling site (L2)
was about 3 km downstream of Konjic. Besides domestic waste
disposal, there is some industry (wood manufacture, textile) in this
area that may affect the water quality. Sampling sites L3 and L4
were located about 2 km upstream and 3 km downstream of Mostar, the main industry zone along the Neretva. Sampling sites L5
and L6 were located in the river delta area, about 2 km upstream
and 3 km downstream of Metkovic, close to the border with Croatia. The area is dominated by agricultural activities, but also includes the protected area, Hutovo Blato (salt marsh/wetland
reserve).
2.2. Instrumentation
An advanced microwave labstation Ethos 1600 (Milestone, Sorisole, Italy) equipped with HPR-1000/10S rotor and 100 ml PFA Teflon vessels was used for sample digestion.
A PerkinElmer Sciex Elan 5000 ICP-MS (Norwalk, CT, USA) was
used for the determination of metals. The ICP-MS operating conditions and measurement parameters are shown in Table 1. The
instrument was tuned daily to adjust the sensitivity distribution
across the mass spectrum. The adjustment was performed in two
steps: (1) by optimising the nebulizer gas ow to achieve a high
rhodium (103Rh) sensitivity (P4.5  104) while maintaining a low
oxide and double charged ions sensitivity (156CeO+/140Ce+ <3%
and 69Ba2+/138Ba+ <2%), and (2) by adjusting the optics (lenses) to
achieve equal sensitivity (P7.5  103) for both the low mass
(24Mg) and high mass (208Pb) ions.
Concentrations were calculated using blank subtraction and a
linear t standardization.

2.3. Reagents
Concentrated (65%) Suprapur nitric acid (Merck, Darmstadt,
Germany) and concentrated (30%) Suprapur hydrogen peroxide
(Merck, Darmstadt, Germany) were used for sample digestion.
High purity deionised water (resistivity 18 MX cm), produced by
a Millipore Elix-5/Milli-Q water purication system (Bedford,
USA), was used throughout.
Calibration standard solutions were prepared from commercial
single-element Spectrascan stock solutions (Teknolab A/S, Drbak,
Norway). Calibration standards were prepared by diluting (with 2%
HNO3) stock solution, to produce concentrations of 1, 5, 10 and
50 lg/l of each element analysed. A daily tuning solution, containing
10 ng/ml of Ba, Ce, Mg, Rh and Pb in 2% HNO3 was also prepared from
single elements standards (Teknolab A/S, Drbak, Norway).
All solutions were stored in high density polyethylene (HDPE)
asks, pre-cleaned by soaking overnight in 5% HNO3, and rinsed
ve times with Milli-Q water.
2.4. Sample collection, preparation and digestion
The shes were collected by angling in October and November,
2003. Samples were placed on ice and transported to the laboratory. The total lengths and weights of sh were taken (Table 4).
Scales were removed by scraping, using the edge of a knife with
a titanium blade (a few scales were saved for estimation of sh
age); the blade was rinsed with distilled water and llets were removed and freed of bones if present. Homogenised dorsal muscle
and liver of sh were packed in clean HDPE containers, labelled
with a unique code and stored at 20 C. The digestion of test samples was performed as follows: 5 ml of concentrated HNO3 and
0.5 ml of concentrated H2O2 were added to approximately 0.25 g
of test sample accurately weighed in the digestion vessel. After a
few minutes, the vessels were closed and the bombs placed in
the microwave oven. The temperature control digestion programme was used (Table 2). The digestates were left to cool, quantitatively transferred to 100 ml volumetric asks, diluted to the
mark with Milli-Q water and stored in a refrigerator prior to ICPMS analysis, which was performed within 24 h after the digestion.
All of the decomposed sample solutions were clear, without visible
residues.
The digestion vessels were cleaned after each run using the
same reagents and program that was applied for sample decomposition, followed by rinsing with Milli-Q water.
2.5. Quality assurance/quality performance
The accuracy and precision of the analytical method was estimated by analysing two certied reference materials (CRMs) for
trace metal determination: DOLT-3 (dogsh liver) and DORM-2
(dogsh muscle), supplied by The National Research Council of
Canada (Ottawa, Canada). Each analytical batch of the digestion
set contained one portion of both CRMs and one procedural blank.
The accuracy was given as the mean percent recovery for each of
the elements measured in 14 CRMs digests prepared on seven
executive days (Table 3). The relative standard deviation (RSD) of
this series of measurements gives the interday precision.
The instrument limit of detection (LoD) was calculated as the
concentration associated with 3.3-times the standard deviation
of the background noise recorded on seven measurements of the
procedural blank. The instrument limit of quantication (LoQ)
was calculated as the concentration associated with 10-times the
standard deviation of the background noise. The method limit of
detection and limit of quantication were calculated in a similar
way, using real sample digest with concentrations of metals in
the range of one to ve times the instrument LoD.

