Journal of Exposure Science and Environmental Epidemiology (2006) 16, 138146

r 2006 Nature Publishing Group All rights reserved 1559-0631/06/$30.00

www.nature.com/jes

Biomonitoring of chromium for residents of areas with a high density

of electroplating factories
FENG-HSIANG CHANG,a,b SHU-LI WANG,a,c YEOU-LIH HUANG,d MING-HSIEN TSAI,e SHENG-TSUNG YUa
AND LOUIS W. CHANGa
a

Division of Environmental Health and Occupational Medicine, National Health Research Institute, Zhunan, Taiwan
Department of Information Management, Tzu Hui Institute of Technology, Pingtung, Taiwan
c
Graduate Institute of Occupational Safety and Health, Kaohsiung Medical University, Kaohsiung, Taiwan
d
Faculty of Biomedical Laboratory Science, Kaohsiung Medical University, Kaohsiung, Taiwan
e
Division of Basic Medicine, Graduate Institute of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan
b

The high density of electroplating factories in the geographic middle of Taiwan has prompted concern over the potential for exposure to harmful metals.
The present study aimed to determine the levels of chromium in whole blood (B-Cr) of residents living in the high vs. low factory-density areas, and to
examine the relations to gender and age. A total of 660 residents who had not moved within the 5 years preceding the study were sampled according to the
stratied random sampling approach, at ages 3544, 4554, and 5564 years, for both genders. Chromium determinations (n 641) were made using a
graphite furnace atomic absorption spectrometer. The geometric mean (95% C.I.) of B-Cr was 0.357 (0.340.38) mg/l. The International Federation of
Clinical Chemistry (IFCC) nonparametric 0.95 reference limits of B-Cr was estimated to be o0.905 mg/l. B-Cr levels decreased with increasing age.
Subjects in the areas with a high density (0.38 mg/l, 95% C.I.: 0.360.40) of electroplating factories had signicantly higher B-Cr levels, compared to
residents of the low-density (0.27, 0.250.30) areas and to the general population from western countries. The health signicance of the elevated B-Cr
remains to be determined.
Journal of Exposure Science and Environmental Epidemiology (2006) 16, 138146. doi:10.1038/sj.jea.7500445; published online 17 August 2005

Keywords: biomonitoring, chromium, whole blood, graphite furnace atomic absorption spectrometer, reference values.

Introduction
Metal contamination of agriculture soil has been documented
in Changhua County, a rural area of approximately
1074 km2 situated in the middle of the island of Taiwan
(ROCEPA, 2002). While the origin of the contaminants is
not known absolutely, the metal-work-related factories in the
region are suspect. Most of the industrial plants in Changhua
County are located in the northern region (e.g. Changhua
City, Hemei, Lugang, Sioushuei and Huatan) (Figure 1) and
served metal work, electroplating, and other metal surface
treatment industries. These plants have long been suspected
of discharging wastewater containing large amounts of heavy
metals including chromium into irrigation channels and rivers
(IDBMOEA, 2002; Lin et al., 2002; ROCEPA, 2002).
Chromium is found in several oxidative and physical forms
that differ substantially in their toxicological potency.

1. Address all correspondence to: Dr. S-L. Wang, Division of Environmental Health and Occupational Medicine, National Health Research
Institutes, 100 Shih-Chuan 1st Road, Kaohsiung 807, Taiwan, ROC.
Tel.: 886-7-3126772 ext. 4015. Fax: 886-7-3221912.
E-mail: slwang@nhri.org.tw
Received 31 December 2004; accepted 30 May 2005; published online 17
August 2005

Among the major oxidative states of chromium encountered

in occupational and environmental settings, chromium (VI)
is a procarcinogen that is reduced intracellularly to form
DNA-damaging species while chromium (III) is considered
to be essential in nutrition and for the maintenance of normal
glucose tolerance (Versieck and Cornelis, 1989; Chi, 1997;
Zhitkovich, 2002). Chromium (VI) does not itself bind to
DNA but is reduced to chromium (III) which does. The
binding of chromium (III) is insufcient to damage DNA in
vitro and it is supposed that damage arises from intermediates
in the reduction process, perhaps intermediate valence states
of Cr itself (Wetterhahn and Hamilton, 1989; Aiyar et al.,
1991; Standeven and Wetterhahn, 1991).
Exposure to chromium (VI) compounds has been
consistently found to be associated with an elevated incidence
of respiratory cancers and other adverse health effects
(IARC, 1990; Langardt, 1990; Sorahan et al., 1998). The
genotoxic potential of chromium (VI) has been conrmed in
animal experiments and in several cell-based assays (Biedermann and Landolph, 1990; Snow, 1992). Chromium (VI) is
the second most potent allergen after nickel and chromiumcontact allergies are frequently found among occupationally
exposed workers. The highest incidence of chromium
sensitivity is found in chromium-plating industry, manufacturing of mineral pigments, shipbuilding, textile industry,

Chromium biomonitoring in exposed populations

Chang et al.

