FENTON, PHOTO-FENTON, , H2O2

PHOTOLYSIS AND TiO 2 PHOTOCATALYSIS

OF DIPYRONE OXIDATION: DRUG
REMOVAL,
MINERALIZATION,BIODEGADABILITY AND
DEGRADATION MECHANISM.

BY
NAME: ANKITA ROY
R0LL NO. : 001610301067
DEPARTMENT OF CHEMICAL ENGINEERING
JADAVPUR UNIVERSITY
SECTION: A2
 PREFACE
Dipyrone (DIPY), an analgesic drug, is very quickly hydrolyzed to 4-methylaminoantipyrine
(4-MAA) in an acidic solution. Batch study was conducted to compare the performance of
different oxidation processes such as Fenton (FP), photoFenton (PFP), UV/H2O2 photolysis
(UVP), and UV/TiO2 photocatalysis (UVPC) for removal of 4-MAA from aqueous solution.
The degradation efficiency was evaluated in terms of total organic carbon (TOC) reduction
and enhancement of biodegradability. Maximum 4-MAA removals of 94.1, 96.4, 74.4, and
71.2% were achieved in FP, PFP, UCP, and UVPC, respectively, against mineralization of
49.3, 58.2, 47, and 24.6%. The proposed mechanisms suggest that the cleavage of three
methyl moieties followed by pyrazolinone ring breakage led to formation of various
intermediates with low errors (−0.88 to 0.11 g/mol). The intermediates primarily were
hydroxylated and carboxylic derivatives. BOD5 to COD (BOD, biochemical oxygen
demand; COD, chemical oxygen demand) ratio of ≥0.4 resulted from DIPY decomposition
in all processes with highest improvement in PFP (BOD5/COD ≈ 1.5). The collapse of
iron(III)-chelates under UV irradiation gave higher biodegradability.

1. INTRODUCTION
Advanced oxidation processes (AOPs) are widely used for removal of pharmaceutical and
personal care products from industrial and municipal wastewater.1 AOPs undergo conditions
through different reacting systems such as homogeneous or heterogeneous phases, in light or
dark, etc. However, they have common characteristics of formation of hydroxyl free radicals
(OH• ).2 It causes consecutive unselective degradation of organic materials. Complete
mineralization and/or oxidization of contaminants occur even at very low concentration, and
the byproducts formed may also be environmentally nonhazardous.3 The Fenton process
(FP) is based on redox reaction between Fe2+ and H2O2 generating OH• radicals,4 whereas
the photoFenton process (PFP) occurs additionally under UV−vis irradiation. Heterogeneous
semiconductor photocatalysis using TiO2 causes oxidation of organic pollutants via
hydroxyl radicals, OH• ad, and valence holes (h+ ) generated when the semiconductor is
exposed to UV irradiation. TiO2 photocatalysis works at ambient conditions and may be
induced by solar irradiation.5 UV photolysis of H2O2 (UVP) and TiO2 photocatalysis
(UVPC) generally show a lesser extent of oxidation than FP and PFP. Both homogeneous
and heterogeneous catalytic processes have shown promising results even at pilot-plant scale
in treatment of nonbiodegradable and toxic compounds. Dipyrone (DIPY) is one of the most
popular analgesics and antipyretic drug. It is also known as Metamizole and Novalgin.
About one-third of it remains unchanged and can be found in urinary excretion. DIPY is
rapidly hydrolyzed into its main metabolite, 4-methylaminoantipyrine (4-MAA). 4-MAA is
absorbed and biotransformed by enzymatic reactions.6 The metabolites of DIPY can be
subdivided into two groups: (i) decarboxylated metabolites and (ii) metabolites with a
degraded pyrazolinone moiety. The metabolic route is shown in Figure 1. 4-MAA is
metabolized to 4-aminoantipyrine (4-AA) in human liver via demethylation and further
acetylated to acetylaminoantipyrine (4-AAA) (Figure 1). 4-Formylaminoantipyrine (4-FAA)
is generated by an uncharacterized oxidation of an n-methyl group of 4-MAA (Figure 1).

Figure 1. Chemical structure of dipyrone drug (sodium [(2,3-dihydro- 1,5-dimethyl-3-

oxo-2-phenyl-1-pyrazol-4-yl) methylamino]- methanesulfonate) and its metabolic
pathways.
The metabolites of DIPY enter into the environment from several anthropogenic sources
such as pharmaceutical industry and infirmary effluent, water disinfection, and waste
incineration facilities. These compounds are not completely eliminated by biological
treatment, and thus their presence has been referenced in sludge treatment plant effluents
and surface water at high concentrations. Proper removal of DIPY and its metabolites
present in water and wastewater has an important role in the prevention of diseases both in
humans and animals. AOPs can be employed for the detoxification of such compounds
until the biodegradability is improved to a level amicable for subsequent biological
treatment.7 However, there is a lacuna of investigation for the selection of suitable
oxidation process(es) to achieve biodegradability to such extent. Therefore, in this study,
four AOPs, i.e., FP, PFP, UVP, and UVPC, are compared based on their performance for
the: (i) oxidation and mineralization of 4-MAA (primary metabolite of DIPY), (ii)
formation of degradation products, and (iii) variation of biodegradability (BOD5/COD
where BOD is the biochemical oxygen demand and COD is the chemical oxygen demand).
A unified mechanism of 4-MAA oxidation is presented. It would help to elucidate the
effect of unreacted drug and degradation products on the variation of biodegradability of
different oxidation processes.
 2. MATERIAL AND METHODS

