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Development of Fluorescence Surrogates to Predict the Photochemical Transformation of Pharmaceuticals in Wastewater Effluents Shuwen Yan, Bo Yao, Lushi Lian, Xinchen Lu, Shane A. Snyder, Rui Li, and Weihua Song Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b05251 • Publication Date (Web): 15 Feb 2017 Downloaded from http://pubs.acs.org on February 15, 2017

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Environmental Science & Technology

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Development of Fluorescence Surrogates to Predict the Photochemical

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Transformation of Pharmaceuticals in Wastewater Effluents

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Shuwen Yan1, Bo Yao1, Lushi Lian1, Xinchen Lu1, Shane A. Snyder2, Rui Li1, and Weihua Song1,*

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Department of Environmental Science & Engineering, Fudan University, Shanghai 200433, P. R.

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China

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USA

Department of Chemical & Environmental Engineering, University of Arizona, Tucson, AZ 85721,

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Resubmitted to Environ. Sci. & Technol.

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*Corresponding author; email: [email protected]; Tel: (+86)-21-6564-2040

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ACS Paragon Plus Environment


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Abstract

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The photochemical transformation of pharmaceutical and personal care products (PPCPs) in

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wastewater effluents is an emerging concern for environmental scientists. In the current study, the

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photodegradation of 29 PPCPs was examined in effluents under simulated solar irradiation. Direct

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photodegradation, triplet state effluent organic matter (3EfOM*)-mediated and hydroxyl radical

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(HO•)-mediated degradation are three major pathways in the removal process. With the

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photodegradation of trace levels of PPCPs, the excitation-emission matrix (EEM) fluorescence

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intensities of the effluents were also gradually reduced. Therefore, fluorescence peaks have been

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identified, for the first time, as appropriate surrogates to assess the photodegradation of PPCPs. The

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humic-like fluorescence peak is linked to direct photolysis-labile PPCPs, such as naproxen,

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ronidazole, diclofenac, ornidazole, tinidazole, chloramphenicol, flumequine, ciprofloxacin,

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methadone, and dimetridazole. The tyrosine-like EEM peak is associated with HO•/CO3•--labile

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PPCPs, such as trimethoprim, ibuprofen, gemfibrozil, atenolol, carbamazepine, and cephalexin. The

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tryptophan-like peak is associated with 3EfOM*-labile PPCPs, such as clenbuterol, metoprolol,

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venlafaxine, bisphenol A, propranolol, ractopamine, salbutamol, roxithromycin, clarithromycin,

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azithromycin, famotidine, terbutaline, and erythromycin. The reduction in EEM fluorescence

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correlates well with the removal of PPCPs, allowing a model to be constructed. The solar-driven

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removal of EEM fluorescence was applied to predict the attenuation of 11 PPCPs in five field

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samples. A close correlation between the predicted results and the experimental results suggests that

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fluorescence may be a suitable surrogate for monitoring the solar-driven photodegradation of PPCPs

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in effluents.

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Introduction

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Pharmaceuticals and personal care products (PPCPs) are a group of emerging contaminants

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and have drawn considerable attention from environmental scientists and engineers. PPCPs are

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primarily discharged into surface water from municipal wastewater treatment plants due to their

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incomplete elimination by existing biological treatment processes.1-3 Consequently these

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wastewater-derived recalcitrant micropollutants have been increasingly detected in aquatic

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environments all over the world. The concentrations of PPCPs were reported in the range of ng L-1 to

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µg L-1 in surface water,4, 5 which poses largely unknown long-term risks to the ecological system.

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This fact has initiated an enormous scientific effort to understand the transformation and fate of

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PPCPs in surface water.

