PDS Treatment of Trimethoprim and

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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 Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b05604 • Publication Date (Web): 03 Feb 2016 Downloaded from http://pubs.acs.org on February 13, 2016

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Environmental Science & Technology

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UV/H2O2 and UV/PDS Treatment of Trimethoprim and

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Sulfamethoxazole in Synthetic Human Urine:

3

Transformation Products and Toxicity

4

Ruochun Zhanga, Yongkui Yanga, Ching-Hua Huangb, Na Lic, Hang Liua, Lin Zhaoa,*, Peizhe Sunb,*

5 6 7 8

a

School of Environmental Science and Engineering, Tianjin University, Tianjin 300072, China b

Georgia 30332, United States

9 10 11

School of Civil and Environmental Engineering, Georgia Institute of Technology, Atlanta,

c

Tianjin Institute of Agriculture Quality Standards and Testing Technology, Tianjin 300381, China

12 13

* Corresponding Authors.

14

Phone: 86-22-27401154. E-mail: [email protected]

15

Phone:1-404-358-4858. E-mail: [email protected]

16 17

Manuscript submitted to

18


19 20

January 22, 2016

21

(Manuscript word account: 7453)

22 1

ACS Paragon Plus Environment


23

ABSTRACT

24

Elimination of pharmaceuticals in source-separated human urine is a promising approach to

25

minimize the pharmaceuticals in the environment. Although the degradation kinetics of

26

pharmaceuticals by UV/H2O2 and UV/peroxydisulfate (PDS) processes has been investigated in

27

synthetic fresh and hydrolyzed urine, comprehensive evaluation of the AOPs such as product

28

identification and toxicity test has not yet been performed. This study identified the

29

transformation products of two commonly used antibiotics, trimethoprim (TMP) and

30

sulfamethoxazole (SMX), by UV/H2O2 and UV/PDS in synthetic urine matrix. The effects of

31

reactive species, including ·OH, SO4·-, CO3·-, and reactive nitrogen species, on product generation

32

were investigated. Multiple isomeric transformation products of TMP and SMX were observed,

33

especially in the reaction with hydroxyl radical. SO4·- and CO3·- reacted with pharmaceuticals by

34

electron transfer, thus produced similar major products. The main reactive species deduced on

35

the basis of product generation are in a good agreement with kinetic simulation of the advanced

36

oxidation processes. A strain identified as a poly-phosphate accumulating organism was used to

37

investigate the antimicrobial activity of the pharmaceuticals and their products. No antimicrobial

38

property was detected for the transformation products of either TMP or SMX. Acute toxicity

39

employing luminescent bacterium Vibrio Qinghaiensis indicated 20% - 40% higher inhibitory

40

effect of TMP and SMX after treatment. Ecotoxicity was estimated by quantitative

41

structure-active relationship analysis using ECOSAR.

42

2


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INTRODUCTION

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Pharmaceutical micropollutants in the environment have been regarded as a major threat to the

45

ecosystem due to their adverse biological effect and the potential of inducing drug-resistant

46

bacteria. Because pharmaceuticals discharged in wastewater cannot be effectively removed by

47

traditional wastewater treatment technologies, this has resulted in widespread occurrence of

48

pharmaceuticals in the aquatic environment worldwide.1-3 Among all the wastewater streams

49

entering into wastewater treatment plants (WWTPs), the treatment of source-separated urine has

50

been viewed as a promising approach to minimize the harm of the pharmaceuticals, because

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urine not only contributes significant portion of the pharmaceuticals in municipal WWTPs,4,5 but

52

also carries high toxic potential to aquatic organisms.6-8 Moreover, recovering nutrients from

53

urine, as a new resource recovery strategy,9 also requires elimination of pharmaceuticals from

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urine. To date, nanofiltration membranes,10 strong-base anion exchange polymer resins,11

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electrodialysis12 and struvite precipitation13 have been investigated for removing pharmaceuticals

56

from urine, all of which involved only physical separation of the pharmaceuticals and further

57

treatment is still needed. Ozone has been investigated for destruction of pharmaceuticals in urine,

58

but high doses of ozone (150 mg·L-1 or higher) were needed to reach satisfactory reduction.14

59

Therefore, more effective processes are needed to fully destruct the pharmaceuticals in urine.

60

Advanced oxidation processes (AOPs) are among the most effective and promising processes in

61

eliminating organic pollutants. Radical-based AOPs, in particular with hydroxyl radical

62

(·OH)15-17 and sulfate radical (SO4·-),18-22 have been successfully applied to destruct

63

micropollutants in wastewater, drinking water, ground water and sediment-slurry system. ·OH 3



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(E° (·OH/H2O) = 1.9−2.7 V)23 is of low selectivity and can react with many pharmaceuticals

65

rapidly (k = 108−1010 M-1⋅s-1).24 SO4·- is also a strong oxidant (E° (SO4·-/SO42-) = 2.5−3.1 V)25

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and react with pharmaceuticals mainly through electron transfer. In urine matrices, which are

67

more complicated and uniquely challenging compared to typical municipal wastewaters and

68

surface waters because of the high concentrations of the organic components and inorganic salts,

69

the efficacy of

70

pharmaceuticals and metabolites was evaluated in our previous research.26 Sulfamethoxazole

71

(SMX), acetyl-sulfamethoxazole (acetyl-SMX) (a metabolite of SMX), and trimethoprim (TMP)

