A convenient antibiotic indicator in the ozone treatment of wastewaters. An experimental and theoretical study

Article abstract of DOI:10.1039/c0nj00171f

N-Acetyl-oxazolidin-2-one has been found to be a convenient indicator in the removal of nitroimidazole-type antibiotic contaminants in wastewaters by ozone since it is the unique product generated in the treatment of waters containing metronidazole, and it is stable against further ozonation in the experimental conditions used. The oxidative conditions imposed by the reaction do not allow isolation of any by-products, nor other stable subproducts. High-performance liquid chromatography coupled with electrospray ionization-time-of-flight mass spectrometry (HPLC-ESI-TOF(MS)) revealed the presence of simple derivatives of both metronidazole and N-acetyl-oxazolidin-2- one, thus indicating that both products are unambiguously connected and that, possibly, a unique reaction pathway would take place in this case. A feasible mechanism for this transformation has then been postulated, which is supported by several experimental findings such as TOC analysis and nitrate titration, both correlating well with the proposed transformation. Furthermore, this mechanism has been evaluated by DFT/B3LYP calculations, showing that the observed product is readily obtained thanks to the high exothermicity displayed by the metronidazole ozonation process.

Full text of DOI:10.1039/c0nj00171f

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PAPER
www.rsc.org/njc | New Journal of Chemistry
A convenient antibiotic indicator in the ozone treatment of wastewaters.
An experimental and theoretical studyw
a
a
b
´
Antonio J. Mota,* Gonzalo Prados-Joya, David Arraez-Roman,
Manuel Sanchez-Polo, Rafael Robles, M Angeles Ferro-Garcıa and
´
a
c
a
a
´
´
´
a
´
Jose Rivera-Utrilla*
Received (in Montpellier, France) 4th March 2010, Accepted 4th May 2010
DOI: 10.1039/c0nj00171f
N-Acetyl-oxazolidin-2-one has been found to be a convenient indicator in the removal of
nitroimidazole-type antibiotic contaminants in wastewaters by ozone since it is the unique product
generated in the treatment of waters containing metronidazole, and it is stable against further
ozonation in the experimental conditions used. The oxidative conditions imposed by the reaction
do not allow isolation of any by-products, nor other stable subproducts. High-performance liquid
chromatography coupled with electrospray ionization-time-of-flight mass spectrometry
(HPLC-ESI-TOF(MS)) revealed the presence of simple derivatives of both metronidazole and
N-acetyl-oxazolidin-2-one, thus indicating that both products are unambiguously connected and
that, possibly, a unique reaction pathway would take place in this case. A feasible mechanism for
this transformation has then been postulated, which is supported by several experimental findings
such as TOC analysis and nitrate titration, both correlating well with the proposed
transformation. Furthermore, this mechanism has been evaluated by DFT/B3LYP calculations,
showing that the observed product is readily obtained thanks to the high exothermicity displayed
by the metronidazole ozonation process.
an annual 3–4% increase in the weight of pharmaceutical
compounds consumed.4
Introduction
The use of technologies based on ozonation process for the
removal of contaminants from waters is an important topic in
current environmental chemistry since new contaminants are
being generated, and their respective maximum permissible
concentrations in the environment are not yet legally regulated.
