Technical profile: the quantitative advantage for mycotoxin testing
October 01, 1993
by Teresa Acklin
Contributed by suppliers, technical profiles feature new technology, products, specific applications or proprietary concepts. This article was prepared by Dr. Thomsen J. Hansen, director of chemistry for VICAM, Watertown, Massachusetts, U.S.
On June 19, 1993, the World Health Organization's International Agency for Research on Cancer (IARC) approved a report classifying aflatoxin as a Group 1 carcinogen because it has been shown to cause cancer in humans.
Aflatoxin is a mycotoxin produced by naturally occurring molds, which may infest agricultural commodities such as grain, maize, peanuts, dairy and other foods. Hot, humid weather conditions promote the growth of the mold so that contamination is chronic in all parts of the world, depending on the climate. Since there is international trade in the affected commodities, exposure to aflatoxin is a worldwide concern.
According to the IARC report, there are elevated incidences of liver cancer in populations exposed to aflatoxin through dietary intake. Studies conducted in China and the Philippines showed excess mortality from heptocellular carcinoma among people consuming foods heavily contaminated with aflatoxin. Occupational exposure also occurs through handling and processing of contaminated crops. A Danish study attributed a higher rate of liver cancer among workers handling imported feed to exposure of aflatoxin contamination.
TESTING FOR MYCOTOXINS.
Traditional methods for mycotoxin analysis use extensive sample extraction and cleanup procedures, followed by thin layer chromatography (TLC) or high pressure liquid chromatography (HPLC). These methods are quantitative and sensitive, but often lengthy, costly and complicated.
Several newer methods are available that are simple and require less than 30 minutes for analysis. Some of the rapid methods are quantitative, providing an instrumental reading of actual mycotoxin content. Quantitative tests are useful over a range of contamination levels.
Other methods are qualitative, indicating the presence or absence of the mycotoxin at a certain level. The pass/fail level for these tests is usually preset by the developers and cannot be easily varied by the user. In a few cases, qualitative tests are available at more than one level.
Decisions on mycotoxin management depend on determining mycotoxin concentration relative to all significant levels, not just one. Following are details on several mycotoxins and the concentration levels that are important for regulatory or toxicology reasons.
Aflatoxin is currently the mycotoxin of greatest concern. Aflatoxin has been found to contaminate many crops, sometimes at very high concentrations. The commodities with a high risk of aflatoxin contamination include maize, peanuts and cottonseed.
Aflatoxin can cause liver damage or cancer, decreased milk and egg production and immune suppression. In the U.S., the Food and Drug Administration has established a limit of 20 parts per billion for aflatoxin in foods and most feeds and feed ingredients.
Maize or cottonseed meal can be used under certain conditions for animal feed at levels up to 300 ppb. Some food processors set their own allowable aflatoxin levels at less than the F.D.A. level. Products in international commerce may be subject to a variety of tolerance levels established by other countries. Common levels range from 5 ppb to 50 ppb.
Fumonisin has become the fastest growing area of mycotoxin research. Fumonisin is isolated from Fusarium moniliforme, a frequent, almost universal, inhabitant of maize.
This mycotoxin is involved in producing leucoencephalomalacia in horses and pulmonary edema in swine. It also is associated with human esophageal cancer and has tumor-promoting activity in rats.
There is much work in progress on occurrence and effects of fumonisin. In 1992, an F.D.A.-U.S. Department of Agriculture Interagency Working Group on Fumonisins recommended maximum levels of 5 parts per million for horses, 10 ppm for swine and 50 ppm for beef cattle. Other recommendations, including allowable levels for human consumption, will be formulated as more information is gathered on fumonisin.
This mycotoxin is produced in commodities by several mold species. Ochratoxin in feed causes kidney damage and decreased egg production at levels below 1 ppm. Concentrations greater than 5 ppm may cause liver and intestinal damage.
Like aflatoxin, ochratoxin is immunosuppressive and carcinogenic. Barley, maize, oats and wheat in the U.S. contain ochratoxin at a low incidence and low level of contamination less than 200 ppb.
Ochratoxin also has been reported in sorghum, rice, figs and green coffee, at levels of less than 500 ppb. Residues of ochratoxin can be found in pork and poultry meat.
The highest reported incidence levels have been in cereal grains produced in northern European or Balkan countries and in India.
Zearalenone is most frequently found in maize and also occurs in wheat, barley and sorghum. Zearal-enone is estrogenic and decreases reproductive efficiency in animals at feed concentrations of less than 1 ppm.
Generally, zearalenone concentrations are well below this level in processed cereal foods, but amounts above 10 ppm can be encountered in some feeds.
FACTORS AFFECTING TOLERANCE LIMITS.
Several factors must be considered when establishing tolerance levels for mycotoxins. Aflatoxin and ochratoxin, and possibly fumonisin, are carcinogens, so human exposure levels must be kept as low as practicable.
Limits in human food are based on levels that do not unduly limit the food supply and can be readily analyzed. Levels in animal feed must be kept low enough so that residues are not transmitted to meat, milk or eggs.
Levels of mycotoxins in feed must be kept low to prevent toxic effects to animals and consequent economic loss. Levels may be different for different animals and throughout the lifetime of the animal.
Not surprisingly, consideration of these factors leads to different allowable mycotoxin levels in different situations. Because of the different significant mycotoxin levels, quantitative testing of mycotoxins is preferable to pass/fail determination at a single level.
BENEFITS OF QUANTITATIVE TESTS.
A quantitative test can distinguish levels, allowing better decision-making when contamination is found. Quantitative analysis also provides a better determination of the value of a commodity.
A lot of maize containing less than 20 ppb aflatoxin can be processed for human consumption and is worth more than a lot containing more than 20 ppb. But a lot containing 50 ppb is worth more than one containing 320 ppb because it can be used in some animal feeds.
A quantitative test can distinguish these levels, allowing better management when contamination is found. A qualitative test that only gives a result of positive at 20 ppb provides no additional information unless the sample is retested using a different pass/fail test.
Surveys of large numbers of samples, such as those done by F.D.A. to monitor human intake, must use quantitative methods. Such surveys are used to estimate the occurrence and levels of the toxin in the total food supply. It is not sufficient to determine merely that a certain percentage of samples contains more or less than one pass/fail level. A good estimate of overall mycotoxin incidence requires information on the quantitative distribution of mycotoxin levels throughout all samples.
The same reasoning can be applied to samples tested on a smaller scale, such as at a grain elevator. For example, shipments from one area may be found to contain 0 to 5 ppb aflatoxin, and shipments from another region contain 10 to 15 ppb. This information can be used to plan future testing practices.
In this example, future shipments from the second source should be checked more extensively. A 20 ppb nonquantitative test would show only that all samples were negative. Without retesting, this would not provide the information needed to make an informed decision.
Quantitative testing of mycotoxins is possible using rapid, simple methods of analysis. The extra information obtained, compared with pass/fail tests, should make a quantitative test the method of choice in most cases.
Quantitative testing products from VICAM use immunoaffinity columns for aflatoxin and fumonisin, as well as for ochratoxin and zearalenone. Most mycotoxin assays using VICAM's columns are made by direct measurement of fluorescence of the affinity purified mycotoxins. This is done by collecting the mycotoxin from the affinity column and placing it in a test tube in the fluorometer.
Affinity columns also provide excellent cleanup for HPLC. HPLC confirms the presence of the mycotoxins and also allows determination of the individual aflatoxins or fumonisins.
VICAM products are approved by the U.S.D.A. and the Association of Official Analytical Chemists.