A Sensitive Method for Determination of Carbendazim Residue In
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Chiang Mai J. Sci. 2015; 42(3) 681 Chiang Mai J. Sci. 2015; 42(3) : 681-690 http://epg.science.cmu.ac.th/ejournal/ Contributed Paper A Sensitive Method for Determination of Carbendazim Residue in Vegetable Samples Using HPLC-UV and Its Application in Health Risk Assessment Buran Phansawan [a,b], Tippawan Prapamontol*[b], Prasak Thavornyutikarn [c], Somporn Chantara [c], Ampica Mangklabruks [d] and Choochad Santasup [e] [a] Environmental Science Program, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand. [b] Environment and Health Research Unit, Research Institute for Health Sciences, Chiang Mai University, Chiang Mai 50200, Thailand. [c] Department of Chemistry, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand. [d] Department of Internal Medicine, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand. [e] Department of Soil Science, Faculty of Agriculture, Chiang Mai University, Chiang Mai 50200, Thailand. *Author for correspondence; e-mail: [email protected] Received: 8 October 2013 Accepted: 28 August 2014 ABSTRACT Carbendazim is a fungicide widely used in vegetable growing and its residue was often detected. We developed a sensitive method using high performance liquid chromatography-UV detection (HPLC-UV) for determining carbendazim residue in vegetables and assessed the health risk from contaminated vegetable consumption. The consumption data was collected from 244 participants aged 35-65 years living in Suthep subdistrict of Chaing Mai city while vegetable samples were collected from Chiang Mai city fresh markets during August to October 2011. The developed method provided good recoveries of carbendazim from spiked pooled vegetable samples ranged from 92.5 % to 96.0 % with the relative standard deviation (RSD) of 2.1 % to 5.9 % at spiked levels of 0.05-0.30 mg kg-1. The limit of detection (LOD: 0.003 mg kg-1) as well as the limit of quantitation (LOQ: 0.030 mg kg-1) of the developed method is sensitive to detect carbendazim residue in vegetables far below the Codex Maximum Residue Limits (MRLs) such as 0.05 mg kg-1 set for cucumber. The health risk assessment of carbendazim from consumption of 5 common kinds of vegetables, i.e. tomato, cucumber, kale, cauliflower, and ginger, was performed by comparing calculated daily intake (CDI) with acceptable daily intake (ADI). It was found that the CDI of carbendazim residue from consumption of tomato (64.1 %), cucumber (59.6 %), and kale (56.7 %) were greater than 50 % of ADI while of cauliflower (19.3 %), and ginger (0.9 %) were much lower. The present results showed that consumption of some contaminated vegetables may pose the health risk though the commination may vary from season to season. Furthermore, the developed method could be used to survey carbendazim 682 Chiang Mai J. Sci. 2015; 42(3) residue in vegetables as well as fruits by using HPLC-UV which is a common apparatus available in toxicology laboratory. Keywords: carbendazim residue, vegetables, consumption, HPLC-UV, health risk assessment 1. INTRODUCTION In recent years, the total amount of rapid effects on meiotic spermatocytes and pesticides used has increased worldwide. latent effects on spermatids, leading to In Thailand, the Department of Agriculture morphological abnormalities and failure of (DOA) reported that pesticides were increased spermatogenesis [5]. It was also found that 1.2 fold in 5 year time from 110,000 tons in subchronic administration of carbendazim 2008 to 134,000 tons in 2013. The major induced testicular alterations, spermatogenic abundant was herbicides, insecticides and inactivity and embryotoxicity [6]. Over the fungicides, respectively [1]. Some pesticide years, research in the field of carbendazim residues were reportedly detected in residue analysis as well as other pesticides in vegetables, fruits, and soil [2-3]. Benzimidazole food has continuously expanded according fungicides which the main compound is to food safety policy and techniques available carbendazim are systemic pesticides widely for determining their content in different fruits used in agriculture for pre- and post-harvest and vegetables [7-9]. Therefore, it is still of protection of crops against fungal diseases [4] interest to develop a sensitive and low cost and structure of carbendazim is shown in analytical method that suitable and applicable Figure 1. Carbendazim is toxic to humans, for toxicology laboratory especially in animals and plants. The toxicity produces developing country. Figure 1. Structure of carbendazim. The common used analytical techniques extraction (SPE), using C18 bonded silica for the analysis of carbendazim include procedure, has been employed for isolation liquid chromatography with UV [4, 10], [19]. Pan et al. (2008) reported that mobile diode-array [11], fluorescence [12, 13] or phase modifiers such as methanol-water mass spectrometric detections [14]. Various or acetonitrile-water were required to extraction solvents such as