Molecular characterization of dissolved organic matter in glacial ice: coupling natural abundance 1H NMR and fluorescence spectroscopy

Glaciers and ice sheets are the second largest freshwater reservoir in the global hydrologic cycle, and the onset of global climate warming has necessitated an assessment of their contributions to sea-level rise and the potential release of nutrients to nearby aquatic environments. In particular, the release of dissolved organic matter (DOM) from glacier melt could stimulate microbial activity in both glacial ecosystems and adjacent watersheds, but this would largely depend on the composition of the material released. Using fluorescence and (1)H NMR spectroscopy, we characterize DOM at its natural abundance in unaltered samples from a number of glaciers that differ in geographic location, thermal regime, and sample depth. Parallel factor analysis (PARAFAC) modeling of DOM fluorophores identifies components in the ice that are predominantly proteinaceous in character, while (1)H NMR spectroscopy reveals a mixture of small molecules that likely originate from native microbes. Spectrofluorescence also reveals a terrestrial contribution that was below the detection limits of NMR; however, (1)H nuclei from levoglucosan was identified in Arctic glacier ice samples. This study suggests that the bulk of the DOM from these glaciers is a mixture of biologically labile molecules derived from microbes.     

Direct hydrodynamic radius measurement on dissolved organic matter in natural waters using diffusion NMR

Dissolved organic matter from natural waters is a complex mixture of various chemical components, which play vital roles in many environmental processes such as the global carbon cycle and the fate of many key anthropogenic pollutants. Despite its environmental significance, dissolved organic matter in natural form has never been studied using nuclear magnetic resonance based hydrodynamic radius measurements due to its extremely low concentration (e.g., a few mg/L) in natural waters. In this study, NMR-based hydrodynamic radius measurements were performed directly on unconcentrated pond, river, and sea waters. The key chemical components of the dissolved organic matters from different sources were identified as carbohydrates, carboxyl-rich alicyclic molecules, and aliphatic molecules. By using the Stokes-Einstein-Sutherland equation, the average hydrodynamic radii of the three key components were calculated.     

Quantification of dissolved organic carbon at very low levels in natural ice samples by a UV-induced oxidation method

The study of chemical impurities trapped in solid precipitation and accumulated in polar ice sheets and high-elevation, midlatitude cold glaciers over the last several hundreds of years provides a unique way to reconstruct our changing atmosphere from the preindustrial era to the present day. Numerous ice core studies of inorganic species have already evaluated the effects of growing anthropogenic emissions of SO(2) or NO(x) on the chemical composition of the atmosphere in various regions of the world. While it was recently shown that organic species dominate the atmospheric aerosol mass, the contribution of anthropogenic emissions to their budget remains poorly understood. The study of organics in ice is at the infancy stage, and it still is difficult to draw a consistent picture of the organic content of polar ice from sparse available data. A UV oxidation method and IR quantification of CO(2) was optimized to obtain measurements of dissolved organic carbon content as low as a few ppbC. Stringent working conditions were defined to prevent contamination during the cleaning of ice. Measurements in various ice cores corresponding to preindustrial times revealed dissolved organic carbon content of less than 10 ppbC in Antarctica and up to 75 ppbC in alpine ice.     

Nature and abundance of organic radicals in natural organic matter: effect of pH and irradiation

Dissolved natural organic matter (NOM) plays an essential role in freshwater geochemical and biochemical processes. A major property, its redox behavior, can be attributed to the chinone building blocks, which can form stable radicals. However, electron paramagnetic resonance (EPR) data indicating free radicals on solid NOM are sparse. Here we present EPR spectra of 23 NOM from European surface waters isolated by reverse osmosis. The organic radical concentrations of NOM ranged from 5 x 10(15) to 1.84 x 10(17) spins g(-1), and g values ranged from 2.0031 to 2.0045. Number and type of organic radicals in solid NOM are significantly influenced by the pH of raw water. EPR experiments indicate the presence of semiquinone-type radicals in coexistence with carbon-centered "aromatic" radicals, with the semiquinone-type radicals dominating at alkaline pH. Basically these processes are reversible. Organic radical concentrations in NOM adjusted to pH 6.5 before freeze-drying correlate with iron and aluminum contents. UV- and VIS-irradiation of solid NOM can lead to more than a 10-fold increase of the concentration of organic radicals. These radicals were long-lived and had the same g value as the original radical. Similar effects were not observed with isolated humic and fulvic acids, demonstrating the limited reflection of environmental properties of organic carbon by the classical isolation procedure.     

