Calibration bias of experimentally determined chlorine isotope enrichment factors: the need for a two-point calibration in compound-specific chlorine isotope analysis.

RATIONALE: The recent development of compound-specific online chlorine isotope analysis (37 Cl-CSIA) methods has fostered dual chlorine-carbon isotope studies to gain better insights into sources and environmental transformation reactions of chlorinated ethenes. One-point and two-point calibration schemes are currently used to convert raw data to the international δ37 ClSMOC scale, but a critical evaluation of best practices to arrive at reliable δ37 ClSMOC signatures and enrichment factors was missing and is presented here. METHODS: Aqueous solutions of neat perchloroethylene and trichloroethylene (TCE) and aqueous samples from a TCE biodegradation experiment with pure cultures of Desulfitobacterium hafniense Y51 were analysed for their chlorine isotope ratios using GC/qMS and GC/IRMS. The δ37 ClSMOC values were obtained using one-point and two-point calibration schemes. Chlorine isotope enrichment factors, εCl , were calculated using both approaches and the corresponding bias of δ37 ClSMOC values introduced by the different types of calibration was determined. RESULTS: Different calibration methods resulted in significant differences (up to 30%) in both δ37 Cl signatures and εCl values. CONCLUSIONS: Our results demonstrate that a two-point calibration together with comprehensive information on reference materials is indispensable and should become standard practice for reliable 37 Cl-CSIA of organic compounds. Copyright © 2016 John Wiley & Sons, Ltd.

Resiliency of Stable Isotope Fractionation (δ(13)C and δ(37)Cl) of Trichloroethene to Bacterial Growth Physiology and Expression of Key Enzymes.

Quantification of in situ (bio)degradation using compound-specific isotope analysis requires a known and constant isotope enrichment factor (ε). Because reported isotope enrichment factors for microbial dehalogenation of chlorinated ethenes vary considerably we studied the potential effects of metabolic adaptation to TCE respiration on isotope fractionation (δ(13)C and δ(37)Cl) using a model organism (Desulfitobacterium hafniesne Y51), which only has one reductive dehalogenase (PceA). Cells grown on TCE for the first time showed exponential growth until 10(9) cells/mL. During exponential growth, the cell-normalized amount of PceA enzyme increased steadily in the presence of TCE (up to 21 pceA transcripts per cell) but not with alternative substrates (<1 pceA transcript per cell). Cultures initially transferred or subcultivated on TCE showed very similar isotope fractionation, both for carbon (εcarbon: -8.6‰ ± 0.3‰ or -8.8‰ ± 0.2‰) and chlorine (εchlorine: -2.7‰ ± 0.3‰) with little variation (0.7‰) for the different experimental conditions. Thus, TCE isotope fractionation by D. hafniense strain Y51 was affected by neither growth phase, pceA transcription, or translation, nor by PceA content per cell, suggesting that transport limitations did not affect isotope fractionation. Previously reported variable ε values for other organohalide-respiring bacteria might thus be attributed to different expression levels of their multiple reductive dehalogenases.

Electron transfer between iron minerals and quinones: estimating the reduction potential of the Fe(II)-goethite surface from AQDS speciation.

Redox reactions at iron mineral surfaces play an important role in controlling biogeochemical processes of natural porous media such as sediments, soils and aquifers, especially in the presence of recurrent variations in redox conditions. Ferrous iron associated with iron mineral phases forms highly reactive species and is regarded as a key factor in determining pathways, rates, and extent of chemically and microbially driven electron transfer processes across the iron mineral-water interface. Due to their transient nature and heterogeneity a detailed characterization of such surface bound Fe(II) species in terms of redox potential is still missing. To this end, we used the nonsorbing anthraquinone-2,6-disulfonate (AQDS) as a redox probe and studied the thermodynamics of its redox reactions in heterogeneous iron systems, namely goethite-Fe(II). Our results provide a thermodynamic basis for and are consistent with earlier observations on the ability of AQDS to "shuttle" electrons between microbes and iron oxide minerals. On the basis of equilibrium AQDS speciation we reported for the first time robust reduction potential measurements of reactive iron species present at goethite in aqueous systems (EH,Fe-GT ≈ -170 mV). Due to the high redox buffer intensity of heterogeneous mixed valent iron systems, this value might be characteristic for many iron-reducing environments in the subsurface at circumneutral pH. Our results corroborate the picture of a dynamic remodelling of Fe(II)/Fe(III) surface sites at goethite in response to oxidation/reduction events. As quinones play an essential role in the electron transport systems of microbes, the proposed method can be considered as a biomimetic approach to determine "effective" biogeochemical reduction potentials in heterogeneous iron systems.

