Mycotoxins are compounds that are produced by molds. Some are very toxic, like aflatoxin and some are beneficial, like penicillin. There are literally hundreds of different mycotoxins and more are being identified all the time. Some are not found in nature, but can be produced in the lab. Of those that do occur in nature, only a few are known for their toxicity.

Molds do not always produce mycotoxins and we do not completely understand why and under what conditions some of them produce toxins. These molds grow and produce mycotoxins on the grain in the field, but under the right conditions, they can also grow and produce them in stored grain. Of all the mycotoxins that have been identified, I am only going to discuss the seven that seem to cause the most concern. They are aflatoxin, deoxynivalenol (DON or vomitoxin), fumonisin, zearalenone, ochratoxin A and T-2.

The following discussions on toxicity are based on feeding trials and experience on the presence of the single toxin being discussed. There is the belief that there is some synergy between the toxins. That means that if you have more than one of the toxins in the feed/grain, the two may work together to cause health problems. Therefore, the allowable levels for each will not be the same. For instance, if the tolerance for aflatoxin for your animal is 200 ppb and you also have 2 ppm fumonisin in the feed/grain, in order to not have a health effect, you may need to set a 100 ppb aflatoxin level and a 1 ppm fumonisin level. These are examples only for illustration and do not relate to the real facts, which have not been determined as yet.

FDA Rules

First, a discussion of the FDA rules concerning mycotoxins is in order. FDA has set "Action Levels" for aflatoxin and "Advisory Levels" for both DON and Fumonisin. FDA uses the term "action levels" to denote levels at which they are prepared to take regulatory action. The burden is still on them to provide the courts with justification for any action they may initiate under these rules. In effect, this makes the blending of contaminated lots with un-contaminated lots to result in a single un-contaminated lot based on the action levels to be illegal. They use the term "advisory levels" when they simply wish to provide guidance to the industry concerning levels at which they believe provide an adequate safety margin for food and feed. They do reserve the right to take regulatory action against anyone that has egregiously and consistently exceeded these levels. They can set "Regulatory Limits" by issuing regulations under the proper public notice and comment rulemaking procedures. Courts will normally find a per se violation of the law when these limits have been exceeded. This means that FDA does not have to prove that the product is adulterated. It is, on the face, adulterated by exceeding the limits. FDA has not set any "regulatory limits" for any mycotoxin at this time.

Aflatoxin

There are three types of aflatoxin found in grain, B1, B2, G1 and G2. They are called B and G because B1 and B2 after extraction from the grain glow blue under a black light and G1 and G2 after extraction from the grain glow green under the black light. B1 is the most common and the most potent. We seldom see the others in grain unless the level of B1 is high. Then the others tend to show up in lessor amounts. They are most often produced by Aspergillus flavus mold but Aspergillus paraciticus mold can also produce aflatoxin. Aflatoxin, itself, is not directly visible on the grain. However, the molds that most often produce it, if present in large enough quantities, will appear as yellow green spore masses on corn kernels plated out in the laboratory as shown in Figure 1. Figure 2 shows A. flavus on an insect damaged ear of corn.

Figure 1. Aspergillus sp. Growing on a corn kernel (R. Friedrich)

Figure 2. A. flavus on an insect damaged ear of corn (J. L. Richard)

The mold spores are always present in the environment and just waiting for the correct conditions to grow and produce aflatoxin. Corn growing in the field can produce aflatoxin when the nighttime growing temperatures are around 75 F and daytime temperatures are over about 90 F. Historically, this has been during a drought when the insects in the field carry the spores with them beneath the husks. The insects then chew the kernels, resulting in an entrance point for the mold spores to start growing. Aflatoxin can be produced by the mold growing on corn in storage provided the conditions are correct, 14% corn at about 68 F can experience additional aflatoxin formation. Of coarse, if the corn is a mixture of high and low moisture content, the mold can grow and produce aflatoxin on the wet kernels. In the SE US, corn harvested late in the day and left overnight in the truck has been known to significantly increase in aflatoxin levels.

Aflatoxin is the most potent natural carcinogen known to man. At high levels, it can cause liver cancer. It has been suspected of causing hemorrhaging of the intestinal tract and kidneys. It, also, suppresses the immune system and can suppress appetite. Different animals are affected at different levels of consumption. The FDA action levels are shown in Table 1.

FDA Action Limits for Aflatoxin

20 ppb for humans, immature animals (including poultry) and all dairy animals and when the end use is not known.

