Anal Bioanal Chem (2013) 405:2825–2831 DOI 10.1007/s00216-013-6745-0 RAPID COMMUNICATION Enantioselective stable isotope analysis (ESIA) of polar herbicides Michael P. Maier & Shiran Qiu & Martin Elsner Received: 3 December 2012 / Revised: 7 January 2013 / Accepted: 13 January 2013 / Published online: 3 February 2013 # Springer-Verlag Berlin Heidelberg 2013 Abstract Assessing the environmental fate of chiral micropollutants such as herbicides is challenging. The complexity of aquatic systems often makes it difficult to obtain hydraulic mass balances, which is a prerequisite when assessing degradation based on concentration data. Elegant alternatives are concentration-independent approaches like compoundspecific isotope analysis or enantiospecific concentration analysis. Both detect degradation-induced changes from ratios of molecular species, either isotopologues or enantiomers. A combination of both—enantioselective stable isotope analysis (ESIA)—provides information on 13C/12C ratios for each enantiomer separately. Recently, Badea et al. demonstrated for the first time ESIA for the insecticide α-hexachlorocyclohexane. The present study enlarges the applicability of ESIA to polar herbicides such as phenoxy acids: 4-CPP ((RS)-2-(4-chlorophenoxy)-propionic acid), mecoprop (2-(4-chloro-2-methylphenoxy)-propionic acid), and dichlorprop (2-(2,4dichlorophenoxy)-propionic acid). Enantioselective gas chromatography–isotope ratio mass spectrometry was accomplished with derivatization prior to analysis. Precise carbon isotope analysis (2σ≤0.5‰) was obtained with ≥7 ng C on column. Microbial degradation of dichlorprop, 2-(2,4-dichlorophenoxy)-propionic acid by Delftia acidovorans MC1 showed pronounced enantiomer fractionation, but no isotope fractionation. In contrast, Badea et al. observed isotope fractionation, but no enantiomeric fractionation. Hence, the two lines of evidence appear to complement each other. They may provide enhanced insight when combined as ESIA. Published in the topical collection Isotope Ratio Measurements: New Developments and Applications with guest editors Klaus G. Heumann and Torsten C. Schmidt. M. P. Maier : S. Qiu : M. Elsner (*) Helmholtz Zentrum München, Institute of Groundwater Ecology, Ingolstädter Landstr. 1, Neuherberg 85764, Germany e-mail: martin.elsner@helmholtz-muenchen.de Keywords Chiral micropollutants . Pesticides . Derivatization . Isotope fractionation . Herbicide . BF3 Introduction Herbicides can help to increase crop yields by killing weeds and pests. However, after application, their residues end up as micropollutants in aquatic ecosystems [1]. Since they have by definition a biological impact, they pose a potential threat to human and environmental health. It is therefore essential to understand how these compounds behave in complex environmental systems. This is a particular challenge for chiral micropollutants, where degradation behavior and toxicity often differ between single enantiomers [2]. However, in aquatic systems such as surface or ground waters, the interpretation of measured concentrations relies on the possibility to obtain a closed hydraulic mass balance. Without this hydraulic information, it is otherwise not possible to distinguish degradation from dilution processes. There are two elegant approaches that circumvent this difficulty by analyzing ratios that are inherent to the target molecules. One approach is enantiospecific concentration analysis. Here, the concentration of the individual enantiomers is analyzed and the enantiomer fraction (EF) is expressed as the ratio between one enantiomer and the sum of both enantiomers (e.g., EFR =R/[R+S]). Nearly all commercial products have either an EF of 0.5 (racemates) or 0 (enantiopure products). During biological processes, this ratio can be shifted. The reason is that many enzymes that are involved in transformation reactions have an enantioselective preference and favor one enantiomer over the other [2]. Hence, shifts in the EF can be a very useful tool to demonstrate that