2.1 Reagents.
Dipyrone (purity > 99%) was obtained from Sigma Aldrich Chemical Ltd. (Hong Kong,
China). The chemical structure of DIPY is illustrated in Figure 1. DIPY is hydrolyzed
rapidly at low concentration. Almost complete hydrolysis of DIPY to 4-MAA in a solution
of 0.01 mM occurs in 30 min at pH 2.5 and temperature 21 °C.8 Methanol (98% (v/ v)
purity) and acetonitrile (99% (v/v) purity) of high performance liquid chromatographic
(HPLC) grade were procured from Merck Specialties Pvt Ltd. (India). Ferrous ammonium
sulfate heptahydrate (99% purity), sulfuric acid (98% purity), titanium dioxide (TiO2; purity,
99% (w/w); crystal type, rutile; specific surface area, 391 m2 /g), H2O2 (50% (v/v) purity),
silver sulfate (Ag2SO4), and K2Cr2O7 (purity > 98%) were obtained from Merck. Milli-Q
water (model Elix 3, Millipore, Billerica, MA, USA) was used to prepare all reagent and
drug solutions.

2.2Analytical Techniques
High performance liquid chromatography was used for the determination of 4-MAA
concentration. A 20 μL sample was injected directly into a C18 column of 4.6 mm inside
diameter and 25 mm length. HPLC instrument of Shimadzu, Japan (model LC-20AD)
equipped with an UV−visible detector was employed for the chromatographic measurement.
Methanol and water at the flow rate of 0.5 mL/min (80:20 (v/v)) was used as the mobile
phase. The scanning was performed at a fixed wavelength of 254 nm. Liquid
chromatography−time-of-flight mass spectrometry (LC-TOFMS; Waters Q-Tof Premier &
Aquity UPLC) system was employed for the identification of drug fragments. The
chromatographic separation was performed on a YMC (Wilmington, NC, USA) hydrosphere
C18 reverse phase column (4.6 mm × 150 mm, 5 μm particle size) following a guard column
(4 mm × 10 mm, 5 μm particle size) using a mobile phase flow rate of 0.8 mL/min at 25 °C.
The mobile phase was consisting of H2O and acetonitrile with 0.1% (v/v) formic acid.

A linear gradient of 95 to 50% H2O was applied over 10 min. A 10 μL aliquot of both
sample and calibration solution was injected from an autoinjector. The samples were
analyzed by electrospray ionization (ESI) method in positive ion mode over the mass range
of 100−400 amu. The parent ion was fragmented on the basis of a suitable range of mass to
charge (m/z) ratio. The most prominent daughter ions were selected for further
fragmentation. Chemical oxygen demand was determined according to the HACH method.
A closed reflux digester of HACH, Loveland, CO, USA (model DRB 200) was used for
digestion. Total organic carbon (TOC) analyzer of O.I. Analytical, College Station, TX,
USA (model 1030C Aurora) was employed for the measurement of TOC by nondispersive
infrared method. A 5 day biochemical oxygen demand (BOD5) was found per the standard
method of analysis. Dissolved oxygen (DO) was measured using a precision dissolved
oxygen meter of Hanna Instruments, Smithfield, RI, USA (model HI 2400). Solution pH was
measured using a precision pH meter of Eutech Instruments, Malaysia (model pH/ion 510).

2.3 Experimental Procedure.

All experiments were conducted in batch mode with continuous stirring. A 1000 mL
capacity cylindrical borosilicate vessel (i.d., 10.5 cm) was used as the reactor system. A drug
solution of 400 mL was taken for the experimentation. The Fenton experiment was carried
out with 50 mg/L DIPY (≈4-MAA) at room temperature (23−25 °C). The initial
concentration of DIPY was chosen based on the maximum concentration reported in
literature.10 pH of the solution was adjusted using 0.05 N H2SO4 prior to addition of ferrous
catalyst. It was noted that the concentration of 4-MAA was almost invariant in 2 h at pH 3.5
and temperature 25 °C. Subsequently the oxidation reaction was performed after 2 h of an
initial premixing period. Until and otherwise outlined, the efficiency of drug degradation and
mineralization is reported with respect to the concentration of 4-MAA.

