Feed-back action of nitrite in the oxidation of nitrophenols by bicarbonate-activated peroxide system

Article abstract of DOI:10.1016/j.apcata.2016.02.021

In this study it was found that the nitrite anion greatly increases the oxidation rate of the substituted phenols by the bicarbonate-activated peroxide (BAP) system at ambient temperature. This feed-back effect was investigated in the BAP oxidation of 2-amino-4-nitrophenol and 4-nitrophenol, where the kinetic analysis showed that the rate determining step was the elimination of a nitro group as nitrite. Complementary oxidation experiments with 2-aminophenol in BAP system, performed in the presence/absence of sodium nitrite confirmed the catalytic role of this anion. High concentrations of nitrite/BAP prevented formation of polymeric species and favoured the degradation to aliphatic fragments such as 3-oxobutanoic and acetic acid. Indirect evidence suggested in-situ generation of reactive oxygen species (ROS), other than hydroxyl, peroxyl and carbonate radicals, during the nitrite/BAP oxidation. Formation of additional ROS - peroxynitrite and nitrosoperoxycarbonate - may account the synergistic action of nitrite/BAP system. The estimated value of the apparent rate constant of the autocatalytic step for 2-amino-4-nitrophenol (k_app = (4.97 ± 0.19) × 10³ M^-3 s^-1) demonstrates that fast degradation of this oxidation-resistant phenolic dye is feasible even in ambient conditions. These findings can be exploited for developing clean, cost-effective methods for the removal of phenolic dyes as alternative to current advanced oxidation processes.

Full text of DOI:10.1016/j.apcata.2016.02.021

Accepted Manuscript
Title: Feed-back action of nitrite in the oxidation of
nitrophenols by bicarbonate-activated peroxide system
Author: Mihaela Puiu Toma Galaon Luiza Bondilnull Adina
Rnullducan Dumitru Oancea
PII:
S0926-860X(16)30087-4
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.apcata.2016.02.021
APCATA 15780
To appear in:
Applied Catalysis A: General
Received date:
Revised date:
Accepted date:
26-12-2015
15-2-2016
18-2-2016
Please cite this article as: Mihaela Puiu, Toma Galaon, Luiza Bondilx1ce;, Adina
Rx1ce;ducan, Dumitru Oancea, Feed-back action of nitrite in the oxidation of
nitrophenols by bicarbonate-activated peroxide system, Applied Catalysis A, General
http://dx.doi.org/10.1016/j.apcata.2016.02.021
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Page 1 of 31
Feed-back action of nitrite in the oxidation of nitrophenols by bicarbonate-activated
peroxide system
Mihaela Puiua, *, Toma Galaonb, Luiza Bondilǎa, Adina Rǎducana, Dumitru Oanceaa
aDepartment of Physical Chemistry, University of Bucharest, 4-12 Elisabeta Blvd., 030018, Bucharest, Romania
bNational Research and Development Institute for Industrial Ecology, 71-73 Drumul Podu Dambovitei Street, 060652
Bucharest, Romania
Corresponding author: Dr. Mihaela Puiu
Department of Physical Chemistry, University of Bucharest, 4-12 Regina Elisabeta Blvd., 030018 Bucharest, Romania,
e-mail: elenamihaela.puiu@g.unibuc.ro
Graphical abstract
Highlights



2-amino-4-nitrophenol and 4-nitrophenol undergo fast oxidation in the BAP system
The oxidation follows an autocatalytic path promoted by the elimination of nitrite
The in-situ generation of ROS led the degradation to aliphatic products
Abstract
In this study it was found that the nitrite anion greatly increases the oxidation rate of the substituted phenols by the
bicarbonate-activated peroxide (BAP) system at ambient temperature. This feed-back effect was investigated in the

Page 2 of 31
BAP oxidation of 2-amino-4-nitrophenol and 4-nitrophenol, where the kinetic analysis showed that the rate determining
step was the elimination of a nitro group as nitrite. Complementary oxidation experiments with 2-aminophenol in BAP
system, performed in the presence/absence of sodium nitrite confirmed the catalytic role of this anion. High
concentrations of nitrite/BAP prevented formation of polymeric species and favoured the degradation to aliphatic
fragments such as 3-oxobutanoic and acetic acid. Indirect evidence suggested in situ generation of reactive oxygen
species (ROS), other than hydroxyl, peroxyl and carbonate radicals, during the nitrite/BAP oxidation. Formation of
additional ROS - peroxynitrite and nitrosoperoxycarbonate - may account the synergistic action of nitrite/ BAP system.
The estimated value of the apparent rate constant of the autocatalytic step for 2-amino-4-nitrophenol (kapp = (4.97±0.19)
∙103 M-3s-1) demonstrates that fast degradation of this oxidation-resistant phenolic dye is feasible even in ambient
conditions. These findings can be exploited for developing clean, cost-effective methods for the removal of phenolic
dyes as alternative to current advanced oxidation processes.
Abbreviations: AOP advanced oxidation processes, BAP bicarbonate activated peroxide, ROS
reactive oxygen species, AP 2-aminophenol, NAP 2-amino-4-nitrophenol, NP 4-nitrophenol, APX
2amino-3H-phenoxazin-3-one, OQI o-quinoneimine
Keywords: 2-amino-4-nitrophenol, reactive oxygen species, bicarbonate activated peroxide system
1. Introduction
The efficient degradation of contaminants from natural waters such as pharmaceuticals, dyes and
detergents is still one of the targets in water- or waste treatment strategies. These xenobiotics,
mostly containing phenolic rings, pose major ecological threats due to their long biological half-life
and accumulation in water, soil and living organisms[1-3]. The treatment of contaminated water
before discharging into the aquatic environment demands additional non-biological technologies as
advanced oxidation processes (AOPs) [4-8]. (AOPs) are considered the most important
technologies for environmental remediation of natural and wastewaters containing recalcitrant
compounds, through the production of reactive oxygen species (ROS) [9, 10]. AOPs are based on
the in-situ generation of highly reactive hydroxyl radicals (·OH) in mild conditions [11-14]. The
hydroxyl radicals can oxidize any compound present in the water matrix, frequently at diffusion

