Food and Fertilizer Technology Center - publications

May. 01, 1999

Rapid Bioassay

In Asia, the marketing and distribution of vegetables and fruit is rapid. Usually it is completed only one or two days after harvest. When produce is tested for chemical residues, the results must be available quickly if consumers are to be protected from contaminated shipments. The rapid bioassay procedure was developed in response to this need. The training course in 1999 continued a series of courses conducted by FFTC on the use of bioassay to test whether agricultural produce contains pesticide residues. Bioassay is not as precise as chemical testing, and is not as widely used. Its advantage is its low cost, and the fact that bioassay gives results very quickly. Contaminated produce can be withdrawn from the market before it is sold.


Prior to this training course, the National Agricultural Cooperative Federation (NACF) had already sent several staff members in 1996 to the Taiwan Agricultural Research Institute for training in rapid bioassay procedures. Those who attended the course were impressed by the method, but felt that the procedures used in Taiwan are not suited to Korea, which has a different climate and a different range of pests. Accordingly, they began to modify the procedures to suit Korean conditions.


They were successful in doing this, and NACF began to establish rapid bioassay stations in its branch offices and member cooperatives throughout Korea. In 1997 there were 65 testing stations. The following year, the number rose to 100. Bioassay testing rapidly spread to other Korean organizations involved with agricultural produce, including the Agricultural Product Inspection Office, Agricultural Technology Centers at a county level, the Agricultural Wholesale Market Corporation, and some commercial food enterprises.


The method is also widely used in Taiwan, with around 80 bioassay stations in producing areas. There is also one in each of the wholesale markets in the three largest cities (Taipei, Kaohsiung and Taichung). Rapid bioassay is cheap enough to allow these three wholesale markets to test 1% of the produce they receive every day. In this way, every supplier is checked at least once every three months.


Now that rapid bioassay stations are becoming common in Korea, NACF felt it was time to review their performance. This meeting was held to discuss the results of the bioassay testing program, and any technical or institutional problems which had arisen. Four resource persons from the Taiwan Agricultural research institute, led by Dr. Edward Cheng, visited a series of rapid bioassay stations where they gave lectures and technical advice.


How Rapid Bioassay Works


The Ache Test


This is an
in vitro
test for insecticides. It uses AChE (acetylcholinesterase) obtained from the heads of a special strain of housefly. These houseflies have been reared in isolation for more than three decades and have never been exposed to chemical pesticides, so they remain extremely sensitive to them. Methods of mass rearing are so simple that two technicians can produce enough housefly AChE for Taiwan and other countries in Asia. To remove the heads, the flies are frozen, then placed in dry ice and shaken so that the heads drop off. After the enzyme is extracted from the heads, it is freeze-dried into a stable dry powder which can be stored indefinitely.


When the AChE reaction solution is mixed with samples from healthy produce, it slowly turns yellow as the level of 5-thio-2-nitrobenzoateanion increases. If insecticide is present, the enzymatic reaction slows down or stops. The rate at which color development is inhibited indicates the quantity of chemicals present, as well as their toxicity. The AChE test is very rapid, taking only 15 minutes from sampling to completion.


The AChE test does not give false positive results i.e. it does not indicate insecticides are present when they are not. However, it does sometimes give a false negative. It cannot detect certain insecticides, for example, synthetic pyrethroids, and gives a negative result even if they are present.


The B.T. Test


The B.t. test uses
Bacillus thuringiensis (B.t.)
to detect fungicide residues.
B.

thuringiensis
is a bacterium which is entomopathogenic (i.e. it causes disease only in insects). It is highly sensitive to some fungicides used on vegetables. Vegetable samples are finely chopped and placed in EDTA solution for five minutes. The extract is incubated with a suspension of
B.

thuringiensis
for 90 minutes (the period of four generations of the bacteria). Then TTC solution is added and left to act for another thirty minutes. Finally, the reaction is halted with Triton-HCI mixture, which acts as a red color signal for
B. thuringiensis
activity.


If fungicide residues are present, there is no metabolic activity by the bacteria and no red color. If by the end of the test period the mixture has not turned red but is still its original pale yellow, it probably contains toxic chemical residues.