High Factory-density Areas:

A

Changhua City

Hemei

Lugang

Sioushuei

Huatan

Changhua
County

Taiwan

Control Areas:
F

Hsicou

Erhshui

Figure 1. Study area of the chromium biomonitoring in middle Taiwan.

and cement-exposed builders (Baruthio, 1992). A previous

study conducted for the electroplating workers exposed to
chromic acid in middle Taiwan has shown the associated
health effects on immunological parameters (Kuo and Wu,
2002).
The present study aimed to determine the levels of
chromium in whole blood (B-Cr) of randomly selected
Changhua residents, assess the signicance of the metal
exposure and parameters including physical location, gender
and age, and to compare the determined chromium reference
values of Changhua residents with those from other countries.

Methods
Study Area and Subjects
Data of soil metal pollution was acquired from the
Environmental Protection Administration of the Republic
of China (ROCEPA, 1989, 1992, 2002) and data of factory
registration downloaded from an online database of Industrial Development Bureau Ministry of Economic Affairs
(IDBMOEA) at website: http://www.moeaidb.gov.tw/
Fidbweb/index.jsp. This allowed areas with both contamiJournal of Exposure Science and Environmental Epidemiology (2006) 16(2)

nated soil and both high and low concentrations of

electroplating factories to be identied.
Subjects were recruited from seven different townships of
Changhua County; Changhua City (A), Hemei (B), Lugang
(C), Sioushuei (D), Huatan (E), Hsicou (F), and Erhshui
(G) (Figure 1). Townships AE were considered representative of the environment encountered by the largest proportion
of the Changhua population. These ve townships containing
most of the metal-work-related factories in Changhua were
considered as the high factory-density areas. The other two
townships, F and G, were the control areas (the low factorydensity areas); few metal-work-related factories were located
in these townships.
The selection process also utilized population census data
of the Ministry of the Interior of the Republic of China. To
be eligible, subjects needed to be 3564 years of age and to
have resided in their present household for at least 5
consecutive years at the time of recruitment. By means of
stratied random sampling approach for each study area, we
further stratied the study population by gender into three
age groups (3544, 4554, and 5564 years) and 22 villages.
Five residents were selected in each stratum (2  3  22=132
strata), thus a total number of 660 subjects were recruited.
139

Chromium biomonitoring in exposed populations

Chang et al.

Home interviews were conducted with the candidates by

local public health nurses. Those who agreed to participate
were given an information sheet outlining the details of
procedures and related outcomes, and were asked to provide
formal written consents. Ethics approval for this study was
obtained from the Human Ethics Committee of the National
Health Research Institutes, Taiwan (approval no.
EC9109002). After obtaining informed consent, a questionnaire was administered by the interviewing nurse for the
purpose of collecting information on residential and occupational history, personal habits, lifestyle, and medical history.
All participants also provided a blood sample (see below),
which was collected by nurses accompanied by a physician at
a local cooperative health center.
A total of 649 people completed the questionnaire and
provided a blood sample. However, the sample volume was
insufcient for eight subjects. Thus, the study results were
based on 641 subjects. The general characteristics of the
study population are summarized in Table 1.

Table 1. Distribution of age and consecutive resident duration by

gender and township.

Sample Collection
Blood samples were collected as previously detailed (Cornelis
et al., 1996) to minimize contamination, deterioration, and
overestimation of chromium levels. The conventional use of a
needle and syringe and heparin anticoagulant was avoided, to
minimize their potential as contributors of chromium
(Versieck et al., 1982; Minoia et al., 1992; Christensen
et al., 1993). Instead, the BD Vacutainers Evacuated Blood
Collection System (Becton Dickinson, Franklin Lakes, NJ,
USA) was used to collect venous blood samples in 7-ml BD
Vacutainers tubes (for trace element tests) (REF 367735,
Belliver Industrial Estate, Plymouth, UK). All participants
were notied not to eat seafood for 3 days and to have fasted
for 10 h prior to specimen collection.
Blood samples were kept on ice during transport to the
laboratory (within 2 days). At the laboratory, samples were
aliquoted and frozen at 201C until analysis was carried out.
Trace metal analysis was conducted within 1 month of blood
collection.
Chemical Analysis
A method for the graphite furnace atomic absorption
spectroscopy analysis of diluted human whole blood (Huang
et al., 2000) was modied as detailed in Table 2. Chromium
analyses of the 641 whole blood samples were performed using
a PerkinElmer SIMAA 6000 Graphite Furnace Atomic
Absorption Spectrometer (GFAAS) with transversely heated
graphite furnace and Zeeman background correction (PerkinElmer, Bodenseewerk, Uberlingen, Germany). Automated
dilutions and injections were made with a PerkinElmer AS-90
autosampler. The spectrometer and autosampler were controlled by AA Winlab software (PerkinElmer).
Our experiments showed that the calibration curve
obtained by adding the standard in the different amounts
140

Township

Age (years)

Consecutive resident
duration (years)

Mean (SD)

Mean (SD)

Changhua City
Male
Female

171
86
85

49.4 (8.3)
49.6 (8.3)
49.1 (8.4)

20.2 (12.7)
21.7 (14.2)
18.7 (10.9)

Hemei
Male
Female

177
88
89

49.2 (8.5)
49.6 (8.8)
48.9 (8.2)

26.6 (16.5)
29.7 (18.2)
23.5 (14.0)

