Arch Environ Contam Toxicol (2009) 57:639–650
DOI 10.1007/s00244-009-9345-4
Dioxin-Like and Endocrine Disruptive Activity
of Traffic-Contaminated Soil Samples
T. Šı́dlová Æ J. Novák Æ J. Janošek Æ P. Anděl Æ
J. P. Giesy Æ K. Hilscherová
Received: 1 August 2008 / Accepted: 11 May 2009 / Published online: 2 June 2009
Ó Springer Science+Business Media, LLC 2009
Abstract Pollution of surface soils by traffic, especially
along major highways, can be a significant issue. Numerous studies have demonstrated traffic to be an important
source of particulate matter and gas-phase organic air
pollutants that produce many types of deleterious effects.
This article brings original information about the presence
of contaminants with specific mechanisms of action in
traffic-influenced soils as determined by bioanalytical
approaches and instrumental analyses. The initial phase of
the study aimed to compare contamination of soils near
highways with those from reference localities, whereas the
second phase of the study investigated the influence of
traffic pollution in soils at various distances from highways. For the reference areas, forest soils contained greater
concentrations of 2,3,7,8-tetrachlorodibenzo-p-dioxin
equivalents (TCDD-EQs; 483 to 2094 pg/g) than did arable
T. Šı́dlová J. Novák J. Janošek K. Hilscherová (&)
RECETOX, Masaryk University, Brno, Czech Republic
e-mail: hilscherova@recetox.muni.cz
P. Anděl
Evernia s.r.o., Liberec, Czech Republic
soils (96 to 478 pg/g), which represent the relevant reference for the studied soils along highways. The total concentration of TCDD-EQs determined in the in vitro
transactivation assay ranged from 225 to 27,700 pg/g in
traffic-affected soils. The greatest concentration of TCDDEQs among the studied sites was observed in soils collected near highway D1, which is the primary thoroughfare
in the Czech Republic. The concentrations of TCDD-EQs
in roadside soils were the greatest and decreased with
increased distance from highways, and this spatial distribution corresponded with the levels of polycyclic aromatic
hydrocarbons (PAHs). Soils collected 100 m away from
highways in most cases contained concentrations of
TCDD-EQs similar to background values. Most TCDD-EQ
presence was caused by nonpersistent compounds in soils,
with a significant contribution from PAHs as well as other
unknown nonpersistent chemicals. Extracts from most soils
collected near highways exhibited antiestrogenic and in
some cases antiandrogenic activities; for several sites the
activity was also detected in soils farther from highways.
The presence of TCDD-EQs and antihormonal activity in
highway-affected soils points to traffic as a source of polluting compounds having specific effects.
J. P. Giesy
Department of Biology and Chemistry, City University of Hong
Kong, Hong Kong SAR, People’s Republic of China
J. P. Giesy
Biomedical Sciences and Toxicology Centre, University
of Saskatchewan, Saskatoon, SK, Canada
J. P. Giesy
Zoology Department, Center for Integrative Toxicology,
Michigan State University, East Lansing, MI 48824, USA
J. P. Giesy
Environmental Science Program, Nanjing University, Nanjing,
China
Pollution from traffic sources is frequently an important
issue in large city agglomerations, but it can also occur
along major highways. Traffic is connected with the
emission of dust, ie, particulate matter (PM) (de Kok et al.
2006), as well as gaseous pollutants, which can be transported to soil by both wet and dry deposition. Many of the
substances released from traffic are insoluble in water, have
high adsorption ability, and tend to bind to mineral and
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640
organic particulates in soil. The pollutants can be stored or
transformed in the soils or subsequently modified by soil
microorganisms (Wesp et al. 2000). Soils located near
major traffic routes can thus serve as media documenting
pollution from traffic sources. Soils are a relatively stable
matrix compared with air, they do not undergo rapid
changes according to actual weather conditions and thus
reflect longer-term contamination.
A number of studies have investigated the release of
pollutants from traffic into air (Klein et al. 2006). Combustion of fossil fuels also in vehicle engines is an
important source of a group of highly abundant pollutants
called ‘‘polycyclic aromatic hydrocarbons’’ (PAHs). PAHs,
which can be found in all compartments of the environment, are known to affect organisms through various
modes of action. In addition to PAHs, traffic can be a
source of their numerous derivatives and degradation
products as well as persistent organic pollutants (POPs).
Some of these contaminants, such as polychlorinated
biphenyls (PCBs) and polychlorinated dibenzo-p-dioxins
and dibenzofurans (PCDD/Fs) (Safe 1986), are hazardous
because of their toxicity and persistence. In addition, POPs
have nonpolar molecules and hence can accumulate in
adipose tissue and cause deleterious cellular effects. The
potential adverse effects of these compounds and their
environmental mixtures include teratogenicity, carcinogenicity (Muto et al. 1996), and effects on normal physiologic
endocrine function of an organism (Ankley et al. 1998).
Some of these contaminants can disturb signaling of cellular receptors, such as the aryl hydrocarbon (AhR) and
hormonal receptors (eg, estrogen receptor [ER], androgen
receptor [AR], glucocorticoid receptor). Effects mediated
via AhR caused by TCDD-(dioxin)-like compounds
(Whyte et al. 2004) include immune system and liver
function disorders as well as endocrine and nervous system
abnormalities (Mukerjee 1998). In particular, compounds
modulating endocrine regulation can influence reproduction or developmental processes (Kelce and Wilson 1997).
