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The human biomonitoring study in Serbia: Background levels for arsenic, cadmium, lead, thorium and uranium in the whole blood of adult Serbian population
Ecotoxicology and Environmental Safety 169 (2019) 402–409
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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
The human biomonitoring study in Serbia: Background levels for arsenic, cadmium, lead, thorium and uranium in the whole blood of adult Serbian population
T
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Aleksandar Stojsavljevića, , Slavica Borković-Mitićb, Ljiljana Vujotićc,d, Danica Grujičićc,d, Marija Gavrović-Jankulovića, Dragan Manojlovića,e University of Belgrade – Faculty of Chemistry, Studentski trg 16, 11000 Belgrade, Serbia Department of Physiology, Institute for Biological Research “Siniša Stanković”, University of Belgrade, Bulevar despota Stefana 142, Belgrade 11060, Serbia Faculty of Medicine, University of Belgrade, Dr Subotića 8, 11000 Belgrade, Serbia d Clinical Center of Serbia, Neurosurgery Division, Dr Koste Torodorića 4, 11000 Belgrade, Serbia e South Ural State University, Lenin prospect 76, 454080 Chelyabinsk, Russia a
b c
A R T I C LE I N FO
A B S T R A C T
Keywords: Toxic trace metals Reference values Serbian population Thorium/Uranium
The purpose of this study was to establish reference values (RVs) for the occupationally- and environmentallyimportant toxic elements in the whole blood of adult Serbian population for the first time. Contaminated drinking water with arsenic, high share of smokers in the country, removing tetraethyl lead from the gasoline and war attack at the end of the twentieth century were some of the reasons to provide background information for arsenic (As), cadmium (Cd), lead (Pb), thorium (Th), and uranium (U) in the blood of the Serbian population. The whole blood samples were collected from the healthy respondents living in the Belgrade and surrounding areas of the capital (n = 305; w/m ratio = 154/151; mean age: 41 ± 2). The concentrations of toxic metals were determined by inductively coupled plasma-mass spectrometry (ICP-MS). Reference values were estimated as the lower limit (LL) and upper limit (UL) of the 95% confidence interval (CI), together with the selected percentiles (P2.5-P97.5). The obtained geometric mean (GM) for As, Cd, Pb, Th, and U were: 0.50 ng/g, 0.32 ng/ g, 20.94 ng/g, 0.30 ng/g, and 0.06 ng/g, respectively. The influences of age, sex and lifestyle on results were considered. Women have significantly higher levels of Cd and Th than men. The increased level of Th was observed in the aged group below 40 years, while smokers had significantly higher levels of Pb and double higher level of Cd in the blood than non-smokers (p < 0.05). In comparison with other population groups worldwide, the Serbian population had significantly higher levels of Th and U (up to 100 times higher). These findings could contribute to better understanding of the molecular basis for the development of various health hazards, including the increased incidence of cancer among the Serbian population which need be confirmed by clinical studies.
1. Introduction The excess of harmful metal/metalloid has negative consequences to the human health. Daily exposure to toxic metals through contaminated air, food or drinking water is of a major cause for concern, because of its abilities to de-regulate the immune system and increase the incidence of various disease states, including cancer (Kim et al., 2017; Avino et al., 2011; Rocha et al., 2016). Today, the majority of the world's population is exposed to various metals from the increasing anthropogenic pollution (Černa et al., 2012; Khlifi et al., 2014; Yedomon et al., 2017).
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Corresponding author. E-mail address: [email protected] (A. Stojsavljević).
https://doi.org/10.1016/j.ecoenv.2018.11.043
0147-6513/ © 2018 Elsevier Inc. All rights reserved.
Throughout the world, biomonitoring has become the standard for assessing exposure of individuals to different chemicals, including toxic metals (Nunes et al., 2010). Human biomonitoring is a scientific technique for assessing human exposures to environmental agents and their effects, based on sampling and analysis of an individual's tissues and fluids. Among the various clinical samples, whole blood is the most used matrices for biomonitoring of toxic metal exposure in the general population, population groups and individuals to environmental/ occupational pollutants (Canas et al., 2014; Bevan et al., 2013; Lee et al., 2012; Gil et al., 2011; Bocca et al., 2011; Forte et al., 2011). Many efforts were invested in order to provide reference values
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(RVs) for the trace elements in the body fluids of the unexposed population groups. In that manner it is possible to compare the trace element levels of unexposed subjects to environment-, occupationally-, or accidentally-exposed peoples as well as to find the physiological changes, the presence of the various diseases or effectiveness of the medical therapies (Avino et al., 2011; Bocca et al., 2011). The sample size of the examined population should be large enough to cover age-, sex- and other differences. The International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) recommended a sample size of at least 120 people. The validity of RVs seriously depends not only from final instrumental determination, but also from the adopted preanalytical procedures. (Saravanabhavan et al., 2017; Alimonti et al., 2000). The RVs for the toxic and essential metals should be established periodically, since they can differ among populations and can be affected by peoples’ lifestyles and dissimilar environmental scenarios (Bocca et al., 2011). A large number of countries determined their own RVs for trace elements. Some of them used the large number of individuals, such as a National Health and Nutrition Examination Survey (NHANES) or a German Environmental Survey (GerES) (Kuno et al., 2013). Despite the growing number of biomonitoring studies worldwide, no previous biomonitoring study on the blood trace element levels in Serbia has been reported. The aim of this biomonitoring study was to establish reference values for occupationally- and environmentally-important toxic elements (arsenic, cadmium, lead, thorium, and uranium) in the blood of adult Serbian population, as well as to find the differences in element profile according to sex, age and smoking habits. In such way it was possible to compare our studied population with other population groups worldwide.
A certified stock standard solution (VHG, Manchester, UK) concentration of 10 mg/L was used to prepare intermediate standard solutions. An internal standard solution 100 μg/mL Li, Sc; 20 μg/mL Bi, Ga, In, Tb, Y (VHG, Manchester, UK) was used. Instrument parameters of ICP-MS were optimized using an iCAP Q tuning solution B containing 1 µg/L of Ba, Bi, Ce, Co, In, Li, and U in a 2% nitric acid (Thermo Scientific, UK). For analytical quality assurance, Seronorm® (Sero AS, Norway) Trace Elements Whole Blood, Level 1 (L-1) (SERO210105) and Level 3 (L-3) (SERO210305) were employed. Microwave digestion was done on the ETHOS 1 Advanced Microwave Digestion System (Milestone, Italy). The element quantifications were carried out by inductively coupled plasma-mass spectrometry, ICP-MS (iCAP Q, Thermo Scientific, UK). The plasma and nebulizer gas used pure argon (99.999%) supplied by the Messer, Serbia. The collision cell of the ICP-MS was filled with pressured pure helium (99.999%; Messer, Serbia). PFA-ST micro-flow nebulizer and glass cyclonic spray chambers was employed. All measurements on ICP-MS were performed in the kinetic energy discrimination mode by helium as the collision cell gas. Internal standardization was included to compensate matrix-induced ion signal fluctuations and instrumental drift. Due to the large mass range, four internal standards (45Sc in a concentration of 50 µg/L, and 89Y, 159Tb and 209Bi in a concentration of 10 µg/L) were applied. This intermediate internal standard (IS) solution was aspirated by the second channel of the peristaltic pump, allowing on-line addition to the calibration blank, standards and the sample solutions. Six calibration solutions were employed to cover the range of analyte concentrations. In the range from 0.01 to 25 µg/L, correlation coefficient (r) was greater than 0.997 for all examined elements. Based on repeated measurements (n = 6), the relative standard deviation (RSD) of all elements was < 5%. The limit of detection (LOD) was calculated using standard deviation (SD) of the responses and the slopes (S) of the calibration curves according to the formula LOD = 3 (SD/S). The LOD values for As, Cd, Pb, Th and U were: 0.04, 0.02, 0.11, 0.004, 0.001 µg/L, respectively. The traceability of the analytical procedure was controlled by analyzing the SRMs. The certified vs. founded values, together with the recovery (R) values in percentiles (%) for As, Cd, Pb, Th and U in the SRM L-1 were: 2.40 vs. 2.15 ng/g (R = 89.6%), 0.36 vs. 0.33 ng/g (R = 91.7%), 10.20 vs. 11.1 ng/g (R = 108.8%), 6.0 vs. 5.1 pg/g (R = 85%) and 46.0 vs. 44.2 pg/g (R = 96.1%), respectively. The certified vs. founded values, together with the recovery values for As, Cd, Pb, Th and U in the SRM L-3 were: 30.4 vs. 29.3 ng/g (R = 96.4%), 12.1 vs. 11.2 ng/g (R = 92.6%), 186 vs. 174 ng/g (R = 93.5%), 9.0 vs. 7.9 pg/g (R = 87.8%), 69.0 vs. 64.2 pg/g (R = 93%), respectively.