Page 3 of 31
controlled rates [5]. Once formed, ·OH reacts unselectively and degrades efficiently the water
contaminants to non-toxic, low molecular weight (LMW) compounds [15]. The use of primary
oxidants (e.g. ozone, hydrogen peroxide, dioxygen) [16], energy sources (ultraviolet light) [17-19]
or catalysts (titanium dioxide, iron (III), cobalt(II), potassium permanganate)[20-26] are the current
−
pathways of generating hydroxyl radicals and other ROS such as superoxide radical (O2 ·), peroxyl
radical and singlet oxygen [9]. Despite the advantage of removing organic compounds in aqueous
phase AOPs hold several drawbacks which prevent their full-scale implementation; most
prominently, AOPs costs are very high, requiring continuous inputs of expensive chemicals in order
to maintain the operability of the oxidative system[16]. Another issue is the need of pre-treatment
procedures, which are costly and technically demanding. The presence of bicarbonate anion
−
(HCO3 ) can significantly reduce the concentration of ·OH due to scavenging processes that lead to
−
−
H2O and to a much less reactive ROS, the carbonate radical ·CO3 [27]. Consequently, HCO3 must
be removed from the contaminated samples before starting the generation of ROS in typical AOPs.
−
The HCO3 /CO2 buffer is ubiquitous in natural waters and stimulate the oxidation, peroxidation and
−
nitration of several biological targets [28]. In addition Richardson et al. [27] used HCO3 as
activator H2O2 in sulphide oxidation in alcohol/water mixtures. The bicarbonate-activated peroxide
(BAP) system has been highly efficient in the epoxidation of alkenes [29] through generation of
−
−
−
·CO3 and O2 ·. Although less oxidizing than ·OH (E0 = 2.3 V vs NHE, pH 7.0), ·CO3 (E0 =1.78
V, pH 7.0) is a very strong one-electron oxidant that acts by both electron transfer and hydrogen
abstraction mechanisms to produce radicals from the reducing substrates [30]. ROS generation from
BAP system occurs according to the sequence (1) - (4):


H2O2  HCO3 HCO4  H2O
(1)
HCO4 CO3 OH
(2)
(3)
(4)
CO3  H2O2  HOOHCO3
HOO  H  O2 

Page 4 of 31
−
·CO3 is a selective ROS, attacking preferentially phenolic residues from tyrosine, indolic moieties
from tryptophan and sulphides from methionine [31] especially when acting in tandem with ·NO2
[32-34].
ArHCO3  ArCO32  H
Ar NO2  ArNO2
(5)
(6)
The BAP system can be used to build-up environmental-friendly oxidizing technologies as
alternatives to current AOPs. However, the BAP system become efficient in the degradation of
phenol-based compounds only when is taken in a large excess (102 -104 fold) with respect to the
target concentration [35]. The issue is to ensure steady concentrations of ROS, enough to prevent
-
polymeric by-products formation. Since both H2O2 and HCO3 are relatively inexpensive and non-
polluting reagents, the required high BAP/target concentration ratios could successfully be ensured
either in batch or continuously-stirred reactors. In the case of nitroaromatics (e.g. nitrophenols), the
elimination of nitro groups may provide supplementary ROS such as peroxynitrite (ONOO-),
-
nitrosoperoxycarbonate (ONOOCO2 ) and NO2 [30, 31, 36, 37], all of them potential oxidizing
agents. In this work we attempted to exploit the positive feed-back action of the nitro group
(consistent with an autocatalytic degradation path) on the BAP oxidation of three phenol-based
dyes: 2-aminophenol (AP), 2-amino-4-nitrophenol, (NAP) and 4-nitrophenol (NP). NAP and NP
are particularly resistant to chemical/biological oxidation due to the electron-withdrawing nitro
group which deactivates the aromatic ring towards the electrophilic/radical attacks [38]. AP
undergoes (bio)-catalysed oxidation to its stable condensation product 2-amino-3H-phenoxazin-3-
one (APX), which is also recalcitrant to conventional oxidative treatments [39, 40]. The oxidative
degradation of NAP is by far the less studied compared to AP and NP; therefore, we focused our
kinetic study on NAP oxidation. We proposed first a simplified kinetic model which highlighted the
autocatalytic effect of the nitro group during the BAP oxidation and the time-evolution of NAP,
-
-
H2O2, HCO3 and NO2 concentrations; an extended kinetic model including the time-evolution of
the reaction intermediates was further developed, because their toxicity can sometimes be even

