Mycotoxins in the Grain Market

C. R. Hurburgh, Jr.

Mycotoxins are poisonous trace organic residues of mold deterioration. Mycotoxins are not alive themselves, but instead are very potent compounds causing, at very low dosages (parts per million (ppm) or parts per billion (ppb)), a variety of human and animal health problems. Individual mycotoxins are produced by specific mold strains under more restrictive growth conditions than for the molds themselves. Mycotoxins are not automatically produced whenever grain becomes moldy. However, from a risk viewpoint, the likelihood of toxins is greater in damaged kernels than in sound kernels.

Although aflatoxin has received the majority of public attention, advances in chemical detection methods have identified several others that can create problems for grain users. All mycotoxins are present in non-uniformly distributed trace concentrations. Normal bulk sample quality detection methods are hard to use for trace levels. Trace toxics in general will present increasing difficulty for bulk grain handlers.

Health and Regulatory Effects

Table 1 summarizes the major mycotoxins, producing molds, grains usually affected and the regulatory limits. The Fusarium and Aspergillus strains account for the majority of toxin problems. Mycotoxins are regulated by Food and Drug Administration (FDA), not Federal Grain Inspection Service (FGIS), although FGIS has a Memorandum of Understanding to report any over-tolerance results it finds to FDA. This understanding applies to white-certificate inspections where Official personnel collect and analyze samples, not to submitted samples where the lot identity is unknown. Although; FGIS can analyze for aflatoxin, vomitoxin and fumonisin, the Memorandum of Understanding applies only to aflatoxin.

The health impacts of mycotoxins are much less precise than regulatory limits or guidelines would suggest. The concept of mycotoxin poisoning was first discovered in England when 100,000 turkeys mysteriously died of what was ultimately identified as aflatoxin. Most regulatory limits for trace toxics are set as a large safety factor (100 or more) applied to toxicological data from laboratory animals. If there are no limits established and the compound is a known carcinogen, then the so-called Delaney Clause sets the limit at zero until more definitive data is available.

Table 2 gives the known effects of the mycotoxins listed in Table 1. Not all are cancer-causing. Aflatoxin is indeed a potent carcinogen, but the immediate effects of the Fusarium toxins are often more striking. Most of the economic impacts are on animal health.

Fusarium Toxins

Cool wet conditions favor the growth of Fusarium species molds, which can produce several mycotoxins detrimental to livestock. Fusarium stains can produce vomitoxin (also known as DON, deoxynivalenol or "refusal factor"), zearalenone (known as "giberella toxin,") and fumonisins. Swine are the food animal species most at risk from these mycotoxins, that usually are found in corn. Horses are extremely sensitive to fumonisins.

Vomitoxin causes a dose related reduced feed intake in swine at levels above 1 ppm in the ration. Higher levels (5-10 ppm) can cause vomiting and nearly complete feed refusal. If clean feed is offered, feed consumption resumes within 24 hours or less. There are no documented reproductive effects of vomitoxin in swine. Cattle have consumed up to 10 ppm vomitoxin with no adverse effects. The common adsorbents used for aflatoxin (e.g. aluminosilicate [Novasil] or bentonite) are of little value against vomitoxin.

Zearalenone is an estrogenic mycotoxin that occurs in corn and wheat in the midwest. Sometimes zearalenone and vomitoxin occur in the same sample, although one usually predominates. Prepubertal gilts show signs of estrus, straining and prolapse at feed levels as low as 1 ppm. In cycling sows, zearalenone above 3 ppm has caused anestrus and pseudopregnancy, delaying the estrus cycle for up to 60 days. Very high levels (>30 ppm) can cause early embryonic death in sows. However, in late gestation zearalenone is not likely to abort sows. Cattle given more than 10 ppm zearalenone may have infertility and interference with the estrus cycle.

Fumonisins, a group of toxins produced predominately, in corn are believed to be most prevalent when cool wet weather at crop maturity follows early season drought stress. Hepatic damaged and elevated serum liver enzymes occur in all livestock. Horses are highly susceptible to liver damage and also to the brain disease leukoencephalomalacia (moldy corn poisoning) at fumonisin levels above 10 ppm. Swine may have liver damage at feed fumonisin concentrations above 25-50 ppm and cattle develop mild liver lesions at concentrations above 100 ppm. Liver damage in these species is transient and liver function returns to normal when exposure stops. Feed concentrations above 100 ppm in swine can cause acute pulmonary edema. Toxin concentration is highest in broken grain, such as corn screenings.

