As. J. Food Ag-Ind. 2012, 5(05), 364-373

Asian Journal of Food and Agro-Industry
ISSN 1906-3040 Available online at www.ajofai.info

Research Article

Kinetics of the reduction of pesticide residues in vegetables by ultrasonic cleaning
Ratikorn Buakham1, Sirichai Songsermpong1* and Chutima Eamchotchawalit2
1

Food Engineering Program, Department of Food Science and Technology, Faculty of Agro-Industry, Kasetsart University, Jatujak, Bangkok 10900, Thailand. Thailand Institute of Scientific and Technological Research. Klong Luang, Pathumthani 12120, Thailand.

2

Email: sirichai.so@ku.ac.th

Abstract The problem of pathogen and pesticide residues in vegetables is a big concern to both consumers and exporters. This study introduced a new method of cleaning vegetables with high frequency sound waves (ultrasonic) as a way to solve the problem. This research aimed at studying the kinetics of reduction of pesticide residues in four kinds of vegetables: coriander, kale, yard long bean and red chili by using the ultrasonic cleaner (60 kHz 140 W) with tap water compared with soaking in tap water. The cleaning was done at 20, 25 and 30oC for 0, 3, 7 and 10 minutes. The determination of the toxicity levels was done by means of cholinesterase inhibition technique. The decimal reduction time, the reaction rate constant and the activation energy were determined. It was found that ultrasonic cleaning at 25oC and soaking in tap water at 30oC was the best combination to reduce pesticide residues. The study concluded that ultrasonic cleaning was a far better method for reducing pesticide residues than soaking in tap water. Keywords: high frequency sound waves, food contamination, toxicity, cholinesterase inhibition, cavitation, Thailand. Introduction Thailand is a large exporter of agricultural produce and processed agricultural products. However, these products are often rejected by trading partners because of the detection of microbial pathogens and pesticide residues, especially coriander, kale, yard long bean and red chili. The Department of Agriculture [1] reported that during January - July 2009, compared to the same period the previous year, exports of agricultural products and agro-industry products decreased, with about 29% of the EU-destined products found to have problems as reported by RASFF (Rapid Alert System for Food and Feed). Products found to have problems increased on the RASFF list from 40 to 74 items.

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This problem occurred because vegetable production currently uses a lot of pesticides and cleaning is less effective. Good cleaning practice also helps to reduce dirt, pesticide residues and microbial contamination from raw materials. Ultrasonic cleaning is a new technology applied to wash vegetables and fruit in the food industry [2]. The frequency of ultrasound causes a reaction known as cavitation which can eliminate the residual pesticide residue, bacteria and dirt better than other methods, such as a spray cleaning, brush cleaning and water and air turbulence etc. [3]. The cavitation is a phenomenon of the fabrication of very small bubbles (not visible to the naked eye) in a liquid. These bubbles will grow and become filled with vapour, gas or a mixture thereof, expand and regularly break out violently under the stimulus of the ultrasound. These bubbles cause mechanical energy as shockwaves as well as particle mixing to remove dirt, pesticide residues and microorganisms [4, 5]. The effectiveness of the cleaning depends on the number of bubbles imploding and the implosion force of bubbles. The latter depends on amount of vapour and gas in each bubble. The less vapour and gas in the bubble, the cavitation intensity is increased and cleaning is more efficient [6]. Further, the cleaning efficiency depends on the temperature, ultrasound frequency, power level, the size, depth and shape of the cleaning bath, properties of the liquid and duration time of cleaning [4]. One detection method for pesticide residues is by measuring the degree of toxicity which be done by GT-test kit which is a semi-fast testing method using the cholinesterase inhibition technique [7]. The theory suggests that the toxicity in the organophosphate and/or carbamate group is distinct in its ability to halt the mechanism of the enzymes in the body. When the body receives these toxic pesticides, the enzymes will not function properly. Toophorm [7], used this fact as a preliminary test for toxicity to filter out the 2 groups of pesticides that are commonly used. The main feature of the basic test kit involves testing the degree of toxicity of the sample, whether it is an insecticide or toxic residue from plant cells and some chemicals that causes toxicity. These toxic substances also have the ability to reduce the performance of the enzyme. If the vegetables have pesticides/toxic residues, they can inhibit the cholinesterase enzyme. The enzyme cannot hydrolyze acetylcholine to choline. The high amount of acetylcholine remaining will give the high intensity of dark colours. In vegetables with higher pesticide residues, cholinesterase will be inhibited to a greater degree, resulting in acetylcholine remaining in large quantities and showing as a very dark colour in the kit. Toxicity of less than 50% is considered as safe for consumption. This technique was used in this research to determine the percentage reduction of toxicity after cleaning by measuring the toxicity of the residue before and after cleaning. The standard curve of the intensity of colour and degree of toxicity was made for this analysis [7]. Food safety is most important to consumers therefore ultrasonic cleaning of vegetables is a new alternative method to make food safe from excessive pesticide residues. The question that needs to be answered is why cleaning with ultrasonics is better than without ultrasonics, as well as how rapid is the reduction of pesticide residues by ultrasonic cleaning and soaking in water. By studying the kinetics of the reduction of pesticide residues, it is possible to compare the effectiveness of the cleaning. Such knowledge will help increase the safety for consumers, not only within this country but also Thailand’s many import partners. Materials and Methods Vegetable treatment Vegetables (coriander, kale, yard long bean and red chili) were purchased from a local market near Kasetsart University. These vegetables were dipped in the pesticide that has the common name “carbosulfan” which is in carbamate group (Sharpmulators Co. Ltd.) in the ratio of pesticide 8 ml to water 6 L soaking for 20 min and then drained and air dried for 30 min.