Moreover, their potential short-, medium- and long-term effects
on human health are almost completely unknown.1–3 Some of
these emerging contaminants have been present in waters for
decades but are only now beginning to be detected and
recognized as potentially dangerous contaminants. Recent
discoveries include compounds present in cosmetics, domestic
products and, especially, novel pharmaceutical products
designed to have enhanced effects and properties—such as those
combating new diseases—and their metabolites. This is mainly
due to the large variety of these compounds and their high
consumption over the past few years. It has been estimated that
approximately 6 million of Pharmaceuticals and Personal Care
Products (PPCPs) are commercially available worldwide, with
This situation has led to the detection of high concentra-
tions of these compounds in urban wastewaters, estimated to
have an antibiotic content of 50 mg LÀ1 5
. Their persistence
in waters means that resistance to antibiotics not only affects
the individuals misusing these products but also the general
population.6 The majority of these antibiotics, e.g.,
quinolones, nitroimidazoles, and sulfonamides, have a low
biodegradability1,7 and high toxicity,7 and show mutagenic
along with carcinogenic properties.8 Among the antibiotics,
nitroimidazoles can appear in waters at concentrations of
0.1–90.2 mg LÀ1.9 These compounds are widely used to treat
infections caused by anaerobic and protozoan bacteria, e.g.,
Trichomonas vaginalis and Giardia lamblia.10,11 Besides their
use as antibiotics in human beings, nitroimidazoles are also
added to poultry and fish feed, leading to their accumulation
in the animals, in the water of fish-farms, and, especially, in
effluents from meat industries.5
There are few data on the capacity of different treatment
systems to remove nitroimidazoles, which were only recently
detected in waters.8,12 Unfortunately, as demonstrated by
previous studies, conventional treatment systems7,13—mainly
based on the use of microorganisms—have been proved to be
inadequate to effectively destroy these types of organic
compounds, mostly due to their complex molecular structure.
It is therefore necessary to seek new technologically viable and
economically feasible alternatives to effectively remove these
contaminants from waters.
a
´
´
Departamento de Quımica Inorganica, Facultad de Ciencias,
Universidad de Granada, Avenida de Fuentenueva, 18002-Granada,
Spain. E-mail: mota@ugr.es, jrivera@ugr.es;
Tel: (+34) 958 24 04 42 (AJM), (+34) 958 24 85 23 (JRU)
b
c
´
´
Departamento de Quımica Analıtica, Facultad de Ciencias,
Universidad de Granada, Avenida de Fuentenueva, 18002-Granada,
Spain
´
´
Departamento de Quımica Organica, Facultad de Ciencias,
Universidad de Granada, Avenida de Fuentenueva, 18002-Granada,
Spain
w Electronic supplementary information (ESI) available: Cartesian
coordinates and intrinsic energies for all the calculated structures.
See DOI: 10.1039/c0nj00171f
The main objective of this study is the analysis of the
metronidazole (1) degradation by ozone, through the
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from 30% to 6% in 1 min. A 6 min re-equilibration time
was used after each analysis. The flow rate used was set at
0.20 mL minÀ1 throughout the gradient. The column temperature
was kept at 25 1C and the injection volume was 10 mL.
The HPLC system was coupled to a micrOTOF, an
orthogonal-accelerated time-of-flight mass spectrometer
(oaTOF-MS), equipped with an electrospray ionization
(ESI) interface. Parameters for analysis were set using positive
ion mode with spectra acquired over a mass range from
m/z 50 to 1000. The optimum values of the ESI-MS
parameters were: capillary voltage À4.5 kV; drying gas
temperature 190 1C; drying gas flow 7.0 L minÀ1 and nebulizing
gas pressure 21.7 psi.
Scheme 1 The ozone-mediated transformation of metronidazole (1)
into N-acetyl-oxazolidin-2-one (2).
evaluation of the formation of stable N-acetyl-oxazolidin-2-
one (2), see Scheme 1. Thus, compound 2 could be considered
as an adequate contaminant indicator for nitroimidazoles in
the ozonation processes of wastewaters, since 1 is the most
frequently detected nitroimidazole therein. The mechanism
involved in this conversion has also been postulated and
calculated by means of DFT/B3LYP calculations in order to
ascertain the most favorable pathway connecting both
products.
The accurate mass data of the molecular ions were
processed with the DataAnalysis 3.3 program, which provided
a list of possible elemental formulas by using the Generate-
MolecularFormulat Editor. This program uses a CHNO
algorithm, which provides standard functionalities such as
minimum/maximum elemental range, electron configuration,
and ring-plus double bonds equivalents, as well as a sophisti-
cated comparison of the theoretical with the measured isotope
pattern (Sigma-Valuet) for increased confidence in the
suggested molecular formula. It should be mentioned that
even with very high mass accuracy (o1 ppm) many possible
chemical formulae could be obtained depending on the mass
regions considered. So, high mass accuracy (o1 ppm) alone is
not enough to exclude enough candidates with complex
elemental compositions. The use of isotopic abundance
patterns as a single further constraint removes >95% of
false candidates. This orthogonal filter can condense several
thousand candidates down to only a small number of
molecular formulas.
