Novel molecular-level evidence of iodine binding to natural organic matter from Fourier transform ion cyclotron resonance mass spectrometry

Science of the Total Environment 449 (2013) 244–252

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Novel molecular-level evidence of iodine binding to natural organic matter from Fourier transform ion cyclotron resonance mass spectrometry Chen Xu a,⁎, Hongmei Chen b, Yuko Sugiyama b, c, Saijin Zhang a, Hsiu-Ping Li a, Yi-Fang Ho a, Chia-ying Chuang a, Kathleen A. Schwehr a, Daniel I. Kaplan d, Chris Yeager e, Kimberly A. Roberts d, Patrick G. Hatcher b, Peter H. Santschi a a

Laboratory for Environmental and Oceanographic Research, Department of Marine Sciences, Texas A&M University, Building 3029, Galveston, TX 77551, United States Department of Chemistry and Biochemistry, Old Dominion University, Norfolk, VA 23529, United States c University of Hyogo, 1-1-12, Shinzaike-honcho, Himeji, Hyogo 670-0092, Japan d Savannah River National Laboratory, Aiken, SC 29808, United States e Los Alamos National Laboratory, Los Alamos, NM 87545, United States b

H I G H L I G H T S

G R A P H I C A L

A B S T R A C T

► IO3− reduced by lignin-, tannin-like compounds/carboxylic-rich alicyclic molecules ► Condensed aromatic and lignin-like compounds generated after iodateiodination ► Aliphatic and less aromatic compounds formed after iodide-iodination ► Organo-iodine identified as unsaturated hydrocarbons, lignin and protein ► Organo-iodine with low O/C ratios imply less environmental mobility

a r t i c l e

i n f o

Article history: Received 10 December 2012 Received in revised form 21 January 2013 Accepted 21 January 2013 Available online 19 February 2013 Keywords: Natural organic matter (NOM) Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) Radioiodine (129I) Iodide Iodate Organo-iodine

a b s t r a c t Major fractions of radioiodine (129I) are associated with natural organic matter (NOM) in the groundwater and surface soils of the Savannah River Site (SRS). Electrospray ionization coupled to Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS) was applied to elucidate the interactions between inorganic iodine species (iodide and iodate) and a fulvic acid (FA) extracted from a SRS surface soil. Iodate is likely reduced to reactive iodine species by the lignin- and tannin-like compounds or the carboxylic-rich alicyclic molecules (CRAM), during which condensed aromatics and lignin-like compounds were generated. Iodide is catalytically oxidized into reactive iodine species by peroxides, while FA is oxidized by peroxides into more aliphatic and less aromatic compounds. Only 9% of the total identified organo-iodine compounds derived from molecules originally present in the FA, whereas most were iodine binding to newly-produced compounds. The resulting iodinated molecules were distributed in three regions in the van Krevelen diagrams, denoting unsaturated hydrocarbons, lignin and protein. Moreover, characteristics of these organo-iodine compounds, such as their relatively low O/C ratios (b 0.2 or b 0.4) and yet some degree of un-saturation close to that of lignin, have multiple important environmental implications concerning possibly less sterically-hindered aromatic ring system for iodine to get access to and a lower hydrophilicity of the molecules thus to retard their migration in the natural aquatic systems. Lastly, ~ 69% of the identified organo-iodine species contains nitrogen, which

⁎ Corresponding author. Tel.: +1 409 740 4530; fax: +1 409 740 4786. E-mail address: [email protected] (C. Xu). 0048-9697/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.scitotenv.2013.01.064

