1

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PHOTOCHEMICAL FLOCCULATION OF TERRESTRIAL DISSOLVED ORGANIC 2

MATTER AND IRON 3

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John R. Helmsa,b*, Jingdong Maoa, Klaus Schmidt-Rohrc, Hussain Abdullaa, and Kenneth Moppera,* 5

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a. Old Dominion University, Department of Chemistry and Biochemistry, 4541 Hampton Blvd., 7

Norfolk, VA, 23529 8

b. Current address: University of North Carolina Wilmington, Department of Chemistry and 9

Biochemistry, 601 S. College Rd., Wilmington, NC 28403 10

c. Iowa State University, Department of Chemistry, Gilman Hall, Ames, Iowa, 50011 11

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*Corresponding Authors: email: helmsj@uncw.edu; phone: 757-581-8003; fax: 910-962-3013. 13

email: kmopper@odu.edu; phone: 757-683-4094; fax: 757-683-5310 14

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Running Head: Photochemical Flocculation of Organic Matter and Iron 16

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ABSTRACT: Dissolved organic matter (DOM) rich water samples (Great Dismal Swamp, Virginia) 18 were 0.1-um filtered and UV-irradiated in a solar simulator for 30 days. During the irradiation, pH 19 increased, particulate organic matter (POM) and particulate iron formed. After 30 days, 7.1% of the 20 dissolved organic carbon (DOC) was converted to POC while 75% was remineralized. Approximately 21 87% of the iron was removed from the dissolved phase after 30 days, but iron did not flocculate until a 22 major fraction of DOM was removed by photochemical degradation and flocculation (>10 days); thus, 23 during the initial 10 days, there were sufficient organic ligands present or the pH was low enough to 24 keep iron in solution. Nuclear magnetic resonance and Fourier transform infrared spectroscopies 25 indicated that photochemically flocculated POM was more aliphatic than the residual non-flocculated 26 DOM. Photochemically flocculated POM was also enriched in amide functionality, while carbohydrate-27 like material was resistant to both photochemical degradation and flocculation. Abiotic photochemical 28 flocculation likely removes a significant fraction of terrestrial DOM from the upper water column 29 between headwaters and the ocean ocean, but has been previously ignored. Preliminary evidence 30 suggests that this process may significantly impact the transport of DOM and POM in ocean margin 31 environments including estuaries. 32

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ABBREVIATIONS 33 34 A- absorbance; optical density 35 a- absorption coefficent 36 a300- absorption coefficient at 300 nm 37 Ads- adsorbed material 38 Arom.- aromatic 39 CDOM- chromophoric (or colored) dissolved organic matter 40 cm- centimeters; 10-2 meters 41 CP- cross polarization 42 CSA- chemical shift anisotropy 43 DIC- dissolved inorganic carbon 44 Diss- dissolved (supernatant fraction of centrifuged samples or the fraction <0.1 µm) 45 DOC- dissolved organic matter 46 DOM- dissolved organic matter 47 DON- dissolved organic nitrogen 48 FTIR- Fourier transform infrared spectroscopy 49 hν- light or photon (shorthand i.e., Planks constant x frequency) 50 l- path length 51 λ- wavelength 52 ln- natural logarithm 53 Me- metal 54 MilliQ- ultrapure deionized water from MilliQ-UV dispenser (Millipore) 55 N/D- not determined 56 NMR- nuclear magnetic resonance spectroscopy 57 PM- particulate material 58 POM- particulate organic matter 59 ppm- chemical shift frequency in parts per milllion (relative to tetramethyl silane standard in delta 60 notation) 61 r2- square of the correlation coefficient 62 S- spectral slope (subscript denotes wavelength interval) 63 SR- spectral slope ratio 64 SUVA254- specific UV absorption at 254 nm 65 tDOM- terrestrial dissolved organic matter 66 TN- total nitrogen 67 TOC- total organic carbon 68 TOSS- total supression of spining sidebands 69 TPPM- two-pulse phase modulation 70

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1. INTRODUCTION 72

Terrestrial dissolved organic matter (tDOM) appears to be efficiently removed in ocean margin 73

environments including coastal zones, estuaries, and buoyant river plumes; yet the physical, chemical, 74

and biological processes that lead to this removal remain poorly understood (Hedges et al., 1997; Del 75

Vecchio and Subramaniam, 2004; Spencer et al., 2009). It has been proposed that significant amounts of 76

riverine DOM are delivered to estuarine, continental shelf, and continental slope sediments by various 77

processes, including: precipitation, co-precipitation, aggregation, coagulation, micelle formation, 78

hydrophobic interactions, adsorption to or encapsulation into pre-existing particles (Stumm and Morgan, 79

1981; Guo et al., 2007). Here, all physico-chemical processes that convert dissolved (<0.1 µm) material 80

to particulate material are referred to as flocculation. Prior studies investigating the role of abiotic 81

flocculation in estuarine biogeochemistry have focused on the influences of salt, mixing, particle 82

interactions, and pH on the solubility, stability, or removal of dissolved and colloidal riverine organic 83

matter (Sholkovitz, 1976; Turner and Rawling, 2001), humic substances (Eckert and Sholkovitz, 1976; 84

Fox, 1983), carbohydrates (Wang et al., 2010), metals (O'melia and Stumm, 1967; Boyle et al., 1977; 85

Sholkovitz, 1978), phosphorus (Forsgren et al., 1996), clay minerals (Black et al., 1965; O'melia and 86

Stumm, 1967), and algal biomass (Sukenik et al., 1988). In spite of the large number of previous 87

studies, the factors resulting in flocculation remain poorly understood. 88

Sondergaard et al. (2003) showed that mixing-induced flocculation caused only a 2-5% decrease 89

in DOC along a salinity gradient of 0-25. In another study based on end-member mixing experiments 90

and estuarine transects, mixing induced flocculation was insufficient to account for the apparent 91

removal (and non-conservative behavior) of humic acids (Fox, 1983). Guo et al. (2007) showed that salt 92

effects alone were not sufficient to significantly alter the chromophoric DOM (CDOM) composition of 93

a river water sample. Flocculation therefore must be accompanied by additional factors, such as 94

adsorption to mineral surfaces, interactions with pre-existing particles, or other uncharacterized 95

processes. 96

In addition to salt effects and mixing-induced flocculation, surface-active materials that collect 97

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on air bubbles may form particles from DOM upon bubble dissolution (Johnson, 1976) or bursting 98

(Baylor and Sutcliffe, 1963). Surface-active compounds, including polysaccharides, 99

mucopolysaccharides and glycoproteins, may also play a significant role in the formation of organic-100

enriched surface micro-layers (Henrichs and Williams, 1985; Compiano et al., 1993), marine snow 101

(Alldredge and Silver, 1988; Mopper et al., 1995), or mineral/organic aggregates (Kranck and Milligan, 102

1980). 103

Photochemical mineralization of DOM (conversion to inorganic forms) and the decrease in 104

ultraviolet and visible absorption, i.e., photobleaching, have been widely studied (Zepp and 105

Schlotzhauer, 1981; Del Vecchio and Blough, 2002). Photochemical mineralization is a possible 106

removal mechanism for humic acids and colored DOM in estuaries (Stedmon and Markager, 2005; 107

White et al., 2010). However, reports of a link between photochemical alteration of DOM and the 108

flocculation of DOM have received relatively little attention. Thus, little is known about the 109

relationship between these two processes and how photo-induced flocculation may impact processes 110

such as transport and removal of dissolved and particulate materials and pollutants, biological utilization 111

of organic matter and the organic content of coastal sediments. 112

Gao and Zepp (1998) observed that after three days of simulated sunlight exposure, dark-colored 113

particles formed in a 0.22 µm filtered and oxygen saturated Satilla river water sample. They reported 114

that particulate matter accounted for 45% of the iron and about 13% of the organic carbon and 115

speculated that the causes of photochemically induced aggregation are changes to double layer repulsion 116

and van der Waals interactions, from decarboxylation, and/or the loss of DOM’s ability to complex iron 117

leading to formation of polymeric iron oxides. Maurice et al. (2002) and Porcal et al. (2013) observed 118

asimilar phase changes for organic carbon and iron in wetland influenced streams. Von Wachenfeldt et 119

al. (2008) found that light exposure correlated with increases in particulate organic carbon (POC) at the 120

expense of DOC for 5 µm filtered humic lake and mire water samples. Thorn et al. (2010) found that 121

humic and fulvic isolates became decarboxylated, more hydrophobic, much less aromatic, and less 122

soluble after extensive photodegradation. Photochemical flocculation of iron and aluminum has also 123

