Advances in Oxygen Isotope Analysis of Phosphate by Electrospray Orbitrap Mass Spectrometry for Studying the Microbial Metabolism of Microorganisms.

Understanding the impact of human activities on the metabolic state of soil and aquatic environments is of paramount importance to implement measures for maintaining ecosystem services. Variations of natural abundance 18O/16O ratios in phosphate have been proposed as proxies for the holistic assessment of metabolic activity given the crucial importance of phosphoryl transfer reactions in fundamental biological processes. However, instrumental and procedural limitations inherent to oxygen isotope analysis in phosphate and organophosphorus compounds have so far limited the stable isotope-based evaluation of metabolic processes. Here, we discuss how recent developments in Orbitrap high resolution mass spectrometry enable measurements of 18O/16O ratios in phosphate and outline the critical mass spectrometry parameters for accurate and precise analysis. Subsequently, we evaluate the types of 18O kinetic isotope effects of phosphoryl transfer reactions and illustrate how novel analytical approaches will give rise to an improved understanding of 18O/16O ratio variations from biochemical processes affecting the microbial phosphorus metabolism.

Hydrogen Peroxide Formation during Ozonation of Olefins and Phenol: Mechanistic Insights from Oxygen Isotope Signatures.

Mitigation of undesired byproducts from ozonation of dissolved organic matter (DOM) such as aldehydes and ketones is currently hampered by limited knowledge of their precursors and formation pathways. Here, the stable oxygen isotope composition of H2O2 formed simultaneously with these byproducts was studied to determine if it can reveal this missing information. A newly developed procedure, which quantitatively transforms H2O2 to O2 for subsequent 18O/16O ratio analysis, was used to determine the δ18O of H2O2 generated from ozonated model compounds (olefins and phenol, pH 3-8). A constant enrichment of 18O in H2O2 with a δ18O value of ∼59‰ implies that 16O-16O bonds are cleaved preferentially in the intermediate Criegee ozonide, which is commonly formed from olefins. H2O2 from the ozonation of acrylic acid and phenol at pH 7 resulted in lower 18O enrichment (δ18O = 47-49‰). For acrylic acid, enhancement of one of the two pathways followed by a carbonyl-H2O2 equilibrium was responsible for the smaller δ18O of H2O2. During phenol ozonation at pH 7, various competing reactions leading to H2O2 via an intermediate ozone adduct are hypothesized to cause lower δ18O in H2O2. These insights provide a first step toward supporting pH-dependent H2O2 precursor elucidation in DOM.

Elucidating the Role of O2 Uncoupling in the Oxidative Biodegradation of Organic Contaminants by Rieske Non-heme Iron Dioxygenases.

Oxygenations of aromatic soil and water contaminants with molecular O2 catalyzed by Rieske dioxygenases are frequent initial steps of biodegradation in natural and engineered environments. Many of these non-heme ferrous iron enzymes are known to be involved in contaminant metabolism, but the understanding of enzyme-substrate interactions that lead to successful biodegradation is still elusive. Here, we studied the mechanisms of O2 activation and substrate hydroxylation of two nitroarene dioxygenases to evaluate enzyme- and substrate-specific factors that determine the efficiency of oxygenated product formation. Experiments in enzyme assays of 2-nitrotoluene dioxygenase (2NTDO) and nitrobenzene dioxygenase (NBDO) with methyl-, fluoro-, chloro-, and hydroxy-substituted nitroaromatic substrates reveal that typically 20-100% of the enzyme's activity involves unproductive paths of O2 activation with generation of reactive oxygen species through so-called O2 uncoupling. The 18O and 13C kinetic isotope effects of O2 activation and nitroaromatic substrate hydroxylation, respectively, suggest that O2 uncoupling occurs after generation of FeIII-(hydro)peroxo species in the catalytic cycle. While 2NTDO hydroxylates ortho-substituted nitroaromatic substrates more efficiently, NBDO favors meta-substituted, presumably due to distinct active site residues of the two enzymes. Our data implies, however, that the O2 uncoupling and hydroxylation activity cannot be assessed from simple structure-reactivity relationships. By quantifying O2 uncoupling by Rieske dioxygenases, our work provides a mechanistic link between contaminant biodegradation, the generation of reactive oxygen species, and possible adaptation strategies of microorganisms to the exposure of new contaminants.

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.

The Apparently Unreactive Substrate Facilitates the Electron Transfer for Dioxygen Activation in Rieske Dioxygenases.

