Environmental Pollution 213 (2016) 600e607

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Environmental Pollution
journal homepage: www.elsevier.com/locate/envpol

Assessment of status of three water bodies in Serbia based on tissue

metal and metalloid concentration (ICP-OES) and genotoxicity (comet
assay)*
Karolina Sunjog a, b, *, Stoimir Kolarevi

c b, Margareta Kracun-Kolarevi

c c,

Zeljka Visnji
c , Stefan Skori

c-Jefti
a
c , Zoran Ga
a
ci

c , Mirjana Lenhardt a, Nebojsa Vasi

a
c d,
Branka Vukovi
c-Gaci
c b

a
Department of Natural Resources and Environmental Sciences, Institute for Multidisciplinary Research, Kneza Viseslava 1, University of Belgrade, 11000
Belgrade, Serbia
b
Center for Genotoxicology and Ecogenotoxicology, Chair of Microbiology, Faculty of Biology, Studenski Trg 16, University of Belgrade, 11000 Belgrade,
Serbia
c
Institute for Biological Research Sinisa Stankovi
c, Despota Stefana 142, University of Belgrade, 11000 Belgrade, Serbia
d
Department of Mineralogy, Crystallography, Petrology and Geochemistry, Faculty of Mining and Geology, University of Belgrade, Djusina 7, 11000 Belgrade,
Serbia

a r t i c l e i n f o a b s t r a c t

Article history: Metals and metalloids are natural components of the biosphere, which are not produced per se by
Received 26 January 2016 human beings, but whose form and distribution can be affected by human activities. Like all substances,
Received in revised form they are a contaminant if present in excess compared to background levels and/or in a form that would
1 March 2016
not normally occur in the environment. Samples of liver, gills, gonads and muscle from European chub,
Accepted 2 March 2016
Squalius cephalus, were analyzed for Al, As, B, Ba, Cr, Cu, Fe, Hg, Mn, Mo, Sr and Zn using inductively
coupled plasma optical emission spectrometry (ICP-OES) to highlight the importance of tissue selection
in monitoring research. The comet assay or single cell gel electrophoresis (SCGE) was selected as an
Keywords:
ICP-OES
in vivo genotoxicity assay, a rapid and sensitive method for measuring genotoxic effects in blood, liver
Comet assay and gills of the European chub. Microscopic images of comets were scored using Comet IV Computer
Genotoxicology Software (Perceptive Instruments, UK).
Fish The objective of our study was to investigate two reservoirs, Zlatar and Garasi, and one river, Pestan by:
Metals (i) determining and comparing metal and metalloid concentrations in sediment, water and tissues of
Metalloids European chub: liver, gills, muscle and gonads (ii) comparing these ndings with genotoxicity of water
expressed through DNA damage of sh tissues.
A clear link between the level of metals in water, sediment and tissues and between metal and
genotoxicity levels at examined sites was not found. This suggests that other xenobiotics (possibly the
organic compounds), contribute to DNA damage.
2016 Elsevier Ltd. All rights reserved.

1. Introduction

Metals are one of the serious pollutants in our natural envi-

ronment due to their toxicity, persistence and bioaccumulation
* problems (Tam and Wong, 2000; Torre et al., 2013; Aliko et al.,
This paper has been recommended for acceptance by Maria Cristina Fossi.
* Corresponding author. Center for Genotoxicology and Ecogenotoxicology, Chair 2015). Although metals are naturally occurring elements that are
of Microbiology, Faculty of Biology, Institute for Multidisciplinary Research, Uni- found throughout the Earth's crust, most environmental contami-
versity of Belgrade, 11000 Belgrade, Serbia. nation and human exposure result from anthropogenic activities
E-mail addresses: sunjogkarolina@imsi.rs, sunjogkarolina@yahoo.com such as mining and smelting operations, industrial production and
(K. Sunjog), stoimirk@bio.bg.ac.rs (S. Kolarevi
c), margareta.kracun@ibiss.bg.ac.rs
(M. Kracun-Kolarevi
c), nebojsa.vasic@rgf.bg.ac.rs (N. Vasi

c), brankavg@bio.bg.ac.rs
use, and domestic and agricultural use of metals and metal-
(B. Vukovi
c-Gaci

