Separation and toxicity of salithion enantiomers

Separation and Toxicity of Salithion EnantiomersSHANSHAN ZHOU,1,2 KUNDE LIN,2 LING LI,2 MEIQING JIN,1 JING YE,1 AND WEIPING LIU2*

1Institute of Environmental Science, Zhejiang University, Hangzhou, China2Research Center of Green Chirality, College of Biological and Environmental Engineering,

Zhejiang University of Technology, Hangzhou, China

ABSTRACT Enantioseletive toxicities of chiral pesticides have become an environ-mental concern recently. In this study, we evaluated the enantiomeric separation of sali-thion on a suite of commercial chiral columns and assessed the toxicity of enantiomerstoward butyrylcholinesterase and Daphnia magna. Satisfactory separations of salithionenantiomers could be achieved on all tested columns, that is, Chiralcel OD, ChiralcelOJ, and Chiralpak AD column. However, the Chiralpak AD column offered the best sep-aration and was chosen to prepare micro-scale of pure salithion enantiomers for subse-quent bioassays. The first and second enantiomers eluted on the Chiralpak AD columnwere further confirmed to be (2)-S-salithion and (1)-R-salithion, respectively. The halfinhibition concentrations to butyrylcholinesterase of racemate, (1)-R-salithion, and (2)-S-salithion were 33.09, 2.92, and 15.60 mg/l, respectively, showing (1)-R-enantiomerbeing about 5.0 times more potent than its (2)-S-form. However, the median lethal con-centrations (96 h) of racemate, (1)-R-salithion, and (2)-S-salithion toward D. magnawere 3.54, 1.10, and 0.36 lg/l, respectively, suggesting that (2)-S-salithion was about3.0 times more toxic than (1)-R-form. Racemic salithion was less toxic than either of theenantiomers in both bioassays, suggesting that antagonistic interactions might occurbetween the enantiomers during the toxication action. This work reveals that the toxic-ity of salithion toward butyrylcholinesterase and D. magna is enantioselective, and thisfactor should be taken into consideration in the environmental risk assessment of sali-thion. Chirality 21:922–928, 2009. VVC 2009 Wiley-Liss, Inc.

KEY WORDS: organophosphorus pesticides; chiral separation; enantioselectivity;butyrylcholinesterase; Daphnia magna

INTRODUCTION

About 25% of currently used pesticides are chiral, andthis ratio is yet increasing as compounds with morecomplex structures are introduced into use.1 Organophos-phorus pesticides (OPs) commonly have asymmetric cen-ter either on phosphorus atom or carbon atom. Accordingto a survey, more than 30% of OPs in the market are chi-ral.2 Biological processes of enantiomers are usually differ-ent because of the fact that the final molecular targets,such as enzymes and biochemical receptors of the smallmolecule chemicals are naturally anisotropic. Numerousstudies have found that chiral OPs are generally enantiose-lective in toxicity. For instance, enantiomers of chiral OPsdiffer in in vitro inhibitory potentials toward variousenzymes such as acetylcholinesterase,3–8 butyrylcholines-terase,3,9,10 carboxyesterase,3 and neuropathy targetesterase,3,11 and in vivo acute3 or delayed3,11 neurotoxiceffects on mammals and/or nonmammals. Recently,increasing studies have demonstrated that enantioselec-tivity also exists in the in vivo acute aquatic toxicity ofchiral OPs.5–7,9,10,12–14

Salithion ((RS)-2-methoxy-4H-1,3,2 k5 benzodioxaphos-phorin 2-sulfide), an organophosphorus pesticide, whichwas first synthesized in 1963, is used for control of a broad

spectrum of insect pests in orchards or vegetables.15,16 Inrecent years, because of its high efficacy, low cost, andcapacity to control pyrethrin-resistant pests,17 salithion hasbeen considered as an effective alternative of the highlytoxic OPs in China. Salithion has a unique asymmetriccenter at the cyclic phosphorus atom and hence containstwo enantiomers (see Fig. 1). The insecticidal activity18–20

of salithion has been testified enantioselective becausehigh optical pure (>98% e.e.) salithion enantiomers weresynthesized.21 The results showed that (2)-S-salithion wasabout two-folds more highly active than its antipodeagainst both larvicidal and adulticidal Musca domestica.18

*Correspondence to: Weiping Liu, Zhejiang University of Technology,Hangzhou 310032, China. E-mail: [email protected] grant sponsor: The National Basic Research Program of China;Contract grant number: 2009CB421603Contract grant sponsor: National Natural Science Foundation of China;Contract grant numbers: 20837002, 30771255Contract grant sponsor: The Program for Changjiang Scholars and Innova-tive Research Teams in Chinese Universities; Contract grant number: IRT0653Received for publication 31 July 2008; Accepted 10 November 2008DOI: 10.1002/chir.20690Published online 22 January 2009 in Wiley InterScience(www.interscience.wiley.com).