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been observed in lakes (Kopáček et al., 2005; Kopáček et al., 2006). The mechanism proposed by 124

Kopáček et al. (2005) proceeds by the iron mediated degradation of organic iron-binding ligands 125

causing release of inorganic iron, which is susceptible to oxidation and/or hydrolysis to form insoluble 126

(hydr)oxides. Dissolved iron in terrestrially dominated waters (and in the open ocean) is dominantly 127

organically complexed (Rue and Bruland, 1995; Powell and Wilson-Finelli, 2003; Laglara and van den 128

Berg, 2009). Additionally, the concentration of carboxyl groups affects the solubility of DOM 129

(VanLoon and Duffy, 2010) and DOM’s ability to form stable complexes with iron (Barbeau, 2006). It 130

is thus anticipated that photochemical decarboxylation of DOM affects solubility of both DOM and 131

iron. Dissolved O2 and photochemically produced reactive oxygen species oxidize free iron (II) to iron 132

(III), leading to the formation of insoluble iron hydroxides (Kuma et al., 1996; Faust and Zepp, 1993) 133

which may prove important when iron binding ligands are actively photodegraded. Iron also participates 134

directly in the degradation of photo-active organic complexes via ligand-metal charge transfer reactions 135

(Faust and Zepp, 1993). 136

The residence time of tDOM within the euphotic zone of the oceans is poorly constrained. Most 137

of the world’s major rivers form large buoyant plumes often becoming entrained in highly stratified 138

surface waters between 30º S and 30º N latitudes (Moore et al., 1986; Dagg et al., 2004). This highlights 139

the importance of photochemical processes because tDOM-containing water masses will receive 140

considerable light exposure upon export to the ocean margins, thus resulting in photodegradation of 141

tDOM and possible photo-flocculation. 142

The present study examines the photochemical changes to DOM composition that result in 143

abiotic flocculation of organic matter in the absence of salt effects, biological interactions, or 144

interactions with pre-existing particles. Conventional and advanced spectroscopic methods were used to 145

characterize both the POM that forms upon light exposure and the DOM that remains in solution in 146

order to gain insights into the flocculation mechanism(s). Our results show that photochemically 147

flocculated particles account for as much as 60% of TOC and CDOM, 25% of total nitrogen, and 87% 148

of total iron remaining after long-term irradiation. POM that forms after 10 days is enriched in aliphatic 149

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and amide functional groups relative to the remaining DOM, while carbohydrate-like moieties are 150

resistant to both photo-mineralization and photo-flocculation. This study raises new questions regarding 151

both the mechanisms and magnitude with which photochemistry and flocculation affect the export of 152

terrestrial DOM and POM to the oceans and sediments. 153

154

2. MATERIALS AND METHODS 155

2.1 Sample collection 156

Glassware was cleaned with dilute HCl (~1 mol L-1) and rinsed with MilliQ –UV ultrapure grade 157

water (Millipore), referred to below as MilliQ water. Glassware was combusted at 450 ºC (>8 h). 158

Plasticware was cleaned with 0.01 mol L-1 NaOH followed by MilliQ water and dilute HCl and then 159

rinsed with MilliQ water. All containers were rinsed several times with sample prior to filling. Filter 160

capsules (0.1 µm pore size, Whatman PolyCap TF) were rinsed with HPLC grade methanol (Alpha 161

Aesar), flushed with > 20 L of MilliQ water, and conditioned with approximately 1 L of sample prior to 162

use. 163

The water sample used for this irradiation experiment was collected at 76°22' W, 36°36' N from 164

the public boat ramp near the confluence of the Dismal Swamp northwest canal and the Lake 165

Drummond feeder ditch. The waters of the Dismal Swamp are DOC rich and low pH (Minor et al., 166

2006). The water was transported back to the laboratory in a 20 L polyethylene jerrycan. The sample 167

was filtered (within 24 hours of collection) through a pre-cleaned 0.1 µm capsule filter into precleaned 168

and combusted 4 L glass Qorpak bottles with acid rinsed Teflon lined caps. Samples were stored (<48 169

h) at room temperature prior to irradiation. Subsamples were stored at 4-5° C prior to analysis; however, 170

samples were stored at room temperature overnight prior to optical spectroscopic measurements to 171

minimize temperature related artifacts. 172

2.2 Irradiation Experiment 173

Aliquots (450 mL) of the filtered sample were transferred to 550 mL quartz round bottom flasks 174

and placed inside a solar simulator containing UVA340 bulbs (Q-panel). These lamps have a spectral 175

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output similar to natural sunlight from ~290 to ~365 nm, but under-represent solar irradiance at 176

wavelengths greater than about 365 nm. UVA lamps were chosen because this region of the solar 177

spectrum is quantitatively the most important for the mineralization of dissolved organic carbon at the 178

surface (Vähätalo et al., 2000), though given the greater depth to which longer wavelengths penetrate 179

the water column (Fichot and Miller, 2010), our results do not reflect the degree to which photochemical 180

degradation occurs at depth. The solar simulator is described elsewhere (Minor et al., 2007). The lamps 181

were kept on constantly during the irradiation. Dark controls were wrapped in foil and placed inside the 182

solar simulator. All samples were irradiated at their natural concentration and pH (i.e., not diluted to 183

optically thin conditions or buffered prior to irradiation) in order to collect sufficient organic material 184

for solid-state NMR and FTIR, to approximately simulate surface conditions in humic lakes during 185

seasonal stratification, and to avoid potential contamination and artifactual photochemical and dark 186

reactions. At the end of each irradiation period (10, 20, and 30 days), three irradiated flasks and one 187

dark control flask, were sacrificed for analysis (two dark control flasks were analyzed after 30 days). An 188

additional ~2 L aliquot was stored in a combusted glass qorpak bottle in the dark at 4 ºC. It was sub-189

sampled and analyzed using the same methodology as for the irradiated samples and dark controls. UV-190

visible absorption of the unfractionated refrigerated/dark sample was measured at 0, 10, and 30 days, 191

while the centrifuged refrigerated/dark sample was analyzed at 10 and 30 days. All other analyses of the 192

refrigerated/dark sample were performed at the same time as the 30-day irradiated and dark control 193

samples. 194

A 30 mL aliquot from each flask was set aside for measuring pH, which was measured at room 195

temperature (22º C ±2º) using an Orion ROSS gel filled pH electrode connected to a Fisher Scientific 196

Accumet pH meter 25. The pH meter and electrode were calibrated using NIST traceable buffers of pH 197

= 4, 7, and 10 (EMD Millipore). Measurements were repeated until replicate analyses agreed to within 198

0.03 pH units. 199

During the course of the irradiation, particles formed in all samples except for the dark controls. 200

The particles were separated from the remaining supernatant by centrifugation at 6000 rpm (~9000 g) 201

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for 15 minutes. For comparison, 15 µL of 50% w/w NaOH (VWR Scientific) was added to one 450 mL 202

flask of un-centrifuged irradiated samples in order to re-dissolve the flocculated and adsorbed DOM. In 203

addition, a sub-sample with particles re-suspended was collected. After emptying the irradiation and 204

control flasks, they were rinsed with MilliQ water to remove remaining solution. One flask was then 205

rinsed with 10 mL of 0.5 mmol L-1 NaOH solution to dissolve wall-adsorbed organic material, while a 206

second flask was rinsed with 0.1 mol L-1 HCl solution to leach adsorbed iron. Adsorbed DOM was 207

collected from the 10-day and 30-day dark control flasks, while adsorbed iron was collected from the 208

20-day and 30-day dark control flasks. As there was insufficient solid material obtained from a single 209

flask to carry out solid state NMR and FTIR analysis, a second 10-day irradiation was performed using 210

5 flasks. The samples from that second 10-day irradiation were centrifuged, as above. The particles were 211

rinsed once with cold (~4ºC) MilliQ water to remove loosely bound DOM. Both dissolved and 212

particulate phases were then freeze-dried. 213

2.3 UV-visible absorption spectroscopy 214

UV-visible absorption spectra (190-900 nm) were measured for all samples and rinses using an 215

