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J O U RN A L OF E N V I RO N ME N TA L S CI EN CE S X X (2 0 1 6 ) XX X–XXX

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1Q2 Degradation of indomethacin in river water under stress and

2 non-stress laboratory conditions: Degradation products,

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3 long-term evolution and adsorption to sediment

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4Q3 Juan J. Jiménez1,2,⁎, María I. Sánchez3 , Rafael Pardo1 , Beatriz E. Muñoz3

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5 1. Department of Analytical Chemistry, Faculty of Sciences, Campus Miguel Delibes, University of Valladolid, Paseo de Belén 7,
6 47011 Valladolid, Spain

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7 2. I.U. CINQUIMA, Campus Miguel Delibes, University of Valladolid, Paseo de Belén 5, 47011 Valladolid, Spain
8 3. Department of Analytical Chemistry, School of Industrial Engineers, University of Valladolid, Francisco Mendizábal 1,

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9 47014 Valladolid, Spain
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12 AR TIC LE I NFO ABSTR ACT
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14 Article history: The pharmaceutical compound indomethacin is not totally removed in wastewater treatment 19
15 Received 14 June 2016 plants, whose effluents flow into aquatic environments; concentrations in the 0.1–100 ng/L 20
16 range are commonly found in surface waters, and its fate is unknown. Here, biological, 21
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Revised 25 August 2016

17 Accepted 1 September 2016 photochemical and thermal degradation assays were conducted under stress and non-stress 22
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18 Available online xxxx conditions to estimate its degradation rate in river water and establish its degradation products 23
over time. The results revealed that direct sunlight irradiation promoted the complete 24
40 Keywords: degradation of indomethacin (2 μg/L) in less than 6 hr, but indomethacin was detected over a 25
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41 Indomethacin period of 4 months when water was kept under the natural day–night cycle and the exposure to 26
42 Degradation products sunlight was partially limited, as occurs inside a body of water. The biological degradation in 27
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43 River water water was negligible, while the hydrolysis at pH 7.8 was slow. Residues were monitored 28
44 Adsorption coefficients by ultra-pressure liquid chromatography/quadrupole time-of-flight/mass spectrometry after 29
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45 High-resolution mass spectrometry solid-phase extraction, and six degradation products were found; their structures were 30
46 proposed based on the molecular formulae and fragmentation observed in high-resolution 31
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tandem mass spectra. 4-Chlorobenzoic and 2-acetamido-5-methoxybenzoic acids were the 32

long-term transformation products, persisting for at least 30 weeks in water kept under 33
non-stress conditions. Furthermore, the degradation in the presence of sediment was also 34
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monitored over time, with some differences being noted. The adsorption coefficients of 35
indomethacin and degradation products on river sediment were calculated; long-term 36
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degradation products did not have significant adsorption to sediment. 37

© 2016 The Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences. 38
Published by Elsevier B.V. 39
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48
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51 Introduction wastewater treatment plants (WWTPs) are the main intro- 56
duction source of pharmaceuticals in surface waters. These 57
53 The presence of pharmaceutical compounds in the environ- compounds are found in the influents of the WWTPs mainly 58
54 ment is a matter of increasing concern because they impact as a result of the inappropriate domestic disposal of unused 59
55 negatively on the environment. The effluents discharged from medicinal products. Indomethacin (INDO) is a non-steroid 60

⁎ Corresponding author. E-mail: jjimenez@qa.uva.es (Juan J. Jiménez).

http://dx.doi.org/10.1016/j.jes.2016.08.021
1001-0742/© 2016 The Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences. Published by Elsevier B.V.

Please cite this article as: Jiménez, J.J., et al., Degradation of indomethacin in river water under stress and non-stress laboratory
conditions: Degradation products, long-term evolution..., J. Environ. Sci. (2016), http://dx.doi.org/10.1016/j.jes.2016.08.021
 2 J O U RN A L OF E N V I RO N ME N TA L S CIE N CE S X X (2 0 1 6 ) XXX –XXX

61 anti-inflammatory drug detected in the primary influents that sediment sample (total organic carbon 1.2%; clay 11%, silt 44%, 118
62 reach the WWTPs in concentrations, generally, of 20–100 ng/L, sand 45%) was collected from the river Pisuerga. Total organic 119
63 although concentrations of about 1 μg/L have also been carbon was measured by a combustion method with a LECO 120
64 reported (Sui et al., 2009; Radjenovic et al., 2009). Concentra- CS-225 elemental analyzer (St. Joseph, MI, USA). Sediment 121
65 tions of about 10–20 ng/L have been found in depurated particle size analysis was based on the Bouyoucos hydrometer 122
66 effluents (Zhou et al., 2009). In river water, INDO concentra- method; soil aggregates were dispersed by chemical means. 123
67 tions between 0.1 and 100 ng/L are frequent (Kim et al., 2009; Cellulose nitrate disks from Sartorius (Barcelona, Spain) 124
68 Lewandowski et al., 2011; Yamamoto et al., 2009; Zhou et al., were used: river water was filtered through 0.2 μm pore-size 125
69 2009). Concerning the efficiency of WWTPs at removing INDO, disks for the estimation of adsorption coefficients, through 126
70 there are contradictory data; some authors have concluded 3 μm pore-size disks to carry out biodegradation experiments, 127
71 that its removal in the global process is non-existent or slight and through 0.45 μm pore-size disks for other degradation 128
72 (Radjenovic et al., 2009; Rosal et al., 2010; Sui et al., 2009; Tran experiments. 129

