Environ. Sci. Techno/. 1995, 29, 2154-2156
Site ReaProbes: [1,2-13C]Chlo6acetic Acid, a Reactivity Probe for Soil CHARLES E. CASTRO,* STEPHEN K. O'SHEA, AND ELEANOR W. BARTNICKI Nematology Department and the Environmental Toxicology Graduate Program, University of California at Riverside, Riverside, California 92521 -0415
acid to glyoxylic acid and COz. Because of these findings and the water solubility of chloroacetic acid, we chose it as a potential substance for development into a reactivity probe for soil.
Methods Dimethylformamide (Aldrich) was distilled under argon. The substance was stored under argon for subsequent use as a solvent for the 13C-labeledprobe. The probe was synthesized by reacting [1,2-13Claceticacid with sulfuryl chloride in the presence of phosphorous pentachloride (eq 1). 0
0
Introduction Assessing the reactivity of a given site or soil for chemical or biochemical conversion should be a valuable asset for predicting the rate and nature of the processes by which xenobiotics may be transformed in the soil-water sphere. A knowledge of this reactivity could also be useful as a guide for choosing pesticides that may be environmentally tolerable or rapidly degraded at a given site. Moreover, reactivityprobes should be a convenient means of assessing the effectiveness of bioremediation efforts. At this time, however, there is no methodologyinplace thatwill monitor soil reactivity directly. Although pH and redox potential can be monitored easily, they do not necessarily relate to a given site's capacity for transformation. The basic chemicalor biochemical processes a substance may undergo in soil are oxidation,reduction, or substitution. Soil microbes are capable of transforming alkyl halides by all of these processes ( 1 ) . As an example, the chemical and biochemical hydrolysis (substitution)of alkyl halides in soils is well known (2-6). Moreover, the potential transformation of substances by exogenous enzymes and metal or metal ion-mediated oxidation or reduction may also be expected. For some time, we have considered the possibility that a simple substance capable of undergoing oxidation, reduction, and hydrolysis might be employed as a reactivity probe for soil. In principal, the substance could be applied to soil, and the chemistry that it undergoes might be used to read the site. The nature of the chemical process or reaction type(s) and the speed of the process could thus be monitored. Essentiallythe probe, as an arbitrary standard, is run against itself in each experiment. Thus, differences in reactivity between sites can be detected, as can the change in reactivity at any given site. Ideally, the probe and the diagnostic products derived from it must be easily analyzed with minimal workup. The chemistry must be quantifiable, and small quantities of the sample should be able to be subjected to the analysis. Our strategy for the development of reactivity probes rests primarily upon studies of the transformations alkyl halides may undergo in the soil-water sphere. In previous studies, we have noted the generation of chloroacetic acid as a product ofmicrobial oxidationof 1,1,2-trichloroethane (7) and ethylene dichloride (5). It was also apparent that Pseudomonas putida (PpG-786)could oxidize chloroacetic * Address correspondence to this author at the Nematology Department.
2154 = ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 29, NO. 8.1995
I1
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This is a relatively slow reaction, but it goes cleanlyunder mild conditions at 80 "C. Only traces of the dichlorinated product are obtained. The 13C NMR spectrum of distilled 11,2-13Clchloroaceticacid (CAI in dimethylformamide exhibits doublets at 45 6 (CH2Cl) and 176 6 (C02H). [1,2-13C]Chlor~aceticAcid. Into a 10-dflaskequipped with reflux condenser and CaCh drying tube, 1.0 g of [1,2l3C1acetic acid (Sigma Chemical Co., St. Louis, MO) (0.016 mol), 1.4 mL of S02C12(0.017mol), 5 mg of PC15, and one drop of acetic anhydride were placed. The mixture was brought to 80 "C and held at that temperature under slight reflux for 1day. At this time, the 13CNMR analysis indicated that an equal amount of acetic acid and chloroacetic acid was present (-50% conversion). An additional 1.4 mL of S02C12was added through the condenser, and the solution was heated at 80 "C for another day. At this time, 13CNMR of the mixture indicated a mix of 2 . 5 CHzCl:CH3 ~ or a 71% conversion to CA. A trace of C12CH at 66 6 corresponding to dichloroacetic acid could be seen. At this point, the mixture was distilled. After bringing off unreacted S02C12, at 69 OC, the flask was warmed to 130 "C until no further distillate (unreactedacetic acid) was obtained. The product, [1,2-13Clchlor~acetic acid, distilled at 100 OC120 mm and crystallized. Weight was 1.0 g. Note: This substance is hydroscopic. A stock 1 M solution of CA in dimethylformamide (DMF)was prepared for testing the probe. CA does not react with this solvent, and solutions have been stable for 2 years. Soil incubations. Three soils from the grounds at the University of California at Riverside (UCR)were employed; a sandy loam, a dump site soil, and a "basic soil". The dump site contained halogenated compounds. After a variety of trials, the following simple protocol was established. One microliter of a stock 1 M solution of [1,2-13C]chloroacetic acid (CA)in DMF is added to a slurry composed of 0.5 g of soil and 1 mL of glass distilled water. The mixture is thus M in chloroacetic acid at the outset. The mixture may be shaken in a small capped reactivial (Pierce)or test tube. After 2 weeks (or any other time), the suspension is filtered and centrifuged in a micro centrifuge at -4500 rpm. Plastic microfuge filter cartridges (0.45pM) are convenient for this purpose. The clear supernatant solution (-0.5 mL) is placed in a 5-mm NMR tube, and 50 pL of deuterium oxide (D20) is added for the lock. The 13CNMR spectrum is acquired for 14-16 h. In this work, we used a General
0013-93SX/95/0929-2154$09.00/0
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SCHEME 1
Primary Reaction Paths and Chloroacetic Acid Conversion
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Products
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FIGURE 2. 13C NMR spectrum showing the hydrolysis of CA in soil and unreacted CA at 3 weeks.
