Continuous-Flow Stable Sulfur Isotope Analysis of Organic and Inorganic Compounds by EA-MC-ICPMS.

Elemental analysis (EA) coupled to isotope ratio mass spectrometry (IRMS) is a well-established method to derive stable isotope ratios of sulfur (34S/32S). Conversion of sulfur to SO2 by EA and measurement of SO2 isotopologues by IRMS represents the simplest and most versatile method to accomplish isotope measurement of sulfur even in bulk samples. Yet, interferences by oxygen isotopes in SO2 often impair the precision and trueness of measured results. In the current study, we coupled EA to multicollector inductively coupled plasma mass spectrometry (MC-ICPMS) to establish a method that avoids such interferences due to direct measurement of S+ ions. In addition, measurement of the 33S/32S isotope ratios is possible, thus representing the first bulk method that is suitable to study mass-independent isotope fractionation (MIF). Analytical precision (σ) of available Ag2S and BaSO4 reference materials (RMs) was, on average, 0.2 mUr for δ33S and δ34S, never exceeding 0.3 mUr within this study (1 mUr = 1‰ = 0.001). Measured δ34S values of reference materials agreed within ±0.2 mUr of officially reported values. Measurement of wood samples yielded good precision (0.2 mUr) for sulfur amounts as low as 3.5 µg, but precision deteriorated for samples at lower sulfur contents due to poor peak shape. Finally, we explored cross-calibration of organic liquids separated via gas chromatography (GC) against solid RMs combusted via EA that avoids challenging offline conversion of RMs. Results indicate good precision (≤0.08 mUr) and acceptable trueness (≤0.34 mUr) for determination of δ34S, demonstrating the future potential of such an approach.

Determination of Stable Hydrogen Isotopic Composition and Isotope Enrichment Factor at Low Hydrogen Concentration.

Determination of stable hydrogen isotopic compositions (δ2H) is currently challenged to achieve a high detection limit for reaching the linear range where δ2H values are independent of concentration. Therefore, it is difficult to assess precise δ2H values for calculating the hydrogen isotope enrichment factor (εH) and for field application where the concentrations of contaminants are relatively low. In this study, a data treatment approach was developed to obtain accurate δ2H values below the linear range. The core concept was to use a logarithmic function to fit the δ2H values below the linear range and then adjust the δ2H values below the linear range into the linear range by using the fitted logarithmic equation. Moreover, the adjusted δ2H values were calibrated by using laboratory reference materials, e.g., n-alkanes. Tris(2-chloroethyl) phosphate (TCEP) and hexachlorocyclohexane (HCH) isomers were selected as examples of complex heteroatom-bearing compounds to develop the data treatment approach. This data treatment approach was then tested using δ2H values from a TCEP transformation experiment with OH radicals. Comparable δ2H values and εH between the low-concentration experiment and the reference experiment were obtained using the developed approach. Therefore, the developed data treatment approach enables a possibility of determining the hydrogen isotopic compositions of organic components in low concentrations. It is especially valuable for determining organic contaminants in environmental samples, which are usually present in low concentrations.

Requirements for Chromium Reactors for Use in the Determination of H Isotopes in Compound-Specific Stable Isotope Analysis of Chlorinated Compounds.

There is a strong need for careful quality control in hydrogen compound-specific stable isotope analysis (CSIA) of halogenated compounds. This arises in part due to the lack of universal design of the chromium (Cr) reactors. In this study, factors that optimize the critical performance parameter, linearity, for the Cr reduction method for hydrogen isotope analysis were identified and evaluated. These include the effects of short and long vertically mounted reactors and temperature profiles on trapping of Cl to ensure accurate and precise hydrogen isotope measurements. This paper demonstrates the critical parameters that need consideration to optimize any Cr reactor applications to ensure the accuracy of δ2H analysis for organic compounds and to enhance intercomparability for both international standards and reference materials run by continuous flow versus an elemental analyzer.

Simultaneous Compound-Specific Analysis of δ33S and δ34S in Organic Compounds by GC-MC-ICPMS Using Medium- and Low-Mass-Resolution Modes.

