Accepted Manuscript Conservative behavior of fluorescence EEM-PARAFAC components in resin fractionation processes and its applicability for characterizing dissolved organic matter Wei He, Jin Hur PII:

S0043-1354(15)30093-2

DOI:

10.1016/j.watres.2015.06.044

Reference:

WR 11386

To appear in:

Water Research

Received Date: 13 May 2015 Revised Date:

23 June 2015

Accepted Date: 26 June 2015

Please cite this article as: He, W., Hur, J., Conservative behavior of fluorescence EEM-PARAFAC components in resin fractionation processes and its applicability for characterizing dissolved organic matter, Water Research (2015), doi: 10.1016/j.watres.2015.06.044. 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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Conservative behavior of fluorescence EEM-PARAFAC components in resin

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fractionation processes and its applicability for characterizing dissolved organic matter

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Wei He and Jin Hur*

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Department of Environment and Energy, Sejong University, Seoul, South Korea, 143-747

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Revised and Resubmitted to Water Research, June, 2015

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*Corresponding author. Tel.:+82-2-3408-3826; fax:+82-2-3408-4320.

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E-mail addresses:[email protected] (J. Hur)

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Abstract

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In this study, the applicability of the fluorescence excitation-emission matrix combined with

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parallel factor analysis (EEM-PARAFAC) was verified for resin fractionation processes, in

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which bulk dissolved organic matter (DOM) is separated into several fractions presumably

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having similar chemical structures. Here, four PARAFAC components, including three humic-

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like and one protein-like components, were identified from the EEMs of all DOM samples

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through fractionation procedures and the subtracted EEMs between before and after resins for

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different DOM sources (effluent, limnic, and riverine). The PARAFAC components exhibited

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conservative behavior upon resin fractionation, as indicated by the minimal difference in the

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PARAFAC components retained on resins calculated based on the direct subtraction of the

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components and the subtracted EEMs. The conservative behavior of PARAFAC components

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was more obvious compared with other fluorescent DOM (FDOM) indicators derived from peak-

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picking and fluorescence regional integration (FRI) methods. Humic-like components were more

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insensitive to resin fractionation than protein-like component. No consistency was found in the

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relative abundances of the PARAFAC components for the same resin fractions with different

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DOM sources, suggesting that the FDOM composition is more affected by DOM sources rather

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than by the resin fractions. Our study demonstrated that EEM-PARAFAC coupled with resin

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fractionation could provide detailed information on DOM by quantitatively comparing the

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individual PARAFAC components within different resin fractions.

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Key words: Natural organic matter; Resin fractionation; Parallel factor analysis (PARAFAC);

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Fluorescence regional integration; Spectral subtraction.

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1. Introduction Fluorescence excitation-emission matrix combined with parallel factor analysis (EEMPARAFAC) has become a popular tool for probing the fate of dissolved organic matter (DOM)

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and understanding its environmental behaviors in natural and engineered systems (Borisover et

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al., 2009; Ishii and Boyer, 2012; Stedmon et al., 2003; Yang et al., 2015). PARAFAC modeling

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makes it possible to extract dissimilar fluorescent components with minimum residuals from a

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given EEM dataset (Stedmon et al., 2003). The identified components have been successfully

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applied for exploring the biogeochemical dynamics of fluorescent DOM (FDOM) in aquatic

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ecosystems, and the temporal and spatial variations. The individual components have their own

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sources and characteristics, displaying different sensitivities to varying environmental factors

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like light, salinity, pH, temperature, and microorganisms (Borisover et al., 2009; Jørgensen et al.,

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2011; Meng et al., 2013; Saadi et al., 2006; Yamashita et al., 2008; Yang and Hur, 2014; Zhang

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et al., 2009). They also have great potential for assessing water quality and the efficiency of

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DOM removal during treatment systems (Cohen et al., 2014; Gone et al., 2009; Henderson et al.,

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2009; Murphy et al., 2011; Seredyńska-Sobecka et al., 2011).

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To date, several techniques have been suggested for fluorescence data decomposition,

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which included PARAFAC, fluorescence regional integration (FRI), principal component

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analysis (PCA), and self-organizing map (SOM). Among those, PARAFAC and FRI have been

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the most popularly used due to the easy quantification of different FDOM components. The

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interpretation and source assignment of PARAFAC components were primarily based on a

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traditional peak-picking method (Coble, 1996) in which different fluorescence peaks were

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selected from several defined wavelength ranges of EEM. The FRI method has been often

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utilized to differentiate different FDOM components within an EEM, in which EEM was divided

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into several assigned regions and their integrated regional volumes under the EEM surface were

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calculated and treated as the individual FDOM components (Chen et al., 2003; Xue et al., 2012;

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Zhou et al., 2014), although it is often criticized for the physical meaningfulness of the

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integration.

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Resin fractionation has been long used as a DOM characterizing method to obtain

relatively homogeneous fractions from a bulk DOM inherently consisting of heterogeneous

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chemical structures and functional groups (He et al., 2011; Schwede-Thomas et al., 2005; Wu et

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al., 2003; Xue et al., 2012; Zhou et al., 2014). The conventional practices for resin fractionation

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involve the use of acid and base solutions for pH control to retain the desired DOM fractions on

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resins or to elute them from the resins (Imai et al., 2001; Thurman and Malcolm, 1981). Recently,

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a new resin fractionation method was proposed to offer more stable DOM fractions with respect

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to the chemical properties without the pH manipulation (Kim and Dempsey, 2012). Through

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both resin fractionation processes, DOM can be separated into hydrophobics (HPO) and/or

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transphilics (TPI), and hydrophilics (HPI), and further into acidic, basic, and neutral fractions

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(Chen et al., 2003; Imai et al., 2001; Kim and Dempsey, 2012; Li et al., 2014). In general,

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dissolved organic carbon (DOC) and ultraviolet (UV) absorbance have been employed to track

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and quantify the DOM fractions retained on resins and/or eluted from the resins (Imai et al.,

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2002; Kim and Dempsey, 2012). However, such DOM parameters represent only the bulk

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quantity of DOM, providing limited information on the chemical composition of resin fractions

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(Kim and Dempsey, 2012). Addition of other DOM analyses would be more beneficial for

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acquiring detailed information on the fraction’s characteristics.

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In resin fractionation, the subtraction of DOM quantity parameters (i.e. DOC and UV) between before and after resins is simply applied to estimate the fractions retained on resins

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based on mass balance (Imai et al., 2001; Imai et al., 2002; Imai et al., 2003; Kim and Dempsey,

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2012). In the same manner, the spectral subtraction for the fluorescent components distributed

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over EEM could be applied to track the FDOM through the resin fractionation. However, no

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effort has been made to extend the simple subtraction approach into EEM-PARAFAC for

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characterizing different DOM fractions obtained from resin fractionation processes. In fact, this

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is an attempt to test the feasibility of EEM-PARAFAC for tracking DOM in fractionation

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systems where DOM constantly interacts with the solid phase (e.g., resins). There may be two

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approaches available to utilize EEM-PARAFAC to track FDOM in resin fractionation processes.

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One is to obtain the PARAFAC components based on the subtracted EEMs between before and

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after resin (Patra and Mishra, 2002) based on the Beer-Lambert law. The other relies on the

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direct subtraction of the PARAFAC components between before and after resins. It is not clear

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whether or not the two different subtraction approaches would produce the same results for the

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DOM retained on resins.

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In an effort to verify the quantitative applicability of fluorescence EEM-PARAFAC for

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resin fractionation, the two types of resin fractionation methods were employed in this study to

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characterize aquatic DOM with diverse sources (i.e., effluent, limnic, and riverine DOM). The

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main objectives of this study were 1) to verify the applicability of EEM-PARAFAC for

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characterizing DOM in resin fractionation processes, and 2) to compare the differences in the

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FDOM composition of DOM resin fractions among different sources via EEM-PARAFAC.

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2. Materials and methods

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2.1. Sample collection and pretreatment

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Effluent, limnic, and riverine waters were collected three times from a water reclamation

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center in Seoul, Lake Uiam in Gangwon province, and Han River in Seoul, Korea, respectively,

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during the period between November 2014 and January 2015. The in-situ water quality

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parameters of the samples are shown in the supplementary materials (Table S1). The samples

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were filtered through a pre-washed 0.45 µm membrane filter (cellulose acetate, Toyo Roshi

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Kaisha, Ltd., Japan) for further fractionation and analyses.

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2.2. Rein fractionation procedures

The two frequently used DOM fractionation procedures, named Imai’s (Imai et al., 2001)

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and Kim’s (Kim and Dempsey, 2008; 2012) methods, were adopted for this study (Fig. 1). In

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Imai’s method, 1.0 L of DOM sample was fractionated into HPO acids (HPO(a)), HPO neutrals

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(HPO(n)), organic bases (i.e. HPO bases, HPI bases, and TPI bases; HPO/HPI/TPI(b)), TPI/HPI

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acids (TPI/HPI(a)), and TPI/HPI neutrals (TPI/HPI(n)) (Fig. 1a). Before the fractionation, the pH

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of samples was adjusted to 2 by adding concentrated HCl solution. HPO(a) and the HPO(n) were

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retained by the first column (7 cm depth, inner diameter 1.8 cm) packed with nonionic Amberlite

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DAX-8 resin (20-60 mesh), and the retained fraction was subsequently eluted by 100 mL of 0.1

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N NaOH. The DAX-8 resin, poly(methyl methacrylate) resin, is known to have nearly the same

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capability of capturing humic substances (HS) as XAD-8 resin (Peuravuori et al., 2002). It was

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previously demonstrated that the recovery rate of HPO(a) by the alkaline solution was

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approximately 100% (Kim and Dempsey, 2012; Thurman and Malcolm, 1981). HPO/HPI/TPI(b)

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were retained by the second column filled with strong cation-exchange resin (Bio-Rad AG-MP-

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50, 50–100 mesh). TPI/HPI(a) were adsorbed onto strong anion-exchange resin (Bio-Rad AG-

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MP-1, 50–100 mesh) in the third column (Fig. 1a). The surface flow rate was maintained at a rate

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of 4 L min-1 m-2. The initial DOM samples and the DOM fractions eluted from each column (i.e.,

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IF0: NaOH-eluted fraction, and IF1, IF2, and IF3 refer to the DOM fractions eluted from XAD-8,

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AG-MP-50, and AG-MP-1 in Fig. 1a, respectively) were collected to determine DOC, UV, and

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fluorescence EEM.

