Environ. Sci. Technol. 2003, 37, 2668-2674
Enantioselective Degradation of Metalaxyl in Soils: Chiral Preference Changes with Soil pH IGNAZ J. BUERGE,* THOMAS POIGER, MARKUS D. MU ¨ LLER, AND HANS-RUDOLF BUSER Plant Protection Chemistry, Swiss Federal Research Station, CH-8820 Wa¨denswil, Switzerland
Chiral pesticides are often degraded enantio-/stereoselectively in soils. Degradation is typically studied with one or a small number of soils so that it is not possible to extrapolate the findings on chiral preference to other soils. For this study, the fungicide metalaxyl was chosen as a “chiral probe” to investigate its enantioselective degradation in 20 different soils, selected primarily to cover a wide range of soil properties (e.g., acidic/alkaline, aerobic/ anaerobic) rather than to consider soils of agricultural importance. Racemic metalaxyl was incubated in these soils under laboratory conditions, and the degradation of the enantiomers as well as the enantioselective formation/ degradation of the primary major metabolite, metalaxyl acid, was followed over time, using enantioselective GC-MS after ethylation with diazoethane. In aerobic soils with pH > 5, the fungicidally active R-enantiomer was degraded faster than the S-enantiomer (kR > kS), leading to residues with a composition [S] > [R]. However, in aerobic soils with pH 4-5, both enantiomers were degraded at similar rates (kR ≈ kS), and in aerobic soils with pH < 4 and in most anaerobic soils, the enantioselectivity was reversed (kR < kS). These considerable soil-to-soil variations were observed with soils from locations close to each other, in one case even within a single soil profile. Liming and acidification of a “nonenantioselective” soil prior to incubation resulted in enantioselective degradation with kR > kS and kR < kS, respectively. While the enantioselectivity (expressed as ES ) (kR - kS)/(kR + kS)) of metalaxyl degradation in aerobic soils apparently correlated with soil pH, no such correlation was found for metalaxyl acid. Reevaluation of published kinetic data for the herbicides dichlorprop and mecoprop indicated similar correlations between soil pH and ES as for metalaxyl.
Introduction Many pesticides are chiral compounds and consist of two or more enantiomers/stereoisomers, which may differ in biological activity, toxicity, effects on beneficial and nontarget organisms, and environmental fate (1-3). Enantio-/stereoselective degradation in soils was observed for various chiral pesticides such as dichlorprop (4-9), mecoprop (5, 6, 8, 9), metalaxyl (10-12), fluazifop-butyl (13), and ruelene (7), whereas other compounds showed a less pronounced enantio-/stereoselectivity, e.g., metolachlor and dimethen* Corresponding author phone: ++41 1 783 63 83; fax: ++41 1 780 63 41; e-mail:
[email protected]. 2668
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amid (10). However, degradation experiments were usually carried out with only one or a small number of soils, and it is not known whether the same enantio-/stereoselectivity would be observed in other soils. The environmental factors which affect degradation processes in soils are far from being understood, even less those that drive the enantio-/stereoselectivity of degradation. For example, incubation of the chiral pesticides methyldichlorprop, dichlorprop, and ruelene in soil suspensions suggested that environmental changes, such as by deforestation, soil warming, or organic nutrient enrichment, can alter the enantioselectivity of pesticide degradation (7). Another study reported that peat amendment to calcareous soils may reverse the enantioselectivity of the degradation of dichlorprop and mecoprop (9). However, in these studies, it remained unclear which soil parameters, responsible for the observed shifts in enantioselectivity, had actually changed. As the enantio-/stereoselective dissipation of a compound is generally attributed to biological degradation, it is reasonable to study the soil parameters which have a major impact on soil microbiology, such as pH, organic carbon, nutrients, redox