J. Djedjibegovic et al. / Food Chemistry 131 (2012) 469476

471

Fig. 1. Sampling sites at Neretva River (L1-L6).

2.6. Statistical analysis

Table 1
The ICP-MS operating conditions and measurement parameters.
Operating conditions
Spray chamber
Nebulizer
RF power (W)
Plasma gas ow rate (l min 1)
Nebulizer gas (Ar) ow rate (l min
Measurement parameters
Scanning mode
Resolution (amu)
Dwell time (ms)
Sweeps/reading
Readings/replicate
No. of replicates
Isotopes

Scott double-pass
Concentric
1000
15.0
0.70.9 (optimised daily)

Statistical analysis of data was performed with the MinitabTM

Release 13.20 software (Minitab Inc., USA, 2000). Mean metal concentrations in different tissues and species were tested for signicance by Students t-test and analysis of variance (ANOVA) with
the multiple comparison Tukeys test.
3. Results and discussion

Peak hop
0.7
1000
5
1
3
63
Cu, 111Cd,

3.1. Method validation parameters

202

Hg,

207

Pb

The calibration standard solutions were analysed after every 10

test solutions to ensure instrument performances. The concentration drifts were always within the acceptable range of 10%.

The parameters of the method validations are shown in Table 3.

The method LoD should be less than 1/10 of the maximum acceptable concentration (MAC) set for Cd, Hg and Pb in food in the EU,
and the method LoQ should be less than 1/5 MAC (EC 333/2007).
The methods LoDs and LoQs in our study were adequate to reliably
quantify analysed metals for the assessment of sh safety for human consumption. Similar LoD and LoQ values were obtained in

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J. Djedjibegovic et al. / Food Chemistry 131 (2012) 469476

Table 2
Microwave digestion programme.
Step

Temperature (C)

Ramp time (min)

Hold time (min)

1
2
3

75
180
Ventilation

3
6
10

1
15

Table 3
Detection limits and concentrations of metals found in certied reference materials.

Instrument LoD
(lg l 1)
Instrument LoQ
(lg l 1)
Method LoD
(lg kg 1)
Method LoQ
(lg kg 1)
DORM-2
Certied value
(mg kg 1)
Measured value
(mg kg 1)a
Recovery (%)
Interday
precision
(RSD%)
DOLT-3
Certied value
(mg kg 1)
Measured value
(mg kg 1)a
Recovery (%)
Interday
precision
(RSD%)
a