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In Kim’s method (Fig. 1b), the original DOM samples (1.0 L) were separated into

HPO/TPI/HPI acids (HPO/TPI/HPI (a)), HPO bases/neutrals (HPO(b/n)), TPI bases/neutrals

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(TPI(b/n)), and HPI bases/neutrals (HPI(b/n)) upon resin fractionation. The diethylaminoethyl

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(DEAE) resin used in Kim and Dempsey (2008) was replaced by a weakly basic anion-exchange

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resin (Amberlite IRA-67, 500-750 µm) for this study because of the higher capacity to retain

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HPO/TPI/HPI(a) (Peuravuori and Pihlaja, 1998). DAX-8 and XAD-4 resins were filled in the

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next two columns sequentially to obtain the HPO(b/n) and the TPI(b/n), respectively. The

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surface flow rate was set at the same rate as in Imai’s method. The initial DOM and the eluted

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fractions from the resins of IRA-67, DAX-8, and XAD-4, denoted as KF1, KF2, and KF3 in Fig.

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1b, respectively, were collected for further analyses.

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In order to minimize adverse effects of the resins on the eluted DOM fractions, all the resins were previously cleaned using Soxhlet extraction with methanol for 24 h. For Imai’s

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method, DAX-8 resin was cleaned with 0.1 N NaOH and pre-conditioned with 0.1 N HCl before

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use. The AG-MP-1 and the AG-MP-50 were converted into free-base and free-acid forms using

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1 N NaOH and 1 N HCl, respectively, and rinsed with ultrapure water to adjust the pH into ~7.0.

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The blank samples for DAX-8, AG-MP-50, and AG-MP-1 (B1, B2, and B3, respectively) were

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also collected from each column. The DAX-8 and XAD-4 resins used in Kim’s method were

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similarly cleaned and conditioned except for the maintenance of the neutral pH condition. The

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IRA-67 was conditioned with 1 N NaOH, and rinsed with ultrapure water. In the same manner as

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in Imai’s method, the blank samples for IRA-67, DAX-8, and XAD-4 (B4, B5, and B6,

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respectively) were collected from each column. After the blank of the resins and the eluted

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volumes were all taken into account, the targeted DOM fractions were quantified using the

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formula provided in Table S2. The information on DOC, fluorescence EEMs, and PARAFAC

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components of the alkaline solution (i.e., 0.1 N NaOH) and the resin blanks are shown in Table

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S3 and Fig. S1. The blank correction was needed because some DOM resin fractions were eluted

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in low concentrations (Table S3).

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2.3. Analytical methods

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The pH of all DOM samples including the resin fractions was re-adjusted to ~ 7.0 prior to further analyses (Yang and Hur, 2014). DOC concentrations were measured by a Shimadzu V-

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CPH TOC analyzer with a relative precision of 0.95 indicate

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the exact match between the two PARAFAC components (Table S4). The related Matlab code

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(namely, comPARAFAC.m) is contained in Script S2. The relative concentration of each

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PARAFAC component was estimated by the Fmax output from DOMFluor.

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The individual FDOM indicators retained on the resins of the fractionation processes were estimated based on the two approaches: by directly subtracting the FDOM quantity

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indicators between before and after elution (Indd), in which the indicators were previously

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determined based on the EEMs of all collected DOM fractions, and by obtaining the indictors

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(Inds) based on the subtracted EEM spectra between DOM before and after resins. The ratios of

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Indd to Inds were used here to evaluate the stability (or conservativeness) of the three indicator

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groups to DOM resin fractionation. If the ratios approach to 1.0 (i.e., Indd is similar to Inds), it is

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assumed that the indicator subtraction has the same effects as the EEM spectral subtraction on

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characterizing the resin-fractionated DOM and also that the FDOM quantity indicator is

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conservative through the resin fractionation processes.

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3. Results and discussion

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3.1. PARAFAC components

Four different PARAFAC components were identified for this study (Fig. 2): three

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humic-like components (C1, C2, and C3) and one protein-like component (C4). The quantitative

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comparison was made using mTCC values for the identified components versus those previously

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reported in the literature (Table S4). The compared components in the literature with mTCC >

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0.95 indicated excellent coincidence with those of this study. C1, with the ex/em ranges of organic bases >

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TPI/HPI(a) ~ HPO(a) > TPI/HPI(n) for EfOM, which was not the case for the other DOM

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sources.

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3.3.2. Fractionation using Kim’s method

The sum of organic acids (HPO/TPI/HPI(a)) and HPI(b/n) dominated in all DOM

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samples (Fig. S6f), constituting more than 68% of the DOM on the basis of DOC. Only ~18% of

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organic acids were found in EfOM, which was similar to the value (15%) previously reported for

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effluent DOM in a previous study (Kim and Dempsey, 2012). Both LiOM and RiOM exhibited

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more abundance of organic acids than EfOM. This may be attributed to the higher conductivity

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or the higher total dissolved solids (TDS) of the effluents versus limnic and riverine water (Table

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S1), because the inorganic matter may participate in the competitive exchange with organic acids

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for the anionic resin (IRA-67). Note that the same type of the resin was placed in the last column

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of Imai’s method, in which the greater abundance of organic acids was exhibited for the same

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DOM source compared with those in Kim’s method. Similar relative abundances of PARAFAC components were found between HPO(b/n)

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and TPI(b/n) fractions in LiOM, and between organic acids and HPO(b/n) fractions in RiOM.

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For both EfOM and RiOM, either C1 or C4 was dominantly present in the HPO/TPI/HPI(a) and

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the HPO(b/n) fractions, and the overall FDOM composition was similar for the HPI(b/n)

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fractions. Among the four resin fractions, the organic acids (HPO/TPI/HPI(a)) discriminated the

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three DOM sources the most as shown by the highest abundance of C4 in EfOM, followed by

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RiOM and LiOM (Figs. 4d-4f).

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Except for EfOM, organic acids dominated in FDOM (Fig. S6g-i), accounting for 44%,

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48%, 38%, and 53% of C1, C2, C3, and C4, respectively, on average for all collected samples.

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Meanwhile, the compositions of C1 and C2 were similar to each other irrespective of DOM

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source, implying the similarity between the two humic-like components with respect to the

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source and/or the tendency of the interactions with the resins. When all collected samples were

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taken into account, the FDOM composition of the organic bases/neutrals were higher on the

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order of HPO > TPI > HPI for C1 and C2, while the opposite trend was found for C3. C4 was

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predominantly present in the organic acid fractions for all the DOM sources.

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DOC-normalized humic-like components did not exhibit major differences in the organic

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acids (HPO/TPI/HPI(a)) among the three DOM sources (Fig. 5e-f) with the average values of

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10.9, 5.7, and 4.7 µg QS mg-1 C-1 for C1, C2 and C3, respectively. In contrast, DOC-normalized

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protein-like components in the organic acids showed large variability with the DOM sources.

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The highest value (52.3 µg QS mg-1 C-1) was found in EfOM, followed by RiOM and LiOM.

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DOC-normalized PARAFAC components in the HPO(b/n) for EfOM and RiOM were similar to

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each other (p > 0.05). The highest normalized PARAFAC components in the TPI(b/n) fractions

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were observed in RiOM, followed by LiOM and EfOM. The HPI(b/n) fractions had the least

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abundance of FDOM per organic carbon. On average, DOC-normalized humic-like components

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were higher on the order of TPI(b/n) ~ HPO(b/n) > organic acids ~ HPI(b/n), while the DOC-

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normalized protein-like component, in the order of organic acids > HPO(b/n) > TPI(b/n) > HPI

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(b/n) (Fig. 5e-f).

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3.4. Implications of conservative behavior of PARAFAC components to resin fractionation

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and its applicability for other environmental systems

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DOM fractionation combined with EEM-PARAFAC has been successfully applied to understand the dynamics of DOM in complex environmental systems, owing to the enhancement

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of the resolution in DOM characterization (Murphy et al., 2011; Xue et al., 2012). This study

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demonstrated that PARAFAC components are stable under spectral subtraction upon resin

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fractionation processes and thus can be quantitatively treated like DOC and a254 for

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characterizing DOM resin fractions with different chemical structures/reactivities. In detail, the

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conservative behavior of PARAFAC components was more pronounced for humic-like versus

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protein-like FDOM as revealed by the relatively smaller differences between the Indd and Inds

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values for the humic-like components. This finding suggests that humic-like fluorescence

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components can be used as a more robust quantity parameter for tracking FDOM when DOM is

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fractionated upon adsorption. This also provides further insight into the potential applicability of

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EEM-PARAFAC for other environmental systems requiring the tracking of the individual DOM

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fractions with different chemical compositions. For example, the approach used for Indd can be

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applied to study the fate and the behaviors of different FDOM components constantly contacting

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with solid phase (e.g., adsorption and membrane filtration). As shown in this study, however,

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FDOM is not always coupled with DOC in resin fractionation because FDOM constitutes only a

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small portion of the total DOM (Table S7). This limitation points out the necessity of combining

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FDOM indicators with DOC for the full understanding of DOM changes upon fractionation.

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Although similar carbon structures were previously reported for the same resin fractions from

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different DOM sources based on nuclear magnetic resonance (NMR) and FT-IR measurements

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(Chen et al., 2003), our results clearly demonstrated that the chemical composition of resin

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fractions revealed by EEM-PARAFAC might differ by DOM sources. EEM-PARAFAC coupled

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with resin fractionation can be a promising tool to provide further information on DOM resin

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fractions with respect to their chemical composition and the environmental functionalities

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associated with the individual PARAFAC components. It would be even more beneficial for

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examining the complex systems with diverse DOM sources mixed together.

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4. Conclusions

Based on the major findings and environmental significance of our results, the following

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conclusions can be made. •

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SP method. However, because of the low tolerance limit to the indicator subtraction, the

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PARAFAC components appear more suitable for the quantity parameter in tracking the

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individual FDOM components.