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were studied due to their high frequency of detection in the environment. Systematic kinetic

73

studies have elucidated the direct photolysis and indirect photolysis of the above pharmaceuticals

74

and metabolites’ degradation rates. In addition to ·OH and SO4·-, carbonate radical (CO3·-) and

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reactive nitrogen species (RNS) also showed reactivity towards the pharmaceuticals and acted as

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the main reactive species under particular conditions. CO3·- is electrophilic and also sufficient for

77

degrading pharmaceuticals (Eº (CO3·-/CO32-) = 1.63 V at pH 8.4).27 Reactivity of CO3·- towards

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pharmaceuticals has been reported but still in its early stage.28,29 RNS, such as amino radical,

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nitrogen dioxide radical, nitric oxide radical and peroxynitrite etc., are weaker oxidants with

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relative high selectivity.26 However, kinetics studies alone were not sufficient to evaluate the

81

performance AOP processes.

UV/H2O2 and

UV/peroxydisulfate (PDS) processes

in

eliminating

82

While AOPs are powerful oxidation processes, often, they cannot completely mineralize

83

pollutants within the typical operation time and a number of products are formed and may persist

84

in the system.30 The product formation may vary with different treatment processes, oxidants and 4


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background constituents involved, and reaction mechanisms.31-34 Product identification can

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provide crucial clues for reaction pathways.35,36 For TMP and SMX, products generated by

87

chlorination,37-40 ozonation,30,41,42 photochemical reactions,43-45 photocatalytic treatment,46-48

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electrochemical process49 and in other oxidation systems50,51 were identified and utilized to

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propose corresponding reaction pathways. However, to our knowledge, products of SMX by

90

UV/PDS and TMP by UV/H2O2 and UV/PDS have not been reported. Information is also scarce

91

regarding oxidation products of pharmaceuticals by CO3·- and RNS. Product analysis coupled

92

with measurement such as total organic carbon (TOC) can be complementary to assess the

93

overall efficiency of the treatment processes.

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In recent years, evaluation of toxicological effect has been recommended52 and applied to

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investigate the biological impact of pharmaceuticals in various environmental matrices.53-57

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Transformation products may retain the properties of the parent compounds or potentially

97

become more biologically active. Therefore, toxicity tests of the products have been increasingly

98

applied as part of the evaluation of the performance of treatment processes.58,59 At least two or

99

multiple bioassays are commonly needed to reveal the overall toxic potential.60,61 Cytotoxicity,62

100

genotoxicity,63 and estrogenicity64 have been applied to test the toxic effect of the

101

pharmaceuticals and transformation products.58 Luminescent bacteria, particularly Vibrio

102

fischeri,65-67 are frequently employed to assess acute toxicity. Crustaceans, algae,41 fish,68 and

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zebrafish embryos69 are also widely used as toxicity indicators. Vibrio Qinghaiensis, a fresh

104

water luminescent bacterium, is now gaining increasing popularity.65,70 As for antibiotics, the

105

antibacterial property of the compounds is commonly monitored using bacterial strains of E. 5



106

coli71,72 and Bacillus subtilis,73,74 while few studies have applied functional bacteria as indicators.

107

Biodegradability of pharmaceuticals and their transformation products in activated sludge have

108

been investigated and samples after AOP treatments are normally more biocompatible.75-77

109

The objective of this study was to better understand the UV/H2O2 and UV/PDS processes in

110

eliminating pharmaceuticals in synthetic source-separated urine solutions with emphasis on the

111

product identification and toxicity evaluation. TMP and SMX were selected because they are

112

widely used antibiotics and have been shown high reactivity toward different reactive species in

113

AOPs in our previous study.26 In the present study, products generated by specific radicals were

114

identified and degradation of TMP and SMX by mixed reactive species was illustrated in

115

synthetic urine solutions. Toxic effects of the parent compounds and the transformation products

116

were evaluated by testing their antimicrobial property and bioluminescence inhibition.

117

Quantitative structure-active relationship (QSAR) analysis was also applied to estimate the

118

eco-toxicity. The major goal was to achieve comprehensive evaluation of AOPs treatment of

119

pharmaceuticals in urine matrices, which can help develop better urine treatment strategies. The

120

research on reacting mechanism by different reactive species and corresponding toxic effects

121

would improve the understanding of AOPs performances.

122 123

MATERIALS AND METHODS

124

Materials. Sources of chemicals and materials are provided in the Supporting Information (SI)

125

Text S1.

126

Experimental setup. UV, UV/H2O2 and UV/PDS experiments were conducted in a reactor with 6


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the same set-up as in the previous research.26 Potassium ferrioxalate was employed as chemical

128

actinometer78 and UV fluence rate was measured at 1.78 × 10−6 Einstein·L-1·s-1 in this study.

129

Reaction solutions were prepared with 100 µM of TMP or SMX in 100 mL fresh urine,

130

hydrolyzed urine or phosphate buffer (PB) solution at pH 6 or 9. The synthetic human urine

131

composition was adapted from the previous recipe (SI Table S1).26 The high initial concentration

132

of TMP and SMX was chosen in order to explore transformation products. H2O2 and potassium

133

PDS were added at 3 mM in PB solutions to generate ·OH- and SO4·--dominant systems,

134

respectively. To create CO3·-- and RNS-dominant systems, 0.5 M sodium bicarbonate and 1 M

135

ammonia solution were added into PB solution at pH 9, respectively, in UV/H2O2 system.