conditions, moisture, and temperature (14). For the present study, the fungicide metalaxyl was chosen as a “chiral probe” to investigate its enantioselective degradation in different soils. Twenty soils were selected, primarily to cover a wide range of soil properties rather than to consider soils of agricultural importance. Metalaxyl has an asymmetrically substituted C atom (“C-chirality”) and consists of a pair of enantiomers, where the fungicidal activity almost entirely originates from the R-enantiomer ((15, 16), Figure 1). In some countries, racemic metalaxyl has been replaced by metalaxyl-M, consisting of >97% of the R-enantiomer, thus allowing lower application rates and reducing potential side-effects (17). Metalaxyl is used to control phytopathogenic fungi (peronosporales) on potatoes, hops, tobacco, vines, and various other crops (18, 19). The systemic fungicide interferes with the protein synthesis by inhibiting the biosynthesis of ribosomal RNA (18, 19). In soils, metalaxyl was found to be degraded enantioselectively with preferential degradation of the R-enantiomer leading to residues with a composition [S] > [R] (10-12). Metalaxyl carboxylic acid (MX-acid) was identified as primary major metabolite, formed by cleavage of the ester bond (18-20). MX-acid is also C-chiral and its formation from metalaxyl occurred with retention of configuration (11). Metalaxyl and MX-acid were enantiomerically stable in soil, i.e., no interconversion of enantiomers was found (11), an important prerequisite for the application of enantiopure compounds. Some observations, however, indicated a faster degradation of S-metalaxyl and thus suggested a “reversed” enantioselectivity under certain conditions. For instance, water from a drainage canal in an agricultural area of Switzerland (10) and groundwater from an agricultural area in Portugal (unpublished data) showed residues of metalaxyl with a composition [R] > [S] from the application of rac-metalaxyl. Direct evidence for a “reversed” enantioselectivity was obtained from incubation of rac-metalaxyl in sewage sludge under anaerobic conditions, which showed a faster degradation of the S-enantiomer (10). This study will help to interpret these findings. It will be shown that soil pH and redox conditions are important factors affecting the enantioselectivity of metalaxyl degradation. 10.1021/es0202412 CCC: $25.00
2003 American Chemical Society Published on Web 05/10/2003
FIGURE 1. Structure of R- and S-metalaxyl.
FIGURE 2. Map of sampling sites in Switzerland from the Midland region (soils # 3-9, 19-20), the Jura region (# 1-2), the Alpine Upland (# 10-12, 15-18), and the Alps (# 13-14, see Table 1).
Experimental Section Chemicals. Analytical standards (purities, >99%) of racmetalaxyl (methyl N-(methoxyacetyl)-N-(2,6-xylyl)-DL-alaninate) and rac-metalaxyl carboxylic acid (N-(methoxyacetyl)N-(2,6-xylyl)-DL-alanine, MX-acid) were obtained from Syngenta (Basel, Switzerland), and alachlor, used as internal standard, was from Monsanto (St. Louis, MO). Soil Samples. Soil samples were collected between October 12 and 18, 2001 in several regions of Switzerland, including the Midland region (altitudes, 365-680 m), the Jura region (430-980 m), the Alpine Upland (960-1110 m), and the Alps (1940-2480 m, Figure 2). The soils, mostly forest soils, belong to various classes such as cambisols, luvisols, rendzinas, gleysols, podzols, and rankers. Geographical coordinates, altitude, soil unit, sampled horizon and depth, and some soil characteristics (texture, organic carbon, and pH) are listed in Table 1. Standard equipment was used for sampling. Soils from water-saturated horizons were immediately placed into polyethylene bags and firmly closed to minimize contact with oxygen. The soils were kept in the dark at ≈4 °C until used within a few days. None of the soils had previously been treated with metalaxyl, which is known to enhance its rate of degradation (21-24). Incubation of Metalaxyl in Soils under Aerobic Conditions. The soil samples intended for incubation of metalaxyl under aerobic