Accepted Manuscript

Effects of Charging on the Chromophores of Dissolved Organic Matter from the RioNegro Basin

Mingquan Yan, Gregory V. Korshin, Francis Claret, Jean-Philippe Croué,Massimiliano Fabbricino, Hervé Gallard, Thorsten Schäfer, Marc F. Benedetti

PII: S0043-1354(14)00234-6

DOI: 10.1016/j.watres.2014.03.044

Reference: WR 10569

To appear in: Water Research

Received Date: 14 December 2013

Revised Date: 17 February 2014

Accepted Date: 17 March 2014

Please cite this article as: Yan, M., Korshin, G.V., Claret, F., Croué, J.-P., Fabbricino, M., Gallard, H.,Schäfer, T., Benedetti, M.F., Effects of Charging on the Chromophores of Dissolved Organic Matter fromthe Rio Negro Basin, Water Research (2014), doi: 10.1016/j.watres.2014.03.044.

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Graphical AbstractGraphical AbstractGraphical AbstractGraphical Abstract

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Carboxylic groups Carboxylic groups

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Effects Effects Effects Effects of of of of Charging Charging Charging Charging on on on on the the the the Chromophores Chromophores Chromophores Chromophores of of of of Dissolved Dissolved Dissolved Dissolved Organic Organic Organic Organic 1

Matter from the Rio Negro BasinMatter from the Rio Negro BasinMatter from the Rio Negro BasinMatter from the Rio Negro Basin 2

Mingquan Yan&, Gregory V. Korshin*, Francis Claret§, Jean-Philippe Croué##, 3

Massimiliano Fabbricino**, Hervé Gallard##, Thorsten Schäfer§§ and Marc F. 4

Benedetti# 5

&Department of Environmental Engineering, Peking University, Key 6

Laboratory of Water and Sediment Sciences, Ministry of Education, Beijing 7

100871, China 8

* Department of Civil and Environmental Engineering, University of 9

Washington, Seattle, WA 98195-2700 United States 10

**Dipartimento di Ingegneria Idraulica ed Ambientale "Girolamo Ippolito", 11

Universitá degli Studi di Napoli Federico II Via Claudio 21, 80125 Naples Italy 12

# Institut de Physique du Globe de Paris – Sorbonne Paris Cité - Université 13

Paris-Diderot , UMR CNRS 7154, Paris, France 14

## Equipe Chimie de l'Eau et Traitement des Eaux 15

Institut de Chimie des Milieux et Matériaux de Poitiers UMR 7285 CNRS 16

Ecole Nationale Supérieure d'Ingénieurs de Poitiers – Université de Poitiers 17

86022 Poitiers Cedex France 18

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§ Bureau des Recherches Géologiques et Minières, Environment and 19

Process Division 3, avenue Claude Guillemin F-45060 Orleans Cedex 2 20

France 21

§§ Forschungszentrum Karlsruhe, Institut für Nukleare Entsorgung (INE) P.O. 22

Box 3640 76021 Karlsruhe, Germany 23

24

& Corresponding author. Address: Department of Environmental Engineering, 25

College of Environmental Sciences and Engineering, Peking University, 26

Beijing 100871, China; Tel: +86 10 62755914-81, Fax: +86 10 62756526. E-27

mail: yanmq@pku.edu.cn 28

29

30

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AbstractAbstractAbstractAbstract 31

This study demonstrates that the deprotonation of dissolved organic matter 32

(DOM) originating from a small creek characteristic for DOM-rich waters 33

located in the Rio Negro basin can be quantified based on measurements of 34

pH effects on its absorbance spectra. The method was ascertained by the 35

data of Near-Edge X-Ray Absorbance Spectroscopy (NEXAFS), 36

potentiometric titration to quantify the structural and compositional differences 37

between the colloidal and hydrophobic fractions that contribute 91% of black-38

water creek DOM. Changes in the absorbance spectra of the DOM fractions 39

caused by deprotonation quantified via numeric deconvolution which indicated 40

the presence of six well-resolved Gaussian bands in the differential spectra. 41

The emergence of these bands was determined to be associated with the 42

engagement of carboxylic and phenolic functionalities and changes of inter-43

chromophore interactions in DOM molecules. Interpretation of the data based 44

on the NICA-Donnan approach showed that behavior of DOM chromophores 45

was consistent with results of potentiometric titrations. Similar trends were 46

observed for changes of the spectral slope of the DOM absorbance spectra in 47

the range of wavelengths 325 to 375 nm (DSlope325-375). The behavior of 48

DSlope325-375 values was modeled based on the NICA-Donnan approach and 49

correlated with potentiometrically-estimated charges attributed to the 50

carboxylic and phenolic groups. The correlations between DSlope325-375 and 51

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charges of low- and high-affinity protonation-active groups in DOM were 52

monotonic but not linear and had important differences between the colloidal 53

and hydrophobic fractions. 54

55

KeywordsKeywordsKeywordsKeywords: : : : Amazon River, absorbance, deprotonation, dissolved organic 56

matter, NEXAFS, NICA-Donnan model 57

58

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1. Introduction 59

Soil organic matter and water-borne dissolved organic matter (DOM) are 60

fundamentally important components of all environmental systems. Because 61

the generation of aquatic DOM is affected by local biogeochemical conditions, 62

many of its properties, e.g. its affinity to the proton and metal ions, sizes and 63

charges of DOM molecules, their surface activity, the presence of redox active 64

functionalities all of which frequently play a crucial role in environmental 65

processes, are site-specific (Milne et al., 2001; Lenoir et al., 2010; 66

Aeschbacher et al., 2012) and affected by seasonal cycles (Milne et al., 2001; 67

Leenheer and Croué, 2003; Ellis et al., 2012). Remarkable progress has been 68

made in the exploration of DOM site-specificity but effects of local 69

environmental conditions and processes on its structure and reactivity remain 70

to be understood on more detail. 71

In this context, the understanding of properties of DOM from the Amazon 72

River basin is especially important because this area contributes ca. 7% of the 73

global flux of DOM to the oceans while transformations of this DOM have 74

been shown to generate a considerable fraction of the regional flux of CO2 75

(Richey et al., 2002; Mayorga et al., 2005). Due these factors, elucidation of 76

the intrinsic properties of Amazonian DOM as well as DOM from other fluvial 77

systems is essential for understanding global and local carbon cycles 78

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(Hedges et al., 1997; Ellis et al., 2012; Ward et al., 2013). Extensive prior 79

research has addressed many important details the genesis and fate of 80

Amazonian DOM (Hedges et al., 1994; Patel et al., 1999; Krusche et al., 2002; 81

Moreira-Turcq et al., 2003; Amaral et al., 2013; Ward et al., 2013), its role in 82

the speciation of major and trace constituents (Maurice-Bourgoin et al., 2003; 83

Rocha et al., 2003; Allard et al., 2004; de Oliveira et al., 2007; Fritsch et al., 84

2009; Perez et al., 2011; Kim et al., 2012), its photochemical transformations 85

(Patel-Sorrentino et al., 2004; Rodriguez-Zuniga et al., 2008; Remington et al., 86