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Phototransformation driven by sunlight is one of the most important natural processes for the

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attenuation of PPCPs in surface water.6 In general, this process includes direct and indirect

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photodegradation.7 Direct photodegradation requires overlap between the absorption spectra of

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PPCPs and the solar irradiation wavelengths, and as a consequence of that light absorption, PPCPs

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undergo transformation. Indirect photodegradation is associated with triplet states of organic matter

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(3OM*)8 and a series of reactive oxygen species (ROS), such as singlet oxygen (1O2), hydroxyl

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radicals (HO•), superoxide anions (O2•-), carbonate radicals (CO3•-), and halogen radicals.9-15 These

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ROS and 3OM* are capable of oxidizing a wide variety of pollutants present in wastewater effluents

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and also facilitate the decomposition of organic matter.16-20 However, modeling the photochemical

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transformation of PPCPs in effluent dominated waters is challenging for several reasons: 1)

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Photodegradation involves complicated processes that are chemical structure dependent and involve

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a variety of ROS. The complete modeling of these processes is daunting work. 2) The analysis of

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trace amounts of PPCPs is labor intensive, time consuming, and requires costly analytic

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instrumentation. 3) With new PPCPs constantly introduced into the market, the PPCPs of interest

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change with time. 3



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The development of indicators and surrogates may be a useful approach to resolving the

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aforementioned challenges. Numerous trace indicator compounds, including caffeine, nicotine, and

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metformin, have been successfully used to indicate the influence of wastewater on receiving water

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bodies.21,

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indicator tailored to monitor the removal efficiency of trace amounts of organic contaminates (TrOCs)

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during advanced oxidation processes (AOPs).23 Dilantin, DEET, meprobamate, and iopromide were

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proposed as four potential indicators to assess the optimized oxidation conditions in the ozonation of

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tertiary-treated wastewaters.24 Nevertheless, costly tandem mass spectrometry is required for

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monitoring the trace amounts of indicator compounds, limiting the applicability of this technique.

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Furthermore, spectroscopic parameters, including true color,25 ultraviolet absorption (UVA)24 and

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fluorescence,26-29 were employed as surrogates to assess the elimination of PPCPs during the

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traditional and advanced wastewater treatments. Our previous study also noted that the decrease in

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protein-like fluorescence is well correlated with the removal of PPCPs from reverse osmosis (RO)

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retentate via HO• oxidation.30 Spectroscopic surrogates, which are much easier to measure than

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indicator compounds, may provide a rapid and inexpensive online method for the quantitative

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estimation of the removal of PPCPs.

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For advanced treatments, Lester et al. recently applied sucralose as a conservative

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While most of the aforementioned outcomes are initiated by ozonation and AOPs, there is an

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opportunity to apply spectroscopic surrogates to evaluate the attenuation of PPCPs in effluents under

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solar irradiation. Both O3/AOPs and photodegradation involve ROS as key intermediates,

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encouraging the investigation of spectroscopic surrogates for the phototransformation of PPCPs. In

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this study, fluorescence surrogates elevated from effluent organic matter (EfOM) have been carefully

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evaluated because they are more sensitive and specific than UVA surrogates. EfOM and PPCPs are

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co-discharged into surface water and exposed under solar irradiation. In general, EfOM contain

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residual natural organic matter (NOM) from the drinking water supply and soluble microbial

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products (SMPs) contributed from biological treatment.31 As a result, the excitation-emission matrix 4


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(EEM) fluorescence of EfOM can be characterized as containing humic-like, tyrosine-like and

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tryptophan-like peaks. Fluorescent humic acids, tyrosine and tryptophan can undergo

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photo-transformations involving both direct and indirect photodegradation. The latter is driven by

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3

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EfOM and the attenuation of PPCPs may provide an alternative for the rapid monitoring of the

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transformation of PPCPs in surface water. To the best of our knowledge, we report herein the first

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study focused on the development of fluorescence surrogates to predict the photochemical

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transformation of pharmaceuticals in wastewater effluents.

OM*, HO•, and 1O2 et al.32-35 The correlation between the decrease of fluorescent surrogates of

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To test this approach, 29 PPCPs were chosen as target contaminates because they have been

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widely detected in wastewater effluents and contain a variety of photochemical properties.36 We

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investigated four pathways composed of direct photodegradation and indirect photodegradation

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reactions with 3OM*, HO•/CO3•-, and 1O2. UVA and fluorescence spectroscopy were applied to

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examine EfOM photo-transformations. Correlations between the removal of PPCPs and the changes

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in wastewater optical properties were examined in detail.