c). containing compounds. Because of their high degree of toxicity,

http://dx.doi.org/10.1016/j.envpol.2016.03.008
0269-7491/ 2016 Elsevier Ltd. All rights reserved.
 K. Sunjog et al. / Environmental Pollution 213 (2016) 600e607
arsenic (As), cadmium (Cd), chromium (Cr), lead (Pb), and mercury
(Hg) rank among the priority metallic elements that are of public
health signicance. These metal and metalloid elements are
considered systemic toxicants that are known to induce multiple
organ damage, even at lower levels of exposure (Tchounwou et al.,
2012). It has been reported that metals such as cobalt (Co), copper
(Cu), chromium (Cr), iron (Fe), magnesium (Mg), manganese (Mn),
molybdenum (Mo), nickel (Ni), selenium (Se) and zinc (Zn) that are
required as essential nutrients for various biochemical and physi-
ological functions (WHO, 1996a) can become toxic when environ-
mental concentrations are increased. However, because they are
natural, they have certain unique properties that distinguish them
from human-produced contaminants as well as from organic sub-
stances in general (Chapman and Wang, 2000).
The sediments are polluted with various kinds of hazardous and
toxic substances, including metals. In some conditions, more than
99% of metals entering the river stream can be accumulated in
sediments in various forms (Salomons, 1995, Peng et al., 2009).
However, metals cannot stay xed in sediment forever. With the
variation of the physicalechemical characteristics of water condi-
tions, part of these xed metals will re-enter the overlying water
and become available to living organisms.
Metals in the sh body are accumulated in different amounts
and these differences result from different afnity to tissues,
different uptake, deposition and excretion rates (Jezierska and
Witeska, 2006). The uptake of metals in sh was found to occur
through absorption across the gill surface, through the gut wall
tract and skin (Obasohan, 2007, Fazio et al., 2014).
The Europian chub, as other sh belonging to the family Cyp-
rinidae, offers possibilities to investigate water bodies with various
ecological status (Durand et al., 1999; Liew et al., 2015; Chromcova
et al., 2015). The use of chub as a bioindicator has already been
reported in eld studies assessing the environmental quality of
inland waters differently impacted by anthropogenic activities
(Winter et al., 2004; Randak et al., 2006; Kr ca et al., 2007; Pavlica
et al., 2011; Sunjog et al., 2013, 2014).
The comet assay or single cell gel electrophoresis (SCGE) was
selected as an in vivo genotoxicity assay, as a rapid and sensitive
method for measuring genotoxic effects in individual cells
(Fairbairn et al., 1995). It combines the simplicity of biochemical
techniques for detecting DNA single-strand breaks (strand breaks
and incomplete excision repair sites), alkali labile sites, and cross-
linking, with the single-cell approach, typical for cytogenetic as-
says (Kumaravel et al., 2009). This assay has found its application in
basic research as well as in applied sciences such as medicine,
ecogenotoxicology, monitoring studies, etc. (Frenzilli et al., 2009;
Meybodi and Mozdarani, 2009; Kolarevi
c et al., 2011; Sunjog
et al., 2014; Ga
ci
c et al., 2014).
The objective of our study was to investigate two reservoirs,
Zlatar and Garasi, and one river, Pestan by: (i) determining and
comparing metal and metalloid concentrations in sediment, water
and tissues of European chub: liver, gills, muscle and gonads (ii)
comparing these ndings with genotoxicity of water expressed
trough DNA damage in tissues of European chub: blood, liver, and
gills.
2. Materials and methods
2.1. Sampling location
Reservoir Zlatar, formed on river Uvac, is a protected natural
area of great importance with very low anthropogenic impact. The
land on which now lake sits is of Triassic and Jurassic products, and
of less exposed Miocene lacustrine sediments. The supply of ma-
terial from basic igneous rocks (spillite, diabase and gabbro) from
601
the diabase-chert formation led to increased content of chromium
and nickel within the recent sediments.
Reservoir Garasi was chosen as the site with low anthropogenic
impact and it is used as a drinking water source. Recent sediments
at reservoir Garasi are feeding from three geological complexes:
Bukulja granitoid, thermo-contact metamorphosed Upper Creta-
ceous sedimentary rocks (phyllite, ne-grained gneisses,
muscovite-biotite schists and marbles) and Miocene sediments
from the Belanovica Lake basin. Each of them carries its own
petrology and geochemical record.
River Pestan, is a polluted site at Kolubara basin, which has rich
deposits of lignite (brown coal), and hence, the whole area is under
intensive mining activity. Geological resources that nourish Pestan
are petrologically highly heterogeneous. Right bank is from Plio-
cene and Early Quaternary deposits. Left bank is much more com-
