Osburn, C.L., Morris, D.P., Thorn, K.A., and Moellar, T.E., 2001, Chemical and optical changes in freshwater dissolved organic matter exposed to solar radiation: Biogeochemistry, v. 54, p. 251-278.
We studied the chemical and optical changes in the dissolved organic matter (DOM) from two freshwater lakes and a Sphagnum bog after exposure to solar radiation. Stable carbon isotopes and solid-state ¹³C NMR spectra of DOM were used together with optical and chemical data to interpret results from experimental exposures of DOM to sunlight and from seasonal observations of two lakes in northeastern Pennsylvania. Solar photochemical oxidation of humic-rich bog DOM to smaller LMW compounds and to DIC was inferred from losses of UV absorbance, optical indices of molecular weight and changes in DOM chemistry. Experimentally, we observed a 1.20/00 enrichment in δ¹³C and a 47% loss in aromatic C functionality in bog DOM samples exposed to solar UVR. Similar results were observed in the surface waters of both lakes. In late summer hypolimnetic water in humic Lake Lacawac, we observed 3 to 4.50/00 enrichments in δ¹³C and a 30% increase in aromatic C relative to early spring values during spring mixing. These changes coincided with increases in molecular weight and UV absorbance. Anaerobic conditions of the hypolimnion in Lake Lacawac suggest that microbial metabolism may be turning over allochthonous C introduced during spring mixing, as well as autochthonous C. This metabolic activity produces HMW DOM during the summer, which is photochemically labile and isotopically distinct from allochthonous DOM or autochthonous DOM. These results suggest both photooxidation of allochthonous DOM in the epilimnion and autotrophic production of DOM by bacteria in the hypolimnion cause seasonal trends in the UV absorbance of lakes.  Journal Homepage   (Journal access may be limited by subscription considerations.)

Nitrite Fixation by Humic Substances: ¹⁵ N Nuclear Magnetic Resonance Evidence for Potential Intermediates in Chemodenitrification. K.A. Thorn and M.A. Mikita. Soil Sci. Soc. Am. J. 2000, 64,568-582.
Studies have suggested that NO₂¯, produced during nitrification and denitrification, can become incorporated into soil organic matter and, in one of the processes associated with chemodenitrification, react with organic matter to form trace N gases, including N₂O. To gain an understanding of the nitrosation chemistry on a molecular level, soil and aquatic humic substances were reacted with ¹⁵N labeled NaNO₂, and analyzed by liquid phase ¹⁵N and ¹³C nuclear magnetic resonance (NMR). The International Humic Substances Society (IHSS) Pahokee peat and peat humic acid were also reacted with ¹⁵N labeled NaNO₂ and analyzed by solid-state ¹⁵N NMR. In Suwannee River, Armadale, and Laurentian fulvic acids, phenolic rings and activated methylene groups underwent nitrosation to form nitrosophenols (quinone monoximes) and ketoximes, respectively. The oximes underwent Beckmann rearrangements to 2° amides, and Beckmann fragmentations to nitriles. The nitriles in turn underwent hydrolysis to 1° amides. Peaks tentatively identified as imine, indophenol, or azoxybenzene nitrogens were clearly present in spectra of samples nitrosated at pH 6 but diminished at pH 3. The ¹⁵N NMR spectrum of the peat humic acid exhibited peaks corresponding with N-nitroso groups in addition to nitrosophenols, ketoximes, and secondary Beckmann reaction products. Formation of N-nitroso groups was more significant in the whole peat compared with the peat humic acid. ¹³C NMR analyses also indicated the occurrence of nitrosative demethoxylation in peat and soil humic acids. Reaction of ¹⁵N labeled NH₃ fixated fulvic acid with unlabeled NO₂¯ resulted in nitrosative deamination of aminohydroquinone N, suggesting a previously unrecognized pathway for production of nitrogen gas in soils fertilized with NH₃.
Abbreviations: IHSS, International Humic Substances Society • LFA, Laurentian soil fulvic acid • NMR, nuclear magnetic resonance • NOE, nuclear Overhauser enhancement • SRFA, Suwannee River fulvic acid.   Journal Homepage   (Journal access may be limited by subscription considerations.)