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Experimental Section

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Chemicals. Deuterium oxide (D2O, 99.9%), furfuryl alcohol (FFA, 99%), terephthalic acid

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(TA), isopropanol (IPA), and isoproturon (IPU) were purchased from Sigma-Aldrich and used as

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received. 2-Hydroxyl terephthalic acid (HOTA) was synthesized using a method reported in the

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literature.37 All PPCPs were purchased from Sigma-Aldrich or TCI Chemicals at the highest

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available purity. The isotopic labeled compounds used as internal standards were purchased from

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Toronto Research Chemicals. The list of PPCPs and isotopic compounds can be found in Table S1 in

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the supporting information (SI). All solutions were prepared using Milli-Q water.

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Tested waters. The photodegradation model was based on a typical secondary treated

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wastewater effluent collected from a municipal sewage plant located in Jiangsu Province, China.

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This sewage plant treats domestic sewage from the eastern district of the city of Taicang using a 5



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circulatory activated sludge treatment system. Samples were collected in acid-cleaned plastic

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containers and transported to the laboratory within 2 h for preparation. The collected effluent was

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immediately filtered through an acid-cleaned membrane (0.22 µm) to remove free bacteria and solids

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and stored at 4 °C. The effluent for pH influence test was collected from the same facility, but 1 year

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later. Sodium hydroxide and phosphoric acid were used to adjust pH. All water quality parameters

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after filtration are provided in Table S2 of the SI.

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Irradiation experiments. Photolysis experiments were conducted in a photo-reactor (Suntest

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XLS+) with a xenon lamp. The lamp was fitted with a special quartz-glass filter to block the

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transmission of wavelengths below 290 nm and to produce simulated natural sunlight. A temperature

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control unit (Suncool®) was employed to fix the temperature at 25 °C. The emission spectrum for the

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lamp, compared to natural sunlight, is shown in Figure S1 in the SI. The irradiation intensity was

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continually measured with an online sensor and fixed at 40 W m-2 in the range of 290 - 400 nm. All

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solutions were prepared in 200 mL cylindrical quartz glass sealed vials that allow full transmission of

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UV light from solar irradiation. The steady-state concentrations of 1O2, HO•, and CO3•-, were

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measured using methods reported in previous studies,38-40 and the detailed procedures are described

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in Text S1 and Figures S2-S5 in the SI. The measurement of bimolecular reaction rate constants for

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carbonate radicals with PPCPs was also illustrated in Text S1 of SI. UV-visible spectra were obtained

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using a spectrophotometer (Cary 60, Agilent). The TOC content of the solutions was acquired using a

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TOC analyzer (Shimadzu, TOC−CPH/CN). The anions were analyzed using an ion chromatograph

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(Metrohm 883).

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Online SPE LC-MS/MS analysis. The chemical structures of 29 PPCPs are shown in Table

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S1 of the SI. Approximately 1 µg L-1 of each PPCP was spiked into the effluents to mimic

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environmental concentrations. The trace levels of PPCPs were analyzed using an automated online

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SPE system coupled to a liquid chromatography triple quadrupole mass spectrometer (LC-MS/MS,

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Agilent 1290-6430 with Flexcube module). Only 2.0 mL of the solutions was removed at the time 6


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intervals for LC-MS/MS analysis to minimize the interference from sample collection. The detailed

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experimental setup is described in Text S2 and Table S3 of the SI. The LC-MS/MS conditions of

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each PPCP are also available in Table S1 in the SI. The current method is substantially time, labor,

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and solvent efficient compared to the traditional offline SPE methods while also increasing the

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reproducibility of analysis.41

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EEM florescence spectroscopy experiments and parallel factor analysis (PARAFAC). All

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3D EEM experiments were performed with a fluorescence spectrometer (Aqualog, Horriba). EEM

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experiments consisted of 226 emission scans (250 - 620 nm) collected over excitation wavelengths

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ranging from 240 to 600 nm at 3 nm increments. The 3D EEM data were processed with inner filter

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correction and Rayleigh scattering elimination. Finally, 1.3 µM of quinine sulfate was used to

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normalize the data to allow inter-laboratory comparison. In this study, parallel factor analysis

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(PARAFAC) was used to disassemble the EEM spectra into their underlying chemical components.

