400
Chem. Res. Toxicol. 2007, 20, 400-405
Stereoisomeric Separation and Toxicity of a New Organophosphorus Insecticide Chloramidophos Shanshan Zhou,†,‡ Kunde Lin,† Huayun Yang,† Ling Li,† Weiping Liu,*,† and Jian Li§ Research Center of Green Chirality, College of Biological and EnVironmental Engineering, Zhejiang UniVersity of Technology, Hangzhou 310032, China, Institute of EnVironmental Science, Zhejiang UniVersity, Hangzhou 310027, China, and School of Chemical Engineering and Pharmacy, Wuhan Institute of Technology, Wuhan 430073, China ReceiVed October 20, 2006
Chloramidophos (CP), O,S-dimethyl-[(2,2,2)-trichloro-1-hydroxyethyl]phosphoramidothioate, is a new organophosphorus pesticide (OP) with two chiral centers each on the phosphorus and carbon atoms. Although CP has been widely used in some provinces of China, it has received very limited attention toward its environmental behaviors, in particular, with regard to stereospecificity. In this study, the stereoisomeric separation and toxicity of CP were investigated. All of the four stereoisomers of CP were successfully separated by high-performance liquid chromatography on a Chiralpak AD column. The stereoisomers (pk 1 to pk 4) were distinguishable on their mass and circular dichroism spectra. The inhibition on acetylcholinesterases (AChE, in vitro) and the acute aquatic toxicity to Daphnia magna (in vivo) tested with optically pure stereoisomers of CP showed its stereoselectivity. The inhibitory potency toward AChE decreased in the order of pk 4 > pk 3 > pk 2 > pk 1. In comparison, the acute toxicity to D. magna was in the order of pk 3 > pk 2 > pk 1 > pk 4. The stereoselectivity was found to be isomer-dependent, with 1.1-18.1-fold differences (in vitro) and 1.2-13-fold differences (in vivo) among the stereoisomers. These results suggest that the overall toxicity of chiral OPs should be assessed using their individual enantiomers. Introduction Chirality is an important concept in many fields of chemistry. Its significance has long been recognized in regard to the differences in biological activity for individual enantiopure isomers of natural and synthetic compounds (1-3). A lesserknown fact is that many modern pesticides also contain chiral structures and thus consist of two or more enantiomers (4). About 25% of currently used pesticides are chiral, and this ratio is increasing as the compounds with more complex structures are being registered for use (5). A number of studies have shown that the biological activities such as toxicity (6, 7), endocrine disruption (8), and fate in the environment (4, 9-11) of chiral pesticides are enantioselective. For some chiral pesticides, only one enantiomer has desired biological effects on target organisms, with other enantiomers being less effective or even completely inactive. It is more important to note, however, that one or more of these noneffective enantiomers may pose adverse effects on some nontarget organisms in the environment. Although this issue has been raised, current knowledge of this aspect on chiral pesticides is mostly derived from their racemates, instead of individual enantiomers, and therefore does not sufficiently describe their actual environmental fate and ecological risks. Chirality is commonly found in organophosphorus pesticides (OPs),1 which were introduced in the 1950s for use on fruits, vegetables, and other crops. Individual enantiomers of several OPs with a single chiral center on phosphorus, carbon, or sulfur * To whom correspondence should be addressed. Tel: +86 571 8832 0666. Fax: +86 571 8832 0884. E-mail:
[email protected]. † Zhejiang University of Technology. ‡ Zhejiang University. § Wuhan Institute of Technology.
