J. Agric. Food Chem. 2006, 54, 9145−9151
9145
Enantiomeric Differences in Permethrin Degradation Pathways in Soil and Sediment SUJIE QIN
AND JAY
GAN*
Department of Environmental Sciences, University of California, Riverside, California 92521
Chirality occurs widely in synthetic pyrethroids. Studies have shown significant differences in both aquatic toxicity and degradation rates between enantiomers from the same diastereomer of selected pyrethroids. To better understand chiral selectivity in biodegradation of pyrethroids, 14C-labeled permethrin was used to characterize enantiomeric differences in the formation of transformation intermediates in two soils and a sediment. Individual enantiomers of permethrin were spiked into soil and sediment samples, and transformation products were identified with known standards. Enantioselectivity was observed in most treatments when the dissipation of the parent enantiomers, the amount of intermediates and bound residues formed, and mineralization rates were compared between the enantiomers. The results show that all enantiomers of permethrin hydrolyzed rapidly and that the hydrolysis products were quickly further transformed. The direct hydrolysis products, cyclopropanic acid (Cl2CA), 3-phenoxybenzyl alcohol (PBalc), and 3-phenoxybenzoic acid (PBacid), were recovered at small percentages, ranging from 1 to 14% for Cl2CA and from 0.2 to 6% for PBalc and PBacid. The R-enantiomer of both cis- and trans-permethrin was mineralized more quickly than the S-enantiomer after hydrolysis. The degradation products from cis-permethrin were more persistent than those from trans-permethrin. As some transformation intermediates of permethrin may have greater acute and chronic toxicity than the parent compound, enantioselectivity in the formation of degradation intermediates may lead to different overall toxicities and merit further investigation. This study suggests that for chiral compounds, enantioselectivity may be reflected not only in the dissipation of the parent enantiomers but also in the kinetics of formation of intermediate transformation products. KEYWORDS: Enantioselectivity; chiral selectivity; enantiomer; pyrethroids; transformation pathways
INTRODUCTION
Chirality in modern pesticides has received an increasing amount of attention over the past decade (1-4). Pyrethroids are synthetic derivatives of the chrysanthemumic acids. They constitute a group of very potent insecticides, which are considered to be good substitutes for organophosphate and carbamate compounds (5). Pyrethroids have been widely used for insect control on agricultural crops, on animals, and around households (5-7). For instance, agricultural use of permethrin in California increased by 50% from 1999 to 2004 (California Pesticide Use Reports, http://www.cdpr.ca.gov/docs/pur/purmain.htm). With the restriction of some organophosphate insecticides, the use of pyrethroids is expected to increase further. Even though pyrethroids show low toxicity to birds and mammals, they are acutely toxic to a wide range of aquatic organisms, including water-column- and sediment-dwelling invertebrates, often at a trace concentration of 100-fold greater than those of the parent compound. The main objective of this study was to determine if enantioselectivity occurred in the biodegradation of pyrethroids in soil and sediment media by comparing the formation of transformation products between enantiomers. Permethrin was selected as a model pyrethroid compound due to its widespread use and the availability of 14C-labeled permethrin and authentic standards of some of its metabolites. MATERIALS AND METHODS Chemicals. Racemic [14C]permethrin [3-phenoxybenzyl (1RS)cis,trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecaboxylate], with 14 C labeled in the acid or alcohol moiety, was provided by FMC (Princeton, NJ). The structure and labeling positions of cis- and transpermethrin are shown in Figure 1. A previously developed HPLC method (16) was used for the separation and preparation of individual 14 C-labeled permethrin enantiomers. The resolution of enantiomers was achieved on a Sumichiral OA-2500I column (Sumika Chemical Analysis Service, Osaka, Japan) by using 99.5% hexane and 0.5% 1,2dichloroethane as the mobile phase. The individual enantiomers were manually collected at the HPLC outlet and enriched. The purity of these enantiomers was determined to be >99% by HPLC and/or GC analysis prior to their use. A similar approach was also used to prepare pure enantiomers from nonlabeled permethrin. By mixing 14C-labeled and nonlabeled enantiomers, the radioactivity of each permethrin enantiomer was adjusted to about 5 × 106 dpm mg-1. Standards of four metabolites, cis-3-(2,2-dicholorovinyl)-2,2-dimethylcyclopropanecarboxylic acid (c-Cl2CA, 98.9%), trans-3-(2,2dicholorovinyl)-2,2-dimethylcyclopropanecarboxylic acid (t-Cl2CA, 99.3%), 3-phenoxybenzyl alcohol (PBalc, 99.5%), and 3-phenoxybenzoic acid (PBacid, 99.2%), were obtained from FMC. Solvents and other chemicals used in this study were of analytical or HPLC grade. Soils and Sediment. Two soils and one sediment were used in this study (Table 1). The soil samples were collected from the surface layer (0-15 cm) of a field plot planted with a low-growing groundcover (spring cinquefoil, Potentilla tabernaemontani) and a field plot planted with tall fescue turfgrass (Festuca arundinacea) at the Agricultural Experiment Station near the University of California, Riverside, campus. The two soil samples were selected because they had been planted to a dicot (spring cinquefoil) and a monocot (grass) for an extended time (about 10 years), and the different planting practices may have
pH
OM (%)
sand (%)
silt (%)
clay (%)
7.9 7.4 7.3
1.1 1.7 1.6
20.0 65.0 62.0
46.0 22.5 24.0
34.0 12.5 14.0