Garlic and ginger can kill bacteria naturally, and cannot be tested by
B.t.
. Some compounds which occur naturally in oranges and tea also interfere with the TTC reaction. Strawberry fruits contain a red pigment, which makes it difficult to distinguish any color change from the bacteria. It is possible to test these crops, but the procedure must be modified.


Application of Rapid Bioassay


The A-test is mainly used for vegetables, which are often sprayed with pesticides. The toxic chemicals found on fruit in Korea are mostly fungicides. Insecticides are more dangerous to humans than fungicides. The A-test can detect two major categories of insecticides, organophosphates and carbamates. Both of these are very toxic to humans. Insecticides other than these two groups should be checked by other methods, not rapid bioassay.


Rice and other grains should be tested by chemical analysis rather than bioassay. They have a long storage life, and there is plenty of time to test them before they are eaten.


Farmers often spray a mixture of several pesticides at the same time. Samples from vegetables or fruit contaminated by several pesticides will demonstrate the combined inhibition rate.


Farmers' Reaction to Bioassay


In the early days of rapid bioassay testing by agricultural cooperatives in Korea, some farmers were worried about what the test results might be, and refused to take samples from their crops. As regular residue testing by government authorities became widespread, farmers came to understand the need for it. They began to bring vegetable samples to bioassay stations of their own accord before harvest. The attitude of farmers now to bioassay is positive, because they feel consumers are more likely to buy produce which has been tested for residues.


The Garak Wholesale Market in Seoul, the largest market in Korea, collects 220 samples of produce every day and tests them by bioassay. If the samples indicate that residues are present, the same samples are immediately sent for chemical analysis in a laboratory located in the same building. If chemical testing indicates that the samples have more than the tolerated level of chemicals, the shipment is destroyed. The dealer responsible for bringing the shipment to market is not allowed to do any business in the market for one month.


Conclusion


Within a short period of time, Korea has established a nationwide system of bioassay suited to Korean conditions. Procedures at the Garak Wholesale Market are ideal, since bioassay is supported by immediate chemical analysis. However, there are still some problems. As yet, the rapid bioassay program is not backed by any legislation. A regular system of compensation is needed for the relatively few farmers whose produce was shown by bioassay to be contaminated, but which chemical testing showed to be within tolerated levels. There also needs to be an efficient system for removing contaminated produce from the market and destroying it, and imposing a penalty on offenders.


Another concern is that the Taiwan Agricultural Research Institute is still the sole source of the enzyme in Asia. Korean scientists are now working to develop an enzyme from honeybees, which are easy to raise and collect. Perhaps enzymes from other insects might also give an effective response to different pesticides. While rapid bioassay is cheaper than chemical analysis, it is still fairly costly for developing countries to use. A wider choice of enzymes at a lower price would help make rapid bioassay more widely available.


Most developing countries lack a monitoring system to detect chemical residues in fresh produce. The main constraint is the cost. Chemical analysis is very accurate but very expensive, and is not generally available in producing areas. Rapid bioassay offers a quick, low-cost method of detecting chemical residues in produce before consumers eat it. Ideally, rapid bioassay should be backed by a system of chemical testing. Bioassay is best used as a way of screening large numbers of samples for residues, while the more accurate chemical testing is best for monitoring. n


FFTC Training Course on Rapid Bioassay for Pesticide Residues in Fruit and Vegetables


Held at the Agricultural Cooperative College, National Agricultural Cooperative Federation, and the Cheju Citrus Research Institute, Korea on May 18-22


No. of Participants: 110


Co-sponsor: National Agricultural Cooperative Federation, Korea


The Training Course was given by Dr. Edward Yun Cheng, Pesticide Research Laboratory, Taiwan Agricultural Research Institute (TARI). He was assisted by Ms. Ching-hua Kao, Ms. Yueh-Chang Li and Ms. Chia-lin Tseng, all of TARI.


For further information contact Dr. Kyung Joo Park , FFTC Agricultural Economist


Index of Images




  • ac1999g1.jpg


    Figure 1 Garak Marketing Center in Seoul, the Largest Wholesale Market in Korea, Where Vegetables Are Tested by Bioassay. Suspect Shipments Are Removed from the Market and Tested Further by Chemical Analysis.



  • ac1999g2.jpg


    Figure 2 Yangjae Sales Center, a Marketing Outlet for Agricultural Cooperatives in Korea. It Uses Rapid Bioassay to Monitor Produce for Pesticide Residues.

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