Lugang
Male
Female

117
57
60

49.5 (9.0)
49.8 (8.9)
49.3 (9.2)

31.3 (18.0)
36.2 (18.6)
26.7 (16.3)

Sioushuei
Male
Female

27
12
15

49.8 (8.6)
50.0 (8.9)
49.7 (8.6)

28.1 (14.9)
35.6 (14.7)
22.1 (12.5)

Huatan
Male
Female

29
10
19

52.1 (8.7)
51.7 (7.8)
52.3 (9.4)

15.8 (8.7)
16.3 (9.3)
15.5 (8.6)

Hsicou
Male
Female

60
30
30

49.7 (8.1)
50.3 (8.0)
49.0 (8.2)

32.4 (16.3)
39.0 (16.8)
25.7 (12.9)

Erhshui
Male
Female

60
28
32

51.3 (8.4)
52.2 (8.5)
50.5 (8.4)

23.3 (14.5)
26.0 (17.5)
20.9 (11.0)

Total
Male
Female

641
311
310

49.7 (8.5)
50.0 (8.5)
49.4 (8.5)

25.6 (16.0)
29.0 (17.8)
22.3 (13.4)

n number of subjects; SD standard deviation.

of chromium to the diluted samples were highly linear, at

least up to 10 mg/l. The slope of the standard addition
method for the ve-fold diluted whole blood was 0.0153,
which was very close to that of the aqueous standard. It
indicated that the standard addition method is not required
for the determination of chromium for the diluted whole
blood when using 5 mg Mg(NO3)2 as the matrix modier.
In this study, chromium standard solutions were prepared
by diluting the 1-g/l chromic acid stock solution in
deionized water to give nal concentrations of 0, 2, 4, 6, 8,
and 10 mg/l.
Table 3 summarizes the performance of the analytical
procedure in terms of recovery, precision, and method
detection limit (MDL) (see Appendix A).

Quality Control
In addition to the rigorous screening and clean sample
collection protocols that were observed, blood-based quality
Journal of Exposure Science and Environmental Epidemiology (2006) 16(2)

Chromium biomonitoring in exposed populations

Chang et al.

Table 2. Operating conditions for chromium determination by GFAAS.

Wavelength
Slit width
Current
Sample volume injected
Lamp type
Signal measurement
Background
Tube type
Gas

357.9 nm
0.7 nm
25 mA
20 ml
Hollow cathode lamp
Peak area
Zeeman background correction
Pyro/Platform
Argon and oxygen

Furnace program for chromium determination of whole blood (1 : 5)a

Step
Drying
Charring
Cooling
Ashing
Atomization
Clean-out

Temperature (1C)

Ramp (s)

Hold (s)

Gas ow (ml/min)

Gas type

110
130
700
20
1600
2400
2600
20
2500

1
5
5
5
10
0
1
1
1

30
45
10
5
30
5
5
5
5

250
250
50
250
250
0
250
250
250

Argon
Argon
Oxygen
Argon
Argon
Argon
Argon
Argon

Diluent 0.2% HNO3, matrix modier 5 mg Mg(NO3)2.

Table 3. Precision, accuracy and determination power of the GFAAS

determination of chromium in whole blood samples.
Item
Precision
Series 1 (2.5-mg/l chromium standard solution used),
n6
Series 2 (5.0-mg/l chromium standard solution used),
n6
Series 3 (10.0-mg/l chromium standard solution used),
n6
Pooled, n 18

Quality control item

Criteria

Calibration check
Blank sample analysis
Duplicate sample analysis

Correlation coefcient, r40.995

Signal o 2 LOD
Relative percent difference,
RPD(%) o25%
Relative error, RE(%): 725%
Recovery, R(%): 75125%

C.V. (%)

9.2
2.1
1.7

Quality sample analysis

Spiked sample analysis
LOD limit of detection.

5.5
Concentration
(mg/l)

Accuracy
Certied value
Measured value (mean7SD), n 9
Method detection limit (MDL)

Table 4. Criteria for the quality control analyses by GFAAS.

11.0a
11.671.0
0.133

n numbers of aliquots.
Seronormt trace elements whole blood (lot no. Ml1256, Billingstad,
Norway).
a

control (QC) materials were included to evaluate the

statistical control and accuracy of the experimental and
analytical procedures. Standard Reference Material (SRM),
SeronormTM Trace Elements Whole Blood (Lot Ml1256,
Billingstad, Norway) was used for QC evaluation. QC
analyses were performed once every 10 samples analyzed
Journal of Exposure Science and Environmental Epidemiology (2006) 16(2)

(except for calibration check) throughout the analysis. If any

QC analysis did not meet the requirements (Table 4), analysis
was halted and both the equipment and all aspects of the
analytical procedure were checked. Subsequently, the failed
QC analysis would be repeated once. If performance
standards were met, analysis continued. If standards were
not achieved, all results that had been obtained between the
successful and failed QC runs were discarded and reanalysis
of the affected samples was performed. In addition, during
the period of sample analysis, control charts were established
to monitor if any of the determinations exceeded the QC
limits based on the statistical probability. The run was
rejected when any of the following conditions occurred. First,
one control observation exceeded the limit of mean73 SD
(standard deviation). Second, two consecutive different
controls exceeded the limit of mean72 SD. Third, 10
consecutive control observations felled on the same side of
the mean.
141

Chromium biomonitoring in exposed populations

Chang et al.

