MONITORING DIOXIN-LIKE AND ESTROGENIC ACTIVITY USING THE EREAND DRE-CALUX BIOASSAY: CASE-STUDY IN THE ZENNE RIVERCROSSING THE BRUSSELS REGION (BELGIUM)
Vandermarken T1*, Van Langenhove K1, Croes K1, Denison MS2, Brion N1, Elskens M1
1

Vrije Universiteit Brussel, Department of Analytical, Environmental and Geo-Chemistry, Pleinlaan 2, 1050
Brussels, Belgium, 2 Department of Environmental Toxicology, University of California, Davis, CA 95616,
USA
Introduction
Emerging pollutants (EPs) are a group of chemical substances that have been recently detected in fresh waters,
as a result of human activities1. According to the NORMAN website these substances are “pollutants currently
not included in routine monitoring programmes (at least at the EU level) but which could pose a significant risk
requiring regulation, depending upon their potential (eco)toxicological and health effects and on their levels inthe (aquatic) environment”.
Among the various substances that can be categorized as EPs, endocrine disrupting chemicals (EDCs) are ofspecial concern because these substances interfere with hormone biosynthesis, metabolism, or their action results
in a deviation from normal homeostatic control or reproduction2. The group of molecules identified as EDCs ishighly heterogeneous and includes synthetic chemicals used as industrial solvents/lubricants and their byproducts, plastic/resin precursors, plasticizers, pesticides, pharmaceutical and personal care products and flame
retardants. The peculiarity of EDCs is that these substances are extremely diverse and do not apparently shareany structural similarity other than usually being of small molecular mass (<1000 Daltons), and often composedof fused aromatic rings that may contain halogen group substitutions by chlorine and bromine. This structural
diversity makes that EDCs can interact with a large number of nuclear receptors as analogs or antagonists2.
Many EDCs are still unregulated or are in process of being regulated. Therefore, water bodies currently containthousands of chemicals about which there is little information regarding levels and occurrence of suchchemicals3. They may or may not pose risks to the aquatic environment and human health, but in the absence ofregulation they are not routinely monitored. Recently in January 2013, the European Commission (IP/12/88)
proposed to add 15 chemicals to the list of 33 pollutants already regulated by the Environmental Quality
Standards Directive (2008/105/EC). Pharmaceuticals are proposed for the first time and include amongst others
the 17 α-ethinylestradiol (EE2) and the 17 β-estradiol (E2). Also found in this amendment are the dioxin-likecompounds. Scientific questions that arise towards their impact on ecosystems and human health are justified by(i) their diffuse and continuous release into the aquatic environment, which despite generally low concentrations(ng/L to µg/L) confers to EDCs a pseudo-persistent character4,5, and (ii) the fact that EDCs are chemicals withthe potential to elicit negative effects on the endocrine systems of humans and wildlife at low dose 2,6.
In order to address these important environmental issues, the main objective of this study is to monitor theoccurrence of EDCs in the Zenne, a previously polluted river crossing the Brussels region (Belgium). Although
instrumental analysis can be used to identify and quantify known EDCs, hazard evaluation based on chemicalmonitoring is complicated. Since EDCs are structurally highly distinct compounds, mixture interactions have tobe taken into account and many compounds responsible for estrogenic activity in sediment samples remain to beelucidated7. Dioxin responsive elements (DRE-) and estrogen responsive elements (ERE-) CALUX-assays(Chemical Activated Luciferase Gene Expression) were developed as mechanism-based, rapid and extremely
sensitive in vitro reporter gene bioassays to assess dioxin-like and estrogenic activity8,9. They provide useful
information about the total dioxin-like and estrogenic potential of complex mixtures of chemicals inenvironmental samples and are thus able to account for both unknown active compounds and mixture
interactions in a sample. In this study, we have used both bioassays and focused on two categories of endocrine
disrupters: (i) those interacting with the aryl hydrocarbon receptor (AhR) such as the halogenated aromatichydrocarbons involving polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs)