2. Materials and methods 2.1. Sample collection This study included 305 clinically healthy subjects (women/men ratio = 154/151). All examined respondents were from Belgrade and the surrounding areas of the capital city (Fig. 1). The selected blood donors consisted only of adults, age ranging from 18 to 65 years (mean age: 41 ± 2). The blood samples were collected within a three month period (March–June 2018). About 38% of subjects were smokers (consuming more than 10 cigarettes per day) and were included in our investigation with the aim to compare results for smokers and nonsmokers. All examined subjects were voluntary blood donors and were selected by medical professionals. From each donor a 5 mL of intravenous blood was drawn in the morning hours and stored in the trace metal free evacuated tube (BD Vacutainer). The gathered data included information on age, gender, place of residence, smoking habits, medical history, fish consumption, and other data that could influence on metal concentrations. After careful consideration of more than 500 subjects, only 305 of them met the requirements to participate in our research. These subjects were reported not to be occupationally exposed to metal compounds or to have a chronic disease. The study was approved by the Ethics Committee of Clinical Centre Belgrade, Serbia. All subjects voluntarily participated in this study and the consent was obtained from each subject.
2.3. Sample preparation After removing the whole blood from the freezer at − 80 °C, samples were kept at room temperature before sampling aliquots (approx. 1.5 mL of each sample). Four milliliters of 65% nitric acid and 1 mL of 30% hydrogen-peroxide were added to each PTFE cuvette and digestion was performed under the following programs: warm up for 2 min to 85 °C, 4 min to 135 °C, 5 min to 230 °C and held for 15 min at that temperature. After cooling samples were quantitatively transferred into a volumetric flask (25 mL) and diluted with ultrapure water. Prior to analysis, the standard reference materials (SRMs) were prepared according to manufacturer recommendation and further prepared in the same way as described for real samples.
2.2. Chemicals, instrumentation and analysis All used chemicals were of analytical grade and were supplied by Merck (Darmstadt, Germany). Concentrated nitric acid was additionally purified by double distillation. Ultrapure water was prepared by passing double de-ionized water of Milli-Q system (18.2 MΩ). In order to minimize contamination, all plastic and glass material was immersed in 10% HNO3 for 24 h and rinsed with ultrapure water before use.
2.4. Data analysis Descriptive statistics and Mann-Whitney U-test was performed by a demo version of the NCSS statistical software Number Crucher Statistical System Kaysville, UT, www.ncss.com). Following the recommendations of IFCC and IUPAC (International Union for Pure and 403
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Fig. 1. The sampling sites of blood collection at a map of Serbia are shaded.
3. Results
Table 1 Parameters of descriptive statistics for all investigated blood samples (ng/g).
Mean Stdev Min Max Median
As
Cd
Pb
Th
U
0.69 0.48 0.02 2.38 0.63
0.46 0.41 0.04 1.86 0.33
23.89 11.54 6.09 47.21 21.17
0.44 0.38 0.03 1.76 0.34
0.06 0.04 0.03 0.20 0.05
By applying ICP-MS method for metal determination, less than 1% of examining element concentrations were below the limit of detection. These samples had low level of arsenic. It was observed that peoples who eat seafood/fish more than twice a week had higher levels of arsenic in the blood (GM = 0.53 ng/g) than peoples who eat seafood/fish less than twice a week (GM = 0.51). However, this difference was not statistically significant (p > 0.05). Parameters of descriptive statistics are presented in Table 1 and Fig. 2. Considering all investigated blood samples, lead was the most abundant element, followed by arsenic, cadmium, thorium and uranium. It is interesting to point out that the level of Th was practically the same as the level of Cd for the total studied population (0.44 ± 0.38 ng/g vs. 0.46 ± 0.41 ng/g). Differences between examining groups are presented in Table 2. In comparison with men, women had higher values of As, Cd and Th. However, this difference was only significant for Th (GM: 0.34 vs.
Applied Chemistry), reference values for toxic metals was expressed as percentiles (P) in the range from 2.5th–97.5th, and was calculated as the lower limit (LL) and the upper limit (UL) of the 95% confidence interval (CI). The metal concentrations below the LOD values were replaced with the value LOD/2. The reference values for each element were computed for men, women, subjects aged < 40 years old, subjects aged ≥ 40 years old, as well as for smokers and non-smokers. The differences between groups were considered significant at p < 0.05. 404
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Fig. 2. The box-plot for As, Cd, Pb, Th, and U.
was observed that the level of As, Cd, and Pb increased with years, while the level of Th and U decreased with years. Statistically significant difference was only found for Th (GM: 0.39 ng/g for group < 40 years vs. 0.26 ng/g for group ≥40 years). The level of Pb and Cd in smokers (that consumed more than 10 cigarettes per day) was significantly higher when compared to non-smokers (GM: 25.72 ng/g vs.
0.22 ng/g). Men had higher levels of lead than women (GM: 23.70 vs. 20.04 ng/g). The level of uranium was not changed in regard to sex (p > 0.05). In order to evaluate differences in the element profiles according to subject's age, the data were separated into two categories. The first category included subjects from 18 to 40 years (< 40 years) and the second category subjects from 40 to 65 years (≥ 40 years). It 405
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Table 2 Blood metal levels (ng/g) in the adult Serbian population. Element
Population
GMa
P2.5 As
Cd
Pb
Th
U
a b c d
Total Sex Women Men Age < 40 years > 40 years Smoking status Non-smoker Smoker Total Sex Women Men Age < 40 years > 40 years Smoking status Non-smoker Smoker Total Sex Women Men Age < 40 years > 40 years Smoking status Non-smoker Smoker Total Sex Women Men Age < 40 years > 40 years Smoking status Non-smoker Smoker Total Sex Women Men Age < 40 years > 40 years Smoking status Non-smoker Smoker
95% CIb
Percentiles P10
P25
P50
P75
P95
P97.5
c
LL
p-value UL
d
0.50
0.04
0.19
0.40
0.63
0.88
1.70
1.89
0.56
0.81
0.53 0.45
0.04 0.04
0.27 0.10
0.40 0.33
0.68 0.56
0.84 0.95
1.57 1.84
1.70 2.11
0.55 0.43
0.83 0.94
0.50 0.51
0.04 0.04
0.16 0.25
0.41 0.38
0.63 0.65
0.83 0.91
1.45 1.89
1.59 2.03
0.50 0.52
0.83 0.91
0.44 0.61 0.32
0.03 0.21 0.06
0.13 0.34 0.13
0.41 0.40 0.17
0.60 0.69 0.33
0.76 0.93 0.63
1.59 1.43 1.37
1.77 1.83 1.70
0.43 0.55 0.35
0.82 0.92 0.56
0.35 0.28
0.11 0.05
0.14 0.09
0.19 0.16
0.35 0.26
0.64 0.57
1.64 0.79
1.78 0.85
0.40 0.27
0.61 0.48
0.30 0.34
0.05 0.10
0.10 0.14
0.15 0.19
0.23 0.36
0.73 0.60
1.66 0.77
1.80 0.99
0.38 0.33
0.59 0.54
0.24 0.49 20.94
0.07 0.12 7.44
0.12 0.19 9.92
0.15 0.31 15.68
0.19 0.45 21.17
0.42 0.79 32.46
0.76 1.75 42.57
0.82 1.81 45.11
0.21 0.55 20.29
0.42 0.77 27.49
20.04 23.70
7.13 9.87
9.88 17.14
12.99 17.74
20.35 27.01
32.88 29.56
42.08 43.13
43.77 44.15
18.76 18.80
27.43 33.46
20.38 21.70
6.73 8.53
10.89 9.88
15.25 15.60
20.86 27.82
31.62 35.10
45.27 41.54
46.24 42.08
17.82 19.40
28.82 30.11
18.04 25.72 0.30
6.73 11.25 0.04
7.47 14.93 0.11
11.53 18.58 0.13
17.51 27.01 0.34
31.62 38.41 0.60
36.41 43.13 1.13
41.81 44.15 1.42
15.72 22.26 0.33
26.57 34.01 0.56
0.34 0.22
0.03 0.08
0.11 0.13
0.13 0.13
0.44 0.26
0.67 0.40
1.28 0.52
1.51 0.52
0.35 0.16
0.66 0.38
0.39 0.26
0.12 0.03
0.13 0.10
0.13 0.13
0.50 0.34
0.79 0.45
1.45 0.67
1.60 0.80
0.34 0.24
0.78 0.46
0.26 0.37 0.06
0.06 0.08 0.03
0.12 0.13 0.03
0.13 0.13 0.04
0.33 0.42 0.05
0.51 0.79 0.08
0.72 1.45 0.13
0.84 1.60 0.13
0.23 0.33 0.05
0.48 0.77 0.07
0.06 0.04
0.03 0.02
0.03 0.03
0.04 0.04
0.06 0.04
0.08 0.05
0.13 0.10
0.15 0.10
0.05 0.03
0.08 0.07
0.06 0.05
0.03 0.03
0.03 0.03
0.04 0.04
0.05 0.05
0.10 0.07
0.13 0.09
0.17 0.10
0.05 0.05
0.09 0.07
0.05 0.06
0.03 0.03
0.03 0.04
0.04 0.04
0.05 0.07
0.06 0.09
0.09 0.13
0.11 0.17
0.04 0.05
0.07 0.09
p > 0.05
p > 0.05
p > 0.05
p > 0.05
p > 0.05
p < 0.05
p < 0.05
p > 0.05
p < 0.05
p < 0.05
p < 0.05
p > 0.05
p > 0.05
p > 0.05
p > 0.05
Geometric mean (GM). Confidence interval (CI) of the 95th percentile. Lower limit (LL) of the 95%CI. Upper limit (UL) of the 95%CI.
