Environ. Sci. Technol. 2002, 36, 4065-4073
Nucleophilic Aliphatic Substitution Reactions of Propachlor, Alachlor, and Metolachlor with Bisulfide (HS-) and Polysulfides (Sn2-) A. R. LOCH,‡ K. A. LIPPA,§ D. L. CARLSON,§ Y. P. CHIN,† S . J . T R A I N A , ‡ A N D A . L . R O B E R T S * ,§ Department of Geography and Environmental Engineering, 313 Ames Hall, The Johns Hopkins University, 3400 North Charles Street, Baltimore, Maryland 21218-2686, and School of Natural Resources and Department of Geological Sciences, The Ohio State University, Columbus, Ohio
Reactions of bisulfide and polysulfides with alachlor, propachlor, and metolachlor were examined in aqueous solution to investigate the role reduced sulfur species could play in effecting abiotic transformations of chloroacetanilide herbicides. Experiments at 25 °C demonstrated that reactions were approximately first-order in HS- concentration and revealed that polysulfides are considerably more reactive than HS-. ∆Hq values for reactions of the three chloroacetanilides with HS- are statistically indistinguishable at the 95% confidence level, as are ∆Sq values, despite significant differences in second-order rate constants (kHS-). Transformation products were characterized by GC/MS (in some cases following methylation) and were found to be consistent with substitution of chlorine by the sulfur nucleophile. Products containing multiple sulfur atoms were observed for the reactions of chloroacetanilides with polysulfides, while products resulting from reaction with HS- only possessed a single sulfur atom. When secondorder rate constants at 25 °C are multiplied by HS- and polysulfide concentrations reported in salt marsh porewaters, predicted half-lives range from minutes to hours. HSand especially polysulfides could thus exert a substantial influence on the fate of chloroacetanilide herbicides in aquatic environments. Oxidation of the resulting sulfursubstituted products could generate ethane sulfonic acid derivatives, previously reported as prevalent chloroacetanilide degradates.
Introduction Chloroacetanilide herbicides are among the most widely applied pesticides worldwide, primarily employed in the United States for preemergence control of annual grasses and broadleaf weeds in corn and soybeans (1). U.S. use of metolachlor in 1997 was estimated at 30 million kg of active ingredient, exceeded only by atrazine (2). Comparable figures for alachlor (currently undergoing replacement by other chloroacetamides), acetochlor, and propachlor were 6 million * Corresponding author phone: (410)516-4387; fax: (410)516-8996; e-mail:
[email protected]. § The Johns Hopkins University. ‡ School of Natural Resources, The Ohio State University. † Department of Geological Sciences, The Ohio State University. 10.1021/es0206285 CCC: $22.00 Published on Web 08/23/2002
2002 American Chemical Society
kg, 15 million kg, and < 2 million kg, respectively (2). Not surprisingly, chloroacetanilide herbicides are also among the most prevalent pesticides encountered in ground and surface waters. For example, the National Water Quality Assessment studies conducted by the U.S. Geological Survey report acetochlor, alachlor, and metolachlor in 89-99% of Midwestern surface water samples analyzed (3), with metolachlor among the top five most frequently detected pesticides in groundwater (4, 5). Alachlor is viewed by the EPA as a “likely” human carcinogen (6); its consumption is also associated with eye, liver, kidney, or spleen problems and anemia (7). This herbicide is therefore regulated in drinking water, with maximum contaminant level (MCL) values of 2 µg/L (7). Alachlor is ranked as having high to moderate chronic toxicity to aquatic vertebrates and invertebrates and high toxicity to aquatic plants (6); concentrations reported in surface waters are sufficiently elevated to have extensive adverse effects on aquatic plants (6). Although MCL values have not been established for other chloroacetanilides, metolachlor and acetochlor are included on EPAs “Drinking Water Contaminant Candidate List” (CCL) of pollutants that may be subject to future regulation by virtue of their likely occurrence at levels of concern to human health (8). In addition to these parent herbicides, “alachlor ethane sulfonic acid and other acetanilide pesticide degradation products” are also included as CCL contaminants. Although chloroacetanilides can undergo biotransformation in surface soils, their removal tends to proceed slowly under the relatively dilute or nutrient-limited conditions more characteristic of subsurface soils, groundwater, and river water (9-11). Indeed, their widespread presence as groundwater contaminants attests to at least a moderate level of environmental persistence. Even when biodegradation does occur, several studies have demonstrated that alachlor and metolachlor are not readily mineralized (as summarized by refs 12 and 13). Chloroacetanilide herbicide degradates are thus likely to represent prevalent micropollutants. Even though several dozen different degradation products of metolachlor, alachlor, and propachlor have been identified in laboratory studies, relatively few have been sought (or identified) in affected water samples, with the most extensive work having been conducted by Potter and Carpenter (10) and USGS researchers (14-16 and references therein). The latter studies have demonstrated that certain chloroacetanilide degradates, such as 2,6-diethylaniline (an alachlor degradate) and the ethane sulfonic acid (ESA) and oxanilic acid (OA) derivatives of alachlor, metolachlor, and acetochlor, can be detected much more frequently in groundwater than the parent compounds. The ESA and OA derivatives are also typically measured at much higher concentrations than the parent compounds (15). They have been hypothesized to result from a process initiated by nucleophilic displacement of chlorine by the sulfhydryl group of glutathione, a conjugation