Dual C-Cl Isotope Analysis for Characterizing the Reductive Dechlorination of α- and γ-Hexachlorocyclohexane by Two Dehalococcoides mccartyi Strains and an Enrichment Culture.

Hexachlorocyclohexanes (HCHs) are persistent organic contaminants that threaten human health. Microbial reductive dehalogenation is one of the most important attenuation processes in contaminated environments. This study investigated carbon and chlorine isotope fractionation of α- and γ-HCH during the reductive dehalogenation by three anaerobic cultures. The presence of tetrachlorocyclohexene (TeCCH) indicated that reductive dichloroelimination was the first step of bond cleavage. Isotope enrichment factors (εC and εCl) were derived from the transformation of γ-HCH (εC, from -4.0 ± 0.5 to -4.4 ± 0.6 ‰; εCl, from -2.9 ± 0.4 to -3.3 ± 0.4 ‰) and α-HCH (εC, from -2.4 ± 0.2 to -3.0 ± 0.4 ‰; εCl, from -1.4 ± 0.3 to -1.8 ± 0.2 ‰). During α-HCH transformation, no enantioselectivity was observed, and similar εc values were obtained for both enantiomers. The correlation of 13C and 37Cl fractionation (Λ = Δδ13C/Δδ37Cl ≈ εC/εCl) of γ-HCH (from 1.1 ± 0.3 to 1.2 ± 0.1) indicates similar bond cleavage during the reductive dichloroelimination by the three cultures, similar to α-HCH (1.7 ± 0.2 to 2.0 ± 0.3). The different isotope fractionation patterns during reductive dichloroelimination and dehydrochlorination indicates that dual-element stable isotope analysis can potentially be used to evaluate HCH transformation pathways at contaminated field sites.

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.

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.

A concept for studying the transformation reaction of hexachlorocyclohexanes in food webs using multi-element compound-specific isotope analysis.

An analytical concept using stable isotope fractionation for analyzing persistent organic pollutants (POPs) in food webs was developed and tested. We have evaluated methods for the extraction and clean-up of hexachlorocyclohexane isomers (HCHs) as the model compounds of POPs from water, soil, plant, milk, fish oil and pork liver in order to study the reactive transport processes of HCHs in food webs using multi-element compound-specific isotope analysis (CSIA). The extraction and clean-up methods were evaluated for recovery efficiency and isotope effects. The precision and accuracy for carbon, hydrogen and chlorine isotope analysis were within the analytical precision of ±0.5‰, ±5‰ and ±0.3‰, respectively. The method was applied for stable isotope analysis of HCHs in possible food webs from soil to plants, and to animals. Isotope compositions of HCHs in cow/buffalo milk and dung, wild animal livers and seal blubber were obtained and compared to the sources of HCHs. The magnitude of isotope enrichment demonstrated the potential of CSIA for analyzing reactive transport processes of HCHs in the food web. The concept using multi-element stable isotope analysis can be applied for source identification, characterization of degradation mechanisms, and particularly contaminant accumulation in the food web, which demonstrates the potential in new scientific areas for CSIA.

Triple-element compound-specific stable isotope analysis of 1,2-dichloroethane for characterization of the underlying dehalogenation reaction in two Dehalococcoides mccartyi strains.

Chlorinated ethanes belong to the most common groundwater and soil contaminants. Of these, 1,2-dichloroethane (1,2-DCA) is a man-made, persistent and toxic contaminant, released due to improper waste treatment at versatile production sites. This study investigated the anaerobic transformation of 1,2-DCA by Dehalococcoides mccartyi strain 195 and strain BTF08 using triple-element compound-specific stable isotope analysis of carbon, chlorine and hydrogen for the first time. Isotope fractionation patterns for carbon (εCBTF08 = -28.4 ± 3.7‰; εC195 = -30.9 ± 3.6‰) and chlorine (εClBTF08 = -4.6 ± 0.7‰; εCl195 = -4.2 ± 0.5‰) within both investigated D. mccartyi strains, as well as the dual-element analysis (ΛBTF08 = 6.9 ± 1.2; Λ195 = 7.1 ± 0.2), supported identical reaction mechanisms for dehalogenation of 1,2-DCA. Hydrogen isotope fractionation analysis revealed dihaloelimination as prevalent reaction mechanism. Vinyl chloride as major intermediate could be excluded by performing the experiment in deuterated aqueous media. Furthermore, evaluation of the derived apparent kinetic isotope effects (AKIECBTF08 = 1.029/AKIEC195 = 1.031; AKIEClBTF08 = 1.005/AKIECl195 = 1.004) pointed towards simultaneous abstraction of both involved chlorine-substituents in a concerted matter. It was shown that D. mccartyi strain BTF08 and strain 195 are capable of complete, direct dihaloelimination of 1,2-DCA to ethene.