4.1. Arsenic (As)
18.04 ng/g for Pb, and 0.49 ng/g vs. 0.24 ng/g for Cd).
Arsenic is a non-essential metalloid for humans. Exposure to inorganic arsenic, As3+ and As5+, mainly occurs through a drinking water and smoking tobacco. Exposure to organic arsenic (arsenocholine or arsenobetaine) primarily occurs through fish or seafood consumption. Chronic exposure to inorganic arsenic has been associated with the skin, lung, and bladder cancers (Freire et al., 2015; Kim et al., 2017; Jovanović et al., 2011; Goulle et al., 2014). It was well known that arsenic in the drinking waters presents a serious public health problem in Serbia, but it was not uniquely present in all drinking waters. Thus, the highest arsenic levels were found in the drinking waters in the Middle Banat region (Fig. 1). The central and southern regions of Serbia
4. Discussion Biomonitoring (BM) of toxic metals in the whole blood has become an important tool for occupational and environmental human's health. Populations from different areas worldwide showed different element exposure profiles and different dietary patterns, which could explain the discrepancies between different countries. According to this, it is very important to compare blood metal levels for the adult Serbian population with other population groups worldwide.
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2017). Our results indicated that B-Cd were higher in the older group of peoples. However, the difference was not statistically significant (Table 2). This finding could be explained by the low rate of urine excretion (0.001%) and long-term accumulation in the body, particularly in the renal cortex, where reach a plateau at 50 years. The half-life of Cd in the blood is approx. 2.5 months. This fact created the basis for Cd toxicity (Forte et al., 2011; Khlifi et al., 2014). The B-Cd concentrations for smokers were twice higher than for non-smokers (p < 0.05). Some authors reported up to three or more times higher values for the B-Cd (Černa et al., 2012; Freire et al., 2015; Forte et al., 2011; Wilhelm et al., 2004; Nunes et al., 2010).
contained much lower levels of arsenic in the drinking waters (Jovanović et al., 2013). Obtained a GM value for B-As (0.50 ng/g) was significantly lower when compared to non-occupationally exposed individuals living in the non-European countries: Brazilian population in Rio Branco, GM = 4.19 ng/g (Freire et al., 2015), Benin population (Cotonou) in West Africa, GM = 5.81 ng/g (Yedomon et al., 2017), general Korean population, GM = 7.19 ng/g (Kim et al., 2017), and Canadian population, GM = 2.0 ng/g (Saravanabhavan et al., 2017). It is interesting to point out that these reported values were similar to those in individuals living near mining and active industrial areas in the South of Tunisia (Khlifi et al., 2014) or in the steel mill workers in Pakistan (Afridi et al., 2011), as well as in the control subjects in a bladder cancer case-control study (Feki-Tounsi et al., 2013). According to the chemometric parameters and in the comparison with other European counties, the Serbian population had lower B-As than the Lombardy region of Northern Italy (max. value: 2.38 vs. 11.9 ng/g) (Minoia et al., 1990), general French population (median value: 0.63 vs. 1.87 ng/g) (Goulle et al., 2014), Northern French population (GM = 1.67 ng/g) (Nisse et al., 2017), and Danish population (mean value: 0.69 vs. 9.0 ng/g) (Poulsen et al., 1994). The similarities in the results were only found by the inhabitants of the Northern German, GM = 0.71 ng/g (Heitland and Koster, 2006). Kim et al. (2017) found that male Koreans had significantly higher As than females. According to our results, there were no significant differences in regard to sex, age or smoking habits (Table 2). These findings were in agreement with Freire et al. (2015), Nunes et al. (2010), Khlifi et al. (2014), Nisse et al. (2017) and other studies.
4.3. Lead (Pb) The predominant sources of lead exposure for the non-smoker population are drinking water, food and contaminated air. Chronic exposure to lead has negative effects on hem-biosynthesis and cardiovascular systems, as well as on the nervous and gastrointestinal systems. The blood is the best sample for assessing short-term individual exposure to lead (half-life in blood is approx. 30 days and 95% of the total lead binds to the erythrocytes) (Forte et al., 2011). The level of lead in the air was drastically reduced in Serbia after removing tetraethyl lead from the gasoline in the 1980s. Unfortunately, there was no information about B-Pb levels since then, so we can only biomonitoring lead level of our population from now. Obtained GM and P95 values for B-Pb in all investigated samples were 20.94 and 42.57 ng/g, respectively (Table 2). In comparison with other population groups worldwide, we found some similarities with the German population studied by Heitland and Koster (2006) (GM = 19 ng/g; P95 = 47 ng/g), and the French population studied by (P95 = 39.2 ng/g) and Goulle et al. (2014) (P95 = 39.3 ng/g). Some European countries had significantly higher values than Serbia, such as Germany (P95 = 71 ng/g) (Wilhelm et al., 2004) and Czech Republic (GM = 29 ng/g) (Černa et al., 2012). On the other hand, our values were up to five times lower when compared to the Italian population studied by, Alimonti et al. (2005), and Forte et al. (2011). The concentration of lead in the blood of men was higher. However, this difference was not statistically significant and could be associated with differences in lifestyles. The higher B-Pb values for men were also reported by Forte et al. (2011), Wilhelm et al. (2004) and others. Our results for older group were higher in comparison with a younger group (Table 2). It was reported that lead increased with age as a result of storage in bones and teeth. In fact, bone holds approx. 90% of the body burden of this heavy metal, where it could remain up to 30 years. During lifespan, lead is released into circulation as a consequence of bone remodeling (Forte et al., 2011) and thus exhibits toxic effects. In our studied population, smokers had significantly higher Pb in blood than non-smokers (p < 0.05) and these findings were in agreement to Lee et al. (2012) and Černa et al. (2012).
