Journal of Hydrology 472–473 (2012) 314–327

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Journal of Hydrology
journal homepage: www.elsevier.com/locate/jhydrol

Degradation of thiamethoxam and metoprolol by UV, O3 and UV/O3 hybrid

processes: Kinetics, degradation intermediates and toxicity
D. Šojić a, V. Despotović a, D. Orčić a, E. Szabó b, E. Arany b, S. Armaković a, E. Illés b, K. Gajda-Schrantz b,c,d,
A. Dombi b, T. Alapi b,c, E. Sajben-Nagy e, A. Palágyi e, Cs. Vágvölgyi e, L. Manczinger e, L. Bjelica a,
B. Abramović a,⇑
a
University of Novi Sad, Faculty of Sciences, Department of Chemistry, Biochemistry and Environmental Protection, Trg D. Obradovića 3, 21000 Novi Sad, Serbia
b
Research Group of Environmental Chemistry, Institute of Chemistry, University of Szeged, Rerrich Béla tér 1, 6720 Szeged, Hungary
c
Department of Inorganic and Analytical Chemistry, University of Szeged, Dóm tér 7, 6720 Szeged, Hungary
d
EMPA, Laboratory for High Performance Ceramics, Überlandstrasse 129, 8600 Dübendorf, Switzerland
e
Department of Microbiology, University of Szeged, Közép fasor 52, 6726 Szeged, Hungary

a r t i c l e i n f o s u m m a r y

Article history: A comprehensive study of the degradation of thiamethoxam (THIA) and metoprolol (MET) was conducted
Received 3 April 2012 by using UV-induced photolysis (k = 254 nm), ozonation, and a combination of these methods. In order to
Received in revised form 10 September 2012 investigate how molecular structure of the substrate influences the rate of its degradation, we compared
Accepted 18 September 2012
these three processes for the insecticide THIA and the drug MET (a b1-blocker). Of the three treatments
Available online 8 October 2012
This manuscript was handled by Andras
applied, the UV photolysis and the combination of UV/O3 were found to be most effective in the degra-
Bardossy, Editor-in-Chief, with the dation of THIA, while the UV/O3 process appeared to be the most efficient in terms of MET decay. The deg-
assistance of Eddy Y. Zeng, Associate Editor radation kinetics was monitored by LC–DAD, and spectrophotometry, while the mineralization of the
substrates was studied by TOC analysis. Reaction intermediates were studied in detail and a number
Keywords: of them were identified using LC–MS (ESI+/ESI). Both parent compounds showed slight toxic effects
Thiamethoxam towards algae Pseudokirchneriella subcapitata and bacteria Vibrio fischeri. However, the toxicity of the
Metoprolol solutions containing also the degradation intermediates appeared to be much higher for all the test
Advanced oxidation processes organisms. The inhibition/mortality rates were reduced most efficiently by the UV/O3 procedure. Ames
Kinetics test and Comet assay were used to follow the genotoxicity during the degradation of the studied com-
Ecotoxicity pounds. Genotoxic intermediates were frequently detected in the case of MET in the UV treatment alone
Mutagenicity
or in the presence of ozone. Treatments of THIA samples resulted less frequently in genotoxic interme-
diates. To our best knowledge, this work is the first genotoxicological investigation dealing with the pho-
tolytic degradation process of the studied compounds.
Ó 2012 Elsevier B.V. All rights reserved.

1. Introduction health of aquatic organisms (Banks et al., 2005). In developing

There are many research articles dealing with the removal of or-
ganic micropollutants (Hu et al., 2008; Li et al., 2008; Swaim et al.,
2008), including pesticides (Rastogi et al., 2009), as well as phar-
maceuticals (Benotti et al., 2009; Vilhunen et al., 2009; Szabó
et al., 2011). Pesticides represent a potential threat to humans as
they can contaminate water supplies and directly impact the
⇑ Corresponding author. Tel.: +381 214852753; fax: +381 21454065.
E-mail addresses: daniela.sojic@dh.uns.ac.rs (D. Šojić), vesna.despotovic@dh.uns. ac.rs
(V. Despotović), dejan.orcic@dh.uns.ac.rs (D. Orčić), eszabo@chem.u-szeged.hu (E. Szabó),
arany.eszter@chem.u-szeged.hu (E. Arany), sanja.armakovic@dh.uns.ac.rs (S. Armaković),
erzsebet.illes@chem.u-szeged.hu (E. Illés), sranc@chem.u-szeged.hu (K. Gajda-Schrantz),
dombia@chem.u-szeged.hu (A. Dombi), alapi@chem.u-szeged.hu (T. Alapi), sajben@gmail.
com (E. Sajben-Nagy), palagyia@freemail.hu (A. Palágyi), csaba@bio.u-szeged.hu (Cs.
Vágvölgyi), manczing@bio.u-szeged.hu (L. Manczinger), bjelica@eunet.rs (L. Bjelica),
biljana.abramovic@dh.uns.ac.rs (B. Abramović).
countries, both pesticides and feces contaminate drinking water
(WR, 1998–1999). Thiamethoxam (THIA) belongs to neonicoti-
noids, a relatively new class of insecticides, which have been
extensively used for crop protection against a broad spectrum of
chewing and sucking pests and for a variety of other purposes such
as seed and pet treatment (Maienfisch et al., 2001). Metoprolol tar-
trate salt (MET) is a selective b1-blocker that is used to treat a vari-
ety of cardiovascular diseases, such as hypertension, coronary
artery disease, and arrhythmias (Ikehata et al., 2006). MET is char-
acterized by an increasing use in recent years and, as a conse-
quence, its occurrence in aqueous effluents is expected to
increase as well (Alder et al., 2010; Rivas et al., 2010). Advanced
oxidation processes (AOPs) have provided a promising alternative
for the remediation of contaminated water when compared to
other treatment methods (Benitez et al., 2002; Chiron et al.,
2000; Venkatadri and Peters, 1993). The most efficient and the

