Sarah G. Pati

Carbon, hydrogen, and nitrogen isotope fractionation associated with oxidative transformation of substituted aromatic N-alkyl amines.

We investigated the mechanisms and isotope effects associated with the N-dealkylation and N-atom oxidation of substituted N-methyl- and N,N-dimethylanilines to identify isotope fractionation trends for the assessment of oxidations of aromatic N-alkyl moieties by compound-specific isotope analysis (CSIA). In laboratory batch model systems, we determined the C, H, and N isotope enrichment factors for the oxidation by MnO(2) and horseradish peroxidase (HRP), derived apparent (13)C-, (2)H-, and (15)N-kinetic isotope effects (AKIEs), and characterized reaction products. The N-atom oxidation pathway leading to radical coupling products typically exhibited inverse (15)N-AKIEs (up to 0.991) and only minor (13)C- and (2)H-AKIEs. Oxidative N-dealkylation, in contrast, was subject to large normal (13)C- and (2)H-AKIEs (up to 1.019 and 3.1, respectively) and small (15)N-AKIEs. Subtle changes of the compounds electronic properties due to different types of aromatic and/or N-alkyl substituents resulted in changes of reaction mechanisms, rate-limiting step(s), and thus isotope fractionation trends. The complex sequence of electron and proton transfers during the oxidative transformation of substituted aromatic N-alkyl amines suggests highly compound- and mechanism-dependent isotope effects precluding extrapolations to other organic micropollutants reacting along the same degradation pathways.

Carbon and Nitrogen Isotope Effects Associated with the Dioxygenation of Aniline and Diphenylamine

Dioxygenation of aromatic rings is frequently the initial step of biodegradation of organic subsurface pollutants. This process can be tracked by compound-specific isotope analysis to assess the extent of contaminant transformation, but the corresponding isotope effects, especially for dioxygenation of N-substituted, aromatic contaminants, are not well understood. We investigated the C and N isotope fractionation associated with the biodegradation of aniline and diphenylamine using pure cultures of Burkholderia sp. strain JS667, which can biodegrade both compounds, each by a distinct dioxygenase enzyme. For diphenylamine, the C and N isotope enrichment was normal with ε(C)- and ε(N)-values of -0.6 ± 0.1‰ and -1.0 ± 0.1‰, respectively. In contrast, N isotopes of aniline were subject to substantial inverse fractionation (ε(N) of +13 ± 0.5‰), whereas the ε(C)-value was identical to that of diphenylamine. A comparison of the apparent kinetic isotope effects for aniline and diphenylamine dioxygenation with those from abiotic oxidation by manganese oxide (MnO(2)) suggest that the oxidation of a diarylamine system leads to distinct C-N bonding changes compared to aniline regardless of reaction mechanism and oxidant involved. Combined evaluation of the C and N isotope signatures of the contaminants reveals characteristic Δδ(15)N/Δδ(13)C-trends for the identification of diphenylamine and aniline oxidation in contaminated subsurfaces and for the distinction of aniline oxidation from its formation by microbial and/or abiotic reduction of nitrobenzene.

Isotope Effects of Enzymatic Dioxygenation of Nitrobenzene and 2-Nitrotoluene by Nitrobenzene Dioxygenase

Oxygenation of aromatic rings is a frequent initial step in the biodegradation of persistent contaminants, and the accompanying isotope fractionation is increasingly used to assess the extent of transformation in the environment. Here, we systematically investigated the dioxygenation of two nitroaromatic compounds (nitrobenzene and 2-nitrotoluene) by nitrobenzene dioxygenase (NBDO) to obtain insights into the factors governing its C, H, and N isotope fractionation. Experiments were carried out at different levels of biological complexity from whole bacterial cells to pure enzyme. C, H, and N isotope enrichment factors and kinetic isotope effects (KIEs) were derived from the compound-specific isotope analysis of nitroarenes, whereas C isotope fractionation was also quantified in the oxygenated reaction products. Dioxygenation of nitrobenzene to catechol and 2-nitrotoluene to 3-methylcatechol showed large C isotope enrichment factors, ϵC, of -4.1 ± 0.2‰ and -2.5 ± 0.2‰, respectively, and was observed consistently in the substrates and dioxygenation products. ϵH- and ϵN-values were smaller, that is -5.7 ± 1.3‰ and -1.0 ± 0.3‰, respectively. C isotope fractionation was also identical in experiments with whole bacterial cells and pure enzymes. The corresponding (13)C-KIEs for the dioxygenation of nitrobenzene and 2-nitrotoluene were 1.025 ± 0.001 and 1.018 ± 0.001 and suggest a moderate substrate specificity. Our study illustrates that dioxygenation of nitroaromatic contaminants exhibits a large C isotope fractionation, which is not masked by substrate transport and uptake processes and larger than dioxygenation of other aromatic hydrocarbons.

