Rapid Bioassay of Pesticide Residue (RBPR) by Using Honey Bee AChE on Fruits and Vegetables

1. Rapid Bioassay of Pesticide Residue (RBPR) by Using Honey Bee AChE on Fruits and Vegetables • Jae-Han Shim, Li-Tai Jin* and Chang-Joo Lee1 • Department of Agricultural Chemistry, Chonnam National University, Kwangju 500-757,Korea 1Department of Environmental Engineering Kwangju University,Kwangju 503-703, Korea Abstract A rapid bioassay for pesticide residue method was developed for the detection of organophosphorous(OP) and carbamates insecticides. Acetylcholinesterase(AChE) of honey bee (Apis mellifera L.) was used for detecting pesticide residues on fruit and vegetables. Optimum extraction conditions of AChE from honey bee were determined. Also, optimum reaction conditions for pesticide residues analysis were examined. The sensitivity of RBPR to pesticides and stability of honey bee AChE were investigated. Among the detection limit of examined pesticides, carbofuran was 15 ppb which is the most sensitive insecticide. Enzyme activity was fairly stable at 0∼4℃ for 20 days. It can be complete the examination of 80 vegetable samples within 4 hours or even faster. Objectives Table 1. Recovery Percent of Carbamate Insectidices by the Fortified ID50 Concentration of Various Vegetables Vegetables Inesecticides Tomato Grape Perilla Lettuce Pepper Cucumber Bendiocab 88.4 86.4 85.2 87.4 86.2 86.4(0.09ppm) Carbaryl 89.0 92.2 88.4 85.0 82.5 85.7(0.17ppm)Carbofuran 83.2 84.4 80.3 86.6 87.0 83.0(0.06ppm)Isoprocarb 81.8 85.6 78.7 80.4 80.2 81.1(1.0ppm)Methiocarb 84.7 92.1 88.3 81.2 81.2 85.6(0.13ppm)Methomyl 81.8 84.2 79.1 81.2 80.0 81.6(1.3ppm)Oxamyl 84.5 87.7 84.2 79.2 79.7 82.6(3.5ppm)Propoxur 81.6 83.0 78.6 80.6 81.3 81.2(1.5ppm) AChE를 이용한 신선 과채류중의 유기인계농약과 카바메이트계 농약의 신속한 농약잔류 분석방법 개발[1] 꿀벌 AChE의 안정성과 감도를 고려한 효소원으로서의 이용가능성 검토 꿀벌 AChE의 최적 추출법과 반응조건의 확립 ◎ ◎ ◎ Materials 0.1M 인산 buffer (PBS) pH 8.60 꿀벌 (Apis mellifera L.) 에서 추출한 AChE 용액 (1.1 units/ml)[2~4] 30가지 농약 (유기인계 19가지,카바메이트계 11가지) Acetylthiocholine iodine (AThCh) 21.6 mg/ml in D.W. 5, 5＇dithio-bis (2-nitrobenzoic acid) (DTNB) 3.96 mg/ml PBS (pH 7.0) ◎ ◎ ◎ ◎ ◎ Methods 30℃ 에서 완충용액 ＋ 효소액 ＋ 농약이 녹아 있는 methanol ＋ AThCh 용액 ＋ DTNB용액을 넣은 후 412 ㎚에서 흡광도 변화를 분당 측정한다[5] . Results Table 2. The Inhibition Percent of AChE by Bendiocarb and Carbaryl from Different Size of Perilla Leaf Size of leaf Bendiocarb %AChE Size of leaf Carbaryl %AChE (cm2) (ppm) inhibition (cm2) (ppm) inhibition 1 0.02 15.0 1 0.04 15.3 3 0.06 35.2 3 0.12 35.9 5 0.1 48.3 5 0.2 50.5 10 0.2 68.2 10 0.4 69.8 20 0.4 94.1 20 0.8 95.7 Conclusion Fig. 1 Inhibition Rate of Honeybee AChE Activity for Various Phosphorus Pesticides. • 유기인계 농약 19가지를 측정한 결과 meta-systox와 cygon를 제외한 17가지 농약은 1ppm 에서 신속한 농약잔류분석이 가능 함을 확인하였다. • 2. 카바메이트계 농약 11가지를 측정한 결과 aldicarb, pirimicarb와 thiobencarb를 제외한 17가지농약은 1ppm 에서 신속한 농약잔류분석이 가능 함을 확인하였다. • 3. 꿀벌 AChE는 0~4℃ 조건에서 20일간 활성을 유지하였다. • 4. 이 방법을 사용할 경우 80시료를 4시간 이내에 측정 가능함을 밝혔다. References • Chiu, C. S., Kao, C. H. and Cheng, E. Y.. 1991. Rapid bioassay of pesticide • residues (RBPR) on fruits and vegetables.Agricultural Research of China. 40: 188 -203. • 2. Suh, Y. T. and Shim, J. H.. 1988. Effects of insecticides on the activity of acetylcholinesterase in the honey bees(Apiscerana F. and A. mellifera L.). Korean J. Apiculture. 3: 48 - 60. • 3. Stepanidou, M., A Koutselinis, A., Pappas, F. and Methenitou, G..1996. Bee head acetylcholinesterase as an indicator of exposure to organophosphate and carbamateonsecticedes. Veterinary and Human Toxicology. 38: 420 - 422. • 4. Belzunces, L. P., Toutant, J. P. and Bounials, M.. 1988. Acetylcholinesterase from Apismellifera head. Biochem. J. 255: 463 - 470. • 5. Ellman, G. L., K. D. Courtney, V. Andres and R. M. Featherstone. 1961. A new and rapid colorimetric determination of acetylcholinesterase activity. Bilchem. Pharmarcol. 7: 88 - 95. Fig. 2 Inhibition Rate of Honeybee AChE Activity for Various Carbamate Pesticides. Corresponding address: http://chonnam.chonnam.ac.kr/~jhshim E-mail address: jhshim@chonnam.chonnam.ac.kr