2011; Amaral et al., 2013) and longitudinal, seasonal or anthropogenically-87

induced changes of its properties (McClain et al., 1997; Aufdenkampe et al., 88

2001; Bernardes et al., 2004; de Oliveira et al., 2007; Salisbury et al., 2011; 89

Amaral et al., 2013). For instance, (Hedges et al., 2000) and ensuing studies 90

(Aufdenkampe et al., 2007) presented a regional “chromatographic” model to 91

account for the evolution of DOM from alluvial soils to the Amazon’s main 92

stem and concluded that selective sorption of DOM onto minerals was the key 93

process that affects the properties of different fractions of organic carbon of 94

the rivers. Prior studies concerned with the evolution of DOM also concluded 95

that further exploration of its composition and reactivity, especially its 96

deprotonation-protonation and charging processes is necessary to understand 97

the partitioning processes in soils and riparian zones (Amon and Benner, 98

1996; Hedges et al., 2000; Alasonati et al., 2010). 99

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While many advanced structure- and compound-specific ex situ methods, e.g. 100

potentiometric titrations have been used to examine the composition, genesis 101

and reactivity of DOM from the Amazon basin and other environmental 102

systems (Hedges et al., 1994; Benner et al., 1995; Hedges et al., 2000; 103

Aufdenkampe et al., 2001; Bernardes et al., 2004; Aufdenkampe et al., 2007; 104

Mopper et al., 2007; Kujawinski et al., 2009; Ellis et al., 2012; Ward et al., 105

2013), results of these studies can be augmented by data of techniques that 106

allow quantification of DOM properties in situ. Examination of absorbance and 107

fluorescence of DOM can play this role since these methods use unaltered 108

waters to produce optical spectra that are sensitive to DOM molecular weight, 109

aromaticity and fluorophore and chromophore speciation (Hoge et al., 1993; 110

Green and Blough, 1994; Peuravuori and Pihlaja, 1997; McKnight et al., 2001; 111

Chen et al., 2003; Del Vecchio and Blough, 2004; Helms et al., 2008; Boyle et 112

al., 2009). Studies that employed these techniques to examine Amazonian 113

DOM have demonstrated the presence of fluorophore and chromophore 114

groups associated with DOM molecules of varying sizes and chemical natures 115

(Mounier et al., 1999; Patel-Sorrentino et al., 2002; Patel-Sorrentino et al., 116

2004; Rodriguez-Zuniga et al., 2008). Variations of pH prominent in the 117

Amazon basin (Do Nascimento et al., 2008) affect the fluorescence of 118

Amazonian DOM (Mounier et al., 1999; Patel-Sorrentino et al., 2002) but the 119

nature of such changes that are common to fresh-water DOM (Tam and 120

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Sposito, 1993; Patel-Sorrentino et al., 2002; Spencer et al., 2007; Do 121

Nascimento et al., 2008) has not been unambiguously determined. 122

Effects of pH variations on the absorbance of DOM (Tam and Sposito, 1993; 123

Andersen et al., 2000; Andersen and Gjessing, 2002; Spencer et al., 2007) 124

have been addressed but because the absorbance spectra of DOM are 125

featureless, these studies has been limited. DOM absorbance spectra can be 126

made more feature-rich via the use of a differential approach that quantifies 127

the evolution of the spectra as a function of any desired reaction parameter, 128

for instance metal complexation, oxidant dose or pH (Korshin et al., 1999; 129

Dryer et al., 2008; Janot et al., 2010; Yan et al., 2013b). 130

In this paper, we present results of the examination of DOM from the basin of 131

the Rio Negro River, one of the most important tributaries of the Amazon, 132

using the method of differential absorbance (DA) and compare its data with 133

those generated using potentiometric titrations and structure-sensitive 134

methods. This study’s objective is to establish in situ measurable 135

spectroscopic markers of the important intrinsic properties of molecules 136

Amazonian DOM without preconcertration and altering DOM properties, 137

notably relationships between pH, charge and, on the other hand, absorbance 138

of these molecules and, ultimately, their reactivity in more complex systems, 139

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for instance in their interactions with mineral phases (Perez et al., 2011; Janot 140

et al., 2012) that define the evolution of DOM in the soil-river continua. 141

2.2.2.2. Materials and Materials and Materials and Materials and MMMMethodsethodsethodsethods 142

2.1.2.1.2.1.2.1. Isolation and fractionation of samples.Isolation and fractionation of samples.Isolation and fractionation of samples.Isolation and fractionation of samples. 143

DOM samples were collected from the Igarapé Bonito, a small creek that 144

flows into the Jau River. The GPS coordinates of the Jau station were S 145

01°52.325', W 61°35.027'. The Jau River, a tributary of the Rio Negro, is fed 146

by groundwater seepages and creeks similar to the Igarapé Bonito (Alasonati 147

et al., 2010). The dissolved organic carbon (DOC) concentration, pH and 148

conductivity in the Igarapé Bonito at the time of sampling were 56.6 mg L-1, 149

3.6 and 65 µS cm-1, respectively. Preparation of the samples included filtration 150

through 1 µm GF/C filters followed by reverse osmosis (RO). RO concentrates 151

were dialyzed against 0.1 M HCl and 0.2 M HF to isolate a colloidal (COLL) 152

fraction with a 3,500 D nominal cutoff from the other DOM fractions. The 153

fraction passing through the dialysis membrane was fractionated using XAD-8 154

and XAD-4 resin columns to obtain the fractions of hydrophobic (HPO) and 155

transphilic (TPH) DOM, respectively (Croue et al., 2000). Prior to this 156

separation, DOM solutions were acidified to pH 2. After pumping each sample 157

through the columns, they were rinsed with a formic acid solution at pH 2. 158

DOM retained on them was eluted using an acetonitrile/water (75%/25% v/v) 159

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mixture. The eluent was evaporated under vacuum at 35-45 °C. 200 mL of 160

acetonitrile were added two or three times during the evaporation to eliminate 161

traces of formic acid. The dialysis/XAD resins procedure isolated over 90% of 162

the DOM in the RO concentrate. The COLL, HPO and TPH fractions 163

constituted 51%, 40% and 9%, respectively, of the total weight of DOM 164

extracted from the sample. DOC concentrations were determined with a 165

Shimadzu TOC-Vcsh carbon analyzer. Effects of acidic agents, e.g. HCl and 166

HF used in DOM isolation on intrinsic properties of the DOM were assumed to 167

be negligible for the purposes in this study (Hamilton-Taylor et al., 2011; 168

Ahmed et al., 2013) . 169

2.2. 2.2. 2.2. 2.2. Potentiometric titrationsPotentiometric titrationsPotentiometric titrationsPotentiometric titrations 170