136

Simulation of radical concentrations under different experimental conditions was conducted by

137

Gepasi 3.0. The rate constants for reactions considered in the simulation were obtained from our

138

previous publication, which considered all relevant reactions under AOP conditions. This model

139

has been successfully employed in a number of studies and proved to be reliable to predict

140

radical concentrations.26,79 Total simulation time was set as 300 s because most reactive species

141

were close to the pseudo-steady state. Small amounts of concentrated NaOH and perchloric acid

142

were added to adjust the pH when needed.

143

Analytical methods. A Waters AcQuity UPLC system equipped with a PDA detector and a BEH

144

C18 column (2.1 × 50 mm, 1.7 µM) was used to monitor the loss of the parent compounds. The

145

UPLC system was connected to a TOF mass spectrometer (Premier, Micromass, UK) with

146

electrospray interface to analyze transformation products. Structure identification was achieved

147

based on the fragmentation pattern. The elemental composition was deduced from the exact m/z 7



148

values obtained from ESI-TOF-MS system (micrOTOF-Q II, Bruker, Germany). The detailed

149

chromatographic and MS conditions are summarized in SI Text S2.

150

Toxicity analysis. The antimicrobial property of TMP and SMX and their transformation

151

products was tested using a poly-phosphorus accumulating bacterium identified as Aeromonas.

152

Optical density (absorbance at 600 nm) was used as an indication of bacterial growth. The acute

153

toxicity assay was carried out against freshwater luminescent bacterium Vibrio Qinghaiensis.

154

Because ammonia and bicarbonate show high toxicity to Vibrio Qinghaiensis, the acute toxicity

155

of products by CO3·- and RNS were not tested. QSAR analysis calculated by the Ecological

156

Structure-Activity Relationship Model (ECOSAR) program30 was also employed to estimate the

157

acute and chronic toxicity for fish, daphnid and green algae. Detail information for toxicity

158

analysis is described in SI text S3.

159 160

RESULTS AND DISCUSSION

161

Contribution of reactive species. With UV/H2O2 and UV/PDS processes in synthetic urine

162

solution, ·OH, SO4·-, CO3·- and RNS were major reactive species to degrade TMP and SMX.26 In

163

order to investigate the products generated by each reactive species, experimental conditions

164

where certain reactive species dominated were designed. Based on the simulation results, the

165

concentrations of above-mentioned reactive species under UV/H2O2 and UV/PDS conditions

166

with different components in solutions are present in SI Table S2. The dominant reactive species

167

generated by UV/H2O2 and UV/PDS in PB solution are ·OH and SO4·-, respectively, for both pH

168

6 and pH 9. Although ·OH can react with SO4·- and generate ·OH so that ·OH concentration 8


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would increase when pH was higher, it was still not sufficient (1.21×10-15 M) for an observable

170

degradation for TMP. More than 99% of TMP was degraded by SO4·-. By adding excess sodium

171

bicarbonate in UV/H2O2 system, the CO3·- concentration was 4 orders of magnitude higher than

172

that of ·OH. Based on the second order rate constants of TMP and SMX with ·OH and CO3·-,26

173

more than 90% of TMP and SMX was destructed by CO3·- in indirect photolysis. Because the

174

second order rate constants of pharmaceuticals with RNS were not available, RNS contribution

175

was not possible to evaluate quantitatively. But according to the simulation result shown in SI

176

Table S2, when excess ammonia solution was added in UV/H2O2 system, the ·OH concentration

177

was 3.72 × 10-15 M which was insufficient to yield an observable degradation of TMP and SMX.

178

Meanwhile, concentrations of several RNS were relatively high thus they may play a role in

179

degrading pharmaceuticals with higher reactivity. Therefore, RNS was assumed to be the

180

dominant reactive species under this condition.

181 182

Identification of transformation products of TMP. Based on the previous results,26 direct

183

photolysis of TMP under low pressure UV irradiation was negligible. Under AOP conditions,

184

TMP was mainly destructed by ·OH, SO4·-, and CO3·-. Therefore, products produced by these

185

radicals were identified. Degradations of TMP by different radicals are shown in Figure 1(a). The

186

accurate m/z values obtained by the ESI-TOF-MS system are shown in SI Table S3. On the basis

187

of accurate mass, empirical chemical formulas of each transformation product were proposed (SI

188

Table S2). In all cases, no cleavage of the methylene group or aromatic rings was observed

189

(Figure 2). It suggested inadequate mineralization of oxidation processes. Indeed, the decrease of 9



190

TOC was negligible under all tested conditions within corresponding reaction time (data not

191

shown). Because standards of the transformation products are not commercially available,

192

accurate quantification of each product is impossible to achieve. Based on the structural

193

similarity of the parent compound and the transformation products, the MS signal responses were

194

assumed to be similar. Therefore, evaluation of the product abundance was on the basis of peak

195

area (shown in SI).

196

Transformation products of TMP by hydroxyl radical. The degradation of TMP by hydroxyl

197

radical (i.e. under UV/H2O2 condition, SI Table S2) produced 13 main products at pH 6 in PB

198

solution (Figure 2). Hydroxylation was the most prominent mechanism, accompanied with

199

demethylation and carbonylation. TP 307-3 (m/z 307, C14H19N4O4) was the most abundant

200

product based on the peak area (Figure 2, SI Figure S2 (a)). The addition of 16 Da to the

201

molecular weight of the parent compound suggested a transformation pathway of hydroxylation.

