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Toxic and essential elements in children's blood (<6 years) from Kinshasa, DRC (the Democratic Republic of Congo)
Journal of Trace Elements in Medicine and Biology 28 (2014) 45–49
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Epidemiology
Toxic and essential elements in children’s blood (<6 years) from Kinshasa, DRC (the Democratic Republic of Congo) J. Tuakuila a,b,∗ , M. Kabamba a , H. Mata a , G. Mata a a
Environmental Chemistry, Faculty of Sciences, University of Kinshasa, Kinshasa, The Democratic Republic of the Congo Louvain Center for Toxicology and Applied Pharmacology (LTAP), Université Catholique de Louvain, Avenue E. Mounier 53, Box 52.02.12, 1200 Brussels, Belgium b
a r t i c l e
i n f o
Article history: Received 16 January 2013 Accepted 6 September 2013 Keywords: Toxic and essential elements Whole blood Children Kinshasa
a b s t r a c t In this study we determined the concentration of 9 trace elements (As, Cd, Cu, Hg, Mn, Mo, Pb, Se and Zn) in whole blood of children (n = 100, 64 girls, 36 boys and median age: 36 months) using inductively coupled plasma mass spectrometry (ICP-MS). The proportion of children potentially deficient in essential elements or poisoned by toxic elements was evaluated. The aging effects on the concentration of these elements were also investigated. The median values were 3.17 g/L (As), 0.15 g/L (Cd), 1.1 mg/L (Cu), 2.1 g/L (Hg), 10.4 g/L (Mn), 17.7 g/L (Mo), 8.7 g/dL (Pb), 10.7 g/L (Se) and 5.0 mg/L (Zn). The concentration of many elements (As, Cd, Hg, Mn, Pb and Zn) showed significant age variations but not sex influence. Regarding levels of the essential elements (Cu, Mn, Mo, Se and Zn), B-Cu, B-Mn, B-Se and B-Zn were in the normal range, whereas exceeded levels were observed for B-Mo. None of these children was deficient in essential elements. Except B-Cd, all toxic elements showed exceeded blood levels. The proportion of children potentially poisoned by toxic elements varies from 10% (n = 10) to 95% (n = 95) and depends on toxic element: 95% for As, 10% for Hg and 35% for Pb. The main health concerns emerging from this study are the high As, Hg and Pb exposures of the Kinshasan children requiring further documentation, corrective actions and the implementation of appropriate regulations. © 2013 Elsevier GmbH. All rights reserved.
Introduction There is need for knowledge of trace element levels in different population groups. One application is as reference values that can be used for detection of changed environmental exposure situations. Moreover, the levels of toxic elements may be close to concentrations where adverse effects can occur. The levels of essential elements may be used to discover deficiency, and in some cases toxicity [1]. It is of importance to investigate various categories within the population and to identify specific risk groups. Children are more likely to be exposed to environmental sources because of their parental exposures, hand-to-mouth behavior and physiology. Because of this special susceptibility, children are more vulnerable to the effects of trace elements than adults [2]. Information on the levels of many trace elements in biological tissues in children is scarce and for many non-essential elements, baseline in the general population of Kinshasa [the capital of Democratic Republic of Congo (DRC)], and especially in children, are lacking. The present study originated from that observation and its main objective was to evaluate the blood levels of nine trace
elements from ≤6 years old children in the city of Kinshasa [i.e., arsenic (As), cadmium (Cd), copper (Cu), manganese (Mn), mercury (Hg), molybdenum (Mo), lead (Pb), selenium (Se) and zinc (Zn)]. Materials and methods Study design This survey was organized by the laboratory of environmental chemistry at the University of Kinshasa. Because Kinshasa does not have a register of population, we applied a systematic sampling approach [3] in order to obtain a representative sample of the Kinshasa population. The methods used for sampling of study population are described in detail by Tuakuila et al. [4,5]. The survey selected 100 children aged 1–5 years (91% of the target number was reached). This study was approved by the Congolese Committee of Medical Ethics and the study results will be informed back to individuals sample donors with proper explanations. Analysis of blood metals
∗ Corresponding author at: Environmental Chemistry, Faculty of Sciences, University of Kinshasa, Kinshasa, Democratic Republic of the Congo. Tel.: +234 819347828. E-mail address: [email protected] (J. Tuakuila). 0946-672X/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.jtemb.2013.09.004
Blood specimens were collected in metal-free tubes (EDTA anticoagulated) in the local health centers after careful cleaning of the
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J. Tuakuila et al. / Journal of Trace Elements in Medicine and Biology 28 (2014) 45–49
skin at the venipuncture site. After sampling, the tubes were frozen and transported in a cool box to the Louvain centre for Toxicology and Applied Pharmacology (Brussels, Belgium), for analysis. In all blood samples, nine trace elements: total arsenic (B-As), cadmium (B-Cd), copper (B-Cu), manganese (B-Mn), mercury (B-Hg), molybdenum (B-Mo), lead (B-Pb), selenium (B-Se) and zinc (B-Zn)) were quantified by means of inductively coupled argon plasma mass spectrometry (ICP-MS) with an Agilent 7500cx ICP-MS using a Babington nebulizer, following 1:10 dilution in a basic diluent: 1-butanol (2%, w/v), EDTA (0.05%, w/v), Triton X-100 (0.05%, w/v), NH4 OH (1%, w/v), internal standards (Sc, Ge, Rh and Ir) and MilliQ water (ISO 15189 accredited method). Table 2 reports the limits of detection (LOD) for all trace elements. For quality control purposes, internal controls and reference materials were run together with the samples on a daily basis. Statistical analysis Data analyses were conducted with the SAS Software package version 9.2 (SAS Institute Inc., Cary, NC). For all trace elements, Geometric means (GM), arithmetic means (AM), percentiles (P) and range were calculated. The limit of detection (LOD) divided by 2 was used for imputation of values lower than the LOD. The Kolmogorov–Smirnov test was used to verify the normality of each distribution and parametric tests were used for the analysis of normally distributed variables. The Pearson correlation test was used to examine the effects of aging on trace element levels. Stepwise multiple linear regression analyses of log-transformed data were used to estimate the influence of independent variables (age and sex) on the trace element levels (Stepwise procedure, criteria: F probability to enter ≤0.05 and F probability to remove ≥0.10). A p-value lower than 0.05 was considered as statistically significant for all tests. Results Populations investigated The 100 children included (64 girls, 36 boys) in this study were between 1 and 60 months of age, with a median of 36 months. The median age by sex was 36 months and 37 months for girls and boys, respectively (Table 1). Trace element levels The AM and GM levels of trace elements among 100 children as a whole are summarized in Table 2 together with percentiles (P)
Table 1 Demographic characteristics of the participants. Number of subjects Age, monthsa Sex Boys, n (%) Girls, n (%) a
100 36 [1–60] 64 (64%) 36 (36%)
Median age [Range].