100 ppb for breeding beef cattle, swine and mature poultry

200 ppb for finishing swine (100 pounds and up)

300 ppb for feeder cattle

Deoxynivalenol

The next mycotoxin on my list is deoxynivalenol, also known as DON or vomitoxin. This mycotoxin is most often produced by Fusarium graminearium but F. culmorum is sometimes the culprit. The grains most often affected are corn, wheat, oats, and barley. The mold over winters in the previous years crop residue, which provides the mold spores that can be blown onto the flowers of the next year's crop. The mold prefers cool, moist conditions and starts growing on the flowers and proceeds into the kernels where the toxin is produced.

Often, the mold will appear as a purple to pink stain on the kernels and sometimes pink mold growth can be seen on the ears of corn. The disease caused by the growth of this mold on wheat is often referred to as scab and FGIS calls the shriveled kernels of wheat "tombstone" damaged. These damaged wheat kernels are shriveled and light in weight and when broken open are chalky in appearance. See Figure 3. Figure 4 shows the disease on the wheat head in the field. Figure 5 shows this mold growing on ears of corn.

Figure 3. Scab Damage

Figure 4. Wheat heads infected with scab.

Figure 5. Gibberella ear rot. (G. Munkvold)

Before the toxin was identified, scientists noticed that when they tried to feed some but not all moldy corn to swine, the swine refused to eat it. They dubbed this the refusal factor. Later, they discovered that if you could somehow trick the swine into eating the moldy corn, the swine then regurgitated the feed hence the name then given it was "vomitoxin". This is not a very human friendly term, so we prefer to use the term "DON". In addition to this type of toxicity, DON, at very high levels, will cause bread to not rise properly and even be a little grayish in color. Malt made from barley with excess DON will cause the beer to have off-flavors and what is termed as "gushing" or "foaming". However, just because your beer bottle or can foams excessively when opened does not mean that DON is the culprit. There are other process related issues that can cause the same thing to happen. It is an immunosuppressant and is suspected to be involved in some kidney problems. The FDA advisory levels for this mycotoxin are shown in Table 2. These same molds can produce Zearalenone. Some end users believe there are other toxins produced by these molds that have not been identified as yet. They use DON as an identifier for other potential problems. Often, they will set much lower tolerances for DON than the FDA advisory levels because of the health effects they have seen at the lower levels of DON which they think was from one of the unknown toxins.

Table 2

FDA Advisory Levels for DON

No advisory limit for raw wheat. It is up to the miller to decide what level he can accept and still make the below levels.

1 ppm for finished wheat products for human consumption

5 ppm for swine and other animals (except cattle and chickens); not to exceed 20% of swine diet and 40% for other animals.

10 ppm for beef and feedlot cattle older than 4 months and for chickens; not to exceed 50% of the diet.

Fumonisins

Fumonisins like aflatoxins consist of several different toxins called FB1, FB2 and FB3 and were first identified in 1988 by a group of South African scientists. The molds that produce these toxins are Fusarium moniliforme and Fusarium proliferatum. These are the molds that are most often the causal agents, but there are other mold strains that can produce these toxins. FB1 is the most often found of the three and is also the most potent. Figures 6 shows this mold growing on grain.

Figure 6. Corn infected by Fusarium sp. (G. Munkvold)

Because the mold grows best in insect or bird damaged kernels and just plain broken kernels, corn screenings are often found to contain high levels of fumonisins. As with all the mycotoxins of concern, fumonisin is most often produced in the field, but under the correct moisture and temperature conditions can be produced in storage bins. FDA has set advisory levels for human consumption as shown in Table 3 and animal feed as shown in Table 4.

Table 3

FDA Advisory Levels for Fumonisins in Human Food
Product	Total Fumonisins(FB1 + FB2 + FB3)parts per million
Degermed dry milled corn products (e.g., flaking grits, corn grits, corn meal, corn flour with fat content of <5%, dry weight basis)	2.0
Whole or partially degermed dry milled corn products (e.g. flaking grits, corn grits, corn meal, corn four with fat content of <2.25% dry weight basis)	4.0
Dry Milled corn bran	4.0
Cleaned corn intended for masa production	4.0
Cleaned corn intended for popcorn	3.0