pathway prevalent in soil microorganisms (17, 18). Similar enzymatic and nonenzymatic conjugations in plants play an important role in the metabolism of chloroacetanilides, conferring a useful selectivity that favors resistant plant species (19). Some degradates exhibit low acute or chronic toxicity; for example, alachlor ESA is nonmutagenic (18). Other alachlor degradates have been shown to be promutagens (such as 2,6-diethylaniline (20)) or mutagenic (such as 2-chloro-2′,6′diethylacetanilide and 2-hydroxy-2′,6′-diethylacetanilide; ref 21). Partial degradation, whether abiotic or biochemically mediated, may therefore fail to reduce the overall risk VOL. 36, NO. 19, 2002 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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associated with use of herbicides, especially if particular pathways lead to degradates that are themselves toxic or that are readily converted to toxic species. Chloroacetanilide degradates can also be of concern because at least some have been demonstrated to exhibit cross-reactivity in enzymelinked immunosorbent assay (ELISA)-based determinations of herbicides (22, 23). Indeed, the cross-reactivity of alachlor ESA contributed to its discovery as a prevalent environmental contaminant (24). At present, relatively little is known about the rates or mechanisms of abiotic transformations that may be important to the fate of chloroacetanilides, such as those involving displacement of chlorine by environmentally relevant nucleophiles. Such information would be invaluable for anticipating which degradates may occur. Even though chloroacetanilides are slow to hydrolyze at ambient pH (for example, the hydrolysis half-life for acetochlor is > 6 years at pH 7 and 25 °C (25)), abiotic reactions could still be important under certain conditions. Wilber and co-workers (26, 27) reported rate constants for the reactions of alachlor and propachlor with HS-, but the temperature at which the experiments were conducted was not specified, nor were the products characterized. Stamper et al. (28) found that alachlor, metolachlor, and propachlor underwent facile abiotic reaction with HS-, but their experimental approach precluded extraction of rate constants from the results. One product of alachlor reaction in the latter study was identified via fast atom bombardment mass spectrometry. Rather than the mercaptoalachlor (RSH) derivative (2-mercapto-2′,6′diethyl-N-(methoxymethyl)acetanilide) that might be anticipated from nucleophilic substitution of chlorine by HS-, it proved to be the mercaptoalachlor RSSR “dimer” bis-2thio-2′,6′-diethyl-N-(methoxymethyl)acetanilide, postulated to result from the oxidative coupling of two molecules of mercaptoalachlor. The primary purpose of this study was to more closely examine rates and products of reactions of three chloroacetanilide herbicides (metolachlor, alachlor, and propachlor) with reduced sulfur nucleophiles. Primary emphasis was placed on reactions with HS- as the most abundant sulfur nucleophile in the environment. Reactions were monitored at varying temperatures and concentrations of HS- so as to obtain activation parameters as well as second-order rate constants. Because prior work (29, 30) has shown that the lesser abundance of polysulfides relative to HS- can be outweighed by their considerably greater reactivity toward relatively simple alkyl halides, this study also included experiments with polysulfides at 25 °C. Products of reactions with HS- and polysulfides were characterized by GC/MS (positive chemical ionization as well as electron impact modes), in some cases following methylation. These reactions may serve as abiotic models of enzymatic conjugations with glutathione or related thiols. They may represent an important “sink” for chloroacetanilides in aquatic environments, and they may give rise to degradation products of unknown degradability or toxicity but potentially high cross-reactivity in ELISA analyses. Oxidation of a sulfursubstituted product resulting from reaction with HS- or polysulfides might even provide an alternate route for formation of ESA derivatives of chloroacetanilides, as previously suggested by Stamper and Tuovinen (13).
Experimental Section Chemicals. Alachlor [2-chloro-2′,6′-diethyl-N-(methoxymethyl)acetanilide; 99%], metolachlor [2-chloro-2′-ethyl6′-methyl-N-(1-methyl-2-methoxyethyl)acetanilide; 99%; mixture of (R,S) enantiomers], propachlor [2-chloro-Nisopropylacetanilide; 99%], and terbuthylazine [2-chloro-4(ethylamino)-6-(1,1-dimethylethylamino))-s-triazine; 99%] were obtained from Chem Service. (S)-Metolachlor (98.5% 4066
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(S) enantiomeric purity) was a gift of Novartis Crop Protection, Inc. (Greensboro, NC). 2-Nitro-m-xylene (99%) and cromatin [N-ethyl-N-(2-methylphenyl)-2-butenamide; 99%] were obtained from Aldrich. All chemicals were used as received. Stock solutions for spiking aqueous samples were prepared in methanol (HPLC grade or better). Reduced Sulfur Solution Preparation and Analysis. Na2S stock solutions were prepared with Na2S‚9H2O, HCl, and deoxygenated high-purity deionized, distilled water. Complete details are given in Loch (31) and Lippa (32). Solutions were handled within an anaerobic glovebox containing a 5% H2/95% N2 or Ar atmosphere. Bisulfide (HS-) working solutions (0.5-50 mM) were prepared by dilution of Na2S stock solutions into 20 mM Na2B4O7‚10H2O pH buffer or 0.5 mM phosphate buffer with sufficient NaCl added to establish an ionic strength of 0.15 equiv/L. Polysulfide solutions were prepared by equilibrating excess S8 with HS- solutions containing 43 mM Na2B4O7‚10H2O as a pH buffer (pH 9.0) as described in ref 32. NaCl was added to polysulfide solutions to establish an ionic strength of 0.25 equiv/L. Bisulfide reacts with S8 to form polysulfides according to the following pHdependent reactions (with n ) 2-5):
HS- + (n-1)/8 S8(s) h Sn2- + H+;
