Characterization of dioxin-like contamination in soil and sediments from the "hot spot" area of petrochemical plant in Pancevo (Serbia).

Abstract

Combinatorial bio/chemical approach was applied to investigate dioxin-like contamination of soil and sediment at the petrochemical and organochlorine plant in Pancevo, Serbia, after the destruction of manufacturing facilities that occurred in the spring of 1999 and subsequent remediation actions.
Soil samples were analyzed for indicator polychlorinated biphenyls (PCBs) by gas chromatography/electron capture detection (GC/ECD). Prioritized soil sample and sediment samples from the waste water channel were analyzed for polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) by high-resolution gas chromatography/high-resolution mass spectrometry (HRGC/HRMS). Microethoxyresorufin o-deethylase (Micro-EROD) and H4IIE-luciferase bioassays were used for monitoring of dioxin-like compounds (DLC) and for better characterization of dioxin-like activity of soil samples.
Bioanalytical results indicated high dioxin-like activity in one localized soil sample, while the chemical analysis confirmed the presence of large quantities of DLC: 3.0 × 10(5) ng/g d.w. of seven-key PCBs, 8.2 ng/g d.w. of PCDD/Fs, and 3.0 × 10(5) ng/g d.w. of planar and mono-ortho PCBs. In the sediment, contaminant concentrations were in the range 2-8 ng/g d.w. of PCDD/Fs and 9-20 ng/g d.w. of PCBs.
This study demonstrates the utility of combined application of bioassays and instrumental analysis, especially for developing and transition country which do not have capacity of the expensive instrumental analysis. The results indicate the high contamination of soil in the area of petrochemical plant, and PCDD/Fs contamination of the sediment from the waste water channel originating from the ethylene dichloride production.

Full text

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RESEARCH ARTICLE
Characterization of dioxin-like contamination in soil
and sediments from the “hot spot” area of petrochemical
plant in Pancevo (Serbia)
Sonja Kaisarevic & Klara Hilscherova & Roland Weber & Kristina L. Sundqvist &
Mats Tysklind & Ernest Voncina & Stanka Bobic & Nebojsa Andric &
Kristina Pogrmic-Majkic & Mirjana Vojinovic-Miloradov & John Paul Giesy &
Radmila Kovacevic
Received: 5 July 2010 /Accepted: 8 November 2010 /Published online: 23 November 2010
# Springer-Verlag 2010
Abstract
Purpose Combinatorial bio/chemical approach was applied
to investigate dioxin-like contamination of soil and sedi-
ment at the petrochemical and organochlorine plant in
Pancevo, Serbia, after the destruction of manufacturing
facilities that occurred in the spring of 1999 and subsequent
remediation actions.
Materials and methods Soil samples were analyzed for
indicator polychlorinated biphenyls (PCBs) by gas chro-
matography/electron capture detection (GC/ECD). Priori-
tized soil sample and sediment samples from the waste
water channel were analyzed for polychlorinated dibenzo-
p-dioxins and dibenzofurans (PCDD/Fs) by high-resolution
gas chromatography/high-resolution mass spectrometry
Responsible editor Ake Bergman
This article belongs to the series “Dioxin and POP contaminated sites”
edited by Roland Weber, Mats Tysklind, and Caroline Gaus.
Electronic supplementary material The online version of this article
(doi:10.1007/s11356-010-0418-8) contains supplementary material,
which is available to authorized users.
S. Kaisarevic (*):N. Andric:K. Pogrmic-Majkic:
M. Vojinovic-Miloradov:R. Kovacevic
Laboratory for Ecotoxicology (LECOTOX), University of Novi
Sad Faculty of Science,
Trg D. Obradovica 2,
21000, Novi Sad, Serbia
e-mail: sonja.kaisarevic@dbe.uns.ac.rs
K. Hilscherova
Research Centre for Environmental Chemistry and Ecotoxicology
(RECETOX), Masaryk University,
Kamenice 3,
625 00, Brno, Czech Republic
R. Weber
POPs Environmental Consulting,
Ulmenstraβe 3,
73035, Göppingen, Germany
K. L. Sundqvist:M. Tysklind
Department of Chemistry, Umeå University,
SE-90187, Umeå, Sweden
E. Voncina
Institute of Public Health,
Prvomajska 1,
Maribor, Slovenia
S. Bobic
Institute of Occupational Health,
Futoska 121,
21000, Novi Sad, Serbia
J. P. Giesy
Department Veterinary Biomedical Sciences,
University of Saskatchewan,
Saskatoon, Canada
J. P. Giesy
Department of Zoology, National Food Safety and Toxicology
Centre, Michigan State University,
East Lansing, MI, USA
J. P. Giesy
Department of Biology and Chemistry,
City University of Hong Kong,
Kowloon, Hong Kong SAR, China
Environ Sci Pollut Res (2011) 18:677–686
DOI 10.1007/s11356-010-0418-8