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The FDOM indicators calculated from FRI and PARAFAC methods both showed higher stability to the indicator subtraction upon resin fractionation compared with those of the

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Humic-like components showed more conservative behavior to resin fractionation than the protein-like component as indicated by the smaller differences between the Indd and

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Inds values. Among the resin fractions, organic acid fractions only exhibited similar

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FDOM composition between Imai’s and Kim’s methods.

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No consistent trends in the relative abundances of PARAFAC components were found for the same resin fractions from different DOM sources, suggesting that the chemical

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composition of DOM revealed by EEM-PARAFAC is more greatly affected by DOM

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sources rather than by the types of resin fractions. •

The relative abundances of different resin fractions in DOM as DOC were completely

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different from those on the basis of the FDOM. Furthermore, the distributions of the

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FDOM resin fractions varied with the individual PARAFAC components, suggesting that

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EEM-PARAFAC could provide additional information on resin fractions with respect to

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the environmental fate and reactivity associated with each PARAFAC component.

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Acknowledgements

This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP) (No.2014R1A2A2A09049496).

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REFERENCES

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Borisover, M., Laor, Y., Parparov, A., Bukhanovsky, N., Lado, M., 2009. Spatial and seasonal patterns of fluorescent organic matter in Lake Kinneret (Sea of Galilee) and its catchment basin. Water Res. 43(12), 3104-3116. Bro, R., 1997. PARAFAC. Tutorial and applications. Chemometr. Intell. Lab. 38(2), 149-171. Chen, W., Westerhoff, P., Leenheer, J.A., Booksh, K., 2003. Fluorescence excitation - Emission matrix regional integration to quantify spectra for dissolved organic matter. Environ. Sci. Technol. 37(24), 5701-5710. Coble, P.G., 1996. Characterization of marine and terrestrial DOM in seawater using excitationemission matrix spectroscopy. Mar. Chem. 51(4), 325-346. Cohen, E., Levy, G.J., Borisover, M., 2014. Fluorescent components of organic matter in wastewater: Efficacy and selectivity of the water treatment. Water Res. 55, 323-334.

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Cook, R.L., Birdwell, J.E., Lattao, C., Lowry, M., 2009. A multi-method comparison of Atchafalaya Basin surface water organic matter samples. J. Environ. Qual. 38(2), 702-711. Cory, R.M., Kaplan, L.A., 2012. Biological lability of streamwater fluorescent dissolved organic matter. Limnol. Oceanogr. 57(5), 1347-1360. Cory, R.M., McKnight, D.M., 2005. Fluorescence spectroscopy reveals ubiquitous presence of oxidized and reduced quinones in dissolved organic matter. Environ. Sci. Technol. 39(21), 8142-8149. Fellman, J.B., Petrone, K.C., Grierson, P.F., 2011. Source, biogeochemical cycling, and fluorescence characteristics of dissolved organic matter in an agro-urban estuary. Limnol. Oceanogr. 56(1), 243-256. Gone, D.L., Seidel, J.L., Batiot, C., Bamory, K., Ligban, R., Biemi, J., 2009. Using fluorescence spectroscopy EEM to evaluate the efficiency of organic matter removal during coagulation-flocculation of a tropical surface water (Agbo reservoir). J. Hazard. Mater. 172(2-3), 693-699. He, X.S., Xi, B.D., Wei, Z.M., Jiang, Y.H., Yang, Y., An, D., Cao, J.L., Liu, H.L., 2011. Fluorescence excitation-emission matrix spectroscopy with regional integration analysis for characterizing composition and transformation of dissolved organic matter in landfill leachates. J. Hazard. Mater. 190(1-3), 293-299. Henderson, R.K., Baker, A., Murphy, K.R., Hambly, A., Stuetz, R.M., Khan, S.J., 2009. Fluorescence as a potential monitoring tool for recycled water systems: A review. Water Res. 43(4), 863-881. Hur, J., Cho, J., 2012. Prediction of BOD, COD, and total nitrogen concentrations in a typical urban river using a fluorescence excitation-emission matrix with PARAFAC and UV absorption indices. Sensors 12(1), 972-986. Imai, A., Fukushima, T., Matsushige, K., Hwan Kim, Y., 2001. Fractionation and characterization of dissolved organic matter in a shallow eutrophic lake, its inflowing rivers, and other organic matter sources. Water Res. 35(17), 4019-4028. Imai, A., Fukushima, T., Matsushige, K., Kim, Y.-H., Choi, K., 2002. Characterization of dissolved organic matter in effluents from wastewater treatment plants. Water Res. 36(4), 859-870. Imai, A., Matsushige, K., Nagai, T., 2003. Trihalomethane formation potential of dissolved organic matter in a shallow eutrophic lake. Water Res. 37(17), 4284-4294. Ishii, S.K.L., Boyer, T.H., 2012. Behavior of reoccurring PARAFAC components in fluorescent dissolved organic matter in natural and engineered systems: a critical review. Environ. Sci. Technol. 46(4), 2006-2017. Jørgensen, L., Stedmon, C.A., Kragh, T., Markager, S., Middelboe, M., Søndergaard, M., 2011. Global trends in the fluorescence characteristics and distribution of marine dissolved organic matter. Mar. Chem. 126(1–4), 139-148. Kim, H.C., Dempsey, B.A., 2008. Effects of wastewater effluent organic materials on fouling in ultrafiltration. Water Res. 42(13), 3379-3384. Kim, H.C., Dempsey, B.A., 2012. Comparison of two fractionation strategies for characterization of wastewater effluent organic matter and diagnosis of membrane fouling. Water Res. 46(11), 3714-3722. Kowalczuk, P., Cooper, W.J., Durako, M.J., Kahn, A.E., Gonsior, M., Young, H., 2010. Characterization of dissolved organic matter fluorescence in the South Atlantic Bight with

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use of PARAFAC model: Relationships between fluorescence and its components, absorption coefficients and organic carbon concentrations. Mar. Chem. 118(1-2), 22-36. Li, W.T., Chen, S.Y., Xu, Z.X., Li, Y., Shuang, C.D., Li, A.M., 2014. Characterization of dissolved organic matter in municipal wastewater using fluorescence PARAFAC analysis and chromatography multi-excitation/emission scan: A comparative study. Environ. Sci. Technol. 48(5), 2603-2609. Massicotte, P., Frenette, J.J., 2011. Spatial connectivity in a large river system: resolving the sources and fate of dissolved organic matter. Ecol. Appl. 21(7), 2600-2617. Meng, F., Huang, G., Yang, X., Li, Z., Li, J., Cao, J., Wang, Z., Sun, L., 2013. Identifying the sources and fate of anthropogenically impacted dissolved organic matter (DOM) in urbanized rivers. Water Res. 47(14), 5027-5039. Murphy, K.R., Butler, K.D., Spencer, R.G.M., Stedmon, C.A., Boehme, J.R., Aiken, G.R., 2010. Measurement of dissolved organic matter fluorescence in aquatic environments: An interlaboratory comparison. Environ. Sci. Technol. 44(24), 9405-9412. Murphy, K.R., Hambly, A., Singh, S., Henderson, R.K., Baker, A., Stuetz, R., Khan, S.J., 2011. Organic matter fluorescence in municipal water recycling schemes: toward a unified PARAFAC model. Environ. Sci. Technol. 45(7), 2909-2916. Murphy, K.R., Stedmon, C.A., Waite, T.D., Ruiz, G.M., 2008. Distinguishing between terrestrial and autochthonous organic matter sources in marine environments using fluorescence spectroscopy. Mar. Chem. 108(1-2), 40-58. Osburn, C.L., Handsel, L.T., Mikan, M.P., Paerl, H.W., Montgomery, M.T., 2012. Fluorescence tracking of dissolved and particulate organic matter quality in a river-dominated estuary. Environ. Sci. Technol. 46(16), 8628-8636. Parr, T.B., Ohno, T., Cronan, C.S., Simon, K.S., 2014. comPARAFAC: a library and tools for rapid and quantitative comparison of dissolved organic matter components resolved by Parallel Factor Analysis. Limnol. Oceanogr. - Meth. 12, 114-125. Patra, D., Mishra, A.K., 2002. Study of diesel fuel contamination by excitation emission matrix spectral subtraction fluorescence. Anal. Chim. Acta 454(2), 209-215. Peuravuori, J., Lehtonen, T., Pihlaja, K., 2002. Sorption of aquatic humic matter by DAX-8 and XAD-8 resins: Comparative study using pyrolysis gas chromatography. Anal. Chim. Acta 471(2), 219-226. Peuravuori, J., Pihlaja, K., 1998. Multi-method characterization of lake aquatic humic matter isolated with two different sorbing solids. Anal. Chim. Acta 363(2-3), 235-247. Saadi, I., Borisover, M., Armon, R., Laor, Y., 2006. Monitoring of effluent DOM biodegradation using fluorescence, UV and DOC measurements. Chemosphere 63(3), 530-539. Schwede-Thomas, S.B., Chin, Y.P., Dria, K.J., Hatcher, P., Kaiser, E., Sulzberger, B., 2005. Characterizing the properties of dissolved organic matter isolated by XAD and C-18 solid phase extraction and ultrafiltration. Aquat. Sci. 67(1), 61-71. Seredyńska-Sobecka, B., Stedmon, C.A., Boe-Hansen, R., Waul, C.K., Arvin, E., 2011. Monitoring organic loading to swimming pools by fluorescence excitation–emission matrix with parallel factor analysis (PARAFAC). Water Res. 45(6), 2306-2314. Stedmon, C.A., Bro, R., 2008. Characterizing dissolved organic matter fluorescence with parallel factor analysis: a tutorial. Limnol. Oceanogr. - Meth. 6, 572-579. Stedmon, C.A., Markager, S., 2005. Resolving the variability in dissolved organic matter fluorescence in a temperate estuary and its catchment using PARAFAC analysis. Limnol. Oceanogr. 50(2), 686-697.