0022-1694/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jhydrol.2012.09.038
 D. Šojić et al. / Journal of Hydrology 472–473 (2012) 314–327
most convenient of them involve the use of UV radiation, which
promotes the absorbing target molecules to their excited triplet
states.
UV-photolysis is widely used for disinfection purposes. It in-
volves the interaction of artificial or natural light with the target
compounds, which then induces a series of photochemical reac-
tions (Gjessing and Källqvist, 1991; Frimmel, 1994, 1998). The di-
rect photolysis is dependent on the ability of the compounds to
absorb the emitted light, and on the relevant quantum yields.
Ozone can also be used for the degradation of organic pollutants
in water. It is a powerful oxidant (electrochemical oxidation poten-
tial of 2.07 versus 2.8 V for hydroxyl radical (HO)), which is gener-
ally produced by an electric discharge in the presence of air or
oxygen. A considerable disadvantage of ozone can be its expensive
production. Similar to other disinfectants for water treatment (e.g.
chlorine or chlorine dioxide), ozone is unstable in water and under-
goes reactions with some water matrix components (Von Gunten,
2003). However, the unique feature of ozone is its decomposition
into HO radicals which are the strongest oxidants in water (Staeh-
elin and Hoigné, 1985). Two reactions of ozone with dissolved or-
ganic substances can be distinguished in water at low pH: highly
selective electrophilic attack of molecular ozone, taking place on
the specific part of organic molecules with C@C bonds and/or aro-
matic ring (Von Gunten, 2003), whereas free radicals from ozone
decomposition (especially HO) can also react non-selectively with
organic compounds (Hoigné and Bader, 1976). Additional free HO
can be produced in aqueous media from ozone by pH modification
(Doré, 1989) or can be introduced by combining ozone either with
hydrogen peroxide (Duguet et al., 1985) or UV irradiation from e.g.
a low pressure mercury vapor lamp (Staehelin and Hoigné, 1985).
When hydroxide ions initiate the reaction, the decomposition of
ozone in water occurs through the following mechanism
(Andreozzi et al., 1999):
O3 þ HO ! O2 þ HO2 ð1Þ
O3 þ HO2 ! HO2 þ O
3 ð2Þ
HO2 ¡Hþ þ O
2 ð3Þ
O 
2 þ O3 ! O2 þ O3 ð4Þ
þ
O 
3 þ H ¡HO3 ð5Þ
HO3 ! HO þ O2 ð6Þ
According to reactions (1) and (2), the initiation of ozone
decomposition can be artificially accelerated by increasing the
pH of the solution.
UV/O3 treatment is also an AOP and it has been used for practi-
cal water treatment (Japan Ozone Association, 2004). The advanced
oxidation effect of UV/O3 treatment is based on the reactions (1)–
(11) (Beltrán, 2004). In the UV/O3 process, O3 absorbs UV radiation
and produces HO and H2O2 (reactions (7)–(9)), which further
decomposes to HO by direct photolysis (reaction (10)) or dissoci-
ates to form HO +
2 and H (reaction (11)) (Vilve et al., 2007).
O3 þ hm ! O2 þ O ð7Þ
O þ H2 O ! 2HO ð8Þ
O3 þ H2 O þ hm ! O2 þ H2 O2 ð9Þ
H2 O2 þ hm ! 2HO ð10Þ
H2 O2 ¡Hþ þ HO2 ð11Þ
315
The generated HO 
2 reacts then with O3 to produce HO (reac-
tions (2)–(6)). Accordingly, two main pathways result in the pro-
duction of HO during the UV/O3 treatment: photolysis of H2O2
(reaction (10)) and HO production via O 3 (reactions (1)–(6)).
THIA (Zheng et al., 2006) and MET (Piram et al., 2008; Liu et al.,
2009) undergo slow direct phototransformation under simulated
sunlight, and UVA, UVB, and simulated sunlight, respectively,
whereas both of them undergo slow hydrolysis. MET is readily re-
moved from water by photocatalytic treatment (Yang et al., 2010;
Abramović et al., 2011; Romero et al., 2011) and by ozonation
(Benner and Ternes, 2009), while the decomposition of THIA has
been monitored only in the conditions of direct photolysis
(254 nm) (De Urzedo et al., 2007).
The main objectives of this study were (a) to determine the
reaction kinetics for THIA (molar absorption maximum at
250 nm) and MET (molar absorption maximum at 225 nm) by
using UV-induced photolysis (k = 254 nm), ozonation, and the
combination of these methods, (b) to investigate how molecular
structure of the substrate influences the rate of its degradation,
bearing in mind their different molar absorption maxima, (c) to
identify the major intermediates of both substrates in the three
studied processes, (d) to investigate ecotoxic effects of THIA, MET
and their degradation intermediates towards algae Pseudokirch-
neriella subcapitata, zooplankton Daphnia magna and bacteria
Vibrio fischeri, and (e) to follow the changes of the genotoxic effect
during different treatments of the parent molecules.
2. Materials and methods
2.1. Chemicals and solutions
All chemicals were of reagent grade and were used without
further purification. The insecticide thiamethoxam {(3-[(2-chloro-
5-thiazolyl)methyl]tetrahydro-5-methyl-N-nitro-4H-1,3,5-oxadia-
zin-4-imine, CAS No. 153719-23-4, C8H10ClN5O3S, Mr = 291.71,
99.7% purity} and the drug (±)-metoprolol(+)-tartrate salt {1-[4-
(2-methoxyethyl)phenoxy]-3-(propan-2-ylamino)propan-2-ol tar-
trate (2:1), CAS No. 56392-17-7, (C15H25NO3)2 C4H6O6,
Mr = 684.81, P99% purity} were purchased from Sigma–Aldrich;
85% H3PO4 was purchased from Merck; 99.8% OptigradeÒ, HPLC
Gradient Grade acetonitrile (ACN) was a product of Scharlau. The
other chemicals used were: NaH2PO4 (P99%, Spektrum 3D),
Na2HPO4 (P99.0%, Fluka), NaOH (P99.0%, Fluka), NaCl (99.5%,
Sigma), formic acid (98–100%, Merck), HCl (37%, Scharlau), boric
acid (99.5%, Sigma), Tris (tris(hydroxymethyl)aminomethane,
99.8%, Sigma), and EDTA (99%, Sigma).
Phosphate buffer solution (pH 7.4) was used for toxicity mea-
surements. The pH of the samples was adjusted with solutions of
NaH2PO4, Na2HPO4, NaOH and HCl.
Ultrapure water, from a Millipore Milli-Q RG system (Milford,
MA, USA), was used to prepare the solutions in all experiments.
2.2. Photochemical reactors
All experiments were carried out using the recirculation photo-
chemical setup shown in Fig. 1. The solution (300 mL) was circu-
lated (375 mL min1) around the low pressure mercury vapor
lamp covered with a Suprasil sleeve (LightTech, 254/185 nm,
GCL307T5VH/CELL, length 307 mm, 20.5 mm external diameter,
17 W electric power and 4.0 W UV output) in a water-cooled, dou-
ble-walled tubular glass reactor (length 340 mm, inner diameter
30 mm).
The solution was continuously stirred with a magnetic bar and
thermostated at 25 ± 0.5 °C. The UV photon flux determined by
potassium ferrioxalate actinometry (Hatchard and Parker, 1956)
316 D. Šojić et al. / Journal of Hydrology 472–473 (2012) 314–327
Fig. 1. Experimental apparatus: (1) power supply, (2) Teflon packing ring, (3) low-pressure mercury vapor lamp, (4) envelope (UV photolysis: non-perforated quartz;
ozonation: perforated glass; combination of UV photolysis with ozonation: perforated quartz), (5) reactor, (6) reservoir, (7) magnetic stirrer, (8) pump, (9) thermostat, (10)
oxygen and (11) flow meter.
was I0 = (3.45 ± 0.09)  105 Einstein s1. The photon flux of the
185 nm component of the UV/VUV light (around 6% of the photon
flux of 254 nm light, according to the manufacturers data) was
estimated to be 2.1  106 Einstein s1. Oxygen (Messer, >99.5%
purity) was bubbled (690 mL min1) through the solution to regu-
late the dissolved oxygen concentration in the reservoir as well as
to generate ozone. For the latter purpose, oxygen was introduced
between the wall of the lamp and the envelope, which separated
the gas phase and the aqueous solution. The generated ozone
(cgO3 = 3.50  105 mol L1) was then bubbled through the perfora-
tion of the envelope in the solution. Agilent 8453 UV–VIS spectro-
photometer was used to determine the O3 concentration in the gas
phase at 254 nm (e254nm = 2952 mol1 L s1 (Atkinson et al., 1997)),
using a flow cell. Depending on the material of the envelope, ozon-
ation (perforated glass sleeve), its combination with UV photolysis
(perforated quartz envelope) or UV photolysis only (non-perfo-
rated quartz sleeve, introducing the oxygen only into the reservoir)
of the target compound was investigated (Alapi and Dombi, 2007).
In this way, the efficiency of these processes could be compared
using the same light source. The kinetic measurements were initi-
ated by switching on the light source. Samples were taken at the
beginning of the experiment and at regular time intervals. All the
presented results are the averages of at least three consecutive
experiments.
2.3. Analytical procedures
2.3.1. Kinetic studies
For the LC–DAD kinetic studies of the THIA and MET degrada-
tion, samples of about 0.50 mL of the reaction solution were taken
at the beginning of the experiment and at regular time intervals. A
volume of 20 lL was then injected and analyzed on an Agilent
Technologies 1100 Series liquid chromatograph, equipped with a
UV/Vis DAD set at the absorption maxima (250 nm for THIA and
225 nm MET), and a Zorbax Eclipse XDB-C18 (150 mm  4.6 mm
i.d., particle size 5 lm, 25 °C) column. The mobile phase (flow rate
0.8 mL min1) was a mixture of ACN and water (2:8, v/v for THIA
and with the following gradient for MET: 0 min 15% ACN which in-
creased to 30% ACN in 5 min, after which 30% ACN was constant for
5 min; post time was 3 min), the water being acidified with 0.1%
H3PO4. The use of the gradient mode to follow the degradation
kinetics of MET was necessary in order to separate the peaks orig-
inating from MET and intermediates, and shorten the time of the
LC–DAD analysis. Reproducibility of repeated runs was around
3–10%.
Absorption spectra were recorded on an Agilent 8453 UV/Vis
spectrophotometer at a fixed slit width (1 nm), using 1 cm quartz
cells and computer-loaded Agilent UV–Vis software. Kinetics of
degradation of the oxadiazine ring was monitored at 250 nm (for
THIA) and that of aromatic ring transformation at 225 nm (for
MET).
For the total organic carbon (TOC) analysis, aliquots of 10 mL of
the reaction solution were taken at regular time intervals, diluted
to 25 mL and analyzed on an Elementar Liqui TOC II analyzer
according to Standard US 120 EPA Method 9060A.
2.3.2. Identification of the reaction intermediates
For the LC–ESI–MS/MS evaluation of intermediates, 20-lL
samples were analyzed on an Agilent Technologies 1200 series
HPLC with Agilent Technologies 6410A series electrospray ioniza-
tion triple-quadrupole MS/MS, using Agilent Technologies Zorbax
XDB-C18 column (50 mm  4.6 mm i.d., particle size 1.8 lm,
40 °C). The mobile phase (flow rate 1.0 mL min1) consisted of
0.05% aqueous formic acid and MeOH (gradient, 0 min 30% MeOH,
10–12 min 100% MeOH, post-time 3 min). Analytes were ionized
using an electrospray ion source, a capillary voltage of 4.0 kV and
with nitrogen as the drying gas (temperature 350 °C, flow
10 L min1) and nebulizer gas (45 psi). High-purity nitrogen was
used as the collision gas. Full scan mode (m/z range 50–800, scan
time 100 ms, fragmentor voltage 100 V) in both polarities was used
to select precursor ions for starting compound and each degrada-
tion intermediate, as well as to examine isotopic peak distribution.
 D. Šojić et al. / Journal of Hydrology 472–473 (2012) 314–327
Then, the product ion scan MS2 mode (fragmentor voltage 100 V,
scan time 200 ms, collision energy 0–30 V in increments of 10 V)
was used to elucidate the structure of each degradation
intermediate.
2.4. Toxicity tests
The toxicity towards P. subcapitata (formerly known as Selena-
strum capricornutum) was determined using Algaltoxkit FTM
(MicroBioTests Inc.) microbiotests according to the ISO Standard
8692 and OECD Guideline 201. In each case, the cell density was
calculated from the optical density of the algae in different
samples, measured in 10 cm path-length cuvettes at 670 nm, using
a Hewlett–Packard (HP8452A) diode array spectrophotometer. The
initial cell density was in each case min. 10,000 mL1. The growth
rate (l) was calculated by subtracting the natural logarithm of the
number of the cells after 72 h incubation (lnx72) from the natural
logarithm of the number of the cells before the incubation (lnx0)
and dividing it with the time of incubation (tI) (12).
lnx72  lnx0
l¼ ð12Þ
tI
