Journal of Environmental Radioactivity 203 (2019) 147–153

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Journal of Environmental Radioactivity

journal homepage: www.elsevier.com/locate/jenvrad

Determinations of uranium concentrations in soil, water, vegetables and T

biological samples from inhabitants of war affected areas in eastern Croatia
(ICP-MS method)
Miroslav Venusa, Dinko Puntarićb, Vlatka Gvozdićc, Domagoj Vidosavljevića,∗, Lidija Bijelića,
Ada Puntarićd, Eda Puntariće, Marina Vidosavljevićf, Jergović Matijanag, Šabarić Jasenkag
a
Faculty of Medicine, J.J. Strossmayer University of Osijek, Cara Hadrijana 10/E, Osijek, Croatia
b
Croatian Catholic University Zagreb, Ilica 242, 10000, Zagreb, Croatia
c
Department of Chemistry, J.J. Strossmayer University of Osijek, Cara Hadrijana 8/A, Osijek, Croatia
d
Faculty of Food Technology and Biotechnology, University of Zagreb, Piertottijeva 6, 10000, Zagreb, Croatia
e
Croatian Agency for Environment and Nature, Radnička cesta 80, 10000, Zagreb, Croatia
f
Vinkovci County General Hospital, Zvonarska 57, 32100, Vinkovci, Croatia
g
Andrija Stampar Teaching Institute of Public Health, Mirogojska 16, 10000, Zagreb, Croatia

ABSTRACT

The occurrence of elevated uranium levels in post-war areas raise concerns among populations, especially in areas affected by heavy bombardment and potential use
of depleted uranium weapons. The aim of this study was to assess public exposure to the uranium Water, soil, vegetables, urine, serum and hair samples were
collected for the first time in eastern Croatia and analysed using an inductively coupled plasma mass spectrometry (ICP-MS) method, in order to try to explain the
possible origins of uranium in the population and environment. Urine, serum and hair samples were collected from 389 inhabitants. A large variation of uranium
concentrations in urine, serum and hair samples was found in this study. The majority of urine, serum and hair samples from our study had uranium concentrations
below the reference literature values. A higher uranium concentration in the hair of 4% of inhabitants, mostly from rural areas, could not be explained at this stage of
research. A further, extended epidemiological study should be made of uranium in the region.

1. Introduction
War in east Croatia was accompanied by major destruction and
killings and lasted from 1991 to 1996, with final war activities hap-
pening in 1999 when NATO forces bombarded the bridges across the
Danube River linking east Croatia and Serbia, with heavy magnitude
weapons, leaving complete destruction.
The use of depleted uranium by the military has been described in
the literature (Duraković, 1999, 2001), and most of the locations of
NATO bombardments in Serbia, Montenegro as well as Bosnia and
Herzegovina have been revealed, and some of them had been explored
and cleared. The use of depleted uranium in such weapons was mainly
to improve the penetration, explosion and destructive capacity of such
projectiles, but they leave radioactive traces in the environment and
exposed individuals (Loppi et al., 2004; Tokonami and Mishra, 2012;
Sahoo et al., 2015; Sarap et al., 2014).
Uranium is not an essential element for life and can be toxic to both
humans and the environment. Approximately 200 minerals contain
uranium as an essential component. Of these, the U (VI) minerals
constitute the largest portion (Finch and Murakami, 1999; Jurković,
2003). Of three uranium isotopes 234U, 235U and 238U, the most abun-
dant naturally occurring uranium isotope, 238U, has the longest half-life
(4.47 × 109 years). Although it exists in a small percentage, because of
its high specific activity, 234U (half-life 2.46 × 105 years), it also con-
tributes as much as 238U to the natural uranium radioactivity
(Duraković, 1999). In nature, uranium is in secular equilibrium with
the daughters of the decay chain, and for this reason, natural uranium
together with its daughters yields 4–5 times as many decays/s as pure
uranium. When uranium is separated from its ores, the decay chain is
broken. In comparison with naturally occurring uranium, Depleted
uranium (DU) has lower relative concentrations of 234U and 235U, and
consequently lower activity. Although depleted uranium has the bio-
logical and chemical properties of natural uranium, it is less radio-
active, this makes depleted uranium attractive for military (Duraković,
1999) use in. Due to its presence in the soil, rocks, surface waters,
groundwater, air, plants and animals, it occurs in trace amounts in food
and drinking water, thus people are continually exposed to uranium.
Uranium in body fluids interacts with a wide range of complexing