CHIRALITY 21:922–928 (2009)

VVC 2009 Wiley-Liss, Inc.

While against Tribolium castaneum, the (1)-R-enantiomercontrarily had a more potent larvicide (LC50 5 9.3 mg/l)than (2)-S-enantiomer (LC50 5 63 mg/l).20 Besides,enantiomeric differences also appeared in the inhibitoryactivities against larval growth,19,20 trehalase-trehalosesystem,19,20 digestive enzymes,19,20 and octopamine-receptor20 of the target organisms, with a discriminationsometimes over than 10-fold. Itoh’s work then found thatdegradation of salithion in two Japanese upload soils wasalso enantioselective, with (2)-S -salithion consistently dis-appearing 1.5–1.7 times faster than the (1)-R-salithion.22,23

However, despite of the universal existence of enantio-specificity in toxicity of chiral OPs, no correspondinginformation with respect to salithion is reported, makingthe current assessment of its actual environmental risksinsufficiently.

One of the biggest challenges in determining the enan-tioselective toxicities of chiral pesticide is the preparationof enantiomer standards. Although high optically puresalithion enantiomers can be obtained by synthesis via anL-proline methyl ester derivative,21 this approach is toocomplicated for environmental or toxicological researcherswho just need milligram scale amount of pure enantio-mers. In recent years, high-performance liquid chromatogra-phy (HPLC) with chiral stationary phase (CSP) columnhas been employed to prepare small scale of individualenantiomers from racemic chiral OPs.5–14 Two chiralHPLC columns, Chiralcel OB [Cellulose tribenzoate]21

and Chiralpak OT(1)[poly(triphenylmethylmethacry-late)]24 have been used for enantiomeric separation ofsalithion. However, we consider that both of them may notbe suitable for preparing salithion enantiomers becausethe majority of labs worldwide may not possess these highanalyte-specific columns. Moreover, the retention times ofthe salithion enantiomers on the Chiralcel OB were twolong,21 which would prevent a semipreparative application.In this work, to make more labs easily obtain the salithionenantiomers, the enantiomeric separation was evaluatedon three most commonly used commercial chiral columns,Chiralpak AD, Chiralcel OD, and Chiralcel OJ, which cangive satisfactory resolution for more than 80% of the race-mates tested.25 The inhibitory potentials of each enan-tiomer and racemate of salithion against butylcholinester-ase (BChE) from human serum, together with their acutetoxicities toward Daphnia magna (D. magna), an aquaticinvertebrate, were studied. Results from this study may

offer useful information for a more comprehensive assess-ment of the environmental risk of salithion.

MATERIALS AND METHODSChemicals

Racemic salithion with purity of 100% was purchasedfrom Wako Pure Chemical Industries (Osaka, Japan).BChE from human serum, butyrylthiocholine iodide(BTCh-I), and 5,50-dithiobis-(2-nitrobenzoic acid) (DTNB)were purchased from Sigma Chemical (St. Louis, MO).Other solvents or chemicals were of HPLC or analyticalgrade. Standard salithion concentration of 5000 mg/l forseparation and bioassay was prepared in ethanol.

Chromatographic Conditions and Preparationof Enantiomers

Enantiomeric separation was performed on a Jasco LC-2000 series HPLC system (Jasco, Tokyo, Japan) equippedwith a PU-2089 quaternary gradient pump, a mobile phasevacuum degasser, an AS-1559 autosampler with a 100-llloop, a CO-2060 column temperature control compartment,a variable-wavelength CD-2095 circular dichroism (CD),an OR-2090 optical rotation detector, and an LC-Net II/ADC data collector. The chromatographic and integrateddata were recorded and processed with the computer-based ChromPass software (version 1.7.403.1, Jasco). Thefollowing commercial chiral columns purchased from Dai-cel Chemical Industries (Tokyo, Japan) were used in thisstudy: Chiralpak AD-H [amylose tris-(3,5-dimethylphenyl-carbamate)], Chiralcel OD-H [cellulose tris-(3,5-dimethyl-phenyl-carbamate)], Chiralcel OJ-H [cellulose tris-(4-meth-ylbenzoate)], and Chiralpak OT(1) [poly(triphenylmethyl-methacrylate)]. All of the columns were 250 mm 34.6 mmi.d. in dimensions. In the preliminary experiments, the mo-bile phase for all polysaccharide columns was n-hexanemodified with 0 to 10% of isopropanol or ethanol. All thesolvents were filtered through a 0.45-lm filter anddegassed in vacuum prior to use. The injection volume,flow rate, temperature, and UV detection wavelength were20 ll, 1.0 ml/min, 258C, and 220 nm, respectively.