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Stedmon, C.A., Markager, S., Bro, R., 2003. Tracing dissolved organic matter in aquatic environments using a new approach to fluorescence spectroscopy. Mar. Chem. 82(3-4), 239-254. Thurman, E.M., Malcolm, R.L., 1981. Preparative isolation of aquatic humic substances. Environ. Sci. Technol. 15(4), 463-466. Wu, F.C., Evans, R.D., Dillon, P.J., 2003. Separation and characterization of NOM by highperformance liquid chromatography and on-line three-dimensional excitation emission matrix fluorescence detection. Environ. Sci. Technol. 37(16), 3687-3693. Xue, S., Zhao, Q.L., Wei, L.L., Hui, X.J., Ma, X.P., Lin, Y.Z., 2012. Fluorescence spectroscopic studies of the effect of granular activated carbon adsorption on structural properties of dissolved organic matter fractions. Front. Env. Sci. Eng. 6(6), 784-796. Yamashita, Y., Jaffe, R., Maie, N., Tanoue, E., 2008. Assessing the dynamics of dissolved organic matter (DOM) in coastal environments by excitation emission matrix fluorescence and parallel factor analysis (EEM-PARAFAC). Limnol. Oceanogr. 53(5), 1900-1908. Yang, L., Hur, J., 2014. Critical evaluation of spectroscopic indices for organic matter source tracing via end member mixing analysis based on two contrasting sources. Water Res. 59, 80-89. Yang, L., Hur, J., Zhuang, W., 2015. Occurrence and behaviors of fluorescence EEMPARAFAC components in drinking water and wastewater treatment systems and their applications: a review. Environ. Sci. Pollut. Res., DOI: 10.1007/s11356-11015-1421411353. Zhang, Y.L., van Dijk, M.A., Liu, M.L., Zhu, G.W., Qin, B.Q., 2009. The contribution of phytoplankton degradation to chromophoric dissolved organic matter (CDOM) in eutrophic shallow lakes: Field and experimental evidence. Water Res. 43(18), 4685-4697. Zhou, S.Q., Shao, Y.S., Gao, N.Y., Li, L., Deng, J., Tan, C.Q., Zhu, M.Q., 2014. Influence of hydrophobic/hydrophilic fractions of extracellular organic matters of Microcystis aeruginosa on ultrafiltration membrane fouling. Sci. Total Environ. 470, 201-207.

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Figure Captions

558

Fig. 1. Schematic diagrams of the resin fractionation procedures based on Imai’s method (a) and

559

Kim and Dempsey (2008, 2012) (b). EEMs of the fractions eluted from resins as well as those

560

retained on resins are shown for effluent DOM (EfOM).

561

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557

Fig. 2. Representative EEMs and the spectral loadings of four identified PARAFAC components.

563

Individual excitation and emission loading are shown for the comparison of the modeled and the

564

split-half (split 1-2 and 3-4) validated results.

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Fig. 3. Comparison of the FDOM indicators from the subtracted EEM spectra (Inds) versus those

567

from the direct subtraction between the two indicators before and after resins (Indd) for Imai’s

568

fractionation method (chart a-c) and Kim’s fractionation method (chart d-f). Chart a and d are

569

based on the FDOM indicators from the peak-picking method; Chart b and e are based the

570

FDOM indicators from the FRI method; Chart c and f are based on PARAFAC components.

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Fig. 4. Relative abundance (%) of four PARAFAC components for different resin fractions of

573

EfOM, LiOM, and RiOM based on Imai’s method (chart a-c) and Kim’s method (chart d-f).

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574

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Fig. 5. DOC-normalized intensities of PARAFAC components in different resin fractions for

576

EfOM, LiOM, and RiOM. Fractions were obtained by Imai’s method (chart a-d) and Kim’s

577

method (chart e-h). “All” in the x-axis represents the average values of each fraction over EfOM,

578

LiOM, and RiOM.

579

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Tables

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Table 1. Ratios of Indd to Inds for the fractions retained on various resins based on Imai’s and Kim’s fractionation methods. FDOM quantity indicator groups are obtained from Coble (1996)’s specific peak-picking (SP) method (SPprotein: the peaks of B and T, SPhumic: the peaks of A, M, and C), fluorescent regional integration (FRI) method (FRIprotein: the regions of B and T, FRIhumic: the regions of A, M, and C), and PARAFAC components (PARAFAChumic: C1, C2, and C3, and PARAFACprotein: C4). Indicator

Fractions on 1st resin

Fractions on 2nd resin

groups

n

n

Imai’s method

HPO (n)

SPtotal

39

0.74 (26)

0.28-0.98

44

0.79 (26)

SPprotein

15

0.68 (28)

0.28-0.98

17

0.68 (40)

SPhumic

24

0.78 (24)

0.31-0.98

27

0.86 (12)

FRItotal

38

0.95 (14)

0.28-1.00

45

FRIprotein

15

0.94 (13)

0.63-1.00

FRIhumic

38

0.95 (14)

0.28-1.00

PARAFACtotal

30

1.07 (36)

0.72-3.04

PARAFACprotein

8

0.96 (10)

0.72-1.00

PARAFAChumic

22

1.11 (39)

0.96-3.04

Kim’s method

HPO/TPI/ HPI (a)

SPtotal

45

0.86 (25)

0.00-1.00

44

0.78 (26)

0.11-1.00

43

0.78 (27)

0.22-1.00

SPprotein

18

0.87 (18)

0.35-1.00

17

0.72 (38)

0.11-1.00

17

0.67 (36)

0.22-1.00

SPhumic

27

0.85 (29)

0.00-0.99

27

0.82 (16)

0.53-0.98

26

0.85 (17)

0.45-1.00

FRItotal

45

0.99 (4)

0.80-1.00

44

0.96 (13)

0.38-1.00

45

0.91 (19)

0.35-1.00

FRIprotein

18

0.98 (5)

0.80-1.00

17

0.89 (20)

0.38-1.00

18

0.79 (28)

0.35-1.00

FRIhumic

45

0.99 (4)

0.80-1.00

44

0.96 (13)

0.38-1.00

45

0.91 (19)

0.35-1.00

PARAFACtotal

35

1.02 (7)

1.00-1.32

36

0.97 (11)

0.42-1.07

36

0.97 (12)

0.42-1.02

PARAFACprotein

9

1.00 (1)

1.00-1.02

9

0.90 (22)

0.42-1.00

9

0.95 (13)

0.62-1.00

PARAFAChumic

26

1.02 (8)

1.00-1.32

27

1.00 (4)

0.83-1.07

27

0.98 (11)

0.42-1.02

Min-Max

Mean(RSD)

HPO/TPI/HPI (b)

n

Mean(RSD)

Min-Max

TPI/ HPI (a) 44

0.86 (16)

0.36-1.00

0.01-1.00

17

0.78 (23)

0.36-1.00

0.61-1.00

27

0.91 (8)

0.70-0.99

0.92 (20)

0.31-1.00

44

0.94 (17)

0.12-1.00

18

0.80 (32)

0.31-1.00

17

0.84 (27)

0.12-1.00

45

0.92 (20)

0.31-1.00

44

0.94 (17)

0.12-1.00

36

0.99 (3)

0.84-1.03

36

0.96 (15)

0.29-1.09

9

0.97 (5)

0.84-1.00

9

0.83 (31)

0.29-1.00

27

1.00 (2)

0.94-1.03

27

1.00 (2)

0.97-1.09

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0.01-1.00

HPO (b/n)

EP

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Min-Max

SC

Mean(RSD)

Fractions on 3rd resin

TPI (b/n)

Note: n – number of samples; RSD – relative standard deviation (%); Min – minimum; Max – maximum.

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Fractionation methods Organic matter

Imai's method HPO (a)a

HPO (n)

HPO/TPI/HPI (b)

RI PT

Table 2. DOC concentrations and the intensities of PARAFAC components for different resin fractions obtained from Imai’s and Kim’s fractionation methods. Kim's method

TPI/HPI (a)

TPI/ HPI (n)

HPO/TPI/HPI (a)

HPO (b/n)

TPI (b/n)

HPI (b/n)

1.68±1.26 22.84±18.36

0.88±0.60 22.70±18.61

1.66±1.24 29.70±15.92

4.03±1.04 23.36±13.47

15.53±12.21

15.42±12.39

13.97±9.69

14.57±8.10

2.04±0.56 0.65±0.80 14.83±7.45 23.86±5.76

1.43±1.35 36.14±6.86

2.41±1.18 29.75±19.29

1.60±0.06 5.06±3.99

C2 (µg QS L-1)

7.76±2.40

14.30±5.82

18.77±1.91

16.59±8.53

2.81±2.08

C3 (µg QS L ) C4 (µg QS L-1)

4.38±2.62 5.76±4.79

16.29±8.94 23.17±7.20

21.49±7.92 16.38±9.17

19.85±6.28 24.65±4.10

3.11±2.51 2.50±2.05

11.59±13.27 31.75±2.22

12.56±11.70 31.55±2.51

10.76±6.99 12.65±7.29

18.79±10.84 10.54±9.63

Limnic DOC (mg L-1) C1 (µg QS L-1)

0.99±0.36 5.78±1.25

0.23±0.05 6.52±8.71

0.36±0.31 6.14±2.34

0.66±0.27 5.03±3.05

0.93±0.44 0.55±0.36

1.28±0.55 12.62±2.11

0.50±0.74 3.07±1.92

0.10±0.14 3.93±1.54

1.16±0.09 2.19±0.93

C2 (µg QS L-1)

3.33±0.49

1.34±2.18

1.99±1.09

2.01±1.35

0.54±0.21

5.27±1.49

1.27±0.88

1.30±0.56

0.79±0.21

C3 (µg QS L ) C4 (µg QS L-1)

1.89±0.53 2.01±0.88

2.10±2.97 4.47±4.36

3.13±0.39 4.04±1.38

2.60±1.41 2.89±3.04

0.44±0.25 0.00±0.00

5.42±0.81 4.92±1.19

1.07±1.09 1.11±1.34

1.67±1.41 1.63±1.07

2.36±1.28 1.49±0.28

Riverine DOC (mg L-1) C1 (µg QS L-1)

1.01±0.26 7.47±1.47

0.43±0.08 6.67±4.10

0.24±0.17 5.07±3.49

0.80±0.14 9.36±5.45

0.96±0.25 0.65±0.77

1.58±0.08 15.74±6.78

0.21±0.14 7.21±5.31

0.24±0.23 5.21±4.26

1.37±0.14 3.45±1.97

C2 (µg QS L-1)

3.14±1.50

1.88±2.03

2.37±1.61

3.82±2.42

0.62±0.52

6.74±2.72

2.50±2.16

1.83±1.61

1.28±0.57

2.54±0.32 3.96±0.57

2.23±1.79 4.97±3.84

2.42±1.02 3.26±2.10

5.02±2.76 6.66±3.72

0.61±0.44 0.18±0.16

6.24±2.48 10.41±5.66

2.57±1.88 4.35±3.70

3.24±2.50 2.28±1.84

3.11±1.93 1.93±0.53

-1

C3 (µg QS L ) C4 (µg QS L-1)

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AC C

-1

SC

Effluent DOC (mg L-1) C1 (µg QS L-1)

Note: Data are expressed by average ± standard deviation.

