Arsenic speciation in human hair: a new perspective for epidemiological assessment in chronic arsenicism
←
→
Page content transcription
If your browser does not render page correctly, please read the page content below
PAPER
Arsenic speciation in human hair: a new perspective for
epidemiological assessment in chronic arsenicism
www.rsc.org/jem
Jorge Yáñez,*a Vladimir Fierro,a Hector Mansilla,b Leonardo Figueroa,c Lorena Cornejoc
and Ramon M. Barnesd
a
Department of Analytical & Inorganic Chemistry, Faculty of Chemical Sciences, University of
Concepción, Concepción, P.O. Box 160-C, Chile
b
Department of Organic Chemistry, Faculty of Chemical Sciences, University of Concepción,
Concepción, P.O. Box 160-C, Chile
c
Department of Chemistry, Faculty of Sciences, University of Tarapacá, Arica, Chile
d
University Research Institute for Analytical Chemistry, Amherst, Massachusetts, USA
Received 5th May 2005, Accepted 6th October 2005
First published as an Advance Article on the web 20th October 2005
The analysis for arsenic in hair is commonly used in epidemiological studies to assess exposure to this toxic
element. However, poor correlation between total arsenic concentration in hair and water sources have been
found in previous studies. Exclusive determination of endogenous arsenic in the hair, excluding external
contamination has become an analytical challenge. Arsenic speciation in hair appears as a new possibility for
analytical assessing in As-exposure studies. This study applied a relative simple method for arsenic speciation
in human hair based on water extraction and HPLC-HG-ICP-MS. The concentration of arsenic species in
human hair was assessed in chronically As(V)-exposed populations from two villages (Esquiña and Illapata)
of the Atacama Desert, Chile. The arsenic concentrations in drinking water are 0.075 and 1.25 mg L1,
respectively, where As(V) represented between 92 and 99.5% of the total arsenic of the consumed waters. On
average, the total arsenic concentrations in hair from individuals of Esquiña and Illapata were 0.7 and 6.1 mg
g1, respectively. Four arsenic species, As(III), DMA(V), MMA(V) and As(V), were detected and quantified in
the hair extracts. Assuming the found species in extracts represent the species in hair, more than 98% of the
total arsenic in hair corresponded to inorganic As. On average, As(III) concentrations in hair were 0.25 and
3.75 mg g1 in Esquiña and Illapata, respectively; while, the As(V) average concentrations were 0.15 and 0.45
mg g1 in Esquiña and Illapata, respectively. Methylated species represent less than 2% of the extracted As
(DMA(V) þ MMA(V)) in both populations. As(III) in hair shows the best correlation with chronic exposure
to As(V) in comparison to other species and total arsenic. In fact, concentrations of As(total), As(III) and
As(V) in hair samples are correlated with the age of the exposed individuals from Illapata (R ¼ 0.65, 0.69,
0.57, respectively) and with the time of residence in this village (R ¼ 0.54, 0.71 and 0.58, respectively).
Introduction than 1 mg L1.3 This situation greatly surpasses World Health
Organization and the U.S. Environmental Protection Agency
Arsenic is a toxic element for humans and is commonly recommendations for As concentrations up to 10 mg L1,
associated with serious health disruptions. The principal man- respectively.4–6
ifestations of arsenicism affecting health are melanosis, kera- It is well known that the toxicity of arsenic is highly
tosis and different forms of cancer (skin, bladder, lung, liver dependent on its chemical form. In fact, As(III) is more toxic
and prostate among others).1 The most common form of than As(V) and methylated compounds that contain trivalent
massive and chronic exposure is by consumption of contami- arsenic are more cytotoxic and genotoxic than arsenite.1,7–9
nated drinking water. Bangladesh, India, Mongolia, China, Other organic compounds of arsenic, such as arsenobetaine,
Taiwan, Mexico, Argentina and Chile are countries where arsenocoline and arsenosugars, can be ingested by seafood and
arsenic poisoning appears as a public health problem resulting seaweed consumption, although their toxicity is lower than
mainly from consumption of As-contaminated water.2 found for inorganic species.1,10
The northern zone of Chile, and especially the Atacama Exposed individuals transform, accumulate, and eliminate
Desert, has been described as an arsenic-rich environment. the ingested arsenic. Inorganic arsenic can be transformed into
Minerals of metallic sulfides containing arsenic are dissolved in organic arsenic, mainly to methylated species such as dimethyl-
the Andes Mountains, affecting superficial and ground waters arsinate (DMA(V)) and monomethylarsenate (MMA(V)).
