Journal of Environmental Chemical Engineering 9 (2021) 106442

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Journal of Environmental Chemical Engineering

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Degradation of hormones in tap water by heterogeneous solar

TiO2-photocatalysis: Optimization, degradation products identification,
and estrogenic activity removal
Rodrigo Nogueira Padovan a, Lucas Sponton de Carvalho b, Patrícia Luisa de Souza Bergo d,
Chubraider Xavier a, *, Andrei Leitão c, Álvaro José dos Santos Neto b, Fernando Mauro Lanças b,
Eduardo Bessa Azevedo a
a
University of São Paulo (USP), Chemistry Institute of São Carlos (IQSC), Laboratório de Desenvolvimento de Tecnologias Ambientais (LDTAmb), Avenida Trabalhador
São-Carlense, 400 – Centro, P. O. Box: 780, São Carlos 13560-970, SP, Brazil
b
University of São Paulo (USP), Chemistry Institute of São Carlos (IQSC), Grupo de Cromatografia (CROMA), Avenida Trabalhador São-Carlense, 400 – Centro, P. O.
Box: 780, São Carlos 13560-970, SP, Brazil
c
University of São Paulo (USP), São Carlos Institute of Chemistry (IQSC), Medicinal & Biological Chemistry Group (NEQUIMED), Avenida Trabalhador São-Carlense,
400 – Centro, P. O. Box: 780, São Carlos 13560-970, SP, Brazil
d
Federal University of São Carlos (UFSCar), Department of Chemistry (DQ), Rodovia Washington Luiz, km 235, P.O. Box: 676, São Carlos 13565-905, SP, Brazil

A R T I C L E I N F O A B S T R A C T

Editor: Dr. G.L. Dotto Hormones are one of the most hazardous classes of Contaminants of Emerging Concern (CEC) found in tap water,
being significantly recalcitrant regarding traditional water treatment processes. In this work, the hormones
Keywords: estrone, 17β-estradiol, 17α-ethinylestradiol, and estriol, present in tap water, were degraded by heterogeneous
estrone solar photocatalysis in a flat-plate photochemical reactor (recycle mode) using TiO2 (P25, Evonik) as the pho­
17β-estradiol
tocatalyst. A fully automated analytical method based on online SPE and LC-UV was developed and validated for
17α-ethinylestradiol
monitoring those molecules. For a fixed amount of sample (125 μL), the limit of quantification was 10 μg L− 1 (for
estriol
estrogenic activity all four hormones) with coefficients of variation smaller than 20%, and accuracy between 80% and 120%, all of
MCF-7 which are acceptable figures for this kind of determination. The reactional system was optimized with the aid of a
22 factorial design. At optimized conditions (pH ≈ 6.8, flow rate 250 mL min− 1), the removal of the four hor­
mones (250 μg L− 1 each) was approximately 85% after 3 h of treatment. However, even after 9 h of treatment, it
was not possible to completely remove the estrogenic activity (MCF-7 cells) of the treated tap water. The
structures of some degradation products were proposed.

1. Introduction essentially, on the physical-chemical process of floc-coagulation and

Water treatment technology is a critical topic of discussion, as the use
of water resources defines how the whole society and the environment
develop. The depletion of freshwater is severe in Africa and Asia, and the
United Nations Educational, Scientific and Cultural Organization
(UNESCO) estimates that only 39% of the world population has access to
safely managed drinking water services, while 48% has no access to
water with any kind of treatment at all [1]. The traditional treatment of
urban waters is focused on the removal of suspended solids and is based,
decantation, followed by disinfection. However, those processes are not
able to completely remove the so-called Contaminants of Emerging
Concern (CECs), once they are not meant for that. CECs are species that
occur in the environment at concentrations ranging from ng L− 1 to μg
L− 1 and are not currently monitored by environmental agencies,
although their chronic effects are being reported in the literature [2].
Several CECs (for instance, drugs and natural hormones excreted by
the population) act as endocrine disruptors (ED). When unwillingly
ingested, those chemicals may be responsible for anomalies in the

