Photochemically Induced Formation of Reactive Oxygen Species

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Photochemically Induced Formation of Reactive Oxygen Species (ROS) from Effluents Organic Matter Danning Zhang, Shuwen Yan, and Weihua Song Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/es5028663 • Publication Date (Web): 06 Oct 2014 Downloaded from http://pubs.acs.org on October 7, 2014

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Environmental Science & Technology

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Photochemically Induced Formation of Reactive Oxygen Species (ROS) from

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Effluent Organic Matter

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Danning Zhang, Shuwen Yan and Weihua Song*

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

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*Corresponding author: email: [email protected]

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Tel: (+86)-21-6564-2040

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Submitted to Environmental Science & Technology

1 ACS Paragon Plus Environment


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Abstract

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The formation of reactive oxygen species (ROS) from effluent organic matter (EfOM) was investigated

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under simulated solar irradiation. In this study, EfOM was isolated into three different fractions based on

21

hydrophobicity. The productivity of ROS in EfOM was measured and compared with that of natural

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organic matter (NOM) isolates, including Suwannee River humic acid/fulvic acid (SRHA/FA) and Pony

23

Lake fulvic acid (PLFA). The hydrophilic (HPI) component had a greater quantum yield of 1O2 than

24

those of the hydrophobic (HPO) and transphilic (TPI) fractions because the HPI contained peptides and

25

proteins. Regarding O2•−, the phenolic moieties acted as electron donating species after photochemical

26

excitation and therefore electron transfer to oxygen. A positive correlation was found between the

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phenolic concentrations and the steady state O2•−concentrations. H2O2 accumulated during the irradiation

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process from superoxide as precursor. Potentially, due to the presence of proteins or other organic

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species in the HPI fraction, the decay rates of H2O2 in the dark for both the effluent wastewater and the

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HPI fraction were significantly faster than the rates observed in the standard NOM isolates, the HPO and

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TPI fractions. Autochthonous NOM showed a higher •OH productivity than terrestrial NOM. The

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[•OH]ss was lowest in the HPI fraction due to the lack of humic fraction and existence of soluble

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microbial products (SMPs), which easily reacted with •OH. Overall, the HPO and TPI fractions were the

34

major sources of superoxide, H2O2 and •OH under simulated solar irradiation. The HPI fraction

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dominated the production of 1O2 and acted as a sink for H2O2 and •OH.


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Introduction Effluent organic matter (EfOM) is discharged into surface water from wastewater treatment

37 38

plants (WWTPs) and has received considerable attention regarding its environmental impacts.1-4 EfOM

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is composed of a combination of background natural organic matter (NOM), soluble microbial products

40

(SMPs) and trace amounts of organic pollutants. Most of the NOM present in drinking water remains in

41

the water after use and enters WWTPs through sewer lines.5 SMPs are derived from biological processes

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in WWTPs and can be categorized as hydrophilic molecules. The presence of SMPs is hypothesized to

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affect the environmental fate of trace organic pollutants, most of which are resistant to biodegradation

44

and exert a negative ecological impact.6-9 Among others, trace pollutants include industrial waste,

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pharmaceuticals and personal care products (PPCPs), endocrine-disrupting chemicals (EDCs),

46

disinfection by-products (DBPs), and herbicides.

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In the last 30 years, the photochemistry of NOM has been extensively studied because NOM is

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ubiquitous in natural water systems and is an important chromophore.10-16 Numerous researchers have

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made significant contributions to the description of the photochemical properties of NOM, and a series

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of well-accepted theories has been established.13, 17-23 Under solar irradiation, NOM can be excited to a

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singlet state and rapidly undergoes intersystem crossing to the triplet excited state (3NOM*). Next, the

52

3

53

(1O2), hydroxyl radicals (•OH), superoxide anions (O2•−), and H2O2. 18, 19, 24, 25

NOM* can react with dissolved oxygen to form reactive oxygen species (ROS), such as singlet oxygen

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Despite the numerous studies on NOM, the environmental impact of EfOM is not fully

55

understood. Relative to NOM, previous studies have indicated that EfOM exhibits distinct properties,

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including a specific SUVA, greater hydrophilic organic matter concentrations, fluorescence index (FI)

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values and polysaccharide contents, and a clear protein-like peak in its EEM.26 Therefore, the

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transformation and fate of EfOM in environmental and engineered systems is different from those of



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NOM. SMPs are common and account for a major portion of dissolved organic carbon (DOC) in

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wastewater effluent.2 SMPs result in more frequent membrane fouling during wastewater reclamation.27

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When a source of drinking water is impaired by municipal wastewater effluent, nitrogen-based

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disinfection byproducts (N-DBPs) are a cause of concern because the organic nitrogen compounds

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present in EfOM are a common precursor for N-DBPs.28

