Impact of ozone treatment on the degradation and activity of endocrine
active substances in wastewater
H. Bielak*,***, F. Itzel**,***, A. Simon*,***, J. Tuerk**,***, E. Dopp*,***
* IWW Rheinisch-Westfaelisches Institut fuer Wasserforschung (IWW Water Center), Moritzstr. 26, 45476
Muelheim a. d. Ruhr, Germany, email@example.com
** Institut für Energie- und Umwelttechnik e. V. (IUTA, Institute of Energy and Environmental Technology),
Bliersheimer Str. 58-60, 47229 Duisburg, Germany
*** Centre for Water and Environmental Research (ZWU), University of Duisburg-Essen, Universitaetsstr. 2,
Abstract: The aim of this study was to investigate the impact of ozone treatment on substances with endocrine
activity in wastewater. More precisely, the degradation of substances with known endocrine potentials was
related to the analysis of the biological activity after ozonation. Therefor wastewater samples were spiked with
model substances, degradation was analysed with mass spectrometry, and endocrine activities were measured
with the cell-based Calux testsystem. The comparison of the chemical and biological analyses revealed that a
degradation of the substances does not always lead to an equivalent change of the biological response, especially
when the samples contain other active substances. Furthermore, it was shown that in some wastewaters masking
effects occur when agonistic and antagonistic substances are present, reinforcing the necessity to include the
investigation of combination effects in wastewater and surface water assessment.
Keywords: combination effects; endocrine disruptors; in-vitro bioassays; ozone treatment
Endocrine active substances (EAS) are commonly used as active ingredients in
pharmaceuticals and personal care products and are known to have a biological activity
already at very low concentrations (picomolar range). A complete removal of these substances
during conventional wastewater treatment is not possible, leading to the emission into surface
water through wastewater treatment plant (WWTP) effluents. There, EAS have been shown to
act disruptive in aquatic non-target organisms. By the advanced treatment with ozone
especially estrogenic substances can be effectively removed (Lee and van Gunten., 2010).
However, the impact of ozone on other EAS and the interaction of different EAS in complex
water samples still need more consideration. An extensive load of wastewater with a variety
of active agents, e.g. from hospitals, leads to the assumption that combination effects occur
which might lead to additive or masking effects (Ihara et al., 2015; Gehrmann et al., 2016).
Therefore, the aspect of hormonal combination effects was investigated in this project. To
understand in which way transformation products by the use of ozone might contribute to an
observed biological effect, the degradation of selected endocrine active substances in
wastewater was determined and the water samples tested in biological test systems
(Bioassays). The aim of this study is to verify whether the ozonation of wastewater causes a
reduction of endocrine effects by the degradation of EAS. On the other hand, effects may
occur that were not measureable earlier due to possible masking or combination effects.
Material and Methods
Wastewater samples were taken as grab samples after biological and post treatment in
municipal or hospital wastewater treatment plants (WWTP A-C). Four pharmaceuticals
(17α-ethinylestradiol, methyltestosterone, toremifene, bicalutamide) with known endocrine
activities were added to each wastewater in a defined concentration, single and as mixture.
Each native and spiked sample, respectively, was treated with ozone in laboratory scale
(approx. 5 mg ozone/L) and enriched using SPE cartridges (Oasis HLB, Waters, Germany)
for the chemical analysis with mass spectrometry. Bicalutamide-spiked samples were
measured directly without prior enrichment. Samples for biological testing were taken just
before the ozone treatment and before SPE (after ozonation, no enrichment). The activation
and inhibition, respectively, of estrogenic or androgenic effects was analysed in genetically
modified osteosarcoma cells (U2-OS) using Calux (chemically activated luciferase gene
expression) assays based on the producer’s instructions (BioDetection Systems, Amsterdam,
NL), the ISO/DIS 19040-3 (2016) and OECD TG455 & 458 (2016) with slight modifications.
Results and Conclusions
The concentrations of all substances were reduced by the ozone treatment, but with variations
between the substances and between wastewaters (table 1).
Table 1 Degradation after ozone treatment of model substances (compared to the spiked initial concentrations)
added individually and as mixture-of-four to three different wastewaters (A-C). Initial concentrations were
different for each substance.
Degradation by ozone (%)
Model substance (biological activity)
direct measurement without prior enrichment
*The half quantification limit was used for calculating values below the quantification limit
The estrogenic effect of the EE2-spiked samples measured in the bioassay was reduced
after ozonation in all three wastewaters with the strongest reduction of the single substance in
wastewater A and C (78% and 99% compared to the initial concentration), which is mainly in
accordance with the results of the chemical analysis, though a direct transfer of values is not
possible. A lower reduction was observed in wastewater B (about 9%). Also, the degradation
of the anti-estrogen toremifene in all three wastewaters led to a decrease of the inhibitory
effect of the toremifene-spiked samples, but the decrease of the effect was lower than
suggested by the chemical degradation (14%-29%). This might be due to still potent
transformation products, possibly in combination with a degradation of estrogenic substances
from the matrix. When both estrogen-modulators were present (mixture sample) even without
ozone treatment both the activation and inhibition response was lower or even not measurable
compared to individually spiked samples. After ozone treatment there was a further reduction
of the estrogenic effect, whereas the anti-estrogenic effect increased (example of wastewater
A shown in Figure 1a).
Figure 1 Endocrine effects of wastewater A spiked with model substances before and after treatment with ozone,
measured in Calux assays. EE2: 17α-Ethinylestradiol; MT: Methyltestosterone; Bic: Bicalutamide; Tor: Toremifene.
Positive control of relative activation: 17β-estradiol (33 pM) (a), dihydrotestosterone (0.1 µM) (b). Negative control
of relative inhibition: 17β-estradiol (3 pM) (a), dihydrotestosterone (0.45 nM) (b).
Although methyltestosterone was partly degraded by ozone, the androgenic activity of the
methyltestosterone-spiked samples (single and mixture) increased slightly in all wastewaters
after ozonation. When bicalutamide was added together with methyltestosterone (mixture
sample) the initial androgenic effect before ozonation was reduced by 57 ± 2 % (see Figure
1b). The anti-androgenic effect of the bicalutamide-spiked sample in the bioassay was almost
not changed by ozone treatment, which is mainly in accordance with the results of the
chemical analysis. The anti-androgenic effect in the bioassay was reduced by 59 ± 7 % when
methyltestosterone was also added to the sample, and slightly modified by ozonation.
The results demonstrate that the combination of chemical and biological analyses is
necessary to assess complex water samples. By the application of this approach to real waste
water samples, information about biological effects and sample composition as well as
formation of transformation products can be provided at the same time.
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