Exposure to diesel exhaust has been correlated with
adverse effects on the reproduction of rodents (Yoshida
et al. 1999; Watanabe and Kurita 2001; Li et al. 2006a) and
birds (Li et al. 2006b). Human fertility has been suggested
to be adversely affected by exposure to pollution from
traffic (de Rosa et al. 2003). Some studies have demonstrated in vitro estrogenic as well as antiestrogenic and
antiandrogenic effects of traffic exhaust particulates and
road dust (Kizu et al. 2003; Misaki et al. 2008; Ueng
and Wang 2004, Okamura et al. 2004; Taneda and Mori
2004).
Few studies exist regarding the potential influence of
traffic on soil contamination. One study that focused on
several major pollutant groups pointed to traffic as a
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Arch Environ Contam Toxicol (2009) 57:639–650
source of organic pollutants, such as PCDD/Fs, PCBs,
PAHs, and heavy metals in the affected soils (Benfenati
et al. 1992). Relatively great concentrations of POPs were
also found in soils near a heavily congested road in
northern Italy (Capuano et al. 2004), with the greatest
concentrations of PCDD/Fs occurring in surface layers of
B10 cm in depth. Bioassays have demonstrated estrogenic, androgenic and/or glucocorticoid-like, and dioxinlike activities in agricultural soils, which was partially
attributed to residues of pesticides, PCBs, and PAHs
(Kannan et al. 2003). In addition, that study indicated that
soil can serve as a secondary source of organochlorine
pesticides (OCPs) and reflect the history of pesticide use
in the area. Estrogenic and AhR-mediated activity were
also found in surface soils from Tianjin in China (Xiao
et al. 2006). The distribution of sites with estrogenic
activity was different than the distribution of sites with
dioxin-like activity, which was mostly observed in urban
areas.
In traffic-affected soils, contaminants are present as
complex mixtures of both known and unknown compounds with various toxic effects. In addition, some
compounds can act through multiple mechanisms of
action (Schrader and Cooke 2003). The interactions
among contaminants present in complex soil mixtures,
such as synergism, antagonism, or additivity, can also
modulate toxic potential (Hilscherova et al. 2000). For
example, some studies have reported additive or even
synergistic effects of estrogenic compounds (Payne et al.
2000; Bergeron et al. 1999).
In vitro bioassays are useful as integrative measures of
effects of individual chemicals or environmental complex
mixtures. These tests assess the total specific toxic potency
of complex mixtures and include interactions between
compounds (Hilscherova et al. 2000). The best characterization of contamination status is obtained by the combined
use of bioanalytic approach and instrumental analyses.
Instrumental analyses provide information on concentrations of selected priority compounds, whereas bioassays
characterize the overall presence of compounds and their
specific modes of action.
This study was conducted to determine if traffic can be a
source of pollutants with specific modes of action in soils
along highways. The study investigated contamination of
soils close to major highways, with a focus on compounds
having potential dioxin-like and hormonal effects. The
research focused on specific mechanisms of action,
including the signaling pathways of AhR, AR, and ER.
Another study goal was to compare residue concentrations
and their combined potential to interact with AhR, ER, and
AR in different types of soil (forest, arable soil) from a
reference area.
Arch Environ Contam Toxicol (2009) 57:639–650
641
Methods
Sample Collection
Sampling sites along major highways, where traffic intensity is regularly monitored (Table 1), were selected to
represent a range of roads with heavy traffic. In 2004, soils
Table 1 Traffic intensity (number of cars/d) in the SB and RS areas
in 2004 and in the CV and MB areas in 2005
Cars
Trucks
Total
2004
SB5
11813
320
12881
SB6
43242
5364
53350
SB7
51593
981
55229
SB8
37363
6307
47415
RS5
55384
2761
61693
RS6
RS7
34121
34011
8522
7023
47651
45011
RS8
83187
10580
99765
CV
21023
17009
38100
MB
24005
7158
31228
2005
were sampled near urban highways and at reference
localities in the broader Prague metropolitan area (Fig. 1).
The composite soil samples were collected in the areas
Ruzyne, Suchdol, and Brezineves in Central Bohemia in
December 2004. One set of sites was located between
Ruzyne and Suchdol (RS), and second set of sites was
located between Suchdol and Brezineves (SB). Eight
samples of arable soils and eight samples of forest soils
were collected in regions where highways will be built in
the future; these were chosen to be reference areas.
Another eight samples were taken immediately adjacent to
existing highways (0 to 1 m distance), and another eight
samples were taken from roadsides (approximately 20 m
away). The samples are labeled by location (RS or SB) and
by numbers 1–4 for reference sites (no highway), 5–8 for
sites along the highway.
In the next part of the study, another group of soil
samples was collected from regions along two major
highways in the Czech Republic in November 2005. The
first sampled region was along the main highway in Czech
Republic D1 in the area of Ceskomoravska Vysocina (CV),
and the second sampled region was along a highway near
the city Mlada Boleslav (MB) (Fig. 1). Samples were
collected from two transects in each region (CV1, CV2,
MB1, and MB2). The composite samples of soils were
Fig. 1 Map of the study sites
within the four regions along
major highways and reference
areas sampled in 2004 and 2005.