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(HRGC/HRMS). Microethoxyresorufin o-deethylase
(Micro-EROD) and H4IIE–luciferase bioassays were used
for monitoring of dioxin-like compounds (DLC) and for
better characterization of dioxin-like activity of soil
samples.
Results Bioanalytical results indicated high dioxin-like
activity in one localized soil sample, while the chemical
analysis confirmed the presence of large quantities of DLC:
3.0×105ng/g d.w. of seven-key PCBs, 8.2 ng/g d.w. of
PCDD/Fs, and 3.0×105ng/g d.w. of planar and mono-ortho
PCBs. In the sediment, contaminant concentrations were in
the range 2–8 ng/g d.w. of PCDD/Fs and 9–20 ng/g d.w. of
PCBs.
Conclusions This study demonstrates the utility of com-
bined application of bioassays and instrumental analysis,
especially for developing and transition country which do
not have capacity of the expensive instrumental analysis.
The results indicate the high contamination of soil in the
area of petrochemical plant, and PCDD/Fs contamination of
the sediment from the waste water channel originating from
the ethylene dichloride production.
Keywords Dioxin-like activity.Ethylene dichloride.
PCBs.PCDD/Fs.Pattern analysis.Petrochemical industry
1 Background, aim, and scope
The petrochemical plant and organochlorine industry
“HIP-Petrohemija” in industrial area of city Pancevo,
20 km south of Belgrade, is the largest producer of
ethylene dichloride (EDC), vinyl chloride monomer
(VCM), polyethylene, polyvinyl chloride (PVC), plastics,
ethylene, propylene, C4 (60% butadiene), and pyrolytic
oil and gasoline in Serbia. As by-products in EDC,
VCM, and PVC production, polychlorinated dibenzo-p-
dioxins and dibenzofurans (PCDD/Fs) can be formed
(Carroll et al. 2001), which can result in contamination
of the affected areas by dioxin-like compounds (DLC;
Fattore et al. 1997; Isosaari et al. 2000; Weber et al. 2008).
The industrial complex in Pancevo also emitted chemicals
as a result of the destruction of plant sections that occurred
during air strikes in the spring of 1999. The releases
included direct emissions of 2,314 t of EDC and burning
of about 500 t of VCM resulting in emissions of VCM,
PCDD/Fs, and polycyclic aromatic hydrocarbons (PAHs)
(Brown 2004). Levels of organic pollutants in different
environmental matrices in the petrochemical plant were
determined by the United Nations Environment
Programme/United Nations Centre for Human Settlements
(UNEP/UNCHS) Balkans Task Force (BTF) in October
1999. The results revealed soil contamination by EDC
(10 mg/kg) and other volatile organic compounds, occur-
rence of a fingerprint of technical mixture of polychlori-
nated biphenyls (PCBs) (up to 1 mg/kg), and one soil
sample contaminated with PCDD/Fs (4.4×103pg TEQ/g
d.w.). On the basis of all obtained data, Pancevo was
designated as one of four “hot spots” in Serbia (UNEP
1999a). Actions for remediation, concerning selection and
putting into operation the best available technology for
soil remediation at VCM plant, were conducted by UNEP
in the period 2000–2003, and mostly focused on remedi-
ation of the EDC pollution. By January 2004, over 400 t of
EDC were treated, and thereafter, UNEP Clean-up
Programme handed over the full-scale remediation system
for soil and groundwater remediation at the VCM plant to
the site-owner. Since then, the petrochemical plant is
responsible for operating and further optimizing the
system until cleanup targets are met (UNEP 2004).
Environmental management and control of persistent
organic pollutants (POPs) is stressed through the Stockholm
Convention, signed by Serbia in 2002, by requiring parties
to develop strategies to identify sites contaminated by these
chemicals (Article 6(1)e SC 2001). European commission
experts of the Fifth Framework Programme (Call identifier
ICFP501A2PR02) have recognized the problem of identi-
fying POP contamination in war-ravaged areas of former
Yugoslavia and approved the proposed investigations under
the Contract APOPSBAL ICA2-CT2002-10007 (Picer et al.
2006; Radonic et al. 2009). Within the frame of this project,
in light of the results reported by UNEP (1999a) and due to
the fact that sediment toxicity evaluation has never been
included into official monitoring programs in Serbia
(Teodorovic 2009), this study was conducted to determine
dioxin-like contamination in soil from the “hot spot” area of
petrochemical plant in Pancevo, and potential release routes
via waste water into waste water channel sediment. The
study reflects the situation at the selected area within the
petrochemical plant in Pancevo, Serbia, after remediation
actions have been taken, by application of low-cost effect-
directed approach.
Soil samples were initially analyzed for indicator
polychlorinated biphenyls (PCBs) by gas chromatography/
electron capture detection (GC/ECD), and microethoxyr-
esorufin o-deethylase (Micro-EROD) bioanalysis was used
as a screening tool for DLC in soil. The prioritized soil
sample from the first screening was further fractionated and
tested by H4IIE–luciferase analysis for better characteriza-
tion of its dioxin-like induction potency. Prioritized soil
sample and sediment samples from the waste water channel
were analyzed by high-resolution gas chromatography/
high-resolution mass spectrometry (HRGC/HRMS) for
congener-specific analysis of planar and mono-ortho PCBs
and PCDD/Fs.
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2 Materials and methods
2.1 Sampling sites
In October 2004, five soil samples (P1–P5) were collected
from the sites selected according to the UNEP report
(1999a), from the area in the vicinity of EDC and VCM
plant that was found to be contaminated by PCDD/Fs, and
sites that suffered from destruction of industrial facilities.
The area is still exposed to input of pollutants due to
manufacturing processes. One control sample (P0) was
collected from residential part of the city Pancevo. Samples
were taken by “T”-type sampling tube from 0 to 0.15 m
depth, transferred to the laboratory, and kept at +4°C until
the extraction.
Sediment samples were collected from the waste water
channel (WWC), collecting waste waters from the petro-
chemical plant, and nearby oil refinery and a chemical
fertilizer factory, and draining into the River Danube.
Sediment cores of 0.4 m length were collected in January
2007 using Eijkelkamp corer, from three points along the
channel: WWC 1—downstream the fertilizer factory outlet;
WWC 2—downstream the petrochemical plant and oil
refinery outlets; and WWC 3—near the mouth of the WWC
to the River Danube. Six samples were collected at each
sampling site, stored at +4°C, and transported to the
laboratory. Samples from the same sampling site were
pooled, freeze-dried, sieved using the 0.056-mm sieve, and
stored at−20°C until the extraction.
2.2 Sample extraction and fractionation
In the first trial, a simple cleanup procedure of soil samples
was performed using acetone:hexane as solvent mixture,
and subsequent purification of hexane fraction by sulfuric
acid, as previously described (Kaisarevic et al. 2007) and as
explained in Online Resource (ESM 1). The applied
procedure resulted in refined hexane extracts (P0–P5), used
in GC/ECD analysis for determination of seven PCB
congeners and in micro-EROD analysis for screening of
dioxin-like activity.
One sample contained relatively high levels of dioxin-
like activity and extremely high concentrations of PCBs in
the initial tests, and was therefore subjected to further
analysis to quantify planar and mono-ortho PCBs and
PCDD/Fs by HRGC/HRMS. The extraction procedure was
conducted as explained in Online Resource (ESM 1).
In the second trial, another portion of the prioritized
sample (P4) was extracted, fractionated into five different
fractions, and tested by H4IIE–luciferase analysis for
better characterization of its dioxin-like activity. The
sample (5 g) was extracted by ultrasound and microwave
in toluene (2×), and thereafter, the whole organic extract
was fractionated into five fractions on sulfuric acid-
treated silica gel column (FLUKA Silica gel 60 (35–70
mesh)). Compounds were eluted from the column by the
solvents with increasing polarity: first fraction (F1) was
eluted with hexane, second (F2) with 10% DCM in
hexane, third (F3) with 50% DCM in hexane, fourth (F4)
with 100% DCM, and fifth (F5) fraction with methanol.
Each fraction was evaporated to dryness, dissolved in
DMSO, and subjected to H4IIE–luc analysis to examine
their contribution to the overall aryl-hydrocarbon receptor
(AhR)-mediated activity.
Sediment samples were prepared according to Sundqvist
et al. (2009) with minor modifications. Briefly, the freeze-
dried samples were Soxhlet-extracted with toluene. After
evaporation, copper granulate was added to remove sulfur.
Cleanup consisted of a multilayer silica column, and
fractionation was achieved on a carbon/Celite column. A
miniature multilayer silica column was used as a final
cleanup step. 13C-labeled congeners were used as internal
and recovery standards.
2.3 Instrumental analysis
Residual PCBs (PCB28, PCB52, PCB101, PCB138,
PCB153, PCB180, and PCB209) in refined hexane extracts
of soil samples P0–P5 from the first trial were determined
by GC/ECD as previously described (Kaisarevic et al.
2007) and as explained in Online Resource (ESM 1).
Identification and quantification of planar and mono-
ortho PCBs and PCDD/Fs in the corresponding extracts of
sample P4 were carried out using HRGC/HRMS according
to Krizanec et al. (2005) and as explained in Online
Resource (ESM 1).
Congener-specific quantification of planar and mono-
ortho PCBs and all tetra- through octa-PCDD/Fs in
sediment samples was carried out using HRGC/HRMS
equipped with a 60-m DB5 capillary column (J&W
Scientific, Agilent Technologies, USA; i.d. 0.25 mm, film
thickness 0.25 μm) according to Sundqvist et al. (2009).
On the basis of PCB and PCDD/F concentrations,
chemically derived toxic equivalents are calculated and
assigned as H4IIE–luc–TEQ and WHO2005–TEQ. H4IIE–
luc–TEQs were calculated by multiplication of measured
congener concentrations with corresponding relative poten-
cies values for H4IIE–luc assay (H4IIE–luc REP (EC50))
according to Behnisch et al. (2003), and subsequent
summation. WHO2005−TEQs were calculated by multipli-
cation of measured congener concentrations with
corresponding toxicity equivalent factors (WHO2005−TEF)
according to van den Berg et al. (2006), and subsequent
summation.
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2.4 Micro-EROD analysis
Micro-EROD analysis was applied for screening of dioxin-
like activity of refined hexane extracts of soil samples (P0–
P5). The analysis was conducted on primary rat hepatocyte
culture as previously described (Kaisarevic et al. 2007).
The assay was performed in 96-well plates, with Williams’
Medium E (WME) and 10% FCS as a culturing medium,
the exposure time of 48 h and PCB126 as a referent
compound. The exposure time was selected on the basis of
our previous investigations, which revealed that after 48 h
of treatment, optimal EROD activity is reached. The assay
procedure is explained in detailinOnlineResource(ESM1).
Bioassay-derived induction equivalents (IEQ), expressed as
picogram PCB126-eq./g d.w., were calculated for each
sample as quotient of PCB126 EC25value and the sample
concentration that induced 25% of the PCB126 maximal
response.
2.5 H4IIE–luciferase analysis
H4IIE–luciferase analysis was applied for testing of
prioritized soil sample P4 (refined hexane extract and five
different fractions). The H4IIE–luc cells culturing and
H4IIE–luciferase analysis have been carried out according
to Bittner et al. (2006). The assay was performed in 96-well
plates, with Dulbecco’s Modified Eagle’s Medium
(DMEM) and 10% FCS as a culturing medium, the
exposure time of 24 h, and 2,3,7,8-tetrachlorodibenzo–p-
dioxin (TCDD) as a referent compound. The exposure time
was selected on the basis of our previous investigations,
which revealed 24-h treatment as an optimal for measure-
ment of luciferase activity. The assay procedure is
explained in detail in Online Resource (ESM 1). IEQ
values, expressed as picogram TCDD-eq./g d.w., were
calculated for each sample as quotient of TCDD EC50
value and the sample concentration that induced 50% of the
TCDD maximal response.
3 Results and discussion
The EDC, VCM, and the former elemental chlorine
production are potential sources for PCDD/Fs and other
unintentionally produced POPs (Weber et al. 2008). In
addition to long-term pollution sources at petrochemical
and organochlorine plant in Pancevo, destruction of
manufacturing facilities followed by open burning of
released chemicals additionally contributed to the pollution
of this area. In order to characterize dioxin-like contamina-
tion in soil from the “hot spot” area of petrochemical plant
in Pancevo, and potential release routes via waste water, a
combined bio/chemical approach was applied. Such approach
has been shown to be a powerful tool for identification of
targetcompoundsincomplextoxicmixtures(Anderssonetal.
2009; Brack 2003; Rastall et al. 2006; Wölz et al. 2008).
3.1 Soil samples from the petrochemical plant
Results from the first phase of investigation revealed
differences in total levels of marker PCBs in soil samples,
with the significantly highest concentrations in sample P4
(3.0×105ng/g d.w.; Table 1). However, the proportion of
PCB congeners in their total amount was similar in all
investigated samples. PCB101, PCB138, PCB153, and
PCB180 accounted for 95–100% of total PCB content in
each sample, with hexa-chlorinated congeners (PCB138
and PCB153) being most abundant. Such uniform PCB
pattern across the investigated area indicates that industri-
ally produced PCBs from transformers and condensers were
responsible for the pollution in all examined soils. Similar
congener profile in soil from petrochemical zone with
Table 1 PCB concentrations in soil samples from residential part of Pancevo (P0) and petrochemical plant (P1–P5), and micro-EROD-derived
IEQ values of their refined hexane fractions
Sample PCB congener (ng/g d.w.)
Σ PCBa(ng/g d.w.)IEQb(pg PCB126-eq/g d.w.)
28 52 101138153 180209
P0
P1
P2
P3
P4
P5
nd
nd
nd
nd
1.6×103
nd
nd
nd
9.8
1.6
1.5×104
16
0.6
0.8
43
13
7.5×104
1.3×102
11
1.7
82
25
1.0×105
1.4×102
1.1
2.0
92
27
7.3×104
2.8×102
0.5
2.5
50
15
3.7×104
75
nd
nd
nd
nd
nd
nd
13
7.0
2.8×102
82
3.0×105
6.4×102
62
3.7×102
2.5×103
1.5×102
1.2×104
3.7×102
nd not detectable
aRiedel-de-Haen standard of seven PCB congeners: PCB28, PCB52, PCB101, PCB138, PCB153, PCB180, and PCB209
bMicro-EROD-derived induction equivalents
680Environ Sci Pollut Res (2011) 18:677–686