Compound-specific isotope analysis of sulfur (δ34S-CSIA) in organic compounds was established in the last decade employing gas chromatography connected to multiple-collector inductively coupled plasma mass spectrometry (GC-MC-ICPMS). However, δ33S-CSIA has not yet been reported so far. In this study, we present a method for the simultaneous determination of δ33S and δ34S in organic compounds by GC-MC-ICPMS applying medium- and also low-mass-resolution modes. The method was validated using the international isotope reference materials IAEA-S-1, IAEA-S-2, and IAEA-S-3. Overall analytical uncertainty including normalization and reproducibility for δ33S and δ34S was usually better than ±0.2 mUr (σ) for analytes containing at least 100 pmol of S. Further, it is demonstrated that, despite small isobaric interferences, results obtained at low mass resolution are indistinguishable from medium mass resolution offering the benefit of increased sensitivity and versatility of this method. Additionally, the method was applied for the δ33S and δ34S isotope analysis of industrially produced organic compounds to investigate potential mass-independent fractionation (MIF). The relation between δ34S and δ33S in these compounds followed a mass-dependent fractionation trend (MDF; Δ33S ≤ ±0.2 mUr). Degradation of dimethyl disulfide by direct photolysis caused a small but significant MIF (Δ33S = 0.55 ± 0.04 mUr, n = 3), demonstrating sufficient sensitivity of the method for these types of studies.

Toward Improved Accuracy in Chlorine Isotope Analysis: Synthesis Routes for In-House Standards and Characterization via Complementary Mass Spectrometry Methods.

Increasing applications of compound-specific chlorine isotope analysis (CSIA) emphasize the need for chlorine isotope standards that bracket a wider range of isotope values in order to ensure accurate results. With one exception (USGS38), however, all international chlorine isotope reference materials (chloride and perchlorate salts) fall within the narrow range of one per mille. Furthermore, compound-specific working standards are required for chlorine CSIA but are not available for most organic substances. We took advantage of isotope effects in chemical dehalogenation reactions to generate (i) silver chloride (CT16) depleted in 37Cl/35Cl and (ii) compound-specific standards of the herbicides acetochlor and S-metolachlor (Aceto2, Metola2) enriched in 37Cl/35Cl. Calibration against the international reference standards USGS38 (-87.90 ‰) and ISL-354 (+0.05 ‰) by complementary methods (gas chromatography-isotope ratio mass spectrometry, GC-IRMS, versus gas chromatography-multicollector inductively coupled plasma mass spectrometry, GC-MC-ICPMS) gave a consensus value of δ37ClCT16 = -26.82 ± 0.18 ‰. Preliminary GC-MC-ICPMS characterization of commercial Aceto1 and Metola1 versus Aceto2 and Metola2 resulted in tentative values of δ37ClAceto1 = 0.29 ± 0.29 ‰, δ37ClAceto2 = 18.54 ± 0.20 ‰, δ37ClMetola1 = -4.28 ± 0.17 ‰ and δ37ClMetola2 = 5.12 ± 0.27 ‰. The possibility to generate chlorine isotope in-house standards with pronounced shifts in isotope values offers a much-needed basis for accurate chlorine CSIA.

Compound Specific Stable Chlorine Isotopic Analysis of Volatile Aliphatic Compounds Using Gas Chromatography Hyphenated with Multiple Collector Inductively Coupled Plasma Mass Spectrometry.

Stable chlorine isotope analysis is increasingly used to characterize sources, transformation pathways, and sinks of organic aliphatic compounds, many of them being priority pollutants in groundwater and the atmosphere. A wider use of chlorine isotopes in environmental studies is still inhibited by limitations of the different analytical techniques such as high sample needs, offline preparation, confinement to few compounds and mediocre precision, respectively. Here we present a method for the δ37Cl determination in volatile aliphatic compounds using gas chromatography coupled with multiple-collector inductively coupled plasma mass spectrometry (GC-MC-ICPMS), which overcomes these limitations. The method was evaluated by using a suite of five previously offline characterized in-house standards and eight chlorinated methanes, ethanes, and ethenes. Other than in previous approaches using ICP methods for chlorine isotopes, isobaric interference of the 36ArH dimer with 37Cl was minimized by employing dry plasma conditions. Samples containing 2-3 nmol Cl injected on-column were sufficient to achieve a precision (σ) of 0.1 mUr (1 milliurey = 0.001 = 1‰) or better. Long-term reproducibility and accuracy was always better than 0.3 mUr if organics were analyzed in compound mixtures. Standardization is carried out by using a two-point calibration approach. Drift, even though very small in this study, is corrected by referencing versus an internal standard. The presented method offers a direct, universal, and compound-specific procedure to measure the δ37Cl of a wide array of organic compounds overcoming limitations of previous techniques with the benefits of high sensitivity and accuracy comparable to the best existing approaches.

