Influence of dissolved organic matter on photogenerated reactive oxygen species and metal-oxide nanoparticle toxicity

Accepted Manuscript Influence of Dissolved Organic Matter on Photogenerated Reactive Oxygen Species and Metal-Oxide Nanoparticle Toxicity Yang Li, Junfeng Niu, Enxiang Shang, John Charles Crittenden PII:

S0043-1354(16)30175-0

DOI:

10.1016/j.watres.2016.03.050

Reference:

WR 11934

To appear in:

Water Research

Received Date: 2 November 2015 Revised Date:

21 March 2016

Accepted Date: 22 March 2016

Please cite this article as: Li, Y., Niu, J., Shang, E., Crittenden, J.C., Influence of Dissolved Organic Matter on Photogenerated Reactive Oxygen Species and Metal-Oxide Nanoparticle Toxicity, Water Research (2016), doi: 10.1016/j.watres.2016.03.050. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Influence

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Photogenerated

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Metal-Oxide Nanoparticle Toxicity

Organic

Reactive

Oxygen

Matter Species

on and

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State Key Laboratory of Water Environment Simulation, School of Environment,

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Beijing Normal University, Beijing 100875, People’s Republic of China

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Dissolved

Yang Li1, Junfeng Niu1∗, Enxiang Shang1, and John Charles Crittenden1,2

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School of Civil and Environmental Engineering and the Brook Byers Institute for Sustainable Systems, Georgia Institute of Technology, Atlanta, GA 30332, United

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States

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Corresponding author: e-mail: [email protected], phone: +86-10-5880 7612, fax: +86-10-5880 7612.

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ACCEPTED MANUSCRIPT ABSTRACT

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The effect of humic acid (HA) or fulvic acid (FA) on reactive oxygen species (ROS)

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generation by six metal-oxide nanoparticles (NPs) and their toxicities toward

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Escherichia coli was investigated under UV irradiation. Dissolved organic matter

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(DOM) decreased •OH generation by TiO2, ZnO, and Fe2O3, with FA inhibiting •OH

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generation more than HA. The generated •OH in NPs/DOM mixtures was higher than

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the measured concentrations because DOM consumes •OH faster than its molecular

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probe. None of NPs/FA mixtures produced O2•−. The generated O2•− concentrations in

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NPs/HA mixtures (except Fe2O3/HA) were higher than the sum of O2•− concentrations

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that produced as NPs and HA were presented by themselves. Synergistic O2•−

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generation in NPs/HA mixtures resulted from O2 reduction by electron transferred

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from photoionized HA to NPs. DOM increased 1O2 generation by TiO2, CuO, CeO2,

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and SiO2, and FA promoted 1O2 generation more than HA. Enhanced 1O2 generation

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resulted from DOM sensitization of NPs. HA did not increase 1O2 generation by ZnO

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and Fe2O3 primarily because released ions quenched 1O2 precursor (3HA*). Linear

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correlation was developed between total ROS concentrations generated by NPs/DOM

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mixtures and bacterial survival rates (R2 ≥ 0.80). The results implied the necessity of

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considering DOM when investigating the photoreactivity of NPs.

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Keywords: Metal-oxide nanoparticles; Humic acid; Fulvic acid; Reactive oxygen

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species; Escherichia coli; Toxicity

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1. Introduction The application of metal-oxide nanoparticles (NPs) leads to their release into

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natural waters and causes toxicity toward bacteria (Li et al. 2012, Cho et al. 2004). It

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have been demonstrated that light exposure (UV lamp, xenon lamp, solar, or

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conventional fluorescent tubes) could enhance the toxicity of metal-oxide NPs toward

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Escherichia coli (E. coli) because of their unique electronic structures and high

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photoactivity (Cho et al. 2004, Brunet et al. 2009). Concerns over the photoinduced

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toxicity of NPs to bacteria have stimulated study on their photochemical reactivity in

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natural waters.

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Reactive oxygen species (ROS) generation is the major phototoxicity mechanism

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of metal-oxide NPs toward E. coli (Li et al. 2012, Adams et al. 2006, He et al. 2015).

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Metal-oxide NPs exposed to light could generate superoxide anion (O2•−), singlet

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oxygen (1O2), and hydroxyl radical (•OH) (Adams et al. 2006, Ireland et al. 1993).

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These three types of ROS may jointly contribute to the toxicity of metal-oxide NPs

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toward E. coli (Adams et al. 2006, Ireland et al. 1993). After released into natural

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waters, NPs will be exposed to dissolved organic matter (DOM) and light from the

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sun or artificial lighting (Carlos et al. 2012, Dasari and Hwang 2013). The high

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surface-to-volume ratios of metal-oxide NPs and the surfactant properties of DOM

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facilitate sorption of DOM on the surface of NPs (Carlos et al. 2012, Dasari and

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Hwang 2013, Lin et al. 2012). Sorption of DOM can subsequently affect the surface

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charge, stability, photochemical reactivity, and bioavailability of metal-oxide NPs

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(Lin et al. 2012, Sousa and Teixeira 2013). Many previous studies have demonstrated

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that metal-oxide NPs can generate ROS (Li et al. 2012, Ireland et al. 1993). However,

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little research has examined the role of DOM in ROS generation type and

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concentration by metal-oxide NPs and their toxicity.

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ACCEPTED MANUSCRIPT After light absorbance in the 300-500 nm range, DOM can form excited triplet

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states (3DOM*) and hydrated electrons (e-) through the photoionization reaction (Lee

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et al. 2013, Dong and Rosario-Ortiz 2012). It has been demonstrated that 3DOM*

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could photosensitize carbon-based NPs and enhance 1O2 photogeneration in the

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C60/DOM mixtures (Li et al. 2015). Metal-oxide NPs could release metal ions (Li et al.