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73 et al., 2014), while other authors find that the removal rates are Indomethacin (99% purity) was obtained from Sigma- 130
74 about 40%–100% (Huang et al., 2011; Matsuo et al., 2011; Zhou Aldrich (St. Louis, MO, USA). LC–MS grade methanol, acetonitrile 131

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75 et al., 2009). Moreover, it has been stated that INDO infiltrates to and formic acid were supplied by Panreac (Barcelona, Spain) 132
76 subsurface waters (1 m depth) from surface waters, as has and ultrapure water was obtained from a Milli-Q plus apparatus 133

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77 occurred for other pharmaceuticals (Lewandowski et al., 2011). (Millipore, Milford, MA, USA). Analysis-grade sodium hydroxide, 134
78 The frequent detection of INDO in surface waters and potassium dihydrogen phosphate and sodium azide were 135
79 WWTP effluents discharged into rivers advises us not only to purchased from Panreac. EBH cartridges (60 mg) for solid- 136

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80 evaluate its persistence in the environment, but also to phase extraction (SPE) and PTFE disposable syringe filter units, 137
81 determine the possible transformation products in order to 0.20 μm pore size, were obtained from Scharlab (Barcelona, 138

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82 obtain an overall perspective and to assess possible risks, Spain). Tryptone soya broth (TSB), a highly nutrient liquid 139
83 because the effects resulting from exposure to the parent culture medium for general purpose use, was purchased from 140
84 pharmaceutical and the degradation products can be different Scharlab; its composition can be seen in the supplementary
D 141
85 (Celiz et al., 2009). So, INDO dissolved in different media has material (Appendix A Table S1). A vacuum centrifuge evapora- 142
86 been subjected to degradation studies under stress conditions tor, Myvac model, was provided by Genevac (Ipswich, UK), a 143
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87 that indicate its photolability, and some degradation products PK120 centrifuge by ALC (Winchester, VA, USA) and a Promax 144
88 generated in these conditions have been described (Temussi 2020 reciprocating platform shaker by Heidolph (Germany). 145
89
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et al., 2011; Yamamoto et al., 2009), but there is no reliable

90 information about its behavior in surface water and especially 1.2. Biological degradation 146
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91 about its long-term fate in non-stress conditions.

92 In this context, river water spiked with INDO at trace levels 1.2.1. Aerobic degradation 147
93 was subjected to degradation studies in this work to ascertain Biological degradation assays were carried out with water 148
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94 the importance of the chemical, photochemical and biological from the river Pisuerga (pH 7.8) which was spiked with INDO 149
95 processes in its degradation in surface water. In addition to to achieve a concentration of 2 μg/L. A volume of 50 mL of 150
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96 assays under stress conditions, non-stress conditions were river water was transferred into a 100 mL Erlenmeyer flask, 151
97 also applied to INDO in aqueous solution in order to simulate its which was then coated with aluminum foil to avoid exposure 152
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98 behavior inside a body of water. Water aliquots were analyzed by to sunlight but allowing the exchange of air with the 153
99 ultra-pressure liquid chromatography/quadrupole time-of-flight/ atmosphere. An INDO control solution was similarly prepared 154
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100 mass spectrometry, and the structures of the degradation in ultrapure water (pH 7.8 adjusted with NaOH) containing 155
101 products found were tentatively elucidated from the molecular 0.02% (W/V) sodium azide as a biocide. Water blanks were 156
102 formulae and fragmentation observed in high-resolution tan- prepared as well. Samples were run in parallel; flasks 157
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103 dem mass spectra. The evolution of the degradation products were shaken in a reciprocating shaker at a rotation speed of 158
104 was also monitored over time to estimate their occurrence, and 130 r/min for 5 weeks, within a temperature range of 18–21°C. 159
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105 a degradation pathway was outlined. In addition, the adsorp- Aliquots of 5 mL were collected each week and subjected 160
106 tion capacity of sediment for INDO and its degradation products to analysis. Evaporation water losses were periodically 161
107 162
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was evaluated by calculating the corresponding adsorption restored by addition of water of the same type. All biological
108 coefficients. experiments were carried out in duplicate. 163