A predetermined amount of Fe2+ was added first and mixed for about 5 min at 260 rpm on a
magnetic stirrer of Tarsons, Kolkata, India (model Spinot 6020; stirring ba:, i.d., 0.8 mm;
length, 40 mm). After that H2O2 was added to 4-MAA/Fe2+ solution. The agitation was
maintained at the same speed. A sample volume of 10 mL was withdrawn at selected time
intervals. A 1 mL aliquot of 0.1 N NaOH was immediately added into the sample to
terminate the oxidation reaction. Addition of NaOH increased the solution pH at ∼12.3.
Sludge was separated out by centrifuging (Remi Instruments Ltd., Mumbai, India; model
R23 8/06) at 2000 rpm for 30 min. Clear liquid was pipetted out for pH, COD, TOC content,
and drug concentration. Clear liquid sample was heated at 70 °C for the destruction of
residual H2O2 (if any) prior to COD measurement. The supernatant was filtered using 0.45
μm cellulose filter (serial no. 08091ID0683) of Pall India Pvt. Ltd. (Bangalore, India), before
LC-MS analysis. 4- MAA sorbed in sludge, if any, was determined by washing it in distilled
water followed by digestion (dissolution) in H2SO4 (1 M).

In the case of PFP, the experiment was performed following the same procedure in the
presence of UV light. An UV lamp of 9 W from Hong Kong Jie Meng International Lighting
Ltd. Company, China (wavelength, 362 nm; intensity, 12 W/m2 ) was employed for this
work. The UVP experiment was conducted with a fixed concentration of H2O2 under UV
irradiation without Fe2+. The UVPC test was carried out using TiO2 photocatalyst. The
above experimental procedure was adopted for the UVPC experiment with a fixed amount of
TiO2 (1 g/L) only. All of the experiments were performed in duplicate, and the average
values are reported.

3. RESULTS AND DISCUSSIONS

3.1 4-MAA Removal and Mineralization.

Concentrations of Fe2+ and H2O2 and pH were varied from 1 to 3 mM, from 5 to 25 mM,
and from 2 to 4, respectively. The results are shown in Figures S1−S3 of the Supporting
Information. Drug removal efficiency increased with rise of one of these parameters, i.e., pH
and Fe2+ and H2O2 doses. It reached a maximum and then fell. So, the catalytic effect of
Fe2+ was reduced at lower as well as at higher concentration of a reactant. The best
performance of Fenton oxidation in terms of 4-MAA and TOC removal is illustrated in
Figure 2.

Figure 2. (a) Removal of 4-MAA in different AOPs with reaction time. (b) Removal of TOC. Initial 4-
MAA concentration, 50 mg/L; TOC = 23.4 mg/L; temperature, 25 °C. FP: Fe2+, 2.25 mM; H2O2, 22.5
mM; pH, 3.5. PFP: Fe2+, 2.25 mM; H2O2, 22.5 mM; pH, 3.5; UV light, 9 W. UVP: H2O2, 22.5 mM;
pH, 3.5; UV light, 9 W. UVPC: TiO2, 1.0 g/L; pH, 2.5; UV light, 9 W.
PFP was carried out with the same operating conditions. UVP was conducted with the same
amount of H2O2 added in the Fenton reaction. In the case of the heterogeneous catalytic
reaction, catalyst dose and pH were varied from 0.5 to 2.5 g/L and from 2 to 4.5,
respectively. Their effects on 4-MAA removal are in Figures S4 and S5 of the Supporting
Information. Increase in the TiO2 dose from 1 g/L had a negative effect on drug removal.
Higher concentration might cause catalyst agglomeration and hindrance to light
penetration.11 pH determines the surface charge of the TiO2 photocatalyst (pHzpc ≈ 6.8).
Lower pH favors 4-MAA adsorption. However, too strong 4-MAA adsorption probably
decreased the absorption efficiency of light quanta at lower pH. The best performance of
TiO2 photocatalysis with these experimental conditions are in Figure 2.

Temperature, solution volume, reaction time, and extent of mixing were the same for all of
the processes in order to compare their efficiencies for degradation of 4-MAA. The
dynamics of 4- MAA and TOC removal are shown in Figure 2a,b. The order of percentage
removal of 4-MAA at any time of the reactor was found to be PFP > FP ≫ UVP > UVPC.
Percentage removal of drug achieved to 96.4, 94.1, 74.4, and 71.1%, respectively, in 45 min.
Production of OH• radicals according to eqs 1−5 causes degradation of 4-MAA as a function
of time based on the oxidation process.