Aspergillus Toxins

The Aspergillus species molds, generally characterized by greenish yellow coverage of kernels or ears, can produce aflatoxin and ochratoxin under stress conditions. Aflatoxin is a known carcinogen. Both cause animal health problems.

Aflatoxin can be produced when maturing corn is under drought and insect stress with prolonged periods of hot weather, (daytime highs above 90 F, nighttime lows above 75 F). Common in the Southern US, aflatoxin outbreaks are a 5-10 year event in the Corn Belt. Aflatoxin is a cancer promoter and an immunosuppressant. Effects are possible on poultry and small swine at 20 ppb. Aflatoxin-contaminated grain should not be fed to lactating dairy cattle, as it will carry to the milk at 1/200-1/400 dilution rates.

Ochratoxin is a relatively uncommon mycotoxin of corn, but has been reported several times in the midwest. Swine are the most likely animals affected by feed levels of 1-3 ppm. Ochratoxin causes increased water consumption and urination with renal tubular damage which may result in permanent scarring of the kidneys.

Mycotoxin Incidence

Mycotoxins create great concern, even panic, among consumers. As the understanding of growth conditions increases, there is an increasing tendency to anticipate outbreaks and to search for the first reports of toxins in new harvest. Most mycotoxin production occurs in the field before harvest, rather than in-storage. Poor storage practice can increase already existing mycotoxin levels.

Table 3 summarizes the weather conditions required for toxin production, as well as is known. Clearly, the Aspergillus and Fusarium toxins are unlikely in the same area in the same year. Some regions are more likely to have favorable conditions than others (Figure 1). However, the real problems occur when abnormal weather spreads the risk to larger areas than the market is accustomed to handling.

For most mycotoxins, there is little survey data available. Aflatoxin has been the most studied. Table 4 gives a summary of several reports of aflatoxin outbreaks. Weather in the southern US clearly promotes aflatoxin formation more often than in the Corn Belt. However, when weather was favorable, approximately 10-30% of samples were affected. This is enough to make random blending a risky proposition. The same incidence rates seem to occur in wheat (fusarium toxins) when weather is overly cool and wet, as for example, 1992 in the south central US and 1993 in the northern US.

The crucial point is that even in situations where mycotoxins have been detected, there are usually as many or more clean lots as contaminated ones. Crops and growing regions must not be unilaterally condemned by rumor. The key element in preventing unjustified extrapolation of problems is an effective screening and follow-up program.

Weather conditions are not a guarantee of mycotoxins. Our knowledge of growth conditions is not precise enough to predict incidence with absolute certainty. For example, Iowa weather in 1993 was seemingly very favorable for fusarium development. Vomitoxin and zearelenone were expected. Table 5 shows what happened, according to a recent Iowa State University survey.

Most samples had slight traces of mycotoxins (the mold was present) but not enough to raise concerns. Apparently the 2-4 days of warm, dry weather in mid-October relieved enough stress on the standing high moisture corn to retard mold growth. Mycotoxin production is highly weather-sensitive, and conditions can change rapidly. An effective scouting-preharvest check program in your trade area is the only way you can assess your local situation.

Mycotoxin Detection and Sampling

Mycotoxins are a kernel-by-kernel situation. A few kernels have very high levels (several thousand times the average), and most have none. Measurement methods require small ground samples (2-50 grams), so sampling and sample handling are obvious problems.

A representative mycotoxin analysis cannot be made with original samples less than 5 lbs. Most labs recommend 10-lb samples. Samples should be either diverter-based or multiple probings. If other tests are run on the sample, no material should be removed before preparing for mycotoxin analysis.

The entire original sample should be ground before subsampling for analysis portions. The reason for this goes back to the kernel-to-kernel nature of mycotoxins. Dividing whole grain samples (e.g. with a Boerner divider) carries a high probability of not detecting the toxin.

Figure 2 depicts an example of this situation. The five contaminated kernels can only appear in five subsamples at most. Sampling and subsequent sample handling are the largest sources of error in mycotoxin tests.

There are several test methods for mycotoxin analysis. They are generally summarized in Table 6, without reference to manufacturers. All the chemical methods require ground samples and the handling of liquids. Mycotoxin tests should be done in a separate area apart from physical factor grading. Recognize that mycotoxin testing will take time, labor, and money. This is why an advance survey/scouting program is valuable.