plex. It consists of Paleozoic rocks, Miocene sediments, acid
volcanics and the Miocene granitoid Brajkovac. Elevated values of
chromium and nickel in Pestan are the consequence of supply from
Cretaceous clastic rocks, which have been signicantly fed from
ultrabasic rocks. Ultrabasic rocks are the primary carriers of chro-
mium, nickel and cobalt.
2.2. Water and sediment sampling
Information regarding the quality of water and sediment based
on chemical analyses (metals and organic pollutants) was obtained
from the Republic Hydrometeorological Service of Serbia.
At reservoirs Garasi water was sampled at 3 depths (0.5, 10,
20 m) and 3 locations and sediment was sampled at 3 locations. At
Uvac water was sampled at 3 depths (0.5, 20, 40 m) and 3 locations
and sediment was sampled at 1 location. At Pestan water and
sediment were sampled at one location and one depth. Sampling
trials for water were at Uvac 07/2009, 08/2010, 09/2011, at Garasi
08/2009, 09/2010, 08/2011, and at Pestan 11/2009, 11/2010, 10/2011.
Sampling trials for sediments were at Uvac in 08/2010 and in 09/
2011; at Garasi in 08/2009, 09/2010 and 08/2011, and at Pestan in
08/2010 and 08/2011.
2.3. Fish sampling
Field sampling was conducted at three sites located in Serbia,
Pestan (N 44
260 05.2600 E 20
150 22.9500 ), Garasi (N 44
170 29.7100 E
20
280 54.7400 ) and Uvac (N 43
290 56.900 E 19
490 27.300 ). Species of
European chub analyzed in this study are occurring naturally in
these water bodies. Total of 18 specimens were caught at all sites,
where at Uvac (7 specimens) and Garasi (5 specimens) were caught
by gillnets (dimensions 10e30 m  1e2 m, 10e80 mm mesh size),
and at Pestan (6 specimens) by electro shing device ELEMAX SHX
2000 (SAWAFUJI). The sampling trails were from October to
November 2011.
2.4. Tissue sample collection and preparation
Fish are anesthetized with clove oil prior to tissue collection and
dissection. The total mass (g) and the total body length (cm) of each
specimen were measured. For metal analysis samples of liver, gills,
muscle, and gonads were quickly removed, washed with distilled
water, and stored at 20
C. For comet assay blood was collected
using a syringe directly from the heart and added to microtubes
with 0.5 mL cooled Heparin-PBS (Phosphate Buffered Saline) so-
lution to prevent clotting and liver and gills were removed and
added to microtube with 3 mL cooled PBS. After that liver and gills
were excised and chopped separately 10 times in 0.2 ml of HBSS
(Hank's Balanced Salt Solution) using two fresh scalpel blades in a
scissor-like movement on a Petri dish, washed off gently into a
602 K. Sunjog et al. / Environmental Pollution 213 (2016) 600e607
15 ml centrifuge tube with a further 2.5 ml HBSS and 0.3 ml of
trypsin (nal conc. 0.05%). The incubations were gently shaken for
10 min at room temperature, after which 10 ml of HBSS was added
and the suspension passed through a sieve to remove any large
fragments that remained. After centrifugation (2000 rpm for
10 min), the supernatant was discarded and the pellet carefully
resuspended in 0.5 mL of HBSS.
2.5. Analysis of metals and metalloids
Frozen tissue samples of liver, gills, muscle, and gonads were
rst measured (wet weight) and then dried by Freeze Dryers
Rotational Vacuum Concentrator, GAMMA 1e16 LSC, Germany.
After that samples were measured again (rst dry weight) and
sample portions between 0.2 and 0.5 g (second dry weight) were
processed in a microwave digester (speed wave MWS-3; Bergof
Products Instruments GmbH, Eningen, Germany), using 6 mL of
65% HNO3 and 4 mL of 30% H2O2 (Merck suprapure) at a food
temperature program (100e170
C). The digested samples were
diluted with distilled water to a total volume of 25 mL, and the
analysis was performed by inductively coupled plasma optical
spectrometry (ICP-OES). The following 12 metals and metalloids
were analyzed: Al, As, B, Ba, Cr, Cu, Fe, Hg, Mn, Mo, Sr and Zn. The
following wavelength lines of the ICP-OES analysis were used: Al
394.401, As 189.042, B 249.773, Ba 233.527, Cr 205.552, Cu 324.754,
Fe 259.941, Hg 194.227, Mn 259.373, Mo 202.095, Sr 460.733, and
Zn 206.191. BCR-185R reference material of bovine liver as well as
IAEA-336 Lichen reference material was used for the assessment of
the accuracy and precision of the analysis. Analysis indicated that
the concentrations were within 90e115% of the certied values for
all measured elements. All metal and metalloid concentrations
were expressed as mg g1 dry weight (dw).
2.6. Comet assay
The alkaline comet assay procedure was performed by the
method described by Singh et al. (1988) with some modications
(Sunjog et al., 2014). Microscopic slides were precoated with two
layers of 1% NMP (normal melting point) agarose after which 30 ml
of cell pellet suspension was gently mixed with 70 ml of 1% LMP
(low melting point) agarose and pipetted on the supportive layer of
