Orbitrap mass spectrometry with electrospray ionization (ESI-Orbitrap MS) enables¹⁸O/¹⁶O ratio measurements in phosphate and organophosphorus compounds, which offers promising avenues for the study of metabolic, biogeochemical, and environmental processes. While the instrumental feasibility of such¹⁸O/¹⁶O ratio measurements has been shown, the applicability of such analyses in aqueous matrices on multipurpose mass spectrometers remains unaddressed. Here, we (i) evaluated the interplay between instrument parameters and the long-term accuracy and precision of ESI-Orbitrap MS for the determination of δ¹⁸O(PO₄) from H₂PO₄^–vs PO₃^–fragments of phosphate, (ii) identified properties of methanolic sample solutions that are critical for accurate and precise measurements, and (iii) propose a sample purification procedure for the elimination of matrix-based interferences. In 10 measurement campaigns over two years, we observed a long-term reproducibility of δ¹⁸O(PO₄) within ±2.8‰. Results of in-source fragmentation experiments of H₂PO₄^–to PO₃^–show excellent agreement of δ¹⁸O and offer promising opportunities to probe for¹⁸O/¹⁶O ratios in organophosphorus compounds. By investigating the effect of the aqueous matrix and interfering anions on¹⁸O/¹⁶O ratio measurements, we found that a water content exceeding 50 vol % and the presence of oxyanions such as nitrate and sulfate limit measurement accuracy due to interferences of matrix constituents in the ESI source. To overcome these challenges, we evaluated selective phosphate extraction with a zirconium-based metal–organic framework (MOF) as sorbent. The resulting purification procedure allowed for successful extraction and recovery of phosphate from nitrate- and sulfate-containing aqueous solutions, resulting in methanolic phosphate samples that enabled accurate analyses of δ¹⁸O(PO₄) by ESI-Orbitrap MS.

Oxygen isotope ratios of O₂ are important tracers for assessing biological activity in biogeochemical processes in aquatic environments. In fact, changes in the ¹⁸O/¹⁶O and ¹⁷O/¹⁶O ratios of O₂ have been successfully implemented as measures for quantifying photosynthetic O₂ production and biological O₂ respiration. Despite evidence for light-dependent O₂ consumption in sunlit surface waters, however, photochemical O₂ loss processes have so far been neglected in the stable isotope-based evaluation of oxygen cycling. Here, we established the magnitude of the O isotope fractionation for abiotic photochemical O₂ elimination through formation of singlet O_2, ¹O₂, and the ensuing oxygenation and oxidation reactions with organic compounds through experiments with rose bengal as the ¹O₂ sensitizer and three different amino acids and furfuryl alcohol as chemical quenchers. Based on the kinetic analysis of light-dependent O₂ removal in the presence of different quenchers, we rationalize the observable O isotope fractionation of O₂ and the corresponding, apparent ¹⁸O kinetic isotope effects (¹⁸O-AKIE) with a pre-equilibrium model for the reversible formation of ¹O₂ and its irreversible oxygenation reactions with organic compounds. While ¹⁸O-AKIEs of oxygenation reactions amount to 1.03, the O isotope fractionation of O₂ decreased to unity with increasing ratio of the rates of oxygenation reaction of ¹O₂ vs ¹O₂ decay to ground state oxygen, ³O₂. Our findings imply that O isotope fractionation through photochemical O₂ consumption with isotope enrichment factors, ¹⁸O-ϵ, of up to −30‰ can match contributions from biological respiration at typical dissolved organic matter concentrations of lakes, rivers, and oceans and should, therefore, be included in future evaluations of biogeochemical O₂ cycling.

The comprehensive evaluation of pollutant abatement during chemical oxidation processes and the identification of potentially hazardous transformation products are fundamental challenges in water and wastewater treatment. Here, we demonstrate how high-throughput computational chemistry enables the elucidation of reaction pathways via automated, quantum-chemistry-based chemical reaction network (CRN) explorations. We evaluated the predictive capabilities of this computational approach using the Software for Chemical Interaction Networks (SCINE) for studying the reactions of ozone with two olefins, ethene and tetramethylethene, in aqueous solution. Following a benchmarking of the quantum chemical methodology for structure optimization and energy calculations, we generated CRNs containing hundreds of compounds and thousands of reactions, identified reaction mechanisms, and evaluated product formation kinetics through microkinetic modeling. These CRN explorations led to the correct reproduction of experimental evidence for mechanisms and products of olefin ozonolysis for reactions of ozone and ethene solely on the basis of defining the reactants and their initial concentrations. The study of reactions of ozone and tetramethylethene also matched experimental data for the main products but revealed consequences of the limited exploration depth and shortcomings of the implicit solvation model. We envision that CRN explorations not only offer novel means for predicting pollutant transformation pathways but also will support chemical analysis and the assessment of effects on human and environmental health.

The analysis of multi-elemental compound-specific stable isotope analysis (CSIA) has been established for the determination of contaminant degradation pathways. For systems with two pathways taking place simultaneously also a quantitative analysis of each pathway’s contribution to total degradation has been introduced using the combined information from the stable isotope fractionation of two elements. Recent experimental approaches also allow for the assessment of stable isotope fractionation of three different elements of a degraded compound, which would provide the opportunity to analyze systems with three simultaneously occurring degradation pathways using stable isotope data. Yet, approaches for a quantitative analysis of such systems are missing. Here we mathematically derive and present an approach to determine the contribution of three different degradation pathways to total degradation of a contaminant compound using the stable isotope fractionation of three different elements in the remaining compound. To verify the accuracy of the computational approach numerical simulations of virtual batch experiments were performed considering the degradation of a compound via three degradation pathways each leading to a stable isotope fractionation of three different elements. Applying the computational approach to the simulated concentration and stable isotope data allowed an exact determination of the contribution of the individual degradation pathways to total contaminant degradation regardless of the considered degradation rates. As application example we apply our approach to experimental data from the literature on the in-situ degradation of 2,4-DNT and the associated stable isotope fractionation of C, H and N. Calculated results of deoxygenation contributing 92%, partial reduction contributing 7% and CH₃-group oxidation contributing 1% to total degradation are in agreement with estimates presented in the literature. The new computational approach provides a novel tool for an improved analysis of multi-element CSIA data and for the quantitative assessment of contaminant degradation processes.

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.

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 O₂ 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 O₂ 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 O₂ uncoupling based on mass balance or kinetic approaches that relate successful oxygenations to total O₂ 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.

Oxygenation of aromatic and aliphatic hydrocarbons by Rieske oxygenases is the initial step of various biodegradation pathways for environmental organic contaminants. Microorganisms carrying Rieske oxygenases are able to quickly adapt their substrate spectra to alternative carbon and energy sources that are structurally related to the original target substrate, yet the molecular events responsible for this rapid adaptation are not well understood. Here, we evaluated the hypothesis that reactive oxygen species (ROS) generated by unproductive activation of O₂, the so-called O₂ uncoupling, in the presence of the alternative substrate exert a selective pressure on the bacterium for increasing the oxygenation efficiency of Rieske oxygenases. To that end, we studied wild-type 2-nitrotoluene dioxygenase from Acidovorax sp. strain JS42 and five enzyme variants that have evolved from adaptive laboratory evolution experiments with 3- and 4-nitrotoluene as alternative growth substrates. The enzyme variants showed a substantially increased oxygenation efficiency toward the new target substrates concomitant with a reduction of ROS production, while mechanisms and kinetics of enzymatic O₂ activation remained unchanged. Structural analyses and docking studies suggest that amino acid substitutions in enzyme variants occurred at residues lining both substrate and O₂ transport tunnels, enabling tighter binding of the target substrates in the active site. Increased oxygenation efficiencies measured in vitro for the various enzyme (variant)-substrate combinations correlated linearly with in vivo changes in growth rates for evolved Acidovorax strains expressing the variants. Our data suggest that the selective pressure from oxidative stress toward more efficient oxygenation by Rieske oxygenases was most notable when O₂ uncoupling exceeded 60%.

The management and mitigation of chemical pollution are key elements of sustainable development initiatives that aim to provide safe and clean water. While environmental scientists are developing the capabilities to assess the fate, (eco)toxicity and risks of a plethora of synthetic chemicals comprehensively, notorious pollution scenarios and decontamination challenges call for targeted and case-specific evaluation of chemical hazards. Here we review the utility and perspectives of compound-specific isotope analysis for obtaining an understanding of environmental processes that allows one to identify pollution sources, assess contaminant (bio)transformation and gain insights into reaction pathways. Using three prototypical scenarios of water contamination, namely point-source pollution of groundwater at contaminated sites, diffuse pollution of soils and surface waters through pesticide use and the abatement of pharmaceuticals and disinfection by-products in water treatment systems, we illustrate both success stories of compound-specific isotope analysis and current developments to address challenges for future applications.

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.

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.

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 Fe^III-(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.

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 ¹⁸O/¹⁶O ratios of O₂ 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, δ¹⁸O, could be overestimated by more than 6‰ because of interferences from argon in air. Here, we systematically evaluated the extent of such Ar interferences on ¹⁸O/¹⁶O ratios of O₂ for measurements by gas chromatography/IRMS and GasBench/IRMS and propose simple instrumental modifications for improved Ar and O₂ separation as well as post-measurement correction procedures for obtaining accurate δ¹⁸O. We subsequently evaluated the consequences of Ar interferences for the quantification of O isotope fractionation in terms of isotope enrichment factors, ϵ_O, and ¹⁸O kinetic isotope effects (¹⁸O KIEs) in samples where O₂ is consumed and Ar:O₂ 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 δ¹⁸O measurements and experimental variability. Ar interferences can become more relevant and bias ϵ_O values by more than 2‰ in aqueous samples where fractional O₂ conversion exceeds 90%. Practically, however, such samples would typically contain less than 25 μM of O₂ at ambient temperature, an amount that is close to the method detection limit of ¹⁸O/¹⁶O ratio measurement by CF-IRMS.

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 O₂ activation. Here, we studied the role of the substrate in the key elementary reaction leading to O₂ activation from a theoretical perspective by systematically considering (i) the 6-coordinate to 5-coordinate conversion of the non-heme Fe^II upon abstraction of a water ligand, (ii) binding of O₂, 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 H₂O reorientation and exothermic O₂ 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 in agreement with a strategy that avoids unproductive O₂ activation.

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 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 Fe^III-(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.

Methods are needed to verify that attenuation of energetic compounds, such as trinitrotoluene (TNT) and hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), in groundwater is occurring, and it must also be possible to verify that strategies to enhance degradation processes are having the desired effects. This document provides a primer on sample collection, processing, and laboratory methods for compound specific isotope analysis (CSIA)methods for nitro explosives. Data evaluation, analysis, and interpretation for both single and dual isotope fractionation is presented. Two case studies are used to demonstrate the procedures for using CSIA to support the identification of the relevant reaction process and calculate the extent of which reaction, versus dilution or sorption, is leading to decreases in concentration.

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, ^ϵ_N, 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).

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 (ε_Cl from -5.0 ± 2.3 to -6.5 ± 0.7‰). The characteristic dual C-Cl isotope fractionation patterns (Λ_C–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 Λ_C–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.

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, ϵ_N, 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.

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.

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, E_H, 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 E_H-dependence of reaction rates involving oxide-bound Fe²⁺ as a reductant.

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.

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 (ε_C) for reactions with ozone (ε_C = -3.6 to -4.6 ‰) and hydroxyl radicals (ε_C = 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.

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 pH_MER of 5.0 and 7.5. Decreases in iron oxide reducibility associated with ferrihydrite transformation into magnetite were accessible at both pH_MER 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 pH_MER 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.

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-Fe^III) were conducted over a range of solution conditions (pH_trans = 6.50 to 7.50 at 5 mM Fe²⁺ and for pH_trans = 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 pH_MER 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 pH_MER 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.

The recent development of insensitive munitions, such as 2,4-dinitroanisole (DNAN), as components of military explosives has generated concern for potential subsurface contamination and created a need to fully characterize their transformation processes. Compound specific isotope analysis (CSIA) has proven to be a useful means of assessing transformation pathways according to characteristic stable isotope fractionation patterns. The C and N isotope fractionation of DNAN associated with abiotic and enzymatic hydrolysis was recently assessed. The extent to which DNAN isotope fractionation will be affected by other potentially competing transformation pathways known for nitroaromatic compounds (e.g., reduction) and if previous knowledge can be extrapolated to other environmental matrices remains to be understood. Here, we investigated the C and N isotope fractionation and reaction rate constants of DNAN during abiotic reduction mediated by mineral-associated Fe(II) species as a function of mineral type, natural organic matter presence, and repeated exposures to DNAN. Though rate constants varied, N and C apparent kinetic isotope effects (AKIEs) remained consistent across all experiments (averaged values of ¹⁵N-AKIE = 1.0317 ± 0.0064 and ¹³C-AKIE = 1.0008 ± 0.0005) and revealed significant ¹⁵N- and minimal ¹³C-enrichment in agreement with previous work on nitroaromatic compounds. Moreover, the observed fractionation was clearly distinct from trends for abiotic and enzymatic hydrolysis. This study provides a strengthened basis for the use of CSIA as a robust tool for monitoring DNAN degradation in complex environmental matrices as a component of future remediation efforts.