4-MAA removal showed two distinct rate periods (Figure 2a), i.e., initial faster drug removal
followed by virtually constant rate even though there was a notable amount of unreacted 4-
MAA present in solution in the case of UVP and UVPC. The transition for these two
processes lasted for a long period (2.5−15 min) compared to the Fenton reaction with and
without UV light (2.5−10 min). It implies that OH• generation is predominant at 2.5 min). In
the case of FP and PFP, drug removal of 73.5 and 83.2% at 2.5 min of oxidation rose to 94.1
and 96.4%, respectively, in 45 min.
 Application of UV light during Fenton reaction generates excess hydroxyl radicals, and
ferric ions are reduced into ferrous leading to further generation of OH• (eqs 1 and 2).
Increase in gross OH• concentration gave a bit higher degradation of 4-MAA in PFP. It is
evident from Figure 2a that UVP and UVPC are less effective compared to the other two
processes. UVP showed higher removal efficiency than UVPC. The first process exhibits a
rate constant of nearly 25 times higher than the UV/TiO2 process.12 The drug removal
achieved was 56.4 and 44.5% within the first 2.5 min in UVP and UVPC, respectively. It
increased to 74.4 and 71.1% in 45 min. OH• radicals are generated by photolysis of the
peroxide bond (eq 3). It formed on the surface of TiO2 (eqs 4 and 5), and adsorbed 4-MAA
on the surface is oxidized. Hence, the reaction occurs at the diffusion controlled regime in
UVPC. The trend of mineralization was similar to that of drug removal. The highest TOC
removal took place in PFP. It was very close in the cases of FP and UVP (Figure 2b). The
initial rapid mineralization was up to 2.5 and 5 min for FP and PFP. However, it was not so
distinct for the other two processes. TOC removal of 28.4 and 42.78% was obtained in 2.5 and
5 min withFP in addition to 53.6 and 56.0% being obtained in 2.5 and 5 min for PFP. The first
stage of TOC removal was very fast due to mineralization of three-methyl moieties. The slow
second stage is related to the opening and mineralization of pyrazolinone ring.14 Usually, large
molecular weight intermediates were either mineralized or broken to lower molecular weight
products like oxalic and acetic acids.15 Kavitha and Palanivelu indicated that carboxylic acids
are eliminated in PFP very quickly during the first stage of oxidation.16 Bauer et al. reported
continuous formation as well as degradation of lightweight carboxylic acids during UVPC.17
There was a difference of about 20% in drug removal between FP and UVP. However TOC
reduction was almost the same. It is necessary to mention that H2O2 (22.5 mM) alone gave
around 12.8% 4-MAA decomposition. However, only UV irradiation did not show notable
effect on drug removal.
There is a significant role of iron-chelation on drug mineralization. Iron(III)-chelate
complexes are more stable in PF because of lower possibility of further degradation.18 PFP
would have higher ability to degrade such complexes under UV luminesce. Knight et al.
suggested that Fe(III)-humate complexes are easily reduced to Fe(II) by H2O2 in PFP.19 In
addition, humic acids themselves are photodegraded through formation Fe(III) complexes.20
Klamerth et al. pointed out that PFP could breakdown metal complexes of molecular acids and
iron through separation between heavy metal and its complexing agent.21 TOC removed in PFP
mostly corresponds to oxidation of hydrophobic components which predominate in natural
organic compounds present in raw water.22 It can be explained by greater aromaticity of the
hydrophobic fraction giving higher reactivity toward oxidizing agents. The refractory
hydrophilic fraction is consisting of short-chain aliphatic amines, alcohols, aldehydes, esters,
ketones, aliphatic amides (<C5), polyfunc- tional alcohols, carbohydrates, cyclic amides, and
polysacchar- ides.22 In the present work, acidic digestion of sludge formed in FP showed
2.74% 4-MAA appearance on sludge phase.

Figure 3. Degradation pathways of 4-MAA and identification of intermediates were obtained in