Reductive dechlorination of TCE by chemical model systems in comparison to dehalogenating bacteria: insights from dual element isotope analysis (13C/12C, 37Cl/35Cl).

Chloroethenes like trichloroethene (TCE) are prevalent environmental contaminants, which may be degraded through reductive dechlorination. Chemical models such as cobalamine (vitamin B12) and its simplified analogue cobaloxime have served to mimic microbial reductive dechlorination. To test whether in vitro and in vivo mechanisms agree, we combined carbon and chlorine isotope measurements of TCE. Degradation-associated enrichment factors ε(carbon) and ε(chlorine) (i.e., molecular-average isotope effects) were -12.2‰ ± 0.5‰ and -3.6‰ ± 0.1‰ with Geobacter lovleyi strain SZ; -9.1‰ ± 0.6‰ and -2.7‰ ± 0.6‰ with Desulfitobacterium hafniense Y51; -16.1‰ ± 0.9‰ and -4.0‰ ± 0.2‰ with the enzymatic cofactor cobalamin; -21.3‰ ± 0.5‰ and -3.5‰ ± 0.1‰ with cobaloxime. Dual element isotope slopes m = Δδ(13)C/ Δδ(37)Cl ≈ ε(carbon)/ε(chlorine) of TCE showed strong agreement between biotransformations (3.4 to 3.8) and cobalamin (3.9), but differed markedly for cobaloxime (6.1). These results (i) suggest a similar biodegradation mechanism despite different microbial strains, (ii) indicate that transformation with isolated cobalamin resembles in vivo transformation and (iii) suggest a different mechanism with cobaloxime. This model reactant should therefore be used with caution. Our results demonstrate the power of two-dimensional isotope analyses to characterize and distinguish between reaction mechanisms in whole cell experiments and in vitro model systems.

Compound-specific chlorine isotope analysis: a comparison of gas chromatography/isotope ratio mass spectrometry and gas chromatography/quadrupole mass spectrometry methods in an interlaboratory study.

Chlorine isotope analysis of chlorinated hydrocarbons like trichloroethylene (TCE) is of emerging demand because these species are important environmental pollutants. Continuous flow analysis of noncombusted TCE molecules, either by gas chromatography/isotope ratio mass spectrometry (GC/IRMS) or by GC/quadrupole mass spectrometry (GC/qMS), was recently brought forward as innovative analytical solution. Despite early implementations, a benchmark for routine applications has been missing. This study systematically compared the performance of GC/qMS versus GC/IRMS in six laboratories involving eight different instruments (GC/IRMS, Isoprime and Thermo MAT-253; GC/qMS, Agilent 5973N, two Agilent 5975C, two Thermo DSQII, and one Thermo DSQI). Calibrations of (37)Cl/(35)Cl instrument data against the international SMOC scale (Standard Mean Ocean Chloride) deviated between instruments and over time. Therefore, at least two calibration standards are required to obtain true differences between samples. Amount dependency of δ(37)Cl was pronounced for some instruments, but could be eliminated by corrections, or by adjusting amplitudes of standards and samples. Precision decreased in the order GC/IRMS (1σ ≈ 0.1‰), to GC/qMS (1σ ≈ 0.2-0.5‰ for Agilent GC/qMS and 1σ ≈ 0.2-0.9‰ for Thermo GC/qMS). Nonetheless, δ(37)Cl values between laboratories showed good agreement when the same external standards were used. These results lend confidence to the methods and may serve as a benchmark for future applications.