During the incubation at 21 °C, the samples containing algae
were illuminated from the bottom with 6000 lx. The inhibition
values were calculated by comparing the growth rate of the algae
in the samples and in the blank solution (l0) (13).
l0  l
I¼  100 ð13Þ
l0
The toxicity towards D. magna was determined using
Daphtoxkit F™ magna (MicroBioTests Inc.) tests according to the
ISO Standard 6341. The ephippia were used after 72–90 h of
hatching time at 21 °C, applying 6000 lx bottom illumination and
after 2 h of pre-nourishment. The zooplankton containing samples
were incubated under the same conditions for 48 h, and 8–10
neonates were added to each sample. The number of the neonates
was counted using a magnifier table-gooseneck 3/8 (VWR).
Mortality values were given as the percentage of the dead and/or
immobilized neonates compared to their initial number.
The toxicity towards V. fischeri luminescent bacteria was
determined by measuring the natural light emission of these
microorganisms using a HACH-LANGE LUMIStox 3000 lumino-
meter. The inhibition of the light emission in the presence of the
sample was determined against a non-toxic control. NaCl was
added to each sample to obtain a 2% solution. The samples were
diluted to the half with phosphate buffer (pH 7.4) to reach a pH
value of 7 ± 0.2. Samples containing bacteria were incubated at
15 °C for 15 min before the toxicity measurements. Samples for
the toxicity tests were taken at the same degradation degree of
the parent compounds for the three treatments.
2.5. Mutagenicity tests
2.5.1. Ames test
Ames tests were performed basically as described by Maron and
Ames (1983). The tester strains employed were Salmonella
typhimurium TA98, TA1535 and TA1537. Strains TA98 and
TA1537 detect mutagens causing frameshift mutations, while
TA1535 detects base substitution mutations.
2.5.2. Comet assay
Comet assay or Single Cell Gel Electrophoresis (SCGE) assay was
developed in the late 1980s, and was described first by Singh et al.
in 1988, as a fast and effective way of measuring DNA damage in
the individual cells. The name comes from the resulting image,
obtained after the process as a ‘‘comet’’, with a distinct head and
317
tail. The head is composed of intact DNA, while the tail consists
of damaged (fragmented) pieces of DNA. The literature describes
the comet assay in an alkaline or in a neutral version. In our study,
the alkaline version was used to estimate the DNA damage of
Escherichia coli cells exposed to various treatments. The
experiments were performed according to Singh et al. (1988), with
some modifications. Briefly, samples of the treated and untreated
E. coli cells were embedded into low melting point agarose gel.
After gelification, the blocks were incubated at 8 °C, in the dark,
in a lysing solution (2.5 mol L1 NaCl, 0.1 mol L1 EDTA,
0.01 mol L1 Tris, 1% Triton X-100, pH 10) for 16 h. Blocks were
washed three times (5 min each) in cold mixture of 10 mmol L1
Tris and 1 mmol L1 EDTA buffer (1 mL) and placed onto the lateral
surface of the teeth of an electrophoresis comb. Agarose solution
(0.76%) was prepared in diluted (10) alkaline buffer and heated
for complete homogenization. The comb with the attached sample
blocks were fitted in a proper electrophoresis tray and the cooled
agarose solution was poured in it to cast a gel. After gelification,
the comb was gently removed, leaving the sample blocks behind
and embedded in the agarose gel slab. The electrophoresis was
carried out in an alkaline buffer (12 g NaOH and 2 mL 0.5 mol L1
EDTA per liter deionized water, pH 8.0) at 7 V cm1 for 20 h. After
electrophoresis, the gel was neutralized by soaking in a solution
containing 30 mmol L1 NaCl and 50 mmol L1 Tris–HCl buffer
(pH 6.0). Finally, the gel was stained in ethidium bromide in
Tris–Borate–EDTA buffer pH 8.0 (10 g Tris base, 5.5 g boric acid,
and 4 mL of 0.5 mol L–1 EDTA per L) and visualized in a UV-
transilluminator.
3. Results and discussion
3.1. Degradation of THIA and MET by UV, O3 and UV/O3
The results of the comparative study of the degradation of THIA
and MET in distilled water under UV, O3 and UV/O3 irradiation for 2
and 10 min, respectively, shown in Fig. 2a and b, reflect the effect
of the structure of the substrate on the rate of its transformation.
The kinetics of the degradation of THIA and MET was followed by
LC–DAD, as the corresponding instrument was constantly avail-
able, whereas the identification of the reaction intermediates was
carried out using the more expensive LC–MS/MS instrument. As
can be seen significant transformation of THIA was observed under
UV-irradiation, which is understandable bearing in mind its
absorption maximum (250 nm) and the emission maximum of
the UV lamp (k = 254 nm). Namely, the concentration of THIA mea-
sured by LC–DAD decreased rapidly in the first 2 min of photolysis,
and the transformation efficiency achieved was about 99%. As far
as MET and similar compounds are concerned, the UV spectra
show that most of the b-blockers absorb only in the UVC range,
thus they cannot be photodegraded in environmental waters by di-
rect photolysis (Piram et al., 2008). Moreover, as can be seen in
Fig. 2, the efficiency of MET photolytic transformation achieved un-
der UVC radiation in the first 10 min was in this case only 7%.
As already mentioned, using the combination of ozone with UV
radiation, several individual reactions take place: direct ozonation,
direct photolysis, and oxidation by HO. These reactions may also
cause synergistic effects. The aim of our adoption of this AOP
was to generate HO, thereby enhancing the oxidation rate of
MET with respect to the single ozonation process. In this case,
HO is generated during the (photo)decomposition of ozone. The
combination of ozone with UV radiation resulted in an almost
complete transformation of MET in 10 min, as 84% of the substrate
was transformed. In the case of THIA, the 2-min ozonation com-
bined with UV-photolysis gave again a high transformation effi-
ciency of about 98%.
318 D. Šojić et al. / Journal of Hydrology 472–473 (2012) 314–327
Fig. 2. The evolution profiles of the transformation of (a) THIA and (b) MET
(c0 = 0.05 mmol L1) in the presence of UV, O3 and UV/O3, monitored by LC–DAD,
SPH and TOC. Treatment time: 2 min for THIA and 10 min for MET.
The O3-based processes can be considered as the result of two
different contributions: the direct ozonation reaction and the rad-
ical pathway (Chang et al., 2009). In the case of THIA removal, the
O3 method was not effective, as only 3.5% degraded during the first
2 min of treatment. The slower degradation of THIA under O3 com-
pared to UV/O3 was expected (Xu et al., 2010; Sillanpää et al.,
2011). On the other hand, the accelerated degradation rate under
UV/O3 can be explained by UV decomposition of O3, resulting in
the formation of H2O2 and HO (reactions (9) and (10)). Concerning
MET and based on the limited information in the literature, it
seems that all b-blockers are fairly reactive with ozone (Ternes,
1998; Benner and Ternes, 2009). These pharmaceuticals contain a
secondary amine group and a weakly/moderately activated aro-
matic ring that are probable the targets of molecular ozone and
HO attacks. During the 10-min ozonation, 27% of MET was
transformed.
In order to identify the type of intermediates formed during
degradation, UV spectra were recorded in the range of 200–
400 nm for both THIA and MET. It was found that the degradation
rate of both substrates measured by spectrophotometry (SPH) was
lower compared to their transformation rates obtained by LC–DAD
under UV, O3 and UV/O3 processes (Fig. 2a and b). This is under-
standable if we bear in mind that SPH monitors the kinetics of deg-
radation of the oxadiazine/aromatic moiety for THIA and MET,
respectively (starting compounds and their degradation intermedi-
ates that retain the respective moiety), whereas LC–DAD measures
only the changes of the THIA or MET concentration. As can be seen
from Fig. 2, the UV/O3 system was the most efficient in this case
too. With such technique, 90% of THIA and its intermediates con-
taining the oxadiazine ring were transformed after 2 min of treat-
ment, while in the case of MET, 50% of the aromatic intermediates
were transformed in 10 min.
A comparison of the mineralization capacities of UV, O3 and
UV/O3 processes shows that mineralization was slower than
transformation of the initial substrates or of the substances with
oxadiazine/aromatic moiety. Namely, in the case of THIA, TOC
measurements showed that about 40% of organic compounds still
remained in the system after 240 min under UV/O3, 55% in the case
of O3, and in the case of UV even 85%. In the case of MET, after
240 min, the TOC content of the solutions was 15% under UV/O3,
75% in the presence of O3, while under UV, practically, no
mineralization occurred.
3.2. LC–MS/MS identification of degradation intermediates
3.2.1. Thiamethoxam
A total of seven peaks (labeled TP1–TP7) of THIA degradation
intermediates were detected (Table 1 and Fig. 3). Collision-induced
dissociation patterns were less informative than those of MET, and
hence only two intermediates (TP1 and TP5) were completely
identified. For the other compounds, only partial identification of
certain structural features was possible. For all peaks except TP2,
TP4 and TP7, signals could be obtained only in the positive mode,
which could be expected due to the presence of numerous nitrogen
atoms with one electron pair. Retention times of all degradation
intermediates (0.48–0.92 min) were shorter than that of THIA
(1.19 min), indicating their increased polarity due to the molecule
cleavage and/or introduction of polar group.
Peak TP1, eluting at 0.48 min, corresponds to a compound with
a monoisotopic molecular mass (Mmi) of 115 units. Based on the
molecular mass and isotopic peaks profile, it contains an odd num-
ber of nitrogen atoms and no chlorine atoms. In the positive ioni-
zation (PI) MS2 spectrum, consecutive losses of CH2@O (Dm/
z = 30) and CH2@NH (Dm/z = 29) were observed. The spectral char-
acteristics are in agreement with those previously reported for 5-
methyl-1,3-thiazol-2(3H)-one (De Urzedo et al., 2007) (Fig. 4).
Peak TP5, eluting at 0.63 min, represents a compound with
Mmi = 246, which is by 45 units lower than the molecular mass of
THIA and indicates the replacement of the nitro group with
hydrogen. The molecular mass also indicates an even number of
nitrogen atoms, while the intensity of the A + 2 isotopic peak
confirms that chlorine atom is preserved in the molecule. In the
PI MS2 spectrum, several fragment ions were detected: m/z 217
[M+H30]+, m/z 174 [M+H73]+, m/z 161 [M+HCH2O56]+, and
m/z 132 [M+H7342]+. The losses of formaldehyde (Dm/z = 30),
CH3N@CHOCH3 (Dm/z = 73), NH@C@NCH3 (Dm/z = 56) and
NH@C@NH (Dm/z = 42) all point out to the 3-methyl-1,3,5-
oxadiazinan-4-imine moiety, i.e. they confirm the removal of the
nitro substituent from THIA. Thus, the peak TP5 represents
3-[(2-chloro-1,3-thiazol-5-yl)methyl]-5-methyl-1,3,5-oxadiazinan-
4-imine, a compound already identified as THIA degradation inter-
mediate (De Urzedo et al., 2007) (Fig. 4).
Peak TP4 (tR = 0.54 min) corresponds to a compound with
Mmi = 218. The intensity of A + 2 isotopic peak confirms the loss
of a chlorine atom, while the even Mmi indicates an even number
of nitrogen atoms. In contrast to the majority of THIA degradation
intermediates, this compound gave a signal in negative ionization
(NI), indicating the presence of acidic functional groups. However,
due to the very simple, non-informative NI MS2 spectrum, featur-
ing only two fragment ions at m/z 97 [MH120] and m/z 79
[MH120H2O], it was not possible to obtain any structural
information.
A compound with a high Mmi of 397 units, designated TP6,
eluted at 0.59 min. On the basis of the molecular mass and isotopic
peaks, it can be concluded that the compound contains one
chlorine atom and an odd number of nitrogen atoms. The molecu-
lar weight, 106 units higher than that of THIA, indicates the
introduction of additional groups in molecule. The absence of
 D. Šojić et al. / Journal of Hydrology 472–473 (2012) 314–327 319