conditions (soils # 1-14, Table 1) were sieved (2 mm) and carefully air-dried at room temperature to obtain a water content of 10-30 g per 100 g dry soil. One soil sample, the Ah horizon of a podzol (soil # 10), was left at field moisture (≈ 200 g/100 g dry soil), because of its high organic carbon content (46%). Incubation experiments were carried out in 300-mL wide-mouth glass jars, covered with aluminum foil and lid. Portions of moist soil (corresponding to 100 g dry weight) were filled into the jars and fortified by dropwise addition of ≈1 mL of an aqueous stock solution containing ≈220 µg of rac-metalaxyl (spike level, ≈2.2 µg/g dry soil). This spike level corresponds up to 5 times the recommended application rate. The soils were carefully mixed and then
incubated at 20 ( 2 °C in the dark for up to 106 days. Separate incubations with rac-MX-acid were carried out with a smaller number of these soils (initial concentration, ≈2.1 µg/g, Table 2). The jars were opened every 1-3 days to ensure aerobic conditions. The water content was regularly checked by weighing and kept constant by addition of distilled water. At appropriate intervals, first immediately after fortification and mixing, aliquots of 2.5-10 g were removed and transferred into glass vials for extraction and analysis (see below). Incubation of Metalaxyl in an Acid- and Base-Treated Soil under Aerobic Conditions. To investigate whether a changed soil pH had any influence on the enantioselectivity of metalaxyl dissipation, a luvisol (soil # 6) was treated by addition of (i) 10 mL of 0.05 M H2SO4 and (ii) 1.0 g of CaCO3, suspended in 10 mL of water, to the field-moist soil (100 g dry weight). The soils were mixed and dried to obtain a water content of ≈15 g/100 g soil, similar to the experiment with the untreated, native soil. After a preincubation time of 14 days, the two treated soils were fortified with rac-metalaxyl and incubated in the same way as the other aerobic soils. The pH values (CaCl2), measured 56 days after acid and basetreatment, were 3.9 and 7.0, respectively (4.0 in the untreated soil). The soil thus showed a high acid neutralizing capacity. The pH of the base-treated soil was primariliy defined by the dissolution of CaCO3. Incubation of Metalaxyl in Soils under Anaerobic Conditions. Water-saturated soils (soils # 15-20, Table 1) were incubated under anaerobic conditions. These experiments were carried out in 60-mL amber screw cap glass bottles with Teflon-lined silicone septum. Portions of 25 g of the field-wet soils were filled into separate bottles (five bottles, each dedicated for one sampling point), in a glovebox under high-purity N2 (O2 < 0.1 ppm) atmosphere. Twentyfive milliliters of a mineral water (Henniez, Switzerland, with a low sulfate concentration), previously sparged with N2 to remove O2, was added to each bottle. To favor anaerobic conditions, the soils were preincubated during 4 weeks prior to addition of rac-metalaxyl. The soil slurries were fortified by adding 20 µL of a N2-sparged, aqueous stock solution of rac-metalaxyl (1 µg/µL) with a Hamilton syringe through the septum (spike level, ≈2 µg/g). The samples were then incubated in the dark at 20 ( 2 °C for up to 88 days. To check the redox conditions, separate soil slurries were spiked with the redox indicator resazurin to a concentration of ≈2 mg/L. The color of the indicator changes from pink/ blue (pH-dependent) to colorless upon reduction (E0′ ) 51 mV, (25)). UV-vis spectra (between 450 and 650 nm) of filtered soil slurries were periodically recorded to determine the redox conditions. Extraction of Metalaxyl and MX-Acid from Soil. Soil samples (2.5-10 g) from aerobic incubations were immediately mixed with 10 mL of methanol and kept at -20 °C until extracted. Prior to extraction, alachlor was added as internal standard (10 µg in 100 µL of methanol). After vigorous shaking (≈1 min), the samples were centrifuged (≈2000 g for 15-60 min) and the supernatants were transferred into glass vials. This procedure was repeated with 10 mL of acetone/ distilled water (1:1) and