Chiral Insecticide Î±-Cypermethrin and Its Metabolites: Stereoselective

Article pubs.acs.org/JAFC

Chiral Insecticide α‑Cypermethrin and Its Metabolites: Stereoselective Degradation Behavior in Soils and the Toxicity to Earthworm Eisenia fetida Guojun Yao, Xu Jing, Wang Peng, Xueke Liu, Zhiqiang Zhou, and Donghui Liu*

Downloaded by UNIV OF NEBRASKA-LINCOLN on September 9, 2015 | http://pubs.acs.org Publication Date (Web): August 27, 2015 | doi: 10.1021/acs.jafc.5b03148

Department of Applied Chemistry, China Agricultural University, No. 2 Yuanmingyuan West Road, Beijing 100193, People’s Republic of China ABSTRACT: The enantioselective degradation of the widely used chiral insecticide α-cypermethrin in soils has been investigated, and its main metabolites cis-3-(2′,2′-dichlorovinyl)-2,2-dimethylcyclopropane carboxylic acid (cis-DCCA) and 3phenoxybenzoic acid (3-PBA), which have potential environmental problems, have also been determined. The enantiomers of αcypermethrin were separated on Chiralcel OD chiral columns by high-performance liquid chromatography (HPLC) under normal phase, and the metabolites were detected by gas chromatography (GC) after derivatization. The results of the degradation showed that α-cypermethrin dissipated in soils with relatively long half-lives of 12.70−47.08 days and obvious stereoselective degradation of the two enantiomers was observed in the five soils, with enantiomeric fraction (EF) from 0.55 to 0.61 after 42 days, indicating that (+)-(1R,cis,αS) enantiomer was preferentially degraded. cis-DCCA and 3-PBA were formed in all of the soils, and it was found that the amount generated was related to the soil pH. cis-DCCA was easily generated in the acidic soils, while more 3-PBA tended to be generated in the soil of pH over 7. To evaluate the impact on soil animals, the toxicity, including the combined toxicity of cis-DCCA, 3-PBA, and α-cypermethrin, to earthworm (Eisenia fetida) was studied. The results of enantioselective transformation of α-cypermethrin in soils and the toxicity of α-cypermethrin and its metabolites to earthworm have some implications for environmental risk and food safety evaluations. KEYWORDS: α-cypermethrin, degradation, metabolite, stereoselectivity, toxicity

1. INTRODUCTION Pyrethroid insecticides, synthetic versions of the natural compound pyrethrin produced by the Chrysanthemum flower, have been extensively used in agricultural and household formulations for more than 30 years.1 On account of the existence of multiple asymmetric carbon positions, a large amount of pyrethroids contain four or eight optical isomers,2 and a good example is cypermethrin ([(RS)-α-cyano-3phenoxybenzyl(1RS)-cis−trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylate]), which has three chiral carbon atoms at 1C and 3C in the cyclopropane carboxylic acid moiety and αC in the alcohol component and, therefore, consists of eight isomers, in which (+)-1R-cis-S and (+)-1R-trans-S are the only two isomers with insecticidal activity.3 Because of its moderate persistence under field conditions, high potency against insects, and safety for mammals, cypermethrin is used to control a wide range of insect pests, particularly Lepidoptera, in cereals, citrus, cotton, fruit, rape, soybeans, tobacco, tomatoes, vegetables, and other crops. Because of these uses, residues of cypermethrin have been reported in many sediment samples.4,5 Recently, cypermethrin has received increasing concern not only because it causes high acute toxicity to fish and invertebrates but also presents risks to human health.6 For instance, it exhibits neurotoxicity, immunotoxicity,7 genotoxicity,8 reproductive toxicity,9 and endocrine disruption effects10 and also is regarded as a possible human carcinogen by the United States Environmental Protection Agency.11 One of the most important processes influencing the environmental behavior of a pesticide is its degradation in soil. Hydrolysis of the ester linkage producing 3-phenox© 2015 American Chemical Society