Table 4

FDA Advisory Levels for Fumonisins in Animal Feed
Animal or Class	Recommended Maximum Level of Total Fumonisins in Corn and Corn By-Products	Feed Factor2	Recommended Maximum Level of Total Fumonisins in the Total Ration (ppm1)
Horse3	5	0.2	1
Rabbit	5	0.2	1
Catfish	20	0.5	10
Swine	20	0.5	10
Ruminants4	60	0.5	30
Mink5	60	0.5	30
Poultry6	100	0.5	50
Ruminant, Poultry & Mink Breeding Stock7	30	0.5	15
All others8	10	0.5	5
1 total fumonisins = FB1 + FB2 + FB32 fraction of corn or corn by-product mixed into the total ration3 includes asses, zebras and onagers4 cattle, sheep, goats and other ruminants that are ³ 3 months old and fed for slaughter		5 fed for pelt production6 turkeys, chickens, ducklings and other poultry fed for slaughter7 includes laying hens, roosters, lactating dairy cows and bulls8 includes dogs and cats

As can be seen, different animals are sensitive to these toxins at different levels. In fact, fumonisins have a different toxic effect on different kinds of animals. Horses are the most susceptible to these toxins. Low levels will cause a softening of the brain, called leukoencephalomalacia. This results in blind staggers and death in a very short time. The effect on swine results in body fluids collecting in the lungs causing suffocation. Chickens and cattle are fairly resistant to fumonisins. There is some demographic evidence that fumonisins may be involved in esophageal cancer in humans but a direct link has not been established.

Zearalenone

Zearalenone is a mycotoxin that, like DON, is produced by Fusarium graminearium. Therefore, infected kernels may have a pinkish color due to the presence of the mold. It is most often present in corn, but can also be in wheat, sorghum, rye and barley. Since it is produced by the same mold and under the same environmental condition that produces DON, the two toxins may be present in the same sample of grain. As in the other mycotoxins, it is most often developed in the growing crop in the field, but under the correct moisture and temperature conditions, may also be produced in storage bins.

Zearalenone is an estrogenic toxin. This means it causes reproductive problems, especially in swine. Figure 7 shows the effect it can have on gilts; these gilts were fed straight grain sorghum with a level of about 400 ppb. If this sorghum had been incorporated at a fairly normal 50% of the feed, the level would have been 200 ppb and the same effect would probably not have been seen. At higher levels, it will cause a prolapse of the vagina and an enlargement of the mammary glands.

It causes atrophy of the testes in male pigs. The literature indicates that at levels around 500 ppb it will cause weak piglets, small litter size and at even higher levels, it will cause abortion. FDA has not set any Advisory Limits for this toxin in the US, but some swine producers claim to see health problems begin at 200 ppb in the feed

Figure 7. Effect of Zearalenone on a gilt's reproductive organs

Ochratoxin A

Ochratoxin, more commonly called Ochratoxin A, is produced by the molds Aspergillus ochraceous and Penicillium verrucosum. Another mold Aspergillus niger can, also, be a producer of this toxin. There is little in the literature to indicate the conditions necessary for the production of this toxin, however, sufficient moisture and temperature must be present for the mold to grow. We seldom see it in the US.

FDA has not set Advisory Limits for ochratoxin. It is associated with kidney disease and at high enough levels may be carcinogenic to both the kidneys and the liver. It is most often a concern in coffee and some countries have set a 5 ppb level for ochratoxin.

T-2

T-2 toxin is included in a group of mycotoxins called "trichothecenes". Others in this group that I will not discuss in this presentation are HT-2 and DAS. The most common mold involved in the production of T-2 is Fusarium sporotrichioides. This mold may also produce the other two, but they are seldom seen in nature.

It has been known to occur in corn, wheat, rye, barley and oats in the field. Here, again, the exact conditions for production are not known. However, high moisture (humidity) and temperatures of 50 F to 75 F seem to be conducive. This mold on corn will often be white in color, but can also be pink to reddish in color.

FDA has not set any Advisory Levels. T-2 is known to inhibit protein synthesis thereby resulting in a disruption of DNA/RNA synthesis. It can suppress antibody levels causing poor weight gain and even weight loss. Poultry seem to be the most susceptible. It causes beak lesions, resulting in less feed consumption and reduced rate of gain. It may cause abnormal feathering in chicks, a drastic and sudden drop in egg production, eggs with thin shells, reduced weight gains, and mortality. The same feed given to turkeys can result in reduced growth, beak lesions, and less immunity to infection. It has been associated with lesions of the intestinal tract, including the esophagus.

