Article

pubs.acs.org/est

UV/H2O2 and UV/PDS Treatment of Trimethoprim and

Sulfamethoxazole in Synthetic Human Urine: Transformation
Products and Toxicity
Ruochun Zhang,† Yongkui Yang,† Ching-Hua Huang,‡ Na Li,§ Hang Liu,† Lin Zhao,*,† and Peizhe Sun*,‡
†
School of Environmental Science and Engineering, Tianjin University, Tianjin 300072, China
‡
School of Civil and Environmental Engineering, Georgia Institute of Technology, Atlanta, Georgia 30332, United States
§
Tianjin Institute of Agriculture Quality Standards and Testing Technology, Tianjin 300381, China
*
S Supporting Information
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ABSTRACT: Elimination of pharmaceuticals in source-sepa-

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rated human urine is a promising approach to minimize the

pharmaceuticals in the environment. Although the degradation
kinetics of pharmaceuticals by UV/H2O2 and UV/peroxydisul-
fate (PDS) processes has been investigated in synthetic fresh and
hydrolyzed urine, comprehensive evaluation of the advanced
oxidation processes (AOPs), such as product identification and
toxicity testing, has not yet been performed. This study identified
the transformation products of two commonly used antibiotics,
trimethoprim (TMP) and sulfamethoxazole (SMX), by UV/
H2O2 and UV/PDS in synthetic urine matrices. The effects of
reactive species, including •OH, SO4•−, CO3•−, and reactive
nitrogen species, on product generation were investigated.
Multiple isomeric transformation products of TMP and SMX
were observed, especially in the reaction with hydroxyl radical. SO4•− and CO3•− reacted with pharmaceuticals by electron
transfer, thus producing similar major products. The main reactive species deduced on the basis of product generation are in
good agreement with kinetic simulation of the advanced oxidation processes. A strain identified as a polyphosphate-accumulating
organism was used to investigate the antimicrobial activity of the pharmaceuticals and their products. No antimicrobial property
was detected for the transformation products of either TMP or SMX. Acute toxicity employing luminescent bacterium Vibrio
qinghaiensis indicated 20−40% higher inhibitory effect of TMP and SMX after treatment. Ecotoxicity was estimated by
quantitative structure−activity relationship analysis using ECOSAR.

■ INTRODUCTION
Pharmaceutical micropollutants in the environment have been
regarded as a major threat to the ecosystem due to their adverse
biological effect and the potential of inducing drug-resistant
bacteria. Because pharmaceuticals discharged in wastewater
cannot be effectively removed by traditional wastewater
treatment technologies, this has resulted in widespread
occurrence of pharmaceuticals in the aquatic environment
worldwide.1−3 Among all the wastewater streams entering into
wastewater treatment plants (WWTPs), the treatment of source-
separated urine has been viewed as a promising approach to
minimize the harm of the pharmaceuticals, because urine not
only contributes a significant portion of the pharmaceuticals in
municipal WWTPs,4,5 it also carries high toxic potential to
aquatic organisms.6−8 Moreover, recovering nutrients from
urine, as a new resource recovery strategy,9 also requires
elimination of pharmaceuticals from urine. To date, nano-
filtration membranes,10 strong-base anion exchange polymer
resins,11 electrodialysis,12 and struvite precipitation13 have been
investigated for removing pharmaceuticals from urine, all of
which involved only physical separation of the pharmaceuticals
and further treatment is still needed. Ozone has been investigated
for destruction of pharmaceuticals in urine, but high doses of
ozone (150 mg·L−1 or higher) were needed to reach satisfactory
reduction.14 Therefore, more effective processes are needed to
fully destruct the pharmaceuticals in urine.
Advanced oxidation processes (AOPs) are among the most
effective and promising processes in eliminating organic
pollutants. Radical-based AOPs, in particular with hydroxyl
radical (•OH)15−17 and sulfate radical (SO4•−),18−22 have been
successfully applied to destruct micropollutants in wastewater,
drinking water, groundwater, and sediment−slurry system. •OH
(E° (•OH/H2O) = 1.9−2.7 V)23 is of low selectivity and can
react with many pharmaceuticals rapidly (k = 108−1010 M−1·
s−1).24 SO4•− is also a strong oxidant (E° (SO4•−/SO42−) = 2.5−
Received: November 13, 2015
Revised: January 22, 2016
Accepted: February 3, 2016
Published: February 3, 2016

© 2016 American Chemical Society 2573 DOI: 10.1021/acs.est.5b05604

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3.1 V)25 and reacts with pharmaceuticals mainly through electron
transfer. In urine matrices, which are more complicated and
uniquely challenging compared to typical municipal wastewaters
and surface waters because of the high concentrations of the
organic components and inorganic salts, the efficacy of UV/
H2O2 and UV/peroxydisulfate (PDS) processes in eliminating
pharmaceuticals and metabolites was evaluated in our previous
research.26 Sulfamethoxazole (SMX), acetyl-sulfamethoxazole
(acetyl-SMX) (a metabolite of SMX), and trimethoprim (TMP)
were studied due to their high frequency of detection in the
environment. Systematic kinetic studies have elucidated the
direct photolysis and indirect photolysis of the above
pharmaceuticals and metabolites’ degradation rates. In addition
to •OH and SO4•−, carbonate radical (CO3•−) and reactive
nitrogen species (RNS) also showed reactivity toward the
pharmaceuticals and acted as the main reactive species under
particular conditions. CO3•− is electrophilic and also sufficient
for degrading pharmaceuticals (E° (CO3•−/CO32−) = 1.63 V at
pH 8.4).27 Reactivity of CO3•− toward pharmaceuticals has been
reported but is still in its early stage.28,29 RNS, such as amino
radical, nitrogen dioxide radical, nitric oxide radical, and
peroxynitrite, etc., are weaker oxidants with relatively high
selectivity.26 However, kinetics studies alone were not sufficient
to evaluate the performance of AOP processes.