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prevalence of penta-CBs and hexa-CBs was also reported
by Nadal et al. (2007).
The results of micro-EROD analysis (Table 1, Figures
S1 and S2 in Online Resource ESM 2) indicate that AhR-
active compounds were present in all investigated samples,
but at different contamination levels. All samples exhibited
EROD-inducing potency, but the IEQ values vary from
1.5×102pg PCB126-eq./g d.w. to as great as 1.2×104pg
PCB126-eq./g d.w. for sample P4. Our previous studies
document that potential threshold IEQ value in the same
experimental conditions is about 30 pg PCB126-eq./g d.w.
(Zoric et al. 2004). In the present study, IEQ concentrations
were greater than this potential effects threshold for all
samples except the control soil (P0). Complex environmen-
tal mixtures can contain different POPs among which are
AhR agonists (Andersson et al. 2009; Hilscherova et al.
2001). EROD-inducing potency indicates the contamina-
tion with prototypic AhR ligands, including PCDD/Fs,
dioxin-like PCBs, polybrominated biphenyls, and polybro-
minated diphenyl ethers. The presence of PAHs, which are
also well-known EROD inducers (Hilscherova et al. 2001;
Kaisarevic et al. 2009), in refined hexane extracts is
questionable due to cleanup procedure with concentrated
sulfuric acid, which is used to eliminate nonpersistent
organic compounds from the extract.
Sample P4 was prioritized for further HRGC/HRMS
analysis in order to quantify PCDD/Fs and dioxin-like
PCBs (Tables 2 and 3). Results demonstrated H4IIE–luc–
TEQ (14×102pg/g d.w.) and WHO2005–TEQ (5.5×102pg/
g d.w.) concentrations for PCDD/Fs about three to eight
times less than the one obtained within BTF investigation
in 1999 (UNEP 1999a). In that investigation, one hot spot
contaminated with dioxins and furans, with instrumentally
derived TEQ value of 4.4×103pg/g d.w., was identified
between the EDC and VCM plant. According to their
analysis, the detected fingerprint of PCDD/Fs and the
presence of chlorinated hydrocarbons (up to C6) indicated a
thermal formation from either EDC or VCM (UNEP
1999a). However, German trigger concentration (concen-
trations in soil that warrant further investigation to
determine if the concentration of the contaminant in soil
is hazardous) for industrial sites (TEQ of 104pg/g d.w.)
Table 2 PCDD/Fs concentrations, their relative potencies for H4IIE–
luc assay (H4IIE–luc REP) and deduced H4IIE–luc–TEQ along with
WHO2005toxicity equivalent factors (WHO2005–TEF) and deduced
WHO2005–TEQ values in P4 soil sample from industrial complex of
petrochemical plant
Compounds Concentration
ng/g d.w.
H4IIE–luc REP
(EC50)a
H4IIE–luc–TEQ
(pg/g d.w.)b
WHO2005–TEFc
WHO2005–TEQ
(pg/g d.w.)d
2,3,7,8-TCDD
1,2,3,7,8-PCDD
1,2,3,4,7,8-H6CDD
1,2,3,6,7,8-H6CDD
1,2,3,7,8,9-H6CDD
1,2,3,4,6,7,8-H7CDD
OCDD
2,3,7,8-TCDF
1,2,3,7,8-PCDF
2,3,4,7,8-PCDF
1,2,3,4,7,8-H6CDF
1,2,3,6,7,8-H6CDF
1,2,3,7,8,9-H6CDF
2,3,4,6,7,8-H6CDF
1,2,3,4,6,7,8-H7CDF
1,2,3,4,7,8,9-H7CDF
OCDF
Sum PCDD/Fs
1.0×10−3
2.0×10−3
1.0×10−3
3.0×10−3
3.0×10−3
1.1×10−1
7.2×10−1
3.2
4.5×10−1
2.0×10−3
1.2
3.7×10−1
2.0×10−1
2.9×10−1
7.1×10−1
1.7×10−1
7.3×10−1
8.2
1
0.54
0.3
0.14
0.066
0.046
0.0005
0.32
0.21
0.50
0.13
0.039
0.11
0.18
0.029
0.041
0.0065
1.0
1.1
0.3
0.4
0.2
5.1
0.4
10×102
94
1.0
1.6×102
14
22
52
21
7.0
4.7
14×102
1
1
0.1
0.1
0.1
0.01
0.0003
0.1
0.03
0.3
0.1
0.1
0.1
0.1
0.01
0.01
0.0003
1.0
2.0
0.1
0.3
0.3
1.1
0.2
3.2×102
13
0.6
1.2×102
37
20
29
7.1
1.7
0.2
5.5×102
aRelative potencies values for H4IIE–luc assay according to Behnisch et al. 2003
bChemically derived toxic equivalents calculated by multiplication of measured congener concentrations with corresponding H4IIE–luc REP (EC50) and
subsequent summation
cToxicity equivalent factors according to van den Berg et al. 2006
dChemically derived toxic equivalents calculated by multiplication of measured congener concentrations with corresponding WHO2005–TEF and
subsequent summation
Environ Sci Pollut Res (2011) 18:677–686681