chiral compounds are degraded in complex environmental systems, particularly if other approaches (e.g., mass balances, metabolite analysis) fail. 2826 Another promising approach to investigate sources and degradation of micropollutants in complex environmental systems is compound-specific isotope analysis (CSIA). Several studies have demonstrated that changes in the isotope ratio of a compound can be used to investigate degradation of xenobiotics [3–7]. The underlying principle is that (bio-)chemical reactions are commonly associated with a kinetic isotope effect, which causes a fractionation of heavy and light isotopes. Using this approach, isotope fractionation of one element can be used to quantify degradation [8, 9]. If several elements are analyzed, even the mechanisms and pathways of transformation processes can be elucidated [6, 10–12] as demonstrated recently for selected pesticides [13–16]. CSIA and EF analysis work on the same principle that degradation can be detected because one species becomes enriched relative to the other. Despite this similarity, both approaches have been applied together in only one study. Recently Badea et al. presented a first example of enantioselective isotope analysis (ESIA) applied to the insecticide α-hexachlorocyclohexane [17]. In a biodegradation experiment, they observed isotope fractionation, but no enantiomer fractionation so that the line of evidence brought forward in this particular example was similar as for conventional CSIA. The aim of this study was to enlarge the applicability of ESIA to a broader range of substances, including polar compounds that are more challenging to analyze via gas chromatography isotope ratio mass spectrometry (GC-IRMS) and for a case of biodegradation where enantiomer fractionation is well established to occur. To this end, an enantiospecific stable isotope method was developed for three polar herbicides, (RS)-2-(4chlorophenoxy)-propionic acid (4-CPP), mecoprop, 2(4-chloro-2-methylphenoxy)propionic acid (MCPP), and dichlorprop, 2-(2,4-dichlorophenoxy)-propionic acid (DCPP) to provide a tool for investigating their fate in environmental systems. For each compound, the precision of the method was tested as well as the limits of precise isotope analysis. Subsequently, the method was applied to investigate microbial degradation, using the enantioselective strain Delftia acidovorans MC1 and the herbicide DCPP as a model compound. Experimental Compounds and chemicals Acetonitrile (1.25 % acetic acid) and n-hexane were purchased from Carl Roth (Karlsruhe, Germany) and had LCMS grade (purity >0.99). Acetic acid (1.25 %) was purchased from Merck (Darmstadt, Germany). MCPP (CAS M.P. Maier et al. RN 7085-19-0) and BF3 (10 % in methanol) were purchased from Sigma-Aldrich (St. Louis, USA). 4-CPP (CAS RN 330739-9) was purchased from Aldrich Chemistry (Milwaukee, USA) and DCPP propionic acid (CAS RN 120-36-5) and RDCPP from Dr. Ehrenstorfer GmbH (Augsburg, Germany). Milli-Q water was generated with a Millipore Advantage A10 system (Millipore, Molsheim, France). (S)-2-(4-chlorophenoxy)-propionic acid methyl ester (S-4-CPP) was synthesized according to the procedure by Nittoli et al. [18] and as described in Milosevic et al. [19]. Derivatization with BF3 Prior to analysis, the polar carboxyl group of the analytes was methylated in a similar way as described by Chivall et al. [20] for fatty acids. To this end, sterile-filtered samples or standards were evaporated in 2-mL amber glass vials to dryness under a gentle stream of N2. Four hundred microliters of BF3 (10 % in methanol) was added, the vials were sealed with screw caps equipped with PTFE seals, and incubated for 1 h at 40 °C. After cooling, the remaining reactant was quenched with 400 μL Milli-Q water and the methylated analytes were extracted three times with 500 μL n-hexane and transferred to a new vial. If necessary, the extracted phase was reduced to 200 μL under a gentle stream of N2 to increase the concentration for isotope analysis. The toxic aqueous