1.2.2. Anaerobic degradation 164

110
109 1. Experimental River water (pH 7.8) spiked at 2 μg/L was placed in 15 mL vials, 165
completely filled to avoid the presence of air in the headspace. 166
111 1.1. Materials and reagents The vials were closed, protected from light by coating them 167
with aluminum foil and kept in a temperature range of 18– 168
112 Water samples were collected from the rivers Pisuerga (pH 21°C during experimentation. Control solutions with INDO in 169
113 value 7.8, chemical oxygen demand value 4.6 mg/L), in the ultrapure water (pH 7.8 adjusted) containing 0.02% sodium 170
114 urban area of the city of Valladolid, and Tuerto (pH value 7.4, azide, and the corresponding blanks, were also run in parallel. 171
115 chemical oxygen demand value 3.9 mg/L), in the rural area of A batch of vials was assembled to withdraw weekly samples 172
116 the La Bañeza, province of León; chemical oxygen demand over a period of 5 weeks; a volume of 5 mL from each 173
117 was determined by the potassium dichromate method. A withdrawn vial was collected for analysis. 174

Please cite this article as: Jiménez, J.J., et al., Degradation of indomethacin in river water under stress and non-stress laboratory
conditions: Degradation products, long-term evolution..., J. Environ. Sci. (2016), http://dx.doi.org/10.1016/j.jes.2016.08.021
 J O U RN A L OF E N V I RO N ME N TA L S CI EN CE S X X (2 0 1 6 ) XX X–XXX 3

175 1.2.3. Degradation in culture medium degradation of the W1 sample was also studied in the presence 231
176 A 20/80 (V/V) mixture of TSB culture medium and river water of sediment by adding river sediment to the container in a 232
177 was spiked with INDO at 2 μg/L, and 100 mL of the mixture was sediment-to-solution ratio of 0.3 g/mL (SED sample), in the 233
178 placed in a 250 mL glass container. A control solution in absence of sunlight by coating the container with aluminum foil 234
179 ultrapure water (pH 7.8 adjusted) containing 0.02% sodium (DARK sample), and in the presence of nutrients by adding 235
180 azide and INDO, and a blank of the medium–water mixture, culture medium at the percentage of 1% (BIO sample). 236
181 were also prepared and run in parallel. Closed vessels were It was verified that the W1 and W2 river water samples 237
182 heated at 35°C in darkness for 5 weeks. Aliquots of 10 mL were were free of INDO residues. First, water blanks were subjected 238
183 sampled weekly and filtered through 0.45 μm pore-size cellu- to analysis to test for the absence of the parent compound. 239
184 lose nitrate; 5 mL samples of filtrate were collected for analysis. Once the degradation products yielded in the stress assays 240
were known, tests were also performed to verify that they 241
185 1.3. Photochemical and thermal degradation were not present in the water extracts. Similarly, the absence 242

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of residues in the sediment sample was confirmed; to this 243
186 River water (pH = 7.8) spiked with INDO (2 μg/L) was placed in aim, sediment was extracted with methanol by mechanical 244

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187 a quartz cuvette, which was closed and placed on the outer shaking and the extract was concentrated for analysis. 245
188 edge of a window, south-facing, to allow its direct exposure to

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189 sunlight. The assay was performed in the city of Valladolid 1.5. Study of adsorption to sediment 246
190 (latitude: 41°38′15″N, longitude: 4°44′17″W) in one day, in the
191 month of January. Aliquots of 0.3 mL were withdrawn at Experiments were conducted to investigate the adsorption 247

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192 regular time intervals and injected into the chromatographic capacity of the sediment for INDO and its degradation 248
193 system. Control samples of INDO in river water, protected products. The adsorption coefficient (Kd) of INDO to sediment 249