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.

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-, β-, 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 (β-HCH), and potentially competing reactions catalyzed by constitutively expressed enzymes might bias the assessment of HCH biodegradation by CSIA at contaminated sites.

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.

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.

Iron (oxyhydr-)oxide reduction has been extensively studied because of its importance in pollutant redox dynamics and biogeochemical processes. Yet, experimental studies linking oxide reduction kinetics to thermodynamics remain scarce. Here, we used mediated electrochemical reduction (MER) to directly quantify the extents and rates of ferrihydrite, goethite, and hematite reduction over a range of negative reaction free energies, Δ_rG, that were obtained by systematically varying pH (5.0 to 8.0), applied reduction potentials (−0.53 to −0.17 V vs SHE), and Fe²⁺ concentrations (up to 40 μM). Ferrihydrite reduction was complete and fast at all tested Δ_rG values, consistent with its comparatively low thermodynamic stability. Reduction of the thermodynamically more stable goethite and hematite changed from complete and fast to incomplete and slow as Δ_rG values became less negative. Reductions at intermediate Δ_rG values showed negative linear correlations between the natural logarithm of the reduction rate constants and Δ_rG. These correlations imply that thermodynamics controlled goethite and hematite reduction rates. Beyond allowing to study iron oxide reduction under defined thermodynamic conditions, MER can also be used to capture changes in iron oxide reducibility during phase transformations, as shown for Fe²⁺-facilitated transformation of ferrihydrite to goethite.

Compound-specific isotope analysis (CSIA) of polar organic micropollutants in environmental waters requires a processing of large sample volumes to obtain the required analyte masses for analysis by gas chromatography/isotope-ratio mass spectrometry (GC/IRMS). However, the accumulation of organic matter of unknown isotopic composition in standard enrichment procedures currently compromises the accurate determination of isotope ratios. We explored the use of molecularly imprinted polymers (MIPs) for selective analyte enrichment for ¹³C/¹²C and ¹⁵N/¹⁴N ratio measurements by GC/IRMS using 1H-benzotriazole, a typical corrosion inhibitor in dishwashing detergents, as example of a widely detected polar organic micropollutant. We developed procedures for the treatment of > 10 L of water samples, in which custom-made MIPs enabled the selective cleanup of enriched analytes in organic solvents obtained through conventional solid-phase extractions. Hydrogen bonding interactions between the triazole moiety of 1H-benzotriazole, and the MIP were responsible for selective interactions through an assessment of interaction enthalpies and ¹⁵N isotope effects. The procedure was applied successfully without causing isotope fractionation to river water samples, as well as in- and effluents of wastewater treatment plants containing μg/L concentrations of 1H-benzotriazole and dissolved organic carbon (DOC) loads of up to 28 mg C/L. MIP-based treatments offer new perspectives for CSIA of organic micropollutants through the reduction of the DOC-to-micropollutant ratios.

Numerous studies have reported that pollutant reduction rates by ferrous iron (Fe²⁺) are substantially enhanced in the presence of an iron (oxyhydr)oxide mineral. Developing a thermodynamic framework to explain this phenomenon has been historically difficult due to challenges in quantifying reduction potential (E_H) values for oxide-bound Fe²⁺ species. Recently, our group demonstrated that E_H values for hematite- and goethite-bound Fe²⁺ can be accurately calculated using Gibbs free energy of formation values. Here, we tested if calculated E_H values for oxide-bound Fe²⁺ could be used to develop a free energy relationship capable of describing variations in reduction rate constants of substituted nitrobenzenes, a class of model pollutants that contain reducible aromatic nitro groups, using data collected here and compiled from the literature. All the data could be described by a single linear relationship between the logarithms of the surface-area-normalized rate constant (k_SA) values and E_H and pH values [log(k_SA) = −E_H/0.059 V – pH + 3.42]. This framework provides mechanistic insights into how the thermodynamic favorability of electron transfer from oxide-bound Fe²⁺ relates to redox reaction kinetics.

The suitability of two different tracers to mimic the behavior of pesticides in agricultural soils and to evidence the potential for preferential flow was evaluated in outdoor lysimeter experiments. The herbicide atrazine [6-chloro-N-ethyl-N'-(1-methylethyl)-1,3,5-triazine-2,4-diamine] was used as a model compound. Two tracers were used: a nonadsorbing tracer (bromide) and a weakly adsorbing dye tracer (uranine). Two soils that are expected to show a different extent of macropore preferential flow were used: a well-drained sandy-loamy Cambisol (gravel soil) and a poorly drained loamy Cambisol (moraine soil). Conditions for preferential flow were promoted by applying heavy simulated rainfall shortly after pesticide application. In some of the experiments, preferential flow was also artificially simulated by injecting the solutes through a narrow tube below the root zone. With depth injection, preferential leaching of atrazine occurred shortly after application in both soil types, whereas with surface application, it occurred only in the moraine soil. Thereafter, atrazine transport was mainly through the porous soil matrix, although contributions of preferential flow were also observed. For all the application approaches and soil types, after 900 d, <3% of the applied amount of atrazine was recovered in the drainage water. Only uranine realistically illustrated the early atrazine breakthrough by transport through preferential flow. Uranine broke through during the first intense irrigation at the same time as atrazine. Bromide, however, appeared earlier than atrazine in some cases. The use of dye tracers as pesticide surrogates might assist in making sustainable decisions with respect to pesticide application timing relative to rainfall or soil potential for preferential flow.

The emerging use of 2,4-dinitroanisole (DNAN) in insensitive munitions formulations has caused concern about future contamination of subsurface environments, generating significant interest in understanding and identifying its transformation processes. Here we characterized the C and N isotope fractionation associated with abiotic and biological DNAN hydrolysis through alkaline hydrolysis at high pH as well as enzymatic hydrolysis by Nocardioides sp. JS1661 and partially purified DNAN O-demethylase. Whereas both reactions generated 2,4-dinitrophenol (DNP), compound-specific isotope analysis (CSIA) of DNAN and DNP revealed that these reactions occur by different mechanisms. Alkaline hydrolysis was associated with apparent ¹³C and ¹⁵N kinetic isotope effects (¹³C-AKIE and ¹⁵N-AKIE) of 1.044 ± 0.003 and 1.0027 ± 0.0004, respectively, reflecting the previously postulated nucleophilic aromatic substitution mechanism. Conversely, enzyme-catalyzed DNAN hydrolysis exhibited a ¹³C-AKIE of 1.027 ± 0.005 and a ¹⁵N-AKIE of 1.0032 ± 0.0003. On the basis of these AKIE values and the C and N isotope fractionation of DNP, our results imply that enzymatic O-demethylation of DNAN occurs through a nucleophilic substitution reaction at the aliphatic C of the methoxy group. This work provides a basis for the assessment of DNAN transformation by CSIA, as the C and N isotope fractionation patterns observed in this work are distinct from other hypothesized degradation pathways.

The Joux Valley (Swiss Jura Mountains) has a rather unusual history of human occupation, characterized by tardive but extensive settlement since the Late Middle Ages, followed by an intensive period of industrial development. To estimate the links between human activities and environmental consequences, sediment cores were retrieved in Lake Joux and submitted to a multiproxy analysis (high-resolution photographs, magnetic susceptibility, density, x-ray fluorescence, grain size, organic geochemistry, ¹⁴C, ²¹⁰Pb and ¹³⁷Cs dating). The diversity of anthropication phases, defined from historical data, is clearly recognized in the lake archive. The record suggests the region was mainly under climatic influence until the end of the 13th century. The growth of settlements in the valley and the associated massive deforestation is recorded by increasing terrestrial inputs, reflecting large-scale soil destabilization, which subsequently persists despite the transition from farming to industrial activities. Autochthonous contributions then dominate the record, both in response to climatic and anthropogenic influences. Construction works conducted at the outlet of the lake affected water flow, sedimentation and aquatic community (macrophytes, ostracods) dynamics. The substantial increase of anthropogenic heavy metals (Fe, Zn, Pb) recorded during the 20th century could reflect the development of the watch-making industry in the area, as well as the use of leaded gasoline. Historical information facilitated interpretation of the observed paleolimnological evolution in the context of varied coexisting human activities. This study highlights the importance of applying an integrated paleolimnological-historical approach in order to establish clear links between well-defined human activities and their subsequent environmental responses through time.

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.

Assessing the precursors and reactions leading to the carcinogenic N-nitrosodimethylamine (NDMA) during drinking water disinfection is a major challenge. Here, we investigate whether changes of ¹³C/¹²C, ²H/¹H, and ¹⁵N/¹⁴N ratios of NDMA give rise to isotope fractionation trends that can be used to infer NDMA formation pathways. We carried out compound-specific isotope analysis (CSIA) of NDMA during chloramination of four tertiary amines that produce NDMA at high yields, namely ranitidine, 5-(dimethylaminomethyl)furfuryl alcohol, N,N-dimethylthiophene-2-methylamine and N,N-dimethylbenzylamine. Carbon and hydrogen isotope ratios of NDMA function as fingerprints of the N(CH₃)₂ moiety from the tertiary amine and exhibit only minor isotope fractionation during the disinfection process. Nitrogen isotope ratios showed that NH₂Cl is the source of the N atom of the nitroso group. The large enrichment of ¹⁵N in NDMA was indicative of the isotope effects pertinent to bond cleavage and bond formation reactions during chloramination of tertiary amines.Correlation of δ¹⁵N vs. δ¹³C values of NDMA resulted in trend lines that were not affected by the type of tertiary amine and treatment conditions suggesting that the observed C and N isotope fractionation in NDMA may be diagnostic for NDMA precursors and formation pathways during chloramination.

N-Nitrosodimethylamine (NDMA) is a carcinogenic disinfection byproduct from water chloramination. Despite the identification of numerous NDMA precursors, essential parts of the reaction mechanism such as the incorporation of molecular O₂ are poorly understood. In laboratory model systems for the chloramination of secondary and tertiary amines, we investigated the kinetics of precursor disappearance and NDMA formation, quantified the stoichiometries of monochloramine (NH₂Cl) and aqueous O₂ consumption, derived ¹⁸O-kinetic isotope effects (¹⁸O-KIE) for the reactions of aqueous O₂, and studied the impact of radical scavengers on NDMA formation. Although the molar NDMA yields from five N, N-dimethylamine-containing precursors varied between 1.4% and 90%, we observed the stoichiometric removal of one O₂ per N, N-dimethylamine group of the precursor indicating that the oxygenation of N atoms did not determine the molar NDMA yield. Small ¹⁸O-KIEs between 1.0026 ± 0.0003 and 1.0092 ± 0.0009 found for all precursors as well as completely inhibited NDMA formation in the presence of radical scavengers (ABTS and trolox) imply that O₂ reacted with radical species. Our study suggests that aminyl radicals from the oxidation of organic amines by NH₂Cl and N-peroxyl radicals from the reaction of aminyl radicals with aqueous O₂ are part of the NDMA formation mechanism.

Quantifying the extent of biodegradation of nitroaromatic compounds (NACs) in contaminated soils and sediments is challenging because of competing oxidative and reductive reaction pathways. We have previously shown that the stable isotope fractionation of NACs reveals the routes of degradation even if it is simultaneously caused by different bacteria. However, it is unclear whether compound-specific isotope analysis (CSIA) can be applied in situations where multiple pollutants are biodegraded by only one microorganism under multi-substrate conditions. Here we examined the C and N isotope fractionation of 2-nitrophenol (2-NP) and 3-nitrophenol (3-NP) during biodegradation by Pseudomonas putida B2 through monooxygenation and partial reductive pathways, respectively, in the presence of single substrates vs. binary substrate mixtures. Laboratory experiments showed that the reduction of 3-NP by Pseudomonas putida B2 is associated with large N and minor C isotope fractionation with C and N isotope enrichment factors, ε_C and ε_N, of −0.3 ± 0.1‰ and −22 ± 0.2‰, respectively. The opposite isotope fractionation trends were found for 2-NP monooxygenation. In the simultaneous presence of 2-NP and 3-NP, 2-NP is biodegraded at identical rate constants and ε_C and ε_N values (−1.0 ± 0.1‰ and −1.3 ± 0.2‰) to those found for the monooxygenation of 2-NP in single substrate experiments. While the pathway and N isotope fractionation of 3-NP reduction (ε_N = −24 ± 1.1‰) are independent of the presence of 2-NP, intermediates of 2-NP monooxygenation interfere with 3-NP reduction. Because neither pH, substrate uptake, nor aromatic substituents affected the kinetic isotope effects of nitrophenol biodegradation, our study illustrates that CSIA provides robust scientific evidence for the assessment of natural attenuation processes.