The immunoassay-based tests are the most common. FGIS has approved the immunoassay kits for aflatoxin and vomitoxin, with fumonisin being evaluated. Operators need training and practice to be accurate. Mycotoxin tests are not as forgiving of operator error as the common bulk sample quality tests.

The net result of typical sampling and analysis errors is that mycotoxin data have an uncertainty of ±25-40% of the reported value. This is the reason why contradictory results can be obtained from the same load or storage. Obviously, repeat analyses on the same small ground sample will be much closer, but the sampling and sample preparation contributes error not associated with the test itself.

Handling and "Reconditioning"

Mycotoxin policy is sensitive and often unclear. Deliberate blending to dilute toxin levels is illegal if the level of regulated toxin is known prior to the blending. Although FDA receives data from FGIS and state laboratory officials, it generally lacks resources to act on bulk grain data before the grain has been moved, sold or used. In practice, user requirements and specifications for the delivered grain control mycotoxin problems more effectively than government agencies can. Aflatoxin is the only mycotoxin regulated and included in the FDA-FGIS agreement.

The higher the percentage of over-tolerance lots, the more likely it is that some resold grain will exceed limits. In the case of aflatoxin, export grain must be tested. Other mycotoxins are often limited by contract. In the years when aflatoxin was over-tolerance in 15-20% or more of the inbound lots, random blending with no segregation of inbound grain failed to protect grain handlers.

"Reconditioning" is now allowed, for aflatoxin-contaminated corn. As of January 8, 1993, reconditioning by mechanical cleaning at less than 50% of the rated capacity of the cleaner is acceptable. Only one attempt and resample is permitted.

There are mixed reports on the effectiveness of cleaning at reducing mycotoxin levels. Generally, in farm grain, the breakage levels are low and cleaning is not as effective. Moldy kernels are weaker, and therefore will break in handling. Cleaning at a later point seems more effective against mycotoxins than cleaning at the farm/country elevator.

Mycotoxin contaminated kernels are less dense than sound kernels. Density-separation is used for peanuts but has not as yet been practical for grain elevators.

Controlled applications of vapor ammonia will breakdown aflatoxin (not other mycotoxins). This has been used for corn and cottonseed. The corn turns dark in the process, and its subsequent use is limited to feed.

Advance Planning

The best method for handling mycotoxin problems is to obtain good advance information as to the potential in your area, then use both visual and objective screening to sort out the worst lots from inbound grain. In the face of a problem is not the time to plan your operating and testing strategy.

The future will hold increasing concerns over trace toxics in general, not just mycotoxins. Analytical technology steadily increases the number of detectable compounds. Customer demands will increase in proportion to knowledge. Preplanning for the problems possible in your area, then periodic review of those plans, is a good investment. Like emergency fire procedures, you hope you never use them, but you know you need them. The list below gives some points to include in your plan.

Know which toxins are possible in your area.
Understand the weather conditions likely to cause toxin development
Have a strategy for getting a pre-harvest estimate of toxin levels
Have a facility-specific testing and handling procedure. Train your personnel.
Know where toxin-contaminated grain could be used.
Have information, options available for producers. Do not leave customers hanging. Arrange for quantitative testing if needed.
Review the plan periodically; update as technology changes.
Hope you never need to use the plan but know you must have one.

Experience has shown that all grain can find a legitimate, safe use if mycotoxin problems are not approached as knee-jerk responses to panic. Be prepared.

Figure 1. Major mycotoxins associated with field crops by general geographic areas in the United States.

A = aflatoxins

O = ochratoxin A

D = deoxynivalenol

T = tremorgens

E = ergot

Z = zearalenone

Source: CAST Task Force Report 116, November 1989.

Figure 2. The effect of subdividing whole corn samples for aflatoxin analysis.

Table 1. Grain mycotoxin information
Mycotoxin	Producing Mold	Grains often affected	Regulatory limits	Utilization limitsc
Aflatoxin	Aspergillus species	Corn, peanuts, rice, cottonseed, milo	20 ppba 20 ppb	0-human animals except dairy
Ochratoxin	Aspergillus, Penicillium	Corn, wheat, rice, mixed feed	.25 ppm 1 ppm	poultry swine
Deoxynivalenol (DON or Vomitoxin)	Fusarium	Corn, wheat, rice, hay, mixed feed, screenings	1 ppm
Zearalenone	Fusarium	Corn, hay, mixed feed, screenings	1 ppm
Fumonisin	Fusarium	Corn, screenings	10 ppm	horses

a ppb- parts per billion; ppm- parts per million
b Aflatoxin is the only mycotoxin with regulatory limits.
c Limits suggested for most sensitive use. Other uses may allow higher levels.