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2012, Bondarenko et al. 2012), which complex with DOM or 3DOM* through their

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carboxyl, phenolic, or other groups (Prado et al. 2006, Pandey et al. 2000). It would

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be of interest to see whether the complexation reaction deactivates 3DOM* and

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impacts 1O2 generation in NPs/DOM mixtures. It has been demonstrated that the

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photo-produced electron of DOM could be trapped by semiconductor NPs

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(Vinodgopal 1994, Vinodgopal and Kamat 1992). The question that arises is how the

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injected electron impacts O2•− generation in the NPs/DOM mixtures.

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Intense efforts have been devoted to investigate the effect of DOM on the

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toxicity of metal-oxide NPs toward E. coli (Ede et al. 2012, Tong et al. 2013, Rincón

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and Pulgarin 2005). It has been demonstrated that the presence of DOM significantly

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decreased the photoinduced toxicity of TiO2 and CuO toward E. coli (Ede et al. 2012,

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Rincón and Pulgarin 2005). Humic acid (HA) and fulvic acid (FA) are main

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components of DOM in surface waters (Lee et al. 2013, Laurentiis et al. 2013). Their

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distinct physicochemical properties (e.g., chemical composition, molecular structure,

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and surface charge) result in their different photochemical reactivity (Li et al. 2015,

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Aguer et al. 1997). The key question that remains to be answered is how the different

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photochemical reactivity of HA and FA impact of ROS generation from metal-oxide

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NPs and E. coli toxicity.

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We have previously investigated the photogeneration of ROS by six metal-oxide

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NPs (TiO2, CeO2, ZnO, CuO, SiO2, and Fe2O3) and their toxicity toward E. coli (Li et

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ACCEPTED MANUSCRIPT al. 2012). E. coli are selected as model organism because they are light sensitive and

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recommended by USEPA as indicators of fecal contamination in waters (Cho et al.

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2004, Adams et al. 2006). In this study, the effect of DOM fractions (HA or FA) on

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the generation types and concentrations of ROS (1O2, •OH, and O2•−) by six NPs

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under UV irradiation (365 nm) was investigated. The ROS generation mechanism in

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the NPs/DOM mixtures was analyzed by the energy and electron transfer between

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NPs and DOM. Furthermore, we studied the toxicity of NPs toward E. coli in the

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presence of HA or FA, which was then correlated with ROS generated in the

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NPs/DOM mixtures. This study should benefit the understanding of toxicity of NPs

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after their entry into UV treatment process or natural waters.

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2. Materials and methods

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2.1. Photochemical experiments

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Chemicals used in this study are provided in section S1 of Supporting

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Information. Suwannee River HA and FA were purchased from the International

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Humic Substances Society (Atlanta, GA, USA). The preparation method of DOM

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stock solutions and the details of NP properties are provided in section S2 of the

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Supporting Information. For all photochemical experiments, we measured ROS

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concentration, assessed toxicity, and measured ion release of NPs/DOM mixtures.

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One hundred mL of the mixtures was irradiated with a 4-W ultraviolet lamp (UVP,

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San Gabrel, CA, USA). The UV lamp has an output spectrum ranging from 315 to

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400 nm with peak intensity at 365 nm (UV-365). The light intensity in the center of

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the reaction solution was 1.4 × 10-6 Einstein·L-1·s-1. The reaction temperature was

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maintained at (22 ± 2) oC by a constant-temperature water bath.

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2.2. ESR detection of •OH, 1O2 and O2•−

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Production of O2•− and •OH in the NPs/DOM mixtures was monitored using 5,

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5-dimethyl-1-pyrroline-N-oxide

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6-Tetramethyl-4-piperidone (TEMP) was used as spin-trapping agent for

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detection. The mixture for O2•− detection was prepared by mixing 20 µL DMPO (0.5

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M), 3 µL NPs dispersed in DMSO (500 mg/L), 3 µL DOM solution (500 mg/L), and

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274 µL DMSO. The mixture for •OH detection was prepared by mixing 20 µL DMPO

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(0.5 M), 3 µL NP suspension (500 mg/L), 3 µL DOM solution (500 mg/L), and 274 µL

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DI water. For 1O2 detection, the mixture was prepared by mixing 6 µL TEMP (4 M), 3

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µL NP suspension (500 mg/L), 3 µL DOM solutions (500 mg/L), and 288 µL DI water.

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The reaction solutions were placed into the cylindrical quartz cell and irradiated by

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the same ultraviolet lamp. After 30 min, the quartz cell was quickly measured by

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electron spin resonance spectrometry (ESR; Bruker ESP-300E, Karlsruhe, Germany).

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The setting of ESR was as follows: (1) microwave power of 10 mW; (2) modulation

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amplitude of 2.071 G; (3) sweep width of 100 G; (4) center field of 3480 G; and (5)

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sweep time of 41.943 s. TEMP was oxidized by

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6-tetramethyl-1-piperdinyloxy

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Accordingly, ESR signals for DMPO-O2•−, DMPO-•OH, and TEMPO adducts were

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used to measure O2•−, •OH, and 1O2 formation, respectively.

