Photocatalytic degradation and mineralization of tramadol pharmaceutical in aqueous TiO2 suspensions: Evaluation of kinetics, mechanisms and ecotoxicity

Article abstract of DOI:10.1016/j.apcata.2016.02.005

In the present study the transformation and mineralization of a common aquatic pollutant, Tramadol (TRA) was investigated for the first time by means of TiO₂ photocatalysis. The degradation kinetics for both TRA and total organic carbon (TOC) followed apparent first-order model with rate constants of k = 15.3 10 min and k = 9.7 10 min and half lives (t) of 4.5 min and 71.4 min, respectively. The transformation products (TPs) of TRA, were identified by high resolution accurate mass liquid chromatography (HR-LC-MS) suggesting that hydroxylation, oxidation and dealkylation are the main transformation pathways. The reactions were found to occur mainly at the surface of the photocatalyst via surface-bound HO radicals rather than by free diffusion into the homogeneous phase. The potential risk of TRA and its TPs to aqueous organisms was investigated using Microtox bioassay before and during the process. The acute toxicity increased in the first stages and then decreased rapidly to very low values within 120 min of the photocatalytic treatment. The increase in the toxicity is associated with near additive or low synergistic effects between two TPs generated during the process i.e. N-desmethyl and N-oxide tramadol.

Full text of DOI:10.1016/j.apcata.2016.02.005

Accepted Manuscript
Title: Photocatalytic degradation and mineralization of
Tramadol pharmaceutical in aqueous TiO suspensions:
2
Evaluation of kinetics, mechanisms and ecotoxicity
Author: nullaria nullntonopoulou Ioannis Konstantinou
PII:
S0926-860X(16)30060-6
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.apcata.2016.02.005
APCATA 15764
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Applied Catalysis A: General
Received date:
Revised date:
Accepted date:
10-9-2015
2-2-2016
5-2-2016
Please cite this article as: x39c;aria x391;ntonopoulou, Ioannis Konstantinou,
Photocatalytic degradation and mineralization of Tramadol pharmaceutical in aqueous
TiO2 suspensions: Evaluation of kinetics, mechanisms and ecotoxicity, Applied
Catalysis A, General http://dx.doi.org/10.1016/j.apcata.2016.02.005
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Photocatalytic degradation and mineralization of Tramadol
pharmaceutical in aqueous TiO
mechanisms and ecotoxicity
2
suspensions: Evaluation of kinetics,
1
,2
1,2*
Μaria Αntonopoulou , Ioannis Konstantinou
1Department of Environmental and Natural Resources Management,
University
of
Patras,
30100,
Agrinio,
Greece.
2Department of Chemistry, University of Ioannina, 45110 Ioannina, Greece
*
Corresponding author:
e-mail: iokonst@cc.uoi.gr; iokonst@upatras.gr
Tel: +30-2651008349

Graphical Abstract
Highlights

The photocatalytic degradation and mineralization of tramadol was studied.

Ηydroxylation, oxidation and dealkylation were found to be the main
transformation pathways.
•


HO are the main reactive species during the photocatalytic process.
Near additive toxicity effects were observed between N-desmethyl, N-oxide
tramadol.

ABSTRACT
Ιn the present study the transformation and mineralization of a common aquatic
pollutant, Tramadol (TRA) was investigated for the first time by means of TiO
2
photocatalysis. The degradation kinetics for both ΤRA and total organic carbon
-2
-1
(
TOC) followed apparent first-order model with rate constants of kapp=15.3x10 min
-3
-1
and kapp=9.7 Χ 10 min and half lives (t1/2) of 4.5 min and 71.4 min, respectively.
The transformation products (TPs) of TRA, were identified by high resolution
accurate mass liquid chromatography (HR-LC-MS) suggesting that hydroxylation,
oxidation and dealkylation are the main transformation pathways. The reactions were
found to occur mainly at the surface of the photocatalyst via surface-bound HO•
radicals rather than by free diffusion into the homogeneous phase. Τhe potential risk
of TRA and its TPs to aqueous organisms was investigated using Microtox bioassay
before and during the process. The acute toxicity increased in the first stages and then
decreased rapidly to very low values within 120 min of the photocatalytic treatment.
The increase in the toxicity is associated with near additive or low synergistic effects
between two TPs generated during the process i.e. N-desmethyl and N-oxide
tramadol.
Keywords: Tramadol, photocatalysis, transformation products, ecotoxicity