Although AOPs are powerful oxidation processes, often they
cannot completely mineralize pollutants within the typical
operation time and a number of products are formed and may
persist in the system.30 The product formation may vary with
different treatment processes, oxidants, background constituents
involved, and reaction mechanisms.31−34 Product identification
can provide crucial clues for reaction pathways.35,36 For TMP
and SMX, products generated by chlorination,37−40 ozona-
tion,30,41,42 photochemical reactions,43−45 photocatalytic treat-
ment,46−48 electrochemical processes,49 and in other oxidation
systems50,51 were identified and utilized to propose correspond-
ing reaction pathways. However, to our knowledge, products of
SMX by UV/PDS and TMP by UV/H2O2 and UV/PDS have
not been reported. Information is also scarce regarding oxidation
products of pharmaceuticals by CO3•− and RNS. Product
analysis coupled with measurement such as total organic carbon
(TOC) can be complementary to assess the overall efficiency of
the treatment processes.
In recent years, evaluation of toxicological effect has been
recommended52 and applied to investigate the biological impact
of pharmaceuticals in various environmental matrices.53−57
Transformation products may retain the properties of the parent
compounds or potentially become more biologically active.
Therefore, toxicity tests of the products have been increasingly
applied as part of the evaluation of the performance of treatment
processes.58,59 At least two or multiple bioassays are commonly
needed to reveal the overall toxic potential.60,61 Cytotoxicity,62
genotoxicity,63 and estrogenicity64 have been applied to test the
toxic effect of the pharmaceuticals and transformation
products.58 Luminescent bacteria, particularly Vibrio f i-
scheri,65−67 are frequently employed to assess acute toxicity.
Crustaceans, algae,41 fish,68 and zebrafish embryos69 are also
widely used as toxicity indicators. Vibrio qinghaiensis, a fresh
water luminescent bacterium, is now gaining increasing popular-
ity.65,70 As for antibiotics, the antibacterial property of the
compounds is commonly monitored using bacterial strains of E.
coli71,72 and Bacillus subtilis,73,74 while few studies have applied
functional bacteria as indicators. Biodegradability of pharma-
ceuticals and their transformation products in activated sludge
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have been investigated and samples after AOP treatments are
normally more biocompatible.75−77
The objective of this study was to better understand the UV/
H2O2 and UV/PDS processes in eliminating pharmaceuticals in
synthetic source-separated urine solutions with emphasis on the
product identification and toxicity evaluation. TMP and SMX
were selected because they are widely used antibiotics and have
been shown high reactivity toward different reactive species in
AOPs in our previous study.26 In the present study, products
generated by specific radicals were identified, and degradation of
TMP and SMX by mixed reactive species was illustrated in
synthetic urine solutions. Toxic effects of the parent compounds
and the transformation products were evaluated by testing their
antimicrobial property and bioluminescence inhibition. Quanti-
tative structure−activity relationship (QSAR) analysis was also
applied to estimate the ecotoxicity. The major goal was to achieve
comprehensive evaluation of AOPs treatment of pharmaceuticals
in urine matrices, which can help develop better urine treatment
strategies. Research on reacting mechanism by different reactive
species and corresponding toxic effects would improve the
understanding of AOPs performances.
■ MATERIALS AND METHODS
Materials. Sources of chemicals and materials are provided in
the Supporting Information (SI) Text S1.
Experimental Setup. UV, UV/H2O2, and UV/PDS experi-
ments were conducted in a reactor with the same setup as in the
previous research.26 Potassium ferrioxalate was employed as
chemical actinometer78 and UV fluence rate was measured at
1.78 × 10−6 Einstein·L−1·s−1 in this study. Reaction solutions
were prepared with 100 μM TMP or SMX in 100 mL of fresh
urine, hydrolyzed urine, or phosphate buffer (PB) solution at pH
6 or 9. The synthetic human urine composition was adapted from
the previous recipe (SI Table S1).26 The high initial
concentration of TMP and SMX was chosen in order to explore
transformation products. H2O2 and potassium PDS were added
at 3 mM in PB solutions to generate •OH- and SO4•−-dominant
systems, respectively. To create CO3•−- and RNS-dominant
systems, 0.5 M sodium bicarbonate and 1 M ammonia solution
were added into PB solution at pH 9, respectively, in UV/H2O2
system. Simulation of radical concentrations under different
experimental conditions was conducted by Gepasi 3.0. The rate
constants for reactions considered in the simulation were
obtained from our previous publication, which considered all
relevant reactions under AOP conditions. This model has been
successfully employed in a number of studies and proved to be
reliable to predict radical concentrations.26,79 Total simulation
time was set as 300 s because most reactive species were close to
the pseudosteady state. Small amounts of concentrated NaOH
and perchloric acid were added to adjust the pH when needed.