Keq ) {Sn2-}{H+}/{HS-} (1)
Formation constants for these reactions have previously been reported (33, 34). Under our experimental conditions (specifically, in the presence of excess S8(s)), the ratio of [S52-]: [S42-]:[S32-]:[S22-] in any given solution will be constant, being dictated by differences in Keq values for individual polysulfide dianions. As in our prior work with chloroazines (35), the total hydrogen sulfide content [H2S]T, representing the sum of all hydrogen sulfide species, ([H2S] + [HS-] + [S2-]), was measured by iodometric titration using a starch endpoint. Bisulfide ion concentrations were computed from [H2S]T and measured pH values via ionization constants for H2S at 25 °C (36) that were corrected for ionic strength using activity coefficients γHS- and γS2- determined from the Davies approximation. Bisulfide ion concentrations at temperatures different from 25 °C were computed using the expression for the temperature dependence of pKa values reported by Millero (36). Methods appropriate for determining individual polysulfide ions in complex matrixes have not, to the best of our knowledge, been developed. The total concentration of polysulfide dianions Σ[Sn2-] was determined as described in detail elsewhere (37) by a modification of the method of Borchardt and Easty (38). This approach involves derivatization of polysulfide sulfur (0) to triphenylphosphine sulfide, which is analyzed by gas chromatography with flameionization detection (GC/FID). Speciation calculations (based on published equilibrium constants (33, 34)) can be used to compute Σ[Sn2-] from the results. The ensuing values of Σ[Sn2-] were used to compute the second-order rate constant (kSn2-) for reactions of chloroacetanilides with polysulfide dianions. As in our prior work with chloroazines (35), kSn2represents an effective second-order rate constant, equivalent 5 to ∑j)2 kSj2-‚Rj, where kSn2- is the second-order rate constant for each individual polysulfide dianion and Rj is the fraction of Σ[Sn2-] present as each dianion. Most solutions prepared for experimental determination of rate constants for reaction of HS- with chloroacetanilides were also analyzed for polysulfides to confirm that concentrations of the latter were below levels that might contribute appreciably to overall rates of reaction. Experimental Systems. Reaction kinetics were measured under pseudo-first-order conditions whenever possible, with
initial chloroacetanilide concentrations typically only 1/100 to 1/1000 that of reduced sulfur nucleophile concentrations. Kinetic (and buffer control) experiments in most cases were conducted in amber glass bottles with Teflon-lined screw caps. These were placed in a water bath maintained at the appropriate temperature (15.0-58.0 ( 0.1 °C) within an anaerobic glovebox. Solutions prepared in the glovebox were preequilibrated at the selected temperature and were then spiked with 50-500 µL aliquots of methanol containing the appropriate chloroacetanilide, yielding initial herbicide concentrations of 20-35 µM. Aliquots of the reaction solution were extracted into n-hexane containing 2 µM terbuthylazine, 5 µM cromatin, and 8 µM nitroxylene as internal standards. At regular intervals, additional aliquots were removed within the anaerobic glovebox for extraction with n-hexane. The resultant extracts were then subjected to either GC analysis with nitrogen-phosphorus detection (GC/NPD) or mass spectrometric (GC/MS) analysis. Control experiments were initially sampled in triplicate for determining extraction efficiencies, which ranged from 95 to 105% for the chloroacetanilides investigated. Kinetic studies of propachlor and alachlor reactions with HS- at 25 °C were conducted in identical 0.02 M sodium tetraborate/0.1 M NaCl solutions, using 120-mL serum vials that were prepared in an Ar-filled glovebox. After sealing, vials were removed from the glovebox, wrapped in foil, and equilibrated in a water bath for 24 h. The kinetic experiments were then initiated by spiking the vials with 400 µL aliquots of a methanolic solution of chloroacetanilide to generate initial herbicide concentrations of 70-100 µM. Aliquots were removed using a gastight syringe after first pressurizing the serum vial with Ar. Propachlor samples were either analyzed immediately via high performance liquid chromatography (HPLC) or were quenched by acidifying with dilute HCl to pH 4. Quenched samples were refrigerated until analysis via HPLC within 72 h of sampling. No appreciable differences were found between samples analyzed immediately and those quenched prior to analysis. Alachlor samples were quenched with dilute HCl, extracted with n-hexane, and analyzed via GC with electron capture detection (GC/ECD). Timecourses were allowed to progress over approximately 3 half-lives. Pseudo-first-order rate constants (kobs) were determined by regressing the natural logarithm of chloroacetanilide concentration versus time. For selected metolachlor experiments, second-order rate constants for both reactive nucleophiles present (i.e., HS- and Sn2-) were determined by simultaneously fitting the observed parent compound degradation data for solutions with known but differing [HS-] and Σ[Sn2-] values using Scientist for Windows, v. 2.01 (MicroMath Scientific Software). Chloroacetanilide Product Derivatization. All chloroacetanilide transformation products were methylated using CH3I (Aldrich, 99%). A 1-mL aliquot of spent reaction mixture (containing in most cases 50-100 µM chloroacetanilide product and up to 25 mM sulfur nucleophile) was added to ∼2 mL of a deoxygenated 0.1 M CH3I/0.02 M Na2B4O7 buffer solution (pH 9) and then heated at 60 °C in a water bath for 1 h. After cooling to room temperature, the solution was extracted with n-hexane, and the extract was analyzed via GC/MS. Additional analyses of spent reaction mixtures were conducted without derivatization, after acidifying samples to pH 4 using deoxygenated phthalate buffer, followed by extraction into n-hexane. GC and GC/MS Analysis. n-Hexane extracts were typically analyzed for chloroacetanilides using a Carlo-Erba Mega 2 or Series 8000 GC equipped with an on-column injector, a flameless NPD or ECD detector, and a 30 m DB-5 J&W, 0.25 mm i.d. × 0.25 µm fused-silica capillary column. Toluene extracts for triphenylphosphine sulfide were analyzed with