* Corresponding author.
E-mail addresses: rodrigopadovan@iqsc.usp.br (R.N. Padovan), lucassponton@gmail.com (L.S. de Carvalho), patriciabergo@gmail.com (P.L. de Souza Bergo),
chubraiderxavier@usp.br (C. Xavier), andleitao@iqsc.usp.br (A. Leitão), alvarojsn@iqsc.usp.br (Á.J. dos Santos Neto), flancas@iqsc.usp.br (F.M. Lanças), bessa@
iqsc.usp.br (E.B. Azevedo).

https://doi.org/10.1016/j.jece.2021.106442
Received 4 August 2021; Received in revised form 16 September 2021; Accepted 21 September 2021
Available online 24 September 2021
2213-3437/© 2021 Elsevier Ltd. All rights reserved.
R.N. Padovan et al.
Fig. 1. Scheme of the used SPE-online chromatograph.
Fig. 2. Scheme of the flat-plate photochemical reactor used in the solar pho­
tocatalytic experiments. Note: a) TiO2-coated glass plate over which water
flows; b) hormone-contaminated water reservoir; c) magnetic stirrer; d) recycle
pump; e) bypass; f) flow control (valve and rotameter); g) reservoir filled with
replacement distilled water; h) solenoid valve for distilled water replacement; i)
automation device for distilled water replacement; j) contaminated-water
level sensor.
Table 1
Factors studied with coded and real values in the 22 full factorial design.
Factors Levels
(–1) (+1)
x1 : pH 4.5
x2 : Flow rate (mL min− 1) 150 300
metabolism [3]. ED-laden water consumption has been reported, in the
literature, as one of the causes for several types of cancer and develop­
mental delay in children [4,5]. Estrone (E1), 17β-estradiol (E2), estriol
(E3), and 17α-ethinylestradiol (EE2) are of worldwide concern [6], as
they may have deleterious effects even at low concentrations. For
instance, few days of exposure to aqueous E2 (1 ng L− 1) or EE2 (0.1 ng
L− 1) caused fish to change their sexes [7].
In this context, alternative water treatment technologies have been
developed as an add-on to the traditional ones. That is the case of the
advanced oxidation processes (AOPs), which are based on the in-situ
generation of radical species, aiming at oxidizing the target-molecules.
Among them, heterogeneous photocatalysis has risen as a prominent
technique, especially with titanium dioxide (TiO2): a recyclable
Journal of Environmental Chemical Engineering 9 (2021) 106442
photocatalyst that presents low cost, low ecotoxicity, and large pH range
usability [8]. TiO2 exhibits three possible crystalline phases: brookite,
the hardest one to be obtained in a pure state; rutile, the one with the
lowest photocatalytic activity; and anatase, the most photoactive one [9,
10]. Despite the higher activity of anatase, literature reports that Evo­
nik’s TiO2 (P25), a mixture of 70:30 anatase:rutile, gives superior re­
sults. Some authors argue that this effect is partially due to the formation
of heterojunctions between those two phases [11].
There are two main ways of working with TiO2 particles in photo­
catalytic experiments: in suspension or immobilized on a solid substrate.
The former leads to a higher specific surface area, but requires a further
step to recover the photocatalyst; the latter, although no separation is
needed, presents a considerably lower specific surface area [12].
Another approach aiming at decreasing treatment costs is the use of
solar-based AOPs. Although TiO2 is only excited by photons whose
wavelengths are greater than 388 nm (UV-A), corresponding to
approximately 4% of the incident solar radiation at the Earth surface,
several works show the feasibility of this kind of system for the removal
of many compounds, hormones included [13–15].
Although degradation studies of CECs, like hormones, in real
matrices, are of utmost importance, they are difficult to be performed
due to the low concentrations involved and severe interferences from
the matrix. This is possible nowadays thanks to advances in analytical
instrumentation for chromatography and mass spectrometry [16,17].
The full automatization of the analytical process, with online extraction,
makes it possible to work with a significant throughput. Another useful
approach for complex analyses is column switching: it is based on the
use of valves, changing the path of the analytes through the chromato­
8.0 graph. It makes it possible to use an extractor column or multidimen­