Analytical Methods. A Waters AcQuity UPLC system
equipped with a PDA detector and a BEH C18 column (2.1 × 50
mm, 1.7 μM) was used to monitor the loss of the parent
compounds. The UPLC system was connected to a TOF mass
spectrometer (Premier, Micromass, UK) with electrospray
interface to analyze transformation products. Structure identi-
fication was achieved based on the fragmentation pattern. The
elemental composition was deduced from the exact m/z values
obtained from the ESI-TOF-MS system (micrOTOF-Q II,
Bruker, Germany). The detailed chromatographic and MS
conditions are summarized in SI Text S2.
Toxicity Analysis. The antimicrobial properties of TMP and
SMX and their transformation products were tested using a
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polyphosphate-accumulating bacterium identified as Aeromonas.

Optical density (absorbance at 600 nm) was used as an indication
of bacterial growth. The acute toxicity assay was carried out
against freshwater luminescent bacterium Vibrio qinghaiensis.
Because ammonia and bicarbonate show high toxicity to Vibrio
qinghaiensis, the acute toxicity of products by CO3•− and RNS
were not tested. QSAR analysis calculated by the Ecological
Structure−Activity Relationship Model (ECOSAR) program30
was also employed to estimate the acute and chronic toxicity for
fish, daphnid, and green algae. Detailed information for toxicity
analysis is described in SI Text S3.

■ RESULTS AND DISCUSSION

Contribution of Reactive Species. With UV/H2O2 and
UV/PDS processes in synthetic urine solutions, •OH, SO4•−,
CO3•−, and RNS were major reactive species to degrade TMP
and SMX.26 To investigate the products generated by each
reactive species, experimental conditions where certain reactive
species dominated were designed. On the basis of the simulation
results, the concentrations of above-mentioned reactive species
under UV/H2O2 and UV/PDS conditions with different
components in solutions are presented in SI Table S2. The
dominant reactive species generated by UV/H2O2 and UV/PDS
in PB solution are •OH and SO4•−, respectively, for both pH 6
and pH 9. Although •OH can react with SO4•− and generate
•OH so that •OH concentration would increase when pH was
higher, it was still not sufficient (1.21 × 10−15 M) for an
observable degradation for TMP. More than 99% of TMP was
degraded by SO4•−. By adding excess sodium bicarbonate in UV/
H2O2 system, the CO3•− concentration was 4 orders of
magnitude higher than that of •OH. On the basis of the
second-order rate constants of TMP and SMX with •OH and
CO3•−,26 more than 90% of TMP and SMX was destructed by
Figure 1. Degradation of (a) TMP and (b) SMX in UV, UV/H2O2, and
CO3•− in indirect photolysis. Because the second-order rate UV/PDS systems at pH 6 and 9 in 5 mM PB solutions, and UV/H2O2
constants of pharmaceuticals with RNS were not available, RNS systems with 0.5 M NaHCO3 or 1 M NH4OH. Oxidant concentration
contribution was not possible to evaluate quantitatively. But was 3 mM. UV fluence = UV fluence rate × exposure time.
according to the simulation result shown in SI Table S2, when
excess ammonia solution was added in UV/H2O2 system, the
•OH concentration was 3.72 × 10−15 M which was insufficient to Therefore, evaluation of the product abundance was on the basis
yield an observable degradation of TMP and SMX. Meanwhile, of peak area (shown in SI).
concentrations of several RNS were relatively high thus they may Transformation Products of TMP by Hydroxyl Radical. The
play a role in degrading pharmaceuticals with higher reactivity. degradation of TMP by hydroxyl radical (i.e., under UV/H2O2
Therefore, RNS was assumed to be the dominant reactive species condition, SI Table S2) produced 13 main products at pH 6 in
under this condition. PB solution (Figure 2). Hydroxylation was the most prominent
Identification of Transformation Products of TMP. On mechanism, accompanied by demethylation and carbonylation.
the basis of the previous results,26 direct photolysis of TMP TP 307-3 (m/z 307, C14H19N4O4) was the most abundant
under low-pressure UV irradiation was negligible. Under AOP product based on the peak area (Figure 2, SI Figure S2a). The
conditions, TMP was mainly destructed by •OH, SO4•−, and addition of 16 Da to the molecular weight of the parent
CO3•−. Therefore, products produced by these radicals were compound suggested a transformation pathway of hydroxylation.
identified. Degradations of TMP by different radicals are shown The position of the hydroxyl group was proposed (Figure 2) due
in Figure 1a. The accurate m/z values obtained by the ESI-TOF- to the presence of the fragment ions m/z 277, m/z 259, and m/z
MS system are shown in SI Table S3. On the basis of accurate 123 (SI Figure S3), which were in accordance with the
mass, empirical chemical formulas of each transformation fragmentation pattern reported by Sirtori et al.80 TP 307-3 was
product were proposed (SI Table S3). In all cases, no cleavage likely produced by direct •OH addition to the benzene moiety.81
of the methylene group or aromatic rings was observed (Figure Two more m/z 307 products, TP 307-1 and TP 307-2, were also
2). This suggested inadequate mineralization of oxidation detected at much lower concentrations. TP 307-1 was a major
processes. Indeed, the decrease of TOC was negligible under product by SO4•− and will be discussed in a later section.