Page 5 of 31
higher than the toxicity of the initial pollutants [5]. The degradation pathways of AP and NP were
depicted elsewhere [9, 11, 39] . For these substrates we developed only simplified kinetic models
describing the time-evolution of spectrophotometrically detectable intermediates.
-2. Materials and methodsl
2.1 Reagents
AP, NAP and NP (99%), 4-nitrocatechol (97%), methanol (HPLC grade), acetonitrile (ACN)
(HPLC grade) formic acid (LC-MS grade), sodium bicarbonate (99.5%), sodium hydroxide (purity
≥97%), sodium nitrite (purity≥97%), sodium nitrate (purity ≥99) and acetic acid (purity ≥99%)
were acquired from Sigma-Aldrich (Germany). H2O2 obtained as a 30 % solution from Merck was
diluted and the concentration was determined spectrophotometrically, using 240 nm = 43.6 M–1cm–1.
APX (m.p. = 249oC) was synthesized upon the oxidation of 2-aminophenol with mercury oxide
followed by recrystallization from ethanol. Ultrapure water from a Millipore Simplicity 185
(conductivity < 6×10−8S·cm−1, max. 5 ppb total organic compounds, TOC) system was used in the
chromatographic experiments. Gaseous dioxygen was purchased from Linde (99.6% purity).
Trametes versicolor laccase was purchased from Fluka (26U/mg specific activity) and was used to
prepare 2-amino-8-nitro-3H-phenoxazin-3-one and 3-amino-8-nitro-1H-phenoxazin-2(10H)-one
from oxidation of NAP with dioxygen. Other chemicals used were of analytical reagent grade.
2.2 Analytical methods
Spectrophotometric assays
The ultraviolet-visible (UV-VIS) measurements during the BAP oxidation of AP, NAP and NP
were made on a Lambda 25 Perkin-Elmer spectrophotometer with a Peltier cell for temperature and
stirring control. For IR analysis of the reaction products of AP and NAP oxidation, the collected
−
solution after 240 min was adjusted to a pH of 2.5 with HCl to completely decompose HCO3 , then
dried at 30◦C. The resulted solid sample was recorded in KBr pellets on a BrukerTensor 37
spectrophotometer.
Differential Scanning Calorimetry (DSC) analysis

Page 6 of 31
The DSC analysis of the reaction products of NAP oxidation was performed with DSC-2 Perkin–
Elmer apparatus connected to a Tektronix TX3 multimeter and optically coupled to the interface RS
232. The scan heating rate employed was 10 K min-1 in the range 300–650 K under an argon
atmosphere; the apparatus sensitivity was 5 mcal s-1 and the multimeter had a maximum display at
50 000 with a 0.03% error on the work domain.
Liquid chromatography–mass spectrometry (LC-MS) analysis
Experiments were conducted in an Agilent 1260 series LC system (Agilent, Waldbronn, Germany)
consisting of: a binary pump, a thermostated autosampler, a thermostated column coupled with an
Agilent 6410B triple-quadrupole mass analyzer with electrospray ionization (ESI) source. Data
acquisition was performed using Mass Hunter software. All chromatographic runs were carried out
on a Zorbax SB-C18 (50 x 2.1 mm, 1.8 µm) column from Agilent at 35°C. Isocratic elution was
performed with a mobile phase composition of aq. 0.1% formic acid and acetonitrile in the
volumetric ratio 90/10. A mobile phase flow-rate of 0.15 mL/min was chosen to enhance ESI
ionization and sensitivity. Injection volume was 10 µL. MS detection of the substrates and their
oxidation products was done in Single Ion Monitoring (SIM) and full scan mode. Both positive and
negative spectra were acquired in full scan mode using the range 50 – 500 Da. ESI source
parameters were: drying gas temperature 300°C, drying gas flow-rate 9 L/min, nebulizer pressure
50 psi. Sampling capillary voltage was set to 2500 V for negative mode and 4000 V for positive
mode. Skimmer (fragmentor) voltage was set to 90 V. First quadrupole (Q1) resolution was set to
Unit when SIM mode was used. Cell accelerator voltage of second quadrupole (Q2) was maintained
at 7V. Linearity of the detector response was determined in the range 1 – 5 µg/mL. Individual stock
standard solutions AP, NAP and NP were prepared at a concentration of 500µg/mL in methanol.
Calibration solutions were diluted in the mobile phase. All standard solutions were kept protected
from light at 4°C.
Total organic carbon (TOC) determinations

Page 7 of 31
TOC analysis of pollutant solutions and final oxidation mixtures was performed on an Apollo 900
Combustion TOC Analyzer (Teledyne Tekmar, Mason, Ohio, USA) with a Non-dispersive Infra
Red (NDIR) detector for CO2 (0 -20 ppm calibration range) . The total combustion of the samples
was achieved through the 680°C combustion catalytic oxidation method. Data processing was
performed with the TOC Talk Apollo 9000HS v 4.2 software.
2.3 Degradation experiments
The oxidations were conducted directly in the spectrophotometer cuvette. All phenolic substrates
were dissolved in degassed bicarbonate solutions at constant temperature (25oC); their stability was
checked by following the UV-VIS spectra during 240 minutes. Since no variation in the UV-VIS
features was noticed, we assumed that no degradation of substrates in bicarbonate solution occurred
in the considered time range. The reaction was initialized by adding different volumes of H2O2
stock solution such as the total H2O2 concentration in the reaction mixtures ranged from 1·10-2 to
3·10-1M. In all assays at least a 100-fold excess of bicarbonate was ensured with respect to the
phenolic substrates, for keeping NP and NAP as coloured phenolates, with sharp and well-defined
absorption peaks in the VIS region of the spectrum (at λ = 399 nm for NP and λ =443 nm for NAP)
and also for keeping the pH at a constant value. The absorbance decrease was used further to
calculate the time-evolution of the total conversion. In the case of AP, with no significant
absorption in the VIS region, the absorbance measurements were achieved at λ = 434 nm, where the
stable oxidation product APX has a molar absorptivity of ε = 23200 cm-1·M-1 or at λ = 401 nm
where the formation of a coloured intermediate was observed. NaHCO3 concentrations were within
1·10-2 to 3·10-1M, while the AP, NAP and NP ranged within 3·10-6 M to 3·10-4 M. No pH shifting
from the value of 8.5 in the reaction solutions was observed during the reaction time. Blank
experiments were performed by replacing NaHCO3 with NaOH, adjusting NaOH concentration to
increase the pH value from 8.5, as in the presence of NaHCO3 to 12, to strong alkaline medium.
While the UV-VIS features of NP and NAP remained unchanged during 240 minutes, a slight

