Improving the accuracy of δ18 O and δ17 O values of O2 measured by continuous-flow isotope-ratio mass spectrometry with a multipoint isotope-ratio calibration.

RATIONALE: Stable isotope analysis of O2 is a valuable tool to identify O2 -consuming processes in the environment; however, reference materials for O2 isotope analysis are lacking. Consequently, a one-point calibration with O2 from ambient air is often applied, which can lead to substantial measurement uncertainties. Our goals were to develop a simple multipoint isotope-ratio calibration approach and to determine measurement errors of δ18 O and δ17 O values of O2 associated with a one-point calibration. METHODS: We produced O2 photosynthetically with extracted spinach thylakoids from source waters with δ18 O values of -56‰ to +95‰ and δ17 O values of -30‰ to +46‰. Photosynthesis was chosen because this process does not cause isotopic fractionation, so that the O isotopic composition of the produced O2 will be identical to that of the source water. The δ18 O and δ17 O values of the produced O2 were measured by gas chromatography coupled with isotope-ratio mass spectrometry (GC/IRMS), applying a common one-point calibration. RESULTS: Linear regressions between δ18 O or δ17 O values of the produced O2 and those of the corresponding source waters resulted in slopes of 0.99 ± 0.01 and 0.92 ± 0.10, respectively. In the tested δ range, a one-point calibration thus introduced maximum errors of 0.8‰ and 3.3‰ for δ18 O and δ17 O, respectively. Triple oxygen isotopic measurements of O2 during consumption by Fe2+ resulted in a δ18 O-δ17 O relationship (λ) of 0.49 ± 0.01 without δ scale correction, slightly lower than expected for mass-dependent O isotopic fractionation. CONCLUSIONS: No significant bias is introduced on the δ18 O scale when applying a one-point calibration with O2 from ambient air during O2 isotope analysis. Both O2 formation and consumption experiments, however, indicate a δ17 O scale compression. Consequently, δ17 O values cannot be measured accurately by GC/IRMS with a one-point calibration without determining the δ17 O scale correction factor, e.g. with the O2 formation experiments described here.

Managing argon interference during measurements of 18O/16O ratios in O2 by continuous-flow isotope ratio mass spectrometry.

Monitoring changes in stable oxygen isotope ratios in molecular oxygen allows for studying many fundamental processes in bio(geo)chemistry and environmental sciences. While the measurement of [Formula: see text]O/[Formula: see text]O ratios of [Formula: see text] in gaseous samples can be carried out conveniently and from extracting moderately small aqueous samples for analyses by continuous-flow isotope ratio mass spectrometry (CF-IRMS), oxygen isotope signatures, [Formula: see text]O, could be overestimated by more than 6[Formula: see text] because of interferences from argon in air. Here, we systematically evaluated the extent of such Ar interferences on [Formula: see text]O/[Formula: see text]O ratios of [Formula: see text] for measurements by gas chromatography/IRMS and GasBench/IRMS and propose simple instrumental modifications for improved Ar and [Formula: see text] separation as well as post-measurement correction procedures for obtaining accurate [Formula: see text]O. We subsequently evaluated the consequences of Ar interferences for the quantification of O isotope fractionation in terms of isotope enrichment factors, [Formula: see text], and [Formula: see text]O kinetic isotope effects ([Formula: see text]O KIEs) in samples where [Formula: see text] is consumed and Ar:[Formula: see text] ratios increase steadily and substantially over the course of a reaction. We show that the extent of O isotope fractionation is overestimated only slightly and that this effect is typically smaller than uncertainties originating from the precision of [Formula: see text]O measurements and experimental variability. Ar interferences can become more relevant and bias [Formula: see text] values by more than [Formula: see text] in aqueous samples where fractional [Formula: see text] conversion exceeds 90%. Practically, however, such samples would typically contain less than 25 [Formula: see text]M of [Formula: see text] at ambient temperature, an amount that is close to the method detection limit of [Formula: see text]O/[Formula: see text]O ratio measurement by CF-IRMS.

Substrate-Specific Coupling of O2 Activation to Hydroxylations of Aromatic Compounds by Rieske Non-heme Iron Dioxygenases.