The 2004 samples were
collected in the broader area of
Prague. Circles indicate the sites
in area Ruzyne–Suchdol (RS),
and squares indicate sites in area
Suchdol–Brezineves (SB).
Filled symbols indicate sites
near existing highways, and
empty symbols indicate
reference localities. Black
triangles mark MB1 and MB2
(region of Mlada Boleslav) as
well as CV1 and CV2 (region of
Ceskomoravska vysocina), all
situated along two major
highways, where samples were
taken in 2005
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642
collected from distances of 100, 50, 20, and 0 to 1 m (ie,
immediately adjacent to highways). All samples were taken
from one side of each highway.
All soil samples were prepared as homogenized composite samples of five individual subsamples collected at
1 9 3–m sampling plots from 0- to 20-cm layers. Soil
samples were quickly transported to the laboratory in
polyethylene black bags and sieved through 2-mm mesh
(with the exception of a portion used for determination of
physicochemical properties). The soil samples were characterized for organic carbon content (total organic carbon
[TOC]) by a High Temperature TOC/TNb Analyzer
LiquiTOC II (Elementar Analysensysteme GmBH, Hanau,
Germany).
Extraction
Dried soil samples were extracted with high-purity dichloromethane (DCM; Burdick and Jackson, Muskegon, MI) by
use of a Soxtec apparatus. Extracts were concentrated to
approximately 5 ml by rotary evaporation and then to 1 ml
under nitrogen stream. A portion of the extracts was transferred to dimethylsufoxide (DMSO) for testing in the bioassays. The final concentration equivalent of extracts was
10 g soil/ml extract. A portion of each soil extract from year
2004 was treated with sulphuric acid to degrade the less
persistent compounds, such as PAHs, to determine the
contribution of persistent compounds to the bioassay
responses. One half of the extract was evaporated under
nitrogen and dissolved in 100 ll DMSO, and the second half
of the extract was vigorously mixed with 3 ml concentrated
sulphuric acid for 30 minutes to degrade less persistent AhR
ligands, such as PAHs. The layers were separated by centrifugation at 1000 g for 10 minutes after which the top
DCM layer was transferred into a clean tube. Mixing was
repeated after adding 4 ml DCM to the tube containing the
sulphuric acid layer. Finally, the top DCM layer was combined with the first fraction, and the samples were concentrated under nitrogen and dissolved in 100 ll DMSO.
Bioassays
The potency of extracts to elicit AhR receptor–mediated
responses was tested in a reporter gene transactivation
assay using a rat hepatoma cell line (H4IIE.luc) stably
transfected with the luciferase gene of firefly (Photinus
pyralis) under transcriptional control of dioxin-responsive
element. This bioassay is a well-established model for the
evaluation of dioxin-like activity (Sanderson et al. 1996).
Cells were maintained in medium containing 10% fetal calf
serum at 37°C in a humidified 5% CO2 incubator. Cells
were plated in 96-well microplates at a density of 15,000
cells/well. These plates were preincubated for 24 hours to
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Arch Environ Contam Toxicol (2009) 57:639–650
attach the cells in wells. The exposure to standard 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD) or soil extracts was
performed on the second day. All microplates contained
TCDD-calibration standards. Full dose-response curve was
established with final TCDD concentrations between 1.23
and 100 pM.
ER-mediated effects were assessed by use of the human
breast carcinoma cell line MVLN transfected with the ERlinked luciferase gene under control of estrogen-responsive
element (Willemsen et al. 2004). This cell line was cultivated in Dulbecco minimal essential medium (DMEM)/
F12 (Sigma–Aldrich) supplemented with 10% fetal calf
serum Mycoplex (PAA, Austria). MVLN cells were seeded
at a density of 15,000 cells/well. MVLN cells were
exposed in DMEM/F12 supplemented with 5% dialyzed
fetal calf serum, which was treated with dextran/charcoal
to further decrease background concentrations of estradiol.
Approximately 24 hours after plating, cells were exposed
to the tested extracts dissolved in DMSO and/or standard
17b-estradiol (E2, dilution series 1.23 to 100 pM; Sigma–
Aldrich, Czech Republic). Effects of soil sample extracts in
MVLN cells were assessed either singly or in combination
with competing endogenous ligand. Antiestrogenicity was
assessed by simultaneous exposure of the sample extract
and E2 (33.3 pM).
The final concentration of solvent did not exceed 0.5%
final volume in both bioassays. The extracts were tested in
triplicate and four dilutions to determine dose-response
curves. During exposure (24 hours), the plates were incubated at 5% CO2 and 37°C. Before measurement of luminescence, cells were checked for possible cytotoxicity. The
mixture of medium, buffer for lysis, and substrate for
luciferase (Promega Steady Glo Kit; Promega) was added
to the wells. After 10 minutes of incubation at room temperature, luciferase activity was measured as luminescence
produced using a microplate-scanning luminometer (Luminoscan Ascent). The intensity of luciferase luminescence
corresponded to the respective receptor’s activation.
Bioluminescent yeast assay was used for detection of
anti/androgenic activity of the soil sample extracts. The
assay is based on genetically modified yeast strain of
Saccharomyces cerevisiae stably transfected with humanandrogen receptor along with firefly luciferase under transcriptional control of androgen-responsive element (ARE).