Fractionation of natural organic matter in drinking water and characterization by 13C cross-polarization magic-angle spinning NMR spectroscopy and size exclusion chromatography

Natural organic matter from drinking water sources was fractionated, and the fractions were characterized by NMR and SEC with the aim of relating NOM structure to treatability. Organic matter was isolated from two Australian surface waters (Horsham, Moorabool) by reverse osmosis and from a groundwater (Wanneroo) by anion exchange. The isolates were fractionated according to polarity and charge by resin adsorption. 13C NMR spectra of the freeze-dried fractions showed the most hydrophobic fraction to be high in aliphatic and aromatic carbon while slightly hydrophobic organics have more carbonyl and alkoxyl carbon. The Horsham and Wanneroo hydrophilic fractions show strong alkoxyl signals attributed to carbohydrate. Moorabool hydrophilics contain aromatic (phenolic) carbon; the apparent absence of carbohydrate appears to be an artifact. Size-exclusion chromatograms were recorded on the original and fractionated organics with both UV and dissolved organic carbon detection. The Horsham and Moorabool organics have similar molecular size distributions while Wanneroo is dominated by strongly absorbing species having large hydrodynamic radii. The hydrophobic and charged hydrophilic fractions also have high apparent MW, while the neutral fraction is higher in low-MW compounds of relatively low specific absorbance, suggestive of carbohydrates.     

Fluorescence spectroscopy reveals ubiquitous presence of oxidized and reduced quinones in dissolved organic matter

Excitation-emission matrixes (EEMs) of 379 dissolved organic matter (DOM) samples from diverse aquatic environments were modeled by parallel factor analysis (PARAFAC). Thirteen components likely representing groups of similarly fluorescing moieties were found to explain the variation in this data set. Seven of the thirteen components were identified as quinone-like based on comparison of their excitation and emission spectra to spectra of model quinones. These quinone-like fluorophores were found to vary in redox state and degree of conjugation. Two components were identified as amino acid-like based on comparison to tyrosine and tryptophan fluorescence spectra. The other four components are not yet associated with any class of molecules. The quinone-like fluorophores account for about 50% of the fluorescence for every sample analyzed, showing that quinone-like fluorophores are an important and ubiquitous fluorescing moiety and in natural waters. Further, the distribution of the quinone-like fluorophores was evaluated as a function of environmental and laboratory redox gradients. Under reducing conditions, the contribution of the reduced quinone-like fluorophores increased concurrentwith a decrease in the oxidized quinone-like fluorophores, indicating that DOM fluorescence is a function of redox state of quinone-like moieties. Lastly, a ratio of two quinone-like fluorophores was found to explain the variation in the fluorescence index. These results provide new insight into the redox reactivity of DOM and have implications for the application of fluorescence spectroscopy as a tool to characterize DOM.     

Production of hydrated electrons from photoionization of dissolved organic matter in natural waters

Under UV irradiation, an important primary photochemical reaction of colored dissolved organic matter (CDOM) is electron ejection to produce hydrated electrons (e-aq). The efficiency of this process has been studied in both fresh water and seawater samples with both steady-state scavenger (S-SS) and time-resolved laser flash photolysis (LFP) methods. However, the apparent quantum yields (AQYs) of e-aq for the same samples using the two methods differ by as much as a factor of 100, necessitating a closer re-examination of how the process is measured. We developed a highly sensitive multipass LFP apparatus that allows detection of transient species at very low and variable UV irradiation intensities. Under single-photon conditions, we measured the AQY of e-aq from Laurentian fulvic acid as 1.3 x 10(-4), and set the upper limit for other CDOM samples at 6 x 10(-5), bringing the LFP results into agreement with those from S-SS methods. We also examined the ionization at elevated irradiation intensities and clearly demonstrated that multiphoton ionization occurs at intensities well below those usually used in LFP experiments, but well above those likely to occur at the earth's surface. This multiphoton ionization is probably the cause of the high AQYs reported by earlier LFP work. In addition, we also observed in real time other photochemical reactions, such as triplet quenching and bleaching, in the single photon regime.     