1
. INTRODUCTION
Pharmaceutical compounds are a group of emerging contaminants which are found in
−
1
surface waters and wastewaters at levels up to a few μg L , raising major concerns
about their potential impact on ecosystems and public health [1-3]. Aquatic toxicity,
resistance development in pathogenic bacteria, genotoxicity and endocrine disruption
are the main possible adverse effects caused by the frequent occurrence of
pharmaceuticals in the aquatic environment [3-6]. Pharmaceuticals are generally
designed with high stability for their intended effects on humans and they are poorly
removed in wastewater treatment plants (WWTPs) thus, enter the environment as
unchanged parent compounds, metabolites or conjugates of both [1, 2]. A
representative example of persistent pharmaceutical contaminant is Tramadol (TRA),
a centrally acting synthetic opioid analgesic, used worldwide to treat moderate to
severe acute or chronic pain. TRA is excreted via urine as an unchanged compound
up to 30% and undergoes extensive metabolism, primarily resulting in N-desmethyl-
(
N-DES) and O-desmethyl- (O-DES) tramadol [6-8].
TRA is partially removed in conventional wastewater treatment plants and as a result
it has been frequently detected in sewage outflows as well as in surface water
worldwide. According to a European-wide monitoring study on the occurrence of
organic micropollutants in WWTP effluents, TRA was one of the most frequent
detected contaminants with mean and maximum concentration levels up to 256 and
-1
1
116 ng L , respectively [5-7].
Advances in water and wastewater treatment have led to the development of
promising technologies termed as advanced oxidation processes (AOPs), involving
•
highly reactive hydroxyl radicals (HO ), for the treatment of many emerging
contaminants, including pharmaceuticals, poorly eliminated by conventional

biological treatment processes [9, 10]. Heterogeneous photocatalysis using titanium
dioxide (TiO ) as a catalyst is a well-established AOP, providing promising results for
2
both removal and total mineralization of various pharmaceuticals and other micro-
pollutants from aqueous phase [11-13]. Heterogeneous photocatalysis typically
•
+
•
−•
1
involves the formation of HO , h , HO 2,
O
2
, and O
2
, etc. as oxidizing species and
either hydrated electrons (e-aq) or hydrogen atoms (H ) as reducing species, which can
contribute to the degradation of the contaminants [13-16]. However, only few studies
have focused on the identification of photocatalytic transformation products and
possible degradation pathways of pharmaceutical compounds [17].
•
Despite the extensive presence of TRA in various environmental matrices, only a few
AOPs such as ozonation and ferrate oxidation [16], electrochemical oxidation [18],
and UV/chloramines/H
2
2
O treatment [19] have been successfully employed for its
degradation in aquatic matrices. In the first study, different pathways and major
transformation products (TPs) between ozonation and ferrate oxidation were reported
while in both cases complete mineralization was not achieved [16]. In the second
study, further TPs were formed in the presence of halogen ions while TRA removal
was achieved but only a minor reduction of total organic carbon (TOC) was reported
[18]. Similarly, different TPs and partial removal of TOC were also observed in the
latter study [19].
In this context, no data is currently known regarding the photocatalytic transformation
process, the degradation mechanisms and pathways of TRA as well as the toxicity
assessment of the treated TRA aquatic solution and its individual transformation
products. Photocatalytic reactions proceed via parallel and often interconnected
pathways that do not lead always to complete mineralization or detoxification of the
treated solution and sometimes more persistent or toxic compounds are formed during

the degradation process [20]; thus the identification and the toxicity assessment of the
transformation products are significant milestones for the assessment of the process.
Such integrated studies are of great importance for the purposes of proper water and
wastewater treatment by photocatalytic process. In addition, oxidation, degradation
and transformation of pharmaceuticals via photo-induced reactions (direct and
indirect photochemical mechanisms) take place after their release in environmental
media. Photocatalytic redox mechanisms are useful for simulating environmental or
metabolic degradation processes, thus providing important knowledge on the
transformation products of the studied pollutants [21].
The objective of this study was to investigate the photocatalytic degradation of TRA,
as a model compound in the opioid analgesics and antidepressants families, providing
a fundamental understanding on the applicability and effectiveness of the process for
removing the parent compound and transformation products as well as for the
detoxification of the aqueous solutions. The transformation kinetics and mechanisms
of TRA TiO photocatalytic degradation and mineralization were studied for the first
2
time. The possible degradation pathways of TRA were proposed by identifying the
major transformation products using high resolution accurate mass liquid
chromatography (HR-LC-MS) and scavenging experiments. The continuous
monitoring/assessment of potential toxicity during the treatment was also followed in
order to ensure the appropriate degree of treatment and to contribute in the
optimization of operating conditions. Moreover, the ecotoxicological potential effects
of the major transformation products were determined, providing important
knowledge regarding their ecotoxicological potency in aquatic media and giving
useful insight on the application of the treatment as additional step in water and
wastewater treatment.