The solvents containing the resolved enantiomers forsubsequent bioassays were manually collected at the col-umn outlet of the Chiralpak AD column. They were evapo-rated to dryness under a nitrogen stream and redissolvedin ethanol. The two preconcentrated enantiomers werethen identified by their absolute configurations. Based ona study of Wu et al.,24 it has been confirmed that the first-eluting enantiomer of salithion on the Chiralpak OT (1)was (1)-S-form, whereas the second one was (2)-R-form.In this study, racemate and two concentrated enantiomersof salithion were injected into the Chiralpak OT(1) col-umn, respectively, and the absolute configurations of indi-vidual enantiomers can therefore be determined accordingto their elution orders on Chiralpak OT(1). The chromato-graphic conditions, which refer to that in Ref. 24 were:mobile phase, methanol; flow rate, 0.5 ml/min; UV wave-length, 220 nm; oven temperature, 258C. By calculatingthe peak areas of enantiomers in HPLC chromatograms,

Fig. 1. Enantiomers of salithion (*indicates chiral center).

923SEPARATION AND TOXICITY OF SALITHION ENANTIOMERS

Chirality DOI 10.1002/chir

the purities of prepared enantiomers were more than 99%.The concentrations of enantiomers were measured byassuming the same response factor for enantiomers asfor the racemate and also by analyzing an aliquot on agas chromatograph coupled with a nitrogen-phosphorusdetector.

Chromatographic Characterization

The chromatographic parameters, including capacityfactors (k0), separation factors (a), and resolutions (Rs) forthe resolved enantiomers of salithion were calculated andused to evaluate the enantioselectivity of the CSPs. Thecapacity factor k0 for each enantiomer was calculated as

k0 ¼ ðtR � t0Þ=t0 ð1Þ

where tR is the average retention time of duplicate injec-tions of the analyte taken at peak maxima, and t0 is the col-umn void time determined by recording the first baselineperturbation. The separation factor a was calculated as

a ¼ k02=k01 ð2Þ

where k01 and k02 are the capacity factors of first and sec-ond eluted enantiomers, respectively. The resolution fac-tor Rs of enantiomers was calculated as

RS ¼ 23t2 � t1w1 þ w2

ð3Þ

where t1 and t2 represent, respectively, the peak retentiontime of the first and second eluted enantiomers, and w1

and w2 are the peak widths measured at the peak base ofthe first and second peaks, respectively.

BChE Inhibitory Potentials

The anti-BChE potentials of racemic salithion and itsenantiomers were determined by calculating their concen-trations resulting in half inhibition of enzyme activity(IC50). Briefly, 180 ll properly diluted enzyme solutions(in which the hydrolysis rate of BTCh-I was �0.03–0.07 ab-sorbance units/min) was treated in the phosphate buffer(pH 8.0) with 20 ll inhibitor at various concentrations thatinhibited enzymatic activity by 10–80%. At the same time,control samples were also prepared by using 20 ll of phos-phate buffer (pH 8.0) in place of the salithion solutions.Final concentrations of solvents (ethanol) in both BChE-in-hibitor solution and BChE-control solution were main-tained at 0.5% (v/v). Each solution was incubated at 378Cfor 30 min. Then, 80 ll of the BChE-inhibitor solution (orBChE-control solution) was taken to measure the residualactivity of BChE by a modified Ellman method.26 Briefly,150 ll of DTNB solution and 20 ll of BTCh-I solution wereadded to the wells on a 96-well microtiter plate. Enzyme-control or enzyme-inhibitor (80 ll) solution was subse-quently added to make the final concentrations of DTNBand BTCh-I at 0.33 and 1.0 mM, respectively. The enzy-matic activities of the mixtures in the 96-well microtiterplate were determined at 405 nm for 6 min at the interval

of 1 min from the addition of enzyme-buffer or enzyme-in-hibitor using a Bio-Rad model 680 microplate reader (Bio-Rad Laboratories).