Page 8 of 31
increase of the absorption peak at 434 nm, assigned to APX formation was noticed during the AP
oxidation. Since the AP oxidation follows the same pattern to APX formation as described
elsewhere [39], whereas NP and NAP underwent no oxidation, we assumed that the fast fading of
NP and NAP solutions occurred in the BAP system only. LC-MS analysis was performed first on
the bicarbonate solutions of AP, NAP and NP, then at different times till the end of the oxidation
experiments, when the total conversion of the substrates remained constant. Samples from the
reaction mixtures with low conversions of substrates were kept at ambient temperature and their
compositions were analysed after 2-5 days. The identification of the aromatic by-products was
performed by comparing with their standard solutions injected in the same LC-MS conditions as the
substrates. The experiments were also conducted in 3 main sets according to the concentrations’
-
ratio of the substrate/BAP system (i.e. equal concentrations of HCO3 and H2O2): 1:102, 1:103, and
1:104 because this shifting caused significant changes in the composition of the final reaction
mixtures. Finally, the oxidation assays of AP, NAP and NP with BAP + NaNO2, were achieved for
1: 1:104 concentrations’ ratio of substrate/BAP system along with blank experiments with NaHCO3
+ NaNO2 and H2O2 + NaNO2 with NaNO2 ranging from 1∙10-5 to 1∙10-2M.
2.4 Synthesis of 2-amino-8-nitro-3H-phenoxazin-3-one and 3-amino-8-nitro-1H-phenoxazin-
2(10H)-one
The enzymatic oxidation of NAP with dioxygen was conducted in aqueous solution in a bubbling
stirred semi-batch reactor at 25oC. The gaseous stream was passed through two saturation vessels
with water to avoid the evaporation of the solution from the reaction vessel; an aqueous solution of
1∙10-3 M 2-amino-4-nitrophenol was incubated with 1∙10-5 M laccase and continuously stirred for 12
h. The yellow-brown precipitate was collected, washed with water and then recrystallized from
ethanol and gently dried under an argon stream.

Page 9 of 31
3. Results and discussion
3.1 Efficiency of the BAP system
The calibration plots from colorimetric and LC-MS analyses were built up to calculate and
eventually compare the conversion of substrates with both methods. The absorbance –
concentration data of NAP and NP standard solutions at pH 8.5 provided the molar absorption
coefficients: at ελ=443 nm = 12621M-1cm-1 for NAP and ελ=399 nm = 18100M-1cm-1 for NP, which were
used further to calculate AP, NAP and NP concentrations for the extended progress curves during
the degradation experiments. The oxidant systems containing 102, 103and 104 fold excesses of BAP
for all 3 phenolic substrates yielded different oxidation products as well as significant changes of
conversion. The half-lives of the phenolic substrates (τ1/2) were calculated with respect to
substrate/BAP molar ratio (Fig.1 A-B) together with the degradation frequencies in initial
conditions, when the catalytic effect of nitrite was negligible (conversions below 5%). The
degradation frequencies were calculated as slopes of linear parts of the extended progress curves
conversion vs. time for the optimized 1:103 and 1:104 substrate/BAP ratio (Fig. 1 B-C).
Figure 1
The 1:103 phenol/BAP ratio was efficient only for AP degradation giving a half-life of 7122 s. NAP
was more resistant to degradation than NP which can be assigned to their different oxidation
mechanisms. Interestingly, for 1:104 ratios the half-life of NAP was shorter than NP (600 s vs. 7200
s), presumably due to the faster generation of nitrite. The degradation frequencies show a much
higher value for AP compared to NAP and NP (Fig. 1 C). Apparently, the amino substituent in the
o- position decreases the stability of the aromatic ring of both AP and NAP and is consistent with
fact that the degradation frequency of NAP is 10 times higher than NP’s (Fig. 1 D).
1:102 substrate /BAP molar ratios provided low conversions of the phenolic substrates, even after
240 minutes. Conversely, for 1:104 substrate/BAP molar ratios, AP and NAP underwent 99.9 %
conversion after 70 minutes, while NP attained only 21 % (Fig. 2).