that cross the Atacama Desert and are used as drinking water Around 60–75% of the inorganic arsenic ingested by a normal
sources. Since 1970, drinking water is specially treated to individual is excreted in urine in a few days, principally as
DMA(V) (60–80%) and MMA(V) (10–15%).11 Also arseno-
DOI: 10.1039/b506313b
remove arsenic in all the large cities of the Atacama Region,
such as Antofagasta.2 However, the populations of several sugars are metabolized by humans into DMA(V), and then
small rural villages remain exposed to arsenic in drinking eliminated through the urine. This fact restricts the use of
water. The problem of chronic arsenicism affects around DMA(V) as a bioindicator of inorganic arsenic exposure,
50 000 people, mainly in rural populations of the Atacama especially in seafood- or seaweed-consuming individuals.10
Desert in northern Chile. The affected populations drink water On the other hand, reduced methylated arsenical species
from small waterfalls and rivers with arsenic contents greater (MMA(III) and DMA(III)) have been measured in urine. Since
This journal is & The Royal Society of Chemistry 2005 J. Environ. Monit., 2005, 7, 1335–1341 1335these species present low stability even at low temperatures arsenic species in human hair collected from As(V)-exposed
(20 1C),12,13 changes in arsenic species composition during populations living in the Atacama Desert, Chile and to explore
the transportation and storage before analysis need to be possible correlation between arsenic species concentration in
considered in data interpretation. This is an important limita- hair with the exposure time (age and time of residence in the
tion of using As-speciation in urine for epidemiological studies selected villages).
especially when sampling takes place in remote areas.14,15 As a
result, sample preservation requires special consideration in Material and methods
order to assure the reliability of speciation analysis in urine.
Part of the ingested arsenic can be found in keratin-rich Reagents
tissues, such as hair and nails. Metal and non-metal elements, All reagents were of analytical grade. Milli-Q water (Millipore,
such as arsenic, are transported in the blood and included in Bedford, MA) was used for all experiments. Standard stock
the fiber. Alpha-keratine of human hair contains about solutions of arsenic species, containing 1000 mg L1 of As, were
10–14% of cysteine,16 offering abundant thiol groups for prepared by dissolving an appropriate amount of the following
reaction with As compounds. Due to the high affinity of arsenic salts: NaAsO2 and Na2HAsO4 7H2O from Merck, Ger-
for keratin, arsenic concentrates in hair much higher than in many, C2H6AsO2Na (DMA(V)) from SIGMA, USA and
other tissues or biological fluids.17 A normal concentration of CH5AsO2Na (MMA(V)) from Chem Service, USA. NaBH4,
arsenic in hair ranges from 0.08 to 0.25 mg g1 in an unexposed HCl, NaOH, NaH2PO4, Na2HPO4 and tetrabutylammonium
population. In contrast, in chronically exposed populations, hydrogen sulfate (TBAHSO4) were purchased from Merck,
concentrations ranging from 1 to greater than 9 mg g1 have Germany. The stock solutions were stored in the dark at
been reported.18 Owing to the hair’s capacity to accumulate 4 1C, except for NaBH4, which was prepared daily in NaOH
arsenic and its slow growth (0.44 mm day1 or 13 mm (0.05%) owing to its low stability in neutral water.
month1), the total As concentration in hair has often been Human hair certified reference material (GBW 09101 No 18)
used for epidemiological studies in chronically exposed popu- from the National Research Center for Certificated Materials,
lations. However, poor correlation between total arsenic con- Beijing, China was kindly provided by Dr Chitra J. Amarasir-
centration in hair and water sources have been found in iwardena. This standard contains a certified total As concen-
different studies.17,18 Possible explanations for this observation tration of 0.59 0.07 mg g1. GBW 09101 No 18 and was used
are the existence of exogenous contamination and the poor to determine the accuracy, precision, and extraction recoveries
efficiency for removing exogenous arsenic from the hair prior for total arsenic and species. The water standard reference
to analysis. material SRM 1643c, trace elements in water was obtained
In the last few years, arsenic speciation in hair has been from the National Institute of Standards and Technology
reported.19–22 One important advantage of arsenic speciation (NIST, USA). The total arsenic concentration is 82.1 1.2
in hair is related to the good stability of arsenic species when mg L1. No individual As species is certified.
compared to other biological samples. Other advantages are
sample size that can be obtained (approximately 1 g) and the
Populations
relative facility for sampling, transport and storage. Addition-
ally, there is the possibility of differentiating endogenous and Two small rural villages, Esquiña and Illapata, were selected
exogenous arsenic species when they are characterized. for this study. They are located in the middle of Atacama
However, there is a lack of information about arsenic species Dessert, Valley of Camarones River, Region of Atacama,
concentrations in hair for exposed populations. Shraim et al. Chile, 250 km southeast of the city Arica. The population of
reported low extraction recoveries and the possibility of species Esquiña consumes drinking water principally from small
changes during extraction steps.20 Recently, Raab and Feld- waterfalls present in the valley, while the population of Illapata
mann described a new method for arsenic speciation in hair by consumes drinking water mainly from the river and waterfalls.