ybenzoic acid (3-PBA) and 3-(2′,2′-dichlorovinyl)-2,2-dimethylcyclopropane carboxylic acid (DCCA) is the main degradation pathway of cypermethrin in soil (structures B and C of Figure 1).12 DCCA and 3-PBA are frequently detected in the environment and humans.13−16 As a general degradation product of pyrethroids, 3-PBA is classified as an endocrine disrupting chemical, owning to its antiestrogenic activity. Furthermore, 3-PBA also has higher mobility than the parent compounds, causes widespread contamination in soil,17,18 and thus, may pose potential influence on soil organisms. α-Cypermethrin is a racemate of two cypermethrin stereoisomers, (S)-cyano-3-phenoxybenzyl-(1R)-cis-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropare carboxylate [(+)-(1R,cis,αS) enantiomer] and (R)-cyano-3-phenoxybenzyl-(1S)-cis-3-(2,2dichlorovinyl)-2,2-dimethylcyclopropane carboxylate [(−)-(1S,cis,αR) enantiomer],19 which are a pair of enantiomers (Figure 1A). It is one of the most common pyrethroids, broadly used in agriculture, and a great part of the applied dose ends up as residuals in soil, posing potential threats to the soil ecosystem. The main degradation metabolites of α-cypermethrin in soil are cis-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylic acid (cis-DCCA) and 3-PBA. Earthworms live in close contact with soil particles and may represent up to 80% of the total soil biomass. Eisenia fetida is the most common species used for acute and chronic Received: Revised: Accepted: Published: 7714

June 25, 2015 August 17, 2015 August 23, 2015 August 24, 2015 DOI: 10.1021/acs.jafc.5b03148 J. Agric. Food Chem. 2015, 63, 7714−7720

Article

Journal of Agricultural and Food Chemistry

ecological risk assessment. The formation of the metabolites cisDCCA and 3-PBA influences non-target organisms and is also an important issue and should also be considered. In this study, the enantiomers of α-cypermethrin were separated and the enantioselective transformation of αcypermethrin in five soils was investigated. The toxicity of cisDCCA and 3-PBA and the combined acute toxicity of cisDCCA, 3-PBA, and α-cypermethrin to earthworms were also determined. This result may be applied to understand the risks of α-cypermethrin application.


2. MATERIALS AND METHODS 2.1. Chemicals and Reagents. The insecticide of α-cypermethrin [≥98.5% purity, enriched in (+)-1R-cis-S and (−)-1S-cis-R] was provided by the Institute for the Control of Agrochemicals, Ministry of Agriculture (ICAMA, Beijing, China). 1,1,1,3,3,3-Hexafluoroisopropanol (HFIP), N,N-diisopropylcarbodiimide (DIC), 3-phenoxybenzoic acid (3-PBA, 98%), and internal standard (chlorpyrifos, 98%) were purchased from Sigma−Aldrich Corp. (St. Louis, MO). cis-DCCA (98%) was purchased from Chem Service (West Chester, PA). Water was purified by a Milli-Q system. Isopropanol, n-hexane, and ethyl acetate (analytical grade) were purchased from commercial sources, distilled, and filtered through a 0.45 μm filter membrane before use. All other chemicals and solvents were of analytical grade and purchased from commercial sources. 2.2. Chiral Separation. The high-performance liquid chromatography (HPLC) analysis was performed on an Agilent 1200 Series HPLC system (Agilent Technology) equipped with a G1322A degasser, G1311A pump, G1314B variable wavelength detector (VWD), and G1329A ALS. An AT-930 heater and cooler column attemperator (Tianjin Automatic Science Instrument Co., Ltd., China) was used to control the column temperature. The signal was processed by Agilent ChemStation software. The enantiomers of α-cypermethrin were separated on a Chiralcel OD column (250 × 4.6 mm, Daicel Chemical Industries, Tokyo, Japan) using n-hexane and isopropanol in a 98:2 (v/v) ratio at a 0.5 mL/min flow rate (Figure 2A). The injection volume was 20 μL, and the ultraviolet (UV) detection wavelength was 230 nm. The chromatographic separation was conducted at 20 °C. No enantiomerization was observed for αcypermethrin under this analytical condition. 2.3. Gas Chromatography (GC) Analysis. Agilent 6890N GC with an electron capture detector (ECD, Agilent Technologies, Palo Alto, CA) was used for quantitative analysis of α-cypermethrin and its metabolites. The detection was performed using a HP-5 capillary column (30 m × 0.25 mm inner diameter and 0.25 um film thickness). Nitrogen (>99.999%) was used as a carrier gas at a constant flow rate of 1.0 mL/min. The operating conditions were as follows: the column was held initially at a temperature of 100 °C for 10 min, then ramped at 5 °C/min to 155 °C, held at 155 °C for 5 min, ramped at 15 °C/