Iron is present in virtually all terrestrial and aquatic environments, where it participates in redox reactions with surrounding metals, organic compounds, contaminants, and microorganisms. The rates and extent of these redox reactions strongly depend on the speciation of the Fe²⁺ and Fe³⁺ phases, although the underlying reasons remain unclear. In particular, numerous studies have observed that Fe²⁺ associated with iron oxide surfaces (i.e., oxide-associated Fe²⁺) often reduces oxidized contaminants much faster than aqueous Fe²⁺ alone. Here, we tested two hypotheses related to this observation by determining if solutions containing two commonly studied iron oxides—hematite and goethite—and aqueous Fe²⁺ reached thermodynamic equilibrium over the course of a day. We measured reduction potential (E_H) values in solutions containing these oxides at different pH values and aqueous Fe²⁺ concentrations using mediated potentiometry. This analysis yielded standard reduction potential (E_H⁰) values of 768 ± 1 mV for the aqueous Fe²⁺–goethite redox couple and 769 ± 2 mV for the aqueous Fe²⁺–hematite redox couple. These values were in excellent agreement with those calculated from existing thermodynamic data, and the data could be explained by the presence of an iron oxide lowering E_H values of aqueous Fe³⁺/Fe²⁺ redox couples.

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 (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 MnO₂ (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 O₃ 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 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.

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 δ¹⁸O 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 δ¹⁸O 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 δ¹⁸O blank correction for the quantification of ¹⁸O/¹⁶O ratios in 20 nmol of injected O₂.
Conclusions: The successful quantification of ¹⁸O-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.

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 ¹³C- and ²H-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.

Compound-specific isotope analysis is a useful approach to track transformations of many organic soil and water pollutants. Applications of CSIA to characterize photochemical processes, however, have hardly been explored. In this work, we systematically studied C and N isotope fractionation associated with the direct photolysis of 4-Cl-aniline used as a model compound for organic micropollutants that are known to degrade via photochemical processes. Laboratory experiments were carried out at an irradiation wavelength of 254 nm over the pH range 2.0 to 9.0 as well as in the presence of Cs⁺ as a quencher of excited singlet 4-Cl-aniline at pH 7.0 and 9.0. We observed considerable variation of C and N isotope enrichment factors, ϵ_C and ϵ_N, between −1.2 ± 0.2‰ to −2.7 ± 0.2‰ for C and −0.6 ± 0.2‰ to −9.1 ± 1.6‰ for N, respectively, which could not be explained by the speciation of 4-Cl-aniline alone. In the presence of 1 M Cs⁺, we found a marked increase of apparent ¹³C-kinetic isotope effects (¹³C-AKIE) and decrease of 4-Cl-aniline fluorescence lifetimes. Our data suggest that variations of C and N isotope fractionation originate from heterolytic dechlorination of excited triplet and singlet states of 4-Cl-aniline. Linear correlations of ¹³C-AKIE vs ¹⁵N-AKIE were distinctly different for these two reaction pathways and may be explored further for the identification of photolytic aromatic dechlorination reactions.

Organic micropollutants containing aniline substructures are susceptible to different light-induced transformation processes in aquatic environments and water treatment operations. Here, we investigated the magnitude and variability of C and N isotope fractionation during the indirect phototransformation of four para-substituted anilines in aerated aqueous solutions. The model photosensitizers, namely 9,10-anthraquinone-1,5-disulfonate and methylene blue, were used as surrogates for dissolved organic matter chromophores generating excited triplet states in sunlit surface waters. The transformation of aniline, 4-CH₃-, 4-OCH₃-, and 4-Cl-aniline by excited triplet states of the photosensitizers was associated with inverse and normal N isotope fractionation, whereas C isotope fractionation was negligible. The apparent ¹⁵N kinetic isotope effects (AKIE) were almost identical for both photosensitizers, increased from 0.9958 ± 0.0013 for 4-OCH₃-aniline to 1.0035 ± 0.0006 for 4-Cl-aniline, and correlated well with the electron donating properties of the substituent. N isotope fractionation is pH-dependent in that H⁺ exchange reactions dominate below and N atom oxidation processes above the pK_a value of the substituted aniline’s conjugate acid. Correlations of C and N isotope fractionation for indirect phototransformation were different from those determined previously for the direct photolysis of chloroanilines and offer new opportunities to distinguish between abiotic degradation pathways.

Isotope fractionation associated with the photochemical transformation of organic contaminants is not well understood and can arise not only from bond cleavage reactions but also from photophysical processes. In this work, we investigated the photolytic dechlorination of 2-Cl- and 3-Cl-aniline to aminophenols to obtain insights into the impact of the substituent position on the apparent ¹³C and ¹⁵N kinetic isotope effects (AKIEs). Laboratory experiments were performed in aerated aqueous solutions at an irradiation wavelength of 254 nm over the pH range 2.0 to 7.0 in the absence and presence of Cs⁺ used as an excited singlet state quencher. Photolysis of 2-Cl-anilinium cations exhibits normal C and inverse N isotope fractionation, while neutral 2-Cl-aniline species shows inverse C and normal N isotope fractionation. In contrast, the photolysis of 3-Cl-aniline was almost insensitive to C isotope composition and the moderate N isotope fractionation points to rate-limiting photophysical processes. ¹³C- and ¹⁵N-AKIE-values of 2-Cl-aniline decreased in the presence of Cs⁺, whereas those for 3-Cl-aniline were not systematically affected by Cs⁺. Our current and previous work illustrates that photolytic dechlorinations of 2-Cl-, 3-Cl-, and 4-Cl-aniline isomers are each accompanied by distinctly different and highly variable C and N isotope fractionation due to spin selective isotope effects.

Redox-active minerals are ubiquitous in the environment and are involved in numerous electron transfer reactions that significantly affect biogeochemical processes and cycles as well as pollutant dynamics. As a consequence, research in different scientific disciplines is devoted to elucidating the redox properties and reactivities of minerals. This review focuses on the characterization of mineral redox properties using electrochemical approaches from an applied (bio)geochemical and environmental analytical chemistry perspective. Establishing redox equilibria between the minerals and working electrodes is a major challenge in electrochemical measurements, which we discuss in an overview of traditional electrochemical techniques. These issues can be overcome with mediated electrochemical analyses in which dissolved redox mediators are used to increase the rate of electron transfer and to facilitate redox equilibration between working electrodes and minerals in both amperometric and potentiometric measurements. Using experimental data on an iron-bearing clay mineral, we illustrate how mediated electrochemical analyses can be employed to derive important thermodynamic and kinetic data on electron transfer to and from structural iron. We summarize anticipated methodological advancements that will further contribute to advance an improved understanding of electron transfer to and from minerals in environmentally relevant redox processes.

Mitigation of N-nitrosodimethylamine (NDMA) and other hazardous water disinfection byproducts (DBP) is currently hampered by a limited understanding of DBP formation mechanisms. Because variations of the stable isotope composition of NDMA can potentially reveal reaction pathways and precursor compounds, we developed a method for the compound-specific isotope analysis (CSIA) of ¹³C/¹²C, ¹⁵N/¹⁴N, and ²H/¹H ratios of NDMA by gas chromatography coupled to isotope ratio mass spectrometry (GC/IRMS). Method quantification limits for the accurate isotope analysis of NDMA, N-nitrosodiethyl-, -dipropyl-, and -dibutylamine as well as N-nitrosopyrrolidine were between 0.18 to 0.60 nmol C, 0.40 to 0.80 nmol N, and 2.2 to 5.8 nmol H injected on column. Coupling solid phase extraction (SPE) to GC/IRMS enabled the precise quantification of C, N, and H isotope ratios of NDMA in aqueous samples at concentrations of 0.6 μM (45 μg L^–1). We validated the proposed method with a laboratory experiment, in which NDMA was formed with stoichiometric yield (97 ± 4%) through chloramination of the pharmaceutical ranitidine (3 μM). δ¹³C and δ²H values of NDMA remained constant during NDMA formation while its δ¹⁵N increased due to a reaction at a N atom in the rate-limiting step of NDMA formation. The δ²H value of NDMA determined by SPE-GC/IRMS also corresponded well to the δ²H value of the N(CH₃)₂-group of ranitidine measured by quantitative deuterium nuclear magnetic resonance spectroscopy. This observation implies that the N(CH₃)₂-moiety of ranitidine is transferred to NDMA without being chemically altered and illustrates the accuracy of the proposed method.

Lysimeter experiments were carried out to evaluate the applicability of compound-specific isotope analysis (CSIA) for assessing transport and fate of selected herbicides in two agricultural soils cropped with corn. Monitoring of target pesticides, metabolites and tracers in drainage water made apparent the significant role of preferential flow in the mobility of the studied compounds through the soil and the vadose zone during the first days after herbicides application. The detection of metabolites in drainage water confirmed herbicides degradation once higher residence times in the soil zone where achieved. An efficient preconcentration procedure was developed for 10-L drainage water samples to achieve enough analyte enrichment for C, N and Cl isotope ratios measurements. Methods for isotope analyses are currently being developed and the applicability of CSIA for assessing the in situ biodegradation of the target pesticides during transport in soil and vadose zone will be evaluated.

The assessment of oxidative pollutant biotransformation by compound specific isotope analysis (CSIA) is often complicated by the variability of kinetic isotope effects associated with carbon oxygenation in enzymatic reactions. Here, we illustrate how information about the kinetics of oxidative biocatalysis by flavin-dependent monooxygenases (FMOs) enables one to assess if CSIA could be applied for tracking contaminant biodegradation. In "cautious" FMOs, which form reactive flavin (hydro)peroxide species after substrate binding, the monooxygenation of organic compounds is not rate-determining and consequently does not lead to substrate isotope fractionation. Conversely, "bold" FMOs generate hydroperoxides regardless of substrate availability, and substrate disappearance is thus subject to isotope fractionation trends, which are typical for hydroxylation reactions. Because monooxygenations of aromatic moieties are often initial steps of organic pollutant transformation, knowledge of the kinetics of FMOs and other oxidative enzymes can support decisions regarding the use of CSIA.

Calculations of alternative oxidation pathways of toluene and its ortho-substituted nitro derivatives by permanganate anion have been performed. The competition between methyl group and ring oxidation has been addressed. Acceptable results have been obtained using IEFPCM/B3LYP/6-31+G(d,p) calculations with zero-point (ZPC) and thermal corrections, as validated by comparison with the experimental data. It has been shown that ring oxidation reactions proceed via relatively early transition states that become quite unsymmetrical for reactions involving ortho-nitrosubstituted derivatives. Transition states for the hydrogen atom abstraction reactions, on the other hand, are late. All favored reactions are characterized by the Gibbs free energy of activation, ΔG^≠, of about 25 kcal mol⁻¹. Methyl group oxidations are exothermic by about 20 kcal mol⁻¹ while ring oxidations are around thermoneutrality.

Um das Schicksal von Pflanzenschutz- und Schädlingsbekämpfungsmitteln in der Umwelt zuverlässig abschätzen zu können, reichen Kenntnisse über diese Stoffe alleine nicht. Vielmehr müssen die zugrunde liegenden biogeochemischen Prozesse verstanden werden. Mit hochempfindlichen Messverfahren und ausgeklügelten Analysewerkzeugen lassen sich die Pestizide und ihr Abbauverhalten immer besser verfolgen.

If the fate of plant protection and pest control agents in the environment is to be reliably assessed, knowledge of the substances concerned is not in itself sufficient – it is also essential to understand the underlying biogeochemical processes. Using highly sensitive measurement techniques and sophisticated analytical tools, it is now becoming possible to track pesticide degradation more effectively.

Pour évaluer correctement le devenir des produits biocides et phytosanitaires dans l’environnement, il ne suffit pas de disposer de connaissances spécifiques à leur sujet. Il faut également comprendre les processus biogéochimiques dans lesquels ils sont engagés. Grâce à des approches ingénieuses et des méthodes d’analyse ultrasensibles, il est aujourd’hui possible de mieux suivre les pesticides et leurs transformations dans le milieu naturel

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. ¹³C/¹²C, ²H/¹H, ¹⁵N/¹⁴N) 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.

Benzotriazoles are widely used domestic and industrial corrosion inhibitors and have become omnipresent organic micropollutants in the aquatic environment. Here, the range of aerobic biological degradation mechanisms of benzotriazoles in activated sludge was investigated. Degradation pathways were elucidated by identifying transient and persistent transformation products in batch experiments using liquid chromatography-high-resolution tandem mass spectrometry (LC-HR-MS/MS). In addition, initial reactions were studied using compound-specific isotope analysis (CSIA). Biodegradation half-lives of 1.0 days for 1H-benzotriazole, 8.5 days for 4-methyl-1H-benzotriazole, and 0.9 days for 5-methyl-1H-benzotriazole with activated sludge confirmed their known partial persistence in conventional wastewater treatment. Major transformation products were identified as 4- and 5-hydroxy-1H-benzotriazole for the degradation of 1H-benzotriazole, and 1H-benzotriazole-5-carboxylic acid for the degradation of 5-methyl-1H-benzotriazole. These transformation products were found in wastewater effluents, showing their environmental relevance. Many other candidate transformation products, tentatively identified by interpretation of HR-MS/MS spectra, showed the broad range of possible reaction pathways including oxidation, alkylation, hydroxylation and indicate the significance of cometabolic processes for micropollutant degradation in biological wastewater treatment in general. The combination of evidence from product analysis with the significant carbon and nitrogen isotope fractionation suggests that aromatic monohydroxylation is the predominant step during the biotransformation of 1H-benzotriazole.