with 10 mL of distilled water in order to improve extraction of MX-acid. The combined extracts (methanol/acetone/water) were acidified with H2SO4 to “pH ≈ 2” and partitioned three times with 5-mL portions of dichloromethane. The combined dichloromethane phases were evaporated at room temperature with a gentle draught of air, and the residues were redissolved in 5 mL of dichloromethane. Anaerobic soil slurries were extracted similarly, except for the sequence of the solvents (immediate centrifugation of water in the sample, 20 mL of water/acetone, 20 mL of methanol) and the volume of dichloromethane used for partitioning (3 × 10 mL). VOL. 37, NO. 12, 2003 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 1. Sampling Sites, Characterization of the Soils Studied, and Kinetics of Metalaxyl Dissipationg coordinates
altitude [m]
Oesterliwald, foresta Schitterwald, foresta Schachen, foresta Wa¨ denswil, gardenb
47°29′N/8°18′E
430
47°16′N/7°29′E
980
rendzina, Ahk
47°03′N/7°38′E
541
47°13′N/8°41′E
Buechberg, foresta Winzlerboden, foresta Rafz 1, forest Rafz 2, forest Steig, foresta Guberwald, foresta Guberwald, foresta Guberwald, foresta Stillberg, alpine grassland Bra¨ mabu¨ el, alpine grassland
soil site 1 2 3 4 5 6 7 8 9 10 11 12 13 14
15 Bru¨ nnli, foresta 16 Heumoosegg 1, foresta 17 Heumoosegg 2, forest 18 Gottschalkenberg, foresta 19 Abist, foresta
depth [cm]
soil unit (FAO)
soil texture
Incubation under Aerobic Conditions rendzina, Ahk ≈20 clay loam
Corg DT50 pH [%] (CaCl2)c [d]
kR [d-1]
kS [d-1]
ES
0.022
0.64
5.2
7.1
9 ≈0.18
loamy sand
2.4
7.1
9
0.155 0.030
0.61
loam
1.6
7.1
13
0.090 0.027
0.52
510
calcic cambisol, ≈20 Ahk cambisol, Ap ≈10
loam
1.6
6.4d
30f 32
0.060 0.015 0.031 0.014
0.51 0.37
47°36′N/8°38′E
425
luvisol, Ah
≈10
loam
1.1
4.1
32f
0.036 0.033
0.05
47°37′N/8°37′E
365
luvisol, Ah
≈10
loamy sand
1.6
4.0
38
0.019 0.018
0.03
47°37′N/8°33′E 47°37′N/8°33′E 47°32′N/8°37′E 47°01′N/8°12′E
535 540 680 960
≈20 ≈20 ≈10 ≈10
67 16 17 76
0.010 0.038 0.033 0.004
960
sandy loam 0.9 silt loam 2.1 loam 3.2 sandy clay 46 loam sand 0.5
4.1 3.7 3.6 3.0
47°01′N/8°12′E
3.5
127
47°01′N/8°12′E
960
46°47′N/9°52′E
1940
46°47′N/9°52′E
2480
luvisol, Ah, EB luvisol, Ah, EB luvisol, Ah orthic podzol, Ah orthic podzol, E orthic podzol, Bs ferric podzol, Ah ranker, Ah
47°07′N/8°33′E 47°06′N/8°33′E 47°06′N/8°33′E 47°09′N/8°40′E 47°38′N/8°39′E
20 Galmitzkanal, 46°58′N/7°09′E sediment of a drainage canal
≈30
≈40 ≈90
0.011 0.049 0.052 0.018
-0.01 -0.12 -0.23 -0.62
0.005 0.006 -0.07
0.5
5.2
66
0.014 0.008
≈10
sandy clay loam sandy loam
6.3
3.4
39
0.012 0.026 -0.36
≈10
loam
4.2
4.6
92
0.006 0.010 -0.26
0.6
5.0e
160
0.003 0.005 -0.25
2.3
5.0e
26
0.014 0.046 -0.53
7.7
5.0e
17
0.034 0.047 -0.16
2.2
6.5e
71
0.003 0.022 -0.71
0.2
7.1e
29
0.025 0.024
0.02
3.5
6.9e
39
0.022 0.015
0.20
Incubation under Anaerobic Conditions 1065 gleyic cambisol, ≈50 sandy loam Bg 1110 humic gleysol, ≈70 silty clay Brg 1110 humic gleysol, ≈20 clay loam Ah 995 humic gleysol, ≈100 clay loam BCrg 410 eutric gleysol, ≈120 silty clay BCr 432 sediment ≈20 loam
0.25
a Soil described in ref 36. b Data from refs 10 and 11. c Suspension of soil in 0.01 M CaCl ,1:2.5 (w/w). d Estimated from pH (H O) ) 7.0. e pH 2 2 (H2O). f Lag phase of ≈ 10 d. g Time for 50% dissipation, DT50, first-order rate constants for dissipation of R- and S-metalaxyl, kR and kS, respectively, and enantioselectivity, ES.
TABLE 2. Kinetics of MX-Acid Dissipatione soil site foresta
1 Oesterliwald, 3 Schachen, foresta 4 Wa¨ denswil, gardenb 8 Rafz 2, forest 9 Steig, foresta
soil unit (FAO)
pH (CaCl2)c
DT50 [d]
kR [d-1]
kS [d-1]
ES
rendzina, Ahk calcic cambisol, Ahk cambisol, Ap luvisol, Ah, EB luvisol, Ah
7.1 7.1 6.4d 3.7 3.6
183 17 33 37 60
0.004 0.038 0.014 0.023 0.014
0.004 0.045 0.030 0.015 0.010
0.05 -0.08 -0.30 -0.21 0.15
a Soil described in ref 36. b Data from ref 11. c Suspension of soil in 0.01 M CaCl ,1:2.5 (w/w). d Estimated from pH (H O) ) 7.0. e Time for 50% 2 2 dissipation, DT50, first-order rate constants for dissipation of R- and S-MX-acid, kR and kS, respectively, and enantioselectivity, ES.
