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Enantioselectivity in Current Chiral Insecticides in Soil and Sediment Environments ABSTRACT Jay Gan 1, Sujie Qin, 1 Mae G. Nillos, 1 Weiping Liu, 2 and Daniel Schlenk 1 1. Environmental Sciences, University of California - Riverside, Riverside, CA, USA. 2. Zhejiang University of Technology, Hangzhou, China. BACKGROUND CONCLUSIONS AND FUTURE INTERESTS RESULTS Many environmental contaminants are chiral compounds consisting of optical isomers called enantiomers. Among the currently used pesticides, chirality exists in all synthetic pyrethroids and a number of organophosphate (OP) insecticides. In addition, the current use patterns and the wide-spectrum aquatic toxicity of these chemicals make them an emerging ecotoxicological concern. However, due to the general lack of enantiomer standards, the separation and identification of enantiomers are among the biggest challenges in studying enantioselectivity in pesticides. In this study, a number of currently used chiral pesticides, including synthetic pyrethroids and organophosphates, were separated by high-performance liquid chromatography using enantioselective columns. The adequate separation allowed the isolation and recovery of enantiomers for use in assessment of enantioselectivity in ecotoxicity. Fish vitellogenin assays were used to evaluate potential for endocrine disruption by the pyrethroids and possible enantioselectivity in this effect. Enantioselective inhibition by chiral OPs of acetylcholinesterase was also investigated. These investigations build on previous results indicating enantioselectivity in acute toxicity of cis-bifenthrin and cis-permethrin to C. dubia and D. magna, where in the (+) enantiomers contributed almost 97% of the observed toxicity in the racemate, and the chiral OP insecticides in which the (-) enantiomer contributed up to 94% of the total toxicity in the racemate. Enantioselectivity was also observed in synthetic pyrethroids during degradation in sediment under either field or laboratory conditions. Enantioselectivity in all these processes could result in ecotoxicological effects that may be important considerations in future risk assessment and regulatory decisions. C=CH CH 3 COOCH 2 CH 3 H H Cl Cl O CF 3 C=CH CH 3 COOCH 2 CH 3 H H CH 3 Cl O Cl Cl H H CH 3 COOCH CH 3 C=CH CN CN C=CH CH 3 COOCH CH 3 H H Cl Cl O F * * * * * * * * * * a. bifenthrin b. permethrin c. cypermethrin d. cyfluthrin Figure 1. Structures of synthetic pyrethroid insecticides used in this study (* indicate chiral position) b. profenofos a. fonofos c. crotoxyphos * * * d. trichloronate * Figure 2 Structures of organophosphorus insecticides used in this study (* indicate chiral position) Synthetic Pyrethroids (SP) are structural derivatives of the natural pyrethrins Function by altering the kinetics of the voltage-gated sodium channels in the insect nerves Highly effective against pest insects but have low mammalian toxicity Highly toxic to fish and other aquatic organisms All members of this group are chiral, and most SPs can have up to 4 to 8 enantiomers Extremely hydrophobic and low aqueous solubility but may accumulate in sediments and become bioavailable or transported through runoff Extensively used in agriculture and urban/household insect control OP compounds are potent cholinesterase inhibitor Highly toxic to aquatic invertebrates but are less persistent in the environment Among the most important chemicals against agriculture and household pests in many countries Some members of this class are chiral, either on a P or C atom in the structure MATERIALS AND METHODS ENANTIOMER PREPARATION Enantiomers of SPs and chiral OPs were separated by enantioselective HPLC. An Agilent 1100 series HPLC system was used for the separation. The instrument was connected to an advanced laser polarimeter detector (PDR-Chiral, FL) for detecting the direction of rotation of separated enantiomers. Separation of SP enantiomers was carried out on a Sumichiral OA-2500-I column or two Chirex OOG-3019-OD columns in series. Mobile phases used are a mixture of hexane, 1, 2-dichloroethane and ethanol. Enantiomers of chiral OPs were separated on a CHIRALCEL OJ column. Hexane, 2-propanol and ethanol were used in the mobile phase. Individual enantiomers were collected at the HPLC outlet and used in biodegradation and toxicological assessment. ACUTE TOXICITY TO AQUATIC INVERTEBRATES Enantioselectivity in aquatic evaluated through 96-h acute assay using C. dubia and D. magna. The bioassay method was based on EPA solutions containing a given enantiomer or mixture of enantiomers at a range of concentration was prepared from the The LC 50 was determined by probit analysis (ToxCalc TM v5.0, Tidepool Scientific Software, McKinleyville, CA). C. dubia ACETYLCHOLINESTERASE