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, ϵ_C, of −4.1 ± 0.2‰ and −2.5 ± 0.2‰, respectively, and was observed consistently in the substrates and dioxygenation products. ϵ_H- and ϵ_N-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 ¹³C-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.

Amino acids (AAs) comprise a large fraction of organic nitrogen (N) in plankton and sedimenting matter. Aquatic studies of organic N compounds in general and of AAs in particular, mostly concentrate on marine environments. In order to study the cycling and fate of organic N and AAs in lakes, we measured the N isotopic composition (δ¹⁵N) of bulk organic matter (OM) and of single hydrolysable AAs in sediment trap and sediment samples from two Swiss lakes with contrasting trophic state: Lake Brienz, an oligotrophic lake with an oxic water column, and Lake Zug a eutrophic, meromictic lake. We also measured the N isotopic composition of water column nitrate, the likely inorganic N source during biosynthesis in both lakes. The δ¹⁵N–AA patterns found for the sediment trap material were consistent with published δ¹⁵N–AA data for marine plankton. The AA composition and primary δ¹⁵N–AA signatures are preserved until burial in the sediments. During early sedimentary diagenesis, the δ¹⁵N values of single AAs appear to increase, exceeding those of the bulk OM. This increase in δ¹⁵N–AA is paralleled by a decreased contribution of AAs to the total OM pool with progressed degradation, suggesting preferential AA degradation associated with a significant N isotope fractionation. Indicators for trophic level based on δ¹⁵N–AAs were determined, for the first time in lacustrine systems. In our samples, the trophic AAs were generally enriched in ¹⁵N compared to source AAs and higher trophic δ¹⁵N–AA values in Lake Zug were consistent with a higher trophic level of the bulk biomass compared to Lake Brienz. Especially the difference between average trophic δ¹⁵N–AAs and average source δ¹⁵N–AAs was sensitive to the trophic states of the two lakes. A proxy for total heterotrophic AA re-synthesis (ΣV), which is strongly associated with heterotrophic microbial reworking of the OM, was calculated based on δ¹⁵N values of trophic AAs. Higher ΣV in Lake Brienz indicate enhanced heterotrophic bacterial reworking of AAs under oligotrophic conditions. Despite changes in the δ¹⁵N–AA values within the sediments, the proxies based on these values were consistent over the studied sediment profile, indicating the preservation of trophic signatures; therefore, our results underscore that δ¹⁵N–AA analysis of sedimentary records represents a promising tool to assess trophic levels and bacterial re-synthesis in lakes.

Structural Fe in clay minerals is an important redox-active species in many pristine and contaminated environments as well as in engineered systems. Understanding the extent and kinetics of redox reactions involving Fe-bearing clay minerals has been challenging due to the inability to relate structural Fe²⁺/Fe_total fractions to fundamental redox properties, such as reduction potentials (E_H). Here, we overcame this challenge by using mediated electrochemical reduction (MER) and oxidation (MEO) to characterize the fraction of redox-active structural Fe (Fe²⁺/Fe_total) in smectites over a wide range of applied E_H-values (−0.6 V to +0.6 V). We examined Fe²⁺/Fe_total – E_H relationships of four natural Fe-bearing smectites (SWy-2, SWa-1, NAu-1, NAu-2) in their native, reduced, and reoxidized states and compared our measurements with spectroscopic observations and a suite of mineralogical properties. All smectites exhibited unique Fe²⁺/Fe_total – E_H relationships, were redox active over wide E_H ranges, and underwent irreversible electron transfer induced structural changes that were observable with X-ray absorption spectroscopy. Variations among the smectite Fe²⁺/Fe_total – E_H relationships correlated well with both bulk and molecular-scale properties, including Fe_total content, layer charge, and quadrupole splitting values, suggesting that multiple structural parameters determined the redox properties of smectites. The Fe²⁺/Fe_total – E_H relationships developed for these four commonly studied clay minerals may be applied to future studies interested in relating the extent of structural Fe reduction or oxidation to E_H-values.

Compound-specific isotope analysis (CSIA) is an important tool for the identification of contaminant sources and transformation pathways, but it is rarely applied to emerging aquatic micropollutants owing to a series of instrumental challenges. Using four different benzotriazole corrosion inhibitors and its derivatives as examples, we obtained evidence that formation of organometallic complexes of benzotriazoles with parts of the instrumentation impedes isotope analysis. Therefore, we propose two strategies for accurate δ¹³C and δ¹⁵N measurements of polar organic micropollutants by gas chromatography coupled to isotope ratio mass spectrometry (GC/IRMS). Our first approach avoids metallic components and uses a Ni/Pt reactor for benzotriazole combustion while the second is based on the coupling of online methylation to the established GC/IRMS setup. Method detection limits for on-column injection of benzotriazole, as well as its 1-CH_3^–, 4-CH_3^–, and 5-CH_3^– substituted species were 0.1–0.3 and 0.1–1.0 mM for δ¹³C- and δ¹⁵N analysis, respectively, corresponding to injected masses of 0.7–1.8 nmol C and 0.4–3.0 nmol N, respectively. The Ni/Pt reactor showed good precision and was very long-lived (>1,000 successful measurements). Coupling isotopic analysis to offline solid-phase extraction enabled benzotriazole-CSIA in tap water, wastewater treatment effluent, activated sludge, and in commercial dishwashing products. A comparison of δ¹³C and δ¹⁵N values from different benzotriazoles and benzotriazole derivatives, both from commercial standards and in dishwashing detergents, reveals the potential application of the proposed method for source apportionment.

Monooxygenation is an important route of nitroaromatic compound (NAC) biodegradation and it is widely found for cometabolic transformations of NACs and other aromatic pollutants. We investigated the C and N isotope fractionation of nitrophenol monooxygenation to complement the characterization of NAC (bio)degradation pathways by compound-specific isotope analysis (CSIA). Because of the large diversity of enzymes catalyzing monooxygenations, we studied the combined C and N isotope fractionation and the corresponding ¹³C- and ¹⁵N-apparent kinetic isotope effects (AKIEs) of four nitrophenol-biodegrading microorganisms (Bacillus spharericus JS905, Pseudomonas sp. 1A, Arthrobacter sp. JS443, Pseudomonas putida B2) in the pH range 6.1–8.6 with resting cells and crude cell extracts. While the extent of C and N isotope fractionation and the AKIE-values varied considerably for the different organisms, the correlated C and N isotope signatures (δ¹⁵N vs δ¹³C) revealed trends, indicative of two distinct monooxygenation pathways involving hydroxy-1,4-benzoquinone or 1,2- and 1,4-benzoquinone intermediates, respectively. The distinction was possible based on larger secondary ¹⁵N-AKIEs associated with the benzoquinone pathway. Isotope fractionation was neither masked substantially by nitrophenol speciation nor transport across cell membranes. Only when 4-nitrophenol was biodegraded by Pseudomonas sp. 1A did isotope fractionation become negligible, presumably due to rate-limiting substrate binding steps pertinent to the catalytic cycle of flavin-dependent monooxygenases.

Oxidation of aromatic rings and its alkyl substituents are often competing initial steps of organic pollutant transformation. The use of compound-specific isotope analysis (CSIA) to distinguish between these two pathways quantitatively, however, can be hampered by large H isotope fractionation that precludes calculation of apparent ²H-kinetic isotope effects (KIE) as well as the process identification in multi-element isotope fractionation analysis. Here, we investigated the C and H isotope fractionation associated with the transformation of toluene, nitrobenzene, and four substituted nitrotoluenes by permanganate, MnO₄^–, to propose a refined evaluation procedure for the quantitative distinction of CH₃-group oxidation and dioxygenation. On the basis of batch experiments, an isotopomer-specific kinetic model, and density functional theory (DFT) calculations, we successfully derived the large apparent ²H-KIE of 4.033 ± 0.20 for the CH₃-group oxidation of toluene from H isotope fractionation exceeding >1300‰ as well as the corresponding ¹³C-KIE (1.0324 ± 0.0011). Experiment and theory also agreed well for the dioxygenation of nitrobenzene, which was associated with ²H- and ¹³C-KIEs of 0.9410 ± 0.0030 (0.9228 obtained by DFT) and 1.0289 ± 0.0003 (1.025). Consistent branching ratios for the competing CH₃-group oxidation and dioxygenation of nitrotoluenes by MnO₄^– were obtained from the combined modeling of concentration as well as C and H isotope signature trends. Our approach offers improved estimates for the identification of contaminant microbial and abiotic oxidation pathways by CSIA.

Assessing the fate of nitroaromatic explosives in the subsurface is challenging because contaminants are present in different phases (e.g., bound to soil or sediment matrix or as solid-phase residues) and transformation takes place via several potentially competing pathways over time-scales of decades. We developed a procedure for compound-specific analysis of stable C, N, and H isotopes in nitroaromatic compounds (NACs) and characterized biodegradation of 2,4,6-trinitrotoluene (TNT) and two dinitrotoluene isomers (2,4-DNT and 2,6-DNT) in subsurface material of a contaminated site. The type and relative contribution of reductive and oxidative pathways to the degradation of the three contaminants was inferred from the combined evaluation of C, N, and H isotope fractionation. Indicative trends of Δδ¹⁵N vs Δδ¹³C and Δδ²H vs Δδ¹³C were obtained from laboratory model systems for biodegradation pathways initiated via (i) dioxygenation, (ii) reduction, and (iii) CH₃-group oxidation. The combined evaluation of NAC isotope fractionation in subsurface materials and in laboratory experiments suggests that in the field, 86–89% of 2,4-DNT transformation was due to dioxygenation while TNT was mostly reduced and 2,6-DNT reacted via a combination of reduction and CH₃-group oxidation. Based on historic information on site operation, our data imply biodegradation of 2,4-DNT with half-lives of up to 9–17 years compared to 18–34 years for cometabolic transformation of TNT and 2,6-DNT.

Compound-specific stable-isotope analysis (CSIA) has greatly facilitated assessment of sources and transformation processes of organic pollutants. Multielement isotope analysis is one of the most promising applications of CSIA because it even enables distinction of different transformation pathways. This review introduces the essential features of continuous-flow isotope-ratio mass spectrometry (IRMS) and highlights current challenges in environmental analysis as exemplified for the isotopes of nitrogen, hydrogen, chlorine, and oxygen. Strategies and recent advances to enable isotopic measurements of polar contaminants, for example pesticides or pharmaceuticals, are discussed with special emphasis on possible solutions for analysis of low concentrations of contaminants in environmental matrices. Finally, we discuss different levels of calibration and referencing and point out the urgent need for compound-specific isotope standards for gas chromatography–isotope-ratio mass spectrometry (GC–IRMS) of organic pollutants.

Clay minerals often contain redox-active structural iron that participates in electron transfer reactions with environmental pollutants, bacteria, and biological nutrients. Measuring the redox properties of structural Fe in clay minerals using electrochemical approaches, however, has proven to be difficult due to a lack of reactivity between clay minerals and electrodes. Here, we overcome this limitation by using one-electron-transfer mediating compounds to facilitate electron transfer between structural Fe in clay minerals and a vitreous carbon working electrode in an electrochemical cell. Using this approach, the electron-accepting and -donating capacities (Q_EAC and Q_EDC) were quantified at applied potentials (E_H) of −0.60 V and +0.61 V (vs SHE), respectively, for four natural Fe-bearing smectites (i.e., SWa-1, SWy-2, NAu-1, and NAu-2) having different total Fe contents (Fe_total = 2.3 to 21.2 wt % Fe) and varied initial Fe²⁺/Fe_total states. For every SWa-1 and SWy-2 sample, all the structural Fe was redox-active over the tested E_H range, demonstrating reliable quantification of Fe content and redox state. Yet for NAu-1 and NAu-2, a significant fraction of the structural Fe was redox-inactive, which was attributed to Fe-rich smectites requiring more extreme E_H-values to achieve complete Fe reduction and/or oxidation. The Q_EAC and Q_EDC values provided here can be used as benchmarks in future studies examining the extent of reduction and oxidation of Fe-bearing smectites.

Structural Fe in clay minerals is an important, albeit poorly characterized, redox-active phase found in many natural and engineered environments. This work develops an experimental approach to directly assess the redox properties of a natural Fe-bearing smectite (ferruginous smectite, SWa-1, 12.6 wt % Fe) with mediated electrochemical reduction (MER) and oxidation (MEO). By utilizing a suite of one-electron-transfer mediating compounds to facilitate electron transfer between structural Fe in SWa-1 and a working electrode, we show that the Fe²⁺/Fe³⁺ couple in SWa-1 is redox-active over a large range of potentials (from E_H = −0.63 V to +0.61 V vs SHE). Electrochemical and spectroscopic analyses of SWa-1 samples that were subject to reduction and re-oxidation cycling revealed both reversible and irreversible structural Fe rearrangements that altered the observed apparent standard reduction potential (E_H^Φ) of structural Fe. E_H^Φ-values vary by as much as 0.56 V between SWa-1 samples with different redox histories. The wide range of E_H-values over which SWa-1 is redox-active and redox history-dependent E_H^Φ-values underscore the importance of Fe-bearing clay minerals as redox-active phases in a wide range of redox regimes.