Ethylation of MX-Acid. Aliquots of 0.5-mL of the dichloromethane extracts were transferred into 2-mL glass vials and carefully evaporated to dryness. The residues were then redissolved in ≈50 µL of methanol, and MX-acid was derivatized using diazoethane in diethyl ether (26, 27). This derivatization allowed distinction of metalaxyl (a methyl ester, unaffected by diazoethane) and MX-acid (derivatized to the corresponding ethyl ester, MX-acid-Et) in the subsequent GC-MS analysis (11). After derivatization and evaporation, the residues were dissolved in 1 mL of ethyl acetate. Enantioselective GC-MS. Aliquots of 1 µL of the final extracts were analyzed by gas chromatography-mass spec2670
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trometry (GC-MS), using a VG Tribrid MS (VG Analytical, Manchester, England) (28) under electron impact ionization (EI, 60 eV, 180 °C) and selected-ion-monitoring (SIM) conditions. Metalaxyl, MX-acid-Et, and alachlor were quantified using the ions m/z 249 (and 220 for confirmatory purposes), 248 (220), and 188, respectively. Separation of the enantiomers was achieved on a homemade column coated with a mixture of OV1701 and 25% (w/w) of an octakis[(bistert-butyldimethylsilyl)]-γ-cyclodextrin (16 m, 0.25 mm i.d., 0.25 µm film, refs 29 and 30). GC conditions were as follows: split/splitless injection (250 °C, 60 s splitless), temperature program: 70 °C, 2 min isothermal, 20 °C/min to 120 °C, 3
ln[R] ) ln[R]t)x - kR × (t - x) ln[S] ) ln[S]t)x - kS × (t - x)
(1)
where [R] and [S] are the concentrations of the respective Rand S-enantiomers. DT50 values, the time needed for 50% dissipation of total metalaxyl, were numerically calculated from kR and kS using the following equation:
exp(-kR × DT50) + exp(-kS × DT50) ) 1
(2)
This equation is valid for the incubation of racemic compounds that dissipate without lag-phase. In cases with lag-phase, this DT50 value was corrected by addition of x. Enantiomer ratios were defined as
ER ) [R]/[S]
(3)
The slope of the corresponding logarithmic plot, ln ER versus t, provided the difference of the rate constants, kR - kS:
ln(ER) ) ln(ER)t)x - (kR - kS) × (t - x)
FIGURE 3. Incubation of rac-metalaxyl in a cambisol (soil # 3, pH 7.1) and in a podzol (# 13, pH 3.4). Chromatograms show elution of S- and R-metalaxyl and S- and R-MX-acid-Et after incubation times of 11 d (cambisol, panel a) and 29 d (podzol, c). Retention times were measured from the start of data acquisition at 120 °C. Panels b (cambisol) and d (podzol) show the first-order dissipation of S-metalaxyl (large circles) and R-metalaxyl (large squares) and the formation and subsequent dissipation of MX-acid (small symbols). °C/min to 220 °C, isothermal hold at this temperature. The enantiomer elution order on this column was S- prior to Rfor metalaxyl and MX-acid-Et, and the enantiomer resolutions were ≈1.5 and ≈1.3, respectively (Figure 3a,c). No enantiomerization had been observed for metalaxyl and its ethyl ester analogue under these analytical conditions (11). The amounts of S- and R-metalaxyl and S- and R-MXacid-Et were determined from peak area ratios relative to the internal standard (alachlor) and in reference to suitable standard solutions of the racemic compounds. Recoveries of metalaxyl from aerobic soils, determined immediately after fortification, ranged from 114 to 169% (spike level, ≈2.2 µg/ g) and were high likely because of incomplete extraction of the internal standard. Recoveries of metalaxyl from anaerobic soils and recoveries of MX-acid from aerobic soils were satisfactory (97-108% and 83-109%, respectively, spike level, ≈2 µg/g). Concentrations were corrected for recoveries. Blank determinations indicated negligible amounts of metalaxyl in MX-acid fortified soils (spike level, ≈2.1 µg/g) and negligible amounts of MX-acid in metalaxyl fortified soils at the beginning of the incubations (spike level, ≈2.2 µg/g). Limits of detection (mean concentration + 3 SD in these blank samples) were ≈0.01 µg/g for both enantiomers of metalaxyl (formation of the methyl ester when samples were ethylated, presumably due to some diazomethane present in the reagent) and ≈0.03 µg/g for both enantiomers of MXacid (traces of MX-acid in metalaxyl (11)). Kinetic Analyses. First-order kinetics was assumed for the dissipation of the enantiomers. Corresponding rate constants kR and kS for the R- and S-enantiomer were determined from the linear range of logarithmic plots, ln[R] and ln[S] versus time t, respectively (data points during lagphases between time t ) 0 and t ) x were not considered)
(4)
The term kR - kS can be determined fairly precisely because the enantiomer ratio is not expected to depend on the recovery of the analytes (virtually the same recovery of R- and S-enantiomers). The enantioselectivity of dissipation was defined as
ES )
kR - kS kR + kS
(5)
Positive values (0 < ES e 1) indicate a more rapid dissipation of the R-enantiomer (for metalaxyl, the fungicidally active enantiomer), negative values (-1 e ES < 0) indicate a more rapid removal of the S-enantiomer (for metalaxyl, the fungicidally less active enantiomer), and at an ES value of 0, dissipation is nonenantioselective.