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194 from sunlight with aluminum foil, were prepared as well. (sieved through a 0.5 mm mesh) was determined by using a 250
195 For thermal degradation, a volume of 100 mL of spiked batch approach. River water (100 mL) spiked with INDO was 251
196 river water (2 μg/L) was placed in a closed 250 mL glass flask. added to sediment (20 g) to achieve a sediment-to-solution
D 252
197 This was coated with aluminum foil and placed in an oven at ratio of 0.20 g/mL; INDO concentrations were 200, 400, 600, 800 253
198 70 °C. Aliquots of 5 mL were collected hourly and subjected to and 1000 ng/L. River water contained 0.02% sodium azide as a 254
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199 analysis. All experiments were done in duplicate. biocide to minimize any possible microbial activity and pH (7.8) 255
was controlled with phosphate buffer (0.02 mol/L). Control 256
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200 1.4. Non-stress degradation assays solutions without sediment were prepared as well. The flasks, 257
protected from sunlight with aluminum foil, were manually 258
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201 A simple, although slow, approach was adopted in this work to shaken for 1 min and left standing at 20 ± 1°C for a period of 259
202 simulate the concurrent natural process in a body of water. 24 hr. Afterwards, an aliquot of 10 mL, previously centrifuged to 260
203 River water was placed in a transparent sodium calcium silicate remove solids, was collected to determine the INDO concentra- 261
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204 glass container with air-tight seal, which was opened weekly to tion at equilibrium. An adsorption isotherm was drawn in 262
205 collect a sample aliquot and replace the air inside in contact duplicate. The concentration adsorbed on the sediment was not 263
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206 with the water surface. The container was kept at laboratory directly measured. 264
207 temperature (18–21°C) under the natural day–night cycle and Analytical standards are unavailable for most of the degrada- 265
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208 directly exposed to sunlight for 30 weeks, at which time the tion products, however their Kd were estimated based on the 266
209 degradation assay was ended. In these conditions, the solar assumption that there is a linear relationship between peak 267
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210 radiation must pass through the laboratory window glass and area and concentration for each compound. Experiments 268
211 the glass of the container to reach the body of water; the glass similar to the above-described were devised by spiking water 269
212 absorbs UV radiation and the behavior of INDO in the glass with a solution containing the degradation products and an 270
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213 container simulates, in a greater or lesser extent, that in a mass appropriate sediment-to-solution ratio. The decrease percent- 271
214 of water where the penetration of solar UV radiation is age of peak area in water was assumed to be the percentage of 272
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215 diminished with depth. The attenuation of the radiation was compound adsorbed onto sediment (A%), and Kd was calculated 273
216 estimated by measurements of transmittance through the two by Eq. (1), which is valid if the adsorption equation is linear 274
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217 types of glass (container and window), the UV–visible absorp- (OECD, 2000). Peak areas of the adsorption experiments were 275
218 tion spectra of the glasses were recorded; so, it was quantified compared with those of control solutions by a t-test (n = 5) to 276
219 that the percentages of radiation transmitted to the body of confirm the existence of significant differences before applying 277
220 water were 40%, 1.3%, 0.02%, 8 × 10−4% and 8 × 10−5% at the equation. 278
221 wavelengths of 350, 320, 310, 305 and 290 nm, respectively. 0 . 1
222 A volume of 2500 mL of river water placed in glass container A%
B 100 C
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223 was spiked with INDO to achieve an initial concentration of log Kd ¼ log@ A− log R ð1Þ
1−A%
224 2 μg/L in each degradation assay. Aliquots of 25 mL were 100
225 collected periodically and subjected to SPE; extracts were 280
279
226 injected in the chromatographic system to follow the degrada- 1.6. Sample preparation 281
227 tion of INDO, identify degradation products and monitor them.
228 Degradation experiments were carried out between the months Except for the photochemical degradation study, river water 282
229 of November and June with waters from the rivers Pisuerga aliquots were eluted through EBH cartridges previously 283
230 (W1 sample) and Tuerto (W2 sample). Simultaneously, the conditioned by successive elution of methanol (6 mL) and 284

Please cite this article as: Jiménez, J.J., et al., Degradation of indomethacin in river water under stress and non-stress laboratory
conditions: Degradation products, long-term evolution..., J. Environ. Sci. (2016), http://dx.doi.org/10.1016/j.jes.2016.08.021
 4 J O U RN A L OF E N V I RO N ME N TA L S CIE N CE S X X (2 0 1 6 ) XXX –XXX

285 water (6 mL). The cartridges were washed with 3 mL of a

286 water–methanol (80:20, V/V) mixture after sample elution.
287 The stationary phase was dried with air for 3 min and the
288 target compounds were eluted with methanol (4 mL) by
289 gravity. Then, the extract was evaporated in 30 min by a
290 vacuum centrifuge evaporator heated at 40 °C, and the dry
291 residue was re-dissolved in 0.5 mL of methanol, which was
292 filtered through a 0.20 μm pore-size PTFE filter for chromato-
293 graphic analysis.

294 1.7. Determination by liquid chromatography – mass

295 spectrometry

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296 An Acquity ultra-pressure liquid chromatograph from Waters

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297 (Milford, MA, USA) coupled to a Maxis Impact quadrupole
298 time-of-flight tandem mass spectrometer from Bruker Daltonics

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299 (Bremen, Germany) was used. Analyses were performed with
300 electrospray ionization in negative mode. The chromatograph
301 was fitted with a Waters BEH ODS column (50 mm × 2.1 mm,

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302 1.7 μm particle size). The mobile phase flow rate was 0.5 mL/min
303 and consisted of 0.1% formic acid in water (A) and 0.1% formic