Proton titrations were performed using a computer-controlled system in a 171

thermostated vessel (25°C) under 99.99% nitrogen (Janot et al., 2010; Janot 172

et al., 2012). DOM solutions were prepared at a 1 g L-1 DOC concentration in 173

the presence of a 0.04 M ionic strength. The pH was read using two pH 174

Metrohm electrodes (6.0133.100) and a Ag/AgCl glass reference Metrohm 175

electrode (6.0733.100) with a salt bridge (same as the solution). The 176

electrodes were calibrated with CO2-free KOH base (0.099 M) and HNO3 177

(0.100 M) at a 0.1 M ionic strength. The pH values read by the duplicate 178

electrodes were averaged. After addition of acid or base, the rate of drift for 179

both electrodes was measured over 1 minute and readings were accepted 180

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when the drift was less than 0.1 mV min-1. For each data point the maximum 181

time for monitoring pH drift was equal to 20 minutes. 182

2.3. 2.3. 2.3. 2.3. Carbon NEXAFS analysis Carbon NEXAFS analysis Carbon NEXAFS analysis Carbon NEXAFS analysis 183

Carbon K-edge Near Edge X-Ray Absorption Fine Structure (NEXAFS) 184

spectra (Jacobsen et al., 1991) were measured at the Scanning Transmission 185

X-ray Microscopy (STXM) beamline X1A1 (NSLS) operated by the State 186

University of New York at Stony Brook. STXM sample preparation was 187

performed by drying 1 µL solution of resuspended freeze-dried DOM on a 100 188

nm thick Si3N4 window. The spectra were measured using the “point spectra” 189

procedure consisting of measurement between 280 to 310 eV in 0.1 eV steps 190

using 120 ms dwell time (Christl and Kretzschmar, 2007). Five consecutive 191

point spectra of a region on the Si3N4 window without sample were averaged 192

to obtain the I0(E) information. I(E) is the average of 15 spectra taken on three 193

different sample locations. Energy calibration of the spherical grating 194

monochromator was achieved by using the photon energy of the CO2 gas 195

adsorption band at 290.74 eV. To compare NEXAFS spectra, they were 196

baseline corrected and normalized to 1 at 295 eV prior to peak fitting. The 197

spectra were then deconvoluted as described in (Schafer et al., 2005). 198

Precision of determinations of carbon functionalities’ contributions based on 199

carbon NEXAFS data is estimated at ±2%. 200

2.4. 2.4. 2.4. 2.4. Spectrophotometric titrationsSpectrophotometric titrationsSpectrophotometric titrationsSpectrophotometric titrations 201

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The method of differential spectrophotometric titration of DOM and 202

interpretation of its data have been described in sufficient detail in preceding 203

relevant publications (Dryer et al., 2008; Janot et al., 2010; Yan et al., 2013b). 204

DOM solutions were prepared at 2 or 5 mg L-1 DOC concentrations in the 205

presence of NaClO4 with ionic strength 0.04 M. Absorbance spectra were 206

recorded with Perkin-Elmer Lambda 18 UV/Vis spectrometer. Dilution effects 207

due to addition of acid and base were corrected for in the final data. Fitting of 208

model calculations was performed using Matlab 2010a. 209

3. Results and Discussion 210

3.1. 3.1. 3.1. 3.1. NEXAFS dataNEXAFS dataNEXAFS dataNEXAFS data 211

The NEXAFS spectra for the COLL, HPO and TPH fractions of Igarapé Bonito 212

DOM are shown in Figure S1 in the Supporting Information (SI) section.... 213

Results of their deconvolution that allowed determining contributions of 214

different functionalities are compiled in Table S1 in the SI section. In view the 215

NEXAFS spectra for HPO and TPH being similar, only the deconvoluted 216

NEXAFS spectra for the two major fractions, COLL and HPO are shown in 217

Figure 1. These data indicate that the COLL fraction is richer in aromatic 218

groups than HPO and TPH (34, 26 and 22%, respectively), while the 219

contributions of carboxylic groups exhibit an opposite trend (30, 38 and 42%, 220

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respectively). Contributions of other functionalities (Figure 1) do not exhibit 221

prominent changes. 222

Figure 1Figure 1Figure 1Figure 1 223

3.2. 3.2. 3.2. 3.2. PPPPotentiometric otentiometric otentiometric otentiometric datadatadatadata 224

Results of potentiometric titration for the COLL, HPO and TPH fractions of 225

Igarapé Bonito DOM are shown in Figure 2. Modeling of the data shown in 226

Figure 2 based on the NICA-Donnan theory (Milne et al., 2001) to determine 227

concentrations of the low and high affinity proton binding sites (denoted 228

henceforth as LAS and HAS, respectively) and other parameters of DOM 229

protonation are compiled in Table S2. They show that in the COLL fraction the 230

HAS (largely associated with phenolic-type groups) are more abundant. The 231

average values of the LAS and HAS protonation constants of the examined 232

fractions of Igarapé Bonito DOM were determined to have identical average 233

pK values (specifically, the pK values were fixed at average 4.43 and 8.10 234

respectively while the other 4 parameters were optimized). The heterogeneity 235

parameters for the examined fractions were also close, having average values 236

of 0.78 and 0.28 for the LAS (mostly carboxylic type groups) and HAS, 237

respectively. The 0.28 value of the heterogeneity parameter for the HAS 238

indicates a larger chemical heterogeneity. This will be explored in more detail 239

based on the spectroscopic data. 240

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Figure Figure Figure Figure 2222 241

Trends in the concentrations of the HAS and LAS discerned based on the 242

potentiometric data agreed with those indicated by the carbon NEXAFS data 243

(Figure 3). This observation reinforces the notion that while the protonation 244

properties of the HPO and TPH fractions were very close, the colloidal fraction 245

was quite distinct. 246

Figure Figure Figure Figure 3333 247

3.3. 3.3. 3.3. 3.3. Differential absorbance resultsDifferential absorbance resultsDifferential absorbance resultsDifferential absorbance results 248

Absorbance experiments were carried out for the COLL and HPO fractions. 249

The TPH fraction was not studied by optical spectroscopy because it was 250

unavailable for the differential absorbance experiments. In addition, its 251

properties determined by NEXAFS and potentiometric titrations were very 252

close to those of the HPO fraction and it had a small contribution (i.e. 9%) to 253

the total concentration of organic carbon in Igarapé Bonito DOM. 254

The absorbance of both COLL and HPO fractions changed in response to pH 255

variations. As the pH increased, the absorbance increased at all wavelengths, 256

but the spectra remained featureless, as demonstrated in Figure S2 in the SI 257

section. To obtain more information on how pH variations affected the 258

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behavior of chromophore groups of the DOM, DA spectra were calculated 259

using the equation below: 260

( ) ( ) ( )[ ]refpHApHAlDOC

pHA λλλ −⋅

=∆ 1 (1) 261

In this equation, ( )pHAλ∆ is the differential absorbance at any specified 262

wavelength, l is the optical cell’s length, ( )pHAλ and ( )refpHAλ are 263

absorbances at any desired pH value and a reference pH, respectively. The 264

spectrum recorded at pH 3.1 was used as the reference, respectively. 265

The intensity of the DA spectra increased monotonically with the pH, and 266

discernible features that had different prominence for the COLL and HPO 267

fractions were present in them (Figure 4). Specifically, peaks with maxima 268

located approximately at 240, 280 and 315 nm, as well a broad structure 269

located at >350 nm were prominent. 270

Figure 4Figure 4Figure 4Figure 4 271

Normalized (by the maximum of their intensity at λ>350 nm) DA spectra 272

(Figure S3) were calculated using the reference spectra recorded at pH 3.3 273

and 8.3 corresponding to the deprotonation of LAS and HAS chromophores, 274

respectively (Dryer et al., 2008). The data show that the deprotonation of LAS 275

chromophores of Igarapé Bonito DOM is associated with the emergence of 276

bands with maxima located at 240, 278, 315 and 375 nm (Figure S3a). These 277

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features are similar for the HPO and COLL fractions, except that located for 278