all tested conditions within corresponding reaction time (data TP 325 (m/z 325, C14H21N4O5) was another major product
not shown). Because standards of the transformation products with peak area similar to that of TP 307-3 (Figure 2, SI Figure
are not commercially available, accurate quantification of each S2a). The fragment ions m/z 221 and m/z 143 suggested that
product is impossible to achieve. On the basis of the structural one oxygen atom was added to C8 atom as a hydroxyl group and
similarity of the parent compound and the transformation one oxygen atom was added to C13 atom (SI Figure S4).
products, the MS signal responses were assumed to be similar. Previous research with ozone and nitrifying activated sludge
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Figure 2. Proposed structures of transformation products of TMP under different conditions. A: UV/H2O2 at pH 6 (•OH-dominant); B: UV/H2O2 at
pH 9 (•OH-dominant); C: UV/PDS at pH 6 (SO4•−-dominant); D: UV/PDS at pH 9 (SO4•−-dominant); E: UV/H2O2 with 0.5 M NaHCO3 (CO3•−-
dominant). Conditions A−D were conducted in 5 mM PB solutions.
processes proposed addition of carbonyl group on C13 atom
with hydrogenation of C9−C10 double bond.82,83 In contrast,
based on our MS2 spectrum of TP 325, there was no strong
evidence to confirm that the oxygen was added as a carbonyl
group. In addition, in a strong oxidation process, hydrogenation
of a double bond was difficult to achieve. Therefore, instead of
forming a carbonyl group, the oxygen was more likely added as a
hydroxyl group. The same hypothesis also applies to TP 341 (m/
z 341, C14H21N4O6), with a hydroxyl group addition to TP 325
on the benzene ring (implied by the fragment ion m/z 197
instead of m/z 181, SI Figure S5). Further study is still needed for
structural confirmation.
Proposed structures of other products are shown in Figure 2.
Structural identification is discussed in SI Text S4. Overall, all the
products by •OH were not accumulated in the solution and
likely degradable by •OH (SI Figure S2a).
At pH 9, TP 307-3 (m/z 307, C14H19N4O4) was also the major
product in the PB solution, whereas the amount of TP 325 (m/z
325, C14H21N4O5) was less than that at pH 6 (SI Figure S2b). In
addition, all the m/z 323 products were not observed at pH 9.
Overall, most products of TMP at pH 9 were of lower relative
abundance and generated via hydroxylation with addition of a
single hydroxyl group. The difference in product speciation at pH
6 and pH 9 was likely due to the reactivity of the products of
TMP. It was expected that products of TMP reacted faster with
hydroxyl radical at higher pH due to two possible reasons. First,
as reported in our previous study,26 the second-order rate
constant of TMP with hydroxyl radical was higher at pH 9 than
that at pH 6. Because the majority of productsresulted from slight
modification of parent TMP, it was expected the product also has
the same trend. Second, hydroxylated TMPs (such as TP 323)
were likely composed of secondary −OH moiety, which is
known to degrade faster under AOP condition at higher pH (SI
Figure S9).84
Transformation Products of TMP by Sulfate Radical. The
degradation of TMP by sulfate radical (i.e., under UV/PDS
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condition, SI Table S2) produced 6 main products at pH 6 in PB
solution (Figure 2). TP 307-1 (m/z 307, C14H19N4O4) (Figure 2,
SI Figure S7a) was a major product, which was an isomer of the
most prominent product produced by •OH (TP 307-3). The
fragment ions, m/z 289 and m/z 274, implied that a hydroxyl
group was added to the bridge methylene group (SI Figure S8).
The same MS 2 spectrum was also found in previous
studies.30,80,83 TP 305 (m/z 305, C14H17N4O4) was another
major product by SO4•− (Figure 2, SI Figure S7a). It was
previously identified as a product under solar-Fenton46 and TiO2
photocatalysis80 conditions. Both TP 307-1 and TP 305 were
likely generated via electron transfer mechanism (SI Figure S9).
The aromatic moieties (i.e., diaminopyrimidine or benzene) on
TMP lose one electron to SO4•− forming a radical intermediate
with a positive charge. The nonpaired electron is then stabilized
on C7 atom, forming a carbon-center radical, which is known to
transform to superoxide with the presence of dissolved oxygen.
Through bimolecular interaction, the superoxide intermediate
transforms to a hydroxyl moiety yielding TP 307-1 and a
carbonyl moiety yielding TP 305. TP 307-1 and TP 305 were also
found in the •OH-dominant system, but at much lower
abundance. TP 323-1,-2,-4 and TP 325 were also generated by
the reaction with SO4•−, whereas other products found in the
•OH-dominant system (i.e., TP 277-1, TP 277-2, TP 307-2, TP
307-3, TP 295, and TP 341) were not observed. These
differences implied different reaction pathways of SO4•− and
•OH. Unlike the •OH products, the products produced by
SO4•− did not degrade throughout the reaction, indicating they
may be persistent in UV/PDS process (SI Figure S7a).