In the majority of previously published photocatalytic degradation studies of various
pharmaceuticals, toxicity has been generally taken as the overall toxicity of the
solution without providing information on potential ecotoxicity of the TPs as well as
additives, synergistic or antagonistic effects between them. In this sense, the present
work provides various new data regarding the systematic elucidation of the
photocatalytic process of this ubiquitous pharmaceutical pollutant in the water.
EXPERIMENTAL SECTION
1
.1.
Materials
Tramadol hydrochloride salt, anisole and phenol analytical grade (99.9 %) were
purchased from Sigma-Aldrich (USA). High purity (99.9 %) standards of N-
desmethyl-tramadol (N-DES), N,N-bidesmethyl-tramadol (Bi-DES) and N-oxide-
tramadol (N-OX) analytical grade (99.9 %), were purchased from LGC Standards
(Germany). Titanium dioxide Degussa P25 (particle size, 20–30 nm; crystal structure,
2
−1
8
0% anatase and 20% rutile; surface area, 56 m g ) was used as photocatalyst.
HPLC-grade solvents (acetonitrile, isopropanol, methanol and water) were supplied
by Merck (Darmstadt, Germany). Sodium azide (NaN ), potassium iodide (KI),
sodium fluoride (NaF) and potassium dichromate (K Cr ) were obtained from
3
2
2
O
7
Sigma-Aldrich. Superoxide dismutase (SOD) was purchased from Carl Roth GmbH+
Co. KG. (Karlsruhe). HA 0.45 μm filters were supplied by Millipore (Bedford, USA).
Oasis HLB (divinylbenzene/N-vinylpyrrolidone copolymer) cartridges (60 mg, 3 mL)
from Waters (Mildford, MA, USA) were used for the by-products evaluation.
1
.2.
Irradiation procedures

Irradiation experiments were performed using a Suntest XLS+ solar simulator (Atlas,
Germany). The illumination was performed using a vapor xenon lamp (2.2 kW)
equipped with special glass filters restricting the transmission of wavelengths below
3
10 nm. Photocatalytic degradation of TRA was conducted in a Pyrex reactor with a
total volume of 250 mL and a double-walled cooling-water jacket to keep the
temperature of the solutions at about 25 °C throughout all experiments. Before
-1
-1
illumination, suspensions of TRA solutions (10 mg L ) containing 100 mg L TiO
2
were stirred at 600 rpm in the dark for 30 min to achieve adsorption–desorption
equilibrium.
1
.3.
Analytical methods
A Dionex P680 HPLC equipped with a Dionex PDA-100 Photodiode Array Detector
was used for TRA determination and quantitation. TRA analysis was performed on a
Discovery C18, (250 mm length × 4.6 mm ID, 5 μm particle size) column from
Supelco (Bellefonte, PA, USA), thermostated at 40 °C. The elution was performed
isocratically using LC-grade water at pH 3 (30 %) and acetonitrile (70%) solution as
-1
the mobile phase at a flow rate of 1 mL min . Quantification was realized at 215 nm.
-1
The detection limit was 40 μg L using a signal-to-noise ratio (S/N) equal to 3. TOC
-
removal was followed via a Shimadzu, TOC V-csh Analyzer. NO
3
ions released
during the process were determined by a Dionex ICS-1500 equipped with ASRS Ultra
II self-regenerating suppressor. Ammonium ions (NH +
4
) were analyzed by a UV-Vis
spectrophotometer (Hitachi, model U-2000) using the colometric method based on
indophenol blue formation [22].
1
.4.
LC-MS analysis

For the identification of TPs, aliquots of 20 mL the irradiated solutions were extracted
by means of solid-phase extraction (SPE), using Oasis HLB (divinylbenzene/N-
vinylpyrrolidone copolymer) cartridges (60 mg, 3 mL) from Waters (Mildford, MA,
USA). SPE was performed using a 12-fold vacuum extraction box (Visiprep, Supelco,
Bellefonte, PA, USA). The cartridges were activated and conditioned with 3 mL of
-1
methanol and 3 mL LC-grade water at a flow rate of 1 mL min . Then, irradiated
-1
samples were added at a flow rate of 10 mL min and subsequently dried both by
nitrogen stream and vacuum for 15 min. Elution was performed by 2 × 2 mL of
methanol. Finally, evaporation of the extracts with nitrogen gas flow to a final volume
of 0.5 mL was performed.
The TPs generated during TRA photocatalysis were characterized by an UPLC–ESI-
MS system in positive ionization mode. The LC system was equipped with an Accela
Autosampler, an Accela LC pump and a LIT Orbitrap mass spectrometer (Thermo
Fisher Scientific, Germany). The chromatographic separations were run on a C18
Hypersil Gold, 100 mm x 2.1 mm i.d., 1.9 μm particle size (Thermo Fisher Scientific,
San Jose, USA), thermostated at 30 ◦C. Injection volume was 10 μL and flow rate 300
-1
μL min . Gradient mobile phase composition was adopted using water/5mM formic
acid as solvent A and methanol/5mM formic acid as solvent B with the following
program: 90/10 (kept constant 3 min) to 75/25 in 3.5 min, to 50/50 in 9 min, to 0/100
in 12 min and in 13 min returns to the initial conditions (90/10) and and re-
equilibration time was set at 7 min. All ESI-MS experiments were acquired in the
positive mode and the ESI-source parameters adopted are stated in our previous work
[15]. N-DES- and N-OX-TRA were identified by comparison of retention time, high
2
resolution mass and MS spectra to the commercially available standards.