Statistical Analyses
Data were calculated and analyzed by using EXCEL
(Microsofts EXCEL 2002, Redmond, WA, USA) and
SPSS software (Version 10.0;. SPSS, Chicago, IL, USA).
Descriptive statistics including arithmetic mean (AM), SD,
geometric mean (GM), median and percentiles were calculated. Concentrations below MDL were set to MDL/2 for
further data treatment. The normality and log-normality of
the results were evaluated by using the Kolmorogov
Smirnov statistics (KS test). One-way analysis of variance
(ANOVA) was used to test the hypotheses that there were no
differences in chromium levels between genders, age groups,
and residential areas. A Po0.05 (two-tailed) was considered
statistically signicant when testing the hypotheses.
The reference limits were estimated according to the
procedures recommended by International Federation of
Clinical Chemistry (IFCC) (Solberg, 1987). An established
protocol was used to reject outliers (Dixon, 1953). In the
latter test, if the observed value of D (the absolute difference
between an extreme observation and the next largest
observation) was equal to or larger than one-third of the
range R, the extreme observation would be deleted (Reed
et al., 1971). Nonparametric methods were preferred because
their estimation was robust to the distribution of the
measurement results. No lower reference limit was reported
if the 0.025 fractile fell below the MDL.

Results
Chromium Levels in Whole Blood
None of the distributions of B-Cr were found to be normal or
log-normal (Figure 2). Therefore, it was unsuitable to present
the results as the mean7SD due to the skewed distribution of
the value obtained. The skewed distribution of the measurement results suggested that nonparametric methods should be
adopted for the latter estimations of IFCC reference limits.
Table 5 summarizes the concentrations of B-Cr of the
Changhua population aged 3564 years. Approximately

300

S. D. = 0.39
Mean = 0.44
N = 641

200

5.5% of measured values for B-Cr were below the MDL

(0.133 mg/l). The estimated GM and the 95th percentile of BCr were 0.357 and 0.790 mg/l, respectively. The median value
and the GM were preferable because of the observed good
agreement between them. The B-Cr levels apparent in the
blood samples collected from the high factory-density area
were signicantly higher than that of the control area.
However, B-Cr levels tended to increase with decreasing age.

Calculated Reference Limits

The nonparametric 0.95 IFCC reference limit of B-Cr was
estimated to be o0.905 mg/l (Table 5).
Comparison between Data from the Literature
Compared with other general populations, the AM, GM and
IFCC reference limit of B-Cr were approximately two times
higher than the values of healthy population samples from
Italy, the United Kingdom and Spain (Table 6).
Inuencing Factors
The inuences of age group and residential area on B-Cr
levels were highly signicant (Po0.05), but there was no
signicant effect from the gender (Figure 3).

Discussion
Biological monitoring is an efcient tool of monitoring
individual exposure for many toxic elements. Unfortunately,
unequivocal reference levels cannot always be established.
Reference levels may differ between countries and regions,
and exposure to the particular noxious compound can change
over time due to uctuations in environmental exposure and
lifestyle changes. Thus, reference intervals must be established at regular intervals and with respect to the appropriate
inuence factors (Kristiansen et al., 1997). In addition, since
many factors inuence the biomonitoring results, characterization of the reference population and strict quality
assurance during sampling and chemical analysis are

120

S. D. = 0.29
Mean = -0.45
N = 641

100

Frequency

80
60
40

100

20
0
-1.3

0
0.2

1.7
3.2
B-Cr (g/l)

4.7

6.2

-1.4

-0.9

-0.4
0.1
Log (B-Cr) (g/l)

0.6

1.1

Figure 2. Percentage distribution of chromium in whole blood (B-Cr). None of the distribution of B-Cr was found to be normal or log-normal.
142

Journal of Exposure Science and Environmental Epidemiology (2006) 16(2)

Chromium biomonitoring in exposed populations

Chang et al.

Table 5. Chromium in whole blood of the study population from Changhua, Taiwan (3564 years).
N

n o MDL

10th

50th

90th

95th

Range

AM (SD)

GM (GSD)

CI GM

B-Cr (mg/l)

641

35

0.16

0.38

0.714

0.79

0.0676.47

0.441 (0.392)

0.357 (1.930)

0.340.38

o0.905

Years of age*
3544 years
4554 years
5564 years

205
213
223

5
15
15

0.198
0.15
0.157

0.415
0.38
0.35

0.76
0.713
0.694

0.905
0.778
0.75

0.0676.47
0.0672.60
0.0673.71

0.498 (0.522)
0.425 (0.312)
0.403 (0.308)

0.405 (1.835)
0.342 (1.997)
0.330 (1.925)

0.370.44
0.310.38
0.300.36

o0.930
o0.808
o0.775

Area*
High factory-density
Control

521
120

8
27

0.17
0.15

0.405
0.29

0.73
0.525

0.815
0.564

0.0676.47
0.0670.97

0.470 (0.423)
0.316 (0.161)

0.379 (1.938)
0.274 (1.771)

0.360.40
0.250.30

o0.930
o0.614

IFCC reference
limit

B-Cr chromium in whole blood; N sample size; MDL method detection limit (0.133 mg/l for B-Cr); n o MDL number of values below MDL (values
below MDL were set to MDL/2); 10th, 50th, 90th, 95th percentiles; AM arithmetic mean; SD standard deviation; GM geometric mean;
GSD geometric standard deviation; CI GM 95%-condence interval for GM.
*: Signicant parameter (Po0.05).
IFCC reference limit: nonparametric, 0.975 fractile is upper limit, 0.025 fractile is lower limit.
If the lower limit is below MDL only the upper limit is presented.

