Chemosphere 124 (2015) 83–91

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Chemosphere
journal homepage: www.elsevier.com/locate/chemosphere

Biomonitoring of arsenic, cadmium, lead, manganese and mercury in

urine and hair of children living near mining and industrial areas
Isabel Molina-Villalba a, Marina Lacasaña b,c,d, Miguel Rodríguez-Barranco b, Antonio F. Hernández a,
Beatriz Gonzalez-Alzaga b, Clemente Aguilar-Garduño e, Fernando Gil a,⇑
a
Department of Legal Medicine and Toxicology, School of Medicine, University of Granada, Spain
b
Andalusian School of Public Health (EASP), Granada, Spain
c
CIBER of Epidemiology and Public Health (CIBERESP), Madrid, Spain
d
Instituto de Investigación Biosanitaria de Granada (IBS. GRANADA), Granada, Spain
e
Center for Public Health Research (CSISP–FISABIO), Valencia, Spain

h i g h l i g h t s

 The study was conducted in an area with large industry/mining activities.

 Urine and hair levels of As, Cd, Hg, Mn and Pb were determined in School children.
 The only significant correlation between urine and hair levels was found for Hg.
 Cd and Hg levels were higher than in children from other European countries.
 Urine Cd and Hg levels between 25% and 50% of the children might represent a health risk.

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

Article history: Huelva (South West Spain) and its surrounding municipalities represent one of the most polluted estu-
Received 28 July 2014 aries in the world owing to the discharge of mining and industrial related pollutants in their proximity.
Received in revised form 5 November 2014 A biomonitoring study was conducted to assess exposure to arsenic and some trace metals (cadmium,
Accepted 6 November 2014
mercury, manganese and lead) in urine and scalp hair from a representative sample of children aged
Available online 27 November 2014
6–9 years (n = 261). This is the only study simultaneously analyzing those five metal elements in children
Handling Editor: A. Gies urine and hair. The potential contribution of gender, water consumption, residence area and body mass
index on urinary and hair metal concentrations was also studied. Urine levels of cadmium and total mer-
Keywords: cury in a proportion (25–50%) of our children population living near industrial/mining areas might have
Children an impact on health, likely due to environmental exposure to metal pollution. The only significant corre-
Biomonitoring lation between urine and hair levels was found for mercury. Children living near agriculture areas
Urine showed increased levels of cadmium and manganese (in urine) and arsenic (in hair). In contrast,
Hair decreased urine Hg concentrations were observed in children living near mining areas. Girls exhibited
Trace metals significantly higher trace metal concentrations in hair than boys. The greatest urine arsenic concentra-
Environmental contamination
tions were found in children drinking well/spring water. Although human hair can be a useful tool for
biomonitoring temporal changes in metal concentrations, levels are not correlated with those found in
urine except for total mercury, thus providing additional information.
Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction organisms, because of their accumulative capacity and potential

Trace metal contamination is a cause for concern because of its
potential accumulation in both the environment and living
⇑ Corresponding author at: Departamento de Medicina Legal y Toxicología,
Facultad de Medicina, Universidad de Granada, C/Avda. Madrid 11, 18071 Granada,
Spain. Tel.: +34 958 24 35 46/958 24 99 30; fax: +34 958 24 61 07.
E-mail address: fgil@ugr.es (F. Gil).
neurotoxic effects (Gil and Hernández, 2009). Low-dose exposure
to environmental pollutants, including trace metals, in non-occu-
pational settings is becoming a serious problem, especially for
pregnant women and children as they are considered as the most
vulnerable subgroups of population (Rodríguez-Barranco et al.,
2013).