Table 1
MS/MS fragmentation data for THIA degradation intermediates.

Peak label tR (min)a Mmi (g mol1) Mode Precursor ion m/z Vcol (V) Product ions m/z (rel. abundance)
THIA 1.19 291 PI 292 0 292(96), 246(11), 211(100), 210(32), 132(6)
10 211(100), 210(17), 181(24), 180(10), 132(22)
20 211(42), 181(100), 180(15), 152(15), 132(56), 122(23), 69(14)
30 181(68), 152(65), 151(22), 132(100), 125(28), 122(53), 108(32), 71(17), 69(41)
TP1 0.48 115 PI 116 0 116(100), 86(8)
10 116(32), 86(26), 57(100)
20 57(100)
TP2 0.52 195b NI 194 0 196(100)
10 196(95), 161(100)
20 161(100)
TP3 0.53 155b PI 156 0 156(100), 126(22)
10 156(14), 126(100), 98(22), 69(15)
20 126(20), 98(12), 69(100), 55(18)
30 69(100), 55(76)
TP4 0.54 218 NI 217 0 217(100), 97(17)
10 97(100)
20 97(100)
30 97(100), 79(46)
TP5 0.63 246 PI 247 0 247(100), 161(6)
10 247(75), 217(16), 174(19), 161(100), 132(28)
20 174(12), 161(46), 132(100), 57(5)
30 161(12), 133(10), 132(100), 71(9), 57(8)
TP6 0.59 397 PI 398 0 398(100)
10 398(100), 325(31), 208(15), 132(12)
20 398(15), 368(14), 325(47), 208(100), 178(52), 132(99)
30 178(35), 132(100)
TP7 0.92 253 PI 254 0 254(100), 167(21), 138(6)
10 254(7), 181(9), 167(100), 138(78), 117(11)
20 138(100), 87(13)
30 138(100), 111(9)
NI 252 0 252(100), 165(79)
10 252(8), 165(100)
20 165(100)
a
The dead time of the column is 0.42 min.
b
Only one ion was visible in MS1 spectrum, thus Mmi assignation is uncertain.