Compound-specific hydrogen isotope analysis of fluorine-, chlorine-, bromine- and iodine-bearing organics using gas chromatography-chromium-based high-temperature conversion (Cr/HTC) isotope ratio mass spectrometry.

RATIONALE: The conventional high-temperature conversion (HTC) approach towards hydrogen compound-specific isotope analysis (CSIA) of halogen-bearing (F, Cl, Br, I) organics suffers from incomplete H2 yields and associated hydrogen isotope fractionation due to generation of HF, HCl, HBr, and HI byproducts. Moreover, the traditional off-line combustion of highly halogenated compounds results in incomplete recovery of water as an intermediary compound for hydrogen isotope ratio mass spectrometry (IRMS), and hence also leads to isotope fractionation. This study presents an optimized chromium-based high-temperature conversion (Cr/HTC) approach for hydrogen CSIA of various fluorinated, chlorinated, brominated and iodinated organic compounds. The Cr/HTC approach is fast, economical, and not affected by low H2 yields and associated isotope fractionation. METHODS: The performance of the modified gas chromatography/chromium-based high-temperature conversion (GC-Cr/HTC) system was monitored and optimized using an ion trap mass spectrometer. Quantitative conversion of organic hydrogen into H2 analyte gas was achieved for all halogen-bearing compounds. The corresponding accuracy of CSIA was validated using (i) manual dual-inlet (DI)-IRMS after off-line conversion into H2 , and (ii) elemental analyzer (EA)-Cr/HTC-IRMS (on-line conversion). RESULTS: The overall hydrogen isotope analysis of F-, Cl-, Br- and I-bearing organics via GC-Cr/HTC-IRMS achieved a precision σ ≤ 3 mUr and an accuracy within ±5 mUr along the VSMOW-SLAP scale compared with the measured isotope compositions resulting from both validation methods, off-line and on-line. The same analytical performance as for single-compound GC-Cr/HTC-IRMS was achieved compound-specifically for mixtures of halogenated organics following GC separation to baseline resolution. CONCLUSIONS: GC-Cr/HTC technology can be implemented in existing analytical equipment using commercially available materials to provide a versatile tool for hydrogen CSIA of halogenated and non-halogenated organics. Copyright © 2017 John Wiley & Sons, Ltd.

Optimization of on-line hydrogen stable isotope ratio measurements of halogen- and sulfur-bearing organic compounds using elemental analyzer-chromium/high-temperature conversion isotope ratio mass spectrometry (EA-Cr/HTC-IRMS).

RATIONALE: Accurate hydrogen isotopic analysis of halogen- and sulfur-bearing organics has not been possible with traditional high-temperature conversion (HTC) because the formation of hydrogen-bearing reaction products other than molecular hydrogen (H2 ) is responsible for non-quantitative H2 yields and possible hydrogen isotopic fractionation. Our previously introduced, new chromium-based EA-Cr/HTC-IRMS (Elemental Analyzer-Chromium/High-Temperature Conversion Isotope Ratio Mass Spectrometry) technique focused primarily on nitrogen-bearing compounds. Several technical and analytical issues concerning halogen- and sulfur-bearing samples, however, remained unresolved and required further refinement of the reactor systems. METHODS: The EA-Cr/HTC reactor was substantially modified for the conversion of halogen- and sulfur-bearing samples. The performance of the novel conversion setup for solid and liquid samples was monitored and optimized using a simultaneously operating dual-detection system of IRMS and ion trap MS. The method with several variants in the reactor, including the addition of manganese metal chips, was evaluated in three laboratories using EA-Cr/HTC-IRMS (on-line method) and compared with traditional uranium-reduction-based conversion combined with manual dual-inlet IRMS analysis (off-line method) in one laboratory. RESULTS: The modified EA-Cr/HTC reactor setup showed an overall H2 -recovery of more than 96% for all halogen- and sulfur-bearing organic compounds. All results were successfully normalized via two-point calibration with VSMOW-SLAP reference waters. Precise and accurate hydrogen isotopic analysis was achieved for a variety of organics containing F-, Cl-, Br-, I-, and S-bearing heteroelements. The robust nature of the on-line EA-Cr/HTC technique was demonstrated by a series of 196 consecutive measurements with a single reactor filling. CONCLUSIONS: The optimized EA-Cr/HTC reactor design can be implemented in existing analytical equipment using commercially available material and is universally applicable for both heteroelement-bearing and heteroelement-free organic-compound classes. The sensitivity and simplicity of the on-line EA-Cr/HTC-IRMS technique provide a much needed tool for routine hydrogen-isotope source tracing of organic contaminants in the environment. Copyright © 2016 John Wiley & Sons, Ltd.