Rieske dioxygenases catalyze the initial steps in the hydroxylation of aromatic compounds and are critical for the metabolism of xenobiotic substances. Because substrates do not bind to the mononuclear non-heme FeII center, elementary steps leading to O2 activation and substrate hydroxylation are difficult to delineate, thus making it challenging to rationalize divergent observations on enzyme mechanisms, reactivity, and substrate specificity. Here, we show for nitrobenzene dioxygenase, a Rieske dioxygenase capable of transforming nitroarenes to nitrite and substituted catechols, that unproductive O2 activation with the release of the unreacted substrate and reactive oxygen species represents an important path in the catalytic cycle. Through correlation of O2 uncoupling for a series of substituted nitroaromatic compounds with 18O and 13C kinetic isotope effects of dissolved O2 and aromatic substrates, respectively, we show that O2 uncoupling occurs after the rate-limiting formation of FeIII-(hydro)peroxo species from which substrates are hydroxylated. Substituent effects on the extent of O2 uncoupling suggest that the positioning of the substrate in the active site rather than the susceptibility of the substrate for attack by electrophilic oxygen species is responsible for unproductive O2 uncoupling. The proposed catalytic cycle provides a mechanistic basis for assessing the very different efficiencies of substrate hydroxylation vs unproductive O2 activation and generation of reactive oxygen species in reactions catalyzed by Rieske dioxygenases.

Comprehensive screening of quaternary ammonium surfactants and ionic liquids in wastewater effluents and lake sediments.

Quaternary ammonium compounds (QACs) are widely applied as surfactants and biocides in cleaning and personal-care products. Because of incomplete removal during wastewater treatment, QACs are present in wastewater effluents, with which they are discharged into natural waters, where they accumulate in sediments. To assess the levels of QACs in aquatic environments, a liquid chromatography high-resolution mass spectrometry method using both target and suspect screening was developed. The water and sediment sample preparation, measurement, and data analysis workflow were optimized for 22 target compounds with a wide range of hydrophobicity, including ionic liquids that have potential use as solvents and QACs common in personal-care and sanitizing products. In wastewater effluents, average concentrations of all target and suspect QACs combined ranged from 0.4 µg L-1 to 6.6 µg L-1. Various homologs of benzylalkyldimethylammonium (BAC) and dialkyldimethylammonium (DADMAC) as well as the ionic liquid butylpyridinium and 15 suspect QACs were detected in at least one wastewater effluent sample. A spatial profile of sediment samples in a lake demonstrated potential inputs from both municipal wastewater effluent and agricultural sources for BACs. In sediment cores, two distinct trends of temporal QAC accumulation were observed. In lakes with large watersheds and mixed domestic and industrial wastewater sources (Lake Pepin and Duluth Harbor), peak concentrations of QACs were found at depths corresponding to deposition in the 1980s and decreases after this time are attributed to improved wastewater treatment and source control. In a smaller lake with predominantly domestic wastewater inputs (Lake Winona), concentrations of QACs increased slowly over time until today.

Increased Use of Quaternary Ammonium Compounds during the SARS-CoV-2 Pandemic and Beyond: Consideration of Environmental Implications.

Quaternary ammonium compounds (QACs) are active ingredients in over 200 disinfectants currently recommended by the U.S. EPA for use to inactivate the SARS-CoV-2 (COVID-19) virus. The amounts of these compounds used in household, workplace, and industry settings has very likely increased, and usage will continue to be elevated given the scope of the pandemic. QACs have been previously detected in wastewater, surface waters, and sediments, and effects on antibiotic resistance have been explored. Thus, it is important to assess potential environmental and engineering impacts of elevated QAC usage, which may include disruption of wastewater treatment unit operations, proliferation of antibiotic resistance, formation of nitrosamine disinfection byproducts, and impacts on biota in surface waters. The threat caused by COVID-19 is clear, and a reasonable response is elevated use of QACs to mitigate spread of infection. Exploration of potential effects, environmental fate, and technologies to minimize environmental releases of QACs, however, is warranted.

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.

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.

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.

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 ((18)O/(16)O) 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.

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.

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.

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.

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 compound's 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.