NO2 loss (Dm/z = 46) early in the fragmentation pathway suggests
the possible absence of the nitro group. Consecutive losses of
CH2@O (Dm/z = 30) and CH3N@CH2 (Dm/z = 43) point out to the
presence of oxadiazinane ring. No further features of molecular
structure could be determined.
Finally, the peak eluting at 0.92 min (TP7) represents a com-
pound with Mmi = 253, with no chlorine atoms and having an
odd number of nitrogen atoms. Both PI and NI MS2 spectra contain
only a few fragment peaks with significant abundance. The only
reaction observed in NI was the unidentified neutral loss of
86 mass units. Similar loss, of 87 mass units, as well as an ion of
m/z 87 formed through a complementary fragmentation pathway,
was observed in the PI chromatograms. As with TP6, no further
identification was possible.
For the peaks TP2 and TP3, it was not possible to unambigu-
ously determine molecular masses nor to obtain relevant
structural information from the mass spectra.
As can be concluded from Fig. 3, the intermediates formed from
THIA in the UV treatment (Fig. 3a) and in its combination with
ozonation (Fig. 3c) are the same and of similar concentrations in
the beginning of the degradation (first 2 min). This could be
expected since the dominant process is direct photolysis, and the
kinetics of THIA degradation is the same as described in Section 3.1.
However, the efficiency of the degradation of the intermediates
formed using UV radiation only was significantly lower, which
was especially pronounced in the case of the intermediate TP1,
whose concentration increased with time in the first 10 min,
whereas in the case of the UV/O3 process its concentration began
to decrease already after 2 min. This is at the same time an
explanation of the great difference in the mineralization capacities
of the UV and UV/O3 processes, although the rate of the degrada-
tion of THIA is practically the same. In the O3-based process
(Fig. 3b), only some of the intermediates (TP1–TP4) that were
identified using the UV and UV/O3 processes formed, and this
was possible at significantly longer treatment times. In this
treatment, the intermediate TP4 formed in a significantly higher
concentration than in the application of the other two processes,
which indicates that the efficiency of the O3 process in the
degradation of this intermediate was significantly lower.
3.2.2. Metoprolol
By the use of the LC–MS/MS technique, a total of 10 peaks (la-
beled MP1–MP10) of MET degradation intermediates were de-
tected (Table 2 and Fig. 5). Since the collision-induced
dissociation patterns of MET degradation intermediates are well-
defined (Borkar et al., 2012), it was possible to identify the majority
of peaks using PI and NI MS2 spectra. The retention times of inter-
mediates MP1–MP9 (0.47–1.28 min) were shorter than that of MET
(1.48 min), which was due to the molecule cleavage and formation
of polar moieties (alcohols and aldehydes). However, intermediate
MP10 eluted after MET (2.18 min), which indicates its hydropho-
bicity, and which is in line with the assumed structure (additional
i-Pr group).
Peak MP1 represents a compound with Mmi = 133, indicating
the presence of one nitrogen atom. In the PI MS2 spectrum, a series
of fragments were observed, corresponding to the losses of alco-
holic OH as water (Dm/z = 18) and of N-bound isopropyl as pro-
pene (Dm/z = 42): 116 [M+HH2O]+, 92 [M+HC3H6]+, 74
320 D. Šojić et al. / Journal of Hydrology 472–473 (2012) 314–327
Fig. 3. Degradation kinetics of THIA and the appearance/disappearance of the intermediates (TP1–TP7) in the presence of (a) UV, (b) O3 and (c) UV/O3 detected by LC–MS/MS
(ESI+).
Fig. 4. Structural formula of THIA and its degradation intermediates.
[M+HH2OC3H6]+, 72 [CH2@NCH(CH3)2+H]+, 56 [M+H2H2
OC3H6]+. Based on the fragmentation pattern and literature
data, it was concluded that the peak MP1 represents
3-(isopropylamino)propane-1,2-diol, already identified as a MET
degradation intermediate (Fig. 6) (Abramović et al., 2011).
For the peak MP2 (tR = 1.05 min), it was not possible to obtain
MS2 spectra either in the PI or NI mode. Based on its low and even
monoisotopic molecular mass of 158 units, it could only be con-
cluded, that it represents an intermediate of chain cleavage and
it does not contain nitrogen atom. Also, based on the fact it gives
rise to the [MH] ion, the presence of an acidic group (phenolic
hydroxyl or carboxylic group) could be expected.
The remaining peaks (MP3–MP9, Fig. 6) share the common frag-
mentation pattern (Borkar et al., 2012), featuring loss of propene
(Dm/z = 42) and consecutive losses of isopropylamine and water
(Dm/z = 77) from the protonated molecule, as well as the series
of low molecular mass fragments: 56 (C3H6N), 72 (C3H6NO), 74
(C3H8NO), 98 (C6H12N), 105 (C8H9), 116 (C6H14NO), and 133
(C9H9O). The fragment of 116 m/z value points out to a preserved
2-hydroxy-3-(isopropylamino)propoxy chain. The mass difference
between the [M+H77]+ fragment and [C6H5OC3H4]+ (m/z 133)
thus provides an insight into the changes in the 2-methoxyethyl
side chain (hereafter: chain B).
The peak MP3 (tR = 0.69 min) corresponds to a compound with
Mmi = 237, which is by 30 units less than that of MET, and the ob-
served loss of CO (161 ? 133) in the MS2 spectrum indicates that
the chain B underwent oxidative cleavage, giving rise to a formyl
 D. Šojić et al. / Journal of Hydrology 472–473 (2012) 314–327 321