Figure 1. Stereoisomers of CP. The configurations connected with solid arrows are the pairs of enantiomers, and those with dashed arrows are the pairs of diastereoisomers.
have been successfully separated by HPLC on polysaccharide (12, 13) or Pirkle model chiral stationary phases (14) in the past few years. Therefore, it is now possible to prepare enantiopure standards and to study their enantioselective environmental behaviors. Over the past few years, enantioselectivity was found in both in vitro and in vivo toxicity for methamidophos (15), leptophos (16), and fenamiphos (17). The acute aquatic toxicity of fonofos, profenofos, and trichloronate to Ceriodaphnia dubia and Daphnia magna was also found to be enantioselective, with (-)-forms of the three OPs being about 10 times more toxic than their corresponding (+)-forms (6, 18). Nonetheless, the enantioisomeric separation and toxicity assay for chiral pesticides remain as challenging tasks. 1 Abbreviations: AChE, acetylcholinesterase; ATCh-I, acetylthiocholine iodide; BSA, bovine serum albumin; BE-AChE, bovine erythrocytes AChE; CD, circular dichroism; DTNB, 5,5′-dithio-bis-2-nitrobenzoic acid; EEAChE, electrophorus electricus AChE; OP, organophosphorus pesticides.
10.1021/tx600281n CCC: $37.00 © 2007 American Chemical Society Published on Web 03/03/2007
A New Organophosphorus Insecticide Chloramidophos
Chem. Res. Toxicol., Vol. 20, No. 3, 2007 401
Table 1. Capacity Factor (k), Separation Factor (r), Resolution (Rs), and CD Signal Using the Chiralpak AD Column capacity factor
separation factor
k1
k2
k3
k4
R12
R23
2.80
3.16
5.10
5.56
1.13
1.62
1.09
a
a
a
b
a
CD signalc
resolution R34b
a
b
Rs12b
Rs23b
Rs34b
pk 1
pk 2
pk 3
pk 4
1.87
8.86
1.79
-
+
+
-
b
Subscripts 1-4 indicate the effluent orders of the four isomers. Subscripts 12, 23, and 34 indicate the effluent orders of two adjacent peaks. c The detection wavelength of CD was set at 230 nm.
Chloramidophos (CP; Figure 1) is an OP with two asymmetric centers at phosphorus and carbon atoms and thus four stereoisomers. In recent years, some highly toxic and persistent OPs, such as methamidophos, parathion, methyl parathion, monocrotophos, and phosphamidon, have been banned for use in China. As an alternative, CP has been widely applied in some provinces in China. However, the stereoisomeric separation and toxicity of racemic CP has not been well-understood and the information with respect to stereospecificity remains scanty. In this study, we first developed an HPLC method to resolve and prepare four stereoisomers of CP. The pure stereoisomers were then used to test their inhibition on both acetylcholinesterases of bovine erythrocytes (BE-AChE) and electrophorus electricus (EE-AChE) and their acute aquatic toxicity to D. magna under static conditions. Because of the lack of the information on CP with respect to its enantioselective environmental behaviors, the data from this study would offer useful information for a more comprehensive assessment of the ecotoxicological risks associated with the use of CP.
acetone. The separated stereoisomers were identified according to their CD and mass spectra. The CD spectra with wavelengths of 220-400 nm were obtained using the on-line CD detector. The MS analysis was carried out on a TRACE DSQ mass spectrometer (ThermoFinnigan, CA) with a dual-stage quadrupole filter operating in electron impact mode. The conditions for MS assay were as follows: source temperature, 250 °C; ionizing energy, 70 eV; and transfer line, 250 °C. The mass spectrometer was scanned at an m/z range from 50 to 300 Da. The purity and concentration of the recovered isomers were determined by HPLC analysis under conditions described above. The purity was calculated to be more than 98% for all isomers prepared for toxicity assays. Assay of AChE Inhibition. It is known that the acute toxicities of OPs are due to their inhibition on AChE. The inhibitory mechanism can be chemically described as (19)
Materials and Methods