The fate of organic micropollutants in soils and natural waters is difficult to track using traditional methods. But with compound-specific isotope analysis, the isotopic composition of contaminants can be studied. This makes it possible, for example, to trace the origin of dishwasher detergents or to determine the degradation pathways of explosives.

Das Schicksal organischer Mikroverunreinigungen in Böden und Gewässern ist mit herkömmlichen Methoden schwierig zu verfolgen. Die Einzelstoff-Isotopenanalyse ermöglicht es, die Isotopenzusammensetzung von Schadstoffen zu untersuchen. Damit lassen sich zum Beispiel die Herkunft von Geschirrspülmitteln nachweisen oder die Abbauwege von Sprengstoff bestimmen.

Polyhalogenated 1′-methyl-1,2′-bipyrroles are natural products that biomagnify into upper trophic levels of marine food webs. Here we demonstrate that they are unusually enriched in ¹⁵N (δ¹⁵N from +19.3‰ to +28.1‰) relative to other biosynthetic organic compounds measured to date and the mammals from which the compounds were isolated. We argue the ¹⁵N enrichment likely stems from enriched precursors and/or fractionation during biosynthesis and is not from MBP degradation. We also consider possible sources of MBPs in light of these results.

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 (MnO₂) 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 Δδ¹⁵N/Δδ¹³C-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 MnO₂ and horseradish peroxidase (HRP), derived apparent ¹³C-, ²H-, and ¹⁵N-kinetic isotope effects (AKIEs), and characterized reaction products. The N-atom oxidation pathway leading to radical coupling products typically exhibited inverse ¹⁵N-AKIEs (up to 0.991) and only minor ¹³C- and ²H-AKIEs. Oxidative N-dealkylation, in contrast, was subject to large normal ¹³C- and ²H-AKIEs (up to 1.019 and 3.1, respectively) and small ¹⁵N-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.

The industrial and domestic use of chemicals has led to large emissions of organic micropollutants into the environment. Knowledge of contaminants’ degradability in environmental systems is essential for proper risk assessment and further development of adequate remediation strategies for contaminated sites. Transformation processes of contaminants can be identified and quantified by means of compound-specific isotope analysis (CSIA). Depending on the chemical bond(s) that are cleaved or formed during a reaction, the isotopic ratio of the reactant changes systematically as a consequence of kinetic isotope effects (KIE). This change can be indicative for the occurring reaction and the derived apparent KIEs provide important information on the reaction mechanism. Although many emerging micropollutants exhibit reactive N-containing functional groups, only few studies have used CSIA to assess their transformation. This PhD thesis focuses on the transformation of aromatic N-alkyl amines, which are widely used in the chemical industry and can also represent the reactive moiety in larger contaminant molecules. Once present in the environment they are subject to oxidative transformation via multiple reaction pathways, which may occur simultaneously. [...]

Der verbreitete Einsatz von Chemikalien in der Industrie, der Landwirtschaft sowie in Haushalten hat zur Belastung der Umwelt mit organischen Mikroverunreinigungen geführt. Die Risikoabschätzung der Umweltverschmutzung sowie die Entwicklung von entsprechenden Sanierungsstrategien setzen fundierte Kenntnisse über das Verhalten der Schadstoffe in der Umwelt voraus. Die Auswirkungen der organischen Schadstoffe auf das Ökosystem und die Menschen wird weitgehend durch deren Abbau bestimmt. Ein qualitativer und quantitativer Nachweis von solchen Abbauprozessen wird durch die Einzelstoff-Isotopenanalyse (Compound-Specific Isotope Analysis, CSIA) ermöglicht. Die CSIA basiert darauf, dass die Bildung und/oder Spaltung einer chemischen Bindung während einer Reaktion dem kinetischen Isotopeneffekt (KIE) unterliegt. Der KIE führt zu einer systematischen Änderung der stabilen Isotopenzusammensetzung des Reaktanden, die charakteristisch für diese Reaktion ist. Obwohl zahlreiche organische Mikroverunreinigungen reaktive stickstoffhaltige funktionelle Gruppen enthalten, existieren bisher nur sehr wenige Studien, die die relevanten Transformationsprozesse mittels CSIA untersucht haben. Diese Arbeit befasst sich mit der Schadstoffklasse der aromatischen N-alkylierten Amine, weil diese in der chemischen Industrie sehr verbreitet eingesetzt werden und weil sie zudem als reaktive Strukturen in grösseren, strukturell komplexeren Schadstoffmolekülen, wie z.B. Antibiotika, vorkommen. Aromatische Amine werden in der Umwelt mittels verschiedener Oxidationsreaktionen zu Transformationprodukten umgesetzt, wobei viele dieser Prozesse auch gleichzeitig stattfinden können. [...]

Degradation of chlorinated organic contaminants by natural and engineered reductive dechlorination reactions can occur via numerous biotic and abiotic transformation pathways giving rise to either benign or more toxic products. To assess whether dechlorination processes may lead to significant detoxification (a) the thermodynamic feasibility of a reaction, (b) rates of transformation, and (c) product formation routes need to be understood. To this end, fundamental knowledge of chlorohydrocarbon (CHC) reaction mechanisms is essential. We review insight from reaction thermodynamics, structure-reactivity relationships, and applications of radical and carbene traps, as well as of synthetic probe molecules. We summarize the state-of-knowledge about intermediates and reductive dechlorination pathways of vicinal and geminal haloalkanes, as well as of chlorinated ethenes. Transformation conditions are identified under which problematic products may be avoided. In an outlook, we discuss the potential of stable carbon and chlorine isotope fractionation to identify initial transformation mechanisms, competing transformation pathways, and common branching points.

Iron-bearing clay minerals are ubiquitous in the environment and have been shown to play important roles in several biogeochemical processes. Previous efforts to characterize the Fe²⁺–Fe³⁺ redox couple in clay minerals using electrochemical techniques have been limited by experimental difficulties due to inadequate reactivity between clay minerals and electrodes. The current work overcomes this limitation by utilizing organic electron transfer mediators that rapidly transfer electrons with both the Fe-bearing clay minerals and electrodes. Here, an Fe-rich source clay mineral (ferruginous smectite, SWa-1) is examined with respect to what fraction of structural Fe participates in oxidation/reduction reactions and the relationship between bulk Fe²⁺/Fe³⁺ ratios to the reduction potential (E_h).

The analysis of variations in stable isotope composition is becoming an essential approach for evaluating enzymatic and abiotic reactions of organic contaminants in soils and aquatic systems. Different, sometimes complementary analytical techniques are currently used and developed to determine stable isotope ratios in individual organic compounds. Anticipating an increasing demand for compound-specific isotope analysis, this survey compiles information for choosing the most promising analytical approach to an isotope-related problem. To this end, we review the principles of instrumentation for compound-specific isotope analysis and show how they can be exploited to assess contaminant transformation processes. Using chlorinated solvents and triazine herbicides as illustrative examples, we discuss how the isotope-sensitive techniques impact the investigation of stable isotope fractionation in environmental chemistry and microbiology.

The quantitative description of enzymatic or abiotic transformations of man-made organic micropollutants in rivers, lakes, and groundwaters is one of the major challenges associated with the risk assessment of water resource contamination. Compound-specific isotope analysis enables one to identify (bio)degradation pathways based on changes in the contaminants’ stable isotope ratios even if multiple reactive and non-reactive processes cause concentrations to decrease. Here, we investigated how the magnitude and variability of isotope fractionation in some priority pollutants is determined by the kinetics and mechanisms of important enzymatic and abiotic redox reactions. For nitroaromatic compounds and substituted anilines, we illustrate that competing transformation pathways can be assessed via trends of N and C isotope signatures.

Redox processes of structural Fe in clay minerals play an important role in biogeochemical cycles and for the dynamics of contaminant transformation in soils and aquifers. Reactions of Fe(II)/Fe(III) in clay minerals depend on a variety of mineralogical and environmental factors, which make the assessment of Fe redox reactivity challenging. Here, we use middle and near infrared (IR) spectroscopy to identify reactive structural Fe(II) arrangements in four smectites that differ in total Fe content, octahedral cationic composition, location of the negative excess charge, and configuration of octahedral hydroxyl groups. Additionally, we investigated the mineral properties responsible for the reversibility of structural alterations during Fe reduction and re-oxidation. For Wyoming montmorillonite (SWy-2), a smectite of low structural Fe content (2.8 wt%), we identified octahedral AlFe(II)–OH as the only reactive Fe(II) species, while high structural Fe content (>12 wt%) was prerequisite for the formation of multiple Fe(II)-entities (dioctahedral AlFe(II)–OH, MgFe(II)–OH, Fe(II)Fe(II)–OH, and trioctahedral Fe(II)Fe(II)Fe(II)–OH) in iron-rich smectites Ölberg montmorillonite, and ferruginous smectite (SWa-1), as well as in synthetic nontronite. Depending on the overall cationic composition and the location of excess charge, different reactive Fe(II) species formed during Fe reduction in iron-rich smectites, including tetrahedral Fe(II) groups in synthetic nontronite. Trioctahedral Fe(II) domains were found in tetrahedrally charged ferruginous smectite and synthetic nontronite in their reduced state while these Fe(II) entities were absent in Ölberg montmorillonite, which exhibits an octahedral layer charge. Fe(III) reduction in iron-rich smectites was accompanied by intense dehydroxylation and structural rearrangements, which were only partially reversible through re-oxidation. Re-oxidation of Wyoming montmorillonite, in contrast, restored the original mineral structure. Fe(II) oxidation experiments with nitroaromatic compounds as reactive probes were used to link our spectroscopic evidence to the apparent reactivity of structural Fe(II) in a generalized kinetic model, which takes into account the presence of Fe(II) entities of distinctly different reactivity as well as the dynamics of Fe(II) rearrangements.

Redox reactions of structural Fe in clay minerals play important roles in biogeochemical processes and for the fate of contaminants in the environment. Many of the redox properties of Fe in clay minerals are, however, poorly understood, thus limiting the knowledge of the factors that make structural Fe participate in electron transfer reactions. This chapter summarizes the current state of knowledge on the redox properties of structural Fe in clay minerals. In the first part, we review the various spectroscopic observations associated with structural Fe reduction and oxidation and how changes in Fe oxidation state affect the clay mineral structure and the binding environment of Fe in the octahedral sheet of planar 2:1 clay minerals. In the second part, we show how information on the structural alterations and arrangement of Fe can be interpreted to assess the apparent reactivity and the thermodynamic redox properties of structural Fe in clay minerals.

Solid-phase microextraction (SPME) coupled to gas chromatography/isotope ratio mass spectrometry (GC/IRMS) was used to elucidate the effects of N-atom protonation on the analysis of N and C isotope signatures of selected aromatic amines. Precise and accurate isotope ratios were measured using polydimethylsiloxane/divinylbenzene (PDMS/DVB) as the SPME fiber material at solution pH-values that exceeded the pK_a of the substituted aniline’s conjugate acid by two pH-units. Deviations of δ¹⁵N and δ¹³C-values from reference measurements by elemental analyzer IRMS were small (<0.9‰) and within the typical uncertainties of isotope ratio measurements by SPME-GC/IRMS. Under these conditions, the detection limits for accurate isotope ratio measurements were between 0.64 and 2.1 mg L⁻¹ for δ¹⁵N and between 0.13 and 0.54 mg L⁻¹ for δ¹³C, respectively. Substantial inverse N isotope fractionation was observed by SPME-GC/IRMS as the fraction of protonated species increased with decreasing pH leading to deviations of −20‰ while the corresponding δ¹³C-values were largely invariant. From isotope ratio analysis at different solution pHs and theoretical calculations by density functional theory, we derived equilibrium isotope effects, EIEs, pertinent to aromatic amine protonation of 0.980 and 1.001 for N and C, respectively, which were very similar for all compounds investigated. Our work shows that N-atom protonation can compromise accurate compound-specific N isotope analysis of aromatic amines.

We explored the N isotope fractionation associated with the oxidation of substituted primary aromatic amines, which are often the position of initial attack in transformation processes of environmental contaminants. Apparent ¹⁵N-kinetic isotope effects, AKIE_N, were determined for the oxidation of various substituted anilines in suspensions of manganese oxide (MnO₂) and compared to reference experiments in homogeneous solutions and at electrode surfaces, as well as to density functional theory calculations of intrinsic KIE_Nfor electron and hydrogen atom transfer reactions. Owing to the partial aromatic imine formation after one-electron oxidation and corresponding increase in C–N bond strength, AKIE_N-values were inverse, substituent-dependent, and confined to the range between 0.992 and 0.999 in agreement with theory. However, AKIE_N-values became normal once the fraction of cationic species prevailed owing to ¹⁵N-equilibrium isotope effects, EIE_N, of 1.02 associated with N atom deprotonation. The observable AKIE_N-values are substantially modulated by the acid/base pre-equilibria of the substituted anilines and isotope fractionation may even vanish under conditions where normal EIE_N and inverse AKIE_N cancel each other out. The pH-dependent trends of the AKIE_N-values provide a new line of evidence for the identification of contaminant degradation processes via oxidation of primary aromatic amino groups.