202

The position of the hydroxyl group was proposed (Figure 2) due to the presence of the fragment

203

ions m/z 277, m/z 259, and m/z 123 (SI Figure S3), which were in accordance with the

204

fragmentation pattern reported by Sirtori et al.80 TP 307-3 was likely produced by direct ·OH

205

addition to the benzene moiety.81 Two more m/z 307 products, TP 307-1 and TP 307-2, were also

206

detected at much lower concentrations. TP 307-1 was a major product by SO4·- and will be

207

discussed in a later section.

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208

TP 325 (m/z 325, C14H21N4O5) was another major product with similar peak areas as TP 307-3

209

(Figure 2, SI Figure S2 (a)). The fragment ions m/z 221 and m/z 143 suggested that one oxygen

210

atom was added to C8 atom as a hydroxyl group and one oxygen atom was added to C13 atom 10


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211

(SI Figure S4). Previous research with ozone and nitrifying activated sludge processes proposed

212

addition of carbonyl group on C13 atom with hydrogenation of C9-C10 double bond.82,83 In

213

contrast, based on our MS2 spectrum of TP 325, there was no strong evidence to confirm that the

214

oxygen was added as a carbonyl group. In addition, in a strong oxidation process, hydrogenation

215

of a double bond was difficult to achieve. Therefore, instead of forming a carbonyl group, the

216

oxygen was more likely added as a hydroxyl group. The same hypothesis also applies to TP 341

217

(m/z 341, C14H21N4O6), with a hydroxyl group addition to TP 325 on the benzene ring (implied

218

by the fragment ion m/z 197 instead of m/z 181, SI Figure S5). Further study is still needed for

219

structural confirmation.

220

Proposed structures of other products are shown in Figure 2. Structural identification is

221

discussed in SI Text S4. Overall, all the products by ‧OH were not accumulated in the solution

222

and likely degradable by ‧OH (SI Figure S2 (a)).

223

At pH 9, TP 307-3 (m/z 307, C14H19N4O4) was also the major product in the PB solution

224

whereas the amount of TP325 (m/z 325, C14H21N4O5) was less than that at pH 6 (SI Figure S2

225

(b)). In addition, all the m/z 323 products were not observed at pH 9. Overall, most products of

226

TMP at pH 9 were of lower relative abundance and generated via hydroxylation with addition of

227

a single hydroxyl group. The difference in product speciation at pH 6 and pH 9 was likely due to

228

the reactivity of the products of TMP. It was expected that products of TMP reacted faster with

229

hydroxyl radical at higher pH due to two possible reasons. First, as reported in our previous

230

study,26 the second-order rate constant of TMP with hydroxyl radical was higher at pH 9 than

231

that at pH 6. Because the majority of products were resulted from slight modification of parent 11



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232

TMP, it was expected the product also has the same trend. Second, hydroxylated TMPs (such as

233

TP 323) were likely comprised of secondary –OH moiety, which is known to degrade faster

234

under AOP condition at higher pH (SI Figure S9).84

235

Transformation products of TMP by sulfate radical. The degradation of TMP by sulfate radical

236

(i.e. under UV/PDS condition, SI Table S2) produced 6 main products at pH 6 in PB solution

237

(Figure 2). TP 307-1 (m/z 307, C14H19N4O4) (Figure 2, SI Figure S7 (a)) was a major product,

238

which was an isomer of the most prominent product produced by ·OH (TP 307-3). The fragment

239

ions, m/z 289 and m/z 274, implied that a hydroxyl group was added to the bridge methylene

240

group (SI Figure S8). The same MS2 spectrum was also found in previous studies.30,80,83 TP 305

241

(m/z 305, C14H17N4O4) was another major product by SO4·- (Figure 2, SI Figure S7 (a)). It was

242

previously identified as a product under solar-Fenton46 and TiO2 photocatalysis80 conditions.

243

Both TP 307-1 and TP 305 were likely generated via electron transfer mechanism (SI Figure S9).

244

The aromatic moieties (i.e. diaminopyrimidine or benzene) on TMP lose one electron to SO4‧-

245

forming a radical intermediate with a positive charge. The non-paired electron is then stabilized

246

on C7 atom, forming a carbon-center radical, which is known to transform to superoxide with the

247

presence of dissolved oxygen. Through bimolecular interaction, the superoxide intermediate

248

transforms to a hydroxyl moiety yielding TP 307-1 and a carbonyl moiety yielding TP 305. TP

249

307-1 and TP 305 were also found in the ⋅OH-dominant system, but at much lower abundance.

250

TP 323-1,-2,-4 and TP 325 were also generated by the reaction with SO4·- whereas other

251

products found in the ·OH-dominant system (i.e., TP 277-1, TP 277-2, TP 307-2, TP 307-3, TP

252

295, and TP 341) were not observed. These differences implied different reaction pathways of 12


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253

SO4·- and ·OH. Unlike the ·OH products, the products produced by SO4·- did not degrade

254

throughout the reaction, indicating they may be persistent in UV/PDS process (SI Figure S7 (a)).