Rieske dioxygenases belong to the non-heme iron family of oxygenases and catalyze important cis-dihydroxylation as well as O-/N-dealkylation and oxidative cyclization reactions for a wide range of substrates. The lack of substrate coordination at the non-heme ferrous iron center, however, makes it particularly challenging to delineate the role of the substrate for productive O 2 activation. Here, we studied the role of the substrate in the key elementary reaction leading to O 2 activation from a theoretical perspective by systematically considering (i) the 6-coordinate to 5-coordinate conversion of the non-heme FeII upon abstraction of a water ligand, (ii) binding of O 2 , and (iii) transfer of an electron from the Rieske cluster. We systematically evaluated the spin-state-dependent reaction energies and structural effects at the active site for all combinations of the three elementary processes in the presence and absence of substrate using naphthalene dioxygenase as a prototypical Rieske dioxygenase. We find that reaction energies for the generation of a coordination vacancy at the non-heme Fe II center through thermoneutral H2 O reorientation and exothermic O 2 binding prior to Rieske cluster oxidation are largely insensitive to the presence of naphthalene and do not lead to formation of any of the known reactive Fe-oxygen species. By contrast, the role of the substrate becomes evident after Rieske cluster oxidation and exclusively for the 6-coordinate non-heme Fe II sites in that the additional electron is found at the substrate instead of at the iron and oxygen atoms. Our results imply an allosteric control of the substrate on Rieske dioxygenase reactivity to happen prior to changes at the non-heme Fe II in agreement with a strategy that avoids unproductive O 2 activation.

Thermodynamic controls on rates of iron oxide reduction by extracellular electron shuttles.

Anaerobic microbial respiration in suboxic and anoxic environments often involves particulate ferric iron (oxyhydr-)oxides as terminal electron acceptors. To ensure efficient respiration, a widespread strategy among iron-reducing microorganisms is the use of extracellular electron shuttles (EES) that transfer two electrons from the microbial cell to the iron oxide surface. Yet, a fundamental understanding of how EES-oxide redox thermodynamics affect rates of iron oxide reduction remains elusive. Attempts to rationalize these rates for different EES, solution pH, and iron oxides on the basis of the underlying reaction free energy of the two-electron transfer were unsuccessful. Here, we demonstrate that broadly varying reduction rates determined in this work for different iron oxides and EES at varying solution chemistry as well as previously published data can be reconciled when these rates are instead related to the free energy of the less exergonic (or even endergonic) first of the two electron transfers from the fully, two-electron reduced EES to ferric iron oxide. We show how free energy relationships aid in identifying controls on microbial iron oxide reduction by EES, thereby advancing a more fundamental understanding of anaerobic respiration using iron oxides.

Triple-Element Compound-Specific Stable Isotope Analysis (3D-CSIA): Added Value of Cl Isotope Ratios to Assess Herbicide Degradation.

Multielement isotope fractionation studies to assess pollutant transformation are well-established for point-source pollution but are only emerging for diffuse pollution by micropollutants like pesticides. Specifically, chlorine isotope fractionation is hardly explored but promising, because many pesticides contain only few chlorine atoms so that "undiluted" position-specific Cl isotope effects can be expected in compound-average data. This study explored combined Cl, N, and C isotope fractionation to sensitively detect biotic and abiotic transformation of the widespread herbicides and groundwater contaminants acetochlor, metolachlor, and atrazine. For chloroacetanilides, abiotic hydrolysis pathways studied under acidic, neutral, and alkaline conditions as well as biodegradation in two soils resulted in pronounced Cl isotope fractionation (εCl from -5.0 ± 2.3 to -6.5 ± 0.7‰). The characteristic dual C-Cl isotope fractionation patterns (ΛC-Cl from 0.39 ± 0.15 to 0.67 ± 0.08) reveal that Cl isotope analysis provides a robust indicator of chloroacetanilide degradation. For atrazine, distinct ΛC-Cl values were observed for abiotic hydrolysis (7.4 ± 1.9) compared to previous reports for biotic hydrolysis and oxidative dealkylation (1.7 ± 0.9 and 0.6 ± 0.1, respectively). The 3D isotope approach allowed differentiating transformations that would not be distinguishable based on C and N isotope data alone. This first data set on Cl isotope fractionation in chloroacetanilides, together with new data in atrazine degradation, highlights the potential of using compound-specific chlorine isotope analysis for studying in situ pesticide degradation.