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Several studies have reported that the photochemical properties of EfOM differ from those of

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NOM in surface waters. When employing EfOM as a photosensitizer, the quantum yields of 1O2 and

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•OH generation were found to be higher than when using NOM.4, 29 The generation of 1O2 and •OH by

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NOM and EfOM fractions with different molecular weights was also investigated.29 To our knowledge,

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no reports have focused on other ROS, such as O2•− and H2O2. Furthermore, in this study EfOM was

69

isolated according to hydrophobicity. Each fraction included functional groups that were more

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specifically clustered than when separated by molecular weight cutoff. The isolation of EfOM will be

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useful for additional studies that aim to determine the specific roles of different functional groups during

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the generation of each ROS. Because the extent of human influence differs depending on the drainage

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basin of each water system, the proportions of EfOM and NOM present may also differ. Thus, it is

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necessary to explore ROS production in EfOM systems, which may have enormous influence on the

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environmental fate and transformation of trace organic pollutants.

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As mentioned above, the current study conducted a comprehensive isolation of EfOM based on

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the hydrophobicity of its different constituents, including the hydrophobic (HPO), transphilic (TPI) and

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hydrophilic (HPI) fractions. Leenheer and Benjamin et al. pioneered the comprehensive isolation of

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NOM in the 1980s. This isolation method was then further developed by subsequent investigators.30, 31

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As a unique component of EfOM, SMPs are derived from biological processes of WWTPs and behave

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as hydrophilic molecules that only remain in the hydrophilic (HPI) fraction following the isolation


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procedure.2 The initial objective of this research was to evaluate the abilities of different EfOM fractions

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for generating the four different ROS relative to NOM isolates under simulated solar irradiation.

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Additional innovative efforts were made to explore the internal correlation between their photochemical

85

behaviors and functional groups.

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Materials and Methods

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Chemicals. Suwannee River humic acid (SRHA), Suwannee River fulvic acid (SRFA) and Pony

88 lake fulvic acid (PLFA) were purchased from the International Humic Substances Society. Furfuryl 89 alcohol, terephthalic acid, folin-phenol, FZ (3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine-4’,4”-disulfonic 90 acid sodium salt), luminol, resins (DAX-8, XAD-4 and MSH-1), gallic acid, trifluoroacetic acid, and 91 hydrochloric acid were purchased from Sigma-Aldrich and were used as received. 2-methyl-692 [p-methoxyphenyl]-3,7-dihydroimidazo [1,2-a] pyrazin-3-one, a methyl cypridina luciferin analog 93 (MCLA) and 10-methyl-9-(ρ-formylphenyl)-acridinium carboxylate trifluoromethanesulfonate (AE) were 94 purchased from TCI Chemicals. Dimethyl sulfoxide-d6 was purchased from Cambridge Isotope 95 Laboratories. 96

EfOM isolation. Wastewater effluent was obtained from a municipal sewage plant located in

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Jiangsu Province, China. This sewage plant treats domestic sewage from the eastern district of the city

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of Taicang using a circulatory activated sludge treatment system. The term “The effluent” in this paper

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represents the whole waste water obtained for the waste water treatment plant. The isolation procedure

100

used here was previously detailed by Pernet-Coudrier et al. for NOM. 31 A schematic diagram is

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displayed in Figure S1, Supporting Information. First, the effluent was adjusted to pH 2.0 using

102

hydrochloric acid and filtered through a 0.45-µm glass fiber membrane. Approximately 70 L of acidified

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effluent was pumped continuously into a glass packed column filled with DAX-8 resin (100 g) and

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allowed to flow into another column packed with XAD-4 resin (100 g) as padding. The DAX-8 and



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XAD-4 resin columns were each eluted with 1.0 L of methanol to desorb the HPO and TPI fractions,

106

respectively. The two eluents were rotary evaporated and freeze-dried to isolate the HPO fraction from

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the DAX-8 resin and the TPI fraction from the XAD-4 resin. Next, the outflow of the XAD-4 column

108

was passed through a nano-filtration system with a molecular weight cutoff (MWCO) membrane (>160

109

Da) to remove most of the inorganic salts and concentrate the solution. The filtrate from the

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nano-filtration system had a relatively low TOC concentration, which implied that loss of organic

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constituent barely occurred during the nano-filtration process. Calculation for the TOC concentration in

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the original wastewater and final three fractions also performs a mass balance. In addition, azeotrophic

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distillation was applied to remove the trace amount of inorganic salts that remained in the concentrated

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solution.30 Finally, the resulting inorganic-ion-free solution passed through a 20-mL bed volume of

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MSH-1 resin. The eluent was vacuum-evaporated to dryness to obtain the HPI fraction. The TOC

