Oxidation of cefalexin by permanganate: Reaction kinetics, mechanism, and residual antibacterial activity

Article abstract of DOI:10.3390/molecules23082015

The oxidation of cefalexin (CFX), a commonly used cephalosporin antibiotic, was investigated by permanganate (PM) in water. Apparent second-order rate constant of the reaction between CFX and PM was determined to be 12.71 ± (1.62) M^-1·s^-1 at neutral pH. Lower pH was favorable for the oxidation of CFX by PM. The presence of Cl^- and HCO₃^- could enhance PM-induced oxidation of CFX, whereas HA had negligible effect on CFX oxidation by PM. PM-induced oxidation of CFX was also significant in the real wastewater matrix. After addition of bisulfite (BS), PM-induced oxidation was significantly accelerated owing to the generation of Mn(III) reactive species. Product analysis indicated oxidation of CFX to three products, with two stereoisomeric sulfoxide products and one di-ketone product. The thioether sulfur and double bond on the six-membered ring were the reactive sites towards PM oxidation. Antibacterial activity assessment indicated that the activity of CFX solution was significantly reduced after PM oxidation.

Full text of DOI:10.3390/molecules23082015

molecules
Article
Oxidation of Cefalexin by Permanganate: Reaction
Kinetics, Mechanism, and Residual
Antibacterial Activity
Yajie Qian 1, Pin Gao 1, Gang Xue 1, Zhenhong Liu 1 and Jiabin Chen 2,
*
1
College of Environmental Science and Engineering, Donghua University, Shanghai 201620, China;
yqian@dhu.edu.cn (Y.Q.); pingao@dhu.edu.cn (P.G.); xuegang@dhu.edu.cn (G.X.); zhhl@dhu.edu.cn (Z.L.)
School of Environmental Science and Engineering, Suzhou University of Science and Technology,
Suzhou 215009, China
2
*
Correspondence: chenjiabincn@163.com; Tel.: +86-0512-6809-6895
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 23 July 2018; Accepted: 8 August 2018; Published: 13 August 2018
Abstract: The oxidation of cefalexin (CFX), a commonly used cephalosporin antibiotic,
was investigated by permanganate (PM) in water. Apparent second-order rate constant of the
reaction between CFX and PM was determined to be 12.71
±
(1.62) M−1 −1 at neutral pH. Lower
s
·
−
pH was favorable for the oxidation of CFX by PM. The presence of Cl− and HCO3 could
enhance PM-induced oxidation of CFX, whereas HA had negligible effect on CFX oxidation by PM
PM-induced oxidation of CFX was also significant in the real wastewater matrix. After addition
of bisulfite (BS), PM-induced oxidation was significantly accelerated owing to the generation of
Mn(III) reactive species. Product analysis indicated oxidation of CFX to three products, with two
stereoisomeric sulfoxide products and one di-ketone product. The thioether sulfur and double
bond on the six-membered ring were the reactive sites towards PM oxidation. Antibacterial activity
assessment indicated that the activity of CFX solution was significantly reduced after PM oxidation.
.
Keywords: permanganate; water treatment; cefalexin; oxidation; product; antibacterial activity
1. Introduction
The
used antibiotics in human and veterinary medicine [
administered dosages are excreted unchanged, thus a substantial amount of
expected to be released into the environment. Because -lactam antibiotics developed at early stage,
e.g., penicillin G, are susceptible to acid/ -lactamase catalyzed hydrolysis, their occurrence in the
environment is scarcely reported. However, with the development of pharmacology, some -lactam
antibiotics are designed to be acid-stable and -lactamase resistant, thus they are frequently detected
in the environment [ ]. For example, cefalexin (CFX), one of the most frequently used first generation
β
-lactam antibiotics, including penicillins and cephalosporins, are among the most extensively
]. After consumption, a large portion of the
-lactam antibiotics is
1
β
β
β
β
β
2
cephalosporins, is relatively stable in aquatic environments. It has been widely detected in different
water matrices, such as wastewater, surface water and even coastal water with the concentration
up to
non-target organisms, pose risk to drinking water supplies and increase the bacterial resistance [
Cephalosporins have the most prevalence of antibacterial resistance in pathogens according to the
WHO 2014 report on antimicrobial resistance surveillance [ ]. Therefore, it is imperative to develop
effective strategies to eliminate CFX in the water environment.
µg
·
L−1
[
3–
5
]. The residual antibiotics in the environment could lead to adverse effects on
6].
7
Chemical oxidation processes involving permanganate (PM, Mn(VII)) are promising technologies
for eliminating contaminants in the water environment. Compared with other conventional oxidation
processes, PM oxidation process is relatively cost-effective and less likely to generate disinfection
Molecules 2018, 23, 2015; doi:10.3390/molecules23082015
www.mdpi.com/journal/molecules