Exploring the Utility of Compound-Specific Isotope Analysis for Assessing Ferrous Iron-Mediated Reduction of RDX in the Subsurface.

Subsurface contamination with the explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) at ordnance production and testing sites is a problem because of the persistence, mobility, and toxicity of RDX and the formation of toxic products under anoxic conditions. While the utility of compound-specific isotope analysis for inferring natural attenuation pathways from stable isotope ratios has been demonstrated, the stable isotope fractionation for RDX reduction by iron-bearing minerals remains unknown. Here, we evaluated N and C isotope fractionation of RDX during reduction by Fe(II) associated with Fe minerals and natural sediments and applied N isotope ratios to the assessment of mineral-catalyzed RDX reduction in a contaminant plume and in sediment columns treated by in situ chemical reduction. Laboratory studies revealed that RDX was reduced to nitroso compounds without denitration and the concomitant ring cleavage. Fe(II)/iron oxide mineral-catalyzed reactions exhibited N isotope enrichment factors, εN, between -6.3±0.3‰ and -8.2±0.2‰, corresponding to an apparent 15N kinetic isotope effect of 1.04-1.05. The observed variations of the δ15N of ∼15‰ in RDX from groundwater samples suggested an extent of reductive transformation of 85% at an ammunition plant. Conversely, we observed masking of N isotope fractionation after RDX reduction in laboratory flow-through systems, which was presumably due to limited accessibility to reactive Fe(II).

Role of Carbonate in Thermodynamic Relationships Describing Pollutant Reduction Kinetics by Iron Oxide-Bound Fe2.

The reduction of environmental pollutants by Fe2+ bound to iron oxides is an important process that determines pollutant toxicities and mobilities. Recently, we showed that pollutant reduction rates depend on the thermodynamic driving force of the reaction in a linear free energy relationship that was a function of the solution pH value and the reduction potential, EH, of the interfacial Fe3+/Fe2+ redox couple. In this work, we studied how carbonate affected the free energy relationship by examining the effect that carbonate has on nitrobenzene reduction rates by Fe2+ bound to goethite (α-FeOOH). Carbonate slowed nitrobenzene reduction rates by inducing goethite particle aggregation, as evidenced by surface charge and particle size measurements. We observed no evidence for carbonate affecting Fe3+/Fe2+ reduction potentials or the mechanism of nitrobenzene reduction. The linear free energy relationship accurately described the data collected in the presence of carbonate when we accounted for the effect it had on the reactive surface area of goethite. The findings from this work provide a framework for determining why common groundwater constituents affect the EH-dependence of reaction rates involving oxide-bound Fe2+ as a reductant.

Carbon Isotope Fractionation of Substituted Benzene Analogs during Oxidation with Ozone and Hydroxyl Radicals: How Should Experimental Data Be Interpreted?

Oxidative processes frequently contribute to organic pollutant degradation in natural and engineered systems, such as during the remediation of contaminated sites and in water treatment processes. Because a systematic characterization of abiotic reactions of organic pollutants with oxidants such as ozone or hydroxyl radicals by compound-specific stable isotope analysis (CSIA) is lacking, stable isotope-based approaches have rarely been applied for the elucidation of mechanisms of such transformations. Here, we investigated the carbon isotope fractionation associated with the oxidation of benzene and several methylated and methoxylated analogs, namely, toluene, three xylene isomers, mesitylene, and anisole, and determined their carbon isotope enrichments factors (εC) for reactions with ozone (εC = -3.6 to -4.6 ‰) and hydroxyl radicals (εC = 0.0 to -1.2‰). The differences in isotope fractionation can be used to elucidate the contribution of the reactions with ozone or hydroxyl radicals to overall transformation. Derivation of apparent kinetic isotope effects (AKIEs) for the reaction with ozone, however, was nontrivial due to challenges in assigning reactive positions in the probe compounds for the monodentate attack leading to an ozone adduct. We present several options for this step and compare the outcome to quantum chemical characterizations of ozone adducts. Our data show that a general assignment of reactive positions for reactions of ozone with aromatic carbons in ortho-, meta-, or para-positions is not feasible and that AKIEs of this reaction should be derived on a compound-by-compound basis.

Assessment of 2,4-Dinitroanisole Transformation Using Compound-Specific Isotope Analysis after In Situ Chemical Reduction of Iron Oxides.