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a. Imai’s fractionation method

b. Kim’s fractionation method 500 KE1(2) (DOM)

500

500

120

40

250

IF0 300

350

DAX-8

20

400 450 Em. (nm)

500

550

500

500

550

500

0

70

Elute with 0.1 M NaOH

100

400

50 40

350

20

500

550

0

60 350

120

60 350

IF2 500

550

KE3(2) (TPI/HPI b/n)

400 450 Em. (nm)

500

550

0

AG-MP-1

0

TPI/HPI (acids)

450 400

100

80

80 60

350 300

120

40

350

300

350

300

80 60

350 40 300

300

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400 450 Em. (nm)

500

300

20

350

400 450 Em. (nm)

500

0

550

400 450 Em. (nm)

500

550

EP

10

90

450 400

XAD-4

60 50

20

300

350

400 450 Em. (nm)

500

550

90

400

TPI (bases/neutrals)

80 70 60 50 40

350

30 20

300

10 90

HPI (bases/neutrals)

70 60 50

30

350

20 10 400 450 Em. (nm)

500

550

250

80

40

KF3

300

70

40

KE(2) (TPI b/n) 450

350

250

80

10

500

300

Fig. 1

20

550

30

550

0

TE D

250

IF3

350

500

HPO (bases/neutrals)

500

10

Ex. (nm)

250

QS µg/L

400

100

400 450 Em. (nm)

350

250

KE4(2) (HPI b/n)

TPI /HPI (neutrals)

450

400

20

KF2

20

IE5(1) (HiN)

30

300

60

40

500

40

70

50

250

50

KE(2) (HPO b/n) 450

DAX-8

400

300

120

IE(1) (BaS)

Ex. (nm)

400 450 Em. (nm)

Ex. (nm)

350

350

500

20

300

300

550

30

40

300 250

QS µg/L

400

AC C

Ex. (nm)

80

500

60

500

10

400 450 Em. (nm)

90

20

100

250

350

450

300

20

KF1 300

500

40

IE4(1) (HiA/HiN) 450

250

80

Ex. (nm)

500

400 450 Em. (nm)

AG-MP-50

400

100

70

350

250

QS µg/L

350

300

HPO/TPI/HPI (bases)

M AN U

300

120 IE(1) (HoN)

450

Ex. (nm)

250

500

40

IF1

QS µg/L

60 350

400

30

QS µg/L

Ex. (nm)

80 400

80

HPO/TPI/HPI (acids)

300

60

SC

Ex. (nm)

120

450

300

80

450

IE3(1) (HiA/BaS/HiN)

IRA-67

90

Ex. (nm)

400 450 Em. (nm)

90

KE(2) (HPO/TPI/HPI a)

Ex. (nm)

350

550

QS µg/L

300

500

450

0

KE2(2) (HPO/TPI/HPI b/n)

250

400 450 Em. (nm)

500

20

QS µg/L

300

40 300

60 350

350

QS µg/L

60 350

QS µg/L

Ex. (nm)

80

400

QS µg/L

100

400

10

300

80 Ex. (nm)

HPO(neutrals)

20

250 100

HPO(acids)

450

IE(1) (HoN) 450

40

300

120 IE2(1) (AHS)

0

550

50

QS µg/L

400 450 Em. (nm)

60

350

QS µg/L

350

70

30

DOM

20

300

400

300

350

400 450 Em. (nm)

500

550

QS µg/L

300

80

Filtrated water (DOM)

QS µg/L

Ex. (nm)

60 350

250

500

80 QS µg/L

Ex. (nm)

400

450

100

Ex. (nm)

Filtrated water (DOM)

40

DOM

90

120 IE1(1) (DOM)

450

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500

M AN U

SC

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ACCEPTED MANUSCRIPT

AC C

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Fig. 2

ACCEPTED MANUSCRIPT

30 20

25

50

c

Protein Humic Fitting Curve

20 15 10

10

10

20 30 Ind (SP, µg QS/L) s

40

0 0

50

Ind (FRI, µg QS/L) d

e 30

20

40

25

Protein Humic Fitting Curve

20 15 10

20 30 40 Ind (SP, µg QS/L) s

50

60

0 0

40

y= 1.00x + -0.03 n= 134, R2= 1.000, p= 0.0000

10

20 30 Ind (FRI, µg QS/L) s

TE D

Fig. 3

EP

f

50

Protein Humic Fitting Curve

30 20 10

M AN U

5

y= 0.85x + 0.17 n= 132, R2= 0.926, p= 0.0000

10

y= 1.00x + -0.05 n= 102, R2= 1.000, p= 0.0000 20 30 40 Ind (PFC, µg QS/L) s

50

10

10

10

SC

Protein Humic Fitting Curve

30

0 0

20

0 0

35

AC C

Ind (SP, µg QS/L) d

40

30

s

50

d

y= 1.00x + -0.09 n= 127, R2= 1.000, p= 0.0000 10 20 30 Ind (FRI, µg QS/L)

Ind (PFC, µg QS/L) d

0 0

5

y= 0.90x + -0.66 n= 127, R2= 0.945, p= 0.0000

Protein Humic Fitting Curve

40

RI PT

Ind (FRI, µg QS/L) d

Ind (SP, µg QS/L) d

40

35

b 30

Protein Humic Fitting Curve

Ind (PFC, µg QS/L) d

50

a

40

0 0

y= 1.00x + -0.01 n= 107, R2= 1.000, p= 0.0000

10

20 30 Ind (PFC, µg QS/L) s

40

50

80 70 60 50 40 30 20

b

100

80

10 Fr1

Fr2

Fr3

HPO(n)

HPO/TPI/ HPI (b)

Fr4

30 20

80

Fr2

HPO(a)

Fr3

HPO(n)

HPO/TPI/ HPI (b)

Fr4

PARAFAC component (%)

e

60 50 40 30 20 10

90 80

HPO/TPI/ HPI (a)

Fr2

HPO (b/n)

Fr3

TPI (b/n)

Fr4

Limnic

C1 C2 C3 C4

60 50 40 30 20

HPI(b/n)

30 20

Fr1

HPO(a)

f

90 80

Fr2

Fr3

HPO(n)

HPO/TPI/ HPI (b)

Fr4

Fr5

TPI/HPI (a) TPI/ HPI (n)

Riverine

C1 C2 C3 C4

70 60 50 40 30 20 10

Fr1

HPO/TPI/ HPI (a)

Fr2

HPO (b/n)

TE D

Fr3

TPI (b/n)

Fig. 4

EP

40

TPI/HPI (a) TPI/ HPI (n)

70

0

50

100

10 Fr1

60

SC

Effluent

C1 C2 C3 C4

70

0

Fr5

Riverine

C1 C2 C3 C4

90

10 Fr1

100

70

0

40

TPI/HPI (a) TPI/ HPI (n)

AC C

PARAFAC component (%)

80

50

0

Fr5

100 90

60

100

10

HPO(a)

d

70

c

PARAFAC component (%)

0

Limnic

C1 C2 C3 C4

90

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Effluent

C1 C2 C3 C4

90

PARAFAC component (%)

100

M AN U

PARAFAC component (%)

a

PARAFAC component (%)

ACCEPTED MANUSCRIPT

Fr4

HPI(b/n)

0

Fr1

HPO/TPI/ HPI (a)

Fr2

HPO (b/n)

Fr3

TPI (b/n)

Fr4

HPI(b/n)

ACCEPTED MANUSCRIPT

200

C2 (ug QS/(mg C)

150

100

50

Effluent

Limnic

Ri verine

HPO(a) HPO(n) HPO/TPI/HPI (b) TPI/HPI (a) TPI/HPI (n)

150

Effluent

Ri verine

HPO/TPI/HPI (a) HPO (b/n) TPI (b/n) HPI (b/n)

100

0

Effluent

250

150

Limnic

Ri verine

All

HPO/TPI/HPI (a) HPO (b/n) TPI (b/n) HPI (b/n)

AC C

100

All

50

0

100

0

f 250

Riverine

All

Effluent

Limnic

h

Effluent

Limnic

Riverine

All

Riverine

All

Riverine

All

HPO/TPI/HPI (a) HPO (b/n) TPI (b/n) HPI (b/n)

150

100

50

0

Effluent

Limnic

250

200

EP

200

Limnic

HPO(a) HPO(n) HPO/TPI/HPI (b) TPI/HPI (a) TPI/HPI (n)

150

200

150

50

C3 (ug QS/(mg C)

Limnic

TE D

C1 (ug QS/(mg C)

200

Effluent

50

C2 (ug QS/(mg C)

0

0

d 250

M AN U

100

e 250

100

200

50

g

All

C4 (ug QS/(mg C)

C3 (ug QS/(mg C)

200

150

50

C4 (ug QS/(mg C)

0

c 250

HPO(a) HPO(n) HPO/TPI/HPI (b) TPI/HPI (a) TPI/HPI (n)

SC

C1 (ug QS/(mg C)

200

b 250 HPO(a) HPO(n) HPO/TPI/HPI (b) TPI/HPI (a) TPI/HPI (n)

RI PT

a 250

HPO/TPI/HPI (a) HPO (b/n) TPI (b/n) HPI (b/n)

150

100

50

Ri verine

All

0

Fig. 5

Effluent

Limnic

ACCEPTED MANUSCRIPT

Highlights

► Rigorous tests on the applicability of EEM-PARAFAC for resin fractionation of

RI PT

DOM.

►PARAFAC components were the superior to other FDOM indicators in the

SC

conservativeness.

M AN U

►Lower sensitivity to spectral subtraction for humic-like vs. protein-like components.