and range. The median blood (P50) values of trace elements were 3.17 g/L (As), 0.15 g/L (Cd), 1.1 mg/L (Cu), 2.1 g/L (Hg), 10.4 g/L (Mn), 17.7 g/L (Mo), 8.7 g/dL (Pb), 10.7 g/dL (Se) and 5.0 mg/L (Zn). Six (6%) Cd levels were less than their LOD. In multivariable analyses, age (continuous log-variable) was the parameter significantly associated with blood concentrations (after logarithmic conversion) of trace elements: As, Cd, and Pb (p ≤ 0.01) and also for Hg, Mn and Zn (p ≤ 0.05) with a limited range for R2 (0.042–0.192) (Table 3). The analysis showed that the sex was not an influential variable for all elements. Influence of aging on trace element levels in blood A positive correlation was observed between age and log As (r = 0.37, p ≤ 0.01), age and log Cd (r = 0.43, p ≤ 0.01), age and log Hg (r = 0.24, p = 0.01), age and log Pb (r = 0.33, p ≤ 0.01) and age and log Zn (r = 0.20, p = 0.04) (Table 4). A negative correlation was also observed between age and log Mn (r = −0.23, p = 0.02). Discussion The evaluation of essential and toxic elements in human fluids is accepted as a useful tool in both scientific research and the diagnoses of disease [6]. During the data sampling, great care was taken to select a representative sample of the Kinshasan children but the exact representativeness of our sample was not checked because Kinshasa does not have a reliable register of population. There is, however, no reason to suspect a selection bias. In studies of trace elements, numerous errors may be introduced during the procedures from sample collection to the ultime detection of the analyte. The evaluation of data is dependent on the normal range of biological variability and on the end use of the results. According to results of the present work, the level ranges are not wide, and the levels in line of previous studies, or higher, or lower, showing that contamination was not a general problem. Children are more susceptible to toxic exposure than adults because they have proportionally more intake of food
Table 2 Blood concentrations of selected trace elements in children from Kinshasa, DRC (n = 100, <6 years old). Parameter
N
LOD
N < LOD
Range
As (g/L) Cd (g/L) Cu (mg/L) Hg (g/L) Mn (g/L) Mo (g/L) Pb (g/dL) Se (g/dL) Zn (mg/L)
100 100 100 100 100 100 100 100 100
0.23 0.04 0.6 0.1 0.5 0.1 0.1 0.8 0.7
0 6 0 0 0 0 0 0 0
1.10–14.68
N = Sample size. LOD = limit of detection. N < LOD: number of sample below the LOD. P5 – 5th percentile, P50 – 50th percentile = median, P95 – 95th percentile. AM = arithmetic mean (CI = 95% confidence interval). GM = geometric mean (CI = 95% confidence interval).
Selected percentiles P5
P50
P95
1.48
3.17 0.15 1.1 2.1 10.4 17.7 8.7 10.7 5.0
8.82 0.30 1.7 6.0 22.2 30.6 15.9 13.6 6.9
AM (95% CI)
GM (95% CI)
4.08 (3.59; 4.58) 0.16 (0.13; 0.19) 1.2 (1.14; 1.23) 2.5 (2.2; 2.9) 12.4 (10.7; 14.1) 16.1 (14.2; 18.0) 9.5 (8.8; 10.3) 10.9 (10.6; 11.2) 5.0 (4.8; 5.2)
3.52 (3.17; 3.92) 0.13(0.11; 0.15) 1.2 (1.1; 1.2) 2.1 (1.9; 2.4) 10.9 (10.0; 12.0) 12.7 (10.9; 14.8) 8.7 (8.0; 9.5) 10.8 (10.5; 11.1) 4.9 (4.7; 5.1)
J. Tuakuila et al. / Journal of Trace Elements in Medicine and Biology 28 (2014) 45–49 Table 3 Multiple regression analysis models. Partial R2 (independent variables)
Parameter (dependent variable)
Agea As Cd Cu Hg Mn Mo Pb Se Zn
Sexb **
(g/L) (g/L) (mg/L) (g/L) (g/L) (g/L) (g/dL) (g/dL) (mg/L)
Total R2
0.137 0.192** – 0.057* 0.056* – 0.110** – 0.042*
– – – – – – – – –
0.137 0.192 – 0.057 0.056 – 0.110 – 0.042
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[17]; globally, deficiency is a larger concern than excess [18]. The normal range of Mn concentrations is approximately 4–14 g/L in whole blood [19], the value found (10.4 g/L) in this study is in this line and similar to 9.74 g/L found in Canadian children but higher than 8.48 g/L reported in South Africa. 25% of B-Cu levels, 20% of B-Mn levels and 70% of B-Mo levels were, respectively, higher than those (P95th values) reported in Italian population (SIVR list, Table 5). Over all, none of the children in the present study was deficient in essential elements. Toxic elements
R2 : explained variance (i.e., the square of the correlation coefficient). Results are given for those variables that correlated, and only when the regression was significant. a Age represented as continuous variable. b Sex represented as 0 for female and 1 for male. * p < 0.05. ** p < 0.01.
