Korean Journal of Environmental Agriculture Korean J Environ Agric (2013) Online ISSN: 2233-4173 Vol. 32, No. 2, pp. 136-141 http://dx.doi.org/10.5338/kjea.2013.32.2.136 Print ISSN: 1225-3537 Research Article Open Access 복숭아재배기간중살균제 Fenarimol과살충제 Flufenoxuron의생산단계잔류허용기준설정 문혜리, 1 박재훈, 1 윤지영, 1 나은식, 2 이규승 1* 1 충남대학교생물환경화학과, 2 한국인삼공사안전성연구센터 Establishment of Pre-Harvest Residue Limits (PHRLs) of Fungicide Fenarimol and Insecticide Flufenoxuron in Peaches during Cultivation Period Hye-ree Moon, 1 Jae-hoon Park, 1 Ji-Yeong Yoon, 1 Eun-shik Na 2 and Kyu-Seung Lee 1* ( 1 Department of Biological Environment Chemistry, Chungnam National University, Daejeon 305-764, Korea, 2 Korea Ginseng Corp Safety Research Center) Received: 25 February 2013 / Revised: 5 April 2013 / Accepted: 2 May 2013 c 2013 The Korean Society of Environmental Agriculture This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract BACKGROUND: This study was performed to establishment the PHRLs of peach during cultivation period, and also to estimate biological half-lives for residues of fenarimol and flufenoxuron. METHODS AND RESULTS: The extracted samples of fenarimol were analyzed by GC-ECD and the flufenoxuron extracted samples were analyzed by HPLC-DAD. Recoveries of fenarimol at two fortification levels of 0.1 mg/kg, 0.5 mg/kg were 93.69±6.56(%) and 94.45±1.60 (%), respectively. And recoveries of flufenoxuron at two fortification levels of 0.1 mg/kg, 0.5 mg/kg were 106.73±5.90(%) and 96.37±6.66(%), respectively. CONCLUSION(S): The biological half-lives of fenarimol in single treatment and triple treatment were 3.5day and 3.8day. that of Flufenoxuron was also 7.1day and 4.9day, respectively. The PHRL of fenarimol were recommended as 1.5 mg/kg for 10day before harvest and the PHRL of flufenoxuron were recommended as 1.4 mg/kg for 10day * 교신저자 (Corresponding author) Phone: +82-42-821-6735; Fax: +82-42-822-5781; E-mail: kslee@cnu.ac.kr before harvest. Key Words: Biological half-life, Fenarimol, Flufenoxuron, Pre-harvest residue limit(phrl) 서론 농촌경제연구원의농업 농촌에대한 2012년국민의식조사결과에따르면미래에중요하게실행하여야할농촌 농업의역할로전문가, 도시민, 농업인모두가안전한식품의안정적공급을 1위로꼽았다 (KREI, 2012). 