1% NMP agarose. Slides were then placed into cold lysis buffer for
1 h. To allow DNA unwinding, slides were put into the electro-
phoresis chamber containing cold alkaline electrophoresis buffer
for 20 min. Electrophoresis was performed at 0.6 V/cm at 4
C for
20 min. After the electrophoresis, slides were placed into freshly
made neutralizing buffer for 15 min. Staining was performed with
20 ml per slide of EtBr (2 mg mL1). Microscopic images of comets
were scored using Comet IV Computer Software (Perceptive In-
struments, UK). Images of 50 cells were captured from each slide
per sample and among the parameters available for analyses the
tail length (tl) tail intensity (ti) and Olive tail moment (tm) were
chosen as parameters to assess the DNA damage, and marked as:
blood (btl, bti and btm), liver (ltl, lti and ltm) and gills (gtl, gti and
gtm).
2.7. Statistical analysis
We have used the principal component analysis (PCA) as an
unsupervised statistical method that summarize the variation of a
data set between samples to a set of uncorrelated components
(each component is a particular linear combination of the original
variables), in order to assess the differentiation among the analyzed
sh tissues, based on the element level.
Inter-group comparisons of element levels and genotoxicity
parameters (between tissues and sites) were performed by the
non-parametric ManneWhitney test for two independent samples.
Detection of multivariate outliers was performed with Hotel-
ling's T2 test.
3. Results and discusion
3.1. Metal, metalloid and organic pollutants analysis in water and
sediment
Average values of heavy metal concentrations in water and
sediment and organic pollutants in sediment for 2009, 2010, and
2011year are presented in Table 1. According to data analysis in
water Mn and Pb were elevated at Garasi, Zn, Cu, Cr and Cd at Uvac,
while Fe, Ni and As was higher at Pestan comparing to Garasi and
Uvac (Table 1a). According to National regulative (Ofcial Gazette
16/2011) all element concentrations are in rst class quality of
water, except As at Pestan which was in second class.
Sediment analysis showed that Cu and Cd were highest at
Garasi, while Fe, Mn, Cr and Ni were elevated at Uvac and Zn, Pb, Hg
and As were highest at Pestan (Table 1b). At Uvac Cr with 255.5 mg/
kg and Ni with 421 mg/kg was higher than their MAC (Maximum
Allowable Concentration) values for sediment (Cr 240 mg/kg,
Ni 44 mg/kg) according to National regulative (Ofcial Gazette
16/2011). At Garasi Cu was 188.22 mg/kg which was also above
MAC value for sediment (Cu 110 mg/kg).
Presented data suggests on relatively uniform distribution of
chalcophile elements: at Uvac 279.5 ppm, at Garasi 364.4 ppm, and
at Pestan 325.8 ppm. However, the total concentrations of the
metals such as Cr and Ni in these three localities were found to be
different: at Uvac 676.5 ppm, at Garasi 78.7 ppm and at Pestan
279.4 ppm. The likely sources of nickel and chromium contami-
nation in recent sediments in the river Uvac are the basic igneous
rocks (spilite, diabase and gabbro) from the diabase-chert forma-
tion. Elevated concentrations for nickel and chromium in Pestan are
the consequence of contribution of material from Cretaceous clastic
rocks, feeder signicantly from ultrabasic rocks. Ultrabasic rocks
are considered the primary carriers of chromium, nickel and cobalt.
Benzo[b]uoronathene and benzo[k]uoronathene was detec-
ted in similar concentration at Garasi and Pestan and elevated at
Uvac. Fluoronathene was highest at Uvac, than Garasi and the
smallest value was detected at Pestan. Alachlor was only elevated at
Garasi and bellow level of detection at Uvac and Pestan. p,p0 DDT
was elevated at Garasi and Uvac and was not detected at Pestan. On
the other hand para-tert-octylphenol and methoxychlor was
elevated at Pestan.
According to National regulative (Ofcial Gazette 16/2011)
naphthalene, uoronathene and benzo[k]uoronathene are bellow
MAC, and only DDT (MAC 9 mg/kg) was above at Uvac and Garasi.
3.2. Metal analysis in tissues
Average body length and mass of individuals (used for this an-
alyses) and average values of heavy metal concentrations are pre-
sented in Table 2. Inter-group comparisons of the extent of
accumulation among analyzed tissues showed that the differences
were statistically signicant for most of the assessed metals and
trace elements (P < 0.05). Most of these inter-site differences for
elements are seen in liver and muscle. Gills showed no difference in
accumulation of B, Cr, Fe, Mo i Sr between localities (P < 0.05).
Gonads were compared only between Garasi and Uvac, because at
Pestan concentrations of elements were above detection threshold
only in two samples, and all elements, except Fe i Hg, showed
signicantly different accumulation. Comparing metal concentra-
tion in different tissues showed that gills from Garasi had the
 K. Sunjog et al. / Environmental Pollution 213 (2016) 600e607 603