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2.3. Measurement of •OH, 1O2 and O2•− concentrations

spin-trapping

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(DMPO)

The molecular probe assays were conducted to confirm the photogeneration of

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ROS and measure their concentrations. One hundred mL of reaction solution

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containing 5 mg/L NPs and 5 mg/L DOM was placed into a beaker and irradiated by

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the same UV lamp. The concentration of metal-oxide NPs was set at 5 mg/L because

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it is difficult to measure the production amount of ROS at lower concentration of NPs

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by the molecular probe assays. DOM concentration was set at 5 mg/L because it was

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the lowest concentration allowing a reliable measurement of ROS generation

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ACCEPTED MANUSCRIPT concentrations by DOM with molecular probe method. In addition, 5 mg/L was within

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the scope of DOM concentrations in natural waters that range from several hundred

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ng/L to around 10 mg/L (Thurman and Malcolm 1994). No buffer solutions were

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added to the reaction solutions to prevent the possibility of colloidal instability during

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the photochemical experiments. Furfuryl alcohol (FFA, 0.85 mM), p-chlorobenzoic

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acid (pCBA, 20 µM), and XTT (200 µM) were used as molecular probes for 1O2, •OH,

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and O2•−, respectively. XTT reduction by O2•− results in the formation of

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XTT-formazan, which has an adsorption peak at 470 nm. Many previous studies have

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demonstrated that XTT and XTT-formazan were stable under UV-365 irradiation

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(Brunet et al. 2009, Li et al. 2014, Cai 2013). After UV illumination, the reaction

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solutions were sampled, prepared, and analyzed with a high-performance liquid

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chromatograph (HPLC, Dionex U3000, USA) or UV-vis spectrophotometer

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(Beckman, DU 7700, Brea, CA, U.S.A.) (Li et al. 2014, Li et al. 2013, Jin et al. 2013).

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The time-averaged molar concentration of each ROS was calculated according to

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published integration function (Li et al. 2012, Li et al. 2014, Li et al. 2013).

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2.4. Assessment of toxicity

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The spread plate method was used to evaluate the effect of DOM on

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phototoxicity of various NPs. The culture, harvest, plate, and counting methods are

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provided in section S3. One hundred mL of bacteria suspension (105 colony-forming

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units (CFU)/mL) supplemented with 5 mg/L NPs and 5 mg/L DOM were irradiated

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by the same UV lamp. After 2 h of irradiation, the suspensions were collected, diluted,

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and plated onto LB agar plates, which were incubated at 37oC overnight before

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counting the number of viable bacterial colonies. The survival rates of E. coli were

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presented as the log values of the percentage of surviving bacteria, which was

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calculated by Eq. (1):

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(1)

where C is the survival rates of E. coli, N0 is the number of colonies on a control plate

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(N0) (no NPs and DOM exposure), and Nt is the number of colonies on the sample

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plate incubated under the same conditions.

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2.5. Measurement of ion release

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One hundred mL of mixture containing 5 mg/L NPs and 5 mg/L DOM was

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irradiated by the same UV lamp. After 2 h of irradiation, 4 mL of the NPs/DOM

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mixture was collected and filtered using an Amicon Ultra-4 centrifugal ultrafilter with

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a nominal particle size limit of 1–2 nm (Millipore, USA) to remove NPs. After

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centrifugation for 30 min at 7000 × g, the filtrates were collected and mixed with

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trace-metal grade HNO3 (67–70%, w/w). The concentrations of metal ions released

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from NPs/DOM mixtures were measured using inductively coupled plasma-mass

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spectrometry (ICP-MS, Elan DRC II, PerkinElmer, USA).

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2.6. Laser flash photolysis

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The transient absorption spectra traced by a nanosecond laser flash photolysis

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instrument (LP920, Edinburgh, England) were obtained to investigate the electron

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transfer between DOM and NPs. The experimental solutions include (a) 5 mg/L NPs

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in acetonitrile; (b) 5 mg/L DOM solution in acetonitrile prepared by diluting 20 µL

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DOM stock solution (1 g/L) with 4 mL acetonitrile; and (c) NPs/DOM mixtures in

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acetonitrile containing 5 mg/L NPs and 5 mg/L DOM. Prior to experiment, the

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solutions were deoxygenated by bubbling ultrapure N2 for 30 minutes. A 500 W

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xenon lamp was used as a monitoring light. A 355 nm laser pulse (10 mJ, pulse width

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of 7 ns) was generated from a Quanta Ray Nd YAG laser system (Continuum, Santa

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Clara, CA, USA) and used as excitation source.

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2.7. Statistical Analysis

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reproducibility. The data were expressed with mean values ± standard deviations. The

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differences between control and other treatments were analyzed by one-way analysis

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of variance. We used p<0.05 for the statistical significance.

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3. Results and discussion

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3.1. Detection of O2•−

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As shown in Fig. 1, six characteristic peaks of the DMPO-O2•− spin adducts

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could be detected using ESR under UV irradiation in TiO2, ZnO, Fe2O3, and CeO2

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suspensions, while no DMPO-O2•− adducts are detected in CuO and SiO2 suspensions.

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These results were consistent with our published works in which O2•− was generated

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only by TiO2, ZnO, Fe2O3, and CeO2 (Li et al. 2012, Li et al. 2014). Fig. S1 shows the

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changes in the absorption spectra of DOM solutions with or without NPs using XTT

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as indicator during a 48-h exposure to UV irradiation. The absorption peak at λ = 470

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nm indicates the production of O2•−. Our previous work and present molecular probe

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results (Fig. S1) have demonstrated that O2•− could be detected in HA solution but not

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in FA solution, primarily because only HA can undergo photoionization and then

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transfer electron to O2 (Li et al. 2015). Effect of comparative light absorption of XTT

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with NPs or DOM for UV-365 light on O2•− generation is provided in section S5.