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is presumably present as \NH2 or \HNCOR groups and a ring-activating functionality to favor the electrophilic substitution. The ESI-FTICR-MS technique provides novel evidence to better understand the reactivity and scavenging properties of NOM towards radioiodine and possible influence of NOM on 129I migration. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Iodine has one stable isotope, 127I and several radioactive isotopes, e.g., 131I (t1/2 = 8 days) and 129I (t1/2 = 15.7 million years). It is a biophilic element, exhibiting a high concentration factor of 104 in brown algae (Bruland, 1983), 5 × 105 in colloidal macromolecular organic matter in the Mississippi River (Oktay et al., 2001), and is found enriched by factors of 2–10 in soil bacteria (Li et al., 2011), and by more than 100 in human thyroid compared to that in plasma. It is an essential element for the biosynthesis of thyroid hormones and for the proper function of human thyroid gland. Recently, concerns exist about uncontrolled releases of radioiodine related to nuclear fission (mainly 131I and 129I) activities, e.g., from nuclear reprocessing, nuclear reactor accidents such as Chernobyl and Fukushima accidents, or from nuclear waste disposal, such as at the Department of Energy's (DOE) Savannah River Site (SRS), Hanford Site, and Idaho National Laboratory. Due to its high perceived mobility in the environment, excessive inventory, high bioaccumulation factor through the food chain and bioconcentration in the human thyroid, 129I is identified as a key risk driver at most DOE sites, with a Maximum Contamination Level (MCL) of 1 pCi/L (0.037 Bq/L) in groundwater that is lower than that for all other radionuclides (Kaplan et al., 2011). Major iodine species in terrestrial systems include iodide (I−), iodate (IO3−) and organo-iodine, of which organo-iodine comprises a major fraction in many environments. In wetland soils and some subsurface aquifers, almost 90% of iodine is present as organo-iodine species, whereas in low organic-matter sediments, such as many aquifers (OCb 0.1%), organo-iodine exists but inorganic iodine species are more prevalent (Hu et al., 2005; Shimamoto et al., 2011; Stipaničev and Branica, 1996; Xu et al., 2011a,b; Yamaguchi et al., 2010, 2006). Related, even in groundwater or aquifer systems of the SRS, where dissolved organic matter (DOM) is low (~0.4 ppm C), organo-iodine can still comprise as much as 66% of the total iodine (Otosaka et al., 2011). Thus, only low concentrations of natural organic matter (NOM) are needed to greatly influence the biogeochemical behavior of iodine (Denham et al., 2009; Schwehr et al., 2009); however, only recently has the molecular mechanism for iodination been explored. By comparing the iodine L3-edge X-ray absorption spectra of humic acids (HA) and fulvic acids (FA) to the simulated spectra of phenolic reference compounds, it was inferred that iodine is covalently bound to aromatic carbon in naturally iodinated humic substances (Schlegel et al., 2006). When investigating the organo-iodine species formation using electrospray mass spectrometry and tandem mass spectrometry, Moulin et al. (2001) described the molecular structure of an iodinated fulvic acid compound. The authors suggested that organo-iodine compounds were formed through electrophilic substitution of aromatic structures in FAs. However, the limited resolving power (~4000 FWHM at m/z 500) of the quadrupole time-of-flight (Q-TOF) mass spectrometry did not allow strict molecular formula assignment. Despite these breakthroughs, there is still a limited understanding of the interactions between iodine species (mainly iodide and iodate) and NOM, and the molecular characteristics of the organo-iodine and other organic compounds resulting from the iodination process. On one hand, abiotic reduction of iodate by NOM and subsequent sequestration into NOM has been noticed previously (Steinberg et al., 2008; Xu et al., 2011b; Yamaguchi et al., 2010). On the other hand, if only iodide and NOM are present, organo-iodine formation is insignificant (≤1%) at pH≥ 5 (Xu et al., 2011b). H2O2, in lieu of peroxide that is naturally

produced by UV and as a metabolite produced by many organisms, needs to be added as an oxidant to convert iodide into reactive iodine species, (e.g., I2 or HOI), which subsequently interacts with FA to form organo-iodine compounds. This is likely one of the main reactions that occurs in the environment (Li et al., 2012). Lactoperoxidase, exhibits haloperoxidase activity and can catalyze the H2O2 oxidation of I− into electrophilic products (e.g., I2 or HOI), which then readily react with organic moieties or molecules possessing electron-donating groups. Once iodine is incorporated into NOM, mobility of the resulting organo-iodine compounds is determined by the associated NOM, which is then determined by the physical chemical properties of the NOM (e.g., molecular weight, functional groups, hydrophobicity, surface charge, interaction with mineral phase, etc.). Electrospray ionization (ESI) coupled to Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) has emerged as a powerful technique that enables molecular formula assignment to individual NOM components (Kujawinski, 2002; Kujawinski et al., 2001; Minor et al., 2012; Pfeifer et al., 2001). The objectives of this study were thus to: 1) compare the spectra of NOM iodinated via IO3− or I− with those of untreated NOM to identify the NOM binding sites reactive to iodine and 2) obtain the molecular composition of the organo-iodine species that are formed through either non-enzymatic (IO3− as the starting species) or 3) enzymatic (I− as the starting species) processes from accurate mass obtained by ESI-FTICR-MS. 2. Materials and methods 2.1. Laboratory iodination experiments with FA F-Area of SRS is a contaminated former nuclear processing site operated by the federal government for the production of nuclear materials for almost three decades, resulting in the production of a millions of liters of aqueous of radioactive waste, including 129I, which was disposed into unlined seepage basins. Groundwater monitoring data has suggested that essentially all the radionuclides and metal concentrations in the F-Area plume are decreasing, except 129I (Kaplan et al., 2011). For example, in the well closest to the seepage basin, total 129I concentrations in the groundwater vary between 19 and 26 Bq/L, greatly exceeding the drinking water limit of 0.037 Bq/L (Kaplan et al., 2011). A representative surface soil, downgradient the seepage basin and along the contaminated groundwater plume, was chosen to extract NOM. This soil was determined to contain elevated 129I due to surface runoff, stormflow, groundwater infiltration/exfiltration (Xu et al., 2011a,b). Fulvic acid, extracted from this soil, was used, as it has better ionization efficiency than other type of NOM, such as humic acid (data not shown). More site description, soil sampling and extraction of FA procedures are described previously (Xu et al., 2011a) and provided in the supplemental content. Stable iodine was used as an analog of 129I in the ESI-FTICR-MS analysis, for experimental convenience. 2.1.1. Non-enzymatic iodination Iodate was allowed to react with FA in the absence of exogenous reductants, oxidants or enzymes. FA dissolved in 20 mM NaHCO3 was diluted with artificial freshwater (see the supplemental contents) with an ionic strength of 1.64 mM to a final concentration of 1.5 mg-FA/mL (OC ~40% of the total mass). The pH of the solution was adjusted to ~3 with 0.05 M phosphate buffer. We selected pH 3 because it is the optimal for the reaction between iodate and NOM (Xu et