where X represents the leaving group in OP, Kd is the dissociation constant between reactant and complex, kp is the phosphorylation constant, and ki is the bimolecular inhibition constant equal to kp/ Kd. Kd is regarded as a measure for binding ability and depends on the structural features of the molecule. kp describes the effect of change in the reactivity of the ester. Besides kp, Kd, and ki, the concentration of inhibitor leading to half inhibition of enzyme activity (IC50) was also calculated to evaluate the inhibition potency of stereoisomers. The theoretical considerations with corresponding equations of these parameters were fully described in a previous study (15). The in vitro inhibition of AChE activities was evaluated using two enzymes, BE-AChE and EE-AChE. In a typical experiment, each of five test tubes containing properly diluted enzyme solutions (180 µL) was treated in the phosphate buffer (pH 8.0) with 20 µL CP solutions at various concentrations that inhibited enzymatic activity by 10-90%. Meanwhile, control samples were also prepared by use of 20 µL of phosphate buffer (pH 8.0) in place of CP solutions. The mixture was incubated at 37 °C. At predetermined time intervals (total time not longer than 20 min), 20 µL of the AChE-inhibitor solution (or AChE-buffer solution) was taken to measure the residual activity of AChE. The AChE activity was spectrophotometrically determined at 37 °C with a Bio-Rad model 680 microplate reader (Bio-Rad Laboratories) according to a modified Ellman method (20). Briefly, 210 µL of DTNB solution and 20 µL of ATCh-I solution were added to the wells on a 96 well microtiter plate. Enzyme-buffer or enzyme-inhibitor (20 µL) solution was subsequently added to make the final concentrations of DTNB and ATCh-I at 0.28 and 0.5 mM, respectively. The enzymatic activities of the mixtures in the 96 well microtiter plate were determined at 405 nm for 5 min at the interval of 1 min from the addition of enzyme-buffer or enzyme-inhibitor. All of the above tests and measurements were performed in four replicates. Acute Aquatic Toxicity. The aquatic toxicity of individual stereoisomers, their equimolar mixtures, and racemate were evaluated through 48 and 96 h acute toxicity assays using D. magna. Stock organisms were originally obtained from the Chinese Academy of Protection and Medical Science (Beijing, China). The test organisms were obtained from continuous culture maintained at 22 ( 1°C in M4 culture medium (21) with a photoperiod of 12 h per day and a density of 98.0% was obtained from Wuhan Institute of Technology (Wuhan, China). Acetylthiocholine iodide (ATCh-I), 5,5′-dithio-bis-2-nitrobenzoic acid (DTNB), bovine serum albumin (BSA), BE-AChE (type XIIS), and EE-AChE (type V-S) were purchased from Sigma Chemicals (St. Louis, MO). Other solvents or chemicals were of HPLC or analytical grade. Working solutions of the enzymes were made in 0.1 M potassium phosphate buffer (pH 8.0), in which the hydrolysis rates of ATCh-I were approximately 0.05-0.10 absorbance units/min. To avoid alcoholysis, standards of CP were dissolved in ether and acetone at 5000 mg L-1, respectively, for separation and bioassays. The alcoholysis in HPLC analysis was assumed to be negligible. Chromatographic Conditions and Identification of Stereoisomers. Stereoisomeric separation was carried out on a Jasco LC2000 series HPLC system (Jasco, Tokyo, Japan) equipped with a PU-2089 quaternary gradient pump, a mobile phase vacuum degasser, an AS-1559 autosampler with a 100 µL loop, a CO-2060 column temperature control compartment, a variable wavelength CD-2095 circular dichroism (CD) detector, and an LC-Net II/ADC data collector. Chromatographic data were acquired and processed with the computer-based ChromPass software (Jasco). Used were the following commercial HPLC columns purchased from Daicel Chemical Industries (Tokyo, Japan): Chiralpak AD [amylose tris(3,5-dimethylphenyl-carbamate)], Chiralpak AS {amylose tris-[(S)1-methylphenyl-carbamate]}, Chiralcel OD [cellulose tris-(3,5dimethylphenyl-carbamate)], Chiralcel OJ [cellulose tris-(4-methyl benzoate)], and Chiralcel OC [cellulose tri-(phenylcarbamate)]. All of the columns were 250 mm × 4.6 mm (i.d.) in dimensions with different enantioselective phases coated onto 5 µm silica gel beads. For all resolution experiments on various columns, the inject volume, temperature, and detection wavelength of CD were fixed at 20 µL, 25 °C, and 230 nm, respectively. The light source for the chiral detector was a 150 W Hg-Xe lamp, and the tapered cell path was 25 mm with a volume of 44 µL. The solvents containing the resolved stereoisomers for subsequent bioassays were manually collected at the column outlet. They were evaporated to dryness under a nitrogen stream and redissolved in
402 Chem. Res. Toxicol., Vol. 20, No. 3, 2007
Zhou et al.