►FDOM composition of resin fractions is more affected by DOM sources rather than

AC C

EP

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resins.

ACCEPTED MANUSCRIPT

Conservative behavior of fluorescence EEM-PARAFAC components in resin fractionation

Wei He and Jin Hur*

RI PT

processes and its applicability for characterizing dissolved organic matter

M AN U

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Department of Environment and Energy, Sejong University, Seoul 143-747, South Korea

Pages: 19; Tables: 7; Figures: 6;

AC C

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Script: 2.

1

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Table S1. In-situ water quality parameters of effluent, limnic, and riverine water samples and selected parameters for the DOM (Mean±standard deviation, n=3). Effluent water

Limnic water

pH EC (µs cm-1) TDS (mg L-1) DOC a254 (m-1) C1 (µg QS L-1) C2 (µg QS L-1) C3 (µg QS L-1) C4 (µg QS L-1) C1 (%) C2 (%) C3 (%) C4 (%)

6.90±0.13 523±35 335±22 8.19±1.41 31.93±14.73 106.09±15.52 59.36±7.13 63.93±8.53 69.67±14.70 35.49±1.14 19.92±0.70 21.44±1.42 23.15±1.82

7.12±0.16 76±2 49±1 3.10±0.29 8.01±1.17 22.20±3.52 8.24±1.20 10.01±1.36 10.13±2.71 43.92±1.12 16.35±1.31 19.90±1.54 19.82±2.86

Riverine water

RI PT

Parameters

M AN U

SC

7.31±0.21 182±4 116±3 3.41±0.24 15.38±13.04 30.52±7.88 11.56±3.44 14.09±3.33 18.57±6.69 41.05±1.23 15.41±0.53 19.09±1.46 24.46±2.24

AC C

EP

TE D

Note: EC – Electrical conductivity; TDS – Total dissolved solids

2

ACCEPTED MANUSCRIPT

Table S2. Formula used for calculating each DOM resin fractions. Fractions

Formula

RI PT

Imai’s method IF0,cor IF1,cor IF2,cor IF3,cor HPO acids HPO neutrals HPO/TPI/HPI bases TPI/HPI acids TPI/HPI neutrals Kim’s method KF1,cor KF2,cor KF3,cor HPO/TPI/HPI acids HPO bases/neutrals TPI bases/neutrals HPI bases/neutrals

M AN U

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(IF0 – BNaOH) × (Velutant) / (Vsample) IF1 – B1 (IF2 – B2) / [(Vsample – Vmonitoring) / Vsample] (IF3 – B3) / [(Vsample – 2Vmonitoring) / Vsample] IF0,cor DOM – IF0,cor – IF1,cor IF1,cor – IF2,cor IF2,cor – IF3,cor IF3,cor

KF1 – B4 (KF2 – B5) / [(Vsample – Vmonitoring) / Vsample] (KF3 – B6) / [(Vsample – 2Vmonitoring) / Vsample] DOM – KF1,cor KF1,cor – KF2,cor KF2,cor – KF3,cor KF3,cor

AC C

EP

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Note: DOM, IF0, IF1, IF2, IF3, KF1, KF2, and KF3 are the fractions shown in Fig. 1. BNaOH denotes the concentration of NaOH. Vsample, Vmonitoring, and Velutant are the volumes of the initial sample (1000 mL), monitored samples (50 mL per time), and elutants (100 mL).

3

ACCEPTED MANUSCRIPT

Table S3. DOC concentrations and the intensities of fluorescence EEM-PARAFAC components for an alkaline solvent and the resin blanks, and the minimum DOC and PARAFAC components of each fraction based on Imai’s and Kim’s methods C2 (µg QS L-1)

5.80 1.37 1.15 0.54 0.43 (7.4%) 0.20 (14.8%) 0.13 (11.6%) 0.12 (21.7%)

44.50 6.74 3.74 0.79 0.99 (2.2%) 0.12 (1.8%) 0.88 (23.6%) 0.78 (98.7%)

16.08 2.42 1.48 0.15 0.35 (2.2%) 0.02 (1.0%) 0.00 (0.0%) 0.00 (0.0%)

1.31 1.07 1.11 0.11 (8.0%) 0.20 (19.0%) 0.16 (16.8%)

5.10 3.80 1.61 0.01 (0.1%) 0.12 (3.3%) 0.20 (14.0%)

1.54 1.22 0.52 0.00 (0.2%) 0.02 (1.9%) 0.01 (2.9%)

C3 (µg QS L-1)

C4 (µg QS L-1)

13.17 4.70 1.80 0.20 0.47 (3.5%) 0.02 (0.3%) 0.07 (4.0%) 0.07 (35.8%)

12.66 3.39 1.77 1.64 2.26 (17.9%) 0.12 (3.6%) 1.66 (93.6%) 1.64 (100.0%)

3.47 2.82 0.80 0.00 (0.0%) 0.02 (0.5%) 0.00 (0.0%)

2.61 2.19 1.43 0.32 (12.2%) 0.12 (5.6%) 0.27 (23.7%)

RI PT

C1 (µg QS L-1)

SC

Imai’s method IF0sample,min IF1sample,min IF2sample,min IF3sample,min BNaOH B1 B2 B3 Kim’s method KF1sample,min KF2sample,min KF3sample,min B1 B2 B3

DOC mg L-1

M AN U

Fractions

AC C

EP

TE D

Note: Values in the brackets denote the percentage of the blank to the minimum quantity of the fractions, i.e., IF0sample,min / BNaOH × 100% or IF1sample,min / B1 × 100%.

4

ACCEPTED MANUSCRIPT

Table S4. Modified Turker’s Congruence Coefficient (mTCC) for the PARAFAC components between the present study and 38 PARAFAC models summarized by Parr et al. 2014. mTCC

RI PT

Sample (Component) / Database (Component)

AC C

EP

TE D

M AN U

SC

MySample (1) / Fellman 2011 (1) 0.968 MySample (1) / Parr 2014 (1) 0.975 MySample (1) / Stedmon 2003 (1) 0.964 MySample (2) / Burrows 2013 (2) 0.959 MySample (2) / Chen 2010 (1) 0.987 MySample (2) / Kowalczuk 2010 (1) 0.957 MySample (2) / Parr 2014 (2) 0.979 MySample (2) / Singh 2013 (1) 0.971 MySample (2) / Yamashita 2011 (2) 0.958 MySample (3) / CM 2005 (10) 0.964 MySample (3) / Kothawala 2012 (2) 0.972 MySample (3) / Murphy 2006 (2) 0.966 MySample (3) / Murphy 2008 (1) 0.953 MySample (3) / Osburn 2012 (2) 0.955 MySample (4) / Cory 2012 (4) 0.952 MySample (4) / Fellman 2011 (4) 0.981 MySample (4) / Kowalczuk 2010 (6) 0.953 MySample (4) / Massicotte 2011 (5) 0.957 MySample (4) / Murphy 2008 (7) 0.957 MySample (4) / Osburn 2011 (5) 0.963 MySample (4) / Stedmon 2005 (7) 0.962 MySample (4) / Yamashita 2011 (5) 0.956 MySample (4) / Yang 2012 (3) 0.986 MySample (4) / Yang 2013 MC (3) 0.954 Note: Reference list of the database names is included in the literature by Parr et al., (2014)

5

ACCEPTED MANUSCRIPT

Table S5. Ratios of Indd to Inds for the resin fractions based on Imai’s and Kim’s methods. The fluorescence indicators are obtained from Coble (1996)’s specific peak method (SP) (including B, T, A, M, and C), fluorescent regional index (FRI) (including B, T, A, M, C and sum of those indicators), and PARAFAC components (including C1, C2, C3, and C4).

n Mean(RSD) Min-Max TPI/ HPI(a)

8 9 9 9 9

8 9 9 9 9

0.76 (29) 0.80 (18) 0.88 (11) 0.93 (6) 0.94 (6)

0.36-1.00 0.58-1.00 0.70-0.98 0.85-0.99 0.81-0.99

0.90 (12) 0.84 (23) 0.82 (27) 0.88 (24) 0.86 (37)

9 9 9 9 9

0.96 (7) 1.00 (0) 1.00 (1) 0.99 (4) 0.99 (4)

9 1.04 (10) 9 1.03 (8) 8 1.00 (0)

M AN U

SC

0.01-1.00 0.51-0.95 0.61-0.98 0.74-0.97 0.87-1.00

0.63 (41) 0.97 (5) 1.00 (0) 1.00 (0) 1.00 (0)

0.31-0.98 0.84-1.00 0.99-1.00 1.00-1.00 1.00-1.00

8 9 9 9 9

0.81 (36) 0.87 (20) 0.99 (1) 1.00 (1) 1.00 (0)

0.12-0.98 0.53-1.00 0.97-1.00 0.98-1.00 1.00-1.00

9 9 9 9

1.00 (1) 0.99 (2) 1.01 (1) 0.97 (5)

0.97-1.01 0.94-1.00 1.00-1.03 0.84-1.00

9 9 9 9

1.00 (1) 1.00 (1) 1.02 (3) 0.83 (31)

0.97-1.02 0.98-1.00 1.00-1.09 0.29-1.00

HPO (b/n)

TPI (b/n)

0.68-0.99 0.35-1.00 0.26-0.98 0.34-0.99 0.00-0.99

8 9 9 9 9

0.69 (42) 0.75 (37) 0.84 (14) 0.79 (20) 0.83 (16)

0.11-0.97 0.11-1.00 0.62-0.96 0.54-0.98 0.53-0.94

8 9 9 8 9

0.58 (46) 0.75 (25) 0.79 (24) 0.89 (8) 0.89 (16)

0.22-1.00 0.42-0.93 0.45-0.98 0.74-0.98 0.62-1.00

0.80-1.00 1.00-1.00 0.96-1.00 0.89-1.00 0.89-1.00

8 9 9 9 9

0.83 (26) 0.93 (14) 1.00 (0) 1.00 (1) 1.00 (0)

0.38-0.98 0.62-1.00 0.99-1.00 0.98-1.00 1.00-1.00

9 9 9 9 9

0.63 (30) 0.96 (11) 1.00 (1) 0.98 (5) 1.00 (0)