We determined in situ reductive transformation rates of tetrachloroethene (PCE) in a contaminated aquifer by combining compound-specific carbon stable isotope analysis (CSIA) of the contaminants with tracer-based (³H−³He) groundwater dating. With increasing distance from the source, PCE was gradually transformed to trichloroethene (TCE), cis-dichloroethene (cDCE), and vinyl chloride (VC). Using the in situ determined carbon isotopic enrichment factor of −3.3 ± 1.2‰ allowed for quantification of the PCE-to-TCE transformation based on isotopic (δ¹³C) shifts. By combining these estimates of the extent of PCE transformation with measured groundwater residence times (between 16 and 36 years) we calculated half-lives of 2.8 ± 0.8 years (k = 0.27 ± 0.09 yr⁻¹) for the PCE-to-TCE transformation. Carbon isotope mass balances including the chloroethenes PCE, TCE, cDCE, and VC (δ¹³C_Σ(CEs)) enabled an assessment of complete PCE dechlorination to nonchlorinated products. Shifts of δ¹³C_Σ(CEs) at the fringe of the plume of more than 25‰ pointed to dechlorination beyond VC of up to 55 ± 17% of the chloroethene mass. Calculated rates for this multistep dechlorination were highly variable throughout the aquifer (k = 0.4 ± 0.4 yr⁻¹), suggesting that PCE reduction to nonchlorinated products occurred only in locally restricted zones of the investigated site.

Water quality issues are a major challenge that humanity is facing in the twenty-first century. Here, we review the main groups of aquatic contaminants, their effects on human health, and approaches to mitigate pollution of freshwater resources. Emphasis is placed on chemical pollution, particularly on inorganic and organic micropollutants including toxic metals and metalloids as well as a large variety of synthetic organic chemicals. Some aspects of waterborne diseases and the urgent need for improved sanitation in developing countries are also discussed. The review addresses current scientific advances to cope with the great diversity of pollutants. It is organized along the different temporal and spatial scales of global water pollution. Persistent organic pollutants (POPs) have affected water systems on a global scale for more than five decades; during that time geogenic pollutants, mining operations, and hazardous waste sites have been the most relevant sources of long-term regional and local water pollution. Agricultural chemicals and waste-water sources exert shorter-term effects on regional to local scales.

Mass transfer of organic contaminants from nonaqueous phase liquids to the aqueous phase can significantly modulate the observable carbon isotope fractionation behavior associated with contaminant transformation. We evaluated the effects of kinetic interphase mass transfer between tetradecane and water on the observable ¹³C enrichment factor, ϵ_obs, pertinent to the reductive dechlorination of trichloroethene (TCE) by Sulfurospirillum sp. in laboratory batch model systems containing organic, aqueous and gaseous phases. We propose a conceptual model, which includes the kinetics of tetradecane−water and gas−water mass transfer, microbial growth, and isotope-sensitive parameters describing dehalorespiration, for quantifying variable ¹³C enrichment factors. While the C isotope fractionation of TCE reduction to cis-dichloroethene (cDCE) in the absence of phase-transfer effects can be characterized by a constant ϵ-value of −18.8 ± 0.6‰, mass-transfer limitations impede describing this process with a constant enrichment factor typically used in Rayleigh equations. Owing to the masking of kinetic isotope effects by the transfer of TCE from tetradecane to the aqueous phase, ϵ_obs-values gradually changed from −18.4‰ to −5.9‰. Such variations may complicate the interpretation of compound-specific isotope analysis in the assessment of chloroethene biodegradation in field applications.

The application of stable Fe isotopes as a tracer of the biogeochemical Fe cycle necessitates a mechanistic knowledge of natural fractionation processes. We studied the equilibrium Fe isotope fractionation upon sorption of Fe(II) to aluminum oxide (γ-Al₂O₃), goethite (α-FeOOH), quartz (α-SiO₂), and goethite-loaded quartz in batch experiments, and performed continuous-flow column experiments to study the extent of equilibrium and kinetic Fe isotope fractionation during reactive transport of Fe(II) through pure and goethite-loaded quartz sand. In addition, batch and column experiments were used to quantify the coupled electron transfer-atom exchange between dissolved Fe(II) (Fe(II)_aq) and structural Fe(III) of goethite. All experiments were conducted under strictly anoxic conditions at pH 7.2 in 20 mM MOPS (3-(N-morpholino)-propanesulfonic acid) buffer and 23 °C. Iron isotope ratios were measured by high-resolution MC-ICP-MS. Isotope data were analyzed with isotope fractionation models. In batch systems, we observed significant Fe isotope fractionation upon equilibrium sorption of Fe(II) to all sorbents tested, except for aluminum oxide. The equilibrium enrichment factor, , of the Fe(II)_sorb–Fe(II)_aq couple was 0.85 ± 0.10‰ (±2σ) for quartz and 0.85 ± 0.08‰ (±2σ) for goethite-loaded quartz. In the goethite system, the sorption-induced isotope fractionation was superimposed by atom exchange, leading to a δ^56/54Fe shift in solution towards the isotopic composition of the goethite. Without consideration of atom exchange, the equilibrium enrichment factor was 2.01 ± 0.08‰ (±2σ), but decreased to 0.73 ± 0.24‰ (±2σ) when atom exchange was taken into account. The amount of structural Fe in goethite that equilibrated isotopically with Fe(II)_aq via atom exchange was equivalent to one atomic Fe layer of the mineral surface (3% of goethite-Fe). Column experiments showed significant Fe isotope fractionation with δ^56/54Fe(II)_aq spanning a range of 1.00‰ and 1.65‰ for pure and goethite-loaded quartz, respectively. Reactive transport of Fe(II) under non-steady state conditions led to complex, non-monotonous Fe isotope trends that could be explained by a combination of kinetic and equilibrium isotope enrichment factors. Our results demonstrate that in abiotic anoxic systems with near-neutral pH, sorption of Fe(II) to mineral surfaces, even to supposedly non-reactive minerals such as quartz, induces significant Fe isotope fractionation. Therefore we expect Fe isotope signatures in natural systems with changing concentration gradients of Fe(II)_aq to be affected by sorption.

Chlorinated Ethene (CEs) such as tetrachloroethene (PCE) and trichloroethene (TCE) have been used for more than 60 years for dry cleaning and metal degreasing. Caused by accidents or leakage they percolated into the subsurface at numerous field sites. Due to their persistence and toxicity, CEs are increasingly threatening the groundwater – and thus drinking water – resources of industrial countries. Since classical remediation strategies are not always feasible at PCE or TCE spill sites, the naturally occurring migrobial degradation of CEs, proceeding via the toxic vinyl chloride (VC) to the unproblematic ethene, represents an alternative to such engineered strategies. For the assessment of this naturally occurring attenuation, both the efficiency of this process (extent of transformation) as also its time scale (determination of reaction rates) are of major interest. New methods to identify and quantify natural attenuation of organic contaminants, based on compound-specific shifts in stable carbon isotopes (compound-specific stable isotope analysis, CSIA) are therefore increasingly important for the assessment of contaminated aquifers. [...]

Chlorierte Ethene (CEs) wie Tetrachlorethen (PCE) und Trichlorethen (TCE) werden seit mehr als 60 Jahren in Industrie und Gewerbe vielseitig verwendet (chemische Reinigung, Metall-Entfettung) und gelangten an vielen Orten durch Unfälle oder Leckagen in den Untergrund. Aufgrund ihrer Persistenz und Toxizität bedrohen diese Stoffe in zunehmendem Masse die Grundwasser- und somit auch Trinkwasser-Ressourcen industrialisierter Länder. Da gängige Sanierungsmassnahmen für PCE- und TCE-Altlastenstandorten oft unpraktikabel sind, stellt natürlicher mikrobieller Abbau von CEs, welcher über die Zwischenstufen des toxischen Vinylchlorids (VC) zum unproblematischen Ethen abläuft, eine Alternative zu technischen Eingriffen dar. Dabei gewinnen neue Methoden zum Nachweis von natürlichem Abbau von Schadstoffen, basierend auf Veränderungen in Verhältnissen stabiler Kohlenstoffisotope der Schadstoffe (’Compound-Specific Stable Isotope Analyis’, CSIA), an Bedeutung in der Altlastenbeurteilung. In Zentrum stehen dabei die Fragen, wie effizient der Abbau ist (Quantifizierung von Abbau) und auf welcher Zeitskala er abläuft (Bestimmung von Transformationsraten). [...]

A direct aqueous injection-gas chromatography/mass spectrometry (DAI-GC/MS) method for trace analysis of 24 volatile organic compounds (VOCs) in water samples is presented. The method allows for the simultaneous quantification of benzene, toluene, ethyl benzene, and xylenes (BTEX), methyl tert-butyl ether (MTBE), tert-butyl alcohol (TBA), as well as a variety of chlorinated methanes, ethanes, propane, enthenes and benzenes. Applying a liquid film polyethylene glycol or a porous layer open tubular (PLOT) divinylbenzene GC capillary column to separate the water from the VOCs, volumes of 1–10 μL aqueous sample are directly injected into the GC. No enrichment or pretreatment steps are required and sample volumes as low as 100 μL are sufficient for accurate quantification. Method detection limits determined in natural groundwater samples were between 0.07 and 2.8 μg/L and instrument detection limits of <5 pg were achieved for 21 out of the 24 evaluated VOCs. DAI-GC/MS offers both good accuracy and precision (relative standard deviations ≤10%). The versatility of our method is demonstrated for contaminant quantification in drinking water disinfection (advanced oxidation of MTBE) and for VOC concentration measurements in a polluted aquifer. The wide range of detectable compounds and the lack of labor-intensive sample preparation illustrate that the DAI method is robust and easily applicable for the quantification of important organic groundwater contaminants.

Compound-specific isotope analysis was used to monitor the δ¹³C signature of chloroform produced upon the chlorination of model compounds representing natural organic matter functional groups (resorcinol, acetylacetone, acetophenone, phenol, and 2,4,6-trichlorophenol) and a natural water sample. For each model compound, a different apparent kinetic isotope effect was found for chloroform formation. Normal isotope effects were found for resorcinol, acetylacetone, and acetophenone, and ranged from 1.009 ± 0.002 to 1.024 ± 0.004. For the two phenols, an inverse effect was found (0.980 ± 0.004). Lake Zürich water also had a inverse effect (0.997 ± <0.001) indicating that phenols are likely chloroform precursors in NOM, but that other functional groups may also participate. The apparent ¹³C kinetic isotope effect for the addition/elimination reaction of 1,1,1-trichloropropanone mediated by OH⁻ to yield chloroform is 1.014 ± 0.002. A comparison of this value to those found for the chlorination of the model precursors and an evaluation of the differences in chloroform production kinetics for the different model precursors argue against a mechanism in which all NOM precursors react via a common intermediate. Compound specific isotope analysis may give additional insights into chloroform formation mechanisms beyond those allowed by current techniques.

The ¹³C, ²H, and ¹⁵N fractionation associated with light-induced transformations of N-containing pesticides in surface waters was investigated using atrazine as a model compound. In laboratory model systems, bulk isotope enrichment factors ϵ_C, ϵ_H, and ϵ_N were determined during the photooxidation of atrazine by excited triplet states of 4-carboxybenzophenone (³4-CBBP*), by OH radicals, and during direct photolysis at 254 nm. Moderately large ²H fractionations, quantified by ϵ_H values of −51.2 ± 2.5‰ and −25.3 ± 1.7‰, were found for the transformation of atrazine by ³4-CBBP* and OH radicals, respectively. ¹³C and ¹⁵N enrichment factors were rather small (−0.3‰ > ϵ_{C, N} > −1.7‰). The combined δ¹³C, δ²H, and δ¹⁵N analysis suggests that isotope effects are most likely due to H abstraction at the N−H and C−H bonds of the N-alkyl side chains. Direct photolysis of atrazine yielding hydroxyatrazine as main product was characterized by inverse ¹³C and ¹⁵N fractionation (ϵ_C = 4.6 ± 0.3‰, ϵ_N = 4.9 ± 0.2‰) and no detectable ²H fractionation. We hypothesize that isotope effects from photophysical processes involving the excited states of atrazine as well as magnetic isotope effect originating from the magnetic interactions of spin-carrying C and N nuclei have contributed to the observed inverse fractionation. Our study illustrates how compound-specific isotope analysis can be used to differentiate between important direct and indirect phototransformation pathways of agrochemicals in the environment.

