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Background: Water deprivation-induced hypoxia stress (WDIHS) has been extensively investigated in numerous fish species due to their adaptation with accessory respiratory organs to respire air but this has not been studied in Indian stinging fish Heteropneustes fossilis . Data regarding WDIHS-induced metabolism in accessory respiratory organ (ARO) and gills and its relationship with oxidative stress (OS) in respiratory organs of air-breathing fish H. fossilis , are limited. So, this study aimed to investigate the effects of WDIHS (0, 3, 6, 12, and 18 h) on hydrogen peroxide (H 2 O 2 ) as reactive oxygen species (ROS), OS, redox regulatory enzymes, and electron transport enzymes (ETC) in ARO and gills of H. fossilis ., Methods: Fish were exposed to air for different hours (up to 18 h) against an appropriate control, and ARO and gills were sampled. The levels of oxygen saturation in the body of the fish were assessed at various intervals during exposure to air. Protein carbonylation (PC) and thiobarbituric acid reactive substances (TBARS) were used as OS markers, H 2 O 2 as ROS marker, and various enzymatic activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), along with the assessment of complex enzymes (I, II, III, and V) as well as the levels of ascorbic acid (AA) and the reduced glutathione (GSH) were quantified in both the tissues., Results: Discriminant function analyses indicate a clear separation of the variables as a function of the studied parameters. The gills exhibited higher levels of GSH and H 2 O 2 compared to ARO, while ARO showed elevated levels of PC, TBARS, AA, SOD, CAT, and GPx activities compared to the gills. The activities of GR and ETC enzymes exhibited similar levels in both the respiratory organs, namely the gills, and ARO. These organs experienced OS due to increased H 2 O 2 , TBARS, and PC levels, as observed during WDIHS. Under WDIHS conditions, the activity/level of CAT, GPx, GR, and GSH decreased in ARO, while SOD activity, along with GR, GSH, and AA levels decreased in gills. However, the activity/level of SOD and AA in ARO and CAT in gills was elevated under WDIHS. Complex II exhibited a positive correlation with WDIHS, while the other ETC enzymes (complex I, III, and V) activities had negative correlations with the WDIHS., Discussion: The finding suggests that ARO is more susceptible to OS than gills under WDIHS. Despite both organs employ distinct redox regulatory systems to counteract this stress, their effectiveness is hampered by the inadequacy of small redox regulatory molecules and the compromised activity of the ETC, impeding their ability to effectively alleviate the stress induced by the water-deprivation condition., Competing Interests: The authors declare there are no competing interests., (©2024 Pati et al.)

Rationale: Stable isotope analysis of O 2 is a valuable tool to identify O 2 -consuming processes in the environment; however, reference materials for O 2 isotope analysis are lacking. Consequently, a one-point calibration with O 2 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 δ 18 O and δ 17 O values of O 2 associated with a one-point calibration., Methods: We produced O 2 photosynthetically with extracted spinach thylakoids from source waters with δ 18 O values of -56‰ to +95‰ and δ 17 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 O 2 will be identical to that of the source water. The δ 18 O and δ 17 O values of the produced O 2 were measured by gas chromatography coupled with isotope-ratio mass spectrometry (GC/IRMS), applying a common one-point calibration., Results: Linear regressions between δ 18 O or δ 17 O values of the produced O 2 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 δ 18 O and δ 17 O, respectively. Triple oxygen isotopic measurements of O 2 during consumption by Fe 2+ resulted in a δ 18 O-δ 17 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 δ 18 O scale when applying a one-point calibration with O 2 from ambient air during O 2 isotope analysis. Both O 2 formation and consumption experiments, however, indicate a δ 17 O scale compression. Consequently, δ 17 O values cannot be measured accurately by GC/IRMS with a one-point calibration without determining the δ 17 O scale correction factor, e.g. with the O 2 formation experiments described here., (© 2023 John Wiley & Sons Ltd.)