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contents of the solutions were acquired using a TOC analyzer (Shimadzu, TOC-CPH/CN). The amount

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that HPO, TPI and HPI fractions accounted for the TOC content in the original effluent was 21.4%, 27.5%

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and 51.1% respectively. Nitrate and chloride ions were analyzed using a Metrohm 883 Basic plus ionic

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chromatogram equipped with a Metrosep A sup-5 column. Water quality of the effluent is listed in Table

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S1, Supporting Information. H-NMR analysis. Solution-state 1H-NMR spectra were obtained using a Bruker DMX 500

121 122

NMR spectrometer. To remove excess metal ions, each isolated EfOM fraction was dissolved in

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ultra-pure water and then passed through an MSH-1cation exchange resin before NMR spectroscopic

124

analysis. Each eluent of the MSH-1 resin was freeze-dried to minimize the signal of water in the

125

1

H-NMR spectrum. Eventually, 20 mg of each fraction was dissolved in 500 µL of DMSO-d6 for

126

1

H-NMR analysis.


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3D Excitation emission matrix fluorescence. EEM spectra were recorded using a fluorometer

128

(Aqualog, Horiba-Jobin Yvon). The fluorometer was set up as follows: the excitation wavelength was

129

incrementally increased from 240 to 450 nm using 3-nm intervals, with emission monitoring from 250 to

130

550 nm at 3-nm intervals for each excitation wavelength. Quinine sulfate standards were used to

131

calibrate the EEM spectra, and the fluorescence intensities were expressed in units of quinine sulfate

132

equivalents (QSE). 32

133

Photochemical experiments. An Atlas solar simulator (Suntest XLS+) equipped with a 1700W

134

xenon lamp and a solar filter was employed for all photochemical experiments. All samples were placed

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in specially made quartz containers and irradiated for a given period. The absolute irradiance spectrum

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of the solar simulator was recorded using a spectrometer (USB-4000, Ocean Optics Inc.). For

137

comparison, natural sunlight was also measured on a summer day at Fudan University in Shanghai,

138

China, as illustrated in Figure S2, Supporting Information. All solutions were buffered to pH 8.0 with

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1.0 mM phosphate to eliminate the pH effects. UV-vis absorbance spectrums of all the samples were

140

obtained using an ultraviolet-visible spectroscopy to get the quantum yield. All UV-vis spectrums are

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presented in Figure S5, Supporting Information.

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Detection of the steady state 1O2 concentrations [1O2]. The selective probe compound, furfuryl

143

alcohol (FFA), was used to measure the steady state concentration of 1O2. The direct photolysis of FFA

144

was negligible due to its low molar absorption coefficient and quantum yield.33, 34 The steady state

145

concentration of 1O2 can be determined using the following equations:

146 147 148

FFA + O → []

(1)

= −k , ! [FFA][ O ]##

(2)

"

Where k , ! = 8.3×107 M-1 s-1. 35 All results are shown and summarized in Figure S3 and Table S2, "

149

Supporting Information. 7 ACS Paragon Plus Environment


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Detection of steady state O2•− concentrations. In consideration of the low concentration and

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short lifetime of superoxide, 2-methyl-6-[p-methoxyphenyl]-3,7-dihydroimidazo [1,2-a] pyrazin-3-one,

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(MCLA) was used as a chemiluminescent probe for superoxide, which selectively reacts with O2•− in a

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purpose-built detector called a flow injection analysis (FIA) system (Waterville Analytical, US). A 200

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µM MCLA stock solution was prepared and subsequently frozen at -4°C. The analytical reagent

155

contained 1.0 µM MCLA buffered with 0.05 M sodium acetate, and the final pH of this reagent was

156

adjusted to 5.5 using quartz-distilled HCl. This reagent was prepared one day before use to ensure its

157

stability. During the measurement, the MCLA reagent and sample were pumped into the flow cell where

158

the reaction occurred. The reaction between O2•− and MCLA results in a chemiluminescent signal at

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455nm then was detected using a photomultiplier (PMT).

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To figure out the quantitative correspondence between the intensity of PMT and the

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concentration of O2•−, standard O2•− solutions with known concentration were spiked into ultra-pure

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water. The standard O2•− solutions were generated through photolysis of a mixture described by Fujii et

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al. 36 that was composed of 41 mM acetone, 12 M absolute ethanol, 15 µM DTPA and buffered to pH 12

164

using 1 mM borate. This mixture was irradiated with 254 nm light to produce standard O2•− that was

165

simultaneously detected by absorbance at 240 nm with a spectrometer (USB-4000, Ocean Optics Inc.).