http://dx.doi.org/10.1016/j.chemosphere.2014.11.016
0045-6535/Ó 2014 Elsevier Ltd. All rights reserved.
84 I. Molina-Villalba et al. / Chemosphere 124 (2015) 83–91
Huelva (South-West Spain) and its surrounding municipalities
represent one of the most polluted estuaries in the world as a con-
sequence of the discharge of smelters plumes and mining related
pollutants to air and rivers, with the industrial activity influencing
the trace element composition of particulate matter (Aguilera
et al., 2010).
Thus, exposure of the population living in this area to environ-
mental pollutants raises potential health concerns. In addition, the
higher mortality ratios in Huelva province as compared to the rest
of Spain has contributed to an increased concern of the population
about the potential adverse health effects posed by environmental
pollution (Benach et al., 2004).
Environmental exposures, including trace metals, can threaten
children’s health in utero exposure and the mobilization of various
toxic compounds from maternal tissues during pregnancy, and at
later stages through breast feeding (Massart et al., 2008). Exposure
continues during childhood through food and water intake, inhala-
tion and/or dermal absorption of metal elements (Rodríguez-
Barranco et al., 2013). Moreover, children are more susceptible
than adults to environmental contaminants as they present strik-
ing differences in terms of exposure (Landrigan et al., 2004). First,
children show immature detoxification mechanisms and their
heightened vulnerability is related to physical features (high sur-
face area), nutritional aspects (children drink more water and eat
more food per unit of body weight than adults) and behavioral pat-
terns (direct contact with the ground, tendency to put everything
into their mouths, etc.). This situation has prioritized children as
a target group for studying exposure to environmental pollutants.
In this regard, subtle cognitive and neurobehavioral changes have
been reported in children exposed to low doses of trace metals,
even below concentrations considered safe for most people
(Callan et al., 2012).
Blood and urine samples are the most widely accepted matrices
for biomonitoring trace metal exposure. As metal cations bind to
the sulfur atoms of the keratin present in hair matrix, this tissue
appears to be an attractive choice for environmental health surveys
(Bencko, 1995). Advantages of human hair include being a stable
matrix, a simple collection and transportation and lack of changes
during storage. In contrast to blood and urine concentrations,
which reflect recent exposure (with the exception of urine Cd,
which represents chronic exposure – body burden), hair levels
reflect past exposure, providing an average of the growth period
(Bermejo-Barrera et al., 1998).
Conversely, hair has several limitations such as its potential for
external contamination that needs be completely removed by dif-
ferent washing procedures (Olmedo et al., 2010). Another disad-
vantage is the lack of sufficient information to define reference
ranges for trace metals, because metal hair concentrations vary sig-
nificantly according to age, sex and racial/ethnic factors (Esteban
and Castaño, 2009). Thus, further studies are needed for hair test-
ing to become a reliable exposure biomarker and to broaden the
database for trace metal levels in hair that supports the establish-
ment of normal ranges.
This study was aimed at assessing levels of certain metals (Cd,
Hg, Mn, Pb) and metalloid (As) in children living near a industrial-
ized region from the Southwestern coast of Spain (Huelva). Trace
elements were selected because of their involvement in environ-
mental pollution in the Ria of Huelva and their potential for
adverse health effects in humans. The contribution of some
determinants of the variability of metal levels in urine and hair
(age, gender, health-related lifestyle, diet habits, residence area. . .)
was also studied. To the best of our knowledge, this is the first
study carried out so far in which five trace elements are simulta-
neously determined in two different matrices from a children
population.
2. Materials and methods
2.1. Design, study areas and selection of participants
A cross-sectional study was conducted between January–March
2012 in Huelva (Andalusia, Southwestern Spain). Thirteen schools
were selected which includes the capital city of Huelva and six
municipalities also located close to industrial and mining areas.
A total of 261 children aged 6–9 years who had been living for
at least one year continuously in any of the study areas were eligi-
ble. Children with pre-and peri-natal problems were excluded.
Children’s parents gave informed and written consent to partici-
pate in the study and the study protocol was approved by the Bio-
medical Research Ethics Committee of Granada.
Two questionnaires (one self-administrated and another by
phone interview) were applied to the mother, father or guardian
to obtain information about demographic and socioeconomic char-
acteristics, environmental and home exposures, parent’s occupa-
tional history, children’s diet and body mass index among other
variables.
2.2. Urine sample collection
A first morning urine sample was collected from each partici-
pant in a sterile polyethylene container pre-treated with HNO3
(20%) and rinsed twice with MilliQ water. Samples were then
stored at 20 °C until analyzed within 3 months, time period that
lasted sample collection.
2.3. Hair samples
Human hair was cut from the back of the head as close as pos-
sible to the scalp. Hair samples were kept in acid pre-cleaned poly-
ethylene containers. Hair was then washed by ultrasonic cleaning
in a non-ionic detergent and then with an ethanol solution. The
cleaned hair was dried at room temperature and digested in a
microwave oven (for more details, see Olmedo et al., 2010).
2.4. Determination of arsenic and trace metals
The validation of analytical procedures used for the determina-
tion of metal compounds in urine and hair samples has been
reported elsewhere (Gil et al., 2006; Olmedo et al., 2010). The ana-
lytical method was controlled by using external certified reference
materials (CRM). Reference sample for urine (ref. 201205) was sup-
plied by Seronorm (Billingstad, Norway) and reference material
NIES No. 5 for hair was obtained from the National Institute for
Environmental Studies, Japan Environment Agency. In addition,
we were enrolled in an external Quality Control for Pb and Hg in
urine organized by the Instituto Nacional de Seguridad e Higiene
en el Trabajo, Ministry of Labour, Spain. Atomic Absorption Spec-
trometry-furnace technique was used for metal ions determination
with the exception of arsenic and mercury, which were measured
through a hydride generation system. Appropriate matrix modifi-
ers were used for Cd [0.3 g L 1 of Mg(NO3)2 + 0.33 g L 1 of
Pd(NO3)2], Pb [10 g L 1 of NH4H2PO4] and Mn [1 g L 1 of Mg(NO3)2]
and prepared in 0.2% (v/v) nitric acid and 0.1% Triton X-100. Uri-
nary creatinine levels were determined by the method of Jaffe
using a Hitachi 917 auto-analyzer. Limit of detections (LOD) for
urine (lg L 1) and hair (lg g 1) were: 0.030 and 0.0033 for As
and Cd, 0.002 and 0.00022 for Hg, 0.120 and 0.0132 for Mn,
0.830 and 0.0913 for Pb. Limits of quantifications (LOQ) for urine
(lg L 1) and hair (lg g 1) were: 0.100 and 0.010 for As, 0.090
and 0.010 for Cd, 0.007 and 0.0007 for Hg, 0.390 and 0.040 for
 I. Molina-Villalba et al. / Chemosphere 124 (2015) 83–91 85

Mn, and 2.710 and 0.274 for Pb, respectively. Coefficients of varia- Table 1
tion (CV) in the analysis were 6.70%, 4.94%, 3.56%, 2.57% and 2.63% Description of the sociodemographic characteristics of the participants.