HILIC-NMR: toward the identification of individual molecular components in dissolved organic matter

This article presents research targeted toward the isolation and detection of unique molecular structures from what is believed to be the world's most complex organic mixture: dissolved organic matter (DOM). Hydrophilic interaction chromatography (HILIC) was used to separate Suwannee River DOM (SRDOM) into 80 fractions, simplified to the extent that detection with nuclear magnetic resonance spectroscopy (NMR) results in many sharp signals that are indicative of individual compounds, some of which are identifiable with multidimensional NMR. Parallel factor analysis (PARAFAC) of fluorescence excitation-emission matrices (EEMs) was additionally employed on HILIC-simplified fractions to further confirm the effectiveness of the HILIC separations as well as draw insight into how structural characteristics relate to DOM fluorescence signals. Findings suggest that material believed to be derived from both cyclic and linear terpenoids was dominant in the most hydrophobic fractions as were the majority of the fluorescence signals, whereas hydrophilic material was highly correlated with carbohydrate-type structures as well as high contributions from amino acid fluorescence. NMR spectra of DOM, typically featureless mounds, are substantially more detailed with HILIC-simplified fractions to the point where hundreds of signals are present and 2D NMR correlations permit significant structural identifications.     

Solid-state and multidimensional solution-state NMR of solid phase extracted and ultrafiltered riverine dissolved organic matter

In this study we used multidimensional solution-state NMR to elucidate the differences in the chemical composition of solid phase extracted and ultrafiltered DOM isolates. DOM was isolated from water sampled from an oligotrophic river, the River Tagliamento (Italy). The recovery of total DOM was up to 42% with both isolation techniques. In addition to 1- and 2-D solution-state NMR, we also applied 1-D solid-state 13C NMR spectroscopy for DOM characterization. 13C NMR spectroscopy only produced broad overlapping resonances, thus allowing a bulk characterization of DOM composition. However, it demonstrated that the bulk chemical composition of the two DOM fractions exhibited minor spatial-temporal changes. The 2-D experiments (TOCSY, HMQC) showed that the solid phase extracted hydrophobic DOM contained predominantly aliphatic esters, ethers, and hydroxyl groups, whereas the ultrafiltered DOM was comprised partially of peptides/protein, with further evidence for a small amount of aliphatic/fatty acid material. Sugars were present in both DOM fractions. The results show the two isolation techniques selected for different suites of compounds within the bulk DOM pool.     

Molecular and structural characterization of dissolved organic matter from the deep ocean by FTICR-MS, including hydrophilic nitrogenous organic molecules

Dissolved organic matter isolated from the deep Atlantic Ocean and fractionated into a so-called hydrophobic (HPO) fraction and a very hydrophilic (HPI) fraction was analyzed for the first time by Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) to resolve the molecular species, to determine their exact masses, and to calculate their molecular formulas. The elemental composition of about 300 molecules was identified. Those in the HPO fraction (14C age of 5100 year) are very similar to much younger freshwater fulvic acids, but less aromatic and more oxygenated molecules are more frequent. This trend continues toward the HPI fraction and may indicate biotic and abiotic aging processes that this material experienced since its primary production thousands of years ago. In the HPI fraction series of nitrogenous molecules containing one, two, or three nitrogens were identified by FTICR-MS. Production spectra of the nitrogenous molecules suggest that the nitrogen atoms in these molecules are included in the (alicyclic) backbone of these molecules, possibly in reduced form. These mass spectrometric data suggest that a large set of stable fulvic acids is ubiquitous in all aquatic compartments. Although sources may differ, their actual composition and structure appears to be quite similar and largely independent from their source, because they are the remainder of intensive oxidative degradation processes.     

Effect of dissolved natural organic matter on the kinetics of ferrous iron oxygenation in seawater

We have investigated the kinetics of Fe(II) oxygenation in seawater in the presence of a variety of natural organic materials obtained from vegetation near Moreton Bay, Queensland. Natural organic matter (NOM) was observed mostly to accelerate Fe(II) oxygenation, but in some cases oxidation was retarded. We fitted a previously developed kinetic model to the experimental data to determine the critical rate constants, kf for the formation of Fe(II)--NOM complexes and kox for the oxygenation of the Fe(II)--NOM complexes, when assumed to be first order with respect to both the concentration of Fe(II) and the dissolved O2. Analysis of the critical model reactions indicated that the process is in general non-pseudo-first-order but approaches pseudo-first-order under certain conditions. These limiting conditions are rapidly approached, which makes it difficult to determine unique values of kf and kox from oxidation data alone. Both parameters varied considerably between the different samples of NOM, with the value of kox ranging from about 2 up to 1000 M(-1) s(-1) as compared with 13 M(-1) s(-1) for inorganic Fe(II) in seawater. Despite large assumptions, the values of kox calculated were consistent with a linear free energy relation.     