Figure 2. Representative HPLC UV chromatograms. Chromatographic conditions: Chiralpak AD column, detection wavelength at 230 nm, n-hexane/ethanol (90/10, v/v), 1.00 mL min-1, 25 °C. Pk1, pk2, pk3, and pk4 represent the first, second, third, and fourth eluted stereoisomers, respectively.
Figure 4. CD spectra of separated stereoisomers of CP. Chromatographic conditions are the same as in Figure 2. Pair 1 represents the equimolar mixture of pk1 and pk3. Pair 2 represents the equimolar mixture of pk2 and pk4. Table 2. Inhibition Parameters for Individual Stereoisomers, Equimolar Mixtures of Enantiomers, and Racemate of CP against BE-AChE and EE-AChE kia (×102 L mol-1 min-1)
kpa (min-1)
Kda (10-4 M)
IC50a,b (mg L-1)
pk 1 pk 3 pair 1 racemate pair 2 pk 2 pk 4
0.70 ( 0.05 8.81 ( 0.74 5.05 ( 0.21 5.21 ( 0.30 6.02 ( 0.26 2.10 ( 0.10 12.8 ( 0.42
BE-AchE 0.34 ( 0.02 0.06 ( 0.02 0.13 ( 0.04 0.21 ( 0.01 0.30 ( 0.03 0.17 ( 0.02 0.06 ( 0.01
48.56 ( 6.58 0.66 ( 0.01 2.60 ( 0.27 4.14 ( 0.28 4.97 ( 0.50 9.12 ( 0.86 0.48 ( 0.03
94.82 ( 6.81 7.78 ( 0.65 12.96 ( 0.62 12.92 ( 0.64 10.86 ( 0.36 32.72 ( 2.63 5.23 ( 0.38
pk 1 pk 3 pair 1 racemate pair 2 pk 2 pk 4
1.11 ( 0.09 14.1 ( 0.10 8.32 ( 0.50 9.33 ( 0.51 14.4 ( 0.42 1.95 ( 0.06 14.9 ( 0.55
EE-AchE 0.31 ( 0.01 0.10 ( 0.02 0.20 ( 0.02 0.23 ( 0.02 0.26 ( 0.04 0.21 ( 0.01 0.12 ( 0.02
27.06 ( 2.51 0.79 ( 0.06 3.76 ( 0.02 3.45 ( 0.11 5.47 ( 0.50 6.86 ( 0.32 0.80 ( 0.22
61.84 ( 6.20 4.83 ( 0.42 7.96 ( 0.53 7.18 ( 0.08 4.62 ( 0.28 34.93 ( 0.07 4.51 ( 0.15
a
All values are means ( SDs of the mean (n ) 4). b IC50 in 30 min.
Table 3. Comparison of in Vitro and in Vivo Toxicity between Enantiomers and Diastereoisomers enantiomer
Figure 3. Mass spectrum of racemic CP and its probable interpretation.