HPO at 240 nm. The most intense feature in the normalized DA spectra of the 279

LAS chromophores is located at λ>350 nm, that is in the range where 280

manifestations of inter-chromophore interactions are located (Del Vecchio and 281

Blough, 2004; Dryer et al., 2008). The engagement of the HAS chromophores 282

is accompanied by the development of two bands with maxima close 247 nm 283

and 315 nm, and a much stronger feature with a maximum located at 385 to 284

390 nm; that feature was especially prominent for the COLL fraction (Figure 285

S3b). This is likely to indicate higher importance of inter-chromophore 286

interactions in the molecules of the COLL fractions of Igarapé Bonito DOM 287

due to their larger molecular weights (Green and Blough, 1994; Del Vecchio 288

and Blough, 2004). 289

To examine the structure of the pH-differential spectra of both COLL and HPO 290

fractions in more detail, they were deconvoluted to determine contributions of 291

discrete bands constituting them. In agreement with the approach presented 292

in prior research (Korshin et al., 1997; Gege, 2000; Yan et al., 2013b), such 293

bands were assumed to have a Gaussian shape when represented against 294

photon energy (measured in eV), calculated as 295

( ) ( )nmeVE

λ1240= (2) 296

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The fitting procedure allowed determining such characteristics of each 297

Gaussian bands as the location of its maximum (E0i), width (Wi) and intensity 298

at E=E0i (A0i). The overall differential spectra (∆A(E)) were modeled as 299

( ) ∑

−−∆=∆i i

ii

W

EEAEA

2

00

2exp (3) 300

Selected results of the application of this concept to the modeling of pH-301

differential spectra of the COLL and HPO fractions of Igarapé Bonito DOM are 302

shown in Figure 5. They demonstrate a very close fit between the observed 303

and modeled spectra (R2>0.995). The pH-differential spectra of Igarapé 304

Bonito DOM were determined to comprise six Gaussian components whose 305

intensities changed with pH while positions of their maxima were practically 306

constant (Table S3). The maxima of the bands referred to, as in prior relevant 307

publications (Yan et al., 2013b) as A0, A1, A2, A3, A4 and A5, were located 308

at ca. 6.00 eV (207 nm), 5.07 eV (245 nm) 4.45 eV (280 nm), 3.97 eV (313 309

nm), 3.25 eV (380 nm) and 2.33 eV (530 nm). The locations of the maxima of 310

these Gaussian bands A1, A2, A3 and A4 were close to those of the bands 311

observed previously for SRFA (Yan et al., 2013b) while slight differences were 312

observed for bands A0 and A5. Because the properties of band A0 were 313

difficult to estimate due to the presence of spectroscopic interferences from 314

hydroxyl ions and the intensity of band A5 was lower than that of all other 315

bands, only the data for A1, A2, A3 and A4 will be discussed henceforth. 316

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Figure Figure Figure Figure 5555 317

Changes of the intensity of band A1, A2, A3 and A4 with pH for COLL and 318

HPO are presented in Figure S4. Although the relative contributions of each 319

Gaussian band that comprise the pH-differential spectra of Igarapé Bonito 320

DOM were different (Figure 5 and Figure S4), trends in the evolution of the 321

intensity of each band caused variations of pH were similar to those typically 322

seen in potentiometric titrations. This was interpreted to indicate that these 323

bands comprise contributions of chromophores associated with both LAS and 324

HAS groups. 325

Prior research has demonstrated that bands A4 especially sensitive to the 326

changes of the intrinsic chemistry DOM molecules caused by their 327

deprotonation or complexation with metal cations (Yan et al., 2013b). 328

However, the relatively low intensity of these bands, especially that of band 329

A5 necessitate that the absorbance spectra of DOM be log-transformed and 330

differential spectra be calculated using the log-transformed data using the 331

expression below: 332

(4) 333

The differential log-transformed absorbance spectra of the COLL and HPO 334

fractions at selected pH values are presented in Figure 6. Similarly to the 335

linear differential spectra show in Figure 4, the intensity of the log-transformed 336

spectra increases with pH. This increase is especially prominent for 337

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wavelengths > 320 nm. The prominence of the features observed in the log-338

transformed differential spectra and located at wavelengths > 320 nm is 339

associated with the development of Gaussian bands A4 that are relatively 340

inconspicuous in terms of their absolute intensities, compared with the 341

intensities of bands A1, A2 and A3. 342

In accord with the approach presented in prior research (Yan et al., 2013a), 343

we used the differential spectral slope in the range of wavelengths 325 to 375 344

nm (DSlope325-375) measured at varying pHs examine effects of pH on the 345

protonation of Igarapé Bonito DOM. DSlope325-375were calculated using the 346

formula given below: 347

325 375 325 375, 325 375,refiDSlope Slope Slope− − −= − (5) 348

In this expression, 325 375,iSlope − and 325 375,refSlope − are the slopes of the linear fit 349

of the log-transformed DOM absorbance in the wavelengths region 325 to 375 350

nm for any selected solution conditions and reference, respectively. The 351

choice of DSlope325-375 parameter reflects the observation that the intensity of 352

the log-transformed spectra undergoes rapid changes in the 325 to 375 nm 353

region corresponding to the location of Gaussian band A4 and these changes 354

are nearly linear vs. the observation wavelength. 355

To determine whether the evolution of the DA spectra of Igarapé Bonito DOM 356

could be described by the NICA-Donnan theory developed to model the 357

potentiometric behavior of DOM, we applied the equation developed in 358

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literatures (Kinniburgh et al., 1999; Ritchie and Perdue, 2003; Dryer et al., 359

2008; Janot et al., 2010) to model the evolution of differential slopes of the 360

absorbance of Igarapé Bonito samples: 361

362

(6) 363

364

where ( )λLASDSlope and ( )λHASDSlope correspond to the maximum change of 365

absorbance associated with the deprotonation of the LAS (mostly carboxylic) 366

and HAS (mostly phenolic) groups, respectively, ( )λLASDSlope and ( )λHASDSlope 367

referred to DSlope value in the range of wavelengths 325 to 375 nm in this 368

study. ~

KLAS

and ~

KHAS

are the median values of the protons affinity distributions 369

for these groups, mLAS and mHAS define the width of these distributions and 370

are measures of the heterogeneity of DOM (Milne et al., 2001; Dryer et al., 371

2008). 372

The behavior of DSlope325-375 values for COLL and HPO vs. pH and their 373

fitting is shown in Figure 7 and Table S2. The data show that excellent 374

agreement could be reached between the DSlope325-375 data for both COLL 375

and HPO samples and the predictions made based on the revised NICA-376

Donnan model (R2>0.99). 377

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The above observations of the highly interpretable response of the 378