sional separation processes [18].
Therefore, one of the objectives of this work was to develop a fully
automated analytical method and use it in the optimized degradation of
estrone, 17β-estradiol, 17α-ethinylestradiol, and estriol, in tap water.
Those hormones were degraded by heterogeneous solar photocatalysis
in a TiO2-coated flat-plate photochemical reactor, following a 22 facto­
rial design. The degradation/energy ratio profile was obtained, some
degradation products were identified by mass spectrometry, and the
estrogenic activity after treatment was determined using the MCF-7 cell
line. Those are hormone-dependent human-breast cancer cells, in which
cell growth is driven by the addition of estradiol, previously described in
[19].
2
R.N. Padovan et al.
Table 2
The four analyzed hormones (SIGMA-ALDRICH, 95% of purity).
Name Structure
Estrone (E1)
17β-Estradiol (E2)
17α-Ethinylestradiol (EE2)
Estriol (E3)
Note: 1Lewis; Archer [25]; 2Marques et al. [26]; 3Chemaxon [27].
2. Materials and methods
2.1. Online-SPE columns manufacture
The extraction columns were manufactured using stainless steel
tubes (60 mm length, 0.500 mm internal diameter). They were filled
with 10 mg StrataX™ phase (Phenomenex, USA) suspended in 95:5
water:methanol (in volume) solution. The suspension was pumped
(methanol 0.5 mL min− 1 for 15 min) into the stainless tube, which was
closed by a 10.0 µm pore size stainless steel screen filter (VICI VALCO,
Switzerland) grid at the exit end. Finally, the particles (33 µm average
diameter and 800 m2 g− 1 specific surface area) were contained by
another screen filter (at the other end) and a pair of end fittings. The SPE
columns were conditioned with 60 μL min− 1 of water for 60 min before
use.
2.2. Liquid chromatography analyses
The chromatographic setup used is shown in Fig. 1. An UltiMate
3000 RSLCnano (Thermo Scientific, USA) chromatograph was used for
this assembling.
The chromatograph was composed of: 125-μL sample loop, six-port
valve, ternary pump for carrying the sample using water as the solvent
(pump A, 60 μL min− 1), binary capillary pump for carrying the mobile
phase (pump B), and variable wavelength UV-Vis detector with four
channels (10-mm optical path length, 45-nL cell volume). The analytical
column was an Acclaim PepMap RSLC C18 (0.003 × 150 mm, 2 µm
particle size).
The six-port valve had two positions, A and B, as shown in Fig. 1. At
position A, 125 μL of sample were carried to the SPE-online column by
pump A, while pump B conditioned the analytical column. After 4 min,
the valve was switched to position B, the sample was carried to the
analytical column by pump B, while pump A cleansed the SPE column.
After 12 min of injection, the valve was switched back to position A,
conditioning the column for a new run.
The chromatographic runs were performed in reverse mode and
gradient elution, using water (A) and acetonitrile (B): 0–4 min, 40% B;
4–6 min, 40–60% B; 6–10 min, 60% B; 10–11 min, 60–70% B;
11–12 min, 70–40% B. The method qualification is detailed in the
Journal of Environmental Chemical Engineering 9 (2021) 106442
Molar mass (g mol− 1) Water solubility (mg L− 1
at 20ºC) logDpH=7.4 pKa
270.37 5.03 4.31 10.771
272.38 13.03 3.753 10.711
296.40 4.83 4.153 10.333
288.38 13.03 2.673 10.42
Supplementary Material.
2.3. Identification of degradation products
Degradation products were determined by LC-MS/MS experiments.
An HPLC Shimadzu Serie 20 A Prominence™ (Shimadzu, Japan) with a
Kinetex™ XB-C18 (100 × 2.1 mm, 2.6 µm Phenomenex, USA) was used.
The separations were performed in reverse mode, gradient elution, with
water (A) and acetonitrile (B): 0–3 min, 5% B; 3–12 min, 5–60% B;
12–14 min, 60–95% B; 14–20 min, 95% B; 20–22 min, 95–5% B;
22–26 min, 5% B (0.25 mL min− 1 flow rate, 40ºC column heater
temperature).
A micrOTOF-Q II (Bruker Daltonics, USA) was coupled with the
HPLC. The analyses conditions were: electrospray ionization source
(capillary voltage 4.5 kV, desorption temperature 200ºC for both nega­
tive and positive modes), dryer gas flow rate 8 L min− 1, nebulizer
pressure 4 bar, m/z range 50–3000 u, acquisition rate 2 Hz, full MS
mode.
2.4. Photochemical reactor
A flat-plate photochemical reactor (Fig. 2) was manufactured ac­