renewed three times a week, and daphnids were fed daily with the alga Scenedesmas obliquus, which was cultured in a nutrient medium. The test animals used in this experiment were juveniles aged between 6 and 24 h. Prior to testing, a sensitive test for daphnids to potassium dichromate (K2Cr2O7) was performed as a positive control, and the EC50 (24 h) value was in the range of 0.6-1.7 mg L-1 (22). The overall testing procedure followed the EPA guidelines (23). Briefly, five neonates were transferred into glass beakers filled with 20 mL of blank or test solutions. Four replicates for each treatment were prepared. The test organisms were fed the alga S. obliquus 2 h before the test began and at 48 h when the test solution was renewed with the freshly spiked
BE-AChEa EE-AChEa D. magnab D. magnac
diastereoisomer
pk 1/ pk 3
pk 2/ pk 4
pk 1/ pk 2
pk 1/ pk 4
pk 2/ pk 3
pk 3/ pk 4
12.2 12.8 8.5 8.0
6.2 7.7 0.3 0.2
2.9 1.8 2.6 3.2
18.1 13.7 0.8 0.6
4.2 7.2 3.2 2.5
1.5 1.1 0.1 0.08
a IC b 50 in 30 min calculated in the assay of AChE inhibiton. LC50 in the 48 h static tests of acute aquatic toxicity. c LC50 in the 96 h static tests of acute aquatic toxicity.
pesticide. The mortality of daphnids of all vials was monitored at 24 h intervals for the 96 h exposure period.
Results and Discussion Stereoisomeric Resolution and Identification. Stereoisomeric separability of CP was evaluated on five chiral columns.
A New Organophosphorus Insecticide Chloramidophos
Chem. Res. Toxicol., Vol. 20, No. 3, 2007 403
Table 4. LC50 of Individual Stereoisomers, Equimolar Mixtures of Enantiomers, and Racemate of CP (mg L-1) to D. magna test time (h)
pk 1
pk 3
pair 1
racemate
pair 2
pk 2
pk 4
48 96
0.34 ( 0.02 0.16 ( 0.05
0.04 ( 0.00 0.02 ( 0.00
0.60 ( 0.10 0.21 ( 0.06
1.31 ( 0.12 1.12 ( 0.14
0.65 ( 0.06 0.36 ( 0.01
0.13 ( 0.02 0.05 ( 0.01
0.40 ( 0.00 0.26 ( 0.03
No satisfactory separation was achieved on the Chiralcel OC, Chiralcel OD, Chiralcel OJ, and Chiralpak AS columns when using the mobile phase of n-hexane combined with 2-propanol or ethanol. However, baseline resolution for the four stereoisomers was obtained when the Chiralpak AD column was employed. The corresponding capacity factors (k), separation factors (R), and resolutions (Rs) are listed in Table 1, and a representative chromatogram is shown in Figure 2. These results illustrate that the enantiomeric resolution of CP by HPLC is highly CSP specific. Because of the complexity of chiral recognition mechanisms, it is difficult to determine the specific reasons for the complete separation of the four CP stereoisomers only on the Chiralpak AD column. The stereoisomeric separability is determined by the interactions such as steric fit, hydrogen bonding, π-π, and dipole-dipole between CSP and analyte (24, 25). The separated stereoisomers were identified by direct probe inlet MS qualitative analysis. As an example, the mass spectrum of racemic CP and its probable interpretation are shown in Figure 3. When comparing the mass spectra of the resolved stereoisomers with that of racemic CP, the main fragment ion peaks were identical (the mass spectra of stereoisomers not shown). The enantiomers of a chiral compound are usually identified with their absolute configuration or optical rotation. HPLC coupled with a CD detector has recently become a powerful tool for determining the optical property of the resolved enantiomers (26). Because of their low sensitivity, optical rotations of the resolved stereoisomers were not able to be determined in this study. The CD spectra with wavelengths ranging between 220 and 400 nm were obtained for identifying stereoisomers using the on-line CD detector (e.g., Figure 4). Theoretically, the spectra of the enantiomers of a pair are mirror images of each other. It may thus be tentatively concluded that the first (pk 1) and third (pk 3) eluted isomers are one pair of enantiomers and the second (pk 2) and fourth (pk 4) isomers are the other pair (Figure 4). We refer to the equimolar mixture of pk 1 and pk 3 as pair 1 and that of pk 2 and pk 4 as pair 2. According to the HPLC quantitative analysis, the molar ratio for the four stereoisomers in racemic CP