Dimer formation during UV photolysis of diclofenac

Chemosphere 93 (2013) 1948–1956

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Dimer formation during UV photolysis of diclofenac Olya S. Keen a,d,1, E. Michael Thurman b, Imma Ferrer b, Aaron D. Dotson c, Karl G. Linden a,⇑ a

Department of Civil, Environmental and Architectural Engineering, University of Colorado, UCB 428, Boulder, CO 80309, United States Center for Environmental Mass Spectrometry, University of Colorado, UCB 428, Boulder, CO 80309, United States c Civil Engineering Department, University of Alaska, 3211 Providence Drive, Engineering Building, Room 214, Anchorage, AK 99508, United States d Department of Civil and Environmental Engineering, University of North Carolina, 9201 University City Blvd, Charlotte, NC 28223, United States b

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

 Photolytic decay of diclofenac was

studied with unexpected findings.  Formation of dimers was detected

during UV photolysis of diclofenac.  Implications for engineered UV

systems and natural water bodies exposed to sunlight.

a r t i c l e

i n f o

Article history: Received 21 February 2013 Received in revised form 26 June 2013 Accepted 28 June 2013 Available online 1 August 2013 Keywords: Ultraviolet photolysis Pharmaceuticals Quantum yield Photosensitized reactions Singlet oxygen Photopolymerization

a b s t r a c t Dimer formation was observed during ultraviolet (UV) photolysis of the anti-inflammatory drug diclofenac, and confirmed with mass spectrometry, NMR and fluorescence analysis. The dimers were combinations of the two parent molecules or of the parent and the product of photolysis, and had visible color. Radical formation during UV exposure and dissolved oxygen photosensitized reactions played a role in dimer formation. Singlet oxygen formed via photosensitization by photolysis products of diclofenac. It reacted with diclofenac to form an epoxide which is an intermediate in some dimer formation pathways. Quantum yield of photolysis for diclofenac was 0.21 ± 0.02 and 0.19 ± 0.02 for UV irradiation from medium pressure and low pressure mercury vapor lamps, respectively. Band pass filter experiments revealed that the quantum yield is constant at wavelengths >200 nm. The same dimers formed in laboratory grade water when either of the two UV sources was used. Dimers did not form in wastewater effluent matrix, and diclofenac epoxide molecules may have formed bonds with organic matter rather than each other Implications for the importance of dimer formation in NOM are discussed. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction Pharmaceuticals primarily enter natural waters via the effluent of wastewater treatment plants (Kolpin et al., 2002). Many phar⇑ Corresponding author. Tel.: +1 (303) 492 4798; fax: +1 (303) 492 7317. E-mail address: [email protected] (K.G. Linden). Current address: Department of Civil and Environmental Engineering, University of North Carolina, 9201 University City Blvd, Charlotte, NC 28223, United States. 1

0045-6535/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.chemosphere.2013.06.079

maceutical compounds have been subject to scrutiny due to associated environmental concerns (Oaks et al., 2004; Vajda et al., 2008; Painter et al., 2009). Because of the increased application of ultraviolet (UV) for effluent disinfection many studies have looked at the potential for UV to transform pharmaceutical pollutants. Several of the commonly detected pharmaceutical compounds are susceptible to degradation by UV at disinfection doses (Pereira et al., 2007; Canonica et al., 2008). Typically, when UV is studied as a means of degrading photolabile contaminants,