a Carlo-Erba Mega 2 Series GC equipped with a flameionization detector (FID), using an on-column injector and a 15 m DB-5 J&W, 0.25 mm i.d. × 0.25 µm fused-silica capillary column. Chloroacetanilide transformation products were analyzed using a Thermo Finnigan Trace quadrupole GC/MS equipped with an on-column injector and a 30 m J&W DB-200, 0.25 mm i.d. × 0.25 µm fused-silica capillary column. Ionization modes for MS analyses of products included electron impact (EI) and positive chemical ionization (PCI) techniques. The EI mass spectra were generated using an electron energy of 70 eV and were monitored for ions m/z 50-780 in full scan mode. A combination EI/PCI/NCI ionization source was used for chemical ionization, and methane (CH4, 99.995%) was used as the reagent gas. PCI spectra were generated using an electron energy of 70 eV and an emission current of 350 µA. The CH4 flow during PCI was set to produce an initial reagent ion ratio for the ions m/z 17+, 29+, and 41+ of 1:1: 0.25, respectively, although this ratio does drift somewhat during analysis. HPLC Analysis. The reaction of propachlor with HS- at 25 °C was followed using a Shimadzu LC-10AT Liquid Chromatograph and a Shimadzu SPD-10A UV-vis detector set at 200 nm. A Waters Nova-Pak C18 column (3.9 × 300 mm) was used to effect separations, in conjunction with an acetonitrile:H2O (40:60) mobile phase.
Results and Discussion Kinetics of Chloroacetanilide Reactions with HS- at 25 °C. Example timecourses for reactions of alachlor, propachlor, and metolachlor (racemic mixture of R and S enantiomers) with HS- at pH 9 are shown in semilogarithmic form in Figure 1. Details pertaining to solution composition are provided in the Supporting Information (Table S1). No discernible reaction occurred in control experiments conducted in the absence of HS-; the rate of reaction of a chloroacetanilide herbicide in a bisulfide solution can hence be represented by the following general expression
- d[chloroacetanilide]/dt ) kobs[chloroacetanilide]b )
kHS-[HS-]a[chloroacetanilide]b (2)
where a and b represent the order of reaction with respect to [HS-] and the chloroacetanilide in question, respectively. If b ) 1
ln [chloroacetanilide] ) ln [chloroacetanilide]o - kobs‚t (3) The high degree of linearity of the semilogarithmic plots shown in Figure 1 over several half-lives is indicative of a first-order dependence on the concentration of the chloroacetanilide herbicide in question. The reaction order in [HS-] was determined by regressing log kobs versus log [HS-]:
log kobs ) log kHS- + a‚log [HS-]
(4)
Linear regression analysis of log kobs versus log [HS-] yielded a slope (a) equal to 0.90 ( 0.12 for propachlor, 1.13 ( 0.27 for alachlor, and 0.87 ( 0.04 for metolachlor (where the stated uncertainties reflect the 95% confidence limits). Although these results might seem to indicate that the reaction order is significantly less than 1.0 with respect to [HS-] in the case of metolachlor, plots of kobs values against [HS-] (Figure 2) nonetheless fit a linear model quite well. Furthermore, the intercepts of the regressions are not significantly different from zero (at the 95% confidence level), VOL. 36, NO. 19, 2002 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 1. Example timecourses for reactions of chloroacetanilide herbicides with HS- at 25 °C and pH 9 (a) propachlor; (b) alachlor; and (c) (R, S) metolachlor. All experiments were conducted in deoxygenated 0.1 M NaCl/0.02 M sodium tetraborate buffer. Note that experiments conducted with (S)-metolachlor (98.5% enantiomeric purity) gave results that were indistinguishable from those obtained using the (R, S) mixture. consistent with the lack of reaction in buffer control experiments. It is reasonable to describe the reaction of theseherbicides with HS- as an overall second-order process, first-order both in HS- and in chloroacetanilide concentrations. A value for the second-order rate constants (kHS-, slope of kobs versus [HS-] plot) was therefore computed by forcing the intercept through zero; this is mathematically equivalent to computing kHS- ) kobs/[HS-] and then averaging the results for each chloroacetanilide. Results are given in Table 1. In the case of metolachlor and alachlor, some experiments were conducted at lower pH values (7.7-8.8); results are included in Figure 2 for purposes of comparison with kobs values measured at pH 9. The dependence of kobs on [HS-] for these solutions was consistent with data for higher pH; the 95% confidence limits of kobs fall within the 95% confidence intervals obtained from the linear regression of kobs versus [HS-] for the pH 9.0 data. This is as would be expected if HS- (rather than S2-) were the principal reactive species in the pH range investigated. The reactivity of the herbicides decreases in the following order: propachlor > alachlor > metolachlor. This parallels the reactivity trend reported for nonenzymatic conjugation by glutathione (39). Wilber and Wang (27) also reported 4068
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FIGURE 2. Plot of pseudo-first-order rate constant kobs versus [HS-] for reactions of HS- with (a) propachlor; (b) alachlor; and (c) (R, S) metolachlor. Solid lines represent linear regressions of kobs values determined at pH 9.0 (solid symbols); dashed lines represent 95% confidence intervals. Pseudo-first-order rate constants for reactions of alachlor and metolachlor in solutions at pH 7.5-8.8 are indicated by open symbols. These are shown for purposes of comparison and are not included in the regressions.