Cd

Cu

Hg

Pb

0.009

0.007

0.010

0.008

0.026

0.021

0.030

0.024

3.30

3.30

5.30

2.70

10.1

10.1

16.2

8.1

0.043 0.008

2.34 0.16

4.64 0.26

0.065 0.007

0.038 0.002

2.35 0.09

4.26 0.15

0.061 0.006

3.2. Cadmium

88
5.3

101
3.9

92
3.5

94
10.6

19.4 0.6

31.2 1.0

3.37 0.14

0.32 0.05

18.6 0.5

28.1 1.4

3.23 0.17

0.30 0.04

96
2.8

90
4.9

96
5.3

93
12.1

Cadmium concentrations were in a range from <LoD to 0.080 mg/

kg in muscle and 0.0040.540 mg/kg in liver samples (Table 5). The
highest mean concentration in muscle (0.055 mg/kg) was found in
brown trout and in chub, while highest mean liver content was
found in mullet (0.198 mg/kg) and chub (0.190 mg/kg). The results
are comparable to those previously reported for Neretva delta
(Table 6) (Bogut & Bukvic, 2003; Has-Schn et al., 2006, 2008) and
lower than those in sh from some other Bosnian rivers, namely
Una, Vrbas and Drina (Alic et al., 2004). In a more recent study, mean
content of cadmium in muscle of sh caught in the Una river and its
tributaries was reported to be in the range of 0.040.07 mg/kg
(Alibabic, Vahcic, & Bajramovic, 2007).
Liver samples generally have somewhat higher concentrations
than has muscle, as Cd tends to accumulate in liver (Kalay & Canli,
2000). Although the difference between muscle and liver concentrations is relatively small compared to what is reported from
other studies (e.g. Andres, Ribeyre, Tourencq, & Boudoum, 2000),
it is still statistically signicant (Students t-test, p = 0.001). The relatively small difference found in this study is consistent with a previous study from the Neretva delta (Has-Schn et al., 2006). Total
Cd concentration (muscle + liver) was found to be highest at location L4 (downstream of Mostar) (Fig. 2). Potential sources of Cd in
this area include industry in Mostar, as well as substantial runoff
from agricultural areas (potential source of Cd from fertilizers).
Cadmium bioconcentration in sh muscle decreased in the following order: chub, brown trout > prussian carp > rudd > gray
mullet > common carp. International consumption limits for Cd in
sh range between 0.05 and 2 mg/kg (Burger & Gochfeld, 2005).
The European Regulative on maximum residue levels in food (Commission Regulation EC 1881/2006), which is adopted in B&H legislation (Sl. list BiH, 37/09) sets the maximum allowed concentration
(MAC) for Cd in edible sh tissue at 0.050 mg/kg wet weight
(0.10 mg/kg for Mugil sp.). This limit was exceeded in 16 sh muscle
samples (mainly trout). According to the United States Environmental Protection Agency (US EPA) risk-based consumption limit tables,
where concentration levels are related to number of sh meals per
month, the concentration level of 0.05 mg/kg is acceptable when
having less than 16 sh meals per month (USEPA, 2000). Although
food represents the main avenue of Cd exposure in non-smokers, this
applies primarily to fruits and vegetables (2950% of daily dose) and
cereals (1848% of daily dose) (Kostka-Rick & Mekel, 2003), while
sh is considered a minor source of human dietary Cd intake (SACN
(Scientic Advisory Committe on Nutrition)., 2004). Similar ndings

Expressed as means standard deviation of 14 determinations.

Table 4
Fish species, number of samples and estimated sh age by sampling sites.
Fish
species

determinations of Cd, Cu and Pb and CVAAS determination of

Hg in sh and crustacean (Yang & Swami, 2007). The LoD and LoQ,
achieved by the method presented herein for Cd and Cu, were
seven and 43 times lower than those obtained with GFAAS determination in the same matrix (Manutsewee, Aeungmaitrepirom,
Varanusupakul, & Imyim, 2007). The LoD values obtained by
Kosanovic et al. (2007) with the ICP-MS determination of Cd and
Cu in sh were ve and three times higher, and LoD for Pb was 1.5
times lower than the LoD reached in our study.
The correlation coefcients (R2) of the calibration curves for all
metals were P0.998 on each day of analysis. The recovery factors
for analysed metals were in acceptable range (between 88% and
101%). The interday precision, expressed as RSD, ranged from
2.8% to 5.3% for Cd, Cu and Hg. The higher RSD recorded for Pb
(10.6% and 12.1% in two CRMs) can only be attributed to low concentrations of this metal in both certied reference materials.
The results for metal concentrations in sh tissues (Table 5)
show variation in a range that seems to be typical for the Neretva
delta and some other Bosnian rivers, but are lower than those in
rivers with considerable anthropogenic impact (Table 6).

Number of
sh

Fish age
(years)

Mean weight
(g)

Mean length
(cm)

Brown trout (Salmo trutta morfa fario)

L1
5
(2+)(3+)
L2
3
(4+)(5+)
L3
5
(1+)(2+)
L4
3
(2+)(3+)

78.8
641.5
143.0
179.3

19.2
37.1
21.7
24.5

Common carp (Cyprinus carpio)

L2
3
(3+)(3+)
L3
3
(4+)

293.0
772.7

25.7
38.4

Chub (Leuciuscus cephalus albus)

L2
3
(2+)(3+)
L4
3
(3+)(4+)

145.5
578.9

23.8
39.2

Prussian carp (Carassius auratus gibelio)

L5
3
(3+)
L6
2
(3+)(4+)

311.4
446.9

26.7
29.9

Rudd (Scardinius erythrophthalmus scardofa)