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was not exceeded. According to Im et al. (2002), total
concentrations of PCDD/Fs in industrial soil from Korea
were up to 9.7×103pg/g d.w., which is similar to
concentrations observed in our study. Sample P4 also
contained extremely high concentrations of planar and
particularly mono-ortho PCBs with H4IIE–luc–TEQ value
of 77×103pg/g d.w., or WHO2005−TEQ value of 11×
104pg/g d.w. These values are about eight to ten times
greater than German trigger concentration for industrial
sites mentioned above and indicate considerable soil
contamination in this area. Based on the high level of
PCBs, high contribution of PCDF among the PCDD/Fs,
and the distribution of the 2,3,7,8-PCDD/F congeners, the
contamination at P4 sampling spot stemmed mainly from
the nearby exploded PCB transformer having resulted in
some PCDF formation. Also, the relatively low OCDF
concentration in the sample demonstrates a minor impact
from the adjacent EDC/VCM production having OCDF as
a marker congener (Isosaari et al. 2000; Torres et al. 2008).
Great dioxin-like activity of P4 sample was also
confirmed by H4IIE–luc analysis, with H4IIE–luc IEQ of
refined hexane extract (44×103pg TCDD-eq/g d.w.) even
significantly higher than the IEQ obtained in micro-EROD
analysis. Since we used different referent compounds in the
two bioassays (PCB126 in micro-EROD and TCDD in
H4IIE–luc analysis) when comparing derived IEQs, previ-
ous findings that PCB126 has ten times lesser potency than
TCDD in primary rat hepatocytes (Zeiger et al. 2001)
should be considered. Although different exposure times
applied in the two bioassays, as well as different effect
levels used for IEQ calculation, might contribute to the
difference in IEQs, it should be stressed that unlike the
H4IIE–luc analysis, the enzyme activity in micro-EROD
analysis can be diminished due to the competitive inhibition
(antagonistic effect) of compounds in the mixture (Brack et
al. 2000) including also, e.g., non-dioxin-like PCBs
(Behnisch et al. 2002). H4IIE–luc bioassay results (Table 4,
Figures S3 and S4 in Online Resource ESM 2) for five
fractions of sample P4 revealed that F2, probably contain-
ing PAHs, their derivatives, and a portion of the PCDD/Fs
and PCBs (Song et al. 2006), most significantly contributed
to the IEQ. More polar compounds eluted in F3–F5 are not
typical AhR agonists, which resulted in their lower
induction potencies. The sum of IEQs from the fractions
is 5.4 times less than the IEQ obtained for the refined
hexane extract by the same analysis. The same phenome-
non was also reported by Song et al. (2006), and the authors
suggest that it can be caused by antagonistic interactions
among compounds and even their retention on the column
during fractionation. In our study, antagonistic interactions
in refined hexane extract are not likely since it revealed
higher dioxin-like activity compared with the fractions.
Table 3 Planar and mono-ortho PCB concentrations, their relative
potencies for the H4IIE–luc assay (H4IIE–luc REP) and deduced
H4IIE–luc–TEQ along with WHO2005 toxicity equivalent factors
(WHO2005–TEF) and deduced WHO2005–TEQ values in P4 soil
sample from industrial complex of petrochemical plant
CompoundsConcentration
(ng/g d.w.)
H4IIE–luc REP (EC50)a
H4IIE–luc–TEQ
(pg/g d.w.)b
WHO2005–TEFc
WHO2005–TEQ
(pg/g d.w.)d
PCB 77
PCB 81
PCB 126
PCB 169
PCB 105
PCB 114
PCB 118
PCB 123
PCB 156
PCB 157
PCB 167
PCB 189
Sum PCBs
1.5×103
18
1.0×103
20
9.5×104
5.1×103
1.4×105
1.6×104
2.8×104
6.2×103
9.6×103
1.1×103
3.0×105
0.0013000
0.0042000
0.0670000
0.0034000
0.0000120
0.0000480
No dose–response
0.0000240
0.0002100
0.0000800
0.0000082
0.0000067
19×102
76
67×103
68
11×102
2.4×102
–
3.8×102
59×102
5.0×102
79
7.4
77×103
0.00010
0.00030
0.10000
0.03000
0.00003
0.00003
0.00003
0.00003
0.00003
0.00003
0.00003
0.00003
1.5×102
5.4
1.0×105
6.0×102
2.8×103
1.5×102
4.2×103
4.8×102
8.4×102
1.9×102
2.9×102
33
11×104
aRelative potencies values for H4IIE–luc assay according to Behnisch et al. 2003
bChemically derived toxic equivalents calculated by multiplication of measured congener concentrations with corresponding H4IIE–luc REP (EC50) and
subsequent summation
cToxicity equivalent factors according to van den Berg et al. 2006
dChemically derived toxic equivalents calculated by multiplication of measured congener concentrations with corresponding WHO2005–TEF and
subsequent summation
682Environ Sci Pollut Res (2011) 18:677–686