for As, Cd, Hg, Mn and Pb, respectively (Gil et al., 2006; Olmedo N (%)
et al., 2010). Sex
Boys 135 (51.7)
Girls 126 (48.3)
2.5. Statistical analysis
Age
6–7 145 (55.6)
Geometric means and percentiles were calculated to describe 8–9 116 (44.4)
metal concentration in samples. Levels below LOD was assigned
Residence areaa
the LOD (expressed as lg g 1 creatinine and lg g 1 for urine and Urban 225 (89.6)
hair samples, respectively) divided by the square root of 2 (CDC, Metropolitan 15 (6.0)
2009). Rural 11 (4.4)
Differences in mean levels of metal compounds as well as the Distance to mining areas
potential influence of predictor variables and potential confound- Less than 200 m 12 (5.7)
ers (age, gender, residence area, body mass index – BMI, type of Between 200–500 m 4 (1.9)
Between 500–1000 m 41 (19.5)
water consumed, physical activity,. . .) on these levels were
More than 1000 m 153 (72.9)
assessed by using the non parametric Mann–Whitney and Krus-
Distance to industrial areas
kal–Wallis tests. The magnitude of the correlation between metal
Less than 200 m 7 (3.6)
concentration in urine and hair samples was assessed by the Spear- Between 200–500 m 1 (0.5)
man rank correlation test. Between 500–1000 m 10 (5.1)
Univariate and multivariate Tobit log-linear regression models More than 1000 m 178 (90.8)
were used to evaluate variables associated to metal levels. The Distance to agricultural areas
use of Tobit models allows for an estimate of the effect despite hav- Less than 200 m 28 (13.5)
Between 200–500 m 13 (6.3)
ing a large number of samples below the LOD. These models are
Between 500–1000 m 46 (22.2)
useful for modeling censored variables, and thus are suitable for More than 1000 m 120 (58.0)
measurements with a detection limit below which the actual value
Mother’s occupation
of the variable is unknown. These models also take into account Employed 112 (44.6)
the uncertainty in the estimates, which will be greater as the num- Housewife 107 (42.6)
ber of censored values increases. The dependent variables (metal Unemployed 32 (12.8)
concentration in lg L 1) were each regressed against potential pre- Father’s occupation
dictors (independent variables) such as life style habits and soci- Employed 168 (75.0)
odemographic characteristics. As recommended by Barr et al. Unemployed 56 (25.0)

(2005), urine creatinine levels were included as an independent a

covariate in the regression models rather than using creatinine-
adjusted metal concentrations (creatinine standardization)
because this approach is less prone to produce biased effect
estimates.
The statistical analyses were developed with the IBM SPSS 19
and Stata 11 statistical package software.
3. Results
A total of 261 children (126 girls and 135 boys) participated in
the study (Table 1). 56% of children aged 6–7 years and 44% were
8–9 year-old. 45% of mothers reported to be working, 43% self-
defined as housewife and 13% were unemployed. Among fathers,
25% reported being unemployed. Most of children (90%) lived in
urban areas.
Table 2 shows the distribution of metal levels in urine and hair
samples from the study population. Because values did not fit a
normal distribution, ranges, percentiles and geometric means are
presented for each element. 90% and 76% of urine samples were
below the LOD for Pb and Mn, respectively. In turn, 61%, 57% and
35% of hair samples were below the LOD for Cd, Pb and As,
respectively.
Table 3 present data for the bivariate analyses for metal concen-
tration in urine and hair and for some potential predictor variables,
including gender, water consumption, residence area and BMI.
Females had higher significant hair levels of Cd, Hg, Mn and Pb,
approximately twice than males. Interestingly, individuals drink-
ing well or spring presented the highest urine levels of As. A direct
correlation was observed between urine Hg levels and living in
urban residence areas.
The most striking finding in Table 4 was the direct correlation
between levels of As and Cd in urine. In turn, both compounds
Urban areas refer to people living in cities (capital of province) whereas
metropolitan areas refer to people living in villages close to these cities within a
radius of 10 km.
exhibited an inverse correlation in hair. Urine As was inversely cor-
related with hair Cd. Urine Pb levels showed a positive correlation
with Cd and As. Levels of Mn and Cd were positively correlated in
hair as well as Hg in urine and hair.
Table 5 shows multiple log-linear regression analyses for metal
ion concentration in urine and hair adjusted for age, gender, BMI,
food consumption (type of fish, water consumption), lifestyle habits
(physical activity, household cleaning frequency, washing hands,
frequency of child bath, pets at home,. . .) and residence area. Girls
exhibited statistically significant higher levels of metal elements
in hair as compared to boys with the exception of As. However,
no association was found between gender and metal compounds
in urine. Urine As levels showed a direct association with water
consumption (well or spring) and with increased physical activity.
Hair As and Cd were associated with frozen fish consumption, but
in a different way as As showed a positive association and Cd a neg-
ative association. Increased urine levels of Cd and Mn were
observed in children living near agriculture areas as also did hair
As. In contrast, decreased urine Hg concentrations were observed
in children living near mining areas. Other interesting association
observed was between hair Pb and BMI as overweighed children
had statistically significant increased hair lead levels.
4. Discussion
4.1. Hair metal concentrations
Human biological monitoring has become an important tool in
environmental and public health for the assessment of internal
86 I. Molina-Villalba et al. / Chemosphere 124 (2015) 83–91

Table 2
Heavy metal and arsenic levels in urine (alg L 1
and blg g 1
creatinine) and hair (clg g 1
) from our children population.