Quantification of dissolved natural organic matter (DOM) mediated phototransformation of phenylurea herbicides in lakes

For many important classes of pesticides including phenylurea herbicides (PUHs) and triazines, photosensitized transformation may be the only relevant elimination process in surface waters. In this study, the dissolved organic matter (DOM) mediated phototransformation of PUHs has been investigated in laboratory and field experiments. The results indicate that, in surface waters, the photosensitized transformation of PUHs may be significant and occurs primarily by an initial one-electron oxidation most likely involving excited triplet states of DOM (3DOM*) constituents. Using isoproturon and diuron as model compounds, it is shown that for a given DOM, quantum yield factors determined in the laboratory at a few selected wavelengths can be used to quantify the overall DOM- mediated phototransformation of a given PUH under sunlight irradiation. Furthermore, it is demonstrated that this process can be modeled for a given surface water, by applying the program GCSOLAR and a simple algorithm for cloud cover for quantification of average daily light intensities. Finally, the model has been successfully applied to predict vertical concentration profiles of isoproturon and diuron in a small lake in Switzerland. To our knowledge, this is the first study in which DOM-mediated phototransformation of organic pollutants has been quantitatively validated in the field.     

Behavior of reoccurring PARAFAC components in fluorescent dissolved organic matter in natural and engineered systems: a critical review

Fluorescence spectroscopy coupled with parallel factor analysis (PARAFAC) has been widely used to characterize dissolved organic matter (DOM). Characterization is based on the intensity and location of independent fluorescent components identified in models constructed from excitation-emission matrices (EEMs). Similar fluorescent components have been identified in PARAFAC studies across a wide range of systems; however, there is a lack of discussion regarding the consistency with which these similar components behave. The overall goal of this critical review is to compare results for PARAFAC studies published since the year 2000 which include one or more of three reoccurring humic-like components. Components are compared and characterized based on EEM location, characteristic ecosystems, and behavior in natural and engineered systems. This synthesis allows PARAFAC users to more confidently infer DOM characteristics based on identified components. Additionally, behavioral inconsistencies between similar components help elucidate DOM properties for which fluorescence spectroscopy with PARAFAC may be a weak predictive tool.     

Quantitative evaluation of noncovalent interactions between glyphosate and dissolved humic substances by NMR spectroscopy

Interactions of glyphosate (N-phosphonomethylglycine) herbicide (GLY) with soluble fulvic acids (FAs) and humic acids (HAs) at pH 5.2 and 7 were studied by (1)H and (31)P NMR spectroscopy. Increasing concentrations of soluble humic matter determined broadening and chemical shift drifts of proton and phosphorus GLY signals, thereby indicating the occurrence of weak interactions between GLY and humic superstructures. Binding was larger for FAs and pH 5.2 than for HAs and pH 7, thus suggesting formation of hydrogen bonds between GLY carboxyl and phosphonate groups and protonated oxygen functions in humic matter. Changes in relaxation and correlation times of (1)H and (31)P signals and saturation transfer difference NMR experiments confirmed the noncovalent nature of GLY-humic interactions. Diffusion-ordered NMR spectra allowed calculation of the glyphosate fraction bound to humic superstructures and association constants (K(a)) and Gibbs free energies of transfer for GLY-humic complex formation at both pH values. These values showed that noncovalent interactions occurred most effectively with FAs and at pH 5.2. Our findings indicated that glyphosate may spontaneously and significantly bind to soluble humic matter by noncovalent interactions at slightly acidic pH and, thus, potentially pollute natural water bodies by moving through soil profiles in complexes with dissolved humus.     