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Since the sample was spiked into the flow line, the O2•− concentration decreased before it entered into

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the PMT. In order to acquire the initial O2•− intensity, the intensity of each specified concentration was

168

extrapolated from its best fit to a log function, as previously described.37

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Determination of hydrogen peroxide (H2O2). The previously mentioned FIA system was also

170

used to measure both, the generation and decay of H2O2 during the irradiation and in the dark. The

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10-methyl-9-(ρ-formylphenyl)-acridinium carboxylate trifluoromethanesulfonate (AE)

172

chemiluminescent reagent was employed to detect trace concentrations of H2O2.38 Because the reaction


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between AE and H2O2 requires alkaline conditions (pH = 11-12), 1.0 M sodium carbonate was used as a

174

buffer solution. The AE reagent and sample solution were mixed before they entered the flow cell. The

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ultrapure water used to prepare the reagent and buffer solutions was pretreated with 3.0 mg L-1 of

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dissolved catalase to quench the trace H2O2. Detection of steady state •OH concentrations. Terephthalic acid (TA) was used to trap •OH,

177 178

followed by the generation of 2-hydroxy-terephthalic acid (2HTA), which can be detected using an

179

HPLC-FLD system.6 The steady state concentration of the •OH can be calculated using the following

180

equations: •OH

181

$[%&]

182 183

$'

Where -·

+

TA

= 0.35 × -·

%,& =

→ %,&

2HTA

(3)

× [TA][• OH]

(4)

3.3×109 M-1 s-1. 39

A reversed-phase HPLC system employing a 2.7-µm C18 packing column (Agilent) was used for

184 185

separation. The mobile phase consisted of 65% trifluoroacetic acid buffer (0.05% v/v) and 35%

186

methanol (v/v) with a flow rate of 1.0 mL min-1. The fluorescence detector was set at an excitation of

187

315 nm and an emission of 425 nm. Determination of the phenolic moieties in the effluent and NOM solutions. The

188 189

Folin-Ciocalteu method was used to determine the total phenolic contents.40 The samples and

190

Folin-Ciocalteu reagents were mixed at a ratio of 1:1 (0.5 mL each) and kept at room temperature for 5

191

min. Next, 1.5 mL of sodium carbonate was added, and the mixture was stirred thoroughly. The final

192

sample volume for detecting absorbance was 10 mL after dilution with ultrapure water. Next, the

193

samples were placed in a 75°C water bath and incubated for 10 min. A UV-vis spectrophotometer was

194

used to read the absorbance of each sample at 760 nm. Gallic acid was employed to establish a standard

195

curve.



196

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Results and discussion 1

197

H-NMR analysis of the fractions isolated from EfOM. 1H-NMR analysis was used to obtain

198

characteristic information about the fractions extracted from the effluent. Details regarding major

199

functional groups are presented in Figure S4, Supporting Information. (CH2)n accounts for 77% of the

200

1

201

Lignin aromatics were found in the HPO and TPI fractions. Carbohydrates (5.6 ppm > δ (1H) > 3.0 ppm)

202

were present as a hydrophilic component and mainly existed in the HPI extract. The hydrophilicity of

203

the three fractions increased as the aliphatic and aromatic lignin contents decreased and the carbohydrate

204

contents increased. Low-field (7.8 ppm > δ (1H) > 7.3 ppm) aromatic amino acids only existed in the

205

HPI fraction due to the presence of SMPs.

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H-NMR integral of HPO, which was considered as the aliphatic portion (2.4 ppm > δ (1H) > 0.5 ppm).

3D fluorescence excitation-emission matrix spectra. In the EEM spectra (as shown in Figure

207

1), three main fluorescent peaks can be observed: a UV humic-like peak (excitation: 240-265 nm,

208

emission: 375-425 nm), a visible humic-like peak (excitation: 280-320 nm, emission: 380-420 nm), and

209

a protein-like peak (excitation: 260-290 nm, emission: 300-350 nm). The TOC concentration of all

210

solutions in present work was set to equal with those in the effluent (6.28 mg L-1). The HPO and TPI

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fractions were composed of UV and visible humic-like peaks with no distinct protein peaks in the EEM

212

spectra. This observation supported the conclusions that were drawn from the 1H-NMR results, which

213

indicated that most proteins /peptides exist in the HPI fraction.

214 215

(Insert Figure 1) Singlet Oxygen (1O2). Initial studies were conducted under simulated solar irradiation to explore

216

the steady state concentrations of 1O2 ([1O2]ss) in the effluent and three different fractions of EfOM

217

relative to SRHA/FA and PLFA. Previous studies investigated the formation of 1O2 based on molecular

218

weight (MW) fractions.29 In this study separation was based on differences in polarity because we


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hypothesized that the photochemical properties of EfOM would vary due to the occurrence of different

220

functional groups/moieties in different fractions.