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The algorithm used was the N-way Toolbox for MATLAB®.42 The number of components

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(fluorophores) that best fit a model for each set of 3D EEM spectra was determined by minimizing

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the sum of squared residuals. The model was validated with the core consistency diagnostic provided

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in the N-way toolbox and split-half analysis.

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Field studies for fluorescence surrogates. Five field samples were collected from local

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rivers (Suzhou River, Huangpu River, Xietang River, Longhuagang River, and Shagang River, all

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located in Shanghai, China), which are dominated by wastewater effluents. The water quality

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parameters are provided in Table S2 of the SI. Field samples were enriched by SPE after 5 h of

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irradiation in the solar simulator and analyzed using LC-MS/MS (Text S3 of the SI). Only 11 of 29

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PPCPs were quantified in this experiment, and their concentrations are shown in Table S4 of the SI.

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The corresponding decrease of EEM fluorescence was then used to predict the photo-attenuation of

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PPCPs.

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Results and Discussion 7



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Grouping PPCPs on the basis of their photochemical properties. In this study, 29 PPCPs

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were selected because they are widely identified in effluents and surface waters. The initial

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experiment was conducted in Milli-Q water at pH 7.0 under simulated solar irradiation. The

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first-order degradation rates of PPCPs were measured as kDI, which represents the attenuation of

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PPCPs under direct photodegradation. The kDI of PPCPs ranged from 5×10-4 to 4.6 h-1, indicating the

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noteworthy diversity of PPCPs involved in direct photodegradation. In the presence of wastewater

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effluents, the first-order degradation rates of PPCPs were observed as kEw, which represents the

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integrated degradation rates from both direct and indirect photodegradation. In this study, kEw are

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observed in the range from 2×10-2 to 8.3 h-1. Furthermore, a series of enhancing/quenching

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experiments were performed to assess the role of ROS in indirect photodegradation. To explore the

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role of 1O2, the effluent was freeze dried and redissolved in D2O because 1O2 has a longer life time

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due to the isotopic effect.43 IPA (0.2 mM) was employed to minimize the effect of HO•, and the

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first-order degradation rates are denoted as kIPA. In the IPA spiked effluents, not only were the steady

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state concentrations of HO• sharply decreased but the effects of CO3•- were also minimized because

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most of the CO3•- originates from HO• through one electron oxidation of bicarbonate.44 Furthermore,

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argon saturated effluents were irradiated to enhance the 3OM* effect because dissolved oxygen can

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act as a 3OM* quencher and yield 1O2.45 The first-order degradation rates of argon-saturated effluents

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are referred to as kAr.

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As shown in Eq. 1, kEw includes direct photodegradation (kDI) and indirect photodegradation.

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The indirect photodegradation is further classified into the contributions of HO•, CO3•-, 3OM* and

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1

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O2. = + • + • + ∗ +

(1)

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All tested PPCPs were classified into three groups based on their photochemical properties, namely,

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Group I: direct photodegradation dominated; Group II: HO•/CO3•- dominated; and Group III: 3OM*

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dominated. As illustrated in Figure S6, 1O2 has minor effects on the photochemical transformation of 8


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the tested PPCPs, therefore it was not considered as a dominated group. The photochemical features

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of each group are following:

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Group I: Figure 1a demonstrates that the direct photodegradation contribution ( ) for the

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group I compounds is greater than 50%, indicating that they are labile to direct photolysis. Group I

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includes naproxen, methadone, ronidazole, dimetridazole, diclofenac, ornidazole, tinidazole,

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chloramphenicol, flumequine, and ciprofloxacin. Their half-lives in the effluent were measured from

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0.08 to 3 h with a median half-life of 0.6 h. Dimetridazole is employed as an example, as illustrated

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in Figure 2a. The pseudo first-order decay rates (k) of dimetridazole did not alter significantly under

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five experimental conditions, including Di-H2O, effluent, 1O2 enhanced (effluent/D2O), HO•/CO3•-

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scavenged (effluent/IPA), and 3OM* enhanced (argon saturated effluent). These results indicated that

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direct photodegradation is the dominant process. The other degradation profiles for the group I

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compounds can be found in Figure S6 in the SI. The results indicate that direct photodegradation is a

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major contributor to the degradation of this group, referred as the “direct photolysis-labile group.” As

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previous studies have reported, all group I compounds present either significant UV absorption that