Experimental
Reagents
All of the chemical reagents used were high-purity analytical
grade reagents. Solutions were performed using ultrapure
water.
Ozonation setup
The experimental system for nitroimidazole ozonation
basically consisted of an ozone generator, a 2 litre reactor
with agitator and UV-Vis spectrophotometer. Ozone was
generated from oxygen by means of an ozonator with a
maximum capacity of 76 mg minÀ1. The reactor was covered
for temperature control and was equipped with gas inlet and
outlets, agitation system, and reagent and sampling dispensers.
In each experiment, the reactor was filled with 2 L of 50 mM
phosphate-regulating solution at pH = 2. Once ozone partial
pressure and temperature (25 1C) were adjusted, ozone was
supplied to the reactor for 35 min until saturation of the
solution. Then, 15 mL of concentrated metronidazole solution
was injected to obtain the desired initial concentration in the
reactor (10 mg LÀ1). The ozonation reaction was stopped
bubbling a stream of N2 (g) for 5 min. In order to detect
oxidation by-products generated in the metronidazole
ozonation process, aqueous solutions obtained after different
treatment times were extracted with Cl2CH2 and concentrated
by using a rotary evaporator.
During the development of the HPLC method, external
instrument calibration was performed using a syringe pump
directly connected to the interface, passing a solution of
sodium formate cluster containing 5 mM sodium hydroxide
in the sheath liquid of 0.2% formic acid in water/isopropanol
1 : 1 (v/v). Using this method, an exact calibration curve based
on numerous cluster masses each differing by 68 Da
(NaCHO2) was obtained. Due to the compensation of
temperature drift in the micrOTOF, this external calibration
provided accurate mass values (better than 5 ppm) for a
complete run without the need for a dual sprayer setup for
internal mass calibration.
Ionic chromatography was used for nitrite and nitrate anion
determinations. Total organic carbon was used for the CO2
determination.
Computational details
Analytical methods
Dissolved ozone concentration was colorimetrically determined
by the karman-indigo method.14
The calculations were carried out at the DFT-B3LYP
level15–17 with the Gaussian 03 program.18 The geometries
were fully optimized by the gradient technique using the
standard 6-311+G(d,p) basis set,19,20 which incorporates
polarized functions for all the atoms and diffuse functions
only for non-hydrogen atoms. The nature of the optimized
structures, either transition states or intermediates, was
assessed through a frequency calculation. We also report
enthalpy and free energy values (not BSSE corrected), which
Separation of ozonation by-products of antibiotics
compounds from waters was performed using HPLC [equipped
with a C18 analytical column (3.9 Â 150 mm, 4 mm)]. Ammonium
acetate 5 mM at pH 4.3 and acetonitrile were used as the
mobile phases A and B, respectively. The mobile phase
gradient elution was programmed as follows: 6% B during
10 min, from 6% to 30% in 10 min and kept during 3 min, and
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were obtained by taking into account the zero-point energies
and the thermal motion at standard conditions (temperature
of 298.15 K, pressure of 1 atm).
chromatogram (BPC) at 0, 2, 3, 10, 20, 40 and 60 min during
the MNZ degradation.
The present method is able to detect three compounds,
namely (a) 2-methyl-1H-imidazole ([M + H]+
83.0598 m/z),
exp.
(b) oxazolidin-2-one ([M + H]+
88.0391 m/z), and
exp.
(c) MNZ (1) ([M + H]+exp. 172.0717 m/z). Many data related
with these compounds such as their migration time,
formula, selected ion, experimental and calculated m/z, error
(ppm and mDa), sigma value, tolerance, and the classification
order considering other possibilities are summarized in Table 1
(see also Fig. 1).
Results and discussion
The addition of 1 to an ozone-saturated, ultrapure aqueous
solution led to the disappearance of the substrate after 10 min,
although the reaction—taking into account the evolution of
the possible by-products—remained completely stable after
about 60 min. The residual ozone was removed by bubbling a
stream of nitrogen for 5 min. and the resulting water solution
was extracted with CH2Cl2. Gas chromatography revealed the
apparition of a single product with a tR = 6.5 min. Coupled
low-resolution mass spectra (LRMS) gave a molecular peak
for the new product formed of 129 amu, displaying a
fragmentation pattern that fitted to oxazolidinone 2.