Identification of copper binding sites in soil organic matter through chemical modifications and 13C CP-MAS NMR spectroscopy

Metal binding to an organic peat soil was probed by paramagnetic doping with copper, chemical modifications of the organic matter in the soil, and 13C CP-MAS NMR spin lattice relaxation rate measurements. Carboxyl and hydroxyl functional groups were determined to be most significant in copper uptake by the unmodified soil. Esterification and acetylation of the soil showed that metal binding by carbohydrate structures occurs independently of other functional groups and may even induce a pseudochelation phenomenon. Sorption isotherms corroborate the importance of carbohydrate structures in metal binding. These results suggest that environmental modeling of metal binding and retention in soils should incorporate estimates of the distributions of all functional groups in the soil organic matter (e.g. aliphatic, carbohydrate, phenolic, carboxyl) and their relative binding strengths.     

Mercury reduction and oxidation by reduced natural organic matter in anoxic environments

Natural organic matter (NOM)-mediated redox cycling of elemental mercury Hg(0) and mercuric Hg(II) is critically important in affecting inorganic mercury transformation and bioavailability. However, these processes are not well understood, particularly in anoxic water and sediments where NOM can be reduced and toxic methylmercury is formed. We show that under dark anoxic conditions reduced organic matter (NOM(re)) simultaneously reduces and oxidizes Hg via different reaction mechanisms. Reduction of Hg(II) is primarily caused by reduced quinones. However, Hg(0) oxidation is controlled by thiol functional groups via oxidative complexation, which is demonstrated by the oxidation of Hg(0) by low-molecular-weight thiol compounds, glutathione, and mercaptoacetic acid, under reducing conditions. Depending on the NOM source, oxidation state, and NOM:Hg ratio, NOM reduces Hg(II) at initial rates ranging from 0.4 to 5.5 h(-1), which are about 2 to 6 times higher than those observed for photochemical reduction of Hg(II) in open surface waters. However, rapid reduction of Hg(II) by NOM(re) can be offset by oxidation of Hg(0) with an estimated initial rate as high as 5.4 h(-1). This dual role of NOM(re) is expected to strongly influence the availability of reactive Hg and thus to have important implications for microbial uptake and methylation in anoxic environments.     

Determination of the mercury complexation characteristics of dissolved organic matter in natural waters with "reducible Hg" titrations

A new method for the determination of the concentration and conditional stability constant of dissolved organic matter that binds mercury (Hg) has been developed using an in vitro assay of reducible Hg. The technique is a wet chemical analogue to electrochemical approaches now in use for ligand studies of many other trace transition metals in natural waters. Ligand characteristics are obtained from additions of ionic Hg to buffered lake, river water, and seawater and determination of the wet chemically reducible fraction following equilibration of the spike. This approach is robust, as demonstrated by (i) analysis using three reducing agents of varying strengths, (ii) replicate analyses, (iii) comparison to well-characterized complexing species (chloride and EDTA) using a competitive ion-exchange resin, and (iv) kinetic studies. Results indicate that Hg-complexing equivalents are present in the dissolved phase (<0.2 microm) ranging from <1 to >60 nN concentrations and with log conditional stability constants (log K') in the range of 21-24. Only one ligand class was found in the natural waters analyzed. There was indirect evidence for a class of organic ligands that formed reducible complexes with Hg in freshwater. Such ligand characteristics indicate that the vast majority of ionic inorganic Hg dissolved in freshwater and coastal saltwaters is associated with organic complexes. Concentrations, affinities, and kinetics implicate multidentate chelation sites as the principal complexing moieties for Hg and discourage the use of humic carboxylic acids as a proxy for the ligands/functional groups.     

Oxidation state and size of Fe controlled by organic matter in natural waters

In a well mixed-stream, in which the iron/organic carbon (OC) ratio varied from 0.333 to 0.05 with sampling point and discharge, 40-70% of the Fe load was found to be present as lightly bound Fe(II). In laboratory simulations of streamwater, after 24 h of aeration at pH 6.5, and with an Fe/OC concentration ratio of 0.417, 97% of Fe(II) was converted to Fe(III) (hydr)oxides, while at a ratio of 0.083, 87% of Fe(ll) remained unoxidized. The particle size distribution of Fe contained < 0.2 microm fractions only when OC was present and comparison of Fe and OC size distributions suggested that there was more than one mechanism by which colloidal Fe was produced. At high Fe/ OC ratios, < 0.2 microm fractions may be predominantly Fe(III) (hydr)oxides stabilized by OC, but at low ratios, they must consist of otherwise soluble Fe(ll) attached to < 0.2 microm OC. The recognition in the field of the consequences of processes demonstrated in the laboratory suggests that OC may be a predominant control of both size and oxidation state of Fe in many natural waters.