Agilent 8453 diode array spectrophotometer with a 1 cm quartz cuvette. MilliQ water was used as the 216

blank. Strongly absorbing samples were diluted with MilliQ water prior to measurement to ensure linear 217

response. Absorbance values were corrected to account for instrument baseline drift, refractive index, 218

and temperature variations according to Green and Blough (1994) and converted to Napierian 219

absorbance coefficients using: 220

(1) 221

where a = absorbance coefficient (m-1), A = absorbance, and l = path length (m). All CDOM UV-visible 222

spectra were assumed to follow an exponential decay approximated by the expression: 223

(2) aλ = aλref e-S(λ-λref) 224

where λ = wavelength (nm), λref = reference wavelength, and S = spectral slope. Spectral slopes were 225

calculated by linear regression of the natural log transformed absorption spectra, and spectral slope ratio 226

!

a = 2.303A / l

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(SR) was calculated as the ratio of S275-295 to S350-400 (Helms et al., 2008). All samples (dissolved i.e., 227

supernatant centrifuged samples, re-dissolved samples, blanks, and dark controls) were analyzed at 228

similar NaOH concentrations (0.5-0.6 mmol L-1; pH=10.7 ±0.2) to avoid pH and refractive index 229

differences between samples and allow direct comparison. 230

First derivative UV-visible absorption spectra were obtained by fitting absorption data to linear 231

segments over 11 nm intervals and plotting slope vs. wavelength. The process was repeated using first 232

derivative data points in order to produce second derivative spectra. 233

2.4 Dissolved organic carbon 234

Non-purgeable organic carbon was measured using high temperature (720°C) catalytic 235

combustion on a Shimadzu TOC-V-CPH carbon analyzer. Samples were acidified with H3PO4 and 236

sparged with carbon-free air prior to injection. The instrument was calibrated using dried primary 237

standard grade potassium hydrogen phthalate (Alpha Aesar) dissolved in MilliQ water. Measurements 238

of non-purgeable organic carbon for dissolved samples (i.e., centrifuged supernatant) will be referred to 239

as DOC, while measurements of non-fractionated samples will be referred to as total organic carbon 240

(TOC). POC was determined as the difference between TOC and DOC. Total Nitrogen (TN) was also 241

measured using the Shimadzu TOC-V-CPH. Calibration standards were prepared from KNO3 in MilliQ 242

water. 243

2.5 Total iron by atomic absorption spectroscopy 244

Total iron concentration was measured using a Hitachi Z8100 polarized Zeeman flame atomic 245

absorption spectrophotometer equipped with an iron hollow cathode lamp (VWR Scientific). Iron 246

standards were prepared from puris p.a., ACS grade, iron (II) sulfate heptahydrate (Fluka) in 0.01M HCl 247

and ranged in concentration from 0 to 100 µmol L-1. Standards were measured in duplicate and samples 248

were measured in triplicate. Samples were adjusted to pH= 1 using ACS grade concentrated 249

hydrochloric acid (Fisher Scientific). Particulate iron concentrations were determined by comparing the 250

iron concentrations of non-centrifuged (total) and dissolved (centrifuged supernatant) samples. 251

2.6 Solid-state nuclear magnetic resonance spectroscopy 252

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Freeze-dried DOM and POM samples from the second 10-day irradiation were analyzed by 253

solid-state 13C cross polarization/total sideband suppression (CP-TOSS) NMR at 100 MHz with a 254

Bruker Biospin DSX-400 spectrometer. Experiments were performed using 4-mm rotors at a spinning 255

speed of 6.5 kHz and a cross polarization (CP) time of 1 ms, with a 1H 90o pulse-length of 4.1 µs and a 256

recycle delay of 0.4 s. Four-pulse TOSS (Dixon, 1982) was applied prior to detection, and two-pulse 257

phase-modulated (TPPM) decoupling was used for optimum resolution (Deazevedo et al., 2000). 258

Between 16k and 64k scans were accumulated for each spectrum. 259

In addition to CP-TOSS experiments, spectra showing signals from nonprotonated carbons and 260

mobile groups, like rotating CH3, were generated using 13C CP-TOSS with 40 µs dipolar dephasing 261

(Opella and Frey, 1979; Hatcher, 1987; Mao et al., 2007). Between 32k and 64k scans were averaged 262

for each spectrum with dipolar dephasing. To differentiate the signals of anomeric carbons (O-C-O) 263

from those of aromatic carbons that resonate between 90 and 120 ppm, the aromatic carbon signals were 264

selectively suppressed with a five-pulse 13C chemical-shift anisotropy (CSA) filter (Mao, 2004). 265

Between 32k and 64k scans were performed for each CSA-filtered spectrum. 266

2.7 Fourier transform infrared spectroscopy 267

FTIR spectra were obtained by collecting 200 scans using a Nicolet 370 FTIR spectrometer 268

(DTGS detector) equipped with N2 purge gas generator (Thermo Scientific). Samples were freeze-dried 269

and introduced as KBr disks. The KBr was combusted (24 h, 450 °C) prior to use. The sample and KBr 270

mixtures (1:100 ratio) were homogenized in a glass vial on a grinding mill (Wig-L-Bug) and 271

compressed between two clean, polished iron anvils in a hydraulic press at 20,000 psi to form KBr 272

windows. Spectra were collected with a resolution of 4 cm-1 ±1 and Happ-Genzel apodization. 273

Background scans consisted of a KBr disk prepared as above, except that no sample was added. Second 274

derivative spectra were obtained using the second order Savitzky-Golay method as described in Abdulla 275

et al. (2010). The 1900-1500 cm-1 (carboxyl) region of the spectrum was deconvoluted using Voigt line-276

shape fitting (Griffiths and De Haseth, 2007; Abdulla et al., 2010). Details of the identification and 277

calibration of the deconvoluted peaks are given in Abdulla et al. (2010). 278

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2.8 Statistical analyses 279

Analytical uncertainty was estimated for UV-visible, DOC, total iron in terms of reproducibility 280

of replicate analyses. Unless otherwise noted, for irradiated samples, three replicate analyses from two 281

flasks (n = 6) were performed and reported below as mean values and one standard deviation. Dark 282

controls were measured at each time point (n=3 from a single flask). Total iron, TOC, and a300 for the 283

unfractionated dark controls were found to be statistically indistinguishable from an unirradiated sample 284

stored at 4 ºC using Student’s t-test (p > 0.1, i.e., a high probability that the null hypothesis pertained 285

and the difference between the dark control and the refrigerated/dark sample was not significant). The 286

values measured for the unfractionated dark controls and refrigerated/dark sample were pooled and Q-287

tested at the 95% confidence level, and the overall mean and standard deviations (n ≥ 10 after removal 288

of outlying values) are reported below as “0-d Total”. Adsorbed material is reported for triplicate 289

analyses from a single flask per time point (n = 3) except in the case of “0-d Ads” which is reported as 290

the overall mean of two dark controls (n=6; 10-day and 30-day dark controls for dissolved iron and 20-291

day and 30-day dark controls for TOC and CDOM). 292

Freeze-dried samples represent combined and homogenized aqueous, particulate, or total (un-293

fractionated) material from multiple flasks (n ≥ 2). The analytical uncertainty of the NMR spectra due to 294

instrumental and environmental noise was estimated by using the signal to noise ratio of each 295

integration region to calculate relative standard deviation. Additional uncertainty arose from the 296

assignment of chemical shift ranges used for integration. The latter was partly alleviated through the 297

consistent application of integration ranges to maintain comparability of the samples and by the use of 298

dipolar dephased or CSA-filtered spectra to differentiate overlapping signals. The analytical uncertainty 299

of the FTIR method was estimated as the standard deviation of individually deconvoluted and integrated 300

triplicate scans of the same sample. For results calculated based on multiple independent methods or by 301

difference from a single method, uncertainties were propagated as one standard deviation according to 302

Harris (2010). 303

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3. RESULTS 305

3.1 pH shift during irradiation 306

As no buffers were added to the water samples prior to or during the irradiation, the pH shifted 307

over the course of the 30 day experiment from ~3.7 to ~5.7 (Fig. 1). The pH of dark controls did not 308

change. While the exclusion of added buffers was necessary to obtain sufficient carbon for solid-state 309

NMR and to avoid artifactual reactions, the gradual increase in pH complicates the interpretation of the 310

iron flocculation mechanism (see 4. Discussion). 311

3.2 Changes in organic matter parameters during irradiation 312

A summary of the parameters measured for each of the samples is given in Table 1. Comparison 313

of these parameters shows that, throughout the course of the irradiation, photo-mineralization and 314

photobleaching caused reductions in both organic matter concentration and absorption at 300 nm 315