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After addition of FA solutions, no DMPO-O2•− adducts or absorption peaks at λ =

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470 nm are detected in all NP suspensions under UV irradiation. The results showed

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that the NPs/FA mixtures do not produce O2•−, which is probably because FA can

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quench O2•− or compete with NPs for light absorption (Alrousan et al. 2009, Selcuk

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2010). Similarly, the addition of HA into Fe2O3 suspension weakened the intensities

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of the DMPO-O2•− adducts and the absorption peak at λ = 470 nm, indicating that HA

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decreased O2•− generation in Fe2O3 suspension. In marked contrast, the addition of HA

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ACCEPTED MANUSCRIPT solutions into the other five NP suspensions strengthened the intensities of the

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DMPO-O2•− adducts and increased the absorption peaks at λ = 470 nm. The reasons

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for the enhanced O2•− generation in the NPs/HA mixtures remain unclear. It could be

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due to O2•− generation by HA or O2 reduction by trapped electron on NP surface from

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HA or combination of these reactions, which will be discussed in the Electron

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Transfer between DOM and NPs section. No DMPO-O2•− signals and absorption

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peaks at λ = 470 nm have been detected in NP suspensions, DOM solutions, or their

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mixtures in the dark. --------------------------------

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Fig. 1 here

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3.2. Detection of •OH

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As shown in Fig. 2, four characteristic peaks of the DMPO-•OH spin adducts, a

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1:2:2:1 quartet pattern, were detected using ESR under UV irradiation in TiO2, ZnO,

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and Fe2O3 suspensions. We did not detect any DMPO-•OH signals with CeO2, CuO

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and SiO2 suspensions. Our previous work has also demonstrated that only TiO2, ZnO

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and Fe2O3 can induce •OH generation under UV irradiation (Li et al. 2012). Fig. S3

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shows the concentrations of pCBA decrease by less than 5% after 48-h UV irradiation

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in HA or FA solutions. No obvious DMPO-•OH adducts were detected in DOM

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solutions (Fig. S4). Both the molecular probe assays and ESR results indicated that

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•OH was not produced in DOM solutions.

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After addition of HA or FA into TiO2, ZnO, and Fe2O3 suspensions, the

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intensities of DMPO-•OH spin adducts were lower. The degradation rates of pCBA in

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TiO2, ZnO, and Fe2O3 suspensions were reduced after addition of HA or FA under

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UV irradiation (Fig. S3). The decreased •OH generation was primarily due to either

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ACCEPTED MANUSCRIPT •OH scavenging and/or competitive light absorption by DOM (Westerhoff et al. 2007,

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Westerhoff et al. 1999). Both the molecular probe assays and ESR results indicated

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that the inhibition effect of FA on •OH generation by TiO2, ZnO, and Fe2O3 was

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higher than that of HA. This was consistent with previous study that the second-order

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rate constant of •OH reaction with Suwannee River FA (2.7 × 104 s-1 (mg of C/L)-1)

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was ~1.7-fold higher than the constant with Suwannee River HA (1.9 × 104 s-1 (mg of

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C/L)-1) (Goldstone et al. 2002). Further, the addition of DOM in the other three NP

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suspensions (CeO2, CuO, and SiO2) did not degrade pCBA significantly or induce the

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generation of DMPO-•OH spin adducts under UV irradiation, indicating that no

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measurable amount of •OH was produced in the mixtures. In the dark, none of these

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NPs produced detectable •OH with or without DOM. --------------------------------

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Fig. 2 here

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3.3. Detection of 1O2

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As shown in Fig. 3, three characteristic peaks of TEMPO spin adducts were

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detected by ESR in TiO2, ZnO, and SiO2 suspensions when irradiated by UV light. In

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contrast, no obvious TEMPO signals were detected in CeO2, CuO, and Fe2O3

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suspensions. These results were consistent with published works in which 1O2 was

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detected only in TiO2, ZnO, and SiO2 suspensions (Li et al. 2012, Li et al. 2014).

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Many previous works have demonstrated that more 1O2 was detected in FA solution

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than HA solution due to higher photosensitization capacity of FA than that of HA (Li

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et al. 2015, Dalrymple et al. 2010, Frimmel et al. 1987).

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After addition of HA or FA, stronger TEMPO signals were observed in TiO2,

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CeO2, SiO2, and CuO suspensions. For ZnO and Fe2O3, only FA enhanced the

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intensities of TEMPO signals. It is also noteworthy that the TEMPO signals in

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NPs/FA mixtures are stronger than those in HA/NPs mixtures. The 1O2 generation

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profile that was measured by the molecular probe assays (Fig. S5) was similar to that

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detected using ESR. The degradation rates of FFA were enhanced after DOM was

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added to TiO2, CeO2, SiO2, and CuO suspensions. In addition, the degradation rates of

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FFA were faster with FA addition than with HA addition under UV irradiation. For

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ZnO and Fe2O3, only FA increased the degradation rates of FFA. The reasons for the

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enhanced 1O2 generation in the NPs/DOM mixtures remain unclear. It may be due to

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reactions, which will be discussed in the “ROS Concentration Generated by

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NPs/DOM Mixtures” section. It is noteworthy that the addition of HA into ZnO and

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Fe2O3 suspensions did not increase 1O2 generation. This is probably because the

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released metal ions complex with carboxyl and phenolic groups of HA (Fujii et al.

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2014, Neubauer et al. 2013, Cheng and Allen 2006, Cheng et al. 2005), efficiently

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deactivate the triplet states of HA (Carlos et al. 2012), and subsequently results in the

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decreased sensitization capacity of HA for 1O2 generation by ZnO and Fe2O3. In the

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dark, no measurable amount of 1O2 was detected for any of these NPs with or without

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DOM (data not shown).

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O2 generation by DOM or DOM sensitization of NPs or combination of these

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3.4. ROS concentration generated by NPs/DOM mixtures

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Table 1 summarizes the time-averaged molar concentrations of the three types of

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ROS (•OH, 1O2, and O2•−) for different NPs with HA or FA. As shown in Table 1, the

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NPs/DOM mixtures generated at least one type of ROS under UV irradiation. The

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time-averaged molar concentrations of total ROS (the sum of the concentrations of

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three types of ROS) in NPs/DOM mixtures followed the order of TiO2/HA > ZnO/HA >

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SiO2/HA> CeO2/HA > CuO/HA > Fe2O3/HA and TiO2/FA > SiO2/FA > CeO2/FA >

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ZnO/FA > CuO/FA > Fe2O3/FA. It was observed that all of the NPs/HA mixtures generated O2•−, while none of

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the NPs/FA mixtures produced measurable amount of O2•−. The generated O2•−

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concentrations in the Fe2O3/HA mixtures were lower than the sum of O2•−

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concentrations that were produced when Fe2O3 and HA were present in water by

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themselves. In marked contrast, for the other five types of NPs, the generated O2•−

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concentrations in the NPs/HA mixtures were higher than the sum of O2•−

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concentrations that were produced when NPs and HA were present by themselves.