extraction with boiling water.22 They determined the species In both villages, the drinking water was not treated prior to
stability during extraction and quantified four species (As(III), consumption. Esquiña and Illapata were selected because of
As(V), DMA(V) and MMA(V)) in exposed populations from previous information reporting the total arsenic concentrations
West Bengal and Central India, where As(V) was the main present in Camarones Valley waters as published by Figueroa
species found in hair (55 and 63%, respectively). et al.3 According to this study, the arsenic concentration in
The most widely used method for arsenic speciation is high drinking water of Esquiña was 39 mg L1. In spite of this
performance liquid chromatography (HPLC) coupled to spe- information, the population of Illapata consumed As-contami-
cific element detectors, usually elemental spectroscopy includ- nated drinking water containing 55 and 1090 mg As L1 from
ing atomic absorption spectrometry (AAS), inductively the waterfall and river, respectively.3
coupled plasma optical emission spectrometry (ICP-OES) or The populations in the Atacama Dessert and in both studied
inductively coupled plasma mass spectrometry (ICP-MS). Re- villages live in similar geological environments, consume the
verse phase ion-pairing HLPC presents the advantages that it same foods, and are exposed to the same sunlight intensity.
can separate with high resolution inorganic and methylated However, the magnitude of arsenic exposure by drinking
species simultaneously in relatively short times (e.g., o10 min). waters is very different.3 Illapata has about 60 inhabitants, 21
Additionally, IP-HPLC has a good robustness for biological of whom were sampled for the present work; 13 adults
matrices23,24 and it have been coupled directly with ICP-MS (5 females and 8 males) and 8 children. Esquiña has a stable
for arsenic speciation in hair.25 Hydride generation improves population of about 50 people, 22 of whom were sampled
sensitivity and detection limits, and it provides supplementary including 11 adults (6 females and 5 males) and 11 children.
selectivity for the most toxic species (As(III), As(V), MMA(V), Children were considered as individuals from 2 up to 12 year
MMA(III), DMA(V) and DMA(V)), excluding non-toxic species old.
that do not form hydrides (arsenocoline (AsC) and arseno- All sampled individuals were interviewed about their general
betaine (AsB)). Although, the determination of AsC and AsB health condition, food consumption, drug use, drinking water
by using on-line photo oxidation HG been reported,26 con- sources, the time of residence in the village, sun exposure and
sidering the detection limits, the most powerful technique for type of activity. As criteria for exclusion, sampled individuals
arsenic speciation is HPLC-HG-ICP-MS. had to be older than 3 years old, have no incapacitating illness,
To the best of our knowledge, there is a lack of available not consume drugs and have lived at least one month in the
information on arsenic speciation in hair from As(V)-exposed village. Clinical examinations were not performed in this study.
populations. The objective of this study is to characterize All individuals selected for sampling were previously informed
1336 J. Environ. Monit., 2005, 7, 1335–1341about the purposes of this work and voluntarily consented to First, 25–100 mg of washed and dried hair was placed in
participate in the study. polyethylene tubes. Then, 10 mL de-ionized water (o18 MO)
was added to the tube with hair. Samples were leached at 90 1C
for 3 h in an oven, with manual shaking every 30 min. After
Sampling and sample pretreatment leaching, the samples were centrifuged at 3000 rpm for 10 min.
Water. Water samples were collected in 1-L polyethylene The leaching solution (supernatant) was carefully separated
containers from drinking water sources (waterfalls and Camar- from the hair (pellet) and stored at 20 1C until speciation
ones River). To assess the total arsenic and speciation analysis, analysis. When leaching was performed at lower temperatures
two different waterfalls were sampled in Esquiña. In Illapata, (room temperature and 50 1C), lower recoveries were obtained.
samples from one waterfall and the river were collected. These By boiling water, non-reproducible recoveries of As occurred
water sources are available to the population by installation of presumably by volatile arsenic compound losses. Accordingly,
public water faucets. Samples were stabilized with 0.1% HCl the leaching temperature for samples and certified reference
and kept in an ice-cooler at 0 1C during the transport to the material (GBW 09101 No 18) was 90 1C.
laboratory. In the laboratory, samples were stored at 20 1C
until analysis. All samples were filtered with 0.5 mm membrane Instrumentation for arsenic speciation
disks prior to analysis.
Arsenic speciation was performed using ion-pair chromatogra-
phy (IP-HPLC) combined with hydride generation (HG) and
Hair. 0.5–1 g hair was collected from different parts of the inductively coupled plasma-mass spectrometry (ICP-MS) as a
scalp using a stainless-still scissors, cutting at a distance of ca. specific arsenic detector. The HPLC system consisted in a
1 cm from scalp. Hair was placed in polyethylene bags for HPLC from Merck-Hitachi, Germany (model L-7100 La-
transport and storage. Since no well-established procedure Chrom), a six-port HPLC valve from Rheodyne, USA (model
exists to differentiate between endogenous and exogenous 7725i) with a 20 mL sample loop and a monolithic HPLC
arsenic, in this work, samples were washed following the column RP-C18 (100 4.6 mm) from Merck, Germany (model
International Atomic Energy Agency (IAEA) protocol of Chromlith). The separation was performed at room tempera-
1978 for removing exogenous As contamination.27 This simple ture and a flow rate of 1 mL min1. The HPLC mobile phase
method does not remove the endogenous arsenic in compar- contained 0.35 mM of tetrabutylammonium hydrogen sulfate
ison to other described methods.28 Briefly, hair samples were (ion pair reagent), and a pH value of 5.75 was regulated using
first washed using a sufficient volume of acetone to cover the phosphate buffer at 0.5 mM. The column was connected
hair sample in a 50-mL polyethylene centrifuge tube and the directly to the hydride generation system, and consisted in a
acetone was separated by centrifugation. The hair (pellet) was homemade flow injection device with two T-joints for contin-
washed using de-ionized water at room temperature with uous flow of HCl (15%) and NaBH4 (0.6%, in NaOH 0.05%).