contaminants, active developmental processes, multiple exposure pathways and susceptible socio-behavioral activities [2,7]. Reference levels of trace elements in blood for children are generally lacking. In the present study, levels of 9 different elements in blood from Kinshasa children are reported. The concentrations of the essential elements were generally in line with previously reported data, and the concentrations of non-essential elements were mostly high. Essential elements Essential elements are generally reported on plasma or serum [8,9]. In blood and serum, the elements Cu, Se, Mn and Zn were correlated [10–12]. In addition, the median ratios (blood/serum) were 0.89, 6.1, 1.1, for Cu, Zn and Se, respectively [12]. In the present work, Cu, Mn, Se and Zn levels were determined in whole blood. Regarding blood levels of the essential elements, the GM concentrations were 1.2 mg/L for Cu, 10.9 g/L for Mn, 12.7 g/L for Mo, 10.8 g/dL for Se and 4.9 mg/L for Zn (Table 2). Zn increased with age but Mn decreased with increasing of age as previously observed [13]. Sex was not an influential variable as shown in some data [14]. In Table 5, results of the present analysis on essential element concentrations in blood of Kinshasan children were compared with the levels reported in the literature. The present median level (P50) of B-Cu, B-Mn and B-Zn were comparable to, but B-Mo was higher than that found in Canadian children [14]. In the study to evaluate exposure to metals in children aged 9–10 years from Katowice district (Poland), the authors found the same results that this study: 1.08 mg/L for B-Cu and 5.101 mg/L for B-Zn [15]. B-Se was in line with 11.8 g/dL found in Poland children [15] but, respectively, 39% and 42% lower than that reported for South African and Canadian children, probably reflecting the lower Se content in soil and drinking water in Kinshasa as observed by Tuakuila et al. [16]. Either selenium deficiency or excess may lead to adverse health effects
The present analysis showed also that the GM concentrations in blood were 3.52 g/L for As, 0.13 g/L for Cd, 2.1 g/L for Hg and 8.7 g/dL for Pb (Table 2). As, Cd, Hg and Pb increased with age as previously observed [20–23]. Sex was not an influential variable as shown in some data [15,24,25]. B-As The public is exposed to arsenic in food, drinking water, soil, and ambient air, with food (particularly meat and fish) representing the major source of intake [25,26]. Exposure may also arise from indoor house dust, as levels in dust can exceed levels in soil [27]. Although shellfish and marine species have been found to bioconcentrate arsenic, it is not biomagnified through the food chain [28–31]. The present median level of 3.17 g/L was at least two times and four times higher than other reported in South African and Canadian children, respectively (Table 5). 95% of B-As values were higher than 1.2 g/L (95th percentile) obtained in Italian population (SIVR list, Table 5). The high As levels in blood of kinshasan children reported probably reflecting dietary differences because fish (“mpiodi”, i.e., Trachurus trachurus) is one of the frequently consumed food in Kinshasa; the measurement of inorganic arsenic in drinking water and soil samples in Kinshasa by Tuakuila et al. (not shown) did not provide evidence of elevated exposure to this element [16]. B-Cd For nonsmokers who are not exposed to cadmium in the workplace, ingestion through food is the largest source of exposure. Typical concentrations in blood are approximately 0.4–1 g/L for non-smokers [32]. The present median level (0.15 g/L) and selected percentiles (≤LOD for P5th and 0.30 g/L for P95th) found are in line of the literature [14,33,34,48]. Drinking water did not appear to be a significant source of exposure to Cd [14]. No child was exposed to environmental tobacco smoking. B-Hg Speciation of Hg into methylmercury (MeHg) and inorganic Hg can indicate the source of exposure, as MeHg originates from fish consumption [35] and inorganic Hg mainly originates from amalgam [36]. In the present study, speciation was not possible due to the analytical technique used. B-Hg is, however, strongly dependent on fish intake [37,38]. The present median level of 2.1 g/L found which was at least 6 times higher than 0.30 g/L
Table 4 Effects of aging on trace element levels in whole blood from children (<6 years old) of Kinshasa. Ya
r
p for r
Slope
log As log Cd log Hg log Mn log Pb log Zn
0.37 0.43 0.24 −0.23 0.33 0.20
<0.01 <0.01 0.01 0.02 <0.01 0.04
0.26 0.32 0.18 −0.14 0.19 0.06
a
Y shows the logarithm of the elements concentration in g/dL, X shows age in months.
(95% range) (0.13–0.39) (0.18–0.45) (0.03–0.33) (−0.25 to −0.02) (008–0.29) (0.00–2.60)
Evaluation Increasing as aged Increasing as aged Increasing as aged Decreasing as aged Increasing as aged Increasing as aged
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J. Tuakuila et al. / Journal of Trace Elements in Medicine and Biology 28 (2014) 45–49
Table 5 Comparison between Kinshasan population and other international studies. Parameter
Kinshasaa [median levels (P5th–P95th)]
Other international studies (reference values) United Statesb [median levels (P95th)
As Cd Cu Hg Mn Mo Pb Se Zn
(g/L) (g/L) (mg/L) (g/L) (g/L) (g/L) (g/dL) (g/dL) (mg/L)
3.17 (1.48–8.82) 0.15 (≤LOD–0.30) 1.1 (0.9–1.7) 2.1 (0.8–5.9) 10.4 (5.7–22.2) 17.7 (3.6–30.6) 8.7 (3.6–15.9) 10.7 (8.5–13.6) 5.0 (3.4–6.9)
Germanyc (median levels)
<0.14 (0.20) 0.30 (1.80)
1.70 (5.10)
1.96
Canadad [median levels (P95th) 0.66 (2.16) 0.10 (0.23) 0.971 (1.197) 0.28 (2.08) 9.74 (16.36) 0.80 (1.60) 0.87 (1.95) 18.54 (23.17) 5.26 (6.51)
South Africae (median levels) 1.53 1.195 8.48 5.64 17.6 3.431
SIVR listf (5th–95th) 1.0–1.2 1.0–1.5 0.6–1.3 1.0–4.5 3.0–8.0 0.5–5.0 0.1–10 0.7–14.5 3.5–7.5
a
This study. US National Health and Nutrition Examination Surveys: Data from CDC [values for total population of survey years 2003–2004] [33]. c Schulz et al. [34]. d Health Canada: results of the Canadian Health Measures Survey Cycle 1 2007–2009 [values for children aged 6–11 years] [14]. e Bazzi et al. [13]. Data from South African school children. f SIVR circuit laboratory or literature evaluation: 5th–95th of reference values in blood of Italian population (www.valoridiriferimento.it accessed 14.04.13) [48], modified by Caroli and Zàray [49]. b
and 0.28 g/L reported in US and Canadian children, respectively [14,33]. 10% of B-Hg values were higher than 4.5 g/L (95th percentile) reported in Italian population (SIVR list, Table 5). The high Hg levels in blood of kinshasan children reported probably reflecting dietary differences [16]. In this study, children did not receive dental amalgams. However, the mother’s contribution in B-Hg levels of children through the uterus or nursing may represent other important source of exposure to this element [39].