O.S. Keen et al. / Chemosphere 93 (2013) 1948–1956

discovered photolysis products are lower molecular weight than the parent (Lam and Mabury, 2005; Lester et al., 2008; Szabó et al., 2011). Products with slightly larger molecular weight than the parent compound are reported occasionally, but are typically due to radical reactions rather than direct photolysis (Lam and Mabury, 2005; Lester et al., 2008). However, it is rarely considered that larger molecular weight products such as dimers can form during irradiation although possible through photopolymerization (Lányi and Dinya, 2003; Yuan et al., 2009). Increase in size and molecular weight affects essential properties of the molecule that impact bioavailability and potential for bioaccumulation, such as KOW, solubility and volatility, and as a result can affect its toxicity. Dimerization also indicates that the compound can become a relatively stable radical with a lifetime long enough to encounter another radical to bond with. Dissolved organic matter in environmental aquatic matrices also forms relatively long-lived excited states when irradiated (Sharpless, 2012). Therefore, contaminants at trace environmental levels can get incorporated into the background dissolved organic matter by quenching the excited state of organic matter rather than forming dimers. Diclofenac, a non-steroidal anti-inflammatory drug, has been investigated extensively in recent years due to its tendency toward bioaccumulation and biomagnification. Environmental studies have shown that diclofenac has the ability to cause renal failure in wildlife (Oaks et al., 2004) and has toxic effects at concentrations detected in the environment (Fent et al., 2006). Specific attention has been devoted to the photolysis of diclofenac as one of the dominant pathways of its degradation. Several studies published proposed pathways of decay of diclofenac exposed to different sources of radiant energy. Moore et al. (1990) used ultraviolet light (UV) at wavelengths >315 nm, Buser et al. (1998) and Agüera et al. (2005) studied solar degradation under natural light and Eriksson et al. (2010) investigated simulated solar degradation. In addition, degradation products have been studied in photo-Fenton process (Perez-Estrada et al., 2005). All previous studies largely agreed on the degradation pathways of the drug. Diclofenac will ultimately lose both chlorine atoms and the ring will close to form a carbazole-1-acetic acid. However, potential formation of dimers as one of the photolysis products was only mentioned without detailed analysis by Agüera et al. (2005) UV irradiation will break chemical bonds when the photonic energy absorbed exceeds the bond energy. When the bond is broken, an unpaired electron remains on each fragment, and radicals form as a result. For example, chlorinated compounds (e.g. diclofenac) under UV can break a relatively weak carbon-chlorine bond (330 kJ mol 1 bond dissociation energy) which results in the formation of a chlorine radical and an unpaired electron on the carbon of the organic molecule. Radiation with wavelength <360 nm has energy >330 kJ mol 1 capable of breaking the C–Cl bond. Unpaired electrons on the radicals can abstract atoms from other molecules and create new radicals in the process or they can combine with each other and form stable molecules (Oppenlander, 2003). This recombination can create dimers. UV disinfection utilizes UV-C (200–280 nm) wavelengths that supply sufficient energy (as calculated by Planck’s equation) to break a wide range of organic bonds, and are therefore capable of creating radicals (470 kJ Es 1 at 254 nm emitted by monochromatic low pressure mercury lamps; up to 570 kJ Es 1 for polychromatic medium pressure mercury lamps). Ability to form dimers indicates that the produced radical is relatively stable and can exist without abstracting an atom from another molecule. When it encounters another radical, the two molecules combine. This study investigated the mechanism of dimer formation. In particular, it looked at the effects of dissolved oxygen on the formation of dimers. The role of diclofenac products as singlet oxygen sensitizers and whether singlet oxygen participates in the dimer-

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ization process was also evaluated. The process of dimerization of diclofenac under UV was analyzed by examining the short-lived and stable products of photolysis using liquid chromatography combined with mass spectrometry and by looking for changes in the organic character of the diclofenac dimers compared to the parent with fluorescence and NMR spectroscopy. 2. Experimental section 2.1. Reagents All compounds used in the study were reagent grade: diclofenac sodium salt and furfuryl alcohol for singlet oxygen detection (both TCI America, Portland, OR), methylene blue as a reference singlet oxygen sensitizer (Sigma–Aldrich, St. Louis MO). Identification of the products and determination of quantum yield, was carried out in ultrapure water (arium611-VF, Sartorius Stedim, Bohemia, NY). Up to 10% HPLC grade methanol (Honeywell Burdick & Jackson, Morristown, NJ) was used to prepare some of the samples. 2.2. Photolysis setup Irradiations were performed with a low pressure mercury vapor lamp emitting monochromatic light at 253.7 nm and a medium pressure mercury vapor lamp emitting a characteristic polychromatic spectrum above 200 nm (spectra shown in Fig. S1 in the Supplementary Data). The low pressure lamp system consisted of four 15 W mercury lamps (ozone-free, General Electric #G15T8). The light was quasi-collimated by two 10 cm circular apertures 1.2 cm apart. The medium pressure lamp system (Calgon Carbon, Pittsburg, PA) was equipped with a single 1 kW lamp, quasi-collimated by a 10 cm long 6.4 cm diameter cylindrical tube. The irradiation was carried out in a 125 mm diameter crystallization dish. Lamp setup diagrams can be seen elsewhere (Bolton and Linden, 2003). The incident irradiance was measured with International Light IL-1700 radiometer (Peabody, MA), Petri Factor was 0.92 for low pressure lamp and 0.90 for medium pressure lamp and sample depth was 1.9 cm. The average irradiance was calculated using the procedure described by Bolton and Linden (2003). Average irradiance for the medium pressure lamp was calculated for the 200– 300 nm wavelength range and was not germicidally weighted. The method accounts for changes in light penetration caused by different water constituents and by different concentrations of the spiked compound. 2.3. Analysis of intermediates The mass to charge ratios (m/z) of the products were determined using an Agilent 1100 Series high performance liquid chromatography tandem mass spectrometer (HPLC/MS) with XCT Plus ion trap (Agilent Technologies, Inc., Santa Clara, CA) using electrospray ionization in positive ion mode [(+)ESI]. HPLC was equipped with a diode array detector (DAD). Column was 4.6  50-XDB-C8 with 1.8 lm particle size and was kept at 30 °C. Mobile phases were acetonitrile (HPLC grade, Honeywell Burdick & Jackson, Morristown, NJ) and lab grade water with 0.1% formic acid (Fluka, St. Louis, MO). Acetonitrile concentration was increased from 10% to 100% over 8 min. The product was distinguished from its fragments created in the ionization source by the sodium adduct. Sodium adducts form in positive mode when sodium instead of hydrogen attaches to the neutral molecule during ionization. Fragmentation patterns of the parent compound repeated within each product peak. Chlorine isotope abundance ratio, m/z and the fragmentation patterns were used to identify the products and to postulate the degradation pathway.