4.2. Cadmium (Cd) For the general population of non-smokers, diet are the main source of cadmium intakes. Cadmium is a highly toxic metal classified as a human carcinogen (Group 1) (Freire et al., 2015). Cigarettes contain 1–2 µg of cadmium, of which 10% is inhaled and approx. 5% is absorbed. Chronic exposure to cadmium has been associated with renal disease and increased risk of bone fractures (Forte et al., 2011). This heavy metal persists in the kidneys and bones for many years, which indicated that the toxicity of cadmium could occur with no additional occupational or environmental exposure (Satarug and Moore, 2004). Obtained GM, P95 and UL values for B-Cd were 0.32, 1.37 and 0.56 ng/g, respectively (Table 2). Our results were in agreement with the general Brazilian population (Nunes et al., 2010), Bremen area in the northern Germany, GM = 0.38 ng/g (Heitland and Koster, 2006), elderly Swedish population (Schultze et al., 2013), and French population studied by Goulle et al. (2005) (GM = 0.31 ng/g), Goulle et al. (2014) (GM = 0.34 ng/g) and Nisse et al. (2017) (GM = 0.39 ng/g). On the other hand, B-Cd for the Serbian population was lower when compared to subjects living in the: Acre State, Brazil (UL = 0.87 ng/g) (Freire et al., 2015), Sardinia, Italy (P95 = 1.82 ng/g) (Forte et al., 2011), Germany (P95 = 2.34 ng/g) (Wilhelm et al., 2004), Tunisia (P95 = 2.31 ng/g), Italy, Rome (P95 = 1.97 ng/g) (Alimonti et al., 2005). It is interesting to point out that our results were higher when compared to the Spanish population occupationally exposed to cadmium, GM = 0.18 ng/g (Gil et al., 2011), but this value was not in agreement with many populations groups worldwide. Some of the mean values reported for professionally exposed peoples were: 9.81 ng/g for adult inhabitants living in the vicinity of a cement factory in the Pakistan (Afridi et al., 2011) and 24.10 ng/g for the Chinese respondents residing in a polluted area (Wang et al., 2011), and these values were significantly lower when compared to the Serbian population (0.46 ng/ g). Women in our study had significantly higher levels of B-Cd than men (Table 2). These results were in agreement with many studies (Forte et al., 2011; Kim et al., 2017; Černa et al., 2012). One of the main reasons for higher B-Cd is twice higher gastrointestinal absorption of dietary cadmium in women than in men (Forte et al., 2011; Kim et al.,
4.4. Thorium (Th) and uranium (U) Thorium and uranium enter into our body via the air, food and drinking water. Thorium is an alternative source of nuclear fuel. Uranium is an alpha-emitting, radioactive and heavy metal (Kumar et al., 2013; Hon et al., 2015; Arzuaga et al., 2015). Bombs with depleted uranium (DU) were used in aircraft operations against the Federal Republic of Yugoslavia (1999), today's Serbia, and more than 31,000 rounds of ammunition comprising about 8.5 t of DU were shot. In some individuals who participated in land operations, as well as in the civil population in the area involved, there was an enhanced incidence of cancer. Epidemiological studies found an increased frequency of chromosomal abnormalities in the exposed population. It was suggested that DU exposure is either a primary cause or related to the main cause of congenital anomalies and increased rate of malignant 407
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diseases (Hon et al., 2015). In the study of Rodushkin et al. (1999) reported range for Th in the blood of 31 healthy subjects was < 0.008–0.20 ng/g (median 0.067 ng/g). Our results were 5 times higher in regard to a median value (0.34 ng/g) (Table 1). The same group of authors pointed out that women showed significantly higher values than men. This is in agreement with our results (Table 2). We also found that level of B-Th significantly decreased with age (Table 2). Heitland and Koster (2006) reported GM value < 0.003 ng/g for the German population (n = 130) and P5-P95 range below < 0.003 ng/g (which is the concentration below detection limit of ICP-MS method). Our GM was 0.30 µg/L, or 100 times higher for the Serbian population. There are few studies that reported uranium level in the whole blood. For the 100 healthy subjects living in France, median blood-U level was 0.004 ng/g (P5-P95 = 0.002–0.006 ng/g) (Goulle et al., 2005). It was up to 12.5 times lower than for the Serbian population (Table 1). Yedomon et al. (2017) reported GM value for B-U in the Benin population 0.002 ng/g and the P5-P95 range was < 0.001–0.008 ng/g. This range was significantly lower when compared to the Serbian population (P5-P95 = 0.004–0.13 ng/g) and 3 times higher for our studied population in terms of GM value of the total examined population. The GM value for B-U value reported by Heitland and Koster (2006) was below 0.003 ng/g. It was 20 times lower when compared to the Serbian population (Table 2).
Chim. Acta 292, 163–173. Alimonti, A., Bocca, B., Mannell, E., Petrucci, F., Zennaro, F., Cotichini, R., D’ipplito, C., Agresti, A., Caimi, S., Forte, G., 2005. Assessment of reference values for selected elements in a healthy urban population. Ann. Ist. Super. Sanita 41, 181–187. Arzuaga, X., Gehlhaus, M., Strong, J., 2015. Modes of action associated with uranium induced adverse effects in bone function and development. Toxicol. Lett. 236, 123–130. Avino, P., Capannesi, G., Manigrasso, M., Sabbioni, E., Rosada, A., 2011. Element assessment in whole blood, serum and urine of three Italian healthy sub-populations by INAA. Microchem. J. 99, 548–555. Bevan, R., Jones, J., Cocker, J., Assem, F.L., Levy, L.S., 2013. Reference ranges for key biomarkers of chemical exposure within the UK population. Int. J. Hyg. Environ. Heal. 216, 170–174. Bocca, B., Madeddu, R., Asara, Y., Tolu, P., Marchal, J.A., Forte, G., 2011. Assessment of reference ranges for blood Cu, Mn, Se and Zn in a selected Italian population. J. Trace Elem. Med. Biol. 25, 19–26. Canas, A.I., Cervantes-Amat, M., Esteban, M., Ruiz-Moraga, M., Perez-Gomez, B., Mayor, J., Castano, A., 2014. Blood lead levels in a representative sample of the Spanish adult population: the BIOAMBIENT.ES project. Int. J. Hyg. Environ. Heal. 217, 452–459. Černa, M., Krskova, A., Čejchanova, M., Spevačkova, V., 2012. Human biomonitoring in the Czech Republic: an overview. Int. J. Hyg. Environ. Heal. 215, 109–119. Feki-Tounsi, M., Olmedo, P., Gil, F., Khlifi, R., Mhiri, M.N., Rebai, A., Hamza-Chaffai, A., 2013. Low level arsenic exposure is associated with bladder cancer risk and cigarette smoking: a case-control study among men in Tunisia. Environ. Sci. Pollut. Res. Int. 20, 3923–3931. Forte, G., Madeddu, R., Tolu, P., Asara, Y., Marchal, J.A., Bocca, B., 2011. Reference intervals for blood Cd and Pb in the general population of Sardinia (Italy). Int. J. Hyg. Environ. Heal. 214, 102–109. Freire, C., Koifman, R.J., Fujimoto, D., Souza Jr, V.C.O., Koifman, S, F.B., 2015. Reference values of cadmium, arsenic and manganese in blood and factors associated with exposure levels among adult population of Rio Branco, Acre, Brazil. Chemosphere 128, 70–78. Gil, F., Hernandez, A.F., Marquez, C., Femia, P., Olmedo, P., Lopez-Guarnido, O., Pla, A., 2011. Biomonitoring of cadmium, chromium, manganese, nickel and lead in whole blood, urine, axillary hair and saliva in an occupationally exposed population. Sci. Total Environ. 409, 1172–1180. Goulle, J.P., Mahieu, L., Castermant, J., Neveu, N., Bonneau, L., Laine, G., Bouige, D., Lacroix, C., 2005. Metal and metalloid multi-elementary ICP-MS validation in whole blood, plasma, urine and hair: reference values. Forensic Sci. Int. 153, 39–44. Goulle, J.P., Saussereau, E., Mahieu, L., Guerbet, M., 2014. Current role of ICP-MS in clinical toxicology and forensic toxicology: a metallic profile. Bioanalysis 6, 2245–2259. Heitland, P., Koster, H.D., 2006. Biomonitoring of 37 trace elements in blood samples from inhabitants of northern Germany by ICP-MS. J. Trace Elem. Med. Biol. 20, 253–262. Hon, Z., Osterreicher, J., Navratil, L., 2015. Depleted uranium and its effects on humans. Sustainability 7, 4063–4077. Jovanović, D., Jakovljević, B., Rašić-Milutinović, Z., Paunović, K., Peković, G., Knezević, T., 2011. Arsenic occurrence in drinking water supply systems in ten municipalities in Vojvodina Region, Serbia. Environ. Res. 111, 315–318. Jovanovic, D., Rašić-Milutinovic, Z., Paunovic, Paunovic, K., Jakovljevic, B., Plavsic, S., Milosevic, J., 2013. Low levels of arsenic in drinking water and type 2 diabetes in Middle Banat region, Serbia. Int. J. Hyg. Environ. Heal. 216, 50–55. Khlifi, R., Olmedo, P., Gil, F., Feki-Tounsi, M., Hammami, B., Rebai, A., Hamza-Chaffai, A., 2014. Biomonitoring of cadmium, chromium, nickel and arsenic in general population living near mining and active industrial areas in Southern Tunisia. Environ. Monit. Assess. 