(a300). Approximately 41% of the original 52.7 mg L-1 DOC was mineralized during the first 10 days 316

of the irradiation experiment and an additional 9% of initial DOC was converted to POC. The organic 317

carbon remaining after 10 days was ~15% particulate (Table 1; Figs 2a and 3a). After 30 days, 95% of 318

the original a300 and 88% of the original DOC were removed (Table 1). Adsorption to the walls of the 319

quartz flasks was responsible for 2.1% of a300 removal and about 6.5% of DOC removal. Flocculation 320

was responsible for 8.3% of a300 removal and 8.0% of DOC removal. After 30 days irradiation, 38% of 321

the remaining TOC was in the particulate phase, and the particulate phase accounted for ~60% of the 322

remaining absorption at 300 nm (Figs. 2a and 3a). The amount of particulate a300 present after 20 days 323

was 29% lower than it was after 10 days, while the concentration of POC was 36% greater indicating 324

photobleaching of the particles. 325

While up to 15% of the total remaining a300 adsorbed to the walls of the irradiated quartz flasks 326

(Table 1; Fig. 2b), very little wall adsorption (≤0.11% of a300) occurred in the dark control. Wall-327

adsorbed material also exhibited relatively low spectral slope ratios (SR) (Table 1). Likewise, for the 10-328

day irradiated sample, the spectral slope ratio for the isolated re-dissolved particles was low, 0.72 329

(±0.03; Table 1), compared to SR values of 0.91 (±0.02) for the dark control samples, and 1.15 (±0.02) 330

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for the dissolved material. In fact, all samples containing re-dissolved POM exhibited lower SR values 331

than their particle free counterparts. 332

While the absorption spectra all appear relatively featureless, the derivative spectra show that 333

considerable changes in spectral shape occurred over the course of photobleaching (Figs. 4 and 5). 334

Dissolved and particulate materials exhibit significantly different derivative spectra. Second derivative 335

spectra of dissolved material show minima between 262 and 271 nm and maxima between 285 and 295 336

nm, while particulate spectra show minima between 273 and 290 nm and maxima between 296 and 315 337

nm, which shifted during the irradiation (Figs. 4c, 4f, 4i, 5c, 5f, and 5i). These two trends are consistent 338

with the findings of Helms et al. (2008). Re-dissolution of isolated particles from the 10-d sample and 339

dilution with MilliQ water (data not shown) confirm the low spectral slopes exhibited in the difference 340

spectra (Figs. 4g and 5g), however, the features observed at ~280 nm and ~340 nm in the difference 341

spectrum were less pronounced. 342

DOC normalized absorption or “specific UV absorbance” at 254 nm (SUVA254), which has been 343

shown to correlate well with aromatic content (Weishaar et al., 2003) and carbon normalized lignin 344

yield (Stubbins et al., 2012), are shown in Table 1. SUVA254 values decreased throughout the irradiation 345

indicating photochemical degradation of aromatic material, in agreement with the NMR results 346

(discussed below). The SUVA254 values were generally higher in supernatants than in the unfractionated 347

samples. 348

Total nitrogen in both the dissolved and unfractionated samples decreased during irradiation 349

(Table 1), while particulate nitrogen increased (Table 1; Fig. 2d). The distribution of dissolved and 350

particulate nitrogen is shown in Figs. 2d and 3d. Total nitrogen includes both organic and inorganic 351

forms of nitrogen. Because inorganic forms (e.g., NH4+, NO3

-, NO2-) are highly soluble, particulate 352

nitrogen, determined by subtracting TN values of dissolved samples from TN values of unfractionated 353

samples, must dominantly exist as organic nitrogen. The carbon to nitrogen ratio (C:N) of the DOM 354

decreased linearly from 28 to 6.3 during the irradiation. Particulate C:N was 17 after 10-d, 18 after 20-d 355

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and 11 after 30-d. The decrease in particulate C:N ratio after 30 days reflects photobleaching of the 356

flocculated material (i.e., preferential loss of carbon; Figs. 2 and 3). 357

3.3 Flocculation of iron during irradiation 358

No detectable flocculation of iron occurred during the first 10 days of irradiation. However, 359

between 10 and 20 days, iron began to flocculate (Figs. 2c and 3c, Table 1) and after 30 days, 87% of 360

the iron was converted to particulate iron. No measurable particle formation occurred in the dark 361

control, and adsorption of iron to the flask wall was negligible (<0.1%). 362

3.4 Spectroscopic characterization of photodegraded DOM and photochemically flocculated POM 363

Solid-state CP-TOSS NMR spectra for the dark control, 10-day irradiated whole sample, 364

supernatant, and particles are shown in Fig. 6 (heavy lines). Photodegradation led to a preferential 365

decrease in the aromatic signal (90-160 ppm), in agreement with the SUVA data (Table 1), a broadening 366

of the carboxyl peak (160-190 ppm) and selective preservation of both O-alkyl (carbohydrate-like) 367

material (60-90 ppm) and aliphatic carbon (0-50 ppm). It is noteworthy that the spectrum of the 368

irradiated organic matter remaining in the dissolved phase upon 10 days of irradiation (Fig. 6c) exhibits 369

pronounced peaks at 35 ppm, 75 ppm, and 175 ppm that are also characteristic of DOM isolated from 370

marine waters (Esteves et al., 2009; Koprivnjak et al., 2009). In comparison with the dissolved material, 371

the flocculated POM from the 10-day irradiated sample (Fig. 4d) contained relatively little aromatic 372

material (90-165 ppm; consistent with SUVA254 results above). It should be noted that this spectrum 373

exhibits two carboxyl peaks, one at ~178 ppm and the other at ~173 ppm. The CP-TOSS spectra were 374

integrated to provide quantitative information, which is given in Table 2. By normalizing the integration 375

data (Table 2) to the TOC concentrations (Table 1), the concentrations of the various carbon classes 376

were estimated in terms of µmol L-1 C, and are given in Tables 3 and 4. 377

The 13C NMR spectra with CSA-filter (Fig. 6 grey lines) show that ~85-88% of carbons detected 378

in the 90-115 ppm region were anomeric, i.e., carbohydrate-like, and carbons with 12-15% aromatic 379

carbons. No CSA-filtered spectrum was collected for the particulate matter sample as the CP-TOSS 380

spectrum contained very little signal in the region where anomeric and aromatic carbon signals overlap. 381

15

CP-TOSS spectra with dipolar dephasing (Fig. 6 narrow lines) show that 80% ±4% of aromatic carbons 382

in the dark control, ~86% ±3% of aromatic carbons in the unfractionated 10-d irradiated sample, ~84% 383

±3% of the aromatic carbons in the 10-d irradiated DOM (supernatant), and ~78% ±5% of the aromatic 384

carbons in the 10-d irradiated POM (flocculated particles) were non-protonated, i.e. substituted ring 385

carbons or carbons in the interior of condensed polyaromatic structures. 386

Insufficient particulate material was available from the 20 and 30 d irradiation time points to 387

carry out NMR analyses. However, FTIR spectra were collected for particulate material from all three 388

irradiation time points and for the dissolved material (supernatant) from the 10-d and 20-d time points 389

(Fig. 7). All DOM FTIR spectra exhibit a prominent peak at 1020-1160 cm-1 (carbohydrate region); 390

however, this peak is not observed in the 10-d POM spectrum and is less prominent in the 20-d and 30-d 391

POM spectra compared to DOM. The carboxyl region (1500-1800 cm-1) of the IR spectrum for the 392

particles that formed during the first ten days (Fig. 7b) is significantly different from that of the 393

corresponding DOM spectra (Fig. 7a). FTIR spectra for particles from the 20-d and 30-d irradations 394

contain major peaks associated with COOH (1720 cm-1), amide (1650 and 1540 cm-1), COO-Fe (1620 395

cm-1), and COO- (1580 cm-1). 396

Five main types of carboxyl groups were identified from the deconvoluted second derivative 397

FTIR spectra and their concentrations for the dark control and 10-d irradiated samples were estimated 398

by combining the TOC normalized carboxyl concentration (Table 3) and the areas obtained by 399

deconvolution and integration of the FTIR spectra according to Abdulla et al. (2010). The deconvoluted 400

spectrum revealed primarily acetate groups (1745 cm-1) and amide groups (1650 cm-1, 1540 cm-1, and 401