different oxidation processes at 10 min of oxidation (photoreaction with an UV lamp of 9 W; FP, PFP,
and UVP conducted at pH 3.5 and UVPC at pH 2.5): (a) Cleavage of 4-MAA; (b) degradation of D2
(m/z = 279.11) continued from panel a; (c) D5 (m/z = 176.07) cleavage continued from panel b. The
exact mass to charge ratio is in parentheses.
3.2 Mechanisms of Drug Degradation.
Fenton, TiO 2 photocatalysis, and their allied processes are characterized by the
formation of a common oxidizing agent ,i.e., OH• radical used for the decomposition of
organic compounds. However, the extent Of the degradation of parent compounds and
the formation of the intermediates largely depend on process parameters such as pH, dose
of Fe 2+ /H 2 O 2 and TiO 2 , etc. A unified mechanism is developed to elucidate the
possible routes of 4-MAA degradation by OH• generated both in homogeneous and
heterogeneous catalytic processes. 4-MAA (m/z=218.11)is formed quickly due to the
presence of a more water-soluble sulfonate group (-SO 3 H). 4-MAA (D 1 ) contains a
pyrazolinone structure. It was first separated on a reverse phase HPLC column using an
isocratic mobile phase. Then the m/z ratio recorded in MS spectra was verified using 4-
isopropylantipyrine (m/z=231.3) as an internal standard. 23 The nitrogen atom instead of
carbon in the pyrazolinone moiety is the most preferred site of OH• attack releasing
aniline. 4 It was formed via hydroxylation of the aromatic ring by addition of OH• as in
Figure1.N-atom is mainly released in the form of N 2 and ammonia from the -NH−NH-
moiety at around 70 and 7% of the stoichiometric amount.
The mass spectra acquired in different oxidation processes are shown in Figures S6−S9
of the Supporting Information. A total 27 (D 1 −D 27 ) intermediate products were
detected in four oxidation processes on the basis of m/z. The error in mass was calculated
from the difference between the exact and proposed structural mass of protonated ions.
Fairly low error values were obtained for most of the proposed compounds (Table S1 of
the Supporting Information). There are two most possible pathways for the cleavage of 4-
MAA as shown in Figure 3a. Path 1 shows the route of formation of D2−D7
corresponding to the proposed m/zvalues of 279.11 ,250.07 ,136.04 ,176.07, 198.11 ,and
199.11, respectively.Path2 presents the formation route of D8−D10 with m/z of 205.11,
189.06, and 225.11, respectively. D 2 was formed by hydroxylation of a double bond in
the pyrazolinone ring of D 1 due to nucleophilic character of the π-electron. D 2 formed a
chelate compound (m/z = 346.07) with Fe 3+ due to the availability of a lone electron
pair on N-atom (Figure 3a).
The opening of the pyrazolinone ring was confirmed by the detection of 1-acetyl-1-
methyl-2-aminomethyl-oxamoyl-2-phe-nylhydrazide (D3). This compound was yielded
by addition of two OH • radicals to the double bond of the pyrazolinone ring of D1
followed by C−C bond breaking. D3 is found as a derivative in the biotransformation of
DIPY in humans. 25 The formation of D4 was via an unstable intermediate with m/z =
220.03 because of the presence of a diketo group more susceptible foward oxidation in
acidic medium. 23 The cleavage of N−N bond in the hydrazine group led to formation of
N-phenylacetamide (D 4 ) with m/z = 136.04 as well as an acetamide (Figure 3a). D 5
was originated in PFP (Figure S7 of the Supporting Information) with low error (0.031
g/mol) by hydroxylation of the carbonyl group followed by C−N bond breaking along
with formation of acetic acid. Attack of OH • radical to the carbonyl group due to its
electrophilic nature(i.e.,electron deficient center)caused aniline formation with complete
elimination of the pyrazolinone ring.D6 was detected inFP,PFP, and UVP with very low
error(−0.04 g/mol). It was originated by addition reaction between D 4 and aniline on
dehydration. Successive hydroxylation of aniline at ortho- and para-positions originated a
compound with m/z =130.27 (Figure 3a). The -NH 2 group activates benzene nucleus for
the electrophilic substitution reaction due to +R effect (mesmeric). D 7 was identified in
PFP and UVP (Figures S7 and S8 of the Supporting Information). The lone electron pair
on the N-atom of the D 6 compound acts as a Lewis base that appeals to protons.
Therefore, D 6 and D 7 are in equilibrium as shown in Figure 3a.
4-Aminoantipyrine (D 8 , 4-AA) was formed with methanol in FP on hydrolysis of D 1 .
D 9 (m/z = 189.06) was detected in MS spectra of FP. The loss of NH 3 from the
pyrazolinone ring of the previous compound yielded D9 (error,−0.06mg/L)becauseofa
shifting of a lone pair electron on N 1 .D 10 was found both in UVP and UVPC (Figures
S8 and S9 of the Supporting Information). A similar explanation outlined for the
formation of D 2 is also valid here. D 2 was traced in mass spectra of UVP and UVPC
(Figures S8 and S9 of the Supporting Information). D 12 , D 14 -D 16 and D 19 formed
in PFP were originated from D 2 (Figure 3b), whereas only D 16 was formed in FP
(Figure S6 of the Supporting Information). It indicates formation of an iron-chelate
complex which is unstable under UV-luminance. 26 Carboxylic (Figure3a) and
dicarboxylic acids (Figure 3c) are also known to form stable iron complexes which
inhibit the reaction with peroxide. 15 Carboxylic acids mainly exist as acetate and
oxalate. 16 Oxalic acid could form a iron(III)-oxalate complex as shown in Figure 3a.
Such a complex got stability through an extensive conjugation with Fe(III) due to the
availability of a vacant 3d orbital. In the presence of UV light, its stability could loose
because of UV light absorption. 27 The ability of light absorption depends on electron
distribution of a molecule. Generally, the light absorption capacity of sigma (σ) bonds