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al., 2011b), and it is within the pH range of the groundwater immediately downgradient of the F-Area basins (Otosaka et al., 2011). The ionic strength of the final solution was maintained below 6 mM to mimic that of the soil porewater. KIO3 was added to the reaction vessel to a final concentration of 10 −4 M, yielding a final I/C ratio similar to the ratio that commonly occurs in groundwaters or surface soils of the SRS (Otosaka et al., 2011; Xu et al., 2011a). A control sample was prepared identically, but without iodate. The mixture was shaken in darkness for 72 h to minimize photo-oxidation and to reach equilibrium (Xu et al., 2011b). 2.1.2. Enzymatically catalyzed iodination FA was dissolved in 20 mM NaHCO3 buffer to achieve maximum dissolution (pH ~8), and diluted with artificial freshwater to a final concentration of 1.5 mg-FA/ml. The pH of the FA mixture was adjusted to 5.0 with 0.05 M acetate buffer. pH 5 was selected because it is the average pH of the uncontaminated portion of the F-Area groundwater (Otosaka et al., 2011) and the optimal pH for lactoperoxidase-catalyzed iodination of the NOM to occur (Xu et al., 2011b). KI was added to the reaction vessel at a concentration of 10−4 M. Lactoperoxidase and H2O2 were then successively added to reach a final concentration of 20 μg/ml and 5 mM, respectively. In order to track the molecular modification of FA by iodination, a control sample was prepared identically, with the addition of lactoperoxidase and H2O2 but without iodide. The mixture was shaken in darkness for 2 h. Xu et al. (2011a) determined previously that the molecular weight of FA extracted from the SRS F-Area surface soil was >1 kDa. Thus, an ultrafiltration Microsep centrifugal device with a cutoff of 1 kDa (Pall Corporation, USA) was used to remove non-bound I−, IO3− or other salts from iodinated FA. Ultrafiltration efficiency was checked by measuring the absorbance of the original, the retentate (>1 kDa fraction) and the permeate (b 1 kDa) at 280 nm with a UV spectrophotomer. We established that FA loss in the permeate accounted for b 10% of the original solution. Iodinated FAs and control FAs were washed of salts and dissolved iodine species (b1 kDa) and then lyophilized for FTICR-MS analysis. The incorporation of iodine into FA was validated by a method of combining furnace combustion and GC–MS analysis (Zhang et al., 2010) 2.2. FTICR-MS instrumentation and data processing approach Control and iodinated FAs, generated by either non-enzymatic or enzymatic means, were analyzed by both negative and positive ion modes using an Apollo II ESI ion source of a Bruker Daltonics 12 Tesla Apex Qe ESI FTICR-MS, at the College of Science Major Instrumentation Cluster (COSMIC), Old Dominion University, Virginia. Compounds with basic nitrogen (e.g., protein) were ionized better in the positive ion mode, whereas the majority of humic substances containing acidic carboxyl and phenolic groups, i.e., those moieties that are more prone to ionize, were identified in the negative ion mode (Kujawinski et al., 2001; Pfeifer et al., 2001). Duplicates were run to ensure reproducibility. After calibration (see the supplemental contents), mass lists of m/z values with an S/N ≥ 4, ranging from 200 to 800 m/z unit and their peak intensities were imported into a spreadsheet. A molecular formula calculator (Molecular Formula Calc version 1.0 ©NHMFL, 1998) generated matching formulas using carbon (C) (range of possible atoms in structure: 1–50), hydrogen (H) (1–100), oxygen (O) (0–30), nitrogen (N) (0–5), sulfur (S) (0–3), phosphorus (P) (0–3) and iodine (I) (0–3) for negative ion mode, and C (1–50), H (1–100), O (0–30), N (0–5), S (0–3), sodium (Na) (0–1) and I (0–3) for positive ion mode. Considering ionization, for the formulas obtained from the calculator, one hydrogen was added in negative ion mode, and one subtracted in positive ion mode if they did not contain Na+. Mass values with more than one possible formula were assigned according to the selection criteria, which were applied in previously published studies (Chen et al., 2011; Stubbins et al., 2010). After that, mass values above 500 Da which still