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•OH was generated only by Fe2O3, TiO2, and ZnO with or without DOM. Both

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HA and FA decreased the generation of •OH by Fe2O3, TiO2, and ZnO. And the

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inhibition effect of FA on •OH generation was higher than that of HA for the same

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NPs due to the higher reaction rate between •OH and FA (Goldstone et al. 2002).

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Among the NPs/HA mixtures, ZnO/HA mixtures generated the most •OH, which was

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approximately 2.0-fold and 2.4-fold more than that generated in the TiO2/HA and

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Fe2O3/HA mixtures, respectively. The •OH concentrations produced by NPs/FA

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mixtures followed the order of ZnO/FA > Fe2O3/FA > TiO2/FA under UV irradiation.

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After addition of HA or FA, the 1O2 concentrations increased in NP suspensions

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except for addition of HA to Fe2O3 and ZnO suspensions. For the same DOM type,

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the generated 1O2 concentrations after their addition into NPs followed the order of

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TiO2/DOM > SiO2/DOM > CeO2/DOM > ZnO/DOM > CuO/DOM > Fe2O3/DOM.

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For SiO2, CeO2, and CuO, 1O2 generation concentrations in the NPs/DOM mixtures

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were 1.2~2.9 times higher than the sum of 1O2 generation that were produced when

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ACCEPTED MANUSCRIPT NPs and DOM were present in water by themselves. The 365 nm UV light can induce

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the generation of 3DOM* because the incident photon energy (3.4 eV) is higher than

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the energy of 3DOM* (180−250 kJ/mol, 1.9−2.6 eV). As far as the photophysics is

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concerned, 3DOM* has a much higher energy level than that of 1O2 (94.3 kJ/mol, 0.98

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eV). Therefore, the synergistic generation of 1O2 in the NPs/DOM mixtures was

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because 3DOM* can transfer energy to O2 as well as NPs. The excess energy

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transferred from 3DOM* to NPs could be subsequently transferred to O2 and generate

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more 1O2.

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For the same NPs, the generated 1O2 concentrations in the NPs/FA mixtures

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were approximately 1.1 to 1.5 times higher than that generated in the NPs/HA

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mixtures. This was consistent with previous work that FA possessed higher

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photosensitization capacity than HA due to the presence of more chromophores (such

334

as phenyl ketone or aromatic quinone) in the lower-molecular-weight fractions of

335

DOM which comprise FA fraction (Li et al. 2015, Dalrymple et al. 2010, Frimmel et

336

al. 1987).

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In this work, TiO2 was selected to investigate ROS generation at lower

338

concentrations (0.05 and 0.5 mg/L) because it is widely used as antimicrobial agent.

339

As shown in section S9, ROS generation profile by less TiO2 was similar to that by 5

340

mg/L TiO2 with or without DOM. Therefore ROS concentrations generated by 5 mg/L

341

NPs with or without DOM could be extrapolated to natural surface water.

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Table 1 here

344

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345

3.5. Electron transfer between DOM and NPs

346

The transient absorption spectra of NPs, DOM, or their mixtures were recorded

347

to investigate the electron transfer between DOM and NPs. As shown in Fig. 4(a), the 14

ACCEPTED MANUSCRIPT spectrum of HA recorded 7 ns after the flash exhibits two narrow (approximately 100

349

nm) absorption bands at 480 nm and 650 nm. As demonstrated previously, the narrow

350

absorption band at approximately 480 nm was attributed to the cation radical HA•+

351

(Vinodgopal 1994, Frimmel et al. 1987). The narrow absorption band at

352

approximately 650 nm was due to the electron (Vinodgopal 1994, Vinodgopal and

353

Kamat 1992, Lin et al. 2014), which was generated through HA photoionization. After

354

saturating HA solution with O2 or N2O, an electron scavenger, the transient absorption

355

at approximately 650 nm disappeared, confirming that the narrow absorption band

356

was due to the electron. Some works have reported that the solvated electron and

357

radical cations of DOM (extracted from valley peat, collected from natural waters, or

358

commercially from Contech ETC, Ottawa, Canada) exhibited broader spectra than the

359

relatively narrow bands observed in our work (Zepp et al. 1987, Fischer et al. 1985,

360

Chaikovskaya et al. 2004). The different results were probably because DOM used in

361

these previous studies was different from Suwannee River HA and FA used in this

362

study.

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hv HA *  → HA•+ + e-

(2)

The transient absorption spectra of six NPs recorded 7 ns after the flash exhibited

365

no obvious absorption bands (data not shown). After addition of NPs, the transient

366

spectra of HA exhibit similar features, but the intensities of the product for the

367

NPs/HA mixtures (except Fe2O3/HA mixture) are significantly higher. The intensity

368

increase of absorption bands at approximately 650 nm is due to electron transfer from

369

the excited singlet or triplet state of HA to the conduction band of metal-oxide NPs

370

(Vinodgopal and Kamat 1992, Frimmel et al. 1987).

371 372

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hv HA * + NPs  → HA•+ + NPs (e-)

(3)

The trapping of electrons at the semiconductor surface reduces the recombination

15

ACCEPTED MANUSCRIPT between electron and cation radical (Vinodgopal 1994, Frimmel et al. 1987). Such a

374

long-lived electron at the NP surface could reduce O2, which is responsible for

375

synergistic O2•− photogeneration in the NPs/HA mixtures (except Fe2O3/HA mixture).