successive shaking in a high-speed lab shaker for 1 min for They were pumped at a flow 1 mL min1 using a peristaltic
proper homogenization followed by manual shaking for 10 pump into the HPLC effluent. Chemical reaction by volatile
min. After the water-cleaning step, the same washing proce- hydride generation took place in a 1-mL loop made of PTFE
dure was repeated twice using acetone as described previously. tubing. Separation of gaseous hydrides from the liquid was
Samples were dried overnight in an oven at 50 1C. The dried performed in a glass gas–liquid separator (GLS). The GLS
hair was cut into small pieces (o1 mm) using stainless-steel design was previously described for HG-AFS (atomic fluores-
scissors, and the pieces were stored in polyethylene tubes at cence spectrometry),30 and it is used here for the first time
room temperature until analysis. Samples were weighed im- applied to HG-ICP-MS. Hydrides were carried from the GLS
mediately before the digestion or extraction procedure. to the ICP-MS using an argon flow rate of 0.75 L min1. A
second makeup gas (argon) was necessary for obtaining opti-
mal sensitivity. For this purpose, 0.5 mL min1 Ar was
Total arsenic in hair
introduced after the GLS and before the plasma torch. The
The digestion method was adapted from Flores et al.29 Briefly, ICP-MS (Agilent Technologies Model 7500a, Wilmington,
0.1 g of sample was digested by adding 3 mL of concentrated Delaware) was operated under optimal conditions. The Ar-
HNO3 and 1 mL H2O2 (30%) in a Teflon PFA vessel using a senic ion signal was monitored at m/z 75. The m/z 77, 82 and 83
microwave oven (CEM MDS-2000). The vessels were closed signals were also monitored for interference correction. RF
and heated following the MW-program: 100 W (5 min), 250 W power, sample depth, plasma and auxiliary gas flow rate were
(3 min), 400 W (5 min), 450 W (3 min), 630 W (1 min). To 1200 W, 6 mm, 16 and 1 L min1, respectively. Chromato-
avoid overpressure, each heating step was followed by 3 min graphy software of the instrument was used for quantification.
without power. After cooling, the solutions were transferred Fig. 1 shows a typical chromatogram of four arsenic in water;
and diluted in 25-mL polyethylene tubes. Determination of As(III), As(V), MMA(V) and DMA(V), 50 mg L1 each, under
total arsenic was performed using hydride generation (HG) the optimized chromatographic conditions. Acceptable separa-
and inductively coupled plasma-mass spectrometry (ICP-MS) tion (resolution) for the four species was achieved to quantify
(see below). target species. As(III) and MMA(V) have higher sensitivity in
comparison with other species, which can be explained due to
the differences of hydride species generation efficiency.
Speciation in hair
The arsenic leaching procedure was performed after washing Results and discussion
the samples. Water has been described as a simple and effective
Arsenic concentration in water sources
solubilization reactive for arsenic species in hair.19,20 In gen-
eral, water incubation softens keratine-rich tissues, increasing The population of Illapata principally consumes water from
leaching agent accessibility and facilitating the dissolution of the Camarones River. The total arsenic concentration in this
bonded compounds and ions. The effectiveness of water as a drinking water source was 1252 mg L1, more than 100 times
leaching agent depends directly on the temperature. Previous the accepted international levels (10 mg L1) and 25 times
work of Mandal et al. found that at room temperature, arsenic greater than the Chilean standard (50 mg L1). Waterfall water
extraction from hair and fingernails was less that 1% of the sampled in Illapata contains 48.7 mg L1 of total arsenic.
total arsenic.19 Increasing the temperature close to boiling However, waterfall water is not the preferred drinking water
improves the leaching of the total arsenic from hair. because of its taste. In Esquiña, the population principally
J. Environ. Monit., 2005, 7, 1335–1341 1337Fig. 1 Typical chromatogram of four arsenic species in water: As(III), DMA(V), MMA(V) and As(V) 50 mg L1 each. Separation conditions are
described in text.
consumes waterfall water from two sources with arsenic con- presenting concentrations over the toxicity level (41 mg g1).
centrations of 74 and 12.2 mg L1, respectively, slightly over the The maximum concentration found in Esquiña was 3.3 mg g1
maximum permitted levels by Chilean Standards. The water for a 9-year-old child. In Illapata, 97% (29 from 30) of the
was consumed without any prior treatment in both villages individuals present values that exceed the normal and toxicity
before sampling. The total arsenic concentrations found in levels (41 mg g1), with 30% of these presenting values more
water sources agree with values reported by Figueroa.3 Small than 10 times the normal concentration. These values demon-
differences can be explained by seasonal changes of arsenic strate the chronic exposure of the Illapata population to
concentration in water. Concentrations of total As and As arsenic in contrast to the concentrations found in the Esquiña
species in drinking water sources of Esquiña and Illapata are population, evidently less chronically exposed than in Illapata.