Figure 1. Chemical structures of α-cypermethrin and its metabolites: (A) α-cypermethrin, (B) cis-DCCA, and (C) 3-PBA.

ecotoxicity assays according to the Organisation for Economic Co-operation and Development (OECD) guidelines (OECD 1984) and is also used to survey the quality of the terrestrial environment. Traditional ecotoxicity assays and risk assessments have routinely focused on individual chemicals.20 However, earthworms in soil are usually exposed to a variety of pollutants simultaneously. Therefore, toxicity assays using mixtures of contaminants may better reflect the real-world exposures. Studies about the toxicity of α-cypermethrin have been studied by Diao et al.21 Little information regarding the toxicity of the metabolites and the combined acute toxicity of α-cypermethrin and its metabolites to earthworms has been reported. The individual enantiomers of chiral pesticides may show differences in bioactivity, toxicity, metabolism, bioaccumulation, and degradation behaviors.22,23 α-Cypermethrin is manufactured and applied to agro-ecosystems as racemic forms. The enantioselective environmental behavior of α-cypermethrin in the environment has not been considered for human health and

Figure 2. (A) Typical chromatograms of the chiral separation of α-cypermethrin and (B) enantioselective degradation of α-cypermethrin in soil 1 after 42 days. 7715

DOI: 10.1021/acs.jafc.5b03148 J. Agric. Food Chem. 2015, 63, 7714−7720

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Journal of Agricultural and Food Chemistry Table 1. Physicochemical Properties of the Soils


particle size number

site

sand (%)

silt (%)

clay (%)

texture

pH (water)

Corg (%)