Table 1
Metal and organic pollutants concentrations in water (a) and sediment (b) in 2009/2010/2011 (mean SD) at three studied localities: Garasi, Uvac and Pestan.

a) Water

Metal and metalloids Garasi Uvac Pestan

Fe (mg/l) 0.08 0.09 0.06 0.05 0.38 0.31

Mn (mg/l) 0.31 0.24 <0.01 0.24 0.09
Zn (mg/l) 7.01 7.12 15.04 4.69 5.6 4.19
Cu(mg/l) 5.26 3.2 5.72 1.33 4.9 3.54
Cr(mg/l) 0.38 0.66 0.78 1 0.32 0.39
Pb(mg/l) 1.28 2.21 0.06 0.11 <0.05
Cd(mg/l) 0.33 0.32 0.61 0.76 0.02 0.02
Hg(mg/l) <0.1 <0.01 <0.1
Ni(mg/l) 3.11 2.75 3 0.46 9.95 4.8
As(mg/l) 1.12 0.35 0.75 0.30 6.4 1.27

b) Sediment

Metal and metalloids (mg/kg) Garasi Uvac Pestan

Fe 37,500.11 9275.93 49,521 36,686,11 44,450 19,127,24

Mn 1014.89 611.4 1763 1336.43 782 272.24
Zn 122.89 42.28 120 0 127 9.9
Cu 188.22 61.92 125 74.95 133 77.78
Cr 46.89 14.13 255.5 301.93 134 36.8
Pb 39 26.32 34 16.97 46.5 16.26
Cd 0.99 1.91 0.25 0.35 <0.3
Hg 0.03 0.05 0.1 0 0.25 0.07
Ni 31.78 11.33 421 496.39 145.5 38.9
As 13.22 11.3 <6 19 1.41
Ni Cr 78.7 676.5 279.5
Chalcophile elements* 364.4 279.4 325.8

Organic pollutants (mg/kg)

Benzo(b)uoronathene 4.82 4.69 9.56 9.12 4.35 1.06

Benzo(k)uoronathene 3.31 2.62 5.7 4.38 2.8 0.56
Fluoronathe 9.32 6.62 17.45 15.63 5.3 7.49
Naphthalene 0.46 0.93 <1 0.6 0.85
Para-tert-Octylphenol <2.5 4.15 1.34 7.9 11.17
Alachlor 1.36 4.1 <2 <2
Methoxychlor <1 <1 91.65 129.61
p.p'DDT 20.32 25.17 155.4 87.68 <1

*Chalcophile elements are presented as a sum of Zn, Cu, Pb, Cd, Hg and As.