presented in Table 1. The speciation analysis shows that the Correlation between the total arsenic in hair and the resi-
principal species is As(V), representing between 92 and 99.5% dence time was found for both populations. In general, the
of the total arsenic of the consumed waters. No organic arsenic longer the stay in town, the higher the arsenic concentration
species were found in any of the analyzed water samples. This found in hair. In the Illapata population, the total As concen-
result permits the conclusion that the Esquiña and Illapata tration in hair is highly correlated with the individual’s age.
populations are exposed almost exclusively to As(V) in drinking For individuals of Illapata, the correlation (R) between total
water, although intake of other arsenic species contained As in hair and age was 0.64, while the correlation of total As
through vegetable consumption is also possible. concentration in hair and the residence time in the village was
0.53. Fig. 2 presents the plotted correlation of total As in hair
between age and time of residence for the Illapata individuals.
Total arsenic in human hair
The total arsenic concentrations in hair samples from Esquiña Stability of As species during leaching from hair samples
and Illapata were determined using HG-ICP-MS after nitric
acid and hydrogen peroxide digestion in MW oven. For quality The stability of the arsenic species was studied during the
control, the total arsenic was determined in the hair CRM leaching procedure. Clean hair was separately incubated at
(GBW 09101 No 18), finding the value 0.62 0.06 mg g1 (N ¼ 90 1C with standard solutions of 100 mg L1 As(III), As(V) and
3), which agrees with CRM that contains 0.59 0.07 mg g1. DMA(V). No significant changes between As(III) and As(V)
The CRM was not washed prior to the total arsenic analysis. were observed up to 3 h of extraction at 90 1C. In samples
In Esquiña and Illapata, the average total arsenic values collected after 4 h of incubation, a partial oxidation of As(III)
were 0.7 and 5.8 mg g1, respectively. The median values for to As(V) (15%) and no reduction of As(V) to As(III) was found.
total arsenic are 0.4 and 4.7 mg g1, respectively. The values in After 6 h of incubation, 16% oxidation of As(III) to As(V) and
Illapata were around ten times greater than the accepted 12% reduction of As(V) to As(III) was found. DMA(V) results
normal values for non-exposed individuals (o0.5 mg g1). A stable up to 10 h incubation. Based on these results, hair
value greater than 1 mg g1 is considered an indication of sample leaching was performed at 90 1C for 3 h, excluding
chronic exposure and toxicity.18 In Esquiña, 8 of the 23 (35%) the possibility of species changes during extraction. Further
individuals studied presented As concentrations over the re- studies about the stability of arsenic species during the extrac-
ferenced concentration (0.5 mg g1) with 4 individuals (17%) tion procedure are presently being performed. The recovery of
arsenic with 3 h leaching at 90 1C in the human hair CRM
GBW 09101 No 18 (0.59 0.07 mg g1) was 66% (0.39 0.09
Table 1 Arsenic species in Esquiña and Illapata water sources
mg g1, N ¼ 3). For all samples (N ¼ 43), the mean recovery
using the same conditions was 66% 21 mg g1. Further
As in water/mg L1
improvements in extraction efficiency could be achieved by
Village Water source As(III) DMA MMA As(V) Sum controlling the size of the hair pieces.
Esquiña Waterfall 1 1.1 N.D. N.D. 72.9 74.0
Waterfall 3 1.0 N.D. N.D. 11.2 12.2 Arsenic species in human hair
Illapata Camarones river 5.0 N.D. N.D. 1247 1252
Waterfall 1 1.4 N.D. N.D. 47.3 48.7 The concentration of the arsenic species in water extracts of
N.D. ¼ not detected. human hair samples from Esquiña and Illapata was assessed.
As(III), As(V), MMA and DMA concentrations were measured
1338 J. Environ. Monit., 2005, 7, 1335–1341g1, respectively. Mean values of As(III) and As(V) were 2.64
and 0.27 mg g1, respectively. MMA(V) and DMA(V) were
detected in 71 and 24% of the samples from highly exposed
individuals with an average concentration of 0.16 and 0.04 mg
g1, respectively. An interesting result was that four of the five
samples where DMA(V) was detected, corresponded to chil-
dren. No conclusion can be made due to the small number of
samples. Table 2 presents the mean values of the four As
species for the two villages studied.
Fig. 3 presents a typical chromatogram of the extracted
species from hair of an exposed individual (male, 75 years
old) containing 15 mg g1 of total arsenic, distributed as 13.7
As(III); oD.L. (0.07) DMA(V); 0.15 MMA(V) and 1.14 As(V)
(all in mg g1). An unidentified As species was detected in hair,
eluting between MMA and As(V).
