Anat Bernstein

Current challenges in compound-specific stable isotope analysis of environmental organic contaminants

AbstractCompound-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. FigureCompound-specific isotope analysis of environmental contaminants: chromatographic separation is followed by online conversion to a suitable measurement gas (M) and subsequent isotope ratio mass spectrometry. Current challenges in the field concern the analysis of multiple elements (C, H, N, O, Cl) in polar compounds, at low concentrations and in the presence of matrix interferences. An urgent need exists for contaminant-specific reference materials.

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.

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.

Chlorine Isotope Effects from Isotope Ratio Mass Spectrometry Suggest Intramolecular C-Cl Bond Competition in Trichloroethene (TCE) Reductive Dehalogenation

Chlorinated ethenes are prevalent groundwater contaminants. To better constrain (bio)chemical reaction mechanisms of reductive dechlorination, the position-specificity of reductive trichloroethene (TCE) dehalogenation was investigated. Selective biotransformation reactions (i) of tetrachloroethene (PCE) to TCE in cultures of Desulfitobacterium sp. strain Viet1; and (ii) of TCE to cis-1,2-dichloroethene (cis-DCE) in cultures of Geobacter lovleyi strain SZ were investigated. Compound-average carbon isotope effects were −19.0‰ ± 0.9‰ (PCE) and −12.2‰ ± 1.0‰ (TCE) (95% confidence intervals). Using instrumental advances in chlorine isotope analysis by continuous flow isotope ratio mass spectrometry, compound-average chorine isotope effects were measured for PCE (−5.0‰ ± 0.1‰) and TCE (−3.6‰ ± 0.2‰). In addition, position-specific kinetic chlorine isotope effects were determined from fits of reactant and product isotope ratios. In PCE biodegradation, primary chlorine isotope effects were substantially larger (by −16.3‰ ± 1.4‰ (standard error)) than secondary. In TCE biodegradation, in contrast, the product cis-DCE reflected an average isotope effect of −2.4‰ ± 0.3‰ and the product chloride an isotope effect of −6.5‰ ± 2.5‰, in the original positions of TCE from which the products were formed (95% confidence intervals). A greater difference would be expected for a position-specific reaction (chloride would exclusively reflect a primary isotope effect). These results therefore suggest that both vicinal chlorine substituents of TCE were reactive (intramolecular competition). This finding puts new constraints on mechanistic scenarios and favours either nucleophilic addition by Co(I) or single electron transfer as reductive dehalogenation mechanisms.

Small and Reproducible Isotope Effects during Methylation with Trimethylsulfonium Hydroxide (TMSH): A Convenient Derivatization Method for Isotope Analysis of Negatively Charged Molecules

Negatively charged analytes must be derivatized prior to gas chromatography-isotope ratio mass spectrometry (GC-IRMS), with stringent control of isotope fractionation. Current methods require offline sample preparation. This study tests for the first time trimethylsulfonium hydroxide (TMSH) as online methylation agent prior to isotope analysis, addressing the herbicides bentazone and MCPA. Fully automated derivatization was achieved in a temperature-programmable GC injector, where reactants were injected into a packed liner, solvents were removed by split flow, and subsequent flash heating triggered the derivatization, thereby transferring derivatives onto the chromatographic column. Stoichiometric addition of TMSH resulted in complete conversion giving accurate and reproducible nitrogen isotope values of bentazone. In contrast, reproducible carbon isotope analysis required TMSH in > or = 250-fold excess. Contrary to expectations, delta(13)C values became more negative at smaller TMSH excess. This indicates that elevated methyl group concentrations in the pore space of the injection liner facilitated close-to-equilibrium rather than kinetic isotope fractionation resulting in reproducible derivatization conditions. delta(13)C results under these conditions compared favorably with liquid chromatography-IRMS: low standard deviations (0.3 per thousand for GC-IRMS, 0.1 per thousand for LC-IRMS) and a comparable offset of 1 per thousand compared to elemental analyzer-IRMS demonstrate that both methods represent expedient ways for online isotope analysis of anionic target compounds.