∗
Corresponding author. Faculty of Medicine, J.J. Strossmayer University Cara Hadrijana 10e, HR, 31000, Osijek, Croatia.
E-mail address: domagoj.vidosavljevic@gmail.com (D. Vidosavljević).

https://doi.org/10.1016/j.jenvrad.2019.03.004
Received 20 September 2018; Received in revised form 12 February 2019; Accepted 3 March 2019
Available online 23 March 2019
0265-931X/ © 2019 Published by Elsevier Ltd.
M. Venus, et al.
agents competing for uranium ions e.g. complexes of uranium and bi-
carbonate ions, which increase the solubility of uranium in serum.
Uranium (IV) retention sites are the bones and kidneys, whereas uranyl
(VI) ions accumulate in the liver and spleen prior their redistribution in
the renal and skeletal system (Duraković, 1999). Hair is influenced by
blood, perspiration, hormones and enzymes. The changes in the ap-
pearance and composition of hair are visible signs of the presence of
stored substances in the body, so it has been recognized as a valuable
tissue for monitoring human environmental exposure (Sahoo et al.,
2015). For comparison, urine and serum tend to show current or recent
body health status. Analysing uranium concentrations in hair only a
single time could substitute for analysing several urine samples over a
lengthy period in order to estimate exposure (Karpas et al., 2005).
Endogenous and exogenous uranium deposition can result in an ele-
vated uranium concentration, thus the efficacy of hair analysis to
monitor environmental exposure often suffers from over interpretation.
If the exposure is by chronic ingestion of uranium in water or by in-
halation of particulate matter, additional information can be obtained
by urine analysis (Joksić and Katz, 2014). DU-enhanced munitions were
the weapon of choice for US forces during the War in the Balkans in
1995 (Bosnia and Herzegovina) and Kosovo (1999). The possible con-
sequences of the use of depleted uranium (DU) for the people and the
environment need special attention for this reason.
The research was also conducted by an organization of the United
Nations and was focused on the study of soil, air and watercourses in
the area of Novi Sad because of NATO air bombardment, which, be-
cause of the nearness to Croatian territory, could have had a negative
effect on the degree of environmental pollution (Ebling et al., 2005).
The Republic of Croatia was affected by combat activities in the
region for the longest period (1991–1995); however, systematic re-
search on the uranium concentration in the environment of Croatia has
never been made. There are is evidence about an increase in the in-
cidence of malignant diseases in post-war east Slavonia residents, and
in neighbouring countries. However, those diseases were, according to
the authors, at least partially connected to stress caused by war, forced
emigration, poverty etc (Ebling et al., 2005; Obralić et al., 2004;
Petrović et al., 2003).
DU munitions were not used in combat during the 1991–1995 war
in Croatia. The results of previous investigations (Jergović et al., 2010;
Ćurković et al., 2013) showed that, in many hair samples, concentra-
tions of uranium were below a limit of 0.01 μg g −1. The kind of ex-
posure to uranium was not indicated. However, because of the intensive
NATO bombardment of the region neighbouring Hungary (Kerekes
et al., 2001), air samplers located in the southern region of Hungary
were used to asses the amount of DU in the air. Even though no de-
pleted uranium was detected in the air, the increased uranium content
compared to its natural percentage as a component of normal soil,
natural gas, etc., has been suggested to originate from well-dispersed
dust (2.5 μm size) emitted to the atmosphere by explosions during
bombing. In addition, this observation is supported by the geographical
distribution and the relatively rapid decrease in pollution after the
bomb attacks. The observation of Kerekes et al. (2001) cannot be ex-
cluded by meteorological considerations and would support the po-
tential for long-range transport of DU aerosols for extended periods
after primary deposition (Bleise et al., 2003). It is not surprising that
there is considerable evidence emerging that the effects of exposure to
DU or aerosolized uranium weapons involve a wide range of adverse
health effects (Kerekes et al., 2001; Bleise et al., 2003; Alaani et al.,
2011; Závodská et al., 2013). Humans are constantly exposed to natural
uranium by inhalation, ingestion and dermal contact. Individuals can
be exposed to depleted uranium in the same way. Approximately five
percent of all known minerals contain uranium as an essential struc-
tural constituent. Thorium, potassium and uranium are the main ele-
ments contributing to natural terrestrial radioactivity (Alaani et al.,
2011). Food and drinking water are the major sources of exposure to
uranium; root crops (potatoes, turnips, parsnips etc.) contribute to the
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Journal of Environmental Radioactivity 203 (2019) 147–153
highest concentrations of uranium in the diet. In the occupational en-
vironment, workers who mine, mill or process uranium are more vul-
nerable to exposure. Phosphate fertilization remains the main source of
uranium contamination, because uranium is natural and common in
phosphates coming from India and Pakistan used in production of fer-
tilizers. Uranium has a very long half-life and can accumulate in the soil
with repeated applications of fertilizers. Consequently, modern cigar-
ettes may contain higher concentrations of 210Po than those measured
40 years ago (Tidd, 2008).
A systematic research of the uranium concentrations in biological