Organic Reference Materials for Hydrogen, Carbon, and Nitrogen Stable Isotope-Ratio Measurements: Caffeines, n-Alkanes, Fatty Acid Methyl Esters, Glycines, L-Valines, Polyethylenes, and Oils.

An international project developed, quality-tested, and determined isotope-δ values of 19 new organic reference materials (RMs) for hydrogen, carbon, and nitrogen stable isotope-ratio measurements, in addition to analyzing pre-existing RMs NBS 22 (oil), IAEA-CH-7 (polyethylene foil), and IAEA-600 (caffeine). These new RMs enable users to normalize measurements of samples to isotope-δ scales. The RMs span a range of δ(2)H(VSMOW-SLAP) values from -210.8 to +397.0 mUr or ‰, for δ(13)C(VPDB-LSVEC) from -40.81 to +0.49 mUr and for δ(15)N(Air) from -5.21 to +61.53 mUr. Many of the new RMs are amenable to gas and liquid chromatography. The RMs include triads of isotopically contrasting caffeines, C16 n-alkanes, n-C20-fatty acid methyl esters (FAMEs), glycines, and l-valines, together with polyethylene powder and string, one n-C17-FAME, a vacuum oil (NBS 22a) to replace NBS 22 oil, and a (2)H-enriched vacuum oil. A total of 11 laboratories from 7 countries used multiple analytical approaches and instrumentation for 2-point isotopic normalization against international primary measurement standards. The use of reference waters in silver tubes allowed direct normalization of δ(2)H values of organic materials against isotopic reference waters following the principle of identical treatment. Bayesian statistical analysis yielded the mean values reported here. New RMs are numbered from USGS61 through USGS78, in addition to NBS 22a. Because of exchangeable hydrogen, amino acid RMs currently are recommended only for carbon- and nitrogen-isotope measurements. Some amino acids contain (13)C and carbon-bound organic (2)H-enrichments at different molecular sites to provide RMs for potential site-specific isotopic analysis in future studies.

Coupling of a headspace autosampler with a programmed temperature vaporizer for stable carbon and hydrogen isotope analysis of volatile organic compounds at microgram per liter concentrations.

One major challenge for the environmental application of compound-specific stable isotope analysis (CSIA) is the necessity of efficient sample treatment methods, allowing isolation of a sufficient mass of organic contaminants needed for accurate measurement of the isotope ratios. Here, we present a novel preconcentration technique--the coupling of a headspace (HS) autosampler with a programmed temperature vaporizer (PTV)--for carbon (δ(13)C) and hydrogen (δ(2)H) isotope analysis of volatile organic compounds in water at concentrations of tens of micrograms per liter. The technique permits large-volume injection of headspace samples, maintaining the principle of simple static HS extraction. We developed the method for multielement isotope analysis (δ(13)C and δ(2)H) of methyl tert-butyl ether (MTBE), benzene, toluene, ethylbenzene, and o-xylene (BTEX), and analysis of δ(13)C for chlorinated benzenes and ethenes. Extraction and injection conditions were optimized for maximum sensitivity and minimum isotope effects. Injection of up to 5 mL of headspace sample from a 20 mL vial containing 13 mL of aqueous solution and 5 g of NaCl (10 min of incubation at 90 °C) resulted in accurate δ(13)C and δ(2)H values. The method detection limits (MDLs) for δ(13)C were from 2 to 60 µg/L (MTBE, BTEX, chlorinated ethenes, and benzenes) and 60-97 µg/L for δ(2)H (MTBE and BTEX). Overall, the HS-PTV technique is faster, simpler, isotope effect-free, and requires fewer treatment steps and less sample volume than other extraction techniques used for CSIA. The environmental applicability was proved by the analysis of groundwater samples containing BTEX and chlorinated contaminants at microgram per liter concentrations.

