page 1 Mycotoxins in Grain and Feed Industries II. Best Practices in Handling and Testing Erin Bowers, Iowa State University, Agricultural Engineering Charles Hurburgh, Iowa State University, Agricultural Engineering Alison Robertson, Iowa State University, Plant Pathology

Learning Objectives This learning module will focus on the sampling, testing, and storage of grains containing mycotoxins Module Objectives: Learn BMP for testing for mycotoxin contamination and for consignment of inbound ingredients (grains) that potentially contain toxins. Learn BMP for preventing the production of mycotoxins in grain or grain sample storage. **BMP=Best Management Practices

Basics: Agriculturally Important Mycotoxins Fungi contaminate grains, food, and feed worldwide Mycotoxins are chemical compounds produced by some fungi More than 400 mycotoxins identified worldwide, 30 concerning for human or animal health 5 principle mycotoxins affect cereal grains (corn, wheat, rye, barley, oats) aflatoxins, fumonisins, deoxynivalenol (vomitoxin), zearalenone, and ochratoxin A Gibberella Ear Rot caused by Gibberella zeae Wheat Scab

Developing Fungus is Dependent on the Environmental Conditions During Pollination and Early Grain Development page 4 Mycotoxin(s)Fungi Favorable Conditions Primary Grains Aflatoxins Aspergillus flavus Aspergillus parasiticus Hot and dry, drought Corn, Durum (in EU) Deoxynivalenol (Vomitoxin) Zearalenone Fusarium graminearum Fusarium culmorum Cool, wet, humid at grain fill Corn, Wheat, Oats, Rye, Barley, Durum Fumonisins Fusarium verticillioides Fusarium proliferatum Warm to hot, dry at and after flowering Corn Ochratoxin A Penicillium verrucosumHarvest conditions determine Corn

Environmental stresses increase susceptibility Planting, harvest, and storage practices also contribute to mycotoxin risk Most mycotoxins remain intact after cooking, drying, freezing or storage conditions. Mycotoxins at very low concentrations (ppm or ppb) are capable of causing serious health problems for humans as well as animals. There is usually no treatment for mycotoxin poisoning (mycotoxicosis). Basics: Mycotoxins in Grain and Feed ppm: parts per million ppb: parts per billion

Poisonous Ingredients in Food Federal Food, Drug, and Cosmetic Act §402 [21 U.S.C. 342] A food shall be deemed adulterated—(a)(1) If it bears or contains any poisonous of deleterious substance which may render it injurious to health; but in case the substance is not an added substance such food shall not be considered adulterated under this clause if the quantity of such substance does not ordinarily render it injurious to health.

Most mycotoxin contamination happens in the field Prevent contaminated grain from entering the facility Pre-harvest preventative controls Scouting and pre-harvest observations Climate and weather conditions At-receiving mycotoxin testing procedure Mycotoxin Management Plan

Output  Laboratory Sample  Analysis Sample  Mycotoxin Result Mycotoxin testing procedure Procedural component 1) Sampling Sample size reduction (if necessary) 2) Sample Preparation Grinding Mixing Subdividing 3) Analysis An example of a grinder that would be used to grind a laboratory sample.

Mycotoxins are not distributed uniformly throughout fields, bins, or transportation containers Localization of moldy grain High variability among individual kernels A few mycotoxin-affected kernels can limit grain use Misclassification of grain Hypothetical distribution of an incoming load of corn with an average aflatoxin level of 10 parts-per-billion (ppb) 0 ppb 100 ppb Heterogeneous distribution

Sampling (the laboratory sample) The process of acquiring a sample of grain (laboratory sample) that is representative of the lot Subject to sampling bias Sample representativeness is increased by taking incremental samples This is a time-consuming practice Not practical to use for every load of grain entering a facility Prescribed for compliance sampling (aflatoxin) Incremental samples are aggregated and mixed to form the laboratory sample

Sampling Equipment x x x x x x x Hand grain probe (trier) Mechanical grain probe sampling a truckload of grain at a receiving point An Ellis sampler can be used to manually sample grain on a conveyor Above: An example of a probing pattern to be used for sampling a flat-bottom truck or trailer. Bottom right: An illustration of the action of a diverter-type sampler. At set intervals a portion of the moving grain is captured and diverted to a sample receiving container by a moving receptacle. Grain flow Receptacle Sample

Corn shelled, whole kernel meal flour snack foods popcorn cleaned, for masa production dry milled bran grits or flaking grits cereals baby cereals Cottonseed whole grain meal Rice cultivated, whole grain wild, whole grain cereals baby cereals Wheat whole kernel whole wheat flour white flour bran, for human consumption cereals baby cereals Oats whole cereals baby cereals Barley whole malt cereals baby cereals Rye flour soya-based baby food products Aflatoxinxxxxxxxxxx Deoxynivalenolxxxx Fumonisinxxxxxxx Ochratoxin Axxxxxxxxxxxxxxxxxxx Products for Surveillance Sampling Abbreviated list adapted from CPG Attachment A

Aflatoxin Compliance Sampling CommodityLot type Number of incremental samples Incremental sample size (lbs) Laboratory sample size (lbs) Corn Shelled, meal, flour, grits Bulk and consumer 101 Oilseed meal e.g., cottonseed Bulk201 Small grains e.g., wheat, barley, sorghum Bulk101 Adapted from FDA Investigations Operations Manual, 2013, Ch. 4, Chart 6

Preparing the laboratory sample for analysis 1.Grinding the entire laboratory sample The smaller the laboratory sample, the higher the uncertainty in the final analytical result 2.Mixing Homogenize 3.Selecting analysis sample Sample preparation An example of a grinder that would be used to grind a laboratory sample.