Results and Discussion Dissipation of Metalaxyl in Soils under Aerobic Conditions. The dissipation of metalaxyl was investigated in 14 soils under aerobic conditions. Cambisols and Rendzinas. A first group of soils included cambisols and rendzinas (soils # 1-4, Table 1). Cambisols represent agriculturally important soils for field crops and grassland, naturally occurring in deciduous forests, in Switzerland mainly in the Midland region. For agricultural production, cambisols have often been ameliorated by fertilization and liming. Rendzinas are characteristic, shallow, alkaline soils in deciduous forests and pastures of the Jura region above limestone. The dissipation of metalaxyl in the surface (A) horizons of these soils was relatively fast with DT50 values ranging from 9 to 32 d (Table 1), which is similar to previously reported data for agricultural soils (18, 31). The concentrations of Rand S-metalaxyl decreased according to first-order kinetics. In these soils, the R-enantiomer dissipated faster than the S-enantiomer, thus showing “normal”, positive enantioselectivities (ES ) 0.37-0.64, Table 1) as observed in previous soil incubations (10-12). For illustration, a typical chromatogram and kinetic data from incubation of metalaxyl in a cambisol are shown in Figure 3a,b. Luvisols, Podzols, and Rankers. Further aerobic incubation experiments were performed with luvisols, podzols, and rankers (soils # 5-14, Table 1). The luvisols selected for this study are quite acidic, deep soils from deciduous forests above glacial or fluvial drift. Podzols are common, acidic soils in coniferous forests of the Alpine Upland and rankers are found in alpine grassland above acidic bedrock. These soils were VOL. 37, NO. 12, 2003 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 5. pH-dependent enantioselective dissipation of (a) metalaxyl and (b) dichlorprop and mecoprop in aerobic soils.
FIGURE 4. Enantiomer ratios of metalaxyl residues (a) in three horizons of a podzol (soil # 10-12) and (b) in a native and acid- and base-treated luvisol (# 6). Curves represent exponential fits. Chromatograms show residues of S- and R-metalaxyl at the end of incubation. selected to include a wide range of soil properties, and most of them are not important for agricultural production. For example, the pH values of the soils were rather low (pH (CaCl2) 3.0-5.2). One soil (# 10) had an extremely high organic carbon content (46%). The soil texture ranged from a “sand” to a “silt loam”. Some soils were from high altitudes of up to 2480 m. Other soil samples were taken from deep horizons, down to 90 cm, which are biologically less active than top soils (14). Overall dissipation times in this second group of soils were generally higher (DT50 ) 16-127 d, Table 1) than those in the first group (cambisols and rendzinas). Surprisingly, five of these soils showed “reversed”, negative enantioselectivities (-0.62 e ES e -0.12, Table 1) with the S-enantiomer dissipating faster than the R-enantiomer. In four soils, the dissipation of metalaxyl was almost nonenantioselective (-0.07 e ES e 0.05). Data from incubation of metalaxyl in a podzol are shown in Figure 3c,d as an example. The lowest ES value of -0.62 was found in the incubation experiment with the surface (Ah) horizon of a podzol (soil # 10). Interestingly, in deeper horizons of this soil, dissipation of metalaxyl was more or less nonenantioselective (ES ) -0.07 in the E horizon in 40 cm depth, soil # 11) or even showed a positive enantioselectivity (ES ) 0.25 in the Bs horizon in 90 cm depth, soil # 12). The residues of metalaxyl in the Ah, E, and Bs horizon of this soil thus had compositions [R] > [S], [R] ≈ [S], and [R] < [S], respectively (Figure 4a). Enantioselectivity of Metalaxyl Dissipation Correlates with Soil pH. In the 14 aerobic soils, metalaxyl dissipated with enantioselectivities ranging from predominant removal of the R-enantiomer to predominant removal of the S2672