Compound-specific hydrogen isotope analysis of heteroatom-bearing compounds via gas chromatography-chromium-based high-temperature conversion (Cr/HTC)-isotope ratio mass spectrometry.

The traditional high-temperature conversion (HTC) approach toward compound-specific stable isotope analysis (CSIA) of hydrogen for heteroatom-bearing (i.e., N, Cl, S) compounds has been afflicted by fractionation bias due to formation of byproducts HCN, HCl, and H2S. This study presents a chromium-based high-temperature conversion (Cr/HTC) approach for organic compounds containing nitrogen, chlorine, and sulfur. Following peak separation along a gas chromatographic (GC) column, the use of thermally stable ceramic Cr/HTC reactors at 1100-1500 °C and chemical sequestration of N, Cl, and S by chromium result in quantitative conversion of compound-specific organic hydrogen to H2 analyte gas. The overall hydrogen isotope analysis via GC-Cr/HTC-isotope ratio mass spectrometry (IRMS) achieved a precision of better than ± 5 mUr along the VSMOW-SLAP scale. The accuracy of GC-Cr/HTC-IRMS was validated with organic reference materials (RM) in comparison with online EA-Cr/HTC-IRMS and offline dual-inlet IRMS. The utility and reliability of the GC-Cr/HTC-IRMS system were documented during the routine measurement of more than 500 heteroatom-bearing organic samples spanning a δ(2)H range of -181 mUr to 629 mUr.

Development and validation of an universal interface for compound-specific stable isotope analysis of chlorine (37Cl/35Cl) by GC-high-temperature conversion (HTC)-MS/IRMS.

A universal application of compound-specific isotope analysis of chlorine was thus far limited by the availability of suitable analysis techniques. In this study, gas chromatography in combination with a high-temperature conversion interface (GC-HTC), converting organic chlorine in the presence of H2 to gaseous HCl, was coupled to a dual-detection system, combining an ion trap mass spectrometer (MS) and isotope-ratio mass spectrometer (IRMS). The combination of the MS/IRMS detection enabled a detailed characterization, optimization, and online monitoring of the high-temperature conversion process via ion trap MS as well as a simultaneous chlorine isotope analysis by the IRMS. Using GC-HTC-MS/IRMS, chlorine isotope analysis at optimized conversion conditions resulted in very accurate isotope values (δ(37)Cl(SMOC)) for measured reference material with known isotope composition, including chlorinated ethylene, chloromethane, hexachlorocyclohexane, and trichloroacetic acids methyl ester. Respective detection limits were determined to be <15 nmol Cl on column with achieved precision of <0.3‰.

On-line hydrogen-isotope measurements of organic samples using elemental chromium: an extension for high temperature elemental-analyzer techniques.

The high temperature conversion (HTC) technique using an elemental analyzer with a glassy carbon tube and filling (temperature conversion/elemental analysis, TC/EA) is a widely used method for hydrogen isotopic analysis of water and many solid and liquid organic samples with analysis by isotope-ratio mass spectrometry (IRMS). However, the TC/EA IRMS method may produce inaccurate δ(2)H results, with values deviating by more than 20 mUr (milliurey = 0.001 = 1‰) from the true value for some materials. We show that a single-oven, chromium-filled elemental analyzer coupled to an IRMS substantially improves the measurement quality and reliability for hydrogen isotopic compositions of organic substances (Cr-EA method). Hot chromium maximizes the yield of molecular hydrogen in a helium carrier gas by irreversibly and quantitatively scavenging all reactive elements except hydrogen. In contrast, under TC/EA conditions, heteroelements like nitrogen or chlorine (and other halogens) can form hydrogen cyanide (HCN) or hydrogen chloride (HCl) and this can cause isotopic fractionation. The Cr-EA technique thus expands the analytical possibilities for on-line hydrogen-isotope measurements of organic samples significantly. This method yielded reproducibility values (1-sigma) for δ(2)H measurements on water and caffeine samples of better than 1.0 and 0.5 mUr, respectively. To overcome handling problems with water as the principal calibration anchor for hydrogen isotopic measurements, we have employed an effective and simple strategy using reference waters or other liquids sealed in silver-tube segments. These crimped silver tubes can be employed in both the Cr-EA and TC/EA techniques. They simplify considerably the normalization of hydrogen-isotope measurement data to the VSMOW-SLAP (Vienna Standard Mean Ocean Water-Standard Light Antarctic Precipitation) scale, and their use improves accuracy of the data by eliminating evaporative loss and associated isotopic fractionation while handling water as a bulk sample. The calibration of organic samples, commonly having high δ(2)H values, will benefit from the availability of suitably (2)H-enriched reference waters, extending the VSMOW-SLAP scale above zero.