1261 cm-1) in the particulate spectrum, but no readily observable carboxylate-iron complexes (1620 cm-402

1). This result is consistent with the atomic absorption measurements as no iron flocculation was 403

observed until after the day 10 time point (Figs. 2c and 3c; Table 1). Acetate and amide increased, while 404

the other carboxylate moieties decreased during the irradiation. Nearly all of the amide functionality 405

was present in the particulate phase along with nearly half of the carboxylic acid and carboxylate 406

functionality (Table 4). The partitioning of amide functional groups into the solid phase is consistent 407

16

with the TN results (Table 1; Fig. 2d). Neither esters nor iron associated carboxylate were apparent in 408

the particle phase after 10 days of irradiation. The FTIR spectra for the 20-d and 30-d samples were not 409

deconvoluted due to their low signal to noise ratios and lack of corresponding NMR data. 410

The distribution of the major types of DOM structural moieties after 10 days of irradiation (Fig. 411

8) shows that the most rapidly removed carbons were aromatic. Esters, carboxylic acid/carboxylate 412

groups, and carbonyls were also extensively mineralized. Nearly all of the amide functionality was 413

present in the particulate phase along with a fraction of the aliphatic and O-alkyl or amine carbons. The 414

residual (unaltered, photodegraded, and photoproduced) DOM compounds after 10 days of irradiation 415

were markedly more aliphatic and less aromatic than the dark control. While carboxyl carbons remained 416

relatively constant as a fraction of total DOC (approximately 20%; Table 2), the distribution of 417

carboxylic groups shifted towards primarily metal associated carboxylate, which was present only in the 418

dissolved phase (Table 4; Fig. 8). 419

420

4. DISCUSSION 421

4.1 Chemical composition of DOM and POM 422

The compositional differences between the photo-flocculated POM and the dissolved phase can 423

be summarized as follows: POM after 10 days of irradiation contained no detectable metal associated 424

carboxylate, was less aromatic, less carbohydrate-like, and contained more amide and aliphatic 425

functionality than the corresponding DOM. UV-visible absorption results (Table 1) suggest that photo-426

flocculated POM (10 day sample) has a higher average molecular weight (lower spectral slope and slope 427

ratio) and less aromatic functionality (lower SUVA254) than the DOM remaining in solution. First and 428

second derivative UV-visible spectra (Figs. 4 and 5) indicates that different chromophores were present 429

in the particulate and dissolved phases. These results are supported by the considerable differences 430

between the NMR and FTIR spectra observed for DOM and POM. The C/N ratio of particulate matter 431

decreased during the 10-d to 30-d portion of the irradiation because of the preferential mineralization of 432

carbon. The low C:N ratio of the POM that remained after 30 days of irradiation (11) compared to the 433

17

high DOM C:N ratio of the starting material (28) indicates that photo-flocculation may play an 434

important role in the transport of organic nitrogen from the water column to the sediments, especially in 435

estuaries and coastal waters. 436

The combined use of TOC, NMR, and FTIR to quantify carboxyl functionalities reveals a net 437

increase in amide functionality during the early stages of photolysis (Table 4; Fig. 8), nearly all of 438

which appears in the flocculate (Fig. 5d and Fig. 8). Amides may have formed via the following 439

mechanism: Ammonia that is produced during photolysis of dissolved organic nitrogen (DON) (Bushaw 440

et al., 1996; Stedmon et al., 2007) reacts with esters within DOM to generate alkyl amides (McKee and 441

Hatcher, 2010). Since the results show that amides preferentially accumulate in the POM (Table 4; Fig. 442

8), this process may provide a pathway by which nitrogen is exported from surface waters to sediments. 443

The observed decrease in ester concentration (Table 4) supports this mechanism for amide production. 444

Interestingly, deep ocean DON is largely (up to 76%) in the form of chemically uncharacterized amides 445

that apparently resist both chemical and biological degradation (McCarthy et al., 1997; Aluwihare et al., 446

2005). An abiotic source of amide functionality within DOM would help explain its refractory nature 447

(McKee and Hatcher, 2010). Further work is needed to determine whether this abiotic mechanism also 448

applies to waters with lower DOM concentrations. 449

The results of this study suggest that changes in the concentrations and types of carboxyl groups 450

within DOM play critical roles in photo-flocculation. The signal in the carboxyl region of the NMR 451

spectrum for POM for the 10-day sample is partially split into two peaks that are narrower than the 452

single peak observed for DOM (Fig. 6) suggesting that the carboxyl groups in the flocculate phase 453

represent a less diverse mixture than the bulk dissolved material. The narrower, more resolved bands in 454

the carboxyl region for the particulate phase (Fig. 4, day 10) may also be due to the lack or low content 455

of paramagnetic iron in this sample (Pfeffer et al., 1984). Alternatively, similar peaks observed for 456

riverine DOM samples have been attributed to carbonate (Bianchi et al., 2004), though bicarbonate and 457

carbonate would be expected to resonate at ~161 and ~169 ppm (Gottlieb et al., 1997) while our peak 458

maxima were observed at ~173 and ~178. 459

18

The combined FTIR and NMR results indicate that carboxylic acids, carboxylate groups and 460

metal associated carboxylate groups are mineralized during the first ten days of irradiation (Fig. 8a). In 461

addition, decarboxylation appears to have occurred as indicated by the increase in pH in the irradiated 462

samples (Fig. 1; consistent with Porcal et al., 2013) and the production of dissolved inorganic carbon 463

during UV exposure of Dismal Swamp water (Minor et al., 2006) and similar samples (Porcal et al., 464

2013). NMR evidence presented here, and by Thorn et al. (2010) suggests that photochemical 465

decarboxylation leads to decreased DOM aqueous solubility, which is supported by the higher aliphatic 466

content of the POM relative to the DOM (Table 2), thus facilitating flocculation. Abdulla et al. (2010) 467

showed that carboxylic groups in the >1 kDa fraction of DOM decreased downstream along an estuarine 468

transect. They suggested that this decrease was due to flocculation/adsorption, and/or 469

photodegradation/photo-decarboxylation. In light of the results of the current study, it is reasonable to 470

speculate that a coupled photochemical-flocculation mechanism contributed to this observed estuarine 471

trend. 472

4.2 Mechanism of photo-flocculation 473

The mechanism of photochemical flocculation has not previously been directly investigated. 474

Based on the spectroscopic characterization of the DOM and POM remaining after irradiation, it 475

appears that photo-flocculation occurs by two or more pathways (Fig. 9), and that initial flocculation of 476

organic and inorganic material may not occur simultaneously. The processes in Fig. 9 have been 477

subdivided according to whether they occur prior to the flocculation of iron (Phase I) or after iron 478

begins to flocculate (Phase II). It is important to consider that in higher pH, lower DOC waters such as 479

large lakes, whitewater rivers, and the open ocean, Phase I may have little or no applicability due to the 480

limited solubility of iron and prior photobleaching. The ‘Continuous’ category includes reactions that 481

likely impact the process of photo-flocculation throughout this experiment, while the ‘Competitive’ 482

category includes reactions and processes that compete with or counteract the process of photochemical 483

flocculation. During Phase I, loss of DOM solubility may have occurred as a result of selective 484

photodegradation as a portion of the aromatic, polyhydroxyl and polycarboxyl humic material within the 485

19

DOM matrix becomes more aliphatic and less acidic rendering it more hydrophobic (Gao and Zepp 486

1998; Thorn et al. 2010). An additional possibility involves the degradation of an amphiphilic fraction 487

of the DOM, which leads to the flocculation of pre-existing hydrophobic “core” material and/or 488

sparingly soluble amphiphiles such as hydrophobic peptides. Similar amphiphilic-hydrophobic 489

interactions have been proposed to explain the enhanced solubility of model pollutants by natural 490

organic matter (Chiou et al., 1986; Abdulla et al., 2009), which suggests that the change in bulk DOM 491

polarity may impact the partitioning of aromatic polutants. In either case, a photochemically inert 492

fraction of DOM carbohydrate-like material (NMR signal at ~75ppm and ~100ppm, IR peaks from 997 493

to 1152 cm-1, and little or no UV-visible absorption) survives long-term UV exposure and remains 494

primarily in the dissolved phase. 495

4.3 Role of particles and iron in photo-flocculation of OM 496

Silica particles have been shown to readily adsorb humic substances (Liang et al., 2011), and the 497

adsorption of surface active DOM to minerals and detrital POM has been cited as a possible route for 498

the preservation of organic matter in the sediments (Hedges and Keil, 1995; Henrichs, 1995; Mead and 499