255

In the UV/PDS process at pH 9 instead of pH 6, the m/z 323 products were hardly detected,

256

similar to that observed in UV/H2O2 process at the two different pHs. TP 305 overweighed TP

257

307-1 in abundance and became the dominant product at pH 9 (SI Figure S7 (b)). As shown in SI

258

Figure S9, TP 307-1 further reacted with sulfate radical and transformed to TP 305. Although

259

such transformation was slow at pH 6, this reaction was accelerated at pH 9 because the

260

elimination of superoxide radical was catalyzed by basic conditions.84

261

Transformation products of TMP by carbonate radical. The degradation of TMP by carbonate

262

radical (i.e. under UV/H2O2/NaHCO3 condition, SI Table S2) produced 4 main products at pH 9

263

in PB solution (Figure 2). Conditions at pH 6 were not considered because carbonate radical only

264

dominated in hydrolyzed urine (pH 9).26 TP 305 (m/z 305, C14H17N4O4) was also a dominant and

265

stable product in the CO3·--dominant system (Figure 2, SI Figure S11), as by SO4·-, suggesting

266

similar electron transfer mechanism of these two radicals (SI Figure S9). TP 307-1 (m/z 307,

267

C14H19N4O4) was found in CO3·--dominant system but not as significant as it was in

268

SO4·--dominant system (at pH 9). This difference can be explained by additional pathway which

269

produced TP 305. As shown in SI Figure S9, besides the same mechanism as SO4·-, CO3· can

270

transfer one of its oxygen to the carbon-center radical forming a carbonyl moiety (i.e. TP 305).85

271

Two new products (TP 277-3 and TP 291) were generated exclusively by CO3·-. TP 277-3 (m/z

272

277, C13H17N4O3) was an isomer of TP 277-1 and TP 277-2 which were produced by ·OH. The

273

retention time of TP 277-3 on UPLC was close to TMP (SI Table S3), suggesting a similar 13



274

structure to the parent compound, which is also supported by the remarkable resemblance

275

between the MS2 fragmentation patterns of TMP and TP 277-3 (SI Figure S12). The fragment

276

ions m/z 261 and m/z 247 corresponded to the loss of a methoxyl group on C2 or C4 atom. The

277

lack of fragment ion m/z 123 suggested the hydroxyl group was added to the diaminopyrimidine

278

ring or the methylene group. An isomer of TMP, TP 291 (m/z 291, C14H19N4O3), was observed

279

and the retention time was longer than all the products and parent TMP. The different

280

fragmentation pattern of TP 291 and TMP indicated a large difference between the structures, but

281

current information was not sufficient to identify the structure of TP 291.

282

Transformation products of TMP in synthetic urine solution. After gathering the information of

283

transformation products of TMP by individual radicals, product identification was performed in

284

synthetic urine solution. To identify products in urine matrices, in hydrolyzed urine, the reaction

285

time was set the same as the experiment identifying products for CO3·- which was regarded as

286

the dominant radical. In fresh urine, reaction time was set longer than in buffer solution (pH = 6)

287

to reach more than 50% TMP removal, since the degradation was inhibited to a large extent.

288

Samples were taken at each time interval and a spectrum that can show all the dominant products

289

were shown in SI Figure S13-S16 for each case.

Page 14 of 42

290

In terms of product identification, in fresh urine with UV/H2O2 process, the presence of TP

291

277-2 and TP 307-3 confirmed that the oxidation by ·OH played an important role (SI Figure

292

S13). With UV/PDS process, the main product of SO4·- TP 307-1 stayed as the most abundant

293

product, indicating the contribution of SO4·- to TMP degradation (SI Figure S14). In hydrolyzed

294

urine, by UV/H2O2, the presence of TP 307-1, TP 305 and TP 277-3 proved the degradation role 14


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295

of CO3·- (SI Figure S15). With UV/PDS process, the abundance of TP 307-1 implies that besides

296

CO3·-, other reactive species, such as SO4·- and RNS, may also play a role (SI Figure S16).

297

Indeed, when CO3·- was the dominant reactive species in the system, TP 305 was more abundant

298

than TP 307-1. The major reactive species deduced from the product presence in the present

299

study are in good agreement with the simulation results of corresponding contributing reactive

300

species in synthetic urine solution.26

301 302

Identification of transformation products of SMX. Degradation of SMX is profiled in Figure

303

1(b) and accurate m/z values of transformation products are shown in SI Table S4. Because of

304

the high direct photolysis rate of SMX, the majority of SMX was degraded via direct photolysis

305

in the UV/H2O2 and UV/PDS processes. As a result, the products of indirect photolysis of SMX

306

were not as prominent as the case of TMP. On the basis of both experimental and simulation

307

results,26 SMX was of higher reactivity towards radical species than TMP and can be degraded

308

by ·OH, SO4·-, CO3·- and RNS. Products of SMX were analyzed respectively for each reactive

309

species. The corresponding radical-dominant systems were generated under the same conditions

310

as for TMP product analysis. Direct photolysis products were generated in PB solutions.

311

Transformation products by direct photolysis. By direct photolysis, an isomer of SMX, SP 254

312

(m/z 254, C10H11N3O3S) was produced as a major product (Figure 3, SI Figure S17). It was

313

produced

314

3-amino-5-methylisoxazole (Figure 3),41,45 which was produced due to the cleavage of the

315

sulfonamide bond. It was observed under every tested condition. An isomer of SP 99-1 (SP 99-2,

due

to

photoisomerization.45

SP 99-1

(m/z

15


99,

C4H7N2O)

was

likely


Page 16 of 42

316

m/z 99, C4H7N2O) was detected as a product only by direct photolysis at pH 9 (Figure 3, SI

317

Figure S17 (b)), which was probably due to the cleavage of the sulfonamide bond of SP 254.