IC50 was calculated by the logit transition model usingthe following equation27:

log it ¼ lnI

100� Ið4Þ

1g½C� ¼ Aþ B log it ð5Þ

where I and [C] represent the percent of inhibition onBChE activity and the corresponding concentration of theinhibitor, respectively. A and B are two constants. Whenlog it equals to zero, the corresponding [C] is IC50.

Acute Aquatic Toxicity

The median lethal concentrations (LC50) for racemicsalithion and its enantiomers were evaluated using a stand-ard bio-indicator D. magna. Stock organisms were origi-nally obtained from the Chinese Academy of Protectionand Medical Science (Beijing, China). The test organismswere obtained from continuous culture maintained at 22 618C in M4 culture medium28 with a photoperiod of 12 h/day and a density of <50 animals per liter. The mediumwas renewed three times a week, and daphnids were feddaily with the alga Scenedesmas obliquus (S. obliquus),which were cultured in the laboratory using a nutrient me-dium. Offspring were separated at regular intervals. Thetest animals used in this experiment were juveniles agedbetween 12 and 24 h. Prior to testing, a sensitive test fordaphnids to potassium dichromate (K2Cr2O7) was per-formed as a positive control, and the EC50 (24 h) valuewas in the range of 0.6 to 1.7 mg/l.29 The overall testingprocedure followed the EPA guidelines.30 Briefly, five neo-nates were transferred into glass beakers filled with 20 mlof blank or test solutions of various concentrations. Thetest animals were fed with alga S. obliquus 2 h before thetest began and at 48 h when the test solution was renewedwith the freshly spiked pesticide. The mortality of daph-nids of all vials was monitored at 24 h intervals for the96 h exposure period. LC50 of the test population wasdetermined by probit analysis.30,31

Statistic Analysis

All of the above tests and measurements were per-formed in four replicates. The Student’s t test at a signifi-cance level of 0.05 was used to compare the differencesbetween groups.

RESULTS AND DISCUSSIONEnantiomer Separation and Identification

Individual enantiomers of chiral OPs that have been suc-cessfully separated before were mainly obtained on thepolysaccharide model chiral stationary phases.5–8,12–14,32,33

In this investigation, satisfactory resolution of salithionenantiomers was achieved on all of the three CSPs of de-rivative cellulose and amylase we selected, among which

924 ZHOU ET AL.


the Chiralpak AD column offered the highest a of morethan 1.20 suggesting a baseline resolution.34 Moreover,the chiral recognition ability of Chiralpak AD also seemsbetter than that of Chiralcel OB and Chiralpak OT(1), thetwo columns that have been used to separate salithionenantiomers previously.21,24 The enantiomeric separationresults are listed in Table 1, and a representative chromat-ogram is shown in Figure 2. Mechanisms for chiral dis-crimination are very complicated, especially for these poly-meric CSPs, which have a number of different bindingsites with different affinities to enantiomers. As a result,the exact reasons for separation of the salithion enantio-mers on these chiral stationary phases were difficult to eluci-date but may be attributable to the following two mecha-nisms. First, the molecule of salithion contains electroneg-ative atoms (such as oxygen), P¼¼S group, and phenylring attached directly or indirectly to the stereogenic cen-ter, which may lead to different preferences in their inter-action with the derivatized polysaccharide-CSPs throughhydrogen bonding, dipole–dipole or p–p interactions. Inaddition, the degree of steric fit into the chiral cavities ofthe CSPs may also play a role in chiral recognition, andthe chiral cavities of the different CSPs may have differentaccessibilities for salithion. Given its substantially betterperformance, the Chiralpak AD column was chosenfor further preparation of optical pure enantiomers ofsalithioin.

The enantiomers of a chiral compound are usually dis-tinguished by their absolute configurations or optical rota-tions. Recently, CD detectors coupled directly to HPLChave become powerful tools for determining the opticalproperties of the resolved enantiomers.35 Because of theirlow sensitivity, optical rotations and CD signs of theresolved enantiomers of salithion were both not able to bedetermined. And then, identification of the individual enan-tiomers, which were collected on the Chiralpak AD col-umn was made by the detection of absolute configurations.As described above, enantiomeric separation of salithionhas been achieved on the Chiralpak OT(1) column, withthe (2)-S-form eluting former than the (1)-R-form.24 Datain Table 1 displayed that the enantiomers of salithion werealso obviously separated, and the whole separation effectwe got was substantially better than before, which is likelybecause the Chiralpak OT(1) column we used is a newone. When the individual enantiomers preprepared on theChiralpak AD column were reinjected into the ChiralpakOT(1) column, a notable result we obtained was that the