In the UV/PDS process at pH 9 instead of pH 6, the m/z 323
products were hardly detected, similar to that observed in UV/
H2O2 process at the two different pHs. TP 305 overweighed TP
307-1 in abundance and became the dominant product at pH 9
(SI Figure S7b). As shown in SI Figure S9, TP 307-1 further
reacted with sulfate radical and transformed to TP 305. Although
such transformation was slow at pH 6, this reaction was
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Figure 3. Proposed structures of transformation products of SMX under different conditions. A: UV/H2O2 at pH 6 (•OH-dominant); B: UV/H2O2 at
pH 9 (•OH-dominant); C: UV/PDS at pH 6 (SO4•−-dominant); D: UV/PDS at pH 9 (SO4•−-dominant); E: UV/H2O2 with 0.5 M NaHCO3 (CO3•−-
dominant); F: UV/H2O2 with 1 M NH3 (RNS-dominant); G: UV at pH 6; H: UV at pH 9. Conditions A−D, G, and H were conducted in 5 mM PB
solutions.
accelerated at pH 9 because the elimination of superoxide radical
was catalyzed by basic conditions.84
Transformation Products of TMP by Carbonate Radical.
The degradation of TMP by carbonate radical (i.e., under UV/
H2O2/NaHCO3 condition, SI Table S2) produced 4 main
products at pH 9 in PB solution (Figure 2). Conditions at pH 6
were not considered because carbonate radical only dominated in
hydrolyzed urine (pH 9).26 TP 305 (m/z 305, C14H17N4O4) was
also a dominant and stable product in the CO3•−-dominant
system (Figure 2, SI Figure S11), as by SO4•−, suggesting similar
electron transfer mechanism of these two radicals (SI Figure S9).
TP 307-1 (m/z 307, C14H19N4O4) was found in CO3•−-
dominant system but not as significant as it was in SO4•−-
dominant system (at pH 9). This difference can be explained by
an additional pathway which produced TP 305. As shown in SI
Figure S9, besides the same mechanism as SO4•−, CO3•− can
transfer one of its oxygen to the carbon-center radical forming a
carbonyl moiety (i.e., TP 305).85 Two new products (TP 277-3
and TP 291) were generated exclusively by CO3•−. TP 277-3 (m/
z 277, C13H17N4O3) was an isomer of TP 277-1 and TP 277-2
which were produced by •OH. The retention time of TP 277-3
on UPLC was close to that of TMP (SI Table S3), suggesting a
structure similar to the parent compound, which is also
supported by the remarkable resemblance between the MS2
fragmentation patterns of TMP and TP 277-3 (SI Figure S12).
The fragment ions m/z 261 and m/z 247 corresponded to the
loss of a methoxyl group on C2 or C4 atom. The lack of fragment
ion m/z 123 suggested the hydroxyl group was added to the
diaminopyrimidine ring or the methylene group. An isomer of
TMP, TP 291 (m/z 291, C14H19N4O3), was observed and the
retention time was longer than all the products and parent TMP.
The different fragmentation patterns of TP 291 and TMP
indicated a large difference between the structures, but current
information was not sufficient to identify the structure of TP 291.
Transformation Products of TMP in Synthetic Urine
Solution. After gathering the information on transformation
products of TMP by individual radicals, product identification
was performed in synthetic urine solution. To identify products
in urine matrices, in hydrolyzed urine, the reaction time was set
the same as the experiment identifying products for CO3•− which
was regarded as the dominant radical. In fresh urine, reaction
time was set longer than in buffer solution (pH = 6) to reach
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more than 50% TMP removal, since the degradation was
inhibited to a large extent. Samples were taken at each time
interval and a spectrum that can show all the dominant products
is shown in SI Figures S13−S16 for each case.
In terms of product identification, in fresh urine with UV/
H2O2 process, the presence of TP 277-2 and TP 307-3 confirmed
that the oxidation by •OH played an important role (SI Figure
S13). With UV/PDS process, the main product of SO4•− TP
307-1 stayed as the most abundant product, indicating the
contribution of SO4•− to TMP degradation (SI Figure S14). In
hydrolyzed urine, by UV/H2O2, the presence of TP 307-1, TP
305, and TP 277-3 proved the degradation role of CO3•− (SI
Figure S15). With UV/PDS process, the abundance of TP 307-1
implies that besides CO3•−, other reactive species, such as SO4•−
and RNS, may also play a role (SI Figure S16). Indeed, when
CO3•− was the dominant reactive species in the system, TP 305
was more abundant than TP 307-1. The major reactive species
deduced from the product presence in the present study are in
good agreement with the simulation results of corresponding
contributing reactive species in synthetic urine solution.26
Identification of Transformation Products of SMX.
Degradation of SMX is profiled in Figure 1b, and accurate m/z
values of transformation products are shown in SI Table S4.
Because of the high direct photolysis rate of SMX, the majority of
SMX was degraded via direct photolysis in the UV/H2O2 and
UV/PDS processes. As a result, the products of indirect
photolysis of SMX were not as prominent as the case of TMP.
On the basis of both experimental and simulation results,26 SMX
was of higher reactivity toward radical species than TMP and can
be degraded by •OH, SO4•−, CO3•−, and RNS. Products of SMX
were analyzed respectively for each reactive species. The
corresponding radical-dominant systems were generated under
the same conditions as for TMP product analysis. Direct
photolysis products were generated in PB solutions.