Ferrous iron-bearing minerals are important reductants in the contaminated subsurface, but their availability for the reduction of anthropogenic pollutants is often limited by competition with other electron acceptors including microorganisms and poor accessibility to Fe(II) in complex hydrogeologic settings. The supply of external electron donors through in situ chemical reduction (ISCR) has been proposed as one remediation approach, but the quantification of pollutant transformation is complicated by the perturbations introduced to the subsurface by ISCR. Here, we evaluate the application of compound specific isotope analysis (CSIA) for monitoring the reduction of 2,4-dinitroanisole (DNAN), a component of insensitive munitions formulations, by mineral-bound Fe(II) generated through ISCR of subsurface material from two field sites. Electron balances from laboratory experiments in batch and column reactors showed that 3.6% to 11% of the total Fe in the sediments was available for the reduction of DNAN and its partially reduced intermediates after dithionite treatment. The extent of DNAN reduction was successfully quantified from its N isotope fractionation measured in the column effluent based on the derivation of a N isotope enrichment factor, εN, derived from a comprehensive series of isotope fractionation experiments with numerous Fe(II)-bearing minerals as well as dithionite-reduced subsurface materials. Our observations illustrate the utility of CSIA as a robust approach to evaluate the success of in situ remediation through abiotic contaminant reduction.

Enzyme Kinetics of Organic Contaminant Oxygenations.

Enzymatic oxygenations initiate biodegradation processes of many organic soil and water contaminants. Even though many biochemical aspects of oxygenation reactions are well-known, quantifying rates of oxidative contaminant removal as well as the extent of oxygenation remains a major challenge. Because enzymes use different strategies to activate O2, reactions leading to substrate oxygenation are not necessarily limiting the rate of contaminant removal. Moreover, oxygenases react along unproductive pathways without substrate metabolism leading to O2 uncoupling. Here, we identify the critical features of the catalytic cycles of selected oxygenases that determine rates and extents of biodegradation. We focus most specifically on Rieske dioxygenases, a subfamily of mononuclear non-heme ferrous iron oxygenases, because of their ability to hydroxylate unactivated aromatic structures and thus initiate the transformation of the most persistent organic contaminants. We illustrate that the rate-determining steps in their catalytic cycles range from O2 activation to substrate hydroxylation, depending on the extent of O-O cleavage that is required for generating the reactive Fe-oxygen species. The extent of O2 uncoupling, on the other hand, is highly substrate-specific and potentially modulated by adaptive responses to oxidative stress. Understanding the kinetic mechanisms of oxygenases will be key to assess organic contaminant biotransformation quantitatively.

Dual-Element Isotope Analysis of Desphenylchloridazon to Investigate Its Environmental Fate in a Systematic Field Study: A Long-Term Lysimeter Experiment.

Desphenylchloridazon (DPC), the main metabolite of the herbicide chloridazon (CLZ), is more water soluble and persistent than CLZ and frequently detected in water bodies. When assessing DPC transformation in the environment, results can be nonconclusive if based on concentration analysis alone because estimates may be confounded by simultaneous DPC formation from CLZ. This study investigated the fate of DPC by combining concentration-based methods with compound-specific C and N stable isotope analysis (CSIA). Additionally, DPC formation and transformation processes were experimentally deconvolved in a dedicated lysimeter study considering three scenarios. First, surface application of DPC enabled studying its degradation in the absence of CLZ. Here, CSIA provided evidence of two distinct DPC transformation processes: one shows significant changes only in 13C/12C, whereas the other involves changes in both 13C/12C and 15N/14N isotope ratios. Second, surface application of CLZ mimicked a realistic field scenario, showing that during DPC formation, 13C/12C ratios of DPC were depleted in 13C relative to CLZ, while 15N/14N ratios remained constant. Finally, CLZ depth injection simulated preferential flow and demonstrated the importance of the topsoil for retaining DPC. The combination of the lysimeter study with CSIA enabled insights into DPC transformation in the field that are superior to those of studies of concentration trends.

Decreases in Iron Oxide Reducibility during Microbial Reductive Dissolution and Transformation of Ferrihydrite.