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Some of the samples were processed using solid phase extraction and then concentrated. Oasis HLB cartridges, 500 mg (Waters Corporation, Milford, MA) were washed with 5 mL of HPLC grade methanol and with 5 mL of HPLC grade water. One liter of sample was run through the cartridge at 1 mL min 1 and eluted with 5 mL of HPLC grade methanol. The final volume of the eluted methanol was reduced to 0.5 mL by evaporation under nitrogen stream. Ion chromatography (Dionex DX5 with CD20 conductivity detector, Sunnyvale, CA) was used to determine generation of chloride ion that was expected to form as a result of the proposed degradation pathway. pH of the unbuffered solution was measured using a calibrated Beckman U 340 pH meter (Beckman Coulter, Inc., Brea, CA) at treatment intervals to confirm the loss of hydrogen atoms by the parent molecule during the treatment. Diclofenac was spiked into water at 34 nM (10 lg L 1) initially to confirm formation of the colored (absorbing at 400 nm) dimer compounds at environmentally relevant concentrations. In the subsequent experiments 340 lM (100 mg L 1) concentration was used to produce a signal of products about 10 times the background noise at the lowest UV fluence of 10 mJ cm 2. It also allowed for direct analysis of the products without preconcentration or extraction step that could have resulted in the loss of some of the polar products. The samples with 34 nM starting diclofenac were concentrated using solid phase extraction described above. Methanol was added to enhance the solubility of the compound at 340 lM. A low concentration of the compound (below kinetic solubility limit of 40 ± 5 mg L 1) (Llinas et al., 2007) was photolyzed with and without methanol to assess any difference in photolysis of diclofenac with and without methanol. 2.4. Quantum yield The quantum yield of degradation of diclofenac was calculated using the method described by Sharpless and Linden (2003). The overall quantum yield for polychromatic UV photolysis was determined and was further analyzed with a long-pass filter blocking wavelengths below 295 nm and with three band-pass filters (Andover Corporation, Salem, NH) allowing a narrow band of wavelengths to pass through to the sample. The band-pass filters were centered at wavelengths of 228 nm, 254 nm and 280 nm, with nominal half-max bandwidths of 9, 7 and 3 nm respectively (spectra shown in Fig. S2 of the Supporting Information). An Ocean Optics spectrometer USB2000 (Ocean Optics, Dunedin, FL) was used to obtain lamp output spectra with and without the bandpass filters for the quantum yield calculations. The irradiations for quantum yield calculations were carried out in 50 mm diameter dishes at incident irradiance of 1.0 mW cm 2. The corresponding Petri Factors were 0.97 and 0.99 for low pressure lamp and medium pressure lamp respectively. Diclofenac concentration in quantum yield experiments was 17 lM (5 mg L 1). 2.5. Dimer formation The effects of dissolved oxygen presence on formation of dimers were investigated. Duplicate samples were tested with and without dissolved oxygen at irradiance of 0.34 mW cm 2. Petri Factor was 0.97. Hach colorimeter and AccuVacÒ High Range and Low Range ampoules (Hach Company, Loveland, CO) were used to confirm oxygen concentration. The high oxygen samples were at equilibrium with the atmospheric oxygen. In the deoxygenated samples, oxygen was sparged by a nitrogen stream. Once the remaining oxygen concentration was confirmed to be <0.5 mg L 1, the sample was promptly covered with a UV-transparent quartz plate. The UV dosimetry was adjusted for the reflectance of the quartz by measuring the irradiance and the spectral irradiance with the sensor covered with the quartz plate. The dish was com-