In anoxic freshwater environments, various organic pollutants can be transformed via reduction by mineral-bound Fe(II) species. Ferrous iron species associated with clay minerals are of particular interest because iron-containing clay minerals are ubiquitously present in subsurface environments and structural Fe(II) can be formed from microbial Fe(III) reduction. Finally, in contrast to Fe(III)oxy(hydr)oxides, Fe(III) in clay minerals is less susceptible to reductive dissolution, thus providing a renewable source of reduction equivalents for contaminant transformation.
This study explores the nature and reactivity of Fe(II) species in chemically reduced smectites that govern the reduction of organic contaminants. Using contaminants, i.e., nitroaromatic compounds (NACs) and polyhalogenated ethanes, as probe compounds and infrared (IR) spectroscopy, five different smectites (Arizona montmorillonite (SAz-1), Wyoming montmorillonite (SWy-2), Ölberg montmorillonite, ferruginous smectite (SWa-1), and synthetic nontronite) were investigated for the predominant reactive Fe(II) species and their possible structural environment. [...]

Structural Fe(II) in clay minerals is an important source of electron equivalents for the reductive transformation of contaminants in anoxic environments. We investigated which factors control the reactivity of Fe(II) in smectites including total Fe content, Fe(II)/total Fe ratio, and excess negative charge localization using 10 nitroaromatic compounds (NACs) as reactive probe molecules. Based on evidence from this work and previous spectroscopic studies on Fe redox reactions in iron-rich smectites, we propose a kinetic model for quantifying the reactivity, abundance, and interconversion rates of two distinct Fe(II) sites in the minerals’ octahedral sheet. Excellent agreement between observed biphasic NAC reduction kinetics and model fits points toward existence of two types of Fe(II) sites exhibiting reactivities that differ by 3 orders of magnitude in iron-rich ferruginous smectite (SWa-1) and Ölberg montmorillonite. Low structural Fe content, as found in Wyoming montmorillonite (SWy-2), impedes the formation of highly reactive Fe sites and results in pseudo-first order kinetics of NAC reduction that originate from the presence of a single type of Fe(II) species of even lower reactivity. Similar correlations of one-electron reduction potentials of the NACs vs their second order reduction rate constants for all smectite suspensions suggest that contaminant-Fe(II) interactions were identical in all smectite minerals.

Solid-phase microextraction (SPME) coupled to gas chromatography/isotope ratio mass spectrometry was used to determine the δ¹⁵N and δ¹³C signatures of selected nitroaromatic contaminants such as the explosive 2,4,6-trinitrotoluene (TNT) for derivation of isotopic enrichment factors of contaminant transformation. Parameters for efficient extraction of nitroaromatic compounds (NACs) and substituted anilines from water samples were evaluated by SPME-GC/MS. δ¹³C signatures determined by SPME-GC/IRMS and elemental analyzer IRMS (EA-IRMS) were in good agreement, generally within +/- 0.7%, except for 2,4-dinitrotoluene (2,4-DNT) and TNT, which showed slight deviations (< 1.3%). Limits of detection (LODs) for δ¹³C analysis by SPME-GC/IRMS were between 73 and 780 μg L^-1 and correlated with the extraction efficiencies of the compounds determined by SPME-GC/MS. Nitrogen isotope measurements by SPME-GC/IRMS were of similar precision (standard deviations < 0.8%) for all NACs except for TNT. δ¹⁵N signatures matched the reference values obtained by EA-IRMS within +/- 1.3% (+2.5% for TNT), but no systematic trend was found for the deviations. LODs of δ¹⁵N measurements ranged from 1.6 to 9.6 mg L^-1 for nitrotoluenes, chlorinated NACs and DNTs (22 mg L^-1 for TNT). The SPME-GC/IRMS method is well suited for the determination of isotopic enrichment factors of various NAC transformation processes and provides so far unexplored possibilities to elucidate behavior and degradation mechanisms of nitroaromatic contaminants in soils and groundwaters.

We investigated the extent and variability of C and Cl isotope fractionation during the reduction of polychlorinated ethanes to evaluate the potential use of Cl isotope analysis for the assessment of contaminant transformation in subsurface environments. Kinetic isotope effects (KIE) for C and Cl for the reductive β-elimination of 1,1,2,2-tetrachloroethane (1,1,2,2-TeCA), pentachloroethane (PCA), and hexachloroethane by Cr(II) used as model reductant in homogeneous solution were compared to KIEs measured for dehydrochlorination of 1,1,2,2-TeCA and PCA. Since isotopic reactions of polychlorinated compounds are complicated by the simultaneous presence of several Cl isotopologues and intramolecular isotopic competition, we present a procedure for the determination of KIEs for Cl from the initial reactant and final product Cl isotope ratios. Despite different reaction mechanisms, that is reduction via dissociative inner-sphere electron transfer by Cr(H ₂O)₆²⁺ and base-catalyzed, concerted elimination, respectively, apparent KIEs for C of both pathways fall within a similar range (1.021-1.031). In contrast, KIEs for Cl are significantly higher for reductive β-elimination (1.013-1.021) than for dehydrochlorination (1.000-1.006). These results suggest that reductive transformations of polychlorinated contaminants might be identified on the basis of combined C and Cl isotope analysis.

Compound-specific isotope analysis (CSIA) is an increasingly important tool for the qualitative and quantitative assessment of transformations of organic compounds in contaminated environments. To date, the use of CSIA has been mainly restricted to the elements C and H, although N constitutes a very important reactive centerfor many priority contaminants. To evaluate the potential use of N isotope effects in the fate assessment of organic contaminants, we investigated the N isotope enrichment during the abiotic reduction of 4 substituted nitroaromatic compounds (NACs), using two abiotic model reductants, namely Fe(II) sorbed to goethite (α-FeOOH) and juglone (8-hydroxy-1,4- naphthoquinone) in the presence of H₂S. Substantial and virtually identical isotope enrichment factors, ε_N, of about -30‰, indicative of the breaking of one N-O bond, were found for all NACs, regardless of the reductant involved and the substitution of the NAC. These results indicate that the ε_N-values determined in our study could be representative for the reduction of aromatic NO₂-groups and thus be used to assess the abiotic transformation of NACs qualitatively and quantitatively in complex anoxic environments.

Reductive transformation reactions involving mineral-bound Fe²⁺ species are of great relevance for the fate of groundwater contaminants. For clay minerals, which are ubiquitously present in soils and sediments, the factors determining the reactivity of structural Fe²⁺ and surface-bound Fe²⁺ are not well understood. We investigated the reactivity and availability of Fe²⁺ species in suspensions of chemically reduced montmorillonite (SAz-1) as well as in suspensions of oxidized and reduced nontronite (SWa-1, ferruginous smectite) using two acetylnitrobenzene isomers as reactive probe compounds. The analyses of the reduction kinetics of the two nitroaromatic compounds (NACs) suggested that Fe²⁺ bound in the octahedral layer of reduced smectites is the predominant reductant and that electron transfer presumably occurs via basal siloxane planes. In contrast, reduction of NACs by Fe²⁺ associated with oxidized nontronite is orders of magnitude slower than reduction by octahedral Fe²⁺. Reductive transformation and reversible, nonreactive electron donor−acceptor (EDA) complexation of NACs at basal smectite surfaces occur simultaneously at reduced montmorillonite exhibiting low structural iron content. In contrast, EDA complexation was not observed in suspensions of reduced iron-rich nontronite. Due to the similar reduction rate constants measured for the two NACs, we propose that the (re)generation of octahedral Fe²⁺ sites, e.g., by electron transfer and/or Fe rearrangement within the octahedral nontronite layers, partly limited the rate of contaminant transformation. Since iron in clay minerals is available for microbial reduction, our study suggests that octahedral Fe²⁺ can contribute to abiotic contaminant transformation in anoxic environments.

The increasing worldwide contamination of freshwater systems with thousands of industrial and natural chemical compounds is one of the key environmental problems facing humanity. Although most of these compounds are present at low concentrations, many of them raise considerable toxicological concerns, particularly when present as components of complex mixtures. Here we review three scientific challenges in addressing water-quality problems caused by such micropollutants. First, tools to assess the impact of these pollutants on aquatic life and human health must be further developed and refined. Second, cost-effective and appropriate remediation and water-treatment technologies must be explored and implemented. Third, usage and disposal strategies, coupled with the search for environmentally more benign products and processes, should aim to minimize introduction of critical pollutants into the aquatic environment.

Perchlorethylen (PCE) und Trichlorethylen (TCE) gehören zu den häufigsten Grundwasserkontaminanten. Sie sind unter oxischen Bedingungen persistent, können aber unter anoxischen Bedingungen mittels mikrobieller reduktiver Dehalogenierung abgebaut werden. Der Abbau kann mit Hilfe von Konzentrationsmessungen oder mit der Einzelstoffisotopenanalyse (CSIA) gemessen werden. Um den Abbau mit CSIA quantifizieren zu können, sind die Anreicherungsfaktoren ε der Kohlenstoffisotope von Interesse. Diese wurden bereits in verschiedenen Laborstudien gemessen, allerdings ist ihre Variabilität zu gross, um CSIA für Felduntersuchungen quantitativ anzuwenden.
Ziel der vorliegenden Arbeit war es daher, den Ursachen dieser Variabilität nachzugehen. Dazu wurde ein Dreiphasen-Batchsystem (Gasraum, Lösungsmittel, wässriges Medium) zur Messung des mikrobiellen Abbaus von PCE und TCE entwickelt. Als Lösungsmittel für die Zugabe der schwer wasserlöslichen Schadstoffe PCE und TCE wurde Hexadecan gewählt, da dieser Stoff von den Mikroorganismen nicht als Kohlenstoffquelle verwendet werden kann. Zudem weisen PCE und TCE hohe Hexadecan-Wasser-Verteilungskonstanten (Kihw) auf, womit Hexadecan als Reservoir für PCE und TCE dient. Zur Dehalogenierung wurden Reinkulturen von Sulfurospirillum sp. verwendet, die aus einem Sediment angereichert worden waren.
Die Probenahme war über den gesamten Abbauprozess hinweg möglich und geschah aus der wässrigen Phase. Der Konzentrationsverlauf der Produkte und Edukte, sowie deren Kohlenstoffisotopensignatur wurde gemessen und zusätzlich mit dem Computer-Programm Matlab modelliert. Sowohl für PCE wie auch für TCE konnten Anreicherungsfaktoren gemessen werden.
Experimente zur Phasentransfer-Geschwindigkeit zeigten, dass die Nachlieferung von PCE oder TCE aus dem Hexadecan limitierend wird, wenn sich die Bakterien in einer exponentiellen Wachstumsphase befinden. Dies kann zur Folge haben, dass der Anreicherungsfaktor maskiert wird. Die Modellierung zeigte eine eindeutige Maskierung ab einem Schadstoffabbau von 75%. Das Ergebnis der Limitierung durch den Phasentransfer wurde in den Abbauexperimenten bestätigt. Trotzdem konnten ε-Werte bestimmt und mit Literaturwerten verglichen werden.
Für den Abbau von PCE bei 30°C wurde ein ε von -0.3‰ (±0.05) ermittelt. In der Literatur wurde für den PCE-Abbau von Sulfurospirillum multivorans ein ε-Wert von -0.4‰ (±0.08‰) publiziert. Beim Abbau von TCE wurde bei 20°C ein ε von -14.6‰ (±2.75) und bei 30°C ein Anreicherungsfaktor von -12.4‰ (2.74‰) gemessen. Diese Werte liegen im oberen Bereich der in der Literatur publizierten ε-Werte. Allerdings wurden diese Anreicherungsfaktoren mit Bakterien-Konsortien und nicht mit Reinkulturen gemessen. Die unterschiedlichen ε-Werte bei 20°C und 30°C sind wahrscheinlich auf die stärkere Maskierung der Werte bei 30°C zurückzuführen.
Obwohl das in dieser Arbeit vorgeschlagene Dreiphasen-Batchsystem eine Maskierung der Anreicherungsfaktoren durch eine Limitierung des Phasentransfers aufweist, konnten ε- Werte der Literatur reproduziert werden.
Die analytischen Methoden zur Messung der Konzentrationen der Produkte und Edukte sowie der Kohlenstoffisotopensignatur erwiesen sich als geeignet. Die Bestimmung eines Anreicherungsfaktors ist auch im kleinen (und somit umweltrelevanten) Konzentrationsbereich möglich.
Interessant wäre nun die Untersuchung des Einflusses der Maskierung. Dazu wäre jedoch ein Batchsystem ohne Phasentransfer-Limitierung als Referenzsystem notwendig. Dies könnte beispielsweise mit der Zugabe der Schadstoffe mittels schadstoffgesättigten Mediums realisiert werden.

Environmental assessments in pesticide product development are generally restricted to plant uptake and emissions of active ingredients. Life-cycle assessment (LCA) enables a more comprehensive evaluation by additionally assessing the impacts of pesticide production and application (e.g. tractor operations). The use of LCA in the product development of pesticides, in addition to the methods commonly applied, is therefore advisable. In this paper a procedure for conducting LCA in early phases of product development is proposed. In a case study, two plant-growth regulators from different product generations were compared regarding their application in intensive production of winter wheat. The results showed that the reduced emissions from active ingredients of the newer pesticide were compensated by higher impacts from the production process. The authors draw the conclusion that it is important to consider environmental objectives in the procurement of precursors, in addition to the classical goals of increasing the efficacy and reducing the nontarget effects of pesticides. Moreover, the case study showed that decisions based on uncertain results in early stages of product development may need to be revised in later stages, e.g. based on investigations of pesticides' effects on crop yield.