주부를대상으로식품안전성에대해조사한결과평소 매우불안 (7.6%) 하거나 불안한편 (57.8%) 이라는응답이 65.4% 로 불안하지않은편 이라고응답한주부 (34.6%) 에비해 2배가까이많은것으로나타났다. 또한식품안전성에불안을느끼는요인으로는 잔류농약 (96.0%) 을꼽았다 (Choe et al., 2005). 이처럼소비자, 전문가, 농업인모두가식품안전성에대한욕구가높은것을알수있다. 우리나라는 2012년 10월현재 431종의농약잔류허용기준 (MRL) 이설정되어있으며 (Korean Food Standards Codex, KFDA, 2012) 농림수산식품부에서는농산물품질관리법시행규칙제 21조에의거 생산단계농산물의농약잔류허용기준 136
Establishment of Pre-Harvest Residue Limits (PHRLs) of Fungicide Fenarimol and Insecticide Flufenoxuron in Peaches during Cultivation Period 137 (Pre-harvest residue limit, PHRL) 을설정하여출하일에농약잔류량이 MRL을초과하지않도록관리하고있다. 작물에살포된농약은강우, 햇빛, 미생물에의한분해등여러요인과작물의증체에따른희석효과에의해그농도가낮아질수있다 (Hill and Inaba, 1990; Bentson, 1990). 그러므로재배기간중에발생될수있는모든요인을종합하여농산물중잔류농약의감소율을평가하는것이필요하다고본다. 특히이를위해살포된농약의잔류량을수확전일정기간동안조사하여잔류농약감소회귀식과생물학적반감기를산출하여경시적인잔류량감소를연구할필요가있다. 이를바탕으로합리적인잔류감소예측식을만들면수확시잔류량을예측하여 MRL을초과할가능성이있는농산물을판단해출하연기또는폐기처분등의조치를취할수있다 (Choi et al., 2002; Kim et al., 2002; Ko et al., 2003). 본연구는복숭아에안전사용기준이설정되어있는 fenarimol 과 flufenoxuron을선정하여실제재배기간중의잔류량변화를통한합리적인회귀식과반감기를산출하며잔류량변화를예측하고생산단계잔류허용기준설정의기초자료를제공하고자수행하였다. 재료및방법 시험약제시약및기구 Fenarimol ( 표준품순도 99.5%, Merck, Germany) 과 flufenoxuron ( 표준품순도 99.3%, Merck, Germany) 은분석용표준품을사용하였다 (Table 1). 분석에사용된시약은모두 pesticide residue analysis (PRA) 급용매를사용하였으며 dichloromethane (J.T Baker, HPLC grade, USA), acetone (J.T Baker, HPLC grade, USA), n-hexane (J.T Baker, HPLC grade, USA), acetonitril (J.T Baker, HPLC grade, USA), sodium chloride (Junsei GR, Japan), sodium sulfate (Junsei GR, Japan), rotary vacuum evaporator (EYELA, Japan), SPE-florisil cartridge Table 1. Physicochemical of fenarimol and flufenoxuron 1 g (Phenomenex, USA), glass column (11 mm I.D. 30 cm L.), Florisil (60-100 mesh, FLUKA, USA) 약제처리횟수및사용량은작물보호제지침서 (Korea Crop Protection Association, 2012) 의안전사용기준에따라하였다. 훼나리 (fenarimol 12% 수화제, 동부팜한농 ( 주 )) 와카스케이트 (flufenoxuron 5% 분산성액제, 성보화학 ( 주 )) 를사용하였다. 약제처리및작물재배복숭아포장은세종특별자치시연서면성제리소재일반농가의노지를임차하였다. 관행재배법에준하여관리하였으며무대재배로품종은 10년수령의홍백으로하였다. 시험포장에처리한약제는 fenarimol 3,000배, flufenoxuron 1,000배희석하여 7일간격으로 1회처리와 3회처리로구분하였다. 전동식분무기 ( 광성, KP-PK 4000P) 를이용하여과실에충분히살포하였다. 