acetone [15], improve the peak shape and/or resolution acetonitrile [16], methanol [4], ethyl acetate [20]. There was effective absorption of [14, 17], and dichloromethane [18] have interferences on strong anion exchange/ been used to extract carbendazim residue primary secondary amine (SAX/PSA) followed by homogenizing, and shaking by dual-layer SPE for the HPLC condition. using sonication [17]. Moreover, a solid-phase A health risk assessment, i.e. carbendazim, Chiang Mai J. Sci. 2015; 42(3) 683 is the process to estimate the nature and and 500 mg/6 mL SPE cartridges were probability of adverse health effects from Varian Inc. (Harbor, USA.). Organic in humans who may be exposed to solvents such as ethyl acetate and methanol carbendazim in contaminated food, now or (HPLC grade) were from J.T. Baker in the future. Basically, there are four major (NJ, USA). Sodium acetate was from steps in human health risk assessment Sigma-Aldrich (MO, USA.) and hydrochloric including hazard identification, dose-response acid (analytical grade) was from Merck assessment, exposure assessment, and risk (Darmstadt, Germany). Water was purified characterization. The first and the second using a Milli-Q water purification system, steps are processed following the Codex ELGA UHQ PSII (Bucks, UK). Mobile phase Alimentarius Commission (Codex) which was filtered through a 13 mm × 0.45 μm evaluates the hazards by measuring both polytetrafluoroethylene VertiClean from quality and quantity. The Joint Meeting of Vertical Chromatography (Nonthaburi, Food and Agriculture Organization and the Thailand). World Health Organization (FAO/WHO) on Pesticide Residues (JMPR), is responsible 2.2 Instrumentation for reviewing residue and analytical aspects The HPLC (Agilent 1100, USA) was of the pesticides in foods. The health risk equipped with a variable UV detector set to assessment of carbendazim exposure will be 280 nm. Analytical column was C18 (250 mm compared with the acceptable daily intake × 4.6 mm i.d. and 5 μm particle size (Supelco (ADI) which is a measure of the amount of Inc., Bellefonte, PA, USA.). The mobile phase a certain substance in food that can be ingested consisted of methanol-water (25:75, v/v). on a daily basis over a lifetime by human The flow rate was 1.0 mL min-1 and the without appreciable health risk. The ADI of injection volume was 20 μL. carbendazim is 0.03 mg kg-1 bw day-1 [21]. Therefore, the result of human health risk 2.3 Sample Preparation assessment from contaminated carbendazim One kilogram of fresh vegetable samples residue in vegetables could be useful for food was randomly selected from four fresh safety policy. markets in Chiang Mai city from August to October 2011. Thirty two vegetable samples 2. MATERIALS AND METHODS including cauliflower, ginger, kale, cucumber, 2.1 Chemicals yard long bean, guinea-pepper, chili and Carbendazim (99% purity) was from tomato were purchased and transported to Dr. Ehrenstorfer (Augsburg, Germany). Toxicology laboratory, Research Institute Stock solution was prepared by dissolving for Health Sciences, Chiang Mai University. 1 mg of carbendazim in 25% methanol up All vegetable samples were chopped and to 100 mL (10 μg mL-1). Working solutions blended into small pieces according to the for spiking in validation studies were Codex Alimentarius [21]. Ten grams’ aliquots prepared by diluting 1.2, 1.9, 2.5, 5.0 and of well blended samples were kept frozen 7.5 mL of stock solution in 25% methanol (-20 °C) until analysis. The frozen vegetable up to 10 mL. The obtained concentrations aliquot samples were thawed and left to of working solutions were 1.2, 1.9, 2.5, 5.0 room temperature (25 °C) before analysis. and 7.5 μg mL-1, respectively. Pooled matrices collecting from several kinds SAX/PSA dual-layer SPE bulk materials of vegetables were spiked to obtain 5 levels 684 Chiang Mai J. Sci. 2015; 42(3) including 0.05, 0.075, 0.10, 0.20 and 0.30 13 mm × 0.45 μm polytetrafluoroethylene mg kg-1, respectively. Spiked pooled samples membrane. Twenty microliters of clean were used to determine the critical parameters extract was injected into the HPLC-UV for such as the linearity, limit of detection (LOD), carbendazim analysis. limit of quantitation (LOQ), accuracy and precision for method validation. 2.6 Data Collection for Human Health Risk Assessment 2.4 Method Validation Suthep subdistrict of Chiang Mai city Linearity was evaluated using 5 spiked was chosen to be the studied area by using pooled samples including 0.05, 0.075, conditional random sampling. Both urban 0.10, 0.20 and 0.30 mg kg-1, respectively. and rural areas of 15 villages, and one The precision was evaluated by relative market in the area of Suthep subdistrict were standard deviation (RSD) of five replications studied. The consumption data was obtained at each five concentrations. The accuracy from 244 participants who were farmers, was studied using 0.05 mg/kg spiked sample general employees, traders, office workers, performing on the same day for five government officers and business owners, replications.