Thorn, K.A., and Aiken, G.R, 1998, Biodegradation of crude oil into nonvolatile organic acids in a contaminated aquifer near Bemidji, Minnesota: Organic Geochemistry, v. 29, p. 909-931.
As the result of a pipeline burst, a body of light aliphatic crude oil floats atop the groundwater in a shallow sand and gravel aquifer in a remote area outside Bemidji, Minnesota. Biodegradation has resulted in the formation of a plume of DOC downgradient from the oil body. Groundwater has also been contaminated in an area known as the spray zone, from vertical infiltration of DOC resulting from biodegradation of crude oil in the overlying unsaturated zone. The majority of DOC in the contaminated groundwater is in the form of nonvolatile organic acids (NVOA's) which represent the partial oxidation products of the crude oil constituents. The NVOA's have been classified into three fractions according to their isolation on XAD resins: hydrophobic neutrals (HPO-N), hydrophobic acids (HPO-A) and hydrophilic acids (HPI-A). These fractions of NVOA's were isolated from a well downgradient from the oil body (well 530; DOC=21 mg C/l), from a well in the spray zone (well 603; DOC=15 mg C/l) and from an uncontaminated well upgradient of the oil body where the naturally occurring DOC is 2.9 mg C/l (well 310). The three sets of NVOA's were characterized by elemental analyses, molecular weight determinations, ¹⁴C ages and liquid phase ¹H and ¹³C NMR. The crude oil and the saturate, aromatic, resin and asphaltene fractions of the crude oil were similarly analyzed by elemental analysis and NMR. The NVOA's from the contaminated wells were clearly distinguishable from the naturally occurring groundwater DOC. Based upon molecular weights, sulfur contents, aromaticities and the presence of methyl groups bonded to aromatic rings, the characterization data suggests that the NVOA's originate from the C18 or greater alkylaromatic, naphthenoaromatic and sulfur-containing constituents of the crude oil, including possibly the resins and asphaltenes.   Journal Homepage  (Journal access may be limited by subscription considerations.)

Weber, E.J., Spidle, D.L, and Thorn, K.A., 1996, Covalent binding of aniline to humic substances: I Kinetic Studies: Environmental Science and Technology, v. 30, no. 9, p. 2755-2763.
The kinetics of covalent binding of aniline to dissolved organic matter (DOM) at concentrations typically found in natural aquatic ecosystems (1-50 mg carbon per litre) were studied using humic and fulvic isolates from the Suwannee River, Georgia, and International Humic Substances Society soil humic and fulvic acid isolates. Data indicated that approximately 10 per cent of the covalent binding sites associated with Suwannee River fulvic acid were highly reactive and that the reaction rate decreased with decreasing pH. The covalent binding of aniline was inhibited by pre-treatment of the fulvic acid with hydrogen sulfide, hydroxylamine or sodium borohydride.  Journal Homepage   (Journal access may be limited by subscription considerations.)

Thorn, K. A.; Pettigrew, P. J.; Goldenberg, W. S,; Weber, E. J. Covalent Binding of Aniline to Humic Substances. 2. ¹⁵N NMR Studies of Nucleophilic Addition Reactions. Environ. Sci. & Technol. 1996, 2764-2775
Aromatic amines are known to undergo covalent binding with humic substances in the environment. Although previous studies have examined reaction conditions and proposed mechanisms, there has been no direct spectroscopic evidence for the covalent binding of the amines to the functional groups in humic substances. In order to further elucidate the reaction mechanisms, the Suwannee River and IHSS soil fulvic and humic acids were reacted with ¹⁵N-labelled aniline at pH 6 and analyzed using ¹⁵N NMR spectrometry. Aniline underwent nucleophilic addition reactions with the quinone and other carbonyl groups in the samples and became incorporated in the form of anilinohydroquinone, anilinoquinone, anilide, imine, and heterocyclic nitrogen, the latter comprising 50% or more of the bound amine. The anilide and anilinohydroquinone nitrogens were determined to be susceptible to chemical exchange by ammonia. In the case of Suwannee River fulvic acid, reaction under anoxic conditions, and pretreatment with sodium borohydride or hydroxylamine prior to reaction under oxic conditions, resulted in a decrease in the proportion of anilinohydroquinone nitrogen incorporated. The relative decrease in the incorporation of anilinohydroquinone nitrogen with respect to anilinoquinone nitrogen under anoxic conditions suggested that inter- or intramolecular redox reactions accompanied the nucleophilic addition reactions.  Journal Homepage  (Journal access may be limited by subscription considerations.)