samples (urine, serum and hair) of eastern Croatian populations has
never been made. A preliminary study was performed in Croatia with
173 participants, but limitations of a study included the small sample
size and that the exposure to uranium was not indicated (Jergović et al.,
2010). Drinking water in eastern Croatia contains high levels of ele-
ments such as As, Mn and Fe. However, there is lack of data about
concentrations of uranium, since the drinking water from the five sites
tested has never been chemically analysed in such detail (Ćurković
et al., 2013; Gvozdić et al., 2015).
2. Experimental
2.1. Area under investigation
The area under investigation comprises the eastern part of Croatia
situated within the alluvial basins of the Sava, Drava and Danube
Rivers. The water, soil, plant, urine, serum and hair samples were taken
at five sampling sites: Vladislavci (settlements), Dalj (settlements),
Čepin (settlements), Našice (town) and Osijek (town) (Figs. 1–3).
2.2. Analytical procedures
2.2.1. Apparatus used in the analysis of the samples
- Analytical scale: EP 214 OHAUS, New Jersey, 2005.
- Microwave for sample burning: Ethos D Microwave Labstation,
Milestone, Brondby, 1996.
- Automatic sampler: AS 93 plus, Perkin Elmer, 2008.
- Mass spectrometer with inductive coupled plasma: ICP-MS ELAN
DRC Perkin Elmer SCIEX, 2008.
2.2.2. Reagents used in the analysis of the samples
- acetone pro analysis, Kemika d.d. Zagreb
- deionization water, 27 MΩ, Milli-Q System, Millipore, USA
- 65% nitric acid (HNO3), Ultraapur, Merck KGaA, Darmstadt;
Germany.
2.2.3. Questionnaire
An interview was implemented with a targeted questionnaire about
elementary demographic indicators and habits, as well as potential
exposures to metals and metalloids in pre-war, war and post-war per-
iods for each of the examinees. In addition, sex, age, area of residence,
smoking, alcohol consumption and the origin of drinking water were
noted. Examinees answered a questionnaire with 14 questions, of which
12 were offered with rounding-off answers and two with open-ended
answers (age and previous diseases). Possible mechanisms of metal
burden, domicile exposures (living near the possible source of burden),
possible professional exposures to metals, pesticides and fossil fuels, as
well as participation in the war were also noted.
2.2.4. Water, soil and plants
Details about sampling and analysis water, soil and vegetables
samples can be found in recent studies (Gvozdić et al., 2015;
Vidosavljević et al., 2014).
M. Venus, et al.
Fig. 1. Map of locations and uranium urine concentrations DL-Detection limit.
2.2.5. Biological materials (urine, serum, hair)
Each of the examinees gave first morning samples of urine, which
were collected in polyethylene bottles for urine (100-mL bottles,
Greiner Bio-One, Frickenhausen, Germany).
One test tube of whole blood was drawn from each of the ex-
aminees; serum was separated by centrifugation. Blood was drawn by
laboratory technicians with needles (Vacuette Blood needle,
38 × 0.9 mm, Greiner Bio-One, Frickenhausen, Germany) and test
tubes (Vacuette serum gel Tube 3.5 mL, Greiner Bio-One,
Frickenhausen, Germany). Supernatant was decanted, and after that,
samples were transferred to the cryo test tubes (Cryotube, 3.8 mL, TPP,
Switzerland) and stored at −30 °C. After defrosting, 10 mL 1% HNO3
were added to 0.5 mL of sample (serum or urine) in each cuvette. Lab
ware previously used in the analysis was washed and drenched for
seven days in 10% HNO3, then rinsed with deionized water before
using. The instrument (ICP-MS) took samples for the analysis with an
automatic sampler from 15-mL polypropylene cuvettes. New cuvettes
were used for all samples. Cuvettes were filled with a HNO3 (1% v/v)
solution, left still at least 24 h before use and were rinsed three times
with 5 mL of the same solution. Sample of the hair from 391 examinees
was taken from the occiput (ca. 1 cm width and 3 cm length) with
stainless steel scissors and stored in polyethylene bags. Hair samples
Fig. 2. Map of locations and uranium serum concentrations DL-Detection limit.
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Journal of Environmental Radioactivity 203 (2019) 147–153
were washed in distilled water, soaked one hour in acetone, rinsed with
deionized water ten times and dried on air on filter paper for 24 h. For
each 0.1 g of the sample, 1 mL of 65% HNO3was added. After two hours
of soaking, the sample was placed in a microwave oven (Ethos D
Microwave Labstation, Milestone, Brondby, 1996), heated, chilled, di-
luted to a volume of 12 mL and transferred to an automatic sampler
cuvette (AS 93 plus, Perkin Elmer, 2008). Operation conditions of the
microwave oven for incineration of samples were: 5 min at 250 W,
5 min at 400 W, 5 min at 500 W and 10 min of ventilation.
Following that was mass spectrometry analysis with inductive
coupled plasma (ICP-MS; ELAN DRC-e, Perkin Elmer, Waltham, MA,
USA).
Operation conditions of the ICP-MS were as follows: voltage power
(RF), 1050 W; argon > 99.99% (Messer, Sulzbach, Germany). The
phases and speeds of flow through the torch were: 1) Phase of plasma
flow between external and middle column, speed 15.00 L/min; 2) Phase
of auxiliary flow of gas, speed 1.20 L/min; 3) Phase of argon gas
nebulizer flow through induction column, speed 0.88 L/min.
All samples were analysed with mass spectrometry with inductive
coupled plasma (ICP-MS ELAN DRC-e, Perkin Elmer, Waltham, MA,
USA). The instrument was calibrated after every twelfth sample, with
an external standard (71-Element Group Multi Element Standard
M. Venus, et al. Journal of Environmental Radioactivity 203 (2019) 147–153