pletely full and when covered remained head-space free throughout the experiment to avoid diffusion of oxygen from the atmosphere. At the end of the experiment it was confirmed that the concentration of dissolved oxygen remained at <0.5 mg L 1. 2.5.1. Singlet oxygen Furfuryl alcohol was used to capture formation of reactive singlet oxygen with diclofenac as a sensitizer (Haag, 1984). Furfuryl alcohol was spiked into the solution at 20 lM concentration with diclofenac at 340 lM concentration and irradiated to UV fluence of 2000 mJ cm 2 with the solution at equilibrium with atmospheric oxygen. A control sample contained furfuryl alcohol in lab grade water and was irradiated to 2000 mJ cm 2 to confirm that there was no detectable photolytic decay of furfuryl alcohol within the experimental UV fluence. 2.5.2. Dimer analysis A sample containing 340 lM of diclofenac was irradiated with approximately 2000 mJ cm 2 of 254 nm UV, at which point the parent compound was no longer detectable. The 2000 mJ cm 2 fluence was calculated based on the initial absorbance of the sample. At 340 lM concentration of diclofenac, the absorbance of the sample at the wavelengths emitted by the lamp changed during the treatment. However, all quantitative experiments were performed at low diclofenac concentrations where the absorbance change had no measurable effect. Brown color formed during irradiation and was confirmed visually and by measuring absorbance at 400 nm. The colored product of diclofenac photolysis was lyophilized using Freezone 2.5 freeze dry system (Labconco, Kansas City, MO) and analyzed with nuclear magnetic resonance (NMR), fluorescence spectroscopy and HPLC-MS. For NMR analysis, approximately 5 mg of the lyophilized substance was dissolved in approximately 0.5 mL of 99.8% pure deuterated methanol (Cambridge Isotope Laboratories, Inc., Andover MA). The samples were analyzed with Varian (Santa Clara, CA) INOVA 400 MHz NMR spectrometer (9.39 Tesla field). For fluorescence analysis, the samples were diluted to absorbance of 0.1 cm 1 at 254 nm and were analyzed using Fluoromax 4 fluorescence spectrometer (Horiba Jobin Yvon, Inc., Edison NJ). Instrument corrections (excitation and emission) were applied and the fluorescence spectrum was normalized to Raman intensity and corrected for inner filter. Fluorescence spectra were normalized to the DOC content. 3. Results and discussion 3.1. Quantum yield of photolysis Dimer formation at trace levels of diclofenac. When trace levels (34 nM) of diclofenac were irradiated, they resulted in formation of colored compounds. To better understand the nature and the process of formation of the colored compounds, higher starting concentrations of the compound had to be used. The equivalence of the product formation at both low and high concentration of diclofenac was confirmed by matching the absorbance spectra after exposure to the same UV dose. As seen in Fig. 1, both samples produced the same absorbance spectrum: the sample with 34 nM diclofenac concentrated to 68 lM after treatment, and the sample with 340 lM diluted to 68 lM after treatment. The dilution was necessary to stay within the measuring range of the spectrophotometer. Diclofenac has an absorbance spectrum with multiple characteristic peaks of UV photolysis products, so matching the spectra for the two samples indicates that products formed at both high (340 lM) and low (34 nM) starting concentration of diclofenac are very similar. This observation confirms that the same photolysis products form at trace levels of diclofenac found in the

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Absorbance, cm-1

3 2.5 2 1.5 1 0.5 0 200

250

300

350

400

Wavelength, nm 100 ppm irradiated with 100 mJ/cm2 and diluted to 20 ppm 10 ppb irradiated with 100 mJ/cm2 and concentrated to 20 ppm Fig. 1. Absorbance spectrum of diclofenac photolysis products at low and high concentration.

Table 1 Quantum yield values. Quantum yielda

Experiment Low pressure UV Medium pressure Medium pressure Medium pressure Medium pressure Medium pressure

UV, UV, UV, UV, UV,

0.19 ± 0.02 0.21 ± 0.02 0.20 ± 0.01 0.30 ± 0.04 0.21 ± 0.00 0.18 ± 0.05

full spectrum long-pass filter >295 nm band-pass filter 228 nm band-pass filter 254 nm band-pass filter 280 nm

a The average value of a duplicate test ± the difference between the average and the measured values.

environment and at higher concentrations used in this study. Slight differences in the intensity of some peaks are likely the result of some of the products having slightly different affinity for SPE extraction medium. SPE recovery was not monitored in the experiment because the goal of the SPE extraction was to concentrate the sample, to measure the absorbance spectrum, and to compare it to the spectrum of the sample with higher starting diclofenac