Molecules 2018, 23, 2015
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byproducts [
is effective for oxidizing a variety of inorganic and organic compounds, such as dissolved iron and
manganese, odors and pharmaceuticals [ 10]. PM is always regarded as a selective oxidizing agent
8]. As an oxidant, PM exhibits a relatively high redox potential (i.e., E0 = 0.59–1.68 V) and
9
,
which exhibits highly variable rates to various contaminants. Generally, the second-order reaction rate
−1 −1
constants between PM and organic contaminants are the range of 10−5–103 M
·
s
[11
,
12], which are
relatively lower than those for the hydroxyl or sulfate radicals based oxidation (k”, 109–1010
M
·
s
−1).
−1
Recently, Sun et al. reported that the activation of PM by bisulfite (BS) can significantly enhance the
oxidation of organic contaminants [13] and Mn(III) is deemed as the major reactive species involved
in the oxidation process. However, it is still unclear whether PM exhibits reactivity towards CFX.
As discussed below, PM was found to show considerable reactivity towards CFX, and the presence of
BS could significantly enhance PM-induced oxidation of CFX.
As an oxidant, PM is expected to react with the moieties with available electrons during the
oxidation of organic contaminants. In the molecular structure of CFX, the primary amine, and the
thioether sulfur and double bond on the six-membered ring are electron-rich moieties, and thus they
are potentially susceptible to electrophilic attack by the oxidants and lose electrons in the reaction.
For example, the phenylglycine primary amine are the potentially reactive sites in the oxidation of
CFX by Cu(II) [
be reactive to peroxymonosulfate [15] and sulfate radicals [16], while the double bond on the ring is
susceptible to ozone oxidation [ ]. The reactivity of PM with the above electron-rich moieties are still
1] and ferrate(VI) [14]. The thioether sulfur on the six-membered ring was reported to
2
unknown, thus need to be further evaluated. Although CFX is susceptible to oxidative degradation by
different oxidants, the products might still exhibit antibacterial activity, and thus pose potential risk to
aquatic environment [17]. For example, biologically active products were found to be generated in the
reactions of PG and CFX with ozone [2]. Ferrate(VI) oxidation is effective to lower the antibacterial
activities of -lactams, but ~24% residual activity is still retained for the treated CFX [14]. Advanced
β
oxidation processes (AOPs) are among the most effective processes for destroying micro-pollutants.
The increase of antibacterial activities is reported in the hydroxyl radical-based AOPs treated water
samples, such as UV/H2O2 and photo-Fenton processes [18,19]. Therefore, it is essential to evaluate
the antibacterial activity of the transformation products of CFX by PM oxidation.
In this work, we investigated the oxidation of CFX by PM. The objectives were: (1) to investigate
the reactivity of PM towards CFX, and determine their reaction kinetics; (2) to evaluate the impact
of reaction parameters and water matrices on PM-induced oxidation of CFX, and then assess the
promoting effect of BS on CFX oxidation by PM; and (3) to identify the reactive sites on CFX towards
PM oxidation, and then evaluate the antibacterial activity of the transformation products. As CFX
shares the basic structure with other cephalosporin antibiotics, the findings of this work can provide
important references for other cephalosporin antibiotics oxidation by PM.
2. Results and Discussion
2.1. Oxidation of CFX by PM
CFX possesses several electron-rich moieties, e.g., double bond and thioether sulfur on the
six-membered ring, thus it is potentially reactive towards oxidants, such as PM. Indeed, rapid oxidation
of CFX was observed when PM was present in the solution. For example, CFX was stable at neutral pH
condition, but was almost completely degraded within 480 s after addition of 400
µM PM (Figure 1).
The oxidation of CFX followed the pseudo first-order kinetics, with the linear relationship (R2) higher
than 0.995. The observed first-order rate constant (kobs in Equation (1)) was calculated to be 0.005 s−1
The effect of PM concentrations on CFX oxidation was further evaluated at pH 7.0. The results showed
that CFX oxidation increased when PM/CFX ratio increased from 5 to 30 (Figure 1A). The degradation
of CFX followed first-order kinetics at different concentrations of PM, and the kobs values increased
from 0.002 to 0.014 s−1 as the ratio increased from 5 to 30. The log (kobs) was linearly increased
with log (CPM/CCFX), with the slope close to 1. Hence, kobs was first-order with respect to PM
.