cording to Vilela et al. [20] using TiO2 (P25, Evonik) catalyst and solar
light for the degradation experiments.
The plates (240 × 445 mm) were cleansed first with detergent and
then with 11 mol L− 1 nitric acid solution. An aqueous TiO2 suspension
with a mass ratio of 1% at pH 3 (HNO3) was sprayed over the plates,
which were then oven-dried at 100ºC. This procedure was repeated 10
times.
The reactor was operated in recycle mode. The hormone-
contaminated water remained under magnetic stirring. The reactor
flow was controlled by a valve/rotameter. A bypass prevented pump
malfunctioning. There was also a system to replace the water lost by
evaporation. Between samples, the reactor was completely drained.
Afterwards, 2 L of tap water were allowed to circulate through the
reactor, under the sun, for at least 15 min.
During the photocatalytic experiments, the incident solar radiation
was continuously monitored (every 15 min) by a StellarNet EPP2000C
spectroradiometer at the same height and angle of the irradiated plates.
3
R.N. Padovan et al.
Fig. 3. Preliminary experiments: (a) dark sorption on the reactor (plates without TiO2); (b) dark sorption on the surface of the catalytic plates (with TiO2); (c) direct
(solar) photolysis; and (d) solar photocatalysis. Experimental conditions: 250 μg L− 1 of each hormone, pH 4.0, and flow rate 100 mL min− 1.
2.5. Preliminary experiments
First, the feasibility of the proposed reactor for degrading the four
hormones studied here was assessed. Moreover, three controls were
performed to be sure that the solar TiO2-photocatalytic degradation
would be significantly higher than direct photolysis and dark sorption.
Although tap water has a slightly acidic nature, pH was set to 4.0, trying
to avoid major changes in water acidity during the experiments.
The initial concentration of each hormone was 250 µg L–1 and the
reactor flow rate was arbitrarily set at 100 mL min− 1. The controls were:
(a) dark sorption of the hormones in the reactor (plates without TiO2);
(b) dark sorption of the hormones on TiO2; and (c) direct photolysis
(plates without TiO2). For comparison purposes, (d) solar photocatalysis
at these very same conditions was performed.
2.6. Factorial design
After the significance of the solar TiO2-photocatalytic degradation
was checked, the reactor performance was optimized using a 22 factorial
design (two factors, two levels) in duplicate (Table 1). The studied
factors were pH (x1 ) and flow rate (x2 ).
pH levels were set at 4.5 and 8.0. Those values are approximately a
hundred times more acidic and more alkaline than the TiO2 isoelectric
4
Journal of Environmental Chemical Engineering 9 (2021) 106442
point (pI), respectively [21]. Flow rate levels were 150 and
300 mL min− 1, based on preliminary experiments (not shown here). All
experiments were randomly performed to avoid systematic errors.
Coding equations: x1 = pH−1.75
6.25
and x2 = Flow rate− 225
.
75
2.7. Estrogenic activity
Adherent MCF-7 cells were cultivated in Dubelco’s modified Eagle
medium (DMEM) supplemented with glucose (3.5 g L− 1), 10% (v/v)
fetal bovine serum (FBS), 1% (v/v) penicillin/streptomycin solution
[22] in a 37 ◦ C incubator with 5% CO2 atmosphere and 90% humidity.
At 80% confluence, cells were washed with PBS and trypsin, incubated
for 10 min, and centrifuged at 250 RCF (relative centrifugal force) for
5 min. The supernatant was removed, and the cells were resuspended
with medium to reach 1.0 × 105 cells mL− 1. Then, 100 μL were trans­
ferred to a 96-well plate, following 24 h incubation. The medium was
replaced by charcoal-treated DMEM without phenol red with the same
supplements previously described. The preparation of the
charcoal-treated medium was described by Gomes et al. [19]. The pos­
itive control was made with 10 nmol L− 1 estradiol in 0.5% (v/v) ethanol,
while the negative control only had the treated medium with 0.5%
ethanol. The same procedure was performed with reactor samples
(containing the analytes), diluted 90 times. The positive control
R.N. Padovan et al.
Fig. 4. Pareto charts for the results of the performed 22 full factorial design: (a) Estrone (E1), (b) 17β-estradiol (E2), (c) 17α-ethinylestradiol (EE2), and (d) Es­
triol (E3).