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enantiomer. The highest “positive” enantioselectivities (ES ) 0.64 and 0.61) were observed in rendzinas, the soils with the highest pH values (soils # 1 and 2, pH (CaCl2) 7.1), and the highest “negative” enantioselectivity was found in a podzol with the lowest pH (soil # 10, pH 3.0, ES ) -0.62). This suggested a dependence of the enantioselectivity on the pH of the soil. In fact, all aerobic soils with pH > 5 showed positive enantioselectivities (faster dissipation of R-metalaxyl), whereas all soils with pH < 4 showed negative enantioselectivities (faster dissipation of S-metalaxyl, Table 1). In soils with pH 4-5, enantioselectivities were less pronounced. When ES was plotted versus pH, a linear, positive correlation was observed (r2 ) 0.87, n ) 15 soils, Figure 5a). Enantioselectivities, however, did not correlate with other soil parameters (organic carbon, texture) (data not shown). The different enantioselectivities found in the Ah, E, and Bs horizon of the above-mentioned podzol (ES ) -0.62, -0.07, and 0.25) may thus be explained by their pH values (3.0, 3.5, and 5.2, respectively). Literature data also fit in the plot of Figure 5a. A recently published study on the enantioselective degradation of metalaxyl in an alkaline soil with pH 8.2 reported an ES value of 0.56 (12). Plots of the absolute rate constants kR and kS versus soil pH showed much more scattering (Figure 6). The kR values varied by a factor of ≈50 and kS values by a factor of ≈10. The data suggest that the rate constants for dissipation of the R-enantiomer may depend on pH, whereas removal of the S-enantiomer seems not to be affected by pH. However, the presumed correlation between pH and kR is statistically not significant. It can be assumed that the absolute rate constants are influenced by many other soil parameters such as biological activity, organic carbon, nutrients, temperature, and moisture (14), although none of these parameters as such did show a correlation with kR or kS. Enantioselectivity Changed by Acidification or Liming of a Soil. To further investigate whether a change in the pH of a soil had any influence on the enantioselectivity of
FIGURE 6. First-order rate constants kR and kS for dissipation of R- (circles) and S-metalaxyl (squares) in aerobic soils plotted versus pH. metalaxyl dissipation, a luvisol (soil # 6) was treated with acid (H2SO4) or base (CaCO3). After an equilibration time of 14 days, the soils were incubated with rac-metalaxyl under the same temperature and moisture conditions as the untreated, native soil. In the native soil, removal of metalaxyl had essentially been nonenantioselective (ES ) 0.03). In the acid-treated soil, the dissipation of the S-enantiomer was now slightly favored (ES ) -0.09), whereas in the base-treated soil, the dissipation of the R-enantiomer was favored (ES ) 0.18). Figure 4b shows the changing enantiomer ratios (ER) in the native and the treated soils versus incubation time. The pH effects found with this soil are consistent with the correlation between pH and enantioselectivity observed in different aerobic soils (Figure 5a). Dissipation of Metalaxyl under Anaerobic Conditions. Strongly acidic conditions in aerobic soils obviously seem to favor the dissipation of S-metalaxyl. However, a “reversed” enantioselectivity (ES < 0) had also previously been observed in sewage sludge at neutral pH (10) and may thus be the result of anaerobic conditions. Additional anaerobic incubation experiments were therefore carried out with soil samples from horizons permanently saturated with ground- or rainwater (Gleysols, soils # 15-20). In these soils, DT50 values ranged between 17 and 160 d (Table 1). The dissipation of the enantiomers followed firstorder kinetics. Four of these soils showed a preferential dissipation of the S-enantiomer (-0.71 e ES e -0.16, Table 1), and in one soil, the dissipation was almost nonenantioselective (ES ) 0.02). The sediment of a drainage canal (soil # 20) showed a faster dissipation of the R-enantiomer (ES ) 0.20). In most anaerobic soils, enantioselectivities were thus “reversed”, even though their pH values were g 5. In contrast to aerobic soils, no linear correlation was observed between pH and ES values (r2 ) 0.13, n ) 6 soil samples, graph not shown). Formation and Subsequent Dissipation of MX-Acid. The concentration of the primary metabolite MX-acid was followed in all experiments. In a previous study using enantiopure R- and S-metalaxyl, the formation of MX-acid was shown to occur with retention of configuration (11). The present incubation experiments with rac-metalaxyl qualitatively confirmed this finding as illustrated by the chromatograms and kinetic curves in Figure 3. Stoichiometries were determined for five soils and ranged from ≈30% to 100% formation of MX-acid from metalaxyl. In cambisols and rendzinas, MX-acid was further degraded (Figure 3b), whereas in most other soils, the concentration of MX-acid reached a plateau and no further removal was observed within