318

Transformation products of SMX by hydroxyl radical. In addition to the direct photolysis

319

products, two products with m/z 270 (SP 270, SP 270-2, C10H11N3O4S) (Figure 3, SI Figure S18)

320

were produced in ·OH-dominant system (i.e. under UV/H2O2 conditions). The 16 Da higher

321

molecular weight suggested hydroxylation occurred on the parent compound. The fragment ion

322

m/z 172, compared with the fragment ion m/z 156 of SMX, indicated that a ·OH was added to

323

the benzene ring (SI Figure S19). A product of SMX with m/z 270 was also observed in TiO2

324

photocatalysis system.47 SP 262 was found exclusively in the ·OH-dominant system. The

325

concentration profile of SP 262 was consistent with that of SP 99-1 (SI Figure S18), suggesting a

326

correlation between the two products. Considering SP 99-1 represented the isoxazole ring of

327

SMX, the other moiety of SMX after cleavage of the sulfonamide bond might recombine with

328

small fragments and generate SP 262. However, the speculation needs further evidence to be

329

confirmed. All the products degraded more rapidly at pH 9 than at pH 6, especially for SP 99-1

330

and SP 262, which accumulated in the solution at pH 6 (SI Figure S18).

331

Transformation products of SMX by sulfate radical. In UV/PDS process in PB solution

332

(SO4·--dominant system), no additional product was found except for the direct photolysis

333

products. However, at pH 6, the abundance of SP 254 decreased dramatically compared with that

334

by direct photolysis (SI Figure S20 (a)). At pH 9, no isomer product was observed (SI Figure S20

335

(b)). Our preliminary results showed that PDS did not react with SP 254, suggesting SP 254 or

336

some intermediates essential for forming SP 254 may be degraded by SO4·-. 16


Page 17 of 42


337

Transformation products of SMX by carbonate radical and RNS. In CO3·--dominant system

338

(UV/H2O2/NaHCO3), there was no product found other than the direct photolysis products. As by

339

SO4·-, the production of SP 254 was also suppressed (SI Figure S21), suggesting the presence of

340

PDS was not the reason for the decrease of SP 254 production. Considering the similarity of the

341

reacting mechanism of SO4·- and CO3·-, SP 254 or some intermediates essential for forming SP

342

254 might be prone to be destructed by electron transfer mechanism.

343

Based on the kinetic simulation (SI Table S2), the oxidation by ·OH can be neglected when 1

344

M ammonia was added into the solution with UV/H2O2 process. Therefore, in addition to the

345

direct photolysis product, SP 270-2 was also observed and speculated to be a product by reaction

346

with RNS (SI Figure S22). As peroxynitrite was identified as the major RNS in hydrolyzed urine

347

under AOP conditions, formation of hydroxylation products of SMX was likely partly due to the

348

reaction with peroxynitrite. Indeed, oxidation of phenolic compound by peroxynitrite was

349

reported through one-electron oxidiation processes that do not involve free hydroxyl radicals,

350

which yielded hydroxylated phenolic products.86

351

Transformation products of SMX in synthetic urine solution. In urine matrices, the reaction time

352

was set the same as the experiment identifying products for the corresponding main reactive

353

species and a spectrum that can show all the dominant products were shown in SI Figure

354

S23-S26. In fresh urine with UV/H2O2, SP 270-2 was observed, indicating ·OH was contributing

355

(SI Figure S23). SP 99 and SP 254 were also found, which was consistent with the product

356

identification result under UV/H2O2 condition in buffer solution. With UV/PDS, SP 99-1 was the

357

only observed product, which is similar to the case in PB solution at pH 6 (SI Figure S24). In 17



358

terms of hydrolyzed urine, SP 254 was hardly detected, which was expected because SO4·- and

359

CO3·- were the major reactive species in these systems (SI Figure S25, S26).

Page 18 of 42

360 361

Toxicity evaluation. Growth inhibition of TMP and SMX and the transformation products on a

362

target strain was tested to investigate the change of antimicrobial activity during degradation.

363

Bioluminescence inhibition was monitored for acute toxicity. To comprehensively evaluate the

364

toxicity, QSAR analysis was also performed to predict the eco-toxicity of individual

365

transformation products.

366

Antimicrobial property. Aeromonas has been accepted as a Phosphorus Accumulating Organism

367

(PAO) and the strain used in this study has been proved to remove phosphorus satisfactorily. It

368

was employed as a representative of the microorganism in the biological wastewater treatment

369

system to test the antimicrobial property of the parent compounds and transformation products,

370

so that the toxic potential of the transformation products on wastewater treatment system can be

371

delineated.