Table 2
MS/MS fragmentation data for MET degradation intermediates.

Peak tR Mmi Mode Precursor ion Vcol Product ions m/z (rel. abundance)
label (min)a (g mol1) m/z (V)
MET 1.48 267 PI 268 0 268(100)
10 268(100), 116 (11)
20 268(52), 191(35), 159(74), 133(73), 121(67), 116(100), 98(59), 74(99), 72(85), 56(77)
MP1 0.47 133 PI 134 0 134(100)
10 134(100), 116(18), 92(28), 74(39), 72(24), 56(17)
20 134(8), 92(11), 74(38), 72(19), 57(7), 56(100)
30 74(8), 56(100)
MP3 0.69 237 PI 238 0 238(100)
10 238(100), 196(21), 161(15), 56(12)
20 161(31), 149(44), 133(26), 105(69), 74(32), 72(29), 56(100)
30 105(72), 56(100)
MP4 0.59 239 PI 240 0 240(100)
10 240(100), 198(11), 163(18), 133(20), 116(10), 98(11), 74(9), 72(16), 56(8)
20 163(35), 133(88), 133(93), 107(39), 105(36), 98(23), 74(67), 72(84), 56(100)
30 133(21), 107(22), 105(100), 79(30), 77(39), 74(31), 72(26), 56(79)
MP5 0.80 251 PI 252 0 252(100)
10 252(100), 210(9), 175(12)
20 252(18), 175(33), 163(64), 149(22), 137(59), 133(56), 116(25), 105(16), 98(19), 74(48),
72(40), 56(100)
30 105(17), 91(18), 74(19), 72(15), 56(100)
MP6 0.63 253 PI 254 0 254(100)
10 254(100), 212(10), 177(11)
20 212(14), 177(59), 151(22), 133(26), 116(14), 105(74), 74(24), 72(37), 56(100)
30 121(10), 105(22), 79(22), 56(100)
MP7 0.72 253 PI 254 0 254(100)
10 254(100), 177(20), 72(22)
20 254(72), 177(93), 159(50), 133(86), 98(100), 72(64), 56(89)
MP8 1.28 281 PI 282 0 282(100)
10 282(100), 205(8)
20 282(74), 205(40), 159(68), 145(100), 133(34), 121(37), 116(30), 98(45), 74(60), 72(59),
56(72)
MP9 0.66 283 PI 284 0 284(100)
10 284(100), 116(22), 74(5)
20 284(78), 207(6), 175(8), 163(15), 145(8), 133(43), 116(20), 105(5), 74(100), 72(41), 56(31)
MP10 2.18 325 PI 326 0 326(100)
10 326(100), 284(13), 132(5), 132(6)
20 326(21), 284(19), 191(24), 159(26), 132(100), 114(21), 88(25)
NI 324 0 324(100), 172(8), 151(6)
10 324(43), 172(89), 151(100)
20 172(13), 151(100)
30 151(100), 119(27), 106(26)
a
The dead time of the column is 0.42 min.

group. Thus, the peak MP3 represents 4-[2-hydroxy-3-(isopropyla- or 1-{4-[2-hydroxy-3-(isopropylamino)propoxy]phenyl}ethanone.