0.35-0.90 0.67-1.00 0.98-1.00 0.85-1.00 1.00-1.00

1.00-1.32 1.00-1.23 1.00-1.00

9 1.00 (0) 9 0.98 (6) 9 1.01 (2)

1.00-1.01 0.83-1.00 0.97-1.07

9 1.00 (0) 9 1.00 (0) 9 0.94 (21)

0.99-1.00 0.99-1.00 0.42-1.02

EP

9 9 9 9 9

0.57 (58) 0.77 (23) 0.78 (17) 0.85 (8) 0.93 (5)

9 9 9 9 9

HPO/TPI/ HPI (a)

AC C

Kim’s method SP B T A M C FRI B T A M C PARAFAC C1 C2 C3

Fractions on 3rd resin

n Mean(RSD) Min-Max HPO/TPI/HPI(b)

TE D

n Mean(RSD) Min-Max Imai’s method HPO(n) SP B 7 0.69 (34) 0.28-0.98 T 8 0.67 (24) 0.46-0.90 A 8 0.77 (21) 0.42-0.92 M 8 0.75 (32) 0.31-0.95 C 8 0.83 (18) 0.60-0.98 FRI B 7 0.89 (18) 0.63-0.99 T 8 0.98 (4) 0.89-1.00 A 8 0.91 (28) 0.28-1.00 M 8 1.00 (0) 0.99-1.00 C 7 0.99 (3) 0.92-1.00 PARAFAC C1 8 1.04 (9) 0.98-1.28 C2 6 0.99 (1) 0.96-1.00 C3 8 1.26 (57) 1.00-3.04 C4 8 0.96 (10) 0.72-1.00

Fractions on 2nd resin

RI PT

Fractions on 1st resin

Indicators

6

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AC C

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9 1.00 (1) 1.00-1.02 9 0.90 (22) 0.42-1.00 9 0.95 (13) 0.62-1.00 C4 Note: n – number of samples; RSD – relative standard deviation (unit, %); Min – minimums; Max – Maximums.

7

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Table S6. DOC concentrations, and the intensities of a254 and PARAFAC components of sequentially fractionated DOM fractions for effluent, limnic, and riverine DOM samples based on Imai’s and Kim’s methods Supernatant

Remaining organic matter

DOM

After DAX-8 After AG-MP-50 After AG-MP-1 HPO(b), TPI and HPI TPI and HPI TPI(a/b/n), and (a/n) (n) HPI(a/b/n) Imai’s method

Supernatant

After IRA-67

After DAX-8

After XAD-4

DOM

HPO, TPI, and HPI (b/n)

TPI and HPI (b/n)

HPI (b/n)

RI PT

Fractionation step

Kim’s method

8.12±1.27 31.91±16.49

5.44±0.93 25.25±16.70

4.22±1.28 19.95±20.20

1.68±0.07 10.63±16.87

8.25±1.84 31.94±16.44

6.58±0.64 24.62±15.62

6.00±1.30 19.44±20.68

4.25±1.10 10.65±16.93

C1 (µg QS L-1)

109.46±13.80 70.89±13.94

34.76±17.76

5.06±3.99

102.86±19.41 80.02±37.64

50.33±21.75

26.97±11.56

C2 (µg QS L ) C3 (µg QS L-1) C4 (µg QS L-1) Limnic DOC (mg L-1) a254 (m-1)

60.14±4.89 65.15±3.23 72.03±15.13

38.15±9.54 44.49±12.92 43.20±9.67

19.40±7.76 22.97±5.10 26.96±3.20

3.16±0.43 8.01±1.32

1.95±0.77 5.97±0.87

1.67±0.57 4.40±1.47

C1 (µg QS L-1)

22.48±5.30

11.69±4.44

C2 (µg QS L ) C3 (µg QS L-1)

7.84±1.76 9.59±1.92

C4 (µg QS L-1)

M AN U

-1

SC

Effluent DOC (mg L-1) a254 (m-1)

58.48±10.04 63.59±13.12 66.86±16.90

42.95±21.36 52.00±23.66 35.11±18.58

28.98±11.69 41.24±16.79 22.47±11.86

14.42±6.25 22.45±12.54 11.96±2.37

0.98±0.46 0.86±0.35

3.04±0.14 8.01±1.29

1.75±0.67 5.47±0.42

1.32±0.19 4.40±1.46

1.22±0.10 0.87±0.35

5.55±2.64

0.55±0.36

21.80±1.50

9.18±3.55

6.10±2.17

2.19±0.93

4.53±1.89 6.26±1.41

2.55±1.16 3.12±1.36

0.54±0.21 0.44±0.25

8.62±0.24 10.51±0.60

3.34±1.58 5.10±1.41

2.09±0.78 4.02±1.64

0.79±0.21 2.36±1.28

11.25±3.63

6.17±2.81

2.21±2.99

0.00±0.00

9.01±1.10

4.10±1.57

3.09±1.36

1.49±0.28

Riverine DOC (mg L-1) a254 (m-1) C1 (µg QS L-1) C2 (µg QS L-1)

3.44±0.24 15.37±14.58 29.30±10.18 10.72±4.22

2.00±0.54 12.94±15.11 14.96±8.40 6.79±3.30

1.85±0.39 12.38±16.45 9.89±5.02 4.44±2.11

1.01±0.26 9.96±17.13 0.65±0.77 0.62±0.52

3.39±0.29 15.39±14.58 31.60±6.87 12.35±3.11

1.81±0.22 12.42±15.56 15.86±6.97 5.61±2.56

1.69±0.10 12.33±16.47 8.65±5.99 3.11±2.19

1.44±0.15 9.97±17.14 3.45±1.97 1.28±0.57

C3 (µg QS L-1) C4 (µg QS L-1)

13.15±3.22 18.18±9.04

8.13±3.13 9.44±4.94

5.69±2.33 6.27±3.43

0.61±0.44 0.18±0.16

15.15±3.82 18.88±5.38

8.92±3.42 8.48±3.95

6.35±3.80 4.15±2.07

3.11±1.93 1.93±0.53

EP

AC C

-1

TE D

2.81±2.08 3.11±2.51 2.50±2.05

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Table S7 Removal efficiencies of different resins used on the basis of DOC and PARAFAC components (based on all the samples). Resins

DOC (%)

C1 (%)

C2 (%)

C3 (%)

C4 (%)

AC C

EP

TE D

M AN U

SC

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Imai's method XAD-8 25.9(11.3) 43.1(19.1)* 36.2(25.9) 34.0(18.4) 40.7(27.8) AG-MP-50 26.7(18.8) 45.9(15.8)* 41.6(13.8) 43.3(12.1)* 47.7(26.9) AG-MP-1 70.8(33.4) 85.1(15.9) 78.7(17.2) 84.4(12.7) 95.5(6.1)* Kim's method IRA-67 35.9(17.6) 44.3(22.3) 48.3(23.1) 37.9(20.1) 53.0(15.4)* XAD-8 15.6(16.3) 37.9(14.0)** 35.6(16.2)* 23.7(14.9) 34.9(21.4)* XAD-4 16.6(15.1) 55.0(15.7)** 54.9(12.1)** 44.8(26.4)* 44.8(19.9)** Note: Values in the bracket is the relative standard deviations. * Significance of independent t-test between PARAFAC components and DOC is < 0.05; ** Significance of independent t-test between PARAFAC components and DOC is < 0.01.

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500

3

2.5

2.5

450

1.5 350

1.5 350

1 300

35 0

400 450 E m. (nm)

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µ g Q S/L Ex. ( nm)

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2 µg Q S/L Ex. (nm)

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µg Q S/L Ex. (nm)

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500

XAD- 4

µg Q S/L

3 XAD-8 DAX-8

1

300

250

µ g Q S/L

500

0.5

30 0

350

400 450 E m. (nm)

500

5 50

0

Fig. S1. Fluorescence EEMs of the solvent blank and the resin blanks

120

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500 450

100

) 400 m n (. x E350

80

EP AC C

300 250

40 M

B

300

60 C

/L S Q g ♦

T 350

20

A 400 450 Em. (nm)

500

550

0

Fig. S2. Five defined regions of fluorescence EEMs. The emission (ex) and the excitation (em) wavelength ranges for B, T, A, M, and C are ex 250-300 / em 280-330, ex 250-300 / em 330-380, ex 250-300 / em 380-480, ex 300-320 / em 380-420, and ex 320-370 / em 420-480.

10

ACCEPTED MANUSCRIPT

1

a. Protein-like indicators (B and T)

0

-0.5

-1 -1

2

-0.5 0 0.5 1 log10(FRI (Inds ), µg QS/L)

1

d. Humic-like indicators (A, M, and C)

0.5 1 1.5 log10(SP (Inds), µg QS/L) g. Protein- and humic-like indicators 2

y= -0.12x + 0.36x + -0.33 2

0.5 n= 127, R = 0.092, p= 0.0000

0

y= -0.19x + 0.33x + -0.11 2

0.5 n= 127, R = 0.539, p= 0.0000

0

TE D

-0.5

2

-1 -1

-0.5 0 0.5 1 log10(FRI (Inds ), µg QS/L)

1.5

f. Humic-like indicators (C1, C2, and C3)

0.5

n= 76, R2= 0.311, p= 0.0000

SC

0.5 1 1.5 log10(FRI (Inds ), µg QS/L)

h. Protein- and humic-like indicators

0 0.5 1 log10(PFC (Inds ), µg QS/L)

y= 0.10x2 + -0.18x + 0.07

log10(Indd/Inds ,%)

-0.5

2

-0.5

0.5 1 1.5 log10(SP (Inds), µg QS/L)

0

1 Pro Hum FC

n= 77, R2= 0.096, p= 0.0000

0.5

-1 -0.5

2

0

-1 0

1

0

-0.5

-1 -0.5

2

1

Pro Hum FC

1.5

log10(Indd/Inds ,%)

-0.5

1

log10(Indd/Inds ,%)

log10(Indd/Inds ,%)

n= 78, R2= 0.156, p= 0.0000

0

-1 0

-0.5

y= -0.07x2 + 0.12x + -0.05

log10(Indd/Inds ,%)

log10(Indd/Inds ,%)