phase containing HF was disposed in a canister of 2 M NaOH to quench its toxicity. Isotope analysis Depending on the concentrations of extracts, between 1 and 4 μL were injected splitless at 230 °C. The gas chromatograph (TRACE GC Ultra Gas Chromatograph, Thermo Fisher Scientific, Milan, Italy) was equipped with a β-6TBDM column (50 m × 0.25 mm, 0.25 μm film; Macherey & Nagel, Düren, Germany). The column was operated with a constant flow velocity of 29 cm s −1 corresponding to a He carrier flow rate of 1.4 mlmin−1 (GC-IRMS) and 1.0 mlmin−1 (GC-TOF-MS). Initial oven temperature was 80 °C (1 min), ramped to 140 °C with a rate of 10 °Cmin−1, then with 1.5 °Cmin−1 to 185 °C and then with 30 °Cmin−1 to 230 °C (held for 4 min). After separation, the analytes were combusted online in a Finnigan GC combustion interface (Thermo Fisher Scientific, Bremen, Germany) to CO2 with a NiO tube/CuO–NiO reactor operated at 1,000 °C (Thermo Fisher Scientific, Bremen, Germany). CO2 isotope values were determined with a Finnigan MAT 253 isotope ratio mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). Before and after every run, three reference gas peaks were measured. These reference gas peaks were used to link isotope values ESIA of polar herbicides 2827 to the international standard for carbon isotopes (Vienna PeeDee Belemnite, Eq. 1).
13  13 C 13 d C¼ 12 C C 12 C Sample Reference ð1Þ 13 C 12 C influenced by the uncertainty of the GC-IRMS analysis (∆GC), whereas the contribution of the methyl group calculation (∆Me) is reduced by a factor of 1/9 in the case of 4-CPP and DCPP and 1/10 in the case of MCPP, respectively. Reference To validate the results of the GC-IRMS method, in addition, pure in-house standards of all compounds were characterized on an elemental analyzer (EURO EA, EuroVector Instruments) coupled to a Finnigan MAT 253 isotope ratio mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). Calibration was performed with the following organic reference materials provided by the International Atomic Energy Agency (IAEA, Vienna): IAEA 600, IAEA CH3 (cellulose), IAEA CH6 (sucrose), and IAEA CH7 (polyethylene). Calculation of the introduced methyl group During methylation, an additional carbon atom is introduced in each herbicide molecule and changes its bulk carbon isotope ratio. This shift of δ13C was kept constant by using the same batch of methanolic BF3 solution for the derivatization of standards and samples. Values were corrected according to the common procedure as described previously [21–23]. First, the isotope ratio of the introduced methyl group was calculated (δ13C(Me)) (Eq. 2). To this end, the isotope ratio of the lab standard of the respective herbicide was determined prior to methylation on EA-IRMS (δ13C(Analyte)EA) and after methylation on GC-IRMS (δ13C(Me-Analyte)GC). With respect to the number of C-atoms of the analyte (n), the weighted difference was then used to calculate the theoretical isotope ratio of the introduced methyl group (Table 1). Subsequently, this value was used to correct samples (δ13C(Sample)) for the introduced methyl group; again, with respect to the number of C-atoms (Eq. 3). The inherent uncertainty of this procedure can be calculated by Gaussian error propagation (Eq. 4). The uncertainty of the sample value (∆δ13C(Sample)) is strongly Table 1 Given are isotope values of the racemate analyzed on EAIRMS, the enantiomers analyzed on GC-IRMS (R- and S-enantiomers are denoted as superscript capital letters R (R) and S (S), respectively), the theoretical value of the racemate calculated as the mean value of all values analyzed on GC-IRMS, the lowest amount needed on column d13 CðMeÞ ¼ ðn þ 1Þ  d13 CðMe AnalyteÞGC ð2Þ n  d13 CðAnalyteÞEA d13 CðSampleÞ ¼ ðn þ 1Þ  d 13 C ðMe AnalyteÞSample d13 CðMeÞ n ð3Þ Δd13 CðSampleÞ s ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi    @f ðx; yÞ 2 @f ðx; yÞ 2 2 ¼  Δx þ  Δ y2 @x @y sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi    2 nþ1 2 1 ¼  ΔGC2 þ  ΔMe2 n n ð4Þ Peak identity Identity of standards