186, 761–779. Kim, H.J., Lim, H.S., Lee, K.R., Choi, M.H., Kang, N.M., Lee, C.H., Oh, E.J., Park, H.K., 2017. Determination of trace metal levels in the general population of Korea. Int. J. Environ. Res. Public Health 14, 1–12. Kuno, R., Roquetti, M.H., Becker, K., Seiwert, M., Gouveia, N., 2013. Reference values for lead, cadmium and mercury in the blood of adults from the metropolitan area of Sao Paulo, Brazil. Int. J. Hyg. Environ. Heal. 216, 243–249. Lee, J.W., Lee, C.K., Moon, C.S., Choi, I.J., Lee, K.J., Yi, S.M., Jang, B.K., Yoon, B.J., Kim, D.S., Peak, D., Sul, D., Oh, E., Im, H., Kang, H.S., Kim, J.H., Lee, J.T., Kim, K., Park, K.L., Ahn, R., Park, S.H., Kim, S.C., Park, C.H., Lee, J.H., 2012. Korea National Survey for Environmental Pollutants in the Human body 2008: heavy metals in the blood or urine of the Korean population. Int. J. Hyg. Environ. Heal. 215, 449–457. Minoia, C., Sabbioni, E., Apostoli, P., Pietra, R., Pozzoli, L., Gallorini, M., Nicolaou, G., Alessio, L., Capodaglio, E., 1990. Trace element reference values in tissues from inhabitants of the Europian community I. A study of 46 elements in urine, blood and serum of Italian subjects. Sci. Total Environ. 95, 89–105. Nisse, C., Tagne-Fotso, R., Howsam, M., Richeval, C., Labat, L., Leroyer, A., 2017. Blood and urinary levels of metals and metalloids in the general adult population of Northern France: the IMEPOGE study, 2008–2010. Int. J. Hyg. Environ. Heal. 220, 341–363. Nunes, J.A., Batista, B.L., Rodrigues, J.L., Caldas, N.M., Neto, J.A.G., Barbosa Jr, F., 2010. A simple method based on ICP-MS for estimation of background levels of arsenic, cadmium, copper, manganese, nickel, lead, and selenium in blood of the Brazilian population. J. Toxic. Environ. Health A 73, 878–887. Poulsen, O.M., Christensen, J.M., Sabbioni, E., Van der Venne, M.T., 1994. Trace element reference values in tissues from inhabitants of the Europian community. V. Review of trace elements in blood, serum and urine and critical evaluation of reference values for the Danish population. Sci. Total Environ. 141, 197–215. Rocha, G.H.O., Steinbach, C., Munhoz, J.R., Madia, M.A.O., Faria, J.K., Hoeltgebaum, D.,
5. Conclusion This human biomonitoring study provides the first background concentrations for As, Cd, Pb, Th and U in Serbia. Reported data represent a new contribution to the knowledge of the blood chemistry for adult population. It was found that women had statistically significantly higher level of Th in the blood. With regard to age, significant increase of the same element was found for a younger group. The investigated smoker's group had significantly higher levels of Cd and Pb in blood than non-smokers group. In comparison with other population group worldwide, Serbian population had significantly higher level of Th and U in the whole blood (up to 100 times higher). These findings could highlight the molecular basis for the increased incidence of the malignant disease among the Serbian population, which need to be confirmed by clinical studies. Also, the obtained reference values could be interpreted as a baseline for further toxicological assessment studies. Acknowledgement This research was financially supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia, project No. 172030. Conflicts of interest The authors declare that they have no conflict of interest. Ethical approval All procedures performed in studies involving human subjects were in accordance with the ethical standards of the institutional and national research committee, as well as with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. References Afridi, H.I., Kazi, T.G., Kazi, A.G., Shah, F., Wadhwa, S.K., Kolachi, N.F., Shah, A.Q., Baig, J.A., Kazi, N., 2011. Levels of arsenic, cadmium, lead, manganese and zinc in biological samples of paralysed steel mill workers with related to controls. Biol. Trace Elem. Res. 144, 164–182. Alimonti, A., Petrucci, F., Laurenti, F., Papoff, P., Caroli, S., 2000. Reference values for selected trace elements in serum of term newborns from the urban area of Rome. Clin.
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Schultze, B., Lind, P.M., Larsson, A., Lind, L., 2013. Whole blood and serum concentrations of metals in a Swedish population-based sample. Scand. J. Clin. Lab. Invest. 1, 1–6. Wang, M., Xu, Y., Pan, S., Zhang, A., Song, H., Ling, W., 2011. Long-term heavy metal pollution and mortality in a Chinese population: an ecologic study. Biol. Trace Elem. Res. 142, 362–379. Wilhelm, M., Ewers, U., Schulz, C., 2004. Revised and new reference values for some trace elements in blood and urine for human biomonitoring in environmental medicine. Int. J. Hyg. Environ. Heal. 207, 69–73. Yedomon, B., Menudier, A., Etangs, F.L.D., Anani, L., Fayomi, B., Druet-Cabanac, M., Moesch, C., 2017. Biomonitoring of 29 trace elements in whole blood from inhabitants of Cotonou (Benin) by ICP-MS. J. Trace Elem. Med. Biol. 43, 38–45.
Barbosa Jr., F., Batista, B.L., Souza, V.C.O., Nerilo, S.B., Bando, E., Mossini, S.A.G., Nishiyama, P., 2016. Trace metal levels in serum and urine of a population in southern Brazil. J. Trace Elem. Med. Biol. 35, 61–65. Rodushkin, I., Odman, F., Branth, S., 1999. Multielement analysis of whole blood by high resolution inductively coupled plasma mass spectrometry. Fresenius J. Anal. Chem. 364, 338–346. Saravanabhavan, G., Werry, K., Walker, M., Douglas, H., Malowany, M., Khourly, C., 2017. Human biomonitoring reference values for metals and trace elements in blood and urine derived from the Canadian Health Measures Survey 2007–2013. Int. J. Hyg. Environ. Heal. 220, 189–200. Satarug, S., Moore, M.R., 2004. Adverse health effects of chronic exposure to low-level cadmium in foodstuffs and cigarette smoke. Environ. Health. Persp. 112, 1099–1103.
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The human biomonitoring study in Serbia: Background levels for arsenic, cadmium, lead, thorium and uranium in the whole blood of adult Serbian population
T
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Aleksandar Stojsavljevića, , Slavica Borković-Mitićb, Ljiljana Vujotićc,d, Danica Grujičićc,d, Marija Gavrović-Jankulovića, Dragan Manojlovića,e University of Belgrade – Faculty of Chemistry, Studentski trg 16, 11000 Belgrade, Serbia Department of Physiology, Institute for Biological Research “Siniša Stanković”, University of Belgrade, Bulevar despota Stefana 142, Belgrade 11060, Serbia Faculty of Medicine, University of Belgrade, Dr Subotića 8, 11000 Belgrade, Serbia d Clinical Center of Serbia, Neurosurgery Division, Dr Koste Torodorića 4, 11000 Belgrade, Serbia e South Ural State University, Lenin prospect 76, 454080 Chelyabinsk, Russia a
b c
A R T I C LE I N FO
A B S T R A C T
Keywords: Toxic trace metals Reference values Serbian population Thorium/Uranium
The purpose of this study was to establish reference values (RVs) for the occupationally- and environmentallyimportant toxic elements in the whole blood of adult Serbian population for the first time. Contaminated drinking water with arsenic, high share of smokers in the country, removing tetraethyl lead from the gasoline and war attack at the end of the twentieth century were some of the reasons to provide background information for arsenic (As), cadmium (Cd), lead (Pb), thorium (Th), and uranium (U) in the blood of the Serbian population. The whole blood samples were collected from the healthy respondents living in the Belgrade and surrounding areas of the capital (n = 305; w/m ratio = 154/151; mean age: 41 ± 2). The concentrations of toxic metals were determined by inductively coupled plasma-mass spectrometry (ICP-MS). Reference values were estimated as the lower limit (LL) and upper limit (UL) of the 95% confidence interval (CI), together with the selected percentiles (P2.5-P97.5). The obtained geometric mean (GM) for As, Cd, Pb, Th, and U were: 0.50 ng/g, 0.32 ng/ g, 20.94 ng/g, 0.30 ng/g, and 0.06 ng/g, respectively. The influences of age, sex and lifestyle on results were considered. Women have significantly higher levels of Cd and Th than men. The increased level of Th was observed in the aged group below 40 years, while smokers had significantly higher levels of Pb and double higher level of Cd in the blood than non-smokers (p < 0.05). In comparison with other population groups worldwide, the Serbian population had significantly higher levels of Th and U (up to 100 times higher). These findings could contribute to better understanding of the molecular basis for the development of various health hazards, including the increased incidence of cancer among the Serbian population which need be confirmed by clinical studies.
1. Introduction The excess of harmful metal/metalloid has negative consequences to the human health. Daily exposure to toxic metals through contaminated air, food or drinking water is of a major cause for concern, because of its abilities to de-regulate the immune system and increase the incidence of various disease states, including cancer (Kim et al., 2017; Avino et al., 2011; Rocha et al., 2016). Today, the majority of the world's population is exposed to various metals from the increasing anthropogenic pollution (Černa et al., 2012; Khlifi et al., 2014; Yedomon et al., 2017).
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Corresponding author. E-mail address: [email protected] (A. Stojsavljević).
https://doi.org/10.1016/j.ecoenv.2018.11.043
0147-6513/ © 2018 Elsevier Inc. All rights reserved.