The arsenic species were also analyzed in human hair CRM
(GBW 09101 No 18), which was used for recovery and
accuracy studies. CRM was not washed prior to the speciation
analysis. Human hair CRM contains a certified total As
concentration of 0.59 0.07 mg g1. Nevertheless the concen-
tration of each arsenic species is not certified in CRM. Nowa-
days there is no available CRM of human hair containing
certified concentration of arsenic species. The concentrations
found in CRM were 0.14, 0.04 and 0.21 mg g1 for As(III),
MMA and As(V), respectively. The sum (0.39 mg g1) repre-
Fig. 2 Plotted regression between total arsenic concentration in hair sents only 66% extraction. This result confirms that the main
(mg g1) and (a) age of the sampled individual (years); (b) residence proportion of As in hair is due to inorganic species, and that
time (years) in Illapata.
organic species represent a minimum fraction. Recently, Raab
and Feldmann have reported the predominance of inorganic
using HPLC-HG-ICP-MS. The results indicate greater abun- arsenic.22 They also found low arsenic recoveries when using
dance of inorganic species (As(III) and As(V)), which repre- boiling water leaching (68.9%).
sented close to 98% of the total arsenic extracted from the hair Moreover, comparing the species distribution obtained in
samples in both populations. In contrast, the organic species of the exposed population and CRM, some differences have been
arsenic have lower concentrations, representing less than 2% of found. As(III) represents only 36% in CRM, contrasting with
the total arsenic in hair. As(III) was detected in hair extracts of 68 and 88% found in Esquiña and Illapata, respectively. The
95% of the individuals from the Esquiña who are exposed to a concentration of As(V) in the hair CRM represents 53% of the
lower arsenic concentration. In all the calculations of arsenic extracted arsenic in contrast to the 30 and 11% found in
species concentration in hair, we have assumed that the species Esquiña and Illapata, respectively. The higher proportion of
found in extracts represent the original species in the hair. As(V) could occur due to exogenous arsenic in CRM (no
Nevertheless changes of the original species can occur when previous washings were performed). No DMA was detected
arsenic is extracted. This issue is still not clear and further in this CRM, coinciding with the low concentration found in
studies are required. Considering that the concentrations in samples, where DMA was detected only in 5 of 43 samples.
extracts represent the original species in the hair, the As(III)
average concentration was 0.25 mg g1 (median value was 0.14
Correlation of arsenic species in human hair with exposure time
mg g1. As(V) was detected in 70% of the individuals with an
average concentration of 0.15 mg g1 (median value 0.11 mg The correlation between inorganic species concentration and
g1). Considering the methylated species, only MMA(V) was the exposure time of the highly exposed population of Illapata
detected in 40% of the samples with an average and media was assessed. Even though the number of sampled individuals
value of 0.02 mg g1. DMA(V) was not detected in any extract (N ¼ 43) is limited, the number of studied individuals repre-
from exposed individuals from Esquiña. sents an important proportion of the total population living in
In the highly exposed population from Illapata, both inor- the studied villages (45 and 35% in Esquiña and Illapata,
ganic arsenic species, As(III) and As(V), were detected in all respectively) validating interpretation regarding with those
extracts, presenting an average concentration 3.75 and 0.45 mg populations.
Table 2 Arsenic species (As(III), As(V), MMA(V) and DMA(V)) in hair of the Esquiña and Illapata populations
As in hair, mg/g average (min. max.)
Village Population As(III) DMA MMA As(V)
Esquiña (n ¼ 22) Children (n ¼ 11) 0.40 (N.D.–1.53) N.D. 0.01 (N.D.–0.02) 0.15 (N.D.–0.37)
Adults (n ¼ 11) 0.13 (0.02–0.71) N.D. 0.02 (N.D.–0.03) 0.17 (N.D.–0.41)
SD 0.37 0.01 0.11
Average Esquiña 0.26 N.D. 0.02 0.15
Illapata (n ¼ 21) Children (n ¼ 08) 2.15 (0.56–3.24) 0.18 (N.D.–0.35) 0.02 (N.D.–0.03) 0.18 (0.1–0.47)
Adults (n ¼ 13) 4.74 (0.23–13.72) 0.07 (N.D.–0.07) 0.05 (N.D.–0.15) 0.614 (0.21–1.52)
SD 3.87 0.11 0.04 0.38
Average Illapata 3.75 0.16 0.04 0.45
For calculation of averages, only values over the detection limits were considered.
J. Environ. Monit., 2005, 7, 1335–1341 1339Fig. 3 Typical chromatogram of water-extracted arsenic species from a highly exposed individual from Illapata (male, 75 years old and 60 years
residence time in Illapata). Insert shows reduced scale of the same chromatogram.
People in both villages have been drinking contaminated reduction of arsenic occurs, as occurs in the As-methylation
water as long as they have lived there. The degree of exposure pathways into the liver. Additionally, external contamination
can be considered to be proportional to the individual’s age due to As(V) increases the possibility of an interpretation error
and residence time in village (exposure time). For this popula- since almost 99% of the external arsenic (from the Camarones
tion, the calculated As(III) and As(V) concentrations in hair River) is in the form of As(V). Further experiments are required
correlates with the age of the sampled individuals, presenting a to explain this phenomenon. Additionally, another study
correlation coefficient of 0.6923 and 0.5678, respectively. The suggested the limitations of hair analysis in exposure assess-
correlations of As(III) and As(V) with the time of residence in ment.31 According to our results, As speciation in human hair
Illapata are 0.7109 and 0.5799, respectively. Fig. 4 shows the offers more complete analytical information that should permit
plotted regression of As(III) and As(V) in relation with the age better assessment in As-exposure studies.