Chlorine isotope analysis of organic contaminants using GC-qMS: method optimization and comparison of different evaluation schemes.

Compound-specific online chlorine isotope analysis of chlorinated hydrocarbons was evaluated and validated using gas chromatography coupled to a regular quadrupole mass spectrometer (GC-qMS). This technique avoids tedious off-line sample pretreatments, but requires mathematical data analysis to derive chlorine isotope ratios from mass spectra. We compared existing evaluation schemes to calculate chlorine isotope ratios with those that we modified or newly proposed. We also tested systematically important experimental procedures such as external vs. internal referencing schemes, and instrumental settings including split ratio, ionization energy, and dwell times. To this end, headspace samples of tetrachloroethene (PCE), trichloroethene (TCE), and cis-dichloroethene (cDCE) at aqueous concentrations in the range of 20-500 µg/L (amount on-column range: 3.2-115 pmol) were analyzed using GC-qMS. The results (³7Cl/³5Cl ratios) showed satisfying to good precisions with relative standard deviations (n = 5) between 0.4‰ and 2.1‰. However, we found that the achievable precision considerably varies depending on the applied data evaluation scheme, the instrumental settings, and the analyte. A systematic evaluation of these factors allowed us to optimize the GC-qMS technique to determine chlorine isotope ratios of chlorinated organic contaminants.

Effects of Zwitterionic buffers on sorption of ferrous iron at goethite and its oxidation by CCl4.

A major factor which controls sorption and oxidation of Fe(II) at the mineral-water interface is pH, hence buffers are commonly used to control pH in experimental studies. Here, we examined the effects of widely used organic buffers (3-morpholinopropane-1-sulfonic acid (MOPS) and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)) on Fe(II) uptake and oxidation by CCl(4) in aqueous suspensions of goethite. Significant sorption of these zwitterionic buffers occurred only at Fe(II)-loaded goethite but not at native goethite. The addition of MOPS and HEPES caused substantial release of Fe(II) from goethite, retarded the oxidation of surface-bound Fe(II) by CCl(4) and changed the reaction pathway as indicated by lower yields of CHCl(3). To explore electrostatic and steric contributions of MOPS and HEPES to the observed phenomena we studied sorption and competitive effects of model sorbates (Ca(2+), sulfonates) which suggest the formation of a complex between surface-bound Fe(II) and MOPS or HEPES. Our study shows for the first time that these frequently used zwitterionic organic buffers may interfere significantly with the surface chemistry and thus with redox reactions of Fe(II) at goethite. Hence, kinetic or mechanistic information obtained in such systems requires careful interpretation.

Organic matter preservation in the sediment of an acidic mining lake.

Sustainable management of acidic mining lakes requires knowledge on the origin and reactivity of its sedimentary organic matter. We identified different pools of organic matter (OM) in the Fe-rich sediment (up to 35 wt %) of an acidic (pH 2.8) mining lake using delta13C-signals, C/N ratios, and the markers alkanes, lignin-derived phenols, and benzenepolycarboxylic acids (BPCA). Additionally, a density fractionation was applied to each sediment layer. Three fractions, aquatic (AOM), terrestrial (TOM), and lignite-derived (LOM) organic matter, were discriminated, of which AOM comprises only a small fraction, with a minimum at the sediment bottom. The terrestrial contribution to sedimentary OM is higher than that of AOM but still low throughout the sediment core, whereas lignite-derived OM constitutes the major C-fraction, even in the upper sediment layers. The size of the carbon pools was quantified with a mass-balance approach, in which the BPCA content was utilized as an estimate for the lignite fraction in combination with the delta13C-signals of the three C fractions. The largest amount of OM was found in the heaviest (>2.4 g cm3) of the three density fractions of the two upper sediment layers, which implies strong interaction with iron hydroxides. Comparisons with C-oxidation rates revealed that besides the refractory origin of the OM, sorptive preservation by solid iron phases controls C-reactivity in the sediment and, hence, the internal neutralization capacity of the lake system.