highest concentration of Al, B, Fe and Mn, at Uvac for As, Mo and Zn,
while Ba, Cu, Hg and Sr were highest in gills at Pestan (Fig. 1A,
Table 2). When analyzing levels in liver we can see that almost all
metals were highest at Uvac, except for Al and Hg, which was
highest at Garasi and Sr which was above detection level only in
Garasi and Pestan (Fig. 1B, Table 2). For muscle highest levels of Ba,
Mn and Sr were found at Pestan, and Hg was highest at Garasi while
Al, As, B, Cu, Fe, Mo and Zn were highest at Uvac (Fig. 1C, Table 2).
For gonads higher concentrations of elements were distributed
between Garasi and Uvac so that Al, As, B, Ba, Fe and Zn were
highest at Uvac, while Cu, Mn were highest at Garasi (Fig. 1D,
Table 2).
In order to compare heavy metal and trace element concen-
trations in muscles with maximum acceptable concentrations
(MAC) in sh meat intended for human consumption, as estab-
lished by the European Commission (1881/2006/EC), Food and
Agriculture Organization of the United Nations (FAO, 1983), and
national legislation (Ofcial Gazette, No 32/2002), concentrations
were recalculated to the mg kg1 tissue wet weight (ww). Ac-
cording to regulations of the European Commission, the MACs for
Cd, Pb and Hg in sh are 0.05, 0.3, 0.5e1 mg per gram of wet weight.
According to regulations of the Republic of Serbia, MAC for As, Zn,
Cu and Fe in fresh meat sh are 2, 100, 30 and 30 mg per gram of wet
weight, respectively. MACs for Cu and Zn recommended by FAO
(1983) amounts to 30 mg per gram of wet weight. Metal concen-
trations expressed as mg per gram of wet weight in the muscles at
Uvac and Pestan are bellow MAC values for Cd, Pb, Hg, As, Zn, Cu
and Fe, while concentration of Hg in muscle at Garasi (in 2
specimens) were within the limits of MAC in sh, given that the
border is 0.5e1 mg/g wet weight, depending on the type of the sh.
Concentrations of Al, Ba, Cr, Mn, and Sr were highest in gills,
which made gills the most bioaccumulative tissue. Cu, Fe, and Mo
were highest in liver, while muscle and gonads had lowest con-
centration in metals from all sites.
3.3. Comet assay
Results of DNA damage measured with comet assay for blood,
liver and gills at all sites for all parameters are presented in Table 3.
Inter-group comparisons of genotoxicity parameters showed that
Uvac has signicantly lower values of til, tml and tig comparing to
Pestan and Garasi (P < 0.05). Pestan and Garasi differ for blood bti,
btm, for liver ltl and for gills gtm (P < 0.05). We can see that highest
values for DNA damage were found in gills for all sites, which is in
correspondence with highest heavy metal values in gills. Uvac had
the lowest values for all tissues in general, which singled out Uvac
as a referent site, while Pestan had highest values for all tissues.
4. Discussion
All metals and metalloids are naturally occurring in the envi-
ronment, as they are natural components of the Earth's crust.
Background concentrations of metals and metalloids in the envi-
ronment are hence above zero and can vary greatly in different
environmental media (e.g., soils, sediments, water) and under
different geological and environmental conditions (Chapman and
604 K. Sunjog et al. / Environmental Pollution 213 (2016) 600e607

Table 2
Metal and metalloids concentrations in different tissues of European chub (means standard deviation). Concentrations are expressed as mg g1 dry weight, while ND indicates
the values below the detection threshold.

Garasi Uvac Pestan

TL (cm) 37.72 3.53 22.86 2.14 31.12 5.57

M (g) 643.8 227.65 119.53 25.64 277.37 127.03
Gills Al 146.76 248.29a 1.68 2.15b 22.03 30.47a,b
As 0.40 0.11a 1.72 2.22b 0.16 0.06c
B 0.73 1.24a 0.39 0.31a 0.36 0.14a
Ba 7.13 2.43a 5.75 1.29a 18.55 5.76b
Cr 0.23 0.32a 0.20 0.24a 0.14 0.09a
Cu 1.29 0.66a,b 0.75 0.50b 1.96 0.95a
Fe 255.49 326.31a 108.59 34.39a 90.73 30.15a
Hg 0.93 0.30a ND 1.28 0.27a
Mn 40.64 38.44a 11.24 2.96b 22.66 16.34ab
Mo 0.14 0.10a 0.25 0.38a 0.03 0.03a
Sr 50.66 12.30a 56.20 8.76a 66.42 14.75a
Zn 266.14 43.32a,b 340.02 91.59a 197.67 43.29b
Liver Al 6.22 13.78a 0.21 0.57b 0.11 0.05a
As 0.61 0.32a 1.61 1.22b 0.19 0.09c
B 0.10 0.11a 1.44 0.97b 0.33 0.33a
Ba 0.02 0.00a 0.60 0.55b 0.03 0.02ab
Cr 0.04 0.07a 0.63 0.92a 0.03 0.01a
Cu 10.51 5.81a 31.17 23.57b 18.65 16.62a,b
Fe 44.24 34.36a 495.39 365.87b 128.05 102.69a
Hg 1.35 0.17a ND 1.01 0.28a
Mn 2.07 0.49a 4.19 1.67b 2.60 0.84a
Mo 0.26 0.14a 1.19 0.44b 0.71 0.42b
Sr 0.11 0.05a ND 0.15 0.07a
Zn 29.63 4.87a 113.39 36.31b 51.36 21.34a
Muscle Al 0.08 0.03a 2.26 3.62a 0.09 0.03a
As 0.5 0.28a 1.94 1.05b 0.12 0.05c
B 0.05 0.02a 0.18 0.20b 0.04 0.01a
Ba ND 0.49 0.16a 0.62 0.29a
Cr 0.01 0.01a 0.01 0.02a ND
Cu 1.31 0.62a,b 2.22 0.64a 0.89 0.19b
Fe 7.6 5.23a 21.80 5.69b 7.06 1.74a
Hg 1.95 0.42a 0.78 0.54b 1.78 0.29a
Mn 0.3 0.09a 0.59 0.07b 0.79 0.29b
Mo ND 0.05 0.11a 0.02 0.03a
Sr 0.23 0.07a 1.06 0.51b 1.61 0.42b
Zn 16.62 10.42a 61.09 18.87b 32.57 8.89a
Gonads Al 0.14 0.18a 0.31 0.83b 0.06, 2.05*
As 0.11 0.00a 3.22 0.95b 0.11, 0.55*
B 0.14 0.06a 0.24 0.09b 0.22, 0.13*
Ba 0.02 0.00a 2.26 1.05b 1.14, 2.46*
Cr ND ND ND
Cu 5.88 0.95a 1.45 0.55b 5.87, 2.88*
Fe 52.02 17.44a 72.52 34.30a 71.03, 46.41*
Hg 0.90 0.08a 0.72 0.44a 1.05, 1.54*
Mn 25.65 7.20a 0.77 0.38b 17.43, 2.67*
Mo 0.13 0.02a 0.03 0.04b 0.17, 0.15*
Sr 0.47 0.06a 0.42 0.54b 0.47, 0.36*
Zn 96.54 18.64a 365.58 152.21b 231.02, 157.90*
a,b,c
The value with a different letter in the same row is different (Man-Whitney U test, P < 0.05).
*Concentrations above detection threshold only in two samples.