concentration. The results were not used to quantify the concentration of diclofenac or any products. The quantum yield of the parent compound was constant across the wavelengths. Low pressure and medium pressure lamps produced virtually the same value for the quantum yield of 0.19 ± 0.02 and 0.21 ± 0.02 respectively (Table 1). Although the quantum yield around k = 228 nm of 0.30 ± 0.04 appears to be higher than the rest of the values, and it is common for the quantum yield to be higher when the compound is exposed to higher energy wavelengths, the difference in this case with the full spectrum quantum yield is not statistically significant (p-value = 0.176). The quantum yield at wavelengths above 295 nm, around k = 254 nm and around k = 280 nm were all consistent with the full spectrum quantum yield (0.20 ± 0.01, 0.21 ± 0.00 and 0.18 ± 0.05 respectively). The quantum yield experiments were performed in unbuffered solution with pH dropping from 7.0 to 4.8 during the treatment. The compound was in the deprotonated form, and the experiment was completed before the pH of the solution approached the pKa of diclofenac = 4.2. At pH 4.8 about 20% of the compound was protonated. The direct photolysis rate constant was affected by <2% when lower pH values were dropped from the analysis. The calculated quantum yield values are lower than those reported previously for UV at 254 nm: 0.384 ± 0.075 for the deprotonated species (Canonica et al., 2008). The same study showed that fluence-based disappearance reaction rate constant for diclofenac did not change appreciably between pH 8 and 5. The compilation of quantum yields at 254 nm from different sources (Wols and Hofman-Caris, 2012) gave the range of values for diclofenac of 0.292 ± 0.086. This collective value has margins of error that include the results of this study. The difference between the results of the previous studies and this study is within the range common between independent studies. 3.2. Degradation pathway Several product peaks were observed by the MS after the sample was exposed to UV at 254 nm from a low pressure UV lamp (Fig. 2).

Product 6 m/z 549 Product 1 m/z 260

Intens.

Signal intensity, arbitrary units

x10

Parent m/z 296

8

Product 4 m/z 310

Product 5 m/z 589 Product 2 m/z 226

Product 3 m/z 256

2

4

6

8

10

Time [min]

Retention time, min Fig. 2. HPLC-(+)ESI-MS chromatogram of diclofenac (initial concentration 340 lM) after exposure to UV fluence of 100 mJ cm proposed chemical structures of the products.

2

from the low pressure Hg vapor lamp with

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The postulated products are numbered in the order they form. Diclofenac loses its first chlorine and closes the middle ring (Product 1, m/z 260, single chlorine signature), then it loses the second chlorine (Product 2, m/z 226, no chlorine signature), and then the molecule can attach 2 oxygen atoms through double bonds to aromatic carbons (Product 3, m/z 256, no chlorine signature). Product 4 with m/z of 310 and a 2-chlorine signature, which suggests the addition of oxygen and loss of 2 hydrogen atoms, elutes after the m/z 256 product but before all other products and the parent. The increased hydrophilic character (shorter retention time) of the polychlorinated moiety despite the increase in size compared to the parent compound suggests the addition of a polar group. Product 5 that elutes after the parent compound is a dimer with the m/z of 589 and a 4-chlorine signature. Its retention time is longer than that of the parent which is indicative of the increased hydrophobicity characteristic of a larger dimer molecule. A second small dimer peak (Product 6) that elutes immediately before the parent compound has m/z = 549 and 2-chlorine signature. The dimer is larger than the parent compound, so the fact that it has a shorter retention time indicates the addition of polar groups – in this case the two additional oxygen atoms, which is consistent with its m/z value. Product 4 appears to be an epoxide based on its m/z 310 which is 14 units higher than m/z 296 of the parent compound. This product is the second most polar of all products formed preceded only by the Product 3 that attaches two oxygen atoms. The fragmentation pattern and the chlorine signature clearly identify the peak as a product of diclofenac photolysis (Fig. S3 in Supplemental Data). Both the parent and Product 4 have the sodium adduct (+22 u), the loss of water ( 18 u), the loss of formic acid ( 46 u) and the loss of formic acid and one chlorine atom ( 81 u). A study of degradation products of diclofenac by photo-Fenton (Perez-Estrada et al., 2005) proposed an alternative structure for the product with m/z 310 as a parent molecule with an oxygen atom attached by a double bond to a single carbon (carbonyl) and the nitrogen forming a double bond instead of a single bond with one of the aromatic rings. However, the product with an oxygen attached to a single carbon through a double bond would be stable. Product 4 was short lived and was not detected when the samples were retested after several days. Its instability supports the identification of this product as an epoxide. It is possible that the product with 310 m/z formed in the photo-Fenton process was different from Product 4 in this study because of formation of different reactive species, such as the hydroxyl radical, during photo-Fenton compared to photolysis. Any unlikely hydroxyl radicals in this study were scavenged by methanol, so the only reaction pathways were through direct photolysis (hydrogen abstraction and C–Cl bond breaking) and oxidation by singlet oxygen.