Page 10 of 31
Figure 2
The oxidation mechanism of AP was widely studied and its conversion to APX in the presence of
mild oxidants is well known [41, 42]; it was expected NAP to follow a similar pattern. The LC-MS
analysis of the final reaction mixtures supported by literature data [43-46] provided mechanistic
insights regarding the BAP oxidation of AP, NAP and NP. The relevant MS spectra of oxidation
intermediates along with the corresponding chromatograms are summarized in the supporting
information (SI) Figs. SI-1-4. The characteristic precursor ions and molecular masses of the
substrates and of the presumable degradation products are given in Tables SI-1 A-C. It is commonly
accepted that the six-electron oxidative condensation of two molecules of AP to form APX occurs
via o-quinoneimine (OQI), a highly reactive intermediate [46, 47] which was not identified with the
LC-MS analysis. However, while the experiments of AP degradation at 1:103 AP/BAP molar ratio
yielded APX (Table SI-1-A), whose formation was monitored by absorbance measurements, the
assays conducted at a 1:104 AP/BAP molar ratio gave LMW products in the final reaction mixture.
It was noticed the fast appearance/disappearance of an absorption band at 401 nm which can be
assigned to OQI formation/decay (Fig.SI-5). At high concentrations of BAP system the
decomposition of the OQI is faster than the condensation with another AP molecule to give APX.
Since no other possible intermediate with less chromophore groups than APX, stable enough to be
observed from absorbance measurements were reported so far, we assumed that the coloured
compound is OQI. The assumption is supported by the fact that the oxidation of NAP yielded the
corresponding 4-nitro-1,2-quinoneimine, a relatively stable by-product, identified with LC-MS
(Table SI-1-B). Its stability might be assigned to the electron-withdrawing nitro group which
enhances the double bond character of the carbon-oxygen bond. In the given reaction conditions
OQI undergoes apparent first-order decay. The rate constant was estimated by non-linear
regression, fitting an exponential decay equation on the experimental data which gave kapparent
=
(0.463±0.021) s-1 with a coefficient of determination r2 =0.9955, for a significance level α = 0.05.
Consequently, the apparent half-life of OQI (1:104 AP/BAP molar ratio) was around 1.5 s, a

Page 11 of 31
plausible value for an active intermediate. The composition of the reaction mixtures of AP
oxidation at different AP/BAP molar ratio corroborated with the formation of the coloured
intermediate absorbing at 401 nm sustain the hypothesis that at high concentrations of BAP, the
formation of APX doesn’t occur and OQI decomposes to aliphatic compounds (Table SI-1-A). To
confirm this hypothesis, solutions with concentrations from 3∙10-6 to 3∙10-5 M were prepared in
1:104 APX/BAP molar ratios and their VIS spectra were recorded during 240 minute when no
decrease of the characteristic sharp peak of APX was observed. Thus, a degradation pathway for
AP involving the decomposition of OQI to aliphatic compounds (Scheme 1) might serve as well to
explain a similar behaviour of NAP oxidation in the BAP system.
Scheme 1
At lower AP/BAP molar ratios the oxidation yield mainly APX, while higher ratios prevent the
formation of polymeric species In the case of NAP, the 4-nitro-quinoneimine is the key
intermediate either to condensation products such as 3-amino-8-nitro-1H-phenoxazin-2(10H)-one
and 2-amino-8-nitro-3H-phenoxazin- 3-one, or to the decomposition products such as 4-hydroxy-
1,2-benzoquinone. These compounds with 257, respectively 259 molecular weights were obtained
in control experiments from oxidation of NAP with dioxygen in the presence of laccase as
previously described. The solid mixture recrystallized from ethanol contained yellow and brown
crystals which were further separated mechanically and their distinct IR spectra were recorded
(Figs. SI-6, 7 and Table SI-2). Solid samples of the two nitrophenoxazones, analyzed with DSC
revealed close melting points (221 and 231.5oC), consistent with the melting points assigned to
phenoxazones. According to all evidence provided by LC-MS, UV-VIS, IR spectral data and DSC
analysis, a reaction path for NAP degradation in the BAP system was proposed (Scheme 2).
Scheme 2
The main degradation product generated in the BAP system during the oxidation of NAP is 4-
hydroxy-1,2-benzoquinone. At higher substrate/BAP molar ratios, the degradation products of NAP

Page 12 of 31
are mostly the same obtained at NP oxidation, while a lower substrate/BAP molar ratios NAP yields
phenoxazones.
3.2 Degradation kinetics of 2-amino-4-nitrophenol and 4-nitrophenol
A simplified kinetic model was developed for NAP and NP taking into account the degradation
path and the chemistry of the BAP system. In order to estimate the apparent kinetic constants, the
conditions implying the large excess of BAP species (1: 104 substrate/BAP molar ratios) were
preferred. The progress curves as total conversions of phenolic substrates vs. time were drawn in
the following experimental set-up (Fig.3):
-
Keeping the initial concentration of substrates at a constant value i.e. 3∙10-5M and first varying
the initial concentration of bicarbonate from 0.001 to 0.3M, then varying the initial
concentration of hydrogen peroxide from 0.001 to 0.3M. The decomposition of both BAP
species was considered negligible with respect to substrate depletion;
-
Keeping the initial concentration of both BAP species at the optimized value of 0.3M and
varying the concentration of the substrates from 3·10-6M to 3·10-4M.
Figure 3
The sigmoidal-shaped curves conversion vs. time, suggest an autocatalytic pattern. Changing
NAP/BAP ratio from 104 to 103caused a sharp drop of conversion this occurring probably because
the generated ROS from the BAP system reacted at low rates with NAP and consequently the
autocatalytic species evolved slowly, at low yields, in insufficient amounts to oxidize the substrate.
The main issue was to identify the possible autocatalytic species derived from NAP. To do this, we
followed the oxidation of 2-aminophenol in a 1:103 AP/BAP molar ratio, one experiment being
-
performed in the presence of NO2 . At this AP/BAP molar ratio the oxidation product is APX as
resulted from the LC-MS analysis.
Figure 4
In the absence of nitrite, the formation of APX follows an apparent first- order exponential growth
while in the presence of nitrite the shape of the progress curve changed to sigmoidal, finally