Thorn, K.A.; Goldenberg, W. S.; Younger, S. J.; Weber, E. J. Covalent Binding of Aniline to Humic Substances: Comparison of Nucleophilic Addition, Enazyme-, and Metal-Catalyzed Reactions by ¹⁵N NMR. In Humic and Fulvic Acids: Isolation, Structure, and Environmental Role; N. A. Marley and S. B. Clark, Eds.; American Chemical Society, 1996; pp 299-326
The covalent binding of ¹⁵N-labelled aniline, in the presence and absence of catalysis by horseradish peroxidase and birnessite, to the fulvic and humic acids isolated from the IHSS Elliot silt loam soil, has been examined by a combination of liquid and solid state ¹⁵N NMR. In the absence of catalysts, aniline undergoes nucleophilic addition reactions with the carbonyl functionality of the fulvic and humic acids and becomes incorporated in the form of anilinohydroquinone, anilinoquinone, anilide, heterocyclic, and imine nitrogens. In the presence of peroxidase and birnessite, aniline undergoes free radical coupling reactions together with nucleophilic addition reactions with the fulvic and humic acids. Among the condensation products unique to the catalyzed reactions are azobenzene nitrogens, iminodiphenoquinone nitrogens, and nitrogens tentatively assigned as imidazole, oxazole, pyrazole, or nitrile. The incorporation of aniline into the organic matter of the whole Elliot silt loam soil and the IHSS Pahokee peat most closely resembled the noncatalyzed nucleophilic addition reactions, as determined by solid state ¹⁵N NMR.  Journal Homepage   (Journal access may be limited by subscription considerations.)

Thorn, K.A., , Pennington, J.C., and Hayes, C.A., 2002, ¹⁵N NMR Investigation of the reduction and binding of TNT in aerobic bench scale reactor simulating windrow composting: Environmental Science and Technology, v. 36, no. 17, p. 3797-3805.
T¹⁵NT was added to a soil of low organic carbon content and composted for 20 days in an aerobic bench scale reactor. The finished whole compost and fulvic acid, humic acid, humin, and lignocellulose fractions extracted from the compost were analyzed by solid-state CP/MAS and DP/MAS ¹⁵N NMR. ¹⁵N NMR spectra provided direct spectroscopic evidence for reduction of TNT followed by covalent binding of the reduced metabolites to organic matter of the composted soil, with the majority of metabolite found in the lignocellulose fraction, by mass also the major fraction of the compost. In general, the types of bonds formed between soil organic matter and reduced TNT amines in controlled laboratory reactions were observed in the spectra of the whole compost and fractions, confirming that during composting TNT is reduced to amines that form covalent bonds with organic matter through aminohydroquinone, aminoquinone, heterocyclic, and imine linkages, among others. Concentrations of imine nitrogens in the compost spectra suggest that covalent binding by the diamines 2,4DANT and 2,6DANT is a significant process in the transformation of TNT into bound residues. Liquid-phase ¹⁵N NMR spectra of the fulvic acid and humin fractions provided possible evidence for involvement of phenoloxidase enzymes in covalent bond formation.  Article in PDF Format   (Journal access may be limited by subscription considerations.)

¹⁵N NMR Investigation of the covlaent binding and reduced TNT amines to soil humic acid, model compounds, and lignocellulose
The five major reductive degradation products of TNT-4ADNT (4-amino-2,6-dinitrotoluene), 2ADNT (2-amino-4,6-dinitrotoluene), 2,4DANT (2,4-diamino-6-nitrotoluene), 2,6DANT (2,6-diamino-4-nitrotoluene), and TAT (2,4,6-triaminotol- uene)-labeled with ¹⁵N in the amine positions, were reacted with the IHSS soil humic acid and analyzed by ¹⁵N NMR spectrometry. In the absence of catalysts, all five amines underwent nucleophilic addition reactions with quinone and other carbonyl groups in the soil humic acid to form both heterocyclic and nonheterocyclic condensation products. Imine formation via 1,2-addition of the amines to quinone groups in the soil humic acid was significant with the diamines and TAT but not the monoamines. Horseradish peroxidase (HRP) catalyzed an increase in the incorporation of all five amines into the humic acid. In the case of the diamines and TAT, HRP also shifted the binding away from heterocyclic condensation product toward imine formation. A comparison of quantitative liquid phase with solid-state CP/MAS ¹⁵N NMR indicated that the CP experiment underestimated imine and heterocyclic nitrogens in humic acid, even with contact times optimal for observation of these nitrogens. Covalent binding of the mono- and diamines to 4-methylcatechol, the HRP catalyzed condensation of 4ADNT and 2,4DANT to coniferyl alcohol, and the binding of 2,4DANT to lignocellulose with and without birnessite were also examined. http://water.usgs.gov/nrp/proj.bib/Publications/thorn.kennedy.pdf   Article in PDF Format