Goñi, 2008). Although the experimental protocol used in the current study excluded pre-existing 500

particulate matter greater than 0.1 µm, the extent to which DOM adsorbed to the walls of the quartz 501

flasks was quantified. Negligible adsorption occurred in the dark control (~0.10% of total a300), while 502

after 20 days of irradiation, 15% of the residual a300 adsorbed to the walls of the irradiated quartz 503

flasks. TN was not measured for the adsorbed material; however, wall adsorption of DON (along with 504

the formation of volatile nitrogen compounds such as ammonia and amines, which would have been lost 505

during freeze drying) may help to explain the observed ~20% decrease in TN during the irradiation 506

experiment (Fig. 4d). The latter show that a fraction of DOM was more susceptible to mineral 507

adsorption, or more strongly adsorbed after photodegradation. This mechanism may be a pathway by 508

which a significant fraction of the tDOM pool is both exported from surface waters to the sediments and 509

subsequently preserved during early diagenesis (Hedges and Keil, 1995; Henrichs, 1995; Mead and 510

Goñi, 2008). If iron flocculated as amorphous hydroxide particles, they may have selectively scavenged 511

20

significant quantities of DOM irrespective of any photochemical changes to the DOM pool (Luider et 512

al., 2003). 513

4.4 Photo-flocculation of iron 514

The role of iron and other metals in the photochemical flocculation of DOM appears to be 515

complex. Both dissolved and particulate forms of iron likely play roles in the photo-flocculation of 516

DOM. Dissolved iron may form metal ion “bridges” between DOM molecules or colloids resulting in 517

aggregation (Verdugo et al., 2004; Orsetti et al., 2010). Polymeric iron (hydr)oxides as well as soluble 518

forms of iron III, have been found to coagulate DOM (Nierop et al., 2002; Pullin et al., 2004; Honghai 519

et al., 2008). Organic metal-binding ligands have been shown to degrade in sunlit surface seawater 520

transforming organically complexed iron to inorganically complexed or colloidal iron on diel time-521

scales (Powell and Wilson-Finelli, 2003; Shank et al., 2006). Thus, photochemically induced 522

flocculation of dissolved and colloidal iron may be driven by photochemical degradation of metal-523

complexing ligands within DOM (Kopáček et al., 2005). 524

Even though photo-flocculation of DOM occurred during the first 10 days of the experiment, all 525

the iron essentially remained in solution (Fig. 3). It either quickly re-cycled back into the dissolved 526

phase by complexation with soluble organic ligands, or it was photo-reduced to iron (II), which is 527

soluble at the experimental pH (Fig. 1). Reduction to iron (II) may have occurred via ligand to metal 528

charge transfer reactions (Faust and Zepp, 1993) resulting in oxidative decarboxylation (Bockman et al. 529

1996; Thorn et al. 2010), which in turn enhanced the hydrophobicity of the residual DOM molecule 530

(Gao and Zepp 1998; Thorn et al. 2010). It is likely that, as the DOM became increasingly mineralized 531

and/or structurally altered, the abundance of ligands and/or electron donors diminished, causing a shift 532

in the speciation of the iron present and allowing less-soluble iron (III) oxyhydroxides to form (Deng 533

and Stumm, 1994). Alternatively or concurrently, iron-binding sites became more abundant in the POM 534

than in the DOM, leading to a transfer of organically complexed iron from the dissolved phase to the 535

particulate phase. Another concurrent possibility is that, as the pH increased during the irradiation (Fig. 536

1), DOM carboxyl groups became progressively deprotonated and thus more readily complexed with 537

21

metals to form insoluble metal DOM complexes via cation bridged aggregates (Nierop et al., 2002) 538

and/or the solubility of iron (III) may simply have decreased as a result of the rising pH (Schwertmann 539

and Murad, 1983; Schwertmann, 1991; Liu and Millero, 2002). More work is needed to determine the 540

degree to which and the conditions under which each of the above are the most important processes. 541

While iron oxidation rates and solubility have been well studied in pure water (King and Farlow, 542

2000), sodium chloride brines (King et al., 1995; Liu and Millero, 1999), and seawater (Liu and Millero, 543

2002), the impact of strong iron (II and III) ligands present in humic-rich and eutrophic freshwater 544

systems (Shapiro, 1964; Emmenegger et al., 1998) make estimating the oxidative lifetime of iron (II) 545

and solubility of iron (III) at sub-neutral pH, based on available literature, problematic. In pure water at 546

pH = 4 and atmospheric equilibrium for example, more than half of the iron initially present would be 547

expected to oxidize and precipitate within 10 days (Morgan and Lahav, 2007); however, under both 548

light and dark experimental conditions, essentially all of the iron remained in solution in Dismal Swamp 549

water samples for a minimum of ten days. Iron is clearly present in the 20 and 30 d irradiated particles 550

(Figs. 3c and 7b), though it is not known whether this association formed primarily in the solution phase 551

through adsorption of DOM to iron (III) oxyhydroxide particles (e.g. Luider et al., 2003) or resulted 552

simply from coincident precipitation. These results suggest that photochemical flocculation and/or the 553

photochemical removal of iron (III)-binding ligands play an important role in the removal of dissolved 554

iron in estuaries and in the transport of riverine iron to the water column and sediments of the ocean 555

margins. 556

4.5 Implications for biogeochemical cycles 557

It should be noted that the POC formation rates of 0.13-0.47 mg C L-1 d-1, reported here, are 558

lower than the rate of 0.50 mg C L-1 d-1 reported by Gao and Zepp (1998) and that all published 559

instances of photochemically mediated DOM flocculation have been reported for high DOC, low pH 560

terrestrial environments with high DOC/TOC ratios. Furthermore, these estimates do not take into 561

account photo-dissolution of the flocculates in the flasks (Table 1; Kieber et al, 2006; Pisani et al., 2011; 562

Porcal et al., 2013) nor possible dilution effects in the environment. It is not yet known if photo-563

22

flocculation affects algal/marine DOM. The need for further investigation in this area of organic matter 564

reactivity is clearly indicated. 565

Photochemically induced flocculation represents a potentially significant source of organic 566

carbon, organic nitrogen, and iron to the sediments (Gao and Zepp, 1998; Scully et al., 2004; Porcal et 567

al., 2013), and thus may impact the geochemical cycling of these elements particularly in estuaries and 568

perhaps in coastal oceanic waters. The abiotic production of amides, which preferentially partition into 569

the particulate phase, may play a role in estuarine N-cycling and may help to explain the presence of 570

biologically refractory carbon and nitrogen in the sediments and deep ocean. Photochemical production 571

of surface-active DOM may significantly contribute to the high rates of organic matter preservation 572

observed in coastal sediments underlying tDOM rich surface waters (Dagg et al. 2004). Preliminary 573

calculations presented in Helms (2012) suggest that as much as 4.5% of terrestrial DOM may photo-574

flocculate before reaching the open ocean, delivering roughly 7.2 x 1012 g C y-1 to the sediments in 575

marginal environments. Thus, photochemical flocculation may be a quantitatively important removal of 576

tDOM from the water column and may partially explain the apparent disconnect between the large 577

riverine source of tDOM and the low abundance of identifiable terrestrial DOM in the oceans (Hedges 578

et al., 1997). 579

580

ACKNOWLEDGMENTS: The authors would like to thank Aron Stubbins, Patrick G. Hatcher, 581

Richard Zimmerman, and Andrew Wozniak, for helpful discussions and comments about an early draft 582

of this manuscript. The input of three anonymous reviewers was instrumental in improving the quality 583

of this manuscript prior to its publication and is greatly appreciated. This research was supported by 584

NSF grant: OCE-0728634 585

586

23

FIGURE CAPTIONS 587

Fig. 1. pH values of water samples measured at the end of each irradiation time point. 588

Fig. 2. Concentrations of dissolved (gray), particulate (black), and adsorbed (white) material as a 589

function of irradiation time: (a) organic carbon, (b) absorption at 300 nm, (c) total iron by atomic 590

absorption, and (d) total nitrogen. Error bars represent the combined standard deviations of the “total,” 591