Molecules 2018, 23, 2015
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concentration. Apparent second-order rate constant (k2,app in Equation (2)) was subsequently calculated
to be 12.71 ± (1.62) M−1·s−1 by dividing kobs by the concentration of PM. The obtained k2,app value of
CFX with PM was comparable to those with peroxymonosulfate [15], but was smaller than those with
ozone [2], ferrate(VI) [14] and radical species, e.g., sulfate radicals [20].
d[CFX]
−
= kobs × [CFX]
(1)
(2)
dt
d[CFX]
−
= k2,app × [CFX] × [PM]
dt
Figure 1. Effect of PM concentration on the oxidation of CFX (A), and log (kobs) vs. log (CPM/CFX) (B).
[CFX] = 40 µM, pH 7.0. Note: the ratio is PM/CFX.
2.2. Effects of pH
The solution pH has been regarded as an important factor influencing oxidation of various organic
contaminants by PM. Hence, the impact of pH was examined for PM-induced oxidation of CFX at pH
5.0–9.0, with the result shown in Figure 2. The oxidation of CFX by PM was pH dependent, and lower
pH was favorable for PM-induced oxidation of CFX. Specifically, the kobs of CFX gradually decreased
from 0.006 to 0.0041 s−1 when pH increased from 5 to 9. Generally, the redox potential of PM decreased
with increasing pH, i.e., acidic pH was favorable for enhancing the oxidation capacity of PM [21].
Hence, the faster oxidation of CFX by PM at lower pH might be attributed to the higher oxidation
capacity of PM. This result was consistent with the previous reports that PM-induced oxidation of
diclofenac decreased with the elevated pH [22]. However, the oxidative reactivity of PM with some
contaminants, e.g., lincomycin and sulfamethoxazole, increased at elevated pH [23
,24], whereas the
oxidation of dichlorvos by PM was found to be unaffected by the solution pH [25]. Hence, pH impact

Molecules 2018, 23, 2015
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on PM-induced oxidation of organic contaminants was complicated, which was not only dependent
on the oxidation capacity of PM, but also on the structure of target contaminants.
Figure 2. Effect of pH on the kobs of CFX by PM oxidation. [CFX]0 = 40 µM, [PM]0 = 400 µM.
The pH-dependent reactivity of PM towards contaminants was always related to the pKa value
of contaminants. For example, the reactivity of PM towards bisphenol A (BPA) was significantly
dependent on pH when the solution pH was close to the pKa value of BPA [26]. When the solution
pH was far away from the pKa value of carbamazepine (CBZ), the reactivity of PM towards CBZ was
almost unaffected by the pH variation [26]. pH could affect the protonation/deprotonation of some
functional group, e.g., primary amine, and thus influenced its electron density and susceptibility to PM
oxidation. The pKa of CFX is 6.86, thus the primary amine on the side chain of CFX was deprotonated
2
at alkaline pHs and possesses higher electron density than the protonated ones at pH 5. If primary
amine was the reactive site on CFX, faster degradation of CFX by PM could be expected at alkaline pHs
which was opposite to the results in this work. Therefore, the primary amine on the side chain might
be not the reactive site for PM oxidation, which is further elucidated in the following sections.
,
2.3. Effects of Water Matrices
The water matrices, e.g., the inorganic and organic solute, might affect the oxidation of target
organic contaminants during PM oxidation. Hence, we further evaluated the effect of common ions,
e.g., Cl− and HCO3−, on PM-induced CFX oxidation. As shown in Figure 3A, the calculated kobs
of CFX oxidation incr−eased with the increasing concentration of Cl− from 0 to 500 mM. Similarly,
the presence−of HCO3 promoted the oxidation of CFX by PM, and the rate constants also increased
when HCO3 concentrations increased from 0 to 50 mM (Figure 3B). This result was consistent with
a previous report that the common ions, e.g., HCO3−, exhibited strong accelerating effect on BPA
oxidation by PM, but the mechanism was still ambiguous and need further investigation [27].
We further investigated the impact of HA on the oxidation of CFX by PM, and the result showed
that PM-induced oxidation of CFX was not affected by various concentrations of HA (Figure 3C). As a
common matrix in the real waters, the effect of HA on PM-induced oxidation of organic contaminants
have been extensively evaluated, and its impact was complicated. HA was regarded as a reductant
to inhibit the oxidation of SMX by PM [23], whereas it was also reported to promote the oxidation of
some organic contaminants when the concentration of HA was lower than PM [28]. In some previous
reports, the impact of HA on the oxidation of contaminants were dependent on HA concentration and
source. For example, HA could accelerate the oxidation of phenol by PM when low concentration
of HA was present in the solution, but gradually inhibited phenol oxidation as HA concentration
increased [29]. At the constant concentration of HA, commercial HA and HA extracted from soils
exhibited different impact on the contaminant degradation, which could be attributed to their different