1 2 3 4 5

Hunan Heilongjiang Beijing Hainan Neimeng

24.0 48.0 61.2 73.2 69.2

44.0 28.0 8.0 16.0 12.0

36.0 24.0 30.8 10.8 18.8

clay loam loam sandy loam sandy loam sandy loam

5.18 5.79 8.14 8.17 7.85

1.15 15.04 2.51 5.43 6.55

min to 290 °C, and held at 290 °C for 5 min. The total analysis time was 45 min. The temperature of the detector was set at 300 °C. 2.4. Sample Extraction. All of the samples were thawed for about 15 min at room temperature. Soil samples were mixed with 5 g of anhydrous sodium sulfate in a 50 mL polypropylene centrifuge tube, and 25 mL of ethyl acetate was added for extraction. The tube was vortexed for 3 min, exposed to ultrasonic vibration for 10 min, and then centrifuged at 3500 rpm for 5 min. The extraction was repeated following the same step, and the liquid phase was combined. The combined extracts were filtered through 5 g of anhydrous sodium sulfate for dehydration, evaporated to dryness in vacuo rotary at 35 °C, and reconstituted to 1 mL with acetonitrile. The solution was divided into two parts: (1) A total of 750 μL was evaporated to dryness under a stream of nitrogen and diluted to 250 μL with n-hexane, and a 20 μL aliquot was injected into HPLC for enantioselectivity determination. (2) A total of 250 μL was used for derivatization for GC analysis. The derivatization was conducted in a 5 mL centrifuge tube, with 30 μL of HFIP solution and 10 μL of DIC solution. After thorough mixing, the tube was placed in the oscillator. Derivatization was exposed to ultrasonic vibration for 10 min. After reaction, 500 μL of ultrapure water and 500 μL of n-hexane were added to the tube, vortexed for 5 min, and then centrifuged at 3500 rpm for 5 min. A total of 1 μL of nhexane solution was subjected for GC/ECD analysis. 2.5. Method Validation. The GC method described above was validated for the quantitative analysis of α-cypermethrin and its metabolites in soils. Good linearities were observed in the range of 0.015−6 mg/kg for α-cypermethrin and in the range of 0.025−0.3 mg/kg for its two metabolites, with mean coefficients of determination (R2) higher than 0.993. The average recoveries for α-cypermethrin at levels between 0.015 and 6 mg/kg ranged between 77.8 and 94.8% in the five soils, with relative standard deviation (RSD) below 10%, and the average recoveries of its metabolites at levels between 0.020 and 0.3 mg/kg ranged between 78.4 and 95.0% based on an acceptable RSD below 10%. The limits of detection (LODs) for α-cypermethrin, cis-DCCA, and 3-PBA in the tested soils were 0.003, 0.008, and 0.008 mg/kg, respectively, and the corresponding limits of quantitation (LOQs) were 0.015, 0.025, and 0.025 mg/kg, respectively. 2.6. Degradation of α-Cypermethrin in Soils. Five test soils from different agriculture regions (0−10 cm) of China that had not been treated with cypermethrin in the last 5 years were used in this study. Their major properties were shown in Table 1. Test soils were adjusted to a water content of 25% and maintained in the darkness at room temperature for 7 days to activate the microorganisms. To add the pesticide to the soil evenly, 10 g of soil was first placed into a 1000 mL Erlenmeyer flask and 0.5 mL of racemic α-cypermethrin solution (3000 μg/mL, acetone) was added. After the solvent was evaporated, another 290 g of soil was added gradually and stirred thoroughly to a spiked concentration of 5 μg/g (each enantiomer). The flasks were sealed with sterile cotton plugs and stored at 25 °C in the dark for 42 days. The moisture content in each flask was checked gravimetrically at each sampling point. The loss of water by evaporation was compensated by adding distilled water. Triplicate samples were removed from each treatment at different time intervals (0, 7, 14, 21, 28, 35, and 42 days) and immediately transferred to a freezer (−20 °C). 2.7. Acute Toxicity of cis-DCCA, 3-PBA, and α-Cypermethrin. According to the OECD guideline 207, the acute toxicity of cis-DCCA and 3-PBA to earthworms was tested by a paper contact toxicity assay (OECD 1984). Before the assay, the earthworms were rinsed with tap

