WHERE TO SEND GRAIN SAMPLES FOR MYCOTOXIN ANALYSIS

P.E. Lipps

Extension Plant Pathologist

Dennis Mills

Extension Associate

The primary reason for moldy grain analysis is to evaluate the quality of the grain and the presence of toxins that are harmful to livestock. Chemical analysis, not a simple mold count, is necessary to evaluate quality for mycotoxin content. To achieve reliable results, these procedures require considerable expertise in the performance and interpretation of the tests, plus sophisticated and expensive equipment. The Ohio State University C. Wayne Ellett Plant and Diagnostic Clinic is not equipped or staffed to provide this service. Commercial, university or government laboratories that are prepared to isolate, purify and quantify the mycotoxins in grain samples for a fee are listed in the table provided. Laboratories on this list are those that have given us their addresses and /or answered our questionnaire. Inclusion on this list does not imply a recommendation of their service. Exclusion of a lab from the list does not imply that it is not to be used.

Sampling procedure

Mycotoxin-contaminated grain is not distributed uniformly throughout the bin. In most cases the toxin level varies from one kernel to another. Moreover, it is possible for highly contaminated kernels to be next to kernels in which the toxin cannot be detected. A single highly contaminated kernel within a 1-pound sample can be a source of significant contamination. Because of this uneven distribution, it is very difficult to obtain representative samples to analyze. In addition, reliability of the methods for mycotoxin analysis are greatly influenced by the sampling procedure and sample size.

To achieve a more accurate estimate of the degree of mycotoxin contamination, it is critical that the collected grain sample actually represent the entire bin or truckload of grain. Follow these tips on how to collect a random sample.

1. Obtain a minimum of 12 samples from different locations in the bin or truck, and bulk all into one. The total weight of the composite sample should be approximately 10 lbs.

2. Hand mix the sample. Subdivide the 10-pound sample into two 5-pound sub samples. The sample size required varies from one lab to another. Studies have shown that the chances of detecting parts per million (ppm) or parts per billion (ppb) levels of toxins in samples are greatly reduced with a smaller sample size. Choose one 5-pound sample to be sent to the laboratory for analysis.

3. Make sure that the moisture content of the sample does not exceed 13 percent. Samples high in moisture cannot be processed immediately. This results in delays and may allow mycotoxin levels to increase during shipment.

4. Pack the sample in a paper bag (not plastic) and place it in a container with a note stating the type of services you wish the laboratory to perform.

5. Information pertaining to the type of analysis, types of mycotoxins that can be analyzed, cost of analysis, size of the sample and time required to report results are provided in the table.

6. In a few special cases there are some advantages of collecting a biased sample. For instance, if there is evidence of moisture damage due to a leak in the bin, it may be more appropriate to collect a sample from the moistened spot and another from the dry area.

Contact the laboratory for shipping instructions and other additional information prior to sending samples. Some laboratories offer a 24-hour emergency service at higher cost, discounts on volume analysis, and a wide variety of other services.

Sampling Truck-Size Volumes of Wheat

An accurate assessment of vomitoxin levels in truck size loads of grain will require careful attention to an appropriate number and depth of probe sampling. Research by Dr. Pat Hart at Michigan State University has shown that increasing the number of probe samples collected per truck increases the probability of an accurate estimate of the vomitoxin level in the wheat. For instance, the vomitoxin level determined from analyzing grain from two probes per truck could be as much as 3.8 ppm different than the actual level of vomitoxin in that truck. Increasing the number of probes increases the accuracy. We recommend testing the grain from a minimum of five probes per truck, and more is better. Five probes should provide an estimate of vomitoxin within 1 ppm of the maximum that could be expected to occur in that truck. Probes should be six feet in length and every probe sample should collect grain from as much of the entire depth of the truck as possible. A one to two pound sample per probe is recommended.

Food and Drug Administration Advisory

Sampling and testing of wheat are necessary to determine that vomitoxin (deoxynivalenol or DON) is below advisory levels in wheat products. The FDA has set the following advisory levels to insure the safety of the food and feed supply.

Intended Use	DON ppm
For bran, flour, and germ intended for human consumption	1
For grain and grain by-products destined for ruminating beef and feedlot cattle older than four months and for chickens with the added recommendation that these ingredients not exceed 50% of the diet of cattle or chickens	10
For grains and grain by-products destined for swine, and all others animals with the added recommendation that these ingredients not exceed 20% of the diet for swine and 40% of the diet for other animals	5

Analytical Methods to Assay for Mycotoxins

Black light, mini-column chromatography (MCC), ELISA quick test, thin-layer chromatography (TLC), gas liquid chromatography (GLC), and high-pressure liquid chromatography (HPLC) are some of the analytical methods used to detect mycotoxins. The accuracy varies from one method to another as well as with the type of agricultural commodity.

Black light. Examining ground corn under an ultraviolet light at a wave length of 365 nm is a quick technique commonly used to determine possible alfaltoxin contamination in corn lots. A bright-green yellow fluorescence (BGYF) under ultraviolet light is indicative of the presence of Aspergillus flavus and/or A. parasiticus, the two alfaltoxin-producing species. The BGYF is emitted not by the alfaltoxin but by the action of enzymes on kojic acid, a metabolite generally co-produced with alfaltoxin. The presence of other organisms, jimson weed seeds, glumes and other foreign material may emit BGYF, leading to misinterpretations. Black light is a presumptive test; it does not tell if alfaltoxin is present. Because of the availability of highly accurate analytical methods to determine the presence of aflatoxin in grain, the black light technique should no longer be used.