Throughout the world, biomonitoring has become the standard for assessing exposure of individuals to different chemicals, including toxic metals (Nunes et al., 2010). Human biomonitoring is a scientific technique for assessing human exposures to environmental agents and their effects, based on sampling and analysis of an individual's tissues and fluids. Among the various clinical samples, whole blood is the most used matrices for biomonitoring of toxic metal exposure in the general population, population groups and individuals to environmental/ occupational pollutants (Canas et al., 2014; Bevan et al., 2013; Lee et al., 2012; Gil et al., 2011; Bocca et al., 2011; Forte et al., 2011). Many efforts were invested in order to provide reference values
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(RVs) for the trace elements in the body fluids of the unexposed population groups. In that manner it is possible to compare the trace element levels of unexposed subjects to environment-, occupationally-, or accidentally-exposed peoples as well as to find the physiological changes, the presence of the various diseases or effectiveness of the medical therapies (Avino et al., 2011; Bocca et al., 2011). The sample size of the examined population should be large enough to cover age-, sex- and other differences. The International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) recommended a sample size of at least 120 people. The validity of RVs seriously depends not only from final instrumental determination, but also from the adopted preanalytical procedures. (Saravanabhavan et al., 2017; Alimonti et al., 2000). The RVs for the toxic and essential metals should be established periodically, since they can differ among populations and can be affected by peoples’ lifestyles and dissimilar environmental scenarios (Bocca et al., 2011). A large number of countries determined their own RVs for trace elements. Some of them used the large number of individuals, such as a National Health and Nutrition Examination Survey (NHANES) or a German Environmental Survey (GerES) (Kuno et al., 2013). Despite the growing number of biomonitoring studies worldwide, no previous biomonitoring study on the blood trace element levels in Serbia has been reported. The aim of this biomonitoring study was to establish reference values for occupationally- and environmentally-important toxic elements (arsenic, cadmium, lead, thorium, and uranium) in the blood of adult Serbian population, as well as to find the differences in element profile according to sex, age and smoking habits. In such way it was possible to compare our studied population with other population groups worldwide.
A certified stock standard solution (VHG, Manchester, UK) concentration of 10 mg/L was used to prepare intermediate standard solutions. An internal standard solution 100 μg/mL Li, Sc; 20 μg/mL Bi, Ga, In, Tb, Y (VHG, Manchester, UK) was used. Instrument parameters of ICP-MS were optimized using an iCAP Q tuning solution B containing 1 µg/L of Ba, Bi, Ce, Co, In, Li, and U in a 2% nitric acid (Thermo Scientific, UK). For analytical quality assurance, Seronorm® (Sero AS, Norway) Trace Elements Whole Blood, Level 1 (L-1) (SERO210105) and Level 3 (L-3) (SERO210305) were employed. Microwave digestion was done on the ETHOS 1 Advanced Microwave Digestion System (Milestone, Italy). The element quantifications were carried out by inductively coupled plasma-mass spectrometry, ICP-MS (iCAP Q, Thermo Scientific, UK). The plasma and nebulizer gas used pure argon (99.999%) supplied by the Messer, Serbia. The collision cell of the ICP-MS was filled with pressured pure helium (99.999%; Messer, Serbia). PFA-ST micro-flow nebulizer and glass cyclonic spray chambers was employed. All measurements on ICP-MS were performed in the kinetic energy discrimination mode by helium as the collision cell gas. Internal standardization was included to compensate matrix-induced ion signal fluctuations and instrumental drift. Due to the large mass range, four internal standards (45Sc in a concentration of 50 µg/L, and 89Y, 159Tb and 209Bi in a concentration of 10 µg/L) were applied. This intermediate internal standard (IS) solution was aspirated by the second channel of the peristaltic pump, allowing on-line addition to the calibration blank, standards and the sample solutions. Six calibration solutions were employed to cover the range of analyte concentrations. In the range from 0.01 to 25 µg/L, correlation coefficient (r) was greater than 0.997 for all examined elements. Based on repeated measurements (n = 6), the relative standard deviation (RSD) of all elements was < 5%. The limit of detection (LOD) was calculated using standard deviation (SD) of the responses and the slopes (S) of the calibration curves according to the formula LOD = 3 (SD/S). The LOD values for As, Cd, Pb, Th and U were: 0.04, 0.02, 0.11, 0.004, 0.001 µg/L, respectively. The traceability of the analytical procedure was controlled by analyzing the SRMs. The certified vs. founded values, together with the recovery (R) values in percentiles (%) for As, Cd, Pb, Th and U in the SRM L-1 were: 2.40 vs. 2.15 ng/g (R = 89.6%), 0.36 vs. 0.33 ng/g (R = 91.7%), 10.20 vs. 11.1 ng/g (R = 108.8%), 6.0 vs. 5.1 pg/g (R = 85%) and 46.0 vs. 44.2 pg/g (R = 96.1%), respectively. The certified vs. founded values, together with the recovery values for As, Cd, Pb, Th and U in the SRM L-3 were: 30.4 vs. 29.3 ng/g (R = 96.4%), 12.1 vs. 11.2 ng/g (R = 92.6%), 186 vs. 174 ng/g (R = 93.5%), 9.0 vs. 7.9 pg/g (R = 87.8%), 69.0 vs. 64.2 pg/g (R = 93%), respectively.
2. Materials and methods 2.1. Sample collection This study included 305 clinically healthy subjects (women/men ratio = 154/151). All examined respondents were from Belgrade and the surrounding areas of the capital city (Fig. 1). The selected blood donors consisted only of adults, age ranging from 18 to 65 years (mean age: 41 ± 2). The blood samples were collected within a three month period (March–June 2018). About 38% of subjects were smokers (consuming more than 10 cigarettes per day) and were included in our investigation with the aim to compare results for smokers and nonsmokers. All examined subjects were voluntary blood donors and were selected by medical professionals. From each donor a 5 mL of intravenous blood was drawn in the morning hours and stored in the trace metal free evacuated tube (BD Vacutainer). The gathered data included information on age, gender, place of residence, smoking habits, medical history, fish consumption, and other data that could influence on metal concentrations. After careful consideration of more than 500 subjects, only 305 of them met the requirements to participate in our research. These subjects were reported not to be occupationally exposed to metal compounds or to have a chronic disease. The study was approved by the Ethics Committee of Clinical Centre Belgrade, Serbia. All subjects voluntarily participated in this study and the consent was obtained from each subject.
2.3. Sample preparation After removing the whole blood from the freezer at − 80 °C, samples were kept at room temperature before sampling aliquots (approx. 1.5 mL of each sample). Four milliliters of 65% nitric acid and 1 mL of 30% hydrogen-peroxide were added to each PTFE cuvette and digestion was performed under the following programs: warm up for 2 min to 85 °C, 4 min to 135 °C, 5 min to 230 °C and held for 15 min at that temperature. After cooling samples were quantitatively transferred into a volumetric flask (25 mL) and diluted with ultrapure water. Prior to analysis, the standard reference materials (SRMs) were prepared according to manufacturer recommendation and further prepared in the same way as described for real samples.
2.2. Chemicals, instrumentation and analysis All used chemicals were of analytical grade and were supplied by Merck (Darmstadt, Germany). Concentrated nitric acid was additionally purified by double distillation. Ultrapure water was prepared by passing double de-ionized water of Milli-Q system (18.2 MΩ). In order to minimize contamination, all plastic and glass material was immersed in 10% HNO3 for 24 h and rinsed with ultrapure water before use.
2.4. Data analysis Descriptive statistics and Mann-Whitney U-test was performed by a demo version of the NCSS statistical software Number Crucher Statistical System Kaysville, UT, www.ncss.com). Following the recommendations of IFCC and IUPAC (International Union for Pure and 403
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Fig. 1. The sampling sites of blood collection at a map of Serbia are shaded.
3. Results
Table 1 Parameters of descriptive statistics for all investigated blood samples (ng/g).
Mean Stdev Min Max Median
As
Cd
Pb
Th
U
0.69 0.48 0.02 2.38 0.63
0.46 0.41 0.04 1.86 0.33
23.89 11.54 6.09 47.21 21.17
0.44 0.38 0.03 1.76 0.34
0.06 0.04 0.03 0.20 0.05
By applying ICP-MS method for metal determination, less than 1% of examining element concentrations were below the limit of detection. These samples had low level of arsenic. It was observed that peoples who eat seafood/fish more than twice a week had higher levels of arsenic in the blood (GM = 0.53 ng/g) than peoples who eat seafood/fish less than twice a week (GM = 0.51). However, this difference was not statistically significant (p > 0.05). Parameters of descriptive statistics are presented in Table 1 and Fig. 2. Considering all investigated blood samples, lead was the most abundant element, followed by arsenic, cadmium, thorium and uranium. It is interesting to point out that the level of Th was practically the same as the level of Cd for the total studied population (0.44 ± 0.38 ng/g vs. 0.46 ± 0.41 ng/g). Differences between examining groups are presented in Table 2. In comparison with men, women had higher values of As, Cd and Th. However, this difference was only significant for Th (GM: 0.34 vs.