TABLE 1. Second-Order Rate Constants for Reactions of Chloroacetanilide Herbicides with HS- and Sn2- at 25.0 °C chloroacetanilide propachlor alachlor metolachlor
kHS- (M-1 s-1)a
kSn2- (M-1 s-1)
5.03(( 0.30) × 10-2 2.90(( 0.31) 1.50(( 0.19) × 10-2 1.63(( 0.06) × 10-1 2.50(( 0.13) × 10-3 8.05(( 0.30) × 10-2 b
kSn2-/ kHS58 11 32
a Second-order rate constant determined from HS- solutions at pH 9 (see Figure 2). b Second-order rate constant determined from multiple regression analysis of kobs values for three polysulfide solutions containing known but different [HS-] and Σ[Sn2-].
propachlor to be more reactive with HS- than alachlor, although a different order (alachlor > propachlor) was noted by Stamper et al. (28). One possible explanation for this difference could be if polysulfides (generated through oxidation of HS- by adventitious O2) were present in some of Stamper’s solutions, as polysulfides are considerably more reactive than HS- (see below). Activation Barriers for Reaction with HS-. Statistically significant differences in kHS- were observed for the three chloroacetanilide herbicides investigated. The most reactive chloroacetanilide, propachlor, was 20-fold more reactive than the least reactive compound, metolachlor. Subtle variations
FIGURE 3. Temperature dependence of reactions of chloroacetanilide herbicides with HS-. Rate constants designated by solid symbols were determined in 0.1 M NaCl/0.02 M sodium tetraborate buffer at pH 9.0. Open symbols for propachlor and alachlor at 37.5 °C were conducted at pH 6.7 in 0.15 M NaCl/0.5 mM phosphate buffer. Solid lines represent linear regressions to all data shown.
TABLE 2. Calculated Activation Barriers for Reactions of Chloroacetanilide Herbicides with HS-a herbicide
∆Gq (kJ/mol)b
∆Hq (kJ/mol)
∆Sq (J/mol‚K)
propachlor alachlor metolachlor
80.4 ( 13.3 82.8 ( 14.6 87.8 ( 14.2
57.7 ( 11.4 57.9 ( 11.9 63.8 ( 11.9
-76.3 ( 23.1 -83.6 ( 28.3 -80.7 ( 26.4
a
Stated uncertainties represent 95% confidence limits. at 298.15 K.
b
Calculated
in structure of the chloroacetanilide herbicides no doubt influence their reactivity. To explore whether these differences result from enthalpic or entropic effects, kHS- was measured in buffered solution (pH 9) over the temperature range of 15.0 to 58.0 °C, and activation parameters were determined. No measurable reaction was observed in the absence of HS- at these temperatures over comparable time periods in buffer control experiments. A few experiments were also conducted at pH 6.7 for propachlor and alachlor at 37.5 °C to test whether the neutral species H2S could be contributing to the observed rate of reaction at higher temperatures. Under these conditions kH2S proved too small to be experimentally accessible, and the effect of pH on kobs was entirely consistent with HSas the sole reactive species. Data for each herbicide were plotted as shown in Figure 3 according to a linearized version of the Eyring expression
ln(kHS- /T) ) ln(k/h) - ∆Hq/RT + ∆Sq/R
(5)
where k is Boltzmann’s constant, h is Planck’s constant, R is the gas constant, T is temperature in Kelvin, and ∆Hq and ∆Sq are the enthalpic and entropic contributions, respectively, to the overall activation barrier ∆Gq. Linear regression analyses of the data yielded ∆Hq and ∆Sq values summarized in Table 2. Visual inspection of Figure 3 reveals a strikingly parallel nature to the plots. Indeed, differences in slopes (and thus ∆Hq values) are not significant for the three herbicides at the 95% confidence level. This would tend to suggest that reactivity contrasts stem primarily from entropic factors, even though our results do not indicate statistically significant
differences in ∆Sq values. In this context, however, precise experimental measures of ∆Sq are notoriously difficult to determine (40). The computed uncertainties in ∆Sq are of sufficient magnitude as to potentially account for appreciable differences in the overall reaction barrier; differences in steric hindrance stemming from variations in N-alkyl (or Nalkoxyalkyl) substituents or phenyl ring substituents could still account for the observed 20-fold difference in reactivity between the three herbicides. Kinetics of Chloroacetanilide Reactions with Sn2- at 25 °C. The dependence of the pseudo-first-order rate constant kobs on polysulfide concentration was determined by conducting experiments at constant pH and temperature but at varying Σ[Sn2-]. Experimental solutions contained substantial concentrations of HS- in addition to Sn2- species. Secondorder rate constants (kSn2-) for propachlor and alachlor were estimated by dividing pseudo-first-order rate constants by Σ[Sn2-], after first correcting kobs to account for the contribution from reaction with HS-. Confidence limits on the secondorder rate constants were calculated by propagating the errors associated with analysis of the reduced sulfur nucleophile and the pseudo-first-order rate constants. Resulting kSn2values are given in Table 1. The validity of computing kSn2- through subtracting kHS[HS-] from kobs was tested by conducting experiments for metolachlor in three solutions with different ratios of [HS-] to Σ[Sn2-]. Results are shown in Figure 4. The second-order rate constants (kSn2- and kHS-) were simultaneously determined through nonlinear regression analysis of the combined data, fitting experimental data to numerically integrated solutions to the following system of differential rate expressions:
d[metolachlor]/dt ) -(kHS-[HS-]i + kSn2-Σ[Sn2-]i)‚[metolachlor];
i ) 1-3 (6)
In computing kSn2- and kHS-, experimentally determined reduced sulfur nucleophile concentrations were used to constrain [HS-] and Σ[Sn2-]. The resulting kHS- value, 2.30 ((0.14) × 10-3 M-1 s-1, is not significantly different from that determined in solutions containing bisulfide alone (2.50 ((0.13) × 10-3 M-1 s-1). For all three chloroacetanilide herbicides, a greater reactivity toward Sn2- than toward HS- was observed. The ratio of kSn2-/kHS- varied from 11 to 58 and was greatest for the least hindered chloroacetanilide, propachlor. Similar relative reactivity has been reported for SN2 reactions of these nucleophiles with other alkyl halides; for example, 1-bromohexane is 60-fold more reactive toward Sn2- than toward HS- (29), and kSn2-/kHS- is 45 for 1-chlorohexane (32). Factors contributing to the greater nucleophilicity of Sn2- relative to HS- are discussed in Lippa and Roberts (35). Products of Chloroacetanilide Reactions with HS-. Propachlor, alachlor, and metolachlor all reacted with HSvia displacement of chlorine to form mercaptoacetanilides. Analysis of n-hexane extracts of aliquots of the reaction mixture that were acidified to pH 4 in all cases yielded mass spectra consistent with the RSH derivatives mercaptopropachlor [2-mercapto-N-isopropylacetanilide; Figure 5a], mercaptoalachlor [2-mercapto-2′,6′-diethyl-N-(methoxymethyl)acetanilide; Figure 5b], and mercaptometolachlor [2-chloro-2′-ethyl-6′-methyl-N-(1-methyl-2-methyoxyethyl)acetanilide; Figure 5c] as the major products. Note that the mass spectrum for mercaptometolachlor compares closely to that provided in ref 41. Although the lack of authentic reference materials precluded definitive identifications or quantitation of mass balances, a yield of 82% can be estimated in the case of mercaptometolachlor by assuming 100% VOL. 36, NO. 19, 2002 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 5. (a) Mass spectra (EI) for major products obtained in reaction of HS- with (a) propachlor; (b) alachlor; and (c) metolachlor, following acidification of reaction solution to pH 4 in deoxygenated phthalate buffer and extraction with n-hexane. Retention times (relative to parent herbicides) on a DB-200 column are 1.004 for propachlor, 1.000 for alachlor, and 0.993 for metolachlor. (Note that better separation of parent compounds and mercapto-substituted products is achieved on a DB-5 column.)
FIGURE 4. Comparison of (R, S) metolachlor reactivity at pH 9 in (a) solution containing 3.6 mM [HS-] and 7.3 µM Σ[Sn2-]; (b) solution containing 6.7 mM [HS-] and 6.9 mM Σ[Sn2-]; and (c) 2.9 mM [HS-] and 3.9 mM Σ[Sn2-]. Note differences in time scale. Solid lines represent predicted decay according to second-order rate constants obtained from simultaneous fit of data from all three timecourses and are not fits to any individual experiment. Solution (a) contains 0.1 M NaCl/0.02 M sodium tetraborate buffer, while solutions (b) and (c) contain 0.1 M NaCl/0.04 M sodium tetraborate buffer. extraction efficiency and a molar response factor (utilizing fragment ion m/z 162) similar to that of metolachlor. An additional, late-eluting product was observed for solutions spiked with higher concentrations (∼100 µM) of propachlor and alachlor; mass spectra were consistent with the thioethers (RSR) 2,2′-thio-bis-[N-(isopropyl)acetanilide] and 2,2′-thiobis-[2′′,6′′-diethyl-N-(methoxymethyl)acetanilide], respectively. Note that the parent ions for the mercapto-substituted chloroacetanilides were not always evident in EI analyses. In the case of the product of alachlor reaction with HS-, the m/z 235 ion might even be interpreted to represent the parent ion for the reductive dechlorination product, deschloroalachlor [2′,6′-diethyl-N-(methoxymethyl)acetanilide], although published mass spectra (10, 42, 43) for the latter differ in several respects from Figure 5b. PCI techniques were therefore employed to corroborate the EI identification of mercaptopropachlor and mercaptoalachlor. Although the resulting mass spectra (32) exhibit protonated and reagent gas adduct fragments [(f + H)+ and (f + C2H5)+, respectively] consistent with the postulated mercapto-substituted derivatives of the two herbicides, the (M + H)+ ion was discernible only for propachlor. 4070
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Identification of the sulfur-substituted products resulting from chloroacetanilide herbicide reactions with HS- was further supported by analyses of methylated reaction mixtures. The EI mass spectra for these derivatized products are provided in the Supporting Information (Figure S1). Although the abundances of the parent ions expected for the mercaptomethyl-substituted derivatives of alachlor and metolachlor are very small relative to the fragment ions, parent ions are nonetheless present substantially above baseline noise, and the mass spectra are consistent with the anticipated mercaptomethyl-substituted (RSCH3) derivatives. The overall second-order nature of the reactions of HSwith chloroacetanilides, along with the nature of the products in which chlorine has been substituted by a sulfhydryl group, is suggestive of a