221

As shown in Figure 2, steady state concentrations of 1O2 in three NOM isolates’ solutions (4.33 ~

222

5.21 × 10-13M) were slightly higher than that in the effluent (4.14 × 10-13 M). Among the three isolated

223

fractions, the HPI fraction had the highest 1O2 concentration (6.97 × 10-13 M). Because SMPs were

224

present, the EEM results of the HPI were dominated by protein-like peaks. The measurement of protein

225

content was conducted using the Lowery method (for details see Supporting Information). The result

226

illustrated that the HPI contained proteins/peptides at 0.73 mg/mg C. In addition, the 1H-NMR results

227

showed that the HPI fraction contained a high percentage of aromatic amino acids (7.64%). Among

228

proteins and peptides, tryptophan (Trp), tyrosine (Tyr) and phenylalanine (Phe) are considered as the

229

three amino acids that are able to produce considerable amounts of 1O2 by photo-excitation. The 1O2

230

quantum yields for these free amino acids have been reported as 6.2%, 13.8% and 6.5%, respectively.41

231

These values are significantly higher than the yields that were reported for humic acids.42, 43 Aromatic

232

amino acids in the effluent are combined in peptides or proteins, which have lower 1O2 reaction rates

233

than their free forms.44 In addition, 1H-NMR results revealed that the percentage of polysaccharides

234

decreased in the order: HPI > TPI > HPO. To our knowledge, the polysaccharide fraction does not react

235

with 1O2. Therefore, the reaction rate of the HPI fraction with 1O2 significantly decreased, which

236

resulted in a higher [1O2]ss.

237 238

(Insert Figure 2) To confirm the contribution of amino acid in generating 1O2, the 1O2 formation quantum yields

239

of three NOM isolates and EfOM were calculated according to Mostafa et al 29, and are shown in Table

240

1. The 1O2 quantum yield of the effluent (2.65%) was slightly higher than that of the NOM isolates’

241

solutions (1.35~1.85%). A similar result was recently reported for the 1O2 quantum yield of the effluent



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from a waste water treatment plant at Colorado, US.29 HPO and TPI had similar quantum yields of 3.24%

243

and 2.45%, respectively. Among the three fractions, the HPI fraction had the highest photochemical

244

productivity for 1O2 (quantum yield of 8.16%), which was three times higher than the average

245

productivity of NOM isolates. As illustrated by the EEM, the HPO and TPI fractions show more

246

distinctive UV and visible humic-like peaks, which are indicative of the major photosensitizers that are

247

present in NOM. The 1H-NMR results indicated that large concentrations of aliphatic groups were

248

present in the HPO and TPI. However, these groups are considered inert regarding 1O2 production and

249

quenching. Thus, the quantum yields of HPO and TPI are similar to those of NOM isolates.

250 251

(Insert Table 1) Overall, the HPI fraction could be considered as a major 1O2 source in the effluent due to its high

252

percentage of proteins/peptides and possibly low 1O2 reaction rates. Furthermore, it can be speculated

253

that the HPO and TPI fractions could be considered as sinks for 1O2.

254

Superoxide radical ion (O2•−). The O2•− is an important species that exists in natural sunlit

255

surface waters. It can be photochemically initiated by electron transfer from an excited-state substrate to

256

oxygen. 45, 46 The chemical probe MCLA was used to selectively quench O2•−, which resulted in the

257

generation of a chemiluminescence signal that was detected using a FIA system.37, 47, 48 The results from

258

the O2•− standard addition of the photochemically generated mixture mentioned above and intensity

259

extrapolation are presented in Figure S6, Supporting Information. A standard curve was employed for

260

real-time monitoring of the steady state O2•− concentrations.

261

Upon illumination, the [O2•−]ss in the effluent was much lower than that of the SRHA/FA and

262

PLFA at the same TOC concentration. The [O2•−]ss in the HPO fraction was slightly higher than that in

263

the TPI fraction, while low O2•− production was observed in the HPI fraction. Previous studies have

264

indicated that certain NOM moieties, particularly in the humic fraction, possess an apparent


Page 13 of 30


265

electron-donating capacity (EDC) 49, 50. Furthermore, it was also shown that such EDC groups that

266

mostly consist of phenolic moieties are depleted during photo bleaching of NOM.51 Thus, phenols and

267

phenolic moieties may be involved in the formation of superoxide.52, 53 Therefore, to better understand

268

these mechanisms, the [O2•−]ss and phenolic moieties were analyzed for correlations. As illustrated in

269

Figure 3, the steady state O2•− concentration was linearly correlated with the phenolic moieties in the

270

diverse EfOM fractions and NOM isolates (R2 = 0.94). This result indicated that the phenolic moieties

271

may play an important role in generating O2•− in the surface water through a photochemical pathway. (Insert Figure 3)

272 273

As listed in Table 1, O2•− formation quantum yield of HPI fraction (2.95 × 10-5) was much lower

274

than those of the HPO and TPI fractions (7.87 × 10-5 and 4.06 × 10-5respectively) in result of lacking

275

phenolic moieties. PLFA gains the lowest quantum yield of O2•− formation (1.41 × 10-5) due to its

276

comparatively lower phenolic moieties and high absorption at UV-visible range.