Fig. 3. Map of locations and hair uranium concentrations DL- Detection limit.

Solution, Inorganic Ventures, USA), with the application of internal Table 1

standards with elements Y, In, Tb and Bi (Inorganic Ventures, USA). Uranium concentrations measured in water samples collected at five sampling
Internal calibration (international laboratory audit) was conducted in sites.
cooperation with IFA Tulin (Department of the University of Natural Water N a
Reference value: 30 μg L-1
Resources and Applied Life Sciences, Vienna, in cooperation with the
Vienna University of Technology and the University of Veterinary Area Mean Median Min. Max. SD
(μg L−1) (μg L−1) (μg L−1) (μg L−1) (μg L−1)
Medicine). Analytical methods were validated with standard referent
materials (ICP Multi Element Standard Solution X CertiPUR for Surface 1.Vladislavci 16 3.14 2.67 < 0.001 9.016 2.720
Water Testin, Merck, Germany) and standard samples (Trace Elements 2.Dalj 19 4.25 3.32 0.021 13.320 3.450
Urine Blank and Trace Elements Urine, SERO AS, Norway). 3.Čepin 12 0.0012 0.037 < 0.001 0.012 0.0034
Polyatomic interferences with elements Fe, As, Cr were removed in 4.Našice 8 0.19 0.195 0.135 0.292 0.0548
5.Osijek 12 0.0023 < 0.001 < 0.001 0.011 0.004
the dynamic reaction chamber of the instrument with reactive methane
gas (CH4). After that, was the analysis with mass spectrometry with a
WHO Guidelines for Drinking-water-Quality-2011.
inductive coupled plasma (ICP-MS; ELAN DRC-e, Perkin Elmer,
Waltham, MA, USA). Operation conditions for the ICP-MS were as
follows: voltage power (RF), 1050 W and argon > 99.99% (Messer, Table 2
Sulzbach, Germany). Phases and speeds of flow through the torch were Uranium concentrations measured in soil samples collected at five sampling
as follows: 1) Phase of plasma flow between external and middle sites.
column, speed 15.00 L/min; 2) Phase of auxiliary flow of gas-speed Soil N Reference value: (0.8–11.0 mg kg−1) (Kabata-Pendias, 2011)
1.20 L/min and 3) Phase of argon gas nebulizer flow through induction
Area Mean mg Median mg Min.mg Max.mg SD mg
column, speed 0.88 L/min.
kg−1 kg−1 kg−1 kg−1 kg−1