Page 7

Moreover, since the five fractions were not obtained from
the refined hexane extract, but by a different extraction and
fractionation procedure, this is probably one of the main
reasons for the observed differences in their dioxin-like
activity. It is also important to mention that both applied
bioassays, micro-EROD and H4IIE–luciferase, are depen-
dent on gene transcription. For this reason, presence of
compounds inhibiting gene transcription would result in a
lower than expected activity given the amount of AhR
active component in the sample.
HRGC/HRMS-derived TEQs are about two times
greater than bioassay-derived IEQ for the refined hexane
extract, and even ten times greater than the sum of IEQs
from the five fractions. The reason for this difference
probably lies in antagonistic interactions between the
compounds present in this specific mixture of pollutants,
which can be observed in bioassays but not in instru-
mental analysis. High levels of PCB153 has been shown
to diminish, e.g., the potency of the agonists 2,3,7,8-
TCDD and PCB126 (Sanderson et al. 1996). Also,
PCB52 and PCB77 show antagonistic activity (Aarts et
al. 1995).
3.2 Contamination levels and possible sources in the waste
water channel
Waste water discharge can be an important release route for
PCDD/Fs from chlorine and organochlorine industry
(Heinisch et al. 2007; Verta et al. 2009; Weber et al.
2008), and extensive contamination of sediments have been
documented for other EDC/VCM production (Fattore et al.
1997; Isosaari et al. 2000). Therefore, in addition to the soil
monitoring, a congener-specific screening of PCDD/Fs and
PCBs in sediments from the waste water channel of the
industrial complex has been performed (Table 5). The
results of total congener-specific analysis for each sample
are given in Online Resource—Table S1 and Table S2
(ESM 2). Total PCDD/Fs levels in the three pooled
sediments had WHO2005–TEQ concentrations between 20
and 100 pg/g d.w. and 2 to 8 ng/g d.w. total PCDD/Fs
concentration. Planar and mono-ortho PCBs had
WHO2005–TEQ concentrations between 6 and 8 pg/g d.w.
and 9 to 20 ng/g d.w. total PCBs concentration (Table 5).
The biologically derived IEQs of these samples have been
evaluated in detail in previous study by effect-directed
analysis and have revealed extremely high levels of dioxin-
like activity resulting mainly from contamination with a
wide range of polyaromatic compounds (Kaisarevic et al.
2009).
In addition to the hot spot soil sample P4, the PCDD/Fs
levels in the sediments from the waste water channel of the
factories were two to four times above the Norwegian
sediment limits for DLC (Norwegian 2005). In all samples,
Table 4 Comparison of H4IIE–luc-derived IEQs of refined hexane extract and different fractions of soil sample P4 and HRGC/HRMS-derived TEQs
H4IIE–luc IEQ
(pg TCDD-eq./g d.w.)a
Total PCDD/Fs+PCBs HRGC/HRMS-derivedTEQ (pg/g d.w.)
Refined hexane extract
F1
F2
F3
F4
F5
Σ F1–F5
H4IIE–luc–TEQb
WHO2005–TEQc
44×103
3.1×102
74×102
1.7×102
0.5×102
2.5×102
82×102
78×103
11×104
aH4IIE–luciferase bioassay-derived induction equivalents
bChemically derived toxic equivalents calculated according to H4IIE–luc REP values (Behnisch et al. 2003)
cChemically derived toxic equivalents calculated according to WHO2005–TEF values (van den Berg et al. 2006)
Environ Sci Pollut Res (2011) 18:677–686683