Substrate and Enzyme Specificity of the Kinetic Isotope Effects Associated with the Dioxygenation of Nitroaromatic Contaminants.

Compound-specific isotope analysis (CSIA) is a promising approach for tracking biotransformation of organic pollutants, but isotope fractionation associated with aromatic oxygenations is only poorly understood. We investigated the dioxygenation of a series of nitroaromatic compounds to the corresponding catechols by two enzymes, namely, nitrobenzene and 2-nitrotoluene dioxygenase (NBDO and 2NTDO) to elucidate the enzyme- and substrate-specificity of C and H isotope fractionation. While the apparent (13)C- and (2)H-kinetic isotope effects of nitrobenzene, nitrotoluene isomers, 2,6-dinitrotoluene, and naphthalene dioxygenation by NBDO varied considerably, the correlation of C and H isotope fractionation revealed a common mechanism for nitrobenzene and nitrotoluenes. Similar observations were made for the dioxygenation of these substrates by 2NTDO. Evaluation of reaction kinetics, isotope effects, and commitment-to-catalysis based on experiment and theory showed that rates of dioxygenation are determined by the enzymatic O2 activation and aromatic C oxygenation. The contribution of enzymatic O2 activation to the reaction rate varies for different nitroaromatic substrates of NBDO and 2NTDO. Because aromatic dioxygenation by nonheme iron dioxygenases is frequently the initial step of biodegradation, O2 activation kinetics may also have been responsible for the minor isotope fractionation reported for the oxygenation of other aromatic contaminants.

Measurement of oxygen isotope ratios (18O/16O) of aqueous O2 in small samples by gas chromatography/isotope ratio mass spectrometry

RATIONALE Oxygen isotope fractionation of molecular O2 is an important process for the study of aerobic metabolism, photosynthesis, and formation of reactive oxygen species. The latter is of particular interest for investigating the mechanism of enzyme-catalyzed reactions, such as the oxygenation of organic pollutants, which is an important detoxification mechanism. METHODS We developed a simple method to measure the δ(18) O values of dissolved O2 in small samples using automated split injection for gas chromatography coupled to isotope ratio mass spectrometry (GC/IRMS). After creating a N2 headspace, the dissolved O2 partitions from aqueous solution to the headspace, from which it can be injected into the gas chromatograph. RESULTS In aqueous samples of 10 mL and in diluted air samples, we quantified the δ(18) O values at O2 concentrations of 16 μM and 86 μM, respectively. The chromatographic separation of O2 and N2 with a molecular sieve column made it possible to use N2 as the headspace gas for the extraction of dissolved O2 from water. We were therefore able to apply a rigorous δ(18) O blank correction for the quantification of (18) O/(16) O ratios in 20 nmol of injected O2 . CONCLUSIONS The successful quantification of (18) O-kinetic isotope effects associated with enzymatic and chemical reduction of dissolved O2 illustrates how the proposed method can be applied for studying enzymatic O2 activation mechanisms in a variety of (bio)chemical processes.

Exploring Trends of C and N Isotope Fractionation to Trace Transformation Reactions of Diclofenac in Natural and Engineered Systems

Although diclofenac ranks among the most frequently detected pharmaceuticals in the urban water cycle, its environmental transformation reactions remain imperfectly understood. Biodegradation-induced changes in 15N/14N ratios (εN = -7.1‰ ± 0.4‰) have indicated that compound-specific isotope analysis (CSIA) may detect diclofenac degradation. This singular observation warrants exploration for further transformation reactions. The present study surveys carbon and nitrogen isotope fractionation in other environmental and engineered transformation reactions of diclofenac. While carbon isotope fractionation was generally small, observed nitrogen isotope fractionation in degradation by MnO2 (εN = -7.3‰ ± 0.3‰), photolysis (εN = +1.9‰ ± 0.1‰), and ozonation (εN = +1.5‰ ± 0.2‰) revealed distinct trends for different oxidative transformation reactions. The small, secondary isotope effect associated with ozonation suggests an attack of O3 in a molecular position distant from the N atom. Model reactants for outer-sphere single electron transfer generated large inverse nitrogen isotope fractionation (εN = +5.7‰ ± 0.3‰), ruling out this mechanism for biodegradation and transformation by MnO2. In a river model, isotope fractionation-derived degradation estimates agreed well with concentration mass balances, providing a proof-of-principle validation for assessing micropollutant degradation in river sediment. Our study highlights the prospect of combining CSIA with transformation product analysis for a better assessment of transformation reactions within the environmental life of diclofenac.