chromophores of Igarapé Bonito DOM to their deprotonation are enhanced by 379

the observation that changes of the spectral slope of the examined DOM 380

fractions are correlated with the charges of HAS and LAS groups in DOM 381

molecules calculated using the NICA-Donnan parameters obtained using 382

conventional potentiometric titration in Table 2. The correlations between LAS 383

and HAS charges and changes of the spectral slope are compared in Figure 384

8b and 8c, respectively. It demonstrates that while both QLAS and QHAS are 385

correlated with DSlope values, these correlations have distinct differences for 386

the LAS and HAS groups. In the former case, the correlations between QLAS 387

and DSlope values are nearly linear but the slope of the correlations are 388

different for the HPO and COLL fractions reflecting the difference in the 389

responses of carboxylic-type chromophores in these fractions to the 390

accumulation of charge. On the other hand, correlations between QHAS and 391

changes of the DSlope for COLL and HPO fractions are similar but the 392

response of the HAS phenolic-type chromophores is characterized by two 393

distinct ranges. In the range of QHAS charges from 0 to ca. 0.7 meq g-1, 394

changes of the slope are relatively small while for QHAS values above ca. 0.7 395

meq g-1, spectral slopes of DOM changes prominently but the accumulation of 396

charge is less rapid. This is likely to be indicative of the engagement of 397

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distinct sub-groups of the HAS chromophores. Their properties need to be 398

examined in more detail in future studies. 399

Given the complexity of the linear or log-transformed DA spectra, the NICA-400

Donnan approach to interpret them appears to be an excellent approximation 401

although it does not address the nature of the distinct spectroscopic features, 402

for instance Gaussian bands A1 to A5. The presence of these bands, their 403

association with the charging of DOM molecules in the HPO and COLL 404

fractions highlights the need to expand the examination of responses of 405

chromophores and fluorophores of a wider range of DOM. On the other hand, 406

this can indicate that mechanisms other the deprotonation of the discrete 407

operationally defined LAS and HAS chromophores may define the evolution of 408

the pH-differential spectra. As mentioned above, these alternative 409

mechanisms are likely to reflect changes of inter-chromophore interactions 410

(Hoge et al., 1993; Green and Blough, 1994; Korshin et al., 1999; Del Vecchio 411

and Blough, 2004) that depend on the molecular weight and conformational 412

status of DOM molecules. pH-differential spectra can also reflect responses of 413

specific functionalities, for instance lignins, terpenoids, bound proteins and 414

others whose deprotonation may yield a distinct signal in the differential 415

spectroscopy. More work is needed to explore this issue further as well as to 416

quantify effects of other system parameters (e.g., those of ionic strength) on 417

chromophores in DOM of varying provenance. 418

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4. Conclusions 419

The data presented above and their interpretation support the following 420

conclusions: 421

(1) The deprotonation of DOM originating from the Rio Negro basin can be 422

quantified based on measurements of pH effects on its absorbance 423

spectra. 424

(2) Changes in the absorbance spectra of the DOM fractions caused by 425

deprotonation quantified via numeric deconvolution which indicated the 426

presence of six well-resolved Gaussian bands in the differential spectra. 427

The emergence of these bands was determined to be associated with the 428

engagement of carboxylic and phenolic functionalities and changes of 429

inter-chromophore interactions in DOM molecules. 430

(3) Interpretation of the data of spectrophotometric titrations based on the 431

NICA-Donnan approach showed that behavior of DOM chromophores was 432

consistent with results of conventional potentiometric titrations. 433

(4) The behavior of DSlope325-375 values was correlated with charges 434

attributed to the carboxylic and phenolic groups. The correlations between 435

DSlope325-375 and charges of low- and high-affinity protonation-active 436

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groups in DOM were monotonic but not linear and had important 437

differences between the HPO and COLL fractions. 438

439

AcknowledgementsAcknowledgementsAcknowledgementsAcknowledgements 440

This study has been partially supported by National Science Foundation 441

(grants 0504447 and 0931676). G. Korshin expresses gratitude to l’Institut de 442

Physique du Globe de Paris/Université de Paris VII and French INSU-CNRS 443

program ECCO for support of his work in Paris, and to the Foreign Experts 444

Program of China (GDW20131100008) for support of his work at Peking 445

University. Use of the National Synchrotron Light Source, Brookhaven 446

National Laboratory, was supported by the U.S. Department of Energy, Office 447

of Science, Office of Basic Energy Sciences, under Contract No. DE-AC02-448

98CH10886. Data were collected using the X1A STXM developed by the 449

group of Janoz Kirz and Chris Jacobsen at SUNY Stony Brook, with support 450

from the office of Biological and Environmental Research, US. DoE under 451

contract DE-FG02-89ER60858, and from the NSF under grant DBI-9605045. 452

Supporting InformationSupporting InformationSupporting InformationSupporting Information 453

This information is available free of charge on the Internet. 454

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674

675

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Table and Table and Table and Table and Figure CaptionsFigure CaptionsFigure CaptionsFigure Captions 676

Figure Figure Figure Figure 1111.... C(1s) NEXAFS spectra of C(1s) NEXAFS spectra of C(1s) NEXAFS spectra of C(1s) NEXAFS spectra of COLLCOLLCOLLCOLL andandandand HPOHPOHPOHPO fractions fractions fractions fractions of Igarapé of Igarapé of Igarapé of Igarapé 677

Bonito Bonito Bonito Bonito DOMDOMDOMDOM. . . . 678

Figure Figure Figure Figure 2222.... Results of potentiometric titration for Results of potentiometric titration for Results of potentiometric titration for Results of potentiometric titration for COLLCOLLCOLLCOLL andandandand HPO fractions of HPO fractions of HPO fractions of HPO fractions of 679

Igarapé Bonito DOMIgarapé Bonito DOMIgarapé Bonito DOMIgarapé Bonito DOM and its modeling using NICAand its modeling using NICAand its modeling using NICAand its modeling using NICA----Donnan modelDonnan modelDonnan modelDonnan model. . . . 680

Figure Figure Figure Figure 3333.... Correlations between percentage of carboxylic and phenolic carbon Correlations between percentage of carboxylic and phenolic carbon Correlations between percentage of carboxylic and phenolic carbon Correlations between percentage of carboxylic and phenolic carbon 681

estimated based on carbon NEXAFS data and concentrations of the estimated based on carbon NEXAFS data and concentrations of the estimated based on carbon NEXAFS data and concentrations of the estimated based on carbon NEXAFS data and concentrations of the 682

protonationprotonationprotonationprotonation----active carboxylic and phenolic sites determined based on theactive carboxylic and phenolic sites determined based on theactive carboxylic and phenolic sites determined based on theactive carboxylic and phenolic sites determined based on the 683

results of potentiometry. results of potentiometry. results of potentiometry. results of potentiometry. 684