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.

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.

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.

Substrate hydrophobicity and cell composition influence the extent of rate limitation and masking of isotope fractionation during microbial reductive dehalogenation of chlorinated ethenes.

This study investigated the effect of intracellular microscale mass transfer on microbial carbon isotope fractionation of tetrachloroethene (PCE) and trichloroethene (TCE). Significantly stronger isotope fractionation was observed for crude extracts vs intact cells of Sulfurospirillum multivorans, Geobacter lovleyi, Desulfuromonas michiganensis, Desulfitobacterium hafniense strain PCE-S, and Dehalobacter restrictus. Furthermore, carbon stable isotope fractionation was stronger for microorganisms with a Gram-positive cell envelope compared to those with a Gram-negative cell envelope. Significant differences were observed between model organisms in cellular sorption capacity for PCE (S. multivorans-K(d-PCE) = 0.42-0.51 L g(-1); D. hafniense-K(d-PCE) = 0.13 L g(-1)), as well as in envelope hydrophobicity (S. multivorans 33.0° to 72.2°; D. hafniense 59.1° to 60.8°) when previously cultivated with fumarate or PCE as electron acceptor, but not for TCE. Cell envelope properties and the tetrachloroethene reductive dehalogenase (PceA-RDase) localization did not result in significant effects on observed isotope fractionation of TCE. For PCE, however, systematic masking of isotope effects as a result of microscale mass transfer limitation at microbial membranes was observed, with carbon isotope enrichment factors of -2.2‰, -1.5 to -1.6‰, and -1.0‰ (CI95% < ± 0.2‰) for no membrane, hydrophilic outer membrane, and outer + cytoplasmic membrane, respectively. Conclusively, rate-limiting mass transfer barriers were (a) the outer membrane or cell wall and (b) the cytoplasmic membrane in case of a cytoplasmic location of the RDase enzyme. Overall, our results indicate that masking of isotope fractionation is determined by (1) hydrophobicity of the degraded compound, (2) properties of the cell envelope, and (3) the localization of the reacting enzyme.

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‰.

Combined C and Cl isotope effects indicate differences between corrinoids and enzyme (Sulfurospirillum multivorans PceA) in reductive dehalogenation of tetrachloroethene, but not trichloroethene.

The role of the corrinoid cofactor in reductive dehalogenation catalysis by tetrachloroethene reductive dehalogenase (PceA) of Sulfurospirillum multivorans was investigated using isotope analysis of carbon and chlorine. Crude extracts containing PceA--harboring either a native norpseudo-B12 or the alternative nor-B12 cofactor--were applied for dehalogenation of tetrachloroethene (PCE) or trichloroethene (TCE), and compared to abiotic dehalogenation with the respective purified corrinoids (norpseudovitamin B12 and norvitamin B12), as well as several commercially available cobalamins and cobinamide. Dehalogenation of TCE resulted in a similar extent of C and Cl isotope fractionation, and in similar dual-element isotope slopes (εC/εCl) of 5.0-5.3 for PceA enzyme and 3.7-4.5 for the corrinoids. Both observations support an identical reaction mechanism. For PCE, in contrast, observed C and Cl isotope fractionation was smaller in enzymatic dehalogenation, and dual-element isotope slopes (2.2-2.8) were distinctly different compared to dehalogenation mediated by corrinoids (4.6-7.0). Remarkably, εC/εCl of PCE depended in addition on the corrinoid type: εC/εCl values of 4.6 and 5.0 for vitamin B12 and norvitamin B12 were significantly different compared to values of 6.9 and 7.0 for norpseudovitamin B12 and dicyanocobinamide. Our results therefore suggest mechanistic and/or kinetic differences in catalytic PCE dehalogenation by enzymes and different corrinoids, whereas such differences were not observed for TCE.