TOF(MS) provides excellent mass accuracy over a wide
dynamic range and allows measurements of the isotopic
pattern, providing important additional information for the
determination of the elemental composition. Therefore, all the
detected compounds exhibit good sigma values (smaller than
0.05) and mass accuracy (ppm and mDa), see Table 1. Note
that the products detected are directly related either with 1 or 2
and, therefore, no competing mechanisms seem to operate in
this case.
Furthermore,
a rapid, direct-infusion electrospray mass
spectrometry (ES-MS) technique was used to identify all the
products formed in this reaction. Only the product 2 could be
detected, obtaining a [M + Na]+ peak of 152.0382 amu, thus
indicating a C5H7NO3 formula for the product. This structure
was completely ascertained by means of the corresponding
1H-NMR spectrum, realized from the mentioned organic
extract.21
We then decided to theoretically study the possible pathway
connecting both products in order to establish not only the
possible reactions involved therein but also the thermo-
dynamics of such a process. For this purpose, a DFT/
B3LYP computational study was performed using the
standard 6-311+G(d,p) basis set, see the above Computational
details section. Our guideline for the probable mechanism has
been based on the postulated mechanism displayed in
Scheme 2.
For this reaction, an enthalpy change of À214.6 kcal molÀ1
has been calculated, indicating an irreversible and relatively
high exothermic process. We have also calculated the free
energy change since a significant gain for this magnitude is
expected due to the change in the number of moles on each
side of the general equation:
The ozone treatment has been always performed on ultra-
pure water solutions at pH = 2, adding t-BuOH (0.1 M) as
radical scavenger. These conditions warrant a direct reaction
of ozone, avoiding by this manner the radical pathway that
may operate at different reaction conditions (neutral or basic
pH, and absence of radical inhibitors). In fact, we were able to
properly discriminate between the two mechanisms since the
velocity constants are very different for each case. Thus, fÀor1
the direct reaction, kO3 for 1 amounted to 82 (Æ1) MÀ1
s
at pH = 2, whereas this value rose to 4.4 (Æ0.2) 1010
M
À1 sÀ1
at pH = 9.21 Even under direct reaction conditions, ozone
readily and irreversibly transformed the reactant (1) into the
final detected product (2), and no other stable subproducts
were detected by changing reaction times, always finding a
perfect correlation between the disappearance of the reactant
and the apparition of the product. This fits with a unique
reaction pathway operating along the potential surface region
in which is located the starting material. These findings moved
us to consider the observed product as a convenient indicator
for the presence of nitroimidazoles in ozone-treated waste-
waters. Indeed, the ozonation of purchased oxazolidinone 2
indicated a very low rate constant for its degradation by
ozone, kO3 being only 0.03 MÀ1 sÀ1. This value is much lower
than that obtained for 1, meaning that, once the decomposition
of 1 takes place, the concentration of compound 2 remains
almost invariable during the ozonation process.
It has been experimentally observed the presence of two
equivalents of nitrate ions (0.31 mmol from 0.15 mmol of 1)
coming from the two processes indicated in Scheme 2, namely
the elimination of a nitrite ion after the ozonide formation
(4 in Scheme 2), and the hydrolysis of the final imine (9 in
Scheme 2) generating ammonia, both species being readily
oxidized by ozone. Moreover, a total organic carbon (TOC)
analysis revealed the presence of 1.28 mg LÀ1 of CO2, just
coming from the theoretical 1.52 mg LÀ1 of CO2 that should
be expelled in the 8 - 9 transformation. The postulated
mechanism seems then to be coherent enough with the experi-
mental findings.