This bioassay is a simple screening system for identification of the effects of complex environmental samples
because of its easy handling, suitability for large-scale
screening, high sensitivity, and low cost (Michelini et al.
2005; Gaido et al. 1997). Colonies of yeast inoculated onto
an agar plate were grown to 1 mm, and then yeast was
added to medium. The medium contained 6.7 g/l yeast
nitrogen base, appropriate amino acids, and carbon source.
The yeast was grown in this medium overnight at 30°C
Arch Environ Contam Toxicol (2009) 57:639–650
with shaking. Yeast culture, 100 ll, was plated into white
96-well microplates, and 1 ll of soil extracts or standard
testosterone (T) was added. Antiandrogenic activities of
soil extracts were tested with the addition of competitive
concentration of standard (10-8 M T); thus, the final concentration of the solvent did not exceed 2% v/v in a single
well. Plates were incubated at 30°C for 2.5 hours. Standard
calibrations were included in each plate. To obtain full
dose-response curves, we used T concentrations of 10-12 to
10-5 M. Every extract was tested in four dilutions, with
each of those done in three replicates. Substrate for luciferase (100 ll 0.1 mM D-luciferin) was added by automatic
dispenser in a luminometer (Luminoscan Ascent) (Michelini et al. 2005). Luciferase activity was measured 2 minutes after the addition of substrate. All samples were tested
with the control strain (luc) in parallel for possible cytotoxicity (Leskinen et al. 2005).
Chemical Analyses
Concentrations of indicator PCBs, PAHs, and OCPs were
assessed. Laboratory blank and reference material were
analyzed with each set of samples. Fractionation of the raw
extracts was achieved on silica gel column; sulfuric acid–
modified silica gel column was used for PCB and OCP
analyses. Samples were analyzed using a gas chromatographer (GC)–electron capture detector (Hewlett-Packard [HP]
5890) supplied with a Quadrex fused silica column 5% pH
for seven indicator PCB congeners and eight OCPs (a-HCH,
b-HCH, c-HCH, d-HCH, p,p0 -DDE, p,p0 -DDD, p,p0 -DDT,
HCH). Sixteen United States Environmental Protection
Agency (USEPA) PAHs (naphthalene, acenaphthylene,
acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene, pyrene, benzo(a)anthracene, chrysene, benzo(b)fluoranthene, benzo(k)fluoranthene, benzo(a)pyrene, dibenzo
(a,h)anthracene, benzo(ghi)perylene, and indeno(1,2,3-cd)
pyrene) were determined in all samples using a GC–mass
spectrometer instrument (HP 6890–HP 5973) supplied with
J&W Scientific fused silica column DB-5MS. The pollutants
were quantified using Pesticide Mix 13 (Dr. Ehrenstorfer)
and PAH Mix 27 (Promochem) standard mixtures. Terfenyl
and PCB 121 were used as internal standards for PAH and
PCB analyses, respectively. The limit of detection for studied compounds was 0.1 ng/g soil.
Quality Assurance and Control
Recoveries were determined by spiking samples with surrogate standards. Recovery of analytes varied from 88% to
103% for PCBs, from 75% to 98% for OCPs, and from
72% to 102% for PAHs. Recovery factors were not applied
to any of the data. Laboratory blanks always contained
\1% of the amount determined in the samples.
643
Data Analysis
Responses of the cell line H4IIE.luc caused by soil extracts
were compared with TCDD standard dose-response curves.
The values of responses from the bioassays were converted
to a percentage of the mean maximum response for the
TCDD standard (TCDDmax). Dioxin-like potencies of
mixtures were calculated as TCDD-EQs based on a
response equivalent up to 50% of the maximal response
produced by the standard (TCDDmax) (Villeneuve et al.
2000). Dioxin-equivalents derived from the chemical
analyses (TEQs) used relative potencies for PAHs
according to Machala et al. (2001). The values of hormonal
activities (antiandrogenicity, antiestrogenicity) were quantified as the percentage of response caused by competitive
concentration of appropriate standards. For the yeast
model, the results from AR-specific yeast strain were
normalized to the results from the constitutively luminescent strain to take into account the effects of the samples on
yeast propagation (Leskinen et al. 2005). However, the
results from sample dilutions that were considered cytotoxic were discarded from the data analyses.
Results
Comparison of Specific Activities in Soils Collected
in 2004 Near Highways and in Reference Areas
The first part of study compared the situation in forest and
arable soils from background region with soils close to
highways. The number of cars traveling on the studied
highways in 2004 ranged from 13,000 (SB5) to 100,000
cars/d (RS8) (Table 1). RS8 had one of the greatest densities of traffic in the Czech Republic. Traffic density was
comparable (from 45,000 to 60,000 cars/d) at most other
sites in both studied regions (SB and RS). Concentrations
of PAHs as well as PCBs and DDTs were greater in soils
immediately adjacent to highways than in soils collected
from 20 m away or from reference areas. Concentrations of
HCHs and hexachlorobenzene (HCBs) were similar to
those observed in the reference soils, and there was no
clear trend among localities. Concentrations of PAHs,
PCBs, and mostly also DDTs, as well organic carbon
content, were greater in forest soils than in arable soils
within the reference areas (Table 2).
Similarly, the dioxin-like potencies of extracts from
forest soils within reference areas were greater (B10-fold)
then those of arable soils from the same area (Fig. 2).