contain multiple formulas were then assigned through the detection of homologous series (Minor et al., 2012). Details on how Cl− and 13C peaks were treated and removed are given in the Supplemental contents. In the identified organo-iodine formulas, iodine was replaced by a hydrogen atom to revert the molecule to the one that only contains C, H, O, N, S and P. All compounds were thus grouped into three categories: 1) reactive (present in the spectra of the un-iodinated control FAs but not in the spectra of the iodinated FAs), 2) resistant (present in both spectra), and 3) produced (present only in the spectra of the iodinated FAs). Additionally, the m/z lists of the iodinated and un-iodinated FAs were also compared and searched for peaks differing by 125.89664±0.0005, the exact mass difference between 127I and 1H, representing one iodine atom ‘conservatively’ replacing one hydrogen atom through electrophilic substitution. Therefore, it is possible that organo-iodine compounds were found in either the “resistant” or “produced” pool. The situation of multiple iodinated species (i.e., more than one hydrogen replaced by iodine) was also considered. UV absorbance at 280 nm and the fluorescence intensity, which were used to indicate the overall change of the sample after iodination, was also measured (see the Supplemental contents). 3. Results and discussion 3.1. Mass spectra of un-iodinated and iodinated FAs The FA incorporated I− in the enzymatic experiment accounted for (23±3)% of the total initially added amounts at 127I− concentrations of 10−4 M, whereas the FA incorporated IO3− in the non-enzymatic experiment accounted for (14±1)% of the total initially added amounts, at 127IO3− concentrations of 10−4 M, corresponding to I/C atom ratios of 4×10−4 and 2×10−4, respectively. Though the FA molecules also contain naturally-bound iodine (Xu et al., 2011a), formulas for those naturally-occurring organo-iodine compound were not observed in the spectra of control FAs, likely due to their relatively low abundance, compared to those formed in laboratory experiments. Fig. 1a shows the negative ion mode mass spectra of un-iodinated and iodinated FAs with iodate through the non-enzymatic reaction, while Fig. 1b displays the control and iodinated FAs with iodide through the enzymatic reaction. Positive ion mode mass spectra of un-iodinated and iodinated FAs through both reactions were provided in the supplemental contents (Fig. A1). Although intensities of ESI FTICR-MS are not quantitative, all of the FA samples were analyzed on the same instrument with the same procedure, and thus the resulting spectra should reflect similar systematic biases (Wozniak et al., 2008). Moreover, it is assumed that alterations of ionization efficiency, one of the main factors that affect the quantitative capability of ESI FTICR-MS, between the control FA and the iodinated FA should be insignificant (Lee, 1967; Moulin et al., 2001). Average molecular weight (MW) normalized by intensity consistently decreased from 439 to 421 m/z and 428 to 422 m/z for all identified peaks illustrated in negative and positive ion mode mass spectra, respectively, during the process of nonenzymatic iodination (Table 1). However, during enzymatic iodination, it slightly increased from 412 to 416 m/z for those detected by both ion modes (Table 1). During the non-enzymatic reaction, peak numbers generally decreased by 15% in both ion modes, and increased by 34% and 6% in the negative ion mode and the positive ion mode during the enzymatic reactions, respectively. The difference in the changes observed for average MWs and peak numbers in the two reactions suggest different molecular alteration of the NOM by the two methods. Contrary to expectations, the iodinated FA did not significantly increase the MW, when compared to the control FA, as the addition of one iodine atom (MW = 127) into the FA molecule should increase the overall MW of FA. Instead, the average MW of FA was somewhat lower in the case of the iodination with iodate. This is likely due to the fact that the average mass loading of iodine

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Fig. 1. a) Negative EST-FTICR mass spectra of fulvic acid (FA) amended with IO3− through non-enzymatic reaction. b) Negative EST-FTICR mass spectra of FA amended with I− through enzymatic reaction.

into FA in both non-enzymatic and enzymatic reactions (I/C atom ratios of 2 × 10 − 4and 4 × 10 − 4, respectively) is too low for one to observe any significant increase in average MW. It was noticed in a previous study that iodination of FA with I2 led to the appearance of a partially resolved shoulder on the low mass side for the iodinated FA (Moulin et al., 2001). However, as non-bound iodide was not isolated from FA in that experiment, it is possible that the presence of excessive anions suppressed the ionization efficiency and thus changed the mass spectra pattern of the iodinated FA, compared to that of the un-iodinated FA (Sleighter and Hatcher, 2007). In the current study, both un-iodinated FA and iodinated FA were extensively

diafiltered against de-ionized water (18.1 MΩ) and therefore any modification of the ionization efficiency of iodinated FA compared to that of un-iodinated FA caused by the presence of salts should be minimal. Nevertheless, caution is still needed when comparing the mass spectra of un-iodinated FA with the iodinated FA because ESI is selective and only semi-quantitative, whereby the signals of some compounds are ‘magnified’, and some are ‘depressed,’ while other signals fall outside the analytical window. Not all the reactive, resistant, and produced compounds can be identified by this approach, and not all the produced compounds were necessarily derived from the reactive pool. Rather, one has to discuss the compositional change of FA after iodination

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Table 1 Molecular alteration of fulvic acid (FA) after reacting with IO3− through non-enzymatic pathway or I− through enzymatic pathway. Negative mode

Iodate: non-enzymatic reaction Before iodination

Iodide: enzymatic reaction After iodination

Before iodination

After iodination

Peak no.