toxic element: 95% for As, 10% for Hg and 35% for Pb. The main health concerns emerging from this study are the high As, Hg and Pb exposures of the Kinshasan children requiring further documentation, corrective actions and the implementation of appropriate regulations.
Conflict of interest statement The authors declare there are no conflicts of interest.
B-Pb The median B-Pb (8.7 g/L) found in this study was higher than those reported in Table 5. The high blood lead levels (BLL) probably reflecting the use of leaded fuel, lead-based paints and lead from acid battery recycling that remain sources of major exposure in Kinshasa [16,23]. Children are known to constitute a group at high risk of Pb poisoning; they ingest more Pb due to their hand-mouth activity, their digestive absorption rate is higher than adults and their developing nervous system is more sensitive to the adverse effects of Pb [40]. According to the CDC definition of elevated BLL (≥10 g/dL) [41], about 35% of Kinshasa children had elevated BLL. Among children less than 5–6 years tested in developing countries, about 7% were reported to have elevated BLL (>10 g/dL) in Nairobi [42], 21% in the rural Philippines [43] and in Kampala [44], 55% in Nigeria and Egypt [45,46]. The prevalence found in industrialized countries is much lower with, for instance, 1.4% in the USA [47]. Over all, the proportion of children potentially poisoned by toxic elements in this study varies from 10% (n = 10) to 95% (n = 95) and depends on toxic element: 95% (n = 95) for As, 10% (n = 10) for Hg and 35% (n = 35) for Pb. A major limitation should be considered in evaluating present results. With regard to sample collection, selection of blood sample donors did not follow rigid sampling strategy (such as random sampling) but by chance, which was practically inevitable under present survey conditions. Despite such limitation, however, it is prudent to conclude that data from the present study constitutes baseline levels or levels generally exceeded in children for 9 elements, including essential and ubiquitous toxic elements. Regarding levels of the essential trace elements (Cu, Mn, Mo, Se and Zn), B-Cu, B-Mn, B-Se and B-Zn were in the normal range, whereas exceeded levels were observed for B-Mo. None of these children was deficient in essential elements. Except Cd, all toxic elements showed exceeded levels. The proportion of children potentially poisoned by toxic elements varies from 10% (n = 10) to 95% (n = 95) and depends on
Acknowledgments We are highly indebted to the study participants and to the staff of investigators, as well as all the local health services and health centers of the Kinshasan Public Health System that supported the field work. We also thank Professors Lison, Hoet, Haufroid and Mrs Deumer for their collaboration. The financial support of the Belgian Technical Cooperation (Coopération Technique Belge-CTB/Belgische Technische Coöperatie-BTC) and LTAP (Louvain centre for Toxicology and Applied Pharmacology) are gratefully acknowledged.
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[31] ATSDR (Agency for Toxic Substances and Disease Registry). Toxicological profile for arsenic; 2007 [accessed 02.04.12] http://www.atsdr.cdc.gov/ toxprofiles/tp2.html [32] ATSDR (Agency of Toxic Substances and Disease Registry). Toxicological profile for cadmium; 1999 [accessed 04.03.10] www.atsdr.cdc.gov/ toxprofiles/tp5.html [33] CDC (Centers for Disease Control and Prevention). National Health and Nutrition Examination Survey 2003–2004. Fourth National Report on Human Exposure to Environmental Chemicals. Atlanta (GA); 2009 [accessed 20.03.12] www.cdc.gov/nchs/nhanes.htm [34] Schulz C, Angerer J, Ewer U, Heudorf U, Wilhelm M. Revised and new reference values for environmental pollutants in urine or blood of children in Germany derived from the German Environmental Survey on Children 2003–2006 (GerES IV). Int J Hyg Environ Health 2009, http://dx.doi.org/10.1016/j.ijheh.2009.05.003. [35] WHO (World Health Organization). Environmental Health Criteria 101. Methylmercury. Geneva: World Health Organization; 1990. [36] WHO (World Health Organization). Environmental health criteria 181. Inorganic mercury. Geneva: World Health Organization; 1991. [37] Svensson BG, Schütz A, Nilsson A, Akesson I, Akesson B, Skerfving S. Fish as a source of exposure to mercury and selenium. Sci Total Environ 1992;126:61–74. [38] Mehaffey