mino)propoxy]benzaldehyde. Similarly, the peak MP4 (tR =
0.59 min, Mmi = 239), has a molecular mass which is by 28 units
lower than that of MET and features the loss of HCHO
(163 ? 133) in the PI MS2 spectrum. This indicates that chain B
has been converted to hydroxymethyl, i.e. the peak MP4 represents
1-[4-(hydroxymethyl)phenoxy]-3-(isopropylamino)propan-2-ol.
Both MP3 (Abramović et al., 2011) and MP4 (Benner and Ternes,
2009) have already been identified as MET degradation interme-
diates. Spectral characteristics of MP4 are in agreement with
reference data (Benner and Ternes, 2009).
The compound MP5, eluting at 0.80 min, has a molecular mass of
251 units, i.e. 16 units lower than that of MET, suggesting either the
loss of an oxygen atom (unlikely) or the loss of a CH2 unit, followed
by a double bond formation. The observed fragmentation pathways
– the loss of ketene (175 ? 133) and HCHO (163 ? 133) – may
suggest that the side chain B has been transformed to either 2-
oxoethyl- or acetyl-group, i.e. that the compound MP5 is either
{4-[2-hydroxy-3-(isopropylamino)propoxy]phenyl}acetaldehyde
However, no definite identification is possible at the moment.
The two peaks of the compounds with Mmi = 253, designated
MP6 and MP7, were detected at 0.63 min and 0.72 min. The
molecular mass, 14 units lower than that of MET, suggests the
loss of a single CH2 unit. Based on the absence of methanol loss
(Dm/z = 32) in the PI MS2 spectrum, it can be concluded that both
compounds lack terminal methyl group from the side chain B. For
both peaks, consecutive losses of H2O (Dm/z = 18) and C2H2
(Dm/z = 26) were observed, although the reaction sequences dif-
fered, with MP7 dehydrating first (177 ? 159 ? 133) and MP6
losing C2H2 first through rearrangement (177 ? 151 ? 133). The
isomer with the abundant fragment 159 (MP7) was previously
identified as 1-[4-(2-hydroxyethyl)phenoxy]-3-(isopropylami-
no)propan-2-ol (Benner and Ternes, 2009), leading to the
conclusion that MP6 may be 1-[4-(1-hydroxyethyl)phenoxy]-
3-(isopropylamino)propan-2-ol.
The compound marked MP8, eluting at 1.28 min, has a molecu-
lar mass which is by 14 units higher than that of MET, implying the
322 D. Šojić et al. / Journal of Hydrology 472–473 (2012) 314–327
Fig. 5. Degradation kinetics of MET and the appearance/disappearance of the intermediates (MP1–MP10) in the presence of (a) UV, (b) O3 and (c) UV/O3 detected by LC–MS/
MS (ESI+).
Fig. 6. Structural formula of MET and its degradation intermediates.
introduction of one oxygen atom and abstraction of two hydrogen
atoms, i.e. the introduction of an oxo group. The observed consec-
utive losses of 46 mass units (formic acid) and 26 mass units (eth-
yne) indicate that the terminal methyl group of the chain B was
oxidized, i.e. the peak MP8 is 2-{4-[2-hydroxy-3-(isopropylami-
no)propoxy]phenyl}ethyl formate. The lack of methyl (Dm/z = 15)
and the loss of methanol (Dm/z = 32), observed in the MET spectra,
support this assumption.
The peak MP9, eluting at 0.66 min, corresponds to a compound
with Mmi = 283, i.e. to a monohydroxylated derivative of MET. The
observed loss of methanol (207 ? 175) indicates that the terminal
methoxy group of the chain B is preserved, while the subsequent
loss of ketene (175 ? 133) indicates that the additional oxygen is
bound to the chain B. The compound with a hydroxyl in the
b-position (relative to the benzene ring) would represent an
hemiacetal, but since it is very unstable, it is more likely that the
peak MP9 represents an a-hydroxy derivative, i.e. 1-[4-(1-hydro-
xy-2-methoxyethyl)phenoxy]-3-(isopropylamino)propan-2-ol.
Finally, the behavior of the compound eluting at 2.18 min (des-
ignated MP10), with Mmi = 325, differed from the other identified
MET degradation intermediates (Fig. 6). Unlike the other com-
pounds, MP10 gave a signal in NI, indicating the presence of acidic
functional groups (phenolic and/or carboxylic). The only abundant
fragment ions in the NI MS2 spectrum were at m/z 151 and 172, the
former probably being 4-(2-methoxyethyl)phenolate anion, and
the latter representing the complementary ion, formed by the loss
of 4-(2-methoxyethyl)phenol from [MH]. The presence of the ion
m/z 119, formed by the loss of methanol molecule from the ion m/z
151, supports the assumed structure. While a greater number of
abundant ions were observed in the PI MS2 spectrum, the only
structural feature that could be deduced was the N-bound isopro-
pyl group (326 ? 284). Based on the molecular mass, it is possible
that the compound MP10 represents 1-[hydroxyl-4-(2-methoxy-
ethyl)phenoxy]-3-(diisopropylamino)propan-2-ol. To the best of
our knowledge, no MET degradation intermediate of a molecular
mass 325 has been identified so far.
As can be seen from Fig. 5, the intermediates formed from MET
in all three treatments are the same, but only the kinetics of their
formation/disappearance is different. This indicates that in a par-
ticular treatment, in contrast to THIA, there is no significant differ-
ence in the kinetics of the disappearance of MET and formation/
disappearance of its intermediates.
3.3. Toxicity measurements
3.3.1. Toxicity towards algae during the degradation
The toxicity of the parent compounds towards the algae culture
was expressed by the normalized inhibition values of the solutions
after 72 h of incubation at 21 °C. THIA itself inhibited slightly the
growth of P. subcapitata, while the effect of MET was not significant
(under 20%). However, at the end of the treatment (when the par-
ent molecules were completely transformed) the toxicity of the
solutions was in both cases (and especially in case of MET), higher
 D. Šojić et al. / Journal of Hydrology 472–473 (2012) 314–327 323

Fig. 7. Normalized inhibition of Pseudokirchneriella subcapitata during the degradation of (a) THIA and (b) MET using UV (D), O3 (s), and UV/O3 (h).

Fig. 8. Mortality rate of Daphnia magna during the degradation of (a) THIA and (b) MET applying UV (D), O3 (s) and UV/O3 (h).
324 D. Šojić et al. / Journal of Hydrology 472–473 (2012) 314–327
Fig. 9. The inhibition values for Vibrio fischeri during the degradation of (a) THIA and (b) MET applying UV (D), O3 (s) and UV/O3 (h).
than the normalized inhibition values of the initial solutions
(Fig. 7). The likely reason for this is the formation of degradation
intermediates that are more toxic than the parent compound.
The formation of some aliphatic acids during the UV, O3 and the
UV/O3 treatments cannot be excluded too, as it was shown before
(Alapi et al., 2008, in press). In accordance with these assumptions,
the pH of the initial solution decreased from pH 7 to 6–5.5 dur-
ing the treatments. Although Newsted (2004) reported that these
values are still within the optimum pH range for algae, according
to the work of Smith et al. (1987), this change in the pH of the solu-
tion can significantly affect the growth rate of algae, which sup-
ports the results of the present study.
During the UV photolysis of THIA the inhibition showed a sharp
maximum. In all other cases for both compounds it was very broad
or not present at all (THIA, O3). The highest effect observed during
the UV treatment of THIA indicates the formation of by-products
that are more toxic to P. subcapitata than the parent compounds.
The decreasing toxicity of THIA solutions treated by UV photolysis
could correlate with the decreasing concentration of TP5 observed
by LC–MS/MS (Figs. 3a and 7a). However, the intermediate TP1
could contribute to the high toxicity of the solution at the end of
the treatment. Among the UV photoproducts of MET, the maximal
concentration of MP2–MP10 was around the detected toxicity
maximum, and the slow decrease of the algal growth inhibition
could be related to the intermediate MP1, present in the solution
even at the end of the photolysis (Figs. 5a and 7b).
It has been shown previously that among the investigated treat-
ments the combined one (UV/O3) was the most efficient in the
mineralization of the target molecules and their degradation inter-
mediates (Alapi et al., in press). A fast increase of the inhibition was
observed in the first 5 and 25 min of the treatment (for THIA and
MET, respectively), and the decrease at the end could be ascribed
to the decomposition of the by-products formed from THIA or
MET (Figs. 3c and 5c, respectively). However, in the case of MET,
the toxicity of the solution was significant even at the end of the
treatment – most probably because of the presence of some ali-
phatic intermediates formed from the target compound (Alibone
and Fair, 1981; Smith et al., 1987; Zhao et al., 1998; Berglind
et al., 2010). During the UV/O3 process, the toxicity maxima and
the maximum detected concentration of the major intermediates
(TP1, TP5 and TP3 of THIA and MP1 of MET) correlated well
(Figs. 3c, 5c and 7).
During the ozonation, the toxicity was continuously high in par-
allel with the high, only slightly decreasing, concentration of TP1,
TP3 and TP4 in the case of THIA, and MP1 in the case of MET
(Figs. 3b, 5b and 7).
3.3.2. Toxicity towards zooplankton during the degradation
The toxicity of the parent compounds towards D. magna was ex-
pressed by the mortality rate of the zooplankton after 48 h of incu-
bation at 21 °C. From the results it could be concluded that D.
magna was more sensitive to the degradation by-products and less
sensitive to the parent compounds THIA and MET than P. subcapi-
tata (Figs. 7 and 8). The mortality rate increased with the treatment
time in almost all cases, suggesting the presence of toxic by-prod-
ucts (TP1, TP3–TP5 and MP1, Figs. 3 and 5) or a weakly acidic pH
(caused by the formation of aliphatic acids), which could have also
a significant effect on these microcrustaceans (Alibone and Fair,
1981; Zhao et al., 1998). The only exception was the UV/O3 treat-
ment of MET. After 120 min, the mortality rate decreased signifi-
cantly, indicating an efficient mineralization of the toxic
compounds in the solution, which is in good agreement with the
LC–MS/MS data (Figs. 5c and 8b).
 D. Šojić et al. / Journal of Hydrology 472–473 (2012) 314–327 325

Table 3
Ames test results for THIA degradation (number of revertants/107 cells).