Isotopic disproportionation during hydrogen isotopic analysis of nitrogen-bearing organic compounds.

RATIONALE: High-precision hydrogen isotope ratio analysis of nitrogen-bearing organic materials using high-temperature conversion (HTC) techniques has proven troublesome in the past. Formation of reaction products other than molecular hydrogen (H(2)) has been suspected as a possible cause of incomplete H(2) yield and hydrogen isotopic fractionation. METHODS: The classical HTC reactor setup and a modified version including elemental chromium, both operated at temperatures in excess of 1400 °C, have been compared using a selection of nitrogen-bearing organic compounds, including caffeine. A focus of the experiments was to avoid or suppress hydrogen cyanide (HCN) formation and to reach quantitative H(2) yields. The technique also was optimized to provide acceptable sample throughput. RESULTS: The classical HTC reaction of a number of selected compounds exhibited H(2) yields from 60 to 90 %. Yields close to 100 % were measured for the experiments with the chromium-enhanced reactor. The δ(2)H values also were substantially different between the two types of experiments. For the majority of the compounds studied, a highly significant relationship was observed between the amount of missing H(2) and the number of nitrogen atoms in the molecules, suggesting the pyrolytic formation of HCN as a byproduct. A similar linear relationship was found between the amount of missing H(2) and the observed hydrogen isotopic result, reflecting isotopic fractionation. CONCLUSIONS: The classical HTC technique to produce H(2) from organic materials using high temperatures in the presence of glassy carbon is not suitable for nitrogen-bearing compounds. Adding chromium to the reaction zone improves the yield to 100 % in most cases. The initial formation of HCN is accompanied by a strong hydrogen isotope effect, with the observed hydrogen isotope results on H(2) being substantially shifted to more negative δ(2)H values. The reaction can be understood as an initial disproportionation leading to H(2) and HCN with the HCN-hydrogen systematically enriched in (2)H by more than 50 ‰. In the reaction of HCN with chromium, H(2) and chromium-containing solid residues are formed quantitatively.

Performance of the wet oxidation unit of the HPLC isotope ratio mass spectrometry system for halogenated compounds.

The performance of liquid chromatography-isotope ratio mass spectrometry (LC-IRMS) for polar halogenated compounds was evaluated. Oxidation capacity of the system was tested with halogenated acetic acids and halogenated aromatic compounds. Acetic acid (AA) was selected as a reference compound for complete oxidation and compared on the molar basis to the oxidation of other analytes. The isotope values were proofed with calibrated δ(13)C values obtained with an elemental analyzer (EA). Correct isotope values were obtained for mono- and dichlorinated, fluorinated, and tribrominated acetic acids and also for aniline, phenol, benzene, bromobenzene, chlorobenzene, 1,2-dichlorobenzene, 2,4,6-trichlorophenol, pentafluorophenol, and nitrobenzene. Incomplete oxidation of trichloroacetic acid (TCA) and trifluoroacetic acid (TFA) resulted in lower recovery compared to AA (37% and 24%, respectively) and in isotopic shift compared to values obtained with EA (TCA Δδ(13)C(EA/LC-IRMS) = 8.8‰, TFA Δδ(13)C(EA/LC-IRMS) = 6.0‰). Improvement of oxidation by longer reaction time in the reactor and increase in the concentration of sulfate radicals did not lead to complete combustion of TCA and TFA needed for δ(13)C analysis. To the best of our knowledge, this is the first time such highly chlorinated compounds were studied with the LC-IRMS system. This work provides information for method development of LC-IRMS methods for halogenated contaminants that are known as potential threats to public health and the environment.