1.Vladislavci 4 0.797 0.725 0.643 1.091 0.203

2.2.6. Data analysis
2.Čepin 3 0.666 0.670 0.630 0.700 0.035
The data were analysed using the Statistica 13.3 software package. 3.Dalj 3 0.673 0.770 0.480 0.770 0.167
The (dis) similarities of the data were examined by means of cluster 4.Osijek 3 0.653 0.650 0.620 0.690 0.035
analysis. To group variables by (dis) similarity, we applied an ag- 5.Našice 4 0.735 0.765 0.540 0.870 0.143
glomerative single linkage algorithm and Euclidean distance.

3. Results Table 3
Uranium concentrations in vegetables (dandelion and cabbage samples).
To provide information about environmental concentrations of ur- Vegetable N Reference value: (< 1–60 μg kg−1) (Kabata-Pendias, 2011)
anium we measured the uranium concentration of urine, serum and
hair samples. Area Mean μg Median μg Min. μg Max. μg SD μg kg−1
Results of the uranium concentrations regarding water, soil, vege- kg−1 kg−1 kg−1 kg−1

tables, serum, urine and hair samples have been previously only par- 1.Vladislavci 6 1.496 0.881 0.040 3.870 1.433
tially presented (Jergović et al., 2010; Ćurković et al., 2013; Gvozdić 2.Čepin 5 2.224 1.622 0.040 6.460 2.499
et al., 2015; Vidosavljević et al., 2014). In this publication, the results of 3.Dalj 4 3.345 3.364 2.460 4.200 0.823
elementary statistical analysis for the concentrations of uranium in 4.Osijek 4 0.025 0.020 0.020 0.030 0.005
5.Našice 5 14.860 2.161 0.020 66.000 28.659
water, soil, vegetables, urine, serum and hair, are presented in detail in
Tables 1–5 for each area explored. Urine, serum and hair samples were
collected from 389 inhabitants from the areas of Vladislavci and Dalj taken for analysis.
villages, and Čepin, Našice and Osijek towns. In the same locations, Although uranium concentrations were significantly higher
samples of water, soil and vegetables (dandelion and cabbage) were (Kruskal-Wallis test) in water samples from Vladislavci, (p = 0.000342;

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M. Venus, et al. Journal of Environmental Radioactivity 203 (2019) 147–153