277

According to previous studies, the O2•− can act as a 1O2 quencher through physical processes.

278

1

O2

+ O2•−

→

O2 (3∑g) + O2•−

k = 5.0 × 109 M-1 s-1

(5)

279

Both HPO and TPI fractions displayed a notably higher [O2•−]ss relative to the HPI fraction (as

280

mentioned above). Thus, it is reasonable to assert that their low 1O2 formation quantum yields partially

281

resulted from the reactions of 1O2 with the O2•−. However, no correlation between the concentration of

282

O2•− and 1O2 was observed (data not shown), suggesting that the formation of 1O2 and O2•− could be

283

resulting from two independent processes.

284

During the irradiation process the O2•− concentration reached a steady state after 20 min.

285

Subsequently, the solutions were placed in the dark, intensities were continuously recorded in order to

286

reveal the decay process of O2•−. The disproportionation reaction of O2•− accounted for the main



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287

degradation pathway, with a reported rate constant of 4.0 × 104 M-1 s-1.54 Furthermore, reactions with

288

trace amounts of ferrous ions may affect the O2•− lifetime.

289

Fe2+

+ O2•−

290

The ferrous ion concentration was measured using the FIA system, luminol reagent was used as a

→

Fe3+

+

H2O2

k = 1.34 × 106 M-1 s-1

(6)

291

chemiluminescent probe.55 The results from the ferrous ion experiments are listed in Table S3,

292

Supporting Information. The concentrations of the samples varied from 1.2 to 5.2 × 10-11 M, which was

293

two or three orders of magnitude lower than the O2•− concentrations. Therefore, the contributions of the

294

ferrous ions to O2•− decay were negligible. As illustrated in Figure S7 in the Supporting Information, the

295

decay curves of O2•− in the SRFA and SRHA were similar, which was nearly equal to the model

296

disproportionation rates when only the disproportionation of O2•− was considered. The decay trend in the

297

HPO fraction was consistent with that in the TPI fraction. Both rates were significantly faster than the

298

disproportionation rate of O2•−. Thus, organic matter was responsible for most of the O2•− quenching in

299

the EfOM solutions, while the disproportionation of O2•− played an important role in the SRHA/FA

300

solutions.

301

Hydrogen peroxide (H2O2). H2O2 accumulated in the EfOM and NOM isolates’ solutions at

302

varying rates during the illumination experiment. It was proposed that the H2O2 originated from the

303

disproportionation of the superoxide according to the following mechanism:

304

2O2•−

+ 2H+

→

H2O2

k=4.0 × 104 M-1 s-1

+ O2

(7)

305

In the present study, EfOM and NOM isolates’ solutions were exposed to simulated solar

306

irradiation for 1 h (144 kJ m-2) to simulate irradiation on a typical sunny day. The formation rate of H2O2

307

( 2" 3" ) was linearly correlated with [O2•−]2 (R2= 0.94), as shown in Figure S8, Supporting Information.

308

This result indicated that the peroxide radical ion was not as reactive as 1O2 and •OH. The

309

self-combination would serve as a major degradation process of O2•− and result in H2O2 formation. The


Page 15 of 30


310

formation rate of H2O2 could be predicted using [O2•−]ss, or simply tracing back through the phenolic

311

moieties content as illustrated in Figure 3. This result, that H2O2 was generated from O2•−, was also

312

supported by a previous field investigation focused on the photo-production of H2O2 in the ocean. 56 As

313

listed in Table 1, the HPI fraction showed a lower H2O2 formation quantum yield than the HPO and TPI

314

fractions. Quantum yield of the effluent was a slightly lower than that of SRHA/FA, but much higher

315

than that of PLFA. This quantitative relationship corresponded well with that of O2•− formation quantum

316

yield, which also supported that O2•− was the main source of H2O2.

317

H2O2 is comparatively stable and has a much longer lifetime than the other three ROS (•OH, 1O2

318

and O2•−). H2O2 was proved to be an important precursor of •OH through non-photochemical pathways.

319

The generation of •OH through H2O2-mediated oxidation of Fe2+ complexed by NOM was observed.14

320

And in the dark, H2O2-mediated oxidation of reduced humic acids can also produce •OH.57

321

Consequently, H2O2 may have a far-reaching influence on the degradation of pollutants in water systems,

322

especially at night or on overcast days. After 1 h of observing H2O2 production, the samples were

323

removed from the solar simulator and placed in the dark. H2O2 in three NOM isolates’ solutions decayed

324

at almost the same rate. The decay of H2O2 in SRHA was presented as an example. The H2O2 content in

325

the effluent decreased faster than the one in the NOM isolates in the dark (as shown in Figure 4).