and samples was verified on a gas chromatograph (DANI Master GC, Milano, Italy) coupled to a time of flight mass spectrometer (DANI Master TOF, DANI, Italy/Switzerland). The system was operated with same flow velocity, temperature conditions, and the same column as described above for the GC-IRMS system, but without combustion of the samples to CO2. One enantiomer of each DCPP and 4-CPP was available as a pure standard and was used to identify the respective enantiomers. There are only small molecular differences in the structure of MCPP in comparison to 4-CPP and DCPP and they are not related to the chiral center. Hence, it was for precise isotope analysis for each enantiomer, and the calculated δ13C value of the methyl group (see Eq. 1) introduced during derivatization; uncertainties represent the standard deviation. Note that the uncertainty of the calculated isotope value of the methyl group has only a minor influence on the isotope value of samples as shown in Eq. 4 Analyte EA-IRMS δ13C [‰] (n=5) GC-IRMS δ13C [‰] (n=9–13) GC-IRMS δ13C [‰] (n=9–13) Lowest amount needed for precise isotope analysis Calculated δ13C of introduced methyl group [‰] 4-CPP −26.5±0.1 −26.9±0.3R −26.4±0.1S −26.7±0.3 62 pmol (7 ngC) −28±3 MCPP −28.6±0.1 −28.9±0.4 58 pmol (7 ngC) −32±4 DCPP −27.3±0.1 −27.3±0.2 53 pmol (6 ngC) −28±2 −29.2±0.2R −28.6±0.2S −27.5±0.1R −27.2±0.2S 2828 M.P. Maier et al. assumed that the elution order of 4-CPP and DCPP, where the R enantiomer elutes first, also applies to MCPP. In addition, this elution order agrees with the elution order Buser and Muller [1] determined for 4-CPP and MCPP with a similar stationary phase. Ideally, this hypothesis remains to be verified with a pure MCPP enantiomer for degradation studies with MCPP. decreased to 30 % (11–12 min) again. The flow rate was 1 mL min−1 and the oven temperature was 45 °C. The sample injection volume was 100 μL and absorbance of DCPP was measured at 280 nm. DCPP transformation study Chromatographic resolution A pure strain of D. acidovorans MC1 was used for degradation of DCPP. MC1 was first pregrown in mineral salt solution (1 L, 30 °C) with 10 mg DCPP [24]. When DCPP was completely consumed by MC1, the final OD of this solution reached 0.4 at 500 nm. Using this solution, biodegradation of DCPP (60 mgL−1) was conducted in duplicate non-shaken batches (200 mL each). Liquid samples (5 mL each) were collected and sterile filtered (0.22 μm). Sample (0.5 mL) was immediately analyzed by LC-UV as described below. The rest was stored frozen at −20 °C for isotope analysis. DCPP samples were quantified using a Shimadzu LC10A series high-performance liquid chromatograph equipped with an Allure C18 column, 150×4.6 mm, 5 μm particle size (Restek, USA), and an ultraviolet detector. The eluting solvents were acetonitrile with 1.25 % acetic acid (solvent A) and deionized water with 1.25 % acetic acid (solvent B). The gradient was composed of 30 % solvent B (1 min) and was increased to 80 % (2–10 min) and finally As shown previously by Buser and Müller [1], chlorinated phenoxy acid herbicides can be separated on a chiral stationary phase after methylation (Fig. 1). We achieved this with a chromatographic resolution of R≥3.0 for the lowest tested concentrations and R≥1.4 for the highest tested concentrations when R is defined as retention time difference/mean peak width [25]. If R is calculated using the peak width at half height [25], a chromatographic resolution between R≥7.2 and R≥6.2 was achieved. Only the highest concentrations of 4CPP (R≥1.4) fall slightly below the conservative definition of baseline separation (R≥1.5; Fig. 1b). This did not influence the isotope values (Fig. 2); however, in cases where values appear to be biased, samples can be diluted. Fig. 1 GC-IRMS chromatogram (m/z 44) of a racemic mix, containing 4-CPP, MCPP, and DCPP. Examples with an injection volume of 1 μL and concentrations per enantiomer of 12.5 mgL−1 (a) and 50 mgL−1 (b) are shown Results Isotope analysis The precision of ESIA for the three