시료채취최종으로약제를살포한시점을기준으로 ( 최종약제살포후 2시간 ) 0일차, 1, 2, 4, 6, 8, 10, 12, 14일차에각처리구별로생육정도가균일한과실을 2 kg 이상채취하여각각의무게를측정한후생장곡선을작성하고 -20 에저장하였다. 복숭아중 fenarimol, flufenoxuron 분석 Fenarimol 세절한복숭아시료 20 g에 acetone 100 ml와 Celite 545를 2 g 가하여 30분간진탕하여감압여과한후여액을 1,000 ml의분액여두에옮겨증류수 300 ml 와포화식염수 50 ml을가한다음 dichloromethane 60 ml을두번가하여유기용매층을 sodium sulfate에통과시켜감압농축하였다. 이를 n-hexane 5 ml로재용하고 dichloromethane 5 ml로활성화시킨 florisil SPE cartridge에전량충진한다. dichloromethane : ethyl acetate (7/3, v/v) 을 15 ml 용출하여감압농축한후건고물을다시 acetone 4 ml로정용하여 GC/ECD로분석하였다. Common names Physical chemistry MRL(Peach), KFDA Structure Fenarimol (Fungicide) Flufenoxuron (Insecticide) M.W : 331.2 M.W : 488.8 Log Pow : 3.69 (ph 7, 25 ) Vapor pressure : 0.065 mpa (25 ) Log Pow : 4.0 (ph 7) Vapor pressure : 6.52 10-9 mpa (20 ) 0.5 mg/kg 1 mg/kg Flufenoxuron 세절한복숭아시료 25 g에 acetone 100 ml와 Celite 545를 2 g 가하여 60분간진탕하여감압여과한후여액을 1,000 ml의분액여두에옮겨증류수 300 ml 과포화식염수 50 ml을가한다음 n-hexane 100 ml, 50 ml 씩차례로가한뒤유기용매층을 sodium sulfate에통과시켜감압농축한후이를 n-hexane 10 ml로재용해하였다. Florisil 10 g을건식충진한 glass column (11 mm I.D. 30 cm L.) 에 n-hexane 50 ml로활성화시킨후재용해한시료를전량충진하였다. acetone : n-hexane (5/95, v/v) 을 100 ml 로세척하고 acetone : n-hexane (3/7, v/v) 로 100 ml 용출하여감압농축하였다. 건고물을다시 water :
138 MOON et al. acetonitrile (4/6, v/v) 5 ml 로정용하여 HPLC/DAD 로분석하였다. 분석기기조건 Table 2. Instrumental conditions of fenarimol and flufenoxuron residue analysis in peach Fenarimol Instrument Agilent 6890 plus Detector μ-electron Capture Detector(μ-ECD) Temperature Injector : 300, Detector : 280 Oven : 120 (2 min)-20 /min-280 (10 min) Flow rate Carrier gas ; N 2, column 1.0 ml/min, total 53.2 ml/min Column Agilent DB-5 (0.25 mm I.D. 30 m L., 0.25 μm) Split ratio 50:1 Injection vol. 1 μl Flufenoxuron Instrument Waters 2690 0-5 (min) ACN : Water = 40 : 60 (v/v) Mobile phase 5-10 (min) ACN 100% 12-14 (min) ACN : Water = 40 : 60 (v/v) Flow rate 0.6 ml/min Oven temp. 40 Column Wavelength Injection vol. Waters Capcell-pak C18 MG (3.0 150 mm, 3 μm) 254 nm 20 μl 회수율시험회수율시험은무처리시료에 fenarimol, flufenoxuron 의표준용액 0.1 mg/kg과 0.5 mg/kg을각각처리한후상기분석과정과동일하게수행하여회수율을산출하였다. 