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304 acid in acetonitrile (B); assays were performed under gradient
305 conditions: from 20% B to 55% B in 4.5 min, and then 60% B in
306 1 min. Re-equilibration time was 1 min. Injection volume was D
307 7 μL.
308 The operating parameters of the electrospray ionization
Fig. 1 – Degradation of indomethacin in river water according
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309 source for the MS and MS/MS experiments were as follows:
to biological (a) or stress (b) assays. Data are the mean of two
310 nebulizing gas pressure, 2 bar; end plate offset voltage,
experiments.
311
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1000 V; capillary voltage, 4500 V; drying gas temperature,

312 200°C; dry gas flow, 6 L/min. Nitrogen was used as drying
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313 and nebulizing gas. Mass calibration adjustments were

314 performed by using a 10 mmol/L sodium formate solution in about 68%. A 1 week lag period was observed in this last 340
315 2-propanol/water. MS/MS experiments based on collision- experiment; if the initial concentration (time 0 weeks) was 341
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316 induced dissociation with nitrogen gas were performed. The excluded, the variation of the remaining experimental 342
317 quantitation of INDO was accomplished using linear calibra- concentrations with time could be fitted to a linear equation 343
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318 tion graphs based on the measurement of peak areas in the (R2 = 0.98), from which a half-life of 3.70 weeks was calculat- 344
319 chromatograms extracted for the fragment-ion [M–H–CO2]− ed. On the other hand, heating at 70°C and UV radiation from 345
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320 generated in the electrospray source by MS experiments, with the sunlight promoted faster degradation of INDO in river 346
321 a mass range of ±0.01 Da. Similarly, peak areas of degradation water; it could be detected for only 11 and 5 hr, respectively. 347
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322 products were integrated in the chromatograms extracted for Degradation data were fitted to a first-order kinetics equation 348
323 the corresponding [M–H]− ions. with half-lives of 1.97 h (R2 = 0.97) and 1.60 h (R2 = 0.97) for the 349
324 As regards the performance of the analytical method, the heating and direct irradiation assays, respectively. The UV 350
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325 mean recovery of INDO (2 μg/L) was about 95% with a radiation is the main factor that boosts the degradation of 351
326 repeatability of 1.8% for W1 sample (n = 5). The repeatability INDO. 352
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327 in the determination of the degradation products varied from The evolution of the INDO concentration over weeks in the 353
328 1.6% to 5.5% (n = 5, Appendix A Table S2). non-stress assays can be seen in Fig. 2 and Appendix A Table 354
355
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S5. INDO was detected for 16 and 15 weeks in water from the
two rivers (W1 and W2 samples); the concentration decreased 356
330
329 2. Results and discussion gradually until it was less than the detection limit achieved by 357
the method, which was estimated to be about 10 ng/L. 358
331 2.1. Degradation of indomethacin Experimental data were also well fitted to first-order kinetics; 359
half-lives were 2.57 (R2 = 0.97) and 2.47 weeks (R2 = 0.94) for 360
332 Fig. 1 shows the variation of the INDO concentration over time W1 and W2 samples, respectively. The degradation of INDO in 361
333 in biological and stress assays; data are the average of two river water at room temperature and in the absence of 362
334 independent experiments, and individual data are shown in sunlight (DARK sample) was slower (half-life: 5.12 weeks, 363
335 Appendix A Tables S3 and S4. After 5 weeks, the degradation R2 = 0.98), being detected throughout the 30 weeks of exper- 364
336 of INDO in river water was negligible under aerobic and imentation. These assays indicate that solar radiation is a 365
337 anaerobic conditions at room temperature, but when a notable factor in the behavior of INDO inside a body of water 366
338 nutrient was added to river water and the mixture was even though the transmission of UV radiation is attenuated. 367
339 incubated at 35°C, the concentration of INDO decreased by However, the photochemical reactions should not be the only 368

Please cite this article as: Jiménez, J.J., et al., Degradation of indomethacin in river water under stress and non-stress laboratory
conditions: Degradation products, long-term evolution..., J. Environ. Sci. (2016), http://dx.doi.org/10.1016/j.jes.2016.08.021
 J O U RN A L OF E N V I RO N ME N TA L S CI EN CE S X X (2 0 1 6 ) XX X–XXX 5

discussion of the fragmentation patterns, interpretation of the 395

fragment-ions and a brief explanation on the assignment of 396
tentative structures to the degradation products can be found in 397
the supplementary material. Table 1 shows the molecular 398
formulae established and the identifications proposed for the 399
compounds. 400
The compounds 5-methoxy-2-methyl-1H-indole-3-acetic 401
acid (MMIA) and 4-chlorobenzoic acid (CBA) were unequivocally 402
identified. They were the two basic constituent moieties of 403
INDO. An unknown degradation product was identified as acid 404
(DHINDO), a derivative of INDO hydroxylated at the C6 and C7 405
Fig. 2 – Degradation of indomethacin in river water under sites of the indole moiety, while the other unknown compound 406