Heavy metal N % < LOD AM (SD) GM P25 Median P75 P95 Maximum
a
Urine
As 261 14.2 1.417(1.294) 0.701 0.500 1.170 1.930 3.900 8.340
Cd 261 8.4 0.319(0.229) 0.215 0.130 0.292 0.456 0.712 1.502
Hg 261 21.1 1.305(1.230) 0.304 0.330 1.130 2.000 3.460 9.850
Mn 261 65.5 <0.120 <0.120 <0.120 <0.120 <0.120 0.946 6.127
Pb 261 90.0 <0.830 <0.830 <0.830 <0.830 <0.830 1.580 7.320
Urineb
As 261 14.2 5.370(6.500) 2.438 1.535 3.398 6.659 20.784 44.059
Cd 261 8.4 1.370(1.610) 0.747 0.400 0.853 1.582 4.839 10.074
Hg 261 21.1 5.880(9.370) 1.059 0.696 3.100 6.914 20.126 74.103
Mn 261 65.5 NC NC NC NC NC 3.781 107.735
Pb 261 90.0 NC NC NC NC NC 9.390 54.806
Hairc
As 220 34.9 0.067(0.104) 0.017 <0.0033 0.021 0.093 0.302 0.575
Cd 220 61.3 <0.0033 <0.0033 <0.0033 <0.0033 0.007 0.067 0.320
Hg 220 7.3 1.282(1.524) 0.407 0.376 0.910 1.726 4.128 15.110
Mn 220 13.0 0.263(0.271) 0.137 0.082 0.199 0.375 0.717 2.624
Pb 220 57.1 <0.0913 <0.0913 <0.0913 <0.0913 0.251 2.089 13.750

AM (SD): Mean (Standard Deviation) GM: Geometric mean; Px: Percentile x. Limits of detection (LODs) in urine (lg L 1) and hair (lg g 1) were: 0.030 and 0.0033 for As and
Cd, 0.002 and 0.00022 for Hg, 0.120 and 0.0132 for Mn, 0.830 and 0.0913 for Pb.
NC: not computable (AM, GM, P25 and Median for urine levels of Mn and Pb (creatinine-adjusted) cannot be shown because of the higher percentage of samples below the
LOD.
a
lg L 1.
b
lg g 1 creatinine.
c
lg g 1.

Table 3
1 1
Description of heavy metal concentrations (mean values ± standard deviation) in urine (lg g creatinine) and hair (lg g ) according to potential confounders.

Heavy metal Biological sample Gender Usual water consumption

Male Female Municipal Municipal filtered Bottled Municipal + Bottled Well or spring
As Urine 5.89 ± 7.34 4.82 ± 5.44 4.38 ± 5.43 2.30 ± 2.83 6.53 ± 6.52 6.23 ± 9.24 13.75 ± 13.96**
Hair 0.06 ± 0.08 0.08 ± 0.12 0.07 ± 0.10 0.08 ± 0.16 0.06 ± 0.09 0.06 ± 0.10 0.17 ± 0.27
Cd Urine 1.34 ± 1.47 1.41 ± 1.75 1.20 ± 1.34 1.69 ± 3.00 1.63 ± 1.80 1.03 ± 1.37 1.99 ± 1.66
Hair <0.0033 <0.0033 <0.0033 <0.0033 <0.0033 <0.0033 <0.0033
Hg Urine 5.60 ± 7.36 6.18 ± 11.16 5.24 ± 7.67 10.95 ± 22.95 5.31 ± 6.35 9.08 ± 15.72 8.54 ± 15.81
Hair 0.98 ± 1.10 1.51 ± 1.75** 1.17 ± 1.21 0.84 ± 0.67 1.26 ± 1.07 2.09 ± 3.47 1.44 ± 2.01
Mn Urine NC NC NC NC NC NC NC
Hair 0.16 ± 0.15 0.35 ± 0.31** 0.25 ± 0.31 0.46 ± 0.31* 0.25 ± 0.22 0.27 ± 0.16 0.45 ± 0.19
Pb Urine NC NC NC NC NC NC NC
Hair <0.0913 <0.0913 <0.0913 <0.0913 <0.0913 <0.0913 <0.0913
Residence area BMI
Urban Metropolitan Rural Low weight Norm weight Overweight Obesity
As Urine 5.24 ± 6.21 5.07 ± 8.48 3.34 ± 2.88 5.9 ± 5.95 5.26 ± 5.84 6.91 ± 8.73 4.85 ± 8.24
Hair 0.06 ± 0.10 0.07 ± 0.06 0.14 ± 0.18 0.22 ± 0.20  0.06 ± 0.10 0.07 ± 0.12 0.05 ± 0.09
Cd Urine 1.34 ± 1.56 0.97 ± 0.94 1.38 ± 1.48 2.06 ± 1.32 1.36 ± 1.67 1.63 ± 1.77 1.15 ± 1.14
Hair <0.0033 <0.0033 <0.0033 <0.0033 <0.0033 <0.0033 <0.0033
Hg Urine 6.30 ± 9.83* 4.14 ± 5.42 2.08 ± 3.70 2.29 ± 3.09 5.77 ± 9.80 7.74 ± 10.65 5.94 ± 6.48
Hair 1.33 ± 1.58 1.29 ± 1.28 0.71 ± 0.94 1.38 ± 1.91 1.22 ± 1.57 1.41 ± 1.31 1.51 ± 1.37
Mn Urine NC NC NC NC NC NC NC
Hair 0.27 ± 0.28 0.18 ± 0.13 0.29 ± 0.24 0.27 ± 0.17 0.26 ± 0.22 0.23 ± 0.25 0.32 ± 0.48
Pb Urine NC NC NC NC NC NC NC
Hair <0.0913 <0.0913 <0.0913 <0.0913 <0.0913 <0.0913 <0.0913