0.5

0

-1 -0.5

1.5

e. Humic-like indicators (A, M, and C)

y= -0.11x2 + 0.27x + -0.21

n= 26, R2= 0.720, p= 0.0000

0.5

RI PT

0.5 1 1.5 log10(SP (Inds), µg QS/L)

n= 50, R2= 0.639, p= 0.0000

0.5

log10(Indd/Inds ,%)

log10(Indd/Inds ,%)

log10(Indd/Inds ,%)

n= 49, R2= 0.189, p= 0.0000

-0.5

1

y= -0.32x2 + 0.66x + -0.31

y= -0.21x + 0.36x + -0.13

0

-1 0

c. Protein-like indicators (C4)

2

y= -0.27x + 0.79x + -0.67 0.5

1

b. Protein-like indicators (B and T)

2

M AN U

1

0.5

0

0.5 1 1.5 log10(PFC (Inds ), µg QS/L)

i. Protein- and humic-like indicators y= 0.04x2 + -0.05x + 0.00 2

n= 102, R = 0.016, p= 0.0407

2

Pro Hum FC

0

-0.5

-1 -0.5

0

0.5 1 1.5 log10(PFC (inds), µg QS/L)

2

AC C

EP

Fig. S3. Relationships between the ratios of Indd/Inds and Inds for three FDOM indicator groups based on Imai’s fractionation procedures. The charts a, b, and c are protein-like indicators in SP, FRI, and PARAFAC; the charts d, e, and f are humic-like indicators in SP, FRI, and PARAFAC methods; the charts g, h, and i are both above indicators in SP, FRI, and PARAFAC methods. Pro, Hum, and FC in the legends denotes protein-like indicators, humiclike indicators, and fitting curves, respectively. The red arrow represents the lowest concentrations with the Indd/Inds ratios approaching 1.0.

11

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2

2

y= -0.13x + 0.19x + -0.04

0

-0.5

0.5 1 1.5 log10(SP (Inds), µg QS/L)

0

-0.5

-1 -1

2

1

d. Humic-like indicators (A, M, and C) 2

1

0

0.5 1 1.5 log10(SP (Inds), µg QS/L)

g. Protein- and humic-like indicators 2

y= -0.23x + 0.59x + -0.41 0.5 n= 132, R2= 0.142, p= 0.0000

0.5 1 1.5 log10(SP (Inds), µg QS/L)

-0.5 0 0.5 1 log10(FRI (Inds ), µg QS/L) h. Protein- and humic-like indicators 2

y= -0.11x + 0.15x + -0.04

-0.5

0

0.5 n= 134, R2= 0.629, p= 0.0000

0

2

-0.5

-0.5 0 0.5 1 log10(FRI (Inds ), µg QS/L)

-0.5

0 0.5 1 1.5 log10(PFC (Inds ), µg QS/L)

2

f. Humic-like indicators (C1, C2, and C3)

y= -0.07x2 + 0.10x + -0.02 0.5 n= 80, R2= 0.569, p= 0.0000

0

-0.5

-1 -2

1.5

1

Pro Hum FC

-0.5

-1 -1

0

SC log10(Indd/Inds ,%)

-0.5

1 Pro Hum FC

0

-1 -0.5

0

-1 -1

2

n= 81, R2= 0.303, p= 0.0000

TE D

log10(Indd/Inds ,%)

log10(Indd/Inds ,%)

-0.5

log10(Indd/Inds ,%)

log10(Indd/Inds ,%)

0

0.5

y= -0.13x2 + 0.23x + -0.08 0.5 n= 27, R2= 0.840, p= 0.0000

1

e. Humic-like indicators (A, M, and C) y= -0.01x + 0.02x + -0.01

0.5 n= 80, R2= 0.066, p= 0.0004

c. Protein-like indicators (C4)

-1 -1

1.5

2

y= -0.09x + 0.31x + -0.29

-1 -0.5

-0.5 0 0.5 1 log10(FRI (Inds ), µg QS/L)

1.5

-1 0 1 log10(PFC (Inds ), µg QS/L)

i. Protein- and humic-like indicators y= -0.08x2 + 0.13x + -0.04

log10(Indd/Inds ,%)

1

0.5 n= 53, R2= 0.729, p= 0.0000

log10(Indd/Inds ,%)

0.5 n= 52, R2= 0.492, p= 0.0000

log10(Indd/Inds ,%)

log10(Indd/Inds ,%)

y= -0.54x + 1.20x + -0.67

-1 0

1

b. Protein-like indicators (B and T)

RI PT

1

a. Protein-like indicators (B and T)

M AN U

1

0.5 n= 107, R2= 0.573, p= 0.0000

2

Pro Hum FC

0

-0.5

-1 -2

-1 0 1 log10(PFC (inds), µg QS/L)

2

AC C

EP

Fig. S4. Relationships between the ratios of Indd/Inds and Inds for three fluorescence indicator groups based on the Kim’s fractionation procedures. The charts a, b, and c are protein-like indicators in SP, FRI, and PARAFAC; the charts d, e, and f are humic-like indicators in SP, FRI, and PARAFAC methods; the charts g, h, and i are both above indicators in SP, FRI, and PARAFAC methods. Pro, Hum, and FC in the legends denotes protein-like indicators, humic-like indicators, and fitting curves, respectively. The red arrow represents the lowest concentrations with the Indd/Inds ratios approaching 1.0.

12

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380 360

Ex (nm)

320 M

300

T B

280

300

350 400 Em (nm)

450

500

M AN U

240 250

SC

A

260

RI PT

C

340

AC C

EP

TE D

Fig. S5. Peak changes in the original (□) and the subtracted (○) EEM spectra upon resin fractionation

13

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45 40

50 40

30

C1 (%)

DOC (%)

35

60

HPO(a) HPO(n) HPO/TPI/HPI (b) TPI/HPI (a) TPI/HPI (n)

25

c 45

HPO(a) HPO(n) HPO/TPI/HPI (b) TPI/HPI (a) TPI/HPI (n)

35 30

30

20 20

15

20 15

10 5

5 Limnic

Riverine

All

d

60 50

C3 (%)

40

Effluent

Limnic

Ri verine

All

HPO(a) HPO(n) HPO/TPI/HPI (b) TPI/HPI (a) TPI/HPI (n)

60

Effluent

Limnic

Riverine

All

Riverine

All

Riverine

All

Riverine

All

HPO(a) HPO(n) HPO/TPI/HPI (b) TPI/HPI (a) TPI/HPI (n)

50

30

20

40 30

M AN U

20

10 0

0

e

SC

Effluent

0

C4 (%)

0

25

10

10

HPO(a) HPO(n) HPO/TPI/HPI (b) TPI/HPI (a) TPI/HPI (n)

40

RI PT

b 50

C2 (%)

a

10

Effluent

Limnic

Ri verine

0

All

Effluent

Limnic

Imai’s method

70 60

h

HPO/TPI/HPI (a) HPO (b/n) TPI (b/n) HPI (b/n)

80 70

30

60

40 30

20

Riverine

0

All

EP

h

60

C3 (%)

AC C

50

Effluent

Limnic

10 Riverine

0

All

i

HPO/TPI/HPI (a) HPO (b/n) TPI (b/n) HPI (b/n)

80 70

Effluent

Limnic

HPO, TPI, and HPI HPO TPI HPI

60

40

C4 (%)

Limnic

40

20

10

Effluent

50

30

20

10 0

HPO/TPI/HPI (a) HPO (b/n) TPI (b/n) HPI (b/n)

50

40

C1 (%)

DOC (%)

50

g

HPO/TPI/HPI (a) HPO (b/n) TPI (b/n) HPI (b/n)

C2 (%)

60

TE D

f

30

50 40 30

20 20 10 0

10 Effluent

Limnic

Riverine

All

0

Effluent

Limnic

Kim’s method Fig. S6. Relative abundances of different resin fractions on the basis of DOC and PARAFAC components. The fractions were obtained by Imai’s (chart a-e) and Kim’s (chart f-i) fractionation methods. The word “All” in the x-axis denotes the average of EfOM, LiOM, and RiOM. 14

ACCEPTED MANUSCRIPT

Script S1. Matlab codes for the peak identification and volume integration in the specific zone.

RI PT

function F_specific=F_specific_FRI(X,Ex,Em,dataname)

M AN U

SC

%% Coble (1996)'s peaks finding and Chen (2003)'s regional integration % ------Description of this code-----% The specific zones, where the peak finding and volume integration are % carried, is shown in Fig. S2 in the supplementary materials. The largest % intensity value in the specific zone will be assigned as the peak. The % volume under the surface of the EEM of specific zone will be integrated % and assigned as the FRI. % ------Description of the input-----% X - 3D data cube (No headers, i.e. samples*Em*Ex) of fluorescence % intensities, which is also the dataset XcQS or XcRU output from % FDOMcorrect.m. % Em - 1D row vector of emission wavelengths corresponding to EEMs. % Ex - 1D row vector of excitation wavelengths corresponding to EEMs. % dataname - 1D row cell of samples' name corresponding to the first % dimension (also known as the samples)

TE D

%% About this Matlab file % Please cite this program as: F_specific_FRI.m % in He and Hur. 2015 ' Conservative hehaviors of fluorescence EEM-PARAFAC % components in resin fractionation processes and its applicability for % characterizing dissolved organic matter% Copyright(C) 2015 W He, % Copyright (C) 2015 W He, % Sejong University % Department of Environment and Energy % Seoul 143-747, South Korea % [email protected]

EP

%% Data pretreatment % Build a similar struct dataset used in the FDOMFluor toolbox. SP=size(X,1); Data_Input=struct('X',X,'Em',Em,'Ex',Ex,'nEm',length(Em),'nEx',length(Ex),'nS ample',SP);

AC C

% Obtain the interval of the emission and excitation wavelength Ex_inv=Ex(2)-Ex(1); Em_inv=Em(2)-Em(1); % Use he EEMCut.m in the FDOMFluor toolbox to cut the 1st and 2nd Rayleigh % scatter [Data_Cut]=EEMCut(Data_Input,25,0,30,0,''); % Cut the Raman scatter from the EEM for w=1:length(Data_Cut.Em); Excitation(w)=1e7/((1e7/Data_Cut.Em(w))+3600); v=find((Data_Cut.Ex>Excitation(w)+0)&(Data_Cut.Ex