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overlaps with the solar irradiation spectrum and/or high quantum yield to be decomposed.7, 46

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Group II: Excluding group I compounds, other compounds are labile to indirect photolysis (>

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50%), as shown in Figure 1a. To explore the indirect photodegradation mechanism, IPA was applied

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to minimize the effects of HO• and associated CO3•-, and the contribution of HO•/CO3•- can be

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calculated as

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gemfibrozil, carbamazepine, ibuprofen, and cephalexin, belong to group II, and HO•/CO3•- is

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responsible for more than 50% of their indirect photodegradation. Gemfibrozil, as an example from

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this group, is shown in Figure 2b. The half-lives of the compounds in group II were measured from 5

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to 35 h, with a median of 26 h. These values present the longest half-lives among all three groups,

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demonstrating that group II compounds are relatively photo-resistant. On the basis of the

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photochemical features within this group, it is referred to as the “HO•/CO3•--labile group.”

, as

shown in Figure 1b. Six pharmaceuticals, including trimethoprim, atenolol,

9



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Furthermore, the contributions of CO3•- for photodegradation of group II were determined

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based on an assessment of the steady-state concentrations of CO3•- and bimolecular reaction rate

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constants with PPCPs. As illustrated in Table S5, the CO3•- reaction rate constants varied from < 105

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to 107 M-1s-1, which are notably lower than the HO• reaction rate constants (~109 M-1s-1). Although

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[CO3•-]ss (6.7 × 10-14 M) was one order magnitude higher than [HO•]ss (1.2 × 10-15 M), CO3•- still

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played a minor role in the photodegradation of group II compounds at neutral pH.

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Group III: The rest of the PPCPs can be classified as group III, including clenbuterol,

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metoprolol, venlafaxine, bisphenol A, propranolol, ractopamine, salbutamol, roxithromycin,

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clarithromycin, azithromycin, famotidine, terbutaline, and erythromycin. Their half-lives were

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measured in the range of 0.8 to 12 h, with a median half-life of 4 h. These compounds are degraded

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more slowly than the compounds of group I and faster than those of group II. One of the most

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obvious characteristics of group III compounds is that their kAr was 2-fold higher than their kEw.

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Erythromycin, as a model compound, is shown in Figure 2c. The enhanced degradation effect of

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argon-saturated conditions was calculated as

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to degradation, this group was referred to as the “3OM*-labile group.” The primary step of 3OM*

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reaction with group III compounds has been proposed as electron transfer or hydrogen abstraction

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processes, which mainly occurs at electron donor groups.

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. Considering the notable contribution of 3OM*

Our aforementioned grouping principle is in close agreement with previous studies,7,

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although most of them focused on the solar-driven attenuation of PPCPs in NOM-enriched solutions.

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In contrast to natural waters, effluents present considerably higher levels of nitrate ion, which is an

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important photosensitizer for HO• generation; therefore, the contribution of HO• in the effluent may

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be more substantial than in the NOM-enriched solutions.

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Phototransformation of EEM spectra of EfOM. In addition to the photodegradation of

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PPCPs, the progressive changes in the fluorescence spectra of effluents were investigated. As

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illustrated in Figure S7 in the SI, the EEM spectra of the effluents undergo considerable alteration 10


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during the simulated solar irradiation. Considering the possible overlap and interference among

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different fluorescence peaks, PARAFAC was used as a mathematical tool to disambiguate the

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fluorescence EEM spectra into several individual fluorescent components that are independent from

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each other. As illustrated in Figure 3, all emission loadings of components are unimodal. The single

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emission maxima suggests that the model was successful at grouping the fluorophores present into

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groups with similar molecular structure and/or fluorescence properties even though the model was

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not constrained to do this. The model was split-half validated and contained three components; each

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component consisted of a pair of well-rounded symmetrical peaks, with fluorescence located at the

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same λem but two different λex. Furthermore, each component was located at the shortest λex peak that

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had the highest fluorescence intensity, as described for pure compounds and recommended for

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PARAFAC modeling.48 Based on the profile of the EEM spectra,31 three peaks were identified: a

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tyrosine-like peak (λex/λem:

Development of Fluorescence Surrogates to Predict the

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