(C−H, C−C, C−O, and O−H) are very less above 200 nm, whereas pi (π) bonds
(carboxylic, carbonyl group) and especially conjugated pi systems boost the ability of a
compound to absorb UV light at 254 nm by lowering the energy barrier between ground
and excited states. 28 Double bond equivalent (DBE) was calculated bythe summations
of the total number of pi bonds (π) and the residual ring(s). It is also referred to as the
degree of unsaturation. 29 PFP generated greater number of compounds having higher
DBE (Table S1 ofthe Supporting Information). Indeed, it can be seen from Supporting
Information Table S1 that compounds formed in UVPC were consisting of lower DBE.
The highest DBE of 9 was calculated for both D 6 and D 7 .
There are also possibilities for cleavage of D 2 following path 3 and path 4 as shown in
Figure 3b. Path 3 shows the route of formation of D 11 and D 12 corresponding to m/z of
186.11 and 171.21, respectively. D 11 and D 12 both were formed from the same
intermediate (m/z =164.11) via oxidation of D 4 originated By hydrolysis of D 3
compound(path1)as presented before.This compound seems to be more stable in
FP.Further attack of OH •at the α-carbon of D 4 caused formation of D11 in the case of
PFP.Thecarbonyl group is very susceptible to being oxidized at lower pH (<3.5). 30 The
valency of the carbon atom in D 12 is saturated by two additional hydroxyl groups (-OH)
due to the availability of a partially vacant p-orbital in the N 1 -atom. Path 4 represents
the formation route of D 13 −D 19 with m/z = 187.19, 152.19, 152.09, 166.09, 155.06,
183.11, and 212.02, respectively. The structures of these compounds are proposed with
low errors (2.11 to −0.22 g/mol; Figure 3b). D 13 was formed by hydroxylation of the α-
carbon of an intermediate with respect to the N 2 -atom. Both D 14 and D 15 compounds
have almost the same mass (m/z = 152.19 and 152.09). Structures of these compounds
are different and have appeared from the same intermediate with m/z = 194.08. The first
compound was originated in PFP by decarboxylation (-CO 2 ). The second was the
hydroxylation product with formation of NH 3 and CO 2 . Identification of D 14 (Figure
S7 of the Supporting Information) is evidence of the opening of pyrazolinone ring by an
alternative route (Figure 3b). The proposed structure suggests oxidative cleavage of N−N
bond to a multiple hydroxylated derivative. OH • radical can easily attack at the N 1
position of the pyrazolinone ring in the presence of a partially vacant p-orbital of the N-
atom. 31 D 16 and D 17 were originated by hydroxylation. Oxidation of α-carbon in D 17
with loss of hydroxylamine is evidence of formation of D 16 . The α-carbon in D 17
behaves like a nucleophilic center because of the lone pair electron on the N 1 position
shifted toward the terminal carbon atom through conjugation. D 18 was formed from D
17 (Figure 3b). A possible explanation similar to that for D 15 is applicable. The primary
alcohol group of the pyrazolinone moiety was oxidized to D 19 found in PFP. Waki et al.
suggested that the primary alcohol in acidic solution gives acid products on oxidation. 32
D 20 was detected in UVPC with a low error (−0.42 g/mol). Direct hydroxylation of D 5
at the N 1 position with the loss of methylamine justifies its formation. D 20 was broken
in three different routes (paths 5−7; Figure 3c). Path 5 represents formation of D 21
molecule by proton loss from D 21 .It is stable as a salt form because of the slightly
acidic nature of the -OH group. D 22 compound was seen both in PFP and UVP yielded
from N-hydroxyl aniline derivative with formation of oxalic acid (path 6). It was
ultimately transformed to CO 2 giving effective TOC reduction. This aniline derivative
by its para-position attack with acetanilide, formed D 22 compound. Electrons are more
localized at the 4-position with respect to the N-substituted amine group due to shifting of
the lone pair electron and OH • attack at the more electrophilic center of the carbonyl
group. D 23 and D 24 were identified in PFP. The N 1 -atom has a vacant p-orbital to
accept the electron of OH • radicals (Figure 1; path 7). This compound exists as a salt of
sodium ion in alkaline medium (pH > 8). It might have formed during pH adjustment at
>12 for the termination of oxidation reaction. Further oxidation of D 23 compound
yielded the corresponding quinone−imine inter-mediate (D 24 ) which is generally
unstable but it appeared in MS spectra (Figure S7 of the Supporting Information).
Hydrox-ylation probably occurred at the para-position due to availability of more
electron density. The enol form of the D 24 compound is less stable due to a positive
charge on the N 1 -atom. It has a greater tendency to be transformed into the quinone
form, 15 i.e., the formation of D 25 compound obtained both in FP and UVP having
positive characteristics of the benzene nucleus.
D 26 and D 27 also appeared from D 25 by its successive hydroxylation (Figure 3c).
First the molecule was detected in FP and UVP, where as these condone was found in
UVPC. D 27 is alicyclic in nature and has resulted by hydroxylation. This compound
lostitsaromaticitybysubstituting two-OHgroupsat the2-and6-positions with respect to
N,N-hydroxylamine group.
3.3 Biodegradability: 4-MAA and Its Degradation Products.
The ratio of biochemical oxygen demand to chemical oxygen demand (BOD/COD) is an
important indicator to study the nature of biodegradability.33 Generally, wastewater contain-
ing pharmaceutical and personal care products are considered to be reasonably biodegradable
with BOD5/COD > 0.4.34 The initial BOD5 and COD were 3.75 and 41.3 mg/L for an
aqueous solution with 50 mg/L DIPY. It implies the nonbiodegradable nature of DIPY
(BOD5/COD ≈ 0.1).