response at time 0 h was used as the reference for the estrogenic activity
of the water samples.
Diluted samples were sterilized by a polyvinylidene difluoride
(PVDF) 0.22 µm membrane and inoculated into the medium. Cells were
cultivated up to six days at 37ºC in an incubator, where the medium was
replaced every 48 h. The estrogenic activity was measured by the MTT
colorimetric assay, which is based on the conversion of 3-(4,5-dime­
thylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, yellow) to
formazan (violet) by alive cells. The violet crystals were solubilized and
the respective absorbances were measured at 570 nm [23].
3. Results and discussion
3.1. Chromatographic method qualification
Table 2 shows the structures and some properties of the four studied
hormones. It is possible to observe that those four molecules have
similar structures and molecular masses, with pKa values showing that
deprotonation occurs in alkaline pH (above 10), which is unlikely to
happen in the studied matrix (tap water). Those molecules are water-
soluble on a milligram-per-liter basis. logDpH=7.4 values suggest that
those molecules interact with both hydrophilic and hydrophobic sub­
stances [24]. Therefore, reverse-phase liquid chromatography is
adequate for separation purposes.
The qualification results of the chromatographic method are detailed
in the Supplementary Material (Figs. S1–S7, Tables S1–S6). In short, the
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Journal of Environmental Chemical Engineering 9 (2021) 106442
Limit of Detection (LoD) and the Limit of Quantification (LoQ) for the
four hormones were determined as 3.2 and 10 μg L− 1, respectively.
Linearity was tested from 10 to 250 μg L− 1, with R2 ranging from 0.987
to 0.997 (weighted linear regression model).
The asymmetries of the chromatographic bands were 1.0–1.2 for all
tested analytes; their respective chromatographic resolution varied from
2.34 to 3.03. Intraday precisions were estimated as the obtained relative
standard deviations (RSD), which ranged from 0.758% to 14.0% for the
four analytes; inter-day precisions varied from 4.5% to 17.6%. Accu­
racies were above 80% for three concentration levels (15, 130, and
230 µg L–1); similar figures were obtained for the inter columns accuracy
(10, 150, and 250 µg L–1). The matrix effect was measured by the re­
covery of the four analytes, which ranged from 80.9% to 90.4%. All
those values are considered acceptable by a series of guidelines [28].
3.2. Preliminary experiments
Fig. 3 shows the results obtained in the control experiments. One can
observe (Fig. 3a) that even after 20 h of dark sorption (without TiO2
plate), hormone removal did not exceed 20%. Regarding dark sorption
on the TiO2 plate and direct (solar) photolysis (Fig. 3b and c), removals
were approximately 5% and 10%, respectively. Those removals can be
neglected when compared to the ones obtained in the photocatalytic
tests (more than 90% for all hormones in 3 h, Fig. 3d).
R.N. Padovan et al.
Fig. 5. Flow rate optimization: (a) Estrone (E1), (b) 17β-estradiol (E2), (c) 17α-ethinylestradiol (EE2), and (d) Estriol (E3).
3.3. Optimization experiments
Table S7 shows the results of the 22 factorial design performed. The
dependent variable (response) was the ratio between the percentual
degradation of each hormone and the average incident energy at each
experiment. As those averages were different for each experimental
condition of the design, normalization of the data was required to allow
for experiments to be compared.
Fig. 4 shows the respective Pareto chart for each response. In all of
them, flow rate was the only statistically significant factor at 95% con­
fidence interval (bars that cross the red line), within tested levels. Flow
rate exhibited a positive value in all cases, meaning that to achieve
higher degradations, this factor must be increased. Up to a certain flow
rate, this is an expected result: as the degradation takes place only on the
plate, the higher the number of passes, the higher the degradation. pH
was not statistically significant at 95% confidence interval. As the pKa of
all tested hormones are higher than 10, they had no charge at the tested
pH (4.5 and 8.0); similarly, as the TiO2 pI (isoelectric point) is 6.25 [21],