Operating characteristics of mycotoxin sampling plans What is the probability that a grain lot will be correctly quantified using a given sampling plan? All parameters the same except Green=1 lb laboratory sample Blue=5 lb laboratory sample Orange=10 lb laboratory sample Lot Aflatoxin Concentration (ppb) Probability of Accepting Lot (%)

Operating characteristics of mycotoxin sampling plans Seller’s risk Commercial-false rejection of good quality grain Regulatory-false sanctions Buyer’s risk Commercial-false acceptance of poor quality grain Regulatory-compromises food and feed safety Seller’s Risk Buyer’s Risk Lot Aflatoxin Concentration (ppb) Probability of Accepting Lot (%)

Using an appropriate method to determine the quantity or confirm the presence or absence of mycotoxins in the analysis sample Qualitative or Quantitative Rapid test or analytical laboratory The choice of detection method will depend on the user’s needs and resources, the commodity being tested, and the decisions which will be made using the analytical result. Analysis

High Performance Liquid Chromatography (HPLC) Require trained personnel to maintain and operate Produces results with high Precision Accuracy Repeatability Sensitivity Standard for mycotoxin confirmation analysis in compliance samples Analysis The image below shows an example of an HPLC instrument set up in a lab

Rapid test methods Rapid relative to analytical chemistry methods Common on-site analysis methods Examples of rapid test methods Enzyme-linked immunosorbent assay (ELISA) Lateral flow assay Flow through assay Analysis Left: an example of an ELISA test Right: examples of lateral flow assays

Analysis Left: an example of an ELISA test Right: examples of lateral flow assays GIPSA provides outside laboratory validation of rapid test kits submitted by manufacturers Not mandatory to use a GIPSA-approved kit More information,

Analysis Blacklighting (IOM g) Screening purposes (aflatoxin only) Few false negatives, but many false positives Direct quantitative testing to at-risk-loads Thin-Layer Chromatography (TLC) Above: an example of bright greenish-yellow fluorescence produced when Aspergillus flavus- infected corn is viewed under blacklight

Benefits of rapid tests vs HPLC Ease of use Cost Compact (size) Minimal calibration Less equipment maintenance/upkeep Faster Downfalls of rapid tests Reduced sensitivity Narrow operating range Higher limits of detection Not approved for regulatory decision-making Confirmation analysis necessary by accepted analytical method Compliance Policy Guide Manual, section Analysis

FSMA: Industry action required based upon reasonable suspicion of food/feed safety threat Documentation to verify safe handling, processing, storage, and end use Performance Monitoring: Check-samples ensure appropriateness of mycotoxin management (best practice) Especially in facilities producing finished feed or food products Analysis

Time requirement for testing is significant Can’t test/sample every load Can’t test for every mycotoxin Know the risks and be strategic Weather and climate are good indicators of mycotoxin risk Cool, wet conditions- risk of deoxynivalenol and zearalenone Hot, drought conditions- risk of aflatoxins Warm, drought conditions-risk of fumonisins Use this as a predictive tool to direct sampling and testing Composite testing is good practice to monitor overall quality of incoming grain and of that which has been accepted Practical Considerations

Reduce the ability of fungi to survive and grow Shelf life of grain is impacted by Quality of stored grain Water availability Grain moisture Grain temperature Drying: Reduce grain moisture to stop fungal growth and mycotoxin production Dry promptly: minimize time between harvest and drying to maximize shelf life of grain Dry carefully: prevent stress cracks and reduce breakage Kernel temperature ≤110°F Grain moisture in equilibrium with 65% RH will prevent fungal activity Storage and Handling

Temperature is an important factor for grain storage Cool temperatures (<60°F) minimize fungal growth Aeration can be used to facilitate temperature control Temperature uniformity Minimize moisture movement Monitor grain for changes in quality Storage and Handling

Blending Aflatoxin is a food/feed adulterant Grain >20 ppb cannot be deliberately blended FDA blending dispensations have been allowed in rare instances when a large portion of the U.S. corn supply is affected State by state basis Resultant grain used only for specific livestock feed >500 ppb aflatoxin grain has never been allowed to be blended page 27

Mycotoxins in grain handling facilities Mycotoxin management can be accomplished using proper storage and handling practices, and through preventing contaminated grain from entering a facility Testing domestic grain for mycotoxins is discretionary, export is mandatory for aflatoxin only Each facility will likely handle mycotoxin management uniquely, depending on their individual structure, capabilities, and contract requirements Look for evidence of proactive action Strategic, informed plans and actions

Proactive approaches Contract terms for suppliers Attention to growing conditions in the area Pre-harvest scouting/survey A mycotoxin sampling plan Sampling Sample preparation Analysis Monitoring the effectiveness of the sampling plan Finished product checks and verification Check sample analysis (third party laboratory) Composite sample testing } in combination with a threshold for acceptance

This training was a joint effort of *Funding for this Grain and Feed Mill Operations course was made possible, in part, by the Food and Drug Administration through grant (1U54FD ), views expressed in written materials or publications and by speakers and moderators do not necessarily reflect the official policies of the Department of Health and Human Services; nor does any mention of trade names, commercial practices, or organization imply endorsement by the United States Government.*