L5
3
(3+)(4+)
225.3
L6
3
(3+)(4+)
130.2

24.1
21.8

Grey mullet (Mugil cephalus)

L6
7
(3+)

28.9

192.0

SF-ICP-MS determination of Cd and Pb in offal (Forte & Bocca, 2007)

and ICP-MS determination of Hg in sh (Kosanovic et al., 2007),
while somewhat higher LoD and LoQ values were obtained with
the ICP-MS determination of Cd, Cu and Pb in craysh (Snchez
Lpez, Gil Garcia, Snchez Morito, & Martnez Vidal, 2003), ICP-MS

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J. Djedjibegovic et al. / Food Chemistry 131 (2012) 469476

Table 5
Heavy metal concentrations (mg/kg wet weight) in tissues of different sh species expressed as means standard deviation (range).
Species

Brown trout (n = 16)

Common carp (n = 6)
Chub (n = 6)
Prussian carp (n = 5)
Rudd (n = 6)
Mullet (n = 7)
MAC (mg/kg)

Cd

Cu

Hg

Pb

Muscle

Liver

Muscle

Liver

Muscle

Liver

Muscle

Liver

0.055 0.028a
(0.0060.079)
0.013 0.007b
(0.0060.023)
0.055 0.029a,b
(<LoD-0.080)
0.045 0.030 a,b
(0.0040.071)
0.023 0.023 a,b
(0.0050.055)
0.018 0.006b
(0.0120.028)
0.050 (0.10)*

0.046 0.048a
(0.0070.136)
0.031 0.020a
(0.0090.062)
0.190 0.040b,c
(0.1490.243)
0.015 0.014a
(0.0040.034)
0.058 0.055a,c
(0.0060.110)
0.198 0.177b,d
(0.0050.540)

0.502 0.431a
(0.1171.504)
0.807 0.738a,b
(0.0171.500)
16.1 25.8b
(0.02362.32)
0.369 0.287a,b
(0.1080.858)
0.068 0.049a
(0.0110.140)
0.337 0.360a
(0.0440.985)

22.2 19.2a
(2.2278.1)
2.01 1.49a
(0.8974.882)
3.48 4.43a
(0.00712.1)
7.98 10.6a
(0.87526.8)
1.90 0.341a
(1.362.27)
56.8 55.6b
(5.42156)

0.101 0.080a
(0.0070.302)
0.083 0.127a
(0.0100.309)
0.138 0.071a
(0.0570.234)
0.050 0.038a
(0.0060.092)
0.080 0.081a
(0.0090.219)
0.401 0.158a
(0.1820.601)
0.50 (1.0)*

0.111 0.067a
(0.0240.283)
0.132 0.073a
(0.0550.238)
0.119 0.041a
(0.0610.161)
0.049 0.039a
(0.0100.105)
0.092 0.037a
(0.0550.152)
0.605 0.278b
(0.1540.893)

0.077 0.031a
(0.0150.137)
0.073 0.012a,b
(0.0550.087)
0.703 1.166b
(0.0502.957)
0.055 0.020 a,b
(0.0350.079)
0.066 0.016 a,b
(0.0400.081)
0.057 0.043 a,b
(0.0070.102)
0.3

0.085 0.060a,b
(0.0060.190)
0.020 0.022c
(0.0050.064)
0.023 0.008c,d
(0.0110.032)
0.122 0.012a,e
(0.1060.136)
0.149 0.044e
(0.0640.191)
0.033 0.020b,c
(0.0040.055)

a,b,c,d,e

Means between species with the same letter in column are not signicantly different according to Tukeys multiple range test (p > 0.05).
Maximum allowed concentration within the European Union (Commission Regulation (EC) 1881/2006), adopted in B&H legislative (Sl. list BiH, 37/09).
MAC for grey mullet.