Limits of detection (LODs) in hair (lg g 1) were: 0.0033 for As and Cd, 0.00022 for Hg, 0.0132 for Mn and 0.0913 for Pb. NC: not computable (mean urine levels for Mn and Pb
(creatinine-adjusted) cannot be shown because of the higher percentage of samples below the LOD.
 
p < 0.10.
*
p < 0.05.
**
p < 0.01.

doses to harmful substances (Gil and Hernández, 2009). Although
blood and urine analysis are traditional approaches for biomonitor-
ing, human hair is an interesting matrix because hair concentra-
tions of metal compounds are up to 10-fold higher than the
levels found in blood or urine samples. Hair testing can be used
as a screening tool for biomonitoring chronic exposure to environ-
mental toxic elements (Bencko, 1995). Nevertheless, there is con-
troversy on whether the measurement of a substance in the hair
accurately reflects external exposure or internal body dose. Except
for mercury – methyl mercury – (and perhaps arsenic), data are
insufficient to reliably indicate the source of exposure and the
internal dose as well as to predict the resultant health effects from
the measurement of a particular substance in hair (ATSDR, 2001;
Harkins and Susten, 2003).
In our study, median trace metal levels in hair showed the fol-
lowing descending order: Hg > Mn > Pb > As > Cd (Table 2). Overall,
 I. Molina-Villalba et al. / Chemosphere 124 (2015) 83–91
Table 4
Spearman correlation coefficients for heavy metal concentrations in the two biological samples studied (urine and hair).
Urinea
As Cd Hg Mn
Urinea
As 0.337**
Cd 0.337**
Hg
Mn
Pb 0.198** 0.275**
Hairb
As
Cd 0.227**
Hg 0.287**
Mn
Pb
*
p < 0.05.
**
p < 0.01.
a
Concentrations in urine (lg L 1).
b
Concentrations in hair (lg g 1).
levels are very similar to those reported by Dongarrà et al. (2012)
and Evrenoglou et al. (2013) in Sicily and Athens, respectively. Con-
centrations of As in hair of unexposed humans usually ranges from
0.02 to 1 lg g 1 (Hindmarsh et al., 1999). Our children population
had a median value of 0.021 lg g 1, similar to the normal concen-
tration of 0.02–1 lg g 1 reported by Hindmarsh et al. (1999) for
humans. The 95th percentile and even the maximum hair arsenic
level found in our study population were below the critical level
of 1 lg g 1 set by the WHO (Hindmarsh, 2000). Since arsenobe-
taine, the major organic arsenic compound in seafood, is not accu-
mulated in hair (Vahter et al., 1983), arsenic levels in hair only
reflect exposure to inorganic arsenic. Hence, our results indicate
a low contribution of environmental arsenic pollution to the body
burden and thus no adverse effects are expected for our children
population.
Cadmium (Cd) is a nonessential toxic metal widely distributed
at low levels in the environment. Despite its well known acute
and chronic toxic effects, potential developmental and other
adverse health effects in children are unknown (ATSDR, 2008). Diet
(especially cereals and vegetables) represents the major source of
Cd exposure. In our study, although the median value was below
LOD, percentiles 75 and 95 (0.007 and 0.067 lg g 1, respectively)
were similar to the median values reported by Dongarrá et al.
(2012) for a non-occupational population living near industrialized
areas in Sicily (0.0005–0.077 lg g 1) and far below compared to
other studies that found median levels of 7.33 and 0.13 lg g 1 in
Chinese (Bao et al., 2009) and Bolivians children (Stassen et al.,
2012), respectively.
Mercury (Hg) is one of the most toxic elements that may poten-
tially impair children’s neurodevelopment (Grandjean et al., 1999).
Hg content in hair seems to be more related to dietary exposure, in
particular fish consumption (Cheng et al., 2009). Even though total
Hg is widely measured in the general population, 80% of total hair
Hg consists of methyl mercury and both of them are linearly
related (McDowell et al., 2004). Because exposure to methyl mer-
cury occurs only through diet – eating contaminated fish – and
not by external sources, this contaminant has been identified as
a unique substance that could be reliably interpreted through hair
analysis as to source and internal dose, assuming the samples are
properly handled and analyzed (ATSDR, 2001; Harkins and
Susten, 2003). While hair can be a useful sample for determining
mercury in the general population, it has less interest for individ-
uals occupationally exposed to mercury compounds. In our study,
total Hg concentration was determined in hair and the median
87
Hairb
Pb As Cd Hg Mn Pb
0.198** 0.227**
0.275**
0.287**
0.173*
0.173* 0.269**
0.269**
value (0.91 lg g 1) was below the safe limit of 1 lg g 1 (NRC,
2000) and the mean level of 1.99 lg g 1 reported by EFSA (2012)
for European children. The NHANES 1999–2000 hair data provide
population-based data on hair mercury concentrations for U.S.
children aged 1–5 years with a geometric mean and 95th percen-
tile of 0.12 and 0.64 lg g 1, respectively (McDowell et al., 2004),
which are comparatively lower than those found in our study.
Several studies have shown that hair Hg levels depend on the
fish consumption (Peplow and Augustine, 2012). Grandjean et al.
(1999) reported a geometric mean of 2.99 lg g 1 in fish-consum-
ing children. Median hair Hg content in children with important
fish consumption was 3.9 lg g 1 (Benefice et al., 2008) and concen-
trations ranging from 9.5 to 11.4 lg g 1 were reported in children
from a traditional riverside population (Malm et al., 2010). Our
results are comparable to other studies from populations with
low to moderate Hg exposure through fish consumption
(Guentzel et al., 2007).
Children and infants may be particularly susceptible to neuro-
toxic effects of environmental exposure to manganese (Mn). Low
level of exposure to Mn has previously been associated with
adverse effects on neurodevelopmental outcomes in children
(Riojas-Rodríguez et al., 2010). Median Mn hair concentration
found in our study population (0.20 lg g 1) is far lower than the
median level reported by Haynes et al. (2012) and Kordas et al.
(2010) (3.52 and 1.45 lg g 1, respectively). In turn, Montes et al.
(2011) reported median levels of 0.6 and 13.2 lg g 1 for non-
exposed and exposed populations, respectively.
Lead (Pb) largely persists in contaminated environments and
may result in learning disability and reduced growth in children.
Watanabe et al. (2000) reported that 59% and 81% of total Pb
uptake in urban and rural areas, respectively, could be attributable
to dietary exposure. Moreover, neurologic and developmental
effects in children are well-known in the literature (ATSDR,
2007). Normal values for Pb content in hair have been reported
to be lower than 6.0 lg g 1 (Miekeley et al., 1998) although the
proposed level of concern for children was set in 9 lg g 1
(Esteban et al., 1999). In our study, although hair Pb median con-
centration was below the LOD, percentiles 75 and 95 (0.251 and
2.089 lg g 1) were below the aforementioned values. Moreover,
these percentiles are within the same range than the median levels
reported by Sanna et al. (2008) for children living in the vicinity of
industrial areas of Sardinia (Italy), which ranged from 0.27 to
1.47 lg g 1 and the median levels reported by Dongarrá et al.
(2012) for children living near industrialized areas in Sicily, which
88 I. Molina-Villalba et al. / Chemosphere 124 (2015) 83–91