the TiO2 surface was positive at pH 4.0 and negative at pH 8.0. There­
fore, it was highly expected that the pH factor showed no statistical
significance, as no electrostatic attraction occurred at both tested levels.
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Journal of Environmental Chemical Engineering 9 (2021) 106442
However, those two pH were tested because they could be important for
the control experiments of the system (dark adsorption and direct
photolysis).
As pH was not statistically significant (within the tested range),
further experiments were performed at the natural pH of the tap water
used, i.e., approximately 6.8. Flow rate was optimized by varying it from
150 to 400 mL min− 1, at 50 mL min− 1 steps (the smallest division of the
rotameter scale). The best flow rate range was between 200 and
250 mL min− 1 (Fig. 5). During the four hours of each experiment, flow
rate decreased a little (approximately 20 mL min− 1), but in a repro­
ducible way. Therefore, it was set at 250 mL min− 1 at the beginning of
the experiments so that, by their end flow rate was always within that
range.
Fig. 5 shows a typical behavior regarding flow rate. As previously
described, as the number of passes increases, so does degradation.
However, when exceedingly high flow rates are applied to the system,
the reduced contact time between the hormones and TiO2 impairs
degradation.
R.N. Padovan et al.
Fig. 6. Degradation/(incident) energy ratio profile for the four hormones (in triplicate): (a) Estrone (E1), (b) 17β-estradiol (E2), (c) 17α-ethinylestradiol (EE2), and
(d) Estriol (E3).
3.4. Degradation/energy ratio profile
Once the parameters were optimized, the degradation/(incident)
energy ratio profiles could be obtained. That is an important measure as
it is not possible to directly compare two degradation experiments
performed under different climatic conditions and/or different places.
Nevertheless, the degradation obtained with a certain dose of energy is
independent of those experimental conditions, therefore being useful for
comparisons. Fig. 6 shows those profiles for the four hormones.
It is possible to observe that with 5.5 J cm− 2, more than 80% of the
hormones were degraded. Degradations with more than that dose were
difficult to measure because concentrations lower than the LoQ were
reached.
As all experiments herein described were performed with a single
TiO2-coated plate, and replicates were always consistent with one
another, one can say that the TiO2-coated plate exhibited extended
reusability.
3.5. Degradation products identification
During mass analyses, the four tested hormones exhibited low
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Journal of Environmental Chemical Engineering 9 (2021) 106442
ionization in the negative mode and few intense signals in the positive
one. In negative mode, the ionization is based on the deprotonation of
hydroxyl groups, which is favored by alkaline pH. As the reverse phase
bonded to the stationary one has low tolerance to high pH values,
0.1 mL min–1 acetonitrile with 1% NH4OH was added post-column using
a Shimadzu LC-20 CE pump, before the ionization source.
Even with those higher analytical signals, it was not possible to
obtain tandem MS experiments. Therefore, to assess the obtained sig­
nals, saturated solutions of individual hormones were degraded as well.
It is noteworthy that no degradation products were identified for 17α-
ethinylestradiol (EE2) with the analytical conditions used. MZmine 2
freeware [29] was used for the alignment and deconvolution of signals.
The degradation products identification was performed by comparing
the data with the literature.
Two potential products were detected during the degradation of E1.
Product A, with m/z 303.1558 Da, was observed after approximately
90 min. That mass-to-charge ratio corresponds (less than 16 ppm error)
to the structures proposed by Mazellier, Méité, and DeLaat [30]
(Fig. 7a). Product B, with m/z 335.1447 Da, was detected since the early
stages of the degradation, reaching a maximum at 120 min. The prob­
able structure for such degradation product is based on Ohko et al. [31]
R.N. Padovan et al. Journal of Environmental Chemical Engineering 9 (2021) 106442