Because most of the samples collected 20 m from highways were arable soils, these soils were used as relevant
reference samples for comparison with the traffic-affected
sites. Samples collected at sites 20 m from highways
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644
Arch Environ Contam Toxicol (2009) 57:639–650
Table 2 Dioxin-like activity determined in in vitro bioassays presented as TCDD-EQs and concentrations of PAHs, PCBs and DDTs and
organic carbon content in soil samples collected in 2004
Samples
Nontreated
TCDD-EQs (pg/g)
H2SO4-treated
TCDD-EQs (pg/g)
PAHs (mg/kg)
PCBs (mg/kg)
DDTs (mg/kg)
Corg (%)
SB1 forest
900
2.9
1.47
0.014
0.022
7.2
SB1 arable
252
3.4
0.32
0.002
0.003
2.4
SB2 forest
SB2 arable
488
201
3.4
1.5
0.7
0.71
0.005
0.004
0.006
0.005
5.8
2.3
SB3 forest
483
7.0
0.33
0.005
0.028
3.8
SB3 arable
96
2.4
0.17
0.003
0.002
2.1
SB4 forest
1310
9.6
0.64
0.002
0.002
5.7
SB4 arable
153
3.1
0.22
0.001
0.001
3.5
SB5 0–1 m
2172
3.7
3.1
0.150
0.016
2.9
SB5 20 m
394
2.3
0.75
0.004
0.010
4.7
SB6 0–1 m
1961
2.9
3.7
0.021
0.007
2.4
SB6 20 m
225
2.9
0.4
0.188
0.010
3.4
SB7 0–1 m
4819
5.6
4.2
0.046
0.011
3.7
SB7 20 m
613
5.3
0.58
0.007
0.003
4.1
SB8 0–1 m
2546
5.1
2.2
0.019
0.007
2.2
SB8 20 m
930
3.2
1.25
0.004
0.027
3.2
RS1 forest
870
2.5
1.6
0.011
0.007
10.3
RS1 arable
RS2 forest
312
2094
2.9
3.4
0.46
3.1
0.005
0.016
0.008
0.029
2.0
8.3
RS2 arable
460
3.6
0.68
0.002
0.005
2.3
RS3 forest
923
2.9
1.5
0.017
0.043
8.4
RS3 arable
478
3.2
0.81
0.001
0.013
2.1
RS4 forest
1454
13.9
0.52
0.008
0.015
8.5
RS4 arable
232
1.6
0.69
0.002
0.004
2.3
RS5 0–1 m
4927
2.4
0.191
0.029
2.7
RS5 20 m
492
3.5
0.75
0.006
0.009
4.3
RS6 0–1 m
592
1.1
2.8
0.048
0.008
2.9
RS6 20 m
306
7.2
0.2
0.003
0.002
5.4
RS7 0–1 m
652
2.2
1.62
0.015
0.003
1.5
RS7 20 m
255
13.9
0.08
0.001
0.001
2.7
RS8 0–1 m
1366
7.8
0.78
0.010
0.003
3.1
RS8 20 m
1828
8.3
1.40
0.003
0.002
2.0
12.0
Concentrations of PAHs, PCBs, DDTs, and organic carbon content in the studied samples
showed significantly lower concentrations of TCDD-EQs
than soils collected immediately adjacent to highways
(3- to 8-fold), but they were still greater than those
observed for the arable soils in reference areas at most sites
(Table 2).
The relatively great AhR-mediated potency of soils
decreased after treatment with sulphuric acid (Table 2).
The proportion of TCDD-EQs that consisted of persistent
compounds was[20 times less than the total concentration
of TCDD-EQs in all samples. In some cases (eg, samples
collected immediately adjacent to highways at RS5), the
proportion of TCDD-EQs contributed by persistent AhRactive compounds was \0.1%.
123
There was a significant correlation between concentrations of TEQs, which were calculated from concentrations
of individual PAHs and their respective REP values, and
concentrations of TCDD-EQs obtained from in vitro assay
(Fig. 3). The concentrations of TEQs calculated based on
concentrations of the 16 priority PAHs established by the
USEPA were approximately three-fold less than the total
concentration of TCDD-EQs, which suggests the presence
of other AhR-active compounds.
There was no androgenicity in any of the soils, whereas
significant antiandrogenic potencies were observed mainly
in soils from traffic-affected regions. Weak antiandrogenic
effect was detected in two forest soils in reference areas,
Arch Environ Contam Toxicol (2009) 57:639–650
Fig. 2 Dioxin-like activities of
the different types of soil
samples (forest soils, arable
soils, soils adjacent to
highways, and soils 20 m away
from highway) collected in
2004 and determined by
H4IIE.luc bioassays
645
6000
TCDD-EQ (pg/g)
5000
4000
3000
2000
1000
0
forest
highway (0-1m)
arable soil
20m distance
SB
log TCDD-EQ (pg/g)
4
forest
highway (0-1m)
arable soil
20m distance
RS
highways exhibited estrogenic effects, whereas no estrogenicity was found in soils from reference areas.