was 1:2.7:1:2.7 (pk 1:pk 2:pk 3:pk 4) (Figure 2). Stereoselectivity in AChE Inhibition. The IC50 is a relatively insensitive index used to evaluate the toxicity of a compound, with a lower value indicating a more toxic potency. Significant differences were observed in IC50 among the stereoisomers of CP (Tables 2 and 3). When comparing IC50 values between the enantiomers of a pair, pk 3 was about 12-fold more potent than pk 1 and pk 4 was about 6-7-fold more toxic than pk 2 (Table 3). Pair 1 and pair 2 had an intermediate potency to AChE as compared to their respective individual enantiomers (Table 2). This result is similar to that of other chiral pesticides with one asymmetric center (15, 29, 33). For diastereoisomers, the difference in IC50 against BE-AChE between pk 3 and pk 4 is only 1.5-fold, and it increases to 18.1-fold between pk 2 and pk 1. Previous studies showed that AChE may be stereoselectively inhibited by other OPs with two chiral centers, one on pentavalent phosphorus and the other on a carbon atom (2732). For example, stereoselectivity was found among the isomers of isomalathion toward AChE from mammalian, avian, and
piscine species (29, 30, 32). The order of potency toward the AChE from Torpedo californica for the isomers of isomalathion was (PR,CR) > (PS,CR) > (PR,CS) > (PS,CS), with 1.4-53.8fold difference in ki values among the isomers (32). In addition, the increasing order of IC50 for the four resolved isomers of CP toward both BE-AChE and EE-AChE was pk 4 < pk 3 < pk 2 < pk 1, indicating that the stereoisomers interact with both enzymes in a similar manner. Furthermore, IC50 values toward EE-AChE for all of the stereoisomers were lower than those toward BE-AChE, suggesting that the EE-AChE is more sensitive than BE-AChE to all forms of CP. To further examine the inhibitory reaction of the stereoisomers with AChE (eq 1), values for Kd and kp were determined. As indicated in Table 2, they were stereospecific. Taking the BEAChE inhibition as an example, the Kd of pk 4 was about the same as that of pk 3 with an average value of 0.57. This average was about 86 and 16 times lower than those of pk 1 and pk 2, respectively. This may be ascribed to the preferential orientation of the pk 3 and pk 4 configurations to complement the active and/or combining sites of AChE. Contrary to some OPs with only one chiral center (15, 29, 33), the kp values varied among the four stereoisomers of CP. In the BE-AChE inhibition process, kp values for pk 3 and pk 4 were virtually identical. However, values of kp for pk 1 and pk 2 were significantly higher than those of pk 3 and pk 4 and differed by two-fold. Similar variations in kp have been observed for the stereoisomers of isomalathion against AChE from rat brain (29); kp values of PRCS and PSCS isomalathion differed by 4.7-fold and were about 2.8-13.2 times higher than those of PRCR and PRCS diastereoisomers. More remarkable stereoselectivity was found among isomers of isomalathion inhibition toward EE-AChE, which displayed more than 100-fold difference in kp between the PS and the PR isomers. On the basis of the kinetic rates in the inhibition process and considering recovery process (29), mass spectral evidence (34), and molecular modeling experiment (35), the stereoselectivity was attributed to different orientations of the isomalathion stereoisomers to the active site of AChE and the subsequent cleavage of different primary leaving groups for PR and PS isomers. As the absolute configuration of the stereoisomers of CP is unknown, we could not elucidate the corresponding mechanisms for CP in the present work. Stereoselectivity in Aquatic Toxicity. To investigate the stereoselective toxicity toward nontarget organisms, the acute aquatic toxicity for the individual stereoisomers, pairs, and racemate to D. magna was measured through 48 and 96 h static tests. The t test indicated significant differences in LC50 values among the stereoisomers of CP (Tables 3 and 4). Judging from the values of LC50, the decreasing order of toxicity to D. magna was pk 3 > pk 2 > pk 1 > pk 4 (Tables 3 and 4). More specifically, the difference between the enantiomeric pairs was 3.1-8.5 times. The difference between the diastereoisomers was 1.2-13 times (Table 3). It is interesting to note that pk 4, the most potent inhibitor against BE- and EE-AChE (in vitro) reported in a previous study (Table 2), was the least toxic to D. magna (in vivo). This reversal of toxicity has also been observed for methamidophos, with (-)-enantiomer being about 8.0-12.4 times more potent to BE- and EE-AChE than its (+)-form in comparison to the (+)-enantiomer being 7.0 times more toxic to D. magna in 48