Table 6. Reference values for whole blood chromium concentration

obtained from the literature (mg/l)
Literature

Analytical
method

AM

Italy (n 519)
(Minoia et al., 1990)

GFAAS

0.23

Spain (n 144)
(Llobet et al., 1998)

ICPMS

United Kingdom
(n 134) (White and
Sabbioni, 1998)

GFAAS

0.19

GFAAS

0.44

Taiwan (n 641)
(this study)

GM

Reference value

0.090.75 (range)

0.2

0.11.1 (range)

0.10.45 (IFCC)

0.36

o 0.905 (IFCC)
o 0.1336.47 (range)

AM arithmetic mean; GM geometric mean; GFAAS graphite furnace atomic absorption spectrometer with Zeeman effect background
correction; ICPMS inductively coupled plasma F mass spectrometry;
IFCC reference limit.

extremely important (Minoia et al., 1990, 1992; Alessio,

1993; Vesterberg et al., 1993; Christensen, 1995). A recent
review on trace element reference values in blood, serum, and
urine in the Danish population (Poulsen et al., 1994) showed
several shortcomings in some studies, mainly due to a low
number of reference persons or insufcient characterization
of the reference group.
Some researchers suggested that urinary chromium
determination was a suitable approach for the biomonitoring
of workers exposed to high-level chromium and its
compounds, mainly due to the established relationships
between chromium exposure and chromium excretion, and
Journal of Exposure Science and Environmental Epidemiology (2006) 16(2)

the easier specimen collection than blood (Lewis, 1997;

Rosenberg and Harrison, 1997; Huang et al., 1999).
However, in recent studies whole blood and plasma were
found to be a more denitive gage of environmental
monitoring than urine chromium levels (Paustenbach et al.,
1997). In this work, we discussed blood chromium mainly
based on three reasons: rstly, our discussion focused on
general populations exposure, not workers. Theoretically,
for the general population investigated there should not be as
high-level exposures as workplace. Also, easiness of specimen
collection was not the only consideration we concerned;
secondly, blood analyses could provide information on not
only blood chromium concentration but also other biological
parameters (Huang et al., 1999). These parameters might be
useful for other researches; and thirdly, blood chromium was
recommended as a biomarker by institutes of occupational
health and safety in some countries as well as urinary
chromium (Harada, 1990; DFG, 1992; HSE, 1992).
The present study is important and valuable because of the
large number of people sampled, their precise characterization and the strict QC measurements enacted. In Taiwan,
there have been few comparable studies, except for those
dealing with blood lead levels of Taiwanese adults and school
children (Liou et al., 1996a, b; Wang et al., 2002; Yang et al.,
2002).
Despite the aforementioned care in sample collection and
analysis, the reference values of B-Cr estimated in this study
are not representative of the entire Changhua population.
This is because we selected subjects almost exclusively from
metal-contaminated and industrial areas, which may have
produced an overestimation of the reference values. Nevertheless, in the absence of alternative sources in Taiwan, it is
still valuable to compare these chromium reference values
with the intervals determined in other studies to clarify the
143

Chromium biomonitoring in exposed populations

Chang et al.

0.7

Whole blood Cr (g/l)

0.6
0.5
0.4
0.3
0.2
0.1
0

Male

Female

35-44 years

45-54 years

55-64 years

High
factorydensity area

Control
area

Influence factor

Figure 3. Geometric means of chromium in whole blood (mg/l) according to gender, age group, and residential area. Mean values795% condence
interval for mean. Age group and residential area signicant at Po0.05. Gender not signicant (ANOVA after logarithmic transformation).

differences between different populations and to identify

trends in the increase or diminution of environmental
exposure to chromium of toxicologically signicant levels of
chromium.
Regarding chromium measurement results below detection
limit, many researchers used half of the detection limit to
replace measurements below the detection limit (Christensen
et al., 1993; Llobet et al., 1998; Paschal et al., 1998; Seifert
et al., 2000). In Paschals study, calculation of the medians
and distribution statistics using DL/2 (half of the detection
limit) and DL/(21/2) as substitute data did not result in
estimates that were statistically signicant different, so the
substitution of DL/2 was used for all missing (less than
DL) data. Regarding the other researchers, White and
Sabbioni considered that if a large proportion of samples
gave values below the MDL, the median value could be
reported (White and Sabbioni, 1998). In addition, Barany
et al. (2002) did not reject or change any results below the
detection limit to avoid distorting the distributions, mean or
median values. In the present study, we did not exclude data
less than DL to avoid overestimating the chromium levels of
the Changhua population but used MDL/2 to replace
measurements below the MDL and made further statistical
analyses. However, only 5.4% of measurements in this work
were below the MDL (Table 5) and neither signicantly
distorted the distributions and descriptive statistics nor
changed the results of hypothesis testing.
A direct comparison of the situation in Changhua, Taiwan
with that in other areas of Taiwan or in other Asian countries
is difcult due to the limited number of large-scale and
general population studies. With regard to the human
biomonitoring data and the year of sampling, the results of
this present study can most fruitfully be compared with
144