KR, Clickner RP, Bodurow CC. Blood organic mercury and dietary intake: National Health and Nutrition Examination Survey, 1999 and 2000. Environ Health Perspect 2004;112(5):562–70. [39] JECFA. Summary and conclusions of the sixty-first meeting of the Joint FAO/WHO Expert Committee on Food Additives (JECFA). JECFA/61/SC, Rome, 10–19 June 2003; 2003. [40] Lidsky TL, Schneider JS. Lead neurotoxicity in children: basic mechanisms and clinical correlates. Brain 2003;126:5–19. [41] CDC (Centers for Disease Control and Prevention). Preventing lead poisoning in young children. Department of Health and Human Services. Atlanta: CDC; 1991. [42] Olewe TM, Mwanthi MA, Wang’ombe JK, Griffiths JK. Blood lead levels and potential environmental exposures among children under five years in Kibera slums: Nairobi. East Afr J Public Health 2009;6(1):6–10. [43] Riddell TJ, Solon O, Quimbo SA, Tan CMC, Butrick CE, Peabodya JW. Elevated blood-lead levels among children living in the rural Philippines. Bull World Health Organ 2007;85:674–80. [44] Graber LK, Asher D, Anandaraja N, Bopp FR, Merrill K, Cullen MR, et al. Children lead exposure after the phaseout of leaded gasoline: an ecological study of school-age children in Kampala, Uganda. Environ Health Perspect 2010;118(6):884–9. [45] Boseila SA, Gabr AA, Hakim IA. Blood lead levels in Egyptian children: influence of social and environmental factors. Am J Public Health 2004;94(1): 47–9. [46] Wright NJ, Thacher TD, Pfitzner MA, Fischer PR, Pettifor JM. Causes of lead toxicity in a Nigerian city. Arch Dis Child 2005;90(3):262–6. [47] Jones RL, Homa DM, Mayer PA, Brody DJ, Caldwell KL, Pirkle JL, et al. Trends in blood lead levels and blood testing among US children aged 1 to 5 years, 1988–2004. Pediatrics 2009;123:376–85. [48] SIVR circuit laboratory or literature evaluation: 5th–95th of reference values in blood of Italian population; 2005 [accessed 14.04.13], modified by Caroli and Zàray, 2012 http://associazione. squarespace.com/pubblico/Seconda %20lista%20SIVR%20metalli.pdf [49] Caroli S, Zàray G. Analytical techniques for clinical chemistry: methods and applications. 1st ed. John Wiley & Sons, Inc.; 2012. p. 102.
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Journal of Trace Elements in Medicine and Biology journal homepage: www.elsevier.de/jtemb
Epidemiology
Toxic and essential elements in children’s blood (<6 years) from Kinshasa, DRC (the Democratic Republic of Congo) J. Tuakuila a,b,∗ , M. Kabamba a , H. Mata a , G. Mata a a
Environmental Chemistry, Faculty of Sciences, University of Kinshasa, Kinshasa, The Democratic Republic of the Congo Louvain Center for Toxicology and Applied Pharmacology (LTAP), Université Catholique de Louvain, Avenue E. Mounier 53, Box 52.02.12, 1200 Brussels, Belgium b
a r t i c l e
i n f o
Article history: Received 16 January 2013 Accepted 6 September 2013 Keywords: Toxic and essential elements Whole blood Children Kinshasa
a b s t r a c t In this study we determined the concentration of 9 trace elements (As, Cd, Cu, Hg, Mn, Mo, Pb, Se and Zn) in whole blood of children (n = 100, 64 girls, 36 boys and median age: 36 months) using inductively coupled plasma mass spectrometry (ICP-MS). The proportion of children potentially deficient in essential elements or poisoned by toxic elements was evaluated. The aging effects on the concentration of these elements were also investigated. The median values were 3.17 g/L (As), 0.15 g/L (Cd), 1.1 mg/L (Cu), 2.1 g/L (Hg), 10.4 g/L (Mn), 17.7 g/L (Mo), 8.7 g/dL (Pb), 10.7 g/L (Se) and 5.0 mg/L (Zn). The concentration of many elements (As, Cd, Hg, Mn, Pb and Zn) showed significant age variations but not sex influence. Regarding levels of the essential elements (Cu, Mn, Mo, Se and Zn), B-Cu, B-Mn, B-Se and B-Zn were in the normal range, whereas exceeded levels were observed for B-Mo. None of these children was deficient in essential elements. Except B-Cd, all toxic elements showed exceeded blood levels. The proportion of children potentially poisoned by toxic elements varies from 10% (n = 10) to 95% (n = 95) and depends on toxic element: 95% for As, 10% for Hg and 35% for Pb. The main health concerns emerging from this study are the high As, Hg and Pb exposures of the Kinshasan children requiring further documentation, corrective actions and the implementation of appropriate regulations. © 2013 Elsevier GmbH. All rights reserved.