Ferrous iron formed during microbial ferric iron reduction induces phase transformations of poorly crystalline into more crystalline and thermodynamically more stable iron (oxyhydr)oxides. Yet, characterizing the resulting decreases in the reactivity of the remaining oxide ferric iron toward reduction (i.e., its reducibility) has been challenging. Here, we used the reduction of six-line ferrihydrite by Shewanella oneidensis MR-1 as a model system to demonstrate that mediated electrochemical reduction (MER) allows directly following decreases in oxide ferric iron reducibility during the transformation of ferrihydrite into goethite and magnetite which we characterized by X-ray diffraction analysis and transmission electron microscopy imaging. Ferrihydrite was fully reducible in MER at both pHMER of 5.0 and 7.5. Decreases in iron oxide reducibility associated with ferrihydrite transformation into magnetite were accessible at both pHMER because the formed magnetite was not reducible under either of these conditions. Conversely, decreases in iron oxide reducibility associated with goethite formation were apparent only at the highest tested pHMER of 7.5 and thus the thermodynamically least favorable conditions for iron oxide reductive dissolution. The unique capability to adjust the thermodynamic boundary conditions in MER to the specific reducibilities of individual iron (oxyhydr)oxides makes this electrochemical approach broadly applicable for studying changes in iron oxide reducibility in heterogeneous environmental samples such as soils and sediments.

Assessing Aerobic Biotransformation of Hexachlorocyclohexane Isomers by Compound-Specific Isotope Analysis.

Contamination of soils and sediments with the highly persistent hexachlorocyclohexanes (HCHs) continues to be a threat for humans and the environment. Despite the existence of bacteria capable of biodegradation and cometabolic transformation of HCH isomers, such processes occur over time scales of decades and are thus challenging to assess. Here, we explored the use of compound-specific isotope analysis (CSIA) to track the aerobic biodegradation and biotransformation pathways of the most prominent isomers, namely, (-)-α-, (+)-α-, ß-, γ-, and δ-HCH, through changes of their C and H isotope composition in assays of LinA2 and LinB enzymes. Dehydrochlorination of (+)-α-, γ-, and δ-HCH catalyzed by LinA2 was subject to substantial C and H isotope fraction with apparent 13C- and 2H-kinetic isotope effects (AKIEs) of up to 1.029 ± 0.001 and 6.7 ± 2.9, respectively, which are indicative of bimolecular eliminations. Hydrolytic dechlorination of δ-HCH by LinB exhibited even larger C but substantially smaller H isotope fractionation with 13C- and 2H-AKIEs of 1.073 ± 0.006 and 1.41 ± 0.04, respectively, which are typical for nucleophilic substitutions. The systematic evaluation of isomer-specific phenomena showed that, in addition to contaminant uptake limitations, diffusion-limited turnover ((-)-α-HCH), substrate dissolution (ß-HCH), and potentially competing reactions catalyzed by constitutively expressed enzymes might bias the assessment of HCH biodegradation by CSIA at contaminated sites.

Solid-phase extraction method for stable isotope analysis of pesticides from large volume environmental water samples.

Compound-specific isotope analysis (CSIA) is a valuable tool for assessing the fate of organic pollutants in the environment. However, the requirement of sufficient analyte mass for precise isotope ratio mass spectrometry combined with prevailing low environmental concentrations currently limits comprehensive applications to many micropollutants. Here, we evaluate the upscaling of solid-phase extraction (SPE) approaches for routine CSIA of herbicides. To cover a wide range of polarity, a SPE method with two sorbents (a hydrophobic hypercrosslinked sorbent and a hydrophilic sorbent) was developed. Extraction conditions, including the nature and volume of the elution solvent, the amount of sorbent and the solution pH, were optimized. Extractions of up to 10 L of agricultural drainage water (corresponding to up to 200 000-fold pre-concentration) were successfully performed for precise and sensitive carbon and nitrogen CSIA of the target herbicides atrazine, acetochlor, metolachlor and chloridazon, and metabolites desethylatrazine, desphenylchloridazon and 2,6-dichlorobenzamide in the sub-µg L-1-range. 13C/12C and 15N/14N ratios were measured by gas chromatography-isotope ratio mass spectrometry (GC/IRMS), except for desphenylchloridazon, for which liquid chromatography (LC/IRMS) and derivatization-GC/IRMS were used, respectively. The method validated in this study is an important step towards analyzing isotope ratios of pesticide mixtures in aquatic systems and holds great potential for multi-element CSIA applications to trace pesticide degradation in complex environments.

Electrochemical Analysis of Changes in Iron Oxide Reducibility during Abiotic Ferrihydrite Transformation into Goethite and Magnetite.