Fig. 7. Temporal profiles of the detected degradation products and their respective proposed structures: E1 (estrone) → (a) Product A (m/z 303.1558 Da) and (b)
Product B (m/z 335.1447 Da); E2 (17β-estradiol) → (c) Product C (m/z 335.1455 Da); and E3 (estriol) → (d) Product D (m/z 319.1544 Da).

Fig. 8. Hydroxylation mechanism of E1 to Product A or E3 to Product D.

(Fig. 7b). Regarding the degradation pathways, it is possible to say that

One potential product was detected in the E2 degradation: Product C,
with m/z 335.1455 Da, which reached its maximum at approximately
120 min. The presented structure is also based on the work of Ohko et al.
[31] (Fig. 7c).
One potential product was detected during the E3 degradation:
Product D, with m/z 319.1544 Da, which was detected since the early
stages of the degradation and whose concentration increased thereafter.
That structure is similar (errors below 5 ppm) to other ones proposed by
Mazellier, Méité, and DeLaat [30] and Pereira et al. [32] (Fig. 7d).
Product A is probably formed by the direct hydroxylation of E1; the
corresponding mechanism is proposed based on the work of Raghavan
and Steeken [33] (Fig. 8). Those authors proposed that the hydroxyl
radical may attack the aromatic double bonds, adding itself to the ring.
Afterwards, radicalar hydrogen is eliminated to restore aromaticity.
Those radicals are mainly captured by hydroxyl radicals, generating
water as a secondary product. The same mechanism explains the for­
mation of Product D, which is probably obtained by the direct hydrox­
ylation of E3.

8
R.N. Padovan et al. Journal of Environmental Chemical Engineering 9 (2021) 106442

Fig. 9. Degradation mechanism of E1 to Product B.

Fig. 10. Estrogenic activity of samples and controls after six days of incubation (given as absorbance). Hormone degradation: (a) approximately 85%; (b) higher than
96% (below LoQ). Note: n = 6 (two independent experiments in triplicate). ANOVA analysis with Dunnet test is shown as *** p < 0.001 and **** p < 0.0001 in
relation to the 10 nmol L–1 estradiol (E2) positive control. Statistical analyses were performed with GraphPad Prism software.