water and dried by absorbent paper, following a depuration period of 24 h on wet filter paper under dark, to evacuate the gut content of the earthworms. A series of concentrations, 10.0, 30.0, 50.0, 70.0, and 100.0 ng/cm2 cis-DCCA and 3-PBA and 80.0, 159.0, 475.0, 632.0, and 790.0 ng/cm2 α-cypermethrin, were spiked on filter papers (5.5 × 11.5 cm) by adding cis-DCCA, 3-PBA, and α-cypermethrin acetone solutions. Controls were set with the solvent acetone. The filter papers spiked with the test substance were placed in a flat-bottomed glass vial (3.6 cm in diameter and 8 cm in length), and 1 mL of deionized water was added to each vial. A total of 10 replicates were performed for each treatment. The earthworms were placed on the filter papers and kept at 20 ± 2 °C. Mortality was checked after an incubation period of 48 h, and on the basis of that, LC50 values were calculated by SPSS, version 18.0 (SPSS, Inc., Chicago, IL). 2.8. Combined Toxicity of cis-DCCA, 3-PBA, and αCypermethrin. After the LC50 values of cis-DCCA, 3-PBA, and αcypermethrin were measured, two kinds of combined toxicity were determined: the binary combination of cis-DCCA + 3-PBA and the ternary combination of cis-DCCA + 3-PBA + α-cypermethrin. We chose the modified toxic unit (TU) approach to model the combined toxicity according to the literature.24 In the TU model, a value of 1 TU is defined as the 50% lethal concentration (LC50) value of the chemical. A series of the sum of the TU (∑TU) was used to set the concentrations of each component at proportions of their respective LC50 in the mixture. Therefore, the summations of the concentrations were set equivalent to five levels: ∑0.2 TU, ∑0.5 TU, ∑1.0 TU, ∑2.0 TU, and ∑4.0 TU for cis-DCCA + 3-PBA and ∑0.3 TU, ∑0.75 TU, ∑1.5 TU, ∑3.0 TU, and ∑6.0 TU for cis-DCCA + 3-PBA + αcypermethrin. For example of a binary mixture of cis-DCCA + 3-PBA, 3.7 ng/cm2 cis-DCCA (0.1 TU) and 5.0 ng/cm2 3-PBA (0.1 TU) made the mixture of ∑0.2 TU. Therefore, to determine the combined toxicity of cis-DCCA + 3-PBA, the series concentrations were 3.7 + 5.0, 9.2 + 12.5, 18.4 + 24.9, 36.7 + 49.8, and 73.4 + 99.6 ng/cm2, and for the determination of the combined toxicity of cis-DCCA + 3-PBA + α-cypermethrin, the concentrations were 3.7 + 5.0 + 19.5, 9.2 + 12.5 + 48.8, 18.4 + 24.9 + 97.6, 36.7 + 49.8 + 195.2, and 73.4 + 99.6 + 390.4 ng/cm2. The calculation of LC50 mix (50% lethal concentration of the mixture) was conducted by SPSS, version 18.0. The mixture exhibits synergism, additive, or antagonism effects if the determined LC50 mix values were lower than 1 TU, equal to 1 TU, or greater than 1 TU, respectively. 2.9. Data Analysis. Degradation curves (concentration in soils versus incubation time) were regressed by Excel 2010 (Microsoft). The first-order polynomial regression analysis model was used to describe the dissipation process for α-cypermethrin enantiomers in the soils. It was assumed that the degradation of α-cypermethrin in five soils followed pseudo-first-order kinetics. The corresponding rate constants (k) were calculated according to the equation C = C0e−kt. The starting point of regressive functions was the maximum value of the enantiomer concentrations in the soils and then decreased in the following days. The half-life (T1/2, day) was estimated from equation T1/2 = In 2/k = 0.693/k. The enantiomeric fraction [EF = (+)/(+) + (−)]25 was used to express the enantioselectivity of a pair of enantiomers. Here, (+) and (−) were peak areas of the (+) and (−) enantiomers of αcypermethrin. The EF values ranged from 0 to 1, with EF = 0.5 representing the racemic mixture. 7716