Figure Figure Figure Figure 4444.... Development of DOCDevelopment of DOCDevelopment of DOCDevelopment of DOC----normalized pHnormalized pHnormalized pHnormalized pH----differential absorbance spectra differential absorbance spectra differential absorbance spectra differential absorbance spectra 685

of the of the of the of the COLLCOLLCOLLCOLL ((((aaaa) and ) and ) and ) and HPOHPOHPOHPO ((((bbbb) fractions of Igarapé Bonito ) fractions of Igarapé Bonito ) fractions of Igarapé Bonito ) fractions of Igarapé Bonito DOMDOMDOMDOM. Reference pH . Reference pH . Reference pH . Reference pH 686

values values values values 3333.1..1..1..1. 687

FIGURE FIGURE FIGURE FIGURE 5555. Gaussian band fitting of the . Gaussian band fitting of the . Gaussian band fitting of the . Gaussian band fitting of the DOCDOCDOCDOC----normalized normalized normalized normalized differential spectra differential spectra differential spectra differential spectra 688

of COLL and HPO fractions of Igarapé Bonito of COLL and HPO fractions of Igarapé Bonito of COLL and HPO fractions of Igarapé Bonito of COLL and HPO fractions of Igarapé Bonito DOMDOMDOMDOM at selected pH. (a), (b) at selected pH. (a), (b) at selected pH. (a), (b) at selected pH. (a), (b) 689

and (c) for and (c) for and (c) for and (c) for COLL at COLL at COLL at COLL at pH7.pH7.pH7.pH7.1111, pH9., pH9., pH9., pH9.1111 and pH11.0and pH11.0and pH11.0and pH11.0;;;; (d), (e) and (f) for (d), (e) and (f) for (d), (e) and (f) for (d), (e) and (f) for HPOHPOHPOHPO at at at at 690

pH7.0, pH9.0 and pH11.0.pH7.0, pH9.0 and pH11.0.pH7.0, pH9.0 and pH11.0.pH7.0, pH9.0 and pH11.0. 691

Figure Figure Figure Figure 6666. Differential log. Differential log. Differential log. Differential log----transformed absorbance spectra of transformed absorbance spectra of transformed absorbance spectra of transformed absorbance spectra of COLCOLCOLCOLLLLL ((((aaaa) and ) and ) and ) and 692

HPOHPOHPOHPO ((((bbbb) fractions of Igarapé Bonito ) fractions of Igarapé Bonito ) fractions of Igarapé Bonito ) fractions of Igarapé Bonito DOMDOMDOMDOM. Reference pH values . Reference pH values . Reference pH values . Reference pH values 3333.1..1..1..1. 693

Figure Figure Figure Figure 7777. Comparison of effects of pH on the experimental . Comparison of effects of pH on the experimental . Comparison of effects of pH on the experimental . Comparison of effects of pH on the experimental differential differential differential differential loglogloglog----694

transformedtransformedtransformedtransformed spectral slope in the range of wavelengths 325 to 375 nm spectral slope in the range of wavelengths 325 to 375 nm spectral slope in the range of wavelengths 325 to 375 nm spectral slope in the range of wavelengths 325 to 375 nm 695

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(DSlope(DSlope(DSlope(DSlope325325325325----375375375375) ) ) ) and theand theand theand their NICAir NICAir NICAir NICA----based fitting for HPO and COLL fractions of based fitting for HPO and COLL fractions of based fitting for HPO and COLL fractions of based fitting for HPO and COLL fractions of 696

Igarapé Bonito Igarapé Bonito Igarapé Bonito Igarapé Bonito DOMDOMDOMDOM.... 697

Figure Figure Figure Figure 8888. Comparison of p. Comparison of p. Comparison of p. Comparison of potentiometric otentiometric otentiometric otentiometric and sand sand sand spectrophotometric resultspectrophotometric resultspectrophotometric resultspectrophotometric results (a) (a) (a) (a) 698

andandandand LAS LAS LAS LAS (b) (b) (b) (b) and HASand HASand HASand HAS (c)(c)(c)(c) inininin HPO and COLL fractions of Igarapé Bonito DOMHPO and COLL fractions of Igarapé Bonito DOMHPO and COLL fractions of Igarapé Bonito DOMHPO and COLL fractions of Igarapé Bonito DOM 699

predicted by NICApredicted by NICApredicted by NICApredicted by NICA----DonnanDonnanDonnanDonnan model model model model using the parameters shown in Table S2.using the parameters shown in Table S2.using the parameters shown in Table S2.using the parameters shown in Table S2.700

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701

Figure Figure Figure Figure 2222.... C(1s) NEXAFS spectra of COLLC(1s) NEXAFS spectra of COLLC(1s) NEXAFS spectra of COLLC(1s) NEXAFS spectra of COLL andandandand HPOHPOHPOHPO fractions of Igarapé fractions of Igarapé fractions of Igarapé fractions of Igarapé 702

Bonito DOM. Bonito DOM. Bonito DOM. Bonito DOM. 703

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704

Figure Figure Figure Figure 2222.... Results of potentiometric titration for COLLResults of potentiometric titration for COLLResults of potentiometric titration for COLLResults of potentiometric titration for COLL andandandand HPO fractions HPO fractions HPO fractions HPO fractions of of of of 705

Igarapé Bonito DOMIgarapé Bonito DOMIgarapé Bonito DOMIgarapé Bonito DOM and its modeling using NICAand its modeling using NICAand its modeling using NICAand its modeling using NICA----Donnan modelDonnan modelDonnan modelDonnan model. . . . 706

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707

Figure Figure Figure Figure 3333.... Correlations between percentage of carboxylic and phenolic carbon Correlations between percentage of carboxylic and phenolic carbon Correlations between percentage of carboxylic and phenolic carbon Correlations between percentage of carboxylic and phenolic carbon 708

estimated based on carbon NEXAFS data and concentrations of the estimated based on carbon NEXAFS data and concentrations of the estimated based on carbon NEXAFS data and concentrations of the estimated based on carbon NEXAFS data and concentrations of the 709

protonationprotonationprotonationprotonation----active cactive cactive cactive carboxylic and phenolic sites determined based on the arboxylic and phenolic sites determined based on the arboxylic and phenolic sites determined based on the arboxylic and phenolic sites determined based on the 710

results of potentiometry. results of potentiometry. results of potentiometry. results of potentiometry. 711

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712

713

Figure Figure Figure Figure 4444.... Development of DOCDevelopment of DOCDevelopment of DOCDevelopment of DOC----normalized pHnormalized pHnormalized pHnormalized pH----differential absorbance spectra differential absorbance spectra differential absorbance spectra differential absorbance spectra 714

of the of the of the of the COLLCOLLCOLLCOLL ((((aaaa) and ) and ) and ) and HPOHPOHPOHPO ((((bbbb) fractions of Igarapé Bonito ) fractions of Igarapé Bonito ) fractions of Igarapé Bonito ) fractions of Igarapé Bonito DOMDOMDOMDOM. Reference pH . Reference pH . Reference pH . Reference pH 715

values values values values 3333.1..1..1..1. 716

(a)

(b)

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717

718

719

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(b)

(c)

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720

721

722

(d)

(e)

(f)