3.5
R2 = 0.67
3
Soil Contamination with Increasing Distance
from Highway (2005)
2.5
2
1.5
1
1.5
2
2.5
3
3.5
log TEQ chem.calculated (pg/g)
Fig. 3 Correlation between log-TEQs calculated from the results of
chemical analyses, and log-TCDD-EQs determined by H4IIE.luc
bioassays
which were remote from any highways. Antiandrogenicity
was observed in soils taken immediately adjacent (0 to
1 m) to highways in area SB (sites SB5 and SB6) and from
the highway in area RS (site RS5). At a 20-m distance from
highways there was significant antiandrogenicity only in
soil from SB5 (Fig. 4a).
The prevailing effects on the interaction of samples with
ER signaling were antiestrogenic. There was no antiestrogenicity in extracts of arable control soil. Some forest
soils from reference areas showed weak antiestrogenic
potency (approximately 75% of the response of competitive concentration of standard). Forest sample RS1 had the
greatest antiestrogenic potency: approximately 30% of
response of corresponding equivalent concentration of
standard alone.
Most samples collected adjacent to highways (SB5,
SB7, and SB8 as well as RS5 and RS7) and some samples
from 20 m away (SB7 as well as RS5 and RS8) exhibited
greater antiestrogenicity (Fig. 4b) compared with reference
arable soils. One sample collected immediately adjacent to
highways (RS6) and several samples collected 20 m from
This part of study assessed traffic pollution in surface soils
with increasing distance (0 to 1, 20, 50, and 100 m) in two
areas (CV and MB) along highways with average traffic
intensities for the Czech Republic. The average transport
density was 38,100 vehicles/d for CV and 31,228 vehicles/
d for MB (Table 1), which corresponds to the mean traffic
intensity of 31,690 vehicle/d in the Czech Republic in
2005.
AhR-mediated potency, as well as concentrations of
most of the pollutants, exhibited a pattern consistent with
traffic being the source of surface soil contamination.
Similarly to the samples from 2004, concentrations of
PAHs, PCBs, and DDTs were greater in soils collected
immediately adjacent to highways than in soils collected
20 m away or in reference areas (Tables 2 and 3), whereas
concentrations of HCHs and HCBs showed no clear trend.
Generally, concentrations of PAHs in soils collected
adjacent to highways in 2005 were greater than those
collected the previous year. However, this difference was
not obvious in soils collected from 20 m away. The
greatest concentration of PAHs was observed in soils
adjacent to highways, with concentrations decreasing with
increasing distance from highways.
Similarly, the greatest dioxin-like potencies were found
in samples taken immediately adjacent to highways, and
there was a dramatic decrease in TCDD-EQs at more distant sites (Table 3). A milder distance-related degressive
trend was found only for the second transect from the
region of MB. Concentrations of TCDD-EQs in soils from
the most distant sites (100 m from highways) were
123
646
Arch Environ Contam Toxicol (2009) 57:639–650
Fig. 4 a Antiandrogenic and b
antiestrogenic activities of soil
samples collected in 2004 from
two areas along highways. The
first area is SB, and the second
area is RS. Samples were
collected next to highways
(0–1 m) and 20 m away.
Responses are expressed as
percentage of response of
competing concentration of
standard T (10-8 M) and E2
(3 9 10-11 M), respectively
% of response of T (10-8M)
(a)
200
180
160
140
120
100
80
60
40
20
0
competitive concentration of testosterone (T)
0-1 m
20 m
control
% of response of E2 (3*10-11M)
(b)
200
SB5
SB6
SB7
SB8
RS5
RS6
RS7
RS8
RS6
RS7
RS8
competitive concentration of 17β-estradiol (E2)
180
0-1 m
160
20 m
140
120
100
80
60
40
20
0
control
SB5
SB6
SB7
SB8
RS5
samples
Table 3 Dioxin-like activity determined in in vitro bioassays presented as TCDD-EQs and concentrations of PAHs, PCBs and DDTs in the soil
samples collected in 2005
Samples
TCDD-EQs (pg/g)
PAHs (mg/kg)
PCBs (mg/kg)
DDTs (mg/kg)
CV1 0–1 m
27700
14.3
0.088
0.025
0.001
CV1 20 m
802
0.24
0.001
CV1 50 m
671
0.23
0.004
0.001
CV1 100 m
487
0.73
0.001
0.003
CV2 0–1 m
10214
0.13
0.025
CV2 20 m
2782
0.005
0.001
10.2
0.32
CV2 50 m
415
2.5
0.024
0.080
CV2 100 m
333
1.66
0.008
0.025
MB1 0–1 m
6807
9.6
0.078
0.029
MB1 20 m
MB1 50 m
715
1511
0.70
1.12
0.031
0.022
0.011
0.060
MB1 100 m
301
0.92
0.012
0.038
MB2 0–1 m
11713
4.2
0.20
0.034
MB2 20 m
7608
0.13
0.017
0.001
MB2 50 m
4590
0.24
0.002
0.002
MB2 100 m
650
0.27
0.012
0.003
Concentrations of PAHs, PCBs and DDTs in the studied samples
comparable with values observed in extracts from arable
soils in the background area in 2004, with somewhat
greater concentrations along transect MB2. Concentrations
of TCDD-EQs in soils collected 50 m from highways were
123
greater than those in soils collected 100 m away. Concentrations of TCDD-EQs in soils collected adjacent to
highways in 2005 were greater than those collected from
roadside soils during the previous year. The greatest
Arch Environ Contam Toxicol (2009) 57:639–650
(a)
% of response of T (10-8M)
Fig. 5 a Antiandrogenic and b
antiestrogenic activities of soil
samples collected in 2005 at
various distances from highway
D1. Two transects are from CV,
and two transects are from MB.