정량한계 (LOQ, Limit of Quantitation) 분석기기의검출 (S/N=3) 및정량한계 (S/N=10) 를측정하고검체용액중의간섭이없다면분석법의검출및정량한계는다음식에의하여계산하였다 (Korean Food Standards Codex, KFDA, 2012). 분석법의검출및정량한계 = (A/B) (C/D) (mg/kg 또는 mg/l) A: 분석기기의검출한계 (ng), B: 검체주입량 (μl), C: 최종검체용액의부피 (ml), D: 분석검체량 (g) 복숭아중 fenarimol과 flufenoxuron의생물학적반감기에서산출한잔류감소회귀식을국립농산물품질관리원에서규정하고있는회귀방정식을근거로하였다. 출하전일자의농약잔류허용기준 = 출하일의잔류허용기준 e ( 감소상수 출하전일자 ) (2) 식 (2) 을 SafeQIN의 SPSS통계프로그램에적용하여설정하였다 (Calculation by SafeQIN program of NAQS). 결과및고찰 복숭아재배기간중기상조건과생체량증체율재배기간중시험포장의기온은 24.9 29.3 이었다. 습도는 74.3 99.8% 이었으며, 최저 0.5, 최고 180 mm/day 의강우가모두 10차례발생하여 288.5 mm의누적강우를기록했다 (Korea Meterological Administration Report, 2012). 복숭아증체는 0일차 203.9 g에서 14일차 222.5 g 으로 18.6 g이증가하여 9.12% 증가율을보였다. 검량선작성시험농약 fenarimol과 flufenoxuron은 0.05-5 mg/kg 의범위에서각각, acetone 과 water : acetonitrile = (4/6, v/v) 으로표준용액을조제하였다. 표준용액을분석하여얻은검량선의회귀방정식은 fenarimol 이 y=4,306.8723x-101.7314 (r 2 =0.9997) 이며, flufenoxuron은 y=6.10e +004x +6.94e +002 (r 2 =0.9996) 으로직선성이상당히양호하였다. 회수율및정량한계상기분석법에따른 fenarimol의크로마토그램상의머무름시간은 12.2 min 이었으며회수율은저농도 (0.1 mg/kg) 에서는 86.6-97.8% 수준이었고고농도 (0.5 mg/kg) 에서는 93.1-96.1% 로나타났다. Flufenoxuron의크로마토그램상의머무름시간은 7.6 min 이었으며회수율은저농도 (0.1 mg/kg) 에서 100.4-113.0%, 고농도 (0.5 mg/kg) 에서는 89.9-102.8% 로나타났다. 이들약제의정량한계 (LOQ) 는 fenarimol 0.05 mg/kg, flufenoxuron 0.01 mg/kg 이었으며이는국내에서허용되는회수율범위 70-120(%), 변이계수 (Coefficient of variance, CV) 20% 이하를만족하는수준이었다. Table 3. Recovery and LOQ for fenarimol and flufenoxuron in peach Pesticide Fenarimol 0.01 Flufenoxuron 0.01 LOQ Fortification level (mg/kg) (mg/kg) Recovery±CV (%) 0.1 93.69±6.56 0.5 94.45±1.60 0.1 106.73±5.90 0.5 96.37±6.66 생산단계농약잔류허용기준
Establishment of Pre-Harvest Residue Limits (PHRLs) of Fungicide Fenarimol and Insecticide Flufenoxuron in Peaches during Cultivation Period 139 복숭아재배기간중잔류량변화와생물학적반감기 농약의이동, 분포, 잔류, 소실의특성은농약자체의이화학적특성, 농약의제제형태, 사용방법, 대사및분해정도등과밀접한관계가있을뿐만아니라환경조건, 즉기상조건, 토양조건등에따라서도크게영향을받는다. 복숭아재배시 fenarimol과 flufenoxuron을 1회처리와 3회처리로살포한후일정일자마다시험농약의잔류량을살펴보았다. Fenarimol은 1회처리구에서초기농도가 0.29 mg/kg으로 MRL (0.5 mg/kg) 에못미치는수준이었으며 14일뒤에는 0.01 mg/kg으로초기농도대비 96.6% 감소하였다. 3회처리구에서초기농도는 MRL보다높은수치인 1.03 mg/kg이었으며 14일뒤에는 0.07 mg/kg 로초기농도대비 93.2% 감소하였다. 