Alkaline Hydrolysis/Polymerization of 2,4,6-Trinitrotoluene: Characterization of Products by ¹⁵NMR.
Alkaline hydrolysis has been investigated as a nonbiological procedure for the destruction of 2,4,6-trinitrotoluene (TNT) in explosives contaminated soils and munitions scrap. Nucleophilic substitutions of the nitro and methyl groups of TNT by hydroxide ion are the initial steps in the alkaline degradation of TNT. Potential applications of the technique include both in situ surface liming and ex situ alkaline treatment of contaminated soils. A number of laboratory studies have reported the formation of an uncharacterized polymeric material upon prolonged treatment of TNT in base. As part of an overall assessment of alkaline hydrolysis as a remediation technique, and to gain a better understanding of the chemical reactions underlying the hydrolysis/polymerization process, the soluble and precipitate fractions of polymeric material produced from the calcium hydroxide hydrolysis of unlabeled and ¹⁵N-labeled TNT were analyzed by elemental analysis and ¹³C and ¹⁵N nuclear magnetic resonance spectroscopy. Spectra indicated that reactions leading to polymerization included nucleophilic displacement of nitro groups by hydroxide ion, formation of ketone, carboxyl, alcohol, ether, and other aliphatic carbons, conversion of methyl groups to diphenyl methylene carbons, and recondensation of aromatic amines and reduced forms of nitrite, including ammonia and possibly hydroxylamine, into the polymer. Compared to the distribution of carbons in TNT as 14% sp³- and 86% sp²-hybridized, the precipitate fraction from hydrolysis of unlabeled TNT contained 33% sp³- and 67% sp²-hybridized carbons. The concentration of nitrogen in the precipitate was 64% of that in TNT. The ¹⁵N NMR spectra showed that, in addition to residual nitro groups, forms of nitrogen present in the filtrate and precipitate fractions include aminohydroquinone, primary amide, indole, imine, and azoxy, among others. Unreacted nitrite was recovered in the filtrate fraction. The toxicities and susceptibilities to microbial or chemical degradation of the polymeric materials remain unknown.   Journal Homepage  (Journal access may be limited by subscription considerations.)

Development and Application of Pyrolysis Gas Chromatogaphy/Mass Spectrometry for the Analysis of Bound Trinitrotouene Residues in Soil.
TNT (trinitrotoluene) is a contaminant of global environmental significance, yet determining its environmental fate has posed longstanding challenges. To date, only differential extraction-based approaches have been able to determine the presence of covalently bound, reduced forms of TNT in field soils. Here, we employed thermal elution, pyrolysis, and gas chromatography/mass spectrometry (GC/MS) to distinguish between covalently bound and noncovalently bound reduced forms of TNT in soil. Model soil organic matter-based matrixes were used to develop an assay in which noncovalently bound (monomeric) aminodinitrotoluene (ADNT) and diaminonitrotoluene (DANT) were desorbed from the matrix and analyzed at a lower temperature than covalently bound forms of these same compounds. A thermal desorption technique, evolved gas analysis, was initially employed to differentiate between covalently bound and added ¹⁵N-labeled monomeric compounds. A refined thermal elution procedure, termed "double-shot analysis" (DSA), allowed a sample to be sequentially analyzed in two phases. In phase 1, all of an added ¹⁵N-labeled monomeric contaminant was eluted from the sample at relatively low temperature. In phase 2 during high-temperature pyrolysis, the remaining covalently bound contaminants were detected. DSA analysis of soil from the Louisiana Army Ammunition Plant (LAAP; ~5000 ppm TNT) revealed the presence of DANT, ADNT, and TNT. After scrutinizing the DSA data and comparing them to results from solvent-extracted and base/acid-hydrolyzed LAAP soil, we concluded that the TNT was a noncovalently bound "carryover" from phase 1. Thus, the pyrolysis-GC/MS technique successfully defined covalently bound pools of ADNT and DANT in the field soil sample.   Journal Homepage  (Journal access may be limited by subscription considerations.)