Responses are expressed as
percentage of response of
competing concentration of
standard T (10-8 M) and E2
(3 9 10-11 M), respectively
647
200
180
160
140
120
100
80
60
40
20
0
competitive concentration of testosterone (T)
0-1 m
20 m
50 m
100 m
(b)
% of response of E2 (3*10-11M)
control
200
180
160
140
120
100
80
60
40
20
0
CV1
CV2
MB1
MB2
competitive concentration of 17β-estradiol (E2)
0-1 m
20 m
50 m
100 m
control
CV1
CV2
MB1
MB2
localities
concentration of TCDD-EQs (27,700 pg/g dry weight
[dw]) was found in soils adjacent to the main highway of
the Czech Republic D1.
None of the samples from 2005 showed androgenic
potency, whereas antiandrogenic potency was observed in
soils adjacent to highways (Fig. 5a). Greater antiandrogenic potency of soils collected adjacent to highways was
observed in the area of CV than in the MB region. No
antiandrogenicity was observed in soils collected at greater
distances from highways.
No estrogenic effects were found in any soil. The
greatest antiestrogenicity was measured in soils collected
adjacent to highways. Soils from the MB region also
exhibited antiestrogenic potencies at greater distances from
the highway, whereas there was less activity in the samples
more distant from the highways in the CV region (Fig. 5b).
Discussion
Soil is a relatively stable environmental medium that
integrates the longer-term influences of pollution, thus
reflecting the pollution status of a region. Therefore, soils
along roads can serve as a medium for the storage of
pollutants from traffic and reflect long-term pollution
effects caused by contamination from traffic. This fact has
been clearly demonstrated by the greatest presence of the
compounds with specific modes of action as well as the
traditionally studied pollutants in soils from sites adjacent
to highways.
Concentrations of approximately 20 ng/g PCBs in soils
adjacent to highways are comparable with concentrations
from industry-polluted areas. Samples from regions with
heavy traffic (RS5, SB5 [20 m away], and SB6 [adjacent to
highway]) were among the most PCB-contaminated soil
samples. The greatest measured concentrations, which
were approximately 200 ng PCB/g, were considerably
high, even for industrial areas (Holoubek et al. 2000).
Therefore, it is likely that the PCBs did not originate from
general traffic but rather from transported materials or
other sources.
The results of our study have shown a dramatic decrease
in all studied specific activities and pollutant concentrations in soils within as few as 20 m from highways. This
corresponds with results of a study of roadside soils in
Italy, in which a significant decrease in concentrations of
PAHs, PCBs, PCDDs, and heavy metals was observed in
soils as few as 10 m from highways (Benfenati et al. 1992).
Concentrations of PAHs were approximately 1,000-fold
greater in soils adjacent to Czech highways than those
adjacent to Italian highways that were studied. This may be
due to the greater intensity of traffic at the Czech sites
(11,500 to 18,000 vehicles/d in the Italian study compared
with 13,000 to 100,000 vehicles/d for the studied Czech
highways; Table 1). In contrast, concentrations of PCBs
were similar in soils from both the Czech and Italian
123
648
studies. This fact suggests that traffic emissions are likely
not the primary source of PCB contamination.
Concentrations of TEQs contributed by the 16 USEPA
priority PAHs were correlated with the concentrations of
TCDD-EQs; however, concentrations of TEQs based on
PAHs were three-fold less than those of TCDD-EQs. The
results of our investigation show a major contribution of
the nonpersistent fraction to TCDD-EQs, with a significant
contribution made by PAHs as well as also some other
nonpersistent compounds. PAH derivates and humic substances probably belong among these compounds (Bittner
et al. 2006).
TCDD-EQ concentrations were greater in soils collected
immediately adjacent to highways than in soils collected
20 m away; thus, the influence of traffic is evident. The
release of numerous organic pollutants, some of them with
significant dioxin-like potency, into the atmosphere, has
been linked to traffic (Ciganek et al. 2004). Lower
molecular–weight PAHs were distributed mostly into the
gaseous phase. Nitrated PAHs, mainly nitronaphthalens,
were associated with particulate matter (PM10) (Ciganek
et al. 2004). The compounds present in PM, such as PAHs
and their derivatives, are to a large extent responsible for
the AhR-mediated potency of PM.
Some soils with greater concentrations of PAHs also
exhibited greater antiandrogenic and antiestrogenic
potency. Activation of AhR by ligands, such as PAHs, can
influence concentrations of hormones, their metabolism,
and their receptors. Diesel exhaust particles have been
shown to posses antiandrogenic potency (Taneda and Mori
2004). PAHs, such as benzo[a]pyrene, may be responsible
for these endocrine effects (Okamura et al. 2004). Extracts
from motorcycle exhaust particles, which should at least
partly represent traffic-derived contamination, were antiestrogenic both in vitro in MCF-7 cell line as well as in
vivo in immature female rats (Ueng and Wang 2004).