From Figure 2a it is evident that the extent of drug decomposition was around 5%
higher in PFP than FP. Nevertheless, PFP resulted in notably higher biodegradability
(Figure 4). A BOD5/COD = 1.51 was achieved in PFP in comparison to 0.62 in FP. It
implies that the first process generated more (gross) biodegradable intermediates. UVPC
exhibited about 20% higher BOD5/COD improvement than UVP (Figure 4). It was about
11% higher than the corresponding drug decomposition. Comparatively lower BOD 5/COD in
UVP and UVPC than FP and PFP was largely due to unreacted 4-MAA and intermediate
products. Hyvonen et al. reported that a higher extent of conjugation due to formation of
chelates of three different heavy metals, i.e., Cd(II), Hg(II), and Pb(II), and
(dicarboxyethoxy)ethyl]aspartic acid (BCA6) improves biode- gradability. Kummerer et al.
reported that newly formed intermediates act as chelating agents that significantly reduce
their biodegradability.

It can be seen from a to c of Figure 3 that D2, D3, D14, and D23 could form chelates with Fe3+. D1,
D2, D6, and D25 traced in FP could exhibit more biodegradability because of extended
conjugation. Therefore, the net effect of chelation and its stability under UV irradiance and
formation of intermediate products and their different extent of conjugation resulted in
higher biodegradability in PFP. The biodegradable nature of a number of intermediates as in
the proposed structure is found out from the earlier reports. It is summarized in Table S1 of the
Supporting Information.

Figure 4. Variation of BOD5/COD in different oxidation processes and its initial value (initial 4-MAA, 50 mg/L; oxidation time, 10 min;
temperature, 25 °C): with FP, 2.25 mM Fe2+, 22.5 mM H2O2, and pH 3.5; with PFP, 2.25 mM Fe2+, 22.5 mM H2O2, pH 3.5, and 9 W UV light;
with UVP, 22.5 mM H2O2, pH 3.5, and 9 W UV light; with UVPC, 1.0 g/ L TiO2, pH 2.5, and 9 W UV light.
 4. CONCLUSIONS
Dipyrone at lower concentration (0.149 mM) was almost completely hydrolyzed to 4-MAA
within 2 h2 at pH 3.5 and temperature of 25 °C. Maximum 4-MAA removals of 94.1, 96.4, 74.4,
and 71.2% were noted against mineralization effciencies of 49.3, 58.2, 47, and 24.6% in Fenton,
photo-Fenton, H2O2 photolysis, and TiO2 photocatalysis, respectively. PFP showed better
mineralization effciency due to additional amount of OH• formation and collapse of iron
complexes under UV irradiation probably because of enhanced decarboxylation. However, in the
case of TiO photocatalysis more hydroxylated products were identified that resulted from
direct attack of OH• ad. A total of 29 intermediate products appeared in the mass spectra within
the mass to charge ratio of 100−400 in four oxidation processes from the same parent molecule.
Pyrazolinone ring was degraded preceded by cleavage of methyl moieties. The proposed
mechanism implies that most of the intermediates were formed by pyrazolinone ring
degradation. The order of biodegradability in different oxidation processes was found to be
PFP≫ FP > UVP> UVPC.

REFERENCES
 (1) Moldovan, Z. Occurrences of pharmaceutical and personal care
products as micropollutants in rivers from Romania. Chemosphere 2006, 64,
1808−1817.

(2) Torres, R.; Sarria, V.; Torres, W.; Peringer, P.; Pulgarin, C.
Electrochemical treatment of industrial wastewater containing 5-amino- 6-methyl-2-
benzimidazolone: toward and electrochemical-biological coupling. Water Res.
2003, 37, 7−13.Malato, S.; Blanco, J.; Vidal, A.; Richter, C. Photocatalysis with
solar energy at a pilot-plant scale: An overview. Appl. Catal. B: Environ. 2001, 37,
1−15.