Table 4 samples (Dalj), 70% of serum samples (Čepin), 62% of serum samples
Uranium concentrations measured in urine samples collected at five sampling (Našice) and in 90% of serum samples collected in Osijek.
sites. The maximum uranium concentrations in urine samples ranged
Urine N Reference value: (0.012–0.016 μg L−1) (Rodushkin et al., 1999; from 0.39 μg L−1 (Dalj) to 0.89 μg L−1 (Vladislavci). The last value
Rodushkin and Ödman, 2001; Rodushkin and Axelsson, 2000; greatly exceeded the upper range of the reference value of
Rodushkinet al., 2001; Rodushkinet al., 2004) 0.012–0.016 μg L−1. A very similar order of magnitude for maximum
uranium concentrations were detected in urine (from 0.39 to
Area Mean Median Min. Max. μg SD 25% μg 75%
μg μg L−1 μg L−1 μg L−1 μg 0.89 μg L−1) and serum samples (from 1.34 to 8.39 μg L−1) in all in-
L−1 L−1 L−1 L−1 vestigated five areas.
Completely different patterns were detected in hair samples. The
1.Vladislavci 88 0.04 < 0.02 < 0.02 0.89 0.10 < 0.02 0.03
maximum hair concentrations spanned over about four orders of
2.Dalj 106 0.03 < 0.02 < 0.02 0.39 0.07 < 0.02 0.03
3.Čepin 51 0.04 < 0.02 < 0.02 0.41 0.09 < 0.02 0.04 magnitude and ranged from 0.029 μg g−1 to 186.76 μg g−1. However,
4.Našice 80 0.05 < 0.02 < 0.02 0.55 0.11 < 0.02 0.05 these last values, exceeded the upper range of literature reference value
5.Osijek 64 0.05 < 0.02 < 0.02 0.87 0.13 < 0.02 0.04 of 0.06–0.436 μg g-by several orders of magnitude. Nevertheless, the
uranium level in 96% of the hair samples was lower than allowed re-
ference maximum, 0.436 μg g−1.
Table 5 The largest number of hair samples (15 of them) with higher con-
Uranium concentrations measured in serum samples collected at five sampling centrations of uranium ranging from 1.25 to 39.907 μg g−1 were found
sites.
in the area of Vladislavci village, four in the range from 1.79 to
Serum N Reference value: (0.014–0.015 μg L−1) (Rodushkin et al., 1999; 8.24 μg g−1 were found in the area of Čepin town, while two samples
Rodushkin and Ödman, 2001; Rodushkin and Axelsson, 2000; with 39.98 and 186.76 μg g−1 values were found in the western part;
Rodushkinet al., 2001; Rodushkinet al., 2004)
the second one with the highest concentration was from the area of
Area Mean Median Min. μg Max. SD 25% μg 75% Našice town.
μg μg L−1 L−1 μg μg L−1 μg The upper uranium concentration range in the area of village Dalj
L−1 L−1 L−1 L−1 and Osijek city was lower than 0.436 μg g−1, which is considered an
upper limit of reference value.
1.Vladislavci 88 0.10 < 0.02 < 0.02 2.18 0.29 < 0.02 0.03
2.Dalj 106 0.54 < 0.02 < 0.02 8.30 1.26 < 0.02 0.54 Since urine, serum and hair samples were available from males and
3.Čepin 51 0.72 0.048 < 0.02 9.39 2.05 < 0.02 0.19 females, by applying Mann Whitney test on these results, it was possible
4.Našice 81 0.12 0.024 < 0.02 1.34 0.22 < 0.02 0.14 to conclude that there no significant difference between concentrations
5.Osijek 64 0.26 0.093 < 0.02 4.32 0.68 < 0.02 0.25 of uranium between male and female samples at all investigated lo-
calities. In addition to standard descriptive statistics, which are pre-
sented in Tables 1–6, we also used cluster analysis. Dendrograms ob-
p = 0.000827) and Dalj (p = 0.000001, p = 0.000004) compared to
tained by cluster analysis are shown in Figs. 4–6. As can be seen in
the samples taken from Čepin and Osijek, the maximum concentrations
Fig. 5, at higher distances, cluster analysis showed two clusters: a single
of uranium in the samples of water collected in all five areas were
member cluster corresponding to the Čepin area and a multimember
below the new provisional WHO guideline value for uranium in
cluster containing the remaining locations. Urine samples had more
drinking water i.e. 30 μg L−1 (Table 1). This study shows relatively
uniform characteristics with respect to all of the analysed areas (Fig. 4).
small variations in the concentrations of uranium in soil. In addition,
Clustering procedures obtained from the hair data highlighted a quite
the measurement indicates that 90% of soil samples analysed had ur-
different situation, because at higher distances, the single–member
anium concentrations of less than 1 ppm (1 mg kg−1), which is an ex-
cluster corresponds to the area of Našice (Fig. 6).
tremely low concentration. The contents of vegetables range from
0.020 μg kg−1 (Osijek and Našice) to 66.0 μg kg−1(Našice). According
to Alina Kabata–Pendias (Kabata-Pendias, 2011), uranium concentra- 4. Discussion
tions in fresh vegetables from the United States range from 4.7 μg kg−1
(cabbage) to 60 μg kg−1 (Parsley). The uranium concentrations in 32% For a non-essential trace element such as uranium, considerable
of urine, 48% of serum and 5% of hair samples were higher than re- biological and geographical variation in concentrations is to be ex-
ferent values found in previous studies (Rodushkin et al., 1999; pected. Although the mean uranium concentrations were observed to
Rodushkin and Ödman, 2001; Rodushkin and Axelsson, 2000; be higher (0.005–2.802 μg g−1), median values were similar to those
Rodushkinet al., 2001; Rodushkinet al., 2004). reported in a previous study conducted in eastern Croatia
The uranium levels in 70% of the urine samples collected in all five (0.01–0.02 μg g−1) (Jergović et al., 2010; Ćurković et al., 2013).
locations investigated were lower than allowed reference maximum, However, the maximum uranium concentrations were observed to be
0.016 μg L−1. Equally depending on location, the uranium levels in very high (186.768 μg g−1) compared to results reported in the pre-
serum samples were lower than the allowed reference maximum of vious study conducted in eastern Croatia (0.01–0.02 μg g−1) (Jergović
0.015 μg L−1 in 70% of serum samples (Vladislavci), 65% of serum et al., 2010; Ćurković et al., 2013). In comparison with DU