Molecules 2018, 23, 2015
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functional groups [30]. In another study, HA also exhibited negligible effect on the PM-induced
oxidation of contaminants [ ], which was consistent with the result in this work. Although the
9
examined HA exhibited negligible effect, a wider diversity of HA sources need to be evaluated on
whether HA influences reaction between CFX with PM [27].
−
−
;
Figure 3. Degradation rate constants (kobs) of CFX under different concentration of: (
A
) Cl ; (
B
) HCO3
and (C) NOM. [CFX]0 = 40 µM, [PM]0 = 400 µM, pH 7.
PM-induced oxidation of CFX was also evaluated in the real water sample. Four hundred
micromolars of PM and 0.2 M of CFX were spiked into wastewater (WW) sample (water quality
µ
is shown in Table S1), which was maintained at pH 7 with PB (10 mM). Before spiking, CFX was
confirmed to be negligible in the WW sample. Results showed that significant degradation of CFX
was observed in WW without addition of PM (Figure S1). The β-lactam ring in CFX was susceptible
to breakage catalyzed by metal ions, alkali and other water matrices [0], hence CFX degradation in
WW was significant even without spiking PM. After addition of PM, the degradation of CFX was
significantly enhanced, and PM-induced degradation of CFX in WW was slightly slower than that
−
in the DI water (Figure S1). In the real WW sample, although the anions, e.g., HCO3 and Cl−,
could promote PM-induced oxidation of CFX, the water matrix, e.g., reduced substances, might also
consume PM, thus lowering the degradation of CFX by PM. Overall, PM-induced oxidation of CFX
was significant in the real wastewater matrix, and thus PM was an efficient oxidant to eliminate CFX
in the real WW samples.
2.4. Impact of BS on PM-Induced Oxidation of CFX
BS has been reported to significantly accelerate the oxidation of organic contaminants, e.g.,
phenol, by PM. Hence, we further evaluated the oxidation of CFX in the PM/BS system. As shown
in Figure S2, degradation of CFX was negligible in the presence of 600
µM BS alone, while about
30% of CFX was degraded within 15 min by 80 M PM. However, complete degradation of CFX
µ
was achieved within 10 min in the combination of PM and BS. The results indicated that PM and BS
had synergistic effect on the oxidation of CFX. Indeed, BS was regarded as an efficient activator for
PM to generate a reactive intermediate, i.e., Mn(III), which could oxidize organic contaminants at an
extremely rapid rate. In PM/BS system, the rapid oxidation of CFX was also likely induced by the
reactive Mn(III) generated from the reduction of PM by BS.
The impact of different dosages of PM and BS on the oxidation of CFX was further investigated
(Figure 4A). In the absence of BS, the oxidation of CFX increased from 4% to 65% after 20 min when
PM dosage increased from 2
increased from 35% to 75% after 20 min with the increasing dosage of PM from 2
degradation of CFX could be achieved when PM further increased to 40 M. However, the degradation
µM to 80
µM (Figure S3). In the presence of 600
µM BS, CFX oxidation
µ
M to 20 M. Complete
µ
µ