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Cleaning Coriander, kale, yard long bean and red chili of 160, 220, 250 and 600 g, respectively [8], were used in the cleaning test of each replication. Each vegetable was immersed in 2 L of tap water with dimensions of 100×300×150 mm in the ultrasonic cleaner (Thailand Institute of Scientific and Technological Research, TISTR) and treated with and without ultrasonics for 0, 3, 7 and 10 min with 20, 25 and 30oC, operating at 60 kHz of frequency, 140W of power and 3 replications were made. Each vegetable was sample tested for degree of toxicity by GT-pesticide test kit [7]. Testing degree of toxicity by GT-pesticide test kit Sampling 10 g of chopped vegetables and adding 10 ml of dichloromethane (solvent-1) and shaking 1 min were done to extract pesticides from vegetables, thereafter standing for 10-15 min. Then 1 ml sample extract was removed and supplemented with 1 ml 5% ethanol in water (solvent-2) and separated into two phases. The under layer is a sample extract and the top layer is a solvent-1. The solvent is evaporated with an air pump for about 3 min at 34oC in water bath. 0.25 ml sample extract was used, adding 0.5 ml cholinesterase (GT-1), after 10 min adding 0.25 ml acetylcholine (GT-2), stand for 60 min. Adding 1 ml hydroxylamine (GT-3), 0.5 ml hydrochloric acid (GT-4) and 0.5 ml ferric chloride (GT-5) was done respectively. The samples were centrifuged and the absorbance measured by a spectrophotometer at 540 nm wavelength and compared with standard curve (Fig. 1.) to determine the % inhibition (degree of toxicity) [7].
0.700 0.600 y = 0.005x + 0.006 R² = 0.995

Absorbance 540 nm

0.500 0.400 0.300 0.200 0.100 0.000 0 20 40 60 80 100 120

% Inhibition Figure 1. Standard curve of absorbance at 540 nm (colour intensity) and % inhibition (degree of toxicity).
(n=5)

Standard curve preparation The standard curve of the intensity of colour and toxicity level of 0, 10, 30, 50, 80 and 100% was made by adding 1 ml of 5% ethanol in water and 0.5 ml cholinesterase (GT-1) in 6 tubes at 34oC in water bath, each tube representing a different level of toxicity. After 10 min, filling 0.25, 0.275, 0.325, 0.375, 0.45 and 0.5 ml acetylcholine (GT-2) for 0, 10, 30, 50, 80 and 100% of degree of toxicity (% inhibition), respectively. Standing 60 min before adding 1 ml hydroxylamine (GT-3), 0.5 ml hydrochloric acid (GT-4) and 0.5 ml ferric chloride (GT-5) was done respectively and 5 duplications were made. Next, the solution was centrifuged and measured for colour intensity in each tube by spectrophotometer at 540 nm. The standard curve was plotted between absorbance at 540 nm and % inhibition as shown in Figure 1.

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Statistical analysis All treatments were evaluated by mean ± standard deviation (SD) and the means between different experimental treatments were compared by Duncan’s multiple range test (p