% relative intensity

Averaged MW

Peak no.

% relative intensity

Averaged MW

Peak no.

% relative intensity

Averaged MW

Peak no.

% relative intensity

Averaged MW

All identified formulate Reactive Resistant Produced Iodinated Aliphatic Aromatics Condensed aromatics CRAM

1711 576 1135 – – 155 237 34 873

100 25 75 – – 15 11 1 47

440/439* 426 480 – – 390 422 408 461

1452 – 1135 317 13 107 328 82 675

100 – 86 14 0.72 15 20 4 40

421/421* – 423 409 435 365 413 396 439

1883 1528 355 – – 109 758 237 548

100 12 88 – – 17 37 10 23

413/412 448 409 – – 312 425 409 451

2517 – 1528 989 22 97 1239 479 425

100 – 75 25 0.49 7 54 19 13

416/416* – 416 417 350 329 418 403 437

Positive mode

Iodate: non-enzymatic reaction Before iodination

All identified formulate Reactive Resistant Produced Iodinated Aliphatic Aromatics Condensed aromatics CRAM

3233 634 2599 – – 702 570 257 997

100 3 97 – – 19 11 4 33

Iodide: enzymatic reaction After iodination

407/428* 470 405 – – 403 409 419 420

2753 – 2599 154 16 659 464 189 801

100 – 99 1 0.09 19 9 3 32

Before iodination 401/422* – 401 415 349 401 398 407 412

2614 291 2323 – – 971 727 569 382

100 4 96 – – 48 15 12 21

After iodination 399/412* 422 398 – – 388 438 446 398

2775 – 2323 452 78 1014 745 577 118

100 – 93 7 0.80 39 17 12 33

401/416* – 401 406 396 390 428 438 397

Note: Average MW is intensity-normalized molecular weight, after correction for ionization, iodination and sodium adduction, thus the formulas only contains C, H, O, N, S, P for negative ion mode and C, H, O, N, S for positive ion mode; * is the intensity-normalized molecular weight actually observed in the FTICR mass spectra; aromatics are molecules with an aromatic index (AI) larger than 0.5. AI=1+C-O-S-0.5H)/(C-O-S-N-P); Condensed aromatics, AI ≥ 0.67; Carboxylic-rich alicyclic molecules (CRAM), which was recently reported as one of the major refractory nonliving components of freshwater DOM and likely derived from terpenoids, are defined as: double bond equivalent (DBE): C=0.30-0.68, DBE: H=0.20- 0.95; DBE: O=0.77-1.75, DBE=1+ 0.5×(2C-H+N+P). %, relative intensity was the percentage of the summed intensity of one category (e.g., all “resistant” compounds) in the total peak intensity of the whole sample.

more qualitatively. In spite of all these limitations, one can still benefit from the spectrum comparison between un-iodinated and iodinated FAs to explore the molecular variations of NOM during the iodination processes that have never before been recognized. 3.2. Molecular composition alteration of FA during non-enzymatic iodination The compositional variation of FA after iodination through both enzymatic and non-enzymatic pathways can be well visualized in the van Krevelen diagrams (Fig. 2a–d and Fig. A2). Van Krevelen diagrams provide a useful approach for assigning different classes of environmental biomolecules based on their elemental ratios (O/C vs. H/C). By comparing the van Krevelen diagrams of both control and iodinated FAs in the non-enzymatic reaction under negative ion mode in one plot (Fig. 2a), compounds that were seen in the un-iodinated FA but not in the iodinated FA, referred to “reactive pool”, clustered mostly in the region of lignins and tannins, or the region of carboxylic-rich alicyclic molecules (CRAM), which were recently reported as the one of the major refractory nonliving components of freshwater DOM and likely derived from terpenoids (Hertkorn et al., 2006). This indicates that these lignin, tannin-like compounds, or the CRAM, are involved in the reduction of iodate into reactive iodine species and “disappeared” in the spectrum of the iodinated FA. The oxidation of diphenols (e.g., hydroquinones and catechols) to corresponding quinones or semiquinones by iodine or bromate has been well established (Kolthoff, 1926; O'Donoghue et al., 1999), whereas the interaction between iodate and hydroquinone type functional groups has only recently been reported (Steinberg et al., 2008). Hydroxylated aromatic electron donors (i.e., hydroquinones) and quinoid electron acceptors (i.e., quinone or semiquinone), which are ubiquitously and abundantly present in DOM, and likely degradation products of lignins and tannins (Aeschbacher et al., 2012; Cory and McKnight, 2005; Del Vecchio and Blough, 2004; Tossell, 2009), might provide free radicals during transformation (Czechowski et al., 2004; Jerzykiewicz et al.,