2
.5. Scavenging experiments for reactive species
.1 M isopropanol (i-PrOH) was used to scavenge HO , 0.1 M methanol was used for
•
0
+
•
+
•
quenching h and HO , 0.1 M KI was used to scavenge both h and surface HO ads, 0.1
mM NaF was used to wash HO•ads into solution as bulk HO•bulk, 50 μΜ K
Cr
O
7
was
•-.
2
2
used to trap e−aq in solution and SOD (100 units mL ) was used to scavenge O
-1
2
Photocatalytic experiments using MeCN solutions were also performed for
•
+
discrimination between the oxidation by HO and h . Finally, aqueous solutions
(for e−aq) was also photocatalytically treated [23-
degassed with high-purity O
2
and N
2
2
7].
2
.6.
Toxicity measurements
For the assessment of the potential impact of TRA and its TPs to aqueous organisms,
Vibrio Fischeri bioassay was performed before and during the process using a
Microtox Model 500 Analyzer (Azur Enviromental). A detailed description of the
procedure has been reported in our previous study [27]. The toxicity of individual
TP’s as well as of their mixtures with concentrations levels as those determined
during the photocatlytic degradation was also measured in order to discriminate which
TP’s are more toxic and potential additive or synergistic effects.
3
3
. RESULTS AND DISCUSSION
.1. Photocatalytic Degradation Kinetics of TRA
Preliminary hydrolysis, adsorption and simulated solar photolysis experiments were
carried out in order to determine the contribution of these effects in the overall
photocatalytic process. Adsorption experiments carried out in dark with TRA at 10

−1
−1
mg L and TiO at 100 mg L , showed no significant adsorption of TRA on the
2
catalyst's surface, i.e. less than 9 %, at equilibrium (30 min). Similarly, hydrolysis
experiments showed no significant degradation of the compound. Photolysis
experiments under simulated solar light (SSL) without catalyst, using the same initial
concentration as in photocatalytic experiments, confirmed that the direct
photodegradation is a relatively slow process, and about 29 % of TRA was degraded
after 20 min while the corresponding mineralization degree was only 3.75 %. During
simulated solar photolysis, TRA molecules can absorb photons as TRA peak
maximum absorption at 271 nm tails well over 500 nm overlapping with solar
spectrum in the 310–500 nm region.
In contrast, within the same time framework (20 min) the investigated pharmaceutical
was degraded very quickly (more than 95% of degradation) in aqueous titanium
dioxide suspensions, irradiated with simulated solar light (Fig. 1). Consequently, the
observed fast decomposition in the presence of TiO is ascribed to the catalyst’s
2
activity. Complete degradation of TRA was achieved within 30 min with a half time
of 4.5 min, following an apparent first-order degradation model. In contrast, TOC
removal followed a slow rate, showing a half time of 71.4 min. and percentages of
approximately 80 % have been achieved only after 240 min irradiation. At this
irradiation period, nitrogen was mainly mineralized into NH +
ions (almost 48 %) and
4
-
(almost 30 %). The higher amount of NH +
in a lesser extent into NO
to NO -
3
4
ions compared
3
can be explained by the chemical structure of TRA bearing alkyl amine group.
According to literature data, the photocatalytic degradation of molecules having alkyl
amines as functional groups lead to the generation of a large amount of NH +
and a
4
-
minor amount of NO
3
[28, 29]. TiO
2
photocatalytic treatment was demonstrated to be