(3) Pignatello, J. J.; Oliveros, E.; MacKay, A. Advanced oxidation

processes for organic contaminant destruction based on the Fenton reaction and
related chemistry. Crit. Rev. Environ. Sci. Technol. 2006, 36, 1−84.Malato, S.;
Fernandez-Ibanez, P.; Maldonado, M. I.; Blanco, J.;
Gernjak, W. Decontamination and disinfection of water by solar photocatalysis:
recent overview and trends. Catal. Today 2009, 147, 1− 59.
(4) Oliveira, R.; Almeida, M.; Santos, L.; Madeira, L. Experimental design
of 2,4-dichlorophenol oxidation by Fenton’s reaction. Ind. Eng. Chem. Res. 2006,
45, 1266−1276.
(5) Morais, J. L.; Zamora, P. P. Use of advanced oxidation processes to
improve the biodegradability of mature landfill leachates. J. Hazard. Mater.
2005, 123, 181−186.Ergun, H.; Fratarelli, D. A. C.; Aranda, J. V. Characterization
of the role of physicochemical factors on the hydrolysis of dipyrone. J. Pharm.
Biomed. Anal. 2004, 35, 479−487.APHA. Standard Methods for the
Examination of Water and Wastewater, 20th ed.; American Public Health
Association: Washington, DC, USA, 1998.
(6) Huber, M. M.; Gobel, A.; Joss, A.; Hermann, N.; Loffler, D.;
Mcardell, C. S.; Ried, A.; Siegrist, H.; Ternes, T. A.; Von Gunten, U. Oxidation of
pharmaceuticals during ozonation of municipal wastewater effluents: A pilot study.
Environ. Sci. Technol. 2005, 39, 4290−4299.
(7) Sohrabi, M. R.; Ghavami, M. Taguchi experimental design used
for Nano photo catalytic degradation of the pharmaceutical agent Aspirin. J.
Chem. Pharm. Res. 2008, 153 (3), 1235−1239.
(8) Jayson, G. G.; Parsons, B. J. Oxidation of ferrous ions by
perhydroxyl radicals. Trans. Faraday Soc. 1972, 68, 236−242.
(9) Petrovic, M. M.; Bianca, F. S.; Aleksandra, J. R. Oxidative
 transformation of fluoroquinolone antibacterial agents and structurally related
amines by manganese oxide. Chemosphere 2011, 85, 1331−1339.
(10) Gomez, M. J.; Martınez-Bueno, M. J.; Lacorte, S.; Fernandez-
Alba, A. R.; Aguera, A. Pilot survey monitoring pharmaceuticals and related
compounds in a sewage treatment plant located on the Mediterranean coast.
Chemosphere 2007, 66, 993−1002.
(11) Leonidas, A.; Perez-Estrada, S. M.; Ana, A. R. Degradation of
dipyrone and its main intermediates by solar AOPs Identification of intermediate
products and toxicity assessment. Catal. Today 2007, 129, 207−214.
(12) Kavitha, V.; Palanivelu, K. The role of ferrous ion in Fenton and
photo-Fenton processes for the degradation of phenol. Chemosphere
2004, 55, 1235−1243.
(13) Bauer, R.; Waldner, G.; Fallmann, H.; Hager, S.; Klare, M.;
Krutzler, T.; Malato, S.; Maletzky, P. The photo-fenton reaction and the TiO2/UV
process for wastewater treatment Novel developments. Catal. Today 1999, 53,
131−144.
(14) Trovo, A. G.; Raquel, F. P.; Nogueira, A. A.; Carla, S. A.
Photodegradation of sulfamethoxazole in various aqueous media: Persistence,
toxicity and photoproducts assessment. Chemosphere 2009, 77, 1292−1298.
(15) Knight, R. J.; Sylva, R. N. Spectrophotometric investigation of
iron (III) hydrolysis in light and heavy water at 25°C. J. Inorg. Nucl. Chem.
1975, 37, 779−783.
(16) Fukushima, M.; Tatsumi, K.; Nagao, S. Degradation Character-
istics of Humic Acid during Photo-Fenton Processes. Environ. Sci. Technol.
2001, 35, 3683−3690.
(17) Klamerth, N.; Malato, S.; Aguera, A.; Fernandez-Alba, A. Photo-
Fenton and modified photo-Fenton at neutral pH for the treatment of emerging
contaminants in wastewater treatment plant effluents: A comparison. Water
Res. 2013, 47, 833−840.
(18) Buchanan, W.; Roddick, F.; Porter, N.; Drikas, M. Fractionation
of UV and VUV pretreated natural organic matter from drinking water.

Environ. Sci. Technol. 2005, 39, 4647−4654.

(19) Knight, R. J.; Sylva, R. N. Spectrophotometric investigation of

iron (III) hydrolysis in light and heavy water at 25°C. J. Inorg. Nucl. Chem.
1975, 37, 779−783.
 (20) Fukushima, M.; Tatsumi, K.; Nagao, S. Degradation Character-
istics of Humic Acid during Photo-Fenton Processes. Environ. Sci. Technol.
2001, 35, 3683−3690.
(21) Klamerth, N.; Malato, S.; Aguera, A.; Fernandez-Alba, A. Photo-
Fenton and modified photo-Fenton at neutral pH for the treatment of emerging
contaminants in wastewater treatment plant effluents: A comparison. Water
Res. 2013, 47, 833−840.
(22) Buchanan, W.; Roddick, F.; Porter, N.; Drikas, M. Fractionation
of UV and VUV pretreated natural organic matter from drinking water. Environ.
Sci. Technol. 2005, 39, 4647−4654.