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FIGURE FIGURE FIGURE FIGURE 5555. Gaussian band fitting of the . Gaussian band fitting of the . Gaussian band fitting of the . Gaussian band fitting of the DOCDOCDOCDOC----normalized normalized normalized normalized differential spectra differential spectra differential spectra differential spectra 723

of of of of COLL and HPO fractions of Igarapé Bonito COLL and HPO fractions of Igarapé Bonito COLL and HPO fractions of Igarapé Bonito COLL and HPO fractions of Igarapé Bonito DOMDOMDOMDOM at selected pH. (a), (b) at selected pH. (a), (b) at selected pH. (a), (b) at selected pH. (a), (b) 724

and (c) for and (c) for and (c) for and (c) for COLL at COLL at COLL at COLL at pH7.pH7.pH7.pH7.1111, pH9., pH9., pH9., pH9.1111 and pH11.0and pH11.0and pH11.0and pH11.0;;;; (d), (e) and (f) for (d), (e) and (f) for (d), (e) and (f) for (d), (e) and (f) for HPOHPOHPOHPO at at at at 725

pH7.0, pH9.0 and pH11.0.pH7.0, pH9.0 and pH11.0.pH7.0, pH9.0 and pH11.0.pH7.0, pH9.0 and pH11.0. 726

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727

728

Figure Figure Figure Figure 6666. Differential log. Differential log. Differential log. Differential log----transformed absorbance spectra of transformed absorbance spectra of transformed absorbance spectra of transformed absorbance spectra of COLLCOLLCOLLCOLL ((((aaaa) and ) and ) and ) and 729

HPOHPOHPOHPO ((((bbbb) fractions of Igarapé Bonito ) fractions of Igarapé Bonito ) fractions of Igarapé Bonito ) fractions of Igarapé Bonito DOMDOMDOMDOM. Reference pH values . Reference pH values . Reference pH values . Reference pH values 3333.1..1..1..1. 730

(a)

(b)

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731

Figure Figure Figure Figure 7777. Comparison of effects of pH on the experimental . Comparison of effects of pH on the experimental . Comparison of effects of pH on the experimental . Comparison of effects of pH on the experimental differential differential differential differential loglogloglog----732

transformedtransformedtransformedtransformed spectral slope in the range of wavelengths 325 to 375 nm spectral slope in the range of wavelengths 325 to 375 nm spectral slope in the range of wavelengths 325 to 375 nm spectral slope in the range of wavelengths 325 to 375 nm 733

(DSlope(DSlope(DSlope(DSlope325325325325----375375375375) ) ) ) and theand theand theand their NICAir NICAir NICAir NICA----based fitting for HPO and COLL fractions of based fitting for HPO and COLL fractions of based fitting for HPO and COLL fractions of based fitting for HPO and COLL fractions of 734

Igarapé Bonito Igarapé Bonito Igarapé Bonito Igarapé Bonito DOMDOMDOMDOM.... 735

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736

737

738

Figure Figure Figure Figure 8888. Comparison of p. Comparison of p. Comparison of p. Comparison of potentiometric otentiometric otentiometric otentiometric and sand sand sand spectrophotometric resultspectrophotometric resultspectrophotometric resultspectrophotometric results (a) (a) (a) (a) 739

andandandand LAS LAS LAS LAS ((((bbbb) ) ) ) and HASand HASand HASand HAS ((((cccc)))) inininin HPO and COLL fractions of Igarapé Bonito HPO and COLL fractions of Igarapé Bonito HPO and COLL fractions of Igarapé Bonito HPO and COLL fractions of Igarapé Bonito DOMDOMDOMDOM 740

predicted by NICApredicted by NICApredicted by NICApredicted by NICA----DonnanDonnanDonnanDonnan model model model model using the parameters shown in Table Susing the parameters shown in Table Susing the parameters shown in Table Susing the parameters shown in Table S2.2.2.2. 741

(b)

(a)

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Figure 1 and Figure 2

Figure Figure Figure Figure 1111.... C(1s) NEXAFS spectra of COLLC(1s) NEXAFS spectra of COLLC(1s) NEXAFS spectra of COLLC(1s) NEXAFS spectra of COLL andandandand HPOHPOHPOHPO fractions of Igarapé Bonito fractions of Igarapé Bonito fractions of Igarapé Bonito fractions of Igarapé Bonito

DOM. DOM. DOM. DOM.

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Figure Figure Figure Figure 2222.... Results of potentiometric titration for COLLResults of potentiometric titration for COLLResults of potentiometric titration for COLLResults of potentiometric titration for COLL andandandand HPO fractions of HPO fractions of HPO fractions of HPO fractions of

Igarapé Bonito DOMIgarapé Bonito DOMIgarapé Bonito DOMIgarapé Bonito DOM and its modeling using NICAand its modeling using NICAand its modeling using NICAand its modeling using NICA----Donnan modelDonnan modelDonnan modelDonnan model. . . .

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HighlightHighlightHighlightHighlightssss

Dissolved organic matter (DOM) from the Rio Negro area was isolated and

characterized

Colloidal and hydrophobic fractions were major DOM constituents.

Deprotonation of these fractions was tracked using differential spectroscopic

methods

Result of this approach is consistent with those of NEXAFS and potentiometric

methods

Changes of spectra were correlated with the charges of phenolic and

carboxylic groups

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Supporting Information Section 1

Effects of Charging on the Chromophores of Dissolved Organic Effects of Charging on the Chromophores of Dissolved Organic Effects of Charging on the Chromophores of Dissolved Organic Effects of Charging on the Chromophores of Dissolved Organic 2

Matter from the Rio Negro BasinMatter from the Rio Negro BasinMatter from the Rio Negro BasinMatter from the Rio Negro Basin 3

Mingquan Yan&, Gregory V. Korshin*, Francis Claret§, Jean-Philippe Croué##, 4

Massimiliano Fabbricino**, Hervé Gallard##, Thorsten Schäfer§§ and Marc F. 5

Benedetti# 6

&Department of Environmental Engineering, Peking University, Key Laboratory 7

of Water and Sediment Sciences, Ministry of Education, Beijing 100871, China 8

* Department of Civil and Environmental Engineering, University of 9

Washington, Seattle, WA 98195-2700 United States 10

**Dipartimento di Ingegneria Idraulica ed Ambientale "Girolamo Ippolito", 11

Universitá degli Studi di Napoli Federico II Via Claudio 21, 80125 Naples Italy 12

# Institut de Physique du Globe de Paris – Sorbonne Paris Cité - Université 13

Paris-Diderot , UMR CNRS 7154, Paris, France 14

## Equipe Chimie de l'Eau et Traitement des Eaux 15

Institut de Chimie des Milieux et Matériaux de Poitiers UMR 7285 CNRS 16

Ecole Nationale Supérieure d'Ingénieurs de Poitiers – Université de Poitiers 17

86022 Poitiers Cedex France 18

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§ Bureau des Recherches Géologiques et Minières, Environment and Process 19

Division 3, avenue Claude Guillemin F-45060 Orleans Cedex 2 France 20

§§ Forschungszentrum Karlsruhe, Institut für Nukleare Entsorgung (INE) P.O. 21

Box 3640 76021 Karlsruhe, Germany 22

23

& Corresponding author. Address: Department of Environmental Engineering, 24

College of Environmental Sciences and Engineering, Peking University, 25

Beijing 100871, China; Tel: +86 10 62755914-81, Fax: +86 10 62756526. 26

E-mail: yanmq@pku.edu.cn 27

Number of Pages (including this cover sheet): 10 28

Number of Tables 3 29

Number of Figures 4 30

31

32

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Table STable STable STable S1111.... Results of deconvolution of NEXAFS data for COLL, HPO and TPH Results of deconvolution of NEXAFS data for COLL, HPO and TPH Results of deconvolution of NEXAFS data for COLL, HPO and TPH Results of deconvolution of NEXAFS data for COLL, HPO and TPH 33

fractions of fractions of fractions of fractions of Igarapé BonitoIgarapé BonitoIgarapé BonitoIgarapé Bonito DOM. DOM. DOM. DOM. 34