a suitable tool to remove TRA from the water leading to almost complete
mineralization.
3
.2. Contribution of Different Reactive Species by scavenging experiments
+
-
•
−•, •O
, 1O
,
Several oxidative and reductive species including h , e , HO , O
2
2
H, H
2
O
2
2
etc. are generated in UV-Vis/TiO photocatalysis that can react with the organic
2
contaminants [14, 15, 23]. The degradation kinetics of TRA in the presence of various
scavengers are depicted in Fig. 2, and the variation of pseudo-first-order kinetic
parameters in the presence of the employed scavengers is also summarized in Table
S1.
•
Isopropanol, a well-established HO scavenger, has been widely used to discriminate
the direct oxidation of substrates by hydroxyl radicals due to its high-rate constant
9
-1 -1
reaction with the radical (1.9 × 10 Μ s ) [15, 25]. The rate constant decreased by
•
9
5.4% when i-PrOH was added indicating significant contribution of HO to TRA
degradation.
Methanol can act as HO radical scavenger with a rate constant of 1 × 10 Μ s [30]
as well as can be adsorbed on TiO surface and act as an electron donor reacting with
photogenerated holes [31]. With MeOH addition, the rate constant decreased notably
•
9
-1 -1
2
-2
−1
to 0.4× 10 min , showing a reduction of 97.4 % in the rate constant originated from
•
+
both HO and h scavenging. The use of MeCN as reaction media could also help to
•
discern the participation of holes and HO in the reaction mechanism. A similar
inhibition (≈95 %) was observed when TRA photocatalytic degradation was
•
performed in MeCN solution, verifying the participation of HO radicals and the
limited contribution (about 2.4 %) of direct positive hole oxidation.

-
+
•
I anions were used to scavenge both h and surface HO ads, as react steadily with
•
-
holes and HO leading to iodine formation [25,31]. When I anions were added to the
TRA solution, an inhibition of 92.1 % was observed, suggesting that TRA
degradation was mostly induced by HO•ads. According to the above mentioned results,
the degradation of TRA was found to take place at the surface or at least in the
•
intimate proximity of the TiO
2
particles whereas the participation of HO in bulk
+
water can be assumed minor. The participation of h on the degradation mechanism is
•
also consistent with the oxidation of TiO
2
surface OH-groups to produce HO radicals.
1
•
With azide addition, a well-known O
2
and HO scavenger [15], the rate constant
1
decreased by 83.1 % (data not shown). This result indicates that O
2
did not
participate apparently in TRA oxidation. In the presence of NaF the observed rate
constant was 9.2 × 10-2 min , corresponding to 39.9 % inhibition due to the
transformation of HO•ads to HO bulk [23]. This reduction in the degradation
inhibition proves once more that TRA degradation takes place at the surface of the
catalyst.
−1
•
Quenching e−aq in the TiO
conduction band under SSL irradiation by adding
2
-2
−1
K
2
Cr
2
O
7
, the rate constant decreased to 4.5× 10 min (70.6 % inhibition). In order
to clarify the direct or indirect contribution of e−aq as well as to exclude the
interference of light absorption by K
2
Cr
2
O
7
further experiments were conducted. A
was irradiated
-
solution containing TRA, I ions and i-PrOH degassed with N
2
excluding all other species except reductive e−aq and a rate constant equal to 0.3× 10-2
−1
−
min was obtained, indicating that direct e aq reaction could contribute only 2 % in
the degradation. To confirm this assumption, TRA degradation was also conducted in
-
to transform e−aq into the relative oxidative
presence of I ions, i-PrOH, and O
2
species, the superoxide radical anion (O −
). A rate constant of 5.2 × 10 min was
•
-2
−1
2

observed, indicating a considerable contribution (66 %) of O −
•
to TRA degradation.
2
Similarly, the addition of SOD to the solution, which has been used to quench O −
•
by
2
a simple electron transfer mechanism provokes 67.3 % inhibition of the photocatalytic
degradation of TRA, proving once more the indirect contribution of e−aq. In aqueous
−•
solutions the reaction of O
2
with protons is favorable giving rise to the formation of
•
H
2
O
2
which can produced further HO radicals. Overall, oxidative species, in
•
−•
particular HO and to a lesser extent O
2
were predominantly responsible for TRA
degradation in aqueous solutions.
Based on the detailed scavenging study, the photocatalytic degradation of TRA
proceeds as follows: In the first steps TRA diffuses from bulk solution to the catalyst
surface. After the photo excitation of TiO
2
with light energy greater than its band gap
−
+
energy, conduction band electrons (e ) and valence band holes (h ) are generated. The
photogenerated holes oxidize adsorbed TRA in a minor extent, but mainly react with
•
•
OH surface groups oxidizing them into HO radicals. Subsequent HO attack on TRA
molecule leads to various transformation products before mineralization. On the other
hand, the photogenerated electrons react with O
2
adsorbed on the TiO -surface,
2
reducing it to O •
−. Finally, O
•−
could attack TRA or produce more OH contributing
•
2
2
to its degradation.
3
.3. Photocatalytic Degradation Mechanism of TRA
The identification of 14 intermediates during TRA degradation by heterogeneous
photocatalysis was allowed using UPLC/MS/MS (Table 2S). Structural assignment
was based on accurate mass analysis of both pseudo-molecular ion peaks and MS/MS
fragmentation patterns with a high grade of accuracy (< 6 ppm error). In UPLC/MS
TIC chromatograms (Fig. S1), most of the identified TPs eluted before TRA,