2002), and thus act as electron shuttle for the reduction of iodate to iodide. Reactive compound formulas are spatially scattered throughout the van Krevelen diagram drawn from the positive ion mode mass spectra for both non-enzymatic and enzymatic reactions (Fig. 2c and d). Indeed, the very different van Krevelen diagram patterns shown in negative and positive ion modes (Fig. 2a vs. 2c and 2b vs. 2d) can be explained by the fact that both ion modes detect different components of FAs. However, the negative ion mode is more representative than the positive ion mode needed for the full characterization of humic substances, as humic substances consisted mainly of negatively-charged acidic functional groups, such as phenolic and carboxylic acids (Piccolo and Spiteller, 2003), thus would favor a negative ionization. Positive ion mode, under which compounds containing a basic nitrogen are better ionized, still represent different components of humic substances from those found using the negative ion mode, whereas any bias would have been caused only if one would have focused on it for the characterization of the whole FA assemblage (Pfeifer et al., 2001). Therefore, data interpretation concerning the overall compositional variation of the whole FA was focused on negative ion mode mass spectra (Sections 3.2– 3.3), whereas data from both modes were investigated to look into the characteristics of organo-iodine compounds (Section 3.4). The newly-produced peaks mostly fell into the region of condensed aromatics and partially in the low H/C value region of lignin (Fig. 2a). The oxidation of hydroquinone to quinone decreased the H/C values, thus moving the compounds towards the lower H/C value region. The oxidation should also have caused a decrease in aromaticity, as the oxidation can break the aromatic ring. Interestingly the intensityaveraged aromatic index (AI=(1+C–O–S–0.5H)/(C–O–S–N–P; (Koch and Dittmar, 2006)) of the overall sample increased from 0.08 to 0.21 after the amendment of iodate, with a higher AI contributed by the newly-produced compounds (0.24), compared with that of the reactive compounds (~0.00) (Table 2). Though the validity of AI to estimate

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Fig. 2. van Krevelen diagram of reactive and produced compounds in the a) negative ion mode mass spectrum for fulvic acid (FA) amended with IO3− through non-enzymatic; b) negative ion mode mass spectrum for FA amended with I− through enzymatic reaction; c) positive ion mode mass spectrum for fulvic acid (FA) amended with IO3−; and d) positive ion mode mass spectrum for FA amended with I−.

aromatics is under debate (Koch and Dittmar, 2006), it is important to realize that ESI is very selective and can have varying ionization efficiencies among classes of compounds. Nevertheless, the overall fluorescence intensity and UV absorbance (λ =280 nm) did not significantly increase from the control FA to the iodinated one (aka Paired Comparisons t-Test, n = 3, p > 0.05, SPSS 17.0) (Table 2), suggesting either the hydroquinone–quinone conversion by iodate was negligible, or it was compensated by the formation of more condensed aromatic compounds after the addition of iodate, of which the mechanism is still not clear. Encouragingly, ESI-FTICR-MS can still capture subtle molecular signature variations and more studies are warranted to examine further this reactive pool towards iodate in the NOM extracted from other contaminated DOE sites. 3.3. Molecular composition alteration of FA during enzymatic iodination During the enzymatic iodination process, both H2O2 and lactoperoxidase were added to the un-iodinated and iodinated FAs. In addition, the interaction between humic substances and iodide is only minimal at pH 5 or higher, if no other catalytic reagents are present (Xu et al., 2011b). Therefore one can assume that iodinated FAs through the enzymatic pathway should undergo minimal molecular modification with respect to its un-iodinated control FA. However, modification of the molecular signature of FA is still observed after catalytic iodination, with the ‘reactive’ pool being distributed in the region of lipids, proteins and lignins and the ‘produced’ pool mostly occurring in the condensed aromatics (Fig. 2b). Another interesting observation to confirm this trend is

a higher intensity-averaged aromatic index of the FA amended with iodide (0.47), compared to that without iodide (0.36) (Table 2). One of the plausible explanations is that the presence of I− in the iodideamended group would compete with peroxidase for H2O2, leading to a partial decomposition of H2O2 by I− or the enzyme-bound hypoiodite (Magnusson et al., 1984). Furthermore, there would be a buildup of H2O2 concentrations in the un-iodinated control FA, where this enzymatic reaction would not occur. This excessive H2O2 further oxidized more of the labile functional groups of un-iodinated FA, and likely transformed them into more saturated, less aromatic compounds, or even decomposed them into CO2. Strictly speaking, in the enzymatic reaction, the uniodinated FA is not a real control group and the ‘reactive’ and the ‘produced’ pools should somehow be inversely related. Regardless, this explains well why aliphatic (lipids and proteins) and partially lignins, were observed in the un-iodinated FA ‘reactive’ pool with a relatively low intensity-averaged aromatic index of 0.14. The ‘reactive’ pool was then the oxidation products of the apparent ‘produced’ pool, consisting mainly of condensed aromatics (AI: 0.64) in the iodinated FA. It also explains why both increasing average MW and peak number were found in the iodide-amended samples due to the oxidation and reworking of the control group by the relatively high H2O2 concentration. In contrast, decreasing average MW and peak number were observed in the iodate-amended sample due to the oxidation and breakdown of FA by IO3− as the oxidant. In support of these observations from the enzymatic assisted iodination, the overall fluorescence intensity and UV absorbance (λ = 280 nm) did not dramatically change after enzymatic iodination,