326 327

(Insert Figure 4) The H2O2 lifetime is influenced by many complicated factors, including thermo decomposition,

328

enzymatic catalysis degradation, and decomposition catalyzed by metal ions or organic metal species. 34

329

Although copper ions were not detected in the effluent water by ICP-MS, iron ions were observed.

330

Fenton’s reaction can potentially cause the decay of H2O2 in the dark when a trace amount of ferrous ion

331

is present (8.3 × 10-10 M). To determine the contribution of the Fenton’s reaction to H2O2 decay, a

332

ferrous quencher FZ was added into the effluent. 54 The effluent with and without the addition of FZ



Page 16 of 30

333

resulted in identical decay rates, which indicated that the Fenton’s reaction minimally affected H2O2

334

decay. The decay rates of H2O2 in the HPO and TPI fractions were nearly the same as in the SRHA,

335

which implied that the HPI fraction was responsible for the fast H2O2 decay in the effluent. Because the

336

generation of H2O2 was negligible in the HPI fraction, the HPI solution was spiked with 9.4 × 10-8 M

337

H2O2 (the same concentration as in the effluent with 1 hour of irradiation). As illustrated in Figure S9,

338

the decay rate of H2O2 in HPI corresponded with that of the effluent. Therefore, it was concluded that

339

the SMP-related organic species (such as enzymes or proteins) that presented in the HPI fraction might

340

serve as a dominant sink for H2O2 and hinder the accumulation of H2O2.58 In contrast, it can be expected

341

that H2O2 can be accumulated in NOM solutions and keep a higher concentration than in EfOM

342

influenced water system.

343

Hydroxyl radical. •OH is a highly reactive oxygen species that can unselectively react with

344

most of organic substrates. This process significantly contributes to the transformation and elimination

345

of organic contaminants in natural water systems. As illustrated in Figure 5, •OH was measured in NOM

346

isolates and EfOM solutions with the same TOC values under illumination (Figure S10 and Table S4,

347

Supporting Information). With existence of nitrate in the effluent, part of •OH derived from nitrate

348

8 7 -1 4 photolysis at a rate of 3.0 × 10-7 M•OH·M5 s . •OH result of the effluent was obtained after 6

349

elimination of the nitrate photolysis. In this context, the [•OH]ss in PLFA, which is considered as a

350

typical NOM of autochthonous origin, was much higher than that in SRHA/FA, which is considered to

351

be a terrestrial type of NOM. The [•OH]ss is much higher in the PLFA than in the SRHA/FA due to their

352

different chemical compositions PLFA has a lower aromaticity and higher nitrogen content.59

353 354 355

(Insert Figure 5) The effluent had a slightly lower [•OH]ss than the terrestrial NOM. To determine the relationships between [•OH]ss and the chemical composition, each EfOM fraction was examined


Page 17 of 30


356

individually. HPO and TPI had similar capacities for generating •OH compared with terrestrial NOM,

357

which was consistent with the observation that both fractions were considered as humic-like.60 In

358

addition, it was reported that the humic fraction of EfOM significantly contributed to the generation of

359

•OH, in contrast to the non-humic fraction.61 As illustrated in the EEM spectra, the lack of a humic

360

fraction in the HPI solution reduced the formation of •OH. Moreover, the lowest observed [•OH]ss in the

361

HPI solution resulted from the inclusion of SMPs, which have higher reaction rates with •OH. In the

362

effluent, it was assumed that the HPO and TPI fractions are the major sources of •OH while the HPI

363

fraction acts as a sink.

364

As for •OH formation quantum yield (shown in Table 1), the effluent has a higher quantum yield

365

(7.94 × 10-5) than NOM isolates (4.81 ~ 7.30 × 10-5). Whereas a similar result was reported previously

366

by a study focusing on H2O2-unrelated generation of •OH from EfOM,4 our study helps to expand this

367

phenomenon to the photochemical generation of •OH with H2O2 participation.

368

Environmental Implications

369

In summary, HPO and TPI were similar to terrestrial NOM considering the generation of ROS.

370

Both fractions are present as background NOM in WWTPs and dominate the production of superoxide,

371

H2O2 and •OH. Because the HPI fraction has a minor humic fraction and an abundance of SMPs, this

372

fraction serves as the main sink for H2O2 and •OH and is very capable of generating 1O2. The dominant

373

form of NOM in water systems differs depending on the extent of human influence. EfOM and NOM

374

show different photochemical properties that can have far-reaching influences on the environmental

375

transportation and fate of trace amounts of refractory organic pollutants.