Page 13 of 31
yielding lower concentrations of APX. The LC-MS analysis of the latest proved the formation of 2-
-
butenoic acid along with APX. This is consistent with the fact that the NO2 favours the
decomposition of AP to LMW compounds and not the oxidative coupling to APX. Another
experiment performed in similar conditions with NAP, gave a decrease of NAP’s half-life from 600
to 124 s (data not shown).
A sigmoidal-shaped dependence was observed for the time-evolution of NP conversion, supporting
the hypothesis of a catalytic effect of nitrite. Accordingly, the time-evolution of the reactant system
for 1:104 substrate/BAP molar ratio, can be described by the two-step model:

k1
(7)
(8)
AP  M
1
k2
A M P  2M
1
Where A is the nitrophenolic substrate, P1 is a stable intermediate, possibly 4-hydroxy-1-2
-

k
benzoquinone and M is the autocatalytic species NO2 ; 1 is the apparent first-order rate constant of
-
the non-catalytic step when a large excess of HCO3 and H2O2 towards nitrophenols was considered
and k2 is the apparent second-order rate constant of the autocatalytic process.
The reaction rate of the autocatalytic step is:
rR  k2[A][M]
2
The rate of the overall process becomes:
d
 
A

rR  
k1 

A
 k2[A][M]
(9)
dt
From the mass-balance one obtains:
[M][M]0  [A]0 [A]  [P]
(10)
(11)
1
If y is the conversion, then:
d[A]
dy
dt

rR  
 [A]0
 k1[A]0

1 y

 k2[A]0
1 y[M]0 [A]0 y
 
dt
and



k1  k2[M]0
dy



 k2[A]0

1 y

 y
(11)

dt
k2[A]0



Page 14 of 31

k1  k2[M]0
If
then:
 
k2  A 0 k;
 
k2[A]0
dy
 k

1 y y

(12)
(13)
dt
The integrated form of the equation becomes:
1



ek t 1

y 
1ek


1 t

Eq. (13) was fitted on the experimental data NAP and NP conversion vs. time and the parameters k
and ρ were estimated through non-linear regression. Eq. (13) represents the analytical form of the
sigmoidal-shaped curve characteristic for autocatalytic processes. The coordinates of the inflection
point are the dimensionless parameter ρ and the induction period τ; the smaller the value of ρ, the
longer the value of τ.
From the dependence of k and ρ on the concentration of bicarbonate and H2O2 at a settled

concentration of NAP, the rate constants k1 and k2, as well as the empirical reaction orders towards
m
n

p

HCO3

H2O2 0
k2  k2

HCO3

H2O2 0
r the
-

k1  k1

HCO3 and H2O2 were estimated. With
and
0
0
expressions of k and ρ become:

p
r

k  k2

HCO3


H2O2 0 NAP 0

(14)
(15)
0
m
n

p
r

HCO3


k2
H2O2 0  k2
HCO3

HCO3


H2O2 0

NO2


k1

0
0
0
 

p
r




H2O2 0 NAP0
0
and
mp
nr

k1

HCO3


H2O2 0


NO2

0
NAP00
 


k2

NAP0
(16)

k
In order to estimate the rate constants k1 and 2 together with the partial empirical reaction orders m,
-
n, p and r, the parameters k and ρ were represented as functions of H2O2 and HCO3 initial
concentrations. Equations (14) and (16) were used along a multiple nonlinear regression analysis

Page 15 of 31
= (4.97±0.19)·103

k2
(Figs. SI-8 A - B). The estimated values were: p = 1.85±0.18, r = 1.15 ± 0.14,
-
L3·mol-3s-1 from the plot of k vs. [H2O2]0 and [HCO3 ]0 (with r2 = 0.9833, for α =0.05); and m =
-
0.974± 0.011, n = 1.06, k1= (4.16±)·10-3 s-1 from the plot of ρ vs. [H2O2]0 and [HCO3 ]0. In the case
of 4-nitrophenol all the extended progress curves conversion vs. time displayed a similar sigmoidal
shape described in eq. (13); for example, at [NP]0 = 3∙10-5M, ρ was (0.737± 0.048) and k was
(9.76±0.41)∙10-5 s-1 (with r2 = 0.9994 for α = 0.05), while for NAP ρ was (0.1691±0.0053) and k
was (3.02±0.012)∙10-3 s-1 (with r2 = 0.9955 for α = 0.05). It appears that the induction period was
longer for NAP oxidation, but the autocatalytic step in the case of NAP was faster than for NP. This
-
may suggest that the generation of NO2 from oxidation of 4-nitro-1,2-quinoneimine is the rate
-
determining step for NAP oxidation; compared to NP, the elimination of NO2 from 4-nitrocatechol
-
occurs at a higher rate. To plausibly explain the catalytic effect of NO2 in the presence of ROS
from the BAP system, an extended kinetic model was further developed suitable for oxidation
undergoing in batch reactors.
3.3 Extended kinetic model
The previous simplified kinetic model described the time-evolution of the overall degradation
process of NAP and NP in the BAP system, provided that the molar ratio substrate /BAP remains
1:104, as long as the degradation route to 4-hydroxy-1,2- benzoquinone is considered the main path
of NAP and NP decomposition. These conditions are difficult to attain in practice. In order to make
the entire process feasible, a more realistic kinetic model was build-up, taking to account the
-
reported data about the generation of ROS in HCO3 /H2O2 system and the LC-MS results from the
AP, NAP and NP oxidation; here SH- represents the 2-amino-4-nitrophenolate, S is the
corresponding 4-nitro-quinoneimine, P1 is 4-hydroxy-1,2-benzoquinone, P2 and P3 are 3-amino-8-
nitro-1H-phenoxazin-2(10H)-one and 2-amino-8-nitro-3H-phenoxazin-3-one, respectively; P4 is 3-
oxobutanoic acid.