376

It is worth mentioning that the addition of Fe2O3 changed the peak intensities of HA

377

slightly. The released concentrations of iron ions, including Fe3+ and Fe2+, were

378

measured by ICP/MS. The concentrations of Fe3+ and Fe2+ were measured by

379

colorimetric

380

o-phenanthroline, respectively. The O2•− generation concentration of HA solution was

381

decreased by iron ions at similar concentration to that released from Fe2O3. This result

382

indicated that the released iron ions could deactivate HA* and inhibit electron transfer

383

from HA* to O2. Therefore, synergistic O2•− generation was not observed in the

384

Fe2O3/HA mixtures.

through

the

complexes

formed

with

KSCN

and

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As shown in Fig. 4(b), the absorption spectrum of FA recorded 7 ns after the

386

flash exhibits two narrow absorption bands (approximately 100 nm) at 480 nm and

387

650 nm. Similarly, the transient absorption bands at approximately 480 nm and 650

388

nm were attributed to the cation radical FA•+ or electron (Vinodgopal and Kamat

389

1992). After addition of NPs, the peak intensities of the transient spectra changed

390

slightly, indicating that FA cannot inject electron into NPs. In addition, FA could

391

consume O2•− and/or compete with NPs for UV light. Thus synergistic O2•−generation

392

was not observed in the NPs/FA mixtures. Our previous study has also demonstrated

393

that HA possessed higher electron donation efficiency than that of FA due to lower

394

aromaticity and phenolic group content of FA than that of HA (Li et al. 2015). Thus,

395

more O2•− was detected in the NPs/HA mixtures than that in the NPs/FA mixtures for

396

the same NPs.

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Fig. 4 here 16

ACCEPTED MANUSCRIPT 399 400

-------------------------------3.6. Effect of DOM on inactivation kinetics of E. coli by NPs To investigate the effect of HA and FA on the phototoxicity of metal-oxide NPs,

402

we performed the bacterial inhibition assay with E. coli under UV irradiation. For

403

exposure to room light, the survival rates (log(Nt/N0)) of E. coli exposed to NPs with

404

or without DOM were all less than -0.2 (Fig. S7), indicating that the cellular

405

inhibition from exposure to NPs/DOM mixtures under room light should be minimal.

406

The control test without exposure to NPs and DOM demonstrated that the value of

407

log(Nt/N0)) was -0.2 for the 2-h UV irradiation (Li et al. 2012, Li et al. 2014). This

408

result indicated that the bacterial survival rate was not significantly compromised by

409

2-h UV irradiation.

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Our previous work has demonstrated that the toxicity of six types of NPs toward

411

E. coli decreased in the order of CuO > TiO2 > ZnO > SiO2 > Fe2O3 > CeO2 under UV

412

irradiation (Li et al. 2012). The kinetics of E. coli inactivation by six types of

413

metal-oxide NPs with HA or FA under UV irradiation is shown in Fig. 5. It is obvious

414

that the inactivation curves of E. coli undergo a 60-min lag-phase, followed by a

415

exponential decrease. After addition of DOM, the phototoxicity of CuO and ZnO

416

toward E. coli decreased (Table S3). FA decreased E. coli survival rate more

417

significantly than HA. For ZnO, the released Zn2+ concentrations in ZnO suspension,

418

ZnO/HA mixture, and ZnO/FA mixture after 2-h UV irradiation were 178.3, 122.8,

419

and 90.1 µg/L, respectively. Our previous work has demonstrated that no obvious

420

inhibition of E. coli activity was observed when Zn2+ was 1 mg/L (Li et al., 2012).

421

This indicated that the released Zn2+ was not responsible for the antibacterial effect of

422

ZnO with or without DOM under UV irradiation. The antibacterial assay was

423

conducted in the presence of ROS scavengers, using

424

superoxide dismutase as scavengers for 1O2, •OH, and O2•−, respectively. The survival

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L-histidine,

t-BuOH, and

ACCEPTED MANUSCRIPT rates of E. coli in ZnO suspension and ZnO/DOM mixtures were all higher than 95%

426

in the presence of scavengers of three ROS, indicating that ROS was the dominant

427

reason for the antibacterial activity of ZnO with or without DOM. The DOM-induced

428

toxicity reduction of ZnO was primarily because the concentration of total ROS

429

decreased in the trend of ZnO > ZnO/HA mixture > ZnO/FA mixture.

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The dissolved Cu2+concentration in CuO suspension was 30.5 µg/L after 2-h UV

431

irradiation. No measurable amount of Cu2+ was detected in CuO suspension after

432

addition of DOM due to strong complexation between DOM and Cu2+ (Maurer et al.

433

2013, Gao and Korshin 2013), indicating that free Cu2+ did not play a role in the

434

antibacterial activity of CuO/DOM mixtures. After addition of ROS scavengers, high

435

survival rates (> 95%) of E. coli in CuO/DOM mixture were observed, indicating that

436

ROS was the dominant reason for the antibacterial activity of CuO/DOM mixtures.

437

The viability of E. coli after exposure to CuSO4 at the same Cu2+ concentration (30.5

438

µg/L) as that released from CuO was investigated under UV irradiation. Similar

439

bacterial inactivation rate after 2-h UV irradiation was found. This indicated that Cu2+

440

played a major role in the strong inhibition effect of CuO toward E. coli. Although the

441

concentrations of total ROS in CuO/DOM mixtures were higher than that in CuO

442

suspension, the phototoxicity of CuO/DOM mixtures was much lower than that in

443

CuO suspension. This was primarily because the toxicity of Cu2+ toward E. coli was

444

more significant than that of ROS. Some toxicity studies using E. coli have

445

demonstrated that DOM resulted in marked decrease in the antibacterial activity of

446

CuO and ZnO (Zhao et al. 2013, Mileyeva-Biebesheimer et al. 2010, Li et al. 2011),

447

which are consistent with our results.