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and dioxin-like polychlorinated biphenyls (DL-PCBs), and (ii) those interacting with or activating the estrogen
receptor (ER) called estrogenic EDCs (EEDCs) such as natural hormones, pharmaceutical estrogens,
phytoestrogens, surfactants, as well as other industrial compounds like bisphenol A.
Materials and methods
Water samples were collected at the following sites: 3 within upstream Brussels, 1 site inside Brussels (at 39km
from Lembeek) and 3 sites in the downstream region. Sediments were collected on 5 sites using a bottom
sediment grab sampler. The samples were well mixed and aliquots were taken for physical, chemical andmicrobiological analyses.
For water samples, only the estrogenic activity was monitored as the ultimate fate of dioxin-like compounds inaquatic environments is their adsorption to sediment and suspended matter because of their high hydrophobicity.
The sediments were dried, lyophilized, and a 2g aliquot was extracted using the Accelerated Solvent Extraction(ASE) with hexane/acetone, 1:1 (v/v). Water samples were extracted using the Oasis HLB cartridge for estrogenlike compounds.
Dioxins and dioxin-like PCBs were analyzed separately with the DRE-CALUX bioassay10 and the overall
estrogen activity is analyzed using the ERE-CALUX11. Briefly, extracts are diluted and dosed in 10 different
concentrations yielding a full dose-response curve fitted with the “non-linear 4 parameter Hill function” tocalculate an Effective Concentration on 50% of the curve (EC 50). The Bioanalytical Equivalent concentration(BEQ), in pg standard-equivalence/g or l sample, is calculated as the ratio of the EC 50 of the standard and thesample curve.
Results and discussion
The main source of estrogenic active compounds in the aquatic environment is the result of their use by patientsand the treatment of domestic and hospital waste in wastewater treatment plants 4,12,13. This estrogenic activity
includes both natural and synthetic estrogens such as xenoestrogens and pseudoestrogens. Figure 1a shows thatthe estrogenic activities, expressed as pg Bioanalytical Equivalent (BEQ) per L oscillate around 200 pg BEQ/L
until km29 (inside Brussels) to increase sharply up to 938 pg BEQ/L at km39 (Vilvoorde, an industrial area).
The activity then decreases to 397 pg BEQ/L at km41 (downstream Brussels).
While most of the literature data deals with the occurrence of estrogenic chemicals in water, these substanceswere also detected in sewage sludge and sediments5,14,15. In particular, steroids are relatively lipophilic; thesemolecules can be adsorbed on suspended solids and sediments where they can contaminate benthic organisms16.
A peak of estrogenic activity in sediment of the Zenne river is found at km39 (Fig. 1b).
A totally different pattern is observed for the dioxin-like compounds. For both fractions (PCDD/Fs and dlPCBs), results are in the range of what has been previously reported in the Scheldt mouth17. The Beersel station
located at km13 shows a markedly higher PCDD/F potency compared to the other sampling stations, while thePCB potency tends to decrease from upstream to downstream Brussels (Fig. 1c).
To highlight relationships between the results of the CALUX tests and some environmental variables, weapplied a principal component analysis (PCA). The PCA loading plot for the water samples (Fig. 2a) displays therelationships between 10 variables at the same time, visualised in a two dimensional plane defined by the firsttwo principal components. The first component axis explains 68% of the variation and the second component
axis 21%. The coordinates of the original variables in the plane express their correlations with the new principalcomponents. Variables carrying similar information or varying in a comparable way are grouped together, i.e.,they are correlated. When negatively correlated, they are positioned on opposite sides of the plot origin, indiagonally opposed quadrants. The further away a variable lies from the origin, the stronger the influence of thatvariable has on the PCA model. For example, T°C, COND, DOC & POC and to a lesser extent SPM & EEDCs
are strongly correlated with PC1, whilst O2 & pH are anti-correlated. In contrast, %C-SPM is mainly correlatedwith PC2 and d50-SPM is in an intermediate situation. Orthogonal vectors in the loading plot indicate low
correlations between variables, and thus a low predictive ability.

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Estrogenic EDCs
WWTP

800

600

400

200

00102040km from Lembeek
50240

Bioanalytical Equivalent concentration (pg/g)30

1b

Estrogenic EDCs
WWTP

220

2.01c

PCDD/Fs
PCBs
WWTP

40

200

1.5

180
30

160

1.0
140
20
120
0.5

100

1080
60

00102030

40

Bioanalytical Equivalent concentration (pg/g)

Bioanalytical Equivalent concentration (pg/L)

1000

0.0
010km from Lembeek

2030

40km from Lembeek

Figure 1: Bioanalytical Equivalent (BEQ) concentration for estrogenic EDCs (EEDCs) and dioxin likecompounds in water (1a) and sediment (1b and 1c) of the Zenne (Belgium) in October 2012

1.0

1.0

A - Water%C-SPM

0.5

d50

OM

0.5
T°C
O2DOC

0.0

POCCondpH

PC2 (31.16%)

PC2 (20.73%)

B - Sedimentd50-SPM

PCBs

%C

0.0

PCDD/Fs

EEDCs

-0.5

-0.5

SPM

EEDCs

-1.0

-1.0
-1.0

-0.5

0.0

0.5

1.0-1.0

PC1 (67.83%)

-0.5

0.0

0.5

1.0

Pc1 (49.94%)

Figure 2: PCA loading plots obtained after Varimax rotation for the whole dataset. The factor loadingsrepresent the correlations between the original variables and the principal components

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For instance, it is observed that d50-SPM is not a powerful predictor for EEDCs in water samples; neither is%C-SPM. On the other hand, COND, SPM, POC & DOC are predictor variables of potential interest for EEDCs
with Pearson’s correlation coefficients ranging from 0.80 to 0.87, p < 0.05. It is not surprising that POC andDOC appear as controlling variables for EEDCs given the lipophilic nature of some of the investigatedcompounds.
For sediment, the PCA loading plot (Fig. 2b) indicates that dioxins like compounds (PCDD/Fs and DL-PCBs)
and EEDCs behave differently. The vectors are located in opposite quadrant. None of these compounds exhibits
significant correlation with the other environmental variables measured so far (OM, %C and d50).
Data on dioxin and EEDCs activities in sediment are very scarce. Most of the available BEQs are originatingfrom sewage sludge analysis and have been established for the DRE-CALUX. In Belgium, values ranged from10 to 132 pg BEQ /g dw with a median of 30 pg/g dw (15 pg/g dw in this study). Dl-PCB levels were much
lower and ranged from 0.8 to 4 pg BEQ/g dw with a median of 1.8 pg BEQ/g dw (0.8 pg/g dw in this study).
One sample reached 14 pg BEQ/g dw but was excluded from the dataset as a probable outlier10.
An increase in estrogenic activity was noticed in both sediment and water samples at km39 (Vilvoorde, anindustrial area), but further investigation is necessary to see if this site could be an active source of EEDCs.
Based on these preliminary results we were able to conclude, for water samples, that COND, SPM, POC andDOC are powerful predictors for EEDCs and that PCDD/Fs and EEDCS behave differently in the sediments.
Acknowledgements
The BG1LUC4E2 and H1L7.5c1 cell lines were developed by the University of California-Davis (USA) withfunding from a Superfund Research Program grant (ES04699) from the National Institute of Environmental
Health Sciences.
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