Introduction There is need for knowledge of trace element levels in different population groups. One application is as reference values that can be used for detection of changed environmental exposure situations. Moreover, the levels of toxic elements may be close to concentrations where adverse effects can occur. The levels of essential elements may be used to discover deficiency, and in some cases toxicity [1]. It is of importance to investigate various categories within the population and to identify specific risk groups. Children are more likely to be exposed to environmental sources because of their parental exposures, hand-to-mouth behavior and physiology. Because of this special susceptibility, children are more vulnerable to the effects of trace elements than adults [2]. Information on the levels of many trace elements in biological tissues in children is scarce and for many non-essential elements, baseline in the general population of Kinshasa [the capital of Democratic Republic of Congo (DRC)], and especially in children, are lacking. The present study originated from that observation and its main objective was to evaluate the blood levels of nine trace
elements from ≤6 years old children in the city of Kinshasa [i.e., arsenic (As), cadmium (Cd), copper (Cu), manganese (Mn), mercury (Hg), molybdenum (Mo), lead (Pb), selenium (Se) and zinc (Zn)]. Materials and methods Study design This survey was organized by the laboratory of environmental chemistry at the University of Kinshasa. Because Kinshasa does not have a register of population, we applied a systematic sampling approach [3] in order to obtain a representative sample of the Kinshasa population. The methods used for sampling of study population are described in detail by Tuakuila et al. [4,5]. The survey selected 100 children aged 1–5 years (91% of the target number was reached). This study was approved by the Congolese Committee of Medical Ethics and the study results will be informed back to individuals sample donors with proper explanations. Analysis of blood metals
∗ Corresponding author at: Environmental Chemistry, Faculty of Sciences, University of Kinshasa, Kinshasa, Democratic Republic of the Congo. Tel.: +234 819347828. E-mail address: [email protected] (J. Tuakuila). 0946-672X/$ – see front matter © 2013 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.jtemb.2013.09.004
Blood specimens were collected in metal-free tubes (EDTA anticoagulated) in the local health centers after careful cleaning of the
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J. Tuakuila et al. / Journal of Trace Elements in Medicine and Biology 28 (2014) 45–49
skin at the venipuncture site. After sampling, the tubes were frozen and transported in a cool box to the Louvain centre for Toxicology and Applied Pharmacology (Brussels, Belgium), for analysis. In all blood samples, nine trace elements: total arsenic (B-As), cadmium (B-Cd), copper (B-Cu), manganese (B-Mn), mercury (B-Hg), molybdenum (B-Mo), lead (B-Pb), selenium (B-Se) and zinc (B-Zn)) were quantified by means of inductively coupled argon plasma mass spectrometry (ICP-MS) with an Agilent 7500cx ICP-MS using a Babington nebulizer, following 1:10 dilution in a basic diluent: 1-butanol (2%, w/v), EDTA (0.05%, w/v), Triton X-100 (0.05%, w/v), NH4 OH (1%, w/v), internal standards (Sc, Ge, Rh and Ir) and MilliQ water (ISO 15189 accredited method). Table 2 reports the limits of detection (LOD) for all trace elements. For quality control purposes, internal controls and reference materials were run together with the samples on a daily basis. Statistical analysis Data analyses were conducted with the SAS Software package version 9.2 (SAS Institute Inc., Cary, NC). For all trace elements, Geometric means (GM), arithmetic means (AM), percentiles (P) and range were calculated. The limit of detection (LOD) divided by 2 was used for imputation of values lower than the LOD. The Kolmogorov–Smirnov test was used to verify the normality of each distribution and parametric tests were used for the analysis of normally distributed variables. The Pearson correlation test was used to examine the effects of aging on trace element levels. Stepwise multiple linear regression analyses of log-transformed data were used to estimate the influence of independent variables (age and sex) on the trace element levels (Stepwise procedure, criteria: F probability to enter ≤0.05 and F probability to remove ≥0.10). A p-value lower than 0.05 was considered as statistically significant for all tests. Results Populations investigated The 100 children included (64 girls, 36 boys) in this study were between 1 and 60 months of age, with a median of 36 months. The median age by sex was 36 months and 37 months for girls and boys, respectively (Table 1). Trace element levels The AM and GM levels of trace elements among 100 children as a whole are summarized in Table 2 together with percentiles (P)
Table 1 Demographic characteristics of the participants. Number of subjects Age, monthsa Sex Boys, n (%) Girls, n (%) a
100 36 [1–60] 64 (64%) 36 (36%)
Median age [Range].
and range. The median blood (P50) values of trace elements were 3.17 g/L (As), 0.15 g/L (Cd), 1.1 mg/L (Cu), 2.1 g/L (Hg), 10.4 g/L (Mn), 17.7 g/L (Mo), 8.7 g/dL (Pb), 10.7 g/dL (Se) and 5.0 mg/L (Zn). Six (6%) Cd levels were less than their LOD. In multivariable analyses, age (continuous log-variable) was the parameter significantly associated with blood concentrations (after logarithmic conversion) of trace elements: As, Cd, and Pb (p ≤ 0.01) and also for Hg, Mn and Zn (p ≤ 0.05) with a limited range for R2 (0.042–0.192) (Table 3). The analysis showed that the sex was not an influential variable for all elements. Influence of aging on trace element levels in blood A positive correlation was observed between age and log As (r = 0.37, p ≤ 0.01), age and log Cd (r = 0.43, p ≤ 0.01), age and log Hg (r = 0.24, p = 0.01), age and log Pb (r = 0.33, p ≤ 0.01) and age and log Zn (r = 0.20, p = 0.04) (Table 4). A negative correlation was also observed between age and log Mn (r = −0.23, p = 0.02). Discussion The evaluation of essential and toxic elements in human fluids is accepted as a useful tool in both scientific research and the diagnoses of disease [6]. During the data sampling, great care was taken to select a representative sample of the Kinshasan children but the exact representativeness of our sample was not checked because Kinshasa does not have a reliable register of population. There is, however, no reason to suspect a selection bias. In studies of trace elements, numerous errors may be introduced during the procedures from sample collection to the ultime detection of the analyte. The evaluation of data is dependent on the normal range of biological variability and on the end use of the results. According to results of the present work, the level ranges are not wide, and the levels in line of previous studies, or higher, or lower, showing that contamination was not a general problem. Children are more susceptible to toxic exposure than adults because they have proportionally more intake of food
Table 2 Blood concentrations of selected trace elements in children from Kinshasa, DRC (n = 100, <6 years old). Parameter
N
LOD
N < LOD
Range
As (g/L) Cd (g/L) Cu (mg/L) Hg (g/L) Mn (g/L) Mo (g/L) Pb (g/dL) Se (g/dL) Zn (mg/L)
100 100 100 100 100 100 100 100 100
0.23 0.04 0.6 0.1 0.5 0.1 0.1 0.8 0.7
0 6 0 0 0 0 0 0 0
1.10–14.68
N = Sample size. LOD = limit of detection. N < LOD: number of sample below the LOD. P5 – 5th percentile, P50 – 50th percentile = median, P95 – 95th percentile. AM = arithmetic mean (CI = 95% confidence interval). GM = geometric mean (CI = 95% confidence interval).