Applied Chemistry), reference values for toxic metals was expressed as percentiles (P) in the range from 2.5th–97.5th, and was calculated as the lower limit (LL) and the upper limit (UL) of the 95% confidence interval (CI). The metal concentrations below the LOD values were replaced with the value LOD/2. The reference values for each element were computed for men, women, subjects aged < 40 years old, subjects aged ≥ 40 years old, as well as for smokers and non-smokers. The differences between groups were considered significant at p < 0.05. 404
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Fig. 2. The box-plot for As, Cd, Pb, Th, and U.
was observed that the level of As, Cd, and Pb increased with years, while the level of Th and U decreased with years. Statistically significant difference was only found for Th (GM: 0.39 ng/g for group < 40 years vs. 0.26 ng/g for group ≥40 years). The level of Pb and Cd in smokers (that consumed more than 10 cigarettes per day) was significantly higher when compared to non-smokers (GM: 25.72 ng/g vs.
0.22 ng/g). Men had higher levels of lead than women (GM: 23.70 vs. 20.04 ng/g). The level of uranium was not changed in regard to sex (p > 0.05). In order to evaluate differences in the element profiles according to subject's age, the data were separated into two categories. The first category included subjects from 18 to 40 years (< 40 years) and the second category subjects from 40 to 65 years (≥ 40 years). It 405
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Table 2 Blood metal levels (ng/g) in the adult Serbian population. Element
Population
GMa
P2.5 As
Cd
Pb
Th
U
a b c d
Total Sex Women Men Age < 40 years > 40 years Smoking status Non-smoker Smoker Total Sex Women Men Age < 40 years > 40 years Smoking status Non-smoker Smoker Total Sex Women Men Age < 40 years > 40 years Smoking status Non-smoker Smoker Total Sex Women Men Age < 40 years > 40 years Smoking status Non-smoker Smoker Total Sex Women Men Age < 40 years > 40 years Smoking status Non-smoker Smoker
95% CIb
Percentiles P10
P25
P50
P75
P95
P97.5
c
LL
p-value UL
d
0.50
0.04
0.19
0.40
0.63
0.88
1.70
1.89
0.56
0.81
0.53 0.45
0.04 0.04
0.27 0.10
0.40 0.33
0.68 0.56
0.84 0.95
1.57 1.84
1.70 2.11
0.55 0.43
0.83 0.94
0.50 0.51
0.04 0.04
0.16 0.25
0.41 0.38
0.63 0.65
0.83 0.91
1.45 1.89
1.59 2.03
0.50 0.52
0.83 0.91
0.44 0.61 0.32
0.03 0.21 0.06
0.13 0.34 0.13
0.41 0.40 0.17
0.60 0.69 0.33
0.76 0.93 0.63
1.59 1.43 1.37
1.77 1.83 1.70
0.43 0.55 0.35
0.82 0.92 0.56
0.35 0.28
0.11 0.05
0.14 0.09
0.19 0.16
0.35 0.26
0.64 0.57
1.64 0.79
1.78 0.85
0.40 0.27
0.61 0.48
0.30 0.34
0.05 0.10
0.10 0.14
0.15 0.19
0.23 0.36
0.73 0.60
1.66 0.77
1.80 0.99
0.38 0.33
0.59 0.54
0.24 0.49 20.94
0.07 0.12 7.44
0.12 0.19 9.92
0.15 0.31 15.68
0.19 0.45 21.17
0.42 0.79 32.46
0.76 1.75 42.57
0.82 1.81 45.11
0.21 0.55 20.29
0.42 0.77 27.49
20.04 23.70
7.13 9.87
9.88 17.14
12.99 17.74
20.35 27.01
32.88 29.56
42.08 43.13
43.77 44.15
18.76 18.80
27.43 33.46
20.38 21.70
6.73 8.53
10.89 9.88
15.25 15.60
20.86 27.82
31.62 35.10
45.27 41.54
46.24 42.08
17.82 19.40
28.82 30.11
18.04 25.72 0.30
6.73 11.25 0.04
7.47 14.93 0.11
11.53 18.58 0.13
17.51 27.01 0.34
31.62 38.41 0.60
36.41 43.13 1.13
41.81 44.15 1.42
15.72 22.26 0.33
26.57 34.01 0.56
0.34 0.22
0.03 0.08
0.11 0.13
0.13 0.13
0.44 0.26
0.67 0.40
1.28 0.52
1.51 0.52
0.35 0.16
0.66 0.38
0.39 0.26
0.12 0.03
0.13 0.10
0.13 0.13
0.50 0.34
0.79 0.45
1.45 0.67
1.60 0.80
0.34 0.24
0.78 0.46
0.26 0.37 0.06
0.06 0.08 0.03
0.12 0.13 0.03
0.13 0.13 0.04
0.33 0.42 0.05
0.51 0.79 0.08
0.72 1.45 0.13
0.84 1.60 0.13
0.23 0.33 0.05
0.48 0.77 0.07
0.06 0.04
0.03 0.02
0.03 0.03
0.04 0.04
0.06 0.04
0.08 0.05
0.13 0.10
0.15 0.10
0.05 0.03
0.08 0.07
0.06 0.05
0.03 0.03
0.03 0.03
0.04 0.04
0.05 0.05
0.10 0.07
0.13 0.09
0.17 0.10
0.05 0.05
0.09 0.07
0.05 0.06
0.03 0.03
0.03 0.04
0.04 0.04
0.05 0.07
0.06 0.09
0.09 0.13
0.11 0.17
0.04 0.05
0.07 0.09
p > 0.05
p > 0.05
p > 0.05
p > 0.05
p > 0.05
p < 0.05
p < 0.05
p > 0.05
p < 0.05
p < 0.05
p < 0.05
p > 0.05
p > 0.05
p > 0.05
p > 0.05
Geometric mean (GM). Confidence interval (CI) of the 95th percentile. Lower limit (LL) of the 95%CI. Upper limit (UL) of the 95%CI.
4.1. Arsenic (As)
18.04 ng/g for Pb, and 0.49 ng/g vs. 0.24 ng/g for Cd).
Arsenic is a non-essential metalloid for humans. Exposure to inorganic arsenic, As3+ and As5+, mainly occurs through a drinking water and smoking tobacco. Exposure to organic arsenic (arsenocholine or arsenobetaine) primarily occurs through fish or seafood consumption. Chronic exposure to inorganic arsenic has been associated with the skin, lung, and bladder cancers (Freire et al., 2015; Kim et al., 2017; Jovanović et al., 2011; Goulle et al., 2014). It was well known that arsenic in the drinking waters presents a serious public health problem in Serbia, but it was not uniquely present in all drinking waters. Thus, the highest arsenic levels were found in the drinking waters in the Middle Banat region (Fig. 1). The central and southern regions of Serbia
4. Discussion Biomonitoring (BM) of toxic metals in the whole blood has become an important tool for occupational and environmental human's health. Populations from different areas worldwide showed different element exposure profiles and different dietary patterns, which could explain the discrepancies between different countries. According to this, it is very important to compare blood metal levels for the adult Serbian population with other population groups worldwide.
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2017). Our results indicated that B-Cd were higher in the older group of peoples. However, the difference was not statistically significant (Table 2). This finding could be explained by the low rate of urine excretion (0.001%) and long-term accumulation in the body, particularly in the renal cortex, where reach a plateau at 50 years. The half-life of Cd in the blood is approx. 2.5 months. This fact created the basis for Cd toxicity (Forte et al., 2011; Khlifi et al., 2014). The B-Cd concentrations for smokers were twice higher than for non-smokers (p < 0.05). Some authors reported up to three or more times higher values for the B-Cd (Černa et al., 2012; Freire et al., 2015; Forte et al., 2011; Wilhelm et al., 2004; Nunes et al., 2010).
contained much lower levels of arsenic in the drinking waters (Jovanović et al., 2013). Obtained a GM value for B-As (0.50 ng/g) was significantly lower when compared to non-occupationally exposed individuals living in the non-European countries: Brazilian population in Rio Branco, GM = 4.19 ng/g (Freire et al., 2015), Benin population (Cotonou) in West Africa, GM = 5.81 ng/g (Yedomon et al., 2017), general Korean population, GM = 7.19 ng/g (Kim et al., 2017), and Canadian population, GM = 2.0 ng/g (Saravanabhavan et al., 2017). It is interesting to point out that these reported values were similar to those in individuals living near mining and active industrial areas in the South of Tunisia (Khlifi et al., 2014) or in the steel mill workers in Pakistan (Afridi et al., 2011), as well as in the control subjects in a bladder cancer case-control study (Feki-Tounsi et al., 2013). According to the chemometric parameters and in the comparison with other European counties, the Serbian population had lower B-As than the Lombardy region of Northern Italy (max. value: 2.38 vs. 11.9 ng/g) (Minoia et al., 1990), general French population (median value: 0.63 vs. 1.87 ng/g) (Goulle et al., 2014), Northern French population (GM = 1.67 ng/g) (Nisse et al., 2017), and Danish population (mean value: 0.69 vs. 9.0 ng/g) (Poulsen et al., 1994). The similarities in the results were only found by the inhabitants of the Northern German, GM = 0.71 ng/g (Heitland and Koster, 2006). Kim et al. (2017) found that male Koreans had significantly higher As than females. According to our results, there were no significant differences in regard to sex, age or smoking habits (Table 2). These findings were in agreement with Freire et al. (2015), Nunes et al. (2010), Khlifi et al. (2014), Nisse et al. (2017) and other studies.