influenced the soil microbial communities in the rhizosphere. The sediment sample (sandy clay) was collected from the 0-10 cm depth in San Diego Creek in Orange County, California. San Diego Creek is the main drainage channel connecting the inland regions and the Newport Bay estuaries. The soil and sediment samples were air-dried for 24 h at room temperature, homogenized while still slightly moist, and then passed through a 2-mm sieve before use. Incubation Experiments. Ten grams of soil or sediment (dry weight equivalent) was placed in 150-mL glass flasks (for aerobic treatments) or 20-mL glass vials (for anaerobic treatments). For the groundcover and turfgrass soils, the water content was adjusted to about 60% of the field holding capacity (w/w) by adding deionized water. For the San Diego Creek sediment, 6 mL of deionized water was added to each sample to immerse the sediment and form a 0.5-cm layer of water. Individual 14C-carbonyl- or 14C-alcohol-labeled permethrin enantiomers were then spiked into the soil or sediment samples, at a rate of 10 µg of chemical in 50 µL of acetone for each sample. This resulted in an initial pesticide concentration of 1.0 mg kg-1 with a radioactivity of 5 × 104 dpm per sample. The treated soil samples were incubated under aerobic conditions, whereas the treated sediment samples were incubated under both aerobic and anaerobic conditions. For aerobic treatments, the sample containers were loosely covered with aluminum foil and kept at room temperature (20 ( 1 °C). For the anaerobic treatment, the sample vials were equilibrated, spiked, crimp sealed, and incubated in a nitrogen-filled plastic chamber at room temperature. All of the samples in the aerobic treatments were checked regularly for water content by weighing and were frequently mixed by hand for aeration. Duplicate samples were removed from each treatment at 14 and 56 days after the pesticide application and immediately transferred into a freezer (-20 °C) to stop degradation. For extraction, each sample was thawed at room temperature and transferred to a preweighed 250-mL glass centrifuge bottle. The sample was mixed with anhydrous sodium sulfate (25 g for soil samples and 50 g for sediment samples, respectively) and 50 mL of hexane/acetone (1:1, v/v) for 1 h on a mechanical shaker and then centrifuged at 1000 rpm for 20 min. The same extraction step was repeated for a total of three times, and the solvent extracts were combined and evaporated to around 2 mL on a vacuumed rotary evaporator at 50 °C. The residues were transferred to a glass evaporation tube and blown down with nitrogen to 0.5 mL. To determine the organic-soluble radioactivity, a 100-µL aliquot of the final extract was added into 5 mL of Ultima Gold cocktail (PerkinElmer, Boston, MA) and the radioactivity was measured on a Beckman LC-5000TD liquid scintillation counter (LSC) (Beckman, Fullerton, CA). The centrifuge bottle with the extracted soil or sediment was weighed again to determine the water content of the solid phase. An aliquot of the extracted soil or sediment was removed, air-dried, and then combusted on a biological oxidizer (OX-500 Biological Oxidizer, R. J. Harvey, Hillsdale, NJ). The released 14CO2 was trapped in 15 mL of cocktail, and the radioactivity was measured by LSC to obtain the activity associated with the nonextractable or bound residues. The recovery for the oxidizer was about 80% and was used to correct for the measured activity. The overall recovery of the added radioactivity was 96.1-104.2% on the basis of analysis of soil and sediment samples immediately following spiking. Thin-Layer Chromatography (TLC). Silica gel glass TLC plates (60 F254, 10 × 20 cm, 0.25-mm thickness, Merck, Darmstadt, Germany) were used for the separation and tentative identification of permethrin and metabolites in the organic-soluble fraction. The solvent system used for developing the TLC plates was hexane/acetone (3:2, v/v). The Rf values of the four metabolites and cis- and trans-permethrin are given in Table 2. For each sample, 100 µL of the final sample extract was spiked on the TLC plate at about 2 cm from the base with
J. Agric. Food Chem., Vol. 54, No. 24, 2006
Permethrin Degradation Pathways Table 2. Rf Values of Permethrin and Metabolites on Thin-Layer Silica Gel Plates Developed in Hexane/Acetone (3:2, v/v) 14C-acid-labeled
compound
cis
c-Cl2CA t-Cl2CA Pbalc Pbacid cis-PM trans-PM
0.64
trans
14C-alcohol-labeled
cis
trans
0.62 0.53 0.81
0.62 0.53
0.61 0.81 0.80
0.80
a microsyringe. Nonlabeled standards of the metabolites and permethrin were spiked in parallel with the sample on the same plate. The spiked plate was then developed in the solvent mixture to about 17 cm from the baseline. After the developed plate was air-dried, silica gel at the same positions as the reference standards was carefully scraped using a spatula. The silica gel was transferred to 5 mL of Ultima Gold cocktail solution (Perkin-Elmer) in 7-mL scintillation vials and mixed at high speed on a mechanical shaker for 30 min. After the silica gel had settled, the sample vials were measured for radioactivity on LSC. The silica gel remaining in the same channel was also scraped and the total radioactivity counted on LSC. This activity was defined as unidentified metabolites in this study. RESULTS AND DISCUSSION
Disappearance of Parent Enantiomer. The fractions of the parent permethrin enantiomer as percent of the applied radioactivity after 14 and 56 days of incubation are given in Table 3. Paired t test showed that there was no difference in the residual parent enantiomer fractions between the two labeling positions for the same treatment. Under aerobic conditions, degradation of the parent enantiomers was consistently faster in the two soils than in the sediment (Tables 4 and 5). In the San Diego Creek sediment, all permethrin enantiomers were degraded more slowly under anaerobic conditions than under aerobic conditions (Table 5). Paired t test for the same time points between the aerobic and anaerobic treatments yielded a p value of 0.04 for the 14C-carbonyl-labeled permethrin enantiomers and