Novel extraction methods and compound-specific isotope analysis of methoxychlor in environmental water and aquifer slurry samples.

Multi-element compound-specific stable isotope analysis (ME-CSIA) allows monitoring the environmental behavior and transformation of most common and persistent contaminants. Recent advancements in analytical techniques have extended the applicability of ME-CSIA to organic micropollutants, including pesticides. Nevertheless, the application of this methodology remains unexplored concerning harmful insecticides such as methoxychlor, a polar organochlorine pesticide usually detected in soil and groundwater. This study introduces methods for dual carbon and chlorine compound-specific stable isotope analysis (δ13C-CSIA and δ37Cl-CSIA) of both methoxychlor and its metabolite, methoxychlor olefin, with a sensitivity down to 10 and 100 mg/L, and a precision lower than 0.3 and 0.5 ‰ for carbon and chlorine CSIA, respectively. Additionally, three extraction and preconcentration techniques suitable for ME-CSIA of the target pesticides at environmentally relevant concentrations were also developed. Solid-phase extraction (SPE) and liquid-solid extraction (LSE) effectively extracted methoxychlor (107 ± 27 % and 87 ± 13 %, respectively) and its metabolite (91 ± 27 % and 106 ± 14 %, respectively) from water and aquifer slurry samples, respectively, with high accuracy (Δδ13C and Δδ37Cl ≤ ± 1 ‰). Combining CSIA with polar organic chemical integrative samplers (POCISs) for the extraction of methoxychlor and methoxychlor olefin from water samples resulted in insignificant fractionation for POCIS-CSIA (Δδ13C ≤ ± 1 ‰). A relevant sorption of methoxychlor was detected within the polyethersulfones membranes of the POCISs resulting in temporary carbon isotope fractionation depending on the sorbed mass fraction during the first deployment days. This highlights the critical role of the interactions of polar analytes with POCIS sorbents and membranes in the performance of this method. Altogether, this study proposes a proof of concept for ME-CSIA of methoxychlor and its metabolites, opening the door for future investigations of their sources and transformation processes in contaminated sites.

Dual C and Cl compound-specific isotope analysis and metagenomic insights into the degradation of the pesticide methoxychlor.

This study investigates the use of multi-element compound-specific isotope analysis (ME-CSIA) to monitor degradation processes of methoxychlor, a persistent organochlorine insecticide. Laboratory experiments examined the kinetics, release of transformation products, and carbon and chlorine isotope effects during methoxychlor degradation through alkaline hydrolysis, oxidation with alkaline-activated persulfate, and biotic reductive dechlorination. Results showed that hydrolysis and oxidation did not cause significant carbon and chlorine isotope fractionation, indicating that C-H rather than C-Cl bond cleavage was the rate-determining step. Conversely, biotic reductive dechlorination by a field-derived microcosm under strictly anoxic conditions displayed significant carbon (εC = -0.9 ± 0.3 ‰) and chlorine (εCl = -1.9 ± 1.0 ‰) isotope fractionation. Its corresponding calculated dual isotope slope (ΛC/Cl = 0.4 ± 0.1) and apparent kinetic isotope effects (AKIEC = 1.014 ± 0.005 and AKIECl = 1.006 ± 0.003) indicate a C-Cl bond cleavage as the rate-determining step, highlighting the difference with respect to the other studied degradation mechanisms. Changes in the microbial community diversity revealed that families such as Dojkabacteria, Anaerolineaceae, Dysgonomonadaceae, Bacteroidetes vadinHA17, Pseudomonadaceae, and Spirochaetaceae, may be potential agents of methoxychlor reductive dechlorination under anoxic conditions. This study advances the understanding of degradation mechanisms of methoxychlor and improves the ability to track its transformation in contaminated environments, including for the first time an isotopic perspective.

Vitamin B12 as a source of variability in isotope effects for chloroform biotransformation by Dehalobacter.