Page 8

the PCDD/Fs pattern was dominated by PCDF with OCDF
as marker congener (ESM 2). For sampling point WWC2
(near the EDC/VCM plant) and WWC3 (downstream the
plant at the inflow to the river Danube), the homologue
pattern and congener pattern of HexaCDF to OCDF
resemble an EDC pattern including the marker con-
geners OCDF and the 1,2,3,4,7,8-HCDF (Figure S5 in
Online Resource ESM 2) (Isosaari et al. 2000; Torres et
al. 2008). Also upstream, at WWC1 downstream the
fertilizer plant, a distinctive EDC pattern was detected. In
the UNEP/UNCHS Balkans Task Force report, one
sediment sample in the waste water channel has been
analyzed, and the authors concluded that the sediments
were impacted by a thermal pattern (UNEP 1999b).
However, also the pattern in the UNEP/UNCHS report
showed a clear EDC pattern similar to our three sediment
samples and the literature, demonstrating that the sedi-
ments were contaminated by the EDC production. The
TEQ contamination levels in this study are less compared
with the contaminated marine sediment in the Finland
EDC contamination case (Isosaari et al. 2000). This
indicates that waste water release was probably not the
major release vector for PCDD/Fs, but that the PCDD/Fs
heavy fraction of the EDC process along with the highly
PCDD/Fs containing catalyst mass were most probably
deposited elsewhere. The TEQ contribution from PCBs in
the sediment of WWC (6 to 8 pg TEQ/g d.w.) was only
approximately 10% of total TEQ and relatively low. The
PCB contamination from the soil in the factory area thus
appears to not have leached significantly into the waste
water channel at present.
4 Conclusions
This study shows the strength of a combined application of
bioassays and instrumental analysis as the practical cost-
effective approach to ensure more complete insight into
levels and biological effects of dioxin-like compounds
(DLC) in the environment. The approach we presented is
rational, effect-directed, and sufficiently informative regard-
ing presence and levels of DLC and is advisable for transition
and developing countries with the lack of expensive and
maintenance intensive HRGC/HRMS. In the regions where
no institutionalized and standard monitoring programs are
routinelybeingappliedfor DLC,suchapproachcanbecomea
useful tool in environmental pollution screening and research,
provide information on type and level of pollutants, and help
in filling the gaps in present knowledge regarding DLC
contamination. For highly contaminated or selected
samples, the PCDD/Fs can then be analyzed by HRGC/
HRMS from a commercial lab or a research collaboration
partner with the respective capacity allowing detailed
interpretation of the PCDD/Fs fingerprint and the
assignment of the responsible sources and processes.
The study also implies that for the assessment of
biological effects of DLC, it is essential that the cleanup
procedure reduces the presence of other components
affecting the bioasays, such as inhibitors of gene
transcription or enzyme activity.
The preliminary screening of the Pancevo site indicates
that there is still high contamination level in the soil of the
factory area. The analysis of samples from waste water
channel of the factory also revealed that PCDD/Fs were
Table 5 Total PCDD/Fs and PCBs concentrations and deduced WHO2005–TEQ values in sediment samples from the waste water channel
Compounds WWC1WWC2WWC3
Concentrationa
(ng/g)
WHO2005–TEQ
(pg/g d.w.)b
Concentration
(ng/g)
WHO2005–TEQ
(pg/g d.w.)
Concentration
(ng/g)
WHO2005–TEQ
(pg/g d.w.)
PCDDsc
PCDFsd
Sum PCDD/Fs
Planar PCBse
Mono-ortho PCBsf
Sum PCBs (planar+mono-ortho)
1.3×10−1
7.9
8.0
3.2×10−1
18
18
1.0
1.0×102
1.0×102
6.4
5.3×10−1
6.9
9.3×10−2
2.9
3.0
2.3×10−1
9.0
9.3
2.1
41
43
5.9
2.5×10−1
6.1
6.4×10−2
1.7
1.8
3.0×10−1
21
21
1.6
21
23
7.1
5.7×10−1
7.7
aCongener-specific profile is given in supplementary information—supplementary Tables S1 and S2 (ESM 2)
bChemically derived toxic equivalents calculated according to WHO2005–TEF values (van den Berg et al. 2006)
c2,3,7,8-TCDD, 1,2,3,7,8-PCDD, 1,2,3,4,7,8-H6CDD, 1,2,3,6,7,8-H6CDD, 1,2,3,7,8,9-H6CDD, 1,2,3,4,6,7,8-H7CDD, OCDD
d2,3,7,8-TCDF, 1,2,3,7,8-PCDF, 2,3,4,7,8-PCDF, 1,2,3,4,7,8-H6CDF, 1,2,3,6,7,8-H6CDF, 1,2,3,7,8,9-H6CDF, 2,3,4,6,7,8-H6CDF, 1,2,3,4,6,7,8-H7CDF,
1,2,3,4,7,8,9-H7CDF, OCDF
ePCB 77, PCB 81, PCB 126, PCB 169
fPCB 105, PCB 114, PCB 118, PCB 123, PCB 156, PCB 157, PCB 167, PCB 189
684Environ Sci Pollut Res (2011) 18:677–686