잔류감소회귀식으로나타내었으며 1회처리구에서는 y=0.3516e -0.199x (r 2 =0.8193 *** ) 3회처리구에서는 y=0.9466e -0.184x (r2=0.9878 *** ) 이었다 (Fig. 1). 이식에의해산출된반감기는 1회처리구에서 3.5일, 3회처리구에서 3.8일이었다. Flufenoxuron 1회처리구에서초기농도는 MRL (1.0 mg/kg) 에못미치는수준인 0.29 mg/kg이었고 14일뒤잔류량은 0.04 mg/kg으로초기농도대비 86.2% 감소하였다. 3회처리구에서초기농도또한 MRL에못미치는수준인 0.66 mg/kg 이었으며 14일뒤농도는 0.07 mg/kg로초기농도대비 89.4% 감소하였다. 잔류감소회귀식에적용해본결과기준량은 y=0.259e -0.098x (r 2 =0.7413 ** ) 배량은 y=0.6828e -0.141x (r 2 =0.9581 *** ) 이었다 (Fig. 1). 이식에의해산출된반감기는 1회처리구에서 7.1일 3회처리구에서 4.9일이나타났다. 복숭아에서의농약잔류연구결과를살펴보면 Lee 등 (2003) 은 procymidone (50%, WP) 을기준량처리하였을때초기농도는 6.34 mg/kg, 배량처리시 17.46 mg/kg으로기준량처리시에는 MRL (10 mg/kg) 보다낮은수준을나타냈다고하였다. 또 15일후감소량을보면기준량처리시 94.6% 감소한 0.34 mg/kg 배량처리시 95.7% 감소한 0.76 mg/kg을나타내어반감기는기준량처리시 3.1일이며배량처리시 3.4일이었다고보고하였다. Procymidone 이 fenarimol 보다초기농도가높은것은유효성분함량이높기때문인것으로보인다. Park 등 (2012) 에의하면살균제 fluquinconazole (10%, SC) 을복숭아에 1회처리시초기부착량이 0.115 mg/kg, 복숭아에 3회처리시 0.251 mg/kg로이역시 MRL(1 mg/kg) 에미치지못하는수준이었다고했다. 14일후복숭아의살균제 fluquinconazole (10%, SC) 감소율은 1회처리에서는 89.57% 로 0.012 mg/kg, 3회처리시 91.24% 로 0.022 mg/kg를나타내었고반감기는 1회처리시 3.9일 3 회처리시 4.1일이었다고했다. 잔류감소회귀식에적용한반감기는 fenarimol의기준량처리시반감기 3.5일과배량처리시반감기 3.8일로매우비슷하게나타났음을알수있는데잎을대상으로하는작물보다열매를대상으로하는작물에대해서는농약의제형에따른부착량의차이는없고 (Kim et al., 1997) 그유효함량이비슷한수준이기때문인것으로보인다. 또한 Lim 등 (2011) 은 fenarimol을취나물에살포했을때 1회처리시초기부착량이 3.59 mg/kg 3회처리구에서 6.56 mg/kg으로 MRL (1.0 mg/kg) 에비해각각 3배와 6 배가나타났다. 1회처리구의반감기는 6일, 배량은 5.9일로복숭아에비해초기부착량이높게나오고반감기는더길었는데작물의형태적특성상열매보다잎을대상으로하는작물에서부착량이높은것은작물체의생체중과비표면적차이때문인것으로본다 (Hill and Inaba, 1990). 강우에따른작물의희석효과과실의비대생장에따른희석효과는중요하다. 특히오이와같은작물은급격히성장하는작물로써약제처리후 10일만에 16배정도의무게가증가하는데농약의순수분해보다가장큰역할을하는것이작물증체량에따른희석효과이다 (Lee et al., 2008). 그밖에농약의잔류성에영향을주는요인으로농약의재배형태, 재배방법, 농약제형, 살포방법및기상등이있는데, 수확을시작한뒤 3일째부터비가내려 6일차수확후에는 76 mm/day 9일차에는 180 mm/day 의많은강우량을기록했으며, 비에따른과실의무게가늘어나면서잔류량은감소하였다. 특히 9일차 (180 mm/day) 의강우가있은직후 8일차에 208.4 g 이던복숭아의평균무게가 10일차에 223.3 g으로늘어난것을볼수있으며, 이에반해 fenarimol 잔류량은 0.16 mg/kg에서 0.07 mg/kg로급격히하락한것을볼수있다 (Fig. 2). 이는 flufenoxuron이 0.16 mg/kg에서 0.15 mg/kg로떨어진것과차이가나는 Fig. 1. Dissipation of fenarimol (A), flufenoxuron (B) on peach during cultivation period.
