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Article
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
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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
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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
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Lake fulvic acid (PLFA). The hydrophilic (HPI) component had a greater quantum yield of 1O2 than
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those of the hydrophobic (HPO) and transphilic (TPI) fractions because the HPI contained peptides and
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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
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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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Environmental Science & Technology
Introduction Effluent organic matter (EfOM) is discharged into surface water from wastewater treatment
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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
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(SMPs) and trace amounts of organic pollutants. Most of the NOM present in drinking water remains in
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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
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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),
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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
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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
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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
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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
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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
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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
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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,
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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
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was passed through a nano-filtration system with a molecular weight cutoff (MWCO) membrane (>160
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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
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analysis. Each eluent of the MSH-1 resin was freeze-dried to minimize the signal of water in the
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1
H-NMR spectrum. Eventually, 20 mg of each fraction was dissolved in 500 µL of DMSO-d6 for
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1
H-NMR analysis.
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Environmental Science & Technology
3D Excitation emission matrix fluorescence. EEM spectra were recorded using a fluorometer
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(Aqualog, Horiba-Jobin Yvon). The fluorometer was set up as follows: the excitation wavelength was
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incrementally increased from 240 to 450 nm using 3-nm intervals, with emission monitoring from 250 to
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550 nm at 3-nm intervals for each excitation wavelength. Quinine sulfate standards were used to
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calibrate the EEM spectra, and the fluorescence intensities were expressed in units of quinine sulfate
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equivalents (QSE). 32
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Photochemical experiments. An Atlas solar simulator (Suntest XLS+) equipped with a 1700W
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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
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comparison, natural sunlight was also measured on a summer day at Fudan University in Shanghai,
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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
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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
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alcohol (FFA), was used to measure the steady state concentration of 1O2. The direct photolysis of FFA
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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, "
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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
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contained 1.0 µM MCLA buffered with 0.05 M sodium acetate, and the final pH of this reagent was
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adjusted to 5.5 using quartz-distilled HCl. This reagent was prepared one day before use to ensure its
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stability. During the measurement, the MCLA reagent and sample were pumped into the flow cell where
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the reaction occurred. The reaction between O2•− and MCLA results in a chemiluminescent signal at
159
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
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using 1 mM borate. This mixture was irradiated with 254 nm light to produce standard O2•− that was
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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
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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
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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)
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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
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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%
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methanol (v/v) with a flow rate of 1.0 mL min-1. The fluorescence detector was set at an excitation of
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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
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Folin-Ciocalteu reagents were mixed at a ratio of 1:1 (0.5 mL each) and kept at room temperature for 5
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min. Next, 1.5 mL of sodium carbonate was added, and the mixture was stirred thoroughly. The final
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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
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used to read the absorbance of each sample at 760 nm. Gallic acid was employed to establish a standard
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curve.
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Results and discussion 1
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H-NMR analysis of the fractions isolated from EfOM. 1H-NMR analysis was used to obtain
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characteristic information about the fractions extracted from the effluent. Details regarding major
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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
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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
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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
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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
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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.
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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
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These values are significantly higher than the yields that were reported for humic acids.42, 43 Aromatic
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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%
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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
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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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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
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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
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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
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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
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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
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545 546
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. 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.
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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
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Page 26 of 30
Table 1. Quantum yield of each ROS formation in solutions with varying forms of organic matters. Photosensitizer
9
Article
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
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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
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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
27
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
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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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Environmental Science & Technology
Introduction Effluent organic matter (EfOM) is discharged into surface water from wastewater treatment
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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
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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
43
affect the environmental fate of trace organic pollutants, most of which are resistant to biodegradation
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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),
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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
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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
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isolated according to hydrophobicity. Each fraction included functional groups that were more
70
specifically clustered than when separated by molecular weight cutoff. The isolation of EfOM will be
71
useful for additional studies that aim to determine the specific roles of different functional groups during
72
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
74
necessary to explore ROS production in EfOM systems, which may have enormous influence on the
75
environmental fate and transformation of trace organic pollutants.
76
As mentioned above, the current study conducted a comprehensive isolation of EfOM based on
77
the hydrophobicity of its different constituents, including the hydrophobic (HPO), transphilic (TPI) and
78
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
97
Jiangsu Province, China. This sewage plant treats domestic sewage from the eastern district of the city
98
of Taicang using a circulatory activated sludge treatment system. The term “The effluent” in this paper
99
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
101
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
104
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,
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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
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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
110
nano-filtration system had a relatively low TOC concentration, which implied that loss of organic
111
constituent barely occurred during the nano-filtration process. Calculation for the TOC concentration in
112
the original wastewater and final three fractions also performs a mass balance. In addition, azeotrophic
113
distillation was applied to remove the trace amount of inorganic salts that remained in the concentrated
114
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
116
contents of the solutions were acquired using a TOC analyzer (Shimadzu, TOC-CPH/CN). The amount
117
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
119
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
123
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
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1
H-NMR spectrum. Eventually, 20 mg of each fraction was dissolved in 500 µL of DMSO-d6 for
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1
H-NMR analysis.
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3D Excitation emission matrix fluorescence. EEM spectra were recorded using a fluorometer
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(Aqualog, Horiba-Jobin Yvon). The fluorometer was set up as follows: the excitation wavelength was
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incrementally increased from 240 to 450 nm using 3-nm intervals, with emission monitoring from 250 to
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550 nm at 3-nm intervals for each excitation wavelength. Quinine sulfate standards were used to
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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
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comparison, natural sunlight was also measured on a summer day at Fudan University in Shanghai,
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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
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obtained using an ultraviolet-visible spectroscopy to get the quantum yield. All UV-vis spectrums are
141
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, "
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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,
152
(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
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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
159
455nm then was detected using a photomultiplier (PMT).
160
To figure out the quantitative correspondence between the intensity of PMT and the
161
concentration of O2•−, standard O2•− solutions with known concentration were spiked into ultra-pure
162
water. The standard O2•− solutions were generated through photolysis of a mixture described by Fujii et
163
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
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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
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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%
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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
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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.
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Results and discussion 1
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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.
206
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
211
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.
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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%
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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
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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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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
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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
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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
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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
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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
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545 546
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. 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.
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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
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557
Page 26 of 30
Table 1. Quantum yield of each ROS formation in solutions with varying forms of organic matters. Photosensitizer
9