Rieske oxygenases are known as catalysts that enable the cleavage of aromatic and aliphatic C-H bonds in structurally diverse biomolecules and recalcitrant organic environmental pollutants through substrate oxygenations and oxidative heteroatom dealkylations. Yet, the unproductive O 2 activation, which is concomitant with the release of reactive oxygen species (ROS), is typically not taken into account when characterizing Rieske oxygenase function. Even if considered an undesired side reaction, this O 2 uncoupling allows for studying active site perturbations, enzyme mechanisms, and how enzymes evolve as environmental microorganisms adapt their substrates to alternative carbon and energy sources. Here, we report on complementary methods for quantifying O 2 uncoupling based on mass balance or kinetic approaches that relate successful oxygenations to total O 2 activation and ROS formation. These approaches are exemplified with data for two nitroarene dioxygenases (nitrobenzene and 2-nitrotoluene dioxygenase) which have been shown to mono- and dioxygenate substituted nitroaromatic compounds to substituted nitrobenzylalcohols and catechols, respectively., Competing Interests: Competing interests The authors declare no competing interests., (Copyright © 2024. Published by Elsevier Inc.)

Information on the oxidative stress physiology parameters (OSPP) in general and as a function of the fluctuation of Mg, Ca and organic carbon present in soil and aquatic physico-chemical factors such as pH, temperature and salinity in particular are scanty in the amphibious snail Pila globosa. A spatio-temporal analysis of redox metabolism (as OSPP) followed by discriminant function analysis of the obtained data were performed in P. globosa sampled from the east-coasts of Odisha state, India (mostly along the Bay of Bengal) for environmental health assessment purposes. Results revealed that the OSPP are susceptible to seasonal synergistic variation of soil and physico-chemical factors. Overall, lipid peroxidation, total antioxidant capacity, activities of catalase, glutathione reductase had positive correlation whereas ascorbic acid, the reduced glutathione and the activity of superoxide dismutase had non-significant correlation with the soil Mg, Ca, organic carbon, and pH, temperature and salinity of water. In the summer season, the snails had a marked 51.83% and 26.41% higher lipid peroxidation level and total antioxidative activity as compared to the other seasons. Spatial variation of OSPP indicates that snails residing away from the Bay of Bengal coast had at least 4.4% lower antioxidant level in winter and 30% higher lipid peroxide levels in summer as compared to the rest of the sampling sites. Results on OSPP in P. globosa may be useful for monitoring the ecotoxic effects of environment using molluscs in general and P. globosa in particular., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

Positive effects of COVID-19-induced lockdowns on environment are well documented although pre-planned experiments for such analyses and appearance of fish species are lacking. We hypothesize that spotting the fish Aplocheilus panchax along the industrial belt of Mahanadi River near Cuttack in a never seen manner could be due to the regenerated environment. Heavy metals, water and air qualities along with spotting A. panchax in up, mid and downstream of Mahanadi River near Jagatpur industrial basins were analysed during pre-(end of March 2020) and after 60 days of lockdowns (last week of May 2020). An overall 45, 61, 79, 100, 97 and 90% reduction of Fe, Cu, Ni, Cd, Pb and Zn was recorded in the studied area after lockdowns, respectively. Similarly, dissolved oxygen and pH were elevated by 26 and 7%, respectively. Water temperature, conductivity and total dissolved solute levels were reduced by 7, 46 and 15%, respectively, which were again elevated during post-lockdowns during 2021 as observed from the Landsat-8 OLI satellite data. Air NO 2 , SO 2 , NH 3 , PM 2.5 , PM 10 and CO levels were alleviated by 58.75, 80.33, 72.22, 76.28, 77.33 and 80.15%, respectively. Finally, for the first time, about 12 A. panchax fish per 100 m shore line in the area were spotted. The observed lockdown-induced environmental healing at the studied area could contribute to the appearance of A. panchax in the study site and therefore a stringent environmental audit is suggested during post-COVID-19 periods to make the regenerated environmental status long lasting in such habitats., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

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., (© 2022. The Author(s).)

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 Fe II center, elementary steps leading to O 2 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 O 2 activation with the release of the unreacted substrate and reactive oxygen species represents an important path in the catalytic cycle. Through correlation of O 2 uncoupling for a series of substituted nitroaromatic compounds with 18 O and 13 C kinetic isotope effects of dissolved O 2 and aromatic substrates, respectively, we show that O 2 uncoupling occurs after the rate-limiting formation of Fe III -(hydro)peroxo species from which substrates are hydroxylated. Substituent effects on the extent of O 2 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 O 2 uncoupling. The proposed catalytic cycle provides a mechanistic basis for assessing the very different efficiencies of substrate hydroxylation vs unproductive O 2 activation and generation of reactive oxygen species in reactions catalyzed by Rieske dioxygenases., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)