(glycolic acid)
Electric (Schenectady,NY) 300-MHzspectrometer (QE-300). Spectra were acquired in the automode. Note: A shift in resonance position of -3 ppm in the NMR spectrum may occur as a result of pH and other factors. For most incubations, conditions and protocols were like that described above and were so simple that they are given briefly in the Results and Discussion. More soil and water can be used in these assays, e.g., 1 g of soil, 2 mL of HzO, and 2 pL of stock 1 M CA in DMF solution or more. To demonstrate reduction, 1 g of sterile soil, 2 mL of water, and 0.2 g of powdered zinc were incubated anaerobically (at a starting pH of 5) and shaken for 3 days. In this run, the initial CA concentration was 2 x M.
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FIGURE 3. 13C NMR spectrum following reduction of CA with Zno in a "basic" soil.
above. The NMR spectrum is shownin Figure 1. Reisolated CA is the only component derived from the probe.
Hydrolysis. A sample incubated with a UCR soil at pH 10 for 3 weeks is shown in Figure 2. Clearly resonances for untransformed chloroacetic acid ClCH2 at 45 6 and COzat 177,along with new resonances for HOCHZ-at 63 6, and the corresponding COZ-at 1756 are discernible. The figure is an NMR "snapshot" of the hydrolysis of chloroacetic to hydroxyacetic or glycolic acid (eq 2).
Results and Discussion Using [1,2-13Clchlor~acetic acid (CAI and 13CNMR analysis, we show that this substance does respond to chemical and biological conversion in soil and slurries and that the process can be monitored easily. Thus, this substance can be used to measure directly a site's capacity for transformation. It is the first site reactivity probe for soil. The primary paths of conversion that chloroacetic acid may undergo and the corresponding products derived from oxidation, reduction, and substitution are shown in Scheme 1. Glyoxylic acid is rapidly oxidized to COZby soil organisms (7, 8 ) . The simple protocol for using the probe is given in the Methods. Essentially, it entails incubating the probe at M in a soil slurry composed of a 2: 1watersoil mixture, centrifuging at the desired time, and running the NMR of the resultant solution. Examples demonstrating the recovery of CA from soil and its hydrolysis, oxidation, and reduction are shown below. Recovery. To demonstrate the reisolation of CA from soil, a M solution was incubated anaerobically with sterile soil and worked up in the simple manner described
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The main point evident from the figure is that both unreacted chloroacetic acid and the hydrolysis product glycolate are directly observable with an initial starting concentration of M chloroacetic acid (chloroacetate), Thus, the probe responds to hydrolysis. Clearly, the doublets for CH2OH and CHzCl are the main diagnostic resonances. Reduction. In this test, the probe was reduced in the soil slurry by the addition of powdered zinc and acid. A basic soil (pH 8 in the initial slurry) was adjusted to pH 5.0 and incubated with ZnO for 3 days. Following centrifugationlfltration, the clear solution (now pH 6.7) exhibited the NMR spectrum in Figure 3. This spectrum is poorly resolved. DMF resonances predominate, and the methyl group of acetic acid is suppressed. It is discernible, but only as a broadened multiplet. The carboxyl resonances are not seen. However, the addition of one drop of concentrated HzS04to the same NMR tube resulted in the clean and well-resolved spectrum shown in Figure 4. Resonances for acetic acid (21 and 178 6) and glycolic acid (60 and 177 6) are clear. Thus, the artificially forced reduction did occur, but the hydrolytic capability of the soil was also manifest. In this case, the probe has responded to both reduction and hydrolysis by the soil/ZnOmixture. Again, it is the CH3 and CH20H resonance that are diagnostic. Another point this experiment makes is that in the event poorly resolved spectra are obtained, they can be greatly improved by the addition of acid to the final filtrate VOL. 29, NO. 8 , 1995 /ENVIRONMENTAL SCIENCE & TECHNOLOGY 12155
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FIGURE 4. 13C NMR spectrum of the products of the Zna reduction experiment (Figure 3) after acidification (to pH 1).