As Product 4 (epoxide) disappeared after several days, the MS signal of the dimer increased (Fig. 3). It is especially evident at the 100 mJ cm 2 UV dose where both the epoxide and the dimer MS signals are well above the background noise. All other photolysis products and the parent compound remained unchanged in the sample at Day 0 and Day 6 of the analysis. Six day window was selected arbitrarily to allow time for any slower reactions with h 1 or d 1 rates to produce a measureable result. As evident from the results, some of the epoxide disappearance was not accounted for by the main dimer formation. This could mean that some of the epoxide product reacted further by other, non-dimer-forming pathways, e.g. a nucleophilic attack by chlorine radical that gets released during photolysis of diclofenac. The figure also highlights the different rates of formation of epoxide and dimer during the photolysis. Dimer forms at a slower rate, possibly because of epoxide being an intermediate step. It must be noted here that diclofenac product signals appear to be on the same scale as the parent signal and thus allow a mass balance of the constituents in the solution to be evaluated (Fig. S4 in Supporting Information). The mass balance was also confirmed by ion chromatography of the chloride that was liberated during the dechlorination of the parent molecule (Fig. S5 in Supporting Information). Estimated chloride was slightly higher than measured chloride for most of the samples. It is possible that the difference is within the experimental error. Alternatively, it is also possible that some minor chlorinated products formed but were not detected. Based on these observations, it appears that epoxide is a precursor for dimer formation. The parent loses one chlorine and then the other chlorine via photolytic decay as one degradation pathway, or it gets attacked by singlet oxygen and forms an epoxide that eventually leads to dimer formation as another pathway (Scheme 1). Singlet oxygen can also attach to the dechlorinated products but through a double bond to a single carbon rather than by forming an epoxide. It is possible that once the compound is dechlorinated, the loss of the strongly electronegative chlorine atoms causes the shift in the electron distribution within the aromatic ring that makes formation of epoxide less favorable than when the ring is chlorinated. Once the dimer forms, it continues to lose chlorines if exposed to UV. The sample that was exposed to high dose of UV (4000 mJ cm 2) for NMR and fluorescence analysis showed that the main product is a dimer but with no chlorines in its isotope signature (m/z 449). Fig. 4 shows the fragment ions characteristic of diclofenac: sodium adduct (+22), loss of water ( 18) and loss of formic acid ( 46). It also appears that the dimer is stable and not susceptible to additional UV degradation beyond dechlorination. Irradiation by polychromatic and monochromatic UV sources showed no difference in product formation.

Dimer

0.120 0.100 0.080 0.060 0.040 0.020 100-6

100-0

75-6

75-0

50-6

50-0

25-0

25-6

10-6

10-0

0-6

0.000 0-0

Product MS signal normalized to parent MS signal

3.3. Proposed mechanism of dimer formation Epoxide

UV Dose (mJ/cm2)-Day of analysis Fig. 3. Products within hours after the irradiation (Day 0 of analysis) and six days after the irradiation (Day 6 of analysis) at increasing UV doses.

When the effect of the dissolved oxygen on the formation of dimers was investigated, dimers only formed in the samples high in oxygen (see Fig. S6 of Supporting Information for chromatogram). The chromatogram showed two dimer peaks: one containing additional oxygen and thus eluting before the parent compound, and one that did not attach additional oxygen eluting after the parent compound. No dimers formed in the deoxygenated samples. Dissolved oxygen can be sensitized by the energy transfer from other constituents in solution and get promoted to an excited singlet state. Formation of singlet oxygen as a result of photosensitization of photolysis products of carprofen – a pharmaceutical structurally almost identical to diclofenac – has been detected previously (Bosca et al., 1997). Carprofen forms a stable methylcarbazole-2-acetic acid product when exposed to UV, similar to carbazole-1-acetic acid that forms during irradiation of diclofenac.

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m/z = 296

m/z = 260 m/z = 310

m/z = 589

m/z = 226

m/z = 549 m/z = 256

one of possible isomers

Scheme 1. Proposed pathway of dimer formation.

Intens.

+MS, 6.6min #385

Signal intensity, arbitrary units

x10 5

449.4

0.8 0.6 0.4

471.4

403.4

0.2 0.0

431.4

390

400

410

420

430

440

450

460

470

480

m/z

m/z Fig. 4. Mass spectrum of the dimer after 4000 mJ cm m/z 589 dimer without chlorines.