Page 9

released from the EDC production into the waste water
channel flowing into the Danube. Although the levels of the
sediment contamination were above the Norwegian stan-
dard for PCDD/Fs, they were more than an order of
magnitude lower compared with a similar contamination
case in Finland.
For the Pancevo plant, further investigations on soil
contamination on the site and runoff into the river Danube,
and screening for deposits in landfills, including risk
assessment for the environment and human heath, are
recommended. This assessment should be a part of the
Stockholm Convention implementation in Serbia.
Acknowledgments
Science Grant No. 143058 and the Project INCHEMBIOL
VZ0021622412. Rolf Anderson at the Department of Chemistry,
Umeå University is acknowledged for performing comprehensive
PCDD/F analysis and PCB analysis of sediments.
This work was supported by Serbian Ministry of
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Journal: ENVIRONMENTAL SCIENCE AND POLLUTION RESEARCH

Manuscript title: Characterization of dioxin-like contamination in soil and
sediments from the “hot spot” area of petrochemical plant in Pancevo (Serbia)

Sonja Kaisarevic1*, Klara Hilscherova2
Ernest Voncina5, Stanka Bobic6, Nebojsa Andric1, Kristina Pogrmic-Majkic1, Mirjana Vojinovic-
Miloradov1, John Paul Giesy7, Radmila Kovacevic1

1University of Novi Sad Faculty of Science, Laboratory for Ecotoxicology (LECOTOX), Trg D. Obradovica 2, 21000 Novi
Sad, Serbia
2Research Centre for Environmental Chemistry and Ecotoxicology (RECETOX), Masaryk University, Kamenice 3, 625 00
Brno, Czech Republic
3POPs Environmental Consulting, Ulmenstraβe 3, 73035 Göppingen, Germany
4Department of Chemistry, Umeå University, SE-90187 Umeå, Sweden
5Institute of Public Health, Prvomajska 1, Maribor, Slovenia
6Institute of Occupational Health, Futoska 121, 21000 Novi Sad, Serbia
7Dept. Veterinary Biomedical Sciences, University of Saskatchewan, Saskatoon, Canada; Dept. Zoology, National Food
Safety & Toxicology Centre, Michigan State University, Dept. Biology & Chemistry, City University of Hong Kong,
Kowloon, Hong Kong SAR, China

*Corresponding author (sonja.kaisarevic@dbe.uns.ac.rs), telephone: ++381 21 485 2675, fax: ++381 21 450 620


ONLINE RESOURCE
SUPPLEMENTARY MATERIAL – MATERIAL AND METHODS

Sample extraction and fractionation

In the first trial, a simple cleanup procedure of soil samples was performed. Briefly, dry soil (25g) was
extracted with 50ml of acetone: hexane mixture (1:1) on a shaker for 24h. After that, acetone and hexane
layers were separated and the remaining hexane extract was purified with concentrated sulfuric acid until
discoloration of the extract, and residues of sulfuric acid were then washed with distilled water. Thereafter,
samples were evaporated to dryness, dissolved in 1 ml of hexane, and concentration of 7 PCB congeners
was determined by GC/ECD analysis. The rest of the extract was evaporated to dryness, dissolved in
DMSO, and tested for toxicity in micro-EROD analysis. Such specimens were marked as refined hexane
extracts (P0-P5).
Since sample P4 contained relatively high levels of dioxin-like activity and extremely high concentrations
of PCBs, this sample was further analyzed for the presence of planar and mono-ortho PCBs and PCDD/Fs.
Dry soil sample (0.5g) was spiked with PCB internal standard (10,000 pg) and PCDD/Fs internal standard
(100 pg). Standards of PCBs and PCDD/Fs were purchased from Cambridge isotope laboratories (CIL,
Andover, MA, USA). Samples were then extracted by ultrasound and microwave in toluene (2x). Extracts
were purified by use of acidic silica gel column (layers: silica gel/conc. sulphuric acid, silica gel/KOH and
silica gel) followed by additional cleaning using adsorption chromatography on a graphitised carbon
column (Carbopack C). Obtained PCB fraction in 100% dichloromethane (DCM; 70 ml) and PCDD/F
fraction in toluene (70 ml) were concentrated to 20 µl and analysed for mono-ortho/planar PCBs and
PCDD/Fs contents. Corresponding injection of standard was added to the final extract for control of
internal standard recovery.


, Roland Weber3, Kristina L. Sundqvist4, Mats Tysklind4,

Page 12

Instrumental analysis

Residual PCBs in refined hexane extracts of soil samples P0-P5 from the first trial were determined by
GC/ECD using HP 6890 gas chromatograph with capillary column HP 19091J-416 HP-5 5% Phenyl-
Methyl-Siloxane (initial working temperature - 130oC, maximum temperature - 300oC), and electron
capture detector Varian 3400. Concentration of PCBs was determined according to Riedel-de-Haen
standard mixture of 7 PCB congeners: PCB28, PCB52, PCB101, PCB138, PCB153, PCB180 and PCB209.
Method detection limit for all compounds was 0.05 ng/g d.w.
The final identification and quantification of PCBs (planar and mono-ortho) and PCDD/Fs in the
corresponding extracts of sample P4 was carried out using high resolution gas chromatographic separation
on a HP 6890 GC (Hewlett-Packard, Palo Alto, CA, USA) coupled with Finnigan MAT 95PL (Finnigan,
Bremen, Germany) high resolution mass spectrometer. An aliquot (2 µL) of sample was injected into the
GC system, equipped with JW-DB-5MS+DG capillary column (60m x 0.25 mm i.d., film thickness 0.25
µm) in splitless mode. The mass spectrometer operates in the electron impact ionization mode using
selected ion monitoring (SIM), at minimum resolution of 10,000. Samples were analyzed for the PCDD/Fs
concentrations using the isotope dilution method based on US EPA 1613 protocol. In addition to daily
sensitivity and relative response factor (RRF) checks, the mean RRF was regularly reevaluated for each
congener. Quality control samples were included in the analysis scheme.