bimolecular nucleophilic substitution (SN2) mechanism. We note, however, that kHS- values for the chloroacetanilides are more than 60× greater than we find (32) for the reaction of HS- with 1-chlorohexane, despite the considerably lesser steric hindrance within the latter organohalide. The mechanism through which chloroacetanilides react with environmental nucleophiles, and the origin of this apparent activating influence, are addressed in greater detail in Lippa (32). Products of Chloroacetanilide Reactions with Polysulfides. Limited attempts were made to identify the substitution products (RSnH) that might be anticipated from reactions with polysulfides. Spent reaction solutions were acidified to pH 4, extracted with deoxygenated n-hexane, and analyzed via GC/MS (EI). One product of alachlor had a mass spectrum similar to mercaptoalachlor, although it displayed a much longer retention time and a very broad peak. Several additional alachlor and propachlor products were observed with retention times and mass spectra suggestive of coupling products (RSnR), including the RSSR derivative bis-2-thio-
SCHEME 1
TABLE 3. Predicted Half-Lives of Chloroacetanilides at 25.0 °C in a Salt Marsh Sediment Porewater at pH 7.2 Containing 3.4 mM [HS-] and 0.33 mM Σ[Sn2-]a,b chloroacetanilide herbicide
half-life (t1/2)c
% of reaction attributable to Sn2- d
propachlor alachlor metolachlor
10 min 1.8 h 5.5 h
85 51 76
a These values represent the maximum concentrations of these reduced sulfur species reported (46) for Great Marsh, DE sediment porewaters. b [HS-] calculated from 5.06 mM [H2S]T at pH 7.2. c t1/2 ) 0.693/(kSn2-Σ[Sn2-] + kHS-[HS-]). d Computed as 100‚kSn2-Σ[Sn2-]/ (kSn2-Σ[Sn2-] + kHS-[HS-]).
FIGURE 6. EI total ion chromatograms (TICs) for derivatized products obtained in reaction of polysulfides with (a) propachlor, (b) alachlor, and (c) metolachlor. Products were extracted into n-hexane following methylation with CH3I. The abbreviations rt denotes retention time (min), and 1S, 2S, and 3S denote the number of sulfur atoms present in the acetanilide products. Under these conditions, propachlor, alachlor, and metolachlor elute at 12.31, 14.62, and 15.35 min, respectively, on the DB-200 column described. Mass spectra are shown in Figures S2-S4 of the Supporting Information. 2′,6′-diethyl-N-(methoxymethyl)acetanilide, previously reported by Stamper et al. (28) to result from alachlor reaction with HS-. Such coupling products could result either from oxidation of RSH or RSnH products or from reaction of RSnwith a second unreacted chloroacetanilide molecule. More definitive studies were conducted by methylating products with excess CH3I, followed by extraction into n-hexane and analysis via GC/MS (EI). Products of all three herbicides exhibited mass spectra consistent with the presence of multiple sulfur atoms (RSnCH3, with n > 1). The total ion chromatograms (TICs) are provided in Figure 6; the corresponding EI mass spectra are provided in the Supporting Information (Figures S2-S4). The major products for reactions with Sn2- in each case appeared to be the disulfur (2S)substituted methyl derivatives (RSSCH3), with minor contributions of the monosulfur (1S)- and the trisulfur (3S)substituted methyl derivatives. Note that the monosulfur substituted product may form through reaction of the chloroacetanilide herbicide with HS- present in the solution. Several unidentified minor peaks (comprising 5-10% of the total peak area) were also observed that were clearly related to acetanilides, with mass spectra similar to the RSnCH3 derivatives and fragments with isotope ratios indicative of multiple sulfur atoms. It is important to recognize that the number of sulfur atoms we measure in these methylated polymercaptoacetanilide derivatives may not reflect the identity of those products initially formed during reaction with Sn2-. For example, reaction of S42- with propachlor may initially generate the tetrasulfur-substituted product, which may further react with HS-, polysulfides, or CH3S- (the latter originating from reaction of CH3I with HS-) in solution to yield a degradate with a shorter sulfur chain (Scheme 1, where RS- ) HS-, Sn2-, or CH3S-):
Sulfur chains are known to have a conjugative character, in which the 3p lone pairs on the sulfur atoms can be delocalized through their empty 3d orbitals in the excited state (44). Delocalization may also occur through hyperconjugation between the 3p lone pair on a sulfur atom and the antibonding σ* molecular orbital of the neighboring S-S bond (45). Such delocalization produces resonance structures in which sulfur atoms may be capable of functioning either as electron donors or as electron acceptors (44). This increases the susceptibility of the polysulfide chain to nucleophilic attack. We note that methylation of HS-/Sn2- solutions (via reaction with excess CH3I) results in dimethylpolysulfides with a distribution of sulfur chain lengths that is greatly altered relative to the unmethylated polysulfides (37). Environmental Significance. Our results suggest HS- and Sn2- are sufficiently reactive as to control the fate of chloroacetanilides within sulfidic environments, provided these reduced sulfur species are present at sufficiently high concentrations (as is common in hypoxic marine environments). This can be demonstrated