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Dioxin-Like and Endocrine Disruptive Activityof Traffic-Contaminated Soil Samples

T. Šı́dlová Æ J. Novák Æ J. Janošek Æ P. Anděl ÆJ. P. Giesy Æ K. Hilscherová

Received: 1 August 2008 / Accepted: 11 May 2009 / Published online: 2 June 2009

� Springer Science+Business Media, LLC 2009

Abstract Pollution of surface soils by traffic, especially

along major highways, can be a significant issue. Numer-

ous 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 high-

ways. 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

soils (96 to 478 pg/g), which represent the relevant refer-

ence for the studied soils along highways. The total con-

centration 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 TCDD-

EQs among the studied sites was observed in soils col-

lected 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 distri-

bution 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 pol-

luting compounds having specific effects.

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 trans-

ported 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

T. Šı́dlová � J. Novák � J. Janošek � K. Hilscherová (&)RECETOX, Masaryk University, Brno, Czech Republic

e-mail: [email protected]

P. Anděl

Evernia s.r.o., Liberec, Czech Republic

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

123

Arch Environ Contam Toxicol (2009) 57:639–650

DOI 10.1007/s00244-009-9345-4

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). Com-

bustion 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 environ-

ment, 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, carcinoge-

nicity (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 cel-

lular 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 reproduc-

tion 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 demon-

strated 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

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 estro-

genic, androgenic and/or glucocorticoid-like, and dioxin-

like 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 com-

pounds 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 character-

ization of contamination status is obtained by the combined

use of bioanalytic approach and instrumental analyses.

Instrumental analyses provide information on concentra-

tions 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.

640 Arch Environ Contam Toxicol (2009) 57:639–650

123

Methods

Sample Collection

Sampling sites along major highways, where traffic inten-

sity is regularly monitored (Table 1), were selected to

represent a range of roads with heavy traffic. In 2004, soils

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

Table 1 Traffic intensity (number of cars/d) in the SB and RS areasin 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 34121 8522 47651

RS7 34011 7023 45011

RS8 83187 10580 99765

2005

CV 21023 17009 38100

MB 24005 7158 31228

Fig. 1 Map of the study siteswithin 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

Arch Environ Contam Toxicol (2009) 57:639–650 641

123

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 com-

posite 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 char-

acterized 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 dichlo-

romethane (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 trans-

ferred to dimethylsufoxide (DMSO) for testing in the bio-

assays. 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 halfof 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 cen-

trifugation 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 com-

bined with the first fraction, and the samples were concen-

trated 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. Cellswere plated in 96-well microplates at a density of 15,000

cells/well. These plates were preincubated for 24 hours to

attach the cells in wells. The exposure to standard 2,3,7,8-

tetrachlorodibenzo-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 ER-

linked luciferase gene under control of estrogen-responsive

element (Willemsen et al. 2004). This cell line was culti-

vated 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 incu-

bated at 5% CO2 and 37�C. Before measurement of lumi-nescence, 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 tem-

perature, luciferase activity was measured as luminescence

produced using a microplate-scanning luminometer (Lu-

minoscan 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 human-

androgen receptor along with firefly luciferase under tran-

scriptional control of androgen-responsive element (ARE).

This bioassay is a simple screening system for identifica-

tion 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


123

with shaking. Yeast culture, 100 ll, was plated into white96-well microplates, and 1 ll of soil extracts or standardtestosterone (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 con-

centration of the solvent did not exceed 2% v/v in a single

well. Plates were incubated at 30�C for 2.5 hours. Standardcalibrations 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 lucif-

erase (100 ll 0.1 mM D-luciferin) was added by automaticdispenser in a luminometer (Luminoscan Ascent) (Miche-

lini et al. 2005). Luciferase activity was measured 2 min-

utes after the addition of substrate. All samples were tested

with the control strain (luc) in parallel for possible cyto-

toxicity (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 chromatogra-

pher (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, fluo-

ranthene, 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 stud-

ied compounds was 0.1 ng/g soil.

Quality Assurance and Control

Recoveries were determined by spiking samples with sur-

rogate 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.

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 quan-

tified 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 lumines-

cent 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 cyto-

toxic 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 den-

sities 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 high-

ways 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


123

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 concentrationof 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 AhR-

active compounds was \0.1%.

There was a significant correlation between concentra-

tions 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,

Table 2 Dioxin-like activity determined in in vitro bioassays presented as TCDD-EQs and concentrations of PAHs, PCBs and DDTs andorganic 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 488 3.4 0.7 0.005 0.006 5.8

SB2 arable 201 1.5 0.71 0.004 0.005 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 312 2.9 0.46 0.005 0.008 2.0

RS2 forest 2094 3.4 3.1 0.016 0.029 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 12.0 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

Concentrations of PAHs, PCBs, DDTs, and organic carbon content in the studied samples


123

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 antiestr-

ogenicity in extracts of arable control soil. Some forest

soils from reference areas showed weak antiestrogenic

potency (approximately 75% of the response of competi-

tive 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

highways exhibited estrogenic effects, whereas no estrog-

enicity was found in soils from reference areas.