Sampling and Sample Preparation

Because high levels of mycotoxins may be in some kernels and none in others, sampling is very important when testing for their presence. For example, 400,000 ppb of aflatoxin has been found in individual kernels directly from the field. As you can see, this makes it very difficult to detect the very low levels set by FDA. However, here is a worst-case scenario, suppose you have a sample containing 2,000 grams of corn (about 6740 kernels) and there is only one contaminated kernel in the sample. If you are trying to meet the 20 ppb FDA limit for human consumption, and all of your kernels are zero aflatoxin except for one kernel containing 134,800 ppb you would have an average level be 20 ppb. However, if you were to divide that sample in half before grinding, one half would be zero ppb and the other half would be 40 ppb. It is highly unlikely that you will encounter this scenario, so the variability between samples will be somewhat less than this.

The larger the sample, the more kernels there are and the better the chance you have of getting a representative sample. This is why FGIS recommends the sample sizes for determining aflatoxin shown in Table 5. The entire sample should be ground. FGIS says that the Romer mill, Bunn Model G3, Viking Hammer mill, Falling Number Mill and UDY grinder are examples of grinders that will do a good job of reducing the particle size of the sample. After grinding the sample should be divided down to a 500-gram sub-sample. This sub-sample is then well mixed and divided down to a 50-gram sample. Be sure to clean all equipment between samples to avoid sample contamination.

Table 5

FGIS's recommended sample sizes for different types of samples
Carrier	Minimum Sample
Trucks	Two pounds(908 grams)
Railcars	Three pounds(1,362 grams)
Vessel Sub-lots/Barges	Ten pounds(4540 grams)
Submitted Samples	10 poundsRecommended

Table 6 shows how the variability of the results will increase with smaller sample sizes. For example, if you know the true average aflatoxin level in a truck is 20 ppb and you take a 10-pound sample, your answer will vary between 11.6 and 28.4 (a range of 16.8 ppb). If you reduce the sample size down to 5 pounds, the result will vary from 8.1 to 31.9 (a range of 23.8 ppb). Knowing this variability, I have often been asked why I do not support taking another sample and accepting the corn if the result shows less than 20 ppb. The problem is that if you only take another shot at the positive samples, you will slowly be increasing the average level of aflatoxin you are taking into your facility. When you start to ship your corn, you are going to find you will be misgrading more often. Another way to look at this is, suppose the true level in the truck is 25 ppb. A 2.5-pound sample, which is probably the largest sample any of us are using for trucks, could appear to have 7.2 to 42.8 ppb in it. But, suppose your result was actually 25 ppb and you decide to take another sample and it comes back under 20 ppb. You would have accepted a load you should not have. Suppose the next truck has a true average level of 35 ppb. And your test result shows it to be under 20, which it certainly could. You now have taken in a truck of 25 and one of 35. You would have to take in a truck of zero aflatoxin to make your average come out to 20 ppb. OR, it would take two trucks of 10 ppb to make your average level in that bin to be 20 ppb. Based on the results above, you think you have an average level below 20 ppb. When you load out that bin and it arrives at your customer's facility, he takes a 2.5-pound sample and has a 50/50 chance of getting a result over 20 ppb. You have a customer relations problem.

Table 6

Variability between samples at different sample sizes taken from a truck containing 20 ppb aflatoxin (Romer Labs, 1995)
Sample Size(pounds)	# of Kernels	Variability(ppb)
10	30,000	11.6 - 28.4
5.0	15,000	8.1 - 31.9
2.5	7,500	3.2 - 38.8
1.0	3,000	1.0 - 46.9

Testing Methodology

The two reference methods used to test for the most common mycotoxins are TLC (Thin Layer Chromatography) and HPLC (High Pressure Liquid Chromatography). FGIS only approves HPLC for their analysis work. Both of these tests require a laboratory, some expensive equipment and take quite a bit of time to conduct.

There are several types of quick test methods. Enzyme Linked Immunosorbent Assay (ELISA for short) is the technology used by the Neogen Veratox, the Romer AccuTox and the R-Biopharm Ridascreen methods. Vicam's Aflatest and Romer's Fluoroquant procedures use a fluorescent technology.

The ELISA method requires the production of an antibody that is specific to the mycotoxin being tested for. This antibody is attached to the bottom of a test well or tube. The mycotoxin is extracted from the grain, mixed with a solution containing a mycotoxin-enzyme conjugate. This mixture is then transferred to the test well/tube. The mycotoxin extracted from the sample and the mycotoxin-conjugate competes for the antibody. After an incubation period, the excess solution is washed out of the tube and a substrate is added. The reaction that follows results in a color change and this change is read on a meter, which is calibrated to a range of levels of the mycotoxin. Different brands use a little different procedure, but the basics are the same.