Selected percentiles P5
P50
P95
1.48
3.17 0.15 1.1 2.1 10.4 17.7 8.7 10.7 5.0
8.82 0.30 1.7 6.0 22.2 30.6 15.9 13.6 6.9
AM (95% CI)
GM (95% CI)
4.08 (3.59; 4.58) 0.16 (0.13; 0.19) 1.2 (1.14; 1.23) 2.5 (2.2; 2.9) 12.4 (10.7; 14.1) 16.1 (14.2; 18.0) 9.5 (8.8; 10.3) 10.9 (10.6; 11.2) 5.0 (4.8; 5.2)
3.52 (3.17; 3.92) 0.13(0.11; 0.15) 1.2 (1.1; 1.2) 2.1 (1.9; 2.4) 10.9 (10.0; 12.0) 12.7 (10.9; 14.8) 8.7 (8.0; 9.5) 10.8 (10.5; 11.1) 4.9 (4.7; 5.1)
J. Tuakuila et al. / Journal of Trace Elements in Medicine and Biology 28 (2014) 45–49 Table 3 Multiple regression analysis models. Partial R2 (independent variables)
Parameter (dependent variable)
Agea As Cd Cu Hg Mn Mo Pb Se Zn
Sexb **
(g/L) (g/L) (mg/L) (g/L) (g/L) (g/L) (g/dL) (g/dL) (mg/L)
Total R2
0.137 0.192** – 0.057* 0.056* – 0.110** – 0.042*
– – – – – – – – –
0.137 0.192 – 0.057 0.056 – 0.110 – 0.042
47
[17]; globally, deficiency is a larger concern than excess [18]. The normal range of Mn concentrations is approximately 4–14 g/L in whole blood [19], the value found (10.4 g/L) in this study is in this line and similar to 9.74 g/L found in Canadian children but higher than 8.48 g/L reported in South Africa. 25% of B-Cu levels, 20% of B-Mn levels and 70% of B-Mo levels were, respectively, higher than those (P95th values) reported in Italian population (SIVR list, Table 5). Over all, none of the children in the present study was deficient in essential elements. Toxic elements
R2 : explained variance (i.e., the square of the correlation coefficient). Results are given for those variables that correlated, and only when the regression was significant. a Age represented as continuous variable. b Sex represented as 0 for female and 1 for male. * p < 0.05. ** p < 0.01.
contaminants, active developmental processes, multiple exposure pathways and susceptible socio-behavioral activities [2,7]. Reference levels of trace elements in blood for children are generally lacking. In the present study, levels of 9 different elements in blood from Kinshasa children are reported. The concentrations of the essential elements were generally in line with previously reported data, and the concentrations of non-essential elements were mostly high. Essential elements Essential elements are generally reported on plasma or serum [8,9]. In blood and serum, the elements Cu, Se, Mn and Zn were correlated [10–12]. In addition, the median ratios (blood/serum) were 0.89, 6.1, 1.1, for Cu, Zn and Se, respectively [12]. In the present work, Cu, Mn, Se and Zn levels were determined in whole blood. Regarding blood levels of the essential elements, the GM concentrations were 1.2 mg/L for Cu, 10.9 g/L for Mn, 12.7 g/L for Mo, 10.8 g/dL for Se and 4.9 mg/L for Zn (Table 2). Zn increased with age but Mn decreased with increasing of age as previously observed [13]. Sex was not an influential variable as shown in some data [14]. In Table 5, results of the present analysis on essential element concentrations in blood of Kinshasan children were compared with the levels reported in the literature. The present median level (P50) of B-Cu, B-Mn and B-Zn were comparable to, but B-Mo was higher than that found in Canadian children [14]. In the study to evaluate exposure to metals in children aged 9–10 years from Katowice district (Poland), the authors found the same results that this study: 1.08 mg/L for B-Cu and 5.101 mg/L for B-Zn [15]. B-Se was in line with 11.8 g/dL found in Poland children [15] but, respectively, 39% and 42% lower than that reported for South African and Canadian children, probably reflecting the lower Se content in soil and drinking water in Kinshasa as observed by Tuakuila et al. [16]. Either selenium deficiency or excess may lead to adverse health effects
The present analysis showed also that the GM concentrations in blood were 3.52 g/L for As, 0.13 g/L for Cd, 2.1 g/L for Hg and 8.7 g/dL for Pb (Table 2). As, Cd, Hg and Pb increased with age as previously observed [20–23]. Sex was not an influential variable as shown in some data [15,24,25]. B-As The public is exposed to arsenic in food, drinking water, soil, and ambient air, with food (particularly meat and fish) representing the major source of intake [25,26]. Exposure may also arise from indoor house dust, as levels in dust can exceed levels in soil [27]. Although shellfish and marine species have been found to bioconcentrate arsenic, it is not biomagnified through the food chain [28–31]. The present median level of 3.17 g/L was at least two times and four times higher than other reported in South African and Canadian children, respectively (Table 5). 95% of B-As values were higher than 1.2 g/L (95th percentile) obtained in Italian population (SIVR list, Table 5). The high As levels in blood of kinshasan children reported probably reflecting dietary differences because fish (“mpiodi”, i.e., Trachurus trachurus) is one of the frequently consumed food in Kinshasa; the measurement of inorganic arsenic in drinking water and soil samples in Kinshasa by Tuakuila et al. (not shown) did not provide evidence of elevated exposure to this element [16]. B-Cd For nonsmokers who are not exposed to cadmium in the workplace, ingestion through food is the largest source of exposure. Typical concentrations in blood are approximately 0.4–1 g/L for non-smokers [32]. The present median level (0.15 g/L) and selected percentiles (≤LOD for P5th and 0.30 g/L for P95th) found are in line of the literature [14,33,34,48]. Drinking water did not appear to be a significant source of exposure to Cd [14]. No child was exposed to environmental tobacco smoking. B-Hg Speciation of Hg into methylmercury (MeHg) and inorganic Hg can indicate the source of exposure, as MeHg originates from fish consumption [35] and inorganic Hg mainly originates from amalgam [36]. In the present study, speciation was not possible due to the analytical technique used. B-Hg is, however, strongly dependent on fish intake [37,38]. The present median level of 2.1 g/L found which was at least 6 times higher than 0.30 g/L
Table 4 Effects of aging on trace element levels in whole blood from children (<6 years old) of Kinshasa. Ya
r
p for r
Slope
log As log Cd log Hg log Mn log Pb log Zn
0.37 0.43 0.24 −0.23 0.33 0.20
<0.01 <0.01 0.01 0.02 <0.01 0.04
0.26 0.32 0.18 −0.14 0.19 0.06
a
Y shows the logarithm of the elements concentration in g/dL, X shows age in months.