Carbon and chlorine isotope effects for biotransformation of chloroform by different microbes show significant variability. Reductive dehalogenases (RDase) enzymes contain different cobamides, affecting substrate preferences, growth yields, and dechlorination rates and extent. We investigate the role of cobamide type on carbon and chlorine isotopic signals observed during reductive dechlorination of chloroform by the RDase CfrA. Microcosm experiments with two subcultures of a Dehalobacter-containing culture expressing CfrA-one with exogenous cobamide (Vitamin B12, B12+) and one without (to drive native cobamide production)-resulted in a markedly smaller carbon isotope enrichment factor (εC, bulk) for B12- (-22.1 ± 1.9‰) compared to B12+ (-26.8 ± 3.2‰). Both cultures exhibited significant chlorine isotope fractionation, and although a lower εCl, bulk was observed for B12- (-6.17 ± 0.72‰) compared to B12+ (-6.86 ± 0.77‰) cultures, these values are not statistically different. Importantly, dual-isotope plots produced identical slopes of ΛCl/C (ΛCl/C, B12+ = 3.41 ± 0.15, ΛCl/C, B12- = 3.39 ± 0.15), suggesting the same reaction mechanism is involved in both experiments, independent of the lower cobamide bases. A nonisotopically fractionating masking effect may explain the smaller fractionations observed for the B12- containing culture.

Quantifying energy expenditure in Göttingen Minipigs with the 13C-bicarbonate method under basal and drug-treated conditions.

Effective treatments of obesity focusing on energy expenditure (EE) are needed. To evaluate future EE-modulating drug candidates, appropriate animal models and methods to assess EE are needed. This study aimed to evaluate the stable isotope 13C-bicarbonate method (13C-BM) for estimating EE in Göttingen minipigs under basal and drug-treated conditions. Four experiments (Expt.1-4) were conducted to assess: 1) the 13C-BM reproducibility using breath sample collection (n = 8), on two consecutive days, 2) the effect of two dose levels (5 and 10 mg/kg body weight (BW)) of the mitochondrial uncoupler dinitrophenol (DNP) in a crossover design (n = 8), 3) sampling method agreement; blood vs. exhaled air (n = 6) and 4) 13C-BM using constant isotope infusion compared with indirect calorimetry (IC) (n = 3). Results correlated significantly (p < 0.001) between days (Expt.1), with an average coefficient of variance of 5.4 ± 2.3%. Administration of 10 mg DNP/kg BW increased (p < 0.01) EE by 33.2 ± 6.4% (Expt.2). Results based on different sampling methods correlated significantly (p < 0.001) and EE increased after 10 mg DNP/kg BW (p < 0.05) in Expt.3. However, results based on blood sampling were significantly higher (p < 0.01) than those of exhaled air. No effect of DNP and significantly different EE results (p < 0.05) was observed in a limited number of animals, when constant isotope infusion and blood sampling was compared with IC (Expt.4). In conclusion, the 13C-BM is useful for investigating treatment effects on EE in minipigs. However, further validation under standardized conditions is needed to provide accurate estimates of the 13C recovery factor and respiratory quotient, both of decisive importance when using the 13C-BM.

Investigation of active site amino acid influence on carbon and chlorine isotope fractionation during reductive dechlorination.

Reductive dehalogenases (RDases) are corrinoid-dependent enzymes that reductively dehalogenate organohalides in respiratory processes. By comparing isotope effects in biotically catalyzed reactions to reference experiments with abiotic corrinoid catalysts, compound-specific isotope analysis (CSIA) has been shown to yield valuable insights into enzyme mechanisms and kinetics, including RDases. Here, we report isotopic fractionation (ε) during biotransformation of chloroform (CF) for carbon (εC = -1.52 ± 0.34‰) and chlorine (εCl = -1.84 ± 0.19‰), corresponding to a ΛC/Cl value of 1.13 ± 0.35. These results are highly suppressed compared to isotope effects observed both during CF biotransformation by another organism with a highly similar RDase (>95% sequence identity) at the amino acid level, and to those observed during abiotic dehalogenation of CF. Amino acid differences occur at four locations within the two different RDases' active sites, and this study examines whether these differences potentially affect the observed εC, εCl, and ΛC/Cl. Structural protein models approximating the locations of the residues elucidate possible controls on reaction mechanisms and/or substrate binding efficiency. These four locations are not conserved among other chloroalkane reducing RDases with high amino acid similarity (>90%), suggesting that these locations may be important in determining isotope fractionation within this homologous group of RDases.