DFT mechanism
In order to evaluate the presence of other intermediates or
side-reactions for the 1 - 2 transformation, we have checked
the decomposition of MNZ (1) performing several HPLC-
ESI-TOF(MS) experiments at different ozonation times. The
detected products always were related products either
with 1 or with 2, also corroborating the unique process
linking the two products. Thus, Fig. 1 shows the base peak
The first step consists of a 1,3-dipolar cycloaddition of ozone
to the nitroimidazole 1. Although many positions may at first
sight appear as accessible for ozone, only one allows a low-
energy process: the C–C double bond. This is the preferred
pathway to form the corresponding molozonide 3. Further-
more, this process is highly stereoselective since the ozone
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Fig. 1 Tridimensional HPLC-ESI-TOF(MS) diagram of the products found in function of the migration time and ozonation time.
Table 1 HPLC-ESI-TOF(MS) data for the detected compounds
Compound
2-Methyl-1H-imidazole
Oxazolidin-2-one
MNZ (1)
HPLC migration time/min
Formula
Selected ion
Exp. m/z
Calc. m/z
7.6
14.0
24.4
C4H7N2
[M + H]+
83.0598
83.0603
6.7
C3H6N1O2
[M + H]+
88.0391
88.0393
2.0
C6H10N3O3
[M + H]+
172.0717
172.0717
0.1
Error
ppm
mDa
0.55
0.18
À0.01
Sigma value
Tolerance (ppm) in GenerateMolecularFormula
Order on considering other possible compounds
0.0364
10
1st of 1
0.0260
5
1st of 1
0.0068
5
1st of 3
decidedly prefers, by 37.6 kcal molÀ1 (enthalpy value), to be
placed at the same side that the –CH2CH2OH chain, thanks to
the hydrogen bond that can be established between them, see
Fig. 2. The corresponding transition state enthalpy barrier for
this process is quite low since the intrinsic ozone instability
places the energy of the starting separated materials only
2.0 kcal molÀ1 (enthalpy value) below, see Table 2. That the
ozone preferably reacts with the C–C double bond can be
drawn from the frontier molecular-orbital (FMO) theory.22
Indeed, this fast, concerted 1,3-dipolar cycloaddition can be
adequately viewed as the in-phase interaction between the
C–C double bond HOMO (at À0.27039 hartree) and the ozone
LUMO (at À0.20441 hartree),23 through a p - p* electron-
donating process, see Fig. 3.
Several rearrangements have been essayed tempting to
describe a low-energy molozonide re-organization to get the
corresponding ozonide 4, finding that the most favorable
pathway corresponds in fact with that proposed by Criegee,24
which in turn is the mechanism most widely accepted. This
route begins with the molozonide rearrangement through the
1,2,3-trioxo unit breaking of the trioxolane ring. However,
owing to the molecular asymmetry along the C–C double
bond, we can consider two breaking ways: either (a) the
Scheme 2 The proposed mechanism for the conversion of 1 to 2.
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Fig. 2 A sequence of the calculated structures with some significant geometric parameters.
O–O fragment goes to the C–H carbon atom and the
remaining O to the C–NO2 carbon atom, or (b) the O–O
fragment goes to the C–NO2 carbon atom and the remaining
O to the C–H carbon atom. The calculations showed that
process running with no activation barrier.25 The reason for
this can be attributed to four interrelated factors: (a) the
1,3-diaxial disposition that the –NO2 and –H groups acquire
as the O atoms of the O–O fragment separate one from the
other, (b) the more carbonyl character that these oxygen
atoms gain with distance, (c) the subsequent leaving character
that the –NO2 group gets, and (d) the high exothermicity
calculated for such a process (Table 2). This can be seen on the
calculated Ocarbonyl–C–NO2 distance values on going from 4 to
7, see Scheme 3. Note that during the process the –NO2 group
promotes a carbon to oxygen proton transfer in order to
get an sp2 CQO bond, all this progression occurring with no
activation barrier.
the most favorable pathway—by about 15 kcal molÀ1
—
corresponded to the (a) route, through the intermediates
3-intera and 3-interb, which are related by a simple CO(NO2)
rotation, see Fig. 2. This rotation has been calculated as
slightly endothermic, see Table 2. The calculated energy values
for the
3 to 4 transformation showed that, although
the formation of the 3-intera intermediate is significantly
exothermic, a very flat, reversible energy surface can be
achieved on going from 3-intera to 4.