Treatment time (min)

0 0.5 1 2 10 30 60 120 180
UV TA1537 8 14 7 19 8
TA1535 12 61 141 243 152
TA98 2 20 14 5 8
O3 TA1537 8 5 11 6 8
TA1535 14 34 38 33 16
TA98 13 84 26 151 463
UV/O3 TA1537 5 9 14 11 10
TA1535 14 16 49 76 72
TA98 6 14 24 12 9
Fig. 10. Comet assay of THIA and MET samples after UV or UV/O3 treatment. X –
untreated samples (controls).

3.4. Mutagenicity
Table 4
Ames test results for MET degradation (number of revertants/107 cells).
3.4.1. Ames tests
Treatment time (min) Some genotoxic degradation intermediates appeared at signifi-
0 30 60 120 240 cant levels in the course of particular treatments. Treatments of
TA1537 5 679 25 80 7 THIA samples resulted in genotoxic intermediates less frequently
UV TA1535 8 304 281 199 95 compared to MET (Tables 3 and 4). The degradation intermediates
TA98 11 568 450 366 34 of THIA produced revertants, first of all with the TA1535 tester
TA1537 8 5 11 6 8 strain: this mutant detects base substitution mutations. In the case
O3 TA1535 11 28 83 71 47 of MET, the highest level of genotoxic compounds was detected
TA98 7 8 11 17 58
when the UV treatment alone or in the presence of ozone was car-
TA1537 4 49 31 112 49 ried out (Table 4). In this case, the degradation intermediates that
UV/O3 TA1535 7 243 39 149 86
appeared at 30 and 120 min proved to be most genotoxic.
TA98 5 63 8 159 51
To our best knowledge, the present work is the first in which
genotoxicological investigations were performed regarding the
photolytic degradation process of these two compounds, so that
our results cannot be compared with those of other studies.
3.3.3. Toxicity towards bacteria during the degradation
The toxicity of the target compounds towards V. fischeri was ex-
pressed by the inhibition values of the solutions (diluted to one 3.4.2. Comet assay
half with phosphate buffer) after 15 min of incubation at 15 °C. The modified alkaline version of the comet assay used in this
From the results it could be deduced that bacteria were the least study proved to be a reliable approach for studying DNA damage
sensitive to the presence of THIA, MET or their degradation by- caused by the treatment substrate. The UV/O3 treatment generated
products (Fig. 9). a substantial amount of DNA-breaking compounds, first of all at
The curves shown in Fig. 9a have a maximum, indicating the the beginning of the treatments (Fig. 10). Petersen et al. (2000)
formation and decomposition of molecules that exhibit a higher studied the role of different reactive oxygen species formed in
toxic effect than THIA. From these results it could be concluded the UVA irradiation, i.e. the UVA-induced DNA breaks. The UVA
that these compounds were present during the whole UV and O3 irradiation increased the intracellular levels of H2O2, detected by
treatment and that they decomposed most efficiently only in the a fluorescent probe, which caused oxidative damage of DNA, single
combined treatment. The mentioned findings are in agreement strand-breaks, and alkali-labile sites, measured by alkaline single
with the previously presented results (Fig. 3). cell gel electrophoresis (comet assay). The exogenous H2O2 was
In the case of MET, similar tendencies could be observed during also able to induce DNA damage. So, it is very likely that the
the UV/O3 treatment (Fig. 9b). Interestingly, the highest inhibition DNA breaks detected in UV-treated samples in our experiments
value was also detected during the combined treatment, although by comet assay could be in part caused by the presence of H2O2,
the concentration of the most significant degradation by-product in addition to the transformation intermediates. Because the molar
of MET (MP1) was the highest during ozonation (Fig. 5). This expe- absorption coefficient of H2O2 at 254 nm is relatively high
rience underlines again the importance of the possible formation of (18.6 mol1 L cm1) (Nicole et al., 1990) and the quantum yield
aliphatic intermediates, which could also affect the biolumines- of its UV photolysis is 0.98 ± 0.05 (Hunt and Taube, 1952), the
cence of V. fischeri (Berglind et al., 2010). The UV photolysis and harmful effect of H2O2 could be overcome by irradiating the solu-
ozonation did not affect significantly the toxicity of the initial solu- tions with 254 nm light after the end of the UV/O3 treatment,
tion of MET, maybe because they were not efficient enough in the which would lead to the degradation of H2O2 formed during the
mineralization of the target molecules, as it can also be seen from UV/O3 process (Arany et al., 2012).
Fig. 5a and b. However, in the case of THIA, the UV treatment and
the combined method resulted in a higher toxicity compared to 4. Conclusions
ozonation. The inhibition values decreased in parallel with the
decomposition of TP5, indicating probably a higher sensitivity of The results of this study clearly indicate that the concentration
bacteria to the degradation intermediate TP5, formed in a measur- of THIA measured by LC–DAD decreased rapidly in the first 2 min
able concentration during the UV and UV/O3 treatments (Figs. 3 of photolysis, and the transformation efficiency achieved was
and 9a). The same sensitivity towards TP5 could not be detected about 99%. However, with this method only 7% of MET was trans-
either with zooplanktons or algae (Figs. 7a and 8a). formed after the first 10 min, which is understandable bearing in
326 D. Šojić et al. / Journal of Hydrology 472–473 (2012) 314–327
mind the absorption maximum for THIA (250 nm) and MET
(225 nm) and the emission maximum of the UV lamp
(k = 254 nm). The combination of ozone and UV radiation resulted
in the 84% efficiency of degradation of MET after 10 min of treat-
ment, and about 98% of THIA after 2 min. However, in the first
2 min of ozonation, only 3.5% of THIA was transformed. In the case
of MET, whose molecule contains a secondary amine group and a
weakly/moderately activated aromatic ring that are probable tar-
gets of molecular ozone and HO, 27% of the substrate was trans-
formed in 10 min of ozonation. The rates of degradation of both
substrates in all three processes measured by SPH were compared
to LC–DAD results, and the mineralization was slower than the
transformation of the initial substrates or substances having oxadi-
azine/aromatic moiety. The LC–ESI–MS/MS data showed that the
degradation of both substrates resulted in the formation of several
intermediates (7 for THIA and 10 for MET).
As far as toxicity tests are concerned, it can be concluded that
algae and bacteria showed sensitivity towards the parent com-
pounds THIA and MET, although their toxic effect was not higher
than 30%. However, the degradation intermediates (primarily
TP1, TP3, TP4 and TP5 in case of THIA, and MP1 in case of MET)
formed during all treatments, were more toxic. Therefore, the
decomposition of the target molecules, aimed at their complete
mineralization, should be performed with great care. Although
the combined UV/O3 treatment was the most effective technique
as far as mineralization is concerned, its advantage from the toxic-
ity point of view is rather small.
The investigations of mutagenicity and DNA breaking revealed
that some very harmful degradation intermediates of both THIA
and MET are produced in the photolytic processes. In this respect,
new ecotoxicological problems can be encountered, especially in
the degradation of MET.
Acknowledgements
This document has been produced with the financial assistance
of the European Union (Project HU-SRB/0901/121/116 OCE-
EFPTRWR Optimization of Cost Effective and Environmentally
Friendly Procedures for Treatment of Regional Water Resources).
The contents of this document are the sole responsibility of the
University of Novi Sad Faculty of Sciences and can under no cir-
cumstances be regarded as reflecting the position of the European
Union and or the Managing Authority.
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