Table 5
Backward stepwise multiple log-linear Tobit regression analysis of metal ion concentration in the different biological samples studied.

Metal ion Biological sample Pseudo R2 Predictor variable b p-value IC 95%

As Hair 0.0156 Distance to industrial area
Less than 1 km 1.02 0.036 (0.07; 1.97)
Type of fish most consumed
Frozen 1.05 0.036 (0.07; 2.04)
Urine 0.0564 Usual water consumption
Municipal filtered 0.59 0.284 ( 1.68; 0.49)
Bottled 0.79 0.001 (0.34; 1.25)
Municipal + Bottled 0.77 0.050 ( 0.00; 1.55)
Well or spring 1.46 0.046 (0.03; 2.90)
Physical activity of the child
Moderately active 0.87 0.030 (0.09; 1.65)
Quite active 1.00 0.014 (0.21; 1.79)
Very active 1.13 0.052 ( 0.01; 2.28)
Household cleaning frequency
Once a week 0.53 0.039 ( 1.03; 0.03)

Cd Hair 0.0603 Gender

Girls 1.69 0.002 (0.65; 2.74)
Type of fish most consumed
Frozen 1.65 0.013 ( 2.95; 0.35)
Pets at home
Yes 1.26 0.016 (0.24; 2.29)
Household cleaning frequency
Once a week 0.99 0.077 ( 0.11; 2.1)
Urine 0.0781 Age (per year increase) 0.17 0.017 (0.03; 0.30)
Distance to agricultural area
Less than 1 km 0.58 <0.001 (0.31; 0.85)
Usual water consumption
Municipal filtered 0.13 0.713 ( 0.84; 0.57)
Bottled 0.39 0.008 (0.10; 0.67)
Municipal + Bottled 0.24 0.332 ( 0.25; 0.73)
Well or spring 0.40 0.353 ( 0.45; 1.25)
Boy/girl washing his hands before eating
Yes 0.44 0.056 ( 0.01; 0.88)

Hg Hair 0.0144 Gender

Girls 1.15 0.002 (0.42; 1.88)
Body mass index (BMI)
Low weight 0.32 0.774 ( 1.89; 2.53)
Overweight 0.09 0.893 ( 1.18; 1.36)
Obesity 0.93 0.088 ( 0.14; 1.99)
Frequency of child bath or shower
4–6 times per week 0.29 0.481 ( 0.51; 1.09)
1–3 times per week 1.32 0.085 ( 0.19; 2.82)
Urine 0.0081 Distance to mining area
Less than 1 km 1.83 0.031 ( 3.49; 0.17)

Mn Hair 0.0399 Gender

Girls 1.02 0.000 (0.64; 1.41)
Frequency of child bath or shower
4–6 times per week 0.43 0.049 ( 0.85; 0.00)
1–3 times per week 0.61 0.138 ( 1.41; 0.20)
Urine 0.0428 Distance to agricultural area
Less than 1 km 0.85 0.049 (0.00; 1.70)
Monthly family income
1001–2000€ 1.68 0.010 ( 2.95; 0.41)
>2000€ 0.99 0.147 ( 2.34; 0.35)
Frequency of child bath or shower
4–6 times per week 0.34 0.478 ( 1.30; 0.61)
1–3 times per week 1.37 0.058 ( 0.05; 2.80)

Pb Hair 0.0223 Gender

Girls 1.15 0.024 (0.15; 2.15)
Body mass index (BMI)
Low weight 1.03 0.448 ( 1.64; 3.71)
Overweight 1.63 0.038 (0.09; 3.17)
Obesity 0.00 0.996 ( 1.41; 1.42)
Urine 0.0557 Distance to agricultural area
Less than 1 km 0.88 0.077 ( 0.10; 1.85)