Soil Contamination with Increasing Distance

from Highway (2005)

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


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 dis-

tant 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

SB

forestarable soil

highway (0-1m)20m distance

0

1000

2000

3000

4000

5000

6000

RS

forestarable soil

highway (0-1m)20m distance

TC

DD

-EQ

(pg

/g)

Fig. 2 Dioxin-like activities ofthe 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

4

3.5

3

2.5

2

1.51 1.5 2 2.5 3 3.5

log

TC

DD

-EQ

(pg

/g)

log TEQ chem.calculated (pg/g)

R2 = 0.67

Fig. 3 Correlation between log-TEQs calculated from the results ofchemical analyses, and log-TCDD-EQs determined by H4IIE.lucbioassays


123

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

greater than those in soils collected 100 m away. Con-

centrations 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

0

20

40

60

80

100

120

140

160

180

200

control SB5 SB6 SB7 SB8 RS5 RS6 RS7 RS8

samples

% o

f res

pons

e of

E2

(3*1

0-11

M)

competitive concentration of 17β-estradiol (E2)

0-1 m

20 m

02040

6080

100120140

160180200

control SB5 SB6 SB7 SB8 RS5 RS6 RS7 RS8

% o

f res

pons

e of

T (

10-8

M)

competitive concentration of testosterone (T)

0-1 m

20 m

(b)

(a)Fig. 4 a Antiandrogenic and bantiestrogenic 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

Table 3 Dioxin-like activity determined in in vitro bioassays presented as TCDD-EQs and concentrations of PAHs, PCBs and DDTs in the soilsamples 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

CV1 20 m 802 0.24 0.001 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 10.2 0.13 0.025

CV2 20 m 2782 0.32 0.005 0.001

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 715 0.70 0.031 0.011

MB1 50 m 1511 1.12 0.022 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


123

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 antiandro-

genic 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 concentra-

tions 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

0

20

40

60

80

100

120

140

160

180

200

control CV1 CV2 MB1 MB2

control CV1 CV2 MB1 MB2

% o

f res

pons

e of

T (

10-8

M)

competitive concentration of testosterone (T)0-1 m20 m

50 m100 m

020406080

100120140160180200

localities

% o

f res

pons

e of

E2

(3*1

0-11

M)

competitive concentration of 17β-estradiol (E2)0-1 m20 m

50 m100 m

(a)

(b)

Fig. 5 a Antiandrogenic and bantiestrogenic 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


123

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 sub-

stances probably belong among these compounds (Bittner

et al. 2006).

TCDD-EQ concentrations were greater 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 an-

tiestrogenic 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-depen-

dent cytochrome induction because it could be eliminated

by cotreatment with AhR and the cytochrome P450

inhibitor a-naphthoflavone. This finding concurs with thefact 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 dem-

onstrated 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

immediately adjacent to highways in 2005, did not corre-

spond 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 com-

pared 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 repre-

sented only 2.5% to 25% of the total vehicles in the sam-

pled 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 traffic-

related 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 imme-

diately 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 contami-

nation values found by roadsides and from those from 20 m

away. They do not correspond to the distribution of emis-

sions 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 indi-

vidual 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 influ-

ences 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 pol-

lutants 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


123

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 con-

tamination 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 pollu-

tion 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 docu-

mented the presence of contaminants with specific modes

action in soils along highways, which can reflect the long-

term 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 nonpersis-

tent chemicals with AhR-mediated activity, such as PAH

derivates, contribute to the observed activities.

Acknowledgements This work was supported by Grant Agency ofCzech 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.

References

Ankley G, Mihaich E, Stahl R, Tillit D, Colborn T, McMaster S et al

(1998) Overview of a workshop on screening methods for

detecting potential (anti-)estrogenic/androgenic chemicals in

wildlife. Environ Toxicol Chem 17:68–87

Benfenati E, Valzacchi S, Mariani G, Airoldi L, Fanelli R (1992)

PCDD, PCDF, PCB, PAH, cadmium, and lead in roadside soil:

Relationship between road distance and concentration. Chemo-

sphere 24:1077–1083

Bergeron JM, Willingham E, Osborn CT, Rhen T, Crews D (1999)