2

of UV showing MH+ at m/z 449, sodium adduct at m/z 471 and formic acid loss at m/z 403. m/z 449 corresponds to the

Based on structural similarity, diclofenac is also likely to be an effective sensitizer. Singlet oxygen can participate in reactions with unsaturated bonds and possibly enhance the formation of radicals that lead to dimerization. The results suggest that dissolved oxygen participates in the diclofenac dimer formation, possibly by creating an epoxide precursor. Epoxide could form from perepoxide which in turn forms by singlet oxygen addition to double bonds. Perepoxide formation as a result of a reaction with singlet oxygen has been long recognized for alkyl compounds (Mitchell, 1985) and more recently has been identified for an unsaturated cyclic organic compound (Leach et al., 2008). Oxygenated species were observed in this study and in the previous studies and confirmed by NMR (Eriksson et al., 2010). It appears that dissolved oxygen is likely to react with diclofenac and attach at the location of the highly reactive unpaired electron formed during photolytic hydrogen abstraction. It is possible that instead of attaching through a double bond to one carbon, it can attach to two adjacent carbons and form a perepoxide. The second oxygen of the diatomic oxygen molecule then is

attacked by the hydrogen radicals that form during the hydrogen abstraction process. The epoxide molecule that forms in the process is not stable and will eventually break the epoxide ring. The resulting radical will recombine with another radical to form a stable molecule. Because of the relative stability of epoxides compared to radicals, the dimerization process could continue when the source of UV is off. When the epoxide ring breaks, it will create a radical which would recombine with another radical that formed in the same manner. The proposed mechanism of epoxide formation and subsequent dimer formation is outlined in Scheme 2. Change of color (measured by absorbance at 400 nm) was observed in the diclofenac solution with increasing treatment. The color peaks measured by diode array detector corresponded to dimer peaks in the LC-(+)ESI-MS chromatogram. The parent compound is colorless. The color can be an indicator of a higher degree of conjugation of the products. For example, it has been noted that the conjugation of more than 3 double bonds will lead to color formation (Thurman, 1985). Its link to dimers was confirmed by comparing the chromatogram of the high oxygen sample in which

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Signal intensity, arbitrary units

Scheme 2. Proposed mechanism of dimer formation through an epoxide precursor.

Dimer m/z 549

Parent

Retention time, min

Fig. 5. Left – HPLC-DAD chromatograms at 400 nm for oxygenated (gray) and deoxygenated (black) irradiated solutions of diclofenac. Additional peaks in the gray chromatogram potentially correspond to a variety of dimers that exist at concentrations too low for detection by ion trap but strongly absorb visible light. Right – an HPLC(+)-ESI-MS chromatogram (black) and HPLC-DAD chromatogram at 400 nm (gray) of the low irradiance high oxygen sample.

dimers were detected and the low oxygen sample with no dimer formation (Fig. 5). Many additional peaks appeared on the HPLCDAD chromatogram at 400 nm including the peak that corre-

sponded to a dimer peak of the MS chromatogram. The peaks in the UV chromatogram that did not correspond to the peaks in the MS chromatogram are attributed to a variety of other possible

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400

120

350

3

80 60 40

300

2

100

350

Excitation

4

300

Excitation

400

5

1

20

300

350

400

450

500

250

250

0

0

300

550

Emission

parent–product and product–product dimers that do not form in quantities detectable by ion trap but produce significant visible color change even at very low concentrations. The most abundant dimer was the dimer made of the two parent molecules followed by the dimer of the parent with a doubly dechlorinated product. Although the dimer peak is much smaller in the MS chromatogram, its absorbance at 400 nm is disproportionally large confirming the hypothesis that the color formation in the treated diclofenac samples is attributed to dimers. 3.4. Singlet oxygen formation Because diclofenac degrades quickly under UV, a study was conducted to confirm that the doubly dechlorinated product (Product 2) is stable up to the UV doses necessary to detect decay in furfuryl alcohol concentration attributable to singlet oxygen. The product increased rapidly to its maximum concentration for UV fluence from 0 to 500 mJ cm 2 and then slowly decreased (about 13 times slower than the increase rate) from 500 to 4000 mJ cm 2. The concentration of the Product 2 was no lower at 4000 mJ cm 2 than at 50 mJ cm 2. The difference between those concentrations and the peak concentration was approximately a factor of 3. Because of this variation, it would be difficult to calculate the quantum yield of singlet oxygen formation from diclofenac. If it is assumed that Product 2 is the primary sensitizer (the parent and other products decrease rapidly) and its average concentration in solution during the experiment is used in calculations ignoring the factor of 3 variability, its effectiveness to sensitize singlet oxygen appears to be approximately 60% of the effectiveness of methylene blue – one of the standard singlet oxygen sensitizers with 0.37 quantum yield (Gerdes et al., 1997). Oxidation rate of furfuryl alcohol by singlet oxygen in the presence of 20 lM methylene blue and in the presence of 57 ± 18 lM of Product 2 differed by a factor of approximately 1.8 (Fig. S7 in the Supporting Information). Thus the quantum yield of singlet oxygen sensitization from Product 2 can be estimated to be 0.2. This corresponds well to the quantum yield 0.18 of singlet oxygen photosensitization during the photolysis of carprofen (Bosca et al., 1997) which forms essentially the same stable photoproduct as diclofenac. 3.5. Dimer analysis The NMR scan revealed that the dimers still retain structural resemblance to the parent compound (Fig. S8 in Supporting

400

450

500

550

Emission 2

(right) solutions of diclofenac. The two plots are on different scales.