Molecules 2018, 23, 2015
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of CFX could not be further enhanced when PM increased to 80
µM. With the increasing dosage of PM,
more Mn(III) reactive species could be generated to oxidize CFX. When high dosage of PM was present,
PM could consume the Mn(III), thus the degradation of contaminant could not be further increased.
Figure 4. Effect of PM concentrations (
A
); BS dosages (
B
); and pH (
C
) on the oxidation of CFX in
M, pH = 7; and ( ) [PM]0
PM/BS system. [CFX]0 = 40
µM; (A
) [BS]0 = 600 M, pH = 7; (
µ
B
) [PM]0 = 40
µ
C
= 40 µM, [BS]0 = 600 µM.

Molecules 2018, 23, 2015
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Compared to the impact of PM concentration, effect of BS concentration on the oxidation of CFX
was more complicated. In the presence of 40 M PM, the oxidation of CFX was slightly inhibited in
µ
the presence of low BS dosage, but was significantly promoted when high concentration of BS was
present (Figure 4B). For example, the degradation efficiency of CFX within 20 min decreased from
39% (PM alone) to 17% and 25% when 40
almost complete degradation of CFX was achieved within 20 min and 15 min in the presence of
400 µM and 600 M BS, respectively. When low concentration of BS was present in PM/BS system,
the generated Mn(III) could be consumed by PM, thus reducing the effective oxidants (i.e., PM and
Mn(III)) responsible for CFX degradation. When the concentration of BS increased to 200 M or even
µM and 80 µM BS were present, respectively. However,
µ
µ
higher, almost all PM was transformed to Mn(III), thus the reactive Mn(III) was the dominant oxidant
in the reaction system.
pH also had a significant impact on CFX oxidation in PM/BS system. As shown in Figure 4C,
CFX degradation was rapid at pH 5.0 and decreased as pH increased from 5.0 to 9.0. As the generated
Mn(III) is unstable and prone to disproportionate under alkaline conditions [13], higher pH was
unfavorable for the oxidation of CFX in PM/BS system. Furthermore, the speciation of BS is strongly
influenced by solution pH (HSO3− ꢀ SO3 + H+, pKa = 7.2), which will convert to less reactive sulfite
2−
under alkaline conditions [31]. Hence, acidic condition was favorable for CFX oxidation.
2.5. Product Analysis
To identify the potential reaction site of CFX towards PM oxidation, we subsequently analyzed
the oxidation products of CFX by PM. Transformation products of CFX by PM were analyzed by
LC/MS/MS. Two products with molecular weight (MW) of 363, and one product with MW of 379
were generated after oxidation of CFX (MW: 347) by PM (Figure S4–S6). The MW 363 (M + 16)
and 379 products (M + 32) were likely generated via the addition of one or two oxygen atoms
on the molecule of CFX, respectively. The two MW 363 products (363a and 363b) might be
isomers since they have different LC retention time but similar MW and MS fragment patterns.
The M + 16 products were also previously reported as the main oxidation products of CFX by
peroxymonosulfate [15], ozone [2], ferrate (VI) [14], and sulfate radical [32]. They were identified as
stereoisomeric CFX-(R)-sulfoxide and CFX-(S)-sulfoxide. We subsequently analyzed the oxidation
products of CFX by peroxymonosulfate with the analytical methods in this work. The result indicated
that CFX-(R)-sulfoxide and CFX-(S)-sulfoxide generated by peroxymonosulfate matched the 363a and
363b products in LC retention time and MS fragment patterns. Hence, the 363a and 363b products
were verified as CFX-(R)-sulfoxide and CFX-(S)-sulfoxide, respectively. Based on the MS spectrum,
379 product contains the fragment of m/z 106, which is characterized by the intact primary amine
moiety on the side chain. Hence, the two oxygen atoms were most likely added on the double bond on
six membered ring, which was also proposed in the oxidation of CFX by ozone [2].
For the sulfoxide products, the thioether sulfur on the six-membered ring of CFX was
the reaction site for PM oxidation. Indeed, the thioether sulfur was susceptible to oxidative
transformation by various oxidants, e.g., peroxymonosulfate [15], ferrate(VI) [14] and sulfate
radicals [32]. The CFX-sulfoxide products were generated by the electrophilic attack of the positively
charged Mn atom in PM on the thioether sulfur to form Mn-sulfur intermediate 1. Subsequently, a
pair of electrons of sulfur were donated to a d-orbital of Mn, inducing the formation of a coordinate
covalent intermediate 2, which were further decomposed to sulfoxide products by rearrangement [33
]
(Figure 5, scheme 1). For the 379 product, the double bond on the six-membered ring was the
reaction site for PM oxidation. The positively charged Mn electrophile attacked the unsaturated C=C
bond on the ring, and then forming cyclic hypomanganate(V) ester (intermediate 3) via an activated
organometallic complex. Afterwards, the cyclic manganite(VI) ester (intermediate 4) was generated
from the cyclic hypomanganate(V) ester by the oxidation of PM, and subsequently hydrolyzed to
generate di-ketone-containing product [33] (Figure 5, scheme 2).