372 373

374

The antimicrobial property of the parent compounds was measured. The inhibition was calculated from the following equation:

 OD600 ( Sample )  Inhibition(%) = 1 −  × 100  OD600 ( Control ) 

375

where OD600 (Sample) was the absorbance at 600 nm of the sample aliquot taken at each selected

376

time interval from a direct photolysis or AOP reaction, OD600 (Control) was the absorbance of DI

377

water replacing the sample. 18


Page 19 of 42


378

Because standards of transformation products are not commercially available, it is difficult to

379

test the toxicity of the products separately. However, it is beneficial to test the inhibition effect of

380

a mixture of transformation products and remaining parent compounds because they practically

381

co-exist in the AOP systems and may exert toxicity jointly. Concentrations of the parent

382

compounds were measured by UPLC and corresponding inhibition ratios were calculated from

383

the reference curve (SI Figure S1). Therefore, the observed inhibition of the sample (dots in

384

Figure 4) subtracted by the inhibition by the parent compounds (lines in Figure 4) represents the

385

inhibition by the products.

386

To be noted, because the presence of bicarbonate could promote the bacteria growth, the

387

inhibitions by remaining parent compounds in CO3·- samples were shown separately (dashed

388

lines in Figure 4). For almost all the data points, the observed inhibition equaled to or slightly

389

higher than the inhibition of the retaining parent compound calculated by the reference curve.

390

This indicated that toward the tested strain, negligible antimicrobial property was retained for the

391

transformation products throughout the reaction.

392

TMP interferes normal bacterial metabolism pathway by binding to dihydrofolate reductase

393

and SMX acts by inhibiting bacterial utilization of para-aminobenzoic acid (PABA) because of

394

their structural similarity with dihydrofolic acid and PABA, respectively.87 After treatment, the

395

parent compounds were hydroxylated, carbonylated, demethylated, isomerized or broke down.

396

The structural modified products may not able to bind to the receptors as the parent compounds

397

did thus PABA and DHF were utilized without inhibition. The structural transformation of parent

398

TMP and SMX by AOPs is likely to disable the competitive antagonism therefore deprive the 19



399

products of antibiotic properties.

400

Acute toxicity. Microtox test was the most frequently applied method to measure the acute

401

toxicity of toxic substances in environmental studies.88 When the target microorganism contacts

402

with toxic substances, its bioluminescence decreases due to disruption of normal metabolism.

403

Although marine bacterium Vibrio Fischeri was the most commonly used indicator of toxicity, it

404

is not suitable in fresh water studies. Therefore, freshwater luminescent bacterium Vibrio

405

Qinghaiensis was selected in this study.

Page 20 of 42

406

Figure 5 shows the acute toxicity of the parent compounds and the products. The y-axis, L/L0,

407

reflected the comparison of the luminescence of the sample with the initial luminescence (i.e.,

408

before treatment at t=0). Data point above the dash line (standing for no change in the

409

luminescence) indicated that the luminescence increased after treatment, which suggested a

410

decline in toxicity. Likewise, data point below the dash line indicated lower luminescence and

411

higher toxicity. Dot-dash lines in Figure 5 represent the L/L0 of the samples only containing

412

different amount of parent pharmaceuticals.

413

Compared with antibiotic-free samples, TMP, at lower than 100 µM concentration, exhibited

414

almost no inhibition to the Vibrio Qinghaiensis (SI Figure S27). However, at both pH 6 and pH 9,

415

the luminescence of the samples decreased to approximately 80% of the initial luminescence,

416

indicating slight toxicity of the products by ·OH. For products by SO4·-, a distinct inhibition was

417

observed because 40% of the luminescence was suppressed, indicating higher toxicity.

418

SMX inhibited approximate 25% luminescence of Vibrio Qinghaiensis at 100 µM at neutral

419

pH compared with antibiotic-free samples (SI Figure S27). With the decrease of SMX, the 20


Page 21 of 42


420

inhibitory effects on Vibrio Qinghaiensis decreased (dot-dash line in Figure 5 (c), (d)). As for the

421

samples at pH 6 and 9, the luminescence of the samples after direct photolysis reduced by around

422

40% compared with the initial luminescence. The observed toxicity of the samples remained

423

almost unchanged with the degradation of SMX, suggesting that the toxicity of the products

424

alone was increasing because of the decrease of toxicity from the remaining SMX (dot-dash line

425

in Figure 5). At the last time point at pH 6, the luminescence of the samples treated by UV/H2O2

426

was back to the initial level whereas the samples treated by UV/PDS retained its toxicity. At pH

427

9, toxicity of samples treated by UV/H2O2 and UV/PDS treatment increased with the degradation

428

of SMX. SO4·--produced products generated approximately 20% higher toxicity than that

429

of ·OH.

430

Reduced flavin mononucleotide (FMNH2) is necessary for Vibrio Qinghaiensis to luminesce.89

431

Hydroxylated compounds are prone to combine with FMNH2 through hydrogen bond to block

432

the bonding between FMNH2 and luciferase (the most significant catalyzer of Vibrio

433

Qinghaiensis for luminescing).90 Most products of TMP were hydroxylated compounds, which

434

resulted in higher acute toxicity of the treated samples. Moreover, products by SO4·- were more

435

abundant and accumulated in solutions whereas products by ·OH were degraded

436

in ·OH-dominant system, which was likely a reason for why toxicity of UV/PDS treated samples

437

was relatively higher. For SMX, the major transformation products (eg. SP 254 and SP 270)

438

retained −NH2 group. In addition, due to the cleavage of the sulfonamide bond to form SP 99 and

439

the rest moiety (probably aniline-3-sulfonic acid), the number of the −NH2 group was elevated.

440

−NH2 group is known to interact with FMNH2, thus the aminated products led to higher acute 21



441

Page 22 of 42

toxicity against Vibrio Qinghaiensis.