Table 6
Selected references of average metal concentrations (mg/kg wet weight) in sh tissues.
Sites

Cu

Hg

Pb

References

Liver

Muscle

Liver

Muscle

Liver

Muscle

Liver

Brown trout
Common carp
Chub
Prussian carp
Rudd
Mullet

0.055
0.013
0.055
0.045
0.023
0.018

0.046
0.031
0.190
0.015
0.058
0.198

0.502
0.807
16.06
0.369
0.068
0.337

22.2
2.01
3.48
7.98
1.90
56.8

0.101
0.083
0.138
0.050
0.080
0.401

0.111
0.132
0.119
0.049
0.092
0.605

0.077
0.073
0.703
0.055
0.066
0.057

0.085
0.020
0.023
0.122
0.149
0.033

Present study

Neretva delta
(Croatia)

Trout
Common carp
Prussian carp
Rudd
Mullet
Common carp
Mullet
Tench
Sval
Eel
Common carp
Prussian carp
Tench
Eel
Pumpkinseed
Trout
Trout
Nase
Nase
Brown trout
Californian trout (wild)
Californian trout (farmed)
Mullet
Micropogonias furnieri
Mullet
Eleginops maclovinus
Mullet
Eleginops maclovinus

0.010
0.010
0.010b
0.010
0.010b
0.075
0.052
0.067
0.027
0.027
0.011
0.01
0.01
0.02
0.01
1.349
1.664
1.269
1.545
0.065b
0.060b
0.055b
0.34
<LoD
NA
NA
NA
NA

0.130
0.137
0.137
0.135
0.136b
0.191
0.117
0.133
0.131
0.139
0.052
0.136
0.13
0.274
0.125
NA
NA
NA
NA
NA
NA
NA
9.15
3.13
NA
NA
NA
NA

NAa
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
1.098
1.295
0.973
0.925
0.850b
0.820b
0.965b
NA
NA
5.9
3.6
6.2
3.8

NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA

0.083
0.003
0.095b
0.021
0.140b
0.190
0.198
0.121
0.078
0.114
0.031
0.147
0.125
0.159
0.159
0.091
0.08
0.316
0.07
NA
NA
NA
0.40
0.11
NA
NA
NA
NA

0.019
0.007
0.021b
0.005
0.031b
0.080
0.091
0.096
0.058
0.081
0.011
0.033
0.028
0.072
0.036
NA
NA
NA
NA
NA
NA
NA
0.53
0.13
NA
NA
NA
NA

<0.10
<0.10
<0.10
<0.10
<0.10
0.317
0.106
0.134
0.122
0.112
0.012
0.1
0.1
0.123
0.1
0.317
0.31
0.347
0.57
0.630b
0.725b
0.960b
NA
NA
4.0
1.9
3.4
1.6

0.118
0.120
0.120b
0.121
0.110b
0.276
0.113
0.119
0.127
0.128
0.017
0.125
0.123
0.21
0.123
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA

Bogut and Bukvic (2003)

Hutovo Blato
(B&H)

Una River (B&H)

Vrbas River (B&H)
Drina River (B&H)
Una River (B&H)

La Plata estuary
(Argentina)
Maule River
(Chile)
Mataquito River
(Chile)
a

Cd
Muscle

Neretva River
(B&H/Croatia)

Neretva delta
(Croatia)

Species

Has-Schn et al. (2006)

Has-Schn et al. (2008)

Alic et al. (2004)

Alibabic et al. (2007)

Marcovecchio (2004)
Tapia et al. (2009)

NA, not analysed.

Geometric mean calculated from arithmetic means presented in the cited references.

are reported from a recent study on cadmium intake in 16 EU countries (EFSA, 2009). According to this report, the highest dietary exposure to Cd is from cereals and cereal products (daily exposure
4.189 lg) and vegetables, nuts and pulses (daily exposure
3.667 lg), followed by edible offals and offal products (daily exposure 3.031 lg), while daily exposure to Cd from sh and seafood is
assessed to be 1.662 lg (EFSA, 2009). The percentages of sh muscle
samples exceeding the maximum acceptable concentration (MAC)
for Cd are reported to be 4.8% (species with MAC of 0.050 mg kg 1)

and 8.2% (species with MAC of 0.10 mg kg 1). Our results show that
16 out of 46 sh muscles (35%) had Cd concentrations above the MAC
levels in the EU and B&H.
3.3. Copper
Copper concentrations in sh muscle ranged from 0.011 to
62.32 mg/kg, with average concentrations in the range 0.068
16.1 mg/kg. Copper was not investigated in the Neretva delta study

474

J. Djedjibegovic et al. / Food Chemistry 131 (2012) 469476

Fig. 2. Mean concentrations of heavy metals in sh tissue by sampling sites.