Quantifying RDX biodegradation in groundwater using δ15N isotope analysis

Isotope analysis was used to examine the extent of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) biodegradation in groundwater along a ca. 1.35-km contamination plume. Biodegradation was proposed as a natural attenuating remediation method for the contaminated aquifer. By isotope analysis of RDX, the extent of biodegradation was found to reach up to 99.5% of the initial mass at a distance of 1.15-1.35km down gradient from the contamination sources. A range of first-order biodegradation rates was calculated based on the degradation extents, with average half-life values ranging between 4.4 and 12.8years for RDX biodegradation in the upper 15m of the aquifer, assuming purely aerobic biodegradation, and between 10.9 and 31.2years, assuming purely anaerobic biodegradation. Based on the geochemical data, an aerobic biodegradation pathway was suggested as the dominant attenuation process at the site. The calculated biodegradation rate was correlated with depth, showing decreasing degradation rates in deeper groundwater layers. Exceptionally low first-order kinetic constants were found in a borehole penetrating the bottom of the aquifer, with half life ranging between 85.0 to 161.5years, assuming purely aerobic biodegradation, and between 207.5 and 394.3years, assuming purely anaerobic biodegradation. The study showed that stable isotope fractionation analysis is a suitable tool to detect biodegradation of RDX in the environment. Our findings clearly indicated that RDX is naturally biodegraded in the contaminated aquifer. To the best of our knowledge, this is the first reported use of RDX isotope analysis to quantify its biodegradation in contaminated aquifers.

Kinetic bromine isotope effect: example from the microbial debromination of brominated phenols.

The increasing use of kinetic isotope effects for environmental studies has motivated the development of new compound-specific isotope analysis techniques for emerging pollutants. Recently, high-precision bromine isotope analysis in individual brominated organic compounds was proposed, by the coupling of gas chromatography to a multi-collector inductively coupled plasma mass spectrometer using strontium as an external spike for instrumental bias correction. The present study, for the first time, demonstrates an application of this technique for determining bromine kinetic isotope effects during biological reaction, focusing on the reductive debromination of brominated phenols under anaerobic conditions. Results show bromine isotope enrichment factors (ε) of −0.76 ± 0.08, −0.46 ± 0.19, and −0.20 ± 0.06 ‰ for the debromination of 4-bromophenol, 2,4-dibromophenol, and 2,4,6-tribromophenol, respectively. These values are rather low, yet still high enough to be obtained with satisfying certainty. This further implies that the analytical method may be also appropriate for future environmental applications.

Biodegradation of the Explosives TNT, RDX and HMX

In the early twentieth century, more than 60 highly explosive compounds were developed and synthesized for military and civilian use. Of these, the most widely used explosives in the world are probably hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), 2,4,6-trinitrotoluene (TNT) and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX).

Insight on RDX degradation mechanism by Rhodococcus strains using 13C and 15N kinetic isotope effects.

The explosive Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is known to be degraded aerobically by various isolates of the Rhodococcus species, with denitration being the key step, mediated by Cytochrome P450. Our study aimed at gaining insight into the RDX degradation mechanism by Rhodococcus species and comparing isotope effects associated with RDX degradation by distinct Rhodococcus strains. For these purposes, enrichment in (13)C and (15)N isotopes throughout RDX denitration was studied for three distinct Rhodococcus strains, isolated from soil and groundwater in an RDX-contaminated site. The observable (15)N enrichment throughout the reaction, together with minor (13)C enrichment, suggests that N-N bond cleavage is likely to be the key rate-limiting step in the reaction. The similarity in the kinetic (15)N isotope effect between the three tested strains suggests that either isotope-masking effects are negligible, or are of a similar extent for all tested strains. The lack of variability in the kinetic (15)N isotope effect allows the interpretation of environmental studies with greater confidence.

Simple digestion procedure followed by the azomethine-H method for accurate boron analysis and discrimination between its fractions in wastewater and soils

Excess boron is a growing environmental problem. It often affects agricultural yields, where reuse of wastewater for irrigation is practiced. This problem raises the need for reliable, simple and economical methods to monitor boron concentrations in wastewater and soil extracts. One such method, the commonly used azomethine-H spectrophotometric method, suffers from color interference, originating from high concentrations of dissolved organic matter, when applied to many wastewater and agricultural soil extracts. Moreover, this method only quantifies free dissolved boron and lacks the ability to quantify boron that is adsorbed to either the dissolved organic matter or suspended solids that are present in the sample. This work suggests a modification of the standard azomethine-H method, in which the solution is digested with potassium persulfate prior to the standard procedure. We show that this pretreatment can overcome the color interference and lead to highly accurate and precise boron analyses in wastewater. In soil extracts, the boron concentrations obtained using the suggested procedure were better correlated to inductively coupled plasma (ICP) spectrometry results than those measured by the standard method, because whereas the standard method quantifies the free dissolved boron only, the modified method, like the ICP method, quantifies the total dissolved boron in the sample. Thus, the suggested modification can be used to quantify the respective distributions of free dissolved boron, boron adsorbed to dissolved organic matter and boron adsorbed to suspended solids in soil extracts and water samples.