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used for NMR analysis. The broadening observed in this experiment was not observed in other incubations. Both of the above incubations were designed to illustrate that the probe can respond. In both cases, the transformations were forced by the addition of reagents to a sterile soil slurry. This was essential to prove that at M in soil the probe can respond to abiotic processes and moreover that the products are discernible. Oxidation. While we have also demonstrated oxidation in soil by chemical means, we show here a microbiological oxidation to COz (HC03-) using an unknown soil from a dump site at UCR. Usingthe standard protocol, incubation for 1weekin this soil sluny showed a complete consumption of probe and the generation of bicarbonate -162 6 (singlet) (Figure 5). In other incubations with this soil at shorter times, both HC03- and CA were visible. These results test the probe with an unknown soil. They demonstrate the probe’sresponse to microbial oxidation. A sterilized sample of this soil incubated for 6 weeks showed only resonances for starting chloroacetic acid (like Figure 1). Clearly, this soil has good oxidative capacity (eq 3).
This process may well entail glyoxylic acid as an intermediate (a,but the steady-state concentration is too low for NMR detection. In this case, as well as with other potential transformations, a conversion to COz (HC03-)is a measure of oxidation capacity. Kinetics. An idea of the speed of any of the transformations can be assessed directly from the NMR spectra. Assuming low background C1- concentrations in 0.5 g of soil, rates may be measured quite accurately by monitoring the C1- potentiometrically (1). The advantage of a two-carbon I3C-labeled substrate is evident from the NMR spectra presented in the figures. The 13C coupling results in a doublet for products derived from CA. Thus, other substances perhaps present in the sample, if detected, will only appear as singlets as the dimethylformamide resonances do in these spectra. An important exception is HI3C03-. This ultimate product of mineralization appears as a singlet. Background spectra 2156
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of soil blanks, in the absence of added CA, do not exhibit this resonance. The broadening and poor resolution initially observed in the reduction experiment deserves some additional comment. The initial centrifugate/filtrate was clear and homogeneous. The greatly enhanced spectrum observed following the addition of acid suggests that the products (inthis case both acetic acid and glycolic acid) were bonded to soluble substances extracted from the slurry. These may have been a composite of soluble organic matter and ZnZf. The broadening was not the result of this ion alone because experiments in the absence of soil did not show it. We infer that soluble matter from soils may interfere with these analyses by complexing and occluding the corresponding acid anions. In any case, acidification of the solution releases the products. Thus, with those soils from which poorly resolved spectra are obtained, acidification of the sample following centrifugationIfiltration may be a requisite. Although this substrate meets all of the criteria we had established for a site reactivity probe, there is a drawback to this methodology. The NMR analysis itself requires a relatively long (14-16 h) acquisition time on a 300-MHz spectrometer for dilute solutions. This time can be shortenedwith better NMR probe design and more powerful machines. Of course, higher concentrations of CA (e.g., M) greatly diminish the acquisition time. We have deliberatelyused low concentrationsin this work to demonstrate the sensitivity of the method. Since the acquisition requires no work on the part of the investigator, the methodology is viable. In our laboratory, usually an “overnight”acquisition was obtained. The reactivity of different sites or the change in reactivity at a fixed site can be assessed by the CA probe. In principle, it is also possible that a correlation of CA reactivity with that of other substrates may be made by employing the vast literature on structure-activity relationships (9). Finally, it should be noted that the CA probe is not limited to soil. The reactivity of any segment of the environment with which it may be admixed in water can be monitored.
Acknowledgments We thank the Kearney Foundation for Soil Science for support of this work. We also thank members of the Western Regional Committee, W-82, for the criticism, advice, and encouragement that aided in the development of this probe.
literature Cited (1) Castro, C. E . Enuiron. Toxicol. Chem. 1993, 12, 1609-1618. (2) Castro, C. E.; Belser, N. O.J. Agric. Food Chem. 1966, 14, 69-70. (3) (3) Castro, C. E.; Wade, R. S.; Riebeth, D. M.; Bartnicki, E. W.; Belser, N. 0. Environ. Toxicol. Chem. 1992, 11, 757-764. (4) Castro, C. E.; Bartnicki, E. W. Biochem. Biophys. Acta 1965,100, 384-392. (5) Riebeth, D. M.; Belser, N. 0.;Castro, C. E.Environ. Toxicol. Chem. 1992, 11, 497-501. (6) Belser, N . 0.;Castro, C. E.J. Agric. Food Chem. 1971,19, 23-26. (7) Castro, C. E.; Belser, N. 0.Enuiron. Toxicol. Chem. 1990,9,707714. (8) Castro, C. E.; Riebeth, D. M. Riebeth; Belser, N. 0. Enuiron. Toxicol. Chem. 1992, 11, 749-755. (9) As an example, see Klumpp, G., Reactiviry in Organic Chemistry; John Wiley and Sons, New York, 1982.
Received for review December 14, 1994. Revised manuscript received May 2, 1995. Accepted May 16, 1995. ES9407608