Waste solvent incineration is an important issue in life cycle assessments (LCAs) dealing with chemical products and/or processes. Nearly all chemical products and processes involve organic solvents, and incineration is often the favoured solution to waste solvent treatment as it can deal with a large variety of solvent types and quantities. At present, there are no generic models for waste solvent incineration which allow integrating this technology as a unit process in LCA. As waste solvents as a rule are incinerated as a mixture of several solvents, an allocation problem occurs: measurements of the consumption of ancillaries and energy carriers, and of emission of pollutants and generation of co-products, always refer to the mixture of waste solvents. However, in LCAs usually waste specific data is needed. To solve this problem we developed a multi-input allocation model of the incineration process. A comprehensive case study on a waste solvent incineration plant from chemical industry provided the necessary data. The results from the multi-input allocation modelling are consumption and emission factors which facilitate the calculation of solvent specific life cycle inventory results.

Goal, Scope, Background
To improve the environmental performance of chemical products or services, especially via comparisons of chemical products, LCA is a suitable evaluation method. However, no procedure to obtain comprehensive LCI-data on the production of fine and speciality chemicals is available to date, and information on such production processes is scarce. Thus, a procedure was developed for the estimation of LCIs of chemical production process-steps, which relies on only a small amount of input data.
Methods
A generic input-output scheme of chemical production process-steps was set up, and equations to calculate inputs and outputs were established. For most parameters in the resulting estimation procedure, default values were derived from on-site data on chemical production processes and from heuristics. Uncertainties in the estimated default values were reflected as best-case and worst-case scenarios. The procedure was applied to a case study comparing the production of two active ingredients used for crop protection. Verification and a sensitivity analysis were carried out.
Results and Discussion
It was found that the impacts from the mass and energy flows estimated by the procedure represent a significant share of the impacts assessed in the case study. In a verification, LCI-data from existing processes yielded results within the range of the estimated best-case and worst-case scenarios. Note that verification data could not be obtained for all process steps. From the verification results, it was inferred that mass and energy flows of existing processes for the production of fine and speciality chemicals correspond more frequently to the estimated best-case than to the worst-case scenario. In the sensitivity analysis, solvent demand was found to be the most crucial parameter in the environmental performance of the chemical production processes assessed.
Conclusion
Mass and energy flows in LCIs of production processes for fine and speciality chemicals should not be neglected, even if only little information on a process is available. The estimation procedure described here helps to overcome lacking information in a transparent, consistent way.
Recommendations and Outlook
Additional verifications and a more detailed estimation of the default parameters are desirable to learn more about the accuracy of the estimation procedure. The procedure should also be applied to case studies to gain insight into the usefulness of the estimation results in different decision-making contexts.

Mineral-bound Fe(II) species represent important natural reductants of pollutants in the anaerobic subsurface. At clay minerals, three types of Fe(II) species in fundamentally different chemical environments may be present simultaneously, i.e., structural Fe(II), Fe(II) complexed by surface hydroxyl groups, and Fe(II) bound by ion exchange. We investigated the accessibility and reactivity of these three types of Fe(II) species in suspensions of two different clay minerals containing either ferrous iron-bearing nontronite or iron-free hectorite. Nitroaromatic compounds (NACs) exhibiting different sorption behavior on clays were used to probe the reactivity of the various types of reduced iron species. The clay treatment allowed for a preparation of nontronite and hectorite surfaces with Fe(II) adsorbed by surface hydroxyl groups at the edge surfaces. Furthermore, hectorite suspensions with additional Fe(II) bound to the ion exchange sites at the basal siloxane surfaces were set up. We found that both structural Fe(II) and Fe(II) complexed by surface hydroxyl groups of nontronite reduced the NACs to anilines. An electron balance revealed that more than 10% of the total iron in nontronite was reactive Fe(II). Fe(II) bound by ion exchange did not contribute to the observed reduction of NACs. Reversible adsorption of the NACs at the basal siloxane surface of the clays strongly retarded NAC reduction, even in the presence of high concentrations of Fe(II) bound by ion exchange to the basal siloxane surfaces. Our work shows that in natural systems a fraction of the total Fe(II) present on clays may contribute to the pool of highly reactive Fe(II) species in the subsurface. Furthermore, this work may help to distinguish between Fe(II) species of different reactivity regarding pollutant reduction. Although structural iron in clays represents only a small fraction of the total iron pool in soils and aquifers, reactive Fe(II) species originating from the reduction of structural Fe(II) in clays may contribute significantly to the biogeochemical cycling of electrons in the subsurface since it is not subject to depletion by reductive dissolution.

Uptake and transfonnation of nitroaromatic compounds (NACs) by microorganisms, fungi, or plants can contribute to the natural attenuation of the compounds in the subsurface and provide the basis for various technologies to remediate contaminated sites (see Chapter 4). In soil and groundwater, however, NACs also interact with non-living matrix constituents, e.g., by sorption to minerals and organic matter or reactions with various dissolved, adsorbed, or solid chemical species. Abiotic and biotic processes that are relevant to the fate of NACs are often interconnected and occur simultaneously. For instance, slow desorption of NACs from the solid to the aqueous phase may limit the bioavailability and thus the rate of biotransformation in the subsurface. Conversely, microbial activity creates an anoxic milieu that favors various abiotic reduction processes of NACs. Abiotic reactions and sorption processes change the product distribution and bioavailability of NACs and thus affect their biodegradation. Therefore, the subsurface chemistry of NACs must be understood to allow assessment of natural or engineered attenuation processes at contaminated sites.
In this chapter we discuss important interactions and reactions of NACs with natural subsurface constituents. To this end, we will start with a brief summary of the properties and the chemistry of aromatic nitro compounds followed by a review of sorption and transport of NACs in porous media as well as of the major routes of abiotic transformation of NACs. Such knowledge will help to assess the relative importance and mutual dependence of biological and abiotic processes that act on NACs in a given system. Furthermore, it provides a scientific basis for designing appropriate remediation strategies as well as for evaluating their performance at contaminated sites.
We will deal with information on the interactions between NACs and the non-living natural matrix constituents obtained from very different scales of observation, ranging from spectroscopic characterization of sorption mechanisms to field scale observations on the fate of NACs in aquifers under complex geochemical conditions. Using illustrative case studies, we will also address the question of how to transfer and scale up concepts of basic theory and mechanistic information to complex natural systems. As a leading principle, we will address the major interactions and reactions of NACs with subsurface constituents from a structure reactivity point of view.

Contamination of soils and aquifers with (poly)nitroaromatic compounds ((P)NACs) is a widespread problem. This work demonstrates that (P)NACs su ch as the explosive 2,4,6-trinitrotoluene (TNT) can be completely reduced to the corresponding aromatic polyamines by Fe(ll) present at the surface of Fe(III)(hydr)oxides or, less efficiently, by hydroquinone moieties of (natural) organic matter in the presence of H₂S. The reduction kinetics of (P)NACs were investigated in sterile batch systems as well as in columns containing either FeOOH-coated sand and a pure culture of the iron-reducing bacterium Geobacter metallireducens or ferrogenic consortia in aquifer sediments. The relative reactivities as well as the competition behavior of (P)NACs in batch and column systems, respectively, correlated well with their one-electron reduction potentials, E_h¹', which we determined for TNT and its aminonitrotoluene transformation products. A similar reactivity pattern of (P)NACs was found irrespective of the processes that (re)generated the surface-bound Fe(II), i.e., adsorption of Fe(II) from aqueous solution or microbial reduction of Fe(III)(hydr)oxides. The apparent stability of the toxic arylamine products under ferrogenic conditions may compromise intrinsic attenuation as an acceptable remediation option for (P)NAC contaminated anoxic aquifers. Iron-reducing conditions would, however, be favorable as a first step in a two-stage anaerobic/aerobic treatment of PNAC contaminated sediments since aromatic polyamines are biodegradable and/or bind irreversibly to the solid matrix under oxic conditions.

Contamination of soils and aquifers with anthropogenic organic chemicals such as solvents, dyes, pesticides, and explosives has become a widespread environmental problem. The accumulation of organic pollutants in the subsurface may be of significant (eco)toxicological concern and may require the remediation of contaminated sites. In order to evaluate the effects of subsurface contaminants on humans and on the environment, the basic processes relevant for transport and transformation reactions of pollutants must be understood. Furthermore, knowledge of the fate of anthropogenic organic chemicals in contaminated environments is essential to assess the need for remediation and may help to develop appropriate remediation methods. This work focuses on transformation processes of mono- and polynitroaromatic compounds, (P)NACs, representing pollutants that are considered to be quite persistent under oxic conditions but may undergo reductive transformation in the anaerobic subsurface. Because of the potential occurrence of iron-reducing conditions in contaminated soils and aquifers abiotic transformation pathways of (P)NACs involving Fe(II) species present under anoxic conditions were studied. In laboratory and field experiments both, the reactivity of (P)NACs with selected iron(II) species as well as the formation of Fe(II) species relevant for pollutant reduction in the contaminated subsurface environments were investigated. [...]

Anthropogene organische Chemikalien wie Lösungsmittel, Farbstoffe, Pestizide und Explosivstoffe sind bekannte Schadstoffe in Böden und Grundwasserleitern. Der Eintrag von organischen Chemikalien in den Untergrund kann aus (öko)toxikolgischer Sicht bedenklich sein und im Sanierungsfall aufwendige Verfahren zur Entfernung der Schadstoffe verursachen. Um die Auswirkungen organischer Chemikalien im Untergrund auf Mensch und Umwelt abzuschätzen, müssen die Prozesse, welche die Verteilung und die Transformation dieser Chemikalien bestimmen, bekannt sein. Dieses Prozessverständnis ist zudem unerlässlich für die Beurteilung des Sanierungsbedarfs kontaminierter Standorte sowie für die Wahl der geeigneten Sanierungsmethoden. In dieser Arbeit werden mono- und polynitroaromatische Substanzen ((P)NAS)) als Vertreter von Schadstoffklassen untersucht, die im oxischen Milieu stabiler sind als unter anaeroben Bedingungen, wo sie reduktiv transformiert werden können. Weil in kontaminierten, anaeroben Böden und Grundwasserleitern eisenreduzierende Bedingungen sehr verbreitet sind, kommt der reduktiven Transformation von (P)NAS mit verschiedenen Eisen(II)-verbindungen in der Umwelt grosse Bedeutung zu. In Labor- und Feldstudien wurden deshalb sowohl die Reaktivität von (P)NAS mit ausgewählten Fe(II)-Spezies als auch die Dynamik der Bildung reaktiver Fe(II)-Spezies untersucht. [...]

The biogeochemical processes controlling the reductive transformation of contaminants in an anaerobic aquifer were inferred from the relative reactivity patterns of redox- sensitive probe compounds. The fate of five nitroaromatic compounds (NACs) was monitored under different redox conditions in a landfill leachate plume of a sandy aquifer. Results of field experiments (continuous injection and in situ microcosms) were compared to the findings of laboratory batch and column experiments (using aquifer matrix and model systems for sulfate-and iron-reducing conditions). NACs were transformed within 2-70 days in the leachate plume as well as in microbially active and in microbially deactivated experiments. Generally, aromatic amines were the predominant reduction products, and these compounds were stable within the time frame and under the conditions of our experiments. Despite the presence of various potential reductants (e.g., H₂S/HS^-, Fe(II)_aq, reduced organic matter, microorganisms), the patterns of relative reactivity of the probe compounds indicated that ferrous iron associated with iron(lll) (hydr)oxide surfaces was the dominant reductant throughout the anaerobic region of the plume. Our results suggest that Fe(ll) associated with ferric iron minerals is a highly reactive reductant in anaerobic aquifers, which may also determine the fate of other classes of reducible contaminants such as halogenated solvents, azo compounds, sulfoxides, chromate, or arsenate.

In complex environmental systems, tracers are indispensable tools for the investigation of various physical, chemical, and biological processes. From the large variety of tracers employed by EAWAG in the aquatic environment, we present some examples relevant to groundwater research. Some tracers (e.g., ²²²Rn, ³H/³He, chlorofluorocarbons) allow the time since groundwater infiltration to be determined; this information can be used to quantify process rates such as flow velocities, recharge and reaction rates. Other tracers (e.g., conductivity, isotopes of oxygen or boron) can be used to quantify mixing ratios between waters of different origin, for instance, to study the admixture of leachate from a landfill to an aquifer. In contrast to these inert tracers, chemically reactive tracers (e.g., nitroaromatic compounds) can be used to study subsurface biogeochemical transformation processes, e.g. the reduction of contaminants under anaerobic conditions. The successful use of tracers in the study of environmental systems requires highly developed analytical facilities for a broad palette of tracer measurements and a careful choice of appropriate tracers for each specific problem.