140 MOON et al. Fig. 2. Weight, residue and precipitation during cultivation period ((A) fenarimol, (B) flufenoxuron). Table 4. Recommended pre-harvest residue limit of fenarimol and flufenoxuron in peach Pesticide 10 9 8 7 Pre-Harvest Residue Limit (mg/kg) 6 5 4 3 2 1 Harvest day Regression coefficient MRL (mg/kg) Fenarimol 1.5 1.4 1.2 1.1 1.0 0.9 0.8 0.7 0.6 0.6 0.5 0.1117 0.5 Flufenoxuron 1.4 1.4 1.3 1.3 1.2 1.2 1.2 1.1 1.1 1.0 1.0 0.0347 1.0 것을볼수있다. Fenarimol과 flufenoxuron의잔류량은강우량에따른무게의증체에따라잔류량이낮아진경향을보이고있는데특히수용해도가큰 fenarimol에서잔류량이더많이감소된것으로나타났다. 즉 Fenarimol의수용해도는 13.7 mg/kg (ph 7, 25 ), flufenoxuron의수용해도는 0.00152 mg/kg (ph 7, 25 ) 이므로 flufenoxuron에비해 fenarimol이물에대한용해도가훨씬크기때문에증체에의한희석효과와더불어수용해도에따른잔류량의감소까지더해져그폭이더크게나타난것으로볼수있다. 그이유는수용해도가클수록농약의유실이많이되기때문이다 (Kim et al, 2005; Kim et al. 2008). 복숭아의농약잔류허용기준 (PHRL) 산출생산단계농약잔류허용기준 (PHRL) 은수확전일정한시점에잔류허용량을설정함으로써수확시잔류량이 MRL을초과하지않도록설정하는기준치이다. 본연구를통해산출한수치는 Table 4에나타냈으며 fenarimol 은출하 10일전 1.5 mg/kg이어야하며 flufenoxuron 은 1.4 mg/kg이어야출하일에안전하게출하할수있어농산물부적합률을줄일수있다. Acknowledgement This study is part of the establishment of Pre-Harvest Residue Limit in 2012. Thanks to the National Agricultural Products Quality Management Service for their support and research fund. References Bentson, K.P., 1990. Fate of xenobiotics in foliar pesticide deposits, Reviews of Environmental Contamination and Toxicology 114, 125-161. Choe, J.S., Chun, H.K., Hwang, D.Y., Nam, H.J., 2005. Consumer perceptions of food-related hazards and correlates of degree of concerns about food, Kor J. Soc Food Sci Nutr. 34(1), 66-74. Choi, K.I., Seong, K.Y., Jeong, T.G., Lee, J.H., Hur, J.H., Ko, K.Y., Lee, K.S., 2002. Dissipation and removal rate of dichlofluanid and iprodione residues on greenhouse cherry tomato, Korean J. Environ Agric. 21, 149-155. Hill, B D. and Dan J. Inaba., 1990. Rate and persistence of residues on wheat used to explain efficacy differences between SC and EC formulations, Korean J. Pestic. Sci. 29, 57-66. Kim, J.B., Song, B.H., Chun, J.C., Im, Y.B., 1997. Effect of sprayable formulations on pesticide adhesion and persistence in several crops, Korean J. Pestic. Sci. 1(1), 35-40. Kim, S.S., Kim, T.H., Lee, S.M., Park, D.S., Zhu, Y.Z., Hur, J.H., 2005. Mobility of pesticides in different slopes and soil collected from Ganwon alpine sloped-land under simulated rainfall conditions, Korean J. Pestic. Sci. 9(4), 316-329. Kim, S.S., Kim, T.H., Lee, S.M., Park, H.R., Park, D.S., Lim, C.K., 2008. Mobility of pesticides from soil in different slope by simulated rainfall under field
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