MicroEROD analysis

After isolation of rat hepatocytes by liver perfusion, cells were seeded in 96-well collagen-coated culture
plates (5µg collagen/well) at the density 25,000 cells/well in 0.1ml of the Williams` Medium E (WME)
supplemented with 10% foetal calf serum (FCS). After 24h recovery period, the medium was removed and
0.1ml of fresh serum-free medium containing different amounts of extracted sample equivalents, or
different concentrations of PCB126 as a reference compound, was added. PCB126 was tested in a
concentration range 10-12M – 10-6M. Refined hexane extracts of soil samples were tested in a concentration
range 1.25 – 1000 mg soil-eq./ml, with the exception of sample P4 which was tested in a concentration
range 500 ng soil-eq/ml – 50 mg soil-eq./ml. The final concentration of solvent (DMSO) did not exceed
1%, and did not cause any background response in the bioassay. After 48h of exposure, medium was
removed, cells were washed with phosphate buffer saline (PBS) and then incubated for 1h at 37°C in 0.1 ml
of WME containing 8 μM 7-ethoxyresorufin and 10 μM dicumarol. At the end of incubation time, 0.07 ml
of medium from each well was transferred to black 96-well plates and 0.13 ml of methanol was added. The
amount of formed resorufin was measured fluorometrically on a Fluoroscan Ascent FL plate reader
(ThermoLabsystems) using 544 nm excitation and 590 nm emission filter, calculated relative to resorufin
standard curve (range 1.2 nM to 78 nM), and normalized to protein content determined for each well by
fluorescamine protein assay. Cells were washed with PBS, and 150 μl of PBS and 50 μl of 1.08 mM
fluorescamine in acetonitrile was added. The amount of formed fluorescamine-protein adduct was
measured fluorometrically using excitation/emission wavelengths of 400/460 nm on Fluoroscan Ascent FL
plate reader (ThermoLabsystems), and calculated relative to bovine serum albumin standard curve. The
most toxic PCB congener - PCB126 was used as the reference compound in micro-EROD bioassay.
PCB126 dose response curve was calculated using logit-log model, while sample dose response curves
were calculated using logit-ln model and PCB126 maximum. Measurement of EROD activity for each
sample concentration was done in quadruplicate wells, and repeated in at least three independent
experiments. In each plate, eight control and eight blank wells were tested. Along with each set of samples,
standard compound PCB126 was investigated in the same analysis. Fixed effect-level of EC25 was chosen
for IEQ calculation on the basis of the shapes of dose-response curves of the referent compound and
samples, and overall intensity of response. Since the slope of the dose-response curves for samples (e.g. P3,
P4, P5) differed from the slope of the dose-response curve for PCB126, although enzyme activity reach
levels higher than 50% vs PCB126, calculation of IEQs on the basis of EC50 effect-level would result in
greater inaccuracy than the use of EC25.

Page 13

H4IIE-luciferase analysis

The H4IIE-luc cells are stably transfected with dioxin-responsive element (DRE) driven firefly luciferase
reporter gene construct, whose transcriptional activation occurs in an AhR-dependent manner (Sanderson et
al. 1996). Recombinant rat hepatoma H4IIE-luc cells were seeded in 96-well plates at the density 15,000
cells/well in 0.1 ml of the Dulbecco`s Modified Eagle’s Medium (DMEM) supplemented with 10% FCS.
Cells were pre-incubated overnight to attach and treated 24h after plating with different concentration of
standard 2,3,7,8-tetrachlorodibenzo–p-dioxin (TCDD) or soil extracts dissolved in DMSO (the final
concentration of solvent was 0.5%). TCDD was tested in a concentration range 10-12M – 5x10-9M. Refined
hexane extract and different fractions of soil sample P4 were tested in a concentration range 0.015 – 150
mg soil-eq./ml. After 24h of exposure to test samples, cells were washed twice with PBS, and luciferase
activity was measured as luminescence at 30oC with Steady-Glo Kit (Promega, USA) according to
manufacturers’ instructions using an automated microplate luminometer Ascent (Thermo Electron Corp.,
Waltham, MA, USA). Dioxin-like activity was measured repeatedly in three independent experiments,
always in triplicate wells for each sample concentration, with blanks, solvent controls and TCDD tested in
each plate. Fixed effect-level of EC50 was chosen for IEQ calculation on the basis of the shapes of dose-
response curves of the referent compound and samples, and overall intensity of response. Since the slope of
dose-response curves of the samples was similar to the slope of the dose-response curve of TCDD, EC50
effect-level was shown to be appropriate for the most precise calculation of IEQs.



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 the detection of polyhalogenated aromatic hydrocarbons. Toxicol
Appl Pharmacol 137: 316-325.

Page 14

Journal: ENVIRONMENTAL SCIENCE AND POLLUTION RESEARCH

Manuscript title: Characterization of dioxin-like contamination in soil and sediments from the
“hot spot” area of petrochemical plant in Pancevo (Serbia)

Sonja Kaisarevic1*, Klara Hilscherova2
Stanka Bobic6, Nebojsa Andric1, Kristina Pogrmic-Majkic1, Mirjana Vojinovic-Miloradov1, John Paul Giesy7,
Radmila Kovacevic1

1University of Novi Sad Faculty of Science, Laboratory for Ecotoxicology (LECOTOX), Trg D. Obradovica 2, 21000 Novi Sad, Serbia
2Research Centre for Environmental Chemistry and Ecotoxicology (RECETOX), Masaryk University, Kamenice 3, 625 00 Brno, Czech Republic
3POPs Environmental Consulting, Ulmenstraβe 3, 73035 Göppingen, Germany
4Department of Chemistry, Umeå University, SE-90187 Umeå, Sweden
5Institute of Public Health, Prvomajska 1, Maribor, Slovenia
6Institute of Occupational Health, Futoska 121, 21000 Novi Sad, Serbia
7Dept. Veterinary Biomedical Sciences, University of Saskatchewan, Saskatoon, Canada; Dept. Zoology, National Food Safety & Toxicology
Centre, Michigan State University, Dept. Biology & Chemistry, City University of Hong Kong, Kowloon, Hong Kong SAR, China

*Corresponding author (sonja.kaisarevic@dbe.uns.ac.rs), telephone: ++381 21 485 2675, fax: ++381 21 450 620

ONLINE RESOURCE
SUPPLEMENTARY MATERIAL – RESULTS AND DISCUSSION



, Roland Weber3, Kristina L. Sundqvist4, Mats Tysklind4, Ernest Voncina5,


Supplementary figure Fig. S1. EROD activity in primary rat hepatocyte culture after 48h
exposure to different concentrations of PCB126. Points represent mean values ± standard error
from nine independent experiments, expressed as % of maximal EROD activity reached at the
concentration 10-8M.

Page 15

Supplementary figure Fig. S2. EROD activity in primary rat hepatocyte culture after 48h exposure to different
concentrations refined hexane extract of soil samples P0-P5. Points represent mean values ± standard error from three
replicates, expressed as % of maximal EROD activity reached in the presence of PCB126 in the same analysis..