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448

In contrast, the addition of DOM into SiO2 and CeO2 suspensions increased the

449

phototoxicity of these NPs toward E. coli. The DOM-induced toxicity increase of

18

ACCEPTED MANUSCRIPT SiO2 and CeO2 was primarily because DOM enhanced the generated ROS

451

concentrations by NPs. We should note that the addition of FA increased but HA

452

decreased the total ROS concentrations generated by Fe2O3, therefore the toxicity of

453

Fe2O3 with or without DOM toward E. coli followed the order of Fe2O3/FA mixture >

454

Fe2O3 > Fe2O3/HA mixture under UV irradiation. The time-averaged concentration of

455

total ROS generated by TiO2 changed slightly after addition of DOM and thus both

456

HA and FA exhibited minor effect on the antibacterial activity of TiO2. Several

457

previous studies have demonstrated that the toxicity of TiO2 toward E. coli was

458

reduced by HA or FA under simulated solar or UV irradiation (Tong et al. 2013,

459

Rincón and Pulgarin 2005, Alrousan et al. 2009). The differences from our results

460

may be attributed to the differences in sources of TiO2, solution chemistry (e.g., pH

461

and ionic strength), or experimental conditions (e.g., light conditions and medium). --------------------------------

463

Fig. 5 here

464

--------------------------------

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3.7. Relationship between the antibacterial potency of NPs/DOM mixtures and ROS

466

generation

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Our previous work has demonstrated that there was a linear correlation between

468

the time-averaged molar concentration of total ROS (•OH, 1O2, and O2•−) generated

469

by metal-oxide NPs and their bacterial activity toward E. coli under UV irradiation

470

(R2=0.84) (Li et al. 2012). To elucidate the effect of different DOM fractions on the

471

relationship between the antibacterial potency of metal-oxide NPs and ROS

472

generation, we summed the time-averaged molar concentrations of each type of ROS

473

for different NPs/DOM mixtures and plotted these against the survival rates (2 h

474

log(Nt/N0)) of E. coli. As shown in Fig. 5, the degradation rate after 2 h clearly shows

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ACCEPTED MANUSCRIPT the survival rate after a lag phase of about 1 h. That was why we plotted the

476

time-averaged ROS concentration versus the survival rate after 2 h in Fig. 6. As

477

shown in Fig. 6, the toxicity of NPs/DOM mixtures monotonically decreases with the

478

increasing time-averaged molar concentration of total ROS. The linear equation was

479

used to fit the relationship between the time-averaged concentrations of total ROS

480

generated by the NPs/DOM mixtures and their bacterial survival rates. The values for

481

A and B are described as the intercept and slope, respectively. The fit parameters A for

482

the NPs/HA and NPs/FA mixtures are -1.3111 and -1.1243, respectively. The p values

483

of parameter A for the NPs/HA and NPs/FA mixtures are 0.0384 and 0.0161 (less than

484

the significance level, 0.05), respectively. This demonstrates that A values are

485

statistically different from 0. The parameter A represents the background bacterial

486

survival rates without any ROS generation. The toxicity of NPs in the absence of ROS

487

is probably due to the dissolved metal ions or other physical damage from NPs

488

attachment on bacterial cell walls (Adams et al. 2006, Thill et al. 2006, Baek and An

489

2011). The fit parameters B for the NPs/HA and NPs/FA mixtures are -0.0077 and

490

-0.0078 with a confidence level of 95%, respectively. The p values of parameter B for

491

the NPs/HA and NPs/FA mixtures are 0.0135 and 0.0045 (less than the significance

492

level, 0.05), respectively. This indicates that B is statistically different from 0, and

493

demonstrates that the bacterial survival rate depends on the total ROS concentration.

494

The values of R2 for the NPs/HA and NPs/FA mixtures are 0.82 and 0.89, respectively,

495

demonstrating that oxidative stress induced by ROS generation plays an important

496

role in the photoinduced toxicity of metal-oxide NPs.

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497

--------------------------------

498

Fig. 6 here

499

--------------------------------

20

ACCEPTED MANUSCRIPT E. coli enters surface fresh water through agricultural runoff, urban stormwater,

501

animal feces, and sewage overflows. Our study demonstrated that E. coli could be

502

inactivated by metal-oxide NPs under UV irradiation. This indicates that

503

photocatalysis is a promising method for disinfection of natural waters contaminated

504

by E. coli under solar irradiation. The different electron and energy transfer capacity

505

of HA and FA results in their different effects on ROS generation and toxicity of

506

metal-oxide NPs toward E. coli. This strongly suggests that the antibacterial activity

507

assessment of metal-oxide NPs should consider both their inherent toxicity and DOM

508

components under natural solar irradiation and UV irradiation during wastewater

509

treatment. Linear regression of the relation between ROS concentrations generated by

510

NPs/DOM mixtures and their bacterial survival rates resulted in an R2 higher than

511

0.80. Therefore, ROS generation could be used as an indicator for antibacterial

512

activity of NPs after their release into natural surface waters where DOM or E. coli is

513

ubiquitous.

514

4. Conclusions

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The present work investigated the effect of DOM fractions (HA or FA) on the

516

generation types and concentrations of •OH, O2•−, and 1O2 by six NPs and their

517

toxicity toward E. coli under UV-365 irradiation. •OH was generated only by Fe2O3,

518

TiO2, and ZnO with or without DOM. Both HA and FA decreased the generation of

519

•OH by Fe2O3, TiO2, and ZnO, and HA decreased •OH generation less than FA due to

520

the lower rate constant of •OH reaction with HA. None of the NPs/FA mixtures

521

produced measurable amount of O2•−, but all of the NPs/HA mixtures produced O2•−.