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Table 2 Variations of intensity-normalized average aromatic index (AI), fluorescence emission intensity integrated from 480 – 800 nm (excitation wavelength: 465 nm) and ultra-violet absorbance at 280 nm of fulvic acid (FA) before and after iodination, from IO3− (non-enzymatic reaction) and I− (enzymatic reaction). All samples were diluted by the same factors for UV and fluorescemce analyses. Error was calculated based on three replicates. Iodate: non-enzymatic reaction Before iodination

Wholeb Reactive Produced a b

Iodide: enzymatic reaction After iodination

Before iodination

After iodination

AIa

Fluorescence

UV

AI

Fluorescence

UV

AI

Fluorescence

UV

AI

Fluorescence

UV

0.08 0.00 –

28121 ± 367 – –

0.513 ± 0.001 – –

0.21 – 0.24

28452 ± 348 – –

0.518 ± 0.000 – –

0.36 0.14 –

31943 ± 19 – –

0.487 ± 0.005 – –

0.47 – 0.64

32484 ± 107 – –

0.495 ± 0.016 – –

AI = (1 + C–O–S–0.5H) /(C–O–S–N–P), spectra obtained under negative ion mode was used to calculate AI, and its error was b5% based on two duplicate ESI-FTICR-MS analyses. ‘Whole’ means the whole sample.

indicating molecular variation, if any, should be minimal. It was also previously reported that degradation of humic substances in the presence of peroxide only is not significant (Wang et al., 2001), when conducted in darkness. 3.4. Characterization of organo-iodine compounds The two duplicate mass spectra of each sample to test reproducibility were quite consistent: 1) standard deviations of individual absolute peak heights wereb 5%; 2) peaks that were different in the two duplicate spectra only accounted for b 2% of the total peaks and their S/N values were mostly lower than 4; 3) the peaks denoting organo-iodine compounds had an average signal-to-noise ratio of ~7 and were indeed found in both duplicate spectra. All available evidence demonstrates that ESI-FTICR-MS spectra are very reproducible and reliable when used to identify iodinated products. A full formula list of the organo-iodine compounds is provided in the supplemental contents. Organo-iodine species formed from IO3− and I − under different ESI modes are specifically displayed in the van Krevelen diagram (Fig. 3a). A total of 129 organo-iodine compounds were identified from both positive ion mode (n= 94) and negative ion mode (n= 35), yet without any identical formula between these two modes. This suggests that it provides complimentary molecular information on organo-iodine compounds formation, although the positive ion mode is not quantitative for characterizing the whole molecular assemblage of humic substances. Thus, organo-iodine compounds detected in the positive ion mode are not a subset of those detected by the negative ion mode (Pfeifer et al., 2001). Instead, more organo-iodine compounds were identified in the positive ion mode than in the negative ion mode. All the identified organo-iodine compounds contained only one iodine, indicating that iodination of humic substances is dominated by mono-substitution. Only 9% of the total identified iodinated compounds were categorized into the resistant pool, which means only a small amount of molecules resulted in ‘conservative’ iodination, i.e., one iodine ‘conservatively’ replacing one hydrogen without any modification of the adjacent molecular signature (Fig. 3b). In most cases (91% of the total identified organoiodine formulas), iodine is covalently bound to the newly-produced components, which may be all or partially derived from the ‘reactive’ pool (Fig. 3a). Generally, the organo-iodine compounds are distributed in three classes, ranking from most to least: unsaturated hydrocarbons > lignin > protein (Fig. 3a). The O/C ratios of these organo-iodine compounds were mostly lower than 0.4 and more than half (~53%) were lower than 0.2. Even those structures defined as lignin-type compounds were skewed towards the lower O/C region (b 0.4) (Fig. 3a,b). Peaks assigned to the region of unsaturated hydrocarbons (Sleighter and Hatcher, 2007) must contain an aromatic structure because other unsaturated structures, such as alkene or alkyne, are not reactive towards iodine (McMurry, 1984). The relatively low O/C values of organo-iodine compounds could have two important implications. On one hand, the presence of a