404 Chem. Res. Toxicol., Vol. 20, No. 3, 2007
h tests than the (-)-form (15). Although the causes for the reversed toxicity to the two different biological targets are not clear, this phenomenon results often from a combination of several factors. First, different species of enzymes may have different sensitivities to stereoisomers. Second, many biological activities, especially metabolism, transfer, and accumulation that affect the toxicity in vivo, have been found to be enantioselective (36). Moreover, the in vivo metabolism such as oxidation by microsome may change the toxicity of the parent compound. For many organophosphate insecticides, the parent compound is activated through the oxidation process and the oxidized forms are often reported to be more active to inhibit AChE activity (37, 38). Thus, the selectivity may result primarily from the stereoselectivity of activated metabolite(s). This also suggests that the information obtained from in vivo toxicity tests would be toxicologically more meaningful than from in vitro enzyme assays. Stereoselectivity was also found for the stereoisomers of other OP compounds with two chiral centers (27, 39). For a demeton analogue, the levorotatory phosphorus isomers were much more toxic to susceptible and resistant houseflies, Culex mosquito larvae, and mice than the dextrorotatory compounds (27). In an acute bioassay of mice, a 20-128-fold difference was observed among the isomers of soman (39). Large differences were also found between isomers of a synthetic pyrethroid insecticide permethrin in the acute toxicity to C. dubia or D. magna. In cis-permethrin (cis-PM), the 1R-cis enantiomer was 15-38 times more active than the 1S-cis enantiomer, while in trans-PM, the 1R-trans enantiomer was substantially more toxic than the 1S-trans enantiomer (6). For the above three chiral OP compounds, all of the racemates showed intermediate toxicity. By comparison, the LC50 of the equimolar enantiomeric mixtures (pair 1 and pair 2) in this study was higher than that of each of the individual enantiomers. For example, the LC50 of D. magna at 48 h was 0.34 and 0.04 mg L-1 for pk 1 and pk 3, respectively, whereas the corresponding value for pair 1 was 0.60 mg L-1. It was interesting that the toxicity of racemic CP was lowest with an LC50 up to 1.31 mg L-1. These results indicate that the toxicological effects of racemate or equimolar mixture of enantiomers cannot be predicted as the simple addition of the effects of individual enantiomer. The antagonistic interactions between enantiomers may exist in biological processes and must be taken into consideration in pesticide risk assessment.
Conclusions All four stereoisomers of racemic CP can be successfully resolved on the Chiralpak AD column, via which pure stereoisomers can be collected. This work reveals that the toxicity of CP to BE- and EE-AChE (in vitro) and to D. magna (in vivo) is stereospecific. The observed stereoselectivity of CP suggests that the environmental safety of both the enantiomers and the diastereoisomers of the pesticide needs to be individually assessed. Because most chiral pesticides are currently marketed as racemates, more comprehensive studies are proposed to be undertaken to evaluate their enantioselective environmental behaviors and ecological risks. Acknowledgment. This work was supported by the Na tional Basic Research Program of China (2003CB114400), the National Natural Science Foundation of China for Distinguished Young Scholars Program to W.L. (No. 20225721), and the National Natural Science Foundation of China (No. 30500304).
Zhou et al.
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