tested herbicides is comparable to other non-enantioselective studies that do not contain a derivatization step [12]. Triplicate analysis ESIA of polar herbicides 2829 Fig. 2 Dependency of δ13C measurements on the peak amplitude. Blue diamonds indicate S enantiomers and red squares R enantiomers. The solid line represents the EAIRMS value of the racemate delivered a standard deviation of 2σ≤0.5‰. Although a methyl group is introduced in the analytes during derivatization, the isotope values of the GC-IRMS analysis were indistinguishable from the reference values derived by elemental analysis-IRMS within the typical precision of the two methods (2σ of 0.5 and 0.2‰, respectively; Table 1). This applies for the single enantiomers as well as for the racemate value, calculated as the mean of both enantiomers. Correspondingly, the isotope ratio of the introduced methyl group was close to the isotopic composition of the target compounds (Table 1), and the methyl group values were indistinguishable within calculated uncertainties. Because the correction scheme described above uses the isotope shift of standards for the correction of samples, it delivers precise isotope values with respect to the introduced carbon atom taking into account any systematic fractionation effects that Fig. 3 DCPP degradation comes along with enantiomeric fractionation (left) and no isotope fractionation (right). Blue diamonds indicate S-DCPP and red squares R-DCPP. The experiment was made as duplicate; the solid line represents the EA-IRMS value of the racemate can appear during derivatization [23]. For the purposes of the linearity test, we refrained from such corrections because its focus was on the correlation of peak amplitude and isotope value and the correction term would have been the same for all samples. For degradation studies, as shown below, the additional methyl group has to be taken into account because position-specific isotope changes are more strongly diluted by the additional carbon atom. The linear range of the method is illustrated in Fig. 2. For all four tested compounds, the lower limit of precise δ13C isotope analysis was around peak amplitudes of 200 mV. Hence, we recommend that for precise ESIA, only peak amplitudes higher than 200 mV should be used. This corresponds to a minimum amount of substance needed on column of ≥7 ng that is even below the manufacturer specification of 10 ng C [12]. 2830 A critical point for isotope data evaluation is the background correction. In most cases, a 5-s interval prior to each peak is used to correct its isotope value. Because enantiomer peaks in ESIA have narrow retention times, the tailing of the first eluting enantiomer could interfere with the background used for the second eluting enantiomer. Consequently, we used the background of the first enantiomer to correct peak areas of both enantiomers. M.P. Maier et al. Acknowledgments Michael Maier is financially supported by the German Federal Environmental Foundation (DBU). The study was supported by the Seventh Framework Program (2007–2013) of the European Commission within the GOODWATER Marie Curie Initial Training Network (grant no. 212683). We thank Heide Bensch for her support in the synthesis of S-4-CPP. Two anonymous reviewers are acknowledged for critical comments. References Application of ESIA to phenoxy acids Biodegradation of DCPP was done in D. acidovorans MC1. Interestingly, enantioselective degradation of DCPP was observed, but no carbon isotope fractionation occurred to either enantiomer (Fig. 3). Two scenarios can explain the absence of isotope fractionation: either the degrading enzyme has no isotopic preference, or the intrinsic kinetic isotope effect of the enzyme was masked. Such a masking effect appears if the enzymatic bond cleavage is not the ratelimiting step [11]. One example could be that transport into the cell is rate limiting [26] as hypothesized for MC1 [24, 27], but this has to be confirmed in further studies. Our findings contrast with the observation of Badea et