of Has-Schn et al. (2006), but comparison with data from elsewhere suggests that the concentrations found in our study are
rather typical. For instance, similar concentrations were reported
from the United States (Saiki, Martin, Thompson, & Welsh, 2001)
and from Greece (Papagiannis, Kagalou, Leonardos, Petridisd, &
Kalfakakou, 2004). Mean copper concentrations reported for Una,

Vrbas and Drina (Table 6) were between 0.820 and 1.30 mg/kg
for trout and nase (Alibabic et al., 2007; Alic et al., 2004). Our results were comparable, except for two chub samples with signicantly higher Cu contents (62.3 and 31.2 mg/kg). Copper content
in llets of sh from the Tagus estuary, Portugal, was reported to
be up to 92.1 mg/kg (Frana, Vinagre, Cacador, & Cabral, 2005).

J. Djedjibegovic et al. / Food Chemistry 131 (2012) 469476

Copper bioconcentration in sh muscle decreased in the order:

chub > common carp > brown trout > prussian carp > rudd > gray
mullet > rudd.
The concentrations in the liver samples were generally much
(more than 60-fold) higher than those in the muscle tissue (Students t-test, p = 0.001). This nding is consistent with some other
study results (de Mora et al., 2004; Qiao-quiao et al., 2007). Very
high Cu concentrations found in grey mullet liver should not be
interpreted as an indicator of contamination, since the concentrations in other sh species from the same location were signicantly lower. Linde, Klein, and Summer (2005) have shown
exceptional accumulation of copper in liver of this particular sh
species. Total Cu concentrations (muscle + liver) show a decreasing
trend from sampling site L1 to L5, while total concentration at site
L6 was higher than at site L1 (Fig. 2).
Copper is an essential element, but toxic at high concentrations.
The proposed permissible daily dose of copper for adults is
0.05 mg/kg body weight (NRC, 1980), which equals 3.5 mg/day
for a man with body weight of 70 kg. Assuming daily intake of
one portion (100 g) of chub from Neretva (copper contents 62.3
and 31.2 mg/kg), daily Cu dose would be 6.2 and 3.1 mg, respectively. These two chub samples could not be judged as safe for human consumption. Although exceptional, these ndings should not
be overlooked. Copper contents in many liver samples were also
above the safe level.
3.4. Mercury
Mercury concentrations in muscle samples were in the range
0.0060.611 mg/kg, with mean concentrations for different species in the range 0.0500.401 mg/kg (Table 5). Mercury concentrations in sh muscle decreased in the following order:
mullet > chub > brown trout > common carp > rudd > prussian
carp. The Hg concentrations in the liver samples (0.010
0.893 mg/kg in individual samples and means 0.0490.605 mg/
kg) were similar to, or slightly higher than those in the muscle
samples (Students t-test, p > 0.05). The highest concentrations
(both in liver and muscle) were recorded in grey mullet from
station L6 (downstream of Metkovic) (Fig. 2). Obviously, the
much higher Hg content in grey mullet tissue than in other analysed sh species is most probably due to the fact that this species spends most of its life in the sea. Mercury content in sea
sh species are often higher than those in sh from fresh waters
(Cheung, Leung, & Wong, 2008).
Mercury concentrations in sh tissue samples were comparable
to those obtained by Bogut and Bukvic (2003) and Has-Schn et al.
(2006), Has-Schn et al. (2008), although our samples of grey mullet had somewhat higher values (Table 6). Comparing our results to
those reported by Alic et al. (2004), it can be concluded that Hg
contents in muscle of sh from Neretva are approximately the
same as those in rivers Drina (mean content 0.08 mg/kg) and
Una (mean content 0.09 mg/kg), and somewhat lower than those
in sh from river Vrbas (mean content 0.2 mg/kg). Alibabic et al.
(2007) did not detect mercury (LoD = 0.5 ppb) in muscle samples
of sh caught in the Una catchment.
Mercury accumulation in sh has received much attention
internationally and many countries have sh consumption advisories related to Hg (e.g. UNEP, 2002). The most commonly recommended limit for consumption is 0.5 mg/kg, although there are
lower limits in some countries (Burger & Gochfeld, 2005). The limit
in Bosnia and Herzegovina is 0.5 mg/kg (1 mg/kg for long-lived
species). Among our samples, the mean grey mullet concentration
was close to, but still below, this limit in the muscle samples and
slightly higher in some of the liver samples. Two out of seven mullets collected had muscle concentrations slightly above 0.5 mg/kg.
According to US EPA risk consumption limit tables (USEPA, 2000),