-
-
k1
H2O2  HCO3 HCO4  H2O
(17)
(18)

-
-
k1
HCO4  H2O H2O2  HCO3

Page 16 of 31
ka2
HCO4 CO3 OH
(19)
(20)
k3
HCO3 OHCO3  H2O
k4
CO3  H2O2 HOOHCO3
(21)
k5
HOO H O2 
(22)
(23)
k6
O-2 HCO3- HOOCO32-
k7
OH  H2O2 HOOH2O
(24)
(25)
k8
SH - CO3-  O2  S CO32-  HOO
k9
CO32-  H HCO3-
(26)
k10
OH OH H2O2
(27)
(28)
k11
SO-2 CO3- P  NO-2 CO32-  H
1
k12
-
H2O2  NO ONOO  H2O
(29)
2

k13
-
-
2
ONOO  CO2 ONOOCO
(30)
(31)
(32)
(33)

k13
-
2
-
ONOOCO ONOO  CO2

k14
ONOOCO CO3- NO2
-
2

k14
CO3- NO2 ONOOCO
-
2
k15
CO3- NO2 CO2  NO3-
(34)
(35)
(36)
(37)
(38)
(39)
-
k16
ONOO NO3-
k17
SH - NO2 CO3- P  2NO-2  CO32-  2H
1
k18
SSH - P2
k19
SSH - P3
k20
CO3-  P P4  CO32-  CO2  H
1

Page 17 of 31
k21
OH  CO32- HO- CO3-
(40)
The reaction steps from eqs. 17-24, 26-27, 39-40 are typical for the BAP system and their rate
constants estimated [4, 28, 34, 35].
Steps 25, 28, 37-39 were derived from the “empirical” kinetics developed in the previous paragraph
−
and consistent with literature data regarding the reactions of ·CO3 with anilines and phenols [13,
48].
The sequence 29-35 includes steps from literature about peroxynitrite ONOO- formation from the
-
reaction of H2O2 and nitrite [33]. Still, since control experiments with NAP and NP in H2O2/NO2
system did not display any transformation of NAP and NP, we assumed that the key species in the
-
overall autocatalytic process is the nitrosoperoxycarbonate (ONOOCO2 ) which decomposes in
−
·CO3 and ∙NO2 [30, 31]. Reactions 25, 28, 37-39 were considered apparent single steps, comprising
several elementary steps where the mass - balance was neglected for H+ and the other degradation
products, because their entire mechanisms are not completely elucidated. The mass- and charge
balance was applied for the species of interest: 4-nitro-1,2-quinoneimine, 4-hydroxy-1,2-
benzoquinone, 3-amino-8-nitro-1H-phenoxazin-2(10H)-one, 2-amino-8-nitro-3H-phenoxazin-3-one
and 3-oxobutanoic acid, in agreement with the previously estimated apparent reaction orders from
the simplified kinetic model.
3.3.1 Simulation assays
The simulation providing the kinetic behaviour of the NAB/BAP system started from a set of rate
constants and initial concentrations of the chemical species involved. The rate constants from
literature are summarized in Table 1.
The initial concentration of CO2 was estimated using the equilibrium constant (KCO2/HCO3- = 4.3
×10-7M)[31], the initial concentration of bicarbonate, the pH of the solution, and the solubility of
CO2 in water at 25oC and 1 atm [49]. Since for all experiments the concentration of CO2 does not
exceed the solubility limit of 3.3∙10-2M, no absorption/desorption equilibrium was considered here.
NAP was considered as 2-amino-4-nitrophenolate at pH 8.5 (pKa = 7.6 [50]). The mass-balance for

Page 18 of 31
H+ and HO- was neglected because the pH remained constant during the kinetic assays; hence the
concentrations of H+ and HO- were kept at 3.16∙10-9M and 3.16∙10-6M respectively in the
simulation runs.
The time-evolution of the NAP/BAP system was described by the differential equations derived
from steps 17 – 40. The rate constants’ optimization was performed with Kintecus [51] for 10
experimental sets.
3.3.2 Sensitivity analysis
The sensitivity analysis was accomplished with Atropos [52] at three different time intervals to
avoid over-parametrization. Accordingly, all rate constants influenced the shape of the simulated
progress curves, therefore all proposed steps were considered kinetically significant. The optimized
rate constants (with α = 0.05) are summarized in Table 2.
The rate constants k3 and k6 were estimated during the optimization process; steps 20 and 23 were
assumed unilateral in the reaction conditions, even if they were reported in literature as bilateral
[27]. The by-and final products appear to accumulate in amount consistent with the data obtained
by LC-MS analysis in the final reaction mixtures (Fig.5).
Figure 5
Simulated and experimental kinetic curves of the reaction intermediate and products confirmed the
disappearance of NAP in less than 2 hours and the efficiency of nitrite/BAP system. The simulated
and experimental progress curves NAP vs. time matched well to obtained data for BAP
-
-
concentrations ranging from 1∙10-3 to 0.3M, either with NO2 generated in reaction or with NO2
added at the beginning of the experiment. Once again, the proposed path is in agreement with the
obtained results and describes satisfactorily the nitrite mediated oxidation of phenolic substrates in
the BAP system. There are some limitations of the extended model regarding the fate of
intermediates comprised in equations 36-39, which is difficult to trace. Therefore, we assume that
the overall equations 36-39 respect the mass-balance for the species of interest being in agreement
with the reaction sequence depicted in Scheme 2.