limited number of oxygen containing functional groups on aromatic structures, such as hydroxyl (HO-Aryl), or an ester functionality through the C\O\C bond (Aryl\O\(C_O)-R) (Xu et al., 2012), add electron density to the π system and make it more favorable for reactive iodine species to attack its ortho- or para-position that is yet not occupied by any substituent (i.e., least sterically hindered). On the other hand, the relatively low oxygen content decreases the overall hydrophilicity of the organo-iodine molecule and likely retards its mobility in the environment. However, further experiments, such as testing the affinity of the iodinated FAs for mineral surfaces and examining their relative sorptivity to obtain direct evidence of potential immobility are needed. An aromatic-core-aliphatic-sidechain structure was earlier suggested from a statistical investigation among various functional groups of several humic substances determined by NMR techniques, their respective naturally-occurring 127I and 129I contents, and the laboratory determined uptake partitioning coefficients (Kd) (Xu et al., 2012). Though our study cannot provide direct and unambiguous evidence for such a structure, it is reasonable to assume that this aromatic-core-aliphatic-sidechain structure lowers the O/C value and retains some degree of un-saturation. Nevertheless, the aromatic core likely acts like a ‘hotspot’ to scavenge radioiodine while the aliphatic chain surrounding it acts as a hydrophobic barrier, rendering the molecule an ideal natural strategy for remediating radioiodine. As organic carbon in this FA accounts for 46% of the total weight (Xu et al., 2011a), and 13.7% of the total organic carbon was characterized as aromatic carbon by 13C DPMAS NMR (Xu et al., 2012), it theoretically has 8.75×10−4 mol C per gram of the FA available for the formation of C\I bonds, assuming that, based on stoichiometric grounds, one out of six aromatic carbons is, ready for iodine electrophilic substitution. Assuming the relative intensities of ESI-FTICR-MS spectra could give an indication of the relative abundance of organo-iodine molecules present in the FA, the peaks of organo-iodine compounds would take up only (0.09–0.80) % of the total intensities of the whole sample (Table 1), when I− or IO3− was amended to FA at a concentration of 10−4 M. This resulted in 1.0×10−5 to 8.5×10−5 mol C per gram of the FA associated with iodine, if one assumes that iodine is bound to a common phenolic core of the building blocks with a formula of Aryl-OH (C6H6O). This rough estimation and comparison suggest that iodination of FAs occurs at unique and very limited sites, about 10 to 88 times lower than all theoretically available binding sites (8.75×10−4 mol C per gram of the FA). This is consistent with the results reported in Xu et al. (2012), in which a negative linear regression was observed between the logarithm of the uptake partitioning coefficients (Kds) of I− or IO3− onto NOM and the logarithm of amended I− or IO3− concentrations. This suggests that there are probably heterogeneous binding sites with different affinities for iodine binding in the NOM. Access of iodine into the aromatic structures of humic substances might also be sterically hindered in the natural environment. It is important to also point out that only a small portion of the organo-iodine was associated with protein-like compounds (Fig. 3a). Phenylalanine, tyrosine and tryptophan are the common amino acids

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Fig. 3. a) van Krevelen diagram of all organo-iodine identified. b) van Krevelen diagram of positive and negative ion mode mass spectra for organo-iodine ‘conservatively’ formed from IO3− or I− and fulvic acid (FA) through non-enzymatic (starting from IO3−) and enzymatic (starting from I−) reactions.

with aromatic structures. This is in agreement with studies published by Xu et al. (2011a), in which no significant correlation between the sum of the aromatic amino acid concentrations measured by HPLC and 127I or 129 I contents in several humic substances was found, indicating these protein-like compounds are not the dominant form, even though they could be possible hosts for radioiodine in the natural environment. One notable feature of the organo-iodine compounds is that ~69% of the identified total organo-iodine species contain nitrogen (Table A1-4). Nitrogen, presumably present as \NH2 or \HNCOR groups in humic substances (Reckhow et al., 1990), is a ring-activating functionality and thus would enhance the nucleophilicity of the aromatic ring. This is consistent with the findings of Xu et al. (2012) that N/C ratios of several humic substances positively correlate with their respective naturally occurring 127I, 129I contents, and laboratory uptake Kds of humic substances amended with I− or IO3− at ambient concentrations. There are still constrains in laboratory iodination experiments, in which iodine is artificially incorporated to the NOM, and it is hard to really mimic the more complex environmental condition. Moreover, it remains a challenge to define all the molecular structures of the resulting organoiodine compounds, as one formula might correspond to a number of isomers. Nevertheless, this study, with the help of ESI-FTICR-MS, for the first time reveals the molecular reaction mechanism of iodine sequestration into NOM as well as the structure nature of resulting organo-iodine compounds. Further studies utilizing other techniques, such as MS/MS, might be needed to fragment these organo-iodine compounds and elucidate their structures.

4. Conclusions This work not only reveals important and novel information on the transformation mechanisms of both NOM and iodine species through non-enzymatic and enzymatic pathways, but it also enriches the previous studies on organo-iodine compounds using ESI Q-TOF mass spectrometry (Moulin et al., 2001) or NMR techniques(Xu et al., 2012). It further provides more in-depth insight and interpretation on the sources and structures of the organo-iodine compounds. Although ESI-FTICR-MS observations were based on stable iodine, it is reasonable to assume that the reaction mechanisms and organo-iodine characteristics discovered in this study should also apply to 129I. Even though this FA was extracted from one particular contaminated site at the Savannah River Site, our results should be considered generally applicable for describing the interactions between NOM and radioiodine in other radionuclide contaminated sites of the similar climate and vegetation covering. Information obtained from this study is thus vital for interpreting

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