Electron transfer to ferric iron in (oxyhydr-)oxides (hereafter iron oxides) is a critical step in many processes that are central to the biogeochemical cycling of elements and to pollutant dynamics. Understanding these processes requires analytical approaches that allow for characterizing the reactivity of iron oxides toward reduction under controlled thermodynamic boundary conditions. Here, we used mediated electrochemical reduction (MER) to follow changes in iron oxide reduction extents and rates during abiotic ferrous iron-induced transformation of six-line ferrihydrite. Transformation experiments (10 mM ferrihydrite-FeIII) were conducted over a range of solution conditions (pHtrans = 6.50 to 7.50 at 5 mM Fe2+ and for pHtrans = 7.00 also at 1 mM Fe2+) that resulted in the transformation of ferrihydrite into thermodynamically more stable goethite or magnetite. The changes in iron oxide mineralogy during the transformations were quantified using X-ray diffraction analysis. MER measurements on iron oxide suspension aliquots collected during the transformations were performed over a range of pHMER at constant applied reduction potential. The extents and rates of iron oxide reduction in MER decreased with decreasing reaction driving force resulting from both increasing pHMER and increasing transformation of ferrihydrite into thermodynamically more stable iron oxides. We show that the decreases in iron oxide reduction extents and rates during ferrihydrite transformations can be linked to the concurrent changes in iron oxide mineralogy.

13C- and 15N-Isotope Analysis of Desphenylchloridazon by Liquid Chromatography-Isotope-Ratio Mass Spectrometry and Derivatization Gas Chromatography-Isotope-Ratio Mass Spectrometry.

The widespread application of herbicides impacts surface water and groundwater. Metabolites (e.g., desphenylchloridazon from chloridazon) may be persistent and even more polar than the parent herbicide, which increases the risk of groundwater contamination. When parent herbicides are still applied, metabolites are constantly formed and may also be degraded. Evaluating their degradation on the basis of concentration measurements is, therefore, difficult. This study presents compound-specific stable-isotope analysis (CSIA) of nitrogen- and carbon-isotope ratios at natural abundances as an alternative analytical approach to track the origin, formation, and degradation of desphenylchloridazon (DPC), the major degradation product of the herbicide chloridazon. Methods were developed and validated for carbon- and nitrogen-isotope analysis (δ13C and δ15N) of DPC by liquid chromatography-isotope-ratio mass spectrometry (LC-IRMS) and derivatization gas chromatography-IRMS (GC-IRMS), respectively. Injecting standards directly onto an Atlantis LC-column resulted in reproducible δ13C-isotope analysis (standard deviation <0.5‰) by LC-IRMS with a limit of precise analysis of 996 ng of DPC on-column. Accurate and reproducible δ15N analysis with a standard deviation of <0.4‰ was achieved by GC-IRMS after derivatization of >100 ng of DPC with 160-fold excess of (trimethylsilyl)diazomethane. Application of the method to environmental-seepage water indicated that newly formed DPC could be distinguished from "old" DPC by the different isotopic signatures of the two DPC sources.

Kinetic Isotope Effects of the Enzymatic Transformation of γ-Hexachlorocyclohexane by the Lindane Dehydrochlorinase Variants LinA1 and LinA2.

Compound-specific isotope analysis (CSIA) can provide insights into the natural attenuation processes of hexachlorocyclohexanes (HCHs), an important class of persistent organic pollutants. However, the interpretation of HCH stable isotope fractionation is conceptually challenging. HCHs exist as different conformers that can be converted into each other, and the enzymes responsible for their transformation discriminate among those HCH conformers. Here, we investigated the enzyme specificity of apparent 13C- and 2H-kinetic isotope effects (AKIEs) associated with the dehydrochlorination of γ-HCH (lindane) by two variants of the lindane dehydrochlorinases LinA1 and LinA2. While LinA1 and LinA2 attack γ-HCH at different trans-1,2-diaxial H-C-C-Cl moieties, the observed C and H isotope fractionation was large, typical for bimolecular eliminations, and was not affected by conformational mobility. 13C-AKIEs for transformation by LinA1 and LinA2 were the same (1.024 ± 0.001 and 1.025 ± 0.001, respectively), whereas 2H-AKIEs showed minor differences (2.4 ± 0.1 and 2.6 ± 0.1). Variations of isotope effects between LinA1 and LinA2 are small and in the range reported for different degrees of C-H bond cleavage in transition states of dehydrochlorination reactions. The large C and H isotope fractionation reported here for experiments with pure enzymes contrasts with previous observations from whole cell experiments and suggests that specific uptake processes by HCH-degrading microorganisms might modulate the observable HCH isotope fractionation at contaminated sites.