“dissolved,” and “adsorbed” terms from which the “particulate” term was calculated. Total nitrogen was 592

not determined for the “adsorbed” material. 593

Fig. 3. Percentage distribution of dissolved (gray), particulate (black), and adsorbed (white) material as 594

a function of irradiation time: (a) organic carbon, (b) absorption at 300 nm, (c) total iron by atomic 595

absorption, and (d) total nitrogen. 596

Fig. 4. (a, d, and g) UV-visible absorption spectra, (b, e, and h) first derivative absorption spectra, and 597

(c, f, and i) second derivative absorption spectra, were obtained for (a, b, and c) total, unfractionated, 598

samples in which flocculated POM was re-dissolved at high pH, (d, e, and f) dissolved samples from 599

which POM was removed, and (g, h, and i) POM obtained by difference. 600

Fig. 5. (a, d, and g) natural log UV-visible absorption spectra, (b, e, and h) first derivative natural log 601

absorption spectra, and (c, f, and i) second derivative natural log absorption spectra, were obtained for 602

(a, b, and c) total samples in which flocculated POM was re-dissolved at high pH, (d, e, and f) dissolved 603

samples from which POM was removed, and (g, h, and i) POM obtained by difference. 604

Fig. 6. Solid-state 13C CP-TOSS NMR spectra (heavy black line) obtained for (a) the dark control 605

sample, (b) the un-fractionated 10 d irradiated sample, (c) dissolved material isolated from the 10 606

d irradiated sample by centrifugation, and (d) the particulate material isolated from the 10 d 607

irradiated sample by centrifugation. Solid-state 13C CP-TOSS NMR spectra with CSA-filter (gray 608

line) and dipolar dephasing (narrow black line) are shown for comparison. The CSA-filtered 609

24

spectra show aliphatic material, while dipolar dephasing shows primarily non-protonated and 610

highly mobile carbons. 611

Fig. 7. FTIR spectra obtained for (a) dissolved organic matter (DOM) and (b) particulate organic matter 612

(POM). Labels to the left of each spectrum refer to the irradiation time point. 613

Fig. 8. Distribution of organic functional groups after 10 days irradiation. “Photolyzed” columns (white) 614

represent organic matter that was completely mineralized or volatilized. “POM” columns (black) 615

represent flocculated organic matter. “DOM” columns (gray) represent organic matter that remained in 616

the dissolved phase after irradiation. Functional group assignments are based on 13C NMR and FTIR 617

spectra. Quantifications are based on TOC measurements normalized to NMR and/or FTIR spectral 618

integrations. Acetate is included in the “COOH/COO-“ category. The carbonyl category refers to 619

aldehydes, ketones, and quinones. 620

Fig. 9. Proposed components of the photochemical flocculation mechanism for organic matter and 621

iron. During “Phase I” organic matter flocculates, but iron does not. During “Phase II” both 622

organic matter and iron flocculate. The transition from Phase I to Phase II during these 623

experiments may have been caused by loss of soluble iron binding ligands, the increase in pH, 624

and/or the build up of iron (III) as the iron photo-reduction rate decreased relative to Fenton’s 625

reaction. Fe(s) represents solid iron oxyhydroxides. The “Continuous” and “Competitive” 626

processes are described in the Discussion. 627

628

25

TABLES 629

Table 1 630 Summary of parameters measured during the irradiation experiment. Total=whole, unfractionated 631 samples; Diss=dissolved, particle free samples; Ads=adsorbed material; PM=particulate values 632 determined by difference except where denoted with *. Values in italics refer to dark control samples. 633 N/D=not determined. Analytical uncertainty is reported as one standard deviation (n ≥ 3). 634

635 636

637 Sample

Irr. Time (days)

TOC (mg L-1)

TN (mg L-1)

Iron (µmol L-1)

a300 (m-1)

S 275-295 (µm-1) SR SUVA254

(L m-1 mg-1)

0d-Total 0 52.7 ±0.2 1.89 ±0.01 34.8 ±0.3 369 ±3 15.1 ±0.2 0.91 ±0.02 11.8 ±0.1

10d-Total 10 31.0 ±0.2 1.69 ±0.02 34.2 ±0.3 211 ±1 10.8 ±0.2 1.12 ±0.02 10.8 ±0.1 0 53.0 ±0.2 1.81 ±0.02 34.3 ±0.4 366±1 13.2 ±0.3 0.92 ±0.03 11.9 ±0.1

20d-Total 20 22.0 ±0.2 1.66 ±0.02 34.6 ±0.3 118 ±1 9.7 ±0.2 1.13 ±0.02 8.3 ±0.1 0 52.2 ±0.4 1.91 ±0.02 34.4 ±0.2 365 ±1 12.7 ±0.4 0.88 ±0.4 11.6 ±0.1

30d-Total 30 10.5 ±0.2 1.37 ±0.01 34.2 ±0.3 48.7 ±0.3 9.4 ±0.1 1.00 ±0.02 6.7 ±0.1 0 52.9 ±0.3 1.91 ±0.02 34.5 ±0.3 363 ±2 12.7 ±0.3 0.93 ±0.02 11.4 ±0.1

10d-Diss 10 26.3 ±0.1 1.41 ±0.01 34.1 ±0.3 189.1 ±0.6 11.4 ±0.1 1.15 ±0.02 11.7 ±0.1 0 52.0 ±0.3 1.90 ±0.02 33.6 ±0.2 370±1 12.5 ±0.2 0.91 ±0.01 11.8 ±0.1

20d-Diss 20 15.6 ±0.1 1.31 ±0.02 26.8 ±0.3 102.9 ±0.5 10.3 ±0.1 1.17 ±0.02 10.5 ±0.1 0 51.5 ±0.2 1.85 ±0.02 34.2 ±0.3 346 ±12 13.5 ±0.3 0.87 ±0.03 11.5 ±0.4

30d-Diss 30 6.5 ±0.2 1.02±0.01 4.5 ±0.1 19.4 ±0.1 18.2 ±0.1 1.72 ±0.03 5.9 ±0.2 0 54.8 ±0.8 1.88 ±0.02 35.1 ±0.4 369 ±2 12.4 ±0.3 0.91 ±0.02 11.7 ±0.1

0d-Ads 0 0.3 ±0.1 N/D 0.02 ±0.01 0.4 ±0.3 13.5 ±0.3 1.15 ±0.02 2.4 ±2.0

10d-Ads 10 2.3 ±0.1 N/D 0.07 ±0.02 5.4 ±0.5 9.2 ±0.3 0.74 ±0.01 2.8 ±0.3 0 0.3 ±0.2 N/D N/D 0.2 ±0.2 13.8 ±0.5 1.16 ±0.02 2.1 ±3.0

20d-Ads 20 1.7 ±0.1 N/D 0.36 ±0.04 1.3 ±0.5 11.5 ±0.3 0.93 ±0.02 1.0 ±0.4 0 N/D N/D 0.02 ±0.01 N/D N/D N/D N/D

30d-Ads 30 2.5 ±0.1 N/D 0.29 ±0.04 7.4 ±0.7 13.6 ±0.4 0.77 ±0.02 4.3 ±0.4 0 0.3 ±0.2 N/D 0.02 ±0.01 0.5 ±0.2 12.8 ±0.5 1.14 ±0.02 2.5 ±2.0

10d-PM 10 4.7 ±0.2 0.29 ±0.02 0.1 ±0.4 22 ±1 8.6* ±0.3 0.72* ±0.03 4.8 ±0.3 0 1.0 ±0.4 -0.09 ±0.03 0.7 ±0.4 -4 ±1 N/D N/D N/D

20d-PM 20 6.4 ±0.2 0.35 ±0.03 7.8 ±0.4 16 ±1 N/D N/D N/D 0 0.7 ±0.4 0.06 ±0.03 0.2 ±0.4 19 ±12 N/D N/D N/D

30d-PM 30 4.0 ±0.3 0.35 ±0.02 29.7 ±0.1 29.3 ±0.3 N/D N/D N/D 0 -1.9 ±0.9 0.03 ±0.03 -0.6 ±0.5 -6 ±3 N/D N/D N/D

26

Table 2 638 Functional group assignments and integrated area for each CP-TOSS spectral region. Integrals are given 639 as percentage of total area (i.e. percentage of total carbon in the sample) for qualitative comparison of 640 each sample. Uncertainties were determined using the signal to noise ratio of each integration region 641 and are given as one standard deviation. 642

Integration

range (ppm)