But whereas the proposed mechanism (Scheme 2) displayed
the intermediates 5 and 6, calculations showed that the
ozonide 4 could directly evolve—through the corresponding
ozonide O–O fragment breaking process—to the carbamic
anhydride 7 by the release of a molecule of HNO2, the latter
Further intramolecular, nucleophilic attack of the oxygen
atom of the –CH2CH2OH chain to the neighboring carbonyl
group on 7—through the somewhat high-energy four-
membered transition state 7-TS1 (see Fig. 2)—led to achieve
the bicyclic intermediate 7-inter. This process produces a
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concomitant proton transfer to the mentioned carbonyl, see
Fig. 2, generating a tertiary alcohol that lies 16 kcal molÀ1
above the carbamic anhydride 7 (Table 2). This intermediate
evolves, through a second four-membered transition state
(7-TS2, which is about 7 kcal molÀ1 lower in energy than
the corresponding 7-TS1 transition state, see Table 2), to the
corresponding isomeric carbamic acid derivative 8, see Fig. 2,
which in turn is almost iso-energetic with the starting carbamic
anhydride 7.
From 8, the subsequent CO2 elimination process happens
when a proton transfer takes place from the oxygen atom to
the neighbor nitrogen atom, see Fig. 2. This then results in the
moderately exothermic conversion of the original carbamic
acid into the corresponding imine 9, whose inevitable
hydrolysis in the experimental conditions led to the final
detected oxazolidinone 2. This hydrolysis process operates
following two steps: (a) the addition of a water molecule
leading to the intermediate 9-inter after a proton transfer
reaction from the incoming water molecule to the present
Fig. 3 Representation of the HOMO–LUMO p - p* electron-
donating process between MNZ and ozone, respectively.
Table 2 Calculated energies (DE), enthalpies (DH), and free energies (DG) for the species involved in the 1 - 2 transformationa,b
System
DE
DH
DG
Imaginary frequencyc
1 + O3
1-TS
3
3-TS1
3-intera
3-interb
3-TS2
4
4-TS
7f
7-TS1
7-inter
7-TS2
8
0.0
+1.6
À38.6
0.0
+2.0
À36.9
0.0
+15.4
À23.4
—
311i
—
À34.7 (+3.9)d
À77.4 (À38.8)d
À74.8 (À36.2)d
À65.2 (À26.6)d
À77.1 (À38.5)d
À51.3 (+25.8)e
À187.2
À34.2 (+2.8)d
À76.1 (À39.1)d
À73.2 (À36.3)d
À64.3 (À27.4)d
À74.7 (À39.1)d
À52.1 (+22.6)e
À186.6
À20.4 (+3.0)d
À63.1 (À39.6)d
À60.1 (À36.7)d
À50.1 (À26.7)d
À60.5 (À39.6)d
À38.7 (+21.8)e
À187.2
246i
—
—
351i
—
301i
—
À137.2 (+50.0)g
À171.1 (+16.1)g
À146.2 (+41.1)g
À186.6 (+0.6)g
À150.0 (+41.1)h
À200.0 (À13.4)h
À165.2 (+34.8)k
À208.1 (À8.2)k
À171.5 (+28.5)k
À214.6 (À14.6)j
À140.0 (+46.6)g
À170.6 (+16.0)g
À147.1 (+39.5)g
À185.6 (+1.0)g
À152.4 (+39.5)h
À200.2 (À14.5)h
À165.2 (+35.0)k
À205.6 (À5.4)k
À172.4 (+27.8)k
À214.6 (À14.4)j
À138.5 (+48.8)g
À169.4 (+17.9)g
À146.7 (+40.5)g
À185.8 (+1.4)g
À153.0 (+40.5)h
À211.1 (À25.3)h
À165.0 (+46.2)k
À204.7 (+6.5)k
À172.3 (+38.9)k
À226.7 (À15.6)j
985i
—
241i
—
8-TS
9i
1710i
—
9-TS1j
9-inter
9-TS2
2l
435i
—
1660i
—
a
b
The values are in kcal molÀ1, the zero of energy (ZOE) for the whole reaction initially refers to the sum 1 + O3. The B3LYP total energy,
enthalpy and free energy (in a.u.) are, respectively, for 1 + O3: E = À849.540145, H = À849.353822, G = À849.431477. In cmÀ1, refers to the
c
d
unique imaginary frequency of the calculated transition state. Referred to 1 + O3. The values in parenthesis refer to 3 as the ZOE for
e
f
comparison. Referred to 1 + O3. The values in parenthesis refer to 4 as the ZOE for comparison. Energy values referred to 1 + O3 À HNO2:
g
h
E = À643.768426, H = À643.606445, G = À643.655907. The values in parentheses refer to 7 as the ZOE for comparison. The values in
i
parentheses refer to 8 as the ZOE for comparison. Energy values referred to 1 + O3 À HNO2–CO2: E = À455.121511, H = À454.974783, G =
j
k
À454.999986. Energy values referred to 1 + O3 À HNO2 À CO2 + H2O: E = À531.579974, H = À531.408184, G = À531.454809. The values
l
in parentheses refer to 9 as the ZOE for comparison. Energy values referred to 1 + O3 À HNO2 À CO2 + H2O À NH3: E = À474.997338, H =
À474.863601, G = À474.887338.