Signal intensity, arbitrary units

Fig. 6. Fluorescence of untreated (left) and treated with 2000 mJ cm

350

Dimer

6

8

10

Time [min]

Retention time, min Fig. 7. Total ion chromatogram of diclofenac photolysis products in laboratory grade water (red) and effluent (green). Black is the wastewater baseline. First dimer peak is m/z 549, second is m/z 589. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Information) and is relatively homogeneous in character. This indicates that there is an energetically preferred location for the unpaired electron involved in dimer formation. Fig. 6 compares the fluorescence excitation–emission matrix for a solution of parent diclofenac and solution of its products after it has been exposed to 2000 mJ cm 2 of 254 nm wavelength UV. The untreated diclofenac exhibits two distinct fluorophores both emitting in the 340–410 nm range with 280–300 nm and 310–340 nm excitation ranges. A minor fluorophore is also present with excitation range 250–260 nm and emission at 350–370 nm. In the treated sample, those peaks are still present but they are overshadowed by the new peak which forms at the low excitation wavelengths (<240–270 nm) with high intensity emission between approximately 340 and 450 nm. There is a clear red shift in emission combined with blue shift in excitation which indicates that the material that forms has a higher degree of conjugation than the parent molecule. SUVA of diclofenac increased from 1.76 before the treatment to 4.41 after the treatment which also confirms an increase in the degree of conjugation of the molecule confirming formation of dimers. All of the results suggest that under UV irradiation smaller organic molecules of diclofenac can combine into bigger ones. The process was driven by formation of radicals which happens when chemical bonds within a molecule break following absorption of

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a photon. Dimerization was enhanced by the presence of dissolved oxygen. These results have implications for potential incorporation of trace organic pollutants into dissolved organic matter during UV irradiation. During UV disinfection of wastewater, singlet oxygen forms because of the oxygen sensitizing properties of dissolved organic matter (Zepp et al., 1977). Micropollutants susceptible to UV photolysis or reactive with singlet oxygen can form dimers as a result. This is especially true with the loss of chlorine, which we hypothesize is a driving mechanism for dimer formation. Larger product molecules such as dimers may be less toxic than the parent compound due to their decreased mobility through cell membranes. Additionally, if the radicals form bonds with the background organic matter instead of each other, they become parts of larger organic matter molecules and lose their identity and as a result their properties of concern. Indeed, when diclofenac irradiation was performed in filtered wastewater treatment plant effluent (see Table S1 for water quality parameters), no dimers were observed similar to the ones forming during experiments in laboratory grade water (Fig. 7). Also the authors note that in spite of widespread over the counter use of diclofenac, it is rarely detected in wastewaters (Ferrer and Thurman, 2010) or at ng/L levels only, which is further evidence of decomposition of the parent compound. 4. Conclusions Singlet oxygen was demonstrated to be instrumental in dimer formation during UV photolysis of diclofenac, via formation of an intermediate epoxide. This could be a potential degradation pathway for other trace organic contaminants. The evidence of formation of larger organic molecules from smaller ones via non-biological processes presented here may be relevant to humification of organic matter in the environment. While humification is a biologically mediated process of smaller organic molecules combining into larger ones, it is possible that formation of radicals via natural sunlight photolysis or during UV disinfection can lead to chemical bonding between molecules of organic matter. Further studies are necessary to demonstrate the photo-initiated humification process for natural organic matter in general. Acknowledgements This article was developed in part under STAR Fellowship Assistance Agreement no. FP917136 awarded by the U.S. Environmental Protection Agency (EPA). It has not been formally reviewed by EPA. The views expressed in this article are solely those of the authors and are not endorsed by EPA. Thurman acknowledges discussions with L. Perez Estrada about color formation (2005), which led to ideas in this study. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.chemosphere. 2013.06.079. References Agüera, A., Pérez Estrada, L.A., Ferrer, I., Thurman, E.M., Malato, S., Fernández-Alba, A.R., 2005. Application of time-of-flight mass spectrometry to the analysis of phototransformation products of diclofenac in water under natural sunlight. J. Mass Spectrom. 40 (7), 908–915. Bolton, J.R., Linden, K.G., 2003. Standardization of methods for fluence (UV dose) determination in bench-scale UV experiments. ASCE J. Environ. Eng., 209–215. Bosca, F., Encinas, S., Heelis, P.F., Miranda, M.A., 1997. Photophysical and photochemical characterization of a photosensitizing drug: a combined

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