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non-stress conditions. Data are the mean of two was thought to be 2-(4-chlorobenzamido)-5-methoxybenzoic 407
experiments. acid (CMBA), which arose from the cleavage of the indole 408

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structure. The other two degradation products were identified 409
as 5-methoxy-2-methyl-1H-indole-3-carbaldehyde (MMIC) and 410

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369 factor considered because the degradation of INDO in dark- 2-acetamido-5-methoxybenzoic acid (AMBA) in agreement with 411
370 ness is not negligible. two described photodegradation compounds (Temussi et al., 412
371 INDO seems to be a non-biodegradable compound in 2011). Thus, a structure was ascribed to each compound con- 413

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372 unmodified river water. However, the degradation study over sidering spectral and bibliographic data. 414
373 time in water whose nutrient content was enhanced (BIO

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374 sample) revealed that the degradation rate was slightly higher 2.3. Occurrence of degradation products in river water 415
375 in comparison with that of the W1 sample. The half-life was
376 now estimated to be 2.04 weeks (R2 = 0.98, first-order kinetics). The peak areas of the degradation products were monitored
D 416
377 Otherwise, in the presence of aquatic sediment (SED sample) during the degradation studies; a value of 100 was assigned to 417
378 the half-life was higher, 3.39 weeks (R2 = 0.96, first-order the initial peak area of INDO (week 0) in each experiment, and 418
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379 kinetics); INDO was found in the aqueous phase for 24 weeks. all other peak areas were referred to this value. Fig. 3 419
380 The measured initial concentrations in the SED sample were illustrates the evolution of compounds in non-stress condi- 420
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381 lower with regard to the previous assays, which suggested tions. Data for all compounds and experiments are provided 421
382 possible adsorption of INDO to sediment. This adsorption as Supplementary material. Four compounds were found in 422
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383 phenomenon could help protect INDO from sunlight exposure W1 and W2 samples (Appendix A Figs. S8 and S9), whose 423
384 and increase its stability in water samples. degradation conditions were intended to simulate the behav- 424
ior of INDO inside a body of water; CBA, AMBA and MMIA were 425
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385 2.2. Detection and identification of degradation products the major degradation products in the first 15 weeks, while 426
CBA together with AMBA were the only compounds detected 427
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386 The presence of compounds related to degradation of INDO was after 30 weeks. CBA was the most abundant degradation 428
387 established by the observation of new chromatographic peaks product in terms of peak area; its amount increased gradually 429
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388 in the MS chromatograms, which were present neither in the until about the 10th week, and then decreased slowly. AMBA 430
389 previous chromatograms nor in blanks, and the gradual was a minor degradation product that seemed to persist over 431
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390 variation of their peak areas over time. In this way, six degrada- time. DHINDO was also a minor compound that reached its 432
391 tion products of INDO were found in the extracts. The [M–H]− maximum occurrence at about the 9th–10th week; like MMIA, 433
392 ion of each degradation product, besides INDO, was isolated DHINDO was not detected after the 19th week. The evolution 434
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393 and fragmented by collision-induced dissociation to obtain profile of the compounds was basically similar in waters from 435
394 structural information. MS/MS spectra (Appendix A Figs. S1–S7), the two rivers (W1 and W2). The joint presence of CBA and 436
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t1:1 Table 1 – Retention times (RT), masses measured in the mass spectra for the [M-H]− ion, errors in the determination of exact
t1:2 masses, molecular formulae and number of rings and double bonds (rdb) of the corresponding structures for the detected
t1:3 degradation products.
t1:4
t1:5 RT Molecular Exact Measured Error rdb Compound Abbreviation
(min) formula mass (Da) mass (Da) (ppm)

t1:6 0.89 C10H10NO4 208.0615 208.0616 −0.5 6.5 2-acetamido-5-methoxybenzoic acid AMBA
t1:7 1.15 C11H10NO2 188.0717 188.0724 −2.3 7.5 5-methoxy-2-methyl-1H-indole-3-carbaldehyde MMIC
t1:8 1.26 C12H12NO3 218.0823 218.0824 −0.5 7.5 5-methoxy-2-methyl-1H-indole-3-acetic acid MMIA
t1:9 2.04 C7H4ClO2 154.9905 154.9905 0.0 5.5 4-chlorobenzoic acid CBA
t1:10 2.82 C19H17ClNO6 390.0750 390.0746 1.0 11.5 1-(4-chlorobenzoyl)-6,7-dihydro-6,7-dihydroxy-5-methoxy- DHINDO
2-methyl-1H-indole-3-acetic acid
t1:11 3.80 C15H11ClNO4 304.0382 304.0378 −1.3 10.5 2-(4-chlorobenzamido)-5-methoxybenzoic acid CMBA
t1:12 4.25 C19H15ClNO4 356.0695 356.0696 −0.3 12.5 1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indole-3-acetic INDO
acid