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Li et al. have reportd the enantioselctive degradation of βcypermethrin in a Tianjin alkaline soil, and the results showed that significant enantioselectivity occurred during the 30 day incubation period, with the enantiomeric ratio (ER) values of one pair of enantiomers of (1R,cis,αS) and (1S,cis,αR) changing from the initial of 1.00 to the final of 0.79.26 The dissipation of cypermethrin has also been reported in a soil at ambient temperature under aerobic conditions, and the enantiomers of (1R,cis,αS) and (1S,cis,αR) were measured separately, which had half-lives of 63 and 71 days, respectively.27 The results indicated an enrichment of (1S,cis,αR) in the soils during the degradation process, which were similar to the results in this work. Enantiomeric selectivity is also a measurement of enantioselectivity, defined as ES = (kR − kS)/(kS + kR) [where kR and kS are rate constants of (+)-(1R,cis,αS) and (−)-(1S,cis,αR) αcypermethrin in degradation regression equations]. Positive values (0 < ES < 1) indicate a more rapid degradation of (−)-(1S,cis,αR), whereas negative values (−1 < ES < 0) indicate a more rapid dissipation of (+)-(1R,cis,αS), and ES value of 0 shows that the dissipation is not enantioselective. The ES values of α-cypermethrin ranged from 0.0702 to 0.227, showing obvious enantioselectivity in all of the soils, with (+)-(1R,cis,αS) being degraded faster (Table 2). It was found that ES values were positively related to soil organic carbon content after correlation analysis (R2 = 0.781). Consequently, it can be concluded that the enantioselectivity dissipation of αcypermethrin was dependent upon the organic carbon content, which may affect chiral signatures by influencing the activity of the microbial community or the level of enzymes in soils.28 It has been reported that high organic matters in soil increased cypermethrin persistence, which was similar to the result in this experiment.29 Some studies reported the enantiomer selectivity of toxicity of cypermethrin. The acute toxicity was measured for the individual enantiomer of α-cypermethrin, and the enantiomer of (+)-(1R,cis,αS) was more toxic than (−)-(1S,cis,αR).21 The incubation in this paper revealed that (+)-(1R,cis,αS) degraded faster than (−)-(1S,cis,αR), which indicates that enrichment of (−)-(1S,cis,αR) may relieve the toxicity from α-cypermethrin to the earthworm. In addition, in aquatic toxicity, (+)-(1R,cis,αS) was also more toxic than (−)-(1S,cis,αR).23 Therefore, it is essential to assess the toxicity of pesticide on an enantiomeric level. 3.2. Formation of cis-DCCA and 3-PBA. The two metabolites of α-cypermethrin were both detected during its dissipation in the five soils (Figure 4). cis-DCCA maintained a higher concentration than 3-PBA through the experimental periods maybe because of the faster degradation of 3-PBA in the soils. cis-DCCA could be detected at the 7th day in all of the soils, and the concentration increased with time or was kept at a steady level until the last sample point of 42 day. The final concentrations of cis-DCCA in soils 3, 4, and 5 with pH > 7 were 0.59, 0.50, and 0.34 μg/kg, respectively, at the 42nd day, which were higher than the concentrations of 0.11 and 0.17 μg/ kg in soils 1 and 5 with pH < 7, respectively. It indicated that cis-DCCA was easily generated in alkaline soils. 3-PBA could also be detected at the 7th day in all of the soils. The concentrations increased with time in 1 and 2 soils, reaching a level around 0.11 mg/kg, but only a small amount was found in soil 3 and was undetectable after 21 days. In soils 4 and 5, the highest level appeared at about 14 day at concentrations of 0.13 and 0.31 mg/kg, respectively, and then it

3. RESULTS AND DISCUSSION 3.1. Enantioselective Degradation in Soils. Table 2 showed the first-order rate constant, half-life (t1/2), and Table 2. First-Order Rate Constants, k, Half-Life (t1/2), Coefficient (R2), and the Enantioselectivity (ES) Values of the Degradation of α-Cypermethrin in Soils soil soil 1 soil 2 soil 3


soil 4 soil 5

α-cypermethrin enantionmer

k (day−1)

t1/2 (day)

R2

ES

(−)-(1S,cis,αR) (+)-(1R,cis,αS) (−)-(1S,cis,αR) (+)-(1R,cis,αS) (−)-(1S,cis,αR) (+)-(1R,cis,αS) (−)-(1S,cis,αR) (+)-(1R,cis,αS) (−)-(1S,cis,αR) (+)-(1R,cis,αS)

−0.052 −0.057 −0.017 −0.027 −0.039 −0.048 −0.029 −0.038 −0.053 −0.051

14.40 13.30 47.08 28.39 16.56 12.70 22.21 17.53 17.12 14.42

0.9593 0.9580 0.9091 0.9837 0.9545 0.9668 0.9664 0.9717 0.8971 0.9248

0.046 0.227 0.103 0.134 0.070

coefficient (R2), and the results showed that there was great difference in the persistence and enantioselectivity of αcypermethrin in the soils. The half-lives of the enantiomers ranged from 12.7 to 47.1 days in the soils, with R2 of 0.8951− 0.9837, and the persistence of α-cypermethrin in soil 2 was about 2−3 times longer than that in the others. The observed differences in the persistence of α-cypermethrin in the five soils may be due to the organic carbon content. Soil 2 had the highest organic carbon content among the five soils, which might decrease the dissipation of α-cypermethrin. Soils 1 and 5 had similar pH values; however, the degradation rate of αcypermethrin in the two soils was totally different, suggesting no significant association between pH values and the degradation rate. The data showed that the two enantiomers dissipated at different rates in the five kinds of soils. The changes of EFs with time have been shown in Figure 3, which increased from the initial value of 0.50 with time in all of the soils, illustrating obvious enantioselectivity with (+)-(1R,cis,αS) being degraded faster than that with (−)-(1S,cis,αR). Figure 2B shows a typical chromatogram of the enantioselective degradation of αcypermethrin in soil 1 after 42 days of incubation.