Products B and C can be obtained from E1 and E2, respectively, after
the double hydroxylation of the aromatic ring and further oxidation,
resulting in the opening of the ring, as described by Hoffmann et al. [34]
(Fig. 9). In this mechanism, the hydroxyl groups can be oxidized to
ketones. Regarding E2, only one of its hydroxyl groups underwent
oxidation. Then, one hydroxyl radical and one dioxygen attacked the
hormone molecule adding a peroxide group to the ring. Finally, the
aromaticity was lost and the ring opened, generating a dicarboxylic acid,
with the elimination of a hydroperoxyl radical.
3.6. Estrogenic activity
The estrogenic activity test was performed using the positive controls
(medium + estradiol) as the endpoint to be compared with the samples.
Positive and negative controls, as well as four samples from a 3-hour
experiment (0, 1, 2, and 3 h) were tested in sextuplicate. Fig. 8a
shows the measured estrogenic activity, where absorbance and estro­
genicity are positively correlated.
The Analysis of Variance (one-way ANOVA, Table S8) shows that the
groups are not homogeneous. Then, the Dunnett multiple comparison
tests (Table S9) were performed showing that the estrogenic activity of
all samples was statistically the same (i.e., no estrogenic activity
decrease could be noticed) except for the negative control (Fig. 10a).
This behavior was expected because, at the end of the degradation ex­
periments, approximately 15% (or 38 μg L− 1) of the initial hormone
concentration remained in all samples. As Purdom et al. [7] state, hor­
mone concentration as low as 1 ng L− 1 can still have estrogenic activity.
To assess the possibility of removing the estrogenic activity, long-
term degradation experiments were performed (9 h), shown in Fig. 9b.
Again, even after the degradation time was tripled, both the ANOVA
(Table S10) and Dunnet test (Table S11) showed that the estrogenic
activity could still be observed. However, in this extended period, the
estrogenic activity was reduced in comparison to the positive control
(Fig. 10b), pointing out that there was a reduction in the estrogenic
activity, even though the hormone concentrations after 9 h of irradia­
tion could not be quantified once they were below the LoQ. It is known
that the degradation was higher than 96% (LoQ/C0 = 0.04). Therefore,
it was not possible to assess whether the degradation products them­
selves caused estrogenic activity, due to the remaining concentration of
the hormone.
4. Conclusions
When the effects of two factors (pH and flow rate) on the degradation
of tap water contaminated with estrone, 17β-estradiol, 17α-ethinyles­
tradiol, and estriol were assessed by a factorial design, pH was not

9
R.N. Padovan et al.
statistically significant within the tested range (4.5 – 8.0). That finding
indicates the robustness of solar TiO2-photocatalysis for treating
hormone-laden tap water.
After careful reactor optimization, solar (TiO2) photocatalysis could
remove high hormone concentrations (250 μg L–1) in a relatively short
period (4 h). Nevertheless, even extending degradation by three times,
the estrogenic activity of the treated tap water could not be eliminated.
The automated, high-throughput chromatographic-UV-mass spec­
trometry procedure herein employed was deemed suitable for quanti­
fication purposes, regarding its calculated figures-of-merit. However, as
far as structural elucidation is concerned, it did not produce enough
information about the formed fragments. Due to the nature of the hor­
mone molecules, electrospray sources produced incipient ionization.
Therefore, the identification of structures was impaired, as the obtained
instrumental signals were too low to allow for fragmenting the ions
found.
CRediT authorship contribution statement
Rodrigo Nogueira Padovan: Methodology, Validation, Formal
analysis, Investigation, Writing – original draft. Lucas Sponton de
Carvalho: Methodology, Validation, Formal analysis, Investigation.
Patrícia Luisa de Souza Bergo: Investigation. Chubraider Xavier:
Formal analysis, Writing – original draft, Visualization. Andrei Leitão:
Resources, Writing – review & editing. Álvaro José dos Santos Neto:
Methodology, Resources, Writing – review & editing. Fernando Mauro
Lanças: Resources. Eduardo Bessa Azevedo: Conceptualization,
Methodology, Resources, Data curation, Writing – review & editing,
Visualization, Supervision, Project administration.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgement
This study was funded by the São Paulo Research Foundation
(FAPESP, grants: 11/24137-0, 18/15904-6).
Appendix A. Supporting information
Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.jece.2021.106442.
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