Please cite this article as: Jiménez, J.J., et al., Degradation of indomethacin in river water under stress and non-stress laboratory
conditions: Degradation products, long-term evolution..., J. Environ. Sci. (2016), http://dx.doi.org/10.1016/j.jes.2016.08.021
6 J O U RN A L OF E N V I RO N ME N TA L S CIE N CE S X X (2 0 1 6 ) XXX –XXX

AMBA in surface water can constitute an indicator of the 437

earlier presence of INDO in that body of water. 438
The evolution of the degradation products when the 439
amount of nutrients in river water was increased to favor 440
the growth of microorganisms (BIO sample), followed the 441
pattern described for W1 and W2 samples, with the difference 442
that the transformation rate of the compounds increased 443
somewhat, as was previously stated for the degradation of 444
INDO (Appendix A Fig. S10). Excepting CBA, all degradation 445
products were detected during a smaller number of weeks; 446
even AMBA was now fully degraded in 23 weeks. 447
Finally, the transformation products found in the SED 448

F
sample (Appendix A Fig. S11) were the same as those in the 449
W1 and W2 samples, although the evolution profile was 450

O
clearly different. Regardless of the lower degradation rate of 451
INDO, MMIA was detected only in extracts for 10 weeks, and 452

O
its peak areas were lower in relation to assays without 453
sediment. Moreover, DHINDO was present in higher relative 454
amounts, which was attributed not only to the lower initial 455

R
peak area of INDO, but also to the higher occurrence of 456
DHINDO, whose formation was enhanced in the presence of 457

P
sediment. Furthermore, the persistence of DHINDO also 458
increased, being detected in water for 28 weeks. However, 459
CBA and AMBA remained as the only compounds detected
D 460
after 30 weeks. 461
As regards the origin of the degradation products, CBA and 462
E
MMIA were the only degradation products detected in the 463
stress assay at 70°C (Appendix A Fig. S12), the DARK sample 464
465
T

(Appendix A Fig. S13), and the water sample incubated at 35°C

with added culture medium (Appendix A Fig. S14). Their 466
C

occurrence in the two first samples, in addition to W1 and W2, 467

could be easily explained by chemical hydrolysis, while 468
biochemical hydrolysis in the water containing nutrients 469
E

could not be discarded. On the other hand, the peak areas of 470
CBA and MMIA in the DARK sample after 30 weeks were not 471
R

very different between them, in contrast to W1 and W2 472

samples, where the amount of MMIA was clearly lower than 473
R

that of CBA at the end of the monitoring; this suggested that 474
MMIA is a relatively stable compound in the absence of 475
O

sunlight. 476
Five degradation products were yielded by photochemical 477
reactions. They were found when river water was directly 478
C

exposed to sunlight, without passing through the glass, which 479

absorbs UV radiation (Appendix A Fig. S15). DHINDO was the 480
N

predominant product in terms of peak area, which was 481

two-fold higher than that of INDO after 6 hr of exposure (see 482
483
U

Supplementary material). CBA and AMBA were also detected,

but not MMIA. Two other photo-induced degradation prod- 484
ucts that resulted were MMIC and CMBA, which were not 485
noted in the samples under non-stress conditions, likely as a 486
consequence of the attenuation of the UV radiation. It is 487
plausible that the amount of DHINDO could be higher in a 488
body of water if the UV radiation was less attenuated with 489
depth. 490
The described photodegradation compounds can be yielded 491
by direct or indirect photolysis; data to differentiate and discuss 492
the two photolysis mechanisms in the degradation of INDO are 493
Fig. 3 – Variation of peak areas of the degradation products in
not available. In direct photolysis, the molecule of INDO in the 494
river water under non-stress conditions. Mean of two
excited state (after absorbing a photon) would be unstable and 495
experiments.
would break down. In indirect photolysis, the radiation is 496
Q4

Please cite this article as: Jiménez, J.J., et al., Degradation of indomethacin in river water under stress and non-stress laboratory
conditions: Degradation products, long-term evolution..., J. Environ. Sci. (2016), http://dx.doi.org/10.1016/j.jes.2016.08.021
 J O U RN A L OF E N V I RO N ME N TA L S CI EN CE S X X (2 0 1 6 ) XX X–XXX 7