several European studies (Minoia et al., 1990; Llobet et al.,

1998; White and Sabbioni, 1998), as listed in Table 6. Even
these comparisons are limited, however, given the variations
in populations, analytical methods and data treatment.
Presently, a statistically signicant correlation was found
between age group and B-Cr concentration, with an agerelated decrease evident. Until further information is
available on occupational history or exposure routes, the
basis of this decreasing trend remains unexplained. In
addition, statistical analysis showed B-Cr levels to be
signicantly higher in the high factory-density areas than in
the control areas. This observation was expected and seems
reasonable. However, further environmental risk assessments
are necessary to yield the scientic evidence linking the
factories to the observed environmental chromium levels.
In conclusion, this study provides valuable data for
environmentally related health monitoring and reporting.
The data serves as a basis to establish chromium reference
values to characterize the populations internal exposure to
environmental chromium contamination. It is anticipated
that the present results will lead to a revision of these
reference values and to the creation of companion reference
values for other substances of concern. Furthermore, despite
our observation of higher B-Cr levels in the Changhua
residents as compared to the general population from western
countries, it is still unknown if the B-Cr levels will be higher
than those of residents residing in other areas of Taiwan.
Selection of other appropriate counties as control areas to be
compared with Changhua may help distinguish whether or
not the higher B-Cr levels in Changhua is a local
phenomenon. Lastly, the question of whether the elevated
chromium levels indeed pose a health risk warrants further
investigation.
Journal of Exposure Science and Environmental Epidemiology (2006) 16(2)

Chromium biomonitoring in exposed populations

Acknowledgements
The nancial support (No. DOH91-TD-B04) of Department
of Health of the Republic of China is gratefully acknowledged. We thank the Changhua population for support and
participation in this study. We thank Dr. S. H. Liou and
Dennis P. H. Hsieh of the Division of Environmental Health
and Occupational Medicine, National Health Research
Institutes (Taiwan), for their valuable suggestions. We are
highly indebted to the Changhua Christian Hospital, the
Changhua Public Health Bureau and the public health
nurses, and the Changhua Environmental Protection Bureau
for their support. We thank Y.C. Wang, C.Y. Peng, C.J. Li
and H.Y. Yu for their valuable assistance in collecting the
samples, and to H.J. Wang for her technical assistance in
analytical work.
Disclaimer
The scientic content of this manuscript has been reviewed
and approved for publication by the Division of Environmental Health and Occupational Medicine of the National
Health Research Institutes. Approval for publication does
not necessarily signify that the content reects the view and
policies of the DEHOM/NHRI, or condemnation or
endorsement and recommendation for use on this issue
presented.

References
Aiyar J., Berkovits J., Floyd R.A., and Wetterhahn K.E. Reaction of chromium
(VI) with glutathione or with hydrogen peroxide: identication of reactive
intermediates and their role in chromium (VI)-induced DNA damage. Environ
Health Perspect 1991: 92: 5362.
Alessio L. Reference value for the study of low doses of metals. Int Arch Occup
Environ Health 1993: 65: S23S27.
Barany E., Bergdahl I.A., Bratteby L.E., Lundh T., Samuelson G., Schutz A.,
Skerfving S., and Oskarsson A. Trace element levels in whole blood and serum
from Swedish adolescents. Sci Total Environ 2002: 286: 129141.
Baruthio F. Toxic effects of chromium and its compounds. Biol Trace Elem Res
1992: 32: 145153.
Biedermann K.A., and Landolph J.R. Role of valence state and solubility of
chromium compounds on induction of cytotoxicity, mutagenesis and
anchorage independence in diploid human broblasts. Cancer Res 1990: 50:
78357842.
Chi X.Z. Microelement and Human Health. Chemical Industry Press, Beijing, 1997
p. 87.
Christensen J.M. Human exposure to toxic metals: factors inuencing interpretation of biomonitoring results. Sci Total Environ 1995: 166: 89135.
Christensen J.M., Holst E., Bonde J.P., and Knudsen L. Determination of
chromium in blood and serum. Evaluation of quality control procedures and
estimation of reference values in Danish subjects. Sci Total Environ 1993: 132:
1125.
Cornelis R., Heinzow B., Herber R.F., Christensen J.M., Poulsen O.M.,
Sabbioni E., Templeton D.M., Thomassen Y., Vahter M., and Vesterberg
O. Sample collection guidelines for trace elements in blood and urine.
IUPAC Commission of Toxicology. J Trace Elem Med Biol 1996: 10:
103127.
DFG (Deutsche Forschungsgemeinschaft). MAK- and BAT-Values. Report No.
28. DFG, VCH, Nurnberg, 1992.
Dixon W.J. Processing data for outliers. Biometrics 1953: 9: 7489.