FIGURE 7. Incubation of rac-MX-acid (a) in a cambisol (soil # 3) and (b) a luvisol (# 9) showing the first-order dissipation of R-MXacid (squares) and S-MX-acid (circles). the time of incubation (Figure 3d). The latter group of soils were all not of agricultural importance. Separate incubations were carried out with a smaller number of soils, fortified with rac-MX-acid. DT50 values for MX-acid ranged from 17 to 183 days (Table 2) and were higher than corresponding DT50 values for metalaxyl in these soils (Table 1). The concentrations of R- and S-MX-acid decreased according to first-order kinetics as shown for a cambisol and a luvisol in Figure 7. Enantioselectivities ranged from -0.30 to 0.15, but no linear correlation was observed between pH and ES values (r2 ) 0.45, n ) 5 soil samples, graph not shown). Enantioselectivity, a Useful Measure To Characterize Biological Degradation of Chiral Compounds. The enantioselective degradation/dissipation of chiral compounds and thus the enantiomer composition of residues may vary considerably from soil to soil as shown in this study with soils from locations close to each other, in one case even within a single soil profile. With regard to metalaxyl, the chiral preference was clearly different in neutral to alkaline soils than in strongly acidic and anaerobic soils. The high enantioselectivities observed in some soils suggest biologically mediated processes. Various fungi, bacteria, and actinomycetes have been described to be responsible for the biodegradation of metalaxyl in soils (21, 22, 32-34), whereas dissipation has been insignificant in autoclaved (12) or azidetreated, sterilized soils (35). On the other hand, the nonenantioselective dissipation observed in some soils does not necessarily indicate the absence or a minor importance of biological degradation. As reported above, the nonenantioselective dissipation of metalaxyl in a luvisol was changed to an enantioselective dissipation after acid- or base-treatment and was thus also biological. The use of ES values as defined in this study was suitable for identifying the soil parameters that were important in the enantioselective degradation of metalaxyl, i.e., soil pH and redox conditions. However, the underlying processes are still unknown and need further research. Results may be rationalized on the level of the organisms degrading metalaxyl (e.g., predominance of microbial populations or consortia preferentially degrading the R- or S-enantiomer) or on a molecular level (e.g., activation/inhibition of enantiomerspecific enzymes). Enzymatic reactions are often pH-dependent (14), and the enantioselective degradation of other chiral compounds may, therefore, also be influenced by this parameter. The data from the experiments with MX-acid did not show a correlation between soil pH and enantioselectivity and thus demonstrate that, as expected, the finding cannot be generalized to all chiral compounds. However, a reevaluation of published kinetic data for the herbicides dichlorprop and mecoprop (4, 5, 8, 9) indicated, in fact, a similar correlation between soil pH and enantioselectivity as for metalaxyl VOL. 37, NO. 12, 2003 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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(Figure 5b, one of six soils, a clay loam, deviated from the correlation). For both herbicides, the correlation suggested a preferential degradation of the S-enantiomers in acidic to neutral soils and a preferential degradation of the R-enantiomers in alkaline soils. The similar trend between soil pH and enantioselectivity for dichlorprop/mecoprop and metalaxyl is, nevertheless, somewhat unexpected as their degradation pathways are different (ether cleavage and ester hydrolysis, respectively (19)), and different microorganisms and enzyme systems presumably are involved in their degradation.
Acknowledgments This research project was sponsored by Syngenta (Basel, Switzerland). Detailed discussions were held with H. Egli and U. Plu ¨ cken and are kindly acknowledged. We thank our colleagues V. Buser, B. Patrian, and A. Zu ¨ rcher for their help in soil sampling and soil extractions and H. Schwager and R. Flisch (Swiss Federal Research Station, Zu¨rich, Switzerland) for determination of some soil parameters.
Note Added in Proof Enantioselective degradation of metalaxyl was also observed in a recently published study with a German and a Cameroonian soil (37). We calculated enantioselectivities for these soils, which nicely fit in the correlation of Figure 5a (German soil: pH (CaCl2) ) 6.75, ES ) 0.320, Cameroonian soil: (pH (CaCl2) ) 4.16, ES ) -0.300).
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Received for review December 9, 2002. Revised manuscript received March 10, 2003. Accepted March 13, 2003. ES0202412