elution order of salithion enantiomers was reversedbetween the Chiralpak AD and Chiralpak OT(1) (seeFig. 3). That is, the enantiomer eluting first on ChiralpakAD contrarily eluted second on Chiralpak OT(1), imply-ing that the first-eluting enantiomer on the Chiralpak ADis (1)-R-salithion. Similarly, the second-eluting enantiomeron the Chiralpak AD could be confirmed as (2)-S-sali-thion. However, the elution order of salithion enantiomerson Chiralcel AD is identical with that on the ChiralcelOB.21 Additionally, the elution order of salithion enantiom-ers on Chiralcel OD and Chiralcel OJ were also determined.Note that the orders of elution showing on all of the poly-saccharide CSPs, that is, Chiralpak AD, Chiralcel OD,Chiralcel OJ, and Chiralcel OB are consistent (Table 1).

TABLE 1. The enantiomeric separations of salithion on different chiral columns under 258C

Chiral column Hexane/isopropanol (v/v)a

Capacity factorSeparation factor

aResolution

Rs Elution orderk01 k02

Chiralcel OJ 95/5 8.47 9.32 1.10 1.56 R/SChiralcel OD 99.5/0.5 6.76 7.40 1.09 1.42 R/SChiralpak AD 99.5/0.5 4.15 5.03 1.21 4.14 R/SChiralpak OT(1) 100 %b 2.02 2.52 1.25 2.28 S/R

aThe flow rates of mobile phase were all 1.0 ml/min.bMethanol, 0.5 ml/min.

Fig. 2. Representative chromatograms for the chiral separation of sali-thion on the tested chiral columns. (a) Chiralcel OJ; (b) Chiralcel OD;and (c) Chiralpak AD. Chromatographic conditions: the composition ofmobile phases for Chiralcel OJ, OD, and Chiralpak AD columns were hex-ane/isopropanol (95/5, v/v), hexane/isopropanol (99.5/0.5, v/v), and hex-ane/isopropanol (99.5/0.5, v/v), respectively; the flow rate, UV detectionwavelength, and temperature were 220 nm, 1.00 ml/min, and 258C,respectively.



Enantioselective Toxicity

As shown in Table 2, the BChE inhibitory activity anddaphnia toxicity of racemate and enantiomers of salithionboth showed significant differences. Judging from thevalues of IC50, (1)-R-enantiomer has a five times strongerinhibition activity toward BChE than (2)-S-enantiomer(Table 2). BChEs are found in the blood of many verte-brate species,36 and although their physiological roleremains uncertain and the inhibition of serum BChE doesnot represent the molecular mechanism of toxicity of OPs,they appear to have a protective function by sequesteringcirculating OP compounds, thereby decreasing the toxiceffect of these compounds on brain acetylcholinesterase(AChE).37,38 In return, it can be assumed that organisms’risk in acute cholinergic crisis (inhibition of AChE) willpotentiate synchronously with the inhibition of BChE

increasing. In addition, (1)-R-enantiomer may be moreacutely toxic to the fetus than its antipode, because BChEappears in the development of the central nervous systembefore AChE, and has been suggested to function as anembryonic acetylcholinesterase.39 For the aquatic toxicassay, the more potent BChE inhibitor (1)-R-enantiomershowed less toxic to D. magna, with the LC50 value about3.0–5.4 times higher than that of (2)-S-enantiomer duringthe 48- and 96-h test (Table 2). This discrepancy in in vitroand in vivo toxicity has also been observed in other chiralOPs, such as methamidohphos and chloramidophs.5,6 Inthe case of methamidophos, the (2)-enantiomer was about8.0–12.4 times more potent to AChE than its (1)-form,whereas the (1)-enantiomer was 7.0 times more toxic toD. magna than the (2)-form. In the other case, the mostpotent AChE inhibitor of chloramidophos stereoisomersshowed the least toxicity to D. magna. Although thecauses for the reversed toxicity to the two different biologi-cal targets are not clear, it may be because of a combina-tion of several factors. Firstly, different species of enzymesmay have different sensitivities to enantiomers. Secondly,many biological processes, especially metabolism, trans-fer, and accumulation that affect the in vivo toxicity havebeen found to be enantioselective.40 Furthermore, becausesalioxon (2-methoxy-4H-l,3,2-benzodioxaphosphorin), thein vivo activated metabolite of salithion, is also chiral,enantioselectivity of salioxon in biological actions may alsoresponsible for the selectivity.