Journal of Exposure Science and Environmental Epidemiology (2006) 16(2)

Chang et al.

Harada A. Biological monitoring in medical surveillance in Japan. In: FiserovaBergerova V., Ogata M. (Eds.). Biological Monitoring of Exposure to
Industrial Chemicals. ACGIH, Cincinnati, 1990, pp. 4554.
HSE (Health and Safety Executive). Biological Monitoring for Chemical
Exposures in the Workplace. UK HSE Guidance Note EH 56. London 1992.
Huang Y.L., Chen C.Y., Sheu J.Y., Chuang I.C., Pan J.H., and Lin T.H. Lipid
peroxidation in workers exposed to hexavalent chromium. J Toxicol Environ
Health 1999: 56: 235247.
Huang Y.L., Chuang I.C., Pan C.H., Hsiech C., Shi T.S., and Lin T.H.
Determination of chromium in whole blood and urine by graphite furnace
AAS. Atom Spectrosc 2000: 21: 1016.
IARC (International Agency for Research on Cancer). IARC Monograph on the
Evaluation of Carcinogenic Risk to Humans: Chromium, Nickel and Welding.
Vol. 49, IARC, Lyon, 1990, p. 677.
IDBMOEA (Industrial Development Bureau Ministry of Economic Affairs).
Handbook of the Integrated Pollution Prevention Techniques for Metal
Surface Treatment Industries F Electroplating Industry, Industrial Development Bureau Ministry of Economic Affairs, Executive Yuan, Republic of
China, Taipei, 2002.
Kristiansen J., Christensen J.M., Iversen B.S., and Sabbioni E. Toxic trace element
reference levels in blood and urine: inuence of gender and lifestyle factors. Sci
Total Environ 1997: 204: 147160.
Kuo H.W., and Wu M.L. Effects of chromic acid exposure on immunological
parameters among electroplating workers. Int Arch Occup Environ Health
2002: 75: 186190.
Langardt S. One hundred years of chromium and cancer: a review of
epidemiological evidence and selected case reports. Am J Ind Med 1990: 17:
189215.
Lewis R. Metals. In: LaDou J. (Ed.). Occupational and Environmental Medicine.
McGraw-Hill Companies, Inc., OH, USA, 1997, Chapter 27.
Lin Y.P., Teng T.P., and Chang T.K. Multivariate analysis of soil heavy metal
pollution and landscape pattern in Changhua County in Taiwan. Landscape
Urban Plan 2002: 62: 1935.
Liou S.H., Wu T.N., Chiang H.C., Yang T., Yang G.Y., Wu Y.Q., Lai J.S., Ho
S.T., Guo Y.L., Ko Y.C., Ko K.N., and Chang P.Y. Threeyear survey of
blood lead levels in 8828 Taiwanese adults. Int Arch Occup Environ Health
1996a: 68: 8087.
Liou S.H., Wu T.N., Chiang H.C., Yang G.Y., Yang T., Wu Y.Q., Lai J.S., Ho
S.T., Lee C.C., Ko Y.C., Ko K.N., and Chang P.Y. Blood lead levels in
Taiwanese adults: distribution and inuencing factors. Sci Total Environ 1996b:
180: 211219.
Llobet J.M., Granero S., Torres A., Schuhmacher M., and Domingo J.L.
Biological monitoring of environmental pollution and human exposure to
metals in Tarragona, Spain. Trace Elem Electrol 1998: 15: 7680.
Minoia C., Sabbioni E., Apostoli P., Pietra R., Pozzoli L., Gallorini M.,
Nocolaou G., Alessio L., and Capodaglio E. Trace element reference values in
tissues from inhabitants of the European community I. A study of 46 elements
in urine, blood and serum of Italian subjects. Sci Total Environ 1990: 95:
89105.
Minoia C., Pietra R., Sabbioni E., Ronchi A., Gatti A., Cavallieri A., and
Manzo L. Trace element reference values in tissues from inhabitants of the
European community. III. The control of preanalytical factors in the
biomonitoring of trace elements in biological uids. Sci Total Environ 1992:
120: 6379.
Paschal D.C., Ting B.G., Morrow J.C., Pirkle J.L., Jackson R.J., Sampson E.J.,
Miller D.T., and Caldwell K.L. Trace metals in urine of United States
residents: reference range concentrations. Environ Res 1998: 76: 5359.
Paustenbach D.J., Panko J.M., Fredrick M.M., Finley B.L., and Proctor D.M.
Urinary chromium as a biological marker of environmental exposures: what
are the limitations? Regul Toxicol Pharmacol 1997: 26: S23S34.
Poulsen O.M., Christensen J.M., Sabbioni E., and Van der Venne M.T. Trace
element reference values in tissues from inhabitants of the European
Community: V. Review of trace elements in blood, serum and urine and
critical evaluation of reference values for the Danish population. Sci Total
Environ 1994: 141: 197215.
Reed A.H., Henry R.J., and Mason W.B. Inuence of statistical method used on
the resulting estimate of normal range. Clin Chem 1971: 17: 275284.
ROCEPA. Survey of heavy metals in the soil samples. In: Statistics Ofce of
Environmental Protection Administration (Ed.). Yearbook of Environmental
Statistics Taiwan Area, the Republic of China. Environmental Protection
Administration of the Republic of China, Taipei, 1989.