475

chronic systemic non-carcinogenic effects of mercury may be expected, even with concentrations much below 0.5 mg/kg. Compared to these guidelines, all seven mullet samples, three out of
six chub samples and four out of 16 trout samples, could not be
considered as safe if up to four sh meals (a 100 g portions) are
consumed per month. Although wild sh from Neretva constitute
only a small portion of the sh market, local habitants consume
it frequently, and more frequently than could be assessed by ofcial statistical records on food consumption. The wild sh are not
regularly monitored and hence this could be an important public
health issue in certain regions in Bosnia and Herzegovina.
3.5. Lead
The Pb concentrations for most muscle samples were between
0.007 and 2.957 mg/kg (mean values between 0.055 and 0.703
mg/kg). The highest concentration was recorded in chub at sampling
site L2 (Table 5). Lead concentrations in muscle samples decreased
as follows: chub > brown trout > common carp > rudd > mullet >
prussian carp. The concentrations in liver samples were generally
in the same range as those in the muscle samples (0.004
0.191 mg/kg in individual samples, mean values between 0.020
and 0.149 mg/kg). Difference between liver and muscle concentrations was not signicant (Students t-test, p > 0.05).
Concentrations in sh tissue at sites L5 and L6 are comparable
to, or somewhat lower than the values reported by Bogut and
Bukvic (2003) and Has-Schn et al. (2006, 2008). On the other
hand, sh samples from rivers Una, Vrbas and Drina had higher
Pb concentrations (Table 6).
Maximum allowed concentration of Pb in sh tissue in the EU
and in B&H is 0.30 mg/kg. This limit was exceeded in two chub
muscle samples from site L2, while Pb concentrations in other
muscle samples were considerably below the limit. The provisory
tolerable daily (PTDI) intake of lead is 25 lg/kg body weight for
adults and 6 lg/kg body weight for children (Dolan, Nortrup,
Bolger, & Capar, 2003). These equals a PTDI of 1.75 mg for adults
and 0.500 mg for children 46 years old (mean body weight
20 kg). Except for two chub samples, mean Pb content in sh muscle was 0.068 mg/kg. Assuming intake of one 100 g sh portion per
day, the daily dose of lead is calculated to be 0.0068 mg, which accounts for 0.4% of PTDI for adults and 1.4% of PTDI for children.
Content of lead recorded in two chub muscle samples caught
downstream of Konjic (site L2) were 1.00 and 2.96 mg/kg. Daily intake of a 100 g portion, in these two cases, would result in a Pb intake of 0.100 mg (5.7% of PTDI for adults and 20.1% of PTDI for
children) and 0.296 mg (16.9% of PTDI for adults and 59.1% of PTDI
for children). Considering lead exposure from other sources
(foods), these results (although exceptional) are disturbing. Total
metals load seems to be higher in chub muscle than in muscle of
other analysed species, while total metals load in liver was higher
in mullet than in other species.

4. Conclusions
(1) The measured metal concentrations in sh tissue are generally moderate, equal to or lower than levels reported for sh
from other B&H rivers.
(2) Although the metal concentrations in sh muscle were in
most cases below suggested limits for sh consumption, a
health risk due to cadmium and mercury intake can not be
excluded.
(3) Total heavy metals load was found to be highest in chub.
Hence, this species is less recommendable for human consumption, but could be valuable as a bioindicator in environmental surveys.

476

J. Djedjibegovic et al. / Food Chemistry 131 (2012) 469476

(4) Bearing in mind limitations of the presented study (onepoint-in time analysis, no data on metal levels in water
and/or sediment) and the results found, future investigation
is required. We also recommend a survey of frequency of sh
consumption among local inhabitants and sports sherman
as an essential input needed for the risk assessment.
Systematic and regular monitoring is recommendable, both
from environmental and public health aspects.
Acknowledgements
We thank Anne Marie Skramstad and Xie Ruikai, Department of
Chemistry, University of Oslo, for technical assistance in this work
and Professor Avdo Sofradzija and his assistants at Faculty of Science, University of Sarajevo, for their help with sh species identication and estimation of sh age. We also thank the Research
Council of Norway and the Norwegian Institute for Water Research
for their participation in nancing this project.
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