Transformation Products by Direct Photolysis. By direct
photolysis, an isomer of SMX, SP 254 (m/z 254, C10H11N3O3S)
was produced as a major product (Figure 3, SI Figure S17); it was
produced due to photoisomerization.45 SP 99-1 (m/z 99,
C4H7N2O) was likely 3-amino-5-methylisoxazole (Figure
3),41,45 which was produced due to the cleavage of the
sulfonamide bond. It was observed under every tested condition.
An isomer of SP 99-1 (SP 99-2, m/z 99, C4H7N2O) was detected
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Figure 4. Inhibition on the tested strain (identified as Aeromonas) by parent compound standard solutions and samples after treatment: (a) TMP and
samples at pH 6, (b) TMP and samples at pH 9, (c) SMX and samples at pH 6, (d) SMX and samples at pH 9. Initial concentration of TMP and SMX in
AOP samples (i.e., 0% removal) was 100 μM. The samples were diluted by a factor of 2 in the inhibition test (i.e., 50 μM at 0% removal). Lines represent
the inhibition of the remaining parent compounds, and dots represent the observed inhibition by the samples. Errors represent the standard deviation (n
= 3).

as a product only by direct photolysis at pH 9 (Figure 3, SI Figure
S17b), which was probably due to the cleavage of the
sulfonamide bond of SP 254.
Transformation Products of SMX by Hydroxyl Radical. In
addition to the direct photolysis products, two products with m/z
270 (SP 270, SP 270-2, C10H11N3O4S) (Figure 3, SI Figure S18)
were produced in •OH-dominant system (i.e., under UV/H2O2
conditions). The 16 Da higher molecular weight suggested
hydroxylation occurred on the parent compound. The fragment
ion m/z 172, compared with the fragment ion m/z 156 of SMX,
indicated that a •OH was added to the benzene ring (SI Figure
S19). A product of SMX with m/z 270 was also observed in TiO2
photocatalysis system.47 SP 262 was found exclusively in the
•OH-dominant system. The concentration profile of SP 262 was
consistent with that of SP 99-1 (SI Figure S18), suggesting a
correlation between the two products. Considering SP 99-1
represented the isoxazole ring of SMX, the other moiety of SMX
after cleavage of the sulfonamide bond might recombine with
small fragments and generate SP 262. However, the speculation
needs further evidence to be confirmed. All the products
degraded more rapidly at pH 9 than at pH 6, especially for SP 99-
1 and SP 262, which accumulated in the solution at pH 6 (SI
Figure S18).
Transformation Products of SMX by Sulfate Radical. In
UV/PDS process in PB solution (SO4•−-dominant system), no
2578
additional product was found except for the direct photolysis
products. However, at pH 6, the abundance of SP 254 decreased
dramatically compared with that by direct photolysis (SI Figure
S20a). At pH 9, no isomer product was observed (SI Figure
S20b). Our preliminary results showed that PDS did not react
with SP 254, suggesting SP 254 or some intermediates essential
for forming SP 254 may be degraded by SO4•−.
Transformation Products of SMX by Carbonate Radical
and RNS. In CO3•−-dominant system (UV/H2O2/NaHCO3),
there was no product found other than the direct photolysis
products. As by SO4•−, the production of SP 254 was also
suppressed (SI Figure S21), suggesting the presence of PDS was
not the reason for the decrease of SP 254 production.
Considering the similarity of the reacting mechanisms of
SO4•− and CO3•−, SP 254 or some intermediates essential for
forming SP 254 might be prone to destruction by electron
transfer mechanism.
On the basis of the kinetic simulation (SI Table S2), the
oxidation by •OH can be neglected when 1 M ammonia was
added into the solution with UV/H2O2 process. Therefore, in
addition to the direct photolysis product, SP 270-2 was also
observed and speculated to be a product by reaction with RNS
(SI Figure S22). As peroxynitrite was identified as the major RNS
in hydrolyzed urine under AOP conditions, formation of
hydroxylation products of SMX was likely partly due to the
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Environ. Sci. Technol. 2016, 50, 2573−2583
Environmental Science & Technology Article

Figure 5. Impact on Vibrio qinghaiensis luminescence by parent compound standard solutions and samples after treatment: (a) TMP and samples at pH
6, (b) TMP and samples at pH 9, (c) SMX and samples at pH 6, (d) SMX and samples at pH 9. Initial concentration of TMP and SMX in AOP samples
(i.e., 0% removal) was 100 μM. The samples were diluted by a factor of 2 in the inhibition test (i.e., 50 μM at 0% removal). Lines represent the inhibition
of the remaining parent compounds and dots represent the observed inhibition by the samples. Errors represent the standard deviation (n = 3).

reaction with peroxynitrite. Indeed, oxidation of phenolic
compound by peroxynitrite was reported through one-electron
oxidiation processes that do not involve free hydroxyl radicals,
which yielded hydroxylated phenolic products.86
Transformation Products of SMX in Synthetic Urine
Solution. In urine matrices, the reaction time was set the same
as the experiment identifying products for the corresponding
main reactive species and spectra that can show all the dominant
products are shown in SI Figures S23−S26. In fresh urine with
UV/H2O2, SP 270-2 was observed, indicating •OH was
contributing (SI Figure S23). SP 99 and SP 254 were also
found, which was consistent with the product identification result
under UV/H2O2 condition in buffer solution. With UV/PDS, SP
99-1 was the only observed product, which is similar to the case
in PB solution at pH 6 (SI Figure S24). In terms of hydrolyzed
urine, SP 254 was hardly detected, which was expected because
SO4•− and CO3•− were the major reactive species in these
systems (SI Figures S25 and S26).