References: Residence over 1000 m of industrial area; Residence over 1000 m of agricultural area; Residence over 1000 m of mining area; Fresh fish consumption; Municipal
water consumption; Indolent physical activity of the child; More than once a week household cleaning; Boys (gender); No pets at home; No washing his hands before eating;
Norm weight (BMI): 18.5–25 kg m 2; once a day child bath or shower; 61000 € monthly family income.
 I. Molina-Villalba et al. / Chemosphere 124 (2015) 83–91
ranged from 0.70 to 1.58 lg g 1. Accordingly, no adverse effects are
expected from environmental lead exposure in our children
population.
4.2. Urine metal concentrations
Urine it is a non-invasive biological matrix currently used for
biomonitoring studies (NHANES/GerES) and particularly interest-
ing for children as blood samples are difficult to obtain and would
lead to a low participation rate. Because the World Health
Organization guidelines considers that biomarker samples with
creatinine values less than 0.3 g L 1 or greater than 3 g L 1 are
unrepresentative, they should be excluded from statistical analysis
in order to minimize both over- and underestimation in calculating
the normalized concentrations. In our study population, no urine
sample showed creatinine levels above 3 g L 1 and 35% of samples
had a urinary creatinine level below 0.3 g L 1. Nonetheless, we
have performed a sensitivity analysis by removing those children
with creatinine levels <0.3 g L 1 in order to compare the coeffi-
cients of the regression models. No significant changes were
observed in the regression coefficients with respect to the whole
analysis that requires the conclusions of the study to be changed.
Overall, our children population had median metal concentra-
tions in urine lower than those found in hair. Our entire children
population had total urinary As concentration below 50 or
100 lg g 1 creatinine, which are the CDC’s and WHO action levels,
respectively (Carrizales et al., 2006). The median value of
3.40 lg g 1 creatinine found in our study is remarkably lower than
the 27.9 lg g 1 creatinine reported by Gamiño-Gutiérrez et al.
(2013), but slightly higher than the median value of 1.80 lg g 1
creatinine reported by Aguilera et al. (2010) in an earlier study
carried out on children living near our study area. As regards geo-
metric means (GM), we found 2.44 lg g 1 creatinine, which is also
far lower than the GM of 44–80 lg g 1 creatinine reported by
Carrizales et al. (2006) and lower than the GM of 12.8–44.5 lg g 1
creatinine reported by Jasso-Pineda et al. (2012) and the GM of
4.01–4.91 lg g 1 creatinine reported by Chiang et al. (2008). In
any case, the maximum urinary As concentration found in our
study was below the reference value of 15 lg L 1 reported by
Wilhelm et al. (2006). Measurement of urinary arsenic levels is
generally accepted as the most reliable indicator of recent arsenic
exposure. However, total urinary arsenic is a less useful biomarker
of exposure to inorganic arsenic, unless ingestion of food from
marine origin can be excluded. This is particularly important when
exposure to inorganic arsenic is low.
As regards cadmium levels, the median value in our children
population (0.85 lg g 1 creatinine) is higher than the median value
of 0.49 lg g 1 creatinine reported by Aguilera et al. (2010) near our
study area. Both figures are below the limit of 1.0 lg g 1 creatinine
for individuals younger than 25 years (Wilhelm et al., 2005). How-
ever, when our data are compared with the revised German refer-
ence level of 0.2 lg L 1 (Schulz et al., 2009), somewhat more than
50% of our population have levels above this value, which can be a
matter of concern as Cd is a known carcinogen. Besides, values of
urine Cd above 1 lg g 1 creatinine have been associated with renal
tubular dysfunction, calcium metabolism disturbances and a
higher risk of lung cancer (Banza et al., 2009). In our study, less
than 50% of the population had urine Cd levels above 1 lg g 1 cre-
atinine, a value associated with the above-mentioned clinical
effects.
Hg in urine is currently used to test for exposure to metallic
mercury vapor and to inorganic forms of Hg. Measurement of Hg
in blood or hair is used to monitor exposure to methyl-Hg; by con-
trast, urine is not useful for determining exposure to methyl-Hg.
Nonetheless, blood, urine, and hair levels may be used together
to predict possible health effects caused by the different forms of
89
Hg (ATSDR, 1999). The GM found in our study (1.06 lg g 1 creati-
nine) was similar to the GM value of 1.23 reported by Levy et al.
(2004) in children aged 4–8 years but lower than the GM of
2.73 lg g 1 creatinine reported by Rojas et al. (2006) for children
exposed to environmental sources of mercury. Nevertheless, less
than 50% of our children population had urine Hg concentration
above 5 lg g 1 creatinine, which is considered as an appropriate
reference level for mercury in children (ATSDR, 2007). Thus, this
subset of children might be at an increased risk for health effects
from mercury exposure.
While low levels of Mn are non-toxic, extremely high levels
may produce adverse health effects on brain development in chil-
dren, including changes in behavior and decreases in the ability to
learn and remember (ATDSR, 2012). Urine Mn concentration may
be useful in detecting groups with above-average current exposure
to this metal. However, measurements in individuals may only be
related to exposure dose after exposure has ceased (ATSDR, 2012).
The GM of 0.42 lg g 1 creatinine found in our study is comparable
to the GM of 0.40 lg g 1 creatinine reported by Banza et al. (2009)
for people (including children <14 years) living near mining activ-
ity areas, and the GM 0.48 found in the NHANES III for approxi-
mately 500 U.S. residents including children >6 year-old (Paschal
et al., 1998). In turn, less than 25% of our children population
had urine Mn levels above the 95th percentile reported by
Heitland and Köster (2006) for German children (0.25 lg L 1).
Although measurements of urinary Pb levels have been used to
assess Pb exposure, they are not as reliable as blood levels, which is
the most common and accurate method of assessing recent Pb