Developmental synergism of steroidal estrogens in sex determi-

nation. Environ Health Perspect 107:93–97

Bittner M, Janosek J, Hilscherova K, Giesy J, Holoubek I, Blaha L

(2006) Activation of Ah receptor by pure humic acids. Environ

Toxicol 21:338–342

Capuano F, Cavalchi B, Martinelli G, Pecchini G, Renna E, Scaroni I

et al (2004) Environmental prospection for PCDD/PCDF, PAH,

PCB and heavy metals around the incinerator power plant of

Reggio Emilia town (Northern Italy) and surrounding main

roads. Chemosphere 58:1563–1569

Chaloupka K (1993) Synergistic activity of polynuclear aromatic

hydrocarbons mixtures as aryl-hydrocarbon (Ah) receptor ago-

nists. Environ Health Perspect 89:141–158

Ciganek M, Neca J, Adamec V, Janosek J, Machala M (2004) A

combined chemical and bioassay analysis of traffic-emitted

polycyclic aromatic hydrocarbons. Sci Total Environ 334–

335:141–148

de Kok TMCM, Driece HAL, Hogervorst JGF, Briedé JJ (2006)

Toxicological assessment of ambient and traffic-related partic-

ulate matter: A review of recent studies. Mutat Res 613:103–122

de Rosa MS, Zarrilli S, Paesano L, Carbone U, Boggia B, Petretta M

et al (2003) Traffic pollutants affect fertility in men. Hum

Reprod 18:1055–1061

Gaido KW, Leonard LS, Lovell S, Gould JC, Babai D, Portier CJ

(1997) Evaluation of chemicals with endocrine modulating

activity in a yeast-based steroid hormone receptor gene tran-

scription assay. Toxicol Appl Pharm 143:205–212

Hilscherova K, Machala M, Kurunthachalam K, Blankenship AL,

Giesy JP (2000) Cells bioassays for detection of aryl

hydrocarbon (AhR) and estrogen receptor (ER) mediated

activity in environmental samples. Environ Sci Pollut Res Int

7:159–171

Holoubek I, Kočan A, Holoubková I, Hilscherova K, Kohoutek J,

Falandysz J et al (2000) Persistent, bioaccumulative and toxic

chemicals in Central and Eastern European countries: state-of-

the-art report. TOCOEN Report 253

Jaffe R (1991) Fate of hydrophobic organic pollutants in the aquatic

environment—review. Environ Pollut 69:237–257

Kannan K, Battula S, Loganathan BG, Hong CS, Lam WH,

Villeneuve DL et al (2003) Trace organic contaminants,

including toxaphene and trifluralin, in cotton field soils from

Georgia and South Carolina, USA. Arch Environ Contam

Toxicol 45:30–367

Kelce WR, Wilson AM (1997) Environmental antiandrogens: Devel-

opmental effects, molecular mechanisms, and clinical implica-

tions. J Mol Med 5:198–207

Kizu R, Okamura K, Toriba A, Mizokami A, Burnstein KL, Klinge

CM (2003) Antiandrogenic activities of diesel exhaust particle

extracts in PC3/AR human prostate carcinoma cells. Toxicol Sci

76:299–309

Klein GP, Hodge EM, Diamond ML, Yip A, Dann T, Stern G et al

(2006) Gas-phase ambient air contaminants exhibit significant

dioxin-like and estrogen-like activity in vitro. Environ Health

Perspect 114:697–703


123

Leskinen P, Michelini E, Picard D, Karp M, Virta M (2005)

Bioluminescent yeast assays for detecting estrogenic and andro-

genic activity in different matrices. Chemosphere 61:259–266

Li C, Taneda S, Suzuki AK, Furuta C, Watanabe G, Taya K (2006a)

Anti-androgenic activity of 3-methyl-4-nitrophenol in diesel

exhaust particles. Eur J Pharmacol 543:194–199

Li C, Takahashi S, Taneda S, Furuta C, Watanabe G, Suzuki AK

(2006b) Impairment of testicular function in adult male Japanese

quail (Coturnix japonica) after a single administration of 3-methyl-4-nitrophenol in diesel exhaust particles. J Endocrinol

189:555–564

Machala M, Vondracek J, Blaha L, Ciganek M, Neca J (2001) Aryl

hydrocarbon receptor-mediated activity of mutagenic polycyclic

aromatic hydrocarbons determined using in vitro reporter gene

assay. Mutat Res 497:49–62

Michelini E, Leskinen P, Virta M, Karp M, Roda A (2005) A new

recombinant cell-based bioluminescent assay for sensitive andro-

gen-like compound detection. Biosens Bioelectron 20:2261–2267

Misaki K, Suzuki M, Nakamura M, Handa H, Iida M, Kato T et al

(2008) Aryl hydrocarbon receptor and estrogen receptor ligand

activity of organic extracts from road dust and diesel exhaust

particulates. Arch Environ Contam Toxicol 55:199–209

Mukerjee D (1998) Health impact of polychlorinated dibenzo-p-

dioxins: A critical review. J Air Waste Manag Assoc 48:157–165

Muto E, Hayashi T, Yamada K, Esaki T, Sagai M, Iguchi A (1996)