Figure 3. EF curves for the degradation of α-cypermethrin in the five soils. 7717


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Figure 4. Degradation curves of α-cypermethrin enantiomers and the formation of its metabolites in the five soils.

and 3-PBA. The LC50 values for cis-DCCA, 3-PBA, and αcypermethrin were 36.72, 49.81, and 195.22 ng/cm2 , respectively (Table 3). Similar toxicity of α-cypermethrin to

underwent further degradation. It was found that 3-PBA was more slowly generated and degraded in soils 1 and 2 with pH < 7 than in soils 4 and 5 with pH > 7. It has been reported that the degradation half-life of 3-PBA in Guangzhou soil was 123.7 days,30 which was much slower than the degradation in soils 3, 4 and 5. This might be caused by the different soil properties. The metabolites were relatively persistent in the soils, which might be a potential problem to the environment for cypermethrin application. Some epidemiological studies have revealed that 3-PBA has adverse impact on health, such as sperm DNA damage31 and effect on male reproductive hormone levels,15 and a number of studies have reported the detection of 3-PBA and cis-DCCA in humans. Berkowitz et al. detected 3-PBA in urine samples from pregnant women from ethnic groups (Caucasian, African American, and Hispanic) in New York City, and generally, high levels of 3-PBA (median = 19.3 μg/g of creatinine) were found.32 Another study in China indicated that the levels of urinary pyrethroid metabolites (3-PBA, cis-DCCA, and transDCCA) were higher than those from the general population of developed countries.33 Humans may contact those metabolites by a variety of approaches, of which transfer of those from soils by the food chain may be a part. 3.3. Acute Toxicity Assay. By the filter paper contact test, the acute toxicity to earthworm was measured for cis-DCCA

Table 3. Calculated LC50 Values of cis-DCCA, 3-PBA, and αCypermethrin for Earthworm chemicals

48 h LC50 (ng/cm2)

95% confidence intervals

R2

α-cypermethrin cis-DCCA 3-PBA

195.22 36.72 49.81

138.12−275.94 28.76−46.87 37.07−66.89

0.9639 0.9794 0.9951

earthworm has been reported with the calculated LC50 value of 165.61 ng/cm2.21 Therefore, the results indicated that the two metabolites were more toxic to the earthworm than the parent compound α-cypermethrin. The rate and amount of the metabolites produced varied among the soils. Application of α-cypermethrin to alkaline soils (3, 4, and 5) might be more dangerous to the terrestrial community because more metabolites are produced. In the acidic soils, the metabolites degraded slowly and would last longer, also posing a potential threat to soil animals. Fast degradation of most pesticides in the environment makes its terrestrial risks acceptable under practical uses. However, evaluations of the ecological risk of corresponding 7718


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metabolites are limited at present. Some metabolites may be more persistent and toxic than their parent compounds, which may pose a more detrimental effect on the environment. The combined toxicities of cis-DCCA + 3-PBA and cisDCCA + 3-PBA + α-cypermethrin on earthworms were determined. The combined effect was defined as either concentration additive (LC50 mix = 1 TU), antagonistic (greater than additive, LC50 mix > 1 TU), or synergetic (less than additive, LC50 mix < 1 TU) effects. The LC50 mix values for cisDCCA + 3-PBA and cis-DCCA + 3-PBA + α-cypermethrin were 1.82 TU (1.14−2.90, 95% limits) and 1.72 TU (1.19− 2.50, 95% limits), respectively. This result indicated that the combined effect of α-cypermethrin and the two metabolites was antagonistic. Therefore, it is necessary to evaluate the toxicity of the metabolites and the combined toxicity when assessing the environmental risks from pesticides.

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AUTHOR INFORMATION

Corresponding Author

*Telephone/Fax: +8610-62731294. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (Contract Grant 21307155).

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REFERENCES

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Chiral Insecticide Î±-Cypermethrin and Its Metabolites: Stereoselective

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