+
indicating the formation of more hydrophilic TPs. Five species with [M+H] ions of
m/z 280.1891-280.1904 (280-A,B,C,D,E,) and molecular formula C16 were
H26NO
3
formed, corresponding to the addition of 15.995 mass units to TRA (m/z 264.1949),
consistent with TRA mono-hydroxylated products (OH-TRA). In some cases the
2
position of the hydroxylation can be proposed from the analysis of the MS spectra,
summarized in Table 2S. All isomeric TPs showed: (a) the formation of a major MS2
product ion at m/z 262.1804 (C16
on the fragmentation of TRA molecule; and (b) the presence of a fragment at m/z
17.1224 with a molecular formula of C14 (loss of C N). The latter loss
H24NO
2
) originating from H
2
O loss, as observed also
2
H
17
O
2
2
H
7
indicates unmodified N,N-dimethylamino group and suggests the hydroxylation of
either the cyclohexane or benzene ring and the methylene group attached to the
cyclohexane ring. Taking into account that TRA presents a pk
a
of 9.41, the tertiary
amine group was protonated at the experimental pH (6.7) and the reactivity of this
•
+
group towards electrophilic species such as HO and h can be considered low in
consistency with the observed intact tertiary amine group based on the MS data. As
regards isomer 280-A, the structural-diagnostic MS ion at m/z 250.1786 (C15
H
24NO )
2
fits well with the loss of CH =O fragment that is common for aromatic methyl ethers
2
and exclude the methoxy group as an hydroxylation site. Isomers 280B-E presented a
2
MS product ion at m/z 149.0230-149.0241 with a chemical formula of C
8
H
5
3
O which
is indicative for hydroxylation of the aromatic ring. Mono-hydroxy derivatives have
also been identified during ozonation [16] as well as electrochemical oxidation [18] of
TRA. Based on NMR analysis of the dehydrated OH-TRA, Eversloh et al. [18]
suggested that hydroxylation of TRA during electrochemical oxidation occurred in the
benzene ring in para and ortho orientation to the methoxy group. On the contrary,
Radjenovic et al. [19] did not observed hydroxylation of the methoxybenzene moiety

during UV/H
2
2
O /chloramine treatment and the elucidated by-products indicated
mostly oxidative transformations at the cyclohexane ring relied on the observed
successive losses of water.
Simultaneously, the three peaks eluted at 4.98, 5.89 and 6.38 min with a molecular
formula of C16
H
26NO and a pseudo-molecular ion of 296.1844 (296A,B,C) were also
4
detected on the basis of the insertion of two hydroxyl groups (31.9990 units mass
difference comparing to original TRA molecule) and attributed to di-hydroxylated
2
TRA. For TPs 296A,B, successive losses of water in MS fragmentation were
observed. Consequently, it is suggested that one OH-group is located in the
cyclohexane moiety as reported also for other dihydroxylated TRA TPs in Radjenovic
et al. [19]. Further hydroxylation of TRA leads to the formation of one TP with
+
[
M+H] ion of m/z 312.1818 (C16
H
26NO ) at the first stages of the photocatalytic
4
treatment. In this case the very small concentration hampered further structural
elucidation. Multiple hydroxylation of TRA has also been reported during
electrochemical oxidation [18]. Oxidized TPs was formed simultaneously or
consecutively with the formation of mono-hydroxylated derivate with [M+H]+
+
2
78.1887 and empirical formula of C16
H24NO
3
. A TP with [M+H] 278.1751 was also
detected during ozone and Fe(VI) treatment of TRA [16] and it is assigned to the
transformation of the methyl group in the amine moiety to an aldehyde group.
The three TPs eluted at 7.90, 7.92 and 5.76 min were identified as N-DES, N-OX- and
O-DES-TRA, three well-known metabolites of TRA. The formation of N-DES and N-
OX during treatment was also confirmed by comparing R , nominal masses as well as
t
2
MS spectra with the authentic reference standards. The above mentioned TPs had
been also previously identified as major transformation products of ozonation [16]
and electro-oxidation of TRA [18]. In addition, they have been detected as