Endothelial-constitutive nitric oxide synthase exists in airways

and diesel exhaust particles inhibit the effect of nitric oxide. Life

Sci 59:1563–1570

Novák J, Jálová V, Giesy J, Hilscherova K (2009) Pollutants in

particulate and gaseous fractions of ambient air interfere with

multiple signaling pathways in vitro. Environ Int 35:43–49

Okamura K, Kizu R, Toriba A, Murahashi T, Mizokami A, Burnstein

KL et al (2004) Antiandrogenic activity of extracts of diesel

exhaust particles emitted from diesel-engine truck under differ-

ent engine loads and speeds. Toxicology 195:243–254

Payne J, Rajapakse N, Wilkins M, Kortenkamp A (2000) Prediction

and assessment of the effects of mixtures of four xenoestrogens.

Environ Health Perspect 108:983–987

Perez N, Pey J, Querol X, Alastuey A, Lopez de Alda MJ, Viana M

(2007) Partitioning of major and trace components in PM10-

PM2, 5-PM1 at an urban site in Southern Europe. Atmos Environ

42:1677–1691

Safe S (1986) Comparative toxicology and mechanism of action of

polychlorinated dibenzo-p-dioxins and dibenzofurans. Annu Rev

Pharmacol Toxicol 26:977–984

Safe S, Wormke M (2003) Inhibitory aryl hydrocarbon receptor–

estrogen receptor a cross-talk and mechanisms of action. Chem

Res Toxicol 16:807–816

Sanderson JT, Aarts JMMJG, Brouwer A, Froese KL, Denison MS,

Giesy JP (1996) Comparison of Ah receptor-mediated luciferase

and ethoxyresorufin-O-deethylase induction in H4IIE cells:

Implications for their use as bioanalytical tools for detection of

polyhalogenated aromatic hydrocarbons. Toxicol Appl Pharm

137:316–325

Schrader TJ, Cooke GM (2003) Effects of Aroclors and individual

PCB congeners on activation of the human androgen receptor in

vitro. Reprod Toxicol 17:15–23

Taneda S, Mori Y (2004) Estrogenic and anti-androgenic activity of

nitrophenols in diesel exhaust particles (DEP). Biol Pharm Bull

27:835–837

Ueng TH, Wang HW (2004) Antiestrogenic effects of motorcycle

exhaust particulate in MCF-7 human breast cancer cells and

immature female rats. Arch Environ Contam Toxicol

Villeneuve DL, Blankenship AL, Giesy JP (2000) Derivation and

application of relative potency estimates based on in vitro

bioassay results. Environ Toxicol Chem 19:2835–2843

Watanabe N, Kurita M (2001) The masculinization of the fetus during

pregnancy due to inhalation of diesel exhaust. Environ Health

Perspect 109:111–119

Wesp HF, Tang X, Edenharder R (2000) The influence of automobile

exhausts on mutagenicity of soil: contamination with, fraction-

ation, separation, and preliminary identification of mutagens in

the Salmonella/reversion assay and effects of solvent fractions

on the sister-chromatid exchanges in human lymphocyte cultures

and in the in vivo mouse bone marrow micronucleus assay.

Mutat Res 472:1–21

Willemsen P, Scippo M, Kausel G, Figueroa J, Maghuin-Rogister G,

Martial J et al (2004) Use of reporter cell lines for detection of

endocrine-disrupter activity. Anal Bioanal Chem 378:655–663

Whyte JJ, Schmitt CJ, Tillitt DE (2004) The H4IIE cell bioassay as an

indicator of dioxin-like chemicals in wildlife and the environ-

ment. Crit Rev Toxicol 34:1–83

Xiao R, Wang Z, Wang C, Yu G (2006) Soil screening for identifying

ecological risk stressors using a battery of in vitro cell bioassays.

Chemosphere 64:71–78

Yoshida S, Sagai M, Oshio S, Umeda T, Ihara T, Sugamata M (1999)

Exposure to diesel exhaust affects the male reproductive system

of mice. Int J Androl 22:307–315


123

Dioxin-Like and Endocrine Disruptive Activity �of Traffic-Contaminated Soil SamplesAbstractMethodsSample CollectionExtractionBioassaysChemical AnalysesQuality Assurance and ControlData Analysis

ResultsComparison of Specific Activities in Soils Collected �in 2004 Near Highways and in Reference AreasSoil Contamination with Increasing Distance �from Highway (2005)

DiscussionConclusionAcknowledgementsReferences

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