Many chiral OPs have enantioselective toxicities.3–14 Inmost cases, the toxicity of the racemate was found to bean additive result of both enantiomers by showing amedial toxicity between enantiomers, although one of theenantiomers may exhibit more potent than the otherone.14 However, the racemic salithion had less toxicity toboth BChE and D. magna than either of enantiomers (Ta-ble 2), suggesting that an antagonistic interaction betweenthe enantiomers may exist during the toxic action. Forinstance, the racemic salithion showed about 3 or 10 timesless toxic to D. magna in 96-h test than (1)-R-salithion or(2)-S-enantiomer, respectively. Actually, salithion is notthe exclusive organophosphorus pesticide with toxicityinteraction for the enantiomers being antagonistic. Similarantagonistic interactions between the enantiomers canalso found in the aquatic toxicities of other four chiralOPs, that is, trichloronate, chloramidophos, fonofos, and

Fig. 3. Representative HPLC chromatograms for the chiral separationof salithion on the Chiralpak OT (1) column (a) racemate; (b) the first-eluting enantiomer collected on the Chiralpak AD column; and (c) thesecond-eluting enantiomer collected on the Chiralpak AD column.

TABLE 2. Half inhibition concentrations (IC50) forbutyrylcholinesterase and median lethal concentrations(LC50) for Daphnia magna of racemate and enantiomers

of salithion

Compound IC50 (mg/l)a

LC50 (lg/l)b

48 h 96 h

rac-salithion 33.09 6 0.97 7.07 (6.62–7.55) 3.54 (3.14–3.98)(1)-R-salithion 2.92 6 0.17 2.51 (2.21–2.84) 1.10 (0.80–1.54)(–)-S-salithion 15.60 6 0.20 0.46 (0.41–0.51) 0.36 (0.31–0.42)

aData are given as mean 6 standard deviation (n 5 4).bData are given as mean and 95 % confidential intervals.

926 ZHOU ET AL.


fosthiazate.15 In another study, an antagonistic effect wasalso observed between the enantiomers of fenamidophosagainst BChE.10 However, no studies have so far attemptedto understand such antagonistic interaction between enan-tiomers, and the mechanism remains unclear.

Although salithion are applied and released into theenvironment in racemic form, an enantiomer enrichmentcan be easily formed because microorganisms in soilspreferentially decompose (2)-S-salithion, resulting in theenrichment of (1)-R-salithion in soil after biodegrada-tion.22,23 And then, the significant differences observed inthe acute toxicity to D. magna among different forms ofsalithion may have some environmental implications. Forexample, an underestimation may occur when using thedata derived from racemic salithion to assess the riskposed only by either of the (1)-(R)- or (2)-S-salithion.Degradation of a toxicant generally leads to detoxication.However, the enantioselective degradation of salithionmay increase its toxicity. For example, supposing that ra-cemic salithion was present at 3.54 lg l21 (each enantiomer1.77 lg l21) in water and (1)-R-salithion were completelydegraded while (2)-S-salithion still remained 1.77 lg l21,the toxicity of the (2)-S-salithion enrichment solution to D.magna would increase by about five times compared withthe original solution. Therefore, when significant enantiose-lectivity exists for a chiral pesticide, it is important to alsoevaluate the interaction of enantiomers in the joint toxicityeffect when enantiomers are present in a mixture

CONCLUSIONS

Satisfactory resolutions of salithion enantiomers couldbe obtained on the Chiralcel OD, OJ, and Chiralpak ADcolumns. The toxicities of salithion to butyrylcholinester-ase (in vitro) and to Daphnia magna (in vivo) were bothenantioselective. It should be noted that racemic salithiondisplayed lower toxicities to both butyrylcholinesteraseand D. magna than either of its enantiomers. This worksuggests that enantioselective toxicity should be takeninto account in the evaluation of environmental risks of sal-ithion. For a more comprehensive and accurate knowledgeon the enantioselective behaviors of salithion in the envi-ronment, more research work should be conducted onother species in the future.

ACKNOWLEDGMENTS

The authors thank Gao Tingyao, Environmental Scienceand Technology Development Foundation of Tongji Uni-versity, Shanghai, China.

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Separation and toxicity of salithion enantiomers