transformation products of TRA after entering the aquatic environment in its
unchanged structure as a result of biotic and abiotic processes [6-8].
The formation of N-OX can be explained by the well-documented in the literature
oxygen transfer mechanism. The oxidation of tertiary amines by oxygen transfer to
the corresponding N-oxide has also been reported for different oxidants such as
Fe(VI), [16] O
3
[16, 32, 33] and MnO [33-34]. The dealkylation mechanism (N-DES,
4
O-DES) involves the initial hydroxyl radical attack on the methyl group and the
subsequent formation of hydroperoxide and aldehyde structures. Further oxidation of
the aldehyde TPs followed by decarboxylation reactions justify the generation of N-
and O- dealkylated TRA [35-37].
Using HPLC–DAD system, later stage TPs, anisole and phenol, were determined. The
formation of both anisole and phenol was validated by the retention times and the
UV-vis spectra of the commercial standards under the same chromatographic
conditions. Anisole is generated via the oxidative opening and subsequent
dealkylation reactions of cyclohexane moiety of TRA. Various TPs with oxidative
cyclohexane ring opening have been identified in Radjenovic et al. [19] for
UV/H
2
2
O /chloramine treatment. Thereafter, phenol is generated from anisole by a
dealkylation reaction or by cleavage of methoxy group and the subsequent
•
electrophilic addition of the HO radical.
TPs evolution as a function of irradiation time was also followed and the evolution
profiles are depicted in Fig. 3. Most of these TPs were readily transformed and after
3
0 min of irradiation they were completely abated. On the basis of calibration curves
created for the TPs with available standards, the maximum concentration of N-OX-
-1
and N-DES-TRA was determined to be 2 and 52 μg L .

Our data shows that mono- and poly- hydroxy TRA displayed maximum
concentrations within the same time scale (15 min) implying that mono- and poly-
hydroxylation took place simultaneously. Oxidised (keto or aldehydes) and
dealkylated derivatives attained their maximum response within the same time-scale
periods, indicating the fast kinetics in the successive reaction network followed during
the photocatalytic reaction. Αnisole and phenol were formed slowly after successive
hydroxylation and oxidative cleavage of cyclohexane ring. Thus, these oxidation
-1
products presented their maximum concentration (14 and 18 μg L for phenol and
anisole, respectively) at prolonged irradiation period of 60 min.
Based on the structures of the TPs discussed above, three different pathways were
proposed as depicted in Scheme 1 and include: (a) oxidation of tertiary amine
functional group by oxygen transfer mechanism; (b) hydroxylation that occurred
mainly in the aromatic and/or cyclohexane ring and (c) oxidation of the alkyl-groups
to the corresponding aldehyde structures and subsequent demethylation of the N- and
O-alkyl chain.
After prolonged irradiation time, these TPs are further decomposed into other low-
molecular-weight products such as anisole and phenol. The last step involves
oxidative opening of the aromatic ring, leading to lower molecular weight aliphatic
products such as carboxylic acids [38]. Although, the final aliphatic transformation
products were not possible to be identified by the employed analytical
instrumentations, the evolution of nitrogen ions and TOC removal confirm the
cleavage of the rings and the formation of nitrogen containing and de-nitrogenated
aliphatic by-products. Carboxylic acids such as maleic, oxalic, formic, fumaric have
been reported as final products of phenol and phenolic-type derivatives degradation.

Total mineralization of these aliphatic carboxylic acids proceeds though the loss of a
CO molecule via the well-established photo-Kolbe reaction in the literature [38].
2
3
.4. Ecotoxicity evolution
The toxicity evolution during photocatalytic degradation is essential for assessing the
feasibility of water treatment techniques [19]. Aquatic toxicity is an important
parameter in assessing the potential adverse effects of chemicals and complex
mixtures on aquatic species and ecosystems, thus the acute toxicity of TRA and its
transformation products was monitored with the Vibrio Fischeri toxicity test. The
toxicity of initial TRA solution and of the treated samples was measured after 5, 15
and 30 min of exposure to the tested organism. Since no difference in the
measurements between different exposure periods was observed, in Fig. 4A the
inhibition measured after 15 min of contact time is depicted. No variability in toxicity
measurements between the different exposure times has also been observed in
previous studies which used Vibrio Fischeri bioassay as toxicity test [27, 39, 40].
Although the original solution is relatively non-toxic to Vibrio Fischeri (9 %
inhibition), toxicity increased during the first stages reaching the inhibition value of
about 40 % up to 15 min of the photocatalytic reaction to progressively decrease
thereafter. The toxicity profile did not follow the trend of the degradation of TRA
indicating the formation of toxic transformation compounds that were eliminated at
longer prolonged irradiation times. Notably, the observed % inhibition was found to
attain its maximum value in 15 min, simultaneously with the highest abundance of the
majority of the identified TPs.
In an attempt to identify the intermediates responsible for increased toxicity, the toxic
effect of two of the most abundant and commercial available TPs identified, N-DES-,