INHIBITION Acetylcholinesterase (AChE) inhibition of racemates and individual enantiomers of selected chiral OP was determined in Daphnia magna following a 24h exposure, and Oryzias latipes following a 96h exposure, as well as in vitro with purified electric eel enzyme. Enzyme activity was assayed as described by Ellman and coworkers (1961), but modified to a microplate assay. The concentration resulting in 50% inhibition of enzyme activity (IC 50 ) was computed using ToxCalcTM v5.0. Preliminary screening of SPs for endocrine disrupting effect was conducted using commercially available ELISA kits (Biosense, Bergen, Norway). Japanese medaka was initially exposed to racemic SPs to determine which ones are likely endocrine disrupters. The SP that induced vitellogenin in either male or juvenile fish was further investigated for enantioselectivity in this effect. ENDOCRINE DISRUPTION DEGRADATION STUDY ACUTE TOXICITY TO AQUATIC INVERTEBRATES ACETYLCHOLINESTERASE INHIBITION ENDOCRINE DISRUPTION C. dubia D. magna cis-BF Racemate 1R-cis 1S-cis Racemate 1R-cis 1S-cis 0.144 0.026 0.076 0.008 1.342 0.022 0.175 0.025 0.081 0.0078 1.803 0.021 cis-PM Racemate 1R-cis 1S-cis Racemate 1R-cis 1S-cis 0.539 0.055 0.156 0.021 >6.0 0.788 0.071 0.388 0.028 >6.0 trans-PM Racemate 1R-trans 1S-trans Racemate 1R-trans 1S-trans 0.519 0.058 0.197 0.053 >6.0 0.738 0.035 0.307 0.024 >6.0 cis trans RRR SSS SSR RRS RSR SRS SRR RSS CP >7.5 >7.5 >7.5 0.775 0.063 >7.5 >7.5 >7.5 0.995 0.089 CF >10 >10 >10 0.104 0.018 >10 >10 >10 0.214 0.018 Table 1. LC 50 values (ug/L) of enantiomers of cis- bifenthrin (BF) and permethrin (PM) for aquatic invertebrates C. dubia and D. magna Table 2. LC 50 values (ug/L) of enantiomers of cypermethrin (CP) and cyfluthrin (CF) for aquatic invertebrate C. dubia DEGRADATION STUDY Degradation of SP was investigated through laboratory incubation experiments. One set of sample was evaluated under anaerobic condition by equilibrating the sample in a nitrogen filled plastic chamber. The other set was incubated under aerobic condition. Another set of sample was sterilized to determine if enantioselective degradation was a result of microbial transformation. Also to determine if a given enantioselective degradation occur in the natural environment, samples were taken from the field. Quantification of SPs was carried out on an Agilent 6890 GC-ECD. HP-5 and BGB-172 columns were used in achiral and chiral analyses, respectively. A B C D F E H I K J G A B C D F E H I K J G A B C D F E H I K J G Figure 3. Stereoisomeric fractions of enantiomers and diastereomers of permethrin (PM) in stream sediment samples (note: SF when PM was applied are R-cis: 0.25; S-cis: 0.25; trans: 0.5) Figure 4. Half-life of degradation of enantiomers of cis- bifenthrin in soil and sediment under laboratory conditions 0 100 200 300 400 500 600 700 AERO BIC A NA EROBIC R-cis S-cis SEDIMENT (T 1/2 , days) 0 100 200 300 400 500 600 700 800 A ERO BIC A NA ERO BIC R -cis S-cis SOIL (T 1/2 , days) Figure 7. Vitellogenin induction in juvenile medaka following exposure to cypermethrin (CP) enantiomers. (CP1: (1R)cis(R); CP2: (1S)cis(S); CP3: (1S)cis(R) + (1R)cis(S); CP4: (1R)trans(R); CP5: (1S)trans(S); CP6: (1S)trans(R) + (1R)trans(S); CP mix: mixture of enantiomers.; E2 (positive control) vtg induction = 0.162 ng vtg/ug protein) 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 IC 50 (uM ) m edaka D .m agna CROTOXYPHOS (+ ) (± ) Figure 6. In vivo inhibition of acetylcholinesterase in Japanese medaka and D. magna by chiral OPs 0 0.05 0.1 0.15 0.2 IC 50 (uM ) m edaka D .m agna FONOFOS (-) (± ) 0 0.03 0.06 0.09 0.12 IC50(uM) m edaka D .m agna PROFENOFOS (-) (± ) Enantioselective degradation was observed for pyrethroids in both field and laboratory investigation Direction and degree of enantioselectivity in environmental degradation is not always predictable There is enantioselectivity in acute toxicity of SPs to aquatic invertebrates Major differences in potency against AChE was seen between enantiomers, and between enantiomers and racemate of chiral OPs Differences in enantioselectivity in in vitro and in vivo AChE assay for chiral OPs have been observed, suggesting stereospecificity in metabolic processes. Preliminary results suggest endocrine disrupting effects of SPs and the possible enantioselectivity in this effect is currently being explored. 0 2 4 6 8 10 12 % rem aining activity 6.25 12.50 25 50 100 200 O P concentration (uM ) CROTOXYPHOS (+) (-) Figure 5. In vitro inhibition of electric eel acetylcholinesterase by chiral OPs 0 10 20 30 40 50 60 70 80 90 % rem aining activity 15.62 31.25 62.5 125 250 500 O P concentration (uM ) PROFENOFOS (+) (-) ACKNOWLEDGEMENT Funding for this research since October 2005 is supported by USDA-CSREES, through NRI Grant 2005-35107- 16189.