145

Chromium biomonitoring in exposed populations

Chang et al.

ROCEPA. Survey of heavy metals in the soil samples. In: Statistic Ofce of
Environmental Protection Administration (Ed.). Yearbook of Environmental
Statistics Taiwan Area, the Republic of China. Environmental Protection
Administration of the Republic of China, Taipei, 1992.
ROCEPA (Environmental Protection Administration of the Republic of China).
Farmland Soil Heavy Metal Investigation and Contaminated Site Control
Plan, Environmental Protection Administration of the Republic of China
(Project No.: EPA-90-GA13-03-90A285) Taipei, 2002.
Rosenberg J., and Harrison R.J. Biological Monitoring. In: LaDou J. (Ed.).
Occupational and Environmental Medicine. McGraw-Hill Companies, Inc.,
OH, USA, 1997, Chapter 38.
Seifert B., Becker K., Helm D., Krause C., Schulz C., and Seiwert M. The
German environmental survey 1990/1992 (GerES II): reference concentrations
of selected environmental pollutants in blood, urine, hair, house dust, drinking
water and indoor air. J Expos Anal Environ Epidemiol 2000: 10: 552565.
Snow E. Metal carcinogenesis: mechanistic implications. Pharamacol Ther 1992:
53: 3165.
Solberg H.E. Approved recommendation on the theory of reference values Part 5:
Statistical treatment of collected reference values: determination of reference
limits. J Clin Chem Clin Biochem 1987: 25: 645656.
Sorahan T., Burges D.C., Hamilton L., and Harrington J.M. Lung cancer
mortality in nickel/chromium platers, 1946-95. Occup Environ Med 1998: 55:
236242.
Standeven A.M., and Wetterhahn K.E. Possible role of glutathione in chromium
(VI) metabolism and toxicity in rats. Pharmacol Toxicol 1991: 68: 469476.
Versieck J., Barbier F., Cornelis R., and Holst J. Sample contamination as a source of
error in trace element analysis of biological samples. Talanta 1982: 29: 973984.
Versieck J., and Cornelis R. Trace Elements in Human Plasma or Serum, CRC
Press, Orlando, FL, USA, 1989.
Vesterberg O., Alessio L., and Brune D. International project for producing
reference values for concentrations of trace elements in human blood and urine
F TRACY. Scand J Work Environ Health 1993: 19: 1926.
Wang C.L., Chuang H.Y., Ho C.K., Yang C.Y., Tsai J.L., Wu T.S., and Wu T.N.
Relationship between blood lead concentrations and learning achievement
among primary school children in Taiwan. Environ Res 2002: 89: 1218.
Wetterhahn K.E., and Hamilton J.W. Molecular basis of hexavalent chromium
carcinogenicity effect on gene expression. Sci Total Environ 1989: 86: 113129.
White M.A., and Sabbioni E. Trace element reference values in tissues from
inhabitants of the European Union. X. A study of 13 elements in blood and
urine of a United Kingdom population. Sci Total Environ 1998: 216: 253270.
Yang T., Wu T.N., Hsu S.W., Lai C.H., Ko K.N., and Liou S.H. Blood lead
levels of primary-school children in Penghu County, Taiwan: distribution and
inuencing factors. Int Arch Occup Environ Health 2002: 75: 528534.
Zhitkovich A. Chromium: exposure, toxicity, and biomonitoring approaches. In:
Wilson S.H., Suk W.A. (Eds.). Biomarkers of Environmentally Associated
Disease: Technologies, Concepts, and Perspectives. Lewis Publishers, Boca
Raton, 2002, Chapter 19.

146

Appendix A
1. According to our sample collection protocol, collect whole
blood from 5 to 10 nonexposed healthy persons and mix
the bloods collected.
2. Take seven aliquots from the above whole blood mixture.
Add chromium stock solution into each aliquot to prepare
seven samples at the same concentration near one-fth of
the lowest-concentration calibration standard.
3. Pretreat the seven samples and analyze them according to
the standard operation procedure, which is the same as
used for subjects specimens.
4. Calculate the standard deviation (Sa) of the seven
readings.
5. Take another seven aliquots from the above whole blood
mixture. Add chromium stock solution into each aliquot
to prepare seven samples at the same concentration of half
of the last additive concentration. Repeat Steps 3 and 4
and calculate another SD (Sb).
6. Use F-test to evaluate the two SDs (let S1 and S2 be the
larger and smaller ones between Sa and Sb). If
F S12 =S22 is less than 3.05, then use the following
equation to calculate the pooled standard deviation,
Spooled.
s
6S12 6S12
Spooled
12
7. If in Step 6 F is not less than 3.05, then repeat Steps 5 and
6 until F is less than 3.05.
8. Method detection limit (MDL) 2.681 Spooled/a. a is
the slope of the calibration curve.

Journal of Exposure Science and Environmental Epidemiology (2006) 16(2)