by multiplying secondorder rate constants given in Table 1 by the maximum concentrations of [HS-] and Σ[Sn2-] reported by Boule`gue et al. (46) to predict the half-lives of chloroacetanilides in hypoxic salt marsh sediment porewaters. The results, summarized in Table 3, indicate half-lives predicted to range from 10 min for propachlor to 5.5 h for metolachlor. Note that even though the concentration of polysulfides is an order of magnitude lower than the bisulfide concentration (0.33 mM versus 3.4 mM, respectively), polysulfides would still account for the major portion of the overall rate of reaction, a reflection of their high nucleophilicity. Such reactions should therefore proceed very rapidly in sulfidic sediment porewaters. The resultant half-lives are much shorter than the characteristic time of 38 days (47) for exchange of a conservative species within the top 10 cm of unconsolidated Chesapeake Bay sediment with the overlying water column. The characteristic time for sediment-water exchange is short relative to the mean hydraulic residence time of ∼8 months for the Chesapeake Bay (48). Abiotic reactions in underlying sediment porewaters could, therefore, substantially influence the fate of chloroacetanilide herbicides in this important estuary. Abiotic reactions with reduced sulfur species may also represent important reactions in hypoxic agricultural soils, such as might develop under waterlogged conditions. Polysulfides are frequently employed as a 30% aqueous VOL. 36, NO. 19, 2002 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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solution in commercial preparations used for agricultural soil conditioning and for fungal, mite, and insect control (49). Elemental sulfur is also commonly added to soil in order to take advantage of its fungicidal qualities as well as its role as an essential nutrient. Average annual use of elemental sulfur as a fungicide in the U.S. from 1990 to 1993 was 38 million kg/yr (50). Under hypoxic conditions, this elemental sulfur could undergo dissimilatory reduction by soil microorganisms to generate polysulfides and HS- (51). To the best of our knowledge, the resulting mercaptoacetanilides (and their analogues possessing multiple sulfur atoms) have never been sought in natural water samples, nor does information exist pertaining to their (eco)toxicity. The monomercapto substituted RSH products will likely be protonated (and hence uncharged) under environmentally relevant conditions (pKa values range from 8.2 to 10.7 for the thiol group of N-alkyl substituted 2-mercaptoacetanilides in aqueous dioxane solutions (52)); this will influence their bioavailability. The RSn- degradates (with n > 1) are more likely to occur in the deprotonated form, as the presence of multiple sulfur atoms is known to markedly increase the acidity of H2Sn and HSn- species relative to H2S and HS- (45). Because antigens used to develop ELISA analyses for chloroacetanilides are generated through displacement of the chlorine atom by sulfhydryl groups of proteins, degradates containing sulfur in the 2-position have been reported by some researchers to display particularly pronounced crossreactivity (22). Even if the degradation products identified herein are found to exhibit low bioavailability or toxicity, they may therefore contribute to “false positives” in immunoassay-based analyses of chloroacetanilides. The ethane sulfonic acid derivatives of chloroacetanilide herbicides have previously been shown to result from biotransformations initiated by enzymatic or nonenzymatic conjugation with glutathione (or related thiols (17, 53)). Proposed steps leading to the formation of the ESA derivatives from the initial conjugates of propachlor and acetochlor (17, 53) involve RSH intermediates identical to those we identify as the products of abiotic reactions of chloroacetanilides with HS-. Reactions with HS- or polysulfides in waterlogged soils could thus provide an entirely abiotic route for formation of ESA derivatives of chloroacetanilides, once the sulfursubstituted degradates undergo oxygenation upon their diffusion into aerobic environments.
Acknowledgments We are grateful for the detailed comments provided by two anonymous reviewers. Partial funding for this work was provided by a USDA National Research Initiative Grant 9735107-4358 to S. J. T., Y. P. C., and A. L. R. Additional funding was provided by an NSF Young Investigator Award (Grant BES-9457260) to A. L. R.; by a dissertation fellowship (19992000) to K. A. L. granted by the American Association of University Women; and by a National Science Foundation Graduate Research Fellowship for D. L. C.
Supporting Information Available A table showing the composition of HS- solutions used to determine second-order rate constants for reaction of propachlor, alachlor, and metolachlor at 25 °C and figures showing EI mass spectra of methylated products obtained in reaction of propachlor, alachlor, and metolachlor with HS- and with Sn2-. This information is available free of charge via the Internet at http://pubs.acs.org.
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Received for review March 1, 2002. Revised manuscript received July 3, 2002. Accepted July 9, 2002. ES0206285
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