Table 6
Uranium concentrations measured in hair samples collected at five sampling sites.
Hair N Reference value: (0.006–0.436 μg g−1) (Rodushkin et al., 1999; Rodushkin and Ödman, 2001; Rodushkin and Axelsson, 2000; Rodushkinet al., 2001;
Rodushkinet al., 2004)

Area Mean μg g−1 Median μg g−1 Min. μg g−1 Max. μg g−1 SD μg g−1 25% μg g−1 75% μg g−1

1.Vladislavci 88 1.453 0.013 < 0.001 39.907 5.116 0.006 0.033

2.Dalj 106 0.003 0.001 < 0.001 0.028 0.005 < 0.001 0.004
3.Čepin 52 0.328 < 0.001 < 0.001 8.246 1.318 < 0.001 0.008
4.Našice 81 2.802 0.003 < 0.001 186.768 21.165 < 0.001 0.007
5.Osijek 64 0.005 0.002 < 0.001 0.029 0.006 < 0.001 0.009

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M. Venus, et al.
Fig. 4. Dissimilarities of the concentrations of urine samples.
Fig. 5. Dissimilarities of the concentrations of serum samples.
Fig. 6. Dissimilarities of the concentrations of serum samples.
contamination areas such as Fallujah, Iraq (0.02–0.40 μg g−1) and Ko-
sovo, Bosnia and Herzegovina (0.0009–0.449 μg g−1, DU-targeted
areas, 1995 and 1999), our maximum values were found to be several
orders of magnitude higher (Alaani et al., 2011; Zunic et al., 2012). A
wide variation in uranium hair concentration (0.0005–140 μg g−1,
152
Journal of Environmental Radioactivity 203 (2019) 147–153
mean 0.216 μg g−1) has also been reported in the unexposed Finnish
working population where the large variations observed in the urine
concentrations were explained by the high natural uranium con-
centrations, i.e. variations in the uranium level in drinking water
(Muikku et al., 2009). The mean hair uranium concentration in this
paper was 0.95 μg g−1, and this is almost four times higher value from
the Finland sample.
Similar to our case, the mean uranium concentration in hair of the
Finnish working population was 3–15-fold higher than the values re-
ported in the literature, while the mean uranium concentration in urine
was similar to those measured elsewhere in Europe. High concentra-
tions of uranium in the hair, similar to those found in this study, were
also found in hair samples in neighbouring Slovenia in occupationally
exposed uranium workers at the Žirinovski Vrh Uranium Mine (Byrne
and Benedik, 1991).
To explain the higher uranium concentrations in biological samples,
we also measured the uranium content of tap water, well water, soil and
vegetable samples. In all analysed areas, the mean concentrations of
uranium in drinking water samples were lower than 30 μg L−1, which is
the new WHO guideline value for uranium in drinking water. Levels of
uranium in the soil of eastern Croatia were not high (8–11 ppm) in
comparison to natural levels of uranium in Europe (Kabata-Pendias,
2011). The maximum content of uranium in European soils has been
determined to be 56 mg kg−1. Concentration of uranium in water, soil
and vegetables (dandelion and cabbage) collected in the eastern Croatia
area showed low concentrations with means of 3.62 μg L−1,
0.71 mg kg−1 and 1.35 μg kg−1, respectively. Therefore, the water, soil
and vegetable uranium concentrations do not explain the wide range of
uranium variation in the urine, serum and hair samples in the study
area. According to Joksić and Katz (2014), uranium concentrations in
the hair below 0.1 μg g−1 are not indicative of exposure to uranium,
while concentrations above 10 μg g−1 may reflect uranium exposure.
The same authors consider that uranium concentrations higher than
10 μg g−1 (100 μg g−1–1000 μg g−1 or more) provide no clear insights
regarding exposures to uranium, thus urine analysis and the biomarkers
for damage to the kidneys may be necessary to determine the potential
of uranium intoxication. The results of our analyses did not indicate the
presence of elevated uranium levels in urine and serum samples in
subjects with elevated uranium concentrations in hair samples. More-
over, in all hair samples with elevated uranium concentrations, urine
and serum uranium concentrations were below the detection limit. The
results of descriptive statistics were extended by the cluster analysis,
which outlined a lack of correlations between serum samples collected
in Čepin and samples collected in remaining areas. The clustering
procedure obtained on the hair data highlighted a quite different si-
tuation because the Našice area had different characteristics compared
to all remaining areas.
For some elements, the occurrence of higher concentrations, re-
sulting in wide concentration intervals, can be explained. For example,
exogenous deposition from sea aerosols during frequent boat tours is
the most probable cause for extreme Sr concentrations (Rodushkin and
Axelsson, 2000).
If we move the focus on exposure rather than uranium intoxication,