Antiestrogenicity was probably produced by AhR-dependent cytochrome induction because it could be eliminated
by cotreatment with AhR and the cytochrome P450
inhibitor a-naphthoflavone. This finding concurs with the
fact that there is direct link between dioxin-like activity
and antiestrogenicity (Safe and Wormke 2003). Our recent
study found greater concentrations of compounds with
antiestrogenic and AhR-mediated activities in air samples
from traffic affected areas compared with two other regions
(Novák et al. 2009). Testing has confirmed the presence of
chemicals, such as PAHs and their derivates. PAHs and
their analogues, such as nitroderivates, belong among the
main traffic contaminants. Others studies have also demonstrated that PAHs and their derivates can be connected to
antiestrogenicity (Chaloupka 1993).
The observation of greater contamination by the studied
pollutants, as well as greater TCDD-EQs in soils collected
123
Arch Environ Contam Toxicol (2009) 57:639–650
immediately adjacent to highways in 2005, did not correspond with overall traffic intensity, which was greater for
the areas sampled in 2004 compared with those sampled in
2005. However, the number of trucks per day was similar
(MB) or greater (CV) in the areas sampled in 2005 compared with those sampled in 2004. In general, there was a
greater proportion of heavy trucks in areas sampled in 2005
than in areas sampled in 2004, namely in the CV region,
where the number of trucks was almost as great as the
number of cars. The two regions sampled in 2005 differed
in the proportion of trucks, which was 45% in the CV
region and 23% in the MB region (the average proportion
for the Czech Republic is 41%). In contrast, trucks represented only 2.5% to 25% of the total vehicles in the sampled area in Prague metropolitan region (Table 1). This
indicates that not just the number of passing vehicles but
also the types of vehicles can strongly influence trafficrelated pollution. Another contributing factor can be the
specific way in which contamination is released into the
soil. The greatest concentrations of residues and potencies
in the three assays were observed in soils sampled immediately adjacent to highways. There are two likely major
sources of this contamination: (1) emissions from fuel
combustion and (2) dust, spills, or releases from vehicles
and transported materials, which, directly or by way of rain
water washout, are transported to roadsides. A causal
relation can be expected between combustion emissions
and traffic intensity, which is the base for the widely used
application of emission load modeling. However, this is not
true for washout from roads, which is related to accidental
releases and spills. The dominant role of this second cause
is confirmed by the great differences among soil contamination values found by roadsides and from those from 20 m
away. They do not correspond to the distribution of emissions because approximately 90% of roadside values would
be expected at the 20-m distance from highway according
to common emission models.
TCDD-like potency, as well as concentrations of individual pollutants in forest soils from background regions,
was greater than that found in arable soils from the same
regions. Background contamination can be contributed by
city pollution because the reference locations are not
directly influenced by traffic but are affected by the nearby
city agglomeration.
The difference between forest and arable soils can be
related to the greater content of organic matter in forest
soils (Table 2). The organic matter content of soils influences biologic processes as well as the fate of pollutants.
The amount and quality of organic carbon matter is an
important parameter regarding the binding of organic pollutants to solid materials (Jaffe 1991) and thus their
potential accumulation. Another possible explanation for
the greater presence of TCDD-EQs in forest soils is the
Arch Environ Contam Toxicol (2009) 57:639–650
soils’ greater content of humic substances. It has been
shown that some humic substances can elicit dioxin-like
potencies; thus, if these compounds are present in greater
amounts, they could significantly contribute to observed
activity (Bittner et al. 2006). In addition, the regular
plowing of arable soils can contribute to the transfer of the
pollutants to the deeper soil layers and thus to lower contamination in the surface soils.
Soil characteristics can also be important parameters
influencing the amount of POPs and other pollutants. These
parameters include the quantity and quality of organic
carbon content as well as the texture; fine soil particles are
known to contain the greatest concentration of POPs
adsorbed onto their surface (Perez et al. 2007). The greater
content of clay particulates increases the adsorption of
organic pollutants in soil. In addition, the fate, mobility,
and half-life of pollutants in soils can be influenced by soil
types and horizons, pH, redox status, and meteorological
conditions. Thus, in view of the wide spectra of chemical
compounds in transport and the randomness of the pollution releases, differences among the affected soils, related
to their chemistry and composition, can be expected.
Conclusion
The results of this study have confirmed that highways
represent important line sources of contamination. Its
impact into surrounding biotopes is not extensive,
approximately several tens of meters. The results documented the presence of contaminants with specific modes
action in soils along highways, which can reflect the longterm integration of pollution. The results from sample
collection along highways across four regions highways
during 2 years point to traffic as a significant source of
compounds, namely with dioxin-like but potentially also
antiestrogenic and antiandrogenic potencies, since greatest
content of compounds with these specific activities was
shown next to highways and decreased with distance from
highways. The results of the study document reproducible
patterns, namely for TCDD-EQ and PAH levels, where
higher concentrations in roadside samples can be clearly
linked to traffic sources. PAHs were determined to be the
main compounds contributing to dioxin-like activity.
However, the difference between chemical TEQs and
TCDD-EQs indicates that also other unknown nonpersistent chemicals with AhR-mediated activity, such as PAH
derivates, contribute to the observed activities.
Acknowledgements This work was supported by Grant Agency of
Czech Republic (Grant No. 525/05/P160) and by the Czech Ministry
of Education (Project ENVISCREEN 2B08036). We acknowledge
Marko Virta (University of Helsinki, Finland) for providing us with
the yeast cell lines.
649
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