PresentationPDF Available

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.
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, h.bielak@iww-online.de
** 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,
45141 Essen.
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
Introduction
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)
spike type
WWTP A
WWTP B
17α-Ethinylestradiol, EE2
single
97*
68
(estrogenic)
mixture
91
61
Toremifene
single
95
62
(anti-estrogenic)
mixture
94
71
Methyltestosterone
single
33
43
(androgenic)
mixture
54
71
Bicalutamide (anti-androgenic),
single
8
11
direct measurement without prior enrichment
mixture
9
1
*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).
a b
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.
References
Gehrmann, L., Bielak, H., Behr, M., Itzel, F., Lyko, S., Simon, A., Kunze, G., Dopp, E., Wagner, M. and Tuerk,
J. (2016), (Anti-)estrogenic and (anti-)androgenic effects in wastewater during advanced treatment: comparison
of three in vitro bioassays. Environ. Sci. Pollut. Res., doi:10.1007/s11356-016-7165-4.
Ihara, M., Kitamura, T., Kumar, V., Park, C.-B., Ihara, M. O., Lee, S.-J., Yamashita, N., Miyagawa, S., Iguchi,
T., Okamoto, S., Suzuki, Y. and Tanaka, H. (2015), Evaluation of Estrogenic Activity of Wastewater:
Comparison Among In Vitro ERα Reporter Gene Assay, In Vivo Vitellogenin Induction, and Chemical
Analysis. Environ. Sci. Technol., 49(10), 6319-6326.
ISO/DIS 19040-3 (under development), Water quality -- Determination of the estrogenic potential of water and
waste water -- Part 3: In vitro human cell-based reporter gene assay. Stage 2014-11-04.
Lee, Y. and van Gunten, U. (2010), Oxidative transformation of micropollutants during municipal wastewater
treatment: comparison of kinetic aspects of selective (chlorine, chlorine dioxide, ferrate VI, and ozone) and non-
selective oxidants (hydroxyl radical). Wat. Res., 44(2), 555-566.
OECD (2016), Test No. 455: Performance-Based Test Guideline for Stably Transfected Transactivation In Vitro
Assays to Detect Estrogen Receptor Agonists and Antagonists. OECD Publishing, doi:10.1787/9789264265295-
en.
OECD (2016), Test No. 458: Stably Transfected Human Androgen Receptor Transcriptional Activation Assay
for Detection of Androgenic Agonist and Antagonist Activity of Chemicals. OECD Publishing,
doi:10.1787/9789264264366-en.
Richard, J., Boergers, A., vom Eyser, C., Bester, K., Tuerk, J. (2014), Toxicity of the micropollutants Bisphenol
A, Ciprofloxacin, Metoprolol and Sulfamethoxazole in water samples before and after the oxidative treatment.
Int. J. Hyg. Environ. Health, 217(4/5), 506-514.
ResearchGate has not been able to resolve any citations for this publication.
Article
Full-text available
(Anti-)Estrogenic and (anti-)androgenic effects in wastewater during advanced treatment: Comparison of three in vitro bioassays L. Gehrmann & H. Bielak et al., 2016 Endocrine disrupting chemicals, which are discharged into the environment mainly by wastewater treatment plants (WWTP), have been shown to induce adverse effects in aquatic life and to be of potential risk for human. Advanced treatment with ozone successfully removes organic micropollutants but, in previous studies, an increase of estrogenic effects after the ozonation of hospital wastewater was observed. In order to investigate this effect, estrogenic and androgenic as well as anti-estrogenic and anti-androgenic activities were determined during treatment of hospital wastewater using three different effect-based reporter-gene bioassays. Despite different matrix influences, sensitivities and test-specific properties, the actual assessment of the obtained results was in accordance in all systems. Estrogenic and androgenic activities were mainly reduced during the biological treatment and further removed during ozonation and sand filtration, resulting in no detectable agonistic activities in the final effluent. Antagonistic effects were removed in the biological treatment by up to 50% without further reduction in the advanced treatment. Therefore, this study showed the relevance of antagonistic activities hospital WWTPs and illustrates the need for a better understanding about antagonistic effects.