Table 2. Animal and human effects of mycotoxins
Mycotoxin	Animal health effects (most sensitive animals)a Human health effectsb
Aflatoxin	Depressed immune system Intestinal hemorrhage Degeneration of liver In extreme, liver deterioration, intestinal disorders, low level, cancer acceleration
Ochratoxin	Kidney degeneration, decreased egg production (swine, poultry)
Vomitoxin	Feed refusal, gastric disorders (monogastric animals)
Zearalenone	Reproductive disorders (monogastric animals)
Fumonisin	Brain damage, degeneration of liver, heart failure (horses, swine, poultry)

a Does not mean other species are insensitive. Consult veterinarian.
b Aflatoxin documented. Others not determined conclusively.

Table 3. Weather conditions favorable for toxin production
Mold	Favorable weather/storage conditions
Aspergillus	Field	Hot, dry at silking (corn), grain fill (all) Nighttime lows above 75 F. Drought stress
Storagea	Moisture over 17-19% Temperature over 60 F
Fusarium	Field	Cool, wet at grain fill. Excessive rainfall.
Storage	Wet ear corn (above 22% moisture) Not competitive in bulk grain storage

a Aspergillus usually crowded out in wetter grain.
Not enough moisture in dry grain.

Table 4. Examples of mycotoxin survey data
Grain	Location	Year	Mycotoxin	Percent of positive samples	Average concentration of all samples
Corn	Iowa	1983	Aflatoxin	37	21 ppb
Iowa	1988	Aflatoxin	30	21 ppb
N. Carolina	1976-1980	Aflatoxin	29	N/A
Feed	Canada	1990	Vomitoxin (DON)	54	0.4 ppm

Sources:
Agricultrual Research Service publication ARS-83, January 1990. pp 37, 38
Mycotoxins in Grain, American Association of Cereal Chemists, 1992. p 475

Table 5. Data from 1993 Iowa Corn Quality Survey
	Mycotoxinsb (Percent of samples analyzed)
Fumonisin	Vomitoxin	ZLNd
Crop Reporting District (n)	Test Weight (lb/bu)	Proteina (%)	0.5-0.9 ppm	1.0+ ppm	0.0-0.5 ppm	0.5-1.0 ppm	1.0+ ppm	0.0-0.5 ppm
1 NW (30) 2 NC (18) 3 NE (46) 4 WC (29) 5 C (34) 6 EC (36) 7 SW (18) 8 SC (7) 9 SE (22) All (240)	51.3 49.0 51.7 55.1 53.4 53.6 56.4 56.1 55.3 53.2	7.5 7.3 7.5 7.8 7.1 7.3 7.5 6.9 6.9 7.4	7 17 22 24 32 20 50 14 9	3 0 4 17 9 11 11 28 32	88 75 83 91 90 80 69 100 83	0 0 0 0 0 0 0 0 0	0 6 2 0 0 0 0 0 0	0 0 8 9 3 3 0 0 0
Maximum Minimum	58.6 41.8	8.9 6.0	NDc	3.5 ppm	NDc		2.7 ppm	TRc NDc

a Basis 15% moisture
b All samples negative for aflatoxin, T-2, zearalenol and ochratoxin.
c ND- None detected. TR- Trace, not quantifiable.
d Zearalenone
Detection limits: Fumonisin, vomitoxin, zearalenol 0.5 ppm; T-2 toxin 1.0 ppm; aflatoxin 3.0 ppm; ochratoxin 0.1 ppm.

Table 6. Methods for mycotoxin analysis.
Method	Type	Time per sample	Comments
Black-light (ultraviolet)	Screening	1-2 min.	Aflatoxin only. 40-60% false positives. Above 4 glowing particles per 5lb. sample, high likelihood of +20 ppb.
Immunoassay test kits	Screening	5-10 min.	Several toxin kits available. Some chemistry involved. Ground, 5-20g samples. Gives yes or no answer
Immunoassay with reader	Quantitative	5-20 min.	Used by FGIS. Several toxins available
Chromatography	Quantitative	2-4 hours over 2 days	Laboratory confirmation Impractical for elevators All toxins can be tested this way.

Screening- does not measure level, only estimates presence or absence.
Quantitative- estimates concentration in ppm or ppb.

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Mycotoxins in the Grain Market