Toxicity Evaluation. Growth inhibition of TMP and SMX
and the transformation products on a target strain was tested to
investigate the change of antimicrobial activity during degrada-
tion. Bioluminescence inhibition was monitored for acute
toxicity. To comprehensively evaluate the toxicity, QSAR
analysis was also performed to predict the ecotoxicity of
individual transformation products.
2579
Antimicrobial Property. Aeromonas has been accepted as a
polyphosphate accumulating organism (PAO) and the strain
used in this study has been proved to remove phosphorus
satisfactorily. It was employed as a representative of the
microorganism in the biological wastewater treatment system
to test the antimicrobial property of the parent compounds and
transformation products, so that the toxic potential of the
transformation products on wastewater treatment systems can be
delineated.
The antimicrobial property of the parent compounds was
measured. The inhibition was calculated from the following
equation:
⎛ OD600 (Sample) ⎞
Inhibition (%) = ⎜1 − ⎟ × 100
⎝ OD600 (Control) ⎠
where OD600 (Sample) was the absorbance at 600 nm of the
sample aliquot taken at each selected time interval from a direct
photolysis or AOP reaction, and OD600 (Control) was the
absorbance of DI water replacing the sample.
Because standards of transformation products are not
commercially available, it is difficult to test the toxicity of the
products separately. However, it is beneficial to test the inhibition
effect of a mixture of transformation products and remaining
parent compounds because they practically coexist in the AOP
systems and may exert toxicity jointly. Concentrations of the
DOI: 10.1021/acs.est.5b05604
Environ. Sci. Technol. 2016, 50, 2573−2583
Environmental Science & Technology
parent compounds were measured by UPLC, and corresponding
inhibition ratios were calculated from the reference curve (SI
Figure S1). Therefore, the observed inhibition of the sample
(dots in Figure 4) subtracted by the inhibition by the parent
compounds (lines in Figure 4) represents the inhibition by the
products.
To be noted, because the presence of bicarbonate could
promote the bacteria growth, the inhibitions by remaining parent
compounds in CO3•− samples are shown separately (dashed
lines in Figure 4). For almost all the data points, the observed
inhibition equaled, or was slightly higher than, the inhibition of
the retaining parent compound calculated by the reference curve.
This indicated that toward the tested strain, negligible
antimicrobial property was retained for the transformation
products throughout the reaction.
TMP interferes with the normal bacterial metabolism pathway
by binding to dihydrofolate reductase, and SMX acts by
inhibiting bacterial utilization of para-aminobenzoic acid
(PABA) because of their structural similarity with dihydrofolic
acid and PABA, respectively.87 After treatment, the parent
compounds were hydroxylated, carbonylated, demethylated,
isomerized, or broke down. The structurally modified products
may not able to bind to the receptors as the parent compounds
did, thus PABA and DHF were utilized without inhibition. The
structural transformation of parent TMP and SMX by AOPs is
likely to disable the competitive antagonism and therefore
deprive the products of antibiotic properties.
Acute Toxicity. Microtox test has been the most frequently
applied method to measure the acute toxicity of toxic substances
in environmental studies.88 When the target microorganism
contacts with toxic substances, its bioluminescence decreases due
to disruption of normal metabolism. Although marine bacterium
Vibrio f ischeri is the most commonly used indicator of toxicity, it
is not suitable in fresh water studies. Therefore, freshwater
luminescent bacterium Vibrio qinghaiensis was selected in this
study.
Figure 5 shows the acute toxicity of the parent compounds and
the products. The y-axis, L/L0, reflects the comparison of the
luminescence of the sample with the initial luminescence (i.e.,
before treatment at t = 0). Data points above the dashed line
(standing for no change in the luminescence) indicate that the
luminescence increased after treatment, which suggests a decline
in toxicity. Likewise, data points below the dashed line indicate
lower luminescence and higher toxicity. Dot−dash lines in Figure
5 represent the L/L0 of the samples containing different amounts
of parent pharmaceuticals.
Compared with antibiotic-free samples, TMP, at lower than
100 μM concentration, exhibited almost no inhibition to the
Vibrio qinghaiensis (SI Figure S27). However, at both pH 6 and
pH 9, the luminescence of the samples decreased to
approximately 80% of the initial luminescence, indicating slight
toxicity of the products by •OH. For products by SO4•−, a
distinct inhibition was observed because 40% of the
luminescence was suppressed, indicating higher toxicity.