Scheme 3 The elimination of HNO2 with no activation barrier from the corresponding O–O breaking transition state. The non-labeled structures
have been obtained on relaxing the corresponding 4-TS transition state.
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Scheme 4 The complete energy profile for the 1 - 2 transformation.
imine group, and (b) the elimination of ammonia thanks to a
second proton transfer from the hydroxyl group to the amino
group generated in the first step, see Fig. 2.
are probably connected by a unique reaction pathway. Indeed,
coupled HPLC-ESI-TOF(MS) revealed the presence of simple
derivatives of both metronidazole and N-acetyl-oxazolidin-2-one,
leading us to think that both substances are strongly inter-
connected and that no other stable species are implied in the
conversion. A possible mechanism for this transformation has
been then postulated and evaluated by DFT/B3LYP calculations,
showing that the observed product should be readily obtained
mainly thanks to the high exothermicity displayed by the ozone
action over the metronidazole to obtain a lactone derivative that
suffers a series of hydrolysis processes leading to the final product.
Note that the most part of the exothermicity of the studied
reaction comes from the ozonation steps, since the energy of the
carbamic anhydride 7—formed just after the elimination of
the HNO2 molecule—is very close to the final energy calculated
for the oxazolidinone 2. It should also be noted that changes in
the free energy values correspond, as expected, with the uptake
and elimination of the small molecules that participate in the
mechanism. The other processes are all intramolecular and,
consequently, it takes place a small change in the free energy.
A complete view of the energy profile for the whole reaction
can be seen in Scheme 4. Note the high exothermicity of the
HNO2 elimination (4 - 7 conversion), and the proton trans-
fer cascade pathway connecting structures 7 and 2.
Acknowledgements
We thank MEC-DGI-FEDER (project CTQ2007-67792-C02-
01/PPQ) and the Junta de Andalucıa (projects RNM3823 and
FQM03705) for financial support. D. Arraez acknowledges
funds from the Consejerıa de Innovacion, Ciencia y Empresa,
Junta de Andalucıa (project P07-AGR-02619). The calcula-
tions have been carried out at the Centro de Supercomputa-
cion de la Universidad de Granada (CSIRC, Granada, Spain).
The authors wish to thank Javier Camino Sanchez from LVM
Cavendish and Alegrıa Carrasco Pancorbo from University of
Granada for their assistance in the analytical determinations
carried out in this work.
Conclusions
The use of N-acetyl-oxazolidin-2-one as a covenient indicator in
the removal of nitroimidazoles—acting as antibiotic contami-
nants in wastewaters—has been studied since it is the sole
degradation product achieved in the treatment of metronidazole-
containing waters carried out by ozone, and it is stable against
further ozonation in the experimental conditions used. This
conversion has been extensively studied by different
techniques in order to trap possible by-products and/or other
subproducts. The ozone treatment, however, does not allow
isolation of any by-products, the reactant being readily and
irreversibly transformed into the final detected product. Nor
were other stable subproducts detected, indicating that both the
starting metronidazole and the final N-acetyl-oxazolidin-2-one
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2212 | New J. Chem., 2010, 34, 2205–2212