Aromatic

285.2 eV

Phenolic

286.2eV

Aliphatic

287.4 eV

Carboxyl

288.5 eV

Carbonyl

289.5 eV

Aromaticity

(ΣAromatic+Phenolic)

Deconvolution

R2

COLL 17 17 15 30 21 33 0.9932

HPO 13 13 12 38 24 26 0.9931

TPH 11 11 9 42 27 22 0.9934

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36

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Table STable STable STable S2222.... Comparison of potentiometric and Comparison of potentiometric and Comparison of potentiometric and Comparison of potentiometric and sssspectrophotometric pectrophotometric pectrophotometric pectrophotometric 37

NICANICANICANICA----Donnan parameters for protonationDonnan parameters for protonationDonnan parameters for protonationDonnan parameters for protonation----active groups in Igarapé Bonito active groups in Igarapé Bonito active groups in Igarapé Bonito active groups in Igarapé Bonito 38

DOM.DOM.DOM.DOM. 39

COLLCOLLCOLLCOLL HPOHPOHPOHPO TPHTPHTPHTPH

Potentiometric dataPotentiometric dataPotentiometric dataPotentiometric data

~

KLAS

4.43 4.43 4.43

~

KHAS

8.10 8.10 8.10

QLAS, meq/g 1.70 2.45 2.64

QHAS, meq/g 2.64 1.96 1.80

mLAS 0.79 0.75 0.81

mHAS 0.28 0.29 0.27

SSSSpectrophotometric datapectrophotometric datapectrophotometric datapectrophotometric data

~

KLAS

3.74 3.75 n/a

~

KHAS

9.13 9.05 n/a

DSlopeLAS (325-375 nm) 0.0031 0.0027 n/a

DSlopeHAS (325-375 nm) 0.0035 0.0025 n/a

mLAS 0.63 0.56 n/a

mHAS 0.49 0.47 n/a

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Table STable STable STable S3333.... Comparison of properties of the Gaussian bands present in the ClComparison of properties of the Gaussian bands present in the ClComparison of properties of the Gaussian bands present in the ClComparison of properties of the Gaussian bands present in the Cl---- 40

and pHand pHand pHand pH----differential spectra of SRFAdifferential spectra of SRFAdifferential spectra of SRFAdifferential spectra of SRFA 41

Properties of Gaussian bands in pH-differential spectra for COLL

Component

Position of maximum

Ei, eV

Position of maximum

λi, nm

Standard deviation of Ei, eV

Bandwidth W i,

eV

Standard deviation of W i, eV

Fit paramete

r (R2)

A0 5.99 207 0.094 0.45 0.079

0.99

A1 5.01 247 0.063 0.22 0.061

A2 4.47 278 0.025 0.20 0.013

A3 3.99 311 0.033 0.24 0.041

A4 3.28 378 0.044 0.37 0.027

A5 2.33 533 0.048 0.32 0.049

Properties of Gaussian bands in pH-differential spectra for HPO

Component

Position of maximum

Ei, eV

Position of maximum

λi, nm

Standard deviation of Ei, eV

Bandwidth W i,

eV

Standard deviation of W i, eV

Fit paramete

r (R2)

A0 6.00 207 0.150 0.36 0.080

0.99

A1 5.11 243 0.051 0.26 0.042

A2 4.43 280 0.011 0.17 0.019

A3 3.95 314 0.030 0.26 0.050

A4 3.23 383 0.058 0.36 0.022

A5 2.34 529 0.040 0.24 0.046

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Figure Figure Figure Figure S1.S1.S1.S1. C(1s) NEXAFS spectra of fractions of Igarapé Bonito DOM. C(1s) NEXAFS spectra of fractions of Igarapé Bonito DOM. C(1s) NEXAFS spectra of fractions of Igarapé Bonito DOM. C(1s) NEXAFS spectra of fractions of Igarapé Bonito DOM. 49

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Figure SFigure SFigure SFigure S2222.... Absorbance spectra of theAbsorbance spectra of theAbsorbance spectra of theAbsorbance spectra of the COLL (a) andCOLL (a) andCOLL (a) andCOLL (a) and HPOHPOHPOHPO (b)(b)(b)(b) fraction of fraction of fraction of fraction of 53

Igarapé BonitoIgarapé BonitoIgarapé BonitoIgarapé Bonito DOM at varying pHs. DOC concentration 5 mgDOM at varying pHs. DOC concentration 5 mgDOM at varying pHs. DOC concentration 5 mgDOM at varying pHs. DOC concentration 5 mg LLLL----1111, cell length 5 , cell length 5 , cell length 5 , cell length 5 54

cm.cm.cm.cm. 55

(b)

(a)

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58 59

Figure Figure Figure Figure SSSS3333.... Comparison of rangeComparison of rangeComparison of rangeComparison of range----specific differential absorbance spectra of specific differential absorbance spectra of specific differential absorbance spectra of specific differential absorbance spectra of COLL and COLL and COLL and COLL and 60

HPOHPOHPOHPO fractions of Igarapé Bonito DOM. (fractions of Igarapé Bonito DOM. (fractions of Igarapé Bonito DOM. (fractions of Igarapé Bonito DOM. (aaaa) carboxylic range (change of pH from 3.5 to ) carboxylic range (change of pH from 3.5 to ) carboxylic range (change of pH from 3.5 to ) carboxylic range (change of pH from 3.5 to 61

4.5); (4.5); (4.5); (4.5); (bbbb) phenolic range, (change of pH from 8.3 to 9.0).) phenolic range, (change of pH from 8.3 to 9.0).) phenolic range, (change of pH from 8.3 to 9.0).) phenolic range, (change of pH from 8.3 to 9.0). 62

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65

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66 FFFFIGUREIGUREIGUREIGURE SSSS4444. . . . Comparison of Comparison of Comparison of Comparison of the the the the intensities of intensities of intensities of intensities of Gaussian bandGaussian bandGaussian bandGaussian bands s s s in the differential spectra in the differential spectra in the differential spectra in the differential spectra 67

as as as as a a a a function of pH function of pH function of pH function of pH for for for for COLL and HPOCOLL and HPOCOLL and HPOCOLL and HPO fractions of Igarapé Bonito DOMfractions of Igarapé Bonito DOMfractions of Igarapé Bonito DOMfractions of Igarapé Bonito DOM.... A1(a);A1(a);A1(a);A1(a); A2(b); A2(b); A2(b); A2(b); 68

A3(c); A4(d).A3(c); A4(d).A3(c); A4(d).A3(c); A4(d).DOC DOC DOC DOC concentration 5.0 mg Lconcentration 5.0 mg Lconcentration 5.0 mg Lconcentration 5.0 mg L----1111.... 69

(d)