al. that isotope fractionation, but no enantiomeric fractionation, occurred in biodegradation of α-hexachlorocyclohexane [17]. Consequently, the information of both methods (CSIA and enantiomer fractionation) appears to be complementary. Outlook With this study, we provide a tool to investigate degradation of selected chiral herbicides based on the isotope analysis of enantiomers. By widening the application of ESIA to another compound class, we obtained the remarkable picture that CSIA and enantiomer fractionation do not seem to be strongly related. In other words, the methods may nicely complement each other in the assessment of contaminated sites. If only one kind of fractionation occurs, i.e., only enrichment of isotopes [17] or only of enantiomers (our study), ESIA presently has no unique advantage compared to CSIA and enantiomer analysis alone. In contrast, the full potential of ESIA will unfold if both processes occur in parallel. First evidence for such a scenario has been obtained in a recent study by Milosevic et al. where enantioselective degradation and carbon isotope fractionation of 4-CPP were observed downstream at a landfill site under anaerobic conditions [19]. There, isotope values of single 4-CPP enantiomers differed up to 3‰. Hence, much can be expected to be learned from ESIA in the future with respect to the complementarity of both lines of evidence in field applications and with respect to a better process understanding. 1. Buser HR, Muller MD (1998) Occurrence and transformation reactions of chiral and achiral phenoxyalkanoic acid herbicides in lakes and rivers in Switzerland. Environ Sci Technol 32(5):626– 633. doi:10.1021/es970652w 2. Wong C (2006) Environmental fate processes and biochemical transformations of chiral emerging organic pollutants. Anal Bioanal Chem 386(3):544–558. doi:10.1007/s00216-006-0424-3 3. Sherwood Lollar B, Slater GF, Sleep B, Witt M, Klecka GM, Harkness M, Spivack J (2001) Stable carbon isotope evidence for intrinsic bioremediation of tetrachloroethene and trichloroethene at Area 6, Dover Air Force Base. Environ Sci Technol 35:261–269 4. Hunkeler D, Aravena R, Butler BJ (1999) Monitoring microbial dechlorination of tetrachloroethene (PCE) using compoundspecific carbon isotope ratios: microcosms and field experiments. Environ Sci Technol 33(16):2733–2738 5. Richnow HH, Annweiler E, Michaelis W, Meckenstock RU (2003) Microbial in situ degradation of aromatic hydrocarbons in a contaminated aquifer monitored by carbon isotope fractionation. J Contam Hydrol 65(1–2):101–120 6. Hofstetter TB, Berg M (2011) Assessing transformation processes of organic contaminants by compound-specific stable isotope analysis. TrAC Trends Anal Chem 30(4):618–627 7. Schmidt TC, Zwank L, Elsner M, Berg M, Meckenstock RU, Haderlein SB (2004) Compound-specific stable isotope analysis of organic contaminants in natural environments: a critical review of the state of the art, prospects, and future challenges. Anal Bioanal Chem 378(2):283–300 8. Meckenstock RU, Morasch B, Griebler C, Richnow HH (2004) Stable isotope fractionation analysis as a tool to monitor biodegradation in contaminated acquifers. J Contam Hydrol 75(3– 4):215–255 9. Thullner M, Centler F, Richnow H-H, Fischer A (2012) Quantification of organic pollutant degradation in contaminated aquifers using compound specific stable isotope analysis: review of recent developments. Org Geochem 42(12):1440–1460 10. Elsner M, Zwank L, Hunkeler D, Schwarzenbach RP (2005) A new concept linking observable stable isotope fractionation to transformation pathways of organic pollutants. Environ Sci Technol 39(18):6896–6916 11. Elsner M (2010) Stable isotope fractionation to investigate natural transformation mechanisms of organic contaminants: principles, prospects and limitations. J Environ Monit 12(11):2005–2031 12. Elsner M, Jochmann MA, Hofstetter TB, Hunkeler D, Bernstein A, Schmidt TC, Schimmelmann A (2012) Current challenges in compound-specific stable isotope analysis of environmental organic contaminants. Anal Bioanal Chem 403(9):2471–2491. doi:10.1007/s00216-011-5683-y 13. Meyer AH, Penning H, Elsner M (2009) C and N isotope fractionation suggests similar mechanisms of microbial atrazine transformation despite involvement of different enzymes (AtzA and TrzN). Environ Sci Technol 43(21):8079–8085. doi:10.1021/es9013618 14. Hartenbach AE, Hofstetter TB, Tentscher PR, Canonica S, Berg M, Schwarzenbach RP (2008) Carbon, hydrogen, and nitrogen ESIA of polar herbicides 15. 