SMX inhibited approximately 25% of luminescence of Vibrio
qinghaiensis at 100 μM at neutral pH compared with antibiotic-
free samples (SI Figure S27). With the decrease of SMX, the
inhibitory effects on Vibrio qinghaiensis decreased (dot−dash line
in Figure 5c and d). As for the samples at pH 6 and 9, the
luminescence of the samples after direct photolysis reduced by
around 40% compared with the initial luminescence. The
observed toxicity of the samples remained almost unchanged
with the degradation of SMX, suggesting that the toxicity of the
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Article
products alone was increasing because of the decrease of toxicity
from the remaining SMX (dot−dash line in Figure 5). At the last
time point at pH 6, the luminescence of the samples treated by
UV/H2O2 was back to the initial level, whereas the samples
treated by UV/PDS retained its toxicity. At pH 9, toxicity of
samples treated by UV/H2O2 and UV/PDS treatment increased
with the degradation of SMX. SO4•−-produced products
generated approximately 20% higher toxicity than that of •OH.
Reduced flavin mononucleotide (FMNH2) is necessary for
Vibrio qinghaiensis to luminesce.89 Hydroxylated compounds are
prone to combine with FMNH2 through hydrogen bond to block
the bonding between FMNH2 and luciferase (the most
significant catalyzer of Vibrio qinghaiensis for luminescing).90
Most products of TMP were hydroxylated compounds, which
resulted in higher acute toxicity of the treated samples. Moreover,
products by SO4•− were more abundant and accumulated in
solutions, whereas products by •OH were degraded in •OH-
dominant system, which was likely a reason toxicity of UV/PDS
treated samples was relatively higher. For SMX, the major
transformation products (e.g., SP 254 and SP 270) retained
−NH2 group. In addition, due to the cleavage of the sulfonamide
bond to form SP 99 and the rest moiety (probably aniline-3-
sulfonic acid), the number of the −NH2 group was elevated.
−NH2 group is known to interact with FMNH2, thus the
aminated products led to higher acute toxicity against Vibrio
qinghaiensis.
Although no growth inhibition toward Aeromonas was
observed, increasing acute toxicity of products with degradation
of parent compounds was observed for both TMP and SMX.
Therefore, single bioassay was not sufficient to comprehensively
evaluate the toxicity of the products.
Ecotoxicity. To estimate the impact of the parent
pharmaceuticals and transformation products on various species,
QSAR analysis was applied to predict the ecotoxicity by
ECOSAR program.
Multiple classes were identified for TMP based on specific
structure features when running ECOSAR. In previous research,
48-h Half Effective Concentration (EC50) value for D. magna91
and 96-h Half Lethal Concentration (LC50) value for O. latipes68
were reported. The class aniline (unhindered) had the closest
corresponding toxicity value and thus was selected for prediction.
Because of the structural similarity of TMP and its products, the
same class was selected for its products. The results are shown in
SI Table S5. Compounds showed different toxicity levels for
different species, in which, daphnid was the most sensitive
species for TMP and the products. For fish and green algae, LC50
values for most transformation products were higher than that
for TMP. However, for daphnid, LC50 values for most products
were half of the value for TMP, suggesting higher toxicity than
TMP (SI Table S5a). In terms of chronic toxicity, the difference
between species was reduced compared to the results of acute
toxicity. For daphnid and green algae, all the products exhibited
lower toxicity than TMP, whereas for fish, most products were
more toxic (SI Table S5b).
For SMX, the class aniline (unhindered) was also selected
based on its closest corresponding toxicity value with the
reported values.68,91 Unlike TMP, the acute and chronic toxicity
for three species showed the same trend for SMX and the
products (SI Table S6). Except for SP 99, all the other products
showed lower toxicity than SMX. Daphid was also the most
sensitive species.
Environmental Significance. Source-separated urine is a
complex matrix where different types of reactive species
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Environmental Science & Technology
interacted with pharmaceuticals simultaneously under UV/H2O2
and UV/PDS conditions. By elucidation of the transformation
products and mechanisms, this study demonstrated significant
product variations of TMP and SMX when they were attacked by
different reactive species. Especially, the transformation products
of pharmaceuticals by carbonate radical and RNS were
investigated for the first time, which provided more insight on
the radical chemistry in aqueous phase. The final products
detected in the synthetic urine after treatment by AOPs was able
to delineated by the simulation results of radical concentrations
and the transformation products generated by the dominant
radicals.
Toxicity evaluation showed that the UV/H2O2 and UV/PDS
processes were able to eliminate the antibacterial properties from
TMP and SMX on the functional bacteria in wastewater
treatment plants, which suggests AOP treatment lowers the
impact of source-separated urine on the performance of WWTP.
However, it is interesting to observe higher acute toxicity of
transformation products than their parent compounds. Notably,
although our previous study suggested that UV/PDS was more
favorable than UV/H2O2 for the removal of parent pharmaceut-
icals in source-separated urine,26 the toxicity results in this study
indicated higher acute toxicity of the transformation products
generated by UV/PDS. Therefore, it is suggested that a
comprehensive evaluation of both kinetics and toxic effect
should be considered when evaluating treatment processes for
degrading target pollutants.
■
ASSOCIATED CONTENT
*
S Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.est.5b05604.
Text S1−S4, Tables S1−S6, and Figures S1−S28 (PDF)
■ AUTHOR INFORMATION
Corresponding Authors
*Phone: 86-22-27401154; e-mail: zhaolin@tju.edu.cn.
*Phone: 1-404-358-4858; e-mail: sunpeizhe@gatech.edu.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
This work was supported by a project from National Natural
Science Foundation of China (21276182).
■
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