and time of residence for the Illapata population. For the
studied population, As(III) provided a better correlation than
the total arsenic and As(V). The better correlation between
As(III) and time of residence indicates that As(III) concentration Conclusions
in hair should be more closely related with the degree of In both villages, the arsenic concentration in drinking water
exposure in the studied individuals. The explanation for this surpasses the recommended WHO value (10 mg L1). The
phenomenon is still not clear, although it is likely that the populations of Esquiña and Illapata are exposed to As-con-
explanation depends on understanding the arsenic inclusion taminated drinking water exceeding between 7.5 and 125 times
processes in hair. It is not known, if during inclusion in hair, the international referenced values. The result of arsenic
Fig. 4 Correlations of As species in hair in highly exposed population of Illapata. Plotted regression between: (A) As(III) in hair (mg g1) and age of
the sampled individual (years); (B) As(III) and residence time (years) in Illapata; (C) As(V) and age of the sampled individual (years); (D) As(V) and
residence time (years).
1340 J. Environ. Monit., 2005, 7, 1335–1341speciation in water sources shows that theses populations are 8 D. J. Thomas, M. Styblo and S. Lin, Toxicol. Appl. Pharmacol.,
principally exposed to inorganic arsenic, mainly As(V). 2001, 176, 127–144.
Arsenic species found in water extracts of human hair are 9 National Research Council (NRC), Subcommittee on Arsenic in
Drinking Water, Arsenic in Drinking Water, National Academy
mainly inorganic arsenic (close to 98%), where As(III) is the Press, Washington, DC, 1999, vol. 2, pp. 16–25.
principal species. This high proportion of inorganic arsenic was 10 M. Ma and X. Le Chris, Effects of arsenosugar ingestion on
found in both studied populations. Considering that the con- urinary arsenic speciation, Clin. Chem., 1998, 44(3), 539–550.
centrations in extracts represent the original species in the hair 11 G. Samantaa, U. K. Chowdhurya, B. K. Mandala, D. Chakra-
(no changes during extraction procedure), mean concentra- bortia, N. C. Sekaranb, H. Tokunagab and M. Ando, High
tions of As(III) were 0.26 mg g1 (68%) and 3.75 mg g1 (88%) performance liquid chromatography inductively coupled plasma
mass spectrometry for speciation of arsenic compounds in urine,
in Esquiña and Illapata, respectively. As(V) was found as well Microchem. J., 2000, 65, 113–127.
in hair extracts, although at lower concentration than As(III) in 12 X. Le Chris, X. Lu, M. Ma, W. R. Cullen, H. V. Aposhian and B.
both studied populations with values of 0.15 mg g1 (30%) and Zheng, Speciation of key Arsenic methabolic intermediates in
0.45 mg g1 (10.5%) in Esquiña and Illapata, respectively. human urine, Anal. Chem., 2000, 72, 5172–5177.
Further studies are necessary to have a better understanding 13 L. M. Del Razo, M. Styblo, W. R. Cullen and D. J. Thomas,
of possible changes during the extraction of original arsenic Determination of trivalent methylated arsenicals in biological
matrices, Toxicol. Appl. Pharmacol., 2001, 174, 282–293.
species from the hair.
14 Y. C. Chen, C. J. Amarasiriwardena, Y. M. Hsueh and D. C.
The presence of inorganic arsenic as the principal arsenic Christiani, Stability of arsenic species and insoluble arsenic
species in hair agrees with the arsenic distribution found in a in human urine, Cancer Epidemiol., Biomarkers Prev., 2002, 11,
hair CRM, although in CRM the major part of arsenic is 1427–1433.
As(V). Standard reference material of human hair with certified 15 Z. Gong, X. Lu, W. R. Cullen and X. Le Chris, Unstable trivalent
arsenic species is strongly needed for better analytical assess- arsenic metabolites, monomethylarsonous acid and dimethylarsi-
nous acid, J. Anal. At. Spectrom., 2001, 16, 1409–1413.
ment.
16 J. Yu, D. W. Yu, D. M. Checkla, I. M. Freeberg and A. P.
In the studied populations, total arsenic, As(III) and As(V) Bertolino, Human hair keratins, J. Invest. Dermatol., 1993,
concentration in hair correlated with the degree of exposure, 101(Suppl 1), 56S–59S.
but the As(III) concentration exhibits better correlation, with 17 J. T. Hindmarsh, Arsenic, its clinic and environmental signifi-
factors such as individual’s age and time of residence in the cance, J. Trace Elem. Exp. Med., 2000, 13, 165–172.
village. The correlations between total arsenic, As(III), As(V) 18 J. T. Hindmarsh, Caveats in hair analysis in chronic hair poison-
ing, Clin. Biochem., 2002, 35, 1–11.