(95% range) (0.13–0.39) (0.18–0.45) (0.03–0.33) (−0.25 to −0.02) (008–0.29) (0.00–2.60)
Evaluation Increasing as aged Increasing as aged Increasing as aged Decreasing as aged Increasing as aged Increasing as aged
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J. Tuakuila et al. / Journal of Trace Elements in Medicine and Biology 28 (2014) 45–49
Table 5 Comparison between Kinshasan population and other international studies. Parameter
Kinshasaa [median levels (P5th–P95th)]
Other international studies (reference values) United Statesb [median levels (P95th)
As Cd Cu Hg Mn Mo Pb Se Zn
(g/L) (g/L) (mg/L) (g/L) (g/L) (g/L) (g/dL) (g/dL) (mg/L)
3.17 (1.48–8.82) 0.15 (≤LOD–0.30) 1.1 (0.9–1.7) 2.1 (0.8–5.9) 10.4 (5.7–22.2) 17.7 (3.6–30.6) 8.7 (3.6–15.9) 10.7 (8.5–13.6) 5.0 (3.4–6.9)
Germanyc (median levels)
<0.14 (0.20) 0.30 (1.80)
1.70 (5.10)
1.96
Canadad [median levels (P95th) 0.66 (2.16) 0.10 (0.23) 0.971 (1.197) 0.28 (2.08) 9.74 (16.36) 0.80 (1.60) 0.87 (1.95) 18.54 (23.17) 5.26 (6.51)
South Africae (median levels) 1.53 1.195 8.48 5.64 17.6 3.431
SIVR listf (5th–95th) 1.0–1.2 1.0–1.5 0.6–1.3 1.0–4.5 3.0–8.0 0.5–5.0 0.1–10 0.7–14.5 3.5–7.5
a
This study. US National Health and Nutrition Examination Surveys: Data from CDC [values for total population of survey years 2003–2004] [33]. c Schulz et al. [34]. d Health Canada: results of the Canadian Health Measures Survey Cycle 1 2007–2009 [values for children aged 6–11 years] [14]. e Bazzi et al. [13]. Data from South African school children. f SIVR circuit laboratory or literature evaluation: 5th–95th of reference values in blood of Italian population (www.valoridiriferimento.it accessed 14.04.13) [48], modified by Caroli and Zàray [49]. b
and 0.28 g/L reported in US and Canadian children, respectively [14,33]. 10% of B-Hg values were higher than 4.5 g/L (95th percentile) reported in Italian population (SIVR list, Table 5). The high Hg levels in blood of kinshasan children reported probably reflecting dietary differences [16]. In this study, children did not receive dental amalgams. However, the mother’s contribution in B-Hg levels of children through the uterus or nursing may represent other important source of exposure to this element [39].
toxic element: 95% for As, 10% for Hg and 35% for Pb. The main health concerns emerging from this study are the high As, Hg and Pb exposures of the Kinshasan children requiring further documentation, corrective actions and the implementation of appropriate regulations.
Conflict of interest statement The authors declare there are no conflicts of interest.
B-Pb The median B-Pb (8.7 g/L) found in this study was higher than those reported in Table 5. The high blood lead levels (BLL) probably reflecting the use of leaded fuel, lead-based paints and lead from acid battery recycling that remain sources of major exposure in Kinshasa [16,23]. Children are known to constitute a group at high risk of Pb poisoning; they ingest more Pb due to their hand-mouth activity, their digestive absorption rate is higher than adults and their developing nervous system is more sensitive to the adverse effects of Pb [40]. According to the CDC definition of elevated BLL (≥10 g/dL) [41], about 35% of Kinshasa children had elevated BLL. Among children less than 5–6 years tested in developing countries, about 7% were reported to have elevated BLL (>10 g/dL) in Nairobi [42], 21% in the rural Philippines [43] and in Kampala [44], 55% in Nigeria and Egypt [45,46]. The prevalence found in industrialized countries is much lower with, for instance, 1.4% in the USA [47]. Over all, the proportion of children potentially poisoned by toxic elements in this study varies from 10% (n = 10) to 95% (n = 95) and depends on toxic element: 95% (n = 95) for As, 10% (n = 10) for Hg and 35% (n = 35) for Pb. A major limitation should be considered in evaluating present results. With regard to sample collection, selection of blood sample donors did not follow rigid sampling strategy (such as random sampling) but by chance, which was practically inevitable under present survey conditions. Despite such limitation, however, it is prudent to conclude that data from the present study constitutes baseline levels or levels generally exceeded in children for 9 elements, including essential and ubiquitous toxic elements. Regarding levels of the essential trace elements (Cu, Mn, Mo, Se and Zn), B-Cu, B-Mn, B-Se and B-Zn were in the normal range, whereas exceeded levels were observed for B-Mo. None of these children was deficient in essential elements. Except Cd, all toxic elements showed exceeded levels. The proportion of children potentially poisoned by toxic elements varies from 10% (n = 10) to 95% (n = 95) and depends on
Acknowledgments We are highly indebted to the study participants and to the staff of investigators, as well as all the local health services and health centers of the Kinshasan Public Health System that supported the field work. We also thank Professors Lison, Hoet, Haufroid and Mrs Deumer for their collaboration. The financial support of the Belgian Technical Cooperation (Coopération Technique Belge-CTB/Belgische Technische Coöperatie-BTC) and LTAP (Louvain centre for Toxicology and Applied Pharmacology) are gratefully acknowledged.
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