Molecules 2018, 23, 2015
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Figure 5. Transformation products of CFX by PM.
2.6. Antibacterial Activity Assessment
Although PM was effective to oxidize CFX, the
β-lactam ring still existed in the transformation
products. To investigate whether the products possess biological activity, we further evaluated the
antibacterial activity of the transformation products of CFX by PM with E. coli as the tested bacteria.
As shown in Figure 6, the growth of E. coli could be completely inhibited in the presence of 40 µM CFX.
After addition of PM, the antibacterial activity of CFX solution gradually decreased along with the
gradual oxidation of CFX. Specifically, the growth inhibition was reduced to 13% after 8 min when
about 92% of initial CFX was oxidatively degraded in the presence of PM. This result indicated that the
biological activity of CFX could be significantly reduced after treatment of PM. Various oxidants have
been investigated to remove the antibacterial activity of CFX. For example, peroxymonosulfate [15],
ozone [34] and ferrate(VI) [14] were found to significantly eliminate the biological activity of CFX.
However, the products of β-lactam antibiotics (e.g., AMX) with UV/TiO2 still exhibited significant
activity to Enterococcus faecalis (ATCC 14506). The results in this work indicated that PM was effective
to eliminate the biological activity of CFX.
Figure 6. Growth inhibition of Escherichia coli. [CFX] = 40
µM, [PM] = 400 µM, and pH = 7 (10 mM PB).
Error bars represent standard deviations of data from triplicate samples.

Molecules 2018, 23, 2015
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3. Materials and Methods
3.1. Chemicals
Cefalexin (CFX,
permanganate (PM,
≥
99%) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Potassium
≥
99.5%) and sodium bisulfite (BS, 99.9%) were obtained from Aladdin (Shanghai,
≥
China). The HPLC grade of phosphoric acid and methanol were obtained from Sigma-Aldrich. Humic
acid (HA) was purchased from ANPEL Laboratory Technologies Inc. (Shanghai, China). Other
chemicals and reagents used were in analytical grade or higher, and obtained from Sigma-Aldrich
(St. Louis, MO, USA) or Aladdin (Shanghai, China). All chemicals were used as received without
any further purification. Reagent grade deionized (DI) water was produced by a Milli-Q Ultrapure
Gradient A10 purification system (Millipore, Burlington, MA, USA).
3.2. Real Water Samples
Wastewater (WW) after biological treatment was sampled from a municipal wastewater treatment
plant located in the south region of China. Samples were filtered through 0.45 µm glass fiber filters
and then stored under 4 ◦C before use. The water parameters of WW are present in Table S1.
3.3. Experimental Setup
Batch experiments were conducted in 100 mL amber serum bottles open to the air. The reaction
solution in the bottles were constantly mixed by a magnetic stirring. Except where mentioned,
phosphate buffer (PB, 10 mM) was used to control the solution pH at 7. PM (400
the solution containing CFX (40 M) and PB to initiate the reaction. The pre-determined concentration
of Cl−, HCO3 or HA was added into the above reaction system to evaluate their impact on CFX
degradation. BS (600 M) was added in the solution containing PM (40 M), CFX (40 M), and PB
µM) were added into
µ
−
µ
µ
µ
to evaluate the effect of BS. To investigate the pH impact, the solution was controlled at pH 5–9 with
10 mM PB. Samples were periodically withdrawn and supplemented with excess sodium thiosulfate
◦
to quench reactive species. The quenched samples were stored at 2 mL amber vial at 4 C and analyzed
within 24 h. All experiments were conducted in duplicates or more.
3.4. Analytical Methods
CFX was analyzed by an Agilent 1200 series HPLC system equipped with a UV diode-array
detector (DAD). Samples were separated using a Zorbax Extend-C18 column (4.6
and detected at 262 nm. Isocratic elution was employed using 10 mM H3PO4 solution and methanol
(70:30, v/v) with a flow rate of 1 mL/min.
×
250 mm, 5 µm)
The transformation products were separated on HPLC system (Utimate 3000, Dionex, Sunnyvale,
CA, USA) with a C18 column (2.1
×
150 mm, 5 µm), and analyzed with a triple quadrupole mass
spectrometry (TSQ Quantum Ultra EMR, Thermo Fisher Scientific, Waltham, MA, USA). The mobile
phase consisted of (A) 0.1% formic acid in water and (B) acetonitrile at a flow rate of 0.2 mL/min: 5%
B was kept for 2 min, then ramped to 30% B over 10 min, kept for 10 min. The mass spectrometer was
operated at positive electrospray ionization (ESI+). Then, product-full-scan mode was used to obtain
sufficient fragment information for structure elucidation.
3.5. Bacterial Growth Inhibition Bioassays
The antibacterial activity of CFX before and after treatment by PM was determined with E. coli
(ATCC 25,922) as reference strains. Briefly, 4 mL aliquots were taken at the predetermined time (i.e., 0,
2, 4, 6, and 8 min), and immediately quenched by Na2S2O3. Then, the sample was subsequently
inoculated with 1 mL of a broth E. coli culture (1
×
106 CFU mL−1), which was incubated overnight
in sterile Mueller–Hinton broth at 37 ◦C. The samples were further incubated for 8 h at 37 ◦C on a
shaking incubator rotating at 170 rpm. Control experiments with Na2S2O3 or DI water dosing 1 mL of