522

Only FA promoted 1O2 generation by ZnO and Fe2O3, and both HA and FA enhanced

523

1

524

between the average concentrations of total ROS generated by the DOM/NPs

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O2 generation by the other four NPs. In addition, a linear correlation was found

21

ACCEPTED MANUSCRIPT mixtures and their toxicity toward E. coli (R2 ≥ 0.80). These results indicate that the

526

different photoreactivity of HA and FA results in their different effect on the

527

generation of ROS by NPs and their toxicity, highlighting the necessity of considering

528

DOM components when investigating the environmental behaviors of NPs.

529

Acknowledgments

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This study was financially supported by the Fund for Innovative Research Group

531

of the National Natural Science Foundation of China (No. 51421065), the National

532

Natural Science Foundation of China (Nos. 51378065 and 21407010), China

533

Postdoctoral Science Foundation (No. 224234), and China Postdoctoral Science

534

Special Foundation (No. 212400234). The authors also appreciate support from the

535

Brook Byers Institute for Sustainable Systems, Hightower Chair and Georgia

536

Research Alliance at Georgia Institute of Technology.

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541 542 543 544 545 546 547 548 549 550 551 552 553 554 555 556

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Charge injection from excited fulvic acid into semiconductor colloids. Environ. Sci. Technol. 26(10), 1963–1966. Westerhoff, P., Aiken, G., Amy, G., Debroux, J., 1999. Relationships between the structure of natural organic matter and its reactivity towards molecular ozone and hydroxyl radicals. Water Res. 33(10), 2265–2276. Westerhoff, P., Mezyk, S.P., Cooper, W.J., Minakata, D., 2007. Electron pulse radiolysis determination of hydroxyl radical rate constants with Suwannee river fulvic acid and other dissolved organic matter isolates. Environ. Sci. Technol. 41(13), 4640–4646. Zepp, R.G., Braun, A.M., Hoigne, J., Leenheer, J.A., 1987. Photoproduction of hydrated electrons from natural organic solutes in aquatic environments. Environ. Sci. Technol. 21(5), 485–490. Zhao, J., Wang, Z., Dai, Y., Xing, B., 2013. Mitigation of CuO nanoparticle-induced bacterial membrane damage by dissolved organic matter. Water Res. 47(12), 4169–4178.

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ACCEPTED MANUSCRIPT Table 1 Time-averaged molar concentrations of ROS generated by NPs, DOM, or their mixtures under UV light irradiation.

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O2 (µM) 35.7 ± 1.5 53.4 ± 2.2 417.3±18.8 427.5±19.6 434.4±18.2 N.D. 104.8±6.6 133.0±6.6 56.5±2.5 106.5±6.2 143±7.8 100.8±6.4 86.5±6.0 117.2±6.2 N.D. 73.8±3.3 112.0±6.6 N.D. N.D. 96.5±6.0

O2•− (µM) 2.5±0.3 N.D. 8.0±0.4 13.3±8.9 N.D. 8.4±0.2 39.7±2.6 N.D. N.D. 50.7±2.2 N.D. 167±8.6 176.2±7.6 N.D. N.D. 65.7±2.6 N.D. 18.1±1.1 10.1±0.5 N.D.

Total (µM) 99.8 ± 4.5 121.6 ± 7.2 442.9±20.0 443.0±28.7 434.4±18.2 8.4±0.2 144.5±9.2 133.0±6.6 56.5±2.5 157.2±8.4 143.0±7.8 277.3±15.6 267.2±14.0 119.1±14.7 0 139.5±5.9 112.0±6.6 20.4±1.2 12.0±0.6 97.9±6.1

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•OH (µM) N.D. N.D. 19.3±0.8 2.2±0.1 N.D. N.D. N.D. N.D. N.D. N.D. N.D. 9.5±0.6 4.5±0.2 1.9±0.1 N.D. N.D. N.D. 2.3±0.1 1.9±0.1 1.4±0.1

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DOM HA FA No HA FA No HA FA No HA FA No HA FA No HA FA No HA FA

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NPs No No TiO2

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N.D. indicated that ROS were not detectable or were not statistically significant. No indicated that neither HA nor FA was added into the NP solutions.

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Fig. 1. ESR spectra recorded at ambient temperature for DMPO adduct with O2•− in metal-oxide NP solutions with or without DOM (UV irradiation at 365 nm, light

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intensity of 1.4 × 10-6 Einstein·L-1·s-1, NPs of 5 mg/L, and DOM of 5 mg/L). Fig. 2. ESR spectra recorded at ambient temperature for DMPO adduct with •OH in metal-oxide NP solutions with or without DOM (other conditions were the same as in Fig. 1).

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Fig. 3. ESR spectra recorded at ambient temperature for TEMP adduct with 1O2 in metal-oxide NP solutions with or without DOM (other conditions were the same as in Fig. 1).

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Fig. 4. Transient absorption spectra obtained following 355 nm laser pulse excitation of (a) HA or (b) FA with or without NPs immediately after the flash (NP concentration of 5 mg/L and DOM concentration of 5 mg/L).

Fig. 5. Kinetics of E. coli inactivation by six types of metal-oxide NPs in the presence or absence of DOM under UV irradiation (other conditions were the same as in Fig.

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Fig. 6. Linear regression between the 2 h log(Nt/N0) values and the time-averaged concentration of total ROS generated by NPs/HA mixtures (left) or NPs/FA mixtures

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(right) with the 95% confidence limits.

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Fig. 1

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Fig. 3

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Highlights

1. Effect of DOM on ROS generation and toxicity of NPs is investigated. 2. FA inhibited •OH generation by TiO2, ZnO, and Fe2O3 more significantly than HA.

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3. None of NPs/FA mixtures generates O2•−, but all NPs/HA mixtures produce O2•−. 4. FA promotes 1O2 generation by TiO2, CuO, CeO2, SiO2 more significantly than HA.

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5. ROS formed by DOM/NPs mixtures is linearly correlated with their toxicity.