Page 19 of 31
3.3.3 Quantification of the mineralization level
The kinetic analysis coupled with the LC-MS results demonstrate that BAP system cannot achieve
complete mineralization, even at high substrate/BAP molar ratios (though 100% conversion of the
nitro-aromatic pollutants was obtained within 240 minutes). The main concern here, as in other
similar oxidative water treatments, is the formation of toxic or less biodegradable
intermediates/final products [53]. However, for high substrate/BAP molar ratios, AP and NP
yielded small aliphatic compounds, mainly acetate, whose toxicity is far less than of the initial
pollutants. Potentially toxic aromatic compounds were identified in the final reaction mixture after
BAP oxidation of NP (Table SI-1 C); therefore, the TOC analysis was performed first on AP, NAP
and NP in bicarbonate solutions without H2O2 (TOC0) and secondly, on reaction mixtures with the
optimized substrate/BAP molar ratio 1:104 after 240 minutes (TOC∞) The TOC percentage removal
was calculated as:
TOC0 TOC
% TOCremoval

100
(40)
TOC0
The results (summarized in Table SI-3) showed that TOC removal was 75 – 78 % for AP and NAP
and 62% for NP. Since high molar ratios of substrate/BAP were already used, and increasing this
ratio does not suit environmentally realistic conditions, we attempted to exploit the catalytic effect
of nitrite in order to achieve complete mineralization. Several experiments were performed using
-
substrate/BAP/NO2 molar ratios from 1:104:102 to 1:104: 1. Surprisingly, all gave around 100%
TOC removal for the three phenolic substrates, confirming the feed-back action of nitrite in the
BAP oxidation system. Consequently, the molar ratio with the lowest content of nitrite to achieve
complete mineralization (Table SI-3) was considered compatible with more environmental friendly
technologies. Thus, it was demonstrated that the final reaction mixtures of the first cycle of BAP
oxidation contain large amounts of nitrate, nitrite and bicarbonate anions. The bicarbonate anion
can be re-used simultaneously with nitrate and nitrite in a subsequent step through the photolysis of
nitrate with UV radiation at λ = 254 nm to generate peroxynitrite, carbonate and hydroxyl radical
together with other ROS species in the absence of hydrogen peroxide [54]. These findings may

Page 20 of 31
-
allow decreasing the molar ratio H2O2/HCO3 required for the TOC removal trough the cyclic
regeneration of ROS species. Another alternative is to remove nitrate from the final reaction
mixtures. Nitrate/nitrite removal from natural or waste water is usually achieved through reverse
osmosis, ion exchange, electrodialysis and activated carbon adsorption coupled with pH adjustment
[55, 56]. On the other hand, the biological denitrification by which denitrifying bacteria use nitrate
as terminal electron acceptor in their respiratory process, finally yielding gaseous nitrogen, may
well compete with the conventional treatment procedures in terms of economic feasibility [57]. It
was shown that autotrophic microorganisms such as Parcoccus could use molecular hydrogen as a
substrate and bicarbonate as carbon source for the complete reduction of nitrate (0.35 mg H2 being
required to complete the reduction of 1 mg nitrate nitrogen at 25oC)[58]. Therefore, we consider
that a working sequence comprising: BAP/nitrite oxidation combined with UV photolysis,
biological removal of nitrate and another subsequent BAP-assisted removal of the denitrifying
microorganisms may be amenable for wastewater treatment technologies.
4. Conclusions
We attempted to demonstrate that BAP system in tandem with nitro-aromatic compounds can
provide a feasible in situ “self-destroying” technology of stable nitro-derivatives in natural and
waste water amenable for batch reactors; The bicarbonate anion, which is the main source of
carbonate radical regenerates during the oxidation cycles and, as long as a stationary concentration
of H2O2 is ensured, the system can be implemented and developed along other AOP technologies
for nitro-derivatives removal. For example, a faster generation of ROS derived either from H2O2 or
from nitrate in the presence of bicarbonate can be achieved in photochemical reactors. The
simplified kinetic model is applicable to any nitro-derivative undergoing oxidation in a large excess
of BAP species, without requiring advanced knowledge of the reaction mechanism; on the other
side, the extended kinetic model accounted for the chemistry of BAP.

Page 21 of 31
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Page 23 of 31
Figure 1 Degradation efficiency of BAP system expressed as: (A-B) half-lives of phenolic
substrates for 1:103 and 1:104 phenolic substrate/BAP molar ratios and (C-D) degradation
frequencies for 1:104 phenolic substrate/BAP molar ratios (pH = 8.5, T = 25oC)

Page 24 of 31
Figure 2 Fractional conversions of the phenolic substrates vs. phenolic substrate/BAP molar ratios
after 70 minutes (pH = 8.5, T = 25oC)

Page 25 of 31
Figure 3 Optimization of initial molar ratios NAP/BAP system according to the time-evolution of
-
NAP conversion: A) Variation of [HCO3 ]0 when [NAP]0 = 3∙10-5M and [H2O2]0 = 0.3M; B)
-
variation of [H2O2]0 when [NAP]0 = 3∙10-5M and [HCO3 ]0 = 0.3M; C) variation of [NAP]0 when
-
[HCO3 ]0 = [H2O2]0 = 0.3M

Page 26 of 31
Figure 4 Evidence of autocatalytic effect of nitrite on 2-aminophenol oxidation in BAP system;
-
[AP]0 = 3∙10-5M, [HCO3 ]0 = [H2O2]0 =3∙10-2M

Page 27 of 31
Figure 5 Simulated and experimental kinetic curves of the reaction intermediate and products
-
([NAP]0 = 1.85∙10-5M, [HCO3 ]0 = 0.3M, [H2O2]0 = 0.3M, pH = 8.5, T = 25oC)

Page 28 of 31
Scheme 1 Proposed degradation pathway of AP for different molar ratios AP/BAP system

Page 29 of 31
Scheme 2 Possible degradation route of NAP in the BAP system based on LC-MS analysis of the
oxidation by-product