Wang, 2000). Direct correlation of elements in sediment, water and
sh tissues cannot be seen for many reasons: geological composi-
tion, physical and chemical properties of water, specic accumu-
lation pattern in various organs in sh, etc. (Demirak et al., 2006),
which is also clear from our results.
As mentioned earlier, Uvac is under state protection for more
than 45 years, without known anthropogenic activities. It could be
argued that all metals in water originate solely from sediment,
which is again a result of geological composition. According to the
sediment analysis Fe, Mn, Cr and Ni were highest at Uvac and water
analysis showed that Zn, Cu, Cr and Cd were highest, compared to
other sites. Analysis of metals and metalloids in tissues from Uvac
showed that majority of elements in liver (As, B, Ba, Cr, Cu, Fe, Mn,
Mo and Zn) and some in gills (As, Mo, Zn) had higher values than
samples from other sites. On the other hand, genotoxicity results
from liver and gills singled out Uvac as the site with the lowest
genotoxicity compared to Garasi and Pestan. These, seemingly
contrary results, may be explained by acclimation/adaptation of
sh at these fairly constant (without anthropogenic input) levels of
elements in water and geochemical composition of the sediments
metal content. This can result in the evolution of resistance, which
may have important implications for decisions regarding safe
ambient toxicant levels indicating that there can be wide variation
in tolerance to toxic agents among different populations of a spe-
cies (Klerks and Weis, 1987; Chapman and Wang, 2000; Messina
et al., 2014). There are two reasons why organisms may be resis-
tant to a pollutant: (1) individual organisms may have acquired a
degree of tolerance by physiological acclimation during exposure to
sublethal concentrations at some prior period of their lives. This
does not confer tolerance to lethal concentrations upon offspring,
 K. Sunjog et al. / Environmental Pollution 213 (2016) 600e607 605

Fig. 1. PCA analysis of metal concentration in gills (a), liver (b), muscle (c), and gonads (d). PCA analysis of metal concentration showed that specimens from Uvac grouped by higher
values for As, Mo and Zn and that from Pestan by higher values for Ba and Sr in gills (a), specimens from Pestan and Garasi by higher values for Al, Hg and Sr and that from Uvac by
higher values for all other analyzed elements in liver (b), specimens from Pestan and Garasi by higher values for Hg and that from Uvac by higher values for Al, As, B, Cu, Fe and Zn in
muscle (c) and specimens from Garasi by higher values for Cu and Mn and that from Uvac by higher values for As, B, Ba, Fe and Zn in gonads (d).