and the age of the exposed individuals were 0.65, 0.69, 0.56,
19 B. K. Mandal, Y. Ogra and K. T. Suzuki, Speciation of arsenic in
respectively. The correlations with the time of residence were human nail and hair from arsenic-affected area by HPLC-induc-
0.54, 0.71 and 0.58, respectively. These results indicate that, at tively coupled argon plasma mass spectrometry, Toxicol. Appl.
least in the studied populations, As(III) was the most accurate Pharmacol., 2003, 189, 73–83.
indicator for chronic As(V) exposure. Because of the better 20 A. Shraim, S. Hirano and H. Yamauchi, Extraction and specia-
correlation found for As(III) in this study, arsenic speciation tion of arsenic in hair using HPLC-ICPMS, Anal. Sci., 2001, 17,
i1729–i1732.
appears as a promissory tool for more complete analytical 21 A. Raab, H. Hansen, L. Zhuang and J. Feldmann, Arsenic
assessment in epidemiological studies on arsenicism. Further accumulation and speciation analysis in wool from sheep exposed
studies are required to confirm our results using larger number to arsenosugars, Talanta, 2002, 58, 67–76.
of samples of exposed populations with well-characterized 22 A. Raab and J. Feldmann, Arsenic speciation in hair extracts,
arsenic intake. Organic arsenic species are unlikely indicators Anal. Bioanal. Chem., 2005, 381, 332–338.
since they are present in very low and variable concentrations 23 E. Hakala and L. Pyy, Selective determination of toxicologically
important arsenic species in urine by high performanceliquid
in comparison to inorganic arsenic and they were not detected
chromatography hydride generation atomic absorption spectro-
in all the samples. metry, J. Anal. At. Spectrom., 1992, 7, 191–196.
24 X. Le Chris, X. Lu and X. G. Li, Arsenic speciation, Anal. Chem.,
Acknowledgements 2004, 1, 27A–33A.
25 A. Shraim, S. Hirano and H. Yamauchi, Extraction and specia-
The authors acknowledge the Fulbright Alumni Initiative tion of arsenic in hair using HPLC-ICPMS, Anal. Sci., 2001, 17,
Award Program (AIA), Grant AIA-FY2001, for its financial i1729–i1732.
support, and the cooperation of the University Research 26 X.-Y. Wei, C. A. Brockhoff-Schwegel and J. T. Creed, Application
of sample pre-oxidation of arsenite in human urine prior to
Institute for Analytical Chemistry (URIAC), Amherst, MA,
speciation via on-line photo-oxidation with membrane hydride
USA. generation and ICP-MS detection, Analyst, 2000, 125, 1215–1220.
27 International Atomic Energy Agency (AIEA), Activation analysis
References of Hair as an Indicator of Contamination on Man by Environmental
Trace Element Pollutants, AIEA/RL/50, Vienna, 1978.
1 Element Speciation in Bioinorganic Chemistry, ed. S. Caroli, John 28 J. Morton, V. A. Carolan and P. H. E. Gardiner, Removal of
Wiley & Sons, Inc., New York, 1196, pp. 445–463. exogenously bound elements from human hair by various washing
2 B. K. Mandal and K. T. Suzuki, Arsenic round the world: a procedures and determination by inductively coupled plasma,
review, Talanta, 2002, 58, 201–235. Anal. Chim. Acta, 2002, 21759, 1–12.
3 Arica inserta en una región arsenical: El Arsénico en el Ambiente 29 E. M. Flores, A. P. F. Saidelles, J. S. Barin, S. R. Mortari and A.
que la Afecta y 45 Siglos de Arsenicismo Crónico, ed. L. Figueroa, Figueiredo Martins, Hair sample decomposition using polypro-
Editorial Universidad de Tarapacá, Arica, Chile, 1st edn, 2001. pylene vials for determination of arsenic by hydride generation
4 A. H. Smith, P. Lopipero, M. N. Bates and C. M. Steinmaus, atomic absorption spectrometry, J. Anal. At. Spectrom., 2001, 16,
Arsenic epidemiology and drinking water standards, Science, 1419–1423.
2002, 296, 2145–2146. 30 Y. Bohari, A. Astruc, M. Astruc and J. Cloud, Improvements of
5 World Health Organization (WHO), Guidelines for Drinking hydride generation for the speciation of arsenic in natural fresh-
Water Quality, 1993, Vol. 1, Recommendations. water samples by HPLC-HG-AFS, J. Anal. At. Spectrom., 2001,
6 Environmental Protection Agency, 40 CFR Parts 9, 141 & 142. 16, 774–778.
National Primary Drinking water Regulations; arsenic and Clari- 31 H. Yamauchi, K. Takahashi, M. Mashiko and Y. Yamamura,
fications to Compliance and New Source Contaminants Monitor- Biological monitoring of arsenic exposure of gallium arsenide- and
ing: Final rule, 2001. inorganic arsenic-exposed workers by determination of inorganic
7 M. Styblo, Z. Drobna, I. Jaspers, S. Lin and D. J. Thomas, arsenic and its metabolites in urine and hair, Am. Ind. Hyg. Assoc.
Environ. Health Perspect., 2002, 110(Suppl. 5), 767–771. J., 1989, 50, 606–612.
J. Environ. Monit., 2005, 7, 1335–1341 1341You can also read