and N-OX-TRA towards Vibrio Fischeri was investigated. The concentrations
investigated were equal to their maximum concentrations determined after 15 min.
According to the results depicted in Fig. 4B, these TPs, showed lower toxicity
compared to TRA, presenting 6 % and 4 % inhibition, respectively.
In order to investigate possible additive or synergistic effects, mixture solutions
containing TRA and the two TPs at concentrations equal to those detected during the
photocatalytic experiments after 15 min were prepared. The solution showed an
inhibition of 26 % indicating near additive or low synergistic effects between them.
After 30 min of photocatalytic treatment toxicity is decreased and at 120 min
detoxification is achieved reaching the value of 3% inhibition. Acute toxicity test
using Vibrio Fischeri bioassay highlights the efficiency of the photocatalytic process
in the detoxification of the irradiated solution. The results clearly demonstrate that the
potential risk of TPs and the toxicity evolution demands special consideration during
the evaluation and planning of water treatment technologies.
4
. CONCLUSIONS
Heterogeneous photocatalysis was demonstrated to be an efficient treatment
technology for removing TRA, a representative pharmaceutical pollutant from the
water. Simultaneously with the total abatement of TRA, high percentages of
•
mineralization were accomplished. HO radicals were found to be the most important
species, attacking TRA molecule non-selectively, during the process. Using advanced
mass spectroscopy techniques, a great number of hydroxylated, oxidized and
dealkylated TPs were identified. The potential adverse effect of the treated solution
during the process was studied using Microtox bioassay. An increase in the toxicity

was observed in 15 min and attributed to near additive or low synergistic effects
between N-desmethyl and N-oxide tramadol.
Acknowledgements
This research has been co-financed by the European Union (European Social Fund –
ESF) and Greek national funds through the Operational Program "Education and
Lifelong Learning" of the National Strategic Reference Framework (NSRF) -
Research Funding Program: THALIS. Investing in knowledge society through the
European Social Fund.
The authors would like to thank the Unit of Environmental, Organic and Biochemical
high resolution analysis-orbitrap-LC-MS analysis of the University of Ioannina for
providing access to the facilities.
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Legend of Figures-Schemes
Figure 1. Kinetics of TRA photocatalytic degradation and mineralization as expressed
-
+
by TOC removal and evolution of NO and NH4 ions (Experimental conditions:
3
-1
initial concentration of Tramadol [C
0
]=10 mg L , concentration of catalyst [TiO ]
2
-
1
-2
=
100 mg L , Irradiation intensity, I=500 W m ) (N/N
s
= determined concentration of
N-species (NH
4
+, NO -) in each sampling interval/theoretical stoichiometric
3
concentration of N-species formed after mineralization of the initial tramadol
concentration).
-1
Figure 2. Degradation kinetics of TRA (C =10 mg L ) in the presence of different
0
-1
scavengers in aqueous TiO suspensions (100 mg L ) under simulated solar light
2
-2
(
I=500 W m ).
Figure 3. Evolution profiles of TPs (structure assignment as shown in Scheme 1)
identified by liquid chromatography- accurate mass spectrometry during the
-1
-1
photocatalytic degradation of TRA (conditions: [C ]=10 mg L , [TiO ] =100 mg L , I
0
2
-
2
=
500 W m ); A), B), C) LC-ESI-MS peak area as a function of irradiation time; D)
concentration as a function of irradiation time.
Figure 4. % Inhibition of luminescence of Vibrio Fischeri marine bacteria; A) as a
-1
-1
function of photocatalytic treatment ([TRA]=10 mg L , [TiO ] =100 mg L , I = 500
2
-2
W m ); B) in the presence of TRA and the detected TPs with available reference
standards in concentrations equal to those detected in 15 min of photocatalytic
treatment.

Scheme 1. Proposed photocatalytic degradation pathways of TRA in aqueous TiO -
2
P25 suspensions.

1
0
0
0
.0
.8
.6
.4
1.0
0.8
0.6
0.4
0.2
0.0
TRA
TOC
-
NO3
+
NH4
0.2
0.0
0
20 40 60 80 100 120 140 160 180
Irradiation time (min)
Figure 1.

1.0
0.8
0.6
0.4
0.2
0.0
TRA
MeOH
i-PrOH
NaF
SOD
MeCN
-
I
Cr (VI)
-
I +i-PrOH+N
2
-
I +i-PrOH+O
2
0
5
10
15
20
25
30
Irradiation time (min)
Figure 2.

7
7
6
6
6
6
6
6
6
6
1.2x10
1.0x10
8.0x10
6.0x10
4.0x10
2.0x10
(A)
4x10
(B)
296A
96B
296C
12
280A
280B
280C
280D
280E
278
2
3
3x10
2x10
1x10
0
0.0
0
5
10
15
20
25
30
0
5
10
15
20
25
30
Irradiation time (min)
Irradiation time (min)
6
0
0
0
0
0
0
0
(
D)
6
6
6
6
6
6
N-OX-TRA
N-DES-TRA
Anisole
6
5
4
3
2
1
x10
x10
x10
x10
x10
x10
(C)
N-OX-TRA
N-DES-TRA
O-DES-TRA
5
4
3
2
1
Phenol
0
0
20 40 60 80 100 120 140 160 180
Irradiation time (min)
0
5
10
15
20
25
30
Irradiation time (min)
Figure 3.