Article
Full-text available
Various oxidants may be applied for micropollutant transformation in water treatment in systems such as wastewater, water reuse and drinking water. The efficiency of these processes depends on the kinetics of the reaction of the oxidants with the target compounds. In addition, the stability of the oxidant in a specific water matrix and the resulting oxidant exposures are important. These two factors together determine the efficiency of a particular oxidation process. Information on the transformation of a target compound (kinetics) has been extended for the last four decades. In this paper we will review and compare kinetic aspects for micropollutant transformations during water treatment by various oxidants, such as chlorine, chlorine dioxide, ferrate VI , OH radicals, and ozone.
Article
The in vitro estrogen receptor (ER) reporter gene assay has long been used to measure estrogenic activity in wastewater. In a previous study, we demonstrated that the assay represents net estrogenic activity in the balance between estrogenic and antiestrogenic activities in wastewater. However, it remained unclear whether the net estrogenic activity measured by the in vitro ERα reporter gene assay can predict the in vivo estrogenic effect of wastewater. To determine this, we measured the estrogenic and antiestrogenic activities of wastewater and reclaimed water by the in vitro ERα reporter gene assay; the expression of vitellogenin-1 (vtg1) and choriogenin-H (chgH) in male medaka (Oryzias latipes) by quantitative real-time PCR; and estrone, 17β-estradiol, estriol, and 17α-ethynylestradiol concentrations chemically to predict estrogenic activity. The net estrogenic activity measured by the in vitro medaka ERα reporter gene assay predicted the in vivo vtg1/chgH expression in male medaka more accurately than the concentrations of estrogens. These results also mean that in vivo vtg1/chgH expression in male medaka is determined by the balance between estrogenic and antiestrogenic activities. The in vitro medaka ERα reporter gene assay also predicted in vivo vtg1/chgH expression on male medaka better than the human ERα reporter gene assay.
Article
The amount of organic micropollutants detected in surface waters increases steadily. Common waste water treatment plants are not built to remove these substances. Thus there is a need for new technologies. A promising technology is the use of advanced oxidation processes through which organic micropollutants can be removed from waste water. However, the formation of oxidation by-products is likely and needs to be investigated since the by-products not only differ from their parent compounds in regard to their chemical and physical properties but they can also differ in toxicity. Therefore this study was designed to combine chemical and toxicological analyses of the advanced oxidation (O3 [5mg/L] or UV/H2O2 [Hg-LP lamp; 15W; 1g/L H2O2]) of waste water treatment plant effluents and pure water. Effluent samples from conventional activated sludge waste water treatment (mechanical treatment, activated sludge basin, and primary as well as secondary treatment steps) and high-purity deionized water (pure water) were spiked with Bisphenol A, Ciprofloxacin, Metoprolol or Sulfamethoxazole and treated with O3 or UV/H2O2. For the toxicological analyses mammalian cells (CHO-9, T47D) were exposed to the water samples for 24h and were tested for cytotoxicity (MTT Test), genotoxicity (Alkaline Comet Assay) and estrogenicity (ER Calux(®)). The results indicate that the oxidative treatment (O3 or UV/H2O2) of Bisphenol A, Metoprolol, Sulfamethoxazole or Ciprofloxacin in waste water did not result in toxic oxidation by-products, whereas the UV/H2O2 treatment of Bisphenol A and Ciprofloxacin in pure water resulted in by-products with cytotoxic but no estrogenic effects after 60min.
Water quality-Determination of the estrogenic potential of water and waste water-Part 3: In vitro human cell-based reporter gene assay
  • Iso Dis
ISO/DIS 19040-3 (under development), Water quality-Determination of the estrogenic potential of water and waste water-Part 3: In vitro human cell-based reporter gene assay. Stage 2014-11-04.
Test No. 455: Performance-Based Test Guideline for Stably Transfected Transactivation In Vitro Assays to Detect Estrogen Receptor Agonists and Antagonists
OECD (2016), Test No. 455: Performance-Based Test Guideline for Stably Transfected Transactivation In Vitro Assays to Detect Estrogen Receptor Agonists and Antagonists. OECD Publishing, doi:10.1787/9789264265295-en.
Test No. 458: Stably Transfected Human Androgen Receptor Transcriptional Activation Assay for Detection of Androgenic Agonist and Antagonist Activity of Chemicals
OECD (2016), Test No. 458: Stably Transfected Human Androgen Receptor Transcriptional Activation Assay for Detection of Androgenic Agonist and Antagonist Activity of Chemicals. OECD Publishing, doi:10.1787/9789264264366-en.