the occurrence of elevated uranium concentration in the hair could be
explained by environmental factors. Other sources of variability could
also be a consequence of an unequal geological uranium distribution.
The values of uranium soil concentrations in eastern Croatia fall within
the lowest range of these measured worldwide by other authors
(mean = 0.70 mg kg−1) (Vidosavljević et al., 2013).
However, certain types of rocks commonly have higher concentra-
tions of uranium, for example uranium rich granites, acidic gneiss,
carbonatites etc. In the area of Papuk (Nature Park near the town of
Našice), there are many old abandoned trenches from which stone ex-
cavations have long been used, mainly for personal needs. Currently
there are nine exploitation fields in Papuk area, of which four are ac-
tive.
M. Venus, et al.
Applying a combination of different methods: X ray diffraction,
scanning electron microscopy, gamma-spectroscopy and X-ray spec-
troscopy, a suite of uranium minerals was identified in metasediments
of the Radlovac series at the Mt. Papuk area, eastern Croatia. The re-
sults showed that some of samples were characterized by extremely
high concentrations of uranium (1500 ppm), actually more than hun-
dred times higher than the background level (16 ppm) that could be
obtained in sandstone sediments (Šćavničar et al., 2007).
During 1982, studies were implemented in the creeks of Kaptol,
Smrdljivac and Cipalovac (Mikulčić et al., 2013). In Smrdljivac creek,
south from the Viljevačka Kosa, two erratic blocks showed 6.000 cps
with maximum content of 1479 ppm U. There are five operating cement
plants in Croatia, and one of them is located in Našice town (Mikulčić
et al., 2013). In fact, limited research efforts in Croatia have been given
to the impact of alkaline dust pollution arising from open-cast mining
and industrial processes such as cement factories. Investigations into
the impact of cement dust on spruce needles collected in the immediate
vicinity of a cement factory in Našice showed that cement dust pro-
vokes the plant to provide a response to the stress situation. Exogenous
deposition from dust can be a probable cause for extreme uranium
concentrations in hair. Thus, the wide range of uranium concentrations
in hair samples values could be solely explained by local uranium de-
posits.
5. Conclusion
Significantly increased uranium concentrations (from 1.25 to
186.75 μg g−1) in hair were found for approximately 10% of the sam-
ples. Sex-related differences in urine, serum and hair sample con-
centrations were not found. The mean uranium concentration in serum
and hair of the eastern Croatian population was, in some locations, 10-
fold higher than the values reported previously in the literature, while
the mean uranium concentrations in urine were similar to those mea-
sured elsewhere in Europe. Depending on the locations investigated, the
mean concentrations in urine, serum and hair samples were between
0.03 and 0.05 μg L−1, 0.10–0.72 μg L−1 and 0.003–2.802 μg g−1, re-
spectively. At this point, depleted uranium from military sources can be
excluded mainly because the environmental samples from locations of
NATO bombardment were lower than others; however, there are other
possible sources of uranium that should be investigated (geological,
agricultural) as well as the potential individual exposure of examinees
to uranium from other, anthropogenic, sources.
Those data provide unique information about water, soil, vegetable,
urine, serum and hair uranium concentration levels of a large popula-
tion group living in eastern part of Croatia. Thus, due to the number of
subjects considered, the results make an important contribution to the
existing world reference database. The results update pre-existing re-
sults, which referred to a small number of participants. This study re-
ported regional variations in the concentrations of uranium in water,
soil, vegetable, and biological samples for certain individuals, which
were probably the consequence an unequal geological uranium dis-
tribution. Future activities will deal with the analysis of water, soil,
plant and biological samples of subjects living in other Slavonian towns
and the Papuk and Psunj Slavonian Mountains. Further extended
153
Journal of Environmental Radioactivity 203 (2019) 147–153
research will focus on an epidemiological study.
Competing interests
This research is part of Project supported by Ministry of Science
Republic of Croatia: “Investigation of long term consequences of war on
the health of population”.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.jenvrad.2019.03.004.
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