4.3. Lead (Pb) The predominant sources of lead exposure for the non-smoker population are drinking water, food and contaminated air. Chronic exposure to lead has negative effects on hem-biosynthesis and cardiovascular systems, as well as on the nervous and gastrointestinal systems. The blood is the best sample for assessing short-term individual exposure to lead (half-life in blood is approx. 30 days and 95% of the total lead binds to the erythrocytes) (Forte et al., 2011). The level of lead in the air was drastically reduced in Serbia after removing tetraethyl lead from the gasoline in the 1980s. Unfortunately, there was no information about B-Pb levels since then, so we can only biomonitoring lead level of our population from now. Obtained GM and P95 values for B-Pb in all investigated samples were 20.94 and 42.57 ng/g, respectively (Table 2). In comparison with other population groups worldwide, we found some similarities with the German population studied by Heitland and Koster (2006) (GM = 19 ng/g; P95 = 47 ng/g), and the French population studied by (P95 = 39.2 ng/g) and Goulle et al. (2014) (P95 = 39.3 ng/g). Some European countries had significantly higher values than Serbia, such as Germany (P95 = 71 ng/g) (Wilhelm et al., 2004) and Czech Republic (GM = 29 ng/g) (Černa et al., 2012). On the other hand, our values were up to five times lower when compared to the Italian population studied by, Alimonti et al. (2005), and Forte et al. (2011). The concentration of lead in the blood of men was higher. However, this difference was not statistically significant and could be associated with differences in lifestyles. The higher B-Pb values for men were also reported by Forte et al. (2011), Wilhelm et al. (2004) and others. Our results for older group were higher in comparison with a younger group (Table 2). It was reported that lead increased with age as a result of storage in bones and teeth. In fact, bone holds approx. 90% of the body burden of this heavy metal, where it could remain up to 30 years. During lifespan, lead is released into circulation as a consequence of bone remodeling (Forte et al., 2011) and thus exhibits toxic effects. In our studied population, smokers had significantly higher Pb in blood than non-smokers (p < 0.05) and these findings were in agreement to Lee et al. (2012) and Černa et al. (2012).
4.2. Cadmium (Cd) For the general population of non-smokers, diet are the main source of cadmium intakes. Cadmium is a highly toxic metal classified as a human carcinogen (Group 1) (Freire et al., 2015). Cigarettes contain 1–2 µg of cadmium, of which 10% is inhaled and approx. 5% is absorbed. Chronic exposure to cadmium has been associated with renal disease and increased risk of bone fractures (Forte et al., 2011). This heavy metal persists in the kidneys and bones for many years, which indicated that the toxicity of cadmium could occur with no additional occupational or environmental exposure (Satarug and Moore, 2004). Obtained GM, P95 and UL values for B-Cd were 0.32, 1.37 and 0.56 ng/g, respectively (Table 2). Our results were in agreement with the general Brazilian population (Nunes et al., 2010), Bremen area in the northern Germany, GM = 0.38 ng/g (Heitland and Koster, 2006), elderly Swedish population (Schultze et al., 2013), and French population studied by Goulle et al. (2005) (GM = 0.31 ng/g), Goulle et al. (2014) (GM = 0.34 ng/g) and Nisse et al. (2017) (GM = 0.39 ng/g). On the other hand, B-Cd for the Serbian population was lower when compared to subjects living in the: Acre State, Brazil (UL = 0.87 ng/g) (Freire et al., 2015), Sardinia, Italy (P95 = 1.82 ng/g) (Forte et al., 2011), Germany (P95 = 2.34 ng/g) (Wilhelm et al., 2004), Tunisia (P95 = 2.31 ng/g), Italy, Rome (P95 = 1.97 ng/g) (Alimonti et al., 2005). It is interesting to point out that our results were higher when compared to the Spanish population occupationally exposed to cadmium, GM = 0.18 ng/g (Gil et al., 2011), but this value was not in agreement with many populations groups worldwide. Some of the mean values reported for professionally exposed peoples were: 9.81 ng/g for adult inhabitants living in the vicinity of a cement factory in the Pakistan (Afridi et al., 2011) and 24.10 ng/g for the Chinese respondents residing in a polluted area (Wang et al., 2011), and these values were significantly lower when compared to the Serbian population (0.46 ng/ g). Women in our study had significantly higher levels of B-Cd than men (Table 2). These results were in agreement with many studies (Forte et al., 2011; Kim et al., 2017; Černa et al., 2012). One of the main reasons for higher B-Cd is twice higher gastrointestinal absorption of dietary cadmium in women than in men (Forte et al., 2011; Kim et al.,
4.4. Thorium (Th) and uranium (U) Thorium and uranium enter into our body via the air, food and drinking water. Thorium is an alternative source of nuclear fuel. Uranium is an alpha-emitting, radioactive and heavy metal (Kumar et al., 2013; Hon et al., 2015; Arzuaga et al., 2015). Bombs with depleted uranium (DU) were used in aircraft operations against the Federal Republic of Yugoslavia (1999), today's Serbia, and more than 31,000 rounds of ammunition comprising about 8.5 t of DU were shot. In some individuals who participated in land operations, as well as in the civil population in the area involved, there was an enhanced incidence of cancer. Epidemiological studies found an increased frequency of chromosomal abnormalities in the exposed population. It was suggested that DU exposure is either a primary cause or related to the main cause of congenital anomalies and increased rate of malignant 407
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diseases (Hon et al., 2015). In the study of Rodushkin et al. (1999) reported range for Th in the blood of 31 healthy subjects was < 0.008–0.20 ng/g (median 0.067 ng/g). Our results were 5 times higher in regard to a median value (0.34 ng/g) (Table 1). The same group of authors pointed out that women showed significantly higher values than men. This is in agreement with our results (Table 2). We also found that level of B-Th significantly decreased with age (Table 2). Heitland and Koster (2006) reported GM value < 0.003 ng/g for the German population (n = 130) and P5-P95 range below < 0.003 ng/g (which is the concentration below detection limit of ICP-MS method). Our GM was 0.30 µg/L, or 100 times higher for the Serbian population. There are few studies that reported uranium level in the whole blood. For the 100 healthy subjects living in France, median blood-U level was 0.004 ng/g (P5-P95 = 0.002–0.006 ng/g) (Goulle et al., 2005). It was up to 12.5 times lower than for the Serbian population (Table 1). Yedomon et al. (2017) reported GM value for B-U in the Benin population 0.002 ng/g and the P5-P95 range was < 0.001–0.008 ng/g. This range was significantly lower when compared to the Serbian population (P5-P95 = 0.004–0.13 ng/g) and 3 times higher for our studied population in terms of GM value of the total examined population. The GM value for B-U value reported by Heitland and Koster (2006) was below 0.003 ng/g. It was 20 times lower when compared to the Serbian population (Table 2).
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5. Conclusion This human biomonitoring study provides the first background concentrations for As, Cd, Pb, Th and U in Serbia. Reported data represent a new contribution to the knowledge of the blood chemistry for adult population. It was found that women had statistically significantly higher level of Th in the blood. With regard to age, significant increase of the same element was found for a younger group. The investigated smoker's group had significantly higher levels of Cd and Pb in blood than non-smokers group. In comparison with other population group worldwide, Serbian population had significantly higher level of Th and U in the whole blood (up to 100 times higher). These findings could highlight the molecular basis for the increased incidence of the malignant disease among the Serbian population, which need to be confirmed by clinical studies. Also, the obtained reference values could be interpreted as a baseline for further toxicological assessment studies. Acknowledgement This research was financially supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia, project No. 172030. Conflicts of interest The authors declare that they have no conflict of interest. Ethical approval All procedures performed in studies involving human subjects were in accordance with the ethical standards of the institutional and national research committee, as well as with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. References Afridi, H.I., Kazi, T.G., Kazi, A.G., Shah, F., Wadhwa, S.K., Kolachi, N.F., Shah, A.Q., Baig, J.A., Kazi, N., 2011. Levels of arsenic, cadmium, lead, manganese and zinc in biological samples of paralysed steel mill workers with related to controls. Biol. Trace Elem. Res. 144, 164–182. Alimonti, A., Petrucci, F., Laurenti, F., Papoff, P., Caroli, S., 2000. Reference values for selected trace elements in serum of term newborns from the urban area of Rome. Clin.
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