Isotope Effects as New Proxies for Organic Pollutant Transformation

Assessing the pathways and rates of organic pollutant transformation in the environment is a major challenge due to co-occurring transport and degradation processes. Measuring changes of stable isotope ratios (e.g. (13)C/(12)C, (2)H/(1)H, (15)N/(14)N) in individual organic compounds by compound-specific isotope analysis (CSIA) makes it possible to identify degradation pathways without the explicit need to quantify pollutant concentration dynamics. The so-called isotope fractionation observed in an organic pollutant is related to isotope effects of (bio)chemical reactions and enables one to characterize pollutant degradation even if multiple processes take place simultaneously. Here, we illustrate some principles of CSIA using benzotriazole, a frequently observed aquatic micropollutant, as example. We show subsequently how the combined C and N isotope fractionation analysis of nitroaromatic compounds reveals kinetics and mechanisms of reductive and oxidative reactions as well as their (bio)degradation pathways in the environment.

Photochemical Transformation of Four Ionic Liquid Cation Structures in Aqueous Solution

Ionic liquids (ILs) are a new class of solvents expected to be used increasingly by the chemical industry in the coming years. Given their slow biodegradation and limited sorption affinities, IL cations have a high potential to reach aquatic environments. We investigated the fate of ILs in sunlit surface water by determining direct and indirect photochemical transformation rates of imidazolium, pyridinium, pyrrolidinium, and piperidinium cations. The photodegradation of all investigated IL cations was faster in solutions containing dissolved organic matter (DOM) than in ultrapure water, illustrating the importance of indirect photochemical processes. Experiments with model sensitizers and DOM isolates revealed that reactions with hydroxyl radicals dominated the transformation of tested IL cations. Bimolecular reaction rate constants with hydroxyl radicals ranged from (2.04 ± 0.37) × 109 to (8.47 ± 0.97) × 109 M-1 s-1 and showed an increase in rate constants with increasing carbon side-chain length. Consequently, average estimated half-lives of IL cations in sunlit surface water ranged from 32 ± 4 to 135 ± 25 days, highlighting the potential of IL cations to become persistent aquatic contaminants.

Reaction rates and product formation during advanced oxidation of ionic liquid cations by UV/peroxide, UV/persulfate, and UV/chlorine

Ionic liquids (ILs) are expected to be used increasingly in the coming years for industrial chemical applications as replacements for volatile organic solvents. The organic cations used in ILs typically contain quaternary ammonium groups and may reach aquatic environments due to their high water solubility and limited biodegradability. Given the persistence of IL cations in aquatic environments and the potential of quaternary ammonium compounds to form nitrosamines, the potential of advanced oxidation processes with UV irradiation (UV/AOP) to remove IL cations from drinking water sources was assessed. We found that IL cations react readily with hydroxyl and sulfate radicals with bimolecular reactions rate constants ranging from (1.2 ± 0.6) × 109 to (8.5 ± 1.0) × 109 M−1 s−1 and from (0.08 ± 0.06) × 109 to (1.7 ± 0.2) × 109 M−1 s−1, respectively. Consequently, half-lives in the order of minutes are expected for all IL cations in UV/AOP applications with hydrogen peroxide, persulfate, or free chlorine. In addition to efficient removal of the parent compounds, most transformation products of IL cations are formed through sequential hydroxylation reactions, which could ultimately lead to small, benign end-products. While we did not find evidence supporting the direct reaction of IL cations with reactive halogen species, a series of chlorinated transformation products were identified in UV/chlorine experiments. We hypothesize that these compounds are formed through the reactions of radical intermediates with free chlorine.

Characterization of substrate, cosubstrate, and product isotope effects associated with enzymatic oxygenations of organic compounds based on compound-specific isotope analysis

Enzymatic oxygenations are among the most important biodegradation and detoxification reactions of organic pollutants. In the environment, however, such natural attenuation processes are extremely difficult to monitor. Changes of stable isotope ratios of aromatic pollutants at natural isotopic abundances serve as proxies for isotope effects associated with oxygenation reactions. Such isotope fractionations offer new avenues for revealing the pathway and extent of pollutant transformation and provide new insights into the mechanisms of catalysis by Rieske non-heme ferrous iron oxygenases. Based on compound-specific C, H, N, and O isotope analysis, we present a comprehensive methodology with which isotope effects can be derived from the isotope fractionation measured in substrates, the cosubstrate O2, and organic oxygenation products. We use dioxygenation of nitrobenzene and 2-nitrotoluene by nitrobenzene dioxygenase as illustrative examples to introduce different mathematical procedures for deriving apparent substrate and product isotope effects. We present two experimental approaches to control reactant and product turnover for isotope fractionation analysis in experimental systems containing purified enzymes, E. coli clones, and pure strains of environmental microorganisms. Finally, we present instrumental procedures and sample treatment instructions for analysis of C, H, and N isotope analysis in organic compounds and O isotope analysis in aqueous O2 by gas and liquid chromatography coupled to isotope ratio mass spectrometry.