Minicolumn is a quick, semi-quantitative technique to detect alfaltoxins. It can detect alfaltoxins as low as 2 ppb but does not distinguish between types of alfaltoxins.

ELISA card or well tests, also known as quick tests, are semi-quantitative tests used by many grain elevators and feed mills. ELISA tests are based on serological identification of mycotoxins in grain samples. Several test kits are commercially available. These tests are popular because they are inexpensive, easy to use, and results can be had in just a few minutes. A positive test indicates that a certain level or more of toxin is present in the grain sample and a negative test indicates that if toxin is present, its concentration is below the detection level of the ELISA test.

Thin-layer chromatography (TLC) is an accurate but expensive method to determine low levels of alfaltoxin. It is also used to test for Zearalenone and trichothecenes. This technique requires special laboratory equipment and trained personnel.

Gas chromatography (GC) is a quantitative method that can accurately identify Deoxynivalenol, Zearalenone and T-2 toxin. It is much more sensitive than the TLC method in testing for the trichothecenes.

High-performance liquid chromatography is the most sensitive method to accurately identify the different types of alfaltoxins.

Laboratory Address Phone and Fax	Types of Mycotoxins Analyzed	Methods Used	Required Sample Size	Time to Report Results	Price Per Sample
East Indiana Grain Inspection (designated by USDA) 7020 N Walnut St Muncie, IN 47303 765-289-1206 voice & fax	Vomitoxin Aflatoxin Fumonisin	ELISA Quantitive	3-5 lb	1 day	$27.00
Holmes Laboratory Box 204 3559 US Rt. 62 Winesburg, OH 44654 800-344-1101 330-893-3094 fax www.holmeslab.com	Alfatoxin Vomitoxin Zearalenone Fumonisin Ochratoxin T-2	ELISA	1 qt	2-4 days	$31.25
Illinois Dept. of Agriculture Animal Disease Laboratory 9732 Shattuc Rd. Centralia, IL 62804 618-532-6701 618-532-1195 fax Steve Kasten skasten@agr.state.il.us	Alfatoxin Zearalenone Deoxynivalenol T-2 Fumonisin B1	TLC, HPLC TLC TLC, HPLC TLC TLC	3-5 lb	2 days 4 days	$40* $40* $40* $40* *All for $100 $40
Iowa State University Veterinary Diagnostic Lab Ames, IA 50011 515-294-1950 515-294-3564 fax Thomas L. Carson DVM, PhD www.vdpam.iastate.edu	Alfatoxin Zearalenone Deoxynivalenol T-2 Ochratoxin Panel includes all the above	TLC, HPLC TLC, HPLC TLC, GC TLC, GC TLC, HPLC	0.5 lb	3 days	$35 $35 $35 $35 $35 $60
Minnesota Valley Testing Laboratories, Inc P.O. Box 249 1126 North Front St. New Ulm, MN 56073-0249 800-782-3557 507-359-2890 fax Kathy Jones	Alfatoxin	ELISA HPLC	0.25 lb	7-10 days	$50 $135

Pennsylvania Department of Agriculture 2305 North Cameron St. Harrisburg, PA 17110 717-787-8808 717-772-3895 fax	Alfatoxin Deoxynivalenol Zearalenone T-2 Fumosin	ELISA TLC GC ELISA HPLC	0.25 lb	2 weeks to screen all	$13 per test
Trilogy Analytical Laboratory 111 West 4^th St. Washington, MO 63090 636-239-1521 636-239-1531 fax www.trilogylab.com	Alfatoxin Deoxynivalenol Ochratoxin T-2 Zearalenone Fumonisin	HPLC and TLC	1 lb	1-5 days	$60-125 $65-130 $65-130 $75-150 $60-125 $80-165
University of Missouri Veterinary Medical Diagnostic Laboratory P.O. Box 6023 Columbia, MO 65205 573-882-6811 573-882-1411 fax George Rottinghaus	Alfatoxin Deoxynivalenol Zearalenone T-2 Vomitoxin Ochratoxin Fumonisin	TLC TLC TLC TLC TLC TLC TLC	0.5 lb	2-3 days	$15* $20 $15* $15 $15* $15* $15 *All for $25
Veterinary Diagnostic Center University of Nebraska-Lincoln Fair St. and East Campus Loop Lincoln, NE 68583 402-472-1434 402-472-3094 fax Michael P. Carlson	Mycotoxin screen includes: Alfatoxin Zearalenone Deoxynivalenol T-2 Ochratoxin A	TLC	5 lb	3-5 days	$36 first sample $18 additional samples
Waters Agricultural Laboratories P.O. Box 382 257 Newton Highway Camilla, GA 31730 229-336-7216 229-336-7967 fax Keith Dominey www.watersag.com	Alfatoxin T-2 Vomitoxin Zearalenone	Immunoassay	1 lb	24 - 48 hours	$25 $46 $46 $46
Woodsen-Tenent Laboratories Inc 313 E Helena St. P.O. Box 164 Dayton, OH 45404 937-222-4179 937-222-7401 fax Dr. Bill Hirt	Alfatoxin T-2 Zearalenone Deoxynivalenol Fumonisin Ochratoxin	TLC ELISA ELISA ELISA ELISA ELISA ELISA	0.1 lb 0.1 lb 0.1 lb 0.1 lb 0.1 lb 0.1 lb	5-7 days 2 days 2 days 2 days 2 days 2 days 2 days	$50 $25 $45 for one sample $30 for five or more samples