Dark Control

10 d Total

10 d Particles

10 d Dissolved

CH3 and CH2 0-28 8.4 ±0.2 12.7 ±0.3 19 ±0.4 10.5 ±0.2 Mostly CH2 and some CH 28-35 3.8 ±0.1 5.1 ±0.2 7.6 ±0.3 5.4 ±0.1 CH and alkyl Cquat. 35-50 9.3 ±0.2 10.6 ±0.3 9.0 ±0.3 11.3 ±0.2 NCH + OCH3 + OCH2 50-65 9.0 ±0.2 9.5 ±0.2 11.3 ±0.5 9.9 ±0.1 Carbohydrate-like C 65-100 21.0 ±0.4 22.0 ±0.4 13.6 ±0.5 23.0 ±0.3 OCHO + arom. C-C-O 100-115 6.3 ±0.2 5.0 ±0.2 3.0 ±0.3 4.8 ±0.1 Aromatic C 115-145 12.1 ±0.3 7.8 ±0.2 6.2 ±0.3 7.9 ±0.1 Aromatic C-O 145-160 4.7 ±0.2 2.8 ±0.2 2.3 ±0.3 2.8 ±0.1 COO +NC=O 160-187 20.5 ±0.4 22 ±0.4 26 ±0.6 20 ±0.3 Aldehyde, Ketone and Quinone C 187-220 4.6 ±0.2 2.7 ±0.2 1.0 ±0.3 4.1 ±0.1

643 644

27

Table 3 645 CP-TOSS integration data from Table 2 normalized to TOC concentrations and reported in 646 concentration of µmol C L-1 for quantitative comparison of the samples. Uncertainties reported as one 647 standard deviation and calculated based on the combined CP-TOSS and TOC uncertainties. 648

Integration

range (ppm)

Dark Control

10 d Total

10 d Particles

10 d Dissolved

CH3 and CH2 0-28 370 ±9 330 ±8 74 ±4 240 ±5 Mostly CH2 and some CH 28-35 170 ±5 130 ±5 30 ±2 120 ±2 CH and alkyl Cquat. 35-50 410 ±9 270 ±8 35 ±2 240 ±4 NCH + OCH3 + OCH2 50-65 400 ±9 250 ±6 43 ±3 220 ±2 Carbohydrate-like C 65-100 920 ±18 570 ±11 55 3 500 ±7 OCHO + arom. C-C-O 100-115 280 ±9 130 ±5 12 ±1 110 ±2 Aromatic C 115-145 530 ±13 200 ±5 24 ±2 170 ±2 Aromatic C-O 145-160 210 ±9 70 ±5 9±1 61 ±2 COO +NC=O 160-187 920 ±18 570 ±11 102 ±5 440 ±7 Aldehyde, Ketone and Quinone C 187-220 200 ±9 70 ±5 4 ±1 90 ±2

649 650 651 652

28

Table 4 653 Estimated concentrations of different carboxylic moieties (µmol C L-1). Quantifications are based on 654 deconvoluted FTIR spectra, NMR integration of the 165-190 ppm region, and TOC measurements. 655 Uncertainty reported as one standard deviation. 656

Peak Center(s)

(cm-1) Dark

Control 10 d Total

10 d Particles

10 d Dissolved

Ester/Lactone 1775 72 ±8 20 ±1 0 ±0.1 16 ±1 Acetate 1745 0 ±0.2 46 ±5 9 ±1 32 ±2 COOH & COO- 1580, 1720 390 ±20 94 ±4 43 ±8 48 ±4 COO-Fe 1620 380 ±20 280 ±6 0 ±0.1 270 ±5 Amide 1650 28 ±4 44 ±4 40 ±4 1 ±0.5

657

658

29

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891 892

34

893 Fig. 1 894 895

3020100

6

5

4

3

2

Irradiation Time (days)

pH

35

896 Figure 2 897 898

Org

anic

Car

bon

(mm

ol L

-1)

5

4

3

2

1

0

DissolvedParticulate

TotalAdsorbed

3020100

40

30

20

10

0

Irradiation Time (days)

Iron

(µm

ol L

-1)

3020100Irradiation Time (days)

a300

(m-1

)

400

300

200

100

0

Tota

l Nitr

ogen

(mm

ol L

-1)

0.14

0.11

0.07

0.04

0

(a)

(c)

(b)

(d)

36

899 Fig. 3 900

Org

anic

Car

bon

100%

75%

50%

25%

0%

3020100Irradiation Time (days)

Iron

3020100Irradiation Time (days)

a300

Tota

l Nitr

ogen

100%

75%

50%

25%

0%

100%

75%

50%

25%

0%

100%

75%

50%

25%

0%

(a)

(c)

(b)

(d)

37

901 Fig. 4 902 903

650550450350250

30

20

100

250 300 350 400 450

300

200

100

0

300

200

100

0

300

200

100

0

(g)

(d)

(a) 10d20d30dDark

Wavelength (nm)

Abso

rptio

n C

oeffi

cien

t (m

-1)

0.02-0.06

-0.22-0.30

1-1

-3

-71

-3

-5-7

da/d

!

-5

-1

-0.14

250 300 350 400 450Wavelength (nm)

d2a/

d!2

0.01

-0.01

0.1

0.0

-0.1

0.1

0.0

-0.1

0.00

250 300 350 400 450Wavelength (nm)

(h)

(e)

(b)

(i)

(f)

(c)0-Order 1st Derivative 2nd Derivative

TOTAL

DISSOLVED

FLOC

38

904 Fig. 5 905 906

250 300 350 400 450

8

4

20

(g)

(d)

(a) 10d20d

30dDark

Wavelength (nm)

ln a

0.00

-0.010

-0.015

0.00

-0.01

-0.02

d(ln

a)/d

!

-0.005

250 300 350 400 450Wavelength (nm)

d2(ln

a)/d

!2

0.001

0.000

-0.001

250 300 350 400 450Wavelength (nm)

(h)

(e)

(b)

(i)

(f)

(c)6

8

4

20

6

8

4

20

6

0.00

-0.01

-0.02

0.001

0.000

-0.0010.001

0.000

-0.001

0-Order 1st Derivative 2nd Derivative

TOTAL

DISSOLVED

FLOC

39

907 Fig. 6 908 909

(a)OCH& NCH

Arom.

Alkyl(CH3 & CH2)

200 150 100 50 0 ppm

(b)

(c)

(d)

O-C-O

Carboxyl, Ester,and Amide

0d(Dark)

10dTotal

10dDissolved

10dParticulate

40

910 Fig. 7 911 912

2000 800Wavenumber (cm-1)

1400 2000 800Wavenumber (cm-1)

1400

Este

r/Lac

tone

CO

OH Am

ide

CO

O-F

eC

OO

-Am

ide

CH

2 an

d C

H3

-O-He.g. Carbohydrates

Este

r/Lac

tone

CO

OH Am

ide

CO

O-F

eC

OO

-Am

ide

CH

2 an

d C

H3

-O-He.g. Carbohydrates

0d

10d

20d

10d

20d

30d

(a) (b)

1700 1100 1700 1100

POMDOM

41

913 Fig. 8 914 915

1.5

1.0

0.5

0.0 Car

bon

(mm

ol L

-1)

Alky

l-CO-a

lkyl &

NCH

Arom

atic-C

COO

- /COOH

COO-

FeEs

ter/La

ctone

Amide

-CCa

rbon

yl

PhotolyzedPOMDOM

42

916 Fig. 9 917

Phase I: only DOM flocculates

Photolysis of DON ! NH4+NH4+ (aq) + Esters (aq) ! amides (s)Fe3+ + R-COO- ! R + Fe2+ + CO2 (LMCT)Fe2+ " Fe3+ + e- (iron cycling)

Phase II: iron and DOM co-flocculate

Fe2+ + O2 ! Fe3+ + O2*- Fe3+ + OH- ! Fe(OH)3 (s)Fe3+ (aq) + DOM ! ‘Bridging’ ! Fe-POM aggregatesFe(OH)3 (s) + DOM ! adsorption ! Fe-POM aggregates

Continuous processes: photo-Fenton & decarboxylation

Fe2+ + O2 ! Fe3+ + O2*- Photolysis of org. ligands ! DOM oxidation & DIC productionFe2+ + HOOH ! Fe3+ +*OH + OH-

Competitive processes: recycling and remineralization

Fe (s) + ! Fe (aq) & POM ! DOM (Recycling)Organic carbon ! DIC (mineralization)

h# h#

h#

h#