The fluorescent method is a process where the mycotoxin is extracted from the sample and purified. The purified toxin is mixed with a developer. In essence, the developer reacts with the mycotoxin making the product fluoresce. The amount of fluorescence is measured with a meter that is calibrated to read the level of the mycotoxin.

Table 7 is a list of rapid tests that have been approved by FGIS for use in determining aflatoxin in the official system. Table 8 is their approved rapid tests for DON.

Table 7

FGIS Approved Rapid Test Methods For Aflatoxin
Method & kit	Approved for		Test Kit Range
	Qualitative	Quantitative
AflaCup(International Diagnostics, Inc.)	X		20 ppb
AgriScreen(Neogen)	X		20 ppb
Veratox AST (Neogen)	X	X	5-300 ppb(quantitative)
Fluoroquant (Romer)	X	X	5-300 ppb(quantitative)
Aflatest (Vicam)	X	X	5-300 ppb(quantitative)
Myco< (Strategic Diagnostics, Inc.)	X	X	5-80 ppb(quantitative)
RIDASCREEN Fast Aflatoxin (r-Biopharm)	X	X	5-50 ppb(quantitative)

Table 8

FGIS Approved Rapid Test Methods For DON
Method & kit	Approved for		Conformance Limit(s)
	Qualitative	Quantitative
Myco< DON (Strategic Diagnostics, Inc.)		X	3 PPM
RIDASCREENÒ FAST DON(r-Biopharm)		X	5 PPM
AgriScreen (Neogen)	X	X	5 PPM(quantitative test)
Veratox (Neogen)	X	X	5 PPM(quantitative test)
Fluoroquant (Romer)		X	5 PPM
AccuToxTM(Romer)		X	5 PPM
EZ-Quant DON (Diagnostix)		X	5 PPM
EZ-Quant DON (Diagnostix)		X	5 PPM*
EZ-Quant DON 0.5 ppm		X	2.5 PPM*
Veratox 5/5 (Neogen)		X	5 PPM**
DON FQ (Vicam)		X	5 PPM

* The EZ-Quant DON Test kit (part number 600312) for barley, malted barley, wheat and corn is approved to a conformance limit of 5 ppm. The EZ-Quant DON 0.5 ppm test kit (part number 600313) is exclusively for barley and malted barley and is approved to a conformance limit of 2.5 PPM.

** The Veratox 5/5 test kit is approved to a conformance limit of 5 ppm. Using the optional extraction procedure limits the conformance level is 2.5 ppm.

FGIS has approved two quantitative rapid Fumonisin tests. They are the Veratox Quantitative Fumonisin test kit to determine fumonisin in corn, corn meal, popcorn, rough rice, corn/soy blend and wheat; and the RIDASCREEENÒFAST for fumonisin in corn, corn meal, sorghum, corn gluten meal, corn germ meal and corn/soy blends. As Norm Abrams of "The New Yankee Workshop" says, a word about shop safety is in order before we start the project. In this case, a word about laboratory safety is in order. You are dealing with toxic substances. It is a good idea to protect your self from inhaling dust during the grinding process and to not come in direct contact with the toxin during the extraction process. In addition, if the extraction liquid is not water, it could cause both a safety concern and a environmental concern. For example, methanol is considered a hazardous waste after being used in the aflatoxin extraction process. You should discuss its proper disposal with your environmental expert.

References

1. GIPSA, Technical Services Division, 10383 N. Executive Hills Blvd., Kansas City, MO 64153. Grain Fungal Diseases & Mycotoxin Reference. 2. RomerTM Labs, Inc., 1301 Stlylemaster Drive, Union, MO 63084. Mycotoxin Resource Guide, Volume 2.

3. RomerTM Labs, Inc., 1301 Stlylemaster Drive, Union, MO 63084. Mycotoxins - An Overview, January, 2000.

4. RomerTM Labs, Inc., 1301 Stlylemaster Drive, Union, MO 63084. A Total Quality Assurance Program for Risk Management of Mycotoxins. John Richard, Ph.D., October, 2000.

5. Rodericks, Joseph V., Clifford W. Hesseltine, Myron A., Mehlman editors. "MYCOTOXINS IN HUMAN AND ANIMAL HEALTH" Park Forest South, Illinois, Pathotox Publishers, Inc., 1977.