Estrogenicity of 75 European waste water effluents
evaluated by in vitro assay
Barbora
1
Jarošová ,
R.
2
Loos ,
B.
2
Gawlik ,
J. P.
3
Giesy ,
L.
1
Bláha
1 Research Centre for Toxic Compounds in the Environment (RECETOX), Masaryk University, Brno, Czech Republic
2 EC JRC Ispra, Italy
3 University of Saskatchewan, Saskatoon, Canada
e-mail: jarosova@recetox.muni.cz
Introduction and aim of the study
 In 2010, effluents from 90 European waste water treatment plants (WWTPs) were collected and analysed in total for 168
organic chemicals and 20 inorganic trace elements to obtain a comprehensive snapshot on many so far only locally
investigated compound classes.
 Steroidal estrogens (17β-estradiol, estrone, and17α-ethinylestradiol), which are known to have the highest estrogenic potential,
were not detected in any of these sample above detection limit of 10 ng/L (report in preparation).
 The analyses were complemented also by applying effect-based monitoring approach. Here we present the results
of estrogenicity of 75 of these samples, which were analysed by MVLN in vitro reporter gene bioassay.
Material and methods
Sampling sites
• 75 European WWTPs effluents from 16 countries
• 2 types of municipal WWTPs :
i) with both domestic and industrial waste waters. These
WWTPs usually served to larger urban areas (results of most
of them are displayed in Figure 1, 2 and partially 3).
ii) with only domestic sources either with or without rain water.
These WWTPs served to smaller municipalities (results
of most of them in Figure 4 and partially 3).
All municipal WWTPs, results of which are reported here,
used primary treatment followed by secondary activated
sludge treatment, mostly including nitrification and
denitrification, and phosphorus removal.
• Industrial WWTPs effluents were sampled. The industrial WWTPs
effluents were taken from different branches of industry (e.g.
pharmaceutical, foot, textile and light industry).
• Not all participants of the campaign (owners of the WWTPs)
provided the information about waste water sources, technology of
treatment and plant capacity and therefore only 51 out of all 75
WWTPs could be categorized.
Sample preparation / Solid-phase extraction (SPE)
-> 0.5 L of 24h composite samples or garb samples
-> Solid phase extraction (6 mL, 500 mg, SDB Waters Oasis, CZ)
-> Methanol elution
-> Nitrogen stream evaporation
-> Final extracts of volumes which corresponded to 1200-times
concentrated original effluents. This aliquot was chosen a as maximal
concentration which was mostly not cytotoxic to the cells in our
previous studies, and enabled detecting estrogenic activity with limit of
detection (LOD) for estrogenicity 0.5 ng/L EEQ (estrogen equivalents).
-> Sample extracts were stored at -18° C until analyses.
In vitro bioassays
• To determine estrogenicity of the sample extracts, MVLN (human
breast carcinoma) cells stably transfected with luciferase gene
under the control of estrogen receptor were used.
• The results were expressed as estrogenic quivalents (EEQ) with
respect to standard estrogen (17β-estradiol, E2).
• Estrogenic potential quantification:
After subtraction of the solvent control response, detected
induction of luminescence was related to the maximal
response of standard ligand (E2max) and converted into
percentages of E2max.
The maximal induction as well as the shape of the curve
differed among samples. We used a EEQ25 estimate (based
on the 25% E2max response) to avoid any predictions beyond
the measured responses with all samples.
EEQ25 values were based on relating the amount of E2
causing 25% of the E2max response (EC25) to the amount of
sample causing the same level of response.
Values were determined from the nonlinear logarithmic
regression (GraphPad Software, San Diego, USA).
MCF – 7 (MVLN)
Results
Twenty seven out of the 75 extracts of WWTP effluents showed significant estrogenic activity higher than the
detection limit 0.5 ng/L EEQ. The positive values ranged from 0.53 to 17.9 ng/L EEQ (median of the positive
values1.2 ng/L EEQ, mean 2.7 ng/L EEQ).
•
Estrogenicity of 4 out of 10 effluents from large municipal WWTPs (capacity > 500,000 equivalent
citizens) was greater than our detection limit (Figure 1). The maximal EEQ was as high as 12.2 ng/L.
•
Five out of 10 effluents from European municipal WWTPs with capacity from 100,000 to 500,000
equivalent population exceeded our LOD (Figure 2). The maximal EEQ concentration was 4.1 ng/L. In case of
2 effluent samples from this type of WWTP cytotoxic or antiandrogenic effects could mask the estrogenicity.
•
Three out of 14 effluents from smaller European municipal WWTPs with capacity from 10,000 to
100,000 equivalent citizens displayed estrogenic activity higher than LOD and one sample was cytotoxic or
antiestrogenic. The maximal EEQ concentration was 3.6 ng/L (Figure 3).
•
Estrogenicity of 3 out of 7 tested effluents from municipal WWTPs with capacity up to 10,000 equivalent
population exceeded our LOD and 1 effluent was cytotoxic or antiestrogenic. The maximal EEQ concentration
was 1.9 ng/L (Figure 4). These effluents originated mostly from waste waters of domestic origin with minimal
industrial sources.
•
Estrogenic activity of 5 out of 11 treated industrial waste waters was higher than our LOD (0.5 n/L EEQ)
but 5 samples were cytotoxic or antiestrogenic so the actual estrogenic activity was not possible to detect. The
maximal detected concentration of EEQ was 3.4 ng/L for this type of effluent.
Figure 1: Estrogenic activity of extracts of effluents from
European municipal WWTPs with capacity > 500,000
equivalent citizens. Numbers without values represent WWTPs
effluents with EEQ < 0.5 ng/L.
Figure 2: Estrogenic activity of extracts of effluents from
European municipal WWTPs with capacity from 100,000 to
500,000 equivalent citizens. Numbers without values represent
WWTPs effluents with EEQ < 0.5 ng/L.
Figure 3: Estrogenic activity of extracts of effluents from
European municipal WWTPs with capacity from 10,000 to
100,000 equivalent citizens. Numbers without values represent
WWTPs effluents with EEQ < 0.5 ng/L.
Figure 4: Estrogenic activity of extracts of effluents from
European municipal WWTPs with capacity lower than 10,000
equivalent citizens. Numbers without values represent WWTPs
effluents with EEQ < 0.5 ng/L.
Conclusions
 Estrogenicity of 75 European waste water effluents was evaluated by MVLN in vitro assay
 27 sample extracts showed significant estrogenic activity higher than the detection limit > 0.5 ng/L EEQ.
 The positive values ranged from 0.53 to 17.9 ng/L EEQ (median of the positive values1.2 ng/L EEQ, mean
2.65 ng/L EEQ).
 Nine of the 48 negative samples were cytotoxic or antiestrogenic, which might mask their estrogenicity.
 The highest activities have been detected in WWTPs at some of the major European capital cities
indicating the importance of this contamination source.