exposure (ATSDR, 2007). However, the use of non-invasive biolog-
ical samples (urine/hair) may provide useful information for bio-
monitoring studies in certain population subgroups (e.g.,
children) where blood testing is more difficult to carry out. The
GM for urine Pb in our children population (2.22 lg g 1 creatinine)
is comparable to 1.28 lg g 1 creatinine reported by Banza et al.
(2009) for non-exposed children but lower than 3.85 lg g 1 creat-
inine found by Chiang et al. (2008).
4.3. Contribution of different determinants of exposure
In the present study, girls exhibited significantly higher concen-
trations of trace metals in hair than boys (Table 3), thus supporting
previous reports (Batista et al., 1996; Fujimura et al., 2012;
Menezes-Filho et al., 2009). This observation can be accounted
for by the different growth rate of hair among individuals since a
larger trace metal accumulation is expected with a slower rate of
hair growth (Sanna et al., 2008). Nevertheless, there are some con-
troversial results as Olivero-Verbel et al. (2008) and Sanna et al.
(2003) found males with significantly greater Hg and Pb levels than
females.
The greatest urine As concentrations were found in children
drinking well or spring water (Table 3). Liu et al. (2010) suggested
that different As concentrations in drinking water could influence
arsenic levels in children. Although trace metal levels have been
related with the area of residence, our study found that living in
urban, metropolitan or rural areas does not contribute significantly
to metal concentrations in urine except for Hg. This finding was
also reported by Aguilera et al. (2010) near our study area.
The correlation between metal levels in hair and other biologi-
cal samples, such as blood or urine, used for the assessment of
environmental exposures, has been previously addressed for bio-
monitoring purposes (Gil et al., 2011). Our results have found a
correlation between urine and hair trace metal concentrations only
for mercury (Table 4). However, while hair content is related to
past exposures, urinary levels reflect recent exposure except for
Cd. This toxicokinetic difference may account for the lack of corre-
lations found in this study.
90 I. Molina-Villalba et al. / Chemosphere 124 (2015) 83–91
Hair Cd showed a significant negative correlation with urine As
(r = 0.227). In contrast, urine Cd was positively correlated with
urine As (r = 0.337) and Pb (r = 0.198) (Table 4). Moreno et al.
(2010) observed a statistically significant positive correlation
between As–Cd (r = 0.35) and Cd–Mn (r = 0.34) in urine samples.
However, Kippler et al. (2010) did not find a significant correlation
between Cd and As in urine. While Bao et al. (2009) reported a
weak positive correlation (r = 0.249) between hair levels of Pb
and Cd, our results do not support this association. Many of these
associations are difficult to explain with the information available
and further studies are required for a better understanding of cor-
relations between different biological samples.
The multiple regression analysis (Table 5) showed an associa-
tion between hair As levels and living near industrial areas (less
than 1 km), where a higher environmental contamination is
expected. Females have significant higher hair Cd, Hg, Mn and Pb
levels than males, suggesting that metal hair content varies accord-
ing to sex, thus supporting previous observations (Menezes-Filho
et al., 2009; Fujimura et al., 2012; Evrenoglou et al., 2013).
Although the rationale for this finding is unclear, toxicokinetic dif-
ferences may play a significant role.
Significant direct associations were found between urine As and
well or spring water consumption as well as between frozen fish
intake and hair As. An inverse association between hair Cd and fro-
zen fish consumption was also found (Table 5). These associations
suggest an impact of diet on metal content in hair and urine. We
also found a direct association between urine As and high physical
activity of children, very likely because a higher respiratory rate
facilitates inhalation of As present in air. Other significant associa-
tions found are difficult to explain and controversy findings have
been also reported (Umbangtalad et al., 2007).
In spite of the large number of industries and smelting factories
located in the study area, annual mean levels of trace metal in par-
ticulate matter measured in air samples (Sánchez de la Campa
et al., 2011) do not appear to be sufficient as to increase the body
burden of all trace metals studied in our children population living
in the vicinity of these industrial areas with the exception of Cd
and Hg.
5. Conclusions
One strengthen of this study is the simultaneous determination
of five metal elements in urine and hair from children. The results
indicate that the body burden of Cd and Hg in a proportion (25–
50%) of our children population living near industrial/mining areas
might have an impact on health. Children are a particularly vulner-
able population with regard to developmental and neurotoxic
effects of metals and deserve special attention in biomonitoring
programs. By recognizing their special vulnerability, a step forward
is preventing potential environmental diseases in children by
diminishing their exposure to environmental pollutants since ref-
erence limits for adults may not necessarily be protective for chil-
dren. Based on the potential accumulation of many metal
compounds in certain human tissues, including hair, hair washed
thoroughly to remove any residue may be used as a useful biolog-
ical matrix for assessing children exposure to metal compounds in
biomonitoring programs as an additional sample to blood or urine.
Acknowledgements
This study had the financial support of the Andalusian Health
Department (Spain) (Project-PI0755/2010). The present article
constitutes the doctoral thesis of Molina-Villalba in the context
of Doctoral Programme in Clinical Medicine and Public Health
(University of Granada). The authors thank J. Santiago-Rodriguez
for technical assistance. The authors would also like to thank the
schools staff and the children and families.
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