16. 17. 18. 19. 20. isotope fractionation during light-induced transformations of atrazine. Environ Sci Technol 42(21):7751–7756 Penning H, Sorensen SR, Meyer AH, Aamand J, Elsner M (2010) C, N, and H isotope fractionation of the herbicide isoproturon reflects different microbial transformation pathways. Environ Sci Technol 44(7):2372–2378. doi:10.1021/es9031858 Reinnicke S, Simonsen A, Sørensen SR, Aamand J, Elsner M (2012) C and N isotope fractionation during biodegradation of the pesticide metabolite 2,6-dichlorobenzamide (BAM): potential for environmental assessments. Environ Sci Technol 46(3):1447– 1454. doi:10.1021/es203660g Badea S-L, Vogt C, Gehre M, Fischer A, Danet A-F, Richnow H-H (2011) Development of an enantiomer-specific stable carbon isotope analysis (ESIA) method for assessing the fate of alphahexachlorocyclohexane in the environment. Rapid Commun Mass Spectrom 25(10):1363–1372. doi:10.1002/rcm.4987 Nittoli T, Curran K, Insaf S, Di-Grandi M, Orlowski M, Chopra R, Agarwal A, Howe AYM, Prashad A, Floyd MB, Johnson B, Sutherland A, Wheless K, Feld B, O’Connell J, Mansour TS, Bloom J (2007) Identification of anthranilic acid derivatives as a novel class of allosteric inhibitors of hepatitis C NS5B polymerase. J Med Chem 50(9):2108–2116. doi:10.1021/jm701232p Milosevic N, Qiu S, Elsner M, Einsiedl F, Maier MP, Bensch HKV, Albrechtsen HJ, Bjerg PL (2013) Combined isotope and enantiomer analysis to assess the fate of phenoxy acids in a heterogeneous geologic setting at an old landfill. Water Res 47(2):637–649 Chivall D, Berstan R, Bull ID, Evershed RP (2012) Isotope effects associated with the preparation and methylation of fatty acids by boron trifluoride in methanol for compound-specific stable hydrogen isotope analysis via gas chromatography/thermal conversion/ isotope ratio mass spectrometry. Rapid Commun Mass Spectrom 26(10):1232–1240. doi:10.1002/rcm.6188 2831 21. Reinnicke S, Bernstein A, Elsner M (2010) Small and reproducible isotope effects during methylation with trimethylsulfonium hydroxide (TMSH): a convenient derivatization method for isotope analysis of negatively charged molecules. Anal Chem 82(5):2013– 2019. doi:10.1021/ac902750s 22. Pelz O, Hesse C, Tesar M, Coffin RB, Abraham WR (1998) Development of methods to measure carbon isotope ratios of bacterial biomarkers in the environment. Isotop Environ Health Stud 34(1–2):131–144. doi:10.1080/10256019808036364, Taylor & Francis 23. Silfer JA, Engel MH, Macko SA, Jumeau EJ (1991) Stable carbon isotope analysis of amino acid enantiomers by conventional isotope ratio mass spectrometry and combined gas chromatography/isotope ratio mass spectrometry. Anal Chem 63(4):370–374 24. Muller RH, Hoffmann D (2006) Uptake kinetics of 2,4-dichlorophenoxyacetate by Delftia acidovorans MC1 and derivative strains: complex characteristics in response to pH and growth substrate. Biosci Biotechnol Biochem 70(7):1642–1654. doi:10.1271/ bbb.60011 25. Hübschmann H-J (2001) Handbook of GC/MS, vol 1. Wiley-VCH Verlag GmbH, Weinheim 26. Nijenhuis I, Andert J, Beck K, Kastner M, Diekert G, Richnow HH (2005) Stable isotope fractionation of tetrachloroethene during reductive dechlorination by Sulfurospirillum multivorans and Desulfitobacterium sp. Strain PCE-S and abiotic reactions with cyanocobalamin. Appl Environ Microbiol 71 (7):3413–3419 27. Zipper C, Fleischmann T, Kohler HPE (1999) Aerobic biodegradation of chiral phenoxyalkanoic acid derivatives during incubations with activated sludge. FEMS Microbiol Ecol 29(2):197–204. doi:10.1111/j.1574-6941.1999.tb00611.x