In the present study, Water Deprived Condition (WPC, up to 18 h) induced hypoxia and altered oxidative stress (OS) physiology along with responses of respiratory chain enzyme in Heteropneustes fossilis are described . The body O 2 saturation level in the fish was declined with respect to air exposure. Higher levels of lipid peroxidation and protein carbonylation were recorded in the tissue of fish exposed to 6 h of WPC stress. The regulation of the mitochondrial complex and antioxidant enzymes, small antioxidant molecules indicated that the fish can moderately survive up to 6 h of air exposure. Probably with the onset of metabolic depression, it can critically resist the dehydration stress up to 18 h. Although the activities of glutathione peroxidase and reductase were elevated, activities of antioxidant enzymes such as superoxide dismutase and catalase were insufficient to combat WPC induced ROS and OS generated under hypoxia. The small antioxidant molecules played a key role in elimination of ROS. The elevated complex II activity was probably responsible for resisting the complex I, II and IV mediated electron leakage events in mitochondria of the fish under WPC. The total H 2 O 2 removing capacity was less under WPC while the total units of all calculated antioxidants were alleviated signifying an interesting mechanism of WPC induced OS in the fish., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The disease COVID-19 caused by SARS-CoV-2 is the third highly infectious human Coronavirus epidemic in the 21 s⁢t century due to its high transmission rate and quick evolution of its pathogenicity. Genomic studies indicate that it is zoonotic from bats. The COVID-19 has led to significant loss of lives and a tremendous economic decline in the world. Generally, the population at risk of a fatal outcome are the elderly and those who are debilitated or are immune compromised. The fatality rate is high, but now is reduced after the development of preventive vaccine although an effective treatment by drug against the virus is yet to be developed. The treatment is narrowed to the use of several anti-viral drugs, or other re-purposed drugs. Social distancing, therefore, has emerged as a putative method to decrease the rate of infection. In this review, we summarize the aspects of the disease that is so far have come to light and review the impact of the infection on our society, healthcare, economy, education, and environment., (© 2021 The Author(s). Published by BRI.)

Exposure to a variety of environmental factors such as temperature, pH, oxygen and salinity may influence the oxidative status in aquatic organisms. The present review article focuses on the modulation of oxidative stress with reference to the generation of reactive oxygen species (ROS) in aquatic animals from different phyla. The focus of the review article is to explore the plausible mechanisms of physiological changes occurring in aquatic animals due to altered salinity in terms of oxidative stress. Apart from the seasonal variations in salinity, global warming and anthropogenic activities have also been found to influence oxidative health status of aquatic organisms. These effects are discussed with an objective to develop precautionary measures to protect the diversity of aquatic species with sustainable conservation. Comparative analyses among different aquatic species suggest that salinity alone or in combination with other abiotic factors are intricately associated with modulation in oxidative stress in a species-specific manner in aquatic animals. Osmoregulation under salinity stress in relation to energy demand and supply are also discussed. The literature survey of >50 years (1960-2020) indicates that oxidative stress status and comparative analysis of redox modulation have evolved from the analysis of various biotic and/or abiotic factors to the study of cellular signalling pathways in these aquatic organisms., (Copyright © 2021 Elsevier Inc. All rights reserved.)

A discrete heterometallic compound consisting of Cu(ii) and Pd(ii) units which mimicks Bovine Cu-ZnSOD has been synthesised in aqueous medium. Its superoxide radical ion scavenging activity is higher than the SOD activity of Scylla serrata tissues and comparable with the liver of Gallus domesticus.

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., Competing Interests: The authors declare no competing financial interest., (Copyright © 2020 American Chemical Society.)

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.

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) × 10 9 to (8.47 ± 0.97) × 10 9 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.

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 O 2 , 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 O 2 by gas and liquid chromatography coupled to isotope ratio mass spectrometry., (© 2017 Elsevier Inc. All rights reserved.)

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 15 N/ 14 N ratios (ε N = -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 MnO 2 (ε N = -7.3‰ ± 0.3‰), photolysis (ε N = +1.9‰ ± 0.1‰), and ozonation (ε N = +1.5‰ ± 0.2‰) revealed distinct trends for different oxidative transformation reactions. The small, secondary isotope effect associated with ozonation suggests an attack of O 3 in a molecular position distant from the N atom. Model reactants for outer-sphere single electron transfer generated large inverse nitrogen isotope fractionation (ε N = +5.7‰ ± 0.3‰), ruling out this mechanism for biodegradation and transformation by MnO 2 . 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.

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.

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., (Copyright © 2016 John Wiley & Sons, Ltd.)

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.

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.

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.

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.