Table 3 Ali, 2008). It is an insecticide preferred to DDT for use on animals,

DNA damage expressed with three parameters, tl, ti and tm, shows the difference in in animal food, and on DDT-sensitive crops such as squash, melons,
level of DNA damage in three tissues (blood, liver and gills) at three water bodies
etc. Since methoxychlor is more unstable than DDT, it has less re-
rivers, Garasi, Uvac and Pestan.
sidual effects. World Health Organization drinking water quality
Parameter Garasi Uvac Pestan standards permit a maximum of 20 mgL1for methoxychlor in water
Blood btl 16.55 4.37a 17.32 4.07a 18.60 4.06a (WHO, 1996b). In sediments from Pestan methoxychlor was found
bti 3.40 3.94ab 3.24 3.49a 5.15 5.06b at a concentration of 91, 65 mg/kg which is potentially hazardous
btm 0.36 0.44ab 0.33 0.38a 0.61 0.61b considering the possibility of re-entering the water and becoming
Liver ltl 19.96 4.75a 22.48 5.05ab 26.49 5.99b
lti 6.77 5.24a 3.74 3.71b 7.02 6.08a
available to living organisms. EPA (1995) has found methoxychlor
ltm 0.82 0.66a 0.49 0.49b 1.08 0.93a to potentially cause central nervous system depression, diarrhea,
Gills gtl 19.88 4.89a 21.87 5.24a 22.01 6.38a and damage to liver, kidney and heart tissue and retard growth
gti 6.79 5.58a 4.34 4.36b 7.36 6.61a from short-term and long-term exposures at levels above the MCL
gtm 0.83 0.70ab 0.56 0.57a 0.93 0.93b
(Maximum Contaminant Level).
a,b,c
The value with a different letter in the same row is different (Man-Whitney U 4-tert-octylphenol is a common environmental pollutant which
test, P < 0.05). acts as an estrogen receptor agonist in sh. Estrogen agonists are
known to interfere with reproduction parameters as well as sexual
development (including changes in sex-ratio) and growth. Expo-
who must also be pre-exposed to acquire it. (2) Populations may
sure to estrogens such as 4-tert-octylphenol may cause long lasting
have evolved genetically based resistance, through the action of
effects. A change in the endocrine feedback system during sensitive
natural selection on genetically based individual variation in
life stages may result in effects during the entire life. In addition to
resistance (Klerks and Weis, 1987). Since the Uvac is a protected
the endocrine disrupting properties, the concern is increased by
area, it is highly likely that the population of sh was adapted to
evidence that 4-tert-octylphenol biodegrades very slowly in the
levels of naturally presented heavy metals and metalloids.
environment and tends to adsorb to sediment and soil. Ferreira-
We cannot say that solely metals and metalloids are responsible
Leach and Hill (2001) reported about biotransformation, bio-
for genotoxicity effects in tissues, but also various mixtures of
concentration and tissue distribution of 4-tert-octylphenol in ju-
organic and other pollution (Sunjog et al., 2012; Bartoskova et al.,
venile rainbow trout (Oncorhynchus mykiss). In a ow-through
2013). Pesticides are considered to be potential chemical muta-
system the sh was exposed with a concentration of 4 m g/l of 4-
gens and potential chronic health hazards. At Pestan methoxychlor
tert-octylphenol for 10 days. The bioconcentration was tissue
(mg/kg) i para-tert-octylphenol (mg/kg) are present in signicant
dependent, where bioconcentration factor in liver and fat were
levels in sediment. Methoxychlor is widely distributed in both
highest. The study suggests that exposure to water-borne
surface water and groundwater throughout the world (Gupta and
606 K. Sunjog et al. / Environmental Pollution 213 (2016) 600e607
alkylphenols results in rapid conjugation and elimination of the
chemical by the liver/bile route, but the high amount of the parent
substance can accumulate in a variety of other sh tissues.
As previously mentioned, heavy metals exhibit different accu-
mulation pattern in different organs. Among different organs, liver
accumulates higher concentrations of metals comparatively and
has been used widely to investigate the process of bioaccumulation.
Exchange of gases and absorption of heavy metals takes place from
external aquatic to internal body environment through gills, so they
are in contact with all pollutants presented in water. Gills and liver
accumulate heavy metals in higher concentration in comparison to
muscles, which exhibit lowest levels of metals accumulation (Jari
c to base analogue 5-FU on the level of DNA damage in haemocytes of freshwater
et al., 2011; Akan et al., 2012; Sunjog et al., 2012; Ja
cimovi

c et al.,
2015). Our results conrmed that gills and liver, along with go-
nads accumulate more metals in comparison to muscle.
5. Conclusion
The implication of natural occurrence of metals and metalloids
in the environment and their effect on the organism has not been
fully appreciated. Specically, environmental concern related to
metals and metalloids should not be triggered only by their pres-
ence in the environment but rather by any change in the level of
their natural presence. In addition, since the background concen-
tration of a metal or metalloid varies greatly in different areas,
background or baseline concentrations should be measured, where
possible, site specically.
Our results pointed out the importance of geological back-
ground of examined sites. A clear link between the level of metals
and genotoxicity at examined sites was not found, which suggest
that other xenobiotics contribute to DNA damage, possibly the
organic compounds. Also it is possible that the same element level
in tissues does not cause the same stress (in this case DNA damage)
in specimens from sites with a different pollution background.
Acknowledgments
This study represents a part of activities within the Project No.
173045, funded by the Ministry of Education, Science and Tech-
nological Development of the Republic of Serbia. The authors are
especially grateful to Luka Ga
ci

c who provided English language
improvement.
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