Molecules 2018, 23, 2015
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E. coli broth culture were also conducted. The growth of E. coli was found to be unaffected by Na2S2O3.
After the incubation, the bacterial growth was evaluated by determining the optical density (OD) at
625 nm on a UV-vis spectrophotometer (Mapada UV-1600 PC), and then comparing this value to the
initial OD value of each sample. The OD value was subsequently converted to growth inhibition, I,
according to
I = (A0 − A)/A0
where A0 represents the maximum absorbance for control, which corresponds to uninhibited culture
growth. The values of I vary from 0 (null inhibition) to 1 (complete inhibition). All experiments were
performed in triplicates, and the results were reported as the mean with standard deviations.
4. Conclusions
PM exhibits high performance in the oxidative degradation of CFX. Apparent second-order rate
constant of the reaction between CFX and PM was calculated to be 12.71
±
1.62 M−1 −1. Lower pH
s
·
was favorable for PM-induced oxidation of CFX. PM-induced CFX oxidation could be promoted in the
presence of Cl− and HCO3−, and the promoting effect increased with the increasing concentration
of coexisted ions, whereas HA had no effect on the oxidation of CFX by PM. Oxidation of CFX by
PM was also significant in the wastewater matrix. The presence of BS could significantly accelerate
the oxidation of CFX by PM. Product analysis indicated that two products with MW of 363 and one
product with MW of 379 were the primary products of CFX by PM oxidation. The thioether sulfur and
double bond on the six-membered ring were proposed as the main reaction sites in CFX towards PM
oxidation. The antibacterial activity of the transformation products was significantly reduced after
treatment of CFX by PM. Therefore, PM oxidation was an effective approach to significantly eliminate
CFX and its biological activity.
Supplementary Materials: The supplementary materials are available online.
Author Contributions: Y.Q. and J.C. conceived and designed the experiments; Y.Q. performed the experiments;
P.G. and G.X. analyzed the data; Z.L. contributed reagents/materials/analysis tools; J.C. wrote the paper.
Funding: This work was supported by the Fundamental Research Funds for the Central Universities
(NO. 2232018D3-08), National Science Foundation of China (No. 51708097 and 51509175), Natural Science
Foundation of Shanghai (17ZR1400400) and China Postdoctoral Science Foundation (No. 2017M611422).
Conflicts of Interest: The authors declare no conflicts of interest.
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