442

Although no growth inhibition toward Aeromonas was observed, increasing acute toxicity of

443

products with degradation of parent compounds was observed for both TMP and SMX.

444

Therefore, single bioassay was not sufficient to comprehensively evaluate the toxicity of the

445

products.

446

Eco-toxicity. To estimate the impact of the parent pharmaceuticals and transformation products

447

on various species, QSAR analysis was applied to predict the eco-toxicity by ECOSAR program.

448

Multiple classes were identified for TMP based on specific structure features when running

449

ECOSAR. In previous research, 48-h Half Effective Concentration (EC50) value for D.magna91

450

and 96-h Half Lethal Concentration (LC50) value for O.latipes68 were reported. The class aniline

451

(unhindered) was with the closest corresponding toxicity value thus selected for prediction. Due

452

to the structural similarity of TMP and its products, the same class was selected for its products.

453

The results are shown in SI Table S5. Compounds showed different toxicity levels for different

454

species, in which, daphnid was the most sensitive species for TMP and the products. For fish and

455

green algae, LC50 values for most transformation products were higher than that for TMP.

456

However, for daphnid, LC50 values for most products were half of the value for TMP, suggesting

457

higher toxicity than TMP (SI Table S5 (a)). In terms of chronic toxicity, the difference between

458

species was reduced compared to the results of acute toxicity. For daphnid and green algae, all

459

the products exhibited lower toxicity than TMP whereas for fish, most products were more toxic

460

(SI Table S5 (b)).

461

For SMX, the class aniline (unhindered) was also selected based on the closest corresponding 22


Page 23 of 42


462

toxicity value with the reported values.68,91 Unlike TMP, the acute and chronic toxicity for three

463

species showed the same trend for SMX and the products (SI Table S6). Except for SP 99, all the

464

other products showed lower toxicity than SMX. Daphid was also the most sensitive species.

465 466

Environmental Significance. Source-separated urine is a complex matrix where different types

467

of reactive species interacted with pharmaceuticals simultaneously under UV/H2O2 and UV/PDS

468

conditions. By elucidation of the transformation products and mechanisms, this study

469

demonstrated significant product variations of TMP and SMX when they were attacked by

470

different reactive species. Especially, the transformation products of pharmaceuticals by

471

carbonate radical and RNS were investigated for the first time, which provided more insight on

472

the radical chemistry in aqueous phase. The final products detected in the synthetic urine after

473

treated by AOPs was able to delineated by the simulation results of radical concentrations and

474

the transformation products generated by the dominant radicals.

475

Toxicity evaluation showed that the UV/H2O2 and UV/PDS processes were able to eliminate

476

the antibacterial properties from TMP and SMX on the functional bacteria in wastewater

477

treatment plants, which suggests AOP treatment lowers the impact of source separated urine on

478

the performance of WWTP. However, it is interesting to observe higher acute toxicity of

479

transformation products than their parent compounds. Notably, although our previous study

480

suggested that UV/PDS was more favorable than UV/H2O2 for the removal of parent

481

pharmaceuticals in source-separated urine,26 the toxicity results in this study indicated higher

482

acute toxicity of the transformation products generated by UV/PDS. Therefore, it is suggested 23



483

that a comprehensive evaluation of both kinetics and toxic effect should be considered when

484

evaluating treatment processes for degrading target pollutants.

Page 24 of 42

485 486

ASSOCIATED CONTENT

487

Supporting Information. Text S1−S4, Tables S1−S6 and Figures S1−S28. This material is

488

available free of charge via the Internet at http://pubs.acs.org.

489 490

ACKNOWLEDGMENTS

491

This work was supported by the project from National Natural Science Foundation of China

492

(No.21276182).

493 494

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495

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Oxidation

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of

guanine

and

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Antimicrob. Chemoth. 2000, 46 (s1), 53-58.

757 758 759 760

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761

1.2

(a) TMP 1.0

C/C0

0.8 UV/PDS at pH 6 UV/PDS at pH 9 UV/H2O2 at pH 6

0.6

UV/H2O2 at pH 9 UV/H2O2 with HCO3-

0.4

0.2

0.0 0.000

0.002

0.004

0.006

0.008

0.010

0.020

0.030

0.040

UV Fluence (Einstein L-1)

762

1.2

(b) SMX UV at pH6 UV at pH9 UV/PDS at pH 6 UV/PDS at pH 9 UV/H2O2 at pH 6

1.0

C/C0

0.8

UV/H2O2 at pH 9 UV/H2O2 with HCO3-

0.6

UV/H2O2 with ammonia 0.4

0.2

0.0 0.000

763 764 765 766 767

0.002

0.004

0.006

0.008

0.010

UV Fluence (Einstein L-1)

Figure 1. Degradation of (a) TMP and (b) SMX in UV, UV/H2O2, UV/PDS systems at pH 6 and 9 in 5 mM PB solutions, and UV/H2O2 systems with 0.5 M NaHCO3 or 0.1 M NH4OH. Oxidant concentration was 3 mM. UV fluence = UV fluence rate × exposure time.

38


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768 769 770 771 772 773

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.

774 775 776 777 778 779 780

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, H were conducted in 5 mM PB solutions.

39



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782 783 784 785 786 787 788 789

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 two 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).

40


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790

791 792 793 794 795 796 797 798

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 two 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).

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PDS Treatment of Trimethoprim and

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