497 absorbed for one or more chemical species in the water solution Table 2 – Kd and Koc mean values (RSDs in parenthesis) t2:1
498 (for instance, the dissolved organic matter), which could then calculated for the degradation products. Mean adsorption t2:2
499 decompose INDO by direct reaction with it or through new percentages (A%) onto sediment and experimental t2:3
sediment to water ratios (R), n = 5. t2:4
500 chemical species derived from those excited compounds.
t2:5
Compound Kd Koc A% R Experimental t2:6
501 2.4. Distribution between water and sediment. Adsorption (kg/L) (kg/L) (kg/L) t-value*
502 coefficients AMBA <0.10 <8.7(–) <5 0.507 0.6 t2:7
MMIC 35 2938 (6.0%) 54.0 0.0333 10.8 t2:8
503 Fig. 4 shows the adsorption isotherm obtained for INDO in the MMIA 33 2756 (6.7%) 52.2 0.0331 18.2 t2:9
504 batch experiments; the resulting Kd and Koc (organic carbon CBA 1.1 89 (8.6%) 30.5 0.409 10.1 t2:10
DHINDO 0.15 12.3 (28%) 7.0 0.509 2.57 t2:11
505 normalized adsorption coefficient) values were 1.79 and
CMBA 1.8 150 (8.1) 27.4 0.210 15.2 t2:12
506 149 kg/L (RSD 5.0%, n = 10), respectively. The Kd value was t2:13

F
INDO 1.8 150 (7.8%) 27.0 0.206 16.9
507 comparable to that already described in the literature
508 (Yamamoto et al., 2009). Adsorption coefficients were also * Critical t-value: 2.3; –: without data. Q1
t2:14

O
509 calculated for the degradation products from the experimen-
510 tal adsorption percentages; the Kd and Koc values and the

O
511 sediment-to-water ratios used are given in Table 2. The
512 adsorption coefficient of INDO was also calculated by this undergo a similar hydrolysis reaction, but its corresponding 540
513 method and was found to be similar to the previous value. indole moiety was not found in this work. 541

R
514 CBA, and particularly AMBA and DHINDO, had a lower It has been reported that AMBA arises from INDO through a 542
515 capacity for adsorption than INDO. In fact, the low adsorption photodegradation process involving the non-prominent in- 543

P
516 percentages (<10%) of the latter two degradation compounds termediate MMIC (Temussi et al., 2011), however it seems 544
517 resulted in a worse precision (28%) in the determination quite possible that MMIA is transformed into AMBA by 545
518 performed for DHINDO, and only a rough estimate of Kd for photo-induced reactions as well. In fact, the amount of
D 546
519 AMBA. On the other hand, the adsorption coefficients of MMIA remained high and relatively constant in the DARK 547
520 MMIA and MMIC were relatively high, about 18-fold higher sample, as already noted above. In order to verify the 548
E
521 than that of INDO, which was consistent with the low peak conversion of MMIA in AMBA, an INDO aliquot was subjected 549
522 areas observed for MMIA in the SED sample. After checking to hydrolysis by heating at 70°C to yield CBA and MMIA, and 550
T

523 the structures of the compounds, the notable capacity for then was placed in a quartz cuvette for direct exposure to 551
524 adsorption of MMIA and MMIC to sediment would be related sunlight. It was observed that MMIA was completely removed 552
C

525 to the presence of a N–H bond in the indole moiety, because in about 4 hr, that MMIC was very abundant after 2 hr, 553
526 higher adsorption coefficients have been reported in amine decreasing quickly afterwards, and that AMBA was effectively 554
527 compounds (Yamamoto et al., 2009). It should be noted that a photoproduct arising from MMIA (Appendix A Fig. S16); the 555
E

528 the water–sediment interaction is not simple and that the CBA amount remained virtually constant in this assay. 556
529 adsorption coefficients were estimated for only a sample of Finally, there were no data to ascertain the fate of CMBA, 557
R

530 water and sediment; consequently, the data provided in this however it is reasonable to hypothesize its transformation to 558
531 work should be considered preliminary. CBA by hydrolysis. 559
R

532 2.5. Degradation pathway

O

533 A main degradation pathway of INDO in river water was

534 outlined (Fig. 5). First, the amide group of INDO is hydrolyzed
C

535 to give its two constituent moieties: MMIA and CBA, whose
536 presence would be expected (Krzek and Starek, 2001). Simul-
N

537 taneously, INDO undergoes a secondary degradation reaction

538 in which the diol derivative DHINDO is formed through a
U

539 photochemical reaction; it is thought that DHINDO could

Fig. 5 – Degradation pathway of indomethacin in river

water. Bold lines denote the possible predominance of the
Fig. 4 – Adsorption isotherm of indomethacin (n = 2). transformation process.

Please cite this article as: Jiménez, J.J., et al., Degradation of indomethacin in river water under stress and non-stress laboratory
conditions: Degradation products, long-term evolution..., J. Environ. Sci. (2016), http://dx.doi.org/10.1016/j.jes.2016.08.021
 8 J O U RN A L OF E N V I RO N ME N TA L S CIE N CE S X X (2 0 1 6 ) XXX –XXX

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Please cite this article as: Jiménez, J.J., et al., Degradation of indomethacin in river water under stress and non-stress laboratory
conditions: Degradation products, long-term evolution..., J. Environ. Sci. (2016), http://dx.doi.org/10.1016/j.jes.2016.08.021