376

EfOM can generate ROS through photochemical paths, and this process accelerates degradation

377

of organic pollutants existing in water systems. From our results, it can be predicted that the pollutants

378

which possess high reaction rate constants with 1O2, could be removed in the effluent faster than in



Page 18 of 30

379

natural water. Also, considering the higher •OH formation quantum yield of effluent, •OH may also play

380

a more significant role in the removal of pollutants. Furthermore, the fast decay and low concentration

381

of H2O2 in effluent makes non-photochemical related •OH stemming from H2O2 infeasible to reaction

382

with pollutants. From the present study, it can be concluded that phenolic moieties are the source of O2•−,

383

and at the same time O2•− is the precursor of H2O2. So this study offers an easy way to assess the steady

384

state concentration of O2•− and the generation rate of H2O2. This can be a valuable water quality index

385

since H2O2 has a strong influence on microbiology systems and helps to remove organic pollutants

386

through producing •OH. O2•− shows obviously different decay rates depending on the background

387

organic matter. It was also observed that certain organic matter dominated the decay of O2•− in EfOM.

388

This result implies that O2•− may also make a non-ignorable contribution to the elimination of organic

389

pollutants. In general, O2•− presents comparatively low reactivity with organic pollutants, but its high

390

steady state concentration in water system is expected to make removal of certain pollutants exercisable.

391 392 393 394 395

Supporting Information This material is available free of charge via the Internet at http://pubs.acs.org.

Acknowledgments We thank partial funding support from the National Natural Science Foundation of China

396

(21107016, 21377030), the Ministry of Science and Technology of China (2012YQ220113-4), and the

397

Science & Technology Commission of Shanghai Municipality (12PJ1400800). In addition, W.S.

398

acknowledges the support from the program for Professor of Special Appointment (Eastern Scholar) at

399

the Shanghai Institution of Higher Learning. We also thank the reviewers for valuable insights and

400

suggestions.

401

References 18 ACS Paragon Plus Environment

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31. Pernet-Coudrier, B.; Clouzot, L.; Varrault, G.; Tusseau-Vuillemin, M.-H.; Verger, A.; Mouchel, J.-M., Dissolved organic matter from treated effluent of a major wastewater treatment plant: characterization and influence on copper toxicity. Chemosphere 2008, 73, (4), 593-599.

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32. Coble, P. G., Characterization of marine and terrestrial DOM in seawater using excitation-emission matrix spectroscopy. Marine Chemistry 1996, 51, (4), 325-346.

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34. Burns, J. M.; Cooper, W. J.; Ferry, J. L.; King, D. W.; DiMento, B. P.; McNeill, K.; Miller, C. J.; Miller, W. L.; Peake, B. M.; Rusak, S. A., Methods for reactive oxygen species (ROS) detection in aqueous environments. Aquat. Sci. 2012, 74, (4), 683-734.

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55. Rose, A. L.; Waite, T. D., Kinetic model for Fe (II) oxidation in seawater in the absence and presence of natural organic matter. Environ. Sci. Technol. 2002, 36, (3), 433-444.

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61. Lee, E.; Glover, C. M.; Rosario-Ortiz, F. L., Photochemical Formation of Hydroxyl Radical from Effluent Organic Matter: Role of Composition. Environ. Sci. Technol. 2013, 47, (21), 12073-12080.

545 546



. 550

550

500

500

400

350

Effluent

300

400

350

1.0

UV humic-like

300

HPO

250

250

300

350

400

450

EX Wavelength (nm)

550

250

300

350

400

450

EX Wavelength (nm)

550

500

500

450

400

350

UV humic-like

300

TPI

0.50 EM Wavelength (nm)

Visible humic-like EM Wavelength (nm)

450

QSE (ppm)

450

250

450

400

Protein-like 350

HPI

300

0.0

250

250 250

300

350

400

250

450

EX Wavelength (nm)

300

350

400

450

EX Wavelength (nm)

548 549

550

1.4

Visible humic-like EM Wavelength (nm)

EM Wavelength (nm)

547

Page 24 of 30

Figure 1. Fluorescence excitation-emission matrix spectra of the effluent, HPO, TPI, and HPI fractions.


Page 25 of 30


551

8 7

1

-13

[ O2]ss (10 M)

6 5 4 3 2 1 0 SRHA

SRFA

PLFA Effluent

HPO

TPI

HPI

552 553

554

Figure 2. Steady state concentration of singlet oxygen in solutions with varying forms of organic matter

555

(with TOC concentration of 6.28 mg L-1).

556



557

Page 26 of 30